Childhood Leukemias

Childhood Leukemias

Childhood Leukemias Second edition

Edited by

Ching-Hon Pui St. Jude Children’s Research Hospital, Memphis, Tennessee, USA

cambridge university press Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, S˜ao Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 2RU, UK Published in the United States of America by Cambridge University Press, New York www.cambridge.org Information on this title: www.cambridge.org/9780521825191
C Cambridge University Press 2006

This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 2006 Printed in the United Kingdom at the University Press, Cambridge A catalog record for this publication is available from the British Library ISBN-13 978-0-521-82519-1 hardback ISBN-10 0-521-82519-9 hardback

Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Every effort has been made in preparing this publication to provide accurate and up-to-date information which is in accord with accepted standards and practice at the time of publication. Although case histories are drawn from actual cases, every effort has been made to disguise the identities of the individuals involved. Nevertheless, the authors, editors and publishers can make no warranties that the information contained herein is totally free from error, not least because clinical standards are constantly changing through research and regulation. The authors, editors and publishers therefore disclaim all liability for direct or consequential damages resulting from the use of material contained in this publication. Readers are strongly advised to pay careful attention to information provided by the manufacturer of any drugs or equipment that they plan to use.

For my parents and all the patients I have been privileged to care for.

Contents

List of contributors Preface

page ix xv

Part I History and general issues 1 Historical perspective Donald Pinkel

3

2 Diagnosis and classification Mihaela Onciu and Ching-Hon Pui

21

3 Epidemiology and etiology Logan G. Spector, Julie A. Ross, Leslie L. Robison, and Smita Bhatia

48

Part II Cell biology and pathobiology 4 Anatomy and physiology of hematopoiesis Connie J. Eaves and Allen C. Eaves 5 Hematopoietic growth factors James N. Ihle 6 Signal transduction in the regulation of hematopoiesis James N. Ihle 7 Immunophenotyping Fred G. Behm 8 Immunoglobulin and T-cell receptor gene rearrangements Jacques J. M. van Dongen and Anton W. Langerak 9 Cytogenetics of acute leukemias Susana C. Raimondi

69

106

125

150

210

235

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Contents

10 Molecular genetics of acute lymphoblastic leukemia Adolfo A. Ferrando, Jeffrey E. Rubnitz, and A. Thomas Look

272

24 Acute leukemia in countries with limited resources Raul C. Ribeiro, Scott C. Howard, and Ching-Hon Pui

625

298

25 Antibody-targeted therapy Eric L. Sievers and Irwin D. Bernstein

639

11 Molecular genetics of acute myeloid leukemia Robert B. Lorsbach and James R. Downing

339

26 Adoptive cellular immunotherapy Helen E. Heslop and Cliona M. Rooney

648

12 Apoptosis and chemoresistance Kirsteen H. Maclean and John L. Cleveland

27 Gene transfer: methods and applications Martin Pule´ and Malcolm K. Brenner

661

28 Minimal residual disease Dario Campana, Andrea Biondi, and Jacques J. M. van Dongen

679

13 Heritable predispositions to childhood hematologic malignancies Doan Le, Kevin Shannon, and Beverly J. Lange

362

Part III Evaluation and treatment Part IV Complications and supportive care

14 Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations Shinji Kishi, William E. Evans, and Mary V. Relling

391

15 Assays and molecular determinants of cellular drug resistance Monique L. den Boer and Rob Pieters

414

29 Acute complications Scott C. Howard, Raul C. Ribeiro, and Ching-Hon Pui

709

30 Late complications after leukemia therapy Melissa M. Hudson

750

31 Therapy-related leukemias Carolyn A. Felix

774

32 Infectious disease complications in leukemia Jeremy A. Franklin and Patricia M. Flynn

805

487

33 Hematologic supportive care Fariba Navid and Victor M. Santana

829

19 Acute myeloid leukemia Jeffrey E. Rubnitz, Bassem I. Razzouk, and Raul C. Ribeiro

499

34 Pain management Alberto J. de Armendi and Doralina L. Anghelescu

850

540

35 Psychosocial issues Raymond K. Mulhern, Sean Phipps, and Vida L. Tyc

858

20 Relapsed acute myeloid leukemia Ursula Creutzig

548

36 Nursing care Pamela S. Hinds, Jami S. Gattuso, and Belinda N. Mandrell

882

21 Myelodysplastic syndrome Henrik Hasle 22 Chronic myeloproliferative disorders Charlotte M. Niemeyer and Franco Locatelli

571

Index

894

23 Hematopoietic stem cell transplantation Rupert Handgretinger, Victoria Turner, and Raymond Barfield

599

16 Acute lymphoblastic leukemia Ching-Hon Pui

439

17 Relapsed acute lymphoblastic leukemia ¨ Gunter Henze and Arend von Stackelberg

473

18 B-cell acute lymphoblastic leukemia and Burkitt lymphoma John T. Sandlund and Ian T. Magrath

The plates are situated between pages 400 and 401.

Contributors

Anghelescu, Doralina L., MD Associate Member Division of Anesthesiology St. Jude Children’s Research Hospital Memphis, TN, USA Barfield, Raymond, MD Assistant Member Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Behm, Fred G., MD Associate Member and Vice Chair Department of Pathology St. Jude Children’s Research Hospital Memphis, TN, USA Bernstein, Irwin D., MD Head, Pediatric Oncology Program Fred Hutchinson Cancer Research Center Professor and Head Pediatric Hematology/Oncology Department of Pediatrics University of Washington School of Medicine Seattle, WA, USA Bhatia, Smita, MD, MPH Director, Epidemiology and Outcomes Research Division of Pediatrics City of Hope National Medical Center Duarte, CA, USA

ix

x

List of contributors

Biondi, Andrea, MD Director, M. Tettamanti Research Center Associate Professor Department of Pediatrics University of Milano-Bicocca Monza, Italy Brenner, Malcolm K., MB, BChir, Ph.D., FRCP, FRCPath Director, Center for Cell and Gene Therapy Professor, Departments of Medicine and Pediatrics Baylor College of Medicine Houston, TX, USA Campana, Dario, MD, Ph.D. Member Departments of Hematology/Oncology and Pathology St. Jude Children’s Research Hospital Professor, Department of Pediatrics College of Medicine University of Tennessee Health Science Center Memphis, TN, USA Cleveland, John L., Ph.D. Member, Department of Biochemistry St. Jude Children’s Research Hospital Memphis, TN, USA Creutzig, Ursula, MD Professor, Pediatric Hematology/Oncology ¨ University Children’s Hospital Munster ¨ Munster, Germany De Armendi, Alberto J., MD Chief and Member, Division of Anesthesiology St. Jude Children’s Research Hospital Memphis, TN, USA den Boer, Monique L., Ph.D. Associate Professor, Molecular Pediatric Oncology Head, Research Laboratory Pediatric Oncology Erasmus MC–Sophia Children’s Hospital University Medical Center Rotterdam Department of Pediatric Oncology and Hematology Rotterdam, the Netherlands Downing, James R., MD Member and Chair, Department of Pathology Scientific Director St. Jude Children’s Research Hospital Memphis, TN, USA

Eaves, Allen C., MD, Ph.D. Director Terry Fox Laboratory Vancouver, British Columbia, Canada

Eaves, Connie J., Ph.D. Deputy Director Terry Fox Laboratory Vancouver, British Columbia, Canada

Evans, William E., PharmD Director, St. Jude Children’s Research Hospital Professor, Department of Clinical Pharmacy College of Pharmacy University of Tennessee Health Science Center Memphis, TN, USA

Felix, Carolyn A., MD Associate Professor of Pediatrics University of Pennsylvania School of Medicine Attending Physician The Children’s Hospital of Philadelphia Abramson Research Center Philadelphia, PA, USA

Ferrando, Adolfo, MD, Ph.D. Assistant Professor of Pathology and Pediatrics Institute for Cancer Genetics Columbia University Irving Cancer Research Center New York, NY, USA

Flynn, Patricia M., MD Member, Department of Infectious Diseases Arthur Ashe Chair in Pediatric AIDS Research St. Jude Children’s Research Hospital Professor, Department of Pediatrics and Preventive Medicine University of Tennessee Health Science Center Memphis, TN, USA

Franklin, Jeremy A., MD Assistant Professor, Department of Pediatrics Division of Infectious Diseases Assistant Professor, Department of Pharmacy Practice Texas Tech University Health Sciences Center Amarillo, TX, USA

List of contributors

Gattuso, Jami S., RN, MSN, CPON Nursing Research Specialist Division of Nursing Research St. Jude Children’s Research Hospital Memphis, TN, USA Handgretinger, Rupert, MD, Ph.D. Director, Bone Marrow Transplantation Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Hasle, Henrik, MD Associate Professor Department of Pediatrics Skejby Hospital, Aarhus University Aarhus, Denmark ¨ nter, MD Henze, Gu Professor and Director Pediatric Oncology/Hematology Charit´e-Campus Virchow Klinikum Augustenburger Platz Berlin, Germany Heslop, Helen E., MD Director, Adult Stem Cell Transplant Program The Methodist Hospital Professor, Center for Cell and Gene Therapy Baylor College of Medicine Houston, TX, USA Hinds, Pamela S., Ph.D., RN, CS Member and Director Division of Nursing Research St. Jude Children’s Research Hospital Memphis, TN, USA Howard, Scott C., MD Assistant Member Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Hudson, Melissa M., MD Member, Department of Hematology/Oncology Director, After Completion of Therapy Clinic St. Jude Children’s Research Hospital Memphis, TN, USA

Ihle, James N., Ph.D. Investigator Howard Hughes Medical Institute Member and Chair Department of Biochemistry St. Jude Children’s Research Hospital Memphis, TN, USA Kishi, Shinji, MD, Ph.D. First Department of Internal Medicine Faculty of Medical Sciences University of Fukui Fukui, Japan Lange, Beverly, J., MD Yetta Dietch Novotny Professor in Clinical Oncology Medical Director of Pediatric Oncology Children’s Hospital of Philadelphia Philadelphia, PA, USA Langerak, Anton W., MD Department of Immunology Erasmus University Rotterdam Rotterdam, the Netherlands Le, Doan, MD, FRCPC Clinical Assistant Professor Alberta Children’s Hospital Calgary, Alberta, Canada Locatelli, Franco, MD Professor of Pediatrics Pediatric Hematology and Oncology IRCCS Policlinico San Matteo Pavia, Italy Look, A. Thomas, MD Professor of Pediatrics Harvard Medical School Vice Chair for Research Department of Pediatric Oncology Dana-Farber Cancer Institute Boston, MA, USA Lorsbach, Robert B., MD, Ph.D. Assistant Member, Department of Pathology St. Jude Children’s Research Hospital Memphis, TN, USA

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List of contributors

Maclean, Kirsteen H., Ph.D. Research Fellow Department of Biochemistry St. Jude Children’s Research Hospital Memphis, TN, USA Magrath, Ian T., MB, FRCP, FRCPath Professor of Pediatrics Uniformed Services University of the Health Sciences Bethesda, MD Director International Network for Cancer Treatment and Research at Institut Pasteur Brussels, Belgium Mandrell, Belinda N., RN, MSN Pediatric Nurse Practitioner Patient Care Services St. Jude Children’s Research Hospital Memphis, TN, USA Mulhern, Raymond K., MD∗ Chief, Division of Behavioral Medicine St. Jude Children’s Research Hospital Memphis, TN, USA Navid, Fariba, MD Assistant Member Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Niemeyer, Charlotte M., MD Professor of Pediatrics Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Freiburg, Germany Onciu, Mihaela, MD Assistant Member, Department of Pathology Director, Hematology and Special Hematology Laboratories St. Jude Children’s Research Hospital Memphis, TN, USA Phipps, Sean, Ph.D. Associate Member Division of Behavioral Medicine St. Jude Children’s Research Hospital Memphis, TN, USA

∗

Deceased

Pieters, Rob, MD, MS, Ph.D. Professor and Head Department of Pediatric Oncology and Hematology Erasmus MC–Sophia Children’s Hospital University Medical Center Rotterdam Rotterdam, the Netherlands Pinkel, Donald, MD Adjunct Professor Biological Sciences Department California Polytechnic State University San Luis Obispo, CA, USA Pui, Ching-Hon, MD Member and Director Leukemia/Lymphoma Division St. Jude Children’s Research Hospital American Cancer Society–F.M. Kirby Clinical Research Professor Professor, Department of Pediatrics College of Medicine University of Tennessee Health Science Center Memphis, TN, USA Pul´e, Martin, MB, MRCP Postdoctoral Research Fellow Center for Cell and Gene Therapy Baylor College of Medicine Houston, TX, USA Raimondi, Susana C., MD Member and Director of Cytogenetics Department of Pathology St. Jude Children’s Research Hospital Memphis, TN, USA Razzouk, Bassem I., MD Associate Member Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Relling, Mary V., PharmD Member and Chair Department of Pharmaceutical Sciences St. Jude Children’s Research Hospital Professor, Department of Clinical Pharmacy College of Pharmacy University of Tennessee Health Science Center Memphis, TN, USA

List of contributors

Ribeiro, Raul C., MD Member Department of Hematology/Oncology Director, International Outreach Program St. Jude Children’s Research Hospital Memphis, TN, USA Robison, Leslie L., MPH, Ph.D. Professor Division of Pediatric Epidemiology & Clinical Research Department of Pediatrics University of Minnesota Medical School Minneapolis, MN, USA Rooney, Cliona M., Ph.D. Professor Departments of Pediatrics and Molecular Virology and Microbiology Center for Cell and Gene Therapy Baylor College of Medicine Houston, TX, USA Ross, Julie A., MPH, Ph.D. Professor Division of Pediatric Epidemiology & Clinical Research Department of Pediatrics University of Minnesota Medical School Minneapolis, MN, USA Rubnitz, Jeffrey E., MD Associate Member Department of Hematology/Oncology Director of Fellowship Program St. Jude Children’s Research Hospital Memphis, TN, USA Sandlund, John T., MD Member Department of Hematology/Oncology St. Jude Children’s Research Hospital Memphis, TN, USA Santana, Victor M., MD Member and Director, Solid Tumor Division Department of Hematology/Oncology

St. Jude Children’s Research Hospital Memphis, TN, USA Shannon, Kevin, MD Roma and Marvin Auerback Distinguished Professor of Molecular Oncology Department of Pediatrics Program Leader, Hematopoietic Malignancies UCSF Comprehensive Cancer Center University of California San Francisco San Francisco, CA, USA Sievers, Eric L., MD Medical Director ZymoGenetics, Inc. Seattle, WA, USA Spector, Logan G., Ph.D. Assistant Professor Division of Pediatric Epidemiology & Clinical Research Department of Pediatrics University of Minnesota Medical School Minneapolis, MN, USA Turner, Victoria E., Ph.D., D (ABHI) Director, HLA Laboratory Department of Pathology St. Jude Children’s Research Hospital Memphis, TN, USA Tyc, Vida L., Ph.D. Associate Member Division of Behavioral Medicine St. Jude Children’s Research Hospital Memphis, TN, USA van Dongen, Jacques J. M., MD, Ph.D. Professor Department of Immunology Erasmus University Rotterdam Rotterdam, the Netherlands von Stackelberg, Arend, MD Pediatric Oncology/Hematology Charit´e-Campus Virchow Klinikum Berlin, Germany

xiii

Preface

The first edition of Childhood Leukemias was produced to meet a growing need for a comprehensive reference that would probe more deeply into important topics only touched upon by general or pediatric oncology texts. In the six years since its publication, I have been gratified by the positive responses of readers from around the globe, who have enthusiastically endorsed the book and have inquired about plans for a second edition. I was therefore pleased when Cambridge University Press asked me to undertake that task. In keeping with the basic editorial premise of the first edition – that readers will benefit most from a judicious blend of clinical and biological topics – this revised text includes eight new chapters. Three deal with the origins and challenges of chemoresistant leukemia, topics that are rapidly moving to the forefront of leukemia research. Two other chapters cover recent advances in therapy-related leukemias and in pain management, while the remaining three add much-needed discussions of the genetic syndromes predisposing to leukemia, treatment in countries with limited resources, and antibody-targeted therapy. The myelodysplastic syndromes, which represent a large fraction of the preleukemic diseases that continue to resist treatment, now occupy a single chapter, as do the chronic myeloproliferative disorders. As you will notice, the emphasis on molecular mechanisms that characterized the first edition has been retained, reflecting my conviction that the path to better patient management lies in serious inquiry into the pathobiology of specific diseases. Nowhere is this more evident than in the acute leukemias of childhood. As skillfully discussed in the chapters on the molecular genetics of acute lymphoblastic leukemia and acute myeloid leukemia, as well as host pharmacogenetics, improved methods of analysis have revealed previously unrecognized genotypes that could be used to increase the power and precision of clinical trials, and eventually to target therapy to tyrosine kinases

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Preface

Donald Pinkel, MD, first director of St. Jude Children’s Research Hospital.

and other key signaling molecules that lie upstream of pathological transcription programs. The development of imatinib for leukemias expressing the BCR-ABL chimeric protein provides a prototype for this direction in treatment innovation. One of the most difficult challenges was to present topics in a way that would appeal to students and trainees as well as experienced oncologists. Thus, all contributors were urged to strike a balance between a scholarly and a practical approach to their topics, and to take particular care in selecting illustrations that would adroitly highlight their major points of emphasis. They were also asked to avoid

technical jargon whenever possible, in favor of terms that would be meaningful to a general biomedical audience. I hope you will agree with me that this challenge has been successfully met, and that Childhood Leukemias is indeed accessible to a diverse readership. During the editing of the chapters, a concerted effort was also made to lessen inconsistencies in coverage. However, as with most books of this type, the final product reflects the unique perspectives of authors working in rapidly evolving fields, so that differences in opinion and interpretation are both frequent and legitimate. Publication of the second edition of Childhood Leukemias would not have been possible without the generous support of St. Jude Children’s Research Hospital, the American Lebanese Syrian Associated Charities (ALSAC), the American Cancer Society (FM Kirby Clinical Research Professorship), the Angel Grant Award from the National Cancer Coalition, and the National Cancer Institute (Cancer Center Core Grant CA-21765). I owe a debt of gratitude to John Gilbert for expert editorial consultation thoughout the preparation of this volume, to Julie Groff for superb scientific drawings, and to Kimberly Meshun Gayden for her excellent word-processing skills. Finally, my heartfelt thanks go to all the contributors – leaders in their respective fields who graciously donated their time so that readers could benefit from their vast knowledge and experience. The pediatric oncology community suffered a tragic loss with the recent untimely death of Dr. Ray Mulhern, whose groundbreaking contributions to the neuropsychology of children with cancer helped to propel this field into the modern era of clinical investigation. Ray was a brilliant author and a valued colleague and friend; I will miss him very much. One of the authors, Dr. Donald Pinkel, deserves special recognition for his pioneering contributions to the treatment of childhood leukemia, which taught us that acute lymphoblastic leukemia is not a death sentence but an imminently curable disease. His inspirational Total Therapy program at St. Jude greatly influenced my decision to pursue a career in leukemia research and ultimately to accept the challenges posed by the editorship of Childhood Leukemias.

Plate 2.2 ALL, L1 (FAB). Small blasts with indistinct nucleoli, with an admixture of some larger blasts. This spectrum of small and larger blasts is common in ALL. (Wright-Giemsa, ×1000.)

Plate 2.4 B-ALL (FAB ALL, L3) with the t(8;14). Blasts are characterized by intensely basophilic cytoplasm, regular nuclear features, prominent nucleoli, and cytoplasmic vacuolization. (Wright-Giemsa, ×1000.)

Plate 2.6 ALL with cytoplasmic granules. Fuchsia-colored granules are present in the cytoplasm of numerous blasts. Such granules may lead to a mistaken diagnosis of AML, but the granules are negative for MPO and myeloid-pattern SBB. Immunophenotyping will confirm a diagnosis of ALL, usually of precursor B-cell lineage. Granular ALL may display granular positivity for esterase stains. (Wright-Giemsa, ×1000.)

Plate 2.3 ALL, L2 (FAB). Blasts with prominent nucleoli and moderate amounts of cytoplasm, with an admixture of smaller blasts. Such cases overlap morphologically with AML and emphasize the importance of ancillary studies to assign the correct lineage in acute leukemia. (Wright-Giemsa, ×1000.)

Plate 2.5 ALL, L1 with prominent cytoplasmic vacuoles. Note the scant, lightly basophilic cytoplasm, and inconspicuous nucleoli (by comparison with Fig. 2.4). Such cases may be mistaken for B-ALL. Vacuolation is not unique to ALL, L3 (Burkitt) leukemia and other cytologic features have to be considered when making this diagnosis. (Wright-Giemsa, ×600.)

Plate 2.7 AML with minimal granulocytic differentiation (FAB M1). Blasts are large and somewhat irregular, with moderate amounts of cytoplasm but little cytoplasmic differentiation. (Wright- Giemsa, ×1000.)

Plate 2.8 Myeloperoxidase positivity in AML, demonstrated by yellow staining against a Romanowsky-stained background. (o-tolidine stain with dilute Giemsa counterstain, ×1000.)

Plate 2.10 Hypergranular acute promyelocytic leukemia (FAB M3, sometimes designated M3h). The neoplastic cells are abnormal hypergranular promyelocytes with reddish granules and occasional clefted nuclei. Several promyelocytes contain multiple Auer rods (so-called “faggot cells”). (Wright-Giemsa, ×1000.)

Plate 2.12 Acute myelomonocytic leukemia (FAB M4). The leukemic cell population includes large blasts, with irregular and reniform nuclei, promonocytes and monocytes. Esterase staining is often positive in such cases. (Wright-Giemsa, ×1000.)

Plate 2.9 AML with granulocytic differentiation (FAB M2). Differentiating granulocyte precursors are admixed with myeloblasts. Several blasts contain Auer rods (arrows). (Wright-Giemsa, ×1000.)

Plate 2.11 Microgranular acute promyelocytic leukemia (FAB M3v). The leukemic process is characterized by cells with bilobed and grooved nuclei, and sparse cytoplasmic granulation. (Wright-Giemsa, ×1000.)

Plate 2.13 Acute monoblastic leukemia (FAB M5). Blasts are large and uniform, with abundant blue-gray cytoplasm, and may have cytoplasmic vacuolation and amphophilic granules. (Wright-Giemsa, ×1000.)

Plate 2.14 Alpha naphthyl butyrate esterase reactivity in acute monoblastic leukemia. ANB positivity is characterized by intense, diffuse reddish-brown cytoplasmic staining, typical of monoblastic leukemia. (ANB stain with hematoxylin counterstain, ×1000.)

Plate 2.15 AML with predominant erythroid differentiation (FAB M6, also termed M6a). An infiltrate of myeloblasts is present admixed with dysplastic erythroid precursors. (Wright-Giemsa, ×1000.)

Plate 2.16 Acute erythroblastic leukemia (also termed FAB M6b). The blasts are large with basophilic cytoplasm, resembling normal erythroblasts, and may show vacuolization. Immunophenotypic analysis confirms erythroid differentiation of the blasts. (Wright-Giemsa, ×1000.)

Plate 2.17 Acute megakaryoblastic leukemia (FAB M7). The blasts have prominent surface blebs, bi- or multinucleation, and may occasionally form cohesive clusters, mimicking metastatic tumor. (Wright-Giemsa, ×1000.)

Plate 2.18 AML with t(8;21). Blasts are large, with basophilic cytoplasm and single needle-shaped Auer rods. Many dysplastic granulocyte precursors are present, some showing diffuse salmon-pink cytoplasmic staining. (Wright-Giemsa, ×600.)

Plate 2.19 AML with inv(16) (FAB AML M4Eo). Acute myelomonocytic leukemia with numerous dysplastic eosinophils that contain coarse basophilic cytoplasmic granules. (WrightGiemsa, ×600.)

Plate 2.20 Dimorphic T/myeloid acute biphenotypic leukemia. A dual leukemic cell population is present, including small blasts with scant cytoplasm, indistinct nucleoli, ‘hand-mirror’ shape, and a small number of larger blasts with apparent myeloid differentiation. Immunophenotypic analysis shows that the blasts uniformly express T-cell-associated (CD2, CD3, CD7) and myeloid-associated (CD13, CD33, MPO) antigens. (Wright- Giemsa, ×1000.)

Plate 2.21 Bilineal acute leukemia. This leukemic process consists of two morphologically and immunophenotypically distinct blast populations: a lymphoid population resembling ALL, L1 and a monoblastic population resembling AML, M4/M5. (Wright-Giemsa, ×600.)

Genes for class distinction (n=588)

Diagnostic BM samples (n=132)

E2APBX1 −3SD

MLL

T-ALL

HD>50

BCRABL

TEL-AML1

+3SD

Plate 2.22 Expression profile of pediatric ALL diagnostic bone marrow blasts. Two-dimensional hierarchical cluster of 132 pediatric ALL diagnostic bone marrow samples; the normalized expression value of each gene is indicated by a color (red, expression above the mean; green, expression below the mean). (From Ross et al.221 Copyright American Society of Hematology, used with permission.)

Plate 9.1 Spectral karyotyping (SKY) analysis of leukemic cells from a patient with AML-M1. G-banded karyotype of bone marrow showed the following abnormalities: 47,XX,add(5)(q33), add(7)(p22), +20[18]/46,XX[2]. SKY established two unbalanced translocations: 47,XX,der(5)t(5;6)(q33;q21), der(7)t(7;13)(p22;q14),+20. Top panel, spectral image; bottom panel, classified image.

Plate 9.2 FISH detection of an MLL rearrangement in a pediatric patient with AML and a t(9;11)(p22;q23). Chromosomes were hybridized with Spectrum Orange- and Spectrum Green-labeled DNA probes homologous to sequences lying telomeric and centromeric to the MLL gene. In the nuclei of normal cells (not shown), hybridization of these two probes produces signals that either overlap (yellow) or are close to one another. In metaphase chromosome spread (A) and interphase nuclei (B) in which the MLL rearrangement is present, the target sequences are “split” (i.e. distant) because of their translocation to different chromosomal positions (C). (D) Partial karyotype. This translocation is predominantly seen in primary and therapy-related AML M5; it is rarely seen in ALL.

Plate 9.3 Partial karyotype of B-lineage ALL blast cells in which the t(12;21)(p13.3;q22) is present. The metaphase chromosomes were stained with 4 , 6-diamidino-2-phenylindole (DAPI). This chromosomal abnormality is the most common recurrent translocation in patients with ALL and is readily detected by FISH (A) and RT-PCR but not by conventional cytogenetic methods. The ETV6 gene on chromosome 12 (green) and the CBFA2 gene on chromosome 21 (red) probes used in FISH detected ETV6-CBFA2 on the der(21)t(12;21) (B, right) and residual of CBFA2 on the der(12)t(12;21) (B, left).

Plate 16.2 Leukemic involvement of the anterior segment with hypopyon in a 9-year-old boy with relapsed ALL.

Plate 16.1 Leukemic retinopathy manifested by disc edema and white-center hemorrhage in an 11-year-old girl with t(4;11)positive, early pre-B ALL and a presenting leukocyte count of 1512 × 109 /L.

Plate 16.4 Enlargement of right parotid gland due to infiltration of leukemic cells at diagnosis in a 5-year-old boy with near-haploid early pre-B ALL.

Plate 16.3 Left testicular relapse in a 12-year-old boy with T-cell ALL.

Plate 16.8 Results of leukapheresis in a 16-year-old boy with T-cell ALL and a presenting leukocyte count of 350 × 109 /L. The cylinders shown contain 2.8 × 1012 leukemia cells, illustrating well the definition of the term leukemia (white blood); the pink tint represents red cells that have not settled out.

Plate 16.6 Telangiectasis on bullar conjunctiva in a 12-year-old girl with ataxia telangiectasia and B-cell ALL.

Plate 16.10 Giant pronormoblast (50 m in diameter) with deeply basophilic cytoplasm, fine chromatin, and a prominent large nucleolus in a 6-year-old girl with ALL and parvovirus B19 infection.

Plate 16.9 Facial maculopapular erythematous skin rash during continuation treatment with mercaptopurine and methotrexate.

Plate 18.1 Histologic section of Burkitt lymphoma. Photo kindly provided by Frederick G. Behm.

Plate 18.3 African child with endemic Burkitt lymphoma of the jaw. Plate 19.4 Gingival hypertrophy in a patient with monoblastic leukemia.

Plate 19.5 Leukemia cutis in congenital myeloid leukemia with the t(9;11).

Plate 21.2 Cytological features of myelodysplasia. Courtesy of Irith Baumann.

Plate 21.4 Transient abnormal myelopoiesis with skin infiltration in Down syndrome. The boy presented at 1 day of age with a WBC of 110 × 109 /L. The blood was dominated by megakaryoblasts. During the following 2 weeks there was increasing hepatomegaly and skin infiltration by myeloid cells. Cytarabine (75 mg/m2 per day) was administered subcutaneously for 4 days. The skin infiltrate disappeared and the WBC normalized within a week.

Plate 23.7 Graft-versus-host disease tends to appear first as a pruritus or erythema on palms (a), soles or ears. Next, a maculopapular rash (b, c) may progress to a total-body erythroderma (d, e).

Plate 23.8 Extensive chronic GVHD in a patient after matchedsibling transplantation. The skin is the most frequently involved organ with hyper- or hypogigmentation, desquamation and a picture similar to scleroderma, including joint contractures. Other features include dysphagia and indolent weight loss. (Photo kindly provided by Dr. G. Vogelsang, John-Hopkins University, Baltimore, MD, USA.)

Plate 29.4 Linear streaks of precipitated uric acid (arrows) in the renal medulla of a 4-year-old boy who died of massive tumor lysis syndrome.

Plate 29.7 Superior vena cava syndrome with venous engorgement of the neck and arms and development of collateral blood vessels in the trunk of a 10-year-old boy with T-cell ALL and a mediastinal mass.

Plate 29.11 Leukoencephalopathy. These magnetic resonance images of a 3.5-year-old girl with acute lymphoblastic leukemia show normal T2 -weighted, fluid attenuation inversion recovery (FLAIR), and color-mapped images at the end of remission induction therapy (upper left, middle, and right panels, respectively). Marked white matter changes (arrows) are seen after four cycles of high-dose methotrexate (lower panels). The patient had no symptoms and a normal neuropsychological evaluation at the time of these studies. She is now 7 years old and has mild deficits in reading comprehension and mathematics, but functions at grade level and has no neurologic deficits. The color-mapped images were generated with digital imaging processing software.340 Yellow denotes normal gray matter; blue denotes cerebrospinal fluid; green denotes normal white matter; and red denotes abnormal white matter. (Courtesy of Dr. Gene Reddick.)

B

A

C

D

Plate 32.1 (A) Target lesions in Pseudomonas aeruginosa sepsis. (B) Ecthyma gangrenosum due to P. aeruginosa. (C) Cutaneous lesions associated with molluscum contagiosum. (D) Cutaneous lesions associated with disseminated candidiasis.

Part I History and general issues

1 Historical perspective Donald Pinkel

Introduction Since its initial recognition 150 years ago, leukemia has been the focus of remarkable research activity and consequent progress. The drama of its manifestations, its frequency in children, its commercial importance in animal husbandry, its usefulness in understanding hematopoiesis, and its ready adaptability as a model for other human cancers are among the reasons for this attention. But perhaps more important for the current generation of its students was the discovery 30 years ago that the most common variety of leukemia could be cured in approximately one-half of children, the first generalized cancer to be cured and the first autologous cancer to be cured with chemicals.1 This chapter summarizes the history of the study of leukemia, particularly childhood leukemia, with regard to description, causation, and treatment. It concludes with comments about the lessons taught by this history.

Description of leukemia Although the first description of a patient with leukemia was published in 1827,2 it was not until 1845 that Virchow3 in Germany (Fig. 1.1) and Bennett4 and Craigie5 in Scotland, in separate case reports, recognized it as a distinct disease, “white blood.” Two years later, Virchow introduced the term “leukemia” for this entity and proceeded on a series of investigations that were summarized in 1856.6 He distinguished leukemia from leukocytosis and described two types: splenic, associated with splenomegaly, and

lymphatic, associated with large lymph nodes and cells in the blood resembling those in the lymph nodes. He also proposed his cellular theory of the origin of leukemia, a concept basic to current understanding of the disease. The following year, acute leukemia was described by Friedreich,7 and in 1878 Neumann8 established the existence of myelogenous leukemia. The close relation between lymphomas and leukemias was defined by Turk9 in 1903. Ehrlich’s introduction of staining methods in 1891 allowed the differentiation of leukocytes and identification of leukemia cell types.10 Splenic and myelogenous leukemias were soon recognized as the same disease, originating from a myeloid precursor. Eventually the leukemic myeloblast, monoblast, and erythroblast were identified. It also became apparent that some acute leukemias were marked only by abnormal leukocytes in the blood, not leukocytosis. By 1913, leukemia could be classified as chronic lymphocytic, chronic myelogenous, acute lymphocytic, myeloblastic or monocytic, or as erythroleukemia.11 Not only did these advances result in refined classification of leukemia, but they shed light on the nature of normal hematopoiesis as well. The prevalence of acute leukemia during childhood, especially between ages 1 and 5 years, was noted in 1917.12 Progress in the description of leukemia has continued to parallel the development of new technologies, such as special staining, electron microscopy, chromosomal analysis, immunophenotyping, and molecular genotyping. With use of electron microscopy, platelet peroxidase staining, and monoclonal antibody reactivity to a platelet glycoprotein, CD41, acute megakaryocytic leukemia became a welldefined entity.13 Although some hematologists and many

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Fig. 1.2 Luis Borella identified T-cell leukemia, introduced immunophenotyping of leukemia, and initiated its classification by biological function in addition to morphology. Fig. 1.1 Rudolf Virchow, the father of leukemia research, established leukemia as a medical entity in the years 1845 to 1856. He also classified leukemia by its pathologic anatomy and cell morphology and postulated its cellular origin.

chemotherapists lumped all childhood acute leukemias into one category as late as the 1960s, the discovery that acute lymphoid and acute myeloid leukemias (ALL and AML) responded differently to prednisone and methotrexate made it necessary to use the new technologies to clearly distinguish them. After the discovery in 1960 of the Philadelphia chromosome in adult chronic myeloid leukemia, and the later introduction of banding techniques, many nonrandom chromosomal abnormalities were found to be associated with specific types of acute leukemia.14,15 Application of DNA probing and amplification methods resulted in molecular genotyping of leukemias, both for diagnosis and for detection of residual cells of the leukemia clone.16 It also became possible to use archived neonatal Guthrie blood spots to trace back the fetal origin of many childhood leukemias.17–24

In 1973, Borella and Sen25 (Fig. 1.2) demonstrated that in some children with acute lymphoid leukemia, the leukemic lymphoblasts were of thymic origin. They further showed that T-cell leukemia was clinically as well as biologically unique.26 As monoclonal antibodies to leukocyte cell surface antigens were developed, further immunophenotypic classification of leukemia cell populations became possible.27 Currently, leukemia is classified as acute or chronic, lymphoid or myeloid, as in the 19th century (see Chapter 2). However, the morphology of acute leukemia is subclassified into three lymphoid varieties and eight myeloid. Myelodysplastic syndromes such as monosomy 7 syndrome and juvenile myelomonocytic leukemia are also recognized. Immunophenotyping of leukemia cells with monoclonal antibodies separates the lymphoid lineage into early and late B-precursor, B-cell, and T-cell (see Chapter 7). It also helps to distinguish anaplastic lymphoid from myeloid cell types and to classify the eight myeloid types, and contributes to identifying the rare biphenotypic variety. Genotypic classification by chromosomal analysis, fluorescent in situ hybridization, DNA probing, and

Historical perspective

polymerase chain reaction techniques allows molecular genetic definition of leukemias (see Chapters 9, 10, and 11). Because leukemia is now recognized as a molecular genetic disorder, and the most effective acute leukemia drugs disrupt molecular genetic processes, this approach to cell characterization may be the ultimate descriptive method. With use of recent technology, it has become clear that the most frequent form of acute leukemia in children is B-precursor cell, often with excessive chromosomes or expression of novel hybrid genes such as ETV6-CBFA2 (TEL-AML1), E2A-PBX1, or BCR-ABL (190 kb) and, in young infants, often demonstrating rearrangement of the MLL (HRX) gene.28–30 Recently, the World Health Organization published a new classification of leukemia based on the advice of numerous experts.31,32 Whether its complexity will be justified by more precise diagnosis, better understanding and improved prognosis is uncertain. During the past 30 years, the importance of describing the leukemia host has also become more apparent. Not only such features as age, gender, and disease extent, but also ethnicity, nutrition, socioeconomic status, and accompanying syndromes and diseases, have been correlated with type of leukemia and outcome of treatment.33–39 For example, children with trisomy 21 (Down) syndrome have a high incidence of leukemia, especially acute megakaryocytic leukemia.38 They also have twice the cure rate of other children with acute myeloid leukemia when treated with chemotherapy.39 The extra 21 chromosome introduces not only increased vulnerability but also better curability. Hispanic youngsters have a high frequency of acute promyelocytic leukemia.40 Host genetic polymorphisms with regard to enzymes such as thiopurine methyltransferase that make available, activate, or detoxify antileukemic drugs are important.41,42 Genetic polymorphisms may also play a role in susceptibility to leukemia among persons exposed to environmental leukemogens or prone to dietary deficiency of folic acid.43,44 Malnutrition, poverty, and underprivileged ethnicity are associated with low cure rates.33–37 In summary, the history of the past 150 years illustrates that progress in the comprehension of leukemia has paralleled the continued application of new ideas and technology to this disease by creative, industrious, and practical clinical investigators.

Causation of leukemia The search for the causation of leukemia has followed several approaches: infectious, genetic, physical, and chemical. Pursuit has been vigorous and often marked by heated controversy. Over time it has become apparent that all

approaches may be correct and that leukemia results from numerous causes, often interacting and varying from cell type to cell type and from one patient to another. Recent studies suggest that childhood leukemia is initiated during fetal life. Rearrangements of either leukemia-associated genes or immunoglobulin heavy-chain genes in childhood leukemia cells have been identified retrospectively in stored neonatal Guthrie blood spots.17–24 However, the frequency of leukemia-associated gene rearrangments, such as TEL-AML1, in surveys of blood spots far exceeds the incidence of childhood leukemia. This indicates that the gene rearrangement alone is insufficient to cause leukemia. Other factors must be contributory.

Infectious causes When “white blood” was identified, some observers considered it the result of severe inflammation, but the new technology of blood microscopy revealed that the white cells of leukemic leukocytosis appeared different from those of inflammatory leukocytosis. However, interest continued in an infectious etiology. Ellerman and Bang’s45 transmission of fowl leukemia by cell-free extracts in 1908, suggesting a viral causation, was a landmark finding that led to extensive searches for the virus etiology of all leukemias, both in animals and humans, throughout the 20th century. In 1951, a mammalian leukemia virus was first demonstrated by Gross46 (Fig. 1.3) by injection of newborn mice with cell-free filtrates from leukemic mice. Subsequently, several leukemia-producing viruses were isolated from cats, cattle, gibbon apes, and humans with adult-type T-cell leukemia.47–50 All were characterized as retroviruses. These single-stranded RNA viruses produce DNA polymerase and integrase, which reverse transcribe the viral RNA genome to DNA and integrate it into the cellular genome. This can result in neoplastic transformation of the cell with or without virus production. In addition, two large DNA viruses of the herpes group were associated with leukemia: Marek disease virus in birds and Epstein–Barr virus (EBV) in B-cell lymphoma/leukemia of African children (Burkitt lymphoma).51,52 Since both EBV-positive and EBV-negative B-cell lymphoma/leukemia have comparable gene rearrangements and postulated mechanisms of leukemogenesis, it is doubtful that the virus is causative.53 Extensive attempts to identify leukemia viruses in children with B-precursor, T-cell, myeloid, and temperate zone Bcell leukemia have been unsuccessful.54 However, the critical experiments that led to identification of murine and feline leukemia viruses, injection of newborn of the same species, cannot be performed.

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Northumberland and Durham, United Kingdom,57–60 but study elsewhere has failed to confirm significant aggregation or other evidence of communicability.61,62 Also cited to support the infection hypothesis was the lower incidence and younger age of acute leukemia in children of lower income families.57 It was speculated that this could fit the pattern of infectious diseases such as paralytic poliomyelitis, in which early exposure and maternal immunity contribute to the appearance of disease at an earlier age and less frequently in underprivileged children. More recently, Kinlen and colleagues63 described excessive leukemia and non-Hodgkin lymphoma rates in children living near large rural construction sites. They suggested that the high risk was related to unaccustomed mixing of rural and urban people and was evidence for an infectious process. Greaves and associates64,65 have further modified and expanded Kellett’s hypothesis based on a newer understanding of the biology of childhood leukemia and international epidemiologic data. In summary, infectious causation of childhood leukemia remains only a hypothesis.

Physical causes

Fig. 1.3 Ludwik Gross described the first mammalian leukemia virus in 1951, initiating research efforts that led to study of the molecular pathology of leukemia.

Despite the failure to identify causative leukemia viruses in children with leukemia, some epidemiologic characteristics have been interpreted in favor of an infectious cause. In 1917, Ward12 reviewed 1457 cases of acute leukemia and concluded that the weight of evidence was against infection. In 1942, Cooke55 collected information on children with acute leukemia from 33 American pediatric services (a harbinger of pediatric cooperative studies) and demonstrated a sharp peak in incidence between ages 2 to 5 years, paralleling peaks in measles and diphtheria incidence. He concluded that acute infections were a factor in causing childhood leukemia. Lending weight to an infection hypothesis was the report by Kellett56 in 1937 of a concentration of cases in Ashington, England. He suggested that an infection, possibly widespread but of low infectivity, might be the causative agent. Subsequent instances of temporospatial proximity of children with leukemia were reported from Erie County, New York; Niles, Illinois; and

Although ionizing radiation probably induced leukemia in Marie Curie, its leukemogenic effects in radiologists only became quantified in 1944.66 In 1952, studies of Japanese children who survived atomic bombing demonstrated a marked increase in acute leukemia, both lymphoid and myeloid.67 Subsequently, Simpson et al.68 reported that children who received neonatal thymic irradiation had an increased risk of thymic lymphoma and acute leukemia as well as thyroid carcinoma. Numerous subsequent studies of prenatal and childhood exposure to diagnostic radiography and medical radiation for benign disease yielded evidence that low-dose radiation can be a factor in the causation of childhood leukemia.69,70 The most recent evidence suggests that low-dose radiation induces a transmissable genetic instability in hematopoietic stem cells.71 This results in diverse chromosomal aberrations in their progeny many cell divisions later. Action was taken in the 1960s and 1970s to reduce fetal, neonatal, and childhood exposure to ionizing radiation. Medical radiation for neonatal thymus, tinea capitis, acne, benign tumors, and even some malignancies was eliminated. Shoe store fluoroscopes were removed, medical and dental radiology equipment and protection upgraded, and diagnostic radiography, especially by fluoroscope, was reduced or replaced with ultrasound imaging. However, as long as nuclear weapons continue to exist, radiation remains a potential cause of leukemia.

Historical perspective

Chemical causes In 1928, Delore and Borgomano72 reported a patient with acute leukemia associated with benzene intoxication. Subsequently, numerous reports confirmed that benzene can produce myelodysplasia and acute myeloid leukemia.73,74 A dose–response relationship was recently found in China.75 Although the hazards have been occupational and the victims adults, the significant yield of benzene in cigarette smoke – three times greater in sidestream than in mainstream smoke – and in automobile exhaust raises the question of whether parental smoking and automobiles are causative factors of leukemia in children.76 Smith has proposed that the phenolic metabolites of benzene are converted to quinones that produce DNA strand breaks, topoisomerase II inhibition and mitotic spindle damage in hematopoietic cells.77 In recent years folic acid deficiency has become associated with the causation of childhood leukemia. An unconfirmed case control study in Australia78 suggested a protective effect of maternal folate supplementation against the risk of childhood B-precursor ALL. In both children and adults, genetic polymorphism of 5,10– methylenetetrahydrofolate reductase, resulting in loss of this enzyme’s activity, appears to reduce the risk of some forms of ALL.44 The suggested mechanism is the increased availability of methyl groups from the folate cycle for conversion of uracil to thymine. This reduces the possibility of uridine incorporation into DNA and consequent genomic instability. Transfer of methyl groups by way of the folic acid cycle is essential to purine synthesis and the suppression of untimely gene expression as well as the methylation of uracil to form thymine. Defects in the folic acid cycle produced by dietary deficiency, impaired absorption or transport, antifolate agents, genetic polymorphism or exposure to nonphysiologic methylating agents, such as the pesticide methyl bromide, might contribute to the pathogenesis of leukemia. The advent of cancer chemotherapy in the 1950s and its extension in the 1960s and 1970s led to the appearance of secondary leukemia both in children and adults. Alkylating agents and drugs that bind topoisomerase II, especially etoposide and teniposide, were found to be leukemogenic in children, most often producing acute leukemia characterized by MLL gene fusions.79,80 This observation of the role of topoisomerase binding is consistent with the Smith hypothesis77 for the mechanism of benzene leukemogenesis. A recent study demonstrated that children who had acute leukemia with MLL fusion genes were more likely to have low function of an enzyme that detoxifies quinones.43 Another study revealed an association between

this leukemia genotype and maternal exposure to certain drugs and pesticides.81 These data suggest that both maternal exposure to potential leukemogens and fetal genetic polymorphisms might contribute to the induction of childhood leukemia.

Genetic causes A genetic cause of leukemia was first suggested in 1876 by Hartenstein,82 who observed lymphoid leukemia in a cow and its mother and speculated that it was hereditary. In 1931, strains of mice with high frequencies of leukemia/lymphoma were identified,83 and by 1935 an inbred strain with a 90% incidence of lymphoid leukemia was produced.84 Extrinsic nonhereditary factors were postulated to explain the 10% failure of this inbred strain to develop leukemia. The evidence for a possible genetic basis of murine leukemia led to studies of the familial incidence of human leukemia. A 1937 report85 of three families with multiple cases was followed by a large study by Videbaek86 in Denmark comparing families of patients with leukemia and families of healthy persons. A significant difference was found and a genetic hypothesis proposed. An institution-based study in Boston in 195787 did not support Videbaek’s findings, but the author acknowledged three families with multiple cases of acute leukemia, two with parental consanguinity, and suggested a recessive gene in these families. Although leukemia in twins was described in 1928,88 the high concordance rates for leukemia in likesex and monozygous twins were uncovered in 1964 by MacMahon and Levy.89 Recent studies by Ford et al.18 using genetic markers indicate that twin concordance probably results from intrauterine metastases from fetus to fetus. In addition to increased familial incidence and twin concordance, the increased risk of leukemia in children with constitutional chromosome abnormalities further supported a genetic hypothesis. The report of a child with Down syndrome and acute lymphoid leukemia in 193090 and subsequent similar reports led to a national survey in 1957 by Krivit and Good38 that demonstrated the high incidence of leukemia in this trisomy disorder. Over the past 40 years, childhood leukemia has become associated with numerous constitutional genetic disorders, including primary immunodeficiency diseases, chromosome instabilities, and inherited cancer syndromes.91 Observation of the distinct Philadelphia chromosome associated with chronic myeloid leukemia by Nowell and Hungerford14 in 1960, and Rowley’s discovery15 that it resulted from a 9;22 chromosomal translocation in 1973,

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were followed by identification of numerous nonrandom chromosomal abnormalities associated with biologically distinct leukemias and hybrid genes. In 1982, the human homologue of the Abelson murine leukemia virus protooncogene abl was found to be relocated from chromosome 9 to 22 in chronic myeloid leukemia, to form its characteristic hybrid gene, BCR-ABL.92 In the same year the human homologue of an avian leukemia oncogene (MYC) was identified on the region of chromosome 8 that is translocated in B-cell lymphoma/leukemia of children.93 By the mid-1980s, there was a clear consensus that leukemia was a somatic genetic disorder of hematopoiesis.94 More important, these translocations became models of the two general mechanisms of leukemogenesis by chromosome/gene rearrangements. The BCR-ABL hybrid gene gives rise to a BCR-ABL fusion protein with excessive and promiscuous tyrosine kinase activity.95 This leads to the activation of myriads of proteins along several signaling pathways and reduced cell adhesion, increased mitoses and inhibition of apoptosis – conditions favorable to leukemogenesis, either chronic myeloid or acute lymphoblastic. The second mechanism is exemplified by the translocation of the MYC oncogene of chromosome 8 to the immunoglobulin heavy-chain region of chromosome 14.96 The consequence is remarkably increased expression of the MYC gene, whose translation product dimerizes with the normal MAX protein. This drives cell replication at the expense of differentiation. B-cell lymphoma and/or leukemia results. Although the ultimate causation of most childhood leukemias remains unknown, the establishment of a genetic mechanism, recognition of the role of homologues of animal leukemia virus oncogenes in human leukemia cells, and the knowledge that ionizing radiation and chemical leukemogens modify genetic DNA appear to reconcile the four historical approaches to causation. The more recent insights about genetic polymorphisms, folic acid and the consequences of leukemia-associated gene rearrangements have introduced new potentials for the prevention and treatment of childhood leukemias.

Treatment Palliative treatment Because of the diffuse nature of leukemia and its catastrophic manifestations, physicians began to treat patients with chemicals shortly after it became recognized as a disease entity. In 1865, Lissauer97 reported a patient with leukemia whose disease remitted after she received Fowler solution (arsenious oxide); arsenicals became a standard but marginally useful palliation. With the discovery of

Fig. 1.4 Sidney Farber and his colleagues discovered that a synthetic antifolate, 4-amino-pteroylglutamic acid, produced remissions of childhood leukemia. This introduced antimetabolite chemotherapy and began the research leading to a cure for many children with leukemia.

roentgen rays in 1896, interest turned to their clinical application in cancer therapy. In 1903, Senn98 reported the response of leukemia to irradiation, and this modality, applied most often to the spleen, largely replaced arsenious oxide as a palliative measure, especially in chronic leukemia. When radioactive nuclides became available in 1940, radioactive phosphorus came into use for chronic myelogenous leukemia and polycythemia vera.99 Based on pathology reports of hematosuppression in mustard gas victims on the Western Front in World War I100 and at the Bari Harbor disaster in World War II,101 nitrogen mustard was synthesized and tested in animals and then patients with lymphoma and leukemia in 1943.102,103 Temporary partial remissions were produced, but toxicity was considerable, especially in patients with acute leukemia. The chemical identification of folic acid in 1941104 as an essential vitamin, its synthesis in 1946,105 and the reversal of megaloblastosis by its administration106 raised the question of whether it might be useful in the treatment of acute leukemia. In 1947, Farber (Fig. 1.4)107,108 and

Historical perspective

use in sequential and combination chemotherapy with a corticosteroid (usually prednisone) and methotrexate, the 4-amino-N10 -methy1-folate analogue that succeeded aminopterin.108 The enthusiasm generated by the discovery of three effective drugs for childhood acute leukemia in 5 years was dampened, however, by the realization that virtually all of the patients eventually died of resistant leukemia or its complications.108 This led to a fixed notion among most pediatricians and hematologists that temporary remissions and prolongation of survival in comfort were the most one could expect from leukemia chemotherapy. In 1959, a prodrug analogue of nitrogen mustard, cyclophosphamide, with less toxicity for platelet production, was introduced and later shown to have value in lymphoid leukemia.113 In 1962, vincristine, an alkaloid from the periwinkle plant with a unique mode of action, was shown to induce complete remissions of childhood lymphoid leukemia resistant to other agents.114 But, as with all the other agents, remissions were temporary and relapse with resistant leukemia ensued.

Fig. 1.5 Gertrude Elion, working with George Hitchings, used an understanding of purine metabolism to develop three drugs important to children with leukemia: mercaptopurine, allopurinol, and acyclovir.

colleagues gave folic acid (pteroylglutamic acid) to children with acute leukemia and were impressed that it might have produced acceleration of the leukemia. Subsequently, a 4-amino antimetabolite of folic acid, aminopterin, synthesized by Seeger et al.,109 was provided to Farber for use in children with acute leukemia. Many of them achieved complete clinical and hematologic remissions that lasted for several months.107 The era of specific leukemia therapy had begun! A year after the report of remissions with aminopterin, a 1949 conference on the newly isolated adrenocorticotrophic hormone (ACTH) revealed that it produced prompt although brief remissions of acute lymphoid leukemia.110 Cortisone and its synthetic analogue, prednisone, had similar activity and soon replaced ACTH. Unlike the folate antagonists, the purine antimetabolites 6-mercaptopurine and thioguanine resulted from a lengthy study of purine metabolism, purine analogue synthesis, and structure-activity relationships by Elion and Hitchings111 (Fig. 1.5) in the 1940s and early 1950s. In 1953, a report by Burchenal and associates112 that 6mercaptopurine produced remissions in patients with acute leukemia, especially children, promptly led to its

Curative therapy The first cure of leukemia was described in 1930 by Gloor,115 who treated an adult with arsenious oxide, mesothorium, irradiation, and blood transfusions from two siblings (presaging current myeloblation and peripheral blood stem cell transplantation?). In 1964, Burchenal and Murphy116 collected 36 cases of 5-year cures of treated childhood acute leukemia by a questionnaire survey of hematologists. Zuelzer117 reported a 3% 5-year cure rate in children with ALL who received cyclic chemotherapy with prednisone, methotrexate, and mercaptopurine. A 5% 5-year cure rate was reported by Krivit et al.118 for sequential or cyclic chemotherapy of ALL with these agents in a Children’s Cancer Group study. Stimulated by the studies of Skipper et al.119 and Goldin et al.120 in treating mouse leukemia with chemotherapy, Leukemia Study Group B121–123 used two-drug combinations and National Cancer Institute investigators124,125 used four-drug combinations that yielded similar low cure rates in patients with ALL. The failure to achieve a significant cure rate in these courageous attempts reinforced the prevailing pessimism about leukemia therapy. Persons who continued to advocate anything beyond palliation were looked upon with skepticism, if not scorn, into the early in 1970s. In 1962, St. Jude Children’s Research Hospital was opened in Memphis, Tennessee, with a mandate to seek prevention or cure of childhood leukemia. The St. Jude investigators defined several specific obstacles to the cure of

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childhood acute leukemia.94 First was drug resistance: initial, as demonstrated by the high proportion of patients who failed to experience remission on single-drug treatment; and acquired, as indicated by eventual relapse in most children despite continued drug administration. The second obstacle was clinically isolated meningeal relapse that occurred with increasing frequency as systemic chemotherapy became more effective and hematologic remissions lasted longer. Meningeal relapse was thought to be due to the inadequate diffusion of methotrexate and mercaptopurine through the blood–cerebrospinal fluid barrier with consequent proliferation of leukemia cells in the leptomeninges. The third obstacle was the overlapping toxicity of antileukemic drugs, especially hematosuppression, immunosuppression, and mucositis, and thus the dilemma of limiting dosage or risking treatment-related death. However, the greatest obstacle was a pessimism that inhibited thoughts of curing patients with leukemia. A curative approach to children with ALL was initiated in 1962. It consisted of four treatment phases: remission induction, intensification or consolidation, preventive meningeal treatment, and prolonged continuation therapy.94,126–128 The main features were the administration of combination chemotherapy for induction, intensification and continuation chemotherapy, the use of different drug combinations for induction and continuation, preemptive irradiation of the cranial or craniospinal meninges, elective cessation of chemotherapy after 2 to 3 years, and most important, the objective of cure rather than palliation. The pilot studies from 1962 to 1965 were fraught with considerable difficulty, including the emergence of Pneumocystis carinii pneumonia due to immunosuppression and the inadequacy of low-dose craniospinal irradiation to prevent meningeal relapse.126–128 However, longer complete remissions were achieved than previously and 7 of 41 children became long-term leukemia-free survivors after cessation of therapy, a higher rate than previously reported, justifying the notion that acute leukemia could no longer be considered incurable. A fourth study129 compared full versus half-dosage continuation chemotherapy and demonstrated that, despite its toxicity, full dosage was required to achieve longer remission. It was clear from this experience that more capability in prevention and control of infection, especially with Pneumocystis carinii and the herpesviruses, was required. With this information, another pilot study1 was inaugurated in December 1967, in which the intensity of continuation chemotherapy was increased and higher-dose cranial irradiation combined with intrathecal methotrexate was used to treat the leptomeninges. Within 6 months, the superiority of this regimen was apparent, and a randomized comparative study of meningeal irradiation was

initiated.130 Both the pilot study and the subsequent randomized study demonstrated a 50% cure rate for children with ALL who had received multiple-agent chemotherapy and effective preventive meningeal therapy. Since 1970, many institutional and collaborative groups throughout the world, using the same four phases of treatment but with modifications of drug selection and dosage schedules, have confirmed the curability of ALL in children.28 Intrathecal methotrexate alone failed to prevent meningeal leukemia in one study.131 However, Sullivan and associates132 demonstrated that repeated administration of three drugs intrathecally during remission induction and continuation therapy was equivalent to meningeal irradiation for this purpose. Radiotherapy and its adverse sequelae could be avoided in most patients. In the 1980s and 1990s, improved cure rates of up to 75% were reported.28,133 National surveys in the United States and United Kingdom demonstrated marked reduction in childhood leukemia mortality.134,135 Much of this improvement was related to more positive attitudes and greater clinical skill with experience, a remarkable increase in hematology-oncology medical and nursing specialists, better means of prevention and treatment of infection, more availability and use of blood components, earlier diagnosis and treatment, increased governmental and private health insurance coverage, improved childhood nutrition, and, in some instances, patient selection. But the discovery and judicious introduction into treatment of additional antileukemic drugs was also important. These included cytarabine, a synthetic pyrimidine antimetabolite (1968),136,137 daunorubicin, a natural DNA-intercalating anthracycline antibiotic (1968),138 asparaginase, an enzyme synthesized by bacteria that lyses the essential amino acid asparagine (1967),139 and the epipodophyllotoxins etoposide and teniposide, topoisomerase-binding agents derived from the mandrake root.140 Modification of drug schedules, such as the intravenous administration of methotrexate in high dosages with delayed leucovorin rescue, was another factor.141 The definition of subtypes of ALL and the successful targeting of specifically designed chemotherapy in children with T-cell or B-cell leukemia or those otherwise at high risk of relapse with B-precursor leukemia have been important also.142,143 From the beginning of leukemia chemotherapy, the morphologic differences in response to chemotherapy were apparent. Although occasional patients with AML experienced remissions with 6-mercaptopurine or thioguanine, a 50% remission rate was first achieved in 1967 when thioguanine was combined with cytarabine.144 Further improvement followed the introduction and inclusion of daunorubicin and etoposide. By intensive administration of these drugs, accompanied by considerable supportive

Historical perspective

therapy, it became possible in the 1980s to cure approximately 25% to 30% of unselected children with AML.145 More recent reports are more optimistic.146,147 In 1957, Barnes and Loutit148 administered lethal doses (LD98) of total-body irradiation to leukemic mice with or without subsequent homologous bone marrow transplants. The mice that received marrow homografts tended to survive without leukemia but died of a wasting disease; those that did not receive grafts had recurrence of leukemia. This led the investigators to suggest that the grafts had an antileukemic effect and stimulated similar experiments in humans. With the introduction of human leukocyte antigen (HLA) typing and matching,149 Thomas and colleagues150 achieved successful treatment of leukemia by myeloablation with total-body irradiation and chemotherapy and subsequent marrow transplantation from an HLA-compatible sibling. Evaluation of the efficacy of this procedure relative to intensive chemotherapy alone for acute leukemia has been hindered by patient selection and lack of randomized comparative studies.151 Also, the sequelae of the procedure in children, such as chronic graft-versus-host disease, multiorgan impairment, and growth failure, often preclude true cure (i.e. restoration of the capacity for normal growth, development, and health as well as freedom from leukemia). On the other hand, experience demonstrated that some types of leukemia were not curable by chemotherapy alone. Treatment with very high dosage chemotherapy and radiotherapy and histocompatible hematopoietic transplant was often successful in eliminating chronic myeloid leukemia152 that otherwise was only palliated by chemotherapy with myleran153 or hydroxyurea.154 Success was reported in some cases of juvenile myelomonocytic leukemia, myelodysplasia/myeloid leukemia associated with chromosomal monosomy 7, and AML that failed to respond to intensive chemotherapy or relapsed despite it.155–157 Evidence, again from nonrandomized comparisons, was reported that implied an advantage of hematopoietic transplantation in eliminating leukemia from children with ALL who develop hematologic relapse during chemotherapy.158 However, recent comparisons employing more acceptable analysis of results indicate no advantage over aggressive chemotherapy in children with ALL in first relapse and children with ALL that demonstrates rearrangements of the 11q23 chromosomal region.159–161 For children with newly diagnosed AML 6-year event-free survival is similar whether treated with transplant or chemotherapy.147,162 In recent years the original concept of hematosuppression and transplant proposed by Barnes and Loutit148 has been rediscovered. Transplants are viewed as immunotherapy and success dependent on graft versus leukemia reaction, not myeloablation.163 Moderate chemotherapy with-

Fig. 1.6 Zhen Yi Wang and his team developed successful therapy with the vitamin tretinoin in acute promyelocytic leukemia, the first effective differentiation agent and gene-targeted drug in cancer treatment.

out radiotherapy is often used instead of “megatherapy.” This reduces treatment-related mortality and morbidity and may improve eventual outcome. In the 1980s, a new class of agents, biological response modifiers, became available. One of them, alpha interferon, was shown by Talpaz and colleagues164 in 1986 to produce remissions of chronic myeloid leukemia, some complete, both hematologic and cytogenetic, and enduring.165 Children with adult-type chronic myeloid leukemia had similar responses.166 This offered an alternative to myeloablation and marrow transplantation. The conclusion in the 1980s that leukemia was a genetic disorder and observations that drugs effective in curing leukemia modified DNA suggested that chemotherapy might focus on genetic targeting.94,167 In 1988, Wang and colleagues (Fig 1.6).168 reported the differentiation of acute promyelocytic leukemia with resultant complete remission after administration of all-trans-retinoic acid (tretinoin). Subsequently, the genetic defect in acute

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promyelocytic leukemia was linked with an abnormal intranuclear retinoic acid receptor.169 When tretinoin was combined with conventional cytotoxic chemotherapy, the cure rate was significantly increased.170 This was the first instance of successful differentiation-inducing therapy for a human cancer, the first successful use of a vitamin to treat a human cancer, and the first specific targeting of a therapeutic agent to a cancer-associated gene rearrangement. This discovery was a major stimulant to searching for other methods of genetic targeting in the leukemias associated with specific gene rearrangements. With the introduction of molecular diagnostic technology in the 1990s, it became possible to classify most childhood leukemias genetically.28–30 For example, TEL-AML1 + leukemia resulting from a t(12;21) translocation can only be identified by molecular technology in most cases.29 The advantage of genetic classification quickly became clear when Druker and colleagues171 showed that BCRABL leukemia, whether myeloid or lymphoid, could be effectively treated by blocking the tyrosine kinase activity of the BCR-ABL fusion protein. The agent currently used, imatinib mesylate, has replaced hematopoietic transplantation and alpha interferon as initial therapy for chronic myelocytic leukemia.172 It is also included in the treatment of BCR-ABL + ALL. Although Southern blotting, the polymerase chain reaction and fluorescent in situ hybridization have been the mainstays of molecular genetic analysis of leukemia, the introduction of microarray techniques has been an important recent advance.173 With this method, one can predict the likely response to chemotherapy as well. In summary, the past 40 years of clinical investigation to identify curative treatment of childhood leukemia have met with mixed success, as demonstrated by the wide variation in cure rates. This lack of uniformity reflects not only differences in leukemia cell biology and the extent of leukemia, but also the economic status, ethnicity, residence, nutrition and constitutional genetics of the patients. The cost and complexity of curative leukemia therapy severely limit its usefulness, placing it beyond the reach of the majority of the world’s children who need it.174 Another and perhaps increasing problem are the serious adverse late sequelae of treatment with alkylating agents, anthracyclines, epipodophyllotoxins, radiotherapy, and allogeneic transplantation of hematopoietic cells, discussed elsewhere in this text (see Chapters 30 and 31).

Supportive therapy During the 100 years between Virchow’s establishment of leukemia as an entity and the advent of alkylating agents, comforting the patient with narcotics and human empathy

was the first consideration. When ionizing radiation was introduced in 1903, it became an important palliative agent for relieving local bone pain and obstructive masses as well as reducing white blood cell counts.98 Since chemotherapy was introduced in the 1940s, radiation has remained important for palliation of painful lesions as well as for curative therapy in management of extramedullary relapse in the meninges and testes and in myeloablation prior to hematopoietic transplantation.150,175,176 In 1828, Blundell177 reported a successful direct blood transfusion in a woman with postpartum hemorrhage. However, severe reactions discouraged further use. Landsteiner’s178 identification of human blood groups in 1901 enabled safer blood transfusion. During World War I, Rous and Turner179 discovered that a citrate dextrose solution and cold would preserve red blood cells. Robertson,180 an American Army surgeon who had recently worked with Rous,181 used this solution and packing boxes containing ice to preserve human red blood cells for prompt transfusion of wounded soldiers near the battlefront. For children with acute leukemia, the introduction of the hospital blood bank in 1937 was the first step in prolonging their lives.182 By the late 1940s, blood transfusions together with the newly available antibacterial agents became generally accepted as a way of maintaining life while families tried to adapt to the prognosis and begin their grieving. In 1954, with the advent of plastic blood transfusion and transfer bags and the use of the refrigerated centrifuge, platelet transfusions became available to control thrombocytopenic bleeding.183,184 This resulted in a remarkable reduction in hemorrhage as a cause of death. Platelet transfusions also provided time for antileukemic drugs to produce remission, especially in patients with AML, leading to increased rates of remission induction. Finally, the availability of platelet transfusions allowed administration of higher or more prolonged dosages of hematosuppressive agents because one could tide patients through periods of drug-induced thrombocytopenia. When effective chemotherapy was first employed in acute leukemia, rapid lysis of leukemic cells often resulted in serious and occasionally fatal metabolic disturbances, especially in florid leukemia with high white blood cell counts or massive organ involvement. The introduction of allopurinol, a synthetic inhibitor of xanthine oxidase, together with skillful fluid and electrolyte therapy, did much to solve this problem.185 More recently, recombinant urate oxidase (rasburicase) was developed as a more potent drug than allopurinol in the prevention and treatment of hyperuricemia.186 As children survived longer in remission, the immunosuppression caused by chemotherapy was more evident.

Historical perspective

Varicella became a major problem, particularly with prednisone therapy.187,188 Many children died of severe disseminated varicella, while others had treatment interrupted for long periods with consequent increased risk of relapse. With recognition that varicella and herpes zoster were caused by the same virus, plasma from adults convalescing from zoster was used both for treatment and for prevention in recently exposed children. After convalescent plasma was found effective for prevention or modification, varicella-zoster immune globulin (VZIG) was prepared and demonstrated to be effective also.189 The availability of VZIG and the education of parents and teachers about the hazard of varicella zoster infection were a major advance in reducing mortality, morbidity, and treatment interruption in exposed children. However, the third contribution of Gertrude Elion to children with leukemia, the introduction of acyclovir in 1980, was perhaps more important.190,191 Shortly after intensive multiagent therapy was introduced for acute leukemia at St. Jude Children’s Research Hospital, a peculiar pneumonia began to appear in many of the children. At first it was called “St. Jude pneumonia” and thought to be related to drug toxicity, viral infection, or both. However, postmortem study of the lungs and pulmonary needle aspiration in patients and methenamine silver nitrate staining revealed Pneumocystis carinii organisms.192 An institutional epidemiologic study performed in collaboration with the federal Communicable Disease Center (CDC) indicated that the disease was solely related to immunosuppression of the patients and not to contagion.193 Again, this disease became a major limiting factor in treating children with acute leukemia because of its occurrence during remission, its mortality and morbidity, and the consequent interruption of chemotherapy, especially in the critical early months of treatment. Pentamidine isethionate was used to treat infantile Pneumocystis pneumonia in Europe, but it was unavailable in the United States.194 It had to be imported with Food and Drug Administration approval for each diagnosed case. Subsequently, the CDC obtained an investigational new drug permit that not only expedited therapy, but eventually was the mechanism by which the acquired immunodeficiency disease syndrome was recognized. Finally, the brilliant studies of Hughes (Fig. 1.7) and colleagues,195 first in rats and then in patients, demonstrated the value of trimethoprim and sulfamethoxazole (cotrimoxazole) not only in treatment but, more important, in prevention of the disease. Early in the combination therapy of acute leukemia, severe and sometimes fatal bacteremia, particularly with gram-negative bacteria, especially Pseudomonas aeruginosa, was a major obstacle.196 Bodey and associates197

Fig. 1.7 Walter Hughes pioneered infectious disease control in children with leukemia; his work virtually eliminated the threat of Pneumocystis pneumonia as a cause of death or interruption of therapy.

showed that neutropenia was the major reason for these infections, although mucositis was an important contributor. They identified critical levels of neutrophils for control of the infections and demonstrated the need for prompt initiation of appropriate antibiotics in patients with fever and severe neutropenia. As effective aminoglycoside antibiotics became available in the 1960s and were used appropriately, mortality and morbidity due to gram-negative bacteremia declined, resulting again in better survival of children with acute leukemia. Infections with resistant gram-positive cocci have become a problem in the past 25 years, prompting the greater use of vancomycin in patients with staphylococcal or enterococcal infections and neutropenia.198 The immunosuppression and mucositis due to chemotherapy, radiation, and poor nutrition in children with leukemia also encouraged serious and sometimes fatal mycoses.199 The introduction of amphotericin B in 1958200 and of fluconazole in 1990201 represented significant

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advances in controlling these infections. However, some mycoses such as aspergillosis and mucormycosis remain resistant to treatment and are major causes of mortality, especially in children with prolonged neutropenia who are receiving extensive antibiotic therapy (see Chapter 32). Psychosocial issues became more important as children began to survive longer. Farber and associates108 recognized early the need for “total care” of children with acute leukemia. In 1964, Vernick and Karon202 introduced truthfulness in communicating with the children. Anticipating the significance of survival quality, Soni and colleagues203 pioneered longitudinal study of the neuropsychological consequences of acute leukemia and its treatment. Other late effects have also been studied extensively with the goal of defining the human cost/benefit ratio for each element of leukemia therapy (Chapter 30).

Lessons from the history of leukemia The value of history is not just in savoring the past but in appreciating how it illuminates the present and guides us into the future. Several lessons can be learned from the study of the history of leukemia, particularly childhood leukemia. One is the importance of heeding new facts and listening to new ideas and hypotheses. At each point in the history of leukemia, there have been instances of lost time and opportunity because of unreasoned resistance to innovation. Ten years after Virchow’s description of leukemia and its verification by others, its existence was still denied by many. In 1958, 8 years after his pivotal discovery, Gross was still criticized for describing the viral etiology of a mouse leukemia. Twenty years elapsed between the establishment of a battlefront blood bank and the first blood bank in an American hospital. When antifolate and antipurine drugs were first introduced, many hematologists and pediatricians refused to prescribe them because they were “too toxic.” Into the 1960s some parents were advised and medical students taught to withhold chemotherapy from childhood leukemia patients: “let the children die in peace.”204 It is important for physicians and scientists to be open to new thinking that challenges conventional wisdom and ways. Another lesson is the significance of the case report describing a patient and what the patient taught the physician. Virchow’s case report of leukemia in 1845, Lissauer’s description of a patient whose leukemia responded to arsenious oxide, Brewster and Cannon’s observation of leukemia in a child with Down syndrome, and Gloor’s patient who was cured of leukemia after arsenious oxide,

mesothorium, irradiation, and sibling blood transfusions eventually led to important knowledge of leukemia biology and treatment. A third lesson is the need to encourage rather than dampen speculation in spoken and printed discussion. Kellett’s idea that the residential aggregation of leukemia cases in Ashington might reflect an infectious agent, widespread but of low infectivity, remains viable, although statistical significance of time-space clustering is dubious. Equally important, however, is the need to clearly identify speculation and to require adequately controlled, scientifically sound investigations before drawing conclusions. Many children with acute leukemia were subjected to BCG injection on the basis of an uncontrolled study before appropriate investigations demonstrated its lack of efficacy.205–207 The relative lack of value and unfavorable risk/benefit ratio of hematopoietic transplantation for children with most types of acute leukemia has taken decades to clarify because proper comparison with optimal treatment omitting transplantation was not performed initially. The most important lesson is the need to encourage original investigator-initiated research of leukemias by clinicians and scientists working together, exchanging ideas and coordinating clinical observations with biological experimentation. For example, after Gross heard a lecture by Gilbert Dalldorf on the use of newborn mice to identify Coxsackie virus, he switched to newborn mice as subjects of his experiments and discovered the first mammalian leukemia virus. Farber’s impression that folic acid accelerated leukemia encouraged development of antifolates and the first effective treatment for childhood leukemia. Robertson’s knowledge of red blood cell preservation gained at the Rockefeller Institute enabled him to initiate blood banking on a Belgian battlefront. Borella’s observation that children with thymomegaly had a more aggressive lymphoid leukemia and his identification of thymic cell leukemia as a distinct entity led to immunophenotyping and initiated classification of leukemia by biological function. It is also important that clinical and laboratory researchers be free to think independently and to pursue goals as they see fit with minimal intervention by managers and committees. The long-term advantage of scientific freedom often exceeds the short-term gain of tightly restricted research. The late Robert Guthrie illustrates this. Assigned to provide microbiological assays of experimental antileukemic drugs, he deviated when he conceived the notion of using such an assay to screen heel-stick blood spots of newborn for high phenylalanine levels. His purpose was early detection of phenylpyruvic oligophrenia so that

Historical perspective

mental retardation could be prevented by dietary deletion of phenylalanine.17 In order to continue this research, Dr. Guthrie was compelled to resign his position for a lesser one elsewhere. Not only did his work result in today’s highly successful neonatal screening programs, but 45 years later stored “Guthrie spots” are used to track fetal origins of leukemia. Good research benefits all eventually. There is an anecdote that an accomplished senior leukemia researcher was asked by a site visit committee for his 5-year plan. He is said to have responded: “Five years? I don’t know what I will do this afternoon. I haven’t looked at my mice today.”

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67 Folley, J. H., Borges, W., & Yamawaki, T. Incidence of leukemia in survivors of the atomic bomb in Hiroshima and Nagasaki, Japan. Am J Med, 1952; 13: 311–21. 68 Simpson, C. L., Hempelman, L. H., & Fuller, L. M. Neoplasia in children treated with x-rays in infancy for thymic enlargement. Radiology, 1955; 64: 840–5. 69 Stewart, A., Webb, J., Gates, D., et al. Malignant disease in childhood and diagnostic irradiation in utero. Lancet, 1956; 2: 447. 70 Ron, E., Modan, B., & Boice, J. D., Jr. Mortality after radiotherapy for ringworm of the scalp. Am J Epidemiol, 1988; 127: 713–25. 71 Kadhim, M. A. & Wright, E. G. Radiation-induced transmissable chromosomal instability in haemopoietic stem cells. Adv Space Res, 1998; 22: 587–96. 72 Delore, P. & Borgomano, C. Leuc´emie aigue au cours de l’intoxication benzenique: sur l’origine toxique de certaines leuc´emies aigues et leurs relations avec les an´emies graves. J M´ed Lyon, 1928; 9: 227–33. 73 Aksoy, M., Erdem, S., & Dincol, G. Leukemia in shoe workers exposed chronically to benzene. Blood, 1974; 44: 837–41. 74 Vigliani, E. C. & Saita, G. Benzene and leukemia. N Engl J Med, 1964; 271: 872–6. 75 Hayes, R. B., Yin, S. N., Dosemeci, M., et al. Mortality among benzene-exposed workers in China. Environ Health Perspect, 1996; 104 (Suppl. 6): 1349–52. 76 Hoffmann, D., Brunnemann, K. D., & Hoffman, I. Significance of benzene in tobacco carcinogenesis. In M. A. Mehlman, ed., Benzene: Occupational and Environmental Hazards: Scientific Update (Princeton, NJ: Princeton Scientific Publications, 1989), pp. 99–112. 77 Smith, M. T. The mechanism of benzene-induced leukemia: a hypothesis and speculations on the causes of leukemia. Environ Health Perspect, 1996; 104(Suppl. 6): 1219–25. 78 Thompson, J. R., Gerald, P. F., Willoughby, L. N., et al. Maternal folate supplementation in pregnancy and protection against acute lymphoblastic leukaemia in childhood: a case-control study. Lancet, 2001; 358: 1935–40. 79 Tucker, M. A., Meadows, A. T., Boice, J. D., et al. Leukemia after therapy with alkylating agents for childhood cancer. J Natl Cancer Inst, 1987; 78: 459–64. 80 Pui, C.-H., Behm, F. G., Raimondi, S. C., et al. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med, 1989; 321: 136–42. 81 Alexander, F. E., Patheal, S. L., Biondi, A., et al. Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion. Cancer Res, 2001; 61: 2542–6. 82 Hartenstein, Ber Veterin¨arw, Sachsen: 1876; 44: 41: As cited by Engelbreth-Holm, J. In Spontaneous and Experimental Leukemia in Animals (Edinburgh, UK: Oliver and Boyd, 1942), p. 130. 83 Slye, M. The relation of heredity to the occurence of spontaneous leukemia, pseudoleukemia, lymphosarcoma and allied diseases in mice. Preliminary report. Am J Cancer, 1931; 15: 1361–86.

84 MacDowell, E. C. & Richter, M. N. Mouse leukemia. IX. The role of heredity in spontaneous cases. Arch Pathol, 1935; 20: 709–24. 85 Ardashnikov, S. N. The genetics of leukemia in man. J Hyg, 1937; 37: 286–302. 86 Videbaek, A. Heredity in Human Leukemia and its Relation to Cancer: A Genetic and Clinical Study of 209 Probands (London: H K Lewis, 1947). 87 Steinberg, A. G. A genetic and statistical study of acute leukemia in children. In Proceedings of the Third National Cancer Conference (Philadelphia, PA: J. B. Lippincott, 1957), pp. 353–6. 88 Siegel, A. E. Lymphocytic leukemia occurring in twins. Atlantic Med Monthly J, 1928; 31: 748–9. 89 MacMahon, B. & Levy, M. A. Prenatal origin of childhood leukemia. Evidence from twins. N Engl J Med, 1964; 270: 1082–5. 90 Brewster, H. F. & Cannon, H. E. Acute lymphatic leukemia: report of a case in an eleventh month mongolian idiot. New Orleans Med Surg J, 1930; 82: 872–3. 91 Miller, R. W. Persons with an exceptionally high risk of leukemia. Cancer Res, 1967; 27: 2420–3. 92 De Klein, A., Kessel, A. G. van, Grosveld, G., et al. A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature, 1982; 300: 765–7. 93 Dalla-Favera, R., Bregni, M., Erikson, J., et al. Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci U S A, 1982; 79: 7824–7. 94 Pinkel, D. Curing children of leukemia. Cancer, 1987; 59: 1683– 91. 95 Deininger, M. W., Goldman, J. M., & Melo, J. V. The molecular biology of chronic myeloid leukemia. Blood, 2000; 96: 3343–56. 96 Blackwood, E. M. & Eisenman, R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science, 1991; 251: 1211–17. 97 Lissauer, H. Zwei f¨alle von leucaemie. Berl Klin Wochenschr, 1865; 2: 403–4. 98 Senn, N. The therapeutical value of the Roentgen ray in the treatment of pseudoleukemia. N Y Med J, 1903; 77: 665–8. 99 Lawrence, J. H. Nuclear physics and therapy: preliminary report on a new method for the treatment of leukemia and polycythemia. Radiology, 1940; 35: 51–60. 100 Krumbhaar, E. B. & Krumbhaar, H. D. The blood and bone marrow in yellow cross (mustard gas) poisoning. Changes produced in the bone marrow of fatal cases. J Med Res, 1919; 40: 497–506. 101 Alexander, A. F. Medical report of the Bari harbor mustard casualties. Mil Surg, 1947; 101: 1–17. 102 Goodman, L. S., Wintrobe, M. W., Dameshek, W., et al. Nitrogen mustard therapy. Use of methyl-bis (beta-chloroethyl) amine hydrochloride and tris (beta-chloroethyl) amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia, and

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certain allied and miscellaneous disorders. JAMA, 1946; 132: 126–132. Karnofsky, D. A. Summary of results obtained with nitrogen mustard in the treatment of neoplastic disease. Ann NY Acad Sci, 1958; 68: 889–914. Mitchell, H. K., Snell, E. E., & Williams, R. J. The concentration of “folic acid”. J Am Chem Soc, 1941; 63: 2284. Angier, R. B., Boothe, J. H., Hutchings, B. L., et al. The structure and synthesis of the liver (L. casei) factor. Science, 1946; 103: 667–9. Spies, T. D. Treatment of macrocytic anemia with folic acid. Lancet, 1946; 1: 225–8. Farber, S., Diamond, L. K., Mercer, R. D., et al. Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-amino-pteroylglutamic acid (aminopterin). N Engl J Med, 1948; 238: 787–93. Farber, S., Toch, R., Sears, E. M., et al. Advances in chemotherapy of cancer in man. Adv Cancer Res, 1956; 4: 1–71. Seeger, D. R., Smith, J. M., & Hultquist, M. E. Antagonist for pteroylglutamic acid. J Am Chem Soc, 1947; 69: 2567. Farber, S. The effect of ACTH in acute leukemia in childhood. In J. R. Mote, ed., First Clinical ACTH Conference (New York: Blakiston, 1950). Elion, G. B., Hitchings, G. H., & Vanderwerff, H. Antagonists of nucleic acid derivatives. VI. Purines. J Biol Chem, 1951; 192: 505–18. Burchenal, J. H., Murphy, M. L., Ellison, R. R., et al. Clinical evaluation of a new antimetabolite, 6-mercaptopurine, in treatment of leukemia and allied diseases. Blood, 1953; 8: 965–99. Fernbach, D. J., Sutow, W. W., Thurman, W. G., et al. Clinical evaluation of cyclophosphamide. A new agent for the treatment of children with acute leukemia. JAMA, 1962; 182: 30–7. Karon, M. R., Freireich, E. J., & Frei, E., III. A preliminary report on vincristine sulfate: a new active agent for the treatment of acute leukemia. Pediatrics, 1962; 30: 791–6. Gloor, W. Ein fall von geheilter myeloblastenleuk¨amie. Munch Med Wochenschr, 1930; 77: 1096–8. Burchenal, J. H. & Murphy, M. L. Long-term survivors in acute leukemia. Cancer Res, 1965; 25: 1491–4. Zuelzer, W. W. Implications of long-term survival in acute stem cell leukemia of childhood treated with composite cyclic therapy. Blood, 1964; 24: 477–94. Krivit, W., Gilchrist, G., & Beatty, E. The need for chemotherapy after prolonged complete remission in acute leukemia of childhood. J Pediatr, 1970; 76: 138–41. Skipper, H. E., Schabel, F. M., Bell, M., et al. On the curability of experimental neoplasms. I. A-methopterin and mouse leukemias. Cancer Res, 1957; 17: 717–26. Goldin, A., Venditti, J. M., Humphreys, S. R., et al. Influence of the concentration of leukemic inoculum on the effectiveness of treatment. Science, 1956; 123: 840. Frei, E., III, Holland, J. F., Schneiderman, M. A., et al. A comparative study of two regimens of combination chemotherapy in acute leukemia. Blood, 1958; 13: 1126–48.

122 Frei, E., III, Freireich, E. J., Gehan, E., et al. Studies of sequential and combination antimetabolite therapy in acute leukemia. 6-mercaptopurine and methotrexate. Blood, 1961; 18: 431–54. 123 Frei, E., III, Karon, M., Levin, R. H., et al. The effectiveness of combinations of antileukemia agents in inducing and maintaining remission in children with acute leukemia. Blood, 1965; 26: 642–56. 124 Henderson, E. S. Combination chemotherapy of acute lymphocytic leukemia of childhood. Cancer Res, 1967; 27: 2570–2. 125 Henderson, E. S. & Samaha, R. J. Evidence that drugs in multiple combinations have materially advanced the treatment of human malignancies. Cancer Res, 1969; 29: 2272–80. 126 George, P., Hernandez, K., Hustu, O., et al. A study of “total therapy” of acute leukemia in children. J Pediatr, 1968; 72: 399–408. 127 Pinkel, D. Five-year follow-up of “total therapy” of childhood lymphocytic leukemia. JAMA, 1971; 216: 648–52. 128 Simone, J. V. Treatment of children with acute lymphocytic leukemia. Adv Pediatr, 1972; 19: 13–45. 129 Pinkel, D., Hernandez, K., Borella, L., et al. Drug dosage and remission duration in childhood lymphocytic leukemia. Cancer, 1971; 27: 247–56. 130 Aur, R. J. A., Simone, J. V., Hustu, H. O., et al. A comparative study of central nervous system irradiation and intensive chemotherapy early in remission of childhood acute lymphocytic leukemia. Cancer, 1972; 29: 381–91. 131 Jacquillat, C., Weil, M., Gemon, M.-F., et al. Combination therapy in 130 patients with acute lymphoblastic leukemia (Protocol O6 LA 66-Paris). Cancer Res, 1973; 33: 3278–84. 132 Sullivan, M. P., Chen, T., Dyment, P. G., et al. Equivalence of intrathecal chemotherapy and radiotherapy as central nervous system prophylaxis in children with acute lymphatic leukemia. A Pediatric Oncology Group study. Blood, 1982; 60: 948–58. 133 Rivera, G. K., Pinkel, D., Simone, J. V., et al. Treatment of acute lymphoblastic leukemia: 30 years experience at St. Jude Children’s Research Hospital. N Engl J Med, 1993; 329: 1289– 95. 134 Miller, R. W. & McKay, F. W. Decline in US childhood cancer mortality, 1950 through 1980. JAMA, 1984; 251: 1567–70. 135 Birch, J. M., Marsden, H. B., Morris Jones, P. H., et al. Improvements in survival from childhood cancer: results of a population based survey over 30 years. BMJ, 1988; 296: 1372–6. 136 Ellison, R. R, Holland, J. F., Weil, M., et al. Arabinosyl cytosine, a useful agent in the treatment of leukemia in adults. Blood, 1968; 32: 507–23. 137 Howard, J. P., Albo, V., Newton, W. A. Cytosine arabinoside. Results of a cooperative study in acute childhood leukemia. Cancer, 1968; 21: 341–5. 138 Holton, C. P., Lonsdale, D., Nora, A. H., et al. Clinical study of daunomycin in children with acute leukemia. Cancer, 1968; 22: 1014–17. 139 Hill, J. M., Roberts, J., Loeb, E., et al. L-asparaginase therapy for leukemia and other malignant neoplasms. JAMA, 1967; 202: 882–8.

Historical perspective

140 Math´e, G., Schwarzenberg, L., Pouillart, P., et al. Two epipodophyllotoxin derivatives, VM 26 and VP 16213, in the treatment of leukemias, hematosarcomas and lymphomas. Cancer, 1974; 34: 985–92. 141 Djerassi, I., Farber, S., Abir, E., et al. Continuous infusion of methotrexate in children with acute leukemia. Cancer, 1967; 20: 233–42. 142 Lauer, S. J., Pinkel, D., Buchanan, G. R., et al. Cytosine arabinoside/cyclophosphamide pulses during continuation therapy for childhood acute lymphoblastic leukemia. Cancer, 1987; 60: 2366–71. 143 Patte, C., Thierry, P., Chantal, R., et al. High survival rate in advanced-staged B-cell lymphomas and leukemias without CNS involvement with a short intensive polychemotherapy. J Clin Oncol, 1991; 9: 123–32. 144 Gee, T. S., Yu, K.-P., & Clarkson, B. D. Treatment of adult acute leukemia with arabinosylcytosine and thioguanine. Cancer, 1969; 23: 1019–32. 145 Dahl, G. V., Kalwinsky, D. K., Mirro, J. et al. A comparison of cytokinetically based versus intensive chemotherapy for childhood acute myelogenous leukemia. Hematol Blood Transfusion, 1987; 30: 83–7. 146 Perel, Y., Aurvrignon, A., Leblanc, T., et al. Impact of addition of maintenance therapy to intensive induction and consolidation chemotherapy for childhood acute myeloblastic leukemia: results of a prospective randomized trial, LAME 89/91. J Clin Oncol, 2002; 20: 2774–82. 147 Woods, W. G., Neudorf, S., Gold, S., et al. A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission. Blood, 2001; 97: 56–62. 148 Barnes, D. W. H., & Loutit, J. F. Treatment of murine leukemia with x-rays and homologous bone marrow: II. Br J Haematol, 1957; 3: 241–52. 149 Dausset, J. Iso-leuco-anticorps. Acta Haematol, 1958; 20: 156–66. 150 Thomas, E. D., Buckner, C. D., Rudolph, R. H., et al. Allogeneic marrow grafting for hematologic malignancy using HL-A-matched donor recipient sibling pairs. Blood, 1971; 38: 267–87. 151 Pinkel, D. Bone marrow transplantation in children. J Pediatr, 1993; 122: 331–41. 152 Fefer, A., Cheever, M. A., Thomas, E. D., et al. Disappearance of Ph1 -positive cells in four patients with chronic granulocytic leukemia after chemotherapy, irradiation and marrow transplantation from an identical twin. N Engl J Med, 1979; 300: 333–7. 153 Galton, D. A. G. Myleran in chronic myeloid leukemia. Results of treatment. Lancet, 1953; 264: 208–13. 154 Fishbein, W. N., Carbone, P. P., Freireich, E. J., et al. Clinical trials of hydroxyurea in patients with cancer and leukemia. Clin Pharmacol Ther, 1965; 5: 574–80. 155 Sanders, J., Buckner, C., Thomas, E. D., et al. Allogeneic marrow transplantation for children with juvenile chronic myelogenous leukemia. Blood, 1988; 71: 1144–6.

156 Bunin, N., Casper, J., Chitambar, C., et al. Partially matched bone marrow transplantation in patients with myelodysplastic syndromes. J Clin Oncol, 1988; 6: 1851–5. 157 Appelbaum, F. R., Clift, R. A., Buckner, C. D., et al. Allogeneic marrow transplantation for acute nonlymphoblastic leukemia after first relapse. Blood, 1983; 61: 949–53. 158 Dopfer, R., Henze, G., Bender-Gotze, C., et al. Allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia in second remission after intensive primary and relapse therapy according to the BFM and Co-ALL protocols; results of the German cooperative study. Blood, 1991; 78: 2780–4. 159 Harrison, G., Richards, S., Lawson, S., et al. Comparison of allogeneic transplant versus chemotherapy for relapsed childhood acute lymphoblastic leukaemia in the MRC UKALL R1 trial. Ann Oncol, 2000; 11: 999–1006. 160 Gaynon, P. S., Harris, R. E., Trigg, M. E., et al. Chemotherapy (CT) vs. BMT for children (pts) with acute lymphoblastic leukemia (ALL) and early marrow relapse (MR): CCG-1941. Blood, 2000; 96: 418a. 161 Pui, C. H., Gaynon, P. S., Boyett, J. M., et al. Outcome of treatment in childhood acute lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region. Lancet, 2002; 359: 1909–15. 162 Pinkel, D. Treatment of children with acute myeloid leukemia. Blood, 2001; 97: 3673. 163 Giralt, S., Estey, E., Albitar, M., et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versusleukemia without myeloablative therapy. Blood, 1997; 89: 4531–6. 164 Talpaz, M., Kantarjian, H. M., & McCredie, K. Hematologic remission and cytogenetic improvement induced by human interferon alpha in chronic myelogenous leukemia. N Engl J Med, 1986; 314: 1065–9. 165 Talpaz, M., Kantarjian, H., Kurzrock, R., et al. Interferon-alpha produces sustained cytogenetic responses in chronic myelogenous leukemia. Ann Intern Med, 1991; 114: 532–8. 166 Dow, L., Raimondi, S., Culbert, S., et al. Response to alpha-interferon in children with Philadelphia chromosomepositive chronic myelocytic leukemia. Cancer, 1991; 68: 1678–84. 167 Pinkel, D. & Granoff, A., eds. Genetic Targeting in Leukemia. Accomplishments in Oncology, vol. 2 (no. 2) (Philadelphia, PA: J. B. Lippincott, 1988). 168 Huang, M. E., Ye, Y. C., Chen, S. R., et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood, 1988; 72: 567–72. 169 De Th´e, H., Lavau, C., Marchio, A., et al. The PML-RAR fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR
. Cell, 1991; 66: 675–84. 170 Fenaux, P., Wattel, E., Archimbaud, E., et al. Prolonged followup confirms that all-trans retinoic acid followed by chemotherapy reduces the risk of relapse in newly diagnosed acute promyelocytic leukemia. Blood, 1994; 84: 666–7.

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171 Druker, B. J. & Lydon, N. B. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest, 2000; 105: 3–7. 172 Mauro, M. J., O’Dwyer, M., Heinrich, M. C., Druker, B. J. STI 571: a paradigm of new agents for cancer therapeutics. J Clin Oncol, 2001; 20: 325–334. 173 Yeoh, E. J., Ross, M. E., Shurtleff, S. A., et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell, 2002; 1: 133–43. 174 Chandy, M. Childhood acute lymphoblastic leukemia in India: an approach to management in a three-tier society. Med Pediatr Oncol, 1995; 25: 197–203. 175 Kun, L. E., Camitta, B. M., Mulhern, R. K., et al. Treatment of meningeal relapse in childhood acute lymphoblastic leukemia. I. Results of craniospinal irradiation. J Clin Oncol, 1984; 2: 359–64. 176 Stoffel, T. J., Nesbit, M. E., Levitt, S. H. Extramedullary involvement of the testes in childhood leukemia. Cancer, 1975; 35: 1203–11. 177 Blundell, J. Successful case of transfusion. Lancet, 1828; 1: 431–2. 178 Landsteiner, K. Ueber agglutinationserscheinungen normalen menschlichen blutes. Wien Klin Wochenschr, 1901; 14: 1132–4. 179 Rous, P. & Turner, J. R. The preservation of living red blood cells in vitro I. Method of preservation. J Exp Med, 1916; 23: 219–37. 180 Robertson, O. H. Transfusion with preserved red blood cells. Br Med J, 1918; 1: 691–5. 181 Rous, P. & Robertson, O. H. The normal fate of erythrocytes I. The findings in healthy animals. J Exp Med, 1917; 25: 651–64. 182 Fantus, B. The therapy of the Cook County Hospital: blood preservation. JAMA, 1937; 109: 128–131. 183 Gardner, F. H., Howell, D. H., & Hirsch, E. O. Platelet transfusion utilizing plastic equipment. J Lab Clin Med, 1954; 43: 196–207. 184 McGovern, J. J. Platelet transfusions in pediatrics. New Engl J Med, 1957; 256: 922–7. 185 Rundles, R. W., Wyngarden, J. B., Hitchings, G. H., et al. Effects of the xanthine oxidase inhibitor, allopurinol, on thiopurine metabolism, hyperuricemia and gout. Trans Assoc Am Phy, 1963; 76: 126–40. 186 Pui, C.-H., Mahmond, H. H., Wiley, J. M., et al. Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma. J Clin Oncol, 2001; 19: 697–704. 187 Pinkel, D. Chickenpox and leukemia. J Pediatr, 1961; 58: 729– 37. 188 Feldman, S., Hughes, W. T., & Daniel, C. B. Varicella in children with cancer. Seventy-seven cases. Pediatrics, 1975; 56: 388–97. 189 Zaia, J. A., Levin, M. J., & Preblud, S. R., et al. Evaluation of varicella-zoster immune globulin: protection of immunosuppressed children after household exposure to varicella. J Infect Dis, 1983; 147: 737–43.

190 Biron, K. K. & Elion, G. B. In vitro susceptibility of varicellazoster virus to acyclovir. Antimicrob Agents Chemother, 1980; 18: 443–7. 191 Prober, C. G., Kirk, L. E., & Keeney, R. E. Acyclovir therapy of chickenpox in immunosuppressed children: a collaborative study. J Pediatr, 1982; 101: 622–5. 192 Johnson, H. D. & Johnson, W. W. Pneumocystis carinii pneumonia in children with cancer. Diagnosis and treatment. JAMA, 1970; 214: 1067–73. 193 Perera, D. R., Western, K. A., Johnson, H. D., et al. Pneumocystis carinii pneumonia in a hospital for children. Epidemiologic aspects. JAMA, 1970; 214: 1074–8. 194 Ivady, G. & Paldy, L. A new method of treating interstitial plasma cell pneumonia in premature infants with pentavalent antimony and aromatic diamidines. Mschr Kinderheilk, 1958; 106: 10–14. 195 Hughes, W. T., Kuhn, S., Chaudhary, S., et al. Successful chemoprophylaxis for Pneumocystis carinii pneumonitis. N Engl J Med, 1977; 297: 1419–26. 196 Frei, E., Levin, R. H., Bodey, G. P., et al. The nature and control of infections in patients with acute leukemia. Cancer Res, 1965; 25: 1511–15. 197 Bodey, G. P., Buckley, M., Sathe, Y. S., et al. Quantitative relationships between circulating leucocytes and infection in patients with acute leukemia. Ann Intern Med, 1966; 64: 328– 40. 198 Pizzo, P. A., Ladisch, S., Simon, R. M., et al. Increasing incidence of gram-positive sepsis in cancer patients. Med Pediatr Oncol, 1978; 5: 241–4. 199 Young, R. C., Bennett, J. E., Geelhoed, G. W., et al. Fungemia with compromised host resistance. Ann Intern Med, 1974; 80: 605–12. 200 Procknow, J. J. & Loosli, C. G. Treatment of the deep mycoses. AMA Arch Intern Med, 1958; 101: 765–802. 201 Galgiani, J. N. Fluconazole, a new antifungal agent. Ann Intern Med, 1990; 113: 177–9. 202 Vernick, V. & Karon, M. Who’s afraid of death on a leukemia ward? Am J Dis Child, 1965; 109: 393–7. 203 Soni, S. S., Marten, G. W., Pitner, S. E., et al. Effects of central nervous system irradiation on neuropsychologic functioning of children with acute lymphocytic leukemia. N Engl J Med, 1975; 293: 113–18. 204 Pinkel, D. Selecting treatment for children with acute lymphoblastic leukemia. J Clin Oncol, 1996; 14: 4–6. 205 Math´e, G., Amiel, J. L., Schwarzenberg, L., et al. Active immunotherapy for acute lymphoblastic leukemia. Lancet, 1969; 1: 697–9. 206 Kay, H. Treatment of acute lymphoblastic leukemia. Comparison of immunotherapy (BCG), intermittent methotrexate, and no therapy after a 5 month intensive cytotoxic regimen (Concord trial). Br Med J, 1971; 4: 189–94. 207 Heyn, R. M., Joo, P., Karon, M., et al. BCG in the treatment of acute lymphocytic leukemia. Blood, 1975; 46: 431–42.

2 Diagnosis and classification Mihaela Onciu and Ching-Hon Pui

Introduction Precise diagnosis and classification are essential to the successful treatment and biologic study of the childhood leukemias. In broadest terms, the leukemias are classified as acute versus chronic and as lymphoid versus myeloid. The terms acute and chronic originally referred to the relative durations of survival of patients with these diseases when effective therapy was not available. With improvements in treatment, they have taken on new meanings. Acute currently refers to leukemia characterized by rapid tumor cell proliferation and a predominance of blast cells, while chronic leukemia encompasses a variety of myeloproliferative and lymphoproliferative disorders in which the predominant tumor cells show variable degrees of differentiation beyond the blast stage. The vast majority of childhood leukemia cases are acute, unlike those in adults. The most common subtype, acute lymphoblastic (also termed lymphocytic or lymphoid) leukemia (ALL) accounts for 75% to 80% of all childhood cases, while acute myeloid (also termed myelocytic, myelogenous, or nonlymphoblastic) leukemia (AML) comprises approximately 20%.1 By contrast, chronic myeloid leukemia (CML) represents only approximately 2% of childhood leukemias1,2 and chronic lymphocytic leukemia (CLL) is reported only rarely in children.3–6 Finally, myelodysplastic syndrome (MDS) designates a heterogeneous group of clonal diseases related to a subset of AML.2 MDS is characterized by peripheral blood cytopenias, normocellular or hypercellular but nonproductive bone marrow (inefficient hematopoiesis), and dysmorphic maturation of hematopoietic precursors. It may evolve into frank AML or result in death due to cytopenic complications.

The modern approach to leukemia classification incorporates morphologic findings, immunophenotype, and genetic lesions, in an attempt to delineate homogeneous and clinically and biologically relevant disease categories. This chapter is an overview of the current principles and techniques used for the diagnosis and classification of the childhood leukemias as a basis for treatment assignment and biologic study. Data regarding immunophenotyping, cytogenetics, molecular genetic analysis and gene expression profiling, are introduced briefly in this context and covered in greater detail in Chapters 7, 9, 10, and 11.

Bone marrow sampling Bone marrow examination is essential to establishing the diagnosis of leukemia because as many as 20% of patients with acute leukemia lack circulating blast cells at diagnosis,7 and the morphology of leukemic cells in peripheral blood may differ from that in marrow. Marrow samples are usually obtained by aspiration, supplemented in selected circumstances by biopsy.8 The aspirated material provides cells for morphologic evaluation and biologic studies, while biopsy specimens are useful for the estimation of marrow cellularity, documentation of bone marrow fibrosis, and assessment of marrow involvement by certain types of nonHodgkin lymphoma or solid tumors. Biopsy samples can also serve as surrogates for aspirated samples when the latter are not available (e.g. due to extreme packing of marrow cells, myelofibrosis, or markedly hypocellular marrow). The site chosen for marrow aspiration depends mainly on the age and size of the patient (Fig. 2.1). Whether one performs aspiration or a biopsy, full knowledge of bone anatomy is essential to success. Faulty positioning of the

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Fig. 2.1 Common anatomic sites of bone marrow sampling, indicated by arrows (anterior iliac crest, posterior iliac crest, sternum, and anterior tibial tuberosity).

needle (e.g. failure to seat the needle on the posterior iliac crest) is the most common cause of failure. Sliding of the needle point on heavily innervated periosteum due to faulty positioning may cause severe pain, especially if analgesia is insufficient. Aspiration should be done with a needle designed specifically for that purpose; substitution with a biopsy needle should be avoided because it has a much larger dead space, leading to wasteful collection and possible clotting of the specimen. Although an 18-gauge needle and a 1- to 5-ml syringe are adequate for surveillance marrow aspiration, a larger needle (16-gauge) and syringe (20- or 50-ml) ensure better results at diagnosis, when the marrow is densely packed and when a larger sample is needed for characterization of the leukemic process. Most marrow aspirates are obtained from the posterior superior iliac crest. The anterior iliac crest can serve as an alternative site in obese children, or if multiple samples are required, for example, in newly diagnosed cases in which large samples are needed. The anteromedial surface of the tibia is occasionally sampled in infants younger than 12 months old. Rarely, the spinous processes of the most prominent vertebral segments (C7, L1, or L2) or the sternum are used in older adolescents. In some circumstances, needle placement is directed radiographically, usually to access a specific bone lesion.

Although generally a safe procedure, bone marrow aspiration can produce serious complications, such as bone penetration with damage to underlying structures. Sterile technique and sterile (preferably disposable) equipment should be used in all situations. No critical structures lie in close proximity to the posterior superior and anterior iliac crests. However, the heart and the ascending aorta are near the sternum, which is only approximately 1-cm thick, rendering sternal aspiration potentially hazardous8 ; a prudent precaution in sternal aspiration is to use a guard that limits the depth of penetration of the needle. Sternal aspiration is contraindicated in young children, and is only rarely necessary in older children and adolescents. Marrow biopsy is usually performed at the posterior superior iliac crest. Other sites may also be used, with the exception of the sternum, where biopsy is absolutely contraindicated. This procedure should be performed before marrow aspiration, or the biopsy should be redirected away from the aspiration site, to avoid hemorrhage and distortion of the biopsy core. The needle should be directed into the marrow cavity (not tangentially along the marrow cortex) by firmly seating it on the posterior superior iliac crest and aiming toward the anterior crest. The operator must take care to obtain a sufficient quantity of bone marrow for analysis; in infants and young children the needle may initially traverse epiphyseal cartilage, which is of no use for the evaluation of marrow disease. The trochar of the biopsy needle should remain in place until bone is contacted, to avoid contamination of the biopsy with skin, muscle, and connective tissue fragments. To avoid distortion of the biopsy, one should rotate the needle on its long axis as it advances to facilitate cutting rather than crushing the bone. The biopsy specimen should be gently pushed out the butt rather than the cutting edge of the needle, as the latter has a slight inward curve to trap the material. For years, the Jamshidi needle has been used for this procedure.9 A recently developed snare-coil device with an internal capturing coil may minimize postinsertion needle manipulations, and hence pain, and yields intact core specimens.10 Touch preparations should be made from all biopsy specimens, to provide air-dried material for Romanowsky and cytochemical staining in the event that aspiration attempts fail.11

Morphologic and cytochemical analysis Specimen preparation Leukemia diagnosis and classification begins with morphologic analysis of air-dried Romanowsky (Wright,

Diagnosis and classification

¨ Wright-Giemsa, or May-Grunwald-Giemsa)-stained peripheral blood and bone marrow smears and/or biopsy touch preparations. The Wright stain, used widely for analysis of peripheral blood smears, is not satisfactory for bone marrow analysis, as it stains granules poorly and does not allow adequate discrimination of immature cells. Preparation of the bone marrow biopsy material requires several steps, including fixation in formalin or a mercurial fixative (such as B5 or Zenker) and decalcification, followed by paraffin-embedding, sectioning (at 4 or 5 microns) and staining. The hematoxylin-eosin (H&E) stain allows for a general assessment of marrow cellularity, myeloid:erythroid ratio, numbers of megakaryocytes and the presence of abnormal infiltrates such as blasts, lymphoma cells, metastatic tumor or granulomatous inflammation. Additional histochemical staining can further highlight the presence of fibrosis (reticulin and trichrome stains) or the expression of chloroacetate esterase by the tumor cells (Leder stain). Immunohistochemical staining using monoclonal antibodies [e.g. antibodies specific for myeloperoxidase, lysozyme, terminal deoxynucleotidyl transferase (TdT), or CD3, CD10, or CD79a] can aid in lineage assignment when there is insufficient aspirate material to perform flow cytometric analysis. Other types of tissue preparation, such as plastic-embedded sections and electron microscopy are expensive, seldom provide information beyond simpler techniques, and hence are not widely used.

Morphologic diagnosis The morphologic diagnosis of leukemia consists essentially of two steps: establishing a diagnosis of leukemia and classifying the leukemic process according to lineage and degree of differentiation. Establishing a diagnosis of leukemia Correlation of the findings in peripheral blood and bone marrow samples is often required to establish a diagnosis of leukemia. The peripheral blood counts are variably abnormal, depending on the type of leukemia. In acute leukemias bone marrow infiltration by the leukemic process often results in anemia and thrombocytopenia, while the leukocyte counts may be decreased or variably increased with a predominance of blasts. Even in the setting of marked leukopenia, a rare circulating blast may still be encountered on the peripheral smear. However, occasional cases of acute leukemia may present with profound cytopenia and no circulating blasts. The presence of dysplastic features is typically associated with AML and MDS, and therefore such a finding makes ALL less likely.

Chronic leukemias, by contrast, are invariably associated with variable degrees of leukocytosis. In chronic myeloproliferative disorders, the leukocytosis is usually due to marked neutrophilia with associated increase in immature myeloid precursors, including myeloblasts. Depending on the subtype of disease, there may be associated monocytosis, basophilia, and eosinophilia. In chronic lymphoproliferative disorders (such as CLL or leukemic presentation of non-Hodgkin lymphoma), circulating atypical lymphocytes usually account for most of the increase in leukocyte counts. The bone marrow examination should include, at a minimum, an assessment of the myeloid-to-erythroid cell ratio, the presence and percentage of blasts, the percentage of monocytes, and the morphologic features of all cell lines. Acute leukemias are by definition characterized by variable replacement of the marrow cellularity by blasts or abnormal promyelocytes (the latter being characteristic of acute promyelocytic leukemia). The minimum percentage of marrow blasts required to establish a diagnosis of acute leukemia varies with the lineage of leukemia and the classification system applied. Hence, one must first establish the blast lineage (lymphoid versus myeloid) before proceeding with the evaluation (see details below). For ALL, the arbitrary cut-off most frequently used is 25% of marrow replacement by leukemic lymphoid blasts. If malignant lymphoblasts account for less than 25%, the disease is staged and treated as lymphoblastic lymphoma involving the bone marrow. For AML, the French-AmericanBritish (FAB) classification12,13 requires at least 30% bone marrow myeloblasts, while in the World Health Organization (WHO) classification,2,14,15 a diagnosis of AML can be established with 20% myeloblasts or more. When there are sufficient myeloblasts to establish a diagnosis of AML, further classification of the disease requires assessment of the degree of differentiation and lineage (detailed below). If myeloblasts represent less than 20% or 30% of the marrow cells, myelodysplastic syndromes and the accelerated phase of a myeloproliferative disorder (such as CML) will have to be considered in the differential diagnosis. If there is no increase in marrow blasts, and depending on the presence of hypercellularity, myeloid and megakaryocyte hyperplasia or dysplasia, a low-grade myelodysplastic syndrome or the chronic (stable) phase of a myeloproliferative disorder are more likely. As discussed above, if there is an increase in bone marrow blasts, the lineage of these cells should be established. A combination of morphologic examination and cytochemical staining is usually relatively accurate in distinguishing between lymphoid and myeloid blasts (Table 2.1). However, in a certain proportion of cases, the morphologic

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Table 2.1 Morphologic and cytochemical characteristics of blasts in the major subtypes of acute leukemia Stain/Feature Romanowsky stain Cell size Nucleus Shape/outline

Chromatin Nucleoli Cytoplasm Color Amount Granules

Auer rods Nuclear:cytoplasm (N/C) ratio Periodic acid Schiff (PAS) stain Myeloperoxidase stain Sudan Black B stain Esterase stains Alpha naphthyl butyrate (ANBE) Alpha naphthyl acetate (ANAE) Naphthol ASD chloroacetate (CAE) Acid phosphatase stain

Lymphoblasts

Myeloblasts

Monoblasts

Erythroblasts

Megakaryoblasts

Variable

Large

Large

Large

Variable

Round or indented, deeply cleaved

Round or indented

Round, indented, lobulated

Round, binucleated or multinucleated

Condensed or finely dispersed 0–2, small and inconspicuous (L1) or prominent (L2) Basophilic (deeply basophilic in L3 subtype) Scant to moderate

Finely dispersed

Finely dispersed

Round, multilobulated, binucleated or multinucleated Finely dispersed

1–4, prominent

1–3, prominent

0–5, prominent

Condensed or finely dispersed 0–3, variable size

Lightly basophilic

Lightly basophilic to blue-gray Moderate to abundant

Deeply basophilic

Basophilic

Scant

Usually absent; amphophilic granules in some cases (“granular ALL”); abundant azurophilic granules in rare cases Absent Variable, high (L1) or low (L2)

Usually present

Usually present, fine azurophilic or amphophilic

Absent

Variable, often with surface budding Usually absent

Usually present Typically low

Absent Low

Absent Low

Absent Variable

Coarse granules or blocks

Negative

Negative Usually negative; weakly positive in granular ALL

Positive Positive

Usually negative; sometimes fine or coarse granulation Positive or negative Positive or negative

Strongly positive, coarsely granular pattern Negative Negative

Negative or fine granular positivity Negative Negative

Negative; rarely weakly positive in granular ALL Negative or weakly positive

Negative

Positive

Negative

Negative

Negative or positive (not inhibited by fluoride) Positive

Diffuse positivity (inhibited by fluoride) Positive or negative

Positive (not inhibited by fluoride) Negative

Localized positivity (partially inhibited by fluoride) Negative

Negative

Negative

Negative

Localized positivity

Negative or weakly positive Positive in T-cell and in some pre-B ALL

Moderate

Diagnosis and classification

Fig. 2.2 ALL, L1 (FAB). Small blasts with indistinct nucleoli, with an admixture of some larger blasts. This spectrum of small and larger blasts is common in ALL. (Wright-Giemsa, ×1000; see color plate 2.2 for full-color reproduction.)

Fig. 2.3 ALL, L2 (FAB). Blasts with prominent nucleoli and moderate amounts of cytoplasm, with an admixture of smaller blasts. Such cases overlap morphologically with AML and emphasize the importance of ancillary studies to assign the correct lineage in acute leukemia (Wright-Giemsa, ×1000; see color plate 2.3 for full-color reproduction.)

and cytochemical findings may be ambiguous, such that immunophenotypic analysis may be required to make this distinction. The morphologic features seen on bone marrow smear examination may suggest either lymphoid or myeloid differentiation of leukemic cells, but with the exception of Auer rods in myeloblasts, none of these findings are lineagespecific. Lymphoblasts tend to be relatively small (identical to or twice the size of small lymphocytes) with scant, often light-blue cytoplasm; a round, clefted or slightly indented nucleus; fine to slightly coarse and clumped chromatin; and inconspicuous nucleoli (Figs. 2.2 to 2.4).12,16 Cytoplas-

Fig. 2.4 B-ALL (FAB ALL, L3) with the t(8;14). Blasts are characterized by intensely basophilic cytoplasm, regular nuclear features, prominent nucleoli, and cytoplasmic vacuolization. (Wright-Giemsa, ×1000; see color plate 2.4 for full-color reproduction.)

Fig. 2.5 ALL, L1 with prominent cytoplasmic vacuoles. Note the scant, lightly basophilic cytoplasm, and inconspicuous nucleoli (by comparison with Fig. 2.4). Such cases may be mistaken for B-ALL. Vacuolation is not unique to ALL, L3 (Burkitt) leukemia and other cytologic features have to be considered when making this diagnosis. (Wright-Giemsa, ×600; see color plate 2.5 for full-color reproduction.)

mic vacuoles (Fig. 2.5)17–19 and granules (Fig. 2.6)20–23 are found in lymphoblasts in some cases of ALL; such granules are usually amphophilic (fuchsia) rather than the deep purple of primary myeloid granules or the color of any of the secondary myeloid granules. In some cases, lymphoblasts have a “hand-mirror” shape,24–26 but this feature is not lineage specific. Myeloblasts have a more heterogeneous morphology, which depends on their lineage and differentiation. Generally, they tend to be larger than

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Fig. 2.6 ALL with cytoplasmic granules. Fuchsia-colored granules are present in the cytoplasm of numerous blasts. Such granules may lead to a mistaken diagnosis of AML, but the granules are negative for MPO and myeloid-pattern SBB. Immunophenotyping will confirm a diagnosis of ALL, usually of precursor B-cell lineage. Granular ALL may display granular positivity for esterase stains. (Wright-Giemsa, ×1000; see color plate 2.6 for full-color reproduction.)

Fig. 2.7 AML with minimal granulocytic differentiation (FAB M1). Blasts are large and somewhat irregular, with moderate amounts of cytoplasm but little cytoplasmic differentiation. (WrightGiemsa, ×1000; see color plate 2.7 for full-color reproduction.)

lymphoblasts, with round or indented nuclei, fine chromatin, one to several distinct nucleoli, blue to blue-gray cytoplasm, and variable numbers of cytoplasmic granules (Figs. 2.7 to 2.9). Auer rods (elongated red crystalline rods consisting of coalesced lysosomal granules) are pathognomonic of malignant myeloblasts (see Fig. 2.9).27 In the hypergranular variant of acute promyelocytic leukemia (APL), the leukemic cells are promyelocytes with abundant cytoplasmic granulation and prominent, often numerous Auer rods (Fig. 2.10). In the microgranular

Fig. 2.8 Myeloperoxidase positivity in AML, demonstrated by yellow staining against a Romanowsky-stained background. (o-Toluidine stain with dilute Giemsa counterstain, ×1000; see color plate 2.8 for full-color reproduction.)

Fig. 2.9 AML with granulocytic differentiation (FAB M2). Differentiating granulocyte precursors are admixed with myeloblasts. Several blasts contain Auer rods (arrows). (Wright-Giemsa, ×1000; see color plate 2.9 for full-color reproduction.)

variant of APL the cytoplasmic granulation is minimal or absent (Fig. 2.11). Monoblasts are large, often with a folded or indented nucleus, fine chromatin, one to three large nucleoli, and abundant blue or blue-gray, frequently vacuolated cytoplasm that may contain fine amphophilic granules (Figs. 2.12 to 2.14). Erythroblasts are large, with centrally located nuclei, sometimes binucleated or multinucleated, deeply basophilic (blue) cytoplasm, fine nuclear chromatin, and prominent nucleoli (Figs. 2.15 and 2.16).28 Megakaryoblasts are highly polymorphic, ranging from small cells with scant cytoplasm and fine or dense chromatin to large cells with abundant cytoplasm, fine chromatin, and one to several nucleoli (Fig. 2.17).29 They

Diagnosis and classification

Fig. 2.10 Hypergranular acute promyelocytic leukemia (FAB M3, sometimes designated M3h). The neoplastic cells are abnormal hypergranular promyelocytes with reddish granules and occasional clefted nuclei. Several promyelocytes contain multiple Auer rods (so-called “faggot cells”). (Wright-Giemsa, ×1000; see color plate 2.10 for full-color reproduction.)

Fig. 2.11 Microgranular acute promyelocytic leukemia (FAB M3v). The leukemic process is characterized by cells with bilobed and grooved nuclei, and sparse cytoplasmic granulation. (Wright-Giemsa, ×1000; see color plate 2.11 for full-color reproduction.)

may be bi- or multinucleated, may have cytoplasmic blebs or platelets on their surface,29,30 and may form blast cell clumps, mimicking metastatic small cell tumors.31,32 Cytochemical staining improves the accuracy and reproducibility of lineage assessment and is required for traditional AML subclassification according to the FAB and WHO criteria.2,12,14,15,33 The advent of flow cytometric analysis has rendered many of the traditional cytochemical stains obsolete. However, myeloperoxidase (MPO), Sudan Black B (SBB), and nonspecific esterase (NSE) stains,

Fig. 2.12 Acute myelomonocytic leukemia (FAB M4). The leukemic cell population includes large blasts, with irregular and reniform nuclei, promonocytes and monocytes. Esterase staining is often positive in such cases. (Wright-Giemsa, ×1000; see color plate 2.12 for full-color reproduction.)

Fig. 2.13 Acute monoblastic leukemia (FAB M5). Blasts are large and uniform, with abundant blue-gray cytoplasm, and may have cytoplasmic vacuolation and amphophilic granules. (Wright-Giemsa, ×1000; see color plate 2.13 for full-color reproduction.)

including alpha naphthyl butyrate esterase (ANB) and alpha naphthyl acetate esterase (ANA), have remained useful in this regard (Table 2.1). MPO staining detects myeloperoxidase in the peroxisomes of neutrophilic, eosinophilic, and monocytic precursors (see Fig. 2.8). Myeloblasts and some monoblasts stain positively with MPO, while lymphoblasts are uniformly negative. Auer rods are usually strongly positive and can often be identified more readily with MPO than with Romanowsky staining.34 By standard criteria of the FAB Cooperative Group, a leukemic process is considered MPO positive if reactivity is present in 3% or more of the blast

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Fig. 2.14 Alpha naphthyl butyrate esterase reactivity in acute monoblastic leukemia. ANB positivity is characterized by intense, diffuse reddish-brown cytoplasmic staining, typical of monoblastic leukemia. (ANB stain with hematoxylin counterstain, ×1000; see color plate 2.14 for full-color reproduction.)

Fig. 2.15 AML with predominant erythroid differentiation (FAB M6, also termed M6a). An infiltrate of myeloblasts is present admixed with dysplastic erythroid precursors. (Wright-Giemsa, ×1000; see color plate 2.15 for full-color reproduction.)

cells.12 This 3% threshold for MPO positivity requires careful interpretation by the reviewer, as it may be exceeded (in some cases of ALL) by staining of normal residual myeloid precursors. Several subtypes of AML (including minimally differentiated AML, megakaryoblastic leukemia and some erythroleukemias, as well as a subset of the acute monoblastic leukemia) lack MPO reactivity.35,36 MPO staining of unfixed, unstained smears must be performed without undue delay, as myeloperoxidase is unstable and may be undetectable after 7 to 10 days of slide storage. This constraint is of less importance to institutional laboratories than to reference centers. SBB is a direct nonenzymatic stain of phospholipids in the membranes of granules, principally those in myeloid

Fig. 2.16 Acute erythroblastic leukemia (also termed FAB M6b). The blasts are large with basophilic cytoplasm, resembling normal erythroblasts, and may show vacuolization. Immunophenotypic analysis confirms erythroid differentiation of the blasts. (Wright-Giemsa, ×1000; see color plate 2.16 for full-color reproduction.)

Fig. 2.17 Acute megakaryoblastic leukemia (FAB M7). The blasts have prominent surface blebs, bi- or multinucleation, and may occasionally form cohesive clusters, mimicking metastatic tumor. (Wright-Giemsa, ×1000; see color plate 2.17 for full-color reproduction.)

precursors; its reactivity parallels that of MPO but is usually more intense. By standard FAB criteria, a leukemic process is considered SBB positive if 3% or more of the blasts are positive. Rare ALL specimens are weakly positive for SBB, although this reactivity is restricted to intensive SBB staining procedures and produces gray granular staining rather than the dense black staining characteristic of myeloid granules. The SBB staining pattern in ALL lacks the Golgi zone staining seen in myeloid precursors. It is most often seen in granular ALL, where it appears to be associated to the membrane of lysosomal granules.37–39 Reactivity to SBB is stable for months in unfixed, unstained air-dried smears.

Diagnosis and classification

Esterase enzymes in monocytic precursors can be stained with either ANB or ANA as the substrate (Fig 2.14).40 Typically, the cytoplasm of monoblasts stains strongly and diffusely. Although the reaction can be completely inhibited with sodium fluoride, this step is unnecessary in most cases. Occasionally, myeloblasts stain weakly with ANA, with no inhibition by sodium fluoride. Megakaryoblasts stain negatively with ANB and typically show multifocal punctate reactivity with ANA, which is incompletely inhibited by sodium fluoride.41–45 Reactivity to these enzymes is stable for months in unfixed, unstained smears. In our experience, other stains are of limited value for the diagnosis and subclassification of leukemia. The naphtholASD-chloroacetate (CAE) or specific esterase stain identifies secondary lysosomal granules in maturing granulocytic precursors.12,46 The cytoplasmic staining pattern is diffuse granular; however, CAE is not as sensitive as MPO or SBB for identifying myeloblasts, as it stains only secondary lysosomes. Importantly, CAE remains stable in paraffin-embedded tissue and may be useful for diagnosis of granulocytic sarcoma (chloroma) in tissue sections (Leder stain).47 Smears from 1% to 2% of ALL cases may show weak granular cytoplasmic staining with CAE.48 These positive results are largely restricted to cases of granular ALL and are not apparent in tissue sections. Normal eosinophils are CAE negative, but abnormal eosinophils are positive in some subtypes of AML. The periodic acid Schiff (PAS) reaction stains glycogen in immature cells and is positive in most ALL cases, producing a fine-to-coarse granular staining pattern.49–51 It is not useful for routine classification of acute leukemia, as it produces a weak and diffuse staining pattern in one-third of AML cases49–51 and may produce intense block positivity in monoblasts and in erythroid precursors of MDS and erythroleukemia.52,53 PAS staining may also be positive in some small round cell tumors. Although normal eosinophils are PAS-negative, their leukemic counterparts may show granular PAS reactivity in some subtypes of AML. Acid phosphatase is present in all blood cell types, eliciting generally strong reactivity within the Golgi region in leukemic T lymphoblasts.54 However, some cases of B-cell precursor ALL may show an acid phosphatase staining pattern similar to that in T-cell cases, rendering the stain useless for ALL subclassification.55

Classification of acute leukemia The modern classification of leukemias requires the integration of morphologic examination, immunophenotypic (flow cytometric) analysis, cytogenetics and molecular findings. The morphologic examination has remained the

“gold standard” in establishing the diagnosis and guiding the selection of further studies. For that purpose, morphologic criteria, combined with cytochemical and immunophenotypic findings as established by the FAB Cooperative Group are still applied.12,13,29,35 However, identification of biologically significant leukemia subtypes and further risk stratification require the knowledge of immunophenotype and of cytogenetic and molecular lesions, as reflected in other leukemia classifications.14,56,57

Acute lymphoblastic leukemia Morphologic classification (the FAB system) The FAB classification system originally defined three subtypes of ALL (L1, L2, and L3),12,16 based solely on morphologic features (Figs. 2.2 to 2.5; Table 2.2). However, subsequent insights into the immunophenotype and biology of Burkitt lymphoma revealed that the L3 subtype of ALL represents the leukemic phase of this high-grade non-Hodgkin lymphoma with a mature B-cell immunophenotype. Furthermore, extensive studies have documented the importance of immunophenotypic, cytogenetic and molecular features of ALL in risk stratification and the lack of correlation between these latter findings and the L1 and L2 morphologic subtypes.58–68 Hence, the FAB classification of ALL has been largely abandoned in practice. Since the features outlined by the FAB classification are important in making the distinction between ALL and the leukemic phase of Burkitt lymphoma, which has major therapeutic implications, we have included a description of these morphologic categories in this chapter. L1-type blast cells are predominantly small (up to twice the diameter of a small lymphocyte) with homogeneous, finely dispersed-to-clumped nuclear chromatin, inconspicuous or absent nucleoli and scant deeply basophilic cytoplasm. ALL classified as L1 consists predominantly of morphologically homogeneous L1 blasts (Figs. 2.2 and 2.5). L2-type blasts are larger than twice the size of small lymphocytes, with prominent heterogeneity in blast size. The nuclear chromatin may be finely dispersed to coarsely condensed, with nuclear outline irregularities, and prominent nucleoli. The cytoplasm is usually abundant, with variable degrees of basophilia. ALL classified as L2 consists predominantly of blasts with considerable morphologic heterogeneity (Fig. 2.3).12 In practice, most ALLs show a morphologic spectrum between the L1 and L2 subtypes and therefore, even with the introduction of a scoring system,16 the distinction between these categories has remained somewhat arbitrary. It is important to note that both L1 and L2 blasts may contain cytoplasmic vacuoles in up to 30% of ALL cases, and in some cases

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Table 2.2 Classification of acute leukemias according to the revised French-American-British (FAB) criteria

FAB category

Percentage within the category

ALL L1

82

L2

15

L3

3

AML M0

2

M1

10–18

M2

27–29

M3

5–10

M4

16–25

M5

13–22

M6

1–3

M7

4–8

Diagnostic criteria

Small blasts with scanty cytoplasm, smooth-to-variably indented nuclear outline, fine-to-condensed nuclear chromatin and inconspicuous nucleoli in most blasts; often a variable percentage of larger blasts present Large and more heterogeneous blasts with moderately abundant cytoplasm, irregularly shaped nuclei, variable chromatin pattern, and prominent nucleoli; often a variable percentage of smaller blasts present Large, generally homogeneous blasts with finely stippled chromatin, round nuclei, prominent nucleoli, moderately abundant deeply basophilic cytoplasm with prominent vacuolization Large, usually agranular blasts, lacking Auer rods; negative for myeloperoxidase and Sudan Black B (SBB) by cytochemistry; expression of at least one myeloid antigen (e.g. CD13, CD33) by flow cytometry Myeloblasts with occasional Auer rods and/or cytochemical positivity for myeloperoxidase or SBB that take up ≥90% of nonerythroid cells; maturing myeloid cells <10% of nonerythroid cells; bone marrow and peripheral blood monocytic component less than that required for AML, M4 (see below) Myeloblasts with Auer rods and/or cytochemical positivity for myeloperoxidase or SBB that represent at least 30% of marrow cellularity; maturing myeloid cells represent at least 10% of nonerythroid cells; bone marrow and peripheral blood monocytic component less than that required for AML, M4 (see below) Abnormal promyelocytes, with hypergranular cytoplasm, often reniform or grooved bilobed nuclei, and Auer rods; a variable number of cells containing bundles of Auer rods (“faggot cells”) usually present (M3h). M3v variant characterized by similar nuclear morphology, but only few fine granules and infrequent Auer rods; all cells strongly positive for myeloperoxidase by cytochemistry Myeloblastic and monocytic differentiation (20–80% of the nonerythroid cells are myeloid and 20–79% of nonerythroid cells are monocytic, including monoblasts, promonocytes, and more mature monocytes); M4Eo variant associated with >5% dysplastic eosinophilic precursors in the bone marrow Predominantly monocytic differentiation (≥80% of the marrow cells are of monocytic lineage, including monoblasts, promonocytes, and more mature monocytes); M5a has predominance of monoblasts (≥80% of leukemic cells); M5b shows maturation of the monocytic precursors (<80% of the leukemic cells are monoblasts; the remainder are more mature monocytic cells) Myeloblastic leukemia with ≥50% of the marrow cells consisting of erythroid precursors, most of which show dyserythropoiesis and megaloblastoid features (M6a); or leukemia with predominantly erythroblastic differentiation (M6b) Megakaryoblastic differentiation documented by immunophenotypic analysis

these may constitute a prominent morphologic feature (Fig. 2.5).12,17–19 Therefore, the presence of cytoplasmic vacuolization is not sufficient to establish a diagnosis of L3 ALL, which should include an assessment of all the other morphologic features of the leukemic cells. Besides vacuoles, a subset of ALL blasts (5–10% of pediatric ALL) may contain amphophilic or azurophilic cytoplasmic granules (so-called granular ALL; Fig. 2.6). This feature may suggest the possibility of AML in the differential diagnosis. The blasts of granular ALL generally have lymphoid (L1) morphology and are negative for MPO. Some cases are positive for SBB or NSE, but the pattern of reactivity is generally restricted to the granules, and differs from the diffuse cytoplasmic pattern typically found in AML blasts.20–23,69 The presence of cytoplasmic granules does not appear to correlate consistently with any specific disease sub-

type, although it is usually associated with precursor B-cell ALL. The L1 and L2 morphologies may be associated with the precursor B-cell or precursor T-cell subtypes of ALL. The L3 (Burkitt subtype) is defined by large blast cells with regular oval-to-round nuclei and dense but finely stippled chromatin, one or more vesicular nucleoli, and moderately abundant intensely basophilic cytoplasm with prominent vacuolization (Fig. 2.4). These tumors have a high mitotic index (at least 5%). The L3 ALL has a mature B-cell immunophenotype, is associated with a set of translocations that dysregulate the MYC locus at the 8q24 chromosomal region, and possesses essentially the same biologic traits and chemosensitivity as Burkitt lymphoma.70 This leukemia comprises only 2% of pediatric ALL cases overall, with the L1 and L2 subtypes accounting for the remainder.

Diagnosis and classification

Immunophenotypic classification Analysis of the immunologic characteristics of leukemic blast cells was introduced in the mid-1970s, when researchers noted that ALL cases with immunologic features of T-cell precursors or mature B cells had exceptionally poor responses to treatment.59,66 Immunologic analysis has remained useful for the assignment of treatment and comparison of blast cell biological features. The current WHO classification14,15 recognizes two major immunophenotypic subtypes of ALL, designated as precursor B-cell and precursor T-cell lymphoblastic leukemia/lymphoma, respectively. This designation also recognizes the biologic continuum between lymphoblastic tumors involving predominantly extramedullary sites and those with prominent bone marrow and blood involvement. For practical purposes, as previously mentioned, ALL is distinguished from lymphoblastic lymphoma by an arbitrary cut-off of at least 25% bone marrow involvement. The B-lymphoblastic immunophenotype is typically characterized by the expression of Tdt, CD34, and HLA-DR, as well as the B-cell antigens CD19, and cytoplasmic CD79a. Most precursor B-cell ALLs are also positive for CD10 and other B-cell antigens including CD22, CD24, and CD20 (the latter with variable intensity). Notably, some precursor B-cell lymphoblastic leukemias may be negative for CD45 (leukocyte common antigen). As opposed to mature B-cell tumors (such as Burkitt lymphoma), immunoglobulin (Ig) heavy-chain (mu) expression, if present, is generally restricted to the cytoplasm, and there is no Ig light-chain expression. Even when weak Ig mu expression is present on the blast surface, there is no associated light-chain expression or restriction. By contrast, mature B-cell leukemias lack expression of Tdt and CD34, show bright surface CD20 and express light chain-restricted Ig molecules on the surface of the leukemic cells. T-cell lymphoblastic leukemias are typically positive for Tdt, CD34, cytoplasmic CD3, surface CD7 and show variable expression of other T-cell-associated antigens such as CD2, CD4, CD5, CD8, and CD1a. Of note, it is not unusual for cases of T-ALL to lack Tdt or CD34 expression, so that such findings should not dissuade one from a diagnosis of T-cell lymphoblastic disease. Although ALLs can be further subclassified according to stage of maturation,62,71–74 the optimal immunologic subclassification of ALL remains controversial (see Chapter 7 for details). Many ALLs aberrantly express myeloid-associated antigens, most commonly CD13 and CD33. The expression of these antigens does not seem to have any impact on treatment outcome.75–81 Molecular (genetic) classification Genetic alterations, as detected by conventional cytogenetics and/or molecular analysis, have been shown

to identify biologically and clinically significant disease subtypes in ALL.82–85 The karyotypes of leukemic cells not only have diagnostic and prognostic importance, but also indicate sites of molecular lesions potentially involved in cell transformation and proliferation.58,60 The morphologic, immunologic, and cytogenetic (MIC) classification was the first attempt to integrate the morphology, immunophenotype and cytogenetic features of acute leukemia and myelodysplasia into a single framework of disease classification.56,57,86 The current WHO classification14,15 has likewise integrated the current knowledge about these features, with the addition of molecular lesions associated with different subtypes of the disease. However, recurrent cytogenetic/molecular abnormalities can be identified in only 60% to 80% of ALL cases with current methods,14,83,87 so that the remaining cases must be classified solely according to their lineage. ALL can be broadly classified according to the modal chromosomal number per leukemic cell (ploidy). Several different ploidy groups have prognostic and therapeutic relevance: hypodiploid (<46 chromosomes), diploid (46 chromosomes), pseudodiploid (46 chromosomes with numeric or structural abnormalities), hyperdiploid with 47 to 50 chromosomes, hyperdiploid with 51 to 65 chromosomes, and triploid/tetraploid with more than 65 chromosomes.67,68,88–94 Assessment of the DNA content of leukemic cells by flow cytometry (DNA index) is a useful adjunct to cytogenetic ploidy analysis because it is automated, rapid,95,96 and sometimes detects near-haploid or tetraploid leukemia lines that were missed by routine karyotyping.90,91 Of these groups, high-hyperdiploid ALL (51–65 chromosomes, DNA index 1.16–1.6) appears to represent a biologically distinct type of ALL, with low leukemic cell counts at diagnosis and good response to therapy. These features are possibly related to an increased propensity of the leukemic blasts to undergo apoptosis, to accumulate higher concentrations of methotrexate polyglutamates, and to a higher sensitivity to methotrexate and mercaptopurine in vitro.83,97 Conversely, hypodiploidy (<45 chromosomes) appears to define a group of ALL with distinctly poor prognosis and is thus a high-risk feature in most risk classification schemas.83,88,91,94,98 Several recurring chromosomal translocations associated with distinct molecular lesions appear to define biologically and clinically distinct subgroups of precursor B-cell ALL. The value of cytogenetic analysis alone in identification of these subgroups is somewhat limited. Often the presence of the corresponding gene rearrangements can only be detected by molecular methods, such as RT-PCR or Southern blotting, as the balanced translocations may be too subtle to be detected by routine cytogenetic analysis.99,100 Also, identical chromosomal translocations may be associated with

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different genetic lesions at the molecular level [e.g. the t(1;19)101–104 and 11q23 abnormalities with or without MLL gene rearrangements].105,106 Because many of these molecular abnormalities have a major prognostic impact, routine molecular analysis with panels of probes is the ideal approach to precise risk stratification in pediatric ALL. ALL with the t(12;21)(p13;q22); TEL/AML1 (or ETV6/CBFA2) fusion transcript is the most common cytogenetic subtype of pediatric ALL (22–25%).99,100,107,108 However, the translocation is too subtle to be detected by conventional cytogenetics in most cases and more sensitive methods such as fluorescence in situ hybridization (FISH) or molecular methods (RT-PCR) should be employed to search for this gene rearrangement in childhood ALL.100,109 ALL with the TEL/AML1 fusion transcript generally arises in patients who are 1 to 10 years of age and is characterized by a precursor B-cell immunophenotype with frequent expression of CD10 and myeloid antigens (such as CD13 and CD33).99,100,110,111 In some clinical trials, this genetic feature was associated with a favorable prognosis,107 a finding that has been attributed to the high sensitivity of these leukemic cells to asparaginase in vitro.112 ALL with the t(1;19)(q23;p13) E2A/PBX1 fusion transcript is frequently associated with a pre-B (cytoplasmic Ig mupositive) immunophenotype, although in some cases the blasts may lack Ig mu expression.113,114 These leukemias represent 3 to 6% of all childhood ALL cases83,87 and 25% of all pre-B ALL cases.113 The adverse prognostic impact of this translocation has been largely abolished by more effective chemotherapy treatments,83 however, in many current clinical trials, these cases continue to be assigned to more intensive chemotherapy. ALL with the t(9;22)(q34;q11.2) BCR/ABL fusion transcript (Philadelphia chromosome-positive ALL) represents 2 to 3% of all childhood ALL cases.79,87,115 Philadelphia-positive ALL generally develops in patients with an older age (>10 years) and is associated with high initial leukocyte counts, a high incidence of CNS leukemia, L1 blast morphology, CD10-positive precursor B-cell immunophenotype (so-called common ALL) and a poor prognosis.79,115 In rare cases, the blasts may have abundant cytoplasm containing prominent coarse azurophilic granules.14,79 A T-cell immunophenotype has been reported in occasional cases.116,117 Allogeneic hematopoietic stem cell transplantation with cells from matched-related donor has improved the outcome of this type of ALL.115 ALL with 11q23 abnormalities. Among MLL gene rearrangements the t(4;11)(q21;q23) with the AF4/MLL fusion transcript is the most common.118–121 This cytogenetic lesion is encountered in 2 to 3% of all pediatric ALLs

and 50 to 80% of ALL cases in infants less than 1 year of age.118,120–122 These leukemias are characterized by high leukocyte counts at presentation, bulky extramedullary disease, frequent CNS involvement and a poor prognosis.119,121 Immunophenotypically, these tumors are usually CD10 negative, cytoplasmic Ig mu negative, surface CD22 negative, CD15 and/or CD65 positive, and positive for the human homologue of the rat 220-kDa chondroitin sulfate proteoglycan NG2, which can be detected with the monoclonal antibody 7.1.118 NG2 is also expressed in acute myeloid leukemias with MLL gene abnormalities.123,124 In addition, infant ALLs with the t(4;11) appear to be preferentially sensitive to cytarabine,125 possibly because of increased expression of cytarabinemetabolizing enzymes.126 Chromosomal abnormalities in T-cell ALL are of less importance in risk stratification than has been the case in precursor B-cell ALL.127 The most common nonrandom genetic defect in childhood T-cell ALL is alteration of the TAL1 locus on chromosome 1p32 (25%). Based on RT-PCR analysis, a substantial proportion of T-ALL cases can be classified into distinct subgroups, according to overexpression of HOX11, HOX11L2, TAL1, LYL1 and of the MLL-ENL fusion transcript (see also Chapter 10).128,129 In fact, one study showed that virtually all T-ALL cases could be clustered into these categories based on their gene expression profiles.129 If the clinical and biologic relevance of these genetic subtypes is confirmed by additional studies, many of T-cell ALL cases could be classified by this system in the future.

Acute myeloid leukemia AML is subclassified according to lineage commitment along the granulocytic, monocytic, erythroid or megakaryocytic lineage. The predominantly granulocytic leukemias are further arbitrarily subdivided according to their degree of maturation. Classification into these categories generally requires integration of morphologic and cytochemical assessment with immunophenotypic data. These criteria underlie the first widely used classification of AML introduced by the FAB cooperative group in 1976 and later modified in 1985 and 1991.12,13,29,35 It later became obvious that in addition to these classification criteria, cytogenetic and molecular abnormalities define distinct clinical and biological entities with prognostic and therapeutic relevance. In addition, the wide-scale use of flow cytometric analysis uncovered entities such as acute biphenotypic and bilineal leukemias that were difficult to place in any of the FAB categories. As a result, newer classifications have attempted to integrate the morphologic,

Diagnosis and classification

Table 2.3 Classification of acute leukemia according to the World Health Organization (WHO) criteria Acute myeloid leukemia (AML) AML with recurrent cytogenetic abnormalities AML with t(8;21)(q22;q22), (AML 1/ETO) AML with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13;q22), (CBFβ/MYH11) Acute promyelocytic leukemia with t(15;17)(q22;q12), (PML/RAR
) and variants AML with 11q23 (MLL) abnormalities AML with multilineage dysplasia Acute ML and myelodysplastic syndrome, therapy related Alkylating agent/radiation-related type Topoisomerase II inhibitor-related type AML not otherwise categorized AML, minimally differentiated AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/acute monocytic leukemia Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia) Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Acute leukemias of ambiguous lineage Bilineal acute leukemia Biphenotypic acute leukemia Undifferentiated acute leukemia Precursor-B and T-cell neoplasms Precursor-B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) Precursor-T-lymphoblastic leukemia/lymphoma (precursor T-cell acute lymphoblastic leukemia)

immunophenotypic, cytogenetic and molecular features. Such classification systems include the morphologic, immunologic and cytogenetic (MIC) working classification of the acute myeloid leukemias57,86 and the most recent WHO classification of hematopoietic neoplasms (see Table 2.3).14,15 The latter classification defined separate entities based on recurrent cytogenetic lesions, concurrent or pre-existing multilineage dysplasia and preceding chemotherapy, but also retained the FAB categories for those cases that lacked any of these features. However, it is not yet clear if some of the numeric cut-off values established by the WHO classification are associated with the same clinical categories as those traditionally defined in the FAB system. As a result, both the FAB and WHO classification systems are currently in use and will be described in detail below.

As previously mentioned, certain numeric cut-off values are used to define AML and differentiate it from other disease processes with increased numbers of myeloblasts, including myelodysplastic syndromes and myeloproliferative disorders. According to the revised FAB criteria,13 the following diagnostic algorithm should be used to make these distinctions: 1. Differential counts are performed on the bone marrow aspirate and peripheral blood. 2. If erythroid precursors represent less than 50% of all the nucleated bone marrow cells, the blasts should be assessed as a percentage of all nucleated marrow cells. If blasts represent 30% or more of the marrow cells or abnormal promyelocytes (of AML M3) are present, then a diagnosis of acute leukemia (AML M0 to M5, M7) can be established; if blasts represent less than 30%, the diagnosis is MDS. 3. If erythroid precursors represent 50% or more of the marrow cells, then blasts should be assessed as a percentage of all nonerythroid cells. If blasts represent 30% or more, then a diagnosis of AML M6 can be made; if blasts represent less than 30%, the diagnosis is MDS. In the WHO classification,14 a similar algorithm is followed, with a cut-off of 20% instead of the 30% required by the FAB criteria. Of note, when following such an algorithm, it is important to be aware of the recent clinical history, as administration of erythropoietin for refractory anemia may be associated with a marked increase in the percentage of erythroid precursors,130 potentially leading to a diagnosis of AML M6 in a process that may otherwise be classified as MDS. Further subclassification relies on the relative proportions of blasts, maturing granulocytes and monocytic cells in the bone marrow. Acute myeloid leukemia with minimal evidence of myeloid differentiation (AML M0) is characterized by the presence of at least 30% bone marrow blasts that do not show evidence of myeloid differentiation by morphology and cytochemistry, but are demonstrated to be myeloblasts by other methods, including immunophenotyping or electron microscopy (ultrastructural cytochemistry).35 Surface antigens typically associated with myeloid differentiation include CD11b, CD13, CD15, CD33, and CD117. In addition, a significant proportion of cases may express the T-cell-associated antigen CD7.131 In some cases, cytoplasmic expression of myeloperoxidase may be detected by flow cytometry. AML M0 is the least frequent subtype of AML. In larger series132 this subtype of leukemia appears to be associated with a higher incidence of AML1 gene mutations than are the other subtypes of AML. Also, unfavorable cytogenetic abnormalities [such as monosomy 7, inv(3q),

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del(9q)] occur more often in this subtype of AML than in all other AML subtypes (50% versus 18%, respectively).133 Acute myeloid leukemia without maturation (AML M1) (Fig. 2.7) is defined by the presence of myeloid blasts that represent 90% or more of the nonerythroid marrow cells. Blasts show myeloid differentiation on morphologic examination (i.e. presence of Auer rods) and/or by cytochemistry (i.e. positivity for myeloperoxidase or Sudan Black B in at least 3% of the blasts) (Fig. 2.8) and are negative for nonspecific esterase activity and monocytic antigens. Acute myeloid leukemia with maturation (AML M2) (Fig. 2.9) can be diagnosed when myeloblasts (identifiable as such by morphology and/or cytochemistry) represent 30% or more of the bone marrow cells and the maturing bone marrow granulocytic component (including promyelocytes to mature granulocytes) represents more than 10% of the nonerythroid cells. The bone marrow monocytic component should be less than that required for a diagnosis of acute myelomonocytic leukemia (see below). Associated morphologic features often include variable degrees of dysplasia in the maturing granulocytic elements, including hypogranular or hypergranular myelocytes, metamyelocytes and segmented granulocytes, nuclear-to-cytoplasm maturation asynchrony and pseudo-Pelger-Hu¨et changes in segmented granulocytes. Variable degrees of dysplasia may also be present in the erythroid and megakaryocytic precursors. In the presence of marked associated trilineage dysplasia, a leukemia is best classified as AML with multilineage dysplasia.133 Acute promyelocytic leukemia (AML M3; APL), AML with the t(15;17)(q22;q12) and PML/RARα or variant translocations is associated with a unique morphologic spectrum. The predominant leukemic cells are neoplastic promyelocytes and not myeloblasts. In the classic hypergranular subtype of acute promyelocytic leukemia (M3h) (Fig. 2.10), the predominant malignant cell population comprises hypergranular promyelocytes with grooved or bilobed nuclei, abundant cytoplasmic granules, often of a reddish color, and prominent Auer rods. In most cases, variable numbers of cells containing multiple Auer rods (so-called “faggot cells”) are present. In the microgranular variant of the M3 subtype (M3v) (Fig. 2.11), basophilic granulation is minimal or absent in the leukemic cells, which frequently have bi- or multilobed nuclei with characteristic nuclear grooving (often raising the differential diagnosis of leukemias of monocytic lineage). Granules are sometimes associated with the nuclear groove, and Auer rods or faggot cells may be present in M3v blast populations.134 Distinguishing M3v from the M3h variant is sometimes difficult when there is a spectrum of morphologic features. The features of M3v are often more prominent in circu-

lating blood than in bone marrow, and M3v cases tend to have higher peripheral leukocyte count than do M3h cases.135,136 Both variants stain positively with MPO or SBB, with virtually 100% of the cells being positive.135,136 The characteristic immunophenotype of APL reflects the promyelocytic differentiation of the leukemic cells, which often express myeloid antigens, including CD33 and CD13, and usually lack expression of CD34 and HLA-DR (the latter typically expressed by myeloblasts). A subset of APL cases aberrantly express the lymphoid-associated antigens CD2 and CD19, the former being more frequent in cases with M3v morphology.137–139 Immunohistochemical staining with anti-PML monoclonal antibodies can be used to demonstrate the PML/RARα translocation, which is associated with a characteristic shift in the pattern of distribution of the nuclear PML-containing granular complexes.140 Rare cases of APL lack the t(15;17) and contain variant translocations that disrupt the RAR
locus, leading to different chimeric transcripts [e.g. t(11;17)(q23;q21); PLZF/RARα141 ]. Some of these cases have atypical morphologic features, ranging from those of AML M2 to M3. Cases with t(11;17) are frequently characterized by leukemic cells that have round nuclei with regular borders and moderate amounts of variably granular cytoplasm.142,143 Acute myelomonocytic leukemia (AML M4) shows a mixture of granulocytic and monocytic features (Fig. 2.12), with cytochemical evidence of myeloid and monocytic differentiation and a monocytic component (monoblasts to mature monocytes) of 20% to 79% in the bone marrow. In many of these cases, cell populations with overlapping features are positive for both MPO and NSE. In the M4Eo variant, abnormal eosinophilic precursors account for 5% or more of nonerythroid marrow cells. Acute monoblastic/monocytic leukemia (AML M5) (Figs. 2.13 and 2.14) shows 80% or more of nonerythroid marrow cells to be monoblasts, promonocytes, or monocytes.12 These cases may be further divided by the degree of monocytic differentiation, with M5a denoting cases with a predominance of monoblasts, and M5b cases with significant maturation to monocytes. Antigens commonly associated with monocytic differentiation include CD15, CD36, CD64, CD65 and, for more mature monocytes, CD14.14,144 Acute myeloid leukemia with predominant erythroid differentiation (AML M6) (Fig. 2.15), sometimes designated as erythroleukemia or M6a, is diagnosed when more than 50% of the marrow nucleated cells are erythroid precursors, and more than 30% of the nonerythroid cells are myeloblasts that show myeloperoxidase positivity and may contain Auer rods.13 Therefore, in these cases the blastic component consists of myeloblasts, while the erythroid component consists of dysplastic erythroid precursors with

Diagnosis and classification

maturation. Both cell populations have been shown to derive from the same malignant clone.145 In rare instances, erythroleukemia consists predominantly of a proliferation of erythroblasts; this variant is sometimes referred to as M6b (Fig. 2.16).28 Acute megakaryoblastic leukemia (AML M7) is characterized by bone marrow infiltration by megakaryoblasts (Fig. 2.17). In addition to the morphologic and cytochemical characteristics described earlier, the megakaryoblastic lineage should be documented by the expression by blasts of at least one platelet-associated antigen, including CD41 (GPIIb/IIIa), CD42b (GPIb), CD61, or platelet peroxidase (the latter necessitating demonstration by electron microscopy).29,30,146 These leukemias are often associated with abnormal and dysplastic megakaryocytes. Extensive myelofibrosis is frequent in this AML subtype; in such cases, marrow aspiration frequently yields a dry tap and the aspirate smears may contain less than 30% blasts, requiring examination of a bone marrow biopsy sample for diagnosis. AML M7 is heterogeneous with respect to cytogenetic abnormalities, with certain cytogenetic subtypes [e.g. t(1;22), OTT-MAL] being strongly associated with the pediatric age group.147 This subtype of leukemia is associated with a poor prognosis.148 Acute myeloid leukemia with the t(8;21)(q22;q22); AML1/ETO fusion transcript. The presence of this genetic abnormality is generally associated with a favorable outcome and relatively frequent extramedullary disease (granulocytic sarcoma or chloroma).149–153 Morphologically, it most often corresponds to the AML M2 FAB subtype, with only rare cases showing either AML M1 or AML M4 features. A characteristic combination of morphologic features may help in predicting this genetic subtype (Fig. 2.18).154–157 The blasts are often large, with a low nuclear-to-cytoplasm ratio and contain single, long, needle-like Auer rods. They commonly have a single nuclear indentation or cleft, significant cytoplasmic basophilia, and a well-circumscribed clear zone abutting the nuclear indentation. In most cases, the maturing granulocytic component shows marked dysplastic features, including a lack of chromatin condensation, pseudo Pelger-Huet anomaly, a diffuse and homogeneous “salmon-pink” hue to the cytoplasm, and in 40 to 50% of the cases, giant Chediak-Higashi-like cytoplasmic granules that may be present in all stages of maturation, from blasts to mature granulocytes. Approximately one-third of the cases are associated with marrow eosinophilia, with morphologically normal or dysplastic eosinophils. Auer rods may be present in mature neutrophils and eosinophils. The blasts of this type of leukemia also have characteristic immunophenotypic features that include

Fig. 2.18 AML with t(8;21). Blasts are large, with basophilic cytoplasm and single needle-shaped Auer rods. Many dysplastic granulocyte precursors are present, some showing diffuse salmon-pink cytoplasmic staining. (Wright-Giemsa, ×600; see color plate 2.18 for full-color reproduction.)

Fig. 2.19 AML with inv(16) (FAB AML M4Eo). Acute myelomonocytic leukemia with numerous dysplastic eosinophils that contain coarse basophilic cytoplasmic granules. (Wright-Giemsa, ×600; see color plate 2.19 for full-color reproduction.)

expression of the lymphoid-associated antigens CD19 and CD56.152,155,158,159 Acute myeloid leukemia with the inv(16)(p13 q22); CBFB/MYH11 fusion transcript. This genetic subtype is associated with the AML M4Eo morphology (myelomonocytic leukemia with bone marrow eosinophilia, the latter consisting of abnormal eosinophils) (Fig. 2.19).160–162 The degree of bone marrow eosinophilia ranges from 1% to 35%, exceeding 4% in most cases. Despite bone marrow eosinophilia, these cases typically lack eosinophilia and

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atypical eosinophils in the peripheral blood. The atypical eosinophils include many immature eosinophilic forms that contain a mixture of eosinophilic and basophilic cytoplasmic granules. The basophilic granules are generally larger and more numerous than those seen in normal immature eosinophils and are also present in eosinophilic bands and segmented forms. Ultrastructurally, the basophilic granules correspond to immatureappearing granules that lack the well-formed central crystalloids seen in the mature eosinophilic granules. Studies combining FISH with morphologic examination have shown that the abnormal eosinophils contain the inv(16) and are therefore a part of the leukemic clone.163 The leukemic cell population also includes “hybrid cells” which have nuclei similar to those of the immature monocytic cells and contain eosinophilic granules. Cytochemically, in up to a third of cases the nonspecific esterase stain is negative or only weakly positive in otherwise monocytic-appearing cells. The abnormal eosinophils show aberrant granule-associated positivity for PAS and CAE. Immunophenotypically, AML M4Eo frequently shows expression of the lymphoid antigen CD2, which may be present on the blasts as well as on the more mature monocytic cells.164 This type of leukemia is associated with a favorable prognosis133,161,162,165–167 that may be due to increased sensitivity of the leukemic cells to cytarabine.168 Acute myeloid leukemia with 11q23 abnormality; MLL gene rearrangements. The AMLs containing these cytogenetic/molecular abnormalities are morphologically more heterogenous than other genetic subtypes and have no distinctive morphologic or immunophenotypic features. Most of these leukemias show monocytic differentiation (AML M4/M5 in the FAB classification), although other subtypes, including AML M0, M1, M2, and M7, may also be encountered.169,170 Despite this heterogeneity, AML harboring MLL gene alterations are considered as a distinct subtype due to their intermediate or poor outcome in some studies.133,150,151,170 In those studies, the outcome differed according to the partner gene involved in the 11q23 translocation, with the t(9;11)(p21;q23) conferring the most favorable outcome.150,170 MLL gene abnormalities may be encountered in cases of de novo AML, as well as in secondary AML related to therapy with topoisomerase II inhibitors (see below). This distinction has both biologic and clinical importance. While both types of leukemia may contain similar chromosomal translocations, de novo leukemias containing the t(9;11) appear to be very sensitive to treatment with epipodophyllotoxins, while secondary AML bearing the same translocation has an extremely poor response to treatment.170,171 Acute myeloid leukemia with associated multilineage dysplasia. This WHO-defined category is reserved for

leukemias preceded by a well-documented myelodysplastic syndrome and/or associated with dysplastic morphologic features in the maturing myeloid, erythroid and megakaryocytic precursors at presentation. As mild dysplastic or dyspoietic features may be present in many leukemic processes, the WHO criteria specify that dysplasia must be present in at least 50% of the cells of at least two cell lines in order for one to establish this diagnosis14 (see section on myelodysplastic syndromes for a detailed description of morphologic features of dysplasia). Morphologically, the marrow is characterized by the presence of myeloblasts (at least 20% in the WHO classification) that generally represent only a subpopulation of the bone marrow cells. Imunophenotypically, the myeloblasts associated with myelodysplasia frequently express lymphoid-associated antigens, such as CD7 and CD56. Cytogenetic abnormalities most commonly associated with these leukemias are similar to those described in myelodysplastic syndromes. Therapy-related AML (t-AML) can occur after treatment with topoisomerase II inhibitors, alkylating agents or irradiation.14,171–178 These AMLs overlap morphologically with de novo AML and MDS. t-AML related to topoisomerase II inhibitors (such as epipodophyllotoxins, anthracyclin, mitoxantrone, dactinomycin) is characterized by a short latency period (12–130 months, median 33–34 months), lacks a preceding myelodysplastic phase, and has frequent involvement of the MLL gene and monocytic differentiation (M4 or M5 morphology). t-AML related to alkylating agents (e.g. melphalan, chlorambucil, cyclophosphamide, procarbazine) and ionizing radiation has a longer latency (10–192 months, median 5–6 years), is usually preceded by a smoldering myelodysplastic syndrome, is frequently associated with unbalanced translocations or deletions involving chromosome 5 and/or chromosome 7, and generally has the morphologic appearance of AML with multilineage dysplasia.179 A third category of t-AML consists of t-AML with recurring cytogenetic abnormalities generally associated with a favorable prognosis. These include acute promyelocytic leukemia with the t(15;17), AML with the t(8;21), AML with inv(16) and the t(8;16). They may occur after therapy with alkylating agents, topoisomerase II inhibitors, or ionizing radiation alone, have a short latency (7–120 months, median 9–33), are rarely preceded by a preleukemic MDS, and have the morphologic features usually associated with their respective chromosomal abnormalities. It appears that these leukemias have the same favorable prognosis as that of the de novo disease, suggesting that their separate classification may not be warranted.180–183 Acute leukemias of ambiguous lineage (mixed lineage acute leukemias or hybrid acute leukemias) are rare

Diagnosis and classification

leukemias that lack sufficient morphologic, cytochemical or immunophenotypic differentiation to be classified as either lymphoid or myeloid.14,184 In most cases, these poorly differentiated leukemias show a morphologic and/or immunophenotypic mixture of lymphoid and myeloid features. In the WHO classification, three types of acute leukemia are grouped within this category (Table 2.3): acute biphenotypic leukemia, bilineal acute leukemia and undifferentiated acute leukemia. Biphenotypic leukemias are defined as leukemias that contain a morphologically homogeneous population of poorly differentiated blasts that uniformly express a mixture of lymphoid (B- and/or T-) and myeloid-associated antigens. As expression of “lineage-inappropriate” antigens is frequently encountered as part of the aberrant immunophenotype of both acute lymphoblastic and myeloid leukemias and has no prognostic significance, it is important to restrict this diagnosis to leukemias that express multiple antigens associated with each lineage. For this purpose, different scoring systems have been designed based on the number and degree of specificity of the markers expressed by the leukemic cells.71 Of note, a predominantly myeloid antigen expression profile in these leukemias has recently been reported to correlate with a less favorable outcome and to require more intensive therapy than a predominantly lymphoid phenotype.185 A significant proportion of the acute biphenotypic leukemias contain the Philadelphia chromosome or abnormalities of chromosome 11q23, suggesting that they arise from an early hematopoietic precursor cell.185–187 A clinically important subtype within this category are leukemias that coexpress myeloid-associated antigens and the T-lineage-associated antigens CD2, CD7, and cytoplasmic CD3.185,188 Morphologically, these leukemias show dual populations of myeloblasts expressing lowlevels of MPO and small, lymphoid-appearing blasts with a hand-mirror appearance (Fig. 2.20). However, by flow cytometry, the above-mentioned antigens are uniformly expressed by all blasts. These cases respond well to therapy directed to both lymphoid and myeloid leukemias. Bilineal acute leukemias are defined as leukemic processes in which two morphologically and immunophenotypically distinct (lymphoid and myeloid) blast populations can be identified (Fig. 2.21). Most commonly, the two populations consist of a precursor B-cell lymphoid population and a monoblastic population. Frequently, these leukemias contain the t(4;11)(q21;q23), and the lymphoid population shows immunophenotypic features similar to ALL with this chromosomal abnormality, including lack of CD10 expression.14 In undifferentiated acute leukemias, the blasts lack expression of lineage-specific markers and express mainly nonspecific early progenitor cell-associated antigens such as CD34, CD38, HLA-DR, and, more rarely

Fig. 2.20 Dimorphic T/myeloid acute biphenotypic leukemia. A dual leukemic cell population is present, including small blasts with scant cytoplasm, indistinct nucleoli, ‘hand-mirror’ shape, and a small number of larger blasts with apparent myeloid differentiation. Immunophenotypic analysis shows that the blasts uniformly express T-cell-associated (CD2, CD3, CD7) and myeloid-associated (CD13, CD33, MPO) antigens. (WrightGiemsa, ×1000; see color plate 2.20 for full-color reproduction.)

Fig. 2.21 Bilineal acute leukemia. This leukemic process consists of two morphologically and immunophenotypically distinct blast populations: a lymphoid population resembling ALL, L1 and a monoblastic population resembling AML, M4/M5. (Wright-Giemsa, ×600; see color plate 2.21 for full-color reproduction.)

TdT and CD7.14 Finally, there are acute leukemias that show lineage switch during the early phase of chemotherapy and are also classified as mixed-lineage leukemias.14,185,189,190 Myeloid sarcoma (also termed granulocytic sarcoma, chloroma, or extramedullary myeloid tumor) consists of myeloblasts or immature myeloid cells involving an extramedullary site. These tumors represent the tissue counterpart of AML and should be treated as such even in the absence of a leukemic component.191 Myeloid sarcoma may accompany AML at the time of initial

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diagnosis or relapse [with a higher frequency in AML with the t(8;21), inv(16), or an 11q23/MLL rearrangement], with or without bone marrow involvement.47,152,191–195 Other disorders associated with this type of tumor include myelodysplastic syndromes and myeloproliferative disorders. Morphologically similar to the subtypes of AML, myeloid sarcomas may consist predominantly of myeloblasts with a variable component of more mature granulocytic precursors, or may consist predominantly of monocytic cells (so-called monoblastic sarcoma).194–196 In tumors associated with inv(16), immature eosinophilic cells may be present in the leukemic infiltrate. The lineage assignment of these tumors is frequently based on immunohistochemical studies. These tumors are consistently positive for CD45 (leukocyte common antigen), CD43, MPO and/or lysozyme, and CD68 and are negative for B-cell and T-cell associated antigens. The tumors that have a significant maturing granulocytic component often express CD15 (Leu M1) and show CAE activity by histochemical staining (Leder stain).47,197–199 When touch imprints are available, cytochemical positivity for MPO and the presence of Auer rods are diagnostic for this entity.

Myelodysplastic syndromes Myelodysplastic syndromes are clonal hematopoietic disorders characterized by peripheral blood cytopenias with marrow hypercellularity (inefficient hematopoiesis) and morphologic dysplasia. A subset of MDS cases show an increase in bone marrow and peripheral blood myeloblasts. In some cases, these myeloblasts may contain Auer rods. The blast percentage, presence of Auer rods, and type and extent of associated dysplasia are the features commonly used for the morphologic classification of MDS according to the FAB criteria.200 In the WHO classification,14 Auer rods are not used for assigning MDS subcategories, as clinical studies have indicated that they might not have the poor prognosis initially attributed by the FAB system.201,202 While these classification systems can be used to predict outcome in adults, their predictive value in the pediatric population remains controversial. Multiple studies have shown that 25% to 50% of MDS cases in children cannot be classified according to these widely accepted criteria.2,203–207 Many of these difficult-to-classify cases are associated with genetic disorders such as mitochondrial disorders, chromosome instability syndromes, Schwachman-Diamond syndrome, and Down syndrome. In addition, some of the MDS subtypes seen in adults are rarely seen in children (e.g. refractory anemia with ring sideroblasts), while the current classifications do not account for MDS subtypes more common in children, such as hypoplastic MDS.203,207–209 As a result of these obser-

vations, alternative classifications have been proposed for pediatric MDS, which take into account the presence of predisposing genetic conditions, morphologic features and cytogenetic abnormalities.2,208 For a detailed discussion regarding these classifications, we refer the reader to Chapter 21. Morphologically, MDS is characterized by a wide range of abnormalities (see Chapter 21 for illustrations). Erythroid precursors may show nuclear lobulation, nuclear outline irregularity, multinucleation, nuclear fragments, or internuclear bridging. They may have cytoplasmic vacuolization, basophilic stippling, and megaloblastoid changes (fine-to-coarse chromatin with asynchronous or excessive cytoplasm).200 In some cases, erythroid precursors may show strong PAS positivity. Iron staining (Prussian blue stain) performed on aspirate smears may highlight the presence of ringed sideroblasts (i.e. normoblasts containing five or more siderotic granules covering at least onethird of their nuclear rim). In the myeloid series, the most frequent morphologic changes associated with MDS are the pseudo-Pelger-Hu¨et change (hyposegmentation of the nucleus with chromatin condensation), hypogranularity, and megaloblastoid maturation similar to that characterizing the erythroid series (large cells with abundant cytoplasm and large nuclei, but with more clumped chromatin than in true megaloblastic changes). Dysplastic megakaryocyte features include the presence of micromegakaryocytes or small forms with single hypolobulated nuclei, multinucleation that may be seen in small or large forms, or large megakaryocytes with very large hyperchromatic nuclei.

Juvenile myelomonocytic leukemia Juvenile myelomonocytic leukemia (JMML) is a disorder with myeloproliferative and myelodysplastic features that include leukocytosis with left shift in myeloid maturation and monocytosis, variable anemia and thromobocytopenia, usually mild dysplastic features, and elevated hemoglobin F, associated with hepatosplenomegaly and frequent skin infiltrates.210,211 Biologically, JMML is characterized by increased selective in vitro sensitivity of myeloid precursors to GM-CSF which appears to result from abnormal growth factor signal transduction through the RAS pathway (including mutations in the RAS, Nf1, PTPN11 genes that appear to be mutually exclusive and are collectively present in approximately 80% of these tumors).206,212–215 JMML shares features with chronic myelomonocytic leukemia (CMML) a disease of older adults.214,216 However, its distinctive clinical as well as laboratory, functional and molecular features provided impetus for its designation as

Diagnosis and classification

Genes for class distinction (n=588)

Diagnostic BM samples (n=132)

E2APBX1 −3SD

MLL

T-ALL

HD>50

BCRABL

TEL-AML1

+3SD

Fig. 2.22 Expression profile of pediatric ALL diagnostic bone marrow blasts. Two-dimensional hierarchical cluster of 132 pediatric ALL diagnostic bone marrow samples; the normalized expression value of each gene is indicated by a color (red, expression above the mean; green, expression below the mean). (From Ross et al.223 Copyright American Society of Hematology, used with permission. See color plate 2.22 for full-color reproduction.)

a separate entity, a recommendation adopted at an international workshop in 1994. Furthermore, in the current WHO classification,2,14 CMML and JMML are classified under the category of myelodysplastic/myeloproliferative diseases. This assignment reflects the spectrum of myeloproliferative features associated with cytopenias and dysplastic morphology that are often encountered in these entities. For a detailed discussion of JMML, please refer to Chapter 22.

Chronic myelogenous leukemia Chronic myeloid leukemia (CML) is a chronic myeloproliferative disorder, characterized in children as well as in adults by leukocytosis with netrophilia and marked left shift in granulocyte maturation. Associated peripheral blood features usually include basophilia, eosinophilia and thrombocytosis. The bone marrow is markedly hypercellular, with myeloid hyperplasia and an increase in the proportion of early granulocyte precursors, including promyelocytes and myelocytes. Basophilia and eosinophilia are frequent, and there is prominent megakaryocytic hyperpla-

sia consisting predominantly of small, dysplastic (hypolobulated) megakaryocytes and micromegakaryocytes.14,217 Establishing the diagnosis of CML requires the identification of the Philadelphia chromosome, resulting from the t(9;22)(q34;q21) chromosomal translocation, and/or of the corresponding BCR/ABL fusion gene.14

Future trends in the diagnosis and classification of acute leukemia Recent technological advances, including DNA microarray technology, have allowed high throughput analysis of gene expression profiles. A single platform of gene expression profiling can accurately identify the major, important subtypes of ALL and appeared to predict treatment outcome. Golub et al.218 first demonstrated that gene expression profiling can separate ALL cases from AML cases, and B-lineage ALL cases from those of T-lineage. Armstrong and colleagues219 subsequently showed that the expression profiles of MLL- rearranged cases were distinct from those with the classical ALL and AML. In a study of 360 ALL

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cases, we found that expression profiles identified the six major subtypes of this disease, including T-cell ALL and precursor B-cell ALL with E2A-PBX1, BCR-ABL, TEL-AML1, and MLL rearrangements, or hyperdiploidy >50 chromosomes, with almost 100% accuracy.220 A later study using higher density oligonucleotide arrays containing probes for most of the identified genes in the human genome has confirmed these data and the feasibility of classifying ALL cases using this approach (Fig. 2.22).221 In a study of T-cell ALL, Ferrando et al.129 identified distinct gene expression signatures of leukemic cells that corresponded to differentiation arrest at specific stages of normal thymocyte development: LYL1 + signature (pro-T), HOX11 + (early cortical thymocyte), and TAL1 + (late cortical thymocyte). Moreover, H0X11 and MLL-ENL subgroups were associated with a favorable prognosis. More recently, using gene expression profiling, we found that different genetic or lineage subtypes of ALL share common pathways of genomic response to the same treatment.222 The changes in gene expression were treatment specific, and the expression profiles could be used to illuminate differences in cellular responses to drug combinations versus single agents. The genes identified to date have also provided insights into the underlying biology of different leukemic subtypes and may become targets for novel therapy.129,218–220,223–225 Similar results have been obtained for pediatric and adult cases of acute myeloid leukemia.226–228 In addition to identifying gene expression profiles that correlate with the known molecular subtypes of AML, such studies have identified new prognostic groups among cases with normal cytogenetics and have shown that some of the AML subtypes (e.g. AML with MLL gene rearrangement and AML with FLT3 internal tandem duplications) are heterogeneous with respect to their gene expression profiles, with potential biologic and prognostic implications.227,229 Thus, analyses of the type described above may become an integral part of the diagnostic work-up of acute leukemia. Expression profiling of micro RNAs, which reflect the lineage and differentiation state of leukemic cells, could further enhance our ability to classify leukemia.230 It appears likely that, as our knowledge of leukemia pathobiology evolves, genetic or pathogenetic classification systems will supplant more traditional strategies, eventually becoming the gold standard for patient management.

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183 Andersen, M. K., Larson, R. A., Mauritzson, N., et al. Balanced chromosome abnormalities inv(16) and t(15;17) in therapyrelated myelodysplastic syndromes and acute leukemia: report from an international workshop. Genes Chromosomes Cancer, 2002; 33: 395–400. 184 Matutes, E., Morilla, R., Farahat, N., et al. Definition of acute biphenotypic leukemia. Haematologica, 1997; 82: 64–6. 185 Reinhardt, D., Zimmermann, M., Langebrake, C., et al. Acute mixed lineage leukemia in childhood. Blood, 2002; 100: 69a. 186 Carbonell, F., Swansbury, J., Min, T., et al. Cytogenetic findings in acute biphenotypic leukaemia. Leukemia, 1996; 10: 1283–7. 187 Killick, S., Matutes, E., Powles, R. L., et al. Outcome of biphenotypic acute leukemia. Haematologica, 1999; 84: 699–706. 188 Pui, C. H., Raimondi, S. C., Head, D. R., et al. Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at diagnosis and at relapse. Blood, 1991; 78: 1327–37. 189 Pane, F., Frigeri, F., Camera, A., et al. Complete phenotypic and genotypic lineage switch in a Philadelphia chromosomepositive acute lymphoblastic leukemia. Leukemia, 1996; 10: 741–5. 190 Bierings, M., Szczepanski, T., van Wering, E. R., et al. Two consecutive immunophenotypic switches in a child with immunogenotypically stable acute leukaemia. Br J Haematol, 2001; 113: 757–62. 191 Tsimberidou, A. M., Kantarjian, H. M., Estey, E., et al. Outcome in patients with nonleukemic granulocytic sarcoma treated with chemotherapy with or without radiotherapy. Leukemia, 2003; 17: 1100–3. 192 Tallman, M. S., Hakimian, D., Shaw, J. M., et al. Granulocytic sarcoma is associated with the 8;21 translocation in acute myeloid leukemia. J Clin Oncol, 1993; 11: 690–7. 193 Jenkin, R. D., Al Shabanah, M., Al Nasser, A., et al. Extramedullary myeloid tumors in children: the limited value of local treatment. J Pediatr Hematol Oncol, 2000; 22: 34–40. 194 Johansson, B., Gray, A., Kullendorff, C. M., et al. Granulocytic sarcoma in body cavities in childhood acute myeloid leukemias with 11q23/MLL rearrangements. Genes Chromosomes Cancer, 2000; 27: 136–42. 195 Peterson, L., Dehner, L. P., & Brunning, R. D. Extramedullary masses as presenting features of acute monoblastic leukemia. Am J Clin Pathol, 1981; 75: 140–8. 196 Bown, N. P., Rowe, D., & Reid, M. M. Granulocytic sarcoma with translocation (9;11)(p22;q23): two cases. Cancer Genet Cytogenet, 1997; 96: 115–7. 197 Menasce, L. P., Banerjee, S. S., Beckett, E., & Harris, M. Extramedullary myeloid tumour (granulocytic sarcoma) is often misdiagnosed: a study of 26 cases. Histopathology, 1999; 34: 391–8. 198 Oliva, E., Ferry, J. A., Young, R. H., et al. Granulocytic sarcoma of the female genital tract: a clinicopathologic study of 11 cases. Am J Surg Pathol, 1997; 21: 1156–65. 199 Ritter, J. H., Goldstein, N. S., Argenyi, Z., & Wick, M. R. Granulocytic sarcoma: an immunohistologic comparison with

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peripheral T-cell lymphoma in paraffin sections. J Cutan Pathol, 1994; 21: 207–16. Bennett, J. M., Catovsky, D., Daniel, M. T., et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol, 1982; 51: 189–99. Estey, E. H., Keating, M. J., Smith, T. L., et al. Prediction of complete remission in patients with refractory acute leukemia treated with AMSA. J Clin Oncol, 1984; 2: 102–6. Seymour, J. F., & Estey, E. H. The prognostic significance of auer rods in myelodysplasia. Br J Haematol, 1993; 85: 67–76. Forty-four cases of childhood myelodysplasia with cytogenetics, documented by the Groupe Francais de Cytogenetique Hematologique. Leukemia, 1997; 11: 1478–85. Hasle, H., Jacobsen, B. B., & Pedersen, N. T. Myelodysplastic syndromes in childhood: a population based study of nine cases. Br J Haematol, 1992; 81: 495–8. Hasle, H., Wadsworth, L. D., Massing, B. G., McBride, M., & Schultz, K. R. A population-based study of childhood myelodysplastic syndrome in British Columbia, Canada. Br J Haematol, 1999; 106: 1027–32. Luna-Fineman, S., Shannon, K. M., Atwater, S. K., et al. Myelodysplastic and myeloproliferative disorders of childhood: a study of 167 patients. Blood, 1999; 93: 459–66. Mielot, F. Childhood myelodysplastic syndromes. Pediatr Hematol Oncol, 1999; 16: 283–4. Mandel, K., Dror, Y., Poon, A., & Freedman, M. H. A practical, comprehensive classification for pediatric myelodysplastic syndromes: the CCC system. J Pediatr Hematol Oncol, 2002; 24: 596–605. Sasaki, H., Manabe, A., Kojima, S., et al. Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan. Leukemia, 2001; 15: 1713–20. Passmore, S. J., Hann, I. M., Stiller, C. A., et al. Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood, 1995; 85: 1742–50. Passmore, S. J., Chessells, J., Kempski, H., et al. Pediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia in the UK: a population-based study of incidence and survival. Br J Haematol, 2003; 121: 758–67. Arceci, R. J., Longley, B. J., & Emanuel, P. D. Atypical cellular disorders. In V. C. Broudy, J. L. Abkowitz, & J. M. Vose, eds. Hematology, American Society of Hematology Education Program Book, 2002, pp. 297–314. http://www.asheducationbook.org/ cgi/content/full/2002/11297. Emanuel, P. D., Shannon, K. M., & Castleberry, R. P. Juvenile myelomonocytic leukemia: molecular understanding and prospects for therapy. Mol Med Today, 1996; 2: 468–75. Niemeyer, C. M., Arico, M., Basso, G., et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood, 1997; 89: 3534–43.

215 Tartaglia, M., Niemeyer, C. M., Song, X., et al. Somatic PTPN11 mutations in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Blood, 2002; 100: 141a. 216 Arico, M., Biondi, A., & Pui, C. H. Juvenile myelomonocytic leukemia. Blood, 1997; 90: 479–88. 217 Castro-Malaspina, H., Schaison, G., Briere, J., et al. Philadelphia chromosome-positive chronic myelocytic leukemia in children. Survival and prognostic factors. Cancer, 1983; 52: 721–7. 218 Golub, T. R., Slonim, D. K., Tamayo, P., et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science, 1999; 286: 531–7. 219 Armstrong, S. A., Staunton, J. E., Silverman, L. B., et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet, 2002; 30: 41–7. 220 Yeoh, E. J., Ross, M. E., Shurtleff, S. A., et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell, 2002; 1: 133–43. 221 Ross, M. E., Zhou, X., Song, G., et al. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood, 2003; 102: 2951–9. 222 Cheok, M. H., Yang, W., Pui, C. H., et al. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nat Genet, 2003; 34: 85–90. 223 Pui, C.-H., Relling, M. V., & Downing, J. R. Acute lymphoblastic leukemia. N Engl J Med, 2004; 350: 1535–48. 224 Cario, G., Stanulla, M., Fine, B. M., et al. Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia. Blood, 2005; 105: 821–6. 225 Zaza, G., Cheok, M., Yang, M., et al. Gene expression and thioguanine nucleotide disposition in acute lymphoblastic leukemia after in vivo mercaptopurine treatment. Blood, 2005; May 19 [Epub ahead of print] PMID: 15905191. 226 Bullinger, L., Dohner, K., Bair, E., et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med, 2004; 350, 1605–16. 227 Ross, M. E., Mahfouz, R., Onciu, M., et al. Gene expression profiling of pediatric acute myelogenous leukemia. Blood 2004; 104: 3679–87. 228 Valk, P. J., Verhaak, R. G., Beijen, M. A., et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med, 2004; 350: 1617–28. 229 Lacayo, N. J., Meshinchi, S., Kinnunen, P., et al. Gene expression profiles at diagnosis in de novo childhood AML patients identify FLT3 mutations with good clinical outcomes. Blood 2004; 104: 2646–54. 230 Lu, J., Getz, G., Miska, E. A., et al. MicroRNA expression profiles classify human cancers. Nature, 2005; 439: 834–8.

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3 Epidemiology and etiology Logan G. Spector, Julie A. Ross, Leslie L. Robison, and Smita Bhatia

Introduction

Incidence and trends

The acute leukemias of childhood are a heterogeneous group of diseases. In reviewing the descriptive and analytic epidemiology of these malignancies, we have emphasized specific subgroups, as defined by morphology [the French-American-British (FAB) classification], cytogenetic features, or molecular markers. There is evidence that specific subtypes of leukemia may have distinct etiologies, and that molecular abnormalities associated with particular subtypes may be linked with specific causal mechanisms. Moreover, the mutations produced at the successive stages of leukemogenesis, from initiation through induction to promotion, may all involve separate etiologic processes. It is also important to note that changes over time in diagnostic practice and precision may account in part for some reported epidemiologic trends. Moreover, changes in terminology and classification schemes for leukemia make it difficult to perform direct comparisons among studies, especially if risk factors differ for different subgroups. However, in assessing risk factors, studies of the childhood leukemias present several methodologic advantages. The interval between exposure to putative risk factors and the onset of leukemia may be shorter, recall of exposures is likely to be better, and intervening factors may be fewer than those associated with adult leukemias. These characteristics of childhood leukemia may facilitate identification of the most likely risk factors for each leukemia subtype. Furthermore, they lend themselves to an approach that includes both population studies and molecular epidemiologic techniques, permitting the design of research to assess genetic-environmental causal interactions.

In the United States, the acute leukemias represent 31% of malignancies occurring among children under the age of 15 years.1 Acute lymphoblastic leukemia (ALL) comprises 85% of childhood acute leukemias. Annual incidence rates (per million population) for ALL and acute myeloid leukemia (AML) are 30.9 and 5.6, respectively. The two diagnostic categories – ALL and AML – are further subdivided based on leukemic cell features. Childhood ALL is classified by FAB morphology (L1, L2, and L3) and by immunophenotype (B cell, early pre-B, pre-B, and T cell). Childhood AML is classified morphologically into eight distinct morphologic subgroups: M0 (myeloid leukemia with minimal differentiation), M1 (acute myeloblastic without maturation), M2 (acute myeloblastic with maturation), M3 (acute promyelocytic), M4 (acute myelomonocytic), M5 (acute monocytic), M6 (erythroleukemia), and M7 (acute megakaryocytic). Age-specific incidence patterns demonstrate a characteristic peak between the ages of 2 and 5 years for childhood ALL (Fig. 3.1). The incidence rates for AML are highest in infancy and are fairly uniform in older children (Fig. 3.1).2 In the United States, during 1986 and 1995, the incidence in the 0 to 4 year age group was 10.3 per million, and 5.0 per million and 6.2 per million in the 5 to 9 and 10 to 14 year age groups, respectively. Comparable rates have been reported elsewhere in Europe and in Britain. In childhood ALL, males are more often affected than females, with the notable exception of a female predominance in infancy. In contrast, there is no clear pattern in the male-to-female ratio for childhood AML. A striking difference in the incidence of ALL exists between white and black children. The excess incidence

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Epidemiology and etiology

Table 3.1 Annual standardized rates (per million population) of acute lymphoblastic leukemia, from birth to 14 years of age, in selected regionsa

Population Costa Rica Finland Canada Hong Kong Sweden Australia, New South Wales Germany Norway US, SEER (whites) Italy Hungary United Kingdom, England and Wales Czechoslovakia, Slovakia Cuba Kuwait (Kuwaiti) Japan Brazil, Goiania New Zealand (Maori) US, SEER (blacks) Brazil, Belem Israel (Jews) China, Tianjin India, Bombay Cancer Registry

Both sexes

Males

Females

46.3 41.9 41.0 40.6 40.1 39.9 39.0 38.3 38.0 37.9 33.5 32.8

51.7 41.8 44.8 50.6 40.9 56.2 43.6 39.3 41.3 38.9 37.0 35.7

40.7 42.0 36.9 29.9 39.3 43.2 34.1 37.3 34.5 36.8 29.9 29.7

28.4 25.4 24.3 22.6 21.9 21.9 20.8 18.8 18.6 17.4 16.0

31.8 27.6 27.1 25.6 21.8 30.3 22.0 20.5 18.7 19.0 19.8

24.8 23.1 21.4 19.5 22.1 13.1 19.6 17.4 18.4 15.7 12.0

Abbreviations: SEER, Surveillance, Epidemiology, and End Results Program. a Data are from Ferlay et al.5

Fig. 3.1 Age-specific incidence and gender and race ratios for childhood acute leukemia in the United States. (Data derived from Gurney et al.1 )

among white children is apparent in most age groups. The white-to-black ratio for AML in the United States is 1.2. The higher rate of AML among Hispanics1,3 is contributed by acute promyelocytic leukemia (APL), raising the question of genetic predisposition to APL and/or exposure to distinct environmental factors.4 Substantial geographic variation exists in childhood leukemia incidence rates.5 Internationally, annual incidence rates of childhood ALL range from 9 to 47 per million for males and from 7 to 43 per million for females

(Table 3.1). Incidence rates for ALL are highest in the United States (among white children), Australia, Costa Rica, and Germany. Rates are intermediate in most European countries and lowest in India and among black children in the United States. By contrast, the incidence of AML is highest in China, Japan, and among the Maori of New Zealand, with intermediate rates in Australia, the United States, and the United Kingdom. India, Kuwait and the Canadian Atlantic Provinces have the lowest reported rates of childhood AML (Table 3.2). Several investigators have examined temporal trends for leukemia, but the findings are difficult to interpret. This is primarily because of the various time periods covered and the different methods of analysis used. Some investigators analyzed the 0- to 14-year age group as a whole, while others have focused on specific age groups for each sex and by

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Table 3.2 Annual standardized rates (per million population) of acute myeloid leukemia, from birth to 14 years of age, in selected regions worldwidea Population

Both sexes

Males

Females

New Zealand (Maori) Costa Rica Australia, New South Wales Norway Italy Japan China, Tianjin Germany Sweden Canada United Kingdom, England and Wales US, SEER (blacks) US, SEER (whites) Cuba Hungary Finland Brazil, Goiania Israel (Jews) Czechoslovakia, Slovakia India, Bombay Cancer Registry Hong Kong Brazil, Belem Kuwait (Kuwaiti)

14.4 8.9 8.0

15.5 8.5 8.3

13.3 9.2 7.7

8.0 7.9 7.2 6.7 6.7 6.7 6.3 6.3

9.4 7.4 7.8 6.8 7.1 6.0 6.9 6.2

6.5 8.3 6.5 6.6 6.3 7.5 5.6 6.5

6.2 6.0 5.7 5.5 5.4 5.3 5.3 5.1 4.8

5.6 5.9 5.1 6.0 4.4 5.1 4.9 4.4 5.2

6.8 6.0 6.4 5.0 6.4 5.5 5.7 5.9 4.4

3.9 3.5 2.0

5.2 4.5 0.6

2.6 2.7 3.4

Abbreviations: SEER, Surveillance, Epidemiology, and End Results Program. a Data are from Ferlay et al.5

leukemia subtype. Some studies on secular trends in the incidence of childhood leukemia have identified increasing rates over time.6 From 1974 through 1991, the average annual percentage increase for all leukemias was 0.9%, with average annual increases of 1.6% and 0.6% for ALL and AML, respectively. This increase may be attributable in part to changes in diagnostic classification during the time period studied. Nevertheless, it is noteworthy that the increase is modest and the largest average annual increases occurred among children diagnosed with ALL and AML during the first 2 years of life (2.4% and 2.5%, respectively). A similar increase in the incidence of childhood acute leukemia was reported from regions of the United Kingdom,7 and the incidence of childhood AML, but not all of ALL, was reported to have increased in Australia.8 Stable childhood leukemia rates have been reported from other

registries, including the Greater Delaware Valley Pediatric Tumor Registry,9 the nationwide German Registry,10 and the SEER Registry, where Linet et al.11 reported no substantial change in the incidence of childhood leukemia diagnosed in the United States, between 1975 and 1995.

Genetic factors Acute leukemia is a clonal disorder of the hematopoietic system, arising from mutations in a single cell that are passed on to all of its descendants. In most cases, the genetic abnormalities that give rise to acute leukemia are acquired rather than inherited. As many as 5% of acute leukemias, however, are associated with inherited genetic syndromes.12 In addition, a variety of normal inherited polymorphisms in genes may contribute indirectly to the risk of leukemia: these include genes that encode enzymes involved in carcinogen metabolism and detoxification and those that are involved in the immune response to infections.13

Cytogenetic abnormalities Many acquired chromosomal abnormalities have been found in childhood leukemia,14 as described in more detail in Chapter 9. Abnormalities are generally more prevalent in, but not restricted to, specific morphologic subtypes. Some of the more common abnormalities include translocations involving the MLL gene at chromosome band 11q23 in infants (both ALL and AML), the t(8;21) in M2 AML, the t(15;17) in M3 AML, trisomy 8 in AML, and the t(9;22) and t(1;19) in ALL.15–21 Recent studies indicate that the most common reciprocal translocation in ALL is the t(12;21)(p13;q22), which occurs in about 25% of cases and fuses the TEL and AML1 genes.22 This translocation is invisible karyotypically and therefore went undetected in early studies. A significant proportion of ALL cases (about 25%) exhibit leukemic cell hyperdiploidy. With the exception of infant leukemias,14,17 almost 80% of which have an abnormality involving the MLL gene at 11q23, there have been no epidemiologic studies exploring associations between environmental exposures and specific chromosomal abnormalities, partly because of the heterogeneous clinical presentation of these abnormalities and their relatively low frequency.

Genetic syndromes Several genetic syndromes have been associated with an increased risk of childhood leukemia.14 Studies suggest a 10- to 20-fold increased risk of leukemia (both ALL and

Epidemiology and etiology

AML) in children with Down syndrome,23,24 and some reports suggest up to a 600-fold increased risk for one subtype of AML (M7).25 The reasons for this increased risk are unclear, although a gene (AML1) associated with certain cases of AML has been identified in a chromosomal site (band 21q22) believed responsible for the Down syndrome phenotype.26 Other genetic syndromes associated with both childhood ALL and AML include Bloom syndrome, neurofibromatosis type 1, Schwachman syndrome, and ataxia telangiectasia.27–32 Kostmann granulocytic leukemia and Fanconi anemia are associated with AML.33,34 In addition, there is a familial form of AML (familial monosomy 7) in which two or more siblings develop leukemia before the age of 20.35 Although specific associations have been described mainly as case reports, data on the proportion of cases of leukemia with a known genetic etiology or an association with specific genetic syndromes are limited. Several studies report that ∼2.5% of the children with leukemia have a recognized genetic condition, which is almost entirely accounted for by Down syndrome.36–38 To evaluate the risk of leukemia associated with congenital anomalies, a series of matched case-control studies have been conducted. In one such study by the Children’s Cancer Group, children with ALL and AML were compared with matched regional population controls. More congenital anomalies were found in the index child with ALL or AML than in control subjects. The congenital anomalies included Down syndrome, congenital heart defect, and multiple birth marks.39 Another study from the United Kingdom reported a lower frequency of congenital anomalies among children with leukemia or lymphoma, when compared with children with solid tumors. They hypothesized that mutations resulting in the development of leukemias and lymphomas occur at a much later stage in development, in the cells committed to hematopoiesis.40

Familial patterns There have been several reports of familial aggregation of childhood leukemia.29,41–44 Although this finding may represent an inherited predisposition, the available studies do not rule out shared environmental factors. One of the most informative associations is the higher degree of concordance of leukemia among twins (particularly monozygotes), which is highly age-dependent, occurring mostly in infants.45–50 However, in a study of leukemia in a pooled series from the United States, Canada and the United Kingdom, only three concordant pairs (1.5%) were found among 197 pairs in which at least one twin had leukemia, with the concordance rate for monozygotic twins reported as

3.9%.51 Thus, although the concordance rate in twins of the same sex (likely monozygotes) is higher than the zero concordance rate reported for twins of unlike sex (dizygotes), the concordance rate in twins of like sex is quite variable.51 There are molecular data to suggest that in twin pairs with leukemia, the leukemic clone develops in one fetus and disseminates to the other via a shared placental circulation rather than resulting from an inherited mutation.52,53 Recent molecular studies also indicate that concordant ALLs in older twin children (3 to 11 years) have an in utero origin, coupled in these cases with a protracted latency.54,55

Cancer in offspring of patients treated for childhood leukemia There are relatively few reports of the incidence of cancer in the offspring of individuals treated for childhood cancer. Hawkins et al.56 estimated that the proportion of heritable cases among the cancer survivors is unlikely to exceed 5%. Furthermore, recent reports indicate that offspring of subjects previously receiving chemotherapy and/or radiotherapy for childhood malignancies do not exhibit latent chromosomal instability.57

Other conditions in relatives of cases with leukemia A recent case-control study provides support for a positive association between childhood acute leukemia and a family history of hematologic neoplasms and solid tumors (particularly gastrointestinal tumors and melanoma), with a stronger association for patients with AML.58 Further studies need to be conducted to corroborate this report and to identify the biologic basis of this association.

Demographic and environmental risk factors Although the specific causes of most pediatric and adult leukemias are not known, several environmental and demographic features (summarized together with predisposing genetic syndromes in Table 3.3) have been associated with increased risk. These include ionizing radiation and exposure to certain chemicals, particularly organic solvents such as benzene.59 Probably the most extensively studied risk factor is in utero exposure to ionizing radiation from diagnostic x-rays, a well-established risk factor for both childhood ALL and AML (although accounting for only a small proportion of cases).60–63 Other factors associated with increased risk include parental occupational exposures to hydrocarbons and pesticides, maternal alcohol use and cigarette smoking during pregnancy, and a

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Table 3.3 Risk factors for childhood acute leukemia, by degree of certainty Degree of certainty

Acute lymphoblastic leukemia

Acute myeloid leukemia

Generally accepted risk modifiers

Males Age (2–5 years) High socioeconomic status Race (whites > blacks) In utero x-ray exposure Postnatal radiation (therapeutic) Down syndrome Neurofibromatosis type I Bloom syndrome Shwachman syndrome Ataxia telangiectasia Increased birth weight Breast feeding Maternal history of fetal loss

Race (Hispanic) Chemotherapeutic agents (alkylating agents, topoisomerase II inhibitors) Down syndrome Fanconi anemia Neurofibromatosis type 1 Bloom syndrome Shwachman syndrome Familial monosomy 7 Kostmann granulocytopenia

Suggestive of increased risk

Limited evidence

Probably not associated

Parental smoking prior to or during pregnancy Parental occupational exposures Postnatal infections Diet Maternal alcohol consumption during pregnancy Electric and magnetic fields Postnatal use of chloramphenicol Vitamin K prophylaxis in newborns Ultrasound Indoor radon

maternal history of prior fetal loss. Both exposure to electromagnetic fields and paternal preconception exposure to radiation have also been proposed as possible risk factors. These associations remain controversial because of inconsistencies in the results of epidemiologic investigations,64–66 and the lack of biologically plausible etiologic theories.

Ionizing radiation The leukemogenic potential of ionizing radiation has been well documented in studies of survivors of the atomic bombing of Japan in 194567 and of occupational exposure incurred by early radiation scientists.68 In children, exposure in utero to diagnostic x-rays is associated with an increased risk of both ALL and AML, with relative risks of 1.5 to 1.7 for ALL.32 It is reasonable to expect that in utero xray exposure levels would have declined substantially over time, given the knowledge of health-related risks associated with radiation exposure and the reduced level of radiation required for diagnostic procedures. Meeting these expecta-

Maternal alcohol consumption during pregnancy Prenatal and child exposure to pesticides Parental solvent exposure Maternal marijuana use during pregnancy Indoor radon Postnatal use of chloramphenicol

tions are reports from studies conducted in Sweden, United Kingdom and the United States that have demonstrated higher risk estimates among children born in earlier eras (e.g. 1930s to 1950s) compared to more recent times.69–71 The causal nature of this association has been called into question,72,73 however, because studies of Japanese survivors of the atomic bomb revealed no increased incidence of leukemia associated with in utero exposure. Moreover, there are concerns regarding the lack of good exposure assessment and the potential for recall bias in case-control studies related to this subject. Nevertheless, a strong argument in favor of causality is provided by the dose–response pattern that is evident for in utero exposure to diagnostic x-rays (i.e. increased risk with increasing numbers of exposures).74 Studies of children who have received radiation therapy for the treatment of Hodgkin disease, Langerhans cell histiocytosis, thymic enlargement, and tinea capitis show a slightly elevated risk of leukemia, particularly AML.75–78 Postnatal diagnostic radiation, in contrast, does not appear to increase risk, although a recent study suggested risk

Epidemiology and etiology

may be increased in the presence of polymorphisms that decrease the effectiveness of DNA repair genes.79–81 Even if leukemia risk is increased, the potential number of cases that currently might be attributed to in utero and/or postnatal x-ray exposure would likely be very small when one considers the modest magnitude of risk and the limited level of exposure. Exposures to ionizing radiation from fallout from atomic bombs or accidentally released by nuclear power plants, from background radiation and radon, and from parental employment in the nuclear power industry have also been proposed to increase the risk of leukemia. Evidence in support of an association between childhood acute leukemia and radiation from nuclear fallout is quite weak. Because of the widespread fallout from Chernobyl, a series of investigations were undertaken in affected countries, including Sweden,82 Finland,83 United Kingdom,84 Scotland,85 Germany,86 and Greece.87 Except for a recent report of a transient increase in infant acute leukemia in northern Greece,88 no increase in the incidence of leukemia has been identified in the areas contaminated by the Chernobyl reactor accident.89 Similarly, reports relating to nuclear fallout follow-up accidents at Three Mile Island have not provided strong evidence of an increased risk of childhood leukemia.90 However, a reactor accident in 1957 in Chelyabinsk in the former Soviet Union resulted in exposure to radiation levels that may have reached 4 Gy. A recent analysis found that this exposure was associated with a subsequent regional increase in the incidence of leukemia.91 Nuclear fallout affecting regions of Nevada and Utah, as a result of nuclear weapon testing in Nevada, led to studies to determine the potential impact on childhood leukemia, utilizing estimates of geographic site-specific radiation doses to the bone marrow. No significant trend between the estimated dose and risk of leukemia mortality was found.92 However, the risk of death from leukemia was found to be higher in persons receiving the highest exposure level (6–30 mGy) compared to the lowest-dose group (0–2.9 mGy). All of these associations are controversial because of the difficulty of extrapolating from the high acute doses of radiation experienced by the Japanese survivors of the atomic bomb to these much smaller or chronic exposures. Also questionable is the evidence for an association with nuclear energy production and nuclear fuel reprocessing. Studies addressing childhood leukemia have been conducted in the United Kingdom,93,94 France,95 United States,96,97 Germany,98 and Canada.99 Overall, these reports do not lend support for an increased incidence of childhood leukemia, although some do report an increased incidence or mortality. In fact, the childhood acute leukemia clusters

around the nuclear reprocessing plants of Sellafield and Dounreay in the United Kingdom are now thought to result from unique sociodemographic features of the local communities, with population mixing and infection probably playing a role, rather than to any direct effect of proximity to the plants themselves.100 A possible role of paternal preconception radiation exposure has been the subject of recent interest. At least two studies have reported an increased risk of childhood leukemia in offspring of fathers exposed to radiation either occupationally101 or diagnostically.102 However, other studies have found no such associations.103,104 Again, the lack of a plausible biological mechanism and the absence of an increased risk of leukemia in the offspring of survivors of the atomic bomb argue against this proposed association. Natural background radiation from terrestrial sources and cosmic radiation is thought to account for about 5% of all leukemia cases in adults and children.73 Although there is no clear association between the risk of leukemia and cumulative radon exposure in uranium miners,73 it has been proposed that some naturally occurring circumstances might result in the accumulation of a leukemogenic dose of radon in fat-containing bone marrow.105 The leukemogenic effects of radon have recently come under investigation. Although ecologic investigations have provided some evidence for a correlation,105,106 case-control studies incorporating measurement of indoor radon levels have not found higher levels within homes of children with ALL107 and AML.108–111

Nonionizing radiation Public concern has been raised regarding the leukemogenic potential of low-energy electromagnetic fields produced by residential power supply and appliances. The concern is prompted in part by the ubiquitous nature of the exposure – a feature that poses methodologic problems because of the difficulty of finding an unexposed comparison population. In addition, the biologic plausibility of the association is questionable.112 Laboratory studies indicate that electromagnetic fields may produce adverse biological effects; however, they do not release sufficient energy to damage DNA. Results from studies to date are inconsistent, and the evaluation of exposure has often relied on surrogate measures, such as the physical configuration of power lines and their distance from homes. Three of the more recently reported investigations from the United States,66 Canada,113 and the United Kingdom,114 provide rather convincing evidence that electric and magnetic field exposure is not associated with a significantly increased risk of childhood ALL. A recent study failed to show any association

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between exposure to magnetic fields inside infant incubators and the risk of childhood leukemia.115 Nonetheless, results of meta-analysis have been interpreted to suggest that risk may be increased at the highest exposure levels (i.e. > 0.4 muT).116,117 However, it is important to note that even if there is an increased risk at this highest level of exposure, the proportion of the population exposed to such high levels is extremely small and thus the attributable risk would be negligible.

Chemical exposures Pesticide exposure (occupational or home use) has been reported to be associated with childhood leukemias in several studies.118–121 In a multicenter case-control study of 204 cases of AML and matched community controls, Buckley et al.120 reported a positive association with maternal occupational exposure to pesticides before, during and after pregnancy and with paternal occupational exposure during the same time period.120 This association was particularly strong for younger children with the M4 (monocytic) or M5 (myelomonocytic) subtype of AML. Furthermore, there was an independent association with maternal exposure to household fly-sprays, pesticides, garden and agricultural sprays, and home treatment by insect exterminators, in the month before the last menstrual cycle and during the index pregnancy, and with direct exposure of the index child to household and garden insecticides. These associations were independent of parental occupational exposure. Although there is a large body of literature on the association between pesticide exposure and childhood leukemia,122,123 the studies are limited by the nonspecific nature of the exposure, by the reliance of these studies on parental self-reporting and by insufficient evidence for a causal relationship. Moreover, use of pesticides may be an indicator of rural isolation, and hence a possible confounding variable because the patterns of exposure to infection may be associated with population mixing. Parental exposure to solvents has also been associated with an increased risk of childhood leukemia in several studies, although other studies report no such association.118,120,124–127 An increased risk of childhood leukemia associated with paternal exposure to chlorinated solvents has been reported by several investigators,118,120,128 but consistent association with maternal exposure to solvents is apparent.129 However, there is some evidence to indicate that maternal occupational exposure to hydrocarbons may be associated with childhood leukemia, which is compatible with the observation that maternal benzene is associated with AML in adults, but these findings need further substantiation.120 No clear association with residential

proximity to industrial sources of hydrocarbons has been observed.130–132 A recent study revealed a significant association between childhood leukemia and substantial participation by household members in some common household activities involving organic solvents.133 Studies of proximity to motor vehicle traffic and, by proxy, of benzenecontaining exhaust have produced mixed results.134,135 A recent study in which the distance measure was confirmed to be correlated with measures of vehicle exhaust found no relationship with leukemia.136 A positive association between total reported duration of paternal occupational exposure to lead and AML has been reported by Buckley et al.,120 as has an association between maternal occupational exposure to metal dusts and fumes and lead.120,124 However, ecological studies have failed to show an association between leukemia and proximity to industrial facilities with an increased exposure to metal or metal fumes.137,138 An association between maternal occupational exposure to wood dust before conception of the index child and childhood leukemia has been reported, although few women were exposed during or after pregnancy.128 Significantly elevated relative risks have been reported for paternal occupational exposure to wood dust before and near conception and during the gestational and postnatal periods.139

Lifestyle Diet and vitamin supplement use In pediatric malignancies, research has focused primarily upon the use of vitamin supplements during pregnancy,140,141 with fewer studies focusing on maternal diet. A recent case-control study in Australia suggested that maternal folate supplementation during pregnancy may be associated with a decreased risk of childhood ALL.142 This study, however, was very small (83 cases) and lacked sufficient detail regarding exposure. Details of studies reporting the association of maternal diet during pregnancy and infant leukemia are described in detail elsewhere in this chapter. The role of diet of the index child in the development of childhood leukemia has not been investigated extensively. Results of investigations exploring the association between the intake of certain food items thought to be precursors or inhibitors of N-nitroso compounds have been controversial. An increased risk has been associated with consumption of processed meats, such as hot dogs, while others have failed to show such an association.118,141,143 No associations have been reported with postnatal use of vitamins

Epidemiology and etiology

by the index child,144 consumption of fish, dried milk, fruit juice or canned foods,60 although a decreased risk with long-term use of cod liver oil has also been reported.119

Alcohol consumption by parents The majority of studies relate solely to alcohol consumption of the mother and only to consumption during the pregnancy leading to the birth of the index child. These studies have reported an increased risk of AML (particularly in very young children) associated with maternal alcohol consumption during pregnancy.145–147 A recent study of infant leukemia147 found a dose–response relationship with AML; risk was most pronounced for the M1 and M2 subtypes. There was only a modest increase in risk of ALL in this study, while another study reported no association between maternal alcohol consumption and ALL.125 This series of reports has implications for discussions of the etiology of infant leukemia later in this chapter. No association between paternal alcohol consumption and childhood leukemia has been found.147,148

independent of associations with occupational and household pesticide exposure, paternal occupational exposure to paints, and pigments, metal dusts and saw dust. There was no significant association with paternal use of marijuana in the year before conception of the index child.

Maternal reproductive history Fetal loss Several studies have reported an increased risk of childhood leukemia (both AML and ALL) in association with a maternal history of fetal loss,125,160–162 while one study reported an inverse relationship.119 Although some data suggest that this association is confined to cases diagnosed at a very young age,161 a recent study was unable to confirm this finding.163 A history of fetal loss may indicate some common environmental exposure, an inherited genetic defect with variable effects on the fetus, or both.

Maternal age and birth order Tobacco smoking by parents Several studies have examined the role of parental smoking in the development of childhood leukemia. However, issues such as reporting bias (greater likelihood of positive associations being published) limit the quality of the reports. The association with maternal cigarette smoking has been inconsistent. Some studies have found an increased risk of leukemia in the children of women who smoked during pregnancy,149–151 whereas others have reported no increased risk of either ALL119,125,152–156 or AML.145,147 Two recent studies have suggested an association between paternal preconception cigarette smoking and the risk of childhood leukemia in offspring.156,157 A recent large case-control study concluded that parental smoking during pregnancy or exposure to cigarette smoke shortly after birth is unlikely to contribute substantially to the risk of childhood leukemia in North America.158 In summary, the literature shows no consistent association between leukemia and parental exposure to tobacco. Sandler et al.159 evaluated the cancer risk from cumulative household exposures to cigarette smoke in a casecontrol study. Cancer risk was greater for individuals with exposure during both childhood and adulthood than for individuals with exposure during one period only.159 An association between AML (M4 or M5) and the reported use of mind-altering drugs (primarily marijuana) by the mother in the year before or during the index pregnancy has been described.140 The association with marijuana exposure was

Accumulation of chromosomal aberrations and mutations during the maturation of germ cells is a mechanism hypothesized for the association between increasing maternal age and cancer in the offspring. Most studies have failed to show an association between leukemia and maternal age.160,163 However, two recent analyses of the Swedish Family-Cancer Database revealed a maternal age effect for childhood leukemia, as did a very large British case-control study.164–166 Importantly, these results were obtained after exclusion of children with Down syndrome, which is associated with both leukemia and advanced maternal age at birth. Although most studies fail to show a positive association between birth order and childhood leukemia, there are reports of a decreasing trend in the incidence of childhood ALL and AML with increasing birth order, adjusted for age, sex, calendar period and maternal age at birth of child.167 A large case-control study, similarly adjusted, found a significant trend in the incidence of ALL, but not AML, with increasing birth order.166

Birth weight and length at birth High birth weight has been found to increase the risk for both ALL and AML before the age of 5 years with a fair consistency in larger studies.160,164,167,168 Birth weight is likely a marker for endogenous risk factors. Accordingly, the level of insulin-like growth factor 1 has been found to

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be highly correlated with birth weight,169 and high levels of thyroid-stimulating hormones, which is inversely correlated with birth weight, has been found to be protective against ALL.170

Unique populations of interest Because the etiology of childhood leukemia is largely unknown, investigations focusing on unique patient or disease subsets may prove particularly fruitful. In this section, we briefly summarize theories that have been proposed to explain the etiology of infant leukemia and the 2- to 5-year age peak in the incidence of childhood ALL.

Infant leukemias Nearly 80% of infant leukemias present with a specific cytogenetic abnormality involving the MLL gene on chromosome band 11q23.17 Molecular analysis of MLL gene rearrangements in acute leukemias in identical twin infants and studies of neonatal blood samples have shown that this abnormality occurs in utero.52,171 The identification of MLL gene abnormalities in treatment-related AMLs that develop following therapy for a histologically distinct primary cancer suggests relationships that are explored in the following sections.

Treatment-related leukemias AML arising after exposure to genotoxic chemotherapy is a heterogeneous collection of diseases characterized by distinct chromosomal abnormalities. One subset of therapyrelated AMLs is associated with exposure to alkylating agents. The chromosomal abnormalities 5q- and monosomy 7 are commonly observed in leukemic cells in this group (as they are in de novo adult AML). The other major subset of therapy-induced leukemia is associated with exposure to the epipodophyllotoxin drugs teniposide (VM-26) and etoposide (VP-16).172 A high proportion of epipodophyllotoxin-associated AMLs are of the M4 (monocytic) or M5 (myelomonocytic) subtype and have abnormalities at chromosome band 11q23 involving rearrangements of the MLL gene.173 The same genetic abnormality is also found in some secondary AMLs associated with exposure to anthracyclines (e.g. daunorubicin and doxorubicin). These two classes of chemotherapeutic agents share a common mechanism of action that involves binding to and inhibition of DNA topoisomerase II. Topoisomerase II is an enzyme that catalyzes breakage and resealing of DNA, a function that prevents tangling of helical,

duplexed DNA strands during replication.174 Binding of an epipodophyllotoxin or other inhibitor to this enzyme introduces the potential for faulty recombination between chromosomal regions undergoing simultaneous breakage. This faulty recombination can result in the 11q23/MLL rearrangement that characterizes some of these therapyinduced AMLs.

The topoisomerase II inhibitor hypothesis Because significant proportions of epipodophyllotoxinassociated AMLs and infant leukemias exhibit the same chromosomal abnormalities, it is reasonable to ask whether these two groups of leukemias share a common causal mechanism. As noted previously, infant leukemia has been causally linked to an abnormality acquired in utero, which likely results from maternal/fetal exposure to carcinogens during pregnancy. In light of the association between the epipodophyllotoxins and MLL gene rearrangements in secondary leukemias, it has been proposed that in utero exposure to agents that inhibit DNA topoisomerase II function (including epipodophyllotoxins found in both medicinal and dietary sources) might also play a causal role in infant leukemia.14 A subset of mothers of infants with leukemia diagnosed at 1 year or less of age (controls, selected by random digit dialing, were from three multicenter case-control studies of childhood leukemia in the United States) was reapproached and supplemental information on maternal diet during index pregnancy was sought. This study attempted to test the hypothesis that infant leukemia characterized by 11q23 abnormalities may result from exposure to naturally occurring topoisomerase inhibitors, such as caffeine, and a variety of fruits and vegetables.14 Although no association emerged between diet and leukemia in general or ALL, there was a statistically significant association for AML with increasing consumption of dietary topoisomerase II inhibitors.175,176 A recent study has shown that bioflavonoids, natural substances in food as well as in dietary supplements, can cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo.177 These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potential translocations in utero leading to infant leukemia. In utero exposures and their association with infant leukemia were also assessed in a recent study. Use of cigarettes and alcohol, the ingestion of certain herbal medicines and drugs classified as “DNA damaging” and exposure to pesticides were associated with an increased risk of MLL fusion-positive leukemias.178 Despite their common chromosomal rearrangement, it is likely that infant leukemias with 11q23 rearrangements

Epidemiology and etiology

are a heterogeneous group of diseases rather than a single entity. In the study referred to previously,175 the majority of significant associations with dietary topoisomerase II inhibitors were with AML rather than ALL. AML and ALL exhibit distinct chromosomal translocations involving the MLL gene and could have different etiologies, as breaks in the MLL gene occur at different distances from the topoisomerase II binding site, depending on the subtype of acute leukemia. Moreover, inherited susceptibility may play a role in leukemogenesis that is mediated by the actions of topoisomerase II inhibitors. Thus, it is clear that the topoisomerase II inhibitor hypothesis requires further investigation.

The childhood ALL age peak A peak in the incidence of CD10+ B-cell precursor ALL (also known as common ALL, or cALL) occurs in developed countries among children between the ages of 2 and 5 years. This pattern was first noted in the United Kingdom and among white children in the United States, appearing later among U.S. black children.179 Epidemiologic evidence supports the view that many childhood leukemias, especially those that occur during this age peak are the consequence of a rare, abnormal response (brought on by unusual timing, perhaps in combination with individual genetic susceptibility) to a common infection.180

Population mixing The evidence, indirect but compelling, for an etiologic role for infection is provided by studies of leukemia in the context of population mixing. To test the idea that population mixing might provide the conditions under which infection could play a role in childhood leukemia, Kinlen181 performed a series of observational studies in the United Kingdom involving relatively isolated populations affected by significant population mixing or movement. In each case, an increased relative risk of childhood acute leukemia was noted subsequent to the population mixing. Moreover, the leukemia risk decreased after the initial epidemiclike increase, suggesting that immunization of the population had occurred. This effect can be interpreted in terms of the epidemiology and population dynamics of common infections.182 Susceptible individuals who live in areas where a specific infection is not endemic are placed at risk when brought into contact with infected carriers. In the present context, leukemia would be the rare end result of infection for some individuals. Whereas Kinlen’s original studies examined extreme instances of population mixing, such as military

encampments, more recent studies have applied various quantitative measures to wide geographic areas. Several such studies have found an increased risk of childhood leukemia (and, in some instances, trends) with greater population growth and with greater diversity of immigrants.183–188 However, the literature is not entirely in agreement, since associations of leukemia with population mixing have been found for rural but not urban areas and vice versa. Also, one study found an inverse association of ALL with the diversity of the migrants’ origins.189 Interpretation is hampered by the aggregation of diverse types of leukemia; to date only one study has analyzed separately the cALL subtype.186 Other lines of evidence also support an infection-related etiology for childhood leukemia. The 2- to 5-year age peak of cALL is found among affluent populations in developed societies. Many lines of evidence (e.g. international incidence rate comparisons, time trends, geographic distributions, and community characteristics) combine to suggest that cALL is a disease of affluent societies involving a rare response to common infection(s). Delayed first exposure is thought to contribute to the pathogenesis of several diseases associated with affluence in which infection is implicated, the pathologic precedent being paralytic poliomyelitis.190 The timing and pattern of exposure to common infections early in life is considered critical to the production of an appropriate immune response. Factors associated with affluence, including relative social isolation and higher levels of hygiene, may lead to a delayed exposure to common infections. Moreover, the practice of prolonged breast feeding (which, in addition to its nutritional and immunologic benefits, may also provide early exposure to common viruses and bacteria191 ) has declined among affluent populations. Breast feeding has been consistently shown to protect against leukemia, the protection increasing with the length of feeding.192 Lack of early exposure to infections may leave the immune system unprepared for infection at a later time and could lead, in some cases, to an abnormal immunologic response that increases the risk of leukemia. Evidence in support of this contention includes the observation of an inverse association of leukemia with time in attendance at day-care centers,193–195 although this observation is not consistent.196,197 Also relevant is the observation of an inverse association of leukemia with birth order.166,167 Studies that have inquired directly about infection history or have searched for serologic evidence of infection, both of which are problematic in case-control studies, have not identified particular candidate microbial agents either serologically198–203 or through surveys.119,144,194,197,204–209 There is no direct evidence as to the nature of the infection

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or of the abnormal response. It could be (as with feline leukemia virus in cats and HTLV-1 in human beings) a single but common transforming virus to which the exposed individual has an inadequate protective response. Alternatively, the immune system might mount an overexuberant or unbalanced response to some microbial antigens or superantigens.210 In the absence of direct biologic evidence for an infectious etiology in childhood leukemia, further epidemiologic studies are required. Future studies should be designed to test whether the risk of cALL increases in association with certain major histocompatibility types,211 with reduced infections in infancy, with the absence of prolonged breast feeding, and with a lack of social contact with other children in infancy. Some of these associations have been reported in smaller case-control studies and caseseries surveys.182,212

Interactions between genetic and environmental factors Biologic markers (biomarkers) related to genetic mutational events can provide a means of evaluating interactions between inherited genetic susceptibility and environmental health hazards in the etiology of childhood acute leukemias. Biomarkers of both exposure and effect not only provide bases for assessing interactions but may be indicative of future disease risk. At present, however, there is little information on the predictive value of these assays for populations or individuals. The following sections discuss some biomarkers currently under investigation that may generate new insights into leukemogenesis.

Microsatellite instability Microstatellite DNA is noncoding DNA consisting of short tandem repeat sequences that are unique to each individual. Microsatellite instability (MSI) is characterized by mutations in these short tandem repeat sequences, which appear to reflect multiple replication errors brought on by defective mismatch repair genes and which contribute to a “mutator phenotype.”213 MSI has been implicated in several human malignancies, including hereditary nonpolyposis colon cancer, gliomas, and lung cancer. Few studies have investigated MSI in childhood leukemia. The largest such study to date examined 48 primary samples from ALL patients and found that five (10%) exhibited MSI.214 Of interest, the authors found that several of the sites of instability were located in chromosomal regions associated with childhood ALL (including regions containing the TEL gene

in chromosome 12p, the p16 gene on chromosome 9, and the long arm of chromosome 6). Although the overall frequency of instability was low, these data suggest that localized MSI may identify a fragile chromosomal region that could result in an alteration of surrounding target genes and thus lead to leukemia in some children. In another study, Baccichet et al.215 demonstrated alterations in microsatellite patterns in five of six patients with childhood T-cell ALL. Further evidence of genomic instability was provided by a loss of heterozygosity in chromosomes 6p, 9p, and 12p. In fact, two-thirds of these patients had deletion of chromosome 9p21, the location of the tumor suppressor gene p16. Larger studies are needed to further characterize the role of MSI in childhood leukemia.

Mutation frequencies Somatic mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene are rare occurrences in the T lymphocytes of normal individuals. Lacking pathogenic significance, these events can serve as biomarkers for assessing environmental genotoxicity. Finette et al.216 demonstrated an early childhood HPRT mutational spectrum that was quite distinct from the adult background spectrum. This age-frequency distribution of HPRT mutations correlates with the age-frequency distribution of childhood ALL and merits further exploration.216

HLA-DR and susceptibility to childhood ALL Since the demonstration of the influence of the major histocompatibility complex (MHC) on mouse leukemia, an HLA association has been considered as a possible genetic risk factor. Dorak et al.217 have demonstrated a moderate association with the most common allele in the HLA-DR53 group, HLA-DRB1*04, that was stronger in males.217 In addition, homozygosity for HLA-DRB4*01, encoding the HLA-DR53 specificity was increased among patients. These associations could possibly suggest a malespecific increase in homozygosity for HLA-DRB4*01. The cross-reactivity between HLA-DR53 and H-2Ek, extensive mimicry of the immunodominant epitope of HLA-DR53 by several carcinogenic viruses, and the extra amount of DNA in the vicinity of the HLA-DRB4 gene argue for the case that HLA-DRB4*01 may be one of the genetic risk factors for childhood ALL. Comparison of DQA1 and DQB1 alleles in a case-control study revealed that male but not female patients had a higher frequency of DQA1*0101/*0104 and DQB1*0501, thus suggesting a male-associated susceptibility haplotype in ALL, supporting an infectious etiology.218

Epidemiology and etiology

Susceptibility to childhood leukemia: influence of CYP1A1, CYP2D6, GSTM1 and GSTT1 genetic polymorphisms Several investigators have examined the role of polymorphisms in genes encoding drug-metabolizing enzymes such as glutathione S-transferases and cytochrome P-450 in the development of pediatric cancers. Both genes are involved in carcinogen metabolism and have been shown to influence the risk of a variety of adult cancers.219–222 Davies et al.223 demonstrated that the GSTM1 null genotype is significantly more frequent among childhood AML, particularly the M3 and M4 FAB groups.223 Chen et al.224 compared the frequency of the null phenotype for GSTTI or GSTM1, or both, in children with ALL with that in healthy controls. Their results showed that the double-null genotype of GSTT1 and GSTM1 is more common among black than white children with ALL. However, Davies et al.225 failed to show any association between GST polymorphisms and the risk of developing childhood ALL.225 Possible links between the risk of ALL and inducibility of the drug-metabolizing enzyme CYP1A1 have been hypothesized.226 The results of these studies indicate a possible role of gene-environment interaction in the etiology of childhood leukemia that needs to be explored in greater detail.

Issues and future directions Impressive advances in the understanding of leukemia cell biology and the treatment of childhood acute leukemia stand in striking contrast to the relatively limited progress toward understanding the etiology of this heterogeneous group of diseases. Epidemiologic research addressing the etiology of childhood cancer, while considerable in volume, has been limited because of several issues. These include the retrospective nature of study designs, restricting the incorporation of biologic and clinical parameters, and the difficulties in identifying a sufficiently large study population to allow subgroup analyses. In the context of casecontrol studies, the design of choice for all childhood cancer epidemiologic studies, recall bias is a major concern. With regard to biologic samples, the main tissue studied has been blood. Studies that seek to use blood for biomarkers are likely to face difficulties with poor participation rates, especially in relation to control subjects. These barriers may be overcome by the development of techniques using very small samples, or the use of saliva. Case-control studies that examine evidence of infection face problematic temporality. There is mounting evidence that the cellular and molecular characteristics of biologically distinct

subgroups may define the most promising approaches for future epidemiologic/etiologic investigations. Specifically, it may be most productive to evaluate environmental and/or genetic hypotheses within well-defined, wellcharacterized, homogeneous groups of leukemias. Standard analytic epidemiologic investigations of more broadly defined populations of childhood leukemia have provided a relatively limited amount of etiologically relevant data over the past three decades. Several large epidemiologic studies – most of which incorporate detailed biological characterization of cases – are currently nearing completion in North America and Europe. If these investigations fail to identify biologically relevant associations, it is possible that the etiology of the majority of childhood acute leukemias may remain elusive for decades to come.

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121 Leiss, J. K. & Savitz, D. A. Home pesticide use and childhood cancer: a case-control study. Am J Public Health, 1995; 85: 249–52. 122 Zahm, S. H. Childhood leukemia and pesticides. Epidemiology, 1999; 10: 473–5. 123 Daniels, J. L., Olshan, A. F., & Savitz, D. A. Pesticides and childhood cancers. Environ Health Perspect, 1997; 105: 1068–77. 124 Shu, X. O., Stewart, P., Wen, W. Q., et al. Parental occupational exposure to hydrocarbons and risk of acute lymphocytic leukemia in offspring. Cancer Epidemiol Biomarkers Prev, 1999; 8: 783–91. 125 Steensel-Moll, H. A. van, Valkenburg, H. A., Vandenbroucke, J. P., & Zanen, G. E. van. Are maternal fertility problems related to childhood leukaemia? Int J Epidemiol, 1985; 14: 555–9. 126 Gold, E. B., Diener, M. D., & Szklo, M. Parental occupations and cancer in children – a case-control study and review of the methodologic issues. J Occup Med, 1982; 24: 578–84. 127 McKinney, P. A., Roberts, B. E., O’Brien, C., et al. Chronic myeloid leukaemia in Yorkshire: a case control study. Acta Haematol, 1990; 83: 35–8. 128 Shaw, G., Lavey, R., Jackson, R., & Austin, D. Association of childhood leukemia with maternal age, birth order, and paternal occupation. A case-control study. Am J Epidemiol, 1984; 119: 788–95. 129 Infante-Rivard, C., Mur, P., Armstrong, B., Alvarez-Dardet, C., & Boulmar, F. Acute lymphoblastic leukaemia among Spanish children and mothers’ occupation: a case-control study. J Epidemiol Community Health, 1991; 45: 11–5. 130 Schuz, J., Kaletsch, U., Meinert, R., Kaatsch, P., & Michaelis, J. Risk of childhood leukemia and parental self-reported occupational exposure to chemicals, dusts, and fumes: results from pooled analyses of German population-based case-control studies. Cancer Epidemiol Biomarkers Prev, 2000; 9: 835–8. 131 Lyons, R. A., Monaghen, S. P., Heaven, M., et al. Incidence of leukaemia and lymphoma in young people in the vicinity of the petrochemical plant at Baglan Bay, South Wales, 1974 to 1991. Occup Environ Med, 1995; 52: 225–8. 132 Sans, S., Elliott, P., Kleinschmidt, I., et al. Cancer incidence and mortality near the Baglan Bay petrochemical works, South Wales. Occup Environ Med, 1995; 52: 217–24. 133 Freedman, D. M., Stewart, P., Kleinerman, R. A., et al.. Household solvent exposures and childhood acute lymphoblastic leukemia. Am J Public Health, 2001; 91: 564–7. 134 Pearson, R. L., Wachtel, H., & Ebi, K. L. Distance-weighted traffic density in proximity to a home is a risk factor for leukemia and other childhood cancers. J Air Waste Manag Assoc, 2000; 50: 175–80. 135 Reynolds, P., Elkin, E., Scalf, R., Von Behren, J., & Neutra, R. R. A case-control pilot study of traffic exposures and early childhood leukemia using a geographic information system. Bioelectromagnetics, 2001; Suppl. 5: S58–68. 136 Reynolds, P., Von Behren, J., Gunier, R. B., et al. Traffic patterns and childhood cancer incidence rates in California, United States. Cancer Causes Control, 2002; 13: 665–73. 137 Alexander, F., Cartwright, R., McKinney, P. A., & Ricketts, T. J. Investigation of spacial clustering of rare diseases: childhood

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malignancies in North Humberside. J Epidemiol Community Health, 1990; 44: 39–46. Wulff, M., Hogberg, U., & Sandstrom, A. Cancer incidence for children born in a smelting community. Acta Oncol, 1996; 35: 179–83. Feychting, M., Plato, N., Nise, G., & Ahlbom, A. Paternal occupational exposures and childhood cancer. Environ Health Perspect, 2001; 109: 193–6. Robison, L. L., Buckley, J. D., Daigle, A. E., et al. Maternal drug use and risk of childhood nonlymphoblastic leukemia among offspring. An epidemiologic investigation implicating marijuana (a report from the Children’s Cancer Study Group). Cancer, 1989; 63: 1904–11. Sarasua, S. & Savitz, D. A. Cured and broiled meat consumption in relation to childhood cancer: Denver, Colorado (United States). Cancer Causes Control, 1994; 5: 141–8. Thompson, J. R., Gerald, P. F., Willoughby, M. L., & Armstrong, B. K. Maternal folate supplementation in pregnancy and protection against acute lymphoblastic leukaemia in childhood: a case-control study. Lancet, 2001; 358: 1935–40. Peters, J. M., Preston-Martin, S., London, S. J., et al. Processed meats and risk of childhood leukemia (California, USA). Cancer Causes Control, 1994; 5: 195–202. Buckley, J. D., Buckley, C. M., Ruccione, K., et al. Epidemiological characteristics of childhood acute lymphocytic leukemia. Analysis by immunophenotype. The Children’s Cancer Group. Leukemia, 1994; 8: 856–64. Severson, R. K., Buckley, J. D., Woods, W. G., Benjamin, D., & Robison, L. L. Cigarette smoking and alcohol consumption by parents of children with acute myeloid leukemia: an analysis within morphological subgroups – a report from the Children’s Cancer Group. Cancer Epidemiol Biomarkers Prev, 1993; 2: 433–9. Duijn, C. M. van, Steensel-Moll, H. A. van, Coebergh, J. W., & Zanen, G. E. van. Risk factors for childhood acute nonlymphocytic leukemia: an association with maternal alcohol consumption during pregnancy? Cancer Epidemiol Biomarkers Prev, 1994; 3: 457–60. Shu, X. O., Ross, J. A., Pendergrass, T. W., et al. Parental alcohol consumption, cigarette smoking, and risk of infant leukemia: a Children’s Cancer Group study. J Natl Cancer Inst, 1996; 88: 24–31. Sorahan, T., Lancashire, R., Prior, P., Peck, I., & Stewart, A. Childhood cancer and parental use of alcohol and tobacco. Ann Epidemiol, 1995; 5: 354–9. Stjernfeldt, M., Berglund, K., Lindsten, J., & Ludvigsson, J. Maternal smoking and irradiation during pregnancy as risk factors for child leukemia. Cancer Detect Prev, 1992; 16: 129–35. Mucci, L. A., Granath, F., & Cnattingius, S. Maternal smoking and childhood leukemia and lymphoma risk among 1,440,542 Swedish children. Cancer Epidemiol Biomarkers Prev, 2004; 13: 1528–33. John, E. M., Savitz, D. A., & Sandler, D. P. Prenatal exposure to parents’ smoking and childhood cancer. Am J Epidemiol, 1991; 133: 123–32.

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152 Buckley, J. D., Hobbie, W. L., Ruccione, K., et al. Maternal smoking during pregnancy and the risk of childhood cancer [letter]. Lancet, 1986; 2: 519–20. 153 Pershagen, G., Ericson, A., & Otterblad-Olausson, P. Maternal smoking in pregnancy: does it increase the risk of childhood cancer? Int J Epidemiol, 1992; 21: 1–5. 154 McKinney, P. A. & Stiller, C. Maternal smoking during pregnancy and the risk of childhood leukaemia. Lancet, 1986; 2: 519. 155 Li, F. P. Maternal smoking during pregnancy and the risk of childhood cancer. Lancet, 1986; 2: 520. 156 Ji, B. T., Shu, X. O., Linet, M. S., et al. Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers. J Natl Cancer Inst, 1997; 89: 238–44. 157 Sorahan, T., Lancashire, R. J., Hulten, M. A., Peck, I., & Stewart, A. M. Childhood cancer and parental use of tobacco: deaths from 1953 to 1955. Br J Cancer, 1997; 75: 134–8. 158 Brondum, J., Shu, X. O., Steinbuch, M., et al. Parental cigarette smoking and the risk of acute leukemia in children. Cancer, 1999; 85: 1380–8. 159 Sandler, D. P., Wilcox, A. J., & Everson, R. B. Cumulative effects of lifetime passive smoking on cancer risk. Lancet, 1985; 1: 312–5. 160 Kaye, S. A., Robison, L. L., Smithson, W. A., et al. Maternal reproductive history and birth characteristics in childhood acute lymphoblastic leukemia. Cancer, 1991; 68: 1351–5. 161 Yeazel, M. W., Buckley, J. D., Woods, W. G., Ruccione, K., & Robison, L. L. History of maternal fetal loss and increased risk of childhood acute leukemia at an early age. A report from the Children’s Cancer Group. Cancer, 1995; 75: 1718–27. 162 Gibson, R. W., Bross, I. D. J., & Graham, S. Leukemia in children exposed to multiple risk factors. N Engl J Med, 1968; 279: 906–9. 163 Ross, J. A., Potter, J. D., Shu, X. O., et al. Evaluating the relationships among maternal reproductive history, birth characteristics, and infant leukemia: a report from the Children’s Cancer Group. Ann Epidemiol, 1997; 7: 172–9. 164 Westergaard, T., Andersen, P. K., Pedersen, J. B., et al. Birth characteristics, sibling patterns, and acute leukemia risk in childhood: a population-based cohort study. J Natl Cancer Inst, 1997; 89: 939–47. 165 Hemminki, K., Kyyronen, P., & Vaittinen, P. Parental age as a risk factor of childhood leukemia and brain cancer in offspring. Epidemiology, 1999; 10: 271–5. 166 Dockerty, J. D., Draper, G., Vincent, T., Rowan, S. D., & Bunch, K. J. Case-control study of parental age, parity and socioeconomic level in relation to childhood cancers. Int J Epidemiol, 2001; 30: 1428–37. 167 Zack, M., Adami, H. O., & Ericson, A. Maternal and perinatal risk factors for childhood leukemia. Cancer Res, 1991; 51: 3696–701. 168 Hjalgrim, L. L., Rostgaard, K., Hjalgrim, H., et al. Birth weight and risk for childhood leukemia in Denmark, Sweden, Norway, and Iceland. J Natl Cancer Inst, 2004; 96: 1549–56 169 Petridou, E., Skalkidou, A., Dessypris, N., et al. Endogenous risk factors for childhood leukemia in relation to the IGF system

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(Greece). The Childhood Haematologists-Oncologists Group. Cancer Causes Control, 2000; 11: 765–71. Lei, U., Wohlfahrt, J., Hjalgrim, H., et al. Neonatal level of thyroid-stimulating hormone and acute childhood leukemia. Int J Cancer, 2000; 88: 486–8. Gale, K. B., Ford, A. M., Repp, R., et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A, 1997; 94: 13 950–4. Pui, C. H., Ribeiro, R. C., Hancock, M. L., et al. Acute myeloid leukemia in children treated with epipodophyllotoxins for acute lymphoblastic leukemia. N Engl J Med, 1991; 325: 1682–7. Broeker, P. L., Super, H. G., Thirman, M. J., et al. Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites. Blood, 1996; 87: 1912–22. Wang, J. C. DNA topoisomerases. Annu Rev Biochem, 1996; 65: 635–92. Ross, J. A., Potter, J. D., Reaman, G. H., Pendergrass, T. W., & Robison, L. L. Maternal exposure to potential inhibitors of DNA topoisomerase II and infant leukemia (United States): a report from the Children’s Cancer Group. Cancer Causes Control, 1996; 7: 581–90. Ross, J. A. Maternal diet and infant leukemia: a role for DNA topoisomerase II inhibitors? Int J Cancer Suppl, 1998; 11: 26–8. Strick, R., Strissel, P. L., Borgers, S., Smith, S. L., & Rowley, J. D. Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc Natl Acad Sci U S A, 2000; 97: 4790–5. Alexander, F. E., Patheal, S. L., Biondi, A., et al. Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion. Cancer Res, 2001; 61: 2542–6. Ross, J. A., Potter, J. D., & Robison, L. L. Infant leukemia, topoisomerase II inhibitors, and the MLL gene. J Natl Cancer Inst, 1994; 86: 1678–80. Greave, M. F. Infant leukaemia biology, aetiology and treatment. Leukemia, 1996; 10: 372–7. Kinlen, L. J. Epidemiological evidence for an infective basis in childhood leukaemia. Br J Cancer, 1995; 71: 1–5. Anderson, R. M. & May, R. M. Immunisation and herd immunity. Lancet, 1990; 335: 641–5. Boutou, O., Guizard, A. V., Slama, R., Pottier, D., & Spira, A. Population mixing and leukaemia in young people around the La Hague nuclear waste reprocessing plant. Br J Cancer, 2002; 87: 740–5. Langford, I. Childhood leukaemia mortality and population change in England and Wales 1969–73. Soc Sci Med, 1991; 33: 435–40. Stiller, C. A. & Boyle, P. J. Effect of population mixing and socioeconomic status in England and Wales, 1979–85, on lymphoblastic leukaemia in children. BMJ, 1996; 313: 1297– 300.

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186 Dickinson, H. O. & Parker, L. Quantifying the effect of population mixing on childhood leukaemia risk: the Seascale cluster. Br J Cancer, 1999; 81: 144–51. 187 Koushik, A., King, W. D., & McLaughlin, J. R. An ecologic study of childhood leukemia and population mixing in Ontario, Canada. Cancer Causes Control, 2001; 12: 483–90. 188 Dickinson, H. O., Hammal, D. M., Bithell, J. F., & Parker, L. Population mixing and childhood leukaemia and non-Hodgkin’s lymphoma in census wards in England and Wales, 1966–87. Br J Cancer, 2002; 86: 1411–3. 189 Parslow, R. C., Law, G. R., Feltbower, R., Kinsey, S. E., & McKinney, P. A. Population mixing, childhood leukaemia, CNS tumours and other childhood cancers in Yorkshire. Eur J Cancer, 2002; 38: 2033–40. 190 Baccate, E. M. Social patterns of antibody to poliovirus. Lancet, 1983; 1: 778–83. 191 Dworsky, M., Yow, M., Stagno, S., Pass, R. F., & Alford, C. Cytomegalovirus infection of breast milk and transmission in infancy. Pediatrics, 1983; 72: 295–9. 192 Parker, L. Breast-feeding and cancer prevention. Eur J Cancer, 2001; 37: 155–8. 193 Ma, X., Buffler, P. A., Selvin, S., et al. Daycare attendance and risk of childhood acute lymphoblastic leukaemia. Br J Cancer, 2002; 86: 1419–24. 194 Perrillat, F., Clavel, J., Auclerc, M. F., et al. Day-care, early common infections and childhood acute leukaemia: a multicentre French case-control study. Br J Cancer, 2002; 86: 1064–9. 195 Gilham, C., Peto, J., Simpson, J. et at. Day care in infancy and risk of childhood acute lymphoblastic leukaemia: findings from UK case-contol study. BMJ, 2005; 330: 1294. 196 Rosenbaum, P. F., Buck, G. M., & Brecher, M. L. Early child-care and preschool experiences and the risk of childhood acute lymphoblastic leukemia. Am J Epidemiol, 2000; 152: 1136–44. 197 Neglia, J. P., Linet, M. S., Shu, X. O., et al. Patterns of infection and day care utilization and risk of childhood acute lymphoblastic leukaemia. Br J Cancer, 2000; 82: 234–40. 198 Gahrton, G., Wahren, B., Killander, D., & Foley, G. E. Epstein– Barr and other herpes virus antibodies in children with acute leukemia. Int J Cancer, 1971; 8: 242–9. 199 Groves, F. D., Sinha, D., Kayhty, H., Goedert, J. J., & Levine, P. H. Haemophilus influenzae type b serology in childhood leukaemia: a case-control study. Br J Cancer, 2001; 85: 337–40. 200 Heegaard, E. D., Jensen, L., Hornsleth, A., & Schmiegelow, K. The role of parvovirus B19 infection in childhood acute lymphoblastic leukemia. Pediatr Hematol Oncol, 1999; 16: 329–34. 201 MacKenzie, J., Gallagher, A., Clayton, R. A., et al. Screening for herpesvirus genomes in common acute lymphoblastic leukemia. Leukemia, 2001; 15: 415–21. 202 MacKenzie, J., Perry, J., Ford, A. M., Jarrett, R. F., & Greaves, M. JC and BK virus sequences are not detectable in leukaemic samples from children with common acute lymphoblastic leukaemia. Br J Cancer, 1999; 81: 898–9. 203 Salonen, M. J., Siimes, M. A., Salonen, E. M., Vaheri, A., & Koskiniemi, M. Antibody status to HHV-6 in children with leukaemia. Leukemia, 2002; 16: 716–9.

204 Dockerty, J. D., Skegg, D. C., Elwood, J. M., et al. Infections, vaccinations, and the risk of childhood leukaemia. Br J Cancer, 1999; 80: 1483–9. 205 McKinney, P. A., Juszczak, E., Findlay, E., Smith, K., & Thomson, C. S. Pre- and perinatal risk factors for childhood leukaemia and other malignancies: a Scottish case control study. Br J Cancer, 1999; 80: 1844–51. 206 Schuz, J., Kaatsch, P., Kaletsch, U., Meinert, R., & Michaelis, J. Association of childhood cancer with factors related to pregnancy and birth. Int J Epidemiol, 1999; 28: 631–9. 207 McKinney, P. A., Cartwright, R. A., Saiu, J. M., et al. The interregional epidemiological study of childhood cancer (IRESCC): a case control study of aetiological factors in leukaemia and lymphoma. Arch Dis Child, 1987; 62: 279–87. 208 Naumburg, E., Bellocco, R., Cnattingius, S., Jonzon, A., & Ekbom, A. Perinatal exposure to infection and risk of childhood leukemia. Med Pediatr Oncol, 2002; 38: 391–7. 209 Chan, L. C., Lam, T. H., Li, C. K., et al. Is the timing of exposure to infection a major determinant of acute lymphoblastic leukaemia in Hong Kong? Paediatr Perinat Epidemiol, 2002; 16: 154–65. 210 Greaves, M. F. & Alexander, F. E. An infectious etiology for common acute lymphoblastic leukemia in childhood? Leukemia, 1993; 7: 349–60. 211 Taylor, G. M. & Birch, J. M. The hereditary basis of human leukemia. In E. S. Henderson, T. A. Lister, & M. F. Greaves, eds., Leukemia (Philadelphia, PA: W. B. Saunders, 1996), pp. 210–45. 212 Petridou, E., Kassimos, D., Kalmanti, M., et al. Age of exposure to infections and risk of childhood leukaemia. BMJ, 1993; 307: 774. 213 Tasaka, T., Lee, S., Spira, S., et al. Microsatellite instability during the progression of acute myelocytic leukaemia. Br J Haematol, 1997; 98: 219–21. 214 Takeuchi, S., Seriu, T., Tasaka, T., et al. Microsatellite instability and other molecular abnormalities in childhood acute lymphoblastic leukaemia. Br J Haematol, 1997; 98: 134–9. 215 Baccichet, A., Benachenhou, N., Couture, F., Leclerc, J. M., & Sinnett, D. Microsatellite instability in childhood T cell acute lymphoblastic leukemia. Leukemia, 1997; 11: 797–802. 216 Finette, B. A., Poseno, T., & Albertini, R. J. V(D)J recombinasemediated HPRT mutations in peripheral blood lymphocytes of normal children. Cancer Res, 1996; 56: 1405–12. 217 Dorak, M. T., Lawson, T., Machulla, H. K., et al. Unravelling an HLA-DR association in childhood acute lymphoblastic leukemia. Blood, 1999; 94: 694–700. 218 Taylor, G. M., Robinson, M. D., Binchy, A., et al. Preliminary evidence of an association between HLA-DPB1*0201 and childhood common acute lymphoblastic leukaemia supports an infectious aetiology. Leukemia, 1995; 9: 440–3. 219 Rothman, N., Wacholder, S., Caporaso, N. E., et al. The use of common genetic polymorphisms to enhance the epidemiologic study of environmental carcinogens. Biochim Biophys Acta, 2001; 1471: C1–10. 220 Nakachi, K., Imai, K., Hayashi, S., & Kawajiri, K. Polymorphisms of the CYP1A1 and glutathione S-transferase genes associated

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with susceptibility to lung cancer in relation to cigarette dose in a Japanese population. Cancer Res, 1993; 53: 2994–9. 221 Hirvonen, A., Nylund, L., Kociba, P., Husgafvel-Pursiainen, K., & Vainio, H. Modulation of urinary mutagenicity by genetically determined carcinogen metabolism in smokers. Carcinogenesis, 1994; 15: 813–5. 222 Chen, H., Sandler, D. P., Taylor, J. A., et al. Increased risk for myelodysplastic syndromes in individuals with glutathione transferase theta 1 (GSTT1) gene defect. Lancet, 1996; 347: 295–7. 223 Davies, S. M., Robison, L. L., Buckley, J. D., et al. Glutathione S-transferase polymorphisms in children with

myeloid leukemia: a Children’s Cancer Group study. Cancer Epidemiol Biomarkers Prev, 2000; 9: 563–6. 224 Chen, C. L., Liu, O., Pui, C. H., et al. Higher frequency of glutathione S-transferase deletions in black children with acute lymphoblastic leukemia. Blood, 1997; 89: 1701–7. 225 Davies, S. M., Bhatia, S., Ross, J. A., et al. Glutathione Stransferase genotypes, genetic susceptibility, and outcome of therapy in childhood acute lymphoblastic leukemia. Blood, 2002; 100: 67–71. 226 Blumer, J. L., Dunn, R., Esterhay, M. D., Yamashita, T. S., & Gross, S. Lymphocyte aromatic hydrocarbon responsiveness in acute leukemia of childhood. Blood, 1981; 58: 1081–8.

Part II Cell biology and pathobiology

4 Anatomy and physiology of hematopoiesis Connie J. Eaves and Allen C. Eaves

Introduction Hematopoiesis refers to all aspects of the process of blood cell production. Understanding this process requires a comprehensive knowledge of both the anatomy and the physiology of the blood-forming system. Here, the anatomy of the hematopoietic system is viewed as the distinguishable stages of differentiation that together make up the complete hierarchy of hematopoietic cells. These stages reflect the changes that initially endow cells in the embryo with hematopoietic differentiation potential (a step referred to as specification), in addition to those that subsequently constitute the processes of lineage restriction and terminal differentiation. The physiology of hematopoiesis refers to the dynamic aspects of these events and covers issues such as the determination of alternate outcomes, at both the cellular and molecular level, as well as their modulation during development and in response to injury or disease. Leukemias arise from clonal accumulations of mutations that impact the production and differentiation of blood cells. Because of the low probability of such events, a large number of divisions is thought to be required for their successive acquisition. It is therefore not surprising that, in many leukemias, the first leukemogenic mutation appears to take place in a hematopoietic stem cell. Moreover, in spite of the acute picture of many leukemias, there is growing evidence that they may result from relatively subtle perturbations of the mechanisms that regulate normal hematopoiesis. A framework for understanding normal hematopoiesis is therefore essential to obtaining new insights into the nature and better management of these diseases.

The concept of a hierarchical model of hematopoiesis Normal adult blood contains large numbers of highly specialized cells that perform critical physiological functions. Because the life-span of most of these cells is relatively short, their replacement and the mechanisms that control new blood cell output are key to survival. Red blood cells (RBCs, also called erythrocytes) are the most numerous cell type in the blood. Their primary role is to transport oxygen from the lung to peripheral tissues and, for this, the concentration of RBCs in the blood needs to be tightly maintained at 5 × 1012 per liter. In the average adult with a 5-liter blood volume, this requires a controlled output of 2 × 1011 new RBCs each day to replace those that have reached the end of their normal 120-day life-span. Each liter of adult blood also contains approximately 7 × 109 white blood cells (WBCs, also called leukocytes). These are responsible for the elimination of bacteria and viruses. Most of the WBCs are either neutrophilic granulocytes (4 × 109 per liter) or different kinds of lymphocytes [B cells, T cells and natural killer (NK) cells, 2 × 109 per liter] with smaller numbers of monocytes, and eosinophilic and basophilic granulocytes making up the remainder. The life-span of human neutrophils in the blood is very short (approximately 1 day), and the number of neutrophils generated each day has been estimated to be approximately 6 × 1010 . Platelets constitute another important blood cell type because of their role in controlling the clotting process. Platelets are produced by fragmentation of megakaryocytes in the bone marrow and are present in the blood of normal adults at a concentration of approximately 300 × 109 per liter. The average life-span of the human platelet is

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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9 days, which dictates a production rate of approximately 25 × 1010 per day (albeit from a much smaller number of megakaryocytes).1 Thus, even with respect to just the myeloid lineages, hematopoiesis in the normal adult provides for a daily output of approximately half a trillion new blood cells of six different lineages. The first insights into how new blood cells are formed came from light microscopic studies of bone marrow preparations. This led to the description of a hierarchy of precursors for each myeloid lineage. The relationship of these morphologically defined cells to successive cell divisions was then deduced from pulse-labeling experiments.2–4 These studies established that the terminal differentiation of erythroid and granulopoietic cells in adults is completed over the course of three to five cell generations that cover a period of 5 to 7 days. At the same time, the number of cells in each of these lineages is progressively amplified, although cell death is used to finetune cell numbers in successive generations. These observations laid the foundation of a model of hematopoiesis in which differentiation is unidirectional and irreversible and leads to a predominance of terminally differentiating cells. The concept of a persisting population of more primitive cells able to produce multiple types of blood cells was first inferred from the observation in 1951 that the bone marrow of patients with myeloproliferative disorders (MPDs) contains elevated numbers of precursors of all lineages, even though only one mature blood cell type is increased in the circulation.5 More definitive evidence of such pluripotent hematopoietic cells was provided a few years later from experiments showing the production in sublethally irradiated mice of lymphoid and myeloid cells carrying the same clonal cytogenetic markers.6 This was accompanied by the related discovery in humans of a consistent unique cytogenetic abnormality, the Philadelphia (Ph) chromosome, in erythroid, granulopoietic and megakaryocytic cells in patients with chronic myeloid leukemia (CML).7,8 Formal evidence that a population of transplantable cells with long-term lympho-myeloid-reconstituting potential is maintained in normal adult human bone marrow was first reported in 1989.9 Together, these findings established the functional identity of pluripotent hematopoietic stem cells; i.e. cells able to produce some undifferentiated progeny that remain competent to produce all blood cell lineages, as well as progeny that have begun to differentiate irreversibly. However, as discussed in more detail below, only in the last 15 years have quantitative assays specific for murine hematopoietic stem cells defined in this way been devised. Analogous assays for human hematopoietic stem cells are still being

refined. In the interim, much confusion has arisen from the common use of the term stem cell to refer to primitive cells that display features that are shared by, but are not exclusive to hematopoietic stem cells (e.g. expression of CD34). Hematopoietic stem cells, in addition to being able to give rise to progenitors of the various blood cell lineages, must have mechanisms for blocking the activation of their latent differentiation potential. These mechanisms allow some of their progeny to remain in the same undifferentiated, but competent state, thereby allowing the production of new cohorts of blood cells to be sustained throughout adult life. Stem cell divisions that produce at least one daughter stem cell are called self-renewal divisions. If both progeny remain as stem cells, or if both begin to differentiate, the division is described as “symmetric.” If only one of the progeny retains its stem cell properties, the division is described as “asymmetric.” The introduction of functional assays that detect the unique developmental profiles of both murine and human hematopoietic cells with more limited proliferative and differentiation abilities also started in the 1960s. These indicated that execution of the full hematopoietic differentiation process can span many cell generations, even before the acquisition of changes that are overt at a morphological level. Accordingly, a much more extensive hierarchy of intermediate, functionally distinguished cell types is now envisaged (Fig. 4.1). The terms precursor and progenitor have been widely adopted to distinguish between intermediate cell types that either already have, or have not yet, reached the point when morphologic features of a particular lineage first appear. It can be seen that the progenitor pool comprises a large and heterogeneous spectrum of cell types. Some of these are transplantable and may play a significant role in the immediate supply of mature blood cells, particularly in the first month of hematologic recovery in myeloablated recipients of hematopoietic transplants. The development of lymphoid cells differs from the development of myeloid cells in several respects. For example, the terminal differentiation of lymphoid cells is often not accompanied by gross morphologic changes. In addition, reproducible procedures for supporting their growth and differentiation in vitro have been more difficult to establish, particularly for cells of human origin. On the other hand, extensive progress has been made in the identification of sequential stages of lymphoid differentiation based on changes in the molecules they display on the cell surface, and in the transcription factors they express. However, this aspect of hematopoiesis is reviewed in detail elsewhere and therefore is not covered further in this chapter.

Anatomy and physiology of hematopoiesis

Myeloblasts

and

Myelocytes

Fig. 4.1 The process of hematopoietic cell differentiation in normal adults viewed as a developmental hierarchy of functionally distinguished cell types. See text for discussion of developmental stages and for abbreviations of cell types. (Reprinted, with permission, from Eaves.10 )

The developmental origin of hematopoiesis During embryogenesis, the first recognizeable blood cells are nucleated RBCs and macrophages that appear in the extraembryonic yolk sac blood islands. In mice, these are derived from primitive erythroid and macrophage progenitors that develop in the proximal regions of the egg cylinder at the primitive streak stage.11 This is followed rapidly by the appearance in the yolk sac of hematopoietic cells with multilineage myeloid potential and an ability to produce definitive RBCs.11–15 Such cells also arise concurrently, but independently, in the developing aorticgonad-mesonephros (AGM) region inside the embryo from primitive mesenchymal cells with both endothelial and hematopoietic potential (hema-angioblasts). Current evidence suggests that the AGM region is the primary source of hematopoietic stem cells with lympho-myeloid differentiation potential and an ability to reconstitute irradiated mice.16 More limited studies of human embryos indicate a similar intraembryonic origin of human hematopoietic stem cells.17 After the circulation has developed and the heart begins to function, the fetal liver becomes the primary hematopoietic organ with subsequent colonization of the developing thymus, spleen and

bone marrow.13,18,19 Throughout adult life, hematopoiesis continues predominantly in the bone marrow. However, reactivation of extramedullary hematopoiesis in the liver and spleen can occur after hyperstimulation with growth factors, during marrow regeneration, or in association with the development of an MPD (e.g. CML). Investigation of the potentialities of the cells produced during the genesis of the hematopoietic system have indicated that the first cells to display the key properties of hematopoietic stem cells (multilineage differentiation potential and an ability to execute many divisions without loss of this potential) are not generated until after the first mature blood cells have already been produced. Thus, the mechanism(s) regulating when the first lineage restriction events begin in a multipotent hematopoietic cell (the selfrenewal decision) and the mechanism(s) regulating how lineage restriction will be effected (the determination decision) can be activated independently in time. This suggests that these mechanisms are likely to be molecularly distinct, as illustrated schematically in Fig. 4.2,20 and serves to introduce the idea of additional complexities in the process by which blood cells are produced in different situations. Many other aspects of hematopoiesis also change during development. These include the particular growth

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Embryo MD

perturbations in the phenotype or behavior of leukemic populations,47 since they may be secondary to an increased turnover rate, or to the reactivation of pathways that are more characteristic of fetal rather than adult hematopoietic cells.

Adult MD

1

3

PC

HSC

5

2

HSC Wnt

4

PC 2

Fig. 4.2 Schematic model of the progressive development of primitive hematopoietic cells. During embryogenesis, cells with pluripotent hematopoietic potential arise (step 1) from mesodermal precursors (MD). These pluripotent cells (PC) generate macrophages and/or erythroid cells (step 2) directly but do not have long-term self-maintaining ability. Later, mesodermal precursors generate a different type of pluripotent cell (step 3) that meets the definition of a hematopoietic stem cell (HSC) because it can divide without immediately activating its latent competence for multilineage differentiation. According to the signals these HSCs receive, they thus either give rise to progeny pluripotent cells (step 4) that then rapidly differentiate, or they can self-renew (step 5). Recent evidence suggests that the Wnt--catenin pathway may be involved in regulating the choice between steps 4 and 5.21 The separately timed appearance of cells with hematopoietic and self-renewal ability suggests that the regulation of lineage selection and self-renewal involve distinct molecular mechanisms. (Reprinted, with permission, from Eaves20 )

factors to which cells at comparable stages of differentiation respond, 22–28 the time taken for differentiating hematopoietic cells to transit the cell cycle,29,30 and the choice of certain lineage-specific genes expressed during terminal blood cell differentiation (e.g. globin genes in erythroid cells31,32 and T-cell receptor genes in developing T-cells33 ). Changes also occur in the proportion of primitive cells that are cycling (i.e. that are not in G0 ) and in the phenotype of these cells, as a result of their altered cycling/activation status (as discussed further below).34–41 Hematopoietic stem cells present at different times during development have been shown to differ in their average clonal output of daughter stem cells42–44 and in the ordering of the lineage restriction process they undergo.45,46 All of these variations are relevant to understanding apparent

Functional assays for different hematopoietic cell types Early steps in hematopoietic cell differentiation that precede the onset of terminal maturation events take place in cells that share a similar “undifferentiated” (blast) morphology. In normal adult bone marrow, such cells constitute less than 5% of all the cells present. Identification of the various steps that hematopoietic stem cells undergo before the appearance of overt morphologic changes characteristic of particular lineages has been made possible by the development of a series of functional assays that detect the growth and differentiation properties of hematopoietic cells when they are stimulated to proliferate and differentiate either in vivo or in vitro. Combining the use of retrospective functional end points with a strategy that allows these to be assigned to individual cells in the original test cell suspension, makes it possible to measure progenitor frequencies, often when these are less than 10−5 . This gives such functional assays the extraordinary sensitivity required to detect very rare progenitors and to monitor their numbers and responses to different manipulations in a quantitative fashion. Much of what we now know of the process of normal hematopoiesis has thus been obtained using functional assays that allow cells at different stages of hematopoietic cell differentiation to be discriminated by virtue of the numbers and types of mature progeny they generate, and the speed and conditions under which this is achieved. Critical to the utility of functional assays for detecting primitive hematopoietic cells is the use of reproducible conditions for stimulating the cells being evaluated, both in terms of assessing their differentiation potential (number of lineages represented among the daughter cells produced) and in assessing their proliferative potential (total number of mature cells produced). The use of well-defined, objective end points for these parameters is also an important issue. In vivo assays generally depend on transplanting various numbers of test cells into hosts given partially or severely myeloablative treatments to create conditions that will strongly promote stem/progenitor cell stimulation while decreasing the competition from endogenous stem/progenitor cells. However, it is difficult to completely

Anatomy and physiology of hematopoiesis

eliminate the host stem cells by such treatments without having lethal effects on other tissues. Therefore, deriving definitive conclusions about the regenerative potential of transplanted cells requires the use of a donor that is compatible, but genetically distinguishable from the recipient so that the donor (versus host) origin of the blood cells regenerated can be unequivocally established. In vitro assays typically make use of defined (or at least standardized) conditions to stimulate particular primitive cell types to divide and differentiate in a reproducible manner. They must also support the terminal differentiation of all lineage(s) whose representation is required to establish the potentiality(ies) of the original cell being tested. This may require the adoption of sequential culture conditions that differ significantly. A key feature of both in vivo and in vitro functional assays for various primitive hematopoietic cell types is their ability to measure the frequency in a given test population of individual cells with the properties of interest. One of the attractions of the first in vivo assay developed (the spleen colony assay, see below) was the fact that progenitor frequency data could be obtained simply by counting the number of macroscopic colonies visible on the surface of the spleen.48 However, for measurements of the frequency of most other types of cells with in vivo reconstituting ability, limiting dilution methods have to be used.49,50 The frequency of the initial cell of interest is then calculated using Poisson statistics and the method of maximum likelihood.51 Both direct colony-scoring and limiting-dilution analysis make use of a minimal cell output criterion to detect the input cells of interest. Accordingly, these methods circumvent the problems of inferring input cell numbers from “activities” measured in bulk assays, which can be skewed by changes in the average output potential of the cells being quantified. Both of these methods for quantifying the frequency of a given cell type rely on the validity of two important assumptions: (i) that the readout is linearly related to the number of cells tested over the range used, and (ii) that a single cell is responsible for a positive score. These assumptions have been validated for some populations48,52–58 but remain a potential concern when the test cells have been manipulated or altered genetically. A careful appreciation of the specificity of a functional assay for a particular type of hematopoietic cell is another important issue in interpreting data derived from the use of such procedures. In fact, many of the functional assays described for hematopoietic cells detect populations of cells with different biologic properties. Thus, a change in the total number of cells detected with a particular assay will not necessarily mean that all subsets in the original

test population identified by that assay will have changed in parallel. For example, rapid production of mature blood cells in vivo is a property of many types of primitive hematopoietic cell types, ranging from those with a very short-term lineage-restricted growth potential to those with life-long multilineage repopulating ability. Thus, early blood cell recovery data can provide misleading information about the numbers of stem cells transplanted. Similarly, biologically distinct types of primitive hematopoietic cells can be stimulated to form colonies in vitro under the same growth factor conditions. In both cases, the most reliable parameters for discriminating between subsets of cells at different stages of hematopoietic differentiation have been the minimal number of divisions that occur prior to the appearance of the first mature progeny and the durability of their continued output. Another limitation of functional assays for primitive hematopoietic cells is their dependence on entire programs of differentiation being faithfully completed. For some assays, this may require waiting for a period of many weeks or even months. In addition, cells that are more primitive than those that may be adversely affected can masquerade as being defective or may fail to be detected even though they, themselves, are functionally normal or present in normal numbers. Such a situation could arise in cells with altered genomes, or in cells subjected to certain nonphysiological treatments. Similarly, the loss of a feature required for a given cell type to be detected, but not key to its intrinsic differentiation status or proliferative potential, will preclude its identification. Either of these situations can lead to confusion about the biologic processes targeted by various treatments or mutations. Functional assays have also been usefully applied to investigations of many types of transformed hematopoietic cell populations, particularly where the process of differentiation is minimally altered. Examples of diseases where morphologically normal blood cells are produced include both the MPDs and the myelodysplastic diseases (MDS). In contrast, the block in differentiation characteristic of acute myeloid leukemia (AML), has made the development of analogous functional assays for these cells more difficult. Nevertheless, in AML, other features of the early stages of differentiation appear frequently to be retained. This has allowed the use of both cell surface phenotyping and functional assays to construct a hierarchical organization of cells within the neoplastic clone of many types of human AML.59,60 Such a model is also consistent with the occurrence of the first leukemogenic mutation(s) in a hematopoietic stem cell.61,62 Because of their central importance to the investigation of both normal and leukemic hematopoiesis, the most

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commonly used functional assays are reviewed in greater detail below.

5% and 20%67,81,82 and may be influenced by a number of cellular and host parameters (reviewed by Lord83 ).

Competitive repopulating units (CRUs)

Cells with in vivo repopulating activity Colony-forming unit-spleen (CFU-S) The spleen colony assay detects a rare subpopulation of primitive murine hematopoietic cells that are stimulated to generate localized colonies of terminally differentiating cells in the spleens of mice given a myeloablative treatment, or in whom endogenous hematopoiesis is genetically compromised.48,63 If cells with this ability are present in the spleen in sufficiently low numbers, their progeny may, within the first week and a half, be visualized on the surface of the spleen as discrete macroscopic colonies containing a few hundred thousand cells. Most such colonies contain maturing cells of a single lineage64–66 and do not contain cells that can generate new spleen colonies in secondary hosts67 or multilineage colonies in vitro.68 After longer periods, larger spleen colonies containing several million cells may be seen. These typically contain both terminally differentiating granulopoietic and erythroid cells,52 as well as cells that can generate secondary multilineage colonies both in vivo67 and in vitro.68,69 The cells from which all of these spleen colonies arise are referred to operationally as “colony-forming units-spleen,” or CFU-S. However, CFU-S are not a biologically homogeneous population. Careful time-course analyses,68,70 and cell-separation71–74 and biologic characterization studies,75 have established that the CFU-S assay detects a spectrum of early hematopoietic cell types. Some CFU-S are lineage-restricted and have quite limited proliferative potential. These properties account for the small size and rapid, but transient, appearance of the spleen colonies they generate. Other CFU-S have multilineage myeloid differentiation potential, extensive proliferative potential and an independently variable self-renewal potential.52,67,69 A subset of CFU-S can also generate cells able to permanently reconstitute lympho-myelopoiesis in irradiated hosts.76,77 However, the converse is not true; i.e. most cells able to permanently reconstitute lymphomyelopoiesis in irradiated hosts do not have CFU-S activity.78,79 CFU-S appear early in ontogeny and are subsequently maintained throughout adulthood, primarily in the marrow.80 When injected intravenously, only a proportion of these cells end up in the spleen and form macroscopically sized colonies within 2 weeks. This proportion (the seeding fraction) has been estimated to range between

Studies of the in vivo-generated progeny of hematopoietic cells that had acquired unique radiation-induced cytogenetic markers provided the first evidence in mice that large, persisting, multilineage clonal populations of hematopoietic cells could be produced.6,84 Subsequently, similar observations were made using transplants of hematopoietic cells that were genetically marked by unique retroviral inserts.85–87 These and additional serial transplantation experiments88 established the remarkable proliferative potential of normal hematopoietic stem cells but were not useful for quantifying them or for investigating their heterogeneity. The competitive repopulating unit (CRU) assay provides the specificity required for the exclusive quantification of hematopoietic stem cells with life-long blood cellproducing activity.49,89 As shown schematically in Fig. 4.3, this assay simply involves injecting serial dilutions of test cells into congenic hosts and then measuring the fraction of mice in each group with detectable levels of circulating B-cell, T-cell and myeloid cells derived from the transplanted cells after a period of at least 4 months. The recipients are also given a supplemental transplant (or a less myelotoxic treatment) to ensure that they all survive independent of whether they receive any CRUs in the test cells injected. The 4-month interval is used to ensure that the multilineage progeny of cells with limited self-renewal ability will have disappeared.74,90,91 The frequency of CRUs in the test cell suspension can then be calculated with Poisson statistics. Although the contribution of individual CRUs to the mature B-, T- and myeloid cells in the peripheral blood varies greatly at any given time and over time,91,92 it is possible to derive average clone sizes for CRUs from different sources. These average output values can then be used for extensive comparative analyses in studies that would not be practical to undertake with limiting dilution methodologies.93 The CRU assay should not be confused with the longterm competitive transplantation methodology developed by Harrison.94,95 This procedure compares the relative stem cell activity of two cell populations (again using genetic markers to distinguish their progeny) but does not measure their frequency. Additional statistical methods can be applied to the variance data obtained from such experiments to calculate the frequencies of the input stem cell populations. However, the validity of the values thus derived is contingent on the assumption that the average

Anatomy and physiology of hematopoiesis

Test cells

Irradiation

> 16 wks

37% negative mice*

No. of test cells/mouse 2 × 105 compromised cells (2 × serial BMT) or 105 normal BM cells or No cells using sublethally irradiated W41 recipients

*Positive = >0.5% Iymphoid and >0.5% myeloid repopulation by test cells

Fig. 4.3 The mouse CRU assay. This procedure uses the principles of limiting-dilution analysis to measure the frequency of cells in a given suspension that have transplantable long-term repopulating ability and can individually generate both lymphoid and myeloid progeny.89 The treatment of the host is chosen to maximize the sensitivity of the assay by reducing the competing endogenous stem cell population to a minimum and creating an environment in which the engrafting stem cells will be optimally stimulated. This can be achieved by pretreating normal mice with a myeloablative treatment (e.g. a lethal dose of radiation) or by giving c-kit mutant mice (whose stem cells are defective27,63 ) a sublethal treatment. In order for a limiting dilution analysis of the stem cell content of the test cell suspension to be performed, the recipients must be able to survive regardless of whether they receive any stem cells in the test cells injected. Survival of normal recipients is assured by cotransplanting them with hematopoietic cells of the same genotype that contain sufficient numbers of short-term repopulating cells but minimal numbers of long-term repopulating cells. Survival of c-kit mutant hosts is similarly assured by pretreating them with a dose of radiation that allows significant numbers of endogenous cells to survive. The differentiated blood cell progeny of the test cells and the recipients must be genetically distinguishable and assessed at a time when they can safely be assumed to represent the exclusive output of cells with life-long stem cell potential. Strains of mice congenic with the C57B1/6 mouse are typically used to allow the blood cell progeny of the test cells to be uniquely identified by CD45 (Ly5) allotype markers or glucose phosphate isomerase isoform differences. Poisson statistics are then used to calculate the frequency of CRUs in the original test cell suspension from the proportions of mice whose blood does not contain both lymphoid and myeloid cells of donor origin at 4 months post-transplant (or longer). Blood cells present after 4 months are assumed to be derived from stem cells with life-long hematopoietic activity. BM, bone marrow; BMT, bone marrow transplantation.

cell outputs are not different, which is not an invariant feature of different stem cell populations. Cell populations in which at least 40% of the cells can be detected as CRUs in single cell transplant experiments have been isolated by a variety of strategies demonstrating the robustness of this assay.92,96,97 These experiments also indicate that the ultimate marrow seeding efficiency of long-term repopulating stem cells must be almost 100%. Nevertheless, only 10% of injected CRUs can be detected in the bone marrow 24 hours after they have been injected intravenously.98 The full explanation for this apparent discrepancy is not yet known. One possibility is that many CRUs initially locate in other tissues, as has been shown to occur in mice assessed at later times post-transplantation.99–101 Alternatively, CRUs may seed the marrow efficiently, but then rapidly lose this ability while still retaining their full developmental properties. Support for this latter concept is provided by the observation that CRUs acquire a reversible engraftment defect when they are in the S/G2 /M phases of the cell cycle,102,103

in contrast to cells with shorter-term repopulating activities, such as those detectable as CFU-S.104,105 Primitive human hematopoietic cells can also home efficiently to the marrow of various xenogeneic hosts, allowing the characterization of human hematopoietic cells with in vivo repopulating ability. Intravenous injection of human hematopoietic cells into neonatal or sublethally irradiated adult mice with various genetically determined immunodeficiency states (and hence containing greatly reduced numbers of B, T and NK cells) can lead to repopulation of the marrow of these mice with large numbers of human lymphoid and myeloid cells.106–111 Intraperitoneal injections of preimmune fetal sheep produce a similar result.112,113 Both types of experimental models take advantage, not only of the immune tolerance of the hosts, but also of the growth-promoting conditions in the fetal and early postnatal marrow, or induced by a myelosuppressive pretreatment. Prior removal of immunocompetent human cells from the cells to be injected is important to prevent the development of graft-versus-host disease, which can be

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fatal.112,114 Nonobese diabetic-scid/scid (NOD/SCID) mice and NOD/SCID-β2microglobulin−/− mice have been the most widely used hosts to date and have the advantage that they breed easily and are efficiently engrafted by specific subsets of human hematopoietic cells,108,115 if these are injected intravenously before the mice reach 10 weeks of age.116 A significant limitation of these particular mice is their rapid development of fatal endogenous thymomas between 6 and 12 months of age, which precludes their use for long-term follow-up studies.114 Hopefully, the alternative use of long-lived Rag2−/− γc−/− mice110,111 or NOD/SCID-nu/nu mice117 may circumvent this problem in the future. In all of these hosts, human cells belonging to multiple hematopoietic lineages are generated, even in the absence of exogenously supplied human growth factors. In addition, human cells capable of repopulating secondary fetal sheep118–120 or secondary immunodeficient mice108,121–123 are produced in the primary hosts. This finding implies that these hosts can be engrafted by human hematopoietic stem cells and that the xenogeneic environment can support the self-renewal of these cells. In sublethally irradiated immunodeficient murine hosts, an early wave of predominantly erythroid cells and some megakaryocytes is seen in the first 2 to 3 weeks. These cells are then rapidly replaced by a larger population of predominantly pre-B cells and cells of the neutrophil and macrophage lineages.108,122,124,125 Species-specific factors are thus also not essential to support some multilineage human hematopoietic cell differentiation. However, growth factor administration can enhance the production of both primitive and mature human cell types, indicating that those produced by the host are likely suboptimal in type and/or amount.121,126–128 In NOD/SCID and NOD/SCID-β2microglobulin−/− mice, the production of mature human erythroid and megakaryocytic cells,129 and mature B cells and T/NK cells122,123,130 is particularly compromised. This may be related in part to the poor survival of human cells in the bloodstream of such mice.126,130 The human cell population regenerated in NOD/SCID mice thus typically contains a disproportionately high number of more primitive (CD34+ ) cells in comparison to primary human hematopoietic tissues. Adult cells appear more sensitive to the mechanisms responsible for the poor terminal differentiation of their progeny than are human cord blood or fetal liver transplants, and the compromised output of differentiated progeny from adult cells can be partially reversed by the administration of pharmacologic doses of human-specific growth factors.126,127

Interestingly, transgenic NOD/SCID mice engineered to produce human interleukin-3 (IL-3), human granulocyte colony-stimulating factor (G-CSF) and human Steel factor have increased numbers of human cells in the blood and enhanced human granulopoiesis in the marrow, with reduced output of primitive cells and more mature human cells of other lineages.131 This suggests that this method of achieving sustained exposure to high levels of these growth factors may lead to premature exhaustion of the graft through a continuous mobilization of primitive cells into the circulation. The time allowed to elapse before assessing the human progeny produced in the mice can be a critical variable when using this end point to infer the differentiated state of the injected cells responsible for their generation.108,123,132,133 This issue is particularly relevant in interpreting data obtained from experiments with immunodeficient murine hosts that cannot be followed for more than 5 months, since studies in the sheep model have suggested that the output of cells from short-term repopulating cells may be significant for 6 to 9 months.119 In addition, the immune status of the host can differentially affect the ability of different subsets of human hematopoietic cells to engraft and/or survive.108 The most immunocompromised mice are engrafted with the greatest spectrum of human repopulating cells, and there is a progressive selection in favor of the most primitive cells as the NK activity of the murine host increases. Thus, only the most primitive cells efficiently engraft NOD/SCID mice and their progeny constitute the majority of the cells present after a few weeks. NOD/SCID mice thus serve as a relatively selective host for the early quantification of the most primitive type(s) of hematopoietic cells in human tissues. In contrast, the NOD/SCID-β2microglobulin−/− mouse is highly permissive for multiple types of human repopulating cells. End points for detecting three distinct subsets of human cells with different short- and long-term repopulating activities in these hosts have been devised (Fig. 4.4). The features used to distinguish these three cell types include differences in their immunophenotypes, differences in their engrafting abilities when passing through S/G2 /M, and differences in their abilities to generate progeny that will repopulate secondary NOD/SCID mice.108,123,132,134 Limiting-dilution approaches, similar to those developed for measuring murine CRUs, are used to measure the frequency of each of these cell types in different sources of human cells.50,108,129,135 Frequency values for normal adult human marrow, mobilized peripheral blood, cord blood and 3- to 4-month gestational fetal liver are given in Table 4.1.

