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Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2008 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-5083-2 (Hardcover) International Standard Book Number-13: 978-0-8493-5083-2 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www .copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Innovative leukemia and lymphoma therapy / edited by Gertjan J. L. Kaspers . . . . [et al.]. p. ; cm. — (Basic and clinical oncology ; 35) Includes bibliographical references and index. ISBN-13: 978-0-8493-5083-2 (hardcover : alk. paper) ISBN-10: 0-8493-5083-2 (hardcover : alk. paper) 1. Leukemia— Treatment. 2. Lymphomas—Treatment. I. Kaspers, G. J. L., 1963- II. Series. [DNLM: 1. Leukemia—therapy. 2. Lymphoma—therapy. 3. Therapies, Investigational. W1 BA813W v.35 2008 / WH 250 I58 2008] RC643.I46 2008 616.990 41906—dc22 2008006553 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 16th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com
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Foreword
The outcome of therapy for leukemia and malignant lymphoma has improved over the years, mainly in younger patients. Yet, there is no question that the challenges in the area of developmental therapeutics have remained formidable. These challenges relate to the patients who, from the start of treatment, fail to respond to the currently available therapies or combinations of drugs. The outlook of these primarily refractory patients is invariably dismal. Many of the responder patients attaining an initial complete remission, unfortunately, will finally present with relapse of disease. The relapses among the leukemias and high-grade lymphomas usually occur early on, i.e., within the first two years. Both groups, initial nonresponders and secondary failures, pose the notorious difficulty of resistance to conventional therapy. These facts provide an overall notion. Acquired somatic genetic abnormalities of the neoplasms provide keys to the nature of the disease and offer important predictors of treatment failure. They allow to pinpoint individual disease-specific features and distinguish variable disease risks as well as identify those patients with the highest probability of failure. The unmet therapeutic need is, by all standards, greatest among the large population of older patients with hematological cancer in whom response rates are comparatively low, relapse rates are high, and comorbidities prohibit the use of classical chemotherapeutic agents at effective dose levels. Scientists are on the way to discovering new drugs with different modes of action that can overcome the limitations of today’s selection of drugs. Numerous new drugs are currently in early clinical development with the aim of circumventing the clinical bottleneck of chemotherapy resistance. In the coming years, several of these compounds are expected to settle as members of the standard armamentarium of drugs available to the patient with a hematological tumor. New drugs may be designed with the deliberate objective of affecting a known molecular lesion or signaling pathway in the cancer cell, thus critically inhibiting
iii
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Foreword
tumor cell survival. These therapeutic compounds may tackle distinct, molecularly defined subtypes of leukemia or lymphoma, and one would anticipate that their greater specificity will allow for application with enhanced efficacy and reduced toxicity. Currently, we are witnessing the development of diagnostic technologies that directly impact decision making in the clinical management of patients with hematological malignancies. These technologies relate, on the one hand, to more precise tissue diagnosis and involve innovative genomic, proteomic, and immunological techniques. On the other hand, they involve improved in vivo imaging methods, enabling a better and more sensitive visualization of neoplastic deposits in the body. These techniques, when appropriately validated for clinical use, will enable the distinction of prognostic disease subcategories and allow for a specific diagnosis according quantitative, sensitive, and objective parameters. This type of information will guide therapeutic decisions at the outset of treatment. It will also provide substantial insights that will be useful in monitoring treatment effects throughout the therapeutic management of patients and redirect treatment choice. An ambitious diagnostic approach makes sense if there is a choice for the physician among a broader scale of available therapeutic options. One of the major objectives of today’s molecular diagnostics relates to the identification of new druggable targets for pharma developments. Innovative Leukemia and Lymphoma Therapy appropriately and critically deals with each of the issues and challenges as regards developmental therapeutics. The book highlights current, clinically relevant diagnostic strategies for high-throughput diagnosis and disease response monitoring. The book covers, in a series of individual chapters, a collection of overviews that highlight clinically relevant novel therapeutic strategies in concise reviews. It also provides updates on therapeutic compounds with new mechanisms of action that currently raise intense interest and are in active development. This book comes as a timely resource of information that furnishes a state-of-the-art and comprehensive compendium, which will be of value to the interested clinician, researcher, and student. Bob Lo¨wenberg Erasmus University Medical Center Rotterdam, The Netherlands
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Preface
The treatment of leukemia and lymphoma is rapidly developing from conventional chemotherapy toward a more tailored and targeted, innovative therapy. However, conventional therapy is making progress as well. Targeted treatment with increased efficacy and less side effects is becoming more and more a reality, facilitated by fascinating developments such as oncogenomic studies and sophisticated drug engineering. Knowledge on determinants of chemosensitivity is also rapidly increasing. Together with pretreatment individualized tumor response testing and with improved monitoring of treatment response by minimal residual disease measurements, treatment will indeed become more tailored and individualized. This book gives a complete and up-to-date overview of exciting new treatment modalities in leukemia and lymphoma that have been introduced in the clinic or will be introduced in the near future. Well-known international experts summarize clinical studies on drugs such as tyrosine kinase inhibitors, monoclonal antibodies, proteasome inhibitors, farnesyl transferase inhibitors, hypomethylating agents, histone deacetylase inhibitors, mTOR targeting agents, Notch pathway inhibitors, and inhibitors of cyclin-dependent kinases. The first few chapters deal with methodological issues such as gene expression profiling to detect new drug targets, individualized tumor response testing aiming at selecting effective drugs, minimal residual monitoring to adapt treatment based on actual treatment response, and statistical issues concerning clinical studies in small subgroups of patients, while some discuss modulation of drug resistance and improvements in allogeneic bone marrow transplantation. Other chapters summarize targeting regulators of apoptosis, radioimmunotherapy, immunotherapy by vaccination, gene-directed therapy, and anti-angiogenesis approaches. The chapters provide a concise summary of the treatment rationale, of the pathways that are involved, and of relevant preclinical research, whenever relevant.
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Preface
We recommend this well-illustrated, comprehensive book to students, scientists, and clinicians with a special interest in innovative therapy who are involved not only in research and/or treatment of leukemia and lymphoma in particular, but in other malignancies as well. G. J. L. Kaspers Bertrand Coiffier Michael C. Heinrich Elihu Estey
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Contents
Foreword Bob Lo¨wenberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1.
2.
Gene Expression Profiling to Detect New Treatment Targets in Leukemia and Lymphoma: A Future Perspective . . . . . . . . . Torsten Haferlach, Wolfgang Kern, and Alexander Kohlmann
1
Individualized Tumor Response Testing in Leukemia and Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrew G. Bosanquet, Peter Nygren, and Larry M. Weisenthal
23
3.
Minimal Residual Disease ........................... Jacques J. M. van Dongen, Tomasz Szczepa nski, and Vincent H. J. van der Velden
45
4.
New Methods for Clinical Trials: AML as an Example Elihu Estey
......
85
5.
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ch. Michel Zwaan and Marry M. van den Heuvel-Eibrink
99
Monoclonal Antibodies in the Treatment of Malignant Lymphomas and Chronic Lymphocytic Leukemia ........ Bertrand Coiffier
125
6.
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viii
Contents
Radioimmunotherapy of Hematological Malignancies Tim Illidge and James Hainsworth
8.
Differentiation Induction in Acute Promyelocytic Leukemia . . . . 185 Adi Gidron and Martin S. Tallman
9.
DNA Methylation and Epigenetics: New Developments in Biology and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . Jesus Duque, Michael L€ ubbert, and Mark Kirschbaum
207
The Emerging Role of Histone Deacetylase Inhibitors in the Treatment of Lymphoma . . . . . . . . . . . . . . . . . . . . . . Matko Kalac and Owen A. O’Connor
233
10.
......
149
7.
. . . 257
11.
Antileukemic Treatment Targeted at Apoptosis Regulators Simone Fulda and Klaus-Michael Debatin
12.
Angiogenesis in Hematological Malignancies . . . . . . . . . . . . . Alida C. Weidenaar, Hendrik J. M. de Jonge, Arja ter Elst, and Evelina S. J. M. de Bont
13.
Nucleic Acid-Based, mRNA-Targeted Therapeutics for Hematologic Malignancies . . . . . . . . . . . . . . . . . . . . . . . . Alan M. Gewirtz
283
311
14.
Active Specific Immunization by the Use of Leukemic Dendritic Cell Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Ilse Houtenbos, Gert J. Ossenkoppele, and Arjan A. van de Loosdrecht
15.
CDK Inhibitors in Leukemia and Lymphoma Yun Dai and Steven Grant
............
353
16.
FLT3: A Receptor Tyrosine Kinase Target in Adult and Pediatric AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark Levis, Patrick Brown, and Donald Small
379
Treatment of Chronic Myeloid Leukemia with Bcr-Abl Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael J. Mauro and Michael C. Heinrich
411
Tyrosine Kinase Inhibitors: Targets Other Than FLT3, BCR-ABL, and c-KIT ............................. Suzanne R. Hayman and Judith E. Karp
429
17.
18.
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Contents
19.
ix
Tyrosine Phosphatases as New Treatment Targets in Acute Myeloid Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . I. Hubeek, K. Hoorweg, J. Cloos, and G. J. L. Kaspers
20.
Proteasome and Protease Inhibitors . . . . . . . . . . . . . . . . . . . N. E. Franke, J. Vink, J. Cloos, and G. J. L. Kaspers
21.
Farnesyltransferase Inhibitors: Current and Prospective Development for Hematologic Malignancies ............. Judith E. Karp
22.
Targeting Notch Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . Jennifer O’Neil and A. Thomas Look
23.
mTOR Targeting Agents for the Treatment of Lymphoma and Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrea E. Wahner Hendrickson, Thomas E. Witzig, and Scott H. Kaufmann
24.
25.
449 469
491 513
525
Allogeneic Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning ...................... Fre´de´ric Baron, Frederick R. Appelbaum, and Brenda M. Sandmaier
539
Modulation of Classical Multidrug Resistance and Drug Resistance in General . . . . . . . . . . . . . . . . . . . . . . . . . Branimir I. Sikic
563
Index
..............................................
581
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Contributors
Frederick R. Appelbaum Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington, U.S.A. Fre´de´ric Baron Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A. Andrew G. Bosanquet
Bath Cancer Research, Royal United Hospital, Bath, U.K.
Patrick Brown Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, U.S.A. J. Cloos Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Bertrand Coiffier Hematology Department, Hospices Civils de Lyon and Claude Bernard University, Pierre-Benite, France Yun Dai Department of Medicine, Virginia Commonwealth University and Massey Cancer Center, Richmond, Virginia, U.S.A. Evelina S. J. M. de Bont Department of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Hendrik J. M. de Jonge Department of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Klaus-Michael Debatin
University Children’s Hospital, Ulm, Germany
Jesus Duque Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany
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Contributors
Elihu Estey Division of Hematology, University of Washington Medical Center, Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A. N. E. Franke Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Simone Fulda
University Children’s Hospital, Ulm, Germany
Alan M. Gewirtz Division of Hematology/Oncology, Department of Medicine & Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Adi Gidron Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, U.S.A. Steven Grant Department of Medicine, Biochemistry, and Pharmacology, Virginia Commonwealth University and Massey Cancer Center, Richmond, Virginia, U.S.A. Torsten Haferlach
Munich Leukemia Laboratory, Munich, Germany
James Hainsworth Paterson Institute of Cancer Research, School of Medicine, University of Manchester, Manchester, U.K. Suzanne R. Hayman Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, U.S.A. Michael C. Heinrich Center for Hematologic Malignancies and Departments of Medicine and Cell and Developmental Biology, Oregon Cancer Institute, Oregon Health & Science University and Portland VA Medical Center, Oregon Health & Science University, Portland, Oregon, U.S.A. K. Hoorweg Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Ilse Houtenbos Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands I. Hubeek Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Tim Illidge Paterson Institute of Cancer Research, School of Medicine, University of Manchester, Manchester, U.K. Matko Kalac Herbert Irving Comprehensive Cancer Center, The New York Presbyterian Hospital, Columbia University, New York, New York, U.S.A. Judith E. Karp Division of Hematologic Malignancies, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, U.S.A.
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Contributors
xiii
G. J. L. Kaspers Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Scott H. Kaufmann Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, U.S.A. Wolfgang Kern
Munich Leukemia Laboratory, Munich, Germany
Mark Kirschbaum Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte, California, U.S.A. Alexander Kohlmann
Roche Molecular Systems, Pleasanton, California, U.S.A.
Michael L€ ubbert Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany Mark Levis Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, U.S.A. A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, U.S.A. Michael J. Mauro Center for Hematologic Malignancies, Oregon Cancer Institute, Oregon Health & Science University, Portland, Oregon, U.S.A. Peter Nygren Department of Oncology, Radiology, and Clinical Immunology, University Hospital, Uppsala, Sweden Owen A. O’Connor Herbert Irving Comprehensive Cancer Center, The New York Presbyterian Hospital, Columbia University, New York, New York, U.S.A. Jennifer O’Neil Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, U.S.A. Gert J. Ossenkoppele Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands Brenda M. Sandmaier Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington, U.S.A. Branimir I. Sikic Oncology Division, Department of Medicine, Stanford University School of Medicine, Stanford, California, U.S.A. Donald Small Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, U.S.A. Tomasz Szczepa nski Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands, and Department of Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze, Poland
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Contributors
Martin S. Tallman Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, U.S.A. Arja ter Elst Department of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Marry M. van den Heuvel-Eibrink Department of Pediatric Oncology/ Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands Arjan A. van de Loosdrecht Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands Vincent H. J. van der Velden Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Jacques J. M. van Dongen Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands J. Vink Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands Andrea E. Wahner Hendrickson Rochester, Minnesota, U.S.A.
Department of Medicine, Mayo Clinic,
Alida C. Weidenaar Department of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Larry M. Weisenthal California, U.S.A. Thomas E. Witzig Minnesota, U.S.A.
Weisenthal Cancer Group, Huntington Beach,
Department of Medicine, Mayo Clinic, Rochester,
Ch. Michel Zwaan Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands
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BASIC AND CLINICAL ONCOLOGY Series Editor Bruce D. Cheson Professor of Medicine and Oncology Head of Hematology Georgetown University Lombardi Comprehensive Cancer Center Washington, D.C.
1. Chronic Lymphocytic Leukemia: Scientific Advances and Clinical Developments, edited by Bruce D. Cheson 2. Therapeutic Applications of Interleukin-2, edited by Michael B. Atkins and James W. Mier 3. Cancer of the Prostate, edited by Sakti Das and E. David Crawford 4. Retinoids in Oncology, edited by Waun Ki Hong and Reuben Lotan 5. Filgrastim (r-metHuG-CSF) in Clinical Practice, edited by George Morstyn and T. Michael Dexter 6. Cancer Prevention and Control, edited by Peter Greenwald, Barnett S. Kramer, and Douglas L. Weed 7. Handbook of Supportive Care in Cancer, edited by Jean Klastersky, Stephen C. Schimpff, and Hans-Jo¨rg Senn 8. Paclitaxel in Cancer Treatment, edited by William P. McGuire and Eric K. Rowinsky 9. Principles of Antineoplastic Drug Development and Pharmacology, rard A. Milano, and Mark J. Ratain edited by Richard L. Schilsky, Ge 10. Gene Therapy in Cancer, edited by Malcolm K. Brenner and Robert C. Moen 11. Expert Consultations in Gynecological Cancers, edited by Maurie Markman and Jerome L. Belinson 12. Nucleoside Analogs in Cancer Therapy, edited by Bruce D. Cheson, Michael J. Keating, and William Plunkett 13. Drug Resistance in Oncology, edited by Samuel D. Bernal 14. Medical Management of Hematological Malignant Diseases, edited by Emil J Freireich and Hagop M. Kantarjian 15. Monoclonal Antibody-Based Therapy of Cancer, edited by Michael L. Grossbard 16. Medical Management of Chronic Myelogenous Leukemia, edited by Moshe Talpaz and Hagop M. Kantarjian
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17. Expert Consultations in Breast Cancer: Critical Pathways and Clinical Decision Making, edited by William N. Hait, David A. August, and Bruce G. Haffty 18. Cancer Screening: Theory and Practice, edited by Barnett S. Kramer, John K. Gohagan, and Philip C. Prorok 19. Supportive Care in Cancer: A Handbook for Oncologists: Second Edition, Revised and Expanded, edited by Jean Klastersky, Stephen C. Schimpff, and Hans-Jo¨rg Senn 20. Integrated Cancer Management: Surgery, Medical Oncology, and Radiation Oncology, edited by Michael H. Torosian 21. AIDS-Related Cancers and Their Treatment, edited by Ellen G. Feigal, Alexandra M. Levine, and Robert J. Biggar 22. Allogeneic Immunotherapy for Malignant Diseases, edited by John Barrett and Yin-Zheng Jiang 23. Cancer in the Elderly, edited by Carrie P. Hunter, Karen A. Johnson, and Hyman B. Muss 24. Tumor Angiogenesis and Microcirculation, edited by Emile E. Voest and Patricia A. D’Amore 25. Controversies in Lung Cancer: A Multidisciplinary Approach, edited by Benjamin Movsas, Corey J. Langer, and Melvyn Goldberg 26. Chronic Lymphoid Leukemias: Second Edition, Revised and Expanded, edited by Bruce D. Cheson 27. The Myelodysplastic Syndromes: Pathology and Clinical Management, edited by John M. Bennett 28. Chemotherapy for Gynecological Neoplasms: Current Therapy and Novel Approaches, edited by Roberto Angioli, Pierluigi Benedetti Panici, John J. Kavanagh, Sergio Pecorelli, and Manuel Penalver 29. Infections in Cancer Patients, edited by John N. Greene 30. Endocrine Therapy for Breast Cancer, edited by James N. Ingle and Mitchell Dowsett 31. Anemia of Chronic Disease, edited by Guenter Weiss, Victor R. Gordeuk, and Chaim Hershko 32. Cancer Risk Assessment, edited by Peter G. Shields 33. Thrombocytopenia, edited by Keith R. McCrae 34. Treatment and Management of Cancer in the Elderly, edited by Hyman B. Muss, Carrie P. Hunter, and Karen A. Johnson 35. Innovative Leukemia and Lymphoma Therapy, edited by G. J. L. Kaspers, Bertrand Coiffier, Michael C. Heinrich, and Elihu Estey
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1 Gene Expression Profiling to Detect New Treatment Targets in Leukemia and Lymphoma: A Future Perspective Torsten Haferlach and Wolfgang Kern Munich Leukemia Laboratory, Munich, Germany
Alexander Kohlmann Roche Molecular Systems, Pleasanton, California, U.S.A.
INTRODUCTION The standard methods for establishing the diagnosis and prognosis of acute leukemias and lymphomas are cytomorphology and cytochemistry in combination with multiparameter immunophenotyping. However, cytogenetics, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR)-based assays add important information with respect to biologically defined and prognostically relevant subgroups. Together, a combination of different methods allows a comprehensive diagnosis with relevant clearly defined subentities. It also leads to a better understanding of the respective clinical course of defined disease subtypes and to a more or less disease-specific therapeutic approach. However, not all patients achieve complete remission during treatment, and many of those who do, later develop relapse and treatment-resistant disease. To overcome these problems, the microarray technology, which quantifies gene expression intensities of thousands of genes in a single analysis, holds the potential to become an essential tool for a strictly molecularly defined classification of leukemias and lymphomas.
1
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It may therefore be used at first as a novel routine method for diagnostic approaches in the near future (1). But even more importantly, it will also reveal new genetic and therapeutically relevant markers and should guide the search for new targets. Gene expression profiling will also improve patient selection to test therapeutic hypothesis most efficiently and may help define dose and schedule determination. This chapter outlines the major steps for gene expression profiling analyses to approach these different goals by starting at a better diagnostic characterization of leukemias and lymphomas hopefully ending up with new targets for individual treatment of the respective patients. MICROARRAYS AND THE ERA OF FUNCTIONAL GENOMICS Both biology and medicine are undergoing a revolution that is based on the accelerating determination of DNA sequences, including the completion of whole genomes of a growing number of organisms (2). In parallel to the sequencing efforts, a wide range of technologies with tremendous potential has been achieved that can take advantage of the vast quantity of genetic information being now available. The field of functional genomics seeks to devise and apply these technologies, such as microarrays, to analyze the full complement of genes and proteins encoded by an organism to understand the functions of genes and proteins (3) (Fig. 1).
Figure 1 Different types of microarray platforms. Microarray platforms vary according to the solid support used (such as glass slides or silicon wafers), the surface modifications with various substrates, the type and length of DNA fragments on the array (such as cDNA or oligonucleotides), whether the gene fragments are presynthesized and deposited, or synthesized in situ, the machinery used to place the fragments on the array (such as ink-jet printing, spotting, mask, or micromirror-based in situ synthesis), and the method of sample preparation. Currently, combinations of these variables are used to generate two main types of microarrays: spotted glass slide arrays (right) and in situ synthesized DNA-oligonucleotide arrays (left).
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Glass Slide Microarrays Glass slide microarrays were first produced in Patrick Brown’s laboratory at Stanford University (4). In glass slide microarray studies, ribonucleic acid (RNA) species from the test sample and from the reference sample are studied pairwise as an equivalent mixture in which the control RNA is the reference for expressing the gene transcript levels in the target sample (Fig. 1). Various direct and indirect labeling methods for the sample have been developed (5). The majority of expression analysis labeling protocols is based on the reverse transcription of mRNA, either from highly purified poly(A) mRNA or total RNA extracts and often include amplification steps. In most protocols, one sample is labeled with the Cy3 (green) fluorochrome, the other with Cy5 (red). The labeled cRNA molecules hybridize to the corresponding cDNA or long oligonucleotides, of which the exact position on the array is known. The binding of the target to the probe is detected by scanning the array, typically using either a scanning confocal laser or a charge coupled device (CCD) camera-based reader. After scanning, software calculations provide the ratios between green and red fluorescence for each spot, corresponding to the relative abundance of mRNA from a particular gene in the target sample versus the reference sample. However, the technical difficulties in the reproducible production of glass slide microarrays should not be underestimated (5). Much of this variation is introduced systematically during the spotting of the DNA onto the slide surface, and many of the initial cDNA clone sets were compromised by contamination with T1 phage, multiple clones in individual wells, and incorrect sequence assignment. Thus, given the lack of a gold standard for the production of glass slide microarrays using current technologies, there is a high degree of variation in the quality of data derived from glass slide microarray experiments. This poor reproducibility not only adds to the cost of a given study but also leads to data sets that are difficult to interpret. MICROARRAYS AS AN INNOVATIVE TECHNIQUE TO DETECT NEW TARGETS For several reasons many investigations using microarrays for biological approaches today are performed on the whole genome Affymetrix U133 set (HG-U133A and HG-U133B or the HG-U133 2.0 plus array; Affymetrix, Santa Clara, California, U.S.). A detailed up-to-date description on sequences and probe selection rules is available as technical note from the manufacturer (www .affymetrix.com). Affymetrix HG-U133A and HG-U133B Microarrays The U133 two-array set provides comprehensive coverage of well-substantiated genes in the human genome. It can be used to analyze the expression level of
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39,000 transcripts and variants, including greater than 33,000 human genes. The two arrays comprise more than 45,000 probe sets and 1,000,000 distinct oligonucleotide features. The sequences from which these probe sets were derived were selected from GenBank, dbEST, and RefSeq. The sequence clusters were created from the UniGene database (Build 133, April 20, 2001) and then refined by analysis and comparison with a number of other publicly available databases, including the Washington University EST trace repository and the University of California, Santa Cruz, Golden-Path human genome database (April 2001 release). In addition, an advanced understanding of probe uniqueness and hybridization characteristics allowed an improved selection of probes based on predicted behavior. The U133 chip design uses a multiple linear regression model that was derived from a thermodynamic model of nucleic acid duplex formation. This model predicts probe binding affinity and linearity of signal changes in response to varying target concentrations. The two arrays are manufactured as standard format arrays with a feature size of 18 mm and use 11 probe pairs per sequence. The oligonucleotide length is 25 mer. Human Genome U133 Plus 2.0 Array In addition to all the sequences represented on the HG-U133A and HG-U133B two-array set, the HG-U133 Plus 2.0 microarray also covers 9921 new probe sets representing approximately 6500 new genes. These gene sequences were selected from GenBank, dbEST, and RefSeq. Sequence clusters were created from the UniGene database (Build 159, January 25, 2003) and refined by analysis and comparison with a number of other publicly available databases, including the Washington University EST trace repository and the NCBI human genome assembly Build 31 (www.affymetrix.com). Thus, in using this comprehensive whole human genome expression array, an extensive coverage of the human genome is reached. HG-U133 Plus 2.0 microarrays are manufactured as standard format arrays with more than 54,000 probe sets of a feature size of 11 mm and use 11 probe pairs per sequence. The oligonucleotide length is 25 mer. MICROARRAY DATA ANALYSIS A wide range of approaches is available for gleaning insights from the data obtained from transcriptional profiling. Data analyses are performed by two different approaches, i.e., the supervised approach and the unsupervised approach (Fig. 2). Unsupervised analyses are used to test the hypothesis whether specific characteristics, e.g., genetic aberrations, are also reflected at the level of gene expression signatures. Supervised analyses identify a minimal set of genes that could be used to stratify those patients after a training of classification engines (6–8). The gene lists from supervised analyses can also be further
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Figure 2 Overview about a common workflow to analyze microarray data. After preparation of corresponding data sets from the main master table, the data are analyzed either unsupervised or supervised. Unsupervised analyses are performed by hierarchical clustering or principal component analysis. In the supervised analyses, differentially expressed genes can be identified by various methods and selected for further interpretations, e.g., visualization by hierarchical clustering, principal component analysis, plotting as bar graphs, or generation of biological networks. In addition, differentially expressed genes can be selected for classification tasks where several different machinelearning approaches have to be applied.
interpreted in terms of underlying biology. For all gene expression profiles, master data tables have to be maintained. In these tables, rows represent all genes for which data have been collected and columns represent microarray experiments from individual patients. Each cell represents the measured fluorescence intensity from the corresponding target probe set on the microarray. Before analyzing the data, it is a routine procedure to normalize the data. This procedure is a mandatory step in the data-mining process to appropriately compare the measured gene expression levels. U133 set microarray signal intensity
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values can be normalized by scaling the raw data intensities to a common target intensity using a recommended mask file. Some Examples of Software to Identify Genes of Interest Several software packages are used for principal data acquisition (GCOS), storage (MicroDB), and analysis (DMT). The following tables give only some examples to approach data. Individual gene expression profiles can also further be prepared as Microsoft Excel tables. Software
Source
Internet
GCOS MicroDB DMT
Affymetrix, Inc. Affymetrix, Inc. Affymetrix, Inc.
www.affymetrix.com/support/ www.affymetrix.com/support/ www.affymetrix.com/support/
The following packages can be applied for the identification of differentially expressed genes and classification: Software
Source
Internet
SAM
Stanford University
Bioconductor q-Value
Open source University of Washington National Taiwan University
www-stat.stanford.edu/~tibs/SAM/ index.html www.bioconductor.org faculty.washington.edu/~jstorey/qvalue/
LIBSVM
www.csie.ntu.edu.tw/~cjlin/libsvm/
SAM is available as Microsoft Excel Add-in (9). Bioconductor is an open source and open development software project for the analysis and comprehension of genomic data. Bioconductor packages provide statistical and graphical methodologies for analyzing genomic data. LIBSVM (Version 2.6) is a software solution for SVM-based classification. The q-value software takes a list of p-values resulting from the simultaneous testing of many hypotheses and estimates their q-values (10). In addition, further third party software packages can be used for statistical analyses and data visualization. Software
Source
Internet
SPSS Pathways Analysis GeneMaths Genomics Suits
SPSS, Inc. Ingenuity Systems Applied Maths, Inc. Partek, Inc.
www.spss.com/ www.ingenuity.com www.applied-maths.com www.partek.com/
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Tools for Pathway Analyses to Detect New Targets and Correlations The identification of diagnostic, prognostic, or therapeutic markers in leukemia and lymphoma following microarray experiments and their biostatistical read outs have to then focus on the discovery of important pathways in these tumors. Several programs exist in order to identify pathways involved. These include Pathway Assist (http://www.ariadnegenomics.com/products/pathway.html), DAVID (http:// apps1.niaid.nih.gov/david/), and Ingenuity (http://www.ingenuity.com/). As one example, Ingenuity enables researchers to model, analyze, and understand complex biological systems foundational to human health and disease. This includes pathways analysis software and knowledge databases for biologists and biostatisticians and enterprise knowledge management infrastructure. Today, Ingenuity is a useful knowledge base of biological networks with curated relationships between proteins, genes, complexes, cells, tissues, drugs, and diseases. Increasingly, also bioinformaticians are interested in developing analytical tools that help scientists interpret experimental data especially in the context of pathways and biological systems. These analytical tools have broad application throughout research and development, from validating targets by uncovering disease-related pathways to predicting pathways perturbed by therapeutic compounds. As one example in Ingenuity, a broad genome-wide coverage of over 25,900 mammalian genes (11,100 human, 5500 rat, and 9300 mouse) can be found and millions of pathway interactions extracted from literature are managed interactively and web based. At a basic level, an understanding of functions and pathways associated with genes identified within an early-stage candidate region may assist in prioritizing portions of this region for further investigation, e.g., targeted association using higher densities of single nucleotide polymorphisms (SNPs). This type of approach may even assist in identifying which genes to resequence in an attempt to identify further SNPs for association studies. This is achievable now with the ability to upload, for example, Affymetrix SNP identifiers directly into pathway software such as Ingenuity. Future developments may increase the mapping coverage of SNPs beyond the simple 1:1 gene to SNP mapping available today. Beyond this, future functionality may even allow for the correlation between multiple regions of the genome identified at a functional level and findings of a genetic association study that identifies multiple, low scoring regions. Previously, these may not have warranted further investigation based solely on association scores. However, functional, process, pathway, or disease annotations may implicate multiple regions as being relevant to a particular phenotype by virtue of their compound effect. Evidence is already emerging from the HapMap project that there are significant SNPs that are genetically indistinguishable across large regions of individual chromosomes or even different chromosomes. It is anticipated that further development of software and pathway analyses tools to approach the huge sets of data generated in microarray experiments will lead to deeper insights.
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DETECTION OF NEW TARGETS IN LEUKEMIA AND LYMPHOMA As has been outlined before, gene expression profiling has been extensively used for tumor classification (8,11–15) and is on the way to add important information to predict response to therapy as well as for outcome in leukemia and lymphoma patients. As these data are not in the focus of this article, they will only be cited, if they add information also for new target detection. Furthermore, there are only limited efforts yet to incorporate microarrays into clinical trials in hematology and oncology because of several reasons: (1) prospective sample acquisition parallel to the gold standard diagnostic procedures is needed, (2) standardized equipment and software has to be used, (3) experienced scientists and technicians with respect to microarray analyses have to be involved, and (4) funding is mostly lacking and would be best if academic institutions and industry combine efforts. Other factors like intra-laboratory and inter-laboratory comparability have also to be taken into account. This leads to the following relation according to Weeraratna (16): More than 9000 references are available that concern microarrays, but only around 20 are clinical trials, and less than 10 of these pertain to cancer. As currently no single prospective trial has been conducted to our knowledge to address the use of microarrays within a clinical trial in leukemia and lymphoma, we only can rely on information that was published in papers referring to diagnostic or prognostic questions. On the basis of their findings, some preliminary statements can also be made for the use of gene expression profiling to define new targets and drugs in leukemia and lymphoma (17). The following chapters will comment on these aspects and will be subdivided disease specifically. Detection of New Targets in Lymphoma Alizadeh et al. (13) defined distinct subtypes of diffuse large B-cell lymphoma (DLBCL) by specific gene expression signatures. Although this paper mostly focuses on newly defined biological subgroups of DLBCL, different prognosis was also detected. This again leads to the detection of genes that are responsible not only for a better and novel subclassification but also transfer into striking differences in prognosis if patients are treated uniformly. Thus, the authors concluded that a respective gene expression pattern and the IPI score for NHL in combination will guide therapeutic decisions including bone marrow transplantation as one option for high-risk patients. Furthermore, expression profiling may also help to detect homogeneous groups of patients to improve the likelihood of observing treatment efficacy in specific disease entities. This study was the first to show that the two DLBCL subgroups differentially expressed entire transcriptional modules composed of hundreds of genes. Polo et al. identified a discrete subset of DLBCL that are reliant on Bcl6 signaling and uniquely sensitive to Bcl6 inhibitors (18). Therefore, successful new therapeutics may be aimed at the upstream signal-transducing molecules and further investigations are needed.
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Microarrays have also been used to study the targets of c-Myc, a transcription factor that plays a role in Burkitt’s lymphoma as c-Myc is involved in the chromosomal translocation t(8;14). In this study genomic targets including genes involved in cell cycle, cytoskeletal organization, cell growth, and adhesion were identified (19). However, these structures have to be tested again as drug targets after having been detected by gene expression profiling. Detection of New Targets in Acute Myeloid Leukemia Yagi et al. (20) analyzed 54 pediatric acute myeloid leukemia (AML) using Affymetrix U95A arrays and focused on the reproducibility of some FAB subtypes and especially on gene patterns to predict outcome. After unsupervised clustering, they were able to differentiate patients with t(8;21) from those with inv(16) and from those demonstrating an AML M4/5 or AML M7 phenotype or immunophenotype by specific gene expression signatures. Within this unsupervised analysis, no specific profile was found that correlated to the prognosis of the patients. Since the inclusion of further cases with other FAB subtypes and cytogenetic abnormalities (no karyotype was available in 9 of 54 cases) resulted in an increased heterogeneity, the authors restricted their further analyses to the genetically and morphologically better-defined subentities. For further calculation, data were analyzed and supervised with respect to outcome and prognosis. A subset of 35 genes that were independent from the morphology or karyotype of the patients was selected; some of them are associated with the regulation of the cell cycle or with apoptosis. By hierarchical cluster analysis, patients could be classified into high-risk and low-risk groups with highly significant differences in event-free survival (EFS) ( p < 0.001). Another approach was described by Qian et al. (21) in therapy-related AML and myeloid cell lines focussing on CD34-positive selected cells. They were the first ones to define a specific pattern of gene expression for t-AML in comparison with other AML subtypes. The most discriminating genes were found to be involved in arrested differentiation of early progenitor cells. A higher expression of cell cycle control genes such as CCNA2, CCNE2, and CDC2 and genes for cell cycle checkpoints such as BUB1 or growth (Myc) were found. Furthermore, downregulation of transcription factors involved in early hematopoiesis (TAL1, GATA1, EKLF) and overexpression of FLT3 was detected. The authors concluded that these genes may be further investigated for new targets and drugs in this very unfavourable subtype of AML. As a further hallmark in AML, Bullinger et al. analyzed 65 peripheral blood and 54 bone marrow samples in patients with AML (12). On the basis of 6283 most variably expressed genes they were able to reproduce cytogenetically defined AML subgroups and, in addition, to define two different groups with highly differing prognosis on the basis of gene expression profiles. While both groups mainly included AML cases with normal karyotypes without differences in many prognostic parameters, it is noteworthy that the group with the poorer
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prognosis included more patients with monosomy 7, complex aberrant karyotypes, and length mutations of FLT3, while the group with the better prognosis included more patients with inv(16). Thus, the observed differences in the prognosis between both groups may be largely due to imbalances in profiles of established prognostic factors rather than due to the identification of a newly characterized biological subgroup of AML. Genes as published by Bullinger et al. should be tested in independent cohorts of AML patients to further support their prognostic power, and further investigations are again warranted for the definition of such genes as new targets in AML treatment. Similar results have been reported by Valk et al. (11) who discovered 16 groups of AML featuring distinct gene expression profiles on the basis of microarray analysis, which, in addition, showed significant differences in clinical course. However, while many of the identified groups were characterized by specific cytogenetic aberrations known to be highly predictive of outcome, none of the groups were restricted to cases without cytogenetic abnormalities. Thus, the task remains to identify markers capable of discriminating prognostically different cases out of the heterogeneous group of AML with normal karyotype and to use these for target testing. An improvement in this direction has been reported by Kern et al. who analyzed gene expression profiles in 205 patients with AML and normal karyotype (22). In order to identify genetically defined subgroups, an unsupervised principal component analysis revealed 79% of cases clustering together, while a subgroup comprising 21% of cases formed another cluster. Importantly, the analysis of known genetic markers, including the presence of length mutations and point mutations of FLT3, partial tandem duplications of MLL, or mutations of CEBPA, NRAS, or CKIT, did not reveal differences between both groups. Significant differences were found, however, in their phenotypes with more monocytic features in the smaller group. Analysis of differentially regulated genetic pathways revealed CD14, WT1, MYCN, HCK, and SPTBN1 as discriminating genes. Stressing the potential impact of this analysis on the clinical management of AML, these two groups significantly differed in the EFS. Thus, it was demonstrated here also that within the group of AML with normal karyotype highly needed novel molecular markers with prognostic impact can be identified by using gene expression profiling. Some of the discriminating structures defined here may also be used for future targets in specific AML subtypes. However, regarding the biological heterogeneity of AML in general and of AML with normal karyotype in particular, it is anticipated that further largescale studies in the context of clinical trials are needed to fully characterize and validate novel and clinically relevant subgroups in AML and by doing so to define new targets for individual treatment. A recent example is the study of Bullinger et al. who further subclassified 93 patients with core binding factor (CBF) leukemias (AML1-ETO and CBFB-MYH11) in different risk groups (23). Another structure identified by gene expression profiling is the ubiquitinactivating enzyme E1-like (UBE1) gene that is induced by all-trans retinoic acid
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(ATRA) in NB4 cells (24). Detailed investigation revealed that ATRA activates the UBE1 promoter and the overexpression of UBE1 therefore triggers the degradation of promyelocytic leukemia-retinoic acid receptor alpha (PMLRARa) and leads to apoptosis in acute promyelocytic leukemia (APL) cells (25). Clinical studies with UBE1 in leukemia are however missing. Andersson et al. in a recent study compared (26) the molecular signatures in childhood acute leukemias and their correlations to expression patterns in normal hematopoietic subpopulations. 87 B-lineage acute lymphoblastic leukemia (ALL), 11 T-cell ALL, 23 AML, and 6 normal bone marrows, as well as 10 normal hematopoietic subpopulations of different lineages and maturations were ascertained by 27K cDNA microarrays. Not surprisingly, segregation according to lineage and primary genetic changes was achieved. However, several genes were identified that were preferentially expressed by the leukemic cells and not by their normal counterparts. These genes suggest an ectopic activation and are likely to reflect regulatory networks that may provide attractive targets for future directed therapies. However, although this study clearly points to the right direction, targets that were defined in this study have to be tested in an independent cohort of patients before they may be used for drug design. This again demonstrates that even if a variety of markers can be defined by gene expression signatures in addition to the diagnostic pattern of a specific leukemia subtype, the use of such information to find therapeutic structures or even targets is still limited, which emphasizes the need for better support of translational research and drug development in the future. A possible approach to use expression profiling in a high-throughput screening was published by Stegmaier et al. (27). They used HL-60 cells in 384-well culture plates and cultivated them with uniform concentrations of 1739 compounds to induce differentiation. By including different gene expression signatures of AML-versus-monocyte and AML-versus-neutrophil distinctions as measured by DNA microarrays, data were complemented by reverse transcriptionpolymerase chain reaction (RT-PCR) and matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF). Because of this approach, finally eight compounds were identified that reliably induced the differentiation signature. As a result, a modest number of genes were sufficient to capture a complex cellular response. However, the authors concluded that further investigations are needed to identify the optimal gene signature. This work points to a possible scenario for the identification of new targets and drugs by gene expression profiling. However, it again demonstrates the complex problem to combine different highly sophisticated methods in a high-throughput investigation to define at the end drugs to be tested in a clinical trial. Detection of New Targets in ALL For sure, one of the most important questions posed by the use of gene expression profiling is the identification of new targets for the further development of highly
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specific antileukemic drugs. One striking example is based on the results from Armstrong et al. who found mutations and high-level expression in the FLT3 tyrosine kinase receptor gene in MLL-rearranged ALLs (28,29). FLT3 is known as a tyrosine kinase receptor that is frequently activated by mutations in patients with AML (30) but is rarely activated in ALL. However, by gene expression profiling it was demonstrated that FLT3 was the gene most strongly associated with the presence of MLL gene rearrangements in ALL. This leads to the idea (28,31,32) to further investigate the potential role of this oncogene in the pathogenesis of MLL tumors per se. Mutational analyses of FLT3 in MLL gene–rearranged leukemias clearly showed the presence of activating mutations in the activation loop of this tyrosine kinase receptor in 5 of 30 cases studied. This was further validated by treating leukemia cells with PKC412, a specific inhibitor of the FLT3 tyrosine kinase. It was shown both in vivo and in vitro that PKC412 has differential cytotoxic effects on MLL rearranged leukemia cells harbouring FLT3 activation (28). Furthermore, it was demonstrated that also in ALL with hyperdiploid cytogenetics, the FLT3 receptor is frequently expressed at a higher level. This again reinforces the value of gene expression profiling as a powerful approach for the identification of novel drugs also in ALL (32–34), which should motivate an urgent translation into clinical trials including high-risk patients. Another approach in ALL to use gene expression for further insights in biology of the disease was described by Zaza et al. (35). After intravenous administration of thioguanine nucleotide (TGN), the TGN concentration was determined in the leukemic blasts of 82 children with newly diagnosed ALL. After analyses of 9600 genes, they identified 60 probes that were significantly associated with TGN accumulation if patients were treated with mercaptopurine (MP) alone and another 75 genes in patients treated with a combination of metotrexate (MTX) and MP. There was no overlap between these two sets of genes. The investigation was performed in parallel in vivo and in vitro and gene expression profiling led to new insights into the genomic basis of interpatient differences with respect to different treatment options. Through gene expression profiling, clear correlations between a specific drug’s level in vivo and increased expression of specific genes were detected. It was even visible that expression profiles correlated to mono or combined treatment modalities. Prospective studies are needed to test these results. Another outstanding investigation was conducted by Holleman et al. (36) who identified a set of differentially expressed genes in B-lineage ALL being sensitive or resistant to several drugs such as prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), and daunorubicin (20 genes). A score of genes combined to define overall sensitivity or resistance to all four drugs was tested in a multivariate analysis and predicted outcome of 173 children investigated ( p ¼ 0.027). Although these genes do not per se define new targets of treatment, gene expression profiling clearly demonstrated in a prospective setting which treatment may or may not be successful. This may serve as an example for the application of gene expression profiling to improve treatment and to define targets
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and drugs against these targets in ALL. The authors further point to the aspect that it may be important to determine whether specific modulation of proteins encoded by genes that were found may describe treatment response best. These proteins may also point to previously unrecognized potential targets and new agents to augment the efficacy of current chemotherapy for ALL. Brown et al. (37) investigated FLT3 inhibition by the selective inhibitor CEP-701 in ALL. In this study eight ALL cell lines and primary ALL cells from 39 patients were evaluated and a high potency for this drug especially in ALL cells overexpressing FLT3, i.e., MLL rearranged cases, as well as ALLs with hyperdiploid karyotypes was identified. Seven of seven sensitive samples examined by immunoblotting demonstrated constitutively phosphorylated FLT3 that was potently inhibited by CEP-701, whereas zero out of six resistant samples expressed constitutively phosphorylated FLT3. The authors concluded that the compound CEP-701, a potent and selective FLT3 inhibitor, effectively suppresses FLT3driven leukemic cell survival and clinical testing of this compound as a novel molecularly targeted agent for treatment of ALL is warranted. However, in most cases the candidate targets identified in expression studies (28) using relapse or treatment outcome as endpoints of their observation and independent verification is missing (38). Therefore, conflicting results are largely due to differences in treatment and biology of enrolled patients. The gap between gene expression profiling to characterize biological entities in leukemia and lymphoma and the targets to be tested is still not closed, and translation from data management to drug design is still missing. However, the characterization of molecular mutations and of pathway alterations in the leukemias proceeds with high velocity as can be demonstrated by the recent study of Mullighan et al. who revealed the PAX gene as the most frequent target of molecular mutation in ALL and showed that direct disruption of pathways controlling B-cell development and differentiation contribute to B-progenitor ALL pathogenesis (39). This is just one more example of the recent progress in the identification of new molecular targets in ALL. Detection of New Targets in Chronic Myeloid Leukemia McLean et al. (40) intended to define specific gene expression profiles in chronic myeloid leukemia (CML) patients all treated with imatinib. In correlation to cytogenetic response data, the expression pattern of a subset of 55 out of more than 12,000 genes was identified that best predicted response to therapy. The sensitivity to predict the individual response was 93.4%; however, the specificity was only 58.3%. The authors further found that many of the genes identified appeared to be strongly related to BCR-ABL transformation mechanisms. Thus, these genes may need further investigation as potential new drug targets in CML. Diaz-Blanco et al. described several novel transcriptional changes in primary CD34 positive CML cells in comparison with normal CD34-positive cells including an upregulation of components of the TGFB signaling pathway or
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candidate genes such as the leptin receptor (LEPR), thrombin receptor (PAR1), or the neuroepithelial cell transforming gene 1 (NET1) (41). It was further possible to define differentially regulated candidate genes discriminating chronic from blast phase of CML such as PRAME (preferentially expressed antigen of melanoma) (42) or CAMPATH (CD52) (43) or deregulation of pathways, e.g., the WNTB catenin signaling system (42). These studies thus might be helpful for definition of new novel stem or progentior cell-associated targets and of mechanisms being responsible for the higher malignant transformation of CML (41,42). Detection of New Targets in Chronic Lymphocytic Leukemia One highlight to establish the use of gene expression profiling to define new targets in leukemia was the detection of ZAP70 to be expressed in a large proportion of chronic lymphocytic leukemia (CLL) (14). As the expression of ZAP70 was high in IgVH-unmutated cases of CLL, this gene was further correlated to distinction within CLL cases with respect to prognosis. This finding also led to the investigation of the ZAP70 antigen expression by antibodies in CLL using multiparameter immunophenotyping (44). Recently, it was demonstrated that ZAP70 can also be successfully screened by a quantitative RT-PCR method (45). After definition of CLL signature genes, the protein products of these genes may represent such new targets for monoclonal antibodies or for vaccine approaches. Another aspect detected in this investigation was the fact that B-cell activation genes were upregulated in Ig-unmutated patients. Thus, pathways downstream of the B-cell receptor may contribute to aggressive clinical cases. It may be beneficial to target these signaling pathways. However, again, gene expression profiling so far was helpful in finding new epitopes in strict correlation to a specific disease or even subgroups within such diseases, but targeted drugs are still under investigation. Future Investigations to Diagnostic and Therapeutic Use of Gene Expression Profiling: The MILE Study The (microarray innovations in leukemia) MILE study is a cooperation of the European Leukemia Network (ELN, work package 13) together with Roche Molecular Systems. This innovative study was designed to test microarrays in parallel to gold standard diagnostics in 4000 patients with leukemia in 11 different sites (7 from ELN, 3 in United States, 1 in Singapore). At least 18 different classes of leukemia shall prospectively be defined for diagnostic use in the MILE study by their respective gene expression signatures. The ELN work package 13 is per se the head of these activities. In order to set up a clearly defined study with comparable sample quality, as a first step, a prephase was conducted to harmonize laboratory workflows. This prephase included tests of similar aliquots of two cell lines and three
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Figure 3 Example for inter-laboratory reproducibility in MILE study: Center 7 versus all other nine centers was calculated, all genes (38,000) were included in calculation.
leukemia samples—AML, CML, and CLL. A first interim analysis was able to demonstrate a very high inter- and intra-laboratory reproducibility (46). Figure 3 is an example of the data generated. Stage I of the study now includes 2000 samples of leukemia all analyzed in parallel with gold standard methods. After clarification of discrepant results between gene expression analyses and gold standard report forms, the most discriminating genes will be used to design a specific custom microarray for the diagnosis of leukemias. This new microarray will then be tested prospectively in stage II of the study by including another set of 2000 leukemia samples. It is further intended to use a subset of this data to address further questions like response to specific treatment as many patients are enrolled in prospective clinical trials. Only studies like this may define new targets for treatment, because information will be available on diagnosis, prognostic parameters, treatment, and response as well as ultimately for treatment outcome. The power of gene expression profiling may help in approaching such data sets from different perspectives and may therefore be used to address several questions in parallel. SUMMARY AND FUTURE TRENDS As new drugs are classically tested in clinical trials, this may be an interesting scenario for further use of microarrays. In many early clinical phase I/II studies response rate is low and many patients have received some other treatment before. However, if one is coupling clinical trials with gene expression profiling, the investigators may enhance their information, as the identification of specific gene expression profiles may correlate to drug response or resistance of the individual patient. Products of such differentially expressed genes represent at least plausible targets for inhibitors that may reverse the drug-resistance
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phenotype. Thus, these markers may be prospectively used to identify those patients who are likely to respond to the new agent. In follow-up studies far fewer patients would then be required to prove efficacy (38,47–50). Today, we are on the way to design and to use specifically developed microarrays with thousands of genes for the subclassification of leukemias and lymphomas. The ongoing MILE study is one example of an international approach to use gene expression profiling for the first time in a routine diagnostic setting. Also, a lymphoma microarray is already investigated for diagnosis (51). Importantly, this information always includes information about a patient’s prognosis, as clearly defined biological entities in leukemia and lymphoma lead to disease-specific treatment and data are therefore also related to prognosis and outcome. Some examples for this thesis are APL to be treated with ATRA or arsenic trioxide, or BCR-ABL-positive leukemias that can be specifically treated with imatinib or other tyrosine kinase inhibitors. Other examples are the use of CD33-targeted treatment in AML with gemtuzumab ozogamicin, or anti-CD20 and anti-CD52 antibody-related treatment in lymphomas. In addition, several studies were able to define a subset of genes that are not linked to a diagnostic profile but can be also used for outcome prediction. These studies can even demonstrate different marker genes that predict response to specific drugs. So far, one has to accept that much less is known about the use of gene expression profiling in finding new targets in leukemia and lymphoma. One nice example may be the detection of ZAP70 in CLL that not only predicts the IgVH status of the disease but can also be used as an antibody target to discriminate patients at diagnosis. However, new treatment opportunities have not been developed for this gene so far. Of course this does not mean that gene expression profiling will never add information for new targets. By identifying new players and pathways for resistance to therapy, DNA repair, and apoptosis, microarrays open up new avenues for any targeted therapy that had not even existed a few years ago. There is no evidence for any other technique today with so much power for specific and less toxic treatment for cancer patients in the future. However, the exact definition of the difference between a normal and a cancer cell in all details is essentially required for the solution. The goal must be to diagnose and stratify patients according to their disease-specific gene expression profile before treatment starts and to treat individually with drugs specific for such clearly defined biological entities. This does not mean that these drugs will be individually defined for each patient but for a newly defined disease not based only on morphology or cytogenetic parameters. Models for the development of new targets in leukemia and lymphoma should be adapted to large-scale clinical trials and have to focus in detail on new medications tested. Thus, strong links between academic and industry initiatives are urgently needed (52,53) to be the driving force behind the science. As cancer pathways such as Ras, Src, or Myc are known and can be linked to several tumors, their interaction and involvement can be studied by gene expression profiling best. Therefore, not only single genes being over- or underexpressed
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but altered pathways in leukemia and lymphoma also may lead to new targets in the near future. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Following its fast integration in hematological research we can expect gene expression profiling to be included in clinical procedures already in the very near future. First, it might soon support the classification and risk stratification of hematological malignancies as it provides a high degree of correlation with other diagnostic methods such as flow cytometry (54,55) or PCR (56) and shows a high diagnostic accuracy and reproducibility (7,57). The robustness of the method is a further argument for its applicability in the clinical field (58). Moreover, gene expression profiling is able to further subclassify distinct entities such as chronic myelomonocytic leukemia, which could not be previously subdivided by classical techniques (59). Although it is improbable that the new technique will substitute all established methods such as cytomorphology, cytogenetics, or PCR in the next years, it has to be expected that gene expression profiling will become part of the diagnostic panel of hematological malignancies and will be increasingly correlated with other methods or support those in case of difficult differential diagnoses or decisions. Second, a further step in the near future might be the inclusion in minimal residual disease strategies. In combination with real-time PCR gene expression profiling is able to serve for the definition of molecular markers, which can be monitored during follow-up of the disease. This might be exemplified in AML in the WT1 and PRAME genes (60). Third, gene expression profiling will probably find its way in individualized treatment planning as specific gene expression signatures are associated with poor chemotherapy response and with drug resistance. These processes are, e.g., mediated by a transcriptional program active in hematopoietic stem and progenitor cells as was demonstrated in AML (61) and being associated with nucleotide metabolism, apoptosis, and oxygen species metabolism (62). The finding of such signatures therefore might be an indication for immediate planning of allogeneic stem cell transplantation. However, such application of gene expression profiling for the definition of chemosensitivity for individualized treatment planning will probably have to be prepared somewhat longer than the above mentioned indications and will probably be only part of research studies rather than of routine strategies in the near future. REFERENCES 1. Haferlach T, Kohlmann A, Kern W, et al. Gene expression profiling as a tool for the diagnosis of acute leukemias. Semin Hematol 2003; 40:281–295. 2. Wheeler DL, Church DM, Edgar R, et al. Database resources of the National Center for Biotechnology Information: update. Nucleic Acids Res 2004; 32:D35–D40.
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40. McLean LA, Gathmann I, Capdeville R, et al. Pharmacogenomic analysis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib. Clin Cancer Res 2004; 10:155–165. 41. Diaz-Blanco E, Bruns I, Neumann F, et al. Molecular signature of CD34(þ) hematopoietic stem and progenitor cells of patients with CML in chronic phase. Leukemia 2007; 21:494–504. 42. Radich JP, Dai H, Mao M, et al. Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S A 2006; 103:2794–2799. 43. Zheng C, Li L, Haak M, et al. Gene expression profiling of CD34þ cells identifies a molecular signature of chronic myeloid leukemia blast crisis. Leukemia 2006; 20: 1028–1034. 44. Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348:1764–1775. 45. Dicker F, Schnittger S, Kern W, et al. Complex aberrant karyotypes and unbalanced translocations in CLL correlate with an unmutated IgVH status: a study on 133 patients studied with chromosome banding analysis, interphase FISH, IgVH mutation status, ZAP-70 RNA exrpession and immunophenotyping. Blood 2005; 106:825a. 46. Haferlach T, Kohlmann A, Basso G, et al. A multi-center and multi-national program to assess the clinical accuracy of the molecular subclassification of leukemia by gene expression profiling. Blood 2005; 106:224a. 47. Cheok MH, Yang W, Pui CH, et al. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nat Genet 2003; 34:85–90. 48. Golub TR. Mining the genome for combination therapies. Nat Med 2003; 9:510–511. 49. Evans WE, Guy RK. Gene expression as a drug discovery tool. Nat Genet 2004; 36:214–215. 50. Corchero J, Fernandez-Salguero PM. Improving cancer therapeutics by molecular profiling. Curr Drug Metab 2005; 6:553–568. 51. Staudt LM. Molecular diagnosis of the hematologic cancers. N Engl J Med 2003; 348:1777–1785. 52. Altman RB, Flockhart DA, Sherry ST, et al. Indexing pharmacogenetic knowledge on the World Wide Web. Pharmacogenetics 2003; 13:3–5. 53. Downward J. Cancer biology: signatures guide drug choice. Nature 2006; 439: 274–275. 54. Kern W, Kohlmann A, Schoch C, et al. Comparison of mRNA abundance quantified by gene expression profiling and percentage of positive cells using immunophenotyping for diagnostic antigens in acute and chronic leukemias. Cancer 2006; 107: 2401–2407. 55. Basso G, Case C, Dell’Orto MC. Diagnosis and genetic subtypes of leukemia combining gene expression and flow cytometry. Blood Cells Mol Dis 2007; 39:164–168. 56. Sala-Torra O, Gundacker HM, Stirewalt DL, et al. Connective tissue growth factor (CTGF) expression and outcome in adult patients with acute lymphoblastic leukemia. Blood 2007; 109:3080–3083. 57. Kohlmann A, Schoch C, Schnittger S, et al. Molecular characterization of acute leukemias by use of microarray technology. Genes Chromosomes Cancer 2003; 37: 396–405.
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58. Kohlmann A, Schoch C, Dugas M, et al. Pattern robustness of diagnostic gene expression signatures in leukemia. Genes Chromosomes Cancer 2005; 42:299–307. 59. Gelsi-Boyer V, Cervera N, Bertucci F, et al. Gene expression profiling separates chronic myelomonocytic leukemia in two molecular subtypes. Leukemia 2007; 21: 2359–2362. 60. Steinbach D, Schramm A, Eggert A, et al. Identification of a set of seven genes for the monitoring of minimal residual disease in pediatric acute myeloid leukemia. Clin Cancer Res 2006; 12:2434–2441. 61. Heuser M, Wingen LU, Steinemann D, et al. Gene-expression profiles and their association with drug resistance in adult acute myeloid leukemia. Haematologica 2005; 90:1484–1492. 62. Eisele L, Klein-Hitpass L, Chatzimanolis N, et al. Differential expression of drugresistance-related genes between sensitive and resistant blasts in acute myeloid leukemia. Acta Haematol 2007; 117:8–15.
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2 Individualized Tumor Response Testing in Leukemia and Lymphoma Andrew G. Bosanquet Bath Cancer Research, Royal United Hospital, Bath, U.K.
Peter Nygren Department of Oncology, Radiology, and Clinical Immunology, University Hospital, Uppsala, Sweden
Larry M. Weisenthal Weisenthal Cancer Group, Huntington Beach, California, U.S.A.
INTRODUCTION Individualized tumor response testing (ITRT) has a long history, with a number of different technologies and many different tumor types tested. Almost all technologies used for hematological malignancies are identical in their logic and similar in their execution. The concepts underlying cell death assays are relatively simple, even though the technical features and data interpretation can be complex. The logic is that if the drug kills tumor cells from an individual patient in a ‘‘test tube,’’ then it is more likely to be effective when administered directly to a patient. Conversely, a drug that does not kill the patient’s cells, even at concentrations significantly higher than can be achieved in the patient, is unlikely to be effective. Considerable work based on these assays has been reported during the past 25 years, and recently an ad hoc group of 50 scientists from 10 countries agreed on the term ‘‘individualized tumor response’’ for these
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tests, describing them as the ‘‘effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients’’ and not including tests with ‘‘subcellular fractions, animals or cell lines’’ (1). We present results for hematological neoplasms, but note that analogous results have been published for a variety of solid tumors in substantial numbers of patients (2). TOTAL CELL KILL/CELL DEATH ASSAYS There is a clear divide between the two main technologies used in this work: an ITRT endpoint can be based either on reduction of cell proliferation or on cell death (3–6). Historically, the cell proliferation endpoint received great attention as a result of studies by Salmon, Von Hoff, and others during the late 1970s and early 1980s (7,8). These studies occurred during the heyday of the oncogene discovery period in cancer research, when oncogene products were frequently found to be associated with cell growth and when cancer was most prominently considered to be a disease of disordered cell growth. In contrast, the concept of apoptosis had yet to become widely recognized. Also unrecognized were the concepts that cancer may be a disease of disordered apoptosis/cell death and that the mechanisms of action of most, if not all, available anticancer drugs are mediated through apoptosis. When problems with cell proliferation assays emerged (9,10), there was little enthusiasm for studying cell death as an alternative endpoint (11). These factors explain many abandoning research into ITRT during the 1980s. As opposed to measuring cell proliferation, there is a family of assays based on the concept of total cell kill or, in other words, cell death occurring in the entire population of tumor cells (3–6). The basic technology concepts are straightforward. Cells are isolated from a fresh specimen obtained from a viable neoplasm. These cells are cultured in the continuous presence or absence of a drug, most often for three to seven days. At the end of the culture period, a measurement is made of cell injury, which correlates directly with cell death, almost always by apoptosis (12–14). Although there are methods for specifically measuring apoptosis per se, there are practical difficulties in applying these methods to mixed (and sometimes clumpy) preparations of tumor cells and normal cells. Thus, more general measurements of cell death have been applied. One of these measurements is the delayed loss of cell membrane integrity, which has been found to be a useful surrogate for apoptosis. This is measured by differential staining in the Differential Staining Cytotoxicity (DiSC) assay method, which allows selective drug effects against tumor cells to be recognized in a mixed population of tumor and normal cells (6,15). More recently the Tumor Response to Antineoplastic Compounds (TRAC) assay was described as a streamlined version of the DiSC assay (16). Other cell death endpoints include loss of mitochondrial Krebs cycle activity, as measured in the Methylthiazol Tetrazolium (MTT) assay (17), loss of cellular adenosine triphosphate (ATP), as measured in the ATP assay (18), and
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loss of cytosolic esterase activity and cell membrane integrity, as measured by the Fluorometric Microculture Cytotoxicity Assay (FMCA) and similar assays (19–21). Most recently, other methods including assays to measure apoptosis more specifically have been described, although it remains to be seen if these will offer any real advantages over the other measurements of cell death (22–26). These four endpoints produce valid and reliable measurements of cell death. They also correlate well with each other on direct comparisons of the different methods (17,19,20,27–29). For instance, Weisenthal and associates have performed direct correlations between the DiSC and MTT assays in approximately 5,500 fresh human tumor specimens, testing an average of 15 drugs per specimen at two different concentrations. Although these endpoints agree with each other in most solid tumors (overall correlation coefficient ¼ 0.85), we consider that the MTT assay is more problematic in hematological neoplasms. For example, correlations between treatment outcomes and assay results have been more consistent in acute nonlymphocytic leukemia (ANLL) with the DiSC assay endpoint (30–32) than with the MTT endpoint (22,33,34). Additionally, there is a clear relationship between prior treatment status and assay results for anthracyclines in the case of the DiSC assay (relapsed patients having blast cells that are clearly more resistant than those in previously untreated patients, Table 1), which was not evident when ANLL was tested with the MTT assay (35). The absolute magnitudes of drug effects (cell kill) are substantially greater when scored in the DiSC assay than in the MTT assay in the case of ANLL (Table 1). Finally, the correlation coefficient between DiSC and MTT assays was weaker in the case of ANLL (median r ¼ 0.75), than in other classes of neoplasms that Weisenthal had tested (median r ¼ 0.85). There are at least two explanations for the greater drug effects detected in the DiSC endpoint. Firstly, the DiSC assay is a more specific endpoint for drug effects on blast cells (as opposed to drug effects on blast cells plus the normal cells frequently present in ANLL specimens). Table 1 In Vitro Activity of Anthracyclines in ANLL As a Function of Prior Treatment Status and Individualized Tumor Response Testing Endpoint
Drug/assay Doxorubicin/DiSC Doxorubicin/MTT Idarubicin/DiSC Idarubicin/MTT
Number untreated
Number treated
Cell fraction surviving (untreated)
Cell fraction surviving (relapsed)
12 12 10 10
16 16 16 16
0.11 0.34 0.06 0.35
0.33 0.42 0.25 0.45
P 0.020 0.428 0.0015 0.180
Abbreviations: ANLL, acute nonlymphocytic leukemia; DiSC, differential staining cytotoxicity; MTT, methylthiazol tetrazolium. Source: From Ref. 35.
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Secondly, it takes longer for cells to lose the ability to produce a signal in the MTT assay than it does for them to be scored as dead in the DiSC assay [e.g. (36)]. It is possible that the MTT assay would be more useful in ANLL (i) were it applied only in cases in which there was a ‘‘pure’’ (>90%) population of blast cells at the end of the assay, and/or (ii) were the duration of the cell culture (and drug exposure) extended beyond the typical 96-hour period of these assays. With regard to the first of these latter possibilities, it is notable that Hongo, et al. (who contributed a disproportionate share of weak clinical correlations in Table 2) did not attempt to determine the percentage of blast cells at the time the MTT endpoint was measured (34). A final point of emphasis is that it is important to rigorously standardize assay conditions, including precisely controlling the duration of drug exposure and cell culture. Thus, the DiSC assay and similar tests have some advantages over the other short-term assays. COMPLETED STUDIES OF CORRELATION BETWEEN CELL DEATH ASSAY RESULTS AND CHEMOTHERAPY RESPONSE As with other laboratory tests, the determination of the efficacy of ITRT is based on comparisons of laboratory results with patient response (commonly referred to as ‘‘clinical correlations’’). The hypothesis to be tested with clinical correlations is a simple one—that above-average drug effects in the assays correlate with above-average drug effects in the patient, as measured by both response rates and patient survival. Table 2 and Figure 1 show that, with respect to response, the above hypothesis has been confirmed to be true in all published studies. At each point in the distribution of overall response rates, patients with test results in the ‘‘sensitive’’ range were more likely to respond than the total patient population as a whole. Conversely, patients with test results in the ‘‘resistant’’ range were less likely to respond than the patient population as a whole. On average, patients with assays in the test sensitive range were threefold more likely to respond than patients with assays in the test resistant range (see the ‘‘Overall relative risk’’ column in Table 2). Considering this evidence as a whole, can it be inferred with confidence that the cell death measured in the assays correlates with tumor cell death measured in the patient? Comparing the chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) data with the more limited but also consistent data in non-Hodgkin’s lymphoma (NHL), a powerful case is made to support the clinical relevance of this testing in human lymphatic neoplasms. Considering the ANLL data in the context of the lymphatic neoplasm data, a powerful case is made to support the clinical relevance of this testing in hematological neoplasms in general. The body of literature supporting cell death assays in lymphatic neoplasms dates to studies in CLL published by Schrek in the 1960s (37,38). Schrek
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measured the in vitro cell death effects of drugs, heat, and radiation on CLL cells by means of phase contrast microscopy. He measured what we would today recognize as apoptosis and undoubtedly being precisely congruent with the DiSC assay. Radiation effects in vitro were strongly correlated with clinical outcome (37,38). In the late 1970s, Durkin compared in vitro drug effects in NHL and CLL by means of trypan blue dye exclusion with clinical drug effects and reported good correlations in a small study (39). The DiSC assay was developed independently as an improved variation of the trypan blue test in which suspension cultures of cells were first exposed to trypan blue, cytocentrifuged onto microscope slides, and counterstained with either Haematoxylin/Eosin or Wright/ Giemsa (to identify the non-trypan blue-stained cells with respect to whether these surviving cells were tumor cells or normal cells). With further improvement (substitution of fast green stain for trypan blue and the addition of acetaldehyde-fixed duck erythrocytes as an internal standard to aid in scoring the Cytospin slides), clinical correlations in CLL and other neoplasms were first reported in an abstract form and at meetings in the United States and Europe in 1981. The first publications of clinical correlations with the DiSC assay, in 1983 and 1984, included studies of the activity of glucocorticoids and standard cytotoxic agents, which were correlated with prior therapy and with clinical outcome in ALL and CLL (3,15,40). In 1986, these were followed by a study showing the clinical relevance of the DiSC assay in CLL, ALL, and NHL using several clinical endpoints: (i) Correlations with known disease-specific activity profiles, (ii) individual patient correlations with clinical response, (iii) greater resistance of specimens from previously treated patients versus previously untreated patients, and (iv) a shift to significantly greater drug resistance in metachronous assays in the presence of intervening chemotherapy, but no shift in the absence of intervening chemotherapy (41). These early findings were subsequently independently confirmed by other investigators in more comprehensive studies (19,28,42–51). Additionally, studies in pediatric ALL reported that resistance to dexamethasone in the DiSC assay predicted for poor survival (52), findings also independently confirmed. By the late 1980s, a number of other scientists were investigating the DiSC assay and related cell death assays. These began with a head-to-head comparison of the DiSC assay with the MTT assay in established cell lines by the National Cancer Institute (NCI) lung cancer group (17). These studies established the comparability of these endpoints in homogeneous cell populations. A group at the VU University Medical Center of Amsterdam carried out a head-to-head comparison of the DiSC endpoint with the MTT endpoint in ALL (29). This group showed that the endpoints were comparable in specimens in which the proportion of leukemia cells (relative to normal cells in the specimen) was greater than 80% (29,53). They found the MTT endpoint to be less labor intensive. They used the same general conditions originally described for the DiSC assay (including a 96-hour continuous drug exposure, followed by
Diagnosis
ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL/CLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL
Author
Beksac, et al. Bosanquet Hongo, et al. Hongo, et al. Kaspers, et al. Lathan, et al. Nygren, et al. Weisenthal, et al. Weisenthal, et al. Kirkpatrick, et al. Beksac, et al. Bosanquet Hongo, et al. Hongo, et al. Yamada, et al. Langkjer and Norgaard Larsson, et al. Lathan, et al. Norgaard, et al. Nygren, et al. Santini, et al. Sargent and Taylor Staib, et al. Staib, et al. Stute, et al. Tidefelt, et al.
3 15 25 65 125 4 36 29 2 55 12 5 14 43 124 11 21 17 59 38 27 21 83 79 33 34
Number of patients 67 87 68 82 90 75 64 76 100 69 58 20 79 58 86 55 52 71 53 66 52 48 89 90 64 62
Clinical response rate (RR) 100 92 76 95 94 100 80 90 100 79 78 0 82 67 93 100 85 92 71 86 82 83 100 96 73 91
Response rate (test sensitive) 0 0 25 10 81 0 38 44 N/A 0 0 50 67 30 76 0 0 20 35 10 0 0 0 0 56 8
Response rate (test resistant)
Infinite 2.72 8.15 1.12 2.34 1.71 Infinite Infinite 1.18 1.94 1.14 Infinite Infinite 3.53 1.48 6.58 Infinite Infinite Infinite Infinite 1.15 7.41
0.80 0.84 0.87 0.75 0.96 0.87 0.93 0.55 0.62 0.77 0.74 0.77 0.63 0.57 0.94 0.89 0.87 0.68
Relative risk (test resistant)
0.87 0.89 0.86 0.97
Relative risk (test sensitive) 92 42 49 59a 93 30 19 41 15 28 92 42 49 59a 34 66 47 30 33 19 94 64 63 32a 95 31
Reference number
28
1.23 2.22 1.23 Infinite Infinite 4.58 2.01 8.57 Infinite Infinite Infinite Infinite 1.32 10.91
Infinite Infinite
2.93 2.03
Infinite 3.05 9.45 1.16
Overall relative risk
Table 2 Correlations of Individualized Tumor Response Test Results with Clinical Response to Chemotherapy and Relative Risk of Failure to Achieve Remission
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CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL/ALL/NHL CML (blastic) Myeloma Myeloma Myeloma Myelofibrosis NHL NHL NHL NHL NHL NHL NHL
Bosanquet Bosanquet, et al. Bosanquet, et al. Bosanquet, et al. Larsson et al. Morabito, et al. Nerenberg, et al. Silber, et al. Weisenthal, et al. Weisenthal Bosanquet Weisenthal, et al. Bosanquet Weisenthal, et al. Weisenthal, et al Larsson, et al. Beksac, et al. Bosanquet Leone, et al. Nygren, et al. Strauss, et al. Weisenthal, et al. Weisenthal, et al. 1929
73 34 66 442 1 31 40 15 15 3 107 9 16 6 5 1 1 10 3 50 8 10 3
Number of patients
70.4
19 76 55 79 100 74 60 80 67 67 71 22 13 50 0 100 100 40 67 58 50 60 0
Clinical response rate (RR)
84.6
48 93 69 84 100 95 94 100 90 100 82 67 29 67 N/A 100 100 50 67 71 100 86 N/A
Response rate (test sensitive)
28.3
4 0 7 31 N/A 36 32 25 20 0 17 0 0 33 0 N/A N/A 25 N/A 17 0 0 0
Response rate (test resistant)
4.26 Infinite
3.48
0.87 0.44
0.82
2.48
2.04 1.89 3.20 3.33
0.78 0.64 0.80 0.74
0.83
Infinite 8.18 4.60 2.54
Relative risk (test resistant)
0.82 0.79 0.40 0.94
Relative risk (test sensitive)
2.99
4.26
Infinite
4.92
2.61 2.97 4.00 4.50
Infinite 10.29 11.52 2.71
Overall relative risk 42 96 70 83 47 71 25 51 41 15 97a 41 42 41 15 47 92 42 98 27 99 41 15
Reference number
Notes: Relative risks are calculated for experiments containing more than 10 patients. a To avoid duplication, clinical correlations recorded here from these papers do not include previously published correlations. The overall results: (TP ¼ 1220, TN ¼ 349, FP ¼ 222, FN ¼ 138) result in: Specificity (for drug resistance) of 0.61 and Sensitivity (for drug resistance) of 0.90.
Totals
Diagnosis
Author
to Achieve Remission (Continued )
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Figure 1 Correlations between ITRT results and clinical response. The data points are the results of each of 33 individual studies that had at least 10 patients per data subset (see Table 2). The data are plotted in order of the increasing response rate in the total patient cohort studied (x-axis). The crosses (all of which lie on the x ¼ y line) represent the response rates of patients in each study if the assay was of no value or not performed. The squares represent the response rates for patients with assay results classified as in vitro sensitive (&) versus resistant (&). The triangles show the weighted mean of all sensitive results (~, n ¼ 1020)) and all resistant results (~, n ¼ 419) from Table 2. The greater the vertical distance between the sensitive and resistant results for an individual study, the more accurate the test results.
comparisons between drug exposed and control cultures with the cell death endpoint). These Dutch authors continue to publish an extensive, elegant, and ongoing series of rigorous studies. They have established that the assay results correlate with and predict both response and survival in ALL and that the assay results are only two factors (the other being minimal residual disease), which independently predict for survival in pediatric ALL (43,44,46,54–57). They have also extended this work to ANLL (22,35,45,58). Taken in the context of the entire literature, these studies in pediatric ALL provide further support for the validity of complementary studies in CLL. Other investigators have also shown strong correlations between cell death assay results and the clinical outcome (response and/or survival) in pediatric ALL (19,49,50,52,55,59), adult ALL and ANLL (20,28,30–32,60–67), CLL
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(42,51,68–71), and adult NHL (15,27,41,48). These studies included further confirmation of the comparability between DiSC and MTT endpoints in assays on clinical specimens. Larsson and Nygren also introduced the fluorescein diacetate cell death endpoint (FMCA), which, like the DiSC endpoint, measures cell membrane integrity and which correlates well with the DiSC endpoint in homogeneous cell populations (19,27). In 1991, Bosanquet, et al. published in Lancet a relatively large number of correlations between clinical response and DiSC assay results, chiefly in CLL (42). He showed, furthermore, highly significant correlations between assay results and patient survival. This paper also confirmed the relevance of the ‘‘EDR’’ (extreme drug resistance) endpoint, which is defined as an assay result greater than 1 SD more resistant than the median of comparison assays. He later described a paradoxical shift toward increased methylprednisolone sensitivity in previously treated CLL and used the DiSC assay to identify high-dose methylprednisolone as an effective treatment for otherwise refractory CLL (72,73). These studies with the DiSC and MTT assays are further supported by studies with the FMCA. Fluorescein diacetate is a lipid soluble material that readily penetrates cell membranes. Viable cells contain cytosolic esterases that cleaves the dye to non–lipid soluble fluorescein, which is concentrated in cells containing a functionally intact membrane. Thus, the assay is conceptually similar to the DiSC assay, which measures the ability of cells with functionally intact membranes to exclude non–lipid soluble dyes. Delayed loss of this membrane integrity is a marker of apoptotic cell death (74). Investigators at Uppsala University in Sweden began work in the 1980s by comparing the DiSC and FMCA assays and establishing their comparability (19,27,48). They proceeded to publish a series of studies showing correlations between assay results and treatment outcomes in NHL and ANLL (19,20,47,48,61,62,75), confirming the specificity of the EDR endpoint in predicting clinical nonresponse (62), and confirming and extending earlier reports on the capability of the cell death endpoint to identify the general disease-specific activity patterns of a diverse spectrum of drugs (76). Within the past several years, additional studies have provided strong support for the clinical relevance of the information provided by cell death assays in hematological neoplasms. Table 2 shows response correlations for ALL, CLL, NHL, and ANLL and may be summarized as follows: l
ALL: n ¼ 304 published correlations between assay results and response l l
l
81% overall response rate for patients studied 91% response rate for patients treated with ITR test–sensitive (ITRþ) drugs; relative risk of failure to achieve remission 0.90 [95% confidence interval (CI) 0.84–0.96, P ¼ 0.002] 49% response rate for patients treated with ITR test–resistant (ITR–) drugs; relative risk 1.65 [95% CI 1.29–2.11, P < 0.0001]
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32 l
Bosanquet et al.
CLL: n ¼ 720 l l
l
l
l
69% overall response rate 83% response rate with ITRþ drugs; relative risk 0.83 [0.78–0.89; P < 0.0001] 19% response rate with ITR drugs; relative risk 3.66 [2.6–5.1; P < 0.0001]
NHL: n ¼ 85 l 54% overall response rate l 73% response rate with ITRþ drugs; relative risk 0.74 [0.58–0.95; P ¼ 0.025] l 12% response rate with ITR– drugs; relative risk 4.69 [1.59–13.8; P < 0.0001] ANLL: n ¼ 621 l 72% overall remission rate l 88% response rate with ITRþ drugs; relative risk 0.82 [0.77–0.90; P < 0.0001] l 36% response rate with ITR drugs; relative risk 2.01 [1.65–2.45; P < 0.0001]
Thus, there is a long, extensive, and consistent body of evidence supporting the clinical relevance of cell death assays in human hematological neoplasms. Gene Expression Profiling Over the last few years, gene expression profiling has been suggested as the best or only way of determining ex vivo drug sensitivity (77), and there has been some recent progress for the concept of prediction of cytotoxic drug activity in individual patients with solid tumors based on genomic signatures (80). However, due to almost all patients being treated with combination chemotherapy, without ITRT, there are calibration difficulties with this methodology. Thus, in one of the best papers on the subject, originating from work with childhood ALL (78), the supervised cluster analysis was ‘‘based on in vitro drug sensitivity’’ (79). This editorial then continued by erroneously suggesting that ITRT was ‘‘more cumbersome’’ than gene expression profiling (79), whilst ITRT is actually integrating all the gene expression into one convenient test. As a result, and because it is as near real time and real life as is possible for a laboratory test, we believe that ITRT may be most clinically relevant to the patient (Fig. 2). COMPLETED STUDIES OF PATIENT SURVIVAL In 1999, in a study of 243 CLL patients (70), Bosanquet identified 66 patients who received fludarabine within a year of the performance of the DiSC assay— 51 fludarabine test–sensitive patients had a 69% response rate (80% for untreated
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Figure 2 Clinical relevance of various endpoints.
patients; 64% for previously treated patients), while 15 fludarabine test–resistant patients had a 7% response rate (25% for previously untreated; 0% for previously treated). None of these 15 fludarabine-resistant patients treated with fludarabine survived 17 months; median survival was 7.9 months. In contrast, the fludarabinesensitive patients treated with fludarabine had an 80% chance of surviving beyond 17 months, a median survival of 41.7 months, and a 43% chance of surviving beyond four years. Patients (n ¼ 42) with DiSC assay resistance to fludarabine but treated with regimens other than fludarabine had a median survival of 16.3 months and 10% survived beyond four years. The relative risk of death for patients with DiSC assay resistance to fludarabine treated with fludarabine versus those treated with a non-fludarabine regimen was 2.9. On multivariate analysis, fludarabine test resistance was a more important determinant of survival in patients treated with fludarabine than was any other clinical characteristic, including gender, Binet stage, prior chemotherapy, and patient age. In a separate analysis, DiSC assay– directed therapy of CLL was calculated to be cost effective at only $2500 per quality life-year saved (81). Other investigators, as noted, have reported that assay results are important predictors of patient survival in pediatric ALL and ANLL (33,49, 56–58,82). Similar studies from a number of different groups have published correlations between ITRT results and survival in adult ANLL (32,33,63). Correlations between DiSC assay results and patient survival in ANLL were first published by a Swedish group in 1989 (31). These results were confirmed and extended by a group at the University of Cologne (32), in a follow-up to their earlier report of strong correlations between DiSC assay results and clinical remission of adult ANLL a decade earlier (63). In their recent studies, the DiSC assay results 100% accurately predicted clinical outcome and identified a group of patients with a 100% early death rate, when treated with conventional induction therapy (32,63). These studies are very analogous to the above-cited work identifying a group of CLL patients in whom conventional treatment is uniformly inactive (70).
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The University of Cologne group followed up with a presentation at the American Society of Hematology meeting in December, 1999, in which multivariate analysis showed DiSC assay results to be the strongest factor predicting for clinical outcome (both complete remission and long-term patient survival) in adult ANLL, using a novel and sophisticated method for calculating sensitive/ resistant cutoff boundaries (32). Additionally, a Danish group reported studies correlating MTT assay results with both overall and relapse-free survival in 85 adult ANLL patients (33). Assay results remained significantly correlated with survival on multivariate analysis. This work on ANLL is analogous and complementary to the studies by the Dutch (Amsterdam) group in pediatric ALL, discussed above. ONGOING CLINICAL STUDIES A major international clinical trial on ITRT in CLL is ongoing. The first randomization has closed and comparisons of ITRT by TRAC assay (83,84) and patient response (85) have been published. The second randomization (at nonresponse or relapse after first treatment) is between ITRT-directed therapy and protocol-guided treatment. This second randomization continues to accrue patients and will determine to what extent ITRT can improve response and survival of CLL patients at first relapse. EXPERT OPINION: CURRENT USE OF ITRT IN CLINICAL ONCOLOGY The American Society of Clinical Oncology (ASCO) working group recommended against the use of ITRT in oncology practice, stating that ‘‘Oncologists should make chemotherapy treatment recommendations on the basis of published reports of clinical trials. . . .’’ (86). In a published objection to this recommendation, Castro wrote: ‘‘Paradoxically, as the number of possible treatment options supported by completed randomized clinical trials increases, the scientific literature becomes increasingly vague in guiding physicians . . . moreover, clinicians are confronted on nearly a daily basis by decisions that have not been addressed by randomized trial evaluation’’ (87). The data in Table 3 support Castro’s point of view. These data are taken from the United States NCI Web site, in which the so-called state-of-the-art, standard therapy options are reviewed. It can be readily seen that 50 years of prospective, randomized trials have failed to identify clear-cut ‘‘best’’ standard therapies, even in the setting of first-line treatment (Table 3). In each class of adult hematological neoplasms, there are a variety of choices, similar in some respects but with key differences. One conservative application of the assays would be to identify the most active of the otherwise equally acceptable regimens. Another would be to eliminate the most inactive of the regimens and choose from among the rest on the basis of other clinical factors, including cost. In the setting of relapsed, refractory disease, ITRT
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Table 3 Equally Acceptable ‘‘Standard’’ Chemotherapy Options, According to the United States National Cancer Institute ALL (adult)
AML
CLL
NHL (indolent)
D/V/P
C
R/F
R
R/F/Ctx F/Ctx Ctx/V/P Ctx/Dox/V/P
R/F R/Ctx/V/P R/Ctx/Dox/V/P R/F/M
F/Chl
Chl (P or Dex)
D/V/P/A C/D D/V/P/A/Ctx C/D/T M/E C/I
R/Ctx/F/M Cda Ctx (P or Dex) F Ctx/V/P C/V/P/Procarb Ctx/Dox/V/P F/M/Dex
NHL (aggressive) R/Ctx/Dox/ V/P Ctx/Dox/V/P D/Ctx/V/B/P Ctx/M/V/P Mtx/B/Dox/ Ctx/V/Dex P/Dox/Ctx/E/ C/B/V/Mtx
Myeloma Dex or P Ctx/P Melph/P Bortezomib V/Carm/ Melph/Ctx/P V/Melph/ Ctx/P/C/ Carm/Dox/P Thalidomide
Abbreviations: A, Asparaginase; B, Bleomycin; C, Cytarabine; Carm, Carmustine; Cda, Cladribine; Ctx, Cyclophosphamide; Chl, Chlorambucil; D, Daunorubicin; Dex, Dexamethasone; Dox, Doxorubicin; E, Etoposide; F, Fludarabine; I, Idarubicin; M, Mitoxantrone; Melph, Melphalan; Mtx, methotrexate; P, Prednisone; Procarb, Procarbazine; R, Rituximab; T, Tioguanine; V, Vincristine.
provides a mechanism for choosing from an even larger number of potential choices, many of which will not be tested in prospective, randomized trials for years to come. Beyond the potential advantage to the patient is the progress, which would be fostered with regard to improving methodology, to make it available for future applications to handle the explosive growth of new, expensive, potentially toxic, and only partially effective drugs. Castro further argued (87): ‘‘Until the trialist approach has delivered curative results with a high success rate, the clinical autonomy to integrate promising insights and methods, including [ITRT], remains an essential component of patient advocacy.’’ The members of the ASCO working group who formulated the ASCO recommendations concerning ITRT agreed with Castro, stating: ‘‘It is certainly each practitioner’s prerogative to order [ITRT] . . . However, it is important to specify to the patient what the treatment would be in the absence of the assay and to be clear about if and how the information will be used to inform treatment decision making’’(88).
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The authors of this chapter agree completely with the above quoted viewpoints, as expressed by Castro and as modified by the ASCO working group. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Progress in the treatment of cancer has been surprisingly modest in light of the rapid progress in tumor biology, although several new ‘‘targeted’’ drugs have been introduced over the last few years. Most of them have so far been developed for use in solid tumors, but new drugs have also emerged for hematological malignancies, for instance imatinib for chronic myeloid leukemia (CML), rituximab for lymphomas and bortezomib for myeloma. With the exception of the new treatment situation in CML, there is little reason to believe that established chemotherapy will not continue to form the basis of medical treatment of hematological as well as solid tumors in the next five years. The new targeted drugs mostly need to be combined with active chemotherapy to provide any benefit and, thus, the need for predictive tests for individualized therapy selection has increased. Disappointingly, the introduction of the new drugs has not been accompanied by specific predictive tests allowing for a rational and economical use of the drugs. On the other hand, preliminary data indicate that ITRTs also adequately reflect the clinical activity of, for instance, various tyrosine kinase inhibitors. Given also the technical and conceptual advantages of ITRTs together with their performance and the quite modest efficacy of therapy prediction based on analysis of genome expression as published so far, there is reason for a renewal in the interest for ITRTs for future optimized use of medical treatment of malignant disease. Thus, the current and potential role of ITRT in the management of patients with hematological neoplasms remains controversial, although, for some years, ITRT has been approved for reimbursement by Medicare in the United States. Specifically excluded from consideration by the two editorial reviews published in the Journal of Clinical Oncology (86,89) were studies which related ‘‘only’’ to the performance characteristics of ITRT—predictive accuracy, sensitivity, and specificity. The only studies considered were the very few which attempted to show if treatment outcomes could be improved through the use of ITRT. These criteria were surprising, as there are virtually no published studies with any other laboratory test in which patients were randomized to treatment with and without test information. The traditional (and heretofore only) criteria on which other laboratory, clinical, and radiographic tests have been judged are the performance characteristics (predictive accuracy, sensitivity, and specificity) and perceived utility in the judgment of the clinician who orders the tests. Only when these assays are widely performed and used and routinely included as an integral part of clinical trials will these already promising technologies be improved and only then will their role in patient management become better defined. But this is true for all complex laboratory technologies. A good example is immunohistochemical staining for batteries of cell antigens, the
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use of which has never been shown by means of randomized controlled clinical trials to improve treatment outcomes. By raising the bar of acceptance to levels unprecedented for a laboratory test, in essence a tariff has been erected to protect the paradigm of the ‘‘best’’ empiric treatment for the average patient, as identified in the all-too-commonlynonproductive clinical trials. This tariff also discourages discovery of new, effective drug regimens through the use of ITRT to guide drug selection. With greater use of these assays in hemato-oncology and the everincreasing list of licensed drugs, it is very likely that the activity of new drugs and new regimens would be identified at a much earlier time than with the current system relying exclusively on usually empiric phase II trials (90,91). If test results are used to assist in the selection of a regimen chosen from a series of otherwise reasonable alternatives, then patients will never be harmed by using the test result and best available evidence strongly indicates they will often be helped. In conclusion, there is a 45-year history of highly positive studies in hematological neoplasms showing consistent, strong correlations between the results of cell death assays and clinical outcomes (both initial response and longterm patient survival). A similarly convincing body of evidence in solid tumors (2) suggests these technologies are relevant for most, if not all, tumor types. Thus, there is strong scientific rationale and good documentation for these tests in a collectively large and diverse literature about hematological neoplasms for the clinical relevance of the information provided by the tests. Their use, particularly where equally effective treatment options are possible, could improve the rationale of treatment choice as well as probability of response and survival. REFERENCES 1. Bosanquet AG, Kaspers GJ, Larsson R, et al. Individualized tumor response (ITR) profiling for drug selection in tailored therapy: meta-analysis of 1929 cases of leukemia and lymphoma. Blood 2007; 108:1017A (abstr). 2. Weisenthal LM, Nygren P. Current status of cell culture drug resistance testing (CCDRT). Human Tumor Assay Journal 2002. Available at: http://www.weisenthal. org/oncol_t.htm. Accessed February 2008. 3. Weisenthal LM, Shoemaker RH, Marsden JA, et al. In vitro chemosensitivity assay based on the concept of total tumor cell kill. Recent Results Cancer Res 1984; 94:161–173. 4. Weisenthal LM, Lippman ME. Clonogenic and nonclonogenic in vitro chemosensitivity assays. Cancer Treat Rep 1985; 69:615–632. 5. Weisenthal LM. Cell culture assays for hematologic neoplasms based on the concept of total tumor cell kill. In: Kaspers GJL, Pieters R, Twentyman PR, et al., eds. Drug Resistance in Leukemia and Lymphoma: The Clinical Value of Laboratory Studies. Chur, Switzerland: Harwood Academic Publishers, 1993:415–432. 6. Weisenthal LM, Kern, DH. Prediction of drug resistance in cancer chemotherapy: the Kern and DiSC assays. Oncology (U.S.A.) 1992; 5:93–103.
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3 Minimal Residual Disease Jacques J. M. van Dongen Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Tomasz Szczepan´ski Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands, and Department of Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze, Poland
Vincent H. J. van der Velden Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
MINIMAL RESIDUAL DISEASE Current cytotoxic treatment protocols induce complete remission (CR) in most acute leukemia patients [both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)], in some patients with chronic lymphocytic leukemia (CLL), and in most non-Hodgkin’s lymphoma (NHL) and chronic myeloid leukemia (CML) patients. Introduction of allogeneic and autologous hematopoietic stem cell transplantation (HSCT) in treatment protocols has further increased the remission rates in ALL, AML, CML, and NHL. Nevertheless, many of these patients ultimately relapse. Apparently, the treatment protocols are not capable of killing all clonogenic malignant cells in these patients, even though they reached CR according to cytomorphological criteria. The detection limit of cytomorphological techniques is not lower than 1% to 5% of malignant cells, implying that these techniques can provide only superficial information
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about the effectiveness of the treatment. More sensitive techniques are required for the detection of low frequencies of malignant cells during and after treatment, i.e., detection of minimal residual disease (MRD). MRD techniques should reach sensitivities of at least 103 (one malignant cell within thousand normal cells), but sensitivities of 104 to 106 are preferred. Such sensitivities allow ‘‘true’’ MRD detection and thereby evaluation of the effectiveness of the total treatment and assessment of the contribution of each treatment phase. TECHNIQUES AND TARGETS FOR MRD MONITORING For the detection of MRD, at present at least three methods are sufficiently sensitive (103), quantitative, and broadly applicable: flow cytometric immunophenotyping, polymerase chain reaction (PCR)-based detection of junctional regions of rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes (mostly in lymphoid malignancies), and PCR-based detection of breakpoint fusion regions of chromosome aberrations (Table 1). Table 1 Applicability of MRD Techniques in Leukemias and Lymphomasa Flow cytometric immunophenotyping
Precursor-B-ALL children adults T-ALL children adults Chronic B-cell leukemias Chronic T-cell leukemias B-NHL T-NHL AML (non-M3) APL CML
PCR analysis
Aberrant immunophenotypes (103–104)
Junctional regions of Ig/TCR genes (103–106)
Chromosome aberrations (104–106)
70–90% 70–80%
*95% *95%
40–45% 40–45%
*95% *95% >95%
>95% >90% >95%
25–30% 10–15% –
–
*95%
–
– 20–25%c 60–90% NR –
70–80%b *95% 10–15% NR –
25–30% 10–15% 15–30% >95% >95%
a The percentages indicate the applicability of the MRD techniques per category of hematopoietic malignancies; J.J.M. van Dongen, unpublished results. b Somatic mutations hamper primer annealing in a part of the patients with B-NHL or B-CLL. c Based on T-ALL-like immunophenotype in lymphoblastic T-NHL and NPM-ALK expression in *50% of large cell anaplastic lymphomas of T-cell lineage. NR No reports on detailed studies.
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MRD Monitoring by Flow Cytometric Immunophenotyping Principles Hematological malignancies can be regarded as malignant counterparts of cells in immature (acute leukemia) or mature (CLL, CML, NHL) stages of hematopoiesis. Although the immunophenotype of the malignant cells is often comparable to their normal counterparts, tumor-associated immunophenotypes can be observed. Such tumor-associated immunophenotypes can be identified in the vast majority of acute leukemias, while they are less common in mature hematological malignancies. Asynchronous antigen expression refers to the coexpression of two or more antigens that are not present at the same time during normal differentiation. Cross-lineage antigen expression represents the expression of typical myeloid antigens on lymphoid cells or vice versa and the presence of B-lineage antigens on T-lineage cells or vice versa. Ectopic antigen expression refers to the presence of particular antigens on cells outside their normal breeding sites or homing areas or to the expression of antigens that are normally only expressed on nonhematopoietic cells. MRD Monitoring in Acute Leukemias Investigation of normal bone marrow (BM) B-cell precursors enabled establishment of templates for normal B-cell development. Malignant precursor-B lymphoblasts frequently display aberrant immunophenotypic features and thereby fall into ‘‘empty spaces’’ outside the normal B-lineage pathways (Fig. 1) (1,2). Flow cytometric investigations based on three- or four-color stainings showed the presence of leukemia-associated phenotypes in 70% to 95% of precursor-B-ALL patients (3,4). It should be noted that the detection of small numbers of precursor-B-ALL cells in regenerating BM after chemotherapy or after HSCT can be hampered by high frequencies of normal, regenerating precursor-B-cells (up to 50%) (5,6). The extent of B-cell regeneration in BM differs per treatment protocol, per phase of treatment, and seems to be dependent on the intensity of the preceding treatment (6). Since nearly all T-ALL express terminal deoxynucleotidyl transferase (TdT) as well as the pan-T-cell antigens CD2, cytoplasmic CD3 (CyCD3), CD5, and CD7, the ectopic TdT expression allows MRD detection in 90% of T-ALL. Flow cytometric analysis based on cross-lineage myeloid antigen expression, asynchronous antigen expression (e.g., CD34-positive/CD3-positive), and antigen overexpression (e.g., CD99 or CD7) can also be used for MRD detection in TALL (7,8). Similar to precursor-B-ALL, multiparameter flow cytometry in T-ALL reveals empty spaces outside normal T-cell development pathways. Together the various leukemia-associated immunophenotypes can be employed for MRD detection in virtually all T-ALL and lymphoblastic T-NHL. In AML, tumor-associated immunophenotypes can be observed in about 70% to 85% of patients (9–13). However, it should be noted that the immunophenotype of the AML cells may be heterogeneous and that several subpopulations
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Figure 1 Flow cytometric detection of minimal residual disease in a precursor-B-ALL patient, 5756, by use of the CD45/CD10/CD19 (A,B) and TdT/CD10/CD19 (C,D) triple labelings. The leukemia-specific immunophenotypic detection was based on CD10 overexpression and CD45 negativity. In the follow-up BM sample, taken 4.5 years from the diagnosis of ALL, the population of cells with leukemia-specific immunophenotype comprised 0.2% of BM cells, i.e., 1.5% of CD19-positive cells. At that time, the patient was in complete clinical remission of the leukemia. However, he underwent the overt hematological relapse of precursor-B-ALL, nine months after this positive MRD test.
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can be present (14). For reliable MRD analysis, all leukemic subpopulations should be monitored. The detection limit of current flow cytometric MRD methods varies between 0.1% and 0.01% for most precursor-B-ALL and AML, while in virtually all T-ALL a detection limit of 0.01% can be reached (7,15–17). It can be expected that the recent introduction of flow cytometers with six to eight colors will further improve the applicability and sensitivity of MRD detection in ALL and AML. Furthermore, on the basis of a comparison of gene expression profiles of normal and leukemic cells, new and widely applicable markers for MRD studies in acute leukemias are being identified. The applicability of this approach has already been proven for CD58, which is now one of the most useful markers to study MRD in precursor-B-ALL (18). MRD Monitoring in Mature B- and T-Cell Malignancies In B-CLL, quantitative differences in the levels of antigen expression as compared with normal B-cells can be observed in the vast majority of patients. This approach, analogous to detection of empty spaces outside the normal B-cell development in ALL, can potentially reach sensitivities of 104 to 105 (19–21). Especially the combination of CD19/CD5/CD20/CD79b or CD43 antibodies seems to be informative for MRD detection in PB of B-CLL patients. For MRD monitoring in BM, at least one of the combinations CD19/CD5/CD20/CD79b, CD19/CD5/CD38/CD79b, CD19/CD5/CD38/CD20 was found sufficiently sensitive and specific (19). A combination of CD81/CD22/CD19/CD5 is particularly useful for MRD detection in patients treated with CD20 antibody (rituximab) (22). In B-NHL, the sensitivity of immunophenotypic MRD analysis is often hampered by the lack of a tumor-associated immunophenotype and the presence of normal B-cells with a comparable immunophenotype. Nevertheless, by the use of markers that are normally only expressed on a subpopulation of B-cells, sensitivities of 103 can be achieved. Examples include the CD103 antigen on hairy cell leukemias and the CD5 antigen on mantle cell lymphoma (MCL) cells (23). Furthermore, protein products from particular chromosome aberrations may also be used as additional markers in flow cytometric analysis. BCL2/B-cell antigen/Ig light chain stainings may be employed for MRD detection in patients with follicular lymphomas (FL), since BCL2 overexpression is observed in this type of B-NHL with t(14;18) (24). Similarly, the overexpression of CyclinD1 in MCL with t(11;14) or of MYC in Burkitt’s lymphomas with t(2;8), t(8;14), or t(8;22) may be employed for MRD detection of these types of B-NHL (25). The vast majority of chronic T-cell leukemias and T-NHL belong to the TCRab lineage, whereas only a minor fraction expresses TCRgd. Antibodies against the protein products of V gene segments of TCR beta (TCRB) V gene families, recognizing 60% to 70% of normal and malignant T-cells with TCRb chain expression, can be used for detecting malignant cells (26). Also, Vg and Vd antibodies might be useful for detecting malignant (clonal) TCRgdþ T-cells, although the presence of normal TCRgdþ T-lymphocytes will interfere with these
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applications (27,28). This especially concerns Vg9/Vd2þ phenotypes, because most normal TCRgdþ T-lymphocytes have this TCR phenotype (29). The application of Vb, Vg, and Vd antibodies in well-chosen multicolor stainings can result in sensitivity levels of approximately 102. Such sensitivities do not allow true MRD detection but may enable monitoring of T-cell leukemia patients during treatment or predicting the possible outgrowth of a dominant subclone in case of oligoclonal T-cell proliferations. Detection of lower MRD levels (*103) in chronic T-cell leukemia or T-NHL is only possible upon analysis of tumor-associated immunophenotypes (e.g., lack of CD2 expression) or translocation-specific fusion proteins such as the NPM-ALK fusion protein in anaplastic large cell lymphoma (ALCL) with t(2;5) (30). Immunophenotypic Shifts A potential pitfall of immunophenotypic MRD detection in hematological malignancies is the occurrence of immunophenotypic shifts during the course of the disease. Differences in immunological marker expression are particularly frequent in acute leukemias and may concern up to 90% of patients (31–33). However, at least one leukemia-specific marker combination is retained by leukemic cells at relapse in at least 80% of patients (31,34). In order to limit the risk of false-negative results, at least two marker combinations per patient should be used for immunophenotypic MRD monitoring. Furthermore, since in AML shifts toward a more immature phenotype of the myeloblasts, consistent with clonal evolution of a leukemic stem cell, are frequently observed (33), antibody panels used for MRD monitoring in AML should preferably not be restricted to the immunophenotype detected at presentation but should also include markers of lineage immaturity. Immunophenotypic shifts may also occur during the early phase of treatment. Such immunophenotypic shifts have been reported in ALL and may be a direct result of the effect of the drugs on the expression level of various antigens or may be related to drug-induced cell kill (35,36).
MRD Monitoring by PCR Analysis of Junctional Regions Principles During early B- and T-cell differentiation the germline V, (D), and J gene segments of the Ig and TCR gene complexes rearrange, and each lymphocyte thereby obtains a specific combination of V-(D-)J segments that codes for the variable domains of Ig or TCR molecules. The random insertion and deletion of nucleotides at the junction sites of V, (D), and J gene segments make the junctional regions of Ig and TCR genes ‘‘fingerprint-like’’ sequences, which
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are different in each lymphocyte and thus also in each lymphoid malignancy. Therefore, junctional regions can be used as tumor-specific targets for MRDPCR analysis. Such targets can be identified (e.g., by PCR heteroduplex analysis or GeneScan analysis) at initial diagnosis in more than 95% of lymphoid malignancies and in approximately 10% of AML (37,38). Subsequently, the precise nucleotide sequence of the junctional regions can be determined. This sequence information allows the design of junctional region-specific oligonucleotides [either allele specific oligonucleotide (ASO) probes or ASO primers], which can be used to detect malignant cells among normal lymphoid cells during follow-up of patients. At present, real-time quantitative PCR (RQPCR) analysis is the most frequently used approach for MRD monitoring in hematological malignancies (Fig. 2) (39). MRD Monitoring in Acute Leukemias By applying appropriate primer sets, Ig/TCR gene rearrangements can be detected in virtually all precursor-B-ALL and T-ALL patients. The number and type of Ig/TCR rearrangements is however dependent on the age of the patient and the presence of fusion gene transcripts, like TEL-AML1 and MLL-AF4 (40–44). In order to limit the risk of false-negative MRD results due to clonal evolution phenomena (e.g., ongoing rearrangements, loss of subclones), preferably two MRD-PCR targets with sufficient sensitivity (104) should be used for each ALL patient (45–47). MRD Monitoring by PCR Analysis of Junctional Regions in Mature B- and T-Cell Malignancies For MRD studies in B-CLL and B-NHL patients, Ig heavy (IGH) chain gene rearrangements are frequently used, because they are present in virtually all lymphoma patients (37). Also Ig kappa (IGK) light chain and Ig lambda (IGL) light chain gene rearrangements can be applied as MRD-PCR target in lymphoma patients (48,49). A limitation of Ig gene rearrangements as MRD-PCR target is the occurrence of somatic hypermutations in part of B-CLL patients and in many B-NHL patients, especially FL and postfollicular B-NHL. This does not seriously hamper initial diagnostics because currently available multiplex PCR assays allow identification of clonal IGH, IGK, and/or IGL gene rearrangements in more than 95% of mature B-cell malignancies (37,50). However, when the somatic mutation process is active like in FL, this might result in the formation of subclones, which are no longer recognized by the applied primer-probe set. Since IGK-Kde and DH-JH rearrangements are not prone to somatic hypermutations, theoretically they are preferred targets for MRD analysis. Clonal evolution phenomena are rare in mature B-cell malignancies, and consequently this does not hamper MRD monitoring in lymphomas.
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TCR gamma (TCRG) gene rearrangements are found in virtually all mature T-lineage malignancies, whereas TCRB gene rearrangements can be detected in all malignancies belonging to the TCRab lineage (51). Thus, MRD studies in mature T-cell malignancies can generally use junctional regions of rearranged TCRG and TCRB genes as PCR targets, whereas TCR delta (TCRD) gene rearrangements are less often available (52). In mature T-cell malignancies, TCR genes are not affected by somatic mutations and are not susceptible to ongoing or secondary rearrangements. Consequently, one MRD-PCR target should be sufficient for reliable monitoring of mature T-cell malignancies during and after treatment. RQ-PCR Analysis of Ig/TCR Gene Rearrangements Several primer-probe sets for RQ-PCR-based detection of tumor-specific IGH gene rearrangements have been described, particularly for application in ALL patients (reviewed by van der Velden et al.) (39). In principle, these sets can also be applied for MRD studies in mature B-cell malignancies, although the presence of somatic hypermutations might particularly hamper efficient annealing of germline VH primers and probes. Application of 30 -minor groove binding (MGB) probes allows design in small areas of the VH gene segments that are less susceptible to somatic hypermutations (53). Also, for IGK, IGL, TCRG, TCRD, and TCRB, several germline primer-probe sets have been designed (39). In order to determine the sensitivity of the RQ-PCR assay, serial dilutions of the diagnostic sample are generally used (Fig. 2). For defining the sensitivity, several criteria (including reproducibility of the measurement, the difference
< Figure 2 RQ-PCR assay for detection of MRD using IGK-Kde gene rearrangement as a patient-specific target. (A) Schematic representation of an IGK-Kde rearrangement. The position and sequences of the primers used for target identification at diagnosis are indicated. (B) Sequences are given of the germline TaqMan1 probe and the germline Kde reverse primer used for RQ-PCR analysis during follow-up of patients. All sequences are given from 50 to 30 . For each patient, a patient-specific forward primer is designed. (C) RQ-PCR analysis of the Vk-Kde rearrangement in a precursor-B-ALL patient. Tenfold dilutions of the diagnostic sample in normal MNC DNA were analyzed at an annealing temperature of 608C; a quantitative range of 104 was reached. Normal MNC DNA did not show amplification in any of the four wells tested. (D) Applicability of RQ-PCR analysis of IGK-Kde rearrangements for MRD detection in follow-up samples of two patients with precursor-B-ALL an MRD high-risk patient, 5257, with high MRD levels (103) at the early time points and a low-risk patient, 5397, with undetectable MRD already at the end of induction treatment. RQ-PCR analysis (black diamonds) was compared with classical dot blot analysis (open squares). Abbreviations: RQ-PCR, real-time quantitative polymerase chain reaction; MRD, minimal residual disease; ALL, acute lymphoblastic leukemia; MNC, mononuclear cells.
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between specific and nonspecific amplification, slope, and correlation coefficient of the standard curve) should be taken into account (39). In order to compare data between different studies and/or different laboratories, it is essential to have uniform guidelines for RQ-PCR data interpretation (54). For Ig/TCR-based MRD data in ALL, such guidelines have recently been developed within the European Study Group on MRD detection in ALL (ESG-MRD-ALL; a consortium of 30 international laboratories, coordinated by JJM van Dongen and VHJ van der Velden) (55). These guidelines should be evaluated for use in monitoring of other hematological malignancies as well. The sensitivity of MRD-PCR analysis of junctional regions is dependent on the type of rearrangement, on the size of the junctional region, and on the ‘‘background’’ of normal lymphoid cells with comparable Ig/TCR gene rearrangements (56). One should be aware that the background of normal lymphoid cells is not constant, but can differ per treatment phase. For example, high frequencies of normal T-cells can be detected in postinduction follow-up samples, and substantial expansions of normal precursor-B-cells can be detected in regenerating BM after cessation of therapy (5,6,57). To check the quantity and amplifiability of the DNA samples, a control gene RQ-PCR should always be used. MRD Monitoring by PCR Analysis of Chromosome Aberrations Principles In several hematological malignancies, chromosome aberrations can be detected and may be used as MRD-PCR target. This includes breakpoint regions of fusion genes, fusion gene transcripts, and aberrantly expressed genes (39). An advantage of using chromosome aberrations as tumor-specific PCR targets for MRD detection is their stability during the disease course. However, many hematological malignancies do not have specific chromosome aberrations, which can be detected by PCR. Nevertheless, new techniques for rapid and efficient screening of relatively large breakpoint regions, such as long-distance PCR and long-distance inverse PCR, should render such genomic breakpoint fusion sites into more feasible MRD-PCR targets (58). MRD Monitoring in Acute Leukemias In about 40% of precursor-B-ALL patients and in about 15% of T-ALL patients, fusion gene transcripts can be detected. These particularly concern MLL-AF4, BCR-ABL, TEL-AML1, E2A-PBX1, CALM-AF10, and SIL-TAL1. In AML, CBFB-MYH11 and AML1-ETO can be found in 10% to 25% of patients, the frequency decreasing with age (59). PML-RARA transcripts can be detected in virtually all acute promyelocytic leukemia (APL) patients. Primer-probe sets for the detection of these fusion gene transcripts have been developed within the Europe Against Cancer program (60,61).
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MRD Monitoring in CML The BCR-ABL p210 fusion gene transcript can be detected in over 95% of patients with CML and thereby is an excellent MRD-PCR target in this group of patients. Moreover, both BM and PB can be applied for clinically relevant MRD monitoring in CML (62). Several efforts to standardize the methodology of BCRABL detection have been undertaken or are currently in progress (60,63,64). MRD Monitoring by PCR Analysis of Chromosome Aberrations in Mature B- and T-Cell Malignancies In approximately 30% of B-NHL patients, chromosome aberrations can be employed as tumor-specific MRD-PCR targets in which the PCR primers are chosen at opposite sides of the breakpoint fusion region (65). One of the most widely studied chromosomal translocations is t(14;18), involving the BCL2 and IGH genes, which occurs in 80% of FL patients, 20% of DLBCL patients, and which is detectable by standard PCR procedures in 60% to 70% of cases with t(14;18). The t(11;14) is characteristic for most MCL and involves the BCL1 and IGH genes. In 30% to 40% of MCL patients, the breakpoints are clustered within a restricted area [the major translocation cluster (MTC) region], allowing easy identification at the DNA level by standard PCR analysis. In the vast majority of Burkitt’s lymphoma patients, chromosomal aberrations involving one of the Ig genes in combination with the MYC gene, e.g., t(8;14), t(2;8), and t(8;22), can be found. In all the above-mentioned B-NHL types the breakpoints generally occur outside coding regions, implying that these translocations are not amenable to reverse transcriptase PCR (RT-PCR) analysis for MRD detection, but should be studied at the DNA level. In some lymphomas, aberrantly expressed genes can theoretically be used for MRD detection, although transcripts in normal cells may limit the sensitivity. Examples include the expression of CCND1 transcripts in MCL with t(11;14), and overexpression of MYC in Burkitt’s lymphomas with t(2;8), t(8;14), or t(8;22). Although RQ-PCR assays for such transcripts have been reported, they have not yet been used for MRD detection. In T-NHL only a few well-defined translocations are known so far. This concerns the NPM-ALK fusion gene that is observed in ALCL with t(2;5), and that can be used for RT-PCR analysis and potentially in some cases for PCR analysis at the DNA level as well (66). RQ-PCR Analysis of Chromosome Aberrations Depending on the type of chromosome aberration, detection limits of 103 to 106 can generally be reached. Because of the high sensitivity of PCR techniques, cross-contamination of RT-PCR products between patient samples is a major pitfall in RT-PCR-mediated MRD studies, resulting in up to 20% of falsepositive results (60). Such cross-contamination is difficult to recognize since
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leukemia-specific fusion gene transcript-derived PCR products and wild-type transcript-derived PCR products are not patient specific. This is in contrast to PCR products obtained from genomic breakpoint fusion regions, such as in t(14;18) and TAL1 deletions, which can be identified by use of patient-specific oligonucleotide probes. For quantification of fusion gene transcript, generally plasmid standard curves are used. For patient-specific fusion genes, the standard curve is usually prepared from serial dilutions of the diagnostic sample. To correct for the amount and quality of the RNA, the use of a control gene is essential (39,61). CLINICAL RELEVANCE OF MRD MONITORING IN LEUKEMIAS AND LYMPHOMAS Clinical Relevance of MRD Monitoring in ALL Clinical Value of MRD Detection During Frontline Treatment of Childhood ALL The most significant application of MRD monitoring in childhood ALL is the evaluation of the initial response to chemotherapy, since numerous studies have demonstrated that low levels or absence of MRD after completion of induction therapy predicts excellent outcome (67,68) (Fig. 3). The level of MRD-PCR positivity after induction therapy is independent of other clinically relevant risk factors (e.g., age, blast count at diagnosis, immunophenotype at diagnosis, presence of chromosome aberrations, response to prednizone, and classical clinical risk group assignment) and is the most powerful prognostic factor. Depending on the treatment protocol, the sensitivity of the MRD technique, and the timing of the follow-up BM samples, MRD negativity is associated with overall relapse rates of only 2% to 10% (15,17,69–72). Moreover,
> Figure 3 (A) Hypothetical graph showing the kinetics of leukemic cell decrease and regrowth in several ALL patients during and after treatment with the I-BFM-SG treatment protocol. MRD curves represent individual patients of the three MRD-based risk groups two patients with slow MRD clearance (high-risk group), two patients with moderate MRD clearance (intermediate-risk group), and one patient with rapid MRD clearance (low-risk group) (69). The detection limit of cytomorphologic techniques as well as the detection limit of flow cytometric immunophenotyping and PCR techniques are indicated: I, induction treatment; C, consolidation treatment; and II, reinduction treatment. (B) Relapsefree survival of the three MRD-based risk groups of children treated for ALL according to protocols of the International BFM Study Group. The three risk groups were defined by combined MRD information at the end of induction treatment and before consolidation treatment (69). (C) EFS plot of patients divided according to the MRD prior to hematopoietic stem cell transplantation. The five-year EFS and number of patients for each group are shown at the end of each curve (based on the report of the Pre-BMT MRD Study Group) (82). Abbreviations: EFS, event-free survival; MRD, minimal residual disease.
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sensitive MRD detection during the induction phase seems capable of identifying 20% to 50% of childhood ALL patients with a very rapid leukemia clearance and long-term relapse-free survival (73,74). On the other hand, several studies proved that high MRD levels at the end of induction treatment are associated with high relapse rates of 70% to 100% (15,17,69,71). MRD analysis at a single time point gives highly significant prognostic information, but a single time point is not sufficiently precise to define MRDbased low-risk and high-risk groups (15,17,69–71). Depending on the MRD study, the end-of-induction MRD status either identifies only patients at low risk of relapse (69,70) or more frequently identifies exclusively high-risk patients (3,71). In contrast, combined information on MRD at the end of induction treatment and before consolidation treatment is significantly superior to single time point measurement, which was first demonstrated by the International BFM Study Group (I-BFM-SG) (69). Such combined MRD information distinguishes patients at low risk with MRD negativity at both time points (5-year relapse rate of 2%); from patients at high risk with an intermediate (103) or high (102) degree of MRD at both time points (5-year relapse rate of 80%), and the remaining patients at intermediate risk (5-year relapse rate of 22%) (Fig. 3) (69). The group of MRD-based high-risk patients is larger than any previously identified high-risk group (*15%) and has an unprecedentedly high five-year relapse rate of 80%. In ongoing frontline protocols with MRD-based intervention, the MRD-based high-risk group is subjected to further intensification of treatment protocols, including HSCT during first remission or novel treatment modalities, e.g., imatinib in t(9;22)-positive cases. On the other hand, the MRD-based low-risk patients make up a group of a substantial size (*45%), comparable to the frequency of survivors of childhood ALL before treatment intensification was introduced. Therefore, low-intensity standard-risk protocols may be sufficiently effective to cure such patients. MRD-based risk-group distribution is even more striking in T-ALL: with fewer (*25%) low-risk patients with virtually no relapses, more (*25%) high-risk patients uniformly relapsing, and approximately 50% intermediate-risk patients with 25% relapses (72). Most ALL patients on conventional frontline chemotherapy protocols reach MRD negativity at some point during the treatment, while approximately 10% of patients remain continuously MRD positive until the end of treatment. These patients are usually at high or intermediate risk of relapse on the basis of MRD monitoring. Future MRD-based protocols should demonstrate whether continuous MRD monitoring in MRD-based high- and intermediate-risk patients could be also used for treatment intervention. Clinical Value of MRD Detection After Relapse of ALL After first relapse MRD monitoring has strong predictive value by assessing early treatment response after second induction treatment, although reported studies involved small groups of patients and need to be confirmed (75,76). In the BFM
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ALL-REZ treatment protocol, patients with MRD levels less than 103 at day 36 had a probability of relapse-free survival of 86%, whereas MRD levels more than or equal to 103 were uniformly predictive of dismal outcome (probability of relapse-free survival of 0%) (75). Similarly, the study from St. Jude’s Children’s Research Hospital showed that MRD investigation at day 36 of their protocol could largely distinguish patients with relatively good prognosis (MRD 104, 2-year incidence of relapse of 28%) and bad prognosis (MRD > 10-4, 2-year incidence of relapse of 70%). Many current ALL relapse treatment protocols include MRD measurements, which can be particularly useful for patients with late (off-therapy) relapses for optimal qualification for and timing of HSCT (76). Clinical Value of MRD Detection Before and/or After Stem Cell Transplantation in Childhood ALL Several studies have demonstrated that MRD monitoring is highly significant for ALL patients undergoing HSCT (77–81). Multicenter data combined by the PreBMT MRD Study Group showed that the level of MRD prior to allogeneic HSCT identifies a group of patients with a high risk of relapse after transplantation (Fig. 3) (82). The five-year event-free survival (EFS) of the group with negative-, low-, and high-level positive-MRD approximated 75%, 40%, and 20%, respectively (82). MRD-PCR positivity in ALL patients after HSCT is also suggestive of impending relapse (83). MRD was shown to occur in post-HSCT samples in 88% of patients who subsequently relapsed, while only 22% of patients in long-term CR showed MRD at any time after HSCT, mostly at low levels (83). Therefore, the treatment of patients with a high MRD burden before HSCT or persistent MRD positivity after HSCT should be modified (e.g., further cytoreduction before HSCT, intensified conditioning, and/or early post-HSCT immunotherapy to induce ‘‘controlled’’ graft-versus-leukemia effects) in order to improve their generally poor outcome (77–80,82). Clinical Value of MRD Detection in Adult ALL Adult ALL is more frequently characterized by high-risk features with greater drug resistance, poorer tolerance of and compliance with treatment as compared with childhood ALL (84) Also the frequencies of MRD positivity and the MRD levels in adult patients are significantly higher than in comparably treated children (85,86) Therefore, MRD information might be particularly important for standard-risk ALL patient without known factors predictive of resistant disease, which was clearly demonstrated by the study of the German Multicenter Study Group for Adult ALL (87). Similar to studies in childhood ALL, MRDbased measurement of early treatment response in adult ALL resulted in a very precise new risk group definition: low-risk group with MRD less than or equal to 104 at day 11 and day 24 (10%, 3-year relapse rate of 0%), high-risk group with MRD more than or equal to 104 or higher until week 16 (23%, 3-year relapse
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rate of 94%), and intermediate-risk group comprising the remaining patients (3-year relapse rate of 47%) (87). Moreover, among patients who reached MRD negativity, a conversion to MRD positivity (especially within the quantitative range) during the follow-up was associated with significantly increased risk of relapse (88) Another prospective study by Vidriales et al. has shown that MRD detection remains highly relevant for the entire adult ALL group. Patients with low/undetectable MRD early during induction treatment have excellent survival rates, while high MRD positivity at the end of induction treatment was associated with dismal outcomes (89). Introduction of imatinib into therapy of t(9;22)-positive adult ALL brought new promises on improving treatment outcome in this otherwise highly resistant disease (90). Therefore, MRD monitoring becomes of high clinical value to analyze the effectiveness of different combinations of chemotherapy, imatinib, and HSCT in t(9;22)-positive adult ALL (91,92). Clinical Relevance of MRD Monitoring in APL The most extensive MRD studies in AML concerned the RT-PCR monitoring of PML-RARA fusion transcripts in APL patients with t(15;17). The results from several retrospective as well as prospective RT-PCR studies in APL patients showed several distinct molecular characteristics of this AML subtype (93,94), leading to the first successful treatment intervention protocol based on MRD information (95). It was known for many years that treatment with alltrans-retinoic acid (ATRA) alone is insufficient to eliminate all leukemic cells (96) Rapid loss of MRD positivity during the first three months of ATRA and cytotoxic treatment was associated with good outcome, whereas continuous positivity was predictive of relapse (97). Nevertheless, MRD status at the end of induction treatment is clinically insufficient to predict the patient’s subsequent outcome as shown by several large prospective studies (98–100). With modern treatment protocols, combining ATRA with consolidation chemotherapy, PCR negativity is achieved at the end of treatment in virtually all patients. A small subset (*5%) of patients with refractory APL are RT-PCR positive even at the end of treatment consolidation (97–99), while the vast majority of relapsing patients (20–30% of total APL patients) is MRD negative at the end of consolidation treatment (98,101,102). To obtain clinically relevant information, continuous prospective MRD monitoring is required during the first 6 to 12 months after consolidation treatment for early identification of patients at increased risk of relapse (103). This is particularly important for patients with high-risk features at presentation such as hyperleukocytosis, while the need for continuous monitoring of patients with low initial white blood counts (i.e., <10 109/L) is now questionable (94). Reappearance of detectable MRD during the maintenance treatment usually precedes hematological relapse at a median time of two to three months (101,102). This information led to the definition of molecular relapse in APL, which is manifested by conversion from
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RT-PCR negativity to positivity in two successive BM samplings during followup (101). Lo Coco et al. (95) demonstrated that patients treated at the time point of molecular relapse have much better two-year EFS as compared with patients treated with the same salvage therapy (ATRA, chemotherapy and subsequent autologous HSCT in MRD-negative cases) at the time of hematological relapse (92% vs. 44%). Allogeneic HSCT remains a valuable treatment option for patients after hematological and/or molecular relapse. Graft-versus-leukemia effect can even overcome molecularly persistent leukemia after intensive second-line chemotherapy (104). For patients ineligible for allogeneic HSCT, autologous HSCT can be considered, provided that MRD-negative grafts could be obtained. Autologous HSCT with MRD-negative grafts might result in longterm clinical remission in the majority of patients, while MRD-positive autologous HSCT grafts carry increased risk of subsequent relapse of disease (94,105). Other treatment options for patients with molecular relapse of APL include chemotherapy regimens containing gemtuzumab ozogamicin or arsenic trioxide, which in selected cases might lead to prolonged clinical and molecular remission (106,107).
Clinical Relevance of MRD Monitoring in Non-M3 AML The only technique of MRD monitoring available for the vast majority of AML patients is multiparameter immunophenotyping. Initial flow cytometric studies already indicated that persistence and/or increase of cells with leukemiaassociated immunophenotypes precede hematological relapse (108). Similar to what was found for ALL patients, several studies showed strong prognostic significance of MRD detection for assessing the (early) response to chemotherapy in AML (12,109–111) In the study by San Miguel et al. (110), the risk for relapse was significantly increased in adult AML patients bearing equal or more than 5 103 residual cells at the end of induction treatment (67% incidence of relapse), while only 20% of the cases with less than 5 103 residual cells relapsed. At the end of intensification treatment, the threshold value of 2 103 residual cells also identified two distinct groups with relapse rates of 69% versus 36%. Multivariate analysis showed that this type of MRD information was independent of the other known prognostic factors like cell counts at diagnosis, age, or multidrug resistance (110). Extension of these analyses on 126 patients resulted in an even more refined classification with four risk groups: very low-risk group (MRD level <104, 8 patients, no relapses), low-risk group (MRD level 104–103, 37 patients, 3-year cumulative relapse rates of 14%), intermediate-risk group (MRD level <103–102, 64 patients, 3-year cumulative relapse rates of 50%), and high-risk group (MRD >102, 17 patients, 3-year relapse rate of 84%) (111). Similarly, Feller and colleagues clearly showed the prognostic significance of MRD measured continuously after the first three chemotherapy blocks and before HSCT. High MRD levels after
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each chemotherapy cycle (at a cutoff level of 1% after the first, 0.14% after the second, and 0.11% after the third cycle) and in peripheral blood stem cell (PB-SC) products (cutoff > 0.13%) were associated with significantly increased relative risk of relapse (9). In contrast, two other prospective studies could not demonstrate significant prognostic value of MRD determined after induction treatment (12,13). Instead, MRD levels or ‘‘log difference diagnosis-to-checkpoint’’ after consolidation treatment in adult AML were significantly related with outcome. While approximately 80% of patients with high MRD levels after consolidation treatment relapsed, only 18% to 25% of MRD low/negative suffered from disease recurrence; patients with low/undetectable MRD had also significantly better overall outcome (12,13). For many AML patients, HSCT is applied as treatment consolidation. The preliminary results of MRD monitoring in AML patients after HSCT showed an unequivocal association between the finding of cells with abnormal phenotype and subsequent relapse. Multiparameter flow cytometry was also shown to be an effective tool for discrimination between normal blasts transiently present in PB after HSCT and leukemic blasts heralding medullar relapse (112). Furthermore, in AML patients subjected to autologous HSCT, detection of leukemia-specific phenotypes in harvested BM was associated with treatment failure due to relapse (113). Accordingly, MRD levels more than or equal to 103 in autologous PB-SC harvests were found to be associated with AML relapse posttransplantation at a median time of six months (114). Two studies summarized the utility of MRD detection for pediatric AML patients (11,115). In one study, the MRD levels at the end of induction treatment were of clinical significance (11), while the second report emphasized the prognostic significance of detecting more than 0.5% at any time point after successful induction treatment (115). Currently, it is clear that detection of MRD is also of high clinical value for AML patients, but the meaning of MRD differs per treatment protocol and/or age group and depends on the laboratory approach and the timing of follow-up sampling. Therefore, clinical significance of MRD detection in pediatric AML should be further refined by future prospective studies and standardized per treatment protocol. The clinical value of the RT-PCR MRD studies in AML patients with either t(8;21) or inv(16) is less certain. These chromosome aberrations characterize a relatively small subset of AML patients (10–20%) with a fairly good prognosis. Moreover, several RT-PCR studies indicate that AML1-ETO or CBFB-MYH11 fusion transcripts remain detectable in BM and PB of patients in long-term remission, while other reports showed disappearance of fusion transcripts in survivors of leukemia in sustained CR (116–119). Preliminary results from several quantitative RT-PCR and/or RQ-PCR studies indicate that gradual reduction to very low fusion mRNA levels or to PCR-negativity throughout the disease course is associated with durable clinical remission. In contrast, persistently high MRD levels during treatment are associated with subsequent hematological relapse (120).