Anatomy and physiology of hematopoiesis

STRC-ML (34 + 38 −)

Human cells/mouse BM

A

Cell cycle-unrestricted engraftment, do not make NOD/SCID repopulating cells

B

NOD/SCID-b2m −/− 107

STRC-M (34 + 38 +)

LTRC-ML (34 + 38 −)

NOD/SCID

105

Cell cycle-restricted engraftment, self-renew 3

6

13

3

Table 4.1 Frequencies of human CRUs (NOD/SCID lympho-myeloid repopulating cells) in various sources of hematopoietic cells

Tissue

106

6

13

77

Fetal liver (12–20 weeks) Cord blood Adult bone marrow G-CSF-mobilized blood

CRU frequency per 105 CD34+ cells (95% CI)

Reference

9 (5–15)

Holyoake et al.129

6 (3–11) 0.8 (0.5–1.3) 0.06 (0.03–0.1)

Holyoake et al.129 Holyoake et al.129 Van der Loo et al.516

Time post-transplant (weeks)

Fig. 4.4 Different types of human cells with repopulating activity in sublethally irradiated immunodeficient mice (reprinted, with permission, from Glimm et al.108 ). (A) Consistently higher levels of human hematopoietic cells present in the marrow of NOD/SCID-β2microglobulin−/− (NOD/SCID-β2m−/− ) mice as compared with NOD/SCID mice after their transplantation with the same number of normal adult human bone marrow cells (depleted of mature cells expressing erythroid, granulopoietic, megakaryopoietic, or lymphoid lineage markers). (B) Explanation for these differences in terms of the differential ability of human cells with short- and long-term repopulating ability (STRC and LTRC) to engraft the two mouse strains. Most of the first human cells produced in the marrow of the more immunodeficient NOD/SCID-β2microglobulin−/− mice are derived from a type of human repopulating cell that is myeloid -restricted and has very short-lived repopulating activity. The progeny of these cells are then superceded by a second cohort of human lymphoid and myeloid cells derived from a cell that is more primitive but still not self-sustaining. Human cells with long-term repopulating ability and self-renewal activity also engraft the NOD/SCIDβ2microglobulin−/− mice but, because of their relatively low numbers, their progeny in the NOD/SCID-β2microglobulin−/− mice are greatly outnumbered by the progeny of the short-term repopulating cells. Neither of the two types of human short-term repopulating cell engrafts NOD/SCID mice efficiently. Therefore, repopulation of NOD/SCID mice can provide a relatively selective early measure of human cells with long-term repopulating activity.

Abbreviations: CRV, competitive repopulating unit; G-CSF, granulocyte colony-stimulating factor; CI, confidence interval.

specific factors required for the growth of the T-ALL cells.148 The pattern of malignant disease that develops in the engrafted mice usually mimics that seen in the patient from whom the malignant cells were taken. In addition, the AML-repopulating cells share many features with normal human CRUs, including a low frequency and a quiescent CD34+ CD38− phenotype.60,149,150 On the other hand, differences between transplantable normal and AML stem cells have also been demonstrated; for example, leukemic stem cells usually do not express detectable Thy-1 in contrast to their normal counterparts.151 Engraftment of mice with cells from patients with MDS152,153 or chronic phase CML154,155 has also been reported. However, the typically low numbers of very primitive neoplastic cells and/or the predominance of residual normal stem cells in diagnostic marrow or blood samples from patients with these diseases has made it very difficult to develop useful xenotransplant models or to characterize the most primitive subsets of malignant cells.

Cells with in vitro hematopoietic activity Long-term culture-initiating cells (LTC-ICs)

Cells from patients with a variety of hematologic disorders have also been used to successfully engraft immunodeficient mice, and in many instances evidence of the production of neoplastic or leukemic human cells has been documented. These xenotransplant experiments include AML,60,136–138 B-lineage acute lymphoid leukemia (ALL),139–141 CML in blast crisis,142,143 myeloma144–146 and chronic lymphocytic leukemia (CLL).147 Successful transplantation of immunodeficient mice with human T-lineage ALL (T-ALL) has required the prior transplantation of other human cells, presumably to produce unknown species-

Another assay for quantifying a very primitive hematopoietic cell population is one that detects a cell that can initiate sustained hematopoiesis when cocultured on stromal feeder layers.54,156,157 The assay for these so-called long-term culture-initiating cells (LTC-ICs) was developed from the observation that mature granulocytes and macrophages can be produced for several months when bone marrow cells are cultured in media containing horse serum and corticosteroids.158–161 Subsequent studies showed that the primary need for serum was to generate a competent feeder layer of stromal cells that

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stimulate the proliferation and differentiation of very primitive hematopoietic cells in the absence of exogenously supplied growth factors.162 To ensure that the latter end point can be quantified independently of the ability of cells in the test suspension to form a competent feeder layer, irradiated pre-established marrow feeders54,156 or irradiated monolayers of a number of human163,164 or murine28,165–168 fibroblast cell lines are used. Many studies have provided evidence of heterogeneity in the phenotypes of different sources of fibroblasts and in their supportive activity,163,164,169–171 but definitive correlations with particular growth factor-producing profiles have not been established. A role of notch-ligands has been suggested,172–175 and the production of specific types of heparan sulfates176 that may help to colocalize primitive hematopoietic cells and growth factors177 may be another feature of some supportive stromal cells. This concept is certainly consistent with the finding that larger numbers of human LTC-ICs, including a more primitive subset, are detected when test cells are cocultured on competent murine fibroblasts that have been engineered to produce ng/ml quantities of human G-CSF, human IL-3, and human Steel factor constitutively.178 Such feeders are, therefore, now routinely used to optimize human LTC-IC detection.179 However, definitive characterization of the mechanisms responsible for the underlying functionality of stromal cells in LTC-IC assays has remained elusive. The principles underlying the LTC-IC assay are similar to those used to develop the in vivo CRU assay. The LTC system, like the irradiated host, supports the proliferation and differentiation of mature cells (granulocytes and macrophages) from progenitors at multiple stages of differentiation. Therefore, sufficient time must be allowed to elapse for all intermediate types of progenitors in the original test suspension to have exhausted their proliferative potential so that the cells ultimately detected can be safely assumed to have been exclusively derived from a very primitive subpopulation. Early studies indicated that this condition was met after an interval of 4 to 5 weeks when the standard in vitro colony-forming cell (CFC) assay (see below) was used to detect the progeny of the input cells.156,157 However, it was subsequently found that simply prolonging this interval to 6 weeks provides significantly greater specificity for very primitive human cells.178,180 LTC-IC frequencies are best determined by limiting-dilution analysis. If, however, the average output of progeny CFCs per LTCIC is known, the total output of CFCs in a bulk culture can simply be divided by this value, assuming first that the number of LTC-ICs used to initiate the assay is linearly related to the number of CFCs they will produce (at the test cell

Table 4.2 Frequencies of human LTC-ICs in various sources of hematopoietic cells

Tissue Fetal liver (12–20 weeks) Cord blood Adult bone marrow G-CSF-mobilized blood Normal adult blood

LTC-IC frequency (± SEM) 760 ± 170 per 106 low-density cells 310 ± 410 per 106 low-density cells 430 ± 280 per 106 low-density cells 270 ± 40 per 106 low-density cells 0.40 ± 0.14 per ml

Reference Pawliuk et al.43 Hogge et al.178 Hogge et al.178 Hogge et al.178 Eaves et al.517

Abbreviations: LTC-IC, long-term culture-initiating cells; G-CSF, granulocyte colony-stimulating factor.

dose tested) and, second, that the total number of LTC-ICs actually assayed is sufficient to accommodate the highly variable CFC output exhibited by individual LTC-ICs.54,55 Both the murine55 and human181–184 LTC-IC assays have been further modified to allow detection of input cells with B and NK lymphoid potential as well as myeloid differentiation potential. Characterization of murine CRUs and LTC-ICs (defined by a 4-week end point) has shown that the cells detected by both of these assays have similar frequencies and properties throughout ontogeny.28,55,157 In addition, some LTCICs generate progeny that are detectable as CRUs.157,185–187 Taken together, these findings indicate a close relationship between the populations detected by both assays. Nevertheless they are not identical, or at least they are not detected at the same efficiency in vivo and in vitro, since a suspension of 40% pure murine CRUs was found to contain only 25% LTC-ICs.92 In addition, the maintenance of mouse bone marrow cells under LTC conditions results in a more rapid loss of cells detectable as CRUs than of cells detectable as LTC-ICs.188 The human LTC-IC assay also detects very primitive hematopoietic cells that appear to overlap with, but are not identical to, CRUs as shown also by immunophenotyping studies and investigations of changes in their numbers under different conditions in vitro.40,50,189–191 Values for LTC-IC frequencies in different normal human tissues are given in Table 4.2. The LTC-IC assay can also detect a rare subpopulation of primitive leukemic progenitor cells present at highly variable frequencies in the marrow and blood of newly diagnosed CML192 and AML patients.193,194 Interestingly, the detection of either CML or AML LTC-ICs is not enhanced

Anatomy and physiology of hematopoiesis

Table 4.3 Frequencies of human CFCs in various sources of hematopoietic cellsa Tissue

CFU-E

BFU-E

CFU-GM

CFU-Mk

BFU-Mk

CFU-GEMM

Fetal liver (12–20 weeks) Cord blood Adult bone marrow Normal adult blood

130 ± 50

2900 ± 800

2500 ± 1400

120 ± 90

21 ± 10

490 ± 320

4±1 4±1

58 ± 10 27 ± 5

120 ± 30 35 ± 6

26 ± 11 35 ± 11

15 ± 8 14 ± 7

8±4 2±1

9±2

340 ± 50

98 ± 12

ND

ND

19 ± 3

Abbreviations: CFU-E/GM/Mk/GEMM, colony-forming unit-erythrocyte/granulocyte-macrophage/ megakaryocyte/granulocyte-erythroid-megakaryocyte-macrophage; BFUMk, burst-forming unitmegakaryocyte. a All values shown are mean numbers ± SEM. For fetal liver, cord blood and adult marrow, these are expressed per 105 low-density cells44 and for normal blood per ml.517

by the use of feeders engineered to produce G-CSF, IL-3, and Steel factor,195,196 presumably because of the autocrine mechanisms that are active in these cells.197,198 Residual normal LTC-ICs are also often prevalent in these samples and the progeny of the leukemic LTC-ICs may not be distinguishable from those produced by the normal LTC-ICs except by genotyping or clonality analyses.195,196,199,200

Colony-forming cells (CFCs) Most, if not all, types of hematopoietic progenitor cells can proliferate and differentiate in liquid suspension culture when provided with appropriate nutrients and growth factors. However, the ability of the most primitive hematopoietic cells (both normal and leukemic) to generate such responses may be compromised if they are immediately suspended in a semisolid matrix.201–204 The acquisition of an ability to proliferate in three-dimensional cultures by later hematopoietic progenitor cell types has thus formed the basis of a variety of clonal assay cultures for their separate detection and quantitation. In most cases, optimization of the sensitivity and specificity of in vitro colony assays has required the identification of culture conditions and endpoints that are unique for colonies from each progenitor type to be assessed. Media supplements to stimulate the production of colonies from all stages of lineage-restricted progenitors on each of the major myeloid pathways as well as some multi-potent progenitor cells have been identified and are now commercially available as standardized reagents. For progenitor quantitation, all components are usually added at concentrations that will saturate the growth requirements of the cells produced throughout the duration of the culture.

These requirements may vary according to the types of colonies being generated, independent of any growth factors that might be endogenously produced. Colony assays have also been used extensively for investigations of the types (or concentrations) of factors that can support, stimulate, enhance or block the proliferation and differentiation of various types of normal and leukemic progenitor cells. The most commonly monitored types of CFCs are described below and their frequencies in different human hematopoietic tissues are given in Table 4.3.

Granulopoietic colony-forming cells (CFU-GMs) Colonies of granulocytes and macrophages were the first to be generated in culture205–207 and remain the easiest to obtain and recognize. The progenitors of such colonies were originally called CFU-Cs (colony-forming units-culture) prior to their characterization in mice as a population distinct from CFU-S.208 When conditions were defined that allowed the proliferation and maturation of other lineages in semisolid culture media, evidence of CFCs with restricted granulopoietic activity was obtained and the term CFU-GM (colony-forming unit-granulocytemacrophage) was widely adopted to refer to these. CFUGMs are stimulated to proliferate and differentiate into colonies of mature granulocytes and/or macrophages by any single or combination of growth factors, including GMCSF, IL-3, G-CSF, IL-6, and Steel factor.209,210 IL-5 selectively stimulates eosinophil progenitors.211,212 Different stages of granulopoietic progenitors can be recognized by their ability to make colonies of different sizes, the ultimate size being fixed by the number of divisions the original progenitor undergoes before

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all progeny have reached a stage of maturity at which they can no longer divide. However, on the granulopoietic pathways, the number and rate of late divisions can be influenced by the growth factors to which the cells are exposed.213 Growth factors may also salvage some early granulopoietic cells from apoptosis.214 Nevertheless, progenitors of large and small colonies of granulocytes and/or macrophages generated under standardized culture conditions, have distinct characteristics suggesting that they represent a sequence of cells with decreasing proliferative potential.215,216 In addition, they appear to be regulated differently in vivo. For example, in the marrow of normal adult humans, the CFC-GMs that produce very large colonies of neutrophils and macrophages belong to a very slowly cycling population, whereas those that make smaller colonies appear to be maintained in a state of rapid turnover.217

Erythroid colony-forming cells (CFU-Es, BFU-Es) Erythroid progenitors are detected by their ability to produce colonies of maturing erythroblasts. When these are of murine origin, the erythroblasts produced are identified by their small size and tendency to adhere to one another to form tight clusters of up to approximately 50 tiny cells.218,219 The hemoglobin they contain usually cannot be visualized directly, although it can be readily demonstrated histochemically.219 Terminally differentiating human erythroid cells are somewhat larger and more robust in vitro and the hemoglobin they produce is sufficient to give them a distinctive reddish color in the living state. This allows colonies containing human erythroblasts to be uniquely recognized in cultures that contain colonies of other types of cells, or colonies that contain both erythroid cells and other types of mature cells. Different stages of erythroid progenitor cell development are readily distinguished by the different-sized colonies of erythroblasts that they can generate. The most differentiated erythroid colony-forming cells are called CFU-Es (colony-forming units-erythroid). They display a limited proliferative potential of three to seven divisions resulting in the rapid formation of tight colonies of mature hemoglobinized erythroblasts and are thought to be the immediate precursors of cells recognized morphologically as proerythroblasts. Erythroid progenitors that produce larger colonies are called BFU-Es (burst-forming unitserythroid). This term was coined to reflect the sudden final “burst-like” appearance of terminally differentiating erythroid cells in larger colonies due to the semisynchronous proliferation and differentiation behavior of the cells from which they arise.218,220–222 Primitive and mature

BFU-Es are readily distinguished by the time they take to produce terminally differentiating erythroid cells and display properties that allow their physical and biologic separation as distinct stages of differentiation, including differences in their dependence on stimulation by factors other than erythropoietin (EPO); e.g. Steel factor, IL-3, IL-6, or GM-CSF.69,217,223,224 In the adult, CFU-Es and terminally differentiating erythroid cells require continued stimulation by EPO to stay alive,225 even though they make some erythropoietin themselves.226 The addition of EPO to erythroid colony assays is thus essential to the detection of CFU-Es and BFUEs with a few notable exceptions. These include certain disorders in which a stem cell has acquired a mutation(s) that allow(s) its differentiating erythroid progeny to bypass the normal requirement for EPO stimulation. The best example of this is polycythemia vera (PV),222,227–231 although an abnormal ability of erythropoietic cells to survive and mature in the absence of exogenously supplied EPO is also seen in cells from some patients with CML232,233 and essential thrombocytosis (ET).234–237 Interestingly, the inability of adult erythroid precursors to survive in the absence of stimulation by EPO is also a developmentally acquired property. Thus, the generation of the first primitive RBCs is completely EPO-independent,24 while the definitive erythroid precursors subsequently produced in the fetus are more responsive to low concentrations of EPO than are their adult counterparts.22,23,231

Megakaryopoietic colony-forming cells (CFU-Mks, BFU-Mks) Megakaryocytopoiesis is unique in its termination in a cell that undergoes successive endomitoses to produce megakaryocytes of increasing ploidy. A mature 32n megakaryocyte would thus correspond to an erythroid colony containing 16 erythroblasts. This is why a criterion of only two megakaryocytes is commonly used as the minimum required to identify a megakaryocyte colony. The progenitors of megakaryocyte colonies are referred to as CFU-Mks (colony-forming cells-megakaryocyte) and BFUMks (burst-forming units-megakaryocyte) for the progenitors of larger megakaryocyte colonies (which often resemble erythroid BFU-E-derived colonies and may overlap with them). Murine megakaryocytes can be specifically identified by their content of acetylcholinesterase, which can be stained histochemically.238 This is not the case for human megakaryocytes; however, these cells can be specifically identified by immunohistochemical methods that detect their expression of platelet-specific antigens, such as CD41

Anatomy and physiology of hematopoiesis

(GpIIb/III). Quantitation of human CFC/BFU-Mks thus requires generating the colonies in a matrix such as agarose or collagen that not only supports their optimal growth but also allows the entire culture to be fixed and stained. Human megakaryopoietic progenitors are exquisitely sensitive to the inhibitory effects of TGF- and hence are best cultured under serum-free conditions.239–241 Their growth and differentiation can be stimulated effectively by a variety of soluble factors, including IL-3, IL-6, GM-CSF, Steel factor, and thrombopoietin (TPO).241–243 Different stages of megakaryocyte progenitor development, like those of their granulopoietic and erythroid counterparts, can be defined on the basis of the number of megakaryocytes the progenitor will produce and, hence, the time required for this process to be complete in a given colony.241,244,245

Pluripotent colony-forming cells (CFU-GEMMs) Cells with multilineage hematopoietic differentiation potentialities can also generate colonies of mature progeny in semisolid cultures.246–249 Those that generate colonies containing erythroid cells and megakaryocytes as well as granulocytes and macrophages are referred to as CFUGEMMs (colony-forming cells-granulocyte, erythroid, megakaryocytic, macrophage). CFU-GEMM-derived colonies are often large, indicative of the high proliferative potential that cells with unrestricted myeloid differentiation potential would be expected to possess. For this reason, a very large colony size has been used by some as the sole criterion for identifying a subset of very primitive progenitors referred to as high proliferative potential-CFCs (HPP-CFCs).250,251 HPP-CFCs likely overlap extensively with those identified as CFU-GEMMs, and a shared feature of the assays for both is the addition of multiple growth factors to the assay cultures.249–252 Because of their uncommitted differentiated status, these primitive progenitors will, during their initial divisions, generate progeny that can be detected as CFCs when replated into secondary semisolid culture assays. The types of daughter cells thus detected may include CFU-GEMMs as well as lineage-restricted CFCs.253–255 A subset of CFUGEMMs also show delayed entry into the first cell cycle in vitro,256,257 allowing their separate transient recognition as small colonies of “blasts” that can represent almost pure populations of CFCs.254,258 In mice, the progenitors of a large proportion of these “blast colonies” can, when appropriately stimulated, generate CFU-S,248 NK cells, B- and T-lymphoid progenitors,259,260 and even occasional CRUs,202 as well as progenitors of all of the myeloid lineages, as long as IL-1 and IL-3 are not present in excessive amounts.261

CFCs in acute leukemia Conditions for obtaining colonies of leukemic blasts in semisolid assays of primary blood and marrow samples have also been available for many years.262–265 Leukemic CFCs are typically cultured in semisolid media containing the same growth factors that are used to stimulate their normal counterparts (i.e. Steel factor, GM-CSF, G-CSF, IL-3, IL-6, and more recently, flk2/flt3-ligand and TPO).196,266–269 Although abnormal autocrine mechanisms are clearly activated in the leukemic CFCs of some patients,197,198 this does not abrogate their dependence on added growth factors to achieve optimal growth in low density cultures.53,196 Many of the colonies generated by sources of highly enriched AML blast populations fail to complete normal differentiation programs, providing early confidence of their origin from leukemic progenitors. However, in AML, the initial transforming event leading to the generation of a dominant (but mutant) clone does not necessarily perturb the subsequent ability of the progeny to differentiate normally.61,62,270 Even in some cases of ALL, where the initial transforming event appears to occur in a lympho-myeloid stem cell, colonies of genetically abnormal, but morphologically normal terminally differentiating cells may be detected.271,272 Thus, not all blast cell colonies are leukemic, and some leukemic progenitors can make colonies of normally differentiated cells. Both of these facts have thus made it difficult to use colony assays for the routine assessment and characterization of leukemic progenitor populations. Nevertheless, blast colony assays have been important in establishing the concept of a progenitor hierarchy within human leukemic populations and have begun to allow their properties and abnormal regulation to be characterized.59,194

Use of phenotypic markers to describe different stages of hematopoietic cell differentiation The power of phenotype analysis The retrospective and often lengthy nature of functional assays for detecting primitive hematopoietic cells has given great impetus to the search for more immediate methods to quantify hematopoietic cell populations, particularly in cases where the cells of interest cannot be distinguished by standard morphologic criteria or are too rare for this approach to be useful. Multiparameter flow cytometry offers great power in this regard because it allows cells to be distinguished on the basis of molecularly determined features in an objective and quantitative

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fashion, with a high degree of specificity and reproducibility, and at high resolution. In addition, this technology can be applied in a sterile manner to live cells and used not only for their analysis, but also for their separation into viable subsets defined by the analysis. These isolated cells can then be assayed for their functional attributes. In this way, the phenotype of different functionally defined cell populations can be identified. In the last few years, as reagents for detecting an increasing number of markers have become available, significant progress has been made in the description of a hierarchy of primitive hematopoietic cell types in terms of changes in their phenotype. The most commonly analyzed markers are cell surface antigens against which specifically reactive monoclonal antibodies have been made. These antibodies can then be labeled either directly or indirectly (via a secondary antibody) with a unique fluorochrome and used to distinguish cells as positive or negative on the basis of their acquired fluorescence. In general, marker antigens have been grouped into two categories. The socalled lineage markers refer to cell surface antigens whose expression on hematopoietic cells is generally confined to particular lineages of terminally differentiating cells. Common examples are: CD3, CD4 and CD8 for T-lineage cells, B220/CD45RA, CD19 and CD20 for B-lineage cells, CD56 for NK-lineage cells, CD13, CD14, CD15, CD66b and CD11b for GM-lineage cells, glycophorin A (Ter119) for erythroid cells and CD41 and CD61 for MK-lineage cells. The use of flow cytometry to monitor cells positive for these markers has greatly facilitated studies of their differentiation under a variety of conditions both in vitro and in vivo. In addition, this approach has provided evidence of some programmatic differentiation in leukemic blasts that do not undergo the additional morphologic changes characteristic of normal terminally differentiating cells. The concept of lineage-specific markers is also widely exploited for obtaining populations of hematopoietic cells that are enriched in the more primitive elements that have not yet begun to express any of these markers.50,167,273 Forward and side (90◦ ) light-scattering characteristics (related to cell size and granularity, respectively) are additional parameters that have been useful in the characterization and separation of subpopulations of hematopoietic cells. Staining with various fluorescent dyes allows primitive hematopoietic cells to be distinguished on the basis of their unique ABC transporter activities.41,77,274–276 Similarly, their differential expression of aldehyde dehydrogenase can be detected by staining with fluorescent substrates for this enzyme.277,278 Analysis of their replication kinetics has been detected by quantitative losses in fluorescence after staining with stable membrane dyes such as

PKH 26279 or stable cytoplasmic dyes such as carboxyfluorescein diacetate succinimidyl ester (CFSE).280,281 Figure 4.5 depicts a hierarchy of phenotypes from adult mouse bone marrow that have been cross-matched with functional (and transcriptional) attributes.282,283 A picture of sequentially changing phenotypes with progressive stages of primitive human hematopoietic cell differentiation is also emerging,108,167,178,284,285 but equivalent functional homogeneity of the phenotypes defined thus far remains to be achieved. Although these studies do suggest that some changes in immunophenotype are shared during hematopoietic cell differentiation in the two species, important differences have also been revealed. Three notable examples discussed in detail below are CD34, CD38 and Flk2/Flt3. Another example is the “side population” (SP) phenotype revealed after staining cells with the lipophilic dye Hoechst 33342.286

Phenotype instability and the limitations of phenotype analysis CD34, an L-selectin-binding sialomucin, is expressed on the surface of a small subpopulation (<90%) of cells in both human and mouse hematopoietic tissues that have progenitor activity in vitro.156,282,287–289 Evidence of CD34 as a candidate stem cell marker on human cells was first prompted by the observation that human LTC-ICs expressed higher levels of CD34 than did the majority of CFCs and that highly purified CD34+ human cells could regenerate blood cell production in xenogeneic,50,118,290 allogeneic291 and autologous292,293 hosts. However, it is important to remember that CD34 expression is not specific to such cells. Therefore, simply monitoring total CD34+ cell numbers can only serve as a useful surrogate indicator of corresponding changes in hematopoietic stem cell numbers when the latter change in parallel with all other types of cells that bear the CD34 marker. This is often not the case because of differences in how hematopoietic stem cell and progenitor populations are regulated or because non-hematopoietic CD34+ cells may be a biologically significant part of the population being manipulated. In addition, CD34 expression is not a consistent feature of all human hematopoietic cells with primitive functional properties. Rare CD34− cells able to engraft xenogeneic hosts and/or produce CD34+ CFCs in vitro can be identified in many sources of freshly isolated populations of human hematopoietic cells.40,120,294,295 In addition, at the level of the human hematopoietic stem cell compartment, expression of CD34 is reversible, expression being determined by the activation status of the cell rather than by its developmental potential.296 A similar control mechanism operates in mice but, in this species,

Anatomy and physiology of hematopoiesis

Pro-T

NK cell

Lymphoid pathway

T cell

IL-7R+

CLP IL-7R+ c-mpl−

SCL (−) GATA-2 (−) NF-E2 (−) GATA-1 (−)

HSC

LT-HSC SCL (++ ++) ++) GATA-2 (++ NF-E2 (−) GATA-1 (±)

C/EBPα (−) PU.1 (+) Aiolos (+) GATA-3 (+)

IL-7R+

B cell c-Kit+, CD34+ FcyRh1, Sca-1−, Lin−

ST-HSC C/EBPα (±) PU.1 (±) Aiolos (±) GATA-3 (±)

Pro-B

GMP Myeloid pathway IL-7R− c-mpl+

CMP

Sca-1+, c-Kit+ IL-7Rα−, Lin−

++) SCL (++ GATA-2 (+) NF-E2 (+) GATA-1 (+)

C/EBPα (+) PU.1 (+) Aiolos (±) GATA-3 (−)

c-Kit+, CD34+ FcyRlo, Sca-1− Lin−

Monocyte

Epo-R−

SCL (+) GATA-2 (−) NF-E2 (−) GATA-1 (−) Epo-R+

++) C/EBPα (++ PU.1 (++ ++) Aiolos (−) GATA-3 (−)

MEP

++) SCL (++ GATA-2 (++ ++) NF-E2 (++ ++) GATA-1 (++ ++)

Granulocyte

Megakaryocyte Erythrocyte

C/EBPα (−) PU.1 (±) Aiolos (−) GATA-3 (−)

c-Kit+, CD34− FcyRlo, Sca-1− Lin-

Fig. 4.5 The process of hematopoietic (myeloid) cell differentiation in normal adult mouse bone marrow viewed as a developmental hierarchy of phenotypically distinguished cell types. Each phenotype has been found to be highly enriched for cells with the functional attributes of decreasing proliferative potential and increasing lineage restriction and associated changes in transcription factor expression, as indicated. LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte–erythroid progenitor. (Reprinted, in modified form with permission, from Akashi et al.282 )

results in the majority of the hematopoietic stem cells in the mature adult being CD34− .58 This difference is likely due to subtle differences in the transcriptional regulation of the CD34 gene in the two species.297,298 In mice, expression of CD34 also typically persists only into the granulopoietic pathway,282 whereas in humans, CD34 expression is a retained feature of all types of CFCs.287 CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+ ) glycohydrolase and a ligand for platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) that is expressed on B-lineage cells and a variety of precursor cells in both humans and mice.299 However, the regulation of CD38 expression in the most primitive subpopulations of human and mouse hematopoietic cells is quite different. In mice, CD38 is not expressed by the activated or proliferating hematopoietic stem cells, and its

expression in these cells is upregulated only when these cells become quiescent.300,301 In contrast, CD38 expression is very low in the most primitive subpopulations of human hematopoietic cells, regardless of their proliferative activity or developmental status.50,166,290,302–304 In addition, expression of CD38 on early human hematopoietic cells can be modulated independently of their differentiation status.305 SP cells are identified by their ability to efflux Hoechst 33342 efficiently at 37◦ C but not in the presence of certain ABC transporter inhibitors.286 The SP phenotype of adult mouse hematopoietic stem cells is determined by the expression of ABCG2 (also known as BCRP1)276,306 and its expression is rapidly downregulated in more differentiated cell populations, both in mice276 and humans.307 Hoechst 33342 staining has thus been useful to devise relatively

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simple strategies for isolating populations that are at least 40% pure CRUs.92,97 The SP phenotype has attracted considerable interest as a candidate general hematopoietic stem cell marker because a small SP population can be demonstrated in hematopoietic cells from many species including humans.308 It selects for all of the NOD/SCID repopulating activity in human fetal liver.191 In the blood of AML patients with high counts of cytogenetically abnormal blasts, cytogenetically normal NOD/SCID repopulating cells are selectively isolated in the SP fraction, in contrast to their abnormal counterparts.309 However, to date, the presence of repopulating cells in the SP fraction of normal adult human hematopoietic tissues or cord blood has not been reported. In addition, in the mouse, the SP phenotype, like the expression of CD34 and CD38, has been found to vary reversibly during development and following activation in the adult.41 Thus, the SP phenotype is also an unstable indicator of the stem cell status of primitive hematopoietic cells. Small populations of SP cells have also been found in a variety of nonhematopoietic tissues.276,310–314 In some cases, functional data have indicated that Hoechst 33342 efflux activity (ABC transporter expression) may be a property shared by stem cells with different developmental potentialities. On the other hand, primitive hematopoietic cells are now known to migrate to and take residence in many organs, including those where SP cells have been found.99–101,315 Thus, some examples of SP cell detection in nonhematopoietic sites are likely to simply reflect the presence of these primitive migrant hematopoietic cells. A number of other markers initially found to be useful for the selective identification and purification of murine CRUs from particular sources have likewise been found to be unstable when other sources of these cells are evaluated. Examples include Mac1 (CD11b),37–39,316 AA4.1,39,317 c-kit,318 FLT2/FLT3,319–321 and Rhodamine123 efflux activity.39,41 Examples of unstable markers of very primitive human hematopoietic cells include HLADR156,322–324 and CD41,325 in addition to CD34 and CD38, described above. Thus, many of the available parameters for discriminating the most primitive hematopoietic cell types are often not reliable when used to enumerate changes in cell numbers, particularly under circumstances where the activation or cycling status of these cells may have been altered. However, the increasing refinement in methodologies for phenotype discrimination, when validated by functional assays, do offer great promise for obtaining highly purified populations that can then be used for gene expression and proteomic studies.

Gene expression changes during hematopoietic cell differentiation The ultimate goal in hierarchical analyses of differentiation processes is to obtain a complete description of sequential cell types at the molecular level. In hematopoiesis, considerable information has already begun to accumulate with the greatest emphasis on transcript profiling. This has mirrored the development of new methods for improving the sensitivity of reverse transcriptase-PCR detection of specific transcripts using nested PCR procedures to amplify cDNAs from very small numbers of cells in a relatively unbiased fashion, and for surveying the entire transcriptome using a variety of hybridization or serial analysis of gene expression (SAGE) approaches. The development and application of these methods have also been greatly facilitated by the sequencing of both the human326,327 and mouse328 genomes. In general, the results of such efforts have corroborated the hierarchical view of hematopoiesis defined by matched functional and phenotypic end points.282,329–336 These strategies have served to identify common program features between primitive cell types in hematopoietic and nonhematopoietic tissues (e.g. neural and embryonic stem [ES] cells335,337,338 ). For more detailed descriptions of the data sets derived from such analyses, the reader is referred to the original papers and associated websites. Another important finding from transcriptome analyses has been the identification in very primitive hematopoietic cells of transcripts previously thought to be exclusive features of highly differentiated cells of particular lineages, a phenomenon initially called “priming.”339 This observation of promiscuous gene expression in primitive hematopoietic cells has been confirmed in more extended studies.329,334,336,340–343 and has revised thinking on the nature of the commitment process as one that involves shutting down alternative transcriptionally accessible programs, rather than the selective activation ab initio of the final gene expression program to be followed.

Regulation of hematopoiesis Basic concepts Four sets of biological response outcomes regulate the number of cells in a given compartment of differentiating populations: survival versus death, proliferation versus quiescence, self-renewal versus differentiation, and persistence in or departure from a particular environment

Anatomy and physiology of hematopoiesis

(“niche”). In this regard it may be useful to draw an analogy between each of these four types of responses and a simple chemical equation in which the direction of the equation, and hence the probability of one outcome over the other, is determined by the number, status and availability of relevant signaling intermediates and downstream transcription factor complexes in the affected cells. The physiology of hematopoiesis might thus be viewed as a complex, but eventually tractable, series of problems in chemical engineering once all of the parameters and “reaction rate constants” are defined. Interestingly, according to such a model the responses of individual members of a cell population will appear to be “stochastic” when the parameters that can influence outcomes are close to equilibrium; but will appear “determined” or “fixed” when a particular outcome is strongly favored. Such influences may be either intrinsic or extrinsic to the target cell and the relative “importance” of these in regulating the various responses of different types of hematopoietic cells has been a subject of interest and debate for many years. Extrinsic control mechanisms refer to those that involve changes in the cellular environment and alter cellular behavior through interactions with receptors on the cell surface or internally. These are typically upstream of intracellular targets and allow the operation of sensitive feedback control loops targeting cells at all stages of differentiation to maintain overall blood cell output at a constant level, or to introduce changes to respond appropriately to developmental or injury-induced demands. Intrinsic control mechanisms refer to those molecular interactions that take place within hematopoietic cells to mediate changes in their subsequent behavior. Conceptually, it has been useful to categorize such mechanisms into those that terminate in changes that are reversible in successive cell cycles (e.g. those associated with cell proliferation or adhesion/migration) and those that are not (e.g. those associated with differentiation or death). For hematopoietic cells, many molecules able to modulate these responses are known and, in some cases, it has been possible to translate information about factors that regulate specific hematopoietic cell responses into clinically useful applications. The cloning and now routine clinical use of hematopoietic growth factors (EPO and G-CSF in particular) to promote RBC output and neutrophil recovery, respectively, or to mobilize stem cells for transplant harvests are prototypic examples of how basic observations have been effectively extended into clinical procedures.209,344 There is also a growing paradigm of mutated or aberrantly expressed genes associated with leukemogenesis that are turning out to be important reg-

ulators of normal hematopoietic cell viability (e.g. BCL2 in lymphoma345 ), stem cell proliferation and self-renewal (e.g. FLK2/FLT3 in AML346 ) and differentiation (e.g. AML1347 and SCL348 ), or genes that perturb these responses (e.g. BCRABL349 ). However, in most cases, our knowledge of how particular hematopoietic responses are regulated is still too fragmentary to predict the consequences of up- or downregulating the expression or activity of a single molecule, or to devise methods for targeting such an action to a particular cell type.

Control of the viability and proliferation of hematopoietic cells The internal molecular pathways that regulate cell viability350–352 are highly conserved, not only between cell types within a given organism but also during evolution. However, as is the case for many vital control processes, this has resulted in multiple related molecules that perform similar roles in different cell types in response to cell-specific external cues. For example, primitive human hematopoietic cells have been found to express low levels of the proapoptotic proteins BAX, BAD and BAK, and high levels of the antiapoptotic protein BCLXL but low levels of its related family member, BCL2.353 In contrast, BCL2 is necessary for the survival of lymphoid cells354,355 and BIM, a proapoptotic protein, for control of their numbers.356 BCLX appears to be important for the survival of maturing erythroid cells357 and A1, another antiapoptotic family member, for neutrophil survival358 and BIM, for platelet output.356 Mechanisms that regulate cell cycle progression, like those that regulate cell viability, are also highly conserved.359–361 Various members of the intracellular cell cycle progression control machinery, including the Dtype cyclins, the cyclin-dependent kinases (CDKs) and their various inhibitors also appear to be differentially expressed and involved at different stages of hematopoietic cell differentiation.362–366 Notably, inactivation of the CDK inhibitor, p21, inhibits the normal arrest of cell cycling of hematopoietic stem cells in the adult and results in their accumulation and premature exhaustion,367 whereas inactivation of p27 has a selective effect in promoting cell expansion only at the level of intermediate progenitors.368 Not surprisingly, overexpression or inactivation of many of these genes have also been implicated in leukemia and lymphoma. Both the regulation of apoptosis and cell cycle progression in hematopoietic cells is dependent on the external activation of specific growth factor receptors the cells express on the cell surface. Ligand binding then

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activates intracellular signaling intermediates culminating in a mitogenic and/or survival response. Many primitive hematopoietic cells appear to require constant growth factor activation to maintain both of these responses,209 although lower levels of receptor binding,369 or binding of fewer types of receptors257,370–374 appear to be sufficient to sustain survival as compared to stimulating progression through the cell cycle. At the level of terminally differentiating erythroid and myeloid cells, regulation of cell numbers appears to be mediated primarily through the maintenance or loss of cell viability controlled by growth factor-receptor interactions.214,225 Most of the cells at this stage of differentiation are either cycling or dying and there is not a large quiescent reserve in vivo. Rather, increases in cell output are achieved rapidly by decreasing the proportion of differentiating cells that die before completing their maturation and successful exodus into the blood stream. However, at the level of more primitive hematopoietic cells, the opposite holds. In adults, the more primitive the population, the greater the fraction that are quiescent.104,217,256,375–378 Nevertheless, when their turnover is examined over prolonged periods (months), it can be seen that a large proportion have, in fact, entered the cell cycle at least once.379–381 Evidence of a continuous slow turnover of the hematopoietic stem cell population in humans is more limited, but may be inferred from the parallel slow age-dependent decline normally seen in the length of the telomers of granulocytes and lymphocytes.382 Much data now indicate that the cycling status of very primitive hematopoietic cells is also regulated in vivo by contact with cytokines that inhibit mitogenesis. Hematopoietic cells are thus continuously activating and deactivating a myriad of signaling events with ultimately opposing actions, the final balance determining the net outcome at the single cell level. The nature and strength of these signaling events are intrinsically regulated by the levels of different signaling intermediates in the target cell and extrinsically by the types and concentrations of positive and negative regulatory cytokines present in the microenvironment. These cytokines are presented either as soluble or extracellular matrix-bound factors (e.g. GM-CSF and IL-3177,383,384 ), or as transmembrane forms of growth factors expressed on the surface of adjacent cells (e.g. Steel factor385,386 and CSF-1/M-CSF387,388 ). The importance of local mechanisms in regulating the turnover of primitive hematopoietic cells has been inferred from early studies showing differences in the timing of proliferative responses of CFU-S in different sites after partial body irradiation389 and an inability to transplant intravenously the Steel factor defect characteristic of Sl/Sld mice.390 These studies were

followed by later experiments demonstrating that the Sl/Sld genotype results in a failure to produce the membranebound form of this growth factor.386 Much redundancy appears to exist in the cytokines or combinations of cytokines that can stimulate primitive hematopoietic cell proliferation. In contrast, considerable hematopoietic target cell specificity is seen in the antiproliferative action of different chemokines, with macrophage inhibitory protein-1
(MIP1
) affecting only intermediate types of progenitors,391–395 macrophage chemoattractant protein-1 (MCP1) also affecting more primitive progenitors395,396 and stromal derived factor-1 (SDF1) being unique in extending its range of inhibitory activity to repopulating cells.397–399

Control of stem cell self-renewal The decision made by individual hematopoietic stem cells to self-renew (or not) has long been thought to be largely determined by loosely regulated intrinsic mechanisms. This concept was first developed from studies showing a large variability in the numbers of CFU-S generated in individually assessed spleen colonies67 and the demonstration that this variability is predicted by a probabilistic model400 in which the likelihood of each CFU-S producing at least one progeny CFU-S throughout the formation of a spleen colony is only slightly higher than 0.5.401 The caveats to this model were, first, that CFU-S were known to be heterogeneous with respect to their self-renewal potential (see above) and, second, that variations in the microenvironment in which each spleen colony developed could be responsible for the variable degrees of CFU-S self-renewal obtained (reviewed in Till and McCulloch402 ). The second caveat was largely discounted by the demonstration of a similar, highly variable pattern of CFU-S generation (and likewise for CFU-GEMMs) in individual colonies produced in vitro from multipotent progenitors.248,258,403,404 Examination of individual LTC-ICs of either murine or human origin also revealed the same high variability in CFC output from one LTC-IC to another.54,55 These observations argue strongly that, even when self-renewal responses appear to be optimally supported by external factors, intrinsic mediators may be limiting, resulting in a stochastic picture of response outcome at the population level. It is important to note that the concept of stem cell self-renewal outcomes being probabilistic at a population level does not mean that the probability of stem cell selfrenewal cannot be influenced. Indeed, there is now considerable evidence suggesting that changes either in the intrinsic make-up of the cell or in the cytokine stimulation it receives can have this effect. During development, the

Anatomy and physiology of hematopoiesis

total size of the murine CRU population increases at least 20-fold.405 This indicates that the regulation of stem cell self-renewal must shift from a mechanism that allows slow expansion of the compartment to one that later reduces the probability of stem cell self-renewal to a maintenance mode. Presumably, this involves a decrease in the proportion of stem cells executing symmetric self-renewal divisions. When small numbers of CRUs are transplanted into myeloablated or preimmune hosts, the increases in CRU numbers that follow may be even larger than those seen during development.43,92,119,405–407 Recent studies of candidate intrinsic factors that may be relevant to regulating stem cell self-renewal point to the HOX family of transcription factors and its upstream regulator BMI1. Many of these genes are expressed at their highest levels in the most primitive subsets of hematopoietic cells and are then rapidly down-regulated.285,408–410 In addition, forced overexpression studies have shown that prevention of the normal downregulation of some of these transcription factors can lead to faster and greater outputs of hematopoietic stem cells.410–415 Experimental examples of extrinsically directed differences in the self-renewal behavior of hematopoietic stem cells stimulated to divide in vitro are also now well documented.57,92,93,261 Furthermore, it has been shown that such changes can occur within a single cell cycle.92 These studies, together with the selective deficiencies displayed by stem cells from various knock-out mice to amplify themselves in vivo, have allowed the identification of an important role of high levels of activation of certain cytokine receptors to promote the self-renewal of hematopoietic stem cells. These include a combination of gp130 (via IL-11 or IL-6 binding)93,416–418 or c-mpl (via TPO binding372,419,420 ) and the activation of c-kit on murine stem cells93 or FLK2/FLT3 on human stem cells.369,421–423 Evidence of Notch-ligands172–175 and Wnt-mediated424,21 signaling as regulators of hematopoietic stem cell selfrenewal responses has also been reported, although these findings remain controversial.425 In addition, factors that selectively reduce the self-renewal probability of stem cells have been identified. These include high levels of IL-3 or IL-1261,369 and tumor necrosis factor (TNF).204,426,427

Control of lineage restriction Molecular events regulating the progressive and eventually irreversible changes in gene expression that constitute the differentiation process in hematopoietic cells are assumed to involve unique as well as generic intracellular mediators. Examples of gene products that fulfill the expectations of unique regulators include such transcription fac-

tors as AML1, SCL/TAL1, GATA1–3, PU1 and PAX5.428–430 These are expressed only, or primarily, in hematopoietic cells and, in some cases, only in certain hematopoietic lineages. Other intrinsic elements that are not unique to hematopoietic cells but display important developmental stage specificity include MYB431–433 and members of the clustered HOX gene family.434,435 In addition, hematopoietic differentiation-associated changes in the expression of genes encoding cell surface receptors and adhesion proteins, as well as certain signaling intermediates, have been described.282,329–332,436–442 These are thought to facilitate the progression of cells into highly restricted gene expression programs that bring about their terminal differentiation into specific blood cell types and cell cycle arrest. Gene knock-out studies have revealed some remarkable examples of important or even essential roles of certain transcription factors for different aspects of hematopoiesis.443–455 In addition, examination of the effects of forced overexpression of particular transcription factors have in some instances produced skewed differentiation patterns.456–463 An interesting concept that has also emerged from such studies is a dual functional role of lineage-specific transcription factors; i.e. they not only actively promote the expression of genes characterisic of one lineage, but also antagonize the expression of alternative lineage programs.464 This was shown dramatically by the discovery that cells from Pax5 null mice that have early B-lineage features have not yet lost an ability to generate T-lineage or myeloid cells.430,465 Other examples of lineage-specific transcription factors that display crossantagonistic activities are GATA1 and PU.1,466–468 and FOG and C/EBP-β.469 This type of transcription factor cross-talk suggests a simple general mechanism whereby “primed” stem cells with a broad spectrum of lineage options could be efficiently and progressively restricted eventually to a single differentiation pathway. Although much has been learned about genes that participate in the process of lineage restriction, it is still very unclear how this process is regulated. Clonal analysis of the production of the earliest types of differentiating progenitors has shown that the distribution of these cells reflects the variability of the stem cell self-renewal process69,470 and that the subsequent sequence of lineage restriction events also appears to reflect a probabilistic mechanism, even in vitro under controlled growth factor conditions.52,255,258,404,471,472 This has led to the concept that lineage restriction may be largely an intrinsically determined process, with extrinsic factors playing a more permissive role, limited to the regulation of proliferative and survival responses of primitive hematopoietic cells. This concept has been further bolstered by additional

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studies that have failed to demonstrate an ability of forced expression of particular receptors to alter the pattern of lineage restriction in normal hematopoietic cells.473–475 Nevertheless, cytokine-directed differentiation has been clearly documented in a number of hematopoietic cell lines,476–479 and in at least one case of primary hematopoietic cells.480 Moreover, analysis of different domains of the G-CSF receptor has identified separate regions of its cytoplasmic domain that transduce proliferation versus differentiation signals.481 In addition, several carefully performed experiments with primary cells have shown that exposure of primitive hematopoietic cells to certain cytokines can influence the particular differentiation pathways subsequently pursued.472,482–484 Taken together, these findings indicate that extrinsic factors can influence the hematopoietic commitment process, although this is unlikely to be a major mechanism regulating lineage selection during normal hematopoiesis.

Stem cell plasticity The discovery that mammals could be cloned by the introduction of adult somatic nuclei into oocytes turned many of the classical rules of developmental biology upside down.485 This finding prompted a broader search for developmental plasticity among various types of mammalian cells, particularly those with known stem cell properties, including the hematopoietic stem cells. No doubt initial interest in the potential plasticity of hematopoietic stem cells was fostered by the knowledge of their high engraftment abilities. Thus, it could be anticipated that any useful findings might be rapidly translated into therapies since clinically established protocols for harvesting and transplanting hematopoietic cells into both autologous and allogeneic recipients were well established. Considerable excitement was thus sparked by the subsequent spate of reports suggesting that primitive hematopoietic cells could “transdifferentiate” into heart muscle and endothelium,486–489 skeletal muscle,310,490,491 hepatocytes,492–499 neural cells,500–503 lung497 and intestinal epithelium.497 Not surprisingly, the failure of many of these findings to be confirmed in a second generation of more rigorously designed experiments triggered enormous controversy.504–508 Two significant and unanticipated discoveries helped to solidify the skepticism that transdifferentiation is not a common physiological event. One of these was the discovery that both hematopoietic stem cells and CFCs are normal residents of a variety of nonhematopoietic tissues100,314,509,510 due to their constant influx from the circulation.99–101,315 A second discovery was the high frequency of cell fusion events that can occur when disparate

types of mammalian cells are cocultured.511,512 Nevertheless, very stringently executed experiments with clonally repopulated mice have now substantiated a few examples of hematopoietic cells acquiring nonhematopoietic phenotypes. The targeted cell types include Purkinje cells in the brain,513 skeletal muscle cells514,515 and hepatocytes,506 although the frequencies of such events are low and, at least in some instances, appear to represent fusion events rather than transdifferentiation.506 These experiments diminish the original hype of a broader competency of hematopoietic stem cells. Nevertheless, they do demonstrate unequivocally that such processes can take place and raise many basic questions about how this occurs and the mechanisms involved.

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system and blocks definitive hematopoiesis. Proc Natl Acad Sci U S A, 1996; 93: 3444–9. Hendriks, R. W., Nawijn, M. C., Engel, J. D., et al. Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus. Eur J Immunol, 1999; 29: 1912–18. Valtieri, M., Tocci, A., Gabbianelli, M., et al. Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program. Cancer Res, 1998; 58: 562–9. Iwasaki, H., Mizuno, S., Wells, R. A., et al. GATA-1 converts lymphoid and myelomonocytic progenitors into the megakaryocyte/erythrocyte lineages. Immunity, 2003; 19: 451–62. Thorsteinsdottir, U., Sauvageau, G., Hough, M. R., et al. Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia. Mol Cell Biol, 1997; 17: 495–505. Sauvageau, G., Thorsteinsdottir, U., Hough, M. R., et al. Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation. Immunity, 1997; 6: 13–22. Kulessa, H., Frampton, J., Graf, T. GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev, 1995; 9: 1250–62. Thorsteinsdottir, U., Mamo, A., Kroon, E., et al. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood, 2002; 99: 121–9. Taghon, T., Stolz, F., De Smedt, M., et al. HOX-A10 regulates hematopoietic lineage commitment: evidence for a monocyte-specific transcription factor. Blood, 2002; 99: 1197– 204. Hirasawa, R., Shimizu, R., Takahashi, S., et al. Essential and instructive roles of GATA factors in eosinophil development. J Exp Med, 2002; 195: 1379–86. Cantor, A. B. & Orkin, S. H. Hematopoietic development: a balancing act. Curr Opin Genet Dev, 2001; 11: 513–19. Rolink, A. G., Nutt, S. L., Melchers, F., & Busslinger, M. Longterm in vivo reconstitution of T-cell development by Pax5deficient B-cell progenitors. Nature, 1999; 401: 603–6. Nerlov, C., Querfurth, E., Kulessa, H., & Graf, T. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood, 2000; 95: 2543–51. Zhang, P., Behre, G., Pan, J., et al. Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1. Proc Natl Acad Sci U S A, 1999; 96: 8705–10. Rekhtman, N., Radparvar, F., Evans, T., & Skoultchi, A. I. Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. Genes Dev, 1999; 13: 1398–411. Querfurth, E., Schuster, M., Kulessa, H., et al. Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors. Genes Dev, 2000; 14: 2515–25.

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470 Wu, A. M., Siminovitch, L., Till, J. E., & McCulloch, E. A. Evidence for a relationship between mouse hemopoietic stem cells and cells forming colonies in culture. Proc Natl Acad Sci U S A, 1968; 59: 1209–15. 471 Suda, T., Suda, J., & Ogawa, M. Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors. Proc Natl Acad Sci U S A, 1984; 81: 2520–4. 472 Takano, H., Ema, H., Sudo, K., & Nakauchi, H. Asymmetric division and lineage commitment at the level of hematopoietic stem cells: inference from differentiation in daughter cell and granddaughter cell pairs. J Exp Med, 2004; 199: 295–302. 473 Pharr, P. N., Ogawa, M., Hofbauer, A., & Longmore, G. D. Expression of an activated erythropoietin or a colony-stimulating factor 1 receptor by pluripotent progenitors enhances colony formation but does not induce differentiation. Proc Natl Acad Sci U S A, 1994; 91: 7482–6. 474 Goldsmith, M. A., Mikami, A., You, Y., et al. Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo. Proc Natl Acad Sci USA, 1998; 95: 7006–11. 475 Stoffel, R., Ziegler, S., Ghilardi, N., et al. Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo. Proc Natl Acad Sci U S A, 1999; 96: 698–702. 476 Dexter, T. M., Heyworth, C. M., Spooncer, E., & Ponting, I. L. O. The role of growth factors in self-renewal and differentiation of haemopoietic stem cells. Philos Trans R Soc Lond B Biol Sci, 1990; 327: 85–98. 477 Borzillo, G. V., Ashmun, R. A., & Sherr, C. J. Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes. Mol Cell Biol, 1990; 10: 2703–14. 478 Martin, M., Strasser, A., Baumgarth, N., et al. A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages. J Immunol, 1993; 150: 4395–406. 479 Klinken, S. P., Alexander, W. S., & Adams, J. M. Hemopoietic lineage switch: v-raf oncogene converts Emu-myc transgenic B cells into macrophages. Cell, 1988; 53: 857–67. 480 Kondo, M., Scherer, D. C., Miyamoto, T., et al. Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature, 2000; 407: 383–6. 481 Fukunaga, R., Ishizaka-Ikeda, E., & Nagata, S. Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor. Cell, 1993; 74: 1079–87. 482 Metcalf, D. Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors. Proc Natl Acad Sci U S A, 1980; 77: 5327–30. 483 Metcalf, D. & Burgess, A. W. Clonal analysis of progenitor cell commitment to granulocyte or macrophage production. J Cell Physiol, 1982; 111: 275–83. 484 Metcalf, D. Lineage commitment in the progeny of murine hematopoietic preprogenitor cells: influence of thrombopoietin and interleukin 5. Proc Natl Acad Sci U S A, 1998; 95: 6408–12.

485 Campbell, K. H., McWhir, J., Ritchie, W. A., & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature, 1996; 380: 64–6. 486 Orlic, D., Kajstura, J., Chimenti, S., et al. Bone marrow cells regenerate infarcted myocardium. Nature, 2001; 410: 701–5. 487 Orlic, D., Kajstura, J., Chimenti, S., et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A, 2001; 98: 10 344–9. 488 Jackson, K. A., Majka, S. M., Wang, H., et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest, 2001; 107: 1395–402. 489 Strauer, B. E., Brehm, M., Zeus, T., et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation, 2002; 106: 1913–18. 490 Ferrari, G., Cusella-De Angelis, G., Coletta, M., et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998; 279: 1528–30. 491 Jackson, K. A., Mi, T., & Goodell, M. A. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A, 1999; 96: 14 482–6. 492 Petersen, B. E., Bowen, W. C., Patrene, K. D., et al. Bone marrow as a potential source of hepatic oval cells. Science, 1999; 284: 1168–70. 493 Lagasse, E., Connors, H., Al-Dhalimy, M., et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med, 2000; 6: 1229–34. 494 Theise, N. D., Badve, S., Saxena, R., et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology, 2000; 31: 235–40. 495 Theise, N. D., Nimmakayalu, M., Gardner, R., et al. Liver from bone marrow in humans. Hepatology, 2000; 32: 11–16. 496 Alison, M. R., Poulsom, R., Jeffery, R., et al. Hepatocytes from non-hepatic adult stem cells. Nature, 2000; 406: 257. 497 Krause, D. S., Theise, N. D., Collector, M. I., et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell, 2001; 105: 369–77. 498 Wang, X., Ge, S., McNamara, G., et al. Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood, 2003; 101: 4201–8. 499 Ishikawa, F., Drake, C. J., Yang, S., et al. Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Ann N Y Acad Sci, 2003; 996: 174–85. 500 Eglitis, M. A. & Mezey, E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci U S A, 1997; 94: 4080–5. 501 Kopen, G. C., Prockop, D. J., & Phinney, D. G. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A, 2000; 96: 10 711–16. 502 Brazelton, T. R., Rossi, F. M., Keshet, G. I., & Blau, H. M. From marrow to brain: expression of neuronal phenotypes in adult mice. Science, 2000; 290: 1775–9.

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503 Mezey, E., Chandross, K. J., Harta, G., Maki, R. A., & McKercher, S. R. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 2000; 290: 1779– 82. 504 Wagers, A. J., Sherwood, R. I., Christensen, J. L., & Weissman, I. L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science, 2002; 297: 2256–9. 505 Castro, R. F., Jackson, K. A., Goodell, M. A., et al. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science, 2002; 297: 1299. 506 Wang, X., Willenbring, H., Akkari, Y., et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature, 2003; 422: 897–901. 507 Balsam, L. B., Wagers, A. J., Christensen, J. L., et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature, 2004; 428: 668–73. 508 Murry, C. E., Soonpaa, M. H., Reinecke, H., et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature, 2004; 428: 664–8. 509 Taniguchi, H., Toyoshima, T., Fukao, K., & Nakauchi, H. Presence of hematopoietic stem cells in the adult liver. Nat Med, 1996; 2: 198–203. 510 Iwatani, H., Ito, T., Imai, E., et al. Hematopoietic and nonhematopoietic potentials of Hoechst/side population cells isolated from adult rat kidney. Kidney Int, 2004; 65: 1604–14.

511 Ying, Q. L., Nichols, J., Evans, E. P., & Smith, A. G. Changing potency by spontaneous fusion. Nature, 2002; 416: 545–8. 512 Terada, N., Hamazaki, T., Oka, M., et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature, 2002; 416: 542–5. 513 Weimann, J. M., Johansson, C. B., Trejo, A., & Blau, H. M. Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol, 2003; 5: 959–66. 514 Corbel, S. Y., Lee, A., Yi, L., et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med, 2003; 9: 1528– 32. 515 Camargo, F. D., Green, R., Capetenaki, Y., Jackson, K. A., & Goodell, M. A. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med, 2003; 9: 1520–7. 516 Van der Loo, J. C. M., Hanenberg, H., Cooper, R. J., et al. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells. Blood, 1998; 92: 2556–70. 517 Eaves, C., Cashman, J., & Eaves, A. Defective regulation of leukemic hematopoiesis in chronic myeloid leukemia. Leuk Res, 1998; 22: 1085–96.

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5 Hematopoietic growth factors James N. Ihle

Introduction Hematopoiesis is regulated through the interaction of one or more of approximately 60 hematopoietic growth factors with their cogent receptors. Over the past several years, virtually all of the biologically defined hematopoietic growth factor activities have been cloned and characterized. In addition, virtually all of the receptors for these growth factors have been cloned and extensively characterized with regard to the signal transduction pathways they activate. The availability of such information has led to a number of important generalizations that provide important insights into the evolution and biology of these growth factors. This chapter focuses on these principles and provides references to reviews that cover individual cytokines in considerably more detail than is possible here. Unfortunately, due to the isolation of hematopoietic cytokines in an environment of scant information relating to their origin and functional relationships, the cytokine nomenclature has presented the most arduous task in dealing with the field. In today’s world, it has become clear that groups of cytokines are related structurally, presumably reflecting their evolutionary relationships. Indeed, cytokines are related with regard to the families of receptors that they utilize and, in turn, have many functionally similar properties (Fig. 5.1). For example, the vast majority of hematopoietic cytokines (examples include interleukin-2 and erythropoietin-3) are structurally characterized by a four
-helical bundle structure and functionally related by the utilization of receptors of the cytokine receptor superfamily. In contrast, hematopoietic growth factors such as colony-stimulating factor-1 (CSF-1) and stem cell factor (SCF) utilize receptors of the receptor tyrosine kinase family. Other factors that

affect hematopoietic lineages include the tumor necrosis factor (TNF) family, which utilize structurally related receptors of the TNF receptor family that function to activate comparable pathways. Interleukin-1 (IL-1) utilizes a quite distinct receptor of another novel receptor family. The tumor growth factors (TGFs) utilize receptors with serine/threonine kinase catalytic domains, and the chemokines [e.g. IL-8 and macrophase inflammatory protein (MIP)-1] utilize receptors of the serpentine (rhodopsin) superfamily of G-protein-coupled receptors. Lastly, a number of cytokines that affect hematopoiesis belong to the TGF- group of cytokines that utilize receptors that have serine/threonine kinase activity. Again, this group of cytokines activates a novel signaling pathway. The alignment of cytokines with their receptor families not only provides a convenient way of subcharacterizing them, but also has important functional implications because receptors within families have striking similarities in function, as noted in Chapter 6. A discussion of hematopoietic growth factors invariably must consider their role in directing differentiation as opposed to supporting differentiation. Moreover, the concept of differentiation can be defined as either primary differentiation or regulation of the functional properties of differentiated progeny. For example, is an erythrocyte an erythrocyte because it expresses the erythropoietin (Epo) receptor and responds to the cytokine? Or, is expression of the receptor merely a property of erythrocytes, with receptor function not being specific for the erythroid lineage? A vast amount of information now supports the concept that, in many cases, the receptor simply supports differentiation by providing signals to prevent apoptosis and to move the cells through the cell cycle, and that any receptor

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Cytokines

Functional differentiation

Cell cycle progression Fig. 5.1 Regulation of hematopoiesis by cytokines and growth factors that utilize functionally distinct receptor families. The regulation of hematopoiesis is mediated through the action of a wide spectrum of cytokines and growth factors belonging to families of structurally related proteins that share the utilization of structurally and functionally distinct receptor systems, as in the diagram. The properties of the growth factors are considered in detail in the text and the structure and function of the receptors are considered in Chapter 6. IL-1, interleukin-1; TNF, tumor necrosis factor; TGF, tumor growth factor.

can provide this function in any lineage in which it is expressed. This conclusion is supported by the observation that expression of the CSF-1 receptor in early myeloid progenitors will allow CSF-1 to support the proliferation and differentiation of all lineages.1 More recently it has been shown that prolactin, through the prolactin receptor, can provide all of the functions of Epo for red cell development.2 On the other hand, cytokines such as IL-12 or IL-4 primarily influence the function of differentiated lymphocytes. Lastly, granulocyte colony-stimulating factor (G-CSF) may have both functions; namely, it is required for expansion of differentiating cells but may also contribute to the expression of genes that determine the phenotype of the cells. Although general statements are often dangerous, the biological activities of cytokines can be generalized to some extent (Fig. 5.2). A number of cytokines appear to have as their primary function the expansion of early lineagenoncommitted or lineage-committed cells and, as noted above, probably function very similarly. In the absence of this group of cytokines, there are dramatic reductions – but not total depletions – of their target populations. Examples include IL-7 and early lymphoid cells, Epo and erythroid lineage cells, G-CSF and granulocytic cells, and thrombopoietin (Tpo) and megakaryocytes and platelets. The degree of specificity and the lack of redundancy that exist among these cytokines are also somewhat remarkable, as has been emphasized by the results of gene knockout studies described below. The mechanisms by which such expansions occur have been speculated to require effects

Suppression of apoptosis Fig. 5.2 Effects of cytokines on hematopoiesis. The activity of cytokines on hematopoietic cells can be grouped according to whether they affect genes that influence cell cycle progression, suppress apoptosis, or cause the expression of genes that contribute to the phenotype of differentiated cells.

on both cell cycle progression genes and genes that prevent the activation of apoptotic pathways. However, many cytokines have as their primary effects the secondary modification of immune or myeloid lineage function in innate or acquired immunity. Indeed, a majority of the T-cell-derived cytokines are of this type and show a striking number of similar functions.

Regulation of hematopoietic stem cells Research over many years has focused on the biological properties of bone marrow-derived cells that have the ability to reconstitute the entire hematopoietic system. Major advances have included the demonstration that single, retrovirally marked stem cells can give rise to longterm reconstitution of the hematopoietic system.3 Subsequently, a number of groups have defined conditions under which the long-term-reconstituting stem cells can be purified to the extent that reconstitution can be obtained with fewer than 100 cells. In spite of the advances, relatively little is known regarding the cytokines that are able to expand and/or maintain long-term-reconstituting cells. Most hematopoietic stem cells are assumed to be in a quiescent (G0/G1) phase of the cell cycle. To date, no conditions or cytokines have been identified that allow the amplification of these cells in tissue culture. However, a number of cytokines can enhance stem cell survival in tissue culture, presumably by inhibiting apoptosis. For example, SCF is routinely utilized to maintain hematopoietic cells in vitro. The properties of SCF and its receptor are the subject of an extensive review, to which the reader is referred.4 The SCF

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receptor, termed c-kit, is a receptor tyrosine kinase that is highly related to the receptors for CSF-1 and plateletderived growth factor (PDGF)
/PDGF, suggesting an evolutionary relationship among these growth factors and their receptors. The patterns of expression of c-kit and SCF during mouse embryogenesis support the hypothesis that these genes are involved in regulating the migration and differentiation of cells in the hematopoietic lineages, as well as melanoblasts and germ cells. A variety of observations support the conclusion that SCF can contribute to the maintenance of hematopoietic stem cells but is not itself indispensable. A wide variety of mutants of both SCF and its receptor, c-kit, support a role in hematopoiesis.5,6 However, complete deletion of the SCF gene only reduces the number of hematopoietic stem cells present in fetal liver by half, as compared with results with wild-type fetal liver preparations. Taken together, the results with mutants support the hypothesis that SCF, although not essential for hematopoietic stem cells, does in fact augment the numbers and possible anatomic distribution of stem cells that are required for normal hematopoiesis. Both the Flk1 (fetal liver kinase 1) and Flt1 (fms-like tyrosine kinase 1) receptors bind vascular endothelial growth factor (VEGF) with high affinity. Early expression of both Flk1 and Flt1 in the developing blood islands of primitive streak embryos is consistent with a dual role for signaling pathways in the onset of vasculogenesis and hematopoiesis in the yolk sac. Consistent with such a role, embryos that are deficient in Flk1 die during early gestation due to a failure to form blood islands during the primitive streak stage of development. Further studies have demonstrated that Flk1 is also required for definitive hematopoiesis.7 The targeted disruption of the Flt1 gene also results in an embryonic lethal phenotype with defects in vascularization.8 Curiously, however, targeted deletion of the kinase domain did not result in a comparable phenotype 9 and has led to a hypothesis that Flt1 may function to bind VEGF and control its access to the other receptors. The studies are consistent with the possibility that VEGF controls the amplification and possible differentiation of a very early common precursor for endothelial cells and hematopoietic precursors. The Flt3/Flk2 receptor tyrosine kinase is closely related to c-kit and c-fms; therefore, it was not unexpected that the ligand would have hematopoietic activity. Like SCF, the Flt3 ligand enhances the responses of stem cells to growth factors such as IL-3, granulocyte-macrophage CSF (GMCSF), and IL-6.7 It can also synergize with CSF and IL-11 to expand early hematopoietic progenitor populations.9 Again, it should be emphasized that the Flt3 ligand, very much like SCF, has little if any mitogenic activity on its own,

and is active only in combination with other cytokines. The basis for this synergy is largely unknown but presumably reflects the unique signaling capabilities of the two families of receptors. The initial studies with Flt3 strongly supported the hypothesis that this receptor would play an essential, nonredundant role in early hematopoiesis. However, the deletion of Flt3 in mice has a very minor consequence that is limited to a small reduction in some stages of B-cell development.10 These results indicate that Flt3 function may be redundant to other receptors such as c-kit. Consistent with this, a deficiency in both receptors results in a more severe phenotype than that seen with c-kit deficiency alone. Although potentially functionally redundant for normal hematopoiesis, mutations of the Flt3 receptor have been shown to occur with a very high frequency in hematopoietic malignancies.11 The most common activating mutation occurs as a consequence of an internal tandem duplication in exons 14 and 15 and is present in cases of acute myeloid leukemia (AML) (15–35%) as well as in cases of myelodysplasia (5–10%). In addition missense mutations and point mutations have been described. The prevalence of mutations of Flt3 in AML has resulted in the search and identification of a number of inhibitors that are currently in clinical trials.12–14

Myeloid lineages In contrast to the rather ambiguous information on the regulation of long-term repopulating hematopoietic stem cells, the cytokines that regulate lymphoid- and myeloidcommitted cells are much better defined. The majority belong to a group of cytokines that utilize receptors of the cytokine receptor superfamily. As noted in Chapter 6, these receptors function very similarly and often mediate identical intracellular signaling events. The cytokines can be further grouped with regard to the composition of their receptor complexes. It is hypothesized that the structure of the receptor reflects the evolution of the various related growth factors. Importantly, grouping of the cytokines in this manner also reflects function, as will become obvious from the descriptions below. A number of cytokines can maintain the viability and support the proliferation of early myeloid lineagecommitted cells. In particular, IL-3 is a major growth factor for early myeloid lineages. This role became readily appreciated when it was first purified and shown to have a plethora of biological activities that were directly due to its ability to support multiple lineages of cells.15 The spectrum of activity directly reflects the fact that the IL-3 receptor

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is widely expressed among early myeloid lineage cells as well as on early lymphoid progenitors, but not generally on lineage-committed cells. The rather striking effects that IL-3 has in vitro in myeloid lineage colony assays would predict a major role for this cytokine in the regulation of myeloid lineages in vivo. Moreover, because IL-3 is exclusively produced by activated T cells, it has been considered the mediator used by the immune system to physiologically regulate myeloid differentiation.16 It was therefore quite striking to find that mutant mice lacking IL-3 had no apparent alterations in any of the hematopoietic lineages.17,18 In particular, when the IL-3 gene was disrupted, the mice crossed with mice that lacked a critical chain of the IL-3 receptor that is shared with the receptors for GM-CSF and IL-5. Thus, the mice were defective in their ability to respond to all three cytokines. Hematopoiesis in these mice was normal, both under control conditions and during stress induced by Listeria or by 5-fluorouracil. Because of the wide range of activity that IL-3 has in vitro, it was anticipated that clinical conditions could be identified in which this cytokine would afford clinical benefit. In spite of numerous clinical trials in a wide variety of settings, little evidence has been presented to support an important clinical role for IL-3.19 Like IL-3, GM-CSF has a wide range of functions that include the ability to promote the expansions of progenitors of granulocytes, monocytes, and eosinophils as well as enhance intracellular activities, such as the killing of parasites.20 Unlike IL-3, GM-CSF has no activity on mast cells or on multilineage hematopoietic progenitors. The spectrum of GM-CSF activity is directly related to the expression of its receptors and, indeed, as discussed below, IL-3 and GM-CSF as well as IL-5 share a common signaling mechanism and pathways, allowing little opportunity for uniqueness in the responses elicited. In mutant mice that lack GM-CSF, 21 hematopoiesis is completely normal; however, the animals develop lung lesions that resemble alveolar proteinosis, suggesting the possibility that GM-CSF plays an important role in normal pulmonary physiology and in resistance to local infections. IL-5, one of the most potent regulators of eosinophil production, regulates both the proliferation of eosinophil progenitors and their differentiation into mature effector cells.22 The only other functions of IL-5 relate to activities on B cells, which support differentiation to IgM- and IgGproducing plasma cells. In combination with IL-4, the IL-5 cytokine enhances IgE production and cluster of differentiation (CD) 23 expression on B cells. Consistent with these in vitro effects, transgenic mice expressing the IL-5 gene have increased levels of IgA, IgM, and IgE and marked increases in numbers of eosinophils. Again, the spectrum of activ-

HSC

Fig. 5.3 Cytokines that are nonredundantly required for the expansion of hematopoietic lineages of cells. G-CSF, granulocyte colony-stimulating factor; Epo, erythropoietin; Tpo, thrombopoietin; IL-7, interleukin-7.

ity reflects the pattern of expression of the IL-5-specific receptor chain. IL-5 is speculated to be associated with humoral- and eosinophil-mediated defense mechanisms against helminthes and allergic reactions. Consistent with a primary role in immune responses, IL-5, like IL-3, is exclusively produced by activated T cells. The triad of IL-3/GM-CSF/IL-5 is clearly not essential for normal hematopoiesis, and it has been difficult to define a current infectious model in which all three cytokines are required. It could be argued that redundant cytokines exist, although there is little evidence to support this hypothesis. The other alternative possibility is that the function of this triad was critical at some point in evolution, either for combating unique pathogens or for allowing the survival of chronic infectious organisms, and that appropriate assays have not been utilized to demonstrate the conditions under which these cytokines are critical. In contrast to hematopoiesis, the role of GM-CSF in pulmonary function is interesting and has been speculated to be due to the lack of macrophage function. In contrast to the above cytokines, several hematopoietic growth factors play essential, nonredundant roles in hematopoiesis as indicated in Fig. 5.3. Epo was originally cloned as a factor capable of inducing the formation of colonies of erythrocytes. Extensive reviews have recently appeared that detail the properties of the growth factor, the regulation of its production, and its mechanism of action.23,24 The structure of Epo is that of a typical cytokine, consisting of four antiparallel
-helices, and its structure is highly related to that of growth hormone. In addition, the receptors and function of the receptors for prolactin and Tpo are highly related to those of Epo and growth hormone, consistent with the possibility that this group of cytokines is evolutionarily related. Epo is produced exclusively in the kidney, where its production is regulated by oxygen concentrations. The cells that respond to Epo are defined by expression of the Epo receptor and consist solely

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of cells committed to the erythroid lineage. Expression of the Epo receptor in other lineages or stages of development allows a fully mitogenic response to Epo without any indication of erythroid differentiation and, indeed, can support the differentiation of multiple lineages of myeloid cells.25 Conversely, erythroid lineage can be fully supported by expression of the highly related receptor for prolactin in Epo receptor negative cells, again emphasizing the lack of unique receptor signaling required for differentiation.2 The critical, nonredundant role for Epo in erythropoiesis is best illustrated by the phenotype of mice in which the Epo gene has been disrupted.26 These mice die during embryogenesis (at 11–13 days) due to their inability to produce erythrocytes in the fetal liver. Importantly, the production of primitive, yolk sac-derived erythrocytes was decreased but clearly present, indicating the unique role of Epo in definitive erythropoiesis. Moreover, the production of burst-forming units-erythroid (BFU-E) and colonyforming units-erythroid (CFU-E) was unaffected, indicating that Epo is not required for the normal differentiation of cells committed to the erythroid lineage-committed cells.2 In addition to its established role in erythropoiesis, a number of studies have implicated Epo and its receptor in brain function27 ; specifically in a neurotopic and neuroprotective function during neuronal damage. Considerable additional studies are required to establish this function definitively however. The regulation of megakaryocyte proliferation and differentiation is mediated by a cytokine, Tpo, which is very similar in structure and function to Epo. The history of Tpo is quite distinct from that of Epo. The search for a factor that could promote megakaryocyte differentiation took place over a considerable period of time during which a variety of cytokine mixtures were described as “the mixture” that is used in vivo. In many regards, the status of the field was very much like the current views of hematopoietic stem cell differentiation. However, during that time a transforming retrovirus was identified that caused expansion of multiple hematopoietic lineages, including megakaryocytes. Characterization of the gene, termed mpl, indicated that it was a member of the cytokine receptor superfamily and that the expression pattern of the cellular gene, c-mpl, was consistent with a role in megakaryopoiesis. Cloning of the ligand bound by c-mpl resulted in the characterization of Tpo.28–30 The properties of Tpo and its receptor have been the subjects of several recent excellent reviews to which the reader is referred.31–33 However, a few points are important to mention. First, mutant mice lacking the receptor for Tpo have been created,34,35 and these mice have severe thrombocytopenia demonstrating the critical, nonredundant role

that Tpo plays in the generation of platelets. In addition, there is a decrease in the numbers of progenitors for other myeloid lineages, including the erythroid lineage, strongly indicating that Tpo may also function to expand early myeloid lineage progenitors. This interpretation is consistent with the known pattern of expression of the Tpo receptor. Unlike the physiologically regulated production of Epo, the production of Tpo appears to be largely constant, and the extent of amplification is directly related to the amount of available Tpo, which in turn is determined by the number of cells expressing receptors that bind the hormone.36–38 One of the unexpected significant roles that Tpo plays is the maintenance of hematopoietic stem cells. Elimination of cytokine or the receptor results in a significant reduction in stem cell numbers. The ability of Tpo to maintain and expand stem cell progenitors has also implicated the dysregulated production of Tpo in myeloproliferative diseases. Granulocyte-specific CSF is uniquely required for the production of granulocytes. Both the ligand and its receptor have been the subject of a detailed review.39 Interestingly, G-CSF and its receptor are structurally more related to the IL-6 family of cytokines than to the IL-3/GM-CSF/IL-5, Epo/Tpo/GM/prolactin, or lymphoid families of cytokines and may reflect a unique evolutionary starting point for this factor. The similarity with the IL-6 family of cytokines is particularly striking with regard to the cellular signaling induced by the receptor. In addition to its ability to support the differentiation of granulocytes, G-CSF may also contribute to the proliferation or maintenance of multipotential hematopoietic progenitors and is frequently utilized in vivo to mobilize bone marrow stem cells to the periphery. The requirements for G-CSF in various phases of hematopoiesis have been studied through the derivation of mice that lack either the cytokine40 or the G-CSF receptor.41 In both cases, the critical role that G-CSF plays in granulocyte production was demonstrated by the profound neutropenia that was observed. However, although reduced, neutrophils were present and functional. The results support the hypothesis that G-CSF is critical for the physiologic production of granulocytes but is not essential for their terminal differentiation. They also demonstrate the lack of a nonredundant role for G-CSF in early hematopoietic cell regulation. Clinically the use of G-CSF has significantly expanded over the last few years. Initially G-CSF was found to be effective for the treatment of neutropenias associated with kidney failure. Subsequently G-CSF was shown to be an effective therapy for virtually all cases of congenital neutropenia.42 However, there is growing evidence that with continued administration, additional genetic lesions involving receptor mutations, or monosomy 7 or Ras mutations can occur leading to myelodysplasic syndromes and

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ultimately malignant transformation. In addition G-CSF is now widely used to mobilize stem cells. One of the major advances in the study of hematopoiesis was the identification of the growth factors that could support colony formation of hematopoietic progenitors in tissue culture. One of the first such factors to be purified to homogeneity and to be characterized was macrophagespecific colony-stimulating factor, or CSF-1.43 The receptor for CSF-1 was subsequently identified as the tyrosine kinase that had been transduced into a transforming feline retrovirus.44 CSF-1 is synthesized by a variety of cell types – including fibroblasts, endothelial cells, keratinocytes, and bone marrow stromal cells, among others. Indeed, biologically saturating concentrations of CSF-1 are normally found in the serum. CSF-1 was initially extensively characterized by colony assays. However, the spectrum of its biological activities was made most obvious from the identification of a mutant strain of mice (op/op) that have a disrupted CSF-1 gene and fail to produce any CSF-1 protein.45,46 The most distinct phenotype displayed by the mice is that of osteopetrosis due to the impaired bone resorption associated with the lack of osteoclasts. The mice also display deficiencies in blood monocytes and certain tissue macrophages, lack incisors, have poor fertility, and have a lower body weight. However, the development of macrophages that are involved in inflammation and immunologic responses occurs normally, suggesting that other factors, such as GM-CSF, may replace CSF-1.

Lymphoid lineages A variety of cytokines have been identified that have relatively specific effects on cells within the lymphoid lineages. Many of these cytokines are related by their use of a specific subset of the cytokine receptor superfamily, in particular those sharing a common receptor chain referred to as the common  c . Specifically, the receptor for IL-2 consists of three chains – termed the
, , and  c chains – while the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21 consist of cytokine-specific receptor chains corresponding to the  and  c chains. Examples of the roles that this subfamily of cytokines has in lymphoid lineages are shown in Fig. 5.4. As noted in Chapter 6, the similarity of the receptor structure results in considerable similarity in the function of these receptors. Both the structure and genetic organization of genes for these cytokines also support a common evolution by gene duplication. Although IL-2 was the first lymphoid growth factor identified, other cytokines play a much more important role in lymphoid development. In particular, it took the creation

Fig. 5.4 Cytokines of the IL-2 subfamily of cytokines regulate many stages of lymphocyte development. A common lymphoid progenitor (CLP) is derived from hematopoietic stem cells (HSCs) and its expansion is dependent upon IL-7 and to a much lesser extent upon thymic stromal lymphopoietin (TSLP). The expansion and functional differentiation of major T and B cells is influenced by many cytokines of this subfamily, as detailed in the text.

of mice in which the gene for IL-7 was disrupted47 to illustrate the unexpected result that this cytokine is essential for the expansion of the earliest T- and B-cell precursors. IL-7 was initially identified during characterization of stromal cell-derived factors that could support the growth of pre-B cells.48 Relative to other lymphoid-active cytokines, IL-7 largely affects immature lymphoid cells. It is expressed on thymic epithelial cells and in spleen, and presumably on stromal elements within the bone marrow. Importantly, the growth of the most immature double-negative subset of thymocytes is stimulated by IL-7 in the absence of any costimulatory signal. The critical role of IL-7 in lymphoid development became clear from the analysis of mice lacking this cytokine as noted above. Such mutant mice are highly lymphopenic, and the development of their bone marrow B cells is blocked at the transition from the pro-B to pre-B stage. In addition, thymic cellularity is dramatically reduced, although the cells that are present retain the normal distribution of CD4 and CD8 cells. Overall, the phenotype observed in these mice is one of severe combined immunodeficiency (SCID), a phenotype that is observed in other mutant mice in which components of the IL-7 signaling system are disrupted, as described below. These observations strongly support the conclusion that IL-7 is critical for the amplification of early lymphoid progenitors that are in

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the process of undergoing the first rearrangements to generate T- and B-cell receptors. Moreover, the fact that the absence of IL-7 affects only the number of cells generated, and not the properties of the cells, suggests that the primary function of IL-7 is to expand this population rather than contribute to the type of differentiation observed. The properties of IL-7 suggest that it would be ideal in clinical settings in which patients would benefit from the generation of additional lymphoid progenitors. Unfortunately, few clinical studies have explored this possibility. IL-2 is, without doubt, the most studied of all the hematopoietic cytokines. Its structure was the first cytokine structure to be determined and consists of six short helical bundles connected by disulfide bonds. IL-2 was originally identified as a product of activated T cells that enhances thymocyte proliferation and supports the growth of T-cell lines.49 In addition to T cells, natural killer (NK) cells and B cells also express the receptor for IL-2 and respond to the protein with a mitogenic response. Given the mitogenic properties of IL-2 on lymphoid cells, it was assumed that IL2 plays a central role in lymphoid development. For this reason the phenotype of mice deficient in IL-2 due to targeted disruption of the IL-2 gene was completely unexpected.50 These animals are normal with regard to the phenotypic development of T cells but exhibit reduced polyclonal T-cell responses and serum immunoglobulin concentrations. IL-4 was initially identified as a co-stimulatory factor that supports the proliferation of anti-IgM-activated B cells and selectively augments IgG1 production.51 Considerably later, IL-13 was isolated as a novel cytokine produced by activated T cells of the Th2 class. As studies proceeded on IL-13, it became clear that this cytokine is functionally and structurally highly related to IL-4. Although IL-13 and IL-4 are only approximately 30% identical in protein sequence, they are structurally very similar, with both containing the typical four
-helical bundle tertiary structures. Moreover, the two genes are genetically linked and reside within the cluster of cytokine genes found on chromosome 5q31, consistent with the possibility that they evolved from gene duplication. One of the unique properties of IL-4 is its ability to induce immunoglobulin locus class switching to promote the production of IgE. Other functions of IL-4 include upregulation of the expression of a number of cell surface proteins on B cells, including myosin heavy chain (MHC) class II antigens, Fcε receptors, CD40 antigen, and the  chain of the IL-2 receptor. The critical role of IL-4 in specific B-cell functions is evident in mutant mice with targeted disruption of the cytokine gene.52,53 IL-4-deficient mice have normal Band T-cell development, indicating that this cytokine is

probably not important in most stages of lymphoid differentiation. However, the mutant mice have greatly reduced levels of IgG1 and, strikingly, the mice have no detectable IgE, nor can they be induced under a wide variety of conditions to make IgE. The basis for this defect is related to the existence of a very unique signaling pathway that is involved in IgE switching. Somewhat strikingly, with the exception of the above defects, the mice are completely normal. IL-13 was initially isolated as a novel, induction-specific cytokine produced by activated Th2 cells. As noted above, the properties of IL-13 are similar to those of IL-4, in that both cytokines stimulate class switching to IgG4 and IgE, and induce the expression of surface antigens such as MHC class II, CD23, and sIgM. There are some differences between the two cytokines, however, in that IL-13 has no detectable effects on T cells, while IL-4 has some activity on T cells. There may also be differences in the conditions that cause their production. Although considerable redundancy exists between the two cytokines, it should be noted that many of the biological responses to IL-4, including class switching in response to various stimuli, are lost; indicating that IL-13 cannot compensate for the absence of IL-4 in these functions. Functionally, IL-13 utilizes a receptor that consists of the IL-4 receptor
chain and a unique IL-13 receptor
chain which accounts for some of the overlapping properties of the two cytokines. IL-13-deficient mice have been generated and depending upon the background more- or less-severe allergic reactions – including airway hypersensitivity, tissue eosinophilia, and elevated IgE levels – are observed in response to immunizations.54 IL-9 is a Th2-derived cytokine which was initially described as supporting the growth of thymocytes and some T-cell lines.55 It was also independently cloned as a factor that enhanced mast cell proliferation. The biological targets of IL-9 include mast cells, T-cell clones, possibly hematopoietic progenitors, and immature neuronal cell lines. Interestingly, IL-9 transgenic mice spontaneously develop lymphomas at a low rate and are highly susceptible to chemically induced tumors.56 It has also been demonstrated that the B cells in Hodgkin disease produce IL-9 and it has been suggested that IL-9 may mediate an autocrine loop in these tumor cells.57,58 The physiological functions for IL-9 have also been explored through the derivation of mice that are deficient for the gene encoding the cytokine.59 However, the absence of altered phenotypes, including allergic responses, indicates that other cytokines can functionally replace IL-9 functions. IL-15 was identified as a T-cell growth factor that was detected in supernatants from epithelial cells and from a

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T-cell leukemia cell line.60 Under normal conditions IL-15 is produced by monocytes and epithelial cells but not by normal T or B cells.61–64 The biological activity of IL-15 is almost indistinguishable from that of IL-2 and, indeed, utilizes the  chain of the IL-2 receptor in addition to the  c chain. The physiological functions of IL-15 have been the focus of a number of studies that have utilized IL-15-deficient mice.65 In the initial characterization, IL-15-deficient mice were shown to lack NK cells, indicating a nonredundant role for this cytokine in NK cell maintenance. The role in maintaining survival of NK cells was subsequently documented.66 IL-15 has also been shown to be required for the proliferative renewal of virus-specific memory CD8 lymphocytes.67 The most recent addition to the family of cytokines that utilize the  c chain is IL-21.68,69 Like the other receptors for this group of cytokines, the IL-21 receptor consists of a IL-21-specific  equivalent chain that associates with the  c chain. Like many of the other cytokines of this group, IL-21 is produced by activated CD4-positive T cells and functionally affects the growth and survival as well as the functional activity of both B and T cells. However, much of the function of IL-21 appears to be redundant to other cytokines since the deletion of the IL-21-specific receptor chain has a relatively minor phenotype consisting largely of altered immunoglobulin isotype production following immunization.70 IL-21 has also been proposed to induce the functional maturation of NK cells.71 Another cytokine that has been postulated to play a role in early lymphocyte development is termed thymic stromal lymphopoietin (TSLP).72 As indicated by the name, it was initially identified as a stromal cell-derived growth factor for early bone marrow lymphoid progenitors. TSLP utilizes a receptor that consists of the IL-7 receptor
(IL-7R
) chain in association with a novel receptor chain with significant similarity to the  c chain. Consequently, it would be anticipated that mice that are deficient in the IL-7R
chain would lack responses for both IL-7 and TSLP. As noted above, the deletion of the IL-7R
chain results in a dramatic reduction in lymphoid progenitors and studies have suggested that TSLP might contribute primarily to fetal pro-B-cell development.73 The contribution of TSLP to this reduction has been evaluated by creating a strain of mice that lack the TSLP-specific  c -like receptor chain. Unlike the deletion of IL-7 or the IL-7R
chain, there is very little consequence for normal lymphocyte development.74 IL-12 is a cytokine that mediates a number of effects in NK cells and activated T cells. It is produced almost exclusively by activated macrophages and is one of the important mediators of toxic shock responses.75–77 Interestingly, the active cytokine consists of two subunits of 35 and 40 kDa.

The 35-kDa chain has the typical structure of a cytokine and is distantly related to IL-6. The other subunit has the structure of a receptor and is most related in sequence to the extracellular domain of the IL-6 receptor
chain. Importantly, as noted below, a soluble complex between IL-6 and the extracellular domain of the IL-6 receptor
chain is a potent cytokine complex that is able to activate the signal-transducing chain gp130. The control for production of IL-12 by activated macrophages resides in the proteolytic cleavage of the p40 subunit. IL-12 was first cloned by virtue of its ability to potentiate NK cytolytic activity.53 The basis of this action is not known, although it has been suggested that IL-12 may regulate the expression of the genes for enzymes associated with cytolytic activity. In addition, IL-12 is a potent inducer of interferon (IFN) production by spleen cells, and this effect is speculated to be an important contributor to the toxic shock response. More recently, studies have shown that IL-12 is a key cytokine in the differentiation of Th1type T lymphocytes, in contrast to cytokines such as IL4, which play a critical role in promoting the differentiation of Th2-type T lymphocytes. Because the spectrum of cytokines produced by activated Th1 and Th2 cells differ, the type of progression of the immune response can be dramatically affected by these cytokines. The spectrum of activities assigned to IL-12 by in vitro studies has been largely confirmed by studies with mice in which the IL-12 gene has been genetically disrupted.75

The IL-6 family of cytokines The IL-6 family of cytokines shares the utilization of receptors that contain the gp130 signal-transducing chain or the highly related leukemia inhibitor factor receptor (LIFR) chain, as described in Chapter 6. The unique biochemical aspects of this cytokine family have been recently reviewed and contrasted with the other cytokine receptor families.78 This group of cytokines affects both myeloid and lymphoid functions as well as a variety of other cell lineages. Indeed, the IL-6 family of cytokines is the most pleiotropic of all the cytokine subfamilies. The most recent member of the family, leptin, has generated considerable excitement as a potential target of body mass control.79 Other members of this group of cytokines include LIF, oncostatin M (OSM), ciliary neurotropic factor (CNTF), IL-11, cardiotropin-1 (CT-1), and cardiotropin-like cytokine (CLC). The diversity of functions of this family of cytokines is clearly evident in the names. It should also be noted that since the receptor for G-CSF is structurally and functionally very related to gp130, G-CSF could also be included in this group of cytokines.

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IL-6 was the first member of the family identified and was purified and ultimately cloned as a T-cell-produced factor that induces the final maturation steps of B cells into antibody-producing cells.80 In addition to this activity, however, IL-6 has been found to have a very pleiotropic spectrum of biological activities. It is a secreted glycoprotein of 187 amino acids with a typical cytokine structure of four
-helices. Within the hematopoietic lineages, IL-6 induces the proliferation of myeloma and plasmacytoma cells. It is also typically used in cytokine mixtures for the expansion of hematopoietic progenitors; however, like many cytokines used in such experiments, IL-6 does not function alone. With certain hematopoietic tumor cell lines, IL-6 is also able to stop cell proliferation and induce terminal differentiation. Lastly, IL-6 is weakly mitogenic for T lymphocytes and has the ability to support the differentiation of cytotoxic T cells. It should be emphasized, however, that a number of cytokines overlap functionally with IL-6. In addition to the hematopoietic functions of IL-6, this cytokine is critical for the induction of acute-phase response proteins in the liver. Receptors for IL-6 are also found in the nervous system and IL-6 has been shown to affect neuronal differentiation as well as neuronal function – including the ability to induce the release of a number of anteriopituitary hormones such as prolactin, growth hormone and luteinizing hormone. The unique, nonredundant functions that IL-6 fulfills have been identified through the derivation of mice that lack IL-6. Such mice develop normally and survive without discernible phenotypic changes. Therefore, IL-6 does not play a nonredundant role in normal development; however, mice deficient in this cytokine do have difficulty in resolving certain viral infections, and the acute-phase response following tissue damage is severely compromised.81 The effects of deleting IL-6 on hematopoietic progenitors have also been examined.82 Although hematopoiesis is largely intact, subtle changes in the ability of progenitors to proliferate or differentiate were identified. Lastly, IL-6-deficient mice have greatly reduced numbers of IgA-producing cells, suggesting a role in class switching or maintenance of IgAproducing B cells. The broad postulated functions of IL-6 in both the production of immune responses and the potential reliance of tumor cells on IL-6 have prompted the development of clinical anti-IL-6 antibodies that have been examined in a variety of settings of both cancer and lymphoproliferative diseases,83 including, more recently, Crohn’s disease.84 In general, the antibodies have been well tolerated and primary responses have been related to the suppression of inflammatory responses.

Another member of the family that has been implicated as being important in hematopoiesis is IL-11. It was identified as a factor that could support primitive hematopoietic progenitors. Through a variety of studies, it was demonstrated to synergize with various other cytokines to enhance lymphohematopoietic stem cells, megakaryocytic progenitors, erythroid progenitors, and monocytic progenitors. Other effects on the functional properties of T and B cells have also been described.85 Indeed, IL-11 was as pleotropic with regards to its effects on hematopoietic cells as was IL-3. Again, the derivation of mutant mice has provided a somewhat different view of the requirement for IL-11 in normal development. In particular, IL-11 utilizes a receptor that consists of an IL-11-specific component, termed the IL-11 receptor
chain, and the signaling transducing chain, initially identified in the IL-6 receptor gp130. To assess the role of IL-11, mutant mice were produced in which the IL-11-specific
-chain component of the receptor was disrupted.86 Cells from such mice failed to respond to IL-11; however, hematopoiesis was completely normal in the mutant mice. Therefore, IL-11 is completely dispensable for normal hematopoiesis, and its effects on hematopoietic cells are redundant with other factors. OSM, initially identified as a cytokine-induced growth factor, has similarly been implicated in the regulation of hematopoiesis. When expressed transgenically, OSM induces a multilineage myeloproliferative disease and the ability of an activated kinase to induce a myeloproliferative disease has been shown to be a consequence of the activation of expression of OSM. However, as with many of the cytokines, OSM does not play a critical, nonredundant role in normal physiology since the deletion of the gene for the OSM-specific receptor chain has only relatively minor consequences, being limited to a small reduction in platelets.87

IFN-related cytokines For a number of years, the IFNs were considered to be a unique group of cytokines, unrelated to the larger cytokine family. However, recent studies have demonstrated a remarkable similarity in receptor structure and function between the IFNs and the other cytokines, and clearly demonstrate a likely evolutionary relationship. The IFNs have diverse effects on lymphoid and myeloid cells, and play a key role in resistance to viral infections. The typeI IFNs consist of at least 14 functional
genes, a  gene, and – in mammals excluding mice – a 
´ gene, all of which are clustered at a single site on human chromosome 9. In addition, the locus contains a number of pseudogenes. All of

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the type-I IFN gene products utilize a single receptor system. The evolution of the type-I IFN gene locus clearly has involved extensive gene duplication.88 The IFNs are produced by a wide variety of cell types. The type-I IFNs affect a variety of cells, in part reflecting the widespread expression of the receptor. The primary effects within the hematopoietic system involve the suppression of proliferation and immunomodulatory activities, in addition to the antiviral effects. Due to the large number of IFN
genes, it has not been possible to obtain mice that lack all type-I IFNs. However, mice have been created that lack one of the receptor chains (IFNAR1) that is required for the function of all the IFN
genes as well as the IFNβ gene.89,90 Such mice are viable, and the only detectable physiological alterations are elevated levels of myeloid lineage cells. In addition, the mice are highly susceptible to viral infections. The results are therefore consistent with the concept that the primary function of the type-I IFNs is to provide an antiviral response. Secondarily, the IFNs modestly suppress hematopoiesis. IFN is produced by T cells, in response to T-cellreceptor engagement, and by NK cells. It is encoded by a single gene on human chromosome 12, distinct from the IFN
/ locus on chromosome 9. The structure of IFN was one of the first cytokine structures solved.91 This IFN was originally identified by its antiviral activity, although over the years it has been shown to have a wide variety of immunomodulatory activities.92 For example, IFN induces the expression of the class I and II MHC antigens, nitric oxide synthase, and other cytokines such as IL-1 and TNF. Like IFN
/, IFN is not mitogenic but rather can suppress the proliferation induced by cytokines that do affect hematopoietic cell proliferation. The antiproliferative effects of IFN extend to tumor cells, a property that has contributed, in part, to clinical use of this cytokine. Mice that are deficient in IFN develop normally and are viable.93 However, they are sensitive to a variety of pathogens and have altered macrophage and lymphoid functions, consistent with a critical role in the regulation of immune responses. IL-10, initially identified as an activity produced by helper T cells, inhibits the synthesis of cytokines by Th1 cells.94 Based on its structure, as well as that of its receptor, IL-10 is a member of the IFN subfamily of cytokines. Like many cytokines, IL-10 was subsequently found to have a plethora of biological activities and affected the growth and differentiation of a variety of cells of the hematopoietic system. In mice lacking IL-10, hematopoiesis is largely normal.95 However, the mice are growth retarded and suffer from chronic enterocolitis. These results are consistent with a critical role for IL-10 in immunoregulation

Table 5.1 Examples of chemokine subfamily members
-Chemokines

-Chemokines

IL-8 Gro/MGSA PF-4 IP-10 MIP-2 NAP-2 GCP-2 EWA-78 PBSF/SDF-1

MCP-1/JE MCP-2 MCP-3 RANTES MIP-1
MIP-1 1309 Eotaxin

Abbreviations: IL-8, interleukin-8; Gro/MGSA, growth-related oncogene protein/csc-chemokine melanoma growth stimulatory activity; PF-4, platelet factor-4; IP-10, interferon-inducible protein-10; MIP-2, macrophage inflammatory protein-2; NAP-2, neutrophil-activating peptide-2; GCP-2, granulocyte chemotactic protein-2; EWA-78, epithelial cell-derived neutrophil-activated peptide; PBSF/SDF-1, pre-B-cell growth-stimulating factor/ stromal cell-derived factor-1; MCP-1/JE, monocyte chemotactic protein-1; MCP-2, monocyte chemotactic protein-2; MCP-3, monocyte chemotactic protein-3; RANTES, C-C chemokine regulated on activation normal T-cell expression and secretion; MIP-1
, macrophage inflammatory protein-1
; MIP-1, macrophage inflammatory protein-1; Eotaxin, eosinophil-selective chemokine.

of the lymphocytes within the context of the intestinal tract. Over the past several years, several additional cytokines have been identified that are related in primary sequence to IL-10, including IL-19, IL-20, IL-22, IL-24, and IL-26.96,97 As with IL-10 these cytokines are produced by activated T cells or in some cases activated monocytes. The cytokines of this subgroup also utilize receptors with shared subunits. Although far-less studied than the above cytokines, in general this subgroup appears to be largely involved in immune regulation and in inflammatory responses.

The chemokines Many cytokines, referred to as chemokines, share a common structure of a triple-stranded, antiparallel  sheet in what is referred to as a “Greek key” motif. The chemokines also share the utilization of receptors of the rhodopsin superfamily of G-protein-coupled receptors, as detailed in Chapter 6. This is a very large family of factors – currently at least 50 members have been identified – which affects a variety of functions. Examples are listed in Table 5.1 and the reader is referred to recent reviews that catalog the chemokine family in detail.98,99 One of the members is IL-8, illustrating the problems that are

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often encountered with cytokine names although efforts are ongoing to develop a uniform naming scheme for the cytokines. The chemokines can be further divided into the
-chemokines, which cluster on human chromosome 4, and the -chemokines, which are located on human chromosome 17. The
-chemokines are structurally distinguished from the -chemokines by the presence of an amino acid between two critical amino-terminal cysteines; hence the
-chemokines are referred to as the C-X-C subfamily, while the -chemokines are the C-C subfamily. In general, the
-chemokines are neutrophil chemoattractants, while the -chemokines are predominantly monocyte attractants. In general, the
-chemokines appear to play a critical role in the accumulation of leukocytes at sites of inflammation. In addition, the  -chemokines (lymphotactin
and ) have a single cysteine (XC). Fractalkine has two cysteines separated by three amino acids (CX 3 C) and constitutes the -chemokines. Although generally associated with cellular migration during inflammation, the derivation of mice lacking one of the chemokines suggests a potentially broader role in normal development.100 Pre-B-cell growth-stimulating factor, also referred to as stromal cell-derived factor-1 (PBSF/ SDF-1), was initially identified by its ability to stimulate proliferation of B-cell progenitors and is constitutively produced by bone marrow-derived stromal cells. Mice deficient in PBSF die perinatally; the numbers of B-cell progenitors in fetal livers were severely reduced, whereas other myeloid lineages and T-cell development were normal. More remarkably, however, the numbers of myeloid progenitors in the bone marrow of late-stage embryos were dramatically reduced. The basis for the unique requirement of myeloid lineage progenitors just in the bone marrow is not known. Gene deletions of other chemokines have supported the biological functions deduced from their activities. For example, mice deficient in MIP-1
have no abnormalities of peripheral blood or of bone marrow-derived lymphoid or myeloid progenitors. However, the mice have a reduced inflammatory response to influenza virus and are resistant to coxsackie virus-induced myocarditis.101 Similarly, eotaxin, a -chemokine, has been implicated in the recruitment of eosinophils and is an eosinophil-specific chemoattractant. Mice lacking the gene have altered numbers of eosinophils under normal, noninduced conditions. Antigen challenge of deficient mice results in a slower recruitment of eosinophils to sites of inflammation, consistent with a role for eotaxin in the early stage of eosinophil recruitment.102 Mice have also been recently derived that lack the receptors for various chemokines and thus can provide

additional insights into the biological role of the various chemokines. For example, mice deficient in CCR1, the chemokine receptor for MIP-1
, as well as several additional related chemokines, develop normally. However, mature neutrophils failed to undergo chemotaxis in response to MIP-1
and failed to mobilize to peripheral blood in vivo. This phenotype was associated with an increased susceptibility to infections that involve neutrophil activation.103 Mice deficient in the chemokine receptor CCR2 similarly developed normally and had no hematopoietic abnormalities. However, these mice failed to recruit macrophages in experimental peritoneal inflammation models.104 Within the context of chemokines, it is also important to point out relationships within the Duffy (Fy) blood group system, which is comprised of four Fy phenotypes: Fy a and Fyb are major alleles, Fyx is a rare serotype allele, and a null allele (termed Fy) also exists. Importantly, the Fy allele has been shown to be associated with resistance to Plasmodium vivax and P. knowlesi in humans. Recent studies have shown that the Fy gene encodes a chemokine receptor that serves as a cellular receptor for the malarial parasites and therefore accounts for the lack of infectivity in individuals in which the gene is not expressed in red cells.105 The receptor binds IL-8 and melanoma growth stimulatory activity (MGSA), also termed Gro or KC, and is widely expressed in neuronal cells as well as erythrocytes. More recently, the lack of expression of the receptor in erythrocytes has been shown to be due to a single base substitution at a critical GATA1 site in the promoter.106

The TGF superfamily Since the discovery of TGFs, more than 35 cytokines have been identified that are structurally related and use receptors that are structurally and functionally related. Again, caution is necessary with regard to cytokine names, because TGF
is structurally and functionally unrelated to the TGFs. Moreover, TGF uses a receptor of the tyrosine kinase family (c-erbB), whereas the TGFs utilize related receptors that have serine/threonine kinase activity. The TGF family members include: TGF1, TGF2, and TGF3; the activins (A, B, AB); the inhibins (A, B); mullerian-inhibiting substance; the bone morphogenic proteins (BMPs); nodals; and growth and differentiation factors (GDFs).107 The cytokines within this family have, as illustrated by their names, a wide variety of biological activities, most of which are unrelated to hematopoiesis. One of the most exciting observations relating to this family of cytokines is the demonstration that they all activate

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members of a novel family of transcription factors, as described in Chapter 6. The relevance of TGF1 to hematopoiesis was illustrated by the derivation of mice that lack this cytokine.108 The deficient mice die within 3–4 weeks of birth with multiorgan inflammation. The infiltrating cells include monocytes, lymphocytes, and neutrophils. Therefore, TGF1 is a critical negative regulator of inflammation. In addition, there is a significant expansion of hematopoietic stem cells in vivo and stem cell lines proliferate more vigorously to cytokines, again suggesting an important role in negatively regulating cell expansion. The negative regulatory role would suggest that inactivation of the TGF1 pathway would contribute to myeloproliferative diseases or leukemias, a concept for which some data have been obtained.109 In contrast, mice that are deficient in TGF2 have multiple developmental defects that affect the heart, lung, craniofacial elements, bones, and urogenital structures among others.110 Mice that are deficient in TGF3 have developmental abnormalities in the lung and have cleft palates.8,111 It is somewhat remarkable that these three highly related cytokines have such strikingly different functions. The creation of deficiencies in other members of the TGF family has shown their roles to be in the development of a wide variety of nonhematopoietic lineages.112

The Notch signaling pathway The Notch signaling pathway was initially identified in developmental mutants in Drosophila, and in flies the system consists of a single receptor (Notch), which is activated by two distinct ligands (Delta and Serrate). Subsequently, the Notch pathway has been found to regulate cell-fate specificity in many developmental systems. The first mammalian homolog of the Drosophila Notch was identified as the target of a chromosomal rearrangement in T-cell ALL (t7: 9)(q34: q34.3). In mammals there are four receptors (Notch-1 to -4) and five ligands termed Jagged-1, Jagged-2, Delta-1, Delta-3, and Delta-4. As noted in Chapter 6, this ligand–receptor system is coupled to a unique signaling pathway in which signaling converts the receptor into a nuclear transcriptional coactivator. Since the discovery of the activation of Notch in leukemias, a variety of studies have focused on the potential roles of the ligands and receptors in hematopoiesis. Among the approaches that have been used, the most informative have been the characterization of mice in which the various genes of interest are either deleted or conditionally deleted, since many of the deletions are embryonic lethals.113,114 At the level of hematopoietic stem cells,

it has been reported that Notch-1 deficiency impairs the developmental capacity of early, AGM-derived stem cells. However, once hematopoiesis is established, there is little direct evidence that Notch signaling plays a critical, nonredundant role. One of the best-established functions of Notch signaling in hematopoiesis is that in T- and Bcell development. With conditional deletion of Notch-1 in the lymphoid lineage, T-cell differentiation is dramatically reduced, and progenitors that enter the thymus develop along the B-cell lineage, consistent with a critical role in T-cell lineage fate specification. Thymic stromal cells express both Delta and Jagged ligands and it is currently unknown which of the ligands is responsible for the Notch requirement. Conditional deletion of Notch-2 in the lymphoid lineage has revealed that its essential role is the generation of splenic marginal zone B cells.115 Importantly, the conditional deletion of Delta-1 also results in the loss of marginal zone B cells, indicating that the ligand–receptor pair Delta-1/Notch-2 is the critical nonredundant signaling pathway.116

The TNF family of cytokines The first member of the family, TNF, was characterized by its ability to induce necrosis in certain tumors in vivo and, independently, as a mediator of parasite-induced wasting disease (cachectin). Subsequently, a variety of factors were identified that are structurally related to TNF and that interact with structurally and functionally related receptors. Importantly, this group of factors are type-II membrane proteins and normally mediate cell-associated signal transduction by interacting with their cognate receptors on neighboring cells. The family consists of approximately 20 factors including: Fas ligand, TNF
, TNF (lymphotoxin, LT
), CD27 ligand, CD30 ligand, TWEAK, LIGHT, RANKL, APRIL, 4-1BBL, and OX40 ligand.117 Again, the nomenclature is not helpful in grouping these structurally and functionally related factors and the problem is further exacerbated by the existence of multiple names for individual factors. The nomenclature emphasizes the diverse manner in which they were identified as well as perhaps some creativeness in naming by the investigators. The functional significance of many of the TNF family members has been demonstrated through both naturally occurring mutants and through the production of mice deficient in the factor or its receptor. Fas and Fas ligand were identified through the characterization of antibodies that were cytolytic for various cell lines.118,119 Characterization of the cellular target resulted in identification of the receptor, and the ligand was purified by its ability to bind

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the receptor. The role of the Fas system became obvious when it was shown that two mouse mutations, gdl and lpr, were due to functional deletion of the Fas ligand and receptor, respectively. Importantly, both mutations resulted in mice with a comparable phenotype, suggesting that the two genes encoded interacting proteins. The consequence of the interaction of Fas ligand and receptor is the activation of a death-inducing pathway that is becoming increasingly well characterized, as described in Chapter 6. The phenotypes of gdl and lpr mice are complex, and the above reviews can provide the full details. However, the defects can be summarized as the inability to induce cell death at appropriate times or in appropriate situations. For example, the mice suffer from pathological conditions associated with autoimmune disease resulting from the inability to eliminate autoreactive, activated T cells. Therefore, the Fas/Fas ligand system is speculated to play a critical role in the negative selection of autoreactive T cells in the periphery. It is also clear that the cytotoxicity of CD8positive T cells relies, in part, on the T-cell system. Because Fas is expressed on a variety of cells, the overproduction of Fas ligand can induce cytotoxicity in a variety of tissues, including liver, thus contributing to some conditions associated with fulminant hepatitis. It should be emphasized, however, that the phenotype of the mutant mice does not indicate any role for the Fas/Fas ligand system in normal development, although apoptosis is thought to be a critical mediator of differentiation and tissue modeling. The importance of the Fas system is also indicated by the association of an autoimmune lymphoproliferative syndrome characterized by mutations of the Fas gene.119,120 In these cases, the patients are heterozygous for a mutation in the Fas gene that is thought to create a dominantnegative form. Somewhat surprisingly, the parents of the patients, who also carry the mutation, do not show a similar phenotype. The possibility exists that the affected individuals carry mutations in other complementing genes. Alternatively, the phenotype may be present only during perinatal development, so that the condition was undiagnosed in the parents of the affected children. Another system within this group of factors that has been well characterized consists of CD40 and its ligand (CD40L). Both CD40- and CD40L-deficient mice have been produced and have been the subject of several papers dealing with various aspects of the phenotype of the mice.121–123 Again, the reader is referred to the above reviews for all of the details; however, several summary points are important. First, the mice exhibit no phenotypes that would suggest that the CD40/CD40L system is required for any aspect of normal development. Yet, mice that lack either

CD40, which is expressed on B cells, or CD40L, which is expressed on T cells, cannot generate germinal centers and undergo immunoglobulin class switching to produce IgG, IgA, or IgE. This defect appears to be intrinsic in the T cells rather than the B cells. Early studies showed that triggering of CD40 can induce B-cell growth and differentiation. CD40L may also be important in regulating the function of macrophages and, in particular, the production of proinflammatory cytokines, as well as cytokines such as IL-12, which influence the development of particular types of immune responses. The functions of TNF can be deduced from the phenotype of the mice lacking the receptors. The TNF receptors include a 55-kDa type-1 receptor and a 75-kDa type-2 receptor, both of which bind TNF with high affinity. Mice lacking the type-1 receptor develop normally, although TNF signaling is disrupted. The major phenotypes consist of a resistance to lethal doses of lipopolysaccharide (LPS) or enterotoxin B. In addition, the mice fail to clear Listeria monocytogenes and succumb to infection.124,125 Mice lacking the type-2 receptor also display normal development, including the lymphoid lineages, but they have an increased resistance to TNF-induced death. The mice also show a dramatic decrease in tissue necrosis in response to TNF injection. The results support the concept that TNF plays a critical role in host defenses and contributes to host inflammatory responses and tissue necrosis in acute infections. Mice deficient in TNF (lymphotoxin) have also been generated.126 Again, normal development of the mice and the majority of immune functions are unaffected. However, TNF-deficient mice showed abnormal development of peripheral lymphoid organs and, specifically, lacked a variety of lymph nodes including the popliteal, inguinal, para-aortic, mesenteric, axillary, and cervical nodes.

The IL-1-related factors The IL-1-related factors are 17- to 20-kDa polypeptides with a range of biological activities. IL-1
and IL-1 are primarily produced by macrophages as prohormones that require proteolytic cleavage to induce their release. In the case of IL-1, this involves a specific protease termed the IL-1-converting enzyme (ICE), a member of the protease family (caspases) that has been implicated in mediating apoptosis under certain conditions. In contrast, the IL-1 receptor antagonist (IL-1Ra) is secreted by normal pathways involving the Golgi complex. Importantly, IL-1Ra is a natural antagonist of IL-1 signaling and can block binding to the IL-1
and IL-1 receptors. The spectrum of reported

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activities of IL-1
and IL-1 is perhaps one of the broadest. These factors have been shown to synergize with other cytokines to induce the differentiation or maintenance of early hematopoietic progenitors, the differentiation of committed progenitors, and the functional responses of mature cells. The reader is referred to an excellent review that tabulates all the activities of IL-1-related factors.127,128 Based on their patterns of expression, however, it can be deduced that these factors function exclusively within the context of an inflammatory response and contribute to the phenotype of the inflammatory response. It should also be noted that the IL-1-related factors can induce the production of a number of other proinflammatory cytokines. The in vivo functions of IL-1, and to some extent IL-1
, have been addressed by characterizing mice in which the ICE gene was disrupted by homologous recombination in embryonic stem cells.129 Consistent with the proposed role of ICE, the production of the proteolytically cleaved, biologically active IL-1 was dramatically reduced. A significant reduction in IL-1
was also seen. The ICE-deficient mice developed normally and displayed no pathology, indicating that IL-1 does play a critical, nonredundant function in hematopoietic or in other lineages. When the mice were challenged with LPS, unlike wild-type mice, the ICEdeficient mice had no detectable serum IL-1 and had dramatically reduced levels of IL-1
. Importantly, this was associated with a dramatic resistance to LPS-induced toxic shock-induced death. The data therefore confirm the role of the IL-1-related factors in inflammatory responses. The roles of IL-1
, IL-1, and IL-1Ra have also been directly addressed by the derivation of mice lacking the genes individually and in combination.130,131 IL-1deficient mice are impaired in acute-phase inflammatory responses and the development of fever and anorexia in response to selective agents such as turpentine. In contrast, the response to various inflammatory agents is not affected by the deletion of the IL-1
gene and the deficiencies seen with IL-1-deficient mice are not enhanced by further deleting the IL-1
gene. In contrast to the normal development of IL-1
-/IL-- deficient mice, mice lacking the gene for IL-1Ra are growth retarded, starting at 2 months. IL-18 is a cytokine that is included in the IL-1 family of factors and is primarily produced by activated macrophages or Kupffer cells, as well as by keratinocytes.132 IL-18 markedly stimulates the production of IFN by splenic T cells in the presence of T-cell-receptor stimulation.133 This property is shared with IL-12, and indeed the combination of IL-12 and IL-18 results in synergy for the induction of IFN . IL-18, also similar to IL-12, can stimulate

the NK cytolytic activity of spleen cells. In many regards the biological properties are similar to those of IL-12. IL-18 has a typical cytokine structure consisting of 157 amino acids with a leader sequence. The structure of the receptor is not known, although the biological similarities might suggest that IL-18 would utilize components that are identical or similar to the IL-12 receptor. Consistent with this interpretation, the phenotype of mice deficient in IL-18 134 is very similar to that of mice lacking IL-12.135 The defects include reduced IFN production in response to LPS, impaired NK-cell activity, and reduced ability to generate Th1 T cells.

The IL-17 family of cytokines IL-17 was cloned as a novel cytokine that is derived from helper T cells.136,137 It is a 155-amino-acid protein that contains an N-terminal signal peptide consistent with the structure of the majority of cytokines. IL-17 exhibits cytokine-like activities on a variety of cell types, including the activation of NF-B, induction of IL-6 secretion in fibroblasts, costimulation of T-cell proliferation, and enhanced surface expression of intracellular adhesion molecule-1 (ICAM-1) on fibroblasts. Interestingly, IL-17 is 57% identical in amino acid sequence to a protein predicted to be encoded by an open reading frame in the Herpesvirus saimiri genome. Since the initial description of IL-17, various approaches have resulted in the identification of five related ligands, the nomenclature of which is also difficult. The initial cytokine is referred to as IL-17A (CTLA-8) and the family members include IL-17B (CX1, NEFR), IL-17C (CX2), IL-17D (IL-27), IL-17E (IL-25), and IL-17F (ML-1). Correspondingly, a number of single-chain receptors have been identified (IL-17R, IL-17RH1, IL-17RL, IL-17RD, IL-17RE). The family of IL-17 cytokines has been reported to mediate a variety of inflammatory responses.

Other cytokines The identification of new cytokines continues, although perhaps not at the rate that typified the field several years ago. With the newer cytokines, sufficient information is not yet available to readily assign them to specific families, although such information can be expected to appear soon. IL-16 was initially identified as a chemoattractant for CD4+ cells, although it is also a powerful chemoattractant for eosinophils. It is produced by helper T cells in response to a variety of stimuli. Although controversial, it has also been proposed to be a T-cell-derived suppressive factor

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for human immunodeficiency virus (HIV) replication. The suppression may occur at the level of viral RNA expression138 and involve repression of HIV promoter activity.139 Recent studies have suggested that IL-16 is produced by proteolytic cleavage of a large precursor, unlike the situation with most cytokines.140 The type of signaling system that is utilized by IL-16 is not yet known, although CD4 is proposed to be the receptor for IL-16.

Summary Any review of the cytokines invariably leads to some degree of cataloging. However, concepts have emerged that allow a rational subdivision of the multiple cytokines that have been identified. First, and perhaps foremost, cytokines can be grouped by their structural similarities that, importantly, probably reflect a common evolutionary origin of the individual families. The second concept is that structurally and evolutionarily related cytokines may have evolved to function in quite distinct lineages, and probably to mediate quite distinct responses in cells. For example, the highly structurally related cytokines Epo, growth hormone, prolactin, and Tpo affect quite different lineages of cells and mediate quite different biological responses. Another example is the quite diverse roles that are played by the IL-6 subfamily of cytokines – which include IL-6, G-CSF, ciliary neurotropic factor (CNTF), and leptin. A major advance in our understanding of the role and significance of cytokines has resulted from the ability to derive mice that lack individual or multiple cytokine genes. Numerous examples exist in which the necessity to reconcile the phenotype of the mutant mice has changed rather drastically our concept of the importance and role of particular cytokines. For example, the role of IL-7 was completely underappreciated until the mutant mice were derived, whereas the role of IL-2 was significantly overinterpreted until the mutants were available. As detailed above, numerous similar examples could be given. Indeed, today it is much quicker and easier to derive mice deficient in the gene for a specific cytokine than it is to examine the plethora of assays that have been used in the past to characterize a cytokine. The gene-deletion experiments have the added advantage of providing a very definitive picture of what might happen if a drug were developed that targeted a specific cytokine. Conversely, one can readily identify the effects of new agents that are not associated with the inhibition of a specific cytokine. Within the context of the myeloid and lymphoid lineages, a large number of cytokines have thus far been identified that play essential, nonredundant roles in supporting differentiation and

inducing the expression of functionally important phenotypes of terminally differentiated cells. Indeed, the number of essential, nonredundant cytokines – including Epo, Tpo, G-CSF, and IL-7 to name only a few – is quite striking and raises the question as to what extent “redundancy” really does exist in the systems. The last general concept is that, in many cases, the unique biological properties of a cytokine are defined by the site of expression of the receptors and not the function of the cytokine itself. This property is further emphasized in Chapter 6, where the concept emerges that many cytokines, through their receptors, activate virtually identical cellular signaling pathways.

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65 Ma, A., Boone, D. L., & Lodolce, J. P. The pleiotropic functions of interleukin 15: not so interleukin-2-like after all. J Exp Med, 2000; 191: 753–6. 66 Cooper, M. A., Bush, J. E., Fehniger, T. A., et al. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood, 2002; 100: 3633–8. 67 Becker, T. C., Wherry, E. J., Boone, D., et al. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J Exp Med, 2002; 195: 1541–8. 68 Habib, T., Nelson, A., & Kaushansky, K. IL-21: a novel IL-2family lymphokine that modulates B, T, and natural killer cell responses. J Allergy Clin Immunol, 2003; 112: 1033–45. 69 Sivakumar, P. V., Foster, D. C., & Clegg, C. H. Interleukin21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumour responses. Immunology, 2004; 112: 177–82. 70 Ozaki, K., Spolski, R., Feng, C. G., et al. A critical role for IL-21 in regulating immunoglobulin production. Science, 2002; 298: 1630–4. 71 Brady, J., Hayakawa, Y., Smyth, M. J., & Nutt, S. L. IL-21 induces the functional maturation of murine NK cells. J Immunol, 2004; 172: 2048–58. 72 Leonard, W. J. TSLP: finally in the limelight. Nat Immunol, 2002; 3: 605–7. 73 Vosshenrich, C. A., Cumano, A., Muller, W., Di Santo, J. P. & Vieira, P. Thymic stromal-derived lymphopoietin distinguishes fetal from adult B cell development. Nat Immunol, 2003; 4: 773–9. 74 Carpino, N., Thierfelder, W. E., Chang, M. S., et al. Absence of an essential role for thymic stromal lymphopoietin receptor in murine B-cell development. Mol Cell Biol, 2004; 24: 2584–92. 75 Schijns, V. E., Haagmans, B. L., Wierda, C. M., et al. Mice lacking IL-12 develop polarized Th1 cells during viral infection. J Immunol, 1998; 160: 3958–64. 76 Chan, S. H., Kobayashi, M., Santoli, D., Perussia, B., & Trinchieri, G. Mechanisms of IFN-gamma induction by natural killer cell stimulatory factor (NKSF/IL-12). J. Immunology, 1992; 148: 92–8. 77 Thierfelder, W. E., Deursen, J. van, Yamamoto, K., et al. Stat4 is required for IL-12 mediated responses of NK and T-cells. Nature, 1996; 382: 171–4. 78 Heinrich, P. C., Behrmann, I., Haan, S., et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J, 2003; 374: 1–20. 79 Zhang, Y., Proenca, R., Maffei, M., et al. Positional cloning of the mouse obese gene and its human homologue [published erratum appears in Nature, 1995; 374 : 479]. Nature, 1994; 372: 425–32. 80 Kishimoto, T. The biology of interleukin-6. Blood, 1989; 74: 1–10. 81 Kopf, M., Baumann, H., Freer, G., et al. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature, 1994; 368: 339–42. 82 Bernad, A., Kopf, M., Kulbacki, R., et al. Interleukin-6 is required in vivo for the regulation of stem cells and committed

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progenitors of the hematopoietic system. Immunity, 1994; 1: 725–31. Trikha, M., Corringham, R., Klein B., Rossi J. F. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence. Clin Cancer Res, 2003; 9: 4653–65. Ito, H. Anti-interleukin-6 therapy for Crohn’s disease. Curr Pharm Des, 2003; 9: 295–305. Yang, Y. C. Interleukin-11 (IL-11) and its receptor: biology and potential clinical applications in thrombocytopenic states. Cancer Treat Res, 1995; 80: 321–40. Nandurkar, H. H., Robb, L., Tarlinton, D., et al. Adult mice with targeted mutation of the interleukin-11 receptor (IL11Ra) display normal hematopoiesis. Blood, 1997; 90: 2148–59. Tanaka, M., Hirabayashi, Y., Sekiguchi, T. et al. Targeted disruption of oncostatin M receptor results in altered hematopoiesis. Blood, 2003; 102: 3154–62. Pestka, S., Langer, J. A., Zoon, K. C., & Samuel, C. E. Interferons and their actions. Ann Rev Biochem, 1987; 56: 727–77. Hwang, S. Y., Hertzog, P. J., Holland, K. A., et al. A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses [published erratum appears in Proc Natl Acad Sci U S A, 1996; 93: 4519]. Proc Natl Acad Sci U S A, 1995; 92: 11 284–88. Muller, U., Steinhoff, U., Reis, L. F., et al. Functional role of type I and type II interferons in antiviral defense. Science, 1994; 264: 1918–21. Ealick, S. E., Cook, W. J., Vijay-Kumar, S., et al. Threedimensional structure of recombinant human interferongamma. Science, 1991; 252: 698–702. Farrar, M. A. & Schreiber, R. D. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 1993; 11: 571–611. Dalton, D. K., Pitts-Meek, S., Keshav, S. et al. Multiple defects of immune cell function in mice with disrupted interferongamma genes [see comments]. Science, 1993; 259: 1739–42. Moore, K. W., O’Garra, A., de Waal, M. R., Vieira, P., & Mosmann, T. R. Interleukin-10. Annu Rev Immunol, 1993; 11: 165–90. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell, 1993; 75: 263–74. Conti, P., Kempuraj, D., Frydas, S., et al. IL-10 subfamily members: IL-19, IL-20, IL-22, IL-24 and IL-26. Immunol Lett, 2003; 88: 171–4. Kotenko, S. V. The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev, 2002; 13: 223–40. Moser, B., Wolf, M., Walz, A., & Loetscher, P. Chemokines: multiple levels of leukocyte migration control. Trends Immunol, 2004; 25: 75–84. Laing, K. J. & Secombes, C. J. Chemokines. Dev Comp Immunol, 2004; 28: 443–60. Nagasawa, T., Hirota, S., Tachibana, K., et al. Defects of Bcell lymphopoiesis and bone-marrow myelopoiesis in mice

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lacking the CXC chemokine PBSF/SDF-1. Nature, 1996; 382: 635–8. Cook, D. N. The role of MIP-1 alpha in inflammation and hematopoiesis. J Leukoc Biol, 1996; 59: 61–6. Rothenberg, M. E., MacLean, J. A., Pearlman, E., Luster, A. D., & Leder, P. Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J Exp Med, 1997; 185: 785–90. Gao, J. L., Wynn, T. A., Chang, Y., et al. Impaired host defense, hematopoiesis, granulomatous inflammation and type 1–type 2 cytokine balance in mice lacking CC chemokine receptor 1. J Exp Med, 1997; 185: 1959–68. Kurihara, T., Warr, G., Loy, J., & Bravo, R. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J Exp Med, 1997; 186: 1757–62. Horuk, R., Chitnis, C. E., Darbonne, W. C., et al. A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science, 1993; 261: 1182–4. Tournamille, C., Colin, Y., Cartron, J. P., & Kim, C. Van. Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nat Genet, 1995; 10: 224–8. De Caestecker, M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev, 2004; 15: 1–11. Shull, M. M., Ormsby, I., Kier, A. B., et al. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature, 1992; 359: 693–9. Kim, S. J. & Letterio, J. Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia, 2003; 17: 1731–7. Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C., et al. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development, 1997; 124: 2659–70. Proetzel, G., Pawlowski, S. A., Wiles, M. V., et al. Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet, 1995; 11: 409–14. Chang, H., Lau, A. L., & Matzuk, M. M. Studying TGF-beta superfamily signaling by knockouts and knockins. Mol Cell Endocrinol, 2001; 180: 39–46. Radtke, F., Wilson, A., Mancini, S. J., & MacDonald, H. R. Notch regulation of lymphocyte development and function. Nat Immunol, 2004; 5: 247–53. Maillard, I., Adler, S. H., & Pear, W. S. Notch and the immune system. Immunity, 2003; 19: 781–91. Saito, T., Chiba, S., Ichikawa, M., et al. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity, 2003; 18: 675–85. Hozumi, K., Negishi, N., Suzuki, D., et al. Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo. Nat Immunol, 2004; 5: 638–44. Gaur, U. & Aggarwal, B. B. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol, 2003; 66: 1403–8.

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118 Nagata, S. & Golstein, P. The fas death factor. Science, 1995; 267: 1449–56. 119 Fisher, G. H., Rosenberg, F. J., Straus, S. E., et al. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell, 1995; 81: 935–46. 120 Rieux-Laucat, F., Deist, F., Hivroz, C., et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science, 1995; 268: 1347–9. 121 Grewai, I. S., Xu, J., & Flavell, R. A. Impairment of antigenspecific T-cell priming in mice lacking CD40 ligand. Nature, 1995; 378: 617–20. 122 Noelle, R. J. CD40 and its ligand in host defense. Immunity, 1996; 4: 415–19. 123 Essen, D. van, Kikutani, H., & Gray, D. CD40 ligand-transduced co-stimulation of T cells in the development of helper function. Nature, 1995; 378: 620–3. 124 Pfeffer, K., Matsuyama, T., Kundig, T. M., et al. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succum to L. monocytogenes infection. Cell, 1993; 73: 457–67. 125 Rothe, J., Lesslauer, W., Lotscher, H., et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNFmediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature, 1993; 364: 798–802. 126 Togni, P., Goellner, J., Ruddle, N. H., et al. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin [see comments]. Science, 1994; 264: 703–7. 127 Dinarello, C. A. Interleukin-1 and interleukin-1 antagonism. Blood, 1991; 77: 1627–52. 128 Dinarello, C. A. Biologic basis for interleukin-1 in disease. Blood, 1996; 87: 2095–147. 129 Li, P., Allen, H., Banerjee, S., et al. Mice deficient in IL-1converting enzyme are defective in production of mature IL-1 and resistant to endotoxic shock. Cell, 1995; 80: 401–11.

130 Zheng, H., Fletcher, D., Kozak, W., et al. Resistance to fever induction and impaired acute-phase response in interleukin1 beta-deficient mice. Immunity, 1995; 3: 9–19. 131 Horai, R., Asano, M., Sudo, K., et al. Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL1alpha/beta, and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever development and glucocorticoid secretion. J Exp Med, 1998; 187: 1463–75. 132 Stoll, S., Muller, G., Kurimoto, M., et al. Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes. J Immunol, 1997; 159: 298– 302. 133 Okamura, H., Tsutsui, M., Komatsu, T., et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature, 1995; 378: 88–91. 134 Takeda, K., Tsutsui, H., Yoshimoto, T., et al. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity, 1998; 8: 383–90. 135 Peritt, D., Aste-Amezaga, M., Gerosa, F., Paganin, C., & Trinchieri, G. Interleukin-10 induction by IL-12: a possible modulatory mechanism? Ann N Y Acad Sci, 1996; 795: 387– 9. 136 Yao, Z., Painter, S. L., Fanslow, W. C., et al. Human IL-17: a novel cytokine derived from T cells. J Immunol, 1995; 155: 5483–6. 137 Yao, Z., Fanslow, W. C., Seldin, M. F., et al. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity, 1995; 3: 811–21. 138 Zhou, P., Goldstein, S., Devadas, K., Tewari, D., & Notkins, A. L. Human CD4+ cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nat Med 1997; 3: 659–64. 139 Maciaszek, J. W., Parada, N. A., Cruikshank, W. W., et al. IL-16 represses HIV-1 promoter activity. J Immunol, 1997; 158: 5–8. 140 Baier, M., Bannert, N., Werner, A., Lang, K., & Kurth, R. Molecular cloning, sequence, expression, and processing of the interleukin 16 precursor. Proc Natl Acad Sci U S A, 1997; 94: 5273–7.

6 Signal transduction in the regulation of hematopoiesis James N. Ihle

Introduction The cloning of receptors for most of the known cytokines and characterization of their functions have provided essential and, to some extent, unexpected insights into the mechanisms by which cytokines mediate their effects on cells. Cytokines have three broadly defined functions. (1) Many are essential for signaling the cell to proliferate and therefore interface directly with the cellular events that control or contribute to cell cycle progression and cell cycle checkpoints. (2) Many others induce cellular signals that contribute to the suppression of apoptosis. (3) Many cytokines regulate the expression of cell lineageand maturation stage-specific genes that contribute directly to the events associated with differentiation or to the cellular functions that characterize a differentiated state. The continual challenge in understanding the biochemical consequences of ligand binding is to understand the significance of specific signaling pathways to these functions. As will become obvious in this chapter, the consequences of the activation of a signaling pathway are rarely known in precise terms. Cytokines function through their interaction with cellular receptors that bind the cytokines with high affinity and, generally, become aggregated as a consequence of ligand binding. Cytokine receptors belong to structurally and functionally related families of proteins that can be defined by the initial type of biochemical reactions that are induced by ligand binding. Many of the ligands that affect hematopoietic cells utilize receptors that couple ligand binding to the induction of tyrosine phosphorylation. This response is mediated by receptors that intrinsically have kinase activity of the receptor tyrosine kinase

family or receptors that associate with cytoplasmic kinases including the cytokine receptor superfamily members, the integrin-coupled receptors, and the large class of Fc receptors. In contrast, members of the tumor growth factor- (TGF-) family of receptors mediate an obligate, initial induction of serine or threonine phosphorylation. Similarly, serine/threonine kinases are critical for the functions of the Toll/interleukin-1 (IL-1) receptors. A large number of cytokines utilize receptors that are related to the tumor necrosis factor (TNF) or Fas receptors and signal through emerging biochemical mechanisms to activate caspase proteolytic pathways and the nuclear factor (NF)-B transcription factors, and possibly other pathways as well. A group of cytokines, generally referred to as chemokines, use structurally related receptors, referred to as the serpentine receptors, which couple ligand binding to the activation of G-protein-coupled signaling pathways. The Frizzled family of receptors transduce signals from a group of factors collectively termed the Wnt proteins. These receptors also consist of seven membrane-spanning proteins but are linked to the activation of transcription factors in the T-cell factor (TCF) family as well as to G-protein-coupled signaling pathways. Lastly, the Notch receptors recognize ligands related to Delta and transmit signals through a unique mechanism initiated by intracellular cleavage of the receptor. Beyond the receptor level exist both receptor familyspecific signaling events and the activation of common signaling pathways. A detailed analysis of all the receptor systems and signaling pathways is beyond the scope of this chapter; therefore, an overview will be given for those receptor systems that do not induce tyrosine phosphorylation, with the reader referred to selected review articles

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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for greater detail. Because tyrosine phosphorylation is central to most of the hematopoietic growth factors, receptor systems that rely on this reaction will be discussed in more detail.

Chemokine receptor signaling The chemokine receptors mediate the function of a large number of chemokines, now including approximately 50 ligands interacting with approximately 20 receptors.1–7 The receptors are named with regard to the structure of the ligands (see Chapter 5) that bind the receptor: CXCR1– 6, CCR1–11, XCR1, and CX3 CR1. Many of the cytokines, such as IL-8, the monocyte chemotactic proteins (MCPs), and the macrophage inhibitory proteins (MIPs) – to name only a few subfamilies – are involved in different aspects of hematopoiesis. Importantly, these receptors have also been shown to serve as receptors for the human immunodeficiency viruses (HIV).8–13 In addition, receptors of this family mediate the cellular responses of a variety of peptide hormones such as glucagon, thrombin, and angiotensin, as well as small molecules such as platelet-activating factor, the cannabinoids, and serotonin. All of the receptors are structurally related and structurally are cell-surface proteins containing seven membrane-spanning domains, the so-called serpentine receptors. Depending on the ligand, one or more of the extracellular domains may be involved in ligand binding, although the large aminoterminal extracellular fragment is consistently involved in ligand binding. The effect of ligand binding is speculated to cause a sequential change in the conformation of the receptor that initiates its function as a signal-transducing protein. The functions of many of the receptors for chemokines have been further elucidated through the derivation of mice strains that lack individual receptors. In general there have been remarkable similarities in the phenotypic changes that occur with the deletion of the ligand or the associated receptor. The deletion of many of the chemokine receptors results in no overt phenotypic change and the consequences are only seen on immunological challenge. For example, the deletion of CXCR2 results in decreased recruitment of neutrophils to sites of inflammation.14 The deletion of CXCR5 results in B-cell migration defects including the abnormal germinal center formation and altered Peyer’s patches.15 CXCR3 deficiency results in resistance to allograph rejection.16 Among the CXCR family of receptors, deletion of CXCR4 results in an embryonic lethality that is associated with several defects generally associated with the loss of cell migration.17

Similar to the deletions of CXCR receptors, the deletion of CCR receptors generally affects cell migration of various inflammatory cells. For example, deficiency of CCR2 is associated with loss of macrophage recruitment,18 deficiency of CCR3 affects trafficking of eosinophils,19 CCR4 deficiency is associated with a resistance to lipopolysaccharide (LPS)-induced toxic shock20 and CCR6 deficiency is associated with defects in dendritic cell migration.21 There is a naturally occuring deletion of CCR5 that exists in the human population and was initially identified as a locus conferring resistance to HIV.13 Deletion of CCR5 in mice results in decreased recruitment of macrophages to sites of inflammation.22 As is evident from the examples given above, the primary functions of chemokines/chemokine receptors are related to inflammatory responses and, in general, are essential for the proper migration or invasion of inflammatory cells. All of the chemokine receptors function by mediating Gprotein-coupled events.23 As a general model, ligand binding induces a conformational change in the receptor, resulting in dissociation of guanosine diphosphate (GDP) from the
subunit of the associated heterotrimeric G-protein. The subsequent binding of guanosine triphosphate (GTP) to the
subunit induces a conformational change that results in dissociation of the
subunit from the receptor as well as the  and  subunits. The GTP-bound
subunit and the – subunit complex are then capable of interacting with various effector proteins. The cycle is completed with hydrolysis of the
-subunit-bound GTP, a reaction that is accelerated by interaction of various effector proteins with GTP and its coupled
subunit, and by reassociation of the latter with the – complex. The diversity of signaling associated with the chemokine receptors is due to the complexity of the G-proteins available for receptor association. For example, 20 mammalian G-protein
subunits have been identified, some of which are highly restricted in their pattern of expression, whereas others are widely expressed. Similarly, in mammals, 5  subunits and 10  subunits have been identified that show both restricted and wide patterns of expression. With some exceptions, various  subunits associate with various  subunits and the – complexes can associate with various
subunits to generate a wide variety of heterotrimeric combinations. Inhibition studies suggest that defined
– – combinations are associated with specific functions. These observations are important because specific receptors can associate with different heterotrimeric complexes and thereby activate a variety of cellular responses. Downstream of the receptor, the dissociation of the GTP–
subunit complex and the – complex is responsible for the activation of various effector functions. The classical

Signal transduction in the regulation of hematopoiesis

targets of the G
-proteins are the adenyl cyclases, which are regulated both positively and negatively through direct interaction. The activated adenyl cyclases then generate cyclic adenosine monophosphate (cAMP), which allosterically activates protein kinase A (PKA). Many chemokine receptors also lead to stimulation of phospholipase C activity and the hydrolysis of phosphatidyl-4,5-bisphosphate to generate inositol-1,4,5-trisphosphate and diacylglycerol. This occurs through regulation of members of the phospholipase C- (PLC) subfamily of PLC enzymes through their interaction, in a family-member-dependent pattern, with G-proteins. As noted below, proteins in the PLC subfamily are also regulated through phosphorylation by other receptor families. As will be discussed in detail, one of the most consistently activated pathways in response to a wide variety of cytokines is the mitogen-activated protein (MAP) kinase cascade. This pathway has been strongly linked to cell growth and is frequently a target for transforming events. Recently, a variety of G-protein-coupled receptors have been shown to lead to the activation of members of the MAP kinase family. Although this finding is still being examined, it may indicate the involvement of src tyrosine kinase and Pyk2.24 The chemokine receptors have also been reported to associate with Janus (Jak) kinases and to activate the Stat (signal transducer and activator of transcription) transcription factors.25 In general these observations have not been substantiated and the gene deletions of Jaks and Stats have not revealed an essential nonredundant function in chemokine signaling.

The Frizzled family of receptors and their signaling Although generally not considered within the context of hematopoietic signal transduction, there is a growing class of receptors (Frizzled) that transduce signals from a number of proteins related to the wingless (wnt) ligand initially identified in Drosophila. To some extent, early interest in this Drosophila factor originated from the observation that the retrovirally transduced, mammary gland-transforming gene int-1 was the mammalian homologue of wnt.26,27 The Frizzled receptors share an amino-terminal signal sequence followed by a domain of approximately 120 amino acids with an invariant pattern of 10 cysteine residues. This in turn is followed by a 7membrane-spanning domain and a conserved carboxylterminal domain. There are currently 19 wnts that function through their interaction with one or more of the 10 known mammalian receptors.

Fig. 6.1 Components of the Frizzled receptor signaling system. The central component is a complex consisting of the tumor suppressor adenomatous polyposis coli (APC) protein complexed with -catenin and the kinase glycogen synthase kinase-3 (GSK-3). In nonstimulated situations -catenin is recruited to the complex and phosphorylated, and thereby targeted for degradation. With engagement of the receptor, the activity of GSK-3 is inhibited, allowing the accumulation of free -catenin, which is available to associate with members of the TCF family of transcriptional factors and to activate their transcriptional activity.

Proteins that participate in signal transduction through the Frizzled receptors have been identified both genetically and by biochemical studies.28–30 The critical components include proteins related to the product of the Drosophila gene armadillo, including -catenin and plakogloblin in vertebrates, as well as proteins related to that encoded by the Drosophila gene dishevelled, including glycogen synthase kinase-3 (GSK-3), the tumor suppressor adenomatous polyposis coli (APC), and members of the TCF family of transcription factors or the related lymphoid enhancerbinding factors (LEFs). The TCF/LEFs alone bind poorly to DNA and activate gene transcription only weakly, if at all. However, binding of -catenin dramatically affects both functions, indicating that the active transcription factor is a heterodimer of TCF/LEFs and -catenin.31,32 The formation of this transcriptionally active complex is dependent on the availability of -catenin (Fig. 6.1). Normally, -catenin exists in a complex with APC and GSK-3 that targets -catenin for degradation. The role of GSK-3 is both to phosphorylate APC and to increase its affinity for catenin, as well as to phosphorylate -catenin and thereby promote its destruction. The engagement of the receptor is thought to modify the function of the disheveled proteins, possibly through phosphorylation. The inhibition of GSK3 function permits the accumulation of -catenin, in turn

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increasing transcriptionally active complexes that contain -catenin. More recently, considerable experimental data have accumulated to indicate that the Frizzled receptors also function in a very similar way to the G-coupled receptors.33–36 Studies have shown that some members of the receptor superfamily can stimulate phosphatidylinositol signaling through a conventional heterotrimeric G-protein complex.37 The ability to activate G
/ and G subunits would suggest that these receptors can couple wnt binding to the activation of a number of additional pathways, including the activation of PKC. Through genetic approaches it has also been demonstrated that Wnt/Frizzled signaling requires one of two members (Lrp5, Lrp6) of the low-density lipoprotein (LDL) receptor family.36 However, precisely how these transmembrane proteins contribute to signaling is not currently known. The potential relevance of this pathway to hematopoiesis is indicated by the phenotype of mice that lack TCF-1.38 Postnatal expression of TCF-1 is restricted to the T-cell lineage. Consistent with a role in T-cell development, mice lacking the gene have defective thymocyte differentiation. The block appears to occur at the transition from immature single-positive cells to the major double-positive population. It is not known what ligands or receptors regulate TCF-1 function in these cells.

Toll/IL-1 receptor-mediated signal transduction The IL-1 receptor was the first mammalian receptor identified that is now a member of the Toll/IL-1 receptor family.39,40 The term Toll is derived from the single receptor (toll) of this family identified in Drosophila. The Drosophila toll receptor is critical for establishment of dorso-ventral polarity during development and is also involved in the recognition of microorganisms in a primitive system of innate immunity. In mammals, the family of Toll/IL-1 receptors consists of IL-1 and IL-18 as well as six receptors termed Toll-like receptors (TLR1–6). Importantly, while IL-1 and IL-18 bind the corresponding protein ligands, the specificity of the other receptors is unknown, other than their ability to bind distinct bacterial cell wall components and therefore – like the Drosophila receptor – they are speculated to be involved in innate immunity. All the receptors of the Toll/IL-1 family function in signaling in a comparable manner. The intracellular signaling of the Drosophila receptor has been well characterized. Irrespective of the receptor, activation ultimately results in the activation of transcription factors of the NF-B family and activation of the C-Jun N-terminal

kinases (JNK). Activation of signal transduction is dependent upon a domain of approximately 200 amino acids of the cytoplasmic domain of the receptor containing 3 conserved boxes termed the Toll/Interleukin-1 receptor (TIR) domain. In the case of the Drosophila receptor, receptor engagement results in the recruitment of an adapter protein termed tube and a serine/threonine kinase termed pelle. With activation of the receptor complex, dorsal, the Drosophila homolog of the NF-B mammalian transcription factors, is activated. The mammalian homolog of tube is MyD88 (myeloid differentiation 88). Importantly, deletion of the MyD88 gene in mice results in the loss of signaling by the Toll/IL-1 receptors. Recently, two MyD88related proteins have been identified,41 termed TIRAP (TIR domain-containing adapter protein) and Trif (TIRdomain-containing adapter inducing interferon-), that may mediate specific functions of the receptors. Another potentially important adapter protein associated with the complex is termed tollip. Recent studies have indicated that it is a critical negative regulator of Toll/IL-1 receptor signaling, possibly through the binding of phosphatidylinositol3-phosphate (PtdIns(3)P) and phosphatidylinositol-3,4,5phosphate [PtdIns(3,4,5)P].42 The mammalian homologs of pelle are the four members of the IRAK (IL-1 receptorassociated kinases) family of serine/threonine kinases (IRAK-1, IRAK-2, IRAK-M, IRAK-4). These kinases are characterized by an amino-terminal death domain (DD) and a centrally located kinase domain. The death domain is critical for interaction with MyD88 and thereby targeting the kinases to the receptor complex. Another adapter protein, TNF receptor-associated factor 6 (TRAF6), identified in the context of the death domain receptors, has also been shown by genetic approaches to be important in the Toll/IL-1 receptor signaling. Specifically, the deletion of the gene in mice results in loss of signaling of the receptors. The role of the various IRAKs has been explored through the derivation of strains of mice lacking the individual genes and this information, along with biochemical analysis, has revealed some unexpected relationships. Firstly, two of the kinases (IRAK-2 and IRAK-M) have a mutation in a critical lysine residue in the ATP-binding pocket which renders them catalytically inactive, suggesting that, if functionally required for signaling, kinase activity is not necessary. In a somewhat similar manner, the genetic deletion of IRAK-1 reduces receptor activity and the reduced activity is restored by providing a catalytically inactive IRAK-1 protein. In contrast, the deletion of IRAK-4 inactivates signaling through all of the receptors.43 The model that is emerging, which takes into consideration the biochemical and genetic data, is one in which receptor engagement results in a complex that is critical for the recruitment and

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Fig. 6.2 Signal transduction by the TGF- receptor family involves the activation of SMAD regulator transcriptional factors. The receptor for TGF--related growth factors consists of the type-I and type-II receptor chains having serine/threonine kinase activity. Following activation, members of the regulatory group of SMAD transcription factors are recruited to the receptor complex and phosphorylated. They then associate with the common SMAD, SMAD-4, and translocate to the nucleus and activate transcription.

activation of IRAK-4. One of the functions of IRAK-4 is to phosphorylate IRAK-1, one of the consequences of which is the recruitment of TRAF6. In an undefined manner, the consequences of these events is the activation of NF-B transcription factors and members of the JNK/p38 family of kinases.

The TGF- receptor family and SMADs in intracellular signaling TGF- was identified as a factor that could promote tumor cell growth a number of years ago. However, since that time, it is increasingly clear that the TGF- factors (TGF-1, 2, and 3) can induce both growth-promoting and -inhibiting functions and these functions may be specific for different cell lineages. Importantly, the TGF- factors are members of a large family of approximately 30 related ligands which also includes the activins, inhibins, and bone morphogenetic proteins (BMPs). The members of the family play essential roles in regulating developmental events as well as contributing to systemic homeostasis. The receptors for the TGF- ligands consist of membrane-spanning proteins containing a cytoplasmic serine/threonine kinase catalytic domain (Fig. 6.2). The receptors mediate the physiological responses to a number of important ligands involved in development and to environmental changes associated with the production of

TGF-. Developmentally, the TGF- receptor family members mediate the responses to the activins, inhibins, and BMPs. The TGF--related ligands bind to receptors that consist of two distinct serine/threonine kinase domaincontaining transmembrane proteins. The type-I receptors are characterized as containing a glycine-/serinerich domain (GS domain) in the juxtamembrane region. The type-II receptors do not contain this domain. Ligand initially binds to the type-II receptor chain and ligation allows the recruitment of the type-I receptor into the complex. Subsequently the type-II receptor phosphorylates the type-I receptor in the GS domain and activates its catalytic activity. Mammals contain seven type-I receptors which can variably associate with one of the five type-II receptors.44 The TGF- receptors activate a number of pathways, many of which are common to many receptor systems including the mitogen-activated protein kinase (MAPK)/JNK/p38 pathways. However, the majority of intracellular signaling mediated by the TGF- receptor family can be accounted for by the activation of members of the SMAD family of transcription factors. Conversely, the SMAD family members exclusively mediate the physiological functions associated with the TGF- receptor family. Much like the Stat proteins, the SMADs translate a membrane signal directly to the nucleus and gene transcription. The term SMAD is a fusion of the names for the related transcription factors identified in either Drosophila, Mad (mothers against decapentaplegic), or in Caenorhabditis elegans, Sma. The family consists of eight mammalian proteins of 40–60 kDa that share a carboxyl-terminal homology domain that is referred to as the Mad homology 2 (MH2) domain. The molecular structure of the MH2 domain of SMAD-4 has been determined.45 In addition several SMADs contain a related amino-terminal domain that has DNAbinding activity and is referred to as the MH1 domain. The MH1 and MH2 domains are connected by a region termed the linker. The SMADs have been divided into three functional groups. The R-SMADs (SMAD-1, -2, -3, -5, and -8) are receptor regulated through phosphorylation, confer the receptor specificity and contain an MH1 domain. The C-SMAD (SMAD-4) is a common SMAD which is required for the function of the R-SMADs as described below. Interestingly, the human SMAD-4 was initially identified as a tumor suppressor gene (DPC4, for deleted in pancreatic cancer, locus 4) on chromosome 18q21.1 that is associated with pancreatic and possibly other human cancers.46 Lastly, inhibitory SMADs (SMAD-6, and -7) function to suppress receptor signaling and are characterized by the absence of the MH1 domain. The MH1 domain has been shown to bind DNA

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while the MH2 domain is required for transcriptional activation. The linker region contains sites of phosphorylation by MAP kinases that are hypothesized to inhibit translocation to the nucleus and thus suppress TGF- signaling. Although not all the details of activation have been elucidated, a remarkably simply picture is emerging for intracellular signaling. The R-SMADs are initially recruited to the receptor complex through the interaction of a small domain in the carboxyl domain of the SMADs with the receptor complex. The receptor recognition is defined by as few as two amino acid differences in the domain.47 Once recruited to the receptor complex, the SMADs are directly phosphorylated by the type-II receptor kinase, then translocate to the nucleus and activate, or in some cases, suppress gene transcription.48,44 Phosphorylation of the R-SMADs occurs in the carboxyl terminal at a characteristic Ser-Ser-X-Ser (SSXS) motif. The translocation and transcriptional activation requires the association of the common SMAD, SMAD4, with the activated SMADs. SMAD-4, unlike the other SMADs, does not contain the SSXS motif and has not been shown to be recruited into the receptor complex. Thus the model that is emerging is that pathway receptor-regulated SMADs (-1, -2, -3, -5, and -9) are recruited, through their ability to recognize specific receptor complexes, to the receptor complex, are phosphorylated and then associate with the common mediator, SMAD-4, to translocate and regulate gene expression. Inhibitory SMADs have also been identified and are characterized by the absence of the MH2 domain. These SMADs are often induced by TGF- signaling and are hypothesized to control the extent and duration of signaling. The mechanism of repression by SMAD-6 and SMAD-7 appears to involve binding to type-I receptors, thereby interfering with the phosphorylation of receptor-specific SMADs.49,50 Their inability to dissociate from the receptor complex is speculated to be due to the absence of the canonical phosphorylation site in the carboxyl terminus.

TNF family receptor signaling The TNF receptor family was initially identified as the receptors for TNFs and for the Fas ligand. The family is characterized by a conserved motif in the extracellular domain that consists of six cysteine-containing domains.51 There are currently approximately 25 receptors that function in the context of 18 ligands including cluster of differentiation 40 (CD40), CD30, CD27, OX-40 and the TNFs and Fas ligand.52 A subgroup of the receptors – that includes receptors such as TNFR1, FAS, NGFR, DR3, DR4, and DR5 – are further characterized by the presence of a conserved cytoplasmic domain that is referred to as the death domain

Fig. 6.3 TNF signal transduction variably results in the induction of cell death through the activation of caspases or the activation of NF-B. The death-inducing receptors contain a death domain (DD) that can interact with FADD or TRADD. These adapter proteins are essential for the recruitment and activation of the proximal activator of cell death, caspase-8. Alternatively, recruitment results in the activation of NF-B through the recruitment and activation of the kinase RIP (receptor-interacting protein). Details are presented in the text.

(DD). The DD is essential for the ability of these receptors to couple to ligand binding in the induction of apoptosis as described below. Importantly, the DDs can be moved around among various receptors and are capable of coupling to the induction of apoptosis in various receptor settings. In general, like other receptor systems, ligand binding results in the aggregation of the receptor complex and assembly of a signaling complex as detailed below. Activation of members of the TNF receptor family variably induces signaling that results in two contrasting responses. In the case of the DD-containing receptors, the predominant response is one of induction of apoptosis. In addition, however, virtually all the receptors are capable of inducing some degree of activation of NF-B transcription factors. These two distinct responses have been recently proposed to be the consequences of the formation of two distinct signaling complexes.53 Many of the molecular details of the activation of these pathways have been elucidated over the last few years and the derivation of mutant strains of mice lacking various components have provided critical insights into the fundamental role of various proteins in signaling. The induction of apoptosis is dependent upon the DD, which consists of a region containing 6
-helical regions in a domain of approximately 60 amino acids. The function of the DDs is to recruit adapter proteins that also contain DDs (Fig. 6.3). Two DD-containing adapter proteins are TRADD

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(TNF-receptor-associated DD) and FADD (Fas-associated DD) proteins. FADD is specifically recruited to Fas and the Trail receptors R1 and R2. TRADD is recruited to the receptors TNFR1 and DR3. FADD contains a death-effecter domain (DED) that interacts with a comparable domain in caspase 8. Recruitment of caspase 8 into the complex results in its proteolytic conversion from a procaspase to an activate caspase which activates downstream caspases. The genetic deletion of FADD supports a role for signaling in apoptosis through several receptors. However, the deletion of FADD also illustrates a role in fundamental developmental events that may be unrelated to its function in the context of TNF receptors. Specifically, deletion of FADD results in an embryonic lethal phenotype that includes defects in heart development. Somewhat more surprisingly, several approaches have demonstrated an essential role in T-cell proliferation including T-cell-specific deletion.54 Importantly, mutant mice lacking caspase-8 phenotypically copy the FADD-deficient mice and similarly are resistant to apoptosis to several death-inducing ligands.55 TRADD functions to induce apoptosis through the recruitment of FADD through interaction of DDs and the subsequent recruitment and activation of caspase 8. However, TRADD can also associate with TNF-receptorassociated factors TRAF1 and TRAF2 and the kinase RIP; this competing complex is responsible for the activation of NK-B with its induction of anti-apoptotic genes. RIP was initially identified as a protein that interacted with the receptor Fas56 and consists of an amino-terminal kinase domain, an
-helical center domain, and a carboxyl DD. The derivation of mice lacking RIP has provided a somewhat different view than was initially derived from the biochemical studies.57 RIP-deficient mice are normal at birth but fail to thrive and die at 1–3 days. Unexpectedly, there was extensive apoptosis in both lymphoid and adipose tissues. Indeed, the cells are highly sensitive to TNF-induced cell death. Importantly, TNF is not capable of inducing NFB activation in the cells. Thus, RIP in the receptor complex is essential for the signaling pathway that results in NF-B activation. In contrast, the genetic deletion of TRAF2 in mice has demonstrated its essential role for activation of JNKs in response to TNF. TRAF2 deletion results in an early lethality and TRAF2-deficient cells show increased apoptosis with TNF stimulation.58 However, this increased sensitivity occurs without any loss in the ability to activate NK-B and may involve JNK-dependent pathways. Thus, distinct components of the complex mediate the activation of specific, and often competing, pathways. The caspases are a family of proteases, consisting of ten murine family members, that are centrally linked to apoptotic programs in intracellular signaling.59 All the

caspases share in common a conserved catalytic site motif that includes a critical cysteine residue that contributes to the active site. The caspases are synthesized as proenzymes that are activated by proteolytic cleavage. Activation and amplification of caspases results in the degradation of a number of target proteins which together cooperate to induce the characteristic pattern of apoptotic cell death. In general individual caspases can be subgrouped based on structure and, more importantly, on function. Several caspases, such as caspase-1 and caspase-11, are involved in the release of inflammatory cytokines and have been termed inflammatory caspases.60 The caspases associated with apoptosis are typically separated into the initiator caspases (caspases -2, -8, -9, -10, and -12), those that are directly activated in signaling, and the effector caspases (caspases -3, -6, and -7), which are activated by the initiator caspases and are involved in the proteolysis of downstream targets. The first member of the caspase family identified was IL-1-converting enzyme (ICE)61 and is the prototypical inflammatory caspase. Shortly after the identification of the mammalian gene, a homolog was identified in nematodes (ced3) as a gene involved in apoptosis.62 The unique role of ICE in regulating IL-1 release is nicely illustrated by the phenotype of mutant mice lacking ICE.63 The mice develop normally but have a major defect in the production of IL-1, and to a lesser extent the production of IL-1
, in response to stimulation. This inability is speculated to be the basis for the resistance of the mice to LPS-induced endotoxic shock. The phenotype also illustrates the lack of a nonredundant role for this member of the family in other cellular systems. In addition to caspase-1, caspase-11 has a very similar function and, for example, is responsible for the production of IL-1 in response to LPS.64 The IL-1related cytokine, IL-18 (also termed interferon- -inducing factor), is similarly dependent upon caspase-1 for release.65 The genes for caspase-1 and caspase-11, along with the gene for caspase-12, all co-localize on murine chromosome 9, suggestive of a series of gene duplications. In humans, the caspase-11 gene is replaced by the genes for caspase-5 and caspase-4, again suggestive of recent gene-duplication events. Caspase-12 has been implicated in endoplasmic reticulum stress-induced responses.66 The other initiator and effector caspases are more directly involved in apoptotic responses. As noted above, caspase-8 is recruited into the DD receptor complexes and is activated with ligand aggregation of the complex. The importance of caspase-8 is best illustrated by the loss of receptor function in cells from mice deficient in the gene.55 It should be noted, however, that caspase-8deficiency results in an embryonic lethality at mid gestation

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for reasons that are not totally explained by the loss of receptor-induced apoptosis. The deletion of caspase-3 similarly supports the role of this caspase as an effecter caspase since specific responses in the apoptotic response, including DNA/nuclear fragmentation, are lost.67 The TRAF (TNF-receptor-associated factor) family of proteins was isolated primarily through yeast two-hybrid protein association approaches. The family is characterized by a conserved carboxyl-terminal domain, often referred to as the TRAF-C domain, and an
-helical region that is termed the TRAF-N domain. Other than TRAF1, all the TRAFs contain an amino-terminal RING finger domain, a domain that is found in a wide variety of proteins. There are currently six members of the TRAF family.68 Overexpression of any of the family members induces the activation of NF-B although the genetic deletion of family members supports much broader roles for these proteins. As noted above, the deletion of TRAF2 specifically affects the activation of JNKs in TNF signaling, although the embryonic lethality associated with the deletion would suggest other functions. Deletion of TRAF3 results in a perinatal lethality in which the mice die shortly after birth.69 The mice are runted and the peripheral white-cell counts decrease dramatically within a few days after birth. Fetal liver cells can completely reconstitute the hematopoietic system but the T cells are defective in such mice. The phenotype of the T cells is consistent with the hypothesis that TRAF3 is critical for a T-cell proliferative response. The phenotype of the T-cell defects has suggested that CD30 may be the receptor with which TRAF3 interacts. As indicated in the section dealing with Toll/IL-1 signaling, the deletion of TRAF6 in mice demonstrated that it functions in the context of the Toll/IL-1 receptors and may have more limited functions in the context of the TNF receptor family and in particular CD40 signaling.70 Several additional gene products control the apoptotic responses initiated by TNF receptor family members. For example, FLIP (c-FLICE inhibitory protein, also termed Casper) is structurally similar to caspase-8 in containing a DD and a caspase-like domain. However, the protein lacks enzymatic activity and thus can function as a dominant negative to block recruitment of functional caspase-8 to the receptor complex. Consistent with this, FLIP-deficient cells are highly sensitive to DD-containing receptors.71 It should be emphasized, however, that both FLIP and FADD have embryonic lethal phenotypes that are not associated with apoptosis, demonstrating that they have functions that are independent of their roles in TNF intracellular signaling. Another example is A20, a zinc finger gene; when deleted, this gene results in a greatly increased sensitivity to TNF-induced apoptosis.72 Although the mechanisms are

not fully detailed, the absence of A20 is associated with a lack of recruitment of RIP and TRAF2 to the complex. The above examples serve to demonstrate the complex protein interactions that occur to initiate the intracellular signaling involved in obtaining a physiological response. They also serve to illustrate the dynamic interactions that can occur, leading to competition for pathway activation, and the dramatic consequences that can occur from subtle shifts in complex formation. The unique position of TNF receptor intracellular signaling pathways to initiate cell death clearly identifies them as targets for drugs that may be useful in treating cancers. Similarly, the critical role that NK-B plays in controlling the proapoptotic responses has prompted studies to determine whether its inhibition may make cells more susceptible to manipulation of the apoptotic pathways.

Protein tyrosine kinase receptors The protein tyrosine kinase receptors are the largest family of cellular ligand receptors.73 The family is characterized by receptors that have a single transmembrane-spanning domain and a cytoplasmic domain that contains a protein tyrosine catalytic domain. The protein tyrosine kinase receptors are further subdivided into subfamilies based on the structure of their extracellular domains. The defining domains include fibronectin-like repeats, epidermal growth factor (EGF)-like domains, immunoglobulin-like domains, cysteine-rich domains, leucine-rich domains, and acidic box domains. The primary function of the ligand is to induce dimerization or oligomerization of the receptor that juxtapositions the cytoplasmic/catalytic domains in such a manner as to allow transphosphorylation and activation of kinase activity.74–77 The ligand can induce oligomerization by one of several mechanisms. It may exist normally as a dimer and therefore may be able to bind two receptor chains in a symmetrical manner, as is the case for colony-stimulating factor-1 (CSF-1) or stem cell factor (SCF). Alternatively, it may have two binding sites for the receptor. Lastly, many ligands are cell associated and thus can induce receptor oligomerization. Irrespective of the mechanism utilized, the key function of the ligand is to drive receptor oligomerization. The mechanism of activation of receptor tyrosine kinases almost invariably involves the transphosphorylation of a critical tyrosine in the kinase domain, in a region referred to as the activation loop. The recent determination of the molecular structure of receptor tyrosine kinase catalytic domains has provided insights into the basis for activation.

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In the case of the insulin receptor,78 the critical regulatory tyrosine resides in a loop that is very near the substratebinding site and therefore can interfere with the ability of the ATP-binding loop to have access to the catalytic site. Phosphorylation of the tyrosine is speculated to result in a change of conformation which results in the swinging out of this inhibitory loop from the catalytic site, thereby allowing substrates to bind and to be phosphorylated. More recently, the structure for the fibroblast growth factor receptor was solved.78,79 Although the orientation of the tyrosine relative to the substrate-binding site and the ATP-binding loop is somewhat different, the unphosphorylated form can be hypothesized to primarily interfere with access of the ATPbinding loop to the catalytic site. The structure of the kinase domain, together with the critical role that tyrosine phosphorylation plays in receptor activation, provides important target sites for small-molecule inhibitors. As noted below, this same theme also appears to apply to the kinases that are associated with receptors of the cytokine receptor superfamily. The activation of kinase activity is followed by phosphorylation of a variety of sites within the cytoplasmic domain of the receptor. These sites are critical for receptor function and serve as “docking” sites for a variety of proteins that are either substrates for the kinases or are activated through their recruitment into the receptor complex.80 The majority of the proteins recruited to the receptor complex are recruited through the interaction of src homology 2 (SH2) domains with specific sites of tyrosine phosphorylation. SH2 domains consist of 100 amino acid domains that contain a binding pocket that recognizes phosphotyrosine and generally 3–6 carboxyl-terminal amino acid residues that provide the specificity of interaction. The structures of several SH2 domains have been solved and provide important insights into the interactions involved in phosphotyrosine binding and specificity.81,82 More recently, a second domain, termed the phosphotyrosine-binding (PTB) domain, has been identified.83,84 The binding properties of PTB domains are quite different from those of SH2 domains and are much less frequently encountered than SH2 domains. The concept of receptor recruitment of substrates has two important consequences. First, the requirement to recruit substrates to a site of active tyrosine phosphorylation provides an essential mechanism for providing the specificity of signaling. Only those pathways for which the components, or a component, can be recruited to the receptor complex will be activated. Secondly, the requirement for receptor tyrosine phosphorylation ensures that phosphorylation of signaling components will only occur within the context of ligand-driven receptor acti-

vation. Thus, the critical requirements for both liganddependent and ligand/receptor-specific activation are obtained. The importance of this duality is readily apparent when one compares the properties of the wild-type receptors with altered receptors that are mutated or fused with aggregation-inducing partners, as occurs in a variety of transformed cells. In these cases, not only is activation of kinase activity independent of ligand, but often results in the phosphorylation and activation of substrates that are not seen in responses to the normal ligand. Signal transduction through the protein tyrosine kinase receptors, depending on the receptor, can involve the recruitment and phosphorylation of a wide variety of proteins, which in turn activate various cellular pathways or functions. For example, virtually all of the receptor tyrosine kinases activate the Ras pathway through one of several possible mechanisms. Similarly, virtually all of the receptors activate phosphoinositol-3-kinase and phospholipase C- 1 or C- 2. Other substrates that are recruited to the receptor complexes include the tyrosine phosphatase SHP-2. The significance of the activation of these various pathways is discussed below. Essential to a regulated response is the ability to deactivate ligand-activated receptor complexes. One mechanism of inactivation is internalization of the ligand– receptor complex, which often involves internalization through caltherin-coated pits and internalization to the endosomes. In the endosomes, receptor–ligand complexes dissociate and are either degraded or the receptor is recycled back to the membrane. In some cases, ligand-induced receptor activation results in ubiquitination of the receptor that targets it for degradation by the proteosomes. Lastly, dephosphorylation of the receptor may be responsible for its downregulation, particularly dephosphorylation of the critical tyrosines in the activation loop. Of particular interest in this regard are the tyrosine phosphatases that contain SH2 domains, and therefore can be specifically recruited to the receptor complex. Although little evidence supports such a mechanism for the tyrosine protein receptor kinases, a critical role for such phosphatases is evident in the function of the cytokine receptor superfamily, as noted below.

Notch receptor signaling Interest in the mammalian Notch receptor family started with the discovery that the chromosomal translocation t(7;9)(q34;q34.3) in T-cell lymphoblastic leukemia involved the fusion of the T-cell receptor  locus with the cytoplasmic domain of a mammalian Notch homolog, now referred to as the mammalian Notch1 gene.85 Prior to identification

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in mammals, the family was identified in Drosophila where it is involved in cell-fate decisions during differentiation.86 Subsequently, three additional mammalian family members have been identified including Notch2, Notch3, and Notch4. Importantly, Notch1 is also activated in thymic lymphomas by retroviral insertions,87 Notch2 has been implicated in a feline leukemia virus-induced lymphoma, and Notch4 was identified as the gene associated with a common mammary tumor virus integration site in tumors (int3). Lastly, Notch3 has been implicated in an hereditary, adult-onset condition causing stroke and dementia.88 The Notch receptors are large transmembrane proteins of approximately 300 kDa that contain 36 tandem epidermal growth factor (EGF)-like repeats and 3 repeats of a motif termed the lin-12 repeat in the extracellular domain. The cytoplasmic domain characteristically consists of: a number of ankyrin-like repeats; a glutamine-rich domain; and a region that is rich in glutamine, serine, and threonines, referred to as the PEST domain. Transformation of various cell types by Notch family members is associated with the overexpression of the cytoplasmic domain. The ligands for the mammalian Notch receptors include Delta, Deltalike-1, -3 and -4 and Jagged-1 and -2. The ligands contain several unique motifs and properties. The amino terminal contains a signal peptide that is followed by a novel motif shared with the Drosophila Serrate (DSL) and 16 EGF-like repeats comparable with those found in the receptors. In addition the extracellular portion contains a cysteine-rich domain followed by the transmembrane domain. The cytoplasmic domain consists of approximately 130 amino acids without distinguishing motifs. Both Jagged-1 and Jagged-2 are expressed in a variety of tissues. The cell membrane association of the ligands and the existence of a relatively large cytoplasmic domain, suggest the interesting possibility that the ligand may also initiate intracellular signaling events. The Notch receptors are speculated to signal by a quite unique mechanism. Ligand binding is proposed to induce an intracellular cleavage, releasing the cytoplasmic domain. This domain then translocates the nucleus and regulates gene transcription through its interaction with a DNA-binding protein variably termed RBP-J (recombination signal sequence binding protein for Jκ genes), CBF1 (C-promoter-binding factor 1), or KBF2 (kappa-binding factor 2). RBP-J binds the promoters of several viral and cellular genes and suppresses their transcription. Importantly, RBP-J is the mammalian homolog of a DNAbinding protein, suppressor of hairless [Su(H)], that has been implicated in the signaling by the Drosophila Notch receptor. The current model89 envisions that the intracellular cleavage product of Notch, through the membrane

proximal region, interacts with a repression domain of RBP-J. Association with RBP-J both suppresses its activity as a repressor of transcription and may provide transcriptional activator activity. Gene disruption studies have demonstrated that RBP-J is critical for early embryonic development.90 However it has been noted that numerous studies have failed to provide convincing evidence for the nuclear localization of the cytoplasmic domain of the Notch receptors.86 The functions of the mammalian Notch receptors are many and are consistent with controlling cell fate in a number of cell lineages. As an example, recent studies have convincingly demonstrated that Notch-1 signaling is essential for commitment of cells to the T-cell lineage at the stage of the common lymphoid precursor.91 Importantly, the proposed target of Notch signaling, CSL/RBP-J, when deleted at this stage of lymphoid lineage development similarly interferes with T-lineage commitment. In addition, however, the targeted mutation of Notch-1 in mice results in embryonic lethality at day 9 due to defects in somite segmentation, and plays a critical role in neurogenesis.92,93

Cytokine receptor superfamily receptors and the Janus protein tyrosine kinases The most significant receptor family for hematopoiesis is the cytokine receptor superfamily of receptors. Members of this receptor family mediate the cellular responses of critical cytokines such as erythropoietin (Epo), thrombopoietin (Tpo), granulocyte-specific colony-stimulating factor (GCSF), and IL-7, among a wide variety of other cytokines. Initially, it was not thought that this group of cytokines would utilize structurally and functionally related receptors. Indeed, it was only with the initial purification and cloning of several of the receptors that it was realized that they mediated their effects through a very distinct family of receptors. The receptors were initially recognized as a family through conserved motifs that were found in the extracellular domains.51,94 These included four positionally conserved cysteine residues and the characteristic tryptophan, serine, any amino acid, tryptophan, serine (WSXWS) motif, normally located near the transmembrane domain. Indeed, these motifs are now used to predict whether a protein will or will not be a receptor. Recent studies of the molecular structure of the receptors have confirmed a remarkable similarity in the extracellular domains, although they have failed to identify the role of the WSXWS motif.95 The structural similarity of the receptors has suggested a common origin in evolution. Indeed, a very strong case can be made for the recent evolution of a number of the receptors. Independent of the work on the cytokine

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receptors, similar efforts were being made to characterize the receptors for the interferons (IFNs). With purification and cloning of the receptors, it became obvious that they were structurally most closely related to the growing family of cytokine receptors, with some variation of the extracellular characteristic motifs. For this reason, the cytokine receptor superfamily is now generally divided into class I and class II cytokine receptors, the latter containing the highly related receptors for IFN-
, IFN-, IFN , and IL-10. In spite of the similarity of the extracellular domains, the cytokine receptors had cytoplasmic domains that varied considerably in both structure and size. Indeed, the most disappointing observation was that none of the receptors contained any obvious catalytic domains in the cytoplasmic domain, thus leaving unresolved how these receptors might function. The only structural similarity was limited to the membrane proximal domain, and to what is often referred to as the box 1 and box 2 motifs.96 These motifs are very loosely defined, and the box 1 “conserved” motif is limited to a pro, any amino acid, pro (P×P) motif. Irrespective of the limited conservation of the sequence, mutational analysis of this region in all of the receptors examined indicated that this region was essential for receptor function. The cytokine receptors can consist of one or more chains. For example, the receptors for Epo, growth hormone, prolactin, and Tpo consist of single chains that are highly related in structure and therefore have been suggested to have been derived from a common progenitor. This is particularly likely in the case of the growth hormone and prolactin receptors, which genetically co-localize and thus can be considered to have arisen from a recent gene duplication event. The receptors for a number of cytokines consist of multiple chains and often involve the utilization of a ligandspecific chain and a common chain that is shared among a number of receptors. For example, the IL-3, granulocyte macrophase (GM)-CSF, and IL-5 receptors all utilize a common  chain that associates with a ligand-specific
chain. Similarly a number of the cytokines that affect lymphoid cells consist of a ligand-specific
chain, and the common  chain initially identified in the IL-2 receptor. Interestingly, the IL-2 receptor contains, in addition to the  c chain, a  chain that is shared with the IL-15 receptor – and has the characteristic receptor motifs – and another ligandspecific
chain (CD25) that is not a member of the cytokine receptor superfamily. Lastly, the IFN receptors each contain two distinct chains that are both required for signal transduction; however, irrespective of the number of chains involved, the common function of the ligand is to drive the aggregation of the receptor chains.

Studies with a variety of the receptors indicated that receptor function was dependent on the ability of the receptor to mediate ligand-induced activation of tyrosine phosphorylation. Moreover, the induction of tyrosine phosphorylation was dependent on the conserved membrane proximal region of the cytoplasmic domains of the receptors. These observations prompted the search for cytoplasmic protein tyrosine kinases that might associate with the receptors and mediate the critical first steps in signal transduction. Identification of the critical kinases came from two independent approaches. The first came from studies directed at defining the pathways involved in IFN-mediated gene regulation. Initially, a number of cell lines were established through an insightful approach to obtaining mutants defective in signaling. These were further defined as constituting a number of independent complementation groups. Identification of the genes involved relied on a complementation screen by genomic DNA transfections. With this approach, it was demonstrated that Tyk2, a member of a newly evolving family of protein tyrosine kinases, was able to complement one mutant.97 This finding led to the concept that the IFN
/ receptor associated with Tyk2 and mediated its activation following IFN binding. Independent research groups were using biochemical approaches to identify the cytoplasmic protein kinases that associated with cytokine receptors and were activated following ligand binding. Although several kinases were implicated by these approaches, many were not significantly activated, nor could they be shown to associate with the receptors, or activation failed to correlate among receptor mutants. It was only with the development of reagents for the Janus kinases (Jaks) that all the criteria were fulfilled. This was initially demonstrated for Jak2 activation in response to Epo98 as well as growth hormone.99 Subsequent to these studies, it has been shown that one or another of the Jak family of kinases associates with all the receptors of the cytokine receptor superfamily. The Jak family of kinases consists of four members, all of which are characterized by a large amino-terminal domain that contains blocks of homology found among family members but not among other proteins. This domain has been shown to associate with the cytoplasmic domains of the cytokine receptors and is critical for function within the context of the receptor. The carboxylterminal region contains a characteristic protein tyrosine kinase catalytic domain. The amino-terminal to the kinase domain is a pseudokinase domain that contains significant homology to the catalytic domain but does not have kinase activity. The role of the pseudokinase domain is not known, although some studies indicate that it may

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Fig. 6.4 Signal transduction through the cytokine receptor superfamily requires the association of Jak kinases with receptor subunits. Three types of receptor complexes are depicted based on the number of cytokine receptor chains. The membrane proximal domain of the receptors is required for their interaction with one of the four mammalian Jak kinases.

negatively affect the kinase domain. Three of the family members, Jak1, Jak2, and Tyk2, are ubiquitously expressed, whereas Jak3 is primarily expressed in hematopoietic cells. The model that has emerged predicts the constitutive association of one or more of the Jaks with specific cytokine receptor chains through the conserved membrane proximal region (Fig. 6.4). For example, the Epo, Tpo, growth hormone, and prolactin receptors associate with Jak2 through the membrane proximal domain. Ligand is envisioned to drive aggregation of the receptor complex, bringing into close proximity the associated Jak2. Transphosphorylation can then occur, leading to the activation of kinase activity. A variety of studies with receptor and Jak mutants support this model of ligand-induced activation. One of the unique receptor systems is that associated with the IL-6 subfamily of cytokines, in which the receptors consist of ligandspecific components and a shared receptor chain referred

to as gp130 or the highly related chain LIFR
(leukemia inhibitory factor receptor-
). These receptor chains can bind multiple Jaks and mediate their ligand-dependent activation. Lastly, several of the receptors that utilize multiple chains also utilize multiple Jaks. For example, the IL-2 subfamily of cytokines activates both Jak1 and Jak3, through the ability of Jak3 to bind to the common  chain and by the binding of Jak1 to the ligand-specific
chains, as in the case of IL-7 or IL-4, or to the  chain in the case of the receptors for IL-2 and IL-15. The activation is still envisioned to involve ligand-induced receptor aggregation that places the Jaks in a position conducive to phosphorylation. The activation of the Jaks, in each case, involves the transphorylation of a critical tyrosine within the activation loop of the kinase domain. Although the molecular structures of the Jaks have not been determined, the sequence of the kinase domain is highly related to that of the receptor

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tyrosine kinases, particularly in the activation-loop region of the kinase domain. Consistent with a role for tyrosine phosphorylation of sites within the activation loop, mutation of the presumed critical sites results in the virtual elimination of catalytic activity.100 Confirmation for the critical role of the Jaks in cytokine signaling has come from studies of humans and mice that lack specific Jaks. In particular, Jak3 is unique among these kinases in that it binds only to the  c chain and thus functions only within the context of the cytokines that use this receptor chain. This group of cytokines – which includes IL-2, IL-4, IL-7, IL-9, and IL-15 – predominantly function within the context of the immune system, and IL-7, in particular, is known to be essential for the amplification of early progenitors. Loss of IL-7, or its receptor chains, results in a phenotype of severe combined immunodeficiency (SCID).101 The observation that mice deficient in Jak3 also have a SCID phenotype of SCIDs gives support to a critical role in Jak3 signaling.102,103 Moreover, the phenotype of mice lacking Jak3 indicates that this kinase is not required for any other functions, including normal hematopoietic-cell development in general or myeloid lineage development in particular. SCID is also a genetically acquired disease in humans. Although the most frequent form of human SCID is associated with mutations in the IL-2 receptor  c chain, it has recently been shown that a subset of SCID patients have inherited mutations that disrupt the Jak3 gene.104–106 Based on the essential role for Jak3 in the amplification of the earliest lymphoid progenitors, it might be anticipated that a deficiency in Jak3 could be corrected through gene therapy. Indeed, recent studies with a murine model have shown that retrovirus-mediated transfer of the Jak3 gene to early hematopoietic stem cells and reconstitution into deficient mice can rapidly correct the immunodeficiency.107 It will be of considerable interest to determine whether the same degree of efficacy is observed in human SCID, Jak3-deficient patients. Studies have focused on the roles of Jak1 and Jak2 through the derivation of mice that are deficient in these family members. Mice deficient in Jak1 are born viable and die shortly thereafter.108 Although the actual cause of lethality is not known, a number of receptor systems have been examined. For example, receptor signaling through the IL-2 subfamily of cytokines is defective, consistent with an essential role for Jak1, as well as Jak3, in the receptors for these cytokines. Similarly, all of the IFN receptors have been shown to activate Jak1 and to have a receptor chain that associates with this kinase. In the knockout mice these functions are also lost. The absence of Jak2 is more profound and results in an embryonic lethality at 10–12 days of development.109 In this

case, lethality is due to the lack of production of definitive erythrocytes and, indeed, the phenotype is similar to but perhaps more profound than the phenotype seen in mice lacking either the Epo receptor or Epo itself.110 The ability of fetal liver cells to respond to cytokines that had been shown to activate Jak2 was also lost – including the responses to Tpo, IL-3, IL-5, and GM-CSF. Importantly, other aspects of embryonic development were normal. Together, the results establish the critical role of Jak2 in a very defined group of cytokines that utilize receptors capable of associating with Jak2. Following receptor aggregation and activation of the Jaks through phosphorylation within the activation loop, phosphorylation of the receptor, as well as a variety of cellular substrates, is observed. The emerging models for receptorcomplex activation and signaling specificity are virtually identical to those proposed for the protein tyrosine kinase receptors: namely, tyrosine phosphorylation sites on the receptor chains are “docking” sites that allow the recruitment of potential substrates and signal transducers to the receptor complex in a receptor-specific manner. In addition, however, the Jaks are themselves tyrosine phosphorylated at multiple sites, and it can be hypothesized that one or more of these sites will also be important in recruiting signaling proteins to the receptor complex, although this notion lacks firm support. Like the tyrosine kinase receptors, it is important to consider the mechanisms by which the cytokine receptor complexes are downregulated. In this case, considerable evidence exists to indicate that one of the tyrosine-specific phosphatases plays a key role. In particular, phosphatase SHP-1 (see homology phosphatase-1) is recruited to many of the receptor complexes through its ability to interact with sites of tyrosine phosphorylation on the receptor through its SH2 domain. Recruitment of the phosphatase to the Epo receptor complex has been shown to be associated with receptor downregulation and specifically with the dephosphorylation of the associated Jak2.111 However, the most dramatic evidence comes from the phenotype of mice in which the SHP-1 gene is mutated.112,113 These mice die within a few weeks after birth and have a variety of defects of the hematopoietic system that collectively are associated with overproliferation. The results are consistent with a broad role for SHP-1 in the regulation of cytokine signaling.

Fc receptor signaling There exists a large group of structurally and functionally related receptors that include the antigen-specific receptors on T and B cells that are referred to as the Fc receptor

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Fig. 6.5 The family of Fc receptors includes a variety of critical receptor complexes that have as a primary function the induction of calcium signaling events. The components that have been genetically implicated in several of the members of this family are indicated. Details of the importance of each component are given in the text descriptions.

family.114–118 In addition to the antigen-specific receptors, this family includes such diverse receptors as the collagen receptor on platelets, the receptors for Fc domains of immunoglobulins on a variety of cells, as well as the receptors utilized by natural killer (NK) cells. As with all tyrosine kinase-based receptor systems, the function of ligand is to drive the aggregation of the receptor complex and allow the activation of receptor-associated kinases. In the case of the Fc receptor family, the ligand-induced receptor complex contains a number of components and is often referred to as a signalsome. Importantly, among the various receptor complexes, the inherent nature of the complex is virtually identical with the diversity largely due to use of different family members of the proteins comprising the complex. Moreover, one of the most important functional consequences of receptor-complex activation is the induction of a calcium flux that, in turn, activates a variety of cellular responses as described elsewhere in the chapter. The components that are involved in the function of members of the Fc superfamily of receptors are indicated in Fig. 6.5. The initiation of tyrosine phosphorylation in Fc receptor complexes is dependent upon ligand-mediated aggregation of the receptor complex and the initial activation of an src family member protein kinase.119 In T-cell receptors, Lck associates with the receptor complex, while Lyn is involved in B-cell receptor signaling. Activation is also dependent upon the presence of members of the Tec family of protein tyrosine kinases including Btk in B-cell receptor complexes and the family members Rlk and Itk, redundantly in the case of the T-cell receptor.120,121 As a consequence of kinase activation, sites are phosphorylated on the receptor chains or receptor-associated chains. Many receptor complexes contain related sites of phosphorylation that are referred to as immunoreceptor tyrosinebased activation domains (ITAMs) or immunoreceptor

tyrosine-based inhibitory domains (ITIMs).122,123 Phosphorylation of ITIM sequences is associated with the recruitment of SH2 domain-containing tyrosine phosphatases and downregulation of the receptor complex while ITAMs recruit SH2 domain-containing adapter proteins that further coordinate the assembly of an activated receptor complex. Two of the essential additional kinases recruited to the complex are the structurally related kinase ZAP-70, in the case of the T-cell receptor complex, or Syk in the case of the B-cell receptor complex as well as other Fc receptor complexes.124 125 The essential role for each of these kinases in receptor function has been established through the derivation of mouse strains deficient in each of the kinases. In addition, a form of hypogammaglobulinemia in humans is associated with mutations in Btk. One of the key consequences of the activation of tyrosine phosphorylation is the recruitment and tyrosine phosphorylation of an adapter protein that, in the case of the B-cell receptor complex, is SLP-56 (also termed Blnk)126,127 and in the case of the T-cell receptor complex is SLP-76.128 The recruitment and phosphorylation of these adapter proteins is essential for the subsequent recruitment and activation of either PLC-1 in T-cell receptors or PLC-2 in B-cell receptors and other Fc receptor complexes. Recruitment is dependent upon the interaction of the SH2 domains of the enzymes with sites of phosphorylation on the adapter proteins and it is hypothesized that tyrosine phosphorylation of the enzymes may also be required for functional activation. The formation of a stable, activated receptor complex is also dependent upon membrane modifications including the phosphorylation of the 3 hydroxyl on phosphotidylinositol. In the case of the B-cell receptor complex, the kinase responsible for PI phosphorylation is p110, which is recruited to the complex by the adapter/regulatory

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subunit p85 through interaction of its SH2 domain with sites of phosphorylation in the receptor complex. Genetic deletion of either p85 or p110 dramatically reduces receptor-induced signaling.129 The phosphorylation of the 3 site of inositol creates a binding site for plecstrin homology (PH) domains. In the context of the Fc-receptor family members this creates membrane docking sites for both Btk and for p110, both of which contain PH domains that are essential for function in the context of the receptor complex. Conversely, the lipid phosphatase PTEN specifically dephosphorylates the 3 inositol site and is essential to ultimately disassemble the receptor complex. Following recruitment and activation, phospholipase C (PLC)- 1 or PLC- 2 hydrolyze membrane lipids releasing inositol trisphosphate (IP3 ) and diacyclglycerol (DAG). The IP3 generated interacts with its intracellular receptor to promote calcium release from endogenous stores, and the DAG and calcium then activate members of the protein kinase C family of serine/threonine kinases. The essential PKC in the case of the B-cell receptor is the classical PKC 1, while in T cells the critical family member is non-typical PKC . The consequences of activation of PKCs in intracellular signaling are likely to be many although, as noted below, one of the consequences is the activation of a kinase complex involved with the regulation of the transcription factors of the NF-B family (details given below). In T cells, the activation of a calcium flux is also critical for the activation of a sequence of events regulating members of the nuclear factor of activated T cells (NFAT) family of transcription factors.

Integrin signaling Sensing the environment has been an essential capability for cells throughout evolution. The possibility that cell adhesion might be important in regulating cell behavior actually first came from studies of tumor cells. Subsequently, the regulation of a number of cellular responses has been shown to be mediated through the recognition of the extracellular environment and specifically through receptor-mediated recognition of extracellular matrix (ECM) proteins. This recognition is provided through a family of integrin receptors that consist of a core recognition unit containing one of 8  subunits associated with one of 18
subunits giving rise to at least 24 distinct integrin receptors. The integrins are unique in their linkage to the actin-based microfilament system and, thereby, in their ability to control cellular properties such as membrane motion, cellular movement, and cell adherence. The integrin receptors are also unique in their bi-directional regulation; namely, intracellular signaling events can modify integrin-receptor structure to allow the receptor to

recognize ligand and, conversely, ligand binding initiates cellular signaling events that control a variety of cellular responses. As with many receptor systems, the initial consequence of ligand binding is to change the receptor structure and, in the case of the integrins, to induce clustering. The clustered integrins rapidly recruit the tyrosine kinase FAK (focal adhesion kinase), which becomes activated by transphosphorylation of an activation loop tyrosine and goes on to phosphorylate a number of additionally recruited substrates. FAK is a widely expressed kinase that is evolutionarily conserved and is related to the FAK-related kinase PYK2. The recruitment of FAK requires a unique carboxylterminal domain termed the focal adhesion targeting (FAT) domain, which interacts with the focal adhesion complex component paxillin, which binds directly to the cytoplasmic domains of integrin receptors. Once recruited to the complex and tyrosine phosphorylated, c-src is recruited to the sites of FAK tyrosine phosphorylation through SH2domain interactions. As with virtually all receptor complexes containing or recruiting tyrosine protein kinases, the integrin receptor complexes recruit a number of adapter/linker proteins that couple to the activation of shared pathways. For example, phosphatidylinositol-3-kinase (PI-3-K), PLC- , Shc, Grb2, and Grb7 are recruited via SH2 interactions, resulting in the activation of pathways that affect cell survival and promote cell growth. However, of particular importance for the integrin receptor systems is the ability to regulate the activity of the Rho family of small GTPases – including Rho, Cdc42, and Rac – all of which play critical roles in the formation and organization of cortical actin networks in cells. Although the precise details of the activation are still being investigated, some participants are emerging. For example the activation of Rac, a critical event promoting cell migration, is dependent upon the FAK/src recruitment and tyrosine phosphorylation of an adapter protein Cas which, in turn, recruits Crk and it in turn is required for the recruitment of DOCK180. Experimental evidence suggests that DOCK180 directly increases GTP-bound Rac. Paxilian, in addition to recruiting FAK, also recruits PAK (p21-activated kinase), an adapter protein Nck, and members of the Cdc42/Rac GTP exchange factors (PIX, COOL), which further contribute to membrane changes.

Pathways in signaling Stat proteins and cytokine receptors The signal transducers and activators of transcription (Stats) were discovered in studies to identify the

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Fig. 6.6 Members of the cytokine receptor superfamily activate members of the Stat family. The Stat proteins are recruited to the receptor complex through their SH2 domain-directed interaction with sites of tyrosine phosphorylation. The proteins are then phosphorylated and as a consequence dimerize and are translocated to the nucleus. In the nucleus they bind promoters in a sequence-specific manner and activate transcription.

IFN-regulated transcription factors that mediated the gene inductions associated with the establishment of an antiviral response.130 Subsequent studies both identified additional members of the family and implicated them in the regulation of gene transcription by a variety of cytokines that utilize cytokine receptors as well as growth factors and cytokines that utilize tyrosine kinase receptors. Although most commonly associated with cytokine responses that utilize receptors of the cytokine receptor superfamily, these proteins can also be activated by members of the tyrosine kinase receptor family. As detailed below, the significance of these observations, if any, is not apparent at this time. Stats consist of a centrally located, conserved DNAbinding domain that binds a palindromic sequence with the consensus sequence of TTT NC N NN NAA, which is referred to as the INF -activated sequence (GAS). This DNA-binding domain, although highly conserved among the Stats, is not found in any other DNA-binding proteins. In addition, the Stats contain a phosphotyrosine binding, SH2 domain that is highly conserved among the various family members. As noted below, this domain is critical for a number of Stats functions. Immediately following the SH2 domain is a positionally conserved tyrosine that is critical for function. The carboxyl domains are highly diverse, but in all cases are required for transcriptional activation. The amino-terminal domain contains conserved elements and is likely to be involved in stabilization of dimers and translocation of the dimer to the nucleus. The model for the Jak–Stat signaling pathway is remarkably elegant in its simplicity (Fig. 6.6). The Stat proteins exist

as latent cytoplasmic proteins that are widely expressed. Ligand binding and receptor-complex activation result in the creation of phosphotyrosine docking sites that are recognized by the SH2 domains of the Stats. Each Stat recognizes different phosphotyrosine sites, based on the flanking sequences, and thus only certain Stats are recruited to a particular receptor complex. This specificity is elegantly illustrated by the ability of such sites to move to different receptors and thereby change the pattern of Stat recruitment.131 Once the Stat is recruited to the complex, it is phosphorylated on the critical tyrosine that is carboxyl to the SH2 domain. This phosphorylation is mediated by the Jaks and requires the Stat SH2 domain for recognition by the Jak. Importantly, there is no apparent specificity of a particular Jak for a particular Stat, thus emphasizing that all the specificity for activation is dependent on the receptor structure. Following tyrosine phosphorylation of the Stats, they form homo- or heterodimers through the interaction of the SH2 domains with the carboxyl phosphotyrosine site. This interaction is of particularly high affinity and drives the reaction toward the accumulation of dimers. As dimers, the Stats can bind to DNA, whereas the monomers have no detectable DNA-binding activity, consistent with their symmetrical DNA-binding motif. As dimers the Stats also translocate to the nucleus, where they contribute to the activation of gene expression. The mechanisms involved in the ultimate turnover of the proteins in the nucleus are not known, but have been speculated to involve either a proteasome-dependent pathway or dephosphorylation and recycling of the protein to the cytoplasm. Certain Stats have been found to heterodimerize with other Stats. For example, a commonly observed heterodimer is one consisting of Stat1 and Stat3; however, the only heterodimers of Stat5 proteins that have been observed are those occurring between proteins encoded by the two very highly related Stat5 genes referred to as Stat5a and Stat5b. Therefore, although heterodimerization has the potential to significantly expand the spectrum of transcriptional complexes that can be generated, the extent to which this occurs is not clear. The significance of Stat activation has come largely from the analysis of mice that lack individual Stat family members. There have been seven mammalian Stat genes identified, and to date mutant mice have been generated for each of these Stats with the exception of Stat2. The first mutant mice generated were deficient in Stat1132 which is activated in the responses to both IFN
and IFN, as well as IFN, and was identified as the IFN-activated transcription factor that mediated the transcriptional activation of genes involved in the establishment of an antiviral response. A variety of

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reports, however, demonstrated that Stat1 could be activated in other responses, in the context of both cytokine receptor superfamily members and members of the receptor tyrosine kinases. The phenotype of deficient mice was quite striking, in that the animals developed completely normally under pathogen-free living conditions. Therefore, Stat1 is either not required or is redundant with another factor for any aspect of normal developmental physiology. However, the mice were extremely sensitive to infection with a variety of viruses or bacteria. The sensitivity to some viruses was increased by a dramatic 10 000-fold. The results have emphasized, in a striking manner, the critical role that the IFN system, through its ability to activate Stat1, plays in our daily effort to survive infectious agents. Stat3 is primarily activated within the context of members of the IL-6 subfamily of cytokines, including leptin and leukemia inhibitor factor (LIF). Within the context of IL-6, Stat3 is required for the expression of a wide variety of genes collectively referred to as the acute-phase response genes. Mutant mice lacking Stat3 die early during embryogenesis, probably prior to gastrulation, indicating an essential role in some aspect of early embryogenesis.133 Although relatively little information is available, it has been suggested that, in these mice, mesoderm fails to develop. It will be of some interest to develop conditional mutant mice to learn the function of Stat3 in adult physiology. Stat4 was initially cloned because of its homology with Stat1, and for a considerable time it was an orphan Stat for which we could not identify a ligand that would induce its tyrosine phosphorylation and activation.134 However, it was ultimately shown that among the approximately 50 cytokines that utilize receptors of the cytokine receptor superfamily, IL-12 induced the activation of Stat4.135,136 In addition to its activation by IL-12, however, Stat4 had a number of interesting properties, including the loss of expression in differentiating hematopoietic cells and high levels of expression during the terminal stages of spermatogenesis. Nonetheless, the Stat4-deficient mice were not deficient in either hematopoiesis or in spermatogenesis. Indeed, the primary defects in these mice were specifically related to the biological functions of IL-12.137,138 The defects included a loss of upregulation of NK cytolytic activity of spleen cells in response to IL-12. In addition, IL12-induced Th1 differentiation was disrupted. Indeed, the phenotype of the mice was virtually identical to that of mice lacking IL-12.139 Stat6 was initially cloned as an IL-4-induced Statlike activity140 and by searching databases of expressed sequences.141 The induction of tyrosine phosphorylation of Stat4 is primarily seen in the response of lymphocytes to

IL-4 or to IL-13. The specificity of Stat4 activation by IL-4 is related to the presence of a docking site for the IL-4 SH2 domain within the IL-4 receptor
chain. To assess the biological function of Stat6, we142 and others143,144 have generated mice that lack Stat4. Such deficient mice are viable and lack any gross abnormalities. However, virtually all of the biological functions associated with IL-4 or IL-13 are deficient in these mice. For example, splenic T cells are unable to respond to IL-4 by generating Th2 cells. More strikingly, a variety of stimuli are unable to induce IgE production in these mice. This observation supports a previous hypothesis that Stat6 regulated the transcription of the nonrearranged IgE isotope region of the heavy-chain locus and that such transcription was required for efficient rearrangement during the process of class switching. In addition, a number of cell-surface antigens that are normally upregulated during immune responses as a consequence of IL4 production are not upregulated in the deficient mice. Again, somewhat remarkably, the phenotype was very similar to that of mice in which the IL-4 gene had been deleted. In contrast to the above Stats, two highly related Stat5 gene products are activated in response to a wide variety of cytokines, including Epo, Tpo, IL-7, growth hormone, prolactin, and IL-2. Therefore, it was anticipated that Stat5 would have a more general, broadly significant spectrum of activity.145 The analysis of Stat5 function was somewhat complicated by the existence of Stat5a and Stat5b, which are approximately 95% identical in amino acid sequence and co-localize genetically. Indeed, it can reasonably be speculated that the two genes encoding these products arose through a relatively recent genomic duplication event. To generate mutant mice for each of the genes, it was necessary to target each gene in embryonic stem cells and then use double targeting in such cells to obtain mice deficient in both genes. Analysis of the various mutants revealed some striking and unexpected phenotypes. Mice deficient in Stat5a146 are defective in mammary gland development, particularly during lactation. This phenotype is consistent with a critical role for Stat5a in prolactin regulation of mammary gland development. Mice deficient in Stat5b147 have two phenotypes. One includes a decreased size of the males, comparable with the size reductions associated with growth hormone defects, whereas the other is an altered pattern of liver enzyme expression, a phenotype that also suggests a role in growth hormone signal transduction. The phenotype of mutant mice in which both genes were disrupted further supported a key role in both prolactin and growth hormone function as well as revealing an essential role in T-cell activation.148 The mutation of both Stat5 genes resulted in a reduced size of both male

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and female mice, now fully consistent with the phenotype seen in growth hormone-deficient mice. In addition, the females were sterile and failed to develop corpus lutea, consistent with another known function of prolactin. Thus, in these mice, virtually all of the physiological functions associated with growth hormone and prolactin were deficient. In addition, however, mature T cells from the mice failed to respond mitogenically to IL-2 in synergy with stimulation of the T-cell receptor. As a consequence the immune system was defective, and with time the mice developed manifestations of an altered immune response. Taken together, the results indicate that the Stat signaling pathways mediate very precise functions, each associated with relatively few cytokines. This contrasts quite distinctly with pathways such as the Ras pathway, which is speculated to provide a common mechanism by which cytokines regulate cell growth and differentiation.

Phosphatidylinositol-3-kinase function in signaling One of the common receptor-mediated events is the activation of phosphoinositide-3-kinase activity. Such activity is seen with protein tyrosine kinase receptors and the cytokine receptor superfamily as well as with several of the G-protein-coupled receptors. The interest in phosphatidylinositol phosphorylation originated with the observation that phosphatidylinositol (PI) kinase activity was associated with the polyoma middle-T complexes with c-src.149 It was speculated that such activity might contribute to the mechanism by which middle T transforms cells. Subsequently, it was demonstrated that an avian-transforming virus had transduced the PI-3-kinase catalytic subunit and that increased activity was required for transforming activity.150 Therefore, the biological data suggest that the ability to regulate the production of 3-phosphoinositols can influence cell proliferation, leading to a focus on how receptor complexes regulate the activity and utilize phospholipids in cellular signaling.151 In mammals there are several different enzymes that can mediate the PI-3-kinase reaction.152,153 Two enzymes mediate a specific PI-3-kinase reaction and are not known to be regulated by any signaling system. However, a p120 PI-3-kinase is known to be activated by both the
and / heterotrimeric G-protein complexes. In addition, two catalytic subunits, p110
and p110, have PI-3-kinase activity that is regulated through the association with a p85 regulatory subunit. Importantly, the p85 regulatory subunit contains two SH2 domains as well as an SH3 domain and two proline-rich domains. In the current model, p85 is recruited to an activated receptor complex through interaction of the p85 SH2 domains with sites of tyrosine phosphorylation

on the receptor. This interaction recruits the catalytic p110 subunit to the membrane-associated receptor complex, so that it comes in contact with its potential substrates. This ultimately results in the generation of PI(3)P, PI(3,4)P, and PI(3,4,5)P. These signaling molecules are then available to propagate a signal. Studies of the role of the PI-3-kinases in cellular responses have been greatly aided by the identification of inhibitors that are remarkably specific. Wortmannin is a steroid-like toxin isolated from a soil bacterium, whereas LY294002 is a bioflavoid derived from quercitin. As with any inhibitors, caution must be used when interpreting these results, because the effects may reflect activity against unknown additional targets. Precisely how the 3-phosphorylated lipids function as second messengers is not known. Studies have identified at least two potential links in signal transduction. First, it has been shown that Ras interacts with the catalytic subunit of PI-3-kinase in a GTP-dependent manner and may influence the catalytic activity of PI-3kinase.154 The Ras-dependent activation of PI-3-kinase is speculated to be essential for the membrane ruffling seen in Ras-transformed cells. The second potential signaling-linked pathway results in apoptosis. Specifically, 3-phosphorylated lipids bind to and activate the Akt serine/threonine kinase (also termed protein kinase B or PKB). Activated Akt has been found to phosphorylate a protein termed Bad, which in its nonphosphorylated form, binds to Bcl2 and inactivates its ability to suppress apoptosis. Thus, ligand-mediated activation of Akt, through the activation of PI-3-kinases, is able to suppress cell death.

Substrates of protein tyrosine phosphorylation In addition to the various pathways described above, there are a large number of substrates of cytokine or growth factor-induced tyrosine phosphorylation for which pathways do not yet exist, but which are presumed to contribute to cellular responses through as yet unidentified pathways. For example, the gene vav was initially identified by its contribution to a fusion protein that was associated with transforming activity.155 Subsequently, it was shown that Vav is rapidly tyrosine phosphorylated in the response to cytokines or growth factors that utilize either cytokine receptor superfamily members or receptor tyrosine kinases. The ability of a particular receptor complex to mediate Vav phosphorylation again is dependent on the existence of receptor-phosphorylated tyrosines that can recruit the protein through the Vav SH2 domain. The biochemical functions of Vav, phosphorylated or unphosphorylated, are not fully defined. Evidence has been

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presented that Vav is a guanine nucleotide exchange factor (GEF) for Ras and thus could potentially link Vav to the Ras signaling pathway.156 Subsequent studies have failed to establish this link, although Vav may function as a GEF for another Ras family member. In addition, it has been suggested that Vav is associated with PI-3-kinase activity and thus may link receptor activation to phosphatidylinositol signaling.157 These types of observations indicate the considerable difficulty that is often faced when trying to determine how a particular protein participates in cellular signaling. Although a variety of studies using various approaches had suggested a broadly significant role for Vav in cytokine and growth factor signaling, the phenotype of mice that lack the vav gene indicates a more proscribed function.158–160 In particular, the only defect that was identified was a loss of function of T and B cells in signaling through the antigenspecific receptors. Conversely, no defects were detectable in hematopoiesis, although a variety of cytokines – such as Epo – induce the tyrosine phosphorylation of Vav. The insulin response substrates (IRS-1 and IRS-2) were identified in studies of the substrates of insulin-induced tyrosine phosphorylation.161,162 This led to the cloning of IRS-1 and the subsequent identification of a highly related protein termed IRS-2. More recently, a smaller protein of 60 kDa, referred to as IRS-3, was identified; structurally, it is related to the IRS proteins and is speculated to be a member of that family. Both the IRS-1 and IRS-2 proteins are common substrates of ligand-induced tyrosine phosphorylation in the responses to a variety of cytokines and growth hormones. The phosphorylation of IRS proteins has been shown to be critical for IL-4-induced proliferation of myeloid lineage cells.163 Both IRS-1 and IRS-2 contain a pleckstrin homology domain at the amino terminus and multiple sites of tyrosine phosphorylation throughout the remainder of the protein. The IRS proteins are generally regarded as adapter proteins that can recruit a variety of signaling proteins to the receptor complex through the interaction of phosphotyrosine-binding domains with sites of IRS phosphorylation, thus providing surrogate “docking” sites. The role of phosphorylation of the IRS proteins in many of the situations in which their phosphorylation is seen is not known. However, mice that are deficient in IRS-1 are severely growth retarded due to resistance to insulin-like growth factor 1 as well as insulin.164,165 More recently, mice with a deletion of the IRS-2 gene166 have been derived and show impaired peripheral insulin signaling and pancreatic -cell function, resulting in a progressive diabetes similar in pathology to human type-2 diabetes. It is anticipated that mice lacking both IRS-1 and IRS-2 may have a more severe

phenotype. Interestingly, mice that are heterozygous for the loss of IRS-1 and the insulin receptor develop a non-insulindependent diabetes mellitus with age.167 Nonetheless, the data suggest that IRS-1 functions primarily within the context of the insulin and insulin-like growth factor receptors. The significance of ligand-induced IRS phosphorylation in the context of other receptors such as Epo or IL-4 is much less clear. The c-cbl protein is tyrosine phosphorylated in the responses to a number of cytokines. The gene for c-cbl was first identified as the origin of the v-cbl oncogene, the transforming gene of the murine Cas NS-1 retrovirus.168 The ccbl protein consists of an amino-terminal PTB domain and a RING finger domain. The carboxyl region, which is deleted in the transforming gene product, contains a proline-rich domain, three major sites of tyrosine phosphorylation, and a binding site for the interaction of 14-3-3 proteins. More recently a related gene, termed cbl-b, was identified.

The Ras pathway to activation of mitogen-activated protein (MAP) kinases One of the most studied pathways activated by cytokines or growth factors is the Ras/MAP pathway. Conceptually, it consists of a series of events initiated at the receptor that ultimately result in the tyrosine and threonine phosphorylation of members of the MAP kinases. These kinases are nuclear and are speculated to phosphorylate and thereby modify the function of a variety of transcription factors or other functions associated with cell cycle progression. It is beyond the scope of this review to cover the numerous components of the Ras/MAP pathway; however, a few comments are warranted to provide concepts that may be useful in further understanding signal transduction. In the simplest current models, the activation of a receptor complex recruits proteins that can initiate the formation of a complex leading to the activation of c-Raf. For example, the ability of most cytokine receptors to activate the Ras pathway begins with the recruitment of the adapter protein SHC to the receptor complex. This is mediated by the interaction of the SH2 domain with SHC, which recognizes specific sites of tyrosine phosphorylation on the receptor. Once recruited to the receptor complex, SHC is tyrosine phosphorylated and, in turn, recruits the Grb-2 adapter protein to the complex via interaction with the SH2 domain Grb-2, to associate with tyrosine-phosphorylated SHC. In the case of many tyrosine kinase receptors, Grb-2 is directly recruited to the receptor complex by interaction with sites of tyrosine phosphorylation within the receptor. The Grb-2/SOS complex then relocates to the membrane where SOS mediates the exchange of GDP for GTP

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on Ras. Once in the active configuration, Ras-GTP activates various signaling pathways including the recruitment and activation of c-Raf. Activated c-Raf phosphorylates and activates the kinases that are directly able to activate the MAP kinases. The MAP kinases then phosphorylate transcription factors and activate their ability to mediate gene expression, with the c-fos gene, for example.

Summary and conclusions The last several years have seen the identification of many of the components of signaling pathways that are activated in the responses to various cytokines. We have gone from a situation of little information to one in which many biochemical changes related to cytokine action have been detected and characterized. As a result, a number of general conclusions are emerging that are providing novel insights into signal transduction and the evolution of cellular signaling. The regulation of cellular responses resides with a large number of cytokines that utilize relatively few functionally related receptor families. Moreover, a number of the receptor families have relatively dedicated signaling pathways. Thus, members of the TGF family of cytokines can be predicted to use serine/threonine kinase receptor systems that activate transcription factors of the SMAD family of proteins. Similarly, the TNF-related factors will activate transcription factors of the NF-B family as well as proteases of the caspase family. Cytokines that use receptors of the cytokine receptor superfamily utilize Jaks and activate members of the Stat family. Indeed, it is quite possible that as new signaling pathways are identified, this theme of dedication to certain families of receptors will become further emphasized. However, some signaling pathways are much more generic. For example, relatively few cytokine systems do not activate one or more members of the large family of kinases related to the MAP kinases. Although many aspects of the biochemical regulation have been elucidated, there is still much to be learned with regard to the biology of the MAP kinase-related signaling pathways. It can be anticipated that the derivation of mice lacking individual members will provide important insights into the role of these kinases. Similarly, a number of receptor systems can activate PI-3kinase activity and induce the tyrosine phosphorylationdependent activation of PLC- function. Again, the significance of these signaling events in hematopoiesis is not clear and, indeed, in the case of the response to Epo, analysis of receptor mutants would suggest that activations of these pathways are not critical, nonredundant events.

Perhaps one of the most surprising lessons has been that not all biochemical responses to ligands have an essential function in all contexts. Again, several examples could be given, but one from recent studies with the IL-7 receptor system is particularly striking: although IL-7 activates a variety of signaling events, its critical function in the expansion of early T-cell progenitors can be rescued by simply introducing a transgene for Bcl-2. This observation also suggests a powerful experimental tool for assessing the role of various signaling pathways in the function of cytokines. In summary, with the introduction of new genetic approaches, studies of signal transduction have moved from solely biochemical analyses to more comprehensive physiological investigations.

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120 Miller, A. T. & Berg, L. J. New insights into the regulation and functions of Tec family tyrosine kinases in the immune system. Curr Opin Immunol, 2002; 14: 331–40. 121 Schaeffer, E. M., Debnath, J., Yap, G., et al. Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity. Science, 1999; 284: 638–41. 122 Isakov, N. ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery. Immunol Res, 1997; 16: 85–100. 123 Watson, S. P. & Gibbins, J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today, 1998; 19: 260–4. 124 Elder, M. E. ZAP-70 and defects of T-cell receptor signaling. Semin Hematol, 1998; 35: 310–20. 125 Chu, D. H., Morita, C. T., & Weiss, A. The Syk family of protein tyrosine kinases in T-cell activation and development. Immunol Rev, 1998; 165: 167–80. 126 Pappu, R., Cheng, A. M., Li, B., et al. Requirement for B cell linker protein (BLNK) in B cell development. Science, 1999; 286: 1949–54. 127 Minegishi, Y., Rohrer, J., Coustan-Smith, E., et al. An essential role for BLNK in human B cell development. Science, 1999; 286: 1954–7. 128 Clements, J. L., Yang, B., Ross-Barta, S. E., et al. Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development. Science, 1998; 281: 416–19. 129 Fruman, D. A. & Cantley, L C. Phosphoinositide 3-kinase in immunological systems. Semin Immunol, 2002; 14: 7–18. 130 Darnell, J. E., Jr., Kerr, I. M., & Stark, G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science, 1994; 264: 1415–21. 131 Stahl, N., Farruggella, T. J., Boulton, T. G., et al. Modular tyrosine-based motifs in cytokine receptors specify choice of stats and other substrates. Science, 1995; 267: 1349–53. 132 Meraz, M. A., White, J. M., Sheehan, K. C. F., et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell, 1996; 84: 431–42. 133 Takeda, K., Noguchi, K., Shi, W., et al. Targeted disruption of the mouse stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci, 1997; 94: 3801–4. 134 Yamamoto, K., Quelle, F. W., Thierfelder, W. E., et al. Stat4: a novel GAS binding protein expressed in early myeloid differentiation. Mol Cell Biol, 1994; 14: 4342–9. 135 Jacobson, N. G., Szabo, S., Weber-Nordt, R. M., et al. Interleukin 12 activates Stat3 and Stat4 by tyrosine phosphorylation in T cells. J Exp Med, 1995; 181: 1755–62. 136 Bacon, C. M., McVicar, D. W., Ortaldo, J. R., et al. Interleukin12 induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus tyrosine kinases by interleukin-2 and interleukin-12. J Exp Med, 1995; 181: 399–404. 137 Kaplan, M. H., Sun, Y.-L., Hoey, T., & Grusby, M J. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature, 1996; 382: 174–7.

138 Thierfelder, W. E., Deursen, J. van, Yamamoto, K., et al. Stat4 is required for IL-12 mediated responses of NK and T-cells. Nature, 1996; 382: 171–4. 139 Wolf, S. F., Sieburth, D., & Sypek, J. Interleukin-12: a key modulator of immune function. Stem Cells, 1994; 12: 154–68. 140 Hou, J., Schindler, U., Henzel, W. J., Wong, S. C., & McKnight, S. L. Identification and purification of human Stat proteins activated in response to interleukin-2. Immunity, 1995; 2: 321– 9. 141 Quelle, F. W., Shimoda, K., Thierfelder, W., et al. Cloning of murine Stat6 and human Stat6, stat proteins that are tyrosine phosphorylated in response to IL-4 and IL-3 but are not required for mitogenesis. Mol Cell Biol, 1995; 15: 3336– 43. 142 Shimoda, K., Deursen, J. van, Sangster, M. Y., et al. Lack of IL4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature, 1996; 380: 630–3. 143 Kaplan, M. H., Schindler, U., Smiley, S. T., & Grusby, M. J. Stat6 is required for mediating responses to IL-4 and for the development of Th2 cells. Immunity, 1996; 4: 313–19. 144 Kopf, M., Le Gros, G., Bachmann, M., et al. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature, 1993; 362: 245–8. 145 Ihle, J. N. STATs: signal tranducers and activators of transcription. Cell, 1996; 84: 331–4. 146 Liu, X., Robinson, G. W., Wagner, K. U., et al. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev, 1997; 11: 179–86. 147 Udy, G. B., Snell, R. G., Wilkins, R. J., et al. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci U S A, 1997; 94: 7239– 44. 148 Teglund, S., McKay, C., Schuetz, E., et al. Stat5a and Stat5b proteins have essential and non-essential, or redundant, roles in cytokine responses. Cell, 1998; 93: 841–50. 149 Whitman, M., Kaplan, D., Roberts, T., & Cantley, L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J, 1987; 247: 165– 74. 150 Chang, H. W., Aoki, M., Fruman, D., et al. Transformation of chicken cells by the gene encoding the catalytic subunit of Pl 3-kinase. Science, 1997; 276: 1848–50. 151 Divecha, N. & Irvine, R. F. Phospholipid signaling. Cell, 1997; 80: 269–78. 152 Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., & Waterfield, M. D. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci, 1997; 22: 267–72. 153 Vanhaesebroeck, B., Stein, R. C., & Waterfield, M D. The study of phosphoinositide 3-kinase function. Cancer Surv, 1996; 27: 249–70. 154 Rodriguez-Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D., & Downward, J. Activation of phosphoinositide 3kinase by interaction with Ras and by point mutation. EMBO J, 1996; 15: 2442–51.

Signal transduction in the regulation of hematopoiesis

155 Katzav, S., Martin-Zanca, D., & Barbacid, M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J, 1989; 8: 2283–90. 156 Gulbins, E., Coggeshall, K., Baier, G., et al. Tyrosine kinasestimulated guanine nucleotide exchange activity of vav in T cell activation. Science, 1993; 260: 822–5. 157 Shigematsu, H., Iwasaki, H., Otsuka, T., et al. Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity. J Biol Chem, 1997; 272: 14 334–40. 158 Fischer, K.-D., Zmuidzinas, A., Gardner, S., et al. Defective Tcell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes. Nature, 1995; 374: 474–7. 159 Tarakhovsky, A., Turner, M., Schall, S., et al. Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav. Nature, 1995; 374: 467–70. 160 Zhang, R., Alt, F. W., Davidson, L., Orkin, S. H., & Swat, W. Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature, 1995; 374: 470–3. 161 Myers, M. G., Jr., Xiao, J. S., & White, M. F. The IRS-1 signaling system. Trends Biochem Sci, 1994; 19: 289–93.

162 Yenush, L. & White, M. F. The IRS-signalling system during insulin and cytokine action. Bioessays, 1997; 19: 491–500. 163 Keegan, A. D., Nelms, K., White, M., et al. An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth. Cell, 1994; 76: 811–20. 164 Araki, E., Lipes, M. A., Patti, M. E., et al. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature, 1994; 372: 186–90. 165 Tamemoto, H., Kadowaki, T., Tobe, K., et al. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature, 1994; 372: 182–6. 166 Withers, D. J., Gutierrez, J. S., Towery, H., et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature, 1998; 391: 900–4. 167 Bruning, J. C., Winnay, J., Bonner-Weir, S., et al. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell, 1997; 88: 561–72. 168 Langdon, W. Y., Hartley, J. W., Klinken, S. P., Ruscetti, S. K., Morse, H. C, III. v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc Natl Acad Sci U S A, 1989; 86: 1168–72.

149

Immunophenotyping Fred G. Behm

Introduction

The diagnosis and treatment of childhood leukemia rest on the recognition of a leukemic cell population and its cell lineage and, sometimes, the stage of maturation. The presence in leukemic blasts of myeloperoxidase, Auer rods, or monocyte-associated esterases readily identify most cases of acute myeloid leukemia (AML). By contrast, the leukemic blasts of acute lymphoblastic leukemia (ALL) have no unique morphologic or cytochemical features. Malignant megakaryoblasts also lack defining cytologic and cytochemical features and may be mistaken for ALL. Although rare in children, chronic lymphoid malignancies, such as large granular lymphocyte leukemia or HTLV-1associated leukemia can be confused with reactive lymphocytosis or acute leukemia. The prognosis and therapy for ALL, AML, and chronic leukemias differ greatly; thus, it is crucial to document the lineage and stage of maturation of leukemias. In the absence of diagnostic morphologic features, accurate diagnosis requires contemporary immunologic and molecular analyses. Immunologic testing, or immunophenotyping, is an essential component of the diagnostic work-up by confirming or establishing the leukemic cell lineage, stage of differentiation, and sometimes clonality. The results of immunophenotyping also correlate with cytogenetic abnormalities, facilitate minimal residual disease studies, and provide prognostic information. The earliest immunophenotyping studies of leukemias were performed with polyclonal antisera produced to T lymphocytes, immunoglobulin (Ig) heavy and light chains, the common acute lymphoblastic leukemia antigen (CALLA), the minor histocompatibility antigen HLA-DR,

and terminal deoxynucleotidyl transferase (TDT) . Normal and leukemic T cells were also identified by their unique property of serving as a nidus for the formation of rosettes by sheep erythrocytes. These rudimentary panels and test methods have been replaced by monoclonal antibodies to lineage-associated antigens and multiparameter flow cytometry and immunohisto chemistry. Several hundred monoclonal antibodies have nowbeen assigned to over 260 clusters of differentiation (CD) groupings by the International Workshops on Leukocyte Differentiation Antigens.1 Table 7.1 lists CD groups and representative examples of antibodies that have proven particularly useful for studies of leukemic processes. A variety of fluorochromes, includingfluoresceinisothiocyanate (FITC), phycoerythrin (PE), PE-Texas red conjugate (ECD), peridinium chlorophyll complex (PerCP), PE-cyanconjugates (PC5), allophycocyanate (APC), allowfor simultaneous detection of multiple lineages and differentiation markers. Modern automated flow cytometers are designed to make full use of these new antibodies and fluorochromes together with enhanced computer software to provide multiparameter analysis for more precise diagnoses. Recent advances in unmasking leukocyte antigens in formalin-fixed tissues such as bone marrow biopsies have also facilitated more accurate diagnoses. This chapter focuses on the diagnostic capabilities of these new technologies and provides a reference for interpreting immunophenotyping studies of leukemias. Lineage development and antigen expression

The antigen expression profile of leukemias and lymphomas parallel normal stages of myeloid and of B- and

Childhood Leukemias, ed. Ching- Hon Pui. Published by Cambridge University Press. © Cambridge University Press 2006.

150

Immunophenotyping

151

Table 7.1 Cluster of differentiation (CD) antigens used in diagnosis and classification of acute leukemia and lymphoma Marker

Description/function

Normal cell expression

Associated disease states

Method of detection

CD1a

49 kDaGP, [3-chainnoncovalentlybinds

Thymic T cells, dendritic and

T-cell ALL and lymphoma,

FC, IHC

[3-2-microglobulin

Langerhans cells, cytoplasm of

Langerhans cell histiocytosis

activated T cells CD2

50 kDa transmembrane GP, LFA-1, LAF-3

Thymic and mature T cells, NK cells,

T-cell ALL and lymphoma, subsets

(CD58) ligand, sheep E-rosette receptor,

thymic B cells

of M3 and M4 AML, some

FC, IHC

MDS-relatedAML

alternative T-cell activation, T- and NK-cell cytolysis CD3

20 to 50 kDa complex of 6 polypeptides

Thymic and mature T cells

T-cell leukemia and lymphoma

FC, IHC

T- and NK-cell leukemia and

FC

(-y/ε, 8/e, t, It, dimers) bind TCRa/g and 7/8, signal tranducer after antigen recognition by TCR CD3

t, chains are expressed independently of

Thymic and mature Tcells, NK cells

lymphoma

other CD3 dimers, signal transducer after antigen recognition by TCR, regulation of cell surface expression of CD3/TCR complex, NK-cell activation CD4

55 kDa transmembrane GP, coreceptor

Thymic and mature T cells

T-cell ALL and lymphoma, M4 and

with TCR for MHC class II

recognizing MHC class II antigens,

M5 AML, some NK-cell blastic

antigen-induced activation, thymic

monocytes, histiocytes, rare NK

malignancies

T-cell differentiation, receptor for HIV

subset

FC, IHC

retrovirus CD5

67 kDa transmembrane GP, signal

Thymic and mature T cells, B-cell

transduction

subset, higher intensity on Th than Tc

T-cell leukemia and lymphoma, CLL

FC, IHC

FC, IHC

cells CD7 CD8

40 kDa transmembrane protein,

Stem cells, thymic and mature T cells,

T-cell ALL and lymphoma, 15% of

costimulatory molecule

NK cells

Ly+AML

32 kDa protein (a-a or a-[3

Thymic and suppressor/cytotoxic T

T-cell ALL and lymphoma, NK-cell

heterodimers), coreceptor with TCR for

cells, NK cells

leukemia

FC, IHC

MHC class I CD10

100 kDa protein; common ALL antigen

Precursor-B and -T cells, germinal

Precursor-B and T-ALL/LBL, Burkitt

(CALLA); neutral endopeptidase,

center B cells, mature neutrophils

and FCC lymphomas

FC, IHC

regulator of B-cell growth and proliferation CD11b

170 kDa GP, phagocytosis, chemotaxis,

Granulocytes, monocytes, NK cells,

AML, precursor-B and T-ALL,

apoptosis

subsets of B and T cells

NK-cell leukemia, absent in patients

150 kDa GP, functions similar to CD11b

Granulocytes, monocytes, NK cells,

AML, precursor-B and T-ALL,

subsets of B and T cells

NK-cell leukemia, absent in patients

FC

with leukocyte adhesion deficiency CD11c

FC

with leukocyte adhesion deficiency CD13

150 kDa GP, aminopeptidase catalyzes,

Early committed progenitors through

removal of amino acids or small

late stages of granulocytic and

peptides, receptor for coronavirus, CMV

monocytic maturation, large granular

uses CD13 to interact with target cells

lymphocytes, bone marrow stromal

Most AML, 20-30% of My+ALL

FC

FC

cells, osteoclasts CD14

55 kDa phosphoinositol-linked GP, LPS

Mature monocytes, macrophages,

M4 and M5 AML with maturing

receptor for endotoxin to release

weak expression by neutrophils

monocytic cells

cytokines CD15

CD16

Carbohydrate (X-hapten; Lewis-X),

Monocytic and myelocytic cells,

AML, granulocytic sarcoma, 10% of

adhesion/ phagocytosis

Langerhans cell

My+ALL, R-S cells

Fc receptor of IgG (Fc-y RIII), receptor for

Granulocytes, monocytes,NKcellNKcell

NK-cell LGLL and lymphoma

FC, IHC

FC

antibody-dependent cellular cytotoxicity (cont.)

152

Fred G. Behm

Table 7.1 (cont.) Marker

Description/function

Normal cell expression

Associated disease states

Method of detection

CD19

95 kDa GP, signal transduction for B-cell development, activation, and differentiation

Precursor and mature B-cells, absent on plasma cells, follicular dendritic cells

Precursor and mature B-cell

FC

leukemia and lymphoma, t(8;21) + AML FC, IHC a

35 to 37 kDa phosphoprotein, cellular

Precursor and mature B cells, absent

Precursor and mature B-cell

activation and proliferation

on differentiated plasma cell

leukemia and lymphoma

CD21

145 kDa GP, receptor for C3d, C3dg, iC3b, and EBV, signal transduction

Surface Ig-positive B cells (lost with activation), thymic T-cell subset, follicular dendritic cell

B-cell leukemia and lymphoma, T-ALL

FC

CD22

135 kDa GP, adhesion and activation

Precursor and mature B cells

Precursor and mature B cell

FC

CD20

leukemia and lymphoma; rare cases ofAML CD24

35 to 45 kDa GP, early B-cell

Throughout B-cell maturation but not

development and apoptosis?

plasma cells; neutrophils

CD30

105 kDa GP, member of the TNF receptor family

CD33

CD34

CD36

Most precursor-B ALL and LBL cases

FC

Activated T, B, NK cells, monocytes

R-S cells, immunoblastic and anaplastic large cell lymphomas, lymphocytes infected with EBV, HTLV1orHIV,ATLL

FC, IHC

67 kDa transmembrane GP, sialoadhesin (lectin activity for sugar chains containing sialic acid)

CFU-GEMM, CFU-GM, CFU-G, BFU-E, myeloblast -> neutrophil maturation, monocytic cells, megakaryoblasts, early erythroblasts

80% of AML, 30% of My+ALL

FC

40 and 116 kDa transmembrane GP,

Lympho/hematopoietic stem cells

ALL, AML, LBL, vascular tumors

FC, IHC b

sialomucin, two forms with one having a truncated cytoplasmic domain, stromal cell adhesion

and progenitors, small-vessel

Platelet gpIV, collagen,

Platelets, megakaryocytes, erythroblasts and RBCs, monocytes, macrophages

M4, M5, M6, and M7 AML

FC

Megakaryocytes, platelets

M7AML, Glanzmann's

FC

P.falciparum

receptor recognition and phagocytosis of apoptotic cells, platelet adhesion and aggregation CD41

gpIIb(asubunit)ofCD41/CD61

endothelium

(gpIIb/gpIIIa) complex, composed of gpllb-a and gpllb-g subunits, platelet aggregation, receptor for fibronectin, fibrinogen, and von Willebrand factor

thromboasthenia

CD42b

Platelet gplba, forms heterodimer with [3 chain (CD42c) and complexes with gpIX/ (CD42a), von Willebrand factor-ristocetin receptor

Megakaryocytes, platelets

M7 AML, Bernard-Soulier syndrome

FC

CD45

180 to 220 kDa GP, leukocyte common antigen (LCA), tyrosine phosphatase, different isoforms characteristic of different subsets of hematopoietic cells, critical for lymphocyte activation

All leukocytes, weakly expressed by

ALL, AML, lymphoma

FC, IHC

very early erythroblasts

CD45RO 180 kDa glycoprotein, CD45 isoform

Thymic and mature T cells, monocytes, neutrophils

T-cell leukemia and lymphoma, M5 AML

FC, IHC

CD56

140 kDa isoform on NK cells, cytotoxic T cells, subset of CD4+ T cells, neural tissues

T- and NK-cell

FC, IHC

175 to 220 kDa transmembrane GP, neural adhesion molecule (N-CAM), many isoforms

leukemia/lymphoma, some t(8;21) + and t(15;17)+ AMLs, M4 and M5 AML, neuroendocrine tumors

Immunophenotyping

153

Table 7.1 (cont.) Marker

Description/function

Normal cell expression

Associated disease states

Method of detection

CD61

gpIIIa([3subunit)ofCD41/CD61 (gpIIb/gpIIIa) and CD51/CD61 complexes, adhesion to diverse matrix proteins

Megakaryocytes and platelets with

M7 AML, Glanzmann's

FC, IHC

CD41/CD61, monocytes c

thromboasthenia

CD64

72 kDa GP, Fc-y RI, endocytosis of IgG antigen complexes, phagocytosis

Monocytes, histiocytes, neutrophils, early myeloid and monocytic precursors, dendritic cells

Myeloid and monocytic AML, neutrophil expression increases in infection

FC

CD65

Carbohydrate carried by lipid and maybe protein, poly-N-acetyl-lactosamine, unknown function

Myeloid and monocytic cells

Myeloid and monocytic AML,

FC

90 kDa GPI-linked GP, member of CEA antigen family, regulator of adhesion activity?

Granulocytes, epithelial cells

Subset of Precursor-BALL

FC

110 kDa transmembrane GP, primarily in

Monocytes, macrophages, mast cells, neutrophils, basophils, dendritic cells, subset of myeloid progenitors, activated T cells, subset of B cells, osteoclasts

M4 and M5 AML, subset of

FC, IHC

CD66c

CD68

cytoplasmic granules, endocytosis, lysosomal trafficking

some My+ALL

precursor-TALL/LBL

CD79a

mb-1 gene product Iga, associates with CD79b, signal transduction for surface immunoglobulin

Precursor and mature B cells

Precursor and mature B-cell leukemia and lymphomas some T-ALLandAML

FC, IHC

CD79b

B29 gene product Igg, associates with CD79a, signal transduction for surface immunoglobulin

Precursor and mature B cells

Mature B-cell leukemia and

FC

43 kDa transmembrane GP

Nonfollicular dendritic cells,

Dendritic leukemia, Langerhans

Langerhans cells

histiocytosis AML, rare T-precursor ALL, mastocytosis

FC, IHC

Precursor-B ALL, rare precursor-T

FC

CD83

lymphoma

CD117

143 kDa transmembrane GP, tyrosine kinase c-kit, stem cell factor receptor, a growth factor receptor; cell adhesion?

Hematopoietic stem and progenitor cells, mast cells

CD179a

16 to 18 kDa polypeptide, VpreB surrogate light-chain component disulfide-linked to Ig(x to form pre-BCR, transduces signals for B-cell differentiation and proliferation

Early pre-B and pre-B cells

lambda 5/14.1 surrogate light-chain component disulfide-linked to Ig(x to form pre-BCR, transduces signals for B-cell differentiation and proliferation

Early pre-B and pre-B cells

Sialoglycoprotein glycophorin A,

Erythroid cells

CD179b

CD235a

FC

ALL and AML

Precursor-B ALL, rare precursor-T

FC

ALL and AML

M6 AML, some M7 AML

FC, IHC

proposed functions include minimizing RBC aggregation and inhibition of lysis Abbreviations: GP, glycoprotein; FC, flow cytometry; IHC, immunohistochemistry; NK, natural killer; TCR, T-cell receptor; MHC, major histocompatibility complex; Th, helper T cells; Tc, cytotoxic T cells; CLL, chronic lymphocytic leukemia; LBL, lymphoblastic lymphoma; FCC, follicular center cell; BCR, B-cell receptor; MDS, myelodysplastic syndrome; LPS; lipopolysaccharide, My+ALL, myeloid antigen-positive AML; R-S, Reed-Sternberg cell; LGLL; large granular lymphocyte leukemia; CFU, colony-forming unit; GEMM, granulocyte/erythoid/myeloid/monocytic; GM, granulocyte/monocytic; G, granulocytic; BFU-E, burst forming unit-erythroid; EBV, Epstein-Barr virus; HIV, h u m a n immunodeficiency virus; HTLV-1, h u m a n T-cell lymphotrophic virus; ATLL, adult T-cell lymphoma/leukemia; Ig, immunoglobulin; GPI, glycosylphosphatidylinisotol. a b c

CD20 antibodies differ for FC and IHC. Different antibodies optimal for blast cells and endothelium. Nonspecific binding to monocytes.

154

Fred G. Behm

Early pre-B (pro-B)

Pre-B

Late pre-B (transitional)

Mature-B (naïve-B)

Fig. 7.1 Schematic of normal stages of B-cell maturation. Numbers on cell surface refer to CD (cluster of differentiation) antigens. The bars below the cell diagram represent the expression of proteins associated with the pre-B-cell receptor (pre-BCR) and the B-cell receptor (BCR). A lymphoid progenitor (not shown) expressing CD34, CD10, and weak CD19 precedes the first identifiable cell committed to B-lineage differentiation. Pseudo-light chains (\J/ LC) CD179a and CD197b first appear on the surface of early pre-B cells (pro-B). The early pre-B cell has rearranged immunoglobulin heavy-chain (HR) and germline immunoglobulin light-chain (LG) genes. The early pre-B cell expresses terminal deoxynucleotidyl transferase (TDT), \\i LC, CD 10, CD 19, CD24, and CD34. The pre-B stage is heralded by the appearance of cytoplasmic |x immunoglobulin (Ig) heavy chains and the movement of CD22 from the cytoplasm to the cell surface. The late pre-B (transitional) cell displays surface pre-BCR that comprises Ig|x complexed to t|< LC and noncovalently bound to CD79a and CD79b (|x/\J/LC/CD79). Rearrangement of Ig light-chain (LR) genes precedes the synthesis of light chains \ and K. The |x heavy chain is bound by disulfide bonds to either \ or K light chains to form IgM. The mature or nai've-B-cell stage is initiated with the movement of IgM to the cell surface. The mature B cell loses TDT and fully expresses the BCR, a complex of IgM/CD79a/CD79b.

T-cell differentiation and maturation and have provided a framework for immunologic classifications of leukemias and lymphomas. The newest classifications of hematopoietic and lymphoid malignancies are based on immunophenotyping, cytogenetic, and molecular genetic studies.2 Immunophenotyping serves to establish or confirm the diagnosis of hematopoietic and lymphoid malignancies. However, its usefulness in predicting treatment response has largely been replaced by the prognostic importance of cytogenetics and molecular genetic studies. B-lineage cells The maturation of bone marrow progenitor cells to mature B lymphocytes proceeds through stages that can be iden-

Pre-BCR complex (lguA|/LC CD79)

BCR complex (IgM CD79)

Fig. 7.2 Schematics of the pre-B-cell receptor (pre-BCR) and B-cell receptor (BCR) complexes. The pre-BCR consists of pseudo-light chains (t|< LC) VpreB and \ 5 (CD179a and CD179b, respectively) bound by disulfide bonds to immunoglobulin (Ig) |x heavy chains. This Ig|x/\J/ LC receptor is noncovalently bound to CD79a and CD79b. The pre-BCR first appears on the surface of late pre-B (transitional) cells and persists into the early stage of mature B-cell maturation. Mature or naive B cells express IgM, the BCR, which is composed of immunoglobulin |x heavy and light chains (either A. or K). CD79a and CD79b serve as the signaling molecules for the BCR. Binding of antigen by IgM results in tyrosyl phosphorylation of the cytoplasmic tails of the CD79 molecules, leading to activation of cellular signaling pathways.

tified by the pattern of cellular Ig protein expression (Fig. 7.1). The earliest bone marrow cells committed to B-lineage development have rearrangements of the diversity and joining (DJ) regions of the Igheavy-chain gene but do not synthesize immunoglobulin proteins. These proB or early-pre-B cells express surface CD 10, CD 19, CD24, CD34, CD45 (commonleukocyte antigen), HLA-DR, cytoplasmic CD22, CD79a, CD79b, and nuclear TDT.3~6 The expression of CD45 is weak initially, but increases with cell maturation.7 The pseudo-light-chain components of the pre-B-cellreceptor,CD179a(preV5) andCD179b(X5),make their debut in the cytoplasm of pro-B cells.8 Functional rearrangement of the Ig heavy-chain gene heralds the appearance of (j, heavy chains in the cytoplasm and promotes the differentiation of pro-B cells to the preB stage (Fig. 7.2).8 Young pre-B lymphocytes are the first cells to express surface CD22, followed by the appearance of CD20. During the late stages of pre-B-cell maturation (transitionalpre-B phase) lymphocytes weakly express surface (j, heavy chains but not K or X Ig light chains. Instead, (j, chains are transported to the cell surface in the company of the noncovalently linked pseudo-light chains CD179a and CD179b (Fig. 7.1).8-12 The CD179a/CD179b/Ig|j, complex

Immunophenotyping

Extra-follicular

Lymphoid follicle and germinal center

na

Progenitor Bcell

Centrocyte CD19 CD20 CD23 CD10 Centroblast IgG

Fig. 7.3 Diagram of B-cell maturation in the bone marrow with further differentiation after the cells have transited to peripheral lymphoid tissues via the circulatory system. Naive B cells leave the bone marrow and upon binding antigen undergo blastogenesis with the production of plasma cells and memory B cells in either extra-follicular or follicular lymphoid tissues. In the lymphoid follicle, B cells pass through several stages of differentiation that result in immunoglobulin isotype switches and a selection process that yields cells capable of producing high-affinity immunoglobulins. A partial list of B-cell-associated antigens by their CD (cluster of differentiation) names is given for the B cells portrayed.

is referred to as the pre-B-cell receptor (pre-BCR) (Fig. 7.2). CD 179a and CD 179b are encoded by the genes on chromosome 22 that also carries the X lglight-chain gene. Although these genes share partial homology with the variable and constant regions, respectively, of the X light chain, they do not undergo rearrangement. Disulfide-linked Iga (CD79a) and Ig(J (CD79b) molecules are noncovalently associated with the pre-BCR in a manner analogous to their association with IgM B-cell receptor (BCR). Expression of the pre-BCR results in termination of further rearrangement of the heavy-chain gene and leads to the induction of active pre-B-cell proliferation. Cells that fail to generate a preBCR undergo apoptosis.8, 13, 14 As thepre-B stage ends, TDT, CD34, and CD 10 disappear. Rearrangement and transcription of Ig light-chain genes leads to the formation of complete Ig molecules and to the maturation of transitional pre-B cells to mature B lymphocytes. The mature B cell is defined by the expression of surface IgM BCR (Fig. 7.2). For a brief time, the young mature B cells may coexpress the pre-BCRs and IgM BCRs.15 The BCR is a multiprotein structure comprising an antigenbinding membrane Ig molecule, that consists of two heavy and two light Ig chains linked by disulfide bonds, noncovalently associated with signal transducing heterodimers

CD79a and CD79b. The short three-amino-acid cytoplasmic tail of the JL heavy chain has no intrinsic signaling capacity. Instead, the antigen-stimulated IgM molecules induces conformational changes in CD79a/CD79b heterodimers that serve as signal transduction molecules via their cytoplasmic domains. The cytoplasmic domains subsequently induce signal transduction by binding to the Src family kinases Lyn and Fyn, protein tyrosine kinase SYK, and other phosphoproteins.10,13,16 B cells unable to produce functional IgM BCRs undergo apoptosis in the bone marrow. The expression of surface IgM is accompanied by an increased CD20 density and appearance of CD21, a receptor for C3d and Epstein-Barr virus. The product of bone marrow lymphopoiesis is an immune-competent B cell commonly referred to as a 'naive' or 'virgin' B cell to distinguish it from mature B cells stimulated by antigen. Naive B cells migrate from the bone marrow to colonize the follicular regions or B-cellzones of lymphnodes, spleen, Peyer's patches, tonsilar and other secondary lymphoid tissues. Germinal centers develop in the lymphoid follicles in response to proliferation of antigen-primed B cells. In the germinal centers, BCR diversification results from somatichypermutationoftheIggenes (Fig.7.3).17,18 Insecondary lymphoid tissues, further rearrangements of the Ig

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Cortex

Medulla TCRαβ

CD34 CD7 HLA-DR CD33 (CD2)

Pro-T

CD34 CD7 HLA-DR cyCD3 CD1a CD2

Pre-T1

CD7 cyCD3 CD1a CD2 CD5 CD4

Pre-T2

Pre-T3

DP

SP

Fig. 7.4 Schematic of T-cell maturation in the cortical and medullary regions of the thymus. Expression of T-cell-associated antigens are listed as CD groups. The lower panel shows expected sequential expressions of T-cell receptor gene (TCR) transcripts: pre-Tαa (pTαa), pre-TCRαb (pTαb), TCRδ, TCRγ, TCRβ, TCRα. Nuclear expression of terminal deoxynucleotidyl transferase (TDT) is indicated in the cell nuclei. Bone marrow derived pro-T cells migrate to the subcapsular region of thymic lobules. Under the influence of thymic chemokines and thymic stromal and epithelial cells, pro-T cells are induced to begin T-lineage differentiation and maturation. The first recognizable T cell, pre-T1, expresses cytoplasmic CD3 and TCR8 transcripts. Pre-T cells transition first through the cortex and then into medullary regions of the lobule before leaving the thymus as mature or naive T cells. Five maturational stages are represented: pre-T1, pre-T2, pre-T3, CD4/CD8 double-positive (DP), and CD4 or CD8 single-positive (SP). Some studies support two additional stages of maturation: late pre-T3 and pre-DP, during which surface CD3 is temporarily lost or internalized.

T-lineage cells

thymus.19"22 The T-cellprecursor is abone marrow-derived cell that expresses CD34, CD7, CD33, CD45RA, HLA-DR, possibly CD2, and little or no CD117 and CD90.20-26 The blood-borne progenitor cells arrive in the outer cortical layer of thymic lobules, move through the cortex to the cortico-medullary junction, and take up residence in the medulla (Fig. 7.4). During this journey a variety of chemokines and stromal cell chemokine receptors are encountered that induce differentiation and proliferation.27"29 The subcapsular progenitor cells or proT cells express CD34, CD7, and HLA-DR, but not CD1a or CD3. Pro-T cells still have their T-cell receptor 8,7, a and (3 genes (TCRG, TCRD TCRA, and TCRB) in germline configuration and retain the capacity to generate T cells, NK cells, dendritic cells, and myeloid elements. 24,25,28

The commitment of bone marrow and fetal liver-derived progenitor cells to T-cell lineage takes place in the

The earliest cells committed to T-cell development, pre-T1 cells, are found in the outer cortical areas of the thymic lobules. They are identified by their expression

heavy-chain gene can result in production of IgD, IgG, or IgA. Germinal B cells (centroblasts and centrocytes) share some similarities with pre-B cells, in that they express CD19, CD22, and CD10 and have a high propensity for apoptosis. However, unlike pre-B cells, germinal B cells do not express TDT or CD34. Centrocytes not induced to die by apoptosis leave the germinal center to become memory B cells or form plasmablasts that home to the bone marrow or medullary region of lymphoid tissues with subsequent maturation to plasma cells. Plasma cells express CD38, CD79a, CD 138, and can secrete large amounts of Ig, but do not display surface Ig, CD20, CD22, or HLA-DR. A subset of plasma cells may weakly express CD 19 and CD45.

Immunophenotyping

Pre-TCR complex pTαβ

TCR complex a p

Fig. 7.5 Pre-T-cell receptor (pre-TCR) and T-cell receptor (TCR) with associated CD3 transmembrane proteins. The pre-TCR complex a a b b consists of TCR(3 and two other proteins, pre-Ta (pTa ) and pre-Ta (pTa ). The pre-TCR and TCR molecules require CD3 for cell signaling. This antigen is a complex of 8, -y, and two ε chains. Two £ chains are also part of, but not unique to, the CD3 complex. Signal transduction by pre-TCR and TCR depends on the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of the CD3 proteins. After binding of the ligand by the TCR, phosphorylation of tyrosines of ITAMs creates binding sites for downstream signaling molecules and initiation of signaling events. of CD34, CD7, CD1a, CD2, cytoplasmic CD3, and TDT, but not CD4 or CD8 (double-negative cells because of the absence of CD4 and CD8 expression) (Fig. 7.4). TCRG and TCRD genes are rearranged at this stage and partial D-/p rearrangement of TCRB also takes place. PreT1 cells lose the potential to produce NK, dendritic, and myeloid cells. The pre-T1 stage is followed by sequential expression of CD4 and CD8aa, identifying pre-T2 and early pre-T3 stages, respectively. During the pre-T3 stage TCR(3 proteins appear in the cytoplasm with productive rearrangement of TCRB genes. The (3 proteins bind to surrogate pre-Ta proteins to form pre-TCRB receptors that are transported to the cell surface in the company of CD3 (Fig. 7.5). Expression of the pre-TCRp results in further pre-T-cell expansion and differentiation to a common thymocyte stage characterized by CD7/CD5/CD2/CD4/ CD8a(3-positive cells (double-positive cells because of the coexpressionof CD4 and CD8). These double-positive cells display low levels of CD3 (or CD3l° cells) and may also express CD10 and CD21. 2 7 , 3 0 , 3 1 The expression of pre-TCRp receptors induces rearrangements at the TCRA locus. 2 7 , 2 8 In the pre-T4 stage, productive rearrangement of TCRA results in the pro-

duction of TCRa molecules that in turn combine with TCR(3 proteins to form TCRa(3 (Fig. 7.5). The TCRa(3/CD3 complex moves to the surface of double-positive T cells. With the appearance of TCRapJ, cell surface CD3 intensity increases, resulting in CD3 hi cells, while expressions of CD1, CD10, CD21, and TDT rapidly diminish. Most of the TCRa(3 double-positive cells die by apoptosis as a result of the TCR receptors not being able to recognize self-peptideMCH complexes on thymic epithelial cells (death by failing positive selection). Surviving medullary CD3+, CD4+ and CD3+, CD8+ cells are releasedfrom the thymus into circulatory system to home to T-cell zones of peripheral lymphoid tissues.

Myeloid and monocytic lineages Myeloid and monocytic lineages exhibit many antigens in common (Fig. 7.6). The earliest myeloblasts weakly express CD34 and HLA-DR, but these are lost before the promyelocyte stage.32 CD 13 first appears on the surface of committed granulocyte-monocyteCD34-positive progenitors, increases with granulocyte maturation, and decreases

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Fred G. Behm

Myeloblast

locyte

Myelocyte

Neutrophil G^J

HLA-DR CD33 CD13± CD4 CD15 CD14

Fig. 7.6 Schematic of myelocytic and monocytic maturation. Early progenitor cells committed to myeloid and monocytic development (CFU-GM) express CD33, CD34, CD117, HLA-DR and weak (±) CD13. At the myeloblast and monoblast stages, CD34 and CD117 disappear and CD13 intensity increases. Cells in the mid-stages of myelocytic and monocytic maturation show the expression of CD15. CD65 and CD66 also appear in the late myelocyte stage. HLA-DR is lost early in myelocytic differentiation but persists with monocytic maturation. Strong expression of CD 14 distinguishes the mature monocyte from myelocytic lineage cells. CD4 is weakly expressed throughout monocytic development.

Fig. 7.7 Comparison of myeloperoxidase (MPO), elastase, and lactoferrin expression with myelocytic lineage maturation. The MPO messenger RNA (mRNA) and the proenzyme form of MPO (proMPO) are present in the earliest stages of myelocytic development. Small quantities of enzymatically active MPO are present in early myeloblasts and increase with myelocytic maturation. Primary granules contain MPO and elastase. Lactoferrin appears in early myelocytes and is packaged in specific or secondary granules.

The CD33 antigen is expressed by stem cells that give rise to mixed hematopoietic colonies with in vitro assays. The expression of CD33 decreases slightly with myeloid mat5 33 uration, but is evident at all stages of monocytic develwith monocytic development.. , CD 13 is also present opment. CD64 or Fc-/RI appears at the CFU-GM stage in secretory granules. Granulocyte activation or apoptoand persists through granulocyte and monocyte maturasis results in release of storage CD13 and increased cell tion, but its intensity is greater on monocytic cells. CD65, surface expression. Density-gradient separations and RBC which is structurally similar to CD 15 and shares its cellular lysis techniques used in immunophenotype testing can distribution, is first detected on granulocytic and monoinduce release of granule-based CD13 and enhance surcytic precursor cells that have lost CD34. CD65 density face CD13 density. increases with maturation to neutrophils and monocytes. Glycoprotein CD14 is expressed strongly on monocytes CD36 (glycoprotein IV) is found on immature and mature and macrophages but only weakly, if at all, on immature monocytes but not the granulocytic series. Two intracellumonocytic cells. Neutrophils also produce small amounts lar molecules, myeloperoxidase and lactoferrin, are useful of CD14. Glycosyl phosphatidylinositol (GPI) links CD14 markers for assessing myeloid differentiation. Myeloperoxto the cell surface of monocytes. Monocytes of patients idase is a component of primary or azurophilic granules; with paroxysmal nocturnal hemaglobulinuria lack funclactoferrin is an enzyme contained in secondary or spetional GPI and hence CD14. cific granules. Late myeloblasts and promyelocytes contain Molecules of CD 15 are saccharide antigens having a myeloperoxidase but not lactoferrin, whereas myelocytes, common terminal pentasaccharide, lacto-N-fucopentose metamyelocytes, and neutrophils produce myeloperoxiIII or the Lewis antigen. The CD 15 family also includes a siadase plus lactoferrin (Fig. 7.7).34 lylated (CD15s) form. Some bone marrow CD34+, CD117+ progenitor cells express CD15 or CD15s. CD15 expression increases with monocyte and granulocyte maturation. Similar to CD 13, CD 15 is present inbothprimary and secondary Megakaryocytic and erythroid lineages granules, and with granulocyte or monocyte stimulation increases in surface expression. Neutrophil CD 15 expres- Mature megakaryocytes express platelet-associated antision is reduced in patients or stem cell donors receiving gens CD9, CD36, CD41, CD42, CD61, and Factor VIII.35,36 G-CSF. Less is known about the surface antigen pattern of

Immunophenotyping

Megakaryoblast

MicromegaK3ryocyt6

Megakaryocyte ^ __^^^

Platelets •

- v

•.••;

CD41/61 CD36 CD42 Factor VIM

CD41/61,CD36 CD42, Factor VIII

RBC

CD34 CD117 CD33 HLA-DR CD41±

CD33± CD71 CD36 CD235a±

CD71 CD36 CD235 Hgb

CD71 CD36 CD235

Hgb

CD71 CD36 CD235

Hgb

CD36 CD235

Hgb

Fig. 7.8 Schematic of megakaryocytic and erythroid maturation. A common progenitor cell (CFU-EM) expresses CD33, CD34, CD117, HLA-DR, and weak (±) CD41. CFU-EM progenitors give rise to megakaryocytic and erythroid lineages. Megakaryocytic maturation is accompanied by increased CD41a expression and sequential appearance of CD36 and CD42b. Factor VIII antigen appears relatively late in megakaryocytic development. The earliest identifiable erythroid precursor, the proerythroblast, expresses CD36 and low amounts of CD235a (glycophorin A). Hemoglobin (Hgb) appears relatively late in erythroid maturation.

megakaryoblasts and their immediate parent cells. Studies of megakaryoblastic leukemia cells and normal marrow cell cultures support an orderly appearance and increasing density of platelet antigens with megakaryocytic maturation (Fig. 7.8) .36,37 Young megakaryoblasts have surface CD4, CD33, CD34, and CD117.38-40 The CD41a (glycoprotein IIb/IIIa complex) and CD61 (glycoprotein IIIa) molecules appear early in megakaryocytic development, followed by the expression of CD42b (glycoprotein lb) and CD36 (glycoprotein IV). Factor VIII-related proteins appear relatively late, being fully developed on recognizable megakaryocytes. Relatively few cell surface antigens are unique to early erythroid precursor cells. Immunophenotypic descriptions of erythropoiesis are derived from studies of cultured normal erythroid precursors and leukemic cell lines and, thus, may not accurately reflect in vivo states.41"44 CD34 is expressed only by the earliest erythroid progenitor cells. The transferrin receptor (CD71) is present on all stages of erythroblastic development and on reticulocytes, but is lost with formation of the mature red blood cells (RBCs) (Fig. 7.8). Proerythroblasts, and basophilic, polychromatic, and orthochromatic normoblasts express

CD36. Glycophorin A (CD235a) appears during the late proerythroblast stage and shows its greatest intensity on the mature RBC. Hemoglobin is a specific marker for erythroid lineage but is detectable only after the basophilic normoblast stage.

Methods used in immunophenotyping Immunohistochemistry

Immunohisto chemical techniques use combined immunologic and chemical reactions to reveal surface and intracellular antigens. A primary antibody to the antigen in question is recognized by a secondary antibody conjugated to horseradish peroxidase or alkaline phosphatase (Fig. 7.9). Alternatively, advantage is taken of the strong binding of avidin substrates to biotin. Secondary antibodies are biotinylated and subsequently detected by labeling with streptavidin conjugated to peroxidase of phosphatase. The enzymes react with diaminobenzidine/H2O2 or an alkaline phosphatase substrate to produce a color reaction. The major advantages of immunohisto chemical

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Goat anti-mouse conjugated with HRP

Goat anti-mouse conjugated with biotin

Avidin

nated this problem. The clinical laboratory now has a wide selection of commercially available antibodies to facilitate the diagnosis of hematopoietic and lymphoid neoplasms. Antigens easily detected by immunohistochemistry and useful in the differential diagnosis of pediatric hematologic and lymphoid malignancies are noted in Table 7.1.

Biotin HRP

Flow cytometry

Fig. 7.9 Diagrams of two different immunohistochemical staining procedures. (A) Indirect immunohistochemical method. A primary antibody is used to detect a cellular antigen (Ag). The primary antibody is identified by a secondary antibody conjugated to horseradish peroxidase (HRP). In the presence of a benzidine substrate and H2O2, HRP catalyzes the production of a colored reaction product that can be observed by routine light microscopy. (B) Avidin-biotin-HRP complex immunohistochemical method. The secondary antibody is conjugated with biotin. Biotin molecules have high affinity for avidin that can be bonded to HRP or alkaline phosphatase. As compared with the indirect immunohistochemical method, the avidin-biotin-HRP complexes provide more HRP molecules per detected antigen and hence a more sensitive immunohistochemical technique.

assays are their excellent sensitivity, retention of cell morphology by light microscopy, minimal sample requirement, and applicability to blood and marrow smears and paraffin-embedded tissue including bone marrow biopsies. Immunohistochemical assays are not commonly used for the initial diagnosis and classification of most leukemias, but are particularly valuable when "dry" bone marrow aspirates and peripheral blood specimens yield insufficient numbers of leukemic cells for flow cytometric analysis. Immunohistochemistry studies of processed bone marrow biopsies enable reliable detection of B- and T-precursor ALLs and AMLs.45"55 This technique can aid in the diagnosis of megakaryoblastic leukemia (M7 AML) from marrow core biopsies when cells cannot be obtain by a needle aspirate.56 Also, these assays are sometimes the only way to establish a diagnosis of granulocytic or monocytic sarcoma in afixedtissue biopsy.57"59 In the past, the deleterious effect of formalin and mercuric tissuefixativeson cellular antigens was the major disadvantage of immunohistochemistry. However, the development of new antibodies and of improved immunologic methods for detecting antigens masked by these tissuefixativeshas largely elimi-

The lineage and maturational stage of neoplastic cells can be examined by immunofluorescent microscopy or flow cytometry with antibodies conjugated to fluorochromes. Immunofluorescent microscopic methods are generally successful, but multiparameter flow cytometry offers the advantages of quantitative measurements of antigen expression and rapid analysis of a large number of cells. A past disadvantage offlowcytometry, especially with samples containing small numbers of malignant cells, was the difficulty of discriminating between neoplastic and normal hematopoietic cells. Current analysis based on CD45 antigen intensity expression and light side scatter largely overcome this limitation.60"63 This analysis takes advantage of the different intensities of CD45 and light side scatter properties of the major hematopoietic lineages. For example, normal lymphocytes strongly express CD45 but produce little light side scatter. By contrast, late myeloid cells express CD45 more weakly than lymphocytes but emit strong light side scatter signals. The simultaneous display of CD45 and light side scatter on a two-parameter histogram permits discrimination between the different cellular components of normal bone marrow (Fig. 7.10). Lymphoid and myeloid leukemias show characteristic histogram patterns of CD45 and light side scatter expressions that largely resemble their normalhematopoietic or lymphoid counterparts and thus, facilitate their identification (Figs. 7.11 and 7.12).

Immunophenotyping panel

With few exceptions, most leukocyte antigens fail to retain their lineage specificity in malignant processes. However, the lineage of over 98% of acute leukemias is discernible with appropriately designed panels of monoclonal antibodies directed toward relatively lineage-restricted antigens.64,65 The antibody screening panel for acute leukemias used at St. Jude Children's Research Hospital (SJCRH) was designed to include at least one very sensitive and one relatively specific marker for each major hematopoietic and lymphoid lineage (Fig. 7.13). Leukemic processes can be analyzed with this panel by use of flow cytometry, immunofluoromicroscopy, or immunohistochemistry. Flow cytometry and immunofluoromicroscopy

Immunophenotyping

B L" IL-

RBC

Light side scatter (SS) intensity

•

Fig. 7.10 Flow cytometric dot plot histograms of CD45 intensity versus light side scatter (SS) for two normal pediatric bone marrow aspirates. Lymphocytic, monocytic, myeloid precursor, and blast populations have distinct intensities of CD45 and light SS, facilitating their identification in a two-dimensional dot plot histogram. In both histograms, discrete populations of bone marrow cells are identified: mature lymphocytes (L), immature B lymphocytes (IL), monocytes (Mo), myeloid precursors (My), and erythroid elements (RBC). (A) Infant marrow containing a large number of immature B lymphocytes. (B) Adolescent marrow with fewer immature B lymphocytes and more myeloid cells.

?'..*-'B

Light side scatter (SS) intensity

Fig. 7.11 Characteristic dot plot histograms of acute leukemias graphed as light side scatter (SS) versus CD45 expression based on Beckman-Coulter flow cytometric analysis. Non-neoplastic lymphocytic, monocytic, and myeloid cell populations are indicated by L, Mo, and My, respectively. The leukemic blast populations are labeled B. The CD45 and light SS signal intensities for leukemic processes are comparable with their normal marrow hematopoietic counterparts (compare with Fig. 7.10). (A) Early pre-B ALL. (B) Precursor-T ALL. (C) Acute myeloblastic leukemia, M1 AML. (D) Acute promyelocytic leukemia, M3 AML. (E) Acute monoblastic leukemia, M5 AML. (F) Acute megakaryoblastic leukemia, M7 AML. All studies were performed on bone marrow samples enriched for mononuclear cells and leukemic blasts by a density-gradient separation technique.

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10 2

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CD45

Fig. 7.12 Characteristic dot plot histograms of acute leukemias graphed as CD45 versus light side scatter (SS) based on Becton-Dickinson flow cytometric analysis. The axes for CD45 and SS are reversed compared with Fig. 7.11. Differences in instrumentation result in slightly different histogram representations of normal and leukemic cell populations. Lymphocytic, myelocytic, monocytic, and erythroid populations are indicated by Ly, My, Mo, and RBC, respectively. The leukemic blast populations are identified by the large arrows and normal marrow cells by thin-line arrows. (A) Early pre-B ALL. Leukemic blasts show a spectrum of negative-to-weak CD45 expression and low intensity light SS. (B) Mature B-cell ALL (Burkitt leukemia). Leukemic cells strongly express CD45 and their light SS properties extend into the monocyte region. (C) Precursor-TALL. In general, blasts of precursor-TALL strongly express CD45 while having relatively low intensity light SS. (D) Acute myeloblastic leukemia (M1 AML). (E) Acute myelomonocytic leukemia (M4 AML). Large, open arrow points to the myeloblast component. Large, shaded arrow identifies the monocytic component. (F) Acute monoblastic leukemia (M5 AML). All studies were performed on bone marrow samples that incorporated a red blood cell lysis step.

Acute leukemia

CD45 CD19 CD22 CD79a* CD7 CD3* MPO* CD13 CD33 CD117 CD61 CD235

± + + + ± ± -

B-lineage

CD45 CD19 CD22 CD79a* CD7 CD3* MPO* CD13 CD33 CD117 CD61 CD235

+ ± + + ± ± -

T-lineage

CD45 CD19 CD22 CD79a* CD7 CD3* MPO* CD13 CD33 CD117 CD61 CD235

+ ± ± ± ± ± ± ± -

+

CD45+ CD19 CD22 CD79a* CD7 CD3* MPO* CD13 CD33 CD117 CD61 CD235

+ _ _ ± _ _ ± ± ± + _

Myeloid/ Megakaryocytic monocytic

CD45 CD19 CD22 CD79a* CD7 CD3* MPO* CD13 CD33 CD117 CD61 CD235

± ± ± ± ± -

Erythroid

Fig. 7.13 Example of a basic acute leukemia immunophenotype screening panel. Immunologic studies are necessary to confirm or establish the lineage of myeloperoxidase (MPO)-negative leukemias. Additional antigenic studies are required to distinguish among subtypes of lymphoid and myeloid leukemias. Antigens are identified by their CD (clusters of differentiation) groups. CD235 represents glycophorin A. Asterisks indicate cytoplasmic antigen expression.

require a prior cell permeabilization step to expose myeloperoxidase (MPO) and for optimal detection of CD3 and CD79a.66-68 The SJCRH panel identifies the lineage of over 98% of childhood acute leukemias with samples rich in neoplastic cells. Examples of frequently encountered immunophenotype profiles of ALL and AML are presented in Table 7.2. If specimens have a small leukemic component, it may be necessary to include antibodies to several other antigen groups to differentiate between normal and neoplastic cells. The subclassification of Band T-precursor ALL and AML requires the study of additional leukocyte antigens. Although CD34 and TDT are not lineage-restricted antigens, screening for their presence is useful in distinguishing blastic from mature cell malignancies. Important aspects of TDT, CD34, and the antigens includedinthe SJCRH immunophenotype screening panel are discussed below.

CD45

The common leukocyte antigen (CD45) is a tyrosine phosphatase expressed by all leukocytes and their progenitors.

Immunophenotyping

Table 7.2 Examples of immunophenotype profiles of acute leukemias and their lineage assignments using the SJCRH screening panel

Immunophenotype profile Example

CD19

CD22

CD79aa

CD7

CD3a

CD13

1 2 3 4 5 6 7 8 9 10 11 12 13

CD33

CD117

MPOa

CD61

+ +

14 15 16 17 18

CD235a

Lineage interpretation B B (My+ALL) B (My+ALL) T T T (My+ALL) Myeloid Myeloid Myeloid (Ly+AML) Myeloid (Ly+AML) Myeloid (Ly+AML) Megakaryocytic Megakaryocytic (Ly+AML) Myeloid/erythroidb Erythroidc Biphenotypic d Biphenotypic d Biphenotypic d

Abbreviations and symbols: My+ALL, myeloid antigen-positive acute lymphoblastic leukemia; Ly+AML, lymphoid antigen-positive acute myeloid leukemia (see text and Table 7.12 for descriptions of these leukemia profiles); +, positive; - , negative; + / - , positive or negative. a Cytoplasmic antigen expression. b Characteristic of acute erythroleukemia, M6 AML. c Characteristic of erythroblastic leukemia with no significant myeloblast component. d Mixed lymphoid-myeloid lineage (see Table 7.12 for definition of biphenotypic leukemia).

It functions in antigen receptor signaling by dephosphorylation of Src kinase.69 CD45 also acts as a Janus kinase (JAK) tyrosine phospatase to regulate cytokine receptor signaling in differentiation and proliferation of hematopoietic cells. CD45 appears very early in B-cell development and persists throughout maturation up to but not through the plasma cell stage.1 Similarly, T and myeloid cells at all stages of maturation express CD45. The cell surface intensity of CD45 increases with B- and T-cell maturation but stays relatively constant with myeloid maturation. Very early erythroblasts express CD45 but at levels that are not easily discernible from cellular autofluoresence by routine flow cytometry. Megakaryocytes and platelets do not display CD45. All acute leukemias, except for approximately 10% of B-precursor ALLs and some M7 AMLs have easily detectable levels of CD45.70 Low-level CD45 expression in ALL correlates with favorable clinical and laboratory features, including lower leukocyte counts, B-cell lineage, and hyperdiploidy (>51 chromosomes).70,71 CD45 is very important in differentiating leukemia and lymphoma from small cell tumors. Small cell tumors (e.g. neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma) can involve the

bone marrow and morphologically mimic leukemia but do not synthesize CD45. CD19, CD22, and CD79a

The synthesis of immunoglobulins is the hallmark of Blineage commitment. However, Ig molecules are insensitive markers for diagnosing precursor-B ALL in that only about 25% of such cases will express these proteins. By contrast, CD 19, CD22, and cytoplasmic CD79a are expressed in almost every case of B-lineage ALL. The sensitivity of these latter markers is compromised by their atypical expression in other types of acute leukemia. Cases of AML and T-ALL can also weakly express CD 19, CD22, or CD79a, but coexpression of CD 19 with CD22 or CD79a is indicative of a B-lineage commitment with the exception of rare examples of biphenotypic leukemia (Table 7.2). Thus, it is recommended that immunophenotyping studies of acute leukemia include CD 19 plus CD22 or CD79a. CD19 alone is expressed by the earliest of precursor B cells and remains detectable through B-cell maturation up to the plasma cell (Figs. 7.1 and 7.3).1 Follicular dendritic cells in lymphoid tissues also express CD 19, which is not

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Fred G. Behm

detectable in epithelial and soft tissue tumors. This antigen is a signal transduction molecule that participates in Blymphocyte development, activation, and differentiation. The intensity of CD19 expressionis useful in distinguishing between precursor-B ALL and other leukemias that aberrantly display this antigen. The aberrant expression of CD 19 by some cases of AML and T-ALL is very weak in comparison to strong expression in precursor-B ALL. CD22 is a B cell restricted glycosylated protein that acts as an adhesion receptor and signaling molecule.1 It is expressed at all stages of B cell differentiation, initially in the cytoplasm of early precursor B cells and on the surface of more mature B cells (Fig. 7.2). Similar to their paucity of CD19, plasma cells do not express CD22, while basophils express CD22 but not CD19. With the appropriate monoclonal antibodies, over 98% of precursor-B ALLs show relatively strong surface expression of CD22. Although normal myeloid- and T-cells and the blasts of T-ALL do not express this antigen, certain anti-CD22 antibodies can react with a non-CD22 cytoplasmic protein in some AMLs.72 A small number of myeloperoxidase-positiveAMLs weakly express surface CD22. CD79a forms a heterodimer with CD79b that is noncovalently bound to immunoglobulin to form the BCR (Fig. 7.2). 1 The heterodimer transmits signals into the cytoplasm upon antigen binding by cell surface immunoglobulin. CD79a appears prior to CD 19 in B-cell ontogeny.73 Current commercially available monoclonal antibodies only detect the cytoplasmic domain of CD79a. With cell permeabilization techniques and flow cytometry over 98% of precursor B ALLs will have detectable CD79a. The leukemic blasts of some cases of precursor-B ALL with the t(4;11) translocation involving the MLL gene can express little or no CD79a. Initial studies found CD79a only in normal and neoplastic B cells,74,75 but more recent investigations show that CD79a is also weakly expressed by a minority of T-ALLs and AMLs.51 , 76 - 80 The CD79b antigen is restricted to B cells, but is an insensitive marker for most cases of precursor-B ALL.75 CD7 and CD3

CD7 is a glycoprotein found on the surface of pluripotent hematopoietic stem cells, thymic and mature T cells, and NK cells.1 CD7 appears to be a coactivator molecule involved in cytokine secretion and cellular adhesion.1 It is a very sensitive marker of precursorT-ALL but lacks lineage specificity. Virtually every case of precursor-T ALL expresses CD7; thus, its absence on leukemic blasts mitigates against that diagnosis. Expression by precursor-T ALL blasts is strong as compared to normal mature T cells. Unfortunately for lineage assignment purposes, CD7 is also weakly expressed by approx-

imately 50% of acute megakaryoblastic leukemias, many acute myeloid leukemias, and a small percentage of 81 84 B-lineage ALLs (Table 7.2). ~ The expression of CD7 by precursor-B ALLappears to lack clinical significance, butits expression by AMLs maybe associated with a lower overall survival rate. 82 " 84 CD3 consists of six polypeptides divided into three dimers (7 /ε, 8/e, and t, It,) that associate with either the TCR 1 22 25 28 ct(J or 78 proteins to form the CD3-TCR complex. , , , CD3 is also associated with a pre-TCR molecule on thymic T cells. Although the ε chain of CD3 has been described in fetal cells and pro thymic T cells with the potential of differentiating along myeloid or natural killer (NK) lineages, the complete CD3 complex is expressed only by T cells in nor85 mal postnatal tissues. The youngest thymic cell committed to T-lineage development contains cytoplasmic but not 26 85 86 surface CD3. , , Because many precursor-T ALL cases express only cytoplasmic CD3, test methodologies must include a procedure that permeabilizes the leukemic cell membrane to expose CD3. With these methods, every case of T-ALL has detectable cytoplasmic CD3, providing an excellent T-lineage-restricted antigen for immunophenotyping studies.86"88 Although many monoclonal CD3 antibodies only detect the ε chain when combined with the 7 or 8 chain of the CD3 complex, some polyclonal antibodies to CD3ε detect CD3ε proteins independently.89 This raises the possibility that rare cases of leukemia reacting with the latter polyclonal antibodies may correspond to a pro-T or early NK-cell stage of development rather than a committed T-lineage process. CD33 and CD13

CD33 is a transmembrane glycoprotein that is expressed by myeloid progenitors (GFU-GEMM, CFU-GM, CFU-G, BFUE), granulocyte precursors, mature granulocytes, monocytes, and macrophages but not normal lymphocytes.1,90 CD33 has no known expression outside of hematopoiesis, and its biologic function is not understood. CD 13 is similar to CD33 in its expression by granulocytic and monocytic precursors and their progeny.1,91 This metallopeptidase is identical to aminopeptidase N, which degrades regulatory pep tides produced by a wide variety of cell types. CD 13 is also expressed on a subpopulation of large granular lymphocytes and cells of vascular endothelium, renal proximal tubules, intestinal brush border, bone marrow stroma, and osteoclasts.1 Small numbers of normal precursor-B cells in the marrow (< 5 x 1CT3) and reactivated mature B cells express CD13.92,93 CD13 expression can be induced by in vitro stimulation with B cell growth factor.94 Antibodies to many myeloid- andmonocytic-associated antigens, including CD13, CD14, CD15, CD33, CD64, and CD65, are available for identifying the blast cells of AML.

Immunophenotyping

However, only CD13 and CD33 have proved to be sensitive markers of AML. Over 95% of AMLs express CD13 and/or CD33. In general, expression of CD33 is more intense and CD 13 less intense in monocytic than in myelogenous acute leukemias. Unfortunately, CD 13 andCD33 are notvery specific, in that 15% to 35% of ALLs also weakly express one or both of these antigens, although the weak specificity of CD13 and CD33 is offset by their sensitivity for AML when interpretedin the context of the expression (or lack thereof) of other antigens listed in the SJCRH screening panel (Table 7.2) .95 Myeloperoxidase The cytoplasmic granules of myeloid, monocytic, and eosinophilic cells contain peroxidases that defend against invading microorganisms. One of these, myeloperoxidase (MPO), a specific marker for myelocytic and monocytic lineages, is indispensable in the diagnosis and classification of acute leukemias.2,96 The MPO gene isfirsttranscribed in CD34-positive progenitor cells that give rise to myeloblasts (Fig. 7.7) ,97~101 while myeloperoxidase is packaged together with other leukocyte enzymes in primary granules. The enzymatic form of MPO is detected by cytochemical reactions with benzidine compounds. Newer approaches use immunofluorescence or immunohistochemical assays with monoclonal antibodies to detect MPO protein.102,103 The cytochemical test for peroxidase requires the presence of functional enzyme, whereas the antibody test needs only the intact protein. The enzymatic activity of MPO decays rapidly with time, but old cytologic preparations retain sufficient antigenic sites for antibody binding. The proenzyme form of MPO that is cleaved to produce the dimeric enzymatic form of MPO is also detected by anti-MPO staining, providing a test for very immature myeloid cells. The anti-MPO test may be more sensitive than the cytochemical assay for MPO. Indeed, cases of acute leukemia cytochemically negative for MPO may test positive with anti-MPO This may be due to reactions with nonfuncassays. tional, degraded, or proenzymatic forms of MPO. Whether the anti-MPO method is more sensitive than the enzymatic reaction is controversial.109 Several investigations detected a surprisingly, if not suspiciously, high percentage of ALLs expressing MPO by immunologic methods.110,111 Significant differences between enzymatic and antigenic MPO expression are not observed at SJCRH, where these tests are performed within hours of each other on aliquots of the same leukemic specimen. CD117

The CD 117 antigen is the product of the proto-oncogene cKITand belongs to a family of growth factor receptors with tyrosine kinase activity.1,112,113 This receptor is found on

over half of all CD34+ cells and megakaryocytic, erythroid, granulocytic, and monocytic lineage-restricted progenitor cells in normal bone marrow. Bone marrow mast cells, a subset of NK cells, and some early prothymic T cells also have detectable CD 117. With its ligand (mast cell factor, stem cell factor, or Steel factor), CD 117 is thought to play a crucial role in early hematopoiesis. Only a rare case of T-precursor ALL and almost no cases of precursor-B ALL express CD117.114"118 Blasts of up to 90% of cases of AML express CD117, and it appears to be more highly associated with AML than either CD13 or CD33.119 All subtypes of AML can express CD117.103'117"121 The close association with AML makes CD 117 a valuable addition to a marker panel designed for lineage determination.65,119 In childhood and adult AML, CD117 is strongly associated with the expression of CD34 and CD7, but not with other clinical features or with prognosis. 108,117,119,122

TDT This nuclear DNA polymerase participates in the addition of nucleotides to the N regions of Ig and TCR genes undergoing rearrangement.123 Thus, TDT is normally detected in the nuclei of immature B and T lymphocytes and disappears with lymphocyte maturation (Figs. 7.1 and 7.4).124,125 In healthy persons, cells bearing TDT are present in the bone marrow and thymus, but in very small numbers in the peripheral blood and lymphoid tissues.126,127 TDT is readily detected with immunofluorescence and immunohistochemical techniques using cell smears, touch preparations, cytospin smears, or frozen tissue sections. All of these tissue preparations rapidly lose TDT if left at room temperature for more than 24 hours. TDT is masked from anti-TDT antibodies in marrow and other tissue biopsies fixed in formalin, but immunologic reactivity can be restored by microwaving or by other antigen retrieval techniques.49 Flow cytometry with methods that partially permeabilize cells to expose nuclear proteins to antibodies to TDT is in common use. The blasts of more than 90% of B- and T-lineage ALLs and lymphoblastic lymphomas harbor easily detectable TDT.128,129 Sensitive flow cytometric and immunohistochemical assays will reveal TDT in 10% to 45% of AMLs.130,131 Although TDT assays are not useful for determining lineage, they are helpful in several diagnostic situations. For example, assays are positive in lymphoblastic but not myeloblastic crisis of chronic myelogenous leukemias.2 The presence of TDT-positive blasts in spinal, pleural, or peritoneal fluid, testicular biopsy specimens, or other nonlymphoid tissues is indicative of a lymphoblastic malignancy. TDT is also helpful in distinguishing lymphoblastic lymphoma from Burkitt and other lymphomas.2 Dual immunofluorescence techniques use anti-TDT plus anti-CD3 to detect minimal residual

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Table 7.3 Immunologic classification of B-lineage ALL

Immunologic marker (% cases positive for marker) Subtype

CD19

CD20

CD22

CD79aa

CD10

cylg|x

slg|x

slgKOrA.

Frequency

Early pre-B (pro-B) Pre-B Transitional pre-B (late pre-B) Mature B

100 100 100 100

35 45 55 99

99 100 100 100

99 100 100 99

95 100 100 50

0 100 100 100

0 1 100 ~95 b

0 0 0 ~95 b

60-65% 20-25% 10-12% 3-5%

Abbreviations: cylg|x, cytoplasmic immunoglobulin mu heavy chain; slg|x, surface immunoglobulin mu heavy chain; slgK or \, surface immunoglobulin kappa or lambda light chain. a Cytoplasmic expression. b From 2% to 5% of cases with t(8;14), t(2;8), or t(8;22) may lack surface immunoglobulin or express only cytoplasmic immunoglobulin. disease in the b o n e m a r r o w of patients with T-lineage ALL. 132

CD34

The transmembrane sialoglycoprotein CD34 is expressed by early hematopoietic progenitors of all lineages, endothelial cells of high endothelial venules, bone marrow stromal cells, peripheral nerve sheath cells, and osteoclasts.133"137 This glycoprotein may play a role in progenitor cell localization and stromal adhesion in the b one marrow. Less than 10% of cells inpostnatalbone marrows express CD34. Primitive multipotent hematopoietic stem cells express CD34 but not CD38 or other leukocyte-associated antigens. However, the majority of normal CD34-positive marrow cells correspond to later stages of stem cell commitment and coexpress CD38, HLA-DR, CD33, and/or CD19.138 Smaller populations of CD34 cells express CD4, CD10, CD7, and/or CD41. In normal bone marrows most CD34+ cells appear committed to the myeloid lineage (CD13+, CD33+) or to lymphoid development (CD19+'", CD10+). CD34 expression progressively decreases as hematopoietic progenitors differentiate. The majority of acute leukemias are CD34-positive, whereas the chronic leukemias are negative.2 About 70% of B-lineage and30% of T-lineage ALLs are CD34-positive. In adult AML, CD34 expression correlates with a M1 or M2 morphology by French-American-British (FAB) criteria, as well as leukemias evolving from myelodysplastic syndrome, karyotypic abnormalities of chromosome 5 or 7, and a lower remission induction rate.139"143 In pediatric AML, CD34 expression also correlates withM1 andM2morphologies but not with chromosome 5 and 7 abnormalities, and appears to have no prognostic significance.144,145 In childhood precursor-B ALL, CD34 expression by leukemic blasts is associated with an age of 1 to 10 years, hyperdiploidy (>50 chromosomes), the absence of central ner-

vous system (CNS) leukemia, a n d a favorable response to therapy. 1 4 6 , 1 4 7

Classification of specific leukemias and lymphomas B-lineage ALL

Several classifications of B-lineage ALL have been proposed. The SJCRH classification recognizes four subtypes discernible by their pattern of immunoglobulin expression (Table 7.3).148 Other classifications include additional subgroups based on schemes of immunoglobulin, CD10, CD179a, or CD179b expression.149"152 Although initial studies demonstrated an association of treatment response with different subtypes of precursorB ALL, improved treatment approaches and recognition of the overriding significance of genotypic abnormalities has marginalized the importance of immunologic subgrouping in these leukemias. The WHO classification divides acute B-lymphoid neoplasms into precursor-B lymphoblastic leukemia/lymphoma and Burkitt leukemia/lymphoma with further subgrouping based on cytogenetic abnormalities.2 Early pre-B ALL

The leukemic blasts of early pre-B ALL resemble a normal marrow B-precursor cell that lacks immunoglobulins (Fig. 7.1). Although Ig heavy-chain genes are usually rearranged in these leukemias, immunoglobulins are not detectable. The leukemic cells of all early pre-B ALL cases expressCD19andHLA-DR(Table7.3).Allbutrarecases display surface CD22 and/or cytoplasmic CD22, and almost all have cytoplasmic CD79a.63,69,81-83 CD10 and TDT are detectable in over 90% of cases, andmore than 75% express CD34.147,148 The CD20 antigen that normally appears with

Immunophenotyping

Table 7.4 Antigen expression profiles typical of B-lineage ALL with various cytogenetic features

Karyotype t(4:11)(q21;q23) t(11;19)(q23;q13.3) t(12;21)(p12;q22) t(1;19)(q23;p13) Hyperdiploidat(1;19) t(9;22)(q34;q11) Hyperdiploida t(8;14)(q24;q32) Normal

Genes involved

ALL subtype

AF4-MLL MLL-ENL TEL-AML1 PBX1-E2A

EPB EPB PB, EPB PB, TBP EPB PB, EPB PB, EPB Mature B EPB, PB

ABL-BCR MYC-IGH

Leukocyte antigen expression profile CD45

CD34

CD22

CD10

CD13

CD15

CD24

/-

-10 + -to +

CD33

CD66c

NG2

-to +

Abbreviations and symbols: NG2, nonhematopoietic chondroitin proteoglycan sulfate; EPB, early pre-B; PB, pre-B; TPB, transitional preB;+, weakly positive; ++, moderately positive, +++, strongly positive; -, negative; - / + , negative more often than positive; + / - , positive more often than negative. a More than 51 chromosomes. the production of (j, heavy chains is present in varying proportions of blasts in many cases. Up to 10% of early pre-B ALLs do not have detectable CD45.70 Leukemias harboring rearrangements of the MLL gene, resulting from the t(4;11), t(11;19), and t(9;11) chromosomal translocations, are usually classified as early pre-B ALL, although examples have been described with a preB and precursor-T immunophenotype. 153,154 The blasts of t(4;11)+ B-lineage ALL usually present with a characteristic antigenic profile: CD19+, CD22+, CD24 lo/ -, and CD10- (Table 7.4).154,155 This differs from other B-lineage cases, which almost always show strong expression of CD10 and CD24. Further, most t(4;11)+ cases express myeloid-associated CD 15 or CD65 antigens. 154 , 155 ThepanT-cell CD7 antigen and myeloid-associated antigens CD 13 and CD33 can also be present. Precursor-B ALLs with a t(9;11) or t(11;19) often have immunophenotypes similar to those associated with the t(4;11) (Table 7.4).156 Cellsurface chondroitin protoglycan sulfate, a nonhematopoietic cellular molecule detected by the monoclonal antibody 7.1, is present in almost all precursor-B ALLs and over one-half ofAMLs having a rearrangedMLL gene. 157~161 T-cell-associated CD2 is rarely expressed by early preB ALL and may be associated with poorer treatment outcome. 162 " 164 Pre-B ALL

About 25% of newly diagnosed ALLs have a preB immunophenotype. 148 Like early pre-B ALL, this subtype expresses CD 19, CD22, CD79a, and HLA-DR (Table 7.3). By definition, the lymphoblasts of pre-B ALL exhibit cytoplasmic Ig (cIg) (j, chains without detectable

surface (sIg) (j, chains. 148,165 Rearrangement of Ig lightchain genes is evident in some of these leukemias, but K and X proteins are not detectable. Over 95% of these leukemias express CD10 and TDT, but only two-thirds express CD34.147,148,166 In contrast to the normalbone marrow pre-B lymphocyte, blasts of pre-B ALL may lack or only weakly express surface CD20, which is thought to function in B-cell activation and proliferation by regulating transmembrane Ca2+ conductance and cell cycle progression. 1 Studies of precursor-B ALL, including early pre-B and preB ALL, suggest that expression of CD20 maybe associated with a poorer treatment response. 167 In comparison to earlypre-B ALL, pre-B ALL is more often associated with higher leukocyte counts, elevated serum lactic acid levels, fewer than 51 chromosomes or a DNA indexless than 1.16, and recurrent chromosome translocations. Between 20% and 25% of pre-B ALLs harbor either the t(1;19)(q23;p13) or der(19)t(1;19)(q23;p13).148,168 Patients with these translocations require more intensive therapy to obtain a satisfactory treatment response. The antigen expression profile of CD19+, CD22+, CD20 ±, CD34~, CD45hi, clgjju+ is characteristic of ALLs with the t(1;19) but lacks specificity (Table 7.4).167,169 A few well-studied cases of early pre-B ALL also carry a t(1;19).167,169,170 However, in those cases the blasts are CD34+, hyperdiploid (chromosome number >51), and lack evidence of the chimeric E2A-PBX1, which is transcript that is uniformly detected in t(1;19)+ pre-BALL.170 Polyclonal and monoclonal antibodies are now available for the flow cytometric detection of the PBX1 portion of the chimeric E2A-PBX1 protein in the nucleus of t(1;19)+leukemic blasts.171 Immunologic testing for E2A-PBX1 provides rapid screening for leukemias with

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Table 7.5 Features of B-lineage ALL expressing surface immunoglobulin

Transitional pre-B Mature B (naive B-cell type) Mature B (GC B-cell type)

Extramedullary mass

FAB subtype

No No Yesb

L1orL2 L1orL2 L3 (L2)

Immunologic marker expression TDT CD34

CD20

CD10

cylg|x

slg|x

slgK or \

Karyotype Variablea Variablea t(8;14)(q24;q32), t(2;8)(p12;q24), or t(8;22)(q24;q11)

Abbreviations and symbols: GC, germinal center; cylg|x, cytoplasmic immunoglobulin mu heavy chain; slg|x, surface immunoglobulin mu heavy chain; slgK or \, surface immunoglobulin kappa or lambda light chain; +, positive; —.negative; +/—, positive or negative; +(—), rarely negative. a t(1;19)(p13;q32) frequent, no t(8;14)(q24;q32), t(2;8)(p12;q24), or t(8;22)(q24;q11), and no MYCrearrangement. b Majority of patients present with lymphomatous masses of the ileo-cecum, gonads, or head and neck. c Less than 5% of cases express IgA or IgG without IgM; <5% lack sIg or sIg plus clg|x.

this translocation and thus maybe of use in studies of minimal residual disease. ALL with the t(12;21)(p13;q22) may also have a CD19+, CD10+, cylgn + , slgjo," phenotype. 172,173 Additionally, the leukemic blasts of most t(12;21)+ ALLs atypical express CD13 or CD33 (Table 7.4). 173~176 Blasts with TEL rearrangements are reportedly more likely to be CD9- / k \ CD2(T, CD13+, CD45+, but this finding has not been confirmed by others. 175 Transitional pre-B ALL Leukemic blasts that express both clgju, and sign heavy chains without K or X light chains have been designated transitional pre-B ALL as they resemble cells in transition from pre-B to mature-B cell stages of maturation (Fig. 7.1 and Table 7.3) .177 As is the case with normal late pre-B bone marrow cells, the sign o n the leukemic blasts is linked to pseudo-lambda light chains, CD179aandCD179b, and the CD79a/CD79b heterodimer (Fig. 7.2).8,10,14 Similar to preB ALL, the blasts express CD 10, usually TDT, and sometimes CD34.177 An initial study found this phenotype in only 1% of childhood ALL cases, 177 but it was performed by immunofluorescence microscopy, which is relatively insensitive for detecting low-density surface pre-BCR complexes. More current studies at SJCRH with sensitive flow cytometric analyses indicate that this immunophenotype is more common than previously reported. Patients with transitional pre-B ALL have leukemic blasts with FAB L1 or L2 morphology and lack the t(8;14), t(8;22), or t(2;8) translocation associated with FAB L3 ALL.177 These children do not usually present with bulky extramedullary masses or CNS leukemia. The leukocyte count and serum levels of lactate dehydrogenase (LDH) are usually low. Although some transitional pre-B ALLs have an associated t( 1; 19), most cases lack characteristic chromosomal abnormality. Patients with this ALL subtype appear to have an

excellent response to the chemotherapy used for early preB and pre-B ALL. Mature B-ALL In 2% to 4% of childhood ALLs, the blasts express sign plus either k or X light chains and are classified as BALL. There are two phenotypically and genotypically distinct types of B-ALL (Tables 7.3 and 7.5). The more common type features L3 blasts and in the WHO classification is classified as Burkitt leukemia. 2 Similar to early pre-B and pre-B ALLs, blasts in L3 B-ALL express CD19, CD22, and frequently CD10, but not TDT and CD34. In contrast to other B-lineage ALLs, L3 B-ALL consistently and strongly expresses CD20 and commonly CD23. The immunophenotypic pattern of antigen and immunoglobulin expression suggests that the normal cellular counterpart of the L3 blasts is a follicular germinal center cell (centroblasts), whereas precursor B-ALL blasts resemble immature marrow B cells (Fig. 7.3). Hypermutation of the Ig gene locus in Burkitt leukemia/lymphoma is further evidence of a germinal center origin. 178 Often these cases represent the leukemic phase of Burkitt lymphoma arising in the abdomen or head and neck. Burkitt lymphoma and leukemia (L3 ALL) have identical immunophenotype profiles. The blasts of this B-ALL subtype have a reciprocal translocation of c-MYC on chromosome 8 with one of the Ig genes on chromosome 2, 14, or 22 [i.e.,t(8;14)(q24;q32),t(2;8)(p12;q24),ort(8;22)(q24;q11)].2 Very infrequently, blasts with L3 morphology or the t(8;14) express TDT, fail to produce sIg or cIg, express cytoplasmic IgK and/or IgX light chains but no surface Ig. 179-191 An infrequent subtype of B-ALL leukemia is characterized by blasts with L1 or L2 morphology (Table 7.5).192~196 Unlike blasts of L3 B-ALL, these leukemias may express TDT or CD34, and they only weakly express CD20.182,183,192-196

Immunophenotyping

The intensity of their sIg expression is very weak compared with that of the L3 subtype of B-ALL, and their immunophenotype resembles a late stage of normal marrow B-cell maturation, or naive B cell. These leukemias lack extramedullary masses at presentation. The t(8;14), t(2;8), and t(8;22) translocations are absent, as are rearrangements of the MYCgene.192-196 It is important to differentiate between these two types of B-ALL, since the treatment for L3 B-ALL with a t(8;14), t(8;22), or t(2;8) is intense, short, and highly successful. Treatment outcomes for pediatric mature B-ALL patients without one of these three translocations are similar to those for early pre-B and pre-B ALL.192

Table 7.6 Immunologic classifications of T-lineage ALL.

T-lineageALL The blasts of all T-ALLs express surface CD7 and surface and/or cytoplasmic CD3 (cyCD3).86-88,148,197 Leukemic blasts of over 90% of cases express CD2, CD5, and TDT. Individually, surface CD1a, CD3, CD4, or CD8 antigens are detected in less than 45% of cases. 197 The HLA-DR antigen, common to all precursor-B ALL and most AMLs, is only occasionally expressed. CD45 intensity is usually stronger than encountered with early pre-B and pre-B ALL.70 Another 40% to 45% of cases display CD 10 and/or CD21.166,197,198 C y t o t o x i c T - a n d NK-cell-associated CD56 antigen is detected in a small subset of cases. 199

Common T

Several classifications of T-ALL have been proposed based on the pattern of T-cell-associated antigen expression or thymic T-cell maturation stage. 200"204 The WHO classification recognizes "precursor T-cell lymphoblastic leukemia/lymphoma" without further subgrouping by maturation stage or by cytogenetic abnormalities. 2 The most commonly used classification divides T-ALLs into three stages: early (CD7+, cyCD3+, surface CD3", CD4", CD8"), mid or common (cyCD3+, surface CD3~ or CD3 weak , CD4+, CD8+, CD1+), and late (surface CD3+, CD1", CD4+ or CD8+). However, up to 25% of T-ALL have antigenic patterns that do not conform to one of these maturation stages (e.g., CD7+, CD1", CD3lo, CD4", CD8", or CD7+, CD1", CD3-, CD8+, CD4~). These may be examples of intralineage antigen infidelity, or may reflect incomplete understanding of cell antigen expression during normal thymic T-cell development. Subclassification schemes of T-ALL used at SJCRH and the European Group for the Characterization of Leukemias (EGIL) are presented in Table 7.6. The clinical significance of dividing T-ALL into early, mid, and late stages (or other paradigms thought to be representative of normal T-cell maturation) is controversial, although the results ofseverallarge studies suggest that patients with the mid or common stage of T-ALL fare best. 201-203"205 Several large studies of T-ALL have reached conflicting conclusions regarding the prognostic impact of surface CD3 expression or the absence of CD2, CD5, or CD10.197,201,206

SJCRHcalscfiaoitnEGIL classification

Subgroup Pre-T

Early T

LateT

Immunophenotype profile CD7+, cyCD3+, SCD3-, CD2 ± , CD5", CDla", CD10", CD4", CD8CD7+, cyCD3+, SCD3-, CD2+, CD5 ± , CDla-, CD10-, CD4", CD8CD7+, cyCD3+, sCD3+, CD2+, CD5+, CD1a±, CD10±, CD4 ± , CD8± CD7+, cyCD3+, sCD3+, CD2+, CD5+, CDla-, CD10-, CD4+ or CD8+

169

EGIL classification

Subgroup

Immunophenotype profile

T-I (pro-T)

cyCD3+/CD7+

T-II (pre-T)

cyCD3+ or sCD3+, CD2+ and/or CD5+ and/or CD8+

T-III (cortical T) cyCD3+ or sCD3+, CD1a+

T-IV (mature T) sCD3+, CDla", a(3(groupa) TCR-a[3+, TCR--y8+ 78 (group b)

Abbreviations: SJCRH, St. Jude Children's Research Hospital; EGIL, European Group for the Immunological Characterization of Leukemia; cyCD3, cytoplasmic CD3; sCD3, surface CD3; TCR, T-cell receptor antigen.

These disparities most likely result from differences in treatment or in the methods of immunophenotyping and the interpretation of results. Other studies have suggested that patients with TCR-/8+, as opposed to TCRa(J+ T-ALL, have distinct clinicopathologic features and poorer treatment outcomes. 207 " 209 A study of children with T-cell ALL at SJCRH examined the prognostic influence of age, leukocyte count, blast morphology, CNS leukemia, presence of a mediastinal mass, cytogenetics, immunophenotype, and other factors. Only older age and lack of CD10 expression were independently associated with apoor clinical outcome. 197 Cytogenetic abnormalities in T-ALL have not been associated with characteristic immunophenotypic subgroups. 148,210,211 Gene expression profile studies of T-ALL have identified novel genetic abnormalities that may correlate with the clinical response to treatment, and lead the way to developing prognostically useful immunophenotyping markers. 212,213

Precursor-NK leukemia/lymphoma Malignancies of natural killer cell precursors, which may present as a blastic lymphoma or acute leukemia, are rare in children and adults. 47-214"225 Cases presenting as lymphoma frequently have cryptic involvement of the marrow and frequently develop a leukemic phase

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Fred G. Behm

Normal Early progenitor CD34+/CD38+/HLA-DR+

Neoplastic Myeloid/T/NK progenitor CD34+/CD7+/CD33+/CD56-

? Myeloid/T/NK progenitor leukemia CD34+/CD7+/CD33+/CD56-

T/NK-cell precursor CD34+/CD7+/cyCD3+/sCD3-/CD56-

Myeloid/NK-cell lymphoma/leukemia CD34±/CD7+/cyCD3±/sCD3-/CD56+

Committed NK precursor CD34-/CD7+/cyCD3±/sCD3-/CD56+

Blastic NK-cell lymphoma/leukemia CD34±/CD7+/CD2±/cyCD3±/sCD3-/CD56+

Mature NK-cell CD7+/cyCD3-/sCD3-/CD56±/CD16±/ TIA-1 +/perforin+

NK large granular lymphocyte leukemia CD7+/CD2±/cyCD3±/sCD3-/CD16±/CD56±/ TIA-1+/perforin+

Fig. 7.14 Stages of natural killer (NK)-cell development and corresponding types of NK-cell malignancies.

shortly after diagnosis. Reported cases of precursor NK cell malignancies appear to fall into either blastic NK cell lymphoma/leukemia or myeloid/NK precursor acute leukemia; however, the WHO classification recognizes only a blastic NK-cell lymphoma/leukemia group.2,215 In theory, the myeloid/NK-cell precursor leukemia/lymphoma may correspond to an earlier stage of normal NK-cell development than blastic NK cell lymphoma/leukemia (Fig. 7.14). For the purposes of this discussion, the two entities are grouped together as precursor NK-cell leukemia/lymphoma. These malignancies are not well characterized because of their rarity and our limited understanding of early NK-cell development. The number of cases reported from Southeast Asia greatly exceeds those from the United States and Europe, suggesting environmental or genetic contributions to oncogenesis. The clinical presentation is often in extramedullary sites, such as skin, soft tissues, and mediastinum, with subsequent spread to bone marrow and lymph nodes. As a group, these malignancies are characterized by blastic cytologic features, an immunophenotype consistent with immature NK cell derivation, the absence of Epstein-Barr virus involvement, and an aggressive clinical course. In Wright-Giemsa stained blood smears, the neoplastic cells have variable amounts of basophilic cytoplasm, finely distributed nuclear chromatin, and small or indistinct nucleoli (Fig. 7.15). In contrast to large granular lymphocyte leukemias of T-cell or NK-cell origin, azurophilic cytoplasmic granules are absent or inconspicuous.

The diagnosis of precursor NK-cell leukemia or lymphoma requires immunophenotyping studies and, in some cases, molecular confirmation that TCR genes are in germline configuration. The malignant cells have a variable but characteristic immunophenotype of CD45+, CD7+/~, CD2±, surface CD3~, cytoplasmic CD3e+/~, CD4+/~, CD5~, CD8±, CD16~, and CD56+. TDT is usually negative. Not infrequently, the blasts may express one or more of the following antigens: CD11b, CD13, CD33, or HLA-DR. Granzyme B, TIA-1, and perforin, all granular enzymes, are predictably negative. The differential diagnosis includes precursor-T ALL, AML-M0, aggressive NK-cell large granular lymphocyte leukemia, and CD56+ small round cell tumors such as the Ewing sarcoma (Table 7.7). Since some cases of surface CD3~, cytoplasmic CD3+ precursor-T ALL may also express CD56, it may be necessary to document the absence of rearrangements of a TCRD, TCRG, TCRA, or TCRB gene if one is entertaining a diagnosis of a precursorNK cell malignancy. In the absence of studies of TCR gene status, the presence of CD5 and TDT would be strongly suggestive of T-ALL. Precursor-NK cell leukemias and lymphomas do not appear to have any characteristic cytogenetic abnormalities. The presence of chromosomal translocations involvingTCR genes as in precursor-T ALL would mitigate against but not entirely exclude an NK-cell malignancy. Some cases of precursor-NK cell leukemia may not express CD56 but may be positive for other NK-cell-associated antigens, including CD94, CD158, CD159, and CD161. How-

Immunophenotyping

101

102

103

104

CD45

104

Fig. 7.15 Example of a case of precursor NK-cell acute leukemia. (A) Undifferentiated blasts of the bone marrow. Four leukemic cells surround a normal neutrophil. (B) Flow cytometric study of marrow cells. CD45 expression versus light side scatter (SS) intensity with leukemic cells designated by open arrow. (C) Blasts shown in panel B do not express terminal deoxynucleotidyl transferase (TDT) or cytoplasmic CD3. Other studies showed expression of CD2, CD4, CD7, CD11b, CD16, and CD34; but not CD1a, CD5, CD8, CD117, or B-cell-associated antigens (not shown). (D) Blasts identified in panel B express NK-restricted NKp44 but not CD56. This case is unusual in that the only detectable NK-associated antigen was NKp44. Flow cytometric studies were performed on a whole bone marrow sample using a red blood cell lysis technique.

ever, these markers are not NK-cell restricted and can be expressed by cytotoxic T cells. We have encountered a case of CD56~ precursor-NK cell leukemia that expressed NKp44, a member of the newly described NK natural cytotoxic cell receptors (i.e., NKp30, NKp44, and NKp46) that reportedly are expressed only by NK cells (Fig. 7.15). However, it is not known if leukemic blasts of some AMLs or precursor-T ALL may also aberrantly express these NKp receptors.

Benign lymphocytosis mimicking lymphoblastic leukemia A mixed population of lymphoblasts and lymphocytes constitute up 15% of the bone marrow cellularity at birth. Their numbers rapidly increase thereafter, and by age 1 month can account for 30% to 55% of the marrow cellularity.225

Active lymphopoiesis continues through the first several years of life and then decreases. Approximately 90% of the lymphocytes in the marrows of healthy children are in the B lineage. During the first year of life, the marrow lymphoid population contains various numbers of B cells with morphologic and immunologic features of precursorB ALL. These immature B cells, sometimes referred to as hematogones, have highnuclear-cytoplasmic ratios, round or irregular nuclei with condensed chromatin, and indistinct nucleoli. 226 Hematogones express CD19, CD22, CD34, and TDT, a phenotype characteristic of most precursor-B ALLs.226,227 Increased proportions of immature B cells, or hematogones, frequently accompany other clinical conditions, including immune thrombocytopenic purpura, 228 , 229 neuroblastoma, 230 transient erythroblastopenia, 231 lymphoma and Hodgkin lymphoma, 232 post bone marrow

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Table 7.7 Laboratory features of precursor-NK leukemia/lymphoma and other potentially NK-cell-associated antigen-positive leukemias PNKL

NK-LGLL

PT-ALL

M0 AML

CD45 CD1a CD2 a sCD3 b cCD3ε CD4 CD5 CD7 CD8 CD11b CD13 CD15 CD16 CD34 CD56 CD57 CD94 CD117 TDT TIA-1 Perforin GB TCRc EBVd

Hypoplastic and aplastic marrows associated wth ALL

Abbreviations and symbols: PNKL, precursor-NK cell leukemia/lymphoma; NK-LGLL, NK-cell large granular lymphocyte leukemia; PT-ALL, precursor-T acute lymphoblastic leukemia; M0 AML, acute myeloid leukemia, M0 subtype; TDT, terminal deoxynucleotidyl transferase; GB, granzyme B; TCR, Tcellreceptor; EBV, Epstein-Barrvirus; +, positive; -, negative; + / - , positive or negative; + ( - ) , occasionally negative; - ( + ) , occasionally positive. a Surface CD3 expression. b Cytoplasmic CD3ε expression. c d

should include antibodies that detect antigens appearing early and late in B-cell differentiation. Benign lymphoid proliferations contain a mixture of early (CD19+, CD10+, TDT±, clg|j,-, sign,"), immature (CD19+, CD20+, CD10±, clg|ju+, slg|j,~), and late (mixed sIg|j,K+ and sIg|j,X+) lymphocytes. Conversely, in B-lineage ALL, the majority of blasts reflect a single stage of lymphocyte maturation. In addition, leukemic blasts usually show asynchronous patterns or abnormal intensities of B-cell-associated Multiparameter flow cytomantigen expression. etry using different combinations of antibodies (e.g., CD45, TDT, CD10, CD19 or CD45, CD19, CD10 or CD45, CD19, CD22, CD34) can be used to differentiate normal 240,242 Thus, immunophenotyping hematogones from ALL. studies combined with a clinical history and bone marrow morphology distinguishes between benign and malignant proliferations of immature B lymphocytes.

Applicable to TCRa, (3, and 8. TCR-y may be rearranged in PNKL. As demonstrated by in situ hybridization or other molecular methods.

transplantation,233 and viral infections.234,235 Hematogones may comprise up to 50% of marrow cells for as long as several months after cessation of chemotherapy and be mistaken for persistent residual leukemic blasts.236"240 In some of the preceding conditions, smallnumbers of immature, TDT+ B cells are detectable in the peripheral blood. Not surprisingly, uninformed or incomplete morphologic and immunopheno typing studies of such patients can lead to an incorrect diagnosis of ALL. It is important to note that transformation to ALL does not occur in these preceding clinical conditions. Immunophenotyping studies readily differentiate benign marrow lymphocytosis from ALL. Such studies

Rarely children who develop ALL will present initially with blood cytopenias, hypocellular bone marrow, and the absence of an overt leukemic cell population.244"250 The hypoplastic phase, variously termed preleukemic syndrome, pre-ALL syndrome, and aplastic presentation of ALL, usually lasts 1 to 4 weeks and is followed by a hematologic recovery. Overt ALL typically develops from 3 to 9 months later. The leukemias emerging after the preALL phase usually have L1 morphology and a precursorB immunophenotype. Initially, it may be difficult or not possible to differentiate a pre-ALL syndrome from other hypoproliferative disorders, such as aplastic anemia, transient erythroblastopenia of childhood, Fanconi anemia, and myelodysplastic syndrome. Multiparameter flow cytometry can identify small lymphoblas t pop ulations with aberrant antigen expressions predictive of ALL in some patients presenting in the aplastic phase of the pre-ALL syndrome. Acute myeloid leukemias In the strictest sense, the term "acute myeloid leukemia" (AML) refers to a malignancy of the myeloid or granulocytic lineage, but is used here to refer to all nonlymphoid acute leukemias including the myelocytic (M0, M1, M2, M3), monocytic (M4, M5), erythroid (M6), and megakaryocytic (M7) subtypes. The leukemic cells of AMLM0 through M5 express various combinations of CD13, CD33, CD15, CD117, and myeloperoxidase (Table 7.8). M6 and M7 AMLs are identified by their expression of erythroid (CD36, CD235a) and megakaryocytic (CD41a,

Immunophenotyping

Table 7.8 Immunophenotypes of AML Immunophenotypic marker FABa

M P O CD34CD13CD14

CD 13

CD14

CD15

CD33

CD36

CD61

CD117

CD235ab

M0 M1 M2 M3 M4 M5 M6d M6e M7 Abbreviations and symbols: MPO, myeloperoxidase; +, positive; - , negative; + / - , positive or negative; + ( - ) , occasionally negative;-(+), occasionally positive. a AML subtype by French-American-British (FAB) classification criteria. b CD235aisglycophorinA. c M5AMLblastscannonspecifically bind platelets and gpIIIa/IIb(CD41a and CD61), resultinginfalse-positive expression. d Erythroleukemia subtype. e Pure erythroid subtype.

CD61 or less commonly CD42b) antigens, respectively. Although almost all AMLs express either CD 13 or CD33, or CD117, none of these markers is present in every case. Thus, immunophenotyping panels should include testing for all three of these markers plus MPO and one or more markers for megakaryocytic and erythroid lineages

the light side scatter signatures by flow cytometry also differ among the subtypes of AML. Taken together, graphic representations of leukemic cell CD45 intensity and light side scatter provide characteristic flow cytometric profiles for each subtype of AML (Figs. 7.11 and 7.12).

(Fig. 7.13) .64,65,251

Acute myelocytic leukemia with little differentiation (M1 AML) Acute myelocytic leukemias with little differentiation commonly express CD13, CD33, CD34, CD117, and HLA-DR in various combinations and intensities. Expression of CD4, CD11b, CD15, or CD65 is less frequent. No single antigenic profile is common to all M1 AMLs. This heterogeneity most likely is due to the inclusion of several different genotypes of AML in the M1 group that are morphologically similar. Cytogenetic studies support this conclusion by showing a variety of clonal chromosomal abnormalities in different cases of M1 AML (e.g., chromosome 7q abnormalities, monosomy7, t(8;21)(q22;q22), t(6;9)(p23;q34), or chromosome 3q26 translocations).

Except for MPO, monocyte-associated CD14, megakaryocyte-associated CD41a, CD42b and CD61, other myeloid- and monocytic-associated molecules including CD 11b, CD11c, CD13, CD15, CD33, CD64, CD65, CD66, and CD 117 - are not useful for distinguishing among the different subtypes of AML (Table 7.8) . 252 - 255 This is partly because the antigenic profiles of AML usually do not reflect a particular stage of normal myeloid, monocytic, or megakaryocytic maturation. 254 - 256 " 260 Key differences between the patterns of antigen expression by leukemic versus normal cells include: (1) asynchronous expression of antigens, such as coexpression of CD34 and CD 15; (2) gain, loss, or inappropriate density of myeloid antigens (e.g., diminished expression of CD 13 andCD33 byAMLM2 cells); and (3) aberrant expression of lymphoid-associated CD2, CD7, CD19, or CD56. More recent methods that simultaneously scrutinize cell characteristics by multiparameter flow cytometry provide representative profiles for most morphologic and several genotypic subtypes of AML (Table 7.9).61,261-265 T h e l e u k e m i c blasts of all myelocytic and monocytic leukemias, and many megakaryocytic leukemias, express CD45 with varied intensity. Similarly,

Acute myelocytic leukemia with differentiation (M2AML) Immunologic and cytogenetic studies identify several different leukemic processes included in M2 AML. About 35% to 40% of childhood M2 AMLs have the t(8;21)(q22;q22). The leukemic cells of the bone marrow display characteristic morphologic features including asynchronous cytoplasmic-nuclear myeloid maturation

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Table 7.9 Correlation of cytogenetic abnormalities in AML with leukocyte antigen expression.

Karyotype

Genes involved

t(8;21)(q22;q22) t(15;17)(q22;q11) t(11;17)(q23:q11) inv(16)(p13q22) t(11q23;V) t(11q23;V) t(6;9)(p23;q34) Monosomy 7

ETO-AML1 PML-RARa PLZF-RARa MYH11-CBFβ MLL MLL DEC-CAN

AML a subtype

Leukocyte antigen expression profile CD34

HLA-DR

M2 M3 M3-like M4Eo M4 M5 M2, M4 M1-M5

CD13 CD14 CD33

CD2

Weak

-

-

+/weak

CD7

CD19 CD56

NG2

b

Symbols: +, positive; -, negative; + / - , positive or negative; -(+), occasionally positive AML subtype by French-American-British (FAB) classification criteria. b NG2, nonhematopoietic chondroitin proteoglycan sulfate. a

with hyper- or hypogranulation, giant inclusion-like secondary granules, and thin Auer rods but without associated erythroid and megakaryocytic dysplasia. 266 Type II blasts with a few primary azurophilic granules frequently exceed the number of Type I blasts that lack no azurophilic granules. A distinct immunopheno type usually complements these morphologic findings (Table 7.9). Leukemic blasts commonly express CD 15, CD34, CD65, and HLA-DR, but their expression of CD13 and CD33 may be very weak and sometimes not detectable. 2 6 7 " 2 7 1 Most cases weakly express B-cell-associated CD 19 and less commonly the NK-cell-associated CD56 antigen. 2 6 7 ' 2 6 8 ' 2 7 1 ^ 2 7 7 By contrast, the leukemic cells of M2 AML without the t(8;21) may also express CD34, CD15, and HLA-DR, but the intensity of CD 13 andCD33 expression usually exceeds that of t(8;21)-positive blasts. In addition, the CD19 antigen is rarely detectable, while T-cell-associated CD2 or CD7 is frequently present. 2 6 7 , 2 7 8 The antigenic profile of MPO+, CD13+, CD33+, CD34+, CD19+, CD56+'- is not sufficiently sensitive to identify all t(8;21)+ AML cases but is useful for selecting cases for molecular screening when cytogenetic studies are not informative. Whether or not CD56 expression has any prognostic value in t(8;21)+ AML remains unsettled. 2 6 7 , 2 7 5 , 2 7 7 Other studies have suggested that MPO expression in combination with increased expression of CD34 may be associated with t(8;21)+ AML and maybe predictive of clinical outcome. 2 5 8

Acute promyelocytic leukemia (M3 and M3v AML) Acute promyelocytic leukemia is a distinct subtype of myeloid leukemia with unique clinical, biologic, and molecular features.2 The microgranular variant M3v morphologically mimics acute monocytic leukemia but has the same clinical and laboratory features as M3 AML. Cells of

M3 and M3vAML strongly express CD9, CD13, CD33, CD65, CD68, and MPO, but characteristically lack CD7, CD14, CD34andHLA-DR(Tables7.7and7.8). 2 5 4 , 2 5 6 , 2 7 9 , 2 8 0 Expression of CD 1 1b, CD 15, and CD 117 is variable. Leukemic cells of M3v AML and an uncommon hyperbasophilic, microgranular variant of M3 AML may express CD34, HLA-DR, CD117, and rarely TDT. 2 8 1 - 2 8 4 Flow cytometric light scatter and immunophenotypic characteristics may be less subjective than morphology in distinguishing M3 from M3v AML.265 Expression of CD34 is associated with increased WBC counts but not with overall survival.284 A minority of M3 AMLs show aberrant expression of Tcell-associated CD2 or NK-cell-associated CD56 that correlates with morphologic, genetic molecular and clinical features which differ from that of typical cases of M3 AML. 2 8 3 " 2 9 5 Expression of CD2 or CD56 is more frequent in promyelocytic leukemias with M3v morphology and/or 15;17 chromosomal translocations with a break at bcr3 of the PML gene. Studies also suggest that aberrant CD2 expression is associated with a better initial treatment response, 2 9 4 whereas CD56 may predict a poorer clinical outcome. 2 9 1 " 2 9 3 These seemingly contradictory observations imply that mechanisms other than the bcr3 type of 15;17 translocation may be influencing CD2 and CD56 expression and treatment response. Although considered a characteristic feature, the antigen expression profile of promyelocytic leukemia does not differ from that of some M1 andM2AMLs. 2 9 6 In addition, rare leukemias that morphologically resemble M3AMLbut have at(11;17)(q24;q21),t(5;17)(q34;q11.2-12),t(3;15)(q21;q22), or t(X;15)(p11;q22) can have antigenic profiles identical to that of AML with the t(15;17) (Table 7.9) . 2 9 7 - 3 0 0 A rare myeloid/NK cell-like leukemia may resemble M3 AML morphologically and immunophenotypically. 301

Immunophenotyping

Immunophenotyping is very helpful in differentiating M4 or M5 AML from M3v AML. That is, cells of monocytic leukemias, unlike those in M3v AML, are strongly positive for HLA-DR and CD64, weak to moderately strong expressors of CD4, and frequently express CD 14, CD34, and CD36.263 Multiparameter flow cytometry is helpful in differentiating M3 AML from a recovering case of acute agranulocytosis. 302-304 ACD9+, CDllb- / l o ( CD117+, CD66c~ phenotype is characteristic of leukemic promyelocytes, whereas regenerating normal promyelocytes are CD9- / k \ CDllb+, CD117-, CD66c+. Antibody reagents such as the PG~ M3 monoclonal antibody detect the aminoterminal portion of the promyelocytic leukemia (PML) protein and can be used with immunofluorescence or immunohistochemical methods to screen for M3 AML with the t(15;17).305-307 Leukemic cells with the t(15;17) display a fine microgranular or speckled nuclear pattern of PML expression, while normal myeloid cells and leukemic cells of M3 AML with t(11;17) andt(5;17) display a coarse globular pattern. 307 Acute myelomonocytic leukemia (M4 AML) Acute myelomonocytic leukemia contains neoplastic myeloid and monocytic components, implying a common precursor cell of origin. The WHO classification criteria for acute myelomonocytic leukemia requires that the bone marrow have at least 20% nonerythroid blasts, 20% or more neutrophils and their precursors, and a minimum of 20% monocytes and their precursors. 2 The leukemic cells express CD4, CD 11b, CD11c, CD13, CD14, CD15, CD33, CD65, and HLA-DR (Table 7.8). The leukemic blasts of most cases display CD34, and sometimes a minor subset of blasts express TDT. However, the antigen expression profile for M4 AML varies depending on the subset of leukemic cells included in the analysis, that is, the analysis can include only blasts or blasts plus maturing myeloid and monocytic elements. Analysis of CD45 versus light side scatter shows a myeloid blast population plus cells with features of both myeloid and monocytic maturation. The three populations frequently overlap, thus making their distinction somewhat subjective for marker analysis. The blast population is typically CD4~/to, CD1 1b+, CD13+, CD 14", CD33+, CD34+, CD36-, CD64- /to , the maturing myeloid population CD4~, CD11b+, CD13+, CD14-, CD33+, CD34~, CD36~, CD64lo, and the monocytic elements CD4+, CD11b+, CD13+, CD14- / + , CD33+, CD34~, CD36+'-, CD64+. Multiparameter flow cytometric analysis may be more accurate than morphology and cytochemical staining in differentiating M4 AML from M2 and M5 AML. For example, AMLs are encountered that morphologically resemble M4 AML but are negative or very weakly positive for nonspecific esterase, while having

flow cytometric profiles characteristic of typical nonspecific esterase-positive M4 AML. A more appropriate classification for these AML would be M4 AML. Examples of M2 AML with the inv(16) (p13q22) chromosomal translocation may represent nonspecific esterase-negative M4 AML. A relatively uncommon variant of M4 AML, M4Eo, is associated with increased numbers of marrow eosinophils with or without peripheral blood eosinophilia. 2 Almost all of these leukemias have an inv(16)(p13q22) or a t(16;16)(p13;q22) chromosome abnormality that results in a CBFfi-MYHll chimeric gene. The monocytic component of M4Eo with the CBFfi-MYHll characteristically expressesT-cell-associated CD2 (Table 7.9).290,308,309 A n t i bodies to the chimeric CBF(J /MYHII protein can be used in flow cytometric analyses to screen for inv(16) and t(16;16) abnormalities. 310 , 311 Eosinophils are part of the malignant clone in M4Eo AML,312 but they have not been shown to aberrantly express CD2 or other lymphoid-associated antigens. The t(6;9)(p23;q34) chromosomal anomaly, which produces a chimeric DEC/CAN protein, is a rare cytogenic finding in M4 and M2 AMLs. Increased numbers of basophils are present in over half of the patients. These leukemias express CD 13, CD33, CD34, and occasionally TDT.313~316 The basophils and possibly some leukemic blasts may express CD22, but a unique antigen expressionprofile identifying this genotype has not been described. The clinical outcome for these patients is uniformly poor, so that at present immunophenotypying lacks prognostic significance. Acute monocytic leukemia (M5 AML) The bone marrows of patients with acute monoblastic leukemia contain 80% or more monoblasts, promonocytes, and monocytes. Leukemias composed of at least 80% monoblasts are termed acute monoblastic leukemia M5a, while those showing monocytic cell maturation or fewer than 80% monoblasts are called acute monocytic leukemia M5b.2,96 The latter subtype is uncommon in childhood. The M5 AMLs have a characteristic, albeit not unique, pattern of surface antigen expression. With rare exceptions, monoblasts express HLA-DR, CD4, CD 11b, CD11c, CD33, CD64, and CD65 (Tables 7.8 and 7.10). Some monoblastic leukemias exhibit CD117, but only rare cases express CD34. The majority of monocytic leukemias also express CD15 and CD36, and not infrequently CD56. CD14 is detected in fewer than one-half of pediatric M5a AMLs. The results of antimyeloperoxidase testing may be positive or negative and parallels findings of enzymatic myeloperoxidase staining. Some cases of AML that are morphologically, cytogenetically, and immunophenotypically identical to M5 AML

175

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Table 7.10 Antigen expression profiles of enzymatic myeloperoxidase-negative acute leukemias AEbL a

Feature

M0AML

M5AML

M7AML

CD45 CD7 CD33 CD36 CD41/CD61 CD42 CD117 CD235ac MPOd Factor VIII Hemoglobin Platelet peroxidased,e Ferritin theta bodiesd

+ +/+(-) -(+) +/+/-

+

+/+/+(-) +/+ +/+/-(+) -

-

+(-)

+(-) + (+) +/+ + +/-

-

-

-

+

(+)

+ +/b -

+ /-

-

+ /-

f -

(+) ?

Abbreviations and symbols: AEbL, acute erythroblastic leukemia; MPO, myeloperoxidase; +, positive; - , negative; + / - , positive or negative; - ( + ) , negative, occasionally positive; + ( - ) , positive, occasionally negative. a Not the same as acute erythroleukemia (M6 AML), see text. b Some monoblasts nonspecifically bind gpIIIA/IIB, resulting in false-positive test. c CD235aisglycophorinA. d Ultrastructural study without osmium staining e Some cases do not demonstrate platelet peroxidase f Rare cases weakly positive.

lack nonspecific esterase activity. Recent gene expression profile studies show that these cases are genotypically identical to nonspecific esterase-positive M5 AML and thus should be classified as M5 AML.317 Often, a variable number of monoblasts weakly express CD41a and CD61 due to adherence of platelets or adsorption of glycoprotein IIb/IIIa to the blast cell surface.318"320 A false-positive CD41a or CD61 test may lead to difficulty in differentiating between monocytic and megakaryocytic lineages if immunophenotyping is interpreted without knowledge of the leukemia's morphologic and cytochemical features. In some cases, it may be necessary to confirm the nonspecific adherence of platelet products to monoblasts by immunofluorescence or immunohisto chemical studies of cytospin preparations of the leukemic sample. Methods to reduce nonspecific binding of platelets to leukemic blasts by incorporating EDTA with the immunolabeling procedure are only partially successful.321 Over 90% of pediatric M5 AMLs have a chromosomal 11q23 abnormality that results in a rearranged MLL gene. Immunophenotyping studies have failed to show a

consistent antigen expression profile that separates M5 AMLs with MLL gene rearrangements from those with other molecular genetic abnormalities.322 CD56 is frequently expressed by monocytic leukemias with rearrangement of the MLL gene.161-323"325 Monoclonal antibody 7.1, which detects the human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, is associated with M4 and M5 AML.158 ,159 A study of adult and childhood AML found that reactivity with the 7.1 antibody is a sensitive though not entirely specific marker for 11q23 abnormalities involving the MLL gene (Table 7.9).161 Expression of the NG2 homologue does not appear to have prognostic significance.158,161 Normal hematopoietic cells, including hematopoietic progenitor or stem cells, do not express the NG2 homologue, thus providing a useful marker for minimal residual disease studies. Acute erythroleukemia and pure erythroid leukemia (M6AML) The WHO classification recognizes two M6 AML subtypes based on the presence or absence of a significant myeloid component (i.e., acute erythroleukemia and acute pure erythroid leukemia respectively).2 In other classifications, the latter subtype is synonymous with erythremic myelosis (DiGuglielmo disease) andAMLM6b.326~329 Leukemias consisting primarily of erythroid precursors are rare in children. Most cases of pediatric acute erythroleukemia appear to be transformations of myelodysplastic syndrome with exuberant erythroid and small myeloid components, or they develop in patients with Down syndrome.330"332 The diagnosis of acute erythroleukemia is readily made by morphologic examination, thus limiting the diagnostic utility of immunophenotyping studies.2,96 The erythroblasts express CD36, CD71, and CD235a (glycophorin A) (Table 7.8). Hemoglobin is produced only in the later stages of erythroid maturation and may not be detectable in leukemic cells corresponding to proerythroblasts. The myeloid component expresses CD 13, CD33, and MPO. Pure erythroid leukemia is very rare in children and should be confirmed by immunophenotyping studies. These leukemias will express erythroid-associated markers CD36, CD71, CD235a, andpossibly hemoglobin with little orno evidence of a MPO-positive blast component. Acute erythroblastic leukemia, a minimally differentiated form of pure erythroid leukemia, presents as a blastic proliferation with little or no morphologic evidence of erythroid differentiation andnomyeloblast component.332"336 This leukemia is exceedingly rare in children and adults. Most pediatric cases have been associated with Down syndrome.331,335 The differential diagnosis of acute erythroblastic leukemia includes ALL and M0 and M7 AML. Expressions of B- or T-lineage-associated antigens readily

Immunophenotyping

identify cases of ALL; however, the distinction from M0 and M7 AML may be difficult, as undifferentiated erythroblasts can have no or few erythroid-associated antigens, or may share antigenic and ultrastructural features with young megakaryoblasts (Table 7.10). Ideally, leukemic erythroblasts should express CD36 and CD235a without CD41a, CD42b, and CD61. However, these latter three megakaryocytic antigens can be expressed in various proportions by neoplastic erythroblasts. 39,41,337 CD235a and carbonic anhydrase I, initially thought to be unique to erythroid lineage cells, may also be expressed by some leukemic megakaryoblasts. 337 The cross-lineage expression of these otherwise lineage-restricted antigens suggests a common committed progenitor for erythropoiesis and megakaryopoiesis. Indeed, numerous molecular similarities exist between erythroid and megakaryocytic lineages, suggesting an initial common pathway of development. Recent studies point to the existence of a subset of CD34+, CD38+/ ~ hematopoietic progenitor cells capable of generating only erythroid and megakaryocytic elements. 338 The presence of Factor VIII and hemoglobin, which are expressed late in maturation, is considered evidence of megakaryoblastic and erythroblastic differentiation, respectively, but their presence in very immature blastic proliferations will be rare. The blasts of some cases of minimally differentiated erythroleukemia may express neuron-specific enolase, a marker of metastatic neuroblastoma and other small cell tumors. 339 Acute megakaryoblastic leukemia (M7 AML) Acute megakaryoblastic leukemia accounts for about 10% of pediatric AML and is more common in children under 2 years of age and in patients with Down syndrome. The differentia tion of M7 AML from ALL, M0 AML, and some times small cell tumors of children, is usually not possible by morphologic and cytochemical studies. The presence of many micro megakaryocytes or dysplastic immature megakaryocytes in the bone marrow would be presumptive morphologic evidence of a megakaryocytic leukemia, but these more mature megakaryocytic elements are uncommon in pediatric M7 AML. The diagnosis of M7 AML can be confirmed by immunophenotyping or ultrastructural studies for platelet peroxidase (Table 7.8 and 7.10). Immunologic marker studies are more readily performed than ultrastructural studies and have largely replaced the latter. 37,340 The leukemic cells in 90% to 95% of pediatric M7 AML cases express CD41a and CD61, and many have detectable CD36 or CD42b.341,342 Suspected cases of M7 AML that are devoid of surface megakaryocytic-associated antigens shouldbe examined for the presence of cytoplasmicCD41a or CD61.343 Most M7 AMLs express CD33, CD45, CD 117 and commonly CD13, CD34, and HLA-DR.344 The blasts

of 50% to 70% of cases express CD7 and not infrequently CD2, CD4, or CD56.37,68,342,344 Antibodies to the thrombopoietin receptor MPL reportedly react with the leukemic blasts of M7 and M6 AMLs, as well as many other subtypes of AML, and thus may not be useful in initial lineage determinations. 345 A unique immunophenotypic profile has not been associated with t(1;22)(p13;q13)+ M7 AMLs.344,346,347 The morphologic differential diagnosis of megakaryoblastic leukemia includes ALL, M0 and myeloperoxidasenegative M5 AML, acute erythroblastic leukemia, and metastatic small cell tumors (Table 7.10). The choice of the appropriate immunologic markers readily aids the identification of these malignancies, but several cautionary remarks are in order. As discussed earlier, cells of acute erythroblastic leukemia can also express plateletassociated antigens, such as CD41a, CD61, and even CD42b, whereas some megakaryoblastic processes may express glycophorin A. The coexpression of megakaryocytic and erythroid associated antigens may be due to the derivation of many M7 AMLs from a progenitor cell capable of megakaryocytic or erythroid differentiation. 338 In support of this theory, a recent study of the CD34+ cells ofAMLM0 through M7 showed that the CD34+ megakaryoblasts expressed CD61 or CD235a but not CD38, whereas the CD34+cells in other AML subtypes were usually CD38+.348 Many cases of M5 AML also bind glycoprotein II/IIIa, resulting in false-positive CD41a and CD61 studies. 318"320 Some cases of M7 AML mimic metastatic small cell tumors when the presentation includes lytic or osteosclerotic bone lesions, extramedullary presentations, or aggregates of megakaryoblasts in bone marrow aspirate smears. 349,350 In addition, antibodies to actin and neuron-specific enolase, expressed by some pediatric small cell tumors, may produce weak reactions with leukemic megakaryoblasts. However, unlike M7 AML, small cell tumors of children do not express CD45 or megakaryocytic-associated CD41a, CD42b, andCD61. Many patients with M7 AML have "dry taps" or marrow aspirates yielding very few leukemic cells. Bone marrow biopsies of these patients show a moderate-to-intense reticulin fibrosis enmeshing collections of megakaryoblasts. Unfortunately, immunohistochemical studies with antibodies to CD41a and CD42b are not usually reactive in formalin- or B5-fixed bone marrow biopsy specimens. Although anti-CD61 can readily react with mature megakaryocytes, the reactivity with megakaryoblasts in formalin-fixed bone marrow biopsies is less reliable. Unlike most cases of adult M7 AML, the megakaryoblasts of pediatric cases rarely express Factor VIII, an antigen that is readily detected in formalin-fixed marrow samples. 35,342 Thus, for patients with fibrotic marrows and dry aspirates,

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Since the 1991 proposal of M0 AML, the development of additional lineage-restricted monoclonal antiFAB Cooperative Group criteriaa SJCRH criteriab bodies and the use of multicolor flow cytometry have 1. <3% of blasts positive for MPO or 1. <3% of blasts positive for MPO or improved the immunologic identification of lymphoid, myeloid, andmegakaryocytic lineages. Several groups have SBB SBB proposed modifications of the FAB criteria for identi2. Blasts negative for all lymphoid 2. <5%ofblastspositivewithANB fying M0 AML.149,357-361 The WHO classification groups antigens except CD2, CD4, or CD7 esterase 3. <10% of blasts express platelet or these leukemias as "AML minimally differentiated" and plus erythroid antigens CD36, CD41a, retains, in large part, the defining criteria of the FAB CD42b, CD61, or CD235a (GPA) proposal. 2 The diagnostic criteria used at St. Jude Chil4. < 10% of blasts express 3a. Ultrastructural evidence of MPO dren's Research Hospital for M0 AML builds on the FAB procytoplasmic CD3, CD79a, or posal, but establishes stricter guidelines for excluding lymCD79b phoblastic and megakaryoblastic processes (Table 7.11). plus In the absence of lineage-restricted lymphoid (CD3, CD22, 5a. Ultrastructural evidence of MPO 3b. Blasts react with anti-MPO CD79a,TCR(J, andimmunoglobulins) andmegakaryocytic antibody (CD42b, CD41a, and CD61 antigens), the expression of or or CD13, CD15, CD33, CD65, or antigenic MPO is presumed 3c. Blasts express 1 or more of the 5b. >10% of blasts react with evidence for myeloid lineage commitment. It is important following antigens: CD11b, CD13, anti-MPO antibody to recognize that leukemias devoid of detectable MPO antiCD14, CD15, orCD33. gen are classified as M0 AML only in the absence lymphoidand megakaryocytic-restricted antigens. Although the 5c. >10% of blasts express 2 or more of the following antigens: CD 13, majority of M0 leukemias express CD13 or CD33, examCD15, CD33, CD65, or CD117 ples that lack these antigens but demonstrate anti-MPO reactivity have been reported. 362 " 364 The leukemic cells of Abbreviations: FAB, French-American-British; SJCRH, St. Jude Children's M0 AMLs often express other less lineage-restricted lymResearch Hospital; MPO, myeloperoxidase; SBB, Sudan Black B; ANB, alpha- phoid antigens, such as CD2, CD4, CD7, CD9, CD 10, CD 1 1b, naphthyl butyrate esterase; GPA, glycophorin A. CD 19, CD34, CD71, andTDT, which may make differentiaa Blasts must meet criteria 1 and 2 plus either 3a, 3b, or 3c. tionfrombiphenotypic leukemias difficult. 357'358-360-364"371 b Blasts must meet criteria 1-4 plus either 5a or 5b or 5c. Some cases of M0 AML may be examples of nonspecific esterase-negative M5 AMLs, as suggestedby the presence of monocyte-associated antigens and chromosomal transloimmunophenotyping studies often must be performed on cations [e.g., t(llq23;V)]. 372 " 374 blasts obtained from peripheral blood, teased bone marrow biopsies, or imprints of bone marrow biopsy tissue. The leukemic cells of M0 AML correspond to very early stages of myelopoiesis or monopoiesis. For example, MPO gene expression is detected in cases that lack detectable Acute myeloblastic leukemia without morphologic or MPO protein, as might be expected for very early myeloid cytochemical evidence of differentiation (M0 AML) cells.375 In addition, most M0 leukemias express CD 117, an In 1976, the FAB Cooperative Group assigned allleukemias antigen that is commonly expressed by acute myeloid but without cytochemical evidence of myeloid or monocytic rarely lymphoblastic leukemias. 101,108,114,370,376 At relapse, differentiation to the lymphoblastic category.351 Howsome M0 AMLs retain the clonal genetic abnormality disever, subsequent studies of some MPO-negative leukemias covered at diagnosis, but display morphologic, cytochemishowed ultrastructural and immunologic evidence of cal, andimmunophenotypic features of differentiatedmyemyeloid development. 352 " 355 Further, the clinical outcomes locytic or monocytic leukemias. 363,364,372 of these minimally differentiated leukemias were less favorable than that of ALL. In 1991, the FAB investigators proM0 AMLs frequently show chromosomal abnormaliposed "M0" for this variant of minimally differentiated ties common to myelodysplastic syndrome (MDS) and myeloid leukemia, and listed morphologic, cytochemisecondary AML, including monosomy 7 or 13, +8, cal, and immunophenotypic diagnostic criteria. 356 These or deletions/translocations of 3q, 7q, 5q, and included less than 3% MPO or Sudan Black B-positive a n d in contrast 1 1 q 2 3 . 363,365,368,370,376-379 L i k e MDS blasts, expression of myeloid antigens, ultrastructural evito more differentiated AMLs, adult M0 AMLs have a dence of MPO, and lack of lymphoid lineage antigens with high percentage of CD34+, CD38~ leukemic clonogenic the exception of CD7 and CD2 (Table 7.11). precursor cells.380 Children and adults with M0 AML have a Table 7.11 Diagnostic criteria for AML with minimal differentiation (M0 AML)

Immunophenotyping

very poor prognosis.268,363,367,370,371,377-383 Several studies of adults show a correlation between M0 AML and several other negative prognostic factors, including the expression of CD7 and CD34, the presence of P-glycoprotein 170, complex cytogenetic abnormalities and abnormalities of chromosomes 5 and 7, and older age.364,377,378 Whether the poor outcome is independent of these latter features is unclear in adults and has not been examined in children.364 Others have not shown prognostic significance for CD34 expression.384 Acute undifferentiated leukemia/acute leukemias of uncertain lineage (AUL)

evidence of myeloid maturation.392 Some cases may be examples of acute leukemias with an NK-cell origin.397 In children, one must be careful not to confuse metastatic small cell tumor with AUL. Diagnostic studies of suspected pediatric cases of AUL should include marker studies for neuroblastoma, the Ewing family of tumors, and other small cell tumors, some of which may express leukocyte antigens including HLA-DR, CD9, CD34, CD 117, CD 10, CD56, or CD57, but not CD45. Acute leukemias with aberrant antigen expression (My+ ALL, Ly+ ALL, and biphenotypic leukemia)

Current evidence strongly supports the concept that leukemia represents the clonal expansion of a single transRare cases of acute leukemia remain difficult to classify formed cell and that most leukemic processes mirror even after extensive morphologic and immunophenotypic stages of normal leukocyte differentiation. Nonetheless, studies. Such leukemias are designated acute undifferentiimmunologic and molecular studies show that some acute ated or stem cell leukemia (AUL).385~394 The WHO classifileukemias display features of one or more hematopoication groups these leukemias with the "acute leukemias of etic lineages (lineage infidelity). Acute leukemias whose ambiguous lineage" as "undifferentiated acute leukemia."2 blasts simultaneously show characteristics of more than The incidence of AUL is difficult to determine from the litone lineage (e.g. lymphoid plus myeloid) have been termed erature because some reports include cases of M0 AML acute mixed lineage, hybrid, chimeric, or biphenotypic and biphenotypic leukemia, while others do not incorpoleukemia.398"403 These diseases should not be confused rate immunophenotyping studies for cytoplasmic antigens. Morphologically, the blasts of AUL may resemble ALL with rare cases bilineal leukemia or leukemias composed of two or more different leukemic cell processes. The or AML, but are negative for myeloperoxidase and Sudan leukemias with mixed lineage, hybrid, or biphenotypic feaBlack B and, with immunophenotyping lack antigens assotures can be defined by morphologic, cytochemical, ultraciated with the lymphoid, myeloid, or megakaryocytic linstructural, and molecular studies, but in most instances eages. To be termed AUL, the leukemic cells must express they are identified by immunologic studies alone. CD45 and lack surface and cytoplasmic antigens associated with the B (CD19, CD24, cytoplasmic CD22, cytoplasImmunophenotyping and molecular genetic investigamic CD79a), T (CD2, cytoplasmic CD3, cytoplasmic TCR(J, tions support the concept of two partially overlapping catCD5, CD6), myeloid/mono cytic (CD13, CD14, CD15, CD64, egories of acute leukemias with disparate expressions of CD65, MPO), and megakaryocytic/erythrocytic (CD36, lineage-associated features. The larger category comprises CD41a, CD42b, CD61, CD235) lineages. The blasts may leukemias with immunologic, genotypic, and clinical feaexpress CD7, CD9, CD33, CD34, CD38, CD71, CD90, CD133, tures characteristic of a strong commitment to a single CD 117, or HLA-DR, similar to what might be expected of a lineage but with one or more features of another lineage very early hematopoietic stem cell. (aberrant antigen expression or lineage infidelity). These include ALL expressing myeloid-associated antigens (My+ The lack of lineage-associated markers in AUL may ALL) and AML expressing lymphoid-associated antigens be due to the immaturity of the leukemic cells (true (Ly+ AML). Over half of ALLs and upwards of one-third of stem cell disorders), to lineage-associated antigen expresAMLs are included in this category, depending on the crisions below levels detectable with current techniques, or teria used to define aberrant antigen expression. The other disordered gene regulation resulting in the absence of category is very small and consists of leukemias with genolineage-associated antigens in a lymphoid- or myeloidtypic and antigenic features that make it unclear whether committed process. In several studies, in vitro stimulations the leukemic blasts are committed to myeloid or lymof AUL blasts with tetradecanoyl-phorbol-acetate induced phoid differentiation, i.e., true mixed, hybrid, or biphenomyelomonocytic but not lymphoid differentiation.395,396 typic leukemias. Although recognition and separation of A recent study of the genomic profile and growth facthese two categories is important in the clinical managetor response of an adult case of AUL demonstrated eryment of the leukemia patient, confusion remains as to throid, myeloid and monocytic differentiation potential of 394 their diagnostic criteria, nomenclature, optimal treatment, the leukemic blasts. At relapse, several AULs showed

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Table 7.12 SJCRH criteria for My+ ALL, Ly+ AML, and biphenotypic leukemia

B-lineageMy+ALLa 1. Blasts are CD19+ plus CD22+ or cyCD79a+ or cyIg |x+ 2. Blasts are cyCD33. Blasts are MPCTb 4. Blasts express one or more myeloid-associated antigens: CD13, CD15, CD33, or CD65

Ly+ AMLa 1. Blasts are MPO+b (or NSE+ if AML M5) 2. Blasts are cyCD3~ 3. Blasts are cylg|x~ and do not coexpress CD22 plus cyCD79a 4. Blasts express one or more lymphoid-associated antigens: CD2, CD5, CD7, CD19, CD22, CD56, or cyCD79a

T-lineageMy+ALLa

Biphenotypic acute leukemia 1. Myeloid/B-lineage biphenotypic acute leukemia: blasts coexpress MPOb and CD22 plus CD19 or cyCD79a 2. Myeloid/T-lineage biphenotypic acute leukemia: blasts coexpress MPOb plus cyCD3 3. Mixed B-/T-lineage acute leukemia: blasts coexpress cyCD3 and cylg|x or cyCD3 and CD22 plus cyCD79a

1. Blasts are CD7+ and cyCD3+ 2. Blasts are CD22~ 3. Blasts are MPCr b 4. Blasts express one or more myeloid-associated antigens: CD13, CD15, CD33, or CD65

Abbreviations: SJCRH, St. Jude Children's Research Hospital; My + ALL, acute lymphoblastic leukemia expressing myeloid-associated antigens; Ly+ AML, acute myeloid leukemia expressing lymphoid-associated antigens; MPO, myeloperoxidase; NSE, nonspecific esterase; cy, cytoplasmic antigen expression. a All four criteria must be fulfilled. b Confirmed by cytochemical, anti-MPO, or ultrastructural study.

and prognostic significance. This unsettled picture results from inconsistencies among investigations, including the patient population studied (pediatric, adult or a mixture of both), different laboratory methodologies, stringency of the immunologic criteria for defining commitment to lymphoid or myeloid differentiation, and treatment approaches.404,405 Chief among these appears to be the immunophenotyping criteria for defining commitment to the lymphoid or myeloid lineage. For example, definitions vary depending on the immunologic methods employed: single or multiparameter flow cytometry; fluorescence microscopy or immunohistochemistry; the number and type of monoclonal antibodies used; inclusion of antigens that are not lineage-restricted (e.g., CD4, CD11b, CD15, CD10, or TDT); source and condition of the leukemic samples (e.g. marrow or blood; fresh, old or cryopreserved cells); and the criteria for positive or negative antigen expression. The cytochemical and immunophenotyping criteria used at St. Jude Children's Research Hospital to define My+ ALL, Ly+ AML, and biphenotypic or "true mixed" leukemia are presented in Table 7.12.406 Leukemic blasts are identified by multiparameter flow cytometry with initial identification of the leukemic blasts by CD45 expression versus light side scatter intensity. If the leukemic cells overlap with normal lymphoid and granulocytic elements, the nonneoplastic cells are excluded from the analysis by Boolean gating techniques. The central feature of this classification

is the identification of antigens that substantiate lymphoid and myeloid lineage commitment. B-lineage ALL is diagnosed when leukemic blasts express CD19 plus CD22 or CD19 plus cytoplasmic CD79a or immunoglobulin, and no cytoplasmic CD3 or MPO. The leukemic cells of T-ALL express CD7 plus either surface or cytoplasmic CD3 but no MPO and no surface CD 19 plus CD22 or CD 19 plus cytoplasmic CD79a. AML is diagnosed when leukemic blasts express MPO by cytochemical or immunologic methods or in its absence, two or more myeloid-associated antigens, including CD13, CD15, CD33, CD64, or CD65 but not cytoplasmic CD3, TCR(J, immunoglobulin, or CD 19 plus CD22 or CD 19 plus cytoplasmic CD79a. Thus, My+ ALLs express the antigenic profile defined for B- or T-lineage ALL plus one or more myeloid-associated antigens, such as CD13, CD15, CD33, CD64, or CD65 but not MPO (Fig. 7.16). Ly+ AMLs display the antigenic profile described above for AML plus one or more lymphoid-associated antigens but not cytoplasmic CD3, TRC(J, or simultaneous expression of surfaceCD19, CD22, and cytoplasmic CD79a (Fig. 7.17). Examples of immunophenotypic profiles for My+ ALL and Ly+ AML are presented in Table 7.2. Large studies of childhood My+ ALL using multivariate analysis show that myeloid-associated antigen expression does not have independent prognostic significance.407"412 Other studies have failed to consider the impact of genetic abnormalities on clinical outcome inMy+ cases.413,414 For example, atypical expression of the

Immunophenotyping

10 1

10 2

10 3

CD19

101

10 2

10 3

10 4

Fig. 7.16 Caricature and flow cytometric analysis of myeloid antigen-positive acute lymphoblastic leukemia (My+ ALL). (A) Depiction of a leukemic lymphoblast expressing lymphoid (CD19) and myeloid (CD13 and CD33)-associated antigens. (B) Flow cytometric study of marrow cells. CD45 versus light side scatter (SS) with the leukemic cells enclosed in a rectangle. (C) Blasts identified in panel B express lymphoid-associated CD19 and myeloid-associated CD13. (D) Atypical expression of CD33 by CD19-positive blasts identified in panel B. Flow cytometric studies were from a patient with early pre-B ALL with a t(9;22) (q34;q11) chromosomal abnormality. Flow cytometric studies were performed on whole bone marrow sample using a red blood cell lysis technique.

myeloid-associated antigen CD15 is common in B-lineage ALL with the t(4;11), a translocation that confers a poor outcome in infants and older children independently of immunophenotype. 154,155 , 415 By contrast, the blast cells of patients with B-lineage ALL with the t(12;21)(p12;q21) usually express CD 13 or CD33 but have a favorable outcome. 173 " 176 The clinical importance of My+ALL in adults is still unclear. 416"419 Most studies of pediatric and adult Ly+ AML have found no significant effect of lymphoid antigen expression on clinical outcome except for CD7+ AML. 144,279,410,420-422 Similar to findings in B-lineage ALL, the aberrant lymphoid antigen expression is largely associated with certain chromosomal abnormalities. For example, clinically favorable cases of AMLwith the t(8;21)(q22;q22) andinv(16)(p13q22) almost always express the lymphoid-associated antigens CD 19 and CD2, respectively; whereas CD7 is associated

with MDS-related and secondary AMLs that frequently have abnormalities of chromosome 7. 267,272,308,309 An immunophenotypic diagnosis of biphenotypic leukemia is considered when the leukemic blasts express MPO plus CD3, MPO plus immunoglobulin, or MPO plus surface CD19 and CD22 or CD19 and cytoplasmic CD79a (Table 7.12). By SJCRH criteria, the blasts are identified by CD45 versus light side scatter flow cytometric parameters. A marker is considered positive when 10% or more of the blasts react with antibodies to that marker with a definite intensity shift greater than a corresponding negative control. The European Group for the Immunological Characterization of Leukemia (EGIL) proposed a scoring system for defining biphenotypic leukemias, in which points are assigned to a lymphoid or myeloid antigen based on arbitrarily determined degrees of lineage specificity (Table 7.13) .149 Biphenotypic leukemia is diagnosed when

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10 2

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104

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104

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CD34 Fig. 7.17 Caricature and flow cytometric analysis of lymphoid antigen-positive acute lymphoblastic leukemia (Ly +AML). (A) Depiction of a leukemic myeloblast containing an Auer rod and expressing myeloid (CD13 and CD33) and lymphoid (CD19 and CD56)-associated antigens. (B) Flow cytometric study of leukemic bone marrow specimen. CD45 expression versus intensity of light side scatter (SS). Leukemic cells circled. CD45 and light SS features are suggestive of a leukemic process with partial myeloid differentiation. (C) Leukemic cells identified in panel B express myeloid- and lymphoid-associated antigens CD33 and CD19, respectively. (D) Leukemic CD34-postive blasts identified in panel B also express NK-associated CD56. Flow cytometric studies are from a M2 AML case with a t(8;21) (q22;q22) chromosomal abnormality. Flow cytometric studies were performed on whole bone marrow sample using a red blood cell lysis technique.

scores exceed 2 for the myeloid lineage plus over 2 for either the B or T lineage. EGIL guidelines for identifying the leukemic blast population are based on light scatter properties alone, and a marker is considered positive when 20% or more of the blasts react with antibodies to that marker. Thus, investigations of acute biphenotypic leukemia using SJCRH and EGIL criteria will result in different interpretations depending on the choice of antibodies and their conjugated fluorochromes, as well as the flow cytometric techniques used to identify leukemic blasts and interpretations of a "positive" marker. Multiparameter flow cytometric analyses reveal that many acute leukemias do not consist of a uniform population of leukemic cells "frozen" in one stage of maturation.

In fact, such studies clearly demonstrate that some cases of suspected biphenotypic leukemia may consist of two or more leukemic blastpopulations that, inpart, share antigen expressions but with one population having a committed lymphoid or myeloid phenotype and the other showing a biphenotypic phenotype. Sometimes, only a minor number of leukemic blasts may have a biphenotypic phenotype, with the greater proportion of blasts demonstrating strong commitment to a single lineage (Fig. 7.18). The treatment dilemma created by these observations is obvious. Thus, while pediatric and adult patients with biphenotypic leukemias appear to have a poor clinical outcome, it will be important to confirm this association with standardized immunophenotyping criteria and strict attention to the contribution of genetic abnormalities. Gene expression

Immunophenotyping

Table 7.13 EGIL scoring system for identifying acute biphenotypic leukemiasa

B-lineage

T-lineage

Myeloid

Points

CD79a cylg|x cy/s CD22

cy/sCD3 TCRa(3 TCR-yS

MPO

Each positive marker = 2 points

CD19 CD10 CD20

CD2 CD5 CD8 CD 10

CD13 CD33 CD65s CD117

Each positive marker = 1 point

TDT CD7 CD1a

CD14 CD15 CD64

Each positive marker = 0.5 point

TDT CD24

Abbreviations: EGIL, European Group for the Immunological Characterization of Leukemia; cy/s, cytoplasmic or surface; cylg|x, cytoplasmic immunoglobulin mu; TCR, T-cell receptor; MPO, myeloperoxidase; TDT, terminal deoxynucleotidyl transferase. a The designation of acute biphenotypic leukemia requires >2 points from the myeloid category and >2 points from the B-lineage or T-lineage category. A marker is considered positive when >20% of the blasts are positive. See Bene et al. 14a and Segeren etal.382

profiling may help in recognizing these unusual leukemias, but problems of standardization and definitive diagnostic criteria will likely hamper these studies initially.

Acute bilineal leukemia

Very rarely, one encounters an acute leukemia consisting of two or more blast populations that differ morphologically and/or immunophenotypically. 423"425 For example, one blast population may have a distinct lymphoid immunophenotype and the other a myeloid lineage phenotype (Fig. 7.19). Bilineal leukemia with both precursor-B and precursor-T lymphoblasts is exceedingly rare.425 The preferred term for these leukemias, which are exceptionally rare and comprise less than 1% of acute leukemias in children, is bilineal leukemia.2 Older reports of bilineal leukemia often are examples of biphenotypic leukemia or treatment-related lineage switches.426"429 Thus, as discussed earlier, it is important not to confuse bilineal leukemia with biphenotypic leukemia or with biclonal or oligoclonal leukemias. Whereas biclonal/oligoclonal leukemias represent a single blast population with two or more cytogenetic or rearranged T-/B-cell receptor clones, a bilineal leukemia may be either monoclonal or biclonal. Separation of bilineal leukemia from biphenotypic leukemia requires multiparameter flow cytometry

to analyze each blast population. Infrequently, a child in remission for ALL relapses with bilineal leukemia.423 Precursor-B ALL with t(9;22) or t(4;11) translocations can have a very small population of myeloid or monocytic blasts that would be missed without immunophenotyping studies.424,429 Although clonality studies of bilineal leukemias have not been reported, it is likely that these leukemias are derived from a single multipotent progenitor cell. Congenital leukemia not associated with Down syndrome

Acute leukemias discovered at birth or during the first month of life are defined as "congenital leukemias".430 Leukemia very early in life implies that the process begins in utero with an exposure to an oncogenic insult. 431,432 Indeed, recent investigations show that congenital leukemia and some leukemias developing later in children can be traced back to chromosomal rearrangements in utero.432"435 Acute leukemias in infants under 1 month of age are rare, occurring in 1 per 5 million births,436 with AML being more common than ALL in the newborn. Congenital leukemias are usually M5, M4, or M7 AML, occasionally precursor-B ALL and rarely precursor-T ALL.436"441 The majority of congenital ALLs and AMLs have chromosome 11q23 translocations including t(4;11)(q21;q23), t(9;11)(p21;q23), t(10;11)(p13;q23), or t(11;19)(q23;p 13) .430 ,431 ,436 ,442 ,443 A significant number of congenital M7 AML have the rare chromosomal translocation t(1;22)(p13;q13).346,347,444Theimmunophenotypicprofiles of congenital leukemias do not differ from those of older children with the same leukemia subtype and cytogenetic abnormality.439 However, infants with congenital leukemia have a poorer prognosis than children older than 1year

. 431,436

Leukemia and transient myeloproliferative disorder in Down syndrome

Children with Down syndrome have a 10- to 20-fold increased risk of developing acute leukemias by comparison with normal children.445 The frequency ofALL and AML is not increased among Down syndrome patients over 5 years of age, butyounger patients are at higher risk of developing AML. The proportion of pre-B ALL cases is significantly higher and T-cell ALL lower in these patients.446 The immunophenotypic profiles of B-lineage ALL and T-ALL, including atypical myeloid antigen expression, do not differ from those of patients without Down syndrome.446 The distribution of AML subtypes among Down patients over 5 years of age is similar to thatinother children, butyounger

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Fig. 7.18 Example of precursor-B ALL with a small population of biphenotypic (mixed B- and myeloid-lineage) leukemic blasts. (A) Bone marrow. A monomorphous population of leukemic blasts (three blasts shown) accounts for 95% of the marrow leukocytes. Normal lymphocytes and granulocytic elements (not shown) account for 5% of the marrow specimen. Flow cytometric studies of the bone marrow specimen shown in panel A are shown in panels B, C, andD. (B) Dot plot histogram of CD19 (Y-axis) versus a negative control (X-axis, mouse IgG1 FITC). CD19+ leukemic blasts account for 95% of the leukocytes. The arrow points to nonleukemic cells (lymphocytes and granulocytes) in the marrow specimen. (C) Dot plot histogram of CD22 (Y-axis) versus CD10 (X-axis). The leukemic blasts are CD22+ and only partially CD10+ (CD10+ blasts identified by the rectangle). (D) Dot plot histogram of CD19 (Y-axis) versus cytoplasmic myeloperoxidase (X-axis, MPO). Leukemic blasts coexpressing CD19 and MPO account for 18% of the leukemic blasts (the rectangle identifies the CD19+/MPO+ blasts). Arrow identifies a few MPO+ nonleukemic granulocytic elements in the bone marrow sample. A myeloperoxidase cytochemical study of the same bone marrow sample showed weak to moderately strong positivity in 5% of the leukemic blasts. Additional flow cytometric studies showed: strong expression of CD22, CD34, and cytoplasmic CD79a; and weak to partial expression of myeloid-associated CD13 and CD33 antigens by all leukemic blasts (studies not shown). Cytogenetic studies detected only one clone of leukemic cells.

children have a markedly increased incidence of M7 AML and a higher frequency of erythroleukemia and acute erythroblastic leukemia. 445-447"452 The immunophenotypic profiles of the myelocytic and monocytic subtypes of AML in Down syndrome do not differ from those of other children. 448 As in cases of childhood M7 AML, the megakaryoblasts commonly express CD33, CD36, CD41a, and CD61, with variable expression of CD2, CD4, CD7, CD 11b, CD 13, CD34, CD42b, CD56, CD 117, CD235a, and HLA-DR.448,449,452,453 Down syndrome patients with M7 AML may have a higher incidence of blast cell CD7 expression.448,453,454

Neonates with Down syndrome and phenotypically normal infants with constitutional or acquired chromosome 21 or i(21q) mosaicism may present with transient but marked leukocytosis, erythrocytosis, leukoerythroblastosis, or thrombocytosis. Approximately 10% of Down infants present with a marked proliferation of blast cells that is indistinguishable from acute leukemia by light microscopy. 445'451'455"458 These processes are referred to as transient myeloproliferative disorder (TMD) or transient leukemia of Down syndrome. 459 The prenatal diagnosis of TMD can be made by flow cytometric analysis of fetal blood.460 Hepatomegaly and splenomegaly are common

Immunophenotyping

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•

D (R1) •• *** *•

\

CM

103

CD45

^ -=

C(R2)

Q

102

CD64

i i i

II

i

i

i

i i i

II

i

W: i

101

T i r i M M

103

i

i

111M

i

104

CD22

Fig. 7.19 Example of bilineal acute leukemia. These rare leukemias comprise a mixed population of neoplastic lymphoblasts and myeloblasts. (A) Bone marrow. The leukemic process consists of small and large leukemic blasts. (B) Flow cytometric study of bone marrow cells shown in panel A. Dot plot histogram of CD45 expression versus light side scatter (SS) intensity. Two leukemic cell populations are indicated by R1 (CD45-negative/low light SS intensity) and R2 (CD45-positive/higher light SS intensity). (C) Blasts in the R2 region express CD34 and CD64. These blasts also expressed CD4, CD11b, and CD14; but not CD19, CD22, or CD79a (not shown). (D) Blasts in the R1 region express CD34 and CD22. These blasts also expressed CD19, CD24, and CD79a; but not CD4, CD 11, or CD64 (not shown). Flow cytometric studies were performed on whole bone marrow sample using a red blood cell lysis technique.

in these infants, but otherwise they appear healthy. The leukocyte count may be as high as 300 x 10 9/L.448,460,461 The percentage of circulating blasts may be 50% or greater, and erythroblasts and immature myeloid cells may accompany the blasts. Blast cell numbers in the marrow may be lower, the same, or higher than in the blood. The blast cells of TMD usually do not react with cytochemical stains for MPO and ANB esterase but frequently produce a granular reactivity pattern for ANA esterase that is NaF-resistant. 445 Ultrastructural and immunologic studies commonly show evidence of a large megakaryocytic component and, less commonly, erythroid, basophilic, or rarely T-cell or early NK-cell-like differentiation. 445,451,441,462-467 Unlike M7 AML, marrow fibrosis is uncommon and hemoglobin and platelet numbers are often normal in TMD.

Although most infants with TMD have a benign clinical course, acute megakaryocytic leukemia develops in the first 12 months of life in 20% of cases. 459 Interestingly, TMD does not appear to predispose to subsequent development of ALL or AML other than the M7 subtype. Recent studies show that AML M7 in Down infants arises from the cells of TMD.459,462 ,463 A mutation in the GATA1 gene has been described in Down patients with M7 AML.468 The GATA1 transcription factor is postulated to be a negative regulator of megakaryocytic precursor proliferation. A mutation in GATA1 may prevent differentiation and lead to proliferation of megakaryocytic precursors. 469 , 470 More recently, GATA1 mutations detected in TMD were shown to be identical to those observedinDownpatientswithM7AML. 47 °- 473 Thus, the TMD cellmay be a "premalignant" process requiring the acquisition of additional genetic mutations for conversion

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Fred G. Behm

to M7 AML. Indeed, chromosomal abnormalities other than the constitutional trisomy 21 are rarely detected in TMD, even though other studies show that the blast proliferation is clonal.474"478 By contrast, chromosomal abnormalities in addition to trisomy 21, p53 mutations, and increased telomerase activity are common in Down infants with M7 AML.479,480 Unfortunately, the antigenic expression profile of megakaryoblastic proliferations of TMD is indistinguishable from that of M7 AML.448,449 Gene profiling studies may reveal upregulated gene products in M7 AMLin Down infants that willbe amenable to flowcytometric analysis that rapidly differentiate TMD from M7 AML. Chronic myelogenous leukemia and blast transformation

Chronic myelogenous leukemia (CML) is a myeloproliferative disease that initially presents as a chronic proliferation of neutrophils and their precursors.2 An absolute basophilia and eosinophilia are common in CML. All cases have a t(9;22)(q34;q21) chromosomal abnormality resulting in a BCL-ABL fusion gene. CMLisbi- ortriphasicwithan initial indolent or chronic phase followed byablastphaseor an accelerated phase between the chronic and blast phase. Very rarely, the pediatric patient may present in the blast phase. In the chronic phase, blasts account for less than 2% of the peripheral blood leukocytes and less than 5% of the marrow cells. Cytogenetic, isoenzyme, and molecular studies confirm that myeloid, monocytic, eosinophilic, basophilic, erythroid, megakaryocytic, and B-cell lineages are involved, supporting a multipotent stem cell of origin. Combined flow cytometric and BCR-ABL in situ hybridization studies have not found evidence of mature T- or NKcell involvement by the BCL-ABL translocation, although studies of marrow blasts demonstrate a BCR-ABL fusion gene in a CD34+, CD7+, CD5+ cell population that may be the equivalents of prothymic T-cells.481"483 Immunophenotyping studies in the chronic phase of the disease have not been clinically useful. The neutrophils have decreased or absent levels of alkaline phosphatase as detected by cytochemical studies or by flow cytometry with antialkalinephosphatase antibodies.484 Other studies show altered expression of FeyRII (CD32) and FeyRIII (CD 16) receptors on neutrophils.485,486 The phagocytic activity of chronic phase neutrophils is abnormal and can be assessed by flow cytometry.487 Immunophenotyping of the blasts in the chronic phase show the existence of a heterogeneous population of myeloid and lymphoid progenitor cells containing the BCR-ABL fusion gene.483,488 Attempts to detect the BCL-ABL fusion protein with antibodies have been unsuccessful.

The chronic phase of CML is invariably interrupted by an accelerated phase or transformation to a blast phase or blast crisis. The accelerated phase is characterized by one or more of the following: 10% to19% blasts in blood or the marrow, blood basophilia of 20% or more, persistent thrombocytopenia unresponsive to treatment, increasing spleen or WBC count unresponsive to treatment, or cytogenetic evidence of clonal evolution.2 The accelerated phase will progress to ablast phase if not effectively treated. More commonly, the blast phase emerges quickly from the chronic phase. The blast phase resembles acute leukemia with blasts accounting for 20% or more of blood or marrow nucleated cells.2 The blast phase may also be heralded by an extramedullary proliferation of blasts or a granulocytic sarcoma. The leukemic blasts may be of myeloid, monocytic, megakaryocytic, erythroid, lymphoid or a mixture of any thereof. The survival of patients following treatment of a lymphoid blast phase is better than that associated with other types of blast transformation, emphasizing the importance of determining the lineage of the blast phase.489 Although a myeloid blast phase can sometimes be determined by morphologic and cytochemical studies, more often immunophenotyping studies are required to determine the involved cell lineages. A myeloid immunophenotype (CD79a-, CD3", CD41", CD13+, CD15+'", CD33+, CD34+, CD117+'", MPO+'", TDT-) is present in 50% to 60% of childhood cases. Another 30% to 40% of pediatric patients develop a lymphoid blast crisis with an early pre-B(TDT+,CD19+,CD10+/-,CD34+,CD79a+,sIg-/cIg-) orless commonly apre-B(cIg|ji,+)immunophenotype. Blast transformations are less commonly megakaryoblastic or erythroblastic, very rarely T-lymphoblastic, and sometimes a mixture of two or more cell lineages.490"496 Similar to findings in My+ALL and Ly+AML, many cases have blasts that aberrantly co-express myeloid- or lymphoid-associated antigens.493-495

Lymphomas other than lymphoblastic and Burkitt types presenting as leukemia

Chronic lymphoid leukemias and lymphomas other than lymphoblastic and Burkitt types that present with a leukemic component are distinctly uncommon in children and adolescents. For the experienced morphologist, the morphologic features of these malignancies are usually sufficient to suggest the appropriate diagnosis. However, even for the experienced morphologist, immunophenotyping studies are required for confirming or assisting in the diagnostic work-up. The following sections present several of the lymphoid malignancies that

Immunophenotyping

Table 7.14 Immunophenotype profiles of leukemic T-cell processes in children

Marker

PTALL

ATLL

T-LCLL

NK-LGLL

HSL

ALCL

TDT CD34 CD10 CD3 CD4

+(-) +/+/+a +/-

+ +

+ +

-

+ -

CD5

+(-)

+

+

-

-

(+)

CD7 CD8 CD16 CD25 CD30 CD56 CD57 ALK CTA

+ +/ +/ -b -

-b + +/ -

+ +/+/+/+/-

+ +/+/+/+/-

+ -

-(+) (+)

+/-

+ + +/-

-

+

+

+

+ +/-

+/+/-

Abbreviations and symbols: PT ALL, precursor-T acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; ATLL, adult T-cell lymphoma/leukemia; T-LGLL, T-cell large granular lymphocyte leukemia; NK-LGLL, NK-cell large granular lymphocyte leukemia; HSL, hepatosplenic T-cell lymphoma (leukemic phase); ALCL, anaplastic large cell lymphoma (leukemic phase); TDT, terminal deoxynucleotidyl transferase; ALK, anaplastic lymphoma kinase; CTA, cytotoxic antigens (e.g., TIA-1, perforin, granzyme B); +, positive; —, negative; +/—, positive or negative; —(+), usually negative; +(—), usually positive. a Cytoplasmic or surface CD3. b Some cases positive.

can mimic in part the clinical presentation and morphologic features of acute leukemia. These include HTLV1-associated leukemia/lymphoma, large granular lymphocyte leukemia, hepatosplenic T-cell lymphoma, and anaplastic large cell lymphoma. Fortunately, immunophenotype profiles readily distinguish these malignancies from the acute leukemias. HTLV-1-associated leukemia/lymphoma (adult T-cell leukemia/lymphoma) A leukemia/lymphoma process with unique clinical, morphologic, and immunophenotypic features was first recognized in adult patients in southwestern Japan who were infected with a human retrovirus. At a joint Japanese and American conference, two terms - "adult T-cell leukemia/lymphoma (ATLL)" and "human Tlymphotropic virus type 1" (HTLV-1) - were proposed for the disease process and the etiologic retroviral agent, respectively.497 Subsequently, ATLL was recognized in

other HTLV-1 endemic areas, parts of central Africa, the Caribbean basin, and parts of South America. The few recognized sporadic cases of pediatric ATLL in the United States come from regions receiving immigrants from endemic areas of the world, including the southeastern states and Hawaii. In children and adolescents, HTLV-1 is acquired through blood product transfusions, the maternal-fetal route, breast feeding, and sexual activity. HTLV-1 infection is also associated with several nonmalignant disorders including tropical spastic paraparesis, infective dermatitis, and uveitis.498 ATLL occurs in only 2% to 4% of HTLV-1-infected adults, usually after a long latency period of 20 to 30 years.499 The incidence of ATLL in HTLV-1-infected children is probably considerably less since long latency results in malignancy in adulthood. The clinical presentation of ATL is diverse and can be categorized as acute, chronic, smoldering, and lymphomatous.500,501 In adults, the disease usually presents with evidence of systemic disease with evidence of lymph node, spleen, liver, skin, lung, gastrointestinal, and/or central nervous system involvement. Marrow involvement is often sparse or patchy. As in adults, the pediatric presentation of ATLL can feature peripheral lymphadenopathy hepatosplenomegaly involvement of skin or central nervous system, elevated serum LDH, and sometimes hypercalcemia.502"509 Pediatric patients may present with a mediastinal mass, an unusual manifestation of endemic cases. Unlike most other childhood leukemias, the bone marrow initially shows little involvement even in the presence of high peripheral blood counts. The leukemic phase consists of abnormal lymphoid cells with characteristic prominent nuclear lobulation (flower-like cells) or cerebriform nuclei resembling Sezary cells, condensed chromatin, and absent to prominent nucleoli. ATLL arises from the CD4 subset of peripheral T-cells. Thus, the immunophenotype resembles that of activated T-helper cells with expression of CD2, CD3, CD4, and CD5 (Table 7.14). Rarely, leukemic cells express CD8 instead of CD4. Unlike T-cell ALL, the leukemic cells are TDT, characteristically lack CD7, and usually express the a chain of the interleukin-2 receptor (CD25). The expression of CD30 in some cases may result in confusion with anaplastic large cell lymphoma; however, the neoplastic cells are negative for ALK, TIA-1, and granzyme B.507,510 Morphologic and immunophenotype features are pathognomic of ATL, but the diagnosis requires conformation by serologic evidence of HTLV-1 or demonstration of viral antigens or DNA in malignant cells by molecular techniques. Adult patients receiving chemotherapy alone fare poorly, but newer treatment approaches have improved survival.499,507

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Fred G. Behm

Large granular lymphocyte leukemia The peripheral blood of children and adults normally contains a small population of relatively large lymphoid cells that possess a small number of cytoplasmic azurophilic granules. These large granular lymphocytes (LGLs) comprise 5% to 15% of peripheral blood leukocytes, either T or NK cells. T-cell LGLs express the CD3/TCR complex. NKcell LGLs are natural killer cells that lack the CD3 /TCR complex. Both types of LGLs express receptors for MHC class I glycoproteins, and can mediate cytolysis of virus-infected tissues or foreign tissues (e.g., cancer cells and organ transplants) . The cytotoxicity of T-cell LGLs is activated by binding to unfamiliar peptides embedded in class I molecules on target tissues. NK-cell LGLs recognize the absence of class I glycoproteins, a common result of viral infection and malignant transformation. Increased numbers of LGLs are associated with viral infections (including cytomegalovirus, HIV, and EpsteinBarr), autoimmune disorders (such as Crohn disease and idiopathic thrombocytopenic purpura), lymphomas, and solid tumors. A syndrome of chronic neutropenia with increased numbers of LGLs was first recognized in 1977,511 and subsequent studies documented infiltrations of LGLs in lymph nodes, bone marrow, spleen, and liver.512 Further investigations revealed clonal LGL proliferations, analogous to their normal CD3+ or CD3" counterparts, in TLGL or NK-LGL leukemia, respectively.512"516 Clonal LGL proliferations have been called T-CLL, Ty lymphoproliferative disorder, and lymphoproliferative disorder of granular lymphocytes, but the prevailing term is "LGL leukemia" (LGLL).2,517 T-LGLL is primarily a chronic disease process of adults, who often present with bacterial infections due to neutropenia. Some patients have Felty syndrome, a triad of rheumatoid arthritis, neutropenia, and splenomegaly. Approximately 15% of LGLL cases have an NK-cell phenotype. 517 Most patients with NK-LGLL do not present with evidence of infection or clinical features of rheumatoid arthritis, and their disease usually follows an accelerated clinical course. 518"521 The WHO classification calls this malignant process "aggressive NK-cell leukemia." 2 Patients are younger and the WBC counts are higher than with T-LGLL. The few detailed descriptions of LGLL in children include both T and NK types with clinical and laboratory features that closely resemble the adult counterparts. 522 " 524 The feature that distinguishes between T- and NK-LGLL is the expression of CD3 and TCRapJ or less frequently TCR-/, and clonal TCR gene rearrangements in the T subtype (Table 7.14).505,506 C e l l s o f T-L GLL have a post-

thymic or mature immunophenotype. Most cases are CD3+, TCRa(J+ (or infrequently TCR78+), CD4", CD7+, CD8+, and variably express CD 11b, CD56, and CD57. Rarely, CD4 with or without CD8 may be present. NK-LGLL cells are usually CD7+, CD2+, CD3", CD11b+, CD16+ or ", CD56+, and usually CD57". Expression of CD8 is characteristic of T-LGLL but is also found in NK-LGLL. Detection of perforin and TIA-1, proteins contained in cytoplasmic azurophilic granules, aids in the diagnosis of both types of LGLL. Some cases of precursor-T ALL express CD56 or CD57, but the expression of TDT, CD1a, CD34, or coexpression of CD4 and CD8 distinguishes them from T-LGLL. Aberrant NK antigen expression and clonal TCR rearrangements distinguish malignant from florid reactive LGL proliferations. Some cases of LGLL do not have detectable CD56, CD57, or CD16, while one or more of these antigens are always expressed by normal LGL cells.513 All cases of T- and NK-LGLL frequently express, in aberrant fashion, other NK antigens including CD158a, CD158b, CD94, and CD161.525 The clonality of T-LGL leukemias can be assessed by molecular or flow cytometric analysis of the TCR. Recent advances in analysis of the TCR-VpJ repertoire by flow cytometry provides a rapid means of establishing clonality in over 60% of T-LGL leukemia. 526,527 Hepatosplenic T-cell lymphoma Hepatosplenic T-cell lymphoma is a rare and aggressive extranodal malignancy with a peak incidence in adolescents and young adults. 2 , 528 Reports of pediatric cases are limited to case discussions or are included in reports of adult investigations. 528"538 Males are more frequently afflicted than females, although a female predisposition is reported for hepatosplenic lymphomas with a TCRapS phenotype. 531 , 532 Patients typically present with enlarged spleens and livers with no or little lymphadenopathy. Thrombocytopenia is common as is anemia. Circulating lymphoma cells are commonly present at diagnosis and often mistaken for atypical lymphocytes or lymphoblasts. An overt leukemic phase may develop as the disease progresses.531,532 In children, this malignancy may be confused with precursor-T ALL, precursor-T lymphoblastic lymphoma, or peripheral T-cell lymphomas. The neoplastic cells are medium-sized with scant-to-moderate amounts of cytoplasm (Fig. 7.20). Azurophilic cytoplasmic granules have been described in a minority of cases. The nuclei may be round or convoluted with condensed chromatin and inconspicuous nucleoli. Mitotic cells are infrequent. As the lymphoma progresses, a blast transformation may occur with the neoplastic cells developing prominent

Immunophenotyping

B

o

o

=

co O

-= =

O

=

O

=

D

•

WM * :•.* 1111MIN

101

102

11111

iii

103

V51 Fig. 7.20 Example of leukemic phase of hepatosplenic T-cell lymphoma mimicking acute leukemia. (A) Photomicrograph of four large bone marrow blast forms with indistinct nucleoli. (B) Flow cytometric study of bone marrow cells represented in panel A. Dot plot histogram of CD45 expression versus light side scatter (SS) intensity. Leukemic cells (open arrows) overlap the regions normally occupied by nonmalignant immature and mature lymphocytes. (C) Leukemic cells (open arrow) identified in panel B express CD3 but not CD5. The few CD3+, CD5+ cells (closed arrow) represent non-neoplastic T cells. The leukemic cells also expressed CD2, CD7, CD8, and TCRa(3 (not shown). (D) The leukemic cells (open arrow) show restricted V s l TCR expression. Cytogenetic studies of leukemic cells showed trisomy 8 and isochromosome 7q chromosome abnormalities characteristic of hepatosplenic T-cell lymphoma. Flow cytometric studies were performed on whole bone marrow sample enriched for mononuclear cells by a density-gradient technique.

nucleoli. 534,539,540 Early in the disease, the malignant cells maybe difficult to appreciate inbone marrow aspirates, but with progressive disease the marrow may resemble acute leukemia. Bone marrow needle biopsies usually show characteristic sinusoidal infiltrates of the malignant lymphocytes.532, 533,540 The spleen and liver involvement also show extensive sinusoidal infiltrates. 532,533 Rarely, hepatosplenic lymphoma can mimic a hemophagocytic syndrome. 536 The lymphoma is usually derived from TRC78 and less commonly from TCRapJ T-cells.2-528"533 Flow cytometry is required to differentiate between TCR78 and TCRa(J lymphoma subtypes. TCRapJ hepatosplenic lymphomas are morphologically, cytogenetically, and clinically similar to the TCR78 forms.532TCRa(J and 78 subtypes have similar

and a fairly consistent immunophenotypic expression pattern of CD2+, CD3+, CD4", CD8+'", CD5", CD7+, CD16+, and CD56+/~ (Table 7.14).2,531,532 C D 5 7 m a y b e m o r e frequently expressed by the a(J than 78 subtype. 532 Cytotoxic granular protein TIA-1 is detected in most cases, whereas granzyme B or perforin is detected in only a minority of cases. 531,532,541-543 The Vsl locus is utilized by the TCR of the majority of 78 cytotoxic T-cells in the sinuses of the normal spleen. This splenic cell may be the origin of 78 hepatosplenic T-cell lymphoma since the majority of hepatosplenic lymphomas also preferentially express V81 (Fig. 7.20).544,545 The EBV genome may be present in the neoplastic cells of some cases but there does not appear to be any causative relationship. 532,545 Isochrome

189

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Fred G. Behm

B

102

CD45

104

Fig. 7.21 Leukemic presentation of anaplastic large cell lymphoma. (A) Peripheral blood showing four lymphoma cells resembling leukemic blasts around a normal lymphocyte (closed arrow). (B) Flow cytometric study of peripheral blood cells. CD45 expression versus light side scatter (SS) intensity showing the leukemic cells (open arrow) in the region normally occupied by lymphocytes. (C) Leukemic cells identified in panel B express T-cell-associated CD7 but not surface CD3. The leukemic cells also expressed CD4, CD11b, CD13, and HLA-DR; but not CD1, CD2, CD5, CD8, CD34; or myeloperoxidase (not shown). (D) Flow cytometric study of intracellular antigens shows weak expression of CD3 but not terminal deoxynucleotidyl transferase (TDT) by the leukemic cells. Cytogenetic studies of the leukemic process revealed a t(2;5)(p23;q35) chromosomal abnormality characteristic of anaplastic large cell lymphoma. Flow cytometric studies were performed on a peripheral blood sample enriched for mononuclear cells by a density-gradient technique.

7q, often with trisomy 8 and other random chromosomal abnormalities, was identified in the majority of reported cases. 532 , 539 , 540 , 546 - 548 Similar to adults, the clinical course of this disease in children is aggressive. Initial responses to chemotherapy are followed shortly by relapse and death in most patients. Leukemic phase of anaplastic large cell lymphoma Leukemic presentation of large cell lymphoma is uncommon in children. Anaplastic large cell lymphoma (ALCL) represents 40% to 60% of the large cell lymphomas in children.549 Its cells characteristicallyshowaT-cellorrarely a B-cell lineage origin, a broad spectrum of histologic features, and expression of CD30 (Ki-1/BerH2) and anaplastic

lymphoma kinase (ALK).2,550 The hallmark of these lymphomas are large, pleomorphic or anaplastic tumor cells with variable numbers of smaller lymphoma and reactive inflammatory cells. In 70% of cases, ALK expression is the result of a t(2;5)(p23;q35) chromosomal translocation that juxtaposes the ALKlocus at 2p23 to the NPM (nucleophosmin) locusat5q35. 551 In the remaining cases a translocation of ALK involves other partner genes. Consequently, ALK is aberrantly expressed in the nucleus and/or cytoplasm of the lymphoma cells.550,551 Clinically, patients with ALCL commonly present with extranodal disease, usually involving skin, bone, soft tissues or lung. Leukemic presentation of ALCL is uncommon, 552 " 558 although the presence of lymphoma

Immunophenotyping

cells in the blood is well documented.559 562 Only one child in 400 cases of non-Hodgkin lymphoma at SJCRH presented with a leukemic phase, another child had a leukemic relapse.558 Although the leukemic presentation has characteristic morphologic and immunophenotype features, it can initially be mistaken for T-ALL. In a recent review of 12 pediatric and adult cases, the peripheral leukocyte count ranged between 15 and 151 x 109/L.558 Leukemic cells in most cases were small-to-medium-sized with indented or cerebriform-like nuclei (Fig. 7.21). Bone marrow involvement typically did not exceed 5%, and not unexpectedly a small cell variant of ALCL was identified. The immunophenotype of the leukemic cells resembled that of the typical lymph node- or tissue-based disease (Table 7.14). All cases showed evidence of T-cell differentiation. In contrast to precursor-T ALL, all cases tested expressed EMA (epithelial membrane antigen), most lacked CD5, many did not have detectable CD7, and all were negative for TDT and CD34. Similar to precursor-T ALL, CD4 and CD8 may be absent, coexpressed, or individually expressed in ALCL. Like the lymphoma tissue counterpart, one-third of the cases express myeloid-associated antigens including CD13 or lysozyme. Finally, in all cases studied, the lymphoma cells obtained from tissue biopsies expressed CD30 and ALK, but these antigens were frequently not detected in circulating lymphoma cells.

Conclusions and future directions

Advances in immunophenotyping have provided a tremendous amount of information about the extrinsic and intrinsic cellular properties of normal and leukemic cells. This information has proven extremely valuable in the rapid and highly accurate diagnosis of leukemia and the development of new classifications. Flow cytometric studies quickly and accurately differentiate acute leukemias from other malignant conditions that can mimic acute leukemia, such as the leukemic phase of hepatosplenic or large anaplastic cell lymphomas. However, areas of uncertainty remain. For example, consensus as to the diagnosis of biphenotypic leukemia and its clinical importance remains elusive. New information from gene expression profiling studies of normal hematopoietic and leukemic cells will provide new flow cytometry markers for managing patients with leukemia. These new markers will encompass costimulatory molecules, chemokine receptors, hematopoietic growthfactor receptors, and molecules involved in apop tosis, drug resistance, and cell adhesion. Assessment of these markers will lead to the development of a functionally and

more relevant classification of acute leukemia. Similarly, studies of these markers in leukemia will contribute to our understanding of normal hematopoietic differentiation and function.

REFERENCES

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cases of granular lymphocyte leukaemia. Br J Haematol, 2003; 120: 1026-36. Langerak, A. W., Beemd, R. van den, Wolvers-Tettero, I. L. M., etal. Molecular andflowcytometric analysis of the Vbetarepertoire for clonality assessment in mature TCRalpha/beta T-cell proliferations. Blood, 2001; 98: 165-73. Lima, M., Almeida, J., Santos, A. H., etal. Immunophenotypic analysis of the TCR-Vbeta repertoire in 98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular lymphocytes. AmJClinPathol, 2001; 159:1861-8. Weidmann, E. Hepatosplenic T cell lymphoma. A review on 45 cases since the first report describing the disease as a distinct lymphoma entity in 1990. Leukemia, 2000; 14: 991-7. Lai, R., Larratt, L. M., Etches, W., et al. Hepatosplenic T-cell lymphoma of alphabeta lineage in a 16-year-old boy presenting with hemolytic anemia and thrombocytopenia. AmJSurg Pathol, 2000; 24:45-63. Suarez, F., Wlodarska, I., Rigal-Huguet, F., etal. Hepatosplenic alphabeta T-cell lymphoma: an unusual case with clinical, histologic, and cytogenetic features of gammadelta hepatosplenic T-cell lymphoma. Am JSurgPathol., 2000; 24:102732. Cooke, C. B., Krenacs, L., Steltler-Stevenson, M., et al. Hepatosplenic T-cell lymphoma: a distinct clinicopathologic entity of cytotoxic gamma delta T-cell origin. Blood, 1996; 88: 4265-74. Macon, W. R., Levy, N. B., Kurtin, E J., etal. Hepatosplenic a(3 T-cell lymphomas. Am JSurgPathol, 2001; 25: 285-96. Farcet, J., Gaulard, P., Marolleau, J., etal. Hepatosplenic T-cell lymphoma: sinusal/sinusoidal localization of malignant cells expressing the T-cell receptor a(3. Blood, 1990; 75: 2213-19. Francosis, A., Lesesve, J.-F., Stamatoullas, A., et al. Hepatosplenic gamma/delta T-cell lymphoma: a report of two cases in immunocompromised patients associated with isochromosome 7q. Am JSurgPathol, 1997; 21: 781-90. Garcia-Sanchez, F., Menarguez, J., Cristobal, E., et al. Hepatosplenic gamma-delta T-cell malignant lymphoma: report of the first case in childhood, including molecular minimal residual disease follow-up. Br J Haematol, 1995; 90: 943^6. Nosari, A., Oreste, P. L., Biondi, A., et al. Hepato-splenic gammadelta T-cell lymphoma: a rare entity mimicking the hemophagocytic syndrome. Am JHematol, 1999; 60: 61-5. Coventry, S., Punnett, H. H., Tomczak, E. Z., et al. Consistency of isochromosome 7q and trisomy 8 in hepatosplenic gamma/delta T-cell lymphoma: detection by fluorescence in situ hybridization of a splenic touch-preparation from apediatric patient. Pediatr Dev Pathol, 1999; 2:478-83. Rossbach, H. C., Chamizo, W., Dumont, D. P., et al. Hepatosplenic gamma/delta T-cell lymphoma with isochromosome 7q, translocation t(7;21), and tetrasomy 8 in a 9-year-old girl. JPediatr Hematol Oncol., 2002; 24:154-7. Wang, C. C., Tien, H. F., Kin, M. T., et al. Consistent presence of isochromosome 7q in hepatosplenic T gamma/delta lymphoma: anewcytogenetic-clinicopathologic entity. Genes Chromosomes Cancer, 1995; 12: 161-4.

Immunophenotyping

540 Vega, F., Medeiros, L. J., Bueso-Ramos, C., etal. Hepatosplenic gamma/delta T-cell lymphoma in bone marrow. A sinusoidal neoplasm with blastic cytologic features. Am J Clin Pathol, 2001; 116:410-9. 541 Salhany, K. E., Feldman, M., Kahn, M. J., etal. Hepatosplenic gamma/ delta T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation. Hum Pathol, 1997; 28: 674-85. 542 Felger, R. E., Macon, W. R., Kinney, M. C., etal. TIA-1 expression in lymphoid neoplasms. Identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation. Am J Pathol, 1997; 150: 1893-1900. 543 Boulland, M. L., Kanavaros, P., Wechsler, J., etal. Cytotoxic protein expression in natural killer cell lymphomas and in alpha beta and gamma delta peripheral T-cell lymphomas. J Pathol, 1997; 183:432-9. 544 Przybylski, G. K., Wu, H., Macon, W. R., et al. Hepatosplenic and subcutaneous panniculitis-like gamma/delta T cell lymphomas are derived from different Vdelta subsets of gamma/delta T lymphocytes. J Mol Diagn, 2000; 2: 1119. 545 Weidmann, E., Hinz, T., Klein, S., et al. Cytotoxic hepatosplenic gammadelta T-cell lymphoma following acute myeloid leukemia bearing two distinct gamma chains of the T-cell receptor. Biologic and clinical features. Haematologica, 2000; 85:1024-31. 546 Ohshima, K., Haraoka, S., Harada, N., et al. Hepatosplenic gammadelta T-cell lymphoma: relation to Epstein-Barr virus and activated cytotoxic molecules. Histopathology, 2000; 36: 127-35. 547 Joneaux, P., Daniel, M. T., Martel, V., et al. Isochromosome 7q and trisomy 8 are consistent primary, non-random chromosomal abnormalities associated with hepatosplenic T gamma/delta lymphoma. Leukemia, 1996; 10: 1453-55. 548 Wlodarska, I., Martin-Garcia, N., Achten, R., et al. Fluorescence in situ hybridization study of chromosome 7 aberrations in hepatosplenic T-cell lymphoma: isochromosome 7q as a common abnormality accumulating in forms with features of cytologic progression. Genes Chromosomes Cancer, 2002; 33: 243-51. 549 Sandlund, J. T. & Behm, F .G. Pediatric non-Hodgkin lymphoma. In J. P. Greer, J. Foerster, J. N. Lukens, et al., eds., Wintrobe's Clinical Hematology, 11th edn. (Philadelphia, PA: Lippincott Williams &Wilkins, 2003).

550 Stein, H., Foss, H.-D., Durkop, H., etal. CD30+ anaplasticlarge cell lymphoma: a review of its histopathologic, genetic and clinical features. Blood, 2000; 96: 3681-95. 551 Morris, S. W., Kirstein, M. N., Valentine, M. B., et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science, 1994; 263:1281-4. 552 Kinney, M. C., Collins, R. D., Greer, J. P., et al. A small-cellpredominant variant of primary Ki-1 (CD30)+ T-cell lymphoma. AmJSurgPathol, 1993; 17: 859-68. 553 Anderson, M. M., Ross, C.W., Singleton, T. P., etal. Ki-1 anaplastic large cell lymphoma with a prominent leukemic phase. Hum Pathol, 1996; 27:1093-5. 554 Villamor, N., Rozman, M., Esteve, J., et al. Anaplastic largecell lymphoma with rapid evolution to leukemic phase. Ann Hematol, 1999; 78:478-82. 555 Bayle, C., Charpentier, A., Duchayne, E., etal. Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases. BrJHaematol, 1999; 104: 680-8. 556 Meech, S. J., McGavran, K., Odom, L. F., etal. Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomysin 4-anaplastic lymphoma kinase gene fusion. Blood, 2001; 98:1209-16. 557 Awaya, N., Mori, S., Takeuchi, H., et al. CD30 and NPMALK fusion protein (p80) are differentially expressed between peripheral blood and bone marrow in primary small cell variant of anaplastic large cell lymphoma. Am J Hematol, 2002; 69: 200^. 558 Onciu, M., Behm, F. G., Raimondi, S. C., et al. ALK-positive anaplastic large cell lymphoma with leukemic peripheral blood involvement. Report of three cases and review of the literature. Am J Clin Pathol, 2003; 120: 617-25. 559 Greer, J. P., Kinney, M. C., Collins, R. D., etal. Clinical features of 31 patients with Ki-1 anaplastic large-cell lymphoma. J Clin Oncol, 1991; 9: 539-47. 560 Gordon, B. G., Weisenburger, D. D., Warkentin, R I., et al. Peripheral T-cell lymphoma in childhood and adolescence. Cancer, 1993; 71: 257-63. 561 Chhanabhai, M., Britten, C., Klasa, R., & Gascoyne, R. D. t(2;5) positive lymphoma with peripheral blood involvement. Leuk Lymphoma, 1997; 28: 415-22. 562 Wong, K. F., Chan, J. K. C., Ng, C. S., etal. Anaplastic large cell Ki-1 lymphoma involving bone marrow: marrow findings and association with reactive hemophagocytosis. Am J Hematol, 1991; 37: 112-19.

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8 Immunoglobulin and T-cell receptor gene rearrangements Jacques J. M. van Dongen and Anton W. Langerak

Introduction

IG/TCR gene rearrangement processes

The ability of the immune system to specifically recognize millions of different antigens and antigenic epitopes is based on the enormous diversity (at least 1012 ) of antigen-specific receptors, that is, surface membranebound immunoglobulin (SmIg) molecules on B lymphocytes and T-cell receptor (TCR) molecules on T lymphocytes.1,2 The antigen-specific receptors differ from lymphocyte to lymphocyte, but each single lymphocyte or lymphocyte clone expresses approximately 105 receptors with identical antigen specificity. The extensive diversity of the antigen-specific receptors of lymphocytes is based on rearrangement processes in the Ig/TCR-encoding genes.3 Since the various types of lymphoid leukemias resemble normal lymphoid (precursor) cells,4–7 most lymphoid leukemias and lymphomas also contain rearranged IG/TCR genes. Being derived from a single malignantly transformed lymphoid cell, all cells of a lymphoid malignancy have their IG/TCR genes rearranged in an identical way. This information can be readily employed for clonality assessment in lymphoproliferations.8 We will discuss the IG/TCR gene rearrangement processes and the methods for detecting clonal IG/TCR gene rearrangements in various types of (childhood) leukemia. Finally, several applications of diagnostic clonality studies in childhood leukemia are presented.

Ig molecules and their encoding genes Ig molecules consist of two disulfide-bonded Ig heavy (IgH) chains and two Ig light chains. The Ig class or subclass is determined by the isotype of the involved IgH chain, irrespective of the type of light chain. Each B lymphocyte or B-lymphocyte clone expresses only one type of light chain (Ig or Ig), whereas multiple IgH chains can be expressed.9 Surface membrane-bound Ig molecules are closely associated with disulfide-linked heterodimers, which consist of CD79a (mb1 or Ig
) and CD79b (B29 or Ig) chains, that together mediate transmembrane signal transduction for the SmIg-CD79 complex.1 Each Ig protein chain is composed of one variable domain, involved in actual antigen recognition, and one (for Ig and Ig chains) or three to four (for IgH chains) constant domains, which mediate the effector function. In order to recognize all antigenic epitopes, an extensive repertoire of antigen-specific variable domains is needed. If this entire repertoire of Ig/TCR molecules would be encoded by separate genes, these would occupy a large part of the human genome. However, due to the fact that distinct combinations of gene segments are formed in each lymphocyte, only a limited number of gene segments are able to encode the receptor diversity.2,3,10,11 Each domain of an Ig chain is encoded by a separate exon. The variable

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Fig. 8.1 Schematic diagram of the human IG gene complexes. The IGH gene complex comprises at least 40 functional V gene segments, 27 D gene segments, 6 functional J gene segments, and several C gene segments encoding the various IgH class and subclass constant domains. The switch region that precedes most C gene segments plays a role in IgH (sub)class switching. The IGK gene complex consists of ∼35 functional V gene segments, 5 J gene segments, and a single C gene segment. The Kde (-deleting element) plays a role in the deletion of the J –C or C gene regions in B cells, which rearrange their IGL genes. The IGL gene complex contains ∼30 V gene segments and 4 functional C genes, all of which are preceded by a J gene segment. Pseudo genes ( ) are indicated as open symbols.

domain of an IgH chain is encoded by the so-called VDJ exon, originating from a combination of V (variable), D (diversity), and J (joining) gene segments. A combination of V and J gene segments encodes the variable domain of an Ig or Ig chain (Fig. 8.1).2,12 Coupling of V, (D), and J gene segments is mediated through gene rearrangement processes, which occur during B-cell differentiation.2,3,13 The constant domains of the Ig chains are encoded by isotypedependent C (constant) exons.14–21

TCR molecules and their encoding genes TCR molecules consist of two chains that generally are disulfide-linked. Two different types of TCRs are known: the “classical” TCR
 receptor, consisting of a TCR
and a TCR chain, and the “alternative” TCR  receptor, composed of a TCR and a TCR chain.2,12 The majority of mature T lymphocytes (85–98%) in peripheral blood and in most lymphoid tissues expresses TCR
, while only a minority (2–15%) are TCR  positive.12 Both types of TCR molecules are closely associated with the CD3 protein chains, that are involved in transmembrane signal transduction for the TCR-CD3 complex.1 Analogous to Ig chains, each TCR chain consists of one variable and one constant domain. The variable domain of TCR
and TCR chains is encoded by joined V and J gene segments, whereas in the case of TCR and TCR chains it is shaped by coupled V, D, and J gene segments (Fig. 8.2).2,12 The constant domains of the TCR chains are encoded by one (TCRA and TCRD loci) or two (TCRB and TCRG loci) C gene segments (Fig. 8.2).22–30

The gene rearrangement process: VDJ coupling During early B- and T-cell differentiation the germline V, D, and J gene segments of the IG/TCR gene complexes rearrange, and each lymphocyte thereby obtains specific combinations of VDJ segments, also known as the VDJ exon.2,3,10,11 Gene rearrangements are complex processes that involve several proteins, which together form the recombinase enzyme system. The recombinase complex contains protein products of the recombinase-activating genes (RAG1 and RAG2) as well as regulatory DNA-binding proteins.31–33 RAG1 and RAG2 recognize specific recombination signal sequences (RSS) flanking the 3 side of V gene segments, both sides of D gene segments and the 5 side of J gene segments.34–36 Such signals consist of conserved palindromic heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by 12- or 23-bp spacer regions.34–36 Following the introduction of doublestrand breaks (dsb) between the RSS and the gene segment, a hairpin structure is formed at the coding end side, which is further processed before religation to another gene segment via several enzymes known to be involved in dsb repair; this coupling is called a coding joint or VDJ exon (Fig. 8.3). During the coupling process, deletion and random insertion of nucleotides can occur, leading to imprecise coupling of gene segments. The 5 phosphorylated RSS ends fuse head-to-head to form the signal joint, which is generally removed from the genomic DNA in the form of an excision circle.2,34–36 Upon inversional rearrangement, which occurs in the case of V gene segments with an inverted orientation (e.g. the V 20 or V 3 gene segments and the distal V gene

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Fig. 8.2 Schematic diagram of the human TCR gene complexes. The TCRA gene complex consists of 42 V gene segments, a stretch of 56 functional J gene segments, and a single C gene segment. The TCRB gene complex contains ∼45 functional V gene segments and 2 C gene segments, both of which are preceded by a D gene segment and 6 or 7 J gene segments. The TCRG gene complex consists of a restricted number of V gene segments (6 functional V gene segments and 9 pseudo genes) and 2 C gene segments, each preceded by 2 to 3 J gene segments. The TCRD gene complex comprises 6 V, 3 D, and 4 J gene segments and a single C gene segment. The major part of the TCRD gene complex is located between the V
and J
gene segments and is flanked by the REC and J
gene segments, which are involved in TCRD gene deletions that occur prior to TCRA gene rearrangements. Pseudo genes ( ) are indicated as open symbols.

segments), the signal joint and other intervening sequences between the two coding elements are not removed as excision circles but rather preserved on the genome.18,29 In Fig. 8.4 an example of an TCRB gene rearrangement is illustrated. First, a coding joint is formed between a D gene segment and one of the J gene segments, whereas the signal joint of the 3 D RSS and the J RSS is present on the excision circle, also known as T-cell receptor excision circle (TREC). Second, one of the many V gene segments is coupled to the D –J complex, resulting in a VDJ exon, while the V RSS and 5 D RSS are present on another TREC. The rearranged gene is subsequently transcribed into a precursor mRNA, which is processed into mature mRNA by splicing out all intronic, noncoding sequences (Fig. 8.4).2 Similar rearrangement and transcription processes occur in all IG/TCR gene loci.

Fig. 8.3 Schematic diagram of the V(D)J recombination mechanism. RAG1 and RAG2 bind to RSS, resulting in doublestrand breaks (dsb). After cleavage, hairpin structures are formed at the coding ends, whereas the RSS blunt ends fuse to form a signal joint generally resulting in a so-called excision circle. Further processing results in opening of the hairpins via several enzymes known to be involved in dsb repair (DNA-PKcs , Ku70/Ku80, and Artemis). Finally, opened hairpins are religated (involving DNA ligase IV and XRCC4) to a coding joint, and further diversified by the action of TdT, which introduces nucleotides in a template-independent way.

Repertoire of Ig/TCR molecules The enormous diversity of antigen-specific receptors of lymphocytes is mediated by the VDJ recombination processes of IG/TCR loci. The extent of the potential primary repertoire of antigen-specific receptors is based on: the combinatorial diversity (number of possible VDJ combinations) and the junctional diversity (due to imprecise joining of the V, (D), and J gene segments).10,11 The combinatorial diversity results from all possible combinations of available functional V, (D), and J gene

Immunoglobulin and T-cell receptor gene rearrangements

Table 8.1 Estimation of potential primary repertoire of human Ig/TCR molecules Ig molecules

Number of functional gene segmentsa V gene segments D gene segments J gene segments Combinatorial diversity Junctional diversity Estimation of total repertoire

TCR
 molecules

TCR  molecules

IGH

IGK

IGL

TCRA

TCRB

TCRG

TCRD

40–46 27b 6

34–37 — 5 >2 × 106 ± >1012

27–30 — 4

45 — 50

44–47 2b 13

6 — 5

6 3b 4

±

>2 × 106 + ++ >1012

++

>5000 +

+++ >1012

a

Numbers are based on the international IMGT (ImMunoGeneTics) database. In TCRD gene rearrangements, multiple D segments might be used; this implies that the number of junctions can vary from one to four. In IGH and TCRB gene rearrangements, generally only one D gene segment is used. b

Fig. 8.4 Schematic diagram of a human TCRB gene rearrangement. In this example D is first joined to J , followed by V to D –J joining; as a result of the gene segment couplings, all intervening DNA sequences are deleted. The rearranged gene complex can be transcribed into precursor mRNA, which is further processed into mature mRNA by splicing out all noncoding intervening sequences.

segments per gene complex and the pairing of two different protein chains per antigen-receptor molecule, i.e. IgH with Ig or Ig, TCR
with TCR, and TCR with TCR.10,11 As the IGH gene complex probably contains at least 40 functional VH gene segments, 27 rearranging DH gene segments, and 6 functional JH gene segments, coupling will result in approximately 6000 possible VH –DH –JH combinations. Together with the estimated 175 and 115 V-J combinations of the IGK and IGL genes, respectively, a potential combinatorial diversity of more than 2 × 106 can be obtained.37 A similar diversity can be obtained for TCR
 molecules (Table 8.1).37 The combinatorial diversity of TCR  molecules is less extensive due to the limited number of functional V, (D), and J gene segments in the encoding gene

complexes.37 Nevertheless, because of multiple D gene segment usage, potentially a combinatorial repertoire of greater than 5000 TCR  molecules can be produced. The aforementioned numbers are based on the assumption of random usage of the available functional V, (D), and J gene segments. However, there are several indications that preferential usage of gene segments occurs. Fetal B cells use a restricted set of VH gene segments, related to JH proximity,38,39 while TCR
+ cells tend to use J 2 gene segments more frequently than J 1 gene segments.40 Preferential rearrangement can be established at three levels: first, proximity of the involved gene segments; second, differential accessibility of the gene segments, and third, the exact sequence of the RSS element. Peripheral TCR +

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T lymphocytes exhibit preferential usage of V 9–J 1.2 and V 2–J 1 gene segments.41,42 Over-representation of certain receptor types can only partly be explained by preferential rearrangements, because clonal selection and expansion of particular receptor specificities play an important role in peripheral tissues.43 The junctional diversity of Ig/TCR molecules is based on deletion of nucleotides at the ends of the rearranging gene segments as well as by random insertion of nucleotides (N region nucleotides) between coupled gene segments. Insertion of N region nucleotides at the 3 ends of DNA breakpoints is mediated by terminal deoxynucleotidyl transferase (TdT) in a template-independent way.44,45 The absence or a decrease in TdT activity during IG/TCR gene rearrangements leads to the virtual absence of N region insertion, as is found in early fetal thymocytes.46,47 Rearranged IGK and IGL genes in mature B cells also have lower levels of N region insertion,10,11,48 suggesting that the IGK and IGL genes rearrange in the presence of decreased TdT activity. This is in contrast to the junctional regions of rearranged TCR genes in late fetal and postnatal thymocytes, which all contain N regions.47 The junctional regions of IG/TCR genes encode the so-called complementarity determining region 3 (CDR3), which is involved in antigen recognition. Therefore, N region insertion drastically increases the diversity of antigen receptors, especially when there are multiple junction sites within a junctional region, such as in IGH, TCRB, and especially TCRD genes (Table 8.1). Because of the random insertion and deletion of nucleotides at the junction sites of V, (D), and J gene segments, the junctional regions function as lymphocytespecific “fingerprint-like” sequences (Fig. 8.5).

Secondary IG/TCR gene rearrangements IG/TCR gene rearrangements are complex processes with imprecise coupling of gene segments due to random insertion and deletion of nucleotides.11 Because of the triplet reading frame of DNA sequences, approximately two out of three couplings will be out-of-frame leading.11 The high frequency of out-of-frame rearrangements and the generation of stop codons at the coupling sites may explain why most B cells have biallelic IGH rearrangements and why most T cells have biallelic TCRB and TCRG gene rearrangements.3,8 In addition to biallelic rearrangements, secondary gene rearrangements appear to occur and are assumed to rescue precursor B and precursor T cells with nonproductive IG/TCR genes. The type of secondary rearrangement depends on the involved IG/TCR locus as well as the type of pre-existing rearrangement (Table 8.2). DH –JH replace-

ments in B cells replace pre-existing DH –JH gene complexes by coupling an upstream DH gene segment to a downstream JH gene segment (Fig. 8.6).49 Such D–J replacements can also occur in TCRB and TCRD genes. In a comparable way V–J replacements replace pre-existing V–J complexes in TCRA, TCRG, IGK, and IGL genes.50 Both types of replacements can occur repeatedly in the same IG/TCR gene complex as long as germline V, (D), and J gene segments are still available (Table 8.2). Another type of secondary rearrangement concerns V gene segment replacement in a complete V(D)J exon by an upstream V gene segment.51,52 This process is mediated via an internal heptamer RSS in the 3 part of the V gene segments in IGH, TCRB, and TCRG genes,51–53 but this heptamer RSS is not found in V gene segments of IGK, IGL, TCRA, and TCRD genes. So far, V replacements have especially been observed in IGH genes and in TCRB genes (Table 8.2). Interestingly, secondary rearrangements have not only been found to replace pre-existing non-productive rearrangements, but also for example productive V
–J
rearrangements.54 This suggests that secondary rearrangements not only rescue precursor B and precursor T cells from nonproductive rearrangements, but also are involved in selection processes of immature B cells in bone marrow and immature T cells in the thymus.50,54

Somatic hypermutations in rearranged IG genes The repertoire of Ig molecules can be further increased and adapted via antigen-induced somatic hypermutations in the VDJ exons of rearranged IG genes.55,56 These point mutations occur in B lymphocytes that are present in secondary follicles (germinal center reaction), and hence are not found in virgin B lymphocytes.55,56 They are assumed to serve affinity maturation and clonal selection and precede or coincide with IgH class switching. Consequently, follicular and postfollicular B-cell malignancies, being the malignant counterparts of (post)follicular B lymphocytes, also have somatically mutated IGH, IGK, and/or IGL genes.57,58

Rearrangement and expression of IG/TCR genes during lymphoid differentiation Rearrangements of IG and TCR genes start early during lymphoid differentiation and occur in an hierarchical order that is tightly regulated by transcription factors, among which are E2A and HEB that have been shown to mediate differential accessibility of IG/TCR loci during differentiation.59,60

Immunoglobulin and T-cell receptor gene rearrangements

1

Fig. 8.5 (A) Schematic diagram of the V 1 gene segment joined to the J 1.3/2.3 gene segment via a junctional region. PCR-mediated amplification of the joined TCRG gene segments and subsequent sequencing of the junctional region in the obtained PCR products can be performed. The presented TCRG junctional region sequences are derived from T-ALL patients and illustrate the deletion of nucleotides from the germline sequences as well as the size and composition of the junctional regions. (B) Schematic diagram of the V 1 gene segment joined to the J 1 gene segment via a junctional region. TCRD junctional regions may contain one, two, or three D gene segments. The presented TCRD junctional region sequences are derived from the same T-ALL patients as in panel A, and illustrate that deletion and insertion of nucleotides is more extensive than in the case of TCRG junctions, resulting in much longer junctional regions. D gene segments and inserted nucleotides are indicated by capital letters and small capital letters, respectively.

During B-cell differentiation IGH genes rearrange first, followed by IGK genes (Fig. 8.7). If the latter rearrangements are nonfunctional, the IGL genes will start to rearrange.3,9,10 Generally, IGL gene rearrangements occur after or coincide with IGK gene deletions.61,62 Virtually all IGK gene deletions are mediated via rearrangement of the so-called kappadeleting element (Kde), which is located downstream of the C gene segment (Fig. 8.8).63,64 This Kde sequence re-

arranges either to an isolated heptamer RSS in the J –C intron, thereby deleting the C gene segment, or to a V gene segment, thereby removing both the J and C gene segments.64,65 Cytoplasmic CD79 (CyCD79) expression is already observed in the early stages of B-cell differentiation,66–68 and is followed by expression of the SmIg–CD79 complex on B cells, once functional IGH and IGK or IGL gene re-

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Table 8.2 Secondary rearrangements in IG/TCR gene complexes IG genes

D–J replacement V–J replacement V replacement

TCR genes

IGH

IGK

IGL

TCRA TCRB TCRG TCRD

+ – +

– + –

– (+) –

– + –

(+) – +

– + (+)

(+) – –

Symbols: +, replacement reported to occur; (+), replacement can potentially occur, but not (yet) reported; −, replacement not likely to occur.

Fig. 8.6 Examples of secondary rearrangements in IGH genes. DH –JH replacement: an upstream DH gene segment rearranges to a downstream JH gene segment, thereby replacing the pre-existing DH –JH rearrangement. VH replacement: the VH gene segment in a complete VH –DH –JH rearrangement is replaced by an upstream VH gene segment via a rearrangement process mediated by a heptamer RSS (indicated as 7) within the VH gene segments.

arrangements are produced. In the pre-B cell differentiation stage, functional IGH gene rearrangements lead to weak cytoplasmic Ig heavy-chain (CyIg ) expression.69,70 Some of the pre-B cells also show weak membrane expression of the so-called pre-B-cell complex (pre-B–SmIg – CD79),1,71 which contains a pseudo-light chain derived from nonrearranging IGL-like gene segments.72 This pre-Bcell complex probably plays a role in regulation of early Bcell development.1 Following antigen-induced activation of B lymphocytes, somatic mutations and IGH isotype rearrangements can occur.3,9 Finally, maturation to the plasma cell stage is characterized by disappearance of the SmIg-CD79 complex coincident with high levels of cytoplasmic Ig (CyIg) molecules for secretion (Fig. 8.7). In T-cell differentiation, the TCRD genes rearrange first, followed by the TCRG genes. This might result in TCR + T lymphocytes, provided that the rearrangements are functional. TCR
+ T lymphocytes most probably develop via a

separate differentiation lineage with TCRB gene rearrangements taking place prior to TCRA gene rearrangements.3,73 TCRA gene rearrangements are preceded by deletion of the TCRD gene, which for the largest part is located between V
and J
gene segments (Fig. 8.2).23,28,29 This TCRD gene deletion process is primarily mediated via rearrangement of the flanking REC and J
gene segments.47,74,75 These rearrangement and deletion processes in the TCRA/TCRD locus probably play a crucial role in the divergence of the TCR  and TCR
 differentiation pathways,12 although it is still unclear in which differentiation stage this divergence occurs. The fact that virtually all TCR
+ T lymphocytes have rearranged TCRG genes and that a large part of the TCR + T lymphocytes have rearranged TCRB genes is remarkable and suggests that these discrete differentiation lineages might share a common origin (Fig. 8.7).3 Surface membrane expression of TCR–CD3 complexes depends on the functional rearrangement of TCRD and TCRG genes or TCRB and TCRA genes. Cytoplasmic expression of CD3 is already observed early during T-cell differentiation. Analogous to the pre-B-cell complex, a preT-cell complex has also been identified, which is weakly expressed on the surface membrane of immature thymocytes of the TCR
 differentiation lineage.76 This pre-T-cell complex contains a surrogate TCR chain (pre-TCR
or preT
) next to a TCR protein chain.76

Clonality detection based on IG/TCR gene rearrangements Rearrangements in IG/TCR genes result in relocation and coupling of gene segments and simultaneous deletion of the intervening gene segments. Because leukemias are clonal cell proliferations, the IG/TCR gene rearrangements are assumed to be identical in all cells of a leukemia. Rearrangement processes can be studied by Southern blot analysis and by polymerase chain reaction (PCR)based techniques. Analysis of deletion and relocation of gene segments by Southern blotting is based on typical changes in distances between the cleavage-sites of restriction enzymes, i.e. endonucleases which reproducibly cut DNA at sites where they recognize a specific nucleotide sequence (e.g. GAATTC for the enzyme EcoRI). PCR analysis of IG/TCR gene rearrangements is based on the amplification of rearranged IG/TCR genes including their junctional regions. Such amplification is only possible when the IG/TCR gene segments are juxtaposed through rearrangement, as the distance between these gene segments in the germline configuration is far too large for efficient amplification. Of note, clonality assessment by Southern

Immunoglobulin and T-cell receptor gene rearrangements

I

Fig. 8.7 Hypothetical diagram of IG and TCR gene rearrangements during lymphoid differentiation. The ordered rearrangement of IG and TCR genes is indicated with bars. The expression of nuclear TdT, cytoplasmic CD3 (CyCD3), cytoplasmic CD79 (CyCD79), pre-B Ig molecules (pre-B-CyIg and pre-B-SmIg –CD79), pre-T complex, and the mature SmIg–CD79 and TCR–CD3 antigen receptor complexes is indicated for each differentiation stage.

blotting exploits the combinatorial diversity (i.e. variation in relocation of gene segments), whereas clonality studies by PCR techniques are mainly based on the junctional diversity of IG/TCR gene rearrangements.

Southern blot analysis of IG/TCR gene rearrangements In Southern blot studies, DNA samples are digested with restriction enzymes.77 The resulting DNA fragments (restriction fragments) are size-separated by agarose gel electrophoresis, transferred (blotted) onto a nitrocellulose or nylon membrane, and immobilized.77 This membrane is incubated with a (radio)labeled DNA probe, which hybridizes to complementary sequences of IG/TCR genes.3 Unbound probe is washed away and the location of the probe (hence the size of the recognized restriction fragments) can be detected by autoradiography or by imaging methods. If appropriate combinations of restriction enzymes and DNA probes are used, the detected restriction

Fig. 8.8 Consecutive rearrangements in the IGK locus, resulting in the two main types of kappa-deleting element Kde rearrangements. Generally, recombination starts with V –J rearrangement. Expression of the V –J rearranged allele can be disrupted by rearrangement of Kde to an intronic RSS, resulting in deletion of the C gene segment, or to any of the V gene segments, resulting in deletion of the entire V –J –C region. Both types of Kde rearrangements result in deletion of the IGK gene enhancers (iE and 3 E ), most probably precluding further rearrangements in the IGK locus.

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Optimally designed IG/TCR probes should be located just downstream of the rearranging gene segments and should be used in combination with at least two different restriction enzyme digests. The germline restriction fragments should not be affected by polymorphisms and should preferably be 10 kb or smaller to prevent comigration of germline and/or rearranged bands.3,29,78–84 Since IG/TCR gene rearrangements are identical in all cells of a leukemia, the (identical) restriction fragments give rise to a clearly visible rearranged band that is different from the germline band.3 Furthermore, two rearranged bands of comparable density will be visible if the clonal cell population has rearranged both IG/TCR alleles (Fig. 8.9). In contrast to clonal cell populations, reactive (polyclonal) lymphoid cell proliferations contain many different IG/TCR gene rearrangements that are detectable as a characteristic background pattern or vague smear consisting of multiple faint rearranged bands.3,78,82 Thus, Southern blot analysis of IG/TCR genes allows discrimination between clonal rearrangements and polyclonal rearrangements.3,8 Leukemic cell populations can be detected with a sensitivity of about 5%, whereas the detection limit is 10% to 15%, if a clonal cell population has to be identified within a background of many polyclonal (reactive) cells. Fig. 8.9 Southern blot analysis of IGH genes. (A) Restriction map of JH –C region. The positions of the relevant EcoRI (E), HindIII (H), BamHI (B), and BglII (Bg) restriction sites are indicated. The location of the hypervariable polymorphic (HVP) region upstream of the JH region as well as the switch region (S ) are also indicated. The solid bar represents the JH probe (IGHJ6). (B) Ethidium bromide-stained agarose gel with size-separated BglII restriction fragments of control DNA and four different B-cell leukemia DNA samples. The two outer lanes contain size markers (left; HindIII-digested DNA; right, EcoRI/HindIII-digested DNA). The DNA fragments were blotted to a nylon filter. (C) X-ray film after hybridization of the nylon filter to the 32 P-radiolabeled IGHJ6 probe. The size of the germline band (G) and the position of the size markers are indicated. The two control lanes contain the 3.9-kb germline band, whereas each of the four B-cell leukemia lanes show two clonally rearranged bands, due to biallelic IGH gene rearrangements.

fragments of rearranged IG/TCR genes will differ from those of germline genes.3,29,78–83 In Fig. 8.9 various aspects of Southern blot analysis of the IGH gene are illustrated: the germline restriction map of the JH –C region with an appropriate JH probe (IGHJ6), separation of restriction fragments in an agarose gel, and hybridization results using the radiolabeled IGHJ6 probe.3,78

PCR amplification of IG/TCR gene rearrangements PCR allows the selective amplification of a particular DNA sequence between two primer sequences.85 In order to design appropriate PCR primers, the nucleotide sequences flanking the target DNA should be known precisely. In a typical PCR assay, genomic DNA is first denatured (94 ◦ C) into single-strand DNA molecules, followed by annealing of the primers (55–65 ◦ C) to complementary sequences on the single-strand molecules, and Taq polymerase-mediated extension (72 ◦ C), using the single-strand genomic DNA as template sequence.85 Because the extended product itself can serve as a template for the other primer in subsequent cycles, each successive PCR cycle essentially doubles the number of PCR products. Although, in principle, target DNA sequences up to 10 kb or more can be amplified, for routine PCR analysis target sequences preferably should not be longer than 2 kb.85,86 PCR primers for amplification of IG/TCR junctional regions are generally designed such that amplicons of maximally 500 bp are produced (Fig. 8.10). Since most IG/TCR PCR studies in leukemias and lymphomas are performed at the DNA level, PCR primers are complementary to exon and/or intron sequences of V, (D), and J gene segments, dependent on the type and completeness of the rearrangement.87–93 Obviously, the choice

Immunoglobulin and T-cell receptor gene rearrangements

Table 8.3 Estimated number of human V, (D), and J gene segments, that can potentially be involved in IG/TCR gene rearrangementsa Gene segment

IGH

IGK

IGL

TCRA

TCRB

TCRG

TCRD

V (family) D (family) J (family)

∼70 (7) ∼27 (7) 6

∼60 (7) – 5

∼40 (11) – 5c

∼60 (32) – 61b

∼65 (30) 2 13

9 (4) – 5 (3)

7b 3 4

a

Numbers are based on the international IMGT (ImMunoGeneTics) database.37 These numbers include the nonfunctional  REC gene segment (TCRD locus) and the J
gene segment (TCRA locus). c Two of the seven J gene segments have never been observed to be involved in IGL gene rearrangements, probably because of their inefficient RSS. b

Fig. 8.10 Schematic diagram of a VH –JH rearrangement with junctional region. Indicated are three sets of family-specific VH framework (VH -FR1, VH -FR2, and VH -FR3) primers, which can be used for multiplex PCR analysis in three different tubes in combination with a consensus JH primer.

of primers depends on the exact IG/TCR locus and the involved rearranged gene segments. It may be possible to design general or consensus primers for the IG/TCR loci, which recognize, for example, all V or J gene segments of a particular IG/TCR gene complex, or family-specific primers, which recognize families of V or J gene segments (Fig. 8.10). A third option is to design specific primers, which recognize individual gene segments.87–95 It should be noted that the IG/TCR genes not only contain functional V, (D), and J gene segments (Table 8.1), but nonfunctional (pseudo) gene segments can be present as well. These segments can also be involved in gene rearrangements, if they are flanked by RSS. Table 8.3 summarizes the estimated number of rearranging gene segments and families of all IG/TCR loci. For detection of all possible (functional and nonfunctional) IG/TCR gene rearrangements, the primer sets should be able to recognize virtually all V, (D), and J gene segments. This would imply that many different primer sets have to be designed, which would not be efficient for routine PCR analysis of certain IG/TCR gene rearrangements. In the case of IGH, IGK, and IGL gene rearrangements, the use of primers for the relatively limited number of VH , V , and V families (see Table 8.3) can lead to a reduction in the number of required primers. The recently developed BIOMED-2 multiplex PCR strategies combine multiple V family primers in one PCR reaction mixture with recognition of virtually all segments, resulting in highly

efficient assays.96,97 PCR analysis of TCRA and TCRB gene rearrangements would also require many primers, especially for the many different V and J gene segments of the TCRA and TCRB loci (Table 8.3 and Fig. 8.2).18,21,23,24,98–101 RT-PCR strategies in which V
J
–C
and V D J –C transcripts are analyzed, only partly solves this problem, as many different V
or V primers still need to be used in combination with a single C
or C primer.98–100 A similar BIOMED-2 multiplex approach as for the IG loci has now also proved very helpful for analyzing TCRB gene rearrangements.96,97 Finally, TCRG and TCRD gene rearrangements can relatively easily be analyzed with only a restricted number of PCR primers, because of the limited number of individual V and J gene segments of TCRG and TCRD loci (see also Table 8.3 and Fig. 8.2).14,16,26,29 As compared to Southern blot analysis, PCR-based IG/TCR gene analysis has several disadvantages. One is the risk of false-negative results due to inappropriate recognition of all IG/TCR gene segments that can potentially be involved in rearrangements, by the applied primer sets. This has now largely been overcome by the recent development of standardized multiplex assays for analysis of IG/TCR gene rearrangements.96,97 Another important pitfall of PCR analysis is the risk of false-positive results due to the fact that not only clonally rearranged IG/TCR genes are amplified, but also IG/TCR gene rearrangements from normal, polyclonal cells. Hence, discrimination

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Fig. 8.11 V 1–J 1 PCR products of T-ALL patients with monoallelic or biallelic V 1–J 1 rearrangements. The PCR products were separated in a polyacrylamide gel, resulting in optimal size separation. The positions of the size markers are indicated in the left margin. The differences in size of the PCR products (115 to 155 bp) are due to differences in size of the junctional regions (see Fig. 8.4).

between monoclonal (leukemia-derived) and polyclonal (reactive) PCR products is difficult, emphasizing the need to further analyze the PCR-amplified rearranged gene products.

Analysis of PCR amplified IG/TCR products PCR-based detection of clonal IG/TCR gene rearrangements is relatively easy, if the percentage of leukemic cells is high (e.g. >90%). In such cell samples, the background of IG/TCR gene rearrangements derived from normal, polyclonal cells generally does not interfere with PCR amplification of those of leukemic cells. This is illustrated by Fig. 8.11, which shows that PCR products of comparable clonal TCRD rearrangements in different T-cell acute leukemias are clearly distinct from each other, based on differently sized junctional regions (cf. Fig. 8.5).90 If, however, a sample contains substantial numbers of polyclonal B or T cells, many polyclonal IG/TCR PCR products will be present as well. Discrimination between monoclonal and polyclonal PCR products with standard gel electrophoresis in that case is hampered by the fact that the clonal PCR products have to be identified as a “dominant” band within a background of multiple “weaker” bands of slightly different sizes, which represent the polyclonal PCR products.91,92 Since junctional regions are “fingerprint-like” sequences that differ between lymphocytes or lymphocyte clones, they also represent specific markers for each individual leukemia.87–90,94,95 Strategies based on this idea have been developed that employ the junctional region sequences of amplified rearranged IG/TCR genes for discrimination

between polyclonal and clonal cell populations.87–90,94,95 Methods that have been applied successfully to solve this background problem include direct sequencing of the PCR products,102 single-strand conformation polymorphism (SSCP) analysis,103,104 denaturing gradient gel electrophoresis (DGGE),105,106 temperature gradient gel electrophoresis (TGGE),107 heteroduplex analysis,108,109 and GeneScan analysis.110,111 The latter two methods, especially, have proved their utility. Originally designed for mutation detection of genetic diseases, heteroduplex analysis, after modification, can also be applied to analysis of IG/TCR PCR products. In heteroduplex analysis, PCR products are denatured (94 ◦ C) and subsequently renatured (4 ◦ C) to induce formation of homoduplexes (with identical, clonal junctions) or heteroduplexes (with different junctional regions), which can then be separated from each other by polyacrylamide gel electrophoresis based on differences in conformation (Fig. 8.12).109 As illustrated in Fig. 8.13A, the application of heteroduplex analysis makes it possible to discern between PCR products derived from monoclonal and polyclonal cell populations, based on the presence of homoduplexes or (a smear of) heteroduplexes, respectively. In GeneScan analysis, fluorochrome-labeled IG/TCR PCR products are analyzed in high-resolution polyacrylamide gels (Fig. 8.12). Monoclonal IG/TCR PCR products give rise to fragments of identical size, whereas polyclonal products result in PCR products showing a Gaussian distribution of junctional region sizes (Fig. 8.13B). Although slightly better detection limits have been found for GeneScan analysis than for heteroduplex analysis, the latter technique is more reliable with IG/TCR targets having relatively small junctional regions. This has to do with the fact that in heteroduplex analysis the heterogeneity of PCR products not only reflects their size, but also the composition of the junctional regions, whereas in GeneScan analysis only size is evaluated. Heteroduplex analysis thus offers a relatively easy, cheap, and highly reliable method for IG/TCR PCR product analysis. Multicenter studies to evaluate the extent to which multiplex PCR heteroduplex and/or GeneScan strategies can replace the more laborious Southern blot assessment of IG/TCR clonality have now shown that this is indeed the case, provided that multiple targets are analyzed to reduce the risk of false negativity caused by improper annealing to gene segments and somatic mutations.96,97

Immunogenotype of childhood leukemias Hematopoietic malignancies represent 40% to 45% of all cancers in children under the age of 15. Approximately 70%

Immunoglobulin and T-cell receptor gene rearrangements

Fig. 8.12 (A) Schematic diagram of VH –DH –JH junctional regions with primers for PCR analysis. The approximate position of the VH -family specific framework 1 (VH -FR1) primers as well as of a JH consensus primer are indicated. The presented junctional region sequences are derived from precursor-B-ALL patients and illustrate deletion of nucleotides from the germline sequences as well as the size and composition of the junctional regions. Nucleotides of VH , DH , and JH gene segments are indicated in capital letters, while small capital letters represent inserted nucleotides. (B) Schematic diagram of heteroduplex PCR analysis, in which the junctional region heterogeneity of PCR products of rearranged IG or TCR genes is exploited to discriminate between PCR products derived from monoclonal and polyclonal lymphoid cell populations. In heteroduplex analysis, PCR products are heat-denatured and subsequently rapidly cooled to induce duplex (homo- or heteroduplex) formation. In cell samples consisting of clonal lymphoid cells, the PCR products of rearranged IG or TCR genes give rise to homoduplexes after denaturation and renaturation, whereas in samples that contain polyclonal lymphoid cell populations the single-strand PCR fragments will mainly form heteroduplexes upon renaturation. In the case of an admixture of monoclonal cells in a polyclonal background, both hetero- and homoduplexes are formed. Because of differences in conformation, homo- and heteroduplexes can be separated from each other by electrophoresis in nondenaturing polyacrylamide gels. Homoduplexes with perfectly matching junctional regions migrate more rapidly through the gel than heteroduplex molecules with less perfectly matching junctional regions. The latter form a background smear of slower migrating fragments. (C) Automated high-resolution fluorescence-based analysis of PCR fragments for identification of clonally rearranged IGH genes (fluorescent GeneScan analysis). Polyclonal VH –DH –JH PCR products form a cluster of peaks reflecting a Gaussian distribution of average junctional region sizes in normal B cells. Monoclonal VH –DH –JH gene rearrangements form single fluorescence peaks, representing products of identical size.

of these are leukemias and the other 30% are malignant lymphomas. The vast majority of childhood hematopoietic malignancies (∼80%) are of lymphoid origin and consist mainly of acute lymphoblastic leukemias (ALL) and non-Hodgkin lymphoma (NHL) (Table 8.4).112 Most lymphoid malignancies in childhood belong to the Blineage category, but it is remarkable that the frequency of T-NHL in childhood is substantially higher than in adults (Table 8.4),113,114 mainly due to T-ALL-related

T-lymphoblastic lymphomas (T-LBL) and large-cell Tlineage anaplastic lymphomas.114 The majority (80–85%) of childhood ALL cases belong to the B-cell lineage and generally have the immunophenotype of precursor B cells, except for a small fraction of SmIg+ ALL, or so-called B-ALL. These cases of SmIg+ B-ALL are closely related to Burkitt lymphoma, because of their comparable cytomorphology, the presence of somatic mutations in rearranged IG loci, and the occurrence of

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the characteristic Burkitt translocations t(8;14), t(2;8), and t(8;22).114 Approximately 15% to 20% of ALL cases belong to the T-cell lineage (so-called T-ALL). The various types of T-ALL and the T-cell lymphoblastic lymphomas (T-LBL) form a continuous spectrum of lymphoblastic T-cell malignancies. CD3+ T-ALL and T-LBL seem to be highly comparable in many features, although there has been a single, unconfirmed report claiming a difference in the type of TCR molecule (
 or  ) that is expressed.115 The observation that the IG/TCR gene rearrangement process starts early during B- and T-cell differentiation and occurs in an hierarchical order (Fig. 8.7) implies that the vast majority of B-lineage and T-lineage leukemias have rearranged IG and TCR genes, respectively.8,116–122 However, leukemias can exhibit curious rearrangement patterns, that are rare or absent in normal lymphoid cells. For example, TCR gene rearrangements can be identified in non-T-cell leukemias and IG gene rearrangements in non-B-cell leukemias.8,123–125 These so-called cross-lineage rearrangements are especially common in acute leukemias124,125 and are probably due to continuous recombinase activity after malignant transformation.

IG/TCR gene rearrangements in B-lineage ALL Virtually all cases of B-lineage ALL (∼97%) have a precursor-B immunophenotype resembling that of normal precursor-B cells in the bone marrow. Nonetheless, greater than 95% of precursor-B ALLs have IGH gene rearrangements and most of them contain IGK gene rearrangements (30%) or deletions (50%).79,126,127 Deletions in the IGK gene are predominantly mediated via the Kde sequence, which rearranges either to one of the V gene segments (J –C deletion) or to a J –C intronic heptamer sequence (C deletion).79 This implies that IGK gene deletions can be identified as Kde rearrangements, which occur on one allele or both alleles in 20% and 30% of precursor-B ALL, respectively. IGL gene rearrangements are observed in 20% of precursor-B ALL cases (Table 8.5).8,79,126,127

Fig. 8.13 (A) Multiplex PCR heteroduplex analysis of IGK–Kde rearrangements using multiple V family primers and an intron RSS primer in combination with a Kde primer. In tonsil, only polyclonal smears are observed whereas in the leukemic samples, homoduplexes representing one (positive control cell line U698

and E-6) or two (G-3 and E-4) clonal Kde rearrangements are found. (B) Multiplex IGK–Kde PCR GeneScan analysis of the same samples as in panel A. Gaussian curves in tonsil represent polyclonal rearrangements, whereas in the leukemic samples one or two clonal peaks are found. Note that the two clonal products in sample G-3 differ by only one nucleotide in the GeneScan analysis, making discrimination in the heteroduplex analysis difficult; however, in heteroduplex analysis, an additional product is visible, which represents double-stranded heteroduplex DNA resulting from cross-annealing of the single-stranded fragments of the two Kde rearrangements.

Immunoglobulin and T-cell receptor gene rearrangements

223

Table 8.4 Estimated incidence rates and relative distribution of B-lineage and T–lineage lymphoid malignancies Acute lymphoblastic leukemia

Relative distribution B-lineage T-lineage Incidence per 100,000b Estimated number/yearb European Union USA

Chronic lymphocytic leukemia

Non–Hodgkin lymphoma

Multiple myeloma

Children <15 years

Adults >15 years

Children <15 years

Adults >15 years

Children <15 years

Adults >15 years

Children <15 years

Adults >15 years

80–85% 15–20% 2.9

75–80% 20–25% 0.7

—a — —

∼95% ∼5% 3.2

55–60% 40–45% 0.8

90–95% 5–10% 14.9

— — —

100% 0% 5.5

2400 1600

2050 1350

— —

3750 2500

675 450

44,500 29,500

— —

16,000 10,750

a

Not applicable.

b

Based on Fifth report of the Netherlands Cancer Registry (1996).112

Table 8.5 Frequencies of detectable IG and TCR gene rearrangements and deletions in the acute leukemias IGH R Precursor-B ALL B-ALL (Burkitt lymphoma) SmIg+ SmIg+ T-ALL (T-LBL) CD3− TCR + TCR
+ AML

IGK D

R

IGL D

R

TCRB R

TCRG R

TCRD R

D

>95%

3%

30%

30%

20%

35%

55%

50%

40%

100% 100%

0% 0%

100% 10%

0% 90%

5% 100%

<5% <5%

<5% <5%

0% 0%

0% 0%

15–20% 5–10% 5–10% 15%

0% 0% 0% 0%

0% 0% 0% ∼2%

0% 0% 0% 0%

0% 0% 0% 0%

85% 95% 100% 5%

90% 100% 100% 5–10%

80% 100% 35% 10%

10% 0% 65% 0%

a

Abbreviations and symbols: ALL, acute lymphoblastic leukemia; LBL, lymphoblastic lymphoma; AML, acute myeloid leukemia; R, at least one allele rearranged; D, both alleles deleted.

Cross-lineage TCR gene rearrangements also occur frequently in precursor-B ALL: TCRB, TCRG, and TCRD gene rearrangements and/or deletions are found in 35%, 60%, and 90% of cases, respectively (Table 8.5).8,117,125 Several studies have shown that at diagnosis, precursorB ALLs are frequently oligoclonal due to multiple IGH gene rearrangements (30–40% of cases) and even multiple IGK gene rearrangements (5–10% of cases).126,128 These multiple IG gene rearrangements have been attributed to continuing rearrangement processes (e.g. VH to DH –JH rearrangements) and secondary rearrangements (e.g. DH –JH replacements, VH replacements, and V –J replacements) (see Table 8.2), which result in one or more subclones.128–132 Bi- or oligoclonality at diagnosis have also been observed for rearrangements involving the TCRB (3% of cases) and

TCRG (10% of cases) loci.133 Based on Southern blot data, subclone formation at diagnosis was thought to be less frequent for the TCRD gene complex.29 However, PCR heteroduplex analysis and sequencing have provided substantial evidence that V 2–D 3 and D 2–D 3 rearrangements in precursor-B ALL at diagnosis are often oligoclonal.134,135 Furthermore, V 2–D 3 rearrangements are also prone to continuing rearrangements, especially to J
gene segments with deletion of the C exons.134–136 This probably explains why 40% of precursor-B ALLs at diagnosis have biallelic TCRD gene deletions (Table 8.5). Comparative studies between cell samples at diagnosis and at relapse revealed that the continuing rearrangements and secondary rearrangements can cause changes in the rearrangement patterns of IG and TCR genes at relapse.129,133,134,137,138

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These changes in rearrangement patterns at relapse are especially prominent in precursor-B ALL cases, that already contain subclones at diagnosis.138 Nevertheless, the occurrence and hence the stability of clones differs per type of rearrangement. For example, Kde rearrangements are more stable than IGH, TCRG, or TCRD rearrangements. This high stability of Kde rearrangements is most probably caused by the fact that these rearrangements delete not only the C region, but also the two enhancers (iE and 3 E ), implying that Kde rearrangements are end-stage processes that cannot easily be subject to continuing recombination.139 So far, the rarely occurring B-ALL entity was assumed to be related to other B-lineage ALLs on the one hand, and to Burkitt lymphoma on the other. However, the IG gene rearrangement patterns in B-ALL are quite different from those in precursor-B ALL (Table 8.5), which is in line with their SmIg expression. Moreover, recently acquired data indicate that both show somatically mutated IG genes,140,141 indicating that these lymphomas and leukemias are derived from or related to (post)follicular B cells. Because of the strong oncogenetic relationship between Burkitt lymphoma and B-ALL, it seems that BALL is not related to precursor-B ALL, but rather represents a more mature B-cell malignancy. The term “B-ALL” should therefore be regarded as a misnomer that should be replaced by “Burkitt leukemia.”141

TCR/IG gene rearrangements in T-ALL The immunophenotypes of a large proportion of the lymphoblastic T-cell malignancies (T-ALL and T-LBL) are fully comparable to those of cortical thymocytes, as evidenced from TdT positivity and CD1 antigen expression.5,7,12 A minority of cases are CD1− immature T-ALL, either CD5− (pro-T-ALL) or CD5+ (pre-T-ALL). Based on surface membrane expression of the TCR-CD3 complex, T-ALL and T-LBL can be classified into CD3− , TCR + , and TCR
+ subgroups, which show major differences in TCR gene rearrangement patterns (Table 8.5)5,8,12 Although the frequency of TCR gene rearrangements in the total group of T-ALL is very high, about 10% of CD3− T-ALLs still have all TCR genes in a germline configuration,5,8,12 mainly the immature CD1− /CD3− TALL cases in the pro-T-ALL subgroup. The TCRD genes in CD3− T-ALL are rearranged in most cases (∼80%) and contain biallelic deletions in about 10% of cases.12,29 As expected, all TCR + T-ALLs have TCRG and TCRD gene rearrangements, while the vast majority (∼95%) also contain TCRB gene rearrangements (Table 8.5).12,29,90,142 All TCR
+ T-ALLs contain TCRB and TCRG gene rearrangements and have at least one deleted TCRD allele (= TCRA

rearrangement); the second TCRD allele is also deleted in two-thirds of cases (Table 8.5).8,29 Cross-lineage IG gene rearrangements are not very common in T-ALL (20%) and only involve IGH genes (particularly incomplete DH –JH rearrangements).8,143 Comparative studies of gene rearrangement patterns at diagnosis and relapse in T-ALL patients revealed that secondary rearrangements can occur in 20% and 15% of cases with TCRB and TCRG gene rearrangements, respectively; TCRD gene rearrangements in T-ALL appeared to be completely stable.133 Oligoclonality is rarely seen at diagnosis in T-ALL,8,133 except for a few CD3− T-ALLs showing  REC – J
rearrangements, normally known to be involved in TCRD gene deletions.47 Detailed studies in these TALL cases revealed that the detected  REC - J
rearrangements were fully polyclonal and that they should be interpreted as continuing rearrangements aiming at TCR
 expression. Indeed, one of the analyzed CD3− T-ALLs contained a leukemic TCR
+ subpopulation as detected by TCR
/TdT double immunofluorescence staining.125

Cross-lineage IG/TCR gene rearrangements in AML In AML cross-lineage IG and/or TCR gene rearrangements are observed in 10% to 15% of cases. This mainly concerns the IGH and TCRD loci, but TCRB and TCRG gene rearrangements,124,144–146 and even a few cases with IGK gene rearrangements have been observed (Table 8.5).124

Aberrant and oncogenic IG/TCR gene rearrangements It has become increasingly clear in recent years that IG and TCR loci cannot only be involved in physiological recombination in ALL, but also in so-called illegitimate or aberrant rearrangements. Classical examples include t(8;14)(q24;q32) and the t(2;8)(q11;q24) and t(8;22)(q24;q11) variants, in which IGH (or IGK or IGL) recombine to the MYC gene and which are found in Burkitt lymphoma, and the t(14;18)(q32;q21) (with coupling of IGH to BCL2), which is seen in follicular lymphoma (reviewed in Willis and Dyer 147 ). One type of aberrant TCR rearrangement is found in ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, as well as T-cell neoplasms that develop in AT and NBS patients. This concerns the socalled trans-rearrangements between TCRB and TCRG loci through t(7;7) or inversion 7.148–151 Though described in a T-ALL case as well,152 these trans-rearrangements are not believed to play a direct role in oncogenesis; rather, they are rather considered to be a general indicator of genomic instability with increased risk of lymphoma development.

Immunoglobulin and T-cell receptor gene rearrangements

Table 8.6 Nonrandom chromosomal aberrations in T-ALL Chromosomal aberration

Relative frequency

Involved gene

Involved TCR gene

1p32 aberrations t(11;14)(p13;q11)/t(7;11)(q35;p13) t(11;14)(p15;q11) t(10;14)(q24;q11)/t(7;10)(q35;q24) t(8;14)(q24;q11) t(7;9)(q34;q32) t(7;9)(q34;q34) t(1;7)(p32;q34) t(7;19)(q34;p13) t(5;14) (q35;q11) or t(5;14)(q35;q32)

20–25% 7% 1% 4% 2% 2% 2% 1% 1% 15–20%

TAL1 LMO2 LMO1 HOX11 MYC TAL2 TAN1 LCK LYL1 HOX11L2/(RANBP17)

TCRD / TCRB TCRD / TCRB TCRD TCRD / TCRB TCRD TCRB TCRB TCRB TCRB TCRD or CTIP2

Another type concerns the oncogenic rearrangements between (mostly) TCR loci and (proto)oncogenes, which are often located on different chromosomes (Table 8.6). Among the best-known examples are the t(1;7)(p32;q34q35) and t(1;14)(p32;q11), both leading to overexpression of the TAL1 transcription factor,153–155 and t(11;14)(p15;q11) and t(11;14)(p13;q11), leading to LMO1 or LMO2 overexpression, respectively.156,157 Recently, another translocation, t(5;14)(q35;q32), resulting in overexpression of the HOX11L2 orphon homeobox gene, has been described; the t(5;14) is a frequent but cryptic translocation, which was shown to be correlated with poor outcome.158,159 It can be predicted that in the coming years yet other TCR gene translocations will be found through new experimental approaches including FISH, SKY analysis, and ligationmediated PCR (LM-PCR) or long-distance inverse PCR (LDI-PCR) methods. Although their exact prognostic value remains to be established, it can be anticipated that these illegitimate TCR gene recombinations will be important for further classification of T-cell malignancies, particularly T-ALL.

Applications of IG/TCR gene analysis in diagnostic clonality studies Rearranged IG/TCR genes are present in virtually all lymphoid leukemias (Table 8.5), making them good targets for Southern blot- and PCR-based clonality studies.3,8,160–162 In our experience, Southern blot analysis of IGH, IGK, and TCRB genes is relatively easy and reveals unique rearrangement patterns in each clonal lymphoproliferation due to the extensive combinatorial repertoire of these genes. Because of their high frequency (Table 8.5), analysis of IGH, IGK, and TCRB gene rearrangements is highly informative in the vast majority of cases.3,78,79 Southern blot analysis of IGL, TCRG, and TCRD genes is less suitable for diagnostic

clonality studies, due to the lower frequencies of IGL gene rearrangements8 and the limited combinatorial repertoire of TCRG and TCRD loci, resulting in only a small number of differently rearranged bands (Table 8.1).26,29 Until recently, PCR-based diagnostic clonality studies largely focused on IGH and TCRG gene rearrangements,91–93,105,108 because of the high frequency of these rearrangements in lymphoid leukemias (see Table 8.5) and because of the relatively limited number of primer sets that are required for the detection of these rearrangements, even at the DNA level. In 2003, the BIOMED-2 multiplex PCR tubes became available for fast and easy detection of IGH, IGK, IGL, TCRB, TCRG, and TCRD rearrangement.96,97 Although cytomorphology and immunophenotyping are generally sufficient for making the appropriate diagnosis in suspect lymphoproliferations, diagnostic problems might arise, especially if the suspect cell population is small or if the proliferation concerns mature T cells. In such cases IG/TCR clonality studies are very reliable for discriminating between polyclonal and monoclonal lymphoproliferations. Additional applications include: detection of two or more subclones within a malignancy at diagnosis; analysis of the clonal relationship between two lymphoid malignancies in a single patient; detection of clonal evolution at relapse; and analysis of the differentiation lineage of a malignancy (Table 8.7).8 Given the detection limits of the Southern blot and PCR assays, small clonal cell populations might be missed, if their relative size is less than 1% to 10% (Table 8.7).3,8

Discrimination between polyclonal and monoclonal lymphoproliferations Flow cytometric immunophenotyping is generally sufficient to study clonality in childhood leukemias. Analysis of IG/TCR gene rearrangements should only be

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Table 8.7 Diagnostic applications of IG/TCR gene rearrangement analysis 1.

2. 3.

4. 5.

Discrimination between polyclonal and monoclonal lymphoid cell populations. Caution: monoclonality does not necessarily imply clinical malignancy [e.g. (oligo)clonal lymphoproliferations in immunodeficient patients]. Detection of two or more subclones within one lymphoid malignancy. Analysis of lymphoid malignancies at diagnosis and subsequent relapses: • detection of identical IG/TCR gene rearrangements; • detection of differences: clonal evolution at first relapse and/or subsequent relapses. Assignment or exclusion of the differentiation lineage of a lymphoid malignancy. Caution: cross-lineage IG/TCR gene rearrangements. Detection of low numbers of malignant lymphoid cells. Caution: detection limit of the Southern blot technique is 5–10%; detection limits of PCR heteroduplex and GeneScan analysis are ∼5% and 1–5%, respectively.a

The sensitivity of the PCR technique can be increased to 10−4 to 10−6 , if junctional region specific probes are used (see Chapter 28). a

considered in cases where no discrimination is possible between, for example, regenerating bone marrow postinduction or postmaintenance therapy and an imminent precursor-B ALL relapse. Regenerating bone marrow can contain high frequencies of TdT+ /CD10+ /CD19+ precursor-B cells that might be mistaken for leukemic cells. In such cases Southern blot and/or PCR heteroduplex or GeneScan analysis of IGH and/or IGK gene rearrangements can solve the problem.

Detection of two or more subclones in a leukemia IGH gene studies in precursor-B ALL reveal that 30% to 40% of cases contain subclones at diagnosis, as deduced from the presence of multiple rearranged bands in Southern blot analysis (Fig. 8.14A).126 Such subclone formation might also be detectable at the IGK gene level, but cross-lineage TCRB and TCRG gene rearrangements generally confirm the common clonal origin of the precursor-B ALL.126,127,133 By contrast, TCRD gene rearrangements generally seem to be monoclonal in Southern blot studies, whereas PCR-based studies indicate that they are frequently oligoclonal.134,135 Although the presence of multiple rearranged IGH or IGK alleles can be studied by PCR-based techniques, it generally remains difficult to conclude whether or not the identified rearrangements belong to subclones. Besides, it is also impossible to discriminate between rearranged bands derived from a subclone or from an additional chromosome based on signal intensity of the PCR product. For identification of subclones at the IGH or IGK gene level, Southern blot analysis is therefore still the preferred method.

Analysis of lymphoid malignancies at diagnosis and subsequent relapse Analysis of IG/TCR gene rearrangement patterns at diagnosis and throughout the disease course can prove whether the recurrent disease represents a true relapse or a secondary malignancy. This is especially true for cases where the histomorphologic and/or clinical characteristics of a B-cell malignancy are changed at relapse, while the IGH, IGK, and IGL gene rearrangement patterns remain identical.133,163,164 However, it should be noted that changes in IG/TCR gene rearrangement patterns at relapse can also occur in precursor-B ALL and T-ALL due to progressive and secondary rearrangements (Fig. 8.14B).57,58,128,130,133,138,165 The issue of true relapse or secondary malignancy is sometimes even more difficult to solve by PCR-based techniques. Use of primer sets for amplification of gene rearrangements identified in the malignancy at diagnosis often also allows amplification of similar rearrangements in a secondary malignancy, making it virtually impossible to distinguish between similar PCR products derived from a relapse and an unrelated malignancy. PCR-based comparison of the original malignancy at diagnosis and the newly identified malignancy, is possible, however, when PCR products from both malignancies are mixed and evaluated in comparative heteroduplex analysis (Fig. 8.14C). In case of a relapse, mixing will result in a single homoduplex band upon heteroduplex analysis, whereas in the case of a secondary malignancy, two distinct homoduplexes as well as two dominant heteroduplexes are found. These latter heteroduplexes result from cross-renaturation of singlestrand molecules of the two different malignant cell populations. Nevertheless, even these mixing experiments at

Immunoglobulin and T-cell receptor gene rearrangements

diagnosis and relapse should be interpreted with caution in ALL cases, because progressive and secondary rearrangements might mask the presence of, for example, a common DH –JH stem in two seemingly different complete VH (DH )JH gene rearrangements.138,166 This issue can be solved only by sequence comparison of the rearrangements at diagnosis and relapse.166

Determination of differentiation lineage In some rare undifferentiated leukemias, immunologic marker analysis might not be sufficient to determine the differentiation lineage of the leukemic cells. It has been suggested that detection of IG/TCR gene rearrangements can be used for lineage assignment, but the cross-lineage IG/TCR gene rearrangements in ALL and AML limit this application.8,117,124,125 On the other hand, the presence of germline IG/TCR genes can occasionally support the nonlymphoid or early (very immature) lymphoid character of a malignancy.

Detection of low numbers of malignant cells

Fig. 8.14 (A) Southern blot analysis of IGH genes in precursor-B ALL at diagnosis, using the IGHJ6 probe in combination with the restriction enzyme BglII. Multiple rearranged bands are present in each precursor-B-ALL lane. In one patient (SL) this is due to trisomy 14, but in all other cases subclone formation (biclonality or oligoclonality) has occurred. (B) Comparative analysis at diagnosis and relapse with BglII digests in combination with the IGHJ6 probe. Multiple rearranged bands of different density are present in both digests at diagnosis. However, at relapse, only two rearranged bands are detectable, which appear identical in size to the two faint rearranged bands seen at diagnosis. (C) Comparative heteroduplex analysis of IGH gene rearrangements. Monoclonal homoduplexes (ho) in patients 5766, 6335, and 6124 found at diagnosis and at relapse were of the same size. Mixing of the PCR products of these disease phases followed by heteroduplex PCR analysis demonstrated no heteroduplex (he) formation, proving that these gene rearrangements had identical junctional regions. In patient 5784, monoclonal homoduplexes found at diagnosis and at relapse

The detection limit of routinely performed Southern blot analysis is approximately 5–10%.78,79,82,83 The PCR heteroduplex and PCR GeneScan strategies have at least a comparable but often slightly lower detection limit, 1–5%, provided that the “background” of normal polyclonal B and T cells is not too high.91,92,105,109,111 For many leukemias, such detection limits can also be reached with cytomorphology and immunophenotyping. Nevertheless, in some situations IG/TCR gene analysis might be quite valuable, as in discrimination between regenerating bone marrow with high frequencies of precursor-B cells after withdrawal of cytostatic treatment and an imminent relapse of precursor-B-ALL167,168 and for diagnosing smoldering leukemias.169 The sensitivity of PCR-based analyses can be increased to 10−4 to 10−6 (100 to 1 leukemic cells between 106 normal cells), if junctional region-specific oligonucleotide probes are used for detection of the leukemia-derived PCR products.87–90,94,95,169,170 This approach might be valuable for evaluation of the effectiveness of the applied treatment in leukemia patients. For this purpose, the junctional regions of rearranged IG/TCR genes of the leukemic slightly differed in size. Mixing of the VH 4-JH PCR products followed by heteroduplex PCR analysis demonstrated clear heteroduplex formation, proving that these VH 4–JH gene rearrangements had different junctional regions; ss, remaining single-strand fragments.

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cells at diagnosis have to be sequenced in order to design leukemia-specific junctional region probes171,172 Currently, real-time quantitative PCR techniques are being used to monitor the kinetics of leukemia reduction more closely.139,171,173 (see also Chapter 28).

Conclusions Both Southern blot analysis and PCR-based strategies allow a detailed study of IG/TCR gene rearrangements in childhood leukemias due to the combinatorial diversity and junctional diversity of the gene rearrangements, respectively. Both techniques can be used for diagnostic clonality studies, if appropriate probe/restriction enzyme combinations and appropriate primer sets are used. PCRbased analysis can be performed in a reliable way only if amplification is followed by heteroduplex or GeneScan analysis to distinguish between polyclonality and monoclonality. Southern blotting can be applied routinely in analysis of IGH, IGK, TCRB, and TCRG loci, whereas PCR analyses historically have focussed on junctional regions of IGH and TCRG gene rearrangements. However, the recently developed BIOMED-2 multiplex PCR tubes now allow fast and easy detection of polyclonal versus monoclonal IGH, IGK, IGL, TCRB, TCRG, and TCRD rearrangements for clonality diagnostics in the various types of lymphoid malignancies.96,97 In precursor-B ALL, IGK (especially Kde), TCRB, IGH, TCRG, and TCRD (V 2–D 3 and D 2–D 3) gene rearrangements are being employed for PCR studies, because of their relatively high frequencies. In T-ALL, PCR studies can additionally include incomplete D 2–J 1 and complete V –J 1 gene rearrangements, which represent approximately 70% of all TCRD gene rearrangements in T-ALL, as well as incomplete and complete TCRB gene rearrangements. In lymphoma diagnostics, IGH and IGK genes are excellent PCR targets for clonality assessment in B-cell lymphomas, whereas TCRG and TCRB genes can be used as PCR targets for clonality assessment in T-cell lymphomas. Analysis of IG/TCR gene rearrangements can be employed for clonality assessment in cases in which discrimination between polyclonality and monoclonality is difficult. Another application concerns further classification of lymphoid malignancies, especially in the case of aberrant and oncogenic IG/TCR rearrangements. Finally, PCR-based detection of leukemia-specific IG/TCR junctional regions is a sensitive method for MRD detection in follow-up samples of patients treated for a lymphoid malignancy (see also Chapter 28).

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isotype gene rearrangements by Southern blot analysis. Leukemia, 1996; 10: 1834–9. Langerak, A. W., Wolvers-Tettero, I. L. M., & Dongen, J. J. M. van. Detection of T cell receptor beta (TCRB) gene rearrangement patterns in T cell malignancies by Southern blot analysis. Leukemia, 1999; 13: 965–74. Moreau, E. J., Langerak, A. W., Gastel-Mol, E. J. van, et al. Easy detection of all T cell receptor gamma (TCRG) gene rearrangements by Southern blot analysis: recommendations for optimal results. Leukemia, 1999; 13: 1620–26. ¨ Tumkaya, T., Burg, M. van der, Garcia Sanz, R., et al. Immunoglobulin lambda isotype gene rearrangements in Bcell malignancies. Leukemia, 2001; 15: 121–7. White, T. J., Arnheim, N., & Erlich, H. A. The polymerase chain reaction. Trends Genet, 1989; 5: 185–9. Newton, C. R. & Graham, A. PCR (Oxford, UK: BIOS Scientific Publishers, 1994). Yamada, M., Hudson, S., Tournay, O., et al. Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third-complementarity-determining region (CDR-III)-specific probes. Proc Natl Acad Sci USA, 1989; 86: 5123–7. d’Auriol, L., Macintyre, E., Galibert, F., & Sigaux, F. In vitro amplification of T cell gamma gene rearrangements: a new tool for the assessment of minimal residual disease in acute lymphoblastic leukemias. Leukemia, 1989; 3: 155–8. Macintyre, E. A., d’Auriol, L., Duparc, N. et al. Use of oligonucleotide probes directed against T cell antigen receptor gamma delta variable-(diversity)-joining junctional sequences as a general method for detecting minimal residual disease in acute lymphoblastic leukemias. J Clin Invest, 1990; 86: 2125–35. Breit, T. M., Wolvers-Tettero, I. L. M., H¨ahlen, K., Wering, E. R. van, & Dongen, J. J. M. van. Extensive junctional diversity of gd T-cell receptors expressed by T-cell acute lymphoblastic leukemias: implications for the detection of minimal residual disease. Leukemia, 1991; 5: 1076–86. Deane, M. & Norton, J. D. Immunoglobulin heavy chain variable region family usage is independent of tumor cell phenotype in human B lineage leukemias. Eur J Immunol, 1990; 20: 2209–17. Deane, M., Pappas, H., & Norton, J. D. Immunoglobulin heavy chain gene fingerprinting reveals widespread oligoclonality in B-lineage acute lymphoblastic leukaemia. Leukemia, 1991; 5: 832–8. Veelken, H., Tycko, B., & Sklar, J. Sensitive detection of clonal antigen receptor gene rearrangements for the diagnosis and monitoring of lymphoid neoplasms by a polymerase chain reaction-mediated ribonuclease protection assay. Blood, 1991; 78: 1318–26. Hansen-Hagge, T. E., Yokota, S., & Bartram, C. R. Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences. Blood, 1989; 74: 1762–7.

95 Jonsson, O. G., Kitchens, R. L., Scott, F. C., & Smith, R. G. Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes. Blood, 1990; 76: 2072–9. ¨ 96 Dongen, J. J. M. van, Langerak, A. W., Bruggemann, M., et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene rearrangements in suspect lymphoproliferations. Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–317. 97 Langerak, A. W., San Miguel, J. F., Parreira, A., et al. Clonality analysis in malignant lymphoma: the BIOMED-2 experience. Histopathology, 2002; 41S2: 506–8. 98 Oksenberg, J. R., Stuart, S., Begovich, A. B., et al. Limited heterogeneity of rearranged T-cell receptor V alpha transcripts in brains of multiple sclerosis patients. Nature, 1991; 353: 94. 99 Broeren, C. P., Verjans, G. M., Eden, W. van, et al. Conserved nucleotide sequences at the 5 end of T cell receptor variable genes facilitate polymerase chain reaction amplification. Eur J Immunol, 1991; 21: 569–75. 100 Doherty, P. J., Roifman, C. M., Pan, S. H., et al. Expression of the human T cell receptor V beta repertoire. Mol Immunol, 1991; 28: 607–12. 101 Wei, S., Charmley, P., Robinson, M. A., & Concannon, P. The extent of the human germline T-cell receptor V beta gene segment repertoire. Immunogenetics, 1994; 40: 27–36. 102 Oostveen, J. W. van, Breit, T. M., de Wolf, J. T., et al. Polyclonal expansion of T-cell receptor-gd+ T lymphocytes associated with neutropenia and thrombocytopenia. Leukemia, 1992; 6: 410–18. 103 Davis, T. H., Yockey, C. E., & Balk, S. P. Detection of clonal immunoglobulin gene rearrangements by polymerase chain reaction amplification and single-strand conformational polymorphism analysis. Am J Pathol, 1993; 142: 1841–7. 104 Koch, O. M., Volkenandt, M., Goker, E., et al. Molecular detection and characterization of clonal cell populations in acute lymphocytic leukemia by analysis of conformational polymorphisms of cRNA molecules of rearranged T-cell-receptorgamma and immunoglobulin heavy-chain genes. Leukemia, 1994; 8: 946–52. 105 Bourguin, A., Tung, R., Galili, N., & Sklar, J. Rapid, nonradioactive detection of clonal T-cell receptor gene rearrangements in lymphoid neoplasms. Proc Natl Acad Sci U S A, 1990; 87: 8536–40. 106 Wood, G. S., Tung, R. M., Haeffner, A. C., et al. Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE). J Invest Dermatol, 1994; 103: 34–41. 107 Linke, B., Pyttlich, J., Tiemann, M., et al. Identification and structural analysis of rearranged immunoglobulin heavy chain genes in lymphomas and leukemias. Leukemia, 1995; 9: 840–7. 108 Bottaro, M., Berti, E., Biondi, A., Migone, N., & Crosti, L. Heteroduplex analysis of T-cell receptor gamma gene

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rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas. Blood, 1994; 83: 3271–8. Langerak, A. W., Szczepanski, T., Burg, M. van der, WolversTettero, I. L. M., & Dongen, J. J. M. van. Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations. Leukemia, 1997; 11: 2192–9. Kneba, M., Bolz, I., Linke, B., & Hiddemann, W. Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis. Blood, 1995; 86: 3930–7. Linke, B., Bolz, I., Fayyazi, A., et al. Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes. Leukemia, 1997; 11: 1055–62. Visser, O., Coebergh, J. W. W., Schouten, L. J., & Dijck, J. A. A. M. Incidence of Cancer in the Netherlands 1995 (Utrecht, the Netherlands: Vereniging van Integrale Kankercentra, 1998). Dongen, J. J. M. van, Szczepanski, T., & Adriaansen, H. J. Immunobiology of leukemia. In E. S. Henderson, T. A. Lister, & M. F. Greaves, eds., Leukemia (Philadelphia, PA: W. B. Saunders, 2002), pp. 85–129. Sandlund, J. T., Downing, J. R., & Crist, W. M. Non-Hodgkin’s lymphoma in childhood. N Engl J Med, 1996; 334: 1238–48. Gouttefangeas, C., Bensussan, A., & Boumsell, L. Study of the CD3-associated T-cell receptors reveals further differences between T-cell acute lymphoblastic lymphoma and leukemia. Blood, 1990; 75: 931–4. Korsmeyer, S. J., Arnold, A., Bakhshi, A., et al. Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins. J Clin Invest, 1983; 71: 301–13. Felix, C. A., Wright, J. J., Poplack, D. G., et al. T cell receptor alpha-, beta-, and gamma-genes in T cell and pre-B cell acute lymphoblastic leukemia. J Clin Invest, 1987; 80: 545–56. Foroni, L., Catovsky, D., & Luzzatto, L. Immunoglobulin gene rearrangements in hairy cell leukemia and other chronic B cell lymphoproliferative disorders. Leukemia, 1987; 1: 389–92. Williams, M. E., Innes, D. J., Jr., Borowitz, M. J., et al. Immunoglobulin and T cell receptor gene rearrangements in human lymphoma and leukemia. Blood, 1987; 69: 79–86. Furley, A. J., Mizutani, S., Weilbaecher, K., et al. Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex. Cell, 1986; 46: 75–87. Foroni, L., Foldi, J., Matutes, E., et al. Alpha, beta and gamma T-cell receptor genes: rearrangements correlate with haematological phenotype in T cell leukaemias. Br J Haematol, 1987; 67: 307–18. Dongen, J. J. M. van, Quertermous, T., Bartram, C. R., et al. T cell receptor-CD3 complex during early T cell differentiation. Analysis of immature T cell acute lymphoblastic leukemias (TALL) at DNA, RNA, and cell membrane level. J Immunol, 1987; 138: 1260–9. Greaves, M. F., Chan, L. C., Furley, A. J., Watt, S. M., & Molgaard, H. V. Lineage promiscuity in hemopoietic differentiation and leukemia. Blood, 1986; 67: 1–11.

124 Adriaansen, H. J., Soeting, P. W., Wolvers-Tettero, I. L. M., Dongen, J. J. M. van. Immunoglobulin and T-cell receptor gene rearrangements in acute non-lymphocytic leukemias. Analysis of 54 cases and a review of the literature. Leukemia, 1991; 5: 744–51. 125 Szczepanski, T., Beishuizen, A., Pongers-Willemse, M. J., et al. Cross-lineage T-cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B-acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease. Leukemia, 1999; 13: 196–205. 126 Beishuizen, A., H¨ahlen, K., Hagemeijer, A., et al. Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursor B-cell origin. Leukemia, 1991; 5: 657–67. 127 Beishuizen, A., Wering, E. R. van, Breit, T. M., et al. Molecular biology of acute lymphoblastic leukemia: implications for detection of minimal residual disease. In W. Hiddeman, ¨ T. Buchner, B. W¨ormann, eds., Acute Leukemias V (Berlin: Springer, 1996), pp. 460–74. 128 Bird, J., Galili, N., Link, M., Stites D., & Sklar, J. Continuing rearrangement but absence of somatic hypermutation in immunoglobulin genes of human B cell precursor leukemia. J Exp Med, 1988; 168: 229–45. 129 Steward, C. G., Goulden, N. J., Katz, F., et al. A polymerase chain reaction study of the stability of Ig heavy-chain and T-cell receptor delta gene rearrangements between presentation and relapse of childhood B-lineage acute lymphoblastic leukemia. Blood, 1994; 83: 1355–62. 130 Wasserman, R., Yamada, M., Ito, Y., et al. VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia. Blood, 1992; 79: 223–8. 131 Kitchingman, G. R. Immunoglobulin heavy chain gene VH-D junctional diversity at diagnosis in patients with acute lymphoblastic leukemia. Blood, 1993; 81: 775–82. 132 Steenbergen, E. J., Verhagen, O. J., Leeuwen, E. F. van, Borne, A. E. von dem, Schoot, C. E. van der. Distinct ongoing Ig heavy chain rearrangement processes in childhood B-precursor acute lymphoblastic leukemia. Blood, 1993; 82: 581–9. 133 Szczepanski, T., Willemse, M. J., Brinkhof, B., et al. Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B-ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease. Blood, 2002; 99: 2315–23. 134 Steenbergen, E. J., Verhagen, O. J., Leeuwen, E. F. van, et al. Frequent ongoing T-cell receptor rearrangements in childhood B-precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease. Blood, 1995; 86: 692–702. 135 Ghali, D. W., Panzer, S., Fischer, S., et al. Heterogeneity of the T-cell receptor delta gene indicating subclone formation in acute precursor B-cell leukemias. Blood, 1995; 85: 2795–801. 136 Hansen-Hagge, T. E., Yokota, S., Reuter, H. J., Schwarz, K., & Bartram, C. R. Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine

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their T-cell receptor delta/alpha regions along a hierarchically ordered pathway. Blood, 1992; 80: 2353–62. Taylor, J. J., Rowe, D., Kylefjord, H., et al. Characterisation of non-concordance in the T-cell receptor gamma chain genes at presentation and clinical relapse in acute lymphoblastic leukemia. Leukemia, 1994; 8: 60–6. Szczepanski, T., Willemse, M. J., Brinkhof, B., et al. Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B-ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease. Blood, 2002; 99: 2315–23. Velden, V. H. J. van der, Willemse, M. J., Schoot, C. E. van der, Wering, E. R. van, & Dongen, J. J. M. van. Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR. Leukemia, 2002; 16: 928–36. Chapman, C. J., Zhou, J. X., Gregory, C., Rickinson, A. B., & Stevenson, F. K. VH and VL gene analysis in sporadic Burkitt’s lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection. Blood, 1996; 88: 3562–8. Burg, M. van der, Barendregt, B. H., Wering, E. R. van, et al. The presence of somatic mutations in immunoglobulin genes of Bcell acute lymphoblastic leukemia (ALL-L3) supports assignment as Burkitt’s leukemia-lymphoma rather than B-lineage ALL. Leukemia, 2001; 15: 1141–3. Langerak, A. W., Wolvers-Tettero, I. L. M., Beemd, M. W. M. van den, et al. Immunophenotypic and immunogenotypic characteristics of TCR gammadelta+ T cell acute lymphoblastic leukemia. Leukemia, 1999; 13: 206–14. Szczepanski, T., Pongers-Willemse, M. J., Langerak, A. W., et al. Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH727 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor ab lineage. Blood, 1999; 93: 4079–85. Breit, T. M., Verschuren, M. C. M., Wolvers-Tettero, I. L. M., et al. Human T cell leukemias with continuous V(D)J recombinase activity for TCR-delta gene deletion. J Immunol, 1997; 159: 4341–9. Schmidt, C. A., Oettle, H., Neubauer, A., et al. Rearrangements of T-cell receptor delta, gamma and beta genes in acute myeloid leukemia coexpressing T-lymphoid features. Leukemia, 1992; 6: 1263–7. Boeckx, N., Willemse, M. J., Szczepanski, T., et al. Fusion gene transcripts and Ig/TCR gene rearrangements are complementary but infrequent targets for PCR-based detection of minimal residual disease in acute myeloid leukemia. Leukemia, 2002; 16: 368–75. Willis, T. G. & Dyer, M. J. The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood, 2000; 96: 808–22. Hinz, T., Allam, A., Wesch, D., Schindler, D., & Kabelitz, D. Cellsurface expression of transrearranged Vgamma-Cbeta T-cell

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receptor chains in healthy donors and in ataxia telangiectasia patients. Br J Haematol, 2000; 109: 201–10. Kobayashi, Y., Tycko, B., Soreng, A. L., & Sklar, J. Transrearrangements between antigen receptor genes in normal human lymphoid tissues and in ataxia telangiectasia. J Immunol, 1991; 147: 3201–9. Retiere, C., Halary, F., Peyrat, M. A., et al. The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR. J Immunol, 1999; 162: 903–10. Stern, M. H., Lipkowitz, S., Aurias, A., et al. Inversion of chromosome 7 in ataxia telangiectasia is generated by a rearrangement between T-cell receptor beta and T-cell receptor gamma genes. Blood, 1989; 74: 2076–80. Bernard, O., Groettrup, M., Mugneret, F., Berger, R., & Azogui, O. Molecular analysis of T-cell receptor transcripts in a human T-cell leukemia bearing a t(1;14) and an inv(7); cell surface expression of a TCR-beta chain in the absence of alpha chain. Leukemia, 1993; 7: 1645–53. Begley, C. G., Aplan, P. D., Denning, S. M., et al. The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif. Proc Natl Acad Sci U S A, 1989; 86: 10 128–32. Breit, T. M., Mol, E. J., Wolvers-Tettero, I. L. M., et al. Sitespecific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes. J Exp Med, 1993; 177: 965–77. Fitzgerald, T. J., Neale, G. A., Raimondi, S. C., & Goorha, R. M. c-tal, a helix-loop-helix protein, is juxtaposed to the Tcell receptor-beta chain gene by a reciprocal chromosomal translocation: t(1;7)(p32;q35). Blood, 1991; 78: 2686–95. Boehm, T., Foroni, L., Kaneko, Y., Perutz, M. F., & Rabbitts, T. H. The rhombotin family of cysteine-rich LIM-domain oncogenes: distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13. Proc Natl Acad Sci U S A, 1991; 88: 4367–71. Garcia, I. S., Kaneko, Y., Gonzalez-Sarmiento, R., et al. A study of chromosome 11p13 translocations involving TCR beta and TCR delta in human T cell leukaemia. Oncogene, 1991; 6: 577–82. Bernard, O. A., Busson-LeConiat, M., Ballerini, P., et al. A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia, 2001; 15: 1495–1504. Mauvieux, L., Leymarie, V., Helias, C., et al. High incidence of Hox11L2 expression in children with T-ALL. Leukemia, 2002; 16: 2417–22. Przybylski, G., Oettle, H., Ludwig, W. D., Siegert, W., & Schmidt, C. A. Molecular characterization of illegitimate TCR delta gene rearrangements in acute myeloid leukaemia. Br J Haematol, 1994; 87: 301–7. O’Connor, N., Gatter, K. C., Wainscoat, J. S., et al. Practical value of genotypic analysis for diagnosing lymphoproliferative disorders. J Clin Pathol, 1987; 40: 147–150.

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162 Korsmeyer, S. J. Antigen receptor genes as molecular markers of lymphoid neoplasms. J Clin Invest, 1987; 79: 1291–5. 163 Kneba, M., Bolz, I., Linke, B., et al. Characterization of clonespecific rearranged T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing. Blood, 1994; 84: 574–81. 164 Siegelman, M. H., Cleary, M. L., Warnke, R., & Sklar, J. Frequent biclonality and Ig gene alterations among B cell lymphomas that show multiple histologic forms. J Exp Med, 1985; 161: 850–63. 165 Wering, E. R. van, Beishuizen, A., Roeffen, E. T., et al. Immunophenotypic changes between diagnosis and relapse in childhood acute lymphoblastic leukemia. Leukemia, 1995; 9: 1523–33. 166 Szczepanski, T., Willemse, M. J., Kamps, W. A., et al. Molecular discrimination between relapsed and secondary acute lymphoblastic leukemia – proposal for an easy strategy. Med Pediatr Oncol, 2001; 36: 352–8. 167 Smedmyr, B., Bengtsson, M., Jakobsson, A., et al. Regeneration of CALLA (CD10+), TdT+ and double-positive cells in the bone marrow and blood after autologous bone marrow transplantation. Eur J Haematol, 1991; 46: 146–51. 168 Wering, E. R. van, Linden-Schrever, B. E. van der, Szczepanski, T., et al. Regenerating normal B-cell precursors during and

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after treatment of acute lymphoblastic leukaemia: implications for monitoring of minimal residual disease. Br J Haematol, 2000; 110: 139–46. Knulst, A. C., Adriaansen, H. J., Hahlen, K., et al. Early diagnosis of smoldering acute lymphoblastic leukemia using immunological marker analysis. Leukemia, 1993; 7: 532–6. Campana, D., Yokota, S., Coustan-Smith, E., et al. The detection of residual acute lymphoblastic leukemia cells with immunologic methods and polymerase chain reaction: a comparative study. Leukemia, 1990; 4: 609–14. Pongers-Willemse, M. J., Verhagen, O. J. H. H, Tibbe, G. J. M., et al. Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia, 1998; 12: 2006–14. Dongen, J. J. M. van, Seriu, T., Panzer-Grumayer, E. R., et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet, 1998; 352: 1731–8. Velden, V. H. J. van der, Wijkhuijs, J. M., Jacobs, D. C. H., et al. T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis. Leukemia, 2002; 16: 1372–80.

9 Cytogenetics of acute leukemias Susana C. Raimondi

Introduction Today, acute leukemia in children is managed through the use of risk-adapted therapy, requiring sensitive methods to detect the presence or absence of particular chromosomal abnormalities, one of the most important factors in stratifying patients by risk groups. Identifying genes involved in recurrent chromosomal abnormalities and understanding the roles of these genes in regulating cell growth and inducing malignant transformation can provide important insights into the altered biology of leukemic cells and potentially lead to improved treatment.1 In this chapter, I review the most common conventional and molecular cytogenetic characteristics of the childhood acute leukemias and discuss their impact on clinical management strategies. Considerable attention is paid to abnormalities that were only recently identified in the lymphoid and myeloid leukemias, including the fusion genes involving the MLL (11q23), ETV6 (12p13), and CBFA2 (21q22) loci.

Conventional cytogenetics Standard chromosomal analysis remains the method of choice for the initial screening for karyotypic abnormalities in leukemic cells. Conventional cytogenetic studies detect chromosomal abnormalities only in clones of mitotically active (metaphase) neoplastic cells and are particularly efficient in identifying abnormalities associated with acute leukemias in children and adolescents. These methods detect an abnormal clone in 90% of patients with acute lymphoblastic leukemia (ALL) and 80% of patients

with acute myeloid leukemia (AML). They also permit the study of all complex cytogenetic changes present in neoplastic cells, although complementary genetic methods are needed to detect cryptic abnormalities or to evaluate equivocal results.

Molecular cytogenetics Unlike conventional cytogenetics, molecular methods of chromosome analysis can be applied to interphase (nonmitotic) nuclei and metaphase chromosomes. Interphase cytogenetic analysis facilitates screening of large numbers of nondividing cells or terminally differentiated cells, or both, in a relatively short time. Because chromosomal breakpoints are highly specific, they can be detected by molecular cytogenetic methods (fluorescence in situ hybridization, FISH) or by molecular genetic methods (such as the reverse transcriptase polymerase chain reaction, RT-PCR, and Southern blot analysis) or by both. The high level of sensitivity of these strategies allows the monitoring of minimal residual disease (MRD) during clinical follow-up. With the exception of multicolor karyotyping, FISH, RT-PCR, and Southern blot analysis focus on selected abnormalities, regardless of the global complexity of the karyotype. In addition, a newly developed method called digital karyotyping quantitatively analyzes DNA copy number at high resolution and systematically detects changes on a genomic scale. The implementation of this technology will improve the resolution to which genetic alterations, especially changes in copy number, can be localized to the human genome.2

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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DNA probes Numerous DNA probes that recognize specific chromosomal rearrangements or target DNA segments or whole chromosomes are commercially available. With these probes, FISH can serve as a distinct diagnostic tool that through its ability to detect subtle chromosomal abnormalities enhances the accuracy and sensitivity of conventional cytogenetic methods. The type of probe used with FISH depends on the clinical setting and differential diagnosis. Centromeric (alpha-satellite) probes are derived from repetitive centromeric sequences unique to specific chromosomes and are used to detect trisomies or monosomies. “Painting” probes comprise multiple or composite fragments labeled with fluorochromes and represent the full length of the target chromosome. Hybridization of painting probes results in the specific visualization of whole or partial chromosomes; for this reason, such probes are used to analyze complex rearrangements. Unique-sequence probes specific for individual genes can detect alterations in gene structure or number, in either metaphase chromosomes or interphase nuclei. Subtelomeric probes are specific to unique subtelomere sequences that are within approximately 300 kb of the end of a particular chromosome. Probes of this type are used to detect the most subtle telomeric chromosome rearrangements.

marrow, because these probes accurately label the sex chromosome complement in a large number of interphase nuclei in hypocellular samples with a low mitotic index. Multicolor karyotyping methods of FISH include multiplex-FISH (M-FISH)8 and spectral karyotyping (SKY),9 in which 24 uniquely labeled human chromosomepainting probes hybridize with their targets, so that each pair of chromosomes in a spread of metaphase chromosomes appears to have a unique color. M-FISH and SKY are effective in evaluating “marker” chromosomes, because the components of the marker chromosomes can be identified on the basis of the hybridizing chromosomepainting probes. In addition, M-FISH and SKY can more accurately identify chromosomal aberrations that cannot be readily identified by conventional cytogenetic analysis (Fig. 9.1).10–14 One limitation of SKY is that it cannot detect subtle or cryptic rearrangements, a drawback that is probably due to the absence of subtelomeric sequences in available probes. A recent modification to M-FISH (IPM-FISH) is the use of interspersed PCR (IRS-PCR) painting probes; this method concurrently generates an R-banding pattern as a result of the combinatorial labeling of probes; therefore, modified M-FISH results in stronger hybridization signals at the telomeric ends, and this signal potentially increases the detection of cryptic translocations,15 such as the recently discovered t(5;14)(q35;q32) in T-cell ALL.16,17

Fluorescence in situ hybridization RT-PCR Fluorescence in situ hybridization (FISH) should be performed with metaphase chromosome preparations, because many variations of each genetic abnormality are encountered at the DNA level and because analysis of interphase nuclei alone may lead to misinterpretation of results. Furthermore, FISH analysis has shown that, in a considerable number of cases, “balanced” translocations detected by conventional cytogenetics are not balanced at all, but rather are associated with submicroscopic deletions. The clinical significance of these deletions remains unknown.3,4 FISH has substantially altered the field of cytogenetics by permitting the detection of numeric and structural aberrations in both hematologic malignancies and solid tumors, the elucidation of cryptic abnormalities (those not detected by conventional methods), the detection of MRD, the evaluation of different cell lineages in acute leukemias, and the analysis of hematopoietic chimerism after sexmismatched bone marrow transplantation (BMT).5–7 Commercially available fluorescent probes for chromosomes X and Y are very useful in quantifying engraftment of bone

The reverse transcriptase-polymerase chain reaction method has substantially improved the cytogeneticist’s ability to detect genotypic changes at diagnosis and to monitor MRD. Chimeric mRNA representing fusion genes at the site of chromosomal rearrangements serves as a leukemiaspecific marker suitable for amplification by RT-PCR. Aberrant expression is quantified by assays such as quantitative competitive PCR (QC-PCR) and real-time quantitative PCR (RQ-PCR). RT-PCR or RQ-PCR is routinely used for diagnostic screening, and in some cases they are combined in multiplex assays to reduce the number of reactions and the amount of test material required. In assessments of newly diagnosed childhood ALL, the multiplex RT-PCR assay includes oligonucleotide probes to simultaneously detect the t(9;22), BCR-ABL encoding p210 and BCR-ABL encoding p190; t(4;11), MLL-AF4; t(1;19), E2A-PBX1; and t(12;21), ETV6-CBFA2 (TEL-AML1). For assessment of newly diagnosed childhood AML, the assay detects t(8;21), AML1-ETO; inv(16), CBFB-MYH11; and t(9;11), MLL-AF9. For the simultaneous assessment

Cytogenetics of acute leukemias

Fig. 9.1 Spectral karyotyping (SKY) analysis of leukemic cells from a patient with AML-M1. G-banded karyotype of bone marrow showed the following abnormalities: 47,XX,add(5)(q33),add(7)(p22), +20[18]/46,XX[2]. SKY established two unbalanced translocations: 47,XX,der(5)t(5;6)(q33;q21),der(7)t(7;13)(p22;q14),+20. Top panel, spectral image; bottom panel, classified image. (See color plate 9.1 for full-color reproduction.)

of possible 11q23/MLL rearrangements, the assay detects t(4;11), MLL-AF4; t(9;11), MLL-AF9; t(10;11), MLL-AF10; and t(11;19), MLL-ENL or MLL-ELL.18,19 Other modified PCR methods include long-template PCR (LT-PCR), which detects breakpoints scattered over large genomic regions20 ; long-distance inverse PCR; panhandle and reverse panhandle PCR, both of which facilitate the cloning of genomic breakpoints for identification

of novel chromosomal partners21,22 ; and paired multiplex RT-PCR (PMRT-PCR), which identifies several MLL fusion genes in a single assay.23 Although these methods are accurate, they are costly and labor-intensive. Conventional cytogenetic methods, RT-PCR, and FISH are complementary and allow reliable identification of clonal rearrangements of genes and subclassification of diseases subtypes. Thus, RT-PCR may be a useful adjunct

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when cytogenetic analysis is not possible because of a lack of dividing cells. It should be noted that cytogenetic analyses of t(8;21), inv(16), or t(15;17) have a low rate of false-positive results, but false-negative results occur in rare cases when cryptic, complex rearrangements are not detected.24,25

Cytogenetic nomenclature The nomenclature of chromosomal aberrations has evolved throughout the years and is updated regularly by the International Standing Committee on Human Cytogenetic Nomenclature (ISCN); the most recent update occurred in 1995.26 Chromosomal abnormalities are defined as numeric (gain or loss of a chromosome) or structural changes. Common terms for structural aberrations are translocation (t), the exchange of material between two or more chromosomes; deletion (del), the loss of DNA from a chromosome; inversion (inv), the breaking of a chromosome into two bands, after which the segment between these two bands is inverted; the development of an isochromosome (i), duplication of an entire arm of a chromosome and loss of the other arm; and duplication (dup), duplication of a segment within a chromosome. A dicentric (dic) chromosome has two centromeres and replaces one or two normal chromosomes. The creation of a dicentric chromosome usually results in the loss of DNA material. A derivative (der) chromosome is generated by either more than one rearrangement within a single chromosome or rearrangements involving two or more chromosomes. A marker (mar) chromosome is an abnormal chromosome in which no part can be identified. The “+” symbol indicates the gain of a chromosome and the “−” symbol the loss of a chromosome. In tumor cells, clonal chromosomal aberrations (i.e. an abnormality that is derived from a single progenitor cell) occur when at least two cells have the same numeric or structural abnormality or when three cells lack the same chromosome. The modal number (MN) is the most common chromosome number in a tumor cell population. The most relevant acquired chromosomal changes that are associated with a cancer’s clinicopathologic presentation are considered primary cytogenetic lesions. Found in hematologic disorders and many solid tumors, these primary lesions may be important in the origin or early stages of tumor development. Secondary chromosomal aberrations reflect evolution of the clonal karyotype and, presumably, the progression of the neoplasm. Much information about chromosomal changes in human neoplasia has been collected and is available through the

Mitelman Database of Chromosome Aberrations in Cancer (http://cgap.nci.nih.gov/Chromosomes/Mitelman).

Acute lymphoblastic leukemia Acute lymphoblastic leukemia (ALL) is the most common subtype of childhood acute leukemia, accounting for 75% of all cases of this disease. Pediatric patients with ALL are usually treated on the basis of the risk of relapse, as defined by clinical and laboratory features. This strategy spares those patients who would have a very good outcome with moderately intensive therapy from more toxic treatment, reserving more aggressive therapy for patients with a lower probability of long-term survival. Abnormal karyotypes are subgrouped according to established chromosome number (ploidy) and structural chromosomal changes. The relative significance of the prognostic features is often treatment-dependent and changes over time. Patients with high-risk ALL have one of the following features: a Philadelphia (Ph) chromosome (the fusion gene BCR-ABL), 11q23/MLL gene rearrangement, or near-haploidy or hypodiploidy (<45 chromosomes). The following sections focus on the most common numeric and structural chromosomal findings and their clinical associations with childhood ALL.

Abnormalities not associated with a particular immunophenotype Numeric chromosomal changes ALL can be classified into five subtypes based on the MN of chromosomes: hyperdiploid with more than 50 chromosomes (25–30% of cases, including near-tetraploid, neartriploid, and hyperdiploid with 51 to 68 chromosomes); hyperdiploid with 47 to 50 chromosomes (10–15% of cases); pseudodiploid (46 chromosomes with structural or numeric abnormalities; about 40% of cases); diploid (46 chromosomes; 10–15% of cases); and hypodiploid (fewer than 46 chromosomes; about 8% of cases, including nearhaploid cases). Recognition of ploidy as a distinctive cytogenetic feature in ALL has improved the ability to predict clinical outcome and devise risk-specific therapy. Near-tetraploidy and near-triploidy Near-tetraploidy (MN range, 82–94) occurs in less than 1% of reported cases of childhood ALL.27 Compared with other ploidy groups of childhood ALL, this subset of hyperdiploidy is more likely to be associated with L2 morphology (30% of cases), a T-cell immunophenotype (47%), and older age at the time of diagnosis (median, 8.6 years).

Cytogenetics of acute leukemias

Near-triploidy (MN range, 69–81) is extremely rare (0.3%) in childhood ALL, and the clinical features of patients in this subgroup do not appear to differ from those of patients in the general ALL population; the main difference is that the pre-B immunophenotype is more common in neartriploidy ALL. A strong association of near-tetraploidy and the cryptic t(12;21) has been observed.28 Hyperdiploidy (>50 chromosomes) Hyperdiploidy with a MN range of 51–68 occurs in 25% to 30% of pediatric patients with lymphoid leukemia; this group of patients has more favorable presenting features and higher cure rates than do other major prognostic groups.29–31 Favorable presenting features commonly associated with hyperdiploidy (>50 chromosomes) include an early pre-B immunophenotype, few T cells, low leukocyte counts, and age between 2 and 10 years. Hyperdiploidy (>50 chromosomes) can be rapidly identified by flow cytometric analysis of the DNA content of leukemic cells; however, flow cytometry does not provide information about numeric or structural chromosomal abnormalities. Hence, this method must be used in conjunction with conventional cytogenetic methods.30,31 Among patients with hyperdiploidy, those whose blast cells have trisomies of chromosomes 4, 10, and 17 have a superior prognosis.32,33 In a study of 182 pediatric patients with hyperdiploidy defined by 51–67 chromosomes, 46% had only numeric changes; the remainder had structural and numeric changes.34 The most common numeric changes were addition of chromosomes 21 (97% of cases), 6 (86%), X (81%), 14 (80%), 4 (76%), 18 (68%), 17 (68%), 10 (56%), 8 (34%), and 5 (26%). The most common structural alterations were duplication of the 1q arm (14%) and the presence of isochromosome 17q (5%). The common region of duplication of chromosome 1 was between q25 and q31.35 Chromosomal translocations, including the t(1;19)(q23;p13.3) and t(9;22)(q34;q11.2), were detected in only 20% of the cases, whereas 50% of all cases of ALL contain translocations.34 The presence or absence of other random structural abnormalities did not influence the probability of eventfree survival (EFS). Patients with 51 to 55 chromosomes per leukemic cell (n = 105; 5-year EFS = 72% ± 5% SE) appeared to fare worse than those with 56 to 67 chromosomes (n = 63; 5-year EFS = 86% ± 5%, P = 0.04 by the stratified log-rank test).34 In the same study, unfavorable features of the hyperdiploid (51–55 chromosomes) subgroup included a higher frequency of isochromosome 17q and a low prevalence of trisomies of chromosomes 4 and 10. Moreover, the proportion of patients whose leukocyte counts were greater than

50 × 109 /L was larger than in the subgroup with 56 to 67 chromosomes. Thus, ALL associated with 51 to 55 chromosomes appears to be a clinicobiologic entity quite distinct from ALL associated with higher MNs.34 Whitehead et al.36 reported higher in vitro accumulation of methotrexate and its polyglutamates in leukemic cells with 56 to 65 chromosomes than in those with 51 to 55 chromosomes, suggesting that leukemic cells possessing 56 to 65 chromosomes are more susceptible to methotrexate-induced cytotoxicity. Heerema et al.33 reported that patients with +10, +17, and a MN of 54 to 58 have a better outcome than do those with hyperdiploidy but no +10 or +17; a +5 was associated with a poor prognosis for patients with this MN. Conventional cytogenetic evaluation of chromosomes in metaphase hyperdiploid cells continues to be technically challenging because of the limited number of spread metaphase chromosomes and poor chromosome morphology. Furthermore, in samples mailed to cytogenetics laboratories, it is not unusual for conventional cytogenetic methods to indicate the presence of only normal metaphase chromosomes and for flow cytometry to indicate a higher-than-normal DNA index (i.e. hyperdiploidy). This discrepancy suggests that dividing hyperdiploid blast cells have a short life-span. In an attempt to resolve such discrepancies in a single assay, the United Kingdom Cancer Cytogenetics Group (UKCCG) developed a slide on which centromeric probes specific for each chromosome were placed. This approach resulted in improved evaluation of ploidy in cases in which the karyotype was either “normal” or could not be determined.37 The Pediatric Oncology Group performed a study in which FISH was successfully used to rapidly and accurately assess the karyotypes of hyperdiploid cases (>50 chromosomes) to determine whether trisomies of chromosomes 4 and 10 were present.32 In another study, SKY was shown to be effective in identifying numeric as well as structural chromosomal abnormalities, including the presence of the Ph chromosome.12 Hyperdiploidy may arise by a simultaneous gain of multiple chromosomes from a diploid karyotype during a single abnormal cell division. Panzer-Grumayer et al.38 showed that hyperdiploidy resulting from nondisjunction of chromosomes in childhood B-cell precursor ALL occurs early during leukemogenesis. The authors first detected nondisjunction in a 2.6-year-old boy whose hyperdiploid leukemic blast cells contained immunoglobulin heavy-chain gene (IGH) rearrangements and an extra copy of chromosome 14; then the authors traced the time of the rearrangements to early in utero development. In that study, retrospective evaluation of neonatal blood spots showed a prenatal origin for the boy’s leukemia; this result extends

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earlier observations on the origins of specific chromosomal translocations in children with ALL.39–42 Hyperdiploidy (47–50 chromosomes) Hyperdiploidy defined as 47–50 chromosomes occurs in 10% to 15% of cases of childhood ALL and was initially recognized because it confers a prognosis that is intermediate to those conferred by other ploidy groups.29 Gains of almost every chromosome have been observed in leukemic cells with this ploidy designation.43 Our analysis of 86 cases revealed that +21 was the most common numeric abnormality (39%); less common were +X (21%), +8 (9%), and +10 (8%).44 The chromosomal arms most often affected by structural abnormalities were 1q (15%), 6q (14%), 12p (21%), and 19p (10%). The clinical outcome of patients with hyperdiploid (47–50 chromosomes) ALL in our Total Therapy Study XI was good (4-year EFS, 77% ± 11% SE). Two specific numeric changes in pediatric ALL bear noting. Trisomy 8 as the sole chromosomal abnormality is infrequent in ALL (0.3–1.2% of cases in a large series) and appears to be associated with a T-cell immunophenotype, although the prognostic significance of this abnormality remains to be determined.44 In a review of 11 cases in which trisomy 21 was the sole numeric abnormality, we found features that reflected lower-risk ALL at the initial examination: young age (median, 3.3 years; range, 1–18 years), low leukocyte count (median, 11.6 × 109 /L; range, 1.8–82 × 109 /L), and a B-cell precursor immunophenotype.45 Most of these patients experienced prolonged periods of relapse-free survival. A Pediatric Oncology Group study confirmed that trisomy 21 in patients without Down syndrome suggests a good prognosis.46 The strong association between +21 and the cryptic t(12;21)(p13.3;q22) may account for the favorable outcome of these patients.28 Pseudodiploidy Patients whose leukemic cells contain numeric abnormalities, structural rearrangements, or both in the context of a diploid net chromosome number (46 chromosomes) comprise the largest and most heterogeneous ploidy group in ALL (approximate incidence, 40%).47 Pseudodiploid leukemic blast cells tend to be associated with bulky disease and high leukocyte counts.48 The clinical outcome of this group was extremely poor until modern multiagent chemotherapy was developed. When treated on appropriately intensified regimens, most patients with pseudodiploid ALL now fare as well as those in other risk groups; however, those with pseudodiploidy and either the t(9;22) or the t(4;11) have highly drug-resistant disease (discussed later in this chapter).

Diploidy The number of patients whose leukemic cells lack apparent cytogenetic abnormalities varies widely, in part because of the equivocal morphology that characterizes leukemic metaphase chromosomes. It is not unusual for leukemic cells in 10% to 15% of cases in a large series to lack chromosomal abnormalities detectable by standard cytogenetic evaluation; the prognosis of patients with diploid ALL is intermediate.48 It is uncertain whether so-called normal- karyotype cases yield metaphase cells of suboptimal quality for analysis of subtle chromosomal rearrangements, mitotically inactive clones, or clones with submicroscopic genetic changes. Current molecular cytogenetic methods have identified chromosomal aberrations that were undetected by conventional cytogenetic methods. An example is a cryptic t(12;21)(p13.1;q22), which was only recently shown to be the most common abnormality in childhood ALL.49,50 The incidence of T-cell ALL cases with normal karyotypes may be as high as 30%.51 In approximately 20% of pediatric patients with T-ALL and leukemic cells with an apparently normal karyotype, FISH revealed the specific subtle rearrangement t(5;14)(q35;q22).16,17 Also, many children with T-ALL and leukemic cells with apparently normal karyotypes, as indicated by conventional cytogenetic methods, have submicroscopic deletions of TAL1 or INK4A that can be detected by FISH, PCR or Southern blot analysis (see below). Hypodiploidy and near-haploidy Hypodiploidy (≤45 chromosomes), representing a heterogeneous subgroup of patients, is found in 7% to 8% of cases of childhood ALL.48,52,53 Most hypodiploid cases (80%) have an MN of 45, and the chromosomal deficiency arises from an unbalanced translocation, the loss of a whole chromosome, or the formation of dicentric chromosomes. In our study of 57 cases with hypodiploidy (≤45 chromosomes), 63% involved the loss of a sex chromosome.54 Monosomy 7 as the only abnormality in hypodiploid or near-haploid ALL cells has been seen in a few instances; however, this recurrent change is associated with Ph+ ALL and a poor prognosis.55 The most frequent dicentric abnormalities are dic(9;12), dic(9;20), and dic(7;9); overall, 94% of the dicentric chromosomes involve the p arm of chromosomes 9, 12, or both.54 Other partial deletions occur in 6q, 11q, 12p, and 13q. Among B-lineage ALL cases with hypodiploidy, 42% contain the ETV6-CBFA2 fusion gene.54 Hypodiploidy of 35–44 chromosomes is usually referred to as low hypodiploidy; this numeric abnormality is extremely rare (approximately 0.8% of patients with ALL).52–54

Cytogenetics of acute leukemias

Table 9.1 Overall incidence of recurrent genetic changes in acute lymphoblastic leukemia Overall frequency (%) Chromosomal abnormality

Children

Adults

Lineage

Genetic lesion

t(1;19)(q23;p13.3) t(9;22)(q34;q11.2) t(4;11)(q21;q23)a t(17;19)(q22;p13.3) t(12;21)(p13;q22)b t(12p13;V)c t(12;17)(p13;q11) t(12;22)(p13;q12) del(6q) t/del(9p) del(11q23) t(11q23;V)d t/del(12p) tan dup(21)(q22) RAS mutation TP53

5–6 3–5 2 <1 20–25 —c <1 <1 4–13 7–12 <1 3–5 10–12 1–2 15 10

3 20–30 3–5 <1 <1 —c <1 <1 5–6 10–15 <1 3–6 5 <1 15 10

Pre-B B-lineage B-lineage Early pre-B B-lineage B-lineage Acute leukemia Acute leukemia Nonspecific Nonspecific Nonspecific Nonspecific Nonspecific B-lineage B-lineage Nonspecific

E2A-PBX1 BCR-ABL MLL-AF4 E2A-HLF ETV6(TEL)-CBFA2(AML1) ETV6-multiple partners EWSR1-CIZ (NMP4) TAF15-CIZ (NMP4) Unknown INK4A, INK4B, ARF Unknown MLL-multiple partners ETV6 (TEL) CBFA2 (AML1) amplification RAS TP53

Abbreviations: V, variable chromosome; tan dup, tandem duplication. a Nearly 80% of infants with ALL have this chromosomal abnormality. b ETV6 gene rearrangements are detected by FISH or RT-PCR. c See Table 9.3. The percentage depends on the subtype of leukemia. d See Table 9.2.

In near-haploid ALL, a distinct subtype within the hypodiploid ALL category, the blast cells contain 24 to 34 chromosomes or, more frequently, 26 to 28 chromosomes; the overall incidence of near-haploidy is low, about 0.5% of all ALL cases. Near-haploid ALL is associated with a poor prognosis (median survival, 10 months from the time of diagnosis), despite the presence of relatively favorable presenting features.52–54 The main clone of near-haploid leukemic cells usually contains at least one copy of each chromosome; in most cases (90%), a second copy of the sex chromosomes or chromosome 21 is present. Other chromosomes found in duplicate include 18 (65%), 10 (45%), and 14 (45%).52 In general, the morphologic features of near-haploid ALL are poorly defined, and few or no structural abnormalities have been discerned. In many near-haploid cases, there is a second abnormal line that has a hyperdiploid karyotype that represents a doubling of the chromosomes in the near-haploid line. Several reported cases have had structural chromosome abnormalities in both cell lines; this finding suggests a common precursor cell with a near-haploid karyotype.52 A near-haploid karyotype undetected by conventional cytogenetics may be misinterpreted as a hyperdiploid clone; in these rare cases, DNA index analysis may aid proper risk assignment.

Patients with 45 chromosomes in their leukemic cells generally fare as well as those with other ploidies,53,54 whereas those whose leukemic cells contain fewer than 45 chromosomes have an inferior outcome when treated on protocols effective for other ALL subgroups; hence, alternative therapy should be considered for patients with the later finding. Structural chromosomal changes The most common rearrangements in the leukemic cells of pediatric patients with ALL are the translocations t(12;21), t(1;19), t(9;22), t(4;11), and t(8;14). Other chromosomal regions, such as 6q, 7q35, 8q24.1, 9p, 12p13, 14q11.2, 11q23, and 21q22, undergo translocations, deletions, and inversions with numerous partners (Table 9.1). Although uncommon recurrent chromosomal rearrangements associated with ALL have been identified in small subgroups of patients, this section focuses on the more common structural abnormalities and their likely contribution to the disease process. t(1;19)(q23;p13.3) In childhood ALL, the t(1;19) is the most frequent translocation detected by conventional cytogenetic methods.56–58 This translocation, with a primarily postnatal origin,

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is found in 5% to 6% of all cases of childhood ALL and in approximately 25% of cases of pre-B cytoplasmic immunoglobulin-positive (cIg+ ) ALL.59,60 It occurs in either a balanced form (25% of cases) or an unbalanced form (75% of cases) as der(19)t(1;19)(q23;p13.3). There is no clinicopathologic difference between these two forms. Some leukemic cells contain both forms, suggesting that the der(1) can be lost through clonal evolution without the loss of the transformed genotype. Although leukemic cells in most t(1;19)+ ALL cases are pseudodiploid and cIg+ , cells in 5% to 10% of cases are cIg− and hyperdiploid.61,62 The t(1;19) has been associated with a poor clinical outcome; however, intensified chemotherapy effectively nullifies the adverse prognosis.60 Uckun et al.63 reported 4-year EFS estimates of 69.5% ± 6.6% for patients with t(1;19)+ ALL and 74% ± 1.3% for patients with t(1;19)− ALL. Furthermore, the 4-year EFS probability among patients with the unbalanced der(19)t(1;19) (80.6% ± 7.1%) was substantially better than the result for patients with the balanced t(1;19) (41.6% ± 13.5%, P = 0.003).63 Although this finding is consistent with an earlier study,64 a significant difference in survival estimates was not found in another analysis of t(1;19)+ ALL.58 The t(1;19) leads to the fusion of E2A, which is on chromosome 19 and encodes a helix-loop-helix (HLH) protein, with PBX1, a homeobox-containing gene on chromosome 1.65,66 The resulting hybrid gene, E2A-PBX1, is a potent oncogene67 and can be detected by RT-PCR.61 The fusion proteins encoded by the balanced and unbalanced forms of the rearrangement are comparable. Additional molecular evaluation of t(1;19)+ leukemic cells identified alternatively spliced and variant fusion transcripts.68,69 RT-PCR has made it possible to distinguish cIg+ cells with typical E2A-PBX1 transcripts from cIg− cells in which the t(1;19) does not yield similar transcripts,61 although the chromosomal abnormality in these cIg− cells appears identical to the more prevalent translocation defined by conventional cytogenetic criteria. However, this difference may be explained by the fact that a recognized t(1;22) resembles the t(1;19).70 Consequently, FISH is recommended as a means to resolve diagnostic uncertainty in cases with equivocal chromosome 19, because such distinctions may be important in planning risk-specific therapy for patients with B-lineage ALL.71 t(17;19)(q22;p13.3) and inv(19)(p13.3q13.4) Other rare, nonrandom chromosomal translocations affecting band p13.3 of chromosome 19 also involve the E2A gene. The t(17;19)(q22;p13.3) is found in approximately 1% of patients with B-lineage leukemia, most of whom do not respond to therapy.72 This translocation results in a

fusion gene consisting of E2A and the hepatic leukemia factor gene (HLF) on chromosome 17, which encodes a protein analogous to leucine zipper-containing transcription factors that regulate developmental stage-specific gene expression.73 The similarity of HLF to the apoptosisinducing ces2 gene of Caenorhabditis elegans suggests that this translocation alters the apoptotic pathway in leukemic cells.74 Other studies of the chimeric transcription factor E2A-HLF have indicated that aberrant transcription of target genes containing HLF-binding sites contributes to leukemogenesis.67 In a few cases in which the t(17;19)(q22;p13.3) is present, neither E2A nor HLF rearrangements have been noted. Thus, this translocation, like the t(1;19), may be heterogeneous at the molecular level.73,74 A cryptic inversion of chromosome 19, inv(19) (p13.3q13.4), fuses E2A to the FB1 gene on 19q13.4. E2AFB1 contains the same region of E2A fused to various portions of the novel FB1 gene, which encodes no identifiable protein motifs.75 The inv(19) was found in 4% of pediatric ALL cases. In contrast to known chromosomal translocations that fuse E2A with different genes, resulting in an oncogenic chimera, the E2A-FB1 fusion generates outof-frame products.75 Its role in leukemogenesis remains unclear. t(9;22)(q34;q11.2) The t(9;22)(q34;q11.2) creates the Ph chromosome. Found in 25% of adults and 3% to 5% of children with ALL, the Ph chromosome confers an unfavorable prognosis, especially when it is associated with either a high leukocyte count or slow early response to initial therapy.76,77 The t(9;22) generates the Ph chromosome by moving the ABL gene, which is normally located at 9q34, to the site of the BCR gene at band 22q11.2. The resultant fusion gene encodes a functional chimeric tyrosine kinase.78 Leukemia phenotypes associated with the Ph chromosome are heterogeneous. The cytogenetic characteristics of the Ph chromosome in ALL are identical to those seen in chronic myeloid leukemia (CML), in which the marker is retained throughout the course of the disease. By contrast, when childhood Ph+ ALL enters complete remission, the Ph chromosome is no longer detectable by cytogenetic analysis. At the molecular level, the breakpoints seen in patients with CML and ALL usually differ; in CML, the fusion gene encodes a 210-kDa fusion protein (p210), whereas in ALL, it generally encodes a 190-kDa fusion protein (p190).78 Both proteins show enhanced tyrosine kinase activity and have been linked to the development of leukemia in a transgenic mouse model.79 Different subsets of childhood ALL are associated with the Ph chromosome, but Ph+ ALL has common

Cytogenetics of acute leukemias

clinicopathologic presenting features, such as older age, high leukocyte count, splenomegaly, and early relapse. Moreover, the Ph chromosome is associated with a poor prognosis.76,80 Although most Ph+ blast cells have a Blineage immunophenotype, isolated pediatric cases with leukemic cells of a T-cell or mixed phenotype have been reported.80,81 Except for monosomy 7, additional chromosomal changes observed in approximately half of patients appear to have minimal prognostic impact. Russo et al.82 reported that monosomy 7, found in 23% of the Ph+ cases, was associated with a greater risk of treatment failure than that of cases without this feature.82 Pediatric patients with Ph+ ALL have a markedly poorer prognosis than those with most other acute leukemias.81 However, Ribeiro et al.76 reported durable long-term responses to intensive chemotherapy in children and adolescents with Ph+ ALL and low initial leukocyte counts (25 × 109 /L), favorable age (1–9 years), or a good initial response to therapy. According to Schrappe et al.77 , a good initial response to steroid therapy is an early predictor of a favorable treatment outcome. In addition to confirming that heterogeneous disease in childhood Ph+ ALL is associated with a poor prognosis, Arico et al.81 showed that the outcome resulting from transplantation of bone marrow from HLAmatched related donors is superior to that resulting from other types of transplantation or intensive chemotherapy alone. Conventional cytogenetic results have indicated that approximately 90% to 95% of patients with Ph+ leukemias have the classic t(9;22), and 5% to 6% harbor a translocation involving the Ph chromosome and other chromosomes (variant Ph). In about 3% to 5% of cases in which a BCR-ABL fusion transcript is detected, conventional cytogenetic methods detect no Ph chromosome; such cases are erroneously described as Ph− . FISH analysis has revealed that more than half of the so-called Ph− cases identified by classic methods contain a cryptic translocation with a BCRABL rearrangement arising from a transposition of ABL to chromosome 22 or of BCR to chromosome 9; the remaining patients have no detectable abnormality in either gene despite the presence of the BCR-ABL fusion gene.78,83 In addition, leukemic cells in 20% of patients with childhood Ph+ ALL are hyperdiploid. These results emphasize the importance of additional molecular testing (either RT-PCR or FISH) in ALL patients whose leukemic cells possess a marker consistent with a Ph chromosome or an abnormality in the distal portion of 9q. Recently, several investigators have described large molecular deletions on either side of the t(9;22) breakpoint in leukemic cells of patients with CML; these deletions are believed to occur at the time of the initial

translocation.84 The deletion, primarily revealed by FISH using a dual-color BCR-ABL probe, is found in approximately 15% to 30% of patients with CML and is associated with a poor prognosis.85 These extensive deletions are several megabases long, have variable breakpoints, and are located 5 and adjacent to the translocation breakpoint on the der(9)t(9;22).86 Similar abnormalities have been found in patients with ALL.3 Such a finding suggests that the deletions contribute to the aggressive clinical course of Ph+ ALL; however, the frequency of deletions in patients with Ph+ ALL was significantly lower (1 among 67 patients [2 children]) than in patients with CML (39 among 253 patients).87 Thus, molecular deletions in der(9) are rare in patients with Ph+ ALL and do not appear to account for the aggressive clinical course of the Ph+ subtype of ALL. The molecularly targeted drug imatinib mesylate (formerly STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, is an effective treatment for the chronic phase of CML. It inhibits the fusion protein by binding to the ATPbinding domain of ABL, a tyrosine kinase whose activity is essential for the survival of Ph+ CML cells but not that of normal cells. The inhibitor is less effective in treating CML in blast crisis and Ph+ ALL in adult patients, because the ATP-binding domain of the target enzyme in the leukemic cells contains amplifications or mutations that prevent imatinib mesylate from binding to the site.88–90 A recent report indicated that imatinib mesylate was effective as salvage therapy in a child with Ph+ acute mixedlineage leukemia refractory to standard chemotherapy.91 Because of the compound’s therapeutic potential, clinical trials are under way to evaluate its various features in children with recurrent Ph+ ALL. Recently, microarray analysis of Ph+ ALL blast cells revealed that imatinib mesylate–sensitive and -refractory leukemic cells can be distinguished on the basis of gene expression profiling and genes relevant to apoptosis and cell cycle control are differentially expressed in these cell populations.92 These findings may be helpful in the design of combined treatment strategies to evade or delay resistance in patients with Ph+ leukemia. 11q23/MLL gene rearrangement The q23 region of chromosome 11 is a relatively common site of structural rearrangements in pediatric patients with hematologic neoplasms. In infants with ALL, the incidence of 11q23 abnormalities ranges from 60% to 70%,93 whereas in children with ALL, the overall incidence ranges from 4.5% to 5.7%.47 Children who have ALL with 11q23 abnormalities are usually young and have high leukocyte counts, organomegaly, and central nervous system (CNS) involvement at the time of diagnosis. The leukemic cells usually

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are not hyperdiploid and have an early pre-B immunophenotype with myeloid-related antigens, although they fail to express CD10. ALL with 11q23 abnormalities, except deletion or inversion, is associated with a poor prognosis.94–96 In patients with ALL, the most frequently occurring 11q23 translocations are t(4;11)(q21;q23) and t(11;19) (q23;p13.3).96,97 Numerous alternative partner chromosome sites have been identified (some only in a small number of patients), and novel genes involved in the translocations have been cloned.43,98 Like the t(4;11) (q21;q23), other recurrent 11q23 translocations, such as t(9;11)(p22;q23), t(6;11)(q27;q23), t(10;11)(p variable;q23), and t(11;19)(q23;p13.1), are typically found in acute myelomonocytic and monocytic leukemias;99 however, the t(9;11)(p22;q23) and t(10;11) can also be found in rare cases of ALL.100,101 Several genes that encode proteins involved in cell signaling have been localized to 11q23, and the gene on 11q23 that is most often rearranged in acute leukemias is MLL (myeloid/lymphoid leukemia or mixed lineage leukemia; also called ALL1, HRX, and HTRX).102,103 MLL is a homolog of the Drosophila trithorax gene, whose function is required for proper expression of homeotic genes and regulation of chromatin structure. Every MLL translocation creates a putative oncogene that encodes nearly the entire translocation partner fused to the C-terminal region of the MLL protein, and each oncogene is associated with a distinct form of leukemia.104 There are more than 50 translocations that target the MLL gene in acute leukemias, and approximately 40 MLL partner genes have been identified (Table 9.2).105 Additional MLL partner genes may be eventually identified through the use of PMRT-PCR, a new method that can identify several fusion genes in a single assay.23 In addition, the advent of microchip array technology may facilitate the identification of chromosome partners for the 11q23 translocations.106 To date, microarray studies of gene expression profiles have revealed that ALL blast cells with various 11q23 translocations involving the MLL gene can clearly be distinguished from other ALL blast cells in which 11q23 abnormalities are absent and from AML cells.107 The distinct gene expression signature -the expression of select multilineage markers and individual HOX genes- may provide unanticipated targets for therapy.107 Some MLL gene rearrangements are not detected by conventional cytogenetic methods. The commercially available dual-color MLL probe allows FISH evaluation of derivatives of a translocation involving MLL in metaphase chromosomes and the splitting of signals in interphase nuclei (Fig. 9.2).108 In rare instances, FISH based on this probe detects not only the reciprocal translocation but also

Table 9.2 The 11q23/MLL translocation partner genes

Abnormality

Disease

MLL partner gene

t(1;11)(p32;q23) t(1;11)(q21;q23) t(2;11)(p15;q23) t(3;11)(p21;q23) t(3;11)(q25;q23) t(3;11)(q28;q23) t(4;11)(q21;q23) t(5;11)(q12;q23) ins(5;11)(q31;q13p23) t(5;11)(q31;q23) t(6;11)(q21;q23) t(6;11)(q27;q23) t(7;11)(q21–22;q23) t(9;11)(p22;q23) ins(11;9)(q23;q34)/ inv(11)(q13q23) t(10;11)(p11.2;q23) t(10;11)(p12;q23) t(10;11)(q22;q23) inv(11)(q14q23) del(11)(q23q23) t(11;14)(q23;q24) t(11;15)(q23;q14) t(11;16)(q23;p13.3) t(11;17)(q23;p13) t(11;17)(q23;q21.1) t(11;17)(q23;q21) t(11;17)(q23;q25) t(11;17)(q23;q25) t(11;19)(q23;p13.1) t(11;19)(q23;p13.3) t(11;19)(q23;p13.3) t(11;22)(q23;q11.2) t(11;22)(q23;q13) t(X;11)(q13;q23) t(X;11)(q22;q23)

AML AML ALL t-AML t-AML t-AML ALL AML ALL AML, MDS t-AML AML ALL AML, t-AML, ALL AML

AF1p/eps15 AF1q LAF4 AF3p21 GMPS LPP AF4 AF5A AF5q31 GRAF AF6q21 AF6 CDK6 AF9 FBP17

Infant AML AML AML AML AML t-AML AML t-AML, t-MDS t-AML AML AML AML, t-AML t-AML AML AML AML, T-ALL AML AML T-ALL AML

ABI1 AF10 LCX CALM LARG GPHN AF15q14 CBP GAS7 AF17 RARA AF17q25 MSF ELL EEN ENL hCDCrel p300 AFX1 SEPTIN6

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; T-ALL, T-cell ALL; t-AML, therapy-related AML; t-MDS, therapy-related MDS.

a deletion of at least 190 kb from the 3 region of the MLL gene.3 Molecular cytogenetic methods have shown that the frequency of MLL gene rearrangements exceeds that of 11q23 translocations detected by conventional cytogenetic methods. In ALL cases in which deletions and inversions affect the 11q23 band, both associated with favorable clinical features and prognoses, FISH should be done to determine whether a cryptic rearrangement of MLL is present.95,109

Cytogenetics of acute leukemias

A

B

C

D

Fig. 9.2 FISH detection of an MLL rearrangement in a pediatric patient with AML and a t(9;11)(p22;q23). Chromosomes were hybridized with Spectrum Orange- and Spectrum Green-labeled DNA probes homologous to sequences lying telomeric and centromeric to the MLL gene. In the nuclei of normal cells (not shown), hybridization of these two probes produces signals that either overlap (yellow) or are close to one another. In metaphase chromosome spread (A) and interphase nuclei (B) in which the MLL rearrangement is present, the target sequences are “split” (i.e. distant) because of their translocation to different chromosomal positions (C). (D) Partial karyotype. This translocation is predominantly seen in primary and therapy-related AML M5; it is rarely seen in ALL. (See color plate 9.2 for full-color reproduction.)

In a few cases, an 11q23 translocation involves genes other than MLL. Therefore, the translocation partners for 11q23 chromosomal region are markedly heterogeneous,110,111 and molecular methods are needed to further assess the MLL gene in patients with an 11q23 abnormality to better stratify treatment groups. In a small series, children aged 1 to 9 years with an 11q23/MLL rearrangement in their ALL cells fared substantially better than infants and older children whose ALL cells lacked this abnormality.93,94 In a recent multivariate analysis, performed as part of a large multinational study of children and young adults with ALL and 11q23 rearrangements, age was the most important prognostic factor: infants fared substantially worse than patients who

were 1 year or older.112 In the infants, any 11q23 abnormality conferred a dismal prognosis, whereas in older patients, the t(4;11) and t(9;11) were associated with a worse outcome than were other 11q23 rearrangements. Furthermore, the outcome of patients with the t(4;11) failed to improve after allogenic transplantation, even with marrow or stem cells from HLA-matched related donors.112 Thus, new therapeutic regimens are needed to cure this subgroup. Recent studies have shown high levels of FLT3 expression in patients with MLL rearrangements; thus, inhibitors of FLT3 (a tyrosine kinase) may prove to be beneficial.107,113 Infant ALL, which represents 3% of childhood ALL, is clinically aggressive and strongly associated with a poor

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prognosis.93,114 Leukemic cells in infants show preferential involvement of the 11q23 region in chromosome abnormalities (up to 70% of chromosomal abnormalities), with the t(4;11) being the most prevalent.115 Overall, the t(4;11) is observed in 2% of children and adults with ALL; it is uncommon but occurs with equal frequency in these two age groups.94,96 The t(4;11) results in the fusion of MLL and AF4, located at band q21 on chromosome 4. Almost all of the breakpoints within the MLL gene are clustered in an 8.5-kb region between two BamHI restriction sites in exons 5 and 11 and therefore can be detected by Southern blot hybridization with a single probe.93 Chromosomal breakpoints are dispersed over 38 kb within the AF4 gene and have been mapped to introns 3 and 4 and, rarely, to intron 5.116 Molecular methods to detect the t(4;11) include Southern blot analysis, RT-PCR, and FISH. As mentioned before, infrequent cryptic insertions and complex and variant t(4;11) have been described.96,117 In a series of 183 patients with hematologic malignancies and a t(4;11), only five variants were found; the fusion resulting in some of the variants may have occurred as a result of a two-step mechanism.96 Although patients with ALL and a t(4;11) have a poor prognosis, those with a favorable age or good initial response to prednisone may have a more favorable outcome.112,114 Another recurrent translocation is the t(11;19)(q23;p13.3), seen in 1% of cases including B-precursor ALL and T-cell ALL. The outcome for infants with t(11;19) is poor, whereas that for children older than 1 year is favorable.97 Abnormalities of 12p Abnormalities of 12p are identified by conventional cytogenetic methods in 8% to 11% of pediatric patients with ALL.118 Most 12p rearrangements involve translocations, including dic(9;12)(p11;p12), dic(7;12)(p11;p11–12), t(7;12)(q36;p13), t(12;13)(p13;q14), t(2;12)(q14;p13), and t(12;17)(p13;q21).72,119–122 In a study of childhood ALL, 94 (11.5%) of 815 patients had structural abnormalities in the 12p11–13 region.123 Conventional cytogenetic analysis revealed that the 12p abnormalities included translocations (66%), deletions (28%), inversions (5%), and the formation of isochromosome 12q (1%). Overall, the 69 translocations affecting the 12p region involved 20 different chromosomes as reciprocal partners and 43 distinct breakpoints. The most frequent exchanges involved 9p11 (n = 8), 7p11 (n = 4), 1q22–23 (n = 3), 13q14 (n = 3), 2q21, 4q21, 8p21, and 10q22 (two cases each). Most of the 94 cases (78%) with a 12p abnormality had additional chromosomal changes and, except for the case with a t(1;19)(q23;p13.3), none had any of the known common recurrent translocations. Compared with the general

population of patients with ALL, the patients with ALL and 12p abnormalities had a significantly lower frequency of hyperdiploidy (>50 chromosomes) (7%) but had a similar distribution of leukemic-cell immunophenotypes. In this study, the clinical outcome of patients with 12p abnormalities (5-year EFS = 70% ± 5% SE) did not differ significantly from that of patients in which no 12p abnormalities were detected (5-year EFS = 64% ± 2%, P = 0.64).123 These findings confirmed that the most frequently observed recurrent 12p abnormality, dic(9;12)(p11;p12), is associated with an excellent prognosis.119 The remarkable heterogeneity of the observed changes in 12p suggests that a gene(s) located on 12p may be relevant to leukemogenesis and can be rearranged with genes at loci from other chromosomes. The neoplastic process may also be triggered by loss of heterozygosity (LOH) through deletion or the formation of dicentric chromosomes. The search for genes involved in 12p abnormalities identified TEL (translocation ETS leukemia), which was later renamed ETV6 (ETS translocation variant gene 6). ETV6 encodes an ETS-like putative transcription factor.124 ETV6 is rearranged in half of the patients with 12p13 translocations and either lymphoid or myeloid leukemia.123,125 ETV6 has multiple fusion partners: approximately 41 chromosome bands are involved in translocations with ETV6, and approximately 20 partner genes have been cloned (Table 9.3). The translocation breakpoints are distributed throughout ETV6, and the gene contributes to the pathogenesis of leukemia by diverse molecular mechanisms that are not well understood. Translocation commonly involves an in-frame fusion between the region encoding the N-terminal region of ETV6 and the coding region for either unrelated transcription factors or tyrosine kinases; the ETV6–tyrosine kinase fusion proteins exhibit constitutive tyrosine kinase activity and have transforming ability. However, in some cases in which ETV6 has been rearranged with another gene, the potential function of the fusion gene appears to be related to leukemogenesis but has not been identified; thus, another mechanism may be involved in these translocations.126 The t(12;21)(p13;q22) results in the fusion of ETV6 to AML1 (renamed RUNX1 and CBFA2), the most common fusion partner of ETV6.49,50,127 The fusion gene consists of the region of ETV6 that encodes a HLH domain and the region of CBFA2 that encodes DNA-binding and transactivation domains. The ETV6-CBFA2 gene fusion is the most common rearrangement in childhood ALL, occurring in 20% to 25% of cases of pediatric B-cell precursor ALL. However, ETV6-CBFA2 is rarely observed in infants with ALL, in pediatric patients with hyperdiploid leukemic cells, or in pediatric patients with T-cell ALL. This genetic abnormality is observed mainly in children 3 to 5 years of

Cytogenetics of acute leukemias

Table 9.3 Rearrangements of ETV6 (TEL) Translocation

Disease

Partner gene

t(1;12)(p36.1;p13) t(1;12)(q21;p13) t(1;12)(q25;p13) t(3;12)(q26;p13) t(4;12)(p16;p13) t(4;12)(q11–12;p13) t(5;12)(q31;p13) t(5;12)(q33;p13) t(6;12)(q23;p13) t(7;12)(q36;p13) t(9;12)(q11;p13) t(9;12)(p24;p13) t(9;12)(q22;p13) t(9;12)(q34;p13) t(12;13)(p13;q12) t(12;14)(p13;q32) t(12;15)(p13;q25)a t(12;21)(p13;q22) t(12;22)(p13;q11.2)

MDS AML, ALL AML MDS, MPD PTCL AML, ALL AML, CML AML, MDS, CMML ALL Infant AML ALL ALL, CMML MDS CMML, ALL, AML AML ALL AML ALL MPD, AML

MDS2 ARNT ARG (ABL2) EVI1 (MDS1/EAP) FGFR3 BTL (CHIC2) ACS2 PDGFRB STL HLXB9 PAX5 JAK2 SYK ABL1 CDX2 IGH TRKC (NTRK3) CBFA2 (AML1) MN1

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MPD, myeloproliferative disorder; PTCL, peripheral T-cell lymphoma. a Also observed in congenital fibrosarcoma and mesoblastic nephroma.

age,50 and occurs in only 1.5% to 4.4% of adult patients with ALL.128 The ETV6-CBF2A fusion occurs in CD10+ , CD19+ , CD34+ B-cell precursor ALL; leukemic cells from approximately half of the patients with this type of ALL express myeloidassociated antigens (CD13, CD33, or both). Little or no expression of CD9 and CD20 is highly predictive of the presence of the fusion gene.129 Abnormalities of 12p, trisomy 21, loss of the X chromosome, and tetraploidy are genetic changes associated with t(12;21).28,130 Occasionally, the leukemic blast cells of patients with Down syndrome contain the t(12;21).130 The prognostic significance of the ETV6-CBFA2 fusion gene is controversial. The early association with an excellent prognosis was subsequently challenged, because some patients with late relapses have ETV6-CBFA2 transcripts.50,127,131 In some clinical trials, these patients had a favorable prognosis, whereas in others, the transcript lacked independent prognostic value. Recent evidence indicates that in a few cases the ETV6-CBFA2 rearrangement may be acquired in utero, but ALL does not develop until years later. Thus, an additional cooperating mutation(s) may be required for leukemogenesis.40–42 This

Fig. 9.3 Partial karyotype of B-lineage ALL blast cells in which the t(12;21)(p13.3;q22) is present. The metaphase chromosomes were stained with 4 , 6-diamidino-2-phenylindole (DAPI). This chromosomal abnormality is the most common recurrent translocation in patients with ALL and is readily detected by FISH (A) and RT-PCR but not by conventional cytogenetic methods. The ETV6 gene on chromosome 12 (green) and the CBFA2 gene on chromosome 21 (red) probes used in FISH detected ETV6-CBFA2 on the der(21)t(12;21) (B, right) and residual CBFA2 on the der(12)t(12;21) (B, left). (See color plate 9.3 for full-color reproductions.)

late development of disease after the occurrence of translocations or rearrangements is characteristic of leukemias associated with rearrangement of the gene for the corebinding factor, as seen in cases with t(8;21). Although cryptic, the t(12;21) can be detected by RT-PCR and FISH. The latter method, using probes for ETV6 and CBFA2, is easy to perform, but the interpretation of findings requires caution, because many secondary genetic events may be observed. When commercially available probes are used to analyze a t(12;21) in leukemic cells, they detect ETV6-CBFA2 on the der(21)t(12;21), a residual signal of CBFA2 on the der(12)t(12;21), and a normal ETV6 or CBFA2 allele on chromosome 12 or 21, respectively (Fig. 9.3). Approximately 50% of patients with the

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ETV6-CBFA2 rearrangement lack the normal CBFA2 allele, which is not involved in the translocation and whose deletion is considered a secondary event; this finding supports the hypothesis that loss of ETV6 function contributes to leukemic transformation.123,132,133 FISH and LOH studies have shown that the extent of ETV6 deletion can vary; in fact, small intragenic deletions not detected by commercially available probes have been observed.132 Although most cases studied by FISH have a single fusion signal on der(21), rare cases with an extra chromosome 21 or isochromosome 21 have a duplicated fusion signal on der(21).133,134 Several cases have been reported in which the ETV6CBFA2 fusion transcript was detected by RT-PCR, but the reciprocal (residual) CBFA2 on 12p was not detected by FISH as expected. Instead, CBFA2 was observed as an insertion on other chromosomes or as a participant in complex rearrangements.133,135 In addition to determining whether CBFA2 has been inserted elsewhere or rearranged, FISH can confirm CBFA2 amplification, which occurs in approximately 1% to 2% of pediatric patients with B-lineage, t(12;21)-negative ALL.133,136 Multiple copies, or tandem duplication, of 21q is a recurrent chromosomal abnormality in childhood ALL; therefore, the marker chromosomes acquire different morphologic features on the basis of the number of segments amplified in the p and q arms of chromosome 21. The 21q amplification can be easily confirmed by FISH using probes that detect the t(12;21). In a study of 12p abnormalities that were visible by conventional cytogenetics, ETV6 rearrangements were observed in 36 (56%) of the 64 patients, and ETV6-CBFA2 chimeric transcripts were detected in 25 (66%) of the 38 cases analyzed by RT-PCR.123 Other reports have described additional cases in which a cytogenetically abnormal 12p13 is present but a t(12;21) is absent.137 These findings support the hypothesis that other genes crucial to leukemogenesis lie in 12p. One candidate, CDKN1B (cyclin-dependent kinase inhibitor 1B; also called p27kip1 ), has been shown by FISH to reside on band 12p13, and one allele is deleted in various hematologic disorders.138 However, extensive sequence analysis has failed to reveal mutations in the retained allele.138,139 Therefore, if it is assumed that hemizygosity for CDKN1B is insufficient to alter growth (as suggested by studies of Cdkn1b knockout mice),140 then CDKN1B is probably not a target for rearrangement.141 dic(9;20)(p13;q11.2) The dic(9;20) has been described as a recurrent chromosomal abnormality found mostly in B-cell precursor ALL.142,143 This chromosomal abnormality is difficult to identify by G-banding alone, appearing as monosomy 20

with or without a deletion of 9p. The dic(9;20) should be confirmed by FISH, because it is not present in all cases containing leukemic cells with monosomy 20.143 In a few cases, the dic(9;20) has been observed concurrently with other recurrent changes, such as t(9;22)(q34;q11.2) or t(8;14)(q24;q11.2) in a patient with T-cell ALL, and an extra chromosome 21 has been noted as an accompanying change.142,143 Patients with the dic(9;20) have a favorable prognosis.

Abnormalities of 6q Deletion of the long arm of chromosome 6 occurs in 4% to 13% of pediatric patients with ALL; most of the breakpoints of these deletions are localized to 6q15 and 6q21. In a review of 412 consecutive cases of ALL for which fully banded karyotypes were obtained, we identified 45 (11%) with a 6q abnormality.144 Most (85%) of the alterations in 6q were deletions, and other types of structural rearrangements in 6q accounted for the remainder. The presenting features and the EFS probability for children with a 6q abnormality did not differ from those of patients lacking this feature. The chromosomal changes did not appear to be associated with immunophenotype; the lack of association suggests that the gene(s) affected by 6q abnormalities is broadly active during lymphoid leukemogenesis or that the 6q abnormalities perhaps arise as molecular epiphenomena of more specific lesions.144 No conclusive molecular evidence for LOH has been associated with 6q deletions.145 In one study of ALL patients in which all or part of 6q had been deleted, most of the patients also lacked a human cyclin C (CCNC) gene, which resides on 6q21.146 In the remaining CCNC allele no mutation was observed. The breakpoints for the smallest regions deleted from 6q have been established, but a tumor suppressor gene(s) in these regions has not been identified.147

Immunophenotype-specific abnormalities During B-cell and T-cell development, the genes that encode the immunoglobulin (Ig) and the T-cell receptors (TCR) undergo rearrangements (Table 9.4). Enzymes that control recombination of these genes recognize signal sequences that activate recombination. However, some genes involved in translocations share the same signal sequences, and the recombinase might erroneously use these signal sequences to recombine these genes with immune receptor genes. For example, a translocation may result in the replacement of a transcriptional regulatory region of an oncogene with that of another gene; such a situation could result in overexpression of the oncogene.

Cytogenetics of acute leukemias

Table 9.4 Immunophenotype-specific rearrangements generated by immunoglobulin (IG) and T-cell receptor (TCR) genes in acute lymphoblastic leukemia Disease and genes involved B-cell ALL MYC (8q24.1)

IGH (14q32)

T-cell ALL TCRA (14q11.2)

TCRB (7q35)

Non-TCR gene Non-TCR gene Non-TCR gene Non-TCR gene Non-TCR gene Non-TCR gene Non-TCR gene

Abnormality

Partner gene

t(8;14)(q24.1;q32) t(2;8)(p12;q24.1) t(8;22)(q24.1;q11.2) t(1;14)(q25;q32) t(5;14)(q31;q32) t(9;14)(p13;q32) t(12;14)(p13;q32)

IGH IGK IGL LHX4 IL3 PAX5 ETV6(TEL)

inv(14)(q11.2q32) t(1;14)(p32;q11.2) t(1;14)(p34;q11.2) t(5;14)(q34;q11.2) t(8;14)(q24.1;q11.2) t(9;14)(p21;q11.2) t(10;14)(q24;q11.2) t(11;14)(p13;q11.2) t(11;14)(p15;q11.2) t(14;14)(q11.2;q32.1) t(14;21)(q11.2;q22) inv(7)(p15q35) t(7;7)(p15;q35) t(1;7)(p32;q35) t(1;7)(p34;q35) t(7;9)(q35;q32) t(7;9)(q35;q34) t(7;10)(q35;q24) t(7;11)(q35;p13) t(7;14)(q35;q32.1) t(7;19)(q35;p13) Submicroscopic del(1)(p32) t(1;3)(p32;p21) t(4;11)(q21;p15) t(5;14)(q35;q32) del(9)(p13–22) t(10;11)(p12;q14) t(11;19)(q23;p13.3)

IGH TAL1 (TCL5/SCL) LCK RanBP17/HOX11L2 MYC INK4A, INK4B, ARF HOX11 LMO2 (RBTN2/TGT2) LMO1 (RBTN1/TGT1) TCL1 (TML1) BHLHB1 TCRG TCRG TAL1 (TCL5/SCL) LCK TAL2 TAN1 HOX11 LMO2 (RBNT2/TGT2) TCL1 (TML1) LYL1 TAL1 (TCL5/SCL) TAL1-TCTA NUP98-NRG CTIP2-HOX11L2 INK4A, INK4B, ARF AF10-CALM MLL-ENL

B-lineage ALL t(8;14)(q24.1;q32) Abnormalities of 8q24.1/MYC are associated with B-cell ALL. The t(8;14)(q24.1;q32) was the first immunophenotype-specific translocation to be identified in surface immunoglobulin–positive (sIg+ ) B-cell neoplasias, mainly Burkitt lymphoma. This translocation,

or infrequently one of its variants, the t(2;8)(p12;q24.1) and t(8;22)(q24.1;q11.2), for example, occurs in 85% to 90% of cases of sIg+ B-cell ALL (L3 morphology).148 This type of ALL occurs predominantly in children (incidence 2%) and is generally considered a systemic manifestation of Burkitt lymphoma. The t(8;14) has been found, although rarely, in other B-lineage malignancies without L3 morphology. Most patients with t(8;14)+ ALL have additional abnormalities, most of which involve chromosome 1 and partial duplication of its long arm. Some patients with L3 B-cell leukemia harbor 1q and 6q abnormalities, although the t(8;14) in such cases appears to be absent, as indicated by conventional cytogenetics.148 The t(8;14) or its variant translocations can be detected by FISH using commercially available probes for the IGH, IGL, and MYC genes.149,150 The treatment for t(8;14)+ ALL differs from that for other forms of childhood ALL, and newer treatment regimens have improved the cure rate of these patients to approximately 90%.151 In rare cases of FAB-L3 ALL with translocations affecting the MYC gene, the leukemic cells lack sIg; such cases should receive the same treatment as that for B-cell ALL.152 The t(8;14)(q24.1;q32) usually results in translocation of the MYC gene (located at 8q24.1) to the IGH locus on chromosome 14. In other instances, mainly in variant translocations, MYC remains on the der(8), and the IGK (2p12) or IGL (22q11.2) light-chain genes move adjacent to 8q24.153 In each case, MYC is placed under the transcriptional influence of the immunoglobulin enhancer, resulting in the overproduction of MYC and, ultimately, in uncontrolled cellular proliferation. Burkitt lymphoma and L3 ALL are among the most rapidly dividing and aggressive hematological malignancies. t(8;14)(q11.2;q32) The rare recurrent t(8;14)(q11.2;q32) occurs in pediatric patients with B-lineage ALL and low-risk features.154,155 There is a noteworthy association between the t(8;14) and the presence of both Down syndrome and the Ph chromosome; however, no apparent association of this translocation with adverse outcome has been established. T-lineage ALL About 15% of cases of ALL are of the T-cell lineage and are frequently associated with hyperleukocytosis, the involvement of the CNS, the presence of a mediastinal mass, male sex, and the lack of hyperdiploid (>50 chromosomes) leukemic cells.51,156 The unfavorable outcome of patients with T-cell ALL has recently improved through the use of highly effective treatment protocols.157 With

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appropriately intensive therapy, children with T-cell ALL have an outcome similar to that of children with Bprecursor ALL.156 Compared with ALL patients who have other immunophenotypes, patients with T-cell leukemia have a lower percentage of cytogenetically detectable abnormal clones.50,157,158 In approximately 50% to 60% of cases with an abnormal karyotype, the cells have nonrandom breakpoints within the TCRA/D loci (14q11.2), the TCRB locus (7q35), or the TCRG locus (7p15).51,158,159 During T-cell differentiation, these four loci undergo structural rearrangement that is analogous to the rearrangement of IG genes during B-cell development; structural aberrations in the TCR genes lead to T-cell malignancies. Schneider et al.158 evaluated 343 cases of T-cell ALL by using a conventional cytogenetic method (G-banding) and identified two recurrent chromosomal abnormalities, del(1)(p22) and t(8;12)(q13;p13), and 10 uncommon aberrations. The following section presents the prominent recurring chromosomal abnormalities of T-cell ALL (Table 9.4); however, the clinical significance of these rearrangements remains largely unknown.156,159 Notably, the most common genetic lesion in T-cell ALL is deletion of the putative tumor suppressor gene INK4A at 9p21, which occurs in as many as 80% of cases.160,161 Although 11q23/MLL gene rearrangements are mostly observed in B-lineage ALL, they have been also observed in T-lineage ALL with a similar incidence among children (4%) and adults (6% to 8%).162,163 Most patients with T-cell ALL and 11q23 rearrangements have a t(11;19)(q23;p13.3).97,162 Unlike patients with Blineage ALL and 11q23/MLL gene rearrangements, patients with T-cell ALL do not have a poor prognosis.95,97 Another recurrent abnormality in T-cell ALL is the t(10;11)(p14;q14), which results in a CALM-AF10 fusion gene.100 Abnormalities of 1p32 The t(1;14)(p32;q11.2) has been observed in 3% of patients with T-cell ALL. In this translocation, TAL1 (SCL and TCL5) on 1p32–p34 is juxtaposed with the TCRA/D loci on chromosome 14.164 The TAL1 gene is dysregulated in other rare translocations by juxtaposition with a TCR locus: these rare translocations include the t(1;7)(p32;q35)165 ; the t(1;3)(p32;p21), which fuses TAL1 with a novel gene on chromosome 3 [the T-cell leukemia translocationassociated gene (TCTA)]166 ; and the t(1;5)(p32;q31).167 However, the most common disruption of TAL1 is a sitespecific interstitial deletion (approximately 90 kb) in which the coding region of the TAL1 gene is juxtaposed with the promoter region of SIL, the SCL-interrupting locus.166,168 The deletion, which occurs in as many as 30% of patients with T-cell ALL, is not detected by conventional karyotypic

analysis but is easily detected by Southern blot analysis, PCR, or FISH.168,169 The common aberration results in ectopic expression of the TAL1 protein.170 Misexpression of the TAL1 gene has been detected in another group of patients with T-cell ALL (approximately 30%), but these patients appear to lack a TAL1 abnormality.170,171 Blast cells in as many as 60% of patients with T-cell ALL ectopically express TAL1170,171 ; therefore, alteration of TAL1 (either by translocation or by other rearrangement) represents one of the most common genetic lesions associated with Tlineage leukemia. Whether the TAL1 rearrangement is a prognostic factor remains controversial. t(5;14)(q35;q32) The t(5;14)(q35;q32) is a recurrent, T cell–specific cryptic translocation found in approximately 20% of children and adolescents whose leukemic cells have normal karyotypes, as determined by conventional cytogenetic methods.16,17 This cryptic translocation was detected by a new type of M-FISH called IPM-FISH. In this method, the probes used to evaluate the translocation are painting probes for chromosomes 5 and 14 or a combination of a YAC probe for chromosome 5 (the breakpoints cluster on 5q35) and a painting probe for chromosome 14 (breakpoints are spread out on 14q32).15,16 The HOX11L2 gene (mapped at 5q35) was found to be transcriptionally activated as a result of the translocation; the gene was probably under the influence of CTIP2 transcriptional regulatory elements.16 Overexpression of HOX11L2 was associated with a poor prognosis in one study,172 whereas in another the outcome of 31 children with HOX11L2/t(5;14)+ T-cell ALL was similar to that of 76 patients with other types of T-cell ALLs.173 Abnormalities of 7q35 The 7qter region containing the TCRB locus is affected by chromosomal aberrations less often than is the TCRA/D region of chromosome 14. The TCRB locus participates in several reciprocal exchanges with different chromosomes.51 One infrequent translocation, the t(7;9)(q35;q32), results in dysregulated expression of TAL2, whereas another, t(7;19)(q35;p13), results in dysregulated expression of LYL.174,175 A t(7;11)(q35;p13) and a t(7;10)(q35;q24), which are variants of the more frequently observed t(11;14)(p13;q11.2) and t(10;14)(q24;q11.2), respectively, also activate expression of the DNA-binding transcription factor genes LMO2 and HOX11. Abnormalities of 9p Rearrangements of the short arm of chromosome 9 have been observed in 7% to 12% of cases of childhood ALL.176,177

Cytogenetics of acute leukemias

Early reports indicated that 9p abnormalities were usually associated with a “lymphomatous” type of ALL, often characterized by leukemic cells with a T-cell immunophenotype, lymphadenopathy, mediastinal enlargement, and splenomegaly. Subsequent reports described 9p abnormalities in a substantial number of patients with B-lineage ALL.176 One earlier study showed a 10% incidence of 9p abnormalities (26 deletions, 9 unbalanced translocations, and 5 balanced translocations) in patients with ALL.177 Compared with patients lacking these abnormalities, the affected patients were older and had higher leukocyte counts, a greater frequency of splenomegaly, leukemic cells with a T-cell immunophenotype (25%), and an increased rate of extramedullary relapse. It was concluded that despite their association with high-risk clinical features, 9p abnormalities were not an indicator of any severe adverse events in the clinical course of T-cell ALL, except for an apparently increased risk of CNS involvement. By contrast, another study found that abnormalities of 9p were an adverse risk factor in B-lineage ALL, and were characteristic of a subgroup of patients with standard-risk disease and an increased risk of treatment failure.178 The key region involved in 9p abnormalities is p21–p22, which contains the interferon gene cluster (IFN); this locus is deleted in some patients with ALL, with or without 9p abnormalities detectable by conventional cytogenetics.179 In addition to IFN, the following putative tumor suppressor genes reside within 9p21: INK4A (also called MTS1, CDKN2, and CDK4I), INK4B (MTS2), and ARF. In these genes, large numbers of homozygous deletions, mutations, or both occur in various malignancies (including pediatric ALL), but whether these genes are true tumor suppressors remains unclear.160,180 As indicated by FISH and other molecular analyses, homozygous deletions of INK4A occur in 60% to 80% of children with T-cell ALL and in 20% of those with B- lineage leukemia,161,181 but the prognostic significance of LOH of INK4A in childhood ALL remains controversial.161,181–183 Besides INK4A, PAX5 is also involved in 9p13 abnormalities. However, the molecular events of leukemogenesis in which altered PAX5 participates are unclear.184 Abnormalities of 14q11.2 Chromosome 14 aberrations in which the breakpoint is located at 14q11.2 are the most frequent chromosomal abnormalities of T-cell ALL.51,158,159 The 14q11.2 can participate in numerous translocations, such as t(11;14)(p13;q11.2), t(11;14)(p15;q11.2), t(10;14)(q24;q11.2), t(1;14)(p34;q11.2), t(5;14)(q34;q11.2), t(8;14)(q24;q11.2), t(X;14)(q28;q11.2), and inv(14) (q11.2q32). Abnormalities affecting the 14q11.2/TCRAD

region can now be detected by FISH using an available YAC probe.185 The t(11;14)(p15;q11.2) is the nonrandom abnormality most frequently detected by conventional cytogenetics in childhood T-cell ALL: approximately 7% of pediatric patients with T-cell ALL have this change.51 At the molecular level, the breakpoint on chromosome 14 is within the TCRA/D loci, and on chromosome 11 at p13 there is a breakpoint-cluster region (T-cell ALLbcr ).186 In patients with this translocation, the LMO2 gene (formerly RBTN2/TTG2) is overexpressed. The t(11;14)(p15;q11.2), found in 1% of patients with T-cell ALL,51 also disrupts the TCRA/D loci; on chromosome 11, the break occurs in a rhombotin-2 – related stagespecific differentiation gene (LMO1 or RBTN1/TTG1).187 The rhombotin genes encode nuclear proteins whose structures are highly conserved among diverse species; this conservation suggests an important role in ontogeny.187 Hence, members of the rhombotin gene family may have particular relevance for T-cell ALL. The t(10;14)(q24;q11.2) is found in 5% to 10% of patients with T-cell ALL or T-cell lymphomas; the products of this translocation occur in the TCRA/D loci on chromosome 14 and in a breakpoint-cluster region on chromosome 10.188 A recent study of 343 children and adolescents with T-cell ALL showed that the t(10;14) is associated with a significantly better probability of survival than are other chromosomal aberrations.158 The t(10;14) results in the deregulation of the homeobox gene HOX11 because of its recombination with TCRA/D.189 Because of the tight clustering of chromosomal breakpoints, the t(10;14) can be detected by PCR, and a dual-color FISH probe can detect translocations involving HOX11 on 10q24.190 In patients with T-cell ALL and the t(10;14), HOX11 (10q24) is translocated to TCR (14q11.2), resulting in overexpression of HOX11, a central player in spleen formation, perhaps through the regulation of cell survival.191 t(8;14)(q24.1;q11.2) This translocation is observed in 2% of patients with T-cell ALL but is not restricted to this lineage.51 At the molecular level, the TCRA/D genes are rearranged with the CMYC oncogene. Thus, transcription of CMYC is deregulated.192

Acute myeloid leukemia The clinicobiologic classification of AML is based on the morphologic, immunophenotypic, cytogenetic, and molecular characteristics of the leukemic blast cells. Like ALL, AML is associated with heterogeneous subgroups of

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Table 9.5 Overall incidence of recurrent genetic changes in primary acute myeloid leukemia Incidence among patient groups (%) Chromosomal abnormality

Pediatric

Adult

FAB subtype

Genetic lesion

t(8;21)(q22;q22) inv(16)(p13.1q22)/t(16;16) t(15;17)(q22;q12–21) t(5;17)(q32;q12) t(11;17)(q23;q21) t(11;17)(q13;q21) dup(17)(q21.3–q23) −7/del(7q) t(1;22)(p13;q13) t(3;5)(q25.1;q34) inv(3)(q21q26)/t(3;3) t(3;21)(q26;q22) t(6;9)(p23;q34) t(9;9)(q34;q34) inv(8)(p11.2q13) t(8;16)(p11.2;p13) t(8;22)(p11.2;q13) t(8p11.2;V)a t(11p15;V)b t(11q23;V)c t(10;11)(p12;q13) t(10;16)(q22;p13) t(16;21)(p11;q22) t(12;22)(p13;q11) Isolated trisomy: 4, 6, 8, 9, 11, 13, 14, 19, 21 MLL partial tandem dup FLT3 internal tandem dup FLT3 point mutation C/EBPa mutation FMS point mutation RAS mutation KIT mutation, del, ins CBFA2 (AML1) mutatione TP53 mutation

12 6 9 <1 <1 <1 <1 5 1 1 1 <1 1 <1 <1 <1 <1 1 <1 18 1 <1 <1 <1 —d

6 8 10 <1 <1 <1 <1 8 0 1 1 1 <1 <1 <1 <1 <1 <1 <1 5 1 <1 <1 <1 —d

M2, M1 M4 M3 M3, variant M3, variant M3, variant M3, variant M2, M4 M7 Nonspecific Nonspecific Nonspecific M2, M4 Nonspecific Nonspecific Nonspecific Nonspecific M4, M5 AML, t-AML M4, M5 M5 Nonspecific Nonspecific Nonspecific Nonspecific

CBFA2(AML1)-ETO CBFB-MYH11 PML-RARA NPM-RARA PLZF-RARA NUMA-RARA STAT5b-RARA Unknown RBM15-MLK1(OTT-MAL) NPM-MLF1 RPN1-EVI1(MDS1/EAP) CBFA2-EVI1/EAP/MDS1 DEK-CAN SET-CAN TIF2 CBP p300 FGFR1-multiple partners NUP98-multiple partners MLL-multiple partners CALM-AF10 MORF-CBP TLS (FUS)-ERG ETV6 (TEL)-MN1 Unknown

<1 10–15 Undetermined Undetermined Undetermined 3 Undetermined Undetermined Undetermined

6–11 20–30 5–10 11 10–20 30 <10 5 5–15

Nonspecific Nonspecific Nonspecific Nonspecific Nonspecific Nonspecific Nonspecific M0 Nonspecific

MLL FLT3 FLT3 C/EBPa FMS RAS KIT CBFA2 (AML1) TP53

Abbreviation: V, variable chromosome. a b c See Table 9.6. See Table 9.7. See Table 9.2. d The percentage depends on the subtype of leukemia. e This molecular aberration is mainly observed in patients with M0 AML.

specific chromosomal aberrations. Conventional cytogenetic analysis has revealed that leukemic cells in 80% to 85% of pediatric patients with AML have an abnormal karyotype (Table 9.5). In children and adults with AML, prognostic factors include initial clinical features, t(8;21), t(15;17), inv(16), Down syndrome (in AML-M7 only), and response to treatment. Factors predicting an unfavorable prognosis include

monosomy 7 and complex aberrations.99,193 Because of the increased intensity of chemotherapy and improved supportive care, AML in 80% to 90% of pediatric patients enters remission, and their probability of 5-year EFS is about 50%. Whether BMT is indicated during the first complete remission for patients other than those at high risk remains controversial.194

Cytogenetics of acute leukemias

Numeric chromosomal changes Near-haploidy and hyperdiploidy (>50 chromosomes) are extremely rare in childhood AML. Numeric changes as sole chromosomal abnormalities involving chromosomes X, 4, 6, 7, 8, 9, 11, 14, 19, 21, and 22 have been occasionally found.43,99 In most patients, leukemic cells with an extra chromosome have recurrent structural chromosomal aberrations and a nonspecific immunophenotype. Because of the rarity of these numeric chromosomal aberrations, their prognostic significance in AML is unknown; however, they may be associated with progression. Cases in which the loss of a chromosome is the sole aberration are rare, except for monosomy 7. Monosomy of the X chromosome in female patients or loss of the Y chromosome is the most common whole-chromosome loss identified in pediatric patients with AML. This numeric abnormality is usually associated with a t(8;21) and AMLM2. The presence of an extra or a missing chromosome in a leukemic clone in patients with AML facilitates monitoring of the disease by FISH with a specific centromeric probe. There is limited knowledge of the mechanisms by which numeric chromosomal abnormalities affect the pathogenesis of AML. A dose effect or a duplication of a small genetic defect with oncogenic potential has been suggested as a possible mechanism; however, the relatively low incidence of trisomies as solitary cytogenetic abnormalities may reflect a numeric change secondary to submicroscopic mutations, such as the association of +22 with inv(16) and of +19 with t(7;12).

and refractory anemia may experience prolonged survival or spontaneous remission.197 The loss of a DNA segment in band 7q22 and, although less common, the loss of DNA in 7q32–34 may be important in leukemogenesis. These crucial regions are presumed to contain a myeloid-specific tumor suppressor gene(s). Trisomy 8 Trisomy 8 (+8) is rarely the sole cytogenetic change in ALL (<1% of all patients) but is seen in 2.1% of pediatric patients with various subtypes of AML, and is the most frequent numeric abnormality in this disease, accounting for 9% of all cases with cytogenetically abnormal leukemic cells. It is a heterogeneous and probably not a primary disease-defining aberration, because it occurs with various other cytogenetic and molecular genetic abnormalities.198 In adults with AML, trisomy 8 is associated with a low probability of overall survival.199 To evaluate the relationship between gene expression and cytogenetic findings, Virtaneva et al.200 compared the expression status of 6,606 genes in cytogenetically normal AML blast cells with that in AML blast cells in which +8 was the sole abnormality. The investigators found that the expression profile of AML blast cells with +8 partially overlapped that of AML blast cells with a normal karyotype. Microarray analysis showed altered expression of apoptosisregulating genes and an overall increase in the expression of genes located on chromosome 8, suggesting a gene-dose effect.