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Clinical Relevance of MRD Monitoring in CML Clinical Relevance of MRD Monitoring During Frontline Treatment of CML Introduction of imatinib has completely changed the treatment strategies in CML and the role of MRD monitoring in this malignancy (121,122). More than 75% of patients with newly diagnosed CML reach complete hematological and cytogenetic response on imatinib treatment (123). In contrast, only 25% to 30% of patients were experiencing complete or partial cytogenetic remission with frontline therapy based on IFN-a (62). Moreover, the degree of tumor reduction is significantly higher in patients treated with imatinib as compared with patients on IFN-a therapy. The International Randomized Study of Interferon and STI571 (IRIS) study demonstrated that after the first year of treatment, at least three-log reduction of tumor load was achieved in 39% of all patients treated with imatinib, but in only 2% of those given IFN-a and cytarabine (124). The probability of progression-free survival for patients with such excellent molecular response to imatinib was 100% at 60 months (125). Another study identified the BCR-ABL/ABL ratio less than 0.1% as the MRD threshold associated with continuous remission on imatinib treatment (126). Sequential MRD monitoring in patients demonstrating complete response either to imatinib or to IFN-a therapy showed gradual decline of MRD levels over time with imatinib superior to IFN-a/cytarabine in terms of the speed and degree of molecular responses (126–128). However, the subgroup of patients in long-term CR who converted into sustained PCR-negativity is very small (126,127,129). Most patients remain persistently RT-PCR positive and in patients treated with imatinib, residual BCR-ABL-positive cells are even more frequent in the CD34-positive stem cell compartment (126,130). These CML stem cells have clonogenic capacities, as demonstrated by in vitro experiments (130). MRD levels increasing at least twofold during imatinib treatment are indicative of the emergence of a resistant CML clone, which is most frequently characterized by acquired ABL kinase point mutation (131). Clinical Relevance of MRD Monitoring After Stem Cell Transplantation in CML Allogeneic HSCT following initial cytoreductive phase can cure selected CML patients. Current indications for such treatment include patient’s age, disease phase, the degree of histocompatibility between the donor and the recipient, and initial response to imatinib (122). Most CML patients are RT-PCR positive in the first six to nine months after allogeneic HSCT, indicating that the conditioning regimens before HSCT cannot eradicate all leukemic cells (132,133). Sustained PCR negativity within one year after HSCT is associated with cure, while patients with PCR positivity after one year or more post HSCT have significantly greater risk of relapse than patients with PCR negativity (132). With serial
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quantitative PCR analyses, it is possible to identify the group of high-risk patients that shows increasing MRD levels several months prior to hematological or cytogenetic relapse (132,134). Patients who remain in remission generally have decreasing or persistently low MRD levels, with some patients being BCRABL mRNA positive even 10 years after allogeneic HSCT (135). Quantitative MRD studies in CML enabled the definition of molecular relapse after allogeneic HSCT, which is equivalent to rising or persistently high MRD levels (BCR-ABL/ABL ratio of >0.02%) in two consecutive specimens more than four months after HSCT (136). When donor lymphocyte infusions (DLIs) are administered at the phase of molecular relapse, the outcome after immunotherapy is more favorable (137,138). In some responders, such early treatment results in conversion into sustained PCR negativity (137). Recently, it was demonstrated that imatinib could be an alternative to DLI for the treatment of molecular relapse of CML after HSCT (139). Interestingly, 40% of patients remained in continuous molecular remission after imatinib discontinuation (139). Clinical Relevance of MRD Monitoring in CLL Clinical Relevance of MRD Monitoring in Chronic B-Cell Leukemias B-CLL has a highly variable clinical course and shows heterogeneity in prognosis. Many patients show a rather indolent disease without requiring treatment, whereas others present with more aggressive forms that often lead to early death. In the last few years, more insight has emerged into the biological prognostic factors that determine differences in the disease course. The currently most relevant parameters associated with unfavorable outcome include cytogenetic aberrations (17p deletions, 11q aberrations), unmutated IGH VH segments, and increased CD38 and ZAP70 expression (140). Together with the increased knowledge on prognostic factors, therapy results have further improved over the years. Moreover, the introduction of newer treatment modalities, such as autologous and allogeneic HSCT and especially therapy with antibodies such as rituximab or alemtuzumab (CD52) or chemo-immunotherapy (combination of chemotherapy and antibodies), has resulted in better responses in a significant proportion of B-CLL patients (141,142). In fact, this has shifted therapeutic goals from palliation to cure, at least in subsets of patients. With eradication of MRD becoming a realistic goal in B-CLL, MRD detection has now become a relevant issue (142). To measure MRD in B-CLL, both qualitative and quantitative approaches have been applied. Qualitative MRD studies show variable sensitivities and generally fail to show increased progression-free survival. Quantitative approaches show higher sensitivities and seem more predictive (19,21). B€ ottcher et al. have shown that four-color flow cytometry and RQ-PCR with ASO primers have largely comparable sensitivities (around 104) (21). Measurement of MRD is not relevant in conventionally treated B-CLL patients, because patients still show a relatively high tumor burden, even in CR. In
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contrast, several studies have demonstrated a role for MRD status evaluation in clinical trials employing newer treatment modalities (142,143). Combination chemotherapy (e.g., fludarabine and cyclophosphamide) has been shown to result in MRD negativity in previously untreated patients or relapsed/refractory patients (144). Furthermore, it has also been demonstrated that achieving an MRD-negative response after alemtuzumab was the best predictor of survival in relapsed or refractory B-CLL. Chemoimmunotherapy (e.g., fludarabine, with or without cyclophosphamide, and rituximab, or fludarabine and alemtuzumab) also leads to high overall response rates, including MRD-negative patients. Other studies showed that molecular remission (MRD negativity) could be achieved upon alemtuzumab consolidation therapy and after autologous and allogeneic HSCT (144,145). Despite the molecular remissions that can be achieved in the above studies, it remains to be shown that MRD negativity also correlates with improved and prolonged survival rates in all these therapeutic approaches. In addition, MRD detection has entered B-CLL treatment protocols only recently and is often limited to some B-CLL patient groups. Two important actions are therefore essential in the coming years. One is the further standardization of sensitive methods for MRD detection in multicenter laboratory networks. The other is the implementation of standardized MRD detection in multicenter clinical trials to prove its clinical application (142). Several initiatives in these directions have already been taken. One interesting trial in this respect could be the Nordic/ HOVON-68 trial in which fludarabine and cyclophosphamide will be compared with fludarabine, cyclophosphamide, and alemtuzumab in biologically defined [mutation status and fluorescence in situ hybridization (FISH) aberrations] highrisk B-CLL patients. Included in the response criteria of this trial is MRD detection via ASO-PCR and multiparameter flow cytometry in patients in CR. Results from this and similar trials should help to reveal the true value of MRD detection in clinical management in B-CLL patients as well as define the most sensitive and practical approach for routine MRD analysis. Clinical Relevance of MRD Monitoring in Chronic T-Cell Leukemias In chronic leukemias of the T-cell lineage, such as T-cell large granular lymphocyte (T-LGL) leukemia, MRD evaluation has never been a goal so far; firstly, because these diseases are mostly very indolent and secondly, as chronic T-cell leukemias are relatively rare, large clinical studies with accurate follow-up analysis have simply not been performed. Recently, a clinical trial for standardized treatment of T-LGL leukemias has been initiated in Germany in which methotrexate and fludarabine are being evaluated. In this study, MRD detection via TCRB/TCRG ASO RQ-PCR and four-color flow cytometry will be implemented and be used to assess the treatment response. Eventually, such clinical studies should reveal whether there is a true value for MRD detection as prognostic factor or for evaluating treatment response in a relatively indolent disease as chronic T-cell leukemia.
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Clinical Relevance of MRD Monitoring in NHL In NHL patients, it is generally not possible to monitor MRD at the original site of disease. However, in a proportion of lymphomas (particularly high and intermediate-grade lymphomas) malignant cells can clearly be detected in BM and PB at diagnosis (Table 2). Immunophenotypic and molecular studies can on one hand contribute to better recognition of minimal BM infiltration at diagnosis, undetectable with cytomorphological analyses (Fig. 4). On the other hand, the presence of MRD in BM and to a lesser extent in PB might be applied as a surrogate marker for treatment effectiveness (Table 2) (146). Detection of BM Involvement During Initial Staging of NHL Immunophenotypic and molecular detection of BM and/or PB involvement has not yet been routinely implemented into clinical staging of NHL. Nevertheless, the presence of aberrant clonal cells was demonstrated in BM of most children
Figure 4 An example of BM staging in a patient with NHL using flow cytometric immunophenotyping. CD19-positive B-cells were gated and the expression of CD38, SmIgk, and SmIgl were evaluated. In (A) a normal bone marrow sample is shown, with an approximately equal distribution of SmIgk and SmIgl in the mature B-cells (black). In (B) a bone marrow sample of a patient with B-NHL is shown; clearly an aberrant SmIglpositive B-cell population can be detected. The cells that are strongly positive for CD38 and SmIg-negative are precursor-B-cells (light gray); the cells that are very strongly positive for CD38 are plasma cells (dark gray).
100% 100% 75% 100% <5% 100% 100% 100% NA NA NA
*70% *70% *10% *40% *40% *60% *20% <5% *30% *40% *10% *40%
>60% 10–30% 30–60% 30–60% 30–60% 10–30% <10% 30–60% 30–60% 10–30% 30–60%
1.2% 7.6% 1.8% 22.1% 6.0% 30.6% 2.4% 2.1% 1.7% 2.4% 7.6%
>50%
Occurrence of somatic hypermutation
>60%
Bone marrow
6.7%
Peripheral blood
Abbreviations: NA, not applicable; ALCL, anaplastic large cell lymphoma. Source: From Ref. 146.
B-cell lymphomas Small lymphocytic lymphoma/ B-CLL Lymphoplasmacytic lymphoma MALT lymphoma Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Mediastinal large B-cell lymphoma Burkitt lymphoma T-cell lymphomas Precursor-T-cell lymphoblastic lymphoma ALCL Mature T-cell lymphomas (except ALCL)
Type of lymphoma
Relative frequency
Dissemination to
75–80% >98%
>95%
>95% 85–90% >95% >95% >98% 90–95% >65% >80%
>98%
Ig/TCR
20% –
15%
– 30% – 70–80% 30–40% – – 70%
–
Chromosome breakpoint fusions
75% –
–
– 30% – – – – – –
–
Fusion gene transcripts
Availability of MRD-PCR targets
Table 2 Possibilities for Molecular Monitoring in Most Frequently Occurring Non-Hodgkin’s Lymphomas
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with T-LBL (147). BM involvement detected by BCL2-IGH PCR analysis is a constant feature, not only of advanced stage FL with t(14;18) but also in patients with localized stages (148). Using long-distance PCR for t(8;14), BM involvement was found in more than one-third of samples from children with Burkitt’s lymphoma, mainly patients without morphological BM involvement but with advanced stage disease (149). Immunophenotypic and/or molecular staging might have prognostic significance. This was demonstrated for DLBCL, where it was possible to identify a subset of patients with negative BM histology and positive Ig PCR results characterized by significantly lower CR rate and significantly poorer overall survival as compared with patients, in whom both BM histology and PCR results were negative (150). Further prospective studies should reveal whether detection of submicroscopic BM involvement with sensitivities of 103 to 105 would improve prediction of clinical outcome in lymphoma patients. If so, patients with detectable high MRD levels in BM at diagnosis might require more intensive treatment. Clinical Relevance of MRD Monitoring in FL Patients The vast majority of clinical studies concentrated on FL with t(14;18) using the BCL2-IGH fusion gene as DNA target for PCR-based MRD analysis (23,151,152). Most FL patients harbor lymphoma cells in BM or PB at initial presentation (148). Moreover, the lower the tumor load in BM at diagnosis (as quantitatively assessed by RQ-PCR), the higher is the chance for the achievement of a complete clinical and molecular response (153). For patients treated with conventional chemotherapy, some studies could show a significant association of MRD-negativity during the cytotoxic treatment with longer relapse-free survival, with BM being more informative for MRD monitoring than PB (153–156). In contrast, several reports could not find an obvious correlation between the presence or absence of t(14;18)-positive cells in the circulation and relapse-free survival (157,158). More recently, the combination of chemotherapy and treatment with rituximab was shown to produce durable clinical remission in a subgroup of FL patients accompanied by PCRnegativity in BM and/or PB (153,159,160). Patients who achieved sustained molecular remission had significantly better clinical outcome at three-year followup as compared with persistently MRD-positive patients or those who converted from negativity to MRD-PCR positivity (153,160). With recently developed high-dose sequential chemotherapy, it is possible to harvest MRD-PCR-negative autologous BM grafts in most FL patients and MRDPCR-negative autologous PB grafts in more than half of the patients (161,162). Such in vivo purging is even more effective after addition of rituximab as it was shown that t(14;18)-positive B-cells can be effectively cleared from PB and/or BM in a subset of patients treated with rituximab as single frontline treatment (163). Preliminary data suggest that the combination of high-dose chemotherapy and rituximab can yield MRD-PCR-negative autografts in virtually all patients (164).
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Initial data suggested that patients transplanted with MRD-PCR-negative autologous grafts showed significantly longer disease-free survival in comparison with those whose BM contained residual clonal lymphoma cells after purging (165). Recent studies also showed that only a small fraction of patients who received MRD-PCR-negative autologous grafts collected after high-dose sequential chemotherapy relapsed, while more than half of the patients who were treated with MRD-positive grafts relapsed (161,162). In contrary, several other studies could not demonstrate a significant correlation between FL outcome and PCR status of the reinfused BM (166,167). RQ-PCR data indicate that a small subset of patients in continuous clinical remission (after high-dose chemotherapy supported by autologous HSCT) become MRD-negative (166), while in most patients BCL2-IGH fusion genes can be persistently found with stable levels within one order of magnitude (166). Sequential MRD monitoring in a group of patients with advanced stage FL, treated with high-dose sequential chemotherapy and autografting, showed that virtually all patients with all follow-up samples PCR negative remained in CR (168). In contrast, most patients who were persistently MRD positive relapsed. These combined MRD studies indicate that intensified high-dose sequential chemotherapy followed by MRD-negative autologous HSCT is a promising treatment modality in FL patients. Multicenter clinical studies using wellstandardized MRD-PCR techniques are required to establish the quantitative criteria for molecular remission in FL and the potential applicability of MRD information for clinical decision–making. Another treatment option for patients with FL might be allogeneic HSCT. Monitoring of the number of BCL2-IGH-positive cells in BM/PB after allogeneic HSCT significantly reflects the clinical remission status. This information might be used to assess the graft-versus-lymphoma effect of allogeneic HSCT and subsequent DLIs in relapsing patients (169). Recently, allogeneic HSCT with a reduced-intensity conditioning regimen including rituximab was shown to be effective in a subset of FL patients. Recent data suggest that such treatment might result in long-term clinical remission and sustained BCL2-IGH negativity (170). Clinical Relevance of MRD Monitoring in MCL Patients Using IGH gene rearrangements and BCL1-IGH fusion genes as DNA targets, MCL patients were found to be continuously MRD-positive in BM and/or PB during chemotherapy (171,172). Conventional induction therapy with CHOP-like regimens does not significantly reduce the tumor load in BM and/or PB when compared with pretreatment MRD levels (172,173). With more intensive treatment, including a combination of the rituximab and conventional chemotherapy, approximately one-third of MCL patients can reach an MRD-PCR-negative status (174). However, in the majority of cases, this conversion to PCR negativity is transient and this molecular remission is not associated with better progressionfree survival.
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Until recently, it was virtually impossible to harvest autologous MRDnegative BM or PB-SC grafts in MCL (168,171–173). Some mobilization regimens before PB-SC even resulted in increased PB contamination with tumor cells (172). Also the purging procedure in MCL was generally unsuccessful (171,172,175). Reinfusion of MRD-positive grafts was frequently associated with the relapse of MCL (171). Recently, significant progress has been achieved by ‘‘in vivo purging’’ of PB CD34-positive autografts using a combination of highdose chemotherapy followed by immunotherapy with Rituximab (176). With such a regimen, it was possible to obtain optimal amounts of PCR-MRD-negative PB-SC in virtually all MCL patients (176,177) On the other hand, with intensive conditioning treatment and pretransplant rituximab immunotherapy, MCL cells conferred with the graft might have no major prognostic impact (173). Recent data suggested that high-dose chemoradiotherapy followed by autologous PB-HSCT might result in molecular remission in a subset of MCL patients (168,173). MRD-PCR negativity after standard debulking chemotherapy followed by high-dose chemotherapy, rituximab and autografting is strongly predictive of durable clinical remission (173,177). Allogeneic HSCT represents another effective treatment regimen for patients with advanced MCL and preliminary data show conversion to MRD negativity after HSCT, which is related to long-term hematological remission (171,178). CONCLUSIONS—CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Despite the complex methodology and the relatively high costs, MRD monitoring is becoming an essential part of modern treatment protocols in different leukemia and lymphoma categories (Table 3). Many ALL frontline treatment protocols now use MRD diagnostics during the first three months for treatment stratification, i.e., for recognition of patients at high risk of relapse who should receive intensified treatment and for recognition of low-risk patients, who should be rescued with standard-intensity or reduced chemotherapy. In addition, MRD information is used for optimal timing of allogeneic HSCT in ALL, with the aim to reach low or undetectable MRD status with pretransplant chemotherapy. Within the next five years, it will become clear whether MRD diagnostics can improve the overall clinical outcome in ALL patients. Hopefully, the treatment reduction in the large group of MRD-based low-risk patients will not affect the low relapse rate but will reduce the medical and psychological sequelae. In current CML and APL treatment protocols, MRD is continuously measured over a clinically relevant disease-specific time span. Such MRD monitoring in CML and APL enables identification of patients at high risk of relapse already at an early stage when the tumor load is relatively low (molecular relapse) and when restarting of treatment is more effective. Within the next five years, MRD monitoring will be routinely used to confirm remission in the vast majority of CML and APL patients. For refractory or relapsing patients, MRD
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Table 3 Prognostic Value and Clinical Applicability of MRD Detection in Leukemias and Lymphomas Type of MRD application Disease category ALL Chronic B-cell leukemias B-NHL Chronic T-cell leukemias T-NHL APL AML (excl. APL) CML
MRD assessment before HSCT
MRD assessment after HSCT
þþ þ*
þþþ þ
þþ þþ
þ* þ*
þ
þþ
þþ þþ
þ* þþþ þþ þþþ
þþ þþ þ
þþþ þ þþþ
Early response Continuous monitoring for to frontline therapy titration treatment þþþ
þþþ, value of MRD detection proven in large prospective studies þþ, potentially clinically relevant (e.g., in a subset of patients) but not yet proven by large prospective studies þ, MRD results are statistically significant but their clinical implication is not yet established *, only relevant for patients treated with more aggressive protocols and/or including CD20 antibody , MRD detection has no additional value as compared with conventional cytomorphological techniques.
monitoring should be helpful to evaluate the effectiveness of alternative treatment approaches, e.g., testing novel tyrosine kinase inhibitors in CML or combination of ATRA, chemotherapy, and arsenic trioxide in APL. The strategy of continuous MRD monitoring for therapy ‘‘titration’’ might also be applicable in other subtypes of AML, CLLs, and different subtypes of NHL. However, large-scale studies are required to fully define the disease-specific MRD applications or ‘‘MRD windows’’ for clinically reliable MRD monitoring in AML. Within next five years, improved strategies of MRD monitoring based on multiparameter (eight-color) flow cytometric immunophenotyping should be developed for AML patients and introduced into prospective clinical trials. Such application of MRD diagnostics should become feasible in high-risk CLL patients and NHL subsets with frequent BM involvement such as Burkitt’s lymphoma, T-LBL, FL, and/or MCL. Furthermore, sensitive MRD techniques will be more extensively applied for other specific diagnostics aims, such as detection of minimal central nervous system involvement in ALL, early diagnosis of ocular lymphomas, early diagnosis of leukemia/lymphoma in patients with unexplained cytopenias, improved staging of lymphomas, and for assessing the effectiveness of newly available cytotoxic or immunotherapeutic drugs.
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115. Sievers EL, Lange BJ, Alonzo TA, et al. Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children’s Cancer Group study of 252 patients with acute myeloid leukemia. Blood 2003; 101:3398–3406. 116. Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission. Blood 1993; 82: 712–715. 117. Satake N, Maseki N, Kozu T, et al. Disappearance of AML1-MTG8(ETO) fusion transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission. Br J Haematol 1995; 91:892–898. 118. Hebert J, Cayuela JM, Daniel MT, et al. Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification. Blood 1994; 84: 2291–2296. 119. Tobal K, Johnson PR, Saunders MJ, et al. Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission. Br J Haematol 1995; 91:104–108. 120. Krauter J, Wattjes MP, Nagel S, et al. Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients. Br J Haematol 1999; 107:80–85. 121. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344:1031–1037. 122. Goldman JM, Melo JV. Chronic myeloid leukemia: advances in biology and new approaches to treatment. N Engl J Med 2003; 349:1451–1464. 123. O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348:994–1004. 124. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349:1423–1432. 125. Druker BJ, Guilhot F, O’Brien SG, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355:2408–2417. 126. Paschka P, Muller MC, Merx K, et al. Molecular monitoring of response to imatinib (Glivec) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission. Leukemia 2003; 17:1687–1694. 127. Hochhaus A, Reiter A, Saussele S, et al. Molecular heterogeneity in complete cytogenetic responders after interferon-alpha therapy for chronic myelogenous leukemia: low levels of minimal residual disease are associated with continuing remission. German CML Study Group and the UK MRC CML Study Group. Blood 2000; 95:62–66. 128. Muller MC, Gattermann N, Lahaye T, et al. Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C. Leukemia 2003; 17:2392–2400. 129. Kurzrock R, Estrov Z, Kantarjian H, et al. Conversion of interferon-induced, long-term cytogenetic remissions in chronic myelogenous leukemia to polymerase chain reaction negativity. J Clin Oncol 1998; 16:1526–1531.
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146. van Dongen JJM, Langerak AW, Szczepan´ski T, et al. Molecular monitoring of lymphoma. In: Canellos GP, Lister TA, Young BD, eds. The Lymphomas. Philadelphia, PA: Saunders Elsevier, 2006:83–109. 147. van Dongen JJM, Hooijkaas H, Adriaansen HJ, et al. Detection of minimal residual acute lymphoblastic leukemia by immunological marker analysis: possibilities and limitations. In: Hagenbeek A, L€ owenberg B, eds. Minimal Residual Disease in Acute Leukemia. Dordrecht, The Netherlands: M Nijhoff Publishers, 1986:113–133. 148. Gribben JG, Freedman A, Woo SD, et al. All advanced stage non-Hodgkin’s lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment. Blood 1991; 78:3275–3280. 149. Mussolin L, Basso K, Pillon M, et al. Prospective analysis of minimal bone marrow infiltration in pediatric Burkitt’s lymphomas by long-distance polymerase chain reaction for t(8;14) (q24;q32). Leukemia 2003; 17:585–589. 150. Mitterbauer-Hohendanner G, Mannhalter C, Winkler K, et al. Prognostic significance of molecular staging by PCR-amplification of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma (DLBCL). Leukemia 2004; 18:1102–1107. 151. Corradini P, Ladetto M, Pileri A, et al. Clinical relevance of minimal residual disease monitoring in non-Hodgkin’s lymphomas: a critical reappraisal of molecular strategies. Leukemia 1999; 13:1691–1695. 152. Buckstein R, Pennell N, Berinstein NL. What is remission in follicular lymphoma and what is its relevance? Best Pract Res Clin Haematol 2005; 18:27–56. 153. Rambaldi A, Carlotti E, Oldani E, et al. Quantitative PCR of bone marrow BCL2/ IgHþ cells at diagnosis predicts treatment response and long-term outcome in follicular non-Hodgkin lymphoma. Blood 2005; 105:3428–3433. 154. Lopez-Guillermo A, Cabanillas F, McLaughlin P, et al. The clinical significance of molecular response in indolent follicular lymphomas. Blood 1998; 91:2955–2960. 155. Gribben JG, Neuberg D, Barber M, et al. Detection of residual lymphoma cells by polymerase chain reaction in peripheral blood is significantly less predictive for relapse than detection in bone marrow. Blood 1994; 83:3800–3807. 156. Fernandez-Ruiz E, Cabrerizo M, Ortega M, et al. High molecular response rate and clinical correlation in patients with follicular lymphoma treated with cyclophosphamidevincristine-prednisone plus interferon alpha 2b. Clin Cancer Res 2003; 9:2497–2503. 157. Lambrechts AC, Hupkes PE, Dorssers LC, et al. Clinical significance of t(14;18)positive cells in the circulation of patients with stage III or IV follicular nonHodgkin’s lymphoma during first remission. J Clin Oncol 1994; 12:1541–1546. 158. Mandigers CM, Meijerink JP, Mensink EJ, et al. Lack of correlation between numbers of circulating t(14;18)-positive cells and response to first-line treatment in follicular lymphoma. Blood 2001; 98:940–944. 159. Czuczman MS, Grillo-Lopez AJ, White CA, et al. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J Clin Oncol 1999; 17:268–276. 160. Rambaldi A, Lazzari M, Manzoni C, et al. Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma. Blood 2002; 99:856–862. 161. Corradini P, Astolfi M, Cherasco C, et al. Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose
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176. Magni M, Di Nicola M, Devizzi L, et al. Successful in vivo purging of CD34containing peripheral blood harvests in mantle cell and indolent lymphoma: evidence for a role of both chemotherapy and rituximab infusion. Blood 2000; 96:864–869. 177. Gianni AM, Magni M, Martelli M, et al. Long-term remission in mantle cell lymphoma following high-dose sequential chemotherapy and in vivo rituximab-purged stem cell autografting (R-HDS regimen). Blood 2003; 102:749–755. 178. Corradini P, Ladetto M, Astolfi M, et al. Clinical and molecular remission after allogeneic blood cell transplantation in a patient with mantle-cell lymphoma. Br J Haematol 1996; 94:376–378.
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4 New Methods for Clinical Trials: AML as an Example Elihu Estey Division of Hematology, University of Washington Medical Center, Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
INTRODUCTION This chapter’s fundamental tenet is that the conventional methodology used to evaluate new drugs is incongruent with clinical reality; in particular, the former frequently underestimates the complexity of the latter. I will point out that this situation is by no means inevitable. Indeed, a large number of papers in statistical literature have pointed out flaws in conventional designs and have proposed alternatives; however, this literature has largely been ignored. By conventional methodology, I refer to: (i) a phase 1 trial whose sole endpoint is toxicity that is quantified using standard criteria and evaluated using a ‘‘3þ3 design’’ in order to determine a dose for a subsequent phase 2 trial; (ii) a single-arm phase 2 trial formally concerned only with a single measure of efficacy, followed by (iii) a large randomized phase 3 trial intended to compare the new drug with standard treatment. Although the chapter’s focus is acute myeloid leukemia (AML), I believe readers can generalize its points. QUICKER PHASE 3 TRIALS Because readers are perhaps most familiar with phase 3 trials, I will begin by considering such a trial, which, in order to compare a new drug X with standard 3þ7 therapy in older patients with untreated AML, typically enrolls 400 patients.
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The trial requires such a large number to detect a relatively small difference between the two treatments with, at the most, a 5% false positive rate and a 10% to 20% false negative rate corresponding to 80% to 90% power. For example, a HOVON-MRC trial comparing standard induction therapy with or without PSC833 considered it important to detect a minimum increase from 9.5% to 18% in a two-year event-free survival (EFS) probability (1), a CALGB study comparing the same treatments wished to uncover a minimum improvement in complete remission (CR) rate from 50% to 65% (2), and an ECOG study comparing different anthracylines was interested in a minimum increment in CR rate of 55 to 75% (3); given the propensity to relapse, it seems fair to say that such improvements in CR rate would result in gains in a two-year EFS probability similar to those of interest in the HOVON-MRS study. I question whether such improvements, while statistically significant given the aforementioned 5% false positive rate, are medically significant in the minds of patients and treating physicians. The counter-argument is that improvement in the treatment of AML will only come in small increments that must not be missed. However, it is worth noting that dramatic advances have also occurred, e.g., arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL), 2 chlorodeoxyadenosine (2-CdA) for hairy cell leukemia, and interferon and imatinib for chronic myelogenous leukemia (CML). The cases of interferon for CML, 2 CdA, and ATO illustrate that a more sophisticated understanding of disease pathogenesis than is currently possible for AML need not underlie therapeutic advances. At any rate, it appears fair to ask whether we would be better served by current phase 3 designs or by designs that by focusing on larger differences require fewer patients and can thus be completed more quickly (4). The stipulation of a 5% significance (i.e., false positive) rate and a 10% to 20% false negative rate is also relevant in this regard. This formulation is used for phase 3 trials in many different diseases that, however, are medically quite different. Thus, while the desire to provide more protection against a false positive result than a false negative one is appropriate in diseases for which there is already reasonably effective treatment, the same desire appears less appropriate in a disease such as AML for which treatment is less effective and, accordingly in which replacement of a standard treatment by a (falsely positive) new treatment is less consequential. Accordingly, I think that phase 3 studies might be designed to provide the same protection say 10% to 15% against both false positive and false negative conclusions. This proposal would also make possible smaller sample sizes and allow more timely completion of studies. ADAPTIVE RANDOMIZATION AND BAYESIAN INFERENCE The word ‘‘adaptive’’ refers to the possibility, in light of current results, to unbalance a 1:1 randomization scheme to favor a treatment arm that is outperforming another arm (5). Although this possibility has intuitive appeal, it is
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rare for phase 3 studies to perform more than one or two interim examinations of accumulating data. The reason is that, as detailed below, p-value-based (‘‘frequentist’’) methods cannot maintain a 0.05 significance level at the conclusion of the trial while supporting numerous interim analyses. In contrast, Bayesian methodology permits (and encourages) such analyses. The Bayesian paradigm is built around observed data and parameters, which I will denote by y. An example of a parameter is the probability of CR. In the Bayesian paradigm, parameters are random quantities, with probability distributions describing one’s uncertainty about them (6–8). The Bayesian paradigm begins with a prior distribution, p(y), that characterizes one’s uncertainty about y before conducting a trial and observing the data. The next object is the likelihood, L(data | y), which describes the probability of observing any specified data given any value of y; the binomial distribution is an example of a likelihood for binary events such as CR. The Bayesian paradigm combines the observed data with the prior to arrive at a ‘‘posterior’’ distribution of y, which describes one’s uncertainty about y after observing the data. Specifically, Bayes’ theorem arrives at the posterior by multiplying the prior by the likelihood of observing the data given the parameter. When making decisions or inferences on the basis of accruing data, Bayes’ theorem may be applied repeatedly, with the posterior at each stage becoming the prior for the next stage. The probability distributions in this sequence become increasingly informative about y as the data accumulate. This process, which is known as ‘‘Bayesian learning,’’ (Fig. 1) is especially useful in sequential data monitoring during a clinical trial. At each of the many times, the current posterior probability distribution may be used to make a variety of decisions, including modifying doses (as in a phase 1 trial), dropping an inferior treatment arm, unbalancing a randomization in favor of a treatment or treatments that have relatively superior performance, or terminating the trial early either because of the superiority or futility of a treatment. The Bayesian approach is properly contrasted with the conventional, frequentist approach, which uses p-values as a basis for inferring strength of evidence. The p-value is defined as the probability under the null hypothesis of observing data as extreme, or more extreme, than that actually observed under a predetermined experimental design. Many scientists erroneously believe that the p-value is the probability that the null hypothesis is true, given the observed data. In fact, this intuitively appealing quantity is a Bayesian posterior probability, not a p-value. This is so because, given its definition, the calculation of a p-value involves both observed and unobserved data and is heavily dependent on a predetermined experimental design (9). An obvious logical flaw is that a given data set could give rise to two or more different p-values, depending on which design was intended. For observational data, where there is no experimental design, the p-value of a given test may be a wide variety of different quantities, depending on what sort of assumptions one makes about how the data arose and the manner in which putative hypotheses are formulated. Indeed if the
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Figure 1 Bayesian learning. On the x-axis are various probabilities of CR following receipt of a drug. On the y-axis are ‘‘densities’’ corresponding to the weight of the evidence in favor of each probability. Each curve is characterized by a mean (the most likely probability) and a dispersion that is proportional to the width of the curve. For example, the left-most curve (the ‘‘prior’’) indicates that 16% is the most likely CR rate, but that some weight should be given to probabilities of 10% to 55%. Such a curve might correspond to a prior probability as based on historical data. As patients are treated and a CR rate greater than 16% is observed succeeding curves (‘‘posteriors’’) are shifted to the right and become less disperse as more patients are treated. For each curve, it is simple to compute the probability that the CR rate is greater than a given percentage with decisions to stop or continue the study on the basis of this probability. An obvious issue is the choice of the prior; in practice the effect of various priors on the posterior are examined.
experimental design is not specified a p-value cannot be calculated. Once a frequentist experimental design is specified, the investigator is permitted to look at the accruing data only if and when an interim test is specified by the design. If this constraint is violated, then the p-value must be adjusted upward to account for the fact that a false positive decision might have been made. Many ‘‘group sequential’’ designs have been described to deal with the issue of interim analyses. Each design’s statistical rules are constructed so that, accounting for the interim analyses, the overall false positive probability (type I error) will be maintained below a desired level, typically, p ¼ 0.05. In practice, this is done by performing the interim tests at p-values much smaller than 0.05. However, the same overall p-value can lead to different decisions depending on the particular design employed. Importantly, the frequentist method punishes the investigator for looking at data. Consider a trial designed to have an overall type I error 0.05. If the investigator looks at the data at any unplanned time and performs a test at each look, then it is possible that each of the planned 0.05 type I error has been spent before the trial is completed. Thus, if new data become available thereafter, the frequentist approach makes no allowance for using it along with the
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previous data! The same problem arises in a frequentist design for a trial in which the final planned test yields p-value 0.051, but additional data are subsequently obtained that strengthen the evidence in favor of a difference. The frequentist approach does not permit these subsequent data to be used, since they were not obtained as part of the planned experiment. In contrast, Bayesian inference utilizes all of the available data, with inferences based on posterior probabilities computed from observed data. In particular, Bayesian inference does not involve unobserved data and is not affected by the experimental design (9). That is, the data enter inferences only through the likelihood function. Consequently, posterior probabilities, unlike p-values, can be used as an explicit measure of support for a hypothesis. An M.D. Anderson trial comparing idarubicin þ ara-C (IA), troxacitabine þ ara-C (TA), and troxacitabine þ idarubicin (TI) provides an example of adaptive randomization (10). A maximum of 75 patients were to be randomized. As each patient after the 16th one entered the trial, we calculated the Bayesian posterior probability that the CR rate with either TA or TI was at least 10% worse than the CR rate with IA; if this probability was greater than 85%, the relevant arm closed, subject to the possibility of reopening depending on results in subsequent patients receiving IA. Using this design, the TI arm closed after each of the first five patients given TI did not achieve CR. The TA arm remained open with patients adaptively randomized between it and IA in proportion to the continuously updated CR rates with each regimen. Once the CR rate with TA was 3 of 11 patients, this arm closed since by then the CR rate with IA was 10 of 18 patients. An obvious question is the risk of a false negative using such a design. This risk is quantified before the trial begins by examining the proposed design’s operating characteristics (OC). A design’s OC consist of quantities that characterize, under various hypothetical clinical scenarios, its average behavior (5), e.g., sample size, probability of early termination for a given treatment if it is truly ineffective and probability of selecting a given treatment if it is truly superior, with ‘‘truly’’ here referring to the case where an infinite number of patients are treated. As suggested by Gooley et al. (11) and Thall and Estey (12), a practical way to choose design parameters in all but the simplest settings is to first simulate the trial on the computer under several design parameterizations and several reasonable clinical scenarios, and obtain the OC in each case. One may then use this as a basis for choosing the design parameters to ensure that it will have desirable OC, and in particular that the trial design is ethical. Table 1 illustrates the OC for the IA versus TA versus TI trial described earlier. In the scenario, where CR rates with TA and IA are 50% and 40%, respectively, the design correctly selects TA in 80% of the 10,000 computer simulations of the scenario. In these 80% IA and TI either drop out or have a lower CR rate than TA, which is thus ‘‘selected,’’ i.e., the design has a power of 80%. Hence, the failure to discover the ‘‘activity’’ of TA (or TI) may have reflected the inadequacies of these medicines rather than of the statistical design,
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Table 1 Operating Characteristics of IA vs. TA vs. TI Trial True probability CR IA
TA
TI
40% 40%
50% 30%
30% 30%
Probability arriving at correct conclusion
Correct conclusion TA better IA better
80% 10%
Abbreviations: IA, idarubicin þ ara-C; TA, troxacitabine þ ara-C; TI, troxacitabine þ idarubicin.
Table 2 Consequences of Adaptive Randomization Pts entered with adaptive randomization IA TA or TI
18 16
Pts entered without adaptive randomization 11 22
Abbreviations: IA, idarubicin þ ara-C; TA, troxacitabine þ ara-C; TI, troxacitabine þ idarubicin; pts, patients.
whose power was comparable to that of a standard frequentist design. In contrast, in the scenario where the IA has the highest CR rate, it is selected only 10% of the time. Thus, the design has a 90% false positive rate, declaring the new therapies TA or TI superior when they are not. If physicians wished a lower false positive rate, the design’s parameters would be changed; for example, accrual to IA would stop only if the probability became greater than 95% (rather than 85%) that the CR rate with IA was better (as opposed to > 10% better) than those with TA or TI. Note however that these changes would also reduce the probability of selecting TA when it is better. These considerations illustrate the importance of interaction between the physician and the statistician, an important feature of Bayesian designs with their emphasis of continual updating of data such as CR rates. Another potential disadvantage of adaptive randomized designs such as those used for the IA versus TA versus TI trial is their failure to account for a possible imbalance in prognostic covariates between the arms. This problem can be ameliorated by the use of ‘‘dynamic allocation’’ when patients are randomized (13). Even if not, accounting for such covariates is possible, but would obviously require entry of more patients before an arm might close; the number is however still less than that used in conventional phase 3 trials. Table 2 illustrates the consequences to patients of employing the adaptively randomized design used for the IA versus TA versus TI trial. The design resulted in the administration of IA to 18 patients and of TA or TI to 16. In contrast, use of a 1:1:1 randomization scheme throughout would have resulted in seven fewer patients receiving the seemingly superior IA regimen than TA or TI. Closing accrual to an arm after the entry of a relatively small number of patients
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could lead to failure to detect a small, perhaps biologically unique, subset of patients who might respond to a treatment, even though the average patient is highly unlikely to respond. It is unclear, however, whether any trial should proceed with such a goal in mind. NEED TO STUDY MORE TREATMENTS: SELECTION DESIGNS Proposals for ‘‘quicker’’ phase 3 trials and adaptive randomization enable study of many more drugs. Thus, for example, 200 patients might be randomized among four or five, rather than two, treatments. This is possibly of value given the large number of ‘‘targeted therapies’’ to be tested—a number made all the more larger by the recognized need to combine these with each other and with chemotherapy. While in principle only a few promising treatments might be selected on the basis of preclinical rationale, experience suggests this may not be possible. In addition to the examples (2 CdA, ATO, IFN) of therapies for which the demand of a ‘‘convincing’’ preclinical rationale might have prevented investigation of the therapy, there are numerous examples of treatments that, despite seemingly sound preclinical rationales, have been clinically ineffective. This has led to the proposal of randomized selection designs (5) in which the above 200 patients would be randomized among four or five treatments, with the treatment selected as best subsequently investigated further. Selection designs are intended to choose the best treatment, among those not dropped due to lack of efficacy regardless of the degree of difference between the best and the second best treatment. This is very different from a design in which the goal is to decide whether the best treatment provides a specified degree of improvement over the others. Hypothesis-test-based designs have the latter goal, and thus require a much larger sample size. The OC of selection designs indicate that in the case of a trial randomizing a maximum of 200 patients among four treatments, the probability of selecting a treatment that is 20% superior to the other three is only about 60%. Thus, such selection trials are often criticized as ‘‘underpowered phase 2 studies.’’ Note however that the nominally high false negative rate of 40% must be compared with what would obtain in the absence of the design. Assuming that is very difficult to select the best treatment in the absence of clinical data such as would emanate from a selection design, the effective false negative rate is 75% if one of four new treatments is randomly selected for comparison with standard therapy in a phase 3 study. Indeed, given the uncertainty inherent in selecting the best arm, it might be said that the worse false negative result obtains if a drug is not studied at all; the selection design is intended to prevent this possibility. ARTIFICIALITY OF THE PHASE 2–PHASE 3 DICHOTOMY The phase 2 selection design randomizes patients thus flying in the face of the concept of the phase 2 trial as single arm. However, in reality all phase 2 trials are inherently comparative. In particular, patients are vitally interested in knowing
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which of the several new treatments they might receive is superior—a question that implies comparison. Stopping rules for single-arm phase 2 trials are based on historical data with standard therapy further emphasizing the comparative nature of phase 2 trials (14). However, using data from single-arm phase 2 trials as a basis for comparison introduces bias in the form of trial-treatment confounding. The need to avoid this problem leads to the use of randomization in phase 3. However, it appears irrational to accept randomization in a phase 3 trial, but not in a phase 2 trial intended to determine whether a new therapy should be investigated in phase 3. Certainly the failure to randomize in phase 2 makes it impossible to use any phase 2 data in phase 3 comparisons. Even with randomization, however, decisions to move from phase 2 to phase 3 typically are based on early outcomes in phase 2 rather than survival or disease-free survival (DFS) time. This requires the implicit assumption that the early outcome is to some extent associated with improved survival or DFS. To address these issues ‘‘seamless phase 2–3 designs’’ have been proposed (15). Such a design randomizes between treatments, for example a standard S and an experimental E, throughout and makes repeated interim decisions during the trial based on both early response and survival time data. These decisions include (i) stopping the trial and concluding that E is associated with longer survival than S, (ii) stopping the trial because of futility, i.e., concluding that E and S are associated with similar survival, (iii) continuing the trial, or (iv) expanding the phase 2 trial to incorporate other centers, at which point the ‘‘phase 3’’ trial begins. Accrual continues while the phase 3 trial is being organized, and the ‘‘seamless’’ nature of the phase 2–3 transition allows for the use of all phase 2 data in all phase 3 decisions. This is illustrated by a trial in which patients with stage 3 or 4 unresectable non–small cell lung cancer receive docetaxel and radiation and are randomized to receive or not receive an intratumor injection of an adenovirus containing wild type p53 gene (Ad-p53). The hypothesis is that the transfected p53 will be proapoptotic and also increase the tumor’s sensitivity to docetaxel and radiation therapy. The data on each patient consist of (i) whether a response, defined as local control (LC) of the tumor evaluated by fine-needle aspiration biopsy, is observed at five months and (ii) the patient’s survival time, T. The overall survival distribution is formulated as a mixture of three components: (i) the survival distribution when LC is achieved the probability of achieving LC, (ii) the survival distribution when LC is not achieved the probability that LC is not achieved, and (iii) the survival distribution for patients who die in less than five months, before LC is evaluated. The Ad-p53 effect on T may vary as a function of the LC rates, the possibility that LC affects survival, and the possibility that there is a direct Ad-p53 effect on T not mediated by LC. In particular, the model does not assume that LC is a surrogate for T. The design specifies a maximum sample size of 900 patients and maximum duration of 72 months. At each of up to 18 interim times during the trial, decisions are made on the basis of predictive probability that survival in the Ad-p53 arm is greater, assuming either that (i) accrual stops immediately but treated patients are followed for an additional 12 months or (ii) all 900 patients are accrued and followed until 72 months.
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Table 3 Operating Characteristics of the Bayesian Phase II–III Design and the Conventional Designs Under the Six-Mixture Model-Based Hypotheses Hypothesized Effects Ad-p53 effect on LC rate
LC effect on survival
Ad-p53 direct effect on survival
No
No
No
Yes
No
No
No
Yes
No
Yes
Yes
No
Yes
Yes
Yes
No
No
Yes
Design Bayesian conventional Bayesian conventional Bayesian conventional Bayesian conventional Bayesian conventional Bayesian conventional
Patients (mean number)
Mean duration (mo)
Probability conclude E>S
425 842 453 842 452 854 640 884 525 861 576 873
20.4 28.1 21.6 28.1 21.7 28.5 30.7 29.5 23.2 28.7 29.2 29.1
0.03 0.05 0.04 0.05 0.03 0.05 0.85 0.83 0.97 >0.99 0.56 0.79
This approach may be compared with a conventional frequentist design that uses a log rank test to reject or accept the null hypothesis at up to four successive times with an O’Brien–Fleming test boundary while maintaining overall significance level 0.05 and power 0.80 to detect a 25% increase in median survival time from the null median of 15.5 months. A comparison of the two designs, assuming the same maximum sample size (900) and maximum duration (72 months), is summarized in Table 3. Six different hypotheses are assumed, the first three being different types of ‘‘null’’ cases in which Ad-p53 does not improve survival, whereas the last three are alternatives in which survival is longer in the Ad-p53 arm. The Bayesian design parameters were calibrated to maintain type I error less than 0.05 and power more than 0.80. In all six cases, the Bayesian design has on average a substantially smaller sample size, and a trial duration that is either shorter than or the same as that of the conventional phase 3 design. Although our example involves a lung cancer trial, this sort of seamless phase 2–3 Bayesian design may easily be applied to trials of hematologic malignancies where CR takes the part of LC. MULTIPLE OUTCOMES At the beginning of this chapter, I noted that commonly used statistical designs pay insufficient regard to the complexities of medical practice. The failure to explicitly recognize the inherently comparative nature of phase 2 trials or the
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assumption of a relation between response and survival are examples, as is the failure to provide for monitoring multiple outcomes in phase 2 (16). Specifically, phase 2 trials focus on response and provide explicit stopping rules based on this outcome. In contrast monitoring of toxicity is done informally, i.e., without explicit stopping rules. The assumption is that toxicity rates are already known from the phase 1 study. However, it has been pointed out that the number of patients entered into typical phase 1 trials is often inadequate to estimate the toxicity rate at any given dose (17). Furthermore, the patients treated on phase 2 trials may be quite different with regard to the likelihood of toxicity than those entered on the corresponding phase 1 trial. The following example illustrates the desirability of monitoring multiple outcomes. We conducted a trial in which untreated AML patients under age 50 received ‘‘double induction’’ consisting of a course of idarubicin and ‘‘high-dose’’ ara-C, followed by a second course of this combination 14 days after beginning the first course, regardless of the status of the marrow at that time. While the objective of intensifying therapy was to improve the 90-day CR rate, this approach also carried the risk of increasing the 90-day mortality rate. Furthermore, the second course was to be given only to patients judged ‘‘eligible,’’ i.e., recovered from first course toxicity, by their attending physicians. Accordingly, it was possible that the number of patients eligible for the second course would be sufficiently small that the results would be of little practical significance. Our design formally monitored three outcomes: the course 2 eligibility, and among eligible patients, the CR rate and the death rate within 90 days after the start of course 1. The 90-day window was selected after considering the anticipated accrual rate of three to four patients per month, both because the risks of treatment failure or death within this timeframe are high and for logistical reasons. To account for prognostic heterogeneity, the early stopping rules were applied separately in two subgroups: patients with abnormalities of chromosomes 5 and/or 7 (–5/–7), and patients with all other karyotypes. For illustration, we will focus attention on the latter subgroup. It was decided that the requisite course 2 eligibility rate was greater than or equal to 67%. On the basis of historical rates, a 4% increase in a 90-day mortality rate (‘‘death’’), from 14% to 18%, was considered an acceptable trade-off for a 15% increase in the 90-day CR rate (‘‘success’’), from 67% to 82%. Death and success were not complementary events because an eligible patient could be alive but not in CR at day 90. The trial was to stop if, after evaluating each cohort of five patients, the probability was too high that (i) the eligibility rate was less than 67% or (ii) the death rate among eligible patients was increased by greater than 4%, or (iii) the probability of a 15% increase in the success rate among eligible patients was too low. Criteria probabilities of 95%, 90%, and 5% for these events quantified the terms ‘‘too high’’ and ‘‘too low.’’ These criteria led to three sets of stopping boundaries, one for each rate being monitored. If early termination did not occur, 50 patients would be entered, which would provide a 90% posterior credibility interval for the 90-day success rate having limits within 0.12 of its mean, assuming a mean success rate of 0.82 and mean eligibility rate of 0.68.
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Table 4 Operating Characteristics of the Double Induction Trial
Clinical scenario Same as historical rates 82% success rate and 18% death rate 67% success rate and 33% death rate 50% ineligibility rate
Probability of early termination
Achieved sample size percentiles 25th
50th
75th
0.69 0.21
15 50
25 50
50 50
0.96
10
15
20
0.89
10
15
30
This design’s OC are summarized in Table 4. For example, under the scenario where the true death rate is the unacceptably high value 33%, the probability of early termination (PET) is 96% and 50% of the simulated trials stopped after 15 patients were entered. For higher true death rates, the median sample size becomes smaller. In contrast, if the minimum study goals were met, the trial ran to completion in 79% of the simulated trials, with this percent increasing for higher eligibility rate, higher success rate, or lower death rate. In fact, the trial closed after 14 of the first 25 patients were ineligible although the observed numbers of deaths (2/7) and success (5/7) within 90 days among eligible patients were both acceptable. Such multiple outcome designs allow the investigator to explicitly specify a trade-off between ‘‘efficacy’’ and ‘‘toxicity,’’ which correspond to 90-day success and 90-day death in the double induction trial. Different investigators might have different trade-offs. For example, in the double induction trial, one might consider a 4% increase in death rate acceptable only given a 25% increase in success rate. In the scenario where the true death rate is 33% (row 3, Table 4), on average, 5 of the median sample size of 15 would die compared to 2 of 15 in the historical situation (14% death rate). If the investigator believes this is unacceptable, the criterion probability could be lowered from 90% to 85%. However, making it easier to stop the trial would also increase the PET if the true success rate is the desired 82%. Our experience suggests that the need to specify such trade-offs encourages dialogue between clinical investigators and statisticians. Moreover, multiple outcome designs encourage the use of a wider range of data in therapeutic decision-making. CONCLUSION Statistical methods for conducting clinical trials have remained essentially static for the past 30 years. This phenomenon is difficult to explain in light of the issues raised above and the profusion of papers in the statistical literature describing new designs; I have referenced just a few of these here. It is difficult to reconcile the eagerness with which physician or scientists have adopted new
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molecular biologic techniques with their reluctance to adopt new statistical techniques. Regardless of the reason for the contrast, I believe the consequences of this reluctance are unfortunate from both academic and clinical perspectives. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Hopefully, the next five years will see the adoption of many new methods for conducting clinical trials. Phase 1 and phase 2 trials will be combined and will monitor both efficacy and toxicity, consistent with patients’ primary reason for entering phase 1 trials: to achieve a response, not ‘‘no toxicity.’’ Factors other than dosage will be considered in assessing toxicity. For example, for purposes of determining the dose for phase 2 trials, it is unrealistic to equate a 70-year-old who has severe toxicity at a given dose with a 50-year-old who has the same toxicity at the same dose. Yet this is what is done currently; ‘‘pharmacogenomics’’ will also be considered as a determinant of toxicity. The 3þ3 design will be replaced with Bayesian methods, such as the clinical reassessment method (CRM), which allow all information to be used, rather than only the information obtained at the most recent dose. Most importantly, given the large number of therapies to be investigated, there will be a shift away from large randomized trials to smaller randomized trials. The invariable need for the ‘‘magic’’ statistical significance level of 0.05 and equally magic power of 80% to 90% will be rethought. The question of whether the optimal biological dose should be investigated rather than the more traditional MTD will be explored, perhaps in separate arms of a randomized trial. Using a single protocol, therapies that do poorly will be replaced by other therapies, while if results are equivocal, more patients will be recruited into the study. In general, there will then be a blurring of the artificial distinctions between phases 1, 2, and 3. The above will only occur given a willingness to forego habit and tradition. However, the recent adoption by the MRC of the small, randomized trial, i.e., the selection design described earlier in this chapter, is encouraging and will hopefully provide a model for others. REFERENCES 1. van der Holt B, Lowenberg B, Burnett A, et al. The value of the MDR1 reversal agent PSC-833 in addition to daunorubicin and cytarabine in the treatment of elderly patients with previously untreated acute myeloid leukemia in relation to MDR1 status at diagnosis. Blood 2005; 106:2646–2654. 2. Baer M, George S, Dodge R, et al. Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood 2002; 100:1224–1232.
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3. Rowe J, Neuberg D, Friedenberg W, et al. A phase 3 study of three induction regimens and of priming with GM-CSF in older adults with acute myeloid leukemia: a trial by the Eastern Cooperative Oncology Group. Blood 2004; 103:479–485. 4. Estey E. Clinical trials in AML of the elderly: should we change our methodology? Leukemia 2004; 18:1772–1774. 5. Estey E, Thall P. New designs for phase 2 clinical trials. Blood 2003; 102:442–448. 6. Berry D. Statistics: A Bayesian Perspective. Belmont, CA: Wadsworth Publishing Company, 1996:124–161. 7. Spiegelhalter D, Abrams K, Myles J. Bayesian Approaches to Clinical Trials and Health-Care Evaluation. Chichester, UK: John Wiley & Sons, 2004. 8. Goodman, SN. Toward evidence-based medical statistics. 2: the Bayes factor. Ann Intern Med 1999; 130:1005–1013. 9. Berger J, Berry D, Statistical analysis and the illusion of objectivity. American Scientist 1988; 76:159–165. 10. Giles F, Kantarjian H, Cortes J, et al. Adaptive randomized study of idarubicin and cytarabine versus troxacitabine and cytarabine versus troxacitabine. J Clin Oncol 2003; 21:1722–1727. 11. Gooley T, Martin P, Fisher L, et al. Simulation as a design tool for phase I/II clinical trials: an example from bone marrow transplantation. Control Clin Trials 1994; 15: 450–462. 12. Thall PF, Estey E. A Bayesian strategy for screening cancer treatments prior to phase II clinical evaluation. Stat Med 1993; 12:1197–1211. 13. Pocock SJ, Simon R. Sequential treatment assignment with balancing for prognostic factors in the controlled clinical trial. Biometrics 1975; 31:102–115. 14. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989; 10:1–10. 15. Inoue LYT, Thall PF, Berry DA. Seamlessly expanding a randomized phase II trial to phase III. Biometrics 2002; 58:823–831. 16. Thall PF, Simon RM, Estey EH. New statistical strategy for monitoring safety and efficacy in single-arm clinical trials. J Clin Oncol 1996; 14:296–303. 17. Edler, L. Overview of phase I clinical trials. In: Crowley J, ed. Handbook of Statistics in Clinical Oncology. New York, NY: Marcel Dekker, 2002:1–34.
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5 Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia Ch. Michel Zwaan and Marry M. van den Heuvel-Eibrink Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands
GENERAL INTRODUCTION Monoconal Antibodies Thirty years have passed since Ko¨hler and Milstein first described the possibility of hybridizing murine cell lines, resulting in the expression and production of specific murine antibodies (1,2). In 1984, they were awarded with the Nobel Prize for their discovery. Currently, more than 20 monoclonal antibody preparations are available for clinical use for various diseases, including cancer and autoimmune diseases, of which several are approved by the regulatory authorities, as summarized in Table 1 (this list is neither meant to be exhaustive nor complete) (2). Monoclonal antibody therapy is a type of immunotherapy and is referred to as passive immunotherapy as the antibodies are not produced by the body’s own immune system. Other examples are the use of cytokines such as interferones, interleukines (ILs), or growth factors. This in contrast to cancer vaccines, which are dependent on the host immune system to conquer the disease, are, therefore, referred to as ‘‘active immunotherapy.’’ Another example of active immunotherapy in hematological malignancies is stem cell transplantation (SCT), aiming
99
ErbB2 CD33 CD52 CD20 CD20
Herceptin Mylotarg
Campath, MabCampath Zevalin
Bexxar
Avastin Erbitux
Trastuzumab Gemtuzumab ozogamicin Alemtuzumab Ibitumomab tiuxetan Tositumomab
Bevacizumab Cetuximab
Iodine
Colorectal cancer Colorectal cancer
Non-Hodgkin’s lymphoma
Chronic lymphocytic leukemia Non-Hodgkin’s lymphoma
Non-Hodgkin’s lymphoma Renal allograft rejection and T-cell leukemia Breast cancer Acute myeloid leukemia
Disease
FDA (2003), not approved by EMEA FDA (2004), EMEA (2005) FDA (2004), EMEA (2004)
FDA (1998), EMEA (2000) FDA (2000), not approved by EMEA FDA (2001), EMEA (2001) FDA (2002), EMEA (2004)
FDA (1997), EMEA (1998) FDA (1997), EMEA (1999)
Market authorization status, (yr of approval)
Abbreviations: VEGF, vascular endothelial growth factor; EGFR, Epidermal growth factor receptor; FDA, Food and Drug Administration; EMEA, European Agency for the Evaluation of Medicinal Products.
no no
131
no 90 Yttrium
no To calicheamicin
no no
Conjugated
100
VEGF EGFR
CD20 CD25
Rituxan, Mabthera Zenapax
Rituximab Daclizumab
Target(s)
Brand name
Antibody generic name
Table 1 Monoclonal Antibodies That Have Received Market Authorization from the Regulatory Authorities for Treatment of Cancer, i.e., Either the Food and Drug Administration in the United States and/or the European Agency for the Evaluation of Medicinal Products. Listed by Year of Approval
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at inducing a graft-versus-leukemia effect, in addition to elimination of the malignant stem cell by high-dose chemotherapy and/or irradiation. Monoclonal antibodies have high specificity and are directed against a single antigen. Ideally, they are directed against antigens that are present selectively on tumor cells, which may result in limited side effects and organ toxicity. Hence, broad tissue typing is required for the antigen of interest to study its expression on normal human tissues. In addition, the expression should preferably be strong and homogeneous throughout the tumor. Antibodies targeting antigens that are also present in soluble form in the circulation may be less effective because of rapid antibody clearance after infusion. The use of murine antibodies in the clinical setting has not been very successful, as they will be recognized by the human immune system as foreign proteins, and generate an human anti-mouse antibody (HAMA) immune reaction (3). This may result in inadequate exposure to the antibody due to diminished stability in the circulation, as well as a severe allergic reaction in the recipient, which interferes with repeated dosing. Murine antibodies also have less capacity to recruit effector cells and complement to destruct cancer cells. They are referred to as ‘‘momabs,’’ such as in ibritumomab, which is directed against the CD20 antigen and used in non-Hodgkin’s lymphoma. The ability to construct so-called ‘‘chimeric’’ or ‘‘humanized’’ antibodies by genetic engineering has markedly improved the possibilities to use monoclonal antibodies in the clinic (4). In chimeric antibodies, the variable region is still from mouse origin, whereas in humanized antibodies this is only the hypervariable region. Chimeric antibodies are approximately 60% to 95% human, whereas humanized antibodies are over 95% human (3). In both instances the murine part is responsible for specificity and antigen recognition. They are referred to as ‘‘ximabs’’ and ‘‘zumabs,’’ respectively, as in rituximab (anti-CD20) and gemtuzumab (anti-CD33) or epratuzumab (anti-CD22). Moreover, fully human antibodies are now available by using transgenic mice that have been engineered to synthesize human antibodies. These antibodies usually allow repeated dosing and do not result in severe allergic reactions. Two different types of monoclonal antibodies can be distinguished, i.e., naked versus conjugated monoclonal antibodies: 1. Naked antibodies bind directly to antigens expressed on tumor cells, and may either stimulate the immune system to destroy the cancer cell by antibody-dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), or by induction of apoptosis. Examples are rituximab, an anti-CD20 antibody used in the treatment of non-Hodgkin’s lymphoma (5), and the anti-CD52 directed antibody alemtuzumab, which may be used as part of the conditioning regimen in SCTs or to eliminate T-cells from grafts (6). Rituximab was actually the first monoclonal antibody registered (in 1997) by the Food and Drug Administration (FDA) for use against cancer. Alternatively, monoclonal antibodies may exert their effect as competitive antagonists, prohibiting a ligand to bind to a certain receptor,
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and thereby blocking the activity of the ligand, subsequently shutting down the intracellular signaling cascades that would normally result from ligand binding. The antiangiogenesis monoclonal antibody bevacizumab, which interferes with vascular-endothelial growth factor receptor signaling, is an example of the latter (7). 2. Conjugated monoclonal antibodies are antibodies linked to drugs, toxins or radioactive compounds, and basically represent targeted delivery of the conjugate to the tumor cell rather than inducing cell death by the aforementioned mechanisms. An important issue to consider in developing such conjugates is whether the conjugate will be internalized after binding to the tumor cell. Examples are gemtuzumab ozogamicin (GO) (anti-CD33 linked to calicheamicin) for the treatment of acute myeloid leukemia (AML) (8) and radiolabeled antibodies directed against CD45 used in the conditioning regimen of advanced leukemia patients (9). These data show that the development of a monoclonal antibody represents rigorous scientific effort and financial resources. Both the target and the antibody need to be selected very carefully to ensure that the in vivo properties will allow therapeutic efficacy. Hematological malignancies are ideally suited to treatment with monoclonal antibodies because of the accessibility of malignant cells in the blood, bone marrow, spleen, skin, and lymph nodes and the availability of targets that are restricted to the hematopoietic system. Rapid and repeated evaluation of target antigen expression is feasible using flowcytometry. In this chapter, we will review the current status of targeted monoclonal antibody therapy in use or development for the treatment of children and adults with AML, with the exception of radiolabeled antibodies. Acute Myeloid Leukemia AML is a heterogeneous group of diseases and basically comprises all other than lymphocyte-precursor derived acute leukemias. Traditionally, classification is based on morphology according to the French-American-British (FAB) classification. Recently, a new classification for myeloid neoplasms has been introduced by the WHO, which differentiates between AML with recurrent cytogenetic abnormalities, AML with multilineage dysplasia, therapy-related AML, and AML not otherwise specified (10). For stratification of patients in clinical trials, cytogenetic abnormalities and early response to treatment are often used, representing the underlying biology of the disease (11,12). Patients with t(8;21), t(15;17), or inv(16)/t(16;16) are considered good risk by most collaborative study groups, whereas abn(3q), monosomy 5 or 7 and deletion 5(q) or 7(q) and complex abnormalities are considered poor risk by most groups (12). Currently, other molecular abnormalities, such as mutations in C/EBPa, or receptor tyrosine kinases such as FLT3, KIT, and others may also be taken into account (13). It is well known that the prognosis decreases with increasing age, which is
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partially due to an increasing frequency of unfavorable biology with increasing age, but also represents unfavorable host characteristics (11). On the basis of this, many study groups have separate protocols for children, younger adults, and elderly people with AML. Currently, induction regimens usually consist of intensive chemotherapy comprised of cytarabine in combination with an anthracycline, with or without a third drug such as etoposide or 6-thioguanine. Subsequently, the consolidation phase consists of intensive courses of chemotherapy in children and younger adults, with or without SCT, aiming at the eradication of minimal residual disease. Children and adults with acute promyelocytic leukemia [(APL) or AML FAB M3] receive adapted treatment protocols incorporating all-trans retinoic acid (ATRA), which results in much better treatment outcome than in non-M3 AML. Overall, 80% to 90% of children with AML reach complete remission (CR) versus approximately 75% of younger adults and 50% of older adults (11,13,14). This results in survival rates of approximately 50% to 70% for children versus approximately 40% to 50% for younger adults and only 10% for elderly AML patients (11,13,14). Relapse and nonresponse mainly contribute to these high failure rates. Hence, outcome for most patients with AML is still unsatisfactory in terms of antileukemic efficacy. Moreover, especially in children, there is concern over the acute and long-term side effects associated with the intensive chemotherapy (15). Therefore, there is a continuous interest in the development of new antileukemic agents, preferably those that exert their action without causing too many side effects. CD33-DIRECTED MONOCLONAL ANTIBODIES IN ADULT AML CD33 is expressed on the cell surface of malignant blast cells in 80% to 90% of AML cases, but not on normal hematopoietic stem cells or nonhematopoietic tissues. Antibody-based therapies for AML have, therefore, focused on CD33 as a suitable target antigen. CD33 is also a useful target for conjugated antibodies, as binding to CD33 results in internalization of the complex. The natural ligand of CD33 and its function are currently not known (16). Lintuzumab (HuM195) At first, studies have been performed with lintuzumab (HuM195), which is a naked antibody. This did not result in significant antileukemic activity in AML, with the exception of molecular disease control in AML FAB M3 (APL), in patients who were reverse transcriptase polymerase chain reaction (RT-PCR) positive for the APL characteristic t(15;17) fusion transcript after induction chemotherapy plus ATRA (17,18). These APL patients were treated with lintuzumab twice weekly for three weeks. Half of the patients evaluable for conversion of the RT-PCR of the fusion transcript became molecularly negative. This is not surprising, as APL is characterized by relatively strong CD33
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expression. A recent randomized phase III study in relapsed/refractory AML patients did not show a survival advantage in patients stratified to chemotherapy plus lintuzumab (4 days of lintuzumab 12 mg/m2 following induction chemotherapy were given, starting at day 6 after completion of induction chemotherapy and repeated at day 16–18) versus chemotherapy alone (pOS 36 vs. 28%, p ¼ 0.28) (19). The addition of lintuzumab did not result in increased toxicity when compared with the ‘‘chemotherapy-only’’ arm. Gemtuzumab Ozogamicin The most promising results have been obtained with GO, Mylotarg1, a humanized anti-CD33 monoclonal antibody linked to calicheamicin, which is a potent enidyne antileukemic antibiotic. Calicheamicin dissociates from the antibodycalicheamicin complex after internalization, binds to the minor groove of DNA, and results in DNA-double strand breaks (Fig. 1). The results of the various phase I/II studies in adults are summarized in Table 2, and some are discussed in more detail below.
Figure 1 Mechanism of action of gemtuzumab ozogamicin. (A) Gemtuzumab ozogamcin consists of an anti-CD33 antibody linked to the antitumor antibiotic calicheamicin. (B) After binding to CD33, the complex is internalized, after which the calicheamicin is released by hydrolysis in lysosomes. (C) Free calicheamicin then translocates to the nucleus and cleaves double-stranded DNA, resulting in apoptosis. (text continues on page 112)
Relapsed/ refractory AML Newly diagnosed AML or MDS Newly diagnosed AML
Relapsed/ refractory AML Phase I study Relapsed/ refractory AMLa
Phase of the disease
Induction
Induction
Single agentb
Single agent
Induction
Single agent
Induction
Induction
Single agent
Single agent
Phase of treatment
GO monotherapy or in combination
Elderly AML, median age 76 yr, n ¼ 40
Elderly AML, median age 71 yr, n ¼ 51
Median age 64 yr, n ¼ 57
median age 61 yr, n ¼ 277
median age 54 yr, n ¼ 40
Population and number of patients Response rate 20%
Overall 26% (CR 13% and CRp 13%)
Overall 33% (CR 26% and CRp 7%) Overall 22%, i.e., GO only 8%c and GO þ IL-11 36% Overall 17% (CR 10% and CRp 7%) Note: in patients aged 61–75 yr 33%, in older patients 5%.
Dosages Dose escalation 0.25–9 mg/m2, up to 3 infusions
GO 2 9 mg/m2, on day 1 þ 15 GO 3 mg/m2, day 1, 4, and 7 GO 2 9 mg/m2, on day 1 þ 8 or day 1 þ 15 GO 2 9 mg/m2, on day 1 þ 15 Followed by 2 more courses in case of responsed
29
28
32
22
8
Ref.
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia (Continued)
All patients 4.3 mo Age 61–75: 11.4 mo Age >75: 1 mo
GO: 8 wk GO þ IL-11: 15 wk
CR patients: 6.4 mo CRp patients: 4.5 mo Median overall survival 8.4 mo
NA
Median duration of response
Table 2 Phase I/II Studies with Gemtuzumab Ozogamicin as Single Agent or in Combination with Regular Chemotherapy in Adults with Acute Myeloid Leukemia
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105
GO combined with idarubicin and cytarabine
median age 61 yr, n ¼ 14
Induction
Induction
Sequential combination therapy: GO monotherapy for induction followed by chemotherapy GO combined with cytarabine
Elderly AML, median age 75 yr, n ¼ 12 Elderly AML, median age 68 yr, n ¼ 64
median age 63 yr, n ¼ 9
Induction
Single agent
Population and number of patients
Induction
Phase of treatment
After GO monotherapy: 35.1% (CR 22.8, CRp 12.3%) After GO followed by 1 course of chemotherapy: overall 54.4% (CR 35.1%, CRp 19.3%) CR rate 55%
GO 2 9 mg/m2, day 1 þ 15
GO 1 6 mg/m2, day 1 þ 15 Idarubicin 12 mg/m2, day 2–4 Ara-C 1.5 g/m2, day 2–5
Overall 42% (CR 21%, CRp 21%)
Overall 27%
GO 2 9 mg/m2, on day 1 þ 15
GO 1 6 mg/m2, day 1 and 4 mg/m2, day 8 Ara-C 100 mg/m2, day 1–7, cont IV
Response rate
Dosages
Median survival time 8 wk, in CR patients 27 wk
NA
Available in 2 patients, 11.2 and 4.0 mo 1 yr pOS 34% (SE 7.2%)
Median duration of response
64
63
33
30
Ref.
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Newly diagnosed and relapsed/ refractory AML Relapsed/ refractory AML
Newly diagnosed AML Newly diagnosed AML
Phase of the disease
GO monotherapy or in combination
Myeloid Leukemia (Continued )
Table 2 Phase I/II Studies with Gemtuzumab Ozogamicin as Single Agent or in Combination with Regular Chemotherapy in Adults with Acute
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GO combined with cytarabine and topotecan
Relapsed/ refractory AML or MDS
Relapsed/ refractory AML
GO combined with liposomal daunorubicin, cytarabine, and cyclosporine A (MDAC) GO combined with fludarabine, cytarabine, and cyclosporine A (MFAC)
Relapsed/ refractory AML
Phase of the disease
GO monotherapy or in combination median age 37 yr, n ¼ 11
median age 53 yr, n ¼ 31
median age 55 yr, n ¼ 17
Induction
Induction
Population and number of patients
Induction
Phase of treatment
Overall 34% (CR 28%, CRp 6%)
GO 1 4.5 mg/m2, day 1 Fludarabine 15 mg/m2, 2/day, day 2–4 Ara-C, 0.5 g/m2, 2/day, day 2–4 CsA 16 mg/kg cont IV GO 1 9 mg/m2, day 1 Ara-C 1 g/m2, day 1–5 Topotecan 1.25 mg/m2, day 1–5 Overall 12% Significant toxicity (transaminites)
Overall 18% (1 CR and 1 CRP)
GO 1 6 mg/m , day 6 Ara-C 1 g/m2, day 1–5 DNX 75 mg/m2, day 6–8 CsA 16mg/kg, day 6–8, cont IV 2
Response rate
Dosages
66
36
65
Ref.
(Continued)
Overall survival 8 wk (all patients)
Median survival time 5.3 mo
Both responding patients relapsed again
Median duration of response
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median age 65 yr, n ¼ 6
median age 57 yr, n ¼ 59
Induction
Induction
GO combined with troxatyl
GO combined with fludarabine, cytarabine, and cyclosporine A (MFAC)
Relapsed/ refractory AML
Newly diagnosed AML or MDS
median age 54 yr, n ¼ 17
Population and number of patients
GO combined with cytarabine and mitoxantrone
Induction
Phase of treatment
Relapsed/ refractory AML
Phase of the disease
GO monotherapy or in combination
Myeloid Leukemia (Continued )
2 patients with VOD, 3 CRp
Median survival time 8 mo
Not given
Overall 76% (CR 70%, CRp 6%)
GO 1 9 mg/m2, day 4 Ara-C 1 g/m2, 2 day, day 1–5 Mitoxantrone 12 mg/m2, day 1–3 GO 2 9 mg/m2, day 1 þ 15, later only day 1 Troxatyl 4 mg/m2 GO 1 6 mg/m2, day 1 (Other drugs see above)
34
67
38
Ref.
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Overall 48% (CR 46% and CRp 2%)
Median survival 11 mo
Response rate
Dosages
Median duration of response
Table 2 Phase I/II Studies with Gemtuzumab Ozogamicin as Single Agent or in Combination with Regular Chemotherapy in Adults with Acute
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Newly diagnosed AML (randomized)
Newly diagnosed AML (pilot study)
Phase of the disease
Several induction and consolidation regimens in combination with GO, followed by regular MRC AML15 chemotherapy Several induction and consolidation regimens in combination with GO, followed by regular MRC AML15 chemotherapy
GO monotherapy or in combination
Induction 43% DA, 43% FLAG-Ida, 14% ADE, GO 3 mg/m2
Induction n ¼ 1115, and/or conmedian age solidation 49 yr
Dosages Induction: DAT, DA or FLAG-Ida with GO 3 mg/m2 Consolidation: MACE or HiDAC with GO 3 mg/m2
Population and number of patients
n ¼ 64 in Induction induction and and/or con31 in solidation consolidation, median age 46.5 yr
Phase of treatment
GO vs. no-GO 85% and 85%
Overall CR 86% of patients
Response rate
40
39
Ref.
(Continued)
Relapse rate: GO 37% vs. noGO 52% at 3 yr, p ¼ 0.01. DFS: GO 51% vs. no-GO 40% at 3 yr, p ¼ 0.008. OS: 53% vs. 46% at 3 yr, p ¼ 0.4
78% in CCR at 8 mo
Median duration of response
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Reinduction median age for 52 yr, n ¼ 16 molecular relapse Consolidation Median age 51 yr, n ¼ 8
Single agent
GO combined with ATRA and arsenic trioxide
Postremission median age therapy 57 yr, n ¼ 22
Population and number of patients
GO combined with fludarabine, cytarabine, and cyclosporine A (MFAC)
Phase of treatment
14/16 patients molecular CR
GO 2 6 mg/m2, day 1 and 15
NA
NA
GO 1 4.5mg/m2, day 1 (Other drugs see above)
Consolidation: – arsenic 0.15 mg/kg 5 days, total 5 courses – ATRA 45 mg/m2, 10 days/mo, for 10 courses – GO 9 mg/m2, once a mo, for 10 courses
Response rate
Dosages
NA
7 patients (50%) remained in molcular CR
Median survival from CR was 16 mo
Median duration of response
43
68
37
Ref.
110
Relapsed APL
Newly diagnosed AML or MDS, in remission after GO containing regimen Molecularly relapsed APL
Phase of the disease
GO monotherapy or in combination
Table 2 Phase I/II Studies with Gemtuzumab Ozogamicin as Single Agent or in Combination with Regular Chemotherapy in Adults with Acute Myeloid Leukemia (Continued )
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GO combined with ATRA
Population and number of patients
Induction and median age postremission 50 yr, n ¼ 19 therapy
Phase of treatment Response rate Overall CR rate 84%
Dosages Induction: GO 1 9 mg/m2, day 5 ATRA 45 mg/m2 until CR Postremission: GO 9 mg/m2, every 4–5 wk, maximum 8 ATRA 45 mg/m2, 2 wk on/off schedule NA
Median duration of response 42
Ref.
b
Combined results of the three phase II studies that followed the initial phase I study. Patients were randomized between GO and GO plus interleukin-11 (IL-11). c There was no difference between GO day 1 and 8 or day 1 and 15. d Only 57% of patients received more than one infusion of GO. In 43% only one infusion was given because of toxicity or progressive disease. Only one patient received the four courses as planned. Abbreviations: CR, complete remission; CRp, complete remission with insufficient platelet regeneration, but platelet transfusion independence; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; GO, gemtuzumab ozogamicin; m2, body surface area in square meter; pOS, probability of overall survival; NA, not available or applicable; cont IV, continuous intravenous; ATRA, all-trans retinoic acid; APL, acute promyelocytic leukemia.
a
Newly diagnosed APL
Phase of the disease
GO monotherapy or in combination
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The results of the adult phase I study in relapsed/refractory AML were published in 1999 and showed that a postinfusion syndrome of rigor and chills was the most frequent side effect (8). Two patients who were treated at the 9 mg/m2 dose level had prolonged neutropenia and thrombocytopenia. Of the 40 patients who were treated, 20% showed a response (i.e., absence of leukemia from bone marrow and peripheral blood). No antibodies against the antibody were detected, but two patients developed antibodies against calicheamicin. The recommended dose for phase II studies was two times 9 mg/m2 with a 14-day interval. At this dose level, greater than 75% saturation of CD33 sites on peripheral blood mononuclear cells was found. Three phase II studies in adults with relapsed/refractory AML were started. It has to be noted that inclusion in these three studies was restricted to a selected population of adults with relapsed CD33-positive AML who had a first CR duration of at least three months, and with a minimum of 80% of blasts staining positive for CD33 at least four times above the background level. On the basis of the preliminary data from 142 patients enrolled in these studies, GO was given accelerated approval by the FDA for patients with CD33-positive AML in first relapse who are 60 years or older and who are not considered candidates for cytotoxic chemotherapy (20,21). Administering two dosages of 9 mg/m2 with a 14-day interval, toxicities were similar as in the phase I study, but in addition hepatotoxicity was noted, including elevated transaminases and hyperbilirubinaemia in 17% and 23%, respectively, as well as one death due to liver failure. Of note, mucositis and severe neutropenic infections were infrequent. CR was obtained in 16% of patients. However, another subset of patients also showed remission but with insufficient platelet recovery, and they were categorized as CR with insufficient platelet regeneration, but platelet transfusion independence (CRp). The duration of response in these two subgroups was similar, and hence the total response rate was considered to be 30% (21). Recently, the final results of these phase II studies were published, now including a total of 277 adults (22). The overall response rate was 26% and included 13% of patients classified as CR and 13% as CRp, but now a difference in median leukemiafree survival time between these two patient categories of 6.4 versus 4.5 months was noted. This suggest that the quality of remission in the CRp patients was less than in patients with sufficient platelet recovery. Toxicities included grade 3 or 4 sepsis in 17% and grade 3 or 4 hyperbilirubinemia in 29%. Approximately 1% of patients who did not undergo SCT (either before or after treatment with GO) also developed hepatic veno-occlusive disease (VOD) following GO treatment. Meanwhile more data became available on the hepatotoxicity of GO, again showing that some patients developed clinical signs of VOD, which is thought to be due to CD33 expression in hepatic sinusoids and perhaps better described as ‘‘sinusoidal obstruction syndrome’’ (SOS) (23). Other factors involved may be the liver leukemia load or circulating soluble CD33 levels. A high incidence of VOD (in this particular single-center study as high as 64% of 14 patients) was noted among patients who were transplanted following reinduction with GO, mainly in patients who were transplanted shortly after GO treatment (24). This
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resulted in the recommendation to delay transplantation for at least 3.5 months following treatment with GO. However, in another series VOD also occurred in approximately 4% of patients without prior SCT history, as was also noted in the phase II studies described above (25). Several case reports suggest that defibrotide may be useful in preventing or treating GO-induced VOD, but no larger prospective or comparative studies have been performed (26,27). Three studies have now been performed using single-agent GO as induction therapy in older patients with newly diagnosed AML. The first study of Estey et al. included 51 adults older than 65 years with either AML or myelodysplastic syndrome (MDS) (28). Patents were randomized to receive GO or GO plus interleukin11 (IL-11). CR rates were 8% and 36%, respectively. In this nonrandomized study, the treatment results were compared with a historical cohort of patients receiving cytarabine and idarubicin, and GO IL-11 was considered inferior to the standard cytarabine/idarubicin schedule. The authors, therefore, argue against using GO as standard regimen in elderly patients with AML. Another phase II study in 40 newly diagnosed AML patients older than 60 years, who were considered not fit for intensive chemotherapy, showed an overall response percentage (CR and CRp) of 17, although responses were mainly restricted to patients 61 to 75 years old (29). Two doses of GO 9 mg/m2 were given, and it was planned to give a third and fourth dose of GO to responding patients. However, the second dose was only given to 57% of patients, either because of toxicity (n ¼ 9) or progressive disease (n ¼ 8). Toxicity occurred mainly in patients older than 75 years of age, with 23% of patients suffering from induction deaths, which led to the suggestion that a reduced dose should be applied for this age group. Only one patient tolerated a third and fourth dose of GO. The median survival in responding patients was 11.4 months. The third study concerned 12 patients over 65 years of age (30). Three out of 12 patients were in CR after two doses of GO at 9 mg/m2. Five patients had nonarrhythmia cardiac adverse events, although it was not clear if they were attributable to treatment with GO. Amadori et al. also mentioned 10% arrhythmias, 7% left ventricular dysfunction, and 5% hypotension as grades 3 to 4 adverse events (29). Most single-agent studies with GO have used the ‘‘classical’’ dosing schedule of 9 mg/m2 on day 1 and 14. However, based on in vitro data showing rapid reexpression of CD33 after internalization of the CD33/GO complex, a more fractionated dosing schedule may be more efficacious (31). These in vitro data were recently ‘‘translated’’ to the clinic in a phase II study in which GO was administered at 3 mg/m2 on day 1, 4, and 7 in 57 patients with relapsed/refractory AML (32). This schedule was very well tolerated, without hepatoxicity. The response rate was 26% CR and 7% CRp. Several studies have been reported on combination chemotherapy regimens including GO, both in relapsed/refractory and in newly diagnosed patients. Amadori et al. treated 57 newly diagnosed elderly AML patients with two infusions of GO 9 mg/m2 as induction therapy, which was followed subsequently by conventional chemotherapy (33). Response to GO was observed in 35.1% of patients, with an additional 10.5% of partial remissions. In total five patients
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suffered from VOD, with three cases during the initial GO treatment phase. Oneyear survival was 34%. The value of this GO-containing regimen is currently being compared in a prospective randomized study with a chemotherapy-only arm. In another report, 59 patients with previously untreated AML or MDS were treated with a single infusion of 6 mg/m2 GO, combined with fludarabine, cytarabine, and cyclosporine A—Mylotarg, fludarabine, cytarabine (ara-C), and cyclosporine (MFAC) regimen, which induced CR in 46% of patients but also resulted in considerable toxicity (34). Cyclosporine A was added as MDR1 reversal agent, as MDR1 has been reported to be involved in GO resistance (35). This regimen was first tested in relapsed/refractory patients, and additionally as postremission therapy in patients achieving CR after GO-containing induction (36,37). Chevallier et al. have combined GO 9 mg/m2 with cytarabine and mitoxantrone in induction, in patients with relapsed/refractory AML, and report a very promising response rate of 76% with acceptable toxicity, but the number of patients was limited (38). Kell et al. reported a feasibility study in newly diagnosed adult AML patients in which GO was combined with several different standard AML induction regimens (39). Several lessons were learned from this study: (i) the maximum dose of GO in these combination was 3 mg/m2 single infusion; (ii) this dose could not be administered in consecutive courses because of hepatotoxocity and delayed hematopoietic reconstitution, but it was possible to use GO in course 1 and 3; and (iii) GO should not be combined with 6-thioguanine because of hepatotoxicity. A very promising CR rate of 85% was noted, and a randomized phase III trial (study AML15) based on this schedule has recently been completed in newly diagnosed patients with AML in the MRC-group. The first results on the 1115 randomized patients were reported by Burnett et al. at the ASH 2006 meeting, showing similar remission induction rates of 85% both in the GO as well as in the non-GO arm. However, there was a significant reduction in the relapse rate in patients included in the GO-arm (37% vs. 52% at 3 years, p ¼ 0.01), resulting in improved disease-free survival (51% vs. 40% at 3 years, p ¼ 0.008), although not yet in overall survival (40). Considering toxicity, there was a significant increase in transaminase elevation in the GO-arm but no difference in bilirubin elevation. Patients in the GO-arm needed significantly more platelet transfusions (19 vs. 14; p < 0.0001), and more days on IV antibiotics (20.6 vs. 18.6 days, p ¼ 0.001), although bone marrow recovery was similar. GO did not increase the number of patients with death in CR, after a median of 15 months of follow-up. Several other cooperative groups have started similar prospective randomized studies. Treatment of APL with GO seems promising, although the data are still limited. Lo-Coco reported 16 patients with a molecular relapse of APL who were salvaged by two (or more in 3 patients) infusions (6 mg/m2) of GO (41). Approximately half of the responses were sustained for a median of 15 months. Quantitative RT-PCR studies showed that responding patients experienced a dramatic decline (at least 2 logs) of the PML/RARalpha transcript after the first GO dose. In a series from MD Anderson, GO was given to nine patients with
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untreated APL, in combination with ATRA and idarubicin in patients with high white counts (42). After two induction courses, patients received eight maintenance courses. GO was tolerated well and induced CR in 16/19 patients. Followup was too short to determine whether these responses were durable. In a recent study, GO (dose of 9 mg/m2) was given as consolidation in combination with ATRA and arsenic trioxide, following arsenic trioxide reinduction, in patients with relapsed APL, again showing good tolerability of this regimen (43). CD33 DIRECTED MONOCLONAL ANTIBODIES IN PEDIATRIC AML The results of the various studies in children are summarized in Table 3. The first data reported on the use of GO in children concerned 15 patients with relapsed/ refractory AML who were treated on compassionate use basis with one to three infusions of GO at a dosage of 4 to 9 mg/m2 (44). Basically, efficacy and safety mimicked the experience in adults. Durable responses were noted in two of the eight patients who achieved a response, defined as ‘‘no evidence of leukemia.’’ Brethon et al. published similar results in another compassionate use series (45). The pediatric phase I study results were recently published by Arceci et al. (46). They initially started at the 6-mg/m2-dose level, administering two infusions with a 14-day interval, and subsequently escalated to 9 mg/m2. At this level, 3 of 13 patients had grades 3 to 4 transaminase elevations, and one patient developed VOD, after which dose de-escalation to the 6-mg/m2 level was issued. The last two patients received 7.5 mg/m2 for two dosages, before the study was closed. Overall, toxicities included grades 3 to 4 hyperbilirubinemia in 7% and elevated hepatic transaminases in 21%; the incidence of grade 3 to 4 mucositis (3%) or sepsis (24%) was relatively low. Eight of 29 patients achieved overall remission (28%). Remissions were comparable in refractory (30%) and relapsed (26%) patients. Versluys et al. published a case series of another seven children treated with GO prior to SCT as reinduction treatment (26). One to four doses of GO 9mg/m2 were administered in these patients. After the second patient suffered from severe VOD at subsequent SCT, routine defibrotide prophylaxis was given to all patients, and none of them developed VOD. Preliminary results from a phase II study with GO (2 doses of 7.5 mg/m2 IV) in 20 patients who received homogenous pretreatment according to the Relapsed AML 2001/01 protocol, but were either refractory to reinduction or suffered from second relapse, showed a response rate of 40% (including both CR and CRp) (47). The median survival of responders was longer than for nonresponders to GO (median 1.04 vs. 0.4 years, p ¼ 0.04). Transaminase and bilirubin elevation was found in 5% of patients. One out of eight transplanted children developed VOD. A recent pilot study addressed the use of GO following reduced-intensity stem cell transplantation (RIC-allo-SCT), in eight children with CD33þ AML, either in first or second CR (48). The first dose of GO was given after reconstitution
Relapsed/ refractory AML, phase I study Relapsed/ refractory AML Relapsed/ refractory AML Relapsed/ refractory AML Relapsed/ refractory AML
Phase of the disease
Induction
Induction
Single agent
Single agent
7.5 mg/m2, day 1 þ 15 3–9 mg/m2, once (n ¼ 3), twice (n ¼ 3), thrice (n ¼ 5), or five times (n ¼ 1)
n ¼ 20, median age 8.2 yr n ¼ 11, median age 5.5 yr
9 mg/m2, up to 4 courses
Dose escalation from 6 to 9 mg/m2, day 1 þ 15 4–9 mg/m2, up to 3 courses
Dosages
3 CRs
Overall 40%
Not given
Overall 33% (33% CRp)
Overall 28% (14% CR, 14% CRp)
Response rate
Median survival 1.04 yr NA
2 patients still alive
2 patients still alive
2 patients still alive
Median duration of response
45
47
26
44
46
Ref.
116
Single agent
Induction, com- n ¼ 15, median age 8.9 yr passionate use Induction n ¼ 7, median age 6 yr
Single agent
n ¼ 29, median age 12 yr
Population and number of patients
Induction
Phase of treatment
Single agent
GO monotherapy or in combination
Chemotherapy in Children with Acute Myeloid Leukemia
Table 3 Phase I/II Studies and Compassionate Use Data with Gemtuzumab Ozogamicin as Single Agent or in Combination with Regular
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Single agent
Relapsed/ refractory AML
Post-SCT, following reduced intensity SCT Induction
Phase of treatment NA
Median duration of response
– Induction: 2CR, NA 4 CRp, 4 PR After consolidation: 2 PR became CR, 1 PR became CRp
– Induction: GO 3 mg/m2, day 1, 4, and 7 Cytarabine 100 mg/m2/day CI for 7 days Consolidation: GO 3 mg/m2 at day 1, plus cytarabine 100 mg/m2/day for 7 days
Response rate
n ¼ 17 induction, n¼6 consolidation
2
4.0–6.0 mg/m , NA 1st dose day 60–180 post-SCT, 2nd dose 8 wk later
Dosages
n ¼ 6, median age 9 yr
Population and number of patients
Abbreviations: NA, not available; SCT, stem cell transplant, GO, gemtuzumab ozogamicin.
Single agent
Relapsed/ refractory AML
Phase of the disease
GO monotherapy or in combination
49
48
Ref.
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of neutrophils and platelets at day þ60-180 post RIC-allo-SCT. The second dose was given after recovery of peripheral blood, usually around eight weeks following the first dose. A dose escalation from 4.5 to 6.0 mg/m2 appeared to be safe, and further dose escalation is planned. Toxicity mainly consisted of bone marrow suppression, infections, and transaminase elevations, but no VODs occurred. Further studies are necessary to demonstrate whether GO is able to reduce the relapse rate following RIC-allo-SCT and whether this approach is superior to myeloablative allo-SCT. Brethon et al. presented the first results of a combination of GO with cytarabine, using a fractionated GO schedule; GO was given at 3 mg/m2/day on day 1,4, and 7 plus cytarabine 100 mg/m2/day using continuous IV infusion for seven days (49). Seventeen children received this course as induction regimen, and six patients subsequently received a consolidation course with GO 3 mg/m2 at day 1, plus cytarabine 100 mg/m2/day for seven days. Responses included 2 CRs, 4 CRps, and 4 PRs. Currently, several pediatric studies are underway applying GO in several phases of treatment. This includes the combination of reduced dosages of GO (3 mg/m2) with induction and/or consolidation chemotherapy as well as testing GO as single agent in postremission setting (following intensive AML treatment) with the aim to eradicate minimal residual disease and reduce the relapse rate. CD45-DIRECTED MONOCLONAL ANTIBODIES CD45 is a tyrosine phosphatase and a common leucocyte antigen, expressed in the membrane of all leucocytes, leukemic cells, and erythrocyte progenitors but not outside the hematopoietic system (50). Different isoforms exist because of alternative splicing. Its expression is more dense on lymphoid when compared with myeloid cells, which explains why this antibody is used in conditioning regimens of SCT to deplete the host from lymphocytes and reduce graft failure. Antibody-bound CD45 tends to remain on the cell surface and does not internalize, which makes it an attractive target for radioimmunoconjugates, as the risk of cleavage of the radioisotope and release in the circulation is limited. Several studies on radiolabel-led anti-CD45 antibodies have been reported, but they are outside the focus of this chapter. Experience with unconjugated anti-CD45 antibodies is limited. In a phase I study, patients who were to receive a SCT were treated with the rat anti-CD45 antibodies YTH24 and YTH54. The MTD was defined as 400 mg/kg/day for four days, with bronchospasm as dose-limiting toxicity (50). This resulted in a decline in lymphocytes and granulocytes from the peripheral blood (approximately 1 log reduction), as well as demonstrable antileukemic efficacy (in the 3 patients with measurable disease before the administration of the antibody). In a subsequent study in patients who could not tolerate myeloablative conditioning, anti-CD45 was used in conjunction with alemtuzumab, fludarabine, and total body irradiation, which resulted in successful engraftment (51).
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OTHER ANTIBODIES A phase I study was performed with an anti-GMCSF monoclonal antibody in eight patients with relapsed AML, which was well tolerated but without significant antileukemic activity (52). FLT3 mutations have been detected in approximately 10% to 15% of pediatric AML and 30% of adult AML cases. Apart from inhibitory small molecules that interfere with signal transduction, monoclonal antibodies against FLT3 have been developed (53). These antibodies interfere with ligand-mediated autocrine FLT3 signaling and may induce antibody-dependent cellular cytotoxicity (ADCC). Currently, these antibodies are in the preclinical phase of development, and clinical studies have to be awaited. RESISTANCE MECHANISMS Similar to conventional chemotherapy, the use of monoclonal antibodies may results in clinical resistance against these compounds (16). General mechanisms include clonal evolution of clones that do not (or not high enough) express the selected target antigen. In case of a high tumor load all the antibody may be trapped in the circulation and hence not result in significant antileukemic efficacy, which has also been described for GO (54). Especially when using naked antibodies, immune deficiencies, which may occur secondary to the disease itself or due to immunosuppressive treatment, may impair with successful killing of tumor cells. Of particular relevance for use of monoclonal antibodies in AML is whether the leukemic stem cell itself expresses the target antigen of interest or whether this is only expressed by the more mature bulk of cells (55). This issue is currently unresolved for GO and CD33, although relapses are usually not characterized by CD33-negativity (16,56). For GO, several other mechanisms have been reported, including MDR1 overexpression (35) and differences in cellular calicheamicin sensitivity (57,58). Cell-line studies have shown that GO-induced cytotoxicity was directly related to CD33 expression levels, although several clinical studies have failed to relate CD33 expression to clinical response to GO (59,60). In cell-line studies, apoptosis was not inhibited in the presence of blocking anti-CD33 antibodies in case of continuous exposure to GO in relatively high concentrations, whereas at lower concentrations apoptosis was inhibited (61). This may be explained by other, CD33-independent uptake mechanisms such as endocytosis. A more recent study, in patients undergoing treatment with single-agent GO, showed that responders had higher median CD33 levels and lower P-glycoprotein activity than nonresponders (62). CLINICAL PERSPECTIVE FOR THE NEXT FIVE YEARS The response rates to GO have not been promising enough to justify its use as a single agent. Therefore, the current interest is to incorporate GO in modern multiagent chemotherapy; however, only reduced dosages of GO can be used in
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this setting because of tolerability. Whether combination chemotherapy containing GO will result in improved overall survival rates has to be awaited, although the MRC AML15 study already showed a reduced relapse rate. It is encouraging that the use of GO upfront did not lead to a significant increase in death in CR in this study. The current, conditional approval for GO by the FDA is restricted to elderly AML patients in first relapse who are deemed unfit to receive more intensive therapy only. In addition, there is currently no approval in Europe by the European Agency for the Evaluation of Medicinal Product. This limited approval status may be subject to change once more results become available. GO may very well obtain an important role in the treatment of APL; for instance, as salvage therapy in patients with detectable minimal residual disease after consolidation chemotherapy or in molecular relapse, although larger prospective studies are still lacking. Other options are to combine GO with ATRA or arsenic trioxide, which might be an alternative to the use of regular chemotherapy in APL. GO is still an attractive drug for palliative treatment in AML, given the lack of severe mucositis and alopecia and the possibility to administer it in outpatient basis in most patients.
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6 Monoclonal Antibodies in the Treatment of Malignant Lymphomas and Chronic Lymphocytic Leukemia Bertrand Coiffier Hematology Department, Hospices Civils de Lyon and Claude Bernard University, Pierre-Benite, France
INTRODUCTION Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of B- and T-cell cancers, with a large variety of patterns of growth, clinical presentations, and responses to treatment. Chronic lymphocytic leukemia (CLL) is a B-cell chronic proliferation not very different from the small lymphocytic lymphoma, and both diseases are often treated identically (1). The outcome depends on histological subtype, tumor characteristics, host responses, and treatment. About 90% of lymphomas have a B-cell phenotype, and for them recent therapeutic progress came from the introduction of monoclonal antibodies (mAb) alone or in combination with chemotherapy (2–4). The first antigen that has been targeted for therapeutic purpose with success was the CD20 antigen, a trans-membrane protein expressed by more than 99% of B-cell lymphomas. Rituximab was the first mAb engineered to target the CD20 antigen and first approved mAb for the treatment of lymphoma patients. Through the last 10 years, clinical trials with rituximab have confirmed its efficacy in follicular lymphoma (FL) as well as in aggressive lymphomas and its use has expanded significantly beyond the initial indication of indolent B-cell lymphomas to virtually any CD20-positive
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lymphoma. The addition of rituximab to chemotherapy was the first real progress in 10 years that has significantly prolonged the survival of patients with B-cell lymphomas (4,5). In recent years, several other mAb targeting CD20 or other lymphocyte antigens appeared, some of them associated with a toxin or a radioisotope. However, most of the data generated today on mechanisms of action or clinical efficacy have been for rituximab. Thus, rituximab will serve as example in this review, and differences with other mAb will be outlined when necessary. MECHANISMS OF ACTION OF mAb The mechanisms of action of mAb differ with the type of antibody, the antigen they target, and their use: alone, in combination with chemotherapy, or conjugated to a toxin or a radionucleide. In case of a naked antibody, different mechanisms have been identified (6). CD20 binding by rituximab is followed by homotypic aggregation, rapid translocation of CD20 into specialized plasma membrane microdomains known as rafts, and induction of apoptosis. Membrane rafts concentrate Src family kinases and other signaling molecules (phospholipases, caspases), and the anti-CD20-induced apoptotic signals are thought to occur as a consequence of CD20 accumulation in rafts (7). Fas-induced apoptosis occurs with the clustering of Fas molecules that leads to the formation of the death-inducing signaling complex (DISC) and the downstream activation of the death receptor pathway (8). The role of complement-dependent cytotoxicity (CDC) is suggested by the consumption of complement observed after rituximab administration, but in vitro CDC does not correlate with clinical response in lymphomas (9,10). However, CDC seems to be the most important mechanism of cell lysis in CLL patients (11). CDC is probably involved in the cytokinerelease syndrome and its toxicity (12). The importance of antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated in vivo when rituximab is used alone (13). The Fc receptor (FcgR) of effector cells has two alleles and the valine/valine (V/V) allele of FcgRIIIa which confers a higher affinity for immunoglobulin G1 (IgG1) and rituximab is associated with an increased responsiveness to rituximab (13,14). If the clinical relevance of the FcgRIIIa receptor dimorphism was established in a number of studies with rituximab used alone, it does not seem to play a major role when rituximab is used in combination with chemotherapy (15) even if one study showed an increased response for patients with the V/V allele without difference for progression-free survival (PFS) or overall survival (OS) (16). The immune mechanisms are probably valid for other naked antigens but only those targeting the CD20 antigen may have the direct action described here. Finally, evidences that rituximab could synergize with chemotherapeutic agents in B-cell killing were provided by Demidem (17). Subsequent investigations have confirmed synergy of rituximab with fludarabine, doxorubicin, and other anticancer drugs (18–20). In one hypothesis, this synergism is mediated, at
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least in part, via downregulation of interleukin-10 (IL-10) by rituximab, which in turn causes downregulation of the antiapoptotic protein bcl2 and increased sensitivity to apoptosis (21). Another mechanism involves the inhibition of the activity of P-glycoprotein and, thus, the efflux of drugs like doxorubicin or vincristine (22). In cell lines, the P-glycoprotein pump is translocated out of the lipids rafts. Studies performed with cell lines as model systems revealed several mechanisms that are involved in chemo/immunosensitization and the development of resistance to rituximab. Rituximab has been shown to inhibit the p38 mitogenactivated protein kinase, nuclear factor-kB (NF-kB), extracellular signal-regulated kinase 1/2 (ERK 1/2) and AKT antiapoptotic survival pathways, all of which result in upregulation of PTEN and Raf kinase inhibitor protein and in the downregulation of antiapoptotic gene products (particularly Bcl-2, Bcl-xL, and Mcl-1), and resulting in chemo/immunosensitization. Further, rituximab treatment inhibits the overexpressed transcription repressor Yin Yang1 (YY1), which negatively regulates Fas and DR5 expression, and its inhibition leads to sensitization to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis (23). If these mechanisms may have a role when mAb are combined with a radionuclide, most of the antitumor effect resides in their capacity to deliver local radiotherapy after the mAb is attached to tumor cells (24). The choice of the antibody and therapeutic radioisotopes are critical for the success of radioimmunotherapy (RIT). Several radiolabeled mAb have been studied in clinical trial but only two, yttrium-90 (90 Y or Y-90) ibritumomab tiuxetan and iodine-131 (131I or I-131) tositumomab, have been registered for the treatment of lymphoma patients. Both radiolabeled antibodies are mouse antibodies reacting with CD20 expressing tumors. Y-90 is a pure b-emitter, with a half-life of 2.7 days (25). It is a link to the antibody by a chelator (tiuxetan). The long pathlength of its b-particles is particularly advantageous in tumor with heterogeneous or low distribution of the antigen (26). I-131 is an a- and b-emitter that has a half-life of eight days. The path length of its b-particles is relatively shorter than Y-90. Table 1 presents the differences between Y-90 and I-131 radiolabeled antibodies. Table 1 Characteristics of the Two Registered Radiolabeled MmAb 90
Linker Isotope radiation decay Half-life, days Path length, mm Energy, MeV Non tumor distribution Urine excretion Imaging Source: From Refs. 25, 106.
Y-ibritumomab
Tiuxetan Beta 2.7 5.0 2.3 Bone Limited Not possible
131
I-tositumomab
None Beta and gamma 8.0 0.8 0.61 Thyroid Substantial Possible
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An alternative approach to increase the activity of mAbs has been the development of immunotoxin, a construct conjugating the antibody to cytotoxic plant, bacterial toxic proteins, or chemotherapy drugs (doxorubicin) (27). The commonly used toxins, ricin or diphtheria toxin, are highly potent natural products that disrupt protein synthesis. Unlike unconjugated mAb, immunotoxins must be internalized after antigen binding to allow the toxin access to the cytosol. Although the conjugation to mAbs confers some target specificity, the toxin continues to mediate nonspecific toxicity to normal tissues. Deglycosylated ricin A-chain has been used to eliminate such nonspecific toxicity.
MECHANISMS OF RESISTANCE If multiple mechanisms of rituximab action have been reported, it remains unclear which is or are most important in patients, and therefore it is difficult to know the relative importance of potential mechanisms of resistance. This is true for the other mAb too. Conceptual approaches of resistance mechanisms may be resumed as was followed (28). As far as events up to antigen binding are concerned, resistance to rituximab may be secondary to low serum levels or rapid metabolism of the mAb; development of human antimonoclonal antibodies (HAMA), most frequent with nonhumanized antibodies than with rituximab, or human antichimeric antibodies (HACA) (not yet demonstrated in patients); possibly different distribution within malignant nodes, blood cells, marrow, and extranodal sites and responsible for poor tumor penetration; high level of soluble antigen target (not yet demonstrated for CD20 antigen); high tumor burden; and poor surface antigen expression. Events that may induce resistance to rituximab after the antigen binding are alteration of induced intracellular signals; reduction of direct apoptosis effect in cases of elevated bcl-2 protein; inhibition of CDC by complement inhibitors; and alteration of cell-mediated immunity. Gene microarray analysis has shown that patients who failed to respond to rituximab have altered patterns of gene expression, with an overexpression of genes important in cell-mediated immunity (29). In vitro, long exposition to rituximab induced rituximab-resistant clones. These clones exhibited constitutive hyperactivation of the nuclear factor-KB and extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of bcl-2 protein and bcl-2-related genes. These clones can be chemosensitized following treatment with pharmacologic inhibitors like bortezomib (30). In CLL patients, one particular mechanism of resistance has been described where there is a high number of circulating B-cells—the mononuclear phagocytic system is rapidly saturated and rituximab-opsonized cells are not cleared anymore. Then the complex rituximab-CD20 is shaved from the cells that become CD20-negative or low and rituximab losses its efficacy (31).
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SAFETY AND TOLERABILITY The safety of mAb is mainly related to their origin and to the compound attached to them. Radiolabeled mAb have a greater hematological toxicity than naked mAb because of the effect of surrounding normal hematopoietic cells in bone marrow. Immunotoxins also have greater toxicity because of the release of the toxin. Some mAb such as alemtuzumab may have larger hematological toxicity because the target antigen (CD52, in case of alemtuzumab) is not restricted to lymphoid cells. The safety of rituximab is mainly related to infusion toxicity, a toxicity most mAb have in common (32). These side effects are observed during the infusion or in the first hours after drug infusion and particularly for the first infusion. These include fever, chills, dizziness, nausea, pruritus, throat swelling, cough, fatigue, hypotension, and transient bronchospasm in a majority of patients. These symptoms are part of the cytokine-release syndrome. Their intensity correlates with the number of circulating malignant cells at the time of infusion. More severe infusional toxicity includes bronchospasm, angioedema, and acute lung injury, which are often associated with high circulating cell counts or pre-existing cardiac or pulmonary disease. Another common toxicity is the rapid depletion of normal antigen-positive B-lymphocytes from blood, bone marrow, and lymph nodes of the recipient, lasting between six and nine months following the last administration of rituximab. In the case of short rituximab treatment, this depletion does not compromise immunity: Immunoglobulins do not decrease significantly, and patients do not have an increased risk for infections during and after rituximab therapy (32,33); except for some viruses like herpes virus, cytomegalovirus, or hepatitis B virus (HBV). Maintenance treatment, particularly after autologous transplant, might be associated with a decrease in immunoglobulins (34) and late toxicity (32). Rare toxic events associated with rituximab comprised delayed neutropenia and pulmonary reactions. Delayed neutropenia usually occurs in patients treated with rituximab alone or in combination with chemotherapy. It appears between one and six months after the last infusion, may be transient, is rarely associated with infection, and resolves spontaneously in most of the cases (35). The mechanisms are not fully understood. Pulmonary reactions are rare and diverse, and usually related to rituximab because of the temporal relation (32). RIT is associated with secondary myelodysplastic syndromes. Rituximab as chemotherapy may induce a reactivation of hepatitis B in inactive HBV carriers. Lamivudine or other antiviral treatment must be use prophylactically during treatment and the following months to prevent this severe complication (36). CLINICAL STUDIES A few mAb have been registered for the treatment of lymphoma patients: rituximab (Rituxan1 or MabThera1), 90 Y-ibritumomab tiuxetan (Zevalin1), 131I-tositumomab (Bexxar1), and denileukin diftitox (OnTak1), the last two only in the United States. However, a lot of other mAb are currently in preclinical, phase I, or phase II studies.
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Rituximab is certainly the mAb where the largest experience exists and the mAb with several demonstrative randomized studies. We will focus on demonstrated activity (phase III studies) and some phase II studies with promising results. RITUXIMAB IN FOLLICULAR LYMPHOMA Rituximab Alone in Relapse When used alone, rituximab is usually given as four weekly injections of 375 mg/m2 (37). The pivotal multicenter phase II study that included 166 patients treated with four infusions of rituximab showed an overall remission rate of 48% [including 6% of complete response (CR)], and a median time to progression (TTP) of 13 months (38). Elevated b2-microglobulin, elevated lactate dehydrogenase (LDH), bulky disease, and age older than 60 years did not appear to impact response, implying that patients regarded as having a poor prognosis may respond to rituximab. Patients relapsing after initial response to rituximab treatment may be retreated with comparable response rates and adverse side effects, but, interestingly, median time for progression might be longer than after first treatment (39,40). Whether prolonged treatment with rituximab or maintenance is able to further improve response rates and prolong remission duration is of considerable interest. Several arguments are in favor of this approach: the success of re-treatment or the strong correlation between rituximab plasma levels and response rates (41). A recent randomized trial showed that adding maintenance doses of rituximab prolonged response duration (42); 202 patients with newly diagnosed or refractory/relapsed FL were treated with rituximab. Patients responding, or with stable disease, were randomized to no further treatment or prolonged rituximab administration (375 mg/m2 every two months for four times). With a median follow-up of 35 months, the median event-free survival (EFS) was prolonged in the treated group, 23 months versus 12 months in the control group. However, patients relapsed within the six months after stopping rituximab treatment. In another randomized study, Hainsworth showed that re-treatment at relapse or prolonged treatment have the same benefit in terms of duration of rituximab efficacy or time to chemotherapy (43). Several questions remain without clear response: What is the optimal prolonged treatment? What is the optimal duration maintenance? Which patients benefit from prolonged treatment? And, finally, is prolonged treatment or re-treatment at progression better in terms of survival or impact on transformation rate? Rituximab Alone in Untreated FL Usually, patients with no adverse prognostic factors are not treated until they develop such adverse parameters (44). However, because of its low-profile
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toxicity, its presumed low rate of secondary malignancy, and its lack of stem cell toxicity, rituximab single agent was investigated in this setting (45): in a series of 50 patients, a relative risk (RR) of 73% was obtained, with a CR of 26% ; 57% of the informative patients in CR reached a molecular remission. However, even patients in CR and in molecular response did not seem to benefit from this treatment because the median TTP was only two years, which is not longer than that without treatment. A randomized study is currently underway in the United Kingdom challenging this finding in these otherwise ‘‘watch and wait’’ patients. Rituximab alone was also studied in patients with a more aggressive presentation, needing treatment at diagnosis, or after some follow-up without treatment (46). The RR, just after four infusions, was comparable with the one observed in relapsing patients (50% and <10% of CR). About 10% of patients had progressive disease during the immediate posttreatment period and progression occurred in less than 12 months among 50% of the responding patients. Rituximab in Combination with Chemotherapy In a phase II study, the combination of six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with rituximab given before, during, and after chemotherapy in 40 patients with predominantly untreated FL increased the RR [(55% CR, 40% partial response (PR)], with no added related toxicity (47). Median TTP was 82 months. Several randomized studies have now demonstrated that the addition of rituximab to a standard chemotherapy regimen results in higher response rates and longer TTP, EFS, and OS for patients treated with a combination of rituximab plus chemotherapy (R-CHEMO) in first-line or in first-relapse patients (Table 2). In first-line patients, four studies have reported a benefit in terms of CR rates, PFS, and OS (48–51). The first study randomized patients between eight cycles of CVP (cyclophosphamide, vincristine, prednisone) and CVP combined with rituximab (R-CVP) (48). At a median follow-up of 53 months, patients treated with R-CVP had a highly significantly prolonged TTP (median 32 months vs. 15 months for CVP; P < 0.0001). Median time to treatment failure (TTF) was 27 months in patients receiving R-CVP and 7 months in the CVP arm (P < 0.0001). OS was longer for R-CVP than CVP (19% vs. 29% patients died, P ¼ 0.03) (52). In the second study, patients were randomized between six cycles of CHOP and CHOP combined with rituximab (R-CHOP) (51). In 428 patients, R-CHOP revealed a significantly higher RR (96% vs. 90%, P ¼ 0.011) and a longer TTF (median not reached vs. 2.6 years, P < 0.0001). In the French study, patients were randomized between CHVP (cyclophosphamide, hydroxydaunomycin, vm 26, prednisone) plus interferon for 18 months and CHVP combined with rituximab (R-CHVP) plus interferon (53). This analysis of all patients demonstrated a significant improvement of response to therapy with R-CHVP plus interferon compared with CHVP plus interferon,
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Table 2 Randomized Studies Comparing Chemotherapy with the Combination of Rituximab and Chemotherapy in Patients with Follicular Lymphoma Setting First-line patients Marcus (48,52) R-CVP CVP Hiddemann (107) R-CHOP CHOP Salles (50,53) R-CHVP-Ifn CHVP-Ifn Herold (108) R-MCP MCP Relapsing patients Forstpointer (54,55) R-FCM FCM Adjuvant rituximab Hochster (57) CVP?R
Response rates
CR rates
EFS
Time to progression
81% 57%b
41% 10%b
27 mo 7 mob
32 mo 15 mob
Not different
97% 90%
20% 17%
68 mo 21 mob
50 mo 15 mob
Not analyzed
Not analyzed 79% 63%b
Not reached
Not reached
Not analyzed
85.5% 65.5%b
42% 20%b
Not reached 19 mob
Not reported Not reported
79% 58%b
33% 13%b
Not analyzed 16 mo 10 ma
Not reached 24 mb
Not reported
30%
Not reported
Trend in favor of R
CVP
22%
a
4.2 yr 1.5
yrb
OS
In parentheses are the references. a P < 0.05. b P < 0.01.
both at six months [CR þ CRru 49% vs. 76%; PR 36% vs. 18%; respectively (P < 0.0001)] and at 18 months [CR þ CRu 79% vs. 63%; PR 5% vs. 10%; respectively (P ¼ 0.004)]. In the control arm, estimated 3.5 years EFS was 46% versus 67% with R-CHVP plus interferon (P < 0.0001). Even if the median followup is only 3.5 years, this study showed a statistically significant OS advantage for patients treated with rituximab (91% compared with 84% surviving at 3.5 years, P ¼ 0.029). In relapsing patients, the FCM (fludarabine, cyclophosphamide, mitoxantrone) study showed that FCM combined with rituximab (R-FCM) is superior to FCM alone for relapsing patients with follicular or mantle cell lymphoma (MCL) (54). An update of this study showed that responding patients, treated either with FCM or R-FCM had a prolonged PFS if they received a maintenance with rituximab (55). The European Organisation for Research and Treatment of
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Cancer (EORTC) study compared R-CHOP with CHOP alone in first or second line patients not previously treated with doxorubicin (56). This last study is particularly interesting because preliminary results showed a benefit of R-CHOP over CHOP and also a benefit of rituximab maintenance after CHOP-only induction. Rituximab maintenance yielded a median PFS from second randomization of 51.5 months versus 15 months with observation [Hazard Ratio (HR), 0.40; P < 0.001]. Improved PFS was found both after induction with CHOP (HR, 0.30; P < 0.001) and R-CHOP (HR, 0.54; P ¼ 0.004). Rituximab maintenance also improved OS from second randomization: 85% at three years versus 77% with observation (HR, 0.52; P ¼ 011). Finally, one study reported that maintenance with rituximab in patients treated with chemotherapy increases CR rates and prolongs PFS (57). However, the role of rituximab maintenance after a combination of rituximab plus chemotherapy in first-line patients remains unclear and it is not currently recommended in CR patients. These different studies have implemented the use of combining rituximab with chemotherapy as standard treatment in patients with FL who need to be treated. Which of the chemotherapy regimens is better is not yet demonstrated but the comparison of CR rates, EFS, PFS, and OS from the different studies seems to show a larger benefit with the R-CHOP regimen. The comparison of results obtained with R-CHOP to those reached with rituximab only in the same type of patients equally favors the use of R-CHOP. However, these conclusions need to be accepted with caution because no randomized study has compared these different regimens.
RIT IN FOLLICULAR LYMPHOMA Two mAb have been combined with a radionucleide and have been registered for the treatment of patients with relapsing/refractory FL. RIT with Y-90 and I-131 labeled anti-CD20 antibodies (ibritumomab tiutexan and tositumomab) was associated with a high response rate in relapsing/refractory patients. Zevalin was not tested in untreated FL patients. In the initial phase I/II study, 90Y-ibritumomab was administered on 51 patients with relapsed and refractory CD20-positive B-cell NHL (58). The overall response rate (ORR) for the 34 patients with indolent lymphoma was 82% (CR 26% and PR 56%). The estimate median TTP for the entire group was 12.9 plus months and the median duration of response was 11.7 plus months. The major toxicity of 90Y-ibritumomab was myelosuppression with thrombocytopenia being the most common. 90Y-ibritumomab has been compared with rituximab in a randomized controlled phase III study (59). The ORR was 80% (CR/CRu 34% and PR 45%) for 90Y-ibritumomab as compared with 56% (CR/CRu 20% and PR 36%) for rituximab (P ¼ 0.002). The estimated TTP was
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12.6 months for 90Y-ibritumomab and 10.2 months for rituximab (P ¼ 0.062). In another study, Witzig treated with 90Y-ibritumomab 54 patients with FL refractory to rituximab (60). The ORR for the entire cohort was 74% (CR 14% and PR 59%). The estimated TTP and response duration for responders were 8.7 and 6.4 months, respectively. Long-term responses are seen in 37% of the patients with a median duration of response of 28 months in these good responders (61). 131 I-tositumomab has been studied for more than 10 years. Vose et al. have reported the final results of a multicenter phase II study with objectives to evaluate the efficacy, dosimetry, methodology, and safety of 131I-tositumomab (62). Forty-seven patients with relapsed/refractory low-grade or transformed NHL were treated with 131I-tositumomab. The ORR for the entire group was 57% with 15 (32%) patients achieving CR. The ORR was similar in patients with indolent (57%) or transformed lymphoma (60%). The median duration of response was 8.2 months and 12.1 months, respectively, for each of these two groups. The treatment was well tolerated and hematologic toxicity was the principal adverse event. In the pivotal study, 60 patients with chemotherapyrefractory indolent or transformed CD20-positive B-cell lymphoma (36 follicular, 23 transformed, and 1 mantle cell) were treated with standard dose 131 I-tositumomab (63). The ORR was 65% (CR 20% and PR 45%). The median duration of response was 6.5 months. Kaminski recently presented the results of a phase II study evaluating 131I-tositumomab alone in untreated patients with FL (64). Of the 76 patients included, more than half did not have any criteria associated with poor outcome and corresponded to patients who are usually not treated. CR was observed in 75% of the patients, but only in 58% of those with a large lymph node. Median PFS was 6.1 years for all patients, but less for patients with criteria associated with poor outcome (details not given in the manuscript). This study only showed that patients without large tumor may respond well to 131 I-tositumomab but it did not allow evaluating the role of this drug in patients with FL. Even though 131I-tositumomab has shown interesting results in the phase II study, duration of response is still limited. For this reason, some investigators are beginning to evaluate 131I-tositumomab in combination with other forms of therapies. In a phase II study conducted by the Southwest Oncology Group (SWOG) (65), 131I-tositumomab was combined with CHOP for the treatment of 90 patients with untreated FL. Patients received six cycles of standard CHOP followed by a consolidation dose of 131I-tositumomab if PR was achieved. The ORR after 131I-tositumomab was 90% (CR/CRu 67% and PR 23%). More interestingly, among patients assessable, 27 (57%) improved their level of response after 131I-tositumomab. The estimated two-year PFS and OS were 81% and 97%, respectively (median follow-up 2.3 years). SWOG is currently conducting a study comparing 131I-tositumomab and rituximab in follicular patients treated with CHOP as first treatment. Only such a study may evaluate the benefit and toxicity of Bexxar in comparison with rituximab in first-line patients.
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A consensus meeting defined the optimal setting of RIT in the therapeutic algorithm of patients with advanced stage of FL. According to the reviewed data, RIT should preferably be used as consolidation after initial tumor debulking. First-line RIT may be applied in patients not appropriate for chemotherapy induction (66,67). OTHER MONOCLONAL ANTIBODIES Several MAb directed against CD20 (hA20, HuMax-CD20, ocrelizumab) or other antigens (epratuzumab for CD22, apolizumab for HLA-DRB chain, galiximab for CD80, SGN-40 for anti-CD40) are currently in phases I or II (68). No definitive conclusion can be arrived at on their activity, toxicity, and benefit compared with rituximab. The real interest of these new antibodies will have to be demonstrated in randomized studies. RITUXIMAB IN DIFFUSE LARGE B-CELL LYMPHOMA The combination regimen R-CHOP, consisting of rituximab plus CHOP, is now considered as the standard treatment for treating young and elderly patients with diffuse large B-cell lymphoma (DLBCL) because of the superior activity demonstrated in three randomized studies (Table 3). Results from the GELA study have been recently updated with a five-year median follow-up and showed a persisting advantage for patients treated with R-CHOP (Table 4) (69,70). In this study, patients with DLBCL aged 60 to 80 years were treated either with eight cycles of CHOP or eight cycles of R-CHOP. The difference observed between the two arms was already statistically significant for EFS, PFS, and OS, with a median follow-up of one year and improvement with follow-up. The MInT study compared in 824 patients six cycles of R-CHOP-like chemotherapy with CHOP-like chemotherapy in young patients with a low-risk DLBCL (71). After a median time of follow-up of 34 months, chemotherapy combined with rituximab (R-CHEMO) patients had a significantly longer TTF (P < 0.00001), with estimated 2-year TTF rates of 60% (CHEMO) versus 76% (R-CHEMO). Similarly, OS was significantly different (P < 0.001), with twoyear survival rates of 87% (CHEMO) and 94% (R-CHEMO), respectively. The American study (ECOG/SWOG/CALGB study) was associated with a statistical benefit in the primary endpoint TTF for R-CHOP versus CHOP alone (72). However, the complicated design of this study makes conclusions difficult compared with the two other studies. The interesting point of this study is the second randomization looking at the effect of rituximab in patients who reached a CR or a PR. If rituximab maintenance may decrease the progression rate in patients treated with CHOP only, it has no effect on patients treated with R-CHOP. The same activity was shown in relapsing patients whether treated with rituximab previously or not (73,74).
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Table 3 Randomized Studies Comparing CHOP with R-CHOP in Patients with DLBCL
Setting Median follow-up CR rates R-CHOP CHOP Early progression rates R-CHOP CHOP Relapses R-CHOP CHOP Event-free survival R-CHOP CHOP Progression-free survival R-CHOP CHOP Overall survival R-CHOP CHOP
Coiffier (69,70)
Habermann (72)
Pfreundschuh (71)
60–80 yr old No stage I CHOP 5 yr
60–80 yr old No stage I Maintenance 2.7 yr
<60 yr old IPI 0–1 CHOP or CHOP-like 2 yr
75% 63%a
78% 77%
85% 65%a
9% 22%a
15% 17%
16% 5%a
34% 20%a
Not reported
Not reported
3.8 yr 1.1 yr a
3.4 yr 2.4 yr
2-yr TTF 81% 58%a
Not reached 1.0 yr a
Not reported
Not reported
Not reached 3.1 yr a
Not different
2-yr OS 95% 85%a
Note: In parentheses are the references. a P < 0.01 Abbreviations: DLBCL, diffuse large B-cell lymphoma; CR, complete response, IPI, International Prognostic Index; TTF, time to treatment failure; OS, overall survival.
Table 4 5-Yr Survivals Observed in the GELA Study Comparing 8 Cycles of R-CHOP and CHOP in Elderly Patients with DLBCL
Median EFS 5-yr EFS Median PFS 5-yr PFS Median OS 5-yr OS
R-CHOP
CHOP
3.8 yr 47% Not reached 54% Not reached 58%
1.1 yr 29% 1 yr 30% 3.1 yr 45%
P value 0.00002 <0.00001 0.0073
Abbreviations: DLBCL, diffuse large B-cell lymphoma; EFS, event-free survival; PFS, progressionfree survival; OS, overall survival. Source: From Ref. 70.
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The experience of other mAb in DLBCL is quite restrained, but 90Yibritumomab tiuxetan has activity in rituximab-free relapsing patients (75). mAb IN OTHER LYMPHOMAS Small Lymphocytic Lymphoma The efficacy of rituximab alone in this lymphoma is not very well known, with few and discordant results. In a European study in relapsing patients, the efficacy was low, with only a 10% RR (76). In untreated patients, in contrast, Hainsworth found a 51% RR after four injections, with only 4% CR, and a median PFS of 18.6 months (77). Marginal Zone Lymphoma Most case reports have shown an efficacy of rituximab in these lymphomas. Efficacy was demonstrated in relapsing mucosa-associated lymphoid tissue (MALT) lymphoma (78). A current International Extranodal Lymphoma Study Group (IELSG) trial randomizes chlorambucil versus chlorambucil plus rituximab in new or relapsing patients with MALT lymphoma. Mantle Cell Lymphoma MCL has indolent lymphoma characteristics, but tends to pursue an aggressive clinical course and is incurable with standard chemotherapy. An interim analysis of a randomized trial comparing FCM with R-FCM has shown a striking improvement in RR with rituximab (65% vs. 33%; CR 35% vs. 0%), with a trend towards longer OS (54). Interestingly, about one-third of the patients achieved a molecular remission. A maintenance with rituximab was scheduled for responding patients and allowed to prolong the duration of response (55). Long-term remissions have been reported with intensive chemotherapy and autologous stem cell transplantation plus rituximab (see ‘‘mAb and High Dose Therapy with Autologous Stem Cell Transplantation.’’). Chronic Lymphocytic Leukemia Rituximab, given weekly as a single agent has low activity in relapsing patients with CLL. A better activity has been observed in untreated patients (77). Dose escalation, achieved by a thrice-weekly dosing schedule, (79) or higher weekly doses, 500 to 2250 mg/m2 (80) is necessary to reach significant clinical activity, with an RR of 45% and 36%, respectively, as a single agent. The concurrent administration of rituximab with fludarabine resulted in better results with an RR rate of 90%, with 47% CR (81). Ongoing clinical studies are examining the use of rituximab associated with fludarabine or pentostatin and cyclophosphamide, which has shown great promise in a single-center phase II study (82–84).
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Alemtuzumab is a humanized monoclonal antibody active against CD52. Compared with CD20, CD52 is expressed at much higher density on the surface of CLL cells. Activity of alemtuzumab in fludarabine-refractory CLL was established in the pivotal trial conducted by Keating (85). In 93 patients, the ORR was 33%, including 2% complete responders. The median time to response was six weeks and the median time of therapy extended up to eight weeks. Median survival of all patients was 16 months, but was 32 months for the responding patients. In heavily pretreated patients with CLL, the ORR is approximately 35%, and in previously untreated patients the ORR is greater than 80%, with a recent randomized study suggesting it is superior to alkylator-based therapy (86). Importantly, alemtuzumab is effective in patients with high-risk del(17p13.1) and del(11q22.3) CLL. Alemtuzumab combination studies with fludarabine and/or mAb such as rituximab have demonstrated promising results. Alemtuzumab is also being studied in CLL patients as consolidation therapy for treatment of minimal residual disease. CLL cells are CD23 positive, and anti-CD23 (lumiximab) has clinical activity in these patients (87). Other Lymphomas In posttransplant lymphoproliferative disease, several phase II have shown good activity with rituximab alone or in combination with chemotherapy (88). The only yet reported randomized study without benefit in the rituximab arm was in patients with HIV-associated lymphoma: The response rate was not statistically different in R-CHOP or CHOP arms (89). However, this study did not have the power to show a significant difference, and the follow-up is extremely short. In another phase II study, R-CHOP produced a CR rate of 77% and a two-year survival rate of 75% without severe infectious complications (90). In Europe, rituximab-containing chemotherapy is the standard for HIV-related lymphomas (91). Rituximab alone or combined with chemotherapy has activity in Waldenstrom macroglobulinemia (92). Alemtuzumab have been reported active in cutaneous T-cell lymphomas and peripheral T-cell lymphoma but activity was low and of short duration in most of the cases (93,94). The vast majority of the immunotoxin trials have been phase I studies designated to determine the maximum tolerated dose. These trials have shown that therapeutic serum levels may be achieved with tolerable toxicity. A relatively uniform toxicity has been observed with vascular leak syndrome, hepatotoxicity, and myalgia. The different trials have shown a low response rate of 10% to 25% PRs without durable efficacy. The only available immunotoxin for patients is OnTak for the treatment of cutaneous T-cell lymphoma; 30% of them responded with a CR of 10% (95). The future of this therapy will depend on decreasing toxicity, decreasing immune response against the construct, and increasing the antitumor activity.
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Intrathecal rituximab was used in meningeal and CNS location of lymphomas. It has no special toxicity and has activity for this difficult location (96). mAb AND HIGH DOSE THERAPY WITH AUTOLOGOUS STEM CELL TRANSPLANTATION Rituximab has been used as an in vivo purging agent before and as maintenance therapy after ASCT, in follicular and MCL (97,98) and in aggressive lymphoma (99) in first-line or in relapse patients, with promising results. An ongoing international trial in relapsed and refractory aggressive lymphoma randomizes rituximab-DHAP (dexamethasone, aracytine, cisplatin) versus rituximab-ICE (ifosfamide, carboplatin, etoposide) before ASCT and with a second randomization between rituximab maintenance and observation (CORAL study) (100). Rituximab given after ASCT might have the interest to complete the remission and to further decrease the relapse rate. However, this treatment may be associated with more infections (101). It had been associated with severe decrease in immunoglobulin levels (102) and more frequent neutropenia (35). A few studies have looked at the potential use of radiolabeled mAb in the context of HDT. As compared with external total body irradiation (TBI), radiolabeled monoclonal antibody could theoretically permit delivering higher dose of radiation to the tumor while limiting radiation dose to normal tissues, thus potentially reduce toxicity and treatment–related mortality (103). Press used myeloablative doses of 131I-tositumomab in combination with chemotherapy (104). In this phase I/II study, 25 Gy was considered the maximum dose of radiation that could be delivered to critical normal organs when combined with cyclophosphamide (100 mg/kg) and etoposide (60 mg/kg). They observed an objective response of 87% in a population of the patients with relapsed B-cell lymphoma (73% indolent lymphoma). The estimated two years’ OS and PFS were 83% and 68%. Ibritumomab tiuxetan followed by BEAM (Z-BEAM regimen) has shown activity in patients with chemotherapy-refractory lymphomas, but its advantage over BEAM alone is not yet demonstrated (105). CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Rituximab has a large activity but not all patients respond. Defining at the molecular level all mechanisms of action will allow defining the different mechanisms involved in the resistance to this antibody. Thus, appropriate response may be developed with the construct of new antibodies, defining new antigens, combination of antibodies or antibodies plus chemotherapy or small molecules. The definitive place of RIT must be defined. Because of the large activity of rituximab, R-CHOP is now the standard for treating patients with B-cell lymphoid malignancies. The role of the new antibodies will have to be defined in comparison with R-CHOP in prospective randomized studies. Because of the high ratio of efficacy/safety for rituximab,
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things will not be easy for the newcomers. Characteristics of patients not responding well to R-CHOP will have to be found so that these new antibodies can be tested on this poor-risk population. T-cell malignancies do not have their antibodies yet, and different antibodies will be tested. Other antigens will have to be found if the current ones do not prove to be good. Whatever the antigen and the antibody, mAb are now a standard part of the treatment of lymphoma. Whether they will replace chemotherapy might be considered, but this will not be the case for the next five years because the main objective is to cure patients, and antibodies alone are not sufficient for that. CONCLUSION Rituximab was the first monoclonal antibody registered in the treatment of lymphomas and it has allowed one of the major progresses for the treatment of lymphoma patients. Alone, it is a very well-tolerated drug and has a great activity in relapsing patients. However, it will hardly result in cure in this setting. In combination with chemotherapy, rituximab allowed for the highest response rates and longest EF and OS survivals ever described in FL and DLBCL. It has activity, but it is less well demonstrated in other B-cell lymphomas. Other mAb targeting CD20 or other antigens are on their way, but their activity is not yet well defined compared with rituximab. RIT may add some specific activity, but here too it is not well demonstrated. Antibodies conjugated with toxins are less used at the moment. REFERENCES 1. Jaffe ES, Harris NL, Stein H, et al., eds. World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001. 2. Coiffier B. Current strategies for the treatment of diffuse large B cell lymphoma. Cur Op Hematol 2005; 12:259–265 (review). 3. Coiffier B. First-line treatment of follicular lymphoma in the era of monoclonal antibodies. Clin Adv Hematol Oncol 2005; 3:484–491. 4. Coiffier B. Rituximab therapy in malignant lymphoma. Oncogene 2007; 26: 3603–3613. 5. Schulz H, Bohlius JF, Trelle S, et al. Immunochemotherapy with rituximab and overall survival in patients with indolent or mantle cell lymphoma: a systematic review and meta-analysis. J Natl Cancer Inst 2007; 99:706–714. 6. Cartron G, Watier H, Golay J, et al. From the bench to the bedside: ways to improve rituximab efficacy. Blood 2004; 104:2635–2642. 7. Janas E, Priest R, Wilde JI, et al. Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis. Clin Exp Immunol 2005; 139:439–446.
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96. Rubenstein JL, Fridlyand J, Abrey L, et al. Phase I study of intraventricular administration of rituximab in patients with recurrent CNS and intraocular lymphoma. J Clin Oncol 2007; 25:1350–1356. 97. Gianni AM, Magni M, Martelli M, et al. Long-term remission in mantle cell lymphoma following high-dose sequential chemotherapy and in vivo rituximabpurged stem cell autografting (R-HDS regimen). Blood 2003; 102:749–755. 98. Belhadj K, Delfau-Larue MH, Elgnaoui T, et al. Efficiency of in vivo purging with rituximab prior to autologous peripheral blood progenitor cell transplantation in B-cell non-Hodgkin’s lymphoma: a single institution study. Ann Oncol 2004; 15: 504–510. 99. Horwitz SM, Horning SJ. Rituximab in stem cell transplantation for aggressive lymphoma. Curr Hematol Rep 2004; 3:227–229. 100. Hagberg H, Gisselbrecht C. Randomised phase III study of R-ICE versus R-DHAP in relapsed patients with CD20 diffuse large B-cell lymphoma (DLBCL) followed by high-dose therapy and a second randomisation to maintenance treatment with rituximab or not: an update of the CORAL study. Ann Oncol 2006; 17(suppl 4): IV31–IV32. 101. Neumann F, Harmsen S, Martin S, et al. Rituximab long-term maintenance therapy after autologous stem cell transplantation in patients with B-cell non-Hodgkin’s lymphoma. Ann Hematol 2006; 85:530–534. 102. Lim SH, Zhang Y, Wang Z, et al. Maintenance rituximab after autologous stem cell transplant for high-risk B-cell lymphoma induces prolonged and severe hypogammaglobulinemia. Bone Marrow Transplant 2005; 35:207–208. 103. Press OW, Eary JF, Appelbaum FR, et al. Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 1995; 346:336–340. 104. Press OW, Eary JF, Gooley T, et al. A phase I/II trial of iodine-131-tositumomab (anti CD-20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas. Blood 2000; 96:2934–2942. 105. Shimoni A, Zwas ST, Oksman Y, et al. Yttrium-90-ibritumomab tiuxetan (Zevalin) combined with high-dose BEAM chemotherapy and autologous stem cell transplantation for chemo-refractory aggressive non-Hodgkin’s lymphoma. Exp Hematol 2007; 35:534–540. 106. Dillman RO. Radiolabeled anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoma. J Clin Oncol 2002; 20:3545–3557. 107. Hiddemann W, Dreyling MH, Forstpointner R, et al. Combined immuno-chemotherapy (R-CHOP) significantly improves time to treatment failure in first line therapy of follicular lymphoma: results of a prospective randomized trial of the German Low Grade Lymphoma Study Group (GLSG). Blood 2003; 102:104a (abstr 352). 108. Herold M, Haas A, Srock S, et al. Rituximab added to first-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: an East German Study Group Hematology and Oncology Study. J Clin Oncol 2007; 25:1986–1992.