The +21 as an acquired or constitutional abnormality Complete or partial monosomy 7 Found in 5% of children with AML, monosomy 7 (−7) and partial deletion of the long arm (7q−) are also nonrandom cytogenetic findings in childhood preleukemic myeloproliferative disorder (MPD) and myelodysplastic syndrome (MDS).195 These abnormalities are grouped together because both may lead to the loss of a putative tumor suppressor gene on 7q. They usually accompany chromosomal changes in leukemic cells with very complex karyotypes and are considered to be secondary changes that occur during clonal evolution. Monosomy 7 as the sole abnormality was found in 1.9% of 478 pediatric patients with AML studied by the Pediatric Oncology Group.99 Despite the improved survival probabilities for children with various malignancies over the past two decades, the prognosis of patients with monosomy 7 remains dismal: most experience a rapid progressive course and respond poorly to conventional chemotherapy.196 However, in rare instances, pediatric patients presenting with monosomy 7

An acquired trisomy 21 (+21) is one of the most common numeric abnormalities in the acute leukemias; in childhood AML, the incidence is 5% overall and 1.8% as the sole aberration.99 In some instances, the formation of an isodicentric 21q results in trisomy 21. Tetrasomy 21 occurs rarely in patients without the constitutional trisomy 21 or in patients with constitutional trisomy 21 (Down syndrome) and AML-M7.201 AML with +21 is associated with mutations and deletions of the CBFA2 gene, which is located on 21q22; thus, a mutated oncogene may be involved in leukemogenesis.202 Compared with children who lack cytogenetic abnormalities, those with constitutional trisomy 21 have a 10to 20-fold increased risk of leukemia. The ratio of ALL risk to AML risk in cases of Down syndrome is typical of that in the absence of this syndrome, except during the first 3 years of life, when the risk of AML (especially AML-M7) is greater than that of ALL.203 This FAB subtype is rare in children without Down syndrome. The outcome of AML in

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Down syndrome patients is excellent; less intensive therapy is effective, and BMT is not warranted.203 A transient myeloproliferative disorder called “transient leukemia” occasionally occurs in phenotypically normal neonates with constitutional or acquired trisomy 21 mosaicism. This disorder is characterized by excessive blastemia and other hematologic abnormalities, and typically resolves within 4 to 6 weeks. These infants are at increased risk of acute leukemia, especially in the first 2 years after birth. Their close monitoring is recommended, because the acquisition of additional cytogenetic abnormalities is indicative of an emerging neoplastic process.204 Mutation of CBFA2 is rarely found in patients with Down syndrome, indicating its probable lack of involvement in the development of hematologic disorders in these patients.205 However, somatic mutations of the GATA1 gene which is located on the X chromosome and is essential for proper growth and maturation of erythroid cells and megakaryocytes, have been found only in patients with Down syndrome and AML-M7.206 A cooperating factor that plays a role in development of this leukemia may also be present on chromosome 21. Miscellaneous numeric changes In some patients with acute leukemia, the leukemic cells contain a partial tandem duplication of MLL and trisomy 11 (+11) without cytogenetic evidence of 11q23 translocation.207 Trisomy 13 (+13) and tetrasomy 13 have been associated with AML in which the leukemic cells are poorly differentiated. These characteristics have been observed mainly in adult patients with poor outcomes.208 A +13 can occur in AML as a component of a clone with other numeric and structural abnormalities. Trisomy 19 (+19) is associated with a subtle t(7;12)(q36;p13) and is seen in children who have AML and are younger than 18 months.209 Trisomy 22 (+22) is frequently found as a cytogenetic change secondary to inv(16). Because the latter alteration is sometimes difficult to ascertain by conventional cytogenetic methods, the presence of a +22 should alert the cytogeneticist to determine whether inv(16) is also present.210

Structural chromosomal changes Common recurrent structural chromosomal changes Approximately 60% of pediatric patients with AML have a recurrent structural chromosomal abnormality, which often serves as part of the basis by which they are randomly assigned to different treatment arms in clinical trials.

t(8;21)(q22;q22) The t(8;21) is the most common recurrent translocation in pediatric AML; it occurs in 12% of pediatric cases and 6% of adult cases. More than 40% of pediatric patients with AMLM2 (acute myeloblastic leukemia with maturation) have a t(8;21).211 Although about 90% of t(8;21) abnormalities have been identified in patients with AML-M2, this translocation has also been found in patients with AML-M1 or AML-M4. The t(8;21) is associated with a favorable outcome, especially when the disease is treated with regimens containing high-dose cytarabine.193,212 Variant forms of the t(8;21) occur in approximately 4% of the cases, and the complex rearrangements often involve 8q22, 21q22, and a third chromosome.213 Sixty percent of pediatric patients with AML and a t(8;21) have additional chromosomal changes. The most frequent loss is that of the sex chromosomes (40% of cases); loss of the Y chromosome occurs more frequently than that of the X chromosome (55% versus 33%).99 Although other numeric abnormalities are rarely found in association with this subgroup, approximately 10% of cases with the t(8;21) undergo deletion of 9q and rearrangement of 7q. The breakpoints of the t(8;21) involve CBFA2 on 21q22 and ETO (also called MTG8 and CDR) on 8q22.214 The chimeric product is consistently detected by RT-PCR and FISH. Both methods can detect the translocation in leukemic cells with an apparently normal karyotype as indicated by conventional cytogenetics.25,215 For example, Andrieu et al.216 found that in a group of 64 adult patients, cytogenetic methods identified a t(8;21) in 8% of the patients, and RT-PCR identified it in 16%. In a Cancer and Leukemia Group B (CALGB) study, RT-PCR detected CBFA2-ETO fusion transcripts in leukemic cells from six patients in which karyotyping had not clearly shown the presence of a t(8;21).25 In addition, CBFA2-ETO fusion transcripts were detected in a patient with a t(8;10)(q22;q26) and in another with a t(1;10;8)(p22;p13;q22); a cryptic ins(8;21) that was detected by FISH may represent a variant of the t(8;21). Rowe et al.217 found a low incidence (0.5%) of cryptic t(8;21) in 142 patients with CBFA2-ETO. Hence, gene-specific FISH and RT-PCR analyses are recommended when cytogenetic analysis fails to detect the t(8;21).218 Recently, FISH analysis revealed cytogenetically undetectable deletions located at the 5 end of the ETO breakpoint in six (9%) of 65 adult patients harboring a t(8;21); the clinical significance of these deletions is unknown.4 RT-PCR can detect CBFA2-ETO transcripts in stem cells of patients whose AML is in complete remission, and only an increasing quantity of these transcripts indicates an impending relapse.219 The transcription of CBFA2-ETO in

Cytogenetics of acute leukemias

bone marrow and cord blood cells of persons who do not have AML suggests that the t(8;21) is generated early in hematopoiesis; few persons may acquire secondary genetic alterations that lead to the development of AML.220 This hypothesis is supported by the detection of CBFA2-ETO in neonatal blood spots of children in whom AML subsequently developed more than 10 years later.221

t(15;17)(q22;q12–21) Acute promyelocytic leukemia (APL) is characterized by a typical morphology (FAB-M3) and a translocation between chromosomes 15 and 17. The t(15;17) is found in 11.5% of pediatric patients with AML, and in the metaphase chromosomes of 87% of these patients, it is the only abnormality.99 About 90% of APL cases have the t(15;17), which is not found in other subtypes of AML; it is therefore an important marker in the diagnosis and treatment of this subtype of leukemia. A consequence of the t(15;17) is the formation of a fusion gene in which the promyelocytic leukemia gene (PML) on 15q22 is juxtaposed to the gene encoding the retinoic acid receptor alpha (RARA) on 17q12–21. Patients with t(15;17)+ AML have a high response rate when treated with conventional anthracycline-based chemotherapy; alltrans-retinoic acid (ATRA) is used to induce differentiation and thus complete clinical remission.222 Arsenic trioxide is another active agent, but published findings on its effectiveness in childhood APL are limited.223 In approximately 10% of patients with APL, the t(15;17) cannot be identified by conventional cytogenetic methods24 ; for such patients, Southern blot analysis, RTPCR, FISH, or a combination of the three methods may be needed. The der(15)t(15;17) contains the PML-RARA fusion gene, which has been implicated in the pathogenesis of APL. The reciprocal RARA-PML fusion gene on the der(17)t(15;17) does not appear to be present in every case, and its prognostic significance is unclear.224 The PMLRARA rearrangement appears to be associated with a normal karyotype (as indicated by conventional cytogenetics) in a small group of patients with APL.24 In most of these cases, RT-PCR has detected PML-RARA transcripts representing a submicroscopic insertion of the RARA gene into the PML gene on chromosome 15, but no reciprocal product has been observed.225 In a few cases, RARAPML transcripts in the absence of a reciprocal product appeared to result from an insertion of the PML gene into the RARA gene.226 Variant rearrangements, including complex translocations or translocations between chromosome 17 and a partner chromosome other than 15, have also been reported.24,227

In a few variant forms of APL, several recurrent chromosomal abnormalities target RARA on chromosome 17: the t(5;17)(q34;q11–q12),228 the t(11;17)(q13;q21),229 the t(11;17)(q23;q21),230 and the der(17).231 The RARA partner genes are NPM (nucleophosmin) on chromosome 5, PLZF (promyelocytic leukemia zinc finger) on 11q23 and NuMA on 11q13, and STAT5b on 17q21.1–q21.2.228–231 The PLZF-RARA fusion occurs in fewer than 1% of APL cases, and rearrangements disrupting NPM, NuMA, and STAT5b appear to be even rarer.24,231,232 Although the morphology of leukemic cells containing these variant translocations is similar to that of leukemic cells with the t(15;17), cells containing fusion genes involving NPM and NuMA appear to be sensitive to ATRA.232,233 By contrast, the t(11;17)(q23;q21)/RARA-PLZF+ AML cells are resistant to ATRA in vitro and in vivo, which may account in part for the adverse clinical outcome associated with this variant.234,235 Therefore, successful identification of translocations in patients with APL is essential for proper treatment assignment. Gene expression profiles of AML blast cells have identified cells that are of the FAB-M3 subtype, have the t(15;17) and express a subset of genes identical to that expressed by cells that are of the M3v subtype and have the t(15;17).236 However, in a subsequent analysis, cells of these two subtypes were distinguished on the basis of their gene expression profiles; this difference suggests that genetic lesions other than the t(15;17) result in the distinct phenotypes. A plausible candidate gene is FLT3, because it is mutated significantly more often in AML-M3v cells than in AML-M3 cells (67% versus 19%, P = 0.001).237 inv(16)(p13.1q22) and t(16;16)(p13.1;q22) The pericentric inversion of chromosome 16 is found in 6% of pediatric patients with AML-M4Eo and in patients with other FAB subtypes. The prognosis of patients with the inv(16) is relatively favorable.99,238 The t(16;16)(p13.1;q22) and other translocations affecting the 16p13.1 or 16q22 breakpoints are rarely observed. Although the inv(16)/t(16;16) is the sole abnormality in 78% of cases presenting with this abnormality, a gain of chromosome 22 frequently accompanies the inv(16).210 The inv(16) fuses the core-binding factor beta gene (CBFB) at 16q22 and the smooth muscle myosin heavy-chain gene (MYH11) at 16p13.1.239 CBFB is a subunit of the corebinding factor (CBF), an important transcription factor in the early hematopoietic stem cell and one that has been implicated in the regulation of the expression of various hematopoiesis-related genes.214,240 CBFB acts as a regulator of the CBFA2 subunit, which binds directly to the core enhancer region of CBF target genes.240 The CBFB-MYH11

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fusion product interferes with the formation of the CBF complex by sequestering CBFA2 in the cytoplasm.241 The action of CBF is thus abrogated, and a block in hematopoietic differentiation ensues. The inv(16) is a subtle rearrangement that is occasionally undetectable by conventional cytogenetic methods. Because most patients with the inv(16) have CBFB-MYH11 transcripts that can be detected by RT-PCR, this method or FISH, or both should be used to evaluate leukemic cells with a suspected inv(16). The application of locus-specific probes detected deletions of 16p in as many as 30% of cases with an inv(16); deletion of 16q has been observed in fewer cases.3,242 Submicroscopic deletions may occur in leukemic cells with an inv(16) or, in rarer instances, in leukemic cells in which a microinsertion of MYH11 into 16q22 creates a masked inv(16) and thus a CBFB-MYH11 fusion gene.243 Qualitative RT-PCR has detected CBFB-MYH11 – expressing cells in patients whose AML has been in remission for prolonged periods without evidence of clinical relapse. Quantitative real-time RT-PCR allows the evaluation of CBFB-MYH11 expression; at the RNA level, this method accurately quantifies the percentage of malignant cells and is a powerful method for monitoring MRD. Because most relapses of AML occur within a year after the completion of induction therapy, it may be beneficial to quantify MRD every 3 months to detect relapse at an early stage.244

Abnormalities of 11q23 Aberrations of 11q23 are among the most frequent chromosomal changes (18%) in childhood AML.99 In topoisomerase II inhibitor-induced AML, as many as 80% of chromosomal abnormalities are aberrations of 11q23.245 However, this rate is probably underestimated, because some 11q23 rearrangements are subtle or cryptic. The most common 11q23 abnormalities are the t(9;11)(p22;q23) (7% of AML cases), the t(11;19)(q23;p13.1) (2%), the t(11;19)(q23;p13.3) (2%), and translocations between 10p and 11q with variable breakpoints (3%, see below).99–101 More than 30 additional alterations involving 11q23, such as the t(1;11), t(6;11), and t(11;17), are included in Table 9.2. Patients with 11q23 abnormalities tend to be young and often have hyperleukocytosis, organomegaly, and myelomonocytic (FAB M4) or monoblastic (FAB-M5) disease. As discussed in the ALL section of this chapter, the MLL gene is often rearranged in leukemic blast cells containing 11q23-associated translocations, regardless of the identity of the reciprocal chromosome.108

The prognostic significance of 11q23 abnormalities in childhood AML is unclear, but it seems to depend on the partner gene involved and the patient’s age.101,246,247 Studies of infants with AML have shown that 50% to 60% of these patients have an 11q23/MLL rearrangement typically associated with M4 or M5 subtype and hyperleukocytosis; however, the prognostic significance of this type of chromosomal abnormality could not be determined.248,249 In older children the t(9;11) confers a favorable outcome, whereas other 11q23 abnormalities do not.250 The better outcome of older children may be related to increased drug sensitivity, especially to etoposide.212,247,250 Another study has shown no difference in the survival probability for patients in distinct subgroups of 11q23 translocations.99 Thus, the prognostic value of 11q23 abnormalities in childhood AML is equivocal because of the small number of cases evaluated and the differences between treatment regimens in the reported series. A serious complication in pediatric patients treated initially for ALL is the development of secondary leukemia, which can arise after treatment with the epipodophyllotoxins [teniposide (VM-26) and etoposide (VP-16)]. These therapy-related leukemias are generally of the monocytic or myelomonocytic subtype; have characteristic 11q23 abnormalities, primarily t(9;11)(p22;q23) and t(11;19)(q23;p13.3); and are associated with an extremely poor prognosis.245,251 The MLL breakpoints in patients with secondary leukemia map to the same breakpoint-cluster region identified in patients with primary MLL/11q23associated acute leukemias. Consensus topoisomerase IIbinding sequences occur near the chromosomal breakpoint of 11q23 rearrangements in patients with secondary leukemia. Therefore, topoisomerase II may have a role in the mechanism(s) leading to these translocations.252 Therapy-related leukemias are reviewed in detail in Chapter 31. Other recently identified translocations, most of which have been found in adult patients, are associated with AML or MDS secondary to treatment: the t(11;16)(q23;p13.3) results in the formation of the MLL-CBP fusion gene; the t(11;22)(q23;q13) in the formation of MLL-p300; and the t(16;21)(q24;q22) in the formation of MTG16-CBFA2.253–255 Further study of these proteins, whose wild-type counterparts are involved in differentiation and cell cycle regulation, may provide new insights into leukemogenesis. Partial tandem duplication of the MLL gene, as detected by Southern blot analysis and RT-PCR, occurs in 6% to 11% of adults with primary AML and a normal karyotype as indicated by conventional cytogenetics.201,256,257 In some patients with acute leukemia, the MLL gene is rearranged in leukemic cells that have +11 but not 11q23 translocations.

Cytogenetics of acute leukemias

MLL amplification detected by FISH appears to be characteristic of a subgroup of adults with AML who have a dismal outcome.258 In hematologic malignancies, most deletions of 11q involve 11q23. Kobayashi et al.259 identified a region of deletion proximal to MLL, suggesting that a gene(s) other than MLL may be the target of deletion. Only 1 of 23 patients with AML or MDS and a del(11q) reviewed by the European 11q23 Workshop retained both MLL alleles.109 In these cases, performing FISH with an MLL probe is highly recommended to rule out a subtle or cryptic translocation that may interrupt the gene. The t(6;11)(q27;q23) is subtle, and under substandard banding of chromosomes, this translocation may go undetected or be classified as del(11)(q23).109,260,261 A recent study of 26 patients with t(6;11)+ AML showed that the median period of EFS was only 7.8 months.261 Monitoring MLL-AF6 fusion transcripts showed that MRD was consistently associated with relapse in patients with AML treated with either chemotherapy alone or autologous BMT.262 The t(10;11)(p11.2;q23), which generates the MLL-ABI1 chimeric fusion, has been found in a few infants with AML and monocytic involvement.263 The perinatal presentation suggests that this genetic event occurs during in utero leukemogenesis.263 The t(10;11)(p12;q23) is a rare translocation seen more frequently in AML-M4 and AML-M5 than in other AML subtypes. In most cases, the t(10;11) leads to the fusion of the 5 end of MLL and the 3 end of AF10.264 Interestingly, these genes on chromosomes 10 and 11 are in opposite orientation to one another; thus, the inversion of one gene is required to form the MLL-AF10 fusion gene. During initial cytogenetic evaluation, many instances of the t(10;11) are seen with complex rearrangements, an association that may indicate the inversion of either chromosome;265 however, the most common inversion is that of the region from 11q13 to 11q23 with a subsequent translocation of the inverted region to 10p12. The most complex rearrangements that involve MLL are generated by the t(10;11), and the resulting fusion gene can encode a chimeric protein in which the leucine-dimerization motif of AF10 is juxtaposed to the N terminus of MLL.264 The t(10;11)(p12;q13) results in the rearrangement of CALM and subsequently the generation of CALM-AF10 chimeric transcripts.266 The presence of MLL-AF10 or CALM-AF10 is equivocal at the cytogenetic level and should be further investigated by molecular methods to monitor MRD. This translocation has been observed in acute leukemias of varied morphology and of the B- and T-cell lineages.267 No prognostic significance is currently associated with this type of chromosomal rearrangement,

because an insufficient number of cases have been studied at the molecular level. Uncommon recurrent structural chromosomal changes t(1;22)(p13;q13). The t(1;22) has been found almost exclusively in infants and children as old as 18 months who have AML-M7 but not Down syndrome. The overall incidence in AML is 1%.99 These patients present with abdominal masses and myelofibrosis, and their prognosis is poor: only about 25% of affected children respond to available treatments.268 The detection of a t(1;22) may clarify the diagnosis of acute megakaryoblastic leukemia, which is characterized by highly pleomorphic, cohesive undifferentiated cells that are found in bone marrow aspirates and resemble cells in metastatic solid tumors. The t(1;22) creates a fusion gene OTT-MAL (RBM15-MLK1), which was cloned and characterized independently.269,270 The OTT-MAL fusion protein may contribute to leukemogenesis through modulation of chromatin organization. Abnormalities of 3q21 and 3q26 These abnormalities develop in patients with AML, MDS, or CML and are associated with normal or high platelet counts, trilineage myelodysplasia, and a poor prognosis. The presence of abnormal multilineage hematopoiesis suggests that stem cells are involved in leukemogenesis. The 3q abnormalities are frequently associated with monosomy 7 and are found mainly in adults.271,272 The inappropriate activation of EVI1 (ecotropic virus integration site) on chromosome band 3q26 is a major factor in leukemia pathogenesis resulting from 3q abnormalities and has been directly implicated in abnormal megakaryopoiesis.273–275 This gene is transcriptionally activated or structurally altered in AML blast cells with alterations of 3q21 and 3q26. In a typical case, EVI1 is rearranged with genes on 3q21 through the inv(3)(q21q26), t(3;3)(q21;q26), or ins(3;3); such rearrangements can also occur as a result of the t(3;21)(q26;q22) or t(3;12)(q26;p13).276,277 The t(3;21) usually occurs in adult patients with therapy-related AML. The oncogenic effects of the rearranged genes on 3q26 are unclear, because the length of the affected region (hundreds of kilobases) has made it difficult to study273,274 ; however, three genes (EAP, EVI1, MDS1) in this region undergo rearrangement with CBFA2 as a result of a t(3;21) and with ETV6 as a result of a t(3;12).276,277 Structural changes capable of generating complex AML1-MDS1-EVI1 fusion genes may also delete genes or prevent gene expression.276 Like the molecular breakpoints at 3q26, those at 3q21 are heterogeneous.273,274 The transcriptional activation of

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the EVI1 gene in the inv(3;3) and t(3;3) may be caused by the introduction of enhancer elements of the ribophorin I gene. A better understanding of the genes affected by abnormalities of the 3q21 and 3q26 regions may resolve the pathogenic mechanisms leading to the region’s distinctive association with abnormal multilineage hematopoiesis.275 t(3;5)(q25;q34) This rare translocation occurs in a subset of young adults who have AML or MDS with multilineage dysplasia and normal platelet counts; the prognosis is unfavorable.278 The t(3;5) generates a fusion gene consisting of NPM (nucleophosmin) and MLF1 (myelodysplasia/myeloid leukemia factor 1).279 Because of its rarity little else is known about the t(3;5) and the pathogenic role of NPM-MLF1. Abnormalities of chromosome 5 Deletions of 5q (5q-) are found in primary and therapyrelated MDS and AML, most often in association with other chromosomal abnormalities. Contrary to its frequency in adult patients, monosomy 5 (−5) or 5q− occurs very infrequently in childhood AML (1% of all cases).99 The t(5;11)(q35;p15.5) is cryptic and associated with del(5q) in childhood AML.280 Recently, a cryptic t(5;11) was identified in three cases of childhood AML by FISH using a whole chromosome-5 painting probe and a 5q subtelomeric probe to determine whether the DNA deleted from the 5q arm was translocated to another chromosome(s); the translocation appeared to affect the 11p15.5 in all three cases.280 Interestingly, none of the 24 adults patients with MDS and 5q− in this analysis had this translocation. The breakpoints of t(5;11) were further defined at the molecular level: NUP98 was fused to the novel gene NSD1; transcripts of this fusion gene were expressed.281 Subsequently, a t(5;11)(q35;p15.5) was identified in two patients by an M-FISH telomere assay.282 This finding represented 3% of the 69 pediatric patients with AML whose leukemic cells had isolated trisomy or an apparently normal karyotype as indicated by conventional cytogenetics.282 A more recent study showed that one of five pediatric patients with an apparently normal karyotype had the t(5;11) without a del(5q).283 The identification of these cryptic translocations and the subsequent identification of affected genes in other patients may improve our understanding of leukemogenesis. t(6;9)(p23;q34) This translocation was originally identified in patients with AML-M2baso, which is characterized by granulocytic maturation and a variable infiltrate of blast cells with basophilic granules. The t(6;9) may also be present in other FAB sub-

Table 9.6 Translocations of 8p11.2/PDGFR1 Translocation

Disease

Partner gene

t(6;8)(q27;p11.2) t(8;9)(p11.2;q33) t(8;13)(p11.2;q12) t(8;12)(p11.2;p11) t(8;12)(p11.2;q15) t(8;17)(p11.2;q25) t(8;19)(p11.2;q13)

CMML CMML CMML CMML CMML CMML CMML

FOP CEP110 ZNF198 Unknown Unknown Unknown Unknown

Abbreviation: CMML, chronic myelomonocytic leukemia.

groups of AML, MDS, and acute myelofibrosis. Its overall incidence in childhood AML is approximately 2%, and it confers an unfavorable prognosis.99 When fused, the involved genes, DEK (6p23) and CAN (9q34), encode a fusion protein that may control DNA transcription.284 CAN is associated with the nuclear pore complex and has a key role in nucleocytoplasmic transport processes and cell cycle progression.285 Abnormalities of 8p11.2 Several recurrent chromosomal alterations that involve 8p11.2 have been found in myeloid malignancies. These chromosomal aberrations are uncommon and target two distinct genes: PDGFR1, a growth factor receptor that acts as a tyrosine kinase, and MOZ, a putative histone acetyltransferase of unknown function. The disruption of PDGFR1 is associated with CMML (Table 9.6). Disruption of MOZ is seen in patients with acute myelomonocytic or monocytic leukemia, in which the leukemic blast cells frequently display erythrophagocytosis; the alteration in MOZ is also associated with a poor prognosis.286,287 In AML blast cells, the t(8;16)(p11.2;p13) fuses MOZ to CBP, the inv(8)(p11.2q13) fuses MOZ to TIF2, and the t(8;22)(p11.2;q13) fuses MOZ to p300.286–288 These three partner genes encode proteins that have histone acetyltransferase activity and probably contribute to leukemogenesis by altering chromatin-mediated transcriptional control of unknown target genes.289 Abnormalities of 11p15.5 Translocations of 11p15.5, mostly observed in childhood therapy-related AML/MDS, involve the NUP98 gene.290 NUP98 is a component of the nuclear pore complex that selectively transports RNA and protein between the nucleus and cytoplasm. Among the newly cloned fusion genes encoding components of the nuclear pore are

Cytogenetics of acute leukemias

treatment.295 The t(12;22) fuses ETV6 to MN1 (at 22q11.2); the resulting fusion gene encodes a protein of unknown function.296

Table 9.7 Translocations of 11p15/NUP98 Chromosomal abnormality

Disease

Partner gene

t(1;11)(q23;p15) t(2;11)(q31;p15) t(2;11)(q31;p15) t(4;11)(q21;p15) t(5;11)(q35;p15.5) t(9;11)(p22;p15) t(7;11)(p15;p15) t(7;11)(p15;p15) t(7;11)(p15;p15) t(8;11)(p11.2;p15) t(9;11)(p22;p15.5) inv(11)(p15q22) t(11;12)(p15;q13) t(11;12)(p15;q13) t(11;20)(p15;q11)

AML AML t-AML T-ALL AML AML AML AML AML AML AML AML, MDS AML, t-AML AML t-AML, MDS

PMX1 HOXD11 HOXD13 RAP1GDS1 (NRG) NSD1 LEDGF HOXA9 HOXA11 HOXA13 NSD3 LEDGF DDX10 HOXC11 HOXC13 TOP1

Abbreviations: AML, acute myeloid leukemia; t-AML, therapyrelated AML; T-ALL, T-cell acute lymphoblastic leukemia; MDS, myelodysplastic syndrome.

NUP98-NSD1, which results from a t(5;11)(q35;p15.5); NUP98-LEDGF, which results from the t(9;11)(p22;p15.5); and NUP98-TOP1, which results from a t(11;20)(p15.5;q11) (Table 9.7).281,291,292 Abnormalities of 12p Recurrent translocations involving ETV6 (12p13) have been found in a relatively small number of children with myeloid cell disorders. The t(7;12) was initially recognized in leukemic cells that had a 12p-; most cells with this translocation also had trisomy 8 or trisomy 19. The t(7;12)(q36;p13) and t(7;12)(q32;p13) were identified in children 18 months of age or younger.121,209 The t(7;12)(q36;p13) is more frequent and was found in approximately 20% of similarly aged Dutch patients with AML.209 Because the t(7;12) may play an adverse role in myeloid disorders, all children 18 months and younger should be screened for MLL and ETV6 rearrangements. Other alterations of ETV6 in patients with myeloid disorders have been reported. The fusion of ETV6 with the homeobox gene HLXB9 has been observed in patients with the t(7;12)(q36;p13).293 ETV6 is also involved in other translocations, including the t(5;12), t(9;12), t(10;12), and t(12;22), which are found primarily in children with MDS or MPD.294 The t(12;22)(p13;q11.2) has been found in only a few cases. Most patients acquired the t(12;22) after previous

Abnormalities of 16p13/CBP and 22q13/p300 The CREB-binding protein (CBP) and p300 are closely related transcriptional coactivators. They mediate target gene activation by modulating transcription initiation by RNA polymerase II complexes.297 Moreover, the two coactivators regulate gene expression in and differentiation of hematopoietic cells via their involvement in leukemia-associated chromosomal translocations.298 For example, CBP-MOZ occurs in patients with AML and the t(8;16)(p11;q13),286 CBP-MORF in those with the t(10;16)(q22;p13),299 and CBP-MLL in those with the t(11;16)(q23;p13).300 The p300-MLL has also been found in AML cases with the t(11;22)(q23;q13).286 t(16;21)(p11.2;q22.2) A poor prognosis is associated with the t(16;21) (p11.2;q22.2) in patients with primary pediatric AML, secondary AML associated with MDS, or CML in blast crisis.301 The t(16;21) juxtaposes FUS (TLS) on chromosome 16 and ERG on chromosome 21 to form the FUS-ERG fusion gene.302 t(16;21)(q24;q22) This rare but recurrent translocation is associated with trisomy 8, an abnormality mostly observed in patients with therapy-related acute leukemias and MDS, although it has been noted in children and adults with primary AML.303 The t(16;21)(q24;q22) fuses CBFA2 with CBFA2T (MTG16) generating a fusion gene similar to CBFA2-ETO, which is created by the t(8;21).255,303,304 Other abnormalities of 21q22 The CBFA2 gene on 21q22 encodes a strong hematopoietic transcription factor and is one of the most frequently mutated genes in the human acute leukemias.305 Its fusion partners include ETV6, ETO, MDS, EVI1, and others.305 As described in a preceding section, CBFA2 binds to DNA and heterodimerizes with CBFB to form a complex called the core-binding factor (CBF). The gene encoding CBFB is located at 16q22 and is disrupted by the inv(16).239 More than a dozen translocations and a similar number of point mutations target genes specific for the subunits of the corebinding factor. CBFA2 and CBFB have now been implicated in about 50% of all cases of childhood acute leukemias. Patients whose leukemic cells contain variants of the corebinding factor appear to respond best to multiple courses of high-dose cytarabine.25,193

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In a comparison of the effectiveness of conventional cytogenetic methods and of RT-PCR in detecting CBF gene alterations that result from chromosomal rearrangements, namely the t(8;21) and inv(16), few differences were seen. Therefore, conventional cytogenetic methods reliably identify chromosomal abnormalities that alter CBF genes.25 As mentioned in the section on acute lymphoblastic leukemia, amplification of CBFA2 has occurred in several cases of pediatric B-lineage ALL, suggesting that the gene has an oncogenic role.133,136 Recent articles report the presence of mutations in the region of CBFA2 that encodes the runt domain. Found predominantly in patients with AMLM0202,306 or with myeloid malignancies and an acquired trisomy 21,307 these mutations have been also implicated in a familial platelet defect that is associated with a propensity for AML development.308 Hence, CBFA2 can be mutated or serve as a partner in a chromosomal translocation in patients with AML. In one study, FISH revealed that 7 (23%) of 30 patients with acute leukemia and a known chromosomal abnormality at 21q22 (excluding the common recurrent translocations mentioned above) had breaks within CBFA2.309 Interestingly, five of these seven patients had been previously treated with topoisomerase II-targeting agents.309 This finding and that of the previously recognized t(3;21) in therapy-related disorders indicate that 21q22 abnormalities are associated with cytotoxic chemotherapy. Common genetic lesions in AML Among the most common molecular markers for subclasses of AML include genetic lesions that are not associated with chromosome abnormalities, as determined by conventional cytogenetics. With a few exceptions, the molecular events do not seem to be restricted to a specific stage of differentiation or lineage commitment within the myeloid compartment (Table 9.5). These lesions are reviewed in detail in Chapter 11.

Summary and future directions Our current understanding of the molecular pathogenesis of the lymphoid and myeloid leukemias has emerged from careful characterization of chromosomal translocation breakpoints and cloning of the affected genes. Many of these rearranged genes encode tyrosine kinases, transcription factors, or other proteins that have relevant activities during the cell cycle and apoptosis. Point mutations in hematologic transcription factors and tyrosine kinases, such as CBFA2, C/EBP, FLT3, and KIT, also contribute to

Table 9.8 Cryptic chromosomal aberrationsa Chromosomal abnormality

Disease

Fusion gene or altered gene

t(1;12)(q25;p13) t(5;11)(q35;p15.5) t(5;14)(q35;q32) t(7;12)(q36;p13) t(9;9)(q34;q34) t(9;12)(q11;p13) 11q23 t(12;15)(p13;q25) t(12;21)(p13;q22) inv(19)(p13.3q13)

AML AML T-ALL Infant AML AML ALL ALL, AML AMLb ALL ALL

ETV6-ARG (ABL2) NUP98-NSD1 CTIP2-HOX11L2 ETV6-HLXB9 SET-CAN ETV6-PAX5 MLL – various partners ETV6-TRKC (NTRK3) CBFA2 (AML1) E2A-FB1

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; T-ALL, T-cell ALL. a Cryptic aberration is defined as a subtle chromosomal abnormality not readily detectable at the 500 level of banding resolution. b Also found in congenital fibrosarcoma and mesoblastic nephroma.

leukemogenesis. The ultimate phenotypic consequence of many of these rearrangements and mutations is the impairment of hematopoietic development. Several genes are involved in more than one translocation and thus in the formation of multiple fusion genes. However, the total number of genes targeted by chromosomal rearrangements appears to be relatively small, leading to the conclusion that only a small group of altered genes can induce leukemia.310 Throughout this chapter, the associations between specific cytogenetic and molecular changes and distinct clinical subtypes of diseases or treatment outcomes have been highlighted. Many of these cytogenetic and molecular changes are now recognized as important diagnostic criteria in the classification system for acute leukemias, or are used to assign patients to riskbased treatment groups, or both.311,312 Approximately 40% of all known fusion transcripts can be detected by routine RT-PCR analysis of samples from pediatric patients at the time of initial diagnosis of B-lineage ALL or AML. Although FISH and RT-PCR have improved the detection of subtle chromosomal aberrations that have eluded conventional cytogenetic methods (Table 9.8), new molecular methods are being developed to comprehensively identify chromosomal abnormalities that lead to the genomic instability that causes cancer. For example, microarray analysis has allowed investigators to differentiate ALL from AML on the basis of the leukemic cells’ gene expression profiles,313 to determine that gene expression of AML blast cells with trisomy 8 as the sole abnormality

Cytogenetics of acute leukemias

differs from that of AML blast cells with normal karyotypes,200 to detect differences in the gene expression profiles of ALL blast cells with or without MLL translocations,107 to identify clinically important T-ALL subgroups,314 and to reveal the differences in expression between genes associated with AML- or ALL-specific translocations.236,315 Integration of the functional genome profile with traditional molecular cytogenetic observations could lead to the identification of genes that play crucial roles in the development and progression of leukemia, and in the discovery of agents that selectively inhibit proteins or molecular pathways required for the clonal evolution of transformed myeloid or lymphoid progenitor cells.

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Cytogenetics Group (UKCCG) study. Leukemia, 2000; 14: 1885– 91. Kobayashi, H., Espinosa, R., III, Fernald, A. A., et al. Analysis of deletions of the long arm of chromosome 11 in hematological malignancies with fluorescence in situ hybridization. Genes Chromosomes Cancer, 1993; 8: 246–52. Kobayashi, H., Espinosa, R., III, Thirman, M. J., et al. Do terminal deletions of 11q23 exist? Identification of undetected translocations with fluorescence in situ hybridization. Genes Chromosomes Cancer, 1993; 7: 204–8. Martineau, M., Berger, R., Lillington, D. M., Moorman, A. V., & Secker-Walker, L. M. The t(6;11)(q27;q23) translocation in acute leukemia: a laboratory and clinical study of 30 cases. EU Concerted Action 11q23 Workshop participants. Leukemia, 1998; 12: 788–91. Takatsuki, H., Yufu, Y., Tachikawa, Y., & Uike, N. Monitoring minimal residual disease in patients with MLL-AF6 fusion transcript-positive acute myeloid leukemia following allogeneic bone marrow transplantation. Int J Hematol, 2002; 75: 298–301. Taki, T., Shibuya, N., Taniwaki, M., et al. ABI-1, a human homolog to mouse ABI-interactor 1, fuses the MLL gene in acute myeloid leukemia with t(10;11)(p11.2;q23). Blood, 1998; 92: 1125–30. Chaplin, T., Bernard, O., Beverloo, H. B., et al. The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene. Blood, 1995; 86: 2073–6. Limbergen, H. van, Poppe, B., Janssens, A., et al. Molecular cytogenetic analysis of 10;11 rearrangements in acute myeloid leukemia. Leukemia, 2002; 16: 344–51. Dreyling, M. H., Schrader, K., Fonatsch, C., et al. MLL and CALM are fused to AF10 in morphologically distinct subsets of acute leukemia with translocation t(10;11): both rearrangements are associated with a poor prognosis. Blood, 1998; 91: 4662–7. Bohlander, S. K., Muschinsky, V., Schrader, K., et al. Molecular analysis of the CALM/AF10 fusion: identical rearrangements in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma patients. Leukemia, 2000; 14: 93–9. Carroll, A. J., Civin, S., Schneider, N. R., et al. The t(1;22) (p13;q13) is nonrandom and restricted to infants with acute megakaryoblastic leukemia: a Pediatric Oncology Group Study. Blood, 1991; 78: 748–52. Mercher, T., Coniat, M. B., Monni, R., et al. Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia. Proc Natl Acad Sci U S A, 2001; 98: 5776–9. Ma, Z., Morris, S. W., Valentine, V., et al. Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia. Nat Genet, 2001; 28: 220–1. Testoni, N., Borsaru, G., Martinelli, G., et al. 3q21 and 3q26 cytogenetic abnormalities in acute myeloblastic leukemia: biological and clinical features. Haematologica, 1999; 84: 690–4. Keung, Y. K., Buss, D., Powell, B. L., & Pettenati, M. Central diabetes insipidus and inv(3)(q21q26) and monosomy 7 in acute

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myeloid leukemia. Cancer Genet Cytogenet, 2002; 136: 78– 81. Morishita, K., Parganas, E., Willman, C. L., et al. Activation of EVI-1 gene expression in human acute myelogenous leukemias by translocation spanning 300–400 kilobases on chromosome band 3q26. Proc Natl Acad Sci U S A, 1992; 89: 3937–41. Levy, E. R., Parganas, E., Morishita, K., et al. EVI-1 DNA rearrangements proximal to the EVI-1 locus associated with rearrangements of the 3q21q26 syndrome. Blood, 1994; 83: 1348–53. Jolkowska, J. & Witt, M. The EVI-1 gene – its role in pathogenesis of human leukemias. Leuk Res, 2000; 24: 553–8. Nucifora, G., Begy, C. R., Kobayashi, H., et al. Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21) (q26;q22) translocations. Proc Natl Acad Sci U S A, 1994; 91: 4004–8. Raynaud, S. D., Baens, M., Grosgeorge, J., et al. Fluorescence in situ hybridization analysis of t(3;12)(q26;p13): a recurring chromosomal abnormality involving the TEL gene (ETV6) in myelodysplastic syndromes. Blood, 1996; 88: 682–9. Raimondi, S. C., Dube, I. D., Valentine, M. B., et al. Clinicopathologic manifestations and breakpoints of the t(3;5) in patients with acute nonlymphocytic leukemia. Leukemia, 1989; 3: 42–7. Yoneda-Kato, N., Look, A. T., Kirstein, M. N., et al. The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene, 1996; 12: 265–75. Jaju, R. J., Haas, O. A., Neat, M., et al. A new recurrent translocation, t(5;11)(q35;p15.5), associated with del(5q) in childhood acute myeloid leukemia. The UK Cancer Cytogenetics Group (UKCCG). Blood, 1999; 94: 773–80. Jaju, R. J., Fidler, C., Haas, O. A., et al. A novel gene, NSD1, is fused to NUP98 in the t(5;11)(q35;p15.5) in de novo childhood acute myeloid leukemia. Blood, 2001; 98: 1264–7. Brown, J., Jawad, M., Twigg, S. R., et al. A cryptic t(5;11) (q35;p15.5) in 2 children with acute myeloid leukemia with apparently normal karyotypes, identified by a multiplex fluorescence in situ hybridization telomere assay. Blood, 2002; 99: 2526–31. Panarello, C., Rosanda, C., & Morerio, C. Cryptic translocation t(5;11)(q35;p15.5) with involvement of the NSD1 and NUP98 genes without 5q deletion in childhood acute myeloid leukemia. Genes Chromosomes Cancer, 2002; 35: 277–81. Lindern, M. von, Fornerod, M., Baal, S. van, et al. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA. Mol Cell Biol, 1992; 12: 1687–97. Fornerod, M., Boer, J., Baal, S. van, et al. Relocation of the carboxyterminal part of CAN from the nuclear envelope to the nucleus as a result of leukemia-specific chromosome rearrangements. Oncogene, 1995; 10: 1739–48.

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286 Borrow, J., Stanton, V. P., Jr., Andresen, J. M., et al. The translocation t(8;16)(p11;q13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat Genet, 1996; 14: 33–41. 287 Carapeti, M., Aguiar, R. C., Goldman, J. M., & Cross, N. C. A novel fusion between MOZ and the nuclear receptor coactivator TIF2 in acute myeloid leukemia. Blood, 1998; 91: 3127–33. 288 Tasaka, T., Nagai, M., Matsuhashi, Y., et al. Secondary acute monocytic leukemia with a translocation t(8;22)(p11;q13). Haematologica, 2002; 87: e-case report 19 (ECR19). 289 Champagne, N., Pelletier, N., & Yang, X. J. The monocytic leukemia zinc finger protein MOZ is a histone acetyltransferase. Oncogene, 2001; 20: 404–9. 290 Lam, D. H. & Aplan, P. D. NUP98 gene fusions in hematologic malignancies. Leukemia, 2001; 15: 1689–95. 291 Ahuja, H. G., Hong, J., Aplan, P. D., et al. t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion between NUP98 and the gene encoding transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF). Cancer Res, 2000; 60: 6227–9. 292 Ahuja, H. G., Felix, C. A., & Aplan, P. D. Potential role for DNA topoisomerase II poisons in the generation of t(11;20)(p15;q11) translocations. Genes Chromosomes Cancer, 2000; 29: 96–105. 293 Beverloo, H. B., Panagopoulos, I., Isaksson, M., et al. Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13). Cancer Res, 2001; 61: 5374–7. 294 Cross, N. C. & Reiter, A. Tyrosine kinase fusion genes in chronic myeloproliferative diseases. Leukemia, 2002; 16: 1207–12. 295 Vieira, L., Marques, B., Ambrosio, A. P., et al. TEL and MN1 fusion in myelodysplastic syndrome: new evidence for a therapy-related event. Br J Haematol, 2000; 110: 238–9. 296 Buijs, A., Sherr, S., Baal, S. van, et al. Translocation (12;22) (p13;q11) in myeloproliferative disorders results in fusion of the ETS-like TEL gene on 12p13 to the MN1 gene on 22q11. Oncogene, 1995; 10: 1511–19. 297 Cho, H., Orphanides, G., Sun, X., et al. A human RNA polymerase II complex containing factors that modify chromatin structure. Mol Cell Biol, 1998; 18: 5355–63. 298 Blobel, G. A. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood, 2000; 95: 745– 55. 299 Panagopoulos, I., Fioretos, T., Isaksson, M., et al. Fusion of the MORF and CBP genes in acute myeloid leukemia with the t(10;16)(q22;p13). Hum Mol Genet, 2001; 10: 395–404. 300 Taki, T., Sako, M., Tsuchida, M., & Hayashi, Y. The t(11;16)(q23;p13) translocation in myelodysplastic syndrome fuses the MLL gene to the CBP gene. Blood, 1997; 89: 3945– 50. 301 Morgan, R., Riske, C. B., Meloni, A., et al. t(16;21)(p11.2;q22): a recurrent primary rearrangement in ANLL. Cancer Genet Cytogenet, 1991; 53: 83–90.

302 Ichikawa, H., Shimizu, K., Hayashi, Y., & Ohki, M. An RNAbinding protein gene, TLS/FUS, is fused in ERG in human myeloid leukemia with t(16;21) chromosomal translocation. Cancer Res, 1994; 54: 2865–8. 303 La Starza, R., Sambani, C., Crescenzi, B., et al. AML1/MTG16 fusion gene from a t(16;21)(q24;q22) translocation in treatment-induced leukemia after breast cancer. Haematologica, 2001; 86: 212–13. 304 Kondoh, K., Nakata, Y., Furuta, T., et al. A pediatric case of secondary leukemia associated with t(16;21)(q24;q22) exhibiting the chimeric AML1-MTG16 gene. Leuk Lymphoma, 2002; 43: 415–20. 305 Friedman, A. D. Leukemogenesis by CBF oncoproteins. Leukemia, 1999; 13: 1932–42. 306 Osato, M., Asou, N., Abdalla, E., et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood, 1999; 93: 1817–24. 307 Michaud, J., Wu, F., Osato, M., et al. In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis. Blood, 2002; 99: 1364–72. 308 Song, W. J., Sullivan, M. G., Legare, R. D., et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat Genet, 1999; 23: 166–75. 309 Roulston, D., Nucifora, G., Dietz-Band, J., Le Beau, M. M., & Rowley, J. Detection of rare 21q22 translocation breakpoints within the AML1 gene in myeloid neoplasms by fluorescence in situ hybridization. Blood, 1993; 82(Supp1. 1): 532a, abstract 2114. 310 Bohlander, S. K. Fusion genes in leukemia: an emerging network. Cytogenet Cell Genet, 2000; 91: 52–6. 311 Harris, N. L., Jaffe, E. S., Diebold, J., et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting – Airlie House, Virginia, November 1997. J Clin Oncol, 1999; 17: 3835–49. 312 Jaffe, E. S., Harris, N. L., Stein, H., & Vardiman, J. W., eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Hematopoietic and Lymphoid Tissues (Lyon, France: IARC Press, 2001). 313 Golub, T. R., Slonim, D. K., Tamayo, P., et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science, 1999; 286: 531–7. 314 Ferrando, A. A., Neuberg, D. S., Staunton, J., et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell, 2002; 1: 75–87. 315 Yeoh, E. J., Ross, M. E., Shurtleff, S. A., et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell, 2002; 1: 133–43.

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10 Molecular genetics of acute lymphoblastic leukemia Adolfo A. Ferrando, Jeffrey E. Rubnitz, and A. Thomas Look

Introduction Although most cases of acute lymphoblastic leukemia (ALL) do not arise from a recognized predisposing genetic condition, this disease is still considered to have a genetic basis. That is, somatically acquired genetic changes contribute in pivotal ways to the genesis of ALL and have important implications for its diagnosis and treatment.1–6 Such acquired lesions, restricted to the leukemic clone, include alterations in both the number (ploidy) and structure of the blast cell chromosomes,4,7 the latter consisting of reciprocal translocations, inversions, deletions, gene amplifications, and point mutations. Although many of these abnormalities can be detected by routine cytogenetic analysis, others require molecular assays.8 Molecular identification of the genetic targets of these alterations has led to the isolation and characterization of numerous oncogenes and tumor suppressors, providing valuable clues to the mechanisms of leukemogenesis.4,5,9–11 In this chapter, we review the molecular basis of childhood ALL, emphasizing the oncogenes, oncoproteins, and tumor suppressors that appear to be critical components of a limited number of transforming pathways.

Proto-oncogene activation Standard karyotyping can identify chromosomal translocations, including both recurrent and random rearrangements, in about 50% of cases of childhood ALL. When molecular assays are used with cytogenetic analysis, evidence of a translocation is seen in about 70% of cases (Fig. 10.1). Translocations in ALL most often disrupt genes that encode transcription factors (Tables 10.1 and 10.2),

defined as nuclear proteins that bind to DNA and regulate the expression of other cellular proteins.4,5,9–11 Transcription factors share a variety of structural motifs (e.g. basic helix-loop-helix, bHLH), suggesting that oncoproteins with a common motif may be involved in similar regulatory processes (Table 10.2). In addition, many of the transcription factors involved in human leukemias share homology with key developmental regulatory proteins of Drosophila (e.g. trithorax and runt) (Table 10.1), suggesting that the oncogenic versions of these proteins act positively to upregulate critical target genes or negatively to interfere with normal developmental pathways.11 The net effect is disruption of regulatory cascades that control and coordinate the expression of the large numbers of proteins required for completion of lymphoid cell growth, differentiation, and survival programs. Leukemia-specific translocations can activate transcription factors by at least two mechanisms. In B-progenitor ALL, translocations fuse discrete portions of two different genes to create chimeric transcription factors with oncogenic properties. Gene fusion is also the mechanism by which the ABL tyrosine kinase becomes activated in ALL. Alternatively, in T- and B-cell acute leukemias, structurally intact transcription factor genes are dysregulated by their juxtaposition with transcriptionally active T-cell receptor (TCR) or immunoglobulin (IG) genes.

Chimeric oncogenes BCR-ABL The first fusion oncogene described in ALL was BCR-ABL, generated by the der(22) of the t(9;22)(q34;q11) and often called the Philadelphia (Ph) chromosome. Cytogenetic

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 10.1 Representative chimeric oncoproteins in acute lymphoblastic leukemia Drosophila homologue

Translocation

Fusion oncoprotein

Structural motif

Tyrosine kinase t(9;22)(q34;q11)

BCR-ABL

Kinase (ABL)

Transcription factors t(1;19)(q23;p13.3) t(4;11)(q21;23) t(11;19)(q23;p13) t(10;11)(p12-p13;q14-q21) t(17;19)(q22;p13.3) t(12;21)(p13;q22)

E2A-PBX1 MLL-AF4 MLL-ENL CALM-AF10 E2A-HLF TEL-AML1

Homeodomain (PBX1) A-T hook (MLL) A-T hook (MLL) Clathrin assembly (CALM) Basic leucine zipper (HLF) ETS-homology/Runthomology (AML1)

None

Extradenticle Trithorax Trithorax Giant Runt

Random

30%

25%

HOX11 LYL1 HOX11L2 TAL1 LMO1 TAL2 LMO2 MYC bHLHB1 LCK 7q35/TCRβ or 14q11/TCRαδ

4%

BCR-ABL

3%

t(9;22)

4% 20%

MYC t(8;14),t(2;8),t(8;22)

2%

TEL-AML1 3%

E2A-PBX1 t(1;19)

E2A-HLF t(17;19)

T cell

t(12;21)

1%

B cell

6%

MLL fusions t(4;11),t(1;11),t(11;19)

Pre-B cell

Pro-B cell

Fig. 10.1 Frequency of translocation-associated oncogenes in childhood ALL. The majority of genes affected by chromosomal rearrangements encode proteins active in transcriptional regulation of cell growth, differentiation, or survival (modified, with permission, from Look6 ).

studies have revealed the Ph chromosome in about 95% of cases of chronic myelogenous leukemia (CML), 25% of adult ALL cases, and 3% to 5% of pediatric ALL cases.7,12 In both CML and ALL, the t(9;22) fuses 5 sequences from BCR to 3 sequences from ABL.13–18 The breakpoints on chromosome 9 are scattered over a nearly 200 kilobase (kb) region within the first intron of ABL, whereas the BCR breakpoints on chromosome 22 are clustered in two areas: a 5.8-kb major breakpoint cluster region (M-bcr) in CML and a minor breakpoint cluster region (m-bcr) in

most cases of childhood Ph-positive ALL.19–23 Adult cases of Ph-positive ALL may contain breakpoints in either Mbcr or m-bcr. Fusion genes created by breaks in M-bcr (CML-type break) encode a 210-kilodalton (kDa) fusion protein (p210), whereas fusions that occur in m-bcr (ALLtype break) encode a 190-kDa protein (p190).24–26 Although the normal function of the ABL protein is not known, it possesses tyrosine kinase activity27 and is distributed in both the nucleus and the cytoplasm of proliferating cells. The ABL gene is activated by DNA

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Table 10.2 Major families of proteins/genes dysregulated by translocation in acute lymphoblastic leukemia Protein family

Translocation

Gene

Disease

t(8;14)(q24;q32) t(2;8)(q12;q24) t(8;22)(q24;q11) t(8;14)(q24;q11) t(1;7)(p32;q35) t(1;14)(p32;q11) t(7;9)(q34;q32) t(7;19)(q34;p13) t(14;21)(q11;q22)

MYC MYC MYC MYC TAL1 TAL1 TAL2 LYL1 BHLHB1

B-cell ALL B-cell ALL B-cell ALL T-cell ALL T-cell ALL T-cell ALL T-cell ALL T-cell ALL T-cell ALL

Homeodomain (Hox)

t(10;14)(q24;q11) t(7;10)(q35;q24) t(5;14)(q35;q32)

HOX11 HOX11 HOX11L2

T-cell ALL T-cell ALL T-cell ALL

Cysteine-rich (LIM)

t(11;14)(p15;q11) t(7;11)(q35;p13) t(11;14)(p13;q11)

LMO1 LMO2 LMO2

T-cell ALL T-cell ALL T-cell ALL

t(1;7)(p34;q34) t(5;14)(q31;q32) t(7;9)(q34;q34)

LCK IL3 TAN1

T-cell ALL Pre-B ALL T-cell ALL

Transcription factors Basic helix-loop-helix (bHLH)

Others Tyrosine kinase Growth factor Notch receptor

damage and appears to stimulate p53-dependent growth arrest.28–31 Such activation requires the function of ATM, a protein kinase that regulates the cell cycle checkpoint, DNA repair and apoptotic responses after exposure to genotoxic agents. ABL-deficient cells can activate cell cycle checkpoints and DNA repair, but show defects in apoptosis due to inefficient induction and activation of p73, whose product is a proapoptotic functional homologue of the p53 tumor suppressor protein.32 Mice deficient in ABL develop a wasting syndrome and die shortly after birth.33,34 In contrast to ABL, both the p190- and p210-kDa forms of BCR have a cytoplasmic location and increased tyrosine kinase activity,27,35 and their expression can transform hematopoietic cells in vitro and can induce a syndrome similar to CML in mice.35–41 Transformation by the BCR-ABL oncoprotein appears to involve the activation of multiple pathways, including those driven by RAS/MAPK, PI-3 and Jun kinases, or by c-CBL and CRKL, JAK-STAT, NF-B, Src, and cyclin D1. 42–49 Of these, the RAS, Jun-kinase, and PI-3 kinase pathways have been shown to play major roles in BCR-ABL induction of cell proliferation and transformation. The BCR-ABL fusion oncogene not only results in transformation by inducing an increase in cell proliferation, but also dysregulates other critical aspects of cell homeosta-

sis, including apoptosis, differentiation, and adhesion. A relevant effect of BCR-ABL is its ability to induce cellular resistance to cytostatic drugs and irradiation. Upon DNA damage, BCR-ABL extends the duration of the G2/M cell cycle checkpoint and enhances DNA repair. It also upregulates BCLXL, an antiapoptotic BCL-2 family member.50 Thus, a combination of mechanisms that include enhanced DNA repair, control of cell cycle checkpoints, and the suppression of apoptotic pathways antagonizes the cytotoxic effects of DNA damaging agents in BCR-ABL-positive cells. In childhood ALL, the BCR-ABL fusion gene is associated with an older age, a higher leukocyte count, and more frequent central nervous system (CNS) leukemia at diagnosis.51 Cases with BCR-ABL expression have an extremely poor prognosis despite treatment with contemporary therapy.12,51,52 However, it appears that a subset of these patients, mainly those with low leukocyte counts at diagnosis, can be cured with the use of intensive chemotherapy.53,54 This finding and the development of imatinib mesylate (STI-571/Gleevec), a novel molecularly tailored drug targeting the BCR-ABL tyrosine kinase, have challenged the idea that the only curative treatment for Ph-positive ALL is allogeneic stem cell transplantation in first remission. By inhibiting a restricted number of tyrosine kinases, including ABL,55,56 imatinib produces

Molecular genetics of acute lymphoblastic leukemia

antiproliferative and pro-apoptotic effects in cells harboring BCR-ABL and reverses drug resistance. Initial phase I clinical studies with this agent showed antileukemic activity in patients with chronic myeloid leukemia57 or BCRABL-positive ALL.58 Its activity in leukemias expressing BCR-ABL has been confirmed in phase II trials59–61 ; however, responses to imatinib as a single agent are limited by the rapid development of tumor resistance.62,63 Further studies are needed to optimize the use of imatinib in therapy for BCR-ABL-positive leukemias, using combinations of more conventional antileukemic drugs64,65 and other novel tyrosine kinase inhibitors.66,67

E2A-PBX1 The most common cytogenetically detected translocation in childhood ALL is the t(1;19)(q23;p13), which is present in about 6% of all B-lineage ALLs and in 25% of cases with a pre-B (cytoplasmic immunoglobulinpositive) immunophenotype.7,68–70 The t(1;19) generates the best- characterized chimeric transcription factor in ALL, whose activities illustrate the importance of homeodomain, or HOX, proteins in leukemogenesis.71 This mammalian family of transcription factors shares a common DNA-binding motif, the homeodomain, and show similarity to Drosophila homeotic complex (HOM) proteins in the sequence of their homeoboxes, chromosomal organization, and patterns of expression. In both mice and Drosophila, these proteins bind DNA via their homeodomains and play major roles in regional embryonic development, regulating anterior-posterior segmental identity. Recent studies, however, indicate that the specificity of HOX proteins is achieved not only through their homeodomains, but also through interactions with other cellular proteins. For example, the Drosophila extradenticle (exd) protein functions in segment identity by direct interactions with specific HOM proteins such as Ultrabithorax and Antennapedia.72–75 The t(1;19)(q23;p13) in pre-B ALL fuses the transactivation domains of the E2A gene, which encodes a bHLH transcription factor on chromosome 19, to PBX1 – an atypical homeobox gene that shares homology with exd .76–78 In Drosophila, exd plays a role in segmental development through its ability to heterodimerize with and alter the downstream regulatory programs of the products of the HOM-C cluster of major homeobox genes.11,72–74,79,80 E2A proteins, which play critical roles in B-cell lymphopoiesis,81 contain a bHLH domain that is responsible for sequence-specific DNA binding and protein dimerization82–85 and two transcriptional transactivation domains in the amino terminal portion of the

molecule.84,86–89 E2A-deficient mice show a severe impairment in the production of mature B cells due to the block of B-cell development at the pro-B cell stage.81,90,91 Since the t(1;19) eliminates one copy of the E2A locus, it has been proposed that this translocation may contribute to leukemic transformation both by inducing haploinsufficiency of E2A and by generating the E2A-PBX fusion oncogene.92 E2A-PBX1 is constitutively expressed in the nucleus, binds DNA in a site-specific manner, and is a strong transcriptional transactivator and oncoprotein: (i) transforming NIH-3T3 fibroblasts in culture; (ii) inducing T-cell lymphomas in transgenic mice; and (iii) producing acute myeloid leukemia (AML) when introduced into murine bone marrow progenitors by retroviral infection.87,88,93–96 The transgenic mouse model also implicates E2A-PBX1 in the induction of apoptosis in lymphoid cells.87 An unexpected result is the demonstration that the DNA-binding domain of PBX1 is not required for transformation.95 Even so, analogous to exd and the HOM-C proteins of Drosophila, the binding of both PBX1 and E2A-PBX1 to consensus DNA targets is stimulated by direct interactions of PBX1 with a subset of HOX proteins.97,98 In addition, this cooperative DNA binding requires the conserved motifs flanking both the PBX1 and HOX homeodomains.97 This motif in PBX1 is not only required for cooperative DNA binding and transactivation, but is also both a necessary and sufficient portion of PBX1 for transformation induced by E2A-PBX1.99 In addition to increasing DNAbinding affinity, dimerization of PBX1 with HOX proteins also appears to alter binding specificity by inducing conformational changes in the HOX homeodomain.100 Further insight into the function of PBX1 has been provided by the analysis of endogenous PBX1 complexes, which has demonstrated that the Meis homeodomain proteins are the preferred partners of PBX1, but do not interact with E2APBX1.101 Because HOX proteins appear to direct E2A-PBX1 to DNA sites recognized by HOX-PBX1 complexes, it is likely that E2A-PBX1 interferes with hematopoietic differentiation by disrupting patterns of gene expression that are normally regulated by HOX proteins (Fig. 10.2).100 In this regard, E2A-PBX1 can induce the aberrant expression of developmentally regulated genes when it is expressed in fibroblasts.102 Surprisingly, B-cell precursors, the target of E2A-PBX1 in human leukemias, cannot be transformed in culture by this oncoprotein.103 Instead, the inducible expression of E2A-PBX1 in B-cell progenitors induces p53independent apoptosis, suggesting that its leukemogenic potential in vivo may depend on cell type-specific resistance to apoptosis.103 In addition, when expressed in

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HOX

Repressed gene A

PBX1 Activated gene B

5′ TGATTGAT 3′

Activated gene C Repressed gene D

E2A HOX

Activated gene A

PBX1 Activated gene B

5′ TGATTGAT 3′

Activated gene C Activated gene D

Fig. 10.2 A model depicting gene expression changes associated with the E2A-PBX1 fusion protein. PBX1, located on chromosome 1, encodes a transcriptional regulator that associates with HOX proteins and controls the expression of multiple genes involved in the development of different hemopoietic lineages. The t(1;19) creates an E2A-PBX1 fusion protein, adding two E2A transactivation domains to the homeodomain of PBX1. This chimeric protein induces high levels of expression of genes normally regulated by HOX-PBX1 complexes.

lymphoid cells, E2A-PBX1 induces the expression of a WNT ligand for frizzled receptors.104 Molecular characterization of the t(1;19) has led to the development of RT-PCR assays that detect the E2A-PBX1 chimeric transcript.68,105 These assays can detect E2APBX1 fusions in patients for whom cytogenetic studies were normal or unsuccessful, and can identify patients who have the t(1;19) but lack the fusion gene. Patients with E2A-PBX1 fusions fare poorly on antimetabolite-based therapies, but do quite well when treated more aggressively,70,106,107 underscoring the importance of the detection of E2A-PBX1 at diagnosis, and suggesting that the adverse prognostic impact of this fusion protein can be overcome with effective intensive chemotherapy.

E2A-HLF In rare cases of ALL, the t(17;19)(q22;p13) fuses the amino-terminal transactivation domains of E2A to the C-terminal DNA-binding and dimerization domains of the basic leucine zipper transcription factor HLF.108,109 HLF is normally expressed in liver but not in lymphoid cells and bears homology to DBP, an albumin gene promoter D-box-binding protein, and TEF, which transactivates thyroid-stimulating hormone gene expression.110–114

E2A-HLF binds to specific DNA sequences and transforms NIH-3T3 fibroblasts in culture.71,115–117 Although E2A-HLF can bind DNA either as a homodimer or as a heterodimer with HLF and related proteins, a mutant E2A-HLF capable only of homodimerization transforms cells as well as wild-type E2A-HLF.118 Leukemic transformation requires both the transactivation domain of E2A and the leucine zipper dimerization domain of HLF, suggesting that the chimeric protein, acting as a homodimer, plays a role in leukemogenesis by aberrantly regulating target genes that control the fate of early lymphoid progenitors.117 A major consequence of the activation of this aberrant gene expression program is a dysregulation of the mechanisms that control cell death in lymphoid progenitors. Inaba et al.119 demonstrated that expression of a dominant-negative suppressor of the E2A-HLF protein caused death by apoptosis in t(17;19)-carrying cell lines. In addition, expression of E2A-HLF in pro-B lymphocytes reversed interleukin-3dependence and p53-mediated apoptosis. HLF is the mammalian homologue of ces-2, a gene involved in the control of cell death during the development of the small nematode, C. elegans.120 Activation of ces-2 is required for the death of the sister cell of a specific pair of serotoninergic NSM neurons in the worm. Ces-2 blocks the expression of ces-1, a prosurvival gene

Molecular genetics of acute lymphoblastic leukemia

that normally prevents apoptosis by inhibiting the activity of the proapoptotic gene egl1.121 This pathway is conserved through evolution and perturbed by the fusion of the HLF DNA-binding and E2A transactivation domains. Thus, while CES-2 induces apoptosis by repressing ces-1 in the worm, the human E2A-HLF oncoprotein activates the expression of SLUG, a ces-1 homologue, normally responsible for the protection of hematopoietic progenitors from DNA damage-induced apoptosis.121–123 E2A-HLF-positive leukemias are more frequent in adolescents and often present as a medical emergency due to disseminated intravascular coagulation and hypercalcemia. These symptoms appear to result from products released by the leukemic cells, because they resolve when patients enter remission. Although complete remissions can be attained, of seven reported patients whose blasts expressed E2A-HLF, each has died of leukemia despite aggressive therapy, suggesting that resistance to chemotherapy in these cases may be mediated by E2A-HLF inhibition of apoptosis.116,124–126

MLL fusion genes Structural lesions involving chromosome 11 band q23 are among the most common cytogenetic abnormalities seen in hematopoietic malignancies, occurring in approximately 80% of infant ALL cases, 5% of AML cases, and 85% of secondary leukemias that occur in patients treated with topoisomerase II inhibitors.127–135 The majority of 11q23 translocations disrupt the MLL gene, which encodes a 431 kDa protein of 3968 amino acids.136–139 Molecular analysis is necessary because not all 11q23 abnormalities involve MLL, and in cases lacking MLL rearrangement, the clinical outcome is similar to that for ALL in general.140 By contrast, B-progenitor ALLs with MLL rearrangement have high-risk disease with long-term EFS rates of less than 20%.133,140–145 The MLL protein contains three regions of homology with the Drosophila trithorax protein: two central zinc-finger domains and a 210-amino-acid C-terminal segment.137,138,146 The N-terminal region of MLL contains three A-T hook motifs, originally described in the highmobility-group (HMG) proteins, a family of basic nuclear proteins believed to play a role in establishing chromatin structure. In the HMG-I(Y) proteins, A-T hooks appear to bind A-T-rich sequences specifically in the minor groove of DNA.147 In MLL, the A-T hook motifs and the zincfinger domains are separated by a 47-amino-acid region with homology to the noncatalytic domains of human DNA-methyltransferase, an enzyme that produces fully methylated double-stranded DNA from a hemimethylated substrate.148 MLL stability and subnuclear localization

are dependent on proteolytic postranslational processing, which cleaves the protein into N-terminal and C-terminal fragments that remain associated through intramolecular interaction domains.149,150 MLL structural features suggest that its normal physiologic function, as well as its role in leukemogenesis, may be mediated by direct physical interactions with DNA.139 Clues to the function of MLL are suggested by its homology to trithorax, a transcription factor that plays a pivotal role in segmental determination during Drosophila development by maintaining the expression levels of members of the Antennapedia and Bithorax homeotic gene complexes.146 Trithorax maintains the transcription of genes in these complexes in an activated state by direct interactions of its zinc-finger domains with cis regulatory sequences, and opposes the effects of proteins of the Polycomb group. Another Drosophila protein, enhancer of zeste, is also involved in the regulation of the Antennapedia and Bithorax gene complexes, possibly through direct protein–protein interactions with trithorax.151 Interestingly, the enhancer-of-zeste, trithorax, and MLL proteins each contain a conserved motif called the SET domain near their C-terminal ends.152 The MLL SET domain contains a histone H3/lysine 4-specific methyltransferase activity and plays a critical role in the regulation of HOX gene expression by MLL.153,154 To investigate a possible role for the loss of MLL function in leukemic transformation, Yu et al. 155 disrupted the murine Mll gene by genetic knockout techniques. Mice heterozygous for this gene were small at birth, had retarded growth, and displayed anemia and thrombocytopenia. Yolk sac progenitor cells from homozygous Mll-knockout mice differentiated into colonies that were smaller in size and fewer in number than those derived from wild-type embryos, indicating that Mll is required for normal murine hematopoiesis.156 Mice lacking Mll also demonstrated segmental abnormalities, including disordered identity of structures in the cervical, thoracic, and lumbar regions. These deformities resulted from shifts in the boundaries of Hox gene expression, indicating a crucial role for Mll in Hox gene regulation. Furthermore, Mll-negative mice died in utero and failed to express specific Hox genes. A direct link between MLL activity and the expression of Hox genes has been established by showing that Mll binds directly to cis-regulatory elements present in the promoters of target Hox genes. The interaction of Mll with HOX gene promoters in this study led to the methylation of lysine 4 in histone H3, a chromatin modification mediated by the Mll SET domain that results in transcriptional activation.153

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The pathogenic importance of MLL is underscored by its ability to associate with many different leukemogenic fusion partners.157 Over 40 reciprocal chromosomal loci participate in 11q23 translocations, including 1p32, 1q21, 2p21, 4q21, 5q31, 6q27, 7p15, 9p22, 10p12, 15q15, 16p13, 17q21, 19p13, and Xq13. The most common translocations are t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13), which fuse MLL to a family of genes (AF-4, AF-9, and ENL) that may contribute similar functional domains to the chimeric proteins. AF-4 (also called FEL) is localized to chromosome 4 band q21 and encodes a 140-kDa serine/proline-rich protein that contains a nuclear targeting sequence and is widely expressed in normal tissues.158 AF-9 (band 9p22) and ENL (band 19p13) also encode proteins that contain nuclear targeting signals and are rich in serine and proline, suggesting that all three proteins may function as transcriptional transactivators. In addition, AF-9 and ENL are 82% identical in their N-terminal 140 amino acids and 82% identical in their C-terminal 67 amino acids. Furthermore, ENL is a nuclear protein capable of transactivating reporter genes, with its transcriptional activation domain localized to its C-terminal region of homology with AF-9.159 Formal proof that MLL fusions are important in the development of leukemia was provided by Corral et al.,160 who used homologous recombination to create chimeric mice that expressed Mll-AF9. AF9 sequences were fused in-frame with the endogenous mouse Mll gene in embryonic stem cells, which were then introduced into blastocysts to create chimeric mice. Compared to transgenic models, this strategy has the advantage of using normal Mll transcriptional control elements to express the fusion gene. After 4 to 12 months, chimeric mice expressing Mll-AF9 developed AML at a high frequency, with morphological features similar to those of human leukemias carrying the t(9;11). Retroviral transduction of the MLL-ENL oncogene, created by the t(11;19), into hematopoietic cells immortalized myelomonocytic precursors, which could then induce myeloid leukemia upon transplantation into recipient mice.161 Similar results were recently obtained with a model in which Mll chromosomal translocations were induced by directed interchromosomal recombination in mice, a strategy that recapitulates early events in the pathogenesis of human MLL-ENL-rearranged leukemias.162 See Chapter 11 for additional information about MLL in childhood leukemias. Recent studies have demonstrated that functional domains present in MLL fusion partners are required for the transforming effects of many MLL fusion oncoproteins: MLL-ENL161 , MLL-ELL,163 MLL-CBP,164 and MLL-AF10.165

Considered together, these data suggest that the oncogenic effects of MLL fusion oncogenes depend on the transcriptional effector properties conferred by MLL partners.139 Recently, a second mechanism has been shown in which the partner protein contributes a dimerization motif, resulting in homo-oligomerization of the N-terminal portion of MLL.166–169 Molecular techniques to detect MLL rearrangements include Southern blot analysis, fluorescence in situ hybridization (FISH), and RT-PCR. Although less sensitive than RT-PCR, Southern blots or FISH can detect essentially all MLL rearrangements, regardless of the partner gene involved.141,170 Alternatively, because several of the most common partner genes of MLL have been cloned and sequenced, RT-PCR assays have been developed that detect most of the (der)11-derived chimeric messages including those encoded by the t(4;11), t(6;11), t(9;11), and t(11;19).171–175 These assays have been further simplified by the development of multiplex RT-PCR assays, which can detect these translocations in a single reaction.173 Gene expression profiling using oligonucleotide microarrays facilitates analysis of the relative expression of thousands of genes and has evolved in recent years into a powerful tool for the molecular characterization of human leukemias. Analysis of the gene expression signatures of MLL-rearranged B-precursor ALLs has shown that these leukemias have a characteristic gene expression profile that includes the specific upregulation of major HOX genes and the expression of numerous myeloid genes.176 These findings support the hypothesis that such cases represent a distinct leukemia entity, separate from recognized subtypes of ALL and AML. Indeed, analysis of a large group of patients studied independently at St. Jude Children’s Research Hospital confirmed that MLLrearranged leukemias are a genetically homogeneous group that differs from other ALLs.177 More recently, a comparison of the gene expression patterns characteristic of early B-and T-cell ALLs expressing MLL fusion genes showed that MLL-rearranged T-cell leukemias are arrested at early stages of thymocyte development and are characterized by upregulation of specific HOX genes, including HOXA9, HOXA10, HOXC6, and the HOX gene regulator MEIS1.178 The importance of HOXA9 in MLL fusion protein-mediated leukemogenesis is further indicated by the fact that bone marrow cells from Hoxa9−/− mice are refractory to transformation by MLL fusion proteins.179 In microarray studies, expression of the FLT3 tyrosine kinase, encoded by a proto-oncogene frequently activated by mutations in AML, was a characteristic finding in MLL-rearranged B-lineage cases and correlated with the identification of a high incidence of FLT3-activating

Molecular genetics of acute lymphoblastic leukemia

mutations.180 Thus, in both animal models and cell culture, FLT3 kinase inhibitors have shown potential as therapeutic agents for MLL-rearranged leukemias.180 Further development of this molecularly targeted therapy may yield significant improvement in outcome for this high-risk group of patients.

TEL-AML 1 One of the most exciting recent developments in ALL research is the identification of TEL-AML1 as the most common genetic alteration in this disease. The TELAML1 fusion is created by the t(12;21), a translocation that is very difficult to detect by standard karyotyping.181 Molecular techniques, however, have demonstrated this fusion gene in approximately one-quarter of childhood ALL cases.70,130,145,182–190 The resulting chimeric protein comprises the helix-loop-helix (HLH) domain of TEL, a member of the ETS-like family of transcription factors, fused to the DNA-binding and transactivation domains of AML-1. Interestingly, the latter domain shares homology with the runt pair-rule protein of Drosophila. Both TEL and AML1 are involved in a variety of other leukemiaassociated translocations. TEL was originally cloned as a fusion partner of the platelet-derived growth factor receptor  gene (PDGFR), resulting from the t(5;12) in chronic myelomonocytic leukemia191 and is involved as a fusion partner with proto-oncogenes, such as MN1, ABL, and EVI1 in AML, and with JAK2 in T-ALL.192 AML1 is the DNAbinding component of the AML1/CBF transcription factor complex, the most frequent target of myeloid-associated translocations, including the t(8;21), t(3;21), and inv(16).193 The prominent role of AML1 (also known as RUNX1) in the pathogenesis of human leukemias has been reinforced by the identification of inherited or acquired inactivating mutations in AML1 in acute myeloid leukemias194,195 ; and the presence of gene amplifications of this locus in childhood ALL.195 It has been proposed that TEL-AML1 may transform cells by interfering with AML1-mediated expression of HOX genes in lymphopoiesis.11,192 In this regard, the fusion of TEL to AML1 converts AML1 from an activator to a repressor of transcription (Fig. 10.3), a feature that is dependent on the helix-loop-helix dimerization motif of TEL,196 which has been identified as a 65-amino-acid region of TEL that is conserved in a subset of ETS proteins and is essential for constitutive activation of the protein kinase activity and mitogenic properties of the TEL-PDGFR fusion protein.197 In contrast with the normal AML1 protein, TEL-AML1 interacts with N-CoR, a component of a nuclear coreceptor complex with histone deacetylase

activity. This interaction results in the remodeling of chromatin to a deacetylated/closed/inactive configuration in those promoters normally activated by AML1.198 Although the targets of TEL are unknown, the role of this protein in normal development has been examined by the targeted disruption of Tel in mouse embryos.199 Teldeficient mice die at approximately day 11 of embryogenesis because of defective yolk sac angiogenesis and apoptosis of neural and mesenchymal cells, thus establishing Tel as an important regulator of embryologic development.199 Recently, studies in mice with Cre-lox-induced inactivation of conditioned Tel alleles have shown that Tel is required for definitive hematopoiesis. In addition, the observation that loss of the normal TEL allele frequently accompanies TEL-AML1 fusion in ALL cases suggests that the leukemogenic effects of TEL-AML1 could be mediated, at least in part, through loss of function of the normal TEL protein.184,197,200,201 The absence of definitive hematopoiesis in mice lacking either AML1 or CBF further supports an essential role for the AML1-CBF complex in normal hematopoiesis.193,202–205 These results also suggest that a lack of expression of genes normally activated by AML1 may be important in leukemogenesis. TEL-AML1 expression is associated with an excellent prognosis, with event-free survival rates approaching 90%.182,184,188,189 In addition, the favorable prognostic impact of TEL-AML1 is independent of age and leukocyte count and was consistently favorable among patients treated on several different protocols.188,189 Thus, TELAML1 expression identifies a large, previously unrecognized, subset of B-precursor ALL patients who may be candidates for less intensive therapy.

Oncogenes created by aberrant regulation T-cell and B-cell acute leukemias are characterized by translocations involving the TCR and IG genes, respectively. The juxtaposition of strong enhancer elements from these genes with various transcription factors leads to protein overexpression and ultimately to leukemic transformation.

Activation of MYC in B-cell ALL In B-cell ALL and Burkitt lymphoma, translocations often involve the MYC gene, which encodes a basic helixloop-helix/leucine zipper (bHLHZip) protein.206–214 The majority of cases contain the t(8;14)(q24;q32), in which MYC is translocated to the IG heavy-chain locus.206–208 In the remaining cases, either the kappa (2p12) or lambda (22q11) light-chain gene is translocated to chromosome

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AML1 Activated gene A Activated gene B

TEL Repressed gene C

Normal cell differentiation

Repressed gene D

TEL

t(12;21)

AML1 Repressed gene A Repressed gene B

Additional 12p deletion

TEL

Differentiation block

Activated gene C Activated gene D

Fig. 10.3 A model depicting gene expression changes associated with the TEL-AML1 fusion protein. According to this model, normal differentiation of B-cell progenitors is regulated through the transcriptional activator function of AML1 and the transcriptional repressor activity of TEL. In t(8;21) leukemias, the TEL-AML1 fusion protein results in repression of genes normally activated by AML1. In addition, loss of the other TEL allele contributes to the pathogenesis as a second leukemogenic event resulting in the expression of genes normally repressed by TEL.

8 adjacent to MYC as a result of the t(2;8)(p12;q24) or t(8;22)(q24;q11).209–214 All three translocations result in the dysregulated expression of MYC, which is normally silent in quiescent cells and is rapidly upregulated when cells are stimulated by mitogens to enter the cell cycle. As a prototypic bHLHZip transcription factor, MYC binds to a canonical hexameric E-box DNA sequence (5 -CACGTG3 ) through its C-terminal domain. Its N-terminal transcriptional activator domain interacts with components of the RNA polymerase transcriptional complex,215 and the bHLHZip region serves as a dimerization domain.216–218 The MYC protein transactivates target genes through complex interactions with multiple other proteins, including its DNA-binding partner MAX. MYC:MAX heterodimers bind to DNA and activate transcription.219 MAX, however, also heterodimerizes with members of an extended family of other bHLHZip proteins, including MAD,220 MXI-1 (MAD2),221 and MNT.222 MYC:MAX complexes activate transcription and promote proliferation, whereas MAD:MAX and other MAX heterodimers antagonize these effects. MAD inhibits MYC function both by competing with MYC for MAX, and by directly inhibiting transcription. MAD, as well as MXI1, represses transcription by interacting with a complex containing at least three proteins: SIN3, the nuclear corepressor N-CoR, and

histone deacetylase.223–228 Interestingly, this complex is required for both MAD- and nuclear receptor-dependent repression, and appears to play a crucial role in the pathogenesis of acute promyelocytic leukemia (discussed in Chapter 11).229–232 As a result of the t(8;14), the dysregulated expression of MYC increases the concentration of MYC:MAX complexes relative to MAX:MAD, leading to transcriptional activation. Genes activated by MYC include proteins that regulate cell growth, division, death, metabolism, adhesion, and motility, some of which may play important roles in cellular transformation.233 Most of these genes are activated by MYC; however, a subset of MYC targets are actually repressed by mechanisms that remain poorly understood. The effect of MYC on the cell cycle is mediated in part by the negative regulation of the cell cycle inhibitors p27 and p21 and in part by the transcriptional activation of CDC25A, a protein phosphatase that activates the cyclin-associated kinases CDK2 and CDK4.234–236 Other target genes possibly involved in the pathogenesis of MYC-induced leukemias are the ARF tumor suppressor,237 the catalytic subunit of human telomerase238,239 and genes involved in diverse metabolic pathways including both nucleotide240–246 and protein biosynthesis pathways.247–249

Molecular genetics of acute lymphoblastic leukemia

Dysregulated expression of transcription factor genes in T-ALL Recurring translocations in T-cell ALL (T-ALL) often involve the transcriptionally active sites of the TCRB locus (7q34) or the TCRA/D locus (14q11), leading to dysregulated expression of transcription factor genes. Like the t(8;14) in B-cell ALL, these rearrangements may result from mistakes in the normal recombination process involved in the generation of functional antigen receptors.250 Transcription factor genes altered in T-ALL include members of the bHLH family (MYC, TAL1, TAL2, LYL1 and BHLHB1),250–256 genes encoding the LIM-only domain proteins (LMO1 and LMO2),257–259 and homeodomain genes (HOX11 and HOX11L2)259–262 (see Table 10.1). The t(1;14)(p32;q11) results in abnormal expression of TAL1 following its translocation into the TCRA/D locus.263–269 Although the t(1;14) is seen in less than 5% of T-ALL patients, approximately 25% of this subgroup carry other rearrangements of TAL1 that are not karyotypically apparent, and lymphoblasts from an even greater percentage of patients aberrantly express TAL1.269 TAL1 and LYL1, which is overexpressed in T-cell leukemias carrying the t(7;19), are members of the class II family of bHLH proteins, which are expressed in a tissue-specific manner and heterodimerize with class I bHLH proteins, such as E2A.270,271 TAL1:E2A complexes have been detected in erythroid cells and T-cell leukemias and bind DNA in a site-specific manner. A role for these complexes in erythroid differentiation is supported by the absence of embryonic erythrocyte formation in mice lacking Tal-1, as well as the increased differentiation of erythroid precursors when Tal-1 is overexpressed.272,273 Additional studies have shown that Tal-1 is required not only for erythropoiesis, but also for the earliest steps in the lineage commitment of pluripotent embryonic hematopoietic stem cells.274,275 TAL1:E2A heterodimers are transcriptionally inactive276,277 and have been proposed to mediate the leukemogenic effect of Tal1.278 That the loss of E2a function induces T-cell leukemia in mice279,280 and that the DNA-binding domain of TAL1 is dispensable for transformation support this hypothesis.281 Other transcription factor genes in T-ALL include LMO1 and LMO2, which encode cysteine-rich proteins that are not normally expressed in lymphoid cells.257–259,282 Cysteine-rich LIM domains function in protein–protein interactions, and their overexpression in T-cells may lead to transformation through interactions with other transcription factors. This model is supported by the ability of LMO2 to bind to TAL1 in experimental systems.283–285 Moreover, expression of LMO1 and LMO2, driven by Lck and other

exogenous promoters, induces lymphomas in transgenic mice286–289 and accelerates the onset of leukemia in TAL1 transgenic animals.290 A third group of transcription factor genes involved in the pathogenesis of T-ALL are the orphan homeobox genes HOX11 and HOX11L2. These homeodomain transcription factors are related to the developmentally important Tlx genes of the mouse and Drosophila.291–293 HOX11 overexpression was initially identified in T-ALL cases with the t(10;14) and t(7;10) translocations. HOX11 is normally expressed during embryogenesis in specific regions of the branchial arches and ectoderm of the pharyngeal pouches of the developing hindbrain in the mouse, but not by developing thymocytes.294 In contrast to LMO1 and TAL1, the development of the hematopoietic system is not affected by the loss of HOX11. However, its homeotic role during development is demonstrated by the lack of spleen formation following homozygous disruption of this gene in mice.294,295 HOX11L2, a homeobox gene closely related to HOX11 is activated by a t(5;14) translocation, a cryptic chromosomal rearrangement detectable only by fluorescence in situ hybridization and chromosome painting techniques.262 Hox11l2 is essential for the normal development of the murine ventral medullary respiratory center, and mice deficient in this protein die early after birth due to a respiratory failure resembling congenital central hypoventilation syndrome in humans.296 Recently, the analysis of gene expression using quantitative RT-PCR and oligonucleotide microarrays has demonstrated that the expression of different T-ALL oncogenic transcription factor genes such as TAL1, LYL1, HOX11 and HOX11L2 is associated with distinct gene expression signatures that are related to the blockage of T-cell differentiation at different stages of thymocyte development.297 The identification of HOX11-positive cases as a favorable prognostic group in T-ALL reinforces the hypothesis that T-ALL transcription factor oncogenes define molecularly and clinically distinct subtypes of leukemia.297

Other proto-oncogenes activated in Band T-cell ALL Transcription factor genes are not the only targets mobilized by translocation to the sites of the immunoglobulin or T-cell receptor genes. A rare but distinct subtype of ALL is characterized by an early B-cell immunophenotype, hypereosinophilia, and the t(5;14) translocation. The latter fuses the IG heavy-chain gene to the promoter of the IL-3 gene, causing overexpression of IL-3.298,299

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This effect is believed to provide a growth stimulatory signal to the leukemic cells through an autocrine loop. In T-ALL, the TAN1 gene, which shares homology with the Drosophila notch gene, shows dysregulated expression and amino-terminal truncation following its relocation to the TCRB locus as a result of the t(7;9), resulting in the constitutive expression of the activated Notch1 intracellular domain. Importantly, activating mutations in NOTCH1 have recently been found in over 50% of T-ALL cases, highlighting the importance of NOTCH1 as a major oncogenic factor in the transformation of T-cell progenitors.300,301 Similarly, relocation to the TCRB locus activates expression of the LCK tyrosine kinase genes in T-ALL cases with the t(1;7).302

Tumor suppressor genes and the cell cycle Tumor suppressor genes – recessive oncogenes whose products normally provide negative control of cell proliferation – may be inactivated by deletion or mutation of both chromosomal alleles, leading to malignant transformation. This model was first proposed by Knudson,303 who suggested that two “hits”, or mutations, now known to cause inactivation of both alleles of a single gene, are needed for the development of retinoblastoma; his prediction was later supported by functional studies of the retinoblastoma protein, pRB.304 More recently identified tumor suppressor genes, whose loss of function also contributes to transformation, include p53,305–309 WT1 (the Wilms tumor gene),310,311 and p16.312,313 The presence of recurring deletions in pediatric ALL suggests that additional tumor suppressor genes will soon be identified. For example, deletions of the long arm of chromosome 6 or the short arm of chromosome 12 each occur in about 10% of childhood ALL cases, indicating the loss of important genes in those regions.130 Several of the known tumor suppressors act at specific points in the cell cycle (Fig. 10.4). For example, the p53 transcription factor gene, located on chromosome 17, encodes a ubiquitously expressed 53-kDa nuclear protein that functions as a cell cycle checkpoint.305–309 Expression of p53 is increased by DNA damage, blocks cell division at G1 in the cell cycle to allow DNA repair, and is also capable of stimulating apoptosis of cells with damaged DNA.314–320 Targeted disruption of p53 in the mouse leads to the development of a variety of tumors.321 Germline mutation of one p53 allele occurs in patients with Li-Fraumeni syndrome, who generally inherit a mutated p53 gene from an affected parent.322–325 Patients with Li-Fraumeni syndrome are predisposed to develop sarcomas, breast cancer, brain tumors,

adrenocortical cell carcinoma and acute leukemia, and have a 50% probability of developing cancer by age 50.326 Although p53 is mutated or deleted in a variety of human malignancies, it does not appear to play a significant role in most cases of pediatric leukemia.327 Mutations in p53 are common in B-cell ALL and Burkitt lymphoma, but are seen in only 1% to 2% of B-precursor and T-cell ALLs at diagnosis.328–332 However, such mutations may be important in the development of drug-resistant disease: p53 inactivation has been detected in about 25% of relapsed T-ALL cases,328,329,333 each of four cases of relapsed t(1;19)containing pre-B ALL cases,331 and 3 of 10 cases of ALL that were resistant to induction therapy or relapsed very early in the clinical course.334,335 In addition, suppression of p53 activity by overexpression of the MDM2 protein has also been associated with early treatment failure.335 Another important class of tumor suppressor genes is the INK4 family of cyclin-dependent kinase inhibitors (CDKIs).336 These proteins are negative regulators of the cell cycle (see Fig. 10.4). The INK4A locus is located on the short arm of chromosome 9 and contains two genes, p16INK4A and p14ARF (p19ARF in mice).337,338 Each of these two genes has a distinct first exon while sharing the second and third exons. However, p16INK4A and p14ARF are transcribed from different promoters and their common exons are translated in different reading frames, resulting in two proteins with totally different amino acid sequences.339 A third cell cycle regulator, the p15INK4B cyclin-dependent kinase inhibitor is encoded by a gene located in the vicinity of the INK4A locus on 9p21.312,313,340 All three of these proteins (p16INK4A , p15INK4B , and p14ARF ) act as tumor suppressors and contribute to cell cycle control. p16INK4A and p15INK4B are direct inhibitors of cyclin D:CDK4/6 complexes, which play a critical role in the control of the cell cycle by phosphorylating the retinoblastoma protein. pRB phosphorylation causes the release of transcription factors, such as E2F, that are necessary for entry into S-phase. Thus, p16 and p15 are predicted to limit the growth of normal cells by inhibiting cyclin D-CDK activity and decreasing the proportion of cells entering S-phase. By contrast, p14ARF expression inhibits MDM2, resulting in the stabilization of p53 activity.312,313,341–343 Thus, the tumor suppressor effects of p14ARF do not result from a direct inhibition of the cell cycle machinery, but from the ability to activate p53 by binding to and promoting the degradation of MDM2 in response to different stimuli.341,342 Direct evidence linking the INK4A locus to tumorigenesis using mouse models was provided by the targeted disruption of exon 2 common to both p16INK4A and p19ARF (the murine homologue of human p14ARF ).344 These mice, deficient for p16INK4A

Molecular genetics of acute lymphoblastic leukemia

Mitogenic signals Ras mutations, BCR-ABL

p16INK4A

9p deletion

RAS Cyclin D CDK RB mutations

RB-E2F

+

E2F

†(8;14)/Myc overexpression

MYC

ARF MDM2 p53 mutations

p53 Cell cycle arrest

S-phase entry BCL2 overexpression

Apoptosis

Fig. 10.4 A schematic representation of the integration of cell cycle control and apoptosis regulation by tumor suppressor genes. Mitogenic signals are integrated through cell signaling pathways, such as the RAS pathway, resulting in the activation of cyclin D/CDK complexes and the phosphorylation of the Rb tumor suppressor. Inactivation of Rb by cyclin/CDK phosphorylation releases E2F transcription factors that together with MYC activate the expression of multiple genes responsible for S-phase entry and cell cycle progression. Two critical tumor suppressor genes, p16INK4A and p14ARF , are encoded from a single locus in the short arm of chromosome 9. p16INK4A inhibits CDK4/6 and restores the ability of Rb to sequester and inhibit E2F transcription factors. In addition, p14ARF stabilizes the p53 tumor suppressor protein by inhibiting MDM2; thus, the proteins encoded by this locus control both cell cycle progression and the induction of programmed cell death. Multiple oncogenic events interfere with these pathways during leukemogenesis, resulting in loss of cell growth control and aberrant cell survival.

and p19ARF , developed tumors (primarily lymphomas and fibrosarcomas) that were enhanced by the topical application of carcinogens and ultraviolet light.344 Further genetic analysis showed that selective disruption of p19ARF reproduced the phenotype described for p16/p19-null mice,337 and also that mice harboring a deletion of both copies of p16INK4A with intact p19ARF had increased susceptibility to cancer,345,346 indicating that both p16INK4A and p19ARF are bona fide tumor suppressor genes. The short arm of chromosome 9 is frequently altered in human cancer. Deletions of the 9p21 region, involving both the p16INK4A /p14ARF locus and the p15INK4B gene, frequently result in the inactivation of these three tumor suppressor genes in hematologic tumors. Alternatively, p16INK4A and p15INK4B can be epigenetically inactivated due to hypermethylation of their promoter sequences. In primary ALL samples, homozygous deletions of p16 and p15 have been detected in 20% to 30% of cases of B-precursor ALL and 70% to 80% of T-cell ALL cases, while hypermethylation

of the p15INK4B promoter is present in 44% of B-lineage ALLs.347–359

Apoptotic mechanisms In follicular lymphomas and diffuse B-cell lymphomas harboring the (14;18)(q32;q21), the BCL2 gene undergoes dysregulated expression as a result of juxtaposition with the IG heavy-chain gene.360–364 BCL2 was the first proto-oncogene found to inhibit programmed cell death (apoptosis) rather than to promote proliferation. Its overexpression in lymphoid cells in vitro and in transgenic mice inhibits apoptosis induced by a variety of stimuli, including antineoplastic drugs.365–368 It also inhibits MYC- and p53-dependent cell death pathways. A BCL2 homologue, BAX, was later identified as an interaction partner of BCL2 that promotes apoptosis.369 Subsequently, a large family of related proteins has been described as either inhibiting (BCL2, BCLXL,

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MCL1, A1) or promoting (BAX, BAD, BCLXS) cell death upstream of the apoptotic effector machinery (see Chapter 12 for details).365,367,370 The role of genes that regulate apoptosis have been investigated in cases of childhood ALL.371 In patients with newly diagnosed ALL, high levels of BCL2 expression were associated with the improved ability of lymphoblasts to survive in serum-free medium, but were not associated with resistance to chemotherapy or a poorer outcome.371 In cell lines established from ALL patients, the expression of BCL2 and BAX, but not BCLXL, was dependent on p53 activation and correlated with radiation-induced apoptosis.372 Mutations of BAX were found in four of eight T-ALL cell lines, suggesting that resistance to apoptosis may play a role in this malignancy.373 However, these results were limited to established cell lines and therefore need to be confirmed in diagnostic lymphoblast samples. Nevertheless, it appears likely that multiple members of the BCL2 family play a role in leukemogenesis or in the development of resistance to chemotherapy.

Clinical implications Although early studies suggested that the presence of any translocation was an adverse prognostic factor in ALL, most of this research was conducted before the use of intensive multiagent chemotherapy.374 It has since been learned that the mere presence or absence of a translocation has little prognostic impact.375,376 However, specific chromosomal and molecular genetic abnormalities in the leukemic blasts of children with ALL do have clinical importance; indeed, they are among the best predictors of response to currently available chemotherapy. Risk-based protocols, in which the intensity of treatment is tailored to the risk of relapse, rely on these prognostic factors for accurate risk assignment. Genetic risk evaluation is based on the modal chromosome number (ploidy) of leukemic blasts as measured by flow cytometry, the presence of trisomies 4 and 10, and the identification of specific chromosomal translocations.7,377–381 Initially performed by time-consuming and laborintensive cytogenetic methods, the classification of chromosomal abnormalities in leukemic lymphoblasts has been aided by flow cytometric techniques (to detect hyperdiploidy) and by clinically applicable molecular techniques, such as quantitative RT-PCR and fluorescence in situ hybridization (FISH).8 In recent years, the molecular characterization of human leukemias has been enhanced by the complete sequencing of the human genome and the development of DNA microarrays for the analysis

of gene expression on a genomic scale. Microarray analysis of pediatric ALL has demonstrated that groups of leukemias with distinct genetic abnormalities have specific gene expression signatures, and gene expression profiling has been used to identify clinically relevant prognostic predictors.177,297 Molecular analysis is more sensitive and more specific than cytogenetics, identifying gene rearrangements that are missed by standard cytogenetic techniques, as well as the absence of important gene rearrangements in patients with cytogenetically identical translocations. Currently, each new case of childhood ALL admitted to many centers is studied for the ploidy of the leukemic stem line, MLL and TEL gene rearrangements, and expression of the MLL-AF4, TEL-AML1, E2A-PBX1, and BCR-ABL fusion transcripts. With the availability of these tests, uniform treatment of pediatric ALL patients is no longer acceptable. Instead, it is critical to employ riskbased therapy to reduce toxicity in good-risk patients and to ensure appropriate therapy for patients at a high risk of relapse. Investigators at St. Jude Children’s Research Hospital have proposed a risk classification scheme for childhood ALL that is based largely on the genetic features of leukemic blasts (see Chapter 16). According to this scheme, Blineage patients with hyperdiploidy (DNA index of 1.16– 1.60), TEL-AML1 gene fusion, or a standard-risk age and leukocyte count as defined by NCI criteria,382 represent a low-risk group and are candidates for antimetabolitebased therapy. The standard-risk group includes patients with high-risk age or leukocyte counts,382 a T-cell phenotype, E2A-PBX1, or a slow early response to therapy. The high-risk group, consisting primarily of patients with MLL gene rearrangements or BCR-ABL expression, is eligible for hematopoietic stem cell transplantation in first remission. This risk classification scheme provides a means to incorporate the molecular genetic findings of the past decade into clinical trials seeking to identify optimal treatment intensities for discrete subgroups of ALL patients. In addition to their value in predicting the risk of relapse and allowing one to treat patients with risk-adapted therapy, the identification of molecular lesions in ALL has led to the development of sensitive assays to detect minimal residual disease (see Chapter 28 for details).383,384 These RT-PCR-based assays can detect low levels of disease in patients who are in clinical and morphologic remission. Although the value of minimal residual disease detection in the treatment of ALL is still under investigation in large prospective studies, it is likely that it will be used in the future to alter therapy before overt relapse occurs.384 The incorporation of microarray-based gene expression

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profiling into the clinic will significantly augment the information now available on the molecular features of leukemic clones. More specifically, these findings will be used to establish the hematopoietic cell lineage and degree of leukemic cell differentiation, host-leukemic cell interactions, transformation pathways, and drug sensitivity characteristics. Finally, our understanding of the role of chimeric and aberrantly regulated transcription factors in leukemogenesis may ultimately lead to effective targeted therapies. For example, novel treatments may be directed toward the ALL oncogenes themselves, as in the case of imatinib (Gleevec) in the treatment of BCR-ABL-positive ALL. It is possible that strategies will be developed to interfere with oncogenic transcription factor function. Alternatively, downstream genes that are regulated by oncogenic transcription factors may prove to be better targets for rational drug design.

Summary and future prospects The acute lymphoblastic leukemias of childhood develop from transformed lymphoid progenitors or, in rare cases, from stem cells capable of generating several different lineages. Relatively recent research has established a genetic basis for at least three-fourths of the acute lymphoid leukemias. Most often, chromosomal translocations disrupt transcription factor genes, rearranging their regulatory and coding regions in ways that give rise to aberrant proteins whose activities interfere with the growth, differentiation, or survival of normal blood cell precursors. An intriguing theme of this research is that many leukemogenic transcription factors have striking homology with proteins known to regulate early developmental phases in primitive organisms, including the fruit fly and nematodes. Thus, the critical genetic lesion in many cases of childhood leukemia likely affects evolutionarily conserved pathways that control specific steps in embryogenesis, including formation of the hematopoietic compartment. The actions of a number of translocation-associated oncoproteins have important connotations for the staging and treatment of children with ALL. Detection of MLL fusion oncogenes, for example, almost always mandates the use of allogeneic stem cell transplantation, whereas the presence of TELAML1 indicates a form of leukemia that responds well to less aggressive therapy. For the future, new molecular insights into the origins of childhood leukemia should enable the development of more specific and ultimately more effective treatments.

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regulation of the cell cycle, MTS1/p16INK4A/CDKN2, MTS2/ p15INK4B, p53, and Rb genes in primary lymphoid malignancies. Blood, 1996; 87: 4949–58. Kawamura, M., Ohnishi, H., Guo, S. X., et al. Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia. Leuk Res, 1999; 23: 115–26. Marks, D. I., Kurz, B. W., Link, M. P., et al. High incidence of potential p53 inactivation in poor outcome childhood acute lymphoblastic leukemia at diagnosis. Blood, 1996; 87: 1155– 61. Marks, D. I., Kurz, B. W., Link, M. P., et al. Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia. J Clin Oncol, 1997; 15: 1158–62. Sherr, C. J. & Roberts, J. M. Inhibitors of mammalian G1 cyclindependent kinases. Genes Dev, 1995; 9: 1149–63. Kamijo, T., Zindy, F., Roussel, M. F., et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF . Cell, 1997; 91: 649–59. Quelle, D. E., Zindy, F., Ashmun, R. A., & Sherr, C. J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 1995; 83: 993–1000. Sidransky, D. Two tracks but one race? Cancer genetics. Curr Biol, 1996; 6: 523–5. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994; 264: 436–40. Zhang, Y., Xiong, Y., & Yarbrough, W. G. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell, 1998; 92: 725–34. Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell, 1998; 92: 713–23. Ashcroft, M. & Vousden, K. H. Regulation of p53 stability. Oncogene, 1999; 18: 7637–43. Serrano, M., Lee, H. W., Chin, L., et al. Role of the INK4a locus in tumor suppression and cell mortality. Cell, 1996; 85: 27–37. Krimpenfort, P., Quon, K. C., Mooi, W. J., Loonstra, A., & Berns, A. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature, 2001; 413: 83–6. Sharpless, N. E., Bardeesy, N., Lee, K. H., et al. Loss of p16Ink4a with retention of the p19Arf predisposes mice to tumorigenesis. Nature, 2001; 413: 86–91. Hebert, J., Cayuela, J. M., Berkeley, J., & Sigaux, F. Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias. Blood, 1994; 84: 4038–44. Quesnel, B., Preudhomme, C., Philippe, N., et al. p16 gene homozygous deletions in acute lymphoblastic leukemia. Blood, 1995; 85: 657–63.

349 Haidar, M. A., Cao, X. B., Manshouri, T., et al. p16INK4A and p15INK4B gene deletions in primary leukemias. Blood, 1995; 86: 311–15. 350 Fizzotti, M., Cimino, G., Pisegna, S., et al. Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features. Blood, 1995; 85: 2685–90. 351 Rasool, O., Heyman, M., Brandter, L. B., et al. p15ink4B and p16ink4 gene inactivation in acute lymphocytic leukemia. Blood, 1995; 85: 3431–6. 352 Okuda, T., Shurtleff, S. A., Valentine, M. B., et al. Frequent deletion of p16INK4a /MTS1 and p15INK4b /MTS2 in pediatric acute lymphoblastic leukemia. Blood, 1995; 85: 2321–30. 353 Hirama, T. & Koeffler, H. P. Role of the cyclin-dependent kinase inhibitors in the development of cancer. Blood, 1995; 86: 841– 54. 354 Iolascon, A., Faienza, M. F., Coppola, B., et al. Homozygous deletions of cyclin-dependent kinase inhibitor genes, p16(INK4A) and p18, in childhood T cell lineage acute lymphoblastic leukemias. Leukemia, 1996; 10: 255–60. 355 Nakao, M., Yokota, S., Kaneko, H., et al. Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage. Leukemia, 1996; 10: 249–54. 356 Cayuela, J. M., Madani, A., Sanhes, L., Stern, M. H., & Sigaux, F. Multiple tumor-suppressor gene 1 inactivation is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia. Blood, 1996; 87: 2180–6. 357 Takeuchi, S., Bartram, C. R., Seriu, T., et al. Analysis of a family of cyclin-dependent kinase inhibitors: p15/MTS2/INK4B, p16/MTS1/INK4A, and p18 genes in acute lymphoblastic leukemia of childhood. Blood, 1995; 86: 755–60. 358 Heyman, M., Rasool, O., Borgonovo, B. L., et al. Prognostic importance of p15INK4B and p16INK4 gene inactivation in childhood acute lymphocytic leukemia. J Clin Oncol, 1996; 14: 1512–20. 359 Drexler, H. G. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leukemia, 1998; 12: 845– 59. 360 Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, P. C., & Croce, C. M. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science, 1984; 226: 1097–9. 361 Tsujimoto, Y., Gorham, J., Cossman, J., Jaffe, E., & Croce, C. M. The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining. Science, 1985; 229: 1390–3. 362 Bakhshi, A., Jensen, J. P., Goldman, P., et al. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell, 1985; 41: 899–906. 363 Cleary, M. L. & Sklar, J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally

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active locus on chromosome 18. Proc Natl Acad Sci U S A, 1985; 82: 7439–43. Cleary, M. L., Smith, S. D., & Sklar, J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl- 2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell, 1986; 47: 19–28. Miyashita, T. & Reed, J. C. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood, 1993; 81: 151–7. Naumovski, L. & Cleary, M. L. Bcl2 inhibits apoptosis associated with terminal differentiation of HL-60 myeloid leukemia cells. Blood, 1994; 83: 2261–7. Yang, E. & Korsmeyer, S. J. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood, 1996; 88: 386–401. Vaux, D. L., Cory, S., & Adams, J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature, 1988; 335: 440–2. Oltvai, Z. N., Milliman, C. L., & Korsmeyer, S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993; 74: 609–19. Kroemer, G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med, 1997; 3: 614–20. Coustan-Smith, E., Kitanaka, A., Pui, C. H., et al. Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia. Blood, 1996; 87: 1140–6. Findley, H. W., Gu, L., Yeager, A. M., & Zhou, M. Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood, 1997; 89: 2986–93. Meijerink, J. P. P., Mensink, E. J., Wang, K., et al. Hematopoietic malignancies demonstrate loss-of-function mutations of BAX. Blood, 1998; 91: 2991–7. Williams, D. L., Harber, J., Murphy, S. B., et al. Chromosomal translocation plays a unique role in influencing prognosis in childhood acute lymphoblastic leukemia. Blood, 1986; 68: 205– 16.

375 Rubin, C. M., Le Beau, M. M., Mick, R., et al. Impact of chromosomal translocations on prognosis in childhood acute lymphoblastic leukemia. J Clin Oncol, 1991; 9: 2183–92. 376 Fletcher, J. A., Kimball, V. M., Lynch, E., et al. Prognostic implications of cytogenetic studies in an intensively treated group of children with acute lymphoblastic leukemia. Blood, 1989; 74: 2130–5. 377 Trueworthy, R., Shuster, J., Look, T., et al. Ploidy of lymphoblasts is the strongest predictor of treatment outcome in B-progenitor cell acute lymphoblastic leukemia of childhood: a Pediatric Oncology Group study. J Clin Oncol, 1992; 10: 606– 13. 378 Look, A. T., Roberson, P. K., Williams, D. L., et al. Prognostic importance of blast cell DNA content in childhood acute lymphoblastic leukemia. Blood, 1979; 65: 1079–86. 379 Williams, D. L., Tsiatis, A., Brodeur, G. M., et al. Prognostic importance of chromosome number in 136 untreated children with acute lymphoblastic leukemia. Blood, 1982; 60: 864–71. 380 Harris, M. B., Shuster, J. J., Carroll, A., et al. Trisomy of leukemic cell chromosomes 4 and 10 identifies children with Bprogenitor cell acute lymphoblastic leukemia with a very low risk of treatment failure: a Pediatric Oncology Group study. Blood, 1992; 79: 3316–24. 381 Pui, C. H. & Crist, W. M. Biology and treatment of acute lymphoblastic leukemia. J Pediatr, 1994; 124: 491–503. 382 Smith, M., Arthur, D., Camitta, B., et al. Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia. J Clin Oncol, 1996; 14: 18–24. 383 Campana, D. & Pui, C. H. Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood, 1995; 85: 1416–34. 384 Moppett, J., Burke, G. A., Steward, C. G., Oakhill, A., & Goulden, N. J. The clinical relevance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia. J Clin Pathol, 2003; 56: 249–53.

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11 Molecular genetics of acute myeloid leukemia Robert B. Lorsbach and James R. Downing

Introduction One of the goals of modern leukemia research has been to gain a clearer understanding of the nature of the underlying molecular genetic lesions responsible for the establishment of the leukemic clone. This pursuit is fueled by the hope that the information obtained will not only help us understand the variability in clinical response that is observed among leukemia patients, but will also lead to the identification of rational molecular targets for novel chemotherapeutic agents. This goal is starting to be realized through recent advances in our understanding of the pathogenesis of acute myeloid leukemia (AML). Specifically, molecular genetic and genomic data are beginning to provide the initial framework for the subclassification of AML into clinically meaningful subgroups, and rationally designed compounds that inhibit novel molecular targets in leukemic cells are beginning to be assessed clinically for their utility as antileukemic therapeutics. In this chapter, we provide a summary of the recent advances that have been made in elucidating the molecular genetic lesions involved in the pathogenesis of de novo AML. Our approach is to describe the structure of identified genetic lesions and explain at a molecular level how these alterations lead to cellular transformation. Because of the distinct clinical and biologic differences that exist between de novo AML and myelodysplasia-related AML, we largely restrict our discussion to de novo AML, which lacks any evidence of a prior or concurrent myelodysplastic phase. In addition, due to space limitations, we do not discuss therapy-related AML and do cover to a limited extent congenital conditions that predispose to the development of

AML. For readers interested in these subjects, we refer them to Chapter 31 and several recently published reviews.1,2

Molecular pathogenesis of AML: general considerations AML is characterized by the expansion of a clonal population of leukemic blasts that possess abnormal proliferation and differentiation properties. Until recently, classification of AML was based solely on the lineage commitment of the leukemic cells and their degree of differentiation, as assessed by morphologic examination and cytochemical enzyme assays. However, the recently proposed WHO scheme classifies at least a subset of AMLs based solely on the presence of certain recurrent chromosomal abnormalities.3 Although the leukemic population primarily consists of blasts that show morphologic or immunophenotypic evidence of myeloid lineage commitment, the initiating molecular genetic lesion in most cases appears to arise not in a committed progenitor, but in a primitive leukemic stem cell that has a CD34+, CD38– immunophenotype.4 This leukemic stem cell comprises less than 1% of the total leukemic population and has an exceptionally high self-renewal capacity and gives rise to the more differentiated blasts that are visible within the marrow and peripheral blood. The fact that the cell of origin in most subtypes of AML is the same irrespective of the specific leukemic phenotype suggests that the initiating molecular genetic event directly determines the differentiation program of the leukemic stem cell. The exception to this model is acute promyelocytic leukemia, in which the

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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initiating cell appears to be a committed myeloid progenitor and not a multipotential stem cell. The somatic acquisition of clonal chromosomal translocations and inversions is a critical pathogenetic event in many AMLs, with greater than 50% of cases containing such aberrations (reviewed in Chapter 9). With the identification of many of the targets of these chromosomal rearrangements, it is now apparent that several of the involved genes have essential roles in normal hematopoietic development.5 In AML, these translocations most frequently lead to the formation of chimeric genes that encode fusion proteins with novel biologic properties. The most common targets of these translocations are genes that encode DNA-binding transcription factors or regulatory components of transcription complexes.5 These gene products normally function as master regulatory switches in intracellular signaling cascades involved in cell fate decisions, including lineage commitment, survival, proliferation, and differentiation. The targeted proteins function either at the initiation of these signaling pathways or as key controlling elements that ensure the appropriate balance of positive and negative signals needed for normal function. Not surprisingly, alterations in only a limited number of pathways appear to result in hematopoietic cell transformation. Thus, the observed phenotypic heterogeneity in AML is likely to result, in part, from differences in the point of alteration within several common signaling pathways. In the following sections, we describe the molecular features of the major chromosomal rearrangements in AML and explain what is known about their mechanism of action and their relationship to the normal signaling pathways that control the cell fate decisions of hematopoietic stem cells. Two points are important to emphasize at the outset. First, cellular transformation leading to AML is a multistep process. Although we focus primarily on the initiating events of this process, the lesions to be discussed are, in most cases, insufficient by themselves to generate leukemia. Thus, cooperating secondary molecular genetic lesions are required; these are also discussed but only briefly. The second point to remember is that only half of AMLs have chromosomal rearrangements. In the remaining cases the underlying molecular genetic lesions remain to be identified. Undoubtedly, some of these cases will prove to have cryptic chromosomal rearrangements that are not detectable by routine cytogenetics, while others will have different genetic lesions that lead to the loss or alteration of normal growth regulatory genes. In some cases, the latter molecular changes may perturb common pathways affected by the cloned chromosomal rearrangements. In others, they are likely to affect other signaling pathways that are critical for normal cellular functions. Future identi-

fication of these lesions will provide critical insights into the range of mechanisms that can lead to hematopoietic cell transformation. Combined with the information gained from elucidation of the molecular biology of the chromosomal rearrangements, these studies will provide a more comprehensive understanding of the molecular genetics of AML.

Acute promyelocytic leukemia The 15;17 translocation Acute promyelocytic leukemia (APL) is characterized by the expansion of a clonal population of malignant myeloid cells blocked at the promyelocyte stage of differentiation. In greater than 95% of cases, a t(15;17)(q22;q21) chromosomal translocation is identified within the leukemic promyelocytes. This rearrangement generates a fusion between the genes for promyelocytic leukemia (PML) on chromosome 15 and the retinoic acid receptor
(RAR
) on chromosome 17.6–11 Two chimeric products result from this translocation, PML-RAR
, expressed in all cases and encoded by the derivative 15 chromosome, and RAR
-PML, expressed in 80% of cases and encoded by the derivative 17 chromosome.12 The development of acute leukemia in transgenic mice expressing PML-RAR
has confirmed the central role of this fusion protein in the pathogenesis of APL (Fig. 11.1). However, recent data indicate that the reciprocal fusion product, RAR
-PML may, in some cases, represent a cooperating event in APL leukemogenesis.13,14 These data are described in more detail below. RAR
is a member of the nuclear hormone receptor superfamily and functions as a ligand-dependent transcription factor that regulates the expression of a large number of target genes, some of which are critical for normal myeloid cell differentiation.15 As shown in Fig. 11.1, RAR
is organized into distinct functional domains, including an N-terminal ligand-independent transactivation domain (A/B), a DNA-binding domain that contains two zinc-finger motifs (C), a region containing a nuclear localization signal (D), a large domain that is responsible for ligand-binding, heterodimerization, and contains a liganddependent transactivation function (E), and a C-terminal domain of undefined function (F). This modular structure is shared with other members of the nuclear hormone receptor superfamily of transcription factors. PML is a multidomain protein. It contains a unique cysteine and histidine rich zinc-binding domain termed a RING finger.16 This domain defines a large group of proteins of diverse functions that includes the tumor suppressor

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Fig. 11.1 Schematic representation of PML, RAR
, and the t(15;17)-encoded chimeric protein. PML contains an N-terminal proline-rich region (P), followed by a RING finger, two B-boxes, a coiled-coil domain, and a serine/proline (S/P)-rich region. RAR
is organized into domains labeled A–F. The function of each is described in the text. The positions of breakpoints are illustrated by arrows.

BRCA1, the lymphoid specific recombinase RAG1, RAD18 (involved in postreplicative repair of UV damaged DNA), members of the polycomb group of transcriptional repressors, and TIF1, a mediator of ligand-dependent transactivation of steroid hormone nuclear receptors (recently reviewed17 ). In each of these proteins, the RING finger domain has been demonstrated to have E3 ubiquitin ligase activity, which mediates the transfer of ubiquitin to heterologous substrates.17 Polyubiquitination is a key signal for targeting proteins for proteasome-mediated degradation. Although the RING finger domain has been demonstrated to be important in the normal function of PML, definitive evidence that this domain mediates PML-E3 ligase activity in vivo is lacking. Thus, it is possible that the domain plays an alternative function in PML. PML is a member of the subgroup of RING finger proteins that contain two additional cysteine/histidine-rich regions known as B-boxes, followed by an
-helical coiledcoil domain, which collectively form the RBCC motif or TRIM (tripartite motif ) (Fig. 11.1).18,19 There are multiple isoforms of PML because of alternative splicing of exons 3, 4, 5 and 6; however, all isoforms contain the RBCC/TRIM motif. Each of these regions appears to play a role in protein–protein interactions, with the
-helical coiled-coil domain being critical for the formation of PML homodimers (see below). In addition to these domains, PML contains an N-terminal proline-rich domain and a C-terminal serine/proline-rich region that varies in length as a result

of alternative splicing.20 Collectively, these domains play an important role in controlling the multitude of proteinprotein interactions that are central to the normal cellular functions of PML. In the PML-RAR
chimeric product, the N-terminal portion of PML including the proline-rich region, RING finger, B-boxes, and a variable amount of the
-helical coiledcoil domain is fused in-frame to RAR
, from the B domain through to the C-terminus (Fig. 11.1). This fusion protein contains nearly all of the key functional domains of each molecule, including those in PML required for homodimerization and protein-protein interaction and the DNA-binding, dimerization, ligand-binding and transcriptional activation domains of RAR
. The breakpoint in RAR
always occurs within the second intron of the gene, suggesting that a recombination hot spot exists within this stretch of DNA.21 By contrast, breakpoints in PML vary, with three breakpoint cluster regions (bcr) identified (Fig. 11.1). In about 70% of patients, the breakpoint in PML occurs in the 3 portion of the gene, either in intron 6 (bcr1) or within exon 6 (bcr2).12,22,23 These breakpoints result in fusion products that contain the N-terminal portion of PML, including the majority of the
-helical coiled-coil domain. In approximately 20% of APL patients, however, the PML breakpoint occurs within intron three (bcr3), resulting in deletion of most of the
-helical coiled-coil domain.12,22 This variation in the amount of PML contained in the chimeric product is likely to lead to functional alterations. Indeed,

Molecular genetics of acute myeloid leukemia

Fig. 11.2 Retinoic acid (RA)-induced release of the corepressor complex from RXR/RAR
heterodimers and its replacement by coactivators. The heterodimer RXR/RAR
binds to the retinoic acid response element (RARE) and interacts with the corepressors N-CoR and, Sin3A, and a histone deacetylase (HDAC). RXR, retinoid-X receptor family member.

a correlation between the type of PML-RAR
fusion protein expressed and the clinical or laboratory features of the leukemia has been demonstrated.24 In a subset of bcr2-type cases, an intronic sequence is incorporated at the fusion junction, encoding an additional binding site for the SMRT corepressor in the resultant fusion protein. Recent evidence indicates that these cases are more refractory to all-transretinoic acid (ATRA) therapy.25 However, the clinical significance of the different PML-RAR
isoforms remains a controversial issue.

Functions of wild-type RAR
and PML RAR
is a ligand-regulated transcription factor that controls the transcription of a large number of target genes, some of which are essential for normal hematopoietic cell development and differentiation.15,26 RAR
normally heterodimerizes with a member of the retinoid-X receptor (RXR) family, an interaction that endows the resultant heterodimer with high DNA-binding affinity (Fig. 11.2).27 In the absence of ligand, RAR
/RXR heterodimers effect transcriptional repression by binding specific retinoic acid response elements (RAREs) in the promoter of target genes. Repression is mediated through the formation of a multisubunit complex consisting of the RAR
/RXR heterodimer, the nuclear receptor-corepressor N-CoR/SMRT, the transcriptional corepressors Sin3A or Sin3B, and histone deacetylases (HDAC).28 –31 This complex deacetylates lysine residues in core histone proteins within the nucleosomes of the transcriptional target gene, ultimately leading to an altered chromatin structure that represses transcription.31 Binding of retinoic acid to the RAR
subunit induces a conformational change that causes disassembly of this multisubunit complex and the recruitment of transcriptional co-activators, including p140, p160, and the CREB (cyclic AMP response element-binding protein)-binding proteins CBP and p300, to the RAR
/RXR heterodimer.32–34

The CBP and p300 proteins contain intrinsic histone acetylase (HA) activity and directly interact with both RAR
and p160.35 In addition, two other HAs, ACTF and P/CAF, are recruited to this complex in a ligand-dependent manner.36 Together, these three HAs act cooperatively as an enzymatic unit to acetylate histone proteins, thereby decreasing their affinity for DNA.36 This in turn results in nucleosome unfolding and an increased ability of transcription factors to productively interact with DNA.31,37,38 In addition, HAs can directly acetylate transcription factors and alter their activity.39 Interestingly, in the context of some promoters, retinoic acid binding to RAR
/RXR fails to induce the release of the corepressor complex, suggesting that conversion of RAR
/RXR from a repressor to an activator is partially dependent on the DNA sequence of the RARE to which the heterodimer is bound.29 Thus, whether RAR
/RXR acts as a repressor or activator of gene transcription depends on both the presence or absence of ligand, and the sequence within the response element. PML is ubiquitously expressed and is a component of subnuclear organelles referred to as PML nuclear bodies (NBs) or PML oncogenic domains (PODs) (Fig. 11.3).40,41 Approximately 10 to 30 PML NBs exist within each cell.42 In addition to PML, NBs contain some 30 proteins, including Sp10043 , NDP55, and PLZF, a transcriptional repressor that is fused to RAR
as a result of the rare 11;17 translocation seen in some ATRA-unresponsive cases of APL (reviewed below).44–46 Within the NBs, covalently modified PML is normally present as a homodimer, with dimerization mediated, in part, through the
-helical coiled-coil domain. Mutations in the RING finger or B box domains of PML eliminate its ability to localize to NBs, suggesting that the protein-protein interactions mediated through these domains are essential for normal localization and presumably function.16 PML exists both in a free form within the nucleoplasm and conjugated to the ubiquitin-like protein SUMO1 (also referred to as PIC1).47 Only the PML-SUMO1

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Fig. 11.3 Schematic representation of the PML nuclear body. Only a subset of the proteins known to reside in the PML nuclear body are shown. These include: Sp100; the Bloom syndrome REC-Q DNA helicase, BLM, and its interactive proteins, RAD51 and RP-A; elF-4E; DAXX; and p53. PML interacts with the p53 tumor suppressor pathway at multiple levels. Within the nuclear body, PML facilitates the acetylation of p53 by the CBP transcriptional coactivator. PML also directly interacts with the MDM2 protein and sequesters it away from p53, thereby increasing the stability of p53. Lastly, recent data suggest that PML is a direct transcriptional target of p53.

conjugate is localized to the NBs.48 Furthermore, studies in PML-deficient cells indicate that SUMOylation of PML is required for the assembly of NBs and for the recruitment by PML of other proteins, including CBP, to NBs.49,50 SUMOylation of PML is likely dynamically regulated as several enzymes, including ULP1 and SENP1, capable of removing SUMO1 moieties from protein substrates have been identified (reviewed in Zhong et al.51 ). Indeed, a recently identified protease, SUMO-1 Protease-1 (SuPr-1), catalyzes the removal of the SUMO-1 moiety from PML.52 Expression of SuPr-1 alters the localization of PML in NBs as well as that of other NB-associated proteins such as DAXX and CBP. Furthermore, SuPr-1 enhanced transcription through a mechanism dependent on PML. This effect of SuPr-1 on transcription is likely indirect and mediated through modulation of the activity or localization of other transcriptional coactivators and repressors. Recent studies have demonstrated that PML plays important roles in several critical cellular processes, including cellular proliferation and senescence and apoptosis.53–55 Overexpression of PML has growth suppressive effects, suggesting that PML may normally function in the NBs to limit the proliferative potential of cells.56,57 Indeed, primary mouse embryo fibroblasts (MEFs) from PMLdeficient animals have an enhanced proliferative capacity

and readily form foci in culture.58 Moreover, chemicallyinduced tumors occur at an increased frequency in these mice, raising the possibility that PML is a true tumor suppressor. PML also plays a role in cellular senescence. It was recently demonstrated that there is a marked upregulation of PML in cells expressing an oncogenic form of RAS, which triggers cellular senescence.59,60 Furthermore, induction of oncogenic RAS-induced senescence in PML-deficient cells is defective, an effect likely mediated through diminished p53 transactivation.60 The retinoblastoma protein (pRB) has been implicated in cell cycle control and cellular senescence. Within NBs, the nonphosphorylated form of the pRB colocalizes and interacts with PML, and overexpression of pRB increases NB formation.61,62 Recent studies demonstrate that PML enhances pRB transcriptional repression and can associate with the corepressor complex comprised of N-CoR/SMRT, Ski/Sno, Sin3a and HDAC, through which pRB and the tumor suppressor MAD effect transcriptional repression. Furthermore, the transcriptional repression effected by pRB and MAD is diminished in PMLdeficient MEFs. Finally, PML plays an important role in apoptosis. PML-deficient mice manifest reduced lethality in response to  -irradiation.63 Additionally, primary cells derived from PML-deficient mice are resistant to apoptosis induced by several stimuli, including  -irradiation,

Molecular genetics of acute myeloid leukemia

Fig. 11.4 Schematic representation of the normal activity of the RAR
and PML proteins in hematopoietic development and their disruption by the PML-RAR
chimeric protein. VDR, vitamin D receptor; TR, thyroid hormone receptor; RXR, retinoid-X receptor family member.

Fas signaling, and TNF. The resistance to  -irradiationinduced apoptosis in PML-deficient cells is due in part to impaired signaling through the p53 pathway, as the induction of direct p53 target genes, including those encoding the proapoptotic bax and the cell cycle inhibitor p21, is reduced in PML-deficient cells. Moreover, the acetylation of p53 following  -irradiation, which enhances its transcriptional function, is significantly reduced in PML-deficient cells.64 More recent experiments indicate that in  -irradiationinduced apoptosis, PML function may be regulated in a p53-independent manner through phosphorylation by the DNA damage checkpoint kinase hCds1/Chk2, a component of the ataxia telangiectasia-mutated (ATM) signaling pathway.65

Molecular mechanism of transformation The PML-RAR
chimeric protein induces leukemia through a dominant-negative mechanism to inhibit the normal biologic activities of both RAR
and PML (Fig. 11.4). Moreover, the unique biologic features of this fusion protein not only help to explain the APL leukemic phenotype, but also its unique response to ATRA. As predicted from its structure, PML-RAR
continues to bind RAREs either as a homodimer or a heterodimer with RXRs. In contrast to the wild-type protein, however, it fails to function as a ligand-induced transcriptional activator, but instead directly represses transcription or only weakly activates it, thus blocking the normal transcriptional activation provided by ligand-bound RAR
.9,10 Moreover, by binding

RXRs, the PML-RAR
fusion protein sequesters RXRs away from interactions with other nuclear hormone receptors, such as those for vitamin D (VDR) and thyroid hormone (TR).66 This leads to inhibition of the normal functions provided by these hormones during myeloid cell differentiation. Together these activities are believed to account for the block in differentiation at the promyelocyte stage in APL.67–69 In addition to its effects on retinoid signaling, PMLRAR
also induces the disruption of normal PML NBs (Fig. 11.4). PML-RAR
retains the RING, B-box, and most of the coiled-coil domain and is therefore able to homodimerize, an interaction critical for its repression of RARainduced transactivation, as well as heterodimerize with wild-type PML. 70,71 The latter interaction results in the disruption of the nuclear bodies and the redistribution of PML into a microparticulate pattern within the nucleus.40,41,72 This change in nuclear localization is believed to result in inhibition of the normal growth regulatory activity of PML. PML-RAR
reduces the frequency of cell death under conditions of growth factor deprivation. 54,73–76 Furthermore, myeloid cells from PML-RAR
transgenic mice are resistant to several proapoptotic stimuli, including  -irradiation, TNF and Fas.63 Human APL cells expressing PML-RAR
are, by definition, haploinsufficient for PML. Recent data indicate that the penetrance and latency of APL development are dramatically increased and the sensitivity of bone marrow cells to proapoptotic stimuli significantly reduced in PML-haploinsufficient PML-RAR
transgenic mice, compared with PML wild-type mice expressing this transgene.77

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Fig. 11.5 All-trans-retinoic acid (ATRA)-induced release of the corepressor complex from the PML-RAR
chimeric protein. Abbreviations are defined in the legend to Fig. 11.2.

Thus, the expansion of malignant cells in APL appears to result, at least in part, from antagonism of the normal sensitizing effect of PML to proapoptotic stimuli, resulting in enhanced cell survival. Overexpression of Bcl-2 also accelerates the development of APL in murine models, providing additional support for a role of apoptosis resistance in the pathogenesis of APL.78 Finally, recent evidence indicates that PML-RAR
can directly interact with and recruit DNA methyltransferases to target promoters, resulting in their hypermethylation and subsequent gene silencing.79 The ability of the PML-RAR
fusion protein to alter in a dominant fashion the normal biologic activity of both wild-type RAR
and PML appears, therefore, to be critical for the transforming activity of this molecule. An interesting twist on our understanding of the molecular changes induced by the expression of the PML-RAR
fusion protein has come from the recent demonstration that a proportion of this fusion protein is proteolytically cleaved by neutrophil elastase, a neutral serine protease expressed at high levels in promyelocytes. Interestingly, PML-RAR
-induced leukemia is markedly impaired in mice that lack this protease, raising the possibility that the PML RAR
cleavage products contribute to leukemia development.80 PML-RAR
, like wild-type RAR
, directly binds to the nuclear corepressor multisubunit complex, resulting in the repression of gene transcription (Fig. 11.5).81–83 In contrast to the wild-type receptor, however, PML-RAR
fails to dissociate from the corepressor complex in the presence of physiologic levels of retinoic acid.81–87 This refractoriness of PML-RAR
to normal levels of retinoic acid results in the dominant inhibition of ligand-responsive, RAR
-induced transcriptional activation. In addition, the persistent binding of nuclear corepressors to the chimeric molecule may lead to a titration of these complexes away from other corepressor dependent pathways. For example, normal growth and differentiation of hematopoietic cells is

dependent on the appropriate regulation of the MYC/MAX and MAD/MAX complexes, the latter requiring interaction with the corepressor complex for normal growth arrest and differentiation. The shifting of the corepressors away from these normal interactions may directly contribute to the growth abnormalities of leukemic cells. Pharmacologic levels of retinoic acid, such as those achieved with ATRA administration, induce a conformational change in PML-RAR
that results in the release of N-CoR and its replacement by transcriptional coactivators. Thus, treatment with ATRA reverses the inhibitory activity of PML-RAR
on both RAR
and PML and consequently effects the specific growth arrest of leukemic promyelocytes and their terminal differentiation.40,41 ATRA also induces the proteolytic cleavage of PML-RAR
, in part through a proteasome-mediated pathway.88,89 The resulting 85-kDa fragment lacks the amino-terminus of the PML moiety, likely including the RING finger and B-boxes, and no longer inhibits signaling by endogenous RAR
(see Jing et al.90 and citations therein). Recently, UBE1L was demonstrated to trigger PML/RAR
degradation. This protein catalyzes the first step in the conjugation of the ubiquitin-like moiety ISG15 to target proteins,91 and its gene is in fact a transcriptional target of retinoic acid.92 While the biochemical consequences of ISG15 modification are not well understood, hematopoietic cells deficient in UBP43, an enzyme that removes ISG15 moieties, are hypersensitive to IFN
/ and manifest increased apoptosis in response to IFN treatment.93 Further elucidation of the pathways responsible for PML/RAR
proteolysis may provide opportunities for the development of novel therapeutics that promote the degradation of this fusion protein. Although treatment with ATRA induces the terminal differentiation of leukemic cells, when used as a single agent it does not lead to the eradication of the leukemic clone. Compounding this problem is the rapid development of

Molecular genetics of acute myeloid leukemia

ATRA-resistant cells. A frequent mechanism underlying this resistance is the acquisition of mutations in the RAR
ligand-binding domain, which eliminates binding.94–96 To circumvent this problem, oncologists now use ATRA in combination with other traditional chemotherapeutic agents (reviewed in Zhou et al.96 and Tallman et al.97 ). This treatment strategy has been found to be far superior to either ATRA or standard chemotherapy alone. Recently, studies from China have demonstrated that both ATRA responsive and resistant APL cases are also sensitive to treatment with arsenic trioxide (As2 O3 ).98 In contrast to retinoic acid and ATRA, however, As2 O3 does not bind to RAR
, but instead binds to the PML portion of the chimeric product.99 This interaction induces the degradation, through a proteasome-dependent mechanism, of both PML-RAR
and PML present in heterodimeric complexes with the fusion protein. Although As2 O3 therapy does cause myeloid differentiation similar to that seen following ATRA administration, induction of apoptosis in malignant promyelocytes is thought to be the primary mechanism by which As2 O3 therapy induces its antileukemic functions.99,100 Future protocols combining the use of ATRA, As2 O3 , and standard chemotherapeutics should provide improvements over presently used approaches.

Lessons from transgenic mice Direct confirmation of the role of the X-RAR
fusion proteins in hematopoietic cell transformation has come from a variety of in vitro cell culture systems and from the generation of transgenic mice.101–104 Such mice expressing PML-RAR
, driven by either the human cathepsin G or the migration inhibitory factor-related protein 8 promoter, both of which are active during early myelopoiesis, develop a myeloproliferative disorder; a small subset of animals ultimately develop a fatal APL-like disorder after a long latency period.104 Similarly, transplantation of bone marrow cells transduced with PML-RAR
-expressing retroviruses results in the development of retinoic acid-sensitive APL in greater than 80% of recipient animals.103 In all murine models generated to date, a long latency is required for the development of overt leukemia, indicating that cooperating events are necessary for full transformation. An obvious candidate cooperating mutation is the reciprocal product RAR
-PML, which is expressed in 70% to 80% of t(15;17) APLs. Transgenic mice expressing RAR
PML alone have normal myeloid development and do not develop leukemia. However, in double transgenic mice expressing both PML-RAR
and RAR
-PML, the penetrance of leukemia development is increased three to four

fold with no effect on the latency of APL development.13 The mechanism by which RAR
-PML potentiates leukemogenesis is unknown, but may involve the perturbation of PMLmediated interaction with pRB and p53. Finally, through spectral karyotyping analysis of leukemic cells from transgenic mice with APL, clonal cytogenetic aberrations have been identified, a subset of which are syntenic to those seen in human APL.105,106 Thus, further analysis of APLs derived from transgenic mouse models may facilitate the identification of cooperating mutations involved in the pathogenesis of APL.

Variant translocations affecting RAR
Several variant translocations that involve the RAR
gene have been identified in APL. These include the t(5;17) encoding a fusion between the nucleolar protein nucleophosmin (NPM) and RAR
,107 the t(11;17)(q23;q21) encoding a fusion between the transcriptional repressor PLZF (for promyelocytic leukemia zinc-finger protein) and RAR
(Fig. 11.6),44 the t(11;17)(q13;q21) encoding a fusion between nuclear mitotic apparatus protein (NUMA) and RAR
,108 and a der(17) encoding a fusion between signal transducer and activator of transcription (Stat5b) and RAR
.109 Due to space limitations, only the PLZF-RAR
will be discussed here; for a discussion of the remaining variant RAR
translocations in APL, the reader is referred to a recent review.110 Important insights into the mechanism of transformation in APL have come from studies of the ATRAunresponsive 11;17 translocation. PLZF is a nuclear protein that is highly expressed in the developing nervous system, limb buds, liver, heart, and kidneys.111,112 It is also expressed in CD34+ hematopoietic progenitors and is downregulated upon terminal differentiation in some hematopoietic cell lines. PLZF contains an N-terminal POZ (for poxvirus and zinc-finger) autonomous repres¨ sion domain and a C-terminal Kruppel-like DNA binding domain. Surprisingly, PLZF not only homodimerizes but also forms heterodimers with PML, colocalizing with PML to the NBs, suggesting a possible role for PLZF in the growth suppressive functions of PML or, alternatively, a role for PML in the normal function of PLZF.45,113 Both interactions appear to be mediated through the POZ domain.114 While not having a role in dimerization, the POZ domain recruits N-CoR, SMRT and HDAC and thus plays a critical role in effecting the transcriptional repression of PLZF.115 As a result of the t(11;17) two reciprocal products are formed, PLZF-RAR
and RAR
-PLZF. The PLZF-RAR
product consists of the N-terminal portion of PLZF, including its POZ repression domain, fused in

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Fig. 11.6 Schematic representation of (A) PLZF and RAR
and (B) the PLZF-RAR
chimeric protein encoded by the t(11;17). PLZF contains an N-terminal poxvirus and zinc-finger (POZ) domain followed by an autonomous repression domain (A), a proline-rich region ¨ (P), and a Kruppel-like zinc finger (Zn) domain. The domains of RAR
are defined in the text.

frame to RAR
(Fig. 11.6). Transgenic mice expressing the PLZF-RAR
protein from the cathepsin G promoter uniformly develop a chronic myeloid leukemia within the first year of life.83 Thus, like PML-RAR
, the PLZF-RAR
chimeric product directly contributes to the development of myeloid leukemia. The reciprocal RAR
-PLZF product that is formed as a result of the t(11;17) is expressed in most cases of APL and contains the putative PLZF DNA binding domain. Interestingly, it was recently demonstrated that PLZF-RAR
/RAR
-PLZF double transgenic mice develop an acute leukemia with morphologic and immunophenotypic features of APL, in contrast to mice transgenic for only PLZF-RAR
.14 RAR
-PLZF single transgenic mice do not develop leukemia, and coexpression of RAR
-PLZF has no effect on the latency of leukemia development in double transgenic animals. Thus, while not functioning as a classical oncogene or tumor modifier per se, coexpression of the reciprocal fusion protein does, nonetheless, appear to be critical for development of the phenotypic features of t(11;17) APL. The PLZF-RAR
chimeric product retains the ability to interact with retinoic acid, heterodimerize with RXR, bind to RAREs in RAR
-responsive genes, and directly interact with the nuclear corepressor complex (Fig. 11.7).81–83,85 Moreover, like PML-RAR
, PLZF-RAR
fails to release the corepressor complex in response to retinoic acid binding. However, in contrast to PML-RAR
, PLZF-RAR
is also unresponsive to ATRA.116 This latter property results from the direct binding of nuclear corepressor complexes to both the RAR
domain and the PLZF POZ domain of the chimeric protein.81–83,86 Thus, the PLZF-RAR
protein contains an ATRA-responsive corepressor complex bound through RAR
, and a PLZF bound corepressor complex that is ATRA-unresponsive. Experimentally, this unre-

sponsiveness can be reversed through direct inhibition of the HDAC activity of the corepressor complex. Treatment of PLZF-RAR
-containing leukemic cells with trichostatin A, a specific inhibitor of HDAC, converts these cells to an ATRA-responsive phenotype. Although these data help to explain the phenotypic differences between these leukemias, recent data also suggest that the DNA-binding specificities differ subtly among PLZF-RAR
, PML-RAR
and RAR
.113 These differences are likely to contribute to the diverse leukemias that develop from expression of the chimeric products. In summary, the extensive data on APL paint a picture in which hematopoietic cell transformation results, in part, from the generation of dominant-acting chimeric transcription factors. These fusion products directly repress the retinoic acid-initiated transcriptional cascade required for normal hematopoietic cell development, and block PMLmediated signals that play an important role in the regulation of cell growth and senescence and apoptosis. The conversion of a transcriptional activator to an active repressor is a recurrent theme that we will see again in leukemias induced as a result of alteration of the RUNX1 transcription factor complex, and indirectly in leukemias that result from alterations in the MLL gene. The critical RAR
-regulated hematopoietic target genes whose expression are disrupted by the dominant-acting RAR
chimeric proteins remain to be identified. The similarity of the mechanisms used by RAR
, RUNX1, and MLL fusion proteins suggests that they may alter the expression of common target genes. A gene family whose altered expression may be mechanistically involved in the leukemias caused by these genetic lesions are the HOX genes.5 This concept will be further explored below. Lastly, with the demonstrated efficacy of ATRA and As2 O3 in the treatment for this malignancy, APL therapy

Molecular genetics of acute myeloid leukemia

Fig. 11.7 Transcriptional activity of the PLZF-RAR
chimeric protein. The abbreviations are defined in the legend to Fig. 11.2.

now serves as the paradigm for the future development of novel antileukemic therapeutics that target the underlying, pathogenetic molecular lesions for each of the molecular subtypes of pediatric AMLs.

Acute myeloid leukemia and the core-binding factor (CBF) complex The heterodimeric core-binding factor transcription complex encoded by the RUNX1 gene on chromosome 21q22 and the CBFβ gene on chromosome 16q22 has been shown to be essential for the normal development of all hematopoietic lineages.117 Somewhat surprising was the observation that the two most common chromosomal rearrangements in de novo AML, t(8;21) and inv(16), result in alterations of RUNX1 and CBF, respectively.118–122 Although the leukemias resulting from these translocations manifest a spectrum of morphologic features, they are linked by a common underlying molecular pathogenesis. This unifying feature correlates closely with a relatively good response to conventional multiagent chemotherapy.123–127 Critical to our understanding of these leukemias is the identification of the normal role of RUNX1/CBF in hematopoiesis and the elucidation of how alterations of this complex lead to hematopoietic cell transformation. Further insight into the role of the core-binding factor complex in AML will likely come from studies of the involvement of this complex in other translocations, including the t(3;21) [RUNX1-EVI1] seen in myelodysplasia and rare cases of blast transformation of chronic myeloid leukemia,128 and the t(12;21) [TEL-RUNX1], the most common chromosomal rearrangement in pediatric acute lymphoblastic leukemia (ALL).129,130

Fig. 11.8 The RUNX1/CBF transcription factor complex functions as an enhancer organizer at the core enhancer sequence, TGTGGT. Proteins that interact with RUNX1/CBF at this site include ETS-1, MYB, C/EBP
, LEF-1, ALY, and ATF/CREB. Target genes regulated by this complex include myeloperoxidase (MPO), the receptor for colony-stimulating factor-1 (CSF-1R), the subunits of the T-cell antigen receptor (TCR), and neutrophil elastase.

Normal function of the RUNX1/CBF transcription factor complex RUNX1, initially cloned as the chromosome 21q22 target of the 8;21 translocation,118 encodes a transcription factor that binds the enhancer core sequence, TGT/cGGT (Fig. 11.8).131–133 RUNX1 forms a heterodimer with CBF, and both its DNA-binding and interaction with CBF are mediated through a central 118 amino acid runt homology domain (RHD), so named because of its high level of homology to the Drosophila pair-rule gene runt (Figs. 11.8 and 11.9).134 The RHD defines a family of three closely related proteins, RUNX1, RUNX2, and RUNX3; alternative designations for RUNX1 include AML1, PEBPA2B and

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Fig. 11.9 Schematic representation of (A) RUNX1 and ETO and (B) the t(8;21)-encoded RUNX1-ETO chimeric protein. RUNX1 contains the Runt homology domain (RHD), a proline/serine/threonine-rich region (PST), a nuclear matrix association site (NM), and a transcription activation domain (TA). ETO is organized into three proline/serine/threonine-rich regions (PST) and four domains with homology to nerve (nerve homology regions, NHR1–4).

CBFA2.135,136 Each of these genes shows unique yet partially overlapping patterns of expression, with most tissues expressing only one or two family members. Although Runx1 is widely expressed in the hematopoietic and lymphoid systems, differential expression of Runx1 occurs in certain instances.137,138 For example, in murine bone marrow, Runx1 is expressed by most myeloid, B lymphoid and megakaryocytic cells, whereas it is expressed only weakly in early erythroid progenitors and is silenced in subsequent stages of erythropoiesis. Finally, RUNX1 is also expressed in a cell type-specific pattern in other embryonic and adult organs, including the brain.139–141 The CBF subunit is a ubiquitously expressed protein that does not directly bind DNA but instead increases the DNA-binding affinity of RUNX1 by decreasing its rate of dissociation (Fig. 11.8).131,142 Furthermore, heterodimerization with CBF stabilizes RUNX1 and renders it more resistant to proteosome-mediated degradation.143 Several alternatively spliced forms of CBF that differ in their C-terminal sequences have been characterized; however, no significant functional differences have been observed among the isotypes.131 CBF is localized to both the cytoplasm and nucleus, and nuclear import appears to be regulated by both phosphorylation and by the level of RUNX1 expressed.144–146 The enhancer core motif, to which RUNX1/CBF binds, has been shown to be critical for the tissue specific expression of a number of different hematopoietic specific genes, including myeloperoxidase (MPO),147 the CSF-1 receptor (CSF-1R),148 the subunits of the T-cell antigen receptor

(TCR),131,149 neutrophil elastase,147 and the cytokines IL-3150 and GM-CSF151 (Fig. 11.8). Although the core enhancer sequence is important for the hematopoieticspecific expression of these genes, expression also depends on the presence of adjacent binding sites for lineagerestricted transcription factors such as MYB, LEF-1, CEBP
, and ETS family members.152–155 This observation suggests that RUNX1/CBF functions as a transcriptional organizer that recruits tissue-specific factors to form a nucleoprotein complex, or enhancesome, that stimulates lineage-restricted transcription (Fig. 11.8).156 The enhancer sequence to which RUNX1 binds is typically 100 nucleotides in length and contains specific binding sites for RUNX1, as well as a number of other tissue-specific and non-specific DNA-binding proteins. Cooperative interactions between the various factors that bind to this sequence result in the enhanced DNA binding of each factor.157 Moreover, these interactions create specific activation surfaces that interact with coactivators and with the basal transcriptional machinery.156 In addition to transcription factors, these complexes contain sequence-specific DNA-bending proteins, such as LEF-1, that bind to the minor groove of DNA and induce a bend that facilitates interactions between RUNX1 and adjacently bound coactivators such as CEBP/CBP. The interaction between RUNX1 and LEF1 is facilitated by the coactivator ALY (Fig. 11.8).158 These interactions collectively add to the stability and function of the final complex. RUNX1-mediated transcription requires an activation domain localized to the C-terminal third of the molecule

Molecular genetics of acute myeloid leukemia

(Fig. 11.9).159 In addition, RUNX1 contains an 80 amino acid domain immediately C-terminal to the RHD that binds Ear2, an orphan member of the nuclear hormone receptor superfamily that functions to inhibit transcriptional activation.160,161 Similarly, a repression domain has been suggested to exist in the C-terminal portion of the molecule162 ; however, the nature of the molecule(s) that interact with this region remain to be identified. Moreover, the C-terminal four amino acids, WRPY, are conserved among all RUNX family members and function as a specific binding site for the transcription corepressor Groucho.160 Interestingly, mice expressing solely a form of RUNX1 lacking the WRPY domain have a transient decrease in thymus size but no other significant hematopoietic perturbations, indicating that this domain is not required for postnatal hematopoiesis in vivo.163 Thus, the transcriptional activity of RUNX1/CBF could lead to either transcriptional activation or repression, depending on the specific target gene being regulated and the cellular context in which this is occurring. If corepressors are bound to RUNX1 then transcriptional repression would be predicted to result. By contrast, if these interactions are prevented, transcriptional activation should occur. In addition to the tissue- and cell type-specific expression of RUNX1, the transcriptional activity of the RUNX1/CBF complex is further regulated by several other distinct mechanisms. Two distinct promoters, designated distal and proximal, for the human and murine RUNX1 genes have been identified. The distal promoter resides some 160 kb upstream of the proximal promoter in the human genome.164 The distal RUNX1 promoter is preferentially used in T cells and other hematopoietic cells and due to differential splicing results in the generation of a RUNX1 isoform that contains a highly conserved 19 residue domain at the amino terminus that may have functional properties distinct from that of the isoform generated by utilization of the proximal promoter.165,166 Several other RUNX1 isoforms exist, resulting from alternative splicing events of downstream exons, in which portions of the functional domains outlined above or portions of the RHD are deleted.167–170 Although these alternatively spliced products appear to comprise only a minority of RUNX1 transcripts, changes in the ratio of these different isotypes could lead to a profound change in the transcriptional activity of the RUNX1/CBF complex. Consistent with this prediction is the observation that G-CSF-induced differentiation of the myeloid cell line 32Dcl3 can be blocked by expression of RUNX1 isoforms that either lack sequences C-terminal to the RHD169 or N-terminal sequences including part of the RHD.170 Alterations in the balance of positive and negative signals that are mediated through this complex are likely to

directly contribute to hematopoietic cell development and transformation. RUNX1 is also the target of post-translational modification events that impact on its transactivational properties. RUNX1 is phosphorylated by activated, cytokine-regulated kinases, a modification that potentiates the transactivating function of RUNX1 without apparently altering its ability to interact with CBF or DNA.171 Recent studies indicate that RUNX1 physically interacts with the corepressor Sin3A.172 This association may protect the former from proteasomemediated degradation, since phosphorylated RUNX1 no longer associates with Sin3A and has a reduced half-life. Thus, phosphorylation may influence RUNX1 function through two mechanisms, namely, regulation of the actual level of RUNX1 within a cell by altering its stability, and through modulation of the intrinsic transcriptional activity of RUNX1. Gene targeting experiments in mice have demonstrated that both RUNX1 and CBF are essential for the formation of the definitive hematopoietic system.173–176 Null mutation in either gene results in an embryonic lethal phenotype at the mid-point of development, characterized by a complete absence of fetal liver-derived hematopoiesis, resulting in anemia and an absence of circulating platelets that leads to soft tissue and lethal central nervous system hemorrhages. Although primitive yolk sac-derived erythropoiesis appears normal, no definitive hematopoietic progenitors of any lineage are present. The hematopoietic defect resulting from the loss of RUNX1 is intrinsic to the definitive hematopoietic stem cell (HSC), since homozygous RUNX1-null ES cells contribute to all tissues except hematopoietic lineages in chimeric mice.173 RUNX1 is highly expressed within the aorta-gonad-mesonephros (AGM), a major site of definitive HSC formation in the developing embryo.177 Histologic analyses of midgestation human,178,179 mouse,180,181 and avian182 embryos have identified CD34+ cells budding into the lumen of the aorta along its ventral aspect, which are thought to represent nascent HSCs. In a similar analysis of RUNX1deficient murine embryos, no intra-aortic HSC clusters were identified;183 additionally, CBF-MYH11 knockin embryos, in which the function of wild-type CBF is inhibited, are deficient in fetal liver HSCs and progenitors.184 Taken together, these findings suggest that the hematopoietic defect in RUNX1- and CBF-deficient embryos is due to defective HSC formation in the AGM and at other sites of HSC generation. Until recently, the role of RUNX1/CBF in postnatal hematopoiesis and lymphopoiesis was unknown. Analyses of murine lines in which RUNX1 could be conditionally deleted using Cre recombinase have been reported.185,186

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Significant thrombocytopenia developed in adult mice following RUNX1 deletion when compared to wild-type. RUNX1-deficient megakaryocytes had lower DNA ploidy and manifested less cytoplasmic maturation when compared with wild-type cells. Surprisingly, neutrophil numbers and hemoglobin levels in peripheral blood were comparable in RUNX1-deficient and wild-type animals.185 RUNX1 also plays an important role in T-cell development. Mice in which RUNX1 is specifically deleted within the thymus have significantly reduced numbers of thymocytes.186 Furthermore, RUNX1 is required for silencing of CD4 expression during the earliest, so-called double-negative stage of thymocyte maturation. Taken together, these data indicate that in contrast to its critical role during developmental hematopoiesis, RUNX1 is dispensable for most aspects of postnatal hematopoiesis and to date has been shown to be required only for megakaryocytic maturation and platelet production and certain aspects of lymphopoiesis. During development, RUNX1/CBF functions as a master regulatory switch that establishes, through both positive and negative regulation, a transcriptional cascade involved in critical cell fate decisions necessary for the generation of the definitive hematopoietic system. Although a number of RUNX1/CBF target genes have been identified, none appear to account for the dramatic effect on developmental hematopoiesis of deficiency of the RUNX1/CBF complex. Thus, the critical targets of RUNX1/CBF within the AGM and other sites of HSC formation remain to be identified. These target genes are likely to be components of a transcriptional cascade critical for the formation, proliferation, survival, or differentiation of definitive hematopoietic stem cells. Interestingly, RUNX2 has been demonstrated to function as a master regulatory switch essential for osteoblast differentiation (see Nakashima & de Crombrugghe187 and references therein). Inactivation of RUNX2 in mice results in the absence of both intramembranous and endochondral bone ossification, and inactivating mutations in humans result in cleidocranial dysplasia. The hematopoietic defects in CBF-deficient embyros can be ameliorated by expression of CBF using hematopoieticspecific transgenic promoters188,189 or through the expression of a hypomorphic form of CBF.190 Interestingly, these mice die at birth and manifest marked skeletal malformation, indicating that heterodimerization of RUNX2 with CBF is required for osteoblast differentiation and function.188–190 RUNX3 is expressed at high levels in cranial and dorsal root ganglia (DRG) within certain neuronal subsets. Recently, two independently developed RUNX3-deficient murine lines have been reported, both of which developed severe ataxia due to diminished DRG neuronal

projections.191,192 Interestingly, one of these RUNX3deficient strains also developed gastric hyperplasia.193 Furthermore, RUNX3 expression is significantly reduced in approximately 50% of human gastric cancers due to either hemizygous gene deletion or RUNX3 promoter hypermethylation. These provocative data suggest a role for RUNX3 in the pathogenesis of gastric carcinoma; however, additional studies are needed to confirm these findings. RUNX3 also appears to play an important role in the epigenetic silencing of CD4 gene expression in thymocytes and antigen responsiveness of peripheral cytotoxic T cells.186,194

The inv(16) and t(16;16) The inv(16)(p13q22) and the variant t(16;16)(p13;q22) together constitute one of the most frequent genetic lesions identified in de novo AML.195–197 Although these abnormalities were initially thought to be restricted to acute myelomonocytic leukemia with abnormal eosinophils, they also occur at a significant frequency in AMLs with other morphologies.198,199 The cloning of these chromosomal rearrangements demonstrated that each targets the gene encoding the CBF subunit of the core-binding factor complex on 16q22 and the MYH11 gene on chromosome 16p13 (Fig. 11.10).122 MYH11 encodes a smooth muscle myosin heavy chain that contains a globular head with actin binding and ATPase activity, a hinge region, and an
-helical rod domain that forms coiled-coil dimers and higher order bipolar myosin filaments.122,200 Although two reciprocal chimeric genes are formed, only the CBFβ-MYH11 gene is consistently expressed in these cases. The fusion product encoded by this gene consists of the N-terminal portion of CBF fused in-frame to a variable amount of the C-terminal
-helical rod domain of MYH11 (Fig. 11.10). The breakpoint occurs in the fourth or fifth intron of CBFβ, but is highly variable in MYH11 and consequently results in significant variation in the amounts of MYH11 contained within the fusion proteins. In each case, however, the RUNX1-binding domain of CBF is retained, and thus the CBF-MYH11 chimeric protein can heterodimerize with normal RUNX1.198,201 In addition, the MYH11 moiety contained within the fusion protein is capable of forming homodimers and higher order multimers through intermolecular interactions of the MYH11 rod domains.122 Experimental evidence has demonstrated the presence of high-molecular-weight nuclear RUNX1/CBF-MYH11 complexes within inv(16)-containing leukemic cells. The genetics of the inv(16) chromosomal rearrangement suggest that CBF-MYH11 functions in a dominant manner to induce leukemia. One mechanism by which CBF-MYH11 appears to do this is through the

Molecular genetics of acute myeloid leukemia

Fig. 11.10 Schematic representation of (A) CBF and MYH11 and (B) the inv(16)-encoded CBF-MYH11 chimeric protein. The C-terminal portion of MYH11 contains a protein–protein interaction domain (Pr-Pr).

cytoplasmic sequestration of RUNX1.202,203 CBF-MYH11 is localized primarily within the cytoplasm and associates with the actin cytoskeleton through domains contributed by MHY11. Heterodimerization of RUNX1 with CBF-MYH11 would result in its retention in the cytoplasm, effectively reducing the intranuclear level of RUNX1 available for transactivation of target promoters. A second mechanism is direct transcriptional repression by CBF-MYH11 within the nucleus. CBF-MYH11 directly represses RUNX1-mediated transcriptional activation in a manner similar to translocation-encoded AML chimeric proteins.204 This activity is dependent on both the RUNX1interaction domain of CBF and domains within MYH11. Although the exact mechanism of transcriptional repression is at present unclear, it appears to involve, at least in part, the abnormal recruitment to target gene promoters of corepressors by RUNX1/CBF-MYH11 complexes.205 Relevant to both of these mechanisms is the recent finding that CBF-MYH11 binds the RHD of RUNX1 with higher affinity than does native CBF.206 This finding provides a molecular basis by which CBF-MYH11 can titrate RUNX1 away from wild-type CBF, effectively reducing RUNX1mediated transactivation. Direct evidence that the CBF-MYH11 fusion product functions in a dominant-negative fashion comes from experiments in which a CBFβ-MYH11 allele was created by targeting the MYH11 gene into the murine CBFβ locus.207 Embryos heterozygous for the CBFβ-MYH11 allele died

during mid-embryonic development from a phenotype that was nearly identical to that observed after the loss of either RUNX1 or CBFβ. However, in contrast to embryos deficient in either subunit of the core enhancer complex, expression of CBF-MYH11 resulted in an abnormality in primitive erythropoiesis that consisted of a shift in differentiation toward immature to mid-mature cells. This result suggests that the chimeric product functions not only to inhibit normal RUNX1/CBF activity, but also provides positive signals that result in abnormalities in cell growth and development. In order to address the role of CBF in leukemogenesis, transgenic mouse strains were developed in which the expression of CBF was driven by hematopoieticspecific promoters.208 However, these initial attempts were unsuccessful, likely because the selected transgene promoters did not yield expression of CBF in the appropriate cellular compartment. To circumvent this obstacle, the role of CBF-MYH11 in leukemogenesis was evaluated in chimeric animals derived from ES cells in which MYH11 had been knocked into the CBFβ locus.209 During the first year of life, no leukemia was detected in these animals; however, 84% of animals succumbed to AML 2–6 months after treatment with the mutagen ethylnitrosourea (ENU), whereas control chimeras remained healthy. Leukemic cells containing both eosinophilic and basophilic granules, characteristic of CBF-MYH11-associated AML in humans, were seen. Interestingly, a significant percentage

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of nonmutagenized CBF-MYH11 chimeric mice developed lymphoid tumors after 1 year,210 raising the possibility that perturbation of the RUNX/CBF pathway may play a role in the pathogenesis of some lymphoid neoplasms. These findings indicate that expression of CBFMYH11 alone is insufficient to induce leukemia, suggesting that cooperating mutations are required for full cellular transformation.

The 8;21 translocation AMLs containing the t(8;21)(q22;q22) have been recognized as a distinct AML subset in the WHO classification3 ; in the FAB classification scheme, nearly all AMLs harboring this translocation have M2 morphology and account for over 40% of these cases.211,212 This translocation results in the fusion between RUNX1 and the eight-twenty-one gene (ETO, also known as MTG8 and CBFA2T1) on chromosome 8 (Fig. 11.9). Complex chromosomal translocations suggest that the RUNX1-ETO chimeric product plays a critical role in the establishment of the leukemic clone. This product consists of the N-terminal portion of RUNX1, including its entire RHD, fused in frame to the C-terminal portion of ETO. ETO is the mammalian homologue of the Drosophila gene nervy; two ETO-related genes, MTGX and MTG16, have been identified.213 ETO-deficient mice manifest several gut abnormalities indicating that ETO is required for formation of a normal gut structure during late embryogenesis.214 ETO is not normally expressed within the hematopoietic system, and ETO-deficient mice manifest no hematopoietic abnormalities. The members of this protein family exist as homo- and heterodimeric complexes within the nucleus.215 Interestingly, MTG16 was identified as part of a chimeric RUNX1-MTG16 product that formed as a result of a t(16;21)(q24;q22) seen in rare cases of therapy-related AML or myelodysplastic syndrome (MDS).213 The members of this family are characterized by four regions of high homology (nervy homology regions, NHR) that include a C-terminal zinc-finger motif (Fig. 11.9). As predicted from its structure, RUNX1-ETO retains the ability to bind the enhancer core sequence and to interact with CBF (Fig. 11.11). However, RUNX1-ETO does not function to activate transcription, but instead acts to dominantly repress normal RUNX1-mediated transcriptional activation.216–219 This function is dependent on both the RHD of RUNX1 and sequences contained within the ETO moiety.220 Transcriptional repression appears to be mediated through the direct interaction of ETO with the nuclear corepressor complex (Fig. 11.11). ETO directly binds N-CoR and through formation of the corepressor complex leads to the deacetylation of histones and the repression of tran-

Fig. 11.11 Interaction of the RUNX1-ETO chimeric protein with the nuclear corepressor complex. Abbreviations are defined in the legends to Figs. 11.2 and 11.8.

scription of normal RUNX1/CBF transcription targets.221 In addition, RUNX1-ETO is able to heterodimerize with other ETO family members and thus may also alter the normal functions of these gene products.215,222 Direct proof of the role of RUNX1-ETO in hematopoietic cell transformation has come from gene targeting experiments in which a RUNX1-ETO chimeric gene was created by knocking ETO into the RUNX1 genomic locus.223,224 As predicted from the biochemical activity of this chimeric product, expression of RUNX1-ETO resulted in an embryonic lethal phenotype almost identical to that observed in RUNX1- or CBF-deficient embryos. However, in contrast to the latter, fetal livers from RUNX1-ETO expressing embryos contained dysplastic multilineage hematopoietic progenitors.225 These cells had an abnormally high self-renewal capacity and readily established immortal cell lines in culture. However, these cells were not leukemic when transplanted into syngeneic or immunocompromised recipients. Additional studies in murine and human experimental systems have extended these findings. In order to circumvent the lethality of embryonic expression of RUNX1ETO, transgenic mice were developed in which the expression of RUNX1-ETO is tetracycline-inducible.226 Somewhat surprisingly, expression of RUNX1-ETO in the bone marrow of these mice caused no bone or peripheral blood abnormalities, and mice expressing RUNX1-ETO failed to develop leukemia. Consistent with the above observations, bone marrow cells from these mice manifested enhanced self-renewal capacity during in vitro culture. Importantly, when expression of RUNX1-ETO was extinguished in late-passage cells by removal of tetracycline, the cells manifested enhanced proliferation and differentiation, findings consistent with in vitro analyses of cell lines expressing RUNX1-ETO; 218,227 however, they quickly lost

Molecular genetics of acute myeloid leukemia

their self–renewal capacity. Analysis of human and murine bone marrow cells transduced with a retrovirus expressing RUNX1-ETO have yielded similar findings.228,229 Collectively, the findings indicate that RUNX1-ETO enhances the self-renewal properties of hematopoietic progenitors, whereas it inhibits to some extent their subsequent proliferation and myeloid differentiation. Furthermore, they indicate that while enhancing the self-renewal of HSCs, RUNX1-ETO does not confer a significant growth advantage on HSCs and progenitors. These findings are in keeping with clinical data demonstrating the presence of detectable RUNX1-ETO-positive cells for up to 10 years before the development of leukemia230 and the long-term persistence of a small percentage of RUNX1-ETO-positive HSCs in t(8;21) AML patients who are in durable remission.231 Importantly, these RUNX1-ETO-expressing HSCs retain the capacity to differentiate into B lymphoid, myeloid and erythroid lineages.232 RUNX1-ETO may predispose to leukemia development simply through the expansion of the HSC and progenitor pool, thereby increasing the likelihood of acquiring secondary mutations. However, it is perhaps more plausible that RUNX1-ETO may also render hematopoietic progenitors, through undefined mechanisms, more susceptible to incurring additional mutations (Fig. 11.12). Relevant to this supposition, p14ARF (the human homolog of p19ARF ) has recently been suggested to be a direct transcriptional target of RUNX1/CBF and to be negatively regulated by RUNX1-ETO.233 p14ARF is a component of the p53 tumor suppressor pathway. Its expression is increased by several activated oncogene products including RAS and MYC, and through the inhibition of MDM2-stabilized p53.234 Therefore, loss of p53-mediated growth arrest is impaired in the absence of p14ARF . Thus, the functional loss of p14ARF in myeloid progenitors expressing RUNX1-ETO may extend their lifespan, thereby allowing them to incur the cooperating mutations necessary for leukemia development. Recently developed murine models provide direct evidence that RUNX1-ETO predisposes to leukemia. To circumvent the embryonic lethality of the RUNX1-ETO knock-in strain, mice have been generated that contain a RUNX1-ETO knock-in allele in which expression of the chimeric protein can be conditionally induced using Cre-mediated deletion of a transcriptional stop cassette inserted upstream of the RUNX1-ETO fusion.235 Following induction of Cre recombinase, RUNX1-ETO protein expression was efficiently induced in bone marrow. Somewhat surprisingly, mice expressing the chimeric protein had normal peripheral blood indices and failed to develop leukemia. However, treatment with ENU induced the for-

mation of AML or granulocytic sarcomas (solid tumors consisting of leukemic myeloblasts) in mice expressing RUNX1-ETO, whereas similar treatment of control animals failed to induce such tumors. Analysis of transgenic mice expressing RUNX1-ETO under control of the human MRP8 promoter yielded similar findings.236 These data provide direct proof that while necessary for leukemia development, expression of RUNX1-ETO alone is insufficient to induce leukemia and that full leukemic transformation requires additional genetic mutations (discussed below) that are able to cooperate with the signal generated by RUNX1-ETO (Fig. 11.12). However, the observation that RUNX1-ETO renders leukemic cells growth factor independent, whereas the growth of RUNX1-ETO-expressing bone marrow cells remains factor-dependent in the above described murine model, indicates that genes encoding components of cytokine and growth factor signaling pathways may represent important candidate loci for secondary mutations.

Lessons from RUNX1 alterations induced by other leukemia-associated translocations RUNX1 is fused to the ETO family member, MTG16, as a result of the t(16;21)(q24;q22) seen in rare cases of therapyrelated AML or MDS.213 The structure of this chimeric protein closely resembles RUNX1-ETO, and it is thus predicted to function through a similar mechanism. Similarly, in the t(3;21) seen in rare cases of MDS and blast transformation of CML, RUNX1 is fused to either the EVI1 gene, which encodes a known transcriptional repressor, or to either of two alternative genes of unknown function, EAP and MDS1, which are located adjacent to EVI1 on chromosome 3 and are frequently spliced to EVI1 sequences in the encoded transcript.237 Each of these products results in the replacement of RUNX1 sequences Cterminal to the RHD, thereby abolishing the ability of the resultant fusion molecules to activate transcription. By contrast, the t(12;21) seen in pediatric ALL results in a chimeric product that contains the helix-loop-helix dimerization domain of TEL linked in frame to almost the entire RUNX1 product.130,238–240 Although this fusion protein retains the activation domain of RUNX1, its expression results in a dominant inhibition of RUNX1/CBF-mediated transcription.241 Thus, the recurrent ability of these translocation products to directly inhibit RUNX1/CBF-mediated signals points to the importance of this mechanism in leukemogenesis. Finally, recent studies indicate that the effects of RUNX1 chimeric oncoproteins are highly dependent on the cellular context in which they are expressed. Transplantation of retrovirally transduced bone marrow

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Fig. 11.12 Molecular pathogesis of AML containing the t(8;21) in which the fusion oncoprotein RUNX1-ETO is expressed. Experimental evidence indicates that expression of RUNX1-ETO alone is insufficient for leukemogenesis, although RUNX1-ETO-positive hematopoietic progenitors may have enhanced self-renewal. Acquisition of additional genetic mutations, for example in RAS and FLT3, leads to development of overt leukemia.

hematopoietic progenitors expressing TEL/RUNX1 into recipient mice induced the accumulation of B-cell progenitors with enhanced self-renewal properties.242,243 This effect of TEL/RUNX1 was specific for B-cell progenitors as it has no discernible effect on myelopoiesis. As discussed above, RUNX1/ETO has been exclusively associated with AML, despite the fact that the t(8;21) is thought to occur in a multipotential hematopoietic progenitor. Taken together, these findings suggest that the biologic features of RUNX1associated leukemias are determined to a large extent by cell context-specific properties of a given RUNX1 chimeric oncoprotein rather than merely reflecting the cell type in which the chromosomal translocation initially occurs. Thus, intrinsic and cell context-dependent differences in the way these translocation products inhibit RUNX1/CBF function are likely to account for the biologic and clinical heterogeneity of these leukemias.

Role of RUNX1 mutations in AML In addition to translocations targeting the RUNX1 gene, recent reports have demonstrated that point mutation

and intragenic deletions of RUNX1 may also contribute to leukemogenesis. Through the analysis of patients with de novo and secondary AML, as well as a several chronic myeloproliferative disorders, several somatically acquired point mutations in RUNX1 have been identified in leukemic cells from these patients.244–246 Most of these reside within the RHD and include both missense mutations and nonsense mutations, the latter causing premature termination of the RUNX1 protein. Most of the missense mutations target amino acid residues within the RHD that are predicted to effect sequence-specific DNA base recognition.247,248 Not surprisingly, these mutations reduce or abolish the DNA-binding activity and transcriptional activation of RUNX1; however, most of these mutants can still heterodimerize with CBF.244,245 By contrast, RUNX1 mutants resulting from premature termination generally lack both DNA-binding and heterodimerization activities. An intriguing observation that has emerged from these studies is that the incidence of RUNX1 RHD point mutations is particularly high in poorly differentiated AMLs (AML-M0 subtype by French-American-British criteria).246

Molecular genetics of acute myeloid leukemia

Further strengthening the role of these point mutations in leukemogenesis is the recent identification of mutations in RUNX1 as the cause of a rare clinical entity, familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). FPD/AML patients characteristically manifest moderate thrombocytopenia as well as functional platelet abnormalities from birth.249–252 Additionally, affected individuals have a markedly increased risk for the development of acute leukemia, primarily AMLs, which typically occurs in adulthood. FPD/AML is an autosomal dominant disorder and is linked to chromosome 21q22.1– 22.2.251 Analysis of several FPD/AML pedigrees revealed the presence of either missense or nonsense mutations or intragenic deletions in one of the RUNX1 alleles, which cosegregated with the disease in each of the six pedigrees examined.253 These mutations resulted in either the complete deletion of one RUNX1 allele, premature termination of the coding sequence yielding a truncated RUNX1 protein, or mutation of critical amino acid residues within the RHD that facilitate DNA binding.247,248 No mutations were identified in the remaining RUNX1 allele in any of the cases analyzed. While some of the mutant RUNX1 proteins are predicted to function in a dominant-negative manner, several observations suggest that this may not be the operative mechanism. As discussed earlier, mice expressing dominant-negative forms of RUNX1 uniformly manifest embryonic lethality223,224 ; whereas patients with FPD/AML are born at the expected frequency. Additionally, steadystate levels of mutant RUNX1 transcripts were much lower than those of wild-type RUNX1, presumably reflecting the reduced stability of truncated mRNA transcripts. Lastly, one pedigree contained an intrachromosomal deletion that eliminated one RUNX1 allele. Although reduced expression of the remaining RUNX1 allele through other mechanisms needs to be excluded, these findings suggest that haploinsufficiency for RUNX1 is sufficient to promote leukemogenesis in FPD/AML. Thus, RUNX1 mutations appear to operate through a mechanism distinct from that of the classic tumor-suppressor paradigm in which there is homozygous gene inactivation. The exact mechanisms by which reduced RUNX1 expression predisposes to AML are unknown. Solution of the crystal structure of the RUNX1 RHDCBF-DNA ternary complex has provided insight into the molecular basis for the effects of these mutations on the biologic function of RUNX1.247,248 The RHD consists of a 12-stranded -barrel in which the long axis is oriented perpendicular to the DNA helix. Several DNA contact points of the RHD are located within the E -F loop and the Cterminal portion of the RHD. Many of the RUNX1 point mutations described above occur within these regions of

the RHD and would therefore be predicted to inhibit or abolish DNA binding.

Secondary mutations in APL and core-binding factor leukemia The above translocation-associated fusion proteins clearly play an essential role in the development of leukemia. However, through the analysis of human leukemic cells and murine leukemic models, it is now well established that in the majority of cases, leukemogenesis is a multistep process and that cooperating genetic lesions, in addition to expression of a translocation-generated chimeric protein, are required for development of a complete leukemic phenotype (Fig. 11.12). In many cases of AML, mutations have been identified in both a transcription factor that normally regulates hematopoiesis and in a protein tyrosine kinase. These observations have led to the hypothesis that AML results from cooperation of at least two classes of mutation, one that impairs hematopoietic cell differentiation and a second mutation that confers a survival or proliferative advantage.254 Mutations in several signaling pathways have been identified in AMLs, with mutations in the RAS proteins and two receptor tyrosine kinases, FLT3 and KIT, being the most extensively studied. The fms-like tyrosine kinase 3 (FLT3) gene is preferentially expressed in hematopoietic stem/progenitor cells. It is a member of the class III receptor tyrosine kinase family, which also includes KIT, FMS and the two PDGF receptors. FLT3 contains an extracellular domain, a juxtamembrane (JM) domain just within the cytoplasmic membrane and two tandem tyrosine kinase domains.255 FLT3 activates several downstream signaling molecules through direct phosphorylation or association, including phospholipase C- , the p85 subunit of phosphotidyl inositol (PI3 kinase), VAV, CBL, SHP2, STAT 5A, and STAT 5B. Mutations in FLT3 are among the commonest in AML and include an internal tandem duplication (ITD) in the JM domain, occurring in about 25% of AML patients, as well as a point mutation in the kinase activation loop, occurring in approximately 7% of patients (see Gilliland and Griffin255 and citations therein). The ITD is of variable length, but the open reading frame of the native protein is invariably retained. Both mutations endow FLT3 with constitutive tyrosine kinase activity, and stable expression of either mutant protein renders hematopoietic cells growth factor independent.256 ,257 Indeed, mice receiving bone marrow cells expressing mutant FLT3 develop an oligoclonal myeloproliferative disorder characterized by splenomegaly and leukocytosis.258 The frequency of FLT3 mutations in AML is somewhat higher in adults than in children, largely

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due to the rarity of these mutations in children younger than 10 years of age. Furthermore, the frequency of FLT3 mutations varies significantly among genetic subtypes of AML, with the highest incidence in APL.259,260 Most studies in children have found that FLT3 mutations are associated with higher leukocyte counts and a significantly poorer survival.261–264 Activating mutations of the receptor tyrosine kinase KIT have been shown to play a critical role in the pathogenesis of mast cell disorders and gastrointestinal stromal tumors (GISTs).265 More recently, KIT mutations have also been identified in AML leukemic blasts.266,267 In children and adults, the incidence of KIT mutations in AML is low overall, although some studies have found a high incidence of mutations in the core-binding factor leukemias.268 The RAS proteins regulate signaling pathways that are critical to the maintenance of normal cell growth.269 The importance of the RAS-regulated pathways is highlighted by the high incidence of activating mutations in the RAS proteins. In pediatric AML, mutations in either N-RAS or K-RAS are frequently identified, with an incidence of 21% to 37%.267,270 Interestingly, a recent analysis of pediatric AMLs revealed that the frequencies of RAS mutations and FLT3 ITD was approximately 48% and 10%, respectively, in cases containing either inv(16) or t(8;21).271 By contrast, RAS and FLT3 mutations were detected in 18% and 50%, respectively, of morphologically matched, non-corebinding factor AMLs. These findings suggest that the specific type of cooperating mutation required varies with the presence and biologic properties of the fusion protein. Our understanding of the impact of these cooperating mutations on disease development and prognosis is evolving. While the identification and analysis of these mutations should enhance our understanding of the pathogenetic mechanisms operative in some AMLs, additional efforts are clearly needed to better define the full spectrum of cooperating mutations in translocation-associated AMLs. The application of nondirected methods, such as retroviral insertional mutagenesis, should permit the identification of potentially novel mutations that cooperate with these translocation chimeric oncoproteins. Indeed, a recent retroviral mutagenesis study in a mouse model of CBF-MYH11-associated AML identified 54 candidate genes that may cooperate with CBF-MYH11 to induce acute leukemia, including several that had not been identified in previous retroviral screens and may thus cooperate specifically with CBF-MYH11.272 In summary, alterations of the RUNX1/CBF transcription factor complex are the most common genetic events implicated in the pathogenesis of acute leukemia. Through biochemical analyses, it is now apparent that

these chimeric core-binding factor proteins perturb the normal function of the RUNX1/CBF complex, most frequently through the aberrant recruitment of corepressor complexes, thereby inhibiting the transcription of target genes. In other instances, repression of RUNX1/CBF transactivation is effected through the sequestration of wild-type RUNX1 or through the generation of dominantnegative RUNX1 molecules. Although a significant amount of information has been learned about the normal role of this complex in hematopoietic development, we still have only an incomplete understanding of how alterations in the complex lead to the initiation of leukemia. Critical to our future understanding will be the further elucidation of the normal biochemical mechanisms through which this transcription factor complex regulates transcription and how the translocation-encoded chimeric products and RUNX1 point mutants alter these interactions. It is now apparent that cooperating mutations are critical to the development of core-binding factor leukemias. Therefore, comprehensive analysis of the transcriptional cascade initiated by RUNX1/CBF and the identification of the pathways that are perturbed during leukemogenesis will be essential for the identification of these cooperating mutations and ultimately for the development of novel, molecularly-targeted therapeutics. Finally, approaches that either disrupt the formation or stability of the RUNX1 or CBF chimeric proteinbound corepressor complexes, or that directly inhibit the activity of the corepressor, may lead to a reversal of the transformed phenotype, similar to that observed following targeted therapy in t(15;17) APL.

Acute myeloid leukemias with alterations of MLL Structural alterations involving chromosome 11, band q23, are among the most common cytogenetic abnormalities seen in AML, occurring in approximately 6% to 8% of de novo AMLs and in up to 85% of secondary AMLs that develop after exposure to topoisomerase II inhibitors.273–276 In addition, chromosome 11q23 is also the most frequently rearranged site detected in acute lymphoblastic leukemias arising in infants.277 Molecular cloning of a large number of these translocations has demonstrated that in the majority of cases the 11q23 target is the mixed lineage leukemia (MLL) gene (also referred to as HRX, ALL-1, and HTRX1).278–283 Over 40 different chromosomal loci can participate in these 11q23 translocations, representative examples of which are detailed in Table 11.1; all the reported MLL translocations in AML and ALL, many of which are rare, have been catalogued and discussed in recent reviews.284–286 These translocations yield

Molecular genetics of acute myeloid leukemia

Table 11.1 Representative AML-associated translocations targeting

65% homology to the comparable region of trx, which contains a 130 amino acid C-terminal segment, referred to as the SET domain, that is highly homologous (34%–75% Translocations Fusion protein Function/homology identity) to domains in the Drosophila genes: suppressor of position-effect variegation [Su(var)3–9], enhancer of t(9;11)(p22;q23) MLL-AF9 RNA polymerase II zeste [E(z)], and trithorax.293 This domain possesses hist(11;19)(q23;p13.1) MLL-ELL RNA polymerase II tone methyltransferase activity and may play an important elongation factor t(11;19)(q23;p13.3) MLL-ENL RNA polymerase II role in the regulation of gene expression of target genes of t(1;11)(p32;q23) MLL-AF1p Homology to MLL. poly(A)ribonuclease In addition to these regions of homology to trithort(6;11)(q21;q23) MLL-AF6q21 Forkhead ax, MLL contains three AT-hook motifs that were origitranscription factor nally described in the high-mobility group (HMG) prot(X;11)(q13;q23) MLL-AFX Forkhead teins. These motifs mediate binding to AT-rich sequences transcription factor within the minor groove of DNA. Adjacent to the AT hooks t(10;11)(p12;q23) MLL-AF10 PHD finger and is a region that contains an acid-basic repeat that is simleucine zipper ilar to those found in several RNA-binding proteins and t(11;17)(q23;q21) MLL-AF17 PHD finger and in DNA methyltransferases, enzymes that produce fully leucine zipper methylated DNA from a hemimethylated substrate. This t(11;16)(q23;q13.3) MLL-CBP HAT coactivator t(11;22)(q23;q13) MLL-p300 HAT coactivator domain is followed by a 47 amino acid cysteine-rich domain t(6;11)(q27;q23) MLL-AF6 RAS-binding protein that is directly homologous to the noncatalytic domain of t(1;11)(q21;q23) MLL-AF1q Cloned – no DNA-methyltransferase (MT). In DNA methyltransferases, homology both of these latter regions function in the discrimination of methylated from unmethylated DNA.294 Lastly, immediately N-terminal to the central zinc-finger domains is a a chimeric message in which the 5 portion consists of lysine-rich region with homology to the C-terminal tail of MLL sequence fused in-frame to the 3 sequence derived the late histone H1. from the partner gene encoded on the reciprocal chromoIn addition to these structural domains, several funcsome (Fig. 11.13).287–289 In addition to these translocationtional domains have been identified within MLL. These induced alterations, MLL has been found to undergo parinclude a region that partially overlaps with the AT-hook tial internal duplications in rare cases of AML that have motifs and mediates binding to both DNA with a cruciform either a normal karyotype or trisomy of chromosome 11 three dimensional structure295,296 and to scaffold attach(Fig. 11.14).290 Due to space limitations, the molecular ment regions within the nuclear matrix.296 Two transcripmechanisms by which translocations target the MLL locus tional regulatory domains have also been identified. These will not be discussed. include a strong transcriptional activation domain within the C-terminal third of the molecule, and a weak transcriptional repression domain that partially overlaps with the The normal functions of MLL methyltransferase and H1-like domains.295 An additional factor regulating the activity of MLL came MLL is an exceptionally large molecule that contains 3969 to light with the recent demonstration that MLL is proamino acids and has a predicted molecular mass of 431 kDa teolytically processed into N-terminal 320 kDa (N320) (Fig. 11.13). It shares several regions of homology with the and C-terminal 180 kDa (C180) fragments by a novel Drosophila trithorax protein, trx, which plays a critical role endopeptidase, Taspase1 (Fig. 11.13).297–299 After processin segment determination during development, in part, by ing, the N320 and C180 fragments associate with each controlling the sustained expression of the antennapedia other through domains present in their amino termini, and bithorax homeotic gene complexes.291,292 The regions including the SET domain in the C-terminus of C180. of homology between MLL and trx include two centrally This interaction has important biochemical consequences placed structural domains that contain several unusual since the resultant N320/C180 heterodimer has dramatizinc-finger-like motifs with features similar to PHD, LIM, cally enhanced stability, when compared to the full-length and RING fingers (Fig. 11.13). These regions are believed MLL protein, and it may regulate their subnuclear localto mediate critical protein–protein interactions that are ization as well. Taspase1-mediated proteolysis of MLL is required for the normal function of these molecules. In also functionally important, since inhibition of Taspase1 addition, the C-terminal 215 amino acids of MLL show the MLL gene

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Fig. 11.13 (A) The MLL protein and the organization of the breakpoint cluster region. MLL is organized into an N-terminal AT-hook domain, regions with homology to RNA-binding proteins, DNA-methyltransferases (MT), histone H1 (H1), plant homeodomain (PHD)-type zinc fingers, and SET domain. The breakpoint cluster region is drawn centromeric (Cen) to telomeric (Tel). MLL is proteolytically processed by Taspase1, the details of which are discussed in the text. (B) Schematic of potential molecular mechanisms of transcriptional activation effected by MLL fusion proteins in which the fusion partner is either a nuclear or a cytoplasmic protein. DD, dimerization domain; TAD, transactivation domain; TC, transcription cofactors.

Molecular genetics of acute myeloid leukemia

Fig. 11.14 AML-associated partial MLL internal duplication. Abbreviations are defined in the legend to Fig. 11.13.

Fig. 11.15 Coordinate regulation of the HOX genes during development. Initiation of gene transcription is controlled by the activity of transient-acting factors such as RUNX1/CBF and RAR
, whereas maintenance is regulated by the coordinate activity of the activator MLL and polycomb group family of repressors.

expression largely blocked expression of the 3 HOXA genes, which are expressed earlier in embryonic development, whereas Taspase1 inhibition had no effect on 5 HOXA gene expression.299 The generation and analysis of mice expressing noncleavable forms of MLL and mice deficient in Taspase1 should provide additional insight into the role of proteolysis in the hematopoietic and oncogenic functions of MLL. Taken together, the structural and function domains identified within MLL suggest that its normal physiologic functions are likely to be mediated at the level of DNA or DNA-associated chromatin proteins. Results obtained

from the genetic analysis of both Drosophila trx and mammalian MLL support this hypothesis.300,301 In Drosophila, trx is the founding member of the so-called trithorax group family, which consists of over 40 proteins. Trx functions to maintain the expression of the homeotic genes by retaining chromatin in an active conformation. It counters the repression of the polycomb group (PcG) protein complex which induces chromatin to be packaged into a more compact form that is inaccessible to transcriptional activators thereby repressing gene expression.302,303 Similarly in mice, Mll is required for maintained expression of class I homeodomain (Hox) genes, and counters the repressive effect on Hox gene expression of the PcG homologues Bmi1, mel18, or M33 (Fig. 11.15). Thus, during early embryogenesis in Mll-deficient mice, the pattern of Hox gene expression is normal; however, after the disappearance of transient initiating transcription factors, the expression pattern is disrupted. Specifically, Hox genes whose expression is normally maintained in posterior segments are no longer expressed. As a result, these posterior segments assume the structure of more anterior regions, so called anterior homeotic transformation. By contrast, loss of PcG proteins or the mammalian homologues results in posterior homeotic transformation – that is conversion of anterior regions to posterior structures due to a persistent expression of Hox gene in segments where they are not normally expressed.304–307 Thus, the delicate balance between the pattern of expression of these genes plays an essential role in normal mammalian development. Interestingly, loss of genes from either family also results in subtle alterations

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recent analyses demonstrate that MLL-deficient ES cells are unable to contribute to hematopoiesis in chimeric murine embryos, a defect that is likely attributable to a block in HSC generation in the AGM and other sites of HSC formation.309 The contribution to hematopoiesis by ES cells heterozygous for a MLL null allele was intermediate between wildtype and MLL-deficient ES cells, suggesting that the normal function of MLL is dose-dependent. Biochemical analyses also indicate that MLL domains interact with and recruit several proteins involved in chromatin modeling and remodeling. The SET domain interacts with INI/hSNF5,310 a component of the SWI/SNF chromatin remodeling complex. More recently, the SET domain was shown to possess intrinsic histone methyltransferase activity, methylating histone H3 on the lysine 4 residue, and may preferentially methylate acetylated histones.311 As methylation inhibits histone deacetylation catalyzed by such complexes as the nucleosome remodeling and deacetylase (NuRD) complex,312 the SET domain may function critically in the chromatin remodeling and gene expression effected by MLL, possibly through the inhibition of PcG complex-mediated histone deacetylation. The MLL repression domain has recently been shown to interact with HDAC1, a member of the histone deacetylase family, and the corepressor C-terminal binding protein. Interestingly, the MLL repression domain also interacts with two PcG proteins, HPC2 and BMI-1.313 The factors that dictate whether MLL functions as either a transcriptional activator or repressor at a given target gene locus remain to be determined.

Leukemia-associated translocation of MLL

Fig. 11.16 Schematic representation of MLL chimeric proteins resulting from AML-associated translocations. Arrows indicate the position of breakpoints. NLS, nuclear localization signal; FKHR, forkhead transcription domain; Leu, leucine; NR, nuclear receptor-binding domain; CREB, cyclic AMP response element binding protein interactive domain; B, bromo-domain; HAT, histone acetylase; EIa, adenovirus EIA interactive region.

in hematopoietic development.300,301,304,308 The loss of MLL results in a decrease in the number of multipotential myeloid and macrophage progenitors, without affecting the ability of these cells to differentiate.301 However, given the pleiotropic effects of MLL deficiency, these effects may be due to hematopoietic cell-independent factors. Indeed,

Cytogenetic studies of the AML-associated 11q23 translocations have implicated the der(11) chromosome as the critical lesion. This derivative encodes a chimeric gene that consists of the 5 portion of MLL fused to the 3 portion of target genes from the reciprocal chromosomes. As described above, over 40 11q23 translocations targeting MLL have been characterized (Table 11.1). The structure of representative AML-associated fusion proteins are illustrated in Fig. 11.16. In each of these translocations, the breaks in MLL localize to a breakpoint cluster region of 8.5 kb located between exons 5 and 11 (Figs. 11.13 and 11.16). Rearrangements in this region result in a chimeric protein that retains the AT-hooks, RNA-binding, and methyltransferase motifs, but deletes the zinc-fingers and transactivation and SET domains. Moreover, the portion of MLL contained within the chimeric product include the binding sites for cruciform DNA and the scaffold attachment site, and the majority of its repression domain.

Molecular genetics of acute myeloid leukemia

The consistent involvement of MLL in these 11q23 translocations clearly attests to the critical role MLL alterations must play in hematopoietic cell transformation. The fact that the translocations result in the formation of inframe fusion proteins, however, suggests that novel functions must be provided by the reciprocal partner. To gain a better understanding of the role of MLL fusion proteins in leukemogenesis, early work focused on the identification of the functions provided by the fusion partner. Although the cloning of a large number of the reciprocal partners revealed a variety of different structural motifs, no consistency of function has emerged. Nevertheless, critical insights have come from this work. Partner genes with known function include both nuclear and cytoplasmic proteins and can be arbitrarily subdivided into several groups. It is important to point out at the outset that like the other AML-associated fusion products that we have discussed above, the MLL chimeric proteins are likely to lead to alteration in the normal functions of both of the reciprocal partners. Members of the first subgroup of genes involved in 11q23 translocations encode proteins that are similar to molecules involved in RNA metabolism and include AF-9, ENL, ELL, and AF-1p (Table 11.1 and Fig. 11.16). AF-9 and ENL are the targets of the two most common AML-associated 11q23 translocations, t(9;11)(p22;q23) and t(11;19)(q23;p13.3), respectively.314,315 The encoded proteins share 82% identity in both their N-terminal 140 amino acids and their C-terminal 67 amino acids, are serine/proline rich proteins containing nuclear localization signals, and have structural features in common with RNA polymerase II.288,316 Moreover, ENL contains a transcriptional activation domain that is localized to its C-terminal portion, the region retained within the MLL-ENL chimeric product.317 The high degree of similarity between these molecules suggests that they may contribute a common function to the MLL fusion protein. Consistent with this notion, MLL-ENL, but not MLL, interacts with a novel SWI/SWF chromatin remodeling complex, suggesting that MLL-ENL may perturb gene expression through the aberrant alteration of chromatin structure. The next member of this group, ELL, the target of the t(11;19)(q23;p13.1), has no direct sequence homology to other members, but normally functions as a novel RNA polymerase II elongation factor.283,318,319 ELL increases the catalytic rate of RNA polymerase II by suppressing transient pausing of the polymerase at many sites along the DNA.320 ELL contains a RNA polymerase II interaction domain and an elongation activation domain. As a result of the translocation, a portion of the ELL interaction domain is deleted, resulting in a molecule that continues to bind RNA polymerase II but has an altered

catalytic activity. Thus, structural changes in ELL that result from this translocation may lead to global alterations in cellular transcription. The last member of this subgroup, AF-1p, is the target of the t(1;11)(p32;q23), a rare translocation seen in AML-M0. AF-1p encodes a protein with low homology to the yeast poly(A) ribonuclease.288,321 Little is known about its normal function or the active domains that it contributes to the MLL-AF-1p fusion protein. The next subgroup contains two proteins, AF-6q21 and AF-X, which are members of the forkhead family of transcription factors, and are involved in the t(6;11)(q21;q23) and t(X;11)(q13;q23), respectively.322,323 Each of these proteins shows a high level of homology to FKHR, the chromosome 13 target of the t(2;13) in alveolar rhabdomyosarcoma.324–326 In each of these translocations, the breakpoints occur within the forkhead DNA binding domain and result in retention of only the C-terminal portion of the molecule. In FKHR, this portion of the molecule has been shown to contain a strong transcriptional activation domain.327 Whether an activation domain is retained in the MLL fusion products remains to be determined. The rare translocation t(10;11)(p12;q23) targets the AF-10 gene.328–330 This gene encodes a protein that contains a N-terminal PHD-type zinc-finger similar to those in MLL, and a C-terminal leucine zipper that probably mediates protein-protein interactions. Moreover, the sequence and position of these domains are conserved in BR140, a protein involved in the function of basal transcriptional activators. The MLL-AF-10 fusion protein formed as a result of this translocation lacks the PHD domain but retains the C-terminal leucine zipper. The cloning of the MLL-CBP chimera encoded by the t(11;16)(q23;p13.3)331–334 and the MLL-p300 fusion protein encoded by the t(11;22)(q23;q13)335 provide interesting insights into the potential role of the partner genes in the MLL fusion proteins. The CREB (cAMP response element-binding protein) binding proteins (CBP and p300) are known transcriptional coactivators that contain intrinsic histone acetylase activity.35 As described above, these proteins function as part of a multisubunit complex that is involved in transcription activation induced by a variety of transcription factors including RAR
. The MLL fusion proteins formed as a result of these translocations retain the regions of CBP and p300 required for histone acetylase activity. Thus, it is likely that these chimeric molecules induce aberrant transcriptional regulation of target genes normally regulated by MLL. This observation suggests that the other fusion products may function in a similar manner. Interestingly, CBP has also been found to be the target of another rare AML-associated translocation, t(8;16)(p11;p13.3),336 which generates a chimeric protein

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through fusion of the N-terminal portion of MOZ, a putative acetyltransferase, and CBP. The MOZ-CBP chimeric protein is believed to function by altering the transcriptional regulation of critical genes involved in hematopoiesis. In addition to these distinct subgroups, several additional partners have been identified that lack homology with any of the other cloned partners. These include AF-6 a putative RAS-binding protein that is the target of the t(6;11)(q27;q23),337 and AF-1q, the target of the t(1;11)(q21;q23), which lacks homology to any known protein sequences.338 Interestingly, the latter translocation results in the incorporation of only minimal sequences from AF-1q into the chimeric product. The role that such a minimal contribution by a MLL fusion partner may play in defining the oncogenic properties of chimeric MLL proteins will be discussed below.

Lessons from genetic models of MLL-induced leukemia A direct role for MLL fusion proteins in the pathogenesis of AML has been demonstrated with use of experimental murine models. The in vivo expression of MLAF9, MLL-ENL, and MLL-GAS7, through an MLL knockin strategy339 or transplantation of retrovirally transduced bone marrow cells,340–343 led to the development of AML. The latency period prior to the development of leukemia was of variable duration, suggesting that in some instances cooperating mutations were required for overt leukemic development. In the case of MLL-ENL and MLL-AF10, both the DNA-binding domains of MLL and the transcriptional activation domain of the fusion partner were required for transformation, consistent with the concept that each component contributes a critical function to transformation.340,341 Interestingly, when enriched multipotent progenitors were transduced with MLL-GAS7, the resultant leukemias expressed markers associated with different hematopoietic lineages.342 This suggests that the so-called lineage infidelity frequently observed in MLLassociated leukemias may simply reflect the fact that the oncogenic event occurred in a multipotent progenitor. Taken together, these studies unequivocally demonstrate a direct role for the MLL chimeric proteins in hematopoietic cell transformation. These genetic systems will undoubtedly serve as important tools to aid in the elucidation of the molecular pathways that are altered as a result of the expression of MLL chimeric proteins.

Molecular mechanism of transformation A plethora of mechanisms have been proposed to explain how the various MLL chimeric proteins might lead to transformation. However, no unifying model was appar-

ent until the recent demonstration that dimerization of MLL may be critical for the oncogenic activity of those MLL chimeric proteins in which the fusion partner is normally a cytoplasmic protein. As described above, transplantation of retrovirally transduced murine bone marrow cells expressing either MLL-GAS7, MLL-AF1p or MLLgephrin induces lethal acute leukemia in recipient mice with high penetrance.344,345 Moreover, these cells manifest increased self-renewal in in vitro colony-forming assays. Each of these proteins contains domains that mediate protein–protein interaction, specifically coiledcoil domains in the case of GAS7 and AF1p. Bone marrow cells expressing MLL chimeric proteins containing only the protein–protein interaction domains of the fusion partner showed increased self-renewal and colony forming activity comparable to that of cells expressing the full-length fusion proteins. Importantly, transplantation of these cells induced acute leukemia in recipient mice, albeit with a longer latency and decreased penetrance when compared to the full-length MLL fusion proteins. Biochemical analyses revealed that these domains contributed by the fusion partner mediate oligomerization of the MLL fusion proteins and that oligomerization is required for the transcriptional activation mediated by these fusion proteins. Similar results were obtained when dimerization of MLL was induced through its fusion to the FK506 binding protein.346 Importantly, MLL-FK506 binding to DNA regulatory sequences in the HoxA9 gene was significantly enhanced by dimerization.346 These findings may explain earlier studies in which leukemia was observed in mice in which the bacterial enzyme -galactosidase (lacZ) gene was fused to MLL and knocked into the MLL locus.347 The development of leukemia in these animals was interpreted as indicating that truncation alone imparted oncogenic properties to MLL. Given that lacZ forms tetramers, a more plausible explanation that takes into consideration the more recent findings described above is that lacZ confers oncogenic properties on the MLL-lacZ fusion protein through provision of an oligomerization domain. Based on these findings, distinct mechanisms have been proposed for MLL chimeric proteins fused to nuclear and cytoplasmic partners (Fig. 11.13).344,348 In the former, the MLL fusion protein may function efficiently as a monomer, binding to DNA regulatory sequences via MLL motifs, and relying on the nuclear fusion partner to recruit transcriptional coactivators. MLL fusion proteins containing cytoplasmic partners similarly bind DNA through MLL motifs. However, the oligomerization of the MLL chimeric protein mediated by motifs in its fusion partner leads to the recruitment of transcriptional coactivators not normally

Molecular genetics of acute myeloid leukemia

recruited by MLL to normal target transcriptional targets of MLL. In approximately 10% of cases, there is internal duplication of the MLL gene, leading to duplication of the MLL domains required for the enhanced cell renewal induced by MLL dimerization, including the CXXC domain (Fig. 11.14).349–351 In these cases, duplication of these critical domains may mimic the effects of the dimerization which occurs in some MLL fusion proteins, ultimately leading to the aberrant recruitment of transcriptional coactivators. An important question regarding the oncogenic properties of MLL fusion proteins is whether these chimeric proteins target the same downstream signaling pathways as wild-type MLL and, if not, does the expression of a novel set of genes induced by MLL fusion proteins account for their transforming function. As discussed above, MLL is required for maintenance of HOX gene expression. The HOX genes are expressed in hematopoietic progenitors; however, their expression is transcriptionally downregulated concurrent with myeloid cell differentiation.352–355 Aberrant expression of several HOX genes has been demonstrated to perturb normal hematopoiesis and, in some instances, induce the transformation of primary myeloid progenitors.356–363 Therefore, altered regulation of HOX gene expression by MLL fusion proteins represents a plausible mechanism to account for their oncogenicity. Consistent with this hypothesis, recent studies demonstrate that expression of several MLL fusion proteins in murine bone marrow progenitors induces Hox gene overexpression.344,346,355,364–366 However, defining the role that such overexpression plays in MLL leukemogenesis has been rather problematic. An initial report indicated that while MLL-ENL could immortalize wild-type murine bone marrow cells in vitro, it was incapable of doing so when either Hoxa9- or Hoxa7deficient bone marrow cells were used.364 Additionally, transplantation of MLL-ENL-expressing, Hoxa9-deficient bone marrow cells did not induce acute leukemia in recipient mice, whereas similar MLL-ENL-expressing, wild-type cells caused leukemia with 100% penetrance. Others similarly found that forced expression of Hoxa9 and the Hox coregulator Meis1 could substitute for the transforming properties of MLL-ENL.365 These data suggest that HOXA9 and HOXA7 are critical downstream targets for MLL-ENL. However, subsequent studies with other MLL fusion proteins, including MLL-GAS7 and MLL-AF9, have not demonstrated a general requirement for these HOX genes in MLL leukemogenesis, since recipients receiving bone marrow cells expressing these latter MLL fusion proteins progressed to AML irrespective of whether they also expressed Hoxa9 or Hoxa7.355,366 In one of these studies, deficiency of either of

these Hox genes did reduce the penetrance and influence the immunophenotype of the acute leukemias that developed in recipient mice.355 Currently, the reason for this disparity in HOX gene dependence between the various MLL fusion proteins is not clear, but it may be attributable to subtle differences in the methods used to prepare bone marrow cells for retroviral transduction. Alternatively, this differential requirement for HOX gene expression may reflect intrinsic differences in the transforming properties of the MLL fusion proteins analyzed. HOX-dependent oncogenesis is unlikely to simply reflect the propensity of a MLL fusion protein to oligomerize, since MLLGAS7 requires oligomerization for its oncogenic properties, whereas MLL-AF9 and MLL-ENL both likely exist as monomers. If the deficiency of a given HOX gene can be fully compensated for by other HOX family members, these findings suggest that the targeted inhibition of a single HOX gene product would be unlikely to yield an effective therapeutic approach for the treatment of MLL leukemias. The deregulation of HOX gene expression is a recurrent theme that has been implicated not only in leukemias with MLL rearrangements, but also in leukemias with alteration of RAR
and RUNX1/CBF (Fig. 11.15). Each of these transcriptional regulators is directly involved in controlling the normal pattern of HOX gene expression. To fully understand the role of HOX gene expression in the leukemias produced as a result of alterations in RAR
, RUNX1/CBF, and MLL it will be necessary to quantitatively define the changes in HOX gene expression that result from these genetic alterations and to assess systematically the effect on leukemogenesis of the loss of expression of single and multiple HOX genes. Comparisons between these specific types of leukemia should provide valuable insights into the underlying mechanisms of hematopoietic transformation. In summary, our understanding of how MLL alterations contribute to leukemia remains incomplete. Similarly, why this gene is so frequently altered in acute leukemias of both myeloid and lymphoid lineage remains a mystery. Further characterization of the signaling pathways through which MLL normally functions should provide valuable insights into normal hematopoietic development and leukemogenesis. Finally, characterization of the mechanisms leading to MLL rearrangements, and the identification of agents that induce such rearrangements, should yield critical insights into the underlying cause of these leukemias.

Acute megakaryoblastic leukemia Acute megakaryoblastic leukemia (AMKL) is an uncommon type of leukemia occurring almost exclusively in children and representing 1% to 15% of AMLs.367,368 This

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Fig. 11.17 Molecular pathogenesis of AMKL arising in the setting of Down syndrome (DS) or associated with the t(1;22). In DS-AMKL, expression of a mutant form of GATA1 (GATA1s) leads to polyclonal expansion of a pool of megakaryocytic progenitors that manifests clinically in some DS infants as transient myeloproliferative disorder (TMD). The presumed acquisition of additional genetic mutations subsequently leads to development of AMKL; the identity of these collaborating mutations is unknown. In t(1;22)-associated AMKL, the translocation is acquired in a hematopoietic progenitor. It is currently unknown if RBM15-MKL1 expression alone is sufficient for leukemogenesis or whether secondary mutations are required for full leukemic development.

heterogeneous disorder has at least three subtypes, each with distinct clinical and biologic behaviors: AMKL associated with Down syndrome (DS), AMKL with t(1;22)(p13;q13), and AMKL lacking t(1;22) and occurring in non-DS patients; the latter is a heterogeneous, poorly characterized group of disorders and will not be discussed further. Children with DS have a 10- to 20-fold increased risk of developing acute leukemia, particularly AMKL.369,370 In addition, approximately 10% of infants with DS develop transient myeloproliferative disorder (TMD; also known as transient leukemia or transient abnormal myelopoiesis), a clonal myeloproliferative condition that develops either prenatally or during early infancy and is characterized by the proliferation of myeloid cells, primarily megakaryoblasts.371 As implied by its name, this disorder is evanescent and usually regresses during the first 3

months of life; however, approximately 20% of DS children with TMD will ultimately develop AMKL (DS-AMKL). Recent studies indicate that mutations in the transcription factor gene GATA1 play an important role in the pathogenesis of these DS-associated megakaryoblastic disorders (Fig. 11.17). GATA1 is located at Xp11 and encodes a zinc-finger transcription factor whose expression is limited to megakaryocytes, erythroid cells, eosinophils, and multipotential hematopoietic progenitor cells.372 Mice with megakaryocyte-specific deficiency of GATA1 develop bone marrow megakaryocytic hyperplasia, and GATA1-deficient megakaryocytic progenitors manifest marked hyperproliferation in colony-forming assays, indicating that GATA1 plays a critical role in regulating megakaryopoiesis.373 Mutations in GATA1 have recently been identified in approximately 85% of cases of DS-AMKL.374–379 These mutations primarily target exon 2, the first coding exon of

Molecular genetics of acute myeloid leukemia

GATA1, and include deletions, insertions, splice mutations and nonsense/missense point mutations, all of which block the expression of a full-length GATA1 mRNA.377–379 Consequently, a truncated form of GATA1 (GATA1s) is expressed either through the use of a cryptic translational start codon or alternative RNA splicing. GATA1s retains DNA-binding activity and interaction with cofactor proteins such as FOG1; however, it is a poor transcriptional activator since it lacks the N-terminal activation domain found in fulllength GATA1. Mutations in the GATA1 gene were restricted to DS-associated AMKL and were not seen in other subtypes of AMKL or in other leukemias in DS patients.378 Mutations in GATA1 have similarly been detected in over 90% of cases of TMD.374–380 Importantly, in those patients who developed both disorders, identical GATA1 mutations have been detected in a given patient in both the TMD and AMKL blasts.374,376,377,381 These observations confirm a clonal relationship between TMD and DS-AMKL, and indicate that the latter evolves from latent TMD clones that persist following its clinical regression. These findings indicate that mutations in GATA1 are early, likely initiating events in the pathogenesis of DS-associated megakaryoblastic proliferative disorders (Fig. 11.17). Furthermore, the presence of GATA1 mutations in both TMD and AMKL indicates that the pathogenesis of DS-AMKL is a multistep process and that additional genetic lesions are required for the development of a fully malignant phenotype. The mechanism by which reduced GATA1 transcriptional activity endows megakaryocytic cells with enhanced proliferation and the identity of the relevant cooperating mutations in AMKL remain to be clarified. Furthermore, GATA1 mutations have only been detected in patients with constitutional or acquired trisomy 21. This suggests that such mutations confer a proliferative or survival advantage only in the context of trisomy 21. If so, what are the gene products encoded on chromosome 21 that cooperate with GATA1 to induce the dysregulated megakaryoblastic proliferation seen in TMD and AMKL? In summary, these data suggest that the pathogenesis of DS-AMKL is indeed distinct from AMKL arising in karyotypically normal children, which may account, in part, for the dramatic difference in the AMKL survival rates in these two patient populations. AMKL with an associated t(1;22)(p13;q13) (T-AMKL) is a disease of infants and young children. This disorder is typically accompanied by significant bone marrow fibrosis and organomegaly and has a poor prognosis with a median survival of only 8 months.382–384 The t(1;22)(p13;q13) characteristic of this malignancy is detected exclusively in AMKL, suggesting that the resultant fusion protein plays a central role in its pathogenesis. An important first step toward the elucidation of the molecular pathogenesis of this disorder

is the recent identification of the two genes involved in this translocation, RBM15 (RNA-binding motif protein-15; also known as MAL) and MKL-1 (megakaryoblastic leukemia-1; also known as OTT), on chromosomes 1 and 22, respectively (Fig. 11.17).385,386 MKL-1 is widely expressed and encodes a protein that contains a SAP DNA-binding domain, a coiledcoil domain that presumably mediates protein–protein interactions, and a proline-rich segment that may effect transcriptional activation. MKL1 has recently been shown to be a transcriptional regulator in skeletal muscle. RBM15 contains three RNA binding domains and a spen paralog and ortholog C-terminal (SPOC) domain. The latter motif is present in the Drosophila protein spen as well as mammalian spen-like proteins, such as Msx-2-interacting nuclear target which modulates the function of HOX8. The biologic functions of the native proteins are unknown. It is plausible that RBM15-MKL1 may deregulate RNA processing and possibly HOX signaling; however, the exact mechanism by which this chimeric protein exerts its oncogenic properties remains undefined.

Rare molecular genetic lesions in AML Besides the common chromosomal rearrangements discussed above, a number of rare translocations have been identified in pediatric and adult cases of de novo AML (reviewed in Chapter 9). These include the t(6;9)(p23;q34) involving the DEK and CAN genes, translocations involving the nucleoporin NUP98 gene, and finally translocations targeting the EVI1 gene. The analysis of these lesions has provided insight into the pathogenesis of acute leukemia; however, space limitations preclude a meaningful discussion of the relevant aspects of the molecular leukemogenesis of these genetic lesions.

Conclusions and future directions Characterization of the major AML-associated chromosomal rearrangements has provided important insights into the molecular pathways involved in the development of human leukemia. Moreover, the identification of key components in these different signaling pathways has provided an initial list of rational targets against which novel therapeutics can be developed. The information gained from these studies has led to the development of new diagnostic approaches that have been a significant aid in the clinical management of patients with these leukemia subtypes. However, enthusiasm for these advances has been tempered by the relatively poor outcome experienced by most children with AML. Five-year event-free survival rates are

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still less that 50%. Thus, future research should focus on exploiting the information now available on the basic biology of AML to develop improved therapeutic approaches. Pursuit of this goal will be aided by continued detailed characterization of the signaling pathways that are altered by chromosomal translocations and by characterization of the molecular lesions in the substantial number of AML cases that lack these gene rearrangements. As the pace of clinical and laboratory research in AML accelerates, we can look forward to further advances in understanding the pathogenetic mechanisms of this disease and ultimately to the development of curative treatments.

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341 Slany, R. K., Lavau, C., & Cleary, M. L. The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol Cell Biol, 1998; 18: 122–9. 342 So, C. W., Karsunky, H., Passegue, E., et al. MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice. Cancer Cell, 2003;3:161–71. 343 Zeisig, B. B., Garcia-Cuellar, M. P., Winkler, T. H., & Slany, R. K. The oncoprotein MLL-ENL disturbs hematopoietic lineage determination and transforms a biphenotypic lymphoid/myeloid cell. Oncogene, 2003; 22: 1629–37. 344 So, C. W., Lin, M., Ayton, P. M., Chen, E. H., & Cleary, M. L. Dimerization contributes to oncogenic activation of MLL chimeras in acute leukemias. Cancer Cell, 2003; 4: 99–110. 345 Eguchi, M., Eguchi-Ishimae, M., & Greaves, M. The small oligomerization domain of gephyrin converts MLL to an oncogene. Blood, 2004; 103: 3876–82. 346 Martin, M. E., Milne, T. A., Bloyer, S., et al. Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell, 2003; 4: 197–207. 347 Dobson, C. L., Warren, A. J., Pannell, R., Forster, A., & Rabbitts, T. H. Tumorigenesis in mice with a fusion of the leukaemia oncogene Mll and the bacterial lacZ gene. EMBO J, 2000; 19: 843–51. 348 Hsu, K. & Look, A. T. Turning on a dimer: new insights into MLL chimeras. Cancer Cell, 2003; 4: 81–3. 349 Yamamoto, K., Hamaguchi, H., Nagata, K., Kobayashi, M., & Taniwaki, M. Tandem duplication of the MLL gene in myelodysplastic syndrome- derived overt leukemia with trisomy 11. Am J Hematol, 1997; 55: 41–5. 350 Kwong, Y. L. Partial duplication of the MLL gene in acute myelogenous leukemia without karyotypic aberration. Cancer Genet Cytogenet, 1997; 97: 20–4. 351 Yu, M., Honoki, K., Andersen, J., et al. MLL tandem duplication and multiple splicing in adult acute myeloid leukemia with normal karyotype. Leukemia, 1996; 10: 774–80. 352 Park, I. K., He, Y., Lin, F., et al. Differential gene expression profiling of adult murine hematopoietic stem cells. Blood, 2002; 99: 488–98. 353 Pineault, N., Helgason, C. D., Lawrence, H. J., & Humphries, R. K. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol, 2002; 30: 49–57. 354 Akashi, K., He, X., Chen, J., et al. Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood, 2003; 101: 383–9. 355 So, C. W., Karsunky, H., Wong, P., Weissman, I. L., & Cleary, M. L. Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9. Blood, 2004; 103: 3192–9. 356 Sauvageau, G., Thorsteinsdottir, U., Hough, M. R., et al. Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation. Immunity, 1997; 6: 13–22.

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357 Sauvageau, G., Thorsteinsdottir, U., Eaves, C. J., et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev, 1995; 9: 1753–65. 358 Perkins, A., Kongsuwan, K., Visvader, J., Adams, J. M., & Cory, S. Homeobox gene expression plus autocrine growth factor production elicits myeloid leukemia. Proc Natl Acad Sci U S A, 1990; 87: 8398–402. 359 Blatt, C., Aberdam, D., Schwartz, R., & Sachs, L. DNA rearrangement of a homeobox gene in myeloid leukaemic cells. EMBO J, 1988; 7: 4283–90. 360 Nakamura, T., Largaespada, D. A., Shaughnessy, J. D. J., Jenkins, N. A., & Copeland, N. G. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. Nat Genet, 1996; 12: 149–53. 361 Kroon, E., Krosl, J., Thorsteinsdottir, U., et al. Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. EMBO J, 1998; 17: 3714–25. 362 Schnabel, C. A., Jacobs, Y., & Cleary, M. L. HoxA9-mediated immortalization of myeloid progenitors requires functional interactions with TALE cofactors Pbx and Meis. Oncogene, 2000; 19: 608–16. 363 Calvo, K. R., Sykes, D. B., Pasillas, M., & Kamps, M. P. Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression. Mol Cell Biol, 2000; 20: 3274–85. 364 Ayton, P. M. & Cleary, M. L. Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev, 2003; 17: 2298–307. 365 Zeisig, B. B., Milne, T., Garcia-Cuellar, M. P., et al. Hoxa9 and Meis1 are key targets for MLL-ENL-mediated cellular immortalization. Mol Cell Biol, 2004; 24: 617–28. 366 Kumar, A. R., Hudson, W. A., Chen, W., et al. Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia. Blood, 2004; 103: 1823–8. 367 Lange, B. J., Kobrinsky, N., Barnard, D. R., et al. Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children’s Cancer Group Studies 2861 and 2891. Blood, 1998; 91: 608–15. 368 Athale, U. H., Razzouk, B. I., Raimondi, S. C., et al. Biology and outcome of childhood acute megakaryoblastic leukemia: a single institution’s experience. Blood, 2001; 97: 3727–32. 369 Zipursky, A., Poon, A., & Doyle, J. Leukemia in Down syndrome: a review. Pediatr Hematol Oncol, 1992; 9: 139–49. 370 Lange, B. The management of neoplastic disorders of haematopoiesis in children with Down’s syndrome. Br J Haematol, 2000; 110: 512–24. 371 Zipursky, A. Transient leukaemia – a benign form of leukaemia in newborn infants with trisomy 21. Br J Haematol, 2003; 120: 930–8.

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12 Apoptosis and chemoresistance Kirsteen H. Maclean and John L. Cleveland

Introduction The development of chemoresistance in tumors is a major obstacle in oncology, particularly in the treatment of acute myeloid leukemia. Resistance to therapy can be broad (multidrug resistance) and arise de novo, but is more frequently acquired following the relapse of disease in patients previously treated with chemotherapy. The chemoresistance phenotype can arise through several mechanisms including: (1) overexpression of the P-glycoprotein family of membrane transporters (MDR1, MRP, LRP), which decrease the intracellular levels of anticancer drugs; (2) changes in cellular enzymes that detoxify or metabolize drugs, such as dihydrofolate reductase and cytochrome p450 enzymes; and (3) changes in enzymes intimately involved in DNA repair, such as DNA topoisomerase II.1–6 A fourth and more general mechanism involves changes in the expression and/or activity of proteins that regulate apoptosis, an endogenous program of cell suicide that disassembles the cell when it has received damage or oncogenic insults. Given the number of regulators that feed into the apoptotic response, understanding this mechanism of resistance is now recognized as a significant challenge; however, targeting these regulators holds great promise of overcoming or bypassing drug resistance in relapsed cancer patients. For many years, radiation treatment and chemotherapy were thought to cause irreparable metabolic or physical damage to cancer cells, resulting in cell necrosis. However, over the past decade, numerous studies have revealed that cancer cells killed by irradiation or any of a wide variety of antineoplastic agents undergo changes typical of apoptosis, including cell shrinkage, condensation of chro-

matin and the cytosol, disruption of mitochondrial functions, DNA fragmentation, and finally membrane blebbing of dying cells into apoptotic bodies, which can be easily engulfed by neighboring phagocytes.7,8 Indeed, it is now recognized that antineoplastic drugs and radiation do not passively kill cells, but rather provoke cell suicide through the agency of intracellular surveillance mechanisms that recognize alterations in cell physiology and then actively kill the cell. This revelation suggested that alterations in programs that regulate apoptosis are a major mechanism responsible for drug resistance in cancer. Here we review this issue and summarize how the expression and/or activity of apoptotic regulators becomes altered or disabled in leukemia and lymphoma and how these changes can lead to radio- and chemoresistance.

Apoptotic pathways Apoptotic programs are essential for the proper development of all metazoans, as they control the ordered formation of vital tissues and organs. Similarly, such programs are required to maintain homeostasis of rapidly dividing tissues, particularly in the control of the hematopoietic compartment, where defects in apoptotic regulators can lead to the development of myelodysplasia, anemia, leukemia, lymphoma, or myeloproliferative or autoimmune syndromes.9–11 Apoptosis also plays an essential role in removing somatic or germ cells that have suffered damage or which bear genetic insults that can trigger inappropriate proliferation. It also plays a role in cell death induced by cytotoxic T lymphocytes or natural killer cells and in the engulfment of dying cells by phagocytes. In all of these

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Sequence homology 12 11 13 4 5 1 2 9 8 6 3 7 10

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Fig. 12.1 Caspases. Although a high degree of homology exists among the different caspases, they are commonly divided into these groups: initiator, effector, and cytokine processor. Initiator procaspases are activated via adaptor molecules (e.g. FADD) that bring them together in close proximity, allowing for autoprocessing. Once activated, initiator caspases are responsible for cleaving and activating effector caspases. Effector caspases lead to the morphologic and biochemical characteristics of apoptosis.

scenarios, apoptotic cell death prevents an inflammatory response. There are two major pathways by which cells commit suicide: intrinsic signals mediated by regulators from within the cell12 and extrinsic signals provoked by the activation of the Fas family of death receptors13 or by the release of toxic proteins from cytotoxic lymphocytes or natural killer cells, e.g. perforin and granzyme B.14,15 The key effectors of both pathways are a collection of highly specific and conserved proteases termed caspases (cysteine-directed aspartate specific proteases), which recognize and cleave their targets at tetra-peptide sites that have characteristic sequences, for example the cleavage at DXXD motifs by caspase-3 (where X represents any amino acid).16 Based on their functions and their temporal sequence of activation during apoptosis, caspases have been divided into three subtypes: initiator (activator) caspases, effector (executioner) caspases, and cytokine processors (which cleave pro-forms of cytokines that play roles in inflammation, e.g. IL-117 and Fig. 12.1). Initiator and executioner caspases play important roles in the resistance of human tumors to chemotherapy. Initiator caspases such as caspases-8 and -9 function as upstream activators of the executioner caspases – designated 3, 6, and 7 (Fig. 12.1),18 which cleave proteins required for cellular integrity, including PARP [poly(ADPribose) polymerase], lamins A and B, actin, fodrin, and ICAD, an inhibitor of caspase-activated deoxyribonucle-

ase (CAD), that directs internucleosomal degradation of chromosomal DNA, a hallmark of apoptosis.19–21 However, these substrates are just the tip of the iceberg, as a recent review has suggested that over 280 different proteins are caspase targets.22 Links of initiator caspases to effector caspases can be either direct or indirect. For example, once activated, caspase-8 cleaves the proenzyme form of caspase-3 to activate the enzyme.18 By contrast, caspase-9 is found in a multisubunit protease activation complex called the apoptosome, which exists as a wheel-like structure containing seven molecules of a regulator termed Apaf-1 and seven caspase-9 dimers, and which is assembled in response to signals that provoke mitochondrial outer membrane permeabilization and the release of cytochrome c into the cytosol. Once activated, the apoptosome triggers caspase-9 activity, which then cleaves and activates the proenzyme forms of effector caspases (Fig. 12.2). Given their central roles in executing cell death during development, it is not surprising that familial mutations in these genes or in their direct regulators, such as Apaf-1, have not been detected in man. What is now clear, however, is that their expression is compromised in many tumor types, and that this can occur at post-transcriptional levels (caspase-10),23 by somatic point mutations (caspase-7 and caspase-5) 24,25 or, more often, by epigenetic means.26 In the latter case, the expression of caspase-8, caspase-1, and Apaf-1, for example, is silenced by hypermethylation of

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Fig. 12.2 Apoptotic signal transduction pathways. Apoptosis is initiated either through the intrinsic pathway involving mitochondria or through the extrinsic pathway triggered by death receptors. Caspase-9 and caspase-2 respond to changes in mitochondria, whereas caspase-8 and caspase-10 sense activation of death receptors. These initiator caspases cleave the proenzyme forms of the effector caspases (caspase-3, caspase-6, and caspase-7), thus permitting the cleavage of targets that affect cell viability.

their regulatory sequences in a number of tumor types27–30 and restoration of their expression in these tumor cells or treatment of the cells with a DNA methyltransferase inhibitor, 5-aza-2 -deoxycytidine, reverses chemoresistance.31 Further, embryonic fibroblasts from caspase-9 knockout mice are highly susceptible to transformation and are inherently resistant to drugs used in conventional chemotherapy,32 yet cell context-specific effects of resistance also come into play, as lymphoid and myeloid cells from caspase-9−/− mice remain sensitive to druginduced apoptosis.33 Finally, studies using both leukemic cell lines and clinical samples from patients with acute myeloid leukemia (AML) have established that simultaneous inhibition of both initiator and effector caspases strongly correlates with chemoresistant disease and a poor outcome.34 Thus, therapeutic protocols incorporating regimens that can stimulate the activity of both initiator and effector caspases hold promise for the treatment of drugresistant hematologic malignancies.

The Bcl-2 family of apoptotic regulators Causal links among cancer onset, disease resistance and apoptosis were first recognized with discovery of the Bcell lymphoma gene-2 (Bcl-2 gene) as the gene activated by the t(14;18)(q32;q21) translocation with immunoglobulin

regulatory loci in 85% to 90% of follicular lymphomas.35,36 The seminal studies of Vaux, Cory, and Adams established that Bcl-2 protects cells from programmed cell death occurring when hematopoietic cells are deprived of their required growth factors,37 leading to the concept that cancer development requires the simultaneous acquisition of deregulated cell division and resistance to apoptosis. Advances regarding the mechanism of action and regulation of Bcl-2 in the 1990’s, made it clear that Bcl-2 overexpression is a hallmark of many cancers with a poor outcome (e.g. B-cell leukemia, lymphomas, colon and prostate cancers, and neuroblastoma). When homologues of Bcl2 were identified, it became apparent that the Bcl-2 family could be defined by the presence of conserved motifs known as the Bcl-2 homology domains (BH1 to BH4, Fig. 12.3). It was further revealed that Bcl-2 family members came in three varieties. First, the anti-apoptotic members of the family were identified as Bcl-2, Bcl-XL , Bcl-w, Mcl-1, A1, and Boo (Diva), and nearly all of these proteins harbor a BH4 domain and all are primarily localized to the mitochondrial outer membrane and/or the endoplasmic reticulum (ER), where they prevent the release of proapoptotic proteins such as cytochrome c from mitochondria, or the release of Ca2+ stores from the ER.38,39 Second, proapoptotic Bcl-2 family members were identified that disrupted the integrity of mitochondria and the ER, and

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Fig. 12.3 The Bcl-2 Family. Bcl-2 family members are classified into three categories: (A) the multidomain antiapoptotic subfamily which share sequence homology within some of the four Bcl-2 homology (BH) domains, BH1 to BH4; (B) the multidomain proapoptotic subfamily, sharing sequence homology at BH1, BH2, and BH3; (C) the BH3-only subfamily, consisting of proteins sharing sequence homology only at the BH3 region. TM, transmembrane domain.

could be divided into two subgroups, both structurally and functionally. One contains the proteins Bax, Bak, Bok, and Bcl-rambo, which harbor BH1, BH2, and BH3 domains; whereas the second, designated the “BH3-only proteins,” include Bad, Bid, Noxa, Puma, Bik, and Bim, to name just a few.12,40 In general the current data are most consistent with a model whereby the combined functions of Bax and Bak are required for almost all forms of apoptosis.38,41 Their functions are actively held in check by binding to antiapoptotic proteins such as Bcl-2 and Bcl-XL ,41 whose function in turn can be compromised by binding to BH3-only proteins, which allows Bax and Bak to initiate the cell death cascade (Fig. 12.4). As such the BH3-only proteins function as true apoptotic signaling proteins and indeed the expression and/or activity of many of these proteins are regulated by apoptotic signals (e.g. irradiation or antineoplastic drugs, or deprivation of required survival factors). Bax and Bak regulate apoptosis by controlling the integrity of mitochondria and the ER, and function as the key linchpins of the intrinsic cell death pathway. While precise details of their functions at the ER are still a bit sketchy, much is now known of how Bax and Bak disrupt the functions of the mitochondria, which have been long recognized as the key target in intrinsic cell death pathways, including those triggered by antineoplastic agents.42 An initial event is a drop in the mitochondrial membrane potential (MMP), which is required to maintain

an asymmetric distribution of charges between the inner and outer mitochondrial membranes.42–44 If disruption of the MMP persists, the integrity of the outer membrane is compromised resulting in the release of proapoptotic factors, including cytochrome c45 and apoptosisinducing factor (AIF), which can induce death independent of caspase activation.46 Release of cytochrome c from the mitochondria into the cytosol is a critical initiator of caspase-dependent death pathways: that is, once released, cytochrome c binds to Apaf-1, thereby triggering activation of the apoptosome and caspase-9, which then cleaves and activates caspase-3 (Fig. 12.4). At least in cell culture systems, all antineoplastic agents induce apoptosis via their ability to disrupt the functions of mitochondria, and in many instances this has been associated with the ability to activate the expression and redistribution of BH3-only proteins such as Bim, Noxa, and Puma.47,48 In vivo assessment of these changes is technically challenging because apoptotic cells are rapidly cleared by phagocytes, yet what is clear is that the expression of BH3-only proteins is certainly affected by anticancer regimens49 so that one presumes the events identified in vitro also take place in target cells in the organism. Bcl-2, and other prosurvival family members, can inhibit cell death due to a wide variety of insults including anticancer drugs,50–52 radiation,53,54 and growth factor withdrawal.37,55 Indeed, genetic studies in E -Myc

Apoptosis and chemoresistance

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Fig. 12.4 Interplay of Bcl-2 family members at the mitochondria. Mitochondrial integrity is regulated by a balance of pro- and antiapoptotic Bcl-2 family members. Under normal circumstances, the BH3-only proteins (such as Bid and PUMA) are inactive; however, upon activation, they act as activators of another group of proapoptotic proteins, including Bax and Bak. These proteins associate with the outer mitochondrial membrane during apoptosis. Antiapoptotic proteins such as Bcl-2 and Bcl-XL operate to sequester the BH3-only proteins into stable complexes and thus prevent the activation of Bax or Bak. During apoptosis, cytochrome c is released and together with dATP and Apaf-1 recruits caspase-9 to form the apoptosome.

transgenic mice, which overexpress the c-Myc oncogene in the B-cell lineage and are a model for human Burkitt lymphomas that bear MYC/Ig translocations,56,57 have shown that Bcl-2 is sufficient to override cell death induced by various chemotherapeutic agents.58 Similarly loss-offunction mutations of Bax and Bak in some select tumor types,24,59–63 including leukemia,64,65 correlate with tumor aggressiveness and resistance,66,67 and complete loss of Bax and/or Bak renders cells completely refractory to these insults.68–70

Alterations in the Bcl-2 family in leukemia and lymphoma Loss of function or silencing of the expression of proapoptotic Bcl-2 family proteins, other than a few rare instances involving Bax, have yet to be described for leukemia or lymphoma. However, overexpression of Bcl-2, and some other antiapoptotic family members, is indeed a hallmark of many resistant hematological malignancies. For example, in addition to its direct activation in follicular lymphoma,35,36 Bcl-2 has been shown to be overexpressed in patients suffering from non-Hodgkin’s lymphomas,71,72

diffuse large-cell lymphoma,73 mantle cell lymphoma,74 AIDS-related lymphomas,75 primary cutaneous large B-cell lymphoma,76 hairy cell leukemia,77 mast cell leukemia,78 B-lineage acute lymphoblastic leukemia,79,80 B-lineage chronic lymphocytic leukemia (CLL),81 and several subtypes of AML82–85 ; in general this overexpression connotes a poor clinical response. While direct evidence for activation of other antiapoptotic Bcl-2 family members in leukemia and lymphoma is less pervasive than for Bcl-2, there is strong evidence that Bcl-XL is overexpressed in AML and chronic myeloid leukemia (CML),86–89 and in Hodgkin lymphoma,90 AIDSrelated lymphoma,91 and multiple myeloma.92 Similarly, Mcl-1 has been shown to be overexpressed in high-grade B-cell non-Hodgkin lymphoma,93 multiple myeloma,94 anaplastic large cell lymphoma,95 high-grade mantle cell lym phoma,96 and B-cell chronic lymphocytic leukemia – some of which bear activating insertions in the Mcl-1 promoter,97 and up-regulation of Mcl-1 has been associated with leukemic relapse.98 Finally, the A1 protein has been implicated as playing an important survival role in AML.99 Collectively, then, the clinical evidence is overwhelming that antiapoptotic Bcl-2 family proteins play a major

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role in the development, aggressiveness and resistance of human lymphoma and leukemia. Indeed, genetic tests of their contribution to the development of lymphoma and leukemia in mice have borne these findings out, showing that overexpression of Bcl-2 can provoke a slow-onset lymphoma.100–102 Further, Bcl-2 and Bcl-XL markedly accelerated Myc-driven lymphomagenesis103–105 and PML-RAR
- and BCR-ABL-induced acute leukemia,106,107 while up-regulation of Bcl-2 is a hallmark of lymphomas arising in E -Myc transgenic mice.108 Finally, as might be expected from such findings, the loss of Bax also accelerates Myc-induced lymphoma development.109 In view of the large body of evidence implicating the antiapoptotic Bcl-2 family members in chemoresistance and a poor clinical outcome, there is an obvious and urgent need to develop agents that specifically disable these proteins. Accordingly over the past few years there has been a surge of interest by several pharmaceutical companies in developing novel agents that target these proteins. First of all, VelcadeTM , a new agent currently in clinical trials (jointly developed by the NCI and Millenium Pharmaceuticals) appears to overcome tumor chemoresistance by targeting the proteosome, and increasing levels of Bax. However, many other proteins are affected by wholesale inhibition of the proteasome, so that one is faced with problems of specificity and a suitable therapeutic index. The issue of specificity does not apply to GenasenseTM , a phosphorothioate antisense oligonucleotide that selectively impairs the expression of Bcl-2 mRNA (developed by Genta pharmaceuticals) that is currently undergoing clinical trials (cosponsored by Genta and Aventis pharmaceuticals).110,111 Moreover, a bispecific Bcl-2/Bcl-X phosphorothioate antisense oligonucleotide has shown efficacy in inducing apoptosis in tumor cells that overexpress both proteins.112,113 On a third front, small molecules that interact with the surface pocket of the BH3 domains of Bcl-2 family proteins, the so-called BH3 mimetics, are also being developed, and perhaps hold the best promise for inactivating Bcl-2 and its related proteins. These include HA-14-1, which interacts with Bcl-2 and induces apoptosis of tumor cells,114 and antimycin A, which binds to the BH3-binding domain of Bcl-2 and Bcl-XL and leads to permeabilization of purified mitochondria overexpressing the latter protein.115 Peptide mimetics of the Bax and Bcl-2 BH3 domains fused to the antennapedia plasma membrane translocation domain, which facilitates cellular entry, have also been developed.116 Notably, the actions of these fusion proteins cannot be disabled by overexpressing Bcl-2 and Bcl-XL , suggesting that these agents may be useful in overcoming chemoresistance in hematologic

malignancies proteins.

that

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Death receptor pathways and chemoresistance The extrinsic cell death pathway regulated by the tumor necrosis factor (TNF) and Fas families of transmembrane “death receptors” is very well characterized.117 These receptors include Fas (CD95/Apo1/DR2), TNF-
receptor 1 (TNFR1/DR1), TRAIL-R1 and TRAIL-R2, as well as CD40, CD27, and RANK,118 whose short (∼80 residue) cytoplasmic domains are directly linked to caspases by adaptor proteins, in particular via the Fas-associated Death Domain (FADD) protein.119 Upon binding to their respective ligands (e.g. Fas ligand, TNF-
, and TRAIL), these receptors trimerize, and on their intracellular face this clustering promotes their interactions with FADD through their respective “death domains”. This interaction signals the formation of the death-inducing signaling complex, (i.e. the DISC,117 through the FADD death effector domain (the DED domain), which interacts with the DED domains of caspase-8, and/or caspase-10. This event triggers selfcleavage of caspase-8 or -10, which then cleave and activate caspase-3. In some cells (so-called Type I cells) caspase8 or 10 can also clip BH3-only proteins such as Bid to a cleaved form, tBid, that relocalizes to the mitochondria and disrupts its function; this pathway thus links the extrinsic and intrinsic cell death pathways (Fig. 12.5).120,121 A number of dedicated proteins harness the activity of the DISC, including the Flice-like inhibitory proteins (FLIP), which contain two DED domains that can bind to the DEDs of FADD and inhibit the recruitment of procaspase-8 or -10 to the DISC.122 Furthermore, the functions of death receptors are also antagonized at another level, by the expression of “decoy receptors”. For example, TRAIL itself can bind to two different death receptors (DR4 and DR5) and to three decoy receptors, DcR1, DcR2, which are transmembrane receptors, and osteoprotegerin, a soluble TNFR family member.123 The contribution of the TNF/Fas death receptor pathways to chemoresistance has been a matter of some debate.124 While it is clear that some antineoplastic drugs upregulate Fas ligand expression on Fas-expressing cancer cells, including leukemia125 and pre-B ALL cells,126 and of DR5 in ALL,127 other studies have shown that drug-induced apoptosis associated with increased Fas or Fas ligand is in fact independent of Fas signaling,128 and analyses of clinical samples from AML and ALL patients have indicated conflicting associations between the expression of Fas in AML patients, and of DR5 in ALL patients, with regard to remission induction,

Apoptosis and chemoresistance

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Apoptosis Fig. 12.5 Linking the intrinsic and extrinsic apoptotic pathways. Apoptotic pathways can be triggered either by crosslinking death receptors or at the mitochondrion. Activation of death receptors by a death ligand results in the recruitment of the adaptor molecule FADD and caspase-8 into a DISC. Caspase-8 becomes activated and initiates cleavage of downstream effector caspases. The mitochondrial pathway is initiated by the release of factors such as cytochrome c or Smac from the mitochondria into the cytosol. The release of cytochrome c into the cytosol triggers downstream caspases through the formation of the apoptosome. Smac can promote caspase activation by neutralizing the inhibitory effects of inhibitor-of-apoptosis proteins (IAPs). Both pathways can be interconnected either through Bid, which regulates cytochrome c activity upon cleavage by caspase-8 or through FLIP, which blocks caspase-8 activation.

chemoresistance, and outcome.127,129,130 However, some types of chemotherapy clearly enhance the activity of TNF
131,132 and especially TRAIL during treatment of resistant leukemia and lymphoma.133–137 Furthermore, these drugs can also modulate the expression of components of the DISC, including, for example, the up-regulation of FADD and procaspase-8 in colon carcinoma cells exposed to doxorubicin or cisplatin.138 Finally, a number of studies have indicated links between chemoresistance and alterations in the expression of death receptor pathway components in leukemia and lymphoma, First, FADD, DR4, and DR5 expression can be silenced in drug-resistant leukemia and lymphoma, whereas other tumor cells overexpress decoy receptors.139–141 Second, upregulation of c-FLIP is a hallmark of Hodgkin lymphoma, and perhaps also of non-Hodgkin B-cell lymphoma,142–144 and agents that alter c-FLIP expression render refractory B-CLL and AML cells sensitive to TRAIL and other proapoptotic agents.145–150

AKT pathways and chemoresistance in leukemia and lymphoma Hemopoietins that play key roles in hematopoietic development and homeostasis, such as interleukin-3 (IL-3), IL-2, IL-7, and erythropoietin, promote survival by inducing the expression of antiapoptotic Bcl-2 family members,151–153 and/or by suppressing the expression of some proapoptotic Bcl-2 family members.154–156 However, their ability to block apoptosis also relies on their activation of the phosphatidylinositol-3 kinase (PI3K)-to-PTEN-to-Akt signaling pathway (Fig. 12.6), which is a frequent target in human malignancies.157–159 PI3K catalyzes the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate.160 The Akt (PKB) family of serine/threonine kinases interact with phospholipids, causing translocation of these kinases to the inner face of the cell membrane, where they are phosphorylated and activated by the serine/threonine kinases

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Fig. 12.6 AKT/PTEN signaling pathway. Growth factor receptors recruit PI3K to the cell membrane and induce the production of second messengers such as PIP3 (phosphatidylinositol 3,4,5-trisphosphate) that convey signals to the cytoplasm from the cell surface. PIP3 signals activate the kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1), which in turn activates the kinase AKT. Proteins phosphorylated by activated AKT promote cell survival. For example, phosphorylation of Ikappa-B kinase leads to activation of the transcription factor NF-kB to oppose apoptosis. Bad is a protein in the Bcl-2 family that opposes Bcl-2 to induce apoptosis. Phosphorylation of Bad by AKT blocks antiapoptotic activity to promote cell survival. Similarly, AKT-induced phosphorylation of the protease caspase-9 or forkhead transcription factors blocks the induction of apoptosis by these factors. PTEN is a PIP3 phosphatase that negatively regulates the PI3K/AKT pathway.

PDK1 and PDK2.161,162 Phosphorylations of Akt trigger its activity and it then phosphorylates several key substrates that regulate cell growth and survival (Fig. 12.6). These include, for example, glycogen synthase kinase3, which regulates diverse cellular processes (including glycogen metabolism) and is inactivated by Akt,163 and tuberin, a protein that activates the mammalian target of rapamycin (mTOR), the master regulatory kinase of cell growth and proliferation164,165 and a promising new target for cancer therapeutics.166 Further, Akt phosphorylates several apoptotic regulators, including the forkhead family (FoxO) of transcription factors,167,168 caspase-9,169 and the Bcl-2 proapoptotic family member Bad.170,171 Akt-mediated phosphorylation of these apoptotic regulators generally disrupts their functions, for example, by initiating the degradation of FoxO proteins by the proteasome,172 inactivating caspase-9 activity,169 or by disrupting the association of Bad with Bcl-XL in favor of its interactions with the 14–3–3 scaffolding protein.173 Conversely, activation of the PI3K-to-Akt pathway is held in check by PTEN (for “phosphatase and tensin homolog deleted on chromosome 10”), a lipid phosphatase. PTEN

is amongst the most frequently mutated tumor suppressors in human cancer,174 and germline mutations of PTEN lead to the development of the related hereditary cancer predisposition syndromes, Cowden disease, and BannayanZonana syndrome.175 PTEN expression is silenced or deleted in some cases of ALL, multiple myeloma, primary cutaneous T-cell lymphoma, B-CLL, and non-Hodgkin lymphoma,176–181 and Pten+/− mice develop lymphoma.182 Finally, PTEN-deficient tumor cell lines and cells derived from the Pten knockout mouse are resistant to an array of apoptotic stimuli,183 and loss of PTEN functions has been associated with resistance to chemotherapeutics in the treatment of some cancers, including ALL.184–186 In addition to alterations targeting PTEN, a widespread role for Akt and PI3K in cancer development and chemoresistance has also recently been established. First, cells engineered to overexpress activated Akt are resistant to both staurosporine and etoposide-induced apoptosis.187,188 Second, several studies have found ATK amplifications in human cancers,189–193 including nonHodgkin lymphomas.194 Furthermore, a hallmark of most relapsed or chemoresistant tumors is the activation of Akt,161,195 and the same scenario has been described

Apoptosis and chemoresistance

in resistant leukemia and lymphoma.196,197 Third, there are also reports of direct amplification of the gene encoding the p110 catalytic subunit of PI3K (PIK3CA, at 3q26.3) in human cancer,198–200 including primary cutaneous T-cell lymphomas.201 Finally, Lowe and colleagues, have recently shown, using the E -Myc mouse model of Burkitt lymphoma, that Akt promotes tumorigenesis and drug resistance.202 Therefore, Akt- and/or PI3K-specific inhibitors would clearly be of potential value in the treatment of resistant leukemia and lymphoma, and indeed several agents targeting these kinases are currently under development.203–205

NF-B and chemoresistance in leukemia and lymphoma At the crossroads of many signaling pathways that regulate apoptosis, including those induced by PI3K/Akt and the death receptors, is the NF-B network of transcription factors. Initially discovered as a dimeric transcription factor that relocalizes to the nucleus to play a role in the mitogenic responses of B-cells,206 this family of transcription factors is now recognized to play pivotal roles in many cellular processes, including survival, cell adhesion, inflammation, differentiation and growth.207–210 Active NF-B complexes are dimers of various combinations of the Rel family of proteins, consisting of p50, p52, c-Rel, v-Rel, and Rel A. These proteins share a conserved 300-aminoacid region within their N-terminus that is responsible for DNA binding, dimerization, nuclear translocation, and interactions with other transcription factors. In most cell types, NF-B is sequestered in an inactive state in the cytoplasm by its interaction with IB proteins.211 Upon stimulation, IB becomes phosphorylated by the IKK signaling complex212,213 and is then degraded via the proteosome, thereby releasing NF-B to the nucleus, where it induces the transcription of many target genes that play direct roles in apoptosis, including A1, Bcl-2, Bcl-X, and FLIP, as well as genes encoding components of the TNFR1 signaling complex, TRAF1 and TRAF2.214 Inactivation of several NF-B transcription factors, and their regulators, in mice has revealed their essential roles in lymphoid and skin development, the immune response, and in the survival of liver cells.215–223 NF-B mediates its antiapoptotic functions by affecting the expression of many proteins that regulate apoptosis, including, for example, Bcl-X208 and the inhibitor-of-apoptosis proteins (IAPs224 ) that function as dedicated inhibitors of caspases, as they directly bind to caspases and inhibit their activity.225 Indeed overexpression of IAPs is now recognized to be a hallmark of many cancers, including drug-resistant leukemia.226

Paradoxically, NF-B can also stimulate the expression of proapoptotic molecules such as FasL.227 Given its critical role in cell survival and growth, NFB has been implicated in tumorigenesis, particularly since NF-B can be activated by a number of carcinogens and tumor promoters.228,229 Several studies have shown that downregulation of NF-B sensitizes cells to apoptosis induced by cytokines and antineoplastic drugs,230,231 and indeed the status of NF-B dictates whether cells will survive following the engagement of death receptors.207,232 On the other hand, it has also been shown that many chemotherapeutic agents, including taxol, doxorubicin, etoposide and cisplatin, can activate NF-B and that this, in turn, can lead to chemoresistance, principally via NF-B’s ability to activate the expression of antiapoptotic proteins including members of the Bcl-2 family.207,233–235 Therefore, while on one hand activating apoptosis, the same chemotherapeutic agent can also activate NF-B, leading to cell proliferation and/or chemoresistance. These conflicting effects of NF-B depend on the nature of the inducing signal and are probably specific for cell context and tumor type. Given their central roles as regulators of apoptosis it is not surprising that NF-B factors are often activated in leukemia and lymphomas236,237 and also play roles in resistance to various chemotherapeutic agents.238 Indeed, amplifications of REL (human c-Rel) are frequent in Hodgkin lymphomas and diffuse large B-cell lymphomas (DLBCL), and in some follicular and mediastinal B-cell lymphomas,239 and rearrangements and deletions of the 3 sequences of NFKB2 (pp52/p100) lead to the production of C-terminally truncated and constitutively active forms of this protein in cutaneous T-cell leukemia and BCLL.240,241 In addition, inactivating mutations in the caspase recruitment domain (CARD) of BCL10 in mucosaassociated lymphoid tissue (MALT) lymphomas, due to the recurrent t(1;14)(p22;q32), lead to constitutive activation of NF-B.242 Further, constitutively active RelA has been reported to play a critical role in the survival of B-CLL,243 and in AML blasts,244,245 where it is associated with increased activity.246 In addition, constitutive activation of NF-B has also been reported for ALL,247 multiple myeloma,248 Hodgkin lymphoma,249 mediastinal large B-cell lymphoma,250 mantle cell lymphoma,251 and in leukemia stem cells.252 Finally, NF-B activation is a critical determinant of chemoresistance in many tumor types,207 including pediatric B-ALL,253 multiple myeloma,254 and perhaps cutaneous T-cell lymphoma.255 Genetic tests of its role have also established that NF-B activation is essential for BCR-ABL-mediated leukemogenesis.256 Given its widespread role in the etiology and radioand chemoresistance of leukemia and lymphoma, NF-B

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Fig. 12.7 p53-mediated responses. Levels of the tumor suppressor protein p53 are kept low in cells by a feedback loop targeting Mdm2. p53 induces Mdm2, which then targets p53 for proteasomal degradation. There are two ways of activating p53: (1) DNA damage activates the ATM/Chk2 pathway, which phosphorylates p53 and Mdm2, thereby breaking up the interaction. This leads to release of p53 transcriptional activities which turns on Puma/Bax (apoptosis) and p21 (G1 arrest). (2) Oncoproteins, such as Myc, induce the expression of Arf, which drags Mdm2 into the nucleus, thereby releasing p53 activities.

clearly represents an attractive target for improving the effects of cancer chemotherapy. Recently it has been shown that abrogation of constitutively active NF-B by overexpressing a dominant negative super-repressor IB mutant enhances the apoptotic potential of drugs such as paclitaxel in chemoresistant cells,257 but obstacles of delivery and other hurdles continue to limit this approach. However, suppression of NF-B can be tackled on many fronts. First, agents such as Bortezomib block proteasomal-mediated degradation of IB,258 and nonsteroidal anti-inflammatory drugs such as aspirin block the phosphorylation of IB by IKK259 and a similar mechanism applies to arsenic trioxide.260 Second, decoy peptides and small molecules are being developed to directly block NF-B activation and DNA binding activity.261,262 Finally, genetic manipulations of proteins such as TRAF2 and TRAF6 that suppress NF-B activity are being pursued.263

p53 and its regulators in leukemia and lymphoma and chemoresistance p53 has been anointed as “the guardian of the genome.” The title appears apt in view of the role of p53 as a sequence-specific transcription factor whose targets include those that direct cell cycle arrest (e.g. the cyclindependent kinase inhibitor p21CIP1,264 ) or that induce apoptosis (e.g. the proapoptotic Bcl-2 family member Puma154 ). Direct inactivation of p53, through missense mutations that generate dominant negative forms of the protein, through biallelic deletions of TP53, or through epigenetic silencing, are the most common alterations in human cancer, and are especially prevalent in relapsed,

chemoresistant tumors,265–269 including leukemia and lymphoma.270–274 In normal cells p53 is present at low concentrations due to a finely-tuned feedback loop that includes Mdm2, whose association with p53 blocks its transactivation functions275 and programs it for destruction by initiating ubiquitination and shuttling it to the cytosol for proteosomal degradation.276 However in certain contexts, DNA damage or hypoxic conditions, the interaction of Mdm2 with p53 is disrupted by inducing phosphorylation of p53277,278 and/or Mdm2,278 or by inducing the ARF nucleolar tumor suppressor (see below), which binds to and inhibits Mdm2 functions.279,280 The net result is stabilization of p53, which then accumulates in the nucleus, where it becomes active and binds (in its tetrameric form) to p53 response elements in its target genes that direct cell cycle arrest, apoptosis, and DNA repair and/or differentiation (Fig. 12.7). Collectively, then, these responses prevent damaged cells from passing on mutations from one generation to the next. For example, in cancer cells carrying the wild type p53 gene, cell cycle arrest may occur after treatment with antineoplastic agents so that the cell can repair the damaged DNA or commit to apoptosis. By contrast, the cancer cell with a mutant p53 gene lacks this option and, consequently, its growth is unchecked, circumstances that ultimately select for cells having more mutations that lead to resistance to apoptosis and chemotherapy. p53 functions in cancer cells can be lost by various mechanisms, including the failure of signals required to activate p53, mutations within the Trp53 itself, and/or mutations in the many downstream mediators of p53 function.281 The importance of p53 loss in cancer was definitively established by creation of the p53 knockout mouse, which

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develops normally but inevitably succumbs to cancer.282 Indeed, by 6 months of age, 70% of p53−/− mice develop tumors and within a year almost all are dead. Important links to chemoresistance have also come from this mouse model, where Lowe and colleagues283 demonstrated that p53-null cells were resistant to the damage induced by many forms of therapy, including irradiation, and these findings led to the notion that p53-deficient tumors are both chemo- and radio-resistant. Moreover, in addition to its role in provoking apoptosis, the p53-null mouse model has also been used to show that the induction of a senescence program by chemotherapy, yet another potentially important aspect of the therapeutic response, also relies on p53 and the cdk inhibitor p16INK4a .284 In humans, about 50% of most cancers appear to possess mutated, dominant-negative forms of TP53 and in many tumor types these mutations are associated with a poor prognosis and treatment failure.285 Analysis of p53 status in AML cell lines representing the blast cells of either de novo leukemia patients in remission or patients with relapsed and chemotherapy-resistant disease confirmed that p53 was in a conformation typical of the mutated protein.286 Furthermore, p53 is frequently mutated in blast crisis CML,272 gastric MALT lymphoma,287 cutaneous lymphomas,288 and Burkitt lymphoma,289 and is often deleted in B-CLL.273 In the case of p53 there are many ways to “skin the cat,” and other regulators of the pathway come into play in cancer. First and foremost is p53’s endogenous inhibitor Mdm2 (HDM2 in humans). HDM2 is frequently amplified in human tumors,290,291 and high levels of Mdm2 inactivate p53 by blocking its transcription functions and by programming its destruction by the proteasome. While direct amplifications of HDM2 are thought to be rare in leukemia and lymphoma,292,293 rearrangements and amplifications of this gene have been detected in CLL294,295 and in Hodgkin disease, multiple myeloma, and DLBCL,296–298 but, more frequently, HDM2 is overexpressed in many hematological malignancies,289,299–305 and this abnormality has been associated with a poor prognosis in pediatric leukemia.306 The ARF (alternative reading frame) tumor suppressor, which, curiously, is encoded by an overlapping reading frame in the Ink4a gene that encodes the cdk inhibitor p16Ink4a , is an important regulator of the p53 pathway.307 ARF is a very small (19-kDa) basic protein that is normally localized to the nucleolus, and Arf activates p53 indirectly, by binding to Mdm2 and preventing its ubiquitination of p53308 and also often by sequestering Mdm2 into the nucleolus.280 The net results are p53 stabilization and activation of its transcription targets.309 Arf-null mice are highly

prone to developing tumors310 and Arf +/− mice lose the wild-type allele in tumors that arise spontaneously or when they are crossed with other tumor prone mice, for example, to E -Myc transgenic mice.311 Behind p53, alterations of the Arf/p16Ink4a locus are the second most common alteration in human malignancies and this generally occurs through epigenetic silencing of Arf and/or p16Ink4a or biallelic deletion of the locus, although point mutations in the two genes have also been reported.312 Importantly, Arf appears to function as a selective guardian against hyperproliferative signals emanating from activated oncogenes in cancer cells, as its transcription and protein levels rise dramatically when cells are faced with such signals.313 From a functional standpoint Arf engages p53 to promote either cell cycle arrest or apoptosis, but in the face of oncogenic insults the latter is the predominant outcome.309 Finally, p53independent functions for Arf have also been described, as Arf is capable of inducing a protracted form of cell cycle arrest in cells lacking p53,314 an effect that has been associated with its ability to impair processing of ribosomal RNAs necessary for the translational machinery,315 and recently it has also been suggested to selectively impair the transcriptional functions of c-Myc, by blocking its induction of targets that drive the cell cycle.316 There is widespread evidence for inactivation of ARF and/or p16Ink4a in human leukemia and lymphoma. First, a hallmark of T-ALL is biallelic deletion of these genes.317–319 Second, although deletions of ARF/p16Ink4a are relatively rare in AML,320 they are frequent in CML,321 nonHodgkin lymphoma,322 primary central nervous system lymphomas,323,324 and relapsed ALL,325,326 and this is associated with poor outcome.327 In addition, translocations that inactivate these genes in B-ALL have also been reported.328 Furthermore, this locus is also frequently inactivated by epigenetic means, generally through hypermethylation of its regulatory regions, which can lead to selective inactivation of ARF, and more often of p16Ink4a , and this also occurs in human leukemia and lymphoma.323,328–332 The p53 pathway can also be affected by upstream activators other than Arf and Mdm2, in particular the ATM (ataxia-telangectasia mutant) kinase, which phosphorylates and activates p53,333 and p53’s family members p63 and p73.334,335 Humans with germ-line mutations in the ATM gene, in addition to suffering from ataxia, are highly prone to the development of lymphoma and acute leukemia336 and somatic inactivation of ATM has been described in mantle cell lymphoma,337 B-CLL,338 and DLCBL.339 Structurally p63 and p73 are similar to p53, yet knockout studies in mice have shown that while p53 acts as a tumor suppressor and in stress response pathways,

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p63 and p73 exert their suppressor functions mostly in pathways related to differentiation and development, and knock-out mice do not exhibit enhanced tumor susceptibility. To date very little is known about the contribution of p63 to the response to chemotherapy or resistance, whereas many studies have shown that p73 is activated in response to a diverse repertoire of chemotherapeutic agents (reviewed in Irwin340 and the references therein) and that interference of its function (e.g. using siRNA technology) leads to chemoresistance in a variety of human cancers.341 Furthermore, mutant p53 can bind to and inactivate p73, suggesting that mutant p53 could, at least in part, confer chemoresistance vis-`a-vis its disabling effects on p73 function.342,343 Importantly, frequent inactivation of the p73 gene by abnormal methylation or loss of heterozygosity has been reported in non-Hodgkin lymphomas,344 while overexpression of an antiapoptotic variant of p73, Np73, appears to be a hallmark of B-CLL.345 Thus, both p73 and p53 are attractive targets for the development of new agents that can reactivate p53 functions and/or augment those of p73, to treat drug-resistant tumors (reviewed in Wang et al.346 and Lane and Hupp347 ). Finally, two other key components of the p53 response appear to have direct connections to leukemia, lymphoma and chemoresistance. First, p21Cip1 , the principal downstream transcriptional target of p53 that mediates its cell cycle arrest response, has been directly implicated in the etiology and chemoresistance of leukemia and lymphoma. For example, high levels of p21Cip1 protein have been associated with chemoresistance in AML348,349 and 5 -CpG island hypermethylation of the p21CIP1 gene in ALL is associated with transcriptional silencing and poor prognosis.350 Second, the death-associated protein kinase (DAPK), a proapoptotic calcium/calmodulinregulated serine/threonine kinase that contributes to death receptor pathways and activates Arf expression to trigger the Arf-p53 apoptotic pathway351 (reviewed in Shohat et al.352 and Ng353 ) is frequently inactivated (by epigenetic means) in Burkitt lymphoma, B-cell non-Hodgkin lymphoma,354,355 plasma cell leukemia356 and NK/T-cell lymphomas,357 and in AML DAP-kinase hypermethylation is associated with myelodysplastic changes at the time of diagnosis and with cytogenetic abnormalities.358

Conclusions and future directions Nearly all antineoplastic drugs kill cancer cells by activating apoptotic pathways. However, in clinical practice, the majority of human tumors show resistance to most therapeutic agents, even when they are used in combi-

nation. It is now evident that chemoresistance can often be attributed to genetic alterations that disable apoptotic regulators. Indeed, the key players in these cell death pathways, especially Bcl-2 family members, as well as components of death receptor, PI-3 K-Akt, NF-B, and the Arf-p53 pathways, are often altered in leukemia and lymphoma, and are now recognized as major culprits in chemoresistance. Therefore, strategies designed to selectively disable these regulators (e.g. Bcl-2, Akt or NF-B), or to reactivate their functions (e.g. Arf or p53), are needed to treat resistant disease. We would argue the true “Achilles heel” of resistant leukemia and lymphomas rests squarely on Bcl-2 family members, as their functions override those of other regulators and they act downstream as essential guardians of organelle integrity; hence disabling their functions puts the cell on an irreversible course towards its destruction. More emphasis needs to be placed on understanding the complexity of the genetic response to specific therapeutic agents in appropriate target cells, how it correlates with resistant disease and patient outcome, and how genetic variations among individual patients, and within populations, can affect this response. Indeed, recent studies by Evans and colleagues underscore the value of genome-wide approaches to define changes in the transcriptome, after treatment with drug combinations versus single agents, or high doses of drugs versus more moderate doses. These findings have implicated a number of factors that may explain leukemia resistance to specific regimens of therapy.359 Obviously, the next step is to take these observations to the level of the proteome and combine them with the results of gene expression profiling and the power of bioinformatics. Only then will it be possible to understand how alterations in apoptotic regulators affect the response of the cancer cell to treatment, and to identify other key molecular targets that might be exploited in therapy for resistant disease. REFERENCES 1 Noskova, V., Dzubak, P., Kuzmina, G., et al. In vitro chemoresistance profile and expression/function of MDR associated proteins in resistant cell lines derived from CCRF-CEM, K562, A549 and MDA MB 231 parental cells. Neoplasma, 2002; 49: 418–25. 2 Bodo, A., Bakos, E., Szeri, F., Varadi, A., & Sarkadi, B. The role of multidrug transporters in drug availability, metabolism and toxicity. Toxicol Lett, 2003; 140–141: 133–43. 3 Ribrag, V., Massaad, L., Janot, F., et al. Main drug-metabolizing enzyme systems in human non-Hodgkin’s lymphomas sensitive or resistant to chemotherapy. Leuk Lymphoma, 1995; 18: 303–10.

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329 Baur, A. S., Shaw, P., Burri, N., et al. Frequent methylation silencing of p15(INK4b) (MTS2) and p16(INK4a) (MTS1) in Bcell and T-cell lymphomas. Blood, 1999; 94: 1773–81. 330 Taniguchi, T., Chikatsu, N., Takahashi, S., et al. Expression of p16INK4A and p14ARF in hematological malignancies. Leukemia, 1999; 13: 1760–9. 331 Christiansen, D. H., Andersen, M. K., & Pedersen-Bjergaard, J. Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia, 2003; 17: 1813–19. 332 Gonzalez-Gomez, P., Bello, M. J., Arjona, D., et al. CpG island methylation of tumor-related genes in three primary central nervous system lymphomas in immunocompetent patients. Cancer Genet Cytogenet, 2003; 142: 21–4. 333 Kastan, M. B., Lim, D. S., Kim, S. T., Xu, B., & Canman, C. Multiple signaling pathways involving ATM. Cold Spring Harb Symp Quant Biol, 2000; 65: 521–6. 334 Melino, G., Lu, X., Gasco, M., Crook, T., & Knight, R. A. Functional regulation of p73 and p63: development and cancer. Trends Biochem Sci, 2003; 28: 663–70. 335 Westfall, M. D. & Pietenpol, J. A. p63: molecular complexity in development and cancer. Carcinogenesis, 2004; 25: 857–64. 336 Gumy-Pause, F., Wacker, P., & Sappino, A. P. ATM gene and lymphoid malignancies. Leukemia, 2004; 18: 238–42. 337 Camacho, E., Hernandez, L., Hernandez, S., et al. ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mutations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbalances. Blood, 2002; 99: 238–44. 338 Pettitt, A. R., Sherrington, P. D., Stewart, G., et al. p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation. Blood, 2001; 98: 814–22. 339 Gronbaek, K., Worm, J., Ralfkiaer, E., et al. ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma. Blood, 2002; 100: 1430–7. 340 Irwin, M. S. Family feud in chemosensitivity: p73 and mutant p53. Cell Cycle, 2004; 3: 319–23. 341 Irwin, M. S., Kondo, K., Marin, M. C., et al. Chemosensitivity linked to p73 function. Cancer Cell, 2003; 3: 403–10. 342 Di Como, C. J., Gaiddon, C., & Prives, C. p73 function is inhibited by tumor-derived p53 mutants in mammalian cells. Mol Cell Biol, 1999; 19: 1438–49. 343 Strano, S., Munarriz, E., Rossi, M., et al. Physical and functional interaction between p53 mutants and different isoforms of p73. J Biol Chem, 2000; 275: 29 503–12. 344 Martinez-Delgado, B., Melendez, B., Cuadros, M., et al. Frequent inactivation of the p73 gene by abnormal methylation

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or LOH in non-Hodgkin’s lymphomas. Int J Cancer, 2002; 102: 15–19. Leupin, N., Luthi, A., Novak, U., et al. P73 status in B-cell chronic lymphocytic leukaemia. Leuk Lymphoma, 2004; 45: 1205–7. Wang, M. L., Tuli, R., Manner, P. A., et al. Direct and indirect induction of apoptosis in human mesenchymal stem cells in response to titanium particles. J Orthop Res, 2003; 21: 697–707. Lane, D. P. & Hupp, T. R. Drug discovery and p53. Drug Discov Today, 2003; 8: 347–55. Zhang, W., Kornblau, S. M., Kobayashi, T., et al. High levels of constitutive WAF1/Cip1 protein are associated with chemoresistance in acute myelogenous leukemia. Clin Cancer Res, 1995; 1: 1051–7. Steinman, R. A. & Johnson, D. E. p21WAF1 prevents downmodulation of the apoptotic inhibitor protein c-IAP1 and inhibits leukemic apoptosis. Mol Med, 2000; 6: 736–49. Roman-Gomez, J., Castillejo, J. A., Jimenez, A., et al. 5 CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia. Blood, 2002; 99: 2291–6. Raveh, T., Droguett, G., Horwitz, M. S., DePinho, R. A., & Kimchi, A. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol, 2001; 3: 1–7. Shohat, G., Spivak-Kroizman, T., Eisenstein, M., & Kimchi, A. The regulation of death-associated protein (DAP) kinase in apoptosis. Eur Cytokine Netw, 2002; 13: 398–400. Ng, M. H. Death associated protein kinase: from regulation of apoptosis to tumor suppressive functions and B cell malignancies. Apoptosis, 2002; 7: 261–70. Katzenellenbogen, R. A., Baylin, S. B., & Herman, J. G. Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood, 1999; 93: 4347–53. Shiramizu, B. & Mick, P. Epigenetic changes in the DAP-kinase CpG island in pediatric lymphoma. Med Pediatr Oncol, 2003; 41: 527–31. Galm, O., Wilop, S., Reichelt, J., et al. DNA methylation changes in multiple myeloma. Leukemia, 2004; 18: 1687–92. Nakatsuka, S., Takakuwa, T., Tomita, Y., et al. Hypermethylation of death-associated protein (DAP) kinase CpG island is frequent not only in B-cell but also in T- and natural killer (NK)/T-cell malignancies. Cancer Sci, 2003; 94: 87–91. Voso, M. T., Scardocci, A., Guidi, F., et al. Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes. Blood, 2004; 103: 698–700. Cheok, M. H., Yang, W., Pui, C. H., et al. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nat Genet, 2003; 34: 85–90.

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13 Heritable predispositions to childhood hematologic malignancies Doan Le, Kevin Shannon, and Beverly J. Lange

Introduction Inherited cancer syndromes were estimated to account for only 0.1% of all malignant neoplasms in 1990; however, a known genetic predisposition was observed in 4.2% of pediatric cases. 1 As new genes and more complex patterns of inheritance and penetrance have been uncovered, the proportion of both childhood and adult cancers arising in patients with a known constitutional susceptibility has continued to rise. A survey performed by Draper and associates 2 identified four clinical syndromes that accounted for approximately 90% of children who developed cancer in the context of an inherited predisposition. Two of these – familial Wilms tumor and retinoblastoma – are caused by high-penetrance germline mutations that are rare in the general population. By contrast, neurofibromatosis type 1 (NF1) and Down syndrome are common genetic disorders that are associated with a much lower overall risk of cancer. However, because each conditon is readily diagnosed on the basis of a constellation of clinical features, the elevated incidence of cancer relative to the general population is readily apparent. Although uncommon, familial cancer syndromes have been extraordinarily informative for defining general mechanisms of tumorigenesis and for discovering the responsible genes. In addition, many of the genes that confer a genetic predisposition to childhood cancer play a central role in normal cellular growth control. Furthermore, because most human cancers carry multiple cooperating mutations, uncovering genetic lesions that are capable of initating tumorigenesis in vivo can be problematic. Identifying the molecular basis of inherited cancer predispositions reveals genes that, by definition, can serve as the initiating event(s) in oncogenesis. Understanding the

genetic basis of inherited cancer predispositions also provides a starting point for probing biochemical pathways that are perturbed in cancer cells and can identify targets for therapeutic intervention. In this chapter, we first review general principles of cancer genetics, and then discuss some of these principles in the context of childhood hematologic malignancies. The notion that a particular disorder is genetic in origin often refers to the fact that it is transmitted within families. While some human cancers are heritable genetic disorders, most patients have no known susceptibility to cancer, and malignant transformation results from a series of somatic mutations in a target cell. Cancer is therefore unique in that acquired (somatic) genetic alterations play a major role in its pathogenesis. While this paradigm appears to apply to most childhood cancers, a significant proportion arise in the context of an inherited predisposition. Familial cancers involve an inherited susceptibility that is passed from parent-to-child. In other cases, a child is predisposed to cancer as a result of a new germline mutation that is not found in other family members. Cancer risks are highly age- and disease-specific. Down syndrome is the only inherited condition that is consistently associated with an elevated risk of B-lineage acute lymphoblastic leukemia (ALL), the most common childhood cancer. This may speak to the importance of environmental exposures in leukemogenesis, which is discussed in other chapters of this book. Children and adolescents with ataxia telangiectasia are strongly predisposed to B-cell lineage lymphomas but do not develop myeloid malignancies. Fanconi anemia and congenital neutropenia show the opposite patterns (i.e. myeloid, but not lymphoid, cancers). A second general consideration is related to the development of myeloid malignancies. Leukemias in patients with

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Shwachman–Diamond syndrome, congenital neutropenia, or Fanconi anemia arise in the context of hematopoietic deficits, and are associated with a high incidence of myelodysplasia and other unfavorable clinical and biologic features, including frequent acquisition of complete or partial deletion of chromosome 7 in the malignant cells. 3 Increased susceptibility to DNA damage and/or global genomic instability are likely to underlie these cases, as well as those associated with Bloom syndrome. By contrast, infants and young children with Down syndrome, NF1, or Noonan syndrome show a strong predilection to develop proliferative disorders, and genetic analysis has implicated hyperactive RAS signaling in some of these diseases. A number of distinct genetic mechanisms underlie inherited cancer predispositions. These include mutations of cellular proto-oncogenes, inherited mutations in both alleles of a recessive gene, gross chromosomal abnormalities, and germline inactivation of one allele of a tumor suppressor gene, with or without somatic loss of the normal allele (Table 13.1). We discuss specific instances in which each of these mechanisms leads to hematologic malignancies later in this chapter. Inherited dominant oncogene mutations are rarely responsible for human cancer syndromes; however, the predilection of patients with Noonan syndrome to develop juvenile myelomonocytic leukemia (JMML) is due to dominant mutations in the PTPN11 gene, which deregulates SHP-2 phosphatase activity. Autosomal recessive disorders that carry an increased risk of hematologic malignancies include ataxia telangiectasia, Fanconi anemia, Shwachman–Diamond syndrome, and Bloom syndrome. In these patients, malignant clones arise as a result of acquired mutations at cooperating loci. The constitutional chromosomal abnormality found in children with Down syndrome confers an increased risk of neonatal myeloproliferative disease and acute leukemia. Haploinsufficiency (inactivation of one allele of a putative tumor suppressor) appears to initiate leukemogenesis in familial platelet disorder/acute myeloid leukemia (AML) syndrome. Germline inactivation of one allele of tumor suppressor genes accounts for the majority of heritable human cancer risk in both children and adults. The susceptibility of children with NF1 to JMML and other myeloid leukemias illustrates this mechanism. Given the overall contribution of tumor suppressor mutations to cancer susceptibility, a brief discussion of this class of genes follows. In 1971, Knudson developed a hypothesis to explain the peculiar epidemiology of retinoblastoma. Clinically, children with retinoblastoma show either early onset disease that is characterized by frequent bilateral involvement and by an autosomal dominant pattern of inheritance in many

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Table 13.1 Genetic mechanisms underlying heritable predispositions to leukemias Syndrome

Inheritance

DS

New mutation Unknown/21q22 Unknown (dominant) Dominant or new NF1/17q11.2 Restrain RAS signaling mutation Dominant PTPN11/12q24 Signal transduction: SH2 phosphatase activation Autosomal SBDS/7q11 Unknown recessive Autosomal BRCA2/13q12.3 Multicomponent DNA recessive repair pathway FANCA/16q24.3 FANCC/9q22.3 FANCD2/3p26 FANCE/6p21–p22 FANCF/11p15 FANCG/9p13 FANCL/2p16.1 Autosomal ElA2/19q or Neutrophil elastase or recessive unknown unknown Autosomal Unknown Unknown dominant Autosomal CBFA2/21q22 Transcriptional dominant regulation of hematopoiesis Autosomal ATM/11q22–23 DNA repair; cell cycle recessive regulation Autosomal NBS/8q21–24 DNA repair; telomere recessive lengthening Autosomal BLM/15q26.1 DNA repair recessive

NF1 NS

SDS/S(B)DS FAa

SCN/KS FM7 FPD/AML

A-T NBS BS

Location

Gene function

Abbreviations: DS, Down syndrome; NF1, von Recklinghausen neurofibromatosis type 1; NS, Noonan syndrome; S(B)DS, Shwachman–(Bodian)– Diamond syndrome; SCN, severe congenital neutropenia; KS, Kostmann syndrome; FA, Fanconi anemia; FM7, familial predisposition to monosomy 7; FPD/AML, familial platelet disorder/ acute myeloid leukemia; A-T, ataxia telangiectasia; NBS, Nijmegan breakage syndrome; BS, Bloom syndrome. a FA involves mutations in genes for at least eight related complementation groups involved in DNA repair, two of which have turned out to be mutations in the BRCA2 gene.

families, or later onset, unilateral disease and a negative family history. According to Knudson’s model, children in the group with early onset, heritable retinoblastoma were at markedly increased risk of cancer because all of their somatic cells carry an inactive copy of a gene that plays a central role in regulating the growth of immature retinal cells (“first hit”). An acquired mutation of the single normal

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Fig. 13.1 Analysis of tumor specimens for loss of heterozygosity (LOH). These assays, which are based on the highly polymorphic nature of human DNA, allow investigators to follow the inheritance and somatic loss of disease-associated specific alleles through family studies. LOH analysis was initially based on ascertaining restriction-fragment-length polymorphisms (RFLPs) by Southern blot analysis. More recently, high throughput methods for genotyping normal and tumor DNA samples have been developed that are based on the use of microsatellite markers, which consist of variable nucleotide tandem repeats (VNTRs), or on the detection of single nucleotide polymorphisms. In this example, amplification using oligonucleotide primers that detect a polymorphic marker at a tumor suppressor locus reveals a larger fragment from one of the father’s chromosomes (labeled B) and a shorter (C) fragment. The fragments amplified from maternal DNA are slightly longer (A) and shorter (D) than the paternal alleles. The son has inherited the B allele from his father and the D allele from his mother (D). LOH is observed when the normal maternal allele and flanking DNA sequences are deleted in the tumor. In cases of familial cancer, such as retinoblastoma or Wilms tumor, the “second hit” invariably involves loss of the allele that was inherited from the unaffected parent with retention of the mutant allele from the affected parent.

genetic mechanisms including structural deletions, point mutations, or gene conversion events. Most of the known tumor suppressor genes were identified by “reverse genetics;” that is, their protein products were only known after the gene was cloned by ascertaining the position of the gene by linkage. Analysis of tumor specimens for loss of constitutional heterozygosity (LOH) has provided a powerful tool for localizing human tumor suppressor genes to specific chromosomal regions. The underlying principle is simple. Somatic inactivation of tumor suppressor genes is frequently caused by structural deletions of DNA and, in turn, can be detected by finding that a heterozygous locus in normal tissues is reduced to homozygosity in the tumor (Fig. 13.1). The availability of highly polymorphic markers whose exact positions are known in the genome has provided powerful tools for studies of LOH. Targeted homologous recombination into murine embryonic stem (ES) cells has proved invaluable in characterizing how tumor suppressor genes function in normal tissues and for generating accurate mouse models of many cancers, including some pediatric hematologic malignancies. Clinicians caring for children with hematologic malignancies should be alerted to the possibility of an underlying predisposition, which might affect how an individual patient is managed as well as donor selection when hematopoietic stem cell transplantation (HSCT) is a therapeutic consideration. Ultimately, insights gained from studies of inherited susceptibilities to leukemia and lymphoma are likely to improve the care of affected individuals and of children who develop hematologic malignancies de novo.