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7 Radioimmunotherapy of Hematological Malignancies Tim Illidge and James Hainsworth Paterson Institute of Cancer Research, School of Medicine, University of Manchester, Manchester, U.K.
GENERAL INTRODUCTION—THE PRINCIPLES OF RADIOIMMUNOTHERAPY The use of monoclonal antibody (mAb) in routine clinical practice is now well established and has arguably led to some of the most significant improvements in outcome for patients in hematological malignancies as well as in a wide range of other malignancies including breast and bowel cancer (1–3). Although the single agent activity of most mAb has been modest when used in combination with other antitumor therapies, an additive or synergistic effect has been seen with both chemotherapy and radiotherapy (2). The impressive increases in clinical response rates seen with the combination of mAb and combination chemotherapy has led to not only highly impressive increases in response rates, relapse-free survival (RFS) but even overall survival (3). Radioimmunotherapy (RIT) is the administration of mAb or mAb-derived constructs, which are chemically conjugated to therapeutic radioisotopes targeted to tumor. Initially, mAbs were regarded simply as direct carriers for the radioisotope, which deliver systemically targeted cytotoxic radiation to areas of disease with relative sparing of normal tissue. It is, however, becoming clearer that the mAb effector mechanisms may also play an important additional role in killing lymphoma cells. The nature of RIT determines that its efficacy depends
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on a number of factors, including the properties of the targeted antigen (specificity, density, availability, shedding, and heterogeneity of expression), the tumor (degree of vascularization, blood flow, and permeability), the mAb (specificity, immunoreactivity, stability, and affinity), and the properties of chosen radioisotope (emission characteristics, half-life, and availability) (4). A wide variety of different mAbs, delivery schedules, radioisotopes, and doses of radioactivity have been used in RIT and have resulted in impressive durable partial and complete responses (PRs and CRs) in the treatment of non-Hodgkin’s lymphoma (NHL) (5). Two RIT drugs namely iodine-131 (131I)-tositumomab and yttrium-90 (90Y)-ibritumomab have been approved by the U.S. Food and Drug Administration (FDA) and 90Y-ibritumomab tiuxetan is approved by the European Union (EU). The use of RIT in leukemias is less well developed but the emerging data looks highly encouraging that RIT may play a useful role in the conditioning regimens for a wide variety of hematological malignancies as part of transplantation strategies. This chapter focuses on the current clinical indications of radioimmunoconjugates in hematological malignancy and provides an overview of the clinical trials and ongoing studies. Finally a perspective will be given of how the use of RIT in lymphoma and leukemia might develop in the next five years.
BRIEF DESCRIPTION OF PATHWAY(S) INVOLVED, ESPECIALLY PARTS RELEVANT TO THE TREATMENT Antigen Targeting The use of radiation therapy in the treatment of hematological malignancy has been well established and is highly effective if the disease is localized, as both lymphomas and leukemias are exquisitely sensitive to cell death by radiation. The systemic nature of the majority of lymphomas and leukemias, however, makes localized irradiation inappropriate for most patients. Therefore, the systemic delivery of RIT is a logical strategy given that these disseminated diseases are highly radiosensitive. The effective delivery of RIT requires the selection of a suitable tumor antigen target. Tumor-specific antigens would be the ideal targets for RIT, but such a degree of specificity is unusual. In practice, tumor-associated antigens, expressed abundantly on tumor cells as well as some normal tissues, represent the majority of potential targets. As most NHL are of B-cell origin, the pan-B-cell antigens such as human leukocyte antigen (HLA-DR), CD19, CD20, CD22, CD37, CD52, and MHC II have been extensively evaluated as targets for RIT (1,3,6–9). Table 1 shows the antigen characteristics that are considered ideal for RIT. From these initial investigations, the CD20 antigen emerged as having many of the characteristics thought to be important for a good tumor target and therefore targeting this antigen has dominated clinical RIT of lymphoma (10). The CD20 antigen is a transmembrane phosphoprotein that is expressed on mature B lymphocytes and to a lower degree on pre-B lymphocytes at a higher
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Table 1 Table of Characteristics Considered Ideal in an Antigen Target for RIT The characteristics of an ideal target antigen Tumor cell specific Highly expressed on tumor cells No tendency to mutation Not secreted or shed Not rapidly modulated on antibody binding Critical for target cell survival Not expressed on critical or nonrenewable host cells
density. The antigen is also expressed on greater than 90% of B-cell NHL. The CD20 complex does not internalize or shed from the cell surface and initiates signal transduction that triggers apoptosis through a caspase-dependent pathway (11). CD20 is highly expressed on the majority of B-cell lymphomas but is not expressed on stem cells or plasma cells and consequently following radiolabeled anti-CD20 mAb RIT, the B-cell pool is replenished over the next few months. Although most clinical RIT work has been targeted against the CD20 antigen, other B-cell antigens such as CD22 are still being actively investigated (12). As regards RIT in leukemia, CD45 and CD33 have been the most extensively investigated antigen targets. The CD45 antigen is the most broadly expressed of the known hematopoietic antigens. CD45 is a tyrosine phosphatase that is expressed in different isoforms ranging in molecular mass from 180 to 220 kD. CD45 is found on nearly all leukocytes, including lymphoid and myeloid precursors. Greater than 90% of acute myeloid leukemia (AML) biopsy samples and most acute lymphoblastic leukemia (ALL) biopsy samples show CD45 antigen expression, where cell surface antigen expression averages 200,000 copies per cell. Importantly in the context of RIT, the antigen does not internalize after mAb binding (13). CD33 is a 67-kDa type 1 transmembrane protein whose expression is restricted to early multilineage hematopoietic progenitors, myelomonocytic precursors, and more mature myeloid cells. CD33 is absent on normal pluripotent hematopoietic stem cells, though 85% to 90% of adult and pediatric cases of AML express CD33 (14). Therefore, CD33 has gained clinical importance as a suitable tumor-associated antigen and target for mAb-based AML therapies. The CD66 antigen is expressed at approximately 200,000 molecules per cell on normal myelopoietic cells from the promyelocyte onward but not on AML blasts. Fortunately for RIT, the CD66 molecule is neither internalized nor shed and aberrant expression is found on a significant fraction of CD10-positive ALL blasts (15). Radioisotopes Used in RIT The physical characteristics considered important for a radioisotope in RIT include half-life, type of radioactive emissions (a, b, or g), and ionization path
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length. Particle energy and mean path length in tissue are important determinants of therapeutic efficacy. The emission profile of the radioisotope not only determines its suitability for therapy, but also the toxicological profile of the radiopharmaceutical. The majority of clinical trials to date have used either 131I or 90Y because of their favorable emission characteristics, availability, and well-documented radiochemistry that permit reliable and stable attachment to mAbs. 131I has the advantage of a long history of successful use in the management of thyroid cancer and a well-documented safety profile. It is readily available, inexpensive, easily conjugated, and emits both b-particles with a path length of 0.8 mm and penetrating b-emissions. The g-photons enable uncomplicated imaging using a gamma camera for dosimetry purposes but also result in a significant nontargeted normal tissue radiation dose as well as radiation protection issues for visitors and medical/nursing staff. 90 Y offers a number of theoretical advantages over 131I, although the radioisotopes have not been directly compared by labeling the same mAbs described in this chapter with either isotope. 90Y is a pure b-emitter that produces higher energy radiation (2.3 MeV vs. 0.6 MeV) at a longer path length than 131I (5.3 mm vs. 0.8 mm). Radiolysis induces cellular damage in both the targeted lymphoma cells and neighboring cells. The increased path length would be expected to enhance the ‘‘cross fire’’ effect and could, therefore, be potentially advantageous in treating bulky, poorly vascularized tumors with heterogeneous antigen expression (4). This longer path length is likely, however, to increase the normal tissue dose when targeting microscopic disease for which the shorter b-particle path length of 131I may be preferable. The physical half-life of 90Y is 64 hours and decays to a stable (nonradioactive) form of zirconium (90Zr). The physical half-life of 64 hours approximates to the biological half-life of murine mAbs and the absence of penetrating g-emissions enables delivery as an outpatient (16). Additionally, if a cell internalizes 90Y, it is likely to be retained within the cell (12). In contrast, once 131I-conjugates are internalized by a cell, there is rapid dehalogenation of the free iodide and subsequent excretion of the iodinated products out of the cell, reducing desired tumor absorbed radiation dose and increasing normal tissue radiation exposure (17). The major disadvantages of 90Y relate to its greater expense, relatively limited availability, and requirement for chelation radiochemistry making radiolabeling a more complex procedure. 90Y does not emit g-photons and, therefore, there is a need to use indium-111 as a surrogate to obtain images for biodistribution and dosimetry studies. Rhenium-186 (186Re) and copper-67 (67Cu) are both b-emitters and have physical and chemical properties that make them attractive alternatives to either 131I or 90Y. Nevertheless, their current limited availability has meant that these radioisotopes have received limited clinical use (18). a-Emitters produce a helium nucleus particle of very high energy but with a very short path length. The high linear energy transfer (LET) radiation of
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a-emitters may be lethal to cells with a single collision; however, the very short path length means that the isotope must be adjacent to, or internalized by, the cell to be effective and is likely to have little or no cross-fire effect. The suitability of a-emitters, therefore, appears limited to readily accessible tumors such as leukemia cells confined to the blood or bone marrow. The short half-life of a-emitters [e.g., astatine-211 (211At)—7 hours or bismuth-213 (213Bi)— 45 minutes] complicates the radiopharmaceutical preparation, meaning that such radioisotopes are likely to require generation on the same site as delivery in the clinic. Despite this logistical hurdle, early clinical data in the treatment of leukemia appear extremely promising (19,20). In practice, the choice of the optimal radioisotope for RIT remains controversial, with proponents advocating the relative merits of 131I, 90Y, 186Re, 67 Cu, and a-emitters such as 211At (16). Comparative studies are difficult to conduct and scientifically robust randomized human trials have not been performed. The ideal properties of a radioisotope for RIT remain unclear and it is likely that the optimal radioisotope for a particular situation will depend upon the bulk and type of tumor being targeted. An important area of potential future research will be to define the optimal radioisotope, or cocktail of isotopes, required for different tumor sizes. BRIEF REVIEW OF RELEVANT TRANSLATIONAL RESEARCH Factors Affecting the Therapeutic Efficacy of RIT in Lymphomas Although RIT has emerged as a highly effective treatment for NHL, the underlying mechanisms of action and in particular the interaction of tumor irradiation and mAb signaling in RIT are still poorly understood (16,21). There remain a number of important questions where further work is required to address important issues required to optimize RIT delivery and where further translational research may further inform our current knowledge. These areas include 1. The optimal predose of cold mAb 2. The relative contribution of targeted radiation and mAb effector mechanisms to the overall response and the mechanisms involved in the durable responses seen in some patients 3. Defining whether a tumor radiation dose response exists in RIT for lymphomas and leukemias Predosing of mAb in RIT There are several factors that may theoretically limit lymphoma targeting of radiolabeled pan-B-cell mAb in RIT. These include the complex formation of administered mAb with free-circulating target antigen, the cross reactivity with
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antigen-positive circulating lymphoma cells, normal B cells in the blood or spleen or nonlymphoid tissues, and finally the nonantigenic binding of mAb such as binding by Fc arm of a mAb. Poor tumor targeting of a radioimmunoconjugate leads to lower tumor to normal tissue radiation-dose ratios resulting in potentially reduced therapeutic efficacy. In order to improve the biodistribution of radiolabeled mAb in RIT, it has become the established practice to give a predose of ‘‘cold’’ or unlabeled antiCD20 mAb prior to the therapeutic dose of the anti-CD20 radioimmunoconjugate (22). The predose is considered to prolong the circulating half-life of the radiolabeled mAb, block ‘‘non-specific’’ binding sites (e.g., circulating and splenic B cells), and result in increased tumor retention of the labeled mAb. Buchsbaum et al. investigated whether a predose of anti-B1 improved the delivery of a subsequent radiolabeled mAb to tumor using in vivo preclinical human xenograft models (23). The anti-B1 (anti-CD20) pan-B-cell mAb that is reactive with human B-cell lymphomas but is not reactive with host mouse B cells was used. A predose of unlabeled anti-B1 was found to significantly increase the tumor-uptake of the subsequent radiolabeled anti-B1 although this improvement in tumor targeting appeared to plateau at the highest predoses of unlabeled anti-B1. Relative Contribution of Antibody Effector Mechanisms and Targeted Radiation to Therapy RIT has emerged as an effective treatment for lymphoma; however, the underlying mechanisms are poorly understood. By using different syngeneic murine B-cell lymphoma models, the relative contributions of mAb and targeted radiation to the clearance of tumor in vivo have been investigated (24). There is now substantial evidence that mAbs can form an active component of RIT and that mAb effector mechanisms may be important in the clearance of tumor in vivo. Although the exact in vivo mechanisms of tumor killing by anti-CD20 mAb remain incompletely understood, preclinical data have suggested that the action of rituximab may include mAb-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and the direct induction of apoptosis through cell surface mediated downstream signal transduction (25). Cragg and Glennie reported that a panel of anti-CD20 mAbs (rituximab, 1F5, and anti-B1) acts through distinctively different mechanisms in the therapy of two lymphoma xenograft models (25). Rituximab and 1F5 redistribute CD20 into membrane rafts and are bound efficiently by the complement component C1q and deposit C3b resulting in CDC, which forms the major therapeutic effect of these two mAbs. In contrast, complement depletion had no effect on the potent therapeutic activity of anti-B1 (tositumomab), a mAb that does not redistribute CD20 into membrane rafts, bind C1q, or cause efficient CDC. F(ab’)2 fragments of anti-B1 (tositumomab), but not 1F5, were observed to be able to provide substantial
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immunotherapy, indicating that non-Fc-dependent mechanisms are involved in the tositumomab action. In accordance with this, tositumomab was shown to induce much higher levels of apoptosis than rituximab and 1F5, suggesting that while complement is important for the action of rituximab and 1F5, this is not the case for tositumomab, which more likely functions through its ability to induce downstream signal transduction that results in apoptosis. The relative importance of mAb effector mechanisms and targeted radiation are difficult to measure and it is practically impossible to dissect the action of the two components in clinical RIT. However, preclinical studies using welldefined syngeneic animal models have helped to further clarify the relative contributions of mAb effector mechanisms and targeted radiation. The ability of some anti-B-cell mAbs to improve survival with targeted radiotherapy appeared to correlate with their ability to initiate intracellular signal transduction. Together these data illustrate that using one mAb to target radiation to tumor and a second to induce cell signaling may be an effective new strategy in lymphoma RIT. Defining Whether a Tumor Radiation Dose Response Exists in RIT for Lymphomas and Leukemias There is currently little preclinical data that demonstrate whether a radiation dose response exists. Using syngeneic B-cell lymphoma models, Du et al. have demonstrated that with mAbs used solely as vectors to deliver radiation to tumor, there does appear to be a radiation dose response (24). To date, despite the high response rates seen in lymphoma RIT, clinical dosimetry studies have thus far failed to show a consistent dose-response relationship (4). More recently, the Michigan group has concluded that there could be a radiation dose response at least for 131I-tositumomab (22,26); however, their conclusions differ to others (27,28). Postema, for example, argues that none of the RIT dosing methods use tumor dosimetry to determine the dose administered to patients because the myelotoxicity of radiolabeled mAb will limit the increments of radioactivity dose but not the tumor absorbed dose (27). More recently, Goldenberg and colleagues commented that because RIT has two potentially therapeutic arms, namely radiation and mAb mechanisms, poor radiation targeting does not exclude a good therapeutic response from the mAb (28). Preclinical Leukemia Modeling The group from Memorial Sloan-Kettering Cancer Center initially described the preclinical administration of 213Bi-HuM195 (anti-CD33) constructs (29). The 213 Bi-HuM195 construct was rapidly internalized into the cell in a time-dependent manner ranging from 50% at 1 hour to 65% at 24 hours. 205Bi/206Bi-labeled constructs (bismuth isotopes with a longer physical half-life) were stable for
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at least two days in vitro in the presence of human serum at 378C. After injection into mice, there was no observed uptake or loss of bismuth to tissues not expressing the CD33 antigen, including the kidney which has a high affinity for free bismuth. Mice were treated by intraperitoneal injection of 213Bi-HuM195. Doses ranging from 18.5 to 740 MBq/kg showed no toxicity, but at the higher dose of 2590 MBq/kg, two-thirds of the mice died within two weeks and a third of the mice showed significant reductions in white blood cell counts. Leukemia cell killing in vitro with 213Bi-HuM195 showed dose- and specific activity– dependent killing of CD33 positive HL60 leukemic cells; approximately 50% killing was observed when two bismuth atoms (50 fM radiolabeled mAb) were initially bound onto the target cell surface. The authors concluded that the physical and biochemical properties are suitable for initial RIT studies in humans. The Seattle group has adopted a different targeting strategy and has tested myeloablative doses of 131I-anti-CD45 mAb prior to autologous stem cell transplantation (ASCT) (30). This strategy was developed to reduce the overall systemic radiation dose from total body irradiation (TBI) and to increase the targeted radiation dose delivered to hematopoietic tissues with the goal of decreasing relapse rates without increasing toxicity. To determine whether the 131I-anti-CD45 mAb was able to provide sufficient immunosuppression for efficient transplantation across allogeneic barriers, T-cell-depleted BALB.c marrow was transplanted into H2-compatible B6-Ly5a mice after 131I-30F11 (rat anti-murine CD45) mAb with or without varying dose levels of TBI. Groups of five or six recipient mice per 131I- or TBI-dose level per experiment were given tail vein injections of 100 mg of 131I30F11 mAb four days before marrow infusion, with or without TBI on day 0. Engraftment, defined as 50% or greater donor B cells at three months posttransplant, was determined by two-color flow cytometric analysis of peripheral blood granulocytes, T cells, and B cells using mAbs specific for donor and host CD45 allotypes and for CD3. Donor engraftment of 80% or more recipient mice was achieved with either 8 Gy of TBI or 27.75 MBq (0.75 mCi) of 131I30F11 mAb, with the radioimmunoconjugate delivering an estimated 26 Gy to bone marrow. Subsequent experiments determined the dose of TBI alone or TBI plus 27.75 MBq (0.75 mCi) of 131I-30F11 mAb necessary for engraftment in recipient mice that had been presensitized to donor antigens before transplant, a setting requiring more stringent immunosuppression. Engraftment was seen in 80% or more of presensitized recipients surviving after TBI (14 to 16 Gy or 12 to 14 Gy) and 27.75 MBq (0.75 mCi) of 131I-30F11 mAb. However, only 28 of 69 (41%) presensitized mice receiving 10 to 16 Gy of TBI alone survived. The authors suggest that targeted radiation delivered by 131Ianti-CD45 mAb provides sufficient immunosuppression to replace an appreciable portion of the TBI dose used in matched sibling hematopoietic stem cell transplant.
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REVIEW OF COMPLETED, ONGOING, AND PLANNED CLINICAL STUDIES Nonmyeloablative RIT in Lymphomas Clinical RIT trials in NHL differ in terms of eligibility criteria, mAb and radioisotope used, dose, number of treatments, doses of unlabeled mAb preinfused or coinfused, and the biodistribution or dosimetry estimations required for administration of a therapeutic dose of radiolabeled mAb. Nevertheless, virtually all clinical studies performed to date have shown high response rates in for RIT in NHL and have been well reviewed (4,31–35). DeNardo et al. initially pioneered RIT for NHL with 131I-anti-HLA-DR mAb (Lym-1) (7). The efficacy of escalating fractionated doses of 131I-Lym-1 ranging from 1480 to 3700 MBq/m2 (40–100 mCi/m2) resulted in an overall response rate (ORR) of 52% in 21 treatment courses administered to 20 patients, with seven patients (33%) achieving CR, and four patients (19%) achieving PR (7). Goldenberg et al. used a 131I-LL2 (anti-CD22) mAb to treat a variety of Bcell lymphomas. In one of their trials, 4 out of 17 patients achieved objective remission including 1 CR (36). In another trial, 90Y-LL2 was administered to seven patients with B-cell lymphomas, two of whom achieved PR (Table 2) (36). Impressive responses have been observed in all of the clinical trials using 90 Y-ibritumomab tiuxetan and 131I-tositumomab in relapsed B-cell lymphomas. Although both 131I-tositumomab and 90Y-ibritumomab tiuxetan bind to the same CD20 antigen, tositumomab binds to a unique epitope of CD20 (37). The radioisotopes also have important differences in their emission characteristics. Table 3 compares the main characteristics of 131I-tositumomab and 90Yibritumomab tiuxetan.
Table 2 Results of a Randomized Controlled Trial of 90Y-Ibritumomab Tiuxetan Vs. Rituximab in Relapsed or Refractory Low-Grade or Transformed Follicular B-Cell NHL
Overall response (%) Median DR (mo) CR, Cru (%) Median DR (mo) Ongoing CR, Cru (%) Median DR (mo) Range Source: From Refs. 43,79.
Phase I/II (n ¼ 51)
Phase II (n ¼ 30)
Phase III (n ¼ 73)
73 11.7 29 28 19 62.1 60þ to 66þ
83 11.5 47 23 14 41.2 40þ to 42þ
80 13.9 34 23 32 42.2 33þ to 48þ
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Table 3 Characteristics of 131I-Tositumomab (Bexxar) and 90Y-Ibritumomab Tiuxetan (Zevalin) 131
U.S. trade name Monoclonal antibody Chelation Isotope Isotope emissions b-energy b-particle path length Isotope half-life g-energy Radiation protection measures Isotope excretion Normal tissue uptake Predose (unlabeled mAb) Dose
90
I-tositumomab
Bexxar tositumomab (anti-B1)-murine Simple 131 I g and b 0.606 MeV 0.8 mm 8 days 0.364 MeV 4- to 6-day inpatient stay in shielded room Renal (variable) Thyroid (preblocked with KI) tositumomab (450 mg/patient) 75 cGy whole body dose Dosimetric dose obligatory
90
Y-ibritumomab tiuxetan
Zevalin ibritumomab (2B8)-murine More complex 90 Y b only 2.293 MeV 5.3 mm 2.6 days None Outpatient Limited Bone rituximab (250 mg/m2) 2 15 MBq/kg (0.4 mCi/kg) Dosimetric dose not required Dose reduction for thrombocytopenia
Y-ibritumomab tiuxetan consists of a monoclonal IgG1K anti-CD20 mAb, the murine parent immunoglobulin of rituximab, covalently attached to a metal chelator molecule (tiuxetan, an isothiocyanatobenzyl derivative of the polyaminocarboxylic acid DTPA), which stabilizes the mAb-isotope complex for delivery to the lymphoma site (38). Biological half-life elimination of 90Yibritumomab tiuxetan is 30 hours. More than 90% of the b-radiation is absorbed within a 5-mm proximity (corresponding to a diameter of 100 to 200 cells) of the radiation source. This facilitates highly targeted delivery of radiation without the need for patient isolation or shielding (16). The tiuxetan chelator molecule provides a stable link between the mAb and the radioisotope, and, therefore, free isotope clearance rates are minimal and predictable with 7.3% 3.2% of the radiolabeled activity being excreted in the urine over seven days (39). Consequently 90Y-ibritumomab tiuxetan may be administered on an outpatient basis. Figure 1 outlines the 90Y-ibritumomab tiuxetan therapeutic regimen. Four clinical trials, including three phase I/II and one randomized study formed the basis of the FDA submission for 90Y-ibritumomab tiuxetan. The initial phase I/II study demonstrated that the dose limiting toxicity was myelotoxicity (39). The maximum-tolerated dose (MTD) was identified as 15 MBq/kg (0.4 mCi/kg) to a maximum of 1184 MBq (32 mCi) for patients with a baseline platelet count of greater than or equal to 150 109/L and 11.1 MBq/kg
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Figure 1 Treatment regimen for 90Y-ibritumomab tiuxetan. Intergroup European firstline follicular lymphoma phase III study. Chemotherapy was left to the discretion of the treating physician, e.g., chlorambucil, CHOP, CVP, fludarabine, CVP-R. **n ¼ 410 (accrual completed on January 2005).
(0.3 mCi/kg) for patients with baseline platelet counts of less than 150 109/L but greater than or equal to 100 109/L. In this study, a high ORR for the intentto-treat population (n ¼ 51) was seen at 67% (26% CR; 41% PR); for low-grade disease (n ¼ 34), 82% (26% CR; 56% PR); for intermediate-grade disease (n ¼ 14), 43%. A phase II study of patients with mild thrombocytopenia (baseline platelet count of 100 109/L to 150 109/L) was conducted using the reduced dose of 11.1 MBq/kg (0.3 mCi/kg). The ORR was 83% (37% CR, 6.7% CR unconfirmed, and 40% PR). Kaplan–Meier estimated median time to progression (TTP) was 9.4 months (range, 1.7–24.6). In responders, Kaplan–Meier estimated median TTP was 12.6 months (range, 4.9–24.6). Toxicity was primarily hematologic, transient, and reversible. The incidence of grade 4 neutropenia, thrombocytopenia, and anemia was 33%, 13%, and 3%, respectively. The conclusions from this study were that reduced-dose ibritumomab tiuxetan is safe and well tolerated and has significant clinical activity in this patient population (40). A further single arm phase II study of 90Y-ibritumomab tiuxetan was undertaken to examine the efficacy of 90Y-ibritumomab tiuxetan in a group with rituximab refractory disease (41). Fifty-four heavily pretreated patients with follicular lymphoma (FL) were recruited who were refractory to or progressed after rituximab. The trial showed an ORR of 74% and a CR rate of 15%, despite a median of four prior therapies and 73% of patients having bulky disease (5 cm diameter). Kaplan–Meier-estimated DR was 6.4 months, with a TTP of 6.8 months in all patients and 8.7 months in responders. The randomizedcontrolled trial has been described earlier and compared 90Y-ibritumomab tiuxetan with rituximab in relapsed or refractory low-grade B-cell NHL. This confirmed that 90Y-ibritumomab tiuxetan results in superior ORR and CR rates to those seen with the ‘‘naked’’ mAb, rituximab. A randomized phase III trial including 143 patients with relapsed or refractory low grade, follicular, or transformed NHL compared efficacy of a single dose of 15 MBq/kg (0.4 mCi/kg) 90Y-ibritumomab tiuxetan with rituximab (375 mg/m2 once weekly for 4 weeks) (42). Response rates were significantly higher in the 90Y-ibritumomab tiuxetan arm with an ORR of 80%
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versus 56% (p ¼ 0.002) and a CR rate of 30% versus 16% (p ¼ 0.004). Subgroup analysis revealed a superior benefit for patients with follicular histology with an ORR of 86% versus 55% (p < 0.001) and a significantly increased (p < 0.04) time to treatment progression for this subgroup. The overall TTP was, however, not different in both treatment groups, but patients treated with 90Y-ibritumomab tiuxetan showed a trend toward longer median DR (14.2 months vs. 12.1 months) and more often achieved responses lasting longer than six months duration (64% vs. 47%). A recent retrospective analysis suggests that treatment with 90Yibritumomab tiuxetan is associated with higher response rates and longer DR when used earlier in the therapy schedule (43). An integrated analysis of 211 patients treated in clinical trials compared efficacy and safety of 90Y-ibritumomab tiuxetan in patients with one prior therapy (n ¼ 63) to patients who received greater than or equal to two prior therapies (n ¼ 148). Patients receiving 90Y-ibritumomab tiuxetan as second-line therapy had greater ORR (86% vs. 72%; p ¼ 0.051) and CR/CRu (CR unconfirmed) rates (49% vs. 28%; p ¼ 0.004) and a significantly longer median time to disease progression (TTP) (12.6 months vs. 7.9 months; p ¼ 0.038). In CR/CRu patients, the median TTP (23.9 months vs. 15.6 months; p ¼ 0.0442) and median DR (22.8 months vs. 14.6 months; p ¼ 0.0429) were both significantly increased in patients with only one prior therapy (n ¼ 53). A large European intergroup study of 90Y-ibritumomab tiuxetan therapy of previously untreated FL has now completed accrual with over 400 patients recruited. Patients were treated initially with chemotherapy (the physician’s choice) and then randomized to 90Y-ibritumomab tiuxetan or no further treatment. The data from this study may provide data in determining whether 90Yibritumomab tiuxetan has a potential beneficial role after primary chemotherapy. There was, however, relatively small numbers of patients treated in the latter part of the study who received rituximab in combination with chemotherapy regimens, which is now a widely adopted approach. Therefore, the additional role of 90 Y-ibritumomab tiuxetan RIT after full course rituximab, chemotherapy combinations will probably not be answered by this study. Shipley and colleagues performed an evaluation of the efficacy of shortduration R-CHOP followed by 15 MBq (0.4 mCi) 90Y-ibritumomab tiuxetan as a first-line treatment of patients with FL (44). The study was a multicenter phase II trial that recruited 42 patients in which 39 completed the entire planned therapy. Of the 40 patients that received chemotherapy, 12 (30%) were found to have a CR/Cru and 28 (70%) showed a PR. After 90Y-ibritumomab tiuxetan RIT, 26 patients (66%) showed a CR/Cru, 12 patients (31%) showed a PR and 1 patient (3%) showed disease progression. The group showed that treatment with 90Yibritumomab tiuxetan after chemotherapy/rituximab increased the CR rate from 30% to 66%. Actuarial progression-free survival (PFS) after one year was 98% (38 patients at risk) and after two years was 85% (15 patients at risk). Clinical responses have also been observed for transformed follicular and relapsed diffuse large B-cell lymphoma (DLBCL) when treated with
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Y-ibritumomab tiuxetan. Within an initial phase I/II study reported a response rate of 58% with a 33% CR rate in a group of just 12 patients that had relapsed following two previous chemotherapy regimens that included CHOP (42). A prospective, single-arm, open-label, nonrandomized, multicenter phase II trial was, therefore, undertaken to evaluate the efficacy and safety of 90Yibritumomab tiuxetan in patients over 60 years of age with relapsed or primary refractory DLBCL not suitable for ASCT. Patients were divided into two groups, with firstly those previously treated with chemotherapy alone (group A, n ¼ 76) and secondly those previously treated with chemotherapy and rituximab (group B, n ¼ 28) (45). All patients received a single dose of 15 MBq/kg (0.4 mCi/kg) of 90Y-ibritumomab tiuxetan up to a maximum dose of 1184 MBq/kg (32 mCi). In total, 103 patients could be evaluated for treatment efficacy and 104 for safety. An ORR of 44% was observed in the entire study population. In Group A, the ORR was over 50%. In Group B, where 37% of patients were refractory to rituximab-CHOP, the ORR was 19%. Adverse events (AEs), with the exception of hematological AEs, were generally mild (grade 1/2) and the incidence of severe infection was low, with only 7% of patients hospitalized for infection during the study. The results of this study were encouraging and clinical trials are now underway in the United States or at an advanced stage of development in the EU to integrate 90Y-ibritumomab tiuxetan into a front-line treatment for DLBC alongside rituximab-chemotherapy schedules. Tositumomab was the first mAb to be produced against a B-cell antigen (46). The 131I-tositumomab regimen is completed within one to two weeks and consists of a tracer dose of the radioimmunoconjugate followed by the therapeutic dose 7 to 14 days later. Each infusion of 131I-tositumomab is preceded by an infusion of a predose of 450 mg ‘‘cold’’ or unlabeled tositumomab and the therapeutic regimen is outlined in Figure 2. Whole body gamma camera imaging is performed three times over the week following the trace labeled infusion to calculate the whole body half-time and the dose required for the therapeutic infusion to deliver a 65 to 75 cGy whole body dose (WBD) (usually 3700 to 5550 MBq (100–150 mCi) (47). Dose adjustments to 65 cGy were made for a baseline platelet count of 100,000/mm3 to less than 150,000/mm3 and for obesity.
Figure 2 Treatment regimen for
131
I-tositumomab.
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Kaminski and colleagues initially conducted a series of trials at the University of Michigan using the 131I-tositumomab, for the treatment of relapsed FL (48,49). In a pivotal study, 60 extensively pretreated patients were given a single administration of 131I-tositumomab (6). Disease responses were compared to their previous responses to chemotherapy for follicular or transformed FL. A PR or CR was observed in 39 patients (65%) after iodine 131I-tositumomab, compared with 17 patients (28%) after their last qualifying chemotherapy (LQC) (p < 0.001). 131I-tositumomab therapy was shown to provide greatly superior RFS compared to the LQC. Recently an integrated efficacy analysis of the five clinical trials presented to the U.S. FDA for registration purposes of the 131I-tositumomab regimen in patients with relapsed or refractory low-grade, follicular, or transformed lowgrade NHL (50). This integrated analysis included 250 patients and response rates in the five trials ranged from 47% to 68% with CR rates between 20% to 38%. With a median follow-up of 5.3 years, the five-year PFS was 17%. Of the 250 patients, 81 (32%) had a TTP of more than or equal to one year (termed durable response population). For the durable response population, 44% had not progressed at more than or equal to 2.5 to more than or equal to 9.5 years and had a median DR of 45.8 months. The median duration of CR was not reached. The durable response population had many poor prognostic characteristics, including bone marrow involvement (41%), bulky disease greater than or equal to 5 cm (49%), and transformed histology (23%). Forty-three percent of the patients had been treated with more than four prior therapies and 36% had not responded to their most recent therapy. The authors concluded that the 131I-tositumomab therapeutic regimen produces high response rates in patients with relapsed or refractory low-grade, follicular, and transformed low-grade NHL, with a sizable subgroup of patients achieving long-term durable responses. Impressive response rates have also been seen in patients that were refractory to rituximab, which were subsequently treated with 131I-tositumomab. Horning and colleagues used 131I-tositumomab to treat 40 patients with lowgrade NHL, 72% of which had received four or more previous lines of therapy and 60% of which had failed to respond to rituximab (51). An ORR of 68% with a CR rate of 30% was noted and a median DR of 14.7 months reported. Of the 12 complete responders, 9 remained in CR at the time of presentation with a range of 12 to 26 months. More recently, an analysis including 230 patients treated with 131I-tositumomab was made. Independently assessed durable CRs were noted with similar frequency in patients with rituximab-refractory disease (28%) and rituximab na€ive patients all of whom had chemotherapy refractory disease (23%). With a median follow-up of 4.6 years, 75% of patients with durable CR continue in complete remission (52). Kaminski et al. have shown highly promising results in the front-line treatment of previously untreated low-grade FL using 131I-tositumomab (32). An encouraging ORR of 95% was seen with 75% achieving CR. PCR (polymerase
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chain reaction) was used to detect rearrangement of the BCL2 gene, which revealed molecular responses in 80% of assessable patients who had a clinical CR. The most recent update included 76 patients with a median follow-up of 5.1 years. The actuarial five-year PFS for all patients was 59%, with a median PFS of 6.1 years. Hematological toxicity was moderate, with no patient requiring transfusion or granulocyte colony stimulating factor (G-CSF) (32,53); though 48 out of 76 (63%) patients developed detectable human anti-mouse antibody (HAMA) responses after a single course of treatment with 131I-tositumomab. More recently Press and colleagues conducted a phase II trial in untreated FL that consisted of six CHOP cycles followed up four to eight weeks later by 131 I-tositumomab (54). A cohort of 90 previously untreated eligible patients with advanced-stage FL tolerated the treatment well. Reversible myelosuppression was the main AE and was more severe during CHOP chemotherapy than after RIT. The ORR over the entire treatment regimen was 90%, including 67% CR and 23% PR. Of the 47 fully evaluable patients who achieved less than a CR with CHOP, 27 (57%) improved their remission status after predosed tositumomab and 131I-tositumomab. With a median follow-up of 2.3 years, the two-year PFS was estimated to be 81%, with a two-year overall survival of 97%. Having established the feasibility and the efficacy of this approach, a randomized phase III trial is currently being undertaken to compare this approach of chemotherapy followed by RIT against immunochemotherapy with six cycles of CHOP-R. CONTRIBUTION OF TARGETED RADIATION TO CLINICAL RESPONSE The contribution of targeted radiation to the overall responses seen in RIT has been addressed with two randomized studies comparing the radioimmunoconjugates 90Y-ibritumomab tiuxetan and 131I-tositumomab with the unlabeled mAbs (31,42). Both studies have shown greatly superior clinical responses of RIT over the unlabeled mAb. The 90Y-ibritumomab tiuxetan versus rituximab is described above and the second study compared treatment outcomes for unlabeled tositumomab (predose) and 131I-tositumomab to an equivalent total dose of unlabeled tositumomab involved 78 patients with refractory/relapsed low-grade NHL (31). The investigators reported an ORR of 55% versus 19% (p ¼ 0.002) with a CR 33% versus 8% (p ¼ 0.012) in 131I-tositumomab versus unlabeled tositumomab groups, respectively. The median duration of the ORR was not reached for 131I-tositumomab versus 28.1 months for unlabeled tositumomab. The median duration of CR was not reached in either arm, and the median TTP was 6.3 months versus 5.5 months (p ¼ 0.031), respectively. Although hematological toxicity was more severe and nonhematological AEs were more frequent after 131I-tositumomab than after tositumomab alone, there were no serious infectious or bleeding complications. The frequency of developing HAMA was similar in the two arms of 27% (131I-tositumomab group) versus 19% (tositumomab-alone group), respectively. This study demonstrated
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that although unlabeled tositumomab showed single agent activity, the conjugation of 131I to tositumomab significantly enhanced the therapeutic efficacy (31). Toxicity and Safety of RIT Adverse Effects of
90
Y-Ibritumomab Tiuxetan
Myelosuppression was found to be the dose-limiting toxicity of RIT for both Y-ibritumomab tiuxetan and 131I-tositumomab. An analysis of all patients treated in 90Y-ibritumomab tiuxetan trials (n ¼ 261) indicated that 28% will experience grade 4 neutropenia and 8% will experience grade 4 thrombocytopenia (55). No significant differences in the incidence of hematological and nonhematological grade 3 to 4 AEs were observed in patients 65 years or older as compared with younger patients. However, despite concerns about the potential of an increased risk of radiation-induced secondary hematological malignancies, the observed rate of secondary myelodysplastic syndromes (MDS)/AML was less than 1% (5/348), which is comparable with a similar patient population treated with alkylating agents (55). Safety data from the four clinical trials were reviewed retrospectively in an integrated analysis encompassing 349 patients of whom 345 patients (99%) completing 90Y-ibritumomab (55). Although 80% of patients reported nonhematological AEs, those were generally mild to moderate in severity with asthenia, nausea, and chills being the most common events that were considered to have probable or possible association related to the treatment. Only 11% (39 patients) of all patients experienced grade 3 to 4 nonhematological toxicity. For 90Y-ibritumomab tiuxetan grades 1 to 2 and 3 to 4 thrombocytopenia occurred in 37% and 63% of patients, respectively. Of the patients with grade 3 to 4 thrombocytopenia, 87% recovered to greater than or equal to 50 109/L by week 12 following therapy. Neutropenia grade 3 to 4 was observed in 30% of patients, with 90% recovering to greater than 1.0 109/L within 12 weeks posttreatment. For patients, who received G-CSF support, the median duration of neutropeania was reduced from 27 to 19 days. Grades 3 and 4 anemia developed in 13% and 4% of patients, respectively. Of all patients, 22% required platelet transfusions and 20% required red blood cell transfusions. 90Y-ibritumomab tiuxetan is dosed according to the patient’s body weight and baseline platelet counts. For patients with platelet counts greater than or equal to 150,000/mm3, 5 MBq/kg (0.135 mCi/kg) body weight is given up to a maximum permissible dose of 1200 MBq (32.4 mCi). For patients with platelet counts of 100 to 149 109/L, 90Y-ibritumomab tiuxetan is dosed at 11 MBq/kg (0.297 mCi/kg), up to a maximum allowable dose of 1200 MBq (32.4 mCi). The incidence of severe thrombocytopenia and neutropenia correlated significantly with degree of bone marrow involvement and platelet counts at baseline, underscoring the importance to exclude patients with greater than or 90
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equal to 25% bone marrow infiltration and inadequate bone marrow reserve. Patients who had more than two prior chemotherapies were twice as likely to develop grade 4 thrombocytopenia, whereas number of prior chemotherapies did not correlate with longer median duration of neutropenia, thrombocytopenia, and anemia. Total number of B cells and levels of IgM declined after treatment but recovered after six to nine months. The median T-cell counts and levels of IgG and IgA remained stable following treatment with 90Y-ibritumomab tiuxetan. Most importantly, treatment with 90Y-ibritumomab tiuxetan was not associated with an excess rate of infections. In fact, incidence of infectious complications was low with upper respiratory and urinary tract infections occurring in 5% and 7% of patients, respectively. Only 8% of all patients received antibiotic therapy during the treatment period. Adverse Effects of
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I-Tositumomab RIT
Short-term nonhematological AEs with 131I-tositumomab administration are generally mild and include typically fatigue, nausea, fever, vomiting, pruritus, and rash, which usually respond well to antihistamines. Follow-up examinations include weekly blood and platelet counts following RIT until hematological recovery occurs. 131 I-tositumomab is susceptible to dehalogenation and, therefore, a preadministration of ‘‘cold’’ iodine is given starting 72 hours prior to the dosimetric dose and again at 14 days after the therapeutic dose in order to block the thyroid from radioactive iodine uptake. Despite these attempts to prevent thyroid uptake of 131I, hypothyroidism appears one of the most consistent long-term adverse effects after 131I-mAb treatment. Hypothyroidism can, however, be easily managed with thyroid stimulating hormone (TSH) replacement once detected and screening is an important component of follow-up. Zelenetz reviewed the multicenter RIT trials using 131I-tositumomab in NHL patients and reported that elevated TSH was observed in 5 out of 59 patients in the phase I study (56). However, after a myeloablative dose of 131I-tositumomab, elevated TSH was observed in 59% of patients (57). HAMA reactions appear to be substantially lower in previously treated NHL patients compared with the rates experienced in solid tumor RIT (4). The incidence of HAMA was observed to be approximately 10% after 131I-tositumomab treatment. The impact of administration of the 131I-tositumomab therapeutic regimen on circulating CD20 positive cells was assessed in two clinical studies (32,49), one conducted in chemotherapy na€ive patients and one in heavily pretreated patients. The assessment of circulating lymphocytes did not distinguish normal from malignant cells. Consequently, assessment of recovery of normal B-cell function was not directly assessed. Lymphocyte recovery began at approximately 12 weeks following treatment. Among patients who had CD20 positive cell counts recorded at baseline and at six months, 8 of 58 (14%) chemotherapy na€ive
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patients had CD20 positive cell counts below normal limits at six months and 6 of 19 (32%) heavily pretreated patients had CD20 positive cell counts below normal limits at six months. There was no consistent effect of the 131I-tositumomab therapeutic regimen on posttreatment serum IgG, IgA, or IgM levels. Secondary malignancy following RIT has fortunately proved to be rare (32,56). Recently, 1071 RIT-treated patients were assessed for treatmentrelated MDS/AML (58). Among these patients, 995 with low-grade and transformed low-grade NHL had been previously treated with a median of three therapies (range, 1 to 13 therapies) prior to RIT. As part of their initial therapy for FL, 76 patients received RIT. For the previously treated patients, the median follow-up from the diagnosis of NHL and RIT was six years and two years, respectively; for the patients who received RIT as their initial therapy, the corresponding median follow-up times were 5.6 years and 4.6 years, respectively. Of the 995 previously treated patients, 35 (3.5%) cases of treatment-related MDS/AML were reported, and 13 cases were confirmed to have developed MDS/AML following RIT. This incidence was found to be consistent with that expected on the basis of patients’ prior exposure to chemotherapy. With a median follow-up approaching five years, no case of treatment-related MDS/AML has been reported in the 76 patients, receiving 131 I-tositumomab as their initial therapy (58).
Subsequent Therapy After RIT Data are emerging to suggest that subsequent therapy can be administered after I-tositumomab and 90Y-ibritumomab tiuxetan (59,60). Chemotherapy or ASCT after prior therapy with 90Y-ibritumomab tiuxetan also appears feasible and reasonably well tolerated. The toxicity with subsequent therapy seems similar to that in patients not treated with 90Y-ibritumomab tiuxetan. 131
RIT After Autologous Transplantation Despite initial concerns, preliminary data suggest that patients after prior bone marrow transplant or stem cell support can be safely treated as long as a significant dose reduction is made. Initially for 90Y-ibritumomab tiuxetan, a reduced dose of 11.1 MBq/kg, was suggested; however, recent data has suggested that a full dose of 15 MBq/kg can be tolerated, although the number of patients in this study was only eight (61). For 131I-tositumomab a WBD of 65 cGy is feasible (62). The delivery of either radioimmunoconjugate after high dose chemotherapy and ASCT is currently outside of the licensed indication. This should not be confused with the application of RIT as part of the conditioning regimen used as part of ASCT, where there is increasingly compelling clinical safety and efficacy data.
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Myeloablative RIT in NHL Myeloablative RIT has the theoretical advantage of delivering higher doses of radiation and overcoming the dose-limiting myelotoxicity with autologous stem cell and bone marrow rescue (56,57). Press and colleagues in Seattle pioneered the myeloablative approach, demonstrating the feasibility of using high activity doses of 131I-anti-B-cell mAbs (mB-1 (anti-CD37) or anti-B1 (anti-CD20) with either ASCT or PBSCT (57). Initially the group assessed the biodistribution, toxicity, and efficacy of high doses of radiolabeled anti-B1 in 43 patients with B-cell lymphoma. The dose limiting toxicity was found to be cardiopulmonary and the lung MTD to be 27 Gy. Patients with a favorable biodistribution were eligible for RIT with 131I-tositumomab according to a phase 1 dose-escalation protocol. Twenty-four patients had a favorable biodistribution and 19 received therapeutic infusions of 8.7 to 28.7 GBq (234–777 mCi) of 131I-mAbs (58– 1168 mg) followed by ASCT, resulting in CRs in 16, PRs in 2, and an MR (25–50% tumor regression) in 1. Of these patients, nine have remained in continuous CR for 3 to 53 months. Toxic effects included myelosuppression, nausea, infections, and two episodes of cardiopulmonary toxicity that were moderate in patients treated with doses of 131I- mAbs that delivered less than 27.25 Gy to normal organs. For patients with a favorable biodistribution (tumor burdens <500 cm3 and without massive splenomegaly), the CR rate was 84% with a PR rate of 11%. These remissions appeared durable and the median DR exceeded 11 months at the time of publication. Finally, the anti-CD20 mAb (B1) was considered to be superior to the anti-CD37 (mB-1) because favorable biodistribution was achieved with 2.5mg/kg, as compared with 10mg/kg for the anti-CD37. In the subsequent study, Press et al. evaluated the combination of highdose 131I-tositumomab, etoposide, and cyclophosphamide (Cy) in conjunction with ASCT in 38 patients with NHL (26 low grade, 12 aggressive) (63). Of the 37 evaluable patients, 33 (89%) were alive and 29 (78%) were progression free after a median follow-up of 1.5 years. Toxicities included grade 4 myelosuppression in all patients, grade 2 to 3 nausea in 26 (70%), pulmonary infiltrate in four, and grade 3 veno-occlusive disease (VOD) in two patients. These results confirmed the feasibility of delivering high-dose RIT in combination with highdose chemotherapy in an ASCT setting for NHL. More recently, the long-term follow-up of the 29 patients treated in phase I and phase II trials was reported (57). Twenty-four of the 29 patients are alive and 15 progression free after a median follow-up of 37 months. At five years, overall survival and PFS are projected at 68% and 42%, respectively. None of the surviving patients has objective impairment of performance status or cardiac function. Late toxicities have been uncommon, except for elevation of the TSH levels seen in 60% of the subjects. The Seattle transplant group performed a multivariable comparison of 125 consecutive patients with FL treated with either high-dose RIT using 131 I-tositomamb (n ¼ 27) or conventional high-dose therapy (C-HDT) (n ¼ 98)
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and ASCT 64. The groups were similar, although more patients treated with high-dose RIT had an elevated pretransplantation level of lactate dehydrogenase (41% vs. 20%, p ¼ 0.03) and elevated international prognostic score (41% vs. 19%, p ¼ 0.02). Patients treated with high-dose RIT received individualized therapeutic doses of 131I-tositumomab [median, 19.7 GBq (531 mCi)] to deliver 17 to 31 Gy (median, 27 Gy) to critical organs. Patients treated with C-HDT received TBI plus chemotherapy (70%) or chemotherapy alone (30%). Patients treated with high-dose RIT experienced improved overall survival and PFS versus patients treated with C-HDT. The estimated five-year overall survival and PFS were 67% and 48%, respectively for high-dose RIT and 53% and 29%, respectively for C-HDT. The 100-day treatment-related mortality was 3.7% in the high-dose RIT group and 11% in the C-HDT group. The probability of secondary MDS/AML was estimated to be 0.076 at eight years in the highdose RIT group and 0.086 at seven years in the C-HDT group. This data suggests that high-dose RIT may improve outcomes when compared with C-HDT in patients with relapsed FL. Nademanee et al. recently studied the feasibility of including high dose 90 Y-ibritumomab with high-dose etoposide (VP-16) 40 to 60 mg/kg (day 4) and Cy 100 mg/kg (day 2) followed by ASCT (65). This phase I/II trial of high-dose 90Y-ibritumomab tiuxetan in combination with etoposide and Cy was conducted in 31 patients with CD20 positive NHL. Patients underwent dosimetry (day 21) with 5 mCi (185 MBq) 111In-ibritumomab tiuxetan following 250 mg/m2 rituximab followed a week later by 90Y-ibritumomab tiuxetan to deliver a target dose of 1000 cGy to highest normal organ. Bone marrow biopsy was later performed (day 7) to estimate radiation dose, and stem cells were reinfused when the radiation dose was estimated to be less than 5 cGy. The median 90Y-ibritumomab tiuxetan dose was 2649.2 MBq (range, 1354.2–3885 MBq) (71.6 mCi; range, 36.6–105 mCi). At a median follow-up of 22 months, the two-year estimated overall survival and RFS rates were 92% and 78%, respectively. The author concluded that high-dose 90 Y-ibritumomab tiuxetan can be combined safely with high-dose etoposide and Cy without an increase in transplant-related toxicity or delayed engraftment. Other myeloablative approaches have investigated the biodistribution and pharmacokinetics of 131I-rituximab in 35 patients with NHL (66). After administration of a 20 to 40 mg mAb dose labeled with 250 MBq of 131I, biodistribution was determined by gamma camera imaging. The half-life of the labeled mAb was 88 hours compared with the murine tositumomab with a halflife of 56 hours. Greater dose was delivered to the whole body and organs, although tumor doses were found to increase over time as opposed to other organs. The authors concluded that patient-specific dosimetry was required and that lower activity doses were required than those delivered by tositumomab because of 131I-rituximab’s longer half-life.
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Clinical RIT Trials in Leukemias RIT studies in leukemia have focused on the CD33, CD45, and CD66 antigens. CD33 is a cell surface glycoprotein found on myeloid leukemia cells. HuM195 is a humanized anti-CD33 mAb that has been used in its native form as well as in a radiolabeled form to treat leukemias. The development over the last decade culminated in a recently reported randomized cohort of 50 adult patients [median age of patients was 62 years (range, 26–86)] with relapsed or refractory AML, which were treated with unconjugated HuM195 (67). CD33 was detected in 95% of patients for whom immunophenotyping was available. All patients received a dose of either 12 or 36 mg/m2 of HuM195 by intravenous infusion over four hours on days 1 to 4 and 15 to 18. Those patients with stable or responding disease received two additional cycles on days 29 to 32 and 43 to 46. Of the 50 patients, 24 were treated with HuM195 as a first salvage therapy and 26 as a second or subsequent salvage therapy. The percentage of blast involvement in the marrow ranged from 5% to 30% in 20 patients with the remainder having blast counts greater than 30%. Only two CRs and one PR were observed from all 49 evaluable patients. All three responses were in patients treated at the 12 mg/m2 dose level, which all had baseline blast percentages of less than 30%. Reductions in blast counts ranging from 30% to 74% were described in nine other patients. HuM195 as a single agent was observed to have minimal but measurable, antileukemic activity in patients with relapsed or refractory AML, though its activity is confined to patients with low-burden disease. No significant differences in clinical efficacy or toxicity were seen between the two dose levels of mAb. HuM195 was well tolerated with the predominant toxicities observed being infusion-related fevers and chills, which were primarily related to the first dose of mAb. The authors concluded that significant clinical efficacy with unconjugated HuM195 may only be realized in patients with minimal residual disease or when combined with chemotherapy. 131 I-M195 and 131I-HuM195 has also been combined with busulfan (Bu)/ Cy as conditioning for allogeneic BMT (68). RIT with 131I-M195 or 131IHuM195 was performed on 31 patients with relapsed/refractory AML (n ¼ 16), accelerated/myeloblastic chronic myeloid leukemia (CML) (n ¼ 14), or advanced MDS (n ¼ 1). Subjects received 4.5 to 16.2 GBq (122–437 mCi) plus Bu (16 mg/kg) and Cy (90120 mg/kg) followed by infusion of related-donor bone marrow (27 first ASCT; 4 second ASCT). Hyperbilirubinemia was the most common extramedullary toxicity, occurring in 69% of patients during the first 28 days after ASCT. Gamma camera imaging showed targeting of the radioisotope to the bone marrow, liver, and spleen, with absorbed radiation doses to the marrow of 272 to 1470 cGy. The median survival was 4.9 months (range, 0.3–90þ months). Following bone marrow transplantation, three patients with relapsed AML remained at the time of publication in CR for greater than 59, 87,
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and 90 months. The authors demonstrated the feasibility of adding CD33targeted RIT to a standard ASCT preparative regimen, though randomized trials are required to demonstrate the benefit of intensified conditioning with RIT. Feldman et al. compared the efficacy of HuM195 immunotherapy in combination plus induction chemotherapy with chemotherapy alone in a randomized study of 191 adults with first relapsed or primary refractory AML (duration of first response, 0–12 months) (67). The two groups were administered with 8-mg/m2 mitoxantrone, 80-mg/m2 etoposide, and 1-g/m2 cytarabine daily for six days in combination with 12 mg/m2 with or without HuM195. Of the cohort treated with the combination treatment, 36% showed a CR or CR without complete platelet recovery, whereas only 28% showed similar results in patients treated with chemotherapy alone (p ¼ 0.28). The overall median survival was 156 days and was not different in the two arms of the study. Apart from mild mAb infusion-related toxicities (fever, chills, and hypotension), no differences in chemotherapy-related AEs were observed with the addition of HuM195 to induction chemotherapy. Though the addition of HuM195 to salvage induction chemotherapy was safe, the authors demonstrated that the combination treatment did not result in a statistically significant improvement in ORR or survival in patients with refractory/relapsed AML. A phase I activity dose escalation study was performed in nine patients using 213Bi-HuM195 in patients with refractory and relapsed myeloid leukemias and the data used to estimate pharmacokinetics and dosimetry (69). This was the first trial using an a-emitter in human leukemia patients. Patient imaging was initiated at the start of each injection of 0.6 to 1.6 GBq (16.2– 43.2 mCi) of activity. A set of 40 images of the liver and spleen were dynamically obtained and blood samples were collected until three hours postinjection and counted in a gamma counter. The percentage injected dose in the liver and spleen volumes increased rapidly over the first 10 to 15 minutes to a constant value for the remaining hour of imaging, yielding a very rapid uptake followed by a plateau in the mAb uptake curves. The absorbed dose equivalent to liver and spleen volumes ranged from 2.4 to 11.2 Sv and 2.9 to 21.9 Sv, respectively. Marrow (or leukemia) mean dose ranged from 6.6 to 12.2 Sv. The study demonstrated that patient imaging of 213Bi, an a-particle emitter, labeled to HuM195 was possible and may be used to derive pharmacokinetics and dosimetry data. 213 Bi-HuM195 was also studied in 18 refractory AML or CML patients and was shown to localize to the marrow, spleen, and liver (19). Five dose levels ranging from 10.36 to 37.0 MBq/kg of 213Bi-HuM195 were administered to 18 patients. All 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days and almost all of the activity was observed to rapidly localize and remain in areas of leukemic involvement, including the bone marrow, liver, and spleen. The reduced whole body and increased target organ doses (bone marrow, liver, spleen) produced absorbed dose ratios approximately
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1000 times higher for 213Bi-HuM195 than those for b-emitting anti-CD33 mAb constructs. Fourteen (93%) of the 15 evaluable patients had reductions in circulating blasts, and 14 (78%) of 18 patients had reductions in the percentage of bone marrow blasts. This study further demonstrates the safety, feasibility, and therapeutic effects of 213Bi-HuM195 in treating leukemia. In the phase I trial, 20% of 31 patients were shown to have a long-term survival rate for patients with AML (68). The same group labeled HuM195 with 90 Y and has started a clinical trial using RIT in combination with TBI (2 Gy) and fludarabine in a reduced intensity-conditioning program. The results of this trial are anticipated but have not been revealed to date. There have also been very encouraging results using 131I-BC8 mAb (anti-CD45) in combination with Cy and TBI as a marrow transplant conditioning regimen for acute leukemia (70). Twenty patients were treated with a radiation dose estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver, with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by 120 mg/kg Cy and 12 Gy TBI. Toxicity was not measurably greater than that of Cy/TBI alone and the MTD was not reached. Results from a phase I study of 131I anti-CD45 combined with 120 mg/kg Cy and 12 Gy TBI in HLAmatched related transplants for AML in first remission were recently reported (70). A biodistribution dose of 0.5 mg/kg 131I-BC8 (murine anti-CD45) mAb was given to 44 patients with advanced MDS/ALL. The mean þ/ SEM estimated radiation absorbed dose delivered to bone marrow and spleen was 6.5 þ/ 0.5 and 13.5 þ/ 1.3 cGy/mCi of 131I, respectively. Liver (2.8 þ/ 0.2 cGy/mCi), lung (1.8 þ/ 0.1 cGy/mCi), kidney (0.6 þ/ 0.04 cGy/mCi), and total body (0.4 þ/ 0.02 cGy/mCi), all received lower doses. Thirty-seven patients (84%) had favorable mAb biodistributions, with a higher estimated radiation absorbed dose to marrow and spleen than to normal organs. Of these patients, 34 received a therapeutic dose of 131I-BC8 mAb labeled with 2.8 to 22.6 GBq (76–612 mCi) of 131I to deliver estimated radiation absorbed doses to liver (normal organ receiving the highest dose) of 3.5 Gy (level 1) to 12.25 Gy (level 6) in addition to Cy and TBI. The MTD was level 5 (delivering 10.5 Gy to liver), with grade 3/4 mucositis in two of two patients treated at level 6. The authors concluded that 131I -anti-CD45 mAb can safely deliver substantial supplemental doses of radiation to bone marrow (approximately 24 Gy) and spleen (approximately 50 Gy) when combined with conventional Cy/TBI. Matthews et al. undertook several studies using 131I-anti-CD45 mAb (70). The phase I study demonstrated mAb safety and acceptable pharmacodynamics after 34 refractory AML/ALL patients, which had been evaluated. The patients received a 2.8 to 22.6 GBq (76–612 mCi) therapeutic dose of 131I-anti-CD45 to bring about calculated radiation-absorbed doses to the liver of 3.5 Gy (level 1 Bearman criteria) to 12.25 Gy (level 6) in addition to Cy (120 mg/kg) and TBI (12 Gy). The red-marrow dose from the labeled mAb was 24 Gy, the spleen dose was 50 Gy, and the LFS was 29%.
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Following the success of the phase I study, two phase II studies have been performed to evaluate the short-term toxicity of the anti-CD45 radiopharmaceutical (68). In the first trial, 17 patients with advanced AML were treated with 131I-anti-CD45, TBI (12 Gy), and Cy (120 mg/kg) to obtain a redmarrow dose of 21 to 22 Gy. Results of the trial showed LFS in 42% of patients, 29% relapsed and 29% died of nonrelapsed mortality. In the second study, 46 AML patients in CR1 with either an intermediate or high-risk cytogenetic features were treated with the same treatment as before but with Bu (16 mg/kg) instead of Cy. Red-marrow doses were 11 Gy, although average LFS was 60% with intermediate patient LFS rates being 68% and average high-risk patient LFS being 40%. Almost inevitably, the high-risk patients had a higher relapse rate than the intermediate patients. A further phase I trial has been initiated by the same group using only 2 Gy TBI in combination with fludarabine (71,72). Thus far, the mean red-marrow dose achieved with this treatment has been 24 Gy and of the 11 patients recruited, 8 are in remission at the time of publication. 188 Reis an attractive radioisotope for RIT as it has a 16.9-hour half-life, and emits a high energy (2.2 MeV b-particle and a 155 keV g-photon) facilitating therapy and imaging. Bunjes et al. used a 188Re- anti-CD66 mAb to intensify the conditioning regimen prior to stem cell transplantation to treat 36 patients with high-risk AML and MDS (73). As a favorable dosimetry was observed in all cases, RIT was administered and provided a mean of 15.3 Gy of additional radiation to the marrow. The normal organ receiving the highest dose was the kidney (mean 7.4 Gy), which resulted in late renal toxicity in 17% of patients. RIT was followed by standard full-dose conditioning with TBI (12 Gy) or Bu and high-dose Cy with or without thiotepa. Patients subsequently received a T-cell-depleted allogeneic graft from an HLA-identical family donor (n ¼ 15) or an alternative donor (n ¼ 17). mAb infusion-related toxicity due was minimal, and no increase in mAb treatment-related mortality was observed. After 30, 100 days and 18 months postadministration, mortalities were 3%, 6%, and 22%, respectively. Of the 15 patients undergoing transplantation in first CR or second CR, the relapse rate was 20% and 21 patients found not to be in remission at the time of transplantation had a 30% relapse rate. Waldmann et al. have investigated the use of anti-Tac mAb (antiinterleukin-2 receptor) in adult T-cell leukemia (ATL) caused by the retrovirus human T-cell lymphotropic virus-I (74). 90Y-anti-Tac was administered to the first nine patients as part of a phase I dose-escalation trial and to the second group of nine as part of a phase II trial involving a uniform 370 MBq (10 mCi) dose. Patients in whom a response was observed were able to receive up to eight additional doses. At the 185 to 555 MBq (5–15 mCi) doses used, 9 of 16 evaluable patients had responses (7 PR and 2 CR). The observed responses appear better than those with unmodified anti-Tac and severe toxicity was largely limited to the hematopoietic system.
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Expert Opinion, Including a Table with up to Five Key Issues I-Tositumomab (Bexxar1) Versus (Zevalin1) 131
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No comparative clinical trial has ever been performed between 90Y-ibritumomab tiuxetan and 131I-tositumomab and is unlikely ever to be either. However, it appears reasonable to surmise that the two drugs have very similar response rates and response durations from the published results. The integration of 90Y-ibritumomab tiuxetan and 131I-tositumomab into routine clinical practice seems, therefore, more likely to depend on the cost and convenience of each therapy rather than perceived differences in clinical efficacy. 131I-tositumomab remains licensed in the United States only and if an EU license is eventually gained, the radiation protection issues with the necessity for five to six days inpatient stay for patients (within the EU) receiving 131Itositumomab may influence clinicians on health economic grounds in favor of 90 Y-ibritumomab tiuxetan. The removal of the dosimetric dose, within the EU, further simplifies the delivery of 90Y-ibritumomab tiuxetan making the whole regimen very easy for the patient. RIT Induces Durable Remissions Perhaps the most impressive finding to emerge from these maturing data using RIT is for FLs, in all of the studies is the remarkable DR enjoyed by some patients. This durability of treatment response was initially seen in patients treated with 131I-tositumomab (50) has also been observed with 90Y-ibritumomab tiuxetan (75). In all of the RIT studies, around 70% of patients who achieve a CR remain in remission for years (43,50,75). Further, some patients treated in the early studies have now been in remission for more than five years after and with a median follow-up of almost four years (Table 2). An analysis of long-term responders underscores the potential of 90Y-ibritumomab tiuxetan to achieve durable remissions with observed median DR approaching two years and responses greater than six years being observed in some patients (43) (Fig. 3). An analysis of prognostic factors has confirmed that this remarkable durability of response is unlikely to be accounted for by patient selection as most of these durable remissions have been achieved in heavily chemotherapy pretreated and chemo-refractory patients with validated poor prognostic factors such as extensive prior therapy (1–9 regimens), bulky disease, high LDH, and extranodal disease. Only disease bulk correlated with the ORR (<5 cm) [89 patients ORR 90% (p < 0.001)] (55) (Fig. 4). What Is the Role for Dosimetry in RIT? One of the fundamental potential advantages of RIT is the ability to deliver higher targeted radiation dose to the tumor than to normal tissue and thus
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Figure 3 Progression-free survival for integrated summary of efficacy population (n ¼ 250).
Figure 4 Progression-free survival for patients within each of the five clinical studies that comprise the integrated summary of efficacy population.
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enhance the specific tumor killing. The fact that radioisotopes emit ionizing radiation not only enables them to be used in therapy but also their emissions to be quantified using radiation dosimetry. However, because of the relative complexity of the radiation dose estimation for RIT, the clinical importance of dosimetry in the RIT of NHL currently remains divisive. While some investigators regard dosimetry as essential component of RIT practice, others disagree that it is necessary (27,28,53,62). It is possible that both groups of advocates may be correct and that the requirement for dosimetry could be dependent on the radioisotopes and the mAb targeting used. As ytrrium-90 is a pure b-emitter, indium-111 is used as a surrogate and chelated to ibritumomab tiuxetan to facilitate gamma camera imaging for dosimetry. Following the rituximab infusion, imaging was performed using a dose of 185 MBq (5 mCi) and 1.6-mg total mAb dose, as part of the registration studies at 2 to 24 hours, at 48 to 72 hours and at 90 to 120 hours. For 131I-tositumomab, gamma camera imaging, as previously described, is an essential part of the regimen and required for the patientspecific dosimetry. There are at least two important issues with regard to dosimetry; namely, dosimetry can predict tumor response, and secondly it can predict normal tissue toxicity. To date, despite the high response rates seen in lymphoma RIT, clinical dosimetry studies have thus far failed to show a consistent dose-response relationship, although some investigators have found a correlation (30). One of the factors that is highly likely to complicate the analysis and may underlie the controversy is the inability of dosimetry to measure the tumoricidal capability of some mAb. Two imaging scans following rituximab injection do, however, form part of the approved schedule within the United States, despite the fact that the dosimetric studies failed to demonstrate a consistent correlation between the estimated bone marrow dose and toxicity. It appears that 90Y-ibritumomab tiuxetan can be safely prescribed according to body weight and platelet count (55), and this lack of positive correlation between imaging and bone marrow toxicity led to imaging studies not being required within the EU (76). INTEGRATION INTO TREATMENT ALGORITHMS FOR NHL The high response rates and durable remissions achieved with either Y-ibritumomab or 131I-tositumomab make single agent RIT an attractive treatment option for many patients with relapsed FL. Furthermore the impressive DRs, seen after achieving a CR are achieved with a treatment that is completed within a week, is very well tolerated and that has minimal nonmyelotoxicity toxicity and easily manageable myelotoxicity. The introduction of RIT has some parallels with the introduction of rituximab into clinical practice in the late 1990s. There is no doubting that RIT drugs are highly active but considerable uncertainty remains as to when and how best to integrate RIT into clinical practice even within the licensed indication of
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Figure 5 A proposed treatment algorithm for follicular lymphoma patients after relapse first relapse following a rituximab-chemotherapy combination. Source: From Ref. 77.
relapsed low grade (United States) or relapsed rituximab failure or refractory FL (within EU). The treatment is well tolerated by older patients and RIT makes this approach a strong recommendation for relapsed FL. In the education session on follicular at the American Society of Hematologists (2004), an algorithm was suggested for FL where RIT was recommended upon relapse first relapse after a rituximab-chemotherapy combination (Fig. 5) (77). Currently this seems a reasonable treatment approach to FL, as it does not exclude transplantation options at a later date, especially if, as recommended, progenitor stem cell collection is performed at the time of the initial remission. Although data is emerging to suggest that RIT can be given after transplantation (see above), this is inevitably at lower doses. Further clinical trials will need to be performed to further define the role of RIT in the treatment of other NHL. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Although the results for single agent RIT are encouraging and make RIT an attractive treatment option for relapsed FL, the future is likely to involve integrating RIT into chemotherapy treatment protocols in an attempt to increase relapse-free and overall survival. The current challenge for clinical investigators is to determine the optimal approach of integrating RIT into chemotherapy schedules. The emerging data using both 131I-tostumomab and
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Y-ibritumomab tiuxetan as consolidation following shortened or full course chemotherapy look extremely promising and suggest that the quality of responses can be substantially increased by this type of approach (44,54,78). Future randomized clinical trials will define whether this type of approach offers similar or perhaps even superior RFS over rituximab-chemotherapy regimens followed by maintenance rituximab. Future clinical studies will indicate whether the ability of RIT to improve the quality of the response from partial to CR will add to RFS and perhaps even overall survival. The initial highly promising phase II studies for 131I-tositumomab suggest that this RIT as a single treatment option may be worth exploring further for some patients and preliminary data for patients using 90Y-ibritumomab, tiuxetan, and rituximab, as a first-line treatment for FL suggest very high response rates and will require further exploration. Given the high single agent activity of 90Y-ibritumomab tiuxetan in DLBC 131 and I-tositumomab in aggressive lymphoma, there are a number of clinical trials that are now underway to integrate either of these two RIT reagents into the front-line treatment of DLBC. There are a number of U.S. phase II studies and a large European intergroup study with a randomization to 90Y-ibritumomab tiuxetan or no further treatment after full course CHOP-R chemotherapy that are underway. Over the next five years the clinical studies that are currently underway are likely to further define whether RIT has a role in further improving the results achieved with R-CHOP immunochemotherapy in DLBCL. Given the selection of higher risk poorer prognosis in patients who fail R-CHOP regimens, it is to be expected that the results after standard BEAM (BCNU, etoposide, cytarabine, melphalan) conditioning will result in less impressive overall survival than prior to the introduction of R-CHOP. There is, therefore, an urgent requirement to improve the results and an intensification of the ‘‘conditioning regimen’’ is required. An area that is, therefore, currently being intensely investigated is the integration of RIT into the conditioning regimens instead of TBI or with reduced dose TBI, followed by ASCT rescue. The studies to date have confirmed that higher myeloablative doses can be safely delivered with ASCT support. While Press and colleagues have demonstrated that myeloablative doses of 131I-tositmomab with and without high dose chemotherapy result in highly impressive RFS, the difficulty has been in reproducing these excellent results given the extremely high doses of 131I that are manipulated in the conjugation process and then subsequently administered. A more practical approach, which will allow the participation of many more transplant centers, is the use of ‘‘standard’’ or escalated doses of 90 Y-ibritumomab tiuxetan. The early results are encouraging and confirm the feasibility of the addition of escalated doses of 90Y-ibritumomab tuxetan with the ‘‘standard’’ conditioning regimen of BEAM (65). Appropriately designed randomized studies will confirm over the next few years whether the addition of RIT to high dose chemotherapy will result in improvement in treatment outcome for patients with relapsed lymphoma.
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Y-ibritumomab tiuxetan is also being investigated in the treatment of other aggressive CD20 positive lymphomas such as mantle cell lymphoma. Preliminary data in patients with relapsed or refractory mantle cell lymphoma suggest therapeutic efficacy of 90Y-ibritumomab tiuxetan with disease control being achieved in around half of the patients treated. Further clinical studies are underway using RIT as consolidation after rituximab chemotherapy regimens, and these studies should help to establish whether the poor results in this disease can be improved upon. The use of RIT in leukemia is less well developed and no radioimmunoconjugate has yet been approved for routine delivery. The exquisite sensitivity of these malignancies to targeted radiation alongside the highly impressive results achieved by the pioneers in this field suggests that this is highly likely to be a productive area for future clinical research and lead to tangible patient benefits. The huge progress made over the last decade with the development of RIT in the treatment of hematological malignancies leads to distinct optimism that further development of RIT over the next five years will lead to significant improvements in clinical outcomes for patients.
REFERENCES 1. Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol 2005; 23:1147–1157. 2. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346:235–242. 3. Robak T. Monoclonal antibodies in the treatment of chronic lymphoid leukemias. Leuk Lymphoma 2004; 45:205–219. 4. Knox SJ, Meredith RF. Clinical radioimmunotherapy. Semin Radiat Oncol 2000; 10:73–93. 5. Press OW, Leonard JP, Coiffier B, et al. Immunotherapy of Non-Hodgkin’s Lymphomas. Hematology (Am Soc Hematol Educ Program) 2001; 2001:221–240. 6. Kaminski MS, Zelenetz AD, Press OW, et al. Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell nonHodgkin’s lymphomas. J Clin Oncol 2001; 19:3918–3928. 7. DeNardo GL, DeNardo SJ, Goldstein DS, et al. Maximum-tolerated dose, toxicity, and efficacy of (131)I-Lym-1 antibody for fractionated radioimmunotherapy of nonHodgkin’s lymphoma. J Clin Oncol 1998; 16:3246–3256. 8. Illidge TM, Cragg MS, McBride HM, et al. The importance of antibody-specificity in determining successful radioimmunotherapy of B-cell lymphoma. Blood 1999; 94:233–243. 9. Press OW, Farr AG, Borroz KI, et al. Endocytosis and degradation of monoclonal antibodies targeting human B-cell malignancies. Cancer Res 1989; 49:4906–4912. 10. Grossbard ML, Press OW, Appelbaum FR, et al. Monoclonal antibody-based therapies of leukemia and lymphoma. Blood 1992; 80:863–878.
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11. Chan HT, Hughes D, French RR, et al. CD20-induced lymphoma cell death is independent of both caspases and its redistribution into triton X-100 insoluble membrane rafts. Cancer Res 2003; 63:5480–5489. 12. Sharkey RM, Behr TM, Mattes MJ, et al. Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22. Cancer Immunol Immunother 1997; 44:179–188. 13. Andres TL, Kadin ME. Immunologic markers in the differential diagnosis of small round cell tumors from lymphocytic lymphoma and leukemia. Am J Clin Pathol 1983; 79:546–552. 14. Griffin JD, Linch D, Sabbath K, et al. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res 1984; 8:521–534. 15. Hanenberg H, Baumann M, Quentin I, et al. Expression of the CEA gene family members NCA-50/90 and NCA-160 (CD66) in childhood acute lymphoblastic leukemias (ALLs) and in cell lines of B-cell origin. Leukemia 1994; 8:2127–2133. 16. Press OW, Rasey J. Principles of radioimmunotherapy for hematologists and oncologists. Semin Oncol 2000; 27:62–73. 17. Press OW, Shan D, Howell-Clark J, et al. Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res 1996; 56:2123–2129. 18. DeNardo GL, Kukis DL, Shen S, et al. 67Cu-versus 131I-labeled Lym-1 antibody: comparative pharmacokinetics and dosimetry in patients with non-Hodgkin’s lymphoma. Clin Cancer Res 1999; 5:533–541. 19. Jurcic JG, Larson SM, Sgouros G, et al. Targeted alpha particle immunotherapy for myeloid leukemia. Blood 2002; 100:1233–1239. 20. McDevitt MR, Sgouros G, Finn RD, et al. Radioimmunotherapy with alpha-emitting nuclides. Eur J Nucl Med 1998; 25:1341–1351. 21. Illidge TM, Johnson PW. The emerging role of radioimmunotherapy in haematological malignancies. Br J Haematol 2000; 108:679–688. 22. Wahl RL. The clinical importance of dosimetry in radioimmunotherapy with tositumomab and iodine I 131 tositumomab. Semin Oncol 2003; 30:31–38. 23. Buchsbaum DJ, Wahl RL, Glenn SD, et al. Improved delivery of radiolabeled antiB1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody. Cancer Res 1992; 52:637–642. 24. Du Y, Honeychurch J, Cragg MS, et al. Antibody-induced intracellular signaling works in combination with radiation to eradicate lymphoma in radioimmunotherapy. Blood 2004; 103:1485–1494. 25. Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents. Blood 2004; 103:2738–2743. 26. Koral KF, Kaminski MS, Wahl RL. Correlation of tumor radiation-absorbed dose with response is easier to find in previously untreated patients. J Nucl Med 2003; 44:1541–1543; (author reply 1543). 27. Postema EJ. Dosimetry and radioimmunotherapy of non-Hodgkin’s lymphoma. J Nucl Med 2004; 45:2126–2127; (author reply 2127). 28. Goldenberg DM, Sharkey RM. Radioimmunotherapy of non-Hodgkin’s lymphoma revisited. J Nucl Med 2005; 46:383–384. 29. Nikula TK, McDevitt MR, Finn RD, et al. Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry. J Nucl Med 1999; 40:166–176.
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30. Ruffner KL, Martin PJ, Hussell S, et al. Immunosuppressive effects of (131)I-antiCD45 antibody in unsensitized and donor antigen-presensitized H2-matched, minor antigen-mismatched murine transplant models. Cancer Res 2001; 61:5126–5131. 31. Davis TA, Kaminski MS, Leonard JP, et al. The radioisotope contributes significantly to the activity of radioimmunotherapy. Clin Cancer Res 2004; 10:7792– 7798. 32. Kaminski MS, Tuck M, Estes J, et al. 131I-tositumomab therapy as initial treatment for follicular lymphoma. N Engl J Med 2005; 352:441–449. 33. Press OW. Radioimmunotherapy for non-Hodgkin’s lymphomas: a historical perspective. Semin Oncol 2003; 30:10–21. 34. Sharkey RM, Goldenberg DM. Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J Nucl Med 2005; 46:S115–S127. 35. Wilder RB, DeNardo GL, DeNardo SJ. Radioimmunotherapy: recent results and future directions. J Clin Oncol 1996; 14:1383–1400. 36. Goldenberg DM, Horowitz JA, Sharkey RM, et al. Targeting, dosimetry, and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody. J Clin Oncol 1991; 9:548–564. 37. Tedder TF, Engel P. CD20: a regulator of cell-cycle progression of B lymphocytes. Immunol Today 1994; 15:450–454. 38. Grillo-Lopez AJ. Zevalin: the first radioimmunotherapy approved for the treatment of lymphoma. Expert Rev Anticancer Ther 2002; 2:485–493. 39. Witzig TE, White CA, Wiseman GA, et al. Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(þ) B-cell nonHodgkin’s lymphoma. J Clin Oncol 1999; 17:3793–3803. 40. Wiseman GA, Gordon LI, Multani PS, et al. Ibritumomab tiuxetan radioimmunotherapy for patients with relapsed or refractory non-Hodgkin lymphoma and mild thrombocytopenia: a phase II multicenter trial. Blood 2002; 99:4336–4342. 41. Witzig TE, Flinn IW, Gordon LI, et al. treatment with ibritumomab tiuxetan radioimmunotherapy in patients with rituximab-refractory follicular non-Hodgkin’s lymphoma. J Clin Oncol 2002; 20:3262–3269. 42. Witzig TE, Gordon LI, Cabanillas F, et al. Randomized controlled trial of yttrium90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin’s lymphoma. J Clin Oncol 2002; 20:2453–2463. 43. Gordon LI, Molina A, Witzig T, et al. Durable responses after ibritumomab tiuxetan radioimmunotherapy for CD20þ B-cell lymphoma: long-term follow-up of a phase 1/2 study. Blood 2004; 103:4429–4431. 44. Shipley DL, Greco FA, Spigel DR, et al. Rituximab with short duration chemotherapy followed by 90Y-ibritumomab tiuxetan as first line treatment for patients with follicular lymphoma: update of a Minnie Pearl Cancer Research Network Phase II Trial. J Clin Oncol 2005; (abstr 6577). 45. Morschhauser F, Illidge T, Huglo D, et al. Efficacy and safety of yttrium-90 ibritumomab tiuxetan in patients with relapsed or refractory diffuse large B-cell lymphoma not appropriate for autologous stem-cell transplantation. Blood 2007; 110: 54–58. 46. Nadler LM, Ritz J, Hardy R, et al. A unique cell surface antigen identifying lymphoid malignancies of B cell origin. J Clin Invest 1981; 67:134–140.
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47. Hohenstein MA, Augustine SC, Rutar F, et al. Establishing an institutional model for the administration of tositumomab and iodine I 131 tositumomab. Semin Oncol 2003; 30:39–49. 48. Kaminski MS, Zasadny KR, Francis IR, et al. Radioimmunotherapy of B-cell lymphoma with [131I]anti-B1 (anti-CD20) antibody. N Engl J Med 1993; 329:459–465. 49. Kaminski MS, Zasadny KR, Francis IR, et al. Iodine-131-anti-B1 radioimmunotherapy for B-cell lymphoma. J Clin Oncol 1996; 14:1974–1981. 50. Fisher RI, Kaminski MS, Wahl RL, et al. Tositumomab and iodine-131 tositumomab produces durable complete remissions in a subset of heavily pretreated patients with low-grade and transformed non-Hodgkin’s lymphomas. J Clin Oncol 2005; 23:7565– 7573. 51. Horning SJ, Younes A, Jain V, et al. Efficacy and safety of tositumomab and iodine131 tositumomab (Bexxar) in B-cell lymphoma, progressive after rituximab. J Clin Oncol 2005; 23:712–719. 52. Coleman M, Kaminski MS, Susan JK, Andrew DZ, et al. The BEXXAR therapeutic regimen (tositumomab and iodine I 131 tositumomab) produced durable complete remissions in heavily pretreated patients with non-Hodgkin’s lymphoma (NHL), rituximab-relapsed/refractory disease, and rituximab-naive disease. Proc Am Soc Hem Blood 2003; 102(11):29a; (abstr 89). 53. Koral KF, Dewaraja Y, Li J, et al. Update on hybrid conjugate-view SPECT tumor dosimetry and response in 131I-tositumomab therapy of previously untreated lymphoma patients. J Nucl Med 2003; 44:457–464. 54. Press OW, Unger JM, Braziel RM, et al. A phase 2 trial of CHOP chemotherapy followed by tositumomab/iodine I 131 tositumomab for previously untreated follicular non-Hodgkin lymphoma: Southwest Oncology Group Protocol S9911. Blood 2003; 102:1606–1612. 55. Witzig TE, White CA, Gordon LI, et al. Safety of yttrium-90 ibritumomab tiuxetan radioimmunotherapy for relapsed low-grade, follicular, or transformed nonHodgkin’s lymphoma. J Clin Oncol 2003; 21:1263–1270. 56. Zelenetz AD. Radioimmunotherapy for lymphoma. Curr Opin Oncol 1999; 11: 375–380. 57. Liu SY, Eary JF, Petersdorf SH, et al. Follow-up of relapsed B-cell lymphoma patients treated with iodine-131-labeled anti-CD20 antibody and autologous stemcell rescue. J Clin Oncol 1998; 16:3270–3278. 58. Bennett JM, Kaminski MS, Leonard JP, et al. Assessment of treatment-related myelodysplastic syndromes and acute myeloid leukemia in patients with nonHodgkin lymphoma treated with tositumomab and iodine I131 tositumomab. Blood 2005; 105:4576–4582. 59. Ansell SM, Ristow KM, Habermann TM, et al. Subsequent chemotherapy regimens are well tolerated after radioimmunotherapy with yttrium-90 ibritumomab tiuxetan for non-Hodgkin’s lymphoma. J Clin Oncol 2002; 20:3885–3890. 60. Dosik AD, Coleman M, Kostakoglu L, et al. Subsequent therapy can be administered after tositumomab and iodine I-131 tositumomab for non-Hodgkin lymphoma. Cancer 2006; 106:616–622. 61. Jacobs SA, Vidnovic N, Joyce J, et al. Full-dose 90Y ibritumomab tiuxetan therapy is safe in patients with prior myeloablative chemotherapy. Clin Cancer Res 2005; 11: S7146–S7150.
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62. Wahl RL. Tositumomab and 131I therapy in non-Hodgkin’s lymphoma. J Nucl Med 2005; 46:S128–S140. 63. Press OW, Eary JF, Gooley T, et al. A phase I/II trial of iodine-131-tositumomab (anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas. Blood 2000; 96:2934–2942. 64. Gopal AK, Gooley TA, Maloney DG, et al. High-dose radioimmunotherapy versus conventional high-dose therapy and autologous hematopoietic stem cell transplantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort analysis. Blood 2003; 102:2351–2357. 65. Nademanee A, Forman S, Molina A, et al. A phase 1/2 trial of high-dose yttrium-90ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poorrisk or relapsed non-Hodgkin lymphoma. Blood 2005; 106:2896–2902. 66. Scheidhauer K, Wolf I, Baumgartl HJ, et al. Biodistribution and kinetics of (131)Ilabelled anti-CD20 MAB IDEC-C2B8 (rituximab) in relapsed non-Hodgkin’s lymphoma. Eur J Nucl Med Mol Imaging 2002; 29:1276–1282. 67. Feldman EJ, Brandwein J, Stone R, et al. Phase III randomized multicenter study of a humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with chemotherapy, versus chemotherapy alone in patients with refractory or first-relapsed acute myeloid leukemia. J Clin Oncol 2005; 23:4110–4116. 68. Burke JM, Caron PC, Papadopoulos EB, et al. Cytoreduction with iodine-131-antiCD33 antibodies before bone marrow transplantation for advanced myeloid leukemias. Bone Marrow Transplant 2003; 32:549–556. 69. Sgouros G, Ballangrud AM, Jurcic JG, et al. Pharmacokinetics and dosimetry of an alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia. J Nucl Med 1999; 40:1935–1946. 70. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 1999; 94:1237–1247. 71. Niederwieser D, Maris M, Shizuru JA, et al. Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLAmatched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases. Blood 2003; 101:1620–1629. 72. McSweeney PA, Niederwieser D, Shizuru JA, et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 2001; 97:3390–3400. 73. Bunjes D, Buchmann I, Duncker C, et al. Rhenium 188-labeled anti-CD66 (a, b, c, e) monoclonal antibody to intensify the conditioning regimen prior to stem cell transplantation for patients with high-risk acute myeloid leukemia or myelodysplastic syndrome: results of a phase I-II study. Blood 2001; 98:565–572. 74. Waldmann TA, White JD, Carrasquillo JA, et al. Radioimmunotherapy of interleukin-2R alpha-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac. Blood 1995; 87:4063–4075. 75. Gordon LI, Witzig T, Molina A, et al. Yttrium 90-labeled ibritumomab tiuxetan radioimmunotherapy produces high response rates and durable remissions in patients with previously treated B-cell lymphoma. Clin Lymphoma 2004; 5:98–101.
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8 Differentiation Induction in Acute Promyelocytic Leukemia Adi Gidron and Martin S. Tallman Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, U.S.A.
INTRODUCTION Acute myeloid leukemia (AML) is a heterogeneous group of diseases typically associated with an aggressive course and generally a poor overall survival (OS). Despite advances in therapy over the past 20 years, long-term survival for patients with AML remains approximately 30% (1). Acute promyelocytic leukemia (APL) represents a distinctive subtype of AML. It accounts for approximately 10% to 15% of all patients with AML and is distinguished from other types by a younger median age (40 vs. 68 years), a unique genetic abnormality, the t(15;17) translocation and the formation of the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) fusion transcript, and most importantly, by the high cure rate achieved with differentiation therapy. Until the late 1980s and early 1990s, APL was considered the most fatal subtype of AML primarily because of a severe coagulopathy often leading to catastrophic hemorrhage early in the natural history of the disease or early in the course of treatment. The discovery that the leukemic promyelocytes from patients with APL were uniquely sensitive to all-trans retinoic acid (ATRA) and that the breakpoint on chromosome 17 that resulted in the development of an abnormal PML-RARa fusion product eventually led to the recognition that disruption of RARa gene product was the major cause of
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maturation arrest in APL (2,3). Currently, APL is associated with several variant chromosomal abnormalities leading to different gene rearrangements. PMLRARa is the product of t(15;17), NPM (nucleophosmin)-RARa is the product of t(5;17)(q35;q21), and is NuMA (nuclear matrix associated)-RARa the product of t(11;17)(q13;q21), all of which lead to a syndrome that is responsive to ATRA. PLZF (promyelocytic leukemia zinc finger)-RARa, the product of t(11;17)(q23; q21), is unresponsive to ATRA. Signal transducer and activator of transcription 5B (STAT5B) is the product of t(17;17)(q11;q21) (4). In addition to cytogenetic variants, molecular variant of the PML-RARa transcript such as bcr1, bcr2, and bcr3 have also been described (5). Advances in the understanding of APL have led to a new strategy in anti-leukemia therapy. Rather than relying on intensive chemotherapy, the treatment of APL focuses now on differentiation therapy to induce remission. Such a strategy results in long-term survival rates of more than 80% (6). This chapter will address the unique biology of APL, the role of ATRA in differentiation therapy and arsenic trioxide (ATO) in inducing apoptosis. Both agents contribute to the remarkable cure rates in APL. In addition, novel strategies such as FLT3 inhibitors, antiangiogenesis agents, monoclonal antibodies, differentiation agents, and histone deacetylase inhibitors will be described. THE PATHOGENESIS OF APL Retinoic acid (RA) is important in embryonic development and in a variety of cellular processes. The activity of RA is mediated by RAR that are part of the nuclear receptor superfamily. Several RARs have been described including RXR, RARa, RARb, and RARg. RA activates RARa to bind to RAR elements (RARE) located in the promoter region of genes important for differentiation such as those of RARa, RARb, and RARg, and RXR. A heterodimer of RARa and RXR forms and in conjunction with other coactivator proteins binds to DNA and stimulates transcription through two domains. The ligand-independent domain (AF-1) forms on the N-terminal of the protein and works in a promoter context-dependent manner (5,7,8). The ligand-dependent domain (AF-2) is associated with the corepressors NCoR, SMRT, Sin3A, and histone deacetylase. Ligand binding to the AF-2 domain releases these corepressors and allows for transcription of the target genes (5,9–11). It was recognized early that vitamin A–deficient mice and humans developed defects in hematopoiesis (5,12,13). However, the role of RA in myeloid differentiation was more clearly demonstrated in cell lines such as HL60 and in cells from patients with APL in the 1980s. HL60 cells that had a dominant negative mutant of RARa were resistant to ATRA and showed a maturation block. When these cells were infected with a retrovirus expressing RARa, RARb, and RARg, differentiation was restored. It was then discovered that RA acts directly through RARa to induce myeloid differentiation (14,15). In APL, the fusion protein PML-RARa retains both the DNA and the ligand-binding domains and inhibits terminal differentiation through abnormal recruitment of
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corepressors and histone deacetylases and acts as a dominant-negative RARa mutant (5). The fusion proteins characteristic of APL not only change the activity of RARa, but also affect the normal function of the partner protein, PML. PML is linked to RARa in 98% of all APL patients. The PML gene is located on chromosome 15q22 and the protein localizes to nuclear bodies (NBs) (16). The PML NB plays a key role in tumor suppression by regulating apoptosis, growth arrest, and cellular senescence (16). The PML-RARa fusion results in delocalizing PML from the NB to microspeckled nuclear structures and exerting a dominant-negative effect. The treatment of APL cells with ATRA not only induces differentiation by releasing coreceptors, the release of RXR, and restoring RA signaling, but also restores the PML-NB and therefore promotes growth arrest and apoptosis (5). CLINICAL TRIALS OF ATRA IN APL The Predifferentiation Era of APL Until the 1990s, combination chemotherapy with an anthracycline and cytosine arabinoside (ara-C) was the standard treatment for APL. While most patients achieved complete remission (CR), there was a high rate of early death and an OS of only 20% to 40% was expected at two years. In the LAP0389 study conducted by the Italian cooperative oncology group, GIMEMA (Gruppo Italiano Ematologiche dell’Adulto), 257 patients were randomized to receive either idarubicin alone or idarubicin and ara-C for induction, followed by consolidation with standard chemotherapy and then either by maintenance therapy with 6-mercaptopurine (6-MP) and methotrexate or observation. CR was achieved in 66% to 76% of patients and early death was reported in 15.3% to 21.4%. The estimated eight-year event-free survival (EFS) was 23% to 35% and OS was 36% to 45% in favor of the maintenance arm. Early death was attributed mainly to bleeding from worsening coagulopathy and resistance to chemotherapy (17). In other studies, similar results were obtained and while it was shown that a higher dose of anthracycline was associated with better OS, early death from bleeding remained high (15–20%) (18). Choice of Anthracycline It is not clear if one anthracycline or the other is more effective for induction in APL. Fenaux et al. prospectively randomized patients to either rubidizone or amsacrine, each with ara-C, without observing a difference between the anthracyclines (19). There was a suggestion in a retrospective analysis that idarubicin was associated with an improved outcome, but no prospective randomized trial compared idarubicin with other anthracyclines (20). Although anthracyclines improve OS, early death from bleeding and high relapse rates characterize the predifferentiation era of APL treatment.
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Sequential Studies of ATRA in the Treatment of APL Early phase II studies conducted in the late 1980s and early 1990s show a high CR rate with ATRA alone. In 1988, Huang et al. were among the first to report the activity of ATRA in 24 patients (16 previously untreated and 8 with resistant disease) with 45 to 100 mg/m2/day of ATRA. All patients achieved CR (2). In 1991, Chen et al. reported 50 (47 previously untreated) patients treated with 60 to 80 mg/day of ATRA and observed a 94% CR rate. However, 40% of patients relapsed (median duration of CR was 14.7 months) (21). Although a high CR rate was achieved with ATRA monotherapy and early death was reduced compared with historical chemotherapy studies (likely by resolution of the coagulopathy with ATRA), some patients developed a rapid increase in the leukocyte (WBC) count, which was often accompanied by the development of the RA syndrome (RAS). The manifestations of this unique syndrome include fever, weight gain, pulmonary infiltrates, pleural and pericardial effusions, renal failure, rash, and occasionally death. In the North American Intergroup study (protocol I0129), ATRA was definitively shown to prolong OS in APL patients who were treated with ATRA at induction as compared with patients treated with chemotherapy alone (22). In this study, 346 patients were randomized to receive induction with chemotherapy (daunorubicin and ara-C) or ATRA. OS at one, two, and three years was significantly improved in the ATRA group (75%, 57%, and 50% vs. 82%, 72%, and 67%, respectively) (22). The APL 91 study by the European APL group showed similar results (23). Subsequently, the European APL group compared concurrent ATRA plus chemotherapy with ATRA followed by chemotherapy. This approach resulted in a two-year EFS of 84% for the patients treated concurrently versus 77% for the patients treated sequentially (24). The incidence of RAS was also reduced with the concurrent approach from 25% to 10 % (25,26). Long-term follow-up to the APL 91 study by the European APL group (ATRA followed by chemotherapy vs. chemotherapy alone) found an estimated EFS and relapse rate at four years of 63% and 31 % in the ATRA group, as compared with 17% and 87% in the chemotherapy group. OS at four years was 76% in the ATRA group as compared with 49% in the chemotherapy group (27). Similarly, the North American Intergroup protocol reported a five-year DFS and OS of 69% in patients who received ATRA for induction, compared with 29% and 45%, respectively, for patients induced with daunorubicin and ara-C (28). A recent update of the Spanish cooperative group PETHEMA experience reported DFS of 81% to 90% at three years in 384 patients treated with ATRA and idarubicin for induction followed by consolidation with idarubicn and mitoxantrone. ATRA was added in consolidation in all patients who did not have low-risk disease (29). Maintenance Therapy The importance of ATRA maintenance was demonstrated by two large randomized trials. In the North American Intergroup trial, patients who were randomized to daunorubicin and ara-C (DA) induction and no maintenance following
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consolidation (no exposure to ATRA) had a disease-free survival (DFS) of only 16% compared with 47% in patients who had DA induction and ATRA maintenance following identical consolidation and 74% in those who received ATRA for both induction and maintenance (28). However, two recent studies have suggested that maintenance therapy with ATRA or chemotherapy may not benefit patients who are molecularly negative after intensive consolidation. The Japanese Adult Leukemia Study Group (JALSG) has reported the results of a trial (APL97) in which patients who were reverse transcriptase polymerase chain reaction (RT-PCR) negative following intensive consolidation were randomized to intensive maintenance chemotherapy (no ATRA) or observation (30). No benefit was observed in the group of patients who received maintenance chemotherapy. In a second trial, conducted by the GIMEMA, 586 patients negative for the PML-RARa fusion transcript after consolidation chemotherapy were randomized to one of three maintenance regimens for two years (low-dose chemotherapy, ATRA, both chemotherapy and ATRA) or observation (31). No benefit was observed for any of the maintenance regimens. These data suggest that earlier studies may not have included optimal consolidation. Treatment of Older Adults with APL ATRA is also effective in both elderly patients and in children. In a trial from Spain, 104 patients aged 60 and older received ATRA and idarubicin followed by three consolidation courses of an anthracycline or anthracenedione followed by ATRA maintenance and low-dose chemotherapy. Eighty-four percent achieved CR, the six-year cumulative incidence of relapse was 8.5%, and DFS was 79% (32). Similarly, the GIMEMA conducted a trial in which 134 patients between the ages of 60 and 75 years received a similar regimen as administered in the Spanish trial. The DFS and OS at three years were between 72% and 83% (33). The outcome for patients older than 60 in the APL93 trial was not as favorable when compared with younger adults, with a lower CR rate of 86% versus 94%, and OS at four years was only 57.6% compared with 78% in younger adults. The higher death rate was attributed to higher mortality during consolidation in the elderly group (34). A reduction in the intensity of consolidation in older adults may be a useful strategy. Treatment of Children with APL In a subgroup analysis of the APL93 trial carried out by the European APL Group, 97% of children achieved CR and five-year EFS and OS were 71% and 90%, respectively. Overall, there was no difference in outcome between children and adults (35). In an analysis of the GIMEMA-AIEOP protocol, OS and EFS were of 89% and 76% at over 10 years (36). Both trials confirm that when treated with similar ATRA-based regimens to those used in adults, children fare as well as adults. Similarly, the PETHEMA group treated 66 children with ATRA and idarubicin, followed by consolidation with idarubicin monotherapy and
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ATRA-based maintenance. The OS and DFS at five years were 87% and 82%, respectively (37). Overall, ATRA treatment in children resulted in results similar to those obtained in adults. Because of potential excessive toxicity of ATRA in children, many investigators use a reduced dose of 25 mg/m2/day (38). Liposomal Formulation of ATRA Although ATRA is an extremely effective drug in APL, this agent has the limitation of being available only in an oral formulation. This limitation excludes patients unable to take pills, such as young children and intensive-care patients on life support. However, the introduction of a liposomal formulation proved to be equally effective as oral ATRA and has the added benefit of overcoming the declining blood levels that occur with the oral formulation after several weeks of use (39). At the present time, this formulation is not commercially available. Extramedullary Disease in APL Before the introduction of ATRA, extramedullary disease (EMD) in APL was rarely reported. However, the occurrence of this phenomenon is increasingly observed after treatment with ATRA. While the incidence of EMD appears to be increased in patients receiving ATRA, a report by the GIMEMA compared the risk of developing EMD in two consecutive studies, the LAP0389 and AIDA. Patients receiving ATRA did not appear to have an overall increased risk of EMD (40). However, an increased risk of central nervous system disease was suggested. It is possible that the prolonged survival achieved by treatment with ATRA may account for the perceived increased occurrence of EMD or possibly lack of exposure to ara-C in many patients. Common sites of EMD are the skin (particularly at sites where vascular disruption occurred), central nervous system, external auditory canal, and testis (41–43). Risk factors for developing EMD in APL appear to be a hyperleukocytosis at diagnosis, microgranular variant of APL, and the bcr3 type of the PML-RARa fusion transcript (41). Patients with EMD at relapse may still be salvaged with differentiation therapy. Tsimberidou and colleagues, reported treating two patients with CNS relapse with a combination of ATRA, arsenic trioxide, and gemtuzumab ozogamicin and both achieved CR (42). Others reported successful treatment of EMD relapses with ATRA containing regimens as well (44,45). In summary, ATRA is the treatment of choice for all patients with newly diagnosed APL. It is now routine practice to combine ATRA with chemotherapy during induction. When the presenting WBC is more than 10,000/mL, ATRA can be started alone for two to four days to ameliorate the coagulopathy before initiating chemotherapy (46). It is also routine to administer concurrent ATRA and chemotherapy in this setting, particularly if the coagulopathy is not severe. Following remission induction, consolidation with chemotherapy and ATRA then maintenance therapy is currently the standard of care. However, despite the
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high rate of CRs and cures, some patients, particularly those with high-risk disease, still relapse and require salvage therapy. ARSENIC TRIOXIDE Arsenic Trioxide (ATO) is one of the oldest medicines known and was used both in Chinese medicine and in ancient Greece as a remedy for various ailments. The use of arsenic in hematological disease was noted in the 1880s by Sir William Osler (47). However, the activity of arsenic in modern medicine was revisited in the last two decades in studies originating in China (48). Mechanism of Action ATO has a dual mechanism of action in APL patients. It induces both differentiation and apoptosis. As discussed previously, the chromosomal abnormality t(15;17) results in the PML-RARa fusion protein. This fusion protein causes a maturation block at the promyelocytic stage and disaggregation of PML oncogenic domains, disrupting the NBs and resulting in the loss of tumor suppression (49). At low concentrations, ATO induces the phosphorylation of SMRT/NCoR through the MAPK (mitogen-activated protein kinase) pathway. The SMRT/ NCoR complex dissociates from the PML-RARa fusion protein and allows for coactivators to bind and for transcription to occur, which in turn results in differentiation. In addition, ATO leads to the degradation of the PML-RARa fusion protein and restoration of the normal RARa-RXR dimer, resulting in differentiation (48–50). ATO also induces apoptosis. PML normally controls apoptosis, cell proliferation, and senescence and is localized to the NB (16). The PML-RARa fusion protein disrupts the normal PML localization and function in a dominant-negative way. ATO induces MAPK-mediated phosphorylation of PML leading to sumoylation and retargeting it back to the NB. Restoring normal function eventually triggers growth arrest and apoptosis (48). An additional mechanism by which ATO promotes apoptosis is the generation of reactive oxygen species (ROS). In APL cells, ATO increases the production of NADPH (nicotinamide adenine dinucleotide) and results in the increase of superoxide production. ROS production can induce phosphorylation and activate Jun Nterminal kinase (JNK), which in turn trigger apoptosis (48). In summary, ATO degrades the PML-RARa fusion protein, leads to the dissociation of the corepressors, and allows for differentiation to occur. At higher doses, ATO restores the normal function of PML and promotes apoptosis. CLINICAL EFFICACY OF ATO IN APL Initial Clinical Trials from China Investigators in Harbin, China treated APL patients with the arsenic-containing remedy Ailing 1 in the early 1970s (51). In initial studies, Ailing 1 was able to induce CR in 27% of APL patients (52,53). A later study, which was conducted
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in the 1970s and 1980s and published in 1992, reported a 65% CR rate in 32 patients with APL with a 50% OS at five years, 19% survived at 10 years (54). The active agent was found to be ATO and it was purified and administered to 72 patients with a CR rate of 73% in 30 previously untreated patients and 52% in 42 relapsed or refractory patients (55). The lack of myelosuppression and crossresistance with ATRA as well as the lack of development of hyperleukocytosis in 34 of 44 patients who achieved CR in this study were confirmed in subsequent trials. In 1999, the results of 242 patients with newly diagnosed, relapsed, or refractory APL treated with ATO at Harbin Medical University demonstrated a CR rate of 75% (56).
Clinical Trials in the United States in Patients with Advanced APL These pioneering results from China led to clinical trials in the United States. Initially, two trials of ATO in relapsed and refractory patients were conducted. A single institution pilot study involving 12 patients who previously received ATRA and chemotherapy were treated with 0.06 to 0.2 mg/kg/day until leukemic cells were no longer detected in the bone marrow. CR was obtained in 11 patients (92%) and 8 patients (73%) became negative for PML-RARa fusion transcript by RT-PCR (57). A follow-up multicenter trial of 40 patients with relapsed or refractory APL after ATRA and anthracycline therapy, who received 0.15 mg/kg/ day ATO until the bone marrow cleared or for a maximum of 60 days, reported an 85% CR rate and 86% of assessable bone marrows (29/40) tested negative for PML-RARa transcripts (58). While ATO was active in patients in first or greater relapse, results were best for patients in first relapse (Fig. 1). Niu et al. reported a CR rate of 85% also among 47 patients with relapsed disease (59). Other studies confirmed these results as well (60). Taken together, these studies and others indicate that in patients with relapsed APL, ATO therapy leads to a high CR rate,
Figure 1 Overall and relapse-free survival for relapsed and/or refractory APL treated with arsenic trioxide.
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induces molecular remission in most patients, and is associated with infrequent drug resistance and reversible mild adverse effects. ATO in Patients with Newly Diagnosed APL As the efficacy of ATO in relapsed and refractory disease became clear, investigators began exploring its role in newly diagnosed patients. Investigators at Harbin University in China pioneered this effort. The 88% CR rate reported by Sun and colleagues in 124 previously untreated patients that received single agent ATO was similar to that seen in relapsed patients. These observations, that single agent ATO leads to a high CR rate, were confirmed by studies from India and Iran (54,56,61,62). In the Iranian study, 94 newly diagnosed patients were treated with 0.15 mg/kg daily dose until CR was obtained or for 60 days. Consolidation was given at the same dose for 28 days. These investigators reported an 86.3% CR and 94.5% and 87.6% one- and three-year OS, respectively, for patients who achieved CR. In this trial, patients did not receive chemotherapy. However, hyperleukocytosis occurred in 58.6% of patients and the APL differentiation syndrome occurred in 20.7% of patients. Ten of twentythree patients with the APL differentiation syndrome died of this complication (62). Additional data from India support these findings. In this trial, 72 patients with newly diagnosed APL were treated with ATO as a single agent. At a median follow up of 31 months, EFS and OS were reported as 70.2% and 81.3%, respectively. Many of these patients, however, received chemotherapy in the form of either an anthracycline (6 patients) or hydroxyurea (53 patients) for the APL differentiation syndrome or hyperleukocytosis (63). Table 1 summarizes the experience with ATO as a single agent for induction. Preclinical evidence suggests ATO and ATRA have different mechanisms of action and that, in vitro, they can be synergistic or antagonistic (49,64,65). However, in vivo, these agents have an additive or synergistic effect when administered concomitantly or sequentially, respectively (65,66,67). Results from a clinical trial in which patients received ATO and ATRA for relapsed disease showed that the agents could be given together without producing more toxicity, but without apparent additive benefit (68). In a study from China, Shen et al. randomly assigned 61 newly diagnosed patients with APL to receive ATRA, Table 1 Induction with Single-Agent Arsenic Trioxide for Patients with Untreated APL Study (Ref.) Zhang (56) Ghavamzadeh (62) Mathews (63)
N 124 94 72
CR (%) 88 86 80
PCR-neg (%) NR 92 95
Postremission therapy Chemotherapy ATO 1 ATO 6
Abbreviations: CR, complete response; PCR, polymerase chain reaction; NR, not reacted; ATO, arsenic trioxide.
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ATO, or the combination of both for induction therapy (69). After achieving CR, all patients received three consecutive courses of consolidation therapy with daunorubicin, ara-C and homoharringtonine followed by maintenance with the induction drug plus methotrexate and 6-MP for five cycles. Although chemotherapy was added when hyperleukocytosis occurred (equal among groups), CR rates were more than 90% in all groups and time to CR was faster (25.5 days) in the combination group compared with 40.5 days and 31 days in the ATRA and ATO groups, respectively. Furthermore, in the combination group, platelets recovered faster and PML-RARa transcript copies (as measured by RT-PCR) at CR were significantly lower (69). All patients in the combination group remained in CR at 18 months. At the MD Anderson Cancer Center, Estey and colleagues treated 32 patients with ATRA and ATO as induction therapy. Patients received chemotherapy (typically gemtuzumab ozogamicin) only if they presented with a WBC count of more than 10,000/mL or if they failed to achieve molecular CR three months after achieving CR. Thirteen patients were high risk and received chemotherapy during induction. An 88% CR rate (85% in high-risk group) was reported. Only two patients had a molecular relapse at 6 and 12 months from CR (70). These studies highlight the finding that administering ATRA and ATO concomitantly is safe and results in a greater reduction in disease burden without increased toxicity as compared with each agent alone. In addition, these data indicate the synergy between the agents as suggested by preclinical studies. Despite the synergy between ATRA and ATO, the two agents may not be sufficient for induction. The rate of hyperleukocytosis and APL differentiation syndrome was significant in patients who received ATO as single agent in the Iranian study (62) and mortality was 9%, highlighting the possible need for a cytotoxic agent. Nevertheless, the results from the trial by Mathews and colleagues are very promising and suggest that some patients at very low risk (defined as WBC < 5 109/L and platelet count > 20 109/L) may be curable without chemotherapy and with single agent ATO (Fig. 2). Gemtuzumab
Figure 2 Kaplan–Meier product limit estimate of (A) OS and of EFS (n ¼ 72). (B) Kaplan– Meier product limit estimate of DFS (n ¼ 62). Abbreviations: OS, overall survival; EFS, event-free survival; DFS, disease-free survival.
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ozogamicin (MylotargTM), an anti-CD33 antibody that is chemically linked to the potent cytotoxic antibiotic calicheamicin, is active in APL. In early trials, using gemtuzumab ozogamicin in conjunction with ATRA was effective in controlling leukocytosis (71). OTHER NOVEL THERAPIES IN APL High-risk patients are the group most likely to relapse, and new therapies are needed. Several new approaches are now under clinical investigation such as FLT3 inhibitors, monoclonal antibodies, antiangiogenesis agents, differentiation agents, and histone deacetylase inhibitors (Table 2). FLT3 Inhibitors FLT3 is a tyrosine kinase receptor expressed in leukemic cells. FLT3 mutations that occur as internal tandem duplication (ITD) are the most common genetic abnormality in AML. In APL patients FLT3 mutations are found in approximately 20% to 40% of patients (72,73). The presence of FLT-3/ITD was associated with high peripheral leukemic cell count at presentation and may play a role in the progression of APL (72). Currently FLT3 inhibitors are under clinical investigation, and it remains to be seen if they will provide a therapeutic option in the treatment of APL. Monoclonal Antibodies Alemtuzumab is a humanized anti-CD52 monoclonal antibody with activity in a number of lymphoproliferative diseases and in transplant. Li and colleagues Table 2 Novel Agents with Potential Activity in APL Agent (Refs.)
Rationale
FLT3 inhibitors (72,73)
FLT3 mutations present in 20–40% of APL patients ATRA increases CD52 expression Increased vessels and VEGF in bone marrow samples of APL patients Inhibits NF-kB and induces differentiation with ATRA Increases cAMP—promotes differentiation with ATRA Overcomes ATRA resistance by releasing nuclear co-repressor complex
Alemtuzumab (74) Antiangiogenesis agents (75,76) Curcumin (78,79) Phosphodiesterase Inhibitors (80) Histone deacetylase inhibitors (81)
Abbreviations: APL, acute promyelocytic leukemia; VEGF, vascular endothelial growth factor; ATRA, all-trans retinoic acid; camp, cyclic adenosine monophosphate.
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demonstrated that ATRA induces the expression of CD52 in NB4 cells. Furthermore, they demonstrated that CD52 expression only happened in leukemic cells that expressed PML-RARa or in cells at the promyelocytic stage (74). Currently, data on the clinical efficacy of alemtuzumab in APL are lacking. Antiangiogenesis Strategies Angiogenesis has a critical role in the pathogenesis of hematologic diseases. In APL, angiogenesis in the bone marrow is increased and is mediated by the vascular endothelial growth factor (VEGF). ATRA therapy inhibits VEGF production and decreases microvessel density in the bone marrow (75,76). Clinical trials are needed to establish the role of antiangiogenesis agents such as thalidomide and lenolidomide and other VEGF inhibitors in the treatment of APL. It is hypothesized that VEGF promotes APL cell survival through external and internal autocrine loops (76). It also appears that ATO may have an antiangiogenesis effect as well (77). Differentiation Agents Agents that enhance the activity of ATRA are currently under investigation. Curcumin, an extract from the spice turmeric, inhibits the activation of NF-kB (78) and induces differentiation in the presence of ATRA. Curcumin was found to enhance the activity of ATRA in cell lines resistant to ATRA. NB4-R1 cells, which are resistant to ATRA, differentiated in the presence of ATRA and curcumin but not in the presence of either agent alone. Clinical data are not available at this time. However, these results suggest that curcumin may be a new unconventional agent in the treatment of APL (79). Another compound that may overcome ATRA resistance is cyclic adenosine monophosphate (cAMP). RA-and cAMP-signaling pathways converge on RARa, and cAMP greatly enhances the differentiation mediated by ATRA and ATO. Phosphodiesterase inhibitors such as theophylline, which increase the levels of cAMP, may prove useful in overcoming ATRA resistance (80). Histone Deacetylase Inhibitors The PML-RARa fusion protein leads to abnormal recruitment of histone deacetylase, which in turn leads to a transcription block (5). Therefore, treatment with an inhibitor of histone deacetylase may help reverse this transcription block. One patient with refractory APL resistant to ATRA alone was treated with sodium phenylbutyrate in addition to ATRA. Twenty-three days after initiation of therapy, the patient achieved clinical and cytogenetic CR. A second course of phenylbutyrate resulted in a molecular CR (81). This experience shows that histone deacetylase inhibitors may prove beneficial in ATRA-refractory APL.
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TREATMENT RECOMMENDATIONS FOR APL AND MONITORING OF MINIMAL RESIDUAL DISEASE Induction Therapy Given the proven efficacy of ATRA and ATO in the treatment of newly diagnosed and relapsed APL, new treatment strategies are evolving. The evolution of therapy with ATRA-based regimens is outlined in Table 3. On the basis of the results of the European APL Group trials (APL91, APL93), the North American Intergroup (28), and others, induction therapy should generally include ATRA at a dose of 45 mg/m2/day in divided doses and at least an anthracycline. Because of the apparent increased retinoid toxicities in younger patients, in general patients younger than 20 years, receive ATRA at a dose of 25 mg/m2/day. There is insufficient data to recommend one anthracycline over the other. Idarubicin has been use more often as a single agent without ara-C, and daunorubicin has been used more often in combination with ara-C. ATRA is given until CR or up to 60 days. It is important to resist premature evaluation of the bone marrow before five to six weeks as responses are not expected earlier. In fact, a day-14 bone marrow evaluation, which is so routine after conventional induction chemotherapy for other subtypes of AML, is not useful in APL and can be abandoned. ATRA should be started as soon as the diagnosis is suspected, before genetic confirmation. If the presenting WBC is more than 10,000/mL, it is reasonable to treat with ATRA and concurrent chemotherapy to minimize the coagulopathy (46). Alternatively, if the WBC is low and the coagulopathy is severe, the administration of ATRA for two to four days is a reasonable approach. Table 3 Evolution of Therapy with ATRA-Based Regimens for Patients with Newly Diagnosed APL Regimen Study (Ref.)
N
Induction
Consolidation
Maint
APL 91 (23)
54
DNR þ Ara-C
No
63
DNR þ Ara-C IDA/Mitox þ Ara-C IDA/Mitox ATRA Chemotherapy DNR þ ATRA ATO ATO
Yes Yes
74 89
Yes
90
Yes Yes
100 77*
Interg 0129 (22) GIMEMA (31)
49 108
ATRA þ DNR þ Ara-C ATRA ATRA þ IDA
PETHEMA (29)
384
ATRA þ IDA
Shanghai (69) Interg C9710
20 500
ATRA þ ATO ATRA þ DNR þ Ara-C ATO
Iran (62)
94
ATO
DFS (%)
63.7
Abbreviations: ATRA, all-trans retinoic acid; DNR, daunorubicin; IDA, idarubicin; Mitox, mitoxantrone; ATO, arsenic trioxide; Interg, North American Intergroup. *Event-free survival.
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Consolidation Therapy Standard consolidation chemotherapy regimens include anthracyclines with the goal of eradicating the leukemic clone. The optimal number of consolidation courses is not well defined, but most clinicians administer two to three courses. The role of ara-C has been questioned in this setting. Data presented by Sanz and colleagues demonstrated equal efficacy between anthracycline-based consolidation therapy with and without ara-C (82). Hence, it appears that ara-C may not have a role in this phase of treatment when idarubicin is given, as the anthracycline and ATRA is administered in consolidation. However, in a randomized trial carried out by the European APL group, newly diagnosed patients were randomized to either ATRA, DA, or ATRA and daunorubicin without ara-C (83). The study was closed early because of an increase in relapses among the patients not receiving ara-C. The North American Intergroup (protocol C9710) studied the role of ATO in consolidation. The results suggested a benefit for early consolidation with two cycles of ATO (84). Several other studies have suggested that the administration of intermediate-dose or high-dose ara-C either in induction or in consolidation may benefit patients with high-risk disease (85,86). Maintenance Therapy Randomized studies have demonstrated the superiority of ATRA containing maintenance regimens over that containing no maintenance in reducing relapse (24,27,28,87). Long-term DFS rate with ATRA-based maintenance was 74% to 90%. The question if chemotherapy such as methotrexate and 6-MP add to ATRA maintenance alone is still under investigation. It is currently recommended to give ATRA-based maintenance for at least one to two years following consolidation. However, recent studies have suggested that patients rendered molecularly negative with intensive consolidation may not benefit from maintenance. Relapsed Disease ATO has excellent activity in relapsed and refractory disease and is considered the treatment of choice for relapsed disease. ATO is given at a dose of 0.15 mg/ kg/day for five days a week for five weeks. ATRA as a single agent in relapsed disease produces inferior results compared with ATO (88). Patients who have refractory disease by morphology or persistent disease by molecular studies and are otherwise suitable candidates should be referred for allogeneic hematopoietic stem cell transplant. If a donor is not identified, patients should enroll in a clinical trial. Monitoring of Minimal Residual Disease The PML-RARa fusion protein allows for monitoring of disease at the molecular level. Achieving complete molecular remission on two or more consecutive
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RT-PCR assays is strongly linked to a decreased rate of relapse and prolonged survival. Conversely, patients with two or more positive RT-PCR were likely to relapse. A molecular relapse may precede clinical relapse by several months (89). As burden of disease is substantially lower at the time of molecular relapse as compared with morphologic relapse, there may be a role for the use of ATO at the time of molecular relapse without waiting for morphologic relapse (90). The suggested molecular testing should consist of at least two successive marrow samples at the end of treatment, then every three months for the first two years of CR, then every six months for the next two to three years (90). Such monitoring may provide the most benefit for high-risk patients. CONCLUSION The treatment of APL has evolved rapidly over the last 15 years. Once a disease with poor OS and often an abrupt catastrophic course, APL has become the most curable of all the subtypes of AML. The unique biology of APL, in which an aberrant fusion protein inhibits differentiation and apoptosis, lends itself to a therapeutic strategy that overcomes the transcription block. ATRA and ATO both target the fusion protein and restore differentiation and apoptosis by degrading the PML-RARa fusion protein. Both agents induce a high rate of remission as single agents. Synergy between ATRA and ATO may permit a reduction or possibly elimination of chemotherapy in most patients. This approach could be used in conjunction with gemtuzumab ozogamicin for induction and potentially offer patients with a curative approach without the use of standard intensive chemotherapy. The role of other novel agents such as FLT3 inhibitors, monoclonal antibodies, antiangiogenesis agents, differentiation agents, and histone deacetylase inhibitors remains to be determined. Insights gained in the molecular events that lead to APL may potentially provide advances in the understanding of leukemogenesis in other AML subtypes. These may lead to the development of other differentiation-inducing therapies or novel strategies for other AML subtypes. Clinical Perspective for the Next Five Years Improvements in the outcome of patients with APL will continue to occur and potential short-term and long-term toxicities are likely to diminish. Further characterization of the molecular genetics and clinical features of APL, particularly high-risk patients, may allow more specifically directed therapy. A significant proportion of high-risk patients appear to express the FLT3/ITD gene mutation raising the possibility of treatment with a FLT3 inhibitor (91). It is clear that therapy in the next five years will involve less, and perhaps minimal, or even no cytotoxic chemotherapy since the combination of ATRA and ATO appear to be extremely effective in inducing a molecular remission. ATO alone may be very effective in low-risk patients. More potent synthetic retinoids such as
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tamibarotene (AM80), which has a low affinity for the cellular RA-binding protein and does not bind to RARg, which may limit toxicities such as rash, may prove to be another important advance (92,93). REFERENCES 1. Yanada M, Suzuki M, Kawashima K, et al. Long-term outcomes for unselected patients with acute myeloid leukemia categorized according to the World Health Organization classification: a single-center experience. Eur J Haematol 2005; 74(5): 418–423. 2. Huang ME, Ye YC, Chen SR, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988; 72(2):567–572. 3. Alcalay M, Zangrilli D, Pandolfi PP, et al. Translocation breakpoint of acute promyelocytic leukemia lies within the retinoic acid receptor alpha locus. Proc Natl Acad Sci U S A 1991; 88(5):1977–1981. 4. Zelent A, Guidez F, Melnick A, et al. Translocations of the RARalpha gene in acute promyelocytic leukemia. Oncogene 2001; 20(49):7186–7203. 5. Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999; 93(10): 3167–3215. 6. Lowenberg B, Griffin JD, Tallman MS. Acute myeloid leukemia and acute promyelocytic leukemia. Hematology (Am Soc Hematol Educ Program) 2003 2003(1): 82–101. 7. Nagpal S, Saunders M, Kastner P, et al. Promoter context- and response elementdependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors. Cell 1992; 70(6):1007–1019. 8. Nagpal S, Friant S, Nakshatri H, et al. RARs and RXRs: evidence for two autonomous transactivation functions (AF-1 and AF-2) and heterodimerization in vivo. Embo J 1993; 12(6):2349–2360. 9. Chen JD, Evans RM. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 1995; 377(6548):454–457. 10. Horlein AJ, Naar AM, Heinzel T, et al. Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 1995; 377 (6548):397–404. 11. Pazin MJ, Kadonaga JT. What’s up and down with histone deacetylation and transcription? Cell 1997; 89(3):325–328. 12. Wolbach SB, Howe PR. Nutrition classics from J Exp Med 42: 753–77, 1925. Tissue changes following deprivation of fat-soluble A vitamin. Nutr Rev 1978; 36(1):16–19. 13. Hodges RE, Sauberlich HE, Canham JE, et al. Hematopoietic studies in vitamin A deficiency. Am J Clin Nutr 1978; 31(5):876–885. 14. Collins SJ, Robertson KA, Mueller L. Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor (RAR-alpha). Mol Cell Biol 1990; 10(5):2154–2163. 15. Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci U S A 1980; 77(5):2936–2940. 16. Salomoni P, Pandolfi PP. The role of PML in tumor suppression. Cell 2002; 108(2): 165–170.
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67. Lallemand-Breitenbach V, Guillemin MC, Janin A, et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J Exp Med 1999; 189(7):1043–1052. 68. Raffoux E, Rousselot P, Poupon J, et al. Combined treatment with arsenic trioxide and all-trans-retinoic acid in patients with relapsed acute promyelocytic leukemia. J Clin Oncol 2003; 21(12):2326–2334. 69. Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci U S A 2004; 101(15):5328–5335. 70. Estey E, Garcia-Manero G, Ferrajoli A, et al. Use of all-trans retinoic acid þ arsenic trioxide as an alternative to chemotherapy for untreated APL. Blood 2006; 107(9): 3469–3473. 71. Estey EH, Thall PF, Giles FJ, et al. Gemtuzumab ozogamicin with or without interleukin 11 in patients 65 years of age or older with untreated acute myeloid leukemia and high-risk myelodysplastic syndrome: comparison with idarubicin plus continuous-infusion, high-dose cytosine arabinoside. Blood 2002; 99(12):4343–4349. 72. Kiyoi H, Naoe T, Yokota S, et al. Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group of the Ministry of Health and Welfare (Kohseisho). Leukemia 1997; 11(9):1447–1452. 73. Au WY, Fung A, Chim CS, et al. FLT-3 aberrations in acute promyelocytic leukaemia: clinicopathological associations and prognostic impact. Br J Haematol 2004; 125(4):463–469. 74. Li SW, Tang D, Ahrens KP, et al. All-trans-retinoic acid induces CD52 expression in acute promyelocytic leukemia. Blood 2003; 101(5):1977–1980. 75. Kini AR, Peterson LA, Tallman MS, et al. Angiogenesis in acute promyelocytic leukemia: induction by vascular endothelial growth factor and inhibition by all-trans retinoic acid. Blood 2001; 97(12):3919–3924. 76. Kini AR, Roychowdhury S, Dziuma A, et al. Vascular endothelial growth factor (VEGF) is a critical autocrine survival factor in acute promyelocytic leukemia (APL). Blood 2005; 106(11):772a. 77. Alimoghaddam K, Shariftabrizi A, Tavangar M, et al. Anti-leukemic and antiangiogenesis efficacy of arsenic trioxide in new cases of acute promyelocytic leukemia. Leuk Lymphoma 2006; 47(1):81–88. 78. Weber WM, Hunsaker LA, Roybal CN, et al. Activation of NFkappaB is inhibited by curcumin and related enones. Bioorg Med Chem 2006; 14(7):2450–2461. 79. Kini AR, Nagabhushan M, Tallman MS, et al. Curcumin enhances differentiation of all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant acute promyelocytic (APL) cells. Blood 2005; 106(11):192b. 80. Guillemin MC, Raffoux E, Vitoux D, et al. In vivo activation of cAMP signaling induces growth arrest and differentiation in acute promyelocytic leukemia. J Exp Med 2002; 196(10):1373–1380. 81. Warrell RP Jr., He LZ, Richon V, et al. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst 1998; 90(21):1621–1625. 82. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracyclinebased consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. PETHEMA group. Blood 1999; 94(9):3015–3021.
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9 DNA Methylation and Epigenetics: New Developments in Biology and Treatment Jesus Duque and Michael Lu¨bbert Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany
Mark Kirschbaum Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte, California, U.S.A.
INTRODUCTION Epigenetic changes are defined as all meiotically and mitotically heritable changes in gene expression that are not encoded in the DNA sequence itself. The field of epigenetics is becoming increasingly prominent because it presents a key to understanding differences between growing and senescent cells, tumor and normal cells, and differentiated and aging cells, which can be exploited for treatment of multiple conditions. Interwoven mechanisms, which regulate the gene expression, like DNA methylation, histone modification, and RNA-associated silencing, are recognized as players in a system of growing complexity. While it appears that DNA methylation is the paramount signal, over time, it has become clear that this is one step of a well-choreographed set of modulations at the histone level. The centrality to life of epigenetic regulation, in processes such as differentiation, apoptosis, and cell replication necessitates tight regulation of the accessibility to transcription of DNA. Whole sections of DNA may need to be made unavailable to transcription, while certain spans may need
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to be activated or silenced rapidly in response to conditions inside and out of the cell, and this is coordinated between changes in methylation at the DNA level and the spectrum of changes at the histone level (1). Our ability to treat cancer with hypomethylating agents will be improved as we unravel the systems with which it collaborates. For this reason, we will review the current biology and treatment paradigms based on DNA methylation and its inhibition as well as survey several important examples of epigenetic coordination between DNA methylation and other histone modifications. DNA Methylation in Normal Hematopoiesis and in Cancer Three to six percent of all cytosines are methylated in the genome of mammalian cells. The reaction of cytosine methylation consists of the incorporation of a methyl group from the S-adenosyl-methionine in a previously unmethylated cytosine and it is catalysed by DNA methyltransferases (DNMTs) (2). There are three functional DNMTs: DNMT1, which maintains the pattern of methylation during replication, maintenance methylation (3), DNMT3A, and DNMT3B, both of which can add methyl groups to previously unmethylated templates—de novo methylation (4). The methylated cytosines are mostly localized in CpG dinucleotides. In the mammalian genome, CpG dinucleotides are not found randomly, but in clusters of CpGs, termed CpG islands, which are often associated with sites where the transcription of DNA into RNA is initiated (5). About half of the genes have CpG islands in their promoters (6) (Fig. 1). The normal physiological function of DNA methylation is multifaceted. The repetitive genomic sequences and noncoding regions of the genome are heavily methylated; DNA methylation plays a role in the protection of chromosomal integrity (7). Genomic imprinting, to establish which parental allele will be expressed, is also determined by DNA methylation (8), and the inactivated X chromosomes in females are heavily methylated (9). An important function of DNA methylation is to regulate the expression of tissue-specific genes (10). In normal granulopoiesis, it was shown that MPO-expression is tightly controlled by its methylation status at different stages of myelopoiesis (11). C/EBPa, an important transcription factor of myelopoiesis, is infrequently methylated in some myeloid leukemias, whereas hypermethylation of its promoter is predominantly found in M2 subtype from FAB classification (12,13). In normal erythropoiesis, the g chain of the fetal hemoglobin (HbF) is silenced by DNA methylation after fetal development and replaced by the b chain, which forms the adult hemoglobin A (HbA) (14). DNA methylation has been related to the normal aging process (15). CpG islands in promoter regions of some genes, which have been studied mostly in colon cells, appear to undergo increasing methylation with age. These changes underscore the potential role of aging in the genesis of colorectal cancer (16,17). Aberrant DNA methylation is implicated in cancerogenesis in several ways. In tumor cells there is an apparent paradox—global genomic hypomethylation, but
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Figure 1 Reversibility of DNA methylation in tumor cells. (A) The distribution of DNA methylation in normal cells is predominantly concentrated in the CpG dinucleotides of transcriptional region of genes. The promoters of genes are hypomethylated, so the transcription could be induced by the presence of transcription factors. (B) In contrast, there is a redistribution of the DNA methylation pattern in tumor cells. The transcriptional regions of genes are hypomethylated and the promoters of tumor suppressor genes are hypermethylated, thus avoiding the transcription. (C) The demethylating drugs, like 5-azacytidine and decitabine, can relieve this situation in tumor cells. They are incorporated in the DNA during the S phase of the cell cycle. The DNMTs bind covalently to these molecules, depleting the amount of active DNMTs in the cells. The consequence is a global DNA hypomethylation and the transcription of genes, which were silenced by DNA hypermethylation, could be restored. Abbreviation: DNMTs, DNA methyltransferases.
with hypermethylation concentrated in the promoters of several genes (18). This leads to a picture of overall dysregulation favoring tumor formation, with both inappropriate silencing and activation. For example, global genomic hypomethylation can lead to gene activation of the so-called cancer testis antigens (CTAs) like MAGE and GAGE family and NY-ESO1 genes (19), but can also lead to chromosomal instability and further genomic aberrations, e.g., Wilms’ tumors (20), and in the rare ICF (immunodeficiency, chromosome instability, and facial anomalies) syndrome. This syndrome is caused by a germline mutation in DNMT3B and subsequent genomic hypomethylation, though without a predisposition to cancer (21–23).
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Aberrant de novo methylation of CpG islands is a hallmark of human cancers and is found early during carcinogenesis. The CpG islands in the promoters of several tumor suppressor genes are hypermethylated and thus silenced in different tumor types, e.g., p16INK4A in several tumor types (24), p15INK4B cyclin-dependent kinase inhibitor in acute myeloid leukemia (AML) (25), RARb2 in colon, lung, head, and neck tumors (26,27), retinoblastoma (Rb) cell cycle inhibitor in sporadic and hereditary Rb (28,29), and MLH1 DNA mismatch repair gene in colon tumors (30). Tumors with especially frequent aberrant gene methylation appear to constitute a specific disease entity labeled CpG island methylation phenotype (CIMP) with a worse prognosis (31). How does hypermethylation and silencing promote tumorogenesis? Disruption of the function of a tumor suppressor gene, as defined by Knudson (32), requires a complete loss of function of both copies of the gene involved. Abnormal promoter methylation can have the same effect as a coding-region mutation in one copy of the gene and in this manner cooperates with somatic and familial mutations in the cancerogenesis. Thus, silencing of p16 does not appear in tumors in which the Rb gene is mutated; similarly, mutations in the VHL gene in renal cancer are found in 60% of the cases, while the gene is hypermethylated in another 20%. Also, mutations and epigenetic silencing of the same gene or different genes with similar functions seem to be mutually exclusive (33). Loss of imprinting (LOI) is another mechanism whereby DNA methylation contributes to genetic activation in some types of cancers. Biallelic expression, caused by LOI of the gene IGF2 is a common feature in normal colonic mucosa in patients with colorectal cancer, and the detection of LOI can be used as a predictive marker of individual risk (34). What underlies these epigenetic changes? An aberrant pattern of methylation in tumor cells may be caused by external factors (e.g., toxins, virus, or diet) or by other genetic aberrations (point mutations or chimeric fusion proteins from chromosomal translocations). Toxins like cadmium may inhibit DNMTs and lead to an acute hypomethylation of the DNA followed by hypermethylation after chronic exposure (35) and, like arsenic, can induce Ras hypomethylation in methyl-deficient C57BL/6J mice (36). The latency of cervical cancer and lymphoma seems to be caused, at least in part, by hypermethylation of the promoter of the human papillomavirus 16 (HPV16) genome (37) and Epstein–Barr virus, respectively (38). A polymorphism of the MTHFR gene, which plays a role in the S-adenosyl methionine synthesis, was associated with increased colorectal cancer in a population-based study, where cancer incidence was lower in patients with high dietary methionine (39). In animal models, methyl-deficiency diets (folic acid, methionine, choline, vitamin B12 deficiency diet) lead to increased DNA hypomethylation and development of hepatomas (40). Point mutations in oncogenes like the MYC gene, which has been shown to associate with DNMT in osteosarcoma cells, might also target DNA methylation pattern in tumor cells. Furthermore, the MYC protein does not bind to its recognition sequence in the methylated condition of that site, suggesting that DNA
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methylation may affect the ability of MYC to bind multiple sites within the genome, thus also affecting the chromatin structure (41). Epigenetic dysregulation of gene expression could also be secondary to chromosomal translocations. In human leukemias, the chimeric fusion protein PML/RARa, which arises from the chromosomal translocation t(15;17) in APL, recruits histone deacetylases (HDACs) and DNMTs to the promoters of RARa target genes, leading to hypermethylation and silencing of the RARb2 tumor suppressor gene (42). Another chimeric fusion protein, AML1/ETO resulting from the t(8;21) in AML, recruits HDACs via the corepressor complex NCoR and m-Sin3a (43,44) and DNMTs (45) to target genes, e.g., IL-3 gene, silencing their expression. Although this may not be a specific mechanism of these diseases, as RARb2 is found to be methylated in several tumors and not only in APL (46,47), it is illustrative of a link between genetic and epigenetic aberrations in cancer. DNA methylation is likely not the sole mediator of gene silencing but seems to be critical in maintaining silencing in tumors; DNA hyper- and hypomethylation in tumors are good markers of epigenetic dysregulation in cancer. It is generally accepted that WT1 functions as a tumor suppressor gene in Wilms’ tumor. However, WT1 gene is highly expressed in other tumors, including colorectal, breast, leukemia, and desmoid, suggesting a function also as an oncogene (48). The WT1 locus was found affected in Wilms’ tumors by intragenic deletions and mutations in about 20% of patients and by LOH because of promoter hypermethylation (49), confirming the revised two hits hypothesis in the tumorogenesis. DNA methylation is causal in maintaining the silenced epigenetic state but reactivation of silenced tumor suppressor genes can be achieved by demethylating agents and by siRNAs to inactivate DNMTs. DNA Methylation and Histone Acetylation Epigenetic changes such as DNA methylation need to be maintained and transmitted to daughter cells, necessitating tight coordination among the various enzymes charged with histone regulation (1), for this reason a brief review of other histone related modifications is necessary to fully explicate the role of DNA methylation changes. Over 60 types of histone modification are described at this time, with a paradigm shift from the study of individual changes to that of signaling based on interrelationships between modification, which is referred to as the ‘‘histone code’’ (50). This implies that the modifications are read in terms of the overall set of changes at the histone tail, beyond the effect of any single modification alone, and at the organization level of nucleosomes. The nucleosomes are structures composed of a core of histone proteins around which the DNA binds. At activated genes, where the promoters of the genes are hypomethylated, the nucleosomes are widely and irregularly spaced, promoting the access to transcription factors to initiate transcription. In contrast, at inactivated genes, the nucleosomes are tightly compacted and regularly spaced (33). This configuration of nucleosomes is controlled by posttranslational modifications in
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amino acid residues of histone tails and by DNA-binding proteins like MBD2, which interacts with the nucleosomal remodeling complex NuRD (51), and MeCP2, which associates with the SWI/SNF chromatin-remodeling complex (52). As a result, it has been increasingly recognized that the histones, rather than merely having a structural function, are dynamic regulators of gene activity through posttranslational modifications like acetylation, methylation, phosphorylation, and ubiquitylation. Acetylation and methylation of conserved lysine residues of histone tail domains are known to be important regulators of gene expression. The biology of histone acetylation will be described in more detail in a separate chapter in this volume; here, a brief overview is presented with a focus on interactions between DNA methylation and the other epigenetic processes. The presence of acetyl groups in the histone alters the charge so that chromatin binds less tightly to the phosphate backbone of DNA. Acetylation, in general, activates, whereas deacetylation is suppressive; these changes, modulated by histone acetyltransferases (HATs) and HDACs are rapidly reversible and subject to multiple signals. There are numerous proteins with HAT activity, worthy of note for their relationship to hematological malignancies. CREB (cyclic AMP response element binding protein) and p300 are frequent partners in leukemia and myelodysplastic syndrome (MDS)-related translocations, fusing with acetyltransferases of the MYST family such as MOZ and MORF and the H3K4 methyltransferase mixed lineage leukemia (MLL) (53). It is of interest that in these cases, the fusion protein often contains two distinct active HAT domains. Mutations within the HAT domains are also associated with leukemias (54), confirming the impression that changes in acetylation represent important moments in leukemogenesis. Deacetylation is accomplished via a large group of enzymes comprising, at this time, four groups (based on homology to yeast proteins) of HDACs, with the class I HDACs, similar to Rpd3, being the dominant group of nuclear HDACs, class II, similar to HdaI, having cytoplasmic activity as well in their role as protein acetylases, the class III HDACs, the Sir2 homologs, having very different structure and activity, now generally referred to as Sirtuins, and the class IV HDAC11. In order to understand their manifold activities, it is important to recognize that HATs and HDACs (as well as many of the other enzymes which act in an epigenetic manner) tend to be recruited to large complexes through which they exert their action, with the presence of chromodomains and bromodomains, domains that bind methyl-lysine and acetyl-lysine, respectively (55). HDAC 1 and 2 complex with mSin3A to form a repressor complex (56,57), and complex with NCoR and SMRT to act as nuclear hormone receptor corepressors (58,59). Complexes seem to facilitate the activity of other complexes, so that the various SWI/SNF chromatinremodeling complexes act to recruit HAT complexes leading to acetylation (60–62) and retinoblastoma-E2F complexes recruit HDACs as part of their repressive activity (63). Interestingly, SWI/SNF complex activity has been shown to bring about loss of DNA methylation as well at targets such as CD44 and E-cadherin (64), introducing further levels of network-like complexity to the interplay between
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the various epigenetic enzymes. Thus, it becomes less surprising that treatment with the hydroxamate HDAC inhibitor (HDACi) LBH589 can lead to decreased histone methylation via the disruption of the polycomb repressor complex 2 (65) or that valproic acid, a drug used for years as an antiepileptic agent and recently found to have HDAC activity (66), can lead to DNA demethylation (67). This may help explain the dramatic activity of HDACi, such as vorinostat and LBH589, as single agents particularly in lymphoid malignancies, with single agent activity seen in Hodgkin’s lymphoma, diffuse large B cell lymphoma, ALL, and indolent lymphomas (68–71, Kirschbaum, personal communication) (Fig 2).
Figure 2 Modulation of gene transcription by HDACis. (A) The aberrant recruitment of HDACs to genes is a common hallmark in cancer, so the balance between HATs and HDACs to control gene transcription is disrupted. The HDAC activity predominate, causing hypoacetylation of histone tails around the transcriptional start site. The conformation of histones is closer, and the distance between nucleosomes is shorter. In this way, the transcription factors are not able to initiate the transcription. (B) However, this scenario could be reverted by HDACis, which inactivate transiently the aberrant activity of HDACs. The HAT activity dominates, and the histone tails become hyperacetylated, opening the structure of histones and spacing the nucleosomes. The transcription factor can start the transcription of genes, which were blocked by HDACs. Abbreviations: TF, transcription factor; HATs, histone acetylases; HDACs, histone deacetylases; HDACis, histone deacetylase inhibitors; Ac, acetyl group.
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A very important discovery in the past years was the finding that DNMTs (DNMT3A and DNMT3B), the enzymes which mediate DNA methylation, as well as methyl-binding proteins (MECP2 and MBD2) were physically associated with HDACs and histone methyltransferases (HMT), leading to a connection between the three major epigenetic events: DNA methylation, histone modification, and nucleosomal remodeling (72–74). It is thought that the first event is DNA methylation of the gene promoters, catalyzed by DNMTs. When a cluster of CpGs is methylated, the methyl-binding proteins can recognize the methylated DNA and interact directly with the DNA, thus repressing the transcription by limiting the access to transcription factors. The attached HDACs and HMTs modify further the histone tails by removing acetyl groups from lysine residues or adding methyl groups to lysine residues, leading to a change in the histone tail into a closed conformation. Generally, DNA methylation is associated with histone hypoacetylation of histones 3 (H3) and 4 (H4) and transcriptional repression, whereas hyperacetylated histone marks transcriptionally active genes (75,76). Interestingly, histone modifications are not only related to epigenetic regulation of transcription, but also cooperate with other proteins to regulate nuclear processes like DNA repair. Certain genes, with tumor suppressor properties such as p21WAF1, are silent at the transcriptional level in the absence of DNA hypermethylation and the presence of H3 and H4 hypoacetylation (77). Moreover, H3 acetylation is functionally linked to H3K4 trimethylation by the MLL4 protein, which has a very high homology to MLL1 gene. H3 acetylation determines the degree and the abundance of H3K4 methylation and this interaction plays an important role in the epigenetic response to the HDACis valproate and butyrate (78). DNA Methylation and Histone Methylation It is important on the one hand to distinguish between DNA methylation and histone methylation, although, as we will demonstrate, there are multiple points of contact between the two. Histone methylation occurs on lysine and arginine, with arginine capable of receiving up to two methyl groups, and lysine up to three; the relevant genes are activated or repressed depending on the configuration of histone methylation. Most known mammalian lysine methylating proteins have homologous catalytic domains, known as SET (SuVar3–9, Enhancer of Zeste and Trithorax), and divide in to families depending upon lysine target. We will review histone methylation with regard to DNA methylation activity, grouped by histone methylation mark. Histone methylation is a marker of both active and inactive genes. Trimethylation of lysine 4 in H3K4 is linked to activated genes (79,80). Methylation at this site is associated with multiple other modifications such as acetylation by acetyltransferases (81) and deacetylation by deacetylases, the latter activity perhaps acting as a brake on genes actively transcribed by H3K4 methylation (82). On the other hand, some H3K4 interactions are specific for unmethylated DNA. The
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MLL enzymes, which can polymethylate H3K4 (83,84), exist as multicomponent complexes containing differing catalytic SET-related units (85). MLL1 in particular interacts with other modifiers such as the acetylases MOF and CBP (86), and along with other MLL enzymes also recruits the homeobox family genes. The Hox transcription factors play a role in embryonic development, as well as in angiogenesis, in which HoxD3, HoxB3, and HoxA9 are essential regulators. MLL1 is responsible for H3K4 trimethylation at the HoxA9 locus (86). It has recently been shown that suppression of MLL will inhibit Hox-related proangiogenic activity (87). A necessary component of the MLL complex that regulates Hox gene expression is menin, which specifically associates with MLL proteins among SET1 homologs. Menin is the protein encoded by Men1, which when mutated leads to multiple neoplasms, particularly in endocrine tissue (88). Hox gene expression is dependent upon the association of menin with MLL (89). It thus comes as no surprise that mutations in MLL would be associated with cancer; over 50 mutated fusion proteins as the result of chromosomal translocations have been described, and this mutation is frequent in ALL of infants (90). It is noteworthy that MLL-related transformation, with the resultant disregulation of Hox proteins, is dependent on its association with menin, implicating the methyltransferase activity as critical for leukemogenesis (91,92). It is thus interesting that the ‘‘repression domain’’ of MLL is specific for unmethylated CpG sequences (93). H3K9 methylation, particularly trimethylation, is believed to be crucial to formation of heterochromatin (80), particularly as this methylation creates a binding site for the chromodomain (chromatin organization modifier) of the repression related protein HP1. Trimethylation at H3K9 is achieved by Suv39h1/ Suv39h2 (94) which was the first HMT identified. This process is involved in several tumor-related signaling pathways, such as suppression achieved by Smads after TGF-b signaling (95), or suppression of cyclin E and E2F by Rb protein, the latter losing this suppressive ability when mutated (96,97). Relevant to our argument is that this repression requires the recruitment of HDACs (98). Furthermore, it appears that this Suv39h-HP1-mediated mechanism is intimately involved with DNMT3A and DNMT3B DNA methylating activity (99). It is worth noting that mice with decreased levels of Suv39h1 showed a high incidence of B-cell lymphomas (94). Of particular interest with regard to leukemia, it has been shown that residues 380–432 and 351–381 in the RUNX1 transcription factor, bind Suv39h1 as well as HDAC1 and 3 (100). RUNX1 is a component of fusion proteins found frequently in AML, such as t(8;21), t(12;21), and related to the activity inv(16); this relationship to H3K9 methylation may explain the release of lysosomal repression in cells with these mutations upon treatment with HDACi and hypomethylating agents (101). SETDB1, another enzyme which di- and trimethylates histones at H3K9, also interacts directly with DNMT3A and DNMT3B (102). Another silencing histone methylation mark with relationship to DNA methylation activity is at H3K27 (103). The complexes that bring about this
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methylation share an EZH2 enzyme, part of the polycomb group (PcG) of proteins, which have an important role in silencing the Hox transcription factors, which as described earlier, are critical modulators of development and angiogenesis (104). Methylated H3K27 leads to further repression through binding of other PcG proteins, as well as specific interaction with all three DNMT (76). This EZH2-based relationship between H3K27 and DNA methylation appears to be particularly marked in cancers as opposed to normal tissue (105). Thus it is indicative of the complexity of attempting epigenetic therapy, that treatment of leukemia cells with the hydroxamic acid HDACi LBH589 led to depletion of EZH2 and decreased histone methylation of H3K27 (65). Clinical Use of DNA Hypomethylating Agents One of the most intriguing and clinically exploitable aspect of epigenetics as a target for anticancer therapy is that epigenetic aberrations, described above, are pharmacologically reversible, as opposed to purely genetic aberrations, which are irreversible. Inhibitors of DNA methylation have been shown to reactivate the expression of genes that have undergone epigenetic silencing, particularly if this silencing has occurred in a pathological situation. 5-azacytidine (Vidaza1) and 5-aza-20 deoxycytidine (Dacogen1, decitabine, DAC), which were initially developed as cytotoxic antineoplasic agents when used at maximally tolerated doses as with standard chemotherapeutic agents, were found to have the clinically more important DNA demethylating activity when used at very low doses (106) leading to the recognition of its clinical activity in MDS and AML (107). Both compounds are cytosine analogs that inhibit DNMT, reverse methylation, and reactivate genes. When used at these doses, these agents were shown to differentiate cells in tissues cultures (106) and to induce gene re-expression. These agents do not have direct demethylation activity, rather, 5-azacytidine and decitabine have to be incorporated in the DNA in the S-phase of the cell cycle, covalently binding DNMTs, and thus depleting the nucleus of their enymatic activity (108,109). DNA replication in the absence of DNMTs leads to global and gene-specific hypomethylation (110). In vitro and in vivo, these compounds are able to demethylate the promoter of hypermethylated genes, as was shown for p15INK4B promoter in MDS patients, leading to a reactivation of silenced genes (25,111). At the cytotoxic doses of these compounds, cell death may be due to DNA damage and apoptosis (108). While decitabine at cytotoxic doses demonstrated significant antitumor activity in hematological malignancies in clinical trials, severe and prolonged myelosuppression was frequently observed (112). In vitro, these substances have shown to produce more hypomethylation at lower doses than at high doses (113,114). In vivo, paradoxically, it is at the hypomethylating doses where they produce significant antitumoral activity after a long latency to response in MDS and AML patients (115–117). Repeated courses of 5-azacytidine and decitabine are required to achieve a clinical response, which
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is understandable from the biology of the activity, which necessitates incorporation and subsequent depletion of DNMTs. Thus, the older trials in hematologic and nonhematologic malignancies from the early 1980s are now understood to have been designed with supotimal dosing plans and premature clinical evaluations (e.g., after only two courses of therapy) (118). The current recognition of continued treatment is predicated upon epigenetic changes being reversible, with aberrant promoter methylation and gene silencing return, once 5-azacytidine or decitabine treatment is stopped (119–121). Clinical responses in the therapy with demethylating agents have been related to hypomethylation during the treatment of the p15 promoter (25) with reactivation of expression of p15 gene (122). Hypomethylation in bone marrow of patients treated with decitabine appeared after karyotype normalization, suggesting clonal demethylation changes and demethylation of normal cells (123). In contrast, shorter overall survival was significantly correlated with baseline hypermethylation of multiple genes like p15 and E-cadherin in a multivariate analysis of a phase III study (124). These studies provide proof of principle, that 5-azacytidine and decitabine can induce responses through gene hypomethylation in AML and MDS but other mechanisms of action should not be excluded. In vitro, expression of p21WAF1 was induced by decitabine in cell lines in association with the release of HDAC1 and increased acetylated H3 at the unmethylated p21WAF1 promoter (125). In vivo, global changes of gene expression of primary AML and MDS cells were analyzed using microarrays before and after decitabine treatment. 80 of 22,000 genes were induced, half of them had a CpG island in the promoter region, and the changes in methylation status of promoters could only be validated in one gene, coding for myeloperoxidase (126). Both azacytadine and decitabine have been approved by the FDA for the treatment of MDS, with registration studies for the treatment of AML ongoing. In future, there will be considerable focus on developing compounds that directly inhibit DNMTs. RG101 is one such lead compound currently in clinical development (127). Hypomethylating Agents in Combination with Histone Deacetylation Inhibition HDACis can induce differentiation, growth arrest and/or apoptosis in transformed cells, and have antitumoral properties (128,129). Accumulation of acetylated proteins, particularly histones, results in the induction of several epigenetically silenced genes, some of them with tumor suppressor properties, like the cell cycle kinase inhibitor p21 (77). The first drug of this type, suberoylanilide hydroxamic acid (SAHA) has very recently been approved by the FDA for the treatment of cutaneous T-cell lymphoma, and very active development programs are ongoing for other new HDACis (130) (Table 1). The link between DNA methylation and histone modification in cancer cells has encouraged several investigators to combine DNMT inhibitors with
2. Hydroxamic acid
No clinical development
Phase II (149,150)
Trichostatin A
suberoylanilide hydroxamic (SAHA/ Vorinostat/ Zolinza1)
Phase I/II (146–148)
Valproic acid
PO, IV
—
Hematological and solid tumors
—
MDS and AML
MDS and AML
IV
Phase I/II (145) In combination with 5-azacytidine, Phase I/II (111)
Sodiumphenylbutyrate
PO
Solid tumors
IV
Phase I (144)
Butyrate
1. Short-chain fatty acids
Disease
PO/IV
Chemical structure
Clinical trial
Compounds
Family
Table 1 Classification of HDAC Inhibitors
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3B. Nonepoxyketonecontaining cyclic tetrapeptides
3A. Epoxyketonecontaining cyclic tetrapeptides
Family
Not available Not available
LBH589
PXD101 Trapoxin A
Romidepsin1 (Depsipeptide, FK228, Romidepsin)
Phase I/II
Not available
LAQ824
Phase I/II (151–153)
No clinical development
IV
—
IV
Phase I
Pyroxamide
Phase I/II (70)
PO/IV
Chemical structure
Clinical trial
Compounds
Table 1 Classification of HDAC Inhibitors (Continued )
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(Continued)
Hematological and solid tumors
—
Hematological and solid tumors Hematological and solid tumors
Hematological and solid tumors
Disease
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Phase I (158)
MGCD0103
PO
PO
In combination with gemcitabine. Phase I (156) and Phase II (157)
CI-994 (NAcetyldinadine)
Not available
PO
Phase I (154) Phase I (155)
—
PO/IV
SDX-2751 (MS275/SNOX-275)
Clinical trial No clinical development
Chemical structure
Apicidin
Compounds
Solid tumors and NHL
Solid tumors
Solid tumors, lymphoma and AML
—
Disease
220
Note: The HDAC inhibitors are classified according to their chemical structure. There are four major groups: short-chain fatty acids, hydroxamic acids, cyclic tetrapeptides, and benzamides. Several compounds are already in phase I and II of clinical trials for the treatment of hematological malignancies and solid tumors. Abbreviations: MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; NHL, non-Hodgkin’s lymphoma.
4. Benzamides
Family
Table 1 Classification of HDAC Inhibitors (Continued )
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HDACis. HDACis do not appear to reactivate hypermethylated genes when they are used alone, but they do exert additive of synergistic effects if some demethylation is first achieved by demethylating agents (131,132). It was already shown in 1982 that 5-azacytidine in combination with butyrate acted synergistically to upregulate the b-adrenergic receptor in HeLa cells (133). Decitabine in combination with the HDACi TSA can synergistically reactivate certain tumor suppressor genes (131), while in a murine lung cancer model, decitabine in combination with the HDACi phenylbutyrate prevents the formation of tumors (134). Multiple clinical trials are currently ongoing with the various combinations of the different demethylating agents and HDACis. Nimer and colleagues showed in a small number of patients that the combination of 5-azacytidine with phenylbutyrate is clinically safe and efficacious (135). Gore and colleagues also demonstrated the clinical response of the combination of 5-azacytidine plus phenylbutyrate being associated with the hypermethylator epygenotype of p15 promoter before treatment in MDS/AML patients (111). Garcia-Manero and coworkers achieved a response rate of 22% with the combination of decitabine and valproic acid in a phase I/II study in AML and MDS patients (136). In summary, the combination of demethylating agents and HDACis is feasible, not excessively toxic and clinically promising. Because there are many different HDAC enzymes, some like the sirtuins, the roles of which are only now being unraveled, the challenge will be to design therapies that can target individual enzymes, thus fine tuning our ability to influence the total epigenetic network with the requisite therapeutic specificity. The HMTs represent another valid target for the discovery of new drugs that can reactivate silenced genes, and compounds with potential clinical relevance are being investigated (137), and it is not being prophetic to predict that these compounds will be studied in combination with DNA hypomethylator and HDAC-inhibiting agents. CONCLUSIONS It is now clear that the ability to intervene in the epigenetic changes associated with malignancy is now a clinical reality. Epigenetically mediated silencing of genes such as tumor suppressors, or those related to proteins associated with cell adhesion, cell cycle, metastasis, and proapoptotic activity have been identified in virtually every type of human malignancy. Two major epigenetic processes can be altered using drugs now approved by the FDA (two azanucleosides for the treatment of MDS, the inhibitor of HDACs Zolinza1/Vorinostat1/SAHA for cutaneous T-cell lymphoma). Valproic acid, long used safely in the treatment of seizure disorders, has been shown to have significant HDAC inhibitory activity with clinically relevant activity in MDS, AML, and lymphoma. Translational studies demonstrating the in vivo inhibition of DNA methylation, and subsequent re-expression of tumor suppressor proteins such as p15INK4B have provided proof of principle that demethylating agents reactivate these genes in vivo. Similarly, histone deacetylation in vivo by treatment with phenylbutyrate, valproic
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acid, depsipeptide, vorinostat, LBH589, and other HDACis demonstrates the efficacy of these drugs in remodeling chromatin structure, with all also in combination studies using hypomethylating agents. Ongoing studies are addressing the epigenetic profile of reactivation and silencing before and after therapy with these agents in different malignancies, both in terms of DNA methylation and histone changes. Combination studies to attack the ‘‘silencer’’ phenotype of malignant cells at the different levels of epigenetic regulation are ongoing. The first wave of success clinically has been in the hematologic malignancies; however, combinations of epigenetic agents with chemotherapy in solid tumors are showing signs of clinical and preclinical activity (Ramalingam, 2006). The activation of epigenetically silenced tumor suppressor miRNAs might allow for new treatment modalities (138,139). It may also be possible to make a prognosis depending on which genes are silenced by hypermethylation or choose an adequate therapy. For example, hMLH1 and O6-MGMT genes, which encode DNA-repair proteins (140,141), are often methylated in cancer. It is known that loss of function of these genes sensitizes cells to the effects of chemotherapy that depend on an alkylating mechanism. Brain tumors that carry hypermethylated O6-MGMT respond better to alkylating agents like carmustine (BCNU) than those that do not (142). The ability to impact upon the epigenetic network has proven itself as a sentinel breakthrough in our fight against cancer. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS More than 40 years after the two azanucleosides 5-azacytidine and 5-aza-20 deoxycytidine were first synthesized, both compounds have been approved as effective therapeutics for a rare and difficult-to-treat disorder, MDS. The decisive step toward recognition of these drugs as biologically effective beyond mere cytotoxic drugs (such as the cytidine analog cytarabine) was only after they were identified as drugs modifying the epigenome. Similarly, only the discovery of valproic acid as an HDACi, decades after it was established in routine clinical use as an anticonvulsant, has led to its recognition as a drug also active in lowrisk MDS. The present momentum, with a plethora of new HDACis about to enter the clinical arena, paves a way for studies of these diverse compounds in many hemato-oncological entities. In the next five years we will see results from combination studies with inhibitors of DNA methylation and HDAC activity, and learn about the additional effects of ‘‘classical’’ cell differentiating agents, such as all-trans retinoic acid, in the context of these epigenetically active combinations. Thus a veritable renaissance of the concept of differentiation therapy, as outlined for instance by Koeffler (143), is presently taking place. Challenges may lie in the rational, safe, and effective combination of epigenetically active drugs with standard chemotherapy. Caution has to be taken as regards drug interactions, unexpected toxicities and/or antagonizing effects (i.e., mediation of a drug-resistance phenotype by epigenetic therapy). Clearly the strongest advances in epigenetic therapy have already been made for the azanucleosides in the treatment of myeloid leukemia
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144. Patnaik A, Rowinsky EK, Villalona MA, et al. A phase I study of pivaloyloxymethyl butyrate, a prodrug of the differentiating agent butyric acid, in patients with advanced solid malignancies. Clin Cancer Res 2002; 8:2142–2148. 145. Gore SD, Weng LJ, Figg WD, et al. Impact of prolonged infusions of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukaemia. Clin Cancer Res 2002; 8:963–970. 146. Pilatrino C, Cilloni D, Messa E, et al. Increase in platelet count in older, poor-risk patients with acute myeloid leukemia or myelodysplastic syndrome treated with valproic acid and all-trans retinoic acid. Cancer 2005; 104:101–109. 147. Raffoux E, Chaibi P, Dombret H, et al. Valproic acid and all-trans retinoic acid for the treatment of elderly patients with acute myeloid leukaemia. Haematologica 2005; 90:986–988. 148. Kuendgen A, Knipp S, Fox F, et al. Results of a phase 2 study of valproic acid alone or in combination with all-trans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia. Ann Hematol 2005; 84 (Suppl 1):61–66. 149. Kelly WK, Richon VM, O’Connor O, et al. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 2003; 9:3578–3588. 150. Kelly WK, O’Connor OA, Krug LM, et al. Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol 2005; 10:3923–3931. 151. Sandor V, Bakke S, Robey RW, et al. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res 2002 8:718–728. 152. Piekarz RL, Robey R, Sandor V, et al. Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood 2001; 98:2865–2888. 153. Byrd JC, Marcucci G, Parthun MR, et al. A phase 1 and pharmacodynamic study of depsipeptide (FK228) in chronic lymphocytic leukemia and acute myeloid leukemia. Blood 2005; 105:959–967. 154. Ryan QC, Headlee D, Acharya M, et al. Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma. J Clin Oncol 2005; 23:3912–3922. 155. Gojo I, Jiemjit A, Trepel JB, et al. Phase 1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias. Blood 2007; 109:2781–2790. 156. Nemunaitis JJ, Orr D, Eager R, et al. Phase I study of oral CI-994 in combination with gemcitabine in treatment of patients with advanced cancer. Cancer J 2003; 9:58–66. 157. Richards DA, Boehm KA, Waterhouse DM, et al. Gemcitabine plus CI-994 offers no advantage over gemcitabine alone in the treatment of patients with advanced pancreatic cancer: results of a phase II randomized, double-blind, placebocontrolled, multicenter study. Ann Oncol 2006; 17:1096–1102. 158. Gelmon K, Tolcher A, Carducci M, et al. Phase I trials of the oral histone deacetylase (HDAC) inhibitor MGCD0103 given either daily or 3x weekly for 14 days every 3 weeks in patients (pts) with advanced solid tumors. J Clin Oncol 2005 ASCO Annual Meeting Proceedings. 23, No. 16S, Part I of II: 3147.
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10 The Emerging Role of Histone Deacetylase Inhibitors in the Treatment of Lymphoma Matko Kalac and Owen A. O’Connor Herbert Irving Comprehensive Cancer Center, The New York Presbyterian Hospital, Columbia University, New York, New York, U.S.A.
BIOLOGICAL BASIS OF REGULATING GENE TRANSCRIPTION Histones are the major structural proteins around which more than 2 m of DNA in every eukaryotic cell is organized. These proteins are considered to be small molecular weight proteins composed of a very high proportion of positively charged amino acids like lysine and arginine. This complex of histone protein, nonhistone protein, and DNA is often referred to as chromatin, the fundamental unit of which is referred to as the nucleosome. The nucleosome consists of a complex of approximately 150 bp of DNA and a histone octamer. Each histone octamer is comprised of a pair of histones including H2A, H2B, H3, and H4 (Fig. 1). Neighboring nucleosomes are linked together by DNA bound to the linker histone (H1). This complex assembly of protein and DNA provides an important organizational structure that helps the cell maintain control over transcription. The control over chromatin structure, and thus transcriptional regulation, is largely mediated through a variety of posttranslational enzymatic reactions that include methylation (arginine), phosphorylation (histidine, serine, and threonine), sumoylation, methylation, ubiquitination, ADP-ribosylation of glutamic acid residues, and acetylation (lysine) (1). While all of the above can have a
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Figure 1 Structure of a nucleosome.
significant impact on gene expression by altering chromatin accessibility for transcriptional factors, the posttranslational modification that appears to be best understood at the moment involves the histone acetylation and deacetylation reactions. Acetylation of the histone is responsible for maintaining chromatin in an ‘‘open’’ and transcriptionally active state, while deacetylation keeps the chromatin condensed or ‘‘closed’’ and, therefore, transcriptionally silent. Acetyl groups are added onto the terminal epsilon (e) amino moieties of the lysine residues by the enzymatic activity of histone acetyltransferases (HATs). This reaction neutralizes the positive charge of the histones, weakening their link with negatively charged DNA, which in consequence moves the histone away from DNA making the chromatin open. The opposing enzymes, histone deacetylases (HDACs), facilitate the removal of the acetyl group from the histone tail. Upon deacetylation, the DNA becomes more condensed and therefore inaccessible to transcription factors. The involvement of the HATs and HDACs in cancer is well established. For example, mutations affecting HAT genes have been well described in colorectal and gastric cancers (2). The MYST family of HATs (MOZ) has been implicated in leukemogenesis, being found fused to a p300 homolog-CBP (a HAT) in acute myeloid leukemia (AML) (3). Mutations in CREB binding protein (CBP) leading to its loss of HAT activity have been found in the Rubinstein–Taybi syndrome (4), a disease characterized, among other things, by an increased incidence of neoplasia. In addition, numerous viruses target HATs: (i) adenovirus E1A oncoprotein, which binds to p300 through a domain required for viral transformation, likely contributes to its oncogenic potential (5); (ii) the HIV Tat protein, which interacts with TAF1 and p300/CBP altering chromatin
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condensation and presumably gene expression (6); and (iii) the EBNA2 protein of the Epstein–Barr virus, which is responsible for B-lymphocyte immortalization, binds to HDAC2 (7). These illustrative examples underscore the importance of these particular reactions and their effects on the transcriptional state of DNA. As such, these same reactions have now become the topic of intense focus, as new drugs, which appear to modulate this biology, become increasingly more available. CLASSIFICATION AND FUNCTION OF HDACs IN NORMAL AND MALIGNANT CELLS The HDAC family comprises three distinct classes of enzymes, classes I, II, and III. The classes I and II HDACs typically require a Zn ion for their activity, while the class III HDACs, also called the SIRT family on the basis of their similarity to yeast homologs known as the sirtuins, are typically regarded as Zn-independent enzymes that require nicotinamide adenine dinucleotide (NAD). To date, 11 members of the classes I and II have been discovered. The class I HDACs include HDAC1, 2, 3, and 8, and are usually smaller proteins compared with class II enzymes, demonstrating a molecular weight in the 42 to 55 kDa range. In contrast, the class II enzymes include HDAC 4, 5, 6, 7, 9, and 10, and usually demonstrate a molecular weight between 120 and 130 kDa (Table 1). The most recently discovered enzyme, HDAC11, contains conserved residues in the catalytic core regions shared by both class I and class II. Some reports have placed this enzyme in its own newly formed class (class IV) (8). In addition, class I enzymes are mostly localized within the nucleus, while class II enzymes can move between the nucleus and cytoplasm, suggesting a potential nonhistone target for this class of enzymes. The class III HDACs are comparatively unique. To date, seven enzymes belonging to this class have been identified, all of which use NAD for their activity, and all of which are generally insensitive to many chemicals that effectively inhibit classes I and II HDACs. In the cell, most HDACs usually express their activity in multiprotein complexes, usually through the complexation with corepressors such as Sn3, nuclear corepressor (NCoR), and silencing mediator for retinoid and thyroid receptors (SMRT). Interestingly, there are no reports yet of mutations in HDAC genes in human cancer, though overexpression of the enzymes has been well established. For example, HDAC2 has been shown to overexpress in tumors in patients with familial adenomatosis polyposis (FAP). Another well-described example has been reported in acute promyelocytic leukemia (APL), in which insensitivity to normal physiological concentrations of retinoic acid (RA) results in a constant proliferation of leukemic cells. Normally, RARa binds to retinoic acid responsive elements (RARE) as a heterodimer with RXRa. In the absence of RA, the RARa/RXRa completely inhibits transcription through the recruitment of corepressors, DNA methyltransferases, and HDACs (9). Physiological concentrations of RA dissociates the HDAC from the heterodimer complex, leading to transcriptional
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Table 1 Classes I and II Histone Deacetylases Enzyme
Class
HDAC 1
I
HDAC 2 HDAC 3 HDAC 4 HDAC 5
HDAC 6
HDAC 7 HDAC 8 HDAC 9
HDAC 10
HDAC 11
Localization
Localized in the nucleus, equally spread throughout various tissues I Coexists in complexes with HDAC 1, various tissues I Localized in the nucleus, various tissues II Interacts with NCoR, found in nucleus and cytoplasm II Moves between nucleus and cytoplasm suggesting potential acetylation of nonhistone target II Moves between nucleus and cytoplasm suggesting potential acetylation of nonhistone target II Highly expressed in CD4 þ CD8 þ thymocytes I Localized in the nucleus, equally spread throughout different tissues II Moves between nucleus and cytoplasm suggesting potential acetylation of nonhistone target II Moves between nucleus and cytoplasm suggesting potential acetylation of nonhistone target Contains conserved residues in the catalytic core regions shared by both class I and class II
Mr 55.9 kDa 60 kDa 49.7 kDa 140 kDa 51 kDa
159 kDa
65 kDa 46.4 kDa 160 kDa
71.4 kDa
39 kDa
Abbreviations: HDAC, histone deacetylase; NCoR, nuclear corepressor; Mr, relative molecular weight.
activation. In comparison, physiological concentrations of RA in acute promyelocytic leukemia (APL) cells, which carry the PML-RARa fusion protein, fail to affect transcription as the PML-RARa fusion protein acts as a dominant negative RARa. Pharmacological concentration of RA induces the release of PML-RARa from the HDAC complex, activating transcription. Collectively, these data support the notion that aberrant recruitment of HDAC to RARE represents a key feature in APL leukemogenesis. In normal cells, RA leads to the recruitment of the HDACSin3-NCoR complex and repression of the genes containing RA-response element. When RA is present, HDAC-Sin3-NCoR complex dissociates allowing RA to bind to its response element. When PML-RARa fusion occurs, HDAC is bound to PML leading to the stable recruitment of HDAC-Sin3-NCoR complex, making the cells therefore insensitive to physiological concentrations of RA. However, in the presence of high, pharmacological concentrations of RA, the HDAC-Sin3-NCoR complex dissociates from PML, resulting in differentiation.
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In AML1-ETO positive AML, the resulting fusion protein recruits HDAC enzymes to AML1-dependent promoters (10), downregulating a host of genes contributing to leukemogenesis (11). In non-Hodgkin’s lymphoma (NHL), mutations in the LAZ3/Bcl-6 (lymphoma-associated zinc-finger-3/B-cell lymphoma-6) gene have been implicated in lymphomagenesis. LAZ3/Bcl-6 is a specific regulator of germinal center formation (12), and its structural alterations (translocations, deletions, and point mutations) are well described in diffuse large B-cell lymphoma (13,14). SMRT acts as a repressor of LAZ3/Bcl-6 in a complex formed with HDAC1, inhibiting the normal development of germinal centers (15). In addition to their effects on transcriptional complexes, it is increasingly clear that both HATs and HDACs influence the activity of numerous nonhistone proteins. For example, P/CAF, MYST, p300/CBP, nuclear hormone receptor coactivators, and TAF1 can all be influenced by these enzymes. Other protein substrates include p21, p27, bcl-6, p53, E2F, GATA1, transcription factors IIE and IIF, and glucocorticoid receptors (16,17). HDAC INHIBITORS—MOLECULAR PHARMACOLOGY There is an ever-growing list of molecules found to have HDAC inhibitory activity (Table 2). The history of HDAC inhibitors began in 1970s when several reports, including a sentinel observation from Riggs et al., noted that butyric acid can stop DNA synthesis and cell proliferation and induce histone acetylation and differentiation in erythroleukemic cells (18). This observation opened the door to a host of other chemical classes with the ability to inhibit HDACs. For example, valproic acid and phenylbutyrate have been studied in clinical trials for the treatment of solid and hematological malignancies. However, in comparison to many of the newer generation inhibitors, they are among the least potent of HDAC inhibitors (19). One natural product, trichostatin A (TSA), is among one of the most potent HDAC inhibitors but cannot be studied in the clinic secondary to its significant toxicity (20). Treatment of malignant lymphoid cells with TSA induces accumulation of cells in G0/G1 or G2/M phases, and causes a concomitant decrease of cells in S phase, eventually leading to apoptosis (21). TSA has served as a structural model for how hydroxamates are thought to exert their activity. Through crystallographic analysis, it has been shown that TSA contains three components: a hydroxamic acid residue that binds to the Zn ion of the HDAC, a hydrophobic spacer that helps in spanning the entire active center, and a hydrophobic cap that covers the active center therefore disabling the HDAC enzymatic activity. It was an understanding of these important structure activity relationships that eventually led to the development of the hydroxamic acids, of which suberolyanilide hydroxamic acid (SAHA, vorinostat) has become the best known representative. Treatment of normal and malignant cells with HDAC inhibitors leads to accumulation of acetylated histones H2A, H2B, H3, and H4
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Table 2 Pharmacological Division of Histone Deacetylase Inhibitors and Their IC50 Values with Type O Cells in Which They Exert Antitumor Activity In Vitro and In Vivo IC50 hr in vitro
Chemical group
Drug
Aliphatic acids
Butyrate and its derivatives
0.2–3 mM
Valproic acid
1–5 mM
Trichostatin A
2–250 nM
Vorinostat
1–6,3 mM
Oxamflatin
16–720 nM
CBHA Scriptaid
0,5–4 mM nanomolar
Hydroxamates
Chemical structure
N/A
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Table 2 (Continued ) Chemical group
Cyclic peptides
Drug
IC50 hr in vitro
Pyroxamide
nanomolar
LAQ824
10–100 nM
LBH589 PXD-101
5–50 nM 0,02–6 mM
N/A
CG-1521 A-161906
5-10 mM 1,2–1,8 mM
N/A
Trapoxins
20-80 mM
Romidepsin
1–50 nM
Chemical structure
(Continued)
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Table 2 Pharmacological Division of Histone Deacetylase Inhibitors and Their IC50 Values with Type O Cells in Which They Exert Antitumor Activity In Vitro and In Vivo (Continued ) Chemical group
Benzamides
Drug
IC50 hr in vitro
Apicidin
100 nM
CHAPs
nanomolar
HC-toxin
nanomolar
Chlamydocin
0,36–45 nM
Diheteropeptin MS-275
20,3 mM nanomolar
Chemical structure
N/A
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Table 2 (Continued ) Chemical group
Other HDAC inhibitors
Drug
IC50 hr in vitro
CI-994
4,7–80 mM
SK-7041
0,6–1,7 mM
MGCD0103
38–232 nM
Depudecin
nanomolar
Chemical structure
Abbreviations: CBHA, carboxycinnamic bis-hydroxamide; CHAPs, cyclic hydroxamic acid–containing peptides.
(22,23). Fortunately however, neoplastic cells seem to be much more sensitive to the growth inhibitory and apoptotic effects of these agents compared with normal cells (24). One potentially interesting feature of these drugs revolves around the percent of the genome they influence. For example, Chiba et al. and Lee et al. found that only about 2% to 5% of the genome changed its expression in RNA level, which is surprisingly small, given the presumed mechanism of action of these compounds (25,26). These data suggest that it is not merely chromatin remodeling and transcriptional activation that accounts for the antineoplastic activity of these agents. This view is supported by several lines of data demonstrating that the effects of HDAC inhibitors on the posttranslational modification of other nonhistone proteins like p21, p27, pRb, p53, bcl6, and E2F may be equally or more important.
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TARGETING NONHISTONE PROTEINS WITH HDAC INHIBITORS As just discussed, HDAC inhibitors target numerous other protein substrates besides histones. One important target includes Bcl-6, one of the most commonly altered genes in diffuse large B-cell lymphoma. Bcl-6 is a POZ/zinc finger sequence–specific transcriptional repressor whose gene is located at chromosome 3q27 (27,28). In a large proportion of B-cell lymphomas, Bcl-6 is constitutively expressed by suppressing the genes involved in the control of lymphocyte activation, differentiation, and apoptosis (28). Bereshchenko et al. have shown that the transcriptional coactivator p300 acetylates Bcl-6 and therefore inhibits the ability of Bcl-6 to recruit HDACs, resulting in a compromise of its ability to repress transcription and induce cell transformation (29). These observations may have a therapeutic application. Unacetylated Bcl-6 is a transcriptional repressor, which turns off a host of genes implicated in lymphomagenesis. Acetylation of Bcl-6 abrogates its effects as a transcriptional repressor, allowing those same genes to be activated. The pharmacologic inhibition of HADCs leads to an accumulation of acetylated Bcl-6, nullifying its transforming effects. Recent observations by Pasqualucci et al. using TSA reveal that modulation of the Bcl-6 acetylation status can lead to the induction of apoptosis in several cell lines of B-NHL (28). HDAC inhibitors are also known to have effects on the tumor suppressor protein p53, which is the primary regulator of the G1 checkpoint (30). Deacetylation of p53 decreases its ability to activate the cell cycle inhibitor p21, allowing the cell to proceed into the S phase. Inhibition of HDAC activity would inhibit deacetylation of p53, prevent the suppression of p21, and cause cell cycle growth arrest and apoptosis. Heat shock proteins (HSP) are another potentially important set of targets of HDAC inhibitors. It has been shown that HSP90 serves as a chaperone protein required for proper folding and maintenance of numerous signaling protein kinases like Bcr-Abl, FLT3, AKT, and c-Raf in their active formations (31). HDAC6 is an enzyme responsible for deacetylation of HSP90, which keeps HSP90 active in its chaperone function. Inhibition of HDAC6 causes HSP90 acetylation and disruption of its chaperone function leading to polyubiquitylation and depletion of pro-growth and pro-survival HSP90 client proteins, making such cells more susceptible to other antineoplastic agents (32). HDAC INHIBITORS—CLINICAL PHARMACOLOGY To date, a number of different HDAC inhibitors have made their way into the clinic. A description of select inhibitors is provided in Table 3. Below we briefly describe the clinical experience with the major classes of HADC inhibitors. Aliphatic Acids Butyrate, phenylbutyrate, and valproic acid were among the first drugs that were shown to have HDAC inhibitory activity, with phenylbutyrate and valproate
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Table 3 HDAC Inhibitors with Significant Experience in Clinical Trials HDAC inhibitor
Clinical trials
Aliphatic acids (phenylbutyrate, valproate)
Numerous solid tumors including melanoma, cervical and breast carcinomas, AML, and diffuse large B-cell lymphoma AML, multiple myeloma, non-Hodgkin’s lymphoma, Hodgkin’s disease, mesothelioma, prostate, breast, and lung cancers Multiple myeloma, T-cell lymphoma, colorectal and ovarian cancers Multiple myeloma, chronic myeloid leukemia, AML, ALL AML, myelodysplastic syndrome, ALL , T-cell lymphoma, pancreatic carcinoma AML, myelodysplastic syndrome, chronic myelomonocytic leukemia Pancreatic carcinoma AML, myelodysplastic syndrome, colorectal, renal, and lung carcinomas
Vorinostat
Belinostat LAQ824 LBH589 MS-275 (MS-27-275) CI-994 (acetyldinaline) MGCD0103
Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia.
having a significant clinical experience in cervical (33) and breast cancers, melanoma, and other solid tumors (34). In hematologic malignancies, valproate has been used in the treatment of AML with all-transretinoic acid (ATRA) producing one complete remission (CR) and a response rate of 5% (35). Other studies in patients with AML studied combinations with valproate and the hypomethylating agent 5-aza-20 -deoxycytidine with an overall response rate (ORR) of 22%, and a remarkable CR rate of 19% (36). A recent report by Zain et al. described a CR in a patient with diffuse large B-cell lymphoma refractory to previous lines of therapy following monotherapy with valproate alone (37). Despite the relatively simple nature of these HDAC inhibitors, it is intriguing to note that these compounds have produced interesting signals in patients with otherwise chemotherapy refractory malignancies. On the basis of these biological effects, new generations of HDAC inhibitors have been developed; many with superior activity against different HDAC enzymes and some with more selective activity against different classes of HDAC. Many of these new HDAC inhibitor drugs generally fall into three major classes of agents including: hydroxamic acids, benzamides, and cyclic macrolides. We will briefly discuss some of the relevant clinical data with each of these different classes of HDAC inhibitors below. Hydroxamates SAHA, vorinostat, is the prototypical hyroxamate that has to date been extensively studied in the clinic and recently approved by the FDA for the treatment of
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cutaneous T-cell lymphoma (CTCL). Vorinostat has the ability to induce differentiation and apoptosis in numerous cell lines, including erythroleukemia, bladder transitional cell carcinoma, breast adenocarcinoma, myeloma, and neuroblastoma (38) causing the accumulation of acetylated 2A, 2B, 3, and 4 histones (23). Phase I clinical trials showed that both IV and oral formulations were well tolerated in patients with both solid tumors and hematologic malignancies, including AML, NHL, Hodgkin’s lymphoma, and myeloma. In addition, promising signals of activity were appreciated in patients with Hodgkin’s lymphoma, T-cell lymphoma, and two patients with transformed lymphoma, [one CR for 10 months, one partial remission (PR) for 6 months]. From a pharmacokinetic perspective, peak vorinostat concentrations occurred approximately 60 minutes after initiating the IV infusion (range 25–120 minutes). The terminal half-life of IV vorinostat ranged from 21 to 58 minutes, while the area under the plasma concentration curve was proportional to the dose administered. At all IV doses of vorinostat studied (75, 150, 300, 600, and 900 mg/m2/day), plasma concentrations exceeded 2.5 mM, a concentration that inhibited cell proliferation in vitro and resulted in accumulation of acetylated histones. In addition, IV vorinostat also inhibited HDAC activity in normal cells (peripheral blood mononuclear cells) as well as in tumor tissue biopsies. As illustrated in several patients, accumulation of acetylated histones in peripheral blood mononuclear cells occurred at the end of a two-hour infusion and was still evident two hours after the infusion ended. To simplify administration, an oral formulation was developed that demonstrated a favorable pharmacokinetic profile while retaining its antitumor activity. The bioavailability of oral vorinostat was relatively high (43%) and was uninfluenced by the consumption of food (39). Oral vorinostat demonstrated linear pharmacokinetics from 200 to 600 mg, although peak concentrations were lower than that seen with the IV formulation (39). The apparent half-life of oral vorinostat ranged from 91 to 127 minutes, values that were two- to threefold higher than those seen after IV administration. Plasma concentrations of vorinostat were detectable at least 10 hours post ingestion, whereas vorinostat was undetectable in plasma four to six hours after IV dosing. As observed following IV administration, oral vorinostat consistently effected accumulation of acetylated histones in peripheral blood mononuclear cells at two hours post dosing, an effect that persisted for up to 10 hours after a single dose of 400 mg or higher (39). Histone acetylation was still apparent in patients receiving oral vorinostat for six months or longer. On the basis of the phase I data, Duvic et al. initiated a phase II study in patients with CTCL. They treated 33 patients who had received a median of five prior therapies. Eight patients achieved a PR (ORR of 24%), including patients with advanced disease and Sezary syndrome (40). Subsequently, Olsen et al. reported an ORR of 29.5% in patients with CTCL, including one CR in a group of patients with heavily pretreated CTCL. Drug-related adverse grade 3 or greater adverse events included fatigue (5%), pulmonary embolism (5%), nausea (4%),
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and thrombocytopenia (4%) (41). These results led to FDA approval of vorinostat in the treatment of CTCLs that have progressed after conventional therapy. Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is another example of a hydroxamic acid with potent HDAC inhibitory activity. It is a known potent inducer of terminal differentiation in erythroleukemia cells and is capable of inducing apoptosis in a variety of different hematologic and solid tumor cell lines in vitro and in vivo studies (42,43). To date, the clinical experience with pyroxamide has been limited, with no real data on ORRs or toxicity other than a small experience in patients with gynecologic malignancies (44). PXD101 (belinostat) is another hydroxamic acid that was synthesized around the understanding that the hydroxamic acid moiety from vorinostat and TSA as the zinc-chelating group was critical for conferring high levels of potency. To date, like other HDAC inhibitors, PXD101 has been shown to induce apoptosis, in vitro and in vivo, in a variety of different solid and hematological malignancies. Treatment of mice bearing human ovarian and colon tumor xenografts caused significant dose-dependent growth delay with no significant signs of toxicity (45,46). On the basis of promising preclinical data, phase I studies have been conducted which demonstrate dose-dependent pharmacodynamic effects in patients with both solid and hematological malignancies, including multiple myeloma, T-cell lymphoma, colorectal cancer, and ovarian cancer that were well tolerated. Both oral and IV formulations of the PXD101 have been evaluated. To date, there has been limited evidence for hematological toxicity, with the prominent nonhematologic toxicities including (grade 3) fatigue, reversible atrial fibrillation, diarrhea, and lethargy, all occurring at doses ranging from 600 to 1200 mg/m2 (47). A phase II experience reported by Sullivan et al. noted stabilization of advanced and progressive disease in patients with multiple myeloma, with 6 of 12 patients being evaluable for response (48). LAQ-824/LBH589 are HDAC inhibitors derived from the hydroxamic acid portion of the TSA molecule (49). Both have been shown to be potent inhibitors of different cell lines in nanomolar concentrations in a variety of cell lines including myeloma, chronic myeloid leukemia, acute lymphoblastic leukemia, AML, and melanoma lines refractory to conventional chemotherapy (50–54). LAQ-824 entered phase I trials in 2002 when it demonstrated evidence of activity in patients with AML and myelodysplastic syndrome (MDS). The drug was shown to have dose-dependent pharmacodynamic effects at doses up to 80 mg/m2, with one dose-limiting toxicity (DLT) in a patient with CLL (cerebral hemorrhage due to thrombocytopenia). LBH589 has also been studied in phase I trial as reported by Prince et al. (55). LBH589 is a member of the hydroxamic acid class of HDAC inhibitors with low nanomolar activity. Patients with advanced solid tumors or NHL, including advanced stage CTCL, with and without prior therapy were treated on study. The dosing regimen and schedule evaluated a flat dose of drug at 15, 20, and 30 mg administered on a thrice-per-week schedule for four consecutive weeks, with no holiday. One treatment cycle was 28 days long. DLTs were
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appreciated in two patients at the 30-mg cohort, including one patient with grade 4 thrombocytopenia and one with grade 3 diarrhea. Other grade 2 or less toxicities included fatigue and nausea. Interestingly, the toxicity profile was remarkably similar to what has been reported for other HDAC inhibitors (56). In particular, the thrombocytopenia was noted to be very transient, with adequate evidence of megakaryopoiesis in patients with drug-associated thrombocytopenia. While early, LBH589 has demonstrated some promising and reproducible activity in patients with CTCL, including 6 of 10 patients with CTCL having a CR (n ¼ 2) or PR (n ¼ 4). While only a few patients with solid tumors have been treated to date, no response has been seen in patients with renal cell carcinoma, melanoma, or mesothelioma. One of the more interesting features of the Prince study has been the effect LBH589 has on the gene expression profile in patients. Acquisition of punch biopsies from patients with CTCL, which were studied by gene expression profiling, demonstrated that LBH589 induced rapid and robust changes in the tumor cell gene expression profile, with changes occurring as rapidly as four hours post treatment. Most of the gene expression changes lasted for at least eight hours, and the spectrum of gene expression profile changes was remarkably consistent with what has been seen in vitro. Surprisingly, only about 1% to 4% of all genes were found altered, with the majority of genes actually being downregulated. In fact, on the basis of six patients studied to date, they found that in two of these patients, 61% and 10% of genes were found to be upregulated, while 39% and 90% of genes were found to be downregulated. From these combined data, a consistent and reproducible 23-gene set was found altered in all patients, including genes involved in cyclin D1 expression, angiogenesis, and immune modulation. While these data offer very promising ideas regarding how these drugs might work in lymphoma, it is clear we are a long way from completely understanding precisely how these agents work in different subtypes of lymphoma. What is clear so far is that there may be important class effects with these agents, and to date, there is a recurring theme that these agents appear to produce a remarkably similar toxicity profile, with most demonstrating at least some reproducible activity in patients with T-cell lymphomas. Clearly, a significant experience with hydroxamic acid–based HDAC inhibitors has evolved over a relatively short period of time. While the experience with vorinostat, LBH589, and PXD101 appear to be the most robust to date, there are other potent HDAC inhibitors in this class. These include other promising molecules like EMBA [diethyl bis-(pentamethylene-N,N-dimethylcarboxamide)] and CBHA (m-carboxycinnamic acid bis-hydroxamide), compounds similar to vorinostat that are able to differentiate erythroleukemia cell lines in micromolar concentration. They also induce accumulation of unphosphorylated pRB, increase level of p21, prolong G1 phase of the cell cycle and cause complete suppression of neuroblastoma xenografts in mice (57). Similar results have also been reported in experiments for oxamflatin (58,59), Scriptaid (60–62), propenamides (63), CG-1521 (64), and A-161906 (65).
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Cyclic Peptides Among the first cyclic peptides were the trapoxins, fungal products isolated in 1990 from the culture broth of Helicoma ambiens (66). These compounds exhibit both potent in vitro cytotoxicity and the ability to cause the accumulation of acetylated histones (67). Depsipeptide (romidepsin, formerly known as FK228, FR901228) has emerged as a structurally similar natural product. It was first isolated from a broth culture of chromobacterium violaceum in 1994, shortly after demonstrating antitumor activity in vitro and in vivo in various malignancies (68,69). In fact, Piekarz and Bates made one of the first reports describing the activity of an HDAC inhibitor in T-cell lymphoma in 2001. This publication was among the first to report responses to depsipeptide in patients with drug-resistant CTCL. Patients with advanced or refractory disease received romidepsin as a four-hour infusion on days 1 and 5 of a 21-day cycle, with the maximum tolerated dose (MTD) being 24.9 mg/m2. DLTs included nausea, vomiting, thrombocytopenia, and atrial fibrillation (70,71). Since this initial report, there have been a number of phase II studies initiated and reported over the past few years, including: an expansion of the original NCI study reported by Peikarz and Bates; a pivotal phase II study in patients with CTCL with one or more prior system therapies; and a recently launched pivotal phase II study in patients with peripheral T cell lymphoma (PTCL) with one or more prior therapies. In these studies, the romidepsin dose and schedule employed was 14 mg/m2 given as a four-hour infusion on days 1, 8, and 15 of a 28-day cycle (72). As of the last report, 71 patients with CTCL and 39 patients with PTCL have been treated. While this original study explored a number of different treatment cohorts on the basis of the type of T-cell lymphoma and the number of prior therapies, more recent analyses have reported the data in the context of the specific disease subtypes, including CTCL and PTCL. The ORR in CTCL is 32%, including 6% of patients who attained a CR. The median duration of response for patients in CR was 19 months (range 8 to 63þ), while the median duration of response for patients in PR was 5.5 plus months (1 to 60 months). An interim update on the CTCL data in the pivotal study was reported by Lerner et al. who reported an ORR of 32% with an 8% CR rate, and a median time to response of about eight weeks. Interestingly, one of the prominent features of the treated patients was the relief in their diseaseassociated pruritis. Of 30 patients with pruritis at baseline, approximately 53% had significant relief, while of 16 patients with severe pruritis at baseline, 14 (88%) reported significant relief. While no data are available on the pivotal study in PTCL, of 39 patients reported to date by the NCI group, there is an ORR of 28%, including 7% of patients attaining a CR and 21% of patients attaining a PR. The median duration of response among these patients appears to be approximately 12 months. Clearly, romidepsin has emerged as a promising HDAC inhibitor with significant activity in patients with both CTCL and PTCL. Continued studies in
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these diseases will better clarify the role of romidepsin in these difficult diseases. Other macrolide HDAC inhibitors include apicidin, an antiprotozoal fungal metabolite found to have HDAC inhibitory activity (73). Apicidin produced significant cell cycle arrest and induced apoptosis in a variety of cell lines, including drug-resistant leukemia cells. Similar results have been attained in experiments with cyclic hydroxamic acid–containing peptides (CHAPs) and chlamydocin. Benzamides MS-275 (MS-27-275) is a benzamide derivative that has been the most extensively studied member of this class both in preclinical and clinical settings. Like all other HDAC inhibitors, it is capable of inducing apoptosis in a large variety of different cell lines (74–77). To date, a phase I and pharmacokinetic study has been reported in patients with advanced and refractory solid tumors and lymphoma. All patients received MS-275 orally on a daily schedule for 26 consecutive days of a six-week cycle, and then daily on a 14-day schedule. Interestingly, the MTD was exceeded at the first dose level, with the MTD being identified as 10 mg/m2, with the major DLT, including nausea, vomiting, anorexia, and fatigue. Increased histone H3 acetylation in peripheral blood monocytes was demonstrated at all dose levels, and no responses were demonstrated in any patient, though only two patients with lymphoma were treated, with no details on the subtype of NHL treated (78). A second phase I study in patients with myeloid leukemia explored dosing once weekly for two consecutive weeks repeated every four weeks, evaluating dose ranges from 4 to 8 mg/m2. The study was then expanded to once weekly for four consecutive weeks repeated every six weeks, evaluating doses from 8 to 10 mg/m2. The MTD was 8 mg/m2 when given weekly for four consecutive weeks, and the major DLT included infections, neurological toxicity manifest as unsteady gait, and somnolence. Treatment with MS275 induced increased histone H3/H4 acetylation, though no responses were seen in this patient population (79). While the activity as a single agent was limited, in combination with 5-azacytidine responses have been seen in patients with MDS, AML, and CMML (total of 27 evaluable patients). Responses occurred at all doses including two CRs, four PRs, and six bilineage hematologic improvement in patients with MDS (80). A phase II study in patients with metastatic melanoma was reported by Hauschild et al. with no objective tumor responses observed (81). These data suggest that combinations of HDAC inhibitors with hypomethylating agents may be an important therapeutic platform for patients with hematologic malignancies, certainly for patients with myeloid leukemias. To date however, the single agent activity of the benzamides remains limited and appears less robust than that seen with the hydroxamic acid derivatives and cyclic macrolides. Whether this is a function of the chemical entity, or some PK-PD variable, remains to be clarified.
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CI-994 (acetyldinaline) is an example of another benzamide, which has been shown to have antiproliferative and apoptotic effects in resistant AML cell lines (82,83). Phase I studies identified 8 mg/m2 as the MTD with the major DLT being thrombocytopenia. CI-994 was also studied in a phase I trial in combination with gemcitabine, which did not reveal any significant increase in the toxicity profile (84,85). On the basis of the phase I data with gemcitabine, a phase II of CI-994 with gemcitabine in patients with advanced pancreatic cancer has been initiated. Results showed no significant difference in overall survival, response rate, or time to progression (86). MGCD0103 is a class I–specific HDAC inhibitor (also known as an isotypeselective HDAC inhibitor) with activity in nanomolar concentrations that is orally bioavailable. Unlike vorinostat, which is a broad class I and II inhibitor, MGCD0103 selectively inhibits HDAC 1, 2, 3, and 11 and does not inhibit to any significant degree HDAC 4, 5, 6, 7, and 8. To date, MGCD0103 has been studied in the clinical trials of phases I and II. These studies have demonstrated a favorable toxicity and pharmacokinetic profile, with the most common side effects being fatigue, nausea, anorexia, vomiting, and diarrhea with no hematological toxicities (87). A phase I/II study reported by Garcia-Manero et al. in patients with AML and MDS analyzed the activity of MGCD0103 in combination with a hypomethylating agent (azacytidine). The MTD was found to be 110 mg in combination, while the DLT was considered to be vomiting, nausea, and anorexia. Antileukemia activity was documented in 7 of 24 patients, including 3 CR, 1 PR, and 3 CR without platelet or neutrophil recovery (88). A recently reported phase II study by Younas et al. evaluated the efficacy of MGCD0103 in Hodgkin’s lymphoma on a 28-day treatment cycle with a starting dose of 110 mg. Of 20 patients treated, the ORR was 40% (n ¼ 8), including two (10%) CRs. The major DLTs included fatigue (n ¼ 1) and pneumonia (n ¼ 3). These data are reminiscent of the activity seen with vorinostat in Hodgkin’s lymphoma, where patients experienced both PRs and prolonged stabilization of their disease with minor responses. While the collective experience in Hodgkin’s lymphoma is to date still small, it is becoming increasingly evident that Hodgkin’s lymphoma may be a disease with some intrinsic sensitivity to HDAC inhibitors. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS After years of research, epigenetic-based approaches to the management of cancer are just now beginning to impact on cancer care. Recent studies in CTCL and various subtypes of leukemia have established an important proof of principle: Modulating gene transcription and promoter methylation can result in potent and durable benefits against malignant cells. Despite the enormous advances made in a relatively short period of time, it is clear the volume of work to be done is growing at an incredible pace. With each agent comes new questions regarding the optimal pharmacokinetic and pharmacodynamic schedules to employ and new questions regarding how these agents mediate cell death.
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Perhaps the greatest prospect for these drugs lies in their ability to modulate important cellular pathways in a fashion that may compliment either conventional or other novel targeted agents. This ability to produce rational drug combinations will be intimately linked to our ability to dissect out some of the cause and effect relationships that occur following exposure to an HDACI. Such strategies will need to rely on a strong preclinical base, as empiric drug combination studies begin to define the optimal combinations and schedules for different subtypes of cancer. Over the next five years, studies will begin to explore the merits of a true ‘‘epigenetic’’ platform for treating many hematologic cancers, including lymphoma. Combinations of HDAC inhibitors and hypomethylating agents will continue to be studied in a many subtypes of lymphoma. It is conceivable this combination could be used as an innovative platform to sensitize lymphoma cells to other more conventional chemotherapy programs. In addition, as our understanding of Bcl-6 biology becomes more clear, it will become apparent how to exploit HDAC inhibitors to modify Bcl-6 dysregulation in diffuse large B-cell lymphomas, offering a new form of targeted therapy for these aggressive diseases. What remains even as exciting are the rapidly growing opportunities for these agents to help patients with a host of diseases other than cancer. Modulating gene expression and learning how to do so in a gene or disease specific manner offers physicians the ability to impact on a panoply of diseases long characterized by abnormal expression of particular genes. For example, a vast array of studies have begun to suggest that altering chromatin remodeling and gene expression, that is, focusing on epigenetic phenomenon, can impact diverse diseases like Friedreich’s ataxia, neurodegenerative disorders, including Huntington’s disease, artificial organ generation, diabetes, Alzheimer’s disease, and even aging. Clearly, at their most fundamental level, cancer and a host of other diseases have their roots in some form of genetic dysregulation. Maintaining a focus on understanding the basic biology will be the key that unlocks the many doors of opportunity. REFERENCES 1. Fischle W, Wang Y, Allis CD. Binary switches and modification cassettes in histone biology and beyond. Nature 2003; 425:475–479. 2. Gayther SA, Batley SJ, Linger L, et al. Mutations truncating EP300 acetylase in human cancers. Nat Gen 2000; 24:300–303. 3. Deguchi K, Ayton PM, Carapeti M, et al. MOZ-TIF2-induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2-mediated recruitment of CBP. Cancer Cell 2003; 3:259–271. 4. Murata T, Kurokawa R, Krones A, et al. Defect of histone acetyltransferase activity of the nuclear transcriptional coactivator CBP in Rubinstein-Taybi syndrome. Hum Mol Genet 2001; 10:1071–1076. 5. Fax P, Lehmkuhler O, Kuhn C, et al. E1A12S-mediated activation of the adenovirus type 12 E2 promoter depends on the histone acetyltransferase activity of p300/CBP. J Biol Chem 2000; 275:40554–40560.
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11 Antileukemic Treatment Targeted at Apoptosis Regulators Simone Fulda and Klaus-Michael Debatin University Children’s Hospital, Ulm, Germany
INTRODUCTION Cellular systems with a high intrinsic proliferative capacity and cell turnover in the body such as hematopoietic cells are in constant need to control cell numbers (1,2). Thus, an excess of precursor cells is produced daily, which mature into effector cells. In the lymphoid system, the vast majority of T- and B-cell precursors die by a default mechanism because of the inefficient production of functional antigen receptors. Apoptosis is also a key mechanism for the termination of an immune response by elimination of unnecessary effector cells to avoid autoimmunity and tissue damage. In the myeloid system, a large number of granulocytes are produced per day, which disappear in the peripheral tissues by a process that at least in vitro involves spontaneous apoptosis. From this intrinsic capacity of hematopoietic cells to undergo apoptosis, it is obvious that too much apoptosis or not enough apoptosis will lead to hematopoietic failure, uncontrolled lymphoproliferation with autoimmunity or leukemia and lymphoma. One of the most important recent advances in cancer research is the recognition that apoptosis plays a major role in both tumor formation and treatment response (3–6). Some oncogenic mutations block apoptosis, leading to tumor initiation and progression (7). To this end, defects in apoptosis pathways may create a permissive environment for genetic instability and accumulation of gene mutations, promote resistance to immune-based destruction, facilitate growth
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factor- or hormone-independent survival, and support anchorage-independent growth during metastasis (8). Conversely, other prototypic oncogenic events such as deregulated expression of the Myc oncogene can promote apoptosis, thereby producing selective pressure on tumor cells to override apoptosis during multistage carcinogenesis (5). In addition, killing of tumor cells by diverse cytotoxic approaches such as anticancer drugs, g-irradiation, suicide genes, or immunotherapy, has been shown to be mediated through the induction of apoptosis in target cells (4,6). Since the same oncogenic alterations and defects in apoptosis programs that suppress cell death during tumor development can also confer resistance to cytotoxic therapies (3), apoptosis provides a conceptual framework to link cancer formation and cancer therapy. THE CORE APOPTOTIC MACHINERY Most apoptosis signaling pathways ultimately result in activation of caspases, a family of cysteine proteases that act as common death effector molecules in various forms of cell death (Fig. 1) (9,10). Caspases involved in apoptosis signaling are categorized into initiator and effector caspases, respectively. Activation of caspases can principally be triggered by two different mechanisms. According to the induced proximity model initiator caspases such as caspase-8 or -9 are activated in a multimeric complex, for example, caspase-8 in the death inducing signaling complex (DISC) and caspase-9 within the apoptosome (11). Alternatively, caspases are activated by catalytic processing of the zymogens at specific cleavage sites. Caspase activation can be initiated through different entry sites such as at the plasma membrane by death receptor mediated signaling (receptor pathway) or at the mitochondria (mitochondrial pathway). Stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily results in receptor aggregation and recruitment of the adapter molecule Fasassociated death domain (FADD) and caspase-8 to form the DISC (12–14). Upon recruitment, caspase-8 becomes activated and initiates apoptosis by direct cleavage of downstream effector caspases. The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac)/ direct ‘‘inhibitor of apoptosis protein’’ (IAP) binding protein with low PI (DIABLO), Omi/high temperature requirement protein A2 (HtrA2), endonuclease G, caspase-2 or caspase-9 from the mitochondrial intermembrane space (15). The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex (16). Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to IAPs (17,18). The receptor and the mitochondrial pathway can be interconnected at different levels through caspase-8-mediated cleavage of the BH3 protein Bid and mitochondria dependent caspase-8 cleavage (19). For most apoptosis regulators, numerous studies have demonstrated their importance for normal growth control in hematopoietic cells. For example, the CD95 system is a key receptor for apoptosis mediated by external signals in both
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Figure 1 Apoptosis signaling pathways. Apoptosis pathways can be initiated through different entry sites, e.g., at the plasma membrane by death receptor ligation (receptor pathway) or at the mitochondria (mitochondrial pathway). Stimulation of death receptors of the TNF receptor superfamily such as CD95 (APO-1/Fas) or TRAIL receptors by CD95 ligand (CD95-L) or TRAIL results in receptor aggregation and recruitment of the adaptor molecule FADD and caspase-8. Upon recruitment, caspase-8 becomes activated and initiates apoptosis by direct cleavage of downstream effector caspases. The mitochondrial pathway is initiated by stress signals through the release of apoptogenic factors such as cytochrome c, AIF, or Smac/DIABLO from the mitochondrial intermembrane space. The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex. Smac/DIABLO promotes caspase activation through neutralizing the inhibitory effects to IAPs, while AIF causes DNA condensation. The receptor and the mitochondrial pathway can be interconnected at different levels, e.g., by Bid, a BH3 domain containing protein of the Bcl-2 family, which assumes cytochrome-c-releasing activity upon cleavage by caspase-8. Activation of caspases is negatively regulated at the receptor level by FLIP, which block caspase-8 activation, at the mitochondria by Bcl-2 family proteins and by IAPs. Abbreviations: TNF, tumor necrosis factor; FADD, Fas-associated death domain; AIF, apoptosis inducing factor; IAPs, inhibitor of apoptosis proteins.
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lymphoid and myeloid cells (13). Also, survival of hematopoietic precursor and effector cells critically depends on the regulation of levels of antiapoptotic Bcl-2 family members (1). ALTERNATIVE FORMS OF CELL DEATH Although caspases are crucial for cell death execution in many systems, caspaseindependent apoptosis as well as nonapoptotic modes of cell death have also to be considered. For example, necrosis, autophagy, paraptosis, or some forms of cell death that cannot be easily classified at present have been described (20). Although the signaling pathways and molecules involved in these alternative forms of cell death have not yet exactly been defined, non-caspase proteases such as calpains or cathepsins may be involved (21). The relative contribution of these diverse cell death mechanisms under various conditions both in vitro and in vivo in malignant cells of the hematopoietic system will be an area of future studies. DEREGULATED APOPTOSIS AND LEUKEMOGENESIS Malignant transformation is a multistep process that involves genetic alterations leading to enhanced proliferation, diminished cell turnover, or both (3,5). The potential contribution of reduced cell death to tumor development was in fact first established in follicular lymphoma by the identification of the Bcl-2 protooncogene at the chromosomal breakpoint of the t(14;18) translocation (22). Alterations in the expression of antiapoptotic or proapoptotic members of the Bcl-2 family proteins have been described in a variety of human cancers, including hematologic malignancies (23) (Table 1). The discovery of the key function of p53 in the regulation of apoptosis and the high incidence of p53 mutations in the majority of human cancers often associated with advanced disease and poor prognosis also revealed the critical role of deregulated apoptosis in the process of malignant transformation (24). In addition to Bcl-2 and p53, genetic and epigenetic alterations of apoptosis regulating genes have been demonstrated to contribute to carcinogenesis (25). In general, with the exception of mutations in p53 or Bax, impaired apoptosis in tumor cells and leukemias seems to be the consequence of increased expression of prosurvival molecules such as NF-kB, IAPs, decreased expression of death receptors, and/or decreased sensitivity for apoptosis induction by death-inducing ligands such as CD95 ligand (1–3,5,8,26,27). Given the central role of caspases for cell death execution, one might expect a high frequency of caspase mutations in tumors. Interestingly however, screening for mutations in initiator or executioner caspases has not revealed a high frequency of genomic aberrations in caspase genes. Despite the fact that leukemia represents a highly aggressive malignant disease in vivo, at least cells from patients with acute leukemia often show increased apoptosis when cultured in vitro (1,2). This observation may indicate that true clonogenic leukemic cells represent only a minority of the malignant
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Table 1 Oncogenic Translocations and Alterations of Apoptosis Genes in Leukemia or Lymphoma Genetic lesion
Mechanism of disturbed function
Translocation and fusion proteins t(9;22) Bcr-Abl Inhibition of apoptosis by various stimuli probably through kinase activity of BCR-Abl protein (e.g., phosphorylation of Bad) t(9;14) (PAX-5/IgH) Regression of p53 by increased levels of PAX-5 t(15;17) (PMl-RARa) Decrease in overall apoptosis sensitivity t(17;19) (E2A/HLF) t(10;14) (Hox-11IgH) t(1;14) (TAL-I (SCL)) Apoptosis regulators p53
Bax
Bcl-2
Apaf-1 CD95 FLIP
Inhibition of apoptosis induced by IL-3 withdrawal Protection from spontaneous apoptosis in normal lymphocytes Resistance to cytotoxic drugs and CD95 triggering Mutation Failure to activate increased expression of proapoptotic molecules Mutation Diminished mitochondrial pathway Overexpression due to translocation t(14;18) or constitutive inhibition of the mitochondrial pathway Mutation or epigenetic silencing Mutation or downregulation Production of soluble variant Constitutive overexpression Inhibition of caspase-8 activation
Disease CML, Ph1 positive ALL
Lymphoma AML-M3 (acute promyelocytic leukemia) Precursor B-cell or p53 leukemia (ALL) T-ALL T-ALL
CLL, Burkitt’s lymphoma
AML
Non-Hodgkin’s lymphoma
AML ALL, ATL, Hodgkin’s lymphoma Burkitt’s lymphoma
CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; ATL, adult T-cell leukemia.
blasts detected in peripheral blood or bone marrow. However, in slowly growing chronic lymphoid leukemia (CLL), decreased apoptosis sensitivity in vitro can be found. Characteristic chromosomal translocations have been discovered in most forms of acute and chronic leukemia and lymphoma (Table 1). In general,
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these translocations lead to the deregulated expression of proliferation accelerating genes such as c-Myc or increased expression of apoptosis inhibiting genes such as Bcl-2. Alternatively, the generation of fusion proteins such as Bcr-Abl or PML-RARa mediate increased cell death resistance in tumor cells (1,2). Constitutive expression of c-Myc is a hallmark of a highly aggressive malignant lymphoma, Burkitt’s lymphoma, as a consequence of the t(8;14) translocation. Interestingly, while increased constitutive expression of c-Myc leads to rapid proliferation of malignant cells, these cells are also prone to apoptosis probably because of the concomitant activation of apoptosis pathways by deregulated c-Myc expression. As a recapitulation of the disease in humans, Myc-transgenic mice rapidly develop aggressive leukemia or lymphoma. The concomitant overexpression of Bcl-2 however, produces an even more aggressive phenotype demonstrating that inhibition of apoptosis in addition to deregulated proliferation control may be a secondary hit in malignant transformation (28). The Bcr-Abl fusion protein is created through the t(9;22) translocation, which is the hallmark of chronic myeloid leukemia (CML). The dysregulated function of the Abl tyrosine kinase leads to the activation of several antiapoptotic molecules and pathways including Ras, Akt, NF-kB, and STAT (29). PMLRARa represents another fusion protein generated through t(15;17) translocation found in specific forms of acute promyelocytic leukemia (M3) (30). Similar to the Bcr-Abl fusion protein, PML-RARa has an impact on a variety of regulatory systems including STATs, most probably through transcriptional regulation leading to increased survival of leukemic cells and apoptosis resistance. Several frequent translocations found in acute lymphoblastic leukemia (ALL) were also found to inhibit apoptosis signaling (1). This is particularly interesting since the precursor B and T-cell compartment is highly prone to spontaneous cell death. The t(17;19) translocation associated with pre B-cell leukemia results in the fusion of two transcription factor genes (E2A, HLF). Ectopic expression of E2A, HLF in nonmalignant pro-B-lymphocytes abrogates apoptosis induction induced by IL-3 withdrawal or p53 expression. Also, recurrent translocations associated with T-cell leukemias seem to lead to an oncogenic transformation that results in protection from apoptosis. While most of these oncogenic translocation appear to have a global impact on apoptosis sensitivity, overexpression of the tal-I (SCL) gene as a result of the t(1;14) translocation found in T-cell acute lymphoblastic leukemia (T-ALL) specifically induces resistance to cytotoxic drugs and CD95 triggering. PROGNOSTIC SIGNIFICANCE OF APOPTOSIS REGULATORS IN LEUKEMIA AND LYMPHOMA Hematologic malignancies, in particular childhood ALL, still represent the tumor entity with the highest incidence of cure rates following cytotoxic therapy. Most therapeutic agents now in clinical use were developed using empirical screening
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designed to identify agents that were ‘‘toxic’’ to tumor cells or inhibited proliferation. It is now well established that the majority of anticancer agents eventually act by triggering apoptosis in tumor cells by different mechanisms, which involve damage to DNA, activation of the cellular stress response, and, ultimately, activation of apoptosis signal transduction pathways (3,4,6,23). Since agents with distinct primary intracellular targets can initiate apoptosis through similar mechanisms, defects in apoptosis programs may produce multidrug resistance. Irrespective of the initial insult (e.g., DNA damage, generation of reactive oxygen species), apoptosis in response to cancer therapy precedes through activation of the core apoptotic machinery consisting of the receptor and the mitochondrial signaling pathway. The relative contribution of the receptor and the mitochondrial pathway to drug-induced apoptosis has been a subject of controversial discussion (5,6,31–36). While a number of initial studies suggested that cancer therapy-triggered apoptosis involves the activation of the CD95 receptor or ligand system, compelling evidence subsequently indicated that the majority of cytotoxic drugs initiate cell death by triggering a cytochrome c/Apaf-1/ caspase-9 dependent pathway through the mitochondria inhibitable by Bcl-2. However, the relative contribution of the death receptor versus the mitochondrial pathway may depend on the cytotoxic drug, dose, and kinetics or on differences between certain cell types similar to the cell type–dependent signaling in the CD95 pathway and multiple crosstalks between the two pathways may amplify the response (37). Most experiments addressing the activation of apoptosis pathways by cytotoxic therapy have been performed in vitro. However, in in vivo experiments, resembling the clinical situation in the patient, activation of caspases has been found during lymphoma treatment, and Bcl-2 overexpression considerably diminished the treatment efficacy of Myc-driven lymphomagenesis in transgenic mice (38). Also, spontaneous p53 negative/negative tumors in mice are less responsive to chemotherapy in vivo (3). This suggests that in vitro studies in fact reflect some of the important features of tumor treatment in vivo. On the basis of these findings, a number of clinical studies have been performed to address the significance of expression of apoptosis regulators in treatment response and outcome, in particular in leukemia and lymphoma (Table 1). In contrast to epithelial carcinomas, p53 mutations in hematologic malignancies are variable and associated with specific entities such as CLL (1,2). In general, p53 alterations are more frequent in aggressive disease and have been associated with drug resistance and poor survival in some instances. Despite increased apoptosis in situ, p53 alterations are found in approximately 5% of myelodysplastic syndrome (MDS) patients probably as a prerequisite for malignant transformation in this disease. In CML, p53 mutations are rare in the chronic phase but increase in frequency during blast crises. The highest proportion of p53 mutations are found in Burkitt’s lymphoma and B-cell type ALL (up to 50%). Interestingly, despite the aggressive nature of the disease, p53 mutations are rare in acute myeloid leukemia (AML) and ALL and may, for
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example, correlate well with the excellent response to cytotoxic drugs for pediatric ALL. Overall, the significance of p53 mutations in leukemia and lymphoma for treatment outcome is not entirely clear, as Burkitt’s lymphoma–harboring p53 mutations may also respond well to DNA damaging therapy. In addition to mutations found in CD95 in acute leukemia, Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL), most CD95 expressing NHL-cells are constitutively CD95 resistant (39). In adult T-cell leukemia, enhanced serum levels of soluble CD95 were found to be associated with reduced survival and outcome (40). Also, CD95 expression has been positively correlated to the response to therapy in AML (41,42). Elevated FLIP-expression counteracting apoptosis signaling through death receptors has been found in clinical samples from several tumors including Burkitt’s lymphoma, as well as in tumor cells that developed resistance to chemotherapy, suggesting that FLIP may play a role in chemoresistance (43). All in vitro studies on the overexpression of Bcl-2 suggest an important role in multidrug resistance. It is therefore surprising that clinical studies on the prognostic impact of Bcl-2 expression in tumors have not shown conclusive results (1,2). In general, in AML, high levels of Bcl-2 are correlated with a low level of response to chemotherapy in vivo (44). However, some studies have also indicated that increased levels of Bcl-2 are not prognostic or are even associated with improved survival (45). This includes studies in breast cancer and childhood ALL (46,47). Also, for Bax, a key modulator of the mitochondrial pathway in vitro, conflicting data from clinical studies in leukemia have been reported. Increased levels of Bax expression correlated with improved rates of overall survival in one large cohort study, while a lack of correlation between Bax expression and response to induction chemotherapy and survival was found in another large cohort study (48,49). Also, the influence of downstream effectors such as caspases, Apaf-1, and IAPs on clinical response and treatment outcome has been studied (1–3, 23,50,51). Interestingly, patients with spontaneously increased levels of active caspase-3 at the time of diagnosis had improved survival, indicating that intact caspase activation pathways may be correlated to better prognosis (52). Also, increased levels of procaspase-3 were found to be associated with improved rates of complete remission in AML and ALL. However, in contrast to these findings, no correlation between levels of procaspases-3, -8, and -9 or Apaf-1 and response to induction chemotherapy was detected in a large study of adult patients with AML and ALL (50). Moreover, both Apaf-1 deficiency as well as increased expression of IAPs, including X-chromosome-linked inhibitor of apoptosis protein (XIAP) and survivin, have been found to confer resistance to drug-induced apoptosis in human leukemic cells in vitro (3,53,54). Furthermore, IAP proteins, for example, are frequently deregulated in human cancers because of increased mRNA or protein expression, or loss of endogenous antagonists such as XAF1 (54–56). In gene profiling studies, survivin was identified as the
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fourth most common transcriptome of the human genome in cancer, whereas it was not expressed in normal adult tissues, indicating that it may contribute to the malignant phenotype of cancer cells (57). Aberrant expression of cIAP2 frequently occurs in mucosa-associated lymphoid tissue(MALT) lymphoma, since the cIAP2 gene is affected by the t(11;18)(q21;q21) translocation, which occurs in 50% of MALT lymphoma (58). Expression of IAPs in tumor samples has been correlated to clinical parameters in a series of retrospective trials. For example, high survivin expression predicted poor prognosis in AML, anaplastic, or diffuse large-cell lymphoma (54,59,60). Unexpectedly however, nuclear survivin expression was also described as a significant independent prognostic indicator of a favorable outcome in invasive primary breast carcinoma (61). This suggests that the subcellular localization of survivin has to be also considered where its predictive prognostic value is concerned. Furthermore, AML patients with lower levels of XIAP protein were found to have significantly longer survival in one study (54); whereas in a subsequent study, expression levels of XIAP turned out to have no prognostic impact in AML patients (62). Also, an inverse correlation of XIAP with proliferation markers and favorable prognosis was found in radically resected, non–small cell lung cancer patients (63). Although high expression levels of IAPs were associated with advanced disease states and negative prognostic factors in the majority of studies, these reports also point to a level of complexity regarding the prognostic significance of IAPs that cannot be explained by the simple equation that high IAP expression equals poor prognosis. The conflicting findings obtained in clinical correlative studies that explored the impact of apoptosis regulators on treatment outcome may reflect the complexity of cell death regulation in human cancers in vivo. Therefore, future studies should include a functional analysis of cell death activation by chemotherapy in patients under chemotherapy in vivo. ANTICANCER THERAPIES TARGETED AT APOPTOSIS REGULATORS On the basis of the concept that resistance to apoptosis is a characteristic feature of human cancers that contributes to tumor formation and progression, strategies designed to restore defective apoptosis programs in cancer cells may overcome intrinsic or acquired resistance of tumor cells (3,64). Also, apoptosis-targeted therapies may enhance the responsiveness of human cancers toward conventional treatments that are currently used in the clinic, e.g., chemo- or radiotherapy, since these therapies primarily exert their antitumor activity by triggering apoptosis in cancer cells (4). In principle, apoptosis-based cancer therapeutics aim at disabling the antiapoptotic function of molecules involved in the leukemogenic process and/or in treatment resistance, or alternatively, directly activating the apoptotic machinery as discussed in detail below.
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ANTILEUKEMIC THERAPY TARGETED AT IAPS One promising therapeutic strategy directed at apoptosis regulators is the neutralization of IAPs. IAPs are a family of endogenous caspase inhibitors and comprise eight human analogs, which are XIAP, cIAP1, cIAP2, survivin, livin or melanoma-IAP (ML-IAP), apollon, NAIP, and ILP-2 (26). Among the IAP family members, XIAP is best known for its antiapoptotic function (65). XIAP blocks apoptosis by binding to active caspase-3 and -7 and also by interfering with caspase-9 activation (26). In addition, XIAP inhibits apoptosis via mechanisms unrelated to its ability to inhibit caspases. For example, XIAP can activate the NF-kB pathway by forming a complex with the TAK1 kinase and its cofactor TAB1 (66–69). Mechanistically, the XIAP-mediated NF-kB activation can be dissociated from its caspase-inhibitory effects and requires the E3 ubiquitin ligase activity of XIAP (66–69). The role of survivin in the regulation of apoptosis and proliferation is more complex compared with other IAP family proteins (70). Besides its role as a regulator of apoptosis, survivin is also involved in the control of mitosis (70). There is mounting evidence that cancer cells including leukemia or lymphoma have an intrinsic drive to apoptosis that is held in check by IAPs. In support of this notion, high basal levels of caspase activity in the absence of apoptosis were detected in tumor cell lines and cancer tissues, but not in normal cells (71). Tumor cells, but not normal cells, simultaneously expressed high levels of IAPs, suggesting that upregulated IAP expression may counteract the high basal caspase activity selectively in tumor cells (71). Thus, targeting aberrant expression of XIAP in leukemia and lymphoma may open new perspectives to trigger the apoptotic machinery selectively in cancer cells. Recently, several strategies to inhibit or downregulate XIAP have been developed for therapeutic purposes. For the design of small molecules to target XIAP, the binding groove of the BIR3 domain of XIAP to which Smac binds to after its release from mitochondria has attracted most attention (72). Ectopic expression of Smac or Smac peptides harboring the N-terminal part of Smac that is essential for the binding of Smac to XIAP, were reported to either directly trigger apoptosis or to sensitize leukemia, lymphoma, or multiple myeloma cells for apoptosis induced by deathreceptor ligation, anticancer drugs, or a cytolytic T-cell attack (73–78). In some studies, Smac peptides were linked to a carrier to facilitate their intracellular delivery, for example, the protein transduction motif of the HIV Tat protein, the Drosophila antennapaedia penetratin sequence, or a polyarginine stretch (79–81). On the basis of the three-dimensional structure of Smac in complex with XIAP BIR3, Smac peptidomimetics, which bind to one or several of the BIR domains of IAP family proteins, were designed (82–87). Furthermore, capped tripeptides targeting the Smac binding site of XIAP BIR3 were developed by the structure-based design of the interaction of Smac with the BIR3 domain of XIAP (88). These XIAP antagonists were reported to bind to the BIR3 domain of XIAP
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with nanomolar affinity and promoted cell death in several human cancer cell lines including leukemia cells (88). In addition, the natural product embelin from the Japanese Ardisia herb was discovered as a cell-permeable, nonpeptidic, small-molecular weight inhibitor of the BIR3 domain of XIAP through the structure-based in-silico screening of a traditional herbal medicine threedimensional structure database (89). Embelin was shown to effectively overcome the protective effect of XIAP in Jurkat cells transfected with XIAP by binding to the XIAP BIR3 domain (89). Besides the BIR3 domain of XIAP, its BIR2 motif has also served as target for the development of small molecule compounds. To this end, nonpeptidic XIAP antagonists were identified by the screening of a polyphenylurea library using a caspase derepression assay (90,91). These compounds caused apoptosis in leukemia cells including primary AML blasts without the requirement of an additional cytotoxic stimulus by derepressing downstream effector caspases (90,92). Of note, these XIAP antagonists also killed acute leukemia cells with high Bcl-2 expression levels, suggesting that they may bypass some forms of resistance (90,92). Furthermore, antisense oligonucleotides were designed to downregulate aberrant XIAP expression in human cancers. Cellular studies have shown the anticancer effects of XIAP antisense oligonucleotides in leukemia, either as single agents or in combination with chemotherapeutic drugs (62,93). In animal models, AEG35156, a synthetic 19-mer, second generation, mixed backbone antisense oligonucleotide to human XIAP, reduced XIAP mRNA and protein levels in representative tissues at therapeutically feasible doses (94–96). Importantly, the antitumor activity of XIAP antisense oligonucleotides correlated with downregulation of XIAP levels in targeted tissues isolated from preclinical models (94,95). In a phase I trial, evidence of antitumor activity has been observed in a patient with NHL that was associated with concomitant XIAP knockdown in lymphoma cells (96). Currently, XIAP antisense oligonucleotides are evaluated in phase I/II clinical trials in combination with chemotherapy, i.e., docetaxel, cytarabine, and idarubicin, in AML, for example (95,96) (Table 2). Taken together, Smac mimetics, small molecule XIAP antagonists or XIAP antisense oligonucleotides are promising approaches to target XIAP to trigger apoptosis or to lower the threshold for apoptosis induction in leukemia and lymphoma cells. ANTILEUKEMIC THERAPY TARGETED AT BCL-2 FAMILY PROTEINS Another approach to target apoptosis pathways for cancer therapy is to antagonize antiapoptotic Bcl-2 family members. The Bcl-2 family of proteins consists of both antiapoptotic members, e.g., Bcl-2, Bcl-XL, and Mcl-1, as well as proapoptotic molecules (97). The later comprise multidomain proteins such as Bax, Bak, and Bad as well as BH3-domain only molecules such as Bim, Bid, Bmf, Noxa, or Puma (97). Bcl-2 family proteins play an important role in the regulation of the mitochondrial pathway of apoptosis, since they are involved in
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Table 2 Examples of Apoptosis Targeted Drugs in Clinical Trials Name
Clinical trial
1. XIAP targeting agents XIAP antisense Phase I/II 2. Bcl-2/Bcl-XL targeting agents Bcl-2 antisense Phase I-III
Bcl-2/Bcl-XL inhibitor
Phase I
3. TRAIL receptor agonists TRAIL Phase TRAIL-R1 mAb Phase TRAIL-R2 mAb Phase TRAIL-R1 mAb Phase
TRAIL-R1 mAb
I I I I
Phase I
Cancer type
Single/combined
Solid tumors, AML
Single
Solid tumors, Leukemia/ lymphoma Leukemia
Combination (chemotherapy)
Solid Solid Solid Solid
tumors, NHL tumors tumors tumors
Solid tumors
Refs. 96 102
Single
Single Single Single Combination (paclitaxel, cisplatin) Combination (gemcitabine, cisplatin)
144 143 145 147
148
Abbreviations: XIAP, X-chromosome-linked inhibitor of apoptosis protein; AML, acute myeloid leukemia; NHL, non-Hodgkin’s leukemia; TRAIL, TNF-related apoptosis-inducing ligand; mAb, monoclonal antibodies.
the control of mitochondrial outer membrane permeabilization (97). There are currently two models on how BH3-only proteins activate Bax and Bak during the course of apoptosis. According to the direct activation model (98), putative activators such as Bim and cleaved Bid (tBid) bind directly to Bax and Bak to trigger their activation, while BH3-only proteins that act as sensitizers, e.g., Bad, bind to the prosurvival Bcl-2 proteins. By comparison, the indirect activation model holds that BH3-only proteins activate Bax and Bak by binding and thus inactivating the various antiapoptotic Bcl-2 proteins, which in turn inhibit Bax and Bak (99,100). Imbalances in the ratio of anti- versus proapoptotic Bcl-2 proteins may tip the balance toward tumor cell survival and thus, may contribute to tumor formation and progression. Since high expression of antiapoptotic Bcl-2 family proteins may confer resistance to chemo- or radiotherapy by blocking the mitochondrial pathway of apoptosis (101), there has been much interest in developing strategies to overcome the cytoprotective effect of Bcl-2 and related molecules. To this end, nuclease-resistant Bcl-2 antisense oligonucleotides downregulating Bcl-2 mRNA were tested in clinical trials for hematologic malignancies or solid tumors as single agent or in combination with
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chemotherapy (102) (Table 2). In addition, BH3 peptides, which mimic BH3-only proteins in activating proapoptotic Bax and Bak proteins, are under preclinical evaluation (98). Recently, the attempt to target the protein-protein interaction site between antiapoptotic Bcl-2 proteins and Bax or Bak has resulted in the generation of the small molecule antagonist ABT-737, which binds to the surface groove of Bcl-2, Bcl-XL, and Bcl-w that normally interacts with the BH3 domain of Bax or Bak (103). ABT-737 has been evaluated in cancer cell lines and preclinical models. By preventing the binding of antiapoptotic Bcl-2 proteins to Bax or Bak, ABT-737 frees Bax and Bak to engage the mitochondrial pathway of apoptosis. In some susceptible cancer types, especially those that critically depend on Bcl-2 for survival, for example, CLL, ABT-737 as single agent has been reported to directly trigger apoptosis (103). In addition, ABT-737 sensitized cancer cells for apoptosis when combined with conventional chemotherapeutics (103–105). Since ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, high expression of Mcl-1 may confer resistance to this novel agent. Indeed, recent reports indicate that Mcl-1 represents a key determinant of ABT-737 sensitivity and resistance in cancer cells (104,105). Consequently, Mcl-1 downregulation by genetic approaches or pharmacologic compounds, including CDK inhibitors (e.g., roscovitine, flavopiridol, seliciclib), Raf/Mek inhibitors (e.g., sorafenib), or proteasome inhibitors, has been demonstrated to dramatically increase ABT-737 cytotoxicity in various malignant cell types (104–108). These findings also show that the multidomain proapoptotic proteins Bax and Bak play important functional roles in ABT-737-mediated apoptosis (105). Of note, ABT-737 exerted potent antitumor effects in vivo in xenograft mouse models and was also active against primary tumor cells, from AML samples for example (103–105). Collectively, these findings suggest that small molecule inhibitors of antiapoptotic Bcl-2 family proteins may open new perspectives to reactivate the mitochondrial pathway of apoptosis in cancer cells. ANTILEUKEMIC THERAPY TARGETING TRAIL RECEPTORS TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L is another prime candidate for translation of apoptosis targeted therapeutics into the clinic. TRAIL was identified in 1995 on the basis of its sequence homology to other members of the TNF superfamily (109,110). It is a type II transmembrane protein and its extracellular domain can be proteolytically cleaved from the cell surface. It is constitutively expressed in a wide range of tissues. Five different receptors have been described for the TRAIL receptor system. TRAIL-R1 and TRAIL-R2, the two agonistic TRAIL receptors, contain a conserved cytoplasmic death domain motif, which enables them to engage the cell’s apoptotic machinery upon ligand binding (111). TRAIL-R3 to R5 are antagonistic decoy receptors, which bind TRAIL, but do not transmit a death signal (111). TRAIL-R3 is a glycosylphosphatidylinositol-anchored cell surface protein that lacks a cytoplasmic tail,
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while TRAIL-R4 harbors a substantially truncated cytoplasmic death domain. In addition to these four membrane-associated receptors, osteoprotegerin is a soluble decoy receptor, which is involved in regulation of osteoclastogenesis (111). The concept to selectively trigger death receptors of the TNF receptor gene superfamily to induce cell death in cancer cells is attractive for a potential clinical application since death receptors are directly linked to the cell’s intrinsic death machinery (111). Among the death receptors, the TRAIL system is considered to be most suitable for clinical development, since TRAIL predominantly kills cancer cells, while sparing normal cells (111). Notably, studies in nonhuman primates such as chimpanzees and cynomolgus monkeys showed no toxicity upon intravenous infusion, even at high doses (112). In addition, no cytotoxic activity of TRAIL was reported on a variety of normal human cells of different lineages including fibroblasts, endothelial cells, smooth muscle cells, epithelial cells, or astrocytes. However, some concerns about potential toxic side effects on human hepatocytes or brain tissue have also been raised (113,114), which may be related to the TRAIL preparations used in these studies. Thus, TRAIL preparations, which are antibody-crosslinked or not optimized for Zn content, may overpass the threshold of sensitivity of normal cells by forming multimeric aggregates (111). Importantly, recent evidence suggest that TRAIL can induce survival and proliferation under certain circumstances in cancer cells resistant toward TRAIL-induced apoptosis, for example, via activation of the transcription factor NF-kB (115). These findings suggest that the death-inducing ligand TRAIL might paradoxically promote tumor growth in TRAIL-resistant tumors. Several strategies have been developed to target TRAIL receptors therapeutically. One approach is the use of trimeric TRAIL itself as a recombinant natural ligand. Recombinant soluble TRAIL triggered apoptosis in a wide range of cancer cell lines including hematologic malignancies and also in vivo in several xenograft models of human cancers (107,116–123). An alternative therapeutic strategy is based on agonistic monoclonal antibodies (mAb) that specifically target one of the agonistic TRAIL receptors TRAIL-R1 and TRAILR2, which demonstrated antitumor activity in cancer cell lines and xenograftbearing mice (124–126). The existence of decoy receptors that can bind TRAIL, yet not deliver a death signal, suggests a potential advantage to the use of antibodies that specifically target one of the two agonistic TRAIL receptor. Another potential advantage of these antibodies is their longer half-life compared with that of recombinant TRAIL. However, it remains to be determined in future studies which of these agents triggering the TRAIL pathway will turn out to be superior for clinical application. Since many human cancer cell lines express both TRAIL-R1 and TRAILR2 and since each of these agonistic receptors can initiate apoptosis independently of the other, the relative contribution of TRAIL-R1 versus TRAIL-R2 to ligand-induced apoptosis has been explored using receptor-selective mutants of TRAIL. TRAIL-R2-selective mutants were found to exert increased apoptosis-inducing activity compared with TRAIL-R1-selective mutants in several
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carcinoma cell lines (127). In contrast, primary cells from patients with CLL and mantle cell lymphoma were reported to signal to apoptosis almost exclusively through TRAIL-R1 despite expression of both agonistic TRAIL receptors (128,129). These studies highlight the necessity to determine whether cancer cells from a particular tumor type signal via TRAIL-R1 or TRAIL-R2 to provide a rational approach for the optimal use of TRAIL receptor agonists in the clinic. Interestingly, TRAIL-R2 antibody-based therapy was reported as an efficient strategy not only to eliminate TRAIL-sensitive tumor cells but also to induce tumor-specific T-cell memory that afforded long-term protection from tumor recurrence (130). Despite the expression of at least one of the two agonistic TRAIL receptors, leukemia or lymphoma cells have been reported to frequently present with primary or acquired resistance to TRAIL (115,123). Although the molecular basis for sensitivity versus resistance toward TRAIL has been an area of intensive investigation over the last few years, the overall dominance of antiapoptotic signals may play an important role. By comparison, genetic alterations in components of the TRAIL receptor pathway have been identified only in a small subset of tumors. For example, somatic mutations of TRAIL-R1 and TRAIL-R2 genes were found in a small proportion of NHL (131). Interestingly, TRAIL-R1 and TRAIL-R2 genes map to chromosome 8p21–22 (132), which is a frequent site of allelic deletions in many types of human tumors including NHL (133,134). In a series of 117 human NHL , eight tumors (6.8%) were found to have two TRAIL-R1 gene mutations or six TRAIL-R2 gene mutations (131). Six of these mutations (two TRAIL-R1 and four TRAIL-R2) were detected in the death domains and one nonsense mutation of TRAIL-R2 was detected just before the death domain (131). This study suggests that somatic mutations of TRAILR1 and TRAIL-R2 genes may play a role in the pathogenesis of some NHL (131). Besides the use of recombinant TRAIL or TRAIL receptor antibodies as single agents, numerous studies have shown that combinations with conventional anticancer therapeutics such as chemotherapy or g-irradiation elicit enhanced antitumor activity. For example, several chemotherapeutics or g-irradiation were reported to synergistically interact with TRAIL in hematologic malignancies (126,135–137). In addition, the concomitant use of TRAIL together with new compounds, e.g., proteasome inhibitors (138), histone deacetylase inhibitors (74,139–141), or kinase inhibitors (142) enhanced the therapeutic effects of TRAIL in leukemia. Currently, recombinant soluble TRAIL and fully human mAb directed at TRAIL-R1 or TRAIL-R2 are evaluated in early clinical trials (Table 2). Results from ongoing trials in patients with advanced solid tumors showed no major dose-limiting toxicities for recombinant TRAIL or fully human mAb to TRAILR1 and defined the maximal tolerated dose for mAb to TRAIL-R2 (143–145). In a phase II trial with HGS-ETR1, a fully human mAb against TRAIL-R1 (mapatumumab; Humane Genome Sciences, Rockville, Maryland, U.S.), in
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patients with NHL, tumor responses were seen in 3 out of 40 patients (8%) (146). Also, the antibody was well tolerated, with little toxicity observed (146). On the basis of preclinical studies showing cooperation of TRAIL receptor agonists with chemotherapeutics, clinical trials using HGS-ETR1 in combination with anticancer agents such as carboplatin or paclitaxel or gemcitabine and cisplatin, were also launched (147,148). CONCLUSIONS A tight regulation of clonal expansion on one side and cell death on the other ensures homeostasis within the lymphohemotopoietic system. Defects in key elements of the basic apoptosis signaling machinery may contribute to tumor formation and progression and may also confer drug resistance. Elucidation of cell death mechanisms in cancer cells over the last two decades has resulted in the identification of various therapeutic targets to reverse malignant phenotypes and to treat leukemia or lymphoma. Some apoptosis targeting drugs have already entered the clinical stage, also for the treatment of hematologic malignancies, opening new perspective for molecular targeted therapies. CLINICAL PERSPECTIVE ON THE NEXT FIVE YEARS However, at this point, a detailed analysis of components of the apoptotic machinery in clinical samples is far from being complete. Also, the premise that alterations in apoptosis regulators dictate cancer formation and drug sensitivity needs to be validated in clinical trials before prognostic parameters can be deduced from such studies. The rapid technological progress in gene expression profiling and proteomics is expected to yield a vast amount of new information in the coming years, which can be exploited for patient stratification and selection. Several apoptosis targeting drugs are currently being tested in (early) clinical trials and it is anticipated that over the next 5 to 10 years many more will have entered the clinical stage. Hopefully, the translation of our basic knowledge on apoptosis signaling into medical application will lead to novel prognostic biomarkers and improved cancer therapeutics for the sake of patients suffering from hematologic malignancies. REFERENCES 1. Wickremasinghe RG, Hoffbrand AV. Biochemical and genetic control of apoptosis: relevance to normal hematopoiesis and hematological malignancies. Blood 1999; 93(11):3587–3600. 2. Schimmer AD, Hedley DW, Penn LZ, Minden MD. Receptor- and mitochondrialmediated apoptosis in acute leukemia: a translational view. Blood 2001; 98(13): 3541–3553. 3. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell 2002; 108(2):153–164.
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12 Angiogenesis in Hematological Malignancies Alida C. Weidenaar, Hendrik J. M. de Jonge, Arja ter Elst, and Evelina S. J. M. de Bont Department of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
INTRODUCTION For more than a decade, the role of angiogenesis and vascular endothelial growth factor (VEGF) in relation to tumor growth has been the object of intense research. From 1939 onwards, it was postulated that ‘‘the rapid growth of tumor transplants is dependent upon the development of a rich vascular supply’’ (1,2). Soluble, diffusible factors were held responsible for this new vessel formation (3,4). In 1971, Folkman proposed that antiangiogenesis might be an effective strategy to treat human cancers (5). The identification of VEGF as a potent, diffusible factor affecting vascular endothelial cells led to ongoing investigations focused on VEGF (also referred to as VEGFA) as a key molecule in physiological and pathological vessel formation (6,7). Despite the knowledge that most of the steps in tumor growth are highly complex and multifactorial processes, VEGFA has been shown to be a prerequisite in tumor growth. ANGIOGENESIS IN NORMAL VASCULATURE Blood vessels are critical for maintaining cellular homeostasis in the human body and therefore all cells must reside within 100 mm to 200 mm of a capillary (8,9). Angiogenesis is defined as blood vessel generation from pre-existing blood
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vessels and already exists in embryogenesis (10). After birth, angiogenesis still contributes to organ growth, but during adulthood most blood vessels remain quiescent and angiogenesis occurs only in the cycling ovary and placenta during pregnancy, in response to exercise training, in wound healing, and in inflammatory processes (9,11,12). Angiogenesis in the adult human body is thought to be the result of a delicate balance between endogenous stimulators and inhibitors (Table 1). Angiogenesis is stimulated by growth hormones such as VEGFA, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and during hypoxic conditions. Endogenous inhibitors of angiogenesis include various antiangiogenic peptides, hormone metabolites, and apoptosis modulators (13,14). In addition, vascular basement membrane components can modulate endothelial cell (EC) behavior to provide structural and functional support (15). Table 1 Pro- and Antiangiogenic Factors Proangiogenic factors
Antiangiogenic factors
Acidic and basic FGF Agiogenin Angiopoietin-1 EGF Folistatin G-CSF GM-CSF HGF IGF Interleukin-2 Interleukin-6 Interleukin-8 Leptin MMP Placenta growth factor Platelet activating factor PDGF Platelet-derived epidermal growth factor Pleiotrophin Proliferin Prostagladins E1, E2 TGF a, b Tumor necrosis factor a VEGF Vascular integrin avb3 (vitaxin)
Angiopoietin-2 Angiostatin Arrestatin Canstatin Endostatin Fibronectin Interferon-a, b, g Interleukin-1 Interleukin-12 Interleukin-18 Maspin Platelet factor 4 Restin Retinoic acid Soluble VEGF receptor Thrombospondin TIMP Tumstatin Vasostatin
FGF, fibroblast growth factor; EGF, epidermal growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metalloproteinases; PDGF, plateletderived derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
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Angiogenesis is a multistep process and several cell types such as ECs and inflammatory cells produce and release angiogenic factors (e.g., VEGFA) upon certain stimuli, for instance hypoxia. High levels of VEGFA are able to initiate vasodilatation and an increased vascular permeability of pre-existing capillaries and postcapillary venules (16–18). This allows extravasation of plasma proteins, which lay down a provisional matrix to which activated ECs migrate. Angiopoietin (ANGPT)2 binding to the Tie2 receptor tyrosine kinase (TEK) is thought to result in the loosening of pericyte coverage (19). The activated ECs start to release matrix metalloproteases (MMPs) and serine proteases, which in turn degrade the basement membrane. After degradation of the basal membrane, ECs will be able to migrate from the original vessel walls towards the angiogenic stimuli originating from the injured tissue. Following this migration, ECs start to proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. A vascular basal lamina is produced around the newly formed blood vessels upon disappearance of ANGPT2 and upregulation of ANGPT1, which attracts and stabilizes pericytes and smooth muscle cells, finally resulting in fully mature blood vessels (20). Normal blood vessels are distributed at regular and closely spaced intervals and are organized into a hierarchy of elastic and muscular arteries, arterioles, capillaries, postcapillary venules and small and large veins (Fig. 1).
ANGIOGENESIS IN PATHOLOGICAL VASCULATURE In contrast to normal physiological angiogenesis, where new vessels rapidly mature and become stable, new blood vessels in pathological angiogenesis are nonuniformly distributed, irregularly branched, and are not formed in a clear hierarchical pattern (Fig. 1). Animal models showed that tumor vessels are dilated, immature (i.e., pericyte coverage is missing or detached, basement membrane is missing or too thick), and the vascular density is heterogeneous (Fig. 1C) (21–23). An important feature of tumor blood vessels is that they fail to become quiescent, enabling the constant growth of new tumor vessels. The vascular network in tumors is often leaky and hemorrhagic, leading to an elevated interstitial pressure in the tumor, resulting in hypoxia and acidosis (24). These vessel characteristics make the delivery of therapeutics to solid tumors highly inadequate (21). Rakesh Jain postulated the hypothesis that correcting the structure and function of tumor vessels could normalize the tumor microenvironment and eventually improve the treatment by a more efficient delivery of drugs and oxygen to the targeted cancer cells (25). Vessel normalization may be achieved by correcting the imbalance of angiogenic and antiangiogenic factors in favor of antiangiogenic factors. The high vascular permeability may be accomplished by the high levels of VEGFA, which result in induction of SRC regulated pathways (26–28). Therefore, blocking the VEGF/VEGFR signaling might normalize tumor vasculature.
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Figure 1 Proposed role of vessel normalization in the response of tumors to antiangiogenic therapy. (A) Tumor vasculature is structurally and functionally abnormal. It is proposed that antiangiogenic therapies initially improve both the structure and the function of tumor vessels. However, sustained or aggressive antiangiogenic regimens may eventually prune away these vessels, resulting in a vasculature that is both resistant to further treatment as well as inadequate for the delivery of drugs or oxygen. (B) Dynamics of vascular normalization induced by VEGFR2 blockade. On the left is a two-photon image showing normal blood vessels in skeletal muscle; subsequent images show human colon carcinoma vasculature in mice at days 0, 3, and 5 after administration of VEGR2specific antibody. (C) Diagram depicting the concomitant changes in pericyte (red) and basement membrane (blue) coverage during vascular normalization. (D) These phenotypic changes in the vasculature may reflect changes in the balance of pro- and antiangiogenic factors in the tissue. Source: From Refs. 21 (for image 1B), 25 (for images 1C,1D), 149 (for image 1B).
ANGIOGENIC SWITCH Spontaneously clustering tumor cells are usually not angiogenic at first (29). For cancer progression, a phenotypic switch to angiogenesis is required, a so-called angiogenic switch. This switch is more than an increase of proangiogenic stimuli; it is thought to be the net result of positive and negative regulators, a crosstalk between these factors and their receptors, as well as interaction with vasculature supporting cells such as endothelial progenitor cells (EPCs), mesenchymal (stem) cells, and niche maker cells. It is hypothesized that genetic control of the physiological levels of endogenous angiogenesis inhibitors is a line of defense against the
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conversion of dormant tumor cells to a malignant phenotype. The underlying phenomenon resulting in this phenotypic switch is still not clear; however, ongoing genetic instability is suggested to have a role (30,31). THE KEY PLAYER VEGF The VEGF family includes VEGFA, placental growth factor (PLGF), VEGFB, VEGFC, VEGFD, VEGFE and VEGFF (32–36). These proteins can bind to and exert their effect on two cell surface receptor families: the tyrosine kinase receptors (VEGFR) and the neuropilin (NRP) receptors. Three VEGF receptors have been identified, namely VEGF receptor 1 (FLT1), VEGF receptor 2 (KDR), and VEGF receptor 3 (FLT4) (37–39). Until now, two neuropilins (NRP1 and NRP2) have been described (40,41). Members of the VEGF family exert their effects by binding to the transmembrane receptors, resulting in the formation of dimers in the plasma membrane. Interaction between dimers is thought to stimulate autophosphorylation of the receptor. In response to phosphorylation of the receptors, intracellular signals are transmitted via signaling pathways such as mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-K/Akt) cascades, or the signal transducers and activators of transcription (STAT) pathway (42–44). These signals are essential to various cellular processes including control of cell growth, differentiation, and migration (45). Through the last decade, most of the research involving angiogenesis has focused on VEGFA. In various malignancies, VEGFA is associated with increased tumor growth (e.g., melanoma, lung cancer, breast cancer, colon cancer, rhabdomyosarcoma). Moreover, VEGFA is found to be an independent prognostic factor for outcome [e.g., breast, lung, colon/rectum, liver, gallbladder, bladder cancer, and hematological malignancies such as acute myeloid leukemia (AML)] (46–53). More recently, several studies showed that high VEGFA expression resulted in tumor growth by autocrine and/or paracrine ways of action. Two different paracrine ways can be described: (1) to induce angiogenesis (chemotactic signals from tumor cells might recruit stromal cells, which produce VEGFA, leading to the process of angiogenesis) and (2) to induce tumor cell proliferation [tumor cell–derived VEGFA results in growth factor production in stromal cells and/or ECs, which results in tumor cell proliferation; for instance described in AML (54)]. BONE MARROW–DERIVED TUMOR GROWTH SUPPORTING CELLS AND CIRCULATING ENDOTHELIAL CELLS Vasculature of tumors is predominantly formed by angiogenesis, but can also be developed by postnatal vasculogenesis: the formation of new blood vessels from EPCs that differentiate into mature ECs (55). These EPCs are defined as CD34þ/CD133þ/CD177þ/KDRþ cells, which originate in the bone marrow. EPC recruitment and mobilization have been positively correlated with an increase in VEGFA levels; in response to increased VEGFA levels at the site of the tumor, an increase in circulating EPCs was seen (Fig. 2) (56–58). Moreover,
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Figure 2 A few of the molecular and cellular players in the tumour/microvascular microenvironment. (A) Tumour cells produce VEGF-A and other angiogenic factors such as bFGF, angiopoietins, interleukin-8, PlGF and VEGF-C. These stimulate resident endothelial cells to proliferate and migrate. (B) An additional source of angiogenic factors is the stroma. This is a heterogeneous compartment, comprising fibroblastic, inflammatory, and immune cells. Recent studies indicate that tumour-associated fibroblasts produce chemokines such as SDF-1, which may recruit bone marrow–derived angiogenic cells (BMC). The various hypotheses on the nature and role of such cells in angiogenesis and tumour progression are discussed in the text. VEGF-A or PlGF may also recruit BMC. Tumour cells may also release stromal cell-recruitment factors, such as PDGF-A, PDGF-C or TGFb. A well-established function of tumour-associated fibroblasts is the production of growth or survival factor for tumour cells such as EGFR ligands, HGF, and heregulin. (C) Endothelial cells produce PDGF-B, which promotes recruitment of pericytes in the microvasculature after activation of PDGFRb. Abbreviations: TGF, transforming growth factor; HGF, hepatocyte growth factor. Source: From Ref. 150.
CD133þ cells differentiate into mature-type adherent ECs and abolish the CD133 expression in response to VEGFA (59). While it has been shown that these bone marrow–derived EPCs contribute to the tumor vessel formation by incorporating into the endothelium (60), a second group of proangiogenic cells was described; this population, rather than the EPCs, was thought to home in specifically to the tumor and was characterized by the expression of CD45þ, TEKþ, CD31-, and CD11bþ (61). Inhibition of those cells resulted in a
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significant reduction of tumor angiogenesis and growth. Finally, the existence of a third proangiogenic population was suggested, which is characterized by CD34, CXCR4 and FLT1 expression and is implicated in the initiation and formation of metastases (62). A related circulating cell population is the group of the circulating endothelial cells (CECs); these cells express CD146þ, CD133-, vWFþ, VEcadherinþ, CD45-, and CD14-. The presence of CECs has been described as a useful marker for vascular damage; mature CECs are thought to have sloughed off the vessel wall, indicating endothelial damage (63). Another approach is monitoring CECs as a marker for anticancer treatment; elevated levels of CECs have been described in several malignancies, including multiple myeloma (MM) and lymphoma. In MM, an elevated number of CECs was described, correlating with serum markers of disease activity (64). High CEC levels at diagnosis of lymphoma are changed to normal when they achieve complete remission (CR) following chemotherapy (65). ANTIANGIOGENIC CONCEPTS The importance of new vessel formation and/or VEGFA production for tumor growth resulted in efforts to inhibit angiogenesis, leading to the development of antiangiogenic concepts. In the past, a differentiation has been made upon the goal of the antiangiogenic treatment; three classes are described: antiendothelial cell strategy (e.g., endostatin, thrombospondin), anti-VEGFA therapy (Bevacizumab or VEGFR inhibitors, such as PTK787/ZK 222584, SU5416, SU6668, GW786034), and vascular targeting. The difference between antiendothelial drugs (Rx) and anti-VEGFA Rx is the target of the strategy; the former are directed to ECs whereas the latter exert their effect on ECs as well as on tumor cells. The third class, vascular targeting, is still a promising goal; it initiates a specific response to the already established tumor vascular supply (66). Until now, however, without the recognition of tumor-specific vessel epitopes, this remains a future perspective. Moreover, vascular disrupting agents will result in rapid necrosis and a chance for the fast regrowth in the viable outlayer of a tumor. Mice models and in vitro studies have demonstrated the feasibility of these antiangiogenic concepts. However, antiangiogenic compounds are often characterized by a short half-life, and the delivery at the tumor site remains difficult. Therefore, treatment results only in marginal changes of angiogenesis. Changes of angiogenesis can also be achieved by chemotherapeutics drugs. Tumor-associated ECs proliferate at a much lower rate than cancer cells and are therefore weakly inhibited by conventional chemotherapeutic protocols. During the normal rest periods of chemotherapy, those ECs may recover rapidly, resulting in the regrowth of the tumor. Therefore, frequent administration of lower doses of cytotoxic agents, called ‘‘metronomic scheduling,’’ might be a better strategy to impede the repair of those ECs. Preclinical models have shown
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that metronomic dosing induced repetitive peaks of EC apoptosis, whereas conventional scheduling showed a single wave of EC damage that was largely repaired at the end of the rest period (67,68). Although the metronomic dosing resulted in a delay of tumor growth, these tumors will eventually escape control and relapse. As a result, metronomic schedules have been administered in combination with antiangiogenic compounds, which resulted in a significant increased therapeutic efficacy (68–70). Another explanation for the repair of tumor vasculature during the prolonged drug-free periods between the cycles of high-dose chemotherapy is the mobilization and viability of bone marrow–derived EPCs as part of an adaptive response to the chemotherapy-induced myelosuppression (71). A few days after treatment with high-dose chemotherapy, a high number of EPCs were mobilized, which mediate repair of the vasculature. In contrast, metronomic dosing schedules suppressed EPC numbers and induced the apoptosis of EPCs (72). Because other studies showed the contribution of CECs to the tumor growth (73,74), the antivasculogenesis process of metronomic scheduling may also be mediated through reduction of EPC mobilization and viability. VEGFA is known to promote the mobilization of bone marrow–derived EPCs, which may subsequently differentiate into CECs (58). This mobilization is mediated by VEGFA binding to both FLT1 and KDR (75). VEGFA is also thought to promote survival by activating antiapoptotic pathways in EPCs and CECs (63,76,77). Therefore, treatment with anti-VEGF/VEGFR antibodies may also result in beneficial effects on tumor vasculature by a reduction of CECs mobilization as well as by an increase in apoptosis (78). Initiation of the development of anti-VEGFA strategies, targeting both ECs and tumor cells, followed the knowledge that VEGFA was an important player in tumor growth. Pharmaceutical compounds for humans interfering with VEGF/ VEGFR signaling are currently under development. At this moment, the Food and Drug Administration has approved a humanized anti-VEGFA monoclonal antibody AvastinTM (rhuMab VEGF, bevacizumab, Genentech, South San Francisco, California, U.S.) as a first-line treatment for metastatic colorectal cancers. Phase II/III studies in various malignancies are already reported to be promising for VEGF/VEGFR-interfering drugs in addition to chemotherapeutic strategies; this accounts for anti-KDR antibodies, small molecules inhibiting KDR signal transduction, and VEGFR chimeric proteins (79,80). For an overview of the studies, see Table 2. THE ROLE OF VEGF AND ANTI-VEGF THERAPY IN HEMATOLOGICAL MALIGNANCIES Multiple Myeloma The prognostic relevance of increased angiogenesis in the field of hematological malignancies was for the first time observed in MM (81,82). Increased VEGFA levels are present in the serum of MM patients with advanced disease stages and
Trial
Phase III, renal cell cancer, Phase III, non-small cell lung cancer Phase II/III, Advanced or metastatic non-small cell lung cancer
Tamoxifen citrate Thalidomide
Phase I-IV, primarily breast cancer Phase I-III, several cancer types primarily multiple myeloma
Drugs that inhibit endothelial cells directly ABT-510 Phase I/phase II, advanced head and neck cancer, phase I, advanced solid tumors NGR_TNF Phase I, advanced solid tumors Comretastatin A4 Phase II, advanced anaplastic thyroid cancer, Phase I, advanced Phosphate solid tumors Dimethylxanthenone Phase II, hormone-refractory metastatic prostate cancer acetic acid lenalidomide Phase I-II, several cancer types, Phase III, multiple myeloma and refractory b-cell chronic lymphocytic leukemia, Phase IV, multiple myeloma LY317615 Phase I-II, several cancer types including, Phase III glioblastoma and lymphoma Soy isoflavone Phase II, bladder cancer, stage IV breastcancer, malignant melanoma, renal clear cell carcinoma and pancreatic cancer
Neovastat BMS-275291
Drugs that block breackdown of extracellular matrix Dalteparin Phase II ovarian cancer, advanced colon cancer Marimastat Phase III, small cell lung, and breast cancer COL-3 Phase I/II, brain
Drugs
Table 2 Drug Mechanisms in Various Malignanices: An Overview of Phase I–IV Trials Angiogenesis in Hematological Malignancies
(Continued)
Blocks the activity of PTK, topoisomerase II, and MMP9 and downregulates the expression 11 genes, including VEGF antioestrogen Unknown
Induces the cytokines TNFa, serotonin, and nitric oxide derivative of thalidomide, immonumodulatory drug, exact mechanism not known Protein kinase Cb–selective Inhibitor
Tumor vasculature TNFa microtubule-disrupting agent
Thrombospondin analog
Inhibition of blood coagulation Synthetic inhibitor of MMPs Synthetic MMP inhibitor. Tetracycline derivative Naturally occurring MMP inhibitor Synthetic MMP inhibitor
Mechanism
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Phase I, advanced cancers, Phase II, non small cell lung cancer, Phase II, ovarian cancer Phase I, solid tumors
Squalamine
Phase I–II, several cancer types, phase II, glioblastima, pancreatic cancer, myelodysplastic syndromes, multiple myeloma, breast cancer, gastrointestinal tumors, and meningioma
VEGF receptor-2 tyrosine kinase inhibitor VEGF receptor-2 tyrosine kinase inhibitor Inhibits heparanase activity and heparan sulfate binding to FGF and VEGF Multikinase inhibitor, targeting VEGF, PDGF, and c-kit receptors
VEGF receptor-2 and 3, FLT3 and c-Kit tyrosine kinase inhibitor VEGF receptor-2 tyrosine kinase inhibitor
VEGF receptor-2 tyrosine kinase inhibitor
Antibody against VEGF
Blocks N-cadherin Multikinase inhibitor targeting VEGF, PDGF, and c-kit receptors Antibody against VEGF
Extract from dogfish or shark liver; inhibits sodium, hydrogen exchanger NHE3 Inhibition of endothelial growth
Mechanism
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PTK787/ZK 222584
Drugs that block the activators of angiogenesis ADH-1 Phase I, incurable solid tumors AMG 706 Phase I–II, several cancer types, including breast cancer and non–small cell lung cancer Bevacizumab Phase I–IV, several cancer types, phase IV primarily colorectal cancer and non–small cell lung cancer Avastin Phase I–IV, several cancer types, phase IV primarily colorectal cancer and non–small cell lung cancer AZD2171 Phase I–III, several cancer types, phase III primarily colorectal cancer, breast cancer, and non-small cell lung cancer Bay 43-9006 Phase I–III, several cancer types, phase III renal cell cancer, non–small cell lung cancer, and melanoma BMS-582664 Phase I, solid tumors, phase II, metastatic hepatocellular cancer, phase II, advanced gastrointestinal malignancies CHIR-265 Phase I, advanced metastatic melanoma GW786034 Phase I–III, several cancer types, phase III, renal cell cancer PI-88 Phase II, metastatic melanoma, phase II, prostate cancer
Endostatin
Trial
Drugs
Table 2 Drug Mechanisms in Various Malignanices: An Overview of Phase I–IV Trials (Continued )
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Phase I–II, breast cancer, Phase I, bladder cancer
Phase I–III, several cancer types, phase III, gastrointestinal stromal tumors, renal cell carcinoma, breast cancer, panreatic island tumors, and colorectal cancer Phase I, advanced malignancies
Phase I–III, several malignancies, phase III, non–small cell lung cancer Phase I–III solid tumors, phase I, AML, phase II, multiple myeloma
Phase I, advanced solid tumors
Phase II–III advanced solid tumors
Suramin
SU11248
ZD6474
SU6668
Interferon-a
Upregulation of interferon g and IP-10 Unknown mechanism
Highly selective COX-2 inhibitor Inhibitor of calcium influx
Integrin antagonist Small molecule blocker of a-v-integrins present on endothelial cell surface
Inhibits MET, VEGF receptor2, FLT3, c-Kit, and TIE2 Inhibits VEGF receptor, and EGF receptor activity Selective inhibition of VEGF receptor activity Blocks VEGF, FGF, and PDGF receptor signaling Inhibition of bFGF and VEGF production
Inhibits growth factors and receptors, including EGF, PDGF, FGF and VEGF Multikinase inhibitor targeting VEGF, PDGF, FLT3, and c-kit receptors
Mechanism Angiogenesis in Hematological Malignancies
Abbreviations: FGF, fibroblast growth factor; PDGF, platelet-derived derived growth factor; VEGF, vascular endothelial growth factor; TNFa, tumor necrosis factor alpha; MMP, matrix metalloproteinases.