Down syndrome Demography

allele in any susceptible cell (“second hit”) inactivates the gene and is required for tumor formation. Because every somatic cell contains the first hit, patients with germline mutations of tumor suppressor genes develop cancer at a relatively young age and are prone to multiple independent tumors. Importantly, although the cancer predisposition is transmitted as a dominant trait, the disease is recessive at the cellular level due to somatic inactivation of the normal allele. Many tumor suppressor genes, such as TP53 and APC, that account for rare familial cases of cancer are frequently mutated in common sporadic cancers. In this instance, both copies of the tumor suppressor gene are affected by independent somatic mutations. Tumor suppressor genes are inactivated by a variety of

Down syndrome (DS) encompasses the vast spectrum of physical, immunologic, metabolic and genetic disorders that accompany constitutional trisomy 21. DS occurs in 1/700 to 1/1000 live births. Most cases harbor new mutations caused by nondysfunction in the first or, less often, second meiotic division in pregnancies of women over age 35 years, with risk increasing with increasing maternal age. 4

Phenotype A short list of physical characteristics of children with DS includes flat facies with a large protruding tongue, oblique palpebral fissures, an inner epicanthic fold that

Heritable predispositions to childhood hematologic malignancies

Table 13.2 Leukemia in patients with and without Down syndrome

Risk Median age Median WBC(×109 /L) – FAB type Immunophenotype CD7 T-lineage Karyotype Normal t(8;21);t(15–17);inv(16) t(1;19);t(4;11);t(9;22) Hyperdiploidy (DNA index > 1.16) Survival

AML

ALL

TMDa DS

DS

NDS

DS

NDS

1/10 Neonate 7.6–9.7 M7

1/100 2 years 19.9–25.2 M7, MDS

1/10,000 7 years M4, M5

1/100 6 years <20 L1

1/2200 4 years <20 L1

– –

84–100% –

19–30% –

– 0–5%

– 10–16%

Most – – – >80%

16–24% 0–2% – – 64–100%

14–24% 24–26% – – 31–56%

44–58% – 0% 7–12% 21–76%

31–55% – 4–10% 26% 28–83%

Abbreviations: TMD, transient myeloproliferative disorder; DS, Down syndrome; NDS, no Down syndrome; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; FAB, French-AmericanBritish Cooperative Group. Percentages are estimates based on ranges in published series.4 a TMD occurs almost exclusively in DS patients.

inserts on the lower lid, speckled iris (Brushfield spots), small ears, brachycephaly, thickened nuchal fold, bilateral palmar creases, wide space between the first and second toe, short femurs often with associated hip dysplasia, broad flat hands, hypoplasia of the middle phalanx of the fifth digit (clinodactyly) and other abnormalities. 5 One-third to one-half of the children with DS have congenital heart disease, most commonly endocardial cushion defects with AV canals, ventricular septal defects, and atrial septal defects. Five percent have major gastrointestinal anomalies including Hirschsprung disease, duodenal atresia, tracheoesophageal fistula and esophageal atresia. Fifteen percent have a cervical spine abnormality and 80% have conductive hearing loss. There is an increased incidence of cataracts and strabismus. Twenty percent develop hypothyroidism. Fertility is reduced in most patients. 5 Down syndrome patients also have many immunologic abnormalities including a significantly lower serum IgG level in newborns and generally higher IgG levels in older children with variability in levels of IgM and IgA; response to vaccine is variable but often reduced. The thymus is morphologically abnormal. The absolute lymphocyte count is high, but there is a significant reduction in the proportion of cells expressing the TCRA and TCRB genes and a reduced proportion of CD4+, CD45 RA + cells compared to controls. 6 T-cell function is reduced: patients show anergy to dinitrochlorobenzene and low responsiveness to mitogens and ubiquitous antigens in vitro. 7 Chemotaxis,

phagocytosis and nitroblue tetrazolium (NBT) reduction are also reduced. 8 The net result of these abnormalities is that children with DS tend to have an increased frequency of infections, particularly respiratory infections. They also manifest persistent antigenemia for hepatitis B, which in turn is associated with increased risk of autoimmune thyroiditis, 9,10 and show an increased risk of celiac disease. 11 The life-span of DS patients has increased from 25 years in 1983 to 49 years in 1997. 12 Congenital anomalies and defective immunity contribute to early death. 10 About 1% of children with DS develop leukemia. 4,13,14 The frequencies of lymphoid and myeloid leukemia are roughly the same. There are three distinct syndromes of leukemia in children with DS: (1) a congenital or neonatal myeloproliferative disorder (TMD) that most often regresses spontaneously; (2) AML, which is usually acute megakaryoblastic or erythromegakaryoblastic leukemia or a myelodysplastic syndrome; and (3) B-lineage ALL. The clinical features of these three syndromes are listed in Table 13.2. In contrast to this remarkable increase in acute leukemia, children with DS have a reduced incidence of other types of cancer. 15

Pathogenesis All of the clinical and phenotypic abnormalities in DS are the consequence of trisomy 21 in some or all cells. Trisomy

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21 is caused by nondisjunction during the first or second meiotic division. In the cases of mosaicism, the nondisjunction is postzygotic. 16 Approximately 90% of the children with DS have trisomy 21 in all cells. The other 10% are phenotypically normal or have only a few features typical of DS, such as DS mosaicism (5%), Robertsonian translocations (1%), ring 21 (0.5%) or partial trisomy of 21 (0.5%). 17 In over 90% of the cases, the nondisjoined chromosome is the maternal 21, the chromosome that is affected in the leukemia population. A critical region for DS has been mapped to chromosome 21q22, distal to the locus D21S55. 18 It was originally postulated that disomy of a putative leukemia-predisposition hematopoiesis gene would seem to be the pathogenic mechanism by which DS patients are at an enhanced risk of leukemia. However, it has not been possible to implicate a single known leukemia predisposing gene on 21 in leukemia in DS. It is likely that the various forms of leukemia in DS patients are a polygenic phenomenon. Among the genes that may potentially predispose to leukemia are those encoding the Alzheimer disease amyloid-associated protein A4, the DS cell adhesion molecule, 19 the multiple proteins involved in the immune response, such as CD18 and interferon gamma, 19 the tumor invasion and metastasis factor, and superoxide dismutase. 20 The groups of Kempski, Cavani, and Seghezzi 21–23 provided some evidence that loss of heterozygosity resulting from excessive crossover at 21q22 or interstitial deletions may cause loss of heterozygosity of a leukemia predisposition gene rather than a gene dose response resulting from trisomy 21. Most recent interest has concentrated on the core binding factor (CBFA2, AML1 or RUNX1) gene located at 21q22.1-22.2. CBFA2 is translocated in de novo and treatment-related AML with t(8;21), ALL with t(12;21) and CML with t(3;21) 24 and in a familial platelet disorder (FPD)/AML syndrome. 25 Deletions within CBFA2 in FPD cause haploinsufficiency and loss of function of this gene. 26 However, a loss-of-function mutation of CBFA2 has not been documented in DS-related AMKL. These negative results suggest that loss of function of CBFA2 is not sufficient to explain the pathogenesis of AML in DS patients. Wechsler et al. 27 demonstrated acquired mutations in the transcription factor gene GATA1 in the megakaryoblastic leukemia associated with Down syndrome. 27 They discovered that acquired mutations of GATA1 on chromosome 17 are a unique and obligatory feature of AMKL in DS cases. 27 These mutations are present in all cases of TMD and AMKL in DS and are absent in ALL and nonAMKL myeloid leukemia in DS and absent in AMKL or MDS in patients without DS. 27–29 Using neonatal blood-

spots and cord blood samples, Ahmed et al. have demonstrated that GATA1 mutations are uniformly present at birth in DS patients who have or who will develop TMD or AMKL. GATA1 mutations are also present at birth in 10% of DS who have not developed AMKL and were absent in 62 cord bloods of children without DS. 30 Thus, both constitutional trisomy 21 and GATA1 mutation are necessary for but not sufficient for the development of the TMD and AMKL in children with Down syndrome. The mutations invariably result in the introduction of a stop codon in the N-terminal activation domain leading to the synthesis of incomplete GATA1 with a reduced transactivation potential. 27 At this time the relation between GATA1 mutation and trisomy 21 is unclear. Wechsler et al. 27 propose that manipulation of the syntenic region on chromosome 16 in the murine mouse model of DS may enable us to understand the action between the trisomy and the mutation of GATA1. There are few leads about the pathogenesis of ALL in DS. There is a relative lack of T-cell ALL and of common translocations including the t(1;19), t(2;8), t(8;14), t(8;22), t(11q23) and t(12:21). 31–36 Recurring abnormalities are an additional acquired 21; however, the favorable high hyperdiploid disease is rare, and an additional X is a unique additional chromosome in DS ALL. 35 TEL/AML1 fusion transcripts have not been detected in DS ALL blasts, 37 although they occur in more than 20% of pediatric ALL cases.

Therapy In the early years of cytotoxic therapy for leukemia, patients with DS were frequently not entered on the protocols of cooperative group clinical trials. However, by the 1980s, changes in the legal and social standards gradually led to their participation. It soon became clear that children with DS responded differently to cytotoxic therapy than did other children. Until the last decade, children with DS and ALL had a higher rate of treatment failure and a higher rate of toxic death than did other children. 4,34,35,38 Although the leukemic cells of patients with DS differ in subtle ways from those of other pediatric ALL cases, these findings do not explain the differences in outcome. During induction therapy, patients with DS are more likely to experience slow early responses, induction failure, pancreatitis, pneumonia and death.34,33, 37,38 After induction their poor tolerance of ALL therapy can be attributed in part to their underlying immunologic and anatomic defects; however, specific proteins coded for by genes on chromosome 21 have also been implicated. Belkov et al. 39 have shown that extra copies of the reduced folate carrier on 21 increased the methotrexate polyglutamate levels in leukemic blasts, 39 which would

Heritable predispositions to childhood hematologic malignancies

account for the cells’ exquisite sensitivity to methotrexate. They have hypothesized that copy number may have the same effect on the nonmalignant cells in DS patients, leading to enhanced toxicity. In recent series reported by the Pediatric Oncology Group (POG) and the Children’s Cancer Group (CCG), the DS and non-DS patients with ALL had similar outcomes when treated according to current protocols. 4 The DS patients more frequently required augmentation of protocol therapy to adjust for slow early responses. The experiences with therapy in AML and DS contrast markedly with that in ALL. The leukemic blasts are more sensitive to conventional AML therapy. Five-year survival rates are frequently 80% or higher, nearly double that for non-DS patients. 40–46 DS patients do not benefit from the more dose-intensive therapies or marrow transplant in first remission. 41,42,44,47 The relatively good outcomes in DS patients with AML are probably the result of both a different disease and a different host. Patients with DS present with a lower white count, a higher rate of myelodysplastic syndrome and a strong predominance of AMKL or AEL/AMKL, and at a younger age compared to children without DS. 4 GATA1 mutations are a unique marker of a different disease. 27 The most common cytogenetic abnormality is a +21 with an acquired 21. Translocations, both favorable and unfavorable, are exceptional. Few patients have monosomy 7 in association with their leukemia, but they tend to have an outcome similar to that of other DS patients with leukemia. Taub et al. 45 have demonstrated that the leukemic blasts of DS patients have extra copies of the cystathionine B synthase (CBS) gene, which correlates with an enhanced sensitivity of the cells to cytarabine. The CBS promoter in DS AMKL blasts has substantially greater activity than in the megakaryoblasts of patients without DS. 48 The blasts consequently have higher levels of ara-CTP. The current practice is to treat children and infants with DS and AML with cytarabine-based chemotherapy of low or moderate intensity. The transient myeloproliferative disorder (TMD) occurs in utero or in the first one or two months of life in about 10% of DS neonates. 23,49–52 It is a clonal megakaryocytic proliferation derived from a common erythromegakaryoblastic progenitor. 53,54 The percentage of blasts in the peripheral blood is typically higher than in the marrow. 13 Acquired cytogenetic abnormalities are rare. In most cases the TMD resolves spontaneously in weeks to months. 55 At least 20% of the DS patients who have TMD in the newborn period develop AMKL in infancy or early childhood. 29 The risk of AMKL appears lower in mosaic DS patients with TMD than in those with complete constitutional

trisomy 21. 56 Sometimes the second leukemia is accompanied by new acquired cytogenetic abnormalities and frequently an acquired fourth chromosome 21. 57 The conventional approach is to withhold cytotoxic therapy and use supportive care, such as leukapheresis, to deal with the problems of tumor burden. There is a subset of patients with TMD who succumb to fatal complications either in utero from hydrops fetalis or in the perinatal period from the complications of visceral fibrosis, most often hepatic fibrosis. 58,59 Usually the hepatic fibrosis is fatal in a matter of weeks to months. 60 Al Kasim et al.61 have described complete resolution of hepatic fibrosis in three infants treated with low-dose cytarabine. This group and Rizzari et al.62 have documented complete resolution after two 3-day courses of cytarabine and daunomycin 4 weeks apart. It is not yet clear why the AMKL in the neonatal period is a transient myeloproliferative syndrome, whereas AMKL not infrequently in the same patient some months later behaves more like AML, requiring cytotoxic therapy albeit of reduced intensity. Holt et al.63 have hypothesized that increased telomerase activity characterizes the aggressive forms of TMD, which may be fatal as a result of tumor burden or visceral fibrosis and acute AMKL in later infancy, showing that the increased telomerase activity correlates statistically with the more aggressive leukemias in DS patients. 61 Taub and Ravindranath52 have advanced a plausible and testable explanation for spontaneous regression of AMKL, based on endogenous folate deprivation leading to the death of megakaryoblasts. 52 Elevated endogenous CBS lowers endogenous levels of folate pathway metabolites: homocysteine, methionine, S-adenosylmethionine and S-adenosylhomocysteine. Sufficient amounts of the latter two metabolites could alter CpG methylation and gene expression. Additionally, in DS, there is an increased frequency of polymorphisms of methylene, tetrahydrofolate reductase (MHFR) and methylene synthase reductase (MTRR). These also could contribute to endogenous folate deficiency. Nonetheless, there is still no connection between GATA1 mutation and folate deprivation and the fact that the age of the patient appears to be a critical host factor.

Neurofibromatosis type 1 Demography With an incidence of 1 in 3000, neurofibromatosis type 1 (NF1) is the most common inherited cancer predisposition syndrome. Approximately 50% of cases are familial, and the

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remaining patients have new mutations. The incidence of NF1 is similar in all ages and ethnic groups.

Phenotype NF1 is characterized by a constellation of clinical findings, including multiple caf´e au lait macules, axillary freckling, and Lisch nodules of the eye. 62 Many children with NF1 have macrocephaly and about 40% have learning disabilities. Persons with NF1 are predisposed to benign and malignant neoplasms, which most often arise in cells derived from the embryonic neural crest. 63 The development of a few to thousands of cutaneous neurofibromas is a hallmark of this disease. Plexiform neurofibromas are distinctive lesions comprised of multiple different cell types that are thought to arise in utero. These histologically benign tumors frequently enlarge during childhood; they are a major cause of morbidity in NF1 and are difficult to excise surgically. Approximately 5% evolve to malignant peripheral nerve sheath tumors. NF1 is associated with an increased risk of other tumors including low- and intermediate-grade astrocytoma, optic track glioma, and pheochromocytoma. Children (but not adults) with NF1 show a 200 to 500-fold increase in the incidence of JMML and are predisposed to myeloid malignancies associated with chromosome 7 deletions. 64–66 Clinical observations also suggest that children who receive mutagenic chemotherapy and external beam irradiation to treat a primary solid tumor are at increased risk of developing therapy-related leukemia. 67 Because the clinical phenotype varies considerably among affected individuals (including members of the same family), NF1 is diagnosed on the basis of criteria set forth by an NIH Consensus Conference. Many signs of NF1 may be absent in young children, and only appear during the first two decades of life. In some instances, the correct diagnosis is not made until adulthood.

Pathogenesis NF1 disease is caused by mutations of the NF1 gene, which is located on the long arm of chromosome 17. NF1 contains over 60 exons, and extensive molecular analysis of patient specimens has shown that about 90% of mutant alleles carry nonsense mutations that truncate the protein. Thus, most NF1 mutations are null and do not act as dominant or dominant-negative alleles. NF1 encodes a large polypeptide called neurofibromin, which is a GTPase- activating protein for p21ras (RAS). 68,69 RAS proteins function as molecular switches in signal transduction by cycling between an active, GTP-bound conformation

and an inactive, GDP-bound form. Most hematopoietic growth factors stimulate growth, at least in part, through RAS activation. RAS proteins have a slow intrinsic rate of GTP hydrolysis, which is accelerated thousands of fold by neurofibromin. Thus, neurofibromin functions as a negative regulator of RAS output. Importantly, missense RAS point mutations are the most common oncogenic lesions found in human malignancies, and are frequently detected in pancreas, lung, and colon cancers as well as in myeloid leukemia. 70 These missense mutations both impair the intrinsic RAS-GTPase and render the mutant proteins resistant to the actions of cellular GTPase-activating proteins, which bind to RAS-GTP and markedly accelerate GTP hydrolysis. The dominantly inherited cancer predisposition in persons with NF1 and the biochemical function of neurofibromin suggested that NF1 might function as a tumor suppressor gene by restraining RAS signaling. Indeed, studies of JMML bone marrows and other tumors from NF1 patients have shown frequent loss of heterozygosity, a hallmark of tumor suppressor gene inactivation. 63,71 As expected, in all cases of familial NF1 studied to date, the allele inherited from the normal parent was lost and the allele from the affected parent was retained. Biochemical analysis of JMML cells from children with NF1 revealed elevated levels of RAS-GTP and activation of downstream effector kinase cascades. 72 Furthermore, mutation detection studies strongly implicate hyperactive RAS in the pathogenesis of JMML. Molecular analysis uncovered mutations in NF1 or RAS in approximately 25% of JMML cases, but these mutations have never been reported in the same specimen. 73 Most recently, somatic mutations in PTPN11, which encodes the SHP-2 signal relay protein, have been detected in JMML samples without alterations in RAS or NF1. As discussed in the next section, PTPN11 mutations are found in about 50% of patients with Noonan syndrome, which is also associated with an increased risk of JMML. The murine Nf1 gene was disrupted by homologous recombination to create mouse models of NF1. 74,75 Heterozygous Nf1 mutant mice are predisposed to specific NF1-associated cancers, including pheochromocytoma and a myeloproliferative disorder (MPD) that resembles JMML. 75 Moreover, although homozygous Nf1-deficient embryos die in utero due to cardiac defects, Nf1−/− myeloid progenitors are hypersensitive to granulocyte-macrophage colony-stimulating factor in vitro and adoptive transfer of mutant fetal liver cells consistently induces a JMMLlike MPD in irradiated recipient mice. 76 In addition, heterozygous Nf1 mutant mice that are exposed to the alkylating agent cyclophosphamide show an increased frequency

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of myeloid malignancies, which develop with reduced latency. 77

what is observed in NF1, where relatively few patients are affected, even though the relative risk is greatly elevated above that in the general population.

Therapy Asymptomatic children with NF1 require no specific treatment or monitoring. Indeed, a perplexing aspect of the association between NF1 and JMML is the low overall incidence of leukemia (<1%), in marked contrast to the high frequency of cancer in patients with germline mutations in RB1, WT1, and some other tumor suppressor genes. Another mystery is why the risk of myeloid malignancies is restricted to young children with NF1 despite the continuous proliferation of hematopoietic cells throughout life. The management of JMML in children with NF1 is the same as in other patients, with HSCT the treatment of choice. Due to the low penetrance of leukemia in NF1, it is reasonable to use a healthy HLA-matched sibling with NF1 as a donor despite the theoretical risk of leukemia arising in the graft due to somatic inactivation of NF1. Nf1 mutant mice provide a tractable experimental model for testing new therapeutic strategies for JMML and other myeloid malignancies associated with hyperactive RAS. The risks of therapy-induced leukemia should be considered when treating other cancers in children with NF1. In particular, alkylating agents should be avoided whenever possible.

Noonan syndrome Demographics Noonan syndrome (NS) is a relatively common dominant genetic disorder with an estimated incidence of between 1 in 1000 and 1 in 2000.

Phenotype Individuals with NS have short stature, facial dysmorphism, skeletal malformations and a variety of congenital heart defects. 78,79 Congenital heart disease is an important cause of morbidity and premature mortality in NS patients. From case reports and small series of patients one can infer that children with NS are predisposed to a spectrum of hematologic abnormalities, including transient monocytosis, thrombocytopenia, transient myeloproliferative disorders, and juvenile myelomonocytic leukemia (JMML). 80,81 The magnitude of this increased risk is not known; however, JMML and other myeloid malignancies are uncommon complications of NS. This low penetrance is similar to

Pathogenesis Linkage analysis mapped the NS locus to chromosome band 12q24 in about 50% of cases. Tartaglia and colleagues80,81 subsequently used a positional candidate gene approach to identify germline missense PTPN11 mutations in affected individuals. SHP-2, the non-receptor tyrosine PTPase encoded by PTPN11, contains two src homology 2 (SH2) domains and a catalytic PTPase domain. The SHP-2 PTPase is activated by binding to phosphotyrosyl peptides through its N-SH2 domain. 82,83 Most of the mutations reported in NS kindreds are found in exons 3 and 8, which encode segments of the N-SH2 and PTPase domains, respectively. The results of molecular modeling suggest that almost all of these exon 3 mutations activate phosphatase activity by altering N-SH2 amino acids that interact with the PTPase domain. 78,79 SHP-2 participates in signal transduction downstream of growth factor receptors to regulate multiple responses including proliferation, differentiation, and migration. 84,85 The protein is expressed at high levels in hematopoietic cells and undergoes rapid tyrosine phosphorylation upon activation of multiple growth factor receptors.87–89 SHP-2 most often plays a positive role in transducing signals, which is mediated, at least in part, through the RAS/RAF/ERK cascade in hematopoietic and nonhematopoietic cells. 84–86 Together, reports of JMML in patients with NS, molecular data implicating hyperactive RAS in the pathogenesis of JMML, and the biochemical function of SHP-2 in relaying signals from hematopoietic growth factor receptors to RAS suggest that mutations in PTPN11 might contribute to the pathogenesis of JMML. Indeed, JMML cells from patients with NS consistently demonstrate mutations in PTPN11, which encode amino acid substitutions that are relatively uncommon in NS patients without JMML. 87,88 Somatic PTPN11 mutations are also found in JMML specimens from about 35% of patients without NS. 87,88 These mutations introduce amino acid substitutions into key positions within the NSH2 domain that make contact with the PTPase domain. Moreover, genetic analysis of JMML bone marrows has uncovered mutations in PTPN11, RAS, or NF1 in about 85% of cases. These results support the idea that proteins encoded by these genes are components of the same signaling pathway deregulated in JMML cells. However, biochemical analyses of RAS activation have shown inconsistent results in hematopoietic and nonhematopoietic cell lines. 87,88 It is uncertain why JMML is relatively rare in NS.

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While generally consistent with the existence of specific mutations that are more leukemogenic than others, the results reported to date do not entirely account for the low penetrance of JMML in NS. It is attractive to speculate that one or more cooperating events are required for full transformation. Consistent with this notion, loss of the normal PTPN11 allele was detected in one case of JMML with a missense mutation in the retained allele, and PTPN11 mutations and monosomy 7 were found to coexist in some cases. 87,88 Although PTPN11 is an attractive candidate oncogene in other hematopoietic malignancies, the screening studies performed to date suggest that it is mutated infrequently in AML, chronic myelomonocytic leukemia, and myelodysplastic syndrome. A number of laboratories are working to engineer strains of mice that express NS and leukemia-associated PTPN11 mutations, but no published data are available at this time.

Therapy As in NF1, asymptomatic children with NS require no specific intervention or monitoring due to the low incidence of hematologic malignancy. The management of JMML in children with NS is the same as in other patients, although treatment may be complicated by intercurrent cardiac disease. It is reasonable to closely monitor infants and very young children with NS who appear well but have evidence of myeloproliferation without specific therapy, as hematologic abnormalities resolve spontaneously in some cases.

Shwachman–Diamond syndrome Demographics Shwachman–Diamond syndrome (SDS) is a rare autosomal recessive disorder. Although it is the second most common cause of pancreatic insufficiency, its precise incidence is unknown.

Phenotype Pancreatic insufficiency and bone marrow dysfunction are sine qua non for a diagnosis of SDS. 89,90 Other findings are hepatomegaly, low birth weight, delayed puberty, short stature, dental abnormalities, metaphyseal chondrodysplasia (65%), rib abnormalities (90%), developmental delay (85%), hypotonia, and, rarely, deafness and retinitis pigmentosa. 91,92 The pancreatic insufficiency is characterized by normal ductal function, preservation of the islets of Langerhans, reduced enzymatic function and

a small or fatty pancreas on magnetic resonance imaging or computed tomography (CT) scans. 93,94 Patients typically maintain normal water and electrolyte excretion, and they have steatorrhea early in life that may improve over time. 95 Hematopoiesis in SDS varies more than pancreatic function, even among siblings with SDS. 96 Neutropenia occurs in all patients, but in about two-thirds it may be cyclic. In a series of 88 SDS patients, Ginzberg et al.96 found neutropenia in 98%, anemia in 45%, and thrombocytopenia in 34%. Anemia is macrocytic with elevated Hgb F. 89,95,97 The marrow shows increased fibrosis and fat and reduced numbers of CD34+ colony forming cells. 98 Trilineage hypoplasia occurs in about one-third of the patients. Dror et al. 99 documented immunologic abnormalities in 11 patients, seven of whom experienced recurrent bacterial infections and six recurrent viral infections. All were neutropenic, and ten had normal lymphocyte counts. Nine patients had B-cell defects including low IgG or IgG subclasses, absence of specific antibody production, and reduced proliferation in vitro. Seven had T-cell abnormalities, and five of six had reduced circulating natural killer cells. Patients with SDS are prone to develop MDS, AML, and rarely ALL. 96,97,100–104 Proposed diagnostic criteria for the SDS pancreatic phenotype are a serum immunoreactive trypsinogen of 15.5 g/L or less at age 3 years or older with a serum isoamylase of less than 16 U/L at any age. 94 Serum trypsinogen frequently increases to normal levels in infancy and early childhood. Proposed criteria to diagnose marrow dysfunction are as follows: (1) neutropenia with an absolute neutrophil count of less than 1500/mm3 on at least three occasions over 3 month or more; (2) anemia; (3) platelet count of less than 150,000/mm3 ; (4) pancytopenia; and (5) MDS documented by marrow examination. 92 Supporting features includes skeletal abnormalities, hepatomegaly, short stature, and a history of frequent respiratory infections. 92

Pathogenesis The clinical manifestations of SDS are the result of the mutation of a single gene. Using linkage studies, investigators at the University of Toronto have mapped the gene to 7q11, 105 where they recently identified recurring mutations in a new gene, designated SBDS. 106 In close proximity to SBDS is a pseudogene, SBDSP, with 97% sequence homology to SBDS. Recombination of SBDS with SBDSP results in gene conversion, an established mechanism of mutagenesis. Of 158 individuals with SDS, 141 had conversion mutations resulting in the genotype 183→184→CT and 258→2T→C, while 44 had conversions with the genotype 258→2T→C and X. 106

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The predisposition to MDS and leukemia is probably a result of a number of intrinsic abnormalities in the marrow progenitors. In one patient a high incidence of increased spontaneous chromosome breakage was demonstrated on three specimens of PHA-stimulated lymphocytes; there was no increase with mitomycin C. 107 Compared to normal controls, the CD 34+, CD34–/CD38+, and CD34–/CD38– fractions of marrow from SDS patients with and without MDS showed significantly higher FAS expression and greater apoptosis in vitro. 98 Although early estimates of the risk of MDS and leukemia ranged from 5% to10%, Smith et al.101 found that among 21 patients followed for 25 years, seven (33%) developed MDS and five AML, making SDS the genetic disorder with the highest predisposition of all for the development of myeloid malignancy. 97 Analysis of patients with SDS and severe congenital neutropenia (SCN) registered in the large Severe Chronic Neutropenia International Registry (SCHNR) revealed that the development of MDS and AML was preceded by a succession of genetic aberrations, including nonrandom clonal cytogenetic abnormalities, activating mutations of RAS and other oncogenes, and G-CSF mutations. Dror et al.103 found that four patients (29%) had cytogenetic abnormalities that were del(20q) in two patients and i(7q) and both del(20q) and i(7q) in one. 99 No patient showed progression to AML or MDS; in one the abnormal clone spontaneously regressed. Smith et al.112 described a boy with SDS who developed i(7q) at age 5 years that persisted through age 14 years, while del (20q) appeared intermittently after age 11 years. 108 Some have proposed that use of recombinant human granulocyte colony-stimulating factor to remedy neutropenia in patients constitutionally predisposed to bone marrow failure may increase their risk of developing MDS and/ or AML. 109 However, the Severe Congenital Neutropenia Registry (SCNR) review of patients with SDS and SCN did not find any significant correlation between the incidence of MDS or AML and dose or duration of G-CSF therapy. 104

Therapy Proceedings of the International Shwachman–Diamond Family Conferences and the First International Scientific Meeting provide concise guidelines for the management of children with SDS. 92 After diagnosis, the report recommends a baseline history; physical examination; nutritional assessment; measurement of the serum aminotransferase and vitamins A, D, and E levels; skeletal survey; 72-hour fecal fat collection; CBC; marrow aspirate; biopsy; and cytogenetics analysis. The initial follow-up should occur as

frequently as every 1 to 3 months to ascertain the efficacy of interventions and every 6 to 12 months thereafter. A formal hematology follow-up with marrow studies should take place yearly or as clinically indicated. The long-term use of G-CSF to treat neutropenia has been controversial because of concerns over provoking leukemia.

Treatment and prognosis Because clonal cytogenetic abnormalities in the marrow may persist for years and may even disappear, pre-emptive use of chemotherapy or marrow transplantation to treat cytogenetic changes does not appear warranted. There is one report in the literature of successful treatment of AML in a 21-year-old woman with SDS using conventional chemotherapy followed by allogeneic marrow transplantation. 110

Fanconi anemia General description Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and a predisposition to myeloid malignancies and other cancers.

Demographics With an incidence of about 1 in 100,000, FA is the most common inherited bone marrow failure syndrome. FA accounts for approximately 20% of all cases of childhood aplastic anemia. The heterozygous mutation frequency is estimated to be 1/300 to 1/600 in the general population and 1/100 in persons of Ashkenazi Jewish ancestry. 111

Phenotype Most patients with FA are identified during the first decade of life due to multiple physical anomalies in association with progressive anemia and thrombocytopenia. The mean age at diagnosis is 6 years old. Approximately 60% of patients have multiple congenital anomalies, which include low birth weight, abnormal skin pigmentation (hyperpigmentation or caf´e au lait spots), skeletal anomalies (thumb anomalies such as aplasia, hypoplasia, and supernumerary), microcephaly, renal malformations (aplasia, duplication, and ectopic or horseshoe kidney), neurologic abnormalities (strabismus, hyper-reflexia, mental retardation), micro-opthalmia, ear

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anomalies, deafness, congenital heart disease, and hypogonadism. However, 25% of patients with FA have no dysmorphic features. 111 The diagnosis is confirmed by performing chromosomal fragility testing with the alkylating agent diepoxybutane or mitomycin C. Cells from patients with FA demonstrate excess chromatic breaks, gaps, formation of radial chromosomes, endoreduplications, and other types of nonhomologous recombination. Diepoxybutane testing identifies affected individuals, but is not reliable for detecting carriers. 111 Furthermore, since the FA phenotype is due to mutations in multiple different genes (see below), a positive diepoxybutane test does not pinpoint the underlying molecular lesions. Leukemia arises in about 10% of FA patients, usually during adolescence. 112 It has been estimated that the relative risk for development of a malignant or premalignant clonal disorder in FA patients is increased 6500 to 15,000 fold above the general population, and the risk of clonal disorders involving chromosome 7 is increased approximately 50,000- to 100,000-fold. 113 Nineteen patients have been diagnosed with FA ex post facto after they have been treated for leukemia. In these cases, the results of bone marrow cytogenetic studies or extraordinarily poor tolerance of cytoreductive therapy prompted testing for FA. 114 Because patients with FA experience severe toxicity when exposed to radiation therapy or alkylating agents, it is crucial to perform diepoxybutane testing before initiating chemotherapy if there is suggestive evidence of FA, such as the presence of dysmorphic features or a prior history of cytopenias or MDS. The International Fanconi Anemia Registry (IFAR), a prospectively collected database on FA patients, published a 20-year perspective in 2003. 115 Of the 754 subjects in the study, 80% had bone marrow failure (BMF) and 23% developed cancer. Hematologic malignancies, specifically MDS and AML, accounted for 60% of these cancers. ALL, CMML, and Burkitt lymphoma were rarely reported. The remaining 40% of the cancers were nonhematologic, mainly squamous cell carcinoma of the head and neck and anogenital region, with rare cases of liver, renal, and brain tumors. By age 40, the cumulative incidence of BMF was 90%, the cumulative incidence of hematologic malignancy was 33%, and the cumulative incidence of solid tumors was 28%. The median survival time was 24 years. Univariate analysis revealed a significantly earlier onset of BMF and poorer survival for FA complementation group C compared to groups A and G (see Pathogenesis section below). There was no significant difference in the time to neoplasm development between these groups. FA patients who survive into the third and forth decades should be followed closely to

screen for solid malignancies, particularly of the head and neck and anogenital regions.

Pathogenesis A hallmark of FA cells is hypersensitivity to genetic damage induced by DNA-cross-linking agents such as diepoxybutane, mitomycin C, and ionizing radiation. FA is a heterogeneous disease at the molecular level with seven distinct complementation groups identified to date. Molecular cloning of the genes for these complementation groups led to the identification of a multicomponent DNA repair pathway comprising distinct FA proteins. 116,117 DNA damage activates a complex consisting of Fanconi proteins A, C, G, and F; this results in monoubiquitination of the FANCD2 protein. 118 The modified FANCD2 protein, in turn, relocalizes in the nucleus at sites of DNA repair, where it directly interacts with the breast cancer susceptibility protein BRCA1. Furthermore, the FANCD2 protein is phosphorylated by the ATM protein kinase in response to ionizing radiation, linking the Fanconi pathway to this checkpoint response. 116 Lastly, mutations in the BRCA2 gene, encoding a DNA repair enzyme, were shown to be the underlying abnormality in FA patients previously assigned to complementation groups B and D1. 119 These data establish a defect in DNA repair as the underlying abnormality in FA. Determining why FA patients develop bone marrow failure followed by MDS and AML is an important experimental priority. Moreover, since MDS and AML in FA patients resembles the sporadic forms of these diseases, it will be important to ascertain if acquired mutations in components of the FA pathways are involved in the pathogenesis of these myeloid malignancies in the adult.

Therapy The treatment of choice for FA patients with marrow failure, MDS, and AML is HSCT from a histocompatible sibling. In some centers, unrelated donor and cord blood transplants are performed for patients without a match. FA complementation group C patients have a more severe clinical course that may warrant earlier HSCT. 115 Modified induction regimens are used to treat AML, and novel conditioning regimens have been devised for performing HSCT in FA patients. 120–122 Some patients with marrow failure show hematologic improvement after treatment with growth factors such as erythropoietin or G-CSF. 123 Other patients respond to androgen therapy. Hirsutism and liver disease are complications of chronic androgen therapy. 123 Patients who survive with hematologic problems or HSCT have a high risk of solid tumors in the third and fourth decade

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of life and close follow-up is essential. 115 Identification of FA gene mutations is important for prenatal diagnosis and carrier detection. Genetic counseling should be provided to FA families.

Severe congenital neutropenia Severe congenital neutropenia (SCN) refers to a group of constitutional disorders of myelopoiesis characterized by low blood neutrophil counts and recurrent infections. Kostmann syndrome (KS) is a subtype of SCN with onset early in life, chronic severe neutropenia defined as an absolute neutrophil count (ANC) of less than 200/ L, and recurrent invasive bacterial infections. 124

Demography There are more than 300 SCN patients described in the literature, and all ethnic groups are affected. The estimated incidence of congenital neutropenia is one to two cases per million with equal distribution for gender. 124 The cases described by Kostmann in 1956 showed an autosomal recessive inheritance pattern, but since then autosomal dominant and many sporadic cases have been reported.

Phenotype Patients with SCN usually present in the first few months of life, and about 90% are diagnosed by 6 months of age. The most common presenting features are severe neutropenia (ANC <200/ L) and infections. The physical examination is usually normal aside from signs of infection. The bone marrows of patients with SCN frequently show a maturation arrest at the promyelocyte stage of development. 124 Pharmacologic doses of recombinant human G-GSF enhance neutrophil production in most patients with SCN, and this alleviates many of the associated life-threatening complications. With longer survival and close observation, it is now clear that SCN should be considered a preleukemic disorder. Evolution to MDS and AML has been observed in 10% to 15% of children with SCN, including occasional patients who never received recombinant G-CSF.125 Importantly, myeloid malignancies have not been reported in children and adults with cyclic neutropenia despite many years of G-CSF treatment. These data suggest that chronic exposure to G-CSF is not inherently leukemogenic, but interacts with the specific mutations that cause SCN. Banerjee and Shannon 125 reviewed 198 patients from the Severe Chronic Neutropenia International Registry with a

diagnosis of SCN who were enrolled in the initial clinical trials that tested the safety and efficacy of G-CSF. Most patients were classified as having Kostmann syndrome; however, a small percentage had other diagnoses such as Shwachman–Diamond syndrome. At the time the data was reviewed, the 198 patients had received G-CSF for an average of almost 8 years, at doses that were adjusted to achieve a target ANC. Twenty-nine patients developed MDS or AML, with a cumulative risk of 15.4% after 8 years of treatment with G-CSF. The risk of transformation was approximately 1% per year during the first 4 years of treatment, and 2% per year thereafter with considerable yearto-year variability. There was no significant relationship between the daily or cumulative doses of G-CSF and the likelihood of developing MDS/AML, and no other clinical risk factors were identified that predicted a high risk of transformation. 125 One potential problem with extrapolating these data to all patients with SCN is that this cohort was older at the start of G-CSF treatment than patients diagnosed more recently.

Pathogenesis Examination of hematopoietic subpopulations of the neutrophil lineage suggests that the myeloid progenitor cells of SCN patients undergo accelerated apoptosis compared to normal controls. 126 Colony-forming unit granulocytemacrophage (CFU-GM) growth in semisolid medium is impaired in response to recombinant G-CSF. 127,128 Other studies have demonstrated normal G-CSF production and G-CSF receptor expression on hematopoietic cells from SCN patients. 129–131 Taken together, these data support an intrinsic defect of myeloid progenitors in the pathogenesis of SCN, which is manifested by impaired responsiveness to growth factor stimulation in vitro and by defective myeloid maturation and neutrophil production in vivo. A positional candidate gene approach revealed mutations in the neutrophil elastase (ELA2) gene in individuals with cyclic neutropenia. 132 Surprisingly, ELA2 mutations were also detected in most patients with SCN.133 The ELA2 mutations identified to date in patients with SCN cluster in different segments of the gene than do those involved in cyclic neutropenia, suggesting an explanation for the clinical differences between these disorders. 134 Additional studies have addressed the molecular lesions that are associated with transformation to MDS/AML in patients with SCN. The most fascinating of these is the acquisition of somatic mutations in the G-CSF receptor that truncate the carboxyl terminus. Heterologous expression of these mutant receptors in myeloid cell lines promotes growth, blocks maturation, and impairs apoptosis. 135,136

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Monosomy 7 is also common in the bone marrows of patients with MDS and AML, and some cases also show RAS mutations. 137 Mice engineered to express the leukemia-associated GCSF receptor mutation did not develop leukemia. Interestingly, a strain of mice that models a neutrophil elastase mutation found in SCN shows normal granulopoiesis and does not develop leukemia. 138 The absence of MDS/AML in the mouse models suggests that truncating mutations of the G-CSFR are probably not leukemogenic, even in the presence of high concentrations of G-CSF unless they occur in a cell that carries another predisposing genetic lesion (i.e. the mutation that causes SCN).

Therapy Before recombinant human G-CSF became available, the treatment for patients with SCN was largely ineffective, with approximately 50% of children surviving the first year of life and 30% reaching age 5. Infections were the major cause of death. Clinical trials performed over the past decade have shown that pharmacologic doses of G-CSF are effective in raising peripheral blood neutrophil counts and reducing the risk of pyrogenic infections in the vast majority of patients. One of these studies was a Phase III trial of 123 patients with chronic severe neutropenia and recurrent infections who were randomized to either receive G-CSF on entry into the study or at 4 months of observation before treatment. The use of G-CSF was associated with a rise in the ANC to more than 1.5 × 109 / L in 90% of patients; this was accompanied by an increase in the degree of myeloid maturation in the bone marrow. Importantly, infection-related morbidity was also reduced significantly among patients who were assigned to treatment with G-CSF.139 Recognized adverse effects of G-CSF therapy included splenomegaly, bone pain, vasculitis, and osteoporosis.124 Patients should receive the lowest dose of G-CSF that is required to maintain an acceptable neutrophil count and should undergo yearly bone marrow examinations with cytogenetics evaluation. HSCT from an HLA-identical sibling is beneficial to patients who are refractory to G-CSF treatment. A more difficult question involves when to perform transplantation in SCN patients who are doing well on G-CSF and have an HLA-matched sibling donor. The limited published data indicate that transplantation cures a high percentage of patients with neutropenia only, but is usually ineffective in children who have progressed to MDS/AML. 140 Patients who acquire a G-CSF receptor mutation appear to have an elevated risk of developing MDS or AML, and this may prove useful for identifying children who should

be transplanted. 141 However, no prospective data are available to address this question.

Familial myeloid disorders with monosomy 7 Demography The myeloid malignancies that arise in the context of many of the inherited predispositions discussed in this chapter, including neurofibromatosis type 1, Fanconi anemia, and severe congenital neutropenia, frequently show bone marrow monosomy 7. Monosomy 7 also occurs in cases of MDS and/or AML that develop in related individuals. This disorder had been termed “familial monosomy 7” in the literature and is very rare with a small number of families reported to date. Monosomy 7 has been reported in at least 10 families in whom myeloid leukemia is the only apparent phenotypic abnormality. 142,143

Phenotype Kwong et al. 144 recently described a new family with myeloid malignancies and monosomy 7 and reviewed the eight previously reported families. The relevant clinical features included a young median age at presentation (8 years), equal incidence in boys and girls, and cytopenias and myelodysplasia in some non-leukemic family members. While there have been no documented multigenerational families with this type of monosomy 7, a few kindreds with one affected child have been reported in which MDS or AML developed in one or more adults. In two other families, the tendency to develop monosomy 7 appears to coinherit with cerebellar ataxia. 145,146 The disease is apparently an autosomal dominant with variable expression such that some members may manifest the neurologic disorder with normal hematopoiesis, while others have the hematologic disorder, and some both. In the first family described, the father and all five children had cerebellar ataxia or atrophy. 146 Familial childhood monosomy 7 differs from another entity that has been termed “monosomy 7 syndrome,” in that females are affected as often as males and the mean age at diagnosis is higher than in de novo cases. Familial cases of monosomy 7 are distinguished from other known predispositions by the inheritance pattern, absence of associated physical anomalies, and by laboratory findings such as the absence of chromosomal fragility on testing.

Pathogenesis Molecular genetic analysis of three families revealed that different chromosome 7 homologs were retained in the

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bone marrows of affected siblings.143 These data argue strongly against the “classic” Knudson model in which one allele of a tumor suppressor gene located on chromosome 7 is inherited in affected persons with loss of the normal allele on the deleted homolog. Inasmuch as mutations of NF1 or FANC genes confer an increased risk of myeloid malignancies associated with monosomy 7, it is plausible that mutations at a locus unlinked to chromosome 7 are responsible for the predisposition in some families. Identifying the relevant gene (or genes) on chromosome 7 is a prerequisite for ascertaining if it undergoes homozygous inactivation or contributes to leukemia by haploinsufficiency.

Therapy Clinicians should always consider the possibility that a child who presents with MDS or AML and monosomy 7 may be the first affected individual in a family. Other children were only identified when their siblings were diagnosed with MDS or AML. 142,143 Siblings of children with de novo monosomy 7 should have a physical examination and complete blood count. Bone marrow examination with cytogenetic studies are indicated if abnormalities are detected on the screening tests and are mandatory if the sibling is being considered as a donor for bone marrow transplantation. The clinical course of MDS is unpredictable, but the ultimate outcome is poor. HSCT should be considered for affected patients. The efficacy of cytoreductive chemotherapy prior to HSCT is unclear, but should probably be used in patients with AML.

Familial platelet disorder/acute myelogenous leukemia Demography Familial platelet disorder/acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disorder in which patients develop thrombocytopenia and have a propensity to develop AML. The incidence of this rare disorder is not known as there are only several families reported in the literature.

or storage pool deficiency. 147,148 In patients who develop leukemia, the subtype is usually AML M1 or M2.

Pathogenesis Germline mutations in the transcription factor gene CBFA2 (core-binding factor alpha subunit 2) on chromosome 21q22 have been detected in multiple families with FPD/AML syndrome. CBFA2 is also known as RUNX1 (Runtrelated transcription factor 1), AML1, and PEBP2AB. Nonsense and missense mutations or intragenic deletions in one allele of the CBFA2 gene have all been reported. 26 Importantly, neither mutations nor deletions of the normal CBFA2 allele have been detected in leukemic cells from patients with FPD/AML. These data suggest that inactivation of one CBFA2 allele results in reduced protein levels that, in turn, predispose to the acquisition of secondary mutations that contribute to leukemic transformation. 149 CBFA2 thus represents the first human gene that is haploinsufficient for tumorigenesis. CBFA2 haploinsufficiency also affects the growth of megakaryocytes, and bone marrow cells from affected patients form fewer and smaller megakaryocyte colonies than do those from unaffected patients. 26

Therapy Supportive care is sufficient therapy for the thrombocytopenia. Treatment for AML consists of chemotherapy and HSCT, if a donor is available. Buijs et al.155 describe a patient with slight thrombocytopenia and AML who underwent a HSCT in which the sister was the donor. A year later, a donor-derived leukemia developed in the recipient, and the donor also developed leukemia. A missense mutation was found within the DNA-binding Runt domain in three of the five siblings. 150 Based on this experience, mutation analysis of the CBFA2 gene should be performed before siblings are used as transplantation donors in families with FPD/AML.

Ataxia telangiectasia (Louis-Bar syndrome)

Phenotype

Demography

Patients have thrombocytopenia, functional platelet abnormalities, and a prolonged bleeding time. Some patients have low platelet ADP levels, while others have abnormal platelet aggregation studies in response to ADP, adrenaline, and collagen. Aggregation patterns in some patients are interpreted as showing an “aspirin-like” effect

The incidence of ataxia telangiectasia (A-T) ranges from 1/40,000 to1/100,000 in the United States to 1/300,000 worldwide. 151 A-T is common where consanguinity is common. Founder effect mutations have been identified in the following ethnic groups or countries: Brazil, Costa Rica, England among the Irish, Iran, Israel, Italy, Norway, Poland,

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Spain, Turkey, and the United States in Amish, Mennonite and Hispanic minorities. 152,153 Heterozygotes comprise 1% to 3% of the population. Between 10% and 38% of patients with a homozygous mutation in the A-T gene develop cancer. Their risk of leukemia is 70-fold and of lymphoma 250-fold higher than normal. 154 Based on the history of cancer in relatives of patients with A-T, A-T heterozygotes were hypothesized to have about a four-fold excess risk of cancer, especially breast cancer, accounting for up to 5% of all cancers in the United States. 155 However, more recent studies based on A-T mutant (ATM) linkage analysis have either shown a much more modest increase in cancer risk or no increase at all in ATM heterozygotes. 156,157 The current hypothesis is that the two-thirds of heterozygotes with truncating null mutations are not at increased risk of cancer, while the remainder have dominant-negative missense mutations that may predispose to an increased cancer risk. 158 Knock-in mice heterozygous for an in-frame dominantnegative deletion in ATM are at increased risk of developing tumors. 159

Phenotype The hallmarks of A-T are progressive neurologic degeneration beginning early in childhood, ocular and cutaneous telangiectasia, immunodeficiency, and a high risk of leukemia or lymphoma. 153,160 Neurologic dysfunction typically presents as cerebellar ataxia between ages 1 and 4 years and progresses so rapidly that most children are in wheelchairs by age 10 years. Slurred speech, drooling, mask facies, saccadic eye movements, choreoathetosis, and difficulty with writing and with self-care emerge in later childhood. The onset of ocular telangiectasia is highly variable, but is usually detectable by 6 years of age. Intelligence is normal. Graying of hair, keratoses and basal cell carcinomas occur prematurely. While there is considerable clinical heterogeneity in time to appearance and severity of these features, the final stages of the disease are remarkably similar in all patients. Most patients who do not develop cancer live to over age 20 with some living as long as 50 or 60 years. Postmortem the cerebellum shows a reduced number of Purkinje cells and axonal loss in posterior columns; all tissues have nucleomegaly. Patients with A-T experience recurrent sinus and pulmonary infections, the latter being the most common cause of death. Infectious complications result from deficiencies of IgA, IgE and IgG2, 161 thymic hypoplasia, lymphopenia with reduced CD4+/CD45RA+, CD8+/CD45RA+ and TCR
+ cells, a sluggish response to phytohemagglutinin, and reduced T-cell response to stimulation through

the T-cell receptor, leading to defective helper and cytotoxic activity. 162,163 An estimated 10% to 38% of patients with A-T develop cancer. Roughly 80% of the cancers are lymphoid leukemia or lymphoma; the others are carcinomas of the stomach, ovaries, uterus, liver, thyroid and parotid. 154,164,165 Lymphoid tumors present at a younger age and at a higher frequency in A-T patients. Children and adolescents with A-T predominantly develop T-cell ALL, while older adolescents or young adults develop T-prolymphocytic leukemia (T-PLL). There is only one reported case of myeloid leukemia. 166 Leukemia in A-T patients is similar in histology, phenotype and karyotype to lymphoma in patients without this disorder. The lymphomas are usually aggressive B-cell lymphomas or Hodgkin disease that present in advanced stages with extranodal involvement.164 Yalcin et al.167 described three Turkish patients with advanced pulmonary parenchymal cavitation who had histologically proven Hodgkin disease rather than infection. 167 Diagnosis of A-T is often made during the evaluation for progressive ataxia in a young child. A family history of A-T is strong confirmatory diagnostic evidence. Supportive clinical laboratory studies include an elevated serum concentration of alpha-fetoprotein (high in >90% of A-T cases), low serum IgA, IgE, and IgG2 (60–80% positive in A-T), presence of t(7;14) in 72-hour cultures of phytohemagglutinin– stimulated peripheral blood lymphocytes (>90% positive), small or absent thymus on chest radiograph, and small cerebellum on neuroimaging. 153 Supportive research laboratory studies include the demonstration on Western blotting of absent or reduced intranuclear serine-protein kinase ATM (85% positive) or colony survival assay after 1 cGy of radiation. 168 Difficulties in differentiating polymorphisms from active mutations make direct sequencing problematic. Diagnosing by demonstration of biallelic mutation of the ATM gene is not always possible because of the large size of the gene. Over 400 mutations have been described. Most patients with A-T are compound heterozygotes for two of these genes; there are no “hot spots” and new or public mutations are rare. 169 Only those familial cases that occur in ethnic groups with established founder effect mutations are amenable to screening. 169 Prenatal testing based on linkage is often successful when mutated alleles of a family member with A-T are known or genetic linkage studies have been established. In those cases, short tandem repeat and single nucleotide polymorphisms at 11q22.3 can be used to rapidly screen for founder heterozygotes or homozygotes. 153 One case of A-T in a child was diagnosed after the onset of ataxia initially attributed to chemotherapy for NHL;

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another was diagnosed after leukoencephalopathy developed following standard prophylactic irradiation to the central nervous system. 170 Some young patients may succumb to malignancies before A-T is diagnosed. A-T should be suspected in any patient who develops a tumor typical of this order at an uncharacteristically young age.

Pathogenesis The clinical features of A-T are a direct consequence of mutation of the ATM gene, which was localized to chromosomal band11q22.3-q23. 171 The gene was cloned in 1995. 172 The gene product of ATM is a serine-protein kinase that has shown considerable homology to PI3 kinase. 172 Over 270 unique mutations have been described. The primary function of the ATM protein is to detect doublestranded DNA breaks and to halt cell cycle progression by phosphorylating cell cycle regulatory genes until the breaks are repaired. The numerous phosphorylation substrates include p53, MDM2, BRCA1, FANCD2 and ABL. 153 Irradiated A-T cells display a characteristic dose–response curve indicative of radioresistant DNA synthesis. They also show delayed radiation-induced p53 activation: p53 normally binds to DNA and activates a signal transduction cascade. 173 The gene product of mutated ATM is unstable: transcripts are present on Northern blot analysis and absent or reduced on Western blotting. In most cases, mutations are autosomal recessive, but some appear to function as dominant negatives. 153 Chromosomal translocation is central to the pathogenesis of leukemia and lymphoma in A-T. The presence of an excessive number of lymphocytes with chromosomal translocations is one manifestation of failure to effect normal DNA repair. In the PHA-stimulated peripheral blood of most A-T patients, there are multiple small clones of immunophenotypically immature cells containing translocations that exclusively involve TCR genes or IGG genes on chromosomes 2, 14, and 22, the most common being t(7;14). 174 Lymphocytes of normal individuals also have these translocations, but their numbers are several logs lower than those seen in A-T. In A-T patients these small clones expand slowly over years or decades without showing changes in peripheral blood counts and without undergoing malignant transformation. The peripheral blood of A-T patients also contains larger clones with translocations of a TCR gene, usually TCRA to a non-TCR, non-IGG gene proximal to the B-cell receptor at 14q32 or to related genes on other chromosomes. 174 Leukemia and lymphoma derive from large clones. The prototypic translocation is inv(14;14)(q11;q32),(iq8), 6q–, in which the TCR gene is translocated to a breakpoint

cluster region at 14q32, 10 megabases proximal to the immunoglobulin heavy chain gene. In T-PLL the 14q32 translocations are all in close proximity to the TCL1 (T-cell leukemia) gene, 175 whose function is to prevent apoptosis. TCL1 is expressed in immature lymphocytes and leukemic lymphoblasts. Another common translocation is t(X;14)(q28;q11). 176 On Xq28 proximal to the Factor VIII gene is c6.1B (also called MTCP-1, mature T-cell proliferation-1), which encodes a mitochondrial protein with homology to TCL1. While the genesis of B-lineage NHL has been less well characterized, the available evidence suggests a similar sequence of events. TCR/TCL1 translocations alone are not sufficient to cause T-PLL; loss of ATM gene function is also necessary. In about two-thirds of cases of T-PLL in patients without A-T, the leukemic cells show either somatic biallelic mutation of ATM or loss of 11q and mutation of the other ATM allele. 177,178 In contrast to studies in T-PLL, studies in T-cell ALL have failed to identify somatic mutations of ATM. 179 Although immunodeficiency is clearly associated with the development of leukemia and lymphoma, studies conducted in the 1980s found no differences in immune profile between ATM patients who developed tumors and those who did not. Epstein–Barr virus is an established cofactor in lymphoproliferative disorders and lymphoma, and A-T patients have an abnormal serologic response to this virus. Nonetheless, lymphoid tumors have not been shown to harbor the Epstein–Barr virus genome. 174

Therapy and prognosis Treatment of leukemia or lymphoma in patients with AT is challenging. Standard dosing of therapy introduces the risk of fatal complications, while reduced dosing may be inadequate to control the tumor. Underlying immunodeficiency predisposes patients to life-threatening or fatal pneumonia. If they have IgA deficiency, they may develop anaphylactic reactions to transfusion products containing IgA, including platelets, plasma, intravenously administered IgG, and even erythrocytes. When patients receive conventional doses of radiation therapy, they invariably suffer complications typical of much higher doses. 180 Vinca alkaloids can accelerate neurologic degeneration. 181 Alkylating agents can cause prolonged pancytopenia with hemorrhagic cystitis occurring in half the patients. 180 Anthracyclines may cause severe mucositis and hemorrhagic colitis. 180 Patients treated with bleomycin may develop severe or fatal pulmonary toxicity. 180,182 The prognosis for patients with A-T and leukemia or lymphoma is grim. There were no survivors among the 19 patients described in the review by Toledano and Lange. 181

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In one Israeli kindred, 10 of 18 children had A-T or a variant syndrome, four of whom developed lymphomas. Two treated with full dose COMP died of sepsis, while one of two treated with low-dose AVB/CVPP survived. 183 There were only two survivors among 32 patients reviewed by Sandoval and Swift, 180 who concluded that standard therapy is preferable because the 21 patients treated with standard therapy had a complete remission rate of 76%, compared to 9% in those treated with reduced-dose therapy, and a median survival of 9 months compared to 5 months. 180 The 2 survivors were among those who received and tolerated conventional doses. Yamada et al. 184 reported a single case of successful therapy of B-cell NHL using half-dose therapy for standard-risk ALL therapy. 184 Irsfeld et al. 185 described fatal complications of reduced-dose BFM-type therapy despite successful control of Hodgkin disease. Tamminga et al. 186 reported successful treatment of one patient by titrating doses of chemotherapy and radiation according to the in vitro sensitivity of her fibroblasts (the cells were three times as sensitive to radiation damage as were normals, and one and a third times as sensitive to doxorubicin. 186 So far there have been two case reports of treatment-related or second malignant neoplasms in children with A-T cured of one cancer. The first was a low grade bilateral renal lymphoma that developed 3 years after successful therapy of a mediastinal lymphoblastic lymphoma at age 7 months. 187 The second was a preleukemic syndrome following low-dose ABV-CVPP for Hodgkin disease in one of the four siblings in the Israeli kindred described above. 183 Successful treatment of one malignancy in a patient with A-T appears to have a high probability of being followed by a second malignancy.

Nijmegen breakage syndrome Demography Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder occurring in persons of Slavic descent. The frequency of the most common mutation of the NBS1 gene in the Czech population is 1:106 (95% CI, 1:46– 1:330). 188 There are over 70 cases in the NBS registry. 189

Phenotype The morphologic features of NBS include microcephaly, a receding forehead, prominent midface, and a receding mandible. 190 Cerebellar agenesis and hypoplasia of the corpus callosum have been described in one patient, renal dysplasia in a second, and cafe-au-lait spots in several. 190 Total

immunoglobulin and IgG levels are low; IgM is normal to elevated; and B lymphocytes, CD3+ T lymphocytes and the mitogenic response to phytohemagglutin are reduced. 190 The immunodeficiency predisposes to chronic pneumonitis, sinusitis and otitis, recurrent infection with herpes simplex virus, and mucocutaneous candidiasis. 190 Patients with NBS have an increased susceptibility to bone marrow aplasia and malignancy, especially leukemia and lymphoma. The precise risk of development of malignancy is not known. In a sample of 47 children with ALL in first relapse, 14.9% had mutations of NBS1 with four being novel mutations. Three of the seven patients had germline mutations. The authors concluded that NBS1 may be involved in the pathogenesis of ALL. 191 In three consecutive German BFM trials for non-Hodgkin lymphoma that included 1569 patients, five had A-T and four had NBS. Histologic evaluation showed diffuse large B-cell lymphoma (seven patients), anaplastic large cell lymphoma (one) and lymphoblastic T-cell lymphoma (one). Primary tumor sites were cervical nodes, paranasal sinuses and epipharynx.

Pathogenesis NBS shares with A-T an underlying defect in the processing of specific types of damaged DNA or intermediate metabolites of normal DNA. 192 NBS1, the gene mutated in NBS, maps to 8q21–24. Most patients with NBS have a characteristic deletion of five nucleotides in exon 6 of the NBS1 gene [657del(5)], suggesting a common origin for most cases. There are, however, unique mutations in other families. 190 NBS1 encodes nibrin, a protein that forms a nuclear complex with two other proteins involved in DNA repair and telomere lengthening, Mre11 and RAD50. In patients with mutated NBS1, the complex remains in the cytoplasm where it is nonfunctional. NBS cells show radiation sensitivities that are in some respects distinct from those in A-T cells. 193

Therapy and outcome In the BFM NHL series, patients received protocolspecified therapy with dose reductions according to individual tolerance. One patient died of toxicity, two died of disease and one suffered a second malignancy. 194,195 In a selected cohort of eight unrelated Russian patients, two had bone marrow aplasia, two had neutropenia, and one had biphenotypic or myeloid leukemia. 190 Both patients with marrow aplasia died of complications, another died from infection, one from amyloidosis, and one from hemothorax; the patient with myeloid malignancy survived. 190 It is anticipated that patients with NBS are

Heritable predispositions to childhood hematologic malignancies

at risk for other malignancies. Two patients with medulloblastoma have been described; one died of pulmonary failure attributed to radiation and the second had hepatitis and gastroesophagitis that was “unresponsive to further medial therapy.” Clearly, radiation is to be avoided in these patients, as in patients with A-T.

Bloom syndrome Demography Bloom syndrome (BS), a rare autosomal recessive disorder, was first described in 1954. 196 Among Ashkenazi Jews the incidence is 2 per 105 live births. 197 The incidence is substantially lower in other ethnic groups. Since 1960, Dr. James German has maintained and reported the Bloom Syndrome Registry, which now contains records on close to 200 patients. 198

Phenotype The defining features of BS are small stature (Fig. 13.2), immunodeficiency, and a predisposition to development of one or more cancers at an early age, especially leukemia, lymphoma and carcinomas.204–206 Small stature, apparent at birth, is the result of a reduced number of cells. In childhood, BS patients typically develop a photosensitive telangiectatic malar erythema similar to that of lupus erythema and are prone to patches of hypopigmentation and hyperpigmentation. Many speak with a characteristic strident, high-pitched voice. Although they have dolicocephaly and microcephaly, patients have normal or low normal intelligence. Males have hypogonadism and azoospermia. Girls experience menarche at a normal age, they can bear children, and they undergo early menopause. Low levels of immunoglobulins and defective cell-mediated immunity of variable severity lead to frequent bouts of diarrhea and respiratory infections in infancy. 199 Other major medical complications include premature development of chronic lung disease, myocardiopathy, hypertriglyceridemia and non-insulin dependent diabetes. Among 170 BS patients described in 1993, there have been 86 cancers. The risk of developing one neoplasm approaches 50%, and among these cases, 10% will develop at least one additional cancer. Roughly 15% of the neoplasms are leukemia, 15% lymphoma, and 30% carcinomas; the remainder comprise a variety of benign and malignant tumors. 198,200 There is one report of a case of hepatocellular carcinoma in a 15-year-old girl and a case of Wilms

Fig. 13.2 Two brothers at ages 17 and 19 years. The young man on the left, identified as 51 in the Bloom Syndrome (BS) Registry, manifests the major clinical features of BS: small body size (height 4 feet 10 inches, weight 90 lb) and a sun-sensitive erythematous facial skin lesion. (The unaffected brother is just under 6 feet tall.) The brother with Bloom syndrome, who is healthy at age 23, was successfully treated for acute undifferentiated leukemia at age 12.

tumor followed by MDS in her 3.5-year-old brother. 201 Among patients with a myeloid malignancy the most common abnormality is del(7). 202 Cancer, immunodeficiency, and other medical complications reduce the life-span of BS patients: one-third have died by age 24 years with a range of 2 to 49 years. 203,204 Heterozygotes are not at increased risk for cancer. 205 The pathognomonic diagnostic test for BS is demonstration of a highly elevated rate of sister chromatid exchange on metaphase spreads: BS cells show an average of 90 sister chromatid exchanges per cell (normal, <10 per cell). There are also increased numbers of triradial and symmetrical

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quadriradial figures. 206 Heterozygotes cannot be detected by this test.

Pathogenesis The clinical and laboratory manifestations of BS are a result of homozygous mutation of the Bloom syndrome gene BLM at chromosomal band 15q26.1. 207 To date, nine mutations have been described. 208 The protein product of BLM is an ATP-dependent 3 -5 DNA helicase. Its function is to resolve Holliday junctions, suppress recombination and repair double-stranded DNA breaks. BLM is homologous to the recQ DEZH-box-containing DNA and RNA helicases. 209 Loss of this helicase activity leads to genomic instability. The mutated protein stays in the cytoplasm while the normal protein is found in the nucleus. Erythrocytes from patients with BS undergo increased mutation and somatic recombination as measured by the glycopharin A assay in peripheral blood. 208 In BS patients adjacent areas of increased and decreased skin pigmentation, reminiscent of “twin spots” in Drosophila, are clinical evidence of somatic recombination. 210 There is now a murine model for Blm mutation. 211 Homozygous mutation (Blmcin/cin ) is lethal to embryos. When challenged with murine leukemia virus, mice with a targeted null mutation for one Blm allele (Blmcin+/+ ) develop lymphomas significantly sooner and in greater numbers than do their wild-type littermates. When crossed with mice with a mutant Apc tumor suppressor, they develop twice the number of intestinal tumors found in their wild-type littermates. Thus, haploinsufficiency of Blm in the mouse predisposes to tumor formation. 211 So far there is no evidence of cancer risk among human heterozygtes for BLM mutation; they are detected only because they are parents of a child with BS. However, studies of haploinsufficiency in mice may have relevance to the 1% of Ashkenazi Jews with one mutant BLM allele. 211

Therapy Supportive care, including use of antibiotic therapy to treat infectious complications, has reduced the early mortality in BS patients. German describes 21 cases of leukemia among the first 170 BS patients. 198,200,212 Of six that clearly had ALL, one is alive, three died of their disease, and two developed MDS or AML. The remaining 15 had a mixture of AML or MDS. The only survivor had a poorly differentiated AML that responded to reduced doses of what would be considered typical ALL therapy. It is not clear if the poor outcome reflects impaired tolerance to therapy in some

patients, empiric use of reduced doses of therapy in others, or inherent resistance of the leukemic population in many. Dr. German concludes that “the similarities of leukemia in BS to that in other people are far more impressive than the differences.” 212 One case of note is of a 16-year-old boy with ALL who had poor hematologic and hepatic tolerance to 6-mercaptopurine and methotrexate, to the point that he developed marrow aplasia 2 months into treatment. He was rescued by two infusions of marrow harvested 7 years earlier. He remained in remission for 4 years, but then developed recurrent ALL followed by AML. 212

Conclusions and future directions With the exception of Down syndrome, the heritable disorders that predispose to leukemia are exceedingly rare. As a group, the children with these rare diseases tolerate standard therapy poorly and require treatments tailored to limiting complications without compromising efficacy. Successful examples are the development of separate clinical trials for DS TMD and AMKL and transplant protocols for children with Fanconi anemia before they develop MDS or AML. Only a minority of patients with these genetic predisposition syndromes develop leukemia. At this point, the study of leukemia in these children has enriched our understanding of the pathogenesis not only of their leukemia but also of leukemia and other cancers in patients with no known genetic predispostion. Among those who are genetically predisposed, both those who do and those who do not develop leukemia constitute an important cohort of readily identifiable children in whom it is possible to observe the evolution of the disease over time. Using the clinical, epidemiological, and the molecular tools at hand, comparisons of the two groups should soon enable us to move from an understanding of pathogenesis to discovery of etiology.

REFERENCES 1 Easton, D. & Peto, J. The contribution of inherited predisposition to cancer incidence. Cancer Surv, 1990; 9: 395–416. 2 Draper, G. J., Sanders, B. M., Lennox, E. L., & Brownbill, P. A. Patterns of childhood cancer among siblings. Br J Cancer, 1996; 74: 152–8. 3 Luna-Fineman, S., Shannon, K. M., & Lange, B. J. Childhood monosomy 7: epidemiology, biology, and mechanistic implications. Blood, 1995; 85: 1985–99. 4 Lange, B. J. The management of neoplastic disorders of haematopoiesis in children with Down’s syndrome. Br J Haematology, 2000; 110: 512–24.

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multipotential progenitor cell. Cancer Genet Cytogenet, 2000; 116: 170–3. Daghistani, D., Curless, R., Toledano, S. R., & Ayyar, D. R. Ataxiapancytopenia and monosomy 7 syndrome. J Pediatr, 1989; 115: 108–10. Li, F. P., Hecht, F., Kaiser-McCaw, B., Baranko, P. V., & Potter, N. U. Ataxia-pancytopenia: syndrome of cerebellar ataxia, hypoplastic anemia, monosomy 7, and acute myelogenous leukemia. Cancer Genet Cytogenet, 1981; 4: 189–96. Ho, C. Y., Otterud, B., Legare, R. D., et al. Linkage of a familial platelet disorder with a propensity to develop myeloid malignancies to human chromosome 21q22.1–22.2. Blood, 1996; 87: 5218–24. Walker, L. C., Stevens, J., Campbell, H., et al. A novel inherited mutation of the transcription factor RUNX1 causes thrombocytopenia and may predispose to acute myeloid leukaemia. Br J Haematol, 2002; 117: 878–81. Michaud, J., Wu, F., Osato, M., et al. In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis. Blood, 2002; 99: 1364–72. Buijs, A., Poddighe, P., Wijk, R. van, et al. A novel CBFA2 singlenucleotide mutation in familial platelet disorder with propensity to develop myeloid malignancies. Blood, 2001; 98: 2856– 8. Swift, M., Morrell, D., Cromartie, E., et al. The incidence and gene frequency of ataxia-telangiectasia in the United States. Am J Hum Genet, 1986; 39: 573–83. Telatar, M., Teraoka, S., Wang, Z., et al. Ataxia-telangiectasia: identification and detection of founder-effect mutations in the ATM gene in ethnic populations. Am J Hum Genet, 1998; 62: 86–97. Gatti, R. A. Ataxia-telangiectasia. Gene Reviews, 2002. http:// geneclinics.org(10/28/2002). Morrell, D., Cromartie, E., & Swift, M. Mortality and cancer incidence in 263 patients with ataxia-telangiectasia. J Natl Cancer Inst, 1986; 77: 89–92. Swift, M., Morrell, D., Massey, R. B., & Chase, C. L. Incidence of cancer in 161 families affected by ataxia-telangiectasia. N Engl J Med, 1991; 25: 1831–6. Olsen, J. H., Hahnemann, J. M., Borresen-Dale, A. L., et al. Cancer in patients with ataxia-telangiectasia and in their relatives in the nordic countries. J Natl Cancer Inst, 2001; 93: 121–7. FitzGerald, M. G., Bean, J. M., Hegde, S. R., et al. Heterozygous ATM mutations do not contribute to early onset of breast cancer. Nat Genet, 1997; 15: 307–10. Khanna, K. K. Cancer risk and the ATM gene: a continuing debate. J Natl Cancer Inst, 2000; 92: 795–802. Spring, K., Ahangari, F., Scott, S. P., et al. Mice heterozygous for mutation in Atm, the gene involved in ataxia-telangiectasia have heightened susceptibility to cancer. Nat Genet, 2002; 31: 185–90. Boder, E. Ataxia-telangiectasia an overview. In R. A. Gatti & M. Swift, eds., Ataxia-Telangiectasia: Genetics, Neuropathy

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and Immunology of a Degenerative Disease of Childhood (New York: Alan Liss, 1985), pp. 1–63. Oxelius, V. A., Laurell, A. B., Lindquist, B., et al. IgG subclasses in selective IgA deficiency: importance of IgG2-IgA deficiency. N Engl J Med, 1981; 304: 1476–7. Schubert, R., Reichenbach, J., & Zielen, S. Deficiencies in CD4+ and CD8+ T cell subsets in ataxia telangiectasia. Clin Exp Immunol, 2002; 129: 125–32. Waldmann, T. A., Misiti, J., Nelson, D. L., & Kraemer, K. H. Ataxia-telangiectasia: a multisystem hereditary disease with immunodeficiency, impaired organ maturation, X-ray hypersensitivity, and a high incidence of neoplasia. Ann Intern Med, 1983; 99: 367–79. Murphy, R. C., Berdon, W. E., Ruzal-Shapiro, C., et al. Malignancies in pediatric patients with ataxia telangiectasia. Pediatr Radiol, 1999; 29: 225–30. Sandoval, C., Schantz, S., Posey, D., & Swift, M. Parotid and thyroid gland cancers in patients with ataxia-telangiectasia. Pediatr Hematol Oncol, 2001; 18: 485–90. Viniou, N., Terpos, E., Rombos, J., Acute myeloid leukemia in a patient with ataxia-telangiectasia: a case report and review of the literataure. Leukemia, 2001; 15: 668–70. Yalcin, B., Kutluk, M. T., Sanal, O., et al. Hodgkin’s disease and ataxia telangiectasia with pulmonary cavities. Pediatr Pulmonol, 2002; 33: 399–403. Sun, X., Becker-Catania, S. G., Chun, H. H., Early diagnosis of ataxia-telangiectasia using radiosensitivity testing. J Pediatr, 2002; 140: 724–31. Telatar, M., Wang, Z., Udar, N., et al. Ataxia-telangiectasia: mutations in ATM cDNA detected by protein-truncation screening. Am J Hum Genet, 1996; 59: 40–4. Loeb, D. M., Lederman, H. M., & Winkelstein, J. A. Lymphoid malignancy as a presenting sign of ataxia-telangiectasia. J Pediatr Hematol Oncol, 2000; 22: 464–7. Gatti, R. A., Berkel, I., Boder, E., et al. Localization of an ataxiatelangiectasia gene to chromsome 11q22–23. Nature, 1988; 336: 577–80. Savitsky, K., Bar-Shira, A., Gilad, S., et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science, 1995; 268: 1749–53. Morgan, S. E. & Kastan, M. B. p53 and ATM: cell cycle, cell death, and cancer. Adv Cancer Res, 1997; 71: 1–25. Taylor, A. M. R., Metcalfe, J. A., Thick, J., & Mak, Y. F. Leukemia and lymphoma in ataxia telangiectasia. Blood, 1996; 87: 423– 38. Virgilio, L., Narducci, M. G., Isobe, M., et al. Identification of the TCL1 gene involved in T cell malignancies. Proc Natl Acad of Sci. U S A, 1994; 91: 12 530–4. Thick, J., Sherrengla, P. D., Fisch, P., Taylor, A. M. R. & Rabbits, T. H. Molecular analysis of a new translocation t(×; 14) (q28; q11) in premalignancy and leukemia associated with ataxia-telangiectasia. Genes Chromosomes Cancer, 1992; 5:321. Stilgenbauer, S., Schaffner, C., Litterst, A., et al. Biallelic mutations in the ATM gene in T-prolymphocytic leukemia. Nat Med, 1997; 3: 1155–9.

178 Stoppa-Lyonnet, D., Soulier, J., Lauge, A., et al. Inactivation of the ATM gene in T-cell prolymphocytic leukemias. Blood, 1998; 91: 3920–6. 179 Luo, L., Lu, F. M., Hart, S., et al. Ataxia-telangiectasia and T-cell leukemias: no evidence for somatic ATM mutation in sporadic T-ALL or for hypermethylation of the ATM-NPAT/E14 bidirectional promoter in T-PLL. Cancer Res, 1998; 58: 2293–7. 180 Sandoval, C., & Swift, M. Treatment of lymphoid malignancies in patients with ataxia-telangiectasia. Med Pediatr Oncol, 1998; 31: 491–7. 181 Toledano, S. R. & Lange, B. J. Ataxia-telangiectasia and acute lymphoblastic leukemia. Cancer, 1980; 45: 1675–8. 182 Chen, R. L., Wang, P. J., Hsu, Y. H., Chang, P. Y., & Fang, J. S. Severe lung fibrosis after chemotherapy in a child with ataxia-telangiectasia. J Pediatr Hematol Oncol, 2002; 24: 77–9. 183 Weyl Ben Arush, M., Rosenthal, J., Dale, J., et al. Ataxia telangiectasia and lymphoma: an indication for individualized chemotherapy dosing – report of treatment in a highly inbred Arab family. Pediatr Hematol Oncol, 1995; 12: 163–9. 184 Yamada, Y., Inoue, R., Fukao, T., et al. Ataxia telangiectasia associated with B-cell lymphoma: the effect of a half-dose of the drugs administered according to the acute lymphoblastic leukemia standard risk protocol. Pediatr Hematol Oncol, 1998; 15: 425–9. 185 Irsfeld, H., Korholz, D., Janssen, G., Wahn, V., & Schroten, H. Fatal outcome in two girls with Hodgkin disease complicating ataxia-telangiectasia (Louis-Bar syndrome) despite favorable response to modified-dose chemotherapy. Med Pediatr Oncol, 2000; 32: 62–4. 186 Tamminga, R. Y., Dolsma, W. V., Leeuw, J. A., & Kampinga, H. H. Chemo- and radiosensitivity testing in a patient with ataxia telangiectasia and Hodgkin disease. Pediatr Hematol Oncol, 2002; 19: 163–71. 187 Overberg-Schmidt, U., Wegner, R. D., Baumgarten, E., et al. Low-grade non-Hodgkin’s lymphoma after high-grade nonHodgkin’s lymphoma in a child with ataxia telangiectasia. Cancer, 1994; 73: 1522–5. 188 Drabek, J., Hajduch, M., Gojova, L., Weigl, E., & Mihal, V. Frequency of 657del(5) mutation of the NBS1 gene in the Czech population by polymerase chain reaction with sequence specific primers. Cancer Genet Cytogenet, 2002; 138: 157–9. 189 Group, INBSS. Nijmegen breakage syndrome. Arch Dis Child, 2000; 84: 400–6. 190 Resnick, I. B., Kondratenko, I., Togoev, O., et al. Nijmegen breakage syndrome: clinical characteristics and mutation analysis in eight unrelated Russian families. J Pediatr, 2002; 140: 355–61. 191 Varon, R., Reis, A., Henze, G., et al. Mutations in the Nijmegen Breakage Syndrome gene (NBS1) in childhood acute lymphoblastic leukemia (ALL). Cancer Res, 2001; 61: 3570–2. 192 Shiloh, Y. Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Annu Rev Genet, 1997; 31: 635–62.

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193 Little, J. B., Nagasawa, H., Dahlberg, W. K., et al. Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation. Radiat Res, 2002; 158: 319– 26. 194 Siwicki, J. D., Degerman, S., Chrzanowska, K. H., & Roos, G. Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients. Exp Cell Res, 2003; 287: 178–89. 195 Seidmann, K., Henze, G., Beck, J. D., et al. Non-Hodgkin’s lymphoma in pediatric patients with chromosomal breakage syndromes (AT and NBS): experience from the BFM trials. Ann Oncol, 2000; 11: 141–5. 196 Bloom, D. Congenital telangiectatic erythema resembling lupus erythematosis in dwarfs. Am J Dis Child, 1954; 88: 754–8. 197 Li, L., Eng, C., Desnick, R. J., German, J., & Ellis, N. A. Carrier frequency of the Bloom syndrome blmAsh mutation in the Ashkenazi Jewish population. Mol Genet Metab, 1998; 64: 286– 90. 198 German, J. Bloom’s syndrome. XX. The first 100 cancers. Cancer Genet Cytogenet, 1997; 93: 100–6. 199 Hutterroth, T. H., Litwin, S. D., & German, J. Abnormal immune responses in Bloom syndrome lymphocytes in vitro. J Clin Invest, 1975; 56: 1–7. 200 German, J. Bloom syndrome: a Mendelian prototype of somatic mutational disease. Medicine, 1993; 72: 393–406. 201 Jain, D., Hui, P., McNamara, J., et al. Bloom syndrome in sibs: first reports of hepatocellular carcinoma and Wilms tumor with documented anaplasia and nephrogenic rests. Pediatr Dev Pathol, 2001; 4: 585–9. 202 Poppe, B., Limbergen, H. van, Roy, N. van, et al. Chromosomal aberrations in Bloom syndrome patients with myeloid malignancies. Cancer Genet Cytogenet, 2001; 128: 39–42.

203 Barakat, A., Ababou, M., Onclercq, R., et al. Identification of a novel BLM missense mutation (2706T>C) in a Moroccan patient with Bloom’s syndrome. Hum Mutat, 2000; 15: 584–5. 204 Dutertre, S., Ababou, M., Onclercq, R., et al. Cell cycle regulation of the endogenous wild type Bloom’s syndrome DNA helicase. Oncogene, 2000; 19: 2731–8. 205 Cleary, S. P., Zhang, W., DiNicola, N., et al. Heterozygosity for the BLM(Ash) mutation and cancer risk. Cancer Res, 2003; 63: 1769–71. 206 Schonberg, S., Louie, E., Chagant, R. S. K., & German, J. Bloom syndrome IV. Sister chromatid exchanges in lymphocytes. Am J Hum Genet, 1977; 29: 248–55. 207 Straughen, J., Ciocci, S., Ye, T. Z., et al. Physical mapping of the Bloom syndrome region by the identification of YAC and PI clones from human chromosome 15 band q26.1. Genomics, 1996; 35: 118–28. 208 Langlois, R. G., Bigbee, W. L., Jensen, R. H., & German, J. Evidence for increased in vivo mutation and somatic recombination in Bloom’s syndrome. Proc Natl Acad Sci U S A, 1989; 86: 670–4. 209 Ellis, N. A., Groden, J., Ye., T. Z., The Bloom’s syndrome gene product in homologous to RecQ helicases. Cell, 1995; 83: 655– 66. 210 Festa, R. S., Meadows, A. T., & Boshes, R. A. Leukemia in a black child with Bloom’s syndrome: somatic recombination as a possible mechanism for neoplasia. Cancer, 1979; 44: 335–8. 211 Goss, K. H., Risinger, M. A., Kordich, J. J., et al. Enhanced tumor formation in mice heterozygous for Blm mutation. Science, 2002; 297: 2051–6. 212 German, J. Bloom’s syndrome: incidence, age of onset, and types of leukemia in the Bloom’s syndrome registry. In C. S. Bartsocas & D. Loukopoulos, eds., Genetics of Hematological Disorders (Washington, DC: Hemisphere, 1992), pp. 241–58.

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Part III Evaluation and treatment

14 Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations Shinji Kishi, William E. Evans, and Mary V. Relling

Introduction Childhood leukemias are among the most drug-responsive of human malignancies. More than 70% of children with acute lymphoblastic leukemia (ALL) can now be cured, largely by systemic chemotherapy.1 Because of their drug responsiveness, childhood leukemias are a good model for evaluating the pharmacodynamics of anticancer drugs. Pharmacokinetics is the study of the absorption, distribution, metabolism, and excretion of drugs. Pharmacodynamics describes the relationship between pharmacokinetics and pharmacologic effect, either adverse or desired. Substantial interindividual variability exists in the pharmacokinetics2–4 and in the pharmacodynamics of many antileukemic agents in children, and these data will not be reviewed herein. Pharmacogenetics is the study of the inherited basis for interindividual differences in response to medications. Thus, individualizing therapy on the basis of germline genetic status may be one means of minimizing interindividual variability in response to antileukemic agents. Interpatient variability characterizes the disposition of many drugs. In the case of drugs with a wide therapeutic index (e.g. penicillins), such variability is unlikely to affect either clinical efficacy or toxicity. In the vast majority of patients, the drugs can be given in high enough doses to assure plasma concentrations that are very likely to produce the desired therapeutic response with little risk of toxicity. With antileukemic drugs, however, there is much less margin for error, due to their very narrow therapeutic index. Many investigations have established the relationship between administered dosage and plasma (or tissue) concentrations of drugs and metabolites, and in

some cases between those concentrations or host genetic polymorphism and pharmacologic effect. It is these latter investigations that establish clinical therapeutic indices for antileukemic agents, and that are the subject of this chapter.

Methotrexate Methotrexate (MTX) is a prodrug requiring metabolism (anabolism) to MTX polyglutamates for maximum cytotoxic effects.5–8 Once inside cells, MTX either binds to target enzymes (e.g. dihydrofolate reductase) or is metabolized by folylpolyglutamate synthetase to polyglutamylated MTX (MTXPG), with up to five additional glutamates sequentially added to the molecule in leukemic lymphoblasts, both in vitro9 and in vivo.10 These longer-chain MTXPGs are retained longer in cells and cause greater inhibition of target enzymes, as compared to MTX; thus, their formation is considered advantageous.6,7,11,12 The relative sensitivity or resistance of cancer cells to MTX is influenced by a number of mechanisms. MTX entry into cells is reduced by impaired reduced folate carrier function,13–15 decreased formation of MTXPG via folylpolyglutamate synthetase (FPGS),16,17 or increased hydrolysis of MTXPG via gamma glutamyl hydrolase (GGH).18,19 Differences in FPGS activities have been clearly related to differences in both MTXPG accumulation and MTX response in ALL and acute myeloid leukemia (AML) cell lines20,21 and leukemic blasts from patients,22–24 whereas the contribution of GGH activities to MTX efficacy is controversial.23–27 When incubated with MTX ex vivo, hyperdiploid leukemic blast cells demonstrate greater MTXPG accumulation than do nonhyperdiploid

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Fig. 14.1 Proportion of children with acute lymphoblastic leukemia in hematologic (upper) or complete (lower) remission, based on their median steady-state methotrexate plasma concentration (MTX Cpss ). (Reprinted with permission, from Evans et al.34 )

ALL blast cells,28 and B-lineage ALL blast cells accumulate more MTXPG than do T-lineage ALL blasts.29 Similar findings were made in patients,10,24,30 consistent with the improved prognoses associated with hyperdiploid and/or B-lineage ALL treated with antimetabolites. High-dose MTX (1.0 g/m2 IV over 24 hours) yields higher concentrations of active MTXPG metabolites in ALL blast cells than does low-dose MTX (30 mg/m2 orally every 6 hours for six doses).10 These higher levels of MTXPG in ALL blast cells have been shown to translate into greater antileukemic effects, such as inhibition of de novo purine synthesis and a decrease in circulating blasts.30,31 These studies have resolved a long-standing debate32 as to whether high-dose MTX (HDMTX) achieves higher MTXPG concentrations in ALL blast cells than does prolonged exposure to low-dose MTX. These pharmacologic findings are consistent with a randomized comparison of the same

doses of IV and oral MTX, indicating improved event-free survival with the former schedule.33 Clinical trials indicate that the level of systemic exposure to HDMTX can have a significant influence on clinical outcome in children with ALL. A significant relation between the steady-state plasma concentrations of MTX and event-free survival was observed in a study of children with “standard-risk” ALL enrolled on our Total-XS protocol (Fig. 14.1).34 Treatment included 15 infusions of HDMTX (1.0 g/m2 over 24 hours) during the first 1.5 years of continuation therapy, in addition to conventional lowdose oral 6-mercaptopurine (50 mg/m2 daily) and MTX (40 mg/m2 weekly). All patients were given the same dosage of HDMTX; therefore, patients with fast systemic clearance of HDMTX had lower steady-state plasma concentrations (Cpss ) of this agent. In a Cox proportional hazard regression analysis, patients with a median MTX Cpss of less than

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

16 M had a significantly greater risk of early treatment failure (relapse) than did patients with a higher MTX Cpss .34 This greater risk remained statistically significant in a multivariate analysis that included other prognostic factors such as white blood cell count and DNA index. In a subsequent analysis after longer follow-up (median, 7.3 years), MTX Cpss remained a significant prognostic factor in patients with “intermediate-risk” ALL (i.e. presenting white cell count 10–100 × 109 /L), while MTX Cpss was a significant prognostic factor only for the risk of early relapse in patients with “very good risk” ALL.35 A similar relation between MTX Cpss during continuation therapy and risk of ALL relapse was also observed in a Pediatric Oncology Group trial.36 Because all patients treated on these trials had Blineage ALL, it is important to recognize that the pharmacodynamic relationships between MTX Cpss and response may differ in T-lineage ALL. In a randomized trial of a single low dose of MTX (40 mg/m2 ) versus a single high dose of MTX (4 or 33 g/m2 ) as initial therapy preceding remission induction in children with ALL, patients receiving the high-dose MTX had a superior 7-year event-free survival rate.37 In a protocol in which patients received low-dose MTX (with 6-mercaptopurine) as part of their continuation therapy, the product of the red blood cell concentrations of MTXPGs and thioguanine nucleotides was a significant predictor of outcome.38 In contrast, plasma MTX AUC (area under the concentration-versus-time curve) measured after three intramuscular or oral doses of low-dose MTX was not predictive of outcome, possibly because MTX concentrations were suboptimal in all patients given lowdose MTX.39 The St. Jude Total XII protocol compared the efficacy of therapy with MTX, teniposide, and cytarabine that was targeted (adjusted based on clearance) versus standard doses of the agents based on body surface area (BSA). The study suggested a benefit of targeting.40 Further analysis revealed that in patients with B-lineage leukemia, the risk of relapse during this period was significantly related to both the average systemic exposure to MTX (P = 0.02) and the proportion of courses with systemic exposures above the target threshold (580 M hour, P = 0.02),40 suggesting that adjusting the dose of MTX to account for the patient’s ability to clear the drug can improve the outcome in children with ALL. Whether this strategy would hold true in other treatment protocols is not known. There are substantial data demonstrating the relationship between pharmacokinetic parameters of MTX exposure and acute toxicity.41–44 Patient and treatment characteristics that significantly influence MTX pharmacokinetics include hydration and alkalinization status; presence of a pleural effusion, gastrointestinal obstruction, ascites, or vomiting; concurrent drugs; and renal function.

Toxicity is known to be affected by the magnitude and duration of cytotoxic MTX plasma concentrations; the timing, dosage, and duration of leucovorin rescue; other concurrent medications; and some underlying factors predisposing to toxicity. Dosage individualization has been recommended to avoid potentially toxic MTX exposures, especially in children with relapsed ALL, many of whom have been heavily pretreated and have subclinical kidney dysfunction.45 Patients with Down syndrome have a higher frequency of delayed plasma MTX excretion46 and greater toxicity than do patients without this complication;47 however, they can tolerate MTX doses of 500 to 1000 mg/m2 with careful monitoring and early leucovorin rescue.46 Because the reduced folate carrier, responsible for MTX transport, is on human chromosome 21,14 it is possible that patients with Down syndrome have enhanced MTX uptake in all tissues, and it has been hypothesized that through alterations in purine metabolism (also related to trisomy 21) leading to folate depletion,48–51 patients with Down syndrome would have enhanced MTX polyglutamylation. Increased polyglutamylation would be consistent with both higher plasma concentrations several hours postinfusion and enhanced MTX toxicity as compared to other children. Receptor or target genetic polymorphisms have been linked to MTX toxicity and relapse. The enhancer polymorphism of 2 versus 3 repeats of a 28-bp region has been linked to the amount of thymidylate synthase (TS) protein52 and to the efficacy of other TS inhibitors.53–55 The TS 2/2 genotype (associated with a lower amount of TS and thus theoretically more susceptible to drugs that target TS) has been associated with improved event-free survival among patients with ALL.56,57 A common polymorphism in methylenetetrahydrofolate (MTHFR), the C-to-T substitution at 677, has been linked to mucositis risk following lowdose MTX.58 The G80A polymorphism in the reduced folate carrier (RFC) has been linked to hyperhomocysteinemia,59 and to lower event-free survival.60 However, this polymorphism was also associated with higher plasma levels of MTX,60 so that the significance of the RFC G80A polymorphism remains uncertain.

6-Mercaptopurine 6-Mercaptopurine (6MP) is anabolized by hypoxanthine phosphoribosyl transferase (HPRT) to thioinosine monophosphate and eventually to mono-, di-, and triphosphates of 6-thioguanosine, which are collectively termed 6thioguanine nucleotides (TGNs) (Fig. 14.2). These metabolites interfere with normal DNA and RNA synthesis and are important to the cytotoxic effects of 6MP,61 which is

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DNA

SH N

N H2N

SCH3

SCH3 N

N SCH3 N

N N

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kinase

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Fig. 14.2 Metabolism of 6-mercaptopurine and 6-thioguanine: TPMT, thiopurine methyltransferase; HPRT, hypoxanthine phosphoribosyl transferase; IMPD, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphate synthetase; PRPP, phosphoribosylpyrophosphate. (Modified, with permission, from Krynetski et al.62 )

catabolized by xanthine oxidase to thiouric acid, and by thiopurine methyltransferase (TPMT) to methylmercaptopurine.62–64 In addition, 6-thioinosine monophosphate is a substrate for TPMT, and the methylated derivative is able to inhibit de novo purine synthesis.65,66 Thus, the precise contribution of TPMT to the desired antileukemic activity of 6MP is not clear. TPMT activity undergoes a common genetic polymorphism. Among most populations studied,67–71 approximately 1 in 300 individuals is homozygous deficient for the enzyme, 10% are heterozygous, and the remainder are homozygous wild-type. Because TPMT activity in circulating red blood cells (RBCs) correlates with liver,72 lymphocyte,73 and leukemic lymphoblast74 TPMT activity, TPMT has been measured in RBCs as an assessment of phenotype. Now that the most common inactivating mutations in TPMT have been identified,75–78 molecular

diagnosis of the TPMT genotype can be used to avoid the confounding effects of allogeneic erythrocyte transfusions and the increases in TPMT activity observed while patients are receiving chemotherapy.79 RBCs contain TPMT and HPRT, and accumulate high levels of thioguanine and methylthioinosine nucleotide metabolites in children receiving continuation therapy with 6MP. A significant inverse relationship between RBC TGNs and TPMT activity has been observed in several studies, following administration of either 6MP or its prodrug, azathioprine.80–83 Acute hematopoietic toxicity (e.g. neutropenia) has been clearly related to RBC TGN concentrations,84 with unacceptable toxicity reported when normal oral doses of 50 to 75 mg/m2 per day are administered to patients with homozygous deficiency of TPMT.85,86 The 10% of the population heterozygous for TPMT mutations are more likely to require a dose

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

reduction to avoid acute myelosuppression87 than are wildtype patients, but not the dramatic (10-fold) dose reduction required in homozygous deficient patients. RBC TGNs can accumulate to very high levels (2000–4000 pmol/8×108 RBCs) in such patients, whereas in most patients receiving 50 to 75 mg/m2 per day orally, the reported median concentrations have ranged from 200 to 400 pmol/8×108 RBCs. There is a significant (>6-fold) over-representation of TPMT deficiency or heterozygosity among patients developing dose-limiting hematopoietic toxicity from therapy containing thiopurines.88 The diagnosis of TPMT deficiency allows the rational adjustment of dosages: i.e. the 6MP dose can be substantially reduced, while other concurrent cytotoxic agents remain at their normal unadjusted doses. Interestingly, even when 6MP dosage is greatly reduced in such deficient patients (as little as 10–30 mg/m2 orally 3 days/week), the RBC TGN level tends to remain much higher (1000–3000 pmol/8×108 RBCs) than the population median.85 With appropriate dosage adjustments, TPMT-deficient and heterozygous patients can be treated with thiopurines, without acute dose-limiting toxicity.87,88 The reason for the increased tolerance of TPMT-deficient patients to high RBC TGNs is not clear, although it has been postulated that the lack of methyl thioinosine monophosphate in such patients may contribute to their better tolerance. This relative lack of methyl activated metabolites following oral 6-thioguanine has also been postulated as a reason why much higher RBC 6TGNs are tolerated following oral administration of 6-thioguanine than after 6-mercaptopurine. Several studies have identified an interaction between exposure to thiopurines and the incidence of secondary malignancies.89–92 In a St. Jude Children’s Research Hospital study, four of the six children with secondary brain tumors had RBC TGN concentrations higher than the 70th percentile of the entire cohort, and three had a genetic defect in TPMT.90 The 8-year cumulative incidence of brain tumors among children with defective TPMT was 42.9% versus 8.3% in wild-type TPMT patients (Fig. 14.3).90 There were also suggestions that lower TPMT activity tended to be associated with shorter time to the onset91 of secondary AML. Low TPMT leads to higher incorporation of thioguanine nucleotides and can thereby affect the magnitude of stabilization of double-strand DNA breaks by topoisomerase II93 ; a possible mechanism by which low TPMT could contribute to secondary cancers. The impact of TPMT status and RBC TGN concentrations on outcome of childhood ALL has been assessed in several studies. The German-based BFM group found better eradication of minimal residual disease among patients with low or intermediate TPMT activity.94 In United Kingdom ALL

P

Fig. 14.3 Estimated cumulative incidence of radiation-associated secondary brain tumors for seven children in the St. Jude Total XII protocol who received preventive cranial radiotherapy and had genetic effects on TPMT, compared with that for 45 with wild-type status. (Reprinted, with permission, from Relling et al.90 )

(UKALL) studies in which 6MP constituted a major component of therapy, TPMT activity was assessed as a possible prognostic factor. Although TPMT activity was not a statistically significant factor, the maximum erythrocyte 6TGN level was related to outcome.95 Children whose 6TGNs exceeded the median for the population had a significantly better event-free survival rate than did those whose 6TGNs were less than the median.95 It is not clear whether high TGNs were indicative of less TPMT activity (or other inactivation of 6MP), greater overall compliance with therapy (both 6MP and weekly MTX were administered by the oral route in these UKALL studies), or some other factor correlated with RBC TGNs. In the NOPHO (Nordic) trials, both RBC TGNs and MTXPGs were measured in children during continuation therapy.38 Again, both drugs were administered orally. The product of the RBC TGN·MTXPG concentrations was associated with a better event-free survival, while the RBC TGN concentration by itself was not.38 Studies at St. Jude showed that event-free survival was related to the dose intensity of 6MP but not to TGNs.96 The salutary effect on leukemia-free survival was somewhat attenuated by the increased risk of secondary tumors in St. Jude studies.90 At this point, it is clear that low RBC TGNs can help to document noncompliance in some patients,88,97 and that RBC TPMT deficiency can identify a subset of patients at risk for unacceptable toxicity if given the usual doses of 6MP. Whether ALL outcome can be improved by adjusting the dose of 6MP to achieve some minimal or optimal level of TGN concentrations is not yet clear.

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Percent in remission

396

Weeks in remission

Fig. 14.4 Percentage of patients remaining in remission based on retention of ara-CTP (cytarabine triphosphate) by AML blast cells 4 hours after a 30-minute ex vivo incubation with ara-C. Remission status in those with high retention (n = 11) is illustrated by the upper solid line; those with lower retention (n = 9) are depicted by the lower dashed line (P < 0.001). (Reprinted, with permission, from Rustum and Preisler.110 )

Cytarabine Cytarabine (ara-C) is a pyrimidine analog commonly used in the remission induction and consolidation therapy for AML. When intrathecally administered, it is effective in the prophylaxis and primary treatment of CNS leukemia.98 AraC, a prodrug, is phosphorylated intracellularly by deoxycytidine kinase (dCK) to 1--D-arabinofuranoxyl cytosine5 -triphosphate (ara-CTP).99 Its cytotoxicity is believed to result from a combination of DNA polymerase inhibition and from incorporation of ara-CTP into DNA,100 in competition with deoxycytidine triphosphate (dCTP). This incorporation causes chain termination, resulting in a block of DNA synthesis.100–102 Cellular uptake and phosphorylation of the parent drug to ara-CTP are the crucial steps for cytotoxicity.103 At standard doses of ara-C (∼100 mg/m2 ), cellular uptake occurs by facilitated diffusion and depends on the number of transmembranous nucleoside carrier sites.104 Once steadystate plasma concentrations exceed the Km for transport (10 M in most leukemic cells), further increases in plasma concentrations do not result in significant increases in intracellular ara-CTP concentrations.105–107 With highdose ara-C therapy (2–3 g/m2 ), cellular uptake by passive diffusion occurs independent of transport capacity.108

The ability of leukemic cells to form and retain intracellular ara-CTP is correlated with clinical response in AML (Fig. 14.4)109,110 ; fewer studies have been performed in ALL.111,112 After infusion of 100 mg/m2 ara-C over 30 minutes, the mean (± SD) ara-CTP retention differed significantly with immunophenotype: B-lineage ALL, 67% ± 25%; T-lineage ALL, 37% ± 15%; and AML, 34% ± 18%.113 A trend toward lower ara-CTP retention in relapsed ALL patients as compared to newly diagnosed patients was also reported.113 The adverse prognostic significance of low ara-CTP retention may be reduced by administering continuous infusions of ara-C, during which the ara-CTP steady-state levels in circulating blast cells increase proportionately to the infusion rate,105 or by using high-dose regimens.107 Trough levels of ara-CTP were significantly higher in responding patients, while the ara-CTP peak levels were comparable in responding and nonresponding patients,109,114 suggesting that the duration of exposure to ara-CTP may be more important than the magnitude of peak concentrations. Target leukemic cell ara-CTP levels have been proposed for high-dose ara-C treatment. Plunkett et al. proposed a minimum effective ara-CTP trough concentration of 75 M following high-dose ara-C short-term infusion,109,114 while Estey et al. observed a median steady-state level of 122 M in responding patients and 63 M in nonresponding AML patients.115 Moreover, high-dose ara-C (3 g/m2 ) was more successful in inducing complete remission than was standard dose ara-C (100 mg/m2 ) in adult patients with de novo AML,116 resulting in higher rates of continuous complete remission when incorporated into continuation therapy regimens.117 The nucleoside activating or inactivating enzymes for ara-C have also been correlated with tumor response or patient outcome. The decreased expression of activity of dCK as a mechanism responsible for clinical resistance to ara-C has been reported.118–120 A high incidence of alternatively spliced forms of dCK in patients with resistant AML and its functional role have also been reported.121,122 The activity of inactivating enzymes (e.g. cytidine deaminase or 5 -nucleotidase) correlate with outcome.120,123–124 The role of genetic polymorphisms in these enzymes is not yet known.125 Cerebrospinal fluid (CSF) concentrations reach about 40% to 50% of the plasma concentrations obtained by continuous intravenous infusions.126 The high CSF levels may result from low cytidine deaminase activity in the CSF. Whereas ara-C has a short retention time in the CSF (half-life 1.8−2.9 hours), ara-CTP accumulates in the CSF lymphoid leukemic cells and has a half-life of 8.1 to 36 hours.111 The rate of elimination of ara-C from

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

the CSF approaches the turnover rate of CSF.106,126 A St. Jude study suggested that early intensification of intrathecal chemotherapy which included ara-C and methotrexate reduced the risk of CNS relapse to a very low level in children with ALL, securing a higher event- free survival rate overall.127 Children with Down syndrome and AML have an increased event-free survival rate and decreased relapse rate as compared to children without Down syndrome.128,129 Down syndrome myeloblasts are approximately 10-fold more sensitive to ara-C than are non-Down syndrome myeloblasts (IC50 of 108.3 ± 66.8 nM and 1040.8 ± 272.9 nM, respectively, on exposure to ara-C for 72 hours).130 Mean levels of ara-CTP were significantly higher in Down syndrome myeloblasts than in non-Down syndrome myeloblasts after incubation with 5 M ara-C (621.4 versus 228.4 pmol/mg protein).130 The enhanced metabolism of ara-C in Down syndrome cells may be a factor contributing to the superior survival rate of children with Down syndrome and AML.131,132 This effect is possibly based on an increased dosage of the genes encoding cystathionine--synthase (CBS) and glycinamide ribonucleotide transformylase on chromosome 21, which alter intracellular reduced folate pools.47,112 The results are decreased deoxycytidine concentrations and consequent decreased feedback inhibition of deoxycytidine kinase and greater generation of ara-CTP. In fact, the CBS transcript levels were significantly higher in Down syndrome compared with non-Down syndrome myeloblasts, and CBS transcript levels correlated with in vitro ara-C sensitivity.133 The most common mutation in the CBS gene is a polymorphism involving a 68-bp insertion (844ins68) in the coding region of exon 8, which produces an alternative splicing site.134 The T-to-C substitution at 833 is also present in all alleles that contain the 844ins68 polymorphism.134 It has been reported that Down syndrome myeloblasts with this polymorphism were more sensitive to ara-C than those with the wild-type CBS allele (P < 0.002).135 The role of the CBS genetic polymorphism in the efficacy of ara-C in non-Down syndrome AML patients is not yet known. Ara-C is associated with a number of drug–drug interactions that have been exploited to enhance the phosphorylation of ara-C in leukemic blasts. The combination of ara-C with 2-chlorodeoxyadenosine (2-CDA), a deoxyadenosine analog, may lead to synergy or antagonism. 2-CDA is phosphorylated intracellularly to 2-CDA monophosphate (2-CDAMP) by deoxycytidine kinase, which is subsequently converted to the triphosphate form, 2-CDATP, by other kinases. 2-CDATP increases deoxycytidine kinase activity through inhibition of ribonucleotide reductase;

thus, administration of 2-CDA and ara-C should lead to increased intracellular ara-CTP levels.136,137 However, because ara-C and 2-CDA use common transporter mechanisms and deoxycytidine kinase phosphorylates both agents with similar efficiency, these drugs may undergo competitive inhibition when given concurrently. When 2CDA and ara-C were given together by continuous intravenous infusion, the rate of ara-CTP accumulation was increased by 40% in seven of nine patients.136 However, it is possible that the activation of ara-C was favored over 2CDA, thus attenuating the effect of 2-CDA on ara-CTP accumulation. In a prospective, randomized trial, the intracellular accumulation of ara-CTP was increased when 2-CDA was given, but no schedule-dependent differences in this effect were seen in 45 pediatric patients with newly diagnosed primary AML who had been randomized to short versus continuous infusions of ara-C with 2-CDA.138 Fludarabine monophosphate (fludarabine), a nucleotide analog of adenine arabinoside, has also been combined with ara-C. Fludarabine is rapidly dephosphorylated to the parent nucleoside (F-ara-A) upon intravenous infusion and is actively transported into the cell.139 The metabolite is rephosphorylated by deoxycytidine kinase to 5 -triphosphate fludarabine (F-ara-ATP). Fludarabine, like 2-CDA, also increases deoxycytidine kinase activity and should lead to a higher rate of ara-CTP accumulation.140–143 F-ara-ATP increases the anabolism of ara-C, whereas araCTP inhibits the phosphorylation of fludarabine. When a second dose of ara-C was administered 4 hours after the start of fludarabine infusion, ara-C caused a dosedependent redistribution of F-ara-A, as evidenced by a transient increase in F-ara-A plasma levels during the ara-C infusion and subsequent reduction in the terminal half-life of F-ara-A. Peak F-ara-ATP concentrations were 43% lower in leukemia cells following ara-C and fludarabine combination therapy as compared to fludarabine monotherapy. Also, the terminal half-life of intracellular F-ara-ATP was reduced after the ara-C infusion in a concentrationdependent manner.144 In contrast, when the first dose of ara-C was given 4 hours after the start of a fludarabine infusion, the rate of ara-CTP synthesis in circulating AML blasts was augmented. The ara-CTP AUC and rate of accumulation in AML cells increased 1.8-fold and 2.0-fold, respectively, after fludarabine infusion.141 The median plasma ara-C concentrations and the rate of ara-CTP elimination from circulating blasts were not affected by fludarabine infusion.141 This combination produced significantly higher remission rates (36%) than either high-dose (23%) or intermediate-dose (25%) ara-C in patients with refractory or relapsed AML, especially if initial remissions had lasted longer than 1 year.145 Even patients who had received

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previous high-dose ara-C therapy responded to the combination, provided the leukemia remained sensitive to araC.145 The maximum tolerated plasma concentration for the combination of continuous infusion of fludarabine followed by continuous infusion of escalated ara-C doses was 10 M F-ara-A for 48 hours followed by 72 hours of 15 M ara-C.146 Nine of 18 evaluable pediatric patients with relapsed AML achieved complete or partial responses, and three of nine evaluable patients with relapsed ALL achieved complete or partial responses.147 The addition of idarubicin to ara-C plus fludarabine has been also proposed as means to improve the outcome of pediatric refractory or relapsed leukemia.147–149

Anthracyclines Daunorubicin and doxorubicin have similar pharmacokinetic properties. Both drugs have long terminal plasma half-lives and display extensive tissue binding. During the distributive phase, plasma drug levels decline rapidly as drug penetrates into tissues, except for the CNS, and binds to DNA. Drug concentrations are proportional to the DNA content of the specific tissue.150 The majority of plasma anthracycline exposure is not exposure to daunorubicin but to its 13-hydroxylated metabolite, daunorubicinol, which is formed by a cytosolic aldo/keto reductase enzyme.151,152 This contrasts with doxorubicin, wherein doxorubicinol concentrations are generally below those of doxorubicin.153 The former metabolite has about 10% the cytotoxic activity of daunorubicin in bone marrow stem cells,154 and the latter has approximately 5% of the antitumor activity of doxorubicin155 but may be a more potent cardiotoxin.156 The peak plasma concentrations are similar in adults and children for both the absolute values and the variability.157 Both daunorubicin and daunorubicinol intracellular concentrations in leukemic myeloblasts have been correlated with responses in AML. In contrast, neither daunorubicin nor daunorubicinol plasma exposure has been predictive of outcome,158 whereas high plasma levels of doxorubicin were associated with both death during remission induction therapy and, for patients who entered remission, long remissions.159 Daunorubicin concentrations in the plasma do not correlate with those in the bone marrow, but drug concentrations in white blood cells are correlated positively with those in nucleated bone marrow cells and correlated negatively with the number of peripheral blast cells at diagnosis.160 The achievement of effective drug levels in peripheral leukemic cells may be a function of total tumor cell burden.160 There were significantly higher

cellular daunorubicin and daunorubicinol AUC values in responders than in non-responders among patients with AML or ALL.158 Similarly, Marie et al.161 found increased cellular daunorubicin concentrations in AML patients achieving a complete remission, as compared with those not responding to treatment. Cardiotoxicity from anthracyclines comprises three general categories. The first manifestation is an acute or subacute phase that can occur immediately after treatment, including transient arrhythmias,162,163 a pericarditismyocarditis syndrome with or without electrophysiologic aberrations, or acute left ventricular failure.164 The second type is a delayed total-dose-related cardiomyopathy,165–167 which typically presents within 1 year of therapy. Third, chronic anthracycline-induced cardiotoxicity is characterized by ventricular dysfunction168–170 or arrhythmias,171–173 with an onset years after the last dose of anthracycline. Potential risk factors for chronic cardiomyopathy include high cumulative doses, prior mediastinal irradiation (>2000 rad), age greater than 70 years, and pre-existing cardiovascular disease.174 In addition, young adults who were treated as infants and children may be more susceptible to chronic anthracycline-induced congestive heart failure, particularly if they were treated in infancy.165,171,175,176 Controlled, prospective comparisons of short bolus versus prolonged infusions have shown no difference in acute cardiotoxicity between the two schedules.177 Newer anthracyclines, such as idarubicin, liposomal daunorubicin or doxorubicin, may be associated with reduced cardiotoxicity.178–180 It is not clear to what extent the degree of anthracycline exposure with each individual dosage, versus cumulative dosage, contributes to an increased risk of anthracycline cardiotoxicity. Also, there are no data available on possible pharmacodynamic or pharmacogenetic relationships that would relate plasma exposure to anthracyclines and subsequent cardiotoxicity.

Epipodophyllotoxins The epipodophyllotoxins, etoposide and teniposide, have been incorporated into both front-line and relapse studies for childhood ALL and AML.181–185 The parent drugs are cytotoxic, exerting their effects through their interference with the normal induction of double-stranded DNA breaks by topoisomerase II. In addition, both drugs are metabolized in humans by cytochrome P450 3A4 and 3A5 (CYP3A4, CYP3A5),186 leading to the formation of reactive catechol, semi-quinone, and quinone metabolites (Fig. 14.5)187 that may also contribute to cytotoxic activity, although the

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

Cytochrome P450 3A4

O -demethylation

Catechol Oxidation

Reduction

Semi-quinone Oxidation

Reduction

Etoposide

Teniposide Quinone

Fig. 14.5 O-Demethylation of etoposide and teniposide to reactive catechol, semi-quinone, and quinone metabolites. (Reprinted, with permission, from Relling et al.187 )

1.0

Response (proportion)

quantitative importance of this reaction has not yet been established. From 10% to 70% of the drugs may be recovered unchanged in the urine,185 with lesser amounts recovered unmetabolized when given as long IV infusions or orally as compared to short IV infusions, and for teniposide as compared to etoposide. Etoposide and teniposide do not accumulate in circulating normal blood cells or leukemic myeloblasts.188 Both are highly plasma-protein bound and are substrates for Pglycoprotein; thus, only about 0.3% of plasma concentrations are achieved in cerebrospinal fluid of children with ALL after administration of IV or oral drug.189 However, because the protein concentration in CSF is very low relative to plasma, it is possible that systemic administration of etoposide could contribute to effective CNS leukemia treatment; some clearing of ALL blasts in cerebrospinal fluid was observed following single-agent oral etoposide189 and after intrathecal administration of very small etoposide doses (e.g. 0.5–1.0 mg).190 Several studies have reported relationships between plasma concentrations of etoposide191–197 or teniposide198 and acute hematopoietic or gastrointestinal toxicity. Toxicity likely depends on both duration of exposure to cytotoxic concentrations and on the magnitude of the concentrations.184,199 In children receiving autologous bone marrow transplants, engraftment was slower when 24-hour post-dose etoposide concentrations were greater than as compared to less than 8.5 M.200 No differences in pharmacokinetics or toxicity were noted in children with ALL randomized to receive 50 mg/m2 per day as a single oral daily dose versus a divided dose every 12 hours; toxicity and antileukemic responses were related to plasma exposure.201 Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. However, no significant differences in etoposide disposition between patients who did and who did not develop secondary AML were shown.91 Teniposide is formulated at 10 mg/mL in a high percentage volume of ethanol (40%); thus, high doses (e.g. 500 mg/m2 ) given over short periods (e.g. <4 hours) to children with low teniposide clearance can result in a syndrome of somnolence, hypotension, and metabolic acidosis.202 Ethanol doses exceeded 20 g/m2 , and plasma ethanol concentrations were greater than 60 mg/dL, in three children with ALL who experienced this syndrome immediately after these higher teniposide dosages.202 Extending the duration of infusion to 8 hours is predicted to minimize the potential for ethanol toxicity from high-dose teniposide in children.202

399

0.8 0.6 0.4 0.2

<600 600--1200 1200--1800 >1800 (mM hour) VM26 Systemic exposure (AUC) Fig. 14.6 Proportion of patients treated with continuous-infusion teniposide (VM26) who experienced an oncolytic response (closed circles) or dose-limiting toxicity (open symbols). (Reprinted, with permission, from Rodman et al.203 )

Teniposide systemic exposure, measured as either AUC or steady-state plasma concentration, correlated positively with both antileukemic response and toxicity in a Phase I– II trial of continuous infusion teniposide in children with relapsed and refractory acute leukemias.203 Fortunately, the response-versus-concentration curve for efficacy lies to the left of the curve for toxicity (Fig. 14.6), suggesting that there may be a useful therapeutic range for teniposide: i.e.

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all-trans-RA (µM)

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Limit of detection

Fig. 14.7 Plasma concentrations of all-trans-retinoic acid (ATRA) versus time after intravenous administration of 50 mg/m2 to four rhesus monkeys on day 1 (•), day 3 (◦), day 5 (
), and day 8 () of drug administration, illustrating the decrease in AUC that occurs with chronic dosing. (Reprinted, with permission, from Adamson et al.206 )

there may a target level of systemic exposure at which a reasonable probability of efficacy and an acceptable probability of toxicity may be predicted. A study demonstrated that children who lacked the glutathione S-transferase theta gene (GSTT1), whose expression plays an important role in determining the cytotoxicity of chemotherapeutic drugs, including alkylating agents and topoisomerase II agents (such as etoposide), had greater toxicity and reduced survival after chemotherapy for AML compared with children with at least one GSTT1 allele.204

Trans-retinoic acid The use of all-trans-retinoic acid (ATRA) in remission induction regimens for acute promyelocytic leukemia (APL) has become widespread, and the drug has interesting pharmacokinetic properties. ATRA is usually administered orally, often for a period of several weeks. The plasma concentrations (AUC) of ATRA decrease substantially with daily dosing (Fig. 14.7), such that the AUC is as much as 8-fold lower after 7 days of ATRA, as compared with the first day.205 This accelerated clearance occurs after both IV and oral dosing.205,206 It has been hypothesized that this increased clearance might be facilitated by the induction of lipid hydroperoxides by ATRA, rather than by a simple induction of P450s or other drug metabolizing enzymes.207 Although other mechanisms undoubtedly play a role, the potent

“auto-induction” of ATRA clearance has been hypothesized to contribute to the development of APL resistance to ATRA.205,207 Intermittent, rather than continuous, administration of ATRA has been proposed to attenuate this accelerated clearance,205,208,209 although whether such a schedule has clinical benefit has not yet been demonstrated. There are some data indicating relationships between systemic and intracellular exposure to ATRA and the antileukemic effect in APL. Oral administration of 30 mg/m2 per day produced lower plasma levels in a single patient who did not achieve a complete remission than in identically treated patients who did achieve a complete remission.208 In addition, adult and pediatric patients with APL whose blasts accumulated higher cellular concentrations of ATRA (>180 pmol/106 cells) when incubated ex vivo with 1 M ATRA were more likely to achieve a complete remission than those with lower accumulation.210 Patients with poor ATRA uptake tended to be older and were more likely to have early toxic deaths,210 so it is not clear whether this enhanced uptake is a variable that could be exploited therapeutically or is simply correlated with a more responsive APL phenotype. A report of patients with adult solid tumors suggested that severe toxicity tended to occur more frequently at higher initial peak plasma ATRA concentrations.211

Asparaginase Asparaginase is an effective chemotherapeutic agent in childhood ALL, and thus is a component in most multiagent remission induction regimens.212 It exploits a metabolic difference between normal and leukemic cells. Normal cells are able to synthesize most amino acids, including asparagine; however, some leukemic cells are deficient in inducing the enzyme asparagine synthetase in response to asparagine depletion.213 Asparaginase does not enter the cell, but hydrolyzes asparagine to aspartic acid and ammonia extracellularly, depriving leukemic cells of their source of asparagine (Fig. 14.8). Asparagine-dependent protein synthesis is halted and nucleic acid synthesis is later inhibited, decreasing leukemic cell proliferation. Different preparations of asparaginase have varied pharmacokinetic properties. Asparaginase is an enzyme isolated from various natural sources: Escherichia coli and Erwinia chrysanthemi. PEG-asparaginase is a conjugate of the native E. coli asparaginase covalently linked to polyethylene glycol at sites not affecting enzymatic activity. This conjugation prevents uptake by the reticuloendothelial system and prolongs the elimination half-life of the drug. Pharmacokinetics have been most extensively

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

Fig. 14.8 Mechanism of action of asparaginase, illustrating the putative dependence of leukemic cells on exogenous asparagine, as compared with normal cells, which are able to synthesize asparagine. This mechanism leads to selective cytotoxicity against leukemic cells, whose extracellular asparagine source is depleted.

studied following intravenous administration of the E. coli preparation,213–216 although the intramuscular route of administration is currently more commonly used. The elimination half-life of asparaginase depends on the preparation used and whether the patient experienced a hypersensitivity reaction to asparaginase. The half-life of Erwinia asparaginase at 25,000 IU/m2 (0.65 ± 0.13 days) was significantly shorter than that of E. coli asparaginase (1.24 ± 0.17 days), and the half-lives of both native preparations were shorter than that of PEG asparaginase 2500 IU/m2 (5.73 ± 3.24 days).217 In patients who have had a hypersensitivity reaction to E. coli asparaginase, the half-lives of both E. coli and PEG preparations are reduced, although the number of patients studied has been small.217 In a CCG study, patients treated with PEG asparaginase had more rapid clearance of lymphoblasts from day-7 and day-14 bone marrow aspirates and more prolonged asparaginase activity than those treated with E. coli asparaginase, although there was no difference in long-term outcome.218 A randomized comparison of 30 weeks of PEG versus E. coli asparaginase showed no differences in outcome between the two forms,219 but outcome was improved in patients able to receive more than 25 weeks of dosing compared to those who received fewer doses. As many as 70% of patients may develop antiasparaginase antibodies, many without clinical evidence of hypersensitivity.220,221 Antibody levels are higher, both before and after the occurrence of the reaction, in patients who develop clinical hypersensitivity to asparaginase. Also, antibody concentrations increase in patients receiving asparaginase over time, regardless of whether patients exhibit clinical allergy.218,222 Low plasma asparaginase

levels are associated with high antibody titers to either E. coli or PEG asparaginase,223 and in a trial for relapsed ALL, the complete remission rate was significantly associated with higher levels of asparaginase.223 It has been suggested that these antibodies may hamper the antileukemic effect of asparaginase by shortening its half-life, preventing or delaying absorption after intramuscular injection, or interfering with enzymatic activity.217,220 No patients with frank allergic reactions during asparaginase treatment had asparagine plasma concentrations below the limit of quantitation.224 However, in settings in which Erwinia asparaginase was used in cases of clinical hypersensitivity to E. coli, asparaginase but not for subclinical development of antibodies, there was no adverse prognostic impact of clinical or subclinical allergy to asparaginase, consistent with better asparagine depletion by Erwinia asparaginase in cases of allergy.225,226 Attenuation of plasma asparagine depletion with successive exposures to asparaginase has been reported.224 For example, asparagine plasma depletion (<7.6 M) was observed in 80% of patients during the first exposure to Erwinia asparaginase and in only 25% of patients during the second or third exposure.224 Asparaginase does not penetrate into the CNS; however, plasma asparagine levels are correlated with those in the CSF.224 As asparagine levels decrease in plasma, levels in CSF also decrease, probably due to a concentration gradient, and thus asparaginase may play a role in the prevention of meningeal leukemia. CSF asparagine levels were below the level of quantitation (<0.2 M) in 75% of children and in 37% of children 3 and 5 days, respectively, after the last dose of a course of Erwinia asparaginase.224 Another study

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demonstrated 73.9% and 0% of children who received E. coli asparaginase had undetectable asparagine (<0.04 M), during and more than 30 days after asparaginase therapy, respectively.227 Interestingly, marked pharmacokinetic and pharmacodynamic differences were noted after doses of two different manufacturers’ preparations of native E. coli asparaginase,228 illustrating the importance of formulation for the pharmacology of asparaginase. Asparaginase significantly affects cellular protein synthesis, resulting in a number of schedule-dependent drugdrug interactions. Inhibition of protein synthesis may attenuate the cytotoxic effects of subsequent methotrexate or cytarabine.229 The asparaginase effect on the former agent may be related to reduction in methotrexate polyglutamylation.230–232 In contrast, administration of either antimetabolite followed by asparaginase resulted in therapeutic synergy.229 The inhibition of protein synthesis caused by asparaginase can cause hypoalbuminemia, which in turn can alter the protein binding of highly bound anticancer drugs such as the epipodophyllotoxins.198 The use of asparaginase has been linked to the risk of teniposide233 and etoposide-associated secondary AML,234 although the mechanism is unclear.

Glucocorticoids Prednisone and dexamethasone are used commonly in induction, reinduction, and continuation drug regimens for childhood ALL. Excellent results of continuation therapy incorporating dexamethasone have been reported.235 When given as pre-induction “window” therapy, there was no difference in the acute bone marrow blast response between 40 mg/m2 of prednisone and 6 mg/m2 of dexamethasone, but increasing dexamethasone doses (18 or 150 mg/m2 ) resulted in a greater response than the standard doses of glucocorticoids (Fig. 14.9).236 In an allogeneic bone marrow-derived stromal support system for shortterm culture (4 days) of patient B-lineage ALL blasts, dexamethasone was a median 5.5 times more potent than prednisone.237 Studies using patient blast cells cultured in the absence of stroma reported an even larger median potency difference of 16.2-fold.238 Thus, based on differences in cytotoxicity to lymphoblasts, the optimal dosage of dexamethasone is not clear. Generally, doses of about 40 mg/m2 per day of prednisone and 6 to 8 mg/m2 per day of dexamethasone have been used in ALL regimens. Controlled trials prospectively comparing the two agents in standard-risk B-lineage ALL showed that dexamethasone at 6 mg/m2 resulted in improved event-free survival compared to prednisone

Percent reduction

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Fig. 14.9 Mean percent reduction in marrow blast cells (day 0 to day 3) according to corticosteroid dose for all 286 assessable patients (All), 105 standard-risk patients (SR), and 181 high-risk patients (HR). PRED-40, prednisolone 40 mg/m2 group; DEX-6, dexamethasone 6 mg/m2 group; DEX-18, dexamethasone 18 mg/m2 group; DEX-150, dexamethasone 150 mg/m2 group. (Modified, with permission, from Schwartz et al.236 )

40 mg/m2 .239,240 A CALGB study241 found that fewer CNS relapses occurred in a group treated with dexamethasone 6 mg/m2 per day than a group who received prednisone 40 mg/m2 per day. Whether these doses were equivalent in terms of toxicity is not clear. The choice of the ratio to determine “equipotent” dosages of dexamethasone and prednisone is controversial, and depends on the pharmacodynamic endpoint used for comparison. Oral doses of dexamethasone have been reported to be 17-fold more potent (in terms of cortisol suppression) than prednisone in humans,242 suggesting that the 6.7-fold difference in dosing currently employed in many ALL regimens may result in greater glucocorticoid exposure with dexamethasone than with prednisone. Whether toxicity will be greater with dexamethasone than with prednisone is not yet clear, but increased toxic death rates have been reported with some remission induction regimens that include dexamethasone.243 However, others have safely used dexamethasone235 and so the precise dose, along with concurrent therapy, is likely to influence toxicity. Although pharmacodynamics of glucocorticoids have not been studied in patients with ALL, there are some data indicating that prednisone clearance was lower (i.e. plasma exposure was higher) in renal transplant patients who developed avascular necrosis than in patients who did not.244 The incidence of avascular necrosis in children over 10 years of age treated for ALL with dexamethasone-containing regimens ranges from 10% to 20%.245 Rare mutations in the glucocorticoid receptor (NR3C1 ) gene have been associated with corticosteroid resistance

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

and hematological malignancies,246 and several NR3C1 gene variants247,248 have been identified that are relatively common in the human populations.249,250 These polymorphisms may affect the toxicity and outcome of leukemia patients who receive glucocorticoids. Glucocorticoids are potent inducers of cytochrome P450 3A and interact with P-glycoprotein, and can thereby affect the disposition of other antileukemic agents (e.g. etoposide), such that polymorphisms affecting CYP3A and MDR1 may influence the inductive effects of steroids.251 Glucocorticoids are partly inactivated by conjugation with glutathione, and an improved early response of childhood ALL has been found in patients who have mutant genotypes for glutathione transferases.252

Vincristine There are few data available on the pharmacokinetics or pharmacodynamics of vincristine, largely due to difficulties in measuring the very low concentrations present in plasma. Radioimmunoassay showed a higher AUC in patients (mostly adults) who experienced vincristineassociated neurotoxicity than in those who did not.253 Significant interindividual and intraindividual variability with vincristine pharmacokinetics in children has been observed,254,255 perhaps related to steroid induction of P450 metabolism or of transporter-mediated excretion. Vincristine clearance for children who received vincristine as monotherapy has been substantially slower than that which has been reported for children receiving vincristine in combination with steroids.256 Other drugs known to interact with vincristine include both phenytoin and carbamazepine,257 which induce CYP3A4 expression and increase vincristine clearance, as well as itraconazole,258 which inhibits CYP3A and P-glycoprotein and enhances vincristine toxicity. Polymorphisms of the CYP3A4, CYP3A5, and MDR1 genes have been reported,259–261 and may affect the toxicity of and response to vincristine. Because increased AUC and neurotoxicity was more common in patients with an elevated alkaline phosphatase,253 and biliary excretion of vincristine appears to represent a significant fraction of vincristine clearance,262 dosage reductions have been recommended for patients with hepatic obstruction. However, neurotoxicity was not clearly related to vincristine AUC in a more recent study using a more specific HPLC assay in children with ALL.263 Because of a high frequency of neurotoxicity reported in very young infants (i.e. those with body surface area of < 0.5 m2 ),264 infants are frequently given lower dosages of vincristine than are older children. Whether this high

frequency of neurotoxicity was due to poor drug clearance or to enhanced tissue sensitivity is not known.

Oxazaphosphorines Cyclophosphamide has been incorporated into continuation and consolidation regimens for ALL, although its benefit as compared to other agents has been questioned.265 In a prospective study in which children were randomly assigned to “full-dose” versus “half-dose” chemotherapy that included mercaptopurine, methotrexate, and cyclophosphamide, remission rates were much more favorable in the full-dose arm.266 Again, however, the contribution of cyclophosphamide cannot be clearly discerned. Although some groups have more recently incorporated ifosfamide into ALL regimens,267 ifosfamide has some pharmacokinetic disadvantages as compared to cyclophosphamide; namely, a much larger percentage of the drug is susceptible to N-dechloroethylation to inactive and potentially toxic metabolites.268 It has been reported that ifosfamide enters the CSF to a greater extent than cyclophosphamide, and the ability of both ifosfamide and cyclophosphamide and their metabolites to cross the blood–brain barrier may be reduced by dexamethasone.269 There are no published pharmacodynamic data available for the oxazophosphorines in acute leukemia. Because of the active metabolism required for these agents, pharmacodynamics should be more likely to relate to metabolite concentrations than to parent drug concentrations. Nonetheless, cardiotoxicity was related to a lower parent drug AUC (and putatively greater active alkylating metabolite AUC) in women with breast cancer who received cyclophosphamide as part of their conditioning regimen.270 Since cyclophosphamide and ifosfamide are prodrugs that undergo extensive CYP-catalyzed metabolism to yield both active and inactive metabolites,271 the polymorphisms that alter their function259,272 may relate to toxicity and response of these drugs. GSTs are involved in inactivation of oxazaphosphorine active metabolites and better survival has been reported among women with breast cancer who were treated with cyclophosphamide and were homozygous for low-activity GSTP1 alleles.273

Conclusion Interindividual differences in the pharmacokinetics of antileukemic agents can affect the efficacy and toxicity of antileukemic therapy.40 Future studies must

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address whether individualizing therapy, based on pharmacokinetic and pharmacogenetic principles, can improve antileukemic outcome in children.

15

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advanced stage lymphoblastic lymphoma: a Pediatric Oncology Group study. Leukemia, 1999; 13: 335–42. Pui, C. H., Relling, M. V., Behm, F. G., et al. L-asparaginase may potentiate the leukemogenic effect of the epipodophyllotoxins. Leukemia, 1995; 9: 1680–4. Veerman, A. J. P., Hahlen, K., Kamps, W. A., et al. High cure rate with a moderately intensive treatment regimen in nonhigh-risk childhood acute lymphoblastic leukemia: results of protocol ALL VI from the Dutch Childhood Leukemia Study Group. J Clin Oncol, 1996; 14: 911–18. Schwartz, C. L., Thompson, E. B., Gelber, R. D., et al. Improved response with higher corticosteroid dose in children with acute lymphoblastic leukemia. J Clin Oncol, 2001; 19: 1040–6. Ito, C., Evans, W. E., McNinch, L., et al. Comparative cytotoxicity of dexamethasone and prednisolone in childhood acute lymphoblastic leukemia. J Clin Oncol, 1996; 14: 2370–6. Kaspers, G. J. L., Veerman, A. J. P., Pop-Snijders, C., et al. Comparison of the antileukemic activity in vitro of dexamethasone and prednisolone in childhood acute lymphoblastic leukemia. Med Pediatr Oncol, 1996; 27: 114–21. Gaynon, P. S., Trigg, M. E., Heerema, N. A., et al. Children’s Cancer Group trials in childhood acute lymphoblastic leukemia: 1983–1995. Leukemia, 2000; 14: 2223–33. Gaynon, P. S., Bostrom, B. C., Hutchinson, R. J., et al. Duration of hospitalization as a measure of cost on Children’s Cancer Group acute lymphoblastic leukemia studies. J Clin Oncol, 2001; 19: 1916–25. Jones, B., Freeman, A. I., Shuster, J. J., et al. Lower incidence of meningeal leukemia when prednisone is replaced by dexamethasone in the treatment of acute lymphocytic leukemia. Med Pediatr Oncol, 1991; 19: 269–75. Meikle, A. W. & Tyler, F. H. Potency and duration of action of glucocorticoids. Effects of hydrocortisone, prednisone and dexamethasone on human pituitary-adrenal function. Am J Med, 1977; 63: 200–7. Hurwitz, C. A., Silverman, L. B., Schorin, M. A., et al. Substituting dexamethasone for prednisone complicates remission induction in children with acute lymphoblastic leukemia. Cancer, 2000; 88: 1964–9. Lausten, G. S., Egfjord, M., & Olgaard, K. Metabolism of prednisone in kidney transplanted patients with necrosis of the femoral head. Pharmacol Toxicol, 1993; 72: 78–83. Mattano, L. A., Jr., Sather, H. N., Trigg, M. E., et al. Osteonecrosis as a complication of treating acute lymphoblastic leukemia in children: a report from the Children’s Cancer Group. J Clin Oncol, 2000; 18: 3262–72. Hurley, D. M., Accili, D., Stratakis, C. A., et al. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest, 1991; 87: 680–6. Weaver, J. U., Hitman, G. A., Kopelman, P. G. An association between a Bc1I restriction fragment length polymorphism of

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the glucocorticoid receptor locus and hyperinsulinaemia in obese women. J Mol Endocrinol, 1992; 9: 295–300. Rossum, E. F. van, Koper, J. W., Huizenga, N. A., et al. A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels. Diabetes, 2002; 51: 3128–34. DeRijk, R. H., Schaaf, M., & de Kloet, E. R. Glucocorticoid receptor variants: clinical implications. J Steroid Biochem Mol Biol, 2002; 81: 103–22. Tissing, W. J., Meijerink, J. P., Noer, M. L. den, & Pieters, R. Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia. Leukemia, 2003; 17: 17–25. Kishi, S., Yang, W., Morand, S., et al. Effects of prednisone and genetic polymorphisms on etoposide disposition in children with acute lymphoblastic leukemia. Blood, 2004; 103: 67–72. Anderer, G., Schrappe, M., Brechlin, A. M., et al. Polymorphisms within glutathione S-transferase genes and initial response to glucocorticoids in childhood acute lymphoblastic leukaemia. Pharmacogenetics, 2000; 10: 715–26. Desai, Z. R., Berg, H. W. van den, Bridges, J. M., et al. Can severe vincristine neurotoxicity be prevented? Cancer Chemother Pharmacol, 1982; 8: 211–14. de Graaf, S. S., Bloemhof, H., Vendrig, D. E., et al. Vincristine disposition in children with acute lymphoblastic leukemia. Med Pediatr Oncol, 1995; 24: 235–40. Gidding, C. E., Meeuwsen-de Boer, G. J., Koopmans, P., et al. Vincristine pharmacokinetics after repetitive dosing in children. Cancer Chemother Pharmacol, 1999; 44: 203–9. Groninger, E., Meeuwsen-De Boar, T., Koopmans, P., et al. Pharmacokinetics of vincristine monotherapy in childhood acute lymphoblastic leukemia. Pediatr Res, 2002; 52: 113–18. Villikka, K., Kivisto, K. T., Maenpaa, H., et al. Cytochrome P450inducing antiepileptics increase the clearance of vincristine in patients with brain tumors. Clin Pharmacol Ther, 1999; 66: 589–93. Kamaluddin, M., McNally, P., Breatnach, F., et al. Potentiation of vincristine toxicity by itraconazole in children with lymphoid malignancies. Acta Paediatr, 2001; 90: 1204–7. Lamba, J. K., Lin, Y. S., Thummel, K., et al. Common allelic variants of cytochrome P4503A4 and their prevalence in different populations. Pharmacogenetics, 2002; 12: 121–32. Kuehl, P., Zhang, J., Lin, Y., et al. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet, 2001; 27: 383–91. Hoffmeyer, S., Burk, O., Richter, O. von, et al. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci U S A, 2000; 97: 3473–8.

Pharmacokinetic, pharmacodynamic, and pharmacogenetic considerations

262 Jackson, D. V., Castle, M. C., & Bender, R. A. Biliary excretion of vincristine. Clin Pharmacol Ther, 1978; 24: 101–7. 263 Crom, W. R., de Graaf, S. S., Synold, T., et al. Pharmacokinetics of vincristine in children and adolescents with acute lymphocytic leukemia. J Pediatr, 1994; 125: 642–9. 264 Woods, W. G., O’Leary, M., & Nesbit, M. E. Life-threatening neuropathy and hepatotoxicity in infants during induction therapy for acute lymphoblastic leukemia. J Pediatr, 1981; 98: 642–5. 265 Niemeyer, C. M., Hitchcock-Bryan, S., Sallan, S. E. Comparative analysis of treatment programs for childhood acute lymphoblastic leukemia. Semin Oncol, 1985; 12: 122–30. 266 Pinkel, D., Hernandez, K., Borella, L., et al. Drug dosage and remission duration in childhood lymphocytic leukemia. Cancer, 1971; 37: 247–56. 267 Reiter, A., Schrappe, M., Wolf-Dieter, L., et al. Chemotherapy in 998 unselected childhood acute lymphoblastic leukemia patients. Results and conclusions of the multicenter trial ALLBFM 86. Blood, 1994; 84: 3122–33.

268 Kamen, B. A., Frenkel, E., & Colvin, O. M. Ifosfamide: should the honeymoon be over? J Clin Oncol, 1995; 13: 307–9. 269 Yule, S. M., Price, L., Pearson, A. D., et al. Cyclophosphamide and ifosfamide metabolites in the cerebrospinal fluid of children. Clin Cancer Res, 1997; 3: 1985–92. 270 Ayash, L. J., Wright, J. E., Tretyakov, O., et al. Cyclophosphamide pharmacokinetics: correlation with cardiac toxicity and tumor response. J Clin Oncol, 1992; 10: 995–1000. 271 Huang, Z., Roy, P., & Waxman, D. J. Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide. Biochem Pharmacol, 2000; 59: 961–72. 272 Lang, T., Klein, K., Fischer, J., et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics, 2001; 11: 399– 415. 273 Sweeney, C., McClure, G. Y., Fares, M. Y., et al. Association between survival after treatment for breast cancer and glutathione S-transferase P1 Ile105Val polymorphism. Cancer Res, 2000; 60: 5621–4.

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15 Assays and molecular determinants of cellular drug resistance Monique L. den Boer and Rob Pieters

Introduction Chemotherapy for children with acute leukemia has improved impressively over the past 40 years, despite the use of similar agents throughout this period. The efficacy of antileukemic drugs depends largely on their dosages and schedules of administration. It also depends on the intrinsic and acquired resistance of leukemic cells to drug treatment. Knowledge of the factors contributing to cellular drug resistance was slow to accumulate, but has grown rapidly over the last two decades. This chapter describes some of the prominent methods for assessing drug resistance in leukemia patients and reviews progress in elucidating the molecular determinants of this phenomenon.

Drug cytotoxicity assays In the early 1980s, drug cytotoxicity was mainly evaluated by clonogenic assays such as colony-forming unit assays and stromal cell layer-supported long-term marrow cultures. These time-consuming assays rely on the in vitro proliferating capacity of cells, a characteristic that in practice is restricted to acute and chronic myeloid blasts.1 The most frequent type of leukemia in children, acute lymphoblastic leukemia (ALL), cannot be tested with assays of this type because the cells lack any in vitro proliferating capacity. Moreover, the resistance of resting, nondividing cells, which may be an important source of treatment failure, is not detectable with these proliferation-based assays.

Dye exclusion or differential staining cytotoxicity assay In 1983, Weisenthal and co-workers2 introduced the dye exclusion assay (DEA), which enables one to test the

cytotoxicity of drugs in nonclonogenic cells.2 This assay exploits trypan-blue dye, which selectively diffuses into damaged cells with permeabilized cell membranes. Since dead cells are often eliminated during cell culture, counting the dead cells would underestimate cell kill. Only counting viable cells gives reliable results. Therefore, an internal standard (usually duck erythrocytes) is added, and the number of viable (dye-excluding) cells per fixed number of erythrocytes is counted by light microscopy at different drug concentrations. This assay was further improved by counterstaining cells with May-Grunwald-Giemsa stain, which facilitates the counting of viable cells on the basis of morphologic features.3,4 An advantage of the dye-exclusion test, also called the differential staining cytotoxicity (DiSC) assay, is that the cytotoxic effect of drugs in leukemic cells can be discriminated from that in normal cells, and only low cell numbers are needed. A disadvantage is the inevitable increase in test-result variability due to technical and/or observer errors.

Methyl tetrazolium assay A second assay that can determine drug toxicity in nonclonogenic cells was developed in the mid 1980s and is based upon the ability of living cells to reduce 3(4,5-dimethylthiazol-2,5-diphenyl-2-yl) tetrazolium bromide (MTT) into formazan.5 This MTT assay determines the cytotoxic effect of a drug after 4 days of culture by measuring the amount of formazan that has been formed in drug-treated compared with untreated control cells. The advantage of this assay is its simplicity and short-term culture conditions; its disadvantage is that contaminating normal cells are highly resistant to drugs and also reduce MTT into formazan. Hence, it is necessary to check the

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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percentage of leukemic cells present in control wells after 4 days of culture.6 In recent years, several MTT analogs (e.g. XTT) have been developed that show better solubility of formazan crystals and/or more stable detection signals.7 Both DiSC and MTT assays yield comparable results, and the data are interchangeable.4,8 This also means that in cases of low leukemic cell numbers (<2.5×106 ), a DiSC assay can be performed, while in cases of sufficient cells the less tedious and time/error-conserving MTT assay can be substituted. Since both assays can be performed with 96-well plates and require the same culture conditions, the decision to perform a DiSC assay can be made as late as the fourth day of culture if the percentage of leukemic cells in the control wells is insufficient.

cells after 7 days of culture to about 90% compared with about 65% after 4 days of culture for the DiSC or MTT assay.12,13 At the end of the incubation interval, the percentage of leukemic cells among surviving cells is determined by flow cytometry using cell surface markers. The disadvantage of this assay is that it is labor intensive and the required cell surface markers are differentially expressed between leukemic cells and normal hematopoietic cells. ATP assay Cytotoxicity can also be determined by analyzing the ATP content of cells that survive the exposure to different drug concentrations, relative to that of untreated control cells. In this so-called ATP assay, a bioluminescent marker is used to quantify the amount of ATP present in the sample.14

Less frequently applied drug cytotoxicity assays Thymidylate synthetase inhibition assay The DiSC and MTT assays cannot be used to measure methotrexate cytotoxicity in primary cells of acute leukemia patients.9 An alternative is the thymidylate synthetase inhibition assay (TSIA), which is based on the principle that methotrexate inhibits the conversion of dUMP into dTMP mediated by thymidylate synthetase. As in the DiSC and MTT assays, leukemic samples are exposed to different concentrations of drug in order to generate dose–response curves; however, in contrast to those assays, the incubation interval is shortened to only 1 day (∼21 hours). The relative cytotoxicity of methotrexate is measured by comparing the relative amount of 3 H-labeled H2 O that is released upon the conversion of [3 H]dUMP to dTMP in methotrexate-treated and control cells.9 Fluorometric microculture cytotoxicity assay (FMCA) This assay differs from the MTT assay mainly in the substrate conversion procedure that is used for detecting the number of viable cells remaining after drug exposure. Since the release of fluorescein upon hydrolysis of fluorescein diacetate by viable cells is measured, the FMCA assay requires a fluorescence reader instead of an absorbance reader.10 Drug cytotoxicity data obtained with the FMCA and DiSC assays in ALL patients showed good correlation.11 Stroma-supported immunocytometric assay (SIA) In the early 1990s, Campana and colleagues developed an in vitro cytotoxicity assay in which precursor B leukemic cells were cultured on top of a stromal layer obtained from allogeneic bone marrow biopsies. The use of a stromal layer improved the survival rate of control (unexposed) leukemic

Nucleotide incorporation assay Radioactive nucleotide precursors, such as [3 H]GTP and [3 H]TTP, are incorporated into the DNA backbone largely during the S-phase of the cell cycle.15 Therefore, nucleotide incorporation assays depend on at least one cell division during culture. Since human (especially lymphoblastic) leukemic cells do not divide after isolation from bone marrow or peripheral blood, nucleotide incorporation assays are not suitable for determining drug toxicity in primary patients-derived cells but can be applied to proliferating leukemic cell lines.

Clinical value of drug cytotoxicity testing in pediatric leukemia Predictive value of cellular drug resistance The DiSC and MTT assays are often used to test the responsiveness of a patient’s leukemic cells to different classes of both novel and already established cytotoxic drugs. Several groups showed independently that children with in vitroresistant ALL cells at initial diagnosis have poorer long-term clinical outcome than do in vitro-sensitive patients.13,16–18 Most discriminative for an unfavorable prognosis in ALL is cellular resistance to glucocorticoids: the 3-year diseasefree survival in Dutch Childhood Leukemia Study Group protocol-ALL7/8 for children with resistant leukemic cells was about 40%, compared with 100% for children with in vitro-sensitive cells.13 Among children with ALL treated on the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocol ALL-92, in vitro dexamethasone resistance at initial diagnosis was also associated with an unfavorable 3-year disease-free survival compared to the rate for sensitive patients.19 Moreover, in vitro resistance to

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prednisolone had a higher predictive value than the widely applied in vivo response to a therapeutic window with this drug before the start of induction chemotherapy.20 As expected, patients with a poor response to prednisone also showed greater in vitro resistance to this drug. Among patients with a good clinical response to prednisone (<1000 leukemic cells/ L of peripheral blood at day 8), however, those with leukemic cells resistant to prednisolone in vitro still had an unfavorable outcome. This study indicates that the cytotoxicity assay has additional discriminative predictive value for relapse in the large group of patients with a good clinical response to prednisone. The presence of residual leukemic cells after 2 to 4 weeks of therapy also correlated with in vitro resistance to prednisolone, whereas no correlation with in vitro vincristine, L-asparaginase and doxorubicin resistance was observed.21,22 At relapse, the leukemic cells of children with ALL were more resistant to glucocorticoids, anthracyclines, L-asparaginase, methotrexate and thiopurines than they were at initial diagnosis, whereas no difference was observed in resistance for vinca alkaloids, cytarabine (ara-C) and epipodophyllotoxins.23,24 Besides glucocorticoids, cellular resistance to other drugs frequently used in therapy for pediatric ALL are associated with an unfavorable outcome. Examples are L-asparaginase, vincristine, daunorubicin and 6-thioguanine.13,18 A resistance profile combining the cellular response to three single drugs – prednisolone (or dexamethasone), vincristine and L-asparaginase – was independently shown by several groups to have the greatest discriminative prognostic value in pediatric ALL. Patients who were most resistant to all three drugs had a two- to seven- fold higher risk of relapse than did relatively sensitive patients.13,25,26 Children with acute myeloid leukemia (AML) are highly resistant to almost all drugs used in their therapy compared with results for ALL patients.27 An exception are the antimetabolites ara-C and thiopurines, to which AML and ALL cells are equally sensitive.27 In contrast to pediatric ALL, cellular drug resistance at initial diagnosis of pediatric AML has not been shown to be discriminative for long-term clinical outcome.28,29 At relapse, children with AML are more resistant to ara-C compared with patients at initial diagnosis, whereas no difference was observed for anthracyclines, thiopurines and epipodophyllotoxins. Paired analysis of initial-relapse samples from the same patients did not indicate that acquired resistance plays a role in pediatric AML.30 In contrast to pediatric AML, resistance to ara-C is associated with an unfavorable outcome in adult AML.31 The increase in degree of cellular resistance to ara-C with increasing age may reflect the more

aggressive nature of adult AML compared with pediatric AML.32

Clinical and biologic risk features Cellular drug resistance is associated with a number of unfavorable risk factors, such as age, (immuno)phenotype and genetic abnormalities in pediatric ALL and AML. Age The relation between age and drug resistance is difficult to define because several important factors, such as immunophenotype and genetic abnormalities are related to age. The frequency of MLL gene rearrangements, for example, is very high in infants and very low in older children with ALL. BCR-ABL fusion is more frequent in adult ALL than in children with ALL, whereas the reverse is true for the prognostically favorable TEL-AML1 fusion and hyperdiploidy.33 Nevertheless, several conclusions can be drawn. Infant ALL cases are relatively resistant to glucocorticoids, both in vitro and in vivo34,35 In addition, infants show relative resistance to L-asparaginase but are remarkably sensitive to ara-C in vitro.34 Infant ALL cells show no defective methotrexate polyglutamation.36 Children with ALL who are older than 10 years are more resistant to glucocorticoids compared with children who are 1.5 to 10 years of age.34 Resistance to drugs increases with age. Adults are more resistant to glucocorticoids (ALL), daunorubicin (ALL), L-asparaginase (ALL) and ara-C (ALL and AML) than are children suffering from the same leukemia type.32,37,38 ALL cells from adults also accumulate less active polyglutamated metabolites of methotrexate.39 Within ALL cases with a BCR-ABL fusion, increasing age of the patient is related to glucocorticoid resistance.40 (Immuno)phenotype Children with T-ALL are more resistant in vitro to glucocorticoids, vinca alkaloids, L-asparaginase, ara-C, anthracyclines and methotrexate compared to children with precursor B-lineage ALL.9,34,41 T-ALL and precursor-B-ALL cases do not differ in their in vitro sensitivity to thiopurines and teniposide.34 Since T-ALL is often associated with age older than 10 years, it is difficult to assess which parameter (immunophenotype or age) contributes the most to drug resistance. However, multivariate analysis (including among other features immunophenotype and age) revealed that cellular drug resistance was an independent prognostic factor, suggesting that both immunophenotype and age both contribute to the resistant phenotype.20,34 Different subtypes of T-ALL can be classified using CD marker expression and gene expression profiling.42,43 Pilot

Assays and molecular determinants of cellular drug resistance

studies in our own laboratory suggest that these subtypes also differ in the level of cellular glucocorticoid resistance. Among children with precursor B-lineage ALL, those with pro-B ALL are more resistant to glucocorticoids, Lasparaginase, thiopurines and anthracyclines but more sensitive to ara-C, than those with common/pre-B ALL.34 Infants with MLL gene rearrangements predominate in the pro-B ALL group.44 Such infants are more sensitive to ara-C than are older children without this rearrangement.45 The frequency of noninfants with pro-B and MLL gene rearrangements is so low that group comparisons have not been performed. In the ongoing Interfant-99 study, the contribution of pro-B phenotype, MLL gene rearrangement and MLL fusion partner to cellular drug resistance is being determined in order to answer this question in infant ALL. Children with AML-M5 are relatively more sensitive in vitro to anthracyclines, ara-C, etoposide, L-asparaginase and vincristine than are children with the less favorable M4 FAB type.27,28 AML-M5 patients are also more sensitive to ara-C and etoposide compared with AMLM1/M2 patients.27 This M5 FAB type is also often found in infants with (MLL-rearranged) AML, which may reflect a similar cause for sensitivity to ara-C in both MLLrearranged ALL and AML. Compared to B-lineage ALL patients, AML patients are more resistant to methotrexate due to impaired polyglutamation of the drug (see also Methotrexate in the section Molecular determinants of cellular drug resistance).9,46,47 Exceptions are AML-M5 and AML-M7, which show relatively effective polyglutamation of methotrexate.48–50 Genetic abnormalities Heterogeneity of cellular drug responsiveness is also observed in ALL subclasses with apparently uniform immunophenotypic features, such as T-ALL and common/pre-B ALL. Deletions in the p16/INK4A gene locus at chromosome 9p21, which encodes two different cell cycle inhibitors p16INK4A and P19ARF by transcription of alternative exons, are found in about 70% of pediatric T-ALL patients. Homozygous deletion of p16 in T-ALL is associated with a poor long-term outcome but not with in vitro drug resistance,51 suggesting that the p16 gene-deleted T-ALL cells are not per se drug resistant but have a regrowth advantage compared with p16 wild-type leukemic cells. About 25% of children with precursor B-lineage ALL have a t(12;21) translocation, resulting in a TEL-AML1 fusion protein. Leukemic cells from these children are relatively sensitive to L-asparaginase but not to the other frequently used drugs.52 Controversial reports on the predictive value of t(12;21) rearrangements may be explained by

the different doses and schedules used for L-asparaginase administration. The increased sensitivity to L-asparaginase is not caused by decreased expression of the gene encoding the L-asparaginase-opposing enzyme asparagine synthetase in t(12;21)-rearranged leukemias (see also L-asparaginase in the section Molecular determinants of cellular drug resistance).53 Polyglutamate levels of methotrexate were lower in t(12;21)-rearranged cases than in children without this rearrangement, suggesting a reduced efficacy of methotrexate in these patients.54 Children with hyperdiploid ALL, defined by greater than 50 chromosomes per leukemic cell, represent about 25% of precursor B-lineage non-t(12;21)-rearranged cases. The favorable prognosis associated with this numeric over-representation of chromosomes is associated with increased sensitivity to the antimetabolites thiopurines and ara-C as well as L-asparaginase, the accumulation of high levels of polyglutamated methotrexate and high levels of spontaneous apoptosis in stroma-supported culture assays, by comparison with nonhyperdiploid cases.54–58 In 3% to 5% of pediatric ALL cases, a translocation occurs between the BCR gene on chromosome 9 and the ABL gene on chromosome 22, resulting in the so-called Philadelphia chromosome. Hongo et al.59 showed that about 60% of Philadelphia chromosome-positive pediatric patients were highly resistant to two or more drugs, a finding associated with an unfavorable outcome compared with that in relatively sensitive patients. Others found no difference in cellular drug resistance patterns between children with or without Philadelphia chromosome-positive ALL.40 Rearrangements involving the MLL gene on chromosome 11q23 are found in about 3% to 5% of children with ALL and in about 6% to 14% of children with AML.60 Within the ALL group, MLL gene rearrangements mainly affect children younger than 1 year of age with a pro-B immunophenotype. These infants are resistant to glucocorticoids and L-asparaginase, show no altered methotrexate polyglutamation but are strikingly more sensitive to araC compared with older/non-MLL-rearranged children with ALL.34,36,45 Among patients with 11q23/MLL-rearranged AML, those with the t(9;11) rearrangement are more sensitive to ara-C.29 This translocation and the AML-M4, M5, and M7 FAB types are predominantly found in infants with AML. Zwaan et al.27 showed that cases of AML-M5 and AML-M7 are also more sensitive to ara-C than are other FAB types in children with AML.27 The striking similarity in ara-C sensitivity between cases of 11q23/MLL-rearranged ALL and AML suggests a common mechanism of sensitivity to this drug in MLL-rearranged leukemias.29,34,45 Leukemic patients with Down syndrome mostly have megakaryoblastic (M7) AML or precursor B-lineage ALL.

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Down syndrome confers a more favorable outcome in patients with AML, but not in those with precursor B-lineage ALL. The Down syndrome-AML patients are in general more sensitive to commonly used drugs such as anthracyclines and ara-C than are patients without this syndrome, whereas Down- and non-Down syndrome-ALL patients are equally sensitive to such agents.61,62 The unfavorable prognosis associated with chromosome 5 or 7 abnormalities in pediatric AML is linked to in vitro resistance to ara-C.29 Although the chromosomal abnormalities t(8;21) and inv(16) are associated with a favorable outcome in pediatric AML, no differences in cellular drug responses between patients with or without the t(8;21) or inv(16)-positive AML were observed.29

Molecular determinants of cellular drug resistance Drug cytotoxicity assays are useful because they focus on cell kill, the end point of many different determinants of response, but they offer no insight into the mechanisms of resistance that may have importance in childhood leukemias. Over the last 20 years, knowledge of the molecular determinants of resistance in the acute leukemias of childhood, especially ALL, has increased tremendously. The following sections review these advances for each of eight major antileukemic drugs or drugs classes.

Glucocorticoids Mechanisms of action Glucocorticoids such as prednisolone and dexamethasone are synthetic derivatives of the naturally occurring steroid hormone cortisol. Glucocorticoids induce cell cycle arrest and apoptosis in sensitive cells (Fig. 15.1). To do so, they enter the cell and bind to the glucocorticoid receptor (GCR). Upon binding, several accessory proteins like heatshock proteins 70 and 90 and BAG-1/RAP46 are released from the GCR, which retains its proper configuration and sequesters the nuclear-localization signaling motifs of the receptor (reviewed by Tissing et al.63 ). The glucocorticoidglucocorticoid receptor (GC-GCR) complex translocates to the nucleus and interferes with gene transcription in either of two ways. First, it activates transcription through binding of GC-GCR homodimers to glucocorticoid responsive elements (GRE) present in promoters of some genes (transactivation). Second, it inhibits transcription (transrepression) through binding to other nuclear transcription factors such as activating protein-1 (AP-1) and nuclear factor B (NF-B).

Resistance mechanisms Figure 15.1 outlines the possible causes of glucocorticoid resistance in childhood ALL.

Glucocorticoid receptor The number of GCRs expressed in leukemic cells may affect the responsiveness to glucocorticoids. Low GCR expression at initial diagnosis is associated with a poor short- and longterm clinical outcome in pediatric ALL. However, high GCR expression at initial diagnosis was not necessarily associated with a good clinical response.64 Most of these receptor number studies determined absolute receptor-binding numbers per cell and analyzed the glucocorticoid-binding at room temperature, whereas recently it was shown that the receptor concentration per cell and receptor-binding of glucocorticoids at 37 ◦ C is more informative.65 Receptor concentration and receptor-affinity for glucocorticoids were lower in T-ALL and AML (which are also in vitro glucocorticoid resistant) compared with B-lineage ALL cells obtained from patients. In addition, glucocorticoidinduced GCR expression may be more important for response to this drug, as was recently shown for leukemic cell lines.66,67 The choice of GCR upregulation or downregulation may depend on the promoter type present in resistant cells. Glucocorticoid exposure upregulated expression of promoter/exon 1A3 receptor transcripts in leukemic cell lines but not lymphoma cell lines.68 In addition to receptor numbers, the ratio between active and inactive splice variants may be important for cellular responses to glucocorticoids. The GCR has five known splice variants alpha, beta, gamma, P (or delta) and A, of which at least the alpha variant is active, while the beta and P/delta variant are incapable of glucocorticoid binding.69,70 The gamma variant differs from other isoforms only by the inclusion of three nucleotides between exons 3 and 4, which encode arginine.71 This variant may be part of both alpha, beta and P/delta and A splice variants, and was reported to have only 50% of the alpha-receptor activity.72 The activity of the A variant is still unknown.73 Knowledge of the clinical relevance of these splice variants (both basal and drug-induced expression) is limited in both leukemias and other malignancies. So far, a decreased alpha/beta ratio has been observed in leukemic cells of T-ALL patients compared with those of precursor B-lineage ALL patients, which may contribute to the relative resistance to glucocorticoids in T-ALL.74 Expression of GCR-gamma was lower in children with ALL sensitive to prednisone.75 Polymorphisms in the GCR gene have been found that are linked with glucocorticoid hypersensitivity (e.g. N363S) and resistance (e.g. R477H) syndromes in humans.63 The resistance or sensitivity of leukemic cells to glucocorticoids

Assays and molecular determinants of cellular drug resistance

GC

GCR (1)

GC GC accessory proteins

(2) (6)

GC

GC

MRP/P-gp

GC

(3) GC

AP-1

NFκ κB GRE

AP-1

NFκ κB

Gene X (4) (5)

X

TF-site

GC-targeted genes: cell cycle-related apoptosis etc.

Gene Y

(5)

Fig. 15.1 Possible causes of resistance to glucocorticoids in ALL: (1) Decreased binding of GC to GCR may be due to decreased GCR number, expression of inactive GCR splice variants, mutations and/or single nucleotide polymorphisms in GCR that affect configuration of the receptor, and/or altered GCR configuration due to changes in the composition of the GCR-accessory protein complex (e.g. Hsp70, Hsp90, BAG-1). (2) Impaired translocation of GCR-dimer to nucleus as a result of alteration in DNA-binding domain sequence and/or nuclear localization sequence (e.g. GCR-gamma splice variant or mutations/polymorphisms), and or altered binding of accessory proteins required for stabilization of GC-bound GCR. (3) Reduced transactivation capacity of GC-GCR complex reflecting an altered composition of the GCR-accessory protein complex, which is required for stabilization and binding to GRE-containing genes; mutations in the DNA-binding domain of GCR; and/or mutations in GRE itself, thereby preventing binding of GCR and subsequent transcription of downstream genes. (4) Reduced transrepression activity of GC-GCR complex due to imbalanced expression of NFB, AP-1 or other transcription factors and/or mutations in GCR affecting the effective binding of NFB, AP-1, etc. (5) Abnormalities in expression of genes targeted by GC due to mutations in downstream genes, imbalance in pro- and antiapoptotic regulatory proteins (BCL2, BAX, MCL1, etc.), and/or expression of GC-targeted genes induced by other signalling pathways. (6) Increased efflux of GC due to overexpression of transmembrane transporter proteins like P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP) and/or conjugation of GC to glutathione. TF site, binding site for transcription factors [such as nuclear factor B (NFB) and activating protein-1 (AP-1)]; GC, glucocorticoid; GCR, glucocorticoid receptor; GRE, glucocorticoid-responsive element.

may therefore be a direct consequence of inherited polymorphisms by patients, although evidence for this hypothesis is still lacking. Alternatively, somatic (leukemic-cell restricted) mutations may occur that determine responsiveness to the drug. A glucocorticoid-resistant leukemic cell line was found to contain a point mutation resulting in a leucine-to-phenylalanine conversion (L753F) in GCR. This mutation was also shown to be present in

a subset of the leukemic cells of the corresponding patient.76 Accessory proteins Heat-shock proteins such as Hsp 70 and 90 were shown to stabilize the configuration of GCR, a requirement for optimal binding of GC. The expression of both heat-shock proteins was decreased in glucocorticoid-resistant leukemic

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cell lines.77 In addition, BAG-1, a negative regulator of Hsp70, is overexpressed in dexamethasone-resistant cell lines.78 In contrast to these cell line studies, expression levels of Hsp90 did not associate with the in vivo prednisone response in pediatric ALL.79 Numerous proteins (e.g. CREB-binding protein) interact with glucocorticoid receptors upon translocation to the nucleus, facilitating binding to transcription factors such as NF-B.80 Whether resistance to glucocorticoids is caused by altered expression and activity of these types of accessory proteins in leukemia remains to be determined. One recent study did not show a relationship between chaperone molecules of the GC and GC resistance in ALL.81 Glucocorticoid efflux by transmembrane transporters Glucocorticoids bound to glutathione were shown to be transported out of the cell by the multidrug-resistance associated protein MRP1, whereas native glucocorticoids may be transported by P-glycoprotein.81,82 Resistance to prednisolone, however, was not associated with the expression levels of MRP1 or P-glycoprotein in pediatric ALL and AML.83 In addition, high glutathione levels were found to be associated with an increased risk of relapse, but not with prednisolone resistance, in the leukemic cells of patients.84 Moreover, expression of glutathione S-transferases, which facilitate the conjugation of glutathione to glucocorticoids, did not differ between glucocorticoid sensitive and resistant leukemic cells of children.85 These data suggest that drug efflux by membrane transporters is not an important mechanism of glucocorticoid resistance in pediatric ALL. Interaction with targeted genes Activated glucocorticoid receptor complexes bind to GREcontaining genes and transcription factors such as AP-1 and NF-B, resulting in transactivation and transrepression of targeted genes, respectively. The introduction of mutations in the DNA-binding domain of the glucocorticoid receptor abrogated binding to GRE-containing genes and resulted in resistance to glucocorticoids in the lymphocytes of mice.86 Resistance to glucocorticoids is not linked to basal expression levels of AP-1 and NF-B in pediatric ALL.87,88 NF-B rearrangements or amplifications found in adult ALL are absent in pediatric ALL.89 However, resistance may be associated with changes in downstream signaling pathways controlled by AP-1 or NF-B. Known downstream targets of NF-B are MYC and inhibitor-of-apoptosis (IAP) genes. Decreased MYC expression correlated with increased glucocorticoid-induced apoptosis in leukemic cell lines.90 Studies in mice showed that overexpression of X-linked IAP proteins resulted into glucocorticoid-resistant thymocytes.91 Resistance to glucocorticoids may therefore

be caused by changes in glucocorticoid-induced transactivation and/or transrepression of proteins involved in apoptosis (see Abnormalities in the apoptosis-signaling pathway in the section Cross-resistance to different classes of drugs).

L-asparaginase Mechanisms of action The toxic action of L-asparaginase results from the rapid systemic depletion of asparagine by hydrolysis into aspartic acid and ammonia. Asparagine is important for the synthesis of proteins, its carbon backbone is used in the synthesis of glucose, and its nitrogen is used for the synthesis of urea, glycine (which is important to purine biosynthesis) and other amino acids. L-asparaginase also possesses glutaminase activity (∼2–3% of the asparaginase activity), so that glutamine levels are also (partly) affected upon treatment. The therapeutic efficacy of L-asparagine therefore resides in the effects on the availability of amino acids and nucleotides required for cell survival and proliferation. Possible causes of L-asparaginase resistance are summarized in Fig. 15.2.

Resistance mechanisms Asparagine synthetase and amino acid metabolism The intracellular enzyme asparagine synthetase (AS) opposes the action of L-asparaginase. Asparagine is formed from aspartic acid and ammonia; the nitrogen may be derived from glutamine, which in turn is converted into glutamic acid. For decades it was assumed that the antileukemic effect of L-asparaginase was caused by a lack of AS in leukemic cells but not in normal cells.92,93 However, it was recently shown that AS is present in leukemic cells and, moreover, that its expression levels are even higher than in normal bone marrow and peripheral blood leukocytes.53 Cell line studies showed that alterations in intracellular amino acid composition regulate the transcription of AS.95 In accord with these cell line findings, L-asparaginase was shown to induce the expression of AS in precursor B-lineage leukemic cells of children with ALL. However, basal and L-asparaginase-induced expression levels were linked with L-asparaginase resistance in TEL-AML1-negative ALL, but not in TEL-AML1-rearranged ALL.53 Whether this association applies to other types of ALL is not yet clear. The activity of the enzyme may be more informative than its expression level in terms of cellular responsiveness to L-asparaginase. AS activity was lower in the leukemic cells of AML-M5 patients as compared with

Assays and molecular determinants of cellular drug resistance

Glutamine

(2)

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amino acid pool serine/threonine kinases (e.g. p70 S6 kinase)

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NH3

NH3

ribosomal proteins

(2) Aspartic acid

Aspartic acid

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plasma

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Fig. 15.2 Proposed basis of resistance to L-asparaginase (ASP) in acute leukemia. (1) Increased expression and/or activity of asparagine synthetase (AS). (2) Increased uptake of glutamine/aspartic acid and/or decreased efflux of asparagine (e.g. due to altered expression of transmembrane transporter proteins). (3) Altered intracellular balance in amino acids interferes with the activity of translation-regulating proteins such as protein serine/threonine kinases and translation repressor proteins, which in turn affect the activity of ribosomal proteins and translation initiation factors, respectively. (4) Imbalanced protein synthesis may protect against apoptosis.

those in other AML subtypes, which may explain the relative sensitivity to L-asparaginase that was observed in these patients.27,97 Leukemic cell lines resistant to L-asparaginase displayed an increased uptake of glutamine, whereas asparagine efflux was decreased compared with that of parental cell lines.98 This increased uptake was accompanied by increased expression of amino acid transmembrane transporters, such as system A, ASC or L. It is unknown whether resistance to L-asparaginase in leukemic cells of patients is caused by an altered expression of these transmembrane transporters. Protein synthesis machinery L-asparaginase inhibits the activity of enzymes functioning in the regulation of protein translation, such as p70 S6 kinase, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal proteins.99,100 The p70 S6 kinase phosphorylates ribosomal proteins involved in

the tRNA/mRNA interaction needed for protein synthesis, while 4E-BP1 participates in the initiation of translation of capped mRNAs. Cell line data suggest that L-asparaginase resistance may be caused by the altered expression and/or activity of enzymes involved in the translation of proteins. Interestingly, recent gene expression profile studies revealed that L-asparaginase resistance in pediatric ALL is linked to overexpression of several members of the ribosomal protein family.101

Vinca alkaloids Mechanisms of action Vinca alkaloids are represented by two naturally occurring compounds, vincristine and vinblastine, and by semisynthetic derivatives such as vindesine. Vinca alkaloids bind to tubulin molecules and interfere with GTP hydrolysis,

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Vinca alkaloids

P-gp MRP1 (3)

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tubulin unit

β α

α

β

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apoptosis

(7)

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reactive oxygen species

O2•

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Topo II (4)

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(8)

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epipodophyllotoxins

Fig. 15.3 Proposed basis of resistance to vinca alkaloids, anthracyclines and epipodophyllotoxins in acute leukemia. Vinca alkaloids: (1) decreased binding of vinca alkaloids to microtubules due to altered expression, mutations and/or post-translational modifications of beta-tubulin isotypes; (2) altered nuclear trafficking by increased expression of microtubule-associated proteins (MAP), such as MAP4 and dynein; (3) increased efflux of drug out of cell by transmembrane transporter proteins, such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). Anthracyclines: (4) increased expression of topoisomerase II enzymes may reduce the chance to induce DNA damage by reducing the relative number of anthracycline–topoisomerase II cleavable complexes that can be formed; (5) increased detoxification of reactive oxygen species (e.g. by glutathione/glutathione S-transferases or the sphingomyelin-ceramide pathway); (6) increased DNA repair after DNA damage due to cleavable complexes or reactive oxygen species; (7) blockade of apoptotic signaling route at or upfront the mitochondrial level prevents cell death; (8) reduced intracellular availability of anthracyclines due to sequestration and/or exocytosis, (e.g. by vaults); (9) increased efflux of anthracyclines mediated by transmembrane transporter proteins such as P-gp and MRP1. Epipodophyllotoxins: (10) increased expression of topoisomerase II enzymes may reduce the chance to induce DNA damage by reducing the relative number of epipodophyllotoxin–topoisomerase II cleavable complexes that can be formed; (11) increased efflux of epipodophyllotoxins mediated by transmembrane transporter proteins such as P-gp and MRP1.

Assays and molecular determinants of cellular drug resistance

resulting in depolymerization of microtubules.102 Since microtubules maintain cell shape, contribute to intracellular transport of molecules, and form the mitotic spindle needed for cell division, disruption of these structures is highly toxic to cells.

Resistance mechanisms The molecular basis of resistance to the vinca alkaloids is outlined in Fig. 15.3. Interaction with microtubules Vinca alkaloids bind to the beta-tubulin subunit of alpha/beta-tubulin heterodimers. Six beta-tubulin isotypes have been identified so far, of which the class I and III isotypes have been linked to vinca alkaloid cytotoxicity. Vincristine-resistant leukemic cell lines demonstrated decreased expression of class III beta-tubulin, whereas class I beta-tubulin and alpha-tubulin expression did not differ from that of parental cells. In class I betatubulin of resistant cells, a point mutation, as well as a post-translational modification, was found in the alphabeta dimerization site of the protein. In addition, expression of the microtubule-associated protein 4 (MAP4) was increased in these cells.103 Recently, vinca alkaloids were shown to stimulate the translocation of the tumor suppressor protein p53 from cytosol to the nucleus via the microtubule-associated protein dynein.104 Hypothetically, vinca alkaloid resistance may therefore be linked to altered (increased or decreased) nuclear trafficking of proteins due to altered isoform expression and/or modifications of tubulin and associated proteins. Whether this hypothesis is valid remains to be determined in the leukemic cells of patients. Vinca alkaloid efflux by transmembrane transporters Vinca alkaloids can be transported out of the cell by the transmembrane transporters P-glycoprotein and multidrug resistance-associated protein 1 (MRP1), thereby lowering the intracellular drug concentration. However, P-glycoprotein and MRP1 expression are not increased at relapse compared to initial diagnosis, and protein expression levels are not associated with vincristine resistance in pediatric ALL and AML.83

Anthracyclines Mechanisms of action The main members of the anthracycline family, which are often used in the treatment of acute leukemia, are daunorubicin, doxorubicin and idarubicin. Anthracycline cytoxicity

can be explained by several mechanisms of action that interfere with the normal physiology of the cell. Direct binding of anthracyclines to DNA and their interaction with DNA helices and DNA topoisomerases influence the transcription, replication and repair of DNA. Anthracyclines can also form free radicals that cause DNA damage and lipid peroxidation. Peroxidation of cardiac lipids may also explain an important side effect of anthracyclines – cardiotoxicity. Finally, but not least important, anthracyclines are thought to induce cell death via activation of caspases and other apoptotic signaling molecules.

Resistance mechanisms Possible avenues of anthracycline resistance are summarized in Fig. 15.3. Accumulation, retention and sequestration of anthracyclines The intracellular concentration of daunorubicin was lower in daunorubicin-resistant compared with daunorubicinsensitive leukemic cells of children with ALL.105 Decreased intracellular daunorubicin concentrations could not be explained by increased expression or activity of drug-efflux mediating transmembrane proteins such as P-glycoprotein and multidrug resistance-associated protein 1 (MRP1) in children with ALL.83,106 Alternatively, (exocytotic) transport and sequestration of anthracyclines by vault proteins may explain resistance to the anthracyclines.107 Expression of vaults, especially the major vault protein or lung resistance protein (MVP/LRP) has been associated with resistance to anthracyclines, vinca alkaloids and epipodophyllotoxins.107 Expression of MVP/LRP was increased in the leukemic cells of children with recurrent ALL and in children with initial AML compared to initial ALL. However, MVP/LRP expression correlated only weakly with daunorubicin resistance in children with ALL and not at all in adults with AML, suggesting that this factor is not a major cause of anthracycline resistance in leukemia.83,108 Additionally, the lack of prognostic value of MVP/LRP in both pediatric and adult acute leukemias implies that it is not a critical determinant of clinical drug resistance.108–114 Free radical scavengers The formation of free radicals or reactive oxygen species by anthracyclines is thought to trigger the sphingomyelinceramide pathway, which ultimately results in apoptosis. Overexpression of protein kinase C (PKC) zeta in myeloid leukemic cell lines counteracted this pathway by detoxifying reactive oxygen species, leading to daunorubicin resistance.115 It is unknown whether anthracycline

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resistance is associated with decreased free radical and/or altered PKC zeta levels in the leukemic cells of patients. Free radicals are also scavenged by glutathione Stransferases. These enzymes can also directly bind anthracyclines and therefore overexpression may protect against the cytotoxic effects of these drugs. However, expression of different glutathione S-transferase isoenzymes did not differ between the daunorubicin-sensitive and resistant leukemic cells of children with ALL.85 The glutathione S-transferase-mediated detoxification of anthracyclines by conjugation to reduced glutathione is less likely to occur due to the chemical structure of these agents. Depletion of reduced glutathione enhanced the intracellular retention of daunorubicin in cell lines, which was attributed to the function of the multidrug resistance-associated protein 1 (MRP1).116,117 In contrast to these cell line studies, reduced glutathione levels are not linked to anthracycline resistance; moreover, MRP1 expression is not linked to reduced daunorubicin retention and resistance in children with ALL.84,105

DNA damage and apoptosis Anthracyclines generate double-stranded DNA breaks by interfering with cleavable complexes of DNA and DNA topoisomerase II enzymes (see also Epipodophyllotoxins). However, in both pediatric ALL and adult AML, the expression and activity of topoisomerase II enzymes were not linked with resistance to anthracyclines, indicating that these enzymes do not play a pivotal role in the anthracycline resistance of acute leukemia.118–120 Anthracycline toxicity may depend on the inability of cells to repair DNA lesions properly, resulting in apoptosis. Anthracyclines can induce cell cycle arrest via upregulation of the cyclin-dependent kinase inhibitor p21 (also called wild-type p53 activated factor, WAF1, or cyclin-dependent kinase-interacting protein, CIP1).121 If DNA repair is not possible, cells enter apoptosis. Resistance to anthracyclines is associated with decreased depolarization of mitochondrial membranes in pediatric ALL.122 In leukemic cell lines, anthracyclines were shown to trigger CD95/Fas death receptors, which in turn activate caspase 8 and depolarize mitochondria.123–125 Constitutive expression of CD95 is not sufficient to induce apoptosis upon exposure to CD95-ligand or CD95-ligand-mimicking antibodies in the leukemic cells of children with ALL.126–128 De novo synthesis of CD95 upon anthracycline exposure is required to trigger apoptosis in leukemic cell lines.123 However, the clinical relevance of CD95 upregulation for drug responsiveness is questionable in the leukemic cells of ALL and AML patients.127,129,130 This suggests that other factors play a role in clinical resistance to anthracycline-induced

apoptosis (see also Cross-resistance to different classes of drugs). Anthracyclines were shown to induce apoptosis via triggering of the sphingomyelin-ceramide signaling pathway in myeloid leukemic cell lines, which resulted in altered expression of ceramide-sensitive genes.131,132 Interestingly, protection against doxorubicin-induced apoptosis by PKC zeta overexpression is associated with decreased ceramide production and inhibited reactive oxygen species formation in leukemic myeloid cell lines.115 It remains to be determined whether this pathway is also important in the leukemic cells of patients, but current gene expression profiling studies comparing daunorubicin-resistant and sensitive patients may give us more insight into the role of this sphingomyelin-ceramide pathway in pediatric ALL.

Epipodophyllotoxins Mechanisms of action Historically, the epipodophyllotoxins, a class represented by etoposide (VP16) and teniposide (VM26), have been identified as double-strand DNA break-inducing agents. The toxicity of the epipodophyllotoxins has been ascribed to the drug-induced stabilization of so-called cleavable complexes between DNA topoisomerase II
and DNA (Fig. 15.3). DNA topoisomerase II
is an enzyme involved in DNA replication, transcription and repair through formation of transient DNA breaks. Etoposide and teniposide induce increased formation of DNA breaks without repair, eventually leading to fragmentation of DNA and cell death.

Resistance mechanisms DNA topoisomerase II Epipodophyllotoxins (as well as anthracyclines) may irreversibly freeze the cleavable complex of topoisomerase II
and DNA, leading to permanent DNA breaks (Fig. 15.3). Transfection studies using leukemic cell lines showed that the increased expression of topoisomerase II
was linked to resistance to epipodophyllotoxins and anthracyclines. However, expression levels of topoisomerase II
did not differ between diagnostic and relapsed ALL samples. In addition, expression levels were not associated with cellular resistance to epipodophyllotoxins and anthracyclines in pediatric ALL, although children in relapse were more resistant to both classes of drugs compared to children with newly diagnosed ALL.119 The activity of topoisomerase may be more important than its expression levels, as recently shown by the fact that phosphorylation of topoisomerase II
at 1106Ser of the catalytic domain is important to the cytotoxicity of

Assays and molecular determinants of cellular drug resistance

etoposide.133 Also, mutations in the ATP-binding and/or DNA-binding site of topoisomerase II
affected the cytotoxicity of epipodophyllotoxins in leukemic cell lines.134,135 Multidrug resistance transmembrane transporters The induction of double-strand breaks and, ultimately, fragmentation of DNA may also be a late feature of apoptosis, so that a reduced number of double-strand DNA breaks may not represent the real cause of resistance to this class of drugs. Theoretically, a reduced intracellular drug concentration due to increased efflux by so-called multidrug resistance proteins like P-glycoprotein (P-gp) and multidrug-resistance-associated protein (MRP1) may explain resistance (Fig. 15.3). However, these proteins are unlikely to play a role in pediatric ALL and AML.83,106,114 Also, drug sequestration or drug exocytosis mediated by the major vault protein/lung resistance protein MVP/LRP seems an unlikely explanation for epipodophyllotoxin resistance in pediatric ALL and AML.83 Since cross-resistance between epipodophyllotoxins and other classes of drugs, especially the vinca alkaloids and anthracyclines, is often observed in the leukemic cells of patients, one might conclude that epipodophyllotoxinspecific targets are lacking and that resistance to this class of drugs is linked to more general pathways related to cell death (see Abnormalities in the apoptosis-signaling pathway in the section Cross-resistance to different classes of drugs).

Methotrexate Mechanism of action Methotrexate possesses antileukemic activity because of its structural resemblance to folic acid. Folic acid metabolites are necessary for the synthesis of DNA, RNA and amino acids – processes that are inhibited by methotrexate. Methotrexate is especially useful against ALL, but presumably lacks activity in AML. Most studies on mechanisms of resistance to methotrexate have therefore been performed in ALL. The mechanism of action of methotrexate is reviewed by Rots et al.136 Methotrexate is actively taken up by cells via the reduced folate carrier (RFC). The membrane folate receptor (MFR) is thought to play a role in folate transport across the cell membrane but not in methotrexate membrane transport due to the low affinity of the MFR for methotrexate. Passive membrane transport occurs at high extracellular methotrexate concentrations that are reached when high-dose methotrexate regimens are applied in ALL. Intracellularly, polyglutamation of methotrexate, catalyzed by folylpolyglutamate synthetase (FPGS), takes place by

addition of glutamate residues, a process that is important for the retention of methotrexate inside the cell. Interestingly, methotrexate polyglutamates (methotrexate-PGs), which are degraded by folylpolyglutamate hydrolase, affect more target enzymes than does methotrexate. The target enzymes of methotrexate and methotrexate-PGs are dihydrofolate reductase (DHFR) and thymidylate synthase (TS), which are involved in the folate metabolism necessary for DNA synthesis. Purine de novo synthesis is inhibited by methotrexate-PG.

Resistance mechanisms Alterations in at least three phases of methotrexate action (Fig. 15.4) could contribute to resistance to this agent. Membrane transport Conflicting results have been published with respect to possible defects in membrane transport as a cause of methotrexate resistance in relapsed ALL and AML.136–138 In these studies methotrexate membrane transport was measured indirectly. Other studies measuring a direct methotrexate uptake assay showed no impaired membrane transport of methotrexate in relapsed ALL.24,139 T-ALL and B-lineage ALL patients do not differ in methotrexate transport or RFC mRNA expression levels.138–140 Extra copies of chromosome 21, often found in hyperdiploid ALL and of course in Down syndrome, correlate with higher levels of RFC mRNA levels.139,140 Polymorphisms in the RFC gene may contribute to the sensitivity of methotrexate. Carriers of the RFC1 A80 polymorphism have a worse outcome than do patients with the GG genotype.141 Higher plasma methotrexate levels were found in patients with homozygosity for A80 , although methotrexate levels and outcome were not related. Future studies should elucidate whether RFC1 polymorphisms are related to intracellular methotrexate levels because of changes in substrate affinity.142 A point mutation at codon 45 (E45K) is related to methotrexate resistance in a transport defective cell line but was not detected in any of the 121 childhood ALL samples analyzed.143 The ratio between methotrexate and normal folate uptake might be important for the response to methotrexate in ALL.144 Recently, cell line studies have shown the importance of the multidrug resistance gene MRP1–4 for methotrexate efflux.145–147 Whether this finding has clinical relevance remains to be demonstrated in leukemic cells from patients. Methotrexate polyglutamation Accumulation of low amounts of methotrexate-PGs in ALL cells correlates with a poor outcome,41,148 although this relationship might depend on the therapy protocol.149

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Fig. 15.4 Mechanisms of resistance to methotrexate (MTX) in acute leukemia. (1) Decreased membrane transport by polymorphisms in the reduced folate carrier (RFC). (2) Increased MTX efflux by increased activity of multidrug resistance-related proteins (MRP). (3) Decreased formation of MTX-polyglutamates (MTX-PG) by decreased activity of folylpolyglutamate synthetase (FPGS). (4) Increased breakdown of MTX-PG by increased activity of folylpolyglutamate hydrolase (FPGH). (5) High levels of the target enzyme dihydrofolate reductase (DHFR); gene amplification of DHFR or DHFR mutations. (6) Polymorphisms in the target gene thymidylate synthase (TS). (7)Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. DNPS, de novo purine synthesis; FHS, dihydrofolate; FH4, tetrahydrofolate.

Hyperdiploid ALL cells accumulate higher levels of methotrexate-PGs than do nonhyperdiploid ALL cells.41,58 T-lineage ALL cases accumulate fewer methotrexate-PGs than do B-lineage ALL cases, both in vitro and in vivo after either low-dose or high-dose exposure.9,39,41 This is due to less effective polyglutamation: T-ALL cells contain fewer pharmacologically important long-chain methotrexatePGs than do B-lineage ALL cells.9,39,57,150 The less efficient methotrexate-PG formation in T-ALL is related to a lower FPGS activity compared to that in B-lineage ALL9,151 and not to an increased activity of FPGH.9 AML cases show impaired methotrexate polyglutamation compared to cases of B-lineage ALL,9,46,47 due to both lower FPGS and higher FPGH activity,9,151 as well as a lower affinity of FPGS for methotrexate.152 Relapsed ALL shows no defective

polyglutamation as compared to ALL at diagnosis.24 Two subtypes of AML, M5 and M7, have shown relatively effective methotrexate-PG formation.48–50 Defects in methotrexate polyglutamation can be overcome both in vitro and in vivo by continuous exposure to high doses of the drug.41,136 This knowledge has led to the incorporation of higher doses of methotrexate into some contemporary treatment protocols for T-ALL. Target enzymes High levels of the target enzyme DHFR also correlated with a relatively poor outcome.138 DHFR gene amplification associated with elevated DHFR mRNA levels and higher DHFR activity were found in relapsed ALL.138,153 DHFR mutations were not detected in clinical samples of

Assays and molecular determinants of cellular drug resistance

relapsed ALL cases.153,154 Cases of T-ALL express higher DHFR levels than do B-lineage ALL cases.138 Polymorphisms in the promoter region of the thymidylate synthase gene were found to be important determinants of outcome in ALL.155 Polymorphisms in the methylenetetrahydrofolate reductase gene might also be associated with sensitivity to methotrexate.156,157

Thiopurines Mechanism of action 6-Mercaptopurine is metabolized via the purine salvage pathway. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) converts 6-mercaptopurine to thioinosine monophosphate, which is then metabolized to thioguanine nucleotides. Alternatively, it can be metabolized to methylated products via thiopurine methyltransferase (TPMT). The methylthioinosine monophosphate is an inhibitor of PRPP amidotransferase. The results of these processes are inhibition of the purine salvage pathway and the de novo purine synthesis (DNPS) by 6-mercaptopurine. 6-Thioguanine produces its effects in a comparable way but has less impact on the DNPS because it is directly converted to 6-thioguanine nucleotides; hence, the production of (methyl-)thioinosine monophosphate is not observed.158,159

Resistance mechanisms Decreased levels of HGPRT lead to thiopurine resistance in cell lines and animal models.160–164 These experimental systems showed an almost complete loss of HGPRT. However, studies on ALL patient samples showed only a 10-fold interindividual variation of HGPRT activity that was not related to thiopurine sensitivity; nor was HGPRT activity different between relapsed and initially diagnosed ALL patients and patients.165,166 Also, activities of the salvage pathway enzymes adenosine deaminase and purine nucleoside phosphorylase were not related to thiopurine cytotoxicity in ALL samples derived from patients.167 Breakdown of the cytotoxic nucleotides by nucleotidases or phosphatases has also been studied as a possible cause of resistance. Ecto-5 -nucleotidase has been suggested to play a role in this breakdown,168,169 but this enzyme has its active site facing the extracellular compartment, which makes breakdown of cytosolic thiopurine nucleotides by ecto-5 -nucleotidase unlikely.170,171 Degradation of thiopurine nucleotides by intracellular nucleotidases and phosphatases might play a role in thiopurine resistance in some ALL patients.163,171,172

In 1989, Lennard and Lilleyman173,174 showed that low intracellular thioguanine nucleotide levels in red blood cells were correlated with a poor treatment outcome.173,174 These variations especially appeared to reflect differences in the inherited activity of TPMT,175,176 although other factors might also contribute to these differences.177,178 The molecular basis for altered TPMT activity was defined by demonstrating polymorphic mutant alleles in the TPMT gene.177 Many papers (reviewed by McLeod et al.179 ) have been published on the clinical relevance of the genetic polymorphism of TPMT in ALL. Lower activities of TPMT lead to lower amounts of methylated TIMP and less inhibition of the DNPS but higher amounts of cytotoxic thionucleotides.158,159 Consequently, the leukemia cells show increased sensitivity to 6-mercaptopurine. However, TPMT-deficient ALL patients treated with 6mercaptopurine are exposed to excessive levels of thioguanine nucleotides in hematopoietic tissues and experience life-threatening side effects due to severe marrow suppression. There was a trend toward a better outcome in TPMTdeficient ALL patients in a study in which dose intensity for 6-mercaptopurine was the most significant prognostic factor.180

Cytarabine Mechanisms of action Cytarabine (ara-C) is a modified deoxynucleoside that can be irreversibly integrated into DNA during DNA repair and replication, leading to apoptosis. Cytarabine enters the cell by the same transmembrane transporters as naturally occurring nucleosides and is activated into its active form, ara-CTP, by the sequential phosphorylating activity of a number of kinases [e.g. deoxycytidine kinase (dCK), deoxycytidylate (dCMP) kinase and nucleoside diphosphate (NDP) kinase]. Opposing reactions are the dephosphorylation of ara-CMP into ara-C by pyrimidine nucleotidase I and the deamination of ara-C into ara-uridine (ara-U) and ara-CMP into ara-UMP by cytidine deaminase and deoxycytidylate deaminase, respectively. These opposing reactions decrease the bioavailability of ara-CTP in the cell.

Resistance mechanisms Metabolic activation of ara-C The most frequently observed cause of resistance to ara-C in cell lines and mouse models is a decreased activity of dCK (Fig. 15.5). Abnormalities in the dCK gene have been reported that affect ara-C sensitivity.181 However, mutations and polymorphisms in dCK are rare in both pediatric and adult acute leukemias.182,183 Increased expression of

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Ara-C (1)

(2) Ara-C (6)

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(5) dCDP

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(8) UTP pyrimidine de novo synthesis

apoptosis

Fig. 15.5 Metabolic activation pathway and putative causes of cytarabine (ara-C) resistance in acute leukemia. (1) Decreased expression of equilibrative nucleoside transporter ENT1 hampers the intracellular accumulation of ara-C. (2) Mutations, polymorphisms and/or expression levels of full-length and/or splice variants may affect the activity of deoxycytidine kinase (dCK), which converts ara-C to ara-C monophosphate (ara-CMP). (3) ara-CMP may be inactivated by dephosphorylation to ara-C, which is catalyzed by pyrimidine nucleotidase-I (PN-I). (4) Decreased activity of deoxycytidylate (dCMP) kinase, which converts ara-CMP to ara-C diphosphate (ara-CDP). (5) Decreased formation of ara-CTP by aberrant activity of broad specificity nucleoside diphosphate kinases (NDP kinase), which interconverts nucleoside diphosphates (NDP) to nucleoside triphosphates (NTD) and vice versa. (6) Inactivation of ara-C to ara-U by increased activity of cytidine deaminases. (7) Increased deamination of ara-CMP to ara-UMP by deoxycytidylate deaminase (dCMPD). (8) Increased CTP pool by de novo synthesis of UTP mediated by CTP synthetase. (9) Increased formation of deoxycytidine diphosphate (dCDP) catalyzed by ribonucleotide reductase, which in turn is converted into dCTP by NDP kinases (5). (10) High levels of dCTP compete with ara-CTP for incorporation into DNA.

dCK was observed in Down syndrome children with AML compared to those with non-Down syndrome, which may contribute to the increased sensitivity for ara-C observed in these children.61,184 Besides full-length dCK, inactive splice variants of dCK have been reported that were expressed at a higher level in adults with refractory and relapsed AML than in patients with newly diagnosed disease. However, the contribution of these splice variants to ara-C resistance is presumably limited since the function of full-length dCK is dominant over the function of splice variants.185,186 Infants with ALL, who are relatively sensitive to ara-C compared to older children with this leukemia, did not express higher levels of dCK than older children.45 In addition, expression levels of dCMP kinase, NDP kinases as well as the inactivating enzymes pyrimidine nucleotidase I, cytidine deaminase and deoxycytidylate deaminase were not linked

with resistance to ara-C in infant ALL.45 However, the amount of accumulated ara-CTP was higher in ara-Csensitive infant ALL cells.45 Intracellular ara-CTP levels were lower in adult AML compared to normal bone marrow cells, in children with T-ALL and AML compared to patients with precursor B-lineage ALL, and in relapsed compared to newly diagnosed patients with ALL.187,188 Decreased araCTP levels may indicate resistance to ara-C since cells in adult AML and T-ALL and in relapsed ALL are more resistant to this drug than cells of newly diagnosed precursor B-lineage ALL.23,27,34 Since the activity of kinases does not seem to be the limiting factor for ara-CTP formation in leukemic cells, decreased intracellular ara-CTP levels may be caused by decreased expression of transmembrane nucleoside transporters. A low expression of the transmembrane

Assays and molecular determinants of cellular drug resistance

nucleoside transporter ENT1 at initial diagnosis of adult AML was associated with an unfavorable outcome.189 In infants with ALL sensitivity to ara-C was linked to higher expression of ENT1 and higher intracellular ara-CTP levels.45 Another mechanism by which cells could circumvent the cytotoxic effects of ara-C may be the de novo synthesis of cytidine triphosphate (CTP) from uridine triphosphate (UTP), mediated by CTP synthetase. CTP is in turn converted to dCTP, which can compete with ara-CTP for incorporation into DNA. However, the activity of CTP synthetase in the leukemic cells of children with ALL did not differ from that of nonmalignant CD34-positive bone marrow cells.190

Cross-resistance to different classes of drugs Drug resistance proteins Numerous groups have determined the prognostic value of the drug-efflux mediating ATP-binding cassette transmembrane transporters P-glycoprotein and MRP1 in acute leukemia, with conflicting data reported for both ALL and AML (reviewed extensively by Van den Heuvel–Eibrink et al.114 ). In general, both proteins have no or only weak prognostic strength in pediatric and adult ALL and pediatric AML, whereas P-glycoprotein but not MRP1 may contribute to an unfavorable prognosis in adult AML. The function of (overexpressed) P-glycoprotein can be inhibited by multidrug resistance modulators such as PSC-833, which is currently being studied in phase III trials in adult AML patients. MRP1 belongs to a family of more than six MRPmembrane transporters with different tissue expression and substrate specificities. In this family, only MRP3 mRNA levels were linked to an unfavorable outcome in pediatric ALL and AML; MRP2, MRP4, MRP5 and SMRP lacked this effect.191,192 In adult AML, mRNA expression of MRP1-3 and MRP5 did not differ in paired samples taken at initial diagnosis and at relapse.193 Since each MRP member has different substrate specificities, MRP3 or one of the other family members may be important for drug resistance in pediatric leukemia. The breast-cancer resistance protein (BCRP) is also a member of the ATP-binding cassette transmembrane transporter-family. BCRP expression levels were higher at relapse than at initial diagnosis of paired adult AML samples, and the expression levels correlated with an unfavorable outcome in pediatric AML.194,195 However, to date, the limited studies addressing BCRP expression and cellular drug resistance do not suggest that BCRP is an important

contributor to cellular resistance. BCRP expression was not linked with anthracycline resistance in adult AML,196 and BCRP expression and activity did not correlate with resistance to ara-C, glucocorticoids, vinca alkaloids, anthracyclines or L-asparaginase in infant ALL.197 Moreover, BCRP expression was not associated with unfavorable prednisolone window response and long-term clinical outcome in pediatric ALL.198

Abnormalities in the apoptosis-signaling pathway A low degree of spontaneous apoptosis in stromasupported cultures of precursor B-lineage cells is related to an unfavorable clinical outcome in pediatric ALL.199 However, our own unpublished data show that the level of spontaneous apoptosis is not associated with cellular drug resistance in pediatric ALL. Resistance to prednisolone, vincristine, L-asparaginase or daunorubicin was linked to decreased phosphatidyl serine exposure at the plasma membrane (marker for early apoptosis) and impaired reduction in the mitochondrial transmembrane potential (trigger to release cytochrome c and other apoptotic factors).122 This suggests that differences in spontaneous apoptosis reflect differences in survival/regrowth advantage of cells, whereas the level of drug-induced apoptosis reflects defects in the execution of the apoptosis-signal transduction pathway. Functional studies showed that apoptotic defects occur in approximately 80% of adult AML patients and correlate with drug-resistant disease in vivo and a poor clinical outcome.200 The apoptotic defect was mainly due to the inability of leukemic cells to trigger both the death receptor/caspase 8-mediated and the mitochondria/cytochrome c release-mediated pathways of apoptosis. This suggests that the inability to induce apoptosis is caused by events upstream of both pathways, (i.e. upstream of mitochondria). We also observed this phenomenon in pediatric ALL: resistance to the abovementioned drugs was always associated with both reduced phosphatidyl serine exposure at the plasma membrane and impaired reduction in mitochondrial transmembrane potential, whereas caspase 3 activation and poly (ADPribose) polymerase (PARP) inactivation occurred later, and the degree of activation or inactivation was drugdependent.122 The genes that contribute to drug crossresistance in pediatric leukemia remain to be identified. The classical theory of an imbalance between anti- and proapoptotic BCL2/BAX family molecules does not seem to be linked to cellular drug resistance in pediatric leukemia. Salomons et al.201 found that neither the BCL2/BAX ratio nor expression levels of anti-apoptotic BCL2, BCLX1

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and MCL1 or pro-apoptotic BAX, BAD and BAK were important for prednisolone, vincristine or L-asparaginase resistance in children with ALL. In accord with these findings, Coustan-Smith et al.202 failed to link BCL2 expression with drug resistance in pediatric ALL,202 while Wuchter et al.203 showed that expression levels of BCL2, BAX, and CD95 and triggering of CD95 activity were not associated with resistance to in vitro prednisolone-and doxorubicininduced apoptosis in the T-ALL cells of children.203 In contrast to cellular drug resistance, altered BCL2/BAX ratios may be important for the regrowth advantage of cells, as reflected by an increase in this ratio at relapse of ALL in children, although such ratios were not associated with longterm clinical outcome in pediatric ALL.201,204 Knowledge of other genes that may be linked with cellular drug resistance and apoptotic defects is lacking. However, ongoing gene expression profiling studies in our laboratories and others, comparing drug resistant and drug-sensitive cases, may reveal new insights into the origins of cross-resistance to drugs in pediatric leukemia.205

Conclusions and future perspectives The prognostic value of biologic and clinical features, such as type of acute leukemia, age, immunophenotype and chromosomal abnormalities, can be explained at least partly by the resistance of leukemic cells to different classes of drugs. Over the past four decades, leukemic cell line studies have uncovered a number of interesting candidate genes that might explain drug resistance in the leukemic cells of patients. In practice, however, results obtained in leukemic cell line models are not comparable to results obtained in the fresh leukemic cells of patients. Resistance to glucocorticoids is not linked with the number of glucocorticoid receptors, but presumably resides in the intracellular signaling pathway mediated by the receptor. Resistance to asparaginase may be related to alterations in amino acid metabolism and protein synthesis in pediatric leukemia. Resistance to vinca alkaloids seems to be associated with altered expression of tubulin isoforms and associated proteins, which influence the cytoskeleton structure and nuclear trafficking of molecules. Studies to find the causes of anthracycline and epipodophyllotoxin resistance have indicated that the contributions of DNA topoisomerase II
, drug-efflux mediating proteins P-glycoprotein and multidrug-resistance associated protein 1, as well as the major vault protein/lung resistance protein, are limited in pediatric leukemia. An interesting new insight points to a role of the sphingomyelinceramide pathway in anthracycline-induced apoptosis, an observation that needs further exploration in clinical

samples. Resistance to antimetabolites is mainly caused by abnormal intracellular accumulation and metabolism of methotrexate (polyglutamation defects), thiopurines (altered metabolic enzyme activities) and ara-C (decreased expression of nucleoside membrane transporter). Crossresistance to different classes of drugs is likely to be associated with defects in apoptosis signaling pathways, especially defects upstream of mitochondrial function, although the nature of these alterations remains to be elucidated. Ongoing gene expression profiling studies may reveal new causes of cellular resistance to separate drugs as well as causes of cross-resistance to different classes of drugs in pediatric leukemia. Most data discussed in this chapter were generated by linking the expression of genes/proteins to cellular resistance to drugs. The real proof of whether molecules contribute to drug resistance will come from, for example, RNA-interference studies in which the gene of interest is selectively downregulated and the effect on drug cytotoxicity can be monitored in the leukemic cells of patients. In vitro cellular drug resistance testing can be used to stratify ALL patients into risk groups, as illustrated by studies of the German COALL group.26 Improved stratification is important for reducing unnecessary side effects due to overtreatment and better recognition of patients who are likely to relapse on current low-risk protocols. Drug resistance profiling of specific risk groups can be helpful for designing more specific therapies. An example of this is the Interfant-99 therapy schedule for infants with ALL, which is partly based upon the finding that infant ALL cells are highly sensitive to ara-C. A very attractive option is the identification of new genes involved in cellular drug resistance by newly developed high-throughput techniques such as microarray expression analysis. In the past, research was limited to a limited number of genes of interest that could be studied one at a time. However, many other, still unknown, genes are likely to be involved in clinical resistance. For example, Cheok et al.206 showed that changes in gene expression in childhood ALL after treatment with methotrexate, 6mercaptopurine, or a combination of both were treatment specific, suggesting that gene expression can elucidate differences in responses to specific drugs.205 We recently showed that resistant and sensitive ALL cells of children show specific differences in gene expression profiles depending on the drug studied.101 Also, microarray analysis has led to specific gene expression profiles that can be used to classify different subtypes of pediatric ALL and AML.207–210 Such advances can lead to the recognition of new targets of therapy in resistant subtypes of acute leukemia, such as the overexpression of FLT3 in MLL gene-rearranged ALL.211 Molecular targeting of these

Assays and molecular determinants of cellular drug resistance

genes may well translate into improved, patient-specific therapies. 15

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104 Giannakakou, P., Nakano, M., Nicolaou, K. C., et al. Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics. Proc Natl Acad Sci U S A, 2002; 99: 10855–60. 105 Boer, M. L. den, Pieters, R., Kazemier, K. M., et al. Relationship between the intracellular daunorubicin concentration, expression of major vault protein/lung resistance protein and resistance to anthracyclines in childhood acute lymphoblastic leukemia. Leukemia, 1999; 13: 2023–30. 106 Boer, M. L. den, Pieters, R., Kazemier, K. M., et al. The modulating effect of PSC 833, cyclosporin A, verapamil and genistein on in vitro cytotoxicity and intracellular content of daunorubicin in childhood acute lymphoblastic leukemia. Leukemia, 1998; 12: 912–20. 107 Siva, A. C., Raval-Fernandes, S., Stephen, A. G., et al. Upregulation of vaults may be necessary but not sufficient for multidrug resistance. Int J Cancer, 2001; 92: 195–202. 108 Legrand, O., Simonin, G., Beauchamp-Nicoud, A., et al. Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia. Blood, 1999; 94: 1046–56. 109 Goasguen, J. E., Lamy, T., Bergeron, C., et al. Multifactorial drug resistance phenomenon in acute leukemias: impact of P170MDR1, LRP56 protein, glutathion-transferases and methallothione systems on clinical outcome. Leuk Lymphoma, 1996; 23: 567–76. 110 Kakihara, T., Tanaka, A., Watanabe, A., et al. Expression of multidrug resistance-related genes does not contribute to risk factors in newly diagnosed childhood acute lymphoblastic leukemia. Pediatr Int, 1999; 41: 641–7. 111 Borg, A. G., Burgess, R., Green, L. M., et al. P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells. Br J Haematol, 2000; 108: 48–54. 112 Tsuji, K., Motoji, T., Sugawara, I., et al. Significance of lung resistance-related protein in the clinical outcome of acute leukaemic patients with reference to P-glycoprotein. Br J Haematol, 2000; 110: 370–8. 113 Leith, C. P., Kopecky, K. J., Chen, I. M., et al. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1 and LRP in acute myeloid leukemia: a Southwest Oncology Group study. Blood, 1999; 94: 1086–99. 114 Heuvel-Eibrink, M. M. van den, Sonneveld, P., & Pieters, R. The prognostic significance of membrane transport-associated multidrug-resistance (MDR) proteins in leukemia. Int J Clin Pharmacol Ther, 2000; 38: 94–110. 115 Bezombes, C., de Thonel, A., Apostolou, A., et al. Overexpression of protein kinase C zeta confers protection against antileukemic drugs by inhibiting the redox-dependent sphingomyelinase activation. Mol Pharmacol, 2002; 62: 1446–55. 116 Batist, G., Schecter, R., Woo, A., et al. Glutathione depletion in human and in rat multi-drug resistant breast cancer cell lines. Biochem Pharmacol, 1991; 41: 631–5.

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174 Lilleyman, J. S. & Lennard, L. Mercaptopurine metabolism and risk of relapse in childhood lymphoblastic leukaemia. Lancet, 1994; 343: 1188–90. 175 McLeod, H. L., Relling, M. V., Liu, Q., et al. Polymorphic thiopurine methyltransferase in erythrocytes is indicative of activity in leukemic blasts from children with acute lymphoblastic leukemia. Blood, 1995; 85: 1897–902. 176 Lennard, L., Welch, J. C., & Lilleyman, J. S. Thiopurine drugs in the treatment of childhood leukaemia: the influence of inherited thiopurine methyltransferase activity on drug metabolism and cytotoxicity. Br J Clin Pharmacol, 1997; 44: 455–61. 177 Krynetski, E. Y., Tai, H. L., Yates, C. R., et al. Genetic polymorphism of thiopurine S-methyltransferase: clinical importance and molecular mechanisms. Pharmacogenetics, 1996; 6: 279– 90. 178 Coulthard, S. A., Howell, C., Robson, J., et al. The relationship between thiopurine methyltransferase activity and genotype in blasts from patients with acute leukemia. Blood, 1998; 92: 2856–62. 179 McLeod, H. L., Krynetski, E. Y., Relling, M. V., et al. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia, 2000; 14: 567–72. 180 Relling, M. V., Hancock, M. L., Boyett, J. M., et al. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood, 1999; 93: 2817–23. 181 Owens, J. K., Shewach, D. S., Ullman, B., et al. Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene. Cancer Res, 1992; 52: 2389–93. 182 Flasshove, M., Strumberg, D., Ayscue, L., et al. Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside. Leukemia, 1994; 8: 780–5. 183 Heuvel-Eibrink, M. M. van den, Wiemer, E. A., Kuijpers, M., et al. Absence of mutations in the deoxycytidine kinase (dCK) gene in patients with relapsed and/or refractory acute myeloid leukemia (AML). Leukemia, 2001; 15: 855–6. 184 Taub, J. W., Huang, X., Matherly, L. H., et al. Expression of chromosome 21-localized genes in acute myeloid leukemia: differences between Down syndrome and non-Down syndrome blast cells and relationship to in vitro sensitivity to cytosine arabinoside and daunorubicin. Blood, 1999; 94: 1393–400. 185 Veuger, M. J., Honders, M. W., Landegent, J. E., et al. High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia. Blood, 2000; 96: 1517–24. 186 Veuger, M. J., Heemskerk, M. H., Honders, M. W., et al. Functional role of alternatively spliced deoxycytidine kinase in sensitivity to cytarabine of acute myeloid leukemic cells. Blood, 2002; 99: 1373–80. 187 Boos, J., Hohenlochter, B., Schulze-Westhoff, P., et al. Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia. Med Pediatr Oncol, 1996; 26: 397–404.

188 Braess, J., Wegendt, C., Feuring-Buske, M., et al. Leukaemic blasts differ from normal bone marrow mononuclear cells and CD34+ haemopoietic stem cells in their metabolism of cytosine arabinoside. Br J Haematol, 1999; 105: 388–93. 189 Galmarini, C. M., Thomas, X., Calvo, F., et al. In vivo mechanisms of resistance to cytarabine in acute myeloid leukaemia. Br J Haematol, 2002; 117: 860–8. 190 Verschuur, A. C., Gennip, A. H., van Leen, R., et al. In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia. Br J Haematol, 2000; 110: 161–9. 191 Steinbach, D., Wittig, S., Cario, G., et al. The multidrug resistance-associated protein 3 (MRP3) is associated with a poor outcome in childhood ALL and may account for the worse prognosis in male patients and T-cell immunophenotype. Blood, 2003; 102: 4493–8. 192 Steinbach, D., Lengemann, J., Voigt, A., et al. Response to chemotherapy and expression of the genes encoding the multidrug resistance-associated proteins MRP2, MRP3, MRP4, MRP5, and SMRP in childhood acute myeloid leukemia. Clin Cancer Res, 2003; 9: 1083–6. 193 Kolk, D. M. van der., De Vries, E. G., Noordhoek, L., et al. Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia. Leukemia, 2001; 15: 1544– 53. 194 Heuvel-Eibrink, M. M. van den., Wiemer, E. A., & Prins, A., et al. Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML). Leukemia, 2002; 16: 833–9. 195 Steinbach, D., Sell, W., Voigt, A., et al. BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia, 2002; 16: 1443–7. 196 Sargent, J. M., Williamson, C. J., Maliepaard, M., et al. Breast cancer resistance protein expression and resistance to daunorubicin in blasts from patients with acute myeloid leukemia. Br J Haematol, 2001; 115: 257–62. 197 Stam, R. W., Heuvel-Eibrink, M. M. van den, Boer, M. L. den, et al. Multidrug resistance genes in infant acute lymphoblastic leukemia; Ara-C is not a substrate for the breast cancer resistance protein (BCRP). Leukemia, 2004; 18: 78–83. 198 Sauerbrey, A., Sell, W., Steinbach, D., et al. Expression of the BCRP gene (ABCG2/MXR/ABCP) in childhood acute lymphoblastic leukaemia. Br J Haematol, 2002; 118: 147–50. 199 Kumagai, M., Manabe, A., Pui, C. H., et al. Stroma-supported culture in childhood B-lineage acute lymphoblastic leukemia cells predicts treatment outcome. J Clin Invest, 1996; 97: 755– 60. 200 Schimmer, A. D., Pedersen, I. M., Kitada, S., et al. Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy. Cancer Res, 2003; 63: 1242–8. 201 Salomons, G. S., Smets, L. A., Verwijs-Janssen, M, et al. Bcl-2 family members in childhood acute lymphoblastic leukemia:

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drug response in human leukemia cells. Nat Genet, 2003; 34: 85–90. Yeoh, E. J., Ross, M. E., Shurtleff, S. A., et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell, 2002; 1: 133–43. Armstrong, S. A., Staunton, J. E., Silverman, L. B., et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet, 2002; 30: 41–7. Ross, M. E., Zhou, X., Song, G., et al. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood, 2003; 102: 2951–9. Yagi, T., Morimoto, A., Eguchi, M., et al. Identification of a gene expression signature associated with pediatric AML prognosis. Blood, 2003; 102: 1849–56. Armstrong, S. A., Kung, A. L., Mabon, M. E., et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell, 2003; 3: 173–83.

16 Acute lymphoblastic leukemia Ching-Hon Pui

Introduction Childhood acute lymphoblastic leukemia (ALL) has served as a model for cancer treatment for approximately five decades. With more precise diagnostic criteria and risk classifications, more effective therapy administered in controlled clinical trials, and better supportive care, the outlook for children with ALL has improved dramatically. Today, approximately 80% of children treated for this disease in developed countries will be cured (no evidence of disease for 10 or more years).1,2 Remarkably, this high cure rate is achieved mainly by optimizing risk-directed therapy, using the drugs that were discovered before 1980. Because of the ease with which samples of leukemic lymphoblasts can be obtained from the bone marrow and blood, laboratory studies of childhood ALL have consistently been at the fore of efforts to elucidate the principles of cancer cell biology. This chapter attempts to integrate advances in the biological understanding of ALL with basic principles of clinical management.

Pathobiology and pathophysiology Leukemic transformation of hematopoietic cells requires subversion of the controls of normal proliferation, a block in differentiation, resistance to apoptotic signals, and enhanced self-renewal.2 The prevailing theory of leukemia pathophysiology is that a single mutant hematopoietic progenitor cell, capable of indefinite self-renewal, gives rise to malignant, poorly differentiated hematopoietic precursors. Several lines of research support the clonal origin of leukemia, including glucose-6-phosphate dehydrogenase

enzyme studies and recombinant DNA analysis based on Xlinked restriction fragment length polymorphisms in heterozygous females (whose normal tissues have a mosaic pattern of X-chromosome expression, yet whose leukemic cells show a single active parental allele).3,4 Other examples are the determination of T-cell receptor (TCR) gene or immunoglobulin (IG) gene rearrangements,5 demonstration of Ig idiotypes and single light-chain types in B-cell malignancies, and analysis of leukemic cell karyotypes.6 Molecular characterization of the genetic alterations in blast cells has contributed greatly to our understanding of the pathogenesis of this disease.7 The general oncogenic mechanisms include aberrant expression of oncoproteins (e.g. MYC, TAL1, LYL1, LMO2, and HOX11) and chromosomal translocations that generate fusion genes encoding active kinases (e.g. BCR-ABL) or altered transcription factors (e.g. TEL-AML1, E2A-PBX1, and MLL linked to one of many fusion partners) (see Chapter 10 for detailed information). Multiple-step models of carcinogenesis predict that the primary oncogenic events are not sufficient by themselves to induce leukemia, and secondary mutations that alter the normal differentiation and survival of hematopoietic progenitors are required.8,9 Among the common secondary mutations are overexpression of FLT3, a receptor tyrosine kinase important for the normal development of hematopoietic stem cells, and genetic and epigenetic changes in interrelated pathways controlled by p53, the tumor suppressor RB (the retinoblastoma protein) and related proteins p130 and p107.2,10–12 Genetic studies of identical twins with concordant leukemia13,14 and the detection of leukemia-specific gene sequences or clonotypic rearrangements of the IG or TCR loci in archived neonatal blood spots (Guthrie cards) of

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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children who later developed leukemia14–16 have indicated that many cases of ALL originate in utero. A high concordance rate (approaching 100%) and a very short latency period (from a few days to a few months) are typical of infant twins with the t(4;11)/MLL-AF4, suggesting that this fusion is leukemogenic by itself or could facilitate acquisition of additional cooperating mutations to cause leukemia.13,14,17 By contrast, in cases with the TEL-AML1 fusion the concordance rate is lower (approximately 10%), the incubation period longer and the presenting features and outcome variable, suggesting that additional postnatal events (e.g. deletion of TEL from the other allele) are required for leukemic transformation.13,14,17 This interpretation is supported by the identification of rare cells expressing t(12;21)/TEL-AML1 fusion transcripts in approximately 1% of normal cord blood samples, a frequency 100-fold higher than the incidence of ALL defined by this fusion transcript.18 However, not all cases of childhood ALL develop in utero. For example, studies of archived neonatal blood spots and patterns of IG and TCR gene rearrangements in leukemic cells support a postnatal origin of ALL with t(1;19)/E2A-PBX1 fusion.19 Another important insight has come from the realization that rearrangements of the MLL gene occur not only in most infant leukemias but also in secondary leukemias induced by antileukemic drugs that function as topoisomerase-II inhibitors.20,21 Exposure of the fetus to substances that affect topoisomerase II could be a crucial leukemogenic event in cases with MLL rearrangement. A variety of natural and synthetic compounds, including quinolone antibiotics, flavonoids in foods and drinks, catechins, podophyllin resin, benzene metabolites, and even estrogens can inhibit topoisomerase.21 Indeed, transplacental exposure to DNA-damaging drugs, a nonsteroidal anti-inflammatory drug (dipyrone), and mosquitocidals (Baygon) have been implicated in the development of infant leukemias with MLL gene fusion in an international epidemiological study.22 Although the leukemogenic effects of dietary, medical and environmental exposures are much weaker than those of anticancer chemotherapy, a reduced ability of fetuses or their mothers to detoxify these agents could enhance susceptibility to ALL.21,23 Several monogenic syndromes (e.g. ataxia telangiectasia and Bloom syndrome) are associated with an increased risk of ALL (see Chapter 13). In a recent study, carriers of germ-line ataxia-telangiectasia mutated (ATM) gene were also shown to have an increased risk of developing T-cell ALL.24 However, these observations explain only a small fraction of cases; in most others, it is likely that there are multiple subtle genetic polymorphisms that interact

with environmental, dietary, maternal and other external factors to affect the development of ALL. For example, inactivating polymorphisms of detoxifying enzymes, such as glutathione S-transferases25,26 and reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase,27,28 have been linked to the development of ALL in infants and children. Low-penetrance polymorphisms of folate-metabolizing enzymes have also been associated with the development of ALL. Polymorphic variants of methylenetetrahydrofolate reductase, which catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (the predominant circulating form of folate), have been associated with a decreased risk of pediatric ALL.29,30 This protective effect may be due to a greater availability of 5, 10-methylenetetrahydrofolate and thymidine pools, and to an increased fidelity of DNA synthesis. An association between the polymorphisms of two other folate-related genes – serine hydroxymethyltransferase and thymidylate synthase – and a lower risk of adult ALL,31 further suggests that the folate pathway plays a role in susceptibility to leukemia. Consistent with these observations are results suggesting that folate supplementation may reduce the risk of common ALL in children,32 but this intriguing finding needs confirmation due to the very small size of the study sample, the use of multiple comparisons, and the lack of details on folate dosages. Progress in genomic33–35 and proteomic36,37 techniques will almost certainly facilitate studies of host genetic polymorphisms in the development of leukemia. Leukemic cells divide more slowly and take a longer time to synthesize DNA than do normal hematopoietic precursors.38 Despite this seemingly unfavorable growth property, leukemic cells accumulate relentlessly in most patients and compete successfully with normal hematopoietic cells. The failure of normal hematopoiesis, leading to anemia, infection and bleeding, is the most serious pathophysiologic consequence of leukemia. Moreover, leukemic cells can infiltrate any organ and cause enlargement and dysfunction. Most often, leukemia appears to begin in the bone marrow and spread to other parts of the body, although in some instances the disease may arise in an extramedullary site and subsequently invade the bone marrow. For example, leukemia is thought to originate in the thymus in T-cell cases that present with an anterior mediastinal mass. Indeed, the remarkably similar levels of minimal residual leukemia in bone marrow and peripheral blood obtained concomitantly in patients with T-cell ALL support the view that T-cell lymphoblasts derive from progenitor cells residing in the thymus and migrate to bone marrow through the circulating blood.39

Acute lymphoblastic leukemia

Clinical presentation The presenting symptoms and signs of ALL are quite variable. Most cases have an acute onset, while in others the initial signs and symptoms appear insidiously and persist for months. The presenting features generally reflect the degree of bone marrow failure and extent of extramedullary spread. Fever is the most common finding, occurring in approximately 50% to 60% of patients. In at least twothirds of these cases, fever is due to the leukemia and will resolve within 72 hours after the start of induction therapy.40 Nonetheless, because patients are generally neutropenic, and the neutrophils may be functionally abnormal, all febrile patients should be treated with broadspectrum intravenous antibiotics until infection can be excluded. Indeed, cellulitis in a neutropenic patient may be caused by a gram-negative microorganism. Fatigue and lethargy are frequent manifestations of anemia. Over onethird of patients, especially young children, may present with a limp, bone pain, arthralgia, or refusal to walk due to leukemic infiltration of the periosteum, bone, or joint or to expansion of the marrow cavity by leukemic cells. Children with prominent bone pain frequently have nearly normal hematologic values, which can contribute to a delay in diagnosis.41 A small proportion of patients have severe bone pain and tenderness, fever, and a very high serum lactate dehydrogenase level due to bone marrow necrosis.42 Many patients have some manifestations of bleeding. Less common signs and symptoms include headache, vomiting, respiratory distress, oliguria, and anuria. Occasionally, patients present with life-threatening infection or bleeding. In rare cases, ALL does not produce early signs and symptoms and is detected during routine examination. Physical examination may reveal pallor, petechiae, and ecchymoses in the skin or mucous membranes, and bone tenderness due to leukemic infiltrations or hemorrhage that stretches the periosteum. Liver, spleen, and lymph nodes are the most common sites of extramedullary involvement and are enlarged in more than one-half of the patients at diagnosis (Table 16.1). Ocular involvement can manifest as retinal hemorrhage; leukemic infiltration of the orbit, optic nerve, retina, iris, cornea, or conjunctiva (Fig. 16.1); or hypopyon (layering of white cells in the anterior chamber of the eye (Fig. 16.2) in association with blurred vision, photophobia, or ocular pain.43,44 While hemorrhage and deposits of leukemic cells in the retina or optic nerve are initial manifestations of ALL, other ocular abnormalities generally occur at relapse. Historically, such involvement affected almost 10% of patients in relapse, but it has become very rare with contemporary treatments.

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Table 16.1 Presenting clinical and laboratory features of 1317 white and 210 black children with newly diagnosed ALL treated consecutively at St. Jude Children’s Research Hospital (1979–2002) Feature Age (years) <1 1–10 >10 Median (range) Male Leukocyte count (×109 /L) <10 10–24 25–49 50–100 >100 Median (range) Platelet count (×109 /L) <10 10–49 50–100 >100 Median (range) Hemoglobin (g/dL) <8 8–10 >10 Median (range) Mediastinal mass Liver edge below costal margin 1–4 cm ≥5 cm Spleen edge below costal margin 1–4 cm ≥5 cm Central nervous system status CNS1 CNS2 CNS3 Traumatic without blasts Traumatic with blasts

White children (%)

Black children (%)

2.7 71.6 25.7 5.2 (0.08–18.9) 56.0

3.3 66.7 30.0 5.7 (0.2–19.5) 59.5

46.2 19.1 10.2 10.3 14.2 11.8 (0.4–1500)

34.9 17.7 12.5 10.5 24.4 20.1 (0.7–651.2)

6.5 41.3 21.3 30.9 53 (0–1050)

5.8 31.2 25.5 37.5 72 (3–831)

51.8 25.2 23.0 7.9 (2.1–17.6) 10.4

52.0 24.0 24.0 7.8 (1.4–18.2) 17.6

36.7 31.6

39.4 33.2

29.4 30.4

24.9 34.2

59.5 28.7 2.7 2.4 6.8

44.1 30.9 4.4 5.9 14.7

Painless enlargement of the scrotum can be a sign of testicular leukemia or hydrocele, the latter resulting from lymphatic obstruction that is readily diagnosed by ultrasonography. At diagnosis, as many as 25% of male patients have biopsy-identified testicular leukemia, generally in association with high leukocyte counts, but this finding is not correlated with clinical outcome.45 Overt testicular disease is relatively rare at diagnosis, occurring in only 2%

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Fig. 16.1 Leukemic retinopathy manifested by disc edema and white-center hemorrhage in an 11-year-old girl with t(4;11)-positive, early pre-B ALL and a presenting leukocyte count of 1512 × 109 /L. (See color plate 16.1 for full-color reproduction.)

Fig. 16.3 Left testicular relapse in a 12-year-old boy with T-cell ALL. (See color plate 16.3 for full-color reproduction.)

Fig. 16.2 Leukemic involvement of the anterior segment with hypopyon in a 9-year-old boy with relapsed ALL. (See color plate 16.2 for full-color reproduction.)

of patients, most often infants or adolescents with T-cell leukemia and a mediastinal mass or hyperleukocytosis.46 Testicular irradiation is not necessary in these patients as local relapse is uncommon with effective systemic chemotherapy. Testicular relapse (Fig. 16.3), once affecting as many as 10% of patients, occurs only in approximately 1% of the boys treated with contemporary effective regimens.47–49 Thus, testicular biopsy is no longer recommended as a surveillance procedure, being reserved only as a means to document isolated relapse or residual disease in rare patients with persistently enlarged testes after treatment. Less common presenting features include subcutaneous nodules (leukemia cutis), enlarged salivary glands

(Mikulicz syndrome; Fig. 16.4), cranial nerve palsy, and priapism (due to leukostasis of the corpora cavernosa and dorsal veins or sacral nerve involvement). Epidural spinal cord compression (Fig. 16.5) is a rare but serious presenting finding and requires immediate chemotherapy and high-dose glucocorticoid therapy to prevent permanent paraparesis or paraplegia.50 Laminectomy or radiotherapy is generally not necessary because leukemias are very sensitive to chemotherapy at diagnosis. Finally, in some patients, infiltration of the tonsils, adenoids, appendix, or mesenteric lymph nodes leads to surgical intervention before leukemia is diagnosed. Patients with cerebellar ataxia, with or without mental or growth retardation, who develop a lymphoid neoplasm, especially a malignant T-cell disorder, should be suspected of having ataxia telangiectasia.51,52 Such cases show considerable heterogeneity in age at onset; rate of disease progression; and severity of cerebellar symptoms, immunodeficiency, and telangiectasia (Fig. 16.6). Hence, some cases may be characterized by mild ataxia, a lack

Acute lymphoblastic leukemia

Fig. 16.4 Enlargement of right parotid gland due to infiltration of leukemic cells at diagnosis in a 5-year-old boy with near-haploid early pre-B ALL. (See color plate 16.4 for full-color reproduction.)

of deficiencies in IgA and IgG2, and minimal telangiectasia. Although the ataxia telangiectasia gene has been cloned and sequenced, detection of increased levels of serum alpha-fetoprotein is still considered a more useful aid in the diagnosis of this disorder.51,52 Early recognition of ataxia telangiectasia is important because these patients have excessive and sometimes fatal complications from treatment with radiation (leukoencephalopathy), bleomycin (pulmonary fibrosis), and cyclophosphamide (hemorrhagic cystitis).53

Laboratory findings Anemia, abnormal leukocyte and differential counts, and thrombocytopenia are usually present at diagnosis, reflecting the degree to which bone marrow has been replaced with leukemic lymphoblasts. The presenting leukocyte counts range widely, from 0.1 to 1500 × 109 /L (median, 12 × 109 /L) and are increased (>10 × 109 /L) in slightly over one-half of the patients (Table 16.1). Hyperleukocytosis (>100 × 109 /L) occurs in approximately 15% of the patients. Profound granulocytopenia (<0.5 × 109 /L) occurs in 40% of patients, rendering them at high risk of infection. The majority of leukocytes in blood smears are lymphoblasts or lymphocytes. Hypereosinophilia, generally reactive, may be present at diagnosis and occasionally will precede the diagnosis of ALL by many months.54 Several patients, mostly males, have been reported to have B-precursor ALL with classic t(5;14)(q31;q32) chromosomal abnormalities accompanied by the hypereosinophilia syndrome (hypereosinophilia, pulmonary infiltration, cardiomegaly,

Fig. 16.5 Epidural mass at T3 to T7 with cord and thecal sac displacement and extension through multiple neural foramen in an 11-year-old boy presenting with paraparesis at diagnosis of Philadelphia chromosome-positive ALL. The arrows indicate the upper and lower margins of the mass.

and congestive heart failure). Patients with this genetic subtype of ALL often do not have circulating leukemic blasts or other cytopenias, and have a relatively low percentage of blasts in the bone marrow. Activation of the interleukin-3 gene on chromosome 5 by the enhancer of the IG heavy-chain gene on chromosome 14 is thought to play a central role in leukemogenesis and associated eosinophilia in these cases.55 Unlike cases of acute myeloid leukemia (AML) with hypereosinophilia and inv(16)(p13q22), t(8;21)(q22;q22), t(8;12)(p21;p13) or t(9;12)(q34;p13), in which the eosinophils belong to the neoplastic clone,56 the increased eosinophil count in these cases represents a reactive process caused by an increase in the amount of interleukin-3 released from leukemic cells. The morphology of eosinophils is typically abnormal in

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Fig. 16.6 Telangiectasis on bullar conjunctiva in a 12-year-old girl with ataxia telangiectasia and B-cell ALL. (See color plate 16.6 for full-color reproduction.)

these cases, perhaps due to intense stimulation mediated by the cytokine. Decreased platelet counts (median, 50 × 109 /L) are usually present at diagnosis and can be readily distinguished from immune thrombocytopenia, as isolated thrombocytopenia is rare in leukemia.57 Routine bone marrow aspiration is not necessary for patients with severe thrombocytopenia and no other hematologic or physical evidence of leukemia. However, in general, bone marrow aspiration should be performed to exclude leukemia in patients who require glucocorticoid treatment for presumed immune thrombocytopenia. Severe hemorrhage is uncommon, even when platelet counts are as low as 20 × 109 /L, provided that infection and fever are absent.58 Occasional patients present with thrombocytosis (>400 platelets × 109 /L).59 Coagulopathy, usually mild, can occur in T-cell ALL and is only rarely associated with severe bleeding.60 More than 75% of patients present with anemia, which is usually normochromic and normocytic and associated with a normal to low reticulocyte count. Anemia or thrombocytopenia is often mild (or even absent) in patients with T-cell ALL.61 Pancytopenia followed by a period of spontaneous hematopoietic recovery may precede the diagnosis of ALL in rare cases.62 In contrast to patients with aplastic anemia, monoclonal IG gene rearrangement can be found in these patients during hypoplasia.63 Elevated serum uric acid levels are common in patients with a large leukemic cell burden, reflecting an increased rate of purine catabolism. The serum lactate dehydrogenase level is also frequently elevated and correlates with the leukemic cell burden.64 Patients with massive renal involvement can have increased levels of creatinine, urea

nitrogen, uric acid, and phosphorus. Although an enlarged kidney can be documented in 30% to 50% of patients by diagnostic imaging studies, kidney size at diagnosis lacks prognostic or therapeutic implications.65 Occasionally, children with T-cell ALL present with acute renal failure, despite a relatively small leukemic cell burden.66 Approximately 0.5% of patients have hypercalcemia at diagnosis, attributable to the release of parathyroid hormone-like protein from lymphoblasts and leukemic infiltration of bone; this complication generally resolves rapidly with hydration and chemotherapy.67 Interestingly, hypercalcemia frequently recurs at the time of relapse in these patients. Liver dysfunction due to leukemic infiltration occurs in 10% to 20% of patients, is usually mild, and has no prognostic consequences.68 Because vincristine and daunorubicin are metabolized primarily through biliary excretion, modifications of the dosage of these agents are recommended if direct bilirubin level is elevated.69 We recommend a 50% reduction in dosage if the direct bilirubin level is between 2 to 4 mg/dL, a 75% reduction if the level is between 4 and 6 mg/dL, or withholding these drugs if the level is greater than 6 mg/dL. Indirect (unconjugated) hyperbilirubinemia is frequently due to inactivating genetic polymorphisms of hepatic UDP-glucuronosyltransferase (Gilbert syndrome), which has little, if any, effect on the drugs used to treat ALL.70,71 Serum Ig levels are modestly low (mostly IgA and IgM classes) in approximately one-third of patients at diagnosis,72 reflecting the decreased number and impaired function of normal lymphocytes. Urinalysis may reveal microscopic hematuria and the presence of uric acid crystals. Chest roentgenography is needed to detect enlargement of the thymus or mediastinal nodes (Fig. 16.7) or pleural effusion. In fact, 50% to 60% of T-cell cases present with an anterior mediastinal mass.61 A bulky anterior mediastinal mass that compresses the great vessels and trachea can lead to superior vena cava syndrome or superior mediastinal syndrome.73 Patients with this syndrome generally present with cough, dyspnea, orthopnea, dysphagia, stridor, cyanosis, facial edema, increased intracranial pressure, and sometimes syncope, and tolerate anesthesia poorly. Diagnostic procedures in patients with severe tracheal compression should be performed only under local anesthesia. Abnormalities of the bone, such as metaphyseal banding, periosteal reactions, osteolysis, osteosclerosis, or osteopenia, can be demonstrated by radiographic studies in one-half of the patients, especially those with low presenting leukocyte counts.74 Vertebral compression fracture can be detected in 2% of the cases who also frequently present with low leukocyte count and hyperdiploidy

Acute lymphoblastic leukemia

Fig. 16.7 An anterior mediastinal mass in a 12-year-old boy with T-cell ALL.

(>50 chromosomes).75 However, routine skeletal surveys are not necessary for optimal patient management. Cerebrospinal fluid (CSF) should be carefully examined. Depending on the degree of vigilance, leukemic blasts can be identified in as many as one-third of patients at diagnosis, the majority of whom lack neurologic symptoms.76 Traditionally, central nervous system (CNS) leukemia is defined by the presence of at least five leukocytes per microliter of CSF, with leukemic blast cells apparent in a cytocentrifuged sample, or by the presence of cranial nerve palsies.77 At most centers, these patients are treated with cranial irradiation. In 1993, we reported that among patients treated in St. Jude Total Therapy Studies XI and XII (1984 to 1991), any number of identifiable leukemic cells in CSF at diagnosis conferred a poor prognosis.78 We proposed a new classification of CNS status at diagnosis: CNS1 denotes the absence of identifiable leukemic blast cells in CSF; CNS2, the presence of leukemic cells in a sample that contains fewer than 5 WBCs/ L; and CNS3, a nontraumatic sample that contains 5 or more WBCs/ L with identifiable blasts, or the presence of a cerebral mass or cranial nerve palsy with leukemic cells in CSF.78 Although the adverse prognosis of a CNS status was confirmed by the Pediatric Oncology Group,79 several studies initially could not establish this association.80–82 The apparent discrepancy can be explained by differences in treatment regimens (e.g. the use of cranial irradiation) and in the patient cohorts studied.83 Indeed, the adverse prognosis of CNS2 status has been shown in several recent studies that included all

patients.84,85 To date, most investigators would intensify intrathecal therapy not only in patients with CNS3 status but also in those with a CNS2 status. More recently, we found an increased risk of CNS relapse and poor event-free survival in association with traumatic lumbar puncture (≥10 erythrocytes/ L of CSF) with identifiable blast cells at diagnosis,86 a finding con¨ firmed by a study of the Berlin-Frankfurt-Munster (BFM) Consortium.82 We have identified several risk factors for traumatic lumbar puncture: low platelet count (<100 × 109 /L), early treatment era (lack of dedicated procedure area and general anesthesia), infant age group, black race, and less experienced practitioners.87 In light of these findings, we now transfuse thrombocytopenic patients who had circulating leukemic blasts with platelets before the initial diagnostic lumbar puncture; the procedure is performed by the most experienced practitioner while the patient is immobile under deep sedation or general anesthesia; and intrathecal therapy is instilled immediately after collection of CSF from the diagnostic procedure. After receiving intrathecal therapy, the patient should lie down in a prone, head-down position for at least 30 minutes to ensure optimal distribution of antileukemic agents within the CSF.88 For patients who suffer from headache after lumbar puncture, we use a blunt “pencil-point” spinal needle to prevent leakage of CSF for subsequent procedures. With close scrutiny of CSF samples for leukemic blast cells, and modifying treatment accordingly, we have been able to reduce the CNS relapse rate in our clinical trials to a negligible level (<2%).76 It should be emphasized that the presence of blast cells (unless they are positive for terminal deoxynucleotidyl transferase) in CSF with a normal cell count during treatment is not generally predictive of CNS relapse.89

Diagnosis and immunophenotypic classification A definitive diagnosis of ALL rests with examination of the bone marrow, which is usually replaced by leukemic lymphoblasts. Fibrosis or tightly packed marrow can lead to difficulties with bone marrow aspiration, necessitating biopsy. A touch preparation of the biopsied tissue can be stained for morphologic diagnosis. Multiple bone marrow aspirates are sometimes needed to obtain viable tissue from patients with bone marrow necrosis. Although the morphologic and cytochemical staining properties of blast cells can be used to distinguish ALL from most cases of AML (see Chapter 2), immunophenotyping is essential in establishing the correct diagnosis.90 Because of the limited heterogeneity of leukemic lymphoblasts by light

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Table 16.2 Comparative features of immunologic subtypes of ALL treated

DNA index of less than 1.16 and a pseudodiploid karyotype, and is less likely to have a hyperdiploid karyotype (>50 chromosomes).90 The high-risk features previously Percentage of cases ascribed to pre-B ALL are closely associated with the pres93,94 Early pre-B Transitionalence of the t(1;19) translocation and E2A-PBX1 fusion. Transitional pre-B cases have a high frequency of hyperFeature T cell cell Pre-B cell pre-B diploidy (DNA index ≥1.16).95 Patients with T-cell leukemia Age (years) tend to be adolescent males with a large leukemic cell ≤1 1 4 5 0 mass, as reflected by a high leukocyte count, an elevated 2–9 46 75 77 82 serum lactate dehydrogenase level, and the presence of ≥10 53 21 18 18 an anterior mediastinal mass or CNS leukemia.61,96 T-cell Median 10.5 4.6 4.2 4.1 cases can be further subclassified immunophenotypically Male gender 71 50 53 41 9 into prothymocyte (CD34+ CD7+ CD2− CD5− CD3− CD4− Leukocyte count (×10 /L) CD8− ), immature thymocyte (CD1+ CD4+ CD8+ CD3− ), <10 17 49 45 41 and mature thymocyte (CD3+ ) stages, each of which is asso10–24 10 21 24 14 25–49 12 10 12 8 ciated with distinct gene expression signatures (discussed 50–99 13 10 11 5 later).97 Despite strikingly different clinical presenting fea≥100 48 10 8 32 tures and even different leukemic cell burdens, prognostic Median 93.8 10.7 10.6 13.9 distinctions among ALL immunophenotypes have weakHemoglobin (g/dL) ened or have been abolished altogether by recent improve>10 64 14 22 23 ments in risk-directed treatment. In fact, T-cell cases and Median 10.9 7.3 7.7 8.4 pre-B cases with the t(1;19) have fared as well as other B9 Platelet (×10 /L) lineage (B-cell precursor) cases in some effective treatment >100 44 31 33 18 protocols.2,98,99 Hence, for therapeutic purposes, one need Median 82.5 52 55 56.5 only to distinguish T-cell and mature B-cell cases from all Mediastinal mass 58 1 1 0 other B-lineage (B-cell precursor) cases.23 CNS leukemia 14 3 4 12 CD10 positivity 42 94 94 83 Aberrant expression of myeloid-associated antigens on Hyperdiploidy >50 3 38 23 36 otherwise typical lymphoblasts has been recognized since chromosomes the early 1980s.100,101 Because of differences in the senDNA index ≥1.16 3 23 17 29 sitivity of the assay, the panel of cell lineage markers, Liver ≥5 cm below costal 42 32 30 32 and diagnostic criteria, the reported frequency of myeloidmargin associated antigen expression in ALL ranges widely, from Spleen ≥5 cm below costal 47 29 28 50 3.5% to 30%.102 Numerous terms have been applied to margin these cases, and standard nomenclature is lacking. We prefer to designate such cases as myeloid antigen-positive ALL, distinguishing them from lymphoid antigen-positive microscopy, the subjective nature of distinctions among AML.102 Cases with myeloid-associated antigen expression different morphologic subtypes of ALL, and the poor corhave a higher frequency of MLL rearrangements or the relation of these findings with immunologic and genetic TEL-AML1 fusion gene (also termed ETV6-CBFA2) than do features, morphologic classification systems have not been other cases.103–105 Once associated with a poor outcome useful in the clinical management of ALL. in some studies, myeloid-associated antigen expression All cases can be classified according to the recognized has no prognostic impact in contemporary risk-directed steps of normal B- and T-cell maturation, and several treatment programs.105 However, the aberrant expression immunophenotypic subtypes are associated with distincof myeloid-associated antigens is useful as a marker for tive clinical features (Table 16.2). For example, B-cell ALL the measurement of minimal residual leukemia by mul(see Chapter 18) is usually associated with L3 blast cell tiparameter flow cytometer (see Chapter 28).106–109 The morphology, male sex, white race, older age at diagnorationale is that expression of certain combinations of sis, elevated serum lactate dehydrogenase, uric acid and lymphoid- and myeloid-associated antigens is a characphosphorus levels, and extramedullary disease (e.g. lymteristic feature of some leukemias and that these antigens phomatous masses or CNS disease).91,92 Compared to early tend to be expressed more strongly on leukemic cells than pre-B ALL, pre-B leukemia develops more often in blacks, on normal cells. has a larger leukemic cell burden, is more likely to have a consecutively at St. Jude Children’s Research Hospital

Acute lymphoblastic leukemia

Genetic classification Leukemia arises from a hematopoietic progenitor cell that has sustained specific genetic damage, leading to abnormal proliferation and malignant transformation. Thus, a genetic classification system could be expected to reflect ALL pathobiology more closely than would other approaches. ALL can be readily classified according to modal chromosomal number (ploidy) and specific genetic abnormalities of the leukemia stem line. Notably, there are racial differences in leukemic subtypes: for example, black children are less likely to have hyperdiploidy greater than 50 chromosomes, and more likely to have the t(1;19) and a T-cell subtype (Table 16.3).110 Of the ploidy groups, hyperdiploidy (>50 chromosomes per cell) and hypodiploidy (<46 chromosomes per cell) have definite clinical relevance. The former is associated with an age of 1 to 10 years, a lower median leukocyte count,111 a tendency to accumulate increased amounts of methotrexate polyglutamates,112 increased sensitivity to antimetabolite agents,113 a marked propensity for spontaneous apoptosis in vitro,114 and a favorable prognosis, even when treatment is based on antimetabolites.115 The vast majority (>97%) of hyperdiploid blasts have 3 or 4 copies of chromosome 21, in which the gene for reduced folate carrier, active transporter of methotrexate, is located.116 In this regard, high expression of reduced folate carrier due to increase in gene dosage has been shown to account partly for the increased accumulation of methotrexate polyglutamates in hyperdiploid blasts.116 While studies of the Children’s Oncology Group showed that the presence of trisomies 4, 10, and 17 characterized a distinct subgroup of ALL with very favorable prognosis,117,118 a study of the Medical Research Council’s Childhood Leukemia Working Group found trisomies 4 and 18 to be good prognostic indicators among hyperdiploid ALLs.119 A hypodiploid karyotype, by contrast, predicts an exceptionally poor outcome.120,121 Flow cytometric determination of cellular DNA content has proven to be a useful adjunct to standard cytogenetic analysis because it is automated, rapid, and inexpensive, and its measurements are not affected by the mitotic index of the cell population (hence, results can be obtained for virtually all cases). Flow cytometric studies can sometimes identify a small but drug-resistant subpopulation of near-haploid cells that was initially missed by routine karyotyping.120 Specific chromosomal translocations involving reciprocal exchanges of DNA appear to be the most biologically and clinically significant karyotypic changes in leukemia.2,23,122–124 Table 16.4 lists some of the more common and better defined nonrandom translocations and

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Table 16.3 Characteristics of leukemic blast cells in 907 white and 160 black children with newly diagnosed ALL treated consecutively at St. Jude Children’s Research Hospital (1984–2002) Feature Immunophenotype Early pre-B Pre-B Transitional pre-B B T DNA index ≥1.16 Karyotype Hypodiploid Pseudodiploid Hyperdiploid 47–50 Hyperdiploid >50 Near triploid/tetraploid Normal t(1;19)/E2A-PBX1 t(9;22)/BCR-ABL t(4;11)/MLL-AF4 t(12;21)/TEL-AML1

White children (%)

Black children (%)

53.7 24.9 4.0 2.0 15.4 22.1

39.0 24.0 3.2 2.0 31.8 8.3

4.9 37.0 14.8 29.0 0.5 13.7 2.9 2.4 3.0 18.9

6.0 40.3 17.9 16.4 0.5 18.9 11.0 5.9 1.5 13.2

their prominent features; other less common rearrangements are described in Chapter 9. One of the most common translocations in childhood ALL is the t(1;19), found in 3% of white patients and 11% of black patients and primarily seen in patients with pre-B ALL.93,110 The negative prognostic impact once associated with this karyotype has been abolished by contemporary treatment.2,23,99 Likewise, the 2% of patients with B-cell ALL and MYC gene overexpression due to the t(8;14), t(2;8), or t(8;22) rearrangement enjoy a relatively high cure rate due to wider use of more intensive and effective treatment.91,92 B-cell precursor cases with the dic(9;12) also have a favorable prognosis.125 On the other hand, cases with the Philadelphia chromosome [t(9;22)]126 or the t(4;11)127,128 generally fare poorly, despite treatment with intensive chemotherapy. Molecular analysis is an essential part of the work-up of childhood ALL and can identify prognostically and therapeutically relevant subgroups that cannot be identified by other means, including karyotyping (see Chapter 10 for details). For example, TEL-AML1 fusion, the most common specific genetic rearrangement in childhood ALL, can be identified only by molecular analysis.104,129 This leukemia subtype was initially suggested to have a relatively good prognosis; however, on further analysis a favorable outcome was observed only in clinical trials featuring intensive chemotherapy, especially those including asparaginase.129 Interestingly, TEL-AML1-expressing ALLs show a unique in

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Table 16.4 Clinical and biological features of the more common genetic subtypes of childhood ALL

Subtype

Frequency (%)

Molecular genetic alterations

Hyperdiploidy > 50 chromosomes

27–29

Unknown

t(12;21)(p12– 13;q22)a

20–25

t(1;19)(q23;p13)

3–6

ETV6-CBFA2 fusion (also termed TEL-AML1) E2A-PBX1 fusion

t(4;11)(q21;q23) and other 11q23 translocations

4–8

t(9;22)(q34;q11)

3–4

t(8;14)(q24;q32.3), t(2;8)(p12;q24), or t(8;22)(q24;q11)

2

dic(9;12) (p11–12;?p12)

1

Associated MYC overexpression with IGH, IGK, or IGL rearrangement Unknown

10q24a

0.5

HOX11

19p13a

1.5

LYL1

1p32a

6.0

TAL1

a

MLL-AF4 fusion (other MLL rearrangements) BCR-ABL fusion

Associated features Predominant B-cell precursor phenotype; age between 1 and 10 years; low leukocyte count; favorable prognosis with antimetabolite-based therapy B-cell precursor phenotype; pseudodiploidy; age 1–10 years; favorable prognosis with antimetabolitebased therapy Pre-B phenotype; pseudodiploidy; increased leukoctye count; black race; CNS leukemia; improved outcome with intensive therapy CD10− /CD15± B-cell precursor phenotype; infant age group predominantly; hyperleukocytosis; CNS leukemia; dismal outcome in infants Predominant B-cell precursor phenotype; older age; increased leukocyte count; dismal outcome in the subgroup with ≥50 × 109 /L or age ≥10 years B-cell phenotype; L3 morphology; male predominance; bulky extramedullary disease; favorable prognosis with short-term intensive chemotherapy with high-dose methotrexate plus cytarabine plus cyclophosphamide B-cell precursor phenotype; male predominance; excellent outcome with antimetabolite-based therapy CD10+ early cortical T-cell phenotype; male predominance; excellent outcome Pro-T phenotype; male predominance; BCL2 overexpression; unfavorable prognosis Late cortical T-cell phenotype; male predominance; unfavorable prognosis

Estimated 5-year event-free survival (%) 85–90

80–90

80–90

30–40

30–40

75–85

80–90

85–90 35–45 35–45

Requires molecular detection.

vitro sensitivity to asparaginase.130 The molecular mechanism for this increased sensitivity remains undefined but is not due to low expression of asparagine synthetase.131 Amplification of the AML1 gene is another recurrent abnormality that can be identified only by fluorescence in situ hybridization (FISH).132,133 It occurs in approximately 1.5% of childhood ALL cases and appears to be associated with a poor prognosis.133 With the exception of T-cell ALL with the t(11;19), the prognosis of cases with 11q23/MLL rearrangements is generally poor.127,128 Because some cases with MLL rearrangements may be missed by routine cytogenetic evaluation,134,135 most centers perform Southern blotting, FISH, or RT-PCR analysis to detect such cases. It should be noted that there is marked heterogeneity in treatment outcome among MLL-rearranged cases, even for infant

cases with the t(4;11).127,128 High-dose cytarabine was credited with improved clinical outcome in some studies of infants and adults with t(4;11)/MLL-AF4 fusion.21,136 The increased sensitivity to cytarabine appears to result from increased expression of the membrane transporter hENT1, which enables cytarabine to cross the cell membrane.137 Cytogenetic lesions that appear identical by standard analysis may prove quite different in critical ways when examined at the molecular level. ALL with the t(1;19) rearrangement illustrates this point well. Thus, among cases with a t(1;19), those without E2A-PBX1 fusion respond well to antimetabolite-based therapy.93,94 Although activating mutations in the RAS oncogenes can be identified in as many as 15% of childhood ALL cases, they do not have prognostic significance.138 Molecular studies have identified somatic missense mutations of the PTPN11 gene, which

Acute lymphoblastic leukemia

encodes the SHP-2 protein tyrosine phosphatase, in up to 6% of children with ALL.139 Although these abnormalities were first identified in juvenile myelomonocytic leukemia and acute monoblastic leukemia, this genetic alteration occurred in ALL cases with no other recognized genetic abnormalities, suggesting that it might represent a novel primary abnormality in ALL. Finally, activated mutations of NOTCH1, a gene encoding a transmembrane receptor that regulates normal T-cell development, are identified in more than 50% of T-cell ALL cases, and provide a strong rationale for targeted therapies that intefere with NOTCH signaling.140 With the development and application of DNA microarrays, the expression of almost all human genes can now be systematically examined in any given sample of leukemia. Gene expression profiling not only can accurately identify known genotypic and phenotypic subtypes of ALL, but can also provide insights into their underlying biology and the nature of their cellular responses to antileukemic treatment.33,34,97,141–144 In T-cell ALL, microarray analysis allowed virtually all cases to be grouped into LYL1 plus LMO2, HOX11, TAL1 plus LMO1 or LMO2, HOX11L2, and MLL-ENL clusters, with the first three groups corresponding to prothymoctye, immature thymocyte, and mature thymocyte stages of development, respectively.97 A highly favorable prognosis was noted for patients in the HOX11 cluster, whose leukemic cells showed a pattern of gene expression associated with increased susceptibility to programmed cell death.97 Despite a gene expression profile resembling that of HOX11L2 cases, HOX11 cases were distinguished by an increased expression of genes involved in signal transduction and chromatin-mediated control of gene expression.97 Expression of HOX11L2 is one of the most frequent abnormalities in childhood T-cell ALL, and its prognostic significance depends on the type of treatment administered.97,145–147 The finding of overexpression and mutations of FLT3 in MLL-rearranged leukemias has led to development of clinical trials of FLT3 inhibitors in patients with these leukemias.148,149 Interestingly, hyperdiploid ALL blasts also often show FLT3 overexpression or mutations.150 It is hoped that such studies will pinpoint specific molecules that could serve as targets for novel therapies.

Prognostic factors and therapeutically relevant risk groups Stringent assessment of the relapse hazard in individual patients is an integral part of the modern approach to ALL therapy, so that only high-risk cases are treated

aggressively, with less toxic therapy reserved for cases at lower risk of failure. Treatment is ultimately the most important prognostic factor in ALL. Indeed, many other variables have emerged as useful prognostic indicators, only to disappear as treatment has improved. For example, patients with T-cell or B-cell ALL, which once were associated with a very poor prognosis, now have a longterm disease-free survival rate of 70% or better with use of intensive chemotherapy.91,92,117,151 Likewise, the poor prognosis attributed to adolescent age and black race can be abolished with effective contemporary therapy,110,152 although in many studies, black race still confers a poor outcome.153–155 Among patients with B-precursor ALL, age and leukocyte count have consistently shown prognostic strength, regardless of the treatment regimen used.156 To facilitate comparison of treatment results in B-cell precursor ALL, participants in a workshop sponsored by the National Cancer Institute (United States) adopted a uniform risk classification based on these two factors.156 Two-thirds of the patients with ages between 1 and 9 years and a leukocyte count less than 50 × 109 /L were considered to be at standard risk of relapse, while the other third were classified as high risk. However, this classification has limited value because up to a third of the so-called standard-risk patients may relapse, and patients at very high risk, who require allogeneic hematopoietic stem-cell transplantation, cannot be reliably distinguished from the high-risk cases by these criteria.23 Moreover, the prognostic impact of age and, to a lesser extent, leukocyte count can be explained partly by their association with specific genetic abnormalities. For example, the overall poor prognosis of infants less than 12 months of age is due to the very high frequency of MLL rearrangements (70% to 80%) in this age group,127,128 and the overall favorable outcome of patients aged 1 to 9 years is related to the preponderance of cases (70%) with hyperdiploidy (>50 chromosomes) or TEL-AML1 fusion, both favorable genetic features.157 Thus, a more reasonable strategy would be to assign cases to different risk groups based on their major immunophenotypic features (i.e. T-cell, mature B-cell, or B-cell precursor), with genetic classification used to further characterize B-cell precursor cases (Table 16.5). Although HOX11 overexpression and MLL-ENL fusion confer a favorable prognosis in T-cell ALL,97,128 these genetic features have yet to be incorporated into current risk classification systems. It should be emphasized that primary genetic features do not entirely account for treatment outcome. For instance, up to 20% of patients with hyperdiploidy >50 chromosomes or TEL-AML1 fusion continue to suffer recurrences of their leukemia.129,157 On the other hand,

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Table 16.5 Current ALL risk classification at St. Jude Children’s Research Hospital Risk category

Features

Type of therapy

Low

B-cell precursor phenotype and age 1–9 years with leukocyte count <50 × 109 /L, DNA index ≥1.16, or ETV6-CBFA2 (TEL-AML1) fusion, without CNS or testicular leukemia, or a t(1;19), t(4;11), t(9;22), or MLL rearrangement; level of minimal residual leukemia <0.01% at the end of 6-week remission induction T-cell phenotype and B-cell precursor cases not classified as low or high risk t(9;22) or BCR-ABL fusion, induction failure, or ≥1% blasts (by flow-cytometry or polymerase chain reaction) at the end of 6-week remission induction

Antimetabolites primarily

Standard High

a substantial proportion of the patients with the t(9;22) and BCR-ABL fusion who are 1 to 9 years old and have low leukocyte counts at diagnosis, or who have good early responses to prednisone therapy, may be cured with intensive chemotherapy alone.126,158 There are also subsets of patients with MLL rearrangements who respond well to chemotherapy: patients with age 1 to 9 years, especially those with good initial prednisone response, and patients with MLL-ENL fusion and a T-cell phenotype.97,127,128 Inactivation of the p53 gene through mutations or overexpression of HDM2, whose product can bind to p53 and induce its degradation, is associated with a poor prognosis in children with ALL.159–161 Functional inactivation of the RB pathway through deletion or epigenetic silencing of P16INK4a and P15INK4b occurs in nearly all cases of T-cell ALL and in a small proportion of B-lineage ALLs162 ; however, the prognostic impact of these findings has been inconsistent.163–167 More recently, gene expression profiling of leukemic cells by the DNA microarray method has proved useful in identifying, within a given leukemia subtype, previously unrecognized genes whose expression may have prognostic significance.141,144 The predictive power of these newly identified expression signatures will require validation in prospective clinical trials. One plausible reason for the unpredictable relation between the biological features of leukemic cells and response to therapy is that pharmacodynamic and pharmacogenomic factors exert a crucial influence on the effectiveness of treatment (see Chapter 14).71,168–170 Indeed, the rates of metabolism and systemic clearance of antileukemic agents and the absorption of orally administered chemotherapy vary widely. Low systemic exposure to methotrexate and a low dose intensity of mercaptopurine have each been associated with a poor treatment outcome.171,172 Thus, treatment is unsuccessful in some patients because they have received inadequate doses of drugs, not because their leukemia is drug-resistant. Concomitant administration of anticonvulsants (phenytoin, phenobarbital, carbamazepine, or

Intensive multiagent chemotherapy Allogeneic hematopoietic stem-cell transplantation

a combination), by inducing cytochrome P450 enzymes, significantly increases the systemic clearance of several antileukemic agents and may adversely affect treatment outcome.173 We now use other anticonvulsants (e.g. gabapentin and valproic acid) that are less likely to induce the activity of cytochrome P450 enzymes. Genetic polymorphisms of several enzymes involved in drug metabolism are also associated with treatment outcome. Patients who have homozygous or heterozygous deficiency of thiopurine methyltransferase, the enzyme that catalyses the S-methylation (inactivation) of mercaptopurine, tend to have better event-free survival, probably because they, in effect, had received a higher dose intensity of mercaptopurine.172 However, the thiopurine methyltransferase genetic polymorphism is also linked to acute doselimiting toxic effects174 and the risk of radiation-induced brain tumors175 and therapy-related AML,176,177 in the context of antimetabolite-based therapy. Therapy must therefore be adjusted in patients with homozygous mutant genotypes of this enzyme and in many heterozygotes. The null genotype (absence of both alleles) for GSTM1 has also been associated with a lower risk of relapse, perhaps because of the reduction in detoxification of cytotoxic chemotherapy.178 Since response to therapy is determined by both leukemic cell genetics and host pharmacogenetics, measurements of this response in vivo should show greater prognostic strength than any other individual biological or hostrelated feature. The independent prognostic importance of a patient’s gross early response to therapy (i.e. initial decrease in leukemic blasts) has been recognized by investigators of the Children’s Cancer Group and the Berlin¨ Frankfurt-Munster Consortium since the early 1980s.179,180 These investigators assessed this response by morphological examination of the bone marrow or peripheral blood. Although their methods can be readily applied at any center, neither measure has great precision because about 20% of patients with a good initial response eventually relapse, and a third of patients with a poor response may

Acute lymphoblastic leukemia

survive long term, when treated with intensive chemotherapy alone.117,180,181 Measurements of minimal residual disease (MRD), by flow-cytometric detection of aberrant immunophenotypes or analysis by polymerase chain reaction (PCR) of clonal antigen-receptor gene rearrangements, afford a level of sensitivity and specificity that cannot be attained by traditional morphological assessment of treatment response (see Chapter 28).106–109 Patients who achieve an immunological or molecular remission, defined as leukemic involvement of less than 10−4 nucleated bone marrow cells on completion of a 6-week induction therapy, have a much more favorable prognosis than do those who do not achieve this status. Patients who are in morphological remission but have a post-induction MRD level of 1% of more, fare as poorly as those who do not achieve clinical remission by conventional criteria (≥5% blast cells).106,107 About half of all patients show a disease reduction to 10−4 or lower after only 2 weeks of remission induction, and they appear to have an exceptionally good treatment outcome.182,183 Although MRD positivity is strongly associated with known presenting risk features, it has independent prognostic strength.184 Sequential monitoring of MRD can improve the precision of risk assessment still further. Thus, the persistence of MRD (≥0.01%) beyond 4 months from diagnosis was associated with an estimated 70% cumulative risk of relapse.185,186 Patients with 0.1% MRD or more at 4 months had an especially dismal outcome.185 Tandem application of flow cytometry and PCR testing has allowed us to study MRD successfully in virtually 100% of cases.106 We have therefore incorporated MRD detection into our current risk-classification system (Table 16.5). Leukemia cell growth in model systems, such as nonobese diabetic/severe combined immunodeficient mice or cultures based on stromal cell layers, correlates with an adverse prognosis and with resistance to chemotherapy.187,188 Other factors related to a poor outcome include male gender,189,190 malnutrition,191 and expression of transporters of xenobiotics of the adenosine triphosphate-binding cassette protein superfamily (see Chapter 15).192–194 Whether these factors are important in programs of contemporary therapy remains to be determined.

Total therapy approach Supportive care At diagnosis, all febrile patients with or without documented infection should be given broad-spectrum

intravenous antibiotics until an infectious disease can be excluded. Because of the need to draw a relatively large volume of blood for laboratory evaluation and because of bone marrow suppression due to leukemia and chemotherapy, most patients require packed red blood cell transfusions to correct anemia (see Chapter 33). Transfusions should be administered slowly in patients with severe anemia to prevent congestive heart failure. In patients with extreme hyperleukocytosis (e.g. leukocyte count ≥400 × 109 /L), packed red blood cell transfusion should be delayed until after leukocyte count is decreased by leukapheresis, exchange transfusion or chemotherapy to prevent complications of leukostosis. Thrombocytopenia is a common presenting feature of ALL, but active bleeding is relatively rare. We transfuse patients who are thrombocytopenic and have circulating leukemic cells with platelets to keep counts close to 100 × 109 /L before diagnostic lumbar puncture because a traumatic procedure in them can increase the risk of subsequent relapse.86 Patients with traumatic lumbar puncture and leukemic blasts in cerebrospinal fluid require intensified intrathecal therapy to avert the increased risk of relapse.76 However, after the clearance of circulating blasts, intrathecal therapy can be safely administered without prophylactic platelet transfusion, especially during prednisone-vincristine-asparaginase remission induction,195 which is associated with a hypercoagulable state.196 All blood products should be irradiated in patients who are receiving immunosuppressive therapy to prevent graft-versus-host disease. Careful attention to fluid and electrolyte balance is essential (see also Chapter 29). All patients require intravenous hydration to prevent or treat hyperuricemia and hyperphosphatemia. Specific measures to treat or prevent hyperuricemia include allopurinol (a xanthine oxidase inhibitor that can prevent uric acid formation) and rasburicase (a recombinant urate oxidase that breaks down uric acid to allantoin – a readily excretable metabolite with 5- to 10-fold higher solubility than uric acid). Rasburicase is more effective than allopurinol in the prevention and treatment of hyperuricemia, and facilitates the excretion of phosphorus, partly because its potent uricolytic effect obviates the need to alkalinize urine and partly because of more rapid improvement in renal function with its use.197,198 In fact, most patients have improved or stabilized renal function during rasburicase treatment despite ongoing chemotherapy-induced tumor lysis.199 Rasburicase is associated with mild acute hypersensitivity reactions in approximately 3% of the patients, and with methemoglobinemia or hemolytic anemia in patients with glucose-6-phosphate dehydrogenase deficiency because hydroxygen peroxide is a byproduct of the uric acid

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oral complications of leukemia and its treatment.207 In many instances, it is difficult to distinguish between oral mucositis and herpes simplex viral infection. Occasionally, patients have nausea and substantial pain on swallowing due to esophageal herpes simplex viral infection or candidiasis or both. Early recognition, diagnosis, and treatment are important to prevent systemic candidiasis.

Principles of treatment

Fig. 16.8 Results of leukapheresis in a 16-year-old boy with T-cell ALL and a presenting leukocyte count of 350 × 109 /L. The cylinders shown contain 2.8 × 1012 leukemia cells, illustrating well the definition of the term leukemia (white blood); the pink tint represents red cells that have not settled out. (See color plate 16.8 for full-color reproduction.)

breakdown.199,200 In many patients, a phosphorus binder (aluminum hydroxide, sevelamer, lanthanum carbonate or calcium carbonate in patients with low serum calcium levels) is needed to treat or prevent hyperphosphatemia and consequent hypocalcemia and renal dysfunction. For patients with extreme hyperleukocytosis, leukapheresis or exchange transfusion (in small children) has been used to reduce the morbidity and mortality from leukostasis or leukemic cell lysis.201 Although these measures are highly effective in reducing the leukemic cell burden (Fig. 16.8), no randomized studies have been performed to demonstrate their short-term or long-term benefits, and some investigators question the need for these procedures.202 We limit the use of such procedures to patients presenting with a leukocyte count of 400 × 109 /L or greater, because they have a high risk of leukostasis and intracranial bleeding.203 Other important supportive care measures include prophylactic use of trimethoprim-sulfamethoxazole, pentamidine or atovaquone (in patients with poor tolerance to trimethoprim-sulfamethoxazole) for Pneumocystis carinii pneumonia, the placement of indwelling catheters, and psychosocial support, topics that are covered elsewhere in this book. Prophylactic use of human granulocyte colonystimulating factor during induction or consolidation therapy shortened the duration of severe neutropenia but failed to reduce episodes of febrile neutropenia or improve treatment outcome.204,205 In one study, granulocyte colonystimulating factor appeared to increase the risk of therapyrelated AML in the context of epipodophyllotoxin-based therapy.206 Dental evaluation at diagnosis and meticulous oral hygiene during chemotherapy will minimize the

Recognition that ALL is a heterogeneous disease and the identification of reliable prognostic factors have led to wide use of risk-directed therapy. In virtually all centers, B-cell ALL cases are treated separately with protocols featuring short-term intensive chemotherapy (including high-dose methotrexate, cytarabine, and cyclophosphamide),91,92 as described in Chapter 18. For all other patients, specific approaches to therapy differ but consistently emphasize remission induction followed by intensification or consolidation therapy to eliminate residual leukemia, eradication or prevention of CNS leukemia, and treatment to ensure continuation of remission. Table 16.6 summarizes effective treatment components identified in recently completed major clinical trials.48,49,76,82,84,117,118,157,181,208–236 In most studies, patients are divided into three risk groups, even though there has not been a consensus on the most useful criteria or the terminology. For example, in the BFM group, patients are classified as having a standard, medium or high risk of failure based on prednisone response, age, leukocyte count, leukemic cell genetics and immunophenotype, and the level of minimal residual disease after remission induction. We also divided the patients into three risk groups using similar criteria (Table 16.5). Having identified a group of patients at very low risk of relapse, the Children’s Oncology Group recently proposed a four-tiered classification: low, standard, high, and very high.237

Remission induction The goal of remission induction therapy is to induce a complete remission by eradicating over 99% of the initial leukemic cell burden, and by restoring normal hematopoiesis (absolute granulocyte count >0.5 × 109 /L and platelet count >100 × 109 /L) and a normal performance status. This phase of treatment typically includes administration of a glucocorticoid (dexamethasone or prednisone), vincristine, and at least a third agent (L-asparaginase or an anthracycline, or both). There are several forms of L-asparaginase, each with different pharmacokinetic profiles and hence potency. Two commercial preparations were derived from Escherichia

Acute lymphoblastic leukemia

453

Table 16.6 Results of recently completed clinical trials in ALL

Number of patients

% 5-year event-free survival (SE)

Study

Year

Eligible age (years)

AIEOP-91

1991–5

<15

1194

70.8 (1.3)

AIEOP-95

1995–9

<18

1485

—

BFM-90

1990–5

≤18

2178

78.0 (0.9)

BFM-95

1995–9

≤18

2021

79.0

CCG-1800

1989–5

≤21

5121

75.0 (1.0)

CCG 1922

1993–5

1 to 10

1060

81 (2)

COALL-CLCG-92

1992–7

≤18

538

76.9 (1.9)

DCLSG ALL-8

1991–6

≤18

467

73.0 (2.0)

DFCI 91–01

1991–5

≤18

377

83.0 (2.0)

EORTC-58881

1989–98 ≤18

2065

70.9 (1.1)

NOPHO ALL92

1992–98 ≤15

1143

77.6 (1.4)

Lessons learned

References

Extended intrathecal therapy can replace cranial irradiation in intermediate-risk cases; intensive high-dose chemotherapy blocks do not improve outcome of high-risk ALL; Erwinia asparaginase used in delayed intensification phase does not improve outcome of intermediate-risk ALL Double 8-drug reinduction therapy improves outcome of high-risk ALL A cranial radiation dose of 12 Gy appears to provide adequate treatment for subclinical CNS leukemia, even in high-risk cases Traumatic lumbar puncture with blasts at diagnosis is associated with an inferior outcome Prolonged second reinduction/intensification phase (“augmented post-induction therapy”) can abolish the adverse prognostic impact of a poor early response; double delayed intensification therapy improves outcome of intermediate-risk ALL; delayed intensification therapy benefits low-risk cases Dexamethasone treatment decreases CNS relapse and improves event-free survival; intravenous mercaptopurine does not improve outcome and is associated with decreased survival after relapse Continuation treatment with thioguanine does not improve outcome and is associated with prolonged myelosuppression and marked thrombocytopenia Omission of cranial irradiation does not jeopardize overall outcome; high-dose intravenous mercaptopurine does not improve outcome of intermediate-risk ALL; Erwinia asparaginase during continuation treatment does not improve outcome of standard-risk cases Prolonged asparaginase intensification and the use of dexamethasone improves outcome; hyperfractionated cranial irradiation appears to be associated with an inferior outcome in high-risk cases; intravenous mercaptopurine does not improve outcome; pegaspargase and E. coli asparaginase yield similar outcomes Intravenous mercaptopurine does not improve outcome; Erwinia asparaginase given at the same dose is inferior to E. coli asparaginase; high-dose cytarabine does not improve outcome in increased-risk ALL Patients with low thiopurine methyltransferase activity are at risk of developing therapy-related leukemia; adding pharmacologically guided treatment intensification to dose adjustment by blood count may not be warranted for girls

209–212

213 48, 181

82 117, 214, 215

216

217, 218

219

49, 208

84, 220, 221

222, 223

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Ching-Hon Pui

Table 16.6 (cont.)

Eligible age (years)

Number of patients

% 5-year event-free survival (SE)

3828

70.9 (0.8)

Study

Year

POG

1986–94 ≤21

SJCRH 13A&B

1991–8

≤18

412

81.0 (8.0)

TCCSG L92-13

1992–5

≤15

347

63.4 (2.7)

UKALL XI

1990–7

≤15

2090

63.0 (1.1)

Lessons learned

References

Intermediate-dose intravenous methotrexate is superior to oral methotrexate for intensification; intravenous mercaptopurine does not improve outcome; intensive chemotherapy blocks does not improve outcome in high-risk ALL; intensive asparaginase consolidation improves outcome of T-cell ALL Early intensification of intrathecal treatment based on CNS status improves outcome; higher-dose methotrexate results in greater methotrexate polyglutamate accumulation in blast cells and greater antileukemic effects; intravenous mercaptopurine produces minimal de novo purine synthesis inhibition and antileukemic effects Early treatment intensification does not compensate for shortened duration of continuation therapy; 6 months of continuation treatment appears adequate for very high-risk cases with good early response to prednisone treatment Intensified treatment benefits all risk groups; dexamethasone decreases CNS relapse and improves event-free survival; transplantation in first remission does not improve outcome in the majority of very high-risk cases

118, 224–227

76, 157, 228, 229, 269

230, 231

232–236

¨ Abbreviations: AIEOP, Associazione Italiana di Ematologia ed Oncologia Pediatrica; BFM, Berlin-Frankfurt-Munster ALL Study Group; CCG, Children’s Cancer Group; COALL, Cooperative ALL Study Group; DCLSG, Dutch Childhood Leukemia Study Group; DFCI, Dana Farber Cancer Institute ALL Consortium; EORTC-CLCG, European Organization for Research and Treatment of Cancer, Children’s Leukaemia Cooperative Study Group; NOPHO, Nordic Society of Pediatric Hematology and Oncology; POG, Pediatric Oncology Group; SJCRH, St. Jude Children’s Research Hospital; TCCSG, Tokyo Children’s Cancer Study Group; UKALL, United Kingdom Medical Research Council Working Party on Childhood Leukaemia.

coli. Leunase (Kyowa Hakko Kogyo, Japan) is more potent and toxic than Elspar (Merck Sharp & Dhome, USA) given at the same dosage.238,239 Elspar has a half-life of only 1.28 ± 0.35 days (SD), while pegaspargase, an alternative form of asparaginase in which the Elspar is covalently bound to monomethoxpolyethylene glycol (PEG), has a long half-life, 5.73 ± 3.24 days.240 There is another product derived from Erwinia chrysanthemi with a short half-life of 0.65 ± 0.13 days.240,241 If the dosages of these four preparations are based on their pharmacokinetic and pharmacodynamic properties, they should yield comparable clinical results. The reported differences in efficacy and toxicity of the various preparations can be attributed to the use of unequivalent doses.221,241 In terms of leukemia control, the dose intensity and duration of asparaginase therapy are more important than the type(s) of asparaginase used. In the EORTC 58881 trial,

children randomized to receive Erwinia asparaginase had inferior event-free survival but fewer toxicities than those given E. coli asparaginase at the same dosage, 10,000 IU/m2 twice weekly, because of the lower dose intensity of treatment with the Erwinia preparation.221 In the Dana-Farber Consortium Protocol 91-01 study, no difference in eventfree survival was observed between patients randomly selected to receive pegaspargase (2500 IU/m2 every 2 weeks for 15 doses) and those who were randomly chosen to receive Elspar (25,000 IU/m2 every week for 30 doses).208 However, the clinical outcome in patients who received fewer than 25 weeks of asparaginase therapy was significantly worse than that of patients who received 26 to 30 weeks.208 In the Pediatric Oncology Group 9310 study of relapsed ALL, patients were randomly assigned to one of two pegaspargase treatment groups; the reinduction rate was significantly higher in patients who received weekly

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pegaspargase than in those who received treatment every 2 weeks.242 Finally, in one randomized trial, one dose of pegaspargase at 2500 IU/m2 produced more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow, more prolonged asparaginase activity and a lower incidence of neutralizing antibodies than did Elspar at 6000 IU/m2 on days 3, 5, 8, 10, and 12.243 There was no difference in adverse reactions between the two treatment arms. Hence, pegaspargase (2500 IU/m2 for one dose), Elspar (25,000 IU/m2 per week for 2 weeks or 10,000 IU/m2 three times weekly for 2 weeks), and Erwinia asparaginase (25,000 IU/m2 twice a week for 2 weeks) appear to be equally effective in patients with newly diagnosed ALL.240,244 Children’s Cancer Group clinical trials for children with B-lineage ALL are primarily using pegaspargase for front-line therapy. If a patient first receives E. coli asparaginase and hypersensitivity reactions occur, we prefer to replace this product with an Erwinia product (if available) because antibodies against the E. coli preparation cross-react with PEG-asparaginase.245 It should be noted that intramuscular administration causes less frequent and less severe hypersensitivity reaction than intravenous injection of L-asparaginase. Although asparaginase is an indispensable agent in the treatment of ALL, its use during remission induction is being challenged. In one randomized trial comparing the relative efficacy and toxicity of asparaginase and epirubicin as a third remission-induction agent in patients with standard-risk ALL, patients treated with asparaginase had a significantly lower rate of successful remission induction due to a high rate of fatal infection,238 probably because the dosage of asparaginase (Leunase product) was too high. Dana-Farber Consortium protocols and COALL studies using asparaginase in the post-induction period had an excellent remission induction rate with low morbidity (especially in terms of thrombotic complications and hyperglycemia), and excellent long-term eventfree survival.208,217 Hence, additional studies are needed to determine the optimal timing of asparaginase treatment. Because of its better penetration into cerebrospinal fluid and longer half-life,246 dexamethasone has been used instead of prednisone or prednisolone in some induction and many continuation regimens. In two recent randomized trials, dexamethasone decreased isolated CNS relapse and yielded a better event-free survival than did prednisone treatment.216,236 A higher frequency of reversible corticosteroid-induced side effects (hyperglycemia, hypertension, myopathy and severe behavior changes) was observed in patients treated with dexamethasone. Another study implicated dexamethasone as a cause of excessive life-threatening infections and septic deaths in the context

of multiple-agent remission induction regimen.247 Ongoing randomized trials of the AIEOP and BFM cooperative study groups are further evaluating the efficacy and toxicities of dexamethasone versus prednisone treatment and should provide guidelines for future studies. To this end, the biologically equivalent doses between dexamethasone and prednisone are not known. The superior outcome of dexamethasone may be due to the use of a higher biological dose. Indeed, a higher dose of corticosteroid treatment was shown to abrogate relative drug resistance in an upfront single-agent window study.248 Various anthracyclines (e.g. daunorubicin, doxorubicin, and epirubicin) have been used in the treatment of ALL, and the superiority of one over another in terms of toxicity or efficacy is uncertain. In one randomized study, dexrazoxane, a cardioprotective agent that binds free and bound iron, thereby reducing the formation of anthracycline-iron complexes and the subsequent generation of reactive oxygen species, reduced or prevented acute cardiac injury, as reflected by the troponin T level.249 Its use did not compromise event-free survival rate at 2.5 years. However, additional studies are needed to determine if it affects long-term cardiac function or leukemia-free survival. Attempts have been made to intensify induction therapy, especially in high-risk and very high-risk cases, on the premise that a more rapid and profound reduction of the leukemic cell burden may forestall the development of drug resistance in leukemic cells. However, several studies have suggested that intensive induction therapy may not be necessary for standard-risk cases, provided that they receive postinduction intensification therapy.217,250 Moreover, intensive induction therapy may lead to a poor overall outcome due to an increase in early morbidity and mortality.238,247,250 With modern chemotherapy and supportive care, 97% to 99% of children can be expected to attain complete remission. Approximately 1% of the patients die of toxicities during remission induction and another 1% of induction failure due to drug-resistant leukemia. Patients who fail to achieve morphological remission (i.e. ≥5% blasts in bone marrow) at the end of remission induction have a short survival or, if remission is eventually achieved, a higher rate of relapse.251,252 Hence, most investigators offer these patients the option of allogeneic hematopoietic stem cell transplantation at the end of extended induction treatment.253 We and others found that patients with 1% blasts identified by MRD studies had an outcome as poor as those with induction failure106,107 and these patients may also be candidates for allogeneic transplantation. Supportive care is especially important during remission induction treatment, during which time there are

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metabolic complications from leukemic cell lysis and an increased risk of infection due to myelosuppression, immunosuppression and mucosal breakdown. Intensive therapy has been associated with the development of disseminated fungal infection and typhlitis. Oral candidiasis occurs frequently, especially in young children. We prefer to use clotrimazole troches for oropharyngeal candidiasis during remission induction because it has few systemic effects, whereas other azole compounds (e.g. fluconazole, itraconazole, ketaconazole) can inhibit cytochrome P450 enzymes and increase the toxicities of various antileukemic agents, especially vincristine.254 Patients with Charcot–Marie–Tooth hereditary neuropathy are at particular risk for vincristine-induced profound neuropathy.255 A family history for these genetic disorders or other peripheral neuropathy should be sought at diagnosis of ALL; molecular genetic testing is now available for the most common forms of these disorders.255 Attention should also be paid to the diets of leukemia patients, to avoid foods that may be contaminated with pathogens256 ; to decrease the risk of hyperglycemia, which is more prevalent in adolescents, obese individuals, and Down syndrome patients;257 and to reduce salt intake, which could induce hypertension and resultant seizure, especially in patients with severe constipation.258 Behavioral changes due to the prolonged illness, hospitalization, and the use of glucocorticoids, especially dexamethasone, are common.

Intensification (consolidation) and reinduction therapy With restoration of normal hematopoiesis, patients in remission become candidates for intensification (consolidation) therapy. The importance of this phase of therapy is no longer disputed, but there is still a lack of consensus on the best regimens and their duration. Delayed intensification (reinduction) therapy, pioneered by investigators ¨ in the Berlin-Frankfurt-Munster (BFM) Consortium, is perhaps the most widely used strategy.259 Basically a repetition of the initial induction therapy at 3 months after remission, this treatment is most beneficial for standard-risk cases. Investigators in the former Children’s Cancer Group confirmed the efficacy of reinduction therapy260,261 and subsequently extended its application by showing that doubledelayed intensification, beginning at week 32 of treatment, improved outcome in patients with high-risk or so-called intermediate-risk leukemia.215 Of further interest, additional pulses of vincristine and prednisone after one reinduction treatment were not beneficial, suggesting that the observed improvement was due to the increased dose intensity of other agents, such as asparaginase, anthracy-

clines, cytarabine, and cyclophosphamide, or perhaps to the timing of the intensification regimen itself.215 Extended and stronger intensification therapy also significantly benefited patients with high-risk ALL and a slow response to initial induction therapy, especially those of younger age.214 Hence, some form of intensification therapy appears useful in all patients with ALL, with double or prolonged intensification reserved for those with high-risk or very-high-risk disease. The use of different intensification regimens in various clinical trials has also led to the identification of effective treatment components for certain subtypes of leukemia. For example, improved outcome of T-lineage ALL in the clinical trials of the Dana-Farber Cancer Institute Consortium and the Children’s Cancer Group has been credited to the intensive use of asparaginase,151,214 a finding that has been corroborated by a randomized study of the former Pediatric Oncology Group.262 As mentioned earlier, intensive asparaginase treatment is also credited with a very low rate of relapse among TEL-AML1-positive cases treated on the protocols of Dana-Farber Cancer Institute Corsortium.208 Very high doses of methotrexate (5 g/m2 ) also seem to improve outcome in patients with T-lineage ALL.48,181,237 This observation is consistent with our finding that T-lineage blast cells accumulate methotrexate polyglutamates (active metabolites of methotrexates) less avidly than do B-lineage blast cells, so that higher serum concentrations of methotrexate are needed for adequate response in T-lineage ALL.112 Nonetheless, high-dose methotrexate (but not intravenous mercaptopurine) also benefits patients with B-lineage ALL226 ; the optimal dose of methotrexate for individual genetic subtypes remains to be determined, but a dose of 2.5 g/m2 should be adequate for most of these patients.171 To this end, our recent study showed that among patients with B-lineage ALL, blasts with either TEL-AML1 or E2APBX2 gene fusion accumulate significantly lower amounts of methotrexate polyglutamate, compared to those with hyperdiploidy or other genetic abnormalities.263 This finding suggests that cases with TEL-AML1 or E2A-PBX1 fusion may also benefit from a higher dose of methotrexate. The most successful postremission intensification regimens are generally administered continuously,208,214 whereas high-dose pulse therapy with long rest periods due to myelosuppression appears to be less effective.48 This observation is consistent with the concept of metronomic dosing for solid tumors, based on the idea that continuous or frequent administration of cytotoxic drugs may improve outcome by abrogating the ability of slowly proliferating endothelial cells, which are essential for tumor-cell

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survival, to repair and recover during the usual rest period.23 Angiogenesis has also been seen in ALL,264 and chemotherapy could affect the recovery of bone-marrow mesenchymal and endothelial cells that provide essential survival factors for leukemic lymphoblasts.265

Treatment of subclinical CNS leukemia The importance of therapy directed to the CNS was first demonstrated by investigators at St. Jude Children’s Research Hospital in the early 1970s.266 Several factors affect the control of leukemia in the CNS: presenting risk features, the amount of leukemic blast cells in the cerebrospinal fluid, and the type of systemic and CNS-directed therapy. Patients with high-risk genetic features, a large leukemic cell burden, T-lineage ALL, and leukemic cells in the CSF (even from iatrogenic introduction through a traumatic lumbar puncture), are at increased risk of CNS relapse and require more intensive CNS-directed therapy.23 High-dose methotrexate, although useful for preventing hematological or testicular relapse, generally has only a marginal effect on the control of CNS leukemia. In a collaborative meta-analysis of 43 randomized trials involving over 13,000 patients, high-dose methotrexate reduced non-CNS rather than CNS relapses.267 By contrast, dexamethasone was definitely shown to improve CNS control.117,216 In an early study, investigators of the Pediatric Oncology Group showed that triple intrathecal treatment with methotrexate, hydrocortisone, and cytarabine, together with effective systemic chemotherapy, could yield results comparable to those produced by cranial irradiation and that intrathecal therapy could be reduced from 3 years to 1 year in patients with good-risk leukemia.268 Two consecutive studies at St. Jude Children’s Research Hospital also suggested that early intensive triple intrathecal therapy decreases CNS relapse and boosts the event-free survival rate.76,269 In a recent randomized trial of the Children’s Cancer Group for patients with standard-risk ALL, triple intrathecal therapy reduced the frequency of isolated CNS relapse but was associated with increased bone marrow and testicular relapse, so that the event-free survival rate was comparable to the rate achieved with intrathecal methotrexate alone.270 Hence, this intriguing result failed to resolve the controversy over the optimal type of intrathecal therapy. Cranial irradiation is the most effective CNS-directed therapy, but this efficacy is offset by substantial rates of neurotoxicity, endocrinopathy and occasional brain tumors (see Chapter 30). In our recent study of 10-year event-free survivors, prior cranial irradiation was associated with a 20.9 ± 3.9% (SE) cumulative risk of second neoplasms at

20 years (30 years from remission induction), a higher mortality rate than in the general population, and an increased unemployment rate.271 Hence, virtually all leukemia therapists limit the use of cranial irradiation. Investigators of the BFM study group showed that among high-risk patients, radiation dose can be lowered to 12 Gy without increasing the risk of CNS relapse, provided effective systemic chemotherapy is used.48 Whether CNS irradiation can decrease the risk of hematological relapse is controversial. In one study, the omission of cranial irradiation was implicated as a cause of increased CNS and hematological relapses in T-lineage ALL with presenting leukocyte counts of more than 100 × 109 /L.209 However, the study involved only a small number of cases, and inadequate system chemotherapy might have contributed to the increased rate of relapse. In a retrospective study of T-lineage ALL with a high presenting leukocyte count (>50 × 109 /L) or CNS leukemia at diagnosis, CNS irradiation reduced the rates of CNS relapse but did not improve event-free survival.272 Two other studies omitted cranial irradiation altogether.84,273 The cumulative risks of isolated CNS relapse were 4.2% and 3.0%, and rates of any CNS relapse (including combined CNS and hematological relapse) were 8.3% and 6.0%, respectively. Patients with a CD10-negative B-lineage (pro-B) phenotype, CNS2 or CNS3 status, and a leukocyte count of greater than 100 × 109 /L had an increased risk of CNS relapse.84,273 Since the overall 8-year event survival rates for the two studies were only 60.7% ± 4.0% and 68.4% ± 1.2%, it remains unclear whether improved systemic chemotherapy can reduce the CNS relapse hazard. Moreover, patients with an isolated CNS relapse who had not received cranial irradiation as initial CNS-directed therapy, had a very high remission retrieval rate; in those who had a long initial remission before the CNS event, the long-term prognosis may even be similar to that of newly diagnosed patients.274 Thus, we275 and Dutch investigators are conducting clinical trials to determine if, in the context of intensive systemic and intrathecal therapy, cranial irradiation can be omitted altogether, irrespective of a patient’s risk features. Special care is being taken to minimize traumatic lumbar punctures, to deliver intrathecal therapy optimally, and to intensify systemic and intrathecal therapy in high-risk cases.83 Cranial irradiation is now reserved for salvage therapy, thus sparing most patients from its toxic effects. While this approach remains under study, most clinical protocols still specify cranial irradiation for patients at particularly high risk of CNS relapse, especially those with a CNS3 status or T-cell leukemia with a high leukocyte count. A randomized trial showed that

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hyperfractionated (twice daily) cranial irradiation provided no benefit in terms of neurocognitive late effects and might have compromised antileukemic efficacy, as compared to conventionally dosed radiation.276

Continuation treatment For reasons that are poorly understood, children with ALL (except those with mature B-cell leukemia) require longterm continuation treatment. Past attempts to shorten the duration of treatment to 24 months or less have resulted in a high risk of relapse after cessation of therapy.277,278 A more recent study, which intensified early therapy, but shortened total treatment duration to 1 year, also resulted in poor overall event-free survival.231 Interestingly, the short therapy seemed to be adequate for a small subset of children with T-cell disease who responded well to prednisolone. This result notwithstanding, the general rule has been to continue therapy for a total of 2.0 to 2.5 years. Many investigators prefer to extend treatment for boys to 3 years because of their generally poorer outcome compared with girls,189,190 although the benefit of this approach remains to be demonstrated. Several studies showed no advantage to prolonging treatment beyond 3 years.279,280 The combination of weekly methotrexate and daily mercaptopurine constitutes the standard ‘backbone’ of ALL continuation regimens. By virtue of its inhibiting effect on de novo purine synthesis, methotrexate is synergistic with 6-mercaptopurine, enhancing the conversion of mercaptopurine to thioguanine, the active metabolite.281 Pharmacokinetic studies of mercaptopurine and methotrexate have revealed wide intrapatient and interpatient variability in drug disposition.282,283 The accumulation of higher intracellular levels of the active metabolites, methotrexate polyglutamates and thioguanine nucleotides, has been associated with a better clinical outcome.284,285 Some studies have demonstrated an improved response when mercaptopurine is given at bedtime to patients with an empty stomach.286 Mercaptopurine should not be taken together with milk or milk products, which contain an enzyme, xanthine oxidase, that can degrade the drug.287 Tailoring of mercaptopurine doses to the limits of tolerance (as indicated by low neutrophil counts) has been associated with a better clinical outcome.288 Overzealous use of mercaptopurine, to the extent that neutropenia precludes further chemotherapy, thus reducing overall dose intensity, is counterproductive.172 Rare patients (1 in 300) with an inherited deficiency of thiopurine-S-methyltransferase show extreme sensitivity to mercaptopurine.289,290 Indeed, the 10% of patients who are heterozygous for this deficiency, and thus have intermediate levels of enzyme activity, may also require moderate

dose reductions, to avert side effects.174 However, the dose of mercaptopurine may need to be increased in patients homozygous for the wild-type enzyme who are treated on protocols specifying a low dose of the drug.291 Identification of the genetic basis of this autosomal codominant trait has enabled molecular diagnosis of these cases.292 Positive identification of the methyltransferase deficiency allows one to selectively lower the dose of mercaptopurine without modifying the dose of methotrexate. Patients with the enzyme deficiency are also at greater risk for the development of radiation-induced brain tumors and chemotherapy-induced AML.20,175,176 In one randomized trial, the use of thioguanine (mean dose, 36 mg/m2 ) failed to improve outcome over the result with mercaptopurine (48 mg/m2 ) and was associated with profound thrombocytopenia.218 In another randomized trial, the use of thioguanine (50 to 60 mg/m2 ) led to improved eventfree survival as compared to the use of mercaptopurine (75 mg/m2 ).270 However, the high incidence (∼20%) of hepatic veno-occlusive disease in patients treated with thioguanine was considered unacceptable and prompted the investigators to terminate the randomization and shift the treatment to mercaptopurine in all patients. Isolated persistent thrombocytopenia appeared to be the earliest indicator of incipient hepatic veno-occlusive disease.293 Although the relative merits of oral versus parenteral administration of methotrexate are uncertain, the latter route of administration offers a way to circumvent problems of decreased bioavailability and poor compliance. Adjusting the dose of intravenous high-dose methotrexate to account for the patient’s ability to clear the drug was shown to improve the outcome of B-cell precursor ALL in one pediatric study.171 It should be recognized that methotrexate treatment can induce encephalopathy, even when given orally, if the treatment is intensive enough or if leucovorin rescue is insufficient294,295 ; and that patients with Down syndrome tolerate methotrexate poorly.296,297 By inhibiting enzymes involved in folate homeostasis and depleting cellular reduced folates,298,299 high-dose methotrexate treatment can cause transient hyperhomocysteinemia, which may be responsible for the neurotoxicity.300 Elevation of serum aminotransferase, a common finding during antimetabolite-based continuation treatment, appears to be caused by methylated metabolites of mercaptopurine, resolves promptly after the completion of therapy, and correlates with a favorable outcome.302,303 If there is no other evidence of liver toxicity (e.g. direct hyperbilirubinemia) or viral hepatitis, it is not necessary to withhold or reduce the doses of continuation chemotherapy. We do, however, postpone the use of high-dose methotrexate if alanine aminotransferase is above 500 U/L. Alternative ways of using this

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Fig. 16.9 Facial maculopapular erythematous skin rash during continuation treatment with mercaptopurine and methotrexate. (See color plate 16.9 for full-color reproduction.)

combination, such as treatment with multiple, fractionated lower doses of oral methotrexate224,301 and high-dose intravenous mercaptopurine,84,208,216,219,220 have proved ineffective. The addition of intermittent pulses of vincristine and a glucocorticoid to the antimetabolite continuation regimen described above improves results and has been widely adopted.278,304 Dexamethasone has been substituted for prednisone during continuation therapy in many clinical trials because of its better clinical efficacy.117,216 However, studies are needed to find the optimal dose and duration of dexamethasone therapy during this phase of treatment. As mentioned earlier, one study showed that additional prednisone and vincristine pulses beyond reinduction treatment failed to improve outcome.215 Glucocortoid treatment, especially with dexamethasone, often causes behavioral changes and pruritus. Occasionally, the dosage of dexamethasone needs to be reduced or held due to psychotic reactions. Antecdotal experience has suggested that a potassium supplement will decrease the level of agitation.216 Extended use of glucocorticoid, especially during reinduction treatment, can result in stunted growth, obesity, osteoporosis and osteonecrosis, which can be debilitating.305–307 Interrupted use of glucocorticoid (e.g. treatment for 1 week followed by discontinuation of treatment for 1 week) during reinduction treatment can reduce the side effects,308 but whether this approach can maintain the efficacy requires additional studies. Pharmacogenetic studies might help to identify patients at particularly high risk of developing osteonecrosis and guide the individualized dosing in the future.309 Photosensitive skin rash can occur during antimetabolite therapy. The rashes are erythematous, maculopapular,

Fig. 16.10 Giant pronormoblast (50 m in diameter) with deeply basophilic cytoplasm, fine chromatin, and a prominent large nucleolus in a 6-year-old girl with ALL and parvovirus B19 infection. (See color plate 16.10 for full-color reproduction.)

similar to atopic eczema, and more prominent on the face (Fig. 16.9). Children with a history of atopy appear to have a higher incidence of this complication. Topical administration of simple emollients or a weak steroid preparation, and avoidance of extended exposure to sunlight should improve the skin condition. The rashes generally improve dramatically during pulse therapy with a glucocorticoid and vincristine. It should be noted that the skin rashes can become worse during the first 3 months after completion of treatment.310 It is prudent to warn parents of this possible effect and reassure them of its benign and self-limited nature. Transient or chronic anemia, with or without thrombocytopenia or neutropenia, can arise from parvovirus infection. The typical skin lesions (“slapped cheeks” exanthem and a lacy macular rash on the thighs) are often absent during immunosuppressive therapy. While immunologic testing may be uninformative because of delayed antibody responses, PCR detection of parvovirus is diagnostic. Detection of typical morphologic abnormalities in the erythrocytic precursors − giant pronormoblasts (Fig. 16.10) − should lead to a presumptive diagnosis of parvovirus infection.311 Some case reports suggest that treatment with immunoglobulin shortens the duration of viremia and symptoms, but there have been no randomized, controlled studies to confirm this impression.311

Allogeneic hemopoietic stem cell transplantation Many advances have been made in stem cell transplantation, such as prevention of graft-versus-host disease, expansion of the pool of suitable unrelated or related

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donors, acceleration of engraftment, enhancement of the graft-versus-leukemia effect, and supportive care. Such topics are discussed in Chapter 23. Because improvements in transplantation tend to parallel those in chemotherapy, the indications for transplantation in newly diagnosed and relapsed patients should be re-evaluated periodically. At present, Philadelphia-chromosome-positive ALL, failure to enter morphologic remission after 4 to 6 weeks of induction therapy and early hematological relapse are clear indications for transplantation,23,126,253 but this procedure has not been shown to improve outcome in other types of very high-risk leukemia, including infant ALL with MLL rearrangements.127,128

Treatment considerations for specific entities Certain subgroups of patients with ALL have unique responses to treatment and deserve special consideration during the formulation of clinical management plans. Only a few larger subgroups are discussed here; all others are reviewed in Chapter 13.

Infant ALL Infant ALL accounts for 2.5% to 5% of the childhood lymphoid leukemias.312 Rearrangements of the MLL gene on chromosome 11q23 are the most common genetic abnormalities, occurring in 70% to 80% of these cases.21 The t(4;11) with MLL-AF4 fusion is by far the most prevalent subtype of 11q23/MLL rearrangements.21 The prognosis of infants with ALL is determined by the presence or absence of certain presenting features. A very young age (especially neonates), high initial leukocyte count, lack of CD10 expression, myeloid-associated antigen expression, any 11q23/MLL rearrangement, and a poor early response to therapy predict an especially poor outcome.127,128,136,313–316 These high-risk factors are closely related to each other; although in different multivariate analyses, an 11q23/MLL rearrangement, a very young age (<3 months) and a poor early response consistently emerged as the most important adverse prognostic indicators.128,313,314 The increased in vitro sensitivity of leukemic blast cells from infants to cytarabine suggested that added clinical benefit might be obtained from chemotherapy regimens based on this agent.317 Investigators of the DanaFarber Cancer Institute Consortium reported an improved outcome in infants treated with intensified therapy that included high-dose cytarabine.136 Two large ongoing international studies of infant ALL (Children’s Oncology Group

and Interfant) are evaluating the efficacy of different intensified therapy that also included high-dose cytarabine. Although the preliminary results of one study appear promising, with a 2-year event-free survival of 60% in infants with MLL rearrangements,318 it remains to be seen if these intensified regimens will improve the historically dismal outlook for infants. Even though infant cases have a high incidence of CNS leukemia at diagnosis, adequate control of CNS disease can be achieved with the use of intensive systemic and intrathecal treatment only; cranial irradiation can be omitted in all infant cases, including those with CNS leukemia at diagnosis.316 Because of its poor prognosis, allogeneic hematopoietic stem cell transplantation has been advocated for infant ALL, in particular for cases with 11q23/MLL rearrangements. However, transplantations performed in the 1990s did not improve clinical outcome, largely due to the failure to eradicate drug-resistant residual leukemic cells.127,128 One study suggested that early introduction of transplantation with a less toxic conditioning regimen may improve outcome.319 Among the new antileukemic agents now under development, FLT-3-targeted tyrosine kinase inhibitors may be useful in these patients,150,320 as recent studies have demonstrated high levels of FLT-3 expression in cases with a rearranged MLL gene.148,149

ALL in children with Down syndrome Children with Down syndrome have a 10- to 20-fold increased risk of developing either ALL or AML. Before the age of 5 years, the risk for AML is four times higher than that for ALL; afterwards, ALL is the predominant leukemia.321 Compared to the general population of childhood ALL patients, Down syndrome cases are more likely to have a pre-B immunophenotype and less likely to have hyperdiploidy, TEL-AML1 fusion, a T-cell immunophenotype, or CNS leukemia at diagnosis.322,323 There is a notable absence of common chromosomal translocations with adverse prognosis, such as the t(1;19), t(9;22) or t(4;11), among patients with Down syndrome.322,323 The results of in vitro cellular drug-sensitivity testing are similar between ALL patients with or without Down syndrome.324 The outcome of contemporary therapy in Down syndrome patients is similar to that in other patients, but they experience more toxicities due to excessive mucositis, myelosuppression and infection.298,323,325 It is well recognized that they tolerate methotrexate poorly due to delayed clearance of the drug and depleted endogenous folate stores.297,298,326 The increased dosage of reduced folate carrier gene, located on chromosome 21, may also contribute to the increased accumulation of methotrexate

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polyglutamates and hence to methotrexate-induced toxicity.327 Successful treatment of children with Down syndrome remains a daunting challenge.

Mixed-lineage leukemia Aberrant expression of myeloid-associated antigens on otherwise typical lymphoblasts, and vice versa, has no prognostic or therapeutic relevance in the context of contemporary treatment regimens.102–105 The only exceptions are cases with co-expression of lymphoid-associated antigens (CD2, CD7 and cytoplasmic CD3) and myeloidassociated antigens (CD13, CD33) as well as low myeloperoxidase positivity (3–25%).102 Morphologically, such cases appear to have two cell populations: large blasts with myeloperoxidase positivity and small blasts with handmirror morphology. When treated with AML-directed therapy, half of these patients become induction failures, but virtually all of this subgroup achieve remission after treatment with prednisone, vincristine and asparaginase102 and remain in long-term disease-free survival (C.-H. Pui, unpublished observation).

Summary and future directions Although modifications of existing treatment protocols may well boost event-free survival rates to as high as 90%, the trend toward increasingly more aggressive therapy will likely reach a point of diminishing returns, where any therapeutic benefits will be outweighed by damage to normal vital tissues. The hope for the future is the development of therapies that take greater advantage of fundamental biological processes. Progress in the molecular characterization of ALL, through wider use of DNA and microRNA expression profiling, 33,34,97,141–144,328 coupled with methods to assess the functional significance of suggestive gene expression profiles,329 or through proteomic techniques,36,37 will almost certainly lead to the identification of new targets for specific treatments. A clear precedent is imatinib mesylate for the treatment of BCR-ABL-positive chronic myeloid leukemia.330 This agent, which inhibits the BCR-ABL fusion protein and other constitutively active tyrosine kinases, has induced transient remissions or consolidated remissions of BCR-ABL-positive ALL,331–333 an effect that may be enhanced by the use of small interfering RNAs which target the BCR-ABL gene.334 In fact, a small subset of T-cell ALL with extra chromosomal amplification of ABL1 could also benefit from this drug.335 Thus, imatinib is regarded as the forerunner of a new generation of molecularly targeted anticancer drugs. Other potentially useful

agents still in a developmental phase include inhibitors of FLT-3 tyrosine kinases for use against leukemias containing activating mutations of this kinase, and inhibitors of histone deacetylase for leukemias such as TEL-AML1positive ALL.336,337 Recent advances in immunology could also lead to effective adoptive cellular immunotherapy.338 More detailed information on the prospects for antibody therapy, immunotherapy and gene therapy in the management of childhood ALL are discussed in Chapters 25, 26, and 27, respectively. Further refinements in the molecular classification of ALL, together with the identification of pharmacogenetic features that affect the efficacy and toxicity of antileukemic therapy, will afford unique opportunities to devise treatment plans for individual patients. The ultimate challenge is to extend our therapeutic advances so that they benefit all leukemia-stricken children of the world.339,341

REFERENCES 1 Jemal, A., Murray, T., Samuels, A., et al. Cancer statistics 2003. CA Cancer J Clin 2003; 53: 5–26. 2 Pui, C.-H., Relling, M. V., & Downing, J. R. Acute lymphoblastic leukemia. N Engl J Med, 2004; 350: 1535–48. 3 Pui, C.-H., Raskind, W. H., Kitchingman, G. R., et al. Clonal analysis of childhood acute lymphoblastic leukemia with “cytogenetically independent” cell populations. J Clin Invest, 1989; 83: 1971–7. 4 Gale, R. E. & Wainscoat, J. S. Annotation: clonal analysis using X-linked DNA polymorphisms. Br J Haematol, 1993; 85: 2–8. 5 Waldmann, T. A., Davis, M. M., Bongiovanni, K. F., et al. Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms. N Engl J Med, 1985; 313: 776–83. 6 Raskind, W. H. & Fialkow, P. J. The use of cell markers in the study of human hematopoietic neoplasia. Adv Cancer Res, 1987; 49: 127–67. 7 Gilliland, D. G. & Tallman, M. S. Focus on acute leukemias. Cancer Cell, 2002; 1: 417–20. 8 Ernst, P., Wang, J., & Korsmeyer, S. J. The role of MLL in hematopoiesis and leukemia. Curr Opin Hematol, 2002; 9: 282–7. 9 Speck, N. A. & Gilliland, D. G. Core-binding factors in haematopoiesis and leukaemia. Nat Rev Cancer, 2002; 2: 502– 13. 10 Sherr, C. J. & McCormick, F. The RB and p53 pathways in cancer. Cancer Cell, 2002; 2: 103–12. 11 Krug, U., Ganser, A., & Koeffler, H. P. Tumor suppressor genes in normal and malignant hematopoiesis. Oncogene, 2002; 21: 3475–95. 12 Chau, B. N. & Wang, J. Y. Coordinated regulation of life and death by RB. Nat Rev Cancer, 2003; 3: 130–8. 13 Greaves, M., Maia, A. T., Wiemels, J. L., & Ford, A. M. Leukemia in twins: lessons in natural history. Blood, 2003; 102: 2321–33.

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268 Sullivan, M. P., Chen, T., Dyment, P. G., et al. Equivalence of intrathecal chemotherapy and radiotherapy as central nervous system prophylaxis in children with acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood, 1982; 60: 948–58. 269 Pui, C.-H., Sandlund, J. T., Pei, D., et al. Improved outcome for children with acute lymphoblastic leukemia: results of Total Therapy Study XIIIB at St. Jude Children’s Research Hospital. Blood, 2004; 104: 2690–6. 270 Stork, L. C., Sather, H., Hutchinson, R. J., et al. Comparison of mercaptopurine (MP) with thioguanine (TG) and IT methotrexate (ITM) with IT “Triples” (ITT) in children with SR-ALL: results of CCG-1952. Blood, 2002; 100: 156a. 271 Pui, C.-H., Cheng, C., Leung, W., et al. Extended followup of long-term survivors of childhood acute lymphoblastic leukemia. N Engl J Med, 2003; 349: 640–9. 272 Laver, J. H., Barredo, J. C., Amylon, M., et al. Effects of cranial radiation in children with high risk T cell acute lymphoblastic leukemia: a Pediatric Oncology Group report. Leukemia, 2000; 14: 369–73. 273 Manera, R., Ramirez, I., Mullins, J., & Pinkel, D. Pilot studies of species-specific chemotherapy of childhood acute lymphoblastic leukemia using genotype and immunophenotype. Leukemia, 2000; 14: 1354–61. 274 Ritchey, A. K., Pollock, B. H., Lauer, S. J., et al. Improved survival of children with isolated CNS relapse of acute lymphoblastic leukemia; a Pediatric Oncology Group Study. J Clin Oncol, 1999; 17: 3745–52. 275 Pui, C.-H., Relling, M. V., Sandlund, J. T., et al. Rationale and design of Total Therapy Study XV for newly diagnosed childhood acute lymphoblastic leukemia. Ann Hematol, 2004; 83(Suppl. 1):S124–6. 276 Waber, D. P., Silverman, L. B., Catania, L., et al. Outcomes of a randomized trial of hyperfractionated cranial radiation therapy for treatment of high-risk acute lymphoblastic leukemia: therapeutic efficacy and neurotoxicity. J Clin Oncol, 2004; 22: 2701–7. 277 Riehm, H., Gadner, H., Henze, G., et al. Results and significance of six randomized trials in four consecutive ALL-BFM studies. Hematol Blood Transfus, 1990; 33: 439–50. 278 Childhood ALL Collaborative Group. Duration and intensity of maintenance chemotherapy in acute lymphoblastic leukaemia: overview of 42 trials involving 12,000 randomized children. Lancet, 1996; 347: 1783–8. 279 Nesbit, M. E., Jr., Sather, H. N., Robison, L. L., et al. Randomized study of 3 years versus 5 years of chemotherapy in childhood acute lymphoblastic leukemia. J Clin Oncol, 1983; 1: 308–16. 280 Miller, D. R., Leikin, S. L., Albo, V. C., et al. Three versus five years of maintenance therapy are equivalent in childhood acute lymphoblastic leukemia: a report from the Children’s Cancer Study Group. J Clin Oncol, 1989; 7: 316–25. 281 Lennard, L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol, 1992; 43: 329–39.

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282 Teresi, M. E., Crom, W. R., Choi, K. E., et al. Methotrexate bioavailability after oral and intramuscular administration in children. J Pediatr, 1987; 110: 788–92. 283 Lennard, L. & Lilleyman, J. S. Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia. J Clin Oncol, 1989; 7: 1816–23. 284 Lennard, L., Lilleyman, J. S., Loon, J. van, et al. Clinical practice. Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet, 1990; 336: 225–9. 285 Schmiegelow, K., Schrøder, H., Gustafsson, G., et al. Risk of relapse in childhood acute lymphoblastic leukemia is related to RBC methotrexate and mercaptopurine metabolites during maintenance chemotherapy. J Clin Oncol, 1995; 13: 345–51. 286 Rivard, G. E., Infante-Rivard, C., Hoyoux, C., et al. Maintenance chemotherapy for childhood acute lymphoblastic leukaemia: better in the evening. Lancet, 1985; 2: 1264–6. 287 Rivard, G. E., Lin, K. T., Leclerc, J. M., & David, M. Milk could decrease the bioavailability of 6-mercaptopurine. Am J Pediatr Hematol Oncol, 1989; 11: 402–6. 288 Chessells, J. M., Harrison, G., Lilleyman, J. S., et al. Continuing (maintenance) therapy in lymphoblastic leukaemia: lessons from MRC UKALL X. Br J Haematol, 1997; 98: 945–51. 289 Evans, W. E., Horner, M., Chu, Y. Q., et al. Altered mercaptopurine metabolism, toxic effects and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia. J Pediatr, 1991; 119: 985–9. 290 Lennard, L., Gibson, B. E., Nicole, T., et al. Congenital thiopurine methyltransferase deficiency and 6-mercaptopurine toxicity during treatment for acute lymphoblastic leukemia. Arch Dis Child, 1993; 69: 577–9. 291 Stanulla, M., Schaeffeler, E., Flohr, T., et al. Thiopurine methyltransferase (TPMT) genotype and early treatment response to mercaptopurine in childhood acute lymphoblastic leukemia. JAMA, 2005; 293: 1485–9. 292 Yates, C. R., Krynetski, E. Y., Loennechen, T., et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med, 1997; 126: 608–14. 293 Stoneham, S., Lennard, L., Coen, P., Lilleyman, J., & Saha, V. Veno-occlusive disease in patients receiving thiopurines during maintenance therapy for childhood acute lymphoblastic leukaemia. Br J Haematol, 2003; 123: 100–2. 294 Winick, N., Bowman, W. P., & Kamen, B. A. Unexpected acute neurologic toxicity in the treatment of children with acute lymphoblastic leukemia. J Natl Cancer Inst, 1992; 84: 252–6. 295 Mahoney, D. H., Shuster, J. J., Nitschke, R., et al. Acute neurotoxicity in children with B-precursor acute lymphoid leukemia: an association with intermediate-dose intravenous methotrexate and intrathecal triple therapy-a Pediatric Oncology Group study. J Clin Oncol, 1998; 16: 1712–22. 296 Kalwinsky, D. K., Raimondi, S. C., Bunin, N. J., et al. Clinical and biological characteristics of acute lymphocytic leukemia

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in children with Down syndrome. Am J Med Genet Suppl, 1990; 7: 267–71. D¨ordelmann, M., Schrappe, M., Reiter, A., et al. Down’s syndrome in childhood acute lymphoblastic leukemia: clinical characteristics and treatment outcome in four consecutive BFM trials. Leukemia, 1998; 12: 645–51. Jackson, R. C. Biological effects of folic acid antagonists with antineoplastic activity. Pharmacol Ther, 1984; 25: 61–82. Baram, J., Allegra, C. J., Fine, R. L., et al. Effect of methotrexate on intracellular folate pools in purified myeloid precursor cells from normal human bone marrow. J Clin Invest, 1987; 79: 692–7. Kishi, S., Griener, J., Cheng, C., et al. Homocysteine, pharmacogenetics, and neurotoxicity in children with leukemia. J Clinic Oncol, 2003; 21: 3084–91. Mahoney, D. H., Jr., Camitta, B. M., Leventhal, B. G., et al. Repetitive low dose oral methotrexate and intravenous mercaptopurine treatment for patients with lower risk B-lineage acute lymphoblastic leukemia. A Pediatric Oncology Group Pilot study. Cancer, 1995; 75: 2623–31. Farrow, A. C., Buchanan, G. R., Zwiener, R. J., Bowman, W. P., & Winick, N. J. Serum aminotransferase elevation during and following treatment of childhood acute lymphoblastic leukemia. J Clin Oncol, 1997; 15: 1560–6. Nygaard, U., Toft, N., & Schmiegelow, K. Methylated metabolites of 6-mercaptopurine are associated with hepatotoxicity. Clin Pharmacol Ther, 2004; 75: 274–81. Bleyer, W. A., Sather, H. N., Nickerson, H. J., et al. Monthly pulses of vincristine and prednisone prevent bone marrow and testicular relapse in low-risk childhood acute lymphoblastic leukemia: a report of the CCG-161 study by the Children’s Cancer Study Group. J Clin Oncol, 1991; 9: 1012– 21. Mattano, L. A., Jr., Sather, H. N., Trigg, M. E., & Nachman, J. B. Osteonecrosis as a complication of treating acute lymphoblastic leukemia in children: a report from the Children’s Cancer Group. J Clin Oncol, 2000; 18: 3262–72. Kaste, S. C., Jones-Wallace, D., Rose, S. R., et al. Bone mineral decrements in survivors of childhood acute lymphoblastic leukemia: frequency of occurrence and risk factors for their development. Leukemia, 2001; 15: 728–34. Strauss, A. J., Su, J. T., Kimball, Dalton, V. M., et al. Bony morbidity in children treated for acute lymphoblastic leukemia. J Clin Oncol, 2001; 19: 3066–72. Mattano, L. A., Sather, H. N., La, M. K., et al. Modified dexamethasone (DXM) reduces the incidence of treatment-related osteonecrosis (ON) in children and adolescents with higher risk acute lymphoblastic leukemia (HR ALL): a report of CCG1961. Blood, 2003; 102: 221a. Relling, M. V., Yang, W., Das, S., et al. Pharmacogenetics risk factors for osteonecrosis of the hip among children with leukemia. J Clin Oncol, 2004; 22: 3930–6. Shaw, N. J. & Eden, O. B. Skin rash after completion of therapy for leukemia in childhood. Pediatr Hematol Oncol, 1989; 6: 31–5.

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311 McNall, R. Y., Head, D. R., Pui, C.-H., & Razzouk, B. I. Parvovirus B19 infection in a child with acute lymphoblastic leukemia during induction therapy. J Pediatr Hematol Oncol, 2001; 23: 309–11. 312 Pui, C.-H., Kane, J. R., & Crist, W. M. Biology and treatment of infant leukemias. Leukemia, 1995; 9: 762–9. 313 Pui, C.-H., Ribeiro, R. C., Campana, D. C., et al. Prognostic factors in the acute lymphoid and myeloid leukemias of infants. Leukemia, 1996; 10: 952–6. 314 D¨ordelmann, M., Reiter, A., Borkhardt, A., et al. Prednisone response is the strongest predictor of treatment outcome in infant acute lymphoblastic leukemia. Blood, 1999; 94: 1209– 17. 315 Chessells, J. M., Harrison, C. J., Kempski, H., et al. Clinical features, cytogenetics and outcome in acute lymphoblastic and myeloid leukaemia of infancy: report from the MRC childhood leukaemia working party. Leukemia, 2002; 16: 776–84. 316 Isaacs, H. Jr. Fetal and neonatal leukemia. J Pediatr Hematol Oncol., 2003; 25: 348–61. 317 Pieters, R., Boer, M. L. den, Durian, M., et al. Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia – implications for treatment of infants. Leukemia, 1998; 12: 1344–8. 318 Dreyer, Z. E., Steuber, C. P., Bowman, W. P., et al. High risk infant ALL-improved survival with intensive chemotherapy. Proc Am Soc Clin Oncol, 1998; 17: 529a. 319 Kosaka, Y., Koh, K., Kinukawa, N., et al. Infant acute lymphoblastic leukemia with MLL gene rearrangements: outcome following intensive chemotherapy and hematopoietic stem cell transplantation. Blood, 2004; 104: 3527–34. 320 Sawyers, C. L. Finding the next Gleevec: FLT3 targeted kinase inhibitor therapy for acute myeloid leukemia. Cancer Cell, 2002; 1: 413–15. 321 Hasle, H., Clemmensen, I. H., & Mikkelsen, M. Risks of leukaemia and solid tumours in individuals with Down’s syndrome. Lancet, 2000; 355: 165–9. 322 Pui, C.-H., Raimondi, S. C., Borowitz, M. J., et al. Immunophenotypes and karyotypes of leukemic cells in children with Down syndrome and acute lymphoblastic leukemia. J Clin Oncol, 1993; 11: 1361–7. 323 Zeller, B., Gustafsson, G., Forestier, E., et al. Acute leukaemia in children with Down syndrome: a population-based Nordic study. Br J Haematol, 2005; 128: 797–804. 324 Zwaan, C. M., Kaspers, G. J. L., Pieters, R., et al. Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome. Blood, 2002; 99: 245–51. 325 Lange, B. The management of neoplastic disorders of haematopoiesis in children with Down’s syndrome. Br J Haematol, 2000; 110: 512–24.

326 Ueland, P. M., Refsum, H., & Christensen, B. Methotrexate sensitivity in DS: a hypothesis. Cancer Chemother Pharmacol, 1990; 25: 384–6. 327 Belkov, V. M., Krynetski, E. Y., Scheutz, J. D., et al. Reduced folate carrier expression in acute lymphoblastic leukemia: a mechanism for ploidy but not lineage differences in methotrexate accumulation. Blood, 1999; 93: 1643–50. 328 Lu, J., Getz, G., & Miska, E. A., et al. MicroRNA expression profiles classify human cancers. Nature, 2005; 435: 834–8. 329 McManus, M. T. & Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002; 3: 737–47. 330 O’Brien, S. G., Guilhot, F., Larson, R. A., 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. 331 Druker, B. J., Sawyers, C. L., Kantarjian, H., et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med, 2001; 344: 1038–42. 332 Lee, S., Kim, D. W., Kim, Y. J., et al. Minimal residual disease-based role of imatinib as a first-line interim therapy prior to allogeneic stem cell transplantation in Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood, 2003; 102: 3068–70. 333 Thomas, D. A., Faderl, S., Cortes, J., et al. Treatment of Philadelphia chromosome-positive acute lymphocytic leukemia with hyper-CVAD and imatinib mesylate. Blood, 2004; 103: 4396– 407. 334 Wohlbold, L., Kuip, H. van der, Miething, C., et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib meslyate (STI571). Blood, 2003; 102: 2236–9. 335 Graux, C., Cools, J., Melotte, C., et al. Fusion of NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia. Nat Genet, 2004; 10: 1084–9. 336 Batova, A., Shao, L. E., Dicciani, M. B., et al. The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia and drug-resistant primary leukemia and cancer cells lines. Blood, 2002; 100: 3319–24. 337 Melnick, A. & Licht, J. D. Histone deacetylase as therapeutic targets in hematologic malignancies. Curr Opin Hematol, 2002; 9: 322–32. 338 Imai, C., Iwamoto, S., & Campana, D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood, 2005; 106: 376– 83. 339 Howard, S. C., Pedrosa, M., Lins, M., et al. Establishment of a pediatric oncology program and outcomes of childhood acute lymphoblastic leukemia in a resource-poor area. JAMA, 2004; 291: 2471–5. 340 Pui, C.-H. & Ribeiro, R. C. International collaboration on childhood leukemia. Int J Hematol, 2003; 78: 383–9.

17 Relapsed acute lymphoblastic leukemia ¨ Gunter Henze and Arend von Stackelberg

Introduction With current treatment, event-free survival rates in acute lymphoblastic leukemia (ALL) are about 75%. Therefore, relapse of ALL is still frequent with an incidence range close to that of neuroblastoma.1,2 Problems in the management of ALL relapse are the resistance of the leukemic cells and the reduced tolerance of patients to a second round of treatment after having already received intensive frontline therapy, resulting in a lower remission rate as well as a higher incidence of subsequent relapse and an inferior outcome overall. Intensified polychemotherapy is essential for induction of a second complete remission (CR). Depending on a variety of prognostic factors, remission may be maintained with chemotherapy and cranial irradiation alone or with intensification of treatment by stem cell transplantation.

Diagnosis of relapse The diagnosis of ALL relapse (i.e. the reappearance of leukemic cells in any anatomic compartment following CR) must be unequivocal. The work-up includes a careful physical examination as well as investigations of the bone marrow (BM), the cerebrospinal fluid (CSF) and, if necessary, biopsies of other involved sites (e.g. the testicles, lymph nodes or any other organs or tissues). As at initial diagnosis, the leukemic cells have to be characterized morphologically and by immunophenotyping, as well as by cytogenetic and molecular genetic procedures. Only this comprehensive information, together with clinical findings, allows one to classify the leukemic subtype adequately and to assess the prognosis of individual patients.

For morphological investigation, peripheral blood and BM smears have to be stained according to Pappenheim or Wright, and the cells are classified by light microscopy according to criteria of the French-AmericanBritish (FAB) Cooperative Group.3,4 An isolated BM relapse of ALL requires the presence of at least 25% lymphoblastic leukemic cells in the BM smear, without evidence of extramedullary leukemia. The most common sites of extramedullary manifestations are the central nervous system (CNS) and the testicles. CNS relapse is diagnosed if the CSF contains at least 5 white blood cells (WBCs) per microliter with blast cell morphology apparent in a cytospin preparation. A clear blast cell pleocytosis is common. Lymphatic pleocytosis with single suspicious cells is not sufficient for the diagnosis of CNS relapse, but this finding does warrant a repeated lumbar puncture after 1 or 2 weeks. MRI testing may be necessary to confirm or rule out CNS leukemia. A testicular relapse is characterized by unilateral or bilateral painless testicular enlargement. It has to be confirmed by biopsy (or orchiectomy) of the involved testicle(s), and involvement of the contralateral testicle, if not enlarged, has to be excluded. Less frequently, a variety of other extramedullary sites may be involved, such as skin, bone and muscle, abdominal organs, or the eye. Extramedullary relapses may be “isolated” (<5% BM blasts) or combined (≥5% BM blasts). Immunophenotyping of cells by flow cytometry and staining with a standard panel of B-cell, T-cell and myeloid markers is necessary to confirm the relapse diagnosis and to discriminate between B-cell precursor and T-cell ALL. Genetic analyses of the leukemic cells at relapse should follow the same guidelines as applied at the first

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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manifestation of ALL. Besides quantitative DNA analysis for ploidy (DNA index) and standard cytogenetic analysis,the cells should be screened for relevant translocations, such as the t(9;22), t(12;21), and t(4;11), by molecular genetic methods. Furthermore, clone-specific rearrangements of T-cell receptor and immunoglobulin genes can be used as markers to monitor patients for minimal residual disease.5 Genetic analyses are helpful in confirming the clonal stability of the leukemic cells and thus in discriminating relapses from secondary leukemias.6,7

Prognostic factors Well-established prognostic factors in relapsed ALL are time from CR induction to relapse, site of relapse and immunophenotype as well as the translocation t(9;22) or its molecular-genetic equivalent, BCR-ABL. Additionally, the leukemic tumor burden as measured by the peripheral blast cell count has some prognostic implications in relapsed ALL. As in newly diagnosed ALL, the dynamics of the response to treatment has been shown to be one of the most meaningful determinants of outcome. The role of the translocation t(12;21), corresponding to the fusion transcript TEL-AML1, remains to be determined. Likewise, the prognostic importance of in vitro drug resistance analyses awaits confirmation by ongoing or future trials. The most evident prognostic factor is the time to relapse,2,8 a term that is not uniformly defined among different study groups. According to some groups, time to relapse is related to the duration of remission after elective cessation of frontline treatment,9 since the length of antileukemic treatment is itself a major prognostic criterion and prolonged maintenance therapy can delay relapse.10,11 Other groups consider the duration of first CR to be independent of the duration of frontline therapy. These different definitions must be taken into account when the results of clinical trials are compared with respect to time to relapse.1 According to experience gained from systematic consecutive trials, the BFM Study Group defined time to relapse as very early (within 18 months after initial diagnosis), early (beyond 18 months after initial diagnosis and up to 6 months after the cessation of frontline treatment) and late (beyond 6 months after the cessation of frontline treatment). The Kaplan-Meier plots for the event-free survival of 910 patients treated between 1983 and 1997 in trials ALLREZ BFM 83, 85, 87, 90, and 95 according to these categories are shown in Fig. 17.1. An early relapse is associated with a higher rate of nonresponse to treatment, a shorter duration of second CR, and a lower event-free survival rate. Similar results have been described by others.2,12,13

Fig. 17.1 Event-free survival probability (pEFS) for children with ALL in relapse according to time to relapse (SCT censored; P < 0.001 by log-rank test). The patients were treated in trials ALL-REZ BFM 83–95. Late: n = 467, censored = 249, pEFS = 0.40 ± 0.03. Early: n = 301, censored = 123, pEFS = 0.20 ± 0.03. Very early: n = 142, censored = 41, pEFS = 0.13 ± 0.04.

Children with extramedullary relapses have a better prognosis compared to those with an isolated BM relapse. In part, extramedullary relapses are likely to originate from leukemic cells having survived frontline treatment in an extracompartmental sanctuary, where they might have been insufficiently exposed to chemotherapy with the consequence of drug resistance. The microenvironment of the CNS and the testes support a slow growth rate of leukemic cells and protect vulnerable cells from external influences.14–16 Hence, manifestations of leukemia in such sanctuaries require specific local treatment. Subsequent systemic relapses occur frequently, demonstrating that the term “isolated” relapse does not readily apply to recurrent leukemia of this type.2,11,17–19 Indeed, sensitive methods such as multiparameter flow cytometry or molecular genetics can detect the presence of occult BM involvement in children with cytologically “isolated” extramedullary relapse.20–22 Therefore, additional intensive systemic treatment is required. The prognostic relevance of occult BM leukemia in cases of an “isolated” extramedullary relapse remains to be determined by prospective evaluation. Interestingly, children with a combined BM relapse have been reported to have a superior prognosis compared to that of children with an isolated BM relapse (Fig. 17.2).23 This result cannot be explained either by a generally longer duration of first remission in patients with combined BM relapse2,24 or by a lower degree of concomitant leukemic metaplasia in the BM. The most likely explanation is that in combined relapses, the BM blasts derive from leukemic cells that have reseeded the marrow from

Relapsed acute lymphoblastic leukemia

Table 17.1 Frequencies of immunophenotypes in children with a

Table 17.2 Multivariate Cox regression analysis of event-free survival

first ALL relapsea

according to relapse categorya,b

Lineage T-cell

B-cell precursor

Biphenotypic No data Total

Maturity Pre-T Intermediate-T Mature-T Pro-B CALLA Pre-B

n

%

34 53 79 79 648 233 3 78

3.0 4.7 7.0 7.0 57.4 20.6 0.3 (6.4)

1207

100

Abbreviation: CALLA, common ALL antigen. a Data from trials ALL-REZ BFM 90, 95 and 96, including pilot studies.

Risk ratio 95% CI

Parameter

Category

Time point

Late Early Very early Isolated extramedullary Combined Isolated BM B-cell precursor (Pre-)T cell

Site

Immunophenotype

475

P

1 3.7 5.9 1

(Reference group) 3.0–4.5 <0.001 4.7–7.4 <0.001 (Reference group)

2.0 3.5 1 2.3

1.5–2.7 <0.001 2.7–4.5 <0.001 (Reference group) 1.8–2.8 <0.001

Abbreviations: CI, confidence interval; BM, bone marrow. a Cox regression model: n = 1102; 642 events; 2 (Wald) = 443.7; P < 0.001. b Survival data from studies ALL-REZ BFM 83–95.

Table 17.3 Stratification groups S1–S4 of trial ALL-REZ BFM 96 defined by the prognostic factors time, site and immunophenotype of relapse B-cell precursor

Time Very early Early Late

(Pre-)T cell

ExtraCombined Isolated ExtraCombined Isolated medullary BM BM medullary BM BM IR

HR

HR

IR

HR

HR

IR SR

IR IR

HR IR

IR SR

HR HR

HR HR

Abbreviations: BM, bone marrow; IR, intermediate risk; SR, standard risk; HR, high risk. Fig. 17.2 Event-free survival probability (pEFS) for children with ALL in relapse according to site of relapse (SCT censored; P < 0.001 by log-rank test). The patients were treated in trials ALL-REZ BFM 83–95. Isolated extramedullary: n = 159; censored = 84, pEFS = 0.47 ± 0.04. Combined: n = 221, censored = 112, pEFS = 0.40 ± 0.04. Isolated bone marrow: n = 530, censored = 217, pEFS = 0.19 ± 0.02.

an extramedullary compartment rather than representing the original site of systemic relapse.25 According to trials ALL-REZ BFM 90-96, including pilot studies, about 14.7% of children with relapses have T-cell ALL (Table 17.1), a known adverse prognostic factor.8,26,27 Frequently, relapses of T-cell ALL occur early, and the rate of nonresponse to salvage treatment is high, suggesting that the cells are highly drug resistant. The duration of second CR, if achieved, is very short. The stage of maturation of the T-cell and B-cell precursor lineages, as well as the

presence of myeloid markers, has no additional prognostic relevance in relapsed ALL. The time to and site of relapse, as well as immunophenotype, are highly significant, independent prognostic factors in relapsed ALL (Table 17.2). Thus, patients can be divided into standard-(SR), intermediate-(IR) and high-risk (HR) groups (Table 17.3). In retrospective analyses, event-free survival (EFS) estimates range from prognostically favorable (SR group) to dismal (HR group) (Fig. 17.3). Patients in the IR group have an intermediate prognosis with an EFS rate of 35% at 6 years. The peripheral blast cell count (PBC) at diagnosis of relapse has been identified as an additional prognostic factor for children with late, isolated non-T-cell BM relapses, who represent the largest subgroup of IR patients. In children with a PBC exceeding 10 × 109 /L, the EFS rate at

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antileukemic treatment and, after another “second” hit, may emerge as an apparent relapse. Thus, there is evidence that such “relapses” may in fact be second leukemias.39,40 TEL-AML1+ leukemias tend to relapse after a long first CR, and even after “relapse” such patients have a significantly better prognosis than do patients without this marker.38 However, new relapses can occur, again after relatively long second CRs.41,42

Treatment of relapse

Fig. 17.3 Event-free survival probability (pEFS) for children with ALL in relapse according to risk-group assignment (SCT censored; P < 0.001 by log-rank test). The patients were treated in trials ALL-REZ BFM 83–95. Standard risk, n = 59; pEFS = 0.75 ± 0.06. Intermediate risk, n = 661, censored = 309, pEFS = 0.37 ± 0.02. High risk, n = 545, censored = 152, pEFS = 0.04 ± 0.01.

5 years is less than 20%, whereas children with no detectable blasts have a more favorable prognosis.28 The Philadelphia chromosome [i.e. the translocation t(9;22)] or its molecular equivalent, the fusion transcript BCR-ABL, can be detected in 2.3% to 3.6% of children and is associated with a poor prognosis and a high relapse rate.29–31 In relapsed ALL, about 10% of patients have BCR-ABLpositive disease32 which confers an extremely poor prognosis. Very often, a second CR cannot be induced, and the probability of EFS at 2 years is less than 10% after chemoradiotherapy alone. BCR-ABL positivity is associated with several adverse risk factors, such as short duration of first CR and high PBC counts; however, in analyses matching these cofactors, as well as in multivariate analyses, BCRABL expression proved to have independent adverse prognostic significance.32 The most frequent genetic aberration in childhood B-cell precursor ALL is the cryptic translocation t(12;21)(p13;q22) with the resulting fusion transcript TEL-AML. At first diagnosis, about 25% of patients have TEL-AML1-positive ALL, which is generally believed to predict a favorable outcome and a low relapse rate.33–35 However, widely different relapse rates of TEL-AML1-positive ALL have been reported so far.36–38 TEL-AML1 can be detected in 1% or 2% of normal cord blood samples and is thought to be a preleukemic genetic alteration requiring a second event in order to progress to true leukemia. Therefore, a preleukemic TEL-AML1-positive clone may persist after

Response to treatment at relapse is much inferior to that in cases of newly diagnosed ALL, in part because of primary drug resistance already present in subclones at initial diagnosis43 or acquired resistance developing as a consequence of exposure to antileukemic drugs.44 The wellknown mechanism of multiple drug resistance (MDR), regulated by P-glycoprotein, is not likely to play a major role in the development of drug resistant ALL. Other potential mechanisms for resistance, such as the expression of glutathione transferase and metallothionein, are described in Chapter 15 . More specific resistance mechanisms, such as an increased activity of dihydrofolate reductase (DHFR), impaired methotrexate (MTX) membrane transport and impaired MTX polyglutamylation, could be responsible for decreased remission rates. The role of the tumor suppressor gene p53 in childhood ALL remains controversial. Some authors found an association between p53 mutations and a poor prognosis at first diagnosis.45 In primary ALL, it is associated with early relapse and anthracycline resistance.46–48 New p53 mutations have been identified at ALL relapse, but lacked prognostic impact.49 The drug resistance of ALL cells can be assessed with the methyl-thiazol-tetrazolium (MTT) assay, which measures the viability of leukemic cells after exposure to a panel of antileukemic drugs in vitro. A significantly higher resistance to glucocorticoids, L-asparaginase, anthracyclines and thiopurines has been observed at relapse compared with first diagnosis of childhood ALL.44 The most prominent differences have been observed for glucocorticoids. Resistance to glucocorticoids is linked to the quantity and function of the glucocorticoid receptor and to postreceptor pathways leading to the induction of apoptosis.50 Since the early 1970s, prospective attempts have been made to treat children with relapse of ALL. Although second remissions could be induced, the remission rates and the duration of subsequent remission have been unsatisfactory. Obviously, for cells that had survived the first round of chemotherapy, more intensive treatment was needed

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Table 17.4 Treatment results of different study groups for children with ALL in relapse

Group

Author

Protocol

Site and time of relapse

Number of patients

EFS/DFS rate

ALL-REZ BFM

Henze et al., 199459

ALL-REZ BFM 83/85/87 ALL-REZ BFM 83/85/87/90 ALL-REZ BFM 83/85/87/90

BM, earlya

146

DFS = 9%

BM, latea CNS, isolated Testis, isolated

183 73 59

DFS = 39% EFS = 42% EFS = 53%

Henze et al., 19971 Wolfrom et al., 199777 CCG

Gaynon et al., 19982

CCG 100 series 1983–9

BM, earlyb BM, intermediateb BM, lateb CNS, isolated Testis, isolated

267 220 275 220 112

DFS = 5–9% DFS = 10–11% DFS = 33–48% DFS = 37% DFS = 64%

MRC/UKALL

Wheeler et al., 199813

UKALL X, 1985–93

Lawson et al., 2000116

MRC/UKALL, R1 1991–9

UKALL, 1972–87

BM, earlyc BM, intermediatec BM, latec BM, earlyc BM, intermediatec BM, lateb CNS, isolated Testis, isolated

106 57 169 29 39 119 26 33

DFS = 0–11% DFS = 14–40% DFS = 33–50% DFS = 0–5% DFS = 25–41% DFS = 51–81% DFS = 58% EFS = 59%

POG 8303, 1982–7 POG 8304, 1983–9 POG 9061, 1990–3 POG 8304, 1983–9

BM, earlya BM, latea CNS, isolated Testis, isolated

297 105 83 80

DFS = 8% EFS = 37% EFS = 70% EFS = 53–84%

Grundy et al., 199779 POG

69

Buchanan et al., 2000 Sadowitz et al., 199361 Ritchey et al., 199972 Wofford et al., 199281

Abbreviations: BM, bone marrow; EFS, event-free survival; DFS, disease-free survival; CNS, central nervous system; BFM, Berlin-Frankfurt¨ Munster; CCG, Children’s Cancer Study Group; MRC, Medical Research Council; POG, Pediatric Oncology Study Group; UKALL, United Kingdom ALL Study Group. Definitions of time to relapse: a Early, <6 months; intermediate, none; late >6 months after end of frontline therapy. b Early, <18 months; intermediate, 18–36 months; late, >36 months after initial diagnosis. c Early, <24 months; intermediate, 24–36 months; late, >36 months after initial diagnosis.

to overcome drug resistance.51–54 Despite the increasing intensity of induction and postremission therapy, the rate of subsequent relapses was high. Thus, stem cell transplantation has been introduced as a highly intensive element for postremission treatment.55,56 Published data on the treatment of children with relapsed ALL are difficult to compare. One reason is that the intensity of frontline protocols, which seems to play an important role in the outcome of relapse therapy, has increased over time, and more recent relapse trials have enrolled very heavily pretreated patients compared to those studied in the past. Moreover, the different approaches to risk stratification taken by the various leukemia study groups further complicate the comparison of treatment results. Finally, many of the published studies comprise only low number of patients, and salvage

treatments at single centers frequently reflect a variety of individual and experimental approaches. Table 17.4 summarizes some representative treatment results from different study groups.

Reinduction therapy for bone marrow relapse Remission rates for children with BM relapses range from 75% to 100%, depending on the time to relapse and size of the patient cohort.10,57–65 Reinduction therapy consists mostly of a 4-week regimen of prednisone, vincristine, and an anthracycline, often supplemented by L-asparaginase. In a large study of the Pediatric Oncology Group (POG), the remisson rate was 83% in patients with early BM relapse (i.e. before 6 months after completion of frontline therapy) who were treated on a 4-week schedule of prednisone,

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vincristine, daunorubicin and L-asparaginase supplemented by triple intrathecal therapy.66 The BFM Relapse Study Group used a more intensive reinduction treatment for children with early BM relapse, resulting in a remission rate of 77%. The protocol consisted of a 4-week schedule of prednisone, vincristine, L-asparaginase, intermediateor high-dose MTX, and high-dose cytarabine, followed by two alternating 5- to 8-day multidrug courses, R1 and R2, containing glucocorticoids, thiopurines, vinca alkaloids, epipodophyllotoxins, oxazaphosphorines, intermediateor high-dose MTX, daunorubicin, cytarabine and intrathecal MTX.11,59 Other groups have published comparable results of reinduction therapy using different but mostly less intensive regimens.58 In children with late BM relapse (i.e. more than 6 months after cessation of frontline therapy), higher remission rates can be achieved. The reinduction schedule of the POG 8304 protocol consisted of a 4-week course of prednisone, vincristine and daunorubicin. The protocols ALL-REZ BFM 83–87 started reinduction treatment directly with the more intensive multiagent courses, R1 and R2. Again, remission rates were comparable: 97% in the POG series and 94% in the BFM series.59,61 Similar results have been reported by other authors in smaller patient groups using reinduction regimens of variable intensity.10,60 Until now, there has been no clear evidence that more intensive reinduction therapy for BM relapse of ALL leads to better remission rates. However, a recent analysis of trial ALL-REZ BFM 95/96 indicates that dose intensity of the reinduction therapy might have a significant influence on remission rates and outcome, as postulated by Hryniuk.67 Patients with a short time interval between the first two chemotherapy courses had a significantly better remission rate and EFS, compared to results for patients with longer intervals.68

Postremission chemotherapy A broad variety of drug combinations and schedules have been exploited as postremission chemotherapy. The POG performed postremission treatment of children with BM relapse on the basis of alternating drug pairs combined with reinduction courses. They consisted of weekly intermediate-dose cytarabine/teniposide alternating with weekly vincristine/cyclophosphamide, with or without standard reinduction courses for children with early BM relapse.69 For children with late BM relapse, alternating pairs of standard maintenance dose MTX/mercaptopurine and vincristine/cyclophosphamide have been used, interrupted by reinduction pulses with prednisone/doxorubicin or, as a randomized alternative,

with teniposide/cytarabine.61 Both protocols contained extended triple intrathecal therapy. Cranial or gonadal irradiation (24 or 26 Gy) were applied in cases of concomitant leukemic involvement at respective sites. In BFM relapse trials, postremission treatment for patients with early or late BM relapse consisted of alternating multidrug courses, R1 and R2, to a total of eight courses, followed by standard-dose MTX/6-thioguanine given for 2 years as maintenance therapy.11,59 Children with an isolated BM relapse received MTX intrathecally during the intensive multidrug courses only. In cases of concomitant CNS involvement, cranial irradiation was administered at the end of intensive treatment at a dose that depended on the previously applied radiation therapy (maximum dose, 24 Gy), and triple intrathecal therapy was continued throughout the first year of therapy. Patients with gonadal involvement received local irradiation at a dose of 24 Gy, if the involved testis had not been surgically removed. In trial ALL-REZ BFM 85 and during the first period of trial 87, an excess of CNS relapses following isolated (particularly late) BM relapse was observed. Therefore, preventive cranial irradiation was introduced for all children with isolated bone marrow relapse during the ALL-REZ BFM 87 study. As a consequence, the rate of CNS relapses in children who received preventive cranial irradiation was markedly reduced, and their outcome was significantly better than in those not treated with irradiation.70 For patients with early BM relapse, long-term results with chemotherapy have been disappointing. In both the POG and BFM Study Group, disease-free survival after 8 years was less than 10%.59,69 These unfavorable results are in agreement with other reports.2,12,13 By contrast, long-term survival rates in the range of 30% to 40% have been achieved with chemotherapy in children with late BM relapse.59–61 Despite many attempts to improve the prognosis for children with BM relapse of ALL, using intensive multiagent chemotherapy, it has not been possible to achieve a major breakthrough. It appears that the well-known prognostic factors identified at relapse of ALL, and probably also the intensity of the frontline protocol, are far more important than the details of the treatment design in determining clinical outcome after retreatment.

Treatment of CNS relapse With contemporary frontline therapy, CNS relapses have become rare events; however, the management of CNS relapse remains one of the major challenges in pediatric oncology because of the treatment-related adverse long-term sequelae. Besides local therapy, curative

Relapsed acute lymphoblastic leukemia

treatment approaches must include multiagent systemic therapy. Since an early trial of the POG in 1985, craniospinal irradiation was thought to be superior to cranial irradiation in children with CNS relapse because of a much higher (25% versus 55%) subsequent relapse rate following cranial irradiation only.71 The authors described a high rate of leukoencephalopathy after intrathecal and systemic chemotherapy following cranial irradiation and excessive toxicity from systemic chemotherapy following spinal irradiation. In the subsequent POG trial 8304, 120 children with isolated CNS relapse received early cranial irradiation at 24 Gy after standard induction therapy, followed by continuation treatment with rotating drug pairs and a late intensification. Standard triple intrathecal therapy was applied weekly during induction therapy and monthly throughout the remaining treatment. The overall EFS rate was 42% ± 8% at 5 years. The 17% rate of leukoencephalopathy associated with substantial acute and chronic neurotoxicity was remarkably high and led to discontinuation of therapy in some patients.18 In the recently published trial POG 9061,72 irradiation was deferred for 6 months to allow the delivery of maximally intensive systemic chemotherapy before craniospinal irradiation. After a standard induction regimen with weekly triple intrathecal therapy, patients received consolidation treatment that included two courses of high-dose cytarabine followed by L-asparaginase. Early intensification therapy consisted of four courses of intermediate-dose MTX (1 g/m2 per 24 hours), high-dose mercaptopurine (1 g/m2 ) as an 8-hour infusion alternating with four courses of etoposide (300 mg/m2 ) and cyclophosphamide (500 mg/m2 ). During consolidation and intensification treatment, triple intrathecal therapy was given at monthly intervals. During radiation therapy, systemic antileukemic treatment was administered with dexamethasone, vincristine and thrice-weekly L-asparaginase. Maintenance treatment after irradiation was of moderate intensity with standard mercaptopurine/MTX alternating with vincristine/cyclophosphamide reinduction courses. Eighty-three patients were included in the study. The overall EFS rate was 70% ± 6% at 5 years. For patients with a first remission duration of less than 18 months, the rate was 46% ± 12% compared with 81% ± 6% for those with a remission duration of more than 18 months. The rate of neurotoxicity, 6 (7%) of 83 patients, turned out to be remarkably reduced compared with results in prior trials and could be linked to intensive chemotherapy before radiation treatment in most cases.72 In two other large published series of patients with isolated CNS relapse, treated with a variety of regimens, the disease-free survival rate

was 37% ± 3% (CCG; n = 220)2 and the EFS rate ranged from 24% to 64% (UKALL; n = 98), depending on the duration of first remission.13 In the BFM relapse trials, patients with isolated extramedullary relapses have been uniformly treated with the same chemotherapy regimens used for systemic relapse, supplemented by local therapy. In one brief analysis including 73 patients with CNS relapse from several consecutive trials, the EFS rate was 42% ± 3% at 10 years.1 Chemotherapy consisted of the R1 and R2 courses, as for patients with BM relapse. However, the number of courses was restricted to four in trial ALL-REZ BFM 83 and to six in trials 85, 87 and 90. Furthermore, triple intrathecal therapy was intensified during the intensive treatment period and extended to 6 months of maintenance therapy. Irradiation was always given at the end of intensive therapy and administered at doses adapted to the previously used dose and age. Cranial or craniospinal irradiation was employed according to preferences of the participating medical centers. In retrospective analyses, no significant difference in EFS rates could be found between the two radiation modalities. In patients treated in trials ALL-REZ BFM 83–96, outcome according to time to relapse was similar to results reported by the UKALL group. Although recent results from the POG 9061 trial are superior to other data, no specific treatment elements could be identified as primarily responsible for the more favorable outcome. Indeed, most CNS protective agents, administered at even higher cumulative doses than in the POG trial, were included in the regimens of other groups, such as the BFM. One possible explanation might be the low number of patients with T-cell ALL: three (14%) in POG 9061 and four (3%) in POG 8304. The delay of irradiation to allow high-dose CNS effective chemotherapy seems to be a reasonable approach and is now employed in most trials. Postirradiation high-dose systemic chemotherapy should be avoided, and intrathecal therapy should be cautiously performed to prevent the occurrence of leukoencephalopathy. Craniospinal irradiation may be advantageous compared to cranial irradiation, but this impression awaits confirmation by conclusive trials based on contemporary high-dose systemic chemotherapy.

Treatment of testicular relapse After BM and the CNS, the testicles are the third most frequent site of relapse. Isolated overt testicular relapses occur significantly later than isolated CNS relapses and comprise a group of relapses with a comparably favorable outcome.21,73 Like any other relapse, an “isolated” testicular relapse has to be considered as a systemic disease.74–76

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In the case of cytologically overt BM involvement, treatment results are superior as compared to isolated BM relapse, suggesting that BM involvement is a consequence of reseeding by cells persisting in the testes that are still sensitive to chemotherapy.77 In most reports, local therapy consisted of bilateral irradiation of the testes. The optimal dose of testicular irradiation is unclear. Most authors recommend bilateral testicular irradiation at doses above 22 Gy,17,78–81 but persistent disease or subsequent local relapses have been reported after doses of 20 to 26 Gy in 5% to 7% of the patients.17,81,82 Severe gonadal dysfunction has been encountered after testicular irradiation at 24 Gy,83,84 in particular if it is given to younger boys.85 After doses of 12 or 15 Gy, Leydig cell function was sufficiently preserved to allow spontaneous pubertal development.86 For the reasons above, the BFM Relapse Study Group recommends orchiectomy of clinically involved (i.e. enlarged) testes and a biopsy of the contralateral testis. If the contralateral testis is histologically free of leukemia, radiation therapy should be given at a reduced dose of 15 Gy.77 With this approach, local recurrence rates of only 2.5% (2/81) after isolated testicular relapse and 1.1% (1/87) after combined testicular relapse have been registered since 1983. This procedure seems to provide safe local control of the disease with the advantage of giving patients with unilateral testicular relapse a chance to undergo spontaneous puberty. Furthermore, implantation of a testicular prosthesis may lead to cosmetically superior results, compared with leaving atrophic testes without any hormonal function after 24 Gy irradiation. Again, in patients with isolated testicular relapse, time to relapse has been found to be the most relevant prognostic factor. Attempts to detect occult testicular leukemia by routinely performed open wedge biopsy of the testes upon completion of frontline therapy were made but have been abandoned.74,87

Stem cell transplantation In most children with relapsed ALL, second remissions can be induced. However, in many patients, chemotherapy is not sufficient to maintain CR. Stem cell transplantation (SCT) has therefore been introduced as substantially intensified postremission treatment. It offers the possibility of administering chemotherapy and total-body irradiation (TBI) at doses that would be lethal without subsequent rescue of marrow function by BM or stem cell infusion. In addition, allogeneic SCT provides an antileukemic effect caused by a nonspecific reaction of donor immune cells against residual leukemic cells in the recipient. This graft-versusleukemia (GVL) effect is thought to prevent subsequent

relapses after allogeneic SCT, but it is also associated with graft-versus-host disease (GVHD), a nonspecific reaction against cells of the recipient and a major reason for the higher treatment-related mortality associated with allogeneic SCT. In general, SCT provides a better relapse-free survival rate than chemotherapy alone, but is also associated with higher treatment-related morbidity and mortality rates. Nevertheless, subsequent relapse is the most common adverse event after allogeneic SCT. The extent of minimal residual disease (MRD) in the bone marrow prior to SCT seems to be a suitable indicator of the risk for relapse after SCT.88,89 Monitoring of diverse recipient- and donor-specific markers allows one to confirm the complete engraftment and to predict subsequent relapses at an early stage in case of increasing mixed chimerism. This ability provides the opportunity to reduce GVHD prophylaxis or to administer donor lymphocyte infusions to increase the GVL-effect and to suppress the impending relapse.90,91 Comparison of the efficacy of SCT versus chemotherapy remains difficult. Most published data are based on retrospective analyses, and are strongly biased by selection criteria, the effects of treatment at particular centers, the gain of experience and changes in treatment methods. Besides overall survival, the quality of life has to be considered. Allogeneic SCT, mostly including TBI, leads to a variety of severe late effects, and a substantial proportion of surviving patients suffer from chronic GVHD. Thus, an important question remains: which patients are likely to benefit from SCT? Allogeneic SCT from HLA-matched related donors Since the early 1970s, allogeneic SCT from HLA-matched related, mostly sibling donors has been established as a treatment option for patients with leukemia in complete remission.92,93 A variety of regimens, mostly TBI combined with high-dose cyclophosphamide, have been used for myeloablative conditioning prior to SCT.94–98 A retrospective analysis of data from the BFM Study Group suggests better results after TBI/etoposide compared with other regimens.97 Since there are no published studies with a prospective randomized comparison between allogeneic SCT and chemotherapy as postremission therapy, one alternative is to compare the results for groups matched according to established risk parameters. In 1994, such a study was published by Barrett et al.99 The two patient groups were matched by sex, age, immunophenotype, initial leukocyte count and duration of first remission. The probability of EFS was significantly better for the SCT group: 40% ± 3% versus 17% ± 3% for patients receiving chemotherapy only. The higher probability of treatment-related death after SCT,

Relapsed acute lymphoblastic leukemia

27% ± 4% compared with 14% ± 4% after chemotherapy alone, was counterbalanced by a significantly lower probability of subsequent relapse, 45% ± 4% compared with 80% ± 3% after chemotherapy. Disease-free survival after SCT was better than after chemotherapy, regardless of any initial risk factor. When results from various study groups are compared, the general trend is that SCT is more effective than chemotherapy in patients with BM relapses, in particular early BM relapses, whereas similar EFS rates are observed in children with late relapse or extramedullary relapse. Recent but as yet unpublished findings of the BFM Relapse Study Group have shown that the EFS rate for children with late marrow relapse is better after matched sibling-donor SCT than after chemotherapy, whereas overall survival is not different. A substantial proportion of children with a subsequent relapse after chemotherapy could be effectively salvaged even in third CR. By contrast, the outcome for patients with a relapse after allogeneic SCT was extremely poor, such that EFS and overall survival rates were not different.100 SCT from HLA-matched related donors is an effective postremission therapy and should be performed in the majority of patients with systemic relapse of ALL if a suitable donor is available. It is associated with a higher treatment-related mortality rate but a substantially lower relapse rate than chemotherapy alone. Allogeneic SCT from HLA-matched unrelated donors In recent years, SCT from matched unrelated donors has been used for an increasing number of patients who lack a suitable related donor.101 Unrelated-donor SCT carries an even higher risk for treatment-related morbidity and mortality, with death rates ranging from 20% to 30%. But it still provides a significantly better relapsefree survival rate than chemotherapy alone in high-risk patient groups.102–105 However, in the intermediate-risk group, EFS was not superior after matched unrelateddonor SCT, as shown by matched-pair analysis.106 More than in matched sibling-donor transplantation, the toxicity and relapse rates associated with SCT from HLA-matched unrelated donors depend on the extent of in vivo and/or ex vivo T-cell depletion and the intensity of prophylaxis for GVHD. Autologous SCT Rescue of marrow function after high-dose myeloablative irradiation and/or chemotherapy can be achieved by the reinfusion of cryopreserved autologous BM or peripheral stem cells. The major disadvantage of this procedure is the lack of an allogeneic GVL effect. A variety of methods of autologous SCT have been used as postremission therapy in

patients without a suitable related donor, including different preparative regimens and autograft purging procedures to remove residual leukemic cells. In general, compared with allogeneic SCT, autologous SCT is associated with a lower transplant-related mortality rate, which is counterbalanced by a higher relapse rate. In particular, in patients with early systemic relapses, the rate of subsequent relapses has been high. At present, in the face of the availability of suitable unrelated donors, autologous SCT for patients with medullary relapse has been largely abandoned, but is still used by some study groups to treat patients suffering from extramedullary relapses, in whom a large proportion of subsequent adverse events are again local recurrences.

Experimental approaches In more recent years, stem cells from donors with variable degrees of HLA mismatch have been used for allogeneic SCT, if fully matched donors have not been available. The degree of HLA mismatch was associated with treatment failure in the setting of related- or unrelated-donor SCT.107,108 SCT from related donors sharing only one haplotype with the recipient resulted in high treatment-related toxicity and mortality rates, or if the allograft was fully T-cell depleted, in a high rate of rejections and subsequent relapses.109 The feasibility of SCT with cord blood as the stem cell source has been demonstrated with donors having different degrees of HLA mismatch.110 Allogeneic SCT after nonmyeloablative conditioning regimens has been described in selected patient groups, without convincing evidence provided for its general clinical applicability.111 Supplementation of autologous SCT by vaccination with gene-manipulated autologous leukemic cells has been considered to induce an antileukemic T-cell reaction mimicking the allogeneic GVL effect.112 Radio- or chemolabeled monoclonal antibodies directed against leukemia-associated antigens have been used for reduction of the tumor burden or for elimination of residual disease.113,114 The feasibility and efficacy of these experimental approaches remain to be evaluated in controlled, prospective, cooperative clinical trials.

Conclusion and perspective With currently used polychemotherapy, roughly one-third of patients who relapse in first remission will be cured with either chemotherapy (with or without radiotherapy) or SCT. Even some patients experiencing second relapses can be cured with a third round of therapy. Thus, the survival

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rate after relapse is approximately 40%, resulting in an overall survival of about 85% of children with ALL. In the case of relapse, a complete and careful work-up is essential, just as in newly diagnosed patients in order to assess the individual risk profile and to determine the adequate treatment strategy, in particular the postremission regimen. Stem cell transplantation is often necessary in systemic relapse because it offers better relapse-free survival than does chemotherapy alone. However, SCT is not always required, and the potential risks of acute and late adverse effects associated with SCT versus chemotherapy should be carefully considered. A number of clinical prognostic factors can be used to estimate the risk for relapse. In frontline studies of ALL, it has been shown that monitoring of MRD is a valuable technology for detecting early responses to treatment and thus to discriminate among good-, intermediate- and poorprognosis patients. This strategy also appears to be an important tool for assessing ALL in relapse.115 The BFM Study Group is addressing the issue of whether in patients with relapsed ALL, early molecular remission may define a group of children who have a good prognosis and do not need SCT as postremission therapy. Furthermore, MRD at a level of 10−3 or greater before allogeneic SCT proved to be highly predictive for subsequent relapse, even after unrelated-donor SCT.88 Thus, MRD monitoring might be able to identify patients in whom attempts to improve the quality of second CR prior to SCT are urgently needed. REFERENCES 1 Henze, G. Chemotherapy for relapsed childhood acute lymphoblastic leukemia. Int J Pediatr Hematol Oncol, 1997; 5:199– 213. 2 Gaynon, P. S., Qu, R. P., Chappell, R. J., et al. Survival after relapse in childhood acute lymphoblastic leukemia: impact of site and time to first relapse – the Children’s Cancer Group experience. Cancer, 1998; 82: 1387–95. 3 Bennett, J. M., Catovsky, D., Daniel, M. T., et al. Proposals for the classification of the acute leukaemias. French-AmericanBritish (FAB) Co-operative Group. Br J Haematol, 1976; 33: 451– 8. 4 L¨offler, H. & Gassmann, W. Morphology and cytochemistry of acute lymphoblastic leukaemia. Baillieres Clin Haematol, 1994; 7: 263–72. 5 Steward, C. G., Goulden, N. J., Katz, F., et al. A polymerase chain reaction study of the stability of Ig heavy-chain and T-cell receptor delta gene rearrangements between presentation and relapse of childhood B-lineage acute lymphoblastic leukemia. Blood, 1994; 83: 1355–62. 6 Vora, A., Frost, L., Goodeve, A., et al. Late relapsing childhood lymphoblastic leukemia. Blood, 1998; 92: 2334–7.

7 Lo Nigro, L., Cazzaniga, G., Di Cataldo, A., et al. Clonal stability in children with acute lymphoblastic leukemia (ALL) who relapsed five or more years after diagnosis. Leukemia, 1999; 13: 190–5. 8 Chessells, J. M., Leiper, A. D., & Richards, S. M. A second course of treatment for childhood acute lymphoblastic leukaemia: long-term follow-up is needed to assess results. Br J Haematol, 1994; 86: 48–54. 9 Miniero, R., Saracco, P., Pastore, G., et al. Relapse after first cessation of therapy in childhood acute lymphoblastic leukemia: a 10-year follow-up study. Italian Association of Pediatric Hematology-Oncology (AIEOP). Med Pediatr Oncol, 1995; 24: 71–6. 10 Rivera, G. K., Hudson, M. M., Liu, Q., et al. Effectiveness of intensified rotational combination chemotherapy for late hematologic relapse of childhood acute lymphoblastic leukemia. Blood, 1996; 88: 831–7. 11 Henze, G., Fengler, R., Hartmann, R., et al. Six-year experience with a comprehensive approach to the treatment of recurrent childhood acute lymphoblastic leukemia (ALL-REZ BFM 85). A relapse study of the BFM Group. Blood, 1991; 78: 1166–72. 12 Schroeder, H., Garwicz, S., Kristinsson, J., et al. Outcome after first relapse in children with acute lymphoblastic leukemia: a population-based study of 315 patients from the Nordic Society of Pediatric Hematology and Oncology (NOPHO). Med Pediatr Oncol, 1995; 25: 372–8. 13 Wheeler, K., Richards, S., Bailey, C., & Chessells, J. Comparison of bone marrow transplant and chemotherapy for relapsed childhood acute lymphoblastic leukaemia: the MRC UKALL X experience. Medical Research Council Working Party on Childhood Leukaemia. Br J Haematol, 1998; 101: 94–103. 14 Kuo, A. H., Yataganas, X., Galicich, J. H., Fried, J., & Clarkson, B. D. Proliferative kinetics of central nervous system (CNS) leukemia. Cancer, 1975; 36: 232–9. 15 Tsuchiya, J., Moteki, M., Shimano, S., et al. Proliferative kinetics of the leukemic cells in meningeal leukemia. Cancer, 1978; 42: 1255–62. 16 Jahnukainen, K., Saari, T., Salmi, T. T., Pollanen, P., & Pelliniemi, L. J. Reactions of Leydig cells and blood vessels to lymphoblastic leukemia in the rat testis. Leukemia, 1995; 9: 908–14. 17 Buchanan, G. R., Boyett, J. M., Pollock, B. H., et al. Improved treatment results in boys with overt testicular relapse during or shortly after initial therapy for acute lymphoblastic leukemia: a Pediatric Oncology Group study. Cancer, 1991; 68: 48–55. 18 Winick, N. J., Smith, S. D., Shuster, J., et al. Treatment of CNS relapse in children with acute lymphoblastic leukemia:a Pediatric Oncology Group study. J Clin Oncol, 1993; 11: 271–8. 19 Ribeiro, R. C., Rivera, G. K., Hudson, M., et al. An intensive re-treatment protocol for children with an isolated CNS relapse of acute lymphoblastic leukemia. J Clin Oncol, 1995; 13: 333–8. 20 Neale, G. A., Pui, C. H., Mahmoud, H. H., et al. Molecular evidence for minimal residual bone marrow disease in children with ‘isolated’ extra-medullary relapse of T-cell acute lymphoblastic leukemia. Leukemia, 1994; 8: 768–75.

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21 Lal, A., Kwan, E., al Mahr, M., et al. Molecular detection of acute lymphoblastic leukaemia in boys with testicular relapse. Mol Pathol, 1998; 51: 277–82. 22 Uckun, F. M., Gaynon, P. S., Stram, D. O., et al. Paucity of leukemic progenitor cells in the bone marrow of pediatric Blineage acute lymphoblastic leukemia patients with an isolated extramedullary first relapse. Clin Cancer Res, 1999; 5: 2415–20. ¨ 23 Buhrer, C., Hartmann, R., Fengler, R., et al. Superior prognosis in combined compared to isolated bone marrow relapses in salvage therapy of childhood acute lymphoblastic leukemia. Med Pediatr Oncol, 1993; 21: 470–6. 24 Chessells, J. M. Relapsed lymphoblastic leukaemia in children: a continuing challenge. Br J Haematol, 1998; 102: 423–38. 25 Jahnukainen, K., Salmi, T. T., Kristinsson. J., et al. The clinical indications for identical pathogenesis of isolated and nonisolated testicular relapses in acute lymphoblastic leukaemia. Acta Paediatr, 1998; 87: 638–43. 26 Henze, G., Fengler, R., Hartmann, R., et al. Chemotherapy for bone marrow relapse of childhood acute lymphoblastic leukemia. Cancer Chemother Pharmacol, 1989; 24: S16–9. 27 Abshire, T. C., Buchanan, G. R., Jackson, J. F., et al. Morphologic, immunologic and cytogenetic studies in children with acute lymphoblastic leukemia at diagnosis and relapse: a Pediatric Oncology Group study. Leukemia, 1992; 6: 357–62. ¨ 28 Buhrer, C., Hartmann, R., Fengler, R., et al. Peripheral blast counts at diagnosis of late isolated bone marrow relapse of childhood acute lymphoblastic leukemia predict response to salvage chemotherapy and outcome. J Clin Oncol, 1996; 14: 2812–17. 29 Crist, W., Carroll, A., Shuster, J., et al. Philadelphia chromosome positive childhood acute lymphoblastic leukemia: clinical and cytogenetic characteristics and treatment outcome. A Pediatric Oncology Group study. Blood, 1990; 76: 489–94. 30 Schlieben, S., Borkhardt, A., Reinisch, I., et al. Incidence and clinical outcome of children with BCR/ABL-positive acute lymphoblastic leukemia (ALL). A prospective RT-PCR study based on 673 patients enrolled in the German pediatric multicenter therapy trials ALL-BFM-90 and CoALL-05-92. Leukemia, 1996; 10: 857–63. 31 Fletcher, J. A., Lynch, E. A., Kimball, V. M., et al. Translocation (9; 22) is associated with extremely poor prognosis in intensively treated children with acute lymphoblastic leukemia. Blood, 1991; 77: 435–9. 32 Beyermann, B., Adams, H. P., & Henze, G. Philadelphia chromosome in relapsed childhood acute lymphoblastic ¨ leukemia: a matched-pair analysis. Berlin-Frankfurt-Munster Study Group. J Clin Oncol, 1997; 15: 2231–7. 33 Borkhardt, A., Cazzaniga, G., Viehmann, S., et al. Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials. Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-FrankfurtMunster Study Group. Blood, 1997; 90: 571–7.

34 Takahashi, Y., Horibe, K., Kiyoi, H., et al. Prognostic significance of TEL/AML1 fusion transcript in childhood B-precursor acute lymphoblastic leukemia. J Pediatr Hematol Oncol, 1998; 20: 190–5. 35 Rubnitz, J. E., Downing, J. R., Pui, C. H., et al. TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J Clin Oncol, 1997; 15: 1150–7. 36 Loh, M. L., Silverman, L. B., Young, M. L., et al. Incidence of TEL/AML1 fusion in children with relapsed acute lymphoblastic leukemia. Blood, 1998; 92: 4792–7. 37 Rubnitz, J. E., Behm, F. G., Wichlan, D., et al. Low frequency of TEL-AML1 in relapsed acute lymphoblastic leukemia supports a favorable prognosis for this genetic subgroup. Leukemia, 1999; 13: 19–21. 38 Seeger, K., Adams, H. P., Buchwald, D., et al. TEL-AML1 fusion transcript in relapsed childhood acute lymphoblastic ¨ leukemia. The Berlin-Frankfurt-Munster Study Group. Blood, 1998; 91: 1716–22. 39 Konrad, M., Metzler, M., Panzer, S., et al. Late relapses evolve from slow-responding subclones in t(12; 21)-positive acute lymphoblastic leukemia: evidence for the persistence of a preleukemic clone. Blood, 2003; 101: 3635–40. 40 Ford, A. M., Fasching, K., Panzer-Grumayer, E. R., et al. Origins of “late” relapse in childhood acute lymphoblastic leukemia with TEL-AML1 fusion genes. Blood, 2001; 98: 558–64. 41 Seeger, K., Buchwald, D., Peter, A., et al. TEL-AML1 fusion in relapsed childhood acute lymphoblastic leukemia. Blood, 1999; 94: 374–6. 42 Seeger, K., Buchwald, D., Taube, T., et al. TEL-AML1 positivity in relapsed B cell precursor acute lymphoblastic leukemia ¨ in childhood. Berlin-Frankfurt-Munster Study Group [letter]. Leukemia, 1999; 13: 1469–70. 43 Langlands, K., Craig, J. I., Anthony, R. S., & Parker, A. C. Clonal selection in acute lymphoblastic leukaemia demonstrated by polymerase chain reaction analysis of immunoglobulin heavy chain and T-cell receptor delta chain rearrangements. Leukemia, 1993; 7: 1066–70. 44 Klumper, E., Pieters, R., Veerman, A. J., et al. In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia. Blood, 1995; 86: 3861–8. 45 Kawamura, M., Kikuchi, A., Kobayashi, S., et al. Mutations of the p53 and ras genes in childhood t(1; 19)-acute lymphoblastic leukemia. Blood, 1995; 85: 2546–52. 46 Marks, D. I., Kurz, B. W., Link, M. P., et al. Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia. J Clin Oncol, 1997; 15: 1158–62. 47 Lam, V., McPherson, J. P., Salmena, L., et al. p53 gene status and chemosensitivity of childhood acute lymphoblastic leukemia cells to adriamycin. Leuk Res, 1999; 23: 871–80. 48 Zhou, M., Gu, L., Abshire, T. C., et al. Incidence and prognostic significance of MDM2 oncoprotein overexpression in relapsed childhood acute lymphoblastic leukemia. Leukemia, 2000; 14: 61–7.

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49 Blau, O., Avigad, S., Stark, B., et al. Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia. Leuk Res, 1997; 21: 721–9. 50 Kaspers, G. J., Pieters, R., Klumper, E., De Waal, F. C., & Veerman, A. J. Glucocorticoid resistance in childhood leukemia. Leuk Lymphoma, 1994; 13: 187–201. 51 Rivera, G., Pratt, C. B., Aur, R. J., Verzosa, M., & Hustu, H. O. Recurrent childhood lymphocytic leukemia following cessation of therapy: treatment and response. Cancer, 1976; 37: 1679–86. 52 Cornbleet, M. A., & Chessells, J. M. Bone-marrow relapse in acute lymphoblastic leukaemia in childhood. Br Med J, 1978; 2: 104–6. 53 Creutzig, U. & Schellong, G. Treatment of relapse in acute lymphoblastic leukaemia of childhood. Dtsch Med Wochenschr, 1980; 105: 1109–12. 54 Behrendt, H., Leeuwen, E. F. van, Schuwirth, C., et al. Bone marrow relapse occurring as first relapse in children with acute lymphoblastic leukemia. Med Pediatr Oncol, 1990; 18: 190–6. 55 Johnson, F. L., Thomas, E. D., Clark, B. S., et al. A comparison of marrow transplantation with chemotherapy for children with acute lymphoblastic leukemia in second or subsequent remission. N Engl J Med, 1981; 305: 846–51. 56 Woods, W. G., Nesbit, M. E., Ramsay, N. K., et al. Intensive therapy followed by bone marrow transplantation for patients with acute lymphocytic leukemia in second or subsequent remission: determination of prognostic factors (a report from the University of Minnesota Bone Marrow Transplantation Team). Blood, 1983; 61: 1182–9. 57 Buchanan, G. R. Diagnosis and management of relapse in acute lymphoblastic leukemia. Hematol Oncol Clin North Am, 990; 4: 971–95. 58 Giona, F., Testi, A. M., Rondelli, R., et al. ALL R-87 protocol in the treatment of children with acute lymphoblastic leukaemia in early bone marrow relapse. Br J Haematol, 1997; 99: 671–7. 59 Henze, G., Fengler, R., & Hartmann, R. Chemotherapy for relapsed childhood acute lymphoblastic leukemia: results of the BFM Study Group. Haematol Blood Transfus, 1994; 36: 374– 9. 60 Pui, C. H., Bowman, W. P., Ochs, J., Dodge, R. K., & Rivera, G. K. Cyclic combination chemotherapy for acute lymphoblastic leukemia recurring after elective cessation of therapy. Med Pediatr Oncol, 1988; 16: 21–6. 61 Sadowitz, P. D., Smith, S. D., Shuster, J., et al. Treatment of late bone marrow relapse in children with acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood, 1993; 81: 602–9. 62 Culbert, S. J., Shuster, J. J., Land, V. J., et al. Remission induction and continuation therapy in children with their first relapse of acute lymphoid leukemia: a Pediatric Oncology Group study. Cancer, 1991; 67: 37–42. 63 Rivera, G. K., Buchanan, G., Boyett, J. M., et al. Intensive retreatment of childhood acute lymphoblastic leukemia in first bone marrow relapse: a Pediatric Oncology Group study. N Engl J Med, 1986; 315: 273–8.

64 Morland, B. J. & Shaw, P. J. Induction toxicity of a modified Memorial Sloan-Kettering-New York II Protocol in children with relapsed acute lymphoblastic leukemia: a single institution study. Med Pediatr Oncol, 1996; 27: 139–44. 65 Rossi, M. R., Masera, G., Zurlo, M. G., et al. Randomized multicentric Italian study on two treatment regimens for marrow relapse in childhood acute lymphoblastic leukemia. Pediatr Hematol Oncol, 1986; 3: 1–9. 66 Buchanan, G. R., Rivera, G. K., Boyett, J. M.,et al. Reinduction therapy in 297 children with acute lymphoblastic leukemia in first bone marrow relapse: a Pediatric Oncology Group study. Blood, 1988; 72: 1286–92. 67 Hryniuk, W. M. The importance of dose intensity in the outcome of chemotherapy. In V. T. Devita, S. Hellman, & S. A. Rosenberg, eds., Important Advances in Oncology (Philadelphia, PA: Lippincott, 1988), pp. 121–44. 68 Herold, R., Stackelberg, A. von, Hartmann, R., Eisenreich, B., & Henze, G. Acute lymphoblastic leukemia-relapse study of the ¨ Berlin-Frankfurt-Munster Group (ALL-REZ BFM) experience: early treatment intensity makes the difference. J Clin Oncol, 2004; 22: 569–70. 69 Buchanan, G. R., Rivera, G. K., Pollock, B. H., et al. Alternating drug pairs with or without periodic reinduction in children with acute lymphoblastic leukemia in second bone marrow remission: a Pediatric Oncology Group study. Cancer, 2000; 88: 1166–74. ¨ 70 Buhrer, C., Hartmann, R., Fengler, R., et al. Importance of effective central nervous system therapy in isolated bone marrow relapse of childhood acute lymphoblastic leukemia. Blood, 1994; 83: 3468–72. 71 Land, V. J., Thomas, P. R., Boyett, J. M., et al. Comparison of maintenance treatment regimens for first central nervous system relapse in children with acute lymphocytic leukemia: a Pediatric Oncology Group study. Cancer, 1985; 56: 81–7. 72 Ritchey, A. K., Pollock, B. H., Lauer, S. J., Andejeski, Y., & Buchanan, G. R. Improved survival of children with isolated CNS relapse of acute lymphoblastic leukemia: a Pediatric Oncology Group study. J Clin Oncol, 1999; 17: 3745–52. 73 Goulden, N., Langlands, K., Steward, C., et al. PCR assessment of bone marrow status in ‘isolated’ extramedullary relapse of childhood B-precursor acute lymphoblastic leukaemia. Br J Haematol, 1994; 87: 282–5. 74 Ortega, J. J., Javier, G., & Toran, N. Testicular infiltrates in children with acute lymphoblastic leukemia: a prospective study. Med Pediatr Oncol, 1984; 12: 386–93. 75 Sullivan, M. P., Perez, C. A., Herson, J., et al. Radiotherapy (2500 rad) for testicular leukemia: local control and subsequent clinical events: a Southwest Oncology Group study. Cancer, 1980; 46: 508–15. 76 Bowman, W. P., Aur, R. J., Hustu, H. O., & Rivera, G. Isolated testicular relapse in acute lymphocytic leukemia of childhood: categories and influence on survival. J Clin Oncol, 1984; 2: 924–9.

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¨ ¨ 77 Wolfrom, C., Hartmann, R., Bruhm uller, S., et al. Similar outcome on boys with isolated and combined testicular acute lymphoblastic leukemia relapse after stratified BFM salvage therapy. Haematol Blood Transfus, 1997; 38: 647. 78 Atkinson, K., Thomas, P. R., Peckham, M. J., & McElwain, T. J. Radiosensitivity of the acute leukaemic infiltrate. Eur J Cancer, 1976; 12: 535–40. 79 Grundy, R. G., Leiper, A. D., Stanhope, R., & Chessells, J. M. Survival and endocrine outcome after testicular relapse in acute lymphoblastic leukaemia. Arch Dis Child, 1997; 76: 190–6. 80 Nachman, J., Palmer, N. F., Sather, H. N., et al. Open-wedge testicular biopsy in childhood acute lymphoblastic leukemia after two years of maintenance therapy: diagnostic accuracy and influence on outcome – a report from Children’s Cancer Study Group. Blood, 1990; 75: 1051–5. 81 Wofford, M. M., Smith, S. D., Shuster, J. J., et al. Treatment of occult or late overt testicular relapse in children with acute lymphoblastic leukemia: a Pediatric Oncology Group study. J Clin Oncol, 1992; 10: 624–30. 82 Uderzo, C., Grazia, Zurlo, M, Adamoli, L., et al. Treatment of isolated testicular relapse in childhood acute lymphoblastic leukemia: an Italian multicenter study. J Clin Oncol, 1990; 8: 672–7. 83 Brecher, M. L., Weinberg, V., Boyett, J. M., et al. Intermediate dose methotrexate in childhood acute lymphoblastic leukemia resulting in decreased incidence of testicular relapse. Cancer, 1986; 58: 1024–8. 84 Freeman, A. I., Weinberg, V., Brecher, M. L., et al. Comparison of intermediate-dose methotrexate with cranial irradiation for the post-induction treatment of acute lymphocytic leukemia in children. N Engl J Med, 1983; 308: 477–84. 85 Leiper, A. D., Grant, D. B., & Chessells, J. M. Gonadal function after testicular radiation for acute lymphoblastic leukaemia. Arch Dis Child, 1986; 61: 53–6. 86 Castillo, L. A., Craft, A. W., Kernahan, J., Evans, R. G., & AynsleyGreen, A. Gonadal function after 12-Gy testicular irradiation in childhood acute lymphoblastic leukaemia. Med Pediatr Oncol, 1990; 18: 185–9. 87 Askin, F. B., Land, V. J., Sullivan, M. P., et al. Occult testicular leukemia: testicular biopsy at three years continuous complete remission of childhood leukemia: a Southwest Oncology Group study. Cancer, 1981; 47: 470–5. 88 Knechtli, C. J., Goulden, N. J., Hancock, J. P., et al. Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia. Blood, 1998; 92: 4072–9. 89 Bader, P., Hancock, J., Kreyenberg, H., et al. Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL. Leukemia, 2002; 16: 1668–72. 90 Bader, P., Klingebiel, T., Schaudt, A., et al. Prevention of relapse in pediatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of

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increasing mixed chimerism: a single center experience of 12 children. Leukemia, 1999; 13: 2079–86. Bader, P., Beck, J., Frey, A., et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant, 1998; 21: 487–95. Storb, R., Bryant, J. I., Buckner, C. D., et al. Allogeneic marrow grafting for acute lymphoblastic leukemia: leukemic relapse. Transplant Proc, 1973; 5: 923–6. Thomas, E. D., Buckner, C. D., Banaji, M., et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood, 1977; 49: 511–33. Brochstein, J. A., Kernan, N. A., Groshen, S., et al. Allogeneic bone marrow transplantation after hyperfractionated totalbody irradiation and cyclophosphamide in children with acute leukemia. N Engl J Med, 1987; 317–24. Weyman, C., Graham-Pole, J., Emerson, S., et al. Use of cytosine arabinoside and total body irradiation as conditioning for allogeneic marrow transplantation in patients with acute lymphoblastic leukemia: a multicenter survey. Bone Marrow Transplant, 1993; 11: 43–50. Uderzo, C., Rondelli, R., Dini, G., et al. High-dose vincristine, fractionated total-body irradiation and cyclophosphamide as conditioning regimen in allogeneic and autologous bone marrow transplantation for childhood acute lymphoblastic leukaemia in second remission: a 7-year Italian multicentre study. Br J Haematol, 1995; 89: 790–7. Dopfer, R., Henze, G., Bender-G¨otze, C., et al. Allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia in second remission after intensive primary and relapse therapy according to the BFM- and CoALL-protocols: results of the German Cooperative Study. Blood, 1991; 78: 2780–4. Moussalem, M., Esperou, Bourdeau, H., Devergie, A., et al. Allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia in second remission: factors predictive of survival, relapse and graft-versus-host disease. Bone Marrow Transplant, 1995; 15: 943–7. Barrett, A. J., Horowitz, M. M., Pollock, B. H., et al. Bone marrow transplants from HLA-identical siblings as compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission. N Engl J Med, 1994; 331: 1253–8. Borgmann, A., Baumgarten, E., Schmid, H., et al. Allogeneic bone marrow transplantation for a subset of children with acute lymphoblastic leukemia in third remission: a conceivable alternative? Bone Marrow Transplant, 1997; 20: 939–44. Beatty, P. G., Hansen, J. A., Longton, G. M., et al. Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies. Transplantation, 1991; 51: 443–7. Kernan, N. A., Bartsch, G., Ash, R. C., et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med, 1993; 328: 593–602.

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103 Weisdorf, D. J., Billett, A. L., Hannan, P., et al. Autologous versus unrelated donor allogeneic marrow transplantation for acute lymphoblastic leukemia. Blood, 1997; 90: 2962–8. 104 Oakhill, A., Pamphilon, D. H., Potter, M. N., et al. Unrelated donor bone marrow transplantation for children with relapsed acute lymphoblastic leukaemia in second complete remission. Br J Haematol, 1996; 94: 574–8. 105 Lausen, B. F., Heilmann, C., Vindelov, L., & Jacobsen, N. Outcome of acute lymphoblastic leukaemia in Danish children after allogeneic bone marrow transplantation. Superior survival following transplantation with matched unrelated donor grafts. Bone Marrow Transplant, 1998; 22: 325–30. 106 Borgmann, A., Stackelberg, A. von, Hartmann, R., et al. Unrelated donor stem cell transplantation compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission: a matched-pair analysis. Blood, 2003; 101: 3835–9. 107 Szydlo, R., Goldman, J. M., Klein, J. P., et al. Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings. J Clin Oncol, 1997; 15: 1767–77. 108 Kawano, Y., Takaue, Y., Watanabe, A., et al. Partially mismatched pediatric transplants with allogeneic CD34(+) blood cells from a related donor. Blood, 1998; 92: 3123–30. 109 Aversa, F., Tabilio, A., Velardi, A., et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med, 1998; 339: 1186–93. 110 Locatelli, F., Rocha, V., Chastang, C., et al. Factors associated with outcome after cord blood transplantation in children

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with acute leukemia. Eurocord-Cord Blood Transplant Group. Blood, 1999; 93: 3662–71. Slavin, S., Nagler, A., Naparstek, E., et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood, 1998; 91: 756–63. Borgmann, A., Stackelberg, A. von, Baumgarten, E., et al. Immunotherapy of acute lymphoblastic leukemia by vaccination with autologous leukemic cells transfected with a cDNA expression plasmid coding for an allogeneic HLA class I antigen combined with interleukin-2 treatment. J Mol Med, 1998; 76: 215–21. Matthews, D. C., Appelbaum, F. R., Eary, J. F., 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: 1237–47. Pinilla-Ibarz, J., Cathcart, K., Korontsvit, T., et al. Vaccination of patients with chronic myelogenous leukemia with bcrabl oncogene breakpoint fusion peptides generates specific immune responses. Blood, 2000; 95: 1781–7. Eckert, C., Biondi, A., Seeger, K., et al. Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia. Lancet, 2001; 358: 1239–41. Lawson, S. E., Harrison, G., Richards, S., et al. The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the Medical Research Council UKALLR1 study. Br J Haematol, 2000; 108: 531–43.

18 B-cell acute lymphoblastic leukemia and Burkitt lymphoma John T. Sandlund and Ian T. Magrath

Introduction B-cell acute lymphoblastic leukemia (B-cell ALL) has historically accounted for approximately 2% of childhood ALLs diagnosed in the United States each year.1 This malignancy has clinical, histologic, immunophenotypic, and cytogenetic features similar to those of Burkitt lymphoma, a high-grade B-cell non-Hodgkin lymphoma (NHL).2,3 Hence, B-cell ALL and Burkitt lymphoma are usually considered to represent a spectrum of the same disease process. In children, the distinction between Burkitt lymphoma with bone marrow involvement and B-cell ALL is arbitrarily based on the degree of marrow infiltration. Patients who have less than 25% replacement of normal marrow with lymphoblasts are considered to have advanced (stage IV) Burkitt lymphoma, whereas those with more extensive marrow replacement are given the diagnosis of B-cell ALL. These two groups are treated with the same protocols used for patients with advanced-stage Burkitt lymphoma that has not disseminated to the marrow (stage III). Although limited-stage Burkitt lymphoma is also thought to be included in this disease spectrum, it is less common in children than are the advanced forms and is treated with much less intensive therapy. Hence, in describing treatment, we will focus on stages III and IV Burkitt lymphoma and B-cell ALL. Advances in our understanding of the molecular pathogenesis of these malignancies and improvements in treatment outcome have been striking over the past 20 years.4–6 Currently, a 75% to 85% event-free survival rate can be expected for children with B-cell ALL. By contrast, less than 50% of these patients were event-free survivors 20 years

ago.4 These laboratory and clinical advances are the focus of this chapter.

Pathology and immunophenotype Burkitt lymphoma was classified as small noncleaved cell NHL of Burkitt and non-Burkitt types in the National Cancer Institute (NCI) Working Formulation, published in 1982.2 This classification was based purely on morphology alone and therefore should not be considered precisely equivalent to the categories of “Burkitt” and “Burkitt-like” in the Revised European-American Lymphoma (REAL) classification system published in 19943 and the more recent WHO Classification of Tumors for hematopoietic and lymphoid tissue,7 both of which utilize immunophenotyping, and where possible, cytogenetics and molecular genetics in diagnostic categorization. The Burkitt and Burkitt-like subgroups differ with regard to cellular pleomorphism, although this distinction, and that between Burkitt-like and large B-cell lymphoma are imprecise at best. The Burkitt-like tumors are characterized by cellular heterogeneity, with a greater proportion of large cells, and a higher proportion with a single prominent nucleolus rather than the two to five nucleoli more typical of Burkitt lymphomas.6,7 There is no clear difference in clinical features or treatment outcome identified between these two subcategories in the childhood age group, although in adults, in which a large B-cell lymphoma is a much more common disease, Burkitt-like lymphomas have clinical features that are intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma. Burkitt-like lymphoma may therefore consist of a mix of atypical Burkitt

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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can be treated effectively with therapy designed for Bcell precursor ALL and therefore are not assigned to the shorter, more intense cyclophosphamide-based regimens designed for B-cell ALL and advanced-stage Burkitt lymphoma.

Cytogenetics and molecular pathology

Fig. 18.1 Histologic section of Burkitt lymphoma. Photo kindly provided by Frederick G. Behm. (See color plate 18.1 for full-color reproduction.)

lymphoma and atypical diffuse large B-cell lymphoma, and possibly some cases of Burkitt lymphoma arising in a background of follicular lymphoma.8,9 Lymphoblasts obtained from bone marrow, malignant ascites, or pleural fluid of patients with B-cell ALL or Burkitt lymphoma are cytologically consistent with the French-American-British L3 category. The lymphoblasts are characterized by a high nuclear-to-cytoplasmic ratio, prominent nucleoli, and vacuolated basophilic cytoplasm (Fig. 18.1).6,7,10 Histologic examination of an involved lymph node or extranodal mass (e.g. a gastrointestinal tract primary tumor) reveals sheets of monomorphic lymphoid cells as described above, with the classic “starry sky” appearance that results from the presence of interspersed tingible-body macrophages. Immunophenotypically, the lymphoblasts are relatively mature B cells that usually express surface IgM, although IgG or IgA can be detected occasionally.6,7 There are rare cases that contain the classic t(8;14) chromosomal translocation (see next section) but lack surface immunoglobulin.11,12 Also characteristic of these malignancies is the expression of CD19, CD20, CD21, CD10, CD79a, and the absence of terminal deoxynucleotidyl transferase expression.6,7 The high proliferative rate of Burkitt lymphomas is detected by positive nuclear reactivity to the Ki-67 antibody in virtually every tumor cell. In this regard, the WHO classification suggests a “Burkittlike” designation should be used for B-cell lymphomas with morphologic features intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma, where the Ki-67 fraction of viable tumor cells is greater than or equal to 99%, or a MYC translocation is present.7 A small percentage of cases of ALL express surface immunoglobulin, but lack L3 morphology. These cases

Three characteristic chromosomal translocations are identified in cases of B-cell ALL and Burkitt lymphoma. The classic t(8;14)(q24;q32) is identified in approximately 85% of cases, while two variant translocations, t(2;8)(p11;q24) and t(8;22)(q24;q11), account for the remainder.13–15 The unifying feature of these chromosomal abnormalities is the juxtaposition of the MYC proto-oncogene on chromosome 8 with either the heavy- or light-chain immunoglobulin genes, resulting in dysregulation of the translocated MYC gene.13–15 MYC is a transcription factor gene whose expression is increased when cells pass from a quiescent to a proliferative state.13,16–18 Under normal circumstances, mitogenic stimulation of a B cell results in a rapid increase in MYC transcription. The MYC protein induces cycle progression from the G1 phase into the S phase through the activation of key target genes.19 It also forms heterodimers through its carboxy-terminal motifs (basic helixloop-helix and leucine zipper) with related proteins (e.g. MAX). These heterodimers in turn bind to DNA and influence cell cycling.20–23 MYC:MAX heterodimers are potent transactivators20–24 ; by contrast, MAX:MAX dimers are transcriptional repressors. MAD, a related protein, can displace MYC from MAX. The resultant MAD:MAX dimer forms a complex with a transcriptional repressor (Sin3), which blocks the expression of MYC target genes.25 In B-cell ALL and Burkitt lymphoma, it is thought that the translocation-induced dysregulation of MYC results in an increased proportion of MYC:MAX complexes, leading to proliferation of the lymphoblasts.26 Various theories exist regarding the mechanism of MYC dysregulation in these tumors.13,26–29 Some stem from the observation of structural abnormalities in the translocated MYC gene. These abnormalities include truncations of the gene at the site of the translocation breakpoint and/or mutations within either the regulatory or coding regions of the gene. For example, mutations in the MYC-inhibiting factor (MIF) binding sites in the first intron (i.e. regulatory region) remove the repressive effect of MIF binding on transcription.30 Coding region mutations have also been described in Burkitt lymphoma,28 and have been associated with gain-of-function activities. Other data suggest

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the possible role of the translocated immunoglobulin gene in lymphomagenesis. It has been speculated that the juxtaposed immunoglobulin gene usurps control over the MYC gene through putative long-range immunoglobulin enhancer sequences.27,29 Thus, it is probable that multiple mechanisms exist whereby MYC is dysregulated in B-cell ALL and Burkitt lymphoma. Studies in a transgenic mouse model demonstrated that a dysregulated MYC gene is an essential but insufficient factor in the pathogenesis of Burkitt lymphoma. In this model, the programmed expression of a MYC gene in the B-cell compartment, mimicking the abnormality in Burkitt tumor, results in the development of B-cell malignancies.31,32 That these tumors are monoclonal and take 6 to 9 months to develop further strengthens the hypothesis that additional factors or molecular events are necessary for malignant transformation. The identity and role of other oncogenes or tumor suppressor genes are under current investigation.33,34 In this regard, recent studies of the ARF-Mdm2-p53 apoptotic pathway in a Eu-myc transgenic mouse model system has provided insights into lymphomagenesis.35–37 In this model, 80% of the lymphomas had an abnormality at some point in the ARFMdm2-p53 pathway, including mutations in Ink4a/ARF (25%), mutations of p53 (30%) or overexpression of Mdm2 (50%).35 Investigations are currently under way to evaluate this pathway in human Burkitt lymphomas. Abnormalities of the p53 tumor suppressor gene have been identified in cases of B-cell ALL and Burkitt lymphoma.38 However, the pattern of p53 mutations in these malignancies differs from that reported in solid tumors such as breast, lung, and colorectal carcinomas. Interactions between the regulatory systems of MYC and p53 suggest a role of p53 in lymphoma pathogenesis.39 Among patients with Burkitt lymphoma, the frequency of p53 abnormalities is approximately 33% in primary biopsies.39 Investigations in another mouse model of Burkitt lymphoma suggested that the role of MYC in pathogenesis may be the induction of chromosomal instability. In this model, MYC does not cause an elevated rate of mutations, but rather facilitates structural changes including deletions, translocations, and inversions.40

Epidemiology Approximately 200 cases of childhood Burkitt lymphoma and B-cell ALL are diagnosed in the United States each year.41–44 Children at increased risk include those with congenital immunodeficiency syndromes [Wiskott–Aldrich

syndrome, ataxia telangiectasia, or X-linked lymphoproliferative (XLP) disease].45–48 It is important to identify these syndromes so that appropriate therapy can be designed. For example, involved-field irradiation and radiomimetics such as bleomycin should be avoided in the management of children with ataxia telangiectasia who develop a neoplasm. Additionally, children with this syndrome are at increased risk for the development of hemorrhagic cystitis following the administration of cyclophosphamide, which may require additional hydration and use of the uroprotectant, Mesna, even when small doses of cyclophosphamide are given. Boys with XLP disease are at increased risk for the development of B-cell lymphomas or fatal infectious mononucleosis. This diagnosis should be entertained in any boy with a B-cell lymphoma whose brother has had either B-cell lymphoma or fatal infectious mononucleosis, or in any boy with two primary B-cell lymphomas. Children with acquired immunodeficiency conditions are also at increased risk for developing Burkitt lymphoma.49–54 This subgroup comprises patients who have received post-transplant immunosuppressive therapy and those with the acquired immunodeficiency syndrome (AIDS) secondary to HIV infection. The majority of HIV-associated non-Hodgkin lymphomas are peripheral B cell, either Burkitt or B-large cell. HIV infection has also been associated with proliferative lesions of mucosaassociated lymphoid tissue (MALT), which may be either benign or malignant.53

Geographic differences There are striking geographic differences in the incidence rates of Burkitt lymphoma/B-cell ALL. In Japan, the incidence of Burkitt lymphoma is very low, whereas in equatorial Africa it is very high.5,41 In northeastern Brazil, there is a predominance of the Burkitt subtype among children with NHL,55 in contrast to central and southern Brazil, where the distribution of NHL subtypes is similar to those in the United States and Western Europe. There are also geographic differences in clinical features. For example, the endemic subtype of Burkitt lymphoma (found in equatorial Africa) frequently involves the abdomen, jaw, orbit, paraspinal area and the central nervous system, whereas the sporadic subtype (found in the United States and Western Europe) is typically associated with involvement of the abdomen, nasopharynx and bone marrow.5 Biologic differences between the endemic and sporadic subtypes of Burkitt lymphoma have also been described. Among

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Table 18.1 Comparison of endemic and sporadic Burkitt lymphomaa Feature

Endemic

Sporadic

Average annual incidence (children below 16 years) Geographic distribution

10 per 100,000

0.2 per 100,000

Malarial belt (equatorial Africa and New Guinea) 95% Upstream of MYC Blast cell antigens, CD10-negative, no IgM secretion Jaw, abdomen, orbit, paraspinal, CNS

Worldwide

Association with EBV Predominant chromosome 8 breakpoints Immunologic features Common sites of tumor

a

15% Within MYC Few blast antigens, CD10-negative, secretion of IgM Abdomen, bone marrow, nasopharynx

Burkitt lymphoma in South America may have features intermediate between the endemic and sporadic subtypes.55

sporadic cases, the predominant chromosome 8 breakpoints are usually within the MYC gene, whereas among endemic cases, the breakpoints are usually upstream of MYC.56 Table 18.1 summarizes these and other differences between the endemic and sporadic subtypes of Burkitt lymphoma. The Burkitt lymphomas occurring in children from northeastern Brazil are associated with features that appear to be intermediate between the endemic and sporadic subtypes.55

Relationship to Epstein–Barr virus Epstein–Barr virus (EBV) has been shown to be associated with Burkitt lymphoma and B-cell ALL; however, the precise role of EBV in pathogenesis has yet to be directly demonstrated.5,13 Geographic differences in the association of EBV and Burkitt lymphoma and in tumor incidence have provided important insights. Epstein–Barr nuclear antigen (EBNA) testing has revealed an almost invariable association between EBV and the endemic subtype of Burkitt tumor in equatorial Africa, where both seroconversion to EBV and the development of Burkitt lymphoma occur at a younger age.5,13 In contrast to endemic Burkitt lymphoma, the sporadic subtype in Western Europe and the United States has been associated with EBV in only a minority of cases (approximately 15%). It has been postulated that EBV, a B-cell mitogen, increases the target pool of cells susceptible to translocations associated with malignant transformation.5,13 This hypothesis is supported by the observation that EBV induces Rag gene expression; thus, theoretically increasing the chance of a translocation occurring in immature B-cell undergoing immunoglobulin gene rearrangement.57 A potential role for EBNA-1 in lymphomagenesis is suggested by the development of lymphomas in mice transgenic for EBNA-1.58

Moreover, an EBNA-1 variant is associated with the majority of cases studied, prompting investigators to speculate that this tumor-associated mutation alters EBNA-1 function in some way that confers a growth advantage to lymphoma cells.59 The hypothesis of a more direct role for EBV in pathogenesis is supported by investigations of the EBV+ Burkitt lymphoma cell line, Akata, which loses its malignant phenotype after spontaneous loss of the EBV episome, but regains it after reinfection with EBV.60 Additionally, aberrant expression of the EBV genome has been identified in some sporadic cases that were EBV-negative by EBNA testing.61 These findings suggest a greater degree of association between EBV and Burkitt lymphoma than previously recognized.61

Clinical features Children with B-cell ALL or Burkitt lymphoma may present with bone pain, pancytopenia, and associated fatigue, pallor, and mucosal bleeding. Sites of disease other than the bone marrow may include the head and neck region (tonsils, nasopharynx, or cervical lymph nodes) or abdomen (see Fig. 18.2).5,6 Gastrointestinal tract primaries usually arise from the ileocecal region, although the appendix and colon may also be involved. Abdominal primaries may be associated with massive ascites and pleural effusions. In contrast to lymphoblastic lymphoma, mediastinal masses are very unusual. Involvement of cortical bone may occur in some cases. CNS involvement [cerebrospinal fluid (CSF) pleocytosis or the presence of a cranial nerve palsy] is present at diagnosis in approximately 10% of cases.62 As noted previously, sites of disease may vary with geographic location (e.g. endemic versus sporadic subtypes). For example, orbital masses and jaw tumors (Fig. 18.3) are seen almost exclusively in the endemic subtype.

B-cell acute lymphoblastic leukemia and Burkitt lymphoma

Fig. 18.2 Computed tomography scan illustrating abdominal Burkitt lymphoma arising from the ileocecal region of the intestinal tract.

Diagnosis and staging The diagnosis is generally established by examination of the bone marrow for L3 lymphoblasts with the immunophenotypic and cytogenetic features described previously. L3 blasts are observed in the peripheral blood smear in some cases. Diagnostic cells may also be obtained from ascitic or pleural fluid. Histologic examination of a lymph node or an abdominal mass will reveal the characteristic features of a small noncleaved cell NHL, as already described (see also Chapter 2). Because of the rapid doubling time of this malignancy, it is important that an expeditious staging work-up be performed so that appropriate therapy can be started as soon as possible. It should always include bone marrow aspiration; and, for Burkitt lymphoma, bilateral bone marrow aspirates and biopsies should be obtained. The workup should also include examination of the cerebrospinal fluid, as the presence of leukemic blast cells may mandate further intensification of therapy. Computed tomography of the head, neck, abdomen and pelvis may identify lymphomatous masses or leukemic infiltrates that need to be followed. Bone scans may identify cortical bone defects. Gallium scans are usually positive at sites of disease, and therefore may be helpful in evaluating treatment response and residual masses.5 Whole-body positron emission tomography, an imaging technique currently under investigation in these malignancies, may prove useful in assessing marrow disease.63 Essential laboratory studies include a complete blood count, a chemistry panel (including electrolytes, calcium, phosphorus, uric acid, lactate dehydrogenase, blood urea nitrogen, and creatinine), and an HIV screen. After completion of the work-up, a

Fig. 18.3 African child with endemic Burkitt lymphoma of the jaw. (See color plate 18.3 for full-color reproduction.)

disease stage (or a diagnosis of B-cell ALL) is assigned, usually according to the St. Jude system described by Murphy.64 Stages I and II Burkitt lymphoma are considered limited-stage disease, whereas stages III and IV are considered advanced disease, and receive more intensive therapy.

Treatment Advances in the treatment of B-cell ALL and advancedstage Burkitt lymphoma represent one of the great success stories in pediatric oncology.4,5 Improvements in outcome are largely the result of sequential protocol-based studies incorporating multiagent chemotherapy and appropriate CNS prophylaxis and therapy. Improvements in initial (prechemotherapy) management, approach to emergency situations, and supportive care have all contributed to the excellent results of contemporary treatment.

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Initial management Prior to the administration of chemotherapy, a serum chemistry panel should be obtained. Many patients with B-ALL or lymphoma present with hyperuricemia, hyperphosphatemia, and renal dysfunction as a result of the rapid turnover of tumor cells. This complication worsens with the delivery of chemotherapy, which induces rapid cell lysis; the release of purines, potassium, and phosphates into the bloodstream; and subsequent deposition of uric acid, xanthines, and phosphates in the renal tubules, leading to further renal dysfunction (tumor lysis syndrome; see also Chapter 29).5 This complication, can be minimized by vigorous hydration (3 to 4.5 L/m2 daily) introduced before chemotherapy. Alkalinization is usually necessary to bring the urine pH up to 7. The excretion of uric acid is impaired at acidic pH, whereas overalkalinization impairs the excretion of phosphorous. The addition of Lasix may be necessary to maintain an appropriate urine output. Allopurinol, a xanthine oxidase inhibitor that blocks the production of uric acid, is also used to prevent or manage hyperuricemia. Urate oxidase, which directly cleaves uric acid and converts it to allantoin, results in a precipitous drop in serum levels of uric acid, although this effect may be associated with severe allergic reactions.65 A recombinant form of this drug, SR29142 (rasburicase), is equally effective in reducing serum uric acid levels, but does not impose a significant risk of allergy.66 Its use precludes the need for alkalinization, preserves renal function (i.e. avoids the need for dialysis or hemofiltration), and permits timely delivery of scheduled chemotherapy.

Chemotherapy Chemotherapy should be started as soon as possible after pretreatment measures have been instituted to lessen or avoid complications. The most effective current treatment regimens for B-cell ALL and advanced-stage Burkitt lymphoma are intensive cyclophosphamide-based drug combinations given over a relatively short time, ranging from 5 to 8 months.67–85 The earliest approaches to treating B-cell ALL were regimens designed for advanced-stage Burkitt lymphoma. Major trials conducted for children with these malignancies are reviewed in Table 18.2. A study performed by the Children’s Cancer Group (CCG) demonstrated that a cyclophosphamide-based regimen was more active in Burkitt lymphoma than was a multiagent regimen (LSA2 L2 ) designed for the treatment of acute lymphoblastic leukemia.81 Subsequent studies demonstrated that treatment outcome could be improved by incorporating

high-dose methotrexate with or without high-dose cytarabine (ara-C), even if the duration of therapy was shortened to 2 to 4 months.69,75 Further improvements in treatment outcome have been achieved over the past 8 years with additional intensification of therapy (escalating doses of cyclophosphamide, methotrexate, and ara-C) and by adding new active agents (etoposide and/or ifosfamide). However, it is still unclear which of these two modifications is the more important.82,84,85 The LMB-89 regimen of the French Society for Pediatric Oncology (SFOP), for example, has produced one of the best results reported to date.84 With this strategy, children with B-cell ALL who have less than 70% replacement of the marrow by leukemic blast cells are treated with a regimen containing high-dose cyclophosphamide, high-dose methotrexate (3 g/m2 per dose), and low-dose ara-C, given over 4 to 5 months. Children with greater than 70% marrow blasts and those with CNS involvement are treated with a further intensified regimen based on increased doses of both methotrexate (to 8 g/m2 per dose) and ara-C (3 g/m2 per dose), and the inclusion of etoposide, given over approximately 8 months. Other equally successful treatment regimens have been ¨ described. For example, the Berlin-Frankfurt-Munster (BFM) cooperative group has reported excellent results for children with B-cell ALL (≥25% bone marrow blasts) following treatment with a multiagent regimen that incorporates high-dose methotrexate (5 g/m2 ), ifosfamide, etoposide, doxorubicin, and steroids.85 Serum lactate dehydrogenase (LDH) was used to assign patients to risk-adapted treatment. Patients with stage III disease and LDH ≥500 U/L were treated more intensively than those with a lower LDH level. Some patients with incomplete responses received further intensification with high-dose ara-C and etoposide. Sequential studies by the National Cancer Institute (NCI) demonstrated that the outcome for children with advanced-stage Burkitt lymphoma or B-cell ALL could be improved by incorporating ifosfamide, etoposide, and high-dose ara-C into the initial strategy, which included cyclophosphamide, doxorubicin, vincristine, and highdose methotrexate.77,82 It is important to emphasize that most of the evidence suggests that adults with B-cell ALL should be treated with the same aggressive regimens designed for children with advanced-stage Burkitt lymphoma and B-cell ALL.77,82,83,86

CNS prophylaxis and therapy The development of CNS prophylaxis and therapy has also been critical to an improved treatment outcome

B-cell acute lymphoblastic leukemia and Burkitt lymphoma

Table 18.2 Treatment outcome for advanced-stage (III, IV) Burkitt lymphoma (BL) and B-cell ALL (B-ALL) Event-free survival Group protocola St. Jude Total B POG 8617 SFOP LMB84c LMB 86c LMB89c

BFM 81 83 86 90

NCI 77-04 CODOX/VIPA Boston Hi-C-COM

Stage BL-III BL-IV/B-ALL BL-IV B-ALL

No. of patients 17 4/8 34 47

Time (Years)

Estimateb

Reference

2 2 4 4

81% 17% 79% ± 9% 65% ± 8%

67 68

BL-III BL-IV + B-ALL (CNS−) B-ALL (CNS−) B-ALL (CNS+) BL-III BL-IV B-ALL

167 34 11 24 278 62 102

2 2 >1 >1 5 5 5

80% (SE, 3%) 68% (SE, 8%) 82% (SD, 12%) 75% (SD, 9%) 91% (95% CI, 87–94%) 87% (95% CI, 77–93%) 87% (95% CI, 79–92%)

69

B-ALL B-ALL B-ALL BL-III BL-IV B-ALL

22 24 41 169 24 56

5 5 5 6 6 6

40% (SD, 6%) 50% (SE, 10%) 78% (SD, 6%) 86% (SD, 3%) 73% (SD, 8%) 74% (SD, 8%)

71 71 71 85

BL-III BL-IV BL-III/IV + B-ALL

30 9 75

3 3 1

57% ± 9% 13% ± 12% 89%

74

BL-III BL-IV + B-ALL

12 8

2 2

95% (CI, 54–99%) 50% (CI, 15%–78%)

70 84

82 75

a

Treatment regimens are described either in the text or in references accompanying the text descriptions. Statistical variability [standard error (SE), standard deviation (SD), or confidence interval (CI)] is indicated if specified in the original source. c These studies include patients with B-cell large cell NHL. b

for children with B-cell ALL. Effective prophylaxis is achieved with a combination of high-dose systemic therapy (methotrexate and ara-C) and intrathecal therapy with methotrexate, hydrocortisone, and ara-C. Patients with overt CNS disease at diagnosis (CSF pleocytosis, cranial nerve palsy, or both) are generally treated more intensively, with high doses of systemic methotrexate and ara-C and frequent delivery of intrathecal chemotherapy. Historically, the use of cranial radiation in the treatment of CNS disease has been somewhat controversial. Cranial radiation was a component of the very successful French (SFOP) LMB-89 regimen,84 although most investigators do not include it in the treatment plan. Moreover, the French SFOP group has

safely eliminated cranial radiation from their most recent protocol.

Emergency situations Various emergency situations can arise during the treatment of children with advanced Burkitt lymphoma or Bcell ALL. Renal dysfunction as a consequence of tumor lysis syndrome may occur despite prompt initial management (see also Chapter 29). In these situations, a nephrologist must be consulted immediately regarding the potential need for hemofiltration or renal dialysis. Mechanical

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ureteral obstruction by a large pelvic mass may complicate the picture. In some of these cases, the placement of ureteral stents or percutaneous nephrostomy tubes may provide a temporary solution to the obstructive nephropathy. The use of ureteric stents, however, is associated with the risk of perforation of the ureter (if it is surrounded by tumor), and nephrostomy tubes may increase the risk of infection; thus, hemofiltration may be preferable. Patients may also present with epidural masses and associated spinal cord compression. The delivery of specific chemotherapy is the optimal treatment option.5

Relapse The prognosis for children with a hematologic relapse of B-cell ALL or Burkitt lymphoma is generally considered to be dismal, particularly in light of the very aggressive initial therapy these patients receive. The most common approach is to proceed with an intensive multiagent reinduction attempt, which may be followed by an autologous or allogeneic bone marrow transplant, depending on whether the relapsed disease proves to be chemosensitive. Various multiagent reinduction regimens have been studied. The NCI demonstrated the activity of VIPA (etoposide, ifosfamide, and high-dose ara-C) in patients who relapsed following treatment on a regimen comprising cyclophosphamide, vincristine, doxorubicin, and high-dose methotrexate.82 Other regimens, including MIED (high-dose methotrexate, ifosfamide, etoposide, and dexamethasone) and ICE (ifosfamide, carboplatin, and etoposide)87 are also active against recurrent nonlymphoblastic non-Hodgkin lymphomas. Children with chemosensitive relapses are generally considered candidates for chemotherapy intensification with either autologous or allogeneic bone marrow transplantation (BMT).88–94 The European Lymphoma Bone Marrow Transplant Registry reported that among children with poor-risk Burkitt lymphoma, those with either a partial response to induction therapy or a chemosensitive relapse responded well to high-dose chemotherapy followed by autologous BMT.92 This group emphasized, however, that with more intensive front-line therapy, salvage with chemotherapy and autologous BMT may be less successful. Therefore, they suggest the study of novel approaches, including immunotherapy and allogeneic BMT with its accompanying graftversus-lymphoma effect. There are limited data on the comparative effectiveness of autologous versus allogeneic BMT strategies. However, most investigators would prefer allogeneic BMT for a hematologic relapse if a matched

related donor were available. The management of an isolated CNS relapse usually includes aggressive intrathecal therapy, either cranial or craniospinal irradiation, and aggressive systemic chemotherapy with either autologous hematopoietic stem cell support or allogeneic hematopoietic stem cell transplantation.

Future directions Even with dramatic improvements in treatment outcome for childhood B-cell ALL and advanced Burkitt lymphoma, approximately 20% of affected children still die of these diseases. Further, survivors may experience severe late effects, which can include anthracycline-associated cardiomyopathy, secondary malignancies, and/or sterility.95 Thus, there remains a need for less toxic therapies that retain or improve on the efficacy of previous treatments. Such strategies may include the use of new active agents, intensification of certain components of existing regimens with or without the incorporation of cytokines (e.g. G-CSF), or further development of promising but still problematic strategies, such as immunotherapy or gene-based therapy. The anti-CD20 antibody, rituxan, has activity in the management of adults with B-cell lymphoma (B-large cell and follicular),96,97 and in some children with either refractory or recurrent Burkitt lymphoma/B-ALL.98 Rituxan is currently being studied in COG (Children’s Oncology Group) studies of patients with newly diagnosed and recurrent B-cell lymphomas (Burkitt and B-large cell). The potential use of antisense strategies is also provocative.99,100 In EBV-associated tumors, it may be possible to target one of the EBV-associated genes. These and other challenges related to future directions in therapy for B-ALL and Burkitt lymphoma have been reviewed extensively, with emphasis on the need for further, careful testing of gene therapy strategies.100 It will also be important to continue studies of the cytogenetic and molecular abnormalities that define the pathogenetic mechanisms of these diseases. Indeed, such investigations may provide tools for refining disease classifications and for monitoring minimal residual disease, while providing insights that will permit the development of novel therapies that target disease-specific molecular lesions.

REFERENCES 1 Pui, C.-H. Childhood leukemias. N Engl J Med, 1995; 332: 1618– 30.

B-cell acute lymphoblastic leukemia and Burkitt lymphoma

2 National Cancer Institute sponsored study of classifications of non-Hodgkin’s lymphoma: summary and description of a working formulation for clinical usage. Cancer, 1982; 49: 2112–35. 3 Harris, N. L., Jaffe, E. S., Stein, H., et al. A revised EuropeanAmerican classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood, 1994; 84: 1361–92. 4 Sandlund, J. T., Downing, J. R., & Crist, W. M. Non-Hodgkin’s lymphoma in childhood. N Engl J Med, 1996; 334: 1238–48. 5 Magrath, I. T. Malignant non-Hodgkin’s lymphomas in children. In P. A. Pizzo & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology, 4th edn. (Philadelphia, PA: Lippincott, 2002), pp. 661–706. 6 Murphy, S. B., Fairclough, D. L., Hutchison, R. E., et al. NonHodgkin’s lymphomas of childhood: an analysis of the histology, staging, and response to treatment of 338 cases at a single institution. J Clin Oncol, 1989; 7: 186–93. 7 Jaffe, E. S., Harris, N. L., Stein, H., & Vardiman, J. W., eds., World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues (Lyon, France: IARC Press, 2001). 8 Hutchison, R. E., Finch, C., Kepner, J., et al. Burkitt lymphoma is immunophenotypically different from Burkitt-like lymphoma in young persons. Ann Oncol, 2000; 11(Suppl. 1): 35–8. 9 Hutchison, R. E., Murphy, S. B., Fairclough, D. L., et al. Diffuse small noncleaved-cell lymphoma in children, Burkitt’s versus non-Burkitt’s types: results from the Pediatric Oncology Group and St. Jude Children’s Research Hospital. Cancer, 1989; 64: 23–8. 10 Behm, F. G. & Campana, D. Immunophenotyping. In C.-H. Pui, ed., Childhood Leukemias (New York: Cambridge University Press, 1999), pp. 111–44. 11 Navid, F., Mosijczuk, A. D., Head, D. R., et al. Acute lymphoblastic leukemia with the (8;14)(q24;q32) translocation and FAB L3 morphology associated with a B-precursor immunophenotype: the Pediatric Oncology Group experience. Leukemia, 1999; 13: 135–41. 12 Loh, M. L., Samson, Y., Motte, E., et al. Translocation (2;8) (p12;q24) associated with a cryptic t(12;21)(p13;q22) TEL/ AML1 gene rearrangement in a child with acute lymphoblastic leukemia. Cancer Genet Cytogenet, 2000; 122: 79–82. 13 Magrath, I. T. & Bhatia, K. Pathogenesis of small noncleaved cell lymphomas (Burkitt’s lymphoma). In I. T. Magrath, ed., The Non-Hodgkin Lymphomas (London: Arnold, 1997). 14 Dalla-Favera, R., Bregni, M., Erikson, J., et al. Human c-myc oncogene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci U S A, 1982; 79: 7824–7. 15 Taub, R., Kirsch, I., Morton, C., et al. Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci U S A, 1982; 79: 7837–41. 16 Kelly, K. & Siebenlist, U. Mitogenic activation of normal T-cells leads to increased initiation of transcription in the c-myc locus. J Biol Chem, 1988; 263: 4828–31.

17 Kelly, K. & Sienbenlist, U. The regulation and expression of cmyc in normal and malignant cells. Annu Rev Immunol, 1986; 4: 317–38. 18 Luscher, B. & Eisenman, R. N. New light on Myc and Myb. Part I. Myc. Genes Dev, 1990; 4: 2025–35. 19 Packham, G. & Cleveland, J. L. Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis. Mol Cell Biol, 1994; 14: 5741–7. 20 Blackwood, E. M. & Eisenman, R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science, 1991; 251: 1211–17. 21 Prendergast, G. C., Lawe, D., & Ziff, E. B. Association of Myn, the murine homolog of Max, with c-Myc stimulates methylationsensitive DNA binding and ras co-transformation. Cell, 1991; 65: 395–407. 22 Ayer, D. E., Kretzner, L., & Eisenman, R. N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell, 1993; 72: 211–22. 23 Zervos, A. S., Gyuris, J., & Brent, R. Mxil, a protein that specifically interacts with Max to bind Myc-Max recognition sites [Erratum published in Cell, 1994; 79: 388]. Cell, 1993; 72: 223– 32. 24 Amati, B. & Land, H. Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. Curr Opin Genet Dev, 1994; 4: 102–8. 25 Ayer, D. E., Lawrence, Q. A., & Eisenman, R. N. Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell, 1995; 80: 767–76. 26 Gu, S., Cechova, K., Tassi, V., et al. Opposite regulation of gene transcription and cell proliferation by c-Myc and max. Proc Natl Acad Sci U S A, 1993; 90: 2935–9. 27 Croce, C. M., Erikson, J., Ar-Rushdi, A., et al. Translocated cmyc oncogene of Burkitt lymphoma is transcribed in plasma cells and repressed in lymphoblastoid cells. Proc Natl Acad Sci U S A, 1984; 81: 3170–4. 28 Gu, W., Bhatia, K., Magrath, I. T., et al. Binding and suppression of the Myc transcriptional activation domain by p107. Science, 1994; 264: 251–4. 29 Sandlund, J. T., Neckers, L. M., Schneller, H. E., et al. Theophylline-induced differentiation provides direct evidence for the deregulation of c-myc in Burkitt’s lymphoma and suggests participation of immunoglobulin enhanced sequences. Cancer Res, 1993; 53: 127–32. 30 Zajac-Kaye, M., Yu, B., & Ben-Baruch, N. Downstream regulatory elements in the c-myc gene. Curr Top Microbiol Immunol, 1990; 166: 279–84. 31 Adams, J. M., Harris, A. W., Pinkert, C. A., et al. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature, 1985; 318: 533–8. 32 Cory, S. & Adams, J. M. Transgenic mice and oncogenesis. Annu Rev Immunol, 1988; 6: 25–48. 33 Packham, G. & Cleveland, J. L. c-Myc and apoptosis. Biochim Biophys Acta, 1995; 1242: 11–28. 34 Henderson, S., Rowe, M., Gregory, C., et al. Induction of bcl-2 expression by Epstein-Barr virus latent membrane

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protein 1 protects infected B cells from programmed cell death. Cell, 1991; 65:1107–15. Eischen, C. M., Weber, J. D., Roussel, M. F., Sherr, C. J. & Cleveland, J. L. Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev, 1999; 13: 2658–69. Eischen, C. M., Roussel, M. F., Korsmeyer, S. J. & Cleveland, J. L. Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis. Mol Cell Biol, 2001; 21: 7653–62. Eischen, C. M., Woo, D., Roussel, M. F. & Cleveland, J. L. Apoptosis triggered by Myc-induced suppression of Bcl-XL or Bcl-2 is bypassed during lymphomagenesis. Mol Cell Biol, 2001; 21: 5063–70. Gaidano, G., Ballerini, P., Gong, J. Z., et al. p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc Natl Acad Sci U S A, 1991; 88: 5413–17. Bhatia, K. G., Guti´errez, M. I., Huppi, K., et al. The pattern of p53 mutations in Burkitt’s lymphoma differs from that of solid tumors. Cancer Res, 1992; 53: 4273–6. Rockwood, L. D., Torrey, T. A., Kim, J. S., et al. Genomic instability in mouse Burkitt lymphoma is dominated by illegitimate genetic recombinations, not point mutations. Oncogene, 2002; 21: 7235–40. Robison, L. L. General principles of the epidemiology of childhood cancer. In P. A. Pizzo & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology, 2nd edn. (Philadelphia, PA: Lippincott, 1993), pp. 3–10. Bleyer, W. A. The impact of childhood cancer on the United States and the world. CA Cancer J Clin, 1990; 40: 355–66. Parker, S. L., Tong, T., Bolden, S. & Wingo, P. A. Cancer Statistics, 1996. CA Cancer J Clin, 1996; 46: 3–4. Percy, C. L., Smith, M. A, Linet, M., et al. Lymphomas and reticuloendothelial neoplasms. In L. A. G. Ries, M. A. Smith, J. G. Gurney, et al., eds., Cancer Incidence and Survival Among Children and Adolescents: United States SEER Program 1975– 1995, NIH Publication 99–4649 (Bethesda, MA: National Cancer Institute, SEER Program, 1999), pp. 35–49. Young, J. L., Jr., Ries, L. G., Silverberg, E., Horm, J. W., & Miller, R. W. Cancer incidence, survival and mortality for children younger than age 15 years. Cancer, 1986; 58: 598–602. Taylor, A. M. R., Metcalfe, J. A., Thick, J., et al. Leukemia and lymphoma in ataxia telangiectasia. Blood, 1996; 87: 423–38. Gatti, R. A. & Good, R. A. Occurrence of malignancy in immunodeficiency disease. A literature review. Cancer, 1971; 28: 89–98. Filipovich, A. H., Heinitz, K. J., Robison, L. L., & Frizzera, G. The immunodeficiency cancer registry. A research resource. Am J Pediat Hematol Oncol, 1987; 9: 183–4. Ellaurie, M., Wiznia, A., Bernstein, L., et al. Lymphoma in pediatric HIV infection. Pediatr Res, 1989; 25: 150A. Parekh, S., Ratech, H., & Sparano, J. A. Human immunodeficiency virus-associated lymphoma. Clin Adv Hematol Oncol, 2003; 1: 295–301.

51 Levine, A. M., Seneviratne, L., Espina, B. M., et al. Evolving characteristics of AIDS-related lymphoma. Blood, 2000; 96: 4084–90. 52 Murphy, S. B., Jenson, H. B., McClain, K. L., et al. AIDS related tumors. Med Pediatr Oncol, 1997; 29: 381. 53 McClain, K. L., Joshi, V. V., & Murphy, S. B. Cancers in children with HIV infection. Hematol Oncol Clin North Am, 1996; 10: 1189–201. 54 Pluda, J. M., Yarchoan, R., Jaffe, E. S., et al. Development of non-Hodgkin lymphoma in a cohort of patients with severe human immunodeficiency virus (HIV) infection on long-term antiretroviral therapy. Ann Intern Med, 1990; 113: 276. 55 Sandlund, J., Fonseca, T., Leimig, T., et al. Predominance and characteristics of Burkitt lymphoma among children with nonHodgkin lymphoma in northeastern Brazil. Leukemia, 1997; 11: 743–6. 56 Gutierrez, M. I., Bhatia, K., Barriga, F., et al. Molecular epidemiology of Burkitt’s lymphoma from South America: differences in breakpoint location and Epstein-Barr virus association from tumors in other world regions. Blood, 1992; 79: 3261–6. 57 Kuhn-Hallek, I., Sage, D. R., Stein, L., et al. Expression of recombination activating genes (RAG-1 and RAG-2) in Epstein-Barr virus-bearing B-cells. Blood, 1995; 85: 1289–99. 58 Wilson, J. B. & Levine, A. J. The oncogenic potential of EpsteinBarr virus nuclear antigen 1 in transgenic mice. Curr Top Microbiol Immunol, 1992; 182: 375–85. 59 Bhatia, K., Raj, A., Gutierrez, M. I., et al. Variation in the sequence of Epstein Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt’s lymphoma. Oncogene, 1996; 13: 177–81. 60 Ruf, I. K., Rhyne, P. W., Yang, H., et al. Epstein-Barr virus regulates c-MYC, apoptosis, and tumorigenicity in burkitt lymphoma. Mol Cell Biol, 1999; 19: 1651–60. 61 Razzouk, B. I., Srinivas, S., Sample, C. E., et al. Epstein–Barr virus DNA recombination and loss in sporadic Burkitt’s lymphoma. J Infect Dis, 1996; 173: 529–35. 62 Sandlund, J. T., Murphy, S. B., Santana, V. M., et al. Central nervous system involvement in children with newly diagnosed non-Hodgkin lymphoma. J Clin Oncol, 2000; 18: 3018–24. 63 Carr, R., Barrington, S. F., Madan, B., et al. Detection of lymphoma in bone marrow by whole-body positron emission tomography. Blood, 1998; 91: 3340–6. 64 Murphy, S. B. Classification, staging and end results of treatment of childhood non-Hodgkin’s lymphomas: dissimilarities from lymphomas in adults. Semin Oncol, 1980; 7: 332–9. 65 Pui, C.-H., Relling, M. V., Lascombes, F., et al. Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies. Leukemia, 1997; 11: 1813–16. 66 Pui, C.-H., Mahmoud, H. H., Wiley, J. M., et al. Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma. J Clin Oncol, 2001; 19: 697–704. 67 Murphy, S. B., Bowman, W. P., Abromowitch, M., et al. Results of treatment of advanced-stage Burkitt’s lymphoma and B cell (sIg+) acute lymphoblastic leukemia with high-dose

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fractionated cyclophosphamide and coordinated high-dose methotrexate and cytarabine. J Clin Oncol, 1986; 4: 1932–9. Bowman, W. P., Shuster, J., Cook, B., et al. Improved survival for children with B cell acute lymphoblastic leukemia and stage IV small noncleaved cell lymphoma: a Pediatric Oncology Group study. J Clin Oncol, 1996; 14: 1252–61. Patte, C., Philip, T., Rodary, C., et al. High survival rate in advanced-stage B-cell lymphomas and leukemias without CNS involvement with a short intensive polychemotherapy: results from the French Pediatric Oncology Society of a randomized trial of 216 children. J Clin Oncol, 1991; 9: 123–32. Patte, C., Leverger, G., Perel, Y., et al. Updated results of the LMB 86 protocol of the French Pediatric Oncology Society (SFOP) for B-cell non-Hodgkin’s lymphomas (B-NHL) with CNS involvement (CNS+) and B-ALL [abstract]. Med Pediatr Oncol, 1990; 18: 397. Reiter, A., Schrappe, M., Ludwig, W. D., et al. Favorable outcome of B-cell acute lymphoblastic leukemia in childhood: a report of three consecutive studies of the BFM group. Blood, 1992; 80: 2471–8. Sposto, R., Meadows, A. T., Chilcote, R. R., et al. Comparison of long-term outcome of children and adolescents with disseminated non-lymphoblastic non-Hodgkin lymphoma treated with COMP or daunomycin-COMP: a report from the Children’s Cancer Group. Med Pediatr Oncol, 2001; 37: 432–41. Cairo, M. S., Sposto, R., Perkins, S. L., et al. Burkitt’s and Burkittlike lymphoma in children and adolescents: a review of the Children’s Cancer Group experience. Br J Haematol, 2003; 120: 660–70 Magrath, I. T., Janus, C., Edwards, B. K., et al. An effective therapy for both undifferentiated (including Burkitt’s) lymphomas and lymphoblastic lymphomas in children and young adults. Blood, 1984; 63: 1102–11. Schwenn, M. R., Blattner, S. R., Lynch, E., et al. HiC-COM: a 2-month intensive lymphoma and B-cell acute lymphoblastic leukemia. J Clin Oncol, 1991; 9: 133–8. Patte, C., Philip, T., Rodary, C., et al. Improved survival rate in children with stage III and IV B cell non-Hodgkin’s lymphoma and leukemia using multi-agent chemotherapy: results of a study of 114 children from the French Pediatric Oncology Society. J Clin Oncol, 1986; 4: 1219–26. Magrath, I., Adde, M., Shad, A., et al. Adults and children with small non-cleaved-cell lymphoma have a similar excellent outcome when treated with the same chemotherapy regimen. J Clin Oncol, 1996; 14: 925–34. Reiter, A., Schrappe, M., Parwaresch, R., et al. Non-Hodgkin’s lymphomas of childhood and adolescence: results of a treatment stratified for biologic subtypes and stage – a report of the Berlin-Frankfurt-Munster Group. J Clin Oncol, 1995; 13: 359–72. Woessmann, W., Seidemann, K., Mann, G., et al. The impact of the methotrexate administration schedule and dose in the treatment of children and adolescents with B-cell neoplasms: a report of the BFM Group Study NHL-BFM95. Blood, 2005; 105: 948–58.

80 Pillon, M., Di Tullio, M. T., Garaventa, A., et al. Long-term results of the first Italian Association of Pediatric Hematology and Oncology Protocol for the treatment of pediatric B-cell non-Hodgkin lymphoma (AIEOP LNH92). Cancer, 2004; 101: 385–94 81 Anderson, J. R., Jenkis, R. D. T., Wilson, J. F., et al. Long-term follow-up of patients treated with COMP or LSA2L2 therapy for childhood non-Hodgkin’s lymphoma: a report of CCG551 from the Children’s Cancer Group. J Clin Oncol, 1993; 11: 1024–32. 82 Adde, M., Shad, A., Venzon, D., et al. Additional chemotherapy agents improve treatment outcome for children and adults with advanced B-cell lymphomas. Semin Oncol, 1998; 25 (Suppl. 4): 33–9. 83 Soussain, C., Patte, C., Ostronoff, M., et al. Small noncleaved cell lymphoma and leukemia in adults. A retrospective study of 65 adults treated with the LMB pediatric protocols. Blood, 1995; 85: 664–74. 84 Patte, C., Auperin, A., Michon, J., et al. The Soci´et´e Franc¸aise d’Oncologie P´ediatrique LMB89 protocol: highly effective multiagent chemotherapy tailored to the tumor burden and initial response in 561 unselected children with B-cell lymphomas and L3 leukemia. Blood, 2001; 97: 3370–9. 85 Reiter, A., Schrappe, M., Tiemann, M., et al. Improved treatment results in childhood B-cell neoplasma with tailored intensification of therapy: a report of the Berlin-Frankfurt¨ Munster Group Trial NHL-BFM 90. Blood, 1999; 94: 3294–306. 86 Mead, G. M., Sydes, M. R., Walewski, J., et al. An international evaluation of CODOX-M and CODOX-M alternating with IVAC in adult Burkitt’s lymphoma: results of United Kingdom Lymphoma Group LY06 Study. Eur Soc Med Oncol, 2002; 13: 1264–74. 87 Kung, F. H., Harris, M. B., & Krischer, J. P. Isofamide/ carboplatin/etoposide (ICE), an effective salvaging therapy for recurrent malignant non-Hodgkin lymphoma of childhood: a Pediatric Oncology Group phase II study. Med Pediatr Oncol, 1999; 32: 225–6. 88 Philip, T., Biron, P., Philip, I., et al. Massive therapy and autologous bone marrow transplantation in pediatric and young adults with Burkitt’s lymphoma (30 courses on 28 patients: a 5-year experience). Eur J Cancer Clin Oncol, 1986; 22: 1015–27. 89 Philip, T., Armitage, J. O., Spitzer, G., et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediategrade or high-grade non-Hodgkin’s lymphoma. N Engl J Med, 1987; 316: 1493–8. 90 Philip, T., Hartmann, O., Biron, P., et al. High-dose therapy and autologous bone marrow transplantation in partial remission after first-line induction therapy for diffuse non-Hodgkin’s lymphoma. J Clin Oncol, 1988; 6: 1118–24. ˜ 91 Bureo, E., Ortega, J. J., Munoz, A., et al. Bone marrow transplantation in 46 pediatric patients with non-Hodgkin’s lymphoma. Bone Marrow Transplant, 1995; 15: 353–9. 92 Ladenstein, R., Pearce, R., Hartmann, O., et al. High-dose chemotherapy with autologous bone marrow rescue in

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children with poor-risk Burkitt’s lymphoma: a report from the European Lymphoma Bone Marrow Transplantation Registry. Blood, 1997; 90: 2921–30. Appelbaum, F., Deisseroth, A. B., Graw, R. G., et al. Prolonged complete remission following high-dose chemotherapy of Burkitt’s lymphoma in relapse. Cancer, 1978; 41: 1059–63. Sandlund, J. T., Bowman, L., Heslop, H. E., et al. Intensive chemotherapy with hematopoietic stem-cell support for children with recurrent or refractory NHL. Cytotherapy, 2002; 4: 253–8. Haddy, T. B., Adde, M. A., McCalla, J., et al. Late effects in longterm survivors of high-grade non-Hodgkin’s lymphomas. J Clin Oncol, 1998; 16: 2070–9. Colombat, P., Salles, G., Brousse, N., et al. Rituximab (antiCD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor

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burden: clinical and molecular evaluation. Blood, 2001; 97: 101–6. Coiffier, B., Haioun, C., Ketterer, N., et al. Rituximab (antiCD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study. Blood, 1998; 92: 1927–32. Veerman, A. J. P., Nuijens, J. H., Schoot, C. E. Van der, Kardos, G. & Zwaan, C. H. M. Rituximab in the treatment of childhood BALL and Burkitt’s lymphoma, report on three cases [abstract]. Blood, 1999; 94(Suppl. 1): 269b. McManaway, M. E., Neckers, L. M., Loke, S. L., et al. Tumourspecific inhibition of lymphoma growth by an antisense oligodeoxynucleotide. Lancet, 1990; 335: 808–11. Magrath, I. T. Prospects for the therapeutic use of antisense oligonucleotides in malignant lymphomas. Ann Oncol, 1994; 5(Suppl. 1): S67–70.

19 Acute myeloid leukemia Jeffrey E. Rubnitz, Bassem I. Razzouk, and Raul C. Ribeiro

Introduction

Epidemiology and risk factors

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies characterized by the proliferation of abnormal leukemic blast cells and impaired production of normal blood cells.1 AML accounts for only 15% to 20% of cases of acute leukemia in children and adolescents, but is responsible for more than one third of the deaths due to leukemia in these age groups. The outcome of treatment for children with AML has improved markedly over the last three decades: about half of all affected children now remain free of disease at 5 years from diagnosis and are probably cured.2–5 This improvement has been accomplished by enrolling pediatric patients in clinical trials, administering more intensive therapy (including hematopoietic stem cell transplantation), and improving supportive care. However, the cure rate for children with AML continues to lag behind that for children with acute lymphoblastic leukemia (ALL). The main reasons for treatment failure are relapse and treatment-related mortality. A major challenge is the development of novel therapies that overcome drug resistance and decrease relapse rates, while reducing the short- and long-term adverse effects of treatment. Recent advances in understanding the molecular heterogeneity of AML and in identifying specific molecular therapeutic targets should provide essential information needed to meet this challenge. Here we update findings on the epidemiology and pathogenesis of AML in children and adolescents and discuss treatment options, supportive care issues, and complications of therapy.

AML accounts for approximately 16% of the acute leukemias in children and adolescents younger than 15 years.6 Approximately 850 new cases of pediatric AML are diagnosed each year in the United States. As in ALL, the incidence of AML varies with age, with the highest rates seen during the first 2 years of life, followed by a decrease that reaches a nadir at about 9 years of age (Fig. 19.1). The ratio of AML to ALL in infants is approximately 1:1; in children aged 1 to 10 years, 1:7; and in adolescents aged 15 to 19 years, 1:3. Incidence rates of AML are approximately equal between boys and girls in all age groups and between white and black children.6 The distribution of pediatric AML cases varies considerably among ethnic groups.7 For example, children of Asian/Pacific island origin appear to have an increased risk of AML, although these observations have not been confirmed.8 The frequency of specific subtypes of AML appears to vary in distinct populations as well. Promyelocytic leukemia is more frequent in persons from Spain, Italy, Mexico, and Central and South America,9 and granulocytic sarcoma is more frequent in children and adolescents from Africa10 and Turkey.11 Because the causes of childhood AML remain elusive, associations between genetic and environmental factors and pediatric AML have been studied to gain insight into disease mechanisms. For example, patients with Down syndrome have a 600-fold greater chance of developing acute megakaryoblastic leukemia (AMKL) than do persons without the syndrome12,13 ; other inherited constitutional

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Fig. 19.1 Incidence of AML according to age at diagnosis. Reprinted, with permission, from Smith et al.6

genetic disorders, including Fanconi anemia,14 Bloom syndrome,15 neurofibromatosis,16 Noonan syndrome,17 and congenital forms of neutropenia,18 have also been associated with an increased risk of myeloid malignancies (see Chapter 13). Familial predisposition to AML has been noted in persons with germline haploinsufficiency of the AML1 gene.19 In other families, a still unidentified inherited genetic abnormality results in a predisposition to monosomy 7 in the hematopoietic progenitor cells, thereby leading to the development of myeloid disorders, including AML.20,21 Nongenetic risk factors include exposure to ionizing radiation,22,23 alkylating agents,24 and topoisomerase II inhibitors.25 Other factors for which suggestive but inconclusive evidence has been gathered are consumption of alcohol and use of recreational drugs during pregnancy26,27 and exposures to insecticide,28 benzene,29 or high concentrations of radon.30 Exposure to electromagnetic fields has been associated with an increased incidence of leukemia in several studies but not in others.31 Given the imperceptibility, pervasiveness, and multiple sources of low-frequency electric and magnetic fields, reliable quantitative data on the relation of these environmental factors to AML induction have been difficult to obtain. In a large pooled analysis, children postnatally exposed to electromagnetic fields that exceeded 0.4 T had a 2-fold increased risk of acute leukemia.31 However, less than 1% of all children were exposed to levels above 0.4 T, suggesting that these results may be partly due to bias.

Recent pharmacogenetic studies have shown a genetic basis for differences among individual responses to specific drugs or environmental xenobiotics.32 Thus, persons who inherit alleles for relatively inactive drug-metabolizing enzymes may be more susceptible to malignancies. A higher prevalence of polymorphisms that result in inactive detoxifying enzymes has been associated with primary leukemia.33–35 However, it is still unclear whether enzyme polymorphisms explain the uneven ethnic and geographic distribution of AML. Because there is evidence that some of the critical leukemogenic events in AML occur in utero,36,37 and that some of these genetic changes are the same as those observed in association with secondary AML induced by topoisomerase II inhibitors, investigators have suggested that intrauterine exposure to naturally occurring topoisomerase II inhibitors could contribute to the development of childhood acute leukemias. This area of active research is discussed fully in Chapter 3.38,39

Pathogenesis The analysis of proteins altered by chromosomal translocations or mutations in AML has provided valuable insights into the underlying pathogenesis of this disease.40–42 Because leukemic cells are characterized by proliferation and survival advantages as well as by impaired differentiation, it is not surprising that the proteins altered in AML

Acute myeloid leukemia

Table 19.1 Proteins altered in AML Proteins involved in proliferation and survival

Gene ABL PDGFRβ FLT3 RAS c-KIT

Mechanism of alteration Fusion (BCR-ABL) Fusion (TEL-PDGFRβ) Mutation Mutation Mutation

Proteins involved in differentiation

Gene AML1

Mechanism of alteration

CBFβ

Fusion (e.g. AML1-ETO) or point mutation Fusion (CBFβ-MYH11)

PML MLL HOX genes C/EBPα PU.1 GATA1

Fusion (PML-RARα) Fusion (e.g. MLL-AF9) Fusion or mutation Mutation Mutation Mutation

form two distinct functional groups (Table 19.1). Constitutively activated tyrosine kinases, such as BCR-ABL, cKIT, and FLT3, impart proliferative and survival signals to leukemic blast cells.42,43 The alteration of proteins involved in transcriptional regulation, such as core binding factor (CBF), retinoic acid receptor alpha (RAR
), CCAATenhancer binding protein alpha (C/EBP-
), MLL, and HOX proteins, leads to impaired differentiation.41,44,45 It has been proposed that the full leukemic phenotype requires the presence of both types of mutation in the same cell.40,46 Although a full discussion of each of the proteins is beyond the scope of this chapter, we will discuss the roles of FLT3 and CBF, proteins that exemplify the two major classes of mutation in AML. The reader is referred to Chapter 11 for a comprehensive discussion of the molecular pathogenesis of AML. FLT3, a receptor tyrosine kinase expressed on early hematopoietic precursors, is essential for normal immune system development.43 Binding of FLT3 by FLT3 ligand causes dimerization of the receptor, activation of its tyrosine kinase, and autophosphorylation. Subsequent signal transduction is imparted by the phosphorylation of a variety of proteins, including RAS-GAP, PLC , STAT5a, and ERK1/2. In most cases of AML, FLT3 is expressed at high levels; in about one third of cases, it is mutated. Approximately 10% of pediatric AML cases and 25% of adult cases have activating internal tandem duplications (ITDs) of FLT3, while an additional 5% to 10% have activating point mutations.47 These mutations lead to ligand-independent dimerization and autophosphorylation of FLT3, activation of downstream targets, and factor-independent growth in culture. In addition, expression of FLT3-ITDs in a murine model of bone marrow transplantation results in a myeloproliferative disorder similar to that caused by other acti-

vated kinases; this result demonstrates that constitutively activated protein kinases provide advantages in proliferation and survival but do not cause the impaired differentiation that characterizes AML. These observations therefore suggest that cooperation between kinase mutations and transcription factor mutations is necessary for the full leukemic phenotype. Because FLT3-ITDs are seen in many different morphologic and cytogenetic subtypes of AML, it is likely that the mutated proteins cooperate with a variety of abnormal transcription factors, including AML1-ETO, CBF-MYH11, PML/RAR
, and others, to cause AML. Core binding factor, a heterodimeric transcription factor composed of AML1 (also referred to as RUNX1, CBFA2, and PEBPA2B) and CBF, is a master regulator of the entire hematopoietic system.40 CBF is also a frequent target of leukemia-associated chromosomal rearrangements, including t(8;21) and inv(16) in AML, t(12;21) in ALL, and t(3;21) in myelodysplastic syndrome (MDS). The fusion proteins created by these rearrangements function as transcriptional repressors or dominant-negative inhibitors of CBF by recruiting complexes that contain the nuclear corepressor NCoR, Sin3A, and histone deacetylase to sites normally activated by CBF. Mice that express AML1-ETO or CBF-MYH11 have a phenotype nearly identical to that of mice that lack AML1 or CBF – they die during embryogenesis because of a lack of definitive hematopoiesis. This result suggests that the fusion proteins cause complete loss of function of CBF. The function of CBF may also be lost as a result of sporadic mutations of AML1 in about 5% of AML cases and by inherited AML1 mutations in the familial platelet disorder with propensity to develop acute myelogenous leukemia (FPD/AML) syndrome. However, like the mutations in FLT3 described above, alterations of CBF alone are not sufficient to cause AML. In patients with FPD/AML, overt AML does not develop for many years, suggesting that AML development requires the acquisition of additional mutations. Similarly, mouse models of AML1ETO and CBF-MYH11 expression demonstrate that these fusions cause defects in differentiation, but that second mutations are required for leukemia to develop. Identification of the secondary events that lead to leukemia in mice expressing FLT3-ITDs, AML1-ETO, or CBF-MYH11 may provide direct evidence for interactions between altered transcription factors and mutated tyrosine kinases, and should therefore lead to a greater understanding of the pathogenesis of AML.

Clinical presentation and diagnosis Acute myeloid leukemia arises from the uncontrolled proliferation and abnormal differentiation of a transformed

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Fig. 19.2 Periorbital chloroma in a 12-year-old boy with AML with the t(8;21).

stem cell that normally gives rise to the myeloid, erythroid, monocytic, or megakaryocytic lineage. Although the term AML is indiscriminately applied to this process, there is considerable clinical and biologic heterogeneity among the various subclasses of this disease, including response to therapy. This heterogeneity is not surprising in view of the diverse roles of bone marrow cells in hematopoiesis. In the last decade, there have been repeated attempts to design therapies for different types of AML. The success of this approach has been amply demonstrated in patients with acute promyelocytic leukemia; therefore, precise characterization of bone marrow disorders involving myeloid, erythroid, monocytic, or megakaryocytic cells is critical for optimal patient management and for evaluation of specific therapies. The diagnosis and classification of AML continues to evolve.48 Specific criteria for the diagnosis and

Fig. 19.3 Extradural cord compression by chloromas in an 18-year-old male with AML and the t(8;21) translocation. Sagittal T1-weighted MRI scan demonstrates a large epidural mass compressing the lower lumbar nerve roots (lower arrows). A smaller epidural deposit of myeloid cells is present at the level of the conus (upper arrow).

classification of AML, myelodysplastic syndromes, and myeloproliferative disorders were initially developed by the French-American-British (FAB) Cooperative Group in 1976.49 These investigators further refined their criteria to include information disclosed by immunophenotype analysis.50,51 The eight FAB categories of AML (M0 to M7) are based on the percentage, lineage commitment, and differentiation of the leukemic cells as revealed by morphologic, cytochemical, and immunophenotypic analyses. Although the FAB classification has gained wide acceptance and has been used in most contemporary clinical trials, it has several shortcomings, particularly in the pediatric age group, where strict application of FAB criteria may fail to diagnose or classify disease for a substantial number of patients.52–54 More recently, an effort has been made to combine the morphologic, cytochemical, and immunologic data previously

Acute myeloid leukemia

Fig. 19.4 Gingival hypertrophy in a patient with monoblastic leukemia. (See color plate 19.4 for full-color reproduction.)

established by the FAB group with those provided by genetic and clinical findings.55–57 It is hoped that this classification of hematologic malignancies (sponsored by the World Health Organization) will have clinical relevance by discriminating between cases with unusual natural histories and responses to therapy.58 The diagnosis of AML is often suggested by the patient’s clinical manifestations and the examination of his or her peripheral blood. Most patients have prodromal signs and symptoms including fever, pallor, weakness, fatigue, anorexia, and weight loss. Less common clinical features include bone pain, inflammation of the mucosal membranes of the upper airways (cough, nasal discharge, sore throat), and abdominal pain. The duration of signs and symptoms before diagnosis is usually 4 to 6 weeks. Severe infections can be the first sign in many pediatric patients with AML. Rarely, the predominant clinical manifestations result from the compressive effects of myeloid “tumors” or extramedullary deposits (granulocytic sarcoma, chloroma, myeloblastoma, megakaryoblastoma) in the CNS. These masses tend to emerge from bones with intense hematopoietic activity and a delicate periosteum, such as those of the orbital floor and vertebral bodies. Therefore, patients with chloromas commonly first appear with ocular-orbital infiltration (Fig. 19.2) and extradural cord compression (Fig. 19.3). Mucous membrane and cutaneous hemorrhage are common at the time of diagnosis of AML; more serious bleeding occurs only rarely. More than half of the children have an enlarged liver, spleen, or lymph nodes, but marked enlargement is rare. Accentuated gingival hypertrophy (Fig. 19.4) is seen in about 10% of the children. Skin and subcutaneous nodules, which are more

common in infants (Fig. 19.5), are seen in about 1% to 2% of all pediatric cases.59,60 The cutaneous lesion can manifest as a solitary or multiple reddish to violaceous papules, plaques, or nodules of varied sizes (0.5 to 3 cm), or as maculopapular rashes. Some of the lesions are indistinguishable from the classic “blueberry muffin” lesion. The skin nodules sometimes develop before involvement of the bone marrow, and in some cases they wax and wane. Complete spontaneous regression of the nodules has been reported.61–63 The median WBC count at diagnosis is approximately 20 × 109 /L and is less than 50 × 109 /L in about 70% of patients (Table 19.2). The median initial hemoglobin concentration is around 8 g/dL. Although the median initial platelet count is about 70 × 109 /L, 15% of children have platelet counts below 10 × 109 /L. Careful examination of the peripheral blood smear reveals leukemic cells in most cases. However, 10% of children, including many of those with chloromas, do not have circulating blasts. Prolonged prothrombin, thrombin, and partial thromboplastin times; decreased fibrinogen levels; and increased circulating fibrin degradation products [laboratory evidence of disseminated intravascular coagulation (DIC)] are seen in about 5% of pediatric patients with AML.64 Factors associated with DIC include the type of AML (M3, M4, M5), hyperleukocytosis, and infection. Hypokalemia, hypophosphatemia, hypocalcemia, and hypoalbuminemia can be associated with FAB M4/M5 AML as a result of renal tubular dysfunction caused by lysozyme released from the leukemic cells. Hyperuricemia, a common problem in patients with acute lymphoid malignancies, is seen less frequently in AML. Radiographs of the chest are usually unremarkable in patients with AML; very rarely is there a mediastinal mass.65,66 In those rare cases, the possibility of myeloid transformation of a germ cell tumor should be considered.67 The diagnosis of AML is confirmed by examining the bone marrow. Bone marrow aspiration and biopsy can be performed safely for most patients, even for those with severe thrombocytopenia. Examination of the bone marrow aspirate provides a qualitative assessment of the leukemic cells, whereas the bone marrow core biopsy provides a quantitative assessment of the bone marrow cellularity. The posterior iliac crest is the preferred site for aspiration and biopsy in most children; in children younger than 3 months, the tibial tuberosity is the preferred site. An effort should be made to obtain enough bone marrow for WrightGiemsa and enzyme cytochemical staining and for cytogenetic, immunophenotypic, and molecular studies. When 20% or more of the nucleated bone marrow cells are blasts, a diagnosis of AML can be made. In more

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Fig. 19.5 Leukemia cutis in congenital myeloid leukemia with the t(9;11). (See color plate 19.5 for full-color reproduction.)

than 80% to 90% of these cases, the myeloid lineage can be determined from morphologic and cytochemical studies. In the remaining cases, immunophenotypic analysis is necessary to establish the lineage. Immunophenotyping is especially helpful in cases of FAB M0 or M7. Conventional cytogenetic studies and molecular genetic studies are crucial for further classification of AML into prognostically relevant categories. The information provided by the genetic analysis of the leukemic cells complements that generated by other techniques and often points to the correct diagnosis. For example, the presence of an AML-specific genetic lesion such as AML1-ETO [t(8;21)] or RBM15-MKL1 [t(1;22)] establishes the diagnosis of acute myeloid and megakaryoblastic leukemia, respectively, regardless of the blast cell count obtained by the bone marrow examination. The current classification system for AML and the identification of additional prognostically relevant indicators will probably be enhanced by sophisticated techniques that characterize detailed gene expres-

sion profiles (genomics)68 and by protein modulation (proteomics). In addition to bone marrow disease, AML may involve the CNS at the time of diagnosis because of meningeal infiltration, chloromas of epidural or brain parenchyma, or leukostasis.69 Affected patients are usually asymptomatic, but those with diffuse leptomeningeal infiltration may have headache, vomiting, photophobia, papilledema, retinal hemorrhages, or cranial nerve palsies at initial examination.70–72 In patients with hyperleukocytosis and cerebral leukostasis, nodular brain masses may develop, and symptoms of discrete brain tumors resulting from blast cell invasion and penetration of vessel walls may be seen. Seizures are not common. The diagnosis of CNS leukemia is usually made by microscopic examination of the cerebrospinal fluid (CSF). The number of white blood cells in the CSF needed to diagnose CSF involvement in AML varies among study groups. Some investigators define CNS involvement

Acute myeloid leukemia

as the presence of any leukemic blast cells in the CSF, regardless of cell count.72–74 Others define CNS involvement as the presence of at least 5 or 10 white blood cells per microliter with leukemic blast cells present.75–77 Depending on the definition used, the incidence of CNS leukemia at diagnosis ranges from 5% to 30%.70–78 The diagnosis of CNS involvement is sometimes based on signs such as cranial nerve palsy or radiologic evidence of leukemic infiltration in the CNS. Involvement of the CNS is more common in infants and in patients with FAB subtypes M4 and M5, as well as in those with hyperleukocytosis. In the differential diagnosis of pediatric AML, several malignant and nonmalignant conditions should be considered. In the newborn, special attention should be given to reactive processes due to congenital infections. Occasionally, syphilis, toxoplasmosis, and cytomegalovirus infections can mimic leukemia in the newborn. In the young child, megakaryoblastic leukemia can first appear as abdominal abnormalities that result from the leukemic cell deposits (megakaryoblastoma) in the abdominal structures. In patients with osteolytic lesions, which are often associated with AMKL, and in patients whose bone marrow possesses leukemic cell aggregates in a background of marrow fibrosis, solid tumors79 or other disorders are often thought to be present.80 Megakaryocytic marker expression and cytogenetic or molecular findings are used to confirm the diagnosis of AML. In some cases of mononucleosis accompanied by anemia and thrombocytopenia, the diagnosis of acute leukemia is often considered. Epstein-Barr virus (EBV) serologic studies and immunophenotyping of the peripheral mononuclear cells establish the diagnosis of EBV infection. In rare cases, severe bacterial infection can cause bone marrow suppression with strong myeloid response in the peripheral blood, but these processes are usually self-limited and respond to antibiotic therapy. Abnormalities of a single or multiple hematopoietic lineages can present diagnostic dilemmas. Aplastic anemia and MDS are the diagnoses most commonly considered for patients with hypoplastic marrow characterized by a paucity of immature cells. Immunophenotyping is helpful in cases in which the bone marrow cells express clonal markers. Cytogenetic examination of the bone marrow may reveal abnormalities specific for AML. Myelodysplastic syndromes sometimes can be difficult to differentiate from AML. A history of refractory anemia with typical changes in the bone marrow morphology is consistent with the diagnosis of MDS. Patients in whom less than 20% of blasts have morphologic dysplastic features but possess translocations specific for AML, such as the t(8;21)(q22;q22), should be considered to have AML regardless of the blast cell count.

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Table 19.2 Clinical and laboratory characteristics of AML patients treated at St. Jude Children’s Research Hospital (1980–96)

Feature Gender Male Female Age ≤2 years >2 years Hemoglobin <8 g/dl 8–10 g/dl >10 g/dl Platelet count ≤50 × 109 /L >50 × 109 /L Hepatomegaly Present Absent Splenomegaly Present Absent CNS statusb CNS1 CNS2 CNS3 NA Leukocyte count (× 109 /L) <50 ≥50 FAB type M1 M2 M3 M4 M5 M7 Other Genetic group t(9;11) inv(16) or CBFβ-MYH11 t(8;21) or AML1-ETO 11q23 or R-MLL Others

Number (%)

5-year event-free survival (% ± SE)a

161 (54) 137 (46)

25.2 ± 3.6 33.0 ± 4.5

58 (19) 240 (81)

32.8 ± 6.2 27.9 ± 2.9

100 (34) 118 (40) 79 (26)

31.0 ± 4.6 27.1 ± 4.2 29.1 ± 5.0

144 (48) 153 (52)

27.1 ± 3.7 30.7 ± 3.8

157 (47) 141 (53)

26.8 ± 3.6 31.2 ± 3.9

123 (41) 175 (59)

23.6 ± 3.9 32.6 ± 3.5

205 (69) 37 (12) 48 (16) 8 (3)

27.8 ± 3.2 24.3 ± 7.5 43.7 ± 7.0

209 (70) 89 (30)

33.2 ± 3.5 18.2 ± 4.5

52 (18) 78 (26) 18 (6) 51 (17) 63 (21) 20 (7) 13 (4)

17.3 ± 5.2 30.7 ± 5.7 44.4 ± 11.0 26.1 ± 6.5 40.6 ± 7.2 10 ± 5.5 15.4 ± 8.2

23 (8) 19 (6) 40 (13) 33 (11) 180 (60)

64.9 ± 11.1 36.1 ± 10.9 32.5 ± 7.4 24.2 ± 9.4 23.5 ± 3.4

P value 0.20

0.55

0.85

0.27

0.41

0.25

0.04

0.02

0.002

0.003

Abbreviations: AML, acute myeloid leukemia; SE, standard error; FAB, FrenchAmerican-British; R-MLL, rearrangements of the MLL gene. a The Kaplan–Meier method was used in the survival analyses, and the log-rank test was used in comparisons between survival distributions. b CNS1, no blast cells in CSF; CNS2, <5 WBC/ L CSF with blast cells; CNS3, ≥5 WBC/ L CSF with blast cells or signs of CNS involvement. NA, not available.

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A M6 (1-5%)

Classification and important subgroups

Granulocytic sarcoma M0 (0-1%) M7 (1-6%) (4-14%)

M1 (11-19%) M2 (25-30%)

M5 (13-29%)

M4Eo (2-6%) M4 (15-23%)

M4Eo (4-6%)

M5 (9-24%)

M3 (3-12%)

M6 (2-4%)

M7 (1-3%)

M0 (1-6%)

M1 (15-24%) M2 (23-36%)

M4 (13-29%)

M3v (0-3%)

B

M3 (2-10%) Inv16 [5-6%]

Other [26-62%]

t(8;21) [12-16%] t(15;17) [9-12%]

Normal [18-32%]

t(9;11) [2-4%] -7 [2-6%]

t(8;21) [5-7%] Inv16 [4-8%]

For many years, pediatric and adult AML were considered similar entities. However, recent evidence has indicated that AML in pediatric patients differs in many ways from the adult disease; this evidence is based on careful clinical observation of children with AML in conjunction with the systematic characterization of the leukemic cells by morphologic, immunophenotypic, and genetic studies. A marked separation between the pediatric and adult forms of AML should come as no surprise, in view of the plasticity of the hematopoietic system during embryonic, fetal, and postnatal development. In fact, many of the transformation events specific to AML may occur in utero.81,82 Some of these differences can be appreciated from a comparison of FAB morphologic and selected genetic subtypes in pediatric and adult AML. Figure 19.6 depicts the median frequencies and ranges of these AML subtypes in children2,5,75,76,83–89 and adults.90–95 Table 19.2 shows the distribution and treatment responses of major genetic subtypes among patients treated at St. Jude Children’s Research Hospital. For example, AML with 11q23 abnormalities comprises more than 80% of infants with AML, 15% of other age groups in children, and only 2% of adults with AML.88,90,96,97 Moreover, some types of AML, such as megakaryoblastic leukemia with the t(1;22) abnormality, are extremely rare, if even present, in adults.98 Below are the descriptions of selected subtypes of AML.

t(15;17) [5-13%]

-5 [0-2%]

t(9;11) [1-3%]

11q23 [6-19%]

11q23 [3-4%]

AML in the infant or young child

-5 [7-9%]

-7 [8-10%]

Others [4-37%] Normal [43-50%]

Fig. 19.6 Morphologic (A) and cytogenetic (B) subtypes of AML in children (upper charts) and adults (lower charts). Percentages in parentheses reflect the variation of these subtypes among several reported clinical trials.

Although infrequent, AML that occurs during the first 2 years of life has distinct clinical and biological characteristics.99–102 About 20% of the cases of childhood AML are in children younger than 24 months; half of these cases occur during the first 12 months.99 Congenital AML, which accounts for less than 5% of the childhood AML seen in the first 24 months (73 cases reported in the world literature between 1975 and 200062 ), is regarded as AML diagnosed during the perinatal period or the first 4 weeks of life. The term infant AML is applied when the disease occurs in patients older than 1 month but younger than 12 months, whereas the term AML in young children is applied when the disease is diagnosed in children older than 1 year but younger than 2 years. Although segregating AML into these three groups on the basis of patient age is arbitrary, this separation has some merit. The hallmark of congenital AML is the very short latency period between unknown leukemogenic events that occur in utero and clinical manifestations of the

Acute myeloid leukemia

disease. Nonmalignant conditions that mimic leukemia in the newborn include transient myeloproliferative disorder associated with children with Down syndrome, severe bacterial infections, fetomaternal blood incompatibilities, intrauterine viral infections, syphilis, and toxoplasmosis. Other diseases include disseminated neuroblastoma, rhabdomyosarcoma, and Langerhans histiocytosis. The clinical and laboratory manifestations of congenital AML may be present at birth or appear in the subsequent weeks. In a review of 15 cases of congenital AML, the disease in 10 patients was diagnosed in the first or second day of life, whereas that in the other 5 cases was diagnosed at uniformly distributed times in the subsequent weeks.96 Skin involvement (leukemia cutis) is seen in up to 70% of cases of congenital AML (Fig. 19.5). Massive hepatosplenomegaly is also common. The median WBC count was 98 × 109 /L (range, 3.4 to 342 × 109 /L) in one study.62 FAB M4 or M5 subtypes are seen in 80% of the cases. Chromosomal abnormalities involving the 11q23 region are found in about 80% of the cases. Central nervous system leukemia is seen in about 50% of these newborns. Although patients with congenital AML have been thought to have a very poor prognosis, it is plausible that intensive chemotherapy with improved supportive care may cure a large proportion of these children. This view is supported by recent observations showing that a substantial number of children with congenital AML can be cured with current AML therapies.99,103 Spontaneous remission of congenital AML, which has been well documented,61,62 adds another dimension to the complexity of caring for these newborns. The decision whether to treat these patients with intensive chemotherapy or to wait to see whether the disease regresses spontaneously is difficult. The characteristic “blueberry muffin” appearance of the newborn, high WBC count, FAB M5 morphology, and nonspecific cytogenetic abnormalities have been reported for cases in which spontaneous regression occurred. However, abnormalities of chromosome band 11q23 have not been observed in patients with disease that spontaneously regressed; this finding suggests that this marker can be used to select newborns who need immediate treatment for leukemia. There is overlap between the presenting features of congenital AML and AML that occurs during the first 2 years of life. However, the relative proportion of cases with FAB M4/M5 subtype and those with 11q23 abnormalities progressively decreases in relation to other subtypes of AML. A study comparing the presenting features of AML in infants (12 months and younger) or children aged 13 to 24 months at diagnosis with those in children older than 24 months at diagnosis showed that hyperleukocytosis, CNS involvement, FAB M4/M5 subtype, and the t(9;11)

and other 11q23 abnormalities were significantly associated with both younger age groups. Except for a slightly higher frequency of AML FAB M4/M5 among infants, the other features at diagnosis did not differ between children 12 months and younger and those aged 13 to 24 months.104

Megakaryoblastic leukemia Acute megakaryoblastic leukemia (FAB M7, AMKL) is truly a disorder of young children. Recognized as a distinct group of leukemia by the FAB group in 1985, AMKL accounts for about 12% to 15% cases of primary childhood AML and 50% of the AML in children with Down syndrome.51,98 AMKL can also occur as secondary AML.98,105 In adults, AMKL comprises less than 1% of cases of primary AML.98,106,107 The morphology associated with pediatric AMKL can be confused with that of ALL of the FAB L2 subtype. In addition, bone marrow fibrosis, which is common in AMKL, makes the diagnosis difficult to establish. Usually, the bone marrow aspirate is described as “dry tap” because it is impossible to obtain bone marrow for testing. Because of the paucity of leukemic cells in the peripheral blood and bone marrow and the complex clinical presentation of the patients, AMKL in many children is initially incorrectly diagnosed as myelosclerosis, MDS, or solid tumor. Bone lesions are relatively common in AMKL, but their characteristics differ from those found in association with other subtypes of childhood leukemia.108 Extramedullary involvement is common (Fig. 19.7). The difficulties in diagnosis partially explain the differences in frequency of AMKL in several pediatric AML clinical trials. In a study conducted at St. Jude Children’s Research Hospital,105 the median age of 29 children with primary AMKL was 22 months; no child was older than 3 years. None of the children had a clinical history consistent with MDS. The median WBC and platelet counts at the initial examination were 12.1 × 109 /L (range, 3.3 to 59.2 × 109 /L) and 40.0 × 109 /L (range, 3.0 to 352.0 × 109 /L), respectively. Bone marrow aspirates were frequently hypocellular. The malignant blasts in the bone marrow showed cytoplasmic surface blebs in all cases, and binucleate blasts were frequent. In many cases, the blast cells tended to clump. The leukemic cells of all children tested showed alpha naphthyl acetate esterase activity with a characteristic multifocal, punctate cytoplasmic staining pattern. Alpha naphthyl acetate esterase activity was usually inhibited by sodium fluoride. In 75% of the cases, bone marrow sections stained positive for reticulin. An apparent relation existed between reticulin fibrosis in bone marrow aspirates and both the presence of spicules and overall cellularity of the samples. All the leukemic cells that were tested were negative for T-cell

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A

B

D C

Fig. 19.7 Extramedullary involvement in a patient with megakaryoblastic leukemia. (A) Axial CT image of the head with bone windows demonstrates multiple sites of poorly defined permeative bone destruction (arrows). (B) Axial CT image of the abdomen through the level of the kidneys demonstrates an enhancing soft tissue mass replacing the right pedicle of a lower thoracic vertebral body (arrow). Note deviation of the thecal sac by the epidural soft tissue component. (C) Posteroanterior chest radiograph with inclusion of a portion of the left shoulder demonstrates periosteal reaction of the proximal humeral diaphysis (arrow). (D) Anteroposterior image of the femurs from a 99m Tc-MDP bone scan shows intense abnormal avidity of the left distal femoral diaphysis (arrow). There is similar but less marked avidity involving the right proximal lateral femur.

markers (CD3, CD5), B-cell markers (CD19, CD79a, CD10, CD20), and the monocyte marker CD14. The CD33 antigen was expressed by leukemic blast cells in most cases. Atypical expression of the lymphoid-associated antigens CD2, CD7, or both was commonly found. At least one plateletassociated antigen (CD36, CD41a, CD41b, or CD61) was present in all cases in which enough malignant cells were available for immunophenotyping. Cytogenetic studies frequently showed multiple complex karyotypic anomalies, abnormalities of chromosome 3, trisomy 8, trisomy 19, and trisomy 21. The specific AMKL-associated translocation, t(1;22), was noted in one infant. Chromosome band 11q23 aberrations were identified in one third of the children.

AML in children with Down syndrome Transient leukemia (TL), also referred to as transient myeloproliferative disorder (TMD) or transient abnormal myelopoiesis, is estimated to occur in about 10% of children with trisomy 21.109 Typically, the abnormal myeloproliferation is noted in an asymptomatic newborn with Down syndrome who incidentally is found to have an abnormal blood cell count and differential count. The child usually remains well and the blood counts return to normal by 3 months of age. Although this process is considered to be specific for Down syndrome, rarely, newborns without Down syndrome stigmata have had TL. Most of these newborns have

Acute myeloid leukemia

a constitutional abnormality including mosaic trisomy 21, trisomy 18, or Noonan syndrome. An estimated 20% of newborns with TL develop overt AMKL by the age of 3 years.12,109,110 The WBC count and the blast cell count are usually increased in patients with TL. Anemia and thrombocytopenia can be seen, and hepatosplenomegaly is common. Bone marrow examination usually shows trilineage representation with an increased number of blasts. The blast cell count in the bone marrow is characteristically lower than that in the peripheral blood. Morphologic and immunophenotypic analyses often indicate that these cells represent a clonal expansion of an erythroblast/megakaryoblast progenitor. Blasts are negative for myeloperoxidase and Sudan Black and often focally positive for alpha-naphthyl nonspecific esterase, which is inhibited by sodium fluoride. Interestingly, blasts are negative for butyrate acetate esterase. Acquired chromosomal abnormalities are found in about 30% of the cases. Numeric or structural abnormalities of chromosome 21 are the most common karyotypic changes. Other clonal chromosomal abnormalities have also been described. Despite these clonal chromosomal abnormalities, spontaneous remission may occur via an ill-defined mechanism.111 Occasionally, TL is associated with life-threatening complications.109 Increased levels of conjugated bilirubin can be a sign of a severe liver failure.112,113 Histologic analysis of liver sections shows marked sinusoidal and lobular fibrosis. Extramedullary hematopoiesis and megakaryoblastic infiltration are noted. Pancreatic fibrosis and megakaryocytic infiltration of other organs can also occur. Fibrosis is thought to be the result of platelet-derived growth factor activity, which is abundant in the leukemic cells. A second life-threatening complication is cardiopulmonary disease that appears as pulmonary edema, pericardial effusions, and ascites. Like the liver disease seen in TL, this process may also be progressive and fatal. The initial management of TL consists of providing supportive care and parental reassurance that the process is self-limited. Occasionally, the WBC count exceeds 150 to 200 × 109 /L, and some form of cytoreductive procedure is needed to avoid complications of leukostasis. Exchange transfusion and low-dose cytarabine can be used to decrease the hemodynamic consequences of increased circulating blasts. Although there are no data to suggest that liver fibrosis can be reversed with chemotherapy, it is reasonable to recommend cytotoxic therapy for children with TL and increasing bilirubin levels. The rationale for this approach is the observation that bone marrow fibrosis in AMKL is reversed with conventional AML therapy.

Because 20% to 30% of the newborns with a history of TL experience AMKL during the first 3 years of life, they should undergo regular blood counts during this risk period. Primary pediatricians and parents should be aware that bone marrow insufficiency (anemia or thrombocytopenia) can be the initial signs of impending AMKL. In general, children with Down syndrome are predisposed to leukemia and myeloproliferative/myelodysplastic syndrome, even in the absence of a history of TL.12,111,114,115 The incidence of leukemia peaks between birth and 4 years of age. The increased incidence of leukemia seen in this age group is due to AMKL, which is 600 times more frequent in children with Down syndrome than in those without. During the first few years of life, the ratio of AML cases to ALL cases is 4, which reflects this increased incidence. The ratio of AML cases to ALL cases decreases to 1.7 in children aged 5 to 15 years, representing a decrease in the incidence of AML in children of this age group. In a study of 307 patients with Down syndrome and AML registered in the Danish Cancer Registry, only five (1.6%) were older than 5 years. This age distribution is clearly distinct from that seen in children without Down syndrome and AML, in which the incidence increases with age. Cytogenetic abnormalities in AML also differ between children with or without Down syndrome. The t(8;21), inv16, t(16;16), t(15;17), and t(9;11), which are found in almost 40% of pediatric patients with AML, are rarely found in those with Down syndrome. In addition, the t(1;22), which is associated with infant AMKL, is rarely observed in children with Down syndrome and AMKL. Several recent reports have shed light on the pathogenesis of abnormal hematopoiesis in children with Down syndrome.116–119 GATA1 is a hematopoietic transcription factor that is essential for normal erythroid and megakaryocytic differentiation. In one report, somatically acquired mutations of GATA1 were detected in AML blasts from six patients with Down syndrome and AMKL, but were not found in blasts from patients with Down syndrome and other types of leukemia or from patients with AMKL who did not have Down syndrome.116 Recently, GATA1 mutations have also been detected in blasts from patients with Down syndrome and TL, suggesting that mutagenesis of GATA1 may be an initiating event in the leukemogenesis of Down syndrome.117–119 Myelodysplastic syndrome is closely associated with AML in children with Down syndrome. In fact, approximately 25% of pediatric patients with MDS also have Down syndrome. A history of prolonged thrombocytopenia and refractory anemia are the most common features at initial examination. Examination of the bone marrow usually reveals erythroid or megakaryocytic dysplasia or both. Leukemia develops within months to years after the

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diagnosis of MDS. If left untreated, MDS almost always progresses to AML. Because AML in patients with Down syndrome has a relatively good prognosis,114,120 pre-emptive treatment of MDS in these patients is controversial. There are no data to support the benefit of bone marrow transplantation for patients with MDS and Down syndrome. Disease in these patients is usually managed with supportive care until it is clear that MDS has progressed to leukemia. Although AML in patients with Down syndrome usually does not harbor one of the favorable cytogenetic abnormalities, and is often preceded by an MDS phase (features generally associated with a poor prognosis in AML), the disease is very sensitive to conventional AML therapy121 ; therefore, a large proportion of children with Down syndrome and AML are usually cured.120 In a recent trial conducted by the Children’s Cancer Group, children with Down syndrome and AML had an excellent outcome, with an estimated 8-year event-free survival of 77%.120

Childhood monosomy 7 syndrome Infantile or childhood monosomy 7 syndrome describes a type of bone marrow dysfunction that is associated with a complete loss of one copy of chromosome 7 in children younger than 4 years. Bone marrow abnormalities range from myelodysplastic/myeloproliferative changes to frank AML.122 There has been evidence of multilineage involvement in this disorder, although the lymphoid progenitor appears to be spared in most cases.20,123–127 In these cases, AML is thought to progress from myelodysplasia. Typically, these children are aged 6 months to 2 years, and most are boys. Features at the initial examination include a history of recurrent infection, fever, pallor, and bleeding. Hepatosplenomegaly and adenopathy are common. Anemia, macrocytosis, thrombocytopenia, and leukocytosis with monocytosis are present. Hemoglobin F concentration is usually in the normal range for similarly aged children but can be increased in rare cases.128 Functional neutrophil defects have been demonstrated.129,130 Familial occurrence of AML or myelodysplasia has been noted in some of these cases. There is no sex predominance in the familial cases. The median age at diagnosis of the familial cases is higher than that of sporadic cases. Another interesting observation is that in some of these situations, monosomy 7 was an incidental finding in the examination of bone marrow of siblings who were being evaluated as potential bone marrow donors.124 In these healthy children, the bone marrow function was only mildly compromised. Molecular studies in familial cases indicated that the parental source of the retained chromosome 7 varies131 ; this variation suggests that the loss of chromosome 7 is

not a primary genetic event and probably is associated with progression of bone marrow dysfunction. This concept is corroborated by the evidence of other constitutional genetic syndromes such as neurofibromatosis and Down and Noonan syndromes, in which loss of chromosome 7 is a common finding when bone marrow dysfunction develops. Added to the puzzling disease course is the observation that in rare cases monosomy 7 of the bone marrow cells has spontaneously disappeared.21 Some of these children had active Epstein–Barr virus infection at the time of the diagnosis of monosomy 7.21 Whether childhood monosomy 7 is a specific childhood bone marrow disorder is controversial. Because monosomy 7 or 7q deletions occur in about 35% of myelodysplastic/myeloproliferative disorders and in about 5% of primary AML in children and because clinical features of these conditions overlap, it has been difficult to characterize a group among them that has a unique biology. Although the data are scanty, it appears that children younger than 4 years with myelodysplastic/myeloproliferative disorder in association with an acquired isolated loss of chromosome 7 have a better prognosis than children with similar clinical diseases but without loss of chromosome 7.132 There are many examples of cases that were managed with intensive chemotherapy alone and in which the patients became long-term survivors. If a sibling is being considered as a possible bone marrow donor, it is important to have a detailed karyotypic assessment to rule out subclinical monosomy 7. It is also worth mentioning that children with primary AML and partial or complete loss of chromosome 7 have a very poor prognosis.128,133

Primary myeloid tumors In about 4% of children with AML, the initial features include a discrete myeloid mass.134 These masses, which are essentially solid tumors composed of myeloid precursors, were initially designated chloromas (from the Greek chloros, meaning green) to reflect the greenish cut surface of the gross specimen. This color, which is due to the presence of myeloperoxidase in the tumor cells, rapidly fades when the specimen is exposed to the oxygen. Because some of the myeloid tumor cells do not contain myeloperoxidase, for example, those of megakaryoblastic or monoblastic lineages, these tumor cells have been described by other terms such as granulocytic sarcoma, myelosarcoma, myeloblastoma, or megakaryoblastoma. Myeloid tumors have been found in almost every organ, but they are much more frequent in bones and perineural tissues. The pattern of distribution of these tumors may be explained in part by the functional diversity of the myeloid cells. For example,

Acute myeloid leukemia

involvement of the skin, gingiva, and meninges is associated with the monocytic lineage, whereas involvement of the bone is frequently seen in megakaryoblastic leukemia. In the latter case, the leukemic cells in the bone marrow reach the periosteum through the haversian canals, cause osteolytic lesions to form, and then spread to soft tissues and organs (Fig. 19.7). Myeloid tumors are seen twice as often in children and adolescents as in adults. The most common locations include skin, gingiva, orbit-ocular tissue, and other CNS regions. Orbit-ocular involvement is particularly frequent in children and adolescents in some Middle Eastern and African countries.10,11 In a series reported by ¨ Berlin-Frankfurt-Munster (BFM) investigators, children with myeloid tumors were significantly younger and had lower WBC counts and FAB M1/M2 or M4/M5 subtypes than did children with AML.134 Results of a few studies suggested an association between isolated myeloid tumors and specific cytogenetic abnormalities including the t(8;21), t(9;11), and inv16.135,136 Radiographic images of myeloid tumors do not have distinctive features and cannot be distinguished from those of other pediatric soft tissue tumors.137–139 When arising from bone, myeloid tumors can appear as focally destructive osteolysis with an adjacent soft-tissue mass. Periosteal reactions can be seen in association with these findings predominantly in patients with AMKL.108 Soft-tissue myeloid tumors tend to be isodense on T1-weighted magnetic resonance (MR) images and mildly hyperdense to muscle on T2-weighted MR images with homogenous enhancement. Intracranial lesions are hyperdense to normal brain tissue on computed tomography (CT) images and slightly hyperintense or isointense to gray matter on both T1- and T2-weighted MR images; homogenous enhancement is noted with contrast in either MR or CT imaging. In many instances, the clinical syndrome caused by a myeloid tumor (proptosis, skin nodule, gingival pain, spinal cord compression) is seen in the absence of conspicuous bone marrow dysfunction or circulating blast cells; this result leads to delayed or incorrect diagnosis. In rare cases, results of bone marrow examination have been completely normal in association with an isolated myeloid tumor.140 If only local therapy – surgical resection and radiotherapy – is provided in these cases, overt leukemia almost always follows within weeks or months.136 The treatment of a patient with a myeloid tumor, regardless of the number of blasts in the bone marrow, should consist of intensive chemotherapy regimens as for AML. The use of radiation therapy should be used in those cases in which compression of vital structures, such as the spinal cord, or intracranial extension is evident.

Acute promyelocytic leukemia A distinct clinical subtype of AML that is seen in both children and adults, acute promyelocytic leukemia (APL) is characterized by arrested maturation of the leukemic cells at the promyelocytic stage, the presence of the PML-RAR
fusion protein that results from by the t(15;17) translocation, a hemorrhagic diathesis and coagulopathy, and a unique responsiveness to all-trans retinoic acid (ATRA) and anthracyclines.141 APL is the first type of acute leukemia for which successful targeted therapy was derived; ATRA binds to the PML-RAR
fusion protein and induces differentiation of the leukemic cells into mature granulocytes. Except for AML in children with Down syndrome, APL has become the most curable subtype of AML.107,142 APL constitutes less than 10% of AML cases in children and adolescents in the United States, but as many as 31% in some areas of Italy.143,144 The incidence of APL is also higher in Hispanic, Latino, and Asian populations.145 Two studies showed that adolescents and adults with APL had a higher body mass index (BMI) and were more obese than patients with other AML subtypes or the general population.146,147 Because high levels of leptin receptor isoforms are expressed in leukemic (but not in normal) promyelocytes, it has been suggested that the proliferation of leukemic promyelocytes is driven, in part, by increased leptin levels in obese patients with APL.148 Most cases of APL are classified as M3 in the FAB system, and a characteristic morphologic appearance reflects the arrest at the promyelocytic stage of myeloid differentiation. The leukemic promyelocytes usually have a reniform or bilobed nucleus, large azurophilic granules, and Auer rods. In some cases, the leukemic blasts contain bundles of Auer rods in the cytoplasm and are called “fagot cells.” Patients with classic APL have cells with a reniform nucleus that may or may not contain very fine granules. Morphologic variants such as the microgranular variant or M3v exist. For more details on this subject, the reader is referred to Chapter 2. As discussed in Chapters 7, 9, and 11, APL cells have distinct immunophenotypic, cytogenetic, and molecular characteristics. Although the classic t(15;17) translocation is not detected by conventional cytogenetics in many cases of APL, the PML-RARα rearrangement can be detected by fluorescence in situ hybridization (FISH) or RT-PCR. Other reported chromosomal aberrations in APL cells are t(11;17)(q23;q21), t(5;17)(q35;q12–21), t(11;17)(q13;q21), and der(17), whereby RARα is fused to the PZLF, NPM, NµMA, and STAT5b genes, respectively.146,149–151 As in APL associated with PML-RARα, APL involving NPM-RARα or NµMA-RARα appears to be sensitive to ATRA. In contrast,

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APL associated with a PZLF-RARα rearrangement is typified by the lack of a differentiation response to retinoids, and patients with this disease treated with ATRA alone have a poor prognosis. A typical clinical feature of APL is a low leukocyte count at diagnosis. Hepatosplenomegaly or CNS leukemia are often absent, but coagulation abnormalities and signs of clinical hemorrhage are common findings.143 We reviewed 21 cases of APL, diagnosed at St. Jude Children’s Research Hospital between 1992 and 2001, in which the chemotherapy regimens included ATRA. These patients, comprising about 10% of all cases of AML diagnosed during that period, had a median age at diagnosis of 15.6 years (range, 1.4 to 17.5 years), a median leukocyte count of 3.3 × 109 /L (range, 0.8 to 36.8 × 109 /L), and a high incidence (86%) of coagulopathy. Obesity, defined as a body mass index (BMI) of 30 or higher, was seen in 8 patients at diagnosis, and the median BMI for the group was 28.7 (range, 15 to 38.5), confirming the previously reported association between APL and increased BMI and obesity.146 Patients with APL often present with thrombocytopenia, prolongation of thromboblastin and thrombin times, increased levels of fibrin degradation products, and hypofibrinoginemia. The coagulopathy may be fatal before or early in the course of therapy and is frequently exacerbated by cytotoxic chemotherapy, which causes leukemic cell lysis and the release of procoagulants.152 Overexpression of annexin II on the leukemic blast cell surface activates plasminogen through its interaction with tissue plasminogen activator, thereby promoting fibrinolysis and contributing to an increased incidence of DIC.153 The addition of ATRA to remission induction therapy reduces the severity of bleeding and decreases blood product consumption and overall mortality rate during induction; however, early fatal hemorrhages still occur.154 Patients with APL and high leukocyte counts are at particular risk for significant hemorrhage.155 Extramedullary disease at initial examination is rare in APL.156 Because of the hemorrhagic diathesis associated with lumbar puncture, this procedure is not routinely performed at diagnosis, unless the patient has signs or symptoms of CNS involvement. Since the introduction of ATRA treatment, several reports have noted an increase in extramedullary disease associated with APL at the time of relapse; the CNS and skin are most frequently involved.157,158 However, in two recent reports, patients with APL who received ATRA in addition to standard chemotherapy had a risk of extramedullary disease at relapse that was no greater that that of patients treated with chemotherapy alone (although results of one study demonstrated a higher incidence of CNS relapse).156,159 We there-

fore routinely perform lumbar puncture in patients with APL at relapse after their coagulopathy has been corrected with transfusions of platelets and fresh frozen plasma.

Rare subtypes of AML in pediatric patients Because of the plasticity of hematopoietic progenitor cells, the lineage of certain types of pediatric leukemias is difficult to establish.160 In those cases, one usually finds discrepancies among the morphologic, cytochemical, immunologic, and cytogenetic data. Establishing an effective treatment plan for such cases, which include the FAB-M0 (minimally differentiated) and myeloid/lymphoid (mixed phenotypic) subtypes, can be a daunting challenge. The FAB M0 subtype was recognized in 1987,161 and criteria for this variant were developed in 1991.161,162 The leukemic cells lack distinctive morphologic or cytochemical features such as granules and Auer rods. By FAB definition, the diagnosis of AML-M0 requires that less than 3% of the blast cells show positivity for myeloperoxidase and Sudan black B, with greater than 20% of the blast cells expressing myeloid antigens and lacking lymphoidspecific markers. Although this type of leukemia was once thought to occur mainly in the elderly, it can occur in children as well, ranging in incidence from 6% to 8%.163,164 AML-M0 arises more often in girls than in boys, and in children younger than 3 years.165 Other clinical and laboratory findings, including hepatosplenomegaly and WBC counts, do not differ from those of children with other AML subtypes. Immunophenotypic analysis usually shows a profile similar to that of early myeloid progenitors with expression of TdT, HLA DR, CD34, and cCD117; CD13 or CD33 is seen in most cases. Nonspecific lymphoid markers such as CD7 and CD2 are common. Anti-myeloperoxidase antibody (3% or more of the cells) is seen in about half of the cases. The NK cell marker CD56 is also found in about half of the pediatric cases. Cytogenetic studies have revealed no specific chromosomal abnormality in association with AML-M0: about 30% of the cases have a normal karyotype, and 20% show multiple chromosomal changes (complex karyotype). Other abnormalities include a nearly tetraploid karyotype and unbalanced abnormalities involving −5, −7, and +8. Balanced translocations involving 11q23 are also common in pediatric patients with this rare AML subtype. In adults with AML-M0, mutations of the AML1 gene are frequently observed.166 The prognostic importance of AMLM0 has not been established because of the small number of patients who experience the disease; however, it appears that these children have a poor prognosis. In about 1% to 2% of cases of pediatric AML, the leukemic cells express markers considered to be specific

Acute myeloid leukemia

for more than one lineage. The characterization of these leukemias usually requires multiparametric flow cytometric analysis.167–169 Two distinctive processes have been recognized. In the first, the disease manifests with evidence of two separate phenotypic clones, each carrying different lineage-specific markers. These leukemias are designated bilineal or biclonal. In the second, the same clone or leukemic cells express more than one lineagespecific marker. These leukemias have been variously called mixed lineage, biphenotypic, hybrid, or chimeric. Mixed myeloid/B-cell lymphoblastic leukemia requires the coexpression of myeloperoxidase (3% or more of the cells detected by histochemical analysis or 10% or more detected by flow cytometry) and B cell – specific antigens. Similarly, mixed myeloid/T-cell lymphoblastic leukemia requires coexpression of myeloperoxidase and T cell-specific antigens. The coexpression of myeloid-specific markers with one or more lymphoid-associated markers is very common and does not have clinical or biologic relevance.170 The clinical characteristics, biologic features, and treatment outcome of patients with biclonal or mixed phenotypic leukemia have not been systematically studied. Anecdotal experience suggests that patients with mixed phenotypic leukemia have a poor prognosis.171–173 The disease in these patients is managed with arbitrarily derived treatment strategies directed to the predominant clone. If the myeloid clone is predominant, AML-like chemotherapy is begun, with a switch to an ALL-type strategy should the leukemia not respond to one or two courses of the first regimen. Conversely, if a lymphoid clone is predominant, ALL-like therapy is attempted first. Hematopoietic stem cell transplantation is usually considered for patients whose disease is in remission and who have a suitable matched donor.

Treatment of childhood AML Clinical trials of AML feature intensive chemotherapy with or without subsequent stem cell transplantation. The treatment of acute promyelocytic leukemia (APL) also includes all-trans retinoic acid and is discussed separately below.

Induction therapy The treatment of AML consists of two general phases: remission induction therapy and postremission consolidation (or intensification) therapy. Some trials have also included a maintenance phase, but its role in therapy of AML remains unproven. Results of clinical trials conducted in the 1960s demonstrated that cytarabine and daunoru-

bicin were effective agents in AML and, when used as single agents, could induce remission in one third to one half of patients.174 In the 1970s, induction regimens that combined these two agents increased remission rates to 60% to 70%. The addition of other agents, such as etoposide and thioguanine, to the cytarabine plus daunorubicin combination further improved remission induction rates to 70% to 85%, although it is unclear whether the improvements were directly related to these agents or to better use of cytarabine and daunorubicin. Although most modern clinical trials (Table 19.3) have achieved similar complete remission (CR) rates despite different dosages or schedules of chemotherapeutic agents, results of several studies suggest that the “quality” or “depth” of remission has a major impact on outcome; thus, the success of an induction regimen should be based on final outcome rather than simply on remission rate.3,75 Recent attempts to improve induction therapy have included the use of new agents,85 the use of alternative anthracyclines,5,86,175 and intensification of therapy.2,3,75,176 In the St. Jude AML91 trial, CR was achieved in 78% of patients after preinduction therapy with cladribine (2-chlorodeoxyadenosine) and induction therapy with two courses of DAV (daunorubicin, cytarabine, etoposide).85 The 5-year event-free survival (EFS) estimate was 40%, superior to that of a previous St. Jude trial (AML87, 5-year EFS estimate, 31%) that did not include cladribine.177 The use of cladribine plus cytarabine before two courses of DAV increased the CR rates to 90% for patients on Arm A and 100% for patients on Arm B in the St. Jude AML97 trial178 ; the impact on outcome awaits longer follow-up, but early results are encouraging (Table 19.3). Compared with daunorubicin, idarubicin is taken up faster by cells, is retained by cells for a longer period, is associated with less in vitro drug resistance, is potentially less cardiotoxic, and is converted to active metabolites that have a longer plasma half-life.179–181 Therefore, investigators in the BFM consortium and the Australian and New Zealand Children’s Cancer Study Group (ANZCCSG) compared the efficacy of these two agents during induction therapy.5,86,175 Although remission rates and EFS estimates were similar for patients treated with daunorubicin or idarubicin in the AML-BFM 93 trial, idarubicin was associated with better clearance of early blast cells in highrisk patients and with a trend toward a better outcome in patients with more than 5% blast cells on day 15.175 Results of the ANZCCSG trial, however, suggest that daunorubicin is as effective as and less toxic than idarubicin.5 Induction therapy may be intensified by increasing drug dosages or drug exposure time per course of chemotherapy, or by decreasing the time between courses. Although

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Table 19.3 Clinical trials for AML Study

Years 177

St. Jude AML87

1987–91

Treatment schedule

n

CR rate

Cycles 1 and 6, araC/VP16

67

76%

Cycles 2 and 5, araC/Dauno Cycle 3, VP 16/Amsa Cycle 4, VP 16/azacytidine

Outcome

Conclusion(s)

5-year EFS, 31%

There was no benefit from individualized dosing of araC and VP16

5-year OS, 41%

St. Jude AML9185

1991–6

Preinduction 2-CdA Induction Dauno/araC/VP 16 × 2 Consolidation Allo-SCT or auto-SCT

73

78%

5-year EFS, 40% 5-year OS, 53%

2-CdA was effective initially in a subset of patients, especially those with monoblastic leukemia

St. Jude AML97178

1997–2002

Preinduction 2-CdA/araC Induction Dauno/araC/VP 16 × 2 Consolidation HDAC/Asp Mito/araC

83

Arm A, 90% Arm B, 100%

3-year EFS, 50% 3-year OS, 57%

2-CdA increased intracellular accumulation of ara-CTP

CCG 213P190

1983–5

Induction DCTER DA vs. DA DCTER Consolidation HDAC/Asp at 7-day vs. 28-day intervals

195

74%

5-year EFS, 35% 5-year OS, 39%

Intensification with HDAC and asparaginase resulted in outcomes better than those of the previous trial

CCG 213193

1986–9

Induction DCTER DA vs. DA DCTER Consolidation Donor: Allo-SCT No donor: Consolidation chemotherapy ± MT

591

78%

5-year DFS Allo-SCT, 46% Chemotherapy, 38% 5-year OS Allo-SCT, 52% Chemotherapy, 46%

Allo-SCT improved DFS but not survival probability. Maintenance therapy resulted in worse survival probability

CCG 28913, 75

1989–95

Induction DCTER × 2 Consolidation DCTER × 2 Post-consolidation Donor: Allo-SCT

652

74%

3-year EFS Intensive timing, 42% Standard timing, 27% 3-year OS Intensive timing, 51% Standard timing, 39%

Intensively timed induction improved outcome regardless of postremission therapy. Allo-SCT resulted in fewer cases of relapse and longer survival than did chemotherapy or auto-SCT

No donor: Allo-SCT vs. chemotherapy POG 849874

1984–8

Induction DAT DAT vs. HDAC Consolidation HDAC/Asp vs. HDAC VP16/Aza × 4 POMP × 4 AraC × 4

285

85%

3-year EFS, 33%

There was a trend toward improved outcome in patients who received additional doses of HDAC

POG 882187

1988–93

Induction DAT HDAC Consolidation Donor: Allo-SCT No donor: Auto-SCT vs. chemotherapy

649

85%

3-year EFS Allo-SCT, 52% Auto-SCT, 38% Chemotherapy, 36% 3-year OS Allo-SCT, 62% Auto-SCT, 40% Chemotherapy, 44%

There was no advantage of auto-SCT over chemotherapy as postremission therapy

Acute myeloid leukemia

515

Table 19.3 (cont.) Study

Years

Treatment schedule

n

CR rate

Outcome

Conclusion(s)

POG 9421

1995–9

Induction DAT vs. HDAT HDAC Consolidation Donor: Allo-SCT No donor: chemotherapy ± CSA

632

90%

3-year EFS HDAT, 40% DAT, 35%

The use of HDAC during induction did not significantly improve CR or EFS estimate

MRC AML102

1988–95

Induction DAT vs. ADE × 2 Consolidation Amsa/araC/VP16 Mito/araC Post-consolidation Donor: Allo-SCT No donor: Auto-SCT vs. chemotherapy

341

92%

7-year DFS Allo-SCT, 70% Auto-SCT, 68% Chemo, 46% 7-year OS Allo-SCT, 70% Auto-SCT, 70% Chemotherapy, 59%

There was no difference in outcome between the DAT and ADE induction regimens. SCT reduced the risk of relapse but did not improve overall survival probability

AML-BFM 8777

1986–91

Induction Dauno/araC/VP16 Consolidation 6-week consolidation Intensification HDAC/VP16 × 2 Continuation 6TG/araC ± CrRt

210

78%

5-year EFS, 41% CrRt, 78% pRFI No CrRt: 41% pRFI

In the context of this chemotherapy regimen, cranial irradiation reduced the risk of bone marrow relapse

AML-BFM 9386,175

1993–8

Induction Dauno/araC/VP16 vs. Ida/araC/VP16 Consolidation 6-week consolidation plus Mito/araC for high-risk patients Intensification HDAC/VP16 Continuation 6TG/araC plus CrRt Allo-SCT for high-risk patients

471

82%

5-year EFS, 51% 5-year OS, 60%

Use of idarubicin resulted in better early blast cell clearance than Dauno. The addition of HDAC/Mito improved outcome of high-risk patients compared with that in the previous trial

LAME 89/9183

1988–96

Induction AraC/Mito Consolidation Donor: Allo-SCT No donor: chemotherapy VP16/araC/Dauno Amsa/HDAC/Asp × 2 ± Maintenance therapy with 6MP/ara-C

268

90%

5-year DFS + MT, 50% − MT, 60% 5-year OS + MT, 58% − MT, 81%

Patients who received maintenance therapy had a worse outcome than those who did not

ANLL91103

1995–8

Induction Mito/VP16/araC Intensification HDAC/VP16/Mito × 2 AraC/VP16/Doxo × 2 HDAC/VP16/aclarubicin × 2 HDAC/VP16/VCR × 2

35 91% infants

3-year EFS, 72% 3-year OS, 76%

Infants had an excellent outcome when treated with intensive chemotherapy

182

(cont.)

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Table 19.3 (cont.) Study 84

NOPHO-AML 84 AML 8884

NOPHO-AML 93390

Years

Treatment schedule

n

CR rate

Outcome

Conclusion(s)

1984–8 1988–92

Induction 6TG/araC/VP16/Doxo Mito/araC 6TG/araC/VP16/Doxo Consolidation HDAC/Mito HDAC/VP16 HDAC HDAC/VP16

105

78%

5-year EFS

118

85%

The addition of Mito and VP16 to HDAC in consolidation therapy did not improve outcome. Girls, infants, and patients with Down syndrome had better outcomes than other patients

Induction 6TG/araC/VP16/Doxo Mito/araC or 6TG/araC/VP16/Doxo Consolidation Same as NOPHO-AML88

219

91%

1993–2000

ANZCCGSG55

Induction

AML 1

1986–92

AML 2

1993–9

32% 42%

7-year EFS, 49%

Allowing patients with hypoplastic marrows to recover after induction therapy reduced the toxic death rate compared to previous trial

5-year EFS

Dauno was as effective as and less toxic than idarubicin during induction therapy

5-year OS

Ida/araC/6TG

102

95%

50%

56%

HDAC

160

91%

41%

55%

Consolidation Ida/araC/VP16 AraC/Amsa Interim therapy AraC/6TG Post-consolidation Allo-SCT or Auto-SCT ¨ Abbreviations: ANZCCSG, Australian and New Zealand Children’s Cancer Group; BFM, Berlin-Frankfurt-Munster Study Group; CCG, Children’s Cancer Group; LAME, Leuc´amie Aiqu¨e My´elo¨ıde Enfant; MRC, Medical Research Council; NOPHO, Nordic Society of Paediatric Haematology and Oncology; POG, Pediatric Oncology Group; SJCRH, St Jude Children’s Research Hospital; n, number of patients; CR, complete remission rate; araC, cytarabine; VP16, etoposide; Dauno, daunorubicin; Amsa, amsacrine; EFS, event-free survival; OS, overall survival; 2-CdA, 2-chlorodeoxyadenosine; allo-SCT, allogeneic stem cell transplantation; auto-SCT, autologous stem cell transplantation; HDAC, high-dose cytarabine; Asp, L-asparaginase; Mito, mitoxantrone; NA, not available; ara-CTP, cytarabinetriphosphate; DCTER, decadron, cytarabine, 6-thioguanine, etoposide, rubomycin (daunorubicin); DA, daunorubicin and cytarabine; MT, maintenance therapy; DFS, disease-free survival; DAT, low-dose cytarabine, daunorubicin, and thioguanine; POMP, prednisone, vincristine, methothrexate, and mercaptopurine; HDAT, high-dose cytarabine, daunorubicin, and thioguanine; CSA, cyclosporine A; ADE, cytarabine, daunorubicin, and etoposide; 6TG, 6-thioguanine; CrRt, cranial radiation; pRFI, probability of relapse-free interval; Ida, idarubicin; 6MP, 6-mercaptopurine; Doxo, doxorubicin; VCR, vincristine.

attempts to increase the intensity of this treatment have not significantly improved remission rates, evidence suggests that induction intensity improves the ultimate outcome. In the Pediatric Oncology Group (POG) 8498 trial, the impact of induction intensity was studied by randomly assigning patients to receive two courses of DAT (daunorubicin, cytarabine, thioguanine) or one course of DAT followed by a course of high-dose cytarabine (HDAC).74 Remission rates were similar in the two treatment groups (85%), but patients who received HDAC had a better outcome than those who did not (3-year EFS estimates, 34% compared with 29%).

In the subsequent POG 9421 study, the remission rates and outcomes of patients who received HDAC were compared with those of patients who received DAT as the initial course of induction.182 Remission rates were again similar (87% for DAT and 91% for HDAC), but patients who received HDAC tended to have a better outcome (3-year EFS estimates, 40.4% compared with 34.5%, P = 0.17). The United Kingdom Medical Research Council (MRC) used a different strategy to intensify induction; rather than increasing dosage, they prolonged drug exposure in their AML 9 and 10 trials.2,176,183 In the MRC AML 9 trial, patients

Acute myeloid leukemia

were randomly assigned to receive DAT (daunorubicin, cytarabine, thioguanine) in a “1 + 5” or a “3 + 10” schedule (daunorubicin given for 1 or 3 days; cytarabine and thioguanine given for 5 or 10 days).183 Because the DAT 3 + 10 schedule resulted in a higher remission rate (85%), it was used in subsequent MRC trials. For example, the MRC AML 10 trial compared DAT with ADE (etoposide substituted for thioguanine) during induction therapy. Although no differences were detected between the two treatments, the outcome was excellent: 56% of patients survived at least 7 years after diagnosis.2,176 Another method of intensification is timed sequential induction, an approach used by the Children’s Cancer Group (CCG).3,75 In the CCG 2891 trial, patients were randomly assigned to groups that received DCTER (dexamethasone, cytarabine, thioguanine, etoposide, daunomycin) in a standard or intensively timed fashion.3,75 In the standard timing group, the second course of DCTER was administered at the time of bone marrow recovery unless leukemia persisted at day 14, whereas in the intensive timing group, the second course was administered on days 10 to 13 regardless of bone marrow status. Although there was no significant difference in remission rates between the two treatment groups, patients who received the timed sequential induction had a superior overall outcome, which suggests that the quality of remission was better after intensive timing than after standard timing. Finally, the advantage of intensive timing as compared with standard timing was maintained regardless of the type of postremission therapy.3,75 Recently, the Dutch-Belgian Hemato-Oncology Cooperative Group tested the hypothesis that granulocyte colonystimulating factor (G-CSF) would sensitize leukemic cells to the cytotoxic effects of chemotherapy.184 Adults with AML were randomly assigned to receive induction chemotherapy with or without concurrent administration of G-CSF. Patients assigned to receive G-CSF had a higher rate of disease-free survival (42% versus 33% at 4 years, P = 0.02) and a lower probability of relapse (relative risk, 0.77; 95% confidence interval, 0.61 to 0.99; P = 0.04) than patients who did not receive G-CSF.184 The beneficial effects of GCSF were most striking for patients with standard-risk AML.

Postremission therapy Since the mid-1970s, the effects of the duration and intensity of postremission therapy on outcome have been evaluated in many AML trials. Early studies performed by the Eastern Cooperative Oncology Group demonstrated that some postremission therapy is necessary.185,186 However, results of the St. Jude AML 76 and CCG 241 trials indicated

that extended maintenance therapy as the only postremission treatment was ineffective: less than one fourth of patients experienced long-term survival.187,188 By contrast, intensive postremission regimens were associated with decreased relapse rates and contributed to improvements in EFS and overall survival (OS) probability. In the Dana-Farber Cancer Institute VAPA trial conducted in the late 1970s, intensive postremission therapy consisting of sequential combinations of non-cross-resistant drugs was administered for 14 months, resulting in an EFS probability of 38%.78,189 In the 1980s, results of the CCG 213P trial also demonstrated that intensive postremission therapy improved the outcome of children and adolescents with AML.190 Other studies supporting the use of intensified postremission chemotherapy include trials performed by the BFM group, POG, St. Jude, and the MRC (Table 19.3).2,74,86,87,191,192 Interestingly, patients on the CCG 213 trial who were randomly assigned to a group receiving two years of maintenance therapy after intensification actually had a worse outcome than those who received no maintenance therapy.193 Similarly, the French Society of Pediatric Hematology and Immunology recently reported that patients randomly assigned to receive 18 months of maintenance therapy had a similar disease-free survival estimate and a worse OS estimate than did those who received no maintenance therapy.83 Today, most cooperative groups, except for the BFM, have abandoned the use of maintenance therapy in AML. The use of HDAC in the postremission setting has had a positive impact on clinical outcome.74,194 Excellent results were obtained in the MRC AML10 trial, in which MidAC (mitoxantrone, 10 mg/m2 daily for 5 days, and cytarabine, 1 g/m2 every 12 hours for 3 days) was given during consolidation.2,176 The German AML Cooperative Group demonstrated that HAM (mitoxantrone, 10 mg/m2 daily for 3 days, and cytarabine, 1 g/m2 every 12 hours for 3 days) given as a second induction course improved the outcome of adults with high-risk AML.195 Intensification with HAM also improved the outcome for pediatric patients with high-risk AML in the AML-BFM 93 trial.86 The Capizzi II regimen (cytarabine, 3 g/m2 every 12 hours on days 1, 2, 8, and 9, and L-asparaginase, 6000 U/m2 3 hours after the fourth and eighth dosages of cytarabine) contributed to the improved outcome seen in the CCG 213 and CCG 213P trials.193,196 The benefit of HDAC is most pronounced in patients with AML and the t(8;21) or inv(16). Results of clinical trials conducted by the Cancer and Leukemia Group B (CALGB) indicated that patients with these abnormalities had a better outcome than did patients with all other types of AML and had a particularly good outcome when they were treated with HDAC.197 The CALGB subsequently

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showed that among patients whose leukemic cells contained a t(8;21), those who received multiple courses of HDAC had an even better outcome than those who received only one course of this agent.198

Role of hematopoietic stem cell transplantation Studies performed more than 20 years ago demonstrated that allogeneic bone marrow transplantation (BMT) is a feasible and effective alternative to chemotherapy as postremission treatment for AML.199,200 In the St. Jude AML80 trial, which accrued patients from 1980 to 1983, 9 of 19 patients who underwent allogeneic BMT remained in continuous CR.201 Many subsequent studies have demonstrated that relapse-free survival probabilities for patients who have AML and undergo matched related donor BMT are better than those of patients who receive only intensive chemotherapy.176,202–205 Some studies have also shown an OS advantage for BMT; however, others have shown no such advantage, primarily because of transplantationrelated mortality.2,194 In the MRC AML10 trial, children with AML received four courses of chemotherapy with or without subsequent transplantation of autologous or allogeneic bone marrow.2 Although BMT reduced the risk of relapse, neither autologous nor allogeneic BMT resulted in an OS advantage.206 Moreover, among patients with a t(8;21) or inv(16), BMT did not reduce the risk of relapse. Similarly, the CCG 213 trial did not show a superiority of BMT over chemotherapy when results were analyzed by the intentto-treat method.196,207 By contrast, results of the CCG 2891 trial – the largest and most recent randomized comparison of BMT and chemotherapy – demonstrated that for patients who achieved CR, survival probability for patients in the allogeneic BMT group was significantly superior to that for patients in the autologous BMT and chemotherapy treatment groups.3 As a consequence of these studies, autologous BMT is no longer widely used in the treatment of AML. Although matched sibling donor BMT is often recommended, considerable controversy remains as to which patients in first remission should undergo this procedure.208,209 Most European investigators agree that intermediate-risk and highrisk patients should undergo matched related donor BMT during first remission of AML but do not recommend BMT for low-risk patients.208 Investigators from the COG, however, recommend allogeneic BMT for all patients in first remission who have a matched related donor.209 At St. Jude, we currently offer matched related-donor BMT to intermediate-risk patients, and matched-related donor or alternative-donor BMT to high-risk patients; BMT is an option for low-risk patients in second remission.

Treatment of the central nervous system Although no pediatric trials have been done in which patients were randomly assigned to receive or not receive prophylactic therapy for CNS involvement, essentially all contemporary pediatric AML trials include intrathecal chemotherapy. Intrathecal medications include cytarabine, methotrexate, or a combination of both with hydrocortisone. This strategy has resulted in CNS relapse rates less than 5%.70,75,176,210 In BFM studies, the use of cranial radiation has had interesting results. In the BFM-83 study, cranial irradiation and intrathecal methotrexate therapy were used for prophylaxis of CNS involvement, and the incidence of CNS relapse was similarly low.210 In the BFM87 study, which attempted to address whether elimination of cranial irradiation from the regimen for low-risk patients resulted in different relapse rates,77 two courses of HDAC were added to consolidation therapy as a method of intensification. Patients were subsequently randomly assigned to groups that received or did not receive cranial radiation therapy; both groups received intrathecal cytarabine therapy. The randomization was stopped early because a reduction in bone marrow relapse resulted in an improved probability of relapse-free survival for patients who received cranial radiation. Ideally, the role of cranial irradiation in CNS prophylaxis and treatment of childhood AML should be addressed by a clinical trial in which the use of CNS radiotherapy is randomly assigned. However, such a trial seems unlikely, given that the outcomes of patients treated in the recent CCG, POG, and MRC trials, none of which included cranial irradiation but uniformly included HDAC, were similar to those of patients treated in a BFM trial that used cranial irradiation to prevent and treat CNS involvement.2–4 Moreover, the conduct of a trial of randomized CNS radiotherapy is further precluded by the accumulation of evidence that cranial irradiation is a source of long-term mortality and morbidity, including the risk of both secondary neoplasms and long-term endocrine and neurologic sequelae.211–214 Results of some studies have suggested that CNS leukemia at diagnosis of AML in pediatric patients is associated with an adverse prognosis.87,215 In a St. Jude study whose findings were later confirmed by several cooperative groups, the presence of CNS leukemia at diagnosis did not adversely affect the rate of remission induction or the duration of complete remission.72 An accepted treatment approach for these patients is intrathecal therapy administered weekly until the CSF is clear of leukemic blast cells and then administered monthly until the end of therapy.76 In some treatment regimens, intrathecal chemotherapy is followed by CNS irradiation, usually at a dose of 24 Gy,

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with concomitant intrathecal treatments near the end of systemic chemotherapy. We recently analyzed the clinical features and outcome of 290 pediatric patients with newly diagnosed AML with or without CNS involvement; the patients were treated between 1980 and 1997 on one of four consecutive protocols at St. Jude (Table 19.3).216 The diagnosis of CNS leukemia, which was established in 85 patients (29.3%), required the identification of leukemic blast cells in Wright-stained cytocentrifuged samples of CSF, signs of cranial nerve palsy or meningeal involvement, or detection of a nonhemorrhagic mass or chloroma by CT. In addition to analyzing clinical features and outcome, this study addressed the prognostic significance of low leukocyte count in CSF with blast cells at diagnosis, because there is growing evidence that children and adolescents with ALL and these CSF findings require more intensive CNSdirected therapy.217,218 Therefore, all patients were reclassified for this purpose according to the scheme used in the classification of pediatric patients with ALL: CNS1 (no blast cells in cytocentrifuged CSF at the time of diagnosis), CNS2 (1 to 4 leukocytes per microliter of CSF with morphologically identifiable blast cells), or CNS3 (5 or more leukocytes per microliter of CSF with blast cells). Patients who met other criteria for CNS involvement (cranial nerve palsy, signs of meningeal disease, or a nonhemorrhagic CNS mass or chloroma) were classified as CNS3, regardless of CSF findings. The EFS estimate for the CNS1 group did not differ significantly from that for the CNS2 group; this lack of a significant difference indicated that in the context of this treatment, the CSF characteristics of the CNS2 group had no prognostic significance. Another interesting finding was the significant association between the CNS3 group and the favorable cytogenetic abnormalities t(9;11) and inv(16). This finding is consistent with the reported increased frequency of CNS involvement in patients with FAB AML M4 and M5 subtypes, because most cases of AML with inv(16) and t(9;11) are of the M4 or M5 subtype, respectively.219,220 The CNS3 group had a significantly greater probability (±SE) of 5-year EFS (43.7% ± 7.0%) than did the CNS1 (27.8% ± 3.2%, P = 0.015) and CNS2 (24.3% ± 7.5%, P = 0.032) groups. However, after adjustment for favorable genetic features, the EFS probability for the CNS3 group did not significantly differ from that for the combined CNS1 and CNS2 groups. The overall 5-year cumulative incidence of CNS relapse was 3.4% ± 1.1%. CNS relapse was not observed in two more recent treatment protocols, in which radiation therapy was administered 8 months after diagnosis (AML87) or was reserved for patients with symptomatic CNS disease (AML91). Moreover, these protocols included antileukemic drugs (HDAC in AML87 and cladribine in AML91) that are known to pene-

trate the CNS better than other agents. In AML91, four of five patients in the CNS3 group who received no cranial irradiation because of the absence of symptoms remained in CR. Although only a relatively small number of patients with CNS3 status were studied, our findings suggest that cranial irradiation may not be a necessary part of contemporary therapy for patients with AML and asymptomatic CNS involvement at diagnosis. This issue was addressed in our recent prospective study in which asymptomatic patients with CNS3 status did not undergo cranial irradiation and did not have an increased incidence of hematologic or CNS relapse.178 In conclusion, CNS involvement had no adverse prognostic significance in this large group of pediatric patients with AML, partly because of the association between CNS3 status and favorable cytogenetic features. Additional studies are needed to confirm our observation that contemporary chemotherapy can offer long-term disease control without the use of cranial irradiation for patients with AML and asymptomatic CNS involvement at diagnosis.

Treatment of APL Acute promyelocytic leukemia is the first disease for which therapy (ATRA) that directly targets the underlying molecular lesion was successfully used in clinical practice. By binding to the PML-RAR
fusion protein, ATRA causes a partial release of the repressor complex, resulting in the expression of genes leading to cell maturation and ultimately apoptosis.221,222 Compared with treatment regimens that do not include ATRA, those incorporating this agent have significantly reduced relapse rates and improved survival probability.223 The results of contemporary treatment of children and adolescents with APL are similar to those reported for adults. Since 1992, pediatric patients with APL in North America have been almost exclusively treated on the same treatment protocols used for adults.223 The reported CR rates for children and adolescents treated with a combination of ATRA and chemotherapy range from 79% to 88%; long-term EFS estimates range between 64% and 76%.223,224 Leukemic cells from patients with APL are especially sensitive to anthracyclines, perhaps because expression of P-glycoprotein and other markers of drug resistance are significantly lower in APL cells than in cells of other AML subtypes.225,226 Daunorubicin or idarubicin as single agents induce CR in 55% of patients.227,228 Results of several studies have shown that total induction doses of daunorubicin greater than 250 mg/m2 were required to achieve these results.229 Despite theoretical considerations suggesting that idarubicin is better than daunorubicin

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because of its longer half-life and better CNS penetration, these agents appear equally effective. The critical factor appears to be the dose rather than the type of anthracycline used. ATRA alone achieved high CR rates in early Phase II trials, but the duration of remission was short (3–5 months).142,230 Patients treated with ATRA alone experienced a rapid increase in WBC counts that was associated with retinoic acid syndrome.223,231,232 In the North American Intergroup trial, children and adults were randomly assigned to receive ATRA (45 mg/m2 per day) or standard induction chemotherapy with daunorubicin and cytarabine (DA).223 The remission rates were not significantly different between the ATRA and DA groups (70% and 73%, respectively), but the 5-year disease-free survival estimate was greater for patients treated with ATRA than for those treated with DA (69% and 29%, respectively). The European APL group conducted a prospective randomized trial for children and adults to compare the outcome after ATRA administered concurrently with chemotherapy with that achieved by a sequential approach in which ATRA was used as induction therapy and chemotherapy as postremission treatment.233 The CR rates were similar in the two groups, but the EFS probability at two years was 84% for patients in the concurrent therapy group and 77% for patients in the sequential therapy group. This difference was attributable to a significant decrease in the risk of relapse (at 2 years, 6% in the concurrent therapy group versus 16% in the sequential therapy group). Although the complete remission rate has not clearly increased and early mortality rate has not clearly decreased with the concurrent therapy approach, the combination of ATRA and chemotherapy as initial therapy has become an attractive strategy for all patients with APL. This approach has the additional benefit of possibly reducing the incidence of retinoic acid syndrome from approximately 25% with ATRA treatment to approximately 10% with ATRA and concurrent chemotherapy.234–236 Induction treatment is usually started first with ATRA for 2 to 4 days to ameliorate the coagulopathy before chemotherapy is initiated, provided the leukocyte count is less than 10 × 109 /L. Evidence from several studies shows that cytarabine can be omitted during remission induction when an anthracycline is given with ATRA.235–238 The European APL group is currently conducting a trial in which patients with APL who have leukocyte counts less than 10 × 109 /L are randomly assigned to receive ATRA and daunorubicin with or without cytarabine. Consolidation chemotherapy after CR is achieved is necessary because early studies showed an unacceptable early relapse rate after treatment with ATRA alone. In most studies, consolidation chemotherapy has been based on

anthracyclines. In the North American Intergroup study, the following protocol was used: one cycle of daunorubicin (45 mg/m2 per day for 3 days) and standard-dose cytarabine (100 mg/m2 per day for 7 days) as a first consolidation course followed by HDAC (2 g/m2 twice daily for 4 days) and daunorubicin (45 mg/m2 per day for 2 days).223 The European APL group study included standard-dose cytarabine in consolidation therapy.233 However, just as there appears to be little if any role for cytarabine during remission induction, emerging data suggest that there is no role for HDAC in consolidation. Results of a prospective nonrandomized study published by the PETHEMA group suggested that patients do as well without cytarabine in either the induction or consolidation phase as they do when cytarabine is present.236 At least two courses of postremission therapy are routinely administered after the completion of induction therapy consisting of ATRA and anthracycline, although, as with all subtypes of AML, there are no prospective data to establish the optimal number of courses of intensive postremission consolidation therapy.239 Currently, there is no role for hematopoietic stem cell transplantation in the treatment of APL in first remission.240 Two large prospective randomized trials now suggest that maintenance therapy with ATRA is useful.223,233 In the North American Intergroup study, patients whose APL was in remission after two courses of consolidation chemotherapy were randomly assigned to one year of daily maintenance therapy with ATRA at standard doses or to observation.223 Results of this study showed a significant benefit when a year of daily maintenance ATRA was administered to patients whether remission was induced with chemotherapy alone or with ATRA. The best outcome was observed in patients who received ATRA during induction and maintenance therapy: the 5-year disease-free survival probability was 74%. In the European APL93 trial, patients whose APL was in remission after anthracycline-based consolidation therapy were randomly assigned to observation or to one of three maintenance regimens: ATRA in standard dosages for 15 days every 3 months, or 6-mercaptopurine (6-MP) at 90 mg/m2 per day and methotrexate at 50 mg/m2 per week, or the combination of ATRA and 6MP and methotrexate.233 Patients receiving both ATRA and chemotherapy had the lowest relapse rate. In addition, OS estimates were improved for patients who received maintenance chemotherapy, and there was a trend toward greater survival probability for patients who received maintenance ATRA therapy. Therefore, patients appear to benefit from maintenance ATRA therapy with or without continuous low-dose chemotherapy. The GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto) Cooperative

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Group is currently assigning patients in a randomized manner to the same three maintenance regimens used in the APL93 trial or to observation, and the North American Intergroup is randomly assigning pediatric and adult patients with APL in CR either to ATRA therapy every other week with oral 6-MP and methotrexate administered for 1 year or to ATRA therapy alone. The results of these studies will aid in determining the optimal maintenance regimen. In the early 1990s, Chinese investigators reported that arsenic trioxide induced CR in patients with relapsed or refractory APL.241,242 Results of preclinical studies suggest that arsenic has several mechanisms of action, including the induction of apoptosis and leukemic cell differentiation.243–245 In a pilot study, CR was achieved in 11 of 12 patients with relapsed APL who were treated with arsenic trioxide at dosages ranging from 0.06 to 0.2 mg/kg per day, and 8 of the 11 patients whose initial results were positive for the PML-RAR
fusion transcript later had test results that were negative.246 This finding suggested a pivotal role for arsenic trioxide in patients with relapsed APL. The high rates of CR (85%) and molecular remission (78%) were confirmed in a multicenter trial of 40 patients (five of whom were younger than 18 years).247 The most important toxicity included prolongation of the QTc interval and APL differentiation syndrome, a cardiorespiratory distress syndrome with pulmonary infiltrates that is reminiscent of retinoic acid syndrome and responsive to dexamethasone.248 Other commonly reported toxicities are abdominal pain, nausea, vomiting, headache, paresthesias, hypokalemia, and hyperglycemia. Two recent reports of sudden cardiac death of patients treated with arsenic trioxide indicated that careful monitoring is warranted.248,249 As initial remission induction therapy, arsenic trioxide has been associated with hepatotoxicity but has been evaluated in only a limited number of patients.241 The role of arsenic during the induction phase of treatment for patients with newly diagnosed APL is being studied in the current North American Intergroup trial in which patients who are older than 15 years and whose APL is in CR are randomly assigned to receive or not receive two courses of arsenic trioxide as a first consolidation cycle. Trials are under way combining arsenic trioxide with ATRA and chemotherapy as remission induction therapy for patients with newly diagnosed APL and evaluating its possible synergistic role in consolidation therapy.250 Another novel agent for treatment of newly diagnosed APL is gemtuzumab ozogamicin [Mylotarg® (Wyeth Ayerst)], the anti-CD33 antibody conjugated to calicheamicin. In a recent study of 19 adults with APL, 84% entered CR after treatment with gemtuzumab ozogamicin and ATRA, with or without idarubicin. Patients in whom CR

was induced received maintenance therapy that included gemtuzumab ozogamicin, and PCR detected no PML-RAR
transcripts in the patients who were tested.251

Pharmacogenomics of AML therapy Host factors, such as pharmacodynamics and pharmacogenomics, have significant effects on treatment outcome in many types of cancer, including AML.32 The effect of host factors is best exemplified by children with Down syndrome and AML, who have cure rates of 80% to 100%.105,252 These extraordinarily high cure rates are at least partly explained by the finding that increased levels of cystathionine-synthetase (CBS) and a high frequency of CBS polymorphisms in the leukemic blast cells of patients with Down syndrome result in altered metabolism of cytarabine.253,254 Polymorphisms or altered expression of other proteins involved in cytarabine metabolism, such as deoxycytidine kinase, cytidine deaminase, DNA polymerase, and es nucleoside transporter, may also play a role in leukemic blast cell sensitivity or resistance to this agent.255–257 In studies from Japan and from the CCG, AML patients with homozygous deletions of the glutathione S-transferase theta (GSTT1) gene had an increased frequency of early toxic death and a worse OS probability than did patients with at least one GSTT1 allele.258,259 Deletions of GST have also been associated with a poor response to therapy.260 Recently, specific polymorphisms of the MDR1 gene were shown to be associated with an increased risk of relapse among patients with AML, possibly through pharmacokinetic effects on drug metabolism.261

Prognostic factors As discussed in detail above, patients with Down syndrome and AML have a favorable prognosis. For patients with other types of AML, certain clinical features, karyotypes, molecular genetic characteristics, and responses to therapy are important predictors of outcome.76,91 However, the impact of any prognostic factor depends largely on therapy, so that even patients with “favorable” karyotypes may have variable outcomes depending on the therapy received. For example, the results of studies performed by the MRC, POG, CALGB, and others have demonstrated a relatively favorable outcome for patients with AML and either inv(16) or t(8;21).88,90,91,197,262 In the CALGB trials, patients with these karyotypes had a better outcome than did those with all other subtypes of AML and had a particularly good outcome when they were treated with multiple courses of HDAC.91,197,198 In the MRC AML10 trial, the 5-year OS estimates for patients with t(8;21) and inv(16)

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were 69% and 61%, respectively.90 In the POG 8821 trial, pediatric patients with t(8;21) had 4-year OS estimate of 52%, and patients with inv(16) had an estimate of 75%.88 Recent trials at St. Jude yielded similar results, with 6-year OS estimates of 55% for children with t(8;21) and of 70% for children with inv(16).219,263 Recently, the French AML Intergroup reported a 5-year OS estimate of 59% for patients with t(8;21) and suggested that the WBC index (the product of the WBC count and the ratio of marrow blast cells) can identify subgroups of patients with a t(8;21) whose outcome is distinct.264 Structural abnormalities involving chromosome band 11q23 are among the most common genetic abnormalities in acute leukemia and are often associated with diverse leukemia subtypes, including ALL, AML, AMKL, biphenotypic leukemia, congenital leukemia, MDS, and secondary leukemia.88,265 In most instances there is a reciprocal translocation involving the MLL gene, mapped in the 11q23 region, and one of over 30 genes located in distinct chromosomal loci.266 Rearrangements of MLL are seen in up to 20% of cases of AML, although the frequency varies among studies.104,120 Most of these abnormalities consist of a reciprocal translocation involving 11q23 and 9p21–22 (AF9), 10p12 (AF10), 19p13.1 (ELL), or 6q27 (AF6). The t(4;11), which is very common in young children with ALL, is rarely observed in AML. Patients with 11q23 abnormalities tend to be young and have high WBC counts at diagnosis. Overall, the prognosis of children whose leukemic cells contain 11q23 abnormalities does not differ significantly from that of children without these translocations. However, some studies suggest that the t(9;11) confers a favorable outcome.133,267–269 In the MRC AML10 trial, patients with a t(9;11) had an intermediate outcome (3-year OS estimate of 50%),90 whereas in the POG 8821 trial, patients with a t(9;11) or other 11q23 abnormalities had a rather poor outcome (4-year OS estimate of 33%).88 Among AML patients treated at St. Jude during the past two decades, those with a t(9;11) had a better outcome (5-year EFS estimate, 65%) than did patients in all other cytogenetic or molecular subgroups (Table ??).104 We have attributed the favorable outcome of these patients to the use of epipodophyllotoxins, agents known to be effective against M5 leukemia,270,271 and of cladribine, which was particularly effective against M5 AML in our recent clinical trial.85 Blasts from patients with a t(9;11) are more sensitive in vitro to cytarabine, etoposide, anthracyclines, and cladribine than are blasts from other patients.272 Certain morphologic, clinical, and genetic features are associated with a particularly poor outcome in AML. For example, results of some studies have suggested that par-

tial tandem duplications of the MLL gene confer a poor prognosis.95,273 We and others have demonstrated that patients with AMKL have significantly worse outcomes than patients with other subtypes of AML.105,107,274 As discussed above, the 5-year survival estimate for patients with AMKL without Down syndrome treated at our institution was only 10%, and no patients were cured by chemotherapy alone.105 However, a few reports have suggested that chemotherapy is effective for selected groups of patients. A study by French investigators suggested that children with AMKL and the t(1;22), but without Down syndrome, had a better outcome than similar children without this karyotypic abnormality.98 Further studies will be necessary to confirm this observation. The outcome of patients with treatment-related AML is also dismal, with survival rates ranging from 10% to 20%.275,276 Likewise, patients with MDS, AML arising from MDS, or AML with monosomy 7 often have resistant disease that is difficult to cure.21,88,90,277–279 Among patients with APL, the presenting leukocyte count is the most important prognostic factor, although the precise threshold is uncertain.238,280,281 Female sex has conferred a favorable outcome in several trials.223,281 Several groups have reported a worse outcome for patients with the short form of the PML-RARα fusion transcripts, but others have observed no difference.234,281–284 Expression of CD56, which reflects the neural crest adhesion molecule believed to be involved in trafficking of leukemic cells, is an unfavorable prognostic factor.285 Results of a recent study suggested that HLA-B13 was significantly associated with relapse.286 Patients at high risk, such as those with a leukocyte count greater than 10 × 109 /L at initial examination and those whose leukemic cells express CD56, may be considered for investigational strategies that include the use of the anti-CD33 antibody HuM195, which has been used to treat patients whose APL is in CR as indicated by morphologic analysis but in whom RT-PCR-detectable levels of PML-RAR
transcripts remain.287 Mutations and internal tandem duplications (ITD) of the FLT3 gene have been associated with a poor prognosis in adults and children with AML.288–294 In a study conducted in Japan, the estimated 5-year OS rate was only 14% for adult patients with FLT3-ITD, whose presence was the strongest prognostic factor in multivariate analysis.288 Similarly, in an analysis of 106 adults with AML treated in MRC trials, 13 of the 14 patients with FLT3-ITD died within 18 months of diagnosis.290 A subsequent study of 854 patients treated in the MRC 10 and 12 trials revealed FLT3-ITD in 27% of cases.291 In this report, the presence of FLT3-ITD was associated with an increased relapse risk and worse probabilities of disease-free survival, EFS, and OS. An analysis

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of 91 children and adolescents with AML treated in CCG trials demonstrated an 8-year EFS estimate of only 7% for the 15 patients with FLT3-ITD.292 Multivariate analysis demonstrated that FLT3-ITD was the most important prognostic factor in this study. Similarly, a study of 234 children with AML indicated that FLT3-ITD was the strongest predictor of relapse.294 Recently, the prognostic importance of FLT3 mutations has been questioned.93,295–297 In an analysis of 82 adults with AML who were treated according to the CALGB 9621 protocol and whose leukemic blast cells had normal cytogenetic features, three genotypes were detected: two wildtype FLT3 alleles (FLT3wt/wt ), which were observed in 59 patients; one FLT3-ITD allele and one wild-type allele (FLT3ITD/wt ), which were observed in 15 patients; and only one FLT3-ITD allele (FLT3ITD/− ), which was observed in eight patients.295 Interestingly, OS and EFS probabilities for patients with the FLT3wt/wt genotype were similar to those for patients with the FLT3ITD/wt genotype. Only those patients with the FLT3ITD/− genotype had a significantly worse outcome. Similarly, in an analysis of 979 patients with AML, those with a high FLT3 mutant:wild-type allele ratio had significantly reduced disease-free survival and OS estimates, whereas those with low ratios had outcomes similar to those of patients without FLT3 mutations.93 Results of these studies, as well as emerging findings from the CCG, suggest that the true prognostic importance of FLT3 mutations depends on the relative amount of wild-type FLT3 protein and that only those patients with a predominance of the mutant protein are at an increased risk of relapse. Other molecular alterations implicated as prognostic factors in AML include CEBPA mutations,94 DCC expression,298 VEGF secretion,299 expression of apoptosis-related genes,300,301 and global gene expression patterns.302,303 However, the value of these factors needs to be confirmed in prospective clinical trials. By contrast, many investigators have studied the prognostic significance of ATP-binding cassette (ABC) transporters in AML.304–306 Studies in children and adults have implicated P-glycoprotein, multidrug resistance-associated protein 1, and lung resistance protein as important proteins in mechanisms of drug resistance. Recent reports have also suggested that expression of the breast cancer resistance protein, another member of the ABC transporter family, may indicate a poor prognosis for patients with AML, but the clinical relevance of expression of this protein remains to be determined.307–310 It is likely that drug resistance in AML is multifactorial, thus hampering our attempts to use expression of drug transporters as prognostic markers or therapeutic targets.

Minimal residual disease Many studies of ALL have demonstrated the prognostic importance of early response to therapy.311 In contrast to morphologic examination, which tends to be subjective and imprecise, minimal residual disease (MRD) assays provide objective and sensitive measurements of low levels of leukemic cells.312–314 Methods of assessing MRD include DNA-based polymerase chain reaction (PCR) analysis of clonal antigen-receptor gene rearrangements, RNA-based PCR analysis of leukemiaspecific gene fusions, and flow cytometric detection of aberrant immunophenotypes.312–314 In AML, early blast clearance based on morphologic examination of the bone marrow is an independent predictor of treatment outcome.315,316 Like measurements of MRD in patients receiving treatment for ALL, measurement of MRD levels in AML patients by flow cytometry is also predictive of relapse.317,318 In one of the first studies reported, detectable leukemic blasts at the time of morphologic remission were predictive of more rapid relapse.319 Recently, in the CCG2961 trial, the impact of MRD in 252 children with AML was evaluated.320 At the end of induction therapy, occult leukemia, defined as greater than 0.5% blasts with an aberrant phenotype, was detected in 16% of the children for whom remission as achieved. Multivariate analysis demonstrated that patients with detectable MRD were 4.8 times more likely to experience relapse (P < 0.0001) and 3.1 times more likely to die (P < 0.0001) than those lacking detectable levels of disease. A study performed at St. Jude Children’s Research Hospital yielded similar findings: the 2-year survival estimate for patients with detectable MRD at the end of induction therapy was 33%, compared to 72% for MRD-negative patients (P = 0.022).318 Flow cytometric studies of MRD in adults with AML have yielded similar results.321–323 In a study of 126 adults with AML, multivariate analysis showed that the level of MRD was the most important predictor of outcome.323 Patients with MRD levels greater than 1% had a particularly poor outcome: the 3-year relapse rate was 84%, whereas no patient with an MRD level less than 0.01% suffered a relapse. Quantitative RT-PCR of leukemia-specific fusion transcripts is an alternative method of MRD detection that can be used in about one half of AML cases.324,325 Although its predictive value and clinical use in the management of APL is well established,326 the role of RT-PCR monitoring in other subtypes of AML is not as clear. However, promising results suggest that quantification of AML1-ETO and CBFβ-MYH11 fusion transcripts may be useful predictors of relapse.235,327–330 Similarly, evidence is emerging that quantitative PCR assessment of WT1 transcripts may prove

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to be useful for monitoring MRD levels in patients with AML.331,332 For patients with APL, monitoring the level of PMLRARα fusion transcripts by RT-PCR is an effective method of detecting MRD.333–338 Approximately 95% of patients are PML-RARα–negative after intensive consolidation chemotherapy. A positive result after consolidation therapy reliably predicts subsequent relapse, whereas repeated negative results for most patients are associated with relatively high long-term survival probability.339 When patients who converted to a PML-RARα-positive status were treated with chemotherapy before the reappearance of overt disease, the patients’ outcome was significantly better than that of patients whose treatment was delayed until morphologic evidence of relapse.326 A reasonable schedule of testing is to obtain at least two successive marrow samples at the end of consolidation treatment, every 3 months for the first 2 years of CR, and then every 6 months for the next 2 to 3 years. Because of its increased sensitivity, quantitative PCR will probably be used in the future to improve the ability to predict relapse.340,341

Complications and supportive care At initial examination, patients with AML may have lifethreatening complications, including bleeding, leukostasis, tumor lysis syndrome, or infection. Hemostasis should be maintained through the use of platelet transfusions and, in patients with DIC, the judicious use of fresh frozen plasma or factor concentrates. Leukostasis, the sludging of leukemic blasts in small vessels, may lead to infarction and hemorrhage in the lungs or CNS of patients with increased leukocyte counts. In such cases, leukapheresis or exchange transfusion can be used to rapidly reduce the leukocyte count before chemotherapy is begun. The hyperuricemia and hyperphosphatemia of tumor lysis syndrome should be prevented or treated by aggressive hydration and the use of oral phosphate binders and recombinant urate oxidase. Infectious complications, both at diagnosis and during therapy, remain a major cause of morbidity and mortality for patients with AML.342,343 Although the results of some studies suggest that certain cancer patients with fever and neutropenia are at low risk of serious infection and may be treated with oral antibiotics, all AML patients with febrile neutropenia should be hospitalized and treated with broad-spectrum intravenous antibiotics, such as a thirdor fourth-generation cephalosporin (e.g. ceftazidime or cefepime).344,345 In addition, because of the high incidence of Streptococcus viridans infection in AML patients who have received high-dose cytarabine,343,346 we recommend the empiric use of vancomycin as well. An aminoglyco-

side such as tobramycin should be administered to patients who have evidence of sepsis or infection with Pseudomonas aeruginosa or who have recently received cephalosporins. Patients with severe abdominal pain, radiologic evidence of typhlitis, or known infection with Bacillus cereus should be treated with a carbapenem (imipenem or meropenem) rather than a cephalosporin. Patients with AML are at increased risk of fungal infections, most commonly candidiasis and aspergillosis. Therefore, patients who remain febrile after 3 to 5 days of antibiotic therapy should receive empiric antifungal therapy. In addition, it may be prudent to treat all AML patients with prophylactic antifungals, although this strategy has not yet been proved to be effective. Currently available antifungals include traditional amphotericin B, lipid formulations of amphotericin B [AmBisome® (Fujisawa Healthcare) and Abelcet® (The Liposome Company, Inc.)], azoles (fluconazole, itraconazole, and voriconazole), and echinocandins (caspofungin and FK463). Results of several recent studies suggest that voriconazole may be as effective as amphotericin B for empiric antifungal treatment of neutropenic patients with persistent fever347 and for treatment of patients with invasive aspergillosis.348 Cytokines such as granulocyte-macrophage colonystimulating factor (GM-CSF) and G-CSF have been used to accelerate neutrophil recovery after courses of chemotherapy in an attempt to reduce morbidity and improve outcome.349 The effects of GM-CSF and G-CSF on time to neutrophil recovery after chemotherapy have been examined in many studies; however, the number of days during which patients received antibiotics, had fever, or were hospitalized were reduced in only a few.350,351 In one study, in which GM-CSF was used in patients with hypoplastic bone marrow after induction therapy was completed, did the number of infections and mortality rate decrease and remission rate improve.352 Recently, CCG investigators demonstrated that G-CSF improved the outcome of patients who had hypercellular bone marrow at day 7 of therapy.353 Currently, we do not recommend the routine use of growth factors in patients who have AML and are undergoing chemotherapy. Patients with APL are at high risk of DIC and bleeding at the time of diagnosis or during early induction therapy. Overt bleeding may involve the oral mucosa, CNS, or large or small bowels; nose bleeding and menorrhagia may also occur. The exacerbation of coagulopathy associated with cytotoxic chemotherapy may be lessened by the use of ATRA in induction therapy, but fatal hemorrhagic complications still occur.154 The goal is to maintain hemostasis, and not necessarily to achieve a specific platelet count, through the use of platelet transfusions and, in patients

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with DIC, the judicious transfusion of fresh frozen plasma or factor concentrates. The major toxicity of ATRA is retinoic acid syndrome, a cardiorespiratory distress syndrome manifested by fever, weight gain, respiratory distress, interstitial pulmonary infiltrates, pleural and pericardial effusion, episodic hypotension, and acute renal failure.354 Among patients whose APL is induced into remission by treatment with ATRA alone, the incidence of retinoic acid syndrome is approximately 25%.234,354,355 The mortality rate for patients with the syndrome has declined over time; this decrease probably reflects earlier recognition of the syndrome and use of dexamethasone. No factors, including WBC count, are clearly predictive of the syndrome, although the presence of microgranular variant (M3v) morphology was predictive in one study.223,232,355 An important issue is the potential difficulty in accurately diagnosing the retinoic acid syndrome because patients may experience toxicity and complications of therapy, such as pneumonia, congestive heart failure, and sepsis, with manifestations that mimic retinoic acid syndrome. The concurrent administration of chemotherapy with ATRA may decrease the incidence of the syndrome. In addition, the Australian Leukemia Study Group has explored the benefits of prophylactic corticosteroid therapy in patients in whom leukocytosis (WBC count > 10 × 109 /L) develops. In a nonrandomized study, 87 patients received prophylactic corticosteroid therapy (prednisone, 75 mg/day); the syndrome developed in 16% of the patients and was fatal in only 3%.356 However, given the potential toxicity of corticosteroid therapy administered for several weeks and the lack of confirmation by a prospective randomized trial, this approach cannot be routinely recommended for all patients. The use of ATRA in young children presents some unique challenges, including the difficulty of administering the drug by mouth (some young patients do not reliably swallow capsules). The pharmacokinetics of nasogastric delivery have not been studied, but remission has been achieved in a few patients who received treatment by this route.357 An alternative approach might be the use of a newly developed liposomal ATRA compound that can be given intravenously.358,359 A second problem with the use of ATRA in pediatric patients, especially those younger than 10 years, is increased neurotoxicity. Both headache and pseudotumor cerebri are more frequent in children than in adults.360,361 Management of this problem may include withholding ATRA for a period of time, using dexamethasone or acetazolamide (or both), and reinstituting ATRA at a lower dosage. Children on maintenance therapy consisting of ATRA and acetazolamide should undergo periodic ophthalmologic examinations, and electrolytes should be

monitored closely. In several European studies, ATRA given at a dosage of 25 mg/m2 per day to children younger than 15 years was as efficacious and more tolerable than higher dosages.236,238 A final consideration in treating children and adolescents with APL relates to the risk of cardiomyopathy associated with dosages of daunorubicin greater than 400 mg/m2 .362 Because of this issue and the additional cardiac toxicity that may be incurred if marrow transplantation is required, the COG has limited the maximum daunorubicin exposure to children enrolled in the current North American Intergroup trial. In addition, frequent assessment of cardiac function is required for children in that trial.

Treatment-related AML With the successful outcome of modern cancer treatments, the incidence of treatment-related leukemia (t-AML) and MDS (t-MDS) has increased.24,363 These devastating complications are of great importance in children and adolescents, who by virtue of their age, remain at risk of treatmentinduced side effects for prolonged periods. Treatment with alkylating agents, such as cyclophosphamide, ifosfamide, nitrogen mustard, chlorambucil, and melphalan, is associated with an increased incidence of t-AML/MDS at 4 to 5 years after the completion of initial treatment, but some cases have occurred 10 to 12 years later.364,365 A recent report of two children with t-MDS/monosomy 7 after treatment for ALL without alkylating agents or radiation therapy, emphasizes the need for close follow-up of childhood ALL survivors to identify therapy-related side effects.366 The association between epipodophyllotoxins and t-AML in children and adolescents is now well established.25,363,367 The outcome of these patients is dismal, with long-term survival rates of 10% to 20%, even after allogeneic bone marrow transplantation.276,363 By contrast, when APL develops as a second malignancy, it responds to ATRA treatment plus chemotherapy as favorably as does primary APL.368,369 In a recent study of adults with secondary APL, the complete remission was 93%, the 4-year EFS probability was 68%, and 4-year OS probability was 75%.368 Conversely, patients in whom t-AML or t-MDS develops after successful treatment for primary APL have a poor outcome.370 Please see Chapter 31 for a comprehensive discussion of therapy-related leukemias.

Future directions Because cure rates for AML remain unacceptably low, novel therapies are urgently needed. New but nonspecific

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agents that have shown promise in Phase I studies include troxacitabine,371 gemcitabine,372,373 and temozolomide.374 Gemtuzumab ozogamicin, an anti-CD33 antibody conjugated to calicheamicin designed to selectively target AML blasts, has shown activity as a single agent and is now being tested in combination with conventional chemotherapeutic agents.375–378 A recent report suggests that exposure to gemtuzumab ozogamicin increases the risk of veno-occlusive disease following BMT; hence, this agent should be used cautiously in patients who are likely to undergo subsequent BMT.379 Because abnormal methylation of tumor suppressor genes has been implicated in the pathogenesis of AML, demethylating agents are also being investigated for use against this disease.380,381 Similarly, histone deacetylase inhibitors can have a profound impact on gene expression and may, alone or in combination with demethylating agents, retinoids, or conventional chemotherapy, play a role in the treatment of AML by relieving the differentiation block that characterizes the leukemic blast.382–384 Most promising of all, however, is the development of specific inhibitors of kinases that are mutated or overexpressed in AML. As discussed above, constitutively activating mutations (internal tandem duplications and point mutations) of the tyrosine kinase FLT3 are common in AML and are associated with a poor prognosis.47 Like BCR-ABL in chronic myelogenous leukemia, FLT3 represents a specific and rational target for therapeutic intervention. In this regard, several FLT3 inhibitors have recently been developed.385–389 In preclinical models, these agents selectively inhibited autophosphorylation of wild-type and mutant FLT3, inhibited downstream targets of FLT3, induced apoptosis in cell lines and primary AML samples harboring FLT3 mutations, and had a therapeutic effect in mouse models of AML.385–387 These inhibitors may also be able to overcome the differentiation block caused by FLT3 activation.388 We anticipate that these kinase inhibitors, which are now in Phase I and II trials, will lead to less toxic and more effective cures for AML. REFERENCES 1 Bonnet, D. & Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997; 3: 730–7. 2 Stevens, R. F., Hann, I. M., Wheatley, K., et al. Marked improvements in outcome with chemotherapy alone in paediatric acute myeloid leukemia: results of the United Kingdom Medical Research Council’s 10th AML trial. MRC Childhood Leukaemia Working Party. Br J Haematol, 1998; 101: 130–40.

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300 Kohler, T., Schill, C., Deininger, M. W., et al. High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML). Leukemia, 2002; 16: 22–9. 301 Del Poeta, G., Venditti, A., Del Principe, M. I., et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood, 2003; 101: 2125–31. 302 Valk, P. J., Verhaak, R. G., Beijen, M. A., et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med., 2004; 350: 1617–28. 303 Bullinger, L., Dohner, K., Bair, E., et al. Use of geneexpression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med, 2004; 350: 1605– 16. 304 Leith, C. P., Kopecky, K. J., Chen, I. M., et al. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood, 1999; 94: 1086–99. 305 Legrand, O., Simonin, G., Beauchamp-Nicoud, A., et al. Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia. Blood, 1999; 94: 1046–56. 306 Boer, M. L. den, Pieters, R., Kazemier, K. M., et al. Relationship between major vault protein/lung resistance protein, multidrug resistance-associated protein, P-glycoprotein expression, and drug resistance in childhood leukemia. Blood, 1998; 91: 2092–8. 307 Steinbach, D., Sell, W., Voigt, A., et al. BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia, 2002; 16: 1443–7. 308 Heuvel-Eibrink, M. M. van den, Wiemer, E. A., Prins, A., et al. Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML). Leukemia, 2002; 16: 833–9. 309 Kolk, D. M. van der, Vellenga, E., Scheffer, G. L., et al. Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute myeloid leukemia. Blood, 2002; 99: 3763–70. 310 Abbott, B. L., Colapietro, A. M., Barnes, Y., et al. Low levels of ABCG2 expression in adult AML blast samples. Blood, 2002; 100: 4594–601. 311 Gaynon, P. S., Desai, A. A., Bostrom, B. C., et al. Early response to therapy and outcome in childhood acute lymphoblastic leukemia: a review. Cancer, 1997; 80: 1717–26. 312 Campana, D. & Coustan-Smith, E. Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia. Best Pract Res Clin Haematol, 2002; 15: 1–19. 313 Pui, C. H. & Campana, D. New definition of remission in childhood acute lymphoblastic leukemia. Leukemia, 2000; 14: 783– 5. 314 Szczepanski, T., Orfao, A., Velden, V. H. van der, et al. Minimal residual disease in leukaemia patients. Lancet Oncol, 2001; 2: 409–17.

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315 Creutzig, U., Zimmermann, M., Ritter, J., et al. Definition of a standard-risk group in children with AML. Br J Haematol, 1999; 104: 630–9. 316 Kern, W., Haferlach, T., Schoch, C., et al. Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 trial. Blood, 2003; 101: 64–70. 317 San Miguel, J. F., Vidriales, M. B., & Orfao, A. Immunological evaluation of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Best Pract Res Clin Haematol, 2002; 15: 105– 18. 318 Coustan-Smith, E., Ribeiro, R. C., Rubnitz, J. E., et al. Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia. Br J Haematol, 2003; 123: 243–52. 319 Sievers, E. L., Lange, B. J., Buckley, J. D., et al. Prediction of relapse of pediatric acute myeloid leukemia by use of multidimensional flow cytometry. J Natl Cancer Inst, 1996; 88: 1483–8. 320 Sievers, E. L., Lange, B. J., Alonzo, T. A., 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: 3398–406. 321 San Miguel, J. F., Martinez, A., Macedo, A., et al. Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients. Blood, 1997; 90: 2465–70. 322 Venditti, A., Buccisano, F., Del Poeta, G., et al. Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood, 2000; 96: 3948–52. 323 San Miguel, J. F., Vidriales, M. B., Lopez-Berges, C., et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood, 2001; 98: 1746–51. 324 Yin, J. A. & Grimwade, D. Minimal residual disease evaluation in acute myeloid leukaemia. Lancet, 2002; 360: 160–2. 325 Schnittger, S., Weisser, M., Schoch, C., et al. New score predicting for prognosis in PML-+, AML1-ETO+, or CBFBMYH11+ acute myeloid leukemia based on quantification of fusion transcripts. Blood, 2003; 102: 2746–55. 326 Lo, C. F., Diverio, D., Avvisati, G., et al. Therapy of molecular relapse in acute promyelocytic leukemia. Blood, 1999; 94: 2225–9. 327 Tobal, K., Newton, J., Macheta, M., et al. Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood, 2000; 95: 815–19. 328 Buonamici, S., Ottaviani, E., Testoni, N., et al. Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state. Blood, 2002; 99: 443–9.