Drugs with non-specific mechanism action Celecoxib Phase II–III, prostate cancer CAI Phase I, studies in combination against solid tumors, Phase II, ovarian cancer, Phase II, metastatic renal cell cancer Interleukin-12 Phase I–II, Kaposi’s sarcoma IM862 Phase I, recurrent ovarian cancer, phase II, metastatic cancers of the colon and rectal, phase III, Kaposi’s sarcoma
Drugs that inhibit endothelial-specific integrin/survival signaling ATN-161 Phase I–II, recurrent intracranial malignant glioma EMD121974 Phase I, advanced solid tumors or lymphoma, phase II, metastatic androgen-independent prostate cancer, phase II, glioblastoma multiforme
SU5416
XL184
Trial
Drugs
Table 2 Drug Mechanisms in Various Malignanices: An Overview of Phase I–IV Trials (Continued )
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associated with increased angiogenesis (83–85). Moreover, VEGFA is identified to play a key role in sustaining angiogenesis in MM (86,87). A paracrine loop has been described: it was shown that VEGFA is expressed and secreted by MM cells and this MM derived VEGFA stimulates IL-6 secretion in bone marrow stem cells (BMSCs). Stromal cell derived IL-6 in its turn enhances MM cell proliferation, migration, survival, and VEGFA production, thereby augmenting MM cell growth, one of the first described paracrine VEGFA loops (88,89). Another autocrine loop has been demonstrated via FLT1. Therefore, it has been suggested that direct and/or indirect targeting of VEGFA and its receptors may provide a potent therapeutic approach for the related angiogenesis as well as for paracrine and autocrine-mediated tumor growth. The knowledge about increased tumor growth augmented by angiogenesis resulted directly in new antiangiogenic treatment strategies. Thalidomide was used as an antiangiogenic drug in MM and resulted in enhanced survival. Nowadays, it is demonstrated that the function of thalidomide seems not restricted to antiangiogenesis; surrounding bone marrow stromal cells as well as myeloma cells themselves seemed to be targets of thalidomide (90). To counteract angiogenesis and/or VEGFA more specifically, a new generation of drugs was used. Several first-generation small molecule VEGF receptor inhibitors, such as the receptor tyrosine kinase inhibitor PTK787/ZK 222584 (a joint development project between Novartis Pharmaceuticals, Basel, Switzerland and Schering AG, Berlin, Germany) and the pan inhibitor of VEGF receptors GW654652 (GlaxoSmithKline, Middlesex, U.K.) showed significant anti-MM effects in vitro (91,92). Recently, the small-molecule tyrosine kinase inhibitor of FLT1, KDR, and FLT4, pazopanib (GW786034B; GlaxoSmithKline), showed for the first time in vivo tumor inhibition in a mouse model (93). At the moment, a multicenter phase I/II study with single agent pazopanib is ongoing in patients with relapsed or refractory MM. Hodgkin’s Lymphoma Angiogenesis and angiogenic factors have also been studied in malignant lymphomas. Although not much is known about the role of angiogenesis in Hodgkin’s lymphoma (HL) one study showed an inverse relationship between microvessel density (MVD) and the stage of disease in patients with HL. It was hypothesized that in HL, the angiogenic phenotype might be an early event, and the angiogenic capacity is tempered and unable to keep pace with the growth of the neoplastic cells (94). The prognostic role of VEGFA has been observed in HL as well, showing that both high pretherapy and posttherapy VEGFA levels were independently predictive for a poor overall survival (95). Another independent prognostic factor for HL is the amount of mast cells: Hodgkin patients with many mast cells in their tumor had a shorter relapse-free survival (96). Those mast cells express several factors such as VEGFA, which can affect
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angiogenesis both directly and indirectly (97). It was therefore hypothesized that the increase in angiogenesis was because of an increase of VEGFA produced by mast cells. However, so far a high microvessel count could not be correlated to mast cell count (98). Until now, no preclinical or clinical trials with anti-VEGF drugs have been reported for the treatment of HLs. Non-Hodgkin’s Lymphoma The role of angiogenesis and angiogenic factors is has been studied more extensively in non-Hodgkin’s lymphomas (NHL) compared with HL. In contrast to the observations in HL, the angiogenesis increases with tumor progression (99). It is known that the lymph node biopsies in patients with NHL show an increased MVD compared with benign lymphadenopathies (100). VEGFA has also been implicated in the overall disease course of patients with NHL; VEGFA levels were elevated in patients with NHL compared with normal individuals, the event-free survival rate was significantly higher in patients with baseline VEGFA levels, and the response to therapy was significantly more adequate in VEGFA-negative patients compared with VEGFA-positive patients (95, 101,102). Furthermore, the serum concentration of VEGFA and/or bFGF is an independent prognostic factor for outcome (103,104). NHL cells secrete VEGFA and express FLT1 and KDR, suggesting the presence of autocrine and paracrine pathways (105). Moreover, most of the malignant lymphomas express VEGFC, which can bind to FLT4 and is involved in lymph angiogenesis. The level of lymph vessel density (LVD) was significantly correlated to the expression levels of VEGFA and VEGFC. In vivo monoexperiments showed that treatment with an inhibitor targeting tumor FLT1 or host VEGFR2 reduced established diffuse large B-cell lymphoma (DLBCL) xenograft growth, whereas targeting tumor KDR and host VEGFR1 had no effect. Decreased tumor volumes correlated with increased tumor apoptosis and reduced vascularization, respectively, suggesting the existence of autocrine FLT1– and paracrine KDR–mediated pathways in lymph angiogenesis (105). Preliminary results of a phase II clinical trial with bevacizumab a recombinant monoclonal antibody against VEGFA) showed a well-tolerated and prolonged stabilization of disease in patients with relapsed, aggressive NHL (106). Another approach is a small-molecule inhibitor; Sorafenib (Bayer AG/Onxy Inc., Leverkusen, Germany) is an inhibitor of several kinases including Raf, VEGFR, and PDGF. Currently a phase I study with sorafenib is ongoing for NHL. Acute Myeloid Leukemia At diagnosis of acute myeloid leukemia (AML), pronounced vessel density is found in the bone marrow compared with remission-status bone marrow samples or normal controls (107–109), whereas in bone marrow specimens of refractory
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AML vessel density remained high. The impact on outcome was described more recently. A study using dynamic contrast-enhanced magnetic resonance imaging (dMRI) (lumbar spine) measuring blood flow and perfusion as marker for angiogenesis showed that high blood flow or perfusion was correlated to a poor outcome (110). A correlation between increased vessel density and high VEGFA was described previously (107,108). The importance of VEGFA in AML was described by various independent studies. These studies demonstrated that VEGFA was an independent prognostic factor for therapeutic outcome (53,111). Biological insights of VEGFA in AML showed that VEGFA was not only a key player in angiogenesis resulting in indirect tumor growth, but that VEGFA was also able to induce and/or support leukemic cell growth in other ways. First, an additional paracrine route of tumor enhancement was described related to the interaction with bone marrow stromal cells (54,112). Two pathways were recognized; leukemic cell–derived VEGFA leads to GM-CSF production of stromal cells, and this GM-CSF, in turn, supports leukemic cell proliferation and/or survival (112). In another paracrine route, leukemic cell–derived growth factor expression resulted in VEGFA production of stromal cells or ECs. When leukemic cells are sensitive for VEGFA stimulation, this route also enhances tumor growth (113). A second way in which VEGFA results in more leukemic cell proliferation and/or survival is an autocrine route (114). It was shown that AML cells can produce VEGFA and express its receptor, which makes AML cells sensitive for VEGFA dependent proliferation. The downstream effects of VEGFA are mainly executed by KDR binding, resulting in increased AML cell survival and proliferation (via MAPK and PI 3-K/Akt signaling) and protection against apoptosis (via Bcl-2 and Mcl-1) (114–117). These biological insights resulted directly in new antiangiogenic and anti-VEGF treatment studies. Clinical trials are ongoing for AML. A phase I study of SU11248, a receptor tyrosine kinase (RTK) inhibitor of c-Kit, FLT3, KDR, and PDGFR in the treatment of patients with refractory or resistant AML induced partial remission of short duration (118). In a phase II trial SU5416, a RTK inhibitor of c-kit, FLT3, and KDR showed modest clinical activity (119). In this particular study, patients with AML blasts expressing high levels of VEGFA had a significantly higher response rate than patients with low VEGFA expression. In another phase I study, no significant responses to treatment with PTK787/ZK 222584 in patients with primary refractory or relapsed AML was shown. PTK787/ZK 222584 was also tested in patients with secondary AML. Monotherapy resulted in a stabilization of the disease; when PTK787/ZK 222584 was combined with chemotherapy one-third of the patients achieved CR (120). Another clinical approach for the treatment of AML was chosen by using bevacizumab, an anti-VEGFA monoclonal antibody, following chemotherapy (121). In adults with relapsed and refractory AML who were resistant to traditional chemotherapy, a combination of cytotoxic chemotherapy followed by
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bevacizumab resulted in a CR in one-third of the patients and an increase in the median disease-free survival (121). Clinical experiences in studies of phases I and II showed that single agents often are suboptimal in the induction of a clinical response (partial or complete remission) in AML. Various independent studies demonstrated the activation of one of the three important signaling transduction pathways; PI3K/Akt pathway, PKCa phosphorylation and RAS/Raf/MEK/ERK pathway, and its relation with outcome (122–125). Recently, it was shown that simultaneous activation of more than one signal transduction pathways confers poor prognosis in AML (126). These results will have broad implications for the field of drug development; by tradition, new agents are evaluated individually. Consequently, cross-activation between these pathways may suggest that a drug can be ineffective, whereas in cooperation with drugs targeting multiple signaling transduction pathways, the result might be more effective. Until now, the possible role of these kinds of strategies related to outcome is still unclear. Acute Lymphoblastic Leukemia The importance of VEGFA and angiogenesis in acute lymphoblastic leukemia (ALL) is controversial. In the marrow of children with ALL, a significantly elevated MVD was found compared with healthy controls. Moreover, the MVD dropped towards normal in remission (127,128). However, there was no difference in MVD at presentation or remission from patients with a poor prognosis (128). VEGFA levels were higher in patients with recurrent disease compared with those with newly diagnosed ALL. Relapse-free survival and overall survival were shorter in ALL patients with high VEGFA levels (129). Recently, Avramis showed that increased VEGFA serum concentrations during induction are correlated with events and poor survival of standard-risk ALL pediatric patients (130). In contrast, Aguayo showed that levels of HGF, TNFa, and bFGF, but not of VEGFA, were found to be elevated in blood samples of patients with acute lymphoblastic leukemia (ALL) (131). In addition, higher levels of interleukin-1 receptor a, interleukin-8, FLT1 and KDR, but not VEGFA, were predictive of poor survival in adult ALL patients (132). Moreover, another study reported that the serum levels of VEGF at the time of diagnosis were significantly lower than in the control group and at the time of remission (133). In conclusion, controversial data are published on the topic of ALL cells and angiogenic factors; a better understanding of the complex interaction is needed. Currently, it is not clear what the potential role of therapeutic (i.e., antiVEGF) interventions in ALL will be. Chronic Myeloid Leukemia An elevated MVD of the bone marrow is also one of the characteristics for patients with chronic myeloid leukemia (CML) (131). It has been shown that the
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morphology of the vessels in the bone marrow of CML patients differed from controls in that the vessels were more tortuous and branched. The characteristics of the microvessels and the MVD also appeared to be predictors of patient survival and progression (134). In addition, patients with newly diagnosed CML were found to have an increased VEGFA plasma concentration compared with healthy controls (135). CML patients with a higher VEGFA level had a shorter overall survival (136). A reduced overall survival was also correlated with an upregulation of KDR (137). Those data support the prognostic value of VEGFA and MVD in CML patients. Moreover, the number of VEGFA-positive bone marrow cells correlated significantly with the MVD (138). One of the drugs used for the treatment of CML is imatinib mesylate (STI571, also known as GleevecTM , Novartis Pharmaceuticals, Basel, Switzerland), a specific inhibitor of Bcr-Abl tyrosine kinase activity. The oncogene Bcr-Abl induces expression of VEGFA (139). An in vitro study demonstrated that imatinib inhibited VEGFA gene transcription by targeting the Sp1 and Sp3 transcription factors (135). Therefore, this drug might also be a potent inhibitor of VEGFA signaling. SU5416, an inhibitor of KDR and other tyrosine kinase receptors, was tested in a phase II clinical trial of patients with myeloproliferative disorders (MPD), including four patients with CML. Unfortunately, patients did not benefit from this study (140).
Chronic Lymphocytic Leukemia Patients with chronic lymphocytic leukemia (CLL) had a significantly higher MVD measured in bone marrow biopsies than controls. This increased MVD also correlated significantly with the clinical stage of the CLL patients (141,142). Furthermore, other parameters were considered to be useful for prognostics; the plasma levels of VEGFA, bFGF, and HGF were significantly increased in CLL (131) and these high serum levels of VEGFA correlated with a poor clinical outcome (143). A significantly shorter overall survival was also correlated with elevated levels of KDR (144). In addition, it is known that CLL cells produce VEGFA (145) and that the receptors for VEGFA (FLT1, KDR, and FLT4) were expressed in the majority of CLL patients (146), suggesting an autocrine loop that stimulates CLL growth. VEGFA was able to decrease the apoptotic potential of CLL B cells significantly (147); interruption of these pathways might therefore contribute to increased leukemic cell death. An in vitro study showed that inhibition of VEGF receptor activation with either tyrosine kinase inhibitors or VEGF neutralizing antibodies inhibited VEGF receptor phosphorylation, decreased p-STAT3 (serine 727), Mcl1, and induced cell death in CLL cells (148). Until now, no clinical trials have been published.
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CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Although anti-VEGF/VEGFR therapy in clinical trials seems promising, the net result of anti-VEGF/VEGFR therapy in human clinical trials is an increased survival for only a few months. The discrepancy between marked antitumor activity of VEGF/VEGFR inhibitors in, for instance, mouse-models and less promising clinical results when used as a single agent has resulted in the use of combination strategies. For the future, the ideal balance for dosage and administration of antiangiogenic drugs in combination with chemotherapeutics will be one of the hallmarks of research in this field; combined treatment with antiangiogenic agents and chemotherapy seems to be more effective. In addition, broad apoptotic drugs against ECs as well as tumor cells appear to function better than the cell cycle–dependent drugs used in clinics nowadays. Combinations of several tyrosine kinase inhibitors covering a broad spectrum of receptors, in order to avoid the occurrence of escape mechanisms, also show promising results, and are therefore another future perspective in the field of antiangiogenic drugs. There are still many challenges in the molecular understanding of tumor vasculature. It was hypothesized that normalization of tumor vessels could result in the improvement of the treatment by a more efficient delivery of drugs to the targeted cancer cells. It still needs to be answered how these vessels will ‘‘normalize.’’ Furthermore, the challenge will be how to measure this normalization in patients. There is a broad debate on the best way of imaging vessels and blood flow; promising results are shown with dMRI, dynamic computed tomography (dynamic CT), or positron emission tomography (PET) nuclear scanning in specific conditions. The general solution for imaging in clinical studies is not available yet. It can be hypothesized that revascularization occurs concomitant with tumor growth during treatment with anti-VEGF Rx due to provoked hypoxia, which in turn results in an upregulation of various angiogenic growth factors (e.g., VEGFA, FGF, ephrins, and angiopoietins) which may ultimately result in resistance. Another theory about the mechanism behind revascularization is the mobilization of EPCs derived from the bone marrow, which may subsequently differentiate into mature CECs. Those cells may home in to the tumor and form new vessels. Mobilization and recruitment of the CECs can be promoted by VEGFA through interaction with its receptors, FLT1, and KDR, which are expressed on the EPCs (75). VEGFA is also thought to promote survival by activating antiapoptotic pathways in EPCs and CECs (77). Therefore, VEGF/ VEGFR antibodies might have beneficial effects on the outgrowth of tumor vessels. Tackling the problem of dosing and timing will be difficult because molecular processes in normal and tumor vessels are still not completely understood. It is generally known that the maximal tolerable dose of anti-VEGF/ antiangiogenic Rx is not always the most optimal dose. The classical definitions
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for phases I and II need to be reset for biological modulators. Moreover, the decision about the most optimal dose is hampered because measurable endpoints are hard to define. Recent studies demonstrated disadvantages of antiangiogenic/ VEGF approaches such as increased tromboembolic events and hypertension. It still remains to be investigated whether these tromboembolic events are mainly age, disease, and/or treatment related. Also, rational timing of antiangiogenic/ VEGF treatment strategies is difficult due to a lack of complete biological understanding. However, promising results ask for more understanding with global, robust techniques to gain more insights in this process. These insights will guide the way for answering dose and time-frame questions. REFERENCES 1. Algire GH, Chalkley HW, Legallais FY, et al. Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst 1945; 6:73–85. 2. Ide AG, Baker NH, Warren SL. Vascularization of the Brown Pearce rabbit epithelioma transplant as seen in the transparent ear chamber. Am J Roentgenol 1939; 42:891–899. 3. Greenblatt M, Shubi P. Tumor angiogenesis: transfilter diffusion studies in the hamster by the transparent chamber technique. J Natl Cancer Inst 1968; 41(1): 111–124. 4. Ehrmann RL, Knoth M. Choriocarcinoma. Transfilter stimulation of vasoproliferation in the hamster cheek pouch. Studied by light and electron microscopy. J Natl Cancer Inst 1968; 41(6):1329–1341. 5. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971; 285 (21):1182–1186. 6. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983; 219(4587): 983–985. 7. Leung DW, Cachianes G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246(4935):1306–1309. 8. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000; 6(4):389–395. 9. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9(6):653–660. 10. Gilbert-Barness E, Debich-Spicer D. Embryo and Fetal Pathology. Cambridge, UK: Cambridge University Press, 2004. 11. Reynolds LP, Killilea SD, Redmer DA. Angiogenesis in the female reproductive system. FASEB J 1992; 6(3):886–892. 12. Prior BM, Yang HT, Terjung RL. What makes vessels grow with exercise training? J Appl Physiol 2004; 97(3):1119–1128. 13. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1(1):27–31. 14. Folkman J. Angiogenesis and apoptosis. Semin Cancer Biol 2003; 13(2):159–167. 15. Crocker DJ, Murad TM, Geer JC. Role of the pericyte in wound healing. An ultrastructural study. Exp Mol Pathol 1970; 13(1):51–65.
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13 Nucleic Acid-Based, mRNA-Targeted Therapeutics for Hematologic Malignancies Alan M. Gewirtz Division of Hematology/Oncology, Department of Medicine & Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
INTRODUCTION While the advent of small molecule kinase inhibitors and cell surface receptor targeted has made an extraordinary difference in the lives of patients with chronic myelogenous leukemia (CML) (1) and many lymphomas (2,3), patients with other hematologic malignancies are yet to enjoy the benefits of these types of tumor cell–specific therapies. In addition, the issue of resistance to drugs like imatinib and rituximab is becoming increasingly important (4–6). The ability to eliminate proteins, which have escaped specific targeting at the protein level or which have demonstated the ability to evolve resistant forms, is the strength of an RNA-targeted, protein-eliminating, therapeutic strategy. In addition, an everexpanding knowledge of the biochemical and molecular pathogenesis of hematologic malignancies continues to suggest new therapeutic targets (7). Finally, ‘‘gene silencing’’ therapies are in principle highly specific, so if the target gene is thoughtfully chosen, damage to nontargeted tissues should be minimized, and a high therapeutic index should result (8–10).
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A BRIEF OVERVIEW OF GENE SILENCING Numerous gene silencing strategies have evolved over the years, and these have been primarily directed either to the genes themselves (11–13) or to their messenger RNAs (mRNAs). Some exceptionally clever techniques for direct gene targeting, such as the use of DNA-binding polyamides, have been developed (13,14), but thus far, they have not proven either specific or reliable enough for therapeutic applications. In contrast, the perceived ease with which mRNA can be targeted has resulted in most therapeutic efforts being directed to this approach (15–17). A number of modalities are available for mRNA targeting, and of these, the ‘‘antisense’’ strategies have been the most widely applied (18–20). All are based on delivery of a reverse complementary, i.e., antisense nucleic acid (ASNA) strand into a cell expressing the gene of interest. By processes still unknown, the ASNA strand and the mRNA target come into proximity and then hybridize if the strands are physically accessible to each other. Stable mRNAASNA duplexes can interfere with splicing of heteronuclear RNA into mature mRNA (21,22), block translation of mature mRNA (23,24), or can lead to the destruction of the mRNA by binding of endogenous nucleases, such as RNase H (25,26), or by intrinsic enzymatic activity engineered into the sequence as is the case with ribozymes (27,28) and DNAzymes (29,30). More recently, posttranscription gene silencing or RNA interference (RNAi) (31,32) has emerged as an exciting potential alternative to these now more classical gene silencing approaches (19,20,33). The RNAi pathway is a mechanism of gene silencing whereby double stranded RNA (dsRNA) interferes with the production of a gene’s encoded protein in a sequence-specific manner (34). The importance of RNAi as a natural mechanism for protecting cells from viral infection, regulating gene expression at the level of translation, and modifying chromatin has developed through a convergence of discoveries in plants, unicellular organisms, invertebrates, and mammals (35). The mechanism by which the RNAi pathway is activated and functions to silence genes is now known in some detail. Briefly, dsRNA is taken up by a cell and processed into 21–22-nucleotides (nt) long small interfering RNA (siRNA) by the endonuclease Dicer (36,37). Similarly, a species of endogenously derived hairpin RNAs called microRNA (miRNA) (38–41) is processed by the enzyme Drosha; these products then become Dicer substrates (40,42). Typically between 19 and 23 base pairs (bp) in length, siRNA has a 2-nt overhang on the 30 end and must have a 50 phosphate and a 30 hydroxyl group to be active [43,44]. The specificity of siRNA is guided by its interaction with an enzyme complex known as the RNAinduced silencing complex (RISC). RISC is activated when it incorporates siRNA. Once incorporated, the siRNA is unwound, and, using the antisense strand, RISC helps promote hybridization and ultimately cleavage of the complementary mRNA sequence. When complementary mRNA is found, RISC cleaves the target mRNA at a position 10 bp upstream of the 50 end of the siRNA. siRNA is the effector molecule of the pathway from a therapeutic point
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of view and is the form of dsRNA most likely to be clinically useful, potent, and specific. Two potential problems associated with the use of dsRNA are that it does not always act in a sequence-specific manner (45), and that exogenously introduced dsRNA can be a potent inducer of interferon and other effectors of innate immunity such as RNA-binding protein kinase (PKR) and RNase L (46,47), initiating a cascade of events that leads to widespread and nonspecific posttranscriptional gene silencing and ultimately cell death (48). This is conventionally thought to occur in the presence of longer dsRNA molecules (>30 bp) via the RNase L and the PKR and/or the toll-like receptor-3, but very new data challenge this dogma, at least in certain cell types. Two groups have recently identified short immunostimulatory sequence motifs that specifically induce an interferon response. Hornung et al. have identified short immunostimulatory sequence motifs (9 bp within a 19 bp fragment) that induce an interferon response in plasmacytoid dendritic cells via the toll-like receptor-7. Judge et al. have also identified sequence motifs that are immunostimulatory in primary human monocytes and plasmacytoid dendritic cells and suggest that the recognition happens within the endosomal pathway (49,50). It is quite clear that many of the considerations that apply to the use of traditional antisense DNA molecules and ribozymes will also apply to RNAi whether in the form of siRNA, miRNA, or short hairpin RNA (shRNA) expressed from viral vectors (51). These issues include (1) the ability to deliver these molecules into target cells (52–54), (2) development of chemical modifications that increase intracellular stability of the targeting molecules without compromising ability to hybridize with the mRNA target and disable or destroy it (54–57), (3) develop reliable, reproducible strategies for targeting a desired mRNA, and (4) keep ‘‘off target’’ or unintended silencing to a minimum (49,58–61). DELIVERING NUCLEIC ACID MOLECULES TO LIVING CELLS It is straightforward that without the ability to deliver material into cells, even the most cleverly designed molecule cannot be effective. As a general rule, oligonucleotides are taken up primarily through a combination of adsorptive and fluid phase endocytosis (62). After internalization, confocal and electron microscopy studies have indicated that the bulk of the oligonucleotides enter the endosome or lysosome compartment where most of the material either becomes trapped or degraded. Biological inactivity is the predictable result of these events. Nevertheless, oligonucleotides can escape from the vesicles intact, enter the cytoplasm, and then diffuse into the nucleus where they presumably acquire their mRNA target. Colocalization of effector strand and target mRNA in nucleoli (28) or cytoplasmic P-bodies (63) appears important for antisense oligonucleotide (AS ON) and siRNA, respectively. In our hands and those of others (64), lipid transfecting agents have proven toxic to hematopoietic cells. Accordingly, we have
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begun to develop alternate means for delivering AS ON and siRNAs to hematopoietic cells including the use of electroporation for ex vivo delivery (65). We, as are others, are also exploring the possibility of using virosomes (reconstituted viral envelopes) whose original DNA or RNA viruses have been replaced with ASNA (66), ovarious depot or protecting agents such as chitosan polymers (67,68) for systemic delivery. The subject of nucleic acid delivery to living cells has been the focus of numerous recent reviews (69–79). While there are many prospects on the horizon, delivery remains a major roadblock to this form of therapy. CHEMICAL MODIFICATION OF NATIVE NUCLEIC ACIDS FOR THERAPEUTIC PURPOSES Developing chemical modifications of nucleic acids has turned out to be a demanding task (80–83). For this reason, so called first generation molecules containing a phosphorothioate backbone continue to be employed clinically (84,85). Initial work with antisense DNA was carried out with unmodified, natural molecules. It soon became clear, however, that native DNA was not only subject to relatively rapid degradation, primarily through the action of 30 exonucleases, but as a result of endonuclease attack as well. Molecules are now routinely modified to enhance their stability as well as the strength of their hybridization with RNA since these physical attributes are required if the molecules are to function as drugs (82). They also need to be able to cross cell membranes and then to hybridize with their intended RNA target. As will be discussed in more detail below, RNA-associated proteins and tertiary structure are important factors governing the ability of oligodeoxynucleotides (ODN) to hybridize with their target (86). Finally, ODN should exert little in the way of nonsequence-related toxicity (87). Accordingly, while oligonucleotide chemistry continues to evolve, one so called first generation molecule containing a phosphorothioate backbone (red arrow in Fig. 1) continues to be employed clinically.
Figure 1 Chemical structure of 20F-ANA compared with RNA and ANA.
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Among its useful characteristics is the ability to activate RNase H, which many feel is important in mRNA destruction, and therefore antisense DNA mechanism of action. Two modifications, which are also of interest, are the peptide nucleic acids (PNAs) (83) and the morpholino oligonucleotides (PMO) (23). These compounds are very nuclease resistant and form extremely tight bonds with their RNA targets. However, neither penetrates cells and the PNAs do not hybridize as well under physiologic conditions. Nevertheless, progress has been made, and several promising new chemical modifications, which address the needs outlined above, have been described recently (88–92). One that is of particular interest to our group is the 20 -deoxy-20 fluoro-D-arabinonucleic acid (20 F-ANA), developed by Damha and coworkers (93–95) (Fig. 1). The attraction of 20 F-ANA and 20 F-ANA-DNA chimeras derives from their nuclease resistance and their ability to simultaneously increase the strength of oligo:mRNA hybrids and elicit efficient RNase H–mediated degradation of target mRNA. The other two proposed chemistries incorporate a sulfur atom in the sugar ring (30 S- and 40 S-20 F ONs), which imparts hybridization affinity (and likely nuclease resistance) while mimicking the native structure of RNAs. It is quite conceivable that these modifications will also improve the efficiency of siRNA (96). Finally, to the chemical modifications being developed to strengthen ASNA and mRNA hybridization and enhance mRNA destruction, we are experimenting with the ability to activate the silencing molecule in a conditional manner. This can be accomplished with ‘‘caged’’ antisense molecules that are uncaged, i.e., activated, when digested by a specific enzyme, or exposed to light of the appropriate wavelength (97). This capability would bring some measure of control to the silencing machinery with resulting enhancement of targeting temporally expressed genes or for targeting a gene in a tissue-specific manner. THERAPEUTIC TARGET SELECTION Chemical optimization will lead to improved nucleic acid molecules, but selection of which mRNA(s), and, of equal importance, which sequences within those mRNAs to target remain important issues. With regard to appropriate gene targets, we have reasoned that since we are targeting mRNA, and not the protein itself, proteins with short-half-lives would make better targets, especially if the presence of such proteins is required during very specific phases of a cell’s development. Transcription factors would seem ideal candidates with these criteria in mind, and our experience in the laboratory and clinic supports this impression. The bulk of development has focused on targeting the obligate hematopoietic transcription factor c-Myb. The mRNA and protein half-lives of c-Myb are approximately 30 to 60 minutes each (98), and c-Myb plays an important role in both G1/S and G2/M cell cycle transitions (99) as well activation of other critical cellular genes (100–106). In addition, c-Myb itself is
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highly expressed in virtually all hematologic neoplasms except chronic lymphocytic leukemia (CLL) (107). Experience with small molecule drugs and antibodies suggest that directing therapeutic efforts toward a single molecular target are less likely to be successful. For that reason, we are developing a strategy of simultaneously inhibiting multiple targets (108). By way of example, we previously reported that in addition to Myb, the Vav proto-oncogene is amenable to silencing with antisense oligodeoxynucleotides (ASODN) and that inhibition of Vav impairs leukemic cell growth (109,110). Since the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562 and primary patient CML cells (n ¼ 9). K562 and primary CML cells were exposed to ASODN, alone or in combination, for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster and BFU-E and colony forming unit (CFU)-GM colonies were determined and correlated with the ability to downregulate the targeted mRNA. After exposure for 24 hours, K562 cell growth was inhibited in a sequence-specific, dose-responsive manner with either Myb or Vav ASODN. Exposure to both ODN simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24- and 72-hour exposures to either anti-Vav, or anti-Myb AS ODN, equivalent CFU inhibition was observed. Exposing cells to both ASODN simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72 hours of incubation with both ODN, colony formation was diminished significantly when compared with either ODN alone (from ~30% to ~78% for CFU-GM; ~50% to ~80% for BFU-E). On the basis of these results, we hypothesize that exposing primary leukemic cells to ASODN targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules. To target the malignant B-cell lymphomas, we are investigating the utility of targeting Bcl6 mRNA. Bcl6 is a zinc finger protein, which acts as a sequencespecific transcriptional repressor (111). Although Bcl6 mRNA is ubiquitous, its expression is highest in germinal center B-cells where it is thought to repress the expression of genes involved in B-cell activation, cell cycle progression, and terminal differentiation. In non-Hodgkin’s lymphomas, Bcl6 is the most frequently deregulated gene, and abnormal expression is found in approximately 30% to 40% of DLCL, and approximately 14% of follicular lymphomas (FL). We found that cells transfected with an appropriately targeted ASODN exhibited an approximately 50% drop in viability within 24 hours. Coincident with the drop in viability, we found a sevenfold decrease in Bcl6 mRNA expression in cells transfected with 1310, and little change in cells transfected with control oligonucleotides (Fig. 2). Corroborating Western Blot data on Bcl6 expression were also obtained. We were also able to identify regions susceptible to siRNA-based silencing. The degree of silencing obtained using either approach was similar. We concluded from these experiments that effective gene silencing oligonucleotides can be
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Figure 2 Bcl-6 in vitro assays. (A) Bcl-6 RNase Hassay usingSQRM1190 1222. RNA was incubated with each SQRM and run on an agarose gel. TheSQRM1190 successfully recruits RNase H to cleave the RNA of the RNA/DNA hybrid. (B) Bcl-6 SQRM1190 was incubated with various targets and the fluorescence signal was measured. Louckes-1 (20 mg), Louckes-2 (40 mg), and K562 are RNA samples isolated from cell extracts. SQRM was also incubated with in vitro transcribed RNA (IVT RNA; a positive control for SQRM/RNA hybridization) and an ODN (positive control for SQRM function).
developed and that they might find utility as novel agents for treating some patients with non-Hodgkin’s lymphomas. DESIGNING THERAPEUTIC OLIGONUCLEOTIDES How to select sequence targets within the mRNA of interest has been highly problematic. Physical structure of mRNAs is known to play an important role in target accessibility for both classical AS ONs (86) and more recently for siRNA molecules as well (56). Informatic approaches to guide antisense DNA and RNA design abound but are not consistently successful (112–118). For this reason, researchers have developed a novel approach to solving this problem, which depends on the use of fluorescent self-quenching reporter molecules (SQRM) to probe mRNA and signal the presence of hybridization-accessible regions (119–121). To begin, we developed a simple computer algorithm that allowed us to search for palindromes within mRNA sequence downloaded from the NCBI site. Palindromes of between four and six nucleotides were specified, along with
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Figure 3 SQRM design and reaction. (A) Concept: to exploit the traditional stem-loop structure of the SQRMs, a computer algorithm (‘AccessSearch’) searches an entire sequence of mRNA for complementary sequences of a desired length (stems) that are separated by a proscribed distance (loop). (B) Chemistry: the complementary sequences are synthesized as SQRM possessing 50-fluorescein and 30 DABCYL groups. In the absence of target, quenching of fluorescence occurs. Once hybridization of the loop sequence to a complementary target takes place, the moieties are separated and fluorescence can be detected.
intervening sequence of approximately 18 to 20 bases. Numerous sites compatible with these criteria could be found in any message we examined. As shown in Figure 3, the SQRM could then be synthesized as complements to these sites and then used as probes in solution hybridization experiments. SQRM were dissolved in hybridization buffer and added to an excess of the target mRNA. Fluorescence intensity increased compared with background when a SQRM probe hybridized with the mRNA. Hybridization results were used to predict ‘‘hot spots,’’ which were confirmed by targeting mRNA with corresponding ODN or siRNA (120–122).
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In Vivo Treatment Modeling ODN drug candidates are tested for potential clinical efficacy in human-SCID mouse chimeras (123,124). In our original leukemia survival study (123), control mice survived 6 three days (mean SD) after development of overt leukemia, and survival was unaltered by treatment with control sequence ODN. Animals treated with c-Myb ASODN survived 3.5 times or longer and had altered disease phenotype. In animals receiving c-Myb ASODN, the central nervous system and ovaries had markedly less leukemic cell infiltration. We also documented response with downmodulation of c-Myb expression (124). To do this, we employed a simple, highly reproducible, slot blot hybridization technique. DNA extracted from patient cells or serum was slot blotted to a filter and detected with a radiolabeled sense probe. To correlate ODN uptake versus effects on c-Myb mRNA expression versus tumor growth, tumorbearing animals were infused with c-Myb ASODN (500 mg/day) for seven days. On days 7, 9, and 11 post infusion, an animal was sacrificed and its tumor excised to determine tissue c-Myb mRNA levels. Phase I Clinical Studies After extensive preclinical development, we successfully initiated two clinical trials to evaluate the effectiveness of anti-Myb ODN drugs for the treatment of refractory human leukemia. A bone marrow purging study for patients with late stage CML was published (105). In that study, an ODN targeted to the c-Myb proto-oncogene was used to purge marrow autografts administered to allograft-ineligible CML patients. CD34-positive marrow cells were purged with ODN for either 24 (n ¼ 19) or 72 (n ¼ 5) hours. After purging, Myb mRNA levels declined substantially in approximately 50% of patients. Analysis of Bcr-Abl expression in long-term cultureinitiating cells suggested that purging had been accomplished at a primitive cell level in more than 50% of patients and was ODN dependent. Day-100 cytogenetics was evaluated in surviving patients who engrafted without infusion of unmanipulated ‘‘backup’’ marrow (n ¼ 14). Whereas all patients were approximately 100% Philadelphia chromosome-positive [Ph(þ)] before transplantation, two patients had complete cytogenetic remissions; three patients had fewer than 33% Ph(þ) metaphases; and eight remained 100% Ph(þ). One patient’s marrow yielded no metaphases, but fluorescent in situ hybridization evaluation approximately 18 months after transplantation revealed approximately 45% Bcr-Abl-positive cells, suggesting that 6 of 14 patients had originally obtained a major cytogenetic response. Conclusions regarding clinical efficacy of ODN marrow purging cannot be drawn from this small pilot study. Nevertheless, these results lead to the speculation that enhanced delivery of ODN, targeted to critical proteins of short half-life, might lead to the development of more effective nucleic acid drugs and the enhanced clinical utility of these compounds in future. An intravenous infusion trial for patients with refractory hematologic malignancies of any kind has just opened and accrued its first two patients.
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CLINICAL PERSPECTIVE FOR THE NEXT FIVE YEARS The use of reverse complementary nucleic acid drugs to inhibit gene expression originated from studies that were initiated almost a quarter of a century ago (125,126). Even though the mechanism by which these drugs modulate gene expression is not always clear (86,127,128), the clinical development of nucleic acid drugs has proceeded to the point at which several of these drugs have entered phase I/II, and in a few cases, phase III trials. Results to date for most of these trials have shown them to be relatively nontoxic but generally disappointing from the point of view of clinical efficacy (129). There have been glimmers, however, of possible clinical efficacy in a subset of CLL patients (130); though unfortunately for the field, these results were not considered sufficient for drug approval by the FDA. Attempts at certification of the antiBcl2 ODN employed in these studies continues however (R. Warrell, CEO, Genta Inc., personal communication. The first clinical trials of RNAi drugs are about to begin, and there is much excitement accompanying the debut of these molecules as well, especially since one that is locally administered to treat macular degeneration has already received FDA approval (131–134). For all the reasons detailed above, no one disputes that the attraction for drugs of this class remains very strong (132,135). There is no doubt that over the next five years the pace of trials involving siRNA molecules for certain, and perhaps a few antisense DNA molecules as well, will increase dramatically. If these are at all successful, then there is also little doubt that some of the technology used in these trials will be applied to patients with hematologic malignancies. It is the hope of everyone in the field that when these new treatments are applied, patients will benefit and the dawn of an often-promised era of mRNA-targeted therapeutics will finally be upon us.
ACKNOWLEDGMENTS Supported in part by grants from the NIH (CA 101859), the Leukemia and Lymphoma Society, and the Pennsylvania Department of Health. The Department specifically disclaims responsibility for analyses, interpretations, or conclusions. REFERENCES 1. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med; 2002; 346(9): 645–652. 2. Cheson BD. Rituximab: clinical development and future directions. Expert Opin Biol Ther 2002; 2(1):97–110. 3. Johnson AJ, Mone AP, Abhyankar V, et al. Advances in the therapy of chronic lymphocytic leukemia. Curr Opin Hematol 2003; 10(4):297–305.
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14 Active Specific Immunization by the Use of Leukemic Dendritic Cell Vaccines Ilse Houtenbos, Gert J. Ossenkoppele, and Arjan A. van de Loosdrecht Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands
INTRODUCTION Tumor Immunology William B. Coley is often credited with first recognizing the potential role of the immune system in cancer treatment. Coley, an early twentieth-century New York City surgeon, observed that some of his patients with sarcoma underwent spontaneous regression of their tumor. He associated this tumor regression with preceding bacterial infection. Coley was the first physician to exploit the power of the immune system to fight cancer. He deliberately infected cancer patients with bacteria and actually developed a vaccine consisting of killed bacteria to attempt tumor killing (1). Indeed, complete tumor regression was achieved in some patients. It was not until the late 1970s, however, that IL-2 was identified and cloned, making it possible to study T cells ex vivo. Expanding the understanding of the complex working of the immune system now enables exploration of its potential role in cancer therapy. Although intensive chemotherapy-based approaches induce complete remission (CR) in 80% of patients with acute myeloid leukemia (AML), still a lot of patients ultimately relapse because of persistence of minimal residual disease (MRD) cells, resulting in survival percentages of 30% to 40% (2).
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Immunotherapy for leukemia patients, aiming at the generation of antileukemic T-cell responses could provide a new therapeutic approach in eliminating MRD cells in leukemia (3,4). Considerable data point out the critical role played by T-cell immunity in the control of leukemia. Most well known is the reinduction of CR after donor lymphocyte infusion (DLI) for patients with relapsed leukemia after allogeneic stem cell transplantation (5,6). T cells present in DLI are held responsible for this graft-versusleukemia effect. Already in the early 1970s, it was shown that a combination of chemotherapy and immunotherapy, consisting of vaccination with irradiated autologous AML blasts, resulted in an increased survival of patients as compared with treatment with chemotherapy alone (7). More recent data emphasize a possible role for immunotherapy in the eradication of MRD cells, by exploring vaccination strategies (8–11). T-Cell Activation At least two signals are mandatory to activate na€1ve T cells. The first signal is provided by the presentation of the antigen, in the context of the major histocompatibility (MHC) complex, to the T-cell receptor (TCR). The MHC complex consists of class I and class II molecules. Endogenous antigens are presented by MHC class I molecules that can be recognized by CD8-positive cytotoxic T cells (CTL). CTL directly kill their target cells by secretion of granzyme B or perforin. Exogenous antigens are presented by class II molecules to CD4-positive helper T cells (Th cells). A Th1 response is characterized by the production of IFNg and occurs in the presence of IL-12 produced by antigen-presenting cells (APC). Th1 cells are able to activate CTL. Presence of IL-4 induces a Th2 response, characterized by IL-4 and IL-10 production, which results in activation of B cells and consequently antibody formation. CD4-positive regulatory T cells are known to induce tolerance to the presented antigen by secreting inhibitory cytokines. They evolve in presence of IL-10 and TGF-b. A second signal termed costimulation is crucial for final T-cell activation. Absence of this second activation signal can lead to T-cell anergy with subsequent tolerance to the presented antigen. A number of molecular interactions contribute to costimulatory signaling. CD28 is the most important costimulatory molecule expressed on na€1ve T cells and interacts with CD80 and CD86 on APC. Upon activation, T cells will proliferate and differentiate towards effector and memory subtypes. Additionally, T cells upregulate the expression of other costimulatory molecules with either a stimulatory or inhibitory function thereby regulating the immune response. As described earlier, the delicate balance of Th1- or Th2-favoring cytokines produced by APC largely determines the type of T-cell response. This is called the third activation signal.
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Table 1 Characteristics of Immature versus Mature DC
Morphology Immunophenotype
Function
Immature DC
Mature DC
Smaller as compared with mature DC CD40þ, CD54þ/, HLA-DRþ, CD80, CD86þ/, CD83, CCR7 Take up and process antigens
Long cytoplasmic protrusions, eccentric nucleus CD40þþ, CD54þ, HLA-DRþþ, CD80þ, CD86þ, CD83þ, CCR7þ Migration towards lymphnodes Activation of antigen-specific T cells
Dendritic Cells Dendritic cells (DCs) are known for their unique antigen-presenting capacity and their ability to activate na€1ve T cells, thereby orchestrating the primary immune response (12,13). DCs reside in peripheral tissues in an immature state where they capture and process antigens for presentation in the context of MHC molecules (14). Upon the encounter with microbial agents, inflammatory stimuli, or T-cell derived stimuli a complex process of morphological, phenotypical, and functional changes is induced, commonly referred to as the DCs maturation process (15,16) (Table 1). Attracted by lymphoid chemokines, mature DCs migrate toward T cell areas in the lymph nodes where they activate na€1ve T cells (17). With their ability to capture antigens and the capacity to present them in an efficient manner to T cells, only a few DCs are necessary to activate na€1ve T cells. These features make DCs ideal candidates for cellular immunotherapy. With the development of recombinant cytokines and advances in culture techniques, optimal culture conditions could be established for the in vitro generation of DCs. The in vitro culture of CD34-positive progenitor cells and CD14-positive monocytes, in the presence of GM-CSF and IL-4, results in differentiation into immature DCs in five to seven days (16,18,19). Activation of various signal transduction pathways caused by, for example, the cytokines TNFa, CD40L or IFNg, the microbial products lipopolysaccharide (LPS), CpG oligonucleotides, and poly-I:C and inflammatory agents such as IL-1b and prostaglandin E2 (PGE2) leads to the induction of DC maturation (Table 2). Cultured DCs can be fused with target cells and pulsed or transformed with target antigens. Although considerable progress has been made in identifying relevant tumor antigens, for the majority of human cancers it remains unclear which antigens represent the most important tumor-rejection antigens (20). As discussed before, leukemic blasts express tumor antigens, capable of eliciting high avidity T-cell responses. Unfortunately, these antigens are not uniformly
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Table 2 DC Maturation–Inducing Agents
Inflammatory cytokines TLR triggering T-cell derived stimuli Other
Maturation agents
GMP approved
TNFa, PGE2, IL-1b, IL-6 LPS, CpG, poly-I:C CD40L, IFNg Heat shock proteins, IFNa
Yes Only CpG Yes No
Abbreviations: GMP, good manufacturing practice; TLR, toll-like receptor; PGE2, prostaglandine E2; LPS, lipopolysaccharide; CpG, CpG oligonucleotides.
expressed by each leukemia subtype. The unique property of leukemic blasts to be able to differentiate into DCs provides, therefore, a distinctive opportunity to generate APC that harbor the full range of potential, still unidentified tumor antigens. Leukemia-derived DCs have shown the capacity to induce leukemiaspecific T-cell responses in vitro as well as in vivo thus offering a potential new immunotherapeutic modality for patients with AML and CML with MRD (8,9,21,22).
IMMUNOGENICITY OF AML BLASTS Immunological Synapse When T cells and APC encounter, a defined sequence of cellular and molecular interactions follow. After the initial scanning of the APC by filopodial projections of the T cell, antigen recognition by the TCR and development of TCRderived signals, such as calcium influx, the T cell stops crawling (23). Interaction of T cells with APC results in the formation of a contact zone called the immunological synapse (IS) (24). Assembly of the IS occurs in several stages. MHC molecules interacting with TCR are first seen to accumulate in a ring surrounding a central cluster of adhesion molecules with LFA-1 and ICAM-1 interactions, forming the immature synapse. This synapse later inverts in a way that a central zone of TCR termed the central-supramolecular activating complex (c-SMAC) is surrounded by a peripheral ring of adhesion molecules (25). The function of this highly organized structure remains controversial. Initially, the synapse was thought to represent a structure necessary for initiating signals, but more recent data have demonstrated that calcium signaling, for example, already occurs before formation of the c-SMAC, when the IS is immature (26). Several other functions have been ascribed to the IS, such as directing secretion of cytokines, enhanced signaling and the balancing of enhancing, and terminating signals to maintain agonist-triggered signals in T cells (27).
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Antigen Presentation A mature IS forms only on recognition of peptides presented by the MHC complex. AML blasts express tumor antigens, which could be qualified as tumor-rejection or tumor-regression antigens, on the basis of their ability to elicit high-avidity T-cell responses and to recruit a large number of T cells with some diversity in TCR (28). The fusion proteins PML/RARa and ETO/AML1 resulting from the chromosomal translocations t(15;17) and t(8;21), respectively, provide potential targets for immunotherapy. Additionally, normal proteins overexpressed in leukemic progenitors such as WT-1 and the serine protease proteinase 3 (PR3), can be efficiently processed and presented by MHC class I molecules (29,30). WT-1-specific antibodies are identified in 15% to 25% of AML patients (31,32). Furthermore, cytotoxic responses elicited by WT-1 and PR3 have been observed (33). MUC1, an epithelial mucin overexpressed in many epithelial malignancies, has also been shown to be overexpressed on AML cells and to be capable of inducing CTL responses (34). These characteristics make AML cells potential inducers of T-cell immunity. AML blasts consistently show a high expression of MHC class I molecules whereas MHC class II molecules are variably expressed (35,36). The role of MHC class II molecules to present tumor antigens to CD4-positive Th cells to obtain antitumor T-cell responses is well established (37). Antigen presentation via the MHC class II pathway is severely hampered by the presence of class IIassociated invariant chain peptide (CLIP) in the antigen-binding groove. CLIP is a small remnant of the invariant chain that serves a chaperone for newly synthesized class II molecules and prevents unwanted antigens from binding in the endoplasmic reticulum (38). CLIP is released upon replacement by the antigen. A high amount of CLIP expressed on AML blasts might serve as a tumor escape mechanism and results in a shortened disease-free survival of AML patients (35). Adhesion of APC and T Cells Contact between APC and T cells can be either adhesive and static or dynamic and migratory. Migratory contacts are driven by the active ameboid T cell crawling across the APC surface, for example, during the initial scanning phase of T cells (23). After TCR triggering, an adhesive and stable phase can be initiated by receptors of the integrin family that couple to the actin cytoskeleton. The outer ring of the mature IS is formed by interactions of LFA-1 and ICAM-1, but also by other adhesion molecules such as DC-SIGN, interacting with ICAM2 and ICAM-3 (39–41). Subsequently, the T cell polarizes toward the APC to optimize signaling between the cells. Clustering of TCR in the central region might also function to balance signaling. This was shown by T cells lacking LFA-2 adapter protein thus being unable to form a stable mature IS. These T cells were found to be unable to downregulate TCR and were hypersensitive to presented antigens (42).
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Although adhesion molecules are clearly expressed, AML blasts display a low and heterogeneous expression of most adhesion molecules, as seen for ICAM1 (43). Therefore, AML cells are likely to be limited in their ability to develop stable contacts with T cells. It was shown that not one single adhesion molecule is responsible for stable encounters with T cells, rather the combination of these molecules plays a crucial role in the recognition of leukemic cells by T cells (43). Costimulatory Signaling CD28 is the most important costimulatory molecule expressed on na€1ve T cells and interacts with CD80 and CD86 on APC. Data showing recruitment of CD28 to the IS suggest that CD28 regulates events that occur soon after T cell–APC interaction (44). CD28 engagement leads to the initiation and maintenance of the calcium signal within the first 60 seconds of that signal and contributes to the formation of stable TCR-APC couples (45). Therefore, it seems that CD28 has a function in the early immature synapse before the formation of the c-SMAC. In contrast to CD28, its inhibitory counterpart CTLA-4, is recruited into the synapse during the late stages of T-cell stimulation. The extent to which this recruitment takes place depends on high levels of T-cell activation, emphasizing its function as a negative feedback system (46). The expression of costimulatory molecules such as CD80, CD86, and CD40, on AML blasts has been described. Attempts to increase the expression of costimulatory molecules on AML blasts have been made, for example by transfecting AML blasts with genes encoding for CD80 (47,48). Cytokine Secretion It is hypothesized that an important function of the IS could be represented by the direction of signaling toward a Th1 or Th2 type response through colocalization of cytokine receptors and cytokine secretion (49). The observation that the IFNg receptor colocalizes with the TCR in the c-SMAC suggests that the function of such copolarization might include the concentration of cytokine receptors at the actual sites where cytokines are produced (49). For na€1ve T cells the IS could provide a platform for cytokine presentation of activating and polarizing factors such as IL-2, IFNg, or IL-12 derived from APC. In these studies, it was observed that IL-4 prevented colocalization of IFNg and TCR, thereby enabling a Th2 response (49). AML blasts have been shown to be able to produce IFNg (50). However, the inability to form a functional IS is likely to hamper efficient T-cell responses because it has been shown that cytokines act locally rather than at a distance (51). One mechanism by which tumors could potentially escape elimination by the immune system is by secretion of cytokines that suppress cells involved in immune surveillance (52,53). The production of factors such as vascular endothelial growth factor (VEGF), IL-6, M-CSF, IL-10, and TGFb are
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associated with inhibited DC function and maturation (54,55). It was shown that the U937 leukemic cell line generates tumor supernatant that inhibits antigen presentation and subsequent T-cell secretion of IFNg and IL-2 (56). DEVELOPMENT OF LEUKEMIC DCs FOR CLINICAL VACCINATION PURPOSES Culture of Leukemic DCs Culture of leukemic blasts for 14 days in the presence of various combinations of cytokines, including GM-CSF, TNFa, SCF, Flt3-L, IL-3, and IL-4 results in the differentiation toward leukemic DC-like APC (8–10,22,57–63). Maturation of cytokine-cultured AML-DCs to an extent where they are comparable to their normal counterparts, i.e., DCs derived from CD34-positive progenitors, can be achieved by a two-day incubation with a mixture of inflammatory cytokines TNFa, IL-1b, IL-6, and PGE2 or by the addition of CD40L (64,65). Apart from monocytes and CD34-positive hematopoietic progenitors, CML cells as well as AML blasts are able to respond to calcium-mobilizing agents such as calcium ionophores (CI), thereby bypassing receptor mediated signaling (9,21,66–69) (Fig. 1). The acquisition of dendritic features upon CI incubation occurs far more rapidly than after incubation with cytokines thereby providing a time and cost effective approach for the generation of leukemic DCs (9,67). CI-cultured AML-DCs reveal a more mature phenotype and were significantly more potent stimulators of T-cell proliferation compared with cytokine-cultured AML-DCs. However, CI-cultured cells were found to be less viable compared with AML-DCs generated in the presence of cytokines (9). Consequently, the CI-based method can only be applied if high amounts of AML blasts are available. In a direct comparative study, it was shown that the serum-free culture of AML-DCs is feasible by the cytokine- as well as CI-based culture methods and that these DCs are comparable to serum-enriched cultured AML-DCs with regard to morphological, phenotypical, and functional features (22). Thus, AML-DCs are
Figure 1 Morphology of AML blasts (A) and cultured AML-DCs in presence of cytokines (B) or calcium ionophore (C) as described in the text. AML-DCs acquire long cytoplasmic protrusions and an eccentric position of the nucleus.
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suitable for the development of clinical vaccination programs that comply with good clinical practice demands. Functional Properties of Leukemic DCs In order to elicit an adequate T-cell response in vivo, AML-DCs need to fulfill certain criteria, which can be assessed by in vitro assays. Leukemic origin of AML-DCs can be confirmed by fluorescent in situ hybridization (FISH) showing the original chromosomal abnormality in AML-DCs (9,58,59,63). AML-DCs have to be able to migrate from the injection site toward the lymph nodes. In migration assays, not only mature AML-DCs but CML-DCs also exhibited potent migratory capacity toward the lymph node-associated chemokines SDF-1 and MIP-3b, implying their ability to migrate toward the lymph nodes (70). Upon arrival in the lymph nodes, AML-DCs should be capable of stimulating T cells. Mature AML-DCs proved to be potent inducers of T-cell stimulation in alloreactivity tests (9,58,63,71). The type of T-cell response can be determined by the cytokine profile of stimulated T cells. Preferably, AML-DCs should evoke a Th1 response since Th1 cells are capable of stimulating CD8-positive cytotoxic T cells, which is important for antitumor immunity. By intracellular cytokine staining and the use of ELISA or ELISPOT assays a Th1 cytokine profile with IFNg production without IL-4 and IL-10 production could be detected (9,64). Most importantly, T cells primed with autologous AML-DCs demonstrated cytolytic capacity toward autologous AML blasts, as assessed by cytotoxicity assays (8,9,71). Thus, in vitro assays confirm the functional potential of AMLDCs that are instrumental in stimulating autologous cytotoxic T-cell responses. MODULATION OF AML-DCs AND T-CELL INTERACTION In order to take full advantage of the potential of AML-DCs to induce a T-cell response in vitro and in vivo through the formation of an IS, additional strategies can be explored that increase AML-DCs yield and their functional effects (Table 3). Table 3 Strategies to Increase the Efficacy of Leukemic DC Vaccination Strategies to optimize leukemic DC vaccination Selection of optimal culture methods Adjuvants (increased immunogenicity) (increased DC life span) Costimulatory pathways (stimulatory) (inhibitory)
CD14, TNFa-RI, Flt-3 internal tandem duplication BCG, KLH, IL-12, GM-CSF CD40L, TLR CD28, 4–1BBL CTLA-4, PD1
Abbreviations: BCG, bacillus Calmette Guerin; KLH, keyhole limpet hemocyanin; TLR, toll-like receptor.
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Selection of Optimal Culture Methods Reflecting the heterogeneity of the disease, cultured AML-DCs harvested from an array of AML patients form a heterogeneous population with a variable expression of, in particular, the costimulatory molecules. To optimize leukemic DCs yield for vaccination purposes, selection of the most optimal DC culture method for each individual patient at diagnosis is of great importance. Surprisingly, in a large cohort of patients it was found that DC differentiation capacity is independent from the French-American-British (FAB) classification subtype. In particular, no significant differences were found between the monocytic subtypes, M4 and M5, and other subtypes (72). However, it is possible to predict AML-DC culture outcome by the expression of defined surface markers on AML blasts thus enabling selection of culture methods (73–75). High TNFa-RI expression on AML blasts is predictive for the DC differentiation capacity of blasts when cultured in the presence of cytokines (75). In addition, it was described that CD14-positive leukemic blasts represent the population that can be induced in vitro into leukemic DCs whereas the CD14-negative population could not (74). Interestingly, induction of DC differentiation in CD14-negative blasts is possible if these blasts express TNFa-RI (76). Alternative culture methods, for example the CI-based method, can be used to induce DC differentiation in CD14-negative and TNFa-RInegative AML samples. Also, the presence of CD86 on AML blasts has been associated with the ability to differentiate into AML-DCs (73). Besides the expression of surface markers, the presence of an Flt-3 internal tandem duplication (ITD) is strongly associated with a diminished DC differentiation capacity in both culture methods (72). This mutation, causing constitutive activation of the tyrosine kinase receptor contributes to leukemogenesis, thus resulting in decreased survival rates (77–79). Other than providing AML blasts a proliferative advantage, it has been demonstrated that Flt-3 ITD can also cause a block in myeloid differentiation (80). Flt-3 ITD signaling represses the expression of two genes required for myeloid differentiation, namely, CCAAT/ enhancer binding protein (C/EBP) and PU.1 (81). PU.1, being a downstream target of C/EBP, has been found to be decisive in DC fate (82). Thus, suppression of C/EBP and PU.1 in Flt-3 ITD-positive AML cells might result in a decreased capacity to differentiate toward DCs. Using these selection parameters, the most optimal culture protocol for the generation of AML-DCs for individual patients can be identified.
Adjuvants In many of the current DC vaccination protocols, Bacillus Calmette Gu erin (BCG) and keyhole limpet hemocyanin (KLH) are added to vaccines in order to enhance immune responses. Innovative molecular techniques are now instrumental to replace bacterial adjuvants by more sophisticated ways to increase the
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immunogenicity of cancer cells. Genetically modified AML cells that express immunomodulatory cytokines used to enhance antigenicity, such as IL-12 or GM-CSF, proved to be potent vaccines that are able to cure leukemia in mice (83–87). In contrast to the systemic administration of IL-12, no systemic toxicities were observed using these vaccines (88). Analogous to this approach, also the transduction of DCs with genes encoding for GM-CSF and IL-12 could provide a way to enhance T-cell stimulation as shown by the induction of strong T-cell responses in a murine melanoma model (89). Transient production of IL-12, caused by exhaustion of cytokine production by DCs, limits the T-cell activation capacity (90). This finding implies that prolonging the duration of interaction of DCs and T cells could increase the immune response. A strategy to extend the contact time between DCs and T cells would be the targeting of CD40 on DCs. Targeting CD40 not only enhances DC maturation state but also increases the expression of antiapoptotic molecules (91). DCs transfected with a drug-inducible CD40 receptor induced prolonged T-cell activation and enhanced antitumor responses (92). Also in vivo targeting of CD40 leads to sustained antitumor responses. Thus, increased potentiation of an AML-DC vaccine could be achieved by simultaneously targeting CD40 (93,94). Similar to CD40 targeting, it was found that persistent coadministration of toll-like receptor (TLR) ligands enhances DC-based vaccines through prolongation of DC life span (95). Additionally, targeting TLR induces maturation of DCs with upregulation of costimulatory molecules and secretion of IL-12. A clinical applicable TLR ligand is the CpG, which is currently being used in DC vaccination trials. Costimulatory Pathways For effective DC vaccination, the vaccine must overcome the intrinsic tolerant state of the patient. It has been shown that especially MRD cells upregulate certain costimulatory pathways that could protect them from the patients immune response. For example, B7-H1, a ligand for programmed death 1 (PD1), was upregulated in MRD cells generated in an AML mouse model (96). Blocking this pathway increased CTL-mediated killing and enhanced the production of IFNg by effector T cells. Enhanced IFNg and decreased IL-10 production have also been reported after PD1 blockade in myeloid DCs (97,98). These findings suggest that modulation of costimulatory pathways might improve leukemic DC vaccine efficacy. T cells cocultured with AML blasts in presence of the CD28 antibody showed increased proliferative capacity. In addition, costimulation provided by the CD28 antibody could be further enhanced synergistically by the addition of IL-12 (99). For AML-DCs, displaying a heterogeneous expression of CD80 and CD86, direct targeting of CD28, thereby circumventing interaction via CD80 and CD86, could provide a way to increase T-cell responses.
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Blocking CTLA-4, the most important inhibitory costimulating pathway, in vivo results in rejection of immunogenic transplanted tumor cell lines, including colorectal carcinoma, renal carcinoma, and lymphoma (100,101). Weak immune responses elicited by tumors can be potentiated once the inhibitory effect of CTLA-4 is removed. However, susceptibility appears to be correlated with the inherent immunogenicity of the tumor. Therefore, a combined approach of increasing immunogenicity and blocking CTLA-4, as could be the case in AML-DC vaccination protocols combined with CTLA-4 blockade, seems preferable. CTLA-4 is expressed not only by T cells but AML blasts have also been reported to express CTLA-4 (102). Anti-CTLA-4 immunotoxins were able to induce apoptosis of AML blasts, suggesting a possible role for CTLA-4 as a target molecule for immunotherapeutic strategies. It can also be hypothesized that CTLA-4-positive AML blasts could interact with CD80 and CD86 on APC with potential transduction of an immune inhibitory signal, thus proposing another possible advantage of CTLA-4 blockade. 4-1BB, a member of the TNFa receptor family, represents an important costimulatory pathway necessary for the development of CTL (103). Triggering of this pathway increases expansion of antigen specific T cells and could prevent activation induced cell death of CD8-positive T cells. 4-1BB ligation prevents and even restores T-cell anergy in vivo (104). Also the combined approach of DC-based vaccines potentiated with coadministration of the 4-1BB antibody proved to improve antitumor responses (105). In vitro studies show that the combination of T cell priming by AML-DCs with 4-1BB targeting results in increased proliferation of CD8-positive T cells capable of producing IFNg without IL-4 production (106). Addition of 4-1BB targeting to AML-DC vaccination protocols could thus potentially augment antileukemic T-cell responses. However, to obtain optimal responses upon 4-1BB targeting, it could be necessary to simultaneously block the B7-H1 pathway since it was shown that expression of B7-H1 confers resistance to 4-1BB costimulation, while blocking this pathway rescues antitumor responses (107). VACCINATION WITH LEUKEMIC DCs In the past few years, numerous clinical studies have been initiated in which DCs are used as a vaccine to boost an immune response against malignant tumors in patients with cancer. In a phase-I pilot study on CML-DC vaccination in advanced-stage disease strong delayed type hypersensitivity (DTH)-responses representing autologous CML-specific T-cell responses could be detected (108). A decrease in the number of Bcr-Abl-positive cells was shown in a CML patient treated with a CML-DC vaccination following autologous peripheral blood stem cell transplantation (109). Additionally, infused CML-DCs, induced T-cell clones expressing the same TCR as a cytotoxic T-cell line suggesting that the immune repertoire included tumor-reactive T cells. In another pilot study on intradermally injected Bcr-Abl pulsed monocyte-derived DCs (MoDCs),
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peptide-specific cellular responses were detected, although without any clinical responses (110). In these early vaccination trials, no toxic or autoimmune adverse effects were detected, suggesting that these vaccines can be administered safely. To date no clinical AML-DC vaccination studies have been reported, although, one clinical study on AML lysate-pulsed MoDCs was published (111). In this study, positive DTH-responses were observed, although the blast percentage did not decrease. Standardization and Optimizing of Clinical Vaccination Protocols In order to enable comparison of DC vaccination trials standardization of clinical protocols and immune monitoring techniques is essential (112). Minimal quality criteria regarding DC vaccines are mainly focused on the necessity to vaccinate with mature DCs, defined morphologically and immunophenotypically, as well as functionally (113). In vitro and in vivo studies provided evidence that antigen-loaded immature DCs silence T cells either by deleting them or by expanding regulatory T cells (114–116). In a comparative study on the use of mature versus immature peptide-pulsed MoDCs for the vaccination of advanced-stage melanoma patients, it was evident that an immunological response could only be detected after vaccination with mature DCs (117). The absence of a T-cell response could partly be explained by the observation that immature MoDCs are unable to efficiently migrate to the T cell areas of the lymph nodes (117). However, considering relevant cytokine secretion, maturation state of DCs is still a matter of debate. Kinetic studies showed that shortly after activation, DCs secrete higher amounts of cytokines and that prolonged maturation periods result in exhaustion of DCs with considerable less cytokine production and impaired capacity to stimulate Th1 responses (118). Although leukemic DCs meet most proposed quality criteria, it is not yet known which level of maturation needs to be achieved to elicit an optimal immune response and whether leukemic DCs are capable of attaining a fully mature state in vivo upon vaccination (113). Another unresolved question in DC vaccination therapy is the optimal route of administration. Intradermal or subcutaneous injections may lead to improved T-cell responses as compared with IV administration (119,120). However, these routes of administration rely on the capacity of injected DCs to migrate toward the lymph nodes. Intranodal administration circumvents this problem and allows delivery of a known amount of DCs to the desired anatomic region, potentially leading to increased T-cell immunity (121). On the other hand, intranodal vaccination requires technical expertise and includes the risk of damaging the architecture of the lymph node. Additionally, route of administration could determine the location of the primary immune response, the distribution of memory cells, and the ability to control the outgrowth of tumors at different sites in the body (122).
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Immunotherapy is thought to be most effective in an MRD situation. Detection of leukemic MRD cells, as characterized by the presence of a LAP, is highly predictive of the occurrence of a relapse (123). A cut off percentage of 0.1% detectable MRD cells, after the third course of chemotherapy, identifies patients at risk for a fast-developing relapse. Patients with a MRD percentage less than 0.1% should be monitored every three months to anticipate a possible relapse (Feller et al. submitted for publication). On the basis of MRD percentages, selection of patients potentially benefiting from additional therapy such as immunotherapy can be performed. However, the need for reconstitution of lymphocyte populations and lymphoid tissue after chemotherapy and SCT treatment is likely to influence the effect of immunotherapy. For example, after SCT CD8-positive T cells reappear more rapid, i.e., within six months, as compared with CD4-positive T cells that show low levels even after one year. Different immune responses were observed after in vitro priming with AML-DCs at different time points during remission. During early remission immune responses seem to be largely MHC-restricted, whereas during a later time it was observed that the immune response shifted toward non-MHC restricted as detected in a cytotoxicity assay (70). Thus, time of vaccination should take into account the pace of immune reconstitution to obtain efficient immune responses. Immunomonitoring From early clinical studies on DC vaccination it is clear that monitoring the immune response is complex but of great importance (119,124). Most techniques represent indirect measurement of cytolytic activity of effector cells. Effector T cells can be isolated either from peripheral blood, lymph node biopsies or DTH reactions. Several clinical vaccination studies in cancer patients have reported T cell responses in peripheral blood but usually only in a minority of patients or after prolonged antigenic restimulation in vitro (125–128). DTH-infiltrated T lymphocytes are able to show antigen-specific responses after short term in vitro cultures without the need for antigen restimulation (117). The tetramer technology enables the sensitive detection of antigen-specific T cells. Also for leukemia, leukemia-associated antigens have been identified for which tetramers can be developed. However, for a large proportion of the leukemia’s, the leukemia-associated antigens are unknown and T cell specificity needs to be determined in a more indirect way. The classical way to detect CTL activity is a measurement of lytic activity against 51Cr-labeled target cells. For the monitoring of the immune response after leukemic DC vaccination, this method might not be suitable due to the high spontaneous release of 51Cr by the leukemic blasts. A flowcytometric assay using Syto-16/7-AAD staining to detect early apoptosis and secondary necrosis might be more suitable to detect heterogeneous cell populations such as AML (70). Other immune monitoring methods involve the detection of
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cytokines released by CTL, by ELISPOT or ELISA to establish the type of T-cell responses. CONCLUSIVE REMARKS AML blasts, though being potentially immunogenic, fail to induce an effective immune response, thus evading attack. The induction of a long-lasting immune response may, however, provide a novel strategy for eradication of disease. AML blasts harbor several ways to escape the patient’s immune system. The inability to form a stable IS in combination with the tolerogenic microenvironment in which AML cells reside make it difficult for T cells to effectively respond to these cells. On the contrary, the presence of CTL directed against leukemic blasts emphasizes their suitability as immunological targets. Increased immunogenicity of AML blasts can be achieved by the differentiation into leukemic DCs that were demonstrated to induce antileukemic T-cell responses in vitro. Although encouraging results have been obtained by the in vivo application of CMLderived DCs, it remains to be proved whether AML-DCs are capable to elicit profound immune responses in vivo (108,129,130). Important lessons can be learnt from early DC-based vaccines targeting other types of malignancies such as melanoma, colon carcinoma, or prostate carcinoma. In a review on the first 1000 DC-vaccinee data are summarized on the types of DC vaccines, route of administration, reported side effects, and clinical efficacy (131). From these data, an important conclusion was that side effects were minimal, indicating that DC vaccines can be administered safely. Concerning clinical responses, data seem rather disappointing with antitumor responses observed in approximately half of the trials (124,131). It should be taken into account, however, that treated patients were often in end-stage disease. In order to enable comparison of DC-based trials, standardization of DC vaccine preparation, schedule and route of administration, and immunomonitoring techniques are important (113). Nevertheless, it might be possible that additional strategies are required to optimize efficacy of DC vaccines. As outlined above, in case of AML, manipulation of maturation as well as costimulatory pathways provide opportunities to increase leukemic specific T-cell responses. The combined strength of increasing immunogenicity of AML blasts by differentiation into leukemic DCs and enhancement of T-cell responses by immunomodulating agents could represent a new powerful treatment for AML patients with MRD. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS This chapter provides a rationale for the clinical application of leukemic DC vaccination for patients with AML. However, clinical trials utilizing AML-DCs are hampered by patient inclusion criteria (132). One of the main problems in
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current clinical trials is vaccination in a late stage of the disease with a high tumor load and a disabled immune system. Meaningful clinical responses provoked by DC vaccines are likely to occur only during an early stage of disease. Since immunotherapy is thought to be the most effective in a MRD situation, ideally, AML-DCs should be administered, after achieving first CR, on the basis of presence of MRD. Current protocols allow selective entering of patients in second CR. We envision that extending inclusion criteria in the next five years to other patient groups, for example, older patients unfit or unwilling to undergo induction chemotherapy or patients undergoing reduced-intensity chemotherapy, allogeneic stem cell transplantation is mandatory to permit clinical evaluation of the AML-DC vaccination strategy.
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93. van Mierlo GJ, den Boer AT, Medema JP, et al. CD40 stimulation leads to effective therapy of CD40(-) tumors through induction of strong systemic cytotoxic T lymphocyte immunity. Proc Natl Acad Sci U S A 2002; 99(8):5561–5566. 94. Stumbles PA, Himbeck R, Frelinger JA, et al. Cutting edge: tumor-specific CTL are constitutively cross-armed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation. J Immunol 2004; 173(10):5923–5928. 95. Hou WS, van Parijs L. A Bcl-2-dependent molecular timer regulates the lifespan and immunogenicity of dendritic cells. Nat Immunol 2004; 5(6):583–589. 96. Saudemont A, Quesnel B. In a model of tumor dormancy, long-term persistent leukemic cells have increased B7-H1 and B7.1 expression and resist CTL-mediated lysis. Blood 2004; 104(7):2124–2133. 97. Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 2003; 170(3):1257–1266. 98. Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat Med 2003; 9(5):562–567. 99. Orleans-Lindsay JK, Deru A, Craig JI, et al. In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy. Clin Exp Immunol 2003; 133(3):467–475. 100. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996; 271(5256):1734–1736. 101. Sotomayor EM, Borrello I, Tubb E, et al. In vivo blockade of CTLA-4 enhances the priming of responsive T cells but fails to prevent the induction of tumor antigenspecific tolerance. Proc Natl Acad Sci U S A 1999; 96(20):11476–11481. 102. Pistillo MP, Tazzari PL, Palmisano GL, et al. CTLA-4 is not restricted to the lymphoid cell lineage and can function as a target molecule for apoptosis induction of leukemic cells. Blood 2003; 101(1):202–209. 103. Hellstrom KE, Hellstrom I. Therapeutic vaccination with tumor cells that engage CD137. J Mol Med 2003; 81(2):71–86. 104. Wilcox RA, Tamada K, Flies DB, et al. Ligation of CD137 receptor prevents and reverses established anergy of CD8þ cytolytic T lymphocytes in vivo. Blood 2004; 103(1):177–184. 105. Ito F, Li Q, Shreiner AB, et al. Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines. Cancer Res 2004; 64(22):8411–8419. 106. Houtenbos I, Westers TM, Dijkhuis A, et al. Leukemia-specific T-cell reactivity induced by leukemic dendritic cells is augmented by 4–1BB targeting. Clin Cancer Res 2007; 13(1):307–315. 107. Hirano F, Kaneko K, Tamura H, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res 2005; 65(3):1089– 1096. 108. Ossenkoppele GJ, Stam AG, Westers TM, et al. Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells. Leukemia 2003; 17(7):1424–1426.
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15 CDK Inhibitors in Leukemia and Lymphoma Yun Dai Department of Medicine, Virginia Commonwealth University and Massey Cancer Center, Richmond, Virginia, U.S.A.
Steven Grant Department of Medicine, Biochemistry, and Pharmacology, Virginia Commonwealth University and Massey Cancer Center, Richmond, Virginia, U.S.A.
CYCLIN-DEPENDENT KINASE FUNCTION Cell Cycle Regulation Cell cycle progression provides a mechanism that allows both normal and neoplastic cells to proliferate and grow. The cell cycle is divided into four distinct but closely related phases, i.e., DNA synthesis (S phase) and mitosis (M phase), which are separated by two gaps (phases G1 and G2). Following growth stimuli, cells traverse the cell cycle through G1?S?G2?M phases and then divide to produce two daughter cells, which then enter G1 phase once again to initiate the next cycle, or exit from the cell cycle into a quiescent G0 phase. The G1 phase contains a transition point referred to as ‘‘the restriction point,’’ which determines whether the cell cycle progression is independent of exogenous stimuli. Cell cycle progression is tightly controlled by the cyclin-dependent kinase (CDK) complex consisting of a catalytic (CDK) and a regulatory (cyclin) subunit, which exist in a 1:1 ratio. CDKs are serine/threonine kinases that are
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activated by their regulatory partners (members of the cyclin family). Binding of cyclins to this complex induces a conformational change in the CDK structure producing a basal, active state (1). CDK complexes are activated by phosphorylation of CDKs at specific, conserved threonine (Thr) residues (e.g., Thr161 in cell division cycle (CDC)2/CDK1, threonine (Thr)160 in CDK2, Thr172 in CDK4, Thr177 in CDK6) catalyzed by the CDK-activator kinase (CAK), and dephosphorylated at conserved tyrosine and Thr residues (Thr14 and Tyr15 in CDK1 and CDK2, which are phosphorylated by mixed-lineage kinases Wee1 and/or Myt1), events catalyzed by the dual-specificity phosphatases CDC25 (A, B, and C) (2). CDK activity is inhibited by interactions with endogenous CDK inhibitors, which are divided into two families—the INK4 (inhibitor of CDK4) family, including p16INK4A, p15INK4B, p18INK4C, and p19INK4D, which inhibits cyclin D–associated kinases (CDKs 4 and 6), and the CIP/KIP (kinase inhibitor protein) family, comprising p21CIP1/WAF1, p27KIP1, and p57KIP2, which inhibit most CDKs (3). Cyclin expression fluctuates through the cell cycle and influences progression from one phase to the next. Cyclins B, A, and E are regulated by an ubiquitin/proteasome-dependent degradation pathway, whereas cyclin D is primarily regulated by transcriptional and translational mechanisms. Of the large number of CDK complexes identified, CDKs 1, 2, 4, and 6 and cyclins A (A1 and A2), B (B1 and B2), D (D1, D2, and D3), and E (E1 and E2) are directly involved in the cell cycle machinery. Generally, cyclins D-CDK4 and D-CDK6 phosphorylate or inactivate the retinoblastoma protein (pRb, a major member of the ‘‘pocket protein’’ family) and release transcriptional factors E2Fs (activated) from an inactive pRb-E2F complex. E2F binds to its heterodimeric partner DP-1 and induces the expression of genes that is responsible for S-phase entry and progression, including cyclin E. In addition, cyclin E-CDK2 also facilitates G1?S transition by further phosphorylating pRb, complete activation of which requires phosphorylation by both cyclin D-CDK4/6 (hypophosphorylation) and cyclin E-CDK2 (hyperphosphorylation) (4). Cyclin D-CDK4, but not cyclin E-CDK2, also phosphorylates p130 and p107 (additional members of the ‘‘pocket protein’’ family), which may interact with certain E2Fs (e.g., E2F1 and 4) and mimic the function of pRb in RB null tumor cells. In the S phase, cyclin A-CDK2 phosphorylates various substrates, which allows DNA replication and also inactivates G1 transcriptional factors (i.e., E2Fs). Cyclin A-CDK1/CDC2 and cyclin B-CDK1/CDC2 govern G2?M transition. The cyclin B-CDK1 complex also regulates the transition of cells into anaphase and through mitosis. In addition, certain CDK complexes, e.g., cyclin A-CDK2 in the S phase and cyclin B1-CDK1 in the G2/M phase, are associated with the DNA replication competent (RC) complex, which may be directly involved in regulation of DNA replication (5). Lastly, cyclin H-CDK7 (also known as CAK) activates CDKs 1, 2, 4, and 6 via phosphorylation at specific threonine residues, events required for full activation of these CDKs.
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CDKs and Regulation of Transcription The transcription of eukaryotic protein-encoding genes is controlled by ribonucleic acid polymerase II (RNAPII) in the elongation phase. Interplay between negative and positive elongation factors (referred to as N-TEF and P-TEF, respectively) regulates the elongation potential of RNAPII. P-TEFb is the first and only known component of P-TEF. The cyclin T-CDK9 complex (P-TEFb) phosphorylates and activates the carboxy-terminal domain (CTD) of RNAPII preferentially at Ser2 and most likely at Ser5 as well (6), leading to promotion of transcriptional elongation, events sensitive to 5,6-Dichloro-1-b-Dribofuranosylbenzimidaloe (DRB), a well-known inhibitor of transcriptional elongation (6). In addition, CDK9 may act as a multifunctional kinase rather than solely as a CTD kinase (or transcriptional CDK) in cell differentiation, apoptosis, and cell cycle regulation pathways. For example, the cyclin T-CDK9 complex phosphorylates pRb (7). In addition, the initiation phase of transcription has been linked to CDK7 activation/initiation (8). An important implication of these findings is that in addition to the effects on cell cycle progression, disruption of CDK function can have profound effects on gene transcription. CELL CYCLE ABNORMALITIES IN LEUKEMIA AND LYMPHOMA Disruption of cell cycle–regulatory genes frequently occurs in human cancers, including leukemia and lymphoma, and provides a growth advantage to neoplastic cells. These abnormalities most frequently include loss or inactivation of endogenous CDK inhibitors, overexpression of CDK partner cyclins, or amplification or active mutations of CDK genes. Such considerations provide a rationale for employing inhibitors of cell cycle progression as anticancer and antilymphoma agents. Aberrations in cell cycle–regulatory molecules in human cancers occur most frequently in molecules associated with control of G1?S transition, a key step that determines initiation of the cell cycle. In fact, dysregulation of cyclin D/CDK4,6/INK4/pRb/E2F signaling pathway has been identified in more than 80% of human cancers (9). In the case of T-cell lymphomas, abnormalities in expression of the endogenous CDK inhibitors p15, p16, and p21 frequently occur (10). Overexpression of cyclin D (primarily cyclin D1) is also common in a variety of human cancers and represents a hallmark of mantle cell lymphoma (11). Aberrant overexpression of cyclin D1 usually stems from gene rearrangement [e.g., of chromosomes 11p15;q13, and t11;14(q13;q32)], gene amplification, or alternative splicing (which generates a cyclin D1b transcript with constitutively nuclear localization and enhanced transforming capacity) (12). Gene amplification and overexpression of cyclins D2 and D3 are also found in some cancers, such as B-cell malignancies including lymphoma. Collectively, these findings strongly support the notion that cell cycle–regulatory CDKs (cyclin D–dependent kinases in particular) represent attractive therapeutic targets in leukemia and lymphoma (13). Figure 1 summarizes the current understanding
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Figure 1 Abnormalities of cell cycle–regulatory proteins in human leukemia/lymphoma (blue and red fonts indicate deletion/ downregulation or overexpression/activation, respectively), and potential mechanism by which current, clinically-relevant CDK inhibitor disrupt cell cycle regulation.
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of cell cycle–related abnormalities in human leukemia and lymphoma as well as known mechanisms of action of clinically relevant CDK inhibitors. MECHANISMS OF ACTION OF CLINICALLY RELEVANT CDK INHIBITORS CDKs and related molecules represent promising targets in the development of cancer therapeutics. Among various CDK inhibitors under development, several (e.g., flavopiridol, CYC202, UCN-01, BMS-387032, SNS-032, etc.) are currently undergoing clinical evaluation on the basis of preclinical evidence of antitumor activity (14). Flavopiridol, as a pan-CDK inhibitor, exerts multiple actions in tumor cells, including inhibition of both cell cycle and transcriptional CDKs (both CDK9 and CDK7), induction of apoptosis, and antiangiogenesis. UCN-01 was initially developed as a protein kinase C (PKC) inhibitor, and later found to act as a CDK inhibitor. However, its antitumor effects appear to be more closely related to the inhibition of Chk1 (checkpoint kinase 1), leading to ‘‘unscheduled’’ activation of CDC2/CDK1 and abrogation of the G2/M and S checkpoints, as well as inhibition of the prosurvival PDK1 (3-phosphoinositidedependent protein kinase-1)/Akt pathway. CYC-202 and BMS-387032 have been developed as CDK2 inhibitors, but also inhibit CDK1 like most inhibitors of CDK2. In addition, CYC202 has been also found to inhibit cyclin T-CDK9 and cyclin H-CDK7, thereby blocking phosphorylation of RNAPII CTD, which is associated with transcriptional repression of proteins with short half-lives. The ability of BMS-387032 to inhibit CDKs 7 and 9 has also been described (15). Flavopiridol (AlvocidibTM) Flavopiridol is a semisynthetic small molecular derivative of rohitukine, an alkaloid isolated from Dysoxylum binectariferum (a plant indigenous to India). In preclinical studies, flavopiridol potently inhibited cell proliferation (IC50 ¼ 66 nM) in all 60 National Cancer Institute (NCI) human tumor cell lines, with no obvious tumor-type selectivity (14). It potently induces apoptosis in vitro in human chronic lymphocytic leukemia (CLL) and myeloid leukemia cells at submicromolar concentrations (e.g., 100–200 nM) (16,17). As the first clinically relevant CDK inhibitor, initial trials employed schedules involving 24- or 72-hour continuous infusions at q2 week. These schedules achieved concentrations associated with preclinical activity. For example, a 72-hour infusion regimen produced 271 to 415 nM steady-state plasma concentrations (Css) (18). However, prolonged infusion of flavopiridol proved largely inactive in trials involving several hematopoietic malignancies. Consequently, a bolus administration (1-hour infusions for 1 to 5 days every 21 days) was designed to achieve higher plasma concentrations. Indeed, the one-hour infusion regimen resulted in 1.7 to 3.8 mM median Cmax levels reflecting postinfusion peak concentrations (18) and a limited number of responses in certain settings. Notably, clinically
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achievable concentrations by either continuous and bolus infusion exceeded the threshold for inhibition of CDKs and cell growth and induction of apoptosis in preclinical studies. However, in striking contrast to its impressive activity in vitro and in various xenograft models, outcomes of most clinical trials were disappointing (19). The failure of flavopiridol to recapitulate its in vitro activity may stem from more than 90% plasma protein binding and inadequate plasma concentrations of free drug. On the other hand, a variety of clinical trials have demonstrated that combinations of flavopiridol and either conventional chemotherpeutic agents (e.g., paclitaxel, fludarabine, cytosine arabinoside/ara-C, and irinotecan/CPT-11) or novel signal transduction modulators may be more promising (see below) (20). Very recently, a pharmacologically directed infusion schedule has been developed in which half of the flavopiridol dose is administered over 30 minutes and the other half over four hours (see below). This schedule was associated with very promising activity in patients with refractory CLL (21). In fact, the major dose-limiting toxicity (DLT) was tumor lysis syndrome (TLS). Trials are currently underway to evaluate this schedule in patients with other hematologic malignancies including non-Hodgkin’s lymphoma (NHL) and mantle cell lymphoma. Cell Cycle Arrest Flavopiridol induces cell cycle arrest by targeting cell cycle–regulatory CDKs. It directly inhibits the activity of most CDKs by occupying the ATP-binding site of these kinases—an effect that can be competitively blocked by excess ATP. Furthermore, by inhibiting CAK (i.e., cyclin H-CDK7), flavopiridol also prevents phosphorylation at active threonine residues of most CDKs (e.g., CDKs 1, 2, 4, and 6) (22), phosphorylations required for full CDK activation. Inhibition of CDKs by flavopiridol leads to cell cycle arrest at the G1/S and G2/M phase transitions as well as delay in S-phase progression (14). Significantly, flavopiridol also induces cell cycle arrest through transcriptional inhibition and downregulation of cyclin D1, although this action requires slightly higher drug concentrations (100–300 nM) than those necessary for the inhibition of cell cycle–regulatory CDKs (23). This action has prompted the evaluation of flavopiridol for the treatment of mantle cell lymphoma, a disorder in which cyclin D1 is thought to play an important pathophysiologic role. Inhibition of Transcription Flavopiridol very potently represses transcription (IC50 < 10 nM) in vitro by blocking transition into productive elongation mediated by RNAPII, which is controlled by P-TEFb (cyclin T-CDK9) (24). Flavopiridol inhibits CTD kinase activity of RNAPII with a Ki value of 3 nM, a concentration significantly lower than that required for the inhibition of most other CDKs (e.g., CDKs 1, 2, and 4 with Ki values between 40 and 70 nM). Furthermore, unlike the inhibition of other CDKs, inhibition of CDK9 by flavopiridol is noncompetitive with respect
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to ATP. In cells, flavopiridol inhibits transcription at concentrations far lower than those required to inhibit CDK1 and CDK2, even in the presence of the physiologic concentration of ATP. Another potential target for transcriptional repression by flavopiridol is CDK7 (catalytic subunit of TFIIH). However, CDK7 inhibition requires higher concentrations of flavopiridol than those necessary for the inhibition of CDK9 (23). Therefore, inhibition of transcription by flavopiridol primarily stems from the direct inhibition of CDK9. Downregulation of Cyclin D1 Cyclin D1 is a multifunctional protein that plays a critical role not only as a partner of CDK4/6 (see above) in the regulation of the cell cycle (e.g., the G1/S transition) but also as a transcriptional regulator by modulating the activity of several transcriptional factors (e.g., STAT3) that are CDK-independent. This may explain why cyclin D1 is not only involved in cell cycle progression but also in cell growth and survival (25). Cyclin D1 binds to transcriptional factors STAT3 and NeuroD and inhibits their transcriptional activity, which may be related to the modulation of cell differentiation. Cyclin D1 also interacts with histone deacetylases and, in so doing, blocks access of transcriptional factors to the promoter and inhibits loading of initiation complex (26). Cyclin D1, as an oncogene, also plays an important role in carcinogenesis, probably by driving cells into the S phase and cooperating with various oncogenes (such as Myc and Ras) in malignant transformation. Rearrangement of the cyclin D1 locus and/or overexpression of cyclin D1 have been reported in many human tumors, particularly mantle cell lymphoma (27). Flavopiridol transcriptionally downregulates expression of cyclin D1 in multiple cancer cell types. For example, exposure of MCF-7 breast cancer cells to flavopiridol results in a decline in cyclin D1 promoter activity, leading to a decrease in messenger RNA (mRNA) and the protein of cyclin D1 (28). In vivo, flavopiridol results in the depletion of cyclin D1 in the HN12 tumor xenograft (29). Cyclin D1 transcriptional repression may stem from inhibition of P-TEFb by flavopiridol. In addition, flavopiridol can directly bind to duplex DNA with the range of equilibrium dissociation constant values similar to that of the DNA intercalators doxorubicin and pyrazoloacridine (30), which may affect the function of DNA as a transcriptional template. Consequently, administration of flavopiridol leads to cell cycle arrest through multiple mechanisms related to the inhibition of CDK activities, such as by direct binding to the ATP-binding sites, by prevention of CDK phosphorylation through the inhibition of CAK (cyclin H-CDK7), or by transcriptional downregulation of cyclin D1. Transcriptional repression of cyclin D1 by flavopiridol may be particularly relevant in mantle cell lymphoma, in which cyclin D1 is overexpressed in 95% of patients. Notably, flavopiridol has been reported to delay disease progression in a substantial fraction of patients with mantle cell lymphoma (see below) (27).
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Repression of Mcl-1 Expression Recently, the focus of interest was on the antiapoptotic protein Mcl-1 as a transcriptional target of flavopiridol, particularly in hematopoietic malignancies. For example, in vitro treatment with flavopiridol induces declines in the expression of Mcl-1 mRNA and/or protein levels, which precedes apoptosis, in a variety of cancer cells, including non–small cell lung cancer cells, multiple myeloma cells, and freshly isolated CD5þ/CD19þ cells from patients with B-cell CLL (B-CLL) and CD138þ cells from patients with multiple myeloma (31,32). Downregulation of Mcl-1 has also been confirmed in vivo in primary leukemic cells from flavopiridol-treated acute myeloid leukemia (AML) patients (33). H1299 (non–small cell lung cancer) and NIH3T3 (transformed fibroblasts) cells constitutively expressing Mcl-1 are resistant to apoptosis induced by flavopiridol (34). Flavopiridol induces Mcl-1 downregulation most likely by inhibiting P-TEFb (23). However, expression of the Mcl-1 gene is controlled by multiple signaling pathways. For example, the expression is negatively regulated by E2F-1 through direct binding to the Mcl-1 promoter and positively regulated by the phosphophatidyliositide-3-OH kinase (PI3K)/Akt pathway, MAPK pathway, as well as by transcriptional factors like STAT3 and CREB (35). Consequently, flavopiridol-mediated downregulation of Mcl-1 may be also related to other mechanisms, including accumulation of E2F-1 and disruption of STAT3/DNA binding (34). Induction of Apoptosis Flavopiridol induces apoptosis in a broad spectrum of malignant cells. For example, in vitro, 6 to 48 hour exposure to 100 to 400 nM of flavopiridol induces apoptosis in a variety of tumor cells, including leukemia, lymphoma, head/neck squamous cell carcinoma (HNSCC), breast cancer, non–small cell lung cancer, prostate carcinoma, gastric carcinoma, esophageal carcinoma, and bladder carcinoma (36). Human leukemia cells, regardless of their origins (i.e., cultured cell lines or freshly isolated primary cells from patients) or subtypes (myeloid, B-cell, or T-cell type), are the most sensitive to induction of apoptosis by flavopiridol (37). Notably, flavopiridol can also induce apoptosis in tumor cells that are resistant to DNA-damaging agents and radiation (36). In vivo, treatment with flavopiridol (i.p. 5 mg/kg daily for 5 days) induced apoptosis in the HNSCC xenograft HN12 as detected by the TUNEL (Terminal transferase dUTP Nick End Labeling) assay, with significant reduction (60–70%) in tumor size (29). Flavopiridol induces apoptosis in resting tumor cells, which exhibit similar sensitivities as proliferating cells even in the same cell lines (38), arguing against the possibility that the cytotoxicity of flavopiridol stems solely from the inhibition of CDKs involved in cell cycle regulation. However, direct binding of flavopiridol to duplex DNA may provide an explanation to the ability of flavopiridol in killing noncycling (resting) cancer cells (39). Moreover, no
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significant difference in the cytotoxic activity of flavopiridol have been found between cells expressing pRb versus those defective in pRb expression, even though flavopiridol treatment induces hypophosphorylation of pRb. In addition, certain cell lines that lack detectable pRb expression exhibit more pronounced apoptosis following flavopiridol treatment (40). There is no direct evidence for the notion that transcriptional downregulation of cyclin D1 contributes to the cytotoxicity of flavopiridol, although repression of cyclin D1 expression by an antisense oligonucleotide approach triggers apoptosis in carcinoma cells. In contrast, overexpression of cyclin D1 sensitizes human pRb-null myeloma cells (e.g., U266) to flavopiridol (40). Recently, the focus of attention has been the transcriptional downregulation of proteins involved in the regulation of apoptosis, which most likely represents a central theme underlying the induction of apoptosis by flavopiridol. In this context, Mcl-1 represents an important target (see above). In addition, flavopiridol also downregulates many other antiapoptotic proteins. For example, administration of flavopiridol results in decreased expression of Bcl-2 in several cell lines, such as B-cell leukemia, ovarian carcinoma, prostate carcinoma, and multiple myeloma cells (32). However, flavopiridol-induced apoptosis appears largely independent of Bcl-2 inasmuch as flavopiridol kills tumor cells displaying Bcl-2 overexpression, an event that confers resistance to conventional chemotherapeutic agents. Moreover, neither ectopic overexpression nor antisense oligonucleotide–mediated downregulation of Bcl-2 affects flavopiridolinduced cell killing (41). However, human leukemia cells displaying ectopic expression of N-terminal phosphorylation loop–deleted Bcl-2 (amino acids 32–80, a region known to negatively regulate its function) are highly resistant to flavopiridol-mediated cleavage of Bid, cytochrome c release, activation of caspases, degradation of poly (ADP-ribose) polymerase (PARP), and apoptosis (42), indicating that posttranslational modifications (e.g., phosphorylation) of Bcl-2 rather than transcriptional regulation may be involved in the flavopiridol-induced apoptosis. Flavopiridol also induces downregulation of Bcl-xL and XIAP (X-chromosome-linked inhibitor of apoptosis protein) in various types of cancer cells, which are events likely associated with the inhibition of NF-kB (43). In addition, downregulation of other antiapoptotic molecules (e.g., BAG-1, a regulator of Hsp70 family that confers resistance to apoptosis induced by a variety of stimuli) has also been reported in B-CLL cells exposed to flavopiridol (37). Other Antitumor Mechanisms Several systems show that flavopiridol displays significant antiangiogenic activity, indicating that inhibition of tumor angiogenesis could play a significant role in the antitumor effects of flavopiridol (44). Flavopiridol antiangiogenic activity may be related to its ability to induce apoptosis in both resting and proliferating endothelial cells through unknown mechanisms independent of CDK (e.g., CDKs 1 and 2) (44). In human peripheral blood mononuclear cells
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and human neuroblastoma cells, it has been shown that flavopiridol completely blocks hypoxia-induced vascular endothelial growth factor (VEGF) mRNA transcription and downregulates VEGF protein levels by dramatically decreasing VEGF mRNA stability (45). Clinical Evaluation of Flavopiridol in Leukemia and Lymphoma Chronic Lymphocytic Leukemia As noted above, a novel pharmacologically driven schedule of flavopiridol has recently been developed in which a 30 mg/m2 loading dose is given in association with a four-hour infusion of 30 mg/m2 on a weekly basis for four to six weeks (21). Response rates of approximately 40% to 45% were obtained, including patients with particularly poor prognostic factors. The DLT of this regimen was a TLS, which could be circumvented with aggressive hydration and other prophylactic measures. Plans are currently underway to further optimize this schedule of administration and to test it in other B-cell malignancies, including NHL (Table 1). Acute Myeloid Leukemia On the basis of preclinical evidence of activity of flavopiridol, either alone or in combination with other agents in AML, Karp and coworkers recently conducted a phase I and subsequent phase II trial of timed sequential therapy in which flavopiridol was sequentially administered in conjunction with ara-C and mitoxantrone in patients with refractory or high-risk AML (Table 1) (20,33). Very high response rates (e.g., 75%) were obtained in patients in certain poorprognostic categories (i.e., secondary AML, short first-remission relapses), although multiply treated relapsed patients did not respond. Efforts are currently underway to incorporate the novel infusional schedule of flavopiridol into this timed sequential strategy. Mantle Cell Lymphoma On the basis of the preceding considerations, as well as preclinical evidence of the activity of flavopiridol in mantle cell lymphoma models (46), several clinical trials of flavopiridol have been conducted in patients with this disease (Table 1). When administered at a dose of 50 mg/m2 as a 72-hour continuous infusion, flavopiridol exhibited minimal single-agent activity (19). In a follow-up phase II trial by the Canadian NCI Clinical trials group, flavopiridol given as a bolus infusion of 50 mg/m2 daily 3 q3 weeks showed modest activity with an 11% partial response rate (47). As in the case of other trials in patients with leukemia and lymphoma, the activity of the novel infusional flavopiridol schedule is currently being investigated in patients with mantle cell lymphoma.
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Table 1 Clinical Trials Involving the Pain-CDK Inhibitor Flavopiridol (AlvocidibTM) Therapy
Disorder
Phase
Clinical trials gov ID
Single agent þVelcade Single agent Single agent Single agent
Lymphoma, multiple myeloma Lymphoma, multiple myeloma Mantle cell lymphoma CLL, prolymphocytic leukemia Leukemia, lymphoma, multiple myeloma CLL, lymphocytic lymphoma AML, ALL, CML AML
I, II I II II I
NCT00112723 NCT00082784 NCT00005074 NCT00098371 NCT00070239
I I
NCT00058240 NCT00101231 NCT00407966
B-CLL, small lymphocytic lymphoma Acute leukemia
I
NCT00377104
I
Bcr-Ablþ hematologic malignancies NHL, mantle cell lymphoma Acute leukemia, CML Lymphoproliferative disorders, mantle cell lymphoma Mantle cell lymphoma, diffuse large B-cell lymphoma CLL, prolymphocytic leukemia Multiple myeloma CLL Lymphoma
I
NCT00470197 NCT00016016 NCT00064285
II I I
NCT00003039 NCT00278330 NCT00058227
I, II
NCT00445341
II II II I
NCT00464633 NCT00047203 NCT00003620 NCT00012181
Single agent Single agent þcytarabine, mitoxantrone Single agent þcytarabine, mitoxantrone þimatinib mesylate Single agent þvorinostat þfludarabine, rituximab Single agent
Single Single Single Single
agent agent agent agent
Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; B-CLL, B-cell chronic lymphocytic leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin’s lymphoma.
UCN-01 (7-hydroxystaurosporine) Background and Mechanisms of Action UCN-01 (7-hydroxystaurosporine, NSC638850 or KW-2401; Kyowa Hakka Kogyo Company Ltd., Tokyo, Japan), a derivative of the nonspecific PKC inhibitor staurosporine (a natural product isolated from Streptomyces staurosporeus), was originally developed as a selective PKC inhibitor. Studies have also reported that UCN-01 inhibits several CDKs. However, recent studies have shown that it exerts other antitumor effects, including inhibition of Chk1, which results in
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‘‘inappropriate’’ activation of CDKs and abrogation of DNA damage–induced cell cycle checkpoints, as well as interference with the PDK1/Akt survival pathway, thus promoting induction of apoptosis. These effects are largely independent of PKC inhibition. UCN-01 displays antitumor activity in in vitro systems and in vivo xenograft models involving multiple human tumor types, with greater antitumor effects observed in longer administration intervals (e.g., 72 hours in in vitro systems) (23). In preclinical studies, UCN-01 has been shown to be a potent inducer of apoptosis in human AML cell models as well as primary CLL cells (48,49). In addition, by virtue of its ability to disrupt the DNA damage–associated G2 or S-phase checkpoints, UCN-01 has been shown to interact synergistically with nucleoside analogs such as ara-C and fludarabine in several human leukemia cell types (50–52). PKC Inhibition UCN-01 selectively inhibits Ca2þ-dependent PKC isozymes (e.g., PKCa, b, and g; IC50 ¼ 4–30 nM) and less potently inhibits Ca2þ-independent PKC isozymes (IC50 *500 nM) (53). In clinical trials, a clear decrease in the phosphorylated cytoskeletal membrane protein adducin, a specific substrate phosphorylated by PKC, was observed in tumor and bone marrow samples following UCN-01 administration (23). However, PKC inhibition appears to be unrelated to various actions of UCN-01, including antiproliferative activity, interference with cell cycle progression, and induction of apoptosis. CDK Inhibition UCN-01 can either inhibit or activate CDKs. It has been reported that UCN-01 induces G1 cell cycle arrest at low concentrations (IC50 ¼ 100–300 nM) (54). However, this effect seems unrelated to direct the inhibition of CDKs, as UCN-01 inhibits CDC2/CDK1 and CDK2 in vitro only at higher concentrations (IC50 ¼ 300–600 nM). Chk Inhibition In normal cells, DNA damage generally induces G1 arrest mediated by accumulation or activation of p53, a major component of the G1 checkpoint machinery (55). In contrast, p53-defective tumor cells primarily arrest in S or G2 phase in the checkpoint response to DNA damage. UCN-01 abrogates the G2 checkpoint selectively in p53-defective cells with 100,000-fold greater (IC50 *50 nM) potency compared with caffeine (23). Chk1 has been defined as a major target in UCN-01-mediated G2 abrogation (56). Pharmacologic concentrations of UCN-01 inhibit the activity of both Chk1 and Chk2 immunoprecipitated from human tumor cells, which may account for
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the observation that UCN-01 abrogates IR-induced p53-independent G2 arrest, whereas Chk1 activity remains unchanged. Inhibition of these events by UCN-01 results in inappropriate activation of CDC2/CDK1, which drives tumor cells through mitosis prior to the repair of DNA damage, resulting in apoptosis (57). Plasma samples isolated from patients who received UCN-01 were found to induce a 40% to 70% abrogation in an ex vivo G2 checkpoint assay (58). UCN-01 also abrogates the S-phase checkpoint (59), but the mechanism(s) responsible for this event appear to be complex. Such findings have created a theoretical basis for developing a therapeutic strategy in which UCN-01 may sensitize tumor cells (particularly p53-defective cells) to DNA-damaging agents and radiation by abrogating the G2 and/or S checkpoints. It is noteworthy that the checkpoint abrogation effects of UCN-01 are manifested at lower drug concentrations (e.g., IC50 *50 nM for G2 checkpoint abrogation) than those responsible for cytotoxicity or inhibition of cell proliferation. PDK1/Akt Inhibition PI3K/Akt cascade represents a critical signaling pathway in cell survival mediated by many growth factors and cytokines. Phosphorylation at Thr308 of Akt is catalyzed by PDK1 and phosphorylation at Ser473 by PDK2. UCN-01 directly inhibits upstream Akt kinase PDK1 with an IC50 less than 33 nM in vitro and in vivo assays, whereas enforced expression of PDK1 restores Akt kinase activity. Overexpression of active Akt diminishes the cytotoxic effects of UCN-01, indicating inhibition of PDK1-Akt pathway attributes to the antitumor activity of this agent (60). Induction of Apoptosis UCN-01 induces apoptosis with IC50 values of 100 to 1000 nM in a panel of HNSCC cell lines in vitro and in the HN12 xenograft in vivo and exhibits enhanced cytotoxicity in cells displaying mutant p53 (61). Although the mechanism underlying UCN-01-induced apoptosis is still unknown, several potential targets have been postulated. First, inhibition of PDK1/Akt has been directly related to the cytotoxicity of UCN-01 (60). Second, as CDK1 is identified as a proapoptotic mediator (62), Chk1/2–CDC25C-mediated CDK1 activation, particularly under inappropriate circumstances, may contribute to the induction of apoptosis by UCN-01 (see above). Finally, UCN-01-induced apoptosis has been associated with downregulation of antiapoptotic proteins such as Mcl-1, XIAP, BAG-1, and Bcl-2 (37). Clinical Trials of UCN-01 in Leukemia and Lymphoma Initial clinical trials of UCN-01 involved a 72-hour continuous infusion schedule every two weeks (63). Unexpectedly, the plasma half-life (*30 days) of UCN-01 in patients was observed to be 100-fold longer than that observed in preclinical models. It was subsequently shown that UCN-01 extensively binds to plasma
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Table 2 CDK Inhibitor in Clinical Trials Compound
Target
Disorder
Phase
Flavopiridol (Alvocidib) UCN-01
pan CDKs
I, II
Chk1
CYC202 (Seliciclib) PD0332991
CDKs 1, 2, 7, and 9 CDK4/6
Various hematologic malignancies and solid tumors Various hematologic malignancies and solid tumors Solid tumors
BMS-387032 (SNS-032) P276-00
CDKs 2, 7, and 9 CDKs 4, 6, and 1, cyclin D
Others AZD5438 R547 SCH727965
CDKs 1 and 2 CDKs 1, 2, and 4 CDKs 1, 2, and 9
I, II II
Multiple myeloma Solid tumors, diffuse large cell NHL Mantle cell lymphoma B-lymphoid malignancies, solid tumors Advanced refractory neoplasms
II I
I
Multiple myeloma
I, II
Normal volunteers Solid tumors Various hematologic malignancies and solid tumors
I I I
I I
Abbreviation: NHL, non-Hodgkin’s lymphoma.
a1-acidic glycoprotein in humans, which accounts for the unique clinical pharmacology of UCN-01 (63). On the basis of these findings, further clinical trials are being conducted using modified UCN-01 schedules (e.g., a 36-hour continuous infusion every 4 weeks). Such schedules result in a mean UCN-01 half-life of approximately 588 hours, with peak plasma concentrations of total drug ranging from 30 to 40 mM with approximately 100 nM concentrations of free UCN-01 detected in saliva (63). Significantly, such concentrations are in excess of those necessary to inhibit Chk1. Several responses have been observed in patients with melanoma and refractory anaplastic large cell lymphoma. In addition, several phase I trials with shorter schedules (e.g., 3-hour infusion) are currently ongoing as combination regimens involving DNA-damaging agents (64–66) (Table 2). Clinical evaluation of UCN-01 in leukemia and lymphoma has been relatively limited. In a recent pilot trial, patients with refractory AML were treated with 1 gm/m2 ara-C as a daily continuous infusion for four days in combination with UCN-01 administered at a dose of 45 mg/m2 as a continuous intravenous infusion starting on day 2 (66). Although UCN-01 potentiated ara-C lethality, in association with abrogation of the DNA-damage checkpoint in blast cells, correlations and clinical responses were not reported in this small pilot study. In a study in which UCN-01 was administered as a continuous 72-hour infusion at dose of 42.5 mg/m2/day 3 days, one patient with Alk-positive
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anaplastic large cell lymphoma had a prolonged period of disease stabilization (2.5 years) following therapy (67). CYC202 CYC202 (R-roscovitine; seliciclib; Cyclacel Ltd., Dundee, U.K.) is a substituted purine analog derived from 6-DMAP and iospentenyladenine. In vitro kinase assays using purified recombinant kinases have revealed that CYC202 inhibits CDK2 (CDK2/cyclin E: IC50 ¼ 100 nM; CDK7/cyclinH: IC50 ¼ 490 nM; CDK2/cyclin A: IC50 ¼ 710 nM), and less potently CDK1 (CDK1/cyclin B: IC50 ¼ 2.69 mM), but neither CDK4 (CDK4/cyclin D1: IC50 ¼ 14.21 mM) nor other kinases (e.g., PKA, PKC) (68). Like most CDK inhibitors, CYC202 inhibits CDKs by competing with ATP for its CDK-binding site (69). In vitro evaluation of antitumor activity demonstrated the cytotoxicity of CYC202 (average IC50 ¼ 15.2 mM) against a panel of 19 human tumor cell lines, including those with cisplatin- and doxorubincin-resistant phenotypes, which were independent of p53 status and cell cycle alterations (69). In vivo administration of CYC202 resulted in significant antitumor effect and reduction in the tumor growth rate in murine xenografts bearing human colorectal carcinoma and uterine cancer (69,70). On the basis of these findings, CYC202, the first oral bioavailable CDK inhibitor, has entered phase I clinical trials in patients with advanced solid tumors (71), and studies in patients with hematologic malignancies are also underway (Table 2). These studies revealed that plasma concentrations (Cmax) of more than 2000 ng/mL at day 1 and day 7 were achievable at 800 mg/kg twice daily for 7 days, a dose that is in the range of the IC50 values reported for seliciclib in vitro activity and without DTL (71). The anticancer activity of CYC202 has been related to (1) inhibition of cell cycle–regulatory CDKs (e.g., CDKs 1, 2, and 7) and downregulation of cyclin D1, leading to a reduction of pRb phosphorylation at multiple sites and cell cycle arrest in G1, S, and G2/M phases (72); (2) inhibition of transcriptional CDKs (e.g., CDK9 in particular, and CDK7), resulting in a decrease in/inactivation of RNAPII and transcriptional repression of short life-time proteins such as cyclin D1, cyclin A, cyclin B1, as well as Mcl-1 and XIAP (72,73); (3) more importantly, induction of apoptosis in tumor cells while largely sparing normal cells (74), which is most likely related to downregulation of antiapoptotic proteins, particularly Mcl-1 (75); and (4) lowering the threshold of cancer cells to cytotoxic or other novel agents (76). Notably, CYC202 has shown activity in preclinical models of NHL (77), mantle cell lymphoma (73), and CLL (74). BMS-387032 (SNS-032) A series of compounds derived from 2-acetamido-thiazolythioacetic ester have been discovered and optimized as small-molecule inhibitors of cyclin E-CDK2. Among those, compound 21 (BMS-387032; SNS-032) has been identified as an oral bioavailable inhibitor of CDKs 2, 7, and 9. This compound potently inhibits
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CDK2 (IC50 ¼ 48 nM, 10- and 20-fold selective over cyclins B-CDK1 and D-CDK4, respectively) with comparable activity toward CDKs 7 and 9. Similar effects were also observed in a panel of tumor cell lines in vitro. BMS-387032 has demonstrated significant antitumor activity in vivo both in murine tumor models and human tumor xenograft models (78). This systematic investigation led to a phase I clinical trial of BMS-387032 where the agent was administered at doses up to 4 mg/m2 as a one-hour infusion weekly for three weeks (15). Initial results from clinical trials showed some objective tumor responses and good tolerability (Table 2), although the maximum tolerated dose (MTD) was not reached. Trials in patients with leukemia and lymphoma are planned. COMBINATION OF CDK INHIBITORS WITH OTHER TARGETED AGENTS IN LEUKEMIA AND LYMPHOMA Flavopiridol In view of its pleiotropic actions, efforts to combine flavopiridol with other targeted agents in various hematologic malignancies have been the focus of attention. Several groups have described synergistic interactions between flavopiridol and TRAIL (TNF-receptor apoptosis-inducing ligand). For example, Rosato et al. reported that synergism between flavopiridol and TRAIL in human leukemia cells stemmed from downregulation of XIAP (79). Such studies provide a rationale for future attempts to combine CDK inhibitors with TRAIL in leukemia and lymphoma therapy. There is also evidence that flavopiridol interacts synergistically with histone deacetylase inhibitors (HDACIs) to induce apoptosis in human leukemia cells. For example, Almenara et al. reported a highly synergistic interaction between flavopiridol and the HDACI vorinostat (SAHA or suberoylanilide hydroxamic acid) in human leukemia cell lines as well as primary AML blasts (80). This interaction stemmed, in part, from the flavopiridol-mediated inhibition of p21CIP1 induction, an event known to promote the lethality of HDACIs. On the basis of these findings, a phase I trial of vorinostat, administered 200 mg orally thrice daily for 14 days in conjunction with flavopiridol administered as a one-hour infusion daily (days 1–5) in patients with refractory AML/high-risk myelodysplastic syndrome has been initiated and is ongoing along with plans to incorporate the new, infusional flavopiridol schedule. On the basis of evidence of synergism between flavopiridol and the BcrAbl kinase inhibitor imatinib mesylate in CML cells, including some resistance to imatinib (81), a phase I trial of imatinib mesylate and flavopiridol has been initiated. Although the MTD for this combination was not identified, further efforts in this direction have been deferred in light of the introduction of secondgeneration Bcr-Abl kinase inhibitors (e.g., dasatinib, nilotinib). Finally, on the basis of evidence of synergistic interactions between flavopiridol and the proteasome inhibitor bortezomib in malignant hematopoietic cells (82), a phase I trial has been initiated in patients with refractory multiple
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myeloma and indolent NHL in which escalating doses of flavopiridol and bortezomib are given as an intravenous infusion on days 1, 4, 8, and 11 q monthly. The regimen has proven to be well tolerated, and the MTD has not yet been reached. Notably, several patients who no longer responded to standard therapy have responded to the combination of bortezomib and flavopiridol (83). Plans are underway to incorporate the four-hour flavopiridol infusion schedule into this regimen in the hope that an improvement in the activity of the regimen can be achieved. CYC202/R-Roscovitine Raje et al. reported that CYC202 interacted synergistically with bortezomib in human multiple myeloma cells (75), but whether the mechanism underlying this interaction is the same as that responsible for the flavopiridol/bortezomib synergism in human leukemia cells and if such findings can be extended to leukemia and lymphoma, remain to be determined. Rosato et al. described highly synergistic interactions between roscovitine and the pan-HDACI LAQ824 in human leukemia cells and related this interaction to downregulation of Mcl-1, p21CIP1, and XIAP as well as induction of oxidative injury (84). More recently, Chen et al. reported highly synergistic interactions between roscovitine and the Bcl-2 antagonist ABT-737 in human leukemia cells (85). The mechanism responsible for this interaction was determined as Mcl-1 downregulation mediated by roscovitine, which cooperated with ABT-737 in the disruption of the function of Bcl-xL to unleash Bak and activate Bax. In view of evidence of in vivo activity of agents like ABT-737 in murine models of lymphoma (86), the concept of combining Bcl-2 antagonists with CDK inhibitors warrants further attention. UCN-01 Combination strategies involving the CDK inhibitor UCN-01 have primarily focused on the inhibitors of the Ras/Raf/MEK1/2/ERK1/2 pathway. For example, exposure of human leukemia as well as multiple myeloma cells to UCN-01 results in the activation of MEK1/2/ERK1/2, and interference with the latter process, i.e., by MEK1/2 inhibitors such as PD184352, results in a dramatic increase in apoptosis (87,88). These events were associated with enhanced activation of CDC2, which was consistent with the ability of UCN-01 to inhibit Chk1. Lethality of the UCN-01/MEK1/2 inhibitor regimen primarily involved activation of the intrinsic, mitochondrial pathway and was substantially blocked in cells overexpressing Bcl-2 or Bcl-xL. However, lethality of the regimen was restored in leukemia cells by agents capable of activating the extrinsic apoptotic pathway, TRAIL (89). Studies also show UCN-01 activity to be dramatically enhanced in human leukemia cells by the mTOR inhibitor, rapamycin, in association with the inactivation of ERK and Akt, and the activation of JNK (90).
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Similar events were observed in human leukemia cells exposed to UCN-01 in conjunction with the farnesyltransferase inhibitor, R115777 (91). Finally, a regimen combining MEK1/2 inhibitors (e.g., PD184352) with UCN-01 was very active in killing Bcr-Ablþ human leukemia cells, including those resistant to imatinib mesylate (92). CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS As an anticancer strategy, targeting cell cycle as well as transcription-regulatory CDKs represents a highly attractive approach, particularly in the case of leukemia and lymphoma, in which dysregulation of cell cycle–related genes appears to play an important role in disease pathogenesis and prognosis. As the first CDK inhibitor to enter clinical trials, flavopiridol has been the most extensively developed of this class of compounds (Table 1). Its antitumor activity has been related to multiple mechanisms, including inhibition of CDKs, induction of apoptosis, blockade of transcription (e.g., cyclin D1, Mcl-1, and VEGF) presumably mediated through inhibition of P-TEFb (CDK9/cyclin T), and antiangiogenesis. Single-agent activity in AML and lymphoma (e.g., mantle cell lymphoma) has been limited to date, although a new infusional flavopiridol schedule appears to have significant activity in CLL. It will be of considerable interest to determine whether this flavopiridol schedule displays increased activity in AML and lymphoma. UCN-01, an agent targeting Chk1 and related proteins that is currently in clinical trials, exhibits its antitumor activity primarily through G2 and S checkpoint abrogation resulting from inhibition of Chk1. Interference with the PDK1/Akt survival pathway may also contribute to the cytotoxicity of UCN-01 in leukemia and other hematologic malignancies. Significantly, the strategy of combining flavopiridol or UCN-01 with conventional chemotherapeutic drugs and, importantly, other novel signal-transduction modulators such as MEK1/2 or proteasome inhibitors, offers the potential for enhanced tumor-selective cytotoxicity and circumvention of drug resistance. In fact, the ultimate role of CDK inhibitors, such as flavopiridol and UCN-01, may be as modulators of the activity of conventional and possibly more novel chemotherapeutic agents. Finally, a variety of more novel small molecule CDK inhibitors have been developed. Among these, CYC202 (seliciclib), BMS387032 (SNS-032), PD033299, and P276-00 have recently entered clinical trials (Table 2), and others are in development. One overarching question is whether the lethality of CDK inhibitors in leukemia and lymphoma stems primarily from cell cycle perturbations, cell survival–related gene trasnscriptional repression due to CDK7 and CDK9 inhibition, or a combination of these actions. In any case, it is likely that therapeutic strategies targeting CDKs will remain the focus of intense interest in the treatment of acute leukemia and NHL for the foreseeable future. As shown in Figure 2, in the future, attention will also focus on the development of novel agents targeting other components of the cell cycle– regulatory machinery, including DNA-damage checkpoints (e.g., ATM, ATR,
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371 Figure 2 A novel model for antileukemia/lymphoma agent targeting cell cycle control (red block indicates the new target involve in cell cycle regulation).
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Chk1, Chk2, Wee1, Myt1, etc.) and mitotic/spindle assembly checkpoints [e.g., aurora kinases (aurora A, B, C), polo-like kinase 1 (Plk1), TTK/hMps1 (a kinetochore-associated protein kinase), etc.]. ACKNOWLEDGMENTS This work was supported by Public Health Service grants CA-63753, CA-93738, CA-100866, and CA88906 from the National Cancer Institute, award 6045-03 from the Leukemia and Lymphoma Society of America, and a translational research award from the V Foundation.
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67. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001; 19(8): 2319–2333. 68. McClue SJ, Blake D, Clarke R, et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 2002; 102(5): 463–468. 69. Tang L, Li MH, Cao P, et al. Crystal structure of pyridoxal kinase in complex with roscovitine and derivatives. J Biol Chem 2005; 280(35):31220–31229. 70. Raynaud FI, Whittaker SR, Fischer PM, et al. In vitro and in vivo pharmacokineticpharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine and CYC202. Clin Cancer Res 2005; 11(13): 4875–4887. 71. Benson C, White J, De Bono J, et al. A phase I trial of the selective oral cyclindependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days. Br J Cancer 2007; 96(1):29–37. 72. Whittaker SR, Walton MI, Garrett MD, et al. The Cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) inhibits retinoblastoma protein phosphorylation, causes loss of Cyclin D1, and activates the mitogen-activated protein kinase pathway. Cancer Res 2004; 64(1):262–272. 73. Lacrima K, Valentini A, Lambertini C, et al. In vitro activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas. Ann Oncol 2005; 16(7):1169–1176. 74. Alvi AJ, Austen B, Weston VJ, et al. A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival. Blood 2005; 105(11): 4484–4491. 75. Raje N, Kumar S, Hideshima T, et al. Seliciclib (CYC202 or R-roscovitine), a smallmolecule cyclin-dependent kinase inhibitor, mediates activity via down-regulation of Mcl-1 in multiple myeloma. Blood 2005; 106(3):1042–1047. 76. Coley HM, Shotton CF, Kokkinos MI, et al. The effects of the CDK inhibitor seliciclib alone or in combination with cisplatin in human uterine sarcoma cell lines. Gynecol Oncol 2007; 105(2):462–469. 77. Lacrima K, Rinaldi A, Vignati S, et al. Cyclin-dependent kinase inhibitor seliciclib shows in vitro activity in diffuse large B-cell lymphomas. Leuk Lymphoma 2007; 48(1): 158–167. 78. Misra RN, Xiao HY, Kim KS, et al. N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl] methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent. J Med Chem 2004; 47(7):1719–1728. 79. Rosato RR, Almenara JA, Dai Y, et al. Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells. Mol Cancer Ther 2003; 2(12): 1273–1284. 80. Almenara J, Rosato R, Grant S. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Leukemia 2002; 16(7):1331–1343.
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16 FLT3: A Receptor Tyrosine Kinase Target in Adult and Pediatric AML Mark Levis, Patrick Brown, and Donald Small Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, U.S.A.
INTRODUCTION Constitutively activated tyrosine kinases have been investigated for decades now for their role in the pathogenesis of different cancers. The importance of these oncogenes in oncogenesis is no longer a subject of debate. Still being debated, however, is how effectively these enzymes can be targeted and whether or not such targeting will have a meaningful impact in treating these diseases. Tyrosine kinases, in particular, seem to be important in the development of hematologic malignancies, especially myeloid leukemias. Starting with the recognition that the product of the Philadelphia chromosome (Bcr-Abl) is an activated tyrosine kinase, to the recent discovery of a PDGFRa fusion protein in hypereosinophilic syndrome (HES) and eosinophilic leukemia, and a gain of function mutation of Janus Kinase 2 (JAK2), mutation-activated kinases continue to be identified as causative factors in hematopoietic disorders (1–5). The first successful clinical use of a small molecule kinase inhibitor, imatinib, was, in fact, in the treatment of chronic myelogenous leukemia (CML) (6). Small molecule kinase inhibitors represent a new, rapidly expanding class of anticancer drugs. Seven different kinase inhibitors have been approved for the treatment of a variety of malignancies since 2001, and many more are under development (7–13). Their use, particularly in patients with activating kinase mutations, often results in dramatic
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clinical responses. Survival is frequently improved, although, with the exception of imatinib in CML, the responses are usually not durable. The targets of these agents are not necessarily known. While all of them are developed with a specific molecular target in mind, some exert their clinical effects through inhibition of either unknown or unanticipated targets. This has led to enthusiasm for multitargeted inhibitors that have activity against a broad array of biologically relevant kinases. Dasatinib, for example, is a potent inhibitor of both imatinib-resistant forms of Bcr-Abl and also of Src-family kinases. The acute leukemias, acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), are malignancies with a generally poor prognosis when treated with conventional chemotherapy. Both are characterized by a relatively frequent occurrence of constitutively activated tyrosine kinases, which confer an even worse prognosis. Thirty percent of adult ALL cases and 5% of pediatric ALL cases harbor the Philadelphia chromosome (which generates the Bcr-Abl fusion gene) (14,15), while in AML, internal tandem duplications of the FLT3 gene (FLT3/ITD mutations) are present in 23% of adult cases and 13% of pediatric cases (16,17). These mutations are associated with significantly inferior outcomes in the face of conventional chemotherapy. However, both also offer molecular targets and an opportunity to improve those outcomes. Which kinases should be targeted for leukemia therapy? Presumably, the focus should be on those kinases that are essential in generating or maintaining the transformed state, such as Bcr-Abl. Given that malignant transformation is probably a multistep process, with many aberrant proteins playing a role (18), the choice of a suitable target is not necessarily obvious. However, some guiding principles can be used to establish the credentials of a kinase as a therapeutic target. First, the highest priority targets should probably be kinases that have increased activity through some form of mutation. The precedence for this approach actually comes both from experience in solid tumors as well as in leukemia. CML, of course, is defined by the presence of the mutant Bcr-Abl kinase. Activating mutations of JAK2 have now been found in a majority of cases of the Philadelphia chromosome–negative myeloproliferative diseases (3–5). Gastrointestinal stromal tumors (GIST), likewise, are characterized by activated KIT and thus have a high response rate to KIT inhibition (19). More subtle examples exist, however. For example, only a small fraction of lung cancer patients respond to treatment with a small molecule inhibitor of the epidermal growth factor receptor (EGFR). A significant fraction of them, however, harbor activating mutations of EGFR (20). Breast cancer patients whose tumors have HER2 amplification represent the only subset of patients to derive clinical benefit from lapatinib, a dual EGFR and HER2 inhibitor, or the anti-HER2 monoclonal antibody, trastuzumab (13,21,22). In general, then, a guiding principle might be to initially target only those kinases with aberrantly upregulated activity. Abnormal activation of RTKs can occur in a variety of ways. Perhaps the simplest is through overexpression of the wild-type receptor. This can occur via gene amplification or possibly through epigenetic alterations affecting
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transcription, translation, or perhaps even receptor turnover. A classic example of this type of gene amplification is found with the epidermal growth factor family of receptors in solid tumors, most notably c-erb-B in breast cancer (21). Point mutations localized to the so-called activation loop of the kinase domain can constitutively activate a receptor by shifting the ATP-binding pocket to a more open, accessible conformation. Such kinase domain mutations have been identified in KIT, MET, FLT3, and PDGFRa, all associated with malignancy (23–26). Crystal structure analyses of the EphB2 receptor and of the FLT3 receptor suggest that mutations within the juxtamembrane region disrupt the inhibitory influence this domain exerts over the kinase domain (27,28). Point mutations, deletions, insertions, and ITDs have all been found in the juxtamembrane domain of the KIT receptor in GIST, all associated with constitutively phosphorylated receptor (29). Likewise, FLT3/ITD mutations constitutively activate FLT3 and are one of the most common molecular abnormalities found in AML (16,30). Activating mutations can also be found within the extracellular domain. Some of these mutations in the KIT receptor in GIST appear to activate the receptor by promoting dimerization via an abnormal disulfide bond (31). For other mutations in the extracellular domain, the mechanism of constitutive activation is unclear (32). Finally, abnormal activation of FLT3 (and likely other kinases) has been reported to occur because of loss of ubiquitin ligase activity of c-CBL (33,34). Activating mutations of the RTK FLT3 are one of the most common molecular abnormalities in AML (16,30,35). Patients with these mutations achieve remission with the same frequency as those lacking the mutations, but the FLT3/ITD patients have a high likelihood of relapse and a low cure rate. Because of this, the study of how to incorporate these FLT3 inhibitors into standard AML therapy has the potential to have the greatest impact on survival in the disease since the introduction of all–trans retinoic acid. FLT3 RECEPTOR AND LIGAND Human FLT3, cloned over a decade ago by two independent groups (36,37), contains 993 amino acids and is visualized as a doublet corresponding to 130 and 160 kD (the larger being the mature, heavily glycosylated form) on electrophoretic gels. It has an extracellular ligand-binding region with five immunoglobulin-like domains and a single transmembrane domain (Fig. 1). The cytoplasmic portion comprises a juxtamembrane domain (the site of the major class of activating mutation) followed by the tyrosine kinase domain, which is interrupted by a kinase-insert domain. These structural features allow FLT3 to be grouped with the PDGF receptor subfamily of receptor tyrosine kinases (RTKs) (38). On binding FLT3 ligand (FL), wild-type FLT3 dimerizes, and its activation loop in the N-terminal kinase domain assumes an open conformation to allow ATP access to the ATP-binding pocket. The activation loop is the site of the other major classes of FLT3-activating mutations. Upon ligand-induced
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Figure 1 Simplified diagram of the FLT3 receptor in monomeric form. The juxtamembrane domain and the activation loop both act as structural regulatory elements for access to the ATP-substrate-binding pocket.
dimerization, FLT3 undergoes autophosphorylation and transduces signals promoting cell growth and inhibiting apoptosis through pathways linked to cytoplasmic proteins such as Ras-GAP, PLC-gamma, PI-3 kinase, STAT5, and ERK1/2 (39–45). FL is expressed by virtually all cell types thus far examined (46,47). In contrast, FLT3 has a fairly narrow range of cell expression, being restricted primarily to hematopoietic and neural tissues, which presumably confines its functions to these cell types (48). In bone marrow, FLT3 is expressed by the CD34þ fraction of hematopoietic cells and in a smaller fraction of CD34 cells destined to become dendritic cells (49,50). FLT3 is clearly an important receptor in hematopoietic development. Its ligand acts in synergy with other cytokines to promote hematopoietic precursor expansion, and targeted disruption of either FLT3 or FL in mice, while not lethal, leads to a reduction in hematopoietic precursors (51–58). FLT3 MUTATIONS Wild-type FLT3 is expressed by the leukemic blasts in the majority of cases of acute leukemia, and the expression is no longer tightly coupled to CD34 expression (59–63). In 1996, FLT3/ITD mutations localized to the juxtamembrane domain were discovered in a significant fraction of AML cases (64). Subsequently, point mutations at aspartate 835 (D835, within the activation loop), analogous to previously described mutations in the KIT gene, were found
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in 7% of cases (25,65). Both types of mutations constitutively activate FLT3 (25,66,67). Crystal structure studies and mutational analyses of other RTKs suggest that the juxtamembrane domain of these enzymes exerts a negative regulatory effect on the tyrosine kinase activity (27,68,69). The FLT3/ITD mutations presumably destabilize this negative regulatory function. The D835 mutations, meanwhile, are precisely analogous to point mutations that have been well characterized for other receptors (23,70,71). These point mutations, localized to the kinase domain, shift the so-called ‘‘activation’’ loop to an open configuration. The signaling properties of an FLT3 receptor with an ITD mutation differ from those of the wild-type receptor in a manner that clearly contributes to the process of leukemogenesis. STAT5 and FOXO transcription factors are abnormally activated in response to FLT3/ITD, but not FLT3 wild type, signaling (72–75). Microarray studies have identified upregulation of the Pim-1 and Pim-2 protooncogenes, as well as interaction with the Wnt signaling pathway, in FLT3/ITD leukemia cells (72,76,77). Finally, c/EBPa, a transcription factor involved in myeloid differentiation, is downregulated by the mutant receptors, indicative of the differentiation block that so characterizes leukemia cells (72,78). This downregulation is possibly mediated by RGS2 (74). FLT3 MUTATIONS IN ADULT AML The only test currently available to confirm the presence of an FLT3 mutation in AML is polymerase chain reaction (PCR) based (64,79). With regard to the FLT3/ITD mutations, however, there are a number of consistent clinical features that offer clues to their presence. Patients harboring FLT3/ITD mutations tend to present with leukocytosis and a packed bone marrow, features consistent with a highly proliferative disease. The blasts tend to have monocytic features, although, possibly, this is related to the nucleophosmin mutation status (80–83). Cytogenetics are most commonly normal, with two exceptions. The most prominent exception is the case of acute promyelocytic leukemia, in which over one-third of cases harbor an FLT3/ITD mutation. While these cases tend to be the microgranular variants, thus far there has been no evidence for an adverse prognosis associated with the mutations in M3 AML (84–88). Another interesting exception to the normal cytogenetics rule is the association of FLT3/ITD mutations with 6;9 translocations (which are known to carry an adverse prognosis) (81,89). The general consensus is that FLT3/ITD mutations do not affect the likelihood of achieving a complete remission but rather increase the likelihood of relapse, a characteristic that points to a resistant population of leukemia stem cells (see below). A large number of studies comprising the results of screening more than 5000 adult AML samples for FLT3 mutations have now been published (81,90–97). The samples in these studies were derived from patients enrolled on a variety of different treatment protocols. From these results, FLT3/ITD mutations
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can be estimated to occur in 22.9% of de novo adult AML (i.e., AML not arising from preexisting myelodysplasia) and their presence clearly confers a worse prognosis (16). In some patients, the wild-type FLT3 allele is present in much lower amounts, or even undetectable, compared with the mutant allele. Such cases appear to have an even more dismal prognosis (81,90). D835 mutations occur in 7% of cases, with a less certain clinical impact (25,65) In fact, a recent study has suggested that in the context of the inv (16) cytogenetic abnormality, FLT3 kinase domain mutations confer a more favorable prognosis (91). Overall, FLT3 mutations now represent one of the most common molecular abnormalities in adult AML, and the large body of data regarding the incidence and prognostic impact of FLT3 mutations establish this receptor as a worthy therapeutic target. FLT3 MUTATIONS IN PEDIATRIC AML FLT3/ITD mutations have a prevalence of 10% to 17% in pediatric AML, and FLT3 kinase domain mutations occur in another 7% of cases (85,92–99). FLT3 mutations occur in all French-American-British (FAB) classification subtypes. FLT3/ITD is most commonly found in cases with normal cytogenetics but may occur together with cytogenetic abnormalities. FLT3/ITD is particularly common in pediatric patients with t(6;9) and is rarely found in association with monosomy 7 or 5q-. FLT3 kinase domain mutations are significantly associated with MLL rearrangements, especially with t(9;11) in younger patients. Incidence of FLT3/ITD increases with increasing age within the pediatric age group. There is no correlation between age and the incidence of FLT3 kinase domain mutations in children. Children with FLT3/ITD are more likely to present with elevated WBC. Several studies have shown that the presence of FLT3/ITD confers an increased risk of relapse and decreased survival in childhood AML (92,93,95,96,99). In a retrospective study of children with de novo AML enrolled on CCG-2891, for example, patients with FLT3/ITD had an eight-year overall and event-free survival of 13% and 7%, respectively, versus 50% and 44% for patients without FLT3/ITD (96). Further studies in pediatric AML have demonstrated that in FLT3/ITDþ samples, the ratio of mutant to wild-type alleles has additional prognostic significance, such that patients with high ITD allelic ratios have a worse prognosis (95). The prognostic significance of FLT3 kinase domain mutations is less clear in pediatrics, but it does not appear to be a significant negative prognostic factor. FLT3 IN CHILDHOOD ALL Gene expression analyses have shown that some of the highest levels of FLT3 mRNA expressions occur in cases of infant and childhood ALL with rearrangements of the MLL gene at chromosome 11q23 (which account for 80% of infant and 5% of childhood ALL cases) and in childhood ALL with hyperdiploidy with more than 50 chromosomes (which account for 30% of cases) (100–102).