329 Guerrasio, A., Pilatrino, C., De Micheli, D., et al. Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RTPCR amplification of fusion transcripts. Leukemia, 2002; 16: 1176–81. 330 Viehmann, S., Teigler-Schlegel, A., Bruch, J., et al. Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement. Leukemia, 2003; 17: 1130–6. 331 Trka, J., Kalinova, M., Hrusak, O., et al. Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry. Leukemia, 2002; 16: 1381–9. 332 Cilloni, D., Gottardi, E., De Micheli, D., et al. Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia, 2002; 16: 2115–21. 333 Lo, C. F., Avvisati, G., Diverio, D., et al. Molecular evaluation of response to all-trans-retinoic acid therapy in patients with acute promyelocytic leukemia. Blood, 1991; 77: 1657–9. 334 Miller, W. H., Jr., Kakizuka, A., Frankel, S. R., et al. Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. Proc Natl Acad Sci U S A, 1992; 89: 2694–8. 335 Diverio, D., Rossi, V., Avvisati, G., et al. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMAAIEOP multicenter “AIDA” trial. GIMEMA-AIEOP Multicenter “AIDA” Trial. Blood, 1998; 92: 784–9. 336 Ikeda, K., Sasaki, K., Tasaka, T., et al. Reverse transcriptionpolymerase chain reaction for PML-RAR alpha fusion transcripts in acute promyelocytic leukemia and its application to minimal residual leukemia detection. Leukemia, 1993; 7: 544–8. 337 Fukutani, H., Naoe, T., Ohno, R., et al. Prognostic significance of the RT-PCR assay of PML-RARa transcripts in acute promyelocytic leukemia. The Leukemia Study Group of the Ministry of Health and Welfare (Kouseisho). Leukemia, 1995; 9: 588–93. 338 Burnett, A. K., Grimwade, D., Solomon, E., et al. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the Randomized MRC Trial. Blood, 1999; 93: 4131–43. 339 Lo, C. F., Diverio, D., Falini, B., et al. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood, 1999; 94: 12–22. 340 Cassinat, B., Zassadowski, F., Balitrand, N., et al. Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia, 2000; 14: 324–8.

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341 Tobal, K., Moore, H., Macheta, M., et al. Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RTPCR method. Leukemia, 2001; 15: 1060–5. 342 Rubnitz, J. E., Lensing, S., Zhou, Y., et al. Death during induction therapy and first remission of acute leukemia in childhood: the St. Jude experience. Cancer, 2004; 101: 1677–84. 343 Okamoto, Y., Ribeiro, R. C., Srivastava, D. K., et al. Viridans streptococcal sepsis: clinical features and complications in childhood acute myeloid leukemia. J Pediatr Hematol Oncol, 2003; 25: 696–703. 344 Pizzo, P. A. Management of fever in patients with cancer and treatment-induced neutropenia. N Engl J Med, 1993; 328: 1323–32. 345 Freifeld, A., Marchigiani, D., Walsh, T., et al. A double-blind comparison of empirical oral and intravenous antibiotic therapy for low-risk febrile patients with neutropenia during cancer chemotherapy. N Engl J Med, 1999; 341: 305–11. 346 Gamis, A. S., Howells, W. B., DeSwarte-Wallace, J., et al. Alpha hemolytic streptococcal infection during intensive treatment for acute myeloid leukemia: a report from the Children’s Cancer Group Study CCG-2891. J Clin Oncol, 2000; 18: 1845–55. 347 Walsh, T. J., Pappas, P., Winston, D. J., et al. Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N Engl J Med, 2002; 346: 225–34. 348 Herbrecht, R., Denning, D. W., Patterson, T. F., et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med, 2002; 347: 408–15. 349 Estey, E. H. Growth factors in acute myeloid leukaemia. Best Pract Res Clin Haematol, 2001; 14: 175–87. 350 Godwin, J. E., Kopecky, K. J., Head, D. R., et al. A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031). Blood, 1998; 91: 3607–15. 351 Amadori, S., Suciu, S., Jehn, U., et al. Use of glycosylated recombinant human G-CSF (lenograstim) during and/or after induction chemotherapy in patients 61 years of age and older with acute myeloid leukemia: final results of AML-13, a randomized phase 3 study of the European Organisation for Research and Treatment of Cancer and Gruppo Italiano Malattie Ematologiche dell’Adulto (EORTC/GIMEMA) Leukemia Groups. Blood, 2005; 106: 27–34. 352 Rowe, J. M., Andersen, J. W., Mazza, J. J., et al. A randomized placebo-controlled phase III study of granulocytemacrophage colony-stimulating factor in adult patients (>55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood, 1995; 86: 457–62. 353 Alonzo, T. A., Kobrinsky, N. L., Aledo, A., et al. Impact of granulocyte colony-stimulating factor use during induction for acute myelogenous leukemia in children: a report from the Children’s Cancer Group. J Pediatr Hematol Oncol, 2002; 24: 627–35.

354 Frankel, S. R., Eardley, A., Heller, G., et al. All-trans retinoic acid for acute promyelocytic leukemia. Results of the New York Study. Ann Intern Med, 1994; 120: 278–86. 355 Tallman, M. S., Andersen, J. W., Schiffer, C. A., et al. Clinical description of 44 patients with acute promyelocytic leukemia who developed the retinoic acid syndrome. Blood, 2000; 95: 90–5. 356 Wiley, J. S. & Firkin, F. C. Reduction of pulmonary toxicity by prednisolone prophylaxis during all-trans retinoic acid treatment of acute promyelocytic leukemia. Australian Leukaemia Study Group. Leukemia, 1995; 9: 774–8. 357 Bargetzi, M. J., Tichelli, A., Gratwohl, A., et al. [Oral Alltransretinoic acid administration in intubated patients with acute promyelocytic leukemia]. Schweiz Med Wochenschr, 1996; 126: 1944–5. 358 Estey, E. H., Giles, F. J., Kantarjian, H., et al. Molecular remissions induced by liposomal-encapsulated all-trans retinoic acid in newly diagnosed acute promyelocytic leukemia. Blood, 1999; 94: 2230–5. 359 Douer, D., Estey, E., Santillana, S., et al. Treatment of newly diagnosed and relapsed acute promyelocytic leukemia with intravenous liposomal all-trans retinoic acid. Blood, 2001; 97: 73–80. 360 Smith, M. A., Adamson, P. C., Balis, F. M., et al. Phase I and pharmacokinetic evaluation of all-trans-retinoic acid in pediatric patients with cancer. J Clin Oncol, 1992; 10: 1666– 73. 361 Mahmoud, H. H., Hurwitz, C. A., Roberts, W. M., et al. Tretinoin toxicity in children with acute promyelocytic leukaemia. Lancet, 1993; 342: 1394–5. 362 Krischer, J. P., Epstein, S., Cuthbertson, D. D., et al. Clinical cardiotoxicity following anthracycline treatment for childhood cancer: The Pediatric Oncology Group Experience. J Clin Oncol, 1997; 15: 1544–52. 363 Pui, C. H. & Relling, M. V. Topoisomerase II inhibitorrelated acute myeloid leukaemia. Br J Haematol, 2000; 109: 13–23. 364 Micallef, I. N., Lillington, D. M., Apostolidis, J., et al. Therapyrelated myelodysplasia and secondary acute myelogenous leukemia after high-dose therapy with autologous hematopoietic progenitor-cell support for lymphoid malignancies. J Clin Oncol, 2000; 18: 947–55. 365 Sandoval, C., Pui, C. H., Bowman, L. C., et al. Secondary acute myeloid leukemia in children previously treated with alkylating agents, intercalating topoisomerase II inhibitors, and irradiation. J Clin Oncol, 1993; 11: 1039–45. 366 Aquino, V. M., Schneider, N. R., & Sandler, E. S. Secondary myelodysplasia with monosomy 7 arising after treatment for acute lymphoblastic leukemia in childhood. J Pediatr Hematol Oncol, 2001; 23: 48–50. 367 Pui, C. H., Relling, M. V., Rivera, G. K., et al. Epipodophyllotoxinrelated acute myeloid leukemia: a study of 35 cases. Leukemia, 1995; 9: 1990–6. 368 Pulsoni, A., Pagano, L., Lo, C. F., et al. Clinicobiological features and outcome of acute promyelocytic leukemia occurring as

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a second tumor: the GIMEMA experience. Blood, 2002; 100: 1972–6. Beaumont, M., Sanz, M., Carli, P. M., et al. Therapy-related acute promyelocytic leukemia. J Clin Oncol, 2003; 21: 2123– 37. Latagliata, R., Petti, M. C., Fenu, S., et al. Therapy-related myelodysplastic syndrome-acute myelogenous leukemia in patients treated for acute promyelocytic leukemia: an emerging problem. Blood, 2002; 99: 822–4. Giles, F. J., Garcia-Manero, G., Cortes, J. E., et al. Phase II study of troxacitabine, a novel dioxolane nucleoside analog, in patients with refractory leukemia. J Clin Oncol, 2002; 20: 656–64. Rizzieri, D. A., Bass, A. J., Rosner, G. L., et al. Phase I evaluation of prolonged-infusion gemcitabine with mitoxantrone for relapsed or refractory acute leukemia. J Clin Oncol, 2002; 20: 674–9. Gandhi, V., Plunkett, W., Du, M., et al. Prolonged infusion of gemcitabine: clinical and pharmacodynamic studies during a phase I trial in relapsed acute myelogenous leukemia. J Clin Oncol, 2002; 20: 665–73. Seiter, K., Liu, D., Loughran, T., et al. Phase I study of temozolomide in relapsed/refractory acute leukemia. J Clin Oncol, 2002; 20: 3249–53. Sievers, E. L., Appelbaum, F. R., Spielberger, R. T., et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood, 1999; 93: 3678–84. Sievers, E. L., Larson, R. A., Stadtmauer, E. A., et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33positive acute myeloid leukemia in first relapse. J Clin Oncol, 2001; 19: 3244–54. Larson, R. A., Boogaerts, M., Estey, E., et al. Antibody-targeted chemotherapy of older patients with acute myeloid leukemia in first relapse using Mylotarg (gemtuzumab ozogamicin). Leukemia, 2002; 16: 1627–36. Zwaan, C. M., Reinhardt, D., Corbacioglu, S., et al. Gemtuzumab ozogamicin: first clinical experiences in children with relapsed/refractory acute myeloid leukemia treated on compassionate-use basis. Blood, 2003; 101: 3868–71. Wadleigh, M., Richardson, P. G., Zahrieh, D., et al. Prior gemtuzumab ozogamicin exposure significantly increases the risk of veno-occlusive disease in patients who undergo myeloab-

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lative allogeneic stem cell transplantation. Blood, 2003; 102: 1578–82. Daskalakis, M., Nguyen, T. T., Nguyen, C., et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2’-deoxycytidine (decitabine) treatment. Blood, 2002; 100: 2957–64. Silverman, L. R., Demakos, E. P., Peterson, B. L., et al. Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol, 2002; 20: 2429–40. Byrd, J. C., Marcucci, G., Parthun, M. R., et al. A phase 1 and pharmacodynamic study of depsipeptide (FK228) in chronic lymphocytic leukemia and acute myeloid leukemia. Blood, 2005; 105: 959–67. Gore, S. D., Weng, L. J., Figg, W. D., et al. Impact of prolonged infusions of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia. Clin Cancer Res, 2002; 8: 963–70. Sandor, V., Bakke, S., Robey, R. W., et al. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res, 2002; 8: 718–28. Brown, P., Meshinchi, S., Levis, M., et al. Pediatric AML primary samples with FLT3/ITD mutations are preferentially killed by FLT3 inhibition. Blood, 2004; 104: 1841–9. Kelly, L. M., Yu, J. C., Boulton, C. L., et al. CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell, 2002; 1: 421–32. Weisberg, E., Boulton, C., Kelly, L. M., et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell, 2002; 1: 433–43. Zheng, R., Friedman, A. D., & Small, D. Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations. Blood, 2002; 100: 4154–61. Stone, R. M., DeAngelo, D. J., 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: 54–60. Lie, S. O., Abrahamsson, J., Clausen, N., et al. Treatment stratification based on initial in vivo response in acute myeloid leukaemia in children without Down’s syndrome: results of NOPHO-AML trials. Br J Haematol, 2003; 122: 217–25.

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20 Relapsed acute myeloid leukemia Ursula Creutzig

Introduction About 80% to 90% of children with newly diagnosed acute myeloid leukemia (AML) achieve complete remission (CR) with intensive induction chemotherapy.1,2 However, 10% to 20% of patients do not achieve remission due to early death by hemorrhage, leukostasis, infection or resistant disease (primary refractory AML), and even after achieving remission, 30% to 50% of patients still relapse. Primary refractory AML and relapsed AML have a poor prognosis, with an overall survival of less than 25%. For patients in first relapse, the prognosis mainly depends on the time of relapse. If it is early, defined as within 1 to 1.5 years after initial diagnosis, the second CR rate is about 50% and overall survival 10% or less. If the time of relapse is late, defined as later than 1.5 years after diagnosis, the second CR rate is 80% to 90% with an overall survival of up to 40%.3 Multiplerelapsed AML has an even worse prognosis. The management of refractory or relapsed AML is quite difficult. Several treatment schedules have been studied recently, and progress seems feasible with the use of new drugs and novel drug combinations followed by hematopoietic stem cell transplantation (HSCT).

Definition Relapse is defined as the reappearance of leukemic cells at any site in the body. Most relapses in AML occur during treatment, mostly during the first year after diagnosis. Relapses after 2 years are rarely seen (Fig. 20.1). Only exceptionally are relapses observed after more than 10 years, and in these patients it is uncertain whether the leukemic cells

are really from the same clone found at diagnosis (relapse) or if they arose from a secondary (therapy-induced) leukemogenic event. This question is often difficult to resolve because of the lack of precise molecular biological and immunophenotypic techniques at the time the leukemia was first diagnosed.

Diagnosis and relapse site After a documented CR, the presence of 5% or more unequivocal leukemic cells in a representative bone marrow (if <20% blasts are found, a second bone marrow should be investigated 1 or 2 weeks later) and/or evidence of leukemic infiltration or recurrence at any site of the body are required for the diagnosis of relapse. Such diagnoses should include not only morphologic but also immunologic and molecular biologic techniques, because in some patients an apparent relapse by morphologic criteria could represent a switch in the leukemic cell lineage or even secondary AML. A lumbar puncture should always be done. More than 5 cells/ L, unequivocal evidence of blasts on cytospin examination, and/or clinical and radiologic evidence of leukemic infiltration in the central nervous system (CNS) are required for the diagnosis of CNS disease or CNS relapse.

Hematologic relapse The bone marrow is the most common site of relapse in AML. Such clinical indices as insufficient hematopoiesis (anemia, leukopenia, and thrombocytopenia, sometimes leukocytosis), enlargement of liver and spleen, bone pain,

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Relapsed acute myeloid leukemia

Fig. 20.1 Postrelapse survival according to time to relapse. All patients were treated with intensive therapy in AML-BFM studies 87 and 93 between January 1993 and July 2002. The data were compared by log-rank analysis.

and fever suggest bone marrow relapse. In children still receiving treatment (e.g. maintenance therapy), prolonged aplasia may be the first sign of relapse.

CNS relapse CNS relapses, either isolated or combined, account for about 10% to 20% of all relapses in children. They are more often seen in children with initial CNS involvement and in those with hyperleukocytosis.

Other extramedullary relapses Beside the CNS, extramedullary relapses can occur in many other sites of the body (Table 20.1). They are frequent in skin, especially for the M4/M5 subtypes of AML. Isolated testicular relapses are rarely seen.

Multiple relapses More than two relapses are rare in AML. Indeed, after a second relapse, new remissions are difficult to achieve. These patients are candidates for phase I or II trials with new drugs or may receive palliative treatment only.

Prognosis and prognostic factors The prognosis for patients with AML refractory to first-line treatment or in first or subsequent relapse is generally poor,

with an overall survival of less than 25%. The duration of first remission is the most important factor in predicting the length of second remission and survival (Fig. 20.1).3 In most cases, the second CR is shorter than the first. Other risk factors, such as favorable or unfavorable cytogenetics, are of minor importance. The probability of achieving a second CR with the same or a more intensive induction chemotherapy, including novel agents, is usually lower in patients in relapse compared with those with de novo AML. Depending on the duration of first remission, 40% to 80% of children will achieve a second remission, and the probability of extended survival after treatment with intensive chemotherapy and HSCT ranges from 20% to 40% (Fig. 20.1).3–5

Minimal residual disease assays for detecting early relapse Neither morphologic analysis nor immunophenotyping nor cytogenetics can reliably identify childhood AML patients with a high risk of relapse. By a molecular biologic strategy based on qualitative real-time RT-PCR, one group detected AML1/ETO fusion transcripts in patients with t(8;21)-positive AML6 in long-term remissions.7 However, qualitative RQ-RT-PCR offers both the advantage of higher sensitivity and the possibility of quantification, so that smaller amounts of residual leukemic cells can be identified, leading to better correlation between negative or low-level PCR results and prognosis. This technique might

541

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Ursula Creutzig

Table 20.1 Sites of relapse in children with AMLa Type of relapse

Relapse site

Isolated

Combined with other

Bone marrow CNS Skin Testes Lymph node

181 13 4 1 —

32 24b 6b 1b 1b

213/231 (92%) 37/231 (16%) 10/231 (4%) 2/231 (1%) 1/231 (0.4%)

Total

199

32

231

Total (%)

a

Data from 778 children in AML-BFM studies 87 and 93 (unpublished data).

b

Always combined with bone marrow.

therefore offer a reasonable tool for follow-up monitoring of patients in remission. Several studies with qualitative, nested competitive and RQ-RT-PCR have been able to show the prognostic value of these techniques in patients expressing the AML1/ETO and CBFß/MYH11 [associated with the (inv16)] fusion transcripts. Nonetheless, the numbers of patients in these studies were small, most of them were adults, and in most instances they were neither analyzed consecutively nor treated uniformly.8,9 In patients with acute promyelocytic leukemia with a detectable RAR
rearrangement, qualitative RT-PCR has been used to show that the persistence of an abnormal gene rearrangement is indicative of later relapse.10 For such patients, salvage treatment is recommended at the time of molecular relapse, defined as the conversion to PCR positivity in two successive bone marrow samples taken during follow-up.11 With newer qualitative RT-PCR methods, the PML-RAR
fusion gene product can be amplified with increased sensitivity to the level of two leukemic cells in 106 normal cells.12 Five of 11 patients in long-term remission who tested negative by standard RT-PCR were positive with the “hot-start” PCR method. This indicates that a low level of PML-RAR
expression may be present in “clinically cured” patients, so that the sensitivity of the minimal residual disease detection method has to be taken into account in decisions to treat molecular relapse.

Drug resistance In AML therapy the development of drug resistance is a major problem. Drug resistance may already be present in de novo AML when the leukemic cells are not responding to initial chemotherapy, but this is rarely seen in children. Acquired drug resistance occurs after the leukemic cells have responded to chemotherapy and is character-

ized by resistance to the same drugs given initially as well as to new drugs. In multiple drug resistance (MDR), there is decreased intracellular drug accumulation leading to resistance to chemotherapy. MDR is mediated by the multidrug resistance protein, P-glycoprotein (Pgp), which is expressed both in de novo and in relapsed AML.13 Overexpression of MRD1, the gene encoding Pgp, is interpreted as a biologic mechanism contributing to treatment failure in AML.14 Indeed, MDR is associated with lower CR rates and with a poor clinical outcome in patients treated with standard therapeutic regimens.13,15 ,16 In attempts to reverse MRD, some pharmacologic inhibitors of Pgp activity have been investigated in patients with AML.17–19 Cyclosporin A, for example, has been extensively tested as an MDR-modulating agent in children and adults.20,21 However, in these studies the cytotoxic drugs showed increased toxicity, necessitating dose reductions.22 A review of current experience19 indicates no advantage from the use of MDR modulators in AML therapy.

Treatment of relapsed/refractory AML Current chemotherapy options The first aim in children with refractory or relapsed AML is to achieve a (second) remission. Many chemotherapy regimens have been administered for this purpose. Currently, reinduction regimens include high-dose cytarabine in combination with well-known drugs of significant antileukemic activity, such as mitoxantrone, etoposide and amsacrine, with or without newer drugs like azacytidine, fludarabine, idarubicin, and 2-chlorodeoxyadenosine (2-CdA).23–29 With these regimens, consisting of two or more blocks of intensive chemotherapy, remission is achieved in 50% to 80% of patients depending on the duration of first remission. Besides cytarabine, anthracyclines are the most useful drugs in AML treatment, not only in first-line treatment but also during relapse. As many children have already received a high cumulative dose of anthracyclines during first-line treatment, the use of these drugs during relapse increases the risk of cardiotoxicity. Therefore, less cardiotoxic anthracyclines are needed. The liposomal formulation of daunorubicin (L-DNR) may afford the opportunity to reapply anthracyclines with a limited risk of cardiotoxicity. As a new formulation of an old drug, L-DNR is expected to retain the antileukemic effectiveness of the original compound with reduced cardiotoxicity. There is also some evidence that this new agent may overcome resistance to multiple anticancer drugs mediated by the MDR-associated protein Pgp. Thus, L-DNR was introduced

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into the AML-BFM 97 relapse study4 and is being tested in a randomized manner in the ongoing InternationalBFM Relapse Trial 2001, in which patients receive intensive induction chemotherapy with or without L-DNR.

New cytotoxic drugs The purine analogs fludarabine, 2-CdA, and recently clofarabine,30 are among the new drugs used in AML, especially in refractory or relapsed cases. These agents inhibit the ribonucleotide reductase and increase the activity of deoxycytidine kinase, leading to a higher rate of accumulation of cytarabine triphosphate.31 In preclinical and clinical studies, synergistic interactions between fludarabine or 2CdA and cytarabine could be seen.31,32 It is also expected that the effects of fludarabine or 2-CdA in combination with cytarabine can be increased with the addition of granulocyte colony-stimulating factor (G-CSF).33,34 Thus, the combination of fludarabine, cytarabine and G-CSF (FLAG) has emerged, although the role of G-CSF remains questionable.35 Gemcitabine (2 -deoxy-2 , 2s -difluorocytidine monohydrochloride, or dFDC) is another antimetabolite and a new analog of deoxycytidine that resembles cytarabine in its structure.36 It has been shown that gemcitabine in combination with 2-CdA has an additive effect on murine leukemias.37,38 The first clinical studies to confirm the activity of these drugs in AML have just started. Other interesting drugs are the specific inhibitors of topoisomerase I, the alkaloid camptothecin and its semisynthetic analog topotecan, a drug with activity against chronic myelomonocytic leukemias, MDS and AML.39 The combination of cyclophosphamide, cytarabine and topotecan (CAT) was active in refractory or relapsed AML.40 CAT in combination with all-trans-retinoic acid or G-CSF induced CRs in 10 of 11 patients with unfavorable cytogenetics.39

geneic HSCT. Clinical outcomes were also similar in an East German study, although autografts were performed late in second CR (median duration 32 weeks) with bone marrow harvested when the patients were in stable remission.42 The lack of a significant difference in outcome by source of allogeneic stem cells (i.e. matched sibling donor versus matched unrelated donor) is probably due to the greater graft-versus-leukemia effect of the latter cells, which also contribute to a higher rate of toxic deaths. Transplantation in early relapse is not recommended, even though preliminary data from the Seattle group have shown relatively good results for such patients with generally low blast counts.43 According to data of the European Bone Marrow Transplantation (EMBT) group, clinical outcome was significantly worse for children transplanted in relapse compared to patients transplanted in second remission.44 The EBMT Pediatric Diseases Working Party concluded that children in second CR have a definitive indication for allografts from matched sibling or matched unrelated donors or for autografts. AML patients who underwent HSCT in second CR had a survival probability of about 40%, while those transplanted in relapse had a survival rate of 15% to 20%.41 It is therefore recommended that a search is started for an allograft donor at diagnosis or early in relapse. A useful strategy after relapse is to base the type of HSCT on risk groups. It is preferable to perform matched-siblingdonor HSCT; otherwise, patients become candidates for HSCT from a matched unrelated donor. Ultimately, if such donors cannot be found, patients with primary refractory disease, early relapse (within 1 year from diagnosis), or a second or subsequent relapse may be candidates for the more experimental haplo-identical donor HSCT. However, patients with a late relapse (>1 year from initial diagnosis) have a better prognosis and should be offered autologous HSCT if matched sibling or unrelated donors are not available.

Hematopoietic stem cell transplantation After achieving a second remission, all patients should be transplanted as soon as possible, because this procedure is thought to be the only curative treatment after relapse. Generally, allogeneic HSCT appears more effective than autologous HSCT, as the graft-versus-leukemia reaction can destroy residual AML blasts. The International Bone Marrow Transplant Registry/Autologous Marrow Transplant Registry has shown that approximately 40% of children in second remission can be rescued by the different types of HSCT irrespective of the stem cell source.41 The Austrian-German-Italian Pediatric Registry found similar results for patients in second remission whether they received autologous or allo-

Preparative regimens and graft manipulation There are not sufficient data to recommend any conditioning regimen for patients in second CR. The EBMT group reported that there was no difference between totalbody irradiation (TBI)-and non-TBI-containing regimens for extending second remission in children. The same observation was made by the AIEOP Cooperative Group.45 The BFM group prefers non-TBI conditioning regimens for use in relapse trials, which include not only busulfan and cyclophosphamide, but also melphalan.46 After allogeneic transplantation, graft-versus-host disease (GVHD) has been found to have a protective effect against relapse. The EBMT group reported that

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transplantation from an identical twin resulted in an increased probability of relapse compared with results in allograft recipients without GVHD, supporting an antileukemic effect of allografts independent of GVHD.47 It has also been shown that the rate of immune recovery correlates with outcome, and that a slow recovery of lymphocytes after allogeneic HSCT is associated with a higher risk of relapse in patients with AML.48

Relapse after HSCT in first CR The prognosis for children relapsing after HSCT in first CR is more unfavorable than in patients who relapse after chemotherapy only. In allografted patients who relapse during immunosuppressive therapy, the withdrawal of immunosuppression may lead to a survival rate of about 10%.49 Donor leukocyte infusions have been tried as alternative therapy, but the results were not as good as in the chronic phase of CML.50 Patients who relapse later (i.e. after more than 6 to 12 months post-transplant in first remission) may benefit from a second HSCT. However, the salvage rate is only about 20% to 25%, and the risk of fatal toxicity is high.51 In the MRC-10 trial, it was reported that outcome for children who had been autografted in first CR was inferior after relapse compared with results for those without an autograft.2 In the EBMT report, the mortality rate was higher than 50% in patients who had been autografted in first CR and allografted later on.41

Differentiation therapy in acute promyelocytic leukemia Another approach to the treatment of resistant AML is differentiation therapy with several agents having different mechanisms of action, including phorbol esters, phenylbutyrate, retinoids and growth factors, which can induce terminal in vitro differentiation of leukemic cells. The first drug successfully used for differentiation therapy was alltrans-retinoic acid (ATRA) given to patients with acute promyelocytic leukemia (FAB type M3). ATRA resistance is rarely seen in M3 leukemia, except in cases with the special molecular rearrangement PLZF–RAR
.52 As so few patients lose sensitivity to ATRA, this drug can be effectively reapplied in relapse. A liposomal formulation of ATRA can be used in cases with resistance to the unmodified compound.53 Recently, arsenic trioxide has proved to be effective in acute promyelocytic leukemia,54 inducing a high CR rate in patients who were resistant to both ATRAand anthracycline/cytarabine-based chemotherapy.55 The

clinical response was associated with incomplete cytodifferentiation and induction of apoptosis in leukemic cells.56 However, neither arsenic trioxide nor ATRA has clinical efficacy in AML subtypes other than M3. Phase I or II trials with arsenic trioxide to study the best application in acute promyelocytic leukemia are still under way.

Signal transduction inhibitors Signal transduction inhibitors can selectively modulate the abnormal signal transduction by tyrosine kinases. Usually, the specific functional domain of these proteins is involved in the signaling pathways that link growth factors and their receptors to the cell membrane. In leukemias like chronic myeloid leukemia (CML), tyrosine kinase mutations lead to a continuous activation or amplification and overexpression of signaling proteins, which is associated with a growth and a survival advantage. STI571 (imatinib) can selectively suppress the growth of leukemic cells in CML patients by inhibiting the ABL protein tyrosine kinase.57 However, this drug does not seem to be effective against AML, as suggested by in vitro studies.58 Intervention in other signaling pathways may be a more promising route for this disease. For example, RAS mutations play a key role in signal transduction, proliferation and malignant transformation in several leukemias, including 25% to 44% of AMLs.59 Agents targeting RAS or tyrosine kinases, such as inhibitors of farnesyl transferase, may have efficacy in AML patients60 and could be expected to act synergistically with standard chemotherapy. However, the optimal dosages of these agents and their long-term effects in AML patients remain to be established.

Immunotherapy Most of the blast cells in AML patients express the CD33 antigen, making it an attractive therapeutic target. Indeed, when given to patients with relapsed or refractory AML, CD33 antibody conjugated with calicheamicin (Mylotarg, gemtuzumab ozogamicin) selectively reduced leukemic blast cells.61 In further studies with higher numbers of patients, remissions were induced in 42 of 142 patients in first relapse of AML.62 Severe mucositis was rarely seen; however, hepatotoxicity with thromboembolic occlusions might be a problem, especially when HSCT has preceded or is planned after administration of this drug.63 Another CD33 antibody (HuM195) was conjugated with 90 Y, while anti-CD45 was conjugated with 131 I, for the treatment of minimal residual disease. Both agents are being tested together with cytostatics and total-body irradiation as myeloablative therapy prior to HSCT.64

Relapsed acute myeloid leukemia

Active immunotherapy is also being evaluated in the treatment of AML. New vaccine strategies to enhance the immunogenicity of the vaccinating tumor cells have been devised by modifying the cells to express immunomodulatory molecules – for example, cytokines. AML vaccinating strategies with leukemia-derived dendritic cells are under investigation with the intent of reducing minimal residual disease in AML patients with poor prognostic factors.65

Antiangiogenic agents Increased angiogenesis in AML was recently reported.66,67 Since AML cells express the vascular endothelial growth factor (VEGF) mRNA, antiangiogenic drugs might afford a novel therapeutic strategy.68 As one example, arsenic trioxide, which is already being used successfully in the treatment of acute promyelocytic leukemia, inhibits VEGF production by leukemic cells and might therefore decrease angiogenesis and leukemia progression.69 However, prospective antiangiogenic drugs must first be tested in phase I trials.

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Summary Despite the poor prognosis associated with relapsed AML, at least 25% of patients can look forward to a new chance for extended survival with use of the therapeutic options described in this chapter. Once a second remission is induced, the probability of survival may increase to 30% or 40%. Unfortunately, the majority of patients cannot be rescued and become candidates for phase I or II trials with new drugs or new methods of stem cell transplantation. On the other hand, one must always keep in mind that too much toxicity is unacceptable in this situation, so that palliative treatment, which can give a prolonged period of goodquality survival, has to be considered as well. Palliative strategies include not only chemotherapy or immunotherapy to control the leukemia, but also supportive care and pain relief.

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diatric acute myeloid leukaemia: results of the United Kingdom Medical Research Council’s 10th AML trial. Br J Haematol, 1998; 101: 130–40. Stahnke, K., Boos, J., Bender-Gotze, C., et al. Duration of first remission predicts remission rates and long-term survival in children with relapsed acute myelogenous leukemia. Leukemia. 1998; 12: 1534–8. Reinhardt, D., Hempel, G., Fleischhack, G., et al. [Liposomal daunorubicine combined with cytarabine in the treatment of relapsed/refractory acute myeloid leukemia in children]. Klin P¨adiatr, 2002; 214: 188–94. Webb, D. K., Wheatley, K., Harrison, G., Stevens R. F., & Hann, I. M. Outcome for children with relapsed acute myeloid leukaemia following initial therapy in the Medical Research Council (MRC) AML 10 trial. MRC Childhood Leukaemia Working Party. Leukemia, 1999; 13: 25–31. Kozu, T., Miyoshi, H., Shimizu, K., et al. Junctions of the AML1/MTG8(ETO) fusion are constant in t(8;21) acute myeloid leukemia detected by reverse transcription polymerase chain reaction. Blood, 1993; 82: 1270–6. Jaeger, U., Kusec, R., & Haas, O. A. Detection of AML1/ETO rearrangements in acute myeloid leukemia with a translocation t(8;21). Haematol Blood Transfus, 1995; 37: 475–7. Tobal, K., Newton, J., Macheta, M., et al. Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood, 2000; 95: 815–19. Wattjes, M. P., Krauter, J., Nagel, S., et al. Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia. Leukemia, 2000; 14: 329–35. Miller, W. H., Kakizuka, A., & Frankel, S. R. Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. Proc Natl Acad Sci U S A, 1992; 89: 2694–8. Diverio, D., Rossi, V., Avvisati, G., et al. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMAAIEOP multicenter “AIDA” trial. GIMEMA-AIEOP Multicenter “AIDA” Trial. Blood, 1998; 92: 784–9. Tobal, K. & Liu, Y. J. RT-PCR method with increased sensitivity shows persistence of PML-RARA fusion transcripts in patients in long-term remission of APL. Leukemia, 1998; 12: 1349–54. Leith, C. P., Kopecky, K. J., Chen, I. M., et al. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood, 1999; 94: 1086–9. List, A. F. The role of multidrug resistance and its pharmacological modulation in acute myeloid leukemia. Leukemia, 1996; 10(Suppl. 1): S36–8.

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15 Leith, C. Multidrug resistance in leukemia. Curr Opin Hematol, 1998; 5: 287–91. 16 Schaich, M., Ritter, M., Illmer, T., et al. Mutations in ras protooncogenes are associated with lower mdr1 gene expression in adult acute myeloid leukaemia. Br J Haematol, 2001; 112: 300–7. 17 List, A. F., Spier, C. S., Grogan, T. M., et al. Overexpression of the major vault transporter protein lung–resistance protein predicts treatment outcome in acute myeloid leukemia. Blood, 1996; 87: 2464–9. 18 Sonneveld, P. Multidrug resistance in acute myeloid leukaemia. Baillieres Clin Haematol, 1996; 9: 185–203. 19 Vossebeld, P. J. & Sonneveld, P. Reversal of multidrug resistance in hematological malignancies. Blood Rev, 1999; 13: 67– 78. 20 Dahl, G. V., Lacayo, N. J., Brophy, N., et al. Mitoxantrone, etoposide, and cyclosporine therapy in pediatric patients with recurrent or refractory acute myeloid leukemia. J Clin Oncol, 2000; 18: 1867–75. 21 Liu Yin, J. A., Wheatley, K., Rees, J. K. H., & Burnett, A. K. Comparison of ‘sequential’ versus ‘standard’ chemotherapy as re-induction treatment, with or without cyclosporine, in refractory/relapsed acute myeloid leukaemia (AML): results of the UK Medical Research Council AML-R trial. Brit. J. Haematol, 2001; 113: 713–26. 22 Smeets, M., Raymakers, R., Muus, P., et al. Cyclosporin increases cellular idarubicin and idarubicinol concentrations in relapsed or refractory AML mainly due to reduced systemic clearance. Leukemia, 2001; 15: 80–8. 23 Fleischhack, G., Hasan, C., Graf, N., Mann, G., & Bode U. IDAFLAG (idarubicin, fludarabine, cytarabine, G-CSF), an effective remission-induction therapy for poor-prognosis AML of childhood prior to allogeneic or autologous bone marrow transplantation: experiences of a phase II trial. Br J Haematol, 1998; 102: 647–55. 24 Miller, L. P., Pyesmany, A. F., Wolff, L. J., et al. Successful reinduction therapy with amsacrine and cyclocytidine in acute nonlymphoblastic leukemia in children. A report from the Childrens Cancer Study Group. Cancer, 1991; 67: 2235–40. 25 Ozkaynak, M. F., Avramis, V. I., Carcich, S., & Ortega, J. A. Pharmacology of cytarabine given as a continuous infusion followed by mitoxantrone with and without amsacrine/etoposide as reinduction chemotherapy for relapsed or refractory pediatric acute myeloid leukemia. Med Pediatr Oncol, 1998; 31: 475–82. 26 Santana, V. M., Mirro, J., Jr., Harwood, F. C., et al. A phase I clinical trial of 2-chlorodeoxyadenosine in pediatric patients with acute leukemia. J Clin Oncol, 1991; 9: 416–22. 27 Steuber, C. P., Krischer, J., Holbrook, T., et al. Therapy of refractory or recurrent childhood acute myeloid leukemia using amsacrine and etoposide with or without azacitidine: a Pediatric Oncology Group randomized phase II study. J Clin Oncol, 1996; 14: 1521–5. 28 Webb, D. K. Management of relapsed acute myeloid leukaemia. Br J Haematol, 1999; 106: 851–9.

29 Whitlock, J. A., Wells, R. J., Hord, J. D., et al. High-dose cytosine arabinoside and etoposide: an effective regimen without anthracyclines for refractory childhood acute nonlymphocytic leukemia. Leukemia, 1997; 11: 185–9. 30 Jeha, S., Gandhi, V., Chan, K. W., et al. Clofarabine, a novel nucleoside analog, is active in pediatric patients with advanced leukemia. Blood, 2004; 103: 784–9. 31 Gandhi, V., Estey, E., Keating, M. J., Chucrallah, A., & Plunkett, W. Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions. Blood, 1996; 87: 256– 64. 32 Szmigielska, A., G´ora-Tybor, J., & Robak, T. Influence of 2chlorodeoxyadenosine alone and in combiantion with cytosine arabinoside on murine leukemias L1210 and P388. Cancer J, 1996; 9: 319–22. 33 Robak, T., Wrzesien-Kus, A., Lech-Maranda, E., Kowal, M., & Dmoszynska, A. Combination regimen of cladribine (2chlorodeoxyadenosine), cytarabine and G-CSF (CLAG) as induction therapy for patients with relapsed or refractory acute myeloid leukemia. Leuk Lymphoma, 2000; 39: 121–9. 34 Visani, G., Tosi, P., Zinzani, P. P., et al. FLAG (fludarabine + high-dose cytarabine + G-CSF): an effective and tolerable protocol for the treatment of ‘poor risk’ acute myeloid leukemias. Leukemia, 1994; 8: 1842–6. 35 Estey, E., Thall, P., Andreeff, M., et al. Use of granulocyte colony-stimulating factor before, during, and after fludarabine plus cytarabine induction therapy of newly diagnosed acute myelogenous leukemia or myelodysplastic syndromes: comparison with fludarabine plus cytarabine without granulocyte colony-stimulating factor. J Clin Oncol, 1994; 12: 671–8. 36 Plunkett, W., Huang, P., Searcy, C. E., & Gandhi, V. Gemcitabine: preclinical pharmacology and mechanisms of action. Semin Oncol, 1996; 23(Suppl. 10): 3–15. 37 Lech-Maranda, E., Korycka, A., & Robak, T. Influence of gemcitabine (2 ,2 -difluoro-deoxycytidine) and 2chlorodeoxyadenosine on growth of normal and leukemic cells in vitro. Eur J Haematol, 2000; 65: 317–21. 38 Maranda, E., Szmigielska, A., & Robak, T. Additive action of gemcitabine (2 ,2 -difluorodeoxycytidine) and 2chlorodeoxyadenosine on murine leukemias L1210 and P388. Cancer Invest, 1999; 17: 95–101. 39 Estey, E. H. New agents for the treatment of acute myelogenous leukemia: focus on topotecan and retinoids. Leukemia, 1998; 12(Suppl. 1): S13–15. 40 Cortes, J., Estey E., Beran, M., et al. Cyclophosphamide, ara-C and topotecan (CAT) for patients with refractory or relapsed acute leukemia. Leuk Lymphoma, 2000; 36: 479–84. 41 Abella, E. & Ravindranath, Y. Therapy for childhood acute myeloid leukemia: role of allogeneic bone marrow transplantation. Curr Oncol Rep, 2000; 2: 529–38. 42 Hermann, J., Schiller, I., Fuchs, D., et al. Autologous bone marrow transplantation in first complete remission as

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intensification therapy in children with high risk AML – results of the Pediatric Cooperative AML Trial 1987–1992 in East Germany. Haematol Blood Transfus, 1998; 39: 803–9. Clift, R. A., Buckner, C. D., Appelbaum, F. R., et al. Allogeneic marrow transplantation during untreated first relapse of acute myeloid leukemia. J Clin Oncol, 1992; 10: 1723–9. Ringden, O., Labopin, M., Frassoni, F., et al. Allogeneic bone marrow transplant or second autograft in patients with acute leukemia who relapse after an autograft. Acute Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant, 1999; 24: 389–96. Amadori, S., Testi, A. M., Aric´o, M., et al. Prospective comparative study of bone marrow transplantation and postremission chemotherapy for childhood acute myelogenous leukemia. J Clin Oncol, 1993; 11: 1046–54. Locatelli, F., Pession, A., Bonetti, F., et al. Busulfan, cyclophosphamide and melphalan as conditioning regimen for bone marrow transplantation in children with myelodysplastic syndromes. Leukemia, 1994; 8: 844–9. Gale, R. P. & Horowitz, M. M. Graft-versus-leukemia in bone marrow transplantation. The Advisory Committee of the International Bone Marrow Transplant Registry. Bone Marrow Transplant, 1990; 6(Suppl. 1): 94–7. Powles, R., Singhal, S., Treleaven, J., et al. Identification of patients who may benefit from prophylactic immunotherapy after bone marrow transplantation for acute myeloid leukemia on the basis of lymphocyte recovery early after transplantation. Blood, 1998; 91: 3481–6. Elmaagacli, A. H., Beelen, D. W., Trenn, G., et al. Induction of a graft-versus-leukemia reaction by cyclosporin A withdrawal as immunotherapy for leukemia relapsing after allogeneic bone marrow transplantation. Bone Marrow Transplant, 1999; 23: 771–7. Singhal, S., Powles, R., Kulkarni, S., et al. Long-term follow-up of relapsed acute leukemia treated with immunotherapy after allogeneic transplantation: the inseparability of graft-versushost disease and graft-versus-leukemia, and the problem of extramedullary relapse. Leuk Lymphoma, 1999; 32: 505–12. De la Rubia, J., Sanz, G. F., Martin, G., et al. Autologous bone marrow transplantation for patients with acute myeloblastic leukemia in relapse after autologous blood stem cell transplantation. Bone Marrow Transplant, 1996; 18: 1167–73. Guidez, F., Ivins, S., Zhu, J., et al. Reduced retinoic acidsensitivities of nuclear receptor corepressor binding to PMLand PLZF-RARalpha underlie molecular pathogenesis and treatment of acute promyelocytic leukemia. Blood, 1998; 91: 2634–42. Douer, D., Estey, E., Santillana, S., et al. Treatment of newly diagnosed and relapsed acute promyelocytic leukemia with intravenous liposomal all-trans retinoic acid. Blood, 2001; 97: 73–80. Niu, C., Yan, H., Yu, T., et al. Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission

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induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood, 1999; 94: 3315–24. Shen, Z. X., Chen, G. Q., Ni, J. H., et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood, 1997; 89: 3354–60. Soignet, S. L., Maslak, P., Wang, Z. G., et al. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med, 1998; 339: 1341–8. Buchdunger, E., Zimmermann, J., Mett, H., et al. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2phenylaminopyrimidine derivative. Cancer Res, 1996; 56: 100– 4. Scappini, B., Onida, F., Kantarjian, H. M., et al. Effects of signal transduction inhibitor 571 in acute myelogenous leukemia cells. Clin Cancer Res, 2001; 7: 3884–93. Reuter, C. W., Morgan, M. A., & Bergmann, L. Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies? Blood, 2000; 96: 1655–69. Willman, C. L. Targeted AML therapy: new biologic paradigms and therapeutic opportunities. Leukemia, 2001; 15: 690–4. Sievers. E. L., Appelbaum, F. R., Spielberger, R. T., et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood, 1999; 93: 3678–84. Sievers, E. L., Larson, R. A., Stadtmauer, E. A. et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33positive acute myeloid leukemia in first relapse. J. Clin Oncol, 2001; 19: 3244–54. Sato, Y., Asada, Y., Hara, S., et al. Hepatic stellate cells (Ito cells) in veno-occlusive disease of the liver after allogeneic bone marrow transplantation. Histopathology, 1999; 34: 66– 70. Matthews, D. C., Appelbaum, F. R., Eary, J. F., 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: 1237–47. Claxton, D. & Choudhury, A. Potential for therapy with AMLderived dendritic cells. Leukemia, 2001; 15: 668–9. Aguayo, A., Kantarjian, H., Manshouri, T., et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood, 2000; 96: 2240–5. Padro, T., Ruiz, S., Bieker, R., et al. Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia. Blood, 2000; 95: 2637–44. Schuch, G., Oliveira-Ferrer, L., Loges, S., et al. Antiangiogenic treatment with endostatin inhibits progression of AML in vivo. Leukemia, 2005, 19: 1312–17. Roboz, G. J., Dias, S., Lam, G., et al. Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis. Blood, 2000; 96: 1525–30.

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21 Myelodysplastic syndrome Henrik Hasle

Introduction Myelodysplastic syndrome (MDS) is a clonal myeloid malignancy characterized by the triad of growth advantage of clonal cells, disturbed differentiation and increased apoptosis. The malignant cells have retained some capacity for differentiation, and have the propensity to undergo apoptosis in the bone marrow; hence, in contrast to acute leukemia, there is a lack of blast cell domination of bone marrow. MDS is much rarer in children than in adults, and most of the literature on this disease is based upon studies in elderly patients; however, there are significant differences between MDS in children and adults (Table 21.1), including the morphologic features and cytogenetic findings at diagnosis. Many children have associated abnormalities (e.g. pre-existing bone marrow failure or congenital abnormalities). The therapeutic aim in children with MDS is primarily a cure, whereas this possibility is often not realistic in adults. The rarity of MDS in children and the lack of a widely accepted classification have contributed to the paucity of reports on this malignancy in the pediatric literature, although in recent years increasing attention has been paid to childhood MDS patients.1–10

Classification of childhood MDS – historical background The classification of childhood MDS has been inconsistent and confusing. MDS was not included in the official classification of childhood malignancies until the revised version published in 2005.11 The rarity and heterogeneous nature of the disease have further contributed to the difficulties in its classification.

A plethora of names have been used over the last decades to designate MDS, reflecting the conceptual and diagnostic ambiguity surrounding this disorder. The first published case of presumed childhood MDS was described as monocytic leukemia in the 1930s.12 One case that retrospectively might be classified as MDS was described in 1958 as chronic monocytic leukemia.13 Several cases were designated acute myeloid leukemia (AML) despite a low number of blasts.14 The term preleukemia was introduced into pediatrics in the early 1970s,15 but the number of reported cases remained extremely low. Only 11 cases were identified in a review published in 1980.16 This term is misleading because it gives the impression that the patient has yet to develop leukemia. MDS is a clonal malignant disease in itself and not a precursor of a malignant condition. The confusion is increased by using the term preleukemia for conditions with an increased risk of malignant hematologic diseases (e.g. aplastic anemia and Fanconi anemia). Furthermore, in the 1980s, MDS in children was often reported together with cases of transient pancytopenia preceding ALL (preALL) and collectively described as preleukemic states.17,18 These two conditions are very distinctive and should be considered separately.19 Monosomy 7 in children has often been considered to represent a distinct hematologic disorder described as the monosomy 7 syndrome, characterized by young age, male predominance, hepatosplenomegaly, and leukocytosis.20–22 The infantile monosomy 7 syndrome has been included as a separate entity in the classification of childhood MDS.1,23 Complete loss of chromosome 7 occurs in all morphologic MDS subgroups.24,25 There is no evidence that monosomy 7 represents a discrete entity,

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 21.1 Major differences between MDS in children and adults

Incidence per million RA with ringed sideroblasts Associated abnormalities Cytogenetic aberrations −7/7q− −5/5q− General aim of treatment

Children

Adults

1–2 2% 30% 60% 30–40% 1–2% Curative

>30 25% <5% 40% 10% 20% Palliative

Abbreviation: RA, refractory anemia.

and it should no longer be referred to as the monosomy 7 syndrome.25,26 The French-American-British (FAB) group proposed in 1982 a classification of MDS comprising five subgroups: refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with an excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML).27,28 AML was defined by the presence of 30% myeloblasts in the bone marrow. The FAB classification was developed on the basis of reviews of smears from adult patients. The FAB classification of AML became rapidly generally accepted in pediatrics, whereas the FAB classification of MDS has only slowly and partly been accepted among pediatricians. The FAB classification of MDS was introduced into pediatrics in the mid 1980s29,30 and has been used by many researchers and clinicians, although some investigators have had major problems in applying the FAB classification in a pediatric population.3 The FAB classification has prognostic impact in children1,25 and has facilitated communication about pediatric MDS, but several problems remain due to the specific diseases and morphologic features in children and the frequent occurrence of associated anomalies. The recent World Health Organization (WHO) classification of neoplastic diseases of the hematopoietic and lymphoid tissues31 incorporates both morphology and genetic changes. The threshold for distinguishing AML from MDS was lowered from 30% to 20% blast cells. Both the FAB and the WHO proposals were based on a review of adult cases. The WHO classification recognizes juvenile myelomonocytic leukemia (JMML), but the classification of MDS does not acknowledge the special features of MDS in children. The WHO classification includes five MDS subtypes, refractory anemia with and without ringed sideroblasts, refractory cytopenia with multilineage dysplasia, RAEB, 5q− syndrome and MDS not otherwise categorized. Ringed sideroblasts are very infrequently found in children, the

importance of multilineage dysplasia is not known in children, the unique 5q− syndrome has not been described in children, and the last category, “MDS not otherwise categorized,” is not very useful. There are no data to indicate whether a blast threshold of 20% is better than the traditional 30% to distinguish MDS from AML in children. The unique features of Down syndrome are not appropriately addressed in the WHO classification. Accordingly, very little is left from the WHO classification for use in pediatrics. The inclusion of CMML in the FAB-defined group of MDS has often been a matter of controversy. The distinct disorder of infancy, originally termed juvenile chronic myeloid leukemia (JCML), was later included as MDS by some investigators. The disease is now referred to as JMML, a name that has been adapted by the WHO classification, with JMML placed in a separate category of disorders bridging features of myeloproliferative and myelodysplastic syndromes.31,32 JMML is presented in Chapter 22 and will not be discussed further.

Classification of childhood MDS – current approach International consensus has been achieved on the classification of MDS in childhood.33 Myelodysplastic and myeloproliferative disorders in children are separated into three main groups: JMML, MDS, and Down syndrome (Table 21.2). MDS, whether arising de novo or secondary to a predisposing condition, is subdivided into refractory cytopenia (RC), RAEB and RAEB-T. The change in nomenclature from RA to RC reflects the exclusion of anemia as a prerequisite for the diagnosis (further details under the section, Clinical and laboratory features). The word refractory is a relic from the 1930s, and clonal cytopenia might be a more appropriate name. Although the RAEB-T entity is retained, it should be emphasized that the blast count is insufficient to differentiate AML from MDS (further details under the section on Differential diagnoss). Myeloid leukemia in children with Down syndrome has unique features and is kept separate as a distinct entity (see section on Myeloid leukemia and Down syndrome). The Toronto group recently proposed a descriptive system designed to assess children with MDS according to category, cytology and cytogenetics (CCC).34 The system excludes JMML but includes patients with Down syndrome according to morphology. Cytology is used to subdivide both RC and RAEB into three subgroups based upon level of dysplasia. The system records associated abnormalities and cytogenetic abnormalities. The CCC system has an infinite number of possible subgroups, making it difficult to use in clinical practice or research.

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Table 21.2 Diagnostic categories of myelodysplastic and myeloproliferative diseases in children Myelodysplastic/myeloproliferative disease Juvenile myelomonocytic leukemia (JMML) Down syndrome (DS) disease Transient abnormal myelopoiesis (TAM) Myeloid leukemia of DS Myelodysplastic syndrome (MDS) Refractory cytopenia (RC) (PB blasts <2% and BM blasts <5%) Refractory anemia with excess blasts (RAEB) (PB blasts 2–19% or BM blasts 5–19%) RAEB in transformation (RAEB-T) (PB or BM blasts 20–29%) Abbreviations: PB, peripheral blood; BM, bone marrow.

The new pediatric modification of the WHO classification33 emphasizes the subtypes of pediatric MDS and eliminates adult subtypes that are rare or unseen. Nonetheless, we will still face borderline cases that are difficult to fit into this classification. Refinements in immunologic, cytogenetic, and molecular typing are expected to improve our understanding of the biologic processes underlying MDS and lead to a more clinically relevant stratification of the patients. Results from microarray studies may change our fundamental concepts of MDS.35

Primary and secondary MDS MDS arising in a previously healthy child is termed de novo or primary. If it develops in a child with a known predisposing condition, it is referred to as secondary. Secondary MDS has been associated with chemo- or radiation therapy (therapy-related MDS), with inherited bone marrow failure disorders, with acquired aplastic anemia and with familial MDS. It should be recognized, however, that children with so-called primary MDS may have an underlying yet unknown genetic defect predisposing them to MDS at a young age. Therefore, the distinction between primary and secondary disease may become arbitrary. Myeloid neoplasia in patients with predisposing conditions almost always shares the biologic characteristics of MDS, regardless of the presenting blast count. The prognosis appears to depend primarily on the cytogenetic profile. It is essential to note whether MDS is primary or secondary, as preceding events may affect treatment decisions and alter outcome. Secondary MDS may provide insight into the pathogenesis of MDS, but it is questionable whether secondary MDS deserves a separate classification. The natural history and therapy of MDS are different when the disease occurs in Fanconi anemia,36 and those

patients should be reported separately. There are no solid data documenting whether MDS in patients with constitutional abnormalities other than Down syndrome and Fanconi anemia differ from MDS in other children. Such patients should be diagnosed according to recognized MDS guidelines and included in the series of MDS patients, reporting the type and frequency of predisposing conditions.

Epidemiology The epidemiologic literature on childhood MDS is very sparse for several reasons: (1) the lack of an overall accepted classification; (2) the indolent nature of the disease, which may not lead to referral to a tertiary center; (3) the failure of cancer registries to register MDS; and (4) derivation of epidemiologic data from multi-institutional studies to which MDS patients were referred only after progression of their disease. Some authors have tried to estimate the frequency of MDS by searching for a preceding “preleukemic” phase among children with AML. The earliest of these reports found MDS in 17% of childhood AML, corresponding to 2.9% of all children with leukemia.37 Other studies have confirmed that 12% to 20% of childhood AML cases are preceded by a preleukemic phase.38–40 Such studies were based upon referrals because of suspected AML, leaving out the less advanced cases of MDS, as illustrated by a German-Italian study in which MDS represented only 1% of all leukemias with a large proportion of cases diagnosed as RAEB-T.41 These studies underestimate the incidence of MDS because AML does not develop in all cases of MDS. Some children die from complications of cytopenia or are treated before progression to AML. The lack of a widely accepted classification may have contributed to the underdiagnosis of MDS in children. Not all reports dealing with childhood AML have specified the diagnostic criteria distinguishing MDS from AML. The recommendation by the FAB group of more than 30% myeloblasts in the bone marrow has not always been strictly used in pediatrics; for example, the Pediatric Oncology Group42 and Children’s Cancer Group43 defined AML as bone marrow myeloblasts of 25% or more. The exclusion of various constitutional abnormalities may also contribute to an underestimation of the frequency of MDS.1,41,44,45

Incidence, sex, age and subtype distribution Combined population-based data from Denmark and British Columbia in Canada identified 38 cases of MDS, representing 4% of all hematologic malignancies in children

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Table 21.3 Annual incidence of MDS and other hematologic

Table 21.4 Abnormalities associated with MDS in children

malignancies in children 0–14 yearsa Denmark and BC

N ALL AMLb MDSb Myeloid leukemia of DS JMML CML PV/ET Unclassified Total

%

Incidence per million

UK, incidence per million

815 115 38 19

79 11 4 2

38.5 5.4 1.8 0.9

ND 5.8 0.8 0.6

25 13 3 3

2 1 0 0

1.2 0.6 0.1 0.1

0.6 0.5 ND ND

1030

100

48.7

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; DS, Down syndrome; JMML, juvenile myelomonocytic leukemia; CML, chronic myeloid leukemia; PV, polycythemia vera; ET, essential thrombocythemia; BC, British Columbia; UK, United Kingdom; ND, not done. a Combined data from Denmark 1980–91 and British Columbia 1982–962,6 and, for comparison, UK data from 1990–9.10 b Excluding Down syndrome.

(Table 21.3), which corresponds to an annual incidence of MDS of 1.8 per million children aged 0–14 years.2,6 MDS and JMML combined constituted 7.7% of the Japanese cases of childhood leukemia8 with a high proportion of therapy-related cases (23%). Recent data from the United Kingdom suggest a considerably lower annual incidence of MDS, 0.8 per million (Table 21.3).10 The UK study excluded secondary MDS, partly explaining the lower incidence. The incidences of RC in the UK, Denmark and British Columbia are very similar, while the incidence of advanced MDS and JMML differ significantly. Possible differences in classification practice can explain only part of the variation in incidence,10 raising the possibility of genuine regional differences. Combined data on 290 mainly primary MDS cases2,5,6,8,10 showed an equal sex distribution (144/146) and a median age at presentation of 6.8 years. The majority of the patients were classified according to FAB criteria as RA (n = 90), RARS (n = 1), RAEB (n = 88) and RAEB-T (n = 60).2,5,6,8,10

Associated abnormalities Constitutional abnormalities are present in about 30% of childhood MDS cases1,3,5,6,8,10,38 (Table 21.4). Down syn-

Constitutional conditions Congenital bone marrow failure Fanconi anemia Severe congenital neutropenia Shwachman–Diamond syndrome Blackfan–Diamond anemia Trisomy 8 mosaicism Familial MDS (at least one first-degree relative with MDS/AML) Acquired conditions Prior chemotherapy/radiation Aplastic anemia

drome is seen in some 25% of children with a morphologic diagnosis of MDS, but should no longer be included in MDS series. A number of constitutional cytogenetic abnormalities other than trisomy 21, have been linked with MDS, but only trisomy 8 mosaicism has provided solid evidence for an increased risk of MDS.46 Trisomy 8 in the leukemic cells may be due to constitutional trisomy 8 in 15% to 20% of cases.47 Reports of MDS in patients with Klinefelter or Turner syndrome have appeared sporadically, but no increased risk has been documented.48 A relatively high number of children with MDS have congenital malformations, but no consistent patterns of association are evident.

Congenital bone marrow failure MDS develops in up to 50% of the patients with Fanconi anemia before they reach the age of 40 years49 and is often associated with monosomy 7 and duplications of 1q.36,50 It is very difficult to diagnose RC in a patient with Fanconi anemia, and the definition of clonality is problematic.36 The presence of a cytogenetic aberration implies a poorer prognosis, but clones may disappear, evolve, or reappear as new clones.51 The special features of MDS in patients with Fanconi anemia justify separate reporting of these patients. The survival of patients with severe congenital neutropenia (SCN) associated with Kostmann syndrome improved significantly following the introduction of granulocyte colony-stimulating-factor (G-CSF) treatment. Studies from the SCN International Register have shown a 9% crude rate of MDS development and an annual progression rate of 2% in patients with congenital neutropenia.52 Partial or complete loss of chromosome 7 is found in more than half the patients who develop MDS, and the development of MDS is almost always preceded by acquired mutations in the G-CSF receptor gene.53 No cause-and-effect relationship between the development of MDS and G-CSF therapy has been demonstrated, and MDS is not seen in cyclic

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or idiopathic neutropenia treated with G-CSF on a similar schedule.52 It is recommended that yearly bone marrow examinations be performed in patients with SCN to search for morphologic signs of MDS or cytogenetic or molecular aberrations. Early hematopoietic stem cell transplantation (HSCT) seems promising,54 but whether it is beneficial in larger groups of patients remains to be seen. MDS occurs in 10% to 25% of patients with Shwachman– Diamond syndrome,55 where it is often associated with chromosome 7 abnormalities of which isochromosome 7q may represent a separate more benign subtype not heralding progression.56 MDS has occasionally been described in patients with Diamond–Blackfan anemia.6,25,57 No estimates of the relative risk are available, but it is evident that the development of malignant myeloid disease in Diamond–Blackfan anemia is very rare. Not all bone marrow failure syndromes predispose to the development of MDS; for example, patients with dyskeratosis congenita develop marrow failure in 95% of cases, but MDS has been documented in only a few cases.58

Acquired aplastic anemia MDS develops in 10% to 15% of patients with aplastic anemia not treated with HSCT.59–61 Patients diagnosed as having nonsevere aplastic anemia may be over-represented among those in whom clonal evolution is observed,62 suggesting that some cases of MDS are misdiagnosed at presentation. MDS appears to occur at the same rate in idiopathic and hepatitis-associated aplastic anemia63 and may occur earlier in children than in adults, being in most cases diagnosed within the first 3 years from presentation.60,61 Whether prolonged treatment with the combination of G-CSF and cyclosporine is associated with development of MDS is a controversial issue.60,64 Studies using a shorter period of G-CSF treatment have shown a lower incidence of MDS,65 in contrast to the high risk of MDS in patients receiving long-term treatment with G-CSF and those with a poor response to G-CSF.66

Familial MDS Numerous families with several members affected by MDS have been described.44,67–70 A large proportion of the patients showed monosomy 7 or deletion 7q. Larger studies found familial MDS in 0% to 10% of childhood MDS with monosomy 7.1,25,71 Familial MDS also occurs without −7/7q.1,72 Some families show a discordance for −7,25 making it uncertain whether −7 per se increases the risk for familial cases. There are no conspicuous clinical characteristics of the familial cases.25,70 The putatively inherited

predisposing locus in familial MDS with –7/7q– does not seem to be located on chromosome 768 or in the commonly deleted portions of 5q.73 This finding agrees with the lack of leukemia among persons with constitutional aberrations of chromosome 774 and the different parental origins of the remaining chromosome 7 in siblings with monosomy 7.75

Therapy-related MDS Children previously treated for another malignancy are at risk for therapy-related MDS.76–80 The risk is significantly increased at 2 to 10 years after treatment for the primary malignancy and peaks at 4 to 5 years following the leukemogenic therapy. There seems to be two different types of therapy-related malignant transformation.81,82 One is related to treatment with alkylating agents leading to MDS after a latency period of 3 to 5 years and is characterized cytogenetically by deletions or loss of whole chromosomes. The other type is associated with the use of epipodophyllotoxins and acquired translocations involving chromosome 11q23, often presenting as frank AML 1 to 3 years from treatment of the primary disease.77 Most cases follow the epipodophyllotoxin model of disease with a short latency period and an accelerated clinical course, regardless of the etiologic agent involved.83 Studies of polymorphisms in drug-metabolizing enzymes may identify individuals with a high genetic susceptibility to therapy-related MDS.84,85 An increased risk among Hispanic children has been suggested.83 The higher frequency of therapy-related MDS in Japan8 may be related to either therapeutic or ethnic factors, or both. Therapy-related MDS constitutes less than 5% of patients with MDS or JMML2,4,6,10 ; a notable exception can be found in a Japanese study in which therapy-related disease represented 23% of cases.86 New intensive treatment protocols may lead to an increased risk of therapy-related MDS in the future.87

Pathophysiology MDS is a clonal disease arising in a progenitor cell restricted to myelopoiesis, erythropoiesis and megakaryopoiesis, occasionally initiating in a more immature cell involving the lymphoid cell line.88–90 The latter circumstance is illustrated by the very rare patient with MDS progressing to ALL.7 The initiating events of MDS remain obscure, in children as well as adults. Because MDS is very heterogeneous, different mechanisms of initiation and progression of the disease are likely to exist. Some form of genetic damage in a pluripotent hematopoietic progenitor cell may give rise to genetic instability with subsequent acquisition of

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Table 21.5 Hematologic data at diagnosis in 67 patients with RCa Parameter

Median

Range

WBC (× 109 /L) ANC (× 109 /L) Platelets (× 10 9 /L) Hb (g/dL) MCV (fL) HbF (%)

3.6 0.9 65 9.5 98 4.5

0.5–13.0 0–6.7 0–446 3.5–14.5 78–140 0.0–38.0

Abbreviations: RC, refractory cytopenia; WBC, white blood cell count; ANC, absolute neutrophil count; Hb, hemoglobin; MCV, mean corpuscular volume. a Adapted from Kardos et al.9 Fig. 21.1 Progression of MDS to MDS-related AML with different rates of blast cell increase. Although any case above the threshold is conventionally defined as AML, those approximating the middle curve are better described as persistent MDS.

numerous molecular and cellular abnormalities.91 Congenital disorders with DNA repair defects, as in Fanconi anemia, or acquired mutations in genes maintaining genetic stability may result in a mutator phenotype predisposing to MDS.92 About 30% of children with MDS have a known constitutional disorder. It may be speculated that an even higher proportion of the children have a congenital abnormality predisposing them to the acquisition of genetic changes. Subsequent events, such as mutations in proto-oncogenes like RAS, p53 or WT1, and karyotypic changes like monosomy 7, may be part of a final common pathway of disease progression.70,93–95 AML often presents with a high blast count without any recognized prophase, while MDS progresses to higher blast counts at very different rates (Fig. 21.1). Even if the 20% or 30% blast threshold is passed, the disease will maintain the characteristic biologic features of MDS, analogous to CML in blast crisis. To distinguish MDS-derived AML from true de novo AML, Head91 has suggested the term MDS-related AML, or MDR-AML.

Clinical and laboratory features In almost all cases of MDS, the presenting features are those of pancytopenia. Single-lineage cytopenia may be an incidental finding in a few patients during routine work-ups. Not all children with RC have anemia, but macrocytosis (elevated MCV) is a characteristic finding (Table 21.5).9 Fetal hemoglobin (HbF) is often slightly elevated, while the leukocyte count is low to normal. Some

patients present with slight hepatosplenomegaly, but most lack organomegaly. In addition to neutropenia, qualitative defects of the neutrophils have been documented in patients with monosomy 7,20,96 and half of these patients present with fever.25 There is a similar proportion of patients with fever or infection at diagnosis of JMML with or without −7,97 and the risk of infection-related death following intensive chemotherapy in MDS seems to be comparable in patients with and without −7.98 It is likely that the neutrophil defects may be related more to MDS itself than to monosomy 7. Extramedullary myeloid tumor may be the presenting feature of MDS,25,99 but blasts are not seen in cerebrospinal fluid.

Bone marrow features The bone marrow is usually normo- or hypercellular, although a decreased cell content has been observed in up to 40% of childhood RC cases.9 Both the peripheral blood and bone marrow display characteristic dysplastic features with megaloblastic erythropoiesis, bizarre small or unusual large megakaryocytes, and dysgranulopoiesis (Fig. 21.2). The percentage of myeloblasts is often increased, with 5% or more blasts being diagnostic of RAEB, although a blast count of 2% to 4% may be pathologic. The presence of characteristic dysplastic features is suggestive of MDS but is neither diagnostic nor a sine qua non.33 Indeed, the degree of dysplasia is not included in the pediatric classification.33 Interobserver variation of dysplasia assessment100 has led to a recommendation for centralized review. The pathology board of the European Working Group on MDS in childhood (EWOG-MDS) has set up definitions of dysplasia, and a quantitative and qualitative dysplasia scoring system was used in a prospective study.101,102 Detailed morphologic evaluation may be a useful adjuvant tool in the diagnosis of MDS. The degree of dysplasia has prognostic relevance

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Fig. 21.2 Cytological features of myelodysplasia. Courtesy of Irith Baumann. (See color plate 21.2 for full-color reproduction.)

in adults with RA,103 but its predictive value in children remains unknown.

Cytogenetics An abnormal karyotype is found in 60% to 70% of children with MDS.4,5,10,104,105 Numeric abnormalities dominate with only 10% of cases showing a translocation, a derivative, or a deletion as the sole abnormality. Structural abnormalities are frequently part of a complex karyotype with numeric abnormalities. This is in contrast to AML, where structural abnormalities are by far the most frequent findings.106,107 Monosomy 7 is the most common cytogenetic abnormality in childhood MDS, being identified in approximately 30% of cases.1,4,104,105 Studies using fluorescent in situ hybridization (FISH) may identify a few patients with monosomy 7 overlooked by standard cytogenetic analysis,108 but in general FISH analysis adds only scant information to G-banded cytogenetic examinations.109 Trisomy 8 and trisomy 21 are the most common numeric abnormalities after monosomy 7. Constitutional trisomy 21 is usually clinically obvious when present, whereas constitutional trisomy 8 mosaicism may remain unrecognized46 and should be tested for when trisomy 8 is found in the bone marrow. There are very few data on the prognostic value of cytogenetic abnormalities in children. Monosomy 7 as the sole

cytogenetic aberration has not been an unfavorable feature in most studies of childhood MDS,5,25,26,110 whereas complex abnormalities involving chromosome 7 are associated with a poor outcome.25 This observation stands in contrast to findings in adults, where −7/7q– is associated with a very poor prognosis,111 whether treatment is with chemotherapy only or with HSCT.112 Nonetheless, monosomy 7 is associated with a shorter time to progression in RC of childhood,9 and two recent studies identified monosomy 7 as a poor prognostic factor in childhood MDS.8,10 Favorable cytogenetic aberrations, −Y, 20q− and 5q–, have been reported in adults, but these aberrations are so infrequent in children that they are of no practical importance. Complex cytogenetic abnormalities are uncommon and associated with a very poor outcome, even after HSCT (EWOG-MDS, unpublished data). AML-specific translocations, including t(8;21)(q22;q22), t(15;17)(q22;q12), or inv(16)-(p13q22), may occur in cases with a low blast cell count.113–116 Their response to therapy is favorable and they should be considered and treated as AML.113 Molecular based analyses are recommended to search for these abnormalities.

Immunophenotype Flow cytometric immunophenotyping has not shown the same diagnostic value in MDS as in acute leukemia. A

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normal flow cytometric examination, for example, does not preclude MDS. Nonetheless, using a pattern recognitionbased strategy, flow cytometry may serve as a useful adjunct in diagnosing difficult cases with nondiagnostic morphology and cytogenetics.117 Immunophenotypic clustering partly discriminates patients with RA from those with RAEB/RAEB-T.118 Published data on the immunophenotypic characteristics of MDS in children are lacking.

Differential diagnosis The two main diagnostic challenges are to distinguish MDS with a low blast count from aplastic anemia and other nonclonal disorders, and MDS with an excess of blasts from AML. The traditional classification has been based on pure morphology, but a number of additional factors need to be considered.

Refractory cytopenia versus aplastic anemia Earlier studies in children and adolescents with MDS showed decreased bone marrow cellularity in 16% of cases;41,44,45,119–121 however, the studies included only a few RC cases. A recent large study on childhood RC showed a decreased cell content in 39% of cases.9 Bone marrow fibrosis, dilution and sampling variation make it difficult to assess cellularity with use of an aspirated sample. A trephine biopsy is therefore essential for the evaluation of a child with suspected aplastic anemia or MDS. Hypoplastic MDS may be difficult to discriminate from aplastic anemia. The presenting mean corpuscular volume (MCV) is higher in MDS than in aplastic anemia.122 Careful sequential morphologic studies including bone marrow biopsies will almost always establish a distinction between MDS and aplastic anemia.123–125 The biopsy in hypoplastic MDS shows minimally scattered granulopoietic cells, patchy islands of immature erythropoiesis and micromegakaryocytes.125 Clonal hematopoiesis is strongly suggestive of MDS, but may occasionally be seen in aplastic anemia.126,127 Cytogenetics evaluation may fail, and FISH or HUMARA assays may be needed to establish clonality. Point mutations of the NRAS oncogene are frequent in MDS but have not been observed in aplastic anemia.128 Overexpression of p53 is a useful marker of MDS.129

MDS versus other nonclonal disorders Myelodysplasia in the bone marrow may occur in a variety of disorders with very different etiologies (e.g. infection and chronic disease). The diagnosis of MDS can only be made

after exclusion of infections that may mimic MDS (such as parvovirus,130,131 HIV,132,133 and visceral leishmaniasis134 ); vitamin B12 deficiency135 ; drug therapy136 ; rheumatoid arthritis137 ; metabolic disorders138 ; and other causes of cytopenia and dysplasia.139–141 It should be emphasized that serologic tests may be unreliable, and only PCR can exclude viral infection.130 Nonclonal chronic disorders with dysplastic features (e.g. mitochondrial disorders such as Pearson syndrome) should not be considered as MDS. A list of diagnostic tests helpful in differentiating MDS from nonclonal disorders is given in Table 21.6. It may be difficult to diagnose MDS in children who have a low blast cell count and no clonal markers. The minimal diagnostic criteria listed in Table 21.7 may help in this situation.33 The WHO classification defines minimal dysplasia as affecting at least 10% of the cell lineage31 ; it is a less useful criterion for children in whom marked dysplasia may be observed in reactive conditions and MDS may present with discrete dysplasia. Accordingly, dysplasia is not considered a mandatory feature for the diagnosis of MDS in children. The length of persistent cytopenia is not defined,33 but it should probably extend to at least 1 month. Since hematopoiesis is often dysplastic in patients with congenital bone marrow failure disorders, diagnosing MDS in these patients is not recommended unless the bone marrow blast count is increased, a persistent clonal chromosomal abnormality is present, or the bone marrow becomes hypercellular in the presence of persistent cytopenia in peripheral blood.33 The finding of sideroblastic anemia should prompt investigation for possible mitochondrial cytopathy or disorders of heme synthesis.142

Separating MDS from AML AML is the major differential diagnosis of MDS. There are significant differences in clinical features, cytogenetics and response to therapy between these two entities,98,104,143 reflecting fundamental biologic differences between them144 (Table 21.8). Whether the proposed redefinition of AML according to the 20% blast criterion will be useful in pediatrics remains untested. A recent British study suggested a better outcome following AML therapy in patients with RAEB-T compared with RAEB145 ; however, this association was not found in a recent American study.26 Experiences of the EWOG-MDS indicated a poor response to chemotherapy in RAEB-T and no benefit from chemotherapy before HSCT.146 The conflicting data reflect the heterogeneity of the morphologically defined RAEBT group and the inadequacy of the blast count in a single specimen to differentiate MDS from AML. It is suggested to retain the RAEB-T category, while recognizing

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Table 21.6 Guidelines for diagnostic work-up in MDS Medical history Known abnormal constitutional karyotype, bone marrow failure syndrome, previous blood counts, previous malignancies, previous chemo- or radiotherapy, other abnormalities or known disorders. Family history of malignancies, hematologic or immunologic diseases or other syndromes. Physical examination Signs of infection, bleeding, skin rash, skin pigmentation, spleen and liver size in centimeters below the costal margin, lymphadenopathy, enlarged tonsils, extramedullary myeloid tumors. Hematologic studies The complete blood count should include hemoglobin, hematocrit, reticulocytes, MCV, WBC with differential count, platelets and HbF. Morphologic evaluation and differential count should be based upon a minimum of 100 cells in peripheral blood and a minimum of 400 cells in bone marrow. A marrow biopsy at diagnosis should be performed in all subtypes of MDS. The diagnosis must be confirmed by at least two marrow examinations. A repeat marrow examination should be performed after an interval of 6–8 weeks, when blasts <5%, 4 weeks with blasts 5–20%, and 2 weeks with blasts >20%. Blood chemistry and serology Lactate dehydrogenase, uric acid, ferritin, IgA, IgM, IgG, erythrocyte folate and s-B12 . IgG and IgM specific for EBV, CMV, HHV6, herpes simplex, and parvovirus B19. Parvovirus-specific PCR on marrow samples is recommended in cases of RC. Cytogenetic and molecular studies G-banded karyotyping of at least 25 mitoses should be performed. In the case of a normal karyotype or no cytogenetic results, FISH and/or molecular genetic screening for −7, +8, +21, t(8; 21), t(15; 17), and inv(16) is recommended. In the case of +8, the constitutional karyotype should be studied in a tissue of nonhematologic origin. Abbreviations: MCV, mean corpuscular volume; WBC, white blood cell count; HbF, fetal hemoglobin; Ig, immunoglobulin; EBV, Epstein– Barr virus; CMV, cytomegalovirus; HHV6, human herpesvirus 6; PCR, polymerase chain reaction; RC, refractory cytopenia; FISH, fluorescent in situ hybridization.

Table 21.7 Minimal diagnostic criteria for MDS At least two of the following: 1. Sustained unexplained cytopenia (neutropenia, thrombocytopenia, or anemia). 2. At least bilineage morphologic myelodysplasia. 3. Acquired clonal cytogenetic abnormality in hematopoietic cells. 4. Increased blasts (≥5%).

Table 21.8 Major differences between MDS and AML in children Parameter

MDS

AML

WBC Hepatomegaly Cytogenetic aberrations Dysplasia Hematopoiesis Cell of origin Response to chemotherapy Iatrogenic model

Low-normal Infrequent Numeric (−7) Multilineage Clonal (including CR) Stem cell Poor Alkylating agents

Low-normal–high Common Structural Infrequent Nonclonal Lineage restricted Intermediate Epipodophyllotoxins

Abbreviation: CR, complete remission.

that biologic features rather than any arbitrary cut-off in blast count may be more important in distinguishing MDS from (chemosensitive) AML.147 An algorithm to facilitate the distinction between MDS and AML is presented in Fig. 21.3. Cases with AML-specific translocations are more appropriately classified as AML regardless of the blast cell count.113–115 The number of recurrent AML-typical translocations may increase with additional studies. Monosomy 7 as the sole cytogenetic aberration is strongly suggestive of MDS.25 AML-M7 may be difficult to distinguish from RAEB or RAEB-T. That is, AML-M7 is often accompanied by marrow fibrosis, rendering it difficult to obtain an adequate aspirate. Assessment of the blast count in a bone marrow biopsy may be helpful. The presence of t(1;22)(p13;q13) may provide an additional diagnostic marker for AML-M7.148,149 Cytogenetics and molecular biology are constantly challenging our understanding of AML, as illustrated by a recent study of AML-M7 in which nine distinctive cytogenetic subgroups were identified.150 In borderline cases with 20% to 30% bone marrow blasts and no cytogenetic clues to the diagnosis, it is recommended that the bone marrow examination be repeated

Myelodysplastic syndrome

Fig. 21.3 Algorithm for distinguishing MDS from AML.

after 2 weeks. At least 400 marrow cells should be counted to decrease sampling error. If the blast count has increased to more than 30%, the case should be regarded as AML (Fig. 21.3). Significant organomegaly or an increased leukocyte count are suggestive of a diagnosis of AML. It should be emphasized that most children with myeloid malignancies have clear-cut AML, some have MDS with low blast count and only a small percentage have borderline features qualifying them for the diagnostic algorithm shown in Fig. 21.3. The major diagnostic pitfall is undue haste in starting therapy.

Prognosis and natural course Progression of MDS to MDS-related AML occurs at different rates (Fig. 21.1). The percentage of blast cells in the bone marrow may increase abruptly after a set of transforming events, while other cases may show a steady rate of progression. Hence, the 20% or 30% threshold is passed only after a period of months or years (Fig. 21.1). Although any case above the threshold is conventionally defined as AML, cases with dysplasia and progression to a higher percentage of blasts are better described as persistent MDS or MDS-related AML. Children with RC or low-grade RAEB may show a long and stable clinical course without treatment. Blood transfusions are required only infrequently, and severe infections are rarely seen. The condition may smolder with unchanged cytopenia for months or even years, but will eventually progress in virtually all patients. In a series of 67 children with primary RC, 4 died from complications of pancytopenia prior to therapy or progression, and 20 progressed to more advanced MDS at a median of 1.7 years from presentation.9 Although RC with monosomy 7 is associated with a higher risk of progression, both RC and RAEB patients with monosomy 7 may show stable disease without treatment for several years.25 Progression may occur rapidly without any preceding symptoms and predicts an inferior outcome even after HSCT.9,151,152

The International Prognostic Scoring System (IPSS) for MDS uses weighted data on bone marrow blast count, cytopenia and cytogenetics to segregate patients into four prognostic groups.111 Data from the EWOG-MDS showed that thrombocytopenia and bone marrow blasts of more than 5% correlated with a poor survival, whereas two- or three- lineage cytopenia and cytogenetics did not provide useful prognostic information.153 This contrasts with data from Japan8 and the UK10 showing the cytogenetic component of the IPSS to be significantly associated with outcome due to a poor prognosis in patients with monosomy 7. The Japanese study, like that of the EWOG-MDS, showed a poor outcome in patients presenting with more than 5% marrow blasts. A pediatric prognostic scoring system (FPC) proposed by a British group1 assigned one point each for HbF more than 10%, platelets less than 40 × 109 /L, and two or more cytogenetic abnormalities. A significantly higher survival was found in children with MDS and a score of zero. Application of this scoring system in other series has been hampered by HbF being available in only a minority of the MDS patients. Data from EWOG-MDS were used to evaluate the FPC score in 65 patients with complete data. The FPC score was not associated with survival.153 There have been occasional reports of spontaneous regression of MDS,154–158 possibly reflecting polyclonal expression of a multiorgan disease.159 However, most of these cases showed clonal hematopoiesis (often as monosomy 7) and no signs of associated abnormalities. The frequency of spontaneous remission is unknown, but is estimated to occur in well below 5% of all cases.

Treatment As a clonal disorder of primitive stem cells, MDS has few residual nonclonal stem cells. Myeloablative therapy is therefore the only treatment option with a realistic curative potential in a significant proportion of the patients. A diversity of treatment strategies, such as hematopoietic growth factors, differentiating agents, hormones, amifostine,

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low-dose cytotoxic drugs, or other experimental agents, have been investigated in adults and in elderly patients who were not candidates for HSCT. None of these approaches have been shown to prolong survival, and they are generally not indicated in children and adolescents. Given the lack of recurrent molecular abnormalities in MDS, the rational development of molecularly targeted therapy is problematic. Immunosuppressive therapy has been successful in some adults with MDS and a low blast count, especially in patients with bone marrow hypoplasia and HLA-DR15 (DR2).160 Other studies have been less optimistic, reporting a significant burden of side effects.161 In a series of 12 children with RC treated with antithymocyte globulin, 5 had a partial response and 4 a complete response lasting for more than 1 year.162 The long-term outcome of immunosuppressive therapy in MDS is not known. Highdose methylprednisolone has been used with some success by a Turkish group,163 but this approach has not been studied in other series. Children with MDS are at high-risk for cytopenia-related complications, so that optimal supportive care should be the primary focus during all phases of the disease course.

AML-type chemotherapy Conventional intensive chemotherapy without HSCT is unlikely to eradicate the primitive pluripotent cells involved in MDS, rendering the therapy noncurative in most patients, although reported results are somewhat conflicting. Most studies found significant morbidity and mortality associated with induction chemotherapy, with a complete remission rate of less than 60%, many relapses, and an overall survival of less than 30%.8,26,98,113 The treatment-related mortality rate has ranged between 10% and 30%.26,98,113,145 A few studies have reported an outcome in MDS patients that was not significantly different from that in AML.145,164 Some studies suggested that patients with RAEB-T or AML following MDS have a superior outcome compared with RAEB patients,26,145 indicating that the RAEB-T subgroup is heterogeneous and that a purely morphologically based classification is insufficient for a therapeutically relevant stratification.33 Autologous HSCT is used in a large proportion of younger adults165 but only infrequently in children. Two studies that included eight children treated with autologous HSCT reported a single survivor.26,145

Allogeneic stem cell transplantation Allogeneic HSCT is the therapy of choice for virtually all forms of MDS in childhood. Results of HSCT in pedi-

atric MDS are often included in larger series of adult patients,166–170 making the interpretation of the outcome in children difficult. Studies specifically addressing the question of HSCT in children have indicated a probability of disease-free survival (DFS) following transplant with an HLA-matched family donor (MFD) of about 50%.171–177 Children receiving a graft from an HLA-matched unrelated donor (MUD) have previously suffered a higher transplantrelated mortality (TRM) and a lower DFS rate, but more recent studies have shown survival following MUD-HSCT comparable to that after MFD-HSCT.178–180 The European Group for Blood and Marrow Transplantation (EBMT) reported the results for a large number of MDS patients less than 20 years of age transplanted between 1983 and 1998.181 For the 163 patients receiving a MFD transplant, the 3-year actuarial probabilities of DFS, TRM and relapse were 45%, 30% and 36%, respectively. MUD transplants were performed in 84 patients with probabilities of 36%, 45% and 35% for DFS, TRM and relapse, respectively. A preparative regimen consisting of busulfan, cyclophosphamide and melphalan showed encouraging initial results.173 The regimen has been studied in a large number of patients under the auspices of EWOG-MDS and EBMT, with DFS rates of 87% for MFD-SCT and 41% for MUDSCT.182 Other large studies have confirmed a favorable outcome in patients conditioned with a busulfan-based regimen.170 Busulfan- and total-body irradiation (TBI)based regimens appear to produce similar outcomes in less advanced MDS.151 Intensive prophylaxis for graft-versus-host disease (GvHD), including T-cell-depleted grafts,183 is associated with an increased risk of relapse. There seems to be a survival benefit from using peripheral blood compared with bone marrow as a stem cell source especially in advanced MDS.180,184 Stage of disease as indicated by FAB type has a significant effect on relapse and outcome following HSCT.151,169,181,183,185 In RC the relapse rate is very low, provided that the conditioning regimen is myeloablative.167,169 HSCT early in the course of the disease has therefore been recommended for all children and adolescents with MDS. However, in children with RC and the absence of profound cytopenia, postponement of HSCT with a “watchand-wait” strategy may be justified, especially in patients with a normal karyotype. Children have lower TRM and higher DFS rates than adults; however, the TRM rate is still too high. A reducedintensity conditioning regimen followed by allogeneic HSCT with peripheral blood showed a promising 1-year progression-free survival of 66% in adults.186 Preliminary results for a fludarabine-based preparative regimen in

Myelodysplastic syndrome

children with RC indicated success in lowering the TRM of MUD-HSCT to 18%.187 For patients with advanced MDS, the potential benefit of AML-type induction chemotherapy prior to HSCT to reduce relapse and improve DFS remains a contentious issue. An analysis by the EWOG-MDS for 53 children with primary advanced MDS showed no benefit from intensive AML-type therapy preceding HSCT.146 The outcome after HSCT was not dependent on the percentage of blasts prior to HSCT. In a small series of patients undergoing transplantation as first-line therapy, survival rates have ranged from 65% to 70%.98,174,188 Prior chemotherapy may increase TRM.189 Considering the significant morbidity and mortality associated with induction chemotherapy and the high rate of TRM following HSCT, most children with MDS may benefit from HSCT as first-line therapy. Children without a matched donor and progressive disease should be considered for haploidentical HSCT.190 Relapse following HSCT is associated with a very grave outcome. Successful donor leukocyte infusions have occasionally been reported.191 Patients with especially early relapse, detected by increased mixed chimerism, may benefit from withdrawal of immunosuppressive therapy and donor leukocyte infusion.192

Secondary MDS Children with MDS secondary to chemo- or radiation therapy generally have a very poor survival rate. AMLtype therapy may induce remission, but very few patients remain in that state; even HSCT offers a low probability of cure, 20% to 30% in most reports.83,177,193–197 The Children’s Cancer Group (CCG) recently reported a superior, although still poor, outcome for intensive-timing versus standard-timing induction therapy (32% versus 0%).83 The frequency of severe treatment-related toxicity is increased in patients with secondary MDS,166,196 while the risk of relapse may be similar to that observed for patients with primary disease.181 Preliminary data on 13 children with MDS secondary to previous chemotherapy and 10 secondary to aplastic anemia, who received myeloablative therapy with busulfan, cyclophosphamide and melphalan, demonstrated an encouraging event-free survival rate of 57%.198 The few published cases of HSCT in MDS arising from congenital bone marrow failure disorders or acquired aplastic anemia indicate a poor outcome for this heterogeneous group of patients. Early HSCT before neoplastic transformation54 or during less advanced MDS199 may be associated with improved survival. Cooperative studies like those of the EWOG-MDS and the EBMT are needed to

provide further information on the appropriate timing, conditioning regimen and GVHD prophylaxis for the different subtypes of MDS in childhood.

Myeloid leukemia and Down syndrome Individuals with Down syndrome (DS) have a strongly agedependent increased risk of leukemia (more than 50-fold during the first 5 years of life and 10-fold from 5–29 years). After 30 years of age, the risk of leukemia is close to that seen in individuals without DS.200 The cumulative risk for leukemia by the age of 5 years is 2.1% and that by 30 years 2.7%. Almost half of the leukemias are myeloid, and most of them occur before 5 years of age, when the relative risk of myeloid leukemia is increased by more than 150fold.200 In addition to myeloid leukemia, a myeloproliferative disorder indistinguishable from leukemia may occur in infants with DS. Children with DS who have been cured of leukemia appear to have a reduced risk of secondary malignancies.201

Transient abnormal myelopoiesis Increased leukocyte counts with circulating blasts often accompanied by anemia and thrombocytopenia may be seen in up to 10% of newborns with DS. The clinical and morphologic picture may be indistinguishable from congenital leukemia. The blast cells almost invariably have cell surface antigens characteristic of megakaryoblasts.202 The percentage of blasts is often higher in blood than in bone marrow and in most cases marrow aspiration is of limited additional diagnostic value. Clonal abnormalities are observed in 35% of cases.203 This condition is referred to as transient abnormal myelopoiesis (TAM), transient leukemic reaction, or transient myeloproliferative disorder. The presentation is indistinguishable from leukemia, and some investigators have therefore favored the name transient leukemia.202,203 Transient leukemia may occasionally occur in an infant with a normal phenotype and trisomy 21 in the blast cells.204 An unknown proportion of these infants may have low-level constitutional trisomy 21 mosaicism detectable only by FISH.205 TAM selectively involves the trisomic cells in individuals with DS mosaicism.206 Leukemic infiltration of the skin may also occur (Fig. 21.4). Life-threatening complications, mainly progressive hepatic dysfunction, may occur in 10% to 20% of the patients with TAM, but spontaneous remission appears in the majority within 1 to 3 months.203 Generally, no chemotherapy is indicated in TAM; however, in those with progressive hepatic or pulmonary problems, a short

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Fig. 21.4 Transient abnormal myelopoiesis with skin infiltration in Down syndrome. The boy presented at 1 day of age with a WBC of 110 × 109 /L. The blood was dominated by megakaryoblasts. During the following 2 weeks there was increasing hepatomegaly and skin infiltration by myeloid cells. Cytarabine (75 mg/m2 per day) was administered subcutaneously for 4 days. The skin infiltrate disappeared and the WBC normalized within a week. (See color plate 21.4 for full-color reproduction.)

course of low-dose cytarabine may be very effective.207 Myeloid leukemia develops 1 to 3 years later in about 25% of the children who have recovered from TAM.202,203 This leukemia is associated with acquired clonal cytogenetic abnormalities.203 The complexity of TAM and myeloid leukemia is illustrated by a report on a pair of identical twins with DS.208 Twin A had TAM with spontaneous regression and did not develop leukemia later on. Twin B had no hematologic abnormalities in the newborn period but developed myeloid leukemia at 24 months of age.

Myeloid leukemia Myeloid leukemia in DS has often been classified as AML,209 despite bone marrow blasts of less than 30% in many patients.6 DS is present in 25% of those with a morphologic diagnosis of RC, RAEB, or RAEB-T,2,6 whereas only two DS patients with a diagnosis of JMML have been reported.210,211 MDS and AML have traditionally been

differentiated on the basis of a blast count in the bone marrow below or above a certain threshold. The bone marrow in myeloid leukemia in children with DS is often fibrotic, and assessment of the blast count may be difficult. The otherwise uncommon AML-M7 subtype is frequently found in DS and in most cases is preceded by a history of MDS.212,213 In contrast to children without DS, there are no biologic or therapeutic differences between MDS and AML in DS; however, there are many distinctive features indicating biologic differences between MDS/AML-M7 in children with or without DS. Recognizing the unique biologic features of AML and MDS the disease may best be described by the unifying term myeloid leukemia of Down syndrome.33 Myeloid leukemia in older children with DS (4 years or older) behaves more like AML in patients without DS but has a poorer prognosis.214 Such patients may present with “true de novo” AML215,216 not fulfilling the criteria for myeloid leukemia of DS.

Myelodysplastic syndrome

Fig. 21.5 Pathogenetic model for myeloid leukemia in Down syndrome. TAM, transient abnormal myelopoiesis.

Pathobiology Leukemia in children with trisomy 21 mosaicism selectively involves the trisomic cells,206,217,218 pointing to the etiologic role of the additional chromosome 21 as the first hit in the multistep process leading to leukemia. The chromosome 21q22-encoded transcription factor AML1 (RUNX1 or CBFA2) is involved in 25% of childhood ALL cases and 15% of childhood AML and is a main regulator of normal hematopoiesis.219 Families have been identified with a constitutional mutation of the AML1 gene, resulting in a bleeding tendency and an increased risk of myeloid leukemia,220 but an AML1 mutation was detected only in 1 of 46 DS patients with TAM or myeloid leukemia.221 It was recently found that all patients with typical myeloid leukemia of DS had an acquired mutation in the GATA1 gene.222 The mutation was not found in cases with the AML-M7 or other AML subtypes in patients without DS. Others have confirmed the results, and most interestingly, a GATA1 mutation was found in the majority of patients with TAM.223,224 The GATA1 gene encodes a transcription factor essential for the normal erythroid and megakaryocytic differentiation, in accord with the selective involvement of these two lineages rather than the granulocytic lineage in myeloid leukemia of DS.225 These studies of GATA1 mutations support the notion of myeloid leukemia of DS as a separate entity. A model of the pathogenic steps in myeloid leukemia of DS is presented in Fig. 21.5. Trisomy 21 represents the first event predisposing cells to a proliferative advantage or further mutations. A GATA1 mutation is found in the majority of patients with TAM and in a few newborns with DS and normal hematologic findings.223 The mechanisms of the

regression of TAM remain unexplained but may be associated with a decreased telomerase activity in TAM not found in myeloid leukemia.226 A large proportion of patients with TAM and an estimated 1% (>100-fold increased risk) of DS without abnormal hematology in the newborn period develop myeloid leukemia.200 The putative third event is not known.

Epidemiology Myeloid leukemia develops in 1% of the children with DS,200 corresponding to an annual incidence of 0.6–1.0 per million children2,6,10 (Table 21.3). The age distribution is very unusual, with 49% of patients being 1 year of age at diagnosis, 34% 2 years of age, and only 2% more than 4 years of age.201 Only a very few patients present before 1 year of age, and there appears to be no age-overlap between TAM and myeloid leukemia of DS. There is a slight predominance of girls, with a male: female ratio of 0.9.209,213,227,228

Clinical and laboratory features Isolated thrombocytopenia is often the presenting feature of myeloid leukemia. At diagnosis both the platelet count and the leukocyte count are lower than in patients without DS,209,228 in contrast to the very high leukocyte count seen in TAM. The blast cells have in most cases the morphologic and antigenic features of megakaryoblasts, although other morphologic variants may be presented. Many patients have a relatively indolent clinical course characterized by a period of thrombocytopenia and dysplasia with relatively few blasts in the bone marrow.

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Fig. 21.6 Progressive improvements in event-free survival for myeloid leukemia in Down syndrome. Results from NOPHO AML 88 and AML 93.

Numeric aberrations, mainly trisomy 8 and an extra chromosome 21 (tetrasomy 21), are the most common acquired cytogenetic abnormalities in children with myeloid leukemia and DS. Structural aberrations are uncommon, especially prognostically favorable abnormalities such as the t(8;21), t(15;17), and inv(16), which occur in 30% of non-DS children with AML but are very uncommon in DS.209,213,229 The t(1;22)(p13;q13) is the hallmark of AML-M7 in infants. This translocation has been reported in only two DS patients aged 6 and 10 years.230 Monosomy 7 is very common in MDS but very uncommon in patients with DS.5,25,225,231 Clonal evolution has been documented in a few DS patients with TAM or myeloid leukemia.232,233 Karyotype is not known to be a prognostic factor in DS. The clonal cells in children with DS are myeloid progenitors with the potential for differentiation along the megakaryocytic and erythroid lineages225 ; in most cases, the granulocytic lineage is not involved in the leukemic process, in contrast to the situation in children without DS.

Treatment In contrast to TAM, myeloid leukemia in children with DS is fatal if untreated but responds well to AML treatment with a very favorable outcome.42,209,234 The prognosis for myeloid leukemia in DS was considered very poor before 1990. Reports from the Nordic Society of Paediatric Haematology and Oncology (NOPHO)235 and the Pediatric Oncology Group (POG)42 and later the CCG 209 showed a surprisingly high survival rate for DS patients receiving AML treatment. DS was later shown to be the most important prognostic factor in AML.234 Several groups have reported long-term survival in DS patients well above

80%.42,213,227,234 The prognosis for myeloid leukemia of DS has improved significantly during the last 10 to 15 years (Fig. 21.6). The main explanation for this advance is the relatively large fraction of patients diagnosed before 1990 who were not treated adequately by current standards. DS patients with myeloid leukemia who are treated on AML protocols have a significantly better outcome than those receiving minimal treatment207 ; however, intensive timing of induction therapy is associated with an increased mortality rate.209 DS children are at low risk for relapse and at high risk for treatment-related toxicity; hence, they benefit from intensive therapy timed to allow recovery before initiation of the next chemotherapeutic course.209 Longterm survival has been reported in a few patients receiving only low-dose chemotherapy,202 but the results are inferior to those following intensive therapy.207 HSCT is associated with excessive toxicity without therapeutic gain and is not indicated in children with DS and myeloid leukemia in first remission.209,213 It is recommended that therapy should commence when the myeloid disorder is first diagnosed, and not delayed until disease progression.207 DS myeloblasts are 10-fold more sensitive to cytarabine in vitro than are non-DS cells.236 This increased sensitivity may be related to the expression of chromosome 21-localized genes such as those encoding cystathionine-synthetase and superoxide dismutase.236 An elevated cystathionine--synthetase activity may modulate cytarabine metabolism by decreasing levels of deoxycytidine triphosphate or decreasing generation of S-adenosylmethionine and hypomethylation of the gene for deoxycytidine kinase.236 The cystathionine--synthetase gene polymorphism (844ins68) is more frequently observed in DS myeloblasts, than in non-DS myeloblasts, and those DS patients with the polymorphism have an increased cytarabine sensitivity compared with those harboring the wild-type gene.237 The increased susceptibility of DS cells to apoptosis may also contribute to their increased chemosensitivity. It is remarkable that only the constitutional and not the acquired trisomy 21 is associated with a superior outcome.209 In vitro studies showed no increased transcript levels of cystathionine--synthetase in samples with acquired trisomy 21.236 Further studies of the molecular mechanism of the increased sensitivity to chemotherapy in DS may lead to new approaches to the treatment of AML.

Conclusions and future directions The current classification of childhood myelodysplastic and myeloproliferative disorders segregates JMML as a

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disease with primarily myeloproliferative features and recognizes that MDS in Down syndrome is a unique entity not sharing the biologic features of other types of MDS. Nonetheless, distinguishing MDS from nonclonal disorders and from AML may still pose a challenge. Pure morphology is insufficient to differentiate between MDS from AML. Currently, hematopoietic stem cell transplantation offers the only realistic chance of cure in MDS. Future research should be aimed at understanding the biologic mechanisms of MDS induction and progression to AML. While efforts are under way to improve transplantation regimens, a wider role for nonmyeloablative therapy should be evaluated in selected patients.

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171 Guinan, E. C., Tarbell, N. J., Tantravahi, R., & Weinstein, H. J. Bone marrow transplantation for children with myelodysplastic syndromes. Blood, 1989; 73: 619–22. ` 172 Uderzo, C., Locasciulli, A., Cantu-Rajnoldi, A., et al. Allogeneic bone marrow transplantation for myelodysplastic syndromes of childhood: report of three children with refractory anemia with excess of blasts in transformation and review of the literature. Med Pediatr Oncol, 1993; 21: 43–8. 173 Locatelli, F., Pession, A., Bonetti, F., et al. Busulfan, cyclophosphamide and melphalan as conditioning regimen for bone marrow transplantation in children with myelodysplastic syndromes. Leukemia, 1994; 8: 844–9. 174 Yusuf, U., Frangoul, H. A., Gooley, T. A., et al. Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience. Bone Marrow Transplant, 2004; 33: 805– 14 175 Stary, J., Locatelli, F., & Niemeyer, C. M. Stem cell transplantation for aplastic anemia and myelodysplastic syndrome. Bone Marrow Transplant, 2005; 35 (Suppl. 1): S13–16. 176 Rubie, H., Attal, M., Demur, C., et al. Intensified conditioning regimen with busulfan followed by allogeneic BMT in children with myelodysplastic syndromes. Bone Marrow Transplant, 1994; 13: 759–62. 177 Leahey, A., Friedman, D. L., & Bunin, N. J. Bone marrow transplantation in pediatric patients with therapy-related myelodysplasia and leukemia. Bone Marrow Transplant, 1999; 23: 21–5. 178 Davies, S. M., Wagner, J. E., Defor, T., et al. Unrelated donor bone marrow transplantation for children and adolescents with aplastic anaemia or myelodysplasia. Br J Haematol, 1997; 96: 749–56. 179 Hongeng, S., Krance, R. A., Bowman, L. C., et al. Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukaemia. Lancet, 1997; 350: 767–71. 180 Deeg, H. J., Storer, B., Slattery, J. T., et al. Conditioning with targeted busulfan and cyclophosphamide for hemopoietic stem cell transplantation from related and unrelated donors in patients with myelodysplastic syndrome. Blood, 2002; 100: 1201–7. 181 de Witte, T., Hermans, J., Vossen, J., et al. Haematopoietic stem cell transplantation for patients with myelodysplastic syndromes and secondary acute myeloid leukaemias: a report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol, 2000; 110: 620–30. 182 Locatelli, F., Zecca, M., Duffner, U., et al. Busulfan, cyclophosphamide and melphalan as pretransplant conditioning regimen for children with MDS and JMML. Interim analysis of the EWOG-MDS/EBMT prospective study[abstract]. Leukemia, 2000; 14: 971. 183 Sierra, J., Perez, W. S., Rozman, C., et al. Bone marrow transplantation from HLA-identical siblings as treatment for myelodysplasia. Blood, 2002; 100: 1997–2004.

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184 Couban, S., Simpson, D. R., Barnett, M. J., et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood, 2002; 100: 1525–31. 185 Anderson, J. E., Appelbaum, F. R., Schoch, G., et al. Allogeneic marrow transplantation for myelodysplastic syndrome with advanced disease morphology: a phase II study of busulfan, cyclophosphamide, and total-body irradiation and analysis of prognostic factors. J Clin Oncol, 1996; 14: 220–6. 186 Martino, R., Caballero, M. D., Simon, J. A., et al. Evidence for a graft-versus-leukemia effect after allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning in acute myelogenous leukemia and myelodysplastic syndromes. Blood, 2002; 100: 2243–5. 187 Strahm, B., Greil, J., Kremens, B., et al. A new conditioning regimen for patients with refractory anemia and congenital disorders[abstract]. Bone Marrow Transplant, 2003; 31(Suppl. 1): S26. 188 Nagatoshi, Y., Okamura, J., Ikuno, Y., Akamatsu, M., & Tasaka, H. Therapeutic trial of intensified conditioning regimen with high-dose cytosine arabinoside, cyclophosphamide and either total body irradiation or busulfan followed by allogeneic bone marrow transplantation for myelodysplastic syndrome in children. Int J Hematol, 1997; 65: 269–75. 189 Copelan, E. A., Penza, S. L., Elder, P. J., et al. Analysis of prognostic factors for allogeneic marrow transplantation following busulfan and cyclophosphamide in myelodysplastic syndrome and after leukemic transformation. Bone Marrow Transplant, 2000; 25: 1219–22. 190 Peters, C., Matthes-Martin, S., Fritsch, G., et al. Transplantation of highly purified peripheral blood CD34+ cells from HLA-mismatched parental donors in 14 children: evaluation of early monitoring of engraftment. Leukemia, 1999; 13: 2070–8. 191 Okumura, H., Takamatsu, H., & Yoshida, T. Donor leucocyte transfusions for relapse in myelodyplastic syndrome after allogeneic bone marrow transplantation. Br J Haematol, 1996; 93: 386–8. 192 Beck, J. F., Klingebiel, T., Kreyenberg, H., et al. Relapse of childhood ALL, AML and MDS after allogeneic stem cell transplantation can be prevented by donor lymphocyte infusion in a critical stage of increasing mixed chimerism. Klin Padiatr, 2002; 214: 201–5. 193 Pui, C. H., Relling, M. V., Rivera, G. K., et al. Epipodophyllotoxin-related acute myeloid leukemia: a study of 35 cases. Leukemia, 1995; 9: 1990–6. 194 Tsurusawa, M., Manabe, A., Hayashi, Y., et al. Therapy-related myelodysplastic syndrome in childhood: a retrospective study of 36 patients in Japan. Leuk Res, 2005; 29: 625–32. 195 Ballen, K. K., Gilliland, D. G., Guinan, E. C., et al. Bone marrow transplantation for therapy-related myelodysplasia: comparison with primary myelodysplasia. Bone Marrow Transplant, 1997; 20: 737–43. 196 Yakoub-Agha, I., de La Salmoniere, P., Ribaud, P., et al. Allogeneic bone marrow transplantation for therapy-related

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myelodysplastic syndrome and acute myeloid leukemia: a long-term study of 70 patients – report of the French society of bone marrow transplantation. J Clin Oncol, 2000; 18: 963–71. Hale, G. A., Heslop, H. E., Bowman, L. C., et al. Bone marrow transplantation for therapy-induced acute myeloid leukemia in children with previous lymphoid malignancies. Bone Marrow Transplant, 1999; 24: 735–9. Niemeyer, C. M., Kontny, H. H., Strahm, B., et al. Stem cell transplantation for children with secondary MDS: report from a multicenter study of the European Working Group of MDS in childhood (EWOG-MDS)[abstract]. Bone Marrow Transplant, 2002; 29(Suppl. 2): S3. Faber, J., Lauener, R., Wick, F., et al. Shwachman-Diamond syndrome: early bone marrow transplantation in a high risk patient and new clues to pathogenesis. Eur J Pediatr, 1999; 158: 995–1000. Hasle, H., Clemmensen, I. H., & Mikkelsen, M. Risks of leukaemia and solid tumours in individuals with Down’s syndrome. Lancet, 2000; 355: 165–9. Hasle, H. Pattern of malignant disorders in individuals with Down’s syndrome. Lancet Oncol, 2001; 2: 429–36. Zipursky, A., Brown, E., Christensen, H., Sutherland, R., & Doyle, J. Leukemia and/or myeloproliferative syndrome in neonates with Down syndrome. Semin Perinatol, 1997; 21: 97– 101. Massey, G., Zipursky, A., Doyle, J. J., et al. A prospective study of the natural history of transient leukemia (TL) in neonates with Down syndrome (DS): a Pediatric Oncology Group (POG) study[abstract]. Blood, 2002; 100: 87a. Slayton, W. B., Spangrude, G. J., Chen, Z., Greene, W. F., & Virshup, D. Lineage-specific trisomy 21 in a neonate with resolving transient myeloproliferative syndrome. J Pediatr Hematol Oncol, 2002; 24: 224–6. Wu, S. Q., Loh, K. T., Chen, X. R., Joo, W. J., & Mascarenhas, L. Transient myeloproliferative disorder in a phenotypically normal infant with i(21q) mosaicism. Cancer Genet Cytogenet, 2002; 136: 138–40. Hayashi, Y., Eguchi, M., Sugita, K., et al. Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down’s syndrome. Blood, 1988; 72: 15–23. Lange, B. The management of neoplastic disorders of haematopoiesis in children with Down syndrome. Br J Haematol, 2000; 110: 512–24. Miller, R. W., & Shurin, S. B. Neonatal myeloproliferative disorder in Down syndrome: transient or preleukemic[abstract]? Proc ASPHO, 1994; 3: 24. Lange, B. J., Kobrinsky, N., Barnard, D. R., et al. Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children’s cancer group studies 2861 and 2891. Blood, 1998; 91: 608–15. Matzke, E., Winkler, K., Grosch, Worner I., et al. Beitrag zur juvenilen chronischen myeloischen Leuk¨amie (jCML)

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Fallbeschreibung und Verlaufsbeobachtungen bei 9 Patienten. Klin P¨adiatr, 1980; 192: 157–68. Crombet, O. & Svarch, E. Down syndrome and juvenile myelomonocytic leukemia. Pediatr Hematol Oncol, 1999; 16: 181–2. Zipursky, A., Thorner, P., De Harven, E., Christensen, H., & Doyle, J. Myelodysplasia and acute megakaryoblastic leukemia in Down’s syndrome. Leuk Res, 1994; 18: 163–71. Creutzig, U., Ritter, J., Vormoor, J., et al. Myelodysplasia and acute myelogenous leukemia in Down’s syndrome. A report of 40 children of the AML-BFM Study Group. Leukemia, 1996; 10: 1677–86. Gamis, A. S., Alonzo, T. E., Lange, B., Woods, W. G., & Smith, F. O. Acute myelogenous leukemia (AML) in Downs Syndrome (DS) patients: outcome, toxicities, and prognostic factors from the CCG 2891 trial[abstract]. Blood, 2001; 98: 720a. Sato, A., Imaizumi, M., Koizumi, Y., et al. Acute myelogenous leukaemia with t(8; 21) translocation of normal cell origin in mosaic Down’s syndrome with isochromosome 21q. Br J Haematol, 1997; 96: 614–16. Litz, C. E., Davies, S., Brunning, R. D., et al. Acute leukemia and the transient myeloproliferative disorder associated with Down syndrome: morphologic, immunophenotypic and cytogenetic manifestations. Leukemia, 1995; 9: 1432–9. Ferster, A., Verhest, A., Vamos, E., De Maertelaere, E., & Otten, J. Leukemia in a trisomy 21 mosaic: specific involvement of the trisomic cells. Cancer Genet Cytogenet, 1986; 20: 109– 13. Simon, J. H., Tebbi, C. K., Freeman, A. I., et al. Acute megakaryoblastic leukemia associated with mosaic Down’s syndrome. Cancer, 1987; 60: 2515–20. Okuda, T., Cai, Z., Yang, S., et al. Expression of a knockedin AML1-ETO leukemia gene inhibits the establishment of normal definitive hematopoiesis and directly generates dysplastic hematopoietic progenitors. Blood, 1998; 91: 3134– 43. Song, W. J., Sullivan, M. G., Legare, R. D., et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat Genet, 1999; 23: 166–75. Taketani, T., Taki, T., Takita, J., et al. Mutation of the AML1/RUNX1 gene in a transient myeloproliferative disorder patient with Down syndrome. Leukemia, 2002; 16: 1866–7. Wechsler, J., Greene, M., McDevitt, M. A., et al. Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet, 2002; 32: 148–52. Ahmed, M., Sternberg, A., Hall, G., et al. Natural history of GATA1 mutations in Down syndrome. Blood, 2004; 103: 2480– 9. Rainis, L., Bercovich, D., Strehl, S., et al. Mutations in the exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21. Blood, 2003; 102: 981–6.

225 Zipursky, A., Wang, H., Brown, E. J., & Squire, J. Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome. Blood, 1994; 84: 2278–82. 226 Holt, S. E., Brown, E. J., & Zipursky, A. Telomerase and the benign and malignant megakaryoblastic leukemias of Down syndrome. J Pediatr Hematol Oncol, 2002; 24: 14–17. 227 Kojima, S., Sako, M., Kato, K., et al. An effective chemotherapeutic regimen for acute myeloid leukemia and myelodysplastic syndrome in children with Down’s syndrome. Leukemia, 2000; 14: 786–91. 228 Zeller, B., Gustafsson, G., Forestier, E., et al. Acute leukaemia in children with Down syndrome: a population-based Nordic study. Br J Haematol, 2005; 128: 797–804. 229 Teigler-Schlegel, A., Baumann, I., Creutzig, U., Niemeyer, C., & Harbott, J. Acquired chromosome aberrations in children with Down syndrome and myelodysplastic syndrome or acute myeloid leukemia. Leukemia, 2000; 14: 966. 230 Trejo, R. M., Aguilera, R. P., Nieto, S., & Kofman, S. A t(1;22) (p13;q13) in four children with acute megakaryoblastic leukemia (M7), two with Down syndrome. Cancer Genet Cytogenet, 2000; 120: 160–2. 231 Bunin, N., Nowell, P. C., Belasco, J., et al. Chromosome 7 abnormalities in children with Down syndrome and preleukemia. Cancer Genet Cytogenet, 1991; 54: 119–26. 232 Kounami, S., Aoyagi, N., Tsuno, H., et al. Myelodysplastic syndrome after regression of transient abnormal myelopoiesis in a Down syndrome infant: different clonal origin? Cancer Genet Cytogenet, 1998; 104: 115–18. 233 Duflos-Delaplace, D., La¨ı, J. J., Nelken, B., et al. Transient leukemoid disorder in a newborn with Down syndrome followed 19 months later by AML: demonstration of the same structural change in both instances with clonal evolution. Cancer Genet Cytogenet, 1999; 131: 166–71. 234 Lie, S. O., Jonmundsson, G., Mellander, L., et al. A populationbased study of 272 children with acute myeloid leukaemia treated on two consecutive protocols with different intensity: best outcome in girls, infants, and children with Down’s syndrome. Nordic Society of Paediatric Haematology and Oncology (NOPHO). Br J Haematol, 1996; 94: 82–8. 235 Slørdahl, S. H., Smeland, E. B., Holte, H., et al. Leukemic blasts with markers of four cell lineages in Down’s syndrome (“Megakaryoblastic leukemia”). Med Pediatr Oncol, 1993; 21: 254–8. 236 Taub, J. W., Huang, X., Matherly, L. H., et al. Expression of chromosome 21-localized genes in acute myeloid leukemia: differences between Down syndrome and non-Down syndrome blast cells and relationship to in vitro sensitivity to cytosine arabinoside and daunorubicin. Blood, 1999; 94: 1393–400. 237 Ge, Y., Jensen, T., James, S. J., et al. High frequency of the 844ins68 cystathionine-beta-synthase gene variant in Down syndrome children with acute myeloid leukemia. Leukemia, 2002; 16: 2339–41.

22 Chronic myeloproliferative disorders Charlotte M. Niemeyer and Franco Locatelli

Introduction In 1951, Dameshek first speculated on the observation that different chronic proliferative disorders share similar clinical and hematologic features and that patients with one of these diseases often develop, during the course of their illness, symptoms more typical of another disease, usually more severe than the original one. He coined the term “myeloproliferative disorders” (MPD) for these, now widely recognized, clonal proliferations of an abnormal hematopoietic stem cell.1 This group of related diseases, characterized by a variable propensity to evolve into acute leukemia, included chronic myeloid leukemia (CML), polycythemia vera, essential thrombocythemia and myelofibrosis with myeloid metaplasia (currently referred to as chronic idiopathic myelofibrosis).2 More than 50 years later, this classification, which is applicable to both adults and children, still maintains its clinical and biologic value. Apart from these acquired hematopoietic neoplasms, rare congenital genetic abnormalities can give rise to myeloproliferative disorders (Table 22.1). Myeloid neoplasms that present with aberrant proliferative and dysplastic features have raised considerable controversy with respect to their classification. The recent classification system of the World Health Organization (WHO) groups these diseases with their variable effective or dysplastic hematopoiesis into a separate category of myelodysplastic/myeloproliferative disorders,3 including juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), atypical CML and a group of otherwise unclassifiable diseases. While JMML represents about 2% to 3% of leukemias in children,4,5 CMML and atypical CML are extremely rare in young

people. CMML is occasionally diagnosed in an adolescent with persistent monocytosis, low blast count, and the absence of genetic features indicating JMML or CML. In addition, a CMML-like morphology accompanied by hepatosplenomegaly may be observed in secondary hematopoietic neoplasms following chemo- or radiation therapy. This chapter presents a detailed review of JMML and CML in childhood; other myeloproliferative disorders will be summarized only briefly.

Juvenile myelomonocytic leukemia JMML is a unique clonal hematopoietic disorder of infancy and early childhood, characterized by hepatosplenomegaly and organ infiltration due to excessive proliferation of cells of the monocytic and granulocytic lineages. Hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and pathologic activation of the RAS-RAF-MAP (mitogen-activated protein) kinase signaling pathway play an important role in the pathophysiology of JMML and can provide the opportunity for a number of novel therapeutic approaches. However, currently, only allogeneic hematopoietic stem cell transplantation (HSCT) offers the possibility of long-term cure.

History and classification The first case of JMML was described in 1924.6 This was followed by a few additional case reports and a small series of infants with chronic myelogenous leukemia.7 In 1962, Jean Bernard and his co-workers in Paris were the first to carefully describe the clinical picture of JMML in a group of 20 children,8 referring to the disorder as subacute

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 22.1 Classification of myeloproliferative disorders of childhood

Table 22.2 Criteria for the diagnosis of juvenile myelomonocytic leukemiaa

Hematopoietic neoplasia (WHO classification: chronic myeloproliferative diseases308 ) Philadelphia-positive chronic myeloid leukemia Polycythemia vera Essential thrombocythemia Chronic ideopathic myelofibrosis Chronic eosinophilic leukemia (hypereosinophilic syndrome) Congenital disorders Hereditary thrombocythemia (familial thrombocythemia) Familial erythrocytosis (primary familial and congenital polycythemia) Mast cell diseases Systemic mastocytosis

Minimal laboratory criteria (all three have to be fulfilled); 1. No Philadelphia chromosome, no BCR-ABL rearrangement 2. Peripheral blood monocyte count >1 × 109 /L 3. Bone marrow blasts <20% Criteria requested for definite diagnosis (at least two) 1. Hemoglobin F increased for age 2. Myeloid precursors on peripheral blood smear 3. White blood count >10 × 109 /L 4. Clonal abnormality (including monosomy 7) 5. GM-CSF hypersensitvity of myeloid progenitors in vitro a

and chronic myelomonocytic leukemia and identifying prognostic factors for survival.9–12 With the discovery of the Philadelphia chromosome, Reisman and Trujillo in 1963 demonstrated that, in contrast to adult-type chronic myeloid leukemia (CML), this chromosomal abnormality was absent in infants with chronic myelomonocytic leukemia.13 Since Hardisty in 1964 had coined the term “juvenile CML” (jCML),14 it became standard practice to contrast this disorder with Philadelphia chromosomepositive CML.15 Reports on greatly raised hemoglobin F (HbF)16 and other fetal red cell characteristics14,17,18 further stimulated interest in this disorder. Since the first reports of a missing C chromosome in children with myeloproliferative disorders,19,20 childhood monosomy 7 had been defined as a separate entity.21,22 An infantile monosomy 7 syndrome with clinical features similar to jCML, but low HbF levels, was proposed.23 Meanwhile, the French-American-British (FAB) group had rationalized the morphologic classification of MDS in adults.24 Some investigators argued for the application of modified FAB criteria and used the term chronic myelomonocytic leukemia (CMML) instead of jCML25,26 To avoid further confusion, an international working group under the direction of R. Castleberry in 1996 proposed the term JMML and established criteria for its diagnosis (Table 22.2).27 JMML incorporates those disorders previously referred to as jCML or CMML of infancy, as well as some cases of the infantile monosomy 7 syndrome.28 To account for both, the myelodysplastic and proliferative features, the recent WHO classification placed JMML in the group of myelodysplastic/myeloproliferative disorders.3

Epidemiology Incidence studies from Denmark and British Columbia show a JMML incidence of 1.2/million children per year, corresponding to 2.4% of all childhood hematologic

Modified, with permission, from Niemeyer et al. 27

Fig. 22.1 Sex and age at diagnosis. Analysis of 270 patients with JMML included in studies of the European Working Group of MDS in Childhood (EWOG-MDS) (date of analysis, January 1, 2003).

malignancies.4,5 A much lower incidence of 0.6/million has recently been reported from the United Kingdom.29 It is unknown whether these differences are due to different geographical incidence rates or to variations in reporting. JMML predominates in infants (median age at diagnosis, 2 years12,25 ; Fig. 22.1). About 9% of patients are diagnosed by the age of 4 months, whereas only 8% are 6 years or older. There is a male predominance with a male:female ratio of 2:1. Familial occurrence of JMML has been reported. Two pairs of twins, one with a normal karyotype30 and one with a constitutional 7;16 translocation23 have been described. In both pairs, the evolutionary patterns of the disease in the individual twins seemed to differ. In addition, three sets of siblings with JMML are reported in the literature.23,31 The association between neurofibromatosis type 1 (NF1) and JMML has long been established.32–34 A clinical diagnosis of NF1 is already known in up to 11% of patients with JMML (Fig. 22.2).12,23 As estimated from these data, the risk

Chronic myeloproliferative disorders

Fig. 22.2 Signs and symptoms at diagnosis. Analysis of 270 patients with JMML included in studies of the European Working Group of MDS in Childhood (EWOG-MDS) (date of analysis, January 1, 2003).

of developing JMML for patients with NF1 is 200- to 350fold higher than in patients without NF1.25,35 While in general about 50% of NF1 cases represent new mutations, the proportion of children with familial versus sporadic NF1 is higher than expected in some JMML series.36 This observation suggests that, due to young age and subtle phenotype, NF1 is underdiagnosed in JMML patients in the absence of a positive family history. In fact, about 15% of JMML patients without the clinical diagnosis of NF1 have mutations in the NF1 gene,36,37 suggesting that approximately 25% to 30% of JMML cases are associated with NF1. Interestingly, in the familial cases, an unbalanced gender distribution of affected parents with maternal transmission in more than 75% of cases has been reported.38,39 A few cases of JMML have been associated with Noonan syndrome (NS),40–45 a developmental disorder characterized by dysmorphic facial features, growth retardation and heart disease. It has recently been shown that NS can be caused by missense mutations in PTPN11, a gene that encodes the nonreceptor protein tyrosine phosphatase SHP-2.46 In the series of the European Working Group of MDS in Childhood (EWOG-MDS), NS is diagnosed in about 2% of JMML patients registered.46 In all cases a heterozygous PTPN11 mutation was observed.

Clinical presentation Pallor, fever, infection, skin bleeding and cough are the most commonly presenting symptoms (Fig. 22.2).25 There is generally marked splenomegaly and hepatomegaly. Occasionally, spleen size is normal at diagnosis, but it rapidly increases thereafter. About half the patients have

lymphadenopathy,25,47 a finding that is relatively uncommon in Philadelphia-positive CML. In addition, leukemic infiltrates may give rise to markedly enlarged tonsils. Dry cough, tachypnea and interstitial infiltrates on chest x-ray are signs of peribronchial and interstitial pulmonary infiltrates. Cachexia is frequently observed in advanced disease. Skin lesions other than petechiae are common. Most often they present as eczematous eruptions or erythematous maculopapules on the face, trunk, and hands.48 Indurated raised lesions with central clearing48–51 and one case of febrile neutrophil dermatosis (Sweet syndrome)52 have been described. Biopsy specimens reveal myelomonocytic infiltrates. In addition to these often nonspecific lesions, juvenile xanthogranulomas composed of numerous foamy cells may be seen in JMML. They are present by the end of the second year of life and are often multiple.53 In some but not all children, xanthogranulomas are associated with multiple caf´e au lait spots and the clinical diagnosis of NF1.33 Abdominal distension and discomfort are generally due to hepatosplenomegaly. Gut infiltrates may predispose to diarrhea and gastrointestinal infections. Unlike acute monoblastic leukemia, JMML rarely involves the central nervous system (CNS). A small number of patients with CNS chloroma25 and with ocular infiltrates54,55 have been described. As in other myeloid neoplasms with monosomy 7, pituitary infiltration and diabetes insipidus, responsive to antileukemic therapy, have also been observed.56 Immunological abnormalities generally do not lead to overt autoimmune disease; only one JMML patient with lupus nephritis and Hashimoto thyroiditis has been reported.57 As mentioned above, the diagnosis of NF1 with multiple caf´e au lait spots can be established in up to 11% of children with JMML.32–34 Various other clinical abnormalities and/or dysmorphisms, including Noonan syndrome, have been observed with a frequency of about 7%.12,25

Hematologic and laboratory features Leukocytosis, anemia and thrombocytopenia are common findings in JMML patients. The median white blood cell count (WBC) is 33 × 109 /L (Fig. 22.3).12,25,47 In contrast to Philadelphia-positive CML, the WBC is usually under 100 × 109 /L (Fig. 22.4). Leukocytosis consists of mature and immature granulocytes and monocytes. A presenting WBC under 10 × 109 /L is occasionally noted, particularly in children with monosomy 7.23,25 Microscopy of the peripheral blood (PB) smear is often the most important step in establishing the diagnosis. Almost all cases show a striking monocytosis, often with dysplastic cell forms. An absolute monocyte count exceeding 1 × 109 /L is required for

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Fig. 22.3 Hematological data in peripheral blood at diagnosis. The box plots show the median, first quartile (25%) and third quartile (75%), the nonoutlier range, outliers (◦) and extreme values (∗). WBC, white blood cell count; Mono, absolute monocyte count; immature G., promyelocytes, myelocytes and metamyelocytes; Hb, hemoglobin; Reti, reticulocyte; Normo, normoblasts. Analysis of 270 patients with JMML included in studies of the European Working Group of MDS in Childhood (EWOG-MDS) (date of analysis, January 1, 2003).

Fig. 22.4 Range of WBC, hemoglobin and platelet counts at diagnosis. Analysis of 270 patients with JMML included in studies of the European Working Group of MDS in Childhood (EWOG-MDS) (date of analysis, January 1, 2003).

the diagnosis of JMML (Table 22.2).27 Immature monocytes, along with myelocytes, metamyelocytes and nucleated red cells, are usually evident. Occasionally, there is eosinophilia and basophilia. The median blast cell percentage in PB smears is less than 2%12,25 and rarely exceeds 20%. In about 14% of children, the platelet count at diagnosis is below 20 × 10 9 /L. Most patients have a hemoglobin

concentration between 7 and 11 g/100 mL. The reticulocyte count and the number of normoblasts vary over a wide range. Red cells are generally normocytic, while macrocytosis is noted in some patients with monosomy 7.25 Microcytosis may be due to iron insufficiency, but a number of children have microcytosis in the absence of the classic laboratory findings of iron deficiency.25 The latter may be due to acquired unbalanced globin synthesis as described in a patient with a hematologic phenotype of severe ß thalassemia.58 Bone marrow (BM) findings in JMML are not by themselves diagnostic but rather are consistent with the diagnosis. The BM aspirate shows a high cell number with predominance of granulocytic cells at all stages of maturation, except for the few cases in which the erythroid series predominates. Monocytosis in BM is generally less impressive than in PB, the median monocyte percentage in BM being less than 10%.12,25 The BM blast count is moderately elevated, but never reaches the level seen in acute leukemia. A blast count of more than 10% is observed in about 10% of patients.25 Dysplasia of granulocytes is usually minimal, with hypogranulation of neutrophil cytoplasm and pseudo-Pelger Hu¨et forms.3 Besides macrocytic differentiation in a few cases, erythroid cells mature normally. Megakaryocytes are reduced or absent in about 75% of children.25 Cytochemical and immunophenotypic studies are not specific, but might be helpful in identifying the monocytic population.3,59–61 Because smears of PB and BM provide sufficient information, BM biopsy may be omitted

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in most cases. Reticulin fibrosis has been noted in biopsies of some patients.12,62 A remarkable feature of many JMML cases with a normal karyotype is a markedly increased synthesis of HbF,16,17 resulting from a high number of circulating F cells.63 The G  /A  ratio of HbF from these patients is similar to that observed in neonates.18,64,65 In addition, other fetal red cell characteristics, such as increased expression of the i antigen and decreased carbonic anhydrase levels, are present.17 Furthermore, in vitro, the expression of embryonic globins ε and  can be detected in BFU-E-derived colonies.65 Therefore, it has been speculated that the transformed erythroid cells in JMML harbor a trans environment supporting expression of developmentally earlier genes.66 Despite these changes, maturation of red cells does not seem to be compromised. The significance of the immunological abnormalities frequently observed in JMML is unknown. An increase in the serum concentrations of IgG, IgM and IgA can be noted in the majority of patients.25,67 Immunoglobulins are of polyclonal origin, although light-chain imbalance has been described.67 Autoantibodies, such as antinuclear antibodies, antibodies against red cells giving rise to a positive antiglobin test and antithyroglobulin antibodies, may be present.25,67 Increased muramidase serum levels are frequently observed in patients with JMML.

Chromosomal studies JMML lacks the Philadelphia chromosome and the BCRABL fusion gene. Chromosomal studies of leukemic cells show monosomy 7 in about 25% of patients, other abnormalities in 10% and a normal karyotype in 65%.23,25,47 When monosomy 7 is present, it is generally the sole abnormality. Clinical characteristics of patients with monosomy 7 do not differ from those of patients with a normal karyotype.25 However, patients with monosomy 7 display some characteristic hematologic features. They present with a lower median WBC but similar absolute monocyte count, as they have a higher percentage of monocytes on the differential count. Red blood cells are often macrocytic, and erythropoiesis in BM is more pronounced than in cases with a normal karyotype. In addition, patients with monosomy 7 present with a normal or only moderately elevated HbF, which is often elevated in patients with normal karyotype (Fig. 22.5).25 Among the chromosomal abnormalities other than monosomy 7, loss of material on the long arm of chromosome 7 is frequent.25 Submicroscopic loss of large segments of chromosome 7 alleles can be excluded in JMML with a normal karyotype.68 Two JMML patients with partial trisomy of 3q69,70 and a case with transloca-

Fig. 22.5 Percentage of hemoglobin F by age for patients with JMML and either a normal karyotype or monosomy 7.

tion t(5;11)(q31;q23) involving the GRAF gene71 have been described in detail.

Clonality JMML involves the myeloid, erythroid and megakaryocytic lineages, as indicated by studies of cytogenetics,72,73 RAS mutations,74 microsatellite polymorphic markers66 and X-chromosome inactivation patterns.75 Clonal involvement of the B-lymphoid lineage is heterogeneous. Two patients with lymphoid blast crisis76–78 and one child with cytogenetically clonal B-cells79 have been described. The leukemic origin of EBV-transformed B-cells was demonstrated in some,74,80 but not all, cases74,81 analyzed. In addition, three of five children studied showed immunoglobulin rearrangements in the joining region of the heavy chain without VH to DH JH or light-chain rearrangements.30 These results suggest that the malignant transformation takes place at a stage of a committed stem cell that has the ability for myeloid, as well as early B-lymphoid, differentiation. Involvement of T-lymphoid precursors in the clonal

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disorder is generally absent72,74,79 and has only been reported in one child with JMML and T-cell lymphoma.82

Differential diagnosis The clinical and morphologic picture of JMML can be mimicked by a variety of infectious agents such as cytomegalovirus,83 Epstein–Barr virus,84 human herpesvirus 685 and parvovirus B19.86 Viruses can even imitate in vitro culture results thought to be characteristic of JMML.85 Careful investigation for an active infection is mandatory in children suspected of suffering from JMML, particularly in the absence of a PTPTN11, NF1 or RAS mutation. In addition to infections, disorders of granulocyte function, such as leukocyte adhesion deficiency variant87 and some metabolic disorders, can give rise to a clinical and hematologic picture resembling JMML. To facilitate the diagnostic process, criteria for a definite diagnosis of JMML have been proposed (Table 22.2).27

Fig. 22.6 Dose–response curve for granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity in JMML. CFU-GM, colony-forming unit-granulocyte-monocyte. (Kindly provided by P. Emanuel, Birmingham, AL, USA.)

Hematopoiesis in cell culture studies The characterization of JMML cells in culture allowed an important insight into the pathophysiology of the disease. When cultured in semisolid media, JMML cells from PB or BM give rise to an excess number of monocyte-macrophage colonies in the absence of added growth factors.88–91 This so-called spontaneous proliferation of JMML myeloid progenitors depends on endogenous production of cytokines like interleukin-1 (IL-1),92 GM-CSF,93 and tumor necrosis factor
(TNF-
)94 by monocytes. It can be suppressed by the appropriate neutralizing antibodies,93–95 soluble receptor96 or receptor antagonists,97 and is completely abolished by prior depletion of adherent monocytes.98 Because pure populations of JMML monocytes failed to consistently overproduce GM-CSF and other cytokines, a primarily paracrine-based growth mechanism driven by monocytes was considered to be unlikely.93,95 Instead, JMML myeloid progenitor cells were shown to display a striking hypersensitivity to GM-CSF, an observation first made by Emanuel and co-workers.99 In fact, GM-CSF hypersensitivity has become the hallmark of the disease, and represents an important diagnostic tool (Table 22.2).27,39,100 In colony assays, vigorously adherent depleted JMML mononuclear cells exhibit a striking left-shift of the GM-CSF dose–response curves compared to normal controls (Fig. 22.6).101 GM-CSF appears to be obligatory for survival of JMML cells. Diphtheria toxin fused to GM-CSF is toxic to JMML blasts,102 and treatment with the GM-CSF receptor antagonist E21R induces apoptosis103 and remission of JMML in engrafted immunodeficient mice.104

JMML progenitor cells depend on basal production of GM-CSF from monocytes.95 The production not only of GM-CSF, but also of TNF-
and IL-1, by JMML monocytes can be inhibited by IL-10, which has been shown to suppress colony formation and cell viability.105 TNF-
appears to act through GM-CSF by specifically modulating its gene expression.94 Experiments with a TNF-
specific ribozyme106 or oligonucleotide-directed triplex formation107 indicate that the aberrant constitutive GM-CSF production of JMML monocytes is maintained by autocrine TNF-
activation of the GM-CSF gene. The effect of IL-1 on the growth of JMML cells is less clear. Studies supporting a growth-promoting effect of IL-192,97 have been challenged.94,95,103 “Spontaneous” growth of JMML myeloid progenitors in vitro can be inhibited by 13-cis108,109 or all-trans110 retinoic acid. One plausible explanation for this modulating effect is the antagonistic effect of retinoic acid on the transcription factor AP-1,111 which is activated by Jun/Fos, oncoproteins shown to be constitutively upregulated in JMML.112 Inhibitory effects on colony formation have also been described for interferon-
(IFN-
)113 and farnesyltransferase inhibitors114 (see below). While the importance of GM-CSF in the pathophysiology of JMML is unchallenged, a more general intrinsic abnormality of JMML progenitor cell response to hematopoietic growth factors may be present. Enhancement of clonogenic myeloid growth has also been described for stem cell factor (SCF)115,116 and thrombopoietin.117 In addition to excessive myeloid growth, some investigators reported a high

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Fig. 22.7 Model outlining the roles of SHP-2, RAS and NF1 in the GM-CSF signal transduction pathway.

proliferative potential of early and late erythroid progenitors in JMML.66,99,118

Genetic features A number of specific molecular events that activate RASdependent signaling pathways and deregulate growth and survival of leukemic cells, have been described in JMML. They include mutations in the genes encoding RAS, NF1 and SHP-2 and are summarized below.

Oncogenic RAS mutations The GM-CSF hypersensitivity of JMML cells has led to the hypothesis that a specific defect in the GM-CSF signal transduction pathway plays a major role in the pathogenesis of this disease. While mutations in the coding sequences of the
and  chain of the GM-CSF receptor could not be demonstrated,119 abnormalities in the RAS/MAPK pathway became evident (Fig. 22.7). Members of the RAS family of signaling proteins regulate cellular proliferation by cycling between an active guanosine triphosphate (GTP)bound state (RAS-GTP) and an inactive guanosine diphosphate (RAS-GDP)-bound state. Mutant RAS alleles encode proteins that accumulate in the GTP-bound conformation because of defective GPT hydrolysis. Such oncogenic point mutations in codons 12, 13 and 61 of NRAS and KRAS are

observed in leukemic cells of about 15% to 20% of children with JMML.43,74,120–122 Neurofibromatosis type 1 and murine model systems The conversion from active RAS-GTP to the inactive RASGDP state is facilitated by GTP-ase activating proteins (GAPs). Neurofibromin, the protein encoded by the gene for NF1, functions as GAP and negatively regulates RAS. As described above, 25% to 30% of JMML cases carry the clinical diagnosis of NF112,23 or are known to harbor NF1 gene mutations.36,37 The role of NF1 in JMML and GM-CSF signaling has been extensively studied by Shannon and co-workers. Primary leukemic cells from children with NF1 show a selective decrease in NF1-like GAP activity and an elevated percentage of RAS in the GTPbound state.123 Consistent with Knudson’s “two-hit” tumor suppressor gene model, involving germline transmission of an inactive allele and subsequent somatic inactivation of the remaining normal allele, homozygous inactivation of the NF1 alleles has been demonstrated in some children with NF1 and JMML124,125 . In murine model systems it could be shown that mice heterozygous for a targeted mutation in Nf1 (the murine homologue of NF1) are predisposed to a myeloproliferative disorder resembling JMML.126 Homozygous Nf1-deficient mice (Nf1−/− ) die in utero, but their hematopoietic fetal liver cells are hypersensitive to GM-CSF123 and hyperactivate the

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RAS-Raf-MAP kinase signaling pathway in response to GM-CSF, IL-3 and SCF.116 Furthermore, transplantation of Nf1−/− hematopoietic cells in lethally irradiated mice gives rise to an aggressive myeloproliferative disorder.127 When these overproliferating cells were transferred into double knock-out Nf1−/− GM-CSF −/− recipients (second transplant), the mice had myeloid cell counts significantly lower than wild-type recipients transplanted with the same BM cells.128 However, some of the Nf1 −/− GM-CSF −/− mice transplanted with Nf −/− cells developed myeloproliferative disease with prolonged latency, indicating that GM-CSF is not absolutely essential. Nf1 −/− hematopoietic cells are also hypersensitive to SCF in culture, and SCF and GM-CSF act syngergistically to enhance myeloid progenitor growth of Nf1−/− cells.116 Experiments with mice doubly mutant for both the SCF receptor c-kit and Nf1 did, however, indicate that reduced c-kit kinase activity does not suppress the capacity of Nf1−/− fetal liver cells to induce a myeloproliferative disease.129 Protein tyrosine phosphatase SHP-2 JMML has occasionally been observed in patients with NS, a heterogenous disorder defined by short stature, facial dysmorphia, cardiac defects (most common pulmonic stenosis and hypertrophic cardiomyopathy), skeletal defects, mental retardation and bleeding diathesis. Recently, it has been shown that NS is caused by germline mutations in PTPN11, the gene encoding the nonreceptor protein tyrosine phosphatase (PTP) SHP-2,130 a member of a small subfamily of cytoplasmic src-homology 2 (SH-2) domaincontaining PTPases.131 It is required for hematopoietic cell development132 and participates in signal transduction of a number of cytokines, including GM-CSF, IL-3 and SCF.131 SHP-2 contains two tandem SH-2 domains at the Nterminus and a catalytic PTP domain at the C-terminus. In its inactive state, PTPase activity is repressed by inhibition of the enzymatic cleft by the N-terminal SH-2 domain.133 Binding of the SH2 domain to phosphorylated tyrosine residues induces a conformational shift that relieves the inhibitory interaction between the SH-2 domain and the catalytic PTP domain. Heterozygous germline mutations of PTPN11 are present in children with NS and JMML.43 Similar germline mutations are noted in some children with JMML and pulmonic stenosis, suggesting that they have a mild phenotype of NS. In addition, and more importantly, PTPN11 mutations represent a major molecular event in nonsyndromic JMML.43 About 35% of patients with JMML harbor somatic mutations in PTPN11. All PTPN11 mutations identified in JMML are missense mutations in the N-terminal

SH-2 or PTP interacting surfaces, predicting gain-offunction in SHP-2 through preferential occupation of the activated state.43,134 PTPN11 mutations in JMML (with or without NS) differ from those observed in isolated NS. Therefore, it has been speculated that gain of function in the isolated NS-related SHP-2 mutants may be insufficient to perturb hematopoiesis, while JMML-related SHP-2 mutants have stronger gain of function, resulting in pronounced dysregulation with embryonic death when inherited through the germline. The functional consequences of the JMML/NS-related mutant may be intermediate. It is of interest that many cases of JMML in children with NS spontaneously resolve,40–42,135,136 and that the only case tested showed polyclonal hematopoiesis.40 Mutations in PTPN11, RAS and NF1 are mutually exclusive in JMML, suggesting that pathologic activation of RASdependent pathways plays a central role in the pathophysiology of the disease.43 The precise role of SHP-2 in these cascades remains to be elucidated. After phosphorylation of the  chain of the GM-CSF receptor, Shc is recruited and it acts in association with Grb2, Gab2, SHP-2 and the p85 subunit of the phosphoinositide-3-kinase (PI3).137–139 Targeting RAS-dependent pathways The understanding of the important role of GM-CSF and its downstream signaling transduction pathway for viability and proliferation of JMML cells has led to the design of therapeutic strategies targeting individual components of the pathway. The GM-CSF receptor has been targeted by the GM-CSF analogue E21R with a single amino acid substitution that selectively binds to the receptor
chain and thereby abolishes interaction with the  chain. In JMML, E21R induces apoptosis in cells carrying the GM-CSF receptor.103 Administration of E21R to immunodeficient mice grafted with primary JMML cells profoundly reduced the JMML cell load in BM and eliminated JMML cells from spleen and PB.140 Studies in mice engrafted simultaneously with cells form both normal donors and JMML patients, indicate that E21R preferentially eliminates leukemic cells.140 In a child with JMML relapsing after HSCT, the administration of two cycles of E21R resulted in a transient clinical and hematologic response lasting for about 60 days.141 In another approach, GM-CSF fused to a truncated diphteria toxin reduced the number of JMML colony-forming units in vitro.102 RAS proteins are synthesized as precursor molecules in the cytoplasm. A series of post-translational modifications of RAS proteins is required for localization to the inner surface of the plasma membrane. The first obligatory step in this process is addition of a farnesyl moiety catalyzed by the enzyme farnesyl-protein transferase. A

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number of inhibitors of this enzyme, termed farneslytransferase inhibitors (FTIs), have been synthesized and are being tested in clinical trials for various cancers. For JMML, FTIs have been shown to inhibit colony formation in vitro.114 In the NF1−/− murine model the FTI L 744,832 induced dose-dependent inhibition of myeloid colony formation, but did not produce responses when used in vivo in mice with myeloproliferative disease.142 Rate of response and acute toxicity of another FTI is currently being studied by the Children’s Cancer Group in an upfront phase II window in newly diagnosed JMML patients (R. Castleberry, personal communication). RAF kinases are direct downstream mediators of RAS proteins. DNA enzymes specifically designed to cleave RAF-1 mRNA can mediate mRNA degradation and thereby suppress RAF-1 protein expression. Addition of an active DNA enzyme abolished the GM-CSF hypersensitivity of JMML myeloid progenitors in vitro.143 Furthermore, continuous treatment with the active DNA enzyme reduced tumor burden in immunodeficient mice transplanted with human JMML cells. Gain-of- function mutations of SHP-2, the most common molecular event in JMML,43 might be ameliorated by strategies directly interfering with the phosphatase activity of SHP-2. A better understanding of the distinct and cooperative effects of signal transduction through the RAS-RAF-MAP and PI3K/protein kinase B114,144 pathways will help to optimize targeted treatments. Although these strategies may not be able to abolish the malignant clone by themselves, they can have an important role in future regimens of multi-modality therapy.

Other genetic events Mice transplanted with homozygous Nf1−/− mutant cells develop a myeloproliferative disorder, but only a subset of these genetically identical reconstituted mice progress to more aggressive disease.127 This observation suggests that additional somatic genetic mutations are required for disease progression. In the murine model, proviral integration at a locus mapping downstream of the Myb gene represents an example of such an additional genetic lesion, which cooperates with the NF1 gene loss in the progression to acute leukemia.145 In human JMML cells, the gene expression of EVI-1, a retroviral integration site frequently disrupted by chromosomal rearrangements, was studied and shown to be normal.146,147 Inactivation of the p53 tumor suppressor gene, combined with a heterozygous NF1 mutation, has been described in one patient with JMML,148 but appears to be an uncommon event.149 Similarly, internal tandem duplication of the Fms-like tyrosine kinase 3 (FLT3) gene is rare or absent in JMML.150

Fig. 22.8 Survival of patients with and without stem cell transplantation (SCT) calculated from the time of diagnosis. Analysis of 274 patients with JMML included in studies of the European Working Group of MDS in Childhood (EWOG-MDS) (date of analysis, January 1, 2003).

Natural course and prognostic factors JMML is a rapidly fatal disorder for most children if left untreated. The median survival time without HSCT is about 1 year. Low platelet count, age above 2 years at diagnosis and high HbF at diagnosis are the main predictors of short survival.12,23,25 In a retrospective series of 110 cases, all children presenting with a platelet count of 33 × 109 /L or greater had died within a year from diagnosis, while those with higher counts and age less than 2 years of age at diagnosis had a median survival of 3 years.25 British investigators devised a scoring system where an HbF of 10% or higher and a platelet count of 33 × 10 9 /L or less negatively affect prognosis.23 Blastic transformation is infrequent in JMML, and most untreated patients die from respiratory failure due to infiltration of mature leukemic cells. Because JMML may spontaneously resolve in NS patients (see sections on Epidemiology and Genetic features), expectant observation is appropriate for these children.

Treatment other than stem cell transplantation Long-term survival has only been achieved with HSCT (Fig. 22.8). The role of antileukemic therapy prior to transplantation is currently uncertain. Comparative evaluation of the efficacy of different clinical protocols is hampered by the lack of uniform criteria for response. In addition, some young children with JMML (i.e. those diagnosed before 1 year of age) may experience a longer course, characterized by temporary clinical improvement in the absence of therapy.

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Low-dose conventional chemotherapy Clinical and hematologic responses in JMML have most consistently been attributed to mercaptopurine, administered either as single agent9,12,39 or in combination with low-dose cytarabine39,151 or etoposide.39 There are, however, no data indicating that therapy with mercaptopurine influences the duration of survival. A great variety of other antineoplastic drugs and combination-type therapies have been applied but are generally associated with poor responses.39,152 As in other malignant disorders, steroids can be helpful in controlling pleural or pericardial effusion.153 Intensive chemotherapy Most approaches to intensive chemotherapy are derived from treatment protocols for acute myeloid leukemia (AML). Clinical remissions and long-term survival after AML-type combination therapy have been reported in small series.154,155 Study CCG 2891 of the Children’s Cancer Study Group for treatment of childhood AML, MDS and JMML included 13 children with JMML.156 Seven of the 12 JMML patients receiving sequential intensive induction chemotherapy achieved hematologic remission. These results may suggest that aggressive chemotherapy can ameliorate the disease in a proportion of patients. The current JMML study of the Children’s Oncology Group prescribes cytoreductive therapy consisting of fludarabine and high-dose cytarabine concomitantly with 13-cis retinoic acid prior to HSCT. Other investigators have pointed out that intensive chemotherapy is notably unsuccessful in patients with aggressive disease,12,39,152 being complicated by longlasting aplasia often leading to death.157,158 Furthermore, true remission may not be achievable. In a group of 72 children with JMML who did not receive HSCT, the overall survival rate at 10 years was 6%.25 There was no difference in survival whether patients did or did not receive intensive chemotherapy. In view of these results, the current study of the EWOG-MDS does not recommend intensive chemotherapy prior to allogeneic HSCT. Interferon-
(IFN-
) The apparently increased sensitivity of JMML cells to IFN-
113 prompted some investigators to use this cytokine in treatment of the disease. Besides some clinical improvements,39,159–161 cases without objective responses162,163 have been reported. A prospective study of the Pediatric Oncology Group with IFN-
, 30,000 units/m2 subcutaneously daily for 14 days, followed by the same dose three times weekly, was stopped for excessive toxicity.164 None of the evaluable patients had either a partial or com-

plete response. By contrast, in a JMML patient relapsing after allogeneic HSCT, IFN-
induced a sustained and complete remission.165 It is unknown whether a direct antileukemic effect or an induced graft-versus-leukemia (GvL) was responsible for this effect.

13-cis retinoic acid As mentioned above, spontaneous growth of JMML myeloid progenitors in vitro can be inhibited by 13-cisretinoic acid (isotretinoin).98,108,109 Based on these laboratory observations, 10 children with JMML were treated with 100 mg/m 2 isoretinoin daily.109 Response was evaluated by the reduction of WBC and decrease of organomegaly. Two children achieved complete remissions (one lasting 83 months), and four had a partial or a minimal response. The remaining four had disease progression. The authors concluded that isotretinoin could induce durable clinical and laboratory responses. This interpretation was questioned because the mean age of the patients was 10 months, and young age is a favorable prognostic feature in JMML.166 In a subsequent phase II trial of the Pediatric Oncology Group, 22 evaluable patients were accrued: five complete and four partial responses were observed.167 Other investigators did not observe significant clinical responses with retinoic acid39,166,168 , and its value in JMML remains to be determined. Splenectomy Gross enlargement of the spleen may give rise to abdominal discomfort and excessive transfusion requirements. Therefore, early splenectomy has been recommended for amelioration of the disease.39,169 In a retrospective study of 72 children with JMML who did receive an allogeneic stem cell graft,25 splenectomy prolonged survival independently of other risk factors (authors’ unpublished observation). It has been common practice to remove large spleens prior to HSCT170,171 ; however, the benefit of splenectomy for prevention of post-transplant relapse is unknown.39,168 In the ongoing study of the Children’s Oncology Group, all clinically stable patients are scheduled for splenectomy, while the current HSCT study of the EWOG-MDS leaves splenectomy to the decision of the local physician. Splenectomy before HSCT may be indicated in patients with very large spleen size in order to accelerate hematologic recovery and reduce the risk of hemorrhagic complications.

Experimental therapeutic approaches The current concepts and clinical trials targeting RASdependent pathways have already been described.

Chronic myeloproliferative disorders

Hematopoietic stem cell transplantation Allogeneic HSCT is the only curative treatment for JMML, resulting in long-term survival in about a third of the patients.170,172,173 The malignant JMML clone is difficult to eradicate even with HSCT, and post-transplant relapse rate is high. Generally, HSCT shortly after diagnosis is recommended, and younger age at HSCT may predict improved survival.170,173 A wide variety of pretransplant therapies, donor selection, graft manipulation, choice of conditioning regimen and graft-versus host disease (GvHD) prophylaxis have been applied, giving rise to a number of open questions. Because long-term survival in children with JMML who are not grafted is less than 10%,25 matched unrelated donor (MUD) transplant is indicated for all children with JMML who lack a matched family donor (MFD).168,171,172,174 In fact, timely HSCT with the best histocompatible match among available related or nonrelated donor candidates is crucial.171,175,176 In vivo T-cell depletion with antilymphocyte globulin is often used for additional prophylaxis of GvHD in MUD transplants. While relapse rates in MUD and MFD transplants are comparable, graft rejection177 and transplant-related mortality170,175 are still significantly higher in MUD grafts. Most transplant-related deaths are due to infectious causes. A number of unrelated umbilical cord transplants have been performed with outcomes similar to those observed in MUD transplants performed with other stem cell sources.178–181 In the first report on successful HSCT in JMML from Seattle, a conditioning regimen including total-body irradiation (TBI) and cyclophosphamide was used.182 A similar regimen is being used in the ongoing JMML trial of the Children’s Oncology Group. Radiation-induced late effects such as severe growth retardation, cataracts, hypothyroidism and neuropsychologic sequelae, may be especially deleterious for very young children. Therefore, preparative regimens without TBI are particularly attractive for children with JMML. There has been some concern that conditioning with chemotherapy alone may not be sufficient to eradicate the malignant clone.183 However, several investigators reported similar outcome for patients conditioned with TBI compared to non-TBI regimens.172,173,173,184 In a retrospective analysis of the EWOG-MDS, busulfan-based myeloablative therapy offered greater antileukemic efficacy than did TBI.170 The current study of the EWOGMDS is evaluating a preparative regimen with busulfan, cyclophosphamide and melphalan (Fig. 22.9). The ability of HSCT to eradicate malignant hematopoietic cells is based on the antineoplastic effects of both the conditioning regimen and the GvL reaction. The latter reflects recognition by donor lymphocytes of host-

Fig. 22.9 Probability of survival and cumulative risk of relapse in patients with JMML transplanted with a conditioning regimen of busulfan, cyclophosphamide and melphalan from a matched family donor (MFD) or an unrelated (MUD) donor. Interim analysis of EWOG-MDS study 98 (date of analysis, is January 1, 2003).

or disease-specific antigens present on residual leukemic cells. Thus, intensive GvHD prophylaxis may impair the GvL effect of donor T lymphocytes. There is clear evidence that in JMML, GvL plays an important role. Children who receive less immunosuppressive therapy for GvHD prophylaxis have a lower relapse rate.170 Similarly, acute or chronic GvHD is associated with a lower risk of relapse.170,172,173 An increased disparity between donor and recipient in the unrelated setting, particularly if HLAmismatched, requires increased immunosuppression to prevent GvHD, which, in turn, could also increase the likelihood of graft rejection and disease relapse due to reduced GvL.174 The interim analysis of the current HSCT study of the EWOG-MDS indicates a similar relapse rate for both MFD and MUD transplants (Fig. 22.9). Re-emerging donor cells and frank hematologic relapse have been successfully eradicated by reduction of ongoing immunosuppressive therapy.39,165,179,185–187 Reducing the intensity and duration of GvHD prophylaxis may significantly contribute to successful leukemia control; however, unlike BCR-ABL positive CML, donor lymphocyte infusion in JMML relapse is largely unsuccessful.172,175 The role of close monitoring of chimerism after allogeneic HSCT to modulate post-transplant immunesuppression remains to be defined. After HSCT, the relapse rate may be as high as 50%.172,175,179 Relapse occurs early, at a median of 2 to 4 months from transplantation170,172 and generally within the first year. Intensive chemotherapy prior to HSCT does not reduce the relapse rate,39,170,175 and the role of prior splenectomy remains to be determined.39,168,172 Older

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age,170,173 increased HbF172 and abnormal karyotype173 have been reported as patient-specific risk factors for relapse. Despite the generally aggressive re-emergence of the malignant clone and the short interval between first and second HSCT, a substantial number of children have been cured by a second HSCT.39,188,189

Chronic myeloid leukemia Epidemiology and genetics Among the MPDs, CML certainly represents the most common variant, accounting for approximately 3% to 5% of all childhood leukemias.190 The estimated incidence of Philadelphia chromosome-positive (Ph+) CML in childhood has been reported to be less than 1 in 100,000191 and is less common under the age of 2 as compared with other age groups. In children, the disease is characterised by the same molecular, cytogenetic, clinical and morphologic features reported in adults with classical CML. More than 90% of patients with CML harbor the Ph chromosome, the first consistent cytogenetic abnormality identified in cancer, which results from the reciprocal exchange of DNA between the long arms of chromosomes 9 and 22.192,193 This translocation juxtaposes the 3 part of the ABL gene on chromosome 22 to the proximal segment of the BCR gene on chromosome 22.194–198 The breaks in the BCR gene on chromosome 22 vary, although they most often occur centrally between exons 12 and 16, whereas all the translocations affect the same site in ABL (from exon 2 to exon 11).195–197 The chimeric BCR-ABL gene resulting from this translocation encodes a protein called p210BCR-ABL , which has constitutive tyrosine kinase activity that deregulates signal transduction pathways. This, in turn, leads to abnormal cell cycling, inhibition of apoptosis and increased proliferation of cells.194,195 The p210BCR-ABL protein is necessary, and seems to be sufficient, for leukemogenesis, as suggested by cogent experiments in animal models,196 although recent intriguing studies have detected the BCRABL fusion transcript at a very low frequency in the blood of healthy people.199 The Ph chromosome is not identified by conventional cytogenetic analysis in approximately 5% to 10% of patients.194,195,200 However, molecular analysis has demonstrated that the BCR-ABL chimeric transcript is detectable in about half of these patients, who show a clinical course similar to that of subjects with Ph+ CML.194,195,200 By contrast, in the rare patient with both Ph-negative and BCRABL-negative CML, disease progression is usually not manifested by the appearance of a blast crisis.200–202

Clinical and hematologic findings Occasionally, CML is diagnosed on the basis of a routine blood count showing leukocytosis. Clinical symptoms of CML in childhood are mainly due to splenomegaly, which is present in almost all cases, and they include abdominal discomfort, dysphagia and increased abdominal girth.190,203 Symptoms attributable to hypermetabolism, such as fever, night sweats and weight loss, may be present in a proportion of patients as well. Seldom, leukostasis may occur in children with CML, leading to papilledema, strokes and priapism. The WBC usually exceeds 100 × 109 /L, while the platelet count is either normal or increased. The WBC differential spans all the myeloid series, with promyelocytes and myelocytes being easily found in the blood smear. Moderate anemia has been reported in most patients with CML. Leukocyte alkaline phosphatase activity is decreased, whereas vitamin B12 binding protein serum levels are increased in patients with CML. Granulocytic hyperplasia is observed in the BM of almost all patients with CML, leading to a very low erythroid/myeloid ratio. Megakaryocytic hyperplasia may also occur. A definitive diagnosis of CML is made in almost all cases on the basis of the demonstration of the Ph chromosome (the hallmark of the disease) through conventional cytogenetics or by molecular analysis to detect the BCR-ABL fusion gene.

Natural course and prognostic factors The natural history of CML involves the inevitable evolution from chronic phase (CP) to blast crisis (BC), the latter resembling acute leukemia (myeloid in two-thirds of patients and lymphoid in the remaining third). Blast crisis may be heralded by a transitional period, referred to as accelerated phase (AP), during which patients show increasing splenomegaly, thrombocytosis, leukocytosis, refractory to therapy previously effective in controlling WBC, basophilia, anemia and weight loss. The evolution of CP either directly to BC or through an AP is usually associated with the appearance of additional complex chromosome abnormalities. Blast crisis has been reported to occur in children with Ph+ CML at a median time of 3 years from diagnosis.190,203 However, the time from CP to a more advanced phase shows great variability, some patients having an aggressive clinical course, with death occurring within 1 year of diagnosis, while others survive for 10 years or more.201 Prognostic scores based on sex, spleen size at diagnosis, platelet count, circulating myeloblasts, age at presentation, and eosinophil and basophil counts have been devised to assess the duration of survival from

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diagnosis for each individual patient with Ph+ CML.204,205 Although these scores proved to be valuable in subdividing adult patients into groups with a low, intermediate or high risk of death, their utility has not yet been tested in pediatric patients.

Treatment Treatment of patients with Ph+ CML has significantly changed over time. In fact, conventional single-agent therapy of this disease was initially based on the use of lowdose busulfan or of hydroxyurea, which, although lowering the WBC count and reducing splenomegaly, modified neither the median survival of patients nor the median time to progression from CP to a more advanced disease phase.190,202 More recently, IFN-
has been used as therapy for Ph+ CML, either alone or in combination with lowdose cytarabine. When used by itself, IFN-
normalizes hematologic parameters and clinical findings in approximately three quarters of patients.206–209 More importantly, it can induce a major (>65% of metaphases Ph negative) or a complete cytogenetic response in about 30% and 10% of patients, respectively.206,208–210 These percentages can be slightly increased by the association with low-dose cytarabine.211,212 IFN-
alone or in combination with lowdose cytarabine significantly prolongs the survival of Ph+ CML patients in comparison to conventional single-agent chemotherapy and the cytogenetic response to IFN-
predicts the overall outcome, patients with major or complete cytogenetic response being those with the longest duration of progression-free survival.206–209,211,212 However, treatment with IFN-
, either alone or in combination with lowdose cytarabine, does not induce molecular remission (i.e. loss of the chimeric BCR-ABL transcript) and has relevant side effects (such as fatigue, depression, myalgias, arthralgias), a significant proportion of patients being unable to tolerate this therapy. Moreover, specific studies addressing the role of IFN-
in childhood CML are lacking. Thus, therapy with interferon has been viewed as a second-line choice, behind HSCT.190,202 The natural history of Ph+ CML was recently profoundly modified by the introduction of the specific BCR-ABL tyrosine protein kinase inhibitor imatinib mesylate, which targets the enzymatic activity of the BCR-ABL protein, occupying the ATP-binding pocket of the molecule.213,214 In this way, access to ATP is blocked and phosphorylation of any substrate involved in the regulation of the cell cycle is prevented.213,214 Phase II–III clinical trials performed in patients with IFN-
-resistant CP CML have documented response rates (both hematologic and cytogenetic) that are clearly superior to those obtained with any other medical

therapy, and sustained over time.215,216 Further studies with imatinib mesylate in subjects with more advanced disease have shown a complete hematologic response in one third of patients with AP CML217 and in approximately 10% to 20% of patients in BC.218,219 In these patients, however, the median duration of response is less than 10 months, and almost all patients suffer a relapse in advanced phase, indicating that in these cases imatinib mesylate may be useful only as a bridge to HSCT. The results obtained in these first studies have suggested the need to evaluate the role of imatinib mesylate as therapy for newly diagnosed CP CML. Recently, a randomized trial comparing imatinib mesylate with the combination of IFN-
plus low-dose cytarabine showed that patients assigned to the treatment with the tyrosine kinase inhibitor had a greater probability of both major and complete cytogenetic response and a superior rate of freedom from progression to AP or BC.220 Moreover, treatment with imatinib mesylate was better tolerated than combination therapy.220 The mechanisms responsible for failure of treatment with imatinib mesylate are being studied. At the moment, they include functional inactivation of the drug, BCR-ABL gene amplification or mutations, as well as upregulation of multidrug-resistance proteins.221,222 A better understanding of these mechanisms should permit the development of alternative innovative approaches, able to directly interfere with BCR-ABL protein function or to enhance imatinib mesylate efficacy. Several crucial questions pertaining to imatinib mesylate still lack an appropriate answer. In particular, can this drug prolong the CP of the disease indefinitely and can it induce a sustained “molecular cure” of the disease? Recently published results indicate that imatinib mesylate does not eliminate malignant primitive progenitors in CML patients achieving complete cytogenetic remission.223 The optimal dosage for each specific patient is another relevant issue to be addressed, and studies evaluating the role of combined treatment with imatinib mesylate and IFN-
or low-dose cytarabine (or both) are warranted. However, despite the fact that data on pediatric patients are not available at the moment, the results reported in adults indicate that, imatinib mesylate reasonably represents the initial treatment of choice for children with Ph+ CML who do not have a suitable stem cell donor or who, for various reasons, are not candidates for transplantation. As in adults, allogeneic HSCT is considered the only proven curative treatment for children with Ph+ CML.190,202,224 Not all the candidates for transplantation have a suitable (either related or unrelated) donor, and despite its curative potential, HSCT carries the risk of death as well as leukemia recurrence. In adults, leukemia-free

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survival (LFS) of CML patients given an allograft has been reported to range from 45% to 80%, depending on the phase of disease, age of the receipient, type of donor employed (i.e. family donor or unrelated volunteer) and time between diagnosis and HSCT.225–229 These four variables, together with the sex combination in the donor/recipient pair, have been included in a prognostic scoring system by Gratwohl and colleagues, which proved to be useful for assessing the risk of treatment failure in candidates for HSCT.230 In some of the most important studies addressing the role of SCT in patients with Ph+ CML, children have been included in adult series in which they represented only a small proportion of the total group and their outcomes were not considered separately.225,228,229 Two studies specifically analyzed the outcome of HSCT in children, but the number of cases was small.231,232 Only recently, the Chronic Leukemia Registry of the European Group for Blood and Marrow Transplantation (EBMT) evaluated the outcome of 314 children with CML transplanted between 1985 and 2001 from either a related donor or an unrelated one, selected on the basis of high-resolution molecular typing of HLA class II antigens only.233 In this study, 3-year overall survival (OS) and LFS were 66% and 55%, respectively. In a multivariate analysis of factors potentially affecting OS and LFS, outcome was superior in CP1 versus the advanced phase of CML, although it is remarkable that more than one third of patients transplanted in AP or BC are alive and disease-free 3 years after the allografting.233 Inferior LFS was also found in children transplanted more than 6 months from diagnosis, a finding that confirms previously published studies, documenting a worse outcome in adults transplanted more than 12 months after diagnosis, as compared to those transplanted earlier.226,229 The transplant-related mortality in the cohort of patients analyzed by the EBMT group was significantly higher for children transplanted from an unrelated volunteer, with these patients having a 35% chance of fatal transplant-related complications as compared to 20% for recipients of sibling allografts. The higher incidence of transplant-related death observed in patients transplanted from an unrelated volunteer is mainly due to a greater incidence of severe GvHD in these transplant recipients as compared to those transplanted from a compatible sibling. A more precise characterization of HLA alleles using high-resolution typing for both class I and class II molecules may permit a more accurate selection of unrelated donors, thus reducing the incidence of immune-mediated complications and fatal events after the allograft.234,235 Thus, for patients transplanted in more recent years, the outcome of patients given HSC transplantation from an unrelated volunteer is comparable to that of patients transplanted from an HLA compatible sibling.227,229

In view of the above results, it is possible to conclude that HSCT is curative for the majority of children with CML, although in the past treatment-related mortality unfavorably affected the outcome of unrelated donor HSCT. Longterm survival is also influenced by the stage of disease at the time of HSCT with a significantly better outcome in CP1; this observation, together with the finding that LFS is significantly better for children transplanted within 6 months of diagnosis, suggests that it is important to proceed to HSCT as soon as an HLA-identical donor has been identified. It is possible that in the future the choice of transplanting children with CML will have to be balanced against the results achieved with novel medical therapy such as tyrosine kinase inhibitors. However, considering the long life expectancy of children, and the inabilitiy of imatinib mesylate to offer, either alone or in combination with other treatment, a sustained “molecular cure” or indefinitely prolonged CP of the disease, HSCT, possibly in the first year after diagnosis, remains the treatment of choice of childhood Ph+ CML, provided that a well-matched donor is available. Treatment of CML relapse after an allograft has significantly benefited from adoptive immunotherapy with donor leukocyte infusions.236 In fact, in patients with CML experiencing hematologic relapse in CP after HSCT, complete remission can be obtained with this treatment in approximately 70% of patients.237–239 Most of these remissions are sustained over time, proving the capacity of donor leukocyte infusions to eradicate clonogenic leukemia cells or to control their regrowth. Patients suffering from either cytogenetic or molecular relapse have even a greater chance of benefiting from donor leukocyte infusions than those experiencing hematologic relapse, especially in advanced phase.239 This finding suggests that strict monitoring and early detection of minimal residual disease through serial quantitative evaluation of the chimeric BCR-ABL mRNA transcript by means of PCR can be extremely useful for ensuring the best chance of a favorable outcome in patients with Ph+ CML who undergo HSCT.

Polycythemia vera Polycythemia vera (PV) is a myeloproliferative disorder caused by clonal expansion of an abnormal multipotent stem cell that produces erythroid progenitors able to proliferate in the absence of erythropoietin (EPO). In contrast, normal fetal erythroid progenitors and those with mutations in the EPO receptor [primary familial and congenital polycythemia, (PFCP)] require EPO for their function. The clonal origin of PV has been demonstrated in women

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Table 22.3 Diagnostic criteria for polycythemia veraa,b Category A A1

A2

A3 A4

A5 Category B B1 B2 B3

B4

Elevated red blood cell mass >99th percentile of method-specific reference range for age, sex, altitude of residence No cause of secondary erythrocytosis, including: absence of familial erythrocytosis No elevation or erythropoietin (EPO) due to: hypoxia (arterial pO2 ≤92%) high oxygen affinity hemoglobin truncated EPO receptor inappropriate EPO production by tumor Splenomegaly Clonal genetic abnormality other than Ph chromosome or BCR-ABL fusion gene in marrow cells Endogenous erythroid colony formation in vitro Thrombocytosis >400 × 109 /L Leukocytosis >12 × 109 /L Bone marrow biopsy showing panmyelosis with prominent erythroid and megakaryocytic proliferation Low serum erythropoietin levels

a

For the diagnosis, the patient must have A1+A2 and any other category of A and B.

b

Modified, with permission, from Pierre et al. 243

heterozygous for a polymorphic X-chromosome marker (G6PD, HUMARA).1,240 Criteria for diagnosis of PV were developed by the Polycythemia Vera Study Group (PSVG)241 and for many years remained the gold standard for clinical trials. The currently applied revised criteria242,243 make use of methods that were not available when the PSVG criteria were defined (Table 22.3). Such criteria should be applied to children, as well as adults, to diagnose PV. Specifically, all pediatric patients should have a normal P50 measurement or, preferably, a normal hemoglobin dissociation curve. Hemoglobin electrophoresis is not a valid test, because abnormal hemoglobins have the same electrophoretic behavior as normal adult hemoglobin. Recently, an acquired somatic mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 ( JAK2) gene has been described in most adult patients with PV, as well as in half of the patients with chronic idiopathic myelofibrosis and in a quarter of patients with essential thrombocythemia.244 The presence of the Jak2V617F mutation is very highly correlated with overexpression of the polycythemia rubra vera 1 (PRV-1) gene and the ability to form endogenous erythroid colonies in vitro in all three subtypes of MPD.245

Polycythemia vera is extremely uncommon in children. The median age of presentation is 60 years, and less than 1% of PV patients are younger than 25 years.246 Patients with PV may present with hepatosplenomegaly; however, the majority of symptoms, including dizziness, headache, fatigue, pruritus and night sweats, are due to the increased red blood cell mass, blood volume and blood viscosity. Bleeding and bruising are also common symptoms, being observed in about one-quarter of patients. The marrow is characteristically hypercellular, with involvement of all lineages and increased megakaryocyte number. Since this is a hemopoietic stem cell disorder, leukocytosis and thrombocytosis may also be encountered. There are no peculiar cytogenetic findings, but clonal chromosome abnormalities are present in up to 25% of cases.247 Long survival after diagnosis is common. The disease usually remains in a “plethoric” phase for many years, after which a “spent” phase, characterized by falling red cell count and progressive splenomegaly, supervenes. Life-threatening complications of PV include bleeding, thrombosis, myelofibrosis with pancytopenia and acute leukemia. In particular, vascular occlusive episodes, related to the high hematocrit, are an important cause of morbidity and mortality. Patients with PV can be stratified into risk groups (high, intermediate and low risk) according to age of presentation (>60 years), presence of cardiovascular risk factors, thrombotic events, and platelet count (>1500 × 109 /L).248 Treatment strategies, including chemotherapy (hydroxyurea, IFN-
) must take into account this stratification. For pediatric patients, who usually belong to the low-risk group, phlebotomy alone or phlebotomy and low-dose aspirin (for thrombotic prophylaxis) may be used. A reasonable target range for the hematocrit is less than 45%.248 If complications such as symptomatic splenomegaly or extreme thrombocytosis occur, it is advisable to add hydroxyurea, an agent not yet associated with significant leukemogenesis, at least in adults.249

Familial erythrocytosis Primary familial and congenital polycythemia (PFCP) is a rare disorder involving bone marrow progenitors of the erythroid lineage.250 This disorder is typically characterized by an autosomal dominant mode of inheritance and, less frequently, by the occurrence of sporadic cases.251 The clinical features include the presence of isolated erythrocytosis, without evolution into leukemia or other myeloproliferative condition, absence of splenomegaly, normal WBC and platelet count, normal hemoglobin– oxygen dissociation curve (normal P50 measurement),

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low plasma erythropoietin level and hypersensitivity of erythroid progenitors to exogenous erythropoietin in vitro.252 Mutations in the gene encoding the erythropoietin receptor (EPOR) have been described in several families with isolated familial erythrocytosis.253

Essential thrombocythemia Essential thrombocythemia (ET) is a rare chronic myeloproliferative disease, characterized by an elevated platelet count, hemorrhages and thrombosis. It has usually been considered a disease of the middle aged, having a slight preponderance in women, with an onset in the fifth or sixth decade of life.1 However, with the advent of automated platelet counting, ET is diagnosed with increasing frequency in young adults and even in children, although less commonly than in adults. Although the precise incidence of ET in children is unknown, recent data indicate a value of 0.09/million children (aged 0 to 14 years).254 Like the other MPDs, ET is a chronic disorder with an origin in a multipotent stem cell. Recent findings have categorically excluded abnormalities in either the thrombopoietin (TPO) receptor or its signaling pathway (STAT 3 and 5) in determining the excessive proliferation and autonomous growth of megakaryocytes.255 A major breakthrough in our understanding of the molecular pathogenesis of ET has come with the recent identification of a JAK2 mutation in about a quarter of patients.244 As already mentioned, ET may be diagnosed as an incidental finding, or patients may present with venous or arterial thrombosis, hemorrhage, severe headache, paresthesias and erythromelalgia. Bleeding is generally mild and is probably related to platelet dysfunction. Mild splenomegaly and leukocytosis are common. Bone marrow is frequently hypercellular with an abundance of megakaryocytes that often appear hyperlobulated and dysplastic with unusual large and early forms. The karyotype is usually normal.256 Essential thrombocythemia is a diagnosis of exclusion. The requirements, according to the PVSG, include: (1) platelet count >600 × 109 /L; (2) absence of conditions associated with reactive thrombocytosis; (3) normal iron stores; (4) normal red blood cell mass; and (5) no Ph chromosome (by molecular analysis). In addition, BM collagen fibrosis should either be absent or, in the absence of both splenomegaly and leuko-erythroblastosis, restricted to less than a third of the cross-sectional area of the biopsy.257 The rarity of ET makes it especially important that, in children, a comprehensive approach to diagnosis be taken. A thorough clinical assessment should be conducted, along with laboratory tests, in order to elicit

evidence of thrombosis at the time of diagnosis. Indeed, there is little information about the coexistence of ET with inherited thrombophilic states in children or in adults (deficiencies of C protein, S protein and antithrombin III, presence of Leiden Factor, prothrombin 20210 variant and homocysteinemia). The clinical course of ET in children is extremely variable, with some patients followed up for many years without problems and others presenting with a high frequency of thrombotic and hemorrhagic events. Adults as well as children have a risk of approximately 2% to 5% of developing AML256 Therefore, ET in children is not always a benign entity. Therapeutic intervention must aim at reaching a compromise between the risk of potentially serious drug toxicity and the need to prevent thrombo-hemorrhagic complications. The crucial question is how to identify patients who are at high risk for such a complication. One reasonable approach is risk-based therapy, which individualizes treatment based on age (>60 years), platelet count (>1500 × 109 /L), presence of cardiovascular risk factors, and previous thrombosis.248 Hydroxyurea, a ribonucleoside reductase inhibitor, is probably the gold standard of cytoreductive therapy for ET in those children for whom treatment is indicated. It is the sole drug whose efficacy in reducing thrombotic manifestations has been demonstrated in a randomized controlled trial.258 However, hydroxyurea is not platelet specific and may have some troublesome side effects, the most important of which is pancytopenia. For those patients who fail to tolerate hydroxyurea, a reasonable alternative is represented by anagrelide. This drug, an imidazol-quinazoline derivative, has the ability to control thrombocytosis in more than 80% of unselected patients. A recent randomized trial indicated that hydroxyurea plus low-dose aspirin is superior to anagrelide plus low-dose aspirin for ET patients at high risk for vascular events.259

Hereditary thrombocythemia (familial thrombocythemia) Hereditary thrombocythemia is characterized by sustained proliferation of megakaryocytes, resulting in elevated platelet counts and thrombotic or hemorrhagic complications. In most kindreds, hereditary thrombocythemia is inherited as an autosomal dominant trait with high penetrance and early age of onset.260 Mutations in the thrombopoietin (TPO) gene causing overproduction of TPO and elevated TPO serum levels have been described, although they do not represent the only mechanism by which thrombocythosis develops. Indeed, in some cases of hereditary thrombocythemia, both TPO and MPL (which codify for

Chronic myeloproliferative disorders

the TPO receptor) gene mutations have been excluded.261 Children with hereditary thrombocythemia seem to have a more benign course than do ET patients.

Chronic idiopathic myelofibrosis Chronic idiopathic myelofibrosis (CIMF),2 formerly referred to as agnogenic myeloid metaplasia or myelosclerosis with myeloid metaplasia, is characterized by anemia, splenomegaly, fibrosis and prominent progressive reticulin and dense collagen deposition in the BM; immature granulocytes, erythroblasts and teardrop-shaped red cells are observed on the PB smear.190,202 This disease occurs predominantly in the elderly, the median age at presentation being 65 years. Pediatric cases are rare and symptoms reported in children include malaise, weight loss, night sweats and discomfort due to splenomegaly.190,202,262,263 Most of the early reports of myelofibrosis in childhood involved underlying or associated disorders such as infections, vitamin D-deficiency rickets, collagen vascular diseases, renal failure, trisomy 21, MDS and AML FAB-M7. Fewer than 30 cases of true pediatric CIMF have been reported so far.190,202,262–265 Bone marrow is difficult to aspirate (punctio sicca), and biopsy shows the typical fibrosis in most patients. Myeloproliferation is usually the dominant abnormality in the myeloid and megakaryocyte lineages resulting in blood leukocytosis and thrombocytosis. Hypoplastic hematopoiesis may be present initially or develop later, leading to pancytopenia. Anemia is a frequent finding as a result of blunted erythropoiesis, massive splenomegaly and shortened red cell survival. Cytogenetic abnormalities have been observed in a high percentage of cases (30– 75%) at diagnosis, with a progressive increase in anomalies reported at the time of malignant transformation (up to 90% of cases).266 In addition, the Jak2V617F mutation is noted in about half of the patients with CIMF.244 The natural history of CIMF can be quite variable, the median overall survival ranging from 1.5 to more than 5 years.267,268 Prognosis has been correlated with chromosome abnormalities, severity of anemia, thrombocytopenia, leukocyte count and age at presentation.266–269 Complications include bleeding, intercurrent infections, portal hypertension and transformation into acute leukemia (up to 15% of cases). Many children diagnosed with the primary form of the disease and reported in this last decade have shown a stable behavior of the disease, only requiring supportive care or iron therapy.264,265 Additionally, the BM karyotype of pediatric cases is often normal. Hence, the clinical course of CIMF in children could be less aggressive than in adults, with less potential for progression

to malignancy. In view of this relatively benign behavior of CIMF in childhood an initial conservative therapeutic approach is reasonable, a more aggressive intervention (such as allogeneic HSCT) being reserved for those children with intractable symptomatic cytopenia or with a rising blast count. In pediatric patients who need treatment, high-dose steroids, hydroxyurea, and allogeneic HSCT may be considered. IFN-
270 as well as, more recently, the antiangiogenic agent thalidomide271 have been successfully employed in adults with CIMF. Allogeneic HSCT has been demonstrated to induce long-term disease-free survival in a substantial proportion of young adults with high-risk CIMF,272 and encouraging preliminary results have been obtained with autologous transplantation.273

Hypereosinophilic syndrome Eosinophilia in the PB may result either from a number of reactive conditions or from a clonal disorder in the BM.274 In some cases however, it may be impossible to prove clonality of eosinophils, in which case, the term “idiopathic hypereosinophilic syndrome” is preferred.275 It appears likely that many of these cases are actually chronic myeloproliferative disorders. Sustained hypereosinophilia, whether reactive or clonal, can lead to organ damage276–281 as a result of release of cytokines, enzymes or other granule proteins (summarized in Brito-Babapulle274 ). It has been estimated that nonclonal BM disorders such as infection or atopic disease account for 99% of cases of eosinophilia.274 Reactive eosinophilia can also be observed in malignant disease such as T-cell lymphoma, Hodgkin lymphoma, Langerhans cell-histiocytosis or mastocytosis.275 In acute lymphoblastic leukemia with eosinophilia, both neoplastic and nonclonal eosinophils have been described.282,283 Chronic eosinophilic leukemia (CEL) is a clonal myeloproliferative disorder characterized by a hypercellular bone marrow with a predominant eosinophil compartment and generally orderly maturation.275 There may be an increased number of myeloblasts (5–19%). CEL is an exceedingly rare entity in childhood.284–287 Other neoplastic disorders in which eosinophils are part of the malignant clone such as Ph+ CML, ET, PV, chronic idiopathic myelofibrosis, AML with t(8;21) or chromosome 16 abnormalities have to be separated.275 If dysplastic features of other cell lineages are present, MDS with eosinophilia may be diagnosed.23,288 Chromosome abnormalities in CEL are heterogeneous. They may include the closely linked gene loci on 5q31 for the three eosinophilic cytokines IL-3, IL-5 and GM-CSF.284,288

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The definition of idiopathic hypereosinophilic syndrome (HES) is based on the criteria of Chusid et al.289 : exclusion of clonal and reactive eosinophilic disorders, blood eosinophil count greater than 1.5 × 109 /l, persistence for at least 6 months, and presence of organ involvement with dysfunction. Some cases have been shown to be due to abnormal release of cytokines by T cells.290,291 Others have suggested that these cases with aberrant T-cell population should no longer be categorized as HES.275 Recently, Cools et al.292 demonstrated that some cases of HES may result from a novel constitutively activated fusion tyrosine kinase generated by the fusion of the genes for Fip1like 1 (FIP1L1) and platelet-derived growth factor receptor
(PDGFRA). The presence of this gene rearrangement, undetectable by cytogenetic studies, indicates that these patients with HES should be reclassified as having CEL. It also helps to understand the clinical response of HES to imatinib mesylate,292,293 because the FIP1L1-PDGFR
tyrosine kinase is analogous to the imatinib-sensitive BCRABL enzyme.292 Management of hypereosinophilia is determined by its cause.274 Therapy is aimed at lowering the eosinophil count in order to reduce organ damage. Steroids, immunosuppressive therapy such as cyclosporin and vincristin, hydroxyurea, and IFN-
as well as stem cell transplantation have all been employed (summarized in BritoBabapulle274 ).

Systemic mastocytosis Systemic mastocytosis (SM) is a heterogeneous disease of myelomastocytic progenitors with clonal expansion and a relationship to myeloproliferative disorders (reviewed in Valent et al.294 ). In contrast to cutaneous mastocytosis (CM), SM is characterized by involvement of at least one extracutaneous organ with or without skin involvement. Symptoms result from release of several mediators from mast cells and may include flushing, headache, nausea, vomiting, abdominal pain, diarrhea, palpitation, hypotension, wheezing and syncope.295 In patients with more aggressive disease, symptoms may also arise from organ infiltration in liver, BM, skeleton, spleen and the gastrointestinal tract.296 Such organopathies are seen in patients with aggressive SM, mast cell leukemia and in patients with an associated clonal hematological non-mast cell lineage disease, but not in those with indolent SM.297,298 Histologically, there are multifocal dense infiltrates of mast cells in BM and other tissues. While all mast cells demonstrate mast cell tryptase,299 neoplastic mast cells may also express an aberrant immunophenotypic antigen

profile.300 Most adults with SM have somatic point mutations in c-kit, the SCF receptor.301 The most commonly observed mutation substitutes Val for Asp at codon 816, resulting in a spontaneous activation of tyrosine kinase, and causes resistance to imatinib mesylate.302 Adults with SM generally have a chronic course. It has been estimated that from 15% to 20% of adults with SM develop an associated hematological malignancy,303 a finding consistent with the notion that SM is a myeloproliferative disorder. In childhood, most patients with mastocytosis have CM, often diagnosed before the age of 6 months.295 A serum tryptase level of less than 20 ng/mL may be a relatively safe indication of CM without systemic involvement.304 In these cases, BM examination is not required.294,295 SM in children usually runs an indolent course. The activating Asp816Val c-kit mutation or associated hematologic disorders have rarely been observed.305–307 In about 50% of pediatric cases, symptoms spontaneously resolve by adolescence.307 If childhood lesions persist into adulthood, the prognosis corresponds to that of adult mastocytosis.295

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257 Murphy, S., Iland, H., Rosenthal, D., et al. Essential thrombocythemia: an interim report from the Polycythemia Vera Study Group. Semin Hematol, 1986; 23: 177–82. 258 Cortelazzo, S., Finazzi, G., Ruggeri, M., et al. Hydroxyurea for patients with essential thrombocythemia and a high risk of thrombosis. N Engl J Med, 1995; 332: 1132–6. 259 Harrison, C. N., Campbell, P. J., Buck, G., et al. Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia. N Engl J Med, 2005; 353: 33–45. 260 Kondo, T., Okabe, M., Sanada, M., et al. Familial essential thrombocythemia associated with one-base deletion in the 5 -untranslated region of the thrombopoietin gene. Blood, 1998; 92: 1091–6. 261 Wiestner, A., Padosch, S. A., Ghilardi, N., et al. Hereditary thrombocythaemia is a genetically heterogeneous disorder: exclusion of TPO and MPL in two families with hereditary thrombocythaemia. Br J Haematol, 2000; 110: 104–9. 262 Tefferi, A. Myelofibrosis with myeloid metaplasia. N Engl J Med, 2000; 342: 1255–65. 263 Boxer, L. A., Camitta, B. M., Berenberg, W., et al. Myelofibrosismyeloid metaplasia in childhood. Pediatrics, 1975; 55: 861–5. 264 Altura, R. A., Head, D. R., & Wang, W. C. Long-term survival of infants with idiopathic myelofibrosis. Br J Haematol, 2000; 109: 459–62. 265 Sekhar, M., Prentice, H. G., Popat, U., et al. Idiopathic myelofibrosis in children. Br J Haematol, 1996; 93: 394–7. 266 Dewald, G. W. & Wright, P. I. Chromosome abnormalities in the myeloproliferative disorders. Semin Oncol, 1995; 22: 341– 54. 267 Varki, A., Lottenberg, R., Griffith, R., et al. The syndrome of idiopathic myelofibrosis. A clinicopathologic review with emphasis on the prognostic variables predicting survival. Medicine (Baltimore), 1983; 62: 353–71. 268 Barosi, G., Berzuini, C., Liberato, L. N., et al. A prognostic classification of myelofibrosis with myeloid metaplasia. Br J Haematol, 1988; 70: 397–401. 269 Dupriez, B., Morel, P., Demory, J. L., et al. Prognostic factors in agnogenic myeloid metaplasia: a report on 195 cases with a new scoring system. Blood, 1996; 88: 1013–18. 270 Tefferi, A., Elliot, M. A., Yoon, S. Y., et al. Clinical and bone marrow effects of interferon alfa therapy in myelofibrosis with myeloid metaplasia. Blood, 2001; 97: 1896. 271 Elliott, M. A., Mesa, R. A., Li, C. Y., et al. Thalidomide treatment in myelofibrosis with myeloid metaplasia. Br J Haematol, 2002; 117: 288–96. 272 Guardiola, P., Anderson, J. E., Bandini, G., et al. Allogeneic stem cell transplantation for agnogenic myeloid metaplasia: a European Group for Blood and Marrow Transplantation, Societe Francaise de Greffe de Moelle, Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson Cancer Research Center Collaborative Study. Blood, 1999; 93: 2831–8. 273 Anderson, J. E., Tefferi, A., Craig, F., et al. Myeloablation and autologous peripheral blood stem cell rescue results in hematologic and clinical responses in patients with

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myeloid metaplasia with myelofibrosis. Blood, 2001; 98: 586– 93. Brito-Babapulle, F. The eosinophilias, including the idiopathic hypereosinophilic syndrome. Br J Haematol, 2003; 121: 203– 23. Bain, B., Pierre, R., Imbert, M., et al. Chronic eosinophilic leukemia/hypereosinophilic syndrome. In E. S. Jaffe, N. L., Harris, H., Stein, & J. W. Vardiman, eds., World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. (Lyon, France: IARC, 2001), pp. 29–34. Aoki, Y., Nata, M., Hashiyada, M., et al. Sudden unexpected death in childhood due to eosinophilic myocarditis. Int J Legal Med, 1996; 108: 221–4. Kumar, K. A., Anjaneyulu, A., & Murthy, J. M. Idiopathic hypereosinophilic syndrome presenting as childhood hemiplegia. Postgrad Med J, 1992; 68: 831–3. Falade, A. G., Darbyshire, P. J., Raafat, F., et al. Hypereosinophilic syndrome in childhood appearing as inflammatory bowel disease. J Pediatr Gastroenterol Nutr, 1991; 12: 276–9. Horenstein, M. S., Humes, R., Epstein, M. L., et al. Loffler’s endocarditis presenting in 2 children as fever with eosinophilia. Pediatrics, 2002; 110: 1014–18. Rauch, A. E., Amyot, K. M., Dunn, H. G., et al. Hypereosinophilic syndrome and myocardial infarction in a 15-year-old. Pediatr Pathol Lab Med, 1997; 17: 469–86. Schulman, H., Hertzog, L., Zirkin, H., et al. Cerebral sinovenous thrombosis in the idiopathic hypereosinophilic syndrome in childhood. Pediatr Radiol, 1999; 29: 595–7. Meeker, T. C., Hardy, D., Willman, C., et al. Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia. Blood, 1990; 76: 285–9. Hogan, M. B., Piktel, D., & Landreth, K. S. IL-5 production by bone marrow stromal cells: implications for eosinophilia associated with asthma. J Allergy Clin Immunol, 2000; 106: 329–36. Darbyshire, P. J., Shortland, D., Swansbury, G. J., et al. A myeloproliferative disease in two infants associated with eosinophilia and chromosome t(1;5) translocation. Br J Haematol, 1987; 66: 483–6. Michel, G., Thuret, I., Capodano, A. M., et al. Myelofibrosis in a child suffering from a hypereosinophilic syndrome with trisomy 8: response to corticotherapy. Med Pediatr Oncol, 1991; 19: 62–5. Sakamoto, K., Erdreich-Epstein, A., deClerck, Y., et al. Prolonged clinical response to vincristine treatment in two patients with idiopathic hypereosinophilic syndrome. Am J Pediatr Hematol Oncol, 1992; 14: 348–51. Bakhshi, S., Hamre, M., Mohamed, A. N., et al. t(5;9)(q11;q34): a novel familial translocation involving Abelson oncogene and association with hypereosinophilia. J Pediatr Hematol Oncol, 2003; 25: 82–4. Jani, K., Kempski, H. M., & Reeves, B. R. A case of myelodysplasia with eosinophilia having a translocation t(5;12) (q31;q13)

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restricted to myeloid cells but not involving eosinophils. Br J Haematol, 1994; 87: 57–60. Chusid, M. J., Dale, D. C., West, B. C., et al. The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature. Medicine (Baltimore), 1975; 54: 1–27. Simon, H. U., Plotz, S. G., Dummer, R., et al. Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia. N Engl J Med, 1999; 341: 1112–20. Markwell, H. S. & Wilson, E. Separation of hypereosinophilic syndrome from acute lymphoblastic leukemia with reactive eosinophilia. West J Med, 1983; 138: 269–70. Cools, J., DeAngelo, D. J., Gotlib, J., et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med, 2003; 348: 1201–4. Gleich, G. J., Leiferman, K. M., Pardanani, A., et al. Treatment of hypereosinophilic syndrome with imatinib mesilate. Lancet, 2002; 359: 1577–8. Valent, P., Akin, C., Sperr, W. R., et al. Diagnosis and treatment of systemic mastocytosis: state of the art. Br J Haematol, 2003; 122: 695–717. Hartmann, K. & Metcalfe, D. D. Pediatric mastocytosis. Hematol Oncol Clin North Am, 2000; 14: 625–40. Metcalfe, D. D. The liver, spleen, and lymph nodes in mastocytosis. J Invest Dermatol, 1991; 96: 45–6S. Valent, P., Akin, C., Sperr, W. R., et al. Aggressive systemic mastocytosis and related mast cell disorders: current treatment options and proposed response criteria. Leuk Res, 2003; 27: 635–41. Valent, P., Horny, H.-P., Li, C. Y., et al. Mastocytosis. In E. S. Jaffe, N. L., Harris, H., Stein, & J. W. Vardiman, eds., World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. (Lyon, France: IARC, 2001), pp. 293–302. Valent, P. 1995 Mack-Forster Award Lecture. Review. Mast cell differentiation antigens: expression in normal and malignant cells and use for diagnostic purposes. Eur J Clin Invest, 1995; 25: 715–20.

300 Orfao, A., Escribano, L., Villarrubia, J., et al. Flow cytometric analysis of mast cells from normal and pathological human bone marrow samples: identification and enumeration. Am J Pathol, 1996; 149: 1493–9. 301 Longley, B. J., Tyrrell, L., Lu, S. Z., et al. Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat Genet, 1996; 12: 312–14. 302 Ma, Y., Zeng, S., Metcalfe, D. D., et al. The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood, 2002; 99: 1741–4. 303 Travis, W. D., Li, C. Y., Bergstralh, E. J., et al. Systemic mast cell disease. Analysis of 58 cases and literature review. Medicine (Baltimore), 1988; 67: 345–68. 304 Sperr, W. R., Jordan, J. H., Fiegl, M., et al. Serum tryptase levels in patients with mastocytosis: correlation with mast cell burden and implication for defining the category of disease. Int Arch Allergy Immunol, 2002; 128: 136–41. ¨ 305 Buttner, C., Henz, B. M., Welker, P., et al. Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior. J Invest Dermatol, 1998; 111: 1227–31. 306 Longley, B. J, Jr., Metcalfe, D. D., Tharp, M., et al. Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci U S A, 1999; 96: 1609–14. 307 Azana, J. M., Torrelo, A., Mediero, I. G., et al. Urticaria pigmentosa: a review of 67 pediatric cases. Pediatr Dermatol, 1994; 11: 102–6. 308 Vardiman, J. W., Brunning, R. D., & Harris, N. L. Chronic myeloproliferative disorders. In E. S. Jaffe, N. L.Harris, H. Stein, & J. W. Vardiman, eds., World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues (Lyon, France: IARC, 2001), pp. 17–44.

23 Hematopoietic stem cell transplantation Rupert Handgretinger, Victoria Turner, and Raymond Barfield

Introduction Hematopoietic stem cell transplantation (HSCT) in children was first performed in March 1969 in a child with leukemia, who received cells from a sibling donor. By 1975, HSCT was being used successfully to cure otherwise incurable leukemias in adults.1 The next two decades produced remarkable advances in our understanding of histocompatibility and brought the development of novel immunosuppressive drugs. With the establishment of international bone marrow donor registries,2 HSCT has become a therapeutic option for an increasing number of patients with otherwise incurable leukemias. With the addition of unrelated cord blood transplantation3 and the possibility of including partially mismatched4 or three loci-mismatched haploidentical family members in the donor pool,5,6 a stem cell donor can now be identified for almost every patient with leukemia for whom allogeneic transplantation is considered to be superior to conventional chemotherapy. This chapter reviews practical aspects of HSCT, its application to children and young adults with leukemia, the acute and late toxicities associated with transplantation, and approaches to exploiting the antileukemic effect of allogeneic transplants while minimizing the short and long-term side effects in children with leukemia.

Donor selection for HSCT The selection of donors for HSCT depends upon the match between the prospective donor and the recipient in terms of the products of a group of genes on chromosome 6, the so-called major histocompatibility complex (MHC). The

products of the MHC tested for histocompatibility purposes are referred to as HLAs or human leukocyte antigens. The most important groups of genes known to be relevant to transplantation histocompatibility are divided into class I (HLA-A, B and Cw) and class II (HLA-DR, DQ and DP) genes, whose products differ in distribution and structure. Class I specificities are found on all nucleated cells of an individual, whereas class II are found primarily on cells of the immune system, such as B lymphocytes and macrophages. All specificities are registered with the World Health Organization (WHO) in the order of discovery. The HLAA locus was the first class I locus to be characterized, while HLA-A1 was the first antigen described. In general, the higher the number assigned to a given specificity, the later its discovery. The specificities first identified by testing antisera largely derived from multiparous women who had developed antibody responses to HLA antigens expressed on fetal tissue were limited in number compared with the numbers of specificities that have been identified by DNA sequencing of relevant genes. A locus name with a one- or two-digit number indicates a serologically defined specificity, such as HLA-A2. A DNA sequence registered with WHO is assigned a number with the first two digits usually related to the closest serologic equivalent such as HLAA*0201. The third and fourth digits represent the order of sequence registration for alleles of that group. HLA-A*0201 is the first registered DNA sequence for an allele of HLAA2. The asterisk indicates that the data are derived from DNA testing. Allele numbers with more than four digits represent either substitutions that do not result in an amino acid change in the protein product or substitutions outside the coding regions. As of 2004, 1325 class I alleles and 763 class II alleles with official names had been registered.7

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Inheritance of HLA Antigens Father A1

Mother A2

A3

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B8

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Possible Children Child 1 A1 Cw2

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DQ6

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Fig. 23.1 Possible haplotype combinations that may occur in children of two parents. Note that the alleles of a given haplotype are inherited together unless recombination occurs, as in child 5. Specificities are shown as serologic equivalents, for simplicity.

The function of the MHC complex is to present peptides derived either from self-proteins or from exogenous proteins to the T-lymphocyte receptor. T lymphocytes have been educated in the thymus to recognize self and nonself and it is this ability that determines the initiation of an immune response if the HLA-peptide molecular complex is identified by the T-lymphocyte receptor as foreign to the host. A given HLA specificity can bind a range of peptides with specific characteristics. The polymorphism of the system permits a wide range of peptide binding in the population as a whole and has evolved to facilitate microbial immunity. The MHC has proven to be the most polymorphic genetic region known in the human genome. An individual inherits a set of HLAs, called a haplotype, from each parent. Both haplotypes are fully expressed. The chance that any child of any two parents would share both haplotypes with a full sibling is 25%, the chance of sharing one haplotype is 50%, and the chance of sharing neither haplotype is 25% (Fig. 23.1). These genes are closely linked, and their recombination rate is limited. (e.g. the rate between HLA-B and HLA-DR is approximately 1%). The optimally matched donor is a full sibling who shares the same parental haplotypes as the recipient. Approximately one-third of patients have a fully matched

sibling. Alternative donors include unrelated donors and partially mismatched related donors. The initial definitions of appropriate matching emphasized HLA-A, B and DR, providing the basis for matching levels, referred to as 6/6 or 5/6 and so on. Studies on additional transplant antigens such as HLA-C8 or HLA-DQB 1,9 suggested a better clinical outcome with higher levels of matching, so that some transplant centers now prefer a match of all HLAs (10/10 match). However, the likelihood of identifying an unrelated donor decreases with the degree of matching. The direction of mismatching is important to consider in the context of graft rejection or graft-versushost disease (GVHD). The recipient’s immune system can potentially generate immune responses to the grafted cells (host-versus-graft vector). A more important consideration may be the immune responses generated by the engrafting immunocompetent cells toward recipient tissues (graftversus-host vector). Outcome is influenced by the degree of matching and mismatching in each direction (Fig. 23.2).

Stem cell source Mature hematopoietic cells are constantly produced from pluripotent hematopoietic stem cells in the BM,

Hematopoietic stem cell transplantation

Direction of Mismatch Donor A3 Cw1 B7

Recipient A1 Cw4

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B8

DR2

DR3

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A1 Cw2

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A1 Cw4 B8

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Fig. 23.2 Donor T lymphocytes recognize recipients’ antigens or alleles that are not shared by the donor (HLA-Cw2 and HLA-DR4 in this example). This constellation is called a mismatch in the graft-versus-host direction. When the donor’s antigens or alleles are not shared by the recipient, the recipient’s T lymphocytes can recognize donor specificities (HLA-A3, Cw1, B7, DR2 and DQ6 in this example) as different, enabling them to serve as targets of rejection (mismatch in host-versus-graft direction). In this example, the degree of mismatch is different in each direction.

and only those stem cells are capable of reconstituting hematopoiesis after the stem cells have been transplanted into a recipient. Most pluripotent stem cells can be identified via expression of the CD34 antigen10 and such stem cells have a high engrafting capacity.11 In the early years of transplantation, BM was exclusively used as a stem cell source for transplantation. The discovery and clinical application of granulocyte colony-stimulating growth factor (G-CSF) and granulocyte-macrophage colonystimulating growth factor (GM-CSF) led to the observation that CD 34+ BM stem cells can be mobilized in large numbers into the peripheral bloodstream12,13 and that mobilized peripheral blood stem cells (PBSCs) can completely reconstitute hematopoiesis.14 BM is still the main stem cell source for matched sibling donor (MSD) and matched unrelated donor (MUD) transplants, but PBSCs are being increasingly collected from such donors.15,16 In pediatric haploidentical transplantations, PBSCs are often used to increase the number of transplanted stem cells.6 Umbilical cord blood (UCB) from related donors17 or from cord blood banks is also being increasingly used as an alternative source of stem cells for allogeneic transplantation.3,18–21 The disadvantage of UCB is that the limited number of stem cells can lead to a delayed time to engraftment, although methods for ex-vivo expansion of UCB cells have been described.22 Immune reconstitution after UCB transplantation seems to be similar to that observed after transplantation with stem cells from other sources.23 BM is harvested from the posterior iliac crest in the operating room under regional or general anesthesia. The harvested volume depends on the recipient’s weight

and should not exceed 1.5 liters. Usually, a cell dose of 2–4 × 108 /kg nucleated cells is adequate for engraftment, although a significant influence of the cell dose on transplant-related mortality and long-term graft function was recently reported.24 Allogeneic PBSCs are mobilized with a short-term application of G-CSF, GM-CSF or both,25 whereas autologous PBSCs are mobilized either with growth factors alone or in combination with chemotherapy and growth factors. For PBSC collections, anesthesia is not necessary, and one or more leukapheresis procedures are performed with a cell separator. All cell separators have a certain extracorporal volume limiting their application in younger children, although PBSC leukapheresis has been performed in small allogeneic donors.26,27 Autologous PBSCs have been successfully collected in small patients with body weights as low as 8 kg.28 The use of growth factors in healthy children for stem cell mobilization is controversial, and a careful risk–benefit analysis must be performed.

Ex-vivo manipulation of stem cells The removal of leukemic cells from an autologous graft or the removal of unwanted cells from an allogeneic graft is the aim of ex-vivo stem cell manipulation. A number of techniques for tumor cell purging for autologous transplantation have been described, and every transplant center has its own preference.29 The removal of T lymphocytes from allogeneic bone marrow grafts for the prevention of GVHD is widely used,30 and a variety of different techniques based on physical separation methods or more or less T-cell-specific reagents have been described.31 Due to technical limitations regarding the processing of the 5- to 10-fold higher cell number of PBSCs, most of these techniques have been restricted to BM. T-cell-depleted PBSCs are rarely used.32 More recently developed techniques, such as positive selection of CD34+ stem cells by magnetically activated cell sorting (MACS) as a method for indirect depletion of T lymphocytes, allow the processing of PBSCs.33,34 A 100,000-fold reduction of T cells can be obtained, thus allowing the transplantation of large numbers of purified CD34+ stem cells in the matched sibling,35 matched unrelated,36 or three-loci mismatched haploidentical situation6 without additional pharmacologic GVHD prophylaxis and without clinically significant acute or chronic GVHD.37 This technology also allows the add-back of a defined number of T lymphocytes to the graft.38 More recently, the MACS technology has been used for the negative depletion of T lymphocytes from allogeneic PBSCs.39

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Preparative regimens The most commonly used preparative regimes prior to allogeneic transplantation for leukemia include various doses (12 Gy to 15.75 Gy) of fractionated total-body irradiation (TBI) and cyclophosphamide, without or with the addition of etoposide, cytarabine, thiotepa or fludarabine. Non-TBIbased regimens with busulfan/cyclophosphamide with or without additional cytotoxic drugs such as etoposide, thiotepa and fludarabine are also used, but no conclusive studies to support either TBI- or non-TBI-based regimens have been reported for children. The preparative regimen should have a cytotoxic antileukemic effect, but should also provide adequate immunosuppression in order to ensure engraftment. Other non-TBI-based myeloablative regimens based on melphalan, fludarabine and thiotepa have been reported to facilitate safe engraftment in threeloci mismatched haploidentical transplants.40 Less aggressive so-called nonmyeloablative stem cell transplantation regimens are attracting increasing interest, especially for patients who otherwise cannot tolerate a conventional myeloablative regimen. Such regimens range from minimal, to facilitate engraftment (fludarabine plus lowdose TBI),41 to more intensive but still not myeloablative (reduced intensity conditioning, or RIC), such as reduced doses of fludarabine plus busulfan.42 The rationale behind nonmyeloablative stem cell transplantation is to induce an optimal graft-versus-leukemia (GVL) effect by donor alloreactive effector cells.43 While this form of transplantation is mostly applied in adult and elderly patients, the data in children with leukemia remains insufficient to conclude that the reduced cytotoxic antileukemic effect of the preparative regimen is counterbalanced by an increased antileukemic effect of the allograft; hence, these regimens should be used only in the context of controlled clinical trials.

HSCT as immunotherapy Although the patient’s preparative regimen itself has a considerable antileukemic effect, it is only one of the two pillars of successful eradication of a patient’s leukemic cells. The other is the elimination of residual malignant cells that have not been affected by the preparative regimen or by the immunologic mechanisms induced by the graft. Major disparities or minor differences in donor and recipient HLAs are the reasons for the more-or-less intensive alloreactive immune responses of graft-containing lymphocytes, either against the recipient’s normal cells, resulting in GVHD, or against the recipient’s residual leukemic cells (GVL). The GVL effect appears to play an increasingly important role in the therapy of patients with leukemia.44–46

Unfortunately, GVL is mostly associated with GVHD but current knowledge does not permit the effects to be separated. The concept that GVHD is associated with GVL is supported by observations of lower relapse rates in patients who developed acute47–51 and/or chronic52–55 GVHD. Additional evidence for the coexistence of a GVL effect comes from clinical experience in which measures to reduce or prevent GVHD were associated with a higher relapse rate. Retrospective and prospective randomized trials in children with acute leukemia showed that a reduced dose of cyclosporine A used as GVHD prophylaxis after matched sibling transplantation is associated with a significant reduction in the leukemia relapse rate.56 This result agrees with previous findings that patients with lower posttransplant cyclosporine A levels had a higher incidence of GVHD.57 In a recent 10-year follow-up of a randomized study, a low-dose of cyclosporine A and a low level of this immunosuppressant during the first 10 days after allogeneic transplantation conferred significant protection against leukemic relapse in patients with early and advanced disease.58 Chronic GVHD is also associated with a reduced relapse rate. In a large retrospective study in children with various malignancies, the probability of relapse was 16% versus 39% in children with or without chronic GVHD, respectively.59 Interestingly, the antileukemic effect of chronic GVHD was mainly observed in patients with acute lymphoblastic leukemia. Because of the mortality related to acute and chronic GVHD, the reduced relapse rate did not translate into an improved overall survival. In line with these observations is the clinical experience that recipients of syngeneic stem cells or of T-depleted allogeneic graft for GVHD prevention have a higher relapse rate than patients whose grafts are immunogenic.50 The existence of GVL is further corroborated by the successful infusion of viable donor lymphocytes for the posttransplant treatment of leukemic relapse.60 By contrast, an increased relapse rate has not been observed after transplantation of large numbers of mobilized peripheral stem cells from matched and partially matched unrelated donors after indirect T-cell depletion by the MACS method for CD 34+ selection.36 The potential antileukemic effect of GVHD is also supported by anecdotal case reports in children.61,62 A reliable way to identify and treat patients at risk for relapse may be the frequent determination of a patient’s post-transplant chimerism status.63 A mixed chimerism (i.e. the simultaneous presence of host- and donor-derived cells) may herald an impending relapse, especially if the proportion of donor cells is decreasing further,64,65 whereas patients with a complete donor chimerism have a higher rate of relapse-free survival.66 Based on these observations,

Hematopoietic stem cell transplantation

strategies such as withdrawal of immunosuppression or chimerism-guided lymphocyte infusions (DLIs) have been used successfully to either maintain a complete donor chimerism or to convert a decreasing mixed to a complete donor chimerism.66 The kinetics of the decrease of donor chimerism may be important, since an increased relapse rate was not observed in a long-term follow-up study of persisting chimerism.67 Alloreactive natural killer (NK) cells represent a new concept to induce an antileukemic effect in the absence of GVHD.68 NK cells are cytotoxic cells that spontaneously induce cytotoxicity against both leukemic blasts and normal hematopoietic tissue.69 Physiologically, NK cells express killer cell immunoglobulin-like receptors (KIRs), which are members of a group of regulatory molecules found on subsets of lymphoid cells. Upon interaction of KIRs with certain HLA-class I alleles, the NK activity is either active or inhibited. The inhibitory effects protect healthy cells from spontaneous destruction by NK-cell mediated cytolysis. Physiologically, all NK cells have at least one inhibitory receptor for self HLA-class I alleles.70 A wellcharacterized interaction between KIR and HLAs is the inhibition of NK cells via CD158a (receptor for HLA alleles Cw 2, 4, 5, 6 and others, group 2) and CD158b (receptor for HLA alleles Cw 1, 3, 7, 8 and others, group 1).71 This biologic feature offers a fascinating way to harness the antileukemic activity of NK cells in the transplant setting, especially if the donor and patient have a KIR-ligand mismatch in the GVHD direction. An example of such a ‘perfect-mismatch,’72 which can often be identified in the haploidentical situation and which results in the development of alloreactive antileukemic effector cells, is illustrated in Fig. 23.3. While alloreactive NK cells might play a less important role in an HLA-matched setting,73 impressive clinical results have been reported after haploidentical transplantation of patients with AML.71 In adults with ALL, the results were less striking.71 In a more recent study in pediatric patients, the presence of KIRs on the donors’ NK cells and the absence of corresponding KIR ligands in the recipients’ HLA repertoire were predictive for the risk of relapse in both AML and ALL.74 Further intensive research will be necessary to better harness alloreactive NK cells for the treatment of ALL. Since alloreactive NK cells also target normal recipient lymphohematopoietic tissue, KIR ligand mismatching may also facilitate engraftment across the HLA barrier and reduce GVHD via cytolysis of alloantigenpresenting host dendritic cells.75 This would allow the use of less myeloablative and immunosuppressive regimens in KIR ligand-mismatched transplantation. Future research focusing on the interaction between immune effector cells and residual leukemic blasts or host cells may lead to

new and more specific strategies to exploit the GVL effect without increasing GVHD-related mortality.76

Indications for transplantation Acute lymphoblastic leukemia International studies of childhood ALL carried out between 1986 and 1998 in developed countries have achieved 5-year event-free survival rates ranging from 63% to 83%.77 Transplantation is considered in patients who have relapsed or have a very high risk of relapse, including those with (MLL-AF 4), hypodiploidy (<45 chromosomes), induction failure (5% or more leukemic cells) and minimal residual disease (MRD) of more than 1% after 4 to 6 weeks of first-line therapy.78–80 Early clearance of leukemic cells, as measured by morphologic criteria81 or flow cytometry, seems to be an important prognostic factor; indeed detection of leukemic cells on day 19 after remission induction was associated with a high relapse rate.82 A recent study reviewing cases of Philadelphia chromosome (Ph)-positive ALL from 10 study groups between 1986 and 1996 showed that transplantation of marrow from an HLA-matched related donor yields a significantly better outcome in these patients than does chemotherapy alone83 (Fig. 23.4). In the absence of a HLAmatched stem cell donor, haploidentical transplantation in first complete remission (CR1) using mobilized and highly purified CD 34+ selected peripheral stem cells can be a useful option.84 The majority of transplants are performed in relapsed patients in CR2 or beyond, given the fact that the probability of leukemia-free survival with chemotherapy alone is only in the range of 10% to 40%.85 Older age at relapse (>10–15 years of age) and a short CR1 result in lower rates of leukemia-free survival (LFS).86 Patients who relapse on therapy or early in the BM after treatment have a poor outcome with chemotherapy alone87,88 ; hence, the intensity and length of chemotherapy for their initial relapse should be planned to accommodate the later HSCT, thus ensuring that the patient is in good clinical condition before transplantation. Patients with early or late relapse of T-ALL have a poor prognosis and transplantation for this subgroup is recommended.88,89 Patients with the previously described high-risk features in CR2 who lack an HLA-matched stem cell donor have a realistic chance for cure after haploidentical stem cell transplantation.84 About two-thirds of patients with a late extramedullary relapse, and about one-third of those with early extramedullary relapse or late non-Tmarrow relapse or early combined non-T relapses, can be rescued by chemotherapy.90 However, the persistence of

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Fig. 23.3 NK alloreactivity is based on the assumption that all NK cells in an individual have at least one killer-inhibitory receptor (KIR) for a HLA-class I self-allele. If, for example, a patient and his or her leukemic cells express HLACw1 and Cw8 and the donor’s HLA type is HLA-Cw2 and Cw8, this donor has a KIR mismatch in the GVHD direction in the HLA-Cw group 1 alleles (HLA-Cw1) and the following situations can occur: (a) The NK alloreactive donor will have NK cells in his or her repertoire that express CD158b (receptor for self-allele Cw8). This NK subset will also be inhibited after transplantation by the recipient’s HLA-Cw8 allele expressed on his leukemic blasts and will have no antileukemic effect. (b) The NK alloreactive donor will also have NK cells in his or her repertoire that express both CD158a and CD158b (receptors for products of the self-alleles Cw2 and Cw8). This NK cell subset will also be inhibited by the recipient’s HLA-Cw group 1 alleles (HLA-Cw1 and Cw8) and will also not exert an anti-leukemic effect. (c) The NK alloreactive donor will have NK cells in his or her repertoire that express CD158a alone (receptor for self-allele product Cw2). The donor’s NK cells expressing CD158a alone cannot be inhibited by the recipient’s HLA-Cw group 1 alleles (HLA-Cw1 and Cw8) expressed on the leukemic cells. Hence, this donor’s NK cell subset will exert a cytotoxic activity against the recipient’s leukemic cells. In contrast to cytotoxic T lymphocytes, the targets of NK cells are either the recipient’s malignant or normal hematopoietic cells, so that the cytotoxic activity does not induce GVHD. NK cells from a donor who shares the same HLA-Cw alleles with the recipient (KIR-matched nonalloreactive donor) will also express CD158b (a) or CD158b and CD158a (b), but will not have NK cells in his or her repertoire that express only CD158a, for which he would not have an inhibitory self-allele (c). Therefore, when expressed on leukemic cells, the recipient’s HLA-Cw1 and Cw8 alleles continuously exert an inhibitory signal via binding to CD158b, and the donor NK cells cannot exert cytotoxic activity against the recipient’s leukemic cells. A more detailed description of NK-alloreactive donor selection is provided in Ruggeri et al.71

high-level MRD after appropriate relapse therapy in these patients has been reported to identify patients at risk for subsequent relapse.91 The importance of MRD determination is further documented by the observation that patients with detectable MRD before HSCT have a poorer outcome than those with no detectable MRD92 (Fig. 23.5). By identifying patients at risk of relapse after HSCT, one might

introduce measures such as low-dose cyclosporine, early withdrawal of immunosuppression after transplantation or others to improve end results. The role of HSCT in infant ALL is controversial. Most of these patients have rearrangements of the MLL gene in chromosome 11q23 and an associated poor outcome,93 and HSCT with matched sibling donors does not seem to

Hematopoietic stem cell transplantation

Fig. 23.4 Estimates of disease-free and overall survival [± standard error (SE)] in 267 children with Philadelphia chromosome-positive ALL treated with transplantation of bone marrow from HLA-matched related donors or chemotherapy only. (From Arico et al.83 with permission.)

Fig. 23.5 Kaplan–Meier analysis of MRD-negative and -positive patients at the time of HSCT. (From Bader et al.92 , with permission.)

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Fig. 23.6 Actuarial survival of patients with AML in remission, comparing the three postremission regimens. Numbers are patients at risk at yearly intervals. Dashed line, allogeneic HSCT; solid line, chemotherapy; dotted line, autologous HSCT. (From Woods et al.100 with permission.)

improve the prognosis for this subgroup.94 Therefore, HSCT might be restricted to research protocols aimed at investigating new approaches for childhood ALL with rearrangements of the 11q23 chromosomal region.

Acute myeloid leukemia With conventional chemotherapy alone, up to 45% of children and adolescents with AML survive for 5 years or longer without relapse.95–98 Certain cytogenetic abnormalities have prognostic value. Patients with AML associated with the t(8;21), t(15;17), or inv(16) have a relatively favorable prognosis with chemotherapy alone, whereas those with complex karyotypes, such as −5, del(5q), –7, or 3q abnormalities, might benefit from transplantation.99 An intermediate prognosis is conferred by 11q23 abnormalities, +8, +21, +22, and del (7q). In the largest randomized trial to date comparing allogeneic BMT, autologous BMT and aggressive post remission chemotherapy for AML, investigators confirmed that in adolescents and children,

allogeneic BMT in first remission is the treatment of choice when a matched related donor is available (Fig. 23.6).100 A similar good survival rate has been reported by the UK MRC group (56% at 7 years) with chemotherapy alone.101 With a better understanding of underlying pathomechanisms and improvements in treatment with differentiating agents, such as all-trans-retinoic acid (ATRA) in the treatment of AML-M3 with t(15;17)102 or other rearrangements,103,104 the risks and benefits of conventional therapy, autologous HSCT and allogeneic HSCT must be carefully weighed and frequently reassessed. Allogeneic HSCT is the therapy of choice for most patients with relapses of AML. In a recent study, an estimated 5-year disease-free survival of 58% was reported for children with AML who received allogeneic HSCT during second complete remission.105 In the absence of a MSD or MUD donor, it is the transplant center’s preference whether a haploidentical, mismatched cord blood or an autologous transplant is performed. The role of purging in autologous HSCT for AML is not clear. The fact that relapses can be

Hematopoietic stem cell transplantation

induced by graft-contaminating leukemic cells106 should be taken into account when this method of transplantation is considered.

Myelodysplastic syndromes The prognosis of most children with a myelodysplastic syndrome (MDS) is poor, and although there is no consensus on the best treatment, HSCT is currently the therapy of choice.107–109 Patients who are most likely to do better without transplantation are also those who respond best to allogeneic transplantation.110 This group includes patients with refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and those with normal cytogenetics. The children most likely to benefit from HSCT are those with refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-t), an age younger than 2 years, and a hemoglobin F level of 10% or higher.111,112 The rarity of this disease requires international studies by cooperative groups, such as the European Working Group of MDS in Childhood (EWOGMDS) in order to find the best treatment options. The timing of transplantation and whether or not induction chemotherapy should precede transplantation is controversial. Patients with RAEB-t have a high relapse rate if transplanted without preceding chemotherapy, whereas those with less than 5% blasts do better with HSCT performed in the absence of induction chemotherapy.107,108 A recent large prospective study of children with MDS found that patients with RAEB-t often do as well as those with AML when treated with AML therapy at diagnosis including HSCT when an HLA-matched sibling is available.113 On the other hand, children with RA or RAEB do very poorly with standard AML therapy and might be considered for HSCT.114,115 The optimal treatment for juvenile myelomonocytic leukemia (JMML) is not clearly established. Conventional chemotherapy is unlikely to eradicate the stem cell abnormality, but it may ameliorate the disease, and HSCT – preferably with an HLA-matched sibling – offers the greatest likelihood for cure.116–119 The success of HSCT is limited primarily by the proclivity of this disease to relapse.120 Therefore, additional post-transplant interventions such as alpha-interferon, biologic differentiation agents such as retinoic acids or farnesyltransferase inhibitors are under investigation.121,122

Therapy-related myelodysplastic syndrome and acute myeloid leukemia Therapy-related MDS (t-MDS) and AML (t-AML) are defined as clonal malignant disorders that arise after expo-

sure to cytoxic agents. While many of the clinical and biologic features of t-MDS and t-AML are similar to those of de novo disorders, patients with t-MDS and t-AML often have a rapidly progressive disease, and their neoplastic clones usually have distinct chromosomal abnormalities.123 Most likely as a result of the high frequency of poor prognostic factors, including unfavorable cytogenetic abnormalities characteristic of secondary disorders, chemotherapy yields fewer and shorter complete remissions.124 Patients with favorable karyotypes such as the t(8:21), inv 16 or t(15:17) translocations might be treated as any other case of de novo AML.123 HSCT seems to be a potential curative treatment, especially for patients who lack poor-risk cytogenetic features125 and might be the only curative option for a small number of patients with primary refractory disease.126

Adult-type chronic myeloid leukemia Adult-type CML with the t(9;22) is optimally treated with hematopoietic stem cells from an HLA-identical family member or, alternatively, those from a one-antigen mismatched family member or HLA-matched unrelated donor.127 A leading prognostic factor is the phase of disease at transplantation. Only 10% to 20% of patients in blast crisis will achieve a durable remission, whereas transplantation in the accelerated or chronic phase can achieve durable remissions in 40% and 80% of patients, respectively. In the chronic phase, a shorter time between diagnosis and HSCT results in a better outcome.128 One study comparing the use of PBSC transplantation with BMT found the former method to have a significant survival advantage (1000-day overall survival of 94% versus 66%).129 For patients with a cytogenetically positive relapse after transplantation, some interesting results have been obtained with alpha-2a-interferon; including a cytogenetic remission rate of 57% in such patients.130 Post-transplant DLIs induce complete and durable remissions in the majority of patients with CML in early-stage relapse.131 The role of imatinib mesylate (Gleevec; formerly STI 571) in the treatment of pediatric patients with CML is not yet clear. This agent has been shown to induce remissions in patients with blast crisis,132 while in the less aggressive chronic phase of the disease; the response rates results were even more dramatic.133 However, drug resistance does occur, and it seems to be associated with reactivation of BCR-ABL signal transduction.134 The results of ongoing pediatric studies, such as the Children’s Oncology Group phase 2 study of imatinib in Ph+ chronic phase myelogenous leukemia,will be useful in determining the role of imatinib in pediatric patients with CML. Strategies used in adult patients, such as initial therapy with imatinib followed by HSCT depending

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on the response to the first treatment, might also be effective in children.135

Second transplants Relapse is the most frequent cause of therapeutic failure after HSCT. Although chemotherapy might offer a short period of remission, the only curative approach is a second allogeneic transplant. The overall disease-free survival rate at 3 to 5 years after such procedures ranges from 26% to 32%.136,137 Two important prognostic factors are length of remission after the first transplant and type of first transplant (autologous or allogeneic).138–140 The major causes of mortality after second HSCT are infection and relapse.141 Newer approaches, such as reduced intensity conditioning (RIC) regimens will hopefully reduce the high transplantrelated mortality encountered in this group of patients.

Factors associated with transplant-related morbidity and mortality The cytotoxic regimen and the infusion of allogeneic stem cells are associated with appreciable rates of non-diseaserelated morbidity and mortality, which significantly influence the overall survival and the quality of life after transplantation.

Graft failure In primary graft failure, the allograft does not resume its function, and the patient remains aplastic after his or her myeloablative preparative treatment. Rejection or secondary graft failure denotes the loss of the allograft after initial engraftment. The risk of rejection is higher in patients who have an HLA mismatch in the host-versusgraft direction and when persistent functional host alloreactive effector cells mount an alloreaction against the graft. This alloreaction is counterbalanced by an alloreaction of graft-containing T lymphocytes against residual host effector cells. Therefore, the incidence of graft failure is low when unmanipulated grafts are transplanted, but is in the range of 10% to 30% in T-depleted transplants,142–144 and the number of donor CD 3+ T cells in the graft has also been reported to be an important factor in graft failure.145 Addition of anti-T-cell reagents146 can reduce the rejection incidence, but may also interfere with immune reconstitution. While graft failure normally is a fatal complication,142 successful second transplant procedures using purified CD 34+ stem cells, either from the same or from alternative donors, have recently been reported.36,147

Graft-versus-host disease Graft-versus-host disease (GVHD) is a complex immunologic reaction of immunocompetent cells in the graft against the host,148 and the reduction or complete avoidance of GVHD is the basis for choosing the best HLAmatched donor. GVHD can still be triggered in a perfect HLA allele-matched situation by the presence of yet unknown transplantation antigens.149 An association between certain HLA alleles and the development of acute and chronic GVHD in MSD transplants150 as well as host defense and inflammatory gene polymorphisms151 has been reported. While all patients receiving a stem cell allograft are at risk to develop GVHD, the incidence of this complication increases with increasing HLA mismatches between donor and recipient.152,153 Although GVHD is a complex immunologic reaction, graft-contaminating T lymphocytes are mainly responsible for the induction and severity of GVHD. Acute GVHD Acute GVHD is defined as the clinical occurrence of symptoms during the first 100 days after stem cell infusion, and its incidence ranges from 10% to 80%, depending on the HLA disparity, number of T lymphocytes in the graft, patient and donor characteristics, and the regimen used for GVHD prophylaxis.138,154 The major target organs are skin, liver and the gastrointestinal tract, but other tissues can also be involved. Acute GVHD is often first seen in the skin, the first signs commonly being erythema of the palms (Fig. 23.7a), soles or ears. A coarse maculopapular rash is almost pathognomonic for skin GVHD (Fig. 23.7b). In some patients, the skin manifestations are less obvious, and the rash can be a fine erythematous papular rash (Fig. 23.7c). In unclear cases, a skin biopsy is helpful in establishing the diagnosis. More severe skin involvement includes either localized (Fig. 23.7d) or whole-body erythroderma (Fig. 23.7e). The most severe form (stage 4) includes bullae and desquamation. GVHD of liver and gut can occur with or without, before, or after skin disease. Impaired liver function including hyperbilirubinemia and elevation of alkaline phosphatase and transaminases are the leading symptoms of liver GVHD, but can be difficult to differentiate from other diseases, such as veno-occlusive disease, infections or toxicity from drugs. Percutaneous or transjugular liver biopsies may be helpful in confirming the diagnosis. Clinical symptoms suggestive of GVHD of the GI tract include nausea, vomiting, green watery diarrhea or paralytic ileus. Both upper and lower endoscopy with biopsies may be necessary to establish the diagnosis. A grading system for acute GVHD was originally

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Table 23.1 Staging and grading of acute graft-versus-host disease (From Glucksberg et al.155 and Przepiorka et al.156 , with permission.) Staging

Grading

Organ

Extent of Involvement

Stage

Grade

Description

Skin

Maculopapular skin rash <25% of body surface area Maculopapular skin rash 25% to 50% of body surface area Maculopapular skin rash >50% of body surface area Generalized erythroderma with bullae and desquamation

1 2 3 4

0

No GVHD

I

Stage 1/2 skin; no gut or liver involvement; no decrease in clinical performance

Bilirubin 2–3 mg/dL Bilirubin 3–6 mg/dL Bilirubin 6–15 mg/dL Bilirubin >15 mg/dL

1 2 3 4

II

Stage 1–3 skin; stage 1 gut and/or stage 1 liver involvement; mild decrease in clinical performance

III

Stage 2/3 skin; stage 2/3 gut and/or stage 2/3 liver involvement; marked decrease in clinical performance

IV

Stage 2–4 organ involvement with extreme decrease in clinical performance

Liver

Intestine

Diarrhea >500 mL/day (10–15 mL/kg per day) Diarrhea >1000 mL/day (16–20 mL/kg per day) Diarrhea >1500 mL/day (21–25 mL/kg per day) Severe abdominal pain, with or without ileus

1 2 3 4

proposed by Glucksberg et al.155 and later slightly modified.156 Table 23.1 outlines the most commonly used staging and grading system. The prophylaxis and treatment of GVHD in children varies greatly among transplant centers.157–159

Chronic GVHD GVHD occurring 100 days or later after stem cell infusion is arbitrarily defined as chronic GVHD, although typical manifestations can occur earlier than 100 days or several months post-transplantation. In adults, its incidence after HLA-identical sibling transplants has been reported to range from 30% to 50%,160 with a further increase after MUD transplants.161 In a recent report, 25% of children with malignant and nonmalignant diseases developed chronic GVHD at a median of 116 days after MSD or MUD transplants.59 Acute GVHD, malignant disease, a recipient age of more than 10 years and a female donor for a male recipient have been identified as risk factors for the development of chronic GVHD in children.162 Prophylactic antithymocyte globulin given before transplants has been reported to reduce the risk of chronic GVHD.163 Clinically, chronic GVHD involves various organ systems and resembles collagen-vascular autoimmune diseases (Fig. 23.8). It can occur either after acute GVHD (progressive chronic GVHD) or after a disease-free interval following acute GVHD (quiescent chronic GVHD), or without preceding acute GVHD (de novo chronic GVHD). Acute grade II–IV GVHD seems to be highly predictive of the

Fig. 23.7 Graft-versus-host disease tends to appear first as a pruritus or erythema on palms (a), soles or ears. Next, a maculopapular rash (b, c) may progress to a total-body erythroderma (d, e). (See color plate 23.7 for full-color reproduction.)

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Table 23.2 Grading system for chronic graft-versus-host disease. (From Shulman et al.169 ) Grade

Description

Subclinical GVHD Limited chronic GVHD Extensive chronic GVHD

Histologically positive, but no clinical symptoms Either localized skin involvement and/or hepatic dysfunction (from chronic GVHD) Either generalized skin involvement or localized skin involvement or hepatic dysfunction from chronic GVHD, or both Plus liver histology indicating chronic aggressive hepatitis, bridging necrosis or cirrhosis Or involvement of eye (Schimer’s test with <5 mm wetting) or involvement of minor salivary glands or oral mucosa demonstrated on labial biopsy Or involvement of any other target organ (lung, gut)

Fig. 23.8 Extensive chronic GVHD in a patient after matched-sibling transplantation. The skin is the most frequently involved organ with hyper- or hypopigmentation, desquamation and a picture similar to scleroderma, including joint contractures. Other features include dysphagia and indolent weight loss. (Photo kindly provided by Dr. G. Vogelsang, Johns-Hopkins University, Baltimore, MD, USA; see color plate 23.8 for full-color reproduction)

chronic form of this disease.59,164–167 The most commonly affected organs are skin, mouth, eye, sinuses and the GI tract.168 Scleroderma-like lesions can result in severe contraction of the joints. Less common sites of involvement are lungs, muscles, tendons, serous surfaces and bone marrow (in association with persistent thrombocytopenia). Exclusive involvement of skin and/or liver is classified as limited, whereas the involvement of other organs is referred to as extensive (Table 23.2).169 The Karnofsky performance score offers a practical means to assess the severity of chronic GVHD.170 Patients with this complication have an impaired and dysfunctional humoral and cellular immune function, auto and alloreactive T cells, autoantibodies, and other dysregulated immune cells all of which may contribute to the complex clinical picture of GVHD. Children with chronic

GVHD also have a decreased growth velocity, which can revert to normal or catch-up growth after the disease is controlled.171 Due to the negative influence on the immune system, patients with chronic GVHD are highly susceptible to infectious complications, including bacterial, viral and fungal infections, which are the most common cause of death in this subgroup.170 Therefore, prolonged supportive therapy, careful surveillance and monitoring of the immune status may be required. Patients receiving unmanipulated PBSC grafts appear to have a higher risk for the development of chronic GVHD than do patients undergoing BMT,172,173 and a high incidence of extensive chronic GVHD has been reported in patients receiving a PBSC graft from HLA-identical young donors.27 Because of the potential impact on growth and quality of life, this observation should be taken into consideration in the risk/benefit analysis for individual patients, when the choice of donor and stem cell source is made. For treatment of both acute and chronic GVHD, first-line drugs such as cyclosporine A and steroids are commonly used, followed by various second-line therapies in the event of steroid resistance,159 such as thalidomide,174 azathioprine,175 tacrolimus,176 mycophenolate mofetil,177 rapamycin,178 anti-TNF-
monoclonal antibodies or anti-interleukin-2 receptor (anti-CD 25) antibodies,179 pentostatin,180 psoralen plus long-wavelength UVA radiation (PUVA)181,182 and extracorporal photopheresis.183–185 Factors associated with poor-prognosis chronic GVHD are thrombocytopenia, progressive onset from acute GVHD, extensive disease and a Karnofsky performance score lower than 50%.162,186,187

Early and late post-transplant complications other than GVHD Major transplant-related complications There are a number of major transplant-related complications (MTCs) that may or may not be related to

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GVHD including infections, hepatic veno-occlusive disease (VOD),188 lung toxicity189 and capillary leakage syndrome.190 Risk factors for MTCs are older age and advanced disease status,86 a more intensive preparative regimen, use of alternative donors, and less effective methods for GVHD prophylaxis.191–193 There may also be an individual sensitivity to the tissue-damaging effects of the preparative regimen, as indicated by certain biologic markers, such as low production of interleukin-10 by mononuclear cells,194 high levels of tumor necrosis factor-alpha (TNF-
) during myeloablative therapy,195 or an early increase of serum bilirubin and/or blood urea nitrogen.196 Host defense and inflammatory gene polymorphisms might also contribute to the outcome of allogeneic transplantation.151 A significantly lower risk for early severe pulmonary complications after allogeneic HSCT has been observed in patients receiving a T-celldepleted graft as compared to that in patients receiving cyclosporine/methotrexate as GVHD prophylaxis.197 The early identification of patients at risk for MTCs is paramount for early intervention strategies to prevent or ameliorate these complications and their associated high mortality rates. An increase in serum levels of C-reactive protein during the first days post-transplantation as a marker of systemic inflammation198 or in levels of serum cholinesterase199 as an early and sensitive marker of GVHD and transplant-related mortality have been reported as independent risk factors for the occurrence of MTC and transplant-related mortality. Early intervention strategies may include the use of high-dose steroids,200 defibrotide for severe VOD,201 recombinant human activated protein C for severe sepsis,202 or more specific reagents such as monoclonal antibodies.203 Noninfectious pulmonary complications Considering the long life expectancy of children who might be cured of their leukemia after allogeneic transplantation, late pulmonary sequelae assume increased importance as post-transplant complications. There is a high prevalence of children with impaired lung function post-transplantation.204,205 The main pulmonary complications include bronchiolitis obliterans (BO), bronchiolitis obliterans-organizing pneumonia (BOOP) and idiopathic pneumonia syndrome (IPS).206 Respiratory symptoms of BO include dry cough, dyspnea and wheezing.207 In Fig. 23.9a, a chest CT scan of a patient with suspicion of BOOP after allogeneic transplantation shows multiple nodules of variable sizes. In the corresponding biopsy sample, the terminal airways are filled with a loose, watery immature connective tissue that spills out into some adjacent alveoli (Fig. 23.9b). This process is often localized into

Fig. 23.9 (a) CT scan of the chest in a patient with bronchiolitis obliterans-organizing pneumonia (BOOP) 3 months after matched-sibling HSCT. Histologic study shows terminal airways filled with loose, watery, immature connective tissue that spills out into some adjacent alveoli. (b) The process is often localized into poorly defined nodules (Masson trichrome stain, ×350).

poorly defined nodules. Unlike the findings in BOOP, fever is absent in BO. Chronic GVHD, use of methotrexate, and serum immunoglobulin deficiency have been recognized as risk factors for the development of BO.197,208,209 Late onset noninfectious pulmonary complications after HSCT occurring beyond 3 months post-HSCT have been reported to be significantly associated with chronic GVHD. Interestingly, the relapse rate of primary malignant disease was lower in the patients who developed late pulmonary complications, suggesting a GVL effect of the chronic GVHD involving the lung.210 The association of pulmonary complications with chronic GVHD suggests that bronchiolar epithelial cells

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are targets for alloreactive donor T lymphocytes.211 There are no prospective clinical trials addressing the management of BO, although anecdotal reports claiming efficacy for the use of steroids and immunosuppressive drugs are available. In contrast, patients with BOOP usually respond to corticosteroid therapy, although the optimal dose and duration of therapy has not established.212 There is no established treatment for patients with IPS, although a few patients may respond to corticosteroids.205 Other studies have not shown a benefit for steroid treatment.213 More recently, a TNF-
-binding protein (Etanercept) has been successfully used in patients with IPS after allogeneic HSCT214 but further clinical studies are needed to establish efficacy. Regular testing of pulmonary function is recommended,215 since 30% of the pediatric transplant survivors who have been studied retrospectively had subclinical abnormalities in the absence of chronic respiratory symptoms, and patients beyond one remission, recipients of an allograft, or patients with a history of pulmonary infections were at higher risk of developing late lung injuries.204 In the case of abnormal lung function, long-term followup and diagnostic procedures, including bronchoscopy and/or biopsy if clinically indicated, are mandatory. These patients should refrain from smoking (as, of course, should everybody) and should have early and aggressive treatment of any pulmonary illness. Infectious complications and opportunistic pathogens Recipients of HSCT experience certain infections at different times post-transplantation, which reflect their defective host-defense mechanisms. The severity of the impairment of the host is defense against infection varies with the type of transplant (autologous > unmanipulated matched donor > mismatched donor > T-cell-depleted transplant). Globally, the reconstitution of host-defense mechanisms can be divided in three phases that dictate the design of prevention strategies. In the pre-engraftment phase I (<30 days post-HSCT), patients have a breakdown of the mucocutaneous barrier due to effects of the preparative regimen, leading to neutropenia and therefore the outbreak of infectious organisms in oral, gastrointestinal and skin flora, including Candida species and other fungi. Therefore, most centers use prophylactic measures to prevent fungal infections, primarily candidiasis, in neutropenic patients. With prolonged neutropenia, patients are at risk for infection with Aspergillus species, while herpes simplex virus (HSV) may become reactivated during this phase. To decrease the duration of neutropenia, growth factors such as G-CSF or GM-CSF may be used,216 although a negative impact of

G-CSF post-transplantation on immune reconstitution in haploidentical transplants has been reported.217 The post-engraftment phase II (30–100 days after HSCT) is characterized by an impaired cell-mediated immunity for HSCT recipients, and reactivation of cytomegalovirus (CMV) is commonly seen during this phase.218 CMVseronegative patients should receive a graft from a CMV-seronegative donor, if possible.219 If a seropositive donor must be used, the risk of transmission has been reported to range from 20% to 40%.220,221 Patients who are seropositive before transplantation have an estimated 75% risk of reactivation, and without pre-emptive measures, an estimated 20% to 30% risk of developing CMV disease. Pre-emptive therapy based on early detection of CMV has become the most commonly used strategy to prevent CMV disease.222 Adenoviral infections occur in 5% to 21% of patients who undergo HSCT, with an associated mortality rate of up to 50%.223,224 Frequent monitoring for adenovirus and early pre-emptive treatment with cidofovir may be helpful in reducing the high mortality rate associated with this infectious complication after allogeneic HSCT.225,226 Other dominant pathogens during this phase are Pneumocystis carinii and Aspergillus species. While prophylaxis for P. carinii with trimethoprim-sulfamethoxazole or, alternatively, pentamidine is standard practice, the role of prophylaxis for aspergillosis is still unclear. In HSCT recipients, 80% to 90% of invasive fungal infections are caused by Aspergillus species.227 Risk factors for developing noncandida fungal infections are steroid prophylaxis, GVHD, delayed engraftment or rejection.228 Prophylactic antifungal strategies might be adapted to the clinical status of the patient and the specific incidences of fungal infections at the transplant center. During late phase III (>100 days after HSCT), the immune reconstitution of patients under going autologous HSCT is rapid and these patients are at lower risk for infection. Recipients of allogeneic grafts still have an impaired immune function, especially after matched unrelated, cord blood or mismatched-family-related transplants, with or without T-cell depletion. In addition, patients with chronic GVHD have humoral and cellular immune defects and are at risk for infections, due to CMV, varicella zoster virus, Epstein–Barr virus, and community-acquired respiratory viruses, among others.218 Infections with encapsulated bacteria, such as H. influenza and Str. Pneumoniae, or with Aspergillus species can also be encountered. Strategies for the early diagnosis of infections or the introduction of prophylactic therapies may need to be extended, depending on the form of transplantation and the clinical status of each patient.

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Epstein–Barr virus-associated lymphoproliferative syndrome (EBV-LPS) EBV-LPS is a serious complication after allogeneic HSCT.229 The incidence is generally less than 2%, but may increase to 20% in patients with established risk factors, such as unrelated HSCT, use of T-cell-depleted grafts, use of antithymocyte globulin (ATG) and immunosuppression to prevent or treat GVHD.230–232 EBV-LPS is associated with a high mortality rate despite the use of anti-B-lymphocyte antibodies233 or the adoptive transfer of EBV-specific Tlymphocytes.234 The most effective way to prevent EBV-LPS is to remove all mature B lymphocytes from the graft, either by negative depletion235 or by positive selection of CD34+ progenitor cells.33,36 As in other viral infections associated with HSCT, early and quantitative detection of EBV-DNA in plasma using real-time polymerase chain reaction (PCR) analysis and pre-emptive therapy with anti-CD20 antibodies might decrease the high mortality rate otherwise associated with this complication.236,237

Immune reconstitution after transplantation Recipients of an allogeneic transplant demonstrate varying periods of immunoincompetence that can last for several years and cause significant morbidity and mortality.238 Immune reconstitution can further be hampered by the occurrence of acute and chronic GVHD. While the innate immune system usually recovers within weeks after transplantation, the impaired and delayed T-cell-mediated immunity characterized by a low CD4+ helper T-cell count is mainly responsible for the high susceptibility to infections.239 After eradication of the recipient’s T-cell compartment by the preparative regimen, it is reconstituted through thymus-independent and thymus-dependent mechanisms.240,241 A thymus-independent reconstitution involves the expansion of mature graft-containing T cells, and a limited diversity of the T-cell receptor (TCR) repertoire is often seen in this thymus-independent pathway.242 A broad and normal TCR repertoire is generated in the thymus and reflects the ability of T lymphocytes to respond to most known pathogens.243 A high number of mature graft-containing T cells can confer a skewed but to a certain extent protective repertoire, although it also increases the risk of GVHD.244 In recipients of a T-cell-depleted graft, the number of cotransplanted mature T lymphocytes is very low in order to prevent GVHD. The peripheral expansion of these few graftcontaining T cells only results in a ‘memory’ phenotype and generates a very limited repertoire,245 which is associated with high susceptibility to infections in the early and

late post-transplant period. The final reconstitution of a T-cell repertoire in all recipients of an unmaniplated or T-depleted allograft most likely depends on the repopulation of the thymus by transplanted hematopoietic precursor cells and the generation of “na¨ıve” T lymphocytes with a broad repertoire.246 The recovery of thymic function can be roughly measured by phenotypic analysis of na¨ıve lymphocyte subsets, such as CD4/CD45RA,245 determination of the diversity of the TCR repertoire by TCR V-beta spectratyping247 or by thymic output through quantification of TCR excision circles (TRECs).248 Resumption of thymic function can be seen earlier after MSD transplants, but may be further delayed in recipients of a T-cell depleted graft.249 Additional factors influencing immune reconstitution are GVHD and cytomegalovirus infection.250,251 Thymic function can also be impaired by the intensity of the preceding chemotherapy.252 Hence, the time to complete restoration of the immune function can differ from patient to patient and these individual differences should be taken into consideration in the post-transplant care of patients in terms of the extent and length of antibacterial, antifungal or antiviral prophylaxis. An association between the number of transplanted purified CD34+ stem cells and the speed of T-cell reconstitution has been observed in three-loci haploidentical transplantation.6 While it is unclear whether this is due to an increased number of transplanted thymusrepopulating stem cells or the lack of (thymic) GVHD, these observations can lead to new strategies to hasten immune reconstitution without increasing the risk of GVHD. Ablation of the host’s system and reconstitution with na¨ıve graft-derived lymphocytes requires a revaccination of the patient. This should only be done after adequate recovery of immune function.

Development, growth and infertility The use of total-body irradiation, high-dose myeloablative chemotherapies or the development of chronic GVHD can cause a variety of nonmalignant late effects on development, growth and fertility.253 While an early study reported a decline in global IQ from baseline in the first year after BMT in children,254 subsequent prospective studies have found either that cognitive and psychosocial functioning is stable from the pretransplant period through 2 years post-transplantation or that some risk of cognitive decline is present, although it occurs primarily in patients younger than 3 years of age.255,256 Growth hormone deficiency after HSCT can also be observed.257 A decrease in height-standard deviation score is most consistently associated with total-body irradiation (TBI), young

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age at transplantation and male gender.258,259 One recent large study found that of 181 pediatric patients undergoing HSCT, 141 reached adult height within the normal range of the general population.258 Due to the improved survival following HSCT, it is important to counsel patients at the appropriate age regarding their later fertility status; knowledge of the impact of different preparative regimens is helpful in predicting the degree of infertility. In a retrospective study, recovery of spermatogenesis was observed in 50% of patients whose conditioning regimen contained cyclophosphamide and busulfan but in only 17% of patients who received cyclophosphamide and TBI.260 Considering the high incidence of azoospermia, semen banking should be offered to all male patients before HSCT, even when their spermatogenesis is already impaired as a consequence of previous cytotoxic therapies.261 Gonadal recovery in women after HSCT depends on age and menarchal status at the time of HSCT.262 Advances in assisted reproduction techniques offer the possibility of cryopreserving gonadal tissue as a means of conserving fertility.263,264

New malignancies With the increase in the number of long-term survivors after HSCT, the development of post-transplant malignancies (solid tumors; hematologic malignancies, primarily t-MDS/AML; and post-transplant lymphoproliferative disorders) is becoming more important.265 Available data on children younger than 10 years of age show a 36.6 times higher-than-expected risk of developing a new malignant disease after HSCT.266 In another study limited to children with acute leukemia undergoing HSCT, the risk was 45 times higher and was inversely correlated with the age of the patients at transplantation.267

Future directions and conclusions Current attempts to improve the outcome of HSCT are organized to achieve several major objectives. First, the refinement and extension of minimal residual diseasebased strategies to patients with diseases other than ALL could lead to earlier identification of patients who are at risk for subsequent relapse and who might benefit from early HSCT. Second, detailed analysis and better understanding of factors determining post-transplant immune reconstitution should lead to better protection of patients from lethal infections in the early and late post-transplant periods. Such strategies might include the adoptive trans-

fer of nonalloreactive T lymphocytes,268 antigen-specific donor T lymphocytes269 or the clinical application of cytokines to improve either peripheral expansion of T cells or thymic function.270 The third and most important goal is to better understand and harness the GVL effect in the absence of GVHD for patients at high risk of relapse. New approaches include the generation and adoptive transfer of antileukemic donor T cells271,272 or the deliberate use of HLA-mismatched donors with an alloreactive NK cells constellation. Increasing the GVL without increasing the risk of GVHD would also allow reduction of the myeloablative preparative regimens without increasing the risk of relapse, thus reducing the major transplant-related complications and the long-term side effects of the preparative regimen on development, growth and fertility. Since every transplant method has advantages and disadvantages, careful analysis of the risk–benefit ratio for each patient, to include the disease itself, the patient’s clinical status and the experience of the transplant center with each transplant method, should be routinely performed to define the best available strategy of HSCT.

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209 Clark, J. G., Schwartz, D. A., Flournoy, N., et al. Risk factors for airflow obstruction in recipients of bone marrow transplants. Ann Intern Med, 1987; 107: 648–56. 210 Sakaida, E., Nakaseko, C., Harima, A., et al. Late-onset noninfectious pulmonary complications after allogeneic stem cell transplantation are significantly associated with chronic graftversus host disease and with the graft-versus-leukemia effect. Blood, 2003; 102: 4236–42. 211 Crawford, S. W. & Clark, J. G. Bronchiolitis associated with bone marrow transplantation. Clin Chest Med, 1993; 14: 741–9. 212 Cordier, J. F. Organising pneumonia. Thorax, 2000; 55: 318–28. 213 Kantrow, S. P., Hackman, R. C., Boeckh, M., et al. Idiopathic pneumonia syndrome: changing spectrum of lung injury after marrow transplantation. Transplantation, 1997; 63: 1079–86. 214 Yanik, G., Hellerstedt, B., Custer, J., et al. Etanercept (Enbrel) administration for idiopathic pneumonia syndrome after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant, 2002; 8: 395–400. 215 Nysom, K., Holm, K., Hesse, B., et al. Lung function after allogeneic bone marrow transplantation for leukaemia or lymphoma. Arch Dis Child, 1996; 74: 432–6. 216 Vose, J. M. & Armitage, J. O. Clinical applications of hematopoietic growth factors. J Clin Oncol, 1995; 13: 1023–35. 217 Volpi, I., Perruccio, K., Tosti, A., et al. Postgrafting administration of granulocyte colony-stimulating factor impairs functional immune recovery in recipients of human leukocyte antigen haplotype-mismatched hematopoietic transplants. Blood, 2001; 97: 2514–21. 218 Ljungman, P. Prevention and treatment of viral infections in stem cell transplant recipients. Br J Haematol, 2002; 118: 44–57. 219 Bowden, R. A., Slichter, S. J., Sayers, M. H., et al. Use of leukocyte-depleted platelets and cytomegalovirusseronegative red blood cells for prevention of primary cytomegalovirus infection after marrow transplant. Blood, 1991; 78: 246–50. 220 Miller, W., Flynn, P., McCullough, J., et al. Cytomegalovirus infection after bone marrow transplantation: an association with acute graft-v-host disease. Blood, 1986; 67: 1162–7. 221 Ruutu, T., Ljungman, P., Brinch, L., et al. No prevention of cytomegalovirus infection by anti-cytomegalovirus hyperimmune globulin in seronegative bone marrow transplant recipients. The Nordic BMT Group. Bone Marrow Transplant, 1997; 19: 233–6. 222 Avery, R. K., Adal, K. A., Longworth, D. L., et al. A survey of allogeneic bone marrow transplant programs in the United States regarding cytomegalovirus prophylaxis and pre-emptive therapy. Bone Marrow Transplant, 2000; 26: 763–7. 223 Hale, G. A., Heslop, H. E., Krance, R. A., et al. Adenovirus infection after pediatric bone marrow transplantation. Bone Marrow Transplant, 1999; 23: 277–82. 224 Chakrabarti, S., Mautner, V., Osman, H., et al. Adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery. Blood, 2002; 100: 1619–27.

225 Legrand, F., Berrebi, D., Houhou, N., et al. Early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children. Bone Marrow Transplant, 2001; 27: 621–6. 226 Ljungman, P., Deliliers, G. L., Platzbecker, U., et al. Cidofovir for cytomegalovirus infection and disease in allogeneic stem cell transplant recipients. The Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Blood, 2001; 97: 388–92. 227 Morrison, V. A., Haake, R. J., & Weisdorf, D. J. The spectrum of non-Candida fungal infections following bone marrow transplantation. Medicine, 1993; 72: 78–89. 228 Martino, R., Subira, M., Rovira, M., et al. Invasive fungal infections after allogeneic peripheral blood stem cell transplantation: incidence and risk factors in 395 patients. Br J Haematol, 2002; 116: 475–82. 229 Shapiro, R. S., McClain, K., Frizzera, G., et al. Epstein–Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation. Blood, 1988; 71: 1234–43. 230 Gross, T. G., Steinbuch, M., DeFor, T., et al. B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: risk factors, treatment and outcome. Bone Marrow Transplant, 1999; 23: 251–8. 231 Curtis, R. E., Travis, L. B., Rowlings, P. A., et al. Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study. Blood, 1999; 94: 2208–16. 232 Hale, G. & Waldmann, H. Risks of developing Epstein– Barr virus-related lymphoproliferative disorders after T-celldepleted marrow transplants. CAMPATH Users. Blood, 1998; 91: 3079–83. 233 Kuehnle, I., Huls, M. H., Liu, Z., et al. CD20 monoclonal antibody (rituximab) for therapy of Epstein–Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood, 2000; 95: 1502–5. 234 Heslop, H. E., Ng, C. Y., Li, C., et al. Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat Med, 1996; 2: 551–5. 235 Cavazzana-Calvo, M., Bensoussan, D., Jabado, N., et al. Prevention of EBV-induced B-lymphoproliferative disorder by ex vivo marrow B-cell depletion in HLA-phenoidentical or non-identical T-depleted bone marrow transplantation. Br J Haematol, 1998; 103: 543–51. 236 Esser, J. W. van, Holt, B. van der, Meijer, E., et al. Epstein– Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-celldepleted SCT. Blood, 2001; 98: 972–8. 237 Wagner, H. J., Rooney, C. M., & Heslop, H. E. Diagnosis and treatment of posttransplantation lymphoproliferative disease after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant, 2002; 8: 1–8. 238 Parkman, R. & Weinberg, K. I. Immunological reconstitution following bone marrow transplantation. Immunol Rev, 1997; 157: 73–8.

Hematopoietic stem cell transplantation

239 Storek, J., Gooley, T., Witherspoon, R. P., et al. Infectious morbidity in long-term survivors of allogeneic marrow transplantation is associated with low CD4 T cell counts. Am J Hematol, 1997; 54: 131–8. 240 Mackall, C. W., Granger, L., Sheard, M. A., et al. T-cell regeneration after bone marrow transplantation: differential CD45 isoform expression on thymic-derived versus thymicindependent progeny. Blood, 1993; 82: 2585–94. 241 Dumont-Girard, F., Roux, E., Lier, R. A. van, et al. Reconstitution of the T-cell compartment after bone marrow transplantation: restoration of the repertoire by thymic emigrants. Blood, 1998; 92: 4464–71. 242 Roux, E., Helg, C., Dumont-Girard, F., et al. Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts. Blood, 1996; 87: 3984–92. 243 Boehmer, H. von. Thymic selection: a matter of life and death. Immunol Today, 1992; 13: 454–8. 244 Kernan, N. A., Collins, N. H., Juliano, L., et al. Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease. Blood, 1986; 68: 770–3. 245 Roux, E., Dumont-Girard, F., Starobinski, M., et al. Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity. Blood, 2000; 96: 2299– 303. 246 Eyrich, M., Croner, T., Leiler, C., et al. Distinct contributions of CD4(+) and CD8(+) naive and memory T-cell subsets to overall T-cell-receptor repertoire complexity following transplantation of T-cell-depleted CD34-selected hematopoietic progenitor cells from unrelated donors. Blood, 2002; 100: 1915–18. 247 Eyrich, M., Lang, P., Lal, S., et al. A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen-disparate haematopoietic stem cells from parental donors. Br J Haematol, 2001; 114: 422–32. 248 Chen, X., Barfield, R., Benaim, E., et al. Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients. Blood, 2005; 105: 886–93. 249 Godthelp, B. C., Tol, M. J. van, Vossen, J. M., et al. T-cell immune reconstitution in pediatric leukemia patients after allogeneic bone marrow transplantation with T-cell-depleted or unmanipulated grafts: evaluation of overall and antigen-specific T-cell repertoires. Blood, 1999; 94: 4358–69. 250 Kook, H., Goldman, F., Giller, R., et al. Reconstruction of the immune system after unrelated or partially matched T-celldepleted bone marrow transplantation in children: functional analyses of lymphocytes and correlation with immunophenotypic recovery following transplantation. Clin Diagn Lab Immunol, 1997; 4: 96–103. 251 Weinberg, K., Blazar, B. R., Wagner, J. E., et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood, 2001; 97: 1458–66.

252 Mackall, C. L. T-cell immunodeficiency following cytotoxic antineoplastic therapy: a review. Stem Cells, 2000; 18: 10–18. 253 Socie, G., Salooja, N., Cohen, A., et al. Non-malignant late effects after allogeneic stem cell transplantation. Blood, 2003; 101: 3373–85. 254 Kramer, J. H., Crittenden, M. R., Halberg, F. E., et al. A prospective study of cognitive functioning following low-dose cranial radiation for bone marrow transplantation. Pediatrics, 1992; 90: 447–50. 255 Thuret, I., Michel, G., Carla, H., et al. Long-term side-effects in children receiving allogeneic bone marrow transplantation in first complete remission of acute leukaemia. Bone Marrow Transplant, 1995; 15: 337–41. 256 Phipps, S., Dunavant, M., Srivastava, D. K., et al. Cognitive and academic functioning in survivors of pediatric bone marrow transplantation. J Clin Oncol, 2000; 18: 1004–11. 257 Shinagawa, T., Tomita, Y., Ishiguro, H., et al. Final height and growth hormone secretion after bone marrow transplantation in children. Endocr J, 2001; 48: 133–8. 258 Cohen, A., Rovelli, A., Bakker, B., et al. Final height of patients who underwent bone marrow transplantation for hematological disorders during childhood: a study by the Working Party for Late Effects-EBMT. Blood, 1999; 93: 4109–15. 259 Bakker, B., Massa, G. G., Oostdijk, W., et al. Pubertal development and growth after total-body irradiation and bone marrow transplantation for haematological malignancies. Eur J Pediatr, 2000; 159: 31–7. 260 Anserini, P., Chiodi, S., Spinelli, S., et al. Semen analysis following allogeneic bone marrow transplantation. Additional data for evidence-based counselling. Bone Marrow Transplant, 2002; 30: 447–51. 261 Lass, A., Akagbosu, F., & Brinsden, P. Sperm banking and assisted reproduction treatment for couples following cancer treatment of the male partner. Hum Reprod Update, 2001; 7: 370–7. 262 Spinelli, S., Chiodi, S., Bacigalupo, A., et al. Ovarian recovery after total body irradiation and allogeneic bone marrow transplantation: long-term follow up of 79 females. Bone Marrow Transplant, 1994; 14: 373–80. 263 Grundy, R., Gosden, R. G., Hewitt, M., et al. Fertility preservation for children treated for cancer (1): scientific advances and research dilemmas. Arch Dis Child, 2001; 84: 355–9. 264 Kim, S. S., Battaglia, D. E., & Soules, M. R. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril, 2001; 75: 1049–56. 265 Baker, K. S., DeFor, T. E., Burns, L. J., et al. New malignancies after blood or marrow stem-cell transplantation in children and adults: incidence and risk factors. J Clin Oncol, 2003; 21: 1352–8. 266 Curtis, R. E., Rowlings, P. A., Deeg, H. J., et al. Solid cancers after bone marrow transplantation. N Engl J Med, 1997; 336: 897–904. 267 Socie, G., Curtis, R. E., Deeg, H. J., et al. New malignant diseases after allogeneic marrow transplantation for childhood acute leukemia. J Clin Oncol, 2000; 18: 348–57.

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268 Andre-Schmutz, I., Le Deist, F., Hacein-Bey-Abina, S., et al. Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: a phase 1/2 study. Lancet, 2002; 360: 130–7. 269 Marijt, W. A., Heemskerk, M. H., Kloosterboer, F. M., et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci U S A, 2003; 100: 2742–7.

270 Mackall, C. L. Enhancing immune reconstitution after stem cell transplants with cytokines. Cytotherapy, 2002; 4: 427–8. 271 Molldrem, J. J., Komanduri, K., & Wieder, E. Overexpressed differentiation antigens as targets of graft-versus-leukemia reactions. Curr Opin Hematol, 2002; 9: 503–8. 272 Falkenburg, J. H., Marijt, W. A., Heemskerk, M. H., et al. Minor histocompatibility antigens as targets of graft-versusleukemia reactions. Curr Opin Hematol, 2002; 9: 497– 502.

24 Acute leukemia in countries with limited resources Raul C. Ribeiro, Scott C. Howard, and Ching-Hon Pui

Introduction Complex interactions between genetic and environmental factors underlie the etiologies of pediatric leukemias and lymphomas. Thus, it is not surprising that the incidence rates for these disorders vary widely across geographic regions (Table 24.1). In contrast to the wealth of etiologic and epidemiologic data on pediatric cancers in the United States,1–5 comparable information is not available for most countries (which lack population-based registries and adequate diagnostic methods); for that reason, only marked discrepancies in cancer incidence can be noted among regions. For example, non-Hodgkin lymphoma (NHL) of the mature B-cell immunophenotype (Burkitt lymphoma) is the most common malignancy in many African countries.6 Environmental factors, including infectious agents such as Epstein–Barr virus and malaria (highly prevalent in these regions), appear to contribute to the etiology of this disease.7 The high relative incidence of Burkitt lymphoma in these regions is even more remarkable in view of the generally lower incidence of acute lymphoblastic leukemia (ALL) in blacks compared to whites.1 Other examples include an increased incidence of Kaposi sarcoma in children in regions with a high rate of endemic HIV infection,8 of acute promyelocytic leukemia in people of Italian or Hispanic heritage,9 of acute myeloid leukemia (AML) presenting with chloroma in Turkey,10 and of T-cell ALL in children from India and Egypt.11,12 Given these empiric observations on a limited number of countries, it is not surprising that the distribution of pediatric cancers shows substantial variation across the world’s regions.13 This variability is greater among malignant diseases of the immune system because of the rapid proliferation of lymphoid cells during childhood and the con-

stant environmental pressure on this system. Importantly, lymphoid malignancies are expected to become the most common types of pediatric cancer in any developing country with limited resources; however, until these regions have population-based registries and adequate diagnoses and reporting systems, we can gain only a glimpse of the true epidemiology of the world’s childhood leukemias and lymphomas. This chapter seeks to portray the conditions affecting attitudes toward pediatric cancer in several developing countries and the progress that has been made in establishing modern approaches to diagnosis and treatment. (See Chapter 3 for additional information on the epidemiology and etiology of acute leukemias in children.)

Social burden of malignant neoplasms in children and adolescents In developing countries, the leading causes of death among children differ substantially from those in developed nations.14,15 Infectious diseases, including tuberculosis, malaria, measles, and AIDS, are usually ranked first, followed by pneumonia, diarrhea, and malnutrition (the diseases of poverty).15 Cancer is not a conspicuous cause of death in these countries and often is omitted from lists of national health priorities. Even if effective pediatric cancer programs were available, the overall rate of childhood mortality in these countries would not be perceptibly improved. Understandably, the first health priority of developing countries and of international agencies must be to eliminate the diseases of poverty. However, as progress is made toward reducing preventable illness and improving overall

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 24.1 Annual age-standardized incidence rates of leukemia and

idiosyncratic views of parents, legal guardians, or influential community groups, a viewpoint not shared by the vast majority of emerging or underdeveloped countries.

lymphoma (per million population) in selected countriesa Leukemia

Lymphoma

Acute Acute Nonlymphoid myeloid Other Hodgkin Other

Region Africa Egypt Uganda Zimbabwe Central and South America Brazil (state of Goiˆania) Costa Rica Cuba North America Canada United States (SEERb ) Asia India (Delhi) Japan China (Tianjin province) Europe Spain Germany United Kingdom Oceania Australia New Zealand (Maori)

6.2 3.3 11.6

0.5 1.7 11.0

13.3 5.6 0.5

15.5 46.8 6.9

15.3 6.1 5.6

21.9 46.3 25.9

5.3 8.9 5.7

6.5 2.7 6.7

4.9 7.0 8.7

18.7 14.9 13.5

41.0 34.0

6.3 7.0

3.6 2.4

7.0 8.5

7.7 5.5

23.1 22.6 17.4

6.1 7.2 6.7

6.8 8.8 16.1

6.4 5.0 5.6

8.2 5.6 6.7

32.5 39.0 32.8

8.1 6.7 6.3

4.6 0.8 1.7

13.3 9.9 6.2

6.2 6.5 5.0

39.9 21.9

8.0 14.4

1.9 5.1

7.9 9.1

5.4 7.4

a

Except for data from the United States, all rates were obtained from Parkin et al.13 b SEER: Surveillance, Epidemiology and End Results, 1990–5.1,2

public health, pediatric cancer becomes a more important cause of mortality. This relationship is most evident in countries that have made significant progress toward eliminating the diseases of poverty but have yet to develop a comprehensive strategy to deal with pediatric cancer. In those countries, children usually do not have access to adequate cancer treatment because the public investment in health is small, health insurance is not available to most families, and the cost of even a standard course of therapy is beyond most people’s means. The net result is an unacceptably low overall survival rate for children with cancer in many developing countries. In developed countries, by contrast, public policies recognize the right of children with cancer to have access to adequate treatment.16 If parents or legal guardians should refuse treatment of a child’s potentially curable malignancy, the child’s right to treatment can be enforced by law. This mechanism reflects a broad societal consensus that children should be protected from the

Patterns of socioeconomic, cultural, and political development Nations broadly categorized as developing, emerging or poor show substantial diversity in their human and natural resources and their socioeconomic, political, religious, and cultural aspects. These complex, interrelated factors make each country unique. Socioeconomic and health indicators, including the gross national product per capita and rates of longevity, fertility, infant mortality, and other variables used to rank these different nations, clearly do not present a complete picture.17 The probability (per 1000 live births) that a child will die before age 5 years, termed the under-five mortality rate, or U5MR, has been used by the United Nations International Children’s Emergency Fund (UNICEF) to reflect the status of children’s health in a given nation.18 Because the U5MR figures are based on several socioeconomic and health indicators – including per capita caloric availability, immunization, level of maternal and child health services, access to clean water, sanitation, and others – this parameter is considered to be a good indicator of child health. There is a very close relationship between the designation “developed country” and the U5MR. All of the industrialized or developed countries (36 of 193) have a U5MR of less than 9. With a U5MR of 8, the United States ranks 158th among 193 nations. Sierra Leone occupies the lowest rank, with a U5MR of 284, and Sweden ranks highest with a rate of only 3.18 The rate of reduction of the U5MR over a given period of time provides a measure of the progress of a country or region toward addressing its most important health priorities. The U5MR has been dramatically reduced in countries that invest resources in improved sanitation, access to clean water, immunization, and widespread use of oral rehydration to treat diarrheal illnesses. Thus although the U5MR is reduced most by diminishing the diseases of poverty, it is not an adequate measure of a country’s or region’s progress toward the treatment of pediatric cancer. Because mortality caused by pediatric neoplasms represents a negligible fraction of the overall U5MR, improved child health parameters in developing countries do not necessarily reflect improved survival of children with cancer. For example, between 1960 and 2002, the U5MR of Chinese children decreased from 225 to 39 per 1000 live births, but the overall survival of Chinese children with cancer during this period remained

Acute leukemia in countries with limited resources

50

El Salvador, GDP $4701

Brazil, GDP $7537

US, GDP $35,182

Disease-related death in children (%)

45 40 35 30 25 20 15 10 5 0 Cancer

Infection

Malnutrition Respiratory* Congenital

© 2004, St. Jude Children's Research Hospital

Other

Data source: www.who.int

Fig. 24.1 Cause-specific disease-related mortality rates in children 1 to 14 years old in three countries with different levels of economic prosperity. These data, provided by the World Health Organization (WHO), illustrate that as economic prosperity increases, public health improves. As infection, respiratory illness, and malnutrition cause fewer deaths, cancer increases in prominence as a cause of mortality. In El Salvador, which has the lowest per capita gross domestic product (GDP) of the countries shown, cancer is responsible for only 7% of disease-related mortality in children. In the United States, which has the highest per capita GDP, cancer causes 27% of the disease-related childhood deaths. These data exclude violence and accidents (the most common cause of childhood death in the United States) and deaths of unknown cause. Deaths attributed to respiratory illness (asterisk in figure) by the WHO include both infectious and noninfectious diseases of the respiratory system. Used with permission of St. Jude Children’s Research Hospital, Memphis, TN, USA (www.cure4kids.org/ums/home/library).

at or below 15%. Only when nations make substantial progress toward reducing the U5MR does pediatric cancer become a noticeable cause of childhood mortality (Fig. 24.1). In many nations, particularly those in which wealth is unequally distributed and resources are concentrated in certain regions, the survival of children with cancer can vary dramatically within a single geopolitical area. Typically, there are excellent medical centers in affluent areas that serve those who can afford to pay or who have health insurance. These medical centers usually report overall survival rates of 60% to 70% for pediatric cancer patients who complete treatment. However, patients who are lost to follow-up are usually censored from the analysis at the time of their last visit, and many children may not even begin treatment due to the prohibitively high cost. Further, children from low-income families, which usually reside in rural areas distant from the affluent regions, are more likely to abandon treatment.19 Even if these patients’ families are contacted after an appointment is missed, it is rarely possible to force them to return for treatment; there

is no infrastructure to accommodate patients and their families, and no laws protect pediatric patients. Therefore, the survival rates reported by single institutions are often inflated and do not reflect the state of the country’s pediatric cancer care. Moreover, in many of these countries, medical centers compete for affluent patients by offering luxury facilities and advanced medical procedures, such as bone marrow transplantation. In some cases, through political or financial influence, public funds are used to support these sophisticated medical centers rather than to improve care in underserved areas. This health care gap, together with competition between physicians who serve the private versus the public system, inhibits cooperation, hampers the establishment of effective collaborative treatment protocols, and discourages advocacy for children with cancer. Progress in the management of childhood cancer in these countries must be measured not only by the survival rates in single centers but also by the proportion of children and adolescents with cancer (population-based registries) who have access to treatment and who start and complete treatment.

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Requirements for the establishment of pediatric cancer units in developing countries Strategies used to fight pediatric cancer are very different from those used to treat the diseases of poverty. Effective cancer treatment is complex and requires a multidisciplinary team, dedicated hospital facilities, clinical laboratories, blood banking, anticancer medications and antibiotics, outpatient infrastructure for close patient monitoring, community participation, and government support. In the United States, children with cancer are managed in pediatric cancer centers staffed by pediatric hematologists/oncologists, pediatric surgeons, urologists, pathologists, orthopedic surgeons, and radiation oncologists. These physicians and other health professionals, including nurse practitioners, pediatric nurses, social workers, pharmacists, nutritionists, child life specialists, and others, comprise a multidisciplinary team necessary for the optimal care of children and adolescents with cancer.20 The creation of a pediatric cancer unit is the first step toward improving the cure rate of children with cancer in developing countries.21,22 However, the creation of a pediatric cancer unit requires more than a dedicated physical location in a pediatric ward; it also requires the broad engagement of hospital and public leaders in the establishment of a national pediatric cancer program. Inherent in this concept is the access of all children with cancer to treatment and the equitable use of public resources. Such a proposition may not be feasible at present in many countries but should be a goal. The establishment of a pediatric cancer center also requires the commitment of hospital leadership to provide a specific location and to allow medical staff to dedicate their full attention to the care of children with malignancy. Trained pediatric hematologists/oncologists and nurses assume leadership roles to organize multidisciplinary teams that develop modern management guidelines. Alliances with intensive care and infectious disease specialists, surgeons, and radiotherapists improve the quality of care and the survival rates. Patients, parents, and relatives should participate in management decisions and understand the benefits and risks involved in the treatment. Parental education is crucial to increase adherence to treatment and reduce abandonment of therapy – a barrier to effective pediatric oncology that is unique to developing countries.23 When resources are fully utilized and experience has been accumulated, the pediatric cancer unit can be further expanded through alliances with physicians at other institutions, community support groups, government, and international funding agencies.

The main goal is to cure an increasing number of children with cancer. We recently described the development of a pediatric cancer unit in Recife, Brazil.24 The event-free survival (EFS) estimates for 375 children with ALL were used as surrogate markers of the improved effectiveness of treatment. Eightythree children had a diagnosis of ALL during an early era (1980–9) when there was no dedicated pediatric oncology unit, protocol-based therapy, specially trained nurses, 24hour on-site physician coverage, or rapid access to intensive care. During the next era (1994–7), 78 children were treated (all on protocols). During the most recent era (1997– 2002), 214 children were diagnosed and treated on protocol in a dedicated pediatric oncology unit staffed 24 hours a day by pediatric oncologists and oncology nurses. The 5-year EFS estimate and 95% confidence interval improved progressively from 32% (21–43%) in the early period to 63% (55–71%) in the most recent period. Figure 24.2 compares the 5-year EFS rates over the three eras. The cumulative incidence of cause-specific treatment failure within 1 year of diagnosis was reduced from 14.0% to 3.8% to 3.3% for relapse and from 16.0% to 1.3% to 0.5% for abandonment of therapy. The risk of death from infection changed from 6.0% to 12.0% to 10.0%, and death due to non infections from 2.4% to 13.0% to 4.2%. Even as outcomes improved at St. Jude over the past 15 years, the gap in eventfree survival between Recife and St. Jude has narrowed (Fig. 24.3). The toxic effects of therapy remain the single most important cause of the inferior outcome among children with ALL treated in Recife (Fig. 24.4), and will be the focus of future interventions. Table 24.2 details the improvements that were implemented during this period. Only 5% of the unit’s funding comes from international sources (St. Jude). Clearly, even in a poor area, establishment and continuous improvement of a pediatric oncology unit can produce objective improvement in outcomes in only a decade.