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Detailed biochemical studies have shown that leukemic blasts from these patients also express high levels of FLT3 at the protein level, and that FLT3 is constitutively phosphorylated in these cases, even in the absence of FLT3 activating mutations, suggesting autocrine activation via coexpression of FL in these cases (103–105). In a recent report, high-level FLT3 mRNA expression was associated with a poorer treatment outcome within a cohort of 32 uniformly treated infants with MLL-rearranged ALL (106). FLT3 mutations occur much less frequently in childhood ALL than in adult or pediatric AML (17). However, FLT3 kinase domain mutations occur in 18% of MLL-rearranged infant and childhood leukemia cases and in 16% of childhood ALL cases with hyperdiploidy (107,108). Small insertion/deletion mutations in the juxtamembrane domain have also been reported in an additional 12% of high-hyperdiploid ALL cases for a total FLT3 mutation rate of 28% in hyperdiploid ALL (107). By contrast, only 1% of all childhood ALL cases have FLT3/ITD mutations, and these mutations do not appear to be more common in MLL-rearranged or hyperdiploid ALL cases (98,109). FLT3 MUTATIONS IN LEUKEMIA STEM CELLS For AML, it is reasonable to speculate that the malignant transforming event occurs in a primitive hematopoietic stem cell and that relapsing or refractory disease occurs because of the persistence of these leukemia stem cells following initial therapy. These difficult to isolate leukemia stem cells have been best studied using the NOD/ SCID mouse model in which human leukemia cells expressing CD34 (a stem cell marker) but lacking CD38 (a lineage commitment marker) have been shown to contain the so-called ‘‘leukemia-initiating population,’’ a population capable of selfrenewal (110,111). In this regard, FLT3 appears to play a prominent role in stem cell biology, where its expression is first detected on short-term (‘‘low-quality’’) stem cells, and recent work implicates this receptor as a marker for commitment to a nonerythroid nonmegakaryocytic lineage. Wild-type FLT3 is expressed on the leukemic blasts in the majority of cases of acute leukemia, and the expression is no longer tightly coupled to CD34 expression (59–63). A reasonable supposition, therefore, is that the FLT3/ITD mutations occur within the leukemia stem cell population. In support of this is a study using adult AML samples, which found that the mutations were present in CD34þ/CD38– NOD/SCID leukemia-initiating cells (112). In a study of pediatric FLT3/ITD AML, 24 primary samples were sorted into progenitor fractions and analyzed for the presence of the mutations (113). All 24 samples had detectable mutation within the CD34þ/CD33þ progenitor cell fraction, but 5 of the 24 cases lacked the mutation in the more primitive CD34þ/CD33– fraction. These same five patients had dramatically better clinical outcomes than those 19 patients harboring the FLT3/ITD mutations in the more primitive (i.e., stem cell) subpopulation. Two clinical observations offer another glimpse of the role these mutations may play in leukemogenesis. The first is that the ratio of FLT3 mutant-to-wild-type
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DNA may vary among patients and may serve as a predictor for clinical outcome, with patients whose AML cells are homozygous for mutant alleles having the most dismal outcomes (90). The second observation is that the FLT3 mutant-to-wild-type ratio may vary from the time of diagnosis to the time of relapse (114–117). In occasional cases, mutations present at diagnosis (typically those with low mutant-towild-type ratios) were lost at relapse. Alternately, in other patients, mutations only appeared at relapse. In most cases, however, the mutant allele was present at diagnosis and the mutant-to-wild-type ratio increased at relapse. This suggests that the pool of leukemia stem cells is heterogeneous in nature (supported by recent data using the NOD/SCID model) (118) and that, in most cases, the FLT3 mutations may provide a selective advantage to the stem cell clones that harbor them. FLT3 INHIBITORS: PRECLINICAL WORK More than 20 different small molecule kinase inhibitors with activity against FLT3 have been introduced into the literature (119). The following details a general discussion of their properties as well as brief descriptions of some of the more widely used compounds (Table 1). Table 1 FLT3 Inhibitors. Also Listed are Other Known Molecular Targets Inhibited by the Compound with a Potency Similar to That for FLT3, As Well As the Stage of Clinical Development (If Any) Inhibitor
Chemical class
Other targets
Clinical development
Refs.
CEP-701 (Lestaurtinib) PKC412 (Midostaurin) SU11248 (Sunitinib) SU5416
Indolocarbazole
Trka
Phase 3
(120)
Indolocarbazole
KIT
Phase 3a
(121)
3-substituted indolinone 3-substituted indolinone Quinazoline
KIT, PDGFR, VEGFR KIT, VEGFR
Phase 1b
(122)
Phase 3
(123)
KIT, PDGFR
Phase 1
(124)
Quinoxaline Quinoxaline 3-substituted indolinone 3-aminoindazole
PDGFR, KIT PDGFR, KIT KIT, PDGFR, VEGFR KIT, CSF-1 PDGFR,VEGFR, Not reported KIT, FMS
None None Phase 1
(125) (126) (127)
Phase 1
(128)
None None
(129) (123)
KIT, PDGFR, VEGFR
None
(130)
MLN518 (Tandutinib) AG1295 AG1296 SU14813 ABT869 KRN383 SU5614 SU11657
Quinoline-urea 3-substituted indolinone 3-substituted indolinone
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Table 1 (Continued ) Clinical development
Refs.
KIT, FMS, FGFR, VEGFR PDGFR, KIT PDGFR, KIT KIT, FMS, PDGFR PDGFR, KIT
Phase 1
(131)
None None None
(132) (132) (133)
None
(134)
Syk
Nonec
(135)
Raf-1, KIT, PDGFR, VEGFR KIT, FMS, LYN
Phase 1b
(136)
None
(137)
KIT, aurora
Phase 1
(138)
Inhibitor
Chemical class
Other targets
CHIR-258
Benzimidalzolequinoline Quinoxaline Quinoxaline Cyclopenta[a]-inden Bis-indlylmethanone Pyrimidinediamine Biaryl urea
AGL-2043 AGL-2033 GTP-14564 D64406 R406 BAY43-9006 (Sorafenib) FI-700 KW-2449
Pyrimidinediamine Not reported
a
Planned. The compound is approved for use with other cancers. c Phase 2 trials for other indications are underway. b
FLT3 Inhibitors: General The FLT3 inhibitors characterized to date are heterocyclic compounds that either act as ATP competitors or structurally resemble the intermediary complex of a tyrosine covalently bound to ATP. Crystal structure data from other drug receptor combinations as well as from studies of the FLT3 receptor allow some speculation about the structure-activity relationships of these inhibitors (139–141). While most of them likely fit into the ATP-binding pocket of FLT3, the exact mechanism probably varies from inhibitor to inhibitor. For example, some compounds may bind via an induced fit mechanism (as is seen with staurosporine binding to CSK), while others might bind in a lock-and-key manner (such as imatinib binding to ABL). The selectivity of an inhibitor for FLT3 may be greatly influenced by a single amino acid change. For example, FLT3 is normally insensitive to inhibition by imatinib mesylate (IC50 > 3 mM). Substitution of Phe-691 with threonine renders the receptor susceptible to the drug (IC50 0.1–0.3 mM) (140). Recently, FLT3 inhibitors were found to have variable potency against different activating mutations (142). AML samples harboring activating mutations consistently respond in vitro more dramatically than wild-type samples to FLT3 inhibition (120,125,143). A recent report examined the level of FLT3 transcripts in primary AML samples using quantitative PCR and concluded that overexpression of wild-type FLT3
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was associated with a poor prognosis and that such cases may potentially respond clinically to FLT3 inhibitors (144). There are a large number of inhibitors that have little clinical potential but are useful as research agents. The ‘‘tyrphostin’’ AG1296 was the first small molecule reported to directly inhibit FLT3 autophosphorylation. This bicyclic quinoxaline, along with the closely related AG1295, was initially described as a PDGFRa inhibitor with an IC50 of 0.8 mM in cell-based autophosphorylation assays of PDGFstimulated cells (145,146). Both AG1296 and AG1295 were subsequently recognized as FLT3 inhibitors, with IC50 ranges similar to those for PDGFRa. In an important early finding, AG1295 was found to be selectively cytotoxic to primary AML blasts harboring FLT3/ITD mutations (125). A number of tricyclic quinoxalines, with very similar potency and selectivity profiles, were developed as FLT3, PDGFRa, and KIT inhibitors (132). Two of these compounds, AGL2033 and AGL2043, are much more water soluble than the original tyrphostins, opening the potential for clinical development. Other agents that have thus far only been used for research purposes are the Bis(1H-2-indolyl)-1-methanones, which inhibit both PDGFR and FLT3 (147). D64406 inhibits FLT3 with an IC50 of 300 nM. It also inhibits PDGFRa and PDGFRb with IC50 ranges, as determined using in vitro kinase assays, of 1.0 mM and 0.2 mM, respectively. The effects of D64406 on EOL 1 cells provide an interesting illustration of the difficulties in interpreting this type of data. EOL 1 cells, derived from a patient with eosinophilic leukemia, express constitutively activated FLT3 as well as a Fip1L1-PDGFRa fusion protein (120,148). In cytotoxicity assays, the IC50 of D64406 for EOL 1 cells is 0.1 mM, which is lower than the IC50 for inhibition of either FLT3 or PDGFRa kinase activity (147). The observed cytotoxic effect may be due to partial inhibition of either both receptors or inhibition of a completely different target. It is entirely possible that greater antileukemic efficacy is achieved by simultaneously inhibiting two or more receptors within a cell. This multitargeted approach to RTK inhibition is being advanced with other FLT3 inhibitors. Indolocarbazoles: CEP-701 and PKC412 The two FLT3 inhibitors in this class, CEP-701 and PKC412, are alkaloids derived from parent compounds of microbial origin. This class of alkaloids is probably the oldest to be recognized as kinase inhibitors. The parent compounds, staurosporine (the precursor of PKC412) and K252a (the precursor of CEP-701), are highly nonselective, with activity against a broad variety of tyrosine and serine/threonine kinases (149–152). They differ only by the sugar moiety linked to the indolocarbazole scaffold; staurosporine contains a pyranose group, while K252a has a furanose residue. Crystal structure studies of staurosporine bound to different kinases reveal that the indolocarbazole inserts into the ATP-binding pocket, while the pyranose group interacts with residues outside the cleft (139). Modification of the sugar residue of either staurosporine or K252a would therefore be expected to influence the selectivity of the derivative.
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CEP-701 and PKC412 were both originally identified as inhibitors of other kinases (120,121). CEP-701 was initially developed as an inhibitor of the Trka receptor, a member of the nerve growth factor receptor subfamily (153). It was subsequently identified as a potent FLT3 inhibitor with an IC50 of 3 nM (120). The drug is cytotoxic to MV4-11 cells, EOL 1 cells, and Ba/F3 cells transfected with an FLT3/ITD construct, all at doses that closely parallel those required for inhibition of FLT3 in cell-based autophosphorylation assays. CEP-701 inhibits wild-type FLT3, FLT3/ITD mutants, and D835-point mutants with equal potency in cell lines and in primary leukemic blasts. It is clearly multitargeted, with inhibitory activity against Trka and VEGFR2. In a murine FLT3/ITD leukemia model, CEP-701 administered subcutaneously inhibited FLT3 autophosphorylation in vivo, and led to prolonged survival (120). The other indolocarbazole FLT3 inhibitor, PKC412, was originally developed as a PKC inhibitor, but actually has only modest potency toward this enzyme (IC50 500 nM) (154). This drug, like CEP-701, is a potent FLT3 inhibitor with an IC50 of approximately 10 nM (121). It inhibits PDGFRa with an IC50 of 80 nM and KIT only at doses above 500 nM (121). PKC412 inhibits FLT3 autophosphorylation in wild-type- and mutant FLT3-transfected Ba/F3 cells, in MV4-11 cells, and in SEMK2-M1 cells, which have been shown to overexpress wild-type FLT3 through gene amplification (103,121). Oral PKC412 also prolongs survival in a murine myeloproliferative disease model in which mice are transplanted with bone marrow cells transduced with an FLT3/ITD-expressing retrovirus (121,155). Indolinones There are a number of indolinone compounds that have been investigated as FLT3 inhibitors, all of which are 3-substituted indolin-2-ones (122,123). SU5416 was the first of these to be developed extensively, both preclinically and clinically. Hydrophilic, and reported to be highly protein bound in plasma, SU5416 was originally developed as an angiogenesis inhibitor because of its activity against VEGFR-2 (IC50 of 250 nM) (156,157). Subsequently, the drug was found to inhibit FLT3 and KIT even more potently, with an IC50 of 100 nM for both receptors in cell-based autophosphorylation assays (123,158). SU5614, more hydrophilic than SU5416 and more potent as well, inhibits FLT3 autophosphorylation with an IC50 of 10 nM (123). Both SU5416 and SU5614 induce cytotoxicity in MV4-11 cells with IC50 ranges slightly higher than the autophosphorylation IC50 ranges, consistent with the effect being mediated through FLT3 inhibition. SU11248 (sunitinib) is a later generation indolinone multitargeted kinase inhibitor that has been approved for use in renal cell carcinoma. The exact target inhibited in this disease is not clear. Sunitinib is more hydrophilic than its predecessors, and also has more identifiable targets within the same IC50 range. It inhibits FLT3 with an IC50 of 10 to 50 nM (122) and also inhibits KIT, PDGFRa, and VEGFR2 with similar potency (122,159,160). In
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cytotoxic assays of MV4-11 and OCI-AML5 cell lines, sunitinib induced a cytotoxic effect with IC50 ranges of 1 to 10 nM. This inhibitor, therefore, causes significant in vitro cytotoxicity at concentrations much lower than those required for maximal FLT3 inhibition. It is possible that, in the setting of simultaneous inhibition of multiple kinases, even partial inhibition of FLT3 activity can result in cell apoptosis. Another interpretation of this data, however, is that the cytotoxic effects induced by this agent may have little or nothing to do with FLT3 inhibition. Regardless of which receptors are being inhibited, sunitinib is effectively cytotoxic to FLT3 mutant AML cells both in vitro and in vivo (122,159). MLN-518 (Tandutinib) MLN-518 (previously known as CT53518), a piperazinyl quinazoline, is an orally available compound that inhibits FLT3, KIT, and PDGFRa in in vitro kinase assays with IC50 ranges of 17 to 220 nM (124,161,162). Cell-based autophosphorylation assays using Molm-14 cells (which express an FLT3/ITD mutation) yielded an IC50 of approximately 30 nM, while proliferation was inhibited with an IC50 of only 10 nM, perhaps reflecting a multitargeted effect (124). MLN-518 prolonged survival in both the FLT3/ITD murine myeloproliferative model used for PKC412 as well as in athymic mice injected with FLT3/ ITD-transformed Ba/F3 cells. Sorafenib Originally known as BAY43-9006, this multitargeted kinase inhibitor has been approved for use in renal cell carcinoma. As is the case with sunitinib, it is not clear if the observed responses are due to inhibition of a single kinase (such as VEGFR) or multiple kinases. Sorafenib has in vitro activity against both wild-type and mutant FLT3 (163,164). FLT3 Inhibition Combined with Chemotherapy The general consensus in the field is that FLT3 inhibitors by themselves will not measurably impact outcomes in AML patients harboring FLT3 mutations, but that the addition of these drugs to chemotherapy holds great therapeutic promise. However, the best way of incorporating these compounds into standard treatment regimens for the disease remains unclear. The indolocarbazoles lestaurtinib (CEP-701) and midostaurin (PKC412) have both been studied in vitro using combinations with chemotherapy. In one study, lestaurtinib (CEP-701) combined with cytarabine, daunorubicin, etoposide, and mitoxantrone was tested on FLT3 mutant cell lines as well as in a primary AML sample harboring an FLT3/ITD mutation (165). Three treatment sequences were examined: (1) pretreatment of leukemia cells with lestuartinib
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followed by one of the chemotherapeutic agents, (2) simultaneous treatment with lestaurtinib and chemotherapy, and, finally, (3) exposure of cells to chemotherapy followed by lestaurtinib. Pretreatment with lestaurtinib antagonized the cytotoxic effects of the chemotherapy. This was presumably because FLT3 inhibition induced a cell cycle arrest, which, in turn, blunted the S-phase selective effects of agents such as cytarabine and etoposide. Simultaneous treatment with chemotherapy, or lestaurtinib exposure after chemotherapy, induced synergistic cytotoxicity. However, a potential pharmacodynamic interaction between lestaurtinib and daunorubicin was identified because both drugs are bound in plasma to a-1 acid glycoprotein (AAG), free levels of the anthracycline could result if they were to be administered simultaneously. Therefore, the conclusion drawn from this work was that chemotherapy treatment followed by the FLT3 inhibitor offered the most potential for safety and efficacy. In another study, lestaurtinib and midostaurin were compared for cytotoxic effect against primary AML cells, alone and in combination with cytarabine (166). Lestaurtinib was found to be synergistic with cytarabine in inducing cytotoxicity in primary samples with mutant, but not wild-type, FLT3. Sunitinib was found to synergize with cytarabine- and daunorubicininduced cytotoxicity in FLT3/ITD-transformed murine cells and MV4-11 cells (167). Synergy was likewise seen with these combinations in primary AML cells harboring FLT3/ITD mutations. The FLT3 inhibitor and chemotherapies were used simultaneously— no specific treatment sequences were studied. In another study, tandutinib (MLN-518) was administered to mice that had received myelosuppressive doses of cyclophosphamide (168). Furthermore, the drug had only minimal effect on recovery of hematopoiesis following marrow transplantation. From these studies it can be concluded that, in general, FLT3 inhibition will synergize with conventional chemotherapeutic agents in killing FLT3 mutant AML cells. However, a number of issues have been brought to light by this work. In vitro studies, performed primarily using rapidly dividing cell lines in culture medium, cannot precisely imitate the conditions encountered in a patient treated with infusional chemotherapy. For example, in contrast to cell lines in culture, only about 6% of AML cells in human marrow are in S phase. Cytarabine is typically administered over three to seven days to allow a maximal fraction of leukemia cells to enter S phase, when the drug will have maximum effect. If an FLT3 inhibitor is administered at the start of a cytarabine infusion, there exists the potential to arrest leukemia cells in G phase, thereby antagonizing the effects of the chemotherapeutic drug. This phenomenon was observed in one study, but it remains to be seen if it might occur in patients. Pharmacokinetic interaction between FLT3 inhibitors and chemotherapy is also an important concern. A number of inhibitors are highly protein bound, and could compete with anthracyclines for plasma protein–binding sites, with the theoretical potential of increasing free levels of anthracyclines. In addition, the indolocarbazoles (lestaurtinib and midostaurin) have activity as inhibitors of P-glycoprotein. This property could lead to in vivo interactions between a number
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of chemotherapy drugs and the inhibitors. An interaction between lestaurtinib and daunorubicin was observed in vitro in one study, and because of this potential interaction, lestaurtinib is administered to patients only after completion of chemotherapy in trials that are currently accruing. A third issue addressed in these studies was that of the effect of combination treatment on the bone marrow. A number of FLT3 inhibitors also inhibit KIT, a receptor important in early myelopoiesis. While transgenic mice with an FLT3 knockout survive with only mild abnormalities in hematopoiesis, a KIT gene knockout is embryonic lethal. There remains a concern, therefore, that recovery from chemotherapy-induced marrow aplasia may be significantly hindered by a combination of FLT3 and KIT inhibition. To test for this possibility, investigators treated mice with cyclophosphamide to induce myelosuppression, and then followed this treatment with tandutinib, an equipotent inhibitor of FLT3 and KIT. The tandutinib-treated mice were noted to have relatively normal recovery of hematopoiesis compared with the control group, suggesting that, at least in mice, combined inhibition of FLT3 and KIT may be tolerable in the setting of chemotherapy-induced aplasia. PRECLINICAL DATA IN PEDIATRIC AML CEP-701 is the only FLT3 inhibitor for which preclinical data are available specifically for pediatric AML (143). In this study, cytotoxicity and apoptosis assays were performed on 44 diagnostic pediatric AML blast samples (14 wildtype FLT3, 15 FLT3/ITD, 15 FLT3 kinase domain mutations) using CEP-701. Pronounced dose-dependent cytotoxicity and induction of apoptosis were observed in a higher percentage of FLT3/ITD samples (93%) than FLT3 kinase domain mutant samples (27%) or FLT3 wild-type samples (29%). The cytotoxicity was greatest in samples with a high FLT3/ITD mutant-to-wild-type allelic ratio. Cytotoxicity was shown to correlate with expression of constitutively activated FLT3 protein and the ability of CEP-701 to inhibit autophosphorylation of FLT3 and its downstream targets (MAPK, STAT5, etc.). The addition of FL enhanced the survival and augmented the sensitivity to FLT3 inhibition for the CEP-701-responsive subset of FLT3 wild-type and FLT3 kinase domain mutation samples. PRECLINICAL DATA IN CHILDHOOD ALL Both CEP-701 and PKC-412 have been used in preclinical studies using childhood ALL samples. PKC-412 demonstrated in vitro and in vivo antileukemic activity against three primary ALL samples and one ALL cell line (all with an MLL rearrangement), suggesting a potential therapeutic role for FLT3 inhibitors in the treatment of infant and childhood ALL (103). A more comprehensive study of CEP-701 in childhood ALL examined in eight ALL cell lines and 39 bone marrow samples obtained at diagnosis from infants and children with
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various subtypes of ALL (104). CEP-701 induced pronounced apoptotic responses in a higher percentage of samples that expressed high levels of FLT3 (74%, n ¼ 23) compared with samples with low levels of expression (8%, n ¼ 13; p ¼ 0.0003). Sensitivity to FLT3 inhibition was particularly high in samples with MLL gene rearrangements (82%, n ¼ 11; p ¼ 0.0005), high hyperdiploidy (100%, n ¼ 5; p ¼ 0.0007), and/or FLT3 mutations (100%, n ¼ 4; p ¼ 0.0021). Seven of seven sensitive samples examined by immunoblotting demonstrated constitutively phosphorylated FLT3 that was potently inhibited by CEP-701, whereas zero of six resistant samples expressed constitutively phosphorylated FLT3. Another study demonstrated that in primary MLL-rearranged ALL samples from infants, high-level expression of wild-type FLT3 is associated with constitutive FLT3 phosphorylation and in vitro sensitivity to PKC412 (105). CEP-701 has also been shown to result in synergistic killing of MLLrearranged ALL cells when combined with multiple chemotherapy agents (169). The degree of synergy is markedly dependent on sequence of exposure to the agents. Exposure to chemotherapy followed by lestaurtinib results in consistent and strong synergistic cell killing, while simultaneous exposure is in most cases additive. Exposure to lestaurtinib followed by chemotherapy is, in many cases, antagonistic. The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, since these effects are not seen in control cells without activated FLT3. FLT3 INHIBITORS: CLINICAL TRIALS IN ADULT AML SU5416 This intravenously administered indolinone with activity against FLT3 and the VEGF receptor family has been studied clinically as a single agent in two phase 2 studies in AML (170,171). In one study, 43 AML patients who either had refractory disease or were too elderly for conventional therapy were treated twice weekly in four-week cycles (170). One patient achieved a reduction in bone marrow blasts to less than 5% (but did not recover neutrophils or platelets), and seven obtained a 50% or greater reduction in bone marrow or peripheral blasts. The reported response rate was therefore 19%, with responses lasting one to five months. While they observed an association between blast VEGF RNA expression and response, they did not assess the in vivo phosphorylation status of either FLT3 or the VEGF receptors in the study population. None of the responding patients harbored FLT3/ITD mutations. It is difficult, therefore, to draw any meaningful conclusions from this particular trial, largely because it is not clear if the targets (FLT3 or VEGF receptors) were affected. In the second trial of 33 AML patients and 22 MDS patients, there were three partial responses and one hematologic improvement (171). No information was obtained on the in vivo activity of the targeted receptors. Nonetheless, in an elegant follow-up study of these patients, the same group demonstrated in vivo inhibition of FLT3 phosphorylation immediately after drug infusion (172). SU5416, however, has a
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relatively short half-life, and only sustained inhibition of FLT3 is predicted to result in leukemia cell death. Thus, it was not surprising that correlative studies from this trial demonstrated short-lived inhibition of FLT3 autophosphorylation, lasting only a few hours after dosing. The important conclusion from this follow-up work was that the major limitation of SU5416 appeared to be pharmacokinetic, not pharmacodynamic. SU11248 (Sunitinib) In a phase 1 study, 29 patients received a single dose, in escalating fashion, of sunitinib (173). The primary endpoint of the study was the all-important one: confirmation of target inhibition. This data was then followed up with a more traditional phase 1 study in which 16 AML patients, including 4 with FLT3 mutations, were treated in a prolonged fashion (174). Six patients had transient reductions in peripheral and bone marrow blasts, including all four patients with FLT3 mutations. While inhibition of FLT3 phosphorylation (and appropriate downstream targets) was achieved with the higher dose levels, it became apparent that the drug would not be tolerated in sustained fashion because of toxicity that likely relates to the larger number of cellular targets inhibited by sunitinib. CEP-701 (Lestaurtinib) This indolocarbazole derivative was initially introduced as an inhibitor of Trka for possible use in prostate cancer (153) but was recognized subsequently as a potent FLT3 inhibitor (120). A clinical-laboratory correlative phase 1 or 2 trial in relapsed or refractory AML patients with FLT3 mutations was initiated (175). The correlative assays from this trial revealed that if a patient had leukemic blasts that died when exposed to CEP-701 in vitro, and if that patient achieved a level of CEP-701 in plasma sufficient to significantly inhibit FLT3 autophosphorylation in a sustained fashion, then a clinical response was observed. Five out of fourteen patients showed a response, typically with reductions in peripheral blasts. One patient achieved a decrease in marrow blasts to less than 5% that persisted for three months. An additional phase 2 study of CEP-701 was conducted in the United Kingdom, in which elderly AML patients deemed not fit for standard therapy were treated with CEP-701 as a single agent (176). Clinical activity, manifest as transient reductions in bone marrow and peripheral blood blasts or longer periods of transfusion independence, was seen in three of five cases with mutated FLT3 and 5 of 22 wild-type FLT3 patients. Correlative data demonstrated that clinical responses occurred where the presence of sustained FLT3-inhibitory drug levels were combined with in vitro cytotoxic sensitivity of blasts to lestaurtinib. Data (discussed above) from in vitro studies combining CEP-701 with chemotherapy provided the basis for a recently launched, multicenter clinical trial (see below) (165). Preliminary results suggested that target inhibition correlated precisely with clinical responses, and that CEP-701 administered sequentially with chemotherapy appeared to be beneficial (177).
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PKC412 (Midostaurin) PKC412 was originally investigated as a protein kinase C inhibitor (hence, ‘‘PKC’’) then subsequently recognized as a potent FLT3 inhibitor and tested in a phase 2 trial of AML patients, again using a relapsed or refractory group that harbored FLT3 mutations (121,178). Twenty patients were treated with a fixed dosing schedule of 75 mg orally three times daily, and 14 of these showed a clinical response, primarily in peripheral blast reduction. Six patients achieved a greater than 50% reduction in the bone marrow blast count, and three achieved less than 5% blasts. Responses correlated with in vivo FLT3 inhibition. A significant part of its activity, however, may have been exerted through the actions of one of its metabolites, CGP52421 (179). PKC412 was administered concomitantly with a ‘‘7 þ 3’’ induction regimen to newly diagnosed AML patients (both wild-type and mutant FLT3), with considerable resultant toxicity and disappointing clinical outcomes (180). These results, however, were predictable. The preclinical studies of CEP-701 and chemotherapy mentioned above identified antagonistic effects when FLT3 inhibition was initiated prior to cell cycle–dependent chemotherapeutic agents like cytarabine. In addition, because both PKC412 and daunorubicin are bound in plasma to the same carrier protein (AAG), simultaneous administration of these two drugs would be predicted to lead to higher free levels of daunorubicin and resultant increased toxicity. Building on this experience, a pilot trial was launched in which newly diagnosed FLT3 mutant AML patients were treated with chemotherapy followed by PKC412. These results were more encouraging in that this sequential combination seemed to be tolerable and possibly lead to an improved CR rate (181). MLN518 (Tandutinib) This compound, previously known as CT53518, inhibits FLT3, KIT, and PDGFRa with a similar potency (124,161). The results of a phase 1 trial in FLT3/ITD AML showed it to be generally tolerable, and two of five patients with FLT3 mutations achieved a reduction in peripheral blasts (182). In a parallel study, tandutinib was administered to newly diagnosed AML patients (irrespective of FLT3 mutation status) simultaneously with chemotherapy (183). The combination was found to be tolerable, although it is not clear if any follow-up studies are planned. FLT3 INHIBITORS: ONGOING AND PLANNED CLINICAL TRIALS Adult At the present time, the Cephalon 204 trial, a pivotal study in which patients with FLT3 mutations in first relapse are treated with salvage chemotherapy and randomized to receive CEP-701 afterward, is open with a target accrual of
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220 patients. Also, currently accruing patients is the MRC AML15/17 trial, in which newly diagnosed FLT3 mutation patients are treated with induction chemotherapy and likewise randomized to sequential CEP-701. An international trial of induction chemotherapy with randomization to PKC412 afterward for newly diagnosed FLT3 mutant AML is planned for the winter of 2008. Pediatric A phase 1 study of single agent CEP-701 in children with refractory neuroblastoma (based on the inhibitory activity of CEP-701 against the Trk family of neurotrophin receptors) is being conducted by the NANT (new approaches for neuroblastoma treatment) consortium (John Maris, personal communication). The drug has been very well tolerated, with no significant toxicity except for reversible transaminase elevations seen at 70 mg/m2/dose twice daily. No hematologic toxicity has been seen at any dose level. Trough plasma samples taken from patients on this trial have consistently demonstrated greater than 90% FLT3 inhibitory activity in ex vivo plasma bioassays (Patrick Brown, unpublished data), a level which has been positively correlated with antitumor response in the adult AML trials (177). Two clinical trials of CEP-701 combined with chemotherapy for children with leukemia are being conducted by the Children’s Oncology Group (COG). The first is a single-arm pilot study of idarubicin/cytarabine followed by CEP-701 for relapsed or refractory FLT3 mutant pediatric AML. The second is a randomized comparison of standard chemotherapy with or without the addition of CEP-701 for infants newly diagnosed with MLL-rearranged ALL. Data are not yet available from either of these trials. RESISTANCE TO FLT3 INHIBITORS Resistance to imatinib is now a well-described phenomenon in CML (184); so investigators have quickly moved to identify mutations in FLT3 that confer resistance to the various inhibitors in development (185–187). Point mutations in the ATP-binding pocket and the activation loop of the kinase domain have been generated that display varying degrees of in vitro resistance to different FLT3 inhibitors. The potential clinical significance of these mutations is as yet unclear. For all FLT3 inhibitors thus far studied in clinical trials, resistance to monotherapy with the inhibitor has emerged relatively rapidly, but only in occasional cases are data available as to the exact mechanism of resistance. For example, an FLT3/ITD AML patient treated with PKC412 developed clinical resistance to the drug and was found to harbor a novel point mutation at asparagine 676 (188). This mutation expressed in murine 32D cells conferred resistance to the inhibitory effects of PKC412 on FLT3 autophosphorylation. With the indolocarbazoles (CEP-701 and PKC412), loss of response has been related to inadequate plasma drug levels, i.e., pharmacokinetic causes (178,179,189).
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Out of eight patients treated with CEP-701, two had blasts that were resistant to the drug in vitro before any therapy, suggesting that even sustained, effective FLT3 inhibition will be ineffective for some FLT3/ITD AML patients. This intrinsic resistance to FLT3 inhibition had been noted in the preclinical evaluation of CEP-701, and it was further noted that those samples that were resistant to CEP-701-mediated cytotoxicity displayed persistent activation of downstream signaling proteins such as STAT5 (120). This implied that parallel signaling pathways unaffected by CEP-701 were active in those leukemia samples. To further investigate this, leukemia cell lines were cultured in increasing concentrations of CEP-701 (190). After two to four months, these cell lines were able to proliferate slowly to 50 to 120 nM of this inhibitor. In all resistant cells, FLT3 phosphorylation continued to be fully inhibited by CEP-701 at low nanomolar concentrations, and there were neither detectable mutations within the FLT3 coding sequence nor any changes in FLT3 protein expression. However, there was continued activation of AKT and MAPK proteins despite complete inhibition of FLT3. N-RAS genes were sequenced from all resistant cell lines, and those lines derived from Molm-14 cells (an AML line with an FLT3/ITD mutation) contained unique N-RAS mutations (Q61K and G12D) that were not present in the parental line. Transfection of either of these mutant N-RAS constructs into the parental line resulted in resistance to CEP-701. In this same study, it was noted that the expression levels of a number of different RTKs (Insulin-R, c-MET, PDGF-R, Eph A7, CSF-R) in the resistant lines were upregulated compared with the parental line (190). These findings strongly suggest that resistance to FLT3 inhibitors will be a multifactorial process with activating mutations and upregulation of parallel signaling pathways and, in some cases, with mutations in the FLT3 gene itself. ANTI-FLT3 ANTIBODIES A number of antibodies have been developed in recent years that target cellsurface proteins on malignant cells (191). Several have been used successfully as ‘‘naked’’ antibodies, while others have required conjugation with other agents to generate a cytotoxic response. Even when not mutated, FLT3 is almost universally expressed on the surface of both lymphoid and myeloid blasts, and, therefore, is a potential target for the development of these types of agents. Such fully humanized antibodies have been developed by phage display selection for binding to FLT3 followed by interference with FL binding to FLT3 (192). These could function as naked antibodies via two mechanisms: (1) interference with ligand-activated signal transduction through FLT3 and (2) antibody-dependent cellular mediated cytotoxicity (ADCC). When used in vitro, these antibodies have been shown to block signal transduction through FLT3 in some cell lines and primary samples, including some with activating FLT3 mutations. This, however, was not a universal finding. In some instances, in fact,
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perhaps because the antibodies are bivalent and can dimerize wild-type receptors, antibody treatment actually activated the receptor. Importantly, the antibodies were shown to prolong survival and decrease engraftment of both myeloid and lymphoid cell lines and primary leukemic blasts when transplanted into NOD/SCID mice (193,194). This effect was independent of the degree of inhibition or activation of FLT3 signaling, implying ADCC as the major mechanism of action. Confirmation of this mechanism was obtained by in vivo experiments showing decreased efficacy with NK depletion and increased efficacy with NK activation (194). Thus, there is preclinical evidence that anti-FLT3 antibodies have activity in vivo through ADCC and might be expected to be even more efficacious when used in patients less immunocompromised than NOD/SCID mice. They might combine nicely with small molecule FLT3 TKIs as independent ways of targeting this receptor. There is some preclinical evidence for using them this way as cells selected for resistance to FLT3 TKI were still sensitive to killing in vivo with anti-FLT3 antibody (190). CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS The next five years will likely see the introduction of several of the inhibitors listed in Table 1 in advanced clinical trials, trials in which the compounds are combined with chemotherapy. During this period, a definitive answer will likely be obtained as the utility of CEP-701 and PKC412 for the treatment of FLT3 mutant AML, with possible FDA approval for one or both drugs. While these indolocarbazoles are farthest along the approval pathway, they likely represent ‘‘first generation’’ FLT3 inhibitors that could be supplanted in the future with more selective compounds that have a better pharmacokinetic profile. Since their discovery nearly a decade ago, FLT3 mutations have come to define an important new subset of AML, much like the 15;17 translocation defines acute promyelocytic leukemia. Unlike APL, FLT3 AML has a rather grim prognosis with currently available therapy. It is important to remember, however, that before the introduction of all-trans retinoic acid (ATRA) to target this molecular defect, APL was likewise a poor-risk disease. Hence, there is great hope that targeting FLT3 will likewise convert FLT3 AML into one of the more curable leukemias. The consensus from the initial clinical trials is that monotherapy with small molecule FLT3 inhibitors can lead to significant but nonsustained clinical effects in relapsed AML patients harboring activating mutations. Extending the analogy further, APL is not generally curable with ATRA alone. FLT3 inhibitors are more likely to be useful either in some form of combination with conventional chemotherapy or as maintenance therapy after remission has been achieved. The challenge will be to identify those inhibitors with a pharmacokinetic profile that would allow sustained FLT3 inhibition in a tolerable fashion when combined with chemotherapy.
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168. Griswold IJ, Shen LJ, La Rose´e P, et al. Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis. Blood 2004; 104(9):2912–2918. 169. Brown P, Levis M, McIntyre E, et al. Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL-rearranged ALL cells in a sequence-dependent manner. Leukemia 2006; 20(8):1368–1376. 170. Fiedler W, Mesters R, Tinnefeld H, et al. A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia. Blood 2003; 102(8):2763–2767. 171. Giles FJ, Cooper MA, Silverman L, et al. Phase II study of SU5416–a small-molecule, vascular endothelial growth factor tyrosine-kinase receptor inhibitor–in patients with refractory myeloproliferative diseases. Cancer 2003; 97(8):1920–1928. 172. O’Farrell AM, Yuen HA, Smolich B, et al. Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia. Leuk Res 2004; 28(7):679–689. 173. O’Farrell AM, Foran JM, Fiedler W, et al. An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients. Clin Cancer Res 2003; 9(15):5465–5476. 174. Fiedler W, Serve H, Dohner H, et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105(3):986–993. 175. Smith BD, Levis M, Beran M, et al. Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood 2004; 103(10):3669–3676. 176. Knapper S, Burnett AK, Littlewood T, et al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood 2006; 108(10):3262–3270. 177. Levis M, Smith BD, Beran M, et al. A randomized, open-label study of lestaurtinib (CEP-701), an oral FLT3 inhibitor, administered in sequence with chemotherapy in patients with relapsed AML harboring FLT3 activating mutations: clinical response correlates with successful FLT3 inhibition. Blood 2005; 106:121a. 178. Stone RM, DeAngelo DJ, Klimek V, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105(1):54–60. 179. Levis M, Brown P, Smith BD, et al. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. Blood 2006; 108(10):3477–3483. 180. Giles F, Schiffer C, Kantarjian H, et al. Phase 1 study of PKC412, an oral FLT3 kinase inhibitor, in sequential and concomitant combinations with daunorubicin and cytarabine (DA) induction and high-dose cytarabine (HDAra-C) consolidation in newly diagnosed patients with AML. Blood 2004; 104:262a. 181. Stone RM, Fischer T, Paquette R, et al. Phase IB study of PKC412, an oral FLT3 kinase inhibitor, in sequential and simultaneous combinations with daunorubicin and cytarabine (DA) induction and high-dose cytarabine consolidation in newly diagnosed patients with AML. Blood 2005; 106:121a. 182. DeAngelo DJ, Stone RM, Heaney ML, et al. Phase 1 clinical results with tandutinib (MLN518), a novel FLT3 antagonist, in patients with acute myelogenous leukemia or high-risk myelodysplastic syndrome: safety, pharmacokinetics, and pharmacodynamics. Blood 2006; 108(12):3674–3681.
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17 Treatment of Chronic Myeloid Leukemia with Bcr-Abl Kinase Inhibitors Michael J. Mauro Center for Hematologic Malignancies, Oregon Cancer Institute, Oregon Health & Science University, Portland, Oregon, U.S.A.
Michael C. Heinrich Center for Hematologic Malignancies and Departments of Medicine and Cell and Developmental Biology, Oregon Cancer Institute, Oregon Health & Science University and Portland VA Medical Center, Oregon Health & Science University, Portland, Oregon, U.S.A.
INTRODUCTION There may be no better example of innovation in the field of leukemia and lymphoma therapeutics than the development of Abelson (Abl) kinase inhibitors for the treatment of chronic myeloid leukemia (CML). The year 2008 will mark a decade since CML patients were first treated with CGP57148 (STI571) now know as imatinib (GleevecTM, Novartis Pharmaceuticals, East Hanover, New Jersey, U.S.; Glivec1, rest of the world). While it is certainly not the first anticancer therapy directed at a known molecular target, the development of Abl kinase inhibitor ‘‘targeted therapy’’ for CML has set a clear example of the proof of principle in validating molecular targets and a high bar of success to which other innovative therapies are compared. The natural history of CML has been irrevocably changed, treatment algorithms shifted to reflect completely different approaches to old and new choices, and further advances in tyrosine kinase
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inhibitor therapy continue to morph approaches to the treatment of CML and Philadelphia chromosome positive (Ph(þ)) leukemias as a whole. For CML currently, we now have very highly active and durable primary therapy (imatinib) and two approved and convincingly proven salvage options, dasatinib and nilotinib. Other developments expanding the roster of novel therapies in Ph(þ) leukemias include additional multikinase (Abl, Src, Lyn, etc) inhibitors such as SKI-606 and INNO-406, and the aurora kinase inhibitor MK0457, which is specifically active against the T315I mutant Abl kinase variant. In this revolutionary decade of development in CML, therapy has universally shifted away from the primary immunotherapy-based options of interferon (IFN) and allografting and adopted use of kinase inhibitor therapy as primary therapy and beyond. This chapter will review the central role of Bcr-Abl in CML pathogenesis, the development of tyrosine kinase inhibitors against Bcr-Abl, and how this pairing of a crucial target with specific and evolving inhibitors has been a paradigm for unprecedented success. THE DISCOVERY OF BCR-ABL In 1960, Nowell and Hungerford (1) described a consistent chromosomal abnormality (an acrocentric chromosome thought to be a chromosomal deletion) in CML patients. This was the first example of a human cancer linked to a specific chromosomal abnormality. With improvements in chromosomal banding techniques, it became apparent that the observed abnormality was a shortened chromosome 22. In time, Rowley (2) determined that the shortened chromosome, the so-called ‘‘Philadelphia (Ph) chromosome,’’ was in fact the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9:22)(q34;q11). The recognition that mutations in normal cellular genes could be oncogenic came from the study of transforming retroviruses (3). The Abl leukemia virus (v-Abl) was initially described in 1970 and led to the cloning of its normal cellular homolog, c-Abl, which was found to map to the long arm of chromosome 9. As further investigation transpired to unravel the abnormality known as the Ph chromosome, it became apparent that c-Abl had been translocated to chromosome 22 into a region known as the breakpoint cluster region, or Bcr (4,5). Northern blots using Abl probes then demonstrated that a larger Abl messenger RNA (mRNA) was observed in CML samples (6). The description of the infamous fusion protein, termed ‘‘Bcr-Abl,’’ followed, and it was found to be produced by the chimeric mRNA resulting from the 9:22 chromosomal translocation (7,8). The protein associated with v-Abl had already been shown to be a tyrosine kinase with its transforming ability intrinsic to its kinase activity (9,10). Subsequently, the Bcr-Abl fusion product proved to display similar tyrosine kinase activity essential for leukemic transformation in CML (11). Unlike the normal c-Abl gene product, which is
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nuclear and cytoplasmic in location and under tight regulation, the Bcr-Abl fusion proteins are exclusively localized to the cytoplasm and have constitutively increased tyrosine kinase activity (12). Several important experimental works established the ability of Bcr-Abl, as a singular oncogenic abnormality, to cause leukemia. Transgenic mice that express the p190 Bcr-Abl fusion protein were shown to develop a rapidly fatal acute leukemia (13). Transduction of p210 Bcr-Abl into murine hematopoetic stem cells, followed by transplantation into syngeneic mice, causes a CML-like syndrome (14,15). Although convincing, there was and remains controversy about the genesis of CML, particularly surrounding the possibility of clonal stem cell events antecedent to 9:22 translocation and Bcr-Abl fusion, which was championed by Fiaklow and others decades ago (16). More recent work has bolstered the transforming potential of Bcr-Abl by demonstrating that mice expressing a Bcr-Abl transgene, under the control of a tetracycline-repressible promoter, develop a reversible leukemia entirely dependent on the presence or absence of tetracycline (17). BCR-ABL IN PH(1) LEUKEMIAS Using classic karyotyping, the Ph chromosome abnormality can be detected in approximately 90% of patients with CML. An additional 10% of patients will have a ‘‘cryptic’’ translocation and will be detected only by molecular testing for Bcr-Abl [fluorescent in situ hybridization (FISH) and/or quantitative reversetranscriptase polymerase chain reaction (qPCR)]. Different Bcr-Abl fusion proteins are produced, depending on the site of the breakpoint in Bcr: p185 (185kDa), p210 (210kDa), or rarely p230. The different fusion proteins, all variants of Bcr-Abl, segregate to a high degree among the different Ph(þ) leukemias. The p210 protein is seen in 95% of patients with CML and up to 20% of adult patients with de novo acute lymphoblastic leukemia (ALL); the p185 form is seen in approximately 10% of patients with ALL and in the majority of pediatric patients with Ph(þ) ALL (5% of all pediatric ALL cases). If the Ph chromosome is suspected or possible while working up the diagnosis of leukemia, several assays may be used to query for the Bcr-Abl fusion: classical karyotyping, FISH analysis for Bcr-Abl gene rearrangements, qPCR for Bcr-Abl mRNA, or less often immunoblotting for Bcr-Abl protein. While Bcr-Abl itself has proven to be central to CML pathogenesis, work continues to determine the signaling pathways that are activated by Bcr-Abl kinase activity. A number of substrates and binding partners have been identified, and efforts are on to link these pathways to the specific pathologic defects that characterize CML as well as to identify strategies to complement Abl kinase inhibitor therapy and circumvent resistance to such therapy. The pathologic defects identified in CML include a degree of increased cellular proliferation, decreased apoptosis of affected hematopoietic stem or progenitor cells (considered most responsible for the massive increase in myeloid cell burden), and
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defects in adherence of myeloid progenitors to marrow stroma. Candidate pathways that have been identified include the likely role of ras in proliferation, apoptotic protection via STAT-5 upregulation of antiapoptotic Bcl-xL and Akt inactivation of proapoptotic BAD, and Crkl phosphorylation–related decrease in adhesion to fibronectin. Despite the seemingly endless expansion of the list of pathways activated by Bcr-Abl and the increasing complexity that is being revealed in these pathways, all of the transforming functions of Bcr-Abl are dependent on its tyrosine kinase activity and resistance to Abl kinase inhibitor therapy is generally characterized by restoration of kinase activity. Of particular interest is the possibility of Bcr-Abl-‘‘independent’’ resistance to Abl kinase inhibitor therapy in light of the fact that dasatinib, the first approved alternate and more potent Abl kinase inhibitor, is also a potent inhibitor of the related Src family of kinases, whereas nilotinib, also a proven alternate, is simply a more potent Abl kinase inhibitor. Overexpression of the Src-related Lyn kinase (18) has been observed in resistant cell lines and patient samples where Bcr-Abl kinase activity remains inactivated; however, data remain sparse in this area, and further inquiry into kinase inhibitor failure patterns and greater response to dasatinib observed in advanced disease, particularly lymphoid type, is needed to define this escape from Bcr-Abl control. Bcr-Abl is central and well suited as a target for therapy in CML. It is the key ‘‘driver’’ of disease pathogenesis, given its function as a constitutively activated tyrosine kinase, and mutagenic analysis has shown that this activity is essential for the transforming function of the protein. Moreover, it is expressed in the majority of patients with CML. Lastly, a more recent and important observation is that Bcr-Abl retains its central role in the setting of resistance to therapy and disease progression evidenced by a high frequency of Bcr-Abl mutations rather than activation of alternative pathways. An inhibitor of the Bcr-Abl kinase for Ph(þ) leukemias would thus be a rational choice and predicted to be effective and selective therapy. CLINICAL DEVELOPMENT OF IMATINIB, THE FIRST ESTABLISHED BCR-ABL INHIBITOR Phase I studies of imatinib were initiated in 1998 (19) and established several key observations regarding the clinical use of Bcr-Abl inhibitors: first, that no limiting toxicity was observed with daily doses ranging from 25 to 1000 mg; second, that a ‘‘threshold’’ effect was seen, with hematologic and cytogenetic responses increasing noticeably at doses of 300 mg per day and higher; and third and most important, that response to imatinib as salvage for patients with IFN failure was superior to historical reports of frontline therapy with IFN. With more than five years’ follow-up of trials for patients initially studied in ‘‘late chronic phase’’ disease with intolerance or failure during prior IFN, a major cytogenetic response (MCyR) was achieved in 66% and a complete cytogenetic response (CCyR) in 55% (20). Plainly stated, imatinib as second-line therapy had
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superior response and much less toxicity than frontline IFN, making direct comparison to change the gold standard reasonable and necessary. At 60 months, data from the IRIS (International Randomized Trial of IFN/Ara-C vs STI571) trial (21), a landmark trial comparing IFN-based therapy and imatinib in newly diagnosed chronic-phase CML, now demonstrates the following cumulative best response rates with imatinib: 98% complete hematologic remission (CHR), 92% MCyR and 87% CCyR. A more global view of the IRIS imatinib cohort must also include the event-free survival rate (83%) and the retention of patients (69%) after 60 months’ study. In a breakdown of the status of patients from the IRIS trial randomized to imatinib, there is a clear subset with imatinib ‘‘failure,’’ necessitating crossover to IFN or discontinuation for inadequate effect (14%), and imatinib intolerance, necessitating discontinuation for adverse events (4%). An additional 3% of patients exited trial for perusal of stem cell transplant (SCT), implying inadequate response to some degree; the remaining reasons for exiting the study are not therapy or response related and encompass an additional 10%. The fact remains, however, that imatinib is a highly active primary therapy for the overwhelming majority of patients and is a durable option for a similar proportion. The most common toxicity observed with primary imatinib therapy is myelosuppression, with upwards of 17% grades 3/4 neutropenia and 9% grades 3/4 thrombocytopenia; elevated liver enzymes are the most common higher grade nonhematologic toxicity at 5%. Toxicity is generally early in the course of therapy and de novo toxicity occurs rarely after 18 to 24 months of imatinib exposure. Of great interest is the kinetics of imatinib failure in the IRIS cohort, now becoming apparent with longer follow-up. The rate of all progression events, including cytogenetic and hematologic relapse within chronic phase and transformation to advanced phase, is 18% after a median of five years. However, such events appear to be most evident in the first three years of imatinib treatment, where progression to advanced phases of disease averaged 2% per year and progression within chronic phase 5% per year. Year 4 then showed diminution in these rates, and in year 5 both risks are less than 1%; for those patients in CCyR, the rate of progression to advanced CML fell to zero at year 5. Such kinetics suggests an impending plateau in progression free survival, directly contradicting early pessimism surrounding selective targeted therapy with imatinib. Indeed the natural history of CML, particularly for patients in the chronic phase, has been dramatically altered with current therapy and will likely continue to a degree with additional advances forthcoming. High dose imatinib early in disease continues to be studied in comparison with standard dose, with randomized trials ongoing and data forthcoming; further update of previously published single center experience (22) now shows similar ultimate depth of response for both 400 and 800 mg dosing, yet increased rapidity of response and also potentially lower risk of progression for higher dose imatinib. Randomized trials continue to answer the important question about the optimal dose of imatinib to utilize in early chronic phase disease.
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RESISTANCE TO ABL KINASE INHIBITORS Primary resistance to imatinib is defined as an inability to achieve landmark response, whereas secondary resistance defines those who achieve but subsequently lose relevant response. Persistence or re-emergence of Ph(þ) hematopoiesis continues to firmly define clinical resistance; when it occurs despite imatinib or Abl kinase inhibitors as a class, a large proportion of cases (generally in the range of ~50%), have a consistent feature—Bcr-Abl kinase domain mutations. Early investigation of CML cases with relapsing disease queried for potential reasons for the reactivation of the kinase. Critical single amino acid substitutions (mutations) within the Bcr-Abl kinase domain were first identified in patients with advanced disease (23) characterized by rapid clinical relapse and reactivation of the Abl kinase supporting their likely role in imatinib resistance, and widespread study in all phases of Ph(þ) disease followed. The spectrum of Abl kinase domain mutations observed spans the entire kinase domain and >50 mutations have been identified (24). Abl kinase mutations cluster within four main regions of the kinase and are associated with particular numbered amino acid residues (25): ATP-binding loop (p-loop), particularly Y253 and E255 mutants; T315 mutants; M351 mutants; and activation loop (a-loop), particularly H396 mutants. Modeling of imatinib and the novel Abl kinase inhibitors with the crystal structure of the Abl kinase (26) suggests that the effect of mutations is to either disrupt critical drug contact points or induce or favor a conformation of the kinase in which drug binding is reduced or precluded. Termed the ‘‘gatekeeper’’ position, mutations at threonine 315 confer resistance both to imatinib as well as nilotinib and dasatinib and represent a unique challenge. Other mechanisms are likely in patients with wild-type Abl; however, the incidence and role of these mechanisms remains unclear. Bcr-Abl amplification at the genomic or transcript level (27,28) has been described, overexpression of other tyrosine kinases such as the Scr-related Lyn kinase has been observed in the case of Bcr-Abl-independent resistance (18), and variability in the amount and function of the drug-influx protein Oct-1 has been linked to relative insensitivity to kinase inhibition by imatinib (29). CML stem cell resistance may also play a role as progenitors may exchange between a cycling and resting or quiescent (G0) state and vary Bcr-Abl expression, resulting in lack of effect of kinase inhibition. The longstanding phenomenon of cytogenetic clonal evolution remains a cause of resistance and likely represents molecular changes potentially active both in the presence or absence of kinase domain mutations. The search for kinase domain mutations should generally be driven by recognition of clinical resistance only. While screening of patients in all phases of CML before imatinib exposure has demonstrated increased likelihood of mutation detection with a more advanced phase of the disease, and correlation has been demonstrated between mutations identified in the setting of clinical resistance with retrospective analysis of pretherapeutic samples, identification of pretherapy high level imatinib resistant mutations did not consistently predict for
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imatinib insensitivity (30). Kinase domain mutations may thus represent a natural element of CML disease progression that may be highlighted by selection pressure with the use of potent Abl kinase inhibitors. RECOGNITION OF CLINICAL RESISTANCE IN CML NCCN guidelines (31) and a recent European LeukemiaNet consensus paper (32) have described target responses at landmark time points and allow triage of patients into categories of failure, suboptimal, and optimal response based on likelihood risk of relapse or progression. CHR by the three-month mark of therapy is considered the minimum initial response and lack of such response as failure. Failure to achieve any reduction in Ph(þ) cells [>95% Ph(þ)] by cytogenetic testing after six months of imatinib and failure to achieve MCyR after 12 months of imatinib therapy suggest a small (<20%) chance of subsequent CCyR and are also defined as failure. Response beyond these minimums, specifically CCyR achieved by 12 months, offers further risk reduction and defines optimal response. Despite the variability and ongoing need for standardization of qPCR, it is clear that one threshold level of Bcr-Abl transcript reduction, agreed upon to be a 3-log or greater reduction below standard baseline (a major molecular response) targeted to occur in the first 12 to 18 months of imatinib therapy in the setting of CCyR, confers maximal protection from progression to advanced disease (projected transformation free survival 100%) and the lowest rate of any disease progression with five years’ follow-up (21). Overall, the inability to achieve hematologic and cytogenetic response by predetermined time points as well as loss of such responses generally constitutes imatinib failure, as does the inability to maintain therapy at the target dose as a result of either severe or chronic moderate toxicity. Inadequate molecular response has been defined and is associated with more subtle risk of progressive disease; molecular relapse remains controversial, yet may herald cytogenetic relapse or predict for detection of kinase mutations and warrants further clarification. CLINICAL DEVELOPMENT AND RESULTS WITH SECOND-GENERATION KINASE INHIBITORS Clinical development of second-generation Abl kinase inhibitors began with phase I studies of dasatinib (formerly known as BMS354825) (33), in late 2004, and nilotinib (formerly AMN107) (34), approximately six months later, for all phases of CML and Phþ ALL. Subjects included those with ‘‘imatinib resistant’’ chronic phase disease (n ¼ 40 for dasatinib, n ¼ 17 for nilotinib), with slightly different entry criteria (allowance for imatinib intolerant patients in the dasatinib study and patients with cytogenetic resistance only in the nilotinib trial). In chronic phase CML the rate of CHR was identical at 92% for both, as was CCyR at 35%. MCyR rates were 45% for dasatinib and 35% for nilotinib, as an additional 10% of patients on dasatinib achieved partial cytogenetic response.
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No dose limiting toxicity was observed for dasatinib, with a range of 15–240 mg per day administered; for nilotinib, dosing at 600 mg twice daily (BID) was limited by liver (grade 3 indirect bilirubin and transaminase) and pancreatic enzyme elevations (including grade 2 pancreatitis), as well as one grade 3 subdural hematoma. Rash, pruritus, and fatigue were also reported as higher grade adverse events on nilotinib. Results of monitoring for electrocardiographic changes from nilotinib were reported and a 5 to 15 millisecond increase in the corrected QT (QTcF) was noted. Fifteen of eighty-four dasatinib-treated patients overall (13% grades 3/4 in the myeloid blast crisis cohort) had pleural effusions deemed ‘‘therapy related’’ and were treated with diuretics and/or drainage. Other higher grade toxicity from dasatinib included gastrointestinal hemorrhage, diarrhea, and elevated transaminase levels; more frequent moderate toxicities included edema, headache, and hypocalcemia. Myelosuppression greater than that reported with imatinib was noted with both agents, and it was most pronounced with dasatinib. Overall 70% of patients treated with dasatinib and 41% with nilotinib had Abl kinase mutations prior to therapy. In both studies, presence of the specific mutation T315I before therapy precluded any response and was a common finding at relapse; those with other mutations responded, as did patients without mutations. On the basis of the convincing efficacy and acceptable toxicity observed in phase I studies, phase II studies for both agents quickly followed. As in phase I, the target population included patients with both imatinib resistance as well as intolerance. Resistance was defined as failure to achieve CHR after 3 months, cytogenetic response after 6 months, MCyR after 12 months, or loss of a hematologic or cytogenetic response at any time during treatment with imatinib at doses of 600 mg or higher in both trials; additionally, patients on less than 600 mg imatinib with known Abl kinase mutations conferring a high degree of imatinib resistance were permitted on dasatinib trials. Imatinib intolerance was defined for dasatinib and nilotinib trials as grade 3 or higher nonhematologic toxicity or persistent (>7 days) grade 4 hematologic toxicity; for the nilotinib phase II trial, patients with grade 2 nonhematologic toxicity that was recurrent (>3 occurrences) or persistent (>1 month) were also eligible, and all patients deemed intolerant to imatinib in the nilotinib trial were without MCyR at trial entry (thus also resistant). In the ‘‘START-C’’ trial (SRC/ABL Tyrosine kinase inhibition Activity Research Trials of dasatinib in Chronic phase CML) of dasatinib 70 mg BID for imatinib resistant or intolerant chronic phase CML, follow-up data is now available after 15 plus months median therapy in 387 patients (35,36). In this trial, 75% of cases were imatinib resistant and the remainder intolerant. Focusing on the imatinib resistant population (n ¼ 288), 72% had high-dose imatinib (>600 mg) exposure, best response on prior imatinib was MCyR or CCyR in 52%, and mutations in the Abl kinase were found in 52% of cases. Overall CHR was 91%; this included maintenance of response rather than recapturing response in a proportion of patients. Overall cytogenetic response was major in 59% and
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complete in 49%; in the imatinib resistant subset, response was slightly lower at 52% MCyR and 40% CCyR. Major molecular responses (3-log or greater reduction in Bcr-Abl) in the resistant population of 13% at 6 months and 15% at 12 months have been reported (37), as well as a progression free survival of approximately 90% at latest follow-up. Cumulative data from phase II studies of nilotinib in chronic-phase CML (n ¼ 318) continues to be updated (38,39) and the results compared. The proportion of patients with intolerance (31%) was similar, but again in the nilotinib trial these cases also demonstrated cytogenetic resistance. Seventy-three percent of patients either resistant or intolerant had exposure to 600 mg imatinib or higher (vs. 55% of the same group in the dasatinib trial). Of patients lacking CHR at study entry (n ¼ 185), 74% achieved this response on nilotinib. Overall cytogenetic responses in patients with a median of at least six months of therapy (n ¼ 280) were 48% major and 31% complete; similar responses in ‘‘intolerant’’ and ‘‘resistant’’ subsets were seen as expected, given that both groups had documented resistance. Nine percent of complete and 13% of partial cytogenetic responses were based on FISH data rather than metaphase cytogenetics (karyotype), the accepted standard in CML trials. The MCyR rate reported is 41% if partial (1–35%) responses based on FISH analysis, potentially discordant with karyotype, are not included. Of patients achieving major cytogenetic response, 4% progressed or died with six months’ follow-up; an additional 12% lost major response but continued on study. These data are consistent with previously reported progression free survival estimates for nilotinib, which appear somewhat lower than with dasatinib. Building on the experience from phase I studies, toxicity observed in phase II studies of dasatinib (35–37) and nilotinib (38,39) differed, as both agents were positioned to be options not only for imatinib failure but also for imatinib intolerance; ‘‘cross-tolerance’’ from one agent to another was studied as well. Fluid retention and myelosuppression continued to be common toxicities with dasatinib therapy; grades 3/4 neutropenia and thrombocytopenia occurred in nearly one half of treated patients and grades 3/4 fluid retention in approximately 10%, with pleural effusions (all grades) occurring in one-quarter of patients. Other higher grade toxicities with dasatinib in phase II included headache and diarrhea. Nilotinib was associated with less myelosuppression, 25% to 30% grades 3/4 neutropenia and thrombocytopenia, and less than 1% of patients experienced higher grade fluid retention. Grades 3/4 hypophosphatemia and hyperglycemia were observed with nilotinib in 11% of patients and lipase elevation in 15%, but less than 1% with associated pancreatitis. Grades 3/4 elevations in bilirubin levels occurred in 8% and other higher grade toxicities included diarrhea, rash, and arthralgias/myalgias. Evaluation for cardiovascular toxicity revealed a minimal (5 millisecond average) effect on the QTc and no apparent increase in ischemic, arrhythmic, or ventricular dysfunction events. Overall, each agent has new observed toxicities warranting monitoring, and at 70 mg BID dosing of dasatinib, there is a significantly greater need for providers to interrupt and modify dose. In the START-C trial, more than 85% of
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patients’ therapy was interrupted and approximately 75% of patients had dose reduction; the actual delivered dose of dasatinib was a median of 100 mg/day. Two other important studies using dasatinib in chronic-phase include a comparison to high-dose imatinib [the ‘‘START-R’’ trial (40)] and a dose optimization trial. The Start R trial randomized patients (n ¼ 150) with resistance to imatinib at doses of 400 to 600 mg, with resistance defined the same as the single arm phase II trials, to high dose imatinib (800 mg target; n ¼ 49) versus a switch to dasatinib 70 mg BID (n ¼ 101). With follow-up now out to a median of 15 months, the dasatinib arm is more favorable, but some caveats bear explanation. The primary endpoint of MCyR three months after randomization was improved with dasatinib over high dose imatinib (36% vs. 29%) but was a trend only; among responding patients however, there was a significant ( p ¼ 0.04) increase in CCyR for dasatinib (22% vs. 8% after 3 months). After a median of 15 months’ follow-up, the gap has widened, and overall MCyR (53% vs. 33%) and CCyR (40% vs. 16%) favor switch to dasatinib. Important subset analysis sorted patients at entry by imatinib dose (400 or 600 mg) and demonstrated that the cytogenetic response advantage of dasatinib was evident mainly in patients entering study on 600 mg imatinib and for whom intervention was a modest increase (200 mg) only; doubling the imatinib dose (400 mg ? 800 mg) overall produced cytogenetic response rates close to that of switching to dasatinib. Perhaps the most relevant advantage to dasatinib is increased durability of response; across all subsets of patients, time to treatment failure and progression free survival were significantly better and support the consideration and use of dasatinib over high dose imatinib in this setting. Dose optimization studies for dasatinib in chronic-phase CML aimed at exploring a modified dose (100 mg) on the basis of the difficulty in delivering 140 mg/day dosing in earlier trials and schedule [once daily (QD) vs. BID], given that responses were schedule-independent in phase I despite the shorter half-life (~4 hours) of dasatinib. A four-arm trial (n ¼ 662 total) for chronic-phase CML (41) distributed evenly doses of 100 mg QD, 50 mg BID, 140 mg QD and 70 mg BID between patients who had demonstrated imatinib resistance [now defined more broadly to include inadequate response on standard (400 mg) imatinib], patients with Abl kinase mutations despite major cytogenetic response, and patients with molecular and low-level cytogenetic relapse. Recent follow-up from this study showed continued equality between the four arms with regard to hematologic, cytogenetic, and molecular response. Response rates were more favorable than the START-C experience likely because of the inclusion of patients with earlier identification of resistance. Comparison of the 100 mg QD arm to all others showed statistically significant reduction in grades 3/4 thrombocytopenia and congestive heart failure events, significant reduction in the need to interrupt, reduce, or discontinue therapy because of toxicity, and superior progression free survival rates. On the basis of this study, the FDA recently approved a change in the recommended dose of dasatinib to 100 mg QD. A summary of the key features and differences between the three currently approved Abl kinase inhibitors is shown in Table 1.
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Table 1 Comparison of Imatinib, Dasatinib, and Nilotinib Imatinib
Dasatinib
Nilotinib
Kinase targets
Abl, Kit, PDGFR
Abl, Kit, PDGFR, Src family (Src, Lyn, Hck, Lck, Yes, Fyn)
Abl, Kit, PDGFR
Potency vs. nonmutated Abl (IC50, nM)
630
<1
25
FDA-approved indications
CP, AP, BC CML, Ph(þ) ALL, newly diagnosed CP CML
CP, AP, BC CML, Ph(þ) ALL with resistance or intolerance to prior therapy, including imatinib
CP, AP CML with resistance or intolerance to prior therapy, including imatinib
Initial daily dosing
400 mg QD (CP) 600 mg QD (AP/BC/Ph(þ) ALL)
100 mg QD (CP) 70 mg BID (AP/ BC/Ph(þ) ALL)
400 mg BID, (CP/AP)
Activity against selected kinase mutations
Gatekeeper (315): none P-loop (248-255): minimal/none
Gatekeeper (315): none P-loop (248-255): activity
Gatekeeper (315): none P-loop (248-255): diminished activity?
Abbreviations: PDGFR, platelet derived growth factor receptor; CP, chronic phase; AP, accelerated phase; BC, blast crisis; CML chronic myeloid leukemia; Phþ ALL, Philadelphia chromosome positive acute lymphoblastic leukemia; FDA, United States Food and Drug Administration; P-loop, phosphate binding loop of Bcr-Abl; QD, once daily; BID, twice daily.
OBSERVATIONS REGARDING ABL KINASE DOMAIN MUTATIONS FROM TRIAL EXPERIENCE At the time of their preclinical evaluation, both dasatinib and nilotinib were able to inhibit CML cells harboring the spectrum of Abl kinase mutations known, except for position 315, considered the gatekeeper position of the kinase domain drug binding pocket. Drug concentrations required to inhibit such clones (except for T315I mutants) were uniformly low with dasatinib and varied but were well within the achievable plasma level of nilotinib. Patient populations on dasatinib and nilotinib trials have uniformly consisted of a split close to 50% each of patients with and without documented Abl kinase domain mutations prior to salvage therapy. Clinical trials to date have borne results mostly concordant with expectations from preclinical data regarding activity. In the START-C trial (36) with dasatinib in chronic-phase CML, hematologic and cytogenetic responses
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were uniform in cohorts with and without kinase mutations, and, more importantly, there was no discernable difference in response among those with kinase mutations conferring modest versus high level resistance to imatinib. In the phase II trial of nilotinib in chronic phase CML (39), response was not limited to certain types of mutations but was affected and relative to the in vitro sensitivity of the mutation type. Across three groups of mutations with low, intermediate, and high IC50 values, hematologic response was 70%, 50%, and 18% and MCyR was 53%, 43%, and 15%, respectively. Mutations in the ‘‘high IC50’’ group include those with substitution in positions 252 through 255, known as the p-loop, and may be a cluster of mutations like T315I that may need special consideration when identified before or during the selection of salvage therapy agents. Antecedent identification of a T315I mutation precluded hematologic or cytogenetic response to both agents. CURRENT STATUS OF TREATMENT FOR ADVANCED PHASE Ph(1) LEUKEMIAS The role of Bcr-Abl remains central to all phases of the Ph(þ) leukemias, but the events surrounding progression of chronic-phase CML and the genesis of de novo Ph(þ) ALL, which may contribute to the difficulty in treating these conditions, remain to be defined. Clonal cytogenetic evolution—discrete karyotype changes creating subclones of the Ph(þ) clone, with examples being (þ)8, iso 17q, and duplication of the Ph chromosome—is historically and at present still common in advanced phase Ph(þ) leukemias. The presence of kinase domain mutations has been confirmed in advanced disease before Abl kinase inhibitor exposure (30), and the likelihood of kinase mutations appearing during therapy and precluding response increases with the phase of the disease. Inquiry into the role of other kinases in the progression of the disease or facilitating resistance has included identification of targets of current therapeutics, namely the Src family of kinases Lyn and Hck in particular (18). A number of ‘‘dual’’ inhibitors exist that are active against Abl and Src kinases, with the prototype being dasatinib and others, including SKI-606 and INNO-406, under active development. While Bcr-Abl-independent proliferation based in Src activation has been demonstrated and enhanced activity observed in lymphoid subtype blast crisis and Ph(þ) ALL for the dual inhibitor dasatinib, the role of these targets remains unclear, and more studies are needed to clarify the potential problem of Bcr-Ablindependent Ph(þ) transformation and proliferation, which is a troubling concept for the specific Abl inhibitors, imatinib and nilotinib. The activity of second-generation Abl inhibitors in advanced disease is more limited than in the chronic phase, and the certainty of stable response remains doubtful, much along the same lines as the early experience with imatinib as a single agent in advanced disease. The use of imatinib in combination with the lymphoid induction program of HyperCVAD has become standard for Ph(þ) ALL on the basis of improvements in response and, in particular,
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reduction in MRD; dasatinib combined with HyperCVAD is under active investigation to examine for further gains. Dasatinib is approved for use in the myeloid and lymphoid blast phase of CML (42), in addition to chronic and accelerated phase disease, while nilotinib has gained approval for chronic and accelerated phase CML. Major hematologic responses for myeloid and lymphoid blast phase CML for dasatinib are 30% to 35%, and for Ph(þ) ALL 42%; major cytogenetic responses are 30%, 50%, and 58%, respectively, reflecting greater activity in the lymphoid subtypes. The spectrum of kinase domain mutations observed in advanced disease, at the time of transformation and particularly after exposure to salvage therapy, strongly favors those in the p-loop and position 315 (43). This may be due to increased clonal instability and proliferation, and more rapid selection of the highly drug resistant clones; however, given the fact that transforming potential and degree of kinase inhibitor drug resistance appear to be ‘‘uncoupled,’’ as seen with the T315I mutation (44) (which allows for greatest drug resistance but no growth advantage over wild type Bcr-Abl), other factors likely contribute beyond observed Abl kinase mutations. Kinase inhibitors certainly have a role in the treatment of Ph(þ) leukemias in advanced stages, but are, as of now, utilized best in combination with other therapies, including co-administration with induction chemotherapy programs in Ph(þ) ALL and as a lower risk means of achieving a second chronic phase or remission prior to allogeniec SCT. WHAT IS THE CURRENT ROLE OF STEM CELL TRANSPLANT? In the period following imatinib’s approval and with broader application in Ph(þ) leukemias, the number of SCTs in these populations dropped dramatically. Questions that have arisen as allografting has begun repositioning into treatment algorithms include the possibility of the negative impact of antecedent imatinib (or other kinase inhibitor) on transplant related morbidity and mortality, the risk of ‘‘delaying’’ transplant while pursuing primary or salvage therapy with kinase inhibitors, and any impaired capacity of the graft versus leukemia (GVL) effect to manage Ph(þ) leukemia harboring kinase domain mutations rather than wildtype Bcr-Abl. To date, as no negative impact has been shown for patients proceeding to transplant after imatinib exposure (45), it is likely that historically prognostic factors remain most relevant, primarily the phase of disease and kinetics of growth of the malignant clone (e.g., degree of disease control). Immediate transplant even in the presence of a known sibling donor has been de-emphasized on the basis of a genetic randomization study [primary transplant, if sibling donor available, vs. nontransplant therapy (IFN and subsequently imatinib), if no sibling donor] showing an eight year or longer period of favorable survival advantage for nontransplant therapy (46). Finally, the first reports of patient outcome for allografting after exposure to multiple kinase inhibitors and in the presence of highly resistant Abl kinase domain mutations are emerging (47); larger numbers of patients would require if there is any
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discrimination of the GVL effect between Ph(þ) leukemia harboring kinase mutations or not. It is thus often the case that allografting should be discussed and options delineated upon diagnosis in patients for whom transplant is feasible, as the donor options and transplant morbidity and mortality estimate will clearly frame the tolerability for a less than ideal response or intolerance of kinase inhibitor therapy, which is generally offered as primary therapy throughout the developed world. Younger patients with high risk disease and low risk transplant options (sibling donors) in whom kinase inhibitor or other non-transplant therapy does not offer deep and protective remissions remain as excellent candidates to proceed to allografting until more is known about the long term stability of salvage approaches such as the second generation kinase inhibitors. Second remissions gained after emergence of unstable disease (advanced phase disease harboring high level resistant Abl kinase mutations) are also increasingly recognized as opportunities to defer long-term disease control to the strength of GVL through transplant rather than relying on the uncertainty of such disease and limited follow-up of salvage approaches. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS The pace of progress may slow regarding Bcr-Abl targeting and optimization of Abl kinase inhibitors over the next five years, as focus shifts to more difficult questions and needs (Table 2). For the bulk of patients, primary imatinib therapy has elicited cytogenetic response and an increasing likelihood of ‘‘Bcr-Abl transcript undetectable’’ status; however, the gaps are clearly identified and BcrAbl, as the key driver of Ph(þ) leukemias, also lies at the heart of resistance with kinase domain mutations playing the most significant role. The long term prospects appear excellent for ongoing Bcr-Abl kinase inhibition, maintaining remission for chronic phase disease; however, there will be constant query into the risk and feasibility of indefinite use of compounds like imatinib and other Abl inhibitors and vigil for new concerning side effects. The long term prospect for Ph(þ) leukemia that has developed resistance is much more uncertain; it may be so that genetic instability may permit continued genesis of evolved kinase domain mutated subclones able to proliferate despite use of sequential inhibitors. The fact that the gatekeeper position mutations (315/317) appear more often in advanced disease and may be increasingly prevalent among cases of dasatinib and nilotinib resistance is proof of such disease ‘‘evolution’’ and represents a new challenge. As well, continued investigation into the means by which the other 50% of patients with clinical resistance (those without kinase domain mutations) evolve may yield other drug targets and complete the puzzle of Ph(þ) leukemia. From the identification of the Ph(þ) translocation t(9:22) forward, the pace of development has been increasingly rapid and the story of Bcr-Abl as a targetable kinase in a human cancer is a tale of rational scientific success and is hoped to be a paradigm for unraveling the basis of many other or all human cancers.
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Table 2 Key Issues in CML in 2008 and Beyond Upfront therapy
High dose imatinib, novel inhibitors still under study; expectation of increased rapidity and depth of response, ongoing evaluation of risk vs. benefit
Role of SCT
Evaluation of potential transplant candidates useful at diagnosis to identify options and frame tolerance for toxicity and suboptimal response; phase of disease and kinetics of proliferation likely most relevant to window of opportunity rather than basis of kinase inhibitor resistance (kinase domain mutations)
Monitoring response
Initial and early monitoring based on metaphase cytogenetics from bone marrow testing; standardization of molecular response reporting (qPCR) for minimal residual disease crucial, IS proposed and implementation forthcoming
Minimal residual disease
Elimination to approximate or effect ‘‘cure’’ elusive; therapy interruption or discontinuation associated with relapse; role of immunomodulatory therapy under investigation (IFN revisited, vaccine strategies)
Salvage therapy
Options expanding with two well studied agents effective with acceptable toxicity; dose optimization ongoing, development of additional alternatives aimed at minimizing toxicity and address gaps such as selective mutations (T315I)
Abbreviations: SCT, stem cell transplant; qPCR, quantitative reverse-transcriptase polymerase chain reaction; IS, international scale; IFN, interferon.
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18 Tyrosine Kinase Inhibitors: Targets Other Than FLT3, BCR-ABL, and c-KIT Suzanne R. Hayman Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, U.S.A.
Judith E. Karp Division of Hematologic Malignancies, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, U.S.A.
INTRODUCTION The development of targeted therapies to disrupt cancer cell signaling is a burgeoning area of experimental therapeutics for both solid tumors and hematologic malignancies. Cell signaling pathways are tightly regulated by the transfer of phosphate from adenosine triphosphate (ATP) to tyrosine residues on substrate proteins in reactions catalyzed by tyrosine kinase (TK) enzymes. Abnormal activation of such TKs by gene mutation or aberrant expression, with the resultant dysregulation of intracellular pathways, has been implicated in oncogenesis. The original drug paradigm that employs the targeted inhibition of an abnormal TK (Bcr-Abl) is that of imatinib mesylate (STI571) in chronic myelogenous leukemia (CML). The development of other TK inhibitors for use in acute leukemias, such as those targeting the overexpression of c-KIT and mutant FLT-3, are discussed in detail in previous chapters. The purpose of this chapter will be to discuss selected agents that are designed to target and modulate the activities of TKs other than Bcr-Abl, FLT-3,
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and c-KIT. We will give particular attention to TK inhibitors that are currently being assessed in clinical trials, namely agents that target heat shock proteins (Hsps) and vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFR), either through direct receptor TK inhibition, inhibition of the VEGF ligand, or downstream targets such as Raf kinase, including the Raf kinase inhibitor BAY 43-9006 (sorafenib). We will also make note of the dual Src/Abl inhibitors that are emerging as major treatment options for imatinib-resistant CML. HEAT SHOCK PROTEINS A cell’s response to injury or environmental stresses such as heat, inflammation, chemotherapy, or the generation of reactive oxygen species (ROS) may result in cell death, either by apoptosis or necrosis. However, if the level of stress is relatively low, the cell may attempt to survive through the initiation of the heat shock or stress response. This is manifested by the suppression of most new protein synthesis, except for the upregulation of gene expression of Hsps. There are six major families of Hsps grouped according to their approximate weights in kDa (Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small Hsps 10–28 kDa). Hsps and their homologs may be found in multiple intracellular compartments including the nucleus, cytosol, mitochondria, and endoplasmic reticulum. Most Hsps are both constitutively expressed and inducible, with increased gene expression resulting from the binding of heat shock transcription factors , particularly heat shock factor (HSF)-1, to heat shock elements located variably upstream from transcription initiation sites. The stress stimulus responsible for this transcriptional activation is thought to be direct protein damage, including oxidative damage by ROS (1). Hsps minimize proteotoxic damage by functioning as molecular chaperones, thereby interdicting the abnormal protein folding and aggregation that may result during heat shock–related changes in redox states and hydration of injured cells. Within this broad class of proteins, there is considerable variability among the various Hsp families and their members with regard to protein targets, subcellular locations, interactions with cochaperones and specific cellular functions. Thus, modulation of apoptosis by Hsps may occur as a result of their chaperone activities, but genes encoding for Hsps are also transcriptionally regulated by the processes of cell differentiation, proliferation, and the cell cycle, consistent with Hsp influence on apoptosis related to but distinct from the stress response (2). For example, while Hsp90 prevents aggregation of denatured proteins (3), its central function is modulation of factors and proteins that regulate multiple signal transduction pathways involved in cell survival. On the other hand, Hsp70 and Hsp27 exert major antiapoptotic effects through mitochondrial interactions. Whatever the specific protein targets, the heat shock protective response against cell injury is evolutionarily conserved, with transient Hsp elevation linked to thermotolerance and, in turn, the cell becoming relatively
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refractory to further injury. In addition, the Hsp ubiquitin targets damaged proteins for degradation by the 26S proteosome. The constitutive expression of Hsps, their roles in abrogating the apoptotic response to cell stress, and their transcriptional modulation by cell cycle events suggest a potential role for Hsps in chemoresistance and oncogenesis. A connection between overexpression of Hsps and tumorigenesis has been recognized, with aberrant expression of Hsps reported in multiple tumor types, although the underlying mechanism(s) is unknown. It has been postulated that abnormally expressed Hsps may facilitate malignant transformation by stabilizing the aberrant protein conformations and/or signaling activities of constitutively activated oncogenic proteins or mutated tumor suppressor proteins. Hsp90 Hsp90 is an ATP-dependent molecular chaperone that is localized primarily in the cytosol, has homologs in the endoplasmic reticulum and mitochondria, and acts as the cornerstone of a heteroprotein complex that exists in both active and quiescent states depending upon ATP hydrolysis and ADP/ATP nucleotide exchange (4). Hsp90 client proteins initially interact with an Hsp70/Hsp40 complex that is linked subsequently to the ADP-bound conformation of Hsp90 by p60HOP. The replacement of Hsp90 ADP by ATP changes the Hsp90 conformation, which results in the release of p60HOP and Hsp70/Hsp40 and allows for recruitment of other cochaperones including p23, certain immunophilins, or p50Cdc37 (5). This ATP-Hsp90 conformation allows for the appropriate folding and stabilization of client proteins. This chaperone complex associates with and participates in the folding, conformational maturation, and regulation of multiple client signal transduction proteins, either by protecting them from or targeting them for ubiquitination and proteosomal degradation on the basis of the nucleotide conformation within the Hsp90 amino terminus ATP/ADP binding site (6). Overexpression of Hsp90 and its homologs has been found in multiple malignancies including gastrointestinal cancers, breast carcinoma, prostate carcinoma, and acute leukemias, with Hsp90 constitutively expressed 2–10 times higher in tumor cells compared with their normal tissue counterparts (7). Tumor cell Hsp90 is present in multichaperone complexes with increased ATPase activity, while Hsp90 from normal tissue exists in an uncomplexed, inactive state with low ATPase activity (8). Multiple oncogenic signal transduction proteins have been identified as Hsp90 clients and include PKB/Akt (9), ErbB2 (10–12), Raf-1 kinase (13), steroid hormone receptors (14–16), v-Src (17), cyclin-dependent kinases (Cdks) 4 and 6 (18), hypoxia-inducible factor 1a (HIF-1) (19), mutated or chimeric proteins mutant p53 (20) and Bcr-Abl (21,22), respectively, and the cell checkpoint signaling protein Chk1 (23). Correspondingly, depletion of these client proteins through Hsp90 inhibition has been found to correlate with cellular antiproliferative activity (24). There is also an increasing body of evidence connecting Hsp90 to the
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regulation of angiogenesis through its association with HIF-1 (25) and the endothelial nitric oxide synthase (eNOS) complex (26). Hsp90 Inhibitors Benzoquinone Ansamycins The benzoquinone ansamycins (BA) have been identified as Hsp90 inhibitors. While this antibiotic family was identified initially as a group of nonspecific TK inhibitors, Whitesell et al. (17) reported that BA treatment of v-Src-mediated transformed cells resulted in the reversion of transformation without direct inhibition of Src phosphorylating activity. BAs were found to inhibit the formation of a Hsp90-Src heterocomplex through the direct inhibition of Hsp90 (17). The activity of the BAs is exerted through their binding within the hydrophobic ATP/ADP site in the amino terminus of Hsp90, thereby replacing the nucleotide at the binding site with much greater affinity than either ADP or ATP and mimicking the ADP-bound Hsp90 heteroprotein complex that favors client protein degradation, with associated reductions in client protein halflives (5,27). Geldanamycin (GA) is considered the BA prototype. The significant hepatotoxicity of GA in preclinical toxicity trials resulted in the development of its analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in which the allyl amino group is replaced by the methoxy at position 17. Hsp90 inhibitors have shown tumor cell Hsp90 selectivity, with tumor cell Hsp90 having a 100fold greater binding affinity for 17-AAG than Hsp90 from normal cells (8). The metabolism of 17-AAG has been studied in vitro in mouse and human hepatic preparations (28), with metabolic activity primarily in the microsomal fraction. Three metabolites have been identified and compatible with 17-(amino)-17demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The microsomal metabolism of 17-AAG in humans is inhibited by ketoconazole, suggesting that the responsible P450 cytochrome isoform is 3A4. 17AG is not metabolized by microsomes and is similar to 17-AAG in terms of decreasing p185erbB2. Additional GA analogs are under preclinical and clinical development. One such analog is 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG; NSC 707545), a hydrophilic BA, that is orally bioavailable and more potent than 17-AAG (29). Initial pharmacokinetic studies in CD2F1 mice and Fischer 344 rats demonstrated good oral bioavailability (50%) and a wide tissue distribution (30). The water solubility of DMAG circumvents the complex vehicles required for administration of 17-AAG. Radicicol Radicicol is a macrocyclic antifungal antibiotic, which is chemically distinct from the BA antibiotic family, but nonetheless targets the ATP/ADP-binding site in the amino terminus of Hsp90, with resultant inhibition of ATPase activity and
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degradation of Hsp90 client proteins (31). While radicicol has a 50-fold greater binding affinity than GA, by itself it has almost no therapeutic activity in animals because of its instability, perhaps due to epoxy and unsaturated carbonyl groups in its chemical structure (32). However, radicicol oxime derivatives, particularly KF25706 and KF58333, have shown potent antitumor activity in vivo against several human tumor xenograft models, including breast, colon, and epidermoid carcinoma when administered intravenously (31). In addition, KF58333 was found to inhibit VEGF secretion, which was accompanied by a decrease in VEGF mRNA expression in breast cancer cell lines and a decrease in angiogenesis in nude mouse xenografts (31). Novobiocin and Related Coumarin Antibiotics Novobiocin and structurally related compounds, chlorobiocin and coumermycin A1, are being evaluated as alternative Hsp90 inhibitors. Unlike the BA and radicicol, the mechanism of action of these coumarin antibiotics does not appear to relate to binding within the hydrophobic ATP/ADP site in the amino terminus of Hsp90 but rather to an interaction with a previously unrecognized domain in the carboxy-terminus of Hsp90 (33). Novobiocin blocks the ability of Hsp90 to complex with either p23 or p70 (32). Marcu et al. demonstrated reductions in the Hsp90-dependent signaling proteins, mutated p53, p185erbB2, and Raf-1 in a dose-dependent fashion using novobiocin in SKBR3 breast cancer cells, with maximum activity occurring at 500 to 800 mM (33). The p60v-src protein was also reduced in v-Src-transformed NIH 3T3 cells after exposure to 600 mM novobiocin for 16 hours (33). The depletion of these oncogenic proteins appears to be independent of the known topoisomerase II inhibition of the coumarins, since treatment with the topoisomerase II inhibitors, etoposide and doxorubicin, did not result in reduced levels of p185erbB2 (33).
Molecular Effects of Hsp90 Inhibitors By virtue of abrogating the protective function of Hsp90 on diverse oncogenic signaling proteins in response to DNA damage of tumor cells, blockade of Hsp90 function may sensitize tumor cells to the cytotoxic effects of traditional chemotherapeutic agents. Cell sensitivity to such therapy has been found to be tumor-specific and cell cycle–dependent, at least in part based on the observation that Hsp90 inhibitors induce G1 and/or G2/M cycle arrest. As a case in point, Hsp90 inhibition was shown to result in retinoblastoma (Rb)-dependent G1 arrest in breast and colon cancer lines. This G1 arrest has been attributed in part to downregulation of cyclin D mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt pathway (34–36). Munster et al. further demonstrated that the Rb-dependent G1 arrest was accompanied by breast cell differentiation and apoptosis, neither of which occurred during treatment of cell lines lacking Rb (37). This Rb-dependent G1 arrest, however, was not found when 17-AAG
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administration was evaluated in several colon carcinoma cell lines, with G2/M arrest noted in three of four cell lines (38). Munster et al. further examined the combination of 17-AAG plus either paclitaxel or doxorubicin on breast cancer cell lines (38). In those cell lines with intact Rb, the addition of 17-AAG after exposure to paclitaxel resulted in a significant increase in apoptosis, while 17-AAG given prior to the taxane resulted in G1 arrest and the abrogation of apoptosis. Schedule dependence was not seen in cell lines with mutated Rb because both agents blocked cells in mitosis. While schedule dependence was also not seen with doxorubicin, the combination of 17-AAG and doxorubicin exhibited synergistic apoptosis. Schedule dependence was also found in multiple non–small cell lung carcinoma cell lines treated with 17-AAG and paclitaxel, particularly those overexpressing p185erbB2 (39). Cells incubated with 17-AAG prior to exposure to the combination of 17-AAG and paclitaxel resulted in refractoriness to paclitaxel toxicity, while exposure to 17-AAG after being incubated with the two agent combination resulted in enhanced cytotoxicity (39). Nimanapalli et al. (40) reported decreased levels of multiple Hsp90 client proteins, including Akt, c-Raf-1, and c-Src in the human leukemia cell line HL-60, when cells were treated with 17-AAG. Correspondingly, there was cytosolic accumulation of cytochrome c, Smac/DIABLO, caspases 9 and 3, and an associated increase in apoptosis. Nonetheless, Bcl-2 and Bcl-XL can overcome the apoptotic effects of 17-AAG, suggesting that the mitochondrial apoptotic pathway is regulated by proteins in the Bcl-2 pathway independent of the Hsp90-regulated signal transduction proteins (40). Hsp90 Inhibitors in Leukemias The ability to block the prosurvival activities of Hsp90 is a provocative concept that is being tested clinically. In this regard, the ability of 17-AAG to impede the repair of cytarabine (ara-C) damaged DNA is of special interest in acute leukemia. Since its introduction in the 1960s, the antimetabolite ara-C remains the single most effective agent for acute myeloid leukemia (AML). A number of mechanisms of resistance to ara-C have been identified, including the decreased incorporation of ara-CTP into DNA as a consequence of diminished passage of cells through S phase (41), and activation of the checkpoint kinase and Hsp90 client protein Chk1 with subsequent repair of ara-C damaged DNA. The net activity of Chk1 is downregulated by 17-AAG, which facilitates Chk1 turnover. 17-AAG has been shown to enhance the cytotoxicity of ara-C in acute leukemia cell lines by diminishing the S phase slowing observed after ara-C treatment. On the basis of these initial findings, the effects of 17-AAG and 17-AG on Chk1 levels and cellular responses to ara-C in AML cell lines and fresh human AML marrow populations were examined (42). Cells were first arrested in S phase after treatment with ara-C alone for 24 hours, followed by the addition of 17-AAG. Depletion of Chk1 was noted with enhancement of ara-C cytotoxicity after treatment with 17-AAG, with significantly more cells undergoing apoptosis
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when ara-C was present during 17-AAG exposure compared with when ara-C was removed. These preliminary data suggest that 17-AAG may reverse checkpoint-mediated ara-C resistance in AML. In addition, this study reported that 17-AG was more potent than 17-AAG in downregulating Hsp90 client proteins, suggesting that both levels need to be taken into consideration when evaluating drug levels and associated effects. These data form the basis for an ongoing clinical trial in patients with refractory acute leukemias. The administration of 17-AAG in conjunction with other novel agents is also being explored. In particular, histone deacetylase inhibitors (HDI) are capable of transcriptional activation of genes involved in cellular differentiation such as the Cdk inhibitor (Cdki) p21CIP1. Blockade of p21CIP1 using antisense mRNA has been demonstrated to result in a significant increase in HDI-mediated apoptosis in U937 cells (43). In addition, the hydroxamic acid analog class of HDI induces acetylation of Hsp90 with a decrease in binding of ATP to Hsp90 and subsequent inhibition of the interaction of Hsp90 with its client proteins. The combination of 17-AAG with the HDIs, suberoylanilide hydroxamic acid (SAHA), or sodium butyrate (SB) has been evaluated for synergy in inducing apoptosis in human myeloid and lymphoid cell lines (43). Coadministration of 17-AAG with either of the HDIs to the HL-60 or lymphoblastic (Jurkhat) leukemic cell lines resulted in marked synergy with respect to mitochondrial damage and associated cell death. This response was associated with disruption in the Raf/ MEK/ERK pathway and abrogation of p21CIP1. Similarly, the coadministration of HDI LBH589 with 17-AAG in MV4-11 cells (which possess an activating length mutation of FLT-3) resulted in greater declines in p-Akt, Akt, and p-STAT5 levels than either drug alone with an associated increase in apoptosis (44). The same group performed a similar experiment combining 17-AAG with the FLT-3 kinase inhibitor, PKC412, in the same cell line, MV4-11 (45). The combination of agents resulted in marked decrease levels of FLT-3, p-FLT-3, p-Akt, p-ERK1/2, and p-STAT-5 and an increase in apoptosis exceeding that induced by either drug administered alone. In addition, the combination induced more apoptosis in cells with mutant FLT-3 than those containing wt-FLT-3. Other Hsps as Targets for Inhibition Additional Hsps likely to be of future clinical importance as therapeutic targets are the two major antiapoptotic proteins, Hsp70 and Hsp27. Both Hsps are known to exert their antiapoptotic effects at the level of the mitochondria but via different mechanisms, as the stimuli that induce their expressions are not completely redundant (3). Hsp27 appears to inhibit apoptosis through Fas and other receptor-mediated pathways, while Hsp70 primarily appears to inhibit apoptosis by agents that directly or indirectly induce oxidative stress, through either the generation of ROS or the depletion of intracellular antioxidants such as glutathione. Both Hsp70 and Hsp27 have been implicated in multidrug resistance (MDR) and have been associated with chemotherapeutic resistance in leukemia
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(46–48). Moreover, Hsp70 can inhibit apoptosis induced by both topoisomerase I- and II-directed drugs (49,50). Finally, agents and conditions that induce HSPs do so through activation of HSFs. As a case in point, Hsp90 binds to and prevents activation of HSF-1 (51). Cellular stress results in dissociation of HSF-1 and Hsp90, thereby permitting HSF-1 phosphorylation and activation. In turn, activated HSF-1 binds to the Hsp70 promoter region to stimulate Hsp70 transcription (52). Thus, HSF-1 may prove to be a useful indirect target to inhibit Hsp70, as some cytotoxic agents have been shown to inhibit Hsp70 expression by altering HSF-1 phosphorylation levels in leukemia cells (53). VEGF AND ITS RECEPTORS Vascular Endothelial Growth Factor VEGF or VEGF-A belongs to the platelet-derived growth factor (PDGF) superfamily, which includes, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF). The human VEGF gene is found on chromosome 6p21.3 and is comprised of eight exons separated by seven introns (54). At least six splice variants exist composed of 121, 165, 189, 206, 145, and 183 amino acids, respectively. These isoforms differ with respect to their affinities for heparan sulfates, which are low in circulating forms (VEGF121 and VEGF165) and high in forms that remain cell or matrix-associated (VEGF189, VEGF206, and partially VEGF165). Native VEGF is a heparin-binding cytokine and homodimeric glycoprotein of 45 kDa that plays a crucial role in vascular endothelial homeostasis as a survival factor stimulating endothelial cell (EC) migration and proliferation while inhibiting EC apoptosis and inducing expression of antiapoptotic proteins Bcl-2 and A1 (54). VEGF plays a critical role in both developmental and physiologic angiogenesis, as well as pathological neovascularization as seen in tumor growth. The regulation of VEGF in angiogenesis is through a paracrine loop (55), while promoting the proliferation and survival of hematopoietic stem cells (HSCs) via an autocrine loop (54). A major mechanism by which the expression of VEGF, and thus angiogenesis, is regulated is by hypoxia, through the HIF-1/von Hippel-Lindau (VHL) tumor suppressor gene pathway (56,57), resulting in the transcription and upregulation of the VEGF gene. Other cytokines and factors that upregulate VEGF mRNA transcription include interleukin (IL)-1b, IL-6, PDGF, epidermal growth factor (EGF), and tumor necrosis factor (TNF)-a. Increased VEGF expression can also be induced by proto-oncogenes Src, Fos, c-Myc, and Bcl-2 as well as mutant p53 and mutant Ras. Likewise, VEGF can activate EC signal transduction pathways including Ras, Raf-1, PI3-Akt, MEK/ERK, p38 MAPK, and Src TKs (55). VEGF Receptors The VEGF family members bind with different affinities to three receptor TKs belonging to the ‘‘7-Ig’’ or FLT gene family, FLT-1 [VEGF receptor (VEGFR)-1],
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KDR/Flk-1 (VEGFR-2), and FLT-4 (VEGFR-3), each of which contains seven extracellular immunoglobulin-like domains, one membrane-spanning segment, and a consensus TK sequence domain interrupted by a kinase-insert domain (54). The receptors are expressed primarily on vascular ECs with many of their actions mediated through PI3-kinase and activation of EC-derived NOS, and by bone marrow-derived cells as well (54). VEGF-A activity primarily is mediated through VEGFR-1 and VEGFR-2. Both VEGFRs are expressed in all adult vascular ECs, with the exception of the brain (58). VEGR-1 is also expressed on HSCs, leukemic blasts, vascular smooth muscle cells, and monocytes. An alternatively spliced soluble form of VEGFR-1 (s-FLT-1) is an inhibitor of VEGF. The functions of VEGFR-1 have not been characterized fully, and they appear to vary depending on cell type and maturational stage (55). VEGFR-2 activates vascular EC differentiation, proliferation, and migration and also induces vascular permeability (55). Most studies have found that VEGFR-1 is more commonly expressed in hematological malignancies than VEGFR-2 (55). VEGFR-1, not VEGFR-2, is associated with inhibition of HSC cycling, differentiation, and hematopoietic recovery in adults (55). VEGF in Acute Leukemia The ability of VEGF to have a multifunctional impact on angiogenesis and HSC survival in combination with the prevalence of VEGFR expression on leukemic blasts suggests that the VEGF pathway may have a pivotal role in leukemia evolution and disease progression. VEGF promotes angiogenesis in the bone marrow, with AML disease progression positively correlated with the extent of angiogenesis (59,60). Studies have compared the bone marrow microvasculature in AML and normal marrows with the AML marrows demonstrating increased angiogenesis as measured by microvessel density (59,60). A significant reduction in marrow microvessel density has been observed at the time of chemotherapyinduced marrow aplasia in patients who ultimately achieved a complete response (CR), but not in those who did not respond to therapy (60). VEGF has been shown to directly stimulate AML cell growth and survival directly as well as indirectly through its effects on the bone marrow stroma and its release of cytokines including, granulocyte-macrophage colony stimulating factor (GM-CSF) and basic fibroblast growth factor (bFGF). These cytokines drive both endothelial and leukemic cell proliferation in a paracrine manner (61–64). A significant proportion of both de novo and secondary AML blast cell populations express VEGF mRNA and secrete VEGF, with an increase in VEGF levels noted in cultured leukemic blasts compared with their counterparts in normal marrow (61). Additionally, in those patients who present with high blast counts, the AML cell VEGF production has been found to be inversely related to the duration of CR as well as survival (65). It has also been demonstrated that VEGF may drive the clonal expansion and transformation of high-risk myelodysplastic syndrome (MDS) in both autocrine and paracrine fashions, as suggested
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by the expression of both VEGF and its receptors in cells at foci of abnormal localized immature myeloid precursors and by decreased production of inflammatory cytokines by bone marrow stromal cells in the presence of an anti-VEGF antibody (66). VEGF may also support survival of AML cells by its detrimental effects on immune response. Studies have shown that VEGF slows the development and functional maturation of dendritic cells from pluripotent CD34-positive cells through the inhibition of NFkB activation (67), thus interfering with antigen presentation and preventing an appropriate and sustained antitumor response. This may be of relevance in AML where the ability to generate functional dendritic cells impacts the development of a leukemia-directed cytotoxic T-cell response (68).
Novel Anti-VEGF Therapies Anti-VEGF Monoclonal Antibodies Bevacizumab is a recombinant humanized IgG monoclonal antibody directed against VEGF, which acts by blocking the binding of VEGF to its cognate receptors. The clinical activity was evaluated initially in solid tumors, specifically, in metastatic colon and renal cell carcinomas. In the randomized study evaluating previously untreated patients with metastatic colorectal carcinoma, the study arm that received bevacizumab, in addition to irinotecan, bolus fluorouracil, and leucovorin when compared with the arm that received chemotherapy alone, had statistically significant improvements in overall survival, progression-free survival, and in the rate and duration of response (69). The randomized, double-blind, phase II trial conducted in metastatic renal cell carcinoma comparing placebo with two different dosing levels of bevacizumab demonstrated that bevacizumab significantly prolonged time to disease progression, although there was not a significant difference in overall survival between the groups (70). Its clinical activity in AML was recently tested in a phase II trial where bevacizumab was administered to adults with relapsed/ refractory AML after initial cytotoxic chemotherapy with ara-C and mitoxantrone in a timed, sequential-therapy approach (71). Of the 48 adult patients who received induction therapy, the overall response rate was 48% (23/48), with CR in 33% (16 patients). Eighteen patients [4 partial response (PR), 14 CR] completed one cycle of consolidation therapy and five patients (2 PR, 3 CR) underwent allogeneic transplant. Median overall and disease-free survivals for CR patients were 16.2 months (64%, 1 year) and 7 months (35% 1 year), respectively. Marrow blasts demonstrated VEGFR-1 (FLT-1) staining before bevacizumab. Bone marrow biopsies on day 15 showed significant decreases in microvessel density in 8 of 11 patients (73%). Of these eight patients, five achieved a CR, one achieved a (PR), and two had no response. Detectable VEGF levels were present in the pretreatment serum in 67% of those tested. The level increased in 52% by day 8, and decreased in 93% (67% undetectable) two hours
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after antibody infusion. This trial yielded a favorable CR rate and disease-free survival in AML patients typically resistant to traditional therapeutic approaches, and serves as a template for further development in AML. Small Molecule VEGFR Inhibitors Several different compounds targeting one or more of the VEGFRs are being actively studied. One of these agents, SU5416 (Semaxinib, Pfizer Inc., New York, NY, U.S.A.), has been tested in a variety of hematological malignancies and has displayed limited clinical activity in multiple myeloma, AML, MDS, and in the myeloproliferative disorders (MPDs). Evidence of biological effects on the phosphorylation of several receptor TKs, including VEGFRs, FLT-3, and KIT, has been demonstrated. SU11248 (Sutent, Pfizer Inc., New York, NY, U.S.A.), which inhibits VEGF receptors 1, 2, PDGFR, and FLT-3, is being tested in a phase I trial in relapsed/refractory AML and is in phase III solid tumor trials treating renal cell carcinoma and gastrointestinal stromal tumors. PTK787/ ZK222584 (Vatalanib, Novartis Pharmaceutical Corp., East Hanover, NJ, U.S.A.) is an orally available TK inhibitor that binds to the ATP-binding sites of VEGFR. It is now in phase II trials for MDS, and in combination with imatinib mesylate in patients with AML, AMM, and CML-BP. ZD6474 (Zactima, AstraZeneca Pharmaceuticals, Wilmington, DE, U.S.A.), a VEGFR-2 TK inhibitor, and the oral pan-VEGFR inhibitor GW786034 (GlaxoSmithKline Inc. Middlesex, UK.), which also inhibits PDGFRa and b, and c-KIT, are in phase I clinical trials in solid tumors. Other receptor TKs including AG013676 (GenBank, NIH, Maryland, U.S.) and CP-547632 (Pfizer Inc., New York, U.S.) are in the early stages of development. Another novel agent that indirectly inhibits VEGFR is the orally available Raf kinase inhibitor, sorafenib (Nexavar, Bayer Pharmaceuticals Corp., West Haven, CT, U.S.A.), which primarily acts through the oncogenic Raf/MEK/ERK pathway. Ras activates this pathway, which results in Raf kinase activation. The genes in the Raf family are recruited by activated Ras and, in turn, aid in Ras membrane localization and stabilization. Once Raf is activated, it phosphorylates and activates dual-specific kinases, MEK, ERK, and c-Jun N-terminal kinase, which, in turn, regulate over 50 cytoplasmic and nuclear regulatory proteins. Raf is also activated by Ras-independent factors such as Bcl-2, b-interferon, and erythropoietin. Mutations of Raf result in constitutive activation of the kinase in tumor cells. Inhibition of Raf kinase in mouse models resulted in the blockage of anchorage-independent tumor cell growth, progression, and metastases. Raf kinase mutations have been described in diverse epithelial cancer cell lines including, papillary thyroid and ovarian carcinomas, and melanoma (72). Additionally, the inhibition of Raf-1 disrupts the Raf-Rb interaction, thereby inhibiting Rb phosphorylation, cell proliferation, and VEGF-mediated neovascularization (73). Because of the potential importance of Raf in the activation of multiple genes implicated in the growth and survival of leukemia cells, sorafenib is currently being tested in patients with refractory acute leukemias and MDS.
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SRC KINASES: A BURGEONING TARGET Src family proteins are nonreceptor protein TKs that represent the cellular forms of the prototypical transforming protein produced by the Rous sarcoma virus (74). These kinases play pivotal roles in integrating signals from multiple pathways that promote cell division and survival, motility, and adhesion in multiple cell types (75,76). Activated Src proteins translocate from the cytoplasm to the cell membrane where they interact with selected receptor TKs such as the EGF receptor (EGFR) family, focal adhesion kinase (FAK), and steroid hormone receptors to trigger downstream activation of multiple signal transduction pathways (75). While not often mutated, these proteins can be overexpressed in a broad spectrum of epithelial and hematologic malignancies and are linked to the processes of metastasis, angiogenesis, and drug resistance (75–77). Activated Src kinases abrogate the function of the critical negative regulator PTEN, thereby permitting activation of the PI3K/Akt pathway (77). Similarly, constitutive activation of the Src kinase Lyn by the Kaposi sarcoma herpsevirus (KSHV) K1 gene product leads to production of both NFkB and VEGF by KSHV-infected B-cell lymphoma cells (78). The Src family kinase Lyn is expressed by B lymphocytes and myeloid cells, with growth factor–driven overexpression in AML and B-cell malignancies including lymphoma and multiple myeloma cell lines and constitutive activation in CML by the Bcr-Abl fusion protein (reviewed in 77). Furthermore, Lyn may be overexpressed in CML independent of Bcr-Abl activity and may confer imatinib resistance without concomitant Bcr-Abl mutations (79–81). These findings plus the notable conformational similarities between activated Abl and activated Lyn (82,83) have led to the design of dual-specific inhibitors of both Abl and Src for treatment of imatinib-resistant CML that can bind to both the activated and inactivated forms of Abl and Lyn (84–86). At present, the dual inhibitor dasatinib (SprycelTM, BMS-254825, Bristol-Myers Squibb Company, New Jersey, U.S.) has major clinical activity with induction of hematologic and cytogenetic responses in imatinib-resistant chronic phase (87,88), accelerated phase (89), and blast crisis (90), including those with Bcr-Abl mutations conferring imatinib resistance other than the highly resistant T315I (87–90).
SUMMARY This chapter focuses on just a few selected novel therapeutic agents that may prove to be of particular benefit in the treatment of acute leukemias. Hsps, VEGF axis, and Src family kinases are convergence points for many pathways that promote cell growth and survival. As such, these molecules and their downstream effectors play formative roles in tumorigenesis and drug resistance. The ability to target one or more of these molecules should have a direct impact on the cell’s ability to survive toxic stresses, including cytotoxic chemotherapy and should lead to enhanced effectiveness of our current antitumor armamentarium.
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The clinical importance of these targeted therapies will be determined ultimately by their abilities to act at the level of the formative malignant HSC. Those that can modulate the net expression of primary mutational events early in the disease course and thereby abrogate intrinsic drug resistance will have the greatest potential for cure. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS The pathways discussed above are critical to cellular survival, and the agents that target them share the common characteristic of being aimed toward specific molecules, yet with direct and indirect effects on multiple parallel, interrelated, and redundant signaling pathways known to influence the evolution and progression of leukemias. It is unlikely that any of these drugs used alone will have great success in treating acute leukemias, but it is reasonable to speculate that each alone or in combination with each other may increase the tumor cell kill of traditional cytotoxic agents either directly or through their ability to sensitize cells to chemotherapy through the modulation of drug resistance mechanisms. The antigenicities of these target molecules may form the basis for immunomodulatory approaches, including vaccines. The rationale behind the development of vaccines directed against Hsps are the observations that Hsp90 and Hsp70 are expressed on the surface of certain tumor cells and are readily accessible to the host immune system. Hsps themselves may be antigenic, but recent data suggest that Hsp-peptide complexes may be what actually evoke the immune response (2). Indirect support of this includes the lack of autoimmunity observed to date, which has been attributed to the immune response probably targeting a Hsp-peptide complex rather than the carrier Hsp. In contrast to the vaccines being evaluated for Hsps, those being developed against VEGFR-2/ KDR target an auto-antigen associated with angiogenesis rather than tumor cells. A variety of vaccine constructs including dendritic cells pulsed with soluble VEGFR-2 and xenogeneic ECs have been used in an attempt to breach immune tolerance and, at the same time, decrease angiogenesis and associated tumor growth at least in part through induction of immune reactivity (91,92). The development of adjuvant vaccine therapy remains a promising area of investigation. The optimal ways in which to evaluate these new therapeutic agents remain a challenge for clinical investigators. Strategies to develop better clinical trial designs may help utilize the limited patient populations and financial resources in the most efficient manner. Estey and Thall have introduced the concept of adaptive randomization for phase II clinical trial design (93). This strategy may represent a more efficient method of conducting randomized trials and allows more patients to be randomized to the therapeutic arm that stands the greatest chance for clinical benefit. By allowing for faster, and perhaps more accurate assessments of the impact of a particular therapeutic intervention on definable subsets of patients within a heterogeneous disease population, adaptive randomization may be a viable option/addition to conventional phase II clinical trial design.
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19 Tyrosine Phosphatases as New Treatment Targets in Acute Myeloid Leukemia I. Hubeek, K. Hoorweg, J. Cloos, and G. J. L. Kaspers Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands
INTRODUCTION Although the treatment of acute leukemia has improved significantly over the past few decades, the prognosis of acute myeloid leukemia (AML) remains relatively poor. For newly diagnosed patients the complete remission (CR) rate reaches 85% to 90% with standard induction chemotherapy (1). However, about 30% to 50% of the patients that achieve CR relapse from minimal residual disease cells that apparently survived chemotherapy, giving rise to a more resistant leukemia. Resistance to chemotherapy therefore remains a major obstacle in the treatment of AML and novel treatment strategies are warranted (2). Significant advances have been made in understanding the molecular basis of leukemia. In 2002, a 2-hit model of leukemogenesis has been postulated in which one class of mutations (class I) confers proliferative and survival advantage to cells and a second class of mutations (class II) serves primarily to interfere with self-renewal and hematopoietic differentiation (3). AML is the consequence of cooperation between class I and class II mutations, resulting in proliferative and survival advantages of hematopoietic cells and impaired differentiation (4). Class I mutations include activating point mutations in receptor tyrosine kinases (RTK) expressed in early myeloid progenitors, such as FLT3 and c-KIT.
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FLT3 plays a significant role in hematopoiesis and a pathogenic role in the onset, development, and progression of AML. In addition, FLT3 are a poor prognostic factor in AML. FLT3 is the most commonly affected gene in AML and results in constitutive activation in 30% of all adult AML patients. In children the incidence is lower, but still 15 to 20% (5). Another example of class I mutations are activating mutations in c-KIT, N-RAS, and K-RAS, which account for another 20% to 30% of AML cases (6). Class II mutations are responsible for aberrant or arrested differentiation but are not sufficient to cause leukemia when expressed alone. These abnormalities can mostly be detected by cytogenetic studies, and approximately 80% of children with AML have clonal chromosome abnormalities. Chromosomal translocations associated with AML typically involve transcription regulators, which after recombination give rise to functional hybrid genes that encode for fusion proteins with abnormal function (7). Transcription factors that are frequently involved in chromosomal translocations in AML include core binding factor (CBF), retinoic acid receptor alpha (RARa), Hox family members, and transcriptional coactivators [e.g., mixed lineage leukemia (MLL)]. The fusion proteins involving AML1 (t(8;21)) and CBF (inv(16)) share a similar molecular pathogenesis and are referred to as ‘‘core binding factor leukemia.’’ These fusion proteins act as dominant-negative inhibitors and impair normal hematopoietic differentiation in a similar fashion as the PML/RARa fusion protein associated with acute promyelocytic leukemia (APL) antibodies: namely the recruitment of nuclear corepressor complexes, including histone deacetylase (HDAC). This multistep model of leukemogenesis has important clinical therapeutic implications and provides a basis for a rational, targeted approach to AML treatment. It may be possible to target both classes of mutations using selected small molecule inhibitors. Class I mutations of RTKs can be targeted in AML, as has been successfully done with imatinib mesylate (ST1571, Gleevec1), which targets Bcr-Abl in chronic myeloid leukemia (CML). Class II mutations can be targeted by agents that are capable of reversing the block in differentiation, as is the case with all-trans retinoic acid (ATRA) in the therapy for APL. Inhibitors of HDACs for instance, target the corepressor complex recruited by CBF translocations (3). The current knowledge of the role of RTKs in the development and prognosis of childhood leukemia encourages the search for other genes involved in the same signal transduction pathways. The phosphorylation of proteins for signal transduction does not only depend on RTK but also on the counteracting enzymes: protein tyrosine phosphatases (PTPs). Indeed, mutations in genes encoding for PTPs have been associated with leukemogenesis. Moreover, some PTPs have an activating function like RTKs. For example, mutations in PTPN11 can result in oncogenic SHP-2 proteins leading to dysregulated hematopoiesis (8). With the current and increasing insights about the roles that PTPs play in leukemogenesis it is a logical to think of PTPs as potential drug targets in acute leukemia, as will be described in the current chapter.
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Figure 1 The protein phosphorylation state is regulated by the relative activity of protein kinases and phosphatases. The addition and removal of the phosphoryl group can lead to structural and therefore functional change of a protein molecule.
PROTEIN TYROSINE PHOSPHORYLATION Signaling pathways involved in the control of cell proliferation, differentiation, adhesion, and migration are highly regulated. The phosphorylation of tyrosine residues of proteins is an important mechanism in regulating these signaling pathways (9,10). The phosphorylation status of proteins is balanced by the action of protein tyrosine kinases (PTKs) and PTPs. PTKs are enzymes that catalyze the transfer of the g-phosporyl group of ATP to the 4-hydrosyl group of tyrosyl residues within specific protein/peptide substrates (Fig. 1). The activity of PTPs is to catalyze the hydrolysis of phosphoester bonds (9). A change in phosphorylation state can be the result of a change in the activity of these enzymes. Abnormalities in the activities of PTKs and PTPs have been described in many malignancies. Strikingly, proportionally much more research has been focused on PTKs and PTPs were neglected. Perturbation of PTK activity by mutations or overexpression has been demonstrated to result in malignant transformation (11) and PTK inhibitors are now established as anticancer drugs (12). Recently however, studies on PTPs have substantially increased and have demonstrated that PTPs have specific and sometimes even dominant roles in determining the net level of tyrosine phosphorylation. These observations have kindled an interest in PTPs as potential pharmacological targets. Classification of PTPs The human genome codes for at least 107 PTP genes, together referred as the PTPome (Table 1) (13). Of the 107 PTP genes, 11 are catalytically inactive, 2 dephosphorylate mRNA, and 13 dephosphorylate inositol phospholipids. Thus,
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Table 1 Classification of PTPs PTPs A. Class I Cys-based PTPs Classical PTPs (38): VH1-like (DSPs) (61):
B. Class II Cys-based PTPs (1) C. Class III Cys-based PTPs (3) D. Asp-based PTPs (4)
Substrate specificity RPTPS (21) NRPTPs (17) MKPs (11) Atypical DSPs (19) Slingshots (3) PRLs (3) CDC14s (4) PTENs (5) Myotubularins (16) LMPTP (1) Cdc25 (3) EyA (4)
pTyr pTyr pTyr, pThr pTyr, pThr, mRNA pSer pTyr pSer, pThr D3-phospoinositides PI(3)p Ptyr pTyr, pThr pTyr, pSer?
Abbreviations: pTyr, phosphotyrosine; pSer, phosphoserine; pThr, phosphothreonine.
81 PTPs are active protein phosphatases with the ability to dephosphorylate phosphotyrosine (pTyr). The PTP superfamily is characterized by the presence of an approximately 250 amino acids long, absolutely conserved signature motif (H/V)C(X)5R(S/T) that falls within the catalytic domain of the enzyme (14). This PTP superfamily can further be divided into four categories on the basis of the amino sequence of the PTP catalytic domain, each with a range of substrate specificities PTPs (13). The first category is the largest family and consists of the class I cysteinebased PTPs. The 38 strictly tyrosine-specific ‘‘classical PTPs’’ belong to this family and can be further divided into trans-membrane, receptor-like PTPs (RPTPs), and the intracellular, nonreceptor PTPs (NRPTPs) (15). The RPTPs have a single trans-membrane and variable extracellular domain. The intracellular parts of most of the RPTPs contain two tandem PTP domains, D1 and D2, with most of the catalytic activity resident in D1. In many cases, the extracellular domains include immunoglobulin like and fibronectin type III domains. These domains are similar to the extracellular domains of cellular adhesion molecules (16). NRPTPs have striking structural diversity and often contain sequences that target them to specific subcellular locations or enable their binding to specific proteins (17). The 61 dual specificity phosphatases (DSPs), or VH-1 like enzymes, also belong to the class I cysteine-based PTPs. DSPs are able to dephosphorylate serine and threonine residues in their protein substrates in addition to tyrosine residues. This group can be further divided into seven subgroups. MKPs are specific for the mitogen-activated protein kinases (MAPKs) (18,19); atypical DSPs (20) include 19 poorly characterized enzymes; phosphatase and tensin homologs (PTENs) and myotubularins dephosphorylate
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the D3-phosphate of inositol phospholipids (21); PRLs and slingshots are poorly understood; and CDC14s are involved in dephosphorylating cyclin-dependent kinases (Cdks) and exit mitosis (22). The second category of PTPs is the class II cysteine-based PTPs. This type of phosphatases is widely distributed in living organisms especially bacteria, in humans however only one gene, ACP1, has been identified. ACP1 encodes for the low molecular weight PTP (LMPTP). Although its function is still unclear, there is evidence linking LMPTP to several common diseases, including allergy, asthma, obesity, myocardial hypertrophy, and Alzheimer’s disease (23). Cell division cycle 25 (Cdc25) phosphatases make up class III of the cyteine-based PTPs. These are involved in cell cycle progression and are able to catalyze the dephosphorylation of both tyrosine and serine/threonine substrates of Cdks. To date Cdks are the only known substrates for Cdc25 phosphatases (24). Phosphatases based on aspartate instead of cysteine as a key catalytic residue form class IV of the PTPs. Members of the haloacid dehalogenase (HAD) superfamily belong to this category (25). PTPs: Structural Features and Catalytic Mechanism PTPs are proteins composed of b barrels flanked by a helices. The catalytic site is ˚) located in a groove at the protein surface, which is deeper for classical PTPs (9 A ˚ than for DSPs (6 A), a difference that explains the higher substrate selectivity of the classical PTPs. The characteristic motif (H/V)C(X)5R(S/T) and a surface loop rich in acidic residues are further elements common to the PTP domain (26). The hydrolysis of phosphoester bonds on tyrosine residues of proteins differs from threonine and serine in that it is metal-ion independent. Furthermore, the catalytic mechanism involves a two-step process with the formation and breakdown of a transient phosphoenzyme intermediate. The process starts with the stabilization of the negatively charged phosphate substrate by hydrogen bonds to residues of the phosphate binding loop (P-loop) and a highly conserved arginine group. A nearby-localized dipole of an a-helix stabilizes the phosphate. Upon binding of the substrate, the enzyme undergoes a conformational change. This event triggers interactions between residues and result in covering the substrate-binding pocket by the movable WPD loop, which is essential for substrate selectivity and catalytic activity. Subsequently, a deprotonated cysteine residue attacks the substrate, which results in a phosphor-enzyme intermediate (27). After transferring the phosphate to a catalytic cysteine residue, the dephosporylated substrate is expelled from the active site using an acidic amino-acid residue to protonate a tyrosine phenolic oxygen (28). Regulation of PTPs Relatively little is known about the regulation of PTPs. The majority of PTPs are posttranslationally modified. It has been described that PTPs themselves can be regulated by phosphorylation, which increases or decreases their activity (15).
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PTP1B has been shown to be regulated by way of proteolytic cleavage, which results in the subcellular relocation of its catalytic domain from membranes to the cytosol (29). Proteolytic cleavage has also shown to be a mechanism of PTP degradation (30). Furthermore, the catalytic cysteines of class I and II PTPs are usually deprotonated and susceptible to oxidative attack, which temporarily inactivates the catalytic site. Indeed, it has been shown that PTPs are regulated by reversible oxidation (31–33). It has also been reported that homodimerization is a mechanism for the negative regulation of RPTPs. It was reported that the catalytic domains of PTPa form dimer-like structures when crystallized, and it was subsequently demonstrated that the protein dimerizes in living cells (34). Dimerization has also been reported for CD45 and it could well be a common mechanism for RPTPs in general (35). The question remains, however, if the dimerization process is constitutive or requires the presence of a ligand. PTPs in Human Disease PTPs exert key regulatory functions, and it is therefore no surprise that they have been linked to various diseases. Inactivating mutations in PTPs have been correlated with genetic disorders like lafora disease and the autoimmune disease, systemic lupus erythematosus. In cancer, many PTPs act as tumor suppressor genes and are mutated or underexpressed in different tumors. Interestingly, PTPs are also able to enhance disease, which means that inhibition of PTPs could be of considerable therapeutic interest (36). In cancer, Cdc25 stimulates the cell cycle and enhances proliferation. The receptor phosphatase CD45 is involved in many autoimmune diseases and allergic reactions. The inhibition of CD45 is especially likely to be a successful approach for the treatment of Alzheimer’s disease (37). PTP inhibition could also constitute a valuable strategy against infectious diseases. Many bacteria like Salmonella typhimurium and Yersinia Pestis use either their own PTPs or host-derived PTPs to infect their host or escape from immune response (38). Another area, which would benefit from a PTP inhibitor, is the treatment of type 2 diabetes and obesity. PTP1B appears to be a very promising target for the treatment of these disorders (39). PTPs in Cancer One of the first cases of PTP dysregulation in cancer was identified by Zanke et al. who, with his co-workers, cloned HePTP and found that its gene was located on chromosome 1p32.1 (40). This locus is a site of frequent abnormalities in the preleukemic myelodyplastic syndrome as well as hematopoietic malignancies (41). Nowadays it has become clear that PTPs have both inhibitory and stimulatory effects on cancer-associated signaling processes and that deregulation of PTP function is associated with tumorigenesis in different types of human cancer, including leukemias (Table 2).
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Cdc25 proteins belong to a high conserved family of DSPs that activate specific Cdk complexes (42). Cdks regulate progression through the cell division cycle and are kept inactive by the phosphorylation of two residues located within the ATP binding loop. Cdc25 phosphatases dephosphorylate these two residues when the Cdks are required for cell cycle progression (43). The expression and activity of Cdc25 proteins is regulated by many mechanisms, since they are involved in the complex mechanisms of the cell cycle. These regulating mechanisms include alternative exon splicing, phosporylation-dephosphorylation cycles, interaction with partner proteins, their intracellular localization, and cell cycle controlled degradation (24). In mammalian cells, three isoforms of Cdc25 are known Cdc25A, Cdc25B and Cdc25C. Two of them, Cdc25A and Cdc25B, are frequently overexpressed in cancers and are associated with poor prognosis. In AML, adhesion to fibronectin has been reported to upregulate Cdc25, leading to enhanced cell proliferation (44). The inhibition of Cdc25 could therefore be useful as a course of anticancer therapy. Another interesting target for cancer therapy that is currently under investigation is the DSP PTP MKP-1, which inactivates the JNK kinase and is overexpressed in many cancers (45). Table 2 PTPs Linked to Cancer PTPs involved
Effect(s) (Refs.)
PTEN PTPa Cdc25A, Cdc25B FAP-1 HePTP
Tumor supressor gene (46) Activates kinases Src/Fyn (47) Stimulate cell cycle progression; oncogenic roles (42) Apoptosis induction (48) Regulates ERK; changes in expression linked to hematopoietic malignancies (49) Tumor metastasis (50)
PRL-3 PTP1B
JSP-1 SAP-1 RPTP-b/PTP-y SHP-1 SHP-2 PTP-g CD45 PacP MKP-1
Dephosphorylates and activates c-Src in human breast cancer cell lines (51) Selective, endogenous inhibitor of p210bcr-abl in CML (52) Activates the MAPK JNK (53) Negatively regulates integrin signaling; downregulated in hepatocellular carcinoma (54) Highly expressed in glioblastoma cell line (55) Tumor supressor (56,57) Mutated in JMML and AML (58,59) Frequently deleted in renal and lung cancers (60) Correlates with proliferation rate of myeloma cells (61) Decreased activity in prostate cancer cell lines (62) Overexpressed in many cancers (63)
Abbreviations: ERK, extracellular signal-related kinases; MAPK, mitogen-activated protein kinases; JNK, c-Jun-amino terminal kinases; CML, chronic myeloid leukemia; JMML, juvenile myelomonocytic leukemia; AML, acute myeloid leukemia. Source: From Ref. 36.
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PTPs Linked to Leukemia SHPs The SH-2 homology containing PTPs (SHPs) belong to a subfamily of (cytoplasmatic) NRPTPs with N-terminal SH2 domains (N-SH2 and C-SH2), a classic PTP domain, and a C-terminal tail. SHP-1 and SHP-2 have two tyrosyl phosphorylation sites in their C-tails, which are phosphorylated differentially by receptor and nonreceptor PTKs (64). When there are no ligands for the tandem SH2 domains, the N-SH2 occupies the catalytic domain to block substrate access. On encountering a tyrosine-phosphorylated ligand, the closed conformation opens and the enzyme is activated. Although SHP-1 and SHP-2 can employ similar or parallel cellular pathways, these proteins are also able to exert opposing effects upon downstream cellular cascades that affect apoptosis. SHP-1 and SHP-2 modulate cellular signals that involve phosphatidylinisotol-3-OH kinase (PI3k), Akt, Janus kinase 2 (JAK2), signal transducer and activator of transcription proteins, MAPKs, extracellular signal-related kinases (ERKs), c-Jun-amino terminal kinases (JNKs), and nuclear factor-kappaB (NF-kB). SHP-1 The gene PTPN6 is located on chromosome 12p13 and encodes two forms of SHP-1 protein because of different translation initiation codons located within exons 1 and 2, respectively. Expression of the two forms of SHP-1 is regulated by two different and mutually exclusive tissue-specific promoters. Promoter 1, located 7 kb upstream of promoter 2, is active in all cells of nonhematopoietic origin, whereas promoter 2 is active exclusively in cells of hematopoietic lineage (57). SHP-1 functions as a tumor suppressor by promoting the degradation of JAK (56) and is mainly expressed in hematopoietic cells and exerts negative regulatory functions in survival and growth signaling pathways (64,65). The importance of SHP-1 expression for the maturation and function of hematopoietic cells was elucidated by the studies of moth-eaten (me/me) and moth-eaten viable (mev/mev) mice carrying mutations in PTPN6. The homozygous me and mev mice exhibited several abnormalities including neutrophilia, lymphopenia, splenomegaly and elevated serum immunoglobulin, severe combined immunodeficiency, and systemic autoimmunity (66,67). SHP-1 is frequently lost in myelodysplastic syndrome and lymphomas due to the dysregulation of leukocyte development (68). In hematopoietic malignancies, SHP-1 expression is often lost due to the methylation of the promoter region of PTPN6 or a posttranscriptional block of SHP-1 protein synthesis (57,69). PTPN6 was for the first time shown to be methylated in T-cell lymphoma cell lines (65). Hypermethylation of PTPN6 was also shown for anaplastic large-cell lymphoma, different leukemia forms, and in multiple myeloma (70,71). A study of Oka et al. (2002) describes that 62.5% (5/8) of acute lymphoblastic leukemia (ALL) 90.0% (9/10) of AML, and 100.0% (11/11) of CML patient specimens showed methylation at the CpG island of the PTPN6 promoter (70). Thus, loss of SHP-1 in hematopoietic malignancies leads to overactive signaling pathways involved in survival and growth. Zhang et al.
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(2005) identified a protein complex that binds to the PTPN6 promoter and induces epigenetic silencing. This complex includes signal transducer and activator of transcription 3 (STAT3) and DNA methyltransferase 1, both of which are thought to be crucial for the silencing of PTPN6. The involvement of STAT3, DNMT1, and, apparently, HDAC1 in the epigenetic silencing of the SHP-1 gene may have therapeutic implications. Demethylating agents and HDAC inhibitors are being evaluated in various malignancies with promising results (72). Furthermore, there are good indications that lowered SHP-1 expression is indicative of a more aggressive hematopoietic disease. In CML for instance, BcrAbl is inhibited by SHP-1. In advanced stage CML patients, SHP-1 levels are markedly decreased compared with patients in chronic phase, possibly by posttranslational modifications. Decrease of SHP-1 levels therefore plays a role in the progression of CML and could provide an explanation for imatinib resistance seen in advanced stage CML patients (73). In solid tumors such as prostate cancer, ovarian, pancreatic and breast cancer, however, overexpression of SHP-1 has been observed, as reviewed by Wu et al. (57). SHP-2 SHP-2 is a PTP encoded by the PTPN11 gene and is involved in the transduction of migratory and mitogenic signals from different kind of receptors (74). As a positive component of most growth factors, cytokine and extracellular matrix receptor signaling pathways, SHP-2 is required for full activation of the Ras–ERK cascade. However, full knowledge on which substrate(s) must be phosphorylated to fully activate this pathway is still lacking, although there are several candidates (75). Depending on the specific receptor or cell type, SHP-2 can also enhance or antagonize PI3k-Akt activation, increase or decrease Rho activity, and might affect the NF-kB or NFAT pathways (64). SHP-2 function is, in the absence of a ligand, inhibited by the blocking mechanism as described above. Disruption of this normal autoinhibitory mechanism by various mutations and/or overexpression of SHP-2 binding proteins contribute to several human diseases, including cancer. In approximately 50% of the cases of the Noonan syndrome (NS) the cause is an inherited dominant autosomal mutation in PTPN11 (76,77). NS is associated with a raised risk for developing hematological malignancies, especially juvenile myelomonocytic leukemia (JMML) (77). Mutated PTPN11 has shown to act as an oncogene (8). In various types of leukemia, particularly in childhood, mutations in PTPN11 have been described (59). In approximately 35% of the cases of sporadic JMML, somatic PTPN11 mutations are the major cause of disease (75). The pathogenesis of AML involves deregulated tyrosyl phosphorylation as a major event (78). Activating mutations in the same gene have been documented to occur as a somatic event in a heterogeneous group of myeloid and lymphoid malignancies. In 2005, Tartaglia et al. reported, a 4.4% prevalence of PTPN11 mutations in AML patients included in the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) trials with a significantly increased prevalence in French-American-British (FAB) M5 (18%) (79). In
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addition, a United States-based series also detected PTPN11 mutation in 4% of pediatric AML samples, and again the prevalence of PTPN11 mutations was significantly higher in FAB M5 compared with non-FAB M5 cases (12% vs. 2.5%) (58). Goemans et al. also detected a PTPN11 mutation in 5.4% of pediatric AML cases but, in contrast to other reports, the prevalence was not increased in FAB M5 AML (59). Another study reports that PTPN11 lesions occur also childhood ALL. Mutations were observed in 23 of 317 B-cell precursor ALL cases, but not among 44 children with T-lineage ALL (80). Although PTPN11 mutations have been linked to human leukemias and in vivo studies also show that PTPN11 mutations evoke leukemia, it is not fully understood how these PTPN11 mutations transform hematopoietic cells (75). Furthermore, SHP-2 is also a key player downstream of other oncogenes. For example, the transforming capacity of constitutively active forms of EGFR, FGFR3, and Bcr-Abl seem to depend on SHP-2 activity (81). Recently, Chen et al. identified NSC-87877 as the first PTP inhibitor capable of inhibiting SHP-2 in cell cultures without detecting an off-target effect in the EGF-stimulated ERK1/2 activation pathway (82). PTP1B PTP1B has been implicated as a negative regulator of multiple signaling pathways downstream of RTKs (83). Gene-targeting studies in mice have established PTP1B as a major target in diabetes and obesity (84). Initially, inhibition of this enzyme was thought to potentially lead to increased oncogenic signaling, but mice lacking PTP1B do not develop tumors (85). However, several biochemical studies have implicated PTP1B in the attenuation of various PTK-signaling pathways such as the epidermal growth factor receptor (EGFR), platelet-derived GFR (PDGFR), insulin receptor (IR), and insulin growth factor I receptor (IGF-IR) as well as p210bcr-abl, JAK2, and TYK2, involving PTP1B in the regulation of a variety of cellular events as reviewed by Dube and Tremblay (2005) (83). Given the central role of PTP1B in the negative regulation of oncogenic signaling through the dephosphorylation of PTKs such as IGF-I, EGF, and PDGF receptors, as well as attenuation of cellular transformation by oncogenic PTKs such as p210bcr-abl and p185v–Erb2, it is often speculated that inhibition of PTP1B could potentially increase tumorigenesis. It was demonstrated by Dube et al. that PTP1B deficiency can lead to impaired Ras signaling and proliferation, suggesting that decreased Ras activation through inhibition of PTP1B could provide a means to treat a subset of cancers (86). Approximately 30% of cancers harbor activate mutations in the Ras gene, and it its unlikely that loss of PTP1B would affect Ras activity in this subset of cancers. However, in breast cancer, Ras mutations are rarely found, and PTP1B has been identified as one of the major PTPs that activate Src in breast cancer cells (51). The p210bcr-abl fusion protein plays a fundamental role as the primary causative factor in CML (87). Expression of PTP1B is enhanced in various cells expressing Bcr-Abl, including
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a cell line derived from a patient with CML. A bcr-abl-responsive sequence (PRS) has been identified in the human PTP1B promoter (88). It was shown that PTP1B is upregulated rapidly and selectively after expression of p210bcr-abl in a number of CML model systems, as well as Philadelphia-chromosome positive (Phþ) cells lines derived from a patient with CML. PTP1B recognizes p210bcrabl as a substrate and inhibits signaling events initiated by this fusion protein (52). PTEN The most commonly lost PTP in human cancer is PTEN, a tumor suppressor gene located on chromosome 10q23. This PTP is a typical class I cysteine-based phosphate but specific for phosphate at the D3-position of inotisol rather than pTyr. PTEN functions as a negative regulator of the PI3k-Akt pathway, which has crucial roles in cell proliferation, survival, differentiation, and migration (89,90). Thus, the loss of PTEN creates a state in which the PI3 pathway is constitutively active. This leads to an alteration in the transcription of downstream, targets of Akt such as NFkB, HIF-1a and the inhibition of GSK3, which leads to increased C-Myc levels (91–93). The enzyme is lost in over half of glioblastoma and in a high portion of breast and prostate cancer, in lymphomas, and other malignancies including leukemias that feature dysregulated hematopoiesis (89,94). Chimeric and heterozygotic PTEN mutant mice generated by Di Cristofano et al. (46) showed a high incidence of tumor formation, with colonic, testicular, thyroid, germ cell, and hematopoietic (AML) neoplasms observed. Other studies showed a high incidence of T-cell lymphomas and leukemia; moreover, T-cell lymphomagenesis in PTEN-positive or -negative mice is markedly potentiated by irradiation (95,96). In 2006, Yilmaz et al. generated mice with Cre-inducible inactivation of PTEN in the murine hematopoietic system (97). By ways of conditionally deleting the PTEN tumor suppressor gene in adult hematopoietic cells, they induced myeloproliferative disease within days and transplantable leukemias (AML and ALL) within weeks. PTEN deletion also promoted hematopoietic stem cell (HSC) proliferation. However, this led to HSC depletion via a cell-autonomous mechanism, preventing these cells from stably reconstituting irradiated mice. In contrast to leukemia-initiating cells, HSCs were unable to maintain themselves without PTEN. These effects were mostly mediated by mTOR as they were inhibited by rapamycin. Rapamycin not only depleted leukemia-initiating cells but also restored normal HSC function. Mechanistic differences between normal and cancer stem cells can thus be targeted to deplete PTEN. PTPs as Drug Targets PTKs are nowadays established drug targets (12). However, there are no PTP inhibitors in the market today. The reason for this is partly historical. The first PTP was molecularly characterized only 10 years after the first PTK was identified (98), and the development of PTPs is therefore relatively young.
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Furthermore, PTPs have long been considered to function as housekeeping proteins. The insight that these enzymes play mostly nonredundant-specific roles is more recent knowledge. Since over half of the PTPs have been implicated in human malignancies, the interest in PTPs as drug targets by pharmaceutical companies and academic research groups has been, and is, rising (99). Among various members of the PTP superfamily, PTP1B has emerged as the bestvalidated drug target for the treatment of both type 2 diabetes and obesity. Because being overweight is such a big problem in our western world, much effort has been put into developing inhibitors against PTP1B (100). The observation that PTP1B-/- mice are hyper-responsive to insulin and to diet-induced obesity first attracted the interest of the pharmaceutical companies to PTPs as a drug target (84). Mounting evidence from biochemical, genetic, and pharmacological studies support a role for PTP1B as a negative regulator in both insulin and leptin signaling. Consistent with the above-mentioned knowledge on PTP1B, antisense oligonucleotides that target PTP1B have shown efficacy in type 2 diabetes and have entered phase II trials. In addition, small-molecule inhibitors can work synergistically with insulin to increase insulin signaling and augment insulin-stimulated glucose uptake. The development of PTP1B inhibitors for clinical use has made tremendous progress and has addressed several problems associated with targeting PTPs for therapeutic development (e.g., potency, selectivity, and bioavailability). It has become apparent that the conserved structure and mechanistic features of the PTP active site present substantial challenges to drug development. The development of PTP inhibitors for clinical use is hampered because researchers face many and complicated challenges. Selectivity is one of the major issues in the development of PTP inhibitors as drugs. Because all PTPs share a high degree of structural conservation in the active site (the pTyr binding-pocket), designing inhibitors with high affinity and selectivity poses a challenge. The observation that not only the pTyr binding-pocket is sufficient for high affinity binding but residues flanking the active site are also important for substrate recognition has led to the development of so-called bidentate ligands. These ligands bind both the active site and a unique adjacent peripheral site, providing opportunities for the development of potent and specific PTP inhibitors. One has to take into account however that PTPs play often nonredundant, specific, highly regulated roles. The nonredundancy in PTP function might even provide an explanation to the high number of PTPs in the human genome (13). An important consideration that has to be made is that monospecific inhibitors could have a small impact compared with closely related PTPs. That the idea of nonredundancy and specificity is not an absolute one and is very complicated is illustrated by PTP1B that phosphorylates more than one PTK and seems to overlap TC-PTP functioning. For instance, the metal-containing PTP1B inhibitor vanadate, an insulin mimetic, has been used to treat diabetes mellitus in vivo and in clinical trials (101–103). However, vanadate appeared to inhibit many PTPs thereby potentially having many side effects. All PTPs are characterized by their
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Table 3 PTP Inhibitors, Their Characteristics and Limitations Protein Tyrosine inhibitors Inorganic PTP inhibitors Natural products and derivatives Peptide-based inhibitors Small molecule inhibitors
Characteristics
Limitation(s)
Mimicking phosphate group of natural substrate Rich source of PTP inhibitors
Not PTP specific
Low Km Rational approach
Need optimization to improve selectivity and increase potency Poor cell-membrane permeability and degradation Multicharged, which decreases cell-membrane permeability
sensitivity to vanadate, ability to hydrolyze p-nitrophenyl phosphate, the insensitivity to okadaic acid, and lack of metal ion requirement for catalysis (99). In addition, the abundance of PTP encoding genes suggests a tissue- or cellspecific expression pattern. However, Tautz and colleagues show the contrary: white blood cell lines express between 65 and 72 PTPs and this expression pattern shifts upon stimuli. Remarkable is the notion that an identical stimulus elicits a cell line–specific response (99). These examples illustrate that the specificity and nonredundancy of PTPs need to be considered at different levels in the development of PTP inhibitors (Table 3). Taking into account the above-mentioned problems, several groups have identified lead compounds with favorable selectivity, making phosphate inhibition plausible. Inorganic PTP Inhibitors These inhibitors mimic the phosphate group of the natural enzyme substrate, the above-discussed vanadate, as well as nitric oxide and phenyl arside belong to this group. The inhibiting effects of these compounds are due to phosphatase inhibition, however other enzymes are targeted as well, which may be a limitation for clinical use (36). Natural Products and Derivatives This group is a rich source of leading compounds that need optimization to improve selectivity and potency (36,104). For example, the stevalins have been isolated as immunosuppressive agents from Penicillium (105). Derivation of the hydroxy group of the threonine residue of the long alkyl chain, which is required for activity against Jurkat cells, showed abolition of effect against Il-2 and Il-3 in these cells in vitro but good inhibitory effects against VHR.
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Peptide-Based Inhibitors These inhibitors have a very low Km, being 10,000 times lower than free pTyr and are therefore used to study PTP-interactions in vitro. However, because of proteolytic susceptibility and weak partitioning across the plasma membrane, peptide-based compounds are not highly desirable for the development of medicinally effective drugs (28,99). Well-known inhibitors of this group belong to the group of phosphonodifluormethyl phenyalanine (F2Pmp)-containing peptides (106). Small-Molecule Inhibitors High throughput screening using natural products as a lead is a common strategy for drug discovery and is very useful for PTPs because relative simple in vitro assays can be performed. But, rationally developing PTP inhibitors by structurebased information is increasing and the strategies used for this research are being optimized (104). Although all Cys-based PTPs share a catalytic domain with a low pKa, the surface topology surrounding this catalytic pocket has numerous unique features, for instance, the differences in charge distribution, which allow for inhibitor specificity and can be utilized for rational structure-based design of highly selective compounds (107). The novel compound carboxylic acid ertiprotafib was synthesized at Wyeth research and appeared to be a good PTP1B inhibitor in vivo (108). This compound even progressed to Phase II clinical trials for treatment of diabetes mellitus but was terminated in 2002 due to unsatisfactory efficacy and doselimiting side effects. Further research elucidated that ertiprofatib improved glycemic control via several mechanisms (109). Recently, ertiprotafib was also shown to be a potent inhibitor of IkappaB kinase beta (IKKb) (110). FIVE-YEAR PERSPECTIVE Targeted agents directed against PTKs have been a major focus of recent drug development, producing now established drugs such as Gleevec and gefitinib (Iressa1). The success of these drugs has prompted the search for other drugable kinases. Given the complementary role that PTKs and PTPs play in regulating a multitude of signal transduction pathways, there has been increasing interest in identifying potent and selective PTP regulators. For inactivating PTPs that are usually downregulated in cancer cells, new strategies have to emerge such as, for instance, demethylating agents. For activating PTPs, inhibitor development is relatively young and researchers in this field have many challenges to deal with. Good reliable PTP inhibitors would also be valuable tools in elucidating the precise and specific roles that PTPs play in many (downstream) pathways. As anticancer drugs, PTP inhibitors for MKP1 and Cdc25 (43) are the main targets of academic and pharmaceutical settings. Since over half of the PTPs have been linked to malignancies including leukemia, PTP inhibitors could offer a great
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spectrum of new drugs. PTPs make up a considerable part (4%) of the drugable genome and given the crucial roles these enzymes play in various biological processes, it appears almost certain that PTP inhibitors will become part of the next generation of drugs. REFERENCES 1. Kaspers GJ, Creutzig, U. Pediatric acute myeloid leukemia: international progress and future directions. Leukemia 2005; 19:2025–2029. 2. Kaspers GJ, Zwaan CM. Pediatric acute myeloid leukemia: towards high-quality cure of all patients. Haematologica 2007; 92: 1519–1532. 3. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100:1532–1542. 4. Deguchi K, Gilliland DG. Cooperativity between mutations in tyrosine kinases and in hematopoietic transcription factors in AML. Leukemia 2002; 16:740–744. 5. Zwaan CM, Meshinchi S, Radich JP, et al. FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance. Blood 2003; 102:2387–2394. 6. Goemans BF, Zwaan CM, Miller M, et al. Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia. Leukemia 2005; 19: 1536–1542. 7. Gilliland DG. Molecular genetics of human leukemias: new insights into therapy. Semin Hematol 2002; 39:6–11. 8. Chan RJ, Feng GS. PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 2007; 109:862–867. 9. Tonks NK, Neel, BG. From form to function: signaling by protein tyrosine phosphatases. Cell 1996; 87:365–368. 10. Bhaduri A, Sowdhamini R. A genome-wide survey of human tyrosine phosphatases. Protein Eng 2003; 16:881–888. 11. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001; 411:355–365. 12. Sawyers C, Targeted cancer therapy. Nature 2004; 432:294–297. 13. Alonso A, Sasin J, Bottini N, et al. Protein tyrosine phosphatases in the human genome. Cell 2004; 117:699–711. 14. Dixon JE. Structure and catalytic properties of protein tyrosine phosphatases. Ann N Y Acad Sci 1995; 766:18–22. 15. Ostman A, Bohmer FD. Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases. Trends Cell Biol 2001; 11:258–266. 16. Ostman A, Hellberg C, Bohmer FD. Protein-tyrosine phosphatases and cancer. Nat Rev Cancer 2006; 6:307–320. 17. Mauro LJ, Dixon JE. ‘Zip codes’ direct intracellular protein tyrosine phosphatases to the correct cellular ‘address.’ Trends Biochem Sci 1994; 19:151–155. 18. Farooq A, Zhou MM. Structure and regulation of MAPK phosphatases. Cell Signal 2004; 16:769–779. 19. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dualspecificity protein phosphatases. Oncogene 2007; 26:3203–3213. 20. Alonso A, Rahmouni S, Williams S, et al. Tyrosine phosphorylation of VHR phosphatase by ZAP-70. Nat Immunol 2003; 4:44–48.
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20 Proteasome and Protease Inhibitors N. E. Franke, J. Vink, J. Cloos, and G. J. L. Kaspers Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands
INTRODUCTION Although treatment of patients suffering from leukemia improved throughout the last decades, new chemotherapeutic agents are still required to further minimize side effects and increase overall survival rates. Many leukemia patients still suffer from a relapse following initial therapy (1–4). Since patients with a relapse often prove more resistant to chemotherapeutics, it is important to develop new drugs that act through other cellular pathways to minimize cross-resistance and increase response. In this context, the use of proteasome inhibitors might prove a huge step forward, since these inhibitors not only act on a very powerful regulatory target but also influence several cellular pathways simultaneously. Moreover, these drugs may sensitize malignant cells to conventional anticancer drugs. Proteasomes are among the most ingenuous key regulators of the functioning cell. The proteasome is responsible for degradation of many intracellular proteins, thereby helping maintain the cellular homeostasis during biological processes such as cell cycle, signal transduction, response to stress and gene transcription. Among other functions, the proteasomal complex rapidly turns over misfolded proteins to avoid accumulation of dysfunctional proteins (5–7). Furthermore, the proteasome generates small peptides to initiate immune responses. These peptides bind to major histocompatibility complex (MHC) class I molecules and are transported to the plasma membrane. If the immune
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system does not tolerate the displayed peptide, cytolytic CD8 T-lymphocytes will eradicate the cell (8). In multiple myeloma (MM), proteasome inhibitors have been shown to be very successful. Not only do these inhibitors act on MM cells themselves, they also downregulate protective interactions with bone marrow stromal cells and inhibit blood vessel development (9,10). Proteasome inhibitors can be more effective than traditional drugs such as glucocorticoids when used as a single drug and interact in an additive or even synergistic way when combined with these drugs (11,12). The therapeutic index for proteasome inhibitors is favorable. MM cells and leukemic cells are significantly more sensitive to proteasome inhibition than CD34-positive bone marrow progenitor cells or lymphocytes from healthy persons (13–17). Furthermore, proteasome inhibitors inhibit leukemic stem cells very specifically (18). Finally, proteasome inhibition increases sensitivity of cancer cells to traditional anticancer agents such as glucocorticoids, anthracyclines, gemcitabine, cisplatin, and radiation (11,19–22). Ubuquitin-Proteasome Pathway More than 80% of all eukaryotic protein degradation is controlled by the ubiquitin-proteasome pathway (23). This pathway regulates protein ubiquitination, and subsequent recognition and degradation by the proteasome (Fig. 1). The proteasome is present in both the cytoplasm and nucleus of cells (24,25). The 26S proteasome is a large intracellular protease (1,500–2,000kDa)
Figure 1 Functional representation of the mechanism of action of the proteasome. Upon degradation, proteins become ubiquitinated by enzymes E1, E2, and E3. After ubiquitination, the protein is targeted to the 19S complex of the proteasome, where it is deubiquitinated and unfolded. Subsequently, the protein is processed to the 20S complex, where it is further degraded into peptides. The ubiquitin components can be recycled (42).
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that consists of a 20S core catalytic complex and two 19S regulatory subunits (26–28). The 20S proteasome complex is a macromolecule of 700 kDa, made up of four stacked rings. The two outer rings contain seven a-subunits, while the two inner rings consist of seven b-subunits. The b1, 2, and 5 subunits contain the postglutamyl peptidyl hydrolytic-, tryptic-, and chymotryptic-like proteolytic activities of the proteasome, respectively (26,27,29). Together, these three can hydrolyze almost all peptide bonds of proteins, thus forming smaller polypeptide units. When combined with the two 19S regulatory units, the 26S proteasome is formed. This form of the proteasome is the most important mediator of protein degradation. The ubiquitin-conjugating system targets proteins for degradation by attachment of poly-ubiquitin (Ub) chains (30). This ubiquitination is mediated by three enzyme families: E1, E2, and E3. The Ub-activating E1 enzyme binds and activates ubiquitin. The E2 and E3 families consist of many members. One of the Ub-conjugating enzymes E2 transfers the activated ubiquitin to an E3 family member, after which this E3 Ub ligase can mediate the attachment of Ub to the desired protein. By repeating this step, a Ub chain is formed (6). After the attachment of Ub chains to a protein, this protein binds to the subunits of the 19S complex, where it is de-ubiquitinated and subsequently unfolded. The Ub components can then be recycled. Following unfolding, the protein is processed to the 20S complex, where peptides of various lengths (3–22 amino acids) are formed (31,32). PROTEASOME INHIBITORS Proteasome inhibitors block cancer progression by interfering with the degradation of regulatory proteins. It is assumed that the ratio of pro- and antiapoptotic proteins within a cell becomes disturbed, thereby resulting in an increased sensitivity to apoptosis (33). Additionally, proteasome inhibition can cause apoptosis by directly affecting the levels of various specific proteins like inhibitory protein IkB, thereby inactivating the survival protein nuclear factor kB (NF-kB) (34,35). Proteasomal inhibition can also lead to increased activity of p53 and proapoptotic Bax protein, and accumulation of cyclin-dependent kinase inhibitors like p27 and p21 (27,36–38). Currently, many proteasome inhibitors have been described, including carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132), N-acetyl leucyl-leucyl norlucinal (ALLnL), lactacystin, epoxomycin, bortezomib (PS-341), and salinosporamide A (NPI-0052) (36,39–42). Features of the most well-described proteasome inhibitors are summarized in Table 1. These inhibitors can be classified into five major groups: peptide aldehydes, peptide vinyl sulfones, peptide boronates, peptide epoxyketones, and b-lactones (43). Epoxyketones seem quite specific but have not been studied very well, while peptide aldehydes, peptide vinyl sulfones and b-lactones lack enzyme specificity, are metabolically instable, or bind irreversible to the proteasome (36). Peptide boronic acids seem
Cathepsins DHEM: Cathepsin B (weak)
Irreversible Irreversible
Irreversible
Irreversible
NLVS, YLVS
Dihydroeponemycin Epoxomycin (eg PR-171) Lactacystin
NPI-0052
Cathepsin A, Tripeptidyl peptidase II Salinosporamide A
Thus far none known
Relatively specific but weak proteasome inhibitors. Bind to b-subunits of the proteasome. Binds to b-subunits of the proteasome.
Selective proteasome inhibitors. Bind to b-subunits of the proteasome.
Interact with the catalytic threonine residue of the proteasome. Selective proteasome inhibitors. Interact with the catalytic threonine residue of the proteasome. Interact with b-subunits of the proteasome.
Specificity and mechanisms
472
Abbreviations: MG-132, Carbobenzoxy-L-leucyl-L-leucyl-leucinal; ALLnL, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; ALLnM, N-acetyl-L-leucyl-L-leucylLmethioninal; LLnV, N-Carbobenzoxy-L-leucyl-L-norvalinal; PSI, N-carbobenzoxy-L-isoleucyl-L-g-t-butyl-L-glutamyl-L-alanyl-L-leucinal; Leu-Leu-vinyl sulfone; MG-262, N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucyl boronic acid. Source: Refs. 7, 27, 36, 40, 44, 92, 93.
b-lactones
Reversible
Calpain I, Cathepsins
Peptide vinyl sulfones Peptide epoxyketones
Peptide boronates
Reversible
MG-132, ALLnL, ALLnM, LLnV, PSI. Bortezomib, MG-262, PS273.
Binding to other targets
Peptide aldehydes
Binding to proteasome
Compounds
Class
Table 1 Proteasome Inhibitors
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most suitable for clinical usage. They dissociate in a slower rate from the proteasome, have up to 1,000-fold higher potency than peptide aldehydes, are selective, and bind reversible to the proteasome (36,44,45). Bortezomib The most frequently described and well-known proteasome inhibitor is bortezomib (Velcade1, PS-341), a dipeptide boronic acid analog with a broad antitumor activity in several cell lines and murine and human tumor models (19,36,42,46,47). It is the first proteasome inhibitor that has been approved by the US Food and Drug Administration (FDA) and by the European Medicines Agency (EMEA) for use in MM. Bortezomib specifically inhibits the proteasome pathway rapidly and in a reversible manner by binding directly to the b-5 subunit of the 20S complex, thereby blocking its enzymatic activity (48). Exposure to bortezomib in vitro leads to stabilization of several intracellular protein levels such as cyclin-dependent kinase inhibitors (e.g., p21) and proapoptotic Bik/NBK (49,50). Cells accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis. In MM, bortezomib could inhibit growth of dexamethasone- and doxorubicin-resistant myeloma cell lines, and induce apoptosis in dexamethasoneresistant primary cells (10,51). Synergistic interactions were found with doxorubicin and melphalan in sensitive MM cells, and with dexamethasone in leukemia cells (11,52). In vivo, approximately one third of patients with relapsed and refractory MM showed significant clinical benefit in a large clinical phase II trial (53). Currently, additional clinical trials for MM are ongoing. Several (pre)clinical studies have evaluated the anticancer role of bortezomib (and other proteasome inhibitors) in other hematological neoplasias and solid tumors as well, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (54,55). In a LOVO xenograft model studying colon cancer, bortezomib has demonstrated increased antitumor effect in combination with several standard chemotherapy agents, including CPT-11, cisplatin, docetaxel, fluorouracil, gemcitabine, irinotecan, and paclitaxel (47). In a PC-3 prostate xenograft model, bortezomib does not seem to enter the brain, spinal cord, testes, or the eye, thereby avoiding treatment-related side effects on these tissues. Preclinical studies showed that the effect of bortezomib was independent of p53 status, and not overlapping with other chemotherapeutic agents (36). PROTEASOME INHIBITORS AND LEUKEMIA Already in 1990, it was shown that human leukemic cells expressed abnormally high levels of proteasomes compared with normal peripheral blood cells (56). Both protein and mRNA proteasome expression were, in comparison to normal monocytes, higher in several lymphoid and myeloid cell lines (Daudi, DG75, CCRF-CEM, MOLT-10, U937, HL-60, and K562). Furthermore, an increase of
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proteasome expression was shown both in leukemic cells from patients with acute lymphoblastic leukemia (ALL), adult T-cell leukemia, and acute myeloid leukemia (AML), as well as in bone marrow cells from patients with chronic lymphocytic leukemia (CLL) and chronic myelocytic leukemia (CML). The latter increase of proteasome expression seemed to be related to cellular proliferation, presumably in a cell cycle–dependent manner. The results mentioned above seem to indicate that dividing cells in particular are sensitive to proteasome inhibition. It has also been shown that induction of differentiation of chronic and acute leukemic cell lines results in rapid and marked downregulation of ubiquitin expression (57). Moreover, human leukemia cells that had been induced to differentiate were significantly less sensitive to proteasomal inhibition than their dividing precursors (58). Leukemic stem cells have many characteristics of normal hematopoietic stem cells, including a highly similar immunophenotype, and a predominantly G0 cell cycle status (59,60). Therefore, preferential proteasomal inhibition of only dividing cells might be insufficient when applied for clinical use. However, it has been shown that proteasome inhibitors can also induce apoptosis in leukemic stem cells, and that furthermore these stem cells are more susceptible to proteasome inhibition than normal stem cells (18). Since leukemic stem cells have a high NF-kB expression, it is thought that the downregulation of NF-kB by proteasome inhibitors is of influence for this specificity, although direct inhibition of NF-kB does not induce the same degree of apoptosis. Overall, the benefits of using proteasome inhibitors in leukemia seem promising. In Vitro Studies of Proteasome Inhibitors in Leukemia Because of the success of proteasome inhibition in MM, studies have been set up to investigate the possible benefits of proteasome inhibitors in the treatment of leukemia. This review will focus on several in vitro studies of these inhibitors in leukemia, summarized in Table 2. Not only the effect of proteasome inhibitors alone but also the combination with other cytostatics has been investigated. Although many proteasome inhibitors are known, the specificity of bortezomib, in combination with the particular achievements of this drug in MM, resulted in an increased use of this inhibitor in the more recently published studies. Proteasome inhibitors seem very successful in inducing apoptosis in leukemic cells. As shown in Table 2, in cell lines (both of myeloid and lymphoid origin) as well as in primary chronic and acute leukemia cells, inhibitors such as PSI and bortezomib successfully induced cell death. Moreover, normal, nonleukemic cells seemed less sensitive to these inhibitors, suggesting a favorable therapeutic index (14,15,61). Proteasome inhibitors already effectively induce apoptosis in leukemic cells as single drug. A number of studies have also investigated the combination
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of proteasome inhibitors with other chemotherapeutics, such as taxol, flavopiridol, and glucocorticoids (11,15,62). All studies showed enhanced sensitivity upon use of proteasome inhibitors. In these studies, drugs were added simultaneously to the cells. Two studies also investigated the importance of sequential addition of the drugs. In one study, the additional effect was only seen after pretreatment with the proteasome inhibitor. Upon coincubations, no enhanced cytotoxic effects were seen (17). The second study showed the opposite; the interactions were synergistic when drugs were given simultaneously, but only additive when given sequentially (11). Since only two studies described the effect of sequential administration, and since these studies result in opposite conclusions, further investigations on this subject are warranted. Several molecular interactions have been investigated to obtain further insights in the pathways that are affected by proteasomal inhibition. Numerous studies show that the mitochondrial apoptotic pathway is affected, including SMAC activation, cytochrome c release, and caspase activation. Furthermore, the survival protein NF-kB is downregulated, and there is an increase of activation of cysteine proteases (58,63,64). Although it is still not known how many pathways, directly or indirectly, are disturbed by proteasome inhibitors, it is clear that these inhibitors can overcome resistance to other cytostatics. Some examples have already been given in the studies described in Table 2 (14,63). In Vivo Studies of Proteasome Inhibitors in Leukemia Many of the initial studies regarding the effect of proteasome inhibition have been performed in in vitro systems. The first in vivo antitumor activity of proteasome inhibitors was demonstrated in a human Burkitt’s lymphoma xenograft model (65). In 2002, a preclinical study was published in which bortezomib was combined with humanized antiTac in a murine model of adult T-cell leukemia (66). In this study, bortezomib alone did not result in prolongation of the survival of the tumor-bearing mice, which was ascribed to a limited dosing schedule. However, in combination with humanized antiTac, bortezomib therapy could be associated with complete response (CR) in several mice, whereas antiTac alone only resulted in a partial response (PR). The last years’ several in vivo studies have been performed in patients in whom the effect of proteasome inhibitors was investigated. Table 3 summarizes such studies that included leukemia patients. All in vivo leukemia studies regarding proteasome inhibition have been performed using bortezomib, as this drug showed a unique toxicity profile in the NCI preclinical assay and is approved for and successful in MM (36). Bortezomib was shown to work in a dose-dependent manner, and recovery of normal proteasome function was seen within 72 hours after the last dose (67). In the two single-drug studies described, patients suffering from leukemia showed hematological improvements, but in these phase I studies no CRs were reached
Primary CLL cells
CML, AML, ALL cell lines
AML cell line U937 Primary CLL cells AML, ALL cell lines, primary AML cells CML, AML, ALL Cell lines Primary CLL cells
Lactacystin
PSI
Lactacystin Bortezomib MG-132, LLnL, lactacystin Bortezomib
(58) (63)
Induction of apoptosis. Increase of p27Kip1. Activation of cysteine proteases. Induction of apoptosis in both GC-sensitive and -resistant cells. Activation of cysteine proteases. Apoptosis is blocked by caspase antagonist zVADfmk. Inhibition of NF-kB. Induction of apoptosis in radiosensitive and -resistant CLL cells but not in normal lymphocytes. Induction of apoptosis in all cell lines. Enhanced taxol and cisplatinum cytotoxicity. PSI was more active on leukemic than on normal CD34þ bone marrow progenitors. Lactacystin combined with PKC activator bryostatin enhanced apoptosis. Induction of apoptosis associated with release of SMAC and cytochrome c. Synergistic interactions between PI and cyclin-dependent kinase inhibitors flavopiridol and roscovitine. Downregulation of XIAP, p21CIP1, and Mcl-1. Synergistic with flavopiridol. Blockade of the IkB/NF-kB pathway. Activation of the SAPK/JNK cascade. Reduction in activity of STAT3 and STAT5. Dose-dependent cytotoxicity of bortezomib. Additive effect with purine nucleoside analogs cladribine and fludaribine. CLL cells more sensitive than normal lymphocytes.
(96)
(95)
(94) (64) (62)
(15)
(13,14)
Ref.
Study results and mechanisms involved
476
Bortezomib
AML cell line HL60 Primary CLL cells
Leukemic cells
Several Lactacystin, MG-132
Proteasome inhibitors
Table 2 Preclinical Studies of Proteasome Inhibitors (PI) in Leukemia
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Primary AML and ALL cells
Pediatric ALL xenocraft model CML, AML cell lines
AML, ALL cell lines, primary pediatric AML, ALL cells AML cell lines
Leukemic cells (11)
Lymphoblastoid, CML and AML cell lines. Bortezomib induced apoptosis and acted at least additive with dexamethasone, vincristine, asparaginase, cytarabine, doxorubicin, geldanamycin, HA14.1 and trichostatin A. Synergistic with tipifarnib. The combination overcomes cell adhesion-mediated drug resistance. In vitro and in vivo activity of bortezomib against primary pediatric ALL cells in a xenocraft mouse model. PSI enhanced toxicity of daunoblastin, taxol, cisplatinum, and bortezomib. PSI suppressed the clonogenic potential of AML and CML more than that of normal bone marrow (NBM) progenitors. Bortezomib inhibited the clonogenic potential of CML and NBM more effectively. Inhibits proliferation and induces apoptosis AML, inhibits proliferation in ALL (61)
(17)
(98)
(97)
Ref.
Study results and mechanisms involved
Abbreviations: PSI, N-carbobenzoxy-L-isoleucyl-L-g-t-butyl-L-glutamyl-L-alanyl-L-leucinal; LLnV, N-Carbobenzoxy-L-leucyl-L-norvalinal; LLnL, N-acetylleucylleucylnorleucinal; MG-132, Carbobenzoxy-L-leucyl-L-leucyl-leucinal; GC, glucocorticoid; PKC, protein kinase C.
PR-171
PSI, Bortezomib
Bortezomib
Bortezomib
Bortezomib
Proteasome inhibitors
Table 2 Preclinical Studies of Proteasome Inhibitors (PI) in Leukemia (Continued )
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Refractory or relapsed acute leukemia
AML, MM and NHL
AML
Recurrent childhood ALL, AML, blastic phase CML, M3
Phase I
Phase I
Phase I
Phase I
Bortezomib, Idarubicin, Cytarabine
Bortezomib
12
Bortezomib with PegLD
24
42
Bortezomib
Bortezomib
27
15
Cytostatics
No. of patients Bortezomib was given twice weekly for 4 wk every 6 wk. The MTD was 1.04 mg/m2. CR in 1 MM patient. PR in 1 patient with MCL and 1 with FL. Bortezomib was given twice-weekly for 4 wk every 6 wk. The MTD was 1.25 mg/m2. No grade 3 toxicities. 5 patients showed hematological improvement. No CR achieved. Bortezomib was given on days 1, 4, 8, 11 and PegLD on day 4. MTD of bortezomib was 1.30 mg/m2. No significant pharmacokinetic and pharmacodynamic interactions between bortezomib and PegLD. 16/22 evaluable MM patients achieved CR, near-CR or PR. One NHL patient achieved CR, another a PR. Both evaluable AML patients achieved a PR. Addition of bortezomib to AML induction chemotherapy. Bortezomib added on days 1, 4, 8, and 11. 14 CRs, 3 patients received remission without complete recovery of platelet count, 3 PRs, 4 nonresponders. Bortezomib well tolerated up to 1.3 mg/m2. Bortezomib was administered twice wk for 2 wk followed by a 1-wk rest MTD of bortezomib was 1.3 mg/m2/dose. 5 patients were fully evaluable. DLTs occurred in 2 patients at the 1.7 mg/m2 dose level. No OR achieved.
Study results and mechanisms involved
(71)
(70)
(69)
(68)
(67)
(Ref.)
478
Note: Information via http://www.clinicaltrials.gov. Abbreviations: MTD, maximum tolerated dose; DLT, dose limiting toxicities; CR, complete response; PR, partial response; OR, objective response; MCL, mantle cell lymphoma; FL, follicular lymphoma; NHL, on-Hodgkin’s lymphoma; PegLD, pegylated liposomal doxorubicin; 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin; PFS, progression-free survival; EFS, event-free survival; OS, overall survival.
Several hematologic malignancies
Malignancy
Phase I
Study model
Table 3 In Vivo Studies of Bortezomib in Leukemia
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(67,68). Overall, although bortezomib seemed to have biological activity, the clinical benefits were limited when given as a single-drug agent. These results might appear somewhat disappointing, and it seems that the use of bortezomib alone might not have advantages in clinical settings. In 2005, the first phase I combination study in several hematological malignancies including leukemia was presented, in which bortezomib was combined with pegylated liposomal doxorubicin (PegLD) (69). Bortezomib was given on days 1, 4, 8, and 11 and PegLD on day 4. Forty-two patients were included, with an overall response rate of 73% in MM patients. Grades 3 or 4 toxicities in this study included thrombocytopenia, lymphopenia, neutropenia, fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea. Both evaluable AML patients in this study achieved a PR. In another study bortezomib was combined with AML induction chemotherapy (idarubicin and cytarabine). Bortezomib was added on days 1, 4, 8, and 11. Thirty patients were included, of which 24 patients were evaluable. The overall response rate was 83%, with 58% of the AML patients reaching a CR (70). Only 2 out of 24 patients suffered from dose-limiting toxicities (prolonged neutropenia and thrombocytopenia). Although the highest dose used was as high as 1.3 mg/m2 bortezomib, the maximum tolerated dose (MTD) was not reached and additional patients are now being enrolled to the study. Bortezomib was also tested in a phase I study in a pediatric cohort of relapsed leukemic patients. Bortezomib was administered twice weekly for two consecutive weeks at either a 1.3- or 1.7-mg/m2 dose followed by a one-week rest. The treatment was well tolerated and the optimal dose was set on 1.3 mg/m2. No objective clinical responses were obtained in this small group of heavily pretreated patients (71). Currently ongoing phase I studies are focusing on the combination of bortezomib with other cytotoxic agents like 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), fludarabine, rituximab, cyclophosphamide, and prednisone. Although the first results of the use of bortezomib in combination studies are very promising, it seems too early to speculate on the final impact of proteasome inhibitors in the treatment of leukemia. An overview of the ongoing clinical studies is given in Table 4.
PROTEASOME INHIBITORS IN LYMPHOMAS Non-Hodgkin’s Lymphomas Bortezomib also shows in vitro antitumor activity against non-Hodgkin’s lymphoma (NHL) by causing cell cycle arrest and induction of apoptosis in MCL and primary effusion lymphoma (PEL) cells. Inhibition of NF-kB, which is constitutively upregulated in these cells, by bortezomib is thought to be responsible for this cytotoxic effect (55,72). In addition, the upregulation of p21, p27, and p53 has been shown after exposure to bortezomib in PEL cell lines (73). Furthermore,
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Table 4 Ongoing In Vivo Studies of Bortezomib in Leukemia Study model
Cytostatics
Patient population
Phase I Phase I
Bortezomib þ idarubicine Bortezomib þ tipifarnib
Phase I Phase I
Bortezomib þ fludarabine Bortezomib þ 17-N-allylamino-17demethoxygeldanamycin (17-AAG) Bortezomib þ dexamethasone þ PEG-asparaginase þ doxorubicin þ cytarabine þ methotrexate þ vincristine Bortezomib þ mitoxantrone þ etoposide Bortezomib
Elderly and relapsed AML Acute Leukemia and CML in blast phase CLL and NHL Relapsed or refractory AML, ALL, CLL, and NHL Pediatric ALL
Phase I/II
Phase I/II Phase II
AML and ALL PhþCML in chronic and accelerated phase
Note: Information via http://www.clinicaltrials.gov. Abbreviations: AML, acute myeloid leukemia; CML, chronic myelocytic leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin’s lymphoma; Phþ, Philadelphia chromosome-positive.
in MCL cell lines bortezomib induces downregulation of cyclin D1, which is upregulated in this subtype of lymphoma, and subsequent cell cycle arrest (74). Bortezomib was also tested on primary NHL cells showing that primary MCL cells are more sensitive to bortezomib than primary folicular lymphoma (FL) cells (75). Additive or synergistic effects were seen in MCL cells when treated with a combination of doxorubicin, vincristine, 4-hydroperoxycyclophosphamide, and bortezomib (76), and in PEL cells treated with a combination of bortezomib and doxorubicin or paclitaxel (72). Several phase II clinical trials have shown the efficacy of bortezomib as single drug. Interim analysis of these studies shows an overall response rate of 18% to 60% in FL and 39% to 56% in MCL. Currently different combinationstudies are ongoing to identify the optimal treatment strategy. An overview of these studies can be found in the review of Leonard et al. (77). Hodgkin’s Lymphomas In Hodgkin’s lymphoma (HL), bortezomib induces apoptosis in a caspasedependant manner, which is associated with reduced c-FLIP expression (78). NF-kB is overexpressed in Hodgkin/Reed-Sternberg cells and can be inhibited by bortezomib. It was shown that the induction of cell cycle arrest and apoptosis by bortezomib seem to be independent of the mutation status of IkB or the activation status of CD30, CD40, and RANK receptor (79,80). In contrast, Boll et al. showed that anti-CD30 antibody 5F11 activates NF-kB and sensitizes lymphoma cells to bortezomib (81). Furthermore, TRAIL antibodies could enhance the in vitro
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sensitivity of primary lymphoma cells to bortezomib (82). So the exact mechanism of bortezomib induced apoptosis in HL is yet to be clarified. A meta-analysis has been published on the single drug treatment of refractory NL with bortezomib showing no or limited activity. The same group showed no additional effect of the addition of dexamethason to bortezomib (83). Several combination studies are currently being performed and no interim data are yet available. RESISTANCE MECHANISMS Despite of promising results from clinical studies using bortezomib, acquired and intrinsic resistance to treatment with bortezomib have been reported (84). Since conventional mechanisms of drug resistance mediated by efflux pumps like MDR1, BRCP1, and MRPs, do not seem to be responsible for bortezomib resistance (85), several studies have focused further on the etiology of bortezomib sensitivity and resistance. Most of the studies have focused on the proteasome subunit composition in relation to bortezomib sensitivity and resistance. The ratio between b2-type and (b1þ b5)-type catalytic subunits has been correlated with bortezomib response in vitro and ex vivo in primary patient hematological malignant cells (86). The importance of the proteasome subunit composition in bortezomib sensitivity is conformed by two bortezomib two resistant cell-lines. The bortezomib resistant AML cell line HL-60 showed upregulation of the b1 and b5 subunits, and the bortezomib resistant Burkitt lymphoma cell line showed upregulation the b1, b2 and b5 catalytic domains of the proteasome (86,87). The pan proteasome inhibitor NPI0052 might be useful in overcoming this resistance. When treating bortezomibresistant MM cells ex vivo with NPI-0052, apoptosis could still be induced (88). Mechanisms distinct of the proteasome itself have also been suggested to be involved in bortezomib sensitivity and resistance. A microarray study has shown that overexpression of activating transcription factor (ATF)3, ATF4, ATF5, c-Jun, JunD, and caspase-3 is correlated with bortezomib in B-cell lymphoma cells (89). Furthermore, overexpression of cyclin D1 might also increase bortezomib sensitivity in vitro and in vivo in a breast cancer model (90). In contrast overexpression of heat shock protein (HSP)27, HSP70, HSP90 and Tcell factor 4 is associated with bortezomib resistance in B-cell lymphoma cells (89). These data together suggest that although the proteasome conformation is very important in bortezomib sensitivity, other factors might also be involved in intrinsic and acquired bortezomib resistance. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Several in vitro studies have shown that proteasome inhibitors have potential beneficial effects for the treatment of leukemia and lymphoma. Not only as a single drug but especially in combination with other, commonly used
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chemotherapeutic agents, exposure to these inhibitors results in an apoptotic response of the leukemic cells. This is also seen in the in vivo studies published thus far. Although the proteasome inhibitor bortezomib by itself showed minor clinical benefits in leukemia and HL, the effects of combination studies prove very promising. Proteasome inhibitors are effective in dividing cancer cells, but apoptosis is also induced in isolated primary leukemic cells, as well as in leukemic stem cells. This generates treatment possibilities for both rapidly proliferating leukemias such as acute leukemia, as well as for more slow-dividing diseases such as CLL and particular types of lymphoma. Furthermore, normal nonleukemic cells can withstand much higher doses of these drugs, thereby generating a favorable therapeutic index. This review has mainly focused on data obtained in leukemic settings. Not many results have been published thus far regarding the different cellular pathways that are affected by proteasome inhibitors. However, when looking at data presented for other tumors, more insight can be obtained about the many ways that proteasome inhibitors induce apoptosis, including activation of JNK, stabilization of p53, Bax and Bid, and NF-kB downregulation (91). Furthermore, insights obtained in MM studies can give directions to future research regarding proteasome inhibition in leukemia. For instance, when focusing on the effect of proteasome inhibitors and the more traditional glucocorticoids (GC), Richardson et al. showed in relapsed and refractory MM that bortezomib was more efficacious than high-dose dexamethasone, resulting in a longer time to progression and a higher overall response rate (12). In vitro, bortezomib could induce apoptosis in dexamethasone-resistant MM cell lines and MM patient cells (10). Previously, Chandra et al. already showed that proteasome inhibitors lactacystin and MG-132 could induce apoptosis in both GC-sensitive and -resistant cells (63). These results make it worthwhile to investigate the effects of proteasome inhibitors in combination with the commonly used GC in leukemia and NHL. Although data obtained in MM can give useful information about the mechanism of action and efficacy of proteasome inhibitors, one has to remain careful when translating these effects directly to other settings. Interactions between MM cells and bone marrow stroma regulate growth, survival, and homing of MM cells. Proteasome inhibitor bortezomib affects MM cells both directly by inhibiting proliferation and inducing apoptosis, and indirectly by decreasing adherence of MM cells to bone marrow stromal cells (10). Therefore, tissue-based diseases might be more susceptible to bortezomib than blood-based diseases, and leukemic cells in the blood are therefore probably less susceptible than expected based on results obtained in MM. This might also explain the minor clinical benefits in leukemia of bortezomib alone. It will be essential to determine whether proteasome inhibitors will work beneficial in both acute and chronic, and lymphoblastic as well as myeloid leukemia, and to determine which therapy strategies will be most successful for these different leukemia lineages. Thus far, encouraging results were seen in all these subgroups, but further work will be necessary.
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In HL, the role of bortezomib as single drug seems also to be limited, which is in contrast to NHLs, which show significant clinical response on bortezomib monotherapy. Future studies with bortezomib in combination with standard chemotherapy is required to elucidate the full clinical potency of bortezomib. Additionally, it will be important to detect whether there are potential markers of susceptibility to proteasome inhibitors. For instance, t(11;14), resulting in the overexpression of cyclin D1, is typical for MCL. These lymphomas are good responders to bortezomib, and it is shown that exposure to this proteasome inhibitor actually downregulates cyclin D1, probably through downregulation of NF-kB (55). Therefore, it might be worthwhile to investigate whether downregulation of cyclin D1 can be used as a readout system for susceptibility to bortezomib in both leukemia and lymphoma. It will be important to elucidate the working mechanisms of proteasome inhibitors to get optimal benefits from the interactions of these inhibitors with other chemotherapeutics. Furthermore, proteasome inhibitors that can be administered orally like NP-0052 would be more convenient, and novel proteasome inhibitors with less side effects (especially concerning neurotoxicity and thrombocytopenia) would be an important improvement. Further understanding of the working mechanisms and improved selectivity of proteasome inhibitors will result in optimized use of proteasome inhibitors as chemotherapeutic agents in leukemia and lymphoma. ACKNOWLEDGMENTS This work was supported by a European Union Grant (EUGIA, nr. QLG1-CT2001-01574) and Stichting Translational Research (STR) VUmc. This chapter is based on the review article of Vink J, Cloos J, and Kaspers GJ (Proteasome inhibition as novel treatment strategy in leukaemia. Br J Haematol 2006; 134(3):253–262.) published by Blackwell Publishing. REFERENCES 1. Laport GF, Larson RA. Treatment of adult acute lymphoblastic leukemia. Semin Oncol 1997; 24(1):70–82. 2. Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J Med 1998; 339(9): 605–615. 3. Appelbaum FR, Rowe JM, Radich J, et al. Acute myeloid leukemia. Hematology Am Soc Hematol Educ Program 2001; 62–86. 4. Kaspers GJ, Creutzig U. Pediatric acute myeloid leukemia: international progress and future directions. Leukemia 2005; 19(12):2025–2029. 5. Hershko A, Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem 1992; 61:761–807. 6. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998; 67: 425–479. 7. Adams J, Palombella VJ, Elliott PJ. Proteasome inhibition: a new strategy in cancer treatment. Invest New Drugs 2000; 18(2):109–121.
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21 Farnesyltransferase Inhibitors: Current and Prospective Development for Hematologic Malignancies Judith E. Karp Division of Hematologic Malignancies, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, U.S.A.
INTRODUCTION Farnesyltransferase inhibitors (FTIs) are potent and selective competitive inhibitors of intracellular farnesyl protein transferase (FTase), an enzyme that catalyzes the transfer of a farnesyl moiety to the cysteine terminal residue of a substrate protein (1). A host of intracellular proteins are substrates for prenylation via FTase, including Ras, RhoB, Rac, membrane lamins, and centromeric proteins (CENPs) that interact with microtubules to promote the completion of mitosis (1–3). Interruption of prenylation may prevent substrates from undergoing maturation, which, in turn, may result in the inhibition of cellular events that depend on the function of those substrates. This finding is the basis for the development of FTIs for therapy of diverse malignancies. This chapter will focus on the current state of knowledge regarding critical molecular targets for FTI action and on the burgeoning application of this class of agents to the treatment of hematologic malignancies, specifically the spectrum of acute and chronic myeloid malignancies and multiple myeloma (MM).
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SELECTED MOLECULAR TARGETS IN HEMATOLOGIC MALIGNANCIES: MOVING BEYOND RAS Initially, FTIs were developed to target the Ras family of oncoproteins, which are overexpressed in both epithelial and hematologic malignancies, either as the result of activating mutations or activation downstream of tyrosine kinase ligand-receptor interactions (2,4–6). Ras polypeptides are synthesized as cytosolic precursors that must attach to the cell membrane in order to transmit signals. Attachment depends upon the addition of a 15-carbon farnesyl group to a C-terminal amino acid sequence motif known as the CAAX box in a reaction that is catalyzed by FT (1,6). Thus, FTIs were initially developed on the premise that FT inhibition would prevent the posttranslational processing of Ras proteins and thereby impede an early junctional process in overall signal transduction (7,8). There is a compelling body of evidence to support the contention that overexpressed Ras proteins can play a critical role in leukemogenesis and leukemic clonal expansion. Such evidence emanates from an in vivo model where a majority of irradiated mice reconstituted with bone marrow transfected with activated N-ras developed severe hematopoietic defects pathologically similar to acute myelogenous leukemia (AML), myelodysplasia (MDS) and myeloproliferative disorders (MPDs) (9). A critical role of Ras in leukemogenesis can be demonstrated in a chronic myelogenous leukemia (CML) model, as well, where leukemic transformation depends upon the presence of a specific fusion bcr-abl gene and it encoded Bcr-Abl encoded protein. In this model, introduction of dominant negative (inactive) Ras into Bcr-Abl transfected hematopoietic or fibroblast cells results in a complete blockage of malignant transformation (10). In a similar light, disruption of the Grb-2/SOS complex, a key guanine nucleotide exchange factor that positively regulates Ras, blocks Ras-mediated cell proliferation in both Bcr-Abl expressing cell lines and freshly isolated CML blasts (11). Nonetheless, while FTIs have well-established preclinical activity in Rastransformed tumors and cell lines, their activity is neither necessarily limited to Ras-mutated tumors nor is the effect equal among tumors bearing different mutated Ras isoforms. As we will discuss below, FTIs are by no means ‘‘selective’’ as they target proteins involved in disparate pathways and thereby exert effects on multiple mechanisms of cellular survival, including angiogenesis, cellular adhesion, and inhibition of apoptosis (Table 1). This notion is further substantiated by recent DNA microarray analyses of selected AML cell lines and primary AML marrow blasts, where tipifarnib modulates the expression of several gene networks, upregulating multiple genes involved in apoptosis, immunity, and cell-cell adhesion and cytoskeletal organization, while downregulating genes involved in proliferation and cell cycle progression (12). Ras-Related Proteins: Rho, Rac, and Rheb The Rho proteins are Ras-related GTP-binding proteins that coordinate growth factor–induced assembly of intracellular focal adhesions and actin stress fiber
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Table 1 Selected Cellular Proteins Undergoing Farnesylation Protein
Cell structure/function
Ras Ras-related proteins Rho Rab Rheb Lamins HDJ-2 Centromeric proteins
Signal transduction Cytoskeletal organization, adhesion Endosomal trafficking Cell cycle progression Nuclear membranes, mitotic spindle assembly Chaperone protein Centromere-microtubule interactions
formation (3,13). Posttranslational prenylation of Rho proteins leads to both farnesylated and geranylgeranylated forms (14,15). RhoB appears crucial for oncogenic Ras transformation, as dominant inhibitory RhoB mutants are capable of suppressing H-Ras-induced transformation of cell lines (16). Recent evidence suggests that RhoB may mediate the antineoplastic effect of FTIs. To this end, it has been shown that FTI treatment blocks RhoB farnesylation, leading to an increase in the geranylgeranylated form of RhoB via a preferential increase in geranylgeranyltransferase (GGT) type I activity and subsequent cell growth inhibition (17,18). Interestingly, the growth inhibitory and apoptotic effects elicited by FTIs in Ras-transformed cells can be abrogated by the introduction of ectopic forms of RhoB, further implicating a role for RhoB as an important target of FTIs (19). Somewhat to the contrary, however, other investigators found that farnesylated and geranylgeranylated RhoB are both potent tumor growth inhibitors, and that FTI-directed prenylation changes of RhoB may not account for the antitumor effects of these agents (20). While the mechanisms by which RhoB and FTIs intersect to block tumor cell growth are not clear, the significance of Rho proteins as FTI targets is substantiated by the recent findings of Raponi et al. (21) that overexpression of a guanine nucleotide exchange factor for Rho proteins known as AKAP13 is linked to clinical tipifarnib resistance in patients with relapsed or refractory AML (discussed below in ‘‘Gene Expression: Insights into FTI Mechanisms of Action and Determinants of Response’’). With respect to other Ras-related proteins, studies in Caenorhabditis elegans demonstrate that FTIs can induce apoptosis by inhibiting the structurally distinct prenyltransferase enzyme Rab-GGT (GGT type II), which prenylates the Ras-related protein Rab (22). Rab proteins regulate endosomal trafficking and, like Ras, require posttranslational prenylation for membrane attachment and function. FTIs abrogate Rab-GGT activity and thereby directly induce p53independent apoptosis. In addition, FTIs may exert cytotoxicity by inhibiting farnesylation of the Ras-related protein RHEB (Ras homologue enriched in brain). RHEB inhibition, in turn, blocks downstream mTor/S6 kinase signaling (2,3,23).
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Phosphatidylinositol-3 Kinase/Akt Pathway The phosphatidylinositol-3 Kinase (PI3K)/Akt pathway occupies a critical position in the transduction of signals that begin with growth-stimulating cytokines and end with cell proliferation and survival (24–26). At least one trigger for this pathway is activated Ras, which interacts directly with the PI3K catalytic subunit as a critical first step in the phosphorylation and activation of the pivotal ‘‘second messenger’’ serine-threonine kinase Akt which, in turn, phosphorylates a panoply of substrates involved in cell proliferation and survival following DNA damage or other cellular stresses (25). The PI3K/Akt pathway has a pivotal impact on net drug resistance and overall cell survival of both normal and leukemic hematopoietic progenitors. As a case in point, PI3K/Akt is constitutively active in primary AML progenitor cells, and selective inhibition of the pathway leads directly to apoptosis and potentiates antileukemic drug cytotoxicity (27). PI3K/Akt pathway may be a critical target for FTIs, as elucidated by Jiang et al. (28), who demonstrated that FTI-277, a peptidomimetic enzyme inhibitor, could block PI3K/Akt activation, thereby abrogating Akt-driven phosphorylation and subsequent inactivation of the proapoptotic BAD protein, impeding integrin-dependent cell survival and inducing apoptosis. In human hepatocellular carcinoma cells, ABT-100 yields growth suppression by decreasing Akt-dependent phosphorylation of the cyclin-dependent kinase (CDK) inhibitor p27kip1 with concomitant nuclear accumulation of p27kip1, association with CDK2 and resultant inhibition of Cyclin E/CDK2 activity (29). Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF) is a critical determinant for angiogenesis and a pivotal growth and survival factor for hematologic malignancies. In this context, Delmas et al. (30) demonstrated that low doses of tipifarnib can overcome radioresistance in U87 wild-type Ras-expressing human glioblastoma cell line xenografts by reducing xenograft hypoxia in conjunction with decreases in the intracellular levels of hypoxia-inducible factor (HIF)-1a and matrix metalloproteinase (MMP)-2, thereby exerting an antiangiogenesis effect with decreased vessel density. Clearly, this effect is independent of Ras mutations; but, precisely which additional FTI targets may be driving this net antiangiogenesis effect are not yet clarified, although one potential target may be RhoB, which may regulate radioresistance in wild-type Ras-expressing glioma cells (31). Furthermore, PI3K/Akt blockade may be one mechanism by which ABT-100 leads to decreases in VEGF mRNA expression, VEGF secretion in vitro, and tumor angiogenesis in vivo in human tumor xenograft and mouse corneal models (32). Mitotic Proteins The inhibition of protein farnesylation interrupts the function of certain proteins, such as the kinetochore-binding CENPs E and F, which exert their maximal
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effects in the G2 and M phases of the cell cycle and are critical to the orderly completion of mitosis (33,34). The mechanisms by which FTIs impair the full execution of mitosis, however, remain poorly defined. One possibility is that unfarnesylated CENPs are not capable of localizing to and associating with microtubules (34). Alternatively, using human lung cancer cells, Crespo et al. demonstrated that FTIs can inhibit formation of a bipolar spindle, which, in turn, is crucial to proper chromosome alignment at the metaphase plate and in an inability of the FTI-treated cell to progress through mitosis, with resultant accumulation and arrest in prometaphase (35). Furthermore, FTIs can induce checkpoint arrest at G2/M in both a p53-dependent and independent fashion (34,36,37). These effects can occur in Ras-activated (but not Ras-mutated) tumor cells, such as astrocytoma cells, CML cells expressing the p210 Bcr-Abl protein, and human p190 Bcr-Abl-positive acute lymphoblastic leukemia (ALL) cells (36,38,39). Targeting of CENPs by FTIs may be one basis for the combination of FTIs and cytotoxic agents that exert their activity in G2 and/or M phase (for instance, topoisomerase II-directed or taxanes) to augment mitotic arrest and resultant cell death (40), which we will discuss below in ‘‘Combinatorial Approaches.’’
CLINICAL TRIALS IN HEMATOLOGIC MALIGNANCIES Hematologic malignancies provide a fertile testing ground for antitumor agents because of the relative ease with which tumor tissue can be obtained throughout the therapeutic course. The ability to obtain target tissue in a longitudinal fashion provides a unique opportunity to define the relevant molecular components that may be modulated by these compounds and to relate those molecular effects to the clinical outcome. At present, there are three nonpeptidomimetic FTIs being tested clinically in a broad spectrum of hematologic malignancies: tipifarnib (R115777, Zarnestra), lonafarnib (SCH66336), and BMS-214662. Tipifarnib and lonafarnib are given orally, whereas BMS-214662 is administered intravenously because of dose-dependent gastrointestinal toxicities. To date, all three exhibit clinical and molecular biologic activities in diverse myeloid malignancies and MM with modest and acceptable toxicities (Table 2).
Acute Myelogenous Leukemia AML affects older adults disproportionately, with a median age at diagnosis of 68 years. Long-term survival rates are low in elderly AML patients, with median survival less than one year (41,42). In addition, the rates of chemotherapyassociated severe toxicities, along with death, are very high in this disease. In adults older than 60 years, the risk of treatment-related death approaches 20% or more during the induction phase of therapy (42,43). Even for those patients who achieve complete remissions (CRs) through chemotherapy, the remission
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Table 2 Clinical Trials of FTIs as Single Agents in Poor-Risk Hematologic Malignancies Disease AML New diagnosis relapsed/refractory MDS (intermediate to high risk) CML (imatinib refractory) Other MPD MM
Complete response (%)
Overall response (%)
Ref.
14a 5–10 15b
24 15–30 32
47 46,48,49 50–54
10–25c
33
55,56
10–20 0
40 20
58,59 67
a
Complete remission duration 7.3 mo, overall survival of CR patients 18.3 mo. Complete remission duration 11.5 mo, time to AML transformation 12.4 mo. c Hematologic and cytogenetic responses. b
duration is generally on the order of only six to nine months (41–43). Adverse karyotypic profiles, including deletions of chromosomes 5 and 7 or complex karyotypes, abound in elderly AML patients and are associated with poor longterm outcomes (44). Chemotherapy failure in elderly patients with AML is also attributable to the presence of multidrug resistance phenotype, which correlates negatively with remission rate (45). For all of these reasons, novel, targeted, and less toxic agents are necessary to improve outcomes in patients with AML. The first clinical testing of FTIs in AML was a phase I trial of the orally bioavailable FTI tipifarnib administered for 21 days in patients with relapsed or refractory AML (46). Consistent inhibition of FTase activity occurred at or above 300 mg BID orally and dose-limiting toxicity (DLT), manifested as readily reversible central neurotoxicity, was observed at 1200 mg BID. Oral absorption was rapid, with linear pharmacokinetics, and there was a dose-dependent increase in drug concentration in marrow with sustained levels two- to threefold higher than concomitant levels in peripheral blood. Clinical responses were observed in 10 of 34 patients (29%), including two CRs in patients with relapsed AML, and occurred across all dosing levels (100–900 mg BID) without strict relationship to the degree of leukemic cell FTase inhibition. Intriguingly, responses were independent of Ras mutational status, as none of the 34 leukemic samples demonstrated an N-Ras mutation. On the basis of these findings, Lancet et al. (47) conducted a unique phase II study of tipifarnib administered at a dose of 600 mg BID for 21 out of 28 to 63 days for 158 older adults with previously untreated, poor-risk AML. The median age was 74 years, 75% had antecedent MDS, and 47% had adverse cytogenetics. Treatment-related mortality was 7%. The CR rate in these elderly, poor-risk patients was 14%, with an additional 10% partial response (PR). Among patients achieving CR, 82% had prior MDS and 40% had adverse cytogenetics. While
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median overall survival (OS) for all 158 patients was 5.3 months, the median CR duration was 7.3 months and median OS for CR patients was 18.3 months. Patients who achieved PR or hematologic improvement (HI) enjoyed a survival advantage as well, with a median OS of 12.6 months. In contrast, the median survival for patients who did not evince any type of response was 3.6 months. Measurements of inhibition of farnesylation of the chaperone protein HDJ-2 in marrow blasts obtained on day 8 of tipifarnib therapy revealed an increase in unfarnesylated protein in 75% of marrow samples, while inhibition of farnesylation of the nuclear membrane protein Lamin A could be detected in 92% of all concomitantly-obtained samplers of buccal mucosa (47). This provocative finding may provide insight into potential mechanisms of FTI resistance, as discussed later in this chapter. Tipifarnib has also been studied in a large, international, multicenter phase II trial in relapsed and refractory AML patients (48). In this study, 17% of evaluable patients with relapsed disease had a reduction in the bone marrow blasts to less than 5%. CR, including CR with incomplete platelet recovery (CRi), however, was only 6% in this heavily pretreated group. Nonetheless, DNA microarrays on 80 pretreatment marrow samples have uncovered a gene expression signature that may predict for response to tipifarnib and have defined the overexpression of the lymphoid blast crisis oncogene (oncoLBC), or AKAP13, as a predictor of tipifarnib resistance (21), to be discussed further in the section on ‘‘Gene Expression: Insights into FTI Mechanisms of Action and Determinants of Response.’’ A recent phase I study of the intravenous nonpeptidomimetic FTI BMS214662 has been conducted by Cortes et al. in patients with relapsed and refractory acute leukemias and high-risk MDS (49). Of 30 patients, 5 (17%) enjoyed clinical responses including 4 of 19 (21%) with AML and complex cytogenetics who achieved either CRi or a morphologic leukemia-free state and one of eight with high-risk MDS. DLT was defined for one-hour infusion at 157 mg/m2 by gastrointestinal symptoms, hypokalemia, and supraventricular tachycardia, but no DLT occurred with 24-hour continuous infusion, where a weekly dose of 300 mg/m2 was established for phase II testing. The kinetics of FTase inhibition also supports the continuous infusion, since the one-hour administration yielded a 60% inhibition FTase but with complete recovery of enzyme activity at 24 hours. Myelodysplasia Several studies have aimed to define the activity of FTIs in MDS. Kurzrock and colleagues have completed phase I and II clinical trials of tipifarnib (50–52). In the original phase I study, tipifarnib was administered twice daily, in a threeweeks-on/one-week-off schedule. DLTs occurred at 900 mg BID with fatigue and confusion (52). Responses were noted in 6 of 20 patients (30%), including two PRs and one CR. As reported in the phase I trial for acute leukemias (46),
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responses were seen across all dosing cohorts, including the very lowest. In the phase II trial, where tipifarnib was administered at 600 mg BID for four weeks out of every six, significant treatment-related toxicity consisting of fatigue, myelosuppression, neurotoxicity, and rash necessitated dose reduction or discontinuation of in over 40% of patients (51). Clinical responses (CR þ PR) occurred in only a minority of patients (11%), due at least in part to the high rate of drug discontinuation. The largest trial of tipifarnib in MDS to date is an international phase II study of 82 patients with intermediate and high-risk MDS who received tipifarnib 300 mg twice daily (50). The CR rate was 15%, an additional 17% had HI for an overall response rate of 32%, and 45% had stable disease. The CRs appeared to be durable, with median time to progression being more than 12 months and over 50% being alive at three years. Nonetheless, grades 3 or 4 myelosuppression occurred in roughly 30% of these patients, suggesting that the optimal dose and schedule of tipifarnib that would minimize toxicity and maximize efficacy remain to be determined. To this end, the same group designed a phase I trial of alternate week dosing of tipifarnib in MDS (53). Treatment was well tolerated, with DLTs occurring at a dose of 1300 mg daily. Fifteen of fiftyone patients (29%) achieved response (3 CR, 12 HI), at a median time of eight weeks. Once again, responses occurred at the lowest dosing level (100 mg BID), with no relation the Ras-mutational status. Lonafarnib is another nonpeptidomimetic FTI undergoing clinical trials in hematologic malignancies. In a phase I/II study of 32 MDS and 35 chronic myelomonocytic leukemia (CMML) patients treated with continuous oral lonafarnib (54), DLT occurred at 300 mg BID with diarrhea and fatigue. Twelve of forty-two (29%) evaluable patients responded, mainly in the form of HI with improvements in erythroid and platelet response but also with two (5%) achieving CR. Interestingly, nearly half of patients presenting with bone marrow blasts more than 5% experienced a 50% or greater reduction while receiving lonafarnib. CML and Other MPDs CML is relevant clinical target for FTI-based therapy, as evidenced by in vitro findings that both peptidomimetic and nonpeptidomimetic FTIs can inhibit proliferation and induce apoptosis in CML cell lines. The first available clinical data regarding FTIs for CML were reported by Cortes and colleagues, in which 7 of 22 patients with previously (and often heavily) treated CML achieved complete or partial, although transient, hematologic response to tipifarnib (55). Interestingly, responses were related to the presence of an elevated serum VEGF level pretreatment and a significant decrease in VEGF during FTI therapy (55). In a related trial of lonafarnib in patients with CML in chronic or acceleratedphase in whom imatinib had failed, 2 of 12 patients achieved hematologic response with evidence of increased peripheral blood myeloid maturation in
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several others (56). The development of combination approaches for imatinibresistant CML are discussed below in the section on ‘‘Combinatorial Approaches.’’ MPDs in addition to CML may also be appropriate disease targets for FTI therapy. This heterogeneous group of disorders includes agnogenic myeloid metaplasia (AMM), polycythemia vera (PV), essential thrombocythemia (ET), and atypical CML and CMML, both of which are considered to be hybrid MPD/ MDS disorders. In AMM, Mesa et al. found that the addition of tipifarnib at low doses (<50 nM) to peripheral blood mononuclear cells results in significant inhibition of both myeloid and megakaryocytic colony formation, with megakaryocytic progenitors being exquisitely sensitive to doses less than 5 nM (57). Such inhibition also occurred in normal controls and patients with other chronic MPDs (PV and ET). Interestingly, similar results were achieved with the PI3K inhibitor LY294002, substantiating the notion that tipifarnib exerts at least part of its effect by suppressing PI3K activity (28). Clinically, tipifarnib given to 34 patient with symptomatic MPDs at a dose of 300 mg BID for 21 of every 28 days yielded significant responses in organomegaly in 33% and in transfusionrequiring anemia in 38%, but without appreciable effect on cytogenetic abnormalities, degree of myelofibrosis, or intramedullary angiogenesis (58). In a phase I/II trial of tipifarnib, led by Gotlib (59), in a cohort of patients with CMML, atypical CML, and unclassifiable hybrid diseases, sustained reductions in the WBC were noted in 24% of patients, while an additional 41% experienced nonsustained reductions. No cytogenetic responses were noted, and no baseline Ras mutations were observed. Likewise, of 25 CMML patients treated with lonafarnib 200 to 300 mg BID continuously, eight (32%) achieved a clinical response (1 CR, 7 HI) (54). However, lonafarnib can induce hyperleukocytosis and pulmonary leukostatis consistent with a ‘‘leukemia differentiation syndrome’’ in 3 of 35 CMML patients, with an additional 12 patients exhibiting a less dramatic leukemoid response (60). The leukocytosis and accompanying respiratory distress occurred predominantly in patients with proliferative CMML and responded to steroids and lonafarnib discontinuation. The exacerbation of proliferation and release may relate to the ability of lonafarnib to modulate integrin activation and thus CMML cell adhesion to vascular endothelium and bone marrow stroma (61). No such syndrome has been detected with tipifarnib to date. Multiple Myeloma MM is a disease that is associated with a significant incidence of Ras mutations, and multiple groups of investigators have demonstrated that both peptidomimetic (FTI-277) and nonpeptidomimetic (tipifarnib) agents can induce apoptosis and growth inhibition in primary MM cells and MM cell lines (62–66). Indeed, tipifarnib impedes the activation of STAT3 and ERKs by IL-6-64 and FTI-277 induces growth inhibition and apoptosis in U266 and 8226 MM cells that exhibited broad drug resistance from multiple mechanisms (63). However, in
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MM, as in the myeloid malignancies, FTI effects do not appear to relate clearly to Ras mutations and overall Ras activity. For instance, FTI-277 abrogates IL-6driven proliferation of ANBL-6 MM cells, even in the absence of Ras mutations, and the induction of apoptosis in these cells is independent of Ras protein processing (66). Similarly, Beaupre et al. (62) demonstrated that tipifarnibinduced apoptosis in U266 MM cells occurs despite the presence of unimpeded Ras protein prenylation. Instead, tipifarnib appears to trigger apoptosis by three independent pathways: (1) cell cycle arrest in late S/G2, which, in turn, induces nuclear stress and activates caspase 2; (2) blockade of nuclear lamin farnesylation, which also induces nuclear stress; and (3) triggering of the intrinsic apoptotic cascade involving Bax activation, loss of mitochondrial membrane integrity, and activation of the endoplasmic reticulum (ER)-stress response with subsequent caspase 9 activation. BMS-214662 also triggers an apoptotic cascade in MM cells by initially increasing intracellular PUMA, which, in turn, causes activating conformational changes in Bax and Bak, decreases in MCL-1 levels, mitochondrial destabilization, and caspase activation (64). Irrespective of the particular mechanism, MM presents a panoply of targets for potential interdiction by FTIs. In a classical correlative clinical-laboratory trial, Alsina and colleagues (67) treated 43 patients with advanced, heavily previously treated MM with tipifarnib 300 mg twice daily for three out of every four weeks. While there were no CRs, 27 patients (64%) experienced disease stabilization, including four patients who achieved 25% to 49% M-protein reductions, with a median time to progression of four months, and 40% of patients enjoying disease stabilization for at least five months (range 2–26 months). As in the original studies in AML (46), MDS (52), and CML (55), these investigators studied MM marrow populations prior to and during tipifarnib therapy (Table 3) and detected FT enzyme inhibition, inhibition of HDJ-2 farnesylation, and decreases in some but not all signaling intermediaries (phospho-Akt and phospho-STAT3, but not Table 3 In Vivo Molecular Correlates in Malignant Marrow Cells Obtained During Tipifarnib Clinical Trials: What Are We Really Targeting? Target
AML
MDS
MPD
MM
Ras mutations FTAse inhibition Inhibition of chaperone protein HDJ2 farnesylation Decreases in signaling intermediaries p-ERK p-AKT p-STAT3
No Yes Yes
No Yes ND
No Yes Yes
No Yes Yes
No No ND
No No No
ND ND ND
No Yes Yes
Abbreviations: AML, acute myelogenous leukemia; MDS, myelodysplasia; MPD, myeloproliferative disorders; MM, multiple myeloma; FTAse, farnesyl protein transferase.
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phospho-ERKs). Again, no relationship between Ras mutations and clinical outcome could be discerned. CONTINUING CLINICAL DEVELOPMENTS Minimal Residual Disease Setting The minimal residual disease state following remission induction and consolidation therapies offers a fertile testing ground for FTIs, as well. To date, postremission chemotherapy for AML in elderly patients or those with other poor-risk features (e.g., MDS/AML, treatment-related AML) has not prolonged diseasefree survival (DFS) or OS (41–43). It is reasonable to speculate that FTIs might suppress regrowth of the malignant clone when the residual tumor burden after cytotoxic chemotherapies has been reduced to a minimal state. This concept is being tested in AML patients in first CR who are expected to relapse in less than one year (age 60 years and/or secondary AML or adverse cytogenetics) (68). In a phase II trial of tipifarnib monotherapy for 48 adults with poor-risk AML in first CR, tipifarnib 400 mg BID for 14 out of 21 days was initiated after recovery from consolidation chemotherapy, for a maximum 16 cycles. Twenty (42%) completed16 cycles, 24 (50%) were removed from study for relapse, and 4 (8%) discontinued drug prematurely for intolerance. Nonhematologic toxicities were rare, but tipifarnib dose was reduced in 58% for myelosuppression. Median DFS was 13.5 months (range 3.5–60þ months), with 30% having DFS for two years or more. Comparison of CR durations for 25 patients who received two-cycle timed sequential therapy followed by tipifarnib maintenance with 23 historically similar patients who did not receive tipifarnib demonstrated that tipifarnib was associated with DFS prolongation for patients with secondary AML and adverse cytogenetics. This study suggests that some patients with poor-risk AML may benefit from tipifarnib maintenance therapy. Future studies should examine reduced tipifarnib dosing and continuation beyond 16 cycles. In addition, the Eastern Cooperative Oncology Group (ECOG) is conducting a study of tipifarnib following achievement of second CR, which is another setting where eventual relapse is all too common. Combinatorial Approaches While FTIs demonstrate reproducible activities as single agents (Table 2), the resultant outcomes are modest. In order to improve the robustness of these results, the full development of FTIs in the therapeutic armamentarium for hematologic (and other) malignancies will require the design and testing of rational combinations of FTIs with cytotoxic, biologic, and immunomodulatory agents in both the laboratory and the clinic. This is a burgeoning area of investigation in both the clinical and the laboratory arenas. As a case in point, in the clinic, Alvarez et al. (69) are examining the addition of tipifarnib 300 mg
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BID for 21 days to ara-C plus idarubicin induction and consolidation, followed by tipifarnib maintenance 300-mg dose twice daily for 14 days every four to six weeks for six months. Early results demonstrate a 77% CR rate (65% CR, 12% CRi) in the first 95 patients including older adults and those with adverse cytogenetics, with reversible diarrhea and hyperbilirubinemia but very low induction mortality. One obstacle to successful combination therapy is the great heterogeneity by which FTIs may inhibit cell cycle progression. For this reason, it is critical to understand which part of the cell cycle machinery is inhibited by a specific FTI in a specific disease setting. For example, an FTI that blocks entry of the cell into S phase would not act synergistically or additively with a chemotherapeutic agent that is S-phase specific. On the other hand, an FTI that inhibits cell progression through the G2/M phase might be useful in combination with G2/Mdirected agents such as a taxane or epipodophyllotoxin. Along these lines, Zhu and colleagues (40) demonstrated selective synergy between tipifarnib and taxanes with regard to blocking mitosis by inducing G2/M arrest and enhancing mitochondrial-based apoptosis. This synergy occurred in tipifarnib-resistant and taxane-resistant MM cell lines and human MM cells in vitro as well as in vivo in a SCID/hu model and appeared to be specific for this combination, suggesting that the synergy could relate specifically to the combined effects of both drugs on G2/M, with FTI inhibiting CENP farnesylation and taxane stabilizing mitotic spindles. Another potential mechanism may be that FTI (as detected with lonafarnib) can inhibit tubulin deacetylase (i.e., histone deacetylase 6), thereby increasing tubulin acetylation, enhancing microtubulin stabilization, and amplifying taxane-induced mitotic arrest and cell death (70). Etoposide is an epipodophyllotoxin with clinical activity against both newly diagnosed and relapsed leukemias, particularly when combined with other cytotoxic agents (71). Etoposide induces DNA double-strand and single-strand breaks by binding to and stabilizing the covalent linkage between topoisomerase II and DNA, and preventing religation of the resultant strand breaks (72). Because it targets topoisomerase II, it exerts its effects predominantly on cells in the S phase, with subsequent arrest in G2/M. In an attempt to increase CR rates in elderly AML patients, Karp et al. (73) have conducted a phase I trial of oral tipifarnib plus etoposide, with escalating doses of both drugs and 14 versus 21 days of tipifarnib every 28 to 63 days (median time to cycle 2 day 31, range 29–43 days). A total of 84 adults 70 years or older (median 77 years, range 71–91 years) were treated at 14 dose levels of tipifarnib (300, 400, or 600 mg twice daily, 14 or 21 days) þ etoposide (100, 150, or 200 mg daily days 1–3 and 8–10) for a median two cycles (range 1–7 cycles). The majority (90%) had at least 1 comorbidity, and 40% had 3 or more comorbidities, 58% had secondary AML, and 54% had adverse cytogenetics. Hospitalization was necessary in 54% during cycle 1, but was only 28% for all cycles. Treatment-related mortality was 11%. Twenty-one (25%) achieved CR and an additional 13(15%) achieved PR/HI, for an overall response rate that approaches 40%. Median duration of CR so far is
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10.5 months. Fifteen of forty-eight (31%) patients receiving tipifarnib greater than or equal to 400 mg BID versus 5/36 (14%) tipifarnib 300 mg BID and 16/54 (30%) receiving tipifarnib for 14 days versus 4/30 (13%) tipifarnib 21 days have achieved CR. Median age of CR patients is 77 years (range 71–86 years), and 8 (40%) had adverse cytogenetics. CR durations to date range from 2 to 27þ months. The mechanisms for the apparent synergistic interactions between tipifarnib and etoposide are not yet clear, but may relate in part to combined effects of both drugs on G2/M arrest and completion of mitosis. Indeed, the targeting of CENPs by tipifarnib may be one basis for the additive and/or synergistic interactions between tipifarnib and etoposide in terms of augmenting G2/M arrest, mitotic arrest and resultant cell death. Recent data suggest that tipifarnib inhibits the drug efflux activity of the prototypical drug-resistance protein P-glycoprotein (P-gp) in a dose-dependent fashion in human acute T-lymphoblastic leukemia and AML cell lines in a manner that is independent of its farnesyltransferase inhibitory activity (74). The combination of tipifarnib and daunorubicin exhibited synergistic induction of apoptosis and cytotoxicity. These data should apply directly to the combination of tipifarnib and etoposide, since both daunorubicin and etoposide are P-gp substrates (74,75). Moreover, the ability of tipifarnib to modulate P-gp activity and the possibility that such modulation contributes to synergy with etoposide may have special relevance to elderly patients with AML, where high levels of P-gp expression are common and correlate with clinical drug resistance (45). The combination of FTIs with other signaling inhibitors is based on the rationale that malignant hematopoietic cells are governed by a diverse and often redundant array of signaling networks, thereby nullifying the ability of a single pharmacologic agent to abrogate the cellular proliferative and survival processes. The combination of FTI with the clinically proven Bcr-Abl inhibitor imatinib is of great interest, particularly in the clinical setting of imatinib resistance. Cortes et al. conducted phase I trials of tipifarnib plus imatinib (TþI) (76) and lonafarnib plus imatinib (LþI) (77). Patients enrolled in both trials had imatinibrefractory disease. Patients with accelerated phase (AP) or CML in blast crisis (BC) were included in the LþI study, as well (76). At the maximal tolerated dose of 400 mg twice daily for both drugs, TþI resulted in blood count normalization in almost all patients, with complete hematologic response (CHR) achieved in 6 of 11 (54%) and cytogenetic responses achieved in 25% including 1 CR and 1 partial cytogenetic remission in a patient with the imatinib-resistant Bcr-Abl T315I mutation (76). For chronic phase CML patients treated with LþI, two of nine (22%) achieved CHR, and 4 (29%) of 14 AP/BC patients achieved hematologic responses (2 CHR, 1 partial, 1 HI) (77). These results of FTI plus imatinib clearly warrant further clinical investigation for patients with all stages of imatinib-resistant CML. MM cell survival is dependent on multiple inflammatory and growthpromoting cytokines, which, in turn, activate diverse signaling pathways including Ras-driven pathways (78). While there is evidence of modest anti-MM
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clinical activity with tipifarnib (67), it is reasonable to speculate that we need to overcome the exuberant and constitutive activation of multiple signaling pathways with agents that target additional intermediaries (78). In this regard, Yanamadra et al. (79) have demonstrated synergy between tipifarnib and the proteasome inhibitor bortezomib in both AML and MM cells that relates to the ability of both drugs to activate endoplasmic reticulin stress and consequent activation of apoptosis cascades that are both dependent and independent of mitochondrial dysfunction. David et al. (80) found the combination of lonafarnib and the bortezomib induces rapid cell death in MM cells in association with increased cleavage of caspases 3, 8, and 9 and with down regulation of phosphoAkt, although cell death occurred even in the presence of active phospho-Akt or BCL-2. These investigators further demonstrated that administration of bortezomib followed by lonafarnib offered optimal apoptosis induction. These studies are templates for clinical trials of bortezomib followed by lonafarnib in MM and a related trial of tipifarnib and bortezomib in AML. Resistance to FTIs Malignant cells have an uncanny ability to become resistant to new antitumor agents, and it is likely that the FTIs will be no exceptions. Such resistance, whether intrinsic or acquired, limits net drug efficacy. To date, resistance to FTIs has been demonstrated in diverse cell lines that have been developed via selective adaptation (81–84) or have been genetically engineered to express FT enzyme mutations (85,86). For example, a tipifarnib-resistant human colon cancer cell, line generated by continuous drug exposure, exhibits a marked reduction in the FT enzyme itself without enzyme mutations or aberrations in activation of enzyme subunits (83). In a similar construct, Zhang et al. (84) created a lonafarnib-resistant Philadelphia chromosome-positive ALL (PhþALL) cell line from transgenic mice by growing those cells on stroma in the presence of increasing lonafarnib concentrations. The resultant lonafarnibresistant cells exhibit marked increase in gene expression of the novel ATP binding cassette (ABC) transporter homolog ATP11a and, interestingly, relatively resistant to imatinib as well. Buzzeo et al. (81) have developed and characterized a novel MM line derived from 8226 (8226/R5) that exhibits resistance to both tipifarnib and bortezomib, with such resistance unrelated to FTase mutations, drug transporters, or heat shock protein expression. In comparison with drug sensitive 8226 cells, gene expression profiling of the resistant 8226/R5 cells revealed increased expression of Jak2 and STAT1 with marked elevation in both phosphorylated STAT1 and STAT3 and, as a result of activated STAT3, an increase in BCL-XL mRNA and protein expression. In addition, 8226/R5 cells were found to overexpress a particular isoform of the PI3K p110 subunit. It is reasonable to speculate that overexpression of key components of the PI3/Akt antiapoptosis pathway by any mechanism might be expected to confer relative resistance to FTI-induced apoptosis. Whether or not any of these
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mechanisms of resistance are clinically relevant is not yet known. The elucidation of these and other mechanisms of resistance at both cellular and humoral levels of drug disposition is an important aspect of the ongoing clinical development of FTIs. In the clinical arena, Goemans et al. (87) examined drug sensitivity profiles of pediatric leukemias, using a methyl-thiazole-tetrazolium (MTT) assay to compare tipifarnib responsiveness with traditional cytotoxic agents. T-cell ALLs and acute monoblastic AMLs exhibited the greatest tipifarnib sensitivity without correlation between Ras mutational status or in vitro drug responsiveness but with correlation in AML samples between resistance to tipifarnib and resistance to anthracyclines or etoposide. These studies may be a useful template for understanding shared mechanisms of drug resistance among structurally diverse compounds and, in turn, providing insights into strategies by which to overcome such resistance factors. Gene Expression: Insights into FTI Mechanisms of Action and Determinants of Response The ability to identify molecular predictors of response to specific drugs or drug combinations should direct the selection of a therapeutic approach that is most likely to induce meaningful clinical response. Toward this end, microarray technology provides a spectrum of gene expression signatures in AML, which, in turn, affords a molecular stratification of patients with respect to disease, biology, and clinical outcome. Raponi et al. (12) identified integrated gene networks whose activities are modulated in an orchestrated fashion to yield net cell death in diverse AML cell lines and in primary AML marrow samples. Further, these investigators conducted gene profiling of marrow blasts from adults with relapsed and refractory AML obtained prior to treatment with tipifarnib in the setting of an international phase II study in relapsed and refractory AML patients (21). In this setting, it appears that eight genes are differentially expressed in responders versus nonresponders, with overexpression of one gene in particular, the oncoLBC, or AKAP13, capable of accurately predicting clinical response to tipifarnib (21). Provocatively, the AKAP13 protein acts a guanine nucleotide exchange factor for Rho proteins (88,89) and contains a region that is homologous to an a-helical domain known to interact with nuclear envelope protein lamin B (90). This is especially intriguing because Rho and lamin proteins are farnesylated, and AKAP13 activates both types of proteins. The recent discovery by Tsai et al. (91) that lamin B is critical to the assembly of the mitotic spindle makes it tempting to speculate that AKAP13 might have an indirect role in promoting or permitting completion of mitosis, perhaps in concert with another group of FTI targets, namely CENPs. More recently, Raponi et al. (92) have analyzed gene expression profiles of 79 marrow blast samples from elderly adults with newly diagnosed AML obtained longitudinally prior to, during, and/ or following tipifarnib treatment on the phase II study in newly diagnosed adults
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with poor-risk AML, discussed previously (47). Tipifarnib treatment in vivo induced alterations in global gene expression in AML marrow blasts that persisted for up to 120 days posttreatment. Roughly 500 genes involved in protein biosynthesis, intracellular signaling, DNA replication, and cell cycle progression appear to undergo significant changes in expression as a result of in vivo exposure to tipifarnib, with a subset of 27 genes differentially expressed in responders versus nonresponders. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS In summary, FTIs inhibit malignant cell growth and survival by interfering with intracellular signaling pathways. As single agents, they have reproducible clinical effects on diverse hematologic malignancies, but to date, those effects are detected in a minority of patient subgroups. As with all other malignancies, the optimal approach is likely to lie in rational combinations of FTIs with cytotoxic, biologic, and/or immunomodulatory agents with non-cross-resistant mechanisms of action. The clinical trials that are currently in progress and under development will provide the critical foundations for defining the optimal roles of FTIs in patients with hematologic malignancies. Nonetheless, there are some important conclusions to be drawn on FTI-based therapy from the experience to date. First of all, both myeloid and certain lymphoid malignancies appear to depend, at least in part, on signaling processes that may be targeted directly or indirectly by FTIs. Secondly, these agents have clinical activity across a broad spectrum of hematologic malignancies, including AML, MDS, CML, MPDs, and MM. Finally, toxicity data highlight a relative paucity of severe adverse effects directly attributable to these agents. These characteristics, in total, are important because they point out the feasibility of eventually combining FTIs with other agents such as chemotherapy as well as highlight the potential of these agents for use over an extended period, longer than would be possible for traditional cytotoxic agents. The precise mechanisms by which FTIs exert their cytotoxicity remain to be defined. The original notion that these agents targeted Ras mutations is clearly not complete, and it is likely that FTIs have an impact on multiple molecules and pathways involved in cellular integrity. Studies to define the mechanisms by which FTIs alter cellular metabolism and modulate the activities of specific signaling pathways in normal and malignant precursors are a pivotal part of this effort. In this regard, the potential impact of FTIs on other disease processes is presaged by the ability of ABT-100 to ameliorate the process of premature aging in a mouse model of progeria which, like the human condition, is characterized by accumulation of an abnormally farnesylated form of the lamin A precursor prelamin A, disruption of orderly nuclear scaffolding, and resultant misshapen nuclei (93). The clinical and correlative laboratory trials in progress and under development will provide the critical foundations for defining the optimal roles of FTIs in patients with cancer and perhaps other diseases of disordered cellular metabolism as well.
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22 Targeting Notch Pathways Jennifer O’Neil and A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, U.S.A.
INTRODUCTION The Notch Signaling Pathway Thomas Hunt Morgan first described a mutant Drosophila melanogaster strain with ‘‘notched’’ wings in 1917. The gene responsible for this phenotype was discovered many years later and named Notch (1). The human Notch1 homolog, also referred to as TAN1, was identified in T-cell acute lymphoblastic leukemia (T-ALL) patients with the t(7;9)(q34;q34.3) chromosomal translocation (2). The mammalian Notch proteins are heterodimeric transmembrane receptors that control cell proliferation, apoptosis, and cell fate during the development of diverse cellular lineages (3). In adults, Notch signaling regulates stem cell maintenance, binary cell-fate decisions such as B- versus T-lineage differentiation, and differentiation of self-renewing organs (4). Notch is synthesized in the endoplasmic reticulum and is transported to the Golgi network where it is posttranslationally modified. A proteolytic clevage (S1) separates the extracellular portion of the protein from the intracellular form. These two parts of Notch then form a heterodimer that is transported to the cell membrane. Binding of Notch ligands, such as Delta and Serrate, initiates a series of additional proteolytic cleavages in Notch. The last of these cleavages, which is catalyzed by g-secretase, results in the release of the Notch intracellular domain (NICD) permitting it to translocate to the nucleus and form part of a
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Figure 1 The Notch signaling pathway. (A) Pre-Notch is transported from the ER to the Golgi where it is fucosylated and glycosylated. (B) Furin cleaves pre-Notch into the extracellular and intracellular domains (S1 cleavage). (C) Ligand binding initiates the S2 and S3 proteolytic cleavages. (D) The intracellular domain of Notch (NICD) translocates to the nucleus where it interacts with the transcription factor CSL and the coactivator Maml1 and activates the transcription of genes including Deltex, Hes1, and Nrarp. (E) NICD is ubiquitinated and targeted to the proteosome by FBW7. Source: From Ref. 58.
multiprotein complex. The NICD interacts with the DNA-binding protein CSL, displaces corepressors and recruits coactivators, thereby converting CSL from a repressor to activator of gene transcription (Fig. 1). Structural studies have demonstrated that the interaction between the Ankyrin domain of Notch1 and the Rel-homology domain of CSL creates a binding site for the Maml1 Notch coactivator that recruits other coactivators such as p300 and PCAF (5). Notch Signaling in Thymocyte Development Notch signaling is critical for several cell fate determinations in mammalian organisms including the T- versus B-lineage choice in early hematopoietic progenitors. Notch signaling promotes the proliferation, differentiation, and survival of early T-cell progenitors (ETPs) (6–8). Conditional deletion of adult
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Figure 2 The role of Notch in T-cell development. Notch signaling may be involved in the self-renewal of HSCs and is essential for the development of ETPs. Notch signaling is also required for the differentiation of DP cells of the ab lineage. Abbreviations: HSCs, hematopoietic stem cells; ETPs, early thymic precursors. Source: From Ref. 58.
mouse bone marrow prevents the development of T cells and promotes the development of B cells in the thymus (9,10). Conversely, overexpression of the NICD results in extrathymic T-cell development and suppression of B-cell development (11). Expression of a dominant negative form of Maml1 results in a block in T-cell development similar to that observed in Notch1 deficient cells, suggesting that Maml1 plays a critical role in Notch signaling in thymocyte development (12). Mouse studies have revealed that Notch signaling is also important for the DN3 to DN4 thymocyte developmental transition, because deletion of Notch1 or RBP-J (CSL) in mouse T cells leads to a DN3 arrest (13,14) (Fig. 2). Although many transcription factors control hematopoietic cell fate, Notch appears to be a dominant factor that controls the action of other lineage factors. For example, Notch signaling prevents myeloid or dendritic cell differentiation even when C/EBP or PU.1 is overexpressed (15,16). The mechanism by which Notch controls T-cell development is not known; however, it has been postulated to act in concert with the basic helix-loop-helix transcription factor E47 that is essential for T-cell development (17). ACTIVATED NOTCH SIGNALING IN T-ALL Notch1 Is Activated by Translocation or by Mutation In T-ALL The t(7:9) translocation that places a truncated form of the Notch gene under the control of the TCRb locus is quite rare, occurring in less than 1% of T-ALLs (2). In this translocation, the extracellular, ligand-binding domain of Notch1 is
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deleted, resulting in a constitutively active ligand-independent allele. Mice transplanted with cells expressing the activated Notch1 allele rapidly develop T-cell leukemia, demonstrating the transforming potential of activated Notch signaling (18). A recent study has demonstrated a broader role of activated Notch signaling in human T-ALL. Activating somatic mutations in Notch1 were found in over 50% of T-ALL patient samples and cell lines (19). The mutations were found in all subtypes of T-ALL and were found in two regions of the Notch1 protein. Missense mutations in the heterodimerization domain activate Notch signaling by altering the interaction between the transmembrane subunit and the inhibitory extracellular subunit of Notch1 (20). Frameshift and point mutations in the C-terminal region of the Notch1 gene are also observed. These mutations delete the PEST domain that targets the Notch1 protein for degradation by the proteosome, thereby leading to increased NICD stability and increased Notch signaling (Fig. 3). Subsequently, similar activating mutations were found in mouse models of T-ALL, demonstrating the evolutionarily conserved role of Notch in T-ALL and providing in vivo models to test therapeutics that target the Notch pathway (21,22). T-ALL patients with mutated Notch1 have a poorer prognosis than patients with the wild-type gene, making therapeutics that targets the Notch pathway particularly attractive (23). Mechanisms of NOTCH1-induced T-ALL Studies in mice have shed light on the mechanism of Notch-induced leukemia. The tumors that arise in mice are clonal, indicating that several hits are required for transformation (18). Retroviral insertional mutagenesis screens have shown that Notch1 can collaborate with other oncogenes that can cause T-ALL, including MYC, E2A-PBX, IKAROS, MYB, and TAL1 (http://rtcgd.abcc.ncifcrf.gov) (24–27). Cyclin D3, which is required for proper T-cell development, is also required for Notch-induced T-ALL (28). Initial studies indicated that Notch1-induced leukemogenesis also required pre-TCR signaling, but recent studies have clarified the role of pre-TCR signaling in Notch-induced T-ALL; it is not absolutely required but can facilitate the onset of leukemia (29,30). Notch may also contribute to transformation by inhibiting the tumor suppressor p53 (31). IMPLICATIONS FOR TREATMENT OF T-ALL AND OTHER CANCERS g-Secretase Inhibitors The multicomponent g-secretase enzyme complex that cleaves Notch to release the NICD also cleaves the amyloid precursor protein, leading to the production of plaques in Alzheimer’s patients. As a result, g-secretase inhibitors have already been developed for use as drugs. Treatment of T-ALL cell lines with g-secretase inhibitors leads to G0/G1 arrest, demonstrating that the cells are dependent on Notch signaling for their growth, and suggesting that activation of
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Figure 3 Two classes of activating Notch1 mutations in T-ALL. Notch1 mutations can occur in either the HD domain or the PEST domain. Missense mutation or in frame insertions or deletions are observed in the HD domain. These mutations result in ligandindependent activation of the Notch signaling pathway. Most of the mutations in the Notch HD domain result in a less stable heterodimer that is more susceptible to cleavage by metalloprotease. One described T-ALL Notch1 mutation results in increased metalloprotease cleavage without an effect on heterodimer stability (20). The PEST domain of Notch1 is essential for binding to FBW7, a protein ubiquitin ligase that controls its degradation. Truncating mutations and mutations resulting in stop codons in this domain disrupt the binding of Notch1 to FBW7 and result in increased NICD half-life. Abbreviation: HD, heterodimerization.
the Notch pathway contributes to T-cell transformation by influencing cell cycle progression (19). Subsequent studies have shown that MYC is a key target of Notch in T-ALL (27,32,33). Clinical trials have been undertaken to determine whether g-secretase inhibitors will be effective in treating T-ALL. In addition to its critical role in thymocyte development, Notch signaling also plays an important role in intestinal epithelial stem cell fate. Studies in mouse models have demonstrated that decreased Notch signaling leads to decreased numbers of progenitor cells with
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proliferative potential at the base of the intestinal villi and in their place increased numbers of terminally differentiated and metaplastic mucin-producing goblet cells (34). Conversely, activation of the Notch pathway in the intestine by overexpression of the NICD leads to decreased numbers of terminally differentiated cells (35). Furthermore, toxicity studies using g-secretase inhibitors in animal models have shown intestinal metaplasia with bloody diarrhea and death (36,37). Therefore, side effects related to the role of Notch signaling in intestinal cell development might be expected for patients treated with effective dosages of g-secretase inhibitors. The schedule of drug delivery may need to be optimized to maximize therapeutic benefit while minimizing adverse side effects. Rational Drug Combinations Studies in both mouse and human T-ALL cells have identified the MYC oncogene as a direct transcriptional target of Notch (27,32,33). Importantly, reintroduction of MYC into cells that have been treated with g-secretase inhibitors rescues their growth demonstrating the importance of MYC in Notchinduced T-ALL. Therefore, one rational drug combination would be to treat patients with g-secretase inhibitors along with other drugs affecting the MYC pathway to synergistically reduce MYC levels and block tumor cell growth. One attractive candidate is TMPyP4, a cationic porphyrin that binds to and stabilizes guanine quadruplexes in DNA. MYC contains a sequence in its promoter that forms a guanine quadruplex, and TMPyP4 has been shown to inhibit MYC transcription and the growth of tumor cells in vivo (38). Thus, TMPyP4 or other agents that inhibit MYC transcription or enhance MYC protein degradation, such as ERK inhibitors, may be useful in combination with g-secretase inhibitors. Because of the expected toxicity of g-secretase inhibitors, possible combination therapies that would enable use of a lower dose of the drug are of great interest. A microarray screen for activation of phosphorylation events downstream of Notch in T-ALL identified the phosphorylation of several components of the mTOR pathway (39). Treatment of T-ALL cells with the mTOR inhibitor, rapamycin, in combination with g-secretase inhibitors synergistically inhibited cell growth, suggesting a rational drug combination to treat T-ALL patients (39). Another group has demonstrated activation of the NF-kB signaling pathway downstream of Notch and shown that the proteosome inhibitor bortezomib can reduce IC50s of g-secretase inhibitors in T-ALL cell lines (40) (Fig. 4). Notch Pathway Activation in Other Cancers Although activating mutations in Notch1 have only been identified in T-ALL patients and rarely in AML patients (19,41), there is evidence that the Notch pathway is activated in other cancers including ovarian cancer (42), breast cancer (43), anaplastic large cell lymphoma, Hodgkin’s disease (44), melanoma (45), gliomas (46), lung carcinomas (47,48), and cancers of the pancreas (49) and
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Figure 4 Strategies for targeting the Notch pathway in human malignancy. The Notch signaling pathway can be activated in cancer by several different mechanisms. In T-ALL, the pathway is activated by mutation of Notch1 or mutation of FBW7, the protein ubiquitin ligase that controls the turnover or the NICD. The Notch pathway is activated by amplification of the Notch3 gene in ovarian cancer. Low levels of the Notch antagonist NUMB have been detected in breast cancer patients. Notch activation results in increased levels of MYC, activation of the mTOR pathway, and activation of the NF-kB signaling pathway. The Notch pathway could be therapeutically targeted at several points. The pathway could be directly inhibited through the use of GSIs or other drugs that inhibit Notch processing, dimerization, or proteolysis. Alternatively, levels of the Notch pathway inhibitors FBW7 and NUMB could be increased. The pathways downstream of Notch could also be targeted. TMPyP4 inhibits MYC transcription and has shown efficacy in xenograft models (38). The mTOR pathway that is activated downstream of MYC in T-ALL can be inhibited by rapamycin. Velcade, a proteosome inhibitor, inhibits the NF-kB pathway by inhibiting the degradation of IkB, which sequesters NF-kB in the cytoplasm. Understanding the pathways downstream of Notch has uncovered rational drug combinations for T-ALL patients.
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prostate (50). Therefore, g-secretase inhibitors and other inhibitors of the Notch pathway may have therapeutic potential in diverse forms of human malignancy. MYC has also been shown to be a target of Notch in mammary tumorigenesis demonstrating similarity in the pathways downstream of Notch that lead to transformation in different cell types and suggesting that combination therapies that target both Notch and MYC may be useful in cancers other than T-ALL (51). g-Secretase Inhibitor Resistance in T-ALL Although several human T-ALL cell lines are sensitive to g-secretase inhibitor treatment, most lines are resistant to the drug and proliferate normally despite evidence of Notch pathway activation. It has been hypothesized that because these lines have been maintained in culture for many years, they have lost their dependence of the Notch signaling pathway (19). The more pronounced effect on cell viability observed in mouse T-ALL cell lines that have been cultured for a much shorter time supports this hypothesis (21). However, the results in human cell lines suggest that all T-ALLs may not be dependent on Notch signaling, which has definite implications for clinical trials. We, and also others, have recently discovered that the FBW7 protein ubiquitin ligase, which has been implicated in regulating the turnover of NICD, is mutated in human T-ALL cell lines and patient samples (52,53). The missense mutations we found disrupt the binding of FBW7 to its substrates. We demonstrate that FBW7 controls the ubiquitination and degradation of Notch and cell lines with mutant FBW7 are resistant to g-secretase inhibitor treatment because Notch signaling is maintained due to decreased turnover of the NICD. Mutation of FBW7 is one mechanism by which tumor cells can become resistant to g-secretase inhibitors. Another group has demonstrated that Notch controls PTEN expression through HES1 and MYC. Furthermore, they find loss of function PTEN mutations in g-secretase resistant T-ALL cell lines resulting in constitutive AKT activation and uncovering another mechanism of g-secretase resistance (54). Further studies will be necessary to determine if resistance can be overcome by using drugs that target another point in the pathway in combination with g-secretase inhibitors. Targeting DLL4 Another therapeutic strategy has recently emerged to inhibit the Notch pathway to treat cancer. Delta-like ligand 4 (DLL4) is induced by VEGF and is expressed in tumor vascular cells but not in tumor cells. Soluble DLL4 or neutralizing antibodies to DLL4 inhibit Notch signaling and have been shown to increase tumor vasculature but decrease tumor growth in several xenograft mouse models (55–57). This paradoxical finding is explained by the observation that the newly formed tumor vessels do not mature and thus do not function properly. This strategy has promise for the treatment of solid tumors since it is more specific than g-secretase inhibitors, and fewer side effects would be expected.
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CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS The discovery that activating mutations occur in over 50% of T-ALL patients has demonstrated the importance of this signaling pathway in the molecular pathogenesis of T-ALL. Inhibition of the Notch signaling pathway has opened up a new area for therapeutic intervention. g-Secretase inhibitors hold promise as therapeutics for T-ALL and other malignances. These compounds will likely be most beneficial in combination with other drugs, either those currently used to treat T-ALL patients or other specific inhibitors of pathways downstream of Notch described in this chapter. Specific inhibitors of the Notch signaling pathway, such as antibodies or small molecules that specifically block Notch1 signaling but not signaling by Notch 2–4, may be more effective and produce fewer side effects. The complexity of the Notch signaling pathway provides several additional points at which the pathway could be targeted including ligand binding, dimerization, proteolysis, and transcriptional activity. REFERENCES 1. Wharton KA, Johansen KM, Xu T, et al. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 1985; 43:567–581. 2. Ellisen LW, Bird J, West DC, et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991; 66:649–661. 3. Wilson A, Radtke F. Multiple functions of Notch signaling in self-renewing organs and cancer. FEBS Letters 2006; 580:2860–2868. 4. Radtke F, Wilson A, Mancini SJ, et al. Notch regulation of lymphocyte development and function. Nat Immunol 2004; 5:247–253. 5. Nam Y, Sliz P, Song L, et al. Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 2006; 124:973–983. 6. Taghon T, Yui MA, Pant R, et al. Developmental and molecular characterization of emerging beta- and gammadelta-selected pre-T cells in the adult mouse thymus. Immunity 2006; 24:53–64. 7. Sambandam A, Maillard I, Zediak VP, et al. Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat Immunol 2005; 6:663–670. 8. Lehar SM, Dooley J, Farr AG, et al. Notch ligands Delta 1 and Jagged1 transmit distinct signals to T-cell precursors. Blood 2005; 105:1440–1447. 9. Wilson A, MacDonald HR, Radtke F. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J Exp Med 2001; 194:1003–1012. 10. Han H, Tanigaki K, Yamamoto N, et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int Immunol 2002; 14:637–645. 11. Pui JC, Allman D, Xu L, et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 1999; 11:299–308. 12. Maillard I, Weng AP, Carpenter AC, et al. Mastermind critically regulates Notchmediated lymphoid cell fate decisions. Blood 2004; 104:1696–1702.
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23 mTOR Targeting Agents for the Treatment of Lymphoma and Leukemia Andrea E. Wahner Hendrickson and Thomas E. Witzig Department of Medicine, Mayo Clinic, Rochester, Minnesota, U.S.A.
Scott H. Kaufmann Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, U.S.A.
INTRODUCTION The past decade has witnessed a revolution in our understanding of the biochemical mechanisms that contribute to the survival and proliferation of neoplastic cells. Several of these mechanisms depend on signaling by the phosphatidylinositol-3kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. This pathway integrates signals from multiple receptor tyrosine kinases and regulates many cellular processes, including proliferation, growth, and survival. Because these processes are critical for oncogenic transformation, the PI3K/mTOR pathway is being extensively studied in the hope that it will lead to promising new treatments for hematological malignancies. In the sections that follow, we summarize current understanding of this pathway and the results of recent studies of mTOR inhibitors in lymphoma and leukemia. IMPORTANCE OF THE PI3K/mTOR PATHWAY The PI3K/mTOR pathway (Fig. 1) consists of a series of kinases and other signaling molecules that collectively play several crucial roles in the regulation 525
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Figure 1 Overview of the PI3K/mTOR pathway. Binding of the p85 regulatory subunit of PI3K to activated, tyrosine phosphorylated receptor tyrosine kinases leads to allosteric activation of PI3K, which converts plasma membrane PIP2 to PIP3. Various kinases, including PDK1 and Akt, bind to PIP3 through their PH domains. This membrane binding results in activation of PDK1, which catalyzes the activating phosphorylation of Akt on 308 Thr. Once activated, Akt phosphorylates a number of substrates, including the
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of cell proliferation and survival (1–4). In particular, Akt, a central kinase in this pathway, inhibits apoptosis in a variety of different ways (1,2,5). Downstream of Akt, mTOR integrates various inputs and regulates cell proliferation by controlling mRNA translation into protein (2–4,6). Dysregulation of this pathway is common in various neoplasms. PI3K, which was first identified because of its physical association with the oncoproteins src (Rous sarcoma pp60v-src protein) and polyoma middle T antigen, is mutated to a constitutively active form in a substantial fraction of solid tumors (7). PTEN, the lipid phosphatase that antagonizes PI3K, is deleted or silenced in a variety of neoplasms, including glioblastomas, melanomas, lung cancer, and lymphomas (1,3). Akt, another crucial kinase in this pathway, is amplified or overexpressed in ovarian, breast, pancreatic, and colon cancer (3,5). These frequent changes suggest that dysregulation of this pathway plays a critical role in neoplastic transformation. Accordingly, extensive effort has gone into understanding signaling by various components of this pathway. THE PI3K/mTOR SIGNALING CASCADE The PI3K/mTOR pathway can be activated by a variety of growth factor receptor tyrosine kinases (1,4), including FLT3 as well as the epidermal growth factor receptor (EGFR), HER-2/neu and insulin-like growth factor I receptor (IGF-1R). Binding of ligand to each of these receptors leads to receptor tyrosine kinase activation, resulting in autophosphorylation of the cytoplasmic domain of each receptor. These phosphorylation events lead to activation of PI3K (1,8), a lipid kinase composed of an 85 kD regulatory subunit (p85) and a 110 kD catalytic
3 proapoptotic Bcl-2 family member Bad, the transcription factors Foxo3a and NFkB, glycogen synthase kinase-3b, the cyclin-dependent kinase inhibitor p27Kip1, and the GTPase activating protein TSC2. Activating and inhibitory phosphorylations are indicated by arrows and truncated lines, respectively; and the net antiapoptotic effects of the various phosphorylation events are indicated to the right. Akt-mediated phosphorylation of TSC2 leads to dissociation of its activating subunit TSC1 and inhibition of GTP cleavage by Rheb. As a consequence, GTP-bound active Rheb accumulates and enhances the activity of mTOR. As indicated in the text, mTOR exists in two complexes, TORC1 and TORC2, which differ in subunit composition, substrate specificity, and drug sensitivity. TORC1-mediated phosphorylation results in the dissociation of 4E-BP1 from eIF4E, allowing eIF4E to bind eIF4A and eIF4G to form an active helicase that facilitates transcription of messages with long 50 untranslated regions. In addition, TORC1 phosphorylates S6K, which in turn phosphorylates ribosomal protein S6 to facilitate translation of messages encoding polypeptides involved in translation. TORC2 modulates the activity of Rac and Rho GTPases, possibly by phosphorylating protein kinase Ca, and catalyzes an activating phosphorylation at 473Ser of Akt. According to current understanding, TORC1 is preferentially sensitive to inhibition by the complex formed when rapamycin or its analogs bind the cytoplasmic protein FKBP12. Abbreviation: PH, pleckstrin homology.
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subunit (p110). Specific phosphotyrosine residues on the intracellular domains of the activated receptors bind the src-homology 2 (SH2) domains of the PI3K p85 subunit and recruit the enzyme to the inner surface of the plasma membrane, where p110 is activated allosterically. p110 can also be activated upon binding of p85 to adapter molecules such as Grb2 and insulin receptor substrate (IRS), upon phosphorylation of p110 by Bcr-Abl or upon binding of p110 to the small GTPase Ras (1,9). Once at the plasma membrane, PI3K phosphorylates phosphatidylinositol4,5-bisphophate (PIP2) at the 3’ position of the inositol ring to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 subsequently recruits serine/ threonine kinases such as phosphoinositide-dependent kinase 1 (PDK-1) and Akt to the cell membrane by binding their pleckstrin homology (PH) domains (1,5). This recruitment to the plasma membrane activates PDK1, which phosphorylates 308 Thr in the activation loop of Akt, resulting in partial activation of the latter kinase. In order to be fully activated, Akt requires additional phosphorylation at 473 Ser, a process that is described in greater detail below. Once activated, Akt in turn phosphorylates numerous polypeptide substrates (1,2,5), including NFkB (activates transcription of antiapoptotic proteins when phosphorylated), Foxo3a (activates the genes encoding Fas ligand and Bim when unphosphorylated) and the cyclin-dependent kinase inhibitor p27Kip1 (inhibits cell cycle progression in its unphosphorylated state). Pertinent to Aktmediated activation of the mTOR pathway are two tumor suppressor proteins, the GTPase activating protein tuberous sclerosis complex 2 (TSC2, also known as tuberin) and its binding partner TSC1 (also known as hamartin). When TSC2 is unphosphorylated, it binds TSC1 to form a holoenzyme that facilitates GTP turnover and inactivation of the Ras homolog enriched in brain (Rheb), a small GTPase that activates mTOR (1,10,11). Activated Akt directly phosphorylates TSC2, causing its dissociation from TSC1, accumulation of GTP-bound (active) Rheb, and activation of mTOR (8,10). mTOR is present in two distinct complexes (4,8,12). TOR complex 1 (TORC1) consists of mTOR, its binding partner Raptor, and mLST8. TOR complex 2 (TORC2) consists of mTOR, the binding partner Rictor, mLST8, Protor-1, and mSin1. These complexes differ in substrate specificity and rapamycin sensitivity (4,8,11,12). Upon mTOR activation, the TORC1 complex facilitates cell cycle progression from G1 into S phase by phosphorylating two proteins, p70S6 kinase (S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These polypeptides facilitate cell cycle progression in two distinct ways (3,6,8). S6K enhances protein synthesis and ribosome biogenesis (6,8). It does this by phosphorylating and activating S6, a ribosomal 40S subunit polypeptide that facilitates translation of 50 terminal oligopyrimidine tract-containing mRNAs encoding components required for mRNA translation, including eukaryotic initiation factor 4B (eIF4B), eukaryotic elongation factor (eEF) 2 protein kinase, and the ribosomal protein S6 itself.
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In addition, TORC1 enhances the translation of a different set of RNAs by phosphorylating 4E-BP1 (2,6). eIF4E is a component of a helicase complex that binds to the 7-methylguanine cap at the 50 end of mRNAs and enhances the ability of ribosome-eIF complexes to scan the mRNA for initiation sites. 4E-BP1, in its unphosphorylated state, binds to eIF4E and inhibits the eIF4E-containing helicase complex. Activation of TORC1 signaling causes hyperphosphorylation of 4E-BP1, diminishing the stability of the 4E-BP1/eIF4E complex, and causing its dissociation. Free eIF4E then binds to the scaffold protein eIF4G and the RNA helicase eIF4A, forming an active helicase that facilitates translation of mRNAs containing long, highly folded 50 untranslated regions. Included in this class of transcripts are messages encoding cyclin D1, c-Myc, hypoxia inducible factor-1a (HIF-1a), vascular endothelial growth factor and fibroblast growth factor as well as ribosomal proteins themselves (2,3,6). These molecules are not only critical for cell survival and proliferation, but also have the potential to be used to monitor therapy. Because HIF-1a regulates the glycolytic pathway and fluorodeoxyglucose positron emission tomography (FDG-PET) detects tumors by their elevated rates of glycolysis, FDG-PET can potentially be used to assess inhibition of this pathway after treatment with mTOR inhibitors (2,3). In contrast to TORC1, TORC2 does not directly activate protein synthesis. Instead, this complex appears to regulate the actin cytoskeleton by regulating Rac and Rho GTPases. In addition, TORC2 catalyzes the activating phosphorylation of Akt on 473Ser (4,11). As a consequence, increased TORC2 activity would be expected to enhance antiapoptotic signaling in cells. Initial reports indicated that the TORC2 complex is resistant to rapamycin (4,11), although more recent studies suggest that it might be inhibited by prolonged rapamycin exposure (12). REGULATION OF THE PI3K/mTOR PATHWAY Because the PI3K/mTOR pathway is so critical, it is tightly regulated at several steps. First, the lipid phosphatase PTEN dephosphorylates PIP3 back to PIP2, thus terminating PI3K signaling (1). When PTEN is deleted or mutated, as it is in a small percentage of lymphomas (13,14), signaling through the PI3K/mTOR pathway is prolonged, leading to increased proliferation (1,3). Interestingly, PTEN-mediated PIP3 dephosphorylation appears to be particularly important for maintaining the normal number of hematopoietic stem cells in the bone marrow. PTEN-deficient mice develop myeloproliferative disorders and leukemia; and treatment with the mTOR inhibitor sirolimus restores normal hematopoietic stem cell development, blocking leukomogenesis in these mutant mice (15,16). Second, the pathway is also regulated at the level of the TSC1/TSC2 complex (2,10,11). This complex is inactivated when TSC2 becomes phosphorylated by activated Akt, protein kinase C, or extracellular signal–regulated kinases (ERKs). The ability of multiple kinases to inactivate the TSC1/TSC2 complex allows cross talk between different signaling pathways involved in cell
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proliferation. As indicated above, inactivation of this complex leads to activation of Rheb and mTOR. Conversely, the TSC1/TSC2 complex receives activating input from a pathway that monitors the nutritional and energy status of the cell (2,6,8,10). In particular, the AMP-activated kinase (AMPK) detects hypoxia or limited energy sources and phosphorylates TSC2 on a different site, thus activating the TSC1/TSC2 complex, increasing GDP-bound Rheb and inhibiting mTOR activity. mTOR INHIBITORS UNDERGOING TESTING AS POTENTIAL ANTICANCER AGENTS A number of mTOR inhibitors are currently undergoing clinical testing as potential anticancer drugs (2–4). The prototype drug in this class is rapamycin (sirolimus), a natural product isolated from a strain of Streptomyces hygroscopicus found in the soil of the Vai Atore region of Easter Island (3,4). Originally identified as an antifungal agent, rapamycin was subsequently shown to inhibit antigen-induced T-cell proliferation, a biochemical effect that presumably contributes to its ability to inhibit graft rejection (17). Sirolimus was approved as an oral immunosuppressant after renal transplantation in the 1990s and was subsequently shown by the National Cancer Institute to inhibit proliferation of several human cancer cell lines. Rapamycin does not directly inhibit mTOR. Instead, it binds to the abundant 12 kD cytoplasmic FK506 binding protein FKBP12. The resulting rapamycinFKBP12 complex binds to the FK-rapamycin binding domain of mTOR, leading to the disruption of TORC1 signaling and preventing phosphorylation of S6K and 4E-BP1 (2–4). Because of the poor solubility and chemical instability of rapamycin, analogs with more favorable chemical characteristics have been studied. Three of these, everolimus (RAD001, Novartis Pharma AG, Switzerland), temsirolimus (Torisel, Wyeth Pharmaceuticals, New Jersey, U.S.; previously called CCI-779), and AP23573 (Ariad, Massachusetts, U.S.), are currently being tested as antineoplastic agents (2–4). These agents differ in their routes of administration but have a similar mechanism of action and similar toxicities. Everolimus contains a 2-hydroxethyl substitution at position 40 of the rapamycin structure. Like rapamycin, it forms a complex with FKB12, which then binds with mTOR to inhibit downstream signaling. Everolimus is administered by mouth and is well tolerated, with only mild to moderate adverse reactions. Temsirolimus, an ester of rapamycin, is available as an intravenous (IV) or oral formulation. Adverse effects that are unique to the IV formulation include rare, acute hypersensitivity reactions during the infusion. AP23573, which is a nonprodrug rapamycin analog, is also available in an oral or IV form. This compound has been studied most extensively in patients with solid tumors, especially sarcomas; and there have been fewer studies in hematological malignancies.
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These rapamycin analogs have been well tolerated as single agents in phase I and phase II trials. Some patients develop small aphthous ulcerations that often clear without dose reduction. All of these agents tend to be myelosuppressive, with greater effects on platelets than on leukocytes or red blood cells. This myelosuppression has been dose limiting in trials of these agents for hematological malignancies, perhaps because the patients are more likely to be heavily pretreated and their bone marrows are more often compromised by tumor infiltration. In addition, these drugs have occasionally been associated with pulmonary infiltrates. These infiltrates are often noted on computed tomography scans and are difficult to distinguish from infection or tumor. Patients who remain on rapamycin analogs for extended periods of time also complain of mild dysgeusia and cracking of their fingernails. They often develop hyperlipidemia and hyperglycemia that require pharmacologic intervention.
DISEASE-SPECIFIC ACTIVITY OF mTOR INHIBITORS Non-Hodgkin’s Lymphoma and Hodgkin’s Disease Several preclinical observations provided the rationale for testing mTOR inhibitors in non-Hodgkin’s lymphoma (NHL). First, the ability of mTOR inhibitors to diminish expression of cyclin D1, a polypeptide with a clear-cut oncogenic role (18) in mantle cell lymphoma (MCL), suggested that it might be productive to examine rapamycin analogs in this lymphoma subtype. Second, a number of studies demonstrated constitutive activation of the PI3K/mTOR pathway, as manifested by phosphorylation of S6K and 4E-BP1 (19,20), in the vast majority of B-cell lymphomas. While the mechanism for pathway activation is not entirely clear in many lymphoma subtypes, recent investigation revealed that the nucleophosminanaplastic lymphoma kinase (ALK) fusion protein involved in the pathogenesis of anaplastic lymphoma signals through Akt and mTOR (21). In view of this evidence for mTOR activation, preclinical studies have examined the effects of rapamycin analogs on lymphoma cell lines and clinical specimens in vitro. Treatment of MCL lines with everolimus inhibits phosphorylation of mTOR substrates and induces G1 arrest at nanomolar concentrations (22). In addition, everolimus sensitizes MCL cell lines to a variety of cytotoxic agents, including doxorubicin and bortezomib. Similar results have been observed in diffuse large B-cell lymphoma (DLBCL) cell lines (23). Additional studies have suggested that this rapamycin analog-induced G1 arrest is followed by autophagy in MCL and Hodgkin’s disease (HD) cell lines in vitro (24,25). On the basis of the ability of mTOR inhibitors to downregulate cyclin D1, the North Central Cancer Treatment Group (NCCTG) performed a phase II trial examining the activity of temsirolimus 250 mg IV weekly in relapsed MCL (26). Patients enrolled in this phase II trial had received a median of three prior therapies; and 54% were refractory to the last treatment. The overall response rate was 38% [13 of 34 patients; 3% CR (complete remission) and 35%
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PR (partial remission)]. The median time to progression in the entire study population was 6.5 months. Hematological toxicities were common, particularly thrombocytopenia. In view of the promising responses, the NCCTG performed a follow-up trial of temsirolimus 25 mg IV weekly for MCL and demonstrated a similar response rate with lower toxicity (27). Subsequent studies have tested rapamycin analogs in other NHL subsets. A phase II trial of everolimus 10 mg PO (orally) daily in patients with relapsed aggressive NHL (28) revealed an overall response rate of 32% (12 of 37 patients; 3% CR and 29% PR). Hematological toxicity, particularly thrombocytopenia, was again common (28). Similarly, temsirolimus 25 mg IV weekly induced remissions in 38% (14% CR, 24% PR) and stable disease in 47% of evaluable patients with relapsed or refractory NHL (29). More mature results from the latter phase II trial are awaited with interest. A phase II trial also examined the activity of everolimus 10 mg PO per day in patients with relapsed or refractory HD. In heavily pretreated patients (median 6 prior therapies), 82% of whom had relapsed after prior stem cell transplant, a response rate of 47% (7 of 15 patients; all PRs) was observed (30). This overall response rate is the highest of any of the lymphoma disease types and may represent a new treatment for this small subset of patients with HD who fail stem cell transplantation. Collectively, these phase II trials demonstrate activity of rapamycin analogs in a wide range of lymphoma subtypes. Further studies are needed to identify potential biological properties of lymphomas most likely to respond to this class of agents. ACUTE LYMPHOBLASTIC LEUKEMIA Preclinical studies also established activity of rapamycin analogs against at least some subsets of human acute lymphoblastic leukemia (ALL). Initial observations demonstrated that sirolimus not only induces apoptosis in pediatric precursor B-cell ALL lines in vitro, but also diminishes lymphoblast counts in the Em-ret mouse model of pre-B leukemia in vivo (31). More recently, temsirolimus (32) and everolimus (33) were reported to induce apoptosis in vitro in blasts isolated from ALL patients. Moreover, these agents diminished blast counts and increased survival in SCID mice engrafted with human lymphoblasts (32,33). Building on these preclinical results, the group at Children’s Hospital of Philadelphia has performed a phase I trial of sirolimus in pediatric patients with relapsed/refractory leukemia. At the time of the last update, stable disease was observed in several patients, although no objective responses were obtained through the first two dose levels (34). ACUTE MYELOGENOUS LEUKEMIA A number of preclinical observations provide the rationale for exploring mTOR inhibitors in acute myelogenous leukemia (AML). Recent work has demonstrated that as many as 80% of clinical AML samples exhibit constitutive PI3K/mTOR
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pathway activation (35,36). Importantly, AML blasts with pathway activation seem addicted to this signaling and exhibit increased susceptibility to mTOR inhibition in vitro (36,37). Additional experiments have demonstrated that sirolimus or everolimus causes dramatic enhancement of the cytotoxicity of the topoisomerase II poison etoposide (37) and the DNA methyltransferase inhibitor 5-azacytidine (38) in AML blasts in vitro. Clinical evaluation of single-agent sirolimus in AML patients has been limited. Recher et al. (36) reported four PRs among nine relapsed AML patients treated with oral sirolimus; but the median response duration was only 38 days (range 35–120). Minor responses have also been reported in AML patients treated with AP23573 (39). On the basis of the observation that sirolimus sensitizes AML blasts to etoposide, Martin Carroll and colleagues have conducted a phase I study combining sirolimus with mitoxantrone, etoposide, and cytarabine. At the maximum-tolerated dose of this combination, inhibition of S6K phosphorylation was observed in serial samples of blasts harvested prior to and during chemotherapy; and a response rate of 30% (2 CR and 1 PR out of 10 patients) was demonstrated (40). Whether these results are better than those observed with the same regimen in the absence of sirolimus remains to be determined. A phase II trial of this regimen will be performed by the Eastern Cooperative Oncology Group. POTENTIAL FOR COMBINATIONS Because the PI3K/mTOR pathway contributes to expression of antiapoptotic polypeptides such as the Bcl-2 homolog Mcl-1 (41), there is substantial interest in combining rapamycin analogs with conventional chemotherapeutic agents (42). On the other hand, rapamycin also inhibits cell cycle progression (22,23), which could conceivably diminish the cytotoxicity of agents such as cytarabine and vinca alkaloids that depend on cell cycle progression for their toxicity. Accordingly, a number of investigations have begun to examine the effect of combining rapamycin analogs with conventional cytotoxic agents (43). Anthracycline-containing combinations appear promising in lymphoma models. Studies performed using tetrazolium dye reduction assays demonstrated that mTOR inhibition enhances the antiproliferative effects of doxorubicin synergistically in MCL cell lines (22) and at least additively in HD cell lines (20). Additional studies in transgenic mice with B-cell lymphomas driven by c-Myc and constitutively active Akt demonstrated that sirolimus synergizes with doxorubicin to prolong survival in vivo (44), giving rise to the suggestion that rapamycin analogs might enhance the activity of certain lymphoma regimens. Everolimus also synergizes with bortezomib, another agent with activity in MCL, in MCL lines in vitro (22). In contrast, antagonism has been reported when DLBCL lines are treated with everolimus and vincristine in vitro (23). Thus, not all antilymphoma agents will necessarily synergize with mTOR inhibitors.
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Rapamycin has also been reported to enhance cytotoxicity of conventional chemotherapeutic agents in human leukemia cell lines. As mentioned above, enhanced cytotoxicity of etoposide has been observed in U937 cells treated with sirolimus in vitro (37). Likewise, sirolimus enhances the induction of apoptosis by arsenic trioxide in Bcr-Abl-expressing chronic myelogenous leukemia cell lines (45). The possibility of combining rapamycin analogs with other signal transduction inhibitors is also appealing. Several studies have demonstrated synergistic induction of apoptosis in B-cell lymphoma cell lines upon treatment with the cytotoxic anti-CD20 antibody rituximab and everolimus (22,23). Likewise, the cytotoxic effects of the receptor tyrosine kinase inhibitor sunitinib in a variety of hematological cell lines are enhanced by everolimus (46). Finally, building on the observation that rapamycin causes Mcl-1 downregulation (41) and the demonstration that Mcl-1 overexpression is a major mechanism of resistance to Bcl-2 antagonists (47,48), synergistic induction of apoptosis in AML blasts in vitro has been reported upon simultaneous treatment with temsirolimus and the Bcl-2 inhibitor ABT-737 (49). Whether these preclinical observations can be translated into successful rapamycin-containing treatments remains to be determined. PROSPECTS FOR DUAL INHIBITORS AND COMBINED TORC1/TORC2 INHIBITORS Studies performed over the last four years have demonstrated that inhibition of TORC1 complexes by rapamycin analogs can lead to increased activation of the PI3K/Akt portion of the pathway either through enhanced receptor tyrosine kinase signaling or through direct TORC2-mediated phosphorylation of Akt on 473 Ser (4,11,12). Because Akt also facilitates cell survival through pathways that do not involve mTOR (Fig. 1) (1–3,5), these observations have led to interest in combining PI3K or Akt inhibitors with rapamycin analogs. Dual specificity inhibitors that abrogate signaling by PI3K and mTOR, two kinases with structurally similar active sites (4), have recently been identified. One of these, PI-103, inhibits proliferation of AML cell lines and induces apoptosis in primary AML samples but not normal CD34-positive cells in vitro (50). Alternatively, several drug companies are currently developing nonrapamycin-based mTOR inhibitors. Because these agents directly target the active site of mTOR, they inhibit both TORC1 and TORC2 complexes. As a result, their effects on cancer cell proliferation and survival might differ from those of rapamycin. Whether they will have the same therapeutic index as rapamycin analogs and the same ability to induce remissions, particularly in lymphoid malignancies, remains to be determined. CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Collectively, the studies described above have not only demonstrated that signaling through the PI3K/mTOR pathway is activated in the majority of lymphomas and leukemias, but also documented tantalizing activity of rapamycin analogs against a
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variety of B-cell lymphomas. In particular, the phase I and II studies have clearly revealed activity of mTOR inhibitors in relapsed MCL, DLBCL, and HD. Larger registration-type trials are required to confirm these early results. For example, a phase III international trial in MCL (Optimal Trial, Wyeth) has compared two different doses of temsirolimus against ‘‘doctor’s choice’’ chemotherapy in the third arm. This trial has met its accrual goals; and the outcome is likely to be reported in 2008. Although activity observed when rapamycin analogs are administered as single agents is encouraging, history would suggest that these drugs might be even more effective when administered with other antilymphoma agents. Many combinations are currently being examined in vitro; and early-phase clinical trials of the most promising combinations are likely to begin over the next two years. The NCCTG, for example, is currently testing temsirolimus with rituximab (Trial N038H). Because of the myelosuppression (especially thrombocytopenia) produced by the rapamycin analogs, these phase I studies will be needed to determine the appropriate doses to be used in subsequent phase II and phase III trials of combinations. Despite the potentially promising results observed to date, it is important to emphasize that not all lymphomas respond to mTOR inhibition. Accordingly, further study is required to identify predictive markers that identify the lymphomas most likely to respond before administration of therapy. Even though a number of investigations have identified potential mechanisms of resistance to rapamycin analogs in preclinical models, further studies are also needed to identify the mechanisms of resistance that are operative in the clinical setting and, if possible, ways to overcome them. Hopefully, these studies will be performed hand in hand with the efficacy studies over the next few years. ACKNOWLEDGMENTS We apologize to the many authors whose important work could not be cited because of space limitations. The secretarial assistance of Deb Strauss is gratefully acknowledged. Work in our laboratories has been supported in part by R21 CA112904 (Witzig), R01 CA127433 (Witzig and Kaufmann) and a Clinician Investigator Program traineeship (Hendrickson) from the Mayo Foundation. REFERENCES 1. Cully M, You H, Levine AJ, et al. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 2006; 6(3): 184–192. 2. Easton JB, Houghton PJ. mTOR and cancer therapy. Oncogene 2006; 25(48):6436–6446. 3. Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006; 5(8):671–688. 4. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends Mol Med 2007; 13(10):433–442. 5. Downward J. PI 3-kinase, Akt and cell survival. Semin Cell Biol 2004; 15:177–182.
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25. Georgakis GV, Yazbeck VY, Li Y, et al. The mTOR inhibitor temsirolimus (CCI-779) induces cell cycle arrest and autophagy in Hodgkin lymphoma (HL) cell lines and enhances the effect of the PI3-kinase inhibitor LY294002. Blood (ASH annual meeting abstracts) 2006; 108:2259. 26. Witzig TE, Geyer SM, Ghobrial I, et al. Phase II trial of single-agent temsirolimus (CCI-779) for relapsed mantle cell lymphoma. J Clin Oncol 2005; 23(23):5347–5356. 27. Ansell SM, Geyer SM, Ghobrial I, et al. A phase II trial of low-dose single-agent temsirolimus for relapsed mantle cell lymphoma 2008 (Submitted for publication). 28. Reeder CB, Gornet MK, Habermann TM, et al. A phase II trial of the oral mTOR inhibitor everolimus (RAD001) in relapsed aggressive non-Hodgkin lymphoma (NHL). Blood (ASH annual meeting abstracts) 2007; 110:121. 29. Smith SM, Pro B, Smith S, et al. Molecular inhibition of mTOR with temsirolimus (TORISELTM, CCI-779) is a promising strategy in relapsed NHL. The University of Chicago Phase II Consortium. Blood (ASH annual meeting abstracts) 2006; 108:2483. 30. Johnston PB, Ansell SM, Colgan JP, et al. mTOR Inhibition for relapsed or refractory Hodgkin lymphoma: promising single agent activity with everolimus (RAD001). Blood (ASH annual meeting abstracts) 2007; 110:2555. 31. Brown VI, Fang J, Alcorn K, et al. Rapamycin is active against B-precursor leukemia in vitro and in vivo, an effect that is modulated by IL-7-mediated signaling. Proc Natl Acad Sci U S A 2003; 100(25):15113–15118. 32. Teachey DT, Obzut DA, Cooperman J, et al. The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL. Blood 2006; 107(3):1149–1155. 33. Crazzolara R, Cisterne A, Thien M, et al. The mTOR inhibitor RAD001 (everolimus) improves survival in preclinical models of primary human ALL. Blood (ASH annual meeting abstracts) 2007; 110:856. 34. Rheingold SR, Sacks N, Chang YJ, et al. A phase I trial of sirolimus (rapamycin) in pediatric patients with relapsed/refractory leukemia. Blood (ASH annual meeting abstracts) 2007:110:2834. 35. Xu Q, Simpson SE, Scialla TJ, et al. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood 2003; 102:972–980. 36. Recher C, Beyne-Rauzy O, Demur C. Antileukemic activity of rapamycin in acute myeloid leukemia. Blood 2005; 105:2527–2534. 37. Xu G, Thompson JE, Carroll M. mTOR regulates cell survival after etoposide treatment in primary AML cells. Blood 2005; 106:4261–4268. 38. Liesveld JL, Rosell K, Lu C, et al. The mTOR inhibitor rapamycin demonstrates activity against AML in combination with imatinib mesylate and with 5-azacytidine. Blood (ASH annual meeting abstracts) 2007; 110:4318. 39. Rizzieri DR, Feldman E, Moore JO. A phase 2 clinical trial of AP23573, an mTOR inhibitor, in patients with relapsed or refractory hematologic malignancies. Blood 2005; 106:2980. 40. Luger S, Perl A, Kemner A, et al. A phase I dose escalation study of the mTOR inhibitor sirolimus and MEC chemotherapy targeting signal transduction in leukemia stem cells for acute myeloid leukemia. Blood (ASH annual meeting abstracts) 2006; 108:161. 41. Wei G, Twomey D, Lamb J, et al. Gene expression-based chemical genomics identifies rapamycin as a modulator of MCL1 and glucocorticoid resistance. Cancer Cell 2006; 10(4):331–342.
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42. Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Natl Cancer Inst Monogr 2005; 4(12):988–1004. 43. Recher C, Dos Santos C, Demur C. mTOR, a new therapeutic target in acute myeloid leukemia. Cell Cycle 2005; 4:1540–1549. 44. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 2004; 428 (6980), 332–337. 45. Yoon P, Giafis N, Smith J, et al. Activation of mammalian target of rapamycin and the p70 S6 kinase by arsenic trioxide in BCR-ABL-expressing cells. Mol Ca Ther 2006; 5(11):2815–2823. 46. Ikezoe T, Nishioka C, Tasaka T, et al. The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling. Mol Cancer Ther 2006; 5(10):2522–2530. 47. van Delft MF, Wei AH, Mason KD, et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 2006; 10:389–399. 48. Konopleva M, Contractor R, Tsao T, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 2006; 10(5):375–388. 49. Zeng Z, Samudio I, Andreeff M, Konopleva M. Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and mTOR/Akt pathways in acute myeloid leukemia. Blood (ASH annual meeting abstracts) 2007; 110:1588. 50. Park S, Chapuis N, Bardet V, et al. PI-103, a dual inhibitor of class I phosphatidylinositide 3-kinase and mTOR, has anti-leukemic activity in acute myeloid leukemia. Blood (ASH annual meeting abstracts) 2007; 110:876.
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24 Allogeneic Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning Fre´de´ric Baron Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
Frederick R. Appelbaum and Brenda M. Sandmaier Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington, U.S.A.
INTRODUCTION High-dose chemo- or chemoradiotherapy followed by allogeneic hematopoietic cell transplantation (HCT) has been recognized as an effective therapy for a number of hematologic malignancies with tumor cells resistant to conventional doses of chemotherapy (1). The aims of the high-dose conditioning are (i) to abolish host immune responsiveness prior to transplantation to avoid graft rejection and (ii) to deliver doses of cytotoxic anticancer agents beyond the range that is toxic to the bone marrow cells, thereby potentially increasing antitumor efficacy (1). The curative potential of allogeneic HCT is not only due to the high-dose chemoradiotherapy but also due to immune-mediated graft-versus-tumor (GVT) effects (2–4). The existence of a GVT effect was first suggested by Barnes et al. in 1956 (5). They observed that mice receiving syngeneic HCT and injection of congenic leukemic cells after total body irradiation (TBI) almost uniformly died from
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leukemia, whereas a number of mice receiving histoincompatible marrow were cured of leukemia but eventually died from the graft-versus-host disease (GVHD). The authors suggested that a reaction of the donor spleen cells might kill cancer cells. This hypothesis was evinced two decades later in humans by studies reporting reduced leukemic relapse rates in allografted patients who developed GVHD compared with those who did not (2,3). The GVT effect was further demonstrated by other investigators who observed increased risks of relapse in patients given T-cell-depleted grafts and in recipients of syngeneic transplants (3). Those observations led several groups of investigators to investigate the curative potential of donor lymphocytes infusions (DLI) in patients who had relapsed after allogeneic HCT (4). Two large multicenter studies, one from the European Group for Blood and Marrow Transplantation (EBMT) (4) and the other from North America (6), have analyzed the efficacy of DLI in more than 400 patients (Table 1). DLI induced complete remissions in more than 60% of patients with chronic myeloid leukemia and 10% to 40% of patients with other hematologic malignancies. Typically, achievement of complete remissions required several weeks. For example, an average time of four to six months was required before molecular remission was achieved in patients with relapsed chronic myeloid leukemia (4). While 50% of patients without acute GVHD showed tumor regression, this increased to 75% and 85% in patients with grade I or grades II to IV acute GVHD, respectively (4). Similarly, chronic GVHD was associated with disease responses (4,6). DLI have been given without any other treatment in patients with indolent disease such as chronic myeloid leukemia in chronic phase, while chemotherapy has been given before DLI in a number of
Table 1 Results of Donor Lymphocyte Infusions as Treatment of Relapse After HLAMatched HCT Following Myeloablative Conditioning North America (6) Complete response/ evaluable patients (%) Chronic myeloid leukemia Cytogenetic/molecular relapse Hematologic relapse Accelerated phase/blast crisis Acute myeloid leukemia/ myelodysplastic syndrome/ polycythemia vera Acute lymphoblastic leukemia Multiple myeloma Non-Hodgkin’s lymphoma
EBMT (4) Complete response/ evaluable patients (%)
3/3 (100) 25/34 (74) 5/18 (28) 8/44 (18)
40/50 (80) 88/114 (77) 13/36 (36) 16/59 (27)
2/11 (18) 2/4 (50) 0/6 (0)
3/20 (15) 5/17 (29) —
Abbreviation: EBMT, European Group for Blood and Marrow Transplantation.
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Figure 1 Diagnosis and survival after DLI given for progressive disease/relapse after nonmyeloablative HCT. Kaplan–Meier plots of survival after DLI depending on diagnoses. Survival estimates at one year were 61% for B-cell malignancies, 51% for chronic leukemia, 50% for solid tumors, 13% for acute leukemia, and again 13% for myelodysplastic syndrome, respectively. Source: From Ref. 7.
patients with more aggressive diseases. Figure 1 shows overall survival in 48 patients given DLI for progressive disease/relapse after nonmyeloablative conditioning (7). Because of regimen-related toxicities, the use of high-dose myeloablative conditionings has been restricted to younger and medically fit patients. This is unfortunate, given that the median age at diagnosis of patients with acute and chronic myeloid leukemias, chronic lymphocytic leukemia, non-Hodgkin’s lymphomas (NHLs), myelodysplastic syndromes, and multiple myeloma ranges from 65 to 70 years (1). In 1971, Santos et al. reported that conditioning with cyclophosphamide alone, although nonmyeloablative enabled sustained engraftment of transplanted allogeneic hematopoietic cells in patients with advanced leukemia (8). Unfortunately, tumor cells were not completely eradicated, and all patients eventually relapsed. While cyclophosphamide became the conditioning regimen of choice for patients with aplastic anemia (1), it was abandoned as the sole conditioning regimen in patients with hematologic malignancies. In 1974, Graw et al. reported a few cures in patients with acute leukemia given allogeneic marrows after a reduced-intensity (9) conditioning regimen combining BCNU, cytarabine, cyclophosphamide, and thioguanine (10). The growing evidence of the power of GVT effects, as demonstrated by the efficacy of DLI, incited several groups of investigators to develop new reducedintensity (11–14) or truly nonmyeloablative conditioning regimens (15–17) allowing older patients and those with comorbidities to benefit from GVT effects (Table 2).
Postgraft immunosuppression
Reduced-intensity regimens MD Anderson Fludarabine 25 mg/ FK506 þ (12) m2/day (or 2-CDA MTX 12 mg/m2) 5 days Melphalan 140– 180 mg/m2 UK consortium Fludarabine 30 mg/ CSP (13) m2/day 5 days Melphalan 140 mg/m2 Alemtuzumab 20 mg/ day 5 days HadassahFludarabine 30 mg/ CSP þ/ Hebrew m2/day 6 days MTX University Busulfan (p.o.) 4 mg/ (11) kg/day 2 days ATG 5–10 mg/kg/ day 4 days
Center (Ref.)
Preparative regimens
55%
Chronic myeloid 75%a leukemia in first chronic phase.
24 (35)
7%c
15%c
Non-Hodgkin’s lymphoma
88 (48)
2-yr OS: 28%. 2-yr DFS: 23%.
Outcome
3 pts (days 116, 499, and 726)
5-yr DFS 85%.
11d–38%e at 3 yr 3-yr OS: 55%
37% (at 100 days)
NRM (days Chronic after transplant) 68%
Acute (grade II–IV)
GVHD
49%
Hematological malignancies
86 (52)
No. of pts (median age in yr) Diseases
Table 2 Examples of Reduced-Intensity or Nonmyeloablative Conditioning Regimens
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TBI 5.5 Gy Cyclophosphamide 120 mg/kg
Center (Ref.)
Washington University (14)
110 (44)
CSP þ MTX þ steroids
20%
64%
Indolent lymphomas.
20 (51)
59%
b
2 (at day 45 and before 300 days)
2 pts (days 59 and 205)
30% at 1 yr
NRM (days Chronic after transplant)
Hematological þ 10/15 NR solid pts. malignancies. 1 after DLI
33%
Acute (grade II–IV)
15 (50)
Hematological malignancies.
No. of pts (median age in yr) Diseases
Postgraft immunosuppression
Nonmyeloablative regimens CSP National Fludarabine 25 mg/ Institutes of m2/day 5 days Health (17) Cyclophosphamide 60 mg/kg/day 2 days MD Anderson Fludarabine 25 mg/ FK506 þ (15) m2/day 5 days MTX or Fludarabine 30 mg/m2/day 3 days Cyclophosmphamide 1g/m2/day 2 days or 750 mg/m2/day 3 days þ/ Rituximab
Preparative regimens
GVHD
(Continued)
8/15 pts survived between 121 and 409 (median, 200) days. 2-yr DFS: 84%.
2 yr DFS 40%f 2 yr DFS 21%g
Outcome
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FHCRC consortiumh (24) 451 (55)
Hematological malignancies.
No. of pts (median age in yr) Diseases 48%
Acute (grade II–IV) 44% .
b
7% at 100 days 22% (at 2 yr)
NRM (days Chronic after transplant)
2-yr OS: 51% 2-yr PFS: 37%
Outcome
b
grades I–IV, extensive chronic GVHD, c before donor lymphocyte infusions given in 36 of 88 (41%) patients, d in patients with low-grade NHL, e in patients with high-grade NHL, f in patients with good-risk diseases, g in patients with high-risk diseases, h the clinical trials were carried out jointly by a group of collaborators located at the Fred Hutchinson Cancer Research Center, University of Washington, Children’s Hospital and Regional Medical Center, and Veterans Administration Medical Center, all in Seattle, Washington, U.S.A.; Stanford University, Palo Alto, California, U.S.A.; City of Hope National Medical Center, Duarte, California, U.S.A.; University of Leipzig, Germany; University of Colorado, Denver, Colorado, U.S.A.; University of Torino, Italy; University of Arizona, Tucson, Arizona, U.S.A.; Baylor University, Dallas, Texas, U.S.A.; University of Utah, Salt Lake City, Utah, U.S.A.; Oregon Health Sciences University, Portland, Oregon, U.S.A.; the Medical College of Wisconsin, Milwaukee, Wisconsin, U.S.A.; and Emory University, Atlanta, Georgia, U.S.A. Abbreviations: NRM, non-relapse mortality; ATG, antithymocyte globulin; CSP, cyclosporine; FK506, tacrolimus; MTX, methotrexate; pts, patients; OS, overall survival; DFS, disease-free survival; PFS, progression-free survival; NR, not reported. Source: From Ref. 19.
a
TBI 2 Gy þ/ CSP þ Fludarabine 30 mg/ MMF m2/day 3 days.
Center (Ref.)
Postgraft immunosuppression
Preparative regimens
GVHD
Table 2 Examples of Reduced-Intensity or Nonmyeloablative Conditioning Regimens (Continued )
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NONMYELOABLATIVE OR REDUCED-INTENSITY REGIMENS Many of the reduced-intensity conditioning regimens do not meet criteria of nonmyeloablative conditioning as first proposed by Champlin et al., which include (i) no eradication of host hematopoiesis, (ii) prompt hematologic revovery (<4 weeks) without transplant, and (iii) presence of mixed chimerism upon engraftment (18,19). Most reduced-intensity conditioning regimens combine modest dose of highly immunosuppressive purine analogs (fludarabine, cladribine, or pentostatin) given to overcome host-versus-graft reactions, with reasonably high-dose of alkylating agents, usually busulfan or melphalan, given to supplement the GVT effects in the task of tumor eradication. Conversely, nonmyeloablative conditioning regimens usually combine two highly immunosuppressive agents together (low-dose TBI, fludarabine, or cyclophosphamide) to overcome host-versus-graft reactions to allow engraftment and tumor eradication via GVT effects (16,20). Although the division of what constitutes a nonmyeloablative versus reduced-intensity conditioning regimen is somewhat arbitrary, the distinction might be important, given that nonmyeloablative conditioning has been associated with a lower degree of donor engraftment, decreased risk of nonrelapse mortality, and perhaps higher risk of relapse in comparison with reduced-intensity regimens (21).
NONMYELOABLATIVE CONDITIONING WITH 2 GY TBI AND FLUDARABINE On the basis of preclinical studies in a canine model (22), we developed a nonmyeloablative conditioning regimen for allogeneic HCT consisting of 2 Gy TBI given on day 0, with postgrafting immunosuppression combining mycophenolate mofetil (MMF) and cyclosporin (CSP) (16). Nine of the first 44 patients (20%, including four of eight patients with chronic myeloid leukemia) given this regimen had nonfatal graft rejections (16,23). In order to reduce the risk of graft rejection, fludarabine 30 mg/m2/day 3 days was added to the 2 Gy TBI, and the rejection rate decreased to 3% (24). The same nonmyeloablative regimen combining fludarabine and 2 Gy TBI was used to condition patients with 10/10-human leukocyte antigen (HLA)-matched unrelated donors (25). Sustained engraftment was observed in 60 of 71 (85%) peripheral blood stem cells (PBSC) recipients and in 10 of 18 (56%) marrow recipients. On the basis of this observation, all subsequent unrelated recipients were given PBSC grafts. Analysis of the first 451 patients with hematologic malignancies transplanted in a multicenter international consortium is shown in Table 1 (24). Median patient age was 55 (range, 5–74) years, and median follow-up was 696 (range, 82–1795) days. All patients were deemed ineligible for myeloablative conditioning because of age and/or comorbidities. Diagnoses included multiple myeloma (n ¼ 114), myelodysplastic syndromes or myeloproliferative disorders (n ¼ 82), NHLs (n ¼ 79), acute myeloid leukemia (n ¼ 59), chronic lymphocytic leukemia
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(n ¼ 44), chronic myeloid leukemia (n ¼ 37), Hodgkin’s disease (n ¼ 26), and acute lymphoblastic leukemia (n ¼ 10). Three hundred and thirty-two patients had measurable disease at transplantation, and 56.5% achieved complete (49%) or partial (7.5%) remissions. The incidences of nonrelapse mortality at 100 days and two years were 7% and 22%, respectively. Main causes of nonrelapse mortality were GVHD and infections. The two-year probabilities of overall and progression-free survivals were 51% and 37%, respectively. KINETICS OF DONOR ENGRAFTMENT AFTER NONMYELOABLATIVE CONDITIONING The engraftment kinetics after nonmyeloablative conditioning regimen were first analyzed by Childs et al. (17). The authors studied chimerism (i.e., proportion of hematopoietic cells of donor origin) evolution in 15 patients conditioned with fludarabine (125 mg/m2) and cyclophosphamide (120 mg/kg). The patterns of engraftment varied between patients, but most often, full donor chimerism was achieved earlier among T-cells than among granulocytes, and achievement of full donor T-cell chimerism preceded GVHD and antitumor responses. Conversely, Ueno et al. studied chimerism evolution in 23 patients with metastatic tumors transplanted after conditioning with fludarabine (125–150 mg/m2) and melphalan (140 mg/m2) (26). All patients had full donor T-cell and granulocyte chimerisms by day 30 after HCT. We analyzed the kinetics of donor engraftment in various peripheral blood hematopoietic subpopulations from 120 patients conditioned with 2 Gy TBI þ/ fludarabine and postgrafting immunosuppression with MMF and CSP (27). On day 14 post transplant, the highest degree of donor chimerism was seen in the NK cells followed by T-cells, monocytes, and granulocytes (Fig. 2A). By day 28, donor granulocyte chimerism had surpassed those in the remaining cell populations. PBSC recipients had higher degrees of donor T-cell chimerism than recipients of marrow, while greater intensity of therapy before HCT was associated with higher degrees of donor chimerisms. Day-14 donor chimerism levels less than 50% among T-cells (p ¼ 0.0007) and NK cells (p ¼ 0.003) predicted graft rejection (Fig. 2B). High donor chimerism levels on day 14 among T-cells were associated with increased risks of grades II to IV acute GVHD (p ¼ 0.02), while high donor T-cell (p ¼ 0.002) and NK cell (p ¼ 0.002) chimerism levels from days 14 to 42 were associated with decreased risks of relapse. In addition, high levels of donor NK cell chimerism early after HCT correlated with better progression-free survival (p ¼ 0.02) and a trend for better overall survival (p ¼ 0.09). These observations suggest that assessing donor chimerism levels among T-cells and NK cells might help identify patients at risk for graft rejection, acute GVHD, and relapse, and thereby allow early interventions with DLI and/or immunosuppressive drugs (7,28).
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Figure 2 (A) Engraftment kinetics after nonmyeloablative conditioning in 108 patients who achieved sustained engraftment. (B) Cumulative incidence of graft rejection according to day-14 T-cell chimerism. Source: From Ref. 27.
GVHD AND GVT EFFECTS AFTER NONMYELOABLATIVE CONDITIONING GVHD remains a major cause of morbidity and mortality after nonmyeloablative or reduced-intensity conditioning (16,25). Mielcarek et al. compared GVHD in 52 patients given myeloablative conditioning with that among 44 patients given nonmyeloablative conditioning (29). Recipients in both groups were age matched, with median ages of 54 years in the myeloablative and 56 years in the nonmyeloablative groups. Grafts were from either related or unrelated donors who were serologically matched for HLA-A, -B, and -C and allele level matched for HLA-DRB1 and -DQB1. Postgrafting immunosuppression consisted of methotrexate (MTX) plus CSP (n ¼ 45) or MMF plus CSP (n ¼ 7) in myeloablative
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recipients, versus MMF plus CSP in all nonmyeloablative recipients. The cumulative incidences of grades II to IV acute GVHD were 85% in myeloablative recipients versus 64% in nonmyeloablative recipients (p ¼ 0.001), but there were no differences in the cumulative incidences of extensive chronic GVHD (71% vs. 73%, respectively). The 15-month cumulative incidences of death with manifestations of GVHD under treatment were 35% and 24% in myeloablative and nonmyeloablative recipients, respectively (NS). Although there is a close relationship between GVHD and GVT responses observed after myeloablative HCT (2–4,6), whether some degree of clinical GVHD was required for accomplishing remissions after nonmyeloablative conditioning was less clear. In order to address this question, we analyzed the impact of either acute or chronic GVHD on HCT outcomes in 322 patients with hematologic malignancies given grafts from HLA-matched related (n ¼ 192) or unrelated (n ¼ 130) donors following conditioning with 2 Gy TBI with or without fludarabine (90 mg/m2) (20). Two hundred and twenty-one patients had measurable malignant disease at the time of transplantation, and 126 of them (57%) achieved complete (n ¼ 98) or partial (n ¼ 28) remissions 27 to 963 days (median, 176 days) after HCT. Extensive chronic GVHD was suggestively associated with a higher probability of achieving complete remissions (HR 1.7, p ¼ 0.07), but no associations between acute GVHD and achievement of complete remissions were seen. Grades II and III to IV acute GVHD did not decrease the risks of progression/relapse but were associated with an increased risk of nonrelapse mortality and decreased progression-free survival. In contrast, extensive chronic GVHD was associated with decreased risk of progression/ relapse (HR 0.4, p ¼ 0.006) and better progression-free survival (HR 0.5, p ¼ 0.003) (Fig. 3). The beneficial impact of chronic GVHD on relapse was seen in all disease groups but was strongest in the group of patients with acute myeloid leukemia or myelodysplastic syndrome (HR 0.2, p ¼ 0.0009). Similarly, a number of other recent reports have shown a negative impact of grades II to IV acute GVHD (30,31) but a beneficial impact of chronic GVHD (31,32) on HCT outcomes in patients given HCT after reduced-intensity or nonmyeloablative conditioning. Some reduced-intensity conditioning regimens have used in vivo T-cell depletion of the grafts [with either antithymocyte globulin (ATG) or alemtuzumab] to decrease the incidence of GVHD. While these strategies achieved their goal (11,13), increased incidences of both infections and disease relapses were observed, resulting in comparable progression-free survival. TOXICITIES AFTER MYELOABLATIVE OR NONMYELOABLATIVE CONDITIONING A number of retrospective studies have compared incidences of toxicity and infection after nonmyeloablative versus myeloablative conditioning (Table 3) (33–37). Nonmyeloablative conditioning was associated with decreased
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Figure 3 Semilandmark plots illustrating progression-free survival among patients with and without extensive chronic GVHD (20). For patients diagnosed with extensive chronic GVHD, survival is plotted as a function of time since onset of GVHD. For patients free of disease progression and without a diagnosis of extensive chronic GVHD at day 135 (the median day of onset for those with extensive chronic GVHD), survival is plotted as a function of time since day 135. For this group the survival is the conditional survival among patients remaining without a diagnosis of extensive chronic GVHD. Abbreviation: GVHD, graft-versus-host disease. Source: From Ref. 20.
transfusion requirements (33), decreased incidence of idiopathic pneumonia syndrome (IPS) (34), decreased incidence of sinusoidal obstruction syndrome (SOS) (35), decreased incidence of acute renal failure (38), and decreased incidence of bacterial and cytomegalovirus (CMV) infections early after HCT (36,37). However, overall CMV reactivations and fungal infections were similarly frequent after nonmyeloablative and myeloablative conditioning (36,37). Sorror et al. analyzed transplantation-related toxicities (graded according to the National Cancer Institute common toxicity criteria) following HLA-matched unrelated HCT in 134 concurrent patients given either nonmyeloablative (n ¼ 60) or myeloablative (n ¼ 74) conditioning (39). Additionally, the effects of pretransplant comorbidities [graded according to the Charlson Comorbidity Index (CCI) score] on outcome were investigated. Lower numbers of gastrointestinal (p < 0.0001), hepatic (p ¼ 0.005), hematologic (p < 0.0001), infection-related (p ¼ 0.02), and hemorrhagic (p ¼ 0.02) grades III to IV toxicities were seen in nonmyeloablative compared with myeloablative recipients, whereas incidences of cardiovascular, metabolic, pulmonary, and renal toxicities were not statistically significantly different between the two groups. The one-year nonrelapse mortality was 32% in patients given myeloablative conditioning compared with 20% in patients given nonmyeloablative conditioning. In multivariate analyses adjusting for disease risk, age, and CCI score at HCT, myeloablative conditioning was
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Table 3 Toxicity After Nonmyeloablative (Consisting of 2 Gy Total Body Irradiation with or Without 90 mg/m2 Fludarabine) Vs. Myeloablative Conditioning Regimens
Toxicity (Refs.)
Nonmyeloablative
Hematological Toxicity (transfusion requirements) (33) Median units red cells 2 (0–50) Median units platelets 0 (0–214) Pulmonary Toxicity (34) 120-day CI of IPS 2.2% Hepatic Toxicity (35,40) 200-day CI of 26% hyperbilirubinemiaa 100-day CI of sinusoidal 0% obstructive syndrome Renal toxicity (38) 100-day CI of grades II–III 47% acute renal failure 100-day CI of dialysis 3% Infections (36,37) 30-day CI of bacterial 9% infection 100-day CI of bacterial 27% infection 1-yr CI of aspergillosis 14% 100-day CI of CMV disease 6% 1-yr CI of CMV disease 24% GVHD (29) 100-day CI of grades II–IV acute GVHD Matched siblings 62% Matched unrelated donors 65% CI of extensive chronic GVHD Matched siblings 77% Matched unrelated donors 68% Mortality from GVHD 24%
Myeloablative
p value
6 (0–34) 24 (4–358)
p ¼ 0.0002 p < 0.0001
8.4%
p ¼ 0.003
48%
ND
18%
ND
73%
p < 0.0001
12%
p < 0.0001
27%
p ¼ 0.01
41%
p ¼ 0.07
10% 19% 25%
p ¼ 0.30 p ¼ 0.06 p ¼ 0.87
77% 95%
p ¼ 0.02 p ¼ 0.01
74% 69% 35%
p ¼ 0.37 p ¼ 0.37 p ¼ 0.07
a 4 mg/dL. Abbreviations: CI, cumulative incidence; IPS, idiopathic pneumonia syndrome; ARF, acute renal failure; CMV, cytomegalovirus; GVHD, graft-versus-host disease.
associated with increased risks of grade IV toxicities (HR 9.4, p ¼ 0.0001) and higher one-year nonrelapse mortality (HR 3.0, p ¼ 0.04). Interestingly, higher pretransplant CCI scores predicted for increased mortality. Comparable results were observed by Diaconescu et al. in patients given grafts from HLA-identical sibling donors (40).
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RELAPSE AND SURVIVAL AFTER MYELOABLATIVE OR NONMYELOABLATIVE CONDITIONING It has remained difficult to compare relapse risk and survival after myeloablative versus nonmyeloablative recipients, given the short follow-up (and relatively low number) of patients given HCT after nonmyeloablative conditioning so far and the fact that nonmyeloablative recipients were generally older and had more comorbidities than patients given myeloablative conditioning. Two randomized studies in the early 1990s demonstrated lower risk of relapse, increased nonrelapse mortality, and similar survival in patients treated with cyclophosphamide and 15.5 Gy versus 12 Gy TBI followed by HLA-identical sibling HCT, demonstrating that dose intensity does matter for both toxicity and antitumor efficacy (41,42). Alyea et al. performed a retrospective analysis of 152 patients (>50 years old) with hematologic malignancies undergoing HCT after myeloablative [mainly cyclophosphamide (3.6 g/m2) and TBI (14 Gy)] or reduced-intensity conditioning combining fludarabine (120 mg/m2) and intravenous busulfan (3.2 mg/kg) (43). Patients given nonmyeloablative conditioning were more likely to receive grafts from unrelated donors (58% vs. 36%, p ¼ 0.009), to have received a prior HCT (25% vs. 4%, p < 0.0001), and to have active disease at the time of transplantation (85% vs. 59%, p < 0.001). With a median follow-up of 18 months, the cumulative incidences of relapse and nonrelapse mortality were 46% and 32% in the reduced-intensity conditioning group, versus 30% and 50%, respectively, in the myeloablative group. Two-year overall survival was perhaps superior in the nonmyeloablative group (39% vs. 29%; p ¼ 0.056). RESULTS IN SPECIFIC DISEASES Acute Myeloid Leukemia and Myelodysplastic Syndrome Hegenbart et al. analyzed outcome of 122 patients with acute myeloid leukemia ineligible for conventional HCT given allogeneic grafts after 2 Gy TBI with or without added fludarabine (90 mg/m2), and postgrafting immunosuppression combining MMF and CSP (44). Two-year probabilities of overall survival were 51% for patients transplanted in first complete remission (n ¼ 51), 61% for those transplanted in second remission (n ¼ 39), and 28% for those transplanted beyond second remission (n ¼ 32) (Fig. 4). High cytogenetic risks predicted for decreased overall survival (HR 2.4, p ¼ 0.008). Using a genetic randomization through a ‘‘donor’’ versus ‘‘no donor’’ comparison, Mohty et al. investigated whether allogeneic HCT after conditioning with fludarabine (180 mg/m2), busulfan (8 mg/kg), and ATG increased survival in adults (median age 52 years) with newly diagnosed high-risk acute myeloid leukemia in first complete remission ineligible for conventional HCT (45). Ninetyfive patients were retrospectively analyzed; 35 had an HLA-identical sibling donor (donor group), while 60 had no related HLA-matched donor (no donor group).
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Figure 4 Example of GVT response in a patient with mantle cell lymphoma relapsing after high-dose radiolabeled antibodies with autologous peripheral blood stem cell support. (A) Pretransplantation CT scan image (day 27) through the upper pelvis demonstrating an 8-cm by 7-cm mass that extended through 12 0.5-cm cuts. (B) CT scan image through the same region demonstrating complete resolution of the mass on day þ74 after nonmyeloablative transplantation from a matched unrelated donor. The patient remains in remission 30 months after transplantation with no evidence of GVHD. Abbreviations: GVT, graft-versus-tumor; CT, computed tomography; GVHD, graft-versus-host disease. Source: From Ref. 50.
Twenty-five of thirty-five patients included in the donor group (71%) received allogeneic HCT. The 10 remaining patients with an identified donor did not receive allogeneic HCT because of patient or donor refusal (n ¼ 6), early relapse (n ¼ 2), or psychiatric disorders appearing before HCT (n ¼ 2). In an intention to
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treat analysis, the four-year probability of progression-free survivals was 54% in the donor group versus 30% in the nondonor group (p ¼ 0.01). Ho et al. analyzed data from 62 patients with myelodysplastic syndrome given allografts from related (n ¼ 24) or unrelated (n ¼ 38) donors after reducedintensity conditioning with fludarabine (150 mg/m2), oral busulfan (8 mg/kg), and alemtuzumab (100 mg total dose) (46). Postgrafting immunosuppression consisted of CSP alone. Median patient age at HCT was 56 years for patients given grafts from siblings and 52 years for patients given grafts from unrelated donors. Sixteen patients had refractory anemia, 19 refractory anemia with blast excess, 23 refractory anemia with blast excess in transformation, and 4 chronic myelomonocytic leukemia. The one-year probabilities of nonrelapse mortality, overall survival, and progression-free survival were 5%, 73%, and 61%, respectively, for patients given grafts from related donors versus 21%, 71%, and 59%, respectively, for patients given grafts from unrelated donors. Chronic Myeloid Leukemia Or et al. reported data from 24 patients (median age 35 years) with chronic myeloid leukemia in first chronic phase given HLA-matched related (n ¼ 19) or unrelated (n ¼ 5) grafts after reduced-intensity conditioning combining fludarabine (180 mg/m2), busulfan (8 mg/kg), and ATG (11). Day-100 mortality was 0%, but three patients died as a consequence of GVHD 116, 499, and 726 days after HCT. The five-year probability of progression-free survival was 85%, with all 21 survivors having negative reverse transcriptase-polymerase chain reaction (RT-PCR) for Bcr-Abl. Kerbauy et al. analyzed data from 24 patients (median age 58 years) with chronic myeloid leukemia in first chronic phase (n ¼ 14) or beyond (n ¼ 10) given PBSC from HLA-matched related donors after conditioning with 2 Gy TBI with (n ¼ 16) or without (n ¼ 8) fludarabine (23). Four of eight patients not given fludarabine experienced nonfatal graft rejection and recurrence of chronic myeloid leukemia, while the 20 remaining patients achieved sustained engraftment. The two-year overall survival rate was 70% for patients transplanted in first chronic phase, and 56% for those with more advanced disease. Nine of ten patients transplanted in first chronic phase after conditioning with 2 Gy TBI with fludarabine achieved molecular remissions 3 to 24 months after HCT. In contrast to what was observed in patients given grafts from HLAmatched sibling donors, a high rate of graft rejection among chronic myeloid leukemia patients receiving grafts from unrelated donors after nonmyeloablative or reduced-intensity conditioning has been reported. We observed graft rejection in 9 of 21 patients given unrelated grafts for chronic myeloid leukemia after 2 Gy TBI and fludarabine (47). Graft rejections were nonfatal in all cases and followed by autologous reconstitution with persistence or recurrence of chronic myeloid leukemia. Seven of eleven patients with sustained engraftment, including all five patients in first chronic phase were alive in complete
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cytogenetic remissions 118–1205 (median 867) days after HCT. Hallemeier et al. observed graft failure in 5 of 22 evaluable patients given unrelated grafts after conditioning with 5.5 Gy TBI and cyclophosphamide (120 mg/kg) (14). Further efforts for reducing the risk of graft rejection in patients with chronic myeloid leukemia given unrelated HCT are directed at increasing the degree of pretransplant immunosuppression. Lymphoma and Chronic Lymphocytic Leukemia Khouri et al. reported results in 20 patients (median age 51 years) with low-grade NHL given grafts from siblings after conditioning with fludarabine (90–125 mg/m2) and cyclophosphamide (2000–2250 mg/m2), with or without added rituximab (15). Postgrafting immunosuppression consisted of tacrolimus and MTX. After a median follow-up of 21 months, the two-year current probability of disease-free survival was 84%. The same authors evaluated the efficacy of nonmyeloablative HCT in 20 patients with NHL recurrence after autologous HCT (48). Ten patients achieved complete remission with salvage chemotherapy before nonablative HCT, eight had a partial response, and two had stable disease. One patient died at 10.5 months from a fungal infection. The three-year progressionfree survival was 95%. Robinson et al. analyzed data from 188 patients (median age 40 years) with lymphoma [low-grade NHL (n ¼ 52), high-grade NHL (n ¼ 62), mantle cell lymphoma (n ¼ 22), or Hodgkin’s disease (n ¼ 52)] given HCT after various reduced-intensity or nonmyeloablative conditioning in EBMT-affiliated centers (49). The one-year probabilities of nonrelapse mortality were 39% and 22% in patients older or younger than 50 years, respectively (p ¼ 0.03). The twoyear probabilities of overall and progression-free survival were 65% and 54% for patients with low-grade NHL, 47% and 13% for patients with high-grade NHL, 13% and 0% for patients with mantle cell lymphoma, and 56% and 42%, respectively, for patients with Hodgkin’s disease. Chemosensitive disease at HCT was associated with better overall (RR, 2.4; p ¼ 0.002) and progressionfree (RR, 2.3; p ¼ 0.007) survivals in multivariate analyses. Morris et al. reported results of 88 patients with NHL given allogeneic HCT after conditioning with fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (100 mg) (13). Sixty-five patients received PBSC from HLAidentical siblings, while 23 received bone marrow from matched unrelated donors. GVHD prophylaxis consisted of CSP alone. Before DLI, grades III to IV acute GVHD were seen in four patients, but two additional patients developed grade IV acute GVHD after DLI. The actuarial three-year probability of current progression-free survival was 65% for patients with low-grade lymphoma (n ¼ 41), 50% for patients with mantle cell lymphoma (n ¼ 10), and 34% for patients with high-grade lymphoma (n ¼ 37) (Table 2). Maris et al. analyzed outcomes of 33 patients with relapsed or refractory mantle cell lymphoma who underwent allogeneic HCT from related (n ¼ 16) or
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unrelated (n ¼ 17) donors after 2 Gy TBI and fludarabine (90 mg/m2) (50). The overall response rate in the 20 patients with measurable disease at the time of HCT was 85% (including 75% complete remissions and 10% partial remissions) (Fig. 4). The two-year probabilities of relapse, nonrelapse mortality, and progression-free survival were 9%, 24%, and 60%, respectively. Sorror et al. described outcomes in 64 patients with chronic lymphocytic leukemia (median age 56 years) given HCT from HLA-matched related (n ¼ 44) or unrelated (n ¼ 20) donors after conditioning consisting of 2 Gy TBI with (n ¼ 53) or without (n ¼ 11) fludarabine (90 mg/m2) (51). Eighty-eight percent of patients were refractory to fludarabine. With a median follow-up of 24 months, the overall response rate was 67% (including 50% with complete remission). The two-year rates of nonrelapse mortality, overall, and progressionfree survivals were 22%, 60%, and 52%, respectively. Bulky lymphoadenopathy (lymph node diameter 5 cm) independently predicted higher incidence of relapse/progression (HR 3.8, p ¼ 0.009), while marrow infiltration with more than 50% leukemic cells was associated with worse survival (HR 2.4, p ¼ 0.05). These data, in agreement with those described in smaller series (52,53), show that chronic lymphocytic leukemia is remarkably susceptible to GVT effects. Multiple Myeloma Crawley et al. reported data from 229 patients given allogeneic HCT after various reduced-intensity conditioning in EBMT-affiliated centers (32). One hundred and ninety-two patients received grafts from related donors and 37 from unrelated donors. Overall, 25% and 48% of patients achieved complete or partial remissions, respectively. The three-year probabilities of overall and progressionfree survivals were 41% and 21%, respectively. Adverse progression-free survival was associated with alemtuzumab-containing conditioning (RR 1.8, p ¼ 0.001) and chemoresistance prior to transplant (RR 2.4, p ¼ 0.0004), suggesting that heavily pretreated patients and those with progressive disease did not benefit from this approach. Chronic GVHD was associated with better progression-free survival (p < 0.0001), while grades III to IV acute GVHD was associated with a worse overall survival (p ¼ 0.0007) and did not decrease the risk of relapse. TANDEM AUTOLOGOUS/ALLOGENEIC HCT To allow older patients with aggressive chemosensitive disease to benefit from both high-dose chemotherapy and GVT effects, it has been proposed to first use high-dose conditioning and autologous transplantation, which can be administered with overall mortality rates of less than 5%, followed one to three months later by allogeneic HCT using nonmyeloablative conditioning (tandem autologous/allogeneic HCT). This strategy, pioneered by Carella et al. in patients with refractory lymphoma (54), was evaluated by Maloney et al. in 54 patients with
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multiple myeloma. Patients were first given autologous HCT after a cytoreductive dose of 200-mg/m2 melphalan; this was followed 40 to 229 (median 62) days later by allogeneic HCT after 2 Gy TBI (55). Patients were 29 to 71 (median 52) years old, and 48% had refractory (35%) or relapsed (13%) disease. Remarkably, the 100-day mortalities after autologous and allogeneic HCT were 2% each, contrasting with the high nonrelapse mortality (ranging from 20% to 50% (56) observed in patients with multiple myeloma given allogeneic HCT after myeloablative conditioning. The two-year overall and progression-free survivals were 78% and 55%, respectively. CONCLUSIONS AND CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Reduced-intensity conditioning and nonmyeloablative regimens have allowed engraftment of allogeneic hematopoietic cells and the development of GVT effects. Antitumor responses have generally required extended periods of time, with a median time of six months required before achievement of complete remissions. In patients with slowly progressing diseases such as chronic myeloid leukemia in first chronic phase, low-grade myelodysplastic syndrome, chronic lymphocytic leukemia, or low-grade NHL, or with more aggressive diseases in complete remission, nonmyeloablative conditioning may be sufficient to achieve cure of the disease. A number of approaches are being explored for patients with more aggressive diseases, such as acute leukemias, high-grade myelodysplastic syndrome, multiple myeloma, or high-grade lymphomas, who are not in complete remission. A first approach is to combine nonmyeloablative HCT with ‘‘diseasetargeted’’ therapy, such as monoclonal antibodies or thalidomide. Khouri et al. reported 17 patients with chronic lymphocytic leukemia given allogeneic grafts from related donors after fludarabine (90 mg/m2) and cyclophosphamide (2250 mg/m2) (53). Ten patients received rituximab in addition to chemotherapy. The two-year overall survivals were 100% and 57%, in patients given or not given rituximab, respectively. We have been studying the administration of I-131 anti-CD45 monoclonal antibody followed by 2 Gy TBI and fludarabine to condition patients with acute myeloid leukemia not in remission and patients with advanced myelodysplastic syndrome (57). This approach has allowed administration of 40 Gy to marrow and 56 Gy to spleen, with a relative sparing of nonhematopoietic organs. Kroger et al. investigated the efficacy of thalidomide (100 mg) combined with DLI in 18 patients with multiple myeloma progressing after reduced-intensity HCT (58). The overall response rate was 67%, including 22% complete remissions. No grades II to IV acute GVHD were seen, while de novo limited chronic GVHD occurred in two patients (11%). The twoyear progression-free survival after DLI was 84%. A second approach might consist of posttransplant infusion of donorspecific cytotoxic T-cells directed against either tumor antigens (such as
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proteinase 3 or Wilms’ tumor-suppressor 1 in case of leukemia or patient paraprotein in case of multiple myeloma) or recipient minor histocompatibility antigens expressed exclusively on hematopoietic cells (such as HA-1 and HA-2 minor histocompatibility antigens), potentially increasing antitumoral efficacy of DLI with a low risk of inducing GVHD (59). A number of prospective phase III studies aimed at better defining the role of nonmyeloablative conditioning in patients with multiple myeloma (BMTCTN 01–02), lymphoma (BMT-CTN 02–02), or acute myeloid leukemia (GOELAMS AML 2001, FHCRC-1992.00) are ongoing in the United States and in Europe. Other randomized studies are focusing on comparing different conditioning regimens (FHCRC-1813.00) or defining the best postgrafting immunosuppression in the nonmyeloablative transplantation setting (FHCRC-1938.00). ACKNOWLEDGMENTS We thank Bonnie Larson, Helen Crawford, and Sue Carbonneau for their help with manuscript preparation. This work was supported by grants CA78902, CA18029, CA15704, and HL36444 from the National Institutes of Health, Bethesda, Maryland, U.S.A. and by a grant from the Leukemia and Lymphoma Society, White Plains, New York, U.S.A. REFERENCES 1. Storb R. Allogeneic hematopoietic stem cell transplantation: yesterday, today, and tomorrow. Exp Hematol 2003; 31:1–10. 2. Weiden PL, Flournoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300:1068–1073. 3. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 4. Kolb HJ, Schmidt C, Barrett AJ, et al. Graft-versus-leukemia reactions in allogeneic chimeras. Blood 2004; 103:767–776. 5. Barnes DWH, Corp MJ, Loutit JF, et al. Treatment of murine leukaemia with x-rays and homologous bone marrow. Preliminary communication. Br Med J 1956; 2:626–627. 6. Collins RH Jr., Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15:433–444. 7. Bethge WA, Hegenbart U, Stuart MJ, et al. Adoptive immunotherapy with donor lymphocyte infusions after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Blood 2004; 103:790–795. 8. Santos GW, Sensenbrenner LL, Burke PJ, et al. Marrow transplantation in man following cyclophosphamide. Transplant Proc 1971; 3:400–404. 9. Appelbaum FR, Herzig GP, Ziegler JL, et al. Successful engraftment of cryopreserved autologous bone marrow in patients with malignant lymphoma. Blood 1978; 52:85–95.
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24. Sandmaier BM, Maris M, Maloney DG, et al. Low-dose total body irradiation (TBI) conditioning for hematopoietic cell transplants (HCT) from HLA-matched related (MRD) and unrelated (URD) donors for patients with hematologic malignancies: a five-year experience. Blood 2003; 102(pt 1):78a–79a (abstr 264). 25. Maris MB, Niederwieser D, Sandmaier BM, et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 2003; 102:2021–2030. 26. Ueno NT, Cheng YC, Rondon G, et al. Rapid induction of complete donor chimerism by the use of a reduced-intensity conditioning regimen composed of fludarabine and melphalan in allogeneic stem cell transplantation for metastatic solid tumors. Blood 2003; 102:3829–3836. 27. Baron F, Baker JE, Storb R, et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004; 104:2254–2262. 28. Sandmaier BM, Maloney DG, Maris MB, et al. Conversion of low donor chimerism following nonmyeloablative conditioning for hematopoietic cell transplantation (HCT) using pentostatin and donor lymphocyte infusion (DLI). Blood 2004; 104(pt 1):57a (abstr 186). 29. Mielcarek M, Martin PJ, Leisenring W, et al. Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 2003; 102:756–762. 30. Sayer HG, Kro¨ger M, Beyer J, et al. Reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia: disease status by marrow blasts is the strongest prognostic factor. Bone Marrow Transplant 2003; 31:1089–1095. 31. Michallet AS, Furst S, Le QH, et al. Impact of chimaerism analysis and kinetics on allogeneic haematopoietic stem cell transplantation outcome after conventional and reduced-intensity conditioning regimens. Br J Haematol 2005; 128:676–689. 32. Crawley C, Lalancette M, Szydlo R, et al. Outcomes for reduced-intensity allogeneic transplantation for multiple myeloma: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood 2005; 105:4532–4539. 33. Weissinger F, Sandmaier BM, Maloney DG, et al. Decreased transfusion requirements for patients receiving nonmyeloablative compared with conventional peripheral blood stem cell transplants from HLA-identical siblings. Blood 2001; 98: 3584–3588. 34. Fukuda T, Hackman RC, Guthrie KA, et al. Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation. Blood 2003; 102: 2777–2785. 35. Hogan WJ, Maris M, Storer B, et al. Hepatic injury after nonmyeloablative conditioning followed by allogeneic hematopoietic cell transplantation: a study of 193 patients. Blood 2004; 103:78–84. 36. Junghanss C, Boeckh M, Carter RA, et al. Incidence and outcome of cytomegalovirus infections following nonmyeloablative compared with myeloablative allogeneic stem cell transplantation, a matched control study. Blood 2002; 99:1978–1985. 37. Junghanss C, Marr KA, Carter RA, et al. Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: a matched control study. Biol Blood Marrow Transplant 2002; 8:512–520.
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38. Parikh CR, Schrier RW, Storer B, et al. Comparison of ARF after myeloablative and nonmyeloablative hematopoietic cell transplantation. Am J Kidney Dis 2005; 45: 502–509. 39. Sorror ML, Maris MB, Storer B, et al. Comparing morbidity and mortality of HLAmatched unrelated donor hematopoietic cell transplantation after nonmyeloablative and myeloablative conditioning: influence of pretransplant comorbidities. Blood 2004; 104:961–968. 40. Diaconescu R, Flowers CR, Storer B, et al. Morbidity and mortality with nonmyeloablative compared to myeloablative conditioning before hematopoietic cell transplantation from HLA matched related donors. Blood 2004; 104:1550–1558. 41. Clift RA, Buckner CD, Appelbaum FR, et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: A randomized trial of two irradiation regimens. Blood 1990; 76:1867–1871. 42. Clift RA, Buckner CD, Appelbaum FR, et al. Allogeneic marrow transplantation in patients with chronic myeloid leukemia in the chronic phase: A randomized trial of two irradiation regimens. Blood 1991; 77:1660–1665. 43. Alyea EP, Kim HT, Ho V, et al. Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age. Blood 2005; 105:1810–1814. 44. Hegenbart U, Niederwieser D, Sandmaier BM, et al. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 2006; 24:444–453. 45. Mohty M, de Lavallade H, Ladaique P, et al. The role of reduced intensity conditioning allogeneic stem cell transplantation in patients with acute myeloid leukemia: a donor vs. no donor comparison. Leukemia 2005; 19:916–920. 46. Ho AYL, Pagliuca A, Kenyon M, et al. Reduced-intensity allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome and acute myeloid leukemia with multilineage dysplasia using fludarabine, busulphan and alemtuzumab (FBC) conditioning. Blood 2004; 104:1616–1623. 47. Baron F, Maris MB, Storer BE, et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with chronic myeloid leukemia. Biol Blood Marrow Transplant 2005; 11:272–279. 48. Escalon MP, Champlin RE, Saliba RM, et al. Nonmyeloablative allogeneic hematopoietic transplantation: a promising salvage therapy for patients with non-Hodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 2004; 22:2419–2423. 49. Robinson SP, Goldstone AH, Mackinnon S, et al. Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation: an analysis from the Lymphoma Working Party of the European Group for Blood and Bone Marrow Transplantation. Blood 2002; 100:4310–4316. 50. Maris MB, Sandmaier BM, Storer BE, et al. Allogeneic hematopoietic cell transplantation after fludarabine and 2 Gy total body irradiation for relapsed and refractory mantle cell lymphoma. Blood 2004; 104:3535–3542. 51. Sorror ML, Maris MB, Sandmaier BM, et al. Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. J Clin Oncol 2005; 23:3819–3829.
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52. Schetelig J, Thiede C, Bornhauser M, et al. Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reduced-intensity conditioning and allogeneic stem-cell transplantation: the Cooperative German Transplant Study Group. J Clin Oncol 2003; 21:2747–2753. 53. Khouri IF, Lee MS, Saliba RM, et al. Nonablative allogeneic stem cell transplantation for chronic lymphocytic leukemia: impact of rituximab on immunomodulation and survival. Exp Hematol 2004; 32:28–35. 54. Carella AM, Cavaliere M, Lerma E, et al. Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin’s disease and non-Hodgkin’s lymphoma. J Clin Oncol 2000; 18:3918–3924. 55. Maloney DG, Molina AJ, Sahebi F, et al. Allografting with nonmyeloablative conditioning following cytoreductive autografts for the treatment of patients with multiple myeloma. Blood 2003; 102:3447–3454. 56. Bensinger WI. The current status of hematopoietic stem cell transplantation for multiple myeloma. Clin Adv Hematol Oncol 2004; 2:700–706. 57. Pagel JM, Appelbaum FR, Sandmaier BM, et al. 131I-anti-CD45 antibody plus fludarabine, low-dose total body irradiation and peripheral blood stem cell infusion for elderly patients with advanced acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). Blood 2005; 106(pt 1):119a (abstr 397). 58. Kroger N, Shimoni A, Zagrivnaja M, et al. Low-dose thalidomide and donor lymphocyte infusion as adoptive immunotherapy after allogeneic stem cell transplantation in patients with multiple myeloma. Blood 2004; 104:3361–3363. 59. Bleakley M, Riddell SR. Molecules and mechanisms of the graft-versus-leukaemia effect. Nat Rev Cancer 2004; 4:371–380.
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25 Modulation of Classical Multidrug Resistance and Drug Resistance in General Branimir I. Sikic Oncology Division, Department of Medicine, Stanford University School of Medicine, Stanford, California, U.S.A.
INTRODUCTION Drug resistance is a major cause for the failure of both chemotherapies and targeted drugs. Among leukemias and lymphomas, intrinsic resistance, i.e., present from the outset of treatment, is much less common than in epithelial malignancies, but acquired resistance often manifests itself during the course of therapy or at relapse. In this chapter, we review reasons for the failure of systemic cancer therapies and attempts to reverse drug resistance, particularly multidrug resistance (MDR) related to expression of P-glycoprotein (P-gp). PHARMACOLOGICAL AND PHYSIOLOGICAL CAUSES OF TREATMENT FAILURE Inadequate Drug Dose and Suboptimal Schedule of Drug Administration Inadequate drug dosing or suboptimal scheduling of drug administration are potential pharmacological causes of treatment failure. Increased efficacy with increasing dose (up to the maximum tolerated dose) has been observed for most cytotoxic anticancer drugs both in animal models and in the clinic (1). Schedule of drug administration may also be important for efficacy, particularly for drugs
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with short half-lives and cell cycle phase–specific mechanisms of action. Thus, cytarabine in the therapy of acute leukemias is most effective when administered by continuous infusion (2,3). For recently developed targeted drugs such as the kinase inhibitors, optimal doses and schedules may not follow the paradigm that dosing should be based on maximal tolerance. Because many kinase inhibitors, such as imanitinib, inhibit multiple kinases at differing potencies, their efficacy and toxicities may vary depending upon dose and schedule of drug administration. Drug Sanctuary Sites (CNS and Testis) The blood-brain and blood-testicular barriers are major potential physiological causes of treatment failure in acute lymphocytic leukemias and high-grade lymphomas and have resulted in the use of prophylactic intrathecal drug administration and radiation. For many anticancer drugs, the multidrug transporter P-gp is a component of these barriers (4,5). Poor Drug Diffusion into Cancer Tissues Some drugs, notably the anthracyclines, have limited diffusing capacity beyond blood capillaries, and therefore tumor vascularization may be a limiting factor in their efficacy (6). This factor may be less important in hematological malignancies than in solid tumors. CELLULAR MECHANISMS OF DRUG RESISTANCE Intrinsic Vs. Acquired Resistance The various types of cellular mechanisms of drug resistance are listed in Table 1. Drug resistance may be intrinsic, i.e., present from the outset of therapy, or Table 1 Cellular Mechanisms of Resistance to Anticancer Drugs Mechanism
Example
Drug efflux transporters Impaired drug uptake Mutation or altered expression of drug target Intracellular redistribution of drug Detoxification of drug or drug product Enhanced DNA repair Decreased drug activation Altered programmed cell death (apoptosis)
P-glycoprotein (MDR1/ABCB1) Folate-carrier protein Point mutations in Bcr-Abl
Abbreviation: MVP, major vault protein.
MVP Glutathione transferases ERCC1 Cytidine kinase BH3 domain proteins: Bcl-2, Bcl-xl, Mcl-1
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acquired, i.e., developing during or after the course of treatment. Acquired resistance may result from the selection of preexisting, resistant mutant clones in the cancer population, or may also be selected or induced by therapies (7,8). Many detoxifying enzymes and drug transporters are expressed in normal epithelial tissues such as colon, kidney, and liver. These resistance mechanisms are also often expressed in epithelial cancers. They are less frequently expressed in hematolymphoid malignancies, except for certain subtypes, e.g., T-cell lymphomas, natural killer (NK)-cell lymphomas, and leukemic stem cells (7–9). Instrinsic resistance of some hematolymphoid cancers involves oncogenic mutations of p53 and high expression of Bcl-2, resulting in the inhibition of normal apoptotic signaling (10–16). The tissue environment of cancers is also an important physiological factor in drug resistance, via cell-cell and cell-matrix interactions that regulate apoptosis (17–19). Genetics of Drug Resistance Genetic instability resulting in clonal heterogeneity is one of the hallmarks of cancers. This genetic instability manifests as genetic aberrations, including aneuploidy, point mutations, deletions, gene amplifications, and chromosomal translocations (20). Goldie and Coldman modeled the genetic diversity of cancers, relating the rates of generation of drug resistant mutations to the number of cells, the probabilities for cure, and various treatment strategies (21–23). Mutation rates for drug resistance of 10–6 to 10–7 per cell generation have been found in human cancer cell lines via the Luria-Delbruck fluctuation analysis (24–27). Abundant evidence from cell lines, animal models, and clinical trials supports the concept of clonal heterogeneity in cancer cell populations as a major cause of treatment failure. The resistant clones may be preexisting as small populations present at diagnosis or arise during the course of therapy and appear as relapses or regrowth during treatment (20–23,25–28). An increase in gene copy number, or gene amplification, was first described as a drug resistance mechanism for the DHFR gene in cells treated with methotrexate (29). Gene amplification is now recognized as a common manifestation of genomic instability in cancer cells, both for oncogenesis (e.g., MYC, HER2, EGFR) as well as drug resistance (20,30). Epigenetic mechanisms have also been implicated in the induction of drug resistance, including cellular stress responses after radiation or chemotherapy, which can increase expression of the MDR gene MDR1/ABCB1 (31–33). Drug Efflux Transporters The human ABC transporter (ATP-binding cassette membrane proteins) gene family consists of approximately 50 genes, several of which are drug transporters (34–36). The major drug-resistance transporter genes include MDR1/ABCB1, several members of the MRP/ABCC subgroup, and ABCG2 (34).
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The most prevalent and extensively studied drug-resistance transporter is P-gp, the product of the MDR1/ABCB1 gene (37–40). P-gp is a transmembrane, pore-forming protein with 12 transmembrane segments and 2 cytoplasmic ATP-binding domains. Drugs are thought to bind to the transporter via diffusion from either the cytoplasm or from within the lipid bilayer of the cell membrane. It is a highly efficient efflux pump with broad substrate specificity, including approximately a third of all anticancer drugs as well as other drugs used in various areas of medicine. The molecular mechanism of P-gp involves an ATP-ase mediated conformational change in the protein after drug binding (39). Gene transfection studies have confirmed the direct role of P-gp in conferring MDR (38). The function of P-gp in normal tissues includes a role in drug excretion via the biliary tract of the liver and the proximal tubules of the kidneys, a barrier to drug absorption in the small and large bowel, and a barrier to tissue entry (endothelial cells of the central nervous system, testis, and placenta)(4,37). The classical MDR phenotype is due to P-gp expression in cancers, conferring cross-resistance to anthracyclines (doxorubicin, daunorubicin, idarubicin, and epirubicin), vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine), taxanes (paclitaxel, docetaxel), epipodophyllotoxins (etoposide, teniposide), mitoxantrone, and dactinomycin (39). Some newer drugs, such as imatinib, are also transport substrate for P-gp. Among hematologic cancers, P-gp expression confers an adverse prognosis in acute myeloid leukemias (AML), acute lymphoid leukemias, lymphomas, and myeloma, (41–46). P-gp is expressed in more than 70% of AML specimens from patients older than 60 years, versus approximately 30% of patients aged up to 60 years, and its expression correlates with reduced rates of complete remission (CR) and shorter survival (41). P-gp expression is more frequent in leukemia specimens of patients who have relapsed from prior therapy with MDR-related chemotherapy drugs (41). In addition to P-gp, several other members of the ABC gene family also function as drug transporters (47–55). MRP1/ABCC1 confers resistance to anthracyclines, vinca alkaloids and epipodophyllotoxins, and transports glutathione conjugates of drugs (47,48,52–55). None of the other ABC transporters are as strongly associated with clinical endpoints as P-gp/MDR1, and clinical strategies for reversing resistance related to these transporters have not been developed. MRP2/ABCC2, also known as the canalicular multiple organic anion transporter (cMOAT), is highly expressed in the biliary tract, and transports glucuronide and glutathione drug conjugates, including anthracyclines. It is the gene whose deficiency results in the Dubin-Jonson syndrome (51). MRP2 is involved in hepatic excretion of anticancer drugs, but has not been implicated as a resistance factor in cancers (49,55). The MRP3/ABCC3 gene confers resistance to epipodophyllotoxins as well as to methotrexate (50,51), while MRP4/ABCC4 and MRP5/ABCC5 confer resistance to nucleotide analogues and their metabolites (51).
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ABCG2 (BCRP) is implicated in clinical resistance to the anthracenedione drug mitoxantrone and the camptothecins (56,57). This transporter is smaller than those in the ABCB and ABCC gene subgroups and functions in dimeric form. It has been reported to be a negative prognostic factor in AML (58–60). ABCG2 and P-gp are constitutively expressed in both normal hematopoietic and leukemic stem cells (61,62). Polymorphisms in the DNA sequence of ABC transporters may affect drug disposition, efficacy, and toxicities (35), but were not found to affect treatment outcomes in patients with AML (63). Resistance to platinum drugs has been related to the expression of two membrane proteins involved in the efflux of copper, ATP7A and ATP7B (64). Impaired Drug Uptake Although most anticancer agents enter cells via passive diffusion, some drugs are transported into cells by membrane proteins, whose expression is a potential determinant of cellular sensitivity or resistance to antifolates. Thus, methotrexate enters cells by means of the reduced folate carrier, and decreased expression of the carrier is associated with resistance to the drug (65). The copper influx transporter, CTR1, is involved in the regulation of intracellular accumulation of the platinum drugs (cisplatin, carboplatin, and oxaliplatin) (64,66). Mutation or Altered Expression of Molecular Targets Amplification of the dihydrofolate reductase (DHFR) gene, the molecular target of antifolate drugs, has been described in cells grown in increasing concentrations of methotrexate (29), but the clinical relevance of this mechanism for conferring resistance to methotrexate has not been established. b tubulin and polymerized microtubules are targets for several important classes of chemotherapeutic drugs, including vinca alkaloids, taxanes, and epothilones (24). Mutations of b tubulins and altered expression of b-tubulin isoforms, particularly the Class III isoform, have been implicated in resistance to taxanes (24,67,68). Other potential mechanisms of resistance to antitubulin drugs include alterations in the expression of microtubule binding proteins, the P-gp transporter, the cell spindle checkpoint control pathway (69), and regulation of programmed cell death or apoptosis (24). The contribution of these mechanisms to antubulin drug resistance in hematologic malignancies has not been extensively studied. The drug targets for camptothecin and epipodophyllotoxin drugs are topoisomerase I and II, respectively, and altered expression or mutation of these enzymes can cause cellular resistance to these drugs (27,70–72). These drugs produce DNA breakage proportional to the amount of target enzyme in the cell, so that high enzyme content contributes to drug sensitivity, and decreased enzyme content is associated with resistance (73–75).
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Point mutations in the kinase domain of Bcr-Abl are an important resistance mechanism for the tyrosine kinase inhibitor, imatinib, in chronic myeloid leukemias (CML) (76). More than 30 such mutations in the target protein for imatinib have been identified. Two other Bcr-Abl inhibitors, dasatinib and nilotinib, have been approved by the U.S. Food and Drug Administration (FDA) because of their clinical activity against most of the imatinib-resistant Bcr-Abl mutants (76). The T315I mutant remains resistant to all three of these drugs, although other agents are being developed with activity against this Bcr-Abl mutation. Intracellular Redistribution of Drug High expression of the major vault protein (MVP), also known as lung resistance protein (LRP), is associated with intracellular drug sequestration and resistance to anthracyclines in cellular models (77). This protein is expressed in some AMLs, and may contribute to clinical drug resistance in that disease (78,79). Detoxification of Drug or Intermediate Drug Product Metabolic inactivation of drugs is an important mechanism of resistance for some drugs used in hematologic malignancies. For example, cytidine deaminase is a determinant of resistance to cytarabine (80). One of the mechanisms of resistance to bleomycin is its metabolic inactivation by an aminopeptidase termed ‘‘bleomycin hydrolase’’ (81). Detoxification via nucleophilic sulphur-containing compounds is an important mode of resistance to alkylating agents and platinums (82). Glutathione transferases catalyze this detoxification (83–88), and the glutathione-drug conjugates are then effluxed by members of the ABC transporter family (53,55). Enhanced DNA Repair DNA repair is a complex and highly regulated cell function involving more than 30 proteins, and plays an important role in determining tumor sensitivity to alkylating agents and platinum drugs (89–91). Genetic diseases such as ataxia telangiectasia, xeroderma pigmentosum, and Bloom’s syndrome provide evidence for the role of several DNA repair genes in cellular responses to DNA damaging drugs as well as ionizing radiation. Decreased Drug Activation Several antimetabolite drugs are metabolically activated to generate their active nucleoside, or nucleotide moiety, via kinases and phosphoribosyl transferases (92). Thus, for cytarabine, the generation of ara-dCTP levels intracellularly is an important determinant of the antitumor efficacy of this drug (92).
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The nitrogen mustard analogs cyclophosphamide and ifosfamide are prodrugs that require activation predominantly in the liver by mixed-function oxidases (CYP enzymes) (91). Although the major mechanisms of resistance to these drugs are thought to be DNA repair and glutathione conjugation, CYP gene expression within cancers may also be a determinant of their activity. Altered Pathways for Programmed Cell Death (Apoptosis) Most anticancer drugs and ionizing radiation exert their therapeutic effects by activating apoptosis or programmed cell death. The expression of proteins involved in apoptosis is a major determinant of sensitivity to therapies (10,11,13–15,93–95). In addition to Bcl-2, which is oncogenic in many B-cell lymphomas, by chromosomal translocations, Bcl-xl and Mcl-1 are also antiapoptotic proteins that can confer resistance to therapies (11,95). Inhibitors of these proteins are being investigated in hematologic cancers. Oblimersen, an antisense phosphorothioate drug targeting the Bcl-2 gene, has shown some activity when combined with fludarabine in chronic lymphocytic leukemias, with remission rates of 7% versus 17% (96). Small molecule inhibitors of the BH3 domain shared by the Bcl-2, Bcl-xl, and Mcl-1 proteins are being studied in lymphomas and multiple myeloma (97–100). The p53 pathway is mutated in the majority of human cancers. Normal p53 is an important regulator of apoptosis in response to DNA-damaging agents. Mutations of p53 can confer resistance to ionizing radiation and chemotherapeutic drugs such as alkylating agents, platinums, anthracyclines, and topoisomerase inhibitors (16,93,101). MODULATION OF MULTIDRUG RESISTANCE The prevalence of P-gp expression in many cancers, and clinical evidence of its adverse prognostic effects have led to attempts to reverse or modulate MDR by combining chemotherapy with inhibitors of P-gp (41,102–104). These trials of MDR modulation have used various inhibitors of P-gp, including verapamil, cyclosporine, quinine, the cyclosporine analog valspodar (PSC-833), and others. Most of these clinical trials have not shown clinical benefit. One of the major reasons for failure of MDR reversal to make a greater clinical impact is the co-expression of other mechanisms of drug resistance in many cancers. Other obstacles to the success of this approach include the inability to achieve adequate concentrations of the MDR-modulating drugs because of toxicities to normal tissues, drug interactions, and off-target effects because of nonspecificity of the modulating drugs and study designs that use an unselected patient population including patients who did not express P-gp. Some MDR inhibitors, particularly the cyclosporins, inhibit CYP3A4 and other drug metabolizing enzymes as well as other drug transporters, resulting in major drug interactions with cytotoxic chemotherapy drugs. These drug interactions may increase the risk of toxicities
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Table 2 Randomized Clinical Trials in AML and/or High-Risk MDS Demonstrating Benefit for MDR Modulation References
Modulating drug
Chemotherapy
Notes
107,108
Quinine
Mitoxantrone/ cytarabine
109
Cyclosporine
Daunorubicin/ cytarabine
110
Cyclosporine
Idarubicin/ cytarabine/ etoposide
High-risk MDS and secondary AML 65 yr old Benefit in P-gpþ only, OS 8 vs. 13 mo ( p ¼ 0.01) CR 18% vs. 52% ( p ¼ 0.02) Secondary AML (no prior therapy) Relapsed and refractory primary AML Benefit in OS and LFS for all patients, but most of the benefit was in the secondary AML subset Secondary AML >60 yr old CR 27 vs. 52% ( p ¼ 0.01) LFS 7 vs. 12 mo ( p ¼ 0.03)
Abbreviations: MDS, myelodysplastic syndromes; AML, acute myeloid leukemia; OS, overall survival, CR, complete remission; LFS, leukemia-free survival.
and require dose reduction of chemotherapy in many cases, further confounding the design and interpretation of the clinical trials (104–106). Despite these issues, some randomized clinical trials in AML have demonstrated clinical benefit for MDR modulation (41,107–110). Table 2 presents the results of three phase III trials, which have shown positive results in AML. The first of these is a French trial reported by Wattel et al., using quinine combined with mitoxantrone and cytarabine in patients with high-risk myelodysplastic syndromes (MDS) and secondary AML (107,108). In this study, statistically significant improvements were observed for the P-gp positive subgroup in both CR rates (18% vs. 52%) and overall survival (OS) (8 vs. 13 months). Notably, patients with P-gp negative MDS and AML did not benefit from the modulating drug in this study. The second trial was from the Southwest Oncology Group, SWOG 9126, and used continuous infusions of high-dose cyclosporine together with daunorubicin and cytarabine (109). Approximately half of the patients had relapsed or refractory primary AML, and half had secondary AML with no prior induction therapy. The overall results of this study were positive for both leukemia-free survival (9% vs. 34% at 2 years, p ¼ 0.031) and OS (12 vs. 24% at 2 years, p ¼ 0.046). Notably, the major effect was seen in prevention of relapse of patients achieving CR. Most of the benefit was found in the subgroup of patients who had secondary AML and no prior induction therapy. The third positive trial was the phase II randomized study of 55 patients aged over 60 years with secondary AML, recently reported by Matsouka et al.
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(110). The modulating drug in this trial was lower-dose cyclosporine, and the chemotherapy was idarubicin, cytarabine, and etoposide. P-gp was not measured in the AML specimens in this study. Despite the small number of patients, a significant improvement in CR was observed (27% vs. 52%, p ¼ 0.01) and leukemia-free survival (7 vs. 12 months, p –0.03). All three of these trials showed clinical activity in high-risk MDS and secondary leukemias, indicating that these study populations may be more amenable to MDR modulation as a therapeutic approach than other settings of leukemia (primary, relapsed, or refractory patients). In contrast to these trials, several studies of MDR modulation in AML have been negative. The Cancer and Leukemia Group B performed a trial of valspodar (PSC-833) combined with daunorubicin, cytarabine, and etoposide in patients with newly diagnosed AML aged 60 and above (111). The trial was halted early because of excess early mortality in the valspodar arm (44% versus 20% in controls), illustrating the problem of drug interactions with cyclosporin drugs and chemotherapies. Despite the higher early death rate, there was a trend in favor of the experimental arm for leukemia-free survival (5 vs. 14 months, p ¼ 0.07). A French trial of quinine in primary AML for patients 65 years and younger, combined with idarubicin and cytarabine, was negative for OS, but reported an improved CR rate for the P-gp positive subset (48% vs. 83%, p ¼ 0.01) (112). The Eastern Cooperative Oncology Group (ECOG) performed a trial of valspodar combined with mitoxantrone, etoposide, and cytarabine, in patients with refractory or relapsed leukemia aged 15 to 70 years, which was negative for all clinical outcomes (113). Novartis sponsored a trial of valspodar, daunorubicin, and cytarabine in patients aged 60 years and above with previously untreated AML, which was also negative for all clinical outcomes (114). Both this trial and the ECOG trial confirmed that P-gp expression was a significant adverse prognostic factor. Therefore, it is likely that other, co-expressed resistance mechanisms mitigated any clinical benefit for MDR modulation in these trials of valspodar. Finally, the Pediatric Oncology Group performed a trial of high-dose cyclosporine with induction therapy in children with AML (115). The negative results of this trial are not surprising, considering fewer than 15% of these pediatric AML specimens expressed P-gp. These trials are summarized in Table 3 (111–115). A more potent and specific inhibitor of P-gp, zosuquidar, is currently being tested more extensively in AML (116–118). This drug is specific for P-gp and thus has less potential for drug interactions with cytotoxins than other MDR modulators (119–121). CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS Drug resistance will always be a feature of cancer therapies, given the genomic instability and phenotypic heterogeneity of cancer populations. Understanding the nature of resistance to various agents and determination of mechanisms of resistance in clinical specimens will continue to be important areas of research. For targeted therapies, non-cross-resistant drugs can be developed via rational
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Table 3 Negative, Randomized Clinical Trials of MDR Modulation in AML Modulating References drug 111
112
113
114
115
Chemotherapy
Notes
Valspodar Daunorubicin/cytarabine/ De novo and secondary AML (PSC-833) etoposide (ADE) (no prior therapy) 60 yr old No improvement in CR, LFS, and OS 60 yr old Excess early deaths with valspodar, 20% vs. 44% Quinine Idarubicin/cytarabine De novo AML 65 yr old CR 48% vs. 83% in P-gpþ subset (p ¼ 0.01) No improvement in OS Valspodar Mitoxantrone/etoposide/ Relapsed/refractory de novo AML (PSC-833) cytarabine (MEC) High-risk MDS 15–70 yr old No improvement in CR, LFS, and OS Valspodar Daunorubicin/cytarabine De novo and secondary AML (PSC-833) (no prior therapy) 60 yr old No improvement in CR, LFS, and OS Cyclosporine Idarubicin/cytarabine/ Pediatric AML 18 yr old etoposide Less than 15% of specimens expressed P-gp
Abbreviations: AML, acute myeloid leukemia; CR, complete remission; OS, overall survival; LFS, leukemia-free survival; MDS, myelodysplastic syndromes.
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Index
[Acute myeloid leukemia (AML)] cytokine secretion, 334–335 dendritic cells (DC) adjuvants and, 337–338 costimulatory pathways, 338–339 culture of, 335–336 and FISH, 336 Flt-3 internal tandem duplication (ITD), 337 and surface markers, 337 detection of molecular targets in, 9–11 flavopiridol for, 362 FLT3 mutations in, 380 in adult patients, 383–384 in pediatric patients, 384 immunological synapse (IS) in, 332 MHC expression on, 333 monoclonal antibodies in adult patients with, 103–115 monoclonal antibodies in pediatric patients with, 115–118 MUC1 expression on, 333 phase 3 trials for, 85–86 p53 mutations in, 263–264 VEGF in, 295–297 Acute nonlymphocytic leukemia (ANLL) cell death assay results and clinical outcome of, 30, 32 testing cell death in, 25
Abelson (Abl) kinase inhibitors, 411 development of second-generation, 417–421 observations from trial experiments, 421–422 Abl kinase mutations, 418 Abl leukemia virus (v-Abl), 412 Abl messenger RNA (mRNA), 412 ABT-737, 269 Active immunotherapy, 99 Acute lymphoblastic leukemia (ALL), 262, 413 cell death assay results and clinical outcome of, 30–31 clinical relevance of the DiSC assay in, 27 detection of molecular targets in, 11–13 FLT3 mutations in, 380 patient survival in, 33 PML-RARa in, 54, 60, 186–187 maturation arrest, 185–186, 191 p53 mutations in, 263–264 VEGF in, 297 Acute myeloid leukemia (AML), 102–103 Bcl-2 in, 264 costimulatory molecules expression on, 334
581
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582 Acute promyelocytic leukemia (APL), 235 pathogenesis of, 186–187 predifferentiation era of, 187 treatment of children with, 189 treatment of elderly patients with, 189 Adaptive randomization, 86–91 Adenosine triphosphate (ATP) assay, 24 Adenovirus E1A oncoprotein, and HAT, 234 Adjuvants, 337–338 Ad-p53 gene, 92–93 Affymetrix SNPs of, 7 software, 6 U95A microarrays, 9 U133 microarrays, 3–4 AG013676, 439 Agnogenic myeloid metaplasia (AMM), 499 AIDA trial, 190 AKAP13, 493 Akt-mediated phosphorylation, of TSC2, 527 Alemtuzumab, 65, 101, 118 for APL patients, 195–196 for CLL patients, 138 hematological toxicity of, 129 Aliphatic acids, 242–243 ALL. See Acute lymphoblastic leukemia; Acute lymphoblastic leukemia (ALL) Allele specific oligonucleotide (ASO), 51 flow cytometry and PCR with, 64–65 Allogeneic hematopoietic cell transplantation (HCT) importance, 539 observations, 540 results of donor lymphocyte infusions as treatment, 540–541 All-trans retinoic acid (ATRA), 11 with consolidation chemotherapy, 60 for extramedullary disease in APL, 190–191 and idarubicin, 115 and induction chemotherapy, 103 liposomal formulation of, 190 maintenance therapy with, 188–189 for treatment of elder patients and children with APL, 188–189
Index 17-allylamino-17-demethoxygeldanamycin (17-AAG), 432 American Society of Clinical Oncology (ASCO), 34–35 American Society of Hematologists, 176 American Society of Hematology, 34 Amino acids, in FLT3, 381 AML. See Acute myeloid leukemia (AML) AML cells, immunophenotypes of, 47. See also Acute myeloid leukemia (AML) AML-DC. See Acute myeloid leukemia (AML), dendritic cells (DC) AMP-activated kinase (AMPK), 530 Anaplastic large cell lymphoma (ALCL), 50, 55 Angiogenesis. See also Vascular endothelial growth factor (VEGF) agents, 186, 196 angiogenic factors, 285 antibody. See Bevacizumab defined, 283–284 and growth hormones, 284 in pathogenesis of hematologic diseases, 196 in pathology, 285 phenotypic switch to. See Angiogenic switch Angiogenic switch, 286–287 ANLL. See Acute nonlymphocytic leukemia (ANLL) Anthracyclines for combination chemotherapy with cytosine, 103, 187 for consolidation therapy in patients with APL, 198 for induction therapy in APL patients, 187 for treatment of patients with ANLL, 25 Antiangiogenesis agents, 186, 196 Antiangiogenic concepts, 289–290 Anti-B1, 154–155 Antibodies, 397–398 Antibody-dependent cellular cytotoxicity (ADCC), 398 of antibodies, 101 of FLT3 signaling, 119 of rituximab, 126, 154
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Index Anti-CD45 antibodies, 118 Anti-CD20 antibody. See Rituximab Anti-CD22 antibody. See Epratuzumab Anti-CD52 antibody. See Alemtuzumab Anti-CD33 antigen. See Gemtuzumab ozogamicin Antigen expression asynchronous, 47 cross-lineage, 47 ectopic, 47 of ZAP70 gene, 14 Antigen-presenting cells (APC) and T cells, 332–334 Anti-GMCSF antibody, 119 Antileukemic therapy, apoptosis regulators in Bcl-2 proteins in, 267–269 IAP in, 266–267 TRAIL receptors, 269–272 Antisense nucleic acid (ASNA), 312 Antisense oligodeoxynucleotides (ASODN), 316 Antisense oligonucleotide (AS ON) in hematopoietic cells, 313–314 AntiTac, 475 Anti-Tac antibody, 172 AP23573, 530 APC. See Antigen-presenting cells (APC) APL. See Acute promyelocytic leukemia (APL) APL cells, 11. See also Acute promyelocytic leukemia (APL) Apoptosis. See also Cell death blocking anti-CD33 antibodies in, 119 caspases in, 258 CD95 system for, 258, 260, 263 cytotoxic therapy and, 263 due to ATO’s mechanism of action, 191 Fas-induced, 126 by flavopiridol, 360–361 and leukemia. See Leukemia overview, 257–258 p53 in, 260 regulators Bcl-2, 264, 267–269 IAP, 264–267 prognostic significance of, 262–265 TRAIL receptors, 269–272
583 [Apoptosis. See also Cell death] by UCN-01 (7-hydroxystaurosporine), 365 Ara-C, 94. See also Cytosine arabinoside for consolidation chemotherapy, 198 for extramedullary disease in APL, 190 for induction therapy, 197 Arsenic trioxide (ATO), 86, 115 clinical trials for the treatment of APL, 191–193 mechanism of action in APL patients, 191 for monitoring of minimal residual disease, 198–199 for patients with newly diagnosed APL, 193–195 for the treatment of relapsed disease, 198 ASCT. See Autologous stem cell transplantation (ASCT) ASNA. See Antisense nucleic acid (ASNA) Asparaginase, 12 ATO. See Arsenic trioxide (ATO) ATRA. See All-trans retinoic acid (ATRA) Aurora kinase inhibitor MK0457, 412 Autologous stem cell transplantation (ASCT), 139, 166. See also Hematopoietic stem cell transplantation (HSCT) Autophosphorylation, of FLT3, 382 5-azacytidine (Vidaza1), 216–217 5-aza-2’deoxycytidine (Dacogen1), 216–217
Bacillus Calmette Gue´rin (BCG), 337 Basic fibroblast growth factor (bFGF), 437 Bax, 264 Bayesian learning, 87 Bayes’ theorem, 87 B-cells, 154 chronic leukemias detection of MRD in, 49 development and differentiation of, 13 lymphoma. See Diffuse large B-cell lymphoma (DLBCL) malignancies in mature, 51 precursors, 47 receptors, 14
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584 Bcl-2, 128, 264 in antileukemic therapies, 267–269 Bcl-6, 242, 316 signaling and inhibitors. See Diffuse large B-cell lymphoma (DLBCL) BCL2 gene, 49, 55, 163 Bcr-Abl inhibitor clinical development, 414–415 discovery, 412–413 future perspectives, 424–425 pathways activated by, 414 in Ph chromosome abnormality, 413–414 recognition of, 417 resistance to, 416–417 treatment for advanced phase Ph(þ) leukemias, 422–423 Bcr-Abl proteins, 262, 413 BEAM therapy, 139 for the development of RIT, 177 b-emitters, 152, 175 Benzamides CI-994, 249 MGCD0103, 249 MS-275, 248 Benzoquinone ansamycins (BA), 432 Bevacizumab, 102, 438 Bexxar. See 131I-tositumomab BH3-only proteins, 268–269 213 Bi-HuM195 immunotherapy in, 170 preclinical administration of, 155–156 Bioconductor, 6 Bismuth isotopes. See 213Bi-HuM195 BMS-214662, 495 BMS-387032 (SNS-032), 367–368 Bone marrow angiogenesis in, 196 B-cell precursors, 47 premature evaluation of, 197 toxicity in, 118, 129 Bortezomib, 128, 471, 473, 479 and CYC202, 369 and flavopiridol, 368–369 Broad tissue typing, 101 Burkitt’s lymphomas, 49, 55 xenograft model, 475
Index CAAX box, 492 c-Abl gene, 412 Cadmium, and DNMT, 210 Caenorhabditis elegans, 493 Calcium ionophores (CI), 335 CALGB study, 86 Calicheamicin, 104, 195 Carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132), 471 Caspases, 258 CD28, 330, 334 CD34þ AML cells, 9 antigen expression, 47 CML cells, 13 stem cell compartment, 63 CD95, in leukemia, 264 CD45 antibodies, 118 CD20 antigen, 150–151 CD33 antigen, 103, 151 CD45 antigen, 151 CD66 antigen, 151 CD20þ cells, 165–166 CDK inhibitors BMS-387032 (SNS-032), 367–368 CYC202, 367, 369 flavopiridol. See Flavopiridol UCN-01 (7-hydroxystaurosporine), 363–367, 369–370 CD58 marker, for MRD detection, 49 Cell cycle checkpoints, genes for, 9 Cell cycle control genes, 9 Cell cycle progression, and CDK, 353–354 cyclins in, 354 Cell cycle-regulatory genes, 355–357 Cell death assays, 24–26 results and the clinical outcome of, 26–32 modes of, 260 Cell division cycle 25 (Cdc25) phosphatases, 453–455 Cellular mechanisms, of drug resistance altered pathways for programmed cell death (apoptosis), 569 to anticancer drugs, 564
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Index [Cellular mechanisms, of drug resistance] detoxification of drug or intermediate drug product, 568 DNA repair, 568 drug activation, 568–569 drug efflux transporters, 565–567 genetic factors, 565 impaired drug uptake, 567 intracellular redistribution of drug, 568 intrinsic vs acquired resistance, 564–565 mutation or altered expression of molecular targets, 567–568 Central-supramolecular activating complex (c-SMAC), 332 CEP-701, 389, 397 in adult AML, 394 in children, 396 inhibition of, 13 CGP57148 (STI571), 411 Chemoimmunotherapy, 65 Chemotherapy with BEAM, 139 and cell death assay results, 26–32 combination with rituximab, 131–133 drugs (doxorubicin), 128 FLT3 inhibitors with, 390–392 2-chlorodeoxyadenosine (2-CdA), 86 CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy, 131 Chromatin, 233 Chromosomal translocations, in leukemia in ALL, 262 and AML, 450 in CLL, 261–262 in CML, 262 Chromosome aberrations, 54–56, 62 Chronic lymphocytic leukemia (CLL), 261–262 cells, 138 in vitro cell death effects of drugs, heat, and radiation on, 27 detection of molecular targets in, 14 DiSC assay in clinical relevance of, 27 clinical response and results of, 31–32 flavopiridol for, 362
585 [Chronic lymphocytic leukemia (CLL)] improving response and survival of, 34 p53 mutations in, 263 VEGF in, 298 Chronic myeloid leukemia (CML), 262 clinical relevance of, 63–64 defined, 380 detection of molecular targets in, 13–14 MRD monitoring in, 55 p53 mutations in, 263 stem cells, 63 treatment of. See Bcr-Abl inhibitor VEGF in, 297–298 CHVP (cyclophosphamide, hydroxydaunomycin, vm 26, prednisone), 131–132 CI-994, 249 Circulating endothelial cells (CEC), 289 Class I mutations, 449–450 Clinical reassessment method (CRM), 96 CLL. See Chronic lymphocytic leukemia (CLL) Clonogenic leukemic cells, 260–261 CML. See Chronic myeloid leukemia (CML) c-Myb, 315–316 c-Myc, 9, 262, 529 Coley, William B., 329 Complement-dependent cytotoxicity (CDC), of mAb, 126, 154 Conjugated antibodies, 102 Consolidation therapy, 198 with ATRA, 60 Copper-67 (67Cu). See B-emitters Core binding factors (CBFs), 10, 54, 62 Coumarin antibiotics, 433 CP-547632, 439 CpG islands, 208 Crkl phosphorylation, 414 CRp. See Platelet transfusions c-SMAC. See Central-supramolecular activating complex (c-SMAC) Curcumin, 196 CVP (cyclophosphamide, vincristine, prednisone) therapy, 131 CYC202, 367 and ABT-737, 369 and bortezomib, 369
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586 Cyclic adenosine monophosphate (cAMP), 196 Cyclic peptides romidepsin, 247–248 Cyclin D1, 49, 529. See also Cyclins and flavopiridol, 359 Cyclin-dependent kinase (CDK) and cell cycle progression, 353–354 cyclin, 354 and gene transcription, 355 inhibitors. See CDK inhibitors Cyclins, 354 mantle cell lymphoma and, 355 Cyclophosphamide, 168, 171, 479, 541 Cyclosporine A, 114 Cytarabine, 63 combination with anthracyclines, 103, 187 and idarubicin, 113 Cytarabine (ara-C), 434–435, 438 Cytokine-release syndrome, 126, 129 Cytokines, use of, 99 Cytosine arabinoside, 187 Cytotoxic T cells (CTL), 330
Dasatinib, 380, 414, 417–418, 421 dose-optimization studies for, 420 Start R trial, 420 toxicity, 419 Data-mining, 5 Daunorubicin, 12, 197–198 DAVID, pathways analysis software, 7 Deacetylation, of histones, 234 and DNA methylation, 212–213 Death-inducing signaling complex (DISC), formation of, 126 Death receptor pathway, activation of, 126 Decitabine. See 5-aza-20 deoxycytidine (Dacogen1) Dendritic cells (DC), 331–332. See also Acute myeloid leukemia (AML) blasts leukemia-derived DC. See Leukemic DC 20 -deoxy-20 -fluoro-D-arabinonucleic acid (20 F-ANA), 315 Depsipeptide. See Romidepsin 5,6-Dichloro-1-b-Dribofuranosylbenzimidaloe (DRB), 355
Index Differential Staining Cytotoxicity (DiSC) assay, 24–26 clinical relevance in cancer cell death, 27 vs. MTT assay, 27, 30 Diffuse large B-cell lymphoma (DLBCL), 8, 135 17-(dimethylaminoethylamino)-17demethoxygeldanamycin, 432 DiSC assay. See Differential Staining Cytotoxicity (DiSC) assay DLBCL. See Diffuse large B-cell lymphoma (DLBCL) DLI. See Donor lymphocyte infusion (DLI) DNA hypomethylating agents 5-azacytidine (Vidaza1), 216–217 5-aza-20 deoxycytidine (Dacogen1), 216–217 with HDAC inhibitors, 217–221 DNA methylation and genomic imprinting, 208 and HDAC, 212–213 and histone acetylation, 211–214 and histone methylation, 214–216 and tissue-specific genes, 208 in tumor cells, 208–211 DNA methyltransferases (DNMT), 208 cadmium and, 210 and histone methyltransferases (HMT), 214 DNMT. See DNA methyltransferases Docetaxel, 92 Donor lymphocyte infusion (DLI), 330 Dosimetry, 173–175 Double induction trial, 95 Double stranded RNA (dsRNA), 312–313 Doxorubicin, 128, 433–434 D3-phosphate of inositol phospholipids, 453 Drug resistance cellular mechanisms altered pathways for programmed cell death (apoptosis), 569 to anticancer drugs, 564 detoxification of drug or intermediate drug product, 568 DNA repair, 568 drug activation, 568–569 drug efflux transporters, 565–567 genetical factors, 565
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Index [Drug resistance cellular mechanisms] impaired drug uptake, 567 intracellular redistribution of drug, 568 intrinsic vs acquired resistance, 564–565 mutation or altered expression of molecular targets, 567–568 clinical perspectives, 571–572 MDR modulation, 569–571 treatment failure, pharmacological and physiological causes drug sanctuary sites (CNS and testis), 564 inadequate drug dose and suboptimal schedule of drug administration, 563–564 poor diffusion into tissues, 564 ‘‘Dynamic allocation’’, 90 Dysoxylum binectariferum, 357
Early T-cell progenitors (ETPs), 514 EBNA2 protein, and HAT, 235 ECOG study, 86 EMD. See Extramedullary disease (EMD) a-emitters to estimate pharmacokinetics and dosimetry in leukemia patients, 170 LET radiation of, 152–153 Endocytosis, 119 Endothelial progenitor cells (EPC) defined, 287 Epigenetics. See also DNA methylation defined, 207 Epoxomycin, 471 Epoxyketones, 471 Epratuzumab, 101 Etoposide, 139, 168, 433 Eukaryotic elongation factor (eEF) 2 protein kinase, 528 Eukaryotic initiation factor 4B (eIF4B), 528 European Agency for the Evaluation of Medicinal Product, 120 European APL Group trials, 188–189, 197–198 European Leukemia Network (ELN), 14
587 European Organisation for Research and Treatment of Cancer (EORTC), 132–133 Everolimus, 530 Extramedullary disease (EMD), 190–191 Extreme drug resistance (EDR) endpoint, 31
FAB (French-American-British) classification, of AML, 102 Farnesylation, cellular proteins undergoing, 493 Farnesyltransferase inhibitors (FTIs) clinical developments, 501–503 clinical perspectives, 506 gene expression, 505–506 for hematologic malignancies acute myelogenous leukemia (AML), 495–497 CML and other MPDs, 498–499 myelodysplasia, 497–498 myeloid malignancies, 499–501 resistance to, 504–505 role in processing of Ras proteins mitotic proteins, 494–495 phosphatidylinositol-3 Kinase (PI3K)/Akt pathway, 494 Rho, Rac and Rheb proteins, 492–493 vascular endothelial growth factor (VEGF), 494 Fas receptor, 126–127 FBW7 protein, 520 FCM (fludarabine, cyclophosphamide, mitoxantrone) study, 132 Fc receptor, 126 Fine-needle aspiration biopsy, 92 FISH. See Fluorescent in situ hybridization (FISH) FISH technique, 413 FL. See Follicular lymphoma (FL) Flavopiridol apoptosis by, 360–361 and Bcr-Abl kinase inhibitors, 368 bortezomib, 368–369 cell cycle arrest and, 358 cyclin D1 downregulation by, 359
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588 [Flavopiridol] gene transcription inhibition, 358–359 Mcl-1 downregulation by, 360 overview, 357–358 and TRAIL, 368 and vorinostat, 368 Flow cytometric immunophenotyping for MRD detection in acute leukemias, 47–49 in B-cell leukemias, 49 in the occurrence of immunophenotypic shifts, 50 in T-cell leukemias, 49–50 Flow cytometry, 17 four-color, 64–65 immunophenotyping. See Flow cytometric immunophenotyping multiparamete, 47, 62 FLT3, 449–450 amino acids in, 381 antibodies, 397–398 autophosphorylation of, 382 inhibitors. See FLT3 inhibitors ligands of, 382 mutations. See FLT3 mutations FLT3 gene, 12, 119, 195 mutations of, 10 FLT3 inhibitors with chemotheapy, 390–392 clinical trials, 393–396 indolinones, 389–390 indolocarbazoles. See Indolocarbazoles MLN-518, 390, 395 resistance to, 396–397 sorafenib, 390 Flt-3 internal tandem duplication (ITD), AML-DC and, 337 FLT3 mutations, 382–383 in ALL, 384–385 in AML in adults, 383–384 in pediatric patients, 384 in stem cells, 385–386 Fludarabine, 114, 118, 479 for CLL, 32–33 Fluorescent in situ hybridization (FISH), 1, 65, 336
Index Fluorodeoxyglucose positron emission tomography (FDG-PET), 529 Fluorometric microculture cytotoxicity assay (FMCA), 25, 31 Follicular lymphoma (FL) MRD monitoring in patients with, 68–69 radioimmunotherapy for the treatment of, 133–135 somatic mutation process in, 51 Food and Drug Administration (FDA), 101, 120 approval of RIT drugs, 150 registration of RIT drugs, 162 Fusion proteins, 262, 450 in leukemia, 211
Gamma camera imaging, 161, 169, 175 Gastrointestinal stromal tumors (GIST), 380 Geldanamycin (GA), 432 Gemtuzumab ozogamicin, 194–195 combination with ATRA and ATO, 115 combination with MFAC regimen, 114 hepatotoxicity of, 112 and IL-11, 113 mechanism of action, 104 results of phase I/II studies with, 105–111 for the treatment of AML patients, 112–115 Gene expression profiling comparison with ITRT, 32 of leukemia, 9–14 of lymphoma, 8–9 microarrays, 3–5 pathway analyses tools for, 7 software packages, 6 Gene microarray analysis, 128 Gene silencing, 312–313 Gene transcription, and CDK, 355 inhibition by flavopiridol, 358 Gene transcription regulation, 233–235 posttranslational modifications in, 233–234 Genomic imprinting, and DNA methylation, 208
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Index GIMEMA group, 187, 189–190 Glass slide microarrays, 3 GleevecTM, 411, 450 Glivec1, 411 Global genomic hypomethylation, 209 Graft-versus-host disease (GVHD), 540 Graft-versus-leukemia (GVL) effect, 423–424 Graft-versus-tumor (GVT) effects, 539–541, 545, 547–548 Granulocyte colony stimulating factor (G-CSF), 163–164 Granulocyte-macrophage colony stimulating factor (GM-CSF), 437 ‘‘Group sequential’’ designs, 88 GW786034, 439
HapMap project, 7 HDAC. See Histone deacetylation HDAC inhibitors, 196, 217–221, 435 aliphatic acids, 242–243 and Bcl-6, 242 benzamides CI-994, 249 MGCD0103, 249 MS-275, 248 classifications of, 218–220 heat shock proteins (HSP), 242 hydroxamates LAQ-824, 245 LBH589, 245–246 PXD101, 245 pyroxamide, 245 vorinostat, 243–245 molecular pharmacology of, 237–241 and p53, 242 TSA, 237 Heat shock proteins (HSP), 242 functions, 430 Hsp27, 435–436 Hsp70, 435–436 Hsp90, 431–432 ATP-Hsp90, 431 Hsp90 inhibitors, 432–435 in leukemias, 434–435 other, 435–436 roles in apoptotic response to cell stress, 431
589 Hematological malignancies, 46 MRD detection in chromosome aberrations, 54 immunophenotypic, 50 radioimmunotherapy of. See Radioimmunotherapy treatment with monoclonal antibodies, 102 Hematopoiesis defects in, 186 mature and immature leukemic stages of, 46 transcription factors of, 9 Hematopoietic stem cell transplantation (HSCT), 59, 63–64. See also Autologous stem cell transplantation (ASCT) allogeneic, 61, 63 autologous, 61, 69 as treatment consolidation for AML patients, 62 Histone acetylation, 234. See also Histone acetyltransferases (HAT) and DNA methylation, 211–214 nucleosomes in, 211–212 Histone acetyltransferases (HAT) adenovirus E1A oncoprotein and, 234 EBNA2 protein, 235 HIV Tat protein and, 234–235 MYST family of, 234 ‘‘Histone code’’, 211 Histone deacetylases (HDAC), 234 classification of, 235–237 inhibitory activity of. See HDAC inhibitors and PML-RARa, 196, 236 Histone deacetylation, and DNA methylation, 212–213 Histone methylation, and DNA methylation, 214–216 Histone methyltransferases (HMT), and DNMT, 214 Histones. See also Histone acetyltransferases (HAT); Histone deacetylases (HDAC) acetylation of, 234 deacetylation of, 234 octamer, 233 posttranslational modifications, 233–234
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590 HIV Tat protein, and HAT, 234–235 HL-60 cells, 11, 186 Hodgkin’s lymphoma (HL), VEGF in, 294–295 HOVON trials, 86 HSCT. See Hematopoietic stem cell transplantation (HSCT) HSP. See Heat shock proteins (HSP) HuM195, clinical trials of, 169 Human antichimeric antibodies (HACA), 128 Human antimonoclonal antibodies (HAMA), 163, 165 development of, 128 immune reaction, 101 Human genome U133 microrray, 4 Hydroxamates LAQ-824, 245 LBH589, 245–246 PXD101, 245 pyroxamide, 245 vorinostat, 243–245 Hyperbilirubinemia, 169 Hyper CVAD, 422–423 Hypothesis-test-based designs, 91 Hypoxia inducible factor-1a (HIF-1a), 529 IA (Idarubicin þ ara-C), 89–90 131 I-anti-CD45 monoclonal antibody, 156 clinical studies of, 171 for treatment of AML patients, 172 IAP. See Inhibitor of apoptosis proteins (IAP) IA vs. TA vs. TI trial, 89–90 131 I-BC8 mAb, 171 Ibritumomab, 101 Idarubicin, 113, 115 131 I-30F11 antibodies, 156 IFN, 414 IFN-a, 63 IL-11. See Interleukin- 11 (IL-11) 131 I-LL2 antibody, 157 Imatinib, 36, 63, 411, 421 clinical development of, 414–415 resistance to, 416 ‘‘Start C’’ trial, 418–419 Start R trial, 420 toxicity, 419
Index Imatinib mesylate, for CML treatment, 298, 429 Immunoglobulin (Ig) genes detection of junctional regions of, 50–51 rearrangement, 53–54 Immunological synapse (IS), 332 Immunophenotypic shifts, 50 Immunotherapy, for leukemia, 330 Immunotoxins, 128 Indium-111. See B-emitters Individualized tumor response testing (ITRT), 24 comparison with gene expression profiling, 32 discovery of new drugs through the use of, 37 requirement for renewal of, 36 Indolinones, 389–390 Indolocarbazoles CEP-701, 389, 394, 396 397 K252a, 388 PKC412, 389, 395 staurosporine, 388 Induction therapy, 197 combination with ATRA, 103 Infusional toxicity, and monoclonal antibodies, 129 Ingenuity, 7 Inhibitor of apoptosis proteins (IAP), 258 survivin, 264–265 XIAP. See X-chromosome-linked inhibitor of apoptosis proteins (XIAP) INNO-406, 412, 422 Inorganic PTP inhibitors, 461 Insulin-like growth factor I receptor (IGF-1R), 527 Interleukin-11 (IL-11), 113 Internalization, 103–104, 113 International BFM Study Group, 58 International Randomized Study of Interferon (IRIS) study, 63 Iodine-131 (I-131). See 131I-tositumomab 131 I radioisotope, 152 IRIS (International Randomized Trial of IFN/Ara-C vs STI571) trial, 415 131 I-tositumomab, 127 in follicular lymphoma, 134 toxic effects of, 165–166
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Index [131I-tositumomab] treatment regimen for, 161–163 vs. 90Y-ibritumomab tiuxetan, 173
Japanese Adult Leukemia Study Group (JALSG), 189 Japanese Ardisia herb, 267 Journal of Clinical Oncology, 36 Jun N-terminal kinase, 190
Kaposi sarcoma herpsevirus (KSHV) K1 gene, 440 Ketoconazole, 432 Keyhole limpet hemocyanin (KLH), 337 KF25706, 433 KF58333, 433
Lactacystin, 471 B-lactones, 471 Lamivudine, 129 LAP0389 study, 187, 190 Last qualifying chemotherapy, 162 LBH589, 245–246 Lenolidomide, 196 Lestaurtinib. See CEP-701 LET. See Radiation, linear energy transfer (LET) Leukemia ALL. See Acute lymphoblastic leukemia Bcl-2 in, 264 CD95 in, 264 cell cycle-regulatory genes and, 355–357 chromosomal translocations in, 261–262 CLL. See Chronic lymphoid leukemia (CLL) CML. See Chronic myeloid leukemia (CML) detection of MRD in, 47–49 in fusion proteins, 211 lymphoblastic. See Acute lymphoblastic leukemia (ALL) myeloid. See Acute myeloid leukemia (AML) nonlymphocytic. See Acute nonlymphocytic leukemia (ANLL)
591 [Leukemia] p53 mutations in, 263–264 preclinical modeling of, 155–156 promyelocytic. See Acute promyelocytic leukemia (APL) therapies for. See Antileukemic therapies Leukemic DC culture of, 335–336 functional properties of, 336 vaccination with, 339–340 clinical protocols, 340–341 immunomonitoring in, 341–342 LIBSVM, 6 Lintuzumab (HuM195), 103–104 Lipid rafts, 126–127 Lonafarnib (SCH66336), 495, 498 LOVO xenograft model, 473 Low molecular weight PTP (LMPTP), 453 Lymphoma anaplastic. See Anaplastic large cell lymphoma (ALCL) B-cell. See Diffuse large B-cell lymphoma (DLBCL) Burkitt’s. See Burkitt’s lymphomas cell cycle-regulatory genes and, 355–357 detection of molecular targets in, 8–9 follicular. See Follicular lymphomas (FL) MRD monitoring in, 51 clinical relevance of, 66–68 non-Hodgkin’s. See Non-Hodgkin’s lymphoma (NHL) xenograft models, 154
mAB. See Monoclonal antibodies (mAb) Maintenance therapy, with ATRA, 188–189, 198 Major histocompatibility complex (MHC), 330 Mantle cell lymphoma (MCL), 49, 355 flavopiridol in, 362 Matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF), 11 M.D. Anderson trial, 89 MD Anderson Cancer Center, 194 Medicare (United States), 36
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592 Memorial Sloan-Kettering Cancer Center, 155 Mercaptopurine (MP), 12 6-Mercaptopurine (6-MP), 187, 194, 198 Methotrexate, 187, 194, 198 Methylprednisolone, 31 Methylthiazol tetrazolium (MTT) assay, 24–26 vs. DiSC assay, 27, 30 Metotrexate (MTX), 12 MFAC (mylotarg, fludarabine, cytarabine (ara-C), and cyclosporine) regimen, 114 MGCD0103, 249 MHC. See Major histocompatibility complex (MHC) Microarray affymetrix, 3–4 data analysis, 4–5 for detection of molecular targets of c-Myc, 9 glass slide, 3 human genome U133, 4 types of, 2 MicroRNA (miRNA), 312 Midostaurin. See PKC412 MILE (microarray innovations in leukemia) study, 14–15 Minimal residual disease (MRD), monitoring, 198–199 clinical relevance of in acute lymphoblastic leukemia (ALL), 56–60 in acute myeloid leukemia (AML), 61–62 in acute promyelocytic leukemia (APL), 60–61 in chronic lymphocytic leukemia (CLL), 64–65 in chronic myeloid leukemia (CML), 63–64 in non-Hodgkin’s lymphoma (NHL), 66–70 by flow cytometric immunophenotyping in acute leukemias, 47–49 in B- and T-cell leukemias, 49–50 in the occurrence of immunophenotypic shifts, 50
Index [Minimal residual disease (MRD)] molecular markers for, 49 by PCR analysis in acute leukemias, 51 in B- and T-cell leukemias, 51–53 of chromosome aberrations, 54–56 of Ig/TCR gene rearrangements, 53–54 Mitogen-activated protein kinase (MAPK) pathway, 191 Mitotic proteins, 494–495 Mitoxantrone, 114 MLN-518, 390 in adult AML, 395 Molecular testing, 199 Monoclonal antibodies (mAb), 99–102 anti-CD20, 154 and ASCT, 139 CD33 directed in adult AML, 103–115 in pediatric AML, 115–118 CD45 directed, 118 cellular cytotoxicity of. See Antibodydependent cellular cytotoxicity (ADCC) in chronic lymphocytic leukemia, 137–138 effector mechanisms, 154–155 131 I-anti-CD45, 171 131 I-BC8, 171 131 I-30F11, 156 131 I-LL2, 157 infusion toxicity of, 129 131 I-tositumomab, 161–163 in killing lymphoma cells, 149 in lymphoma cells, 137 mechanisms of action of, 126–128 naked vs. conjugated, 101–102 in posttransplant lymphoproliferative disease, 138 principles of, 149–150 resistance mechanisms of, 118 safety and efficacy of, 129 therapeutic efficacy of, 102 for the treatment of APL. See Alemtuzumab 90 Y-ibritumomab tiuxetan, 158–161 MP. See Mercaptopurine (MP) MRD. See Minimal residual disease (MRD)
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Index MS-275, 248 mTOR inhibitors activation, 528 in acute lymphoblastic leukemia (ALL), 532 in acute myelogenous leukemia (AML), 532–533 as anticancer agents, 530–531 disease-specific activity, 531–532 non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma, 531–532 MUC1, 333 Mucosa-associated lymphoid tissue (MALT), 137 Multiple myeloma (MM), 290, 294 CEC in, 289 Murine antibodies, 101 MYC gene, 49, 55 Myelodysplastic syndrome (MDS), 113, 263. See also Acute myeloid leukemia (AML) Myeloid neoplasms, classification of, 102–103 Myelosuppression, 163–164, 170 Mylotarg1. See Gemtuzumab ozogamicin Myotubularins dephosphorylate, 452 MYST family, of HAT, 234
N-acetyl leucyl-leucyl norlucinal (ALLnL), 471 Naked antibodies, 101–102 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), 479 National Cancer Institute (NCI) lung cancer froup, 27 website, clinical data analysis of, 34–35 NB4 cells, 11, 196 NB4-R1 cells, 196 Neoplasms hematological studying clinical data of cell death assays in, 31–32 lymphatic studying clinical data of cell death assays in, 26–27 NHL. See Non-Hodgkin’s lymphoma (NHL); Non-Hodgkin’s lymphomas (NHL)
593 Nilotinib, 417–418, 421 Non-Hodgkin’s lymphoma (NHL) clinical relevance of the DiSC assay in, 27 myeloablative RIT in, 167–168 Non-Hodgkin’s lymphomas (NHL), 295 Bc16 in, 316 Nonmyeloablative conditioning regimens in acute myeloid leukemia and myelodysplastic syndrome, 551–553 clinical perpspectives, 556–557 in CML, 553–554 examples, 542–544 GVHD and GVT effects after, 547–548 with 2 Gy TBI and fludarabine, 545–546 kinetics of donor engraftment after, 546–547 in lymphoma and chronic lymphocytic leukemia, 554–555 multiple myeloma, 555 relapse and survival after, 551 tandem autologous/allogeneic HCT, 555–556 toxicities after, 548–550 North American Intergroup trials, 188, 197–198 Notch intracellular domain (NICD), 513–514, 516, 518 Notch signaling activated, in T-ALL mechanisms, 516 by translocation or mutation, 515–516 clinical perspectives, 521 implications for treatment of T-ALL in cancers, 518–520 drug combinations, 518 inhibition on delta-like ligand 4 (DLL4), 520 g-secretase inhibitors, 516–518, 520 pathway, 513–514 in thymocyte development, 514–515 Novobiocin, 433 NPM-ALK fusion gene, 50, 55 Nucleic acids chemical modification of, 314–315 delivery into living cells, 313–314 Nucleosomes, 233
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594 O’Brien–Fleming test, 93 ODN. See Oligodeoxynucleotides (ODN) Oligodeoxynucleotides (ODN), 314 in vivo treatment model for, 319 Oligonucleotides, 314 Osler, William, 191
p53, 242 mutations in leukemia, 263–264 Paclitaxel, 434 Palindromes, in mRNA, 317–318 Pan-B-cell antigens, 150 Pathway Assist, 7 PAX gene, 13 P190 Bcr-Abl fusion protein, 413 Pegylated liposomal doxorubicin (PegLD), 479 Peptide-based Inhibitors, 462 Peptide boronates, 471 Peptide boronic acids, 471–472 Peptide epoxyketones, 471 Peptide vinyl sulfones, 471 PETHEMA group, 188 P-glycoprotein, 119, 127 Ph chromosome abnormality, 413–414 Phenylbutyrate, 242–243 Philadelphia (Ph) chromosome. See Ph chromosome abnormality Phosphatase and tensin homologs (PTENs), 452, 459 Phosphatidylinositol-3 Kinase (PI3K)/Akt pathway, 494 Phosphatidylinositol- 3,4,5-trisphosphate (PIP3), 528 Phosphodiesterase inhibitors. See Theophylline PI3K, 527 PI3K/mTOR pathway clinical perspectives, 534–535 combinations, 533–534 importance, 525–526 overview of, 526 regulation of, 529–530 signaling cascade, 526–529 PKC412, 389 in adult AML, 395 PKC isozymes, inhibition by UCN-01, 364
Index Platelet-derived growth factor (PDGF), 436 Platelet transfusions, 112, 114 PML. See Promyelocytic leukemia (PML) PML-RARa, 262. See Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) Polycythemia vera (PV), 499 Polymerase chain reaction (PCR), 163 for MRD detection in acute leukemias, 51 in B- and T-cell leukemias, 51–53 Posttranslational modifications, 233–234 Posttransplant lymphoproliferative disease, 138 Prednisolone, 12 Prednisone, 479 Probability distributions, 87 Probability of early termination (PET), 95 Promyelocytic leukemia (PML) fusion with RARa, 186–187 MAPK-mediated phosphorylation of, 190 Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa), 11 in APL, 54, 60, 186–187 maturation arrest in, 185–186, 191 and HDAC, 196, 236 Proteasome inhibitors, 469–470 bortezomib, 473 clinical perspectives, 481–483 and leukemia preclinical studies, 476–477 in vitro studies, 474–475 in vivo studies, 475–479 in lymphomas Hodgkin, 480–481 non-Hodgkin, 479–480 in multiple myeloma (MM), 470 resistance mechanisms, 481 Proteasomes, 469 Protein tyrosine phosphatases (PTP) in cancer, 454–459 classification, 451–453 as drug targets, 459–462 future perspective, 462–463 in human disease, 454 regulation, 453–454 structural features and catalytic mechanism, 453
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Index PTEN, 527, 529 PTK787/ ZK222584 (Vatalanib), 439 PTP. See Protein tyrosine phosphatases (PTP) PTP1B, 458–459 Pulmonary reactions, 129 P-value (probability), 87–89 PXD101, 245 Pyroxamide, 245
Quantitative reversetranscriptase polymerase chain reaction (qPCR) technique, 413 Q-Value, 6
RA. See Retinoic acid (RA) Rab protein, 493 Radiation therapy, 92, 150 linear energy transfer (LET), 152 Radicicol, 432–433 Radioimmunoconjugate, 154 Radioimmunotherapy (RIT), 127 administration of subsequent therapy followed by, 166 antibody biodistribution in, 167 antigen targets for, 150–151 clinical trials in, 169–172 dosimetry in, 173–175 durable remissions of, 173 for follicular lymphoma, 133–135 integration with chemotherapy, 176–178 in NHL, 167–168 predosing of mAb in, 153–154 radiation dose response in, 155 radioisotopes in, 151–153. See also 188 Reis toxicity and safety of, 164–166 Radiolysis, 152 Rapamycin analogs, 532, 534 RA syndrome (RAS), development of, 188 Real-time quantitative PCR (RQ-PCR). See Polymerase chain reaction (PCR) Receptor tyrosine kinases (RTK), 449–450
595 Reduced-intensity regimens. See Nonmyeloablative conditioning regimens Reduced-intensity stem cell transplantation (RIC-allo-SCT), 117–118 188 Reis, 172 Relapsed disease, 198 Retinoblastoma (Rb)-dependent G1 arrest, 433 Retinoic acid (RA), 186 in myeloid differentiation, 186 syndrome, 188 Retinoic acid receptor (RARa) fusion with PML, 186–187 myeloid differentiation induced by, 186 Reverse transcriptase polymerase chain reaction (RT-PCR), 11, 199 for fusion transcript in APL, 103 RHEB protein, 493 Rheb protein, 528 Rhenium-186 (186Re). See B-emitters Ribonucleic acid polymerase II (RNAPII), 355 Ricin, 128 RIT. See Radioimmunotherapy (RIT) Rituximab, 36, 49, 101, 125–126, 479 in combination with chemotherapy, 131–133 in DBCL, 135–137 depletion of antigen-positive B-lymphocytes, 129 as a drug, 130 in follicular lymphoma, 130–131 and high dose ASCT, 139 prolonged administration of, 130 resistance mechanisms of, 128 synergism with chemotherapeutic agents in B-cell killing, 126–127 RNA-induced silencing complex (RISC), 312 RNA (ribonucleic acid) species, for microarray, 3 Rohitukine, 357 Romidepsin, 247–248 R-roscovitine. See CYC202 Rubinstein-Taybi syndrome, 234
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596 S-adenosyl-methionine, 208 SAHA. See Suberoylanilide hydroxamic acid (SAHA) Salinosporamide A (NPI-0052), 471 Salmonella typhimurium, 454 SAM, software for gene expression profiling analysis, 6 Second mitochondria-derived activator of caspase (Smac) binding to XIAP, 266 ectopic expression of, 266 Self-quenching reporter molecules (SQRM), 317 in hybridization, 318 Serum-free culture, of AML-DC, 335–336 SH-2 homology containing PTPs (SHPs), 456 SHP-1 protein, 456–457 SHP-2 proteins, 457–458 Single nucleotide polymorphisms (SNP), 7 Sinusoidal obstruction syndrome (SOS), 112. See also Veno-occlusive disease (VOD) SKI-606, 412, 422 Small interfering RNA (siRNA), 312–313 SNP. See Single nucleotide polymorphisms (SNP) Sodium phenylbutyrate, 196 Software packages and tools for indentification of gene expression profiles, 6 for pathways analysis, 7 Sorafenib, 390, 439 Southwest Oncology Group (SWOG), 134 Src-homology 2 (SH2), 528 Src kinases, 440 Src-related Lyn kinase, 414 ‘‘Start C’’ trial, 418, 421–422 Stem cell transplantation (SCT), 423–425. See also Autologous stem cell transplantation (ASCT); Hematopoietic stem cell transplantation (HSCT) Streptomyces hygroscopicus, 530 SU5416, 298, 389, 439 in adult AML, 393–394
Index SU11248. See Sunitinib Suberoylanilide hydroxamic acid (SAHA), 217, 243–245, 368, 435 Sumoylation, 191 Sunitinib, 389–390, 439 in adult AML, 394 Survivin, 264–266
TA. See Troxacitabine þ ara-C (TA) T-ALL. See T-cell acute lymphoblastic leukemia (T-ALL) Tamibarotene (AM80), 200 TAN1, 513 Tandutinib. See MLN518 TBI. See Total body irradiation (TBI) T-cell acute lymphoblastic leukemia (T-ALL), 262, 513 T-cell leukemias, chronic detection of MRD in, 49–50 T-cell lymphomas, 355 antibodies in, 138 T-cell receptor (TCR) genes, 49–50 detection of junctional regions of, 50–51 T cells activation of, 330 and APC, 332–334 Temsirolimus, 530 Thalidomide, 196 Theophylline, 196 6-Thioguanine, 103 Thyroid stimulating hormone (TSH), 165 TI. See Troxacitabine þ idarubicin (TI) T315I mutant Abl kinase, 412 Tipifarnib (R115777, Zarnestra), 495, 497–499 Tissue-specific genes, and DNA methylation, 208 Tiuxetan, chelator molecule, 158 TMPyP4, 518 TNF-related apoptosis-inducing ligand (TRAIL) receptors, 269–272 Topoisomerase II inhibitors, 433 TORC1-mediated phosphorylation, 527 TORC1/TORC2 inhibitors, 534
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Index Tositumomab, 161. See also Anti-B1 Total body irradiation (TBI), 156, 171 TRAIL receptors. See TNF-related apoptosis-inducing ligand (TRAIL) receptors Treatment failure, pharmacological and physiological causes drug sanctuary sites (CNS and testis), 564 inadequate drug dose and suboptimal schedule of drug administration, 563–564 poor diffusion into tissues, 564 Trichostatin A (TSA), 237 Troxacitabine þ ara-C (TA), 89–90 Troxacitabine þ idarubicin (TI), 89–90 Trypan blue test, 27 TSA. See Trichostatin A (TSA) Tuberous sclerosis complex 2 (TSC2), 528 Tumor cells, DNA methylation in, 208–211 Tumor immunology, 329–330 Tumor necrosis factor (TNF)-a, 436 Tumor response to antineoplastic compounds (TRAC) assay, 24, 34 Tyrosine kinase enzymes clinical perspectives, 441 heat shock proteins (Hsp) Hsp90, 431–432 Hsp90 inhibitors, 432–435 other, 435–436 inhibitor therapy, 411–412 Src kinases, 440 VEGF and, 436–437 in acute leukemia, 437–438 novel therapy, 438–439
Ub-conjugating enzymes, 471 Ubiquitinactivating enzyme E1-like (UBE1) gene, 10–11 Ubiquitin-proteasome pathway, 470–471 UCN-01 (7-hydroxystaurosporine) Akt kinase PDK1 inhibition by, 365 apoptosis by, 365 and CDK, 364 and Chk inhibition, 364–365
597 [UCN-01 (7-hydroxystaurosporine)] combination strategy for, 369–370 overview of, 363–364 PKC isozymes’ inhibition, 364 U133 gene chip, design and modelling of. See Affymetrix microarrays UniGene database, 4
Valproate, 242–243 Vascular endothelial growth factor (VEGF), 196, 283, 436–437, 494 in acute leukemia, 437–438 in ALL, 297 in AML, 295–297 in CLL, 298 in CML, 297–298 in Hodgkin’s lymphoma, 294–295 key players in, 287 in multiple myeloma (MM), 290, 294 in NHL, 295 novel therapy, 438–439 VEGF. See Vascular endothelial growth factor (VEGF) VEGFA, 287. See also Vascular endothelial growth factor (VEGF) bone marrow-derived EPC and, 290 Veno-occlusive disease (VOD), 112, 167 Vincristine, 12, 127 Vitamin A deficiency, and hematopoiesis, 186 Vorinostat, 243–245 and flavopiridol, 368
Waldenstrom macroglobulinemia, 138 WHO, 102 WT1 gene, 211
X-chromosome-linked inhibitor of apoptosis proteins (XIAP), 265 antisense oligonucleotides and, 267 BIR3 domain of, 266–267 Smac binding to, 266 XIAP. See X-chromosome-linked inhibitor of apoptosis proteins (XIAP)
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598 Yersinia Pestis, 454 90 Y-ibritumomab, 127 in follicular lymphoma, 133–134 tiuxetan toxic effects of, 164–165 treatment regimen for, 158–161 vs. 131I-tositumomab, 173
Index Yin Yang1 (YY1), 127
ZAP70 gene, antigen expression of, 14, 16, 64 ZD6474 (Zactima), 439 Zevalin1. See 90Y-ibritumomab tiuxetan
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Hematology and Oncology
Innovative Leukemia and Lymphoma Therapy gives a complete and up-to-date overview of the exciting new treatment modalities in leukemia and lymphoma that are being introduced in the clinic today. Written by international experts in the field, this volume examines clinical studies on topics such as: • tyrosine kinase inhibitors, histon deacetylase inhibitors, and farnesyl transferase inhibitors • radioimmunotherapy, gene-directed therapy, and immunotherapy by vaccination • monoclonal antibodies • anti-angiogenesis approaches Within each chapter, this well-illustrated, comprehensive source provides a summary of the rationale for treatment, the pathways that are involved, and the translational research to students, scientists, and clinicians. In addition, this book focuses on the concerns of conventional but novel anticancer agents, modulation of classical multidrug resistance, and modulators of single agent drug resistance. about the editors... G. J. L. KASPERS is Professor and Head of the Division of Oncology/Hematology, VU University Medical Center, Amsterdam, Netherlands. Dr. Kaspers received his M.D. from the Vrije Universiteit, Amsterdam, Netherlands, and his Ph.D. from the VU University Medical Center, Amsterdam, Netherlands. His current research covers a randomized phase III study on the treatment of children and adolescents with refractory or relapsed acute myeloid leukemia, a phase II study on Mylotarg in childhood relapsed and refractory AML, an international study on the treatment of Pediatric Acute Promyelocytic Leukemia, clinical phase II studies with clofarabine-containing regimens in pediatric acute myeloid leukemia, and a clinical phase II study with bortezomib in pediatric acute lymphoblastic leukemia in preparation. BERTRAND COIFFIER is Professor and Chief of Hematology, Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon and Claude-Bernard University, Pierre-Benite, France. Dr. Coiffier has written more than 300 publications in internationally recognized, leading journals in the field of hematology on lymphomas, lymphoproliferative diseases, chemotherapy, high-dose therapy, and autotransplant, biological response modifiers. MICHAEL C. HEINRICH is Professor of Medicine, Oregon Health & Science University, Division of Hematology & Medical Oncology, Portland, Oregon, USA; Chairman, Portland VA Medical Center Tumor Board, Section Chief, Hematology Oncology, Portland VA Hematology/Medical Oncology Service, Portland, Oregon, USA; and Interim Head, Hematology & Medical Oncology, Oregon Health & Science University, Portland, Oregon, USA. Dr. Heinrich received his M.D. from the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. ELIHU ESTEY is Professor of Hematology in the Division of Medicine at the University of Washington Medical Center and a Member at the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. Dr. Estey received her A.B. from Yale University, New Haven, Connecticut, USA, and her M.D. From Johns Hopkins University, Baltimore, Maryland, USA.
Printed in the United States of America
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Innovative Leukemia and Lymphoma Therapy
about the book…
Innovative Leukemia and Lymphoma Therapy
Kaspers •
Coiffier •
Heinrich •
Estey
Edited by
G. J. L. Kaspers Bertrand Coiffier Michael C. Heinrich Elihu Estey
nC nM nY nK Kaspers_978-0849350832.indd 1
4/21/08 12:43:26 PM