Barriers to implementation of pediatric cancer units in developing countries The main barriers to the implementation of effective pediatric programs differ markedly among countries and, often, among regions within a country.25–28 The scarcity of resources is the dominant theme. For example, the annual operating cost of a general pediatric oncology unit that manages approximately 180 to 200 new cases per year is about $3 million in San Salvador, El Salvador (Miguel Bonilla, personal communication, 2004). This cost is beyond the reach of most resource-poor nations. The lack

Acute leukemia in countries with limited resources

629

Fig. 24.2 Event-free survival in children with acute lymphoblastic leukemia in Recife, Brazil, according to treatment era. The 5-year EFS estimate (with 95% Confidence Interval) was 32% (21–43%) in the early era (1980–9), 47% (36–58%) in the middle era (July 1994 to March 1997), and 63% (55%–71%) in the recent era (April 1997 to December 2002). The pediatric oncology service was discontinued during the years not accounted for. Reprinted, with permission, from Howard et al.24

14 12 10 Percent

of economic resources is an even greater barrier in India, where 60 million families live in poverty.25 Other factors in these countries add to the complexity and cost of pediatric care. Diagnosis is often delayed in poor regions, either because parents first seek traditional folk remedies or because community physicians fail to recognize the disease. For these reasons, patients often have advanced-stage disease, huge masses, and severe medical complications at the time of presentation. The lack of financial resources often prevents the completion of treatment. In many countries where the majority of the population is impoverished and uninsured, the government may provide hospital beds but not anticancer medications. Thus, even if the children are brought to hospitals, the parents are usually unable to afford the necessary medications. Further, those who can pay for the initial phase of treatment often cannot afford the expense of continued therapy, which requires travel to the hospital, lodging, food, and unpaid leave from work. The lack of adequate infrastructure in public pediatric hospitals is an important factor in the high mortality rates of children who undergo intensive myelosuppressive and immunosuppressive treatment (Fig. 24.4). Hospital rooms are usually overcrowded, each housing as many as 10 patients and parents. Infection control is lacking, and systematic hand-washing is rare. Fatal

8 Recife St. Jude

6 4 2 0 Abandonment

Toxicity

Relapse

Fig. 24.3 Comparison of the causes of treatment failure within 1 year of diagnosis of childhood acute lymphoblastic leukemia in Recife, Brazil, and at St. Jude Children’s Research Hospital. Used with permission of St. Jude Children’s Research Hospital, Memphis, TN, USA (www.cure4kids.org/ums/home/library).

infectious, hemorrhagic, and metabolic complications are the norm. Well-trained clinicians are scarce and receive little compensation for the care of children with cancer. For that reason, physicians usually must divide their time between caring for cancer patients and working at

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Rationale for implementation of pediatric cancer programs in developing countries

40 35 30 Percent

25 Recife St. Jude

20 15 10 5 0 1980s

Early 1990s

Late 1990s

Fig. 24.4 Comparison of treatment failure within 1 year of diagnosis of childhood acute lymphoblastic leukemia in Recife, Brazil, and at St. Jude Children’s Research Hospital, according to treatment era. Used with permission of St. Jude Children’s Research Hospital, Memphis, TN, USA (www.cure4kids.org/ ums/home/library).

other, more remunerative jobs. Overworked and undertrained nurses are often unable to provide satisfactory care for clinically complex cases. Among their other duties, in many centers the nurses must mix and administer the chemotherapy. Because pain management is ineffective and psychosocial support nonexistent, parents, and often health-care providers, are disheartened by the suffering of patients who so often die of complications. Not surprisingly, such an environment encourages the abandonment of therapy and fosters an attitude of hopelessness among parents and health-care providers. The lack of laboratory facilities and trained personnel necessary for the correct diagnosis of malignancies is yet another factor associated with low cure rates and poor resource utilization. In many cases, only simple, conventional pathology methods are available for the diagnosis of leukemia and solid tumors. Immunohistochemistry, flow cytometry, and cytogenetics and molecular genetics are not available or must be purchased from private laboratories at prohibitive cost. Several other factors contribute to the lack of progress in reducing the pediatric cancer mortality rate in developing countries. Some of these factors are unique to particular countries. In Honduras, for example, the distance between a patient’s home and the tertiary medical center is the single factor most predictive of abandonment of therapy.19 However, the abandonment rate is still high in El Salvador, where the pediatric cancer unit is relatively close to all points in the country. Finally, the irregular availability of anticancer drugs is a barrier to standardized treatment, the lack of which increases toxicity and reduces efficacy.

The increasing recognition that pediatric cancer is usually curable when treated adequately has created a strong ethical and rational basis for efforts to provide treatment to affected children worldwide.29–31 Moreover, the Convention on the Rights of the Child, adopted by United Nations General Assembly, states: “State Parties recognize that the child has the right to the enjoyment of the highest attainable standard of health possible and to have access to health and medical services.” It can be argued that a child with low-risk ALL who does not have access to adequate treatment is suffering from medical neglect.32 However, most developing countries lack a national health policy for pediatric cancer and lack the expertise to develop such a program. Confronted by meager resources and immense health needs, policy makers usually do not consider pediatric cancer to be an important cause of childhood mortality and thus do not include it in their health agendas. Although only 3% to 7% of childhood deaths in these countries are caused by cancer, malignant neoplasms become a very visible cause of death as the diseases of poverty are conquered, and there is increasing public demand for the establishment of pediatric cancer centers. Hence, developing countries should invest resources in the implementation of pediatric oncology as soon as the U5MR drops substantially or is projected to approximate the rates reported in developed countries (Fig. 24.1). This process is under way in the majority of developing countries.

Establishing pediatric cancer units in developing countries Establishment of an effective pediatric cancer program in a developing country is a complex and daunting task.29,33–40 It is very unlikely that the national government alone can support an adequate program, which should include hospitalization, specialized personnel, ancillary and diagnostic laboratory procedures, medications, and follow-up. In addition, because so many low-income families live far from hospitals, support systems to provide food, lodging, and transportation are necessary. However, the task is worth doing, since an organized pediatric cancer program can facilitate stepwise improvement in all aspects of cancer care and hospital care in general that lead to improved outcomes (Table 24.2).24 A model of economic and technical support has recently been developed for countries that are improving economically and are reducing the mortality caused by diseases of

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Table 24.2 Conditions affecting pediatric cancer outcomes by treatment era in Recife, Brazil Early era (1980–9)

Middle era (1994a –March 1997)

Recent era (April 1997–2002)

Until 1984, not available; from 1985–9, basic lodging available for up to 5 families

Basic lodging for up to 20 families

Money for transportation

Not provided

Provided in a few cases

Subsidized food

None

Provided in a few cases

Parent and volunteer group Fund-raising foundation Social workers

None None One social worker for all patients at the general hospital

Active Active locally One social worker employed by the pediatric oncology unit

Patient tracking

None

Patients contacted after missing several clinic visits.

Lodging for 120 families, with on-site social work, vocational training for patients and parents, physical and occupational therapy, and dentistry Round-trip transportation paid by NACCb for all patients. One month’s supply of basic food (rice, beans) provided at the time of the clinic visit for patients living outside Recife Very active Active regionally Three social workers and a psychologist employed by the pediatric oncology unit Patients contacted within 24 hours of a missed clinic visit

Spread through the general hospital Almost no access None

Embedded oncology ward Delayed access 1

Separate oncology ward Immediate access 7

No night coverage

24-hours coverage by physicians in training

24-hours coverage by a pediatric intensive care physician

Rapid, appropriate treatment in most cases

Rapid, appropriate treatment in most cases

Intermittently prescribed

Prescribed for most patients Transfusion delays usually >6 hours, platelets available

Pharmacy

Delays commonly >10 hours for transfusion; platelets rarely available Hospital pharmacy

Rapid, appropriate treatment in all cases based on written supportive care guidelines Prescribed for all patients based on written supportive care guidelines Transfusion delays usually <6 hours, platelets available

Hospital pharmacy

Separate pharmacy for pediatric oncology

Treatment Drug supply Treatment regimen

Occasional shortages Various

Reliable St. Jude Total XI44

Reliable St. Jude Total XIIB45

Patient and family support Guest housing for families

Hospital and personnel Oncology service ICU Nurses with oncology training Physician availability Supportive care Management of febrile neutropenia Pneumocystis carinii pneumonia prophylaxis Blood products

a b

The pediatric oncology service was not available during the 5 years not accounted for. ´ NACC, Nucleo de Apoio a Crianc¸a com Cˆancer.

poverty. This model has been termed “twinning” because it consists of a partnership between an institution in a developed country and an institution in a country with limited resources. It was pioneered by Italian physicians who partnered with an institution in Nicaragua, with the goal of curing the maximum possible number of children

with cancer.34 Variations of this model exist but are less well characterized. The full implementation of such programs requires several years of persistent and continuous effort to alter established societal beliefs and expectations and challenge policy makers to re-examine the health-care needs and priorities of their country or their community.

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In 1994, St. Jude Children’s Research Hospital established an International Outreach Program (IOP) based on three premises41–43 : (1) most developing nations will continue to improve their general health status over the next 10 to 20 years, bringing the U5MR to less than 15 per 1000 live births per year (for example, the U5MR in El Salvador fell 3.9% each year between 1990 and 2001); (2) cancer will become the most common cause of disease-related death in children aged 2 to 19 years of age in these countries; and (3) most developing countries have untapped resources that can be used to develop and maintain effective pediatric cancer programs. The mission of the St. Jude IOP is to improve the survival rates of children with catastrophic illness worldwide, through the transfer of knowledge, technology, and organizational skills. Analysis of the St. Jude IOP partnerships over the past 10 years has revealed several factors that are essential to the establishment of an effective pediatric oncology program. First, community participation through the development of a non-governmental organization (NGO) is likely to be the single most important element in implementing an effective anticancer program. This factor is particularly crucial in countries where medications or social support for the families of children with cancer are not funded by the government. Typically, these NGOs provide shelter, food, and transportation for patients and their families and purchase medications and other services. They also educate the community and families about childhood cancer and raise general awareness of the needs of these patients and families. In many cases, NGOs exert intense pressure on governments to include childhood cancer programs as a health priority. Moreover, some of the NGOs have developed rather sophisticated fund-raising techniques that allow them to expand their services and their visibility in the community. As these nations’ economies improve further, the role of the NGOs may shift to support specific services such as bone marrow transplantation or research projects. The second most important factor is the availability of at least one full-time pediatric hematologist/oncologist to lead the development and implementation of the pediatric cancer unit. This individual is responsible for organizing the unit and establishing its medical priorities. Working closely with NGOs, hospital leadership, and international partners, these medical directors occupy a pivotal role in the governance of the cancer unit. Third is the promotion of nursing to a prominent role in patient care and family education. It is vital to improved patient care that nurses receive specific training in pediatric oncology and that these trained nurses be retained in the unit. Continuing education, through participation in meetings or other professional activities, motivates the nurses and

provides them with a sense of ownership. Fourth, continuing communication between the twinning partners is needed. This communication can have different forms and may include discussion of clinical cases or treatment protocols or workshops to review treatment outcomes. Fifth is access to essential resources such as up-to-date textbooks and medical literature. The importance of this factor can be verified by the utilization of the St. Jude educational website (www.cure4kids.org), which now has about 2000 users in 88 countries. Sixth is the need for detailed records of patients’ demographic, clinical, and outcome information and development of a database that allows data analysis. Finally, trained individuals must be available to provide correct diagnosis of pediatric malignancies; diagnostic procedures should include immunohistochemistry for solid tumors and flow cytometric immunophenotyping for leukemias.

Results of treatment of acute leukemias and lymphomas in selected countries Acute lymphoblastic leukemia Since 1994, the St. Jude IOP has collaborated with pediatric hematologist/oncologists of several countries to develop effective treatment of ALL in those countries, with the main objective of curing an increasing number of children. Responses to therapy are carefully recorded and periodically analyzed in order to apply evidence-based medicine and evaluate the use of available local resources, including medications and improved supportive care measures. Because ALL is highly curable and the treatment is relatively well tolerated, the rates of survival and the types of treatment failure in children with ALL offer a reliable outcome measure of the progress of the twinning program between St. Jude IOP and the partner sites. The first ALL treatment protocol developed through the collaboration of St. Jude IOP and a partner site was the El Salvador (ELS)-I protocol, a modified version of the St. Jude Total XI protocol.36 This treatment had been very well tolerated at St. Jude, but it was associated with an unacceptable early toxicity rate when used in El Salvador.41 These results were attributed in part to the health care providers’ lack of experience in managing the complications of intensive chemotherapy and possibly to these patients’ increased sensitivity to daunorubicin, which was used weekly during the first 2 weeks of treatment. After the protocol was modified to eliminate daunorubicin and the health-care providers were given further training, tolerance of chemotherapy increased and the deaths caused

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Table 24.3 Guatemala, Honduras, El Salvador (GHS) II treatment

Table 24.4 Guatemala, Honduras, El Salvador (GHS) II treatment protocol:

protocol: induction, intensification, and consolidation

continuation therapy for standard-risk ALLa–d

Induction Vincristine Prednisone Asparaginase

Weeks 1–3

Daunomycin

1.5 mg/m2 IV weekly × 4 40 mg/m2 PO daily × 28 10,000 U/m2 IM on days 2, 3, 5, 8, 10, 12 (plus days 15, 17, 19)a 25 mg/m2 IV on day 1 (plus day 8)a

Intensification Cyclophosphamide Cytarabine 6-Mercaptopurine

1 g/m2 IV on day 22 75 mg/m2 IV on days 23–26, 30–33 60 mg/m2 PO on days 22–36

Consolidation Methotrexate 6-Mercaptopurine

2 g/m2 (3 g/m2 )a IV on days 44 and 51 75 mg/m2 PO on days 44–58

CNS-directed therapy Triple IT therapy

Week 4 Week 5

Week 6 Weeks 7–11 Weeks 12–14 Week 15 Weeks 16–18

Days 1, 15, 29, 44, 51 (plus days 8 and 22 for patients with CNS disease

Week 19 Week 20

Abbreviations: IV, intravenous; PO, per os; IM, intramuscular; IT, intrathecal; CNS, central nervous system. a Patients at high risk only.

Weeks 21–23 Week 24

by toxicity were reduced substantially. The long-term overall survival rate of about 50% (compared with the 5% survival rate before the start of the program) was encouraging. Early mortality due to infection, metabolic complications, or abandonment of therapy was as frequent a cause of failure as relapsed disease.37 Five patients developed secondary acute myeloid leukemia. This rate (1.8%) was similar to that observed in one of the arms of the St. Jude Total XI protocol44 and to that seen in Recife, Brazil (2.1%), where a slightly modified St. Jude Total XIIIB regimen was used.45 Although the rate of secondary AML was relatively low in the ELS-I trial, and is potentially avoidable, it represents patients who have survived the induction and consolidation phases of therapy; thus, it contributes substantially to the rate of adverse events during remission. Moreover, because hematopoietic stem cell transplantation is not available in newly developed pediatric cancer programs, secondary AML is a death sentence for these children. The second protocol developed for use in El Salvador (designated Guatemala, Honduras, El Salvador-II, or GHS-II) (Tables 24.3 and 24.4) was based on the St. Jude Total XIIIB protocol and was also tested in Guatemala and Honduras. During the time that ELS-I was being implemented, several new treatment strategies proved to be effective in increasing the cure rate of ALL. First was

Methotrexate 40 mg/m2 IM weekly Mercaptopurine 75 mg/m2 PO daily Dexamethasone 8 mg/m2 PO daily × 7 Vincristine 1.5 mg/m2 IV Methotrexate 40 mg/m2 IM Mercaptopurine 75 mg/m2 PO daily Vincristine 1.5 mg/m2 IV Methotrexate 40 mg/m2 IV/IM Mercaptopurine 75 mg/m2 PO daily Repeat induction and consolidation therapy Methotrexate 40 mg/m2 IV/IM Mercaptopurine 75 mg/m2 PO daily × 7 Methotrexate 2 g/m2 IV Mercaptopurine 75 mg/m2 PO daily × 7 Methotrexate 40 mg/m2 IM weekly Mercaptopurine 75 mg/m2 PO daily × 7 Dexamethasone 8 mg/m2 PO daily × 7 Vincristine 1.5 mg/m2 IV Methotrexate 40 mg/m2 IV/IM weekly Mercaptopurine 75 mg/m2 PO daily × 7 Vincristine 1.5 mg/m2 IV Methotrexate 40 mg/m2 IV/IM weekly Mercaptopurine 75 mg/m2 PO daily × 7 Methotrexate 2 g/m2 IV Mercaptopurine 75 mg/m2 PO daily × 7

Abbreviations as in Table 24.3. Chemotherapy given between weeks 16 and 24 is repeated during the first year (a total of six doses of high-dose methotrexate are administered during continuation). c Triple IT given on weeks 1, 2, 3, 7, 10, 12, 15, 24, 31, 36, 39, 43, 47, and 55. d Dexamethasone and vincristine are stopped after week 100. e Continuation therapy extended to 3 years for boys. b

augmentation of the intensity of chemotherapy early in remission for patients with high-risk leukemia. A double reinduction phase was added to the trial for patients at high risk of treatment failure, and one reinduction was added for patients with standard-risk ALL. In the ongoing GHS-II, standard-risk ALL requires the presence of B-lineage ALL, age 1 to 10 years, and a WBC count of less than 50 × 109 /L, or a DNA index of 1.16 or greater (but less than 1.60). All other patients, including those with CNS or testicular involvement (or both) at diagnosis or the persistence of lymphoblasts on day 15 or 29 of therapy, are considered at high risk of relapse regardless of other features. Second, to reduce the risk of secondary AML, GHS-II no longer specified the use of epipodophyllotoxin drugs. Finally, because of concerns over neuropsychologic and endocrine sequelae and an

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increased risk of brain tumors after CNS irradiation, intensive systemic chemotherapy featuring dexamethasone in the reinduction phases together with triple intrathecal chemotherapy was substituted for CNS irradiation in most cases. CNS irradiation (1800 cGy at week 56) was reserved for patients with CNS leukemia at diagnosis, defined as the presence of 5 or more cells (including lymphoblasts) in cytocentrifuged cerebrospinal fluid. Moreover, patients considered to be at high risk of CNS relapse (i.e. those with B-lineage ALL with a presenting WBC count greater than 100 × 109 /L, T-lineage ALL with a presenting WBC count greater than 50 × 109 /L, or a hypodiploid chromosome complement) received cranial radiation (1200 cGy) at week 56. Between December 2000 and April 2004, 161 Salvadoran children were enrolled in the GHS-II study; 37% were considered to be at high risk of failure. One hundred forty seven (91%) entered remission. Seven patients died during induction and another seven refused additional treatment. Nine of the 147 patients have relapsed, six have died during remission of infectious complications, and 12 have abandoned treatment. The estimated 2-year EFS rate (considering abandonment as a cause of failure) is 71% (Dr. Miguel Bonilla, personal communication, 2004). El Salvador was chosen as an example because it clearly illustrates many of the problems that commonly exist in developing countries. The hospital Benjamin Bloom in San Salvador is the only public pediatric tertiary care facility in a country with about 6.6 million inhabitants. It has 300 beds and all of the basic pediatric subspecialties, including intensive care and surgery. Approximately 90 new cases of leukemia develop in El Salvador each year, most of which are seen at the hospital Benjamin Bloom (in 2003, it accrued 73 of the expected 90 cases of ALL). No patient is denied treatment. Nineteen hospital beds are dedicated to the pediatric cancer unit, and the occupancy rate is 88%. The unit is staffed by two full-time pediatric hematologists/oncologists, 24 nurses (ratio, 3.6 patients/nurse), two residents, and three medical students, as well as three parttime psychologists and two full-time social workers. Outpatient facilities can accommodate 20 patients for ambulatory chemotherapy, and there is a domiciliary facility for patients and families. Medications have been consistently available, with the occasional exception of L-asparaginase. Program activities are funded by the nongovernmental Foundation Ayudame a Vivir and by the national government. Reimbursement from third-party payers or from the patients’ families is virtually nonexistent. The Foundation pays for domiciliary care, transportation, salary supplementation for health-care providers, and anticancer medications. Government funds support hospital charges,

including partial support for chemotherapy and personnel. The Foundation has an active fund-raising program with a large body of volunteers, and it cooperates with two other NGOs in Honduras and Guatemala, respectively, to purchase medications at discounted prices. The current survival rates can be improved by introducing measures designed to reduce abandonment of therapy and the frequency of fatal infectious complications during remission. In El Salvador, abandonment is likely to reflect the parents’ perception that cancer is incurable. After remission is induced, parents commonly assume that the diagnosis of cancer is wrong and remove the child from the hospital against medical advice. There is no legal recourse to protect the child in this situation. When the disease relapses, most of the parents return, seeking medical care. Abandonment remains a challenge and appears to reach a plateau at approximately 15% of cases. Measures to reduce this rate require identification of families at high risk of treatment abandonment, the establishment by civil institutions of policies that ensure the child’s access to treatment, and the infrastructure to enforce these policies. Death from infectious complications can be further reduced by modifying the intensity of induction therapy for selected patients who arrive with active infection, malnutrition, and very advanced disease and by identifying treatment facilities closer to the patients’ homes that could initiate antibiotic therapy promptly at the onset of fever. The GHS-II treatment protocol has also been used in Honduras and Guatemala, with overall results similar to those obtained in El Salvador. Abandonment of therapy and death from infectious complications remain the main causes of failure in all three countries, although in Honduras the main reason for abandonment appears to be the inability of parents who live far from the hospital to keep the appointment for chemotherapy.19 The outcome of treatment of ALL in Recife, Brazil, has been similar, although the treatment program is slightly different.24

B-cell (Burkitt) lymphoma The treatment of Burkitt NHL in countries with limited resources merits special consideration. Patients with this highly chemosensitive disease require optimal supportive care to fully benefit from specific treatment, particularly during the initial phase. At presentation, it is very common for these children to have advanced-stage disease, large abdominal masses, organ dysfunction, metabolic aberrations, malnutrition, and active infection. Modifications of treatment guidelines established by various investigators,46,47 including reduction of methotrexate,

Acute leukemia in countries with limited resources

cytarabine, and cyclophosphamide dosages, have been successfully used in countries with limited resources.48,49 In one extreme example, children with Burkitt lymphoma of the head and neck or abdomen were treated with cyclophosphamide alone.50 With a mean follow-up time of 59 months (29 to 109 months), 63% of the children with head and neck primary disease and 33% of those with abdominal primary disease were alive. A subsequent study by the same investigators used a multidrug regimen in Malawian children with stage I–III disease (Murphy classification).49 Of 75 children diagnosed with Burkitt lymphoma, 31 were excluded because of disease-stage IV (12 patients), incomplete disease-stage procedures (5 patients), HIV infection (4 patients), parental refusal (3 patients), or other reasons (7 patients). Forty-four children (10 with stage I, 5 with stage II, and 29 with stage III disease) were treated with a simplified version of the French LMB89 protocol.46 Seventy-seven percent of these patients were below the fifth percentile for weight, 23% had circulating Plasmodium falciparum, and 5% had Schistosoma haematobium infestation. Thirty-nine of these children entered remission (2 had resistant disease and 3 died of complications of therapy). Nine of 10 children with stage I disease, 2 of 5 with stage II disease, and 16 of 29 with stage III disease were alive, and many were likely to be cured.46 These observations have broad implications, one of which is that the biology (responsiveness to chemotherapy) of Burkitt lymphoma may differ depending on geographic region.51 Further, even in locales with minimal resources, patients who have advanced-stage B-cell NHL and comorbid conditions, including malnutrition and active infection, can be cured of their cancer. Modifications of the regimen proposed by French investigators have been used in Recife. These modifications include: (1) reduction of high-dose methotrexate from 3 or 5 g/m2 to 1 g/m2 in groups B and C46 ; (2) no escalation of the cyclophosphamide dose beyond 500 mg/m2 per day during COPADM courses; and (3) two courses of CYM rather than two courses of CYVE in consolidation therapy for group C. Between 1995 and 1997, 29 children were treated (1, stage II; 27, stage III; 1, stage IV) on the modified LMB-89 regimen in Recife, Brazil. Reasons for treatment failure included sepsis (n = 10) and recurrent disease (n = 2). The 1-year survival estimate was 62%. No adverse event occurred later than 1 year after diagnosis, suggesting that most treatment failures in Burkitt lymphomas occur within the first year after diagnosis. With improved supportive care, an unmodified LMB-89 regimen was implemented. Since 1997, 72 children (2 stage I, 3 stage II, 58 stage III, 9 stage IV) have been treated on this regimen, which has been well tolerated. Reasons for

treatment failure included sepsis (n = 11) and metabolic complications (n = 1). The 3-year EFS estimate is 82% ± 3% (Dr. Francisco Pedrosa, personal communication, 2004). No failure has been attributed to persistent or relapsed disease. The experience of treating Burkitt lymphoma in Recife has shown that improved supportive care can dramatically reduce the frequency of early treatment-related mortality. Moreover, it clearly establishes that effective methotrexate chemotherapy can be successfully delivered in the absence of serum drug monitoring, if close attention is paid to clinical and laboratory information (Table 24.5). The importance of close clinical monitoring is illustrated by the outcomes of 9 patients with B-cell leukemia (6 patients) or stage IV NHL lymphoma whose chemotherapy included 8 g/m2 of methotrexate infused over 4 hours. These patients experienced no clinical toxicity that could be attributed to methotrexate, although methotrexate serum levels were not available.

Acute myeloid leukemia The management of childhood AML is very challenging in countries with limited resources. Effective treatment requires intensive myelosuppressive regimens and support with an uninterrupted supply of blood products and various antimicrobials, including antifungals. Even in the developed countries, which can offer optimal treatment, including hematopoietic stem cell transplantation, the overall 5-year survival of children and adolescents with AML does not exceed 60%.1 However, despite these obstacles, the same infrastructure that supports the treatment of ALL and NHL in developing countries is adequate to allow the delivery of treatment that produces a 30% to 40% long-term disease-free survival rate in children with AML. There have been very few reports of the outcome of treatment for children with AML in developing countries52,53 ; however, it is reasonable to devise treatment approaches for these children. Recently, the Association of Pediatric Oncologists of Central America (AHOPCA) has implemented a treatment plan based on the Berlin¨ Frankfurt-Munster (BFM)-93 AML protocol.54 Since February 1999, 115 patients with AML (excluding patients with promyelocytic leukemia and Down syndrome) have been enrolled in a modified BFM-93 protocol. The most common FAB subtype was AML M2 (37%), followed by AML M4/M5 (36%). Twelve patients (10%) presented with chloromas and another 12 with CNS leukemia. Eight patients died of bleeding or infection before starting treatment. Of 107 evaluable patients, 82 (77%) entered complete remission. Causes of adverse events during remission induction (23%) included infectious complications in 14 patients, abandonment of

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Table 24.5 Guidelines for use of high-dose methotrexate in Recife, Brazil 1.

2.

3. 4. 5.

6.

Patients are evaluated at 24 hours before admission and proceed to chemotherapy if they are in good clinical condition. All medications that are nephrotoxic or may interfere with MTX excretion (e.g. salicylates, sulfonamides, ketoconazole) are discontinued. General anesthesia is avoided on the day patients are admitted for MTX chemotherapy. Two hours before administration of MTX, an infusion of 5% dextrose with 40 mEq/L NaCO3 (250 mL/m2 per hour for BSA < 0.75 m2 , 150 mL/m2 per hour for BSA ≥ 0.75 m2 ) is started. MTX, 3 g/m2 dissolved in the same solution, is then infused over 3 hours (4 hours when MTX dose is 8 g/m2 ). After completion of the MTX infusion, dextrose 5% with normal saline (1: 1) plus 40 mEq/L NaCO3 is administered for 72 hours at 125 mL/m2 per hour. NaCO3 is given for at least 48 hours if the urine pH is ≤ 7.0. Leucovorin 15 mg/m2 (rounded to the higher multiple of 5) is begun exactly 24 hours after the start of MTX and given every 6 hours. Twelve doses are administered; the first four are given intravenously. Patient remains in the hospital for 48 hours after completion of MTX. Clinical manifestations (skin and mucosa, strict fluid balance) and laboratory values (BUN, creatinine, urine pH) are closely monitored. Patients with clinical or laboratory abnormalities remain in the hospital and receive intravenous hydration and leucovorin. In cases of grade IV toxicity, the leucovorin dose is increased to 250 mg/m2 IV every 6 hours until the toxicity resolves. Intrathecal medication (methotrexate alone or in combination) is given several hours before the start of the MTX infusion.

Abbreviations: BSA, body surface area; MTX, methotrexate.

therapy in 7 patients, and resistant disease in 4 patients. Thirty-two patients had relapses. Only two deaths were attributed to infection during intensification and consolidation phases. As of February 2004, the 3-year event-free survival estimate was 26% (Miguel Bonilla, personal communication, 2004). Another clinical study, which used a modification of the St. Jude AML-97 protocol to treat children with AML in Recife, Brazil, has recently been analyzed. The 5-year EFS estimate (29% ± 7%) is very similar to that obtained with the AHOPCA protocol described above (Francisco Pedrosa, personal communication, 2004). These studies indicate that even AML can be successfully treated in regions with limited resources, although the survival rates are likely to lag behind those obtained in ALL and NHL.

Summary and future direction The successful management of lymphoid malignancies constitutes one of the most spectacular achievements of modern medicine. Despite the heterogeneity of these diseases and the relative lack of knowledge concerning their pathogenesis, the cure rate is now approaching 90%. These results have been possible through a series of wellconducted, hypothesis-driven clinical trials.55,56 Remarkably, this progress has been achieved with antileukemic drugs that were discovered more than a decade ago. Over the past 40 years, clinical investigators have refined treatment by balancing the potential benefits of therapeutic interventions with their adverse sequelae. Concurrently, laboratory investigations have disclosed that the leukemic

cell genotype and the patients’ genetic makeup influence the outcome of treatment and the risk of adverse sequelae.57 Therefore, the development of effective treatment programs for pediatric leukemias and lymphomas in different countries must consider the distribution of leukemia subtypes, the environmental characteristics, and the population genetics of the specific country. The first step in improving cancer cure rates in a particular country or region is the careful documentation of the patients’ demographic characteristics, types of malignancies, and clinical toxicity profiles. Essential to this effort is the correct diagnosis of these disorders. Our experience in Central American countries showed that until recently, about 30% of the malignancies were incorrectly diagnosed. After the establishment of a regional diagnostic laboratory (including training, implementation, maintenance, and quality control), correct diagnosis of all cases of leukemia and lymphoma in that region became available.58 Preliminary analysis of more than 1000 cases of acute leukemia has revealed that the distribution of immunophenotypes may differ from that in the United States. Moreover, the types of adverse events observed during treatment differ widely in developed compared with developing countries. These observations may influence decisions about how these patients are treated. The biologic differences among leukemia subtypes may also provide clues to the basic mechanisms of leukemogenesis, thereby contributing new knowledge that can be generally applied in clinical practice. For these reasons, clinical investigations based on sound scientific methods are crucial for the development of effective treatment in countries with limited resources59 and for the optimal use of the information obtained.

Acute leukemia in countries with limited resources

REFERENCES 1 Smith, A. S., Ries, L. A. G., Gurney, J. G., & Ross, J. A. Leukemia. In L. A. G. Ries, M. A. Smith, J. G. Gurney, et al., eds., Cancer Incidence and Survival Among Children and Adolescents: United States SEER Program 1975–1995, NIH Publication 99-4649 (Bethesda, MD: National Cancer Institute, SEER Program, 1999), pp. 17–34. 2 Percy, C. L., Smith, M. A., Linet, M., Ries, L. A. G., & Friedman, D. L. Lymphomas and reticuloendothelial neoplasms. In L. A. G. Ries, M. A. Smith, J. G. Gurney, et al., eds., Cancer Incidence and Survival Among Children and Adolescents: United States SEER Program 1975–1995, NIH Publication 99-4649 (Bethesda, MD: National Cancer Institute, United States SEER Program, 1999), pp. 35–50. 3 Wheldon, E. G., Lindsay, K. A., Wheldon, T. E., & Mao, J. H. A two-stage model for childhood acute lymphoblastic leukemia: application to hereditary and nonhereditary leukemogenesis. Math Biosci, 1997; 139: 1–24. 4 Greaves, M. F., Maia, A. T., Wiemels, J. L., & Ford, A. M. Leukemia in twins: lessons in natural history. Blood, 2003; 102: 2321–33. 5 Greaves, M. F. Biological models for leukaemia and lymphoma. IARC Sci Publ, 2004; 157: 351–72. 6 Magrath, I. T. African Burkitt’s lymphoma. History, biology, clinical features, and treatment. Am J Pediatr Hematol Oncol, 1991; 13: 222–46. 7 Thorley-Lawson, D. A. & Gross, A. Persistence of the Epstein– Barr virus and the origins of associated lymphomas. N Engl J Med, 2004; 350: 1328–37. 8 Cook-Mozaffari, P., Newton, R., Beral, V., & Burkitt, D. P. The geographical distribution of Kaposi’s sarcoma and of lymphomas in Africa before the AIDS epidemic. Br J Cancer, 1998; 78: 1521–8. 9 Douer, D. The epidemiology of acute promyelocytic leukaemia. Best Pract Res Clin Haematol, 2003; 16: 357–67. 10 Cavdar, A. O., Babacan, E., Gozdasoglu, S., et al. High risk subgroup of acute myelomonocytic leukemia (AMML) with orbitoocular granulocytic sarcoma (OOGS) in Turkish children. Retrospective analysis of clinical, hematological, ultrastructural and therapeutical findings of thirty-three OOGS. Acta Haematol, 1989; 81: 80–5. 11 Rajalekshmy, K. R., Abitha, A. R., Pramila, R., Gnanasagar, T., & Shanta, V. Immunophenotypic analysis of T-cell acute lymphoblastic leukaemia in Madras, India. Leuk Res, 1997; 21: 119– 24. 12 Kamel, A. M., Assem, M. M., Jaffe, E. S., et al., Immunological phenotypic pattern of acute lymphoblastic leukaemia in Egypt. Leuk Res, 1989; 13: 519–25. 13 Parkin, D. M., Kram´arov´a, E., Draper, G. J., et al. International Incidence of Childhood Cancer (Lyon, France: IARC, 1998). 14 Arias, E., MacDorman, M. F., Strobino, D. M., & Guyer, B. Annual summary of vital statistics – 2002. Pediatrics, 2003; 112: 1215–30. 15 World Health Organization. The World Health Report, 2004: Changing History (Geneva: World Health Organization, 2004). 16 Eys, J. van. Ethical and medicolegal issues in pediatric oncology. Hematol Oncol Clin North Am, 1987; 1: 841–8.

17 Yaris, N., Mandiracioglu, A., & Buyukpamukcu, M. Childhood cancer in developing countries. Pediatr Hematol Oncol., 2004; 21: 237–53. 18 UNICEF. The State of the World’s Children 2004 (New York: UNICEF, 2004). 19 Metzger, M. L., Howard, S. C., Fu, L. C., et al. Outcome of childhood acute lymphoblastic leukaemia in resource-poor countries. Lancet, 2003; 362: 706–8. 20 Corrigan, J. J. & Feig, S. A. Guidelines for pediatric cancer centers. Pediatrics, 2004; 113: 1833–5. 21 Wagner, H. P. & Antic, V. The problem of pediatric malignancies in the developing world. Ann N Y Acad Sci, 1997; 824: 193– 204. 22 Magrath, I., Petrilli, S., Gad-el-Mawla, N., et al. Pediatric oncology in less developed countries. In P. A. Pizzo, & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology (Philadelphia, PA: Lippincott, 1993), pp. 1225–51. 23 Spinetta, J. J., Masera, G., Eden, T., et al. Refusal, noncompliance, and abandonment of treatment in children and adolescents with cancer: a report of the SIOP Working Committee on Psychosocial Issues in Pediatric Oncology. Med Pediatr Oncol, 2002; 38: 114–17. 24 Howard, S. C., Pedrosa, M., Lins, M., et al. Establishment of a pediatric oncology program and outcomes of childhood acute lymphoblastic leukemia in a resource-poor area. JAMA, 2004; 291: 2471–5. 25 Agarwal, B. & Dalvi, R. Treatment of childhood leukemias in underprivileged countries. In C. H. Pui, ed., Treatment of Acute Leukemias: New Directions for Clinical Research (Totowa, NJ: Humana Press, 2003), pp. 321–9. 26 Howard, S. C., Ribeiro, R. C., & Pui, C. H. Strategies to improve outcomes of children with cancer in low-income countries. Eur J Cancer, 2005); 41: 1584–7. 27 Barr, R. D., Ribeiro, R. C., Agarwal, B. R., et al. Pediatric oncology in countries with limited resources. In P. A. Pizzo & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology (Philadelphia, PA: Lippincott, 2002), pp. 1541–52. 28 Usmani, G. N. Pediatric oncology in the third world. Curr Opin Pediatr, 2001; 13: 1–9. 29 Pui, C. H. & Ribeiro, R. C. International collaboration on childhood leukemia. Int J Hematol, 2003; 78: 383–9. 30 Pui, C. H., Schrappe, M., Masera, G., et al. Ponte di Legno Working Group: statement on the rights of children with leukemia to have full access to essential treatment. Report on the Sixth International Childhood Acute Lymphoblastic Leukemia Workshop. Leukemia, 2004; 18: 1043–53. 31 Antillon, F., Baez, F. L., Barr, R., et al. AMOR: a proposed cooperative effort to fight childhood cancer in Central America. Pediatric Blood Cancer, 2005; 45: 107–10. 32 Ertem, I. O., Bingoler, B. E., Ertem, M., Uysal, Z., & Gozdasoglu, S. Medical neglect of a child: challenges for pediatricians in developing countries. Child Abuse Negl, 2002; 26: 751– 61. 33 Eden, T. Translation of cure for acute lymphoblastic leukaemia to all children. Br J Haematol, 2002; 118: 945–51.

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34 Masera, G., Baez, F., Biondi, A., et al. North–South twinning in paediatric haemato-oncology: the La Mascota programme, Nicaragua. Lancet, 1998; 352: 1923–6. 35 Masera, G., Baez, F., Biondi, A., et al. Bridging the childhood cancer mortality gap between economically developed and lowincome countries: lessons from the MISPHO experience. In S. Tanneberger, F. Cavalli, & F. Pannuti, eds., Cancer in Developing Countries (New York: W. Zuckschwerdt, 2003). 36 Bonilla, M., Moreno, N., Marina, N., et al. Acute lymphoblastic leukemia in a developing country: preliminary results of a nonrandomized clinical trial in El Salvador. J Pediatr Hematol Oncol, 2000; 22: 495–501. 37 Greenberg, P. L., Gordeuk, V., Issaragrisil, S., et al. Major hematologic diseases in the developing world-new aspects of diagnosis and management of thalassemia, malarial anemia, and acute leukemia. Hematology (Am Soc Hematol Educ Program), 2001: 479–98. 38 Lilleyman, J. Simple deliverable therapy needed for childhood leukaemia. Lancet, 2003; 362: 676–7. 39 Ribeiro, R. C. & Pui, C. H. Saving the children – improving childhood cancer treatment in developing countries. N Engl J Med, 2005; 352: 2158–60. 40 Sala, A., Barr, R. D., Masera, G., et al. A survey of resources and activities in the MISPHO family of institutions in Latin America: a comparison of two eras. Pediatr Blood Cancer, 2004; 43: 758– 64. 41 Ribeiro, R. C., Marina, N., & Crist, W. M. St Jude Children’s Research Hospital’s International Outreach Program. Leukemia, 1996; 10: 570–4. 42 Ribeiro, R. C. & Bonilla, M. A leukaemia treatment programme in El Salvador. Lancet, 2000; 356(Suppl.): S7. 43 Wagner, H. Alliances in pediatric oncology: where do we go from here? Med Pediatr Oncol, 2001; 36: 310–11. 44 Rivera, G. K., Pui, C. H., Hancock, M. L., et al. Update of St Jude Study XI for childhood acute lymphoblastic leukemia. Leukemia, 1992; 6(Suppl. 2): 153–6. 45 Pui, C. H., Boyett, J. M., Rivera, G. K., et al. Long-term results of Total Therapy studies 11, 12 and 13A for childhood acute lymphoblastic leukemia at St Jude Children’s Research Hospital. Leukemia, 2000; 14: 2286–94.

46 Patte, C. Treatment of mature B-ALL and high grade B-NHL in children. Best Pract Res Clin Haematol, 2002; 15: 695–711. 47 Reiter, A., Schrappe, M., Tiemann, M., et al. Improved treatment results in childhood B-cell neoplasms with tailored intensification of therapy: a report of the Berlin-Frankfurt-Munster Group Trial NHL-BFM 90. Blood, 1999; 94: 3294–306. 48 Hesseling, P. B. The SIOP Burkitt lymphoma pilot study in Malawi, Africa. Med Pediatr Oncol, 2000; 34: 142. 49 Hesseling, P. B., Broadhead, R., Molyneux, E., et al. Malawi pilot study of Burkitt lymphoma treatment. Med Pediatr Oncol, 2003; 41: 532–40. 50 Kazembe, P., Hesseling, P. B., Griffin, B. E., Lampert, I., & Wessels, G. Long-term survival of children with Burkitt lymphoma in Malawi after cyclophosphamide monotherapy. Med Pediatr Oncol, 2003; 40: 23–5. 51 Sandlund, J. T., Fonseca, T., Leimig, T., et al. Predominance and characteristics of Burkitt lymphoma among children with nonHodgkin lymphoma in northeastern Brazil. Leukemia, 1997; 11: 743–6. 52 Viana, M. B., Cunha, K. C., Ramos, G., & Murao, M. Acute myeloid leukemia in childhood: 15-year experience in a single institution. J Pediatr (Rio J), 2003; 79: 489–96. 53 Felice, M. S., Zubizarreta, P. A., Alfaro, E. M., et al. Good outcome of children with acute myeloid leukemia and t(8;21)(q22;q22), even when associated with granulocytic sarcoma: a report from a single institution in Argentina. Cancer, 2000; 88: 1939–44. 54 Creutzig, U., Berthold, F., Boos, J., et al. Improved treatment results in children with AML: results of study AML-BFM 93. Klin Padiatr, 2001; 213: 175–85. 55 Pinkel, D. Lessons from 20 years of curative therapy of childhood acute leukaemia. Br J Cancer, 1992; 65: 148–53. 56 Simone, J. V. A history of St Jude Children’s Research Hospital. Br J Haematol, 2003; 120: 549–55. 57 Pui, C. H., Relling, M. V., & Downing, J. R. Acute lymphoblastic leukemia. N Engl J Med, 2004; 350: 1535–48. 58 Howard, S. C., Campana, D., Coustan-Smith, E., et al. Development of a regional flow cytometry center for diagnosis of childhood leukemia in Central America. Leukemia, 2005; 19: 323–5. 59 Magrath, I. & Litvak, J. Cancer in developing countries: opportunity and challenge. J Natl Cancer Inst, 1993; 85: 862–74.

25 Antibody-targeted therapy Eric L. Sievers and Irwin D. Bernstein

Introduction No one stands to benefit more from targeted therapy strategies than children with cancer. Although conventional chemotherapy induces pancytopenia and mucositis that augment the risk of life-threatening infections in patients of all ages, younger patients are particularly vulnerable to late toxicities due to a lack of chemotherapy specificity for malignant cells. Such effects include, but are not limited to, cardiotoxicity, pulmonary insufficiency, sterility, hormone deficiency, and the development of secondary malignancies. Although the dose intensity of treatment regimens for acute lymphoblastic leukemia (ALL) and Hodgkin disease has declined substantially in recent years, children with acute myeloid leukemia (AML) currently receive the highest dose intensity of chemotherapy that has ever been delivered. While a higher proportion of children with AML treated in this manner experienced prolonged disease-free survival, compared with those who received a less aggressive induction regimen, their overall survival was somewhat reduced by a higher induction death rate in this randomized arm.1 Strategies that target therapies directly to malignant cells could conceivably spare normal tissues from injury, resulting in a substantial improvement in the long-term quality of life of children who achieve remissions. The use of monoclonal antibodies as a means of delivering either chemotherapy or radiation directly to leukemic blast cells or, in the setting of hematopoietic stem cell transplantation (HSCT), as a means of targeting increased doses of radiation to sites of normal and malignant hematopoiesis (Table 25.1) will be reviewed in this chapter. Over 25 years ago, K¨ohler and Milstein2 showed that monoclonal antibodies could be continuously produced in quantities sufficient for clinical applications. Publication

of their method induced speculation that antibody therapy might begin to replace conventional cytotoxic treatments. Unfortunately, few antibodies have found clinical utility because leukemia-specific antigens are exceedingly uncommon. For this reason, normal hematopoietic cell surface antigens that are restricted to leukemic blast cells and their normal counterparts have been chosen as alternative targets. While normal hematopoietic cells expressing the antigen of interest are ablated with this strategy, injury to nonhematopoietic tissues can be avoided. The most successful example of this approach is rituximab, an unconjugated anti-CD20 monoclonal antibody that has been shown to be both effective and safe in patients with advanced lymphoma. In 2000, the US Food and Drug Administration approved gemtuzumab ozogamicin (discussed in detail below) for the treatment of older patients with relapsed CD33-positive AML. Pediatric patients appear to tolerate gemtuzumab reasonably well, and several antileukemic responses have been observed.3

Categories of antibody therapies Unconjugated antibody Several mechanisms have been proposed to account for tumor ablation using unconjugated antibody (Fig. 25.1). In some instances, cell surface binding to FAS (CD95) or obligate growth factor receptors can directly induce apoptosis. More commonly, antibody-dependent cell-mediated cytotoxicity (ADCC) occurs after binding of the antibody to antigen, inducing a conformational change in the antibody Fc receptor. Subsequently, natural killer (NK) cells, monocytes and tissue macrophages can eliminate target cells bound by antibody through binding of the Fc receptor. The Fc

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 25.1 Clinical studies of monoclonal antibodies for acute leukemias

Target antigen and agent studied

Pts. (no.)

Indicationa

Responses among evaluable patients

Reference

CD33 Unconjugated antibody M195 (dose escalation) HuM195 (as consolidation therapy) HuM195 (pilot study of supersaturating doses) HuM195 (randomized study of supersaturating doses) Chemotherapy plus or minus HuM195

10 31 10 50 191

Advanced AML, CMML APL in first remission Advanced AML Advanced AML Advanced AML

0 11/22b 1/10 2/50 40/94c

10 16 13 14 15

Antibody conjugated to calicheamicin (gemtuzumab ozogamicin) Phase I dose escalation Phase I dose escalation (pediatric) Phase II studies Gemtuzumab combined with chemotherapy Gemtuzumab combined with chemotherapy

40 29 142 55 42

Advanced AML Advanced AML AML in first untreated relapse De novo AML De novo AML

8/40 8/29 42/142 41/55 15/18

20 3 21 23 24

Advanced AML, MDS, and CML Advanced AML, CMML Advanced AML

17/24

18

0/18 1/19

26 25

Radiolabeled antibody given as monotherapy 131 Iodine-M195 dose escalation 213 99

Bismuth-HuM195 dose escalation Yttrium-HuM195 dose escalation

CD45 Radiolabeled antibody given with transplant preparative regimen 131 Iodine BC8 dose escalation combined with TBI and cyclophosphamide 131 Iodine BC8 combined with busulfan and cyclophosphamide

24 18 19

44

10/34

28

24

Advanced ALL, AML, and MDS AML in first remission

18/24

29

CD19 Antibody conjugated with genistein B43-genistein immunoconjugate

15

Advanced ALL

1/14

31

Antibody conjugated with blocked ricin Anti-B4-blocked ricin Anti-B4-blocked ricin during intensification

19 46

Advanced ALL and lymphoma Untreated de novo ALL

0/19 No obvious benefit

32 33

a

AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia. b c

Negative for t(15;17) RT-PCR product after treatment.

Among patients treated with HuM195.

portion of antibody bound to cells may also induce apoptosis by complement fixation in complement-dependent cellular cytotoxicity (CDC). Although CDC is thought to be clinically less important, Campath-1, a monoclonal antibody directed against CD 52-expressing mature B and T cells, appears to use CDC to achieve a marked depletion of these cells in vivo. Unfortunately, similar approaches using unconjugated antibody to leukemic cells, such as CD33,

have been less effective in patients harboring large tumor burdens. However, this approach might have some benefit for patients with minimal disease (discussed below).

Immunoconjugates, immunotoxins, and radiolabeled antibodies Since treatment with supersaturating doses of unconjugated anti-CD33 antibody showed limited efficacy among

Antibody-targeted therapy

Fig. 25.1 Simplified mechanisms of antibody-targeted tumor killing. (A) Unconjugated monoclonal antibody binding can initiate host-mediated complement-dependent or antibody-dependent cytotoxicity. (B) Unconjugated antibody binding can interrupt cellular signal transduction, leading to subsequent apoptosis. (C) Radiolabeled antibody can remain external when bound to CD45 or become internalized after binding to the CD33 antigen. Irreparable radiation-induced DNA damage then induces cell death. (D) Internalization of the antibody-antigen complex can deliver antibody-bound chemotherapy or toxins to the interior of the cell. Impaired protein synthesis or irreparable double-stranded DNA damage subsequently results in cell death.

patients with high leukemic cell burdens, newer antibodies have been engineered to deliver cytotoxic agents to cells expressing target antigen. After binding antibody, some cell surface antigens such as CD33 translocate to the interior of the cell, delivering an antibody-drug conjugate into the cytoplasm (Fig. 25.1). Because some cytotoxic agents

require cytoplasmic entry to be effective, it would be necessary to select a surface antigen that internalizes upon antibody binding for tumor ablation to occur. Equally important is the selection of a surface stable antigen as a target if radioactive isotopes are attached to an antibody; otherwise, these isotopes can be cleaved

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Fig. 25.2 Expression of myeloid antigen CD33 during hematopoiesis. The antigen is first expressed as hematopoietic stem cells begin to lose self-renewal capacity, maturing into more differentiated progenitor cells that eventually give rise to neutrophils and monocytes. Pluripotent hematopoietic stem cells (PSC) or cells of T or B lymphoid differentiation do not express CD33. CFU -MIX, colony forming unit - mixed; BFU -E, burst-forming unit – erythroid; CFU-GM, colony-forming unit – granulocyte/macrophage; CFU-Meg, colony-forming unit – megakaryocyte; L, lymphoid progenitor.

from antibody and rapidly excreted from a cell’s cytoplasm (Fig. 25.1). CD45 represents a class of antigens that remain stable on the cell surface. Although leukemic cells are vulnerable to the DNA-damaging effects of ionizing radiation, systemic toxicity can be problematic. For this reason, monoclonal antibodies have been proposed as a means of delivering high doses of radiation to hematopoietic tissues while minimizing toxicity to normal tissues. Because radioiodinated antibodies that target CD33 in the context of AML remission induction therapy have been only partially effective, 90 Y-and 213 Bi-labeled anti-CD33 antibodies are being actively explored as a means of reducing the tumor burden in patients with advanced leukemia. In an alternative strategy, radioiodinated antibodies that target CD45 have been used to augment the radiation dose delivered to hematopoietic tissues in the context of total body irradiation given prior to HSCT.

Clinical studies targeting normal hematopoietic cell surface antigens AML expressing CD33 CD33 biology CD33 expression is restricted to hematopoietic cells and is tightly choreographed during normal development (Fig. 25.2). Hematopoietic stem cells do not express significant amounts of CD33,4 which can be detected only as these

cells begin to lose self-renewal capacity to mature into more differentiated progenitor cells.5,6 High levels of CD33 are expressed by unsorted leukemic blast cells from more than 80% to 90% of AML patients,5,6 and experimental data suggest that leukemic progenitor cells in some AML patients express CD33. In long-term marrow culture experiments, selective elimination of CD33-expressing cells from leukemic marrow aspirates resulted in the growth of normal, nonclonal granulocytes and monocytes in a proportion of patients.7,8 Although selectively targeting and eliminating CD33-positive cells was hypothesized to induce clinical responses in patients with AML, compelling data generated by other investigators suggest that the more infrequent clonogenic leukemic cell lacks significant CD33 expression. Bonnet and Dick9 demonstrated the growth of AML in an immunodeficient mouse model after infusion of isolated primitive (CD34+ CD38− ) precursors from human leukemia samples obtained from patients with AML.9 While it is difficult to reconcile these apparently conflicting findings, it is plausible that selective elimination of CD33-expressing cells by antibody might result in clinical remissions without fully ablating rare progenitor cells from which the leukemia arose. In fact, the majority of AML patients who achieved remissions after gemtuzumab monotherapy (described below) relapsed at a median of 2 months, but whether or not the leukemic cells escaped therapy because they lacked CD33 expression is unclear. Unconjugated anti-CD33 antibody: HuM195 Early clinical studies employing trace radioiodinated antiCD33 antibody demonstrated selective targeting and rapid saturation of leukemic blast cells in patients with advanced AML.10,11 Because the murine antibodies used in these preliminary studies were somewhat immunogenic, humanized versions of anti-CD33 antibodies were synthesized. In collaboration with Protein Design Labs, investigators from Memorial Sloan-Kettering Cancer Center (MSKCC) performed several clinical evaluations of HuM195 (Zamyl), a humanized monoclonal antibody of subtype IgG1.12 One of 10 evaluable patients with advanced leukemia achieved remission in a preliminary clinical study testing supersaturating doses of HuM195,13 and 2 complete remissions were documented in a larger randomized study that enrolled 50 patients with advanced leukemia.14 Although infusionrelated fevers and chills were frequently observed, serious organ toxicity was not reported. Humanization of the antibody was successful; no immune responses to HuM195 were observed. Because only those patients with minimal tumor burden experienced a clinical benefit from HuM195 monotherapy, further evaluation of unconjugated CD33 antibody

Antibody-targeted therapy

strategies have sought to determine whether antibody therapy might augment the quality of remissions achieved with conventional chemotherapy induction. Feldman et al.15 performed a prospective randomized study of mitoxantrone, cytarabine and etoposide, with or without HuM195, for patients with AML that was initially refractory to therapy or had relapsed with a remission duration of less than 1 year.15 The study enrolled 191 patients with a median age of 57 years. Despite the fact that the two randomized cohorts were well matched for demographic features, the antibody treatment group included a disproportionately large number of patients with high-risk clinical features. Patients who were randomized to receive HuM195 tolerated it well and entered remission at a rate of 36% compared with 28% among those not treated with antibody. However, this difference was not statistically significant (P = 0.28). Disappointingly, a survival difference was not observed between the two treated populations. Although unconjugated antibody failed to significantly improve survival among patients receiving chemotherapy for advanced AML, findings in patients with acute promyelocytic leukemia (APL) in complete remission suggest an effect of HuM195 “maintenance” monotherapy on the residual tumor burden. Among 27 APL patients induced into first remission with all-trans-retinoic acid (ATRA), followed by idarubicin and cytarabine consolidation therapy, 25 had continued presence of APL cells by reverse transcription-polymerase chain reaction (RT-PCR).16 After patients received 6 months of maintenance therapy with HuM-195 given monthly, 11 of 22 evaluable patients converted to RT-PCR negativity. Given the fact that the leukemic progenitor cell in APL tends to be more likely to express CD33, APL may be uniquely sensitive to antibody strategies that target CD33-expressing cells.

Anti-CD33 antibody conjugated with calicheamicin: gemtuzumab ozogamicin Given the limited activity of unconjugated antibody to CD33, investigators sought to conjugate cytotoxic agents to the antibody as a means of improving clinical efficacy. This strategy is based upon the fact that the CD33 antibody-antigen complex translocates into the cell when the antibody complexes with CD33 on the cell surface – a characteristic enabling a targeted intracytoplasmic delivery of a cytotoxic agent. Because anti-CD33 antibody targets CD33-positive cells in marrow and peripheral blood in a rapid and specific manner, calicheamicin (a potent cytotoxic agent) was conjugated to the antibody to create gemtuzumab ozogamicin (Mylotarg).5,7,8,11,17–19 While chemotherapy agents in current use elicit single- or double-

strand lesions through radical intermediates or topoisomerases, calicheamicin cleaves both strands of DNA simultaneously, akin to ionizing radiation. Since the initial p67.6 murine anti-CD33 antibody was potentially immunogenic, a humanized monoclonal IgG4 isotype antibody containing approximately 98% human amino acid sequences was generated. The IgG4 antibody isotype was chosen because it was associated with a lower likelihood of complement activation and a reasonably long half-life in vivo. Gemtuzumab was shown in vitro to lyse leukemic cells with high relative specificity and eliminate tumor xenografts in vivo. A phase I study of gemtuzumab in patients with relapsed or refractory CD33+ AML showed that the agent was reasonably well tolerated, ablating leukemia from the blood and marrow of 8 (20%) of 40 evaluable patients.20 Similar to the infusion syndrome observed with unconjugated antibody, a postinfusion syndrome of fever and chills was the most common side effect. A series of prospective, international phase II studies in 142 patients with CD33-positive AML in first untreated relapse showed that the agent induced clinical responses in 30% of patients.21 Among the 142 patients, 46% had fewer than 5% blasts in the bone marrow after 1 dose of gemtuzumab. Twenty-three patients (16%) achieved complete remission, and 19 (13%) attained this state with incomplete platelet recovery to 100 × 109 /L. Similar remission induction rates were observed among favorable, intermediate and unfavorable risk cytogenetic groups. Not surprisingly, severe neutropenia and thrombocytopenia were universally observed because gemtuzumab ablates normal myeloid and megakaryocytic precursors. However, because the agent is targeted to a normal hematopoietic antigen, mucositis occurred in only 4% of patients. No treatment-related cardiotoxicity, cerebellar toxicity, or alopecia was seen. Furthermore, no patients in the phase II studies developed antiglobulin or anticonjugate immune responses. Although the mechanism remains obscure, gemtuzumab treatment commonly induced moderate but typically reversible hepatic transaminase and bilirubin elevations. Based on these clinical and toxicity data, the US Food and Drug Administration approved gemtuzumab ozogamicin as monotherapy for the treatment of patients with CD33-positive AML in first relapse who are older than 60 years and not considered candidates for cytotoxic chemotherapy.22 Remission durations after gemtuzumab monotherapy tended to be brief unless patients received consolidation with HSCT. Among these 142 patients with recurrent AML (Fig. 25.3), the median relapse-free survival was at least 8.9 months in the cohort receiving allogeneic (n = 10) or

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Fig. 25.3 Relapse-free survivals following gemtuzumab ozogamicin monotherapy, with (
, n = 15) or without ( , n = 23) subsequent HSCT, in patients with CD33+ AML in first relapse. The median result for the group receiving both gemtuzumab and HSCT was 8.9 months, compared with 2.1 months for patients given gemtuzumab alone.

•

autologous (n = 5) HSCT.21 By contrast, in the group receiving no additional therapy (n = 23), the median relapsefree survival was only 2.1 months. These findings suggest that postremission therapy, particularly in the form of allogeneic HSCT, is necessary for patients to retain extended remissions. Recent studies evaluating gemtuzumab in combination with conventional chemotherapy in patients with de novo AML are beginning to demonstrate both safety and remarkable efficacy.23,24 In a pilot trial conducted by the Medical Research Council, 67 adult patients with de novo AML received gemtuzumab at varied dose levels in combination with daunomycin and cytarabine. The overall survival of all patients receiving gemtuzumab at 3 mg/m2 with the first course of therapy was 68% at 12 months.23 To formally corroborate these early tantalizing data, phase III randomized studies of gemtuzumab combination induction regimens are being performed.

Radiolabeled anti-CD33 antibody for advanced AML Investigators at MSKCC performed a therapeutic doseescalation study of M195, a murine antibody directed against CD33 labeled with iodine-131, in patients with relapsed or refractory myeloid leukemias.18 In 22 evaluable patients, whole-body gamma imaging showed considerable uptake of antibody into bone marrow. Twentythree of 24 evaluable patients (96%) experienced decreases in peripheral blood white blood cell counts. Bone marrow biopsies examined quantitatively showed substantial decreases in the number of blasts in the vast majority of patients. Human antimouse antibody developed in 37% of

the patients treated. A maximum-tolerated dose was not reached. Because clinically significant degrees of cytoreduction were achievable with radiolabeled antibody treatment, and the therapy was well tolerated, the investigators sought to humanize the antibody to diminish the immunogenic potential of the treatment, using 213 Bi or 90 Y in further studies in patients with advanced AML.25,26 213 Bismuth emits an alpha particle with a path length of 0.06 mm and a 46-minute half-life. In a phase I study, 18 patients with advanced leukemia received escalating doses of 213 Bi-HuM195.26 Specific targeting of bone marrow, liver and spleen was documented within 10 minutes of administration by gamma camera. In accord with the ablation of CD33-positive cells, myelosuppression lasting from 8 to 34 days was also observed. No complete remissions were obtained, but reductions in peripheral blood leukemic blast cell counts and percentages of marrow myeloblasts were documented in almost all patients.

Acute leukemias expressing CD45 CD45 biology Almost all normal white blood cells, virtually all AMLs and most ALLs densely express the CD45 cell surface antigen. Because it is present on both lymphoid and myeloid cells, radiolabeled anti-CD45 antibody can deliver radiation to sites of leukemic involvement in marrow, lymph nodes, and spleen. Distinct from the CD33 antigen, the CD45 antigen remains stable on the cell surface after antibody binding.27 Therefore, while CD33 represents an excellent target for antibody-drug conjugates because it modulates upon antibody binding, the lack of internalization and of antibodybound CD45 antigen provides a potential clinical advantage when 131 I-labeled antibody is used. Radiolabeled anti-CD45 antibody followed by HSCT for advanced acute leukemias 131 I-labeled BC8 (murine anti-CD45) antibody has been evaluated in patients with AML, myelodysplastic syndrome, and ALL, in combination with conventional marrow transplant preparative regimens. A phase I study enrolled 44 patients with advanced leukemias to determine the maximum amount of radiation that could be delivered by 131 IBC8 antibody in combination with cyclophosphamide and 12 Gy total-body irradiation followed by matched related or autologous transplantation.28 Thirty-four patients who appeared to receive a higher estimated radiation dose to marrow and spleen than to liver, lung or kidney then received a therapeutic dose of BC8 antibody labeled with 131 I. The maximum tolerated dose of radiation delivered

Antibody-targeted therapy

by antibody was determined to be 10.5 Gy. Veno-occlusive disease of the liver occurred in 1 of 6 patients treated at this dose level. With regard to clinical efficacy, 10 of 34 patients who received a therapeutic dose of 131 I-BC8 antibody were alive and disease-free 33 to 107 months post-transplantation. Similarly, in a study in patients with AML in first remission receiving a matched related HCT, 131 I-labeled anti-CD45 antibody was combined with busulfan and cyclophosphamide.28,29 At the time of the report, 18 of 24 patients with AML transplanted in first remission were alive and disease free from 13 to 66 months (median, 45 months) after transplantation.

Acute leukemias expressing CD19 CD19 biology The 95-kDa cell surface antigen CD19 appears to help establish signaling thresholds for antigen receptors and other surface receptors on normal B lineage lymphocytes. It is expressed by virtually all B lymphocytes, including early B progenitor cells, and is also expressed by leukemic blasts of most patients with ALL. Anti-CD19 conjugated with genistein Genistein, a naturally occurring PTK inhibitor, was conjugated to a monoclonal antibody directed against CD19 (B43).30 In a phase I clinical study targeting the membraneassociated CD19-Src family PTK complex with B43genistein immunoconjugate, 15 patients aged 4 to 60 years with advanced CD19+ B-lineage ALL received escalating doses of B43-genistein.31 The drug was reasonably well tolerated, and the most common infusion-related adverse event was fever. Among the patients enrolled, 1 of 14 evaluable ALL patients achieved a complete remission, and 2 additional patients experienced reductions in percentages of blasts in sequential bone marrow aspirates. Anti-CD19 conjugated with blocked ricin Ricin consists of a cytotoxic protein that inactivates ribosomes, thereby disrupting protein and leading ultimately to apoptosis. Using blocked ricin conjugated to an antibody directed against CD19, Dinndorf et al.32 performed a phase I trial of anti-B4-blocked ricin in 19 children with advanced ALL or lymphoma. Capillary leak syndrome proved to be dose-limiting, and reversible elevations in hepatic transaminases were also seen. Five patients developed human antimouse antibodies or human antiricin antibodies. Unfortunately, no complete or partial remissions were observed. Because anti-B4-blocked ricin is capable of killing residual leukemic cells that are resistant to initial chemotherapy,

the CALGB hypothesized that this agent might be particularly effective for patients who achieve clinical remissions but harbor minimal residual disease. In this study, 82 adults with untreated ALL were enrolled, and 66 achieved remission with a previously evaluated standard chemotherapeutic regimen.33 The 46 patients whose leukemias expressed CD19 were treated with anti-B4-blocked ricin during remission, while patients with CD19-negative ALL received highdose cytarabine. The most common toxicity observed with ricin treatment was asymptomatic and transient elevation of hepatic function tests in 72% of patients. Additionally, lymphopenia was observed in 46% of patients. Antibodies to the anti-B4-blocked ricin were detected in 2 patients as well. Unfortunately, monitoring for minimal residual disease both before and after the anti-B4-blocked ricin intensification failed to show any clinical benefit from this therapeutic approach.

Summary and future study directions The last decade has witnessed slow but steady progress in the creation of targeted therapies for children with cancer, warranting three general observations. First, although impressive efficacy has been observed for rituxan in adult patients with non-Hodgkin lymphoma, unconjugated monoclonal antibodies directed against normal hematopoietic antigens have demonstrated only minimal efficacy in children with acute leukemias. Because unconjugated antibodies appear unable to ablate large tumor burdens, recent clinical studies have sought to augment targeted cytotoxicity by conjugating antibodies with radionuclides or cytotoxic agents. A first generation of antibody-targeted chemotherapy has made its way to the clinic, undoubtedly to be followed by second-generation strategies that will expand antileukemic efficacy without damaging normal tissues. Monotherapy with one such agent, gemtuzumab ozogamicin, has induced remissions in 30% of adult patients with relapsed AML with what appears to be a different toxicity profile than that observed in the setting of conventional combination chemotherapy. Children appear to tolerate this agent reasonably well,3 and strategies using anti-CD33 or anti-CD45 antibodies to target radiation to hematopoietic tissues have shown similar successes in patients with advanced acute leukemias. Second, although some clinical responses have been observed with antibody conjugate monotherapy, remission rates and the quality of remissions are not likely to improve until antibody conjugates are combined with conventional chemotherapy regimens. While emerging data have shown that combinations of conventional chemotherapy and

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gemtuzumab result in a relatively high remission induction rate for patients with newly diagnosed AML, these findings must be regarded as highly preliminary. As noted above, prospective randomized studies are being performed to determine whether the addition of gemtuzumab to standard induction regimens can improve both the remission rate and the quality of remissions achieved without increasing overall toxicity. Finally, it is also recognized that the great appeal of targeted therapy for pediatric cancer lies in the potential for significant reductions of systemic toxicity, a goal that cannot be realized through strategies that simply augment current intensive chemotherapy regimens. Unfortunately, monotherapy with the antibody conjugates described in this chapter has not yet been sufficiently efficacious to replace conventional chemotherapy. However, once mature data are available from larger numbers of antibody conjugates, it is conceivable that multiple targeted antibody conjugate combinations might be developed and one day take the place of conventional chemotherapy regimens. REFERENCES 1 Woods, W. G., Kobrinsky, N., Buckley, J. D., et al. Timedsequential induction therapy improves postremission outcome in acute myeloid leukemia: a report from the Children’s Cancer Group. Blood, 1996; 87: 4979–89. 2 K¨ohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975; 256: 495–7. 3 Arceci, R. J., Sande, J., Lange, B., et al. Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33+ acute myeloid leukemia. Blood, 2005; 106: 1183–8. 4 Andrews, R. G., Singer, J. W., & Bernstein, I. D. Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med, 1989; 169: 1721–31. 5 Griffin, J. D., Linch, D., Sabbath, K., Larcom, P., & Schlossman, S. F.. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res, 1984; 8: 521–34. 6 Dinndorf, P. A., Andrews, R. G., Benjamin, D., et al. Expression of normal myeloid-associated antigens by acute leukemia cells. Blood, 1986; 67: 1048–53. 7 Bernstein, I. D., Singer, J. W., Andrews, R. G., et al. Treatment of acute myeloid leukemia cells in vitro with a monoclonal antibody recognizing a myeloid differentiation antigen allows normal progenitor cells to be expressed. J Clin Invest, 1987; 79: 1153–9. 8 Bernstein, I. D., Singer, J. W., Smith, F. O., et al. Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia. Blood, 1992; 79: 1811–16.

9 Bonnet, D. & Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997; 3: 730–7. 10 Scheinberg, D. A., Lovett, D., Divgi, C. R., et al. A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol, 1991; 9: 478–90. 11 Appelbaum, F. R., Matthews, D. C., Eary, J. F., et al. The use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia. Transplantation, 1992; 54: 829–33. 12 Co, M. S., Avdalovic, N. M., Caron, P. C., et al. Chimeric and humanized antibodies with specificity for the CD33 antigen. J Immunol, 1992; 148: 1149–54. 13 Caron, P. C., Dumont, L., & Scheinberg, D. A. Supersaturating infusional humanized anti-CD33 monoclonal antibody HuM195 in myelogenous leukemia. Clinical Cancer Res, 1998; 4: 1421–8. 14 Feldman, E., Kalaycio, M., Weiner, G., et al. Treatment of relapsed or refractory acute myeloid leukemia with humanized antiCD33 monoclonal antibody HuM195. Leukemia, 2003; 17: 314– 18. 15 Feldman, E. J., 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: 4110–16. 16 Jurcic, J. G., DeBlasio, T., Dumont, L., Yao, T. J., & Scheinberg, D. A. Molecular remission induction with retinoic acid and antiCD33 monoclonal antibody HuM195 in acute promyelocytic leukemia. Clin Cancer Res, 2000; 6: 372–80. 17 Scheinberg, D. A., Tanimoto, M., McKenzie, S., et al. Monoclonal antibody M195: a diagnostic marker for acute myelogenous leukemia. Leukemia, 1989; 3: 440–5. 18 Schwartz, M. A., Lovett, D. R., Redner, A., et al. Dose-escalation trial of M195 labeled with iodine 131 for cytoreduction and marrow ablation in relapsed or refractory myeloid leukemias. J Clin Oncol, 1993; 11: 294–303. 19 Hinman, L. M., Hamann, P. R., Wallace, R., et al. Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res, 1993; 53: 3336–42. 20 Sievers, E. L., Appelbaum, F. A., Spielberger, R. T., et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood, 1999; 93: 3678–84. 21 Sievers, E. L., Larson, R. A., Stadtmauer, E. A., et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33positive acute myeloid leukemia in first relapse. J Clin Oncol, 2001; 19: 3244–54. 22 Bross, P. F., Beitz, J., Chen, G., et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res, 2001; 7: 1490–6.

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23 Kell, J. W., Burnett, A. K., Chopra, R., et al. Mylotarg (gemtuzumab ozogomycin: GO) given simultaneously with intensive induction and/or consolidation therapy for AML is feasible and may improve the response rate [abstract]. Blood, 2002; 100: 199. 24 De Angelo, D., Schiffer, C., Stone, R., et al. Interim analysis of a Phase II study of the safety and efficacy of gemtuzumab ozogamicin (Mylotarg®) given in combination with cytarabine and daunorubicin in patients <60 years old with untreated acute myeloid leukemia [abstract]. Blood, 2002; 100: 198. 25 Jurcic, J., Divgi, C., McDevitt, M., et al. Potential for myeloablation with yttrium-90-HuM195 (anti-CD33) in myeloid leukemia [abstract]. Proc ASCO, 2000; 19: 8. 26 Jurcic, J. G., Larson, S. M., Sgouros, G., et al. Targeted alpha particle immunotherapy for myeloid leukemia. Blood, 2002; 100:1233–9. 27 Jagt, R. H. van der, Badger, C. C., Appelbaum, F. R., et al. Localization of radiolabeled antimyeloid antibodies in a human acute leukemia xenograft tumor model. Cancer Res, 1992; 52: 89–94. 28 Matthews, D. C., Appelbaum, F. R., Eary, J. F., et al. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total

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body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood, 1999; 94:1237–47. Matthews, D. C., Appelbaum, F. R., Eary, J. F., et al. [131I]-antiCD45 antibody plus busulfan/cyclophosphamide in matchrelated transplants for AML in first remission [abstract]. Blood, 1996; 88: 142. Uckun, F. M., Evans, W. E., Forsyth, C. J., et al. Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases. Science, 1995; 267: 886–91. Uckun, F. M., Messinger, Y., Chen, C. L., et al. Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor. Clin Cancer Res, 1999; 5: 3906–13. Dinndorf, P., Krailo, M., Liu-Mares, W., et al. Phase I trial of anti-B4-blocked ricin in pediatric patients with leukemia and lymphoma. J Immunother, 2001; 24: 511–16. Szatrowski, T. P., Dodge, R. K., Reynolds, C., et al. Lineage specific treatment of adult patients with acute lymphoblastic leukemia in first remission with anti-B4-blocked ricin or high-dose cytarabine. Cancer, 2003; 97: 1471–80.

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26 Adoptive cellular immunotherapy Helen E. Heslop and Cliona M. Rooney

Introduction The possibility that the immune system can be harnessed to play a role in eradicating leukemia has long been an attractive concept. Numerous experiments in animal models have convincingly shown that T lymphocytes recognize and kill malignant cells. However, human immunotherapy with nonspecific stimulants, such as BCG (Calmette– Gu´erin bacillus), has not had a successful history. In the last few years, improved knowledge of the molecular basis of antigen presentation and T-cell recognition of antigen has made it clear that many tumors possess antigens that could be targets for activated T cells.1 Interest in cellular immunotherapy has also been stimulated by clinical studies showing the efficacy of unmanipulated donor T cells as therapy for relapse after allogeneic bone marrow transplantation. In this chapter we review clinical immunotherapy strategies now being applied in the treatment of leukemia.

Immune system recognition of tumor cells Recent advances in basic immunology have provided important insights into the mechanisms by which the immune system recognizes tumor cells. Dissection of the processes of antigen presentation and T-cell recognition of antigen has yielded especially useful information in this regard. Advances in genomics have also simplified the identification of putative tumor antigens through the use of new informatics tools to deduce epitopes from candidate genes.

Antigen presentation Antigen-presenting cells recognize either endogenous cellular proteins or exogenous proteins, such as tumor debris,

process them into short peptide fragments, and then present these fragments on the cell surface in association with MHC molecules for subsequent presentation to T cells. Classically, cytosolic proteins are thought to undergo proteolytic cleavage into peptides that are transported to the endoplasmic reticulum by transporter-ofantigen processing (TAP) proteins. They then bind to MHC class I molecules and are transported to the cell surface, where the peptide–MHC complex is presented to CD8+ T cells. By contrast, exogenous antigen that has been ingested by antigen-presenting cells is processed in intracellular vesicles before association with class II molecules and subsequent presentation to CD4+ T cells. At least in specialized antigen-presenting cells, some exogenous antigens may gain access to the cytosolic pathway, so that they are presented by class I molecules. The most efficient antigen-presenting cell is the dendritic cell, which can ingest exogenous protein for subsequent presentation in association with class I or class II MHC molecules.2 Dendritic cells also express a large number of cell adhesion and costimulatory molecules to facilitate their interaction with immune system effectors. Unlike other types of cells that can present antigen effectively, such as B cells and macrophages, dendritic cells can induce both primary and secondary immune responses in vitro, although the molecular basis of this characteristic is unknown. The processing of proteins may also influence which peptides are available to associate with MHC molecules. The Epstein–Barr virus (EBV)-encoded antigen EBNA-1, which is expressed in EBV-positive Burkitt’s lymphoma cells, is prevented from entering the class I processing pathway by an internal glycine alanine repeat region.3

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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In addition, the selectivity of TAP proteins can influence which peptides are presented on the cell surface.4 The binding specificity of the peptide to allelic forms of MHC molecules also influences antigen presentation. Because of MHC polymorphism, different individuals present different peptides derived from tumor antigens. The immunogenicity of a peptide fragment also correlates with the duration of its presentation by MHC molecules.5

Table 26.1 Potential targets for immunotherapy Target

Examples

Malignancies

Viral antigens

EBV

Non-Hodgkin lymphoma99 Hodgkin disease99 Non-Hodgkin lymphoma9 CML, AML100 Acute leukemia21 Testicular and many hematologic malignancies13 CML, acute leukemia27 Many types of malignancies15 CML24

Differentiation antigens Cancer–testis antigens

Requirements for T-cell activation T lymphocytes recognize cellular proteins as short fragments of processed peptides presented in conjunction with MHC molecules on the surface of malignant or virusinfected cells or on “professional” antigen-presenting cells. After initial recognition, a second signal must be generated, usually from interaction of one of the B7 family of molecules on antigen-presenting cells with its CD28 or CTLA4 ligand. If antigens and T-cell receptors interact in the absence of a second signal, then anergy rather than activation results. The type of antigen-presenting cell can therefore influence whether activation will occur. Most tumor cells present antigen poorly, and it is likely that tumor-derived antigens are transferred in vivo to antigen-presenting cells derived from bone marrow. The prior experience of the responding T cell also influences the likelihood of activation. The requirements for induction of a secondary or recall immune response are simpler than those for induction of a primary response from a naive T cell. Finally, immune activation requires a certain threshold number of interactions between the T-cell receptor and the peptide– MHC complex.6

Requirements for tumor recognition by immune system cells If a tumor is to be a target for cytotoxic T lymphocytes (CTLs), several conditions must be met. First, the tumor must contain unique proteins capable of providing epitopes for specific immune responses. The tumor cells must also express MHC antigens, present relevant peptides frequently enough and for sufficient durations to engage responder T cells, and express costimulatory molecules to induce T-cell activation. Failure to meet the last criterion may lead to anergy. The requirement for costimulatory molecules on tumor cells may be overcome if tumor debris is phagocytosed and presented to T cells by a specialized antigen-presenting cell. An additional requirement is that the peptide–MHC complex be within the immunologic repertoire of the individual.

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Minor antigens Tumor-specific antigens

SV40 PR-1 CD45 MAGE-1 NY-ESO HA1, HA2 Telomerase BCR-ABL

Abbreviations: EBV, Epstein–Barr virus; SV40, Simian virus 40; PR-1, proteinase-1; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; MAGE-1, melanoma antigen-1; HA1, histocompatability antigen 1.

Potential CTL targets on leukemic cells In recent years, several groups have identified a number of novel immunogenic tumor proteins by screening tumorderived expression libraries or tumor cells using autologous sera.7 The identification of these antigens, and the mapping of specific epitopes recognized by CD4+ and CD8+ T cells, has led to the identification of a large number of candidate antigens.8 Potential antigens for targeting on leukemia cells fall into several major categories (Table 26.1).

Virus-associated proteins Many malignancies are associated with viruses that will present unique epitopes. Epstein–Barr virus is associated with lymphoma in immunodeficient patients as well as with a subset of patients with Hodgkin disease or nonHodgkin lymphoma. Simian virus 40 (SV40) has recently been reported to be significantly associated with some types of non-Hodgkin lymphoma.9 Unlike most malignant cells, which probably express small quantities of a single modified peptide as a CTL target, virus-transformed cells may express a range of viral antigens that can be presented for CTL recognition. These cells would therefore be much more accessible for T-lymphocyte-mediated immunotherapy than would tumor cells transformed by other mechanisms.

Differentiation antigens Another category of targets comprises differentiation antigens that are selectively expressed in tumor cells, such as proteinase 3 (PR-3), a serine protease overexpressed in progenitor cells of chronic and acute myeloid leukemias,10

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and Wilms tumor antigen 1 (WT-1) which is expressed at a higher level in leukemic compared with normal hematopoietic cells.11 Two distinct HLA-A0201-presented epitopes of WT-1 will distinguish between normal and leukemic progenitors.12 Cancer–testis antigens, or CTAs, represented by proteins with restricted expression among tumor cells and germinal tissues, including the family of MAGE genes (MAGE-1 to -10), BAGE, GAGE, SSX-1 to -9 and NY-ESO-1,13 are also overexpressed in some hematologic malignancies.

Tumor-specific antigens Perhaps the most attractive target for immunotherapy would be a tumor cell-specific antigen crucial to the malignant behaviour of the cell, so that its downregulation would result in tumor cell death. Candidates for such proteins include tumor-specific proteins resulting from chromosomal translocations, such as BCR-ABL14 or proteins overexpressed in tumor cells, such as telomerase.15 Several groups have evaluated universal tumor antigens. One of these, hTERT,15 is expressed in up to 90% of cancer patients and plays an important role in cell growth and development; however, it is also expressed in some hemopoietic stem cells. Alloantigens In the setting of allogeneic bone marrow transplantation (BMT), alloantigens that differ between donor and recipient are potential targets for T-cell recognition. Differences in MHC molecules due to the use of a mismatched family member or a serologically matched unrelated donor for BMT afford a good example. Even when MHC antigens are identical, minor histocompatibility antigens can provide targets for allorecognition, although mismatch is associated with an increased risk of graft-versus-host disease (GVHD).16 These naturally processed peptides, derived from normal cellular proteins, may evoke a strong MHC-restricted response when different polymorphisms are present in donor and recipient. The degree of disparity in minor antigens will vary with different donor recipient pairs and will probably be higher in cases of unrelated donor transplantation, in which the genetic background of donor and recipient are different. Some of these minor antigens have been identified, including the HA2 antigen, which encodes a member of the myosin family,17 and the HY antigen, which encodes a peptide derived from the SMCY protein selectively expressed in male cells.18 In most cases, nucleotide polymorphisms in the respective genes are responsible for immunogenicity, although a recent report details one antigen that is immunogenic because of

differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion.19 Polymorphisms of the adhesion molecule CD31 are also a target for allorecognition, and the risk of GVHD is increased when the donor and recipient have different variants.20 In all of these cases, alloreactivity results in GVHD as well as graft-versus-leukemia (GVL) reactions. However, there is evidence that GVHD can be distinguished from GVL. Differences in the susceptibility of leukemic cells and normal hemopoietic progenitors to minor antigen-specific CTLs have been observed in vitro and may contribute to a selective GVL effect. The pattern of tissue expression of minor antigens varies, and those selectively expressed on hemopoietic cells or on particular lineages would provide specific targets for recognition. In the pretransplant setting, CTLs specific for a hemopoietic antigen may also provide antileukemic activity. For example, allospecific CTLs specific for CD45- derived peptides induce potent activity against leukemic progenitors.21

Can T cells respond to tumor antigens? There is evidence that the immune system can recognize unique peptide sequences contained within malignant cells. For example, one can generate CD4+ T-cell clones that specifically proliferate, both in vitro and in vivo, in response to a RAS peptide with a substitution at residue 12 but not in response to the wild-type peptide.22 Similarly, peptides derived from the fusion portion of BCR-ABL protein generated by the t(9;22) translocation in chronic myeloid leukemia (CML) cells elicit proliferative responses in vitro.23 However, evidence for the processing and presentation of tumor-specific peptides on leukemic cells in quantities sufficient to induce T-cell activation is difficult to find.24 There is circumstantial evidence that T-cell lines able to selectively recognize leukemic cells can be generated, although several groups have shown that CTLs specific for tumor-derived peptides will recognize target cells only when the protein has been overexpressed. For example, CTLs induced by a peptide derived from the PMLRAR
protein generated by the t(15;17) in acute promyelocytic leukemia will subsequently recognize the same peptide expressed on autologous lymphoblastoid cells25 but do not recognize malignant blasts that express this protein.26 This finding is not surprising, as the majority of peptides are never presented on the cell surface, or they may be presented at very low frequency or for short times. Thus, while specific CTLs may be elicited by saturation of antigen-presenting cells with peptide in vitro, malignant cells naturally expressing the protein may not present adequate quantities of the required peptide for recognition.

Adoptive cellular immunotherapy

Table 26.2 Clinical trial of adoptively transferred T cells Cell type

Clinical application

Unmanipulated donor T cells T-cell subsets

Relapsed hematologic malignancies PT32 CD4-selected37 or CD8-depleted39 cells for relapsed leukemic post transplantation Th2 and Tc2 cells (Phase I trial)41 Donor T cells briefly activated ex vivo and transduced with a suicide gene to treat relapsed hematologic malignancies post transplantation 77,81 T cells activated ex vivo with CD3 and CD28 to treat relapsed/refractory NHL post transplantation Expanded cytokine-induced killer or CD8+ NK-T cells in humans42 Treatment after allogeneic transplantation to reduce risk of relapse44,45 EBV-specific CTLs for prophylaxis and treatment of EBV lymphoma after BMT48–50 or SOT52,54 LMP1- or 2-specific CTLs for Hodgkin’s disease61 Leukemia-specific CTLs for relapsed CML101,102 Minor antigen-specific CTLs for relapsed leukemia 66 CD20 chimeric receptor-transduced T cells for NHL91

T-cells nonspecifically activated ex vivo

Allodepleted T cells Antigen-specific CTLs

Chimeric receptor-transduced T cells

Abbreviations: NHL, non-Hodgkin lymphoma; CTL, cytotoxic T lymphocyte; CML, chronic myeloid leukemia; BMT, bone marrow transplantation; SOT, solid organ transplantation.

More recently, studies with tetramers have provided evidence to support the candidacy of some of the antigens described in the previous section; that is, an increase in PR1-specific cells has been shown in patients with CML responding after BMT,10 and an increase in HA1- or HA2specific T cells has been demonstrated in patients responding to donor leukocyte infusions.27

Clinical experience with cellular immunotherapy Selected clinical trials of adoptively transferred T cells are summarized in Table 26.2.

Use of unmanipulated donor T cells Although it has long been known that T cells will eradicate virus-transformed or malignant cells in animal models, the clinical benefit to humans from adoptively transferred T cells became apparent only recently. Much of this evidence comes from the setting of allogeneic BMT, where several studies have demonstrated that T cells may help eradicate cancer and viral infections.28 Leukemia patients who develop GVHD after allogeneic BMT have a lower probability of relapse than do patients who lack this complication.29 Conversely, patients with CML who receive marrow from an HLA-matched sibling that has been depleted of mature T cells have a much higher risk of relapse than do patients who receive unmanipulated grafts.30 The likely explanation for these observations is that the alloreactive T cells in

the donor graft are able to destroy residual leukemic cells in the host. In support of this concept are the results of clinical studies showing that infusions of donor leukocytes can eradicate relapsed leukemia after transplantation31,32 as well as EBV-associated post-transplant lymphoproliferative disease.33,34 The use of donor T cells as immunotherapy was first described in 1990, when this strategy induced remissions in three patients with relapsed CML.35 Updates of the European and US experience32,36 have shown that approximately 70% of CML patients who are treated for cytogenetic or hematologic relapse in the chronic phase after BMT can attain durable remissions when treated with donor mononuclear cells. The mechanism of this response is incompletely understood but is likely to be T-cell-mediated, as selected CD4+ cells can also induce remissions.37 Although donor mononuclear cells produce an impressive therapeutic response, they can also produce aplasia and GVHD due to alloreactive cells in the administered product. Attempts have been made to separate the GVL effect from unwanted alloreactivity by administering either smaller doses of cells or lymphocyte subsets. There is some evidence from MacKinnon and colleagues38 that the risk of alloreactivity can indeed be reduced by administering smaller doses of cells: some patients who received 1 × 107 CD3+ cells/kg attained remission without developing GVHD. However, the degree of alloreactivity varies among donor-recipient pairs, so that it may not be possible to identify a dose that uniformly produces GVL without GVHD. An alternative approach is to administer

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T-cell subsets. Two studies have shown that either CD8depleted or CD4-selected cells can induce remission in CML patients with only a low incidence of GVHD.37,39 Although these results suggest that CD4+ cells may be important in mediating antileukemic effects, it is also possible that small numbers of NK or CD8+ cells were able to expand in vivo and contribute to the antileukemic effects. Adoptive immunotherapy with donor mononuclear cells is less successful in BMT recipients with either CML in blast crisis or acute leukemia, where the remission rate is lower and remissions are less durable. This approach rarely succeeds in patients with acute leukemia unless the leukemia burden can be reduced by prior chemotherapy. There is some evidence that the use of immunostimulatory cytokines such as interleukin-2 (IL-2) may amplify GVL mechanisms and induce remissions in patients who have failed to respond to donor lymphocyte infusions.40 GM-CSF (granulocyte-macrophage colony-stimulating factor) has also been administered to improve antigen presentation. The effectiveness of donor lymphocytes as therapy has stimulated interest in using this therapy prophylactically. In animal studies, delayed administration of donor immune system cells produced antileukemic activity without alloreactivity. However, the unpredictable degree of alloreactivity in human clinical transplantation makes it difficult to rely on this approach, which also may be complicated by the induction of GVHD. Because the use of these crude alloreactive lymphocyte preparations is frequently complicated by severe GVHD, it is important to determine if the observed antitumor effect of allografts and infused donor T cells is simply another manifestation of GVHD, in which the tumor cells are targets of alloreactions. If so, any clinical applications will be limited. If, however, certain Tlymphocyte clones can recognize tumor-specific antigens in a more specific manner then one would predict from current knowledge, it should be possible to use these cells to eradicate malignancy without damaging normal tissue through a GVHD mechanism.

Expanded cells To overcome the problem of GVHD, investigators have evaluated whether expansion of T cells ex vivo may allow GVL and GVHD to be separated. In animal models, the culture of functionally defined T-cell subsets, such as Tc2 or Th2 cells, has allowed antitumor effects to be produced in the absence of alloreactivity, and this approach is being tested in human trials.41 Expanded cells have also been used in the autologous setting to treat relapse or to augment T-cell function against minimal residual

disease. A population of cytokine-induced, CD3- and CD28-activated cells have shown promise in mediating antitumor effects in murine models and are being evaluated in patients with lymphoma.42 CD3- and CD2-activated cells have been administered to patients with relapsed, refractory or chemotherapy-resistant, aggressive nonHodgkin lymphoma following high-dose chemotherapy and CD34-selected autologous hematopoietic cell transplantation (HCT). Preliminary results suggest that this approach is associated with a rapid recovery of lymphocyte counts, but as yet there are no data on antitumor activity.43

Allodepleted T cells An alternative approach to the problem of alloreactivity is to selectively deplete the T-cell product of alloreactive cells expressing activation markers in response to alloantigen. Several studies are evaluating this strategy with an immunotoxin directed against the activation marker CD25.44–46 Preclinical studies have shown that this procedure can deplete alloreactive cells while preserving T cells reactive with viruses such as CMV and EBV and tumor antigens such as PR1 and HA1.45 In a Phase I clinical study that accrued 15 patients, early T-cell expansion in the absence of GVHD was seen in patients who were treated at higher doses.44

Therapeutic applications of antigen-specific CTL transfer The use of antigen-specific CTLs may overcome the problems of alloreactivity and a low frequency of such immune cells in any situation where an antigen recognized by the cytotoxic lymphocytes is known. To generate these cells ex vivo requires that several conditions be met: (1) there must be a defined antigen expressed by the putative target cell; (2) there must be an antigen-presenting cell that can effectively present the antigen to T cells; and (3) an immune donor is helpful because of the difficulty of generating a primary response ex vivo. EBV-induced lymphoproliferative disease (EBV-LPD) occurring after allogeneic BMT is an excellent model for testing cellular immunotherapy. In the absence of virusspecific CTLs, EBV-infected B cells can grow in an uncontrolled fashion both in vitro and in vivo. Tumor cells from lymphomas developing after transplantation have the same phenotype and pattern of EBV latent-cycle gene expression as do B cells immortalized by EBV in vitro and growing as lymphoblastoid cell lines (LCLs). Because LCLs express viral antigens as well as costimulatory molecules, they function as potent APC and can be used as stimulators to generate virus antigen-specific CTLs. LCLs are

Adoptive cellular immunotherapy

normally highly immunogenic and are susceptible to killing by immune system effector cells, including virusspecific CTLs. Factors predisposing to the outgrowth of EBV-infected B cells after BMT include the use of a mismatched family member donor or a matched unrelated donor, in vitro T-cell depletion of the donor marrow, and the degree of post-transplant immunosuppression. EBVinduced lymphoproliferation affords an ideal system with which to evaluate the efficacy of adoptively transferred antigen-specific CTLs, for several reasons. First, the tumor cells express eight unique virus-encoded antigens, most of which are targets for virus-specific immune responses. Second, EBV-transformed LCLs can be readily prepared from any donor and provide a source of antigen-presenting cells that endogenously express the appropriate viral antigens. Third, most donors are immune to EBV and harbor an abundance of EBV-specific CTL precursors. By contrast, in diseases associated with nonpersistent viruses, virus-specific memory CTLs become increasingly difficult to reactivate as time after infection increases, and the generation of primary responses to antigens in vitro is difficult. Finally, LCLs also provide a source of autologous target cells bearing the tumor antigens for CTL testing. In our study evaluating the efficacy of prophylactic administration of EBV-specific CTLs to hematopoietic stem cell transplant recipients at high risk for EBV-LPD, 60 patients were treated and none developed lymphoproliferative disease, compared with an incidence of 11.5% in a comparable untreated historical control group.47–49 Nine patients with levels of EBV-DNA in peripheral blood high enough to be highly predictive for onset of EBV-LPD, had a rapid decrease in EBV-DNA levels that correlated with an increase in EBV-specific CTL precursor frequency. To track the fate of EBV-specific CTL in recipients and to evaluate adverse effects, we marked cells with a retroviral vector encoding the neomycin-resistance gene before infusion. Gene-marked EBV-specific CTLs have persisted in the patients for longer than 7 years, and there has been no toxicity attributable to expression of the marker gene. In one patient, re-emergence of high EBV-DNA blood levels was accompanied by increased numbers of gene-marked EBVspecific CTLs followed by a decline of both indicators to baseline.48 These observations illustrate the importance of CTL in controlling EBV infection and the effectiveness of this strategy in preventing EBV-LPD. A study from Sweden confirmed the efficacy of EBV-specific CTLs in reducing the viral load in patients with high EBV-DNA levels after hematopoietic stem cell transplantation.50 The patients received 4 weekly injections of 1 × 107 cells/m2 ; however, one of six patients who received EBV-specific CTLs subsequently developed overt EBV-LPD and died of progressive

disease. In vitro testing of the donor CTL line of this patient showed that it lacked a strong EBV-specific component, which may explain this failure of CTL therapy. lmmunotherapy with EBV-specific CTL was also used to treat six hematopoietic stem cell recipients who developed overt lymphoma. Five of these patients responded well with complete regression of bulky tumors, one of which had a biopsy-proven accumulation of gene-marked CTL at the site of disease.49 One of the responders required temporary mechanical ventilation due to airway compromise from the inflammatory response at the disease site. The final patient did not respond and died of progressive disease 24 days after CTL therapy. This failure was attributed to a deletion in the EBNA-3B molecule in the tumor virus, which removed immunodominant epitopes, causing resistance to CTL-mediated killing.51 Analysis of EBV polymorphisms demonstrated that before CTL infusion more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant EBV strain in vivo.51 Thus, escape mutants may be a problem even when polyclonal CTL lines are used, particularly when the tumor burden is large. This success in treating bone marrow transplant recipients has led to evaluation of the immunotherapeutic strategy in solid organ transplant (SOT) recipients, who are also at risk for EBV-LPD. The generation of EBV-specific CTLs for these recipients poses challenges not encountered with HSCT recipients. First, SOT recipients and donors are not HLA matched, and LPD occurring after SOT results from infected recipients’ cells; hence, the use of donorderived CTLs is not appropriate. The options are therefore to use closely matched allogeneic CTLs or autologous CTLs. Comoli et al.52 described seven patients undergoing solid organ transplantation who showed an increase of EBV-specific cytotoxicity after the prophylactic infusion of autologous EBV-specific CTLs, coincident with a decrease in EBV-DNA levels in five patients. Khanna et al.53 used autologous EBV-specific CTLs to treat a renal transplant recipient with EBV-LPD and observed significant regression after the CTL infusion. The emergence of new lymphoma lesions requiring retreatment suggested that the transferred CTLs may not survive in solid organ recipients who receive continuous immunosuppressive therapy. An additional cautionary note is that the patient died with evidence of necrosis and hemorrhage in a pulmonary vein at autopsy. This case history reinforces experience with inflammatory reactions in stem cell transplant recipients and argues for caution when adoptively transferred T cells are used to treat patients with bulky disease. One limitation of using autologous CTLs is the time required for

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generation. A recent report describes eight patients with post-transplant LPD who received partly matched allogeneic EBV-specific CTLs from a frozen cell bank.54 Three of the five patients who completed treatment had a complete response, although the patients also had a reduction of immunosuppression, and the authors did not show persistence of the adoptively transferred allogeneic CTLs. Another study reported two SOT patients who responded to allogeneic CTLs.55 EBV-specific CTLs have therefore shown efficacy for the prophylaxis and therapy of post-transplant LPD, after both HSCT and solid organ transplantation, providing “proof of principle” for immunotherapy approaches; however, with the availability of humanized monoclonal antibodies there are now alternative strategies available to prevent and treat these malignancies.56 Having shown that infusions of donor-derived, EBV-specific CTLs can eliminate post-transplant EBV-LPD in immunodeficient patients, we are evaluating the efficacy of this treatment for other EBVassociated malignancies, using allogeneic or autologous CTLs. Not all EBV-associated malignancies are so obviously amenable to cellular immunotherapy, particularly outside the setting of BMT, where fully immunocompetent donors are usually available. Although Hodgkin and Burkitt-type lymphomas are potential candidates for such adoptive therapy, the tumor cells express only a limited set of subdominant EBV latent cycle antigens and therefore are less immunogenic than cells associated with EBVrelated lymphoproliferation. EBV-infected Reed–Sternberg cells express only the LMP-1, LMP-2, and EBNA-1 antigens, whereas Burkitt lymphoma cells express only EBNA-1. For these tumors it may be necessary to induce CTL lines specific for a single antigen. In diseases in which the patients are not profoundly immunosuppressed, likely defects in antigen presentation by the tumor must be overcome. Some of these defects could result from tumor-derived immunosuppressive effects (which might be overcome ex vivo in the absence of tumor-derived inhibitory effects), while others could reflect poor antigen presentation by the tumor cells that might be overcome by stimulating the CTLs with dendritic cells, whose genes have been modified to express the appropriate stimulatory antigen. We are now studying the potential of autologous polyclonal and LMP-specific CTLs as therapy for Hodgkin disease. From 40% to 50% of cases in immunocompetent individuals are associated with expression of EBV-derived antigens in malignant Reed–Sternberg (H-RS) cells and their variants.57,58 EBV-specific CTLs have been used to treat 13 patients with EBV-positive Hodgkin disease, and while the results have been promising (the CTLs home to tumor tissues, persist for prolonged periods of time, and

produce resolution of B symptoms and partial tumor remissions or disease stabilization), none of the patients with bulky disease have been cured.59,60 This lack of CTL efficacy could be due in part to immunosuppressive factors secreted by H-RS cells or may simply reflect the current method of EBV-specific CTL generation, in which the CTL lines are dominated by clones reactive to viral proteins not expressed in Hodgkin disease. Thus, improvement of EBVspecific CTL therapy for Hodgkin disease will require the development of methods to expand CTLs specific for the EBV proteins expressed in Hodgkin disease61 and to modify genetically the expanded CTLs to render them resistant to inhibitory factors. In preclinical studies, LMP2-specific CTLs from PBMCs have been generated by using dendritic cells (DCs) as antigen-presenting cells that express LMP2. In contrast to the standard LCL protocol, this DC-based CTL stimulation strategy led to preferential expansion of LMP2-specific CTLs.62 The resultant CTLs killed not only autologous fibroblasts infected with a vaccinia construct expressing LMP2, but also autologous LCLs, in which LMP2 is solely expressed by EBV. On the basis of these encouraging results, we are currently modifying our clinical protocols to administer LMP2-specific CTLs to patients with relapsed EBVpositive Hodgkin disease. The toxicity of wild-type LMP1 has prevented the use of DCs for CTL stimulation, but this problem can be overcome by expressing an inactive LMP1 mutant that is non-toxic and enables the generation of LMP1-specific CTLs.61 Another challenge is to generate T cells specific for leukemia antigens when the malignant cells present antigen poorly, and the putative target antigens are weak. An approach similar to that described above for LMP2, in which the target antigen is expressed by DCs, has been used to generate HA1-specific T cells.63 Another possibility, when the target antigens are unknown, is to use leukemic cells alone or cultured with dendritic cells64 as the antigen presenting cells. Although this technique is cumbersome, one report describes a patient with relapsed CML who attained remission after infusion of leukemia-specific CTL lines.65 Clinical studies are also under way with minor histocompatibility antigen-specific CTLs.66

Immunotherapy with dendritic cells The potency of DCs as antigen-presenting cells has stimulated interest in their immunotherapeutic potential. Although the frequency of DCs in peripheral blood is low, the availability of methods to expand these populations, using cytokines such as GM-CSF, IL-4, and stem cell

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factor (SCF), has made this strategy feasible. Dendritic cells have been successfully used in clinical pilot trials to induce tumor-specific immunity as well as clinical responses in selected patients.67 These studies have used DCs pulsed with peptides or tumor cell lysates, and transduced with vectors encoding tumor antigens or tumor RNA. In one of the largest published series, 35 patients with follicular lymphoma were vaccinated with DCs pulsed with tumor-derived Id protein.68 Of ten patients with measurable lymphoma, there were eight anti-Id responses and four clinical responses. Subsequently, 25 additional patients were vaccinated after chemotherapy, with 65% mounting T-cell or humoral anti-Id responses. Sixteen of 23 patients (70%) survived without tumor progression for a median of 43 months after chemotherapy. These results show that that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and tumor regression.68 Current studies are focusing on ways to optimize the source of DCs, the choice of antigen, antigen loading, and the mode of injection.

Natural killer (NK) cells NK cells may also produce antileukemic effects, particularly in the setting of haploidentical transplantation. NK cells are normally inhibited by signals delivered through their surface immunoglobulin-like killer inhibitory receptors (KIR) following interaction with HLA class I molecules on target cells.69 If the target cells are mismatched for Class I molecules, the NK cells are able to produce antileukemic activity and may be particularly effective at inducing GVL activity after T-cell-depleted haploidentical transplantation.70 Several studies have shown that KIR mismatch is associated with a reduced risk of relapse particularly in patients with myeloid maligancies.70,71 Many investigators are therefore evaluating adoptive transfer of NK cells to reduce the risk of post-transplant relapse.

Improving immunotherapy While T-cell therapies have produced definite benefits in the treatment of post-transplant relapsed leukemias and EBV-associated malignancies, clinical studies have also identified clear limitations of such therapies including inadequate persistence or expansion of CTLs. Among the strategies that may overcome these obstacles are the use of novel antigen-presenting cells and pretreatment of the patient to allow T-cell expansion. Gene transfer technologies also afford the opportunity to increase the effectiveness of CTL therapy, for example, by gene-marking the CTLs and

tracking their fate, and by confering drug sensitivity to these cells so that they can be destroyed should adverse effects occur. Finally, it may be possible to genetically manipulate CTLs in ways that will optimize their specificity and augment their cytolytic function.

Antigen presentation One of the chief limitations of adoptive immunotherapy is the lack of a convenient source of the antigenpresenting cells needed to generate antigen-specific CTLs. Several different methods have been explored to circumvent this requirement. These include artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB costimulatory surface molecules72 ; mouse fibroblasts retrovirally transduced with a single HLA-peptide complex together with the human accessory molecules B7.1, ICAM-1, and LFA-373 ; and beads coupled to the soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody.74 There has also been much recent effort to identify the optimum phenotype of infused T cells. It seems likely that for optimum persistence, a T-cell population containing both effector and memory cells will be required, although this prediction will need to be confirmed by correlating in vivo function with the types of products generated with different antigen-presenting cells.

Expansion of T cells in vivo Clinical studies of the adoptive transfer of activated lymphocytes to treat cancer have in many cases been limited by poor lymphocyte survival or function. An exception has been in patients surviving after stem cell transplantation, in whom the proliferative environment favors expansion of infused CTLs. Recently, Rosenberg and colleagues75 described how a proliferative environment could be artificially induced, by administration of fludarabine and cyclophosphamide. Patients with advanced melanoma received lymphoreductive doses of these cytotoxic drugs and were then infused with autologous tumor-infiltrating lymphocytes (TILs). In six patients there was marked expansion of the infused cells that correlated with tumor responses: in two patients the tumor responses were complete, and the infused TILs came to dominate the lymphoid compartment, suggesting a relationship between cell expansion and antitumor activity. Selective expansion of infused T cells might also be obtained by using monoclonal antibodies to deplete the lymphoid compartment prior to T-cell infusion.

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Gene transfer to confer drug sensitivity A potential hazard of therapy with allogeneic T cells not rigorously prepared to exclude alloreactive cells is that there may be residual reactivity against MHC polymorphisms, resulting in GVHD. A second hazard is that therapy of established disease with T cells can result in tissue damage due either to an inflammatory response or to direct damage to virus-infected tissues. We have observed this effect in a patient with bulky disease who was successfully treated with EBV-specific CTLs (see preceding section). Similarly, in a murine model, adoptively transferred CTLs that recognized a CD45 polymorphism expressed on all recipient hemopoietic cells caused pneumonitis.76 The transduction of CTL with a gene encoding a prodrug-metabolizing enzyme, such as the herpesvirus thymidine kinase (TK ) gene, would allow CTLs to be eliminated with chemotherapy should adverse effects occur. The suicide gene used most frequently is the herpes simplex virus 1-thymidine kinase (HS-tk) gene, which renders transduced cells sensitive to ganciclovir. This strategy which has been tested in several clinical trials,77,78 has not been associated with any acute toxicity, and alloreactive T cells appear to be sensitive to ganciclovir. Bonini and colleagues77,79 have described studies in which they used donor T cells to treat patients with EBV-LPD or leukemia in relapse after allogeneic BMT. With their protocol, lymphocytes are transfected after a brief primary stimulation (24–48 hours) with leukemic cells or EBV-infected cells, using a construct that contains the TK suicide gene and a truncated version of the low-molecularweight nerve growth factor gene that acts as a selectable marker. Transduced T cells expressing the nerve growth factor receptor are immunoselected with magnetic beads and then infused into the patient. Should GVHD develop, the cells can theoretically be killed with ganciclovir. The infused T cells in this protocol are not classic CTLs, as they are harvested after a brief primary stimulation, leading to retention of alloreactive T cells. Indeed, when recipient leukemic cells are used as stimulator cells, alloreactive cells recognizing minor histocompatibility polymorphisms between donor and recipient will be induced to proliferate. However, this strategy has certain limitations, including the immunogenicity of the TK gene product,80 and a reduction in the immune function of the gene-modified T cells.81,82 An alternative “suicide gene” approach relies on a chimeric human protein expressing the Fas intracellular domain, with two copies of an FK506-binding protein.83 In vitro, primary human T lymphocytes retrovirally transduced to express the Fas/FK506 chimeric protein functioned identically to nontransduced cells. However, the

transduced cells rapidly underwent apoptosis with the addition of subnanomolar concentrations of AP1903, a bivalent “dimerizer” drug that binds FK506 binding protein and induces Fas cross-linking. T cells were eliminated regardless of their proliferation state, and this AP1903/Fas system contains only human components, suggesting that it may be a useful alternative to the HSV-tk-based method for the removal of alloreactive T cells and the prevention or treatment of GVHD.83

Genetic modification of T cells to improve survival or overcome tumor evasion mechanisms Clinical studies of adoptive transfer of activated lymphocytes to treat HIV and cancer have in many cases been limited by poor lymphocyte survival or function.84,85 Several approaches are being explored to promote survival or enhance the function of adoptively transferred cells. IL-2 administration might improve survival of infused lymphocytes but is excessively toxic at the requisite doses.86 Genetically modifying lymphocytes to produce IL-2 should result in high local concentrations of the cytokine and could result in their improved persistence without systemic toxicity. Liu and Rosenberg 87 described the transduction of melanomareactive human T cells to secrete IL-2. After stimulation, these T cells proliferated without the addition of exogenous IL-2. Furthermore, transduction did not alter their specificity and degree of recognition of tumor cells. Alternatively, T cells could be transduced with an antiapoptotic gene as illustrated by the transduction of T cells with a BCL-X retroviral vector, leading to partial resistance to antibody-induced apoptosis.88 Two recent studies have attempted to overcome inhibition of the immune response by the immunosuppressive cytokine-transforming growth factor-beta (TGF), which is secreted by many tumors. In murine models of both thymoma and malignant melanoma, transgenic mice genetically engineered so that all of their T cells are insensitive to TGF signaling were able to eradicate tumors.89 In a preclinical human study, EBV-specific CTLs were transduced with a retroviral vector expressing a mutant dominantnegative TGF type II receptor (DNR) that prevents the formation of the functional tetrameric receptor. Cytotoxicity, proliferation and cytokine release assays showed that exogenous TGF inhibitory to wild-type CTLs had minimal inhibitory effects on DNR-transduced CTLs.90 If murine studies show that DNR-transduced CTLs are not tumorigenic, this approach may be useful not only in Hodgkin disease, but also in the wide variety of other cancers, that secrete TGF.

Adoptive cellular immunotherapy

Gene transfer to modify T-cell growth, target cell recognition, and activity Use of natural CTL for immunotherapy has several disadvantages, including the requirements for an immune donor, a CTL generation period of 4 to 10 weeks, and expression of an appropriate antigen by an effective antigen-presenting cell. In addition the generation of tumor-specific T cells, either ex vivo or by immunization, is limited by the poor antigenicity of most tumors. A novel way of circumventing this problem is the transduction of T cells with chimeric surface proteins that transmit TCR signals in response to target cells. Such proteins are composed of an extracellular domain (ectodomain), usually derived from immunoglobulin variable chains, that recognizes and binds target antigen. This ectodomain is attached via a spacer to an intracytoplasmic signaling domain (endodomain), usually the cytoplasmic segment of the TCR chain, that transmits an activation signal to the T cell. CTL expressing a chimeric gene that encodes a singlechain antibody variable region specific for tumor antigen, linked to the intracellular gamma or zeta chain of the Ig or T-cell receptor, should acquire the ability to recognize and kill the tumor cell via the chimeric receptor. Clinical studies of chimeric receptors targeting the CD20 antigen are under way in patients with non-Hodgkin lymphoma,91 and CD19 is also being evaluated as a target.92,93 Clinical trials in patients with solid tumors have shown that T cells expressing transgenic antigen-specific chimeric receptors have limited therapeutic activity, in part because engagement of the chimeric receptor alone is insufficient to sustain T-cell growth and activation. One means of solving this problem is to transduce antigenspecific T cells rather than nonspecifically activated cells and take advantage of the costimulation provided to the native TCR by antigen. In one recent study investigating this possibility, Rossig et al.94 transduced a chimeric receptor specific for the CD20 antigen into EBV-specific CTLs and showed that there was stimulation of native TCR by EBVpositive LCLs, while killing of leukemic target cells occurred via the chimeric GD2 TCR.94 Kershaw et al.95 additionally generated dual-specific T cells by genetic modification of alloreactive T cells with a chimeric receptor recognizing folate-binding protein.95 More recently CMV-specific T cells were reprogrammed into leukemia-reactive T cells by transferring a T-cell receptor specific for the minor histocompatibility antigen HA2.96 An alternate means of combining an activation and a costimulatory signal is to generate a construct containing both the TCR and CD28 signaling elements97 or the TCR and the signaling domain of 4-1BB.98

Conclusions T-cell therapies have produced clear benefits in the treatment of post-transplant relapsed leukemias and EBVassociated malignancies. However, clinical studies have identified restrictive limitations of such therapies, including inadequate persistence or expansion of CTLs. With increased knowledge of the optimum methods for generating specific T-cell populations and identifying additional antigen targets, and with optimization of gene therapy approaches that enhance the function of adoptively transferred T cells, the list of successful applications will undoubtedly grow. REFERENCES 1 Rosenberg, S. A. Progress in human tumour immunology and immunotherapy. Nature, 2001; 411: 380–4. 2 Banchereau, J. & Steinman, R. M. Dendritic cells and the control of immunity. Nature, 1998; 392: 245–53. 3 Levitskaya, J., Coram, M., Levitsky, V., et al. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature, 1995; 375: 685–8. 4 Androlewicz, M. & Cresswell, P. How selective is the transporter associated with antigen processing? Immunity, 1996; 5: 1–5. 5 Levitsky, V., Zhang, Q.-J., Levitskaya, J., & Masucci, M. G. The life-span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two Class-I restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4. J Exp Med, 1996; 183: 915–26. 6 Viola, A. & Lanzavecchia, A. T cell activation determined by T cell receptor number and tunable thresholds. Science, 1996; 273: 104–6. 7 Dodley, M. E. & Rosenberg, S. A. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer, 2003; 3: 666–75. 8 Pardoll, D. M. Spinning molecular immunology into successful immunotherapy. Nat Rev Immunol. 2002; 2: 227–38. 9 Vilchez, R. A., Madden, C. R., Kozinetz, C. A., et al. Association between simian virus 40 and non-Hodgkin lymphoma. Lancet, 2002; 359: 817–23. 10 Molldrem, J. J., Lee, P. P., Wang, C., et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med, 2000; 6: 1018–23. 11 Gao, L., Bellantuono, I., Elsasser, A., et al. Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT1. Blood, 2000; 95: 2198–203. 12 Bellantuono, I., Gao, L., Parry, S., et al. Two distinct HLA-A0201presented epitopes of the Wilms tumor antigen 1 can function as targets for leukemia-reactive CTL. Blood, 2002; 100: 3835–7. 13 Zendman, A. J., Ruiter, D. J., & Muijen, G. N. Van. Cancer/testisassociated genes: identification, expression profile, and putative function. J Cell Physiol, 2003; 194: 272–88.

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of antiviral or anti-leukemic responses. Blood, 2003; 102: 2292–9. Solomon, S. R., Mielke, S., Savani, B. N., et al. Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation. Blood, 2005; 106: 1123–29. Rooney, C. M., Smith, C. A., Ng, C., et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virusrelated lymphoproliferation. Lancet, 1995; 345: 9–13. Heslop, H. E., Ng, C. Y. C., Li, C., et al. Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat Med, 1996; 2: 551–5. Rooney, C. M., Smith, C. A., Ng, C. Y. C., et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood, 1998; 92: 1549–55. Gustafsson, A., Levitsky, V., Zou, J. Z., et al. Epstein–Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. Blood, 2000; 95: 807–14. Gottschalk, S., Ng, C. Y. C., Smith, C. A., et al. An Epstein–Barr virus deletion mutant that causes fatal lymphoproliferative disease unresponsive to virus-specific T cell therapy. Blood, 2001; 97: 835–43. Comoli, P., Labirio, M., Basso, S., et al. Infusion of autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication. Blood, 2002; 99: 2592–8. Khanna, R., Bell, S., Sherritt, M., et al. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc Natl Acad Sci U S A, 1999; 96: 10 391–6. Haque, T., Wilkie, G. M., Taylor, C., et al. Treatment of EpsteinBarr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet, 2002; 360: 436–42. Sun, Q., Burton, R., Reddy, V., & Lucas, K. G. Safety of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for patients with refractory EBV-related lymphoma. Br J Haematol, 2002; 118: 799–808. Kuehnle, I., Huls, M. H., Liu, Z., et al. CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood, 2000; 95: 1502–5. Herbst, H., Dallenback, F., Hummel, M., et al. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc Natl Acad Aci U S A, 1991; 88: 4766– 70. Pileri, S. A., Ascani, S., Leoncini, L., et al. Hodgkin’s lymphoma: the pathologist’s viewpoint. J Clin Pathol, 2002; 55: 162– 76.

59 Gottschalk, S., Heslop, H. E., & Rooney, C. M. Treatment of Epstein-Barr virus-associated malignancies with specific T cells. Adv Cancer Res, 2002; 84: 175–201. 60 Bollard, C. M., Aguilar, L., Straathof, K. C., et al. Cytotoxic T lymphocyte therapy for Epstein–Barr virus+ Hodgkin’s disease. J Exp Med, 2004; 200: 1623–33. 61 Gottschalk, S., Edwards, O. L., Sili, U., et al. Generating CTL against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies. Blood, 2003; 101: 1905–12. 62 Gahn, B., Siller-Lopez, F., Pirooz, A. D., et al. Adenoviral gene transfer into dendritic cells efficiently amplifies the immune response to the LMP2A-antigen: a potential treatment strategy for Epstein-Barr virus-positive Hodgkin’s lymphoma. Int J Cancer, 2001; 93: 706–13. 63 Mutis, T., Ghoreschi, K.., Schrama, E., et al. Efficient induction of minor histocompatibility antigen HA-1-specific cytotoxic T-cells using dendritic cells retrovirally transduced with HA-1-coding cDNA. Biol Blood Marrow Transplant, 2002; 8: 412–19. 64 Montagna, D., Maccario, R., Locatelli, F., et al. Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy. Blood, 2001; 98: 3359–66. 65 Falkenburg, J. H., Wafelman, A. R., Joosten, P., et al. Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes. Blood, 1999; 94: 1201–8. 66 Warren, E. H., Tykodi, S. S., Murata, M., et al. T-cell therapy targeting minor histocompatibility Ags for the treatment of leukemia and renal-cell carcinoma. Cytotherapy, 2002; 4: 441. 67 Banchereau, J. & Palucka, A. K. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol, 2005; 5: 296–306. 68 Timmerman, J. M., Czerwinski, D. K., Davis, T. A., et al. Idiotypepulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood, 2002; 99: 1517– 26. 69 Parham, P. & McQueen, K. L. Alloreactive killer cells: hindrance and help for haematopoietic transplants. Nat Rev Immunol, 2003; 3: 108–22. 70 Ruggeri, L., Capanni, M., Urbani, E., et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science, 2002; 295: 2097–100. 71 Giebel, S., Locatelli, F. W., Lamparelli, T., et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood, 2003; 102: 814–19. 72 Maus, M. V., Thomas, A. K., Leonard, D. G., et al. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat Biotechnol, 2002; 20: 143–8. 73 Latouche, J. B. & Sadelain, M. Induction of human cytotoxic T lymphocytes by artificial antigen presenting cells. Nat Biotechnol, 2000; 18: 405–9. 74 Oelke, M., Maus, M. V., Didiano, D., et al. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by

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HLA-Ig-coated artificial antigen-presenting cells. Nat Med, 2003; 9: 619–25. Dudley, M. E., Wunderlich, J. R., Robbins, P. F., et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science, 2002; 298: 850–4. Chen, W., Chatta, K., Rubin, W. D., et al. T cells specific for a polymorphic segment of CD45 induce graft-versus-host disease with predominant pulmonary vasculitis. J Immunol, 1998; 161: 909–18. Bonini, C., Ferrari, G., Verzeletti, S., et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft versus leukemia. Science, 1997; 276: 1719–24. Tiberghien, P., Ferrand, C., Lioure, B., et al. Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft. Blood, 2001; 97: 63–72. Ciceri, F., Bonini, C., Gallo-Stampino, C., & Bordignon, C. Modulation of GvHD by suicide-gene transduced donot T lymphocytes: clinical applications in mismatched transplantation. Cytotherapy, 2005; 7: 144–9. Riddell, S. R., Elliot, M., Lewinsohn, D. A., et al. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat Med, 1996; 2: 216–23. Sauce, D., Bodinier, M., Garin, M., et al. Retrovirus-mediated gene transfer in primary T lymphocytes impairs their antiEpstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms. Blood, 2002; 99: 1165–73. Tiberghien, P. Use of suicide gene-expressing donor T-cells to control alloreactivity after haematopoietic stem cell transplantation. J Intern Med, 2001; 249: 369–77. Thomis, D. C., Marktel, S., Bonini, C., et al. A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease. Blood, 2001; 97: 1249–57. Rosenberg, S. A., Aebersold, P., Cornetta, K., et al. Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med, 1990; 323: 570–8. Brodie, S. J., Patterson, B. K., Lewinsohn, D. A., et al. HIVspecific cytotoxic T lymphocytes traffic to lymph nodes and localize at sites of HIV replication and cell death. J Clin Invest, 2000; 105: 1407–17. Rosenberg, S. A., Lotze, M. T., Yang, J. C., et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg, 1989; 210: 474–84. Liu, K. & Rosenberg, S. A. Transduction of an IL-2 gene into human melanoma-reactive lymphocytes results in their continued growth in the absence of exogenous IL-2 and maintenance of specific antitumor activity. J Immunol, 2001; 167: 6356–65. Eaton, D., Gilham, D. E., O’Neill, A. & Hawkins, R. E. Retroviral transduction of human peripheral blood lymphocytes

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with Bcl- X(L) promotes in vitro lymphocyte survival in proapoptotic conditions. Gene Ther, 2002; 9: 527–35. Gorelik, L. & Flavell, R. A. Immune-mediated eradication of tumors through the blockade of transforming growth factorbeta signaling in T cells. Nat Med, 2001; 7: 1118–22. Bollard, C. M., Rossig, C., Calonge, M. J., et al. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood, 2002; 99: 3179–87. Jensen, M., Cooper, L., Wu, A., Forman, S., & Raubitschek, A. Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy. Cytotherapy, 2003; 5: 131–8. Cooper, L. J., Topp, M. S., Serrano, L. M., et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood, 2003; 101: 1637–44. Roessig, C., Scherer, S. P., Baer, A., et al. Targeting CD19 with genetically modified EBV-specific human T lymphocytes. Ann Hematol, 2002; 81(Suppl. 2): S42–3. Rossig, C., Bollard, C. M., Nuchtern, J. G., Rooney, C. M., & Brenner, M. K. Epstein-Barr virus-specific human T lymphocytes expressing antitumor chimeric T-cell receptors: potential for improved immunotherapy. Blood, 2002; 99: 2009–16. Kershaw, M. H., Westwood, J. A., & Hwu, P. Dual-specific T cells combine proliferation and antitumor activity. Nat Biotechnol, 2002; 20: 1221–7. Heemskerk, M. H., Hoogeboom, M., Hagedoorn, R., et al. Reprogramming of virus-specific T cells into leukemiareactive T cells using T cell receptot gene transfer. J Exp Med, 2004; 199: 885–94. Maher, J., Brentjens, R. J., Gunset, G., Riviere, I., & Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor. Nat Biotechnol, 2002; 20: 70–5. Imai, C., Mihara, K., Andreansky, M. et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia, 2004; 18: 676–84. Ambinder, R. F., Lemas, M. V., Moore, S., et al. Epstein-Barr virus and lymphoma. Cancer Treat Res, 1999; 99: 27–45. Molldrem, J. J., Lee, P. P., Wang, C., Champlin, R. E., & Davis, M. M. A PR1-human leukocyte antigen-A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia. Cancer Res, 1999; 59: 2675–81. Mutis, T. & Goulmy, E. Hematopoietic system-specific antigens as targets for cellular immunotherapy of hematological malignancies. Semin Hematol, 2002; 39: 23–31. Falkenburg, J. H., Wafelman, A. R., Joosten, P., et al. Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes. Blood, 1999; 94: 1201–8.

27 Gene transfer: methods and applications Martin Pule´ and Malcolm K. Brenner

Introduction The concept of using gene transfer techniques to express a new gene in the somatic cells of a patient has stimulated considerable interest, speculation, and hyperbole. The inevitable backlash against promises that so far have not been fulfilled has led to much confusion about the aims and achievements of gene transfer and to a lurking suspicion that the entire field is simply a South Sea bubble waiting to burst. This chapter seeks to provide a balanced account of the current status of gene transfer as applied to leukemia and related disorders, and to review the accomplishments of the field as well as the impediments to progress. Most importantly, it will try to give an idea of the incremental way in which gene transfer technologies will supplement, long before they supplant, current therapeutic approaches to hematologic malignancies. There are two broad strategies of gene transfer applicable to the treatment of leukemia and lymphoma. First, the tumor cell itself can be genetically modified to “repair” its intrinsic molecular defect. Alternatively, a toxic gene can be introduced to destroy the tumor cell, or it can be transduced to express molecules that trigger an immune response against it. Second, the host’s T cells can be redirected, their antitumor activity augmented, or they can be transduced with suicide genes to terminate potentially harmful immune reactions. The drug sensitivity of normal host tissues can be decreased by delivering cytotoxic drugresistance genes to sensitive tissues, thereby increasing the therapeutic index of chemotherapy. Host cells may also be transduced with marker genes, not for any direct therapeutic benefit, but simply as a means to track their behavior and persistence.

Because clinical studies of gene therapy must show that any potential benefits outweigh the potential risks, most protocols for the treatment of malignant diseases are open only to patients with a poor prognosis, in whom the risk-to-benefit ratio is most likely to be favorable. It must be emphasized that these patients are ultimately not likely to prove the most suitable group for gene therapy. Instead, many of the approaches to be described will probably be most valuable for the eradication of minimal residual disease remaining after conventional therapies.

Gene transfer vectors Table 27.1 summarizes the properties of the common types of virus-based gene-therapy vectors

Retroviruses Figure 27.1 A shows the structure of a classic retroviral vector.1 The structural and replicative genes (gag, pol, and env) of an oncoretrovirus are replaced by one or more genes of interest, driven either by the retroviral promoter in the 5 long-terminal repeat (LTR) or by an internal promoter. Currently, only oncoretrovirus-based vectors have been used clinically in cancer therapy. The retroviral constructs are made in cell lines in which the missing retroviral genes are present in trans and thus reproduce and package a vector that is not replication competent. Retroviral vectors have a wide target cell range, and the genetic information they convey is integrated into the host cell DNA. Thus, the transferred gene not only survives for the entire

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 27.1 Main gene-therapy vectors in clinical use Vector Packaging genome limit

Tropism

Immune Genome in response transduced cells

RNA

8 kb

Only dividing cells

Weak

Integrated

Lentivirus

RNA

8 kb

Wide

Weak

Integrated

HSV-1

dsDNA

40–150 kb

Strong for neurons

Strong

Episomal

Large packaging capacity

AAV

ssDNA

< 5 kb

Wide, with exception of hematopoietic cells

Weak

Episomal and integrated

Adenovirus

dsDNA

8–30 kb

Wide

Strong

Episomal

Simple and have capacity to integrate Very efficient transduction of nearly all cell types

Vector Retrovirus

Advantages

Disadvantages

Simple, integrating; hence results in persisting gene transfer in daughter cells Same as retrovirus but can transduce non-dividing cells also

Integration may cause oncogenesis More complex to generate than retrovirus; integration may cause oncogenesis Inflammatory response, only transient transgene expression in most cell types Small packaging limit

Only transient expression; potent immune response to capsid

Abbreviations: HSV-1 herpes-simplex virus-1; AAV, adeno-associated virus; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

life-span of the transduced cell, but is also present in that cell’s progeny. Hence, these vectors are ideal for transferring genes into rapidly dividing cell populations (e.g. leukemic cells or normal lymphocytes) or for gene therapy in which hemopoietic stem cells (HSC) are the intended target. Provided that replication-competent virus is absent, the vector preparations appear to be nontoxic. However, oncoretroviral vectors have several disadvantages. Because expression of the transferred gene requires viral integration of the genome, and hence a population of dividing cells, the efficiency of transfer to many types of cell may be low.2 These vectors are not well suited for use in vivo since they are generally unstable in complement and cannot be targeted to specific cell types. More recently, murine retroviruses have been associated with the induction of leukemia in two children receiving corrective therapy for X-linked severe combined immunodeficiency (X-SCID) caused by defects in the common gamma chain for lymphoid cytokine receptors.3,4 The leukemia appears linked to insertional mutagenesis caused by retroviral integration. While these events have dampened enthusiasm for the use of murine retroviruses to correct immunodeficiency, there are no data to suggest any similar ill effects in the many hundreds of patients who have received retrovirally transduced normal or malignant cells of the hemopoietic lineage. This issue is discussed in more detail, in the section Gene therapy as a cause of leukemia.

Lentiviruses Lentiviral vectors have also been evaluated as clinical vectors.5 They can transduce hemopoietic progenitors6 and primary acute lymphoblastic leukemia (ALL) cells,7 and may be more effective than murine retroviruses when primitive HSC’s (e.g. CD34+ CD38− ) are the target population.8 Unlike oncoretroviruses, lentiviruses can infect and replicate in nonmitotic cells because of the karyophilic properties of the nucleoprotein preintegration complex. However, several technical problems remain before lentiviruses can be widely used for clinical application. For example, the toxicity of some HIV proteins has made the generation of stable packaging cells difficult.9 In addition, there are safety concerns with these vectors. In particular, the use of HIV-derived vector systems raises the possibility that wild-type HIV may be generated during vector production. Newer (third generation) lentiviruses have self-inactivating features, making them the type of vector most likely to prove clinically useful.

Adenoviruses Most adenoviral vectors are E1 (early protein) deletion mutants and therefore are not replication competent.10,11 Early genes in the E3 region may also be deleted to increase the “space” for insertion of new genes.10 Adenoviruses

Gene transfer: methods and applications

infect a wide range of cell types and, unlike retroviruses, can transfer genes into nondividing cells. The vectors are reasonably stable in vivo and can be used to infect cells in situ. Examples include gene transfer into respiratory epithelium (the CFTR gene in cystic fibrosis10 ) or liver (genes encoding Factor VIII and Factor IX in hemophilia A or B12 ). However, adenoviral vectors are generally nonintegrating, so that the gene products are expressed from episomal DNA. The episome is often lost after cell division and can be inactivated or lost even in a nondividing cell. Thus, adenoviral vectors are unsuited for any application that requires longterm expression in a rapidly turning-over cell population or transfer into a stem cell and expression in that cell’s progeny. Another limitation is that most adenoviral vectors are immunogenic. That is, immune responses are generated against the vector proteins themselves, often preventing readministration of the vector. More significantly, cellular and humoral immune responses may be generated against low levels of adenoviral proteins, expressed even when cells are transduced by defective viruses. These responses may destroy the target cell or simply downregulate expression of the transgene along with other virus associated proteins. In addition, the entry of adenoviruses into many cell types will trigger the release of cytokines, such as interleukin-8, that induce a nonspecific but potentially highly destructive local inflammatory response.13 Although these immunostimulatory attributes of adenoviruses may render them inherently unsuited to chronic application in diseases such as hemophilia A or B, they may be an asset when the intent is to prepare a tumor vaccine (see below), emphasizing the importance of matching vector characteristics to intended use. Aside from issues of immunostimulation, there are also concerns about recombination with endogenous adenoviruses, potentially leading to the release of novel variants into the environment. Finally, the wide host cell range of adenoviral vectors may hinder in vivo targeting to a specific cell type.

Adeno-associated vector Adeno-associated viruses (AAV) are members of the Parvoviridae genus Dependovirus14 (Fig. 27.1B). As the genus name implies, AAVs typically depend upon coinfection with a helper virus, such as adenovirus, for efficient replication in the productive phase. The most unique feature of AAVs, however, is the latent phase of their life cycle. When AAVs infect a permissive cell in the absence of helper virus, stable latency is established without obvious consequences for the host cell. In this phase, the viral genome can assume a number of integrated and episomal forms, but the predominant form in AAV2 latency in human cells appears

Fig. 27.1 (A) Retrovirus and retroviral vector. The gag (reverse transcriptase), polymerase (pol), and envelope coding (env) sequences are removed, and supplied in trans by a producer cell. One or more genes of interest (GOI) are inserted, driven from the viral long-terminal repeat (LTR) promoter, or from an internal promoter (P). The viral packaging signal ( ) remains in the vector, so that it is appropriately packaged by the producer cell. (B) Structure of AAV. The AAV genome is a linear single-standed DNA molecule. The viral genome is transcribed within three overlapping regions that produce seven primary transcripts. The transcripts obtained from each gene are shown as black lines. The virus has two palindromic inverted terminal repeats (designated ITR), which in combination with products of the rep region are responsible for site-specific integration. The rep products are also required for replication during coinfection with adenovirus. Genes labeled VP1–3 encode the viral capsid proteins. Promoter regions for these genes are boxed (P5, P19, P40, IVS).

to be a tandem head-to-tail integration within a region of chromosome 19 that has been termed the AAVS1 site. In their native form, AAVs are composed only of 145 nucleotides at each end of the genome and two internal genes – rep and cap. Recombinant AAV vectors are generated by replacing rep and cap with the transgene of interest and retaining the ITR sequences. Replication and packaging of these vectors is accomplished by providing permissive cells with rep, cap and adenovirus helper functions. Helper function can now be accomplished without adenoviral infection but rather with plasmids supplying the necessary genes, enabling difficulties in generating a packaging line with cap and rep stably integrated to be overcome.15 A recombinant herpes virus that supplies rep,

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cap and helper genes has also been generated.16 In parallel with methods for producing virus, purification techniques have also improved.17 The first rAAV2 vector developed for clinical studies was the cystic fibrosis transmembrane conductance regulator (CFTR) which has been used in several phase I and phase II studies in cystic fibrosis patients.17–20 These studies have demonstrated safe, long-term, dose-dependent gene transfer, although lung-inflammation creates an intrinsic barrier to gene transfer in this disease. AAVs may also be useful in cancer gene therapy, for example, to transduce myeloma cells to express B7.1 and B7.221 genes to generate a tumor vaccine. Selective killing of hepatocellular carcinoma cells by AAV-mediated transfer of the herpessimplex virus thymidine kinase gene has been reported in a mouse model.22 Whereas some of the major advantages of AAV vectors include stable integration, low immunogenicity, longterm expression and ability to infect dividing and nondividing cells, the major limitations include variations in infectivity among different cell types, the small size of the recombinant genome that can be packaged and the significant immunogenicity in clinical application. Future improvements will probably include retargeting of AAVs, increasing the packaging size, and use of different serotypes.

Liposomes and other physical methods Clinical experience with the available physical methods of gene transfer has primarily involved cationic liposome/DNA complexes23,24 which fuse with the cell membrane and enter the endosomal uptake pathway. DNA released from these endosomes may then pass through the nuclear membrane and be expressed. The main advantage of liposomes is that they are nontoxic and can be given repeatedly. In some cell types, high levels of gene transfer have been obtained by this method23 and successful systemic administration has also been reported in large animal models.25 However, the DNA transferred by liposomes does not integrate the genome, and despite the incorporation of a variety of ligands into the liposomeDNA complex,26 the ability to target these vectors is still quite limited. More recently, successful gene transfer in vivo has been reported with use of a bio-ballistic (“gene gun”) technique in which DNA coated onto colloidal gold particles is driven at high velocity by gas pressure into the cell.27 Indeed, improved methods of electroporation may also be valuable for transduction of normal and malignant hemopoietic progenitor cells with sufficient efficiency to allow therapeutic application.28

Other vectors Herpesviruses have been proposed as high-efficiency transducers of normal and malignant hemopoietic cells, and will likely enter clinical trials within the next few years. However, although these viruses may become future substitutes for currently available vector systems, most investigators now accept that no naturally occurring virus and no simple physical vector will ever prove suitable for all gene therapy purposes. Ultimately, therefore, entirely new synthetic or semisynthetic vectors will have to be developed. Possibilities include the generation of hybrid viral vectors, that may combine, for example, the in vivo stability of adenoviruses and the integrating capacity of retroviruses.29 Alternative, fully synthetic vectors will be developed by combining components from multiple different vectors, allowing safe, efficient, and specific gene transfer and regulation. In the meantime, however, gene therapy protocols for leukemia will require investigators to circumvent the limitations of current vectors and to choose their agents on the basis of the most important feature required.

Modifying the tumor cell Tumor correction or deletion of underlying defects There is a seductive elegance to the strategy of introducing genetic material into a malignant cell to correct the genetic defect causing its neoplastic phenotype or to directly kill the cell. A number of mutant oncogenes and fusion transcripts have been identified in leukemias and lymphomas that are specific to the malignant clone and frequently form a critical component of the malignant process (see Chapters 10 and 11). Alternatively, toxic genes can be selectively expressed in tumor cells. For example, efforts are being made to neutralize fusion transcripts such as BCR-ABL or activated oncogenes such as MYB (in chronic myeloid leukemia, CML), using ribozymes, antisense RNA, or wild-type genes.30,31 Similarly, nonfunctional anti-oncogenes such as p53 may be replaced by wildtype genes in patients with acute myeloid leukemia (AML) or myelodysplasia.32 Interest is also increasing in targeting the genetic pathways involved in the control of apoptosis. Experimental models suggest that even minor perturbations in these pathways can greatly modify the sensitivity of cancer cells to chemotherapy. This approach faces a series of difficulties. The first set arise from the genetic change itself. Unless correction of a single defect is subsequently lethal to the malignant cell, transfer of an individual corrective gene to a patient with 1011 or 1012 leukemia or lymphoma cells will leave

Gene transfer: methods and applications

many cells that are effectively premalignant, with a high risk of later transformation. Moreover, many relevant gene defects produce molecules with “transdominant” effects that will continue to produce a malignant phenotype, even if a corrective wild-type gene is introduced. Hence, most approaches to correction attempt to silence transdominant malignant genes. This can be achieved with ribozymes, antisense oligonucleotides, double-standed RNA or small interfering RNAs, or by homologous recombination with a wild-type gene.32,33 These “subtractive” approaches to gene transfer are designed to destroy the function of an expressed gene rather than add a new activity. Ribozymes cleave specific sequences in targeted mRNA molecules. For clinical use, ribozymes that form hairpin or hammerhead structures are preferred because of their stability even in the absence of substrate, and their activity under physiologic conditions. The original function of these molecules in viruses and other microorganisms is probably to autocatalyze their own cleavage into functional RNA, and perhaps also to destroy the RNA of invading organisms. For applications in gene transfer, ribozymes may be used to destroy transcripts originating from the unwanted host cell DNA sequence while leaving intact the mRNA originating from the transgene. Some ribozymes are currently under clinical investigation, e.g. a VEG-F targeted ribozyme is being investigated for its role in breast, lung, and colon cancer and a phase I trial of a ribozyme directed to human epidermal growth factor 2 (HER-2) is planned in patients with breast and ovarian carcinoma. Antisense oligonucleotides are short, synthetic stretches of DNA that prevent translation of target genes by hybridizing with and then inducing RNAse H digestion of specific mRNAs. Early in vivo studies of oligonucleotides found them to be susceptible to natural phosphodiester backbone degradation by cellular nucleases. Several sugar, base and backbone modifications have been investigated to improve stability. Replacement of the PO moiety by phosphorothioates results in compounds with serum stability and high RNA binding affinity. Several toxic effects are apparent at higher dose levels including complement activation, thrombocytopenia and heptotoxicity. Toxicity may be reduced by delivery in liposomes. Further modifications have concentrated on substitutions at the 2 position with electronegative substitutions such as the 2 O-C-methylene bridge,34 which enhance RNA binding. The sequence has to be carefully chosen to avoid hybridization of full-length oligonucleotides with unrelated targets. Preclinical studies and initial clinical results did not substantiate concerns that non-specific inhibition of RNA would produce toxic effects. Normal healthy cells tolerate the transient loss of function better than cancer cells which carry the proapop-

totic burden of multiple genetic alteration and genomic instability. In several clinical studies, antisense oligonucleotides were used to target the MYB or p53 gene, either as marrow-purging agents for patients with chronic or accelerated phase CML, or intravenously in patients with refractory AML or CML in blast crisis, without significant clinical responses or associated toxicities.35,36 An antisense BCL-2 oligonucleotide has also been administered in patients with refractory non-Hodgkin lymphoma, with objective clinical and biological responses.37 RNA interference is an ancient mechanism of posttranscriptional gene silencing38 in which short segments of double-stranded RNA direct RNAases to digest specific sequences of mRNA. Initial attempts to harness this reaction for experimental manipulation of mammalian cells were foiled by nonspecific antiviral defense mechanisms. The field advanced when synthetic duplexes of short RNAs were found to mediate specific and potent gene silencing.39 Delivery of such molecules (for example, in a lipid envelope) may facilitate application of this form of gene-targeting to cancer therapy. Since RNA itself is fragile and costly, expression systems that allow the generation of short interfering RNA (siRNA) inside the cell may be preferable alternatives,40 and could be made part of a vector. Genes that directly kill tumor cells are frequently used as cancer therapy.41 For example, when expressed in cancer cells, a gene encoding a prodrug-metabolizing enzyme will convert the prodrug to an active moiety, which then kills the transduced cells. The active molecule may also diffuse either through intercellular gap junctions or in the extracellular space and destroy adjacent tumor cells. In this way, transduction of even a small proportion of tumor cells can produce a large “bystander” effect in adjacent tumor tissue. Unlike the corrective approach the transgene is not specifically toxic to the tumor but may affect any transduced normal cells. For this reason the main success has been in treating localized disease including brain tumors. More recently, Hurwitz and colleagues42–44 injected bilateral retinoblastomas with adenovirus type 5 encoding Tk, followed by administration of ganciclovir. Three of five patients have shown significant responses to the adenoviral vector as long as one-year post therapy. Even with bystander effects, a high proportion of tumor cells must be transduced if tumor modification is to be useful. Current vectors cannot be targeted to tumor tissue and thus are relatively inefficient, limiting most gene therapy approaches to the treatment of accessible local disease – rarely an option with hematologic malignancies. Several developments may ultimately overcome this obstacle. Extensive efforts have been made to alter the envelope proteins of vectors to ablate their natural cell

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tropisms and replace them with new tumor-associated binding specificities.45,46–49 It is also possible to construct promoters that are active only in specific cell or tissue types. An example of this “transcriptional targeting” is the use of the carcinoembryonic antigen CEA promoter to drive a prodrug gene, resulting in selective gene expression in CEA-producing tumors.50 A combination of specific envelope (transductional targeting) and specific promoter (transcriptional targeting) may provide optimal specificity for tumor cells.51 The efficiency of gene transfer to tumors may be increased by the use of conditionally replicationcompetent viruses. Viruses have been developed that will replicate in malignant but not in normal cells,52 thereby providing multiple rounds of infection of tumor targets. Alternatively, the cells themselves may be used as vectors if they track to sites of tumor. The most obvious choice among cell carriers would be T cells or natural killer (NK) cells. Promoters that activate viral gene transcription upon TCR binding or after encountering co-stimulatory or other ligands could be used to trigger virus production and release.53,54 Systemic administration of vectors, particularly those derived from viruses, is also bedeviled by innate and adaptive immune responses. Inflammatory responses, specific antibodies and cellular immune responses rapidly diminish the activity of even the largest doses of vector.55 Efforts to avoid these effects through the induction of selective and transient immunosuppression are only in their infancy. Despite these limitations, several tumor correction protocols have been proposed for solid tumors.

Tumor vaccines One of the most commonly used approaches to gene therapy for cancer is to attempt to enhance the immunogenicity of weak tumor antigens by transducing tumor cells to express immune-activating molecules. In murine model systems, tumor cells expressing such molecules have augmented immunogenicity. Injection of neoplastic cells (including those derived from lymphoid and myeloid malignancies) in doses that would normally establish a tumor instead recruits immune system effector cells and eradicates injected tumor cells.56–59 Often the animal is then resistant to challenges by further local injections of nontransduced parental tumor. The transduced tumor has therefore acted like a vaccine. In some models, established, nontransduced, parental malignant cells are also eradicated.58 A number of different molecules have been successfully exploited as animal models. These include chemokines (e.g. lymphotactin,60 agents that enhance anti-

gen presentation (e.g. GM-CSF),61–63 and cytokines that enhance CD4 cell activity (e.g. TNF and interleukin 7)64–67 or increase expression of class I MHC antigens (e.g. gamma interferon)67 or amplify T-cell responses (e.g. IL-2).68 Additionally, efforts have been made to express costimulatory molecules on tumor cells, including CD40 ligand69–73 and B7.1,74 or intercellular adhesion molecules such as ICAM 1 and ICAM 3.75 Translation of this approach to human hematologic malignancies is not straightforward. Although human leukemic cells are often readily available, they are difficult to grow in vitro (making murine retroviral transduction difficult), and they are resistant to transduction with most currently available vectors. Leukemic cells may be heterogenous so that the population used to vaccinate may not express critical target antigens.76 The use of an allogeneic cell line simplifies the logistics of this strategy, but may result in the patient receiving a vaccine that does not express the appropriate target antigens. To date, a phase I study of autologous adult AML cells engineered to secrete GM-CSF has recently commenced and is now being extended to pediatric AML.63 It has also proved feasible to express costimulator molecules such as CD40, CD40 ligand or B7.1 on primary tumor cell surfaces. We are currently using a combination of CD40 ligand and IL-2 gene transfer into pediatric ALL cells in an effort to generate an antitumor immune response in patients with high-risk disease who have entered remission. To date, this study has proved to be safe and has generated both cellular and humoral antileukemic immune responses in 5 of 9 patients treated so far.77 Because these patients are treated in remission, it may be difficult to confirm whether there is any antileukemic activity. A similar study has been completed for adults with chronic lymphocytic leukemia.78 As an alternative to inducing expression in tumor cells directly, and as a throw-back to the vaccine adjuvant days, some groups are testing vaccines composed of autologous tumor cells, mixed with GM-CSF producing bystander cells.79 In conclusion, genetic modification of tumor cells appears safe and is capable of generating specific humoral and cellular antitumor cytotoxic responses. There have been at least some tumor regressions and this strategy is now being evaluated in a wider range of tumors and in larger numbers of patients, including those with leukemia.

DNA-based vaccines Nucleic acid vaccines induce an immune response targeted to a protein expressed in vivo after administration of the naked encoding gene. Nucleotide-based vaccines have a number of appealing features: they provide prolonged

Gene transfer: methods and applications

antigen expression that can continuously stimulate the immune system, probably through an intracellular antigenic reservoir resistant to antibody-mediated clearance. This may favor induction of immune memory even in the absence of booster immunization.80 Co-delivery with plasmids encoding cytokines or costimulatory molecules can further enhance the immune response.81 Moreover, nucleotide vaccination leads to antigen presentation to both specific cytotoxic T lymphocytes (CTL) and helper T cells. Preclinical experiments conducted in a mouse model of adult T-cell leukemia induced by the human T-cell leukaemia virus-1 (HTLV-1) indicated a promising effect for a Tax-coding DNA vaccine against HTLV-1-induced tumors in vivo.82 Immunization with DNA constructs encoding the idiotype (Id) of a murine B-cell lymphoma induced specific anti-Id antibody responses and protected mice against tumor challenge. Furthermore, use of DNA encoding an Id/GM-CSF fusion protein improved vaccine efficacy.83 Introduction of unmethylated CpG motifs into the transferred genes can further enhance the host immune response, as these motifs are one of the most immunogenic components of bacterial DNA recognized by the vertebrate immune system.84 Clinical trials of DNA vaccines are underway in patients with cutaneous T-cell lymphoma or melanoma.

Modifying the host Modifying the immune system While vaccine studies have so far produced meager clinical responses, the effects of adoptive transfer of T cells are more dramatic. Recent studies of SV40 large T-antigendependent mouse tumor models of hepatocellular carcinoma and prostate cancer have shown that adoptive transfer of T cells from non-transgenic mice for whom SV40 is non-self results in dramatic benefit.85,86 In humans, allogeneic donor leukocyte infusion can induce remissions in relapse after bone-marrow transplantation by rejection of minor histocompatability antigens that are foreign to the donor T cell.87 Other human studies by Rooney et al.88 have shown that adoptive therapy with virus-specific T cells is safe, feasible and active as prophylaxis and treatment of post-transplant Epstein–Barr virus (EBV) lymphoproliferative disorder. Other potential targets for the immune system are present in human malignancies, including mutated oncogenes or fusion proteins generated by chromosomal translocations.89,90 Some lymphomas may express immunogenic virally encoded proteins.91 Even normal pro-

teins may be suitable targets if they are expressed in higherthan-usual quantities; tyrosinase and the MAGE series of proteins in melanoma cells are two good examples.92 If tumor cells either express or are able to process and present these tumor-specific peptides, then malignancy-specific T cells administered in large enough doses should be effective treatment. Generating therapeutic doses of specific T cells is difficult, however, except in special cases in which the tumors express viral proteins.

Artificial T-cell receptors One way of circumventing the lack of tumor antigenspecific T cells is to transduce T cells with genes encoding chimeric surface proteins that transmit TCR signals in response to target cells. Such proteins are composed of an extracellular domain (ectodomain), usually derived from immunoglobulin variable chains, which recognizes and binds target antigen. This region is attached via a spacer to an intracytoplasmic signaling domain (endodomain) usually the cytoplasmic segment of the TCR- chain, which transmits an activation signal to the T cell.93 (Fig. 27.2). A wide variety of domains have been combined94 and the generation of these chimeras seems generally robust, probably reflecting the isolating effects of the lipid bilayer, which forces the extracellular and intracellular domains to remain structurally independent of each other. Immunoglobulin T-cell receptors (also known as IgTCR or T bodies) are the most commonly described constructs. They are created by joining the variable domain of an Ig with an intracytoplasmic signaling molecule. They unite the specificities of antibodies with the potency of cellular killing by transposing antibody recognition to T cells. T cells bearing these chimeric TCRs are redirected to kill tumor cells expressing antigen on their surface. The generation of T lymphocytes with an antibody-dictated specificity allows targeting toward any tumor-associated antigen for which a monoclonal antibody exists. In contrast to the lengthy process of selection and expansion of lymphocytes with native specificity for target antigens, large populations of antigen redirected T lymphocytes can be obtained by simple retroviral transduction. Since chimeric IgTCRs provide T-cell activation in an MHC-unrestricted manner, mechanisms of tumor escape from T-cell recognition, such as downregulation of HLA class I molecules and defects in antigen processing, are bypassed. T-cellmediated effector functions are much more likely to result in tumor cell lysis than humoral immune responses alone. Cytokine secretion upon T-cell activation by tumor antigen will result in the recruitment of additional components of the immune system, amplifying the antitumor immune

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Fig. 27.2 Typical artificial T-cell receptor. An artificial T-cell receptor can be imagined in two connected parts: an internal and an external segment. The external segment recognizes target antigen. This region is often derived from the variable chains of an immunoglobulin, which when linked together by a flexible linker form a single-chain variable fragment (scFv) that is connected to a flexible spacer to allow antigen recognition in multiple orientations. The spacer is usually derived from the CH2-CH3 domains of immunoglobulin. The internal segment, most commonly derived from the transmembrane and intracytoplasmic domains of CD3- , is involved in signal transmission. The isolating effect of the lipid bilayer allows many different types of external and internal segments to be functional when joined together.

response. Furthermore, unlike many intact antibodies, T cells can migrate through microvascular walls, extravasate and penetrate the core of solid tumors to exert their cytolytic activity. These predictions have been tested in vivo. In a mouse model of ERB-2 expressing ovarian carcinoma, autologous lymphocytes transduced to express scFv- TCRs directed against ERB-2 were infused into tumor xenografted mice, causing total regression of tumors.95 Similarly, lymphocytes expressing scFv against CEA fused with FcεIg were effective at rejecting colon carcinomas in a mouse model of this tumor.96 Chimeric receptors also promote homing – ERB2-targeted T-cells labeled with fluorescent dye were specifically detected in tumor tissue of ERB-2 + tumor-bearing SCID mice.97

The first clinical trials of a chimeric TCR were performed with an HIV-env-recognizing CD4- construct expressed in either autologous or syngeneic T cells.98 No specific effect on HIV viral load could be demonstrated and transduced T cells persisted only at very low levels. The outcome of this study is representative of many others, and demonstrates the limitation of chimeric TCRs. The signaling function of engineered chimeric proteins is much less effective than that of the native TCR, during which CD4 or CD8 coreceptors are recruited to the signaling complex via their interaction with class I and II MHC molecules. These coreceptors bring SRC kinases to the TCR, promoting phosphorylation of TCR ITAMs and enhancing signaling several fold.99 Furthermore, the engagement of just the TCR is not sufficient to achieve full T-cell activation. A co-stimulatory signal from CD28 is required to cause clonal expansion and full activation of T cells. Chimeric receptors with only a TCR- -chain intracellular sequence do not fully activate na¨ıve T cells in transgenic mice.100 In essence, chimeric TCRs act as crude triggers of cell killing but lack the signaling components to fully activate and induce T-cell proliferation. Deficient signaling may be overcome in a number of ways. The endodomains can be modified to transmit a more complete signal. For example, constructs containing endodomains composed of the intracellular segment of CD28 joined to that of CD3- were able to activate T cells and trigger killing of targets as well as proliferation.101,102 More complex endodomains involving portions of different costimulatory molecules have also been described.103 Alternatively, investigators have generated “bi-specific” T cells. Autologous T cells that recognize a powerful antigen unrelated to the tumor (e.g. allo or viral antigens) are expanded by repeated stimulation and subsequently transduced with a chimeric receptor gene before being returned to the patient. Activation of the T cells via their native TCR leads to their activation and persistence, while recognition of tumor by their chimeric TCR triggers killing of targets. In other words, tumor killing is “piggy-backed” onto a more potent antigen response.104,105

Modification of T cells Adoptively transferred T cells still have to face tumor evasion strategies and hence frequently persist and function poorly in vivo.106–107 Genetic modification may circumvent their tumor evasion strategies. For example, IL-2 administration improves the survival of infused lymphocytes but is toxic at required doses.108 Genetically modified lymphocytes producing IL-2 should result in high local concentration of T cells and could improve their persistence without systemic toxicity. Liu and Rosenberg109 transduced

Gene transfer: methods and applications

melanoma-reactive human T cells to secrete IL-2, and after stimulation these cells proliferated without addition of exogenous IL-2. Moreover, transduction did not alter their specificity or ability to recognize tumor cells. An alternative strategy to promote survival is to transduce T cells with an antiapoptotic gene. Transduction of T cells with a BCL-X retroviral vector results in partial resistance to Fas antibody-induced apoptosis.110 Unfortunately, concerns about secondary oncogenesis (see below: Gene therapy as a cause of leukemia?) make it unlikely that these types of growth promoting activities will be evaluated clinically in the near future. Immunosuppressive transforming growth factor-beta (TGF-) is secreted by many tumors. In murine models of both thymoma and malignant melanoma, transgenic mice genetically engineered so that all of their T cells are insensitive to TGF- signaling were able to eradicate tumors.111 In a preclinical human study, EBV specific CTLs were transduced with a retroviral vector expressing a mutant dominant-negative TGF- type II receptor (DNR) that prevents the formation of the functional tetrameric receptor. Cytotoxicity, proliferation and cytokine release assays showed that levels of exogenous TGF- inhibitory to wild-type CTLs had minimal inhibitory effects on DNRtransduced CTLs.112 If long-term murine toxicity studies continue to confirm that DNR transduced CTLs are not tumorigenic, this approach may be used not only for Hodgkin disease but also for other lymphomas and solid tumors that secrete TGF-.

Suicide genes Suicide genes make transduced cells susceptible to an agent that is not ordinarily toxic. More than a dozen examples of these systems exist, but the most widely used clinically is the Tk gene from herpes-simplex virus-1 (HSV-tk), whose kinase product phosphorylates the prodrug form of ganciclovir, which subsequently causes the death of dividing cells. These genes have been used to treat relapsed leukemia after allogeneic stem cell transplantation. After this procedure, donor T cells contribute to a graft-versus-leukemia effect to reduce relapse, facilitate engraftment and reduce opportunistic infections. Nevertheless, in a subset of patients, donor T cells can cause potentially lethal graftversus-host-disease (GVHD). The introduction of a “suicide gene” in donor T cells that can be activated in vivo allows for their positive effects to be manifest with the option of their possible subsequent deletion should GVHD ensue. Donor T lymphocytes transduced with HSV-tk have been administered to patients as infusions after T-cell-depleted

marrow transplantation.113,114 In these studies, administration of ganciclovir alone resulted in resolution of GVHD in five out of seven patients. Although these studies demonstrated proof-of-principle, the HSV-tk strategy has several limitations. Ganciclovir must often be administered to treat cytomegalovirus infection or reactivation after transplantation, resulting in deletion of the needed donor T cells. As a virus-derived protein, HSV-tk is strongly immunogenic – only in immunosuppressed patients are these cells likely to survive. Even then an immune response may be detected.113 In more immunocompetent patient groups HSV-tk transduced cells are rapidly deleted.115 More useful suicide genes encoding nonimmunogenic proteins with low basal toxicity but high efficacy after triggering are under development. For example, a suicide system has been developed that is based on endogenous proapoptotic molecules linked to modified FK506-binding proteins (FKBPs). These molecules contain a binding site for a lipid permeable, chemical inducer of dimerization.116 Administration of this dimerizing drug results in the aggregation of two or more chimeric proapoptotic molecules, leading to their activation and thus apoptosis. Similarly, the inducible Fas system is based on a self-protein and should be nonimmunogenic.117 Its inducer, AP1903, seems to lack toxicity.118 Another possibility is the use of inducible caspase molecules. These constructs may improve function since caspases are downstream of Fas in the apoptosissignaling cascade, distal to many antiapoptotic molecules such as c-FLIP and BCL-2. Human CD20 has also been proposed as a nonimmunogenic suicide molecule. Exposure to a monoclonal chimeric anti-CD20 antibody (Rituximab), in the presence of complement, rapidly killed transduced cells.119 This strategy precludes the need for coexpression of a surface marker for sorting. However, Rituximab can cause the unwanted loss of B cells as well as donor T cells. Since transduction can never be 100% efficient in any of these systems, coexpression of a nonimmunogenic selectable marker with the suicide gene is required to allow for selection of suicide gene-expressing cells. An internal ribosomal entry site (IRES) or a linker sequence containing a cleavage site that will be cut by endogenous proteases after protein translation (e.g. FMDV 2A peptide) may be useful in securing reliable coexpression of both genes.120

Modification of host cytotoxic drug sensitivity If HSCs could be rendered resistant to one or more cytotoxic drugs, it might enable them to resist the myelosuppressive effects of cytotoxic drugs during leukemia therapy, allowing longer or more intensive therapy that could cure additional patients.121–123

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The MDR gene has been the most widely considered for human therapy. Its product, P-glycoprotein, functions as a drug efflux pump and confers resistance to many chemotherapeutic agents.124 The feasibility of using MDR to protect hemopoietic cells has been demonstrated by transgenic mouse experiments,125,126 and retroviral transfer of MDR to murine clonogenic progenitors conferred drug resistance both in vitro and in vivo.126 These experiments with MDR-containing vectors prove the principle that drug resistance genes can be used to attenuate drug-induced myelosuppression. It is likely that other drug resistance genes could function analogously. DNAmethylguanine methyltransferases (MGMT) are enzymes that repair DNA damage induced by the nitrosoureas, a class of alkylating agents used widely in cancer chemotherapy. Preliminary data suggest that retrovirally mediated gene transfer of the human MGMT gene to mouse bone marrow cells results in protection of murine progenitors from toxicity produced by BCNU.74. Other drug resistance genes, including those for dihydrofolate reductase and topoisomerase II, are also under consideration for clinical testing. In a human breast cancer study a significant proportion of hematopoietic cells were protected with expression levels adequate to reduce the sensitivity of these stem cells to chemotherapeutic agents.127 The clinical application of drug-resistance gene transfer has several potential pitfalls. The low efficiency of stem cell transduction observed with current clinical protocols predicts that drug-induced myelosuppression cannot be ameliorated unless there is dramatic progress in vector targeting. There is also the risk of transferring the genes to neoplastic cells that contaminate the HSC graft and produce drug-resistant relapse. Finally, toxicity to nonprotected organs-including gut, heart, and lungs may rapidly supervene when marrow resistance allows intensification of cytotoxic drug dosages.

Gene marking The essential principle of gene marking is the transfer of a unique DNA sequence into a host cell to permit its easy subsequent detection, thereby serving as a marker for these labeled cells. Gene marking is not intended for direct therapeutic benefit, but rather to obtain information regarding the biology and function of adoptively transferred cells.

Gene marking for autologous stem cell transplantation Autologous HSC rescue has shown promise as effective treatment for leukemias and lymphomas.128–131 Disease

recurrence continues to be the major cause of posttransplantation treatment failure, but whether it originates from harvested stem cells or residual disease remains unclear.128–133 Concern that the HSC infusion may contain residual malignant cells has led to extensive evaluation of techniques for purging these cells.134–137 However, no method has been unequivocally shown to reduce the risk of relapse in naturally occurring disease133–138 and purging techniques usually slow engraftment due to damage to normal progenitor cells. By marking stem cells prior to stem cell infusion it has been possible to determine if contaminating malignant cells in the stem cell harvest contribute to relapse following autologous stem cell transplantation.139 The HSC product is marked at the time of harvest with retroviral vectors encoding the neomycin resistance gene (neo). Then, at relapse, it is possible to detect whether the marker gene is present in the malignant cells. Since 1991, this approach has been used to study a variety of malignancies treated by autologous HSC transplantation139–143 including AML, CML, ALL, neuroblastoma, and lymphoma. In pediatric patients undergoing autologous BMT as part of therapy for AML, 4 of 12 patients who received marked marrow relapsed. In 3 of the 4 patients, detection of both the transferred marker and of a tumor-specific marker in the same cells at the time of relapse provided unequivocal evidence that the residual malignant cells in the marrow were a source of leukemic recurrence. More difficult questions can be answered by simultaneously marking cells with different vectors. For instance what are the effects of purging on relapse and engraftment? Comparison of two purging methods hydroperoxycylocphosphamide (4-HC) and culture with IL-2, by differential marking revealed a greater contribution to hematopoietic reconstitution by 4-HC purged marrow.144 Notably, in these studies, marker gene levels were lower than in studies with unpurged marrow. No in vitro assay can yet assess the capacity of stem cells to repopulate the hemopoietic compartment. Differential marking in single patients allows comparison of these properties between different sources of stem cells (e.g. peripheral blood and marrow), the functions of different subpopulations and the consequences of ex vivo manipulation, such as stromal support or cytokines. For instance, it is possible to use growth factors such as IL-1, IL-3, and stem cell factor to increase by 10- to 50-fold the numbers of hemopoietic progenitor cells.145,146 It is not certain that such ex vivo data will be reflected by results in vivo. In primate and human studies, transplantation of marrow treated ex vivo with growth factor combinations that greatly augment both progenitor numbers and gene transfer rates has yielded

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disconcertingly low levels of long-term gene expression in vivo.146 The likeliest explanation for this seeming paradox is that many of the growth factors intended only to induce cycling in marrow stem cells also induce their differentiation and the loss of self-renewal capacity. Another useful comparison is the repopulating capabilities of autologous bone marrow CD34+ cells and peripheral blood stem cells.147 The results of judging by differential marking indicate that the latter cells contributed to hemopoiesis earlier and for longer periods than did bone-marrow CD34 cells. These marking studies also provided information on the transfer of marker genes to normal hemopoietic cells and showed that marrow autografts contribute to long-term hemopoietic reconstitution after transplantation.148 Longterm transfer for more than 10 years has been seen in the mature progeny of marrow precursor cells, including peripheral blood T and B cells and neutrophils.144

Gene marking of T cells Several studies have also shown the feasibility of gene marking CTL to track their expansion, persistence and homing potential to sites of disease.88,149,150 For example, gene marking of EBV-specific CTL, for the prophylaxis and treatment of post-transplant lymphoproliferative disorder has demonstrated the persistence of gene marked CTL to 78 months post infusion. In addition, gene-marked EBV-CTL given as treatment for relapsed Hodgkin disease have been shown to traffic to tumor sites.149

New methodology Two new techniques will allow even more informative marking trials. Quantitative real-time PCR allows for simple, highly accurate quantification of the integrant copy number that can easily be standardized against an internal control. This is a significant improvement over the semiquantitative PCR techniques previously used. In addition, every retroviral integration event results in a unique integration site in genomic DNA. The progeny of a transduced cell will bear the same integration site and cutting of PCR amplified flanking DNA with a restriction endonuclease yields multiple fragments whose lengths are unique to the integration site. The number of fragments from a given sample indicates “clonality.” Individual fragments can be sequenced and the precise location of integration elucidated.151 Detection systems specific to a particular provirus-flanking region can be developed and allow precise tracking of an individual clone. This strategy allows one to follow the expansion of clones over time and to assess the pluripotency of transduced cells. We can even begin to

Table 27.2 Distribution of cancer gene-therapy protocols to date Therapeutic approach

Protocol numbers

Immunotherapy Pro-drug/HSV-TK Tumor suppressor gene Vector-mediated cell lysis Chemoprotection Oncogene downregulation Antisense Single chain receptor Dominant-negative mutation

155 43 38 20 12 10 7 4 2

Source: Recombinant DNA Advisory Committee.

hypothesize about such things as the number of true stem cells in harvested populations. Importantly, a reduction in the number of clones with rising levels of gene marking may herald emergence of integration-induced leukemogenesis, as described in the following section.

Gene therapy as a cause of leukemia Any vector that randomly inserts its genetic information into the genome of target cells is, by definition, causing insertional mutagenesis. The risk that insertional mutagenesis will disrupt normal gene function, inactivate tumor suppressor genes or activate oncogenes should be directly proportional to the number of insertion/integration events. However, the activation of a single proto-oncogene by itself should not be sufficient to convert a normal cell into a tumor cell; rather multiple gene disruptions would be required. Since current integrating vectors are inefficient, they transduce only a small proportion of cells that harbor a low retroviral copy number. Hence, the risk of inducing malignant change – while finite – has always been considered to be small. Until recently the only description of retroviral vector related tumorigenesis in humans or nonhuman primates was in a monkey who developed T-cell lymphoma following transduction with a replication competent retroviral vector. Analysis of the tumor revealed multiple integration sites due to chronic productive retroviral infections,152 further supporting the belief that the replication incompetent retroviruses now used clinically would not share this property. Unfortunately, this confidence was shattered by reports of two cases of T-cell ALL in 11 patients with commongamma chain deficiency (X-SCID) who received retrovirally corrected autologous stem cells.3 Subsequent integration

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Table 27.3 Some examples of current cancer gene-therapy protocols that have potential application in childhood leukemia Study ID

Title, institution, and comments

0210–553

Treatment of Chronic Lymphocytic B-Leukemia (B-CLL) with Human IL-2 and Human CD40 Ligand Transduced Autologous Tumor Cells. Baylor College of Medicine, Houston, TX, USA. Investigators generate tumor vaccine by modifying autologous tumor cells (in this case CLL) to express both IL-2 and CD40 ligand. Expression is facilitated by adenoviral vectors.

9705–188

Autologous Transplantation for Chronic Myelogenous Leukemia with Stem Cells Transduced with a Methotrexate-Resistant DHFR and Anti-BCR/ABL-Containing Vector and Post Transplant Methotrexate Administration. University of Minnesota, Minneapolis, MN, USA. This is an example of a study where the retroviral vector provides anti-sense sequences which directly target the genetic cause of the malignancy – in this case BCR/ABL. Transduced marrow is also rendered resistant to methotrexate.

0310–608

Administration of LMP2a-specific Cytotoxic T-Lymphocytes to Patients with Relapsed EBV-Positive Hodgkin’s Disease. Baylor College of Medicine, Houston, TX, USA. In this study, gene-transfer technology is used to facilitate ex-vivo selection and expansion of tumor-specific T cells. Dendritic cells are transduced with an adenovirus coding for a tumor antigen – LMP2. These transduced dendritic cells are used to selectively stimulate LMP2-specific T cells from peripheral blood mononuclear cells. Neither transduced cells nor vector are directly administered to the patient.

0005–400

Transfer of the Multidrug Resistance Gene, MDR-1, to Hematopoietic Progenitors from Patients with High Risk Lymphoma. University of Massachusetts Medical School Cancer Center, Worcester, MA, USA. This is an example of a chemoprotection study. Transduced, re-administered marrow should be resistant to subsequent chemotherapy.

9907–330

Autologous CD8+ T Lymphocyte Clones Transfected to Express CD20-Specific scFvFc-Zeta as Therapy for Recurrent/Refractory CD20+ Lymphoma. City of Hope National Medical Center, Duarte, CA, USA. Investigators re-target T cells to recognize CD20 with an artificial T-cell receptor. This study will have the potential side effect of depleting normal B cells as well as tumor cells.

0205–535

Phase I Study to Evaluate the Safety of Cellular Immunotherapy for High-Risk CD19+ Acute Lymphoblastic Leukemia after Autologous Hematopoietic Stem Cell Transplantation Using Genetically Modified CD19-redirected Autologous Cytolytic T-Cell Clones. City of Hope National Medical Center, Duarte, CA, USA. This study is similar to 9907–330, except here CD19 is targeted by an artificial receptor instead of CD20. As well as an artificial receptor, cells are engineered to express a suicide gene – HSV-tk. This renders T cells susceptible to ganciclovir. If unacceptable side effects occur, the investigators have the option of administering ganciclovir, which will delete transduced T cells.

0301–564

Autologous T cells Retrovirally Transduced to Express Anti-CEA-scFv-CD28 Zeta as Therapy for Adenocarcinoma. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. The first proposed clinical study of second-generation artificial TCRs that incorporate a CD28 signal as well as a zeta signal.

analysis revealed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, a gene known to be involved in T-cell growth and differentiation.4 The common-gamma chain is involved in transmitting potent proliferative signals. In the construct used, gene expression was controlled by the retroviral LTR rather than by the gene’s native promoter system. Altered expression or regulation of the common gamma chain due to its placement into a retroviral vector together with activation of

LMO-2 at some stage of T-cell development may enhance leukemogenesis.153 The relatively long latency period (i.e. approximately 3 years) in these two cases suggested a multistep mutagenesis event. Indeed, one of the patients also had acquired mutation of another T-cell oncogene, TAL/SCL.4,154 A similar observation was made when mice transplanted with stem cells transduced with truncated nerve-growth factor receptor (dLNGFR) developed AML with a single integration involving EVI-1.155 Like LMO2,

Gene transfer: methods and applications

disruption of EVI1 was not felt to be sufficient on its own to cause AML. In other words, the transgene itself may represent a “second hit,” and investigators are studying ways in which signaling proteins may be expressed more physiologically. If this hypothesis is correct, the approaches we have outlined for the gene therapy of leukemia are unlikely to prove problematic, since few involve the introduction of unregulated T-cell growth factors. While continued caution is essential, it is also important to remember that many hundreds of cancer patients have received retrovirally transduced cells, without subsequent leukemogenesis.

Conclusion Table 27.2 shows the distribution of attempts at cancer gene-therapy thus far, and Table 27.3 lists some current studies of interest. We have many obstacles to overcome before the extraordinary potential of gene transfer for therapy of hematologic malignancies can be fully exploited. However, most advances in medicine proceed incrementally, and gene transfer is already being used successfully to complement conventional therapies for leukemias and other hematologic cancers. It is facilitating the development of cytotoxic T-cell therapies for leukemias and lymphomas, and is being explored as a means of generating leukemia vaccines. The benefits of this new technology can only increase as current limitations are progressively – albeit slowly – surmounted. REFERENCES 1 Bender, M. A., Palmer, T. D., Gelinas, R. E., & Miller, A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol, 1987; 61: 1639–46. 2 Brenner, M. K., Rill, D. R., Holladay, M. S. et al. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet, 1993; 342: 1134–7. 3 Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., et al. Sustained correction of X-linked severe combined immunodeficiency by ex-vivo gene therapy. N Engl J Med, 2002; 346: 1185–93. 4 Hacein-Bey-Abina, S., Kalle, C. von, Schmidt, M., et al. LMO2associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003; 302: 415–19. 5 Amado, R. G. & Chen, I. S. Lentiviral vectors – the promise of gene therapy within reach? Science, 1999; 285: 674–6. 6 Sutton, R. E., Wu, H. T., Rigg, R., Bohnlein, E., & Brown, P. O. Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells. J Virol, 1998; 72: 5781–8.

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130 Goldstone, A. H., Anderson, C. C., Linch, D. C., et al. Autologous bone marrow transplantation following high dose chemotherapy for the treatment of adult patients with acute myeloid leukaemia. Br J Haematol, 1986; 64: 529–37. 131 Shpall, E. J. & Jones, R. B. Release of tumor cells from bone marrow. Blood, 1994; 83: 623–5. 132 Brugger, W., Bross, K. J., Glatt, M., et al. Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood, 1994; 83: 636–40. 133 Rill, D. R., Santana, V. M., Roberts, W. M., et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood, 1994; 84: 380–3. 134 De Fabritiis, P., Ferrero, D., Sandrelli, A., et al. Monoclonal antibody purging and autologous bone marrow transplantation in acute myelogenous leukemia in complete remission. Bone Marrow Transplant, 1989; 4: 669–74. 135 Gambacorti-Passerini, C., Rivoltini, L., Fizzotti, M., et al. Selective purging by human interleukin-2 activated lymphocytes of bone marrows contaminated with a lymphoma line or autologous leukaemic cells. Br J Haematol, 1991; 78: 197– 205. 136 Gorin, N. C., Aegerter, P., Auvert, B., et al. Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: a European survey of the role of marrow purging. Blood, 1990; 75: 1606–14. 137 Santos, G. W., Yeager, A. M., & Jones, R. J. Autologous bone marrow transplantation. Annu Rev Med, 1989; 40: 99–112. 138 Gribben, J. G., Freedman, A. S., Neuberg, D., et al. Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med, 1991; 325: 1525–33. 139 Brenner, M., Krance, R., Heslop, H. E., et al. Assessment of the efficacy of purging by using gene marked autologous marrow transplantation for children with AML in first complete remission. Hum Gene Ther, 1994; 5: 481–99. 140 Cornetta, K., Tricot, G., Broun, E. R., et al. Retroviral-mediated gene transfer of bone marrow cells during autologous bone marrow transplantation for acute leukemia. Hum Gene Ther, 1992; 3: 305–18. 141 Cai, Q., Rubin, J. T., & Lotze, M. T. Genetically marking human cells – results of the first clinical gene transfer studies. Cancer Gene Ther, 1995; 2: 125–36. 142 Santana, V. M., Brenner, M. K., Ihle, J., et al. A phase I trial of high-dose carboplatin and etoposide with autologous marrow support for treatment of stage D neuroblastoma in first remission: use of marker genes to investigate the biology of marrow reconstitution and the mechanism of relapse. Hum Gene Ther, 1991; 3: 257–72. 143 Deisseroth, A. B., Kantarjian, H., Talpaz, M., et al. Autologous bone marrow transplantation for CML in which retroviral markers are used to discriminate between relapse which arises from systemic disease remaining after preparative therapy versus relapse due to residual leukemia cells in autologous marrow: A pilot trial. Hum Gene Ther, 1991; 2: 359–76.

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144 Rill, D. R. & Smith, S. Long term in vivo fate of human hemopoietic cells transduced by moloney-based retroviral vectors [abstract]. Blood, 2000; 84: 96. 145 Moritz, T., Mackay, W., & Feng, L. J. Gene transfer of 0-6-methylguanine methyltransferase (MGMT) protects hematopoietic cells (HQ) from nitrosourea (NU) induced toxicity in vitro and in vivo [abstract]. Blood, 1993; 81: 18a. 146 Dunbar, C. E., Bodine, D. M., Sorrentino, B., et al. Gene transfer into hematopoietic cells. Implications for cancer therapy. Ann N Y Acad Sci, 1994; 716: 216–24. 147 Dunbar, C. E., Cottler-Fox, M., O’Shaughnessy, J. A., et al. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood, 1995; 85: 3048–57. 148 Brenner, M. K., Rill, D. R., Heslop, H. E., et al. Gene marking after bone marrow transplantation. Eur J Cancer, 1994; 30A: 1171–6. 149 Roskrow, M. A., Rooney, C. M., Heslop, H. E., et al. Administration of neomycin resistance gene marked EBV specific cytotoxic T-lymphocytes to patients with relapsed EBV-positive Hodgkin disease. Hum Gene Ther, 1998; 9: 1237–50.

150 Rooney, C. M., Smith, C. A., Ng, C. Y. C., et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood, 1998; 92: 1549–55. 151 Schmidt, M., Hoffmann, G., Wissler, M., et al. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum Gene Ther, 2001; 12: 743–9. 152 Vanin, E. F., Kaloss, M., Broscius, C., & Nienhuis, A. W. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol, 1994; 68: 4241–50. 153 Dav´e, U. P., Jenkins, N. A., & Copeland, N. G. Gene therapy insertional mutagenesis insights. Science, 2004; 303: 333. 154 McCormack, M. P. & Rabbitts, T. H. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency expectations. N Engl J Med, 2004; 350: 913–22. 155 Li, Z., Dullmann, J., Schiedlmeier, B., et al. Murine leukemia induced by retroviral gene marking. Science, 2002; 296: 497.

28 Minimal residual disease Dario Campana, Andrea Biondi, and Jacques J. M. van Dongen

Introduction A multitude of clinical and biologic factors have been associated with a variable response to treatment in patients with acute leukemia,1,2 but their predictive power is far from absolute, and their usefulness for guiding clinical decisions in individual patients is inherently limited. Rather than predicting treatment response, in vivo measurements of leukemia cytoreduction provide direct information on the effectiveness of treatment in each patient. This information should have great clinical utility, but estimates by conventional morphologic techniques have a relatively low sensitivity and accuracy: in most cases, leukemic cells can be detected in bone marrow with certainty only when they constitute 5% or more of the total cell population. These limitations are overcome by methods for detecting minimal (i.e. submicroscopic) residual disease (MRD), which can be 100 times more sensitive than morphology and allow a more objective assessement of treatment response. The definition of “remission” in patients with acute leukemia by these methods is becoming the standard at many cancer centers. Initial reservations regarding the clinical utility of MRD testing arose from concerns regarding the heterogeneous distribution of leukemia during clinical remission.3,4 Another concern was that MRD signals may not correspond to viable leukemic cells with the capacity for renewal. As discussed in this chapter, several correlative studies of MRD and treatment outcome have now firmly established that MRD studies can be highly informative. The first MRD studies in patients with leukemia were made soon after antibodies for leukocyte differentiation antigens became available. In early studies, it was also

noted that T-lineage acute lymphoblastic leukemia (TALL) cells (and normal thymocytes) expressed terminal deoxynucleotidyl transferase (TdT) and T-cell markers, whereas lymphoid cells in bone marrow and peripheral blood did not.5 This finding allowed the first MRD studies in patients with acute leukemia and remains the basis of MRD studies by immunologic techniques in T-ALL. Over the following two decades, many methods of studying MRD have been tested. The most reliable include flow cytometric profiling of aberrant immunophenotypes, polymerase chain reaction (PCR) amplification of fusion transcripts and chromosomal breakpoints, and PCR amplification of antigen-receptor genes. Conventional karyotyping and fluorescence in situ hybridization (FISH), which have limited sensitivity (approximately 1% to 5%), are occasionally useful in elucidating the nature of morphologically suspicious blast cells, but cannot consistently detect submicroscopic leukemia.6 Improvements in image analysis technology allowing simultaneous visualization of morphologic, immunophenotypic and FISH features may enhance the utility of this approach.7 Methods based on the differential properties of normal and leukemic cells in culture have been successful in a few laboratories,8,9 but are not sufficiently reliable for clinical application.

Polymerase chain reaction Principles of the technique PCR allows exponential and selective amplification of defined DNA regions.10,11 Two oligonucleotide primers (generally 18 to 25 bp), produced with an automated DNA

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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synthesizer, are designed to complement the sequences of opposing DNA strands at opposite sites of the target regions. The two primers are the starting point for DNA synthesis by the thermostable Taq DNA polymerase, which uses both strands of the target sequence as a template. DNA amplification occurs through a series of temperatureregulated cycles. Each cycle starts with the denaturation of the double-stranded DNA at a high temperature (94–95 ◦ C), followed by primer annealing at a lower temperature (55–65 ◦ C); the temperature is then raised (72 ◦ C) to allow primer extension by the Taq polymerase. The PCR products of the previous cycles are the templates in the subsequent cycles. Thus, each PCR cycle essentially doubles the number of PCR products, and continuation of the PCR procedure for 20 to 30 cycles theoretically amplifies the DNA target region 220 to 230 times. To increase the specificity and sensitivity of PCR analysis, one can use an aliquot of PCR product after a series of cycles and add it to a fresh reaction mixture with internal primers (“nested” primers). In this way, the second (nested) PCR uses template sequences, which were enriched via the initial PCR. This nested PCR procedure can eliminate the background of nonspecific PCR products, provide much clearer final products, and increase the sensitivity of the PCR test. In principle, DNA sequences of up to 20 kb can be amplified by the PCR technique, but in routine PCR studies the target generally should not be longer than 2 kb. When the target sequences are spread over two or more exons at the DNA level, the distance between the primers might be too large for efficient amplification. In such cases, it is possible to target mRNA, in which the large intervening intron sequences have been spliced out. Before the PCR procedure can start, the mRNA molecules must be transcribed into complementary DNA (cDNA) by reverse transcriptase (RT), thus the term RT-PCR. PCR products can be easily visualized by electrophoresis in ethidium bromide-stained gels. The products are identified through dot blotting or gel-electrophoresis blotting followed by hybridization with a oligonucleotide probe, which specifically recognizes the amplified (c)DNA region. Current molecular techniques also allow direct sequencing of the (c)DNAs obtained with PCR techniques.

Leukemia-specific targets Breakpoint fusion genes Breakpoint fusion regions of chromosomal aberrations can be used as tumor-specific targets for MRD detection by PCR. Amplification of these hybrid sequences with “standard range” PCR using DNA as a starting material is feas-

ible only when the breakpoints of different leukemias cluster in relatively small breakpoint areas, preferably <2 f kb. This is the case for the submicroscopic 1p32 (TAL1) deletions, which are found in 5% to 15% of T-ALL patients12–14 and occur within a region a few hundred base pairs long. There is marked heterogeneity in the sequence at the junctional region, due to random deletion and insertion of nucleotides mediated via illegitimate V(D)J recombination. The diversity of the fusion region allows the design of matching patient-specific oligonuceotide probes.12,13 New techniques for rapid and efficient screening of relatively large breakpoint regions, such as long-distance PCR and long-distance inverse PCR, allow the amplification of genomic fragments many kilobase-pairs long. Therefore, it is now possible to identify the fusion genes of several translocations at the DNA level.14–19 For most of the translocations listed in Table 28.1, the breakpoints found in leukemic cells of different patients are scattered over segments of up to 200 kb. In these cases, chimeric mRNA and the resulting cDNA after reverse transcription are the preferred target for PCR analysis (reviewed in van Dongen et al.20 ). This approach requires the extraction of total or messenger RNA from bone marrow mononuclear cells, reverse transcription of RNA into cDNA and molecular assay by PCR, followed by agarose gel electrophoresis. The sensitivity of the method depends on the target and can be assessed by amplification of serial dilution of diagnostic RNA into RNA extracted from cells of healthy individuals. The presence of a very small number of target cells, in the range of 1 in 105 or 106 , can be consistently detected in appropriate conditions.21 This sensitivity is approximately 1000 to 10,000 times higher than that of Southern blot analysis.22 A single PCR test is sufficiently sensitive (1 leukemia cell in 102 to 103 normal cells) to detect fusion transcripts at diagnosis. The higher sensitivity required for MRD studies can be achieved by a second round of PCR (“nested” PCR) using internal primers. In this way, 1 leukemia cell in 104 to 105 normal cells can be detected in most cases. Extra primer sets must be designed to cover fusion gene transcripts with different exon compositions.20 Until now, most PCR-based MRD studies have used semiquantitative methods for the detection of clonespecific translocations (reviewed in Szczepanski et al.23 and Cazzaniga et al.24 ). Amplification of target DNA/cDNA by standard PCR eventually reaches a plateau; after 35 to 40 cycles the precise quantitation of the initial amount of target material is impossible. Attempts to improve quantitation with competitive PCR and limiting dilution require serial dilutions and the analysis of multiple replicates, both of which introduce variability and are too difficult and too time-consuming to be performed routinely.25

Minimal residual disease

Real-time quantitative PCR technology (RQ-PCR) circumvents these problems, because the accumulation of the PCR product is monitored throughout the PCR process (reviewed in van der Velden et al.26 ). Numerous studies, reviewed by van der Velden et al.,26 have demonstrated the capacity of RQ-PCR to quantify chimeric transcripts. The principles of RQ-PCR are the same whether DNA or RNA is being analysed, the RT step being the major difference when RNA is used. In this case, it is necessary to correct variations linked to differences in the RNA amount taken for the reaction or, more importantly, in efficiency (or inhibition) during reverse transcription. For this reason, the number of target gene copies must be normalized using a ubiquitously expressed housekeeping gene as a reference (e.g. ABL, b2M, GUS, and PBGD). Thus, the number of chimeric transcripts is expressed as a ratio of the number of copies of the reference gene transcripts. There are several potential causes of false-positive and false-negative results by RT-PCR and RQ-PCR.27 RNA degradation must be prevented and the quality of RNA preparation assessed by evaluating ribosomal RNA bands. The RT step should be checked by parallel amplification of an appropriate housekeeping gene from the same cDNA preparation tested for fusion genes. Suitable control genes should have no highly related homologues and/or pseudogenes in the human genome. For PCR analysis of most leukemia-associated fusion gene transcripts, the ABL and GUS genes appear to meet these requirements.26 Primers selected for the control gene should prevent amplification of genomic DNA by spanning a large intron sequence. A positive test for a certain patient should be verified by testing an independent sample from the same patient with a different analytic technique. In RT-PCR assays, the control gene and the target transcripts should have similar stability. Aberrantly expressed genes The Wilms’ tumor gene (WT1), located on chromosome 11p13, encodes a zinc-finger transcription factor that was originally identified because of its involvement in the pathogenesis of Wilms’ tumor.28 The WT1 protein is a potent transcriptional repressor of several growth factors and its expression is strongly regulated in a time- and tissue-specific manner. This transcription factor is highly expressed in most acute leukemias, and its detection in bone marrow has been associated with the presence, persistence, or reappearance of leukemia.29–31 More recently, WT1 gene expression was shown to be a useful marker for tracking disease progression in adults and children with myelodysplastic syndrome (MDS).32–34 Of note, quantitative analysis of WT1 gene expression in peripheral blood appeared to be a useful tool for detecting MRD in patients

681

Table 28.1 Molecular targets of PCR analysis Chromosomal abnormality

Molecular target for PCR

Frequency (%)a

T-ALL — t(5;14)(q35;q32) t(11;14)(p13;q11) t(10;14)(q24;q11) t(1;14)(p32;q11)

TAL (DNA) or SIL-TAL (RNA) HOX11L2 (RNA) RHOM2-TCRD (DNA) HOX11-TCRD (or TCRA) (DNA) TAL1-TCRD (or TCRA) (DNA)

15–25 20 7 4 3

B-lineage ALL t(12;21)(p13;q22) t(1;19)(q23;p13.3) t(9;22)(q34;q11) t(4;11)(q21;q23) t(8;14)(q24;q32.3) t(5;14)(q31;q32) t(11;19)(q23;p13) t(9;11)(p21–22;q23) t(17;19)(q22;p13) — —

TEL-AML1 (RNA) E2A-PBX1 (RNA) BCR-ABL (RNA) MLL-AF4 MYC-IGH (DNA) IL3-IGH (DNA) MLL-AF9 (RNA) MLL-AF9 (RNA) E2A-HLF (RNA) PRAME (RNA) WT1 (RNA)

25 6 4 2 2 <1 <1 <1 <1 40 60–95

AML t(8;21)(q22;q22) t(15;17)(q21;q21) inv(16)(p13;q22)/ t(16;16) t(9;11)(p21–22;q23) t(3;5)(q25;q34) t(9;22)(q34;q11) t(6;9)(p23;q34) t(16;21)(p11;q22) t(7;11)(p15;p15) t(8;16)(p11;p13) — — —

AML1-ETO (RNA) PML-RARA (RNA) CBFB-MYH11 (RNA)

8–15 7–20 7–12

MLL-AF9 (RNA) NPM-MLT1 (RNA) BCR-ABL (RNA) DEK-CAN (RNA) FUS-ERG (RNA or DNA) NUP98-HOXA9 (RNA) MOZ-CBP (RNA) FLT3/ITD (DNA or RNA) WT1 (RNA) PRAME (RNA)

7–10 1 <1 <1 <1 <1 <1 5–35 70–95 60

CML t(9;22)(q34;q11)

BCR-ABL (RNA)

95

a

Based on literature review.

with juvenile myelo-monocytic leukemia or MDS, suggesting that it could be an ideal target for post-transplant monitoring.33 In some cases of acute myeloid leukemia (AML), cells have an internal tandem duplication of the FLT3 gene (FLT3/ITD),35 in which a fragment of the JM domain coding sequence is duplicated in direct head-to-tail orientation. The length of the FLT3/ITD varies, most often including exon 11 and sometimes intron 11 and exon 12. Additional nucleotides are randomly inserted, resulting in a patientspecific sequence.35,36 The mutated fragments were always

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found in frame. Detected in approximately 25% of adult AML cases,37 the reported incidence of FLT3/ITD in childhood AML ranges widely, from 5.3% to 22.2%.36,38–43 In adult AML, the FLT3/ITD has been associated with leukocytosis at onset and a poor outcome.37 Recently, a high incidence of this abnormality (36%) has been reported in adult and children with APL.41,43,44 Although a sensitive and patient-specific method for detecting FLT3/ITD has been described,45 the observation that this marker may not be stable during the course of the disease46,47 argues against its use as MRD target. Final conclusions await further studies. A T-ALL-specific cryptic chromosomal translocation, t(5;14)(q35;q32), present in approximately 20% of childhood T-ALL cases,48 transcriptionally activates HOX11L2, a HOX11-related gene. HOX11 is an orphan homeobox gene that was first identified because of its transcriptional activation by the t(10;14)(q24;q11) or t(7;10)(q35;q24) translocations of T-ALL. HOX11L2 expression was detected in T-ALL, but not in B-lineage ALL and AML cases, and has been used as a marker for MRD.49 PRAME (preferentially expressed antigen of melanoma) is highly expressed in cells of various solid tumors and normal testis, and was first isolated as a melanoma-specific antigen recognized by cytotoxic T cells. It has also been found to be expressed in some hematologic malignancies and has potential as a target for MRD studies.50–52 Junctional regions of IG and TCR genes The rearrangement and joining of the multiple variable (V), diversity (D), and joining (J) gene segments of the IG and TCR gene loci produce their repertoire of diversity (see Chapter 8 for details).53 These rearrangements are mediated by recombination signal sequences (RSS) that flank the 3 side of V gene segments, both sides of D gene segments, and the 5 side of J gene segments.53 Deletion of nucleotides from the germline sequences of the rearranging gene segments and random insertion of nucleotides at the junctional sites of the gene segments further increase diversity.54,55 The junctional regions of rearranged IG and TCR genes are unique “fingerprintlike” sequences, assumed to be different in each lymphoid cell and in each lymphoid malignant clone. Hence, these regions can be used as tumor-specific targets for PCR analysis (reviewed in Szczepanski et al.56 ). The PCR primers for amplification of junctional regions are matched to opposite sides of the junctions, generally within a distance of less than 500 bp. The size limitation of PCR products (usually less than 2 kb) is an advantage for PCR analysis of junctional regions of rearranged IG and TCR genes, because the distance between germline gene seg-

ments is usually far too large to be covered by PCR amplification, whereas joined gene segments are easily amplified. For almost every possible V–(D–)J rearrangement, a primer pair can be made. However, to reduce the number of PCR primers and PCR tests for detection of all IG and TCR gene rearrangements, general or consensus primers are used. These primers recognize virtually all V or J gene segments of a particular Ig or TCR gene complex. Alternatively, one can use family-specific primers that recognize families of V or J gene segments, or member-specific primers that recognize individual V or J gene segments (reviewed in van Dongen et al.57 ). For PCR studies, the various IG and/or TCR gene rearrangements must be identified in each lymphoid malignancy at diagnosis by using the appropriate PCR primer sets. It should be determined whether the PCR products derive from the malignant cells and not from contaminating normal cells with similar (but polyclonal) IG or TCR gene rearrangements. For this purpose, the PCR products are analyzed for their clonal origin, e.g. by heteroduplex analysis or fluorescent GeneScanning (see Chapter 8).58,59 Monoclonal PCR products can be used for direct sequencing of the junctional regions of the IG/TCR gene rearrangements. This sequence information allows the design of junctional region-specific oligonucleotides, so called allele-specific oligonucleotides (ASO).

PCR analysis of junctional regions in precursor-B-ALL Based on their immunophenotypic characteristics, cases of precursor-B-ALL are regarded as clonal malignant derivatives of normal B-cell precursors. More than 95% of childhood precursor-B-ALL cases have IGH gene rearrangements, and most of them contain IGK gene rearrangements (30%) or deletions (50%); 20% of precursorB-ALL cases have IGL gene rearrangements (Table 28.2).60–64 Most IGH gene rearrangements involve complete VH –(DH )–JH joining. One study identified incomplete DH –JH rearrangements in 22% of patients; in 5% they were the only IGH rearrangement.65 Deletions in the IGK genes are predominantly mediated by the IGK-deleting element (Kde) sequence so that such deletions can be identified as Kde rearrangements. Kde rearranges either to a heptamer RSS in the J –C intron, thereby deleting the C gene segment, or to a V gene segment, thereby deleting a large part of the IGK gene (including the J and C gene segments), or, very rarely, to one of the RSS flanking J gene segments.66,67 IGK-Kde rearrangements occur on one allele or both alleles in 20% and 30% of precursor-B-ALL cases, respectively.62 Cross-lineage TCR gene rearrangements occur at high frequency in childhood precursor-B-ALL: TCRB, TCRG, and

Minimal residual disease

Table 28.2 Frequencies of identifiable IG and TCR gene rearrangements as MRD-PCR targets in ALLa

Gene IGH

IGK

TCRG TCRD

Rearrangement type

Precursor-B ALL (%)

T-ALL (%)

VH –JH DH –JH Total IGH V -Kde Intron RSS-Kde Total IGK-Kde V –J V –J 1 or D –J 1 V 2–D 3 or D 2–D 3 Total TCRD

93 20 98 45 25 50 55 <1 40 40

5 23 23 0 0 0 95 50 5 55

>95b ∼90b ∼65

>95b ∼90b ∼50

At least one PCR target At least two PCR targets At least three PCR targets a

The indicated frequencies refer solely to the presence of PCRdetectable rearrangements. b When a high sensitivity (≤10−4 ) is included as an extra criterion, the frequency of at least one sensitive PCR target drops to 85% to 90% and the frequency of at least two sensitive PCR targets drops to approximately 80%.

TCRD gene rearrangements and/or deletions are found in 35%, 60%, and 90% of cases, respectively.68,69 However, the spectrum of cross-lineage TCR gene rearrangements in precursor-B ALL is limited as compared to that in normal and malignant T cells. For instance, 80% of TCRD gene rearrangements are incomplete V 2–D 3 or D 2–D 3 joinings (Fig. 28.1); precursor-B-ALL cases rarely contain complete V –J rearrangements,70 which are typically found in normal T cells and T-ALL blasts, particularly TCR + T-ALL.71 Furthermore, V 2–D 3 rearrangements are prone to continuing rearrangements, particularly to J
gene segments with concomitant deletion of the C exons and subsequent V
–J
recombination.72,73 In fact, 40% of TCRD gene deletions in childhood precursor-B-ALL result from a V 2–J
recombination, while the remaining 60% of C deletions are most probably caused by V
–J
rearrangements.69 Moreover, more than a half of all V 2–J
employs the J
29 segment (Szczepanski et al., unpublished observations). Interestingly, the occurrence of cross-lineage TCR gene rearrangements seems to be age-dependent.74–76 For example, the incidence of incomplete V 2–D 3 gene rearrangements significantly decreases with the age of patients, while TCRG gene rearrangements are rarely found in patients younger than 2 years of age and are virtually absent in infant ALL.74,75,77

In summary, junctional regions of IGH, IGK (especially IGK-Kde), TCRG, and TCRD gene rearrangements can be identified with only a limited number of PCR primer sets and can be applied in MRD monitoring in the vast majority (>95%) of precursor-B-ALL patients (Table 28.1).13,65,69,78

PCR analysis of junctional regions in T-ALL Leukemic T cells are the clonal counterparts of normal cortical thymocytes. Subclassification of T-ALL into CD3− , TCR + , and TCR
+ subgroups reveals essential differences in TCR gene rearrangement patterns.60,79 Approximately 10% of CD3− T-ALL cases have all of their TCR genes in germline configuration.60,79 These typically have the immature CD1− CD3− phenotype of the T-ALL of the prothymocytic/pre-T-ALL subgroup, which might arise from an early common myeloid/T-cell precursor. The TCRD genes in CD3− T-ALL are rearranged in approximately 80% of cases and contain biallelic deletions in approximately 10% of cases.79,80 Approximately 80% of CD3− T-ALL cases have rearranged TCRG and/or TCRB genes.81,82 As expected, all TCR + T-ALLs have TCRG and TCRD gene rearrangements, and approximately 95% also have TCRB gene rearrangements.71,80 All TCR
+ TALL cases contain TCRB and TCRG gene rearrangements and have at least one deleted TCRD allele (due to a TCRA rearrangement); the second TCRD allele is also deleted in two-thirds of cases.60,80 In more than 95% of childhood TALL cases, TCRG and/or TCRD junctional regions can be identified with PCR techniques and are potential targets for MRD monitoring (Table 28.1).80,81 Newly designed multiplex PCR strategy to sequence the junctional regions of TCRB gene rearrangements should soon allow their routine use as MRD targets.83 Cross-lineage IG gene rearrangements occur in approximately 20% of T-ALL cases and involve virtually only IGH genes (Table 28.2).60 Heteroduplex PCR analysis showed incomplete DH –JH rearrangements in approximately 80% of cases, as well as preferential usage of DH 6–19 and the most downstream DH 7–27 gene segments.84 By contrast, complete VH –JH recombinations comprised only 18% of cross-lineage IGH gene rearrangements in T-ALL patients.84

PCR analysis of junctional regions in AML Cross-lineage IG and TCR gene rearrangements can be also identified in approximately 10% of AML patients and most frequently concern IGH and TCRD genes.85–88 Because of their low prevalence in AML, IG/TCR gene rearrangements are not suitable for routine MRD detection but

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Fig. 28.1 Monitoring of a patient with B-lineage ALL for the presence of MRD in bone marrow samples using a V 2–D 3 PCR target. PCR analysis with V 2 and D 3 primers was performed on 10-fold dilutions of the DNA isolated at diagnosis and on DNA from bone marrow samples during follow-up (in weeks). The junctional region sequence had a deletion of six germline nucleotides and a random insertion of seven nucleotides. Hybridization with the patient-specific probe resulted in a sensitivity of 10−5 . The follow-up samples taken at weeks 5, 15, and 25 were found to be MRD-positive with decreasing leukemic infiltrates. Subsequent samples were MRD-negative.

might occasionally be used as supplementary MRD targets in addition to flow cytometric immunophenotyping and leukemia-specific chromosomal aberrations.

Pitfalls of PCR analysis of IG/TCR genes – oligoclonality, clonal evolution IG and TCR genes in ALL might undergo continuing or secondary rearrangements mediated via the active recombinase enzyme system (reviewed in Szczepanski et al.89 ). This results in oligoclonality, with subclones having distinct clonal IG/TCR gene rearrangements. Oligoclonality at diagnosis creates uncertainty about the clone that will emerge at relapse and that should be monitored with PCR. IGH genes in precursor-B ALL are particularly prone to subclone formation; multiple gene rearrangements are found in 30% to 40% of cases at diagnosis, indicating the presence

of biclonality or oligoclonality.62 Most continuing IGH gene rearrangements represent ongoing VH to DH –JH rearrangements or VH replacements.90,91 During these rearrangements, the DH –JH junctional region remains unaffected, leading to the concept of designing the primers around the relatively stable DH –JH region to prevent false-negative PCR results.90–92 By contrast, secondary VH –JH rearrangements that use upstream VH and downstream JH gene segments will cause false-negative PCR results.93 TCR gene oligoclonality is rarely seen at diagnosis in TALL,60,81,94 in contrast to the more than 30% frequency among cross-lineage TCR rearrangements in precursor-BALL.69 False-negative results due to clonal evolution are a major drawback of using IG/TCR gene rearrangements as PCR targets for MRD detection.93–96 Monoclonal MRDPCR targets in childhood precursor-B ALL are very stable, with approximately 90% of all targets detectable at relapse.

Minimal residual disease

By contrast, only 40% of the oligoclonal MRD-PCR targets are preserved at relapse.93 Therefore, it is important to discriminate between monoclonal and oligoclonal IG/TCR rearrangements with a combined Southern blot and PCR approach. At present, the IGK-Kde gene rearrangements are considered to be the most stable MRD-PCR targets, probably because they are rarely oligoclonal at diagnosis and represent end-stage rearrangements, hence do not allow continuing or secondary rearrangements.93,97 Despite the high frequency of immunogenotypic changes in childhood ALL at relapse, at least one rearranged IGH, TCRG and/or TCRD allele remains stable in 75% up to 90% of precursor-B-ALL and in 90% of T-ALL cases.93–96,98,99 More importantly, in most ALL patients, at least two suitable PCR targets are available. Therefore, it is now generally accepted that MRD-PCR studies should preferably use at least two IG/TCR targets per patient, a strategy that should result in a reduction of false-negative results.

Sensitivity of MRD measurements by PCR amplification of IG/TCR gene rearrangements If recognition of patients with high MRD levels is the aim, sensitivities of 10−2 to 10−3 might be sufficient. Such results might be achieved by detection of clonal IG/TCR gene rearrangements with high-resolution electrophoresis systems, such as radioactive fingerprinting or fluorescent GeneScanning, without the need for patient-specific oligonucleotides.59,100 For more precise discrimination among patients with high, intermediate or low MRD levels, PCR detection should rely on patient-specific oligonucleotides, which allow one to achieve sensitivities of at least 10−4 .

RQ-PCR-based quantification of MRD Currently, the most frequently used quantitative PCR methodology is RQ-PCR, which permits accurate, rapid and easily standardized assessments during the exponential phase of PCR amplification (reviewed in van der Velden et al.26,101 ). Because of the real-time detection, post-PCR processing is not necessary. The method has a very large dynamic detection range, thereby eliminating the need for serial dilutions of follow-up samples. Among several available RQ-PCR techniques, many laboratories use the TaqMan technology, based on the 5 → 3 nuclease activity of the Taq polymerase. Upon amplification, an internal target-specific TaqMan probe (hydrolysis probe) conjugated with a reporter and a quencher dye is degraded, resulting in emission of a fluorescent signal by the reporter dye that accumulates during the consecutive PCR cycles

(Fig. 28.2). For each RQ-PCR method, a real-time amplification plot is generated (Fig. 28.2). The cycle at which the fluorescence signal exceeds a certain background fluorescence level, referred to as the threshold cycle (CT ), is directly proportional to the amount of target DNA present in the sample. The amount of residual leukemic cells in follow-up samples can be calculated by using the standard curve of the diagnostic sample. A control gene (e.g. albumin) should be used to correct for the total amount of DNA and its potential for amplification. If junctional regions of IG and TCR gene rearrangements are used as PCR targets for MRD detection, the TaqMan probe can be used in either two ways. It can be positioned at the junctional region (ASO probe) and used in combination with germline primers, so that the probe will detect leukemia-specific PCR products between the background of PCR products derived from polyclonal IG or TCR gene rearrangements of normal cells. This method requires the design of a new TaqMan probe for each rearrangement, making it labor-intensive and expensive. Alternatively, the TaqMan probe and one of the primers can be positioned at germline sequences, while the other primer is located at the junctional region (ASO primer). This method leads to specific amplification of the leukemia-specific junctional region and is less expensive than the approach described above. Because the ASO primer strategy results in competition for primers between amplification of the leukemic and normal IG/TCR gene rearrangements, it is also more sensitive than the ASO probe method.102 With current RQ-PCR methodology, a reproducible sensitivity of 10−4 can be reached for the majority of currently used MRD-PCR targets. Germline TaqMan probes can, in principle, be used for all IG and TCR gene rearrangements, which use the gene segments that are recognized by the TaqMan probe (Table 28.3). In fact, three consensus germline JH probes,102 one Kde probe,97 one D 3 probe,103 one J 1 probe, and two J probes104 are sufficient for all classically identified IG/TCR MRD-PCR targets in childhood ALL (Tables 28.2 and 28.3).

Sensitivity of RQ-PCR-based MRD detection The theoretical detection limit of the RQ-PCR technique is 10−5 to 10−6 . The sensitivity of MRD-PCR analysis of junctional regions generally ranges from 10−3 to 10−5 , depending on the type of rearrangement. Junctional regions of complete V–D–J rearrangements are extensive, whereas junctional regions of V–J rearrangements are generally three to four times smaller. Accordingly, RQ-PCR-based MRD detection of complete IGH gene rearrangements,

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frequently containing two extensive N-regions, is 5- to 10fold more sensitive than detection based on the smaller junctional regions of IGK-Kde targets (Table 28.3).97,102 The sensitivity of MRD-PCR analysis of IG/TCR junctional regions is also influenced by the “background” of normal lymphoid cells with comparable IG or TCR gene rearrangements. For instance, V 1–J 1 rearrangements not only occur frequently in T-ALL, but also can be found in a small fraction (0.1%–2%) of normal peripheral blood T cells. V1–J 1.3 and V1–J 2.3 joinings constitute 50% to 60% of TCRG gene rearrangements in ALL, and are found in a large fraction (70–90%) of normal T lymphocytes. Taking into account the abundance of normal T lymphocytes with polyclonal V –J joinings, particularly in postinduction follow-up samples,105 it is not surprising that RQ-PCR analysis of short V –J junctional regions is generally less sensitive (10−2 to 10−4 ) than MRD-PCR analysis of long V 1–J 1 junctional regions (10−3 to 10−5 ).13 In fact, a maximum sensitivity of 10−4 can be reached in less than half of TCRG gene rearrangements in precursor-B ALL patients and in only two thirds of TCRG gene rearrangements in TALL patients.104 Similarly, substantial expansions of normal precursor-B cells with polyclonal IGH gene rearrangements in regenerating bone marrow after cessation of chemotherapy might affect the sensitivity of MRD detection using IG gene rearrangements as PCR targets. Nevertheless, it should be possible to identify two sufficiently sensitive targets (≤10−4 ) for RQ-PCR-based MRD detection in at least 80% of patients.56 By adding new targets such as TCRB or V 2–J
gene rearrangements, the proportion of cases with two adequately sensitive MRD-PCR targets should reach 90%.

Fig. 28.2 Real-time quantitative PCR analysis by use of the TaqMan technique. (A) The TaqMan probe contains a reporter dye and a quencher dye, which prevents emission of the reporter dye as long as the two dyes are closely linked. During the extension phase of each PCR cycle, the annealed TaqMan probe is hydrolyzed by the 5 →3 exonuclease activity of the Taq polymerase, thereby separating the reporter dye from the quencher dye. This results in a fluorescent signal (delta Rn), which further increases during each subsequent PCR cycle. (B) Example of an RQ-PCR analysis using the TaqMan approach with IGH rearrangement and an ASO primer. The curves represent real-time amplification plots of 10-fold dilutions of the diagnostic sample in normal mononuclear cell (MNC) DNA. With this IGH gene rearrangement, a reproducible sensitivity of 10−5 was reached. Normal MNC DNA shows amplification in all four wells tested although clearly outside the reproducible range. (C) Standard curve with linear correlation between the cycle threshold (CT ) and the initial amount of DNA (tumor load) for the experiments.

Minimal residual disease

Flow cytometry

687

Table 28.3 RQ–PCR for MRD detection via IG/TCR targets using the allele-specific oligonucleotide (ASO)-primer approach

General methodologic considerations Sensitivity

The discovery that leukemic cells expressed immunophenotypes not expressed by normal bone marrow and peripheral blood cells provided one of the first opportunities to study MRD.5,106,107 Over the years, MRD assays based on immunophenotyping have been consistently improved by advances in the quality and variety of antibodies and fluorochromes, by the refinement of flow cytometers, and by the enormous progress in informatics that has occurred during the last decade. The striking correlations of MRD results obtained by flow cytometry with clinical features and treatment outcome, discussed later in this chapter, provided much credibility to this approach.108–113 Flow cytometry allows the simultaneous analysis of several cellular parameters. By combining information on cell size and granularity, and intensity of expression of surface and intracellular molecules, one can identify an immunophenotypic signature that distinguishes leukemic cells from their normal counterparts. Other features of flow cytometry that are useful when applied to the study of MRD include the rapid analysis of a high number of cells, indefinite storage of data, and capacity of transferring data for analysis in a remote laboratory.114 With the currently available reagents, it is rare when a suitable immunophenotype cannot be found to monitor MRD. Because of the possibility of immunophenotypic shifts, it is advisable to use multiple sets of markers targeting all immunophenotypic abnormalities identified on the leukemic cells to prevent false-negative MRD results. It has been suggested that MRD studies could be done even without the knowledge of the diagnostic immunophenotypic profile, by searching for cells with immunophenotypes that deviate from the established patterns of normal and regenerating lympho-hematopoiesis.115 This approach has the practical advantage of dispensing with the phenotype at diagnosis or relapse, which is often unavailable to referral centers. However, it may be difficult to interpret a negative result without knowing the immunophenotype of the leukemic cells, and this lack of knowledge prevents determining the sensitivity of the MRD assay.

Immunophenotypes for MRD studies Proteins that are produced or dysregulated by gene fusions, such as BCR-ABL, AML1-ETO, or PBX-1 in E2A-PBX1 offer ideal targets for immunophenotypic studies of MRD in acute leukemia, but antibodies that allow reliable detection of these proteins by flow cytometry are not widely

Background

Frequency Gene

Frequency

Germline probe(s)

Reproducible

(%)

Criteria

at 60 ◦ C (%)

IGH

Three JH probes102

10−4 –10−5

85

CT 6

30–40

IGK

One Kde probe97

10−4

85

CT 3

∼30

TCRG

Two J probes104

10−4

30

CT 3

∼90

TCRD

One D 3 probe103

10−4

70

CT 3

∼70

One J 1 probe

10−4

NA

CT 3

NA

Abbreviation: NA, not yet available, experiments in progress.

available.116 Nonetheless, leukemic cells may express leukocyte differentiation markers in combinations and/or at levels quite distinct from those of normal bone marrow and peripheral blood cells. Recognition of these differences forms the basis for current immunophenotypic studies of MRD. To identify immunophenotypes for effective MRD studies, one must consider the variations in the cellular composition and immunophenotype of normal bone marrow that occur with age and exposure to drugs. For example, early lymphoid progenitors (or “hematogones”) are scarce in the bone marrow of healthy adults and rare in patients receiving corticosteroids or chemotherapy.117 By contrast, these cells are abundant in the bone marrows of young children118–120 or of patients with malignancies after transplantation or cessation of chemotherapy.121–124 These conditions may expose normal cells expressing phenotypes that are undetectable in samples obtained from healthy people. Hence, a solid definition of leukemia-associated immunophenotypes requires a thorough analysis of bone marrow cells collected under a variety of conditions.

Markers for MRD studies in ALL Table 28.4 summarizes the combinations of markers used at St. Jude Children’s Research Hospital to study MRD in children with ALL. Other investigators have reported marker combinations found to be useful in their laboratories.108,125–129 The normal equivalents of T-lineage ALL cells are immature T cells. Since these are confined to the thymus, whereas leukemic T lymphoblasts can circulate, MRD studies in patients with T-lineage ALL consist of searching for cells with the phenotype of immature T cells in the bone marrow or peripheral blood. The most widely applicable immunophenotypes used to detect MRD in T-lineage ALL

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Table 28.4 Marker combinations used at St. Jude Children’s Research

Table 28.5 Marker combinations used at St. Jude Children’s

Hospital for monitoring MRD in childhood ALL

Research Hospital for monitoring MRD in childhood AML

Leukemia subtype

Marker combination

Applicability (%)a

Marker combination

Applicability (%)a

T-ALL

Anti-TdT/ CD5/ CD3 CD34/CD5/CD3 CD19/CD34/CD10 /CD58 CD19 /CD34/CD10/CD38 CD19/CD34/CD10/CD45 CD19/CD34/CD10/anti-TdT CD19/CD34/CD10/CD22 CD19/CD34/CD10/CD13 CD19/CD34 / anti-TdT/anti-IgM CD19/CD34/CD10/CD66c CD19/CD34/CD10/CD33 CD19/CD34/CD10/CD65 CD19/CD34/CD10/CD15 CD19/CD34/CD10/CD21 CD19/CD34/CD10/anti-NG2

90–95 30–50 40–60 30–50 30–50 30–50 20–30 10–20 10–20 10–20 5–10 5–10 5–10 5–10 3–5

CD13/CD117/CD34/CD33 CD15/CD117/CD34/CD33 CD13/CD133/CD34/CD33 CD13/CD56/CD34/CD33 HLA-Dr/CD117 / CD34/CD33 CD11b/CD13/CD34/CD33 CD38/CD13/CD34/CD33 CD15/CD13/CD34/CD33 CD7/CD13/CD34/CD33 CD45/CD13/CD34/CD33 CD19/CD13/CD34/CD33 CD11b/CD117/CD34/CD33 HLA-Dr/CD13/CD34/CD33 CD13/anti-NG2/CD34/CD33

30–50 30–50 25–35 20–30 20–30 15–20 15–20 10–15 10–15 10–15 5–10 5–10 5–10 5–10

B-lineage ALL

a

Percentage of cases within a leukemia subtype in which the indicated marker combination allows MRD studies with a sensitivity of 10−4 .

are the co-expression of either CD3 and TdT or CD5 and TdT.106,130 Other authors indicated that antibody combinations, including CD7 and CD3 with CD2 or CD5, may also be aberrantly expressed in a proportion of T-ALL patients.125,126 The normal equivalents of B-lineage ALL cells are B-cell progenitors that normally reside in the bone marrow, and can also be found in low proportions in peripheral blood. B-lineage ALL cells can be distinguished from their normal counterparts on the basis of several molecules expressed at abnormally high or low levels in leukemic cells.122,127,129,131–133 For example, the myeloid-associated markers CD13, CD15, CD33 and CD65, and the mature Bcell-associated marker CD21 can be expressed by CD19+ CD34+ B-lineage ALL cells, whereas normal CD19+ CD34+ B-cell progenitors do not express these markers or express them very weakly.133 Expression of CD19, CD10, TdT and CD34 in B-lineage ALL may be markedly higher or lower than that of their normal counterparts,108,133,134 and CD38 and CD45 (or CD45RA) may be underexpressed in leukemic cells.129,133

Markers for MRD studies in AML Phenotypic abnormalities in AML include expression of markers normally not expressed on myeloid cells and coexpression of markers normally expressed at different stages of maturation, as well as overexpression and underexpres-

a Percentage of cases within a leukemia subtype in which the indicated marker combination allows MRD studies with a sensitivity of 10−3 .

sion of myeloid markers.135 The markers used at St. Jude Children’s Research Hospital are shown in Table 28.5.136 Detection of MRD by flow cytometry in AML presents some specific difficulties. Due to their immunophenotypic heterogeneity, AML cells usually spread across many areas of each dot plot instead of forming the tight cluster typical of ALL cells.137 Therefore, with any given marker combination, only a fraction of cells may appear to be phenotypically abnormal. In addition, AML cells often have lightscattering properties similar to those of normal cells with high autofluorescence. These features introduce complexity in the analysis, and may reduce the sensitivity of the assay. Nevertheless, it is feasible to perform sensitive MRD studies in AML (Fig. 28.3). In a recent St. Jude study, the leukemic cell immunophenotypes of 54 children with newly diagnosed AML were determined by four-color flow cytometry and compared with those of bone marrow mononuclear cells from 21 healthy donors and 38 patients undergoing treatment for ALL or lymphoma.136 In 46 of 54 patients (85.2%), we detected abnormal immunophenotypic features. The leukemic immunophenotypes of 26 patients (48.1%) were sufficiently distinct from those of normal and regenerating bone marrow cells to allow 1 leukemic cell to be detected among 10,000 or more normal bone marrow mononuclear cells (i.e. sensitivity of ≥ 0.01%); in the other 20 patients (37.0%), a partial overlap between the immunophenotypes of regenerating marrow cells and leukemic cells limited the sensitivity to 1 in 1000 (i.e. 0.1%).

Minimal residual disease

Fig. 28.3 Detection of MRD in AML by flow cytometry. Bone marrow mononuclear cells from three healthy donors (top panels) and one patient with AML studied at different stages of treatment (bottom panels) were labeled with antibodies to CD38, CD33, CD34, and CD13. The same number (>105 ) of mononuclear cells were studied in all samples. Flow cytometric dot plots show CD33 and CD38 expression in CD34+ CD13dim cells. In normal samples, these cells also express CD38. In the AML patient, most leukemic cells at diagnosis were CD34+ CD13dim CD33+ but lacked CD38 (dashed square). Cells with this phenotype were detectable after remission induction therapy, a finding that was followed by cytogenetic (Cytogen.) relapse first and then by clinical relapse.

San Miguel et al.110 found that 175 of 233 adult patients with AML expressed leukemia-associated immunophenotypes, while Venditti et al.138 detected them in 65 of 93 patients. A recent study by Sievers et al.139 indicated that residual disease could be studied by flow cytometry in all patients, if a sensitivity of 0.5% was deemed acceptable.

Identification of new markers Microarrays that allow genome-wide analysis of gene expression offer new opportunities to identify markers for MRD studies.140 To test the validity of this concept, we compared the gene profile of ALL cells with that of purified normal B-cell progenitors.141 Among the estimated 4000 genes studied, we found over 250 that were overexpressed in more than one leukemic sample. We selected nine of

these genes for which antibodies were easily available and measured expression of the encoded proteins by flow cytometry. Seven proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19+ CD10+ B cell progenitors. The results with CD58 were in line with a previous report indicating overexpression of this molecule in leukemic cells.142 CD58 is now one of the most useful markers for the study of MRD in B-lineage ALL (Table 28.4). These results suggest that a comparison of the gene profiles of normal and leukemic cells will identify new, widely applicable markers for MRD studies in ALL and in AML, and should ultimately allow the design of simple antibody panels for practical, reliable, and universal monitoring of MRD.

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Sensitivity and measurement of MRD The main variables that influence the detection of rare cells by flow cytometry are the degree of morphologic/phenotypic difference between the target cells and all remaining cells, and the number of cells that can be analyzed. Under ideal conditions [i.e. a large number (107 or more) of very distinct target cells] the sensitivity of flow cytometry is similar to that of PCR.143 During analysis of MRD in clinical samples, however, the number of cells that can be analyzed for each set of markers in children is usually less than 1 × 106 . Because a distinct cluster of at least 10 to 20 dots is necessary to interpret suspect flow cytometric events, the maximum sensitivity achievable in these circumstances would be 1 in 105 cells. The phenotype of primary leukemic cells may not be as distinct as that of cell lines used in an experimental setting and, in the case of Blineage ALL, the sensitivity of detection may be influenced by the treatment interval at which the sample is taken, because of the variable proportion of normal B-cell precursors. Therefore, a sensitivity of 1 in 104 is probably the maximum that can be consistently achieved during routine MRD testing. Flow cytometry has the potential for a very accurate quantitation of MRD. In experiments with serial dilutions of KOPN-57bi pro-B ALL cells admixed with normal peripheral blood mononuclear cells at ratios of 3 in 105 or higher, the estimate of the leukemic cell content in each mixture was extremely accurate (r2 = 0.999).144 In other experiments, the results of multiple measurements of residual leukemia in the same cell mixture was compared.145 In 23 tests of mixtures containing 1 leukemic cell in 104 normal cells, results were remarkably similar (coefficient of variation = 15%); in 22 tests of mixtures containing 1 leukemic cell in 103 cells, the coefficient of variation was 10%.

sistently by flow cytometry but is well within the range of PCR. Such high sensitivity may be desirable, for example, in studies seeking MRD in patients who have a patchy distribution of the disease, or in cell harvests for autografting. Another limitation is that the immunophenotype of leukemic cells may change during the progression of the disease.146–155 If these changes affect markers used for monitoring MRD, a false-negative finding may result.145,153,154 The potential adverse effect of this phenomenon is inversely related to the number of marker combinations that can be applied to each patient. For example, a recent St. Jude study found that at least one of the leukemia-associated immunophenotypes determined at diagnosis and used to monitor residual disease was retained at relapse in 10 of 10 children with AML.136 However, in 1 of the 10 patients, two of three immunophenotypic abnormalities seen at diagnosis were not detectable at relapse, and in another 2 patients, one of three abnormalities disappeared, underscoring the benefit of using multiple marker combinations.136 Finally, a general limitation of flow cytometric assays is that the results may not appear as unequivocal as those of PCR. This is because the distinguishing immunophenotypic features of leukemia are often, although not always, the product of quantitative differences in antigen expression between leukemic and normal cells. Nevertheless, objective MRD estimates are possible if one determines the limits of normal antigenic expression using a variety of normal samples, and avoids the use of immunophenotypes that partially overlap those of normal cells.

Clinical studies in acute lymphoblastic leukemia Molecular genetic abnormalities

Specific advantages and disadvantages Because of its wide availability, flow cytometry is probably the most accessible method for MRD detection. One specific advantage of flow cytometry over PCR-based assays is that it allows direct quantitation of MRD, rather than an extrapolation from amounts of PCR product. This feature makes quantitation easier and, typically, more accurate.144 Moreover, flow cytometry allows the identification of dying cells and cellular debris. Finally, flow cytometric analysis also allows one to establish the status of nonleukemic hematopoietic cells. Flow cytometry also has some specific limitations. Extreme sensitivity, such as detection of 1 leukemic cell among 105 or more normal cells, is difficult to achieve con-

Fusion transcripts generated by the t(9;22), t(4;11), t(1;19) and t(12;21) have been used as targets for RT-PCR to assess MRD in children and adults with ALL (reviewed in Cazzaniga et al.24 and Foroni et al. 156 ). Some of the reported data are controversial, and it is not completely clear if MRD assessment based on tumor-specific translocations helps the prediction of outcome. PCR positivity almost invariably persists during chemotherapy in patients with t(9;22) ALL, in accord with the high relapse rate of this disease subtype in both adults and children.157,158 However, more recent MRD studies following intensive combination chemotherapy (without transplantation) provide intriguing preliminary evidence that achievement of a BCR-ABL negative state, as determined by PCR, may predict durable remissions

Minimal residual disease

in t(9;22) ALL in adults.159,160 PCR studies reveal considerable heterogeneity with respect to MRD levels, even within the t(9;22) ALL subgroup with good early responses to steroids.161 More recently RQ-PCR evaluation of remission has been used as a surrogate marker of response to imatinib.162 Using primer sets from the MLL and AF4 oncogenes, several investigators163–165 have reported results for small series indicating that early conversion to PCR negativity or persistent PCR negativity (particularly after 3 months) is associated with prolonged continuous complete remissions. Similar results were recently confirmed in a prospective series of t(4;11) ALL patients.166 As reviewed by Foroni et al.,156 MRD analysis of ALL patients with the t(1;19) translocation has been performed in 73 children and 3 adults. Although the vast majority of patients achieved a molecular remission, MRD persisted as long as 8 to 12 months (in 25 patients) or 24 to 27 months or longer (in four patients) without the occurrence of clinical relapse. These results raise concerns about the utility of this RT-PCR test for clinical decision making. Residual disease has been investigated in patients with t(12;21) ALL.167–170 Several cases tested positive between 2 and 4 months after remission induction therapy; relapse was observed in cases with persistent MRD positivity at levels greater than 1 × 10−3 . However, relapse also occurred in patients with previous negative tests. Larger prospective studies are needed to fully assess the prognostic value of the t(12;21) translocation as well as its value as a marker for monitoring MRD in childhood ALL.

Antigen receptor gene rearrangements Assessment of early response to remission induction treatment Early retrospective studies and small prospective studies indicated that, in children with ALL, detection of MRD by PCR at the end of remission induction treatment could predict outcome (reviewed in Szczepanski et al.56 ). These studies represented the logical continuation of early treatment response evaluations by morphology, indicating that peripheral blood blast counts of less than 1000/ L after a week of corticosteroid therapy, the absence of circulating blasts after 7 days of multiagent induction chemotherapy, or less than 5% bone marrow blasts at the end of the induction treatment were important prognostic factors.171 Likewise, low levels or the absence of MRD after completion of remission induction therapy predicts a favorable outcome.98,172–177 A review of published MRD studies by PCR amplification of antigen receptor gene rearrangements showed that approximately 50% of children with

ALL are MRD-positive at the end of induction treatment, and approximately 45% of these patients will ultimately relapse with a risk that is proportional to the detected MRD levels.98,156,172,174,176 Multivariate analyses showed that the prognostic value of MRD-PCR levels of positivity after induction therapy is independent and superior to that of other clinically relevant risk factors, including age, blast count at diagnosis, immunophenotype at diagnosis, presence of chromosomal aberrations, response to prednisone, and classical clinical risk group assignment, provided that MRD quantification is accurate and bone marrow samples are adequate.98,172,173 Moreover, several recent large prospective studies showed that estimation of the initial response to chemotherapy via MRD detection can be used to refine risk assignment in current childhood ALL treatment protocols.98,145,172,173,178,179 However, the analysis of MRD at a single time point may not be sufficient for recognition of patients with a poor prognosis as well as those patients with good prognosis.98 A combination of data on the kinetics of tumor load decrease at the end of induction treatment and before consolidation treatment appeared to be highly informative in the prospective MRD study of the International BFM Study Group (I-BFM-SG). In this study, such combined information distinguished among patients at low risk with MRD negativity at both time points (5-year relapse rate, 2%), patients at high risk with an intermediate (10−3 ) or high (≥10−2 ) degree of MRD at both time points (5-year relapse rate, 80%), and the remaining patients at intermediate risk (5-year relapse rate, 22%; see Fig. 28.4).98 The MRD-based low-risk patients made up a substantial group (approximately 45% of all patients), comparable to the proportion of long-term survivors of childhood ALL before treatment intensification was introduced.181 These low-risk patients, half of whom already had low (≤10−4 ) or undetectable MRD levels after 2 weeks of treatment,182 might be good candidates for treatment reduction. On the other hand, MRD-based high risk group was larger than any previously identified high-risk group (approximately 15% of all patients) and had a 5-year relapse rate of 80%. These patients might benefit from further intensification of treatment protocols, including hematopoietic stem cell transplantation during first remission. The MRD data obtained by PCR suggest slower kinetics of leukemia clearance in T-ALL as compared to precursorB-ALL.178,179,183 In a study by Dibenedetto et al.,183 16 of 17 patients were MRD-positive at the end of induction treatment. Assays at the beginning of maintenance treatment were the most prognostically informative: seven of eight MRD-positive patients at this time point subsequently relapsed, while all eight MRD-negative patients remained in continuous complete remission.183 A larger study

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Minimal residual disease

of the I-BFM-SG showed that PCR-based measurement of early treatment response at two consecutive time points after induction treatment and before consolidation treatment yields highly prognostic information (∼25% low-risk patients with 0% relapses, ∼50% intermediate-risk patients with 25% relapses, and ∼25% high-risk patients with 100% relapses).179

The value of continuous MRD monitoring in childhood ALL Continuous MRD monitoring in childhood ALL has shown that a steady decrease of MRD levels to negative PCR results during treatment is associated with a favorable prognosis,184 whereas the persistence of high MRD levels or a steady increase of MRD levels generally leads to clinical relapse.98,177,185,186 Hence, persistent MRD levels during treatment can be regarded as an indicator of in vivo resistance to treatment, suggesting that PCR-based MRD monitoring could be used to identify “poor responders” likely to experience early relapse during maintenance treatment.98,172,173 Sequential sampling generally shows positive MRD-PCR results prior to clinical relapse; however, the time between the detection of MRD positivity and overt hematologic relapse might be too short for effective use of reinduction treatment.187 Low levels of MRD after therapy might be associated with the late development of relapse, but the absence of MRD at the end of treatment is not sufficient to ensure that the patient is cured.98,177 One report claimed that multiple PCR analyses applied to a higher-than-average cell number gave evidence for residual leukemia at very low levels in 15 of 17 patients remaining in long-term clinical remission.188 In 7 of these 15 patients, the positive PCR result was confirmed in a blast colony assay. So far, these data have not been confirmed by other investigators, even using PCR analyses with sensitivities of 10−6 to 10−7 .98 By contrast, large

prospective studies showed MRD positivity rates of 0% to 10% at the end of treatment; most of the patients with a positive result relapsed later on.98,177 Taken together, these data suggest that continuous MRD monitoring in childhood ALL has limited value and might only be useful for a subgroup of patients with a relatively slow early response to treatment, and therefore a high or intermediate risk of relapse. MRD during treatment of relapsed ALL The clinical value of MRD monitoring by PCR amplification of antigen-receptor genes also extends to patients with relapsed ALL.189,190 The BFM group demonstrated that low MRD levels (<10−3 ) after 36 days of treatment were associated with a probability of relapse-free survival of 86%, whereas higher levels (≥10−3 ) were uniformly predictive of a dismal outcome (probability of relapse-free survival, 0%).190 However, the patient numbers are still small and the results need further confirmation. MRD monitoring in childhood ALL patients undergoing hematopoietic stem cell transplantation MRD monitoring using antigen-receptor gene rearrangements as targets was shown to be highly informative for ALL patients undergoing hematopoietic stem cell transplantation (HSCT).100,191–193 This applies to high-risk patients transplanted in first remission as well as to patients undergoing HSCT in second remission after leukemic relapse. In patients receiving T-cell-depleted grafts, high levels of MRD-PCR positivity (10−2 to 10−3 ) before allogeneic transplantion were invariably associated with relapse after transplantation.100,192,193 The 2-year event-free survival in patients with a low level of MRD positivity (10−3 to 10−5 ) was 35% to 50%, irrespective of graft manipulation.100,192,193 It has been suggested that significant graft-versus-host disease associated with nondepleted grafts might overcome

Fig. 28.4 Schematic representation of V III-Kde rearrangement as PCR target in a precursor-B ALL patient treated on the International BFM Study Group treatment protocol. The junctional region sequence of this rearrangement had a deletion of 18 germline nucleotides and a random insertion of 9 nucleotides. Sequences and positions of the germline V III and Kde primers are indicated as well as the patient-specific TaqMan probe (with junctional region nucleotides underlined), which was used for RQ-PCR analysis (see Pongers – Willemse et al.180 for details). (B) MRD kinetics during the follow-up of the patient measured by TaqMan-based RQ-PCR technology using the V III-Kde gene rearrangement. Based on high MRD levels during the first two time points this patient was assigned to an MRD-based high-risk group. Although the patient achieved an MRD-negative status during treatment, he relapsed after maintenance treatment was stopped. Abbreviations: D, diagnosis; I, induction treatment; C, consolidation treatment; II, reinduction treatment; CR, complete remission; R, relapse. (C) Relapse-free 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 (time point 1) and before consolidation treatment (time point 2).49 Patients in the low-risk group were MRD negative at both time points (43% of patients); patients in the high-risk group had MRD at levels ≥10−3 at both time points (15% of patients); while the remaining patients form the MRD-based intermediate-risk group (43% of patients). The numbers of patients for each group are given in parentheses.

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MRD positivity, even at high levels.192,193 By contrast, MRDnegativity before allogeneic transplantation significantly correlated with a better outcome and 2-year event-free survival rate higher than 70%.100,191–193 MRD-PCR positivity in ALL patients after HSCT is associated with impending relapse.194 For example, MRD was detected in the post-transplant samples of 88% of patients who subsequently relapsed, contrasted with only 22% in long-term complete remission, most of whom had only low levels of MRD at any time after HSCT.194 In conclusion, patients with a high MRD burden prior to HSCT and/or persistent MRD positivity after HSCT are candidates for alternative treatment (e.g. further cytoreduction before transplant, intensified conditioning, and/or early post-transplant immunotherapy).100,191 Other applications of PCR-based detection of MRD in ALL In addition to the classical application of MRD-PCR techniques for evaluation of the kinetics of ALL cell disappearance, PCR studies can be applied for many other purposes. For instance, IG/TCR gene rearrangements can be used as clonality markers for confirmation or exclusion of the common origin of two phenotypically different malignancies.70,93 Another specific application of MRDPCR techniques is the early diagnosis of ALL in cases of “smoldering” leukemia, a condition in which patients with initially hypoplastic bone marrow and less than 25% of lymphoblasts subsequently develop overt ALL. A few case reports have demonstrated the presence of identical clonal cell populations during the hypoplastic phase, the subsequent recovery phase, and the overt leukemia phase using patient-specific IG/TCR gene rearrangements as PCR targets.195,196 By contrast, monoclonal gene rearrangements could not be detected in patients with idiopathic hypoplastic anemia.196 MRD techniques can be also useful for detecting bone marrow involvement during “isolated” extramedullary relapse of ALL (e.g. in CNS or testis). Such submicroscopic bone marrow involvement is confirmed in most ALL patients with isolated extramedullary relapse,197–199 in accord with the clinical observation that full systemic reinduction therapy is required in these patients to prevent hematologic relapse. Nevertheless, some ALL patients with isolated CNS relapse do not have detectable MRD levels in bone marrow.197,200 The MRD positivity of histologically normal testicular biopsies obtained at the end of treatment was shown to be followed by overt testicular relapse.201 Moreover, PCRbased MRD assays at the time of a unilateral testicular relapse allowed reliable exclusion of occult leukemic blasts

in the histologically normal contralateral testis.201 Nevertheless, some patients with MRD-negative testicular biopsies did develop testicular relapse. Another application of MRD detection is the evaluation of autologous stem cell grafts for contamination with leukemic blasts. PCR studies showed that MRD positivity of the autologous bone marrow graft before purging was the most predictive factor of treatment failure in patients with high-risk ALL transplanted in second remission, regardless of a seemingly successful purging procedure (MRDnegative graft).202 In fact, the remission duration after autologous stem cell transplantation significantly correlated with MRD levels before the purging procedure. On the other hand, infusion of MRD-negative purged grafts in patients with MRD-negative pre-transplantation bone marrow was associated with durable clinical remissions.203 Finally, PCR-based MRD studies can be used to assess the disappearance of transplacentally migrating maternal ALL blasts from the blood of a newborn child.204 Transplacental migration of leukemic cells has also been observed in monozygotic twins with monochorionic placenta developing ALL at different ages (reviewed by Greaves205 ). The finding of identical DNA sequences of IG/TCR gene rearrangements and translocation breakpoints proved the prenatal origin of ALL in one of the twins with subsequent transplacental dissemination to the second twin. Using MRD-PCR techniques, it is also possible to detect or exclude the presence of the ALL clone in the unaffected monozygotic twin of an ALL patient.206 Extensive MRD-PCR analyses of Guthrie cards of childhood ALL patients showed the presence of clonotypic ALL sequences at birth in the vast majority of cases, confirming the in utero origin of childhood ALL.174,205

Flow cytometry MRD in childhood ALL and prognosis In studies at St. Jude Children’s Research Hospital, flow cytometry was used to prospectively study MRD in 195 children with newly diagnosed ALL enrolled in a single chemotherapy program (Total Therapy study XIII).109,112,145 Detectable MRD (i.e. ≥0.01% leukemic mononuclear cells) at each time point (day 19 of remission induction therapy, end of remission induction and weeks 14, 32 and 56 of continuation therapy) was significantly associated with a higher rate of relapse (Fig. 28.5). Patients with high levels of MRD at the end of the remission induction therapy (≥1%) or at week 14 of continuation therapy (≥0.1%) had a particularly poor outcome. The incidence of relapse among patients with MRD at the end of induction was 7% ± 7% if MRD became undetectable at week 14 of

Fig. 28.5 Detection of MRD in ALL by flow cytometry. (A) Bone marrow mononuclear cells were collected at diagnosis and at the end of remission induction from two patients with T- linease ALL (left four panels) and two patients with B-lineage ALL (right four panels). In the patients with T-lineage ALL, cells were labeled with antibodies to TdT, CD3, CD5, and a cocktail of anti-HLA-Dr, CD19, and CD33. Flow cytometric dot plots show TdT and CD3 expression in CD5+ cells negative for anti-HLA-Dr, CD19 and CD33. In the patients with B-lineage ALL, cells were labeled with antibodies to CD45, CD10, CD34, and CD19. Dot plots show CD45 and CD10 expression in CD19+ cells. The same number (>105 ) of mononuclear cells were studied in all samples. In the two patients illustrated in the top row, cells with immunophenotype characteristic of ALL (dashed rectangles) were detectable at day 46 of therapy. By contrast, MRD was negative in the two patients illustrated in the bottom row. (B) Cumulative incidence of relapse according to presence or absence of MRD in childhood ALL. MRD was measured by flow cytometry at day 19 of remission induction therapy, at the end of remission induction therapy (day 46), and at weeks 14 and 32 of continuation therapy.109,112,145,206

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continuation therapy, compared with 68% ± 16% (SE) if it persisted. Notably, 53 of the 112 patients studied at day 19 of remission induction therapy had achieved MRD negativity (P < 0.01%) despite the short chemotherapy period.112 The 3-year cumulative incidence of relapse was 1.9% ± 1.9%, as compared with 28.4% ± 6.4% for patients who were MRD+ at this time point. At all time points, the prognostic value of MRD was independent from that of other known clinical and biologic predictors of outcome. Additional findings demonstrating the value of immunologic MRD monitoring in patients with ALL were reported by Farahat et al.108 These authors used antibodies to TdT, CD10, and CD19 to detect MRD in six of nine patients 5 to 15 weeks before relapse. By contrast, 43 patients who remained in continuous complete remission, with a median follow-up of 23 months, were consistently free of MRD by flow cytometry. In a study of 53 ALL patients (37 B- and 16 T-lineage ALL), Ciudad et al.126 used three-color flow cytometry to study MRD. Patients who had a gradual increase in MRD levels showed a higher relapse rate (90% versus 22%) and shorter median relapse-free survival than those with stable or decreasing MRD levels. The adverse predictive value of MRD was also observed when children and adults were analyzed separately. Dworzak et al.111 reported the correlation between MRD detected during treatment and outcome in 108 children enrolled in Berlin¨ Frankfurt-Munster 95 protocol in Austria. Sequential monitoring at day 33 and week 12 of treatment was found to be particularly useful: patients with persistent disease [≥1 blast/ L (percentages of MRD were converted to number of blasts among mononucleated cells in this study)] had a 100% probability of relapse, compared with 6% in all others. It was found, however, that the sensitivity of the test with the markers used was limited when regenerating bone marrow samples were studied. Detection of MRD in peripheral blood A St. Jude study compared MRD measurements in 747 pairs of bone marrow and peripheral blood samples collected from 231 children during treatment for newly diagnosed ALL.113,206 MRD was detected in both marrow and blood in 78 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 602 pairs. Findings in marrow and blood were completely concordant in the 179 paired samples from patients with T-lineage ALL: for each of the 41 positive marrow samples, the corresponding blood sample was positive. In B-lineage ALL, however, only 37 of the 104 positive marrow samples had a corresponding positive blood sample. Results reported by other investigators are in agreement with the remarkable concordance of MRD results in marrow and blood of

patients with T-ALL.208 In the St. Jude study, MRD in the peripheral blood of B-lineage ALL patients was associated with a very high risk of relapse.113 MRD in children with first relapse ALL In a recent study at St. Jude Children’s Research Hospital, flow cytometry was applied to study the significance of MRD after remission reinduction in children with first-relapse ALL.209 At the end of remission reinduction, 41 patients had a bone marrow sample adequate for MRD studies; 35 of these were in morphologic remission. Nineteen of the 35 patients (54%) had MRD at levels of 0.01% or greater, a finding that was associated with subsequent leukemic relapse. The 2-year cumulative incidence of second leukemic relapse was 70.2% ± 12.3% for the 19 MRD-positive patients compared with 27.9% ± 12.4% for the 16 MRD-negative patients (P = 0.008). Among patients with a first relapse off therapy, 2-year second relapse rates were 49.1% ± 17.8% in the 12 MRD-positive and 0% in the 11 MRD-negative patients (P = 0.014); among those who received only chemotherapy after first relapse, the 2-year second relapse rates were 81.5% ± 14.4% (n = 12) and 25.0% ± 13.1% (n = 13), respectively (P = 0.004). Time of first relapse and MRD were the only two significant predictors of outcome in a multivariate analysis. Hence, flow cytometric studies of MRD can be used to guide the selection of postremission therapy in patients with ALL in first relapse.

Clinical studies in acute myeloid leukemia Molecular genetic abnormalities There is general agreement that in patients with acute promyelocytic leukemia (APL), a positive PML-RARa test after consolidation therapy is a strong predictor of subsequent hematologic relapse. By contrast, repeatedly negative results are associated with long-term survival in the majority of the patients. However, these correlations are not absolute, as some patients may remain PCR-positive in long-term remission, while others may relapse after negative tests (reviewed in Grimwade.210 ). The advent of alltrans retinoic acid (ATRA) therapy has led to a dramatic improvement in survival among patients with APL, such that the relapse risk has decreased to less than 20%.210–212 It is still unclear whether more sensitive RT-PCR assays for PML-RARA would be useful in identifying patients at a higher risk of relapse and whether quantitative PCR could enhance the informative value of earlier monitoring.213,214 The benefit of earlier treatment at the stage of molecular

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relapse also remains to be proven, although preliminary findings support this strategy.215 RT-PCR studies for molecular detection of AML1-ETO in t(8;21) AML have yielded controversial results. Using sensitive RT-PCR methods, several groups have reported the persistence of AML1-ETO fusion transcripts in patients in complete remission for as long as 94 months.216–218 Moreover, the persistence of residual disease was also detected in patients who underwent autologous or allogeneic bone marrow transplantation.218,219 In contrast to these findings, other authors reported the absence of AMLI-ETO transcripts in a significant number of long-term remitters.220,221 More recently, several studies (mostly with adult patients) have reported the use of RQ-PCR to monitor AML1/ETO fusion transcripts.222–224 In a limited series of children prospectively enrolled and uniformly treated, the monitoring of MRD by RQRT-PCR analysis was helpful in identifying patients at a higher risk of relapse.225 The experience with molecular monitoring of patients carrying the inv(16) is quite limited. Some AML/inv(16) patients may convert to an RT-PCR-negative status and remain in continuous complete remission.226 Although limited to adult patients, most recent data on quantitative PCR indicate that MRD monitoring for the CBFbeta/MYH11 gene identifies patients at a higher risk of relapse.226–228

Flow cytometry A study from the Children’s Cancer Group found that abnormal profiles by three-color flow cytometry in bone marrow samples obtained during treatment was associated with an increased risk of treatment failure.139 Perhaps because of the limited sensitivity of detection in this study (0.5%), the proportion of patients with responsive disease (defined by the authors as those with <30% blasts after one course of therapy) who were found to have occult disease was only 13%. In a recently reported study performed at St. Jude, aberrant immunophenotypes identified at diagnosis were applied to measure residual disease in 230 bone marrow samples obtained from 46 patients at various times during and after treatment.136 To ensure a definition of residual disease positivity applicable to all 46 patients, a threshold of 0.1% was used to define MRD positivity. Of the 230 bone marrow samples studied, 61 (26.5%) were MRD-positive. In contrast to the CCG study, 34.1% and 27.8% of patients had residual disease after induction 1 and induction 2, respectively. Among the various karyotypic and molecular subtypes examined, patients with core binding factor-AML [i.e. those with t(8;21)/AML1-ETO or inv(16)/CBFB-MYH11] had a particularly good early response to treatment. After

window therapy, residual disease was detectable in only 2 of 11 such patients as compared to 18 of the 30 with other karyotypes (P = 0.033). After induction 1, residual disease was detected in 1 of 11 of the former group and in 16 of 33 of the latter group (P = 0.031).136 In the St Jude study, detection of residual disease was associated with a poorer treatment outcome.136 The mean (±SE) 2-year overall survival estimate among patients with no detectable disease by morphologic methods was 30.0% ± 17.7% for patients who had residual disease (≥0.1% level) in the postinduction 1 sample and 72.1% ± 11.5% for those with fewer or no detectable leukemic cells (P = 0.044). Significant differences were also observed for postinduction 2 samples: 2-year survival estimate of 31.7% ± 18.5% for patients with residual disease (≥0.1% level) compared with 80.0% ± 10.3% for those with fewer or no detectable leukemic cells (P = 0.048). Patients in remission with flow cytometrically detectable cells in postinduction 1 samples were 3.79 times more prone to die than were those with undetectable disease (P = 0.037). Similar observations were made for postinduction 2 samples: patients with detectable AML cells while in remission were 6.15 times more prone to die than were those with undetectable disease (P = 0.028).136 In studies performed in adult patients with AML, the first bone marrow in morphologic remission obtained after induction treatment was found to be very informative.110 Of the 126 patients studied, 8 had fewer than 0.01% leukemic cells, none of whom had relapsed at the time of the report; 37 had from 0.01% to 0.1% leukemic cells and a 3-year cumulative relapse rate of 14%; 64 had from 0.1% to 1% leukemic cells and a relapse rate of 50%; and 17 had more than 1% residual cells and a relapse rate of 84%. In another study of 51 patients in whom MRD was examined after consolidation, the most predictive MRD cut-off value determined retrospectively was 0.035%: 17 of 22 patients with that level of MRD or higher levels relapsed, compared with 5 of 29 patients with lower MRD levels.138 In patients with AML receiving autologous bone marrow transplantation, levels of MRD measured by flow cytometry in the autograft correlated with disease recurrence.229

Concluding remarks As discussed in this chapter, each of the three principal methodologies for studying MRD in children with leukemia has relative advantages and disadvantages. The use of multiple approaches simultaneously can increase the number of patients that can be studied and offset the limitations of individual methods.230 At St. Jude

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Children’s Research Hospital, for example, PCR amplification of antigen-receptor genes and flow cytometric detection of aberrant immunophenotypes are applied simultaneously and independently to study MRD in children with ALL.231 This approach allowed the study of MRD with a sensitivity of 1 leukemic cell in 104 in 196 of the 197 consecutive patients enrolled in the current Total Therapy XV protocol; the single patient with no suitable immunologic markers or antigen receptor-gene rearrangements could be monitored by using RT-PCR amplification of MLL-AF4.231 However, combined approaches of this type are impractical at many institutions. Because of their complexity, methods for MRD detection are routinely performed at only a few specialized laboratories. Multicenter studies incorporating MRD assays require the shipment of specimens. While this is generally not a problem for PCR amplification of DNA sequences, which are stable over time and may be detectable even after cells have undergone apoptosis, it can seriously affect RNA integrity and hence the precision of RT-PCR-based MRD assays.232 Flow cytometric studies may also yield imprecise results in samples that are not studied immediately after collection. Nonetheless, because of its wide availability, flow cytometry is amenable to decentralization, so that methods for rapid exchange of flow cytometric files between centers may be invaluable for ensuring homogeneity among MRD measurements in cooperative trials.114 Treatment intensification for patients with slow clearance of leukemia and persistent or resurgent MRD has a strong theoretical basis, since the size of tumor burden and the curability of cancer are related, and this strategy is well supported by the results of correlative studies. Indeed, one study indicated the effectiveness of early administration of salvage therapy to patients with APL after the reappearance of RT-PCR positivity.215 At St. Jude Children’s Research Hospital, MRD detection has been used as a criterion for treatment intensification in children with newly diagnosed ALL since 1998, and it was incorporated into the recently initiated AML02 protocol for children with AML. Conversely, patients with profound cytoreductions at the early stages of therapy are clear candidates for less intensive (hence less toxic) treatment regimens. MRD-based treatment intervention studies that address the benefit of using MRD for both treatment intensification and reduction in childhood ALL have been initiated within the BFM group. MRD studies are becoming an integral part of the modern management of patients with leukemia. Selection of the methods to be used in each center depends on the expertise and facilities available and on collaborative links that can be established with laboratories proficient in MRD detection.

The main challenge for MRD investigators is to identify new robust markers of leukemia that would allow the simplification and export of current methods while maintaining or increasing their reliability, thus ensuring that the potential benefits of MRD monitoring extend to all patients.

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patients with t(4;11) acute lymphoblastic leukemia. Blood, 2000; 95: 96–101. Cayuela, J. M., Baruchel, A., Orange, C., et al. TEL-AML1 fusion RNA as a new target to detect minimal residual disease in pediatric B-cell precursor acute lymphoblastic leukemia. Blood, 1996; 88: 302–8. Nakao, M., Yokota, S., Horiike, S., et al. Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia. Leukemia, 1996; 10: 1463–70. Satake, N., Kobayashi, H., Tsunematsu, Y., et al. Minimal residual disease with TEL-AML1 fusion transcript in childhood acute lymphoblastic leukaemia with t(12;21). Br J Haematol, 1997; 97: 607–11. Zuna, J., Hrusak, O., Kalinova, M., et al. TEL/AML1 positivity in childhood ALL: average or better prognosis? Czech Paediatric Haematology Working Group. Leukemia, 1999; 13: 22–4. Riehm, H., Reiter, A., Schrappe, M., et al. [Corticosteroiddependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83)]. Klin Padiatr, 1987; 199: 151–60. Cave, H., Werff ten Bosch, J. van der, Suciu, S., et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer – Childhood Leukemia Cooperative Group. N Engl J Med, 1998; 339: 591–8. Jacquy, C., Delepaut, B., Daele, S. van, et al. A prospective study of minimal residual disease in childhood B-lineage acute lymphoblastic leukaemia: MRD level at the end of induction is a strong predictive factor of relapse. Br J Haematol, 1997; 98: 140–6. Brisco, M. J., Condon, J., Hughes, E., et al. Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction. Lancet, 1994; 343: 196–200. Wasserman, R., Galili, N., Ito, Y., et al. Residual disease at the end of induction therapy as a predictor of relapse during therapy in childhood B-lineage acute lymphoblastic leukemia. J Clin Oncol, 1992; 10: 1879–88. Gruhn, B., Hongeng, S., Yi, H., et al. Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome. Leukemia, 1998; 12: 675–81. Goulden, N. J., Knechtli, C. J., Garland, R. J., et al. Minimal residual disease analysis for the prediction of relapse in children with standard-risk acute lymphoblastic leukaemia. Br J Haematol, 1998; 100: 235–44. Nyvold, C., Madsen, H. O., Ryder, L. P., et al. Precise quantification of minimal residual disease at day 29 allows identification of children with acute lymphoblastic leukemia and an excellent outcome. Blood, 2002; 99: 1253–8. Willemse, M. J., Seriu, T., Hettinger, K., et al. Detection of minimal residual disease identifies differences in treatment response between T-ALL and precursor B-ALL. Blood, 2002; 99: 4386–93.

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180 Pongers-Willemse, M. J., Verhagen, O. J., Tibbe, G. J., et al. Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia, 1998; 12: 2006–14. 181 Rivera, G. K., Pinkel, D., Simone, J. V., Hancock, M. L., & Crist, W. M. Treatment of acute lymphoblastic leukemia. 30 years’ experience at St. Jude Children’s Research Hospital. N Engl J Med, 1993; 329: 1289–95. 182 Panzer-Grumayer, E. R., Schneider, M., Panzer, S., Fasching, K., & Gadner, H. Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia. Blood, 2000; 95: 790–4. 183 Dibenedetto, S. P., LoNigro, L., Mayer, S. P., Rovera, G., & Schiliro, G. Detectable molecular residual disease at the beginning of maintenance therapy indicates poor outcome in children with T-cell acute lymphoblastic leukemia. Blood, 1997; 90: 1226–32. 184 Nizet, Y., Daele, S. Van, Lewalle, P., et al. Long-term follow-up of residual disease in acute lymphoblastic leukemia patients in complete remission using clonogeneic IgH probes and the polymerase chain reaction. Blood, 1993; 82: 1618–25. 185 Neale, G. A., Menarguez, J., Kitchingman, G. R., et al. Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse. Blood, 1991; 78: 739–47. 186 Yokota, S., Hansen-Hagge, T. E., Ludwig, W. D., et al. Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients. Blood, 1991; 77: 331–9. 187 Biondi, A., Yokota, S., Hansen-Hagge, T. E., et al. Minimal residual disease in childhood acute lymphoblastic leukemia: analysis of patients in continuous complete remission or with consecutive relapse. Leukemia, 1992; 6: 282–8. 188 Roberts, W. M., Estrov, Z., Ouspenskaia, M. V., et al. Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N Engl J Med, 1997; 336: 317–23. 189 Steenbergen, E. J., Verhagen, O. J., Leeuwen, E. F. van, et al. Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or first relapse predicts poor outcome in childhood B-precursor acute lymphoblastic leukemia. Leukemia, 1995; 9: 1726–34. 190 Eckert, C., Biondi, A., Seeger, K., et al. Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia. Lancet, 2001; 358: 1239–41. 191 Velden, V. van der, Joosten, S. A., Willemse, M. J., et al. Real-time quantitative PCR for detection of minimal residual disease before allogeneic stem cell transplantation predicts outcome in children with acute lymphoblastic leukemia. Leukemia, 2001; 15: 1485–7. 192 Bader, P., Hancock, J., Kreyenberg, H., et al. Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL. Leukemia, 2002; 16: 1668–72. 193 Uzunel, M., Mattsson, J., Jaksch, M., Remberger, M., & Ringden, O. The significance of graft-versus-host disease and pre-

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transplantation minimal residual disease status to outcome after allogeneic stem cell transplantation in patients with acute lymphoblastic leukemia. Blood, 2001; 98: 1982–4. Knechtli, C. J., Goulden, N. J., Hancock, J. P., et al. Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia. Br J Haematol, 1998; 102: 860–71. Ishikawa, K., Seriu, T., Watanabe, A., et al. Detection of neoplastic clone in the hypoplastic and recovery phases preceding acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain gene. J Pediatr Hematol Oncol, 1995; 17: 270–5. Morley, A. A., Brisco, M. J., Rice, M., et al. Leukaemia presenting as marrow hypoplasia: molecular detection of the leukaemic clone at the time of initial presentation. Br J Haematol, 1997; 98: 940–4. Goulden, N., Langlands, K., Steward, C., et al. PCR assessment of bone marrow status in ‘isolated’ extramedullary relapse of childhood B-precursor acute lymphoblastic leukaemia. Br J Haematol, 1994; 87: 282–5. Neale, G. A., Pui, C. H., Mahmoud, H. H., et al. Molecular evidence for minimal residual bone marrow disease in children with ‘isolated’ extra-medullary relapse of T-cell acute lymphoblastic leukemia. Leukemia, 1994; 8: 768–75. O’Reilly, J., Meyer, B., Baker, D., et al. Correlation of bone marrow minimal residual disease and apparent isolated extramedullary relapse in childhood acute lymphoblastic leukaemia. Leukemia, 1995; 9: 624–7. Cave, H., Guidal, C., Rohrlich, P., et al. Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes. Blood, 1994; 83: 1892– 902. Lal, A., Kwan, E., al Mahr, M. et al. Molecular detection of acute lymphoblastic leukaemia in boys with testicular relapse. Mol Pathol, 1998; 51: 277–81. Vervoordeldonk, S. F., Merle, P. A., Behrendt, H., et al. PCRpositivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia. Br J Haematol, 1997; 96: 395–402. Balduzzi, A., Gaipa, G., Bonanomi, S., et al. Purified autologous grafting in childhood acute lymphoblastic leukemia in second remission: evidence for long-term clinical and molecular remissions. Leukemia, 2001; 15: 50–6. Velden, V. van der, Willemse, M. J., Mulder, M. F., et al. Clearance of maternal leukaemic cells in a neonate. Br J Haematol, 2001; 114: 104–6. Greaves, M. Molecular genetics, natural history and the demise of childhood leukaemia. Eur J Cancer, 1999; 35: 1941– 53. Wiemels, J. L., Ford, A. M., Wering, E. R. van, Postma, A., & Greaves, M. Protracted and variable latency of acute lymphoblastic leukemia after TEL-AML1 gene fusion in utero. Blood, 1999; 94: 1057–62.

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207 Campana, D. Determination of minimal residual disease in leukemia patients. Br J Haematol, 2003; 121: 823–38. 208 Velden, V. van der, Jacobs, D. C., Wijkhuijs, A. J., et al. Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL. Leukemia, 2002; 16: 1432–6. 209 Coustan-Smith, E., Gajjar, A., Hijiya, N., et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia after first relapse. Leukemia, 2004; 18: 499–504. 210 Grimwade, D. The pathogenesis of acute promyelocytic leukaemia: evaluation of the role of molecular diagnosis and monitoring in the management of the disease. Br J Haematol, 1999; 106: 591–613. 211 Mandelli, F., Diverio, D., Avvisati, G., et al. Molecular remission in PML/RAR alpha-positive acute promyelocytic leukemia by combined all-trans retinoic acid and idarubicin (AIDA) therapy. Gruppo Italiano-Malattie Ematologiche Maligne dell’Adulto and Associazione Italiana di Ematologia ed Oncologia Pediatrica Cooperative Groups. Blood, 1997; 90: 1014–21. 212 Burnett, A. K., Grimwade, D., Solomon, E., Wheatley, K., & Goldstone, A. H. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the Randomized MRC Trial. Blood, 1999; 93: 4131–43. 213 Slack, J. L., Bi, W., Livak, K. J., et al. Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction. J Mol Diagn, 2001; 3: 141–9. 214 Cassinat, B., Zassadowski, F., Balitrand, N., et al. Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia, 2000; 14: 324–8. 215 Lo Coco, F. Diverio, D., Avvisati, G., et al. Therapy of molecular relapse in acute promyelocytic leukemia. Blood, 1999; 94: 2225–9. 216 Nucifora, G., Larson, R. A., & Rowley, J. D. Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission. Blood, 1993; 82: 712–15. 217 Kusec, R., Laczika, K., Knobl, P., et al. AML1/ETO fusion mRNA can be detected in remission blood samples of all patients with t(8;21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation. Leukemia, 1994; 8: 735–9. 218 Saunders, M. J., Tobal, K., & Yin, J. A. Detection of t(8;21) by reverse transcriptase polymerase chain reaction in patients in remission of acute myeloid leukaemia type M2 after chemotherapy or bone marrow transplantation. Leuk Res, 1994; 18: 891–5. 219 Jurlander, J., Caligiuri, M. A., Ruutu, T., et al. Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood, 1996; 88: 2183–91.

220 Kwong, Y. L., Chan, V., Wong, K. F., & Chan, T. K. Use of the polymerase chain reaction in the detection of AML1/ETO fusion transcript in t(8;21). Cancer, 1995; 75: 821–5. 221 Satake, N., Maseki, N., Kozu, T., et al. Disappearance of AML1MTG8(ETO) fusion transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission. Br J Haematol, 1995; 91: 892–8. 222 Sugimoto, T., Das, H., Imoto, S., et al. Quantitation of minimal residual disease in t(8;21)-positive acute myelogenous leukemia patients using real-time quantitative RT-PCR. Am J Hematol, 2000; 64: 101–6. 223 Marcucci, G., Livak, K. J., Bi, W., et al. Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia, 1998; 12: 1482–9. 224 Barragan, E., Bolufer, P., Moreno, I., et al. Quantitative detection of AML1-ETO rearrangement by real-time RT-PCR using fluorescently labeled probes. Leuk Lymphoma, 2001; 42: 747– 56. 225 Viehmann, S., Teigler-Schlegel, A., Bruch, J., et al. Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement. Leukemia, 2003; 17: 1130–6. 226 Marcucci, G., Caligiuri, M. A., Dohner, H., et al. Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia, 2001; 15: 1072–80. 227 Buonamici, S., Ottaviani, E., Testoni, N., et al. Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state. Blood, 2002; 99: 443– 9. 228 Reijden, B. A. van der, Simons, A., Luiten, E., et al. Minimal residual disease quantification in patients with acute myeloid leukaemia and inv(16)/CBFB-MYH11 gene fusion. Br J Haematol, 2002; 118: 411–18. 229 Reichle, A., Rothe, G., Krause, S., et al. Transplant characteristics: minimal residual disease and impaired megakaryocytic colony growth as sensitive parameters for predicting relapse in acute myeloid leukemia. Leukemia, 1999; 13: 1227–34. 230 Pui, C. H. & Campana, D. New definition of remission in childhood acute lymphoblastic leukemia. Leukemia, 2000; 14: 783– 5. 231 Neale, G. A. M., Coustan-Smith, E., Stow, P., et al. Comparative analysis of polymerase chain reaction and flow cytometry for the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia, 2004; 18: 934–8. 232 Muller, M. C., Merx, K., Weibetaer, A., et al. Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization. Leukemia, 2002; 16: 2395–9.

Part IV Complications and supportive care

29 Acute complications Scott C. Howard, Raul C. Ribeiro, and Ching-Hon Pui

Introduction The most common cause of early treatment failure among patients with childhood leukemia is death due to acute complications of the leukemia itself or its initial treatment.1–3 Despite the increasing intensity of treatment for acute lymphoblastic leukemia (ALL) in children, improvements in supportive care have reduced the rate of death due to acute complications from 10% in the early 1970s4 to less than 2% in the 1990s,1,2 and these improvements have had an important impact on eventfree survival estimates for these patients. In fact, studies of the Medical Research Council (MRC)5,6 found that the rate of treatment-related death among children with ALL decreased from 9% in the 1980s (UKALL VIII trial) to 2% in the 1990s (UKALL X and XI trials).3 Hence, the 6% improvement in the 5-year event-free survival estimate during the same period (from 55% to 61%) can be attributed largely to advances in supportive care. The rate of toxic death associated with therapy for acute myeloid leukemia (AML) and relapsed ALL has also decreased over time but remains unacceptably high at 10% or greater in many studies.7–9 In countries with limited resources, death from toxicity accounts for more cases of treatment failure than does relapse in both AML and ALL.10 Acute complications include “early” complications (those occurring within the first 2 weeks of therapy) and “on-therapy” complications (those occurring after the first 2 weeks of therapy). “Late” complications are those occurring after recovery from the final dose of chemotherapy (Table 29.1). Early complications generally are caused by the leukemia itself, while on-therapy and late complications reflect the toxicity of leukemia therapy. This chapter

discusses early and on-therapy complications. The most common early complications include metabolic disturbances, central airway compression, coagulopathy, hyperviscosity/leukostasis, and neurologic dysfunction; whereas on-therapy complications include thrombosis, endocrine disorders, gastrointestinal disorders, osteonecrosis, and neurologic dysfunction. The incidence of early complications depends on patient factors (age, nutritional status, concurrent organ dysfunction, and genetic influences), disease factors (leukemic cell burden, tumor mass, type of leukemia, rate of leukemic cell division and cell death), and treatment factors (rapidity of response to therapy and the drugs used); in contrast to the incidence of ontherapy complications, which is influenced primarily by the treatment regimen employed and host pharmacogenetics (see Chapter 14). Here we review the pathophysiology, clinical manifestations, diagnosis, and management of these problems, because their early recognition and prompt treatment can reduce early morbidity and mortality rates. Chapter 30 discusses late complications such as growth retardation and osteopenia, which may begin during treatment but manifest later. Infectious complications are discussed in Chapter 32 and hematologic supportive care in Chapter 33.

Risk factors Patient factors Nutritional status Nutritional status at the time of leukemia diagnosis can affect the risk of complications and the ultimate outcome.11–16 Malnutrition is rarely a problem for patients

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 29.1 Incidence and risk factors for complications of childhood leukemia by phase of therapy Incidencea Phase of therapy Early

Period included

Complication

From before initiation of therapy until 2 weeks after

Tumor lysis syndrome Uncommon Common

Diabetes insipidus Hypercalcemia Coagulopathy Superior vena cava syndrome Central airway compression syndrome Leukostasis

On-therapy

Late

AML

ALL

Risk factors Large tumor burden, renal insufficiency, initial treatment with highly cytolytic chemotherapy

Rare Rare Common

Rare CNS infiltration of leukemia Rare Dehydration, T-cell leukemia Uncommon Acute promyelocytic leukemia, acute monoblastic leukemia Rare Uncommon Mediastinal mass, rapid rate of leukemia cell growth, inherited prothrombotic conditions, central venous catheter Uncommon Uncommon Mediastinal mass, rapid rate of leukemic cell growth

Uncommon Rare

Elevated white blood cell count, myeloid leukemia, monoblastic leukemia (FAB M4, M5), microgranular variant of APL, age <1 year, dehydration, red blood cell transfusion in the setting of hyperleukocytosis Longer than 2 weeks after Adrenal insufficiency Rare Common Depends on the dosage, schedule, and choice of initiation of therapy glucocorticoid. Rarely clinically important Hyperglycemia Rare Common Treatment with glucocorticoids and L-asparaginase, use of intravenous fluids that contain dextrose, Down syndrome age >10 years, obesity, family history of diabetes Hypoglycemia Rare Rare Mercaptopurine therapy and prolonged fasting in young children SIADH Rare Rare Vincristine therapy Seizure Rare Uncommon High-dose methotrexate therapy, high-dose cytarabine therapy, CNS thrombosis Leukoencephalopathy Rare Uncommon High-dose methotrexate therapy, CNS irradiation Thrombosis Rare Uncommon Inherited prothrombotic conditions, treatment with glucocorticoids and L-asparaginase, central venous catheter use, immobilization Allergic reaction Rare Common L-asparaginase therapy Mucositis Common Common High-dose methotrexate therapy, high-dose cytarabine, anthracycline therapy Pancreatitis Rare Rare L-asparaginase therapy Hepatitis Rare Common High-dose methotrexate therapy Osteonecrosis Rare Common High-dose glucocorticoid, especially dexamethasone treatment After recovery from the final dose of chemotherapy (see Chapter 30)

a Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CNS, central nervous system; SIADH, syndrome of inappropriate antidiuretic hormone.

Very common, >25% of patients are affected; common, 10% to 25% of patients are affected; uncommon, 2% to 9% of patients are affected; rare: <2% of patients are affected. Hemorrhage is discussed in Chapter 33 and osteopenia in Chapter 30.

b

Acute complications

from industrialized countries but was an important contributor to early death resulting from toxicity in children from countries with limited resources,17 such that modification of induction therapy was necessary.15,16 Obesity can also increase the risk of complications.18 Because obese patients have greater caloric needs, nausea and vomiting can cause a relatively greater protein-calorie deficiency. Furthermore, because chemotherapy doses are calculated on the basis of body surface area, morbidly obese children may receive excessively high (or low) effective doses relative to those given to smaller children. There is no consensus as to the appropriate methods for adjusting chemotherapy doses that are calculated on the basis of body surface area for adults, much less for children, especially those with malnutrition, obesity, an altered metabolic rate, or weight gain due to edema.19–26

Patient age Age is also an important consideration. Although leukemia is rare among children younger than 1 year (about 32 cases per million live births per year),27 careful attention must be paid to fluid and electrolyte balance in children, as well as to their nutritional support. Because venous access can become problematic when treating small children, placement of an indwelling central venous catheter should be considered. The pharmacologic and pharmacodynamic aspects of chemotherapy given to infants are not as well documented as in older children and adults, and may be affected by differences in drug absorption, distribution, metabolism, and excretion.28 Because the body water compartment changes rapidly during the first year of life (from 75% of total body weight in the newborn period to 60% at 1 year), the same chemotherapy dose may achieve different plasma levels of drug as the infant grows.28,29 Furthermore, neonates and infants differ from older children and adults in the avidity of serum protein binding of drugs, the activity of P-450 enzymes, renal tubular function, and glomerular filtration.28,30 Another major age difference is the ratio of body weight (kg) to body surface area (m2 ), which is 18 in neonates and 40 in adults.31 Hepatic metabolism of certain drugs also varies with age.32,33 The net effect of these differences is an enhanced exposure to certain antileukemic drugs (e.g. vincristine34 ) when they are dosed by body surface area; hence, the dose of each agent in clinical trials of therapy for infant leukemia must be adjusted accordingly. Adolescents also present distinct problems. For example, they understand the life-threatening nature of their disease and the risks of treatment – neuropsychological toxicity, infertility, and second malignancies – and thus are at greater risk of anxiety, depression, substance abuse, and

nonadherence to treatment than are younger children (see Chapter 35). Concurrent organ dysfunction Children with renal, hepatic, and infectious complications at the time of diagnosis of leukemia are less able to tolerate intensive treatment regimens, resulting in an increased risk of morbidity. Patients with renal insufficiency or decreased urine output are less able to excrete the potassium, phosphorus, and uric acid that are released by lysis of leukemic cells and are more likely to experience overt renal failure.35 Renal insufficiency can lead to excessive toxicities from many renally excreted drugs. In this regard, the plasma concentration of methotrexate remains high for an extended time after intrathecal therapy in children with renal dysfunction, and these patients require prolonged leukovorin rescue to prevent excessive toxicity.36 Children with hepatic insufficiency or cholestasis metabolize chemotherapy erratically; increasing the potential for overdosing or underdosing. Those being treated for infection have an increased risk of adverse drug interactions (e.g. fluconazole and vincristine or all-trans-retinoic acid).37–39 Genetic conditions Certain genetic conditions that predispose patients to leukemia development (e.g. Down syndrome, ataxiatelangiectasia, and Fanconi anemia) also predispose them to complications. Children with Down syndrome have a greater risk of toxicity related to methotrexate or other antileukemic drugs40,41 ; while those with ataxiatelangiectasia or Fanconi anemia are hypersensitive to the effects of radiation and chemotherapy.42,43 In addition to these conditions, common enzyme deficiencies or polymorphisms also affect the rate of complications. Glucose6-phosphate dehydrogenase deficiency, which affects 11% of African-American children in the United States and as many as 30% of boys of other non-Caucasian races, predisposes affected children to hemolysis when they are treated with urate oxidase or other oxidizing drugs.44–49 If possible, male patients from high-risk ethnic groups should be routinely tested before oxidant agents are administered to them. Similarly, children with homozygous thiopurine IS/Imethyltransferase deficiency are at high risk of complications resulting from mercaptopurine treatment.50–52

Leukemia factors Tumor burden and tumor masses At the time of diagnosis, some patients have a large tumor burden and an increased risk of metabolic complications,

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including tumor lysis syndrome. An elevated white blood cell (WBC) count is associated with an increased risk of leukostasis, an important risk factor for early death, especially in patients with AML. Leukemic infiltration of bone marrow, thymus, spleen, and liver is frequently present at the time of diagnosis. Less frequently, the kidneys, testicles, and ovaries may be involved as well. In the past, the degree of organ infiltration has been correlated with mortality risk.53 Furthermore, large leukemic masses in the anterior mediastinum, seen in 60% of children with T-lineage ALL54 and occasionally in patients with AML,55,56 may compress the tracheobronchial tree or the superior vena cava sufficiently to cause symptoms. Extramedullary leukemic tumors (chloromas) of the orbit, of the paraspinal region, or within the cranium can cause symptomatic compression of adjacent structures in occasional patients with AML and rarely in those with ALL, and may require urgent therapy to prevent neurologic injury.57–59 In this regard, these tumors are chemosensitive in newly diagnosed patients and generally respond promptly to chemotherapy.60 Leukemia subtype and biology The incidence and severity of very early complications depend primarily on the type of leukemia. The pathobiology of the ALL or AML case determines the rate of tumor cell division and spontaneous apoptosis, which in turn affect the degree of hyperuricemia, hyperphosphatemia, and the probability of renal dysfunction. Coagulopathy is common at the time of diagnosis among children with acute promyelocytic leukemia (APL) but occurs in only 3% of children with ALL, most of whom have T-cell ALL.61 Symptomatic respiratory and central nervous system (CNS) dysfunction can occur, particularly among children with myelomonoblastic leukemia, in which the leukemic cells express more surface adhesion molecules and have a greater propensity for leukostasis.62,63 Acute myelomonocytic leukemia with eosinophilia can cause pneumopathy and vascular leak syndrome when tumor cells lyse or degranulate in response to chemotherapy.62 Although treatment with antihistamines and leukotriene inhibitors, which inhibit some of the contents of eosinophilic granules, has a biologic rationale, it has not been proven to reduce symptoms or prevent complications.

Treatment factors Besides leukemia pathobiology, the choice of initial therapy affects the rate of leukemic cell lysis and, therefore, the probability of tumor lysis syndrome. The goal of initial therapy is to reduce the leukemic cell burden gradually, allowing the kidneys sufficient opportunity to excrete potas-

sium, uric acid, and phosphorus and maintain homeostasis of calcium and phosphorus. The Berlin-Frankfurt¨ Munster (BFM) group pioneered the use of single-agent glucocorticoid therapy for 7 days before other agents are added, partly to assess early response as a prognostic factor and partly to reduce the probability of tumor lysis syndrome.64,65

Early complications Metabolic abnormalities Rapid spontaneous or chemotherapy-induced lysis of large numbers of malignant cells can cause several biochemical abnormalities and dysfunction of multiple organs. These acute complications, collectively termed tumor lysis syndrome, occur when the rapid destruction of tumor cells releases cellular breakdown products that exceed the patient’s hepatic and renal catabolic and excretory capabilities. Thus, patients with a large tumor burden, as reflected by hyperleukocytosis, high serum lactate dehydrogenase levels, or massive organomegaly, are at especially high risk of tumor lysis syndrome. A high sensitivity of the leukemic cells to chemotherapy is also a risk factor for tumor lysis syndrome.66–68 After remission has been achieved and the risk of tumor lysis syndrome has passed, chemotherapy can cause other metabolic complications, including hyponatremia and hyperglycemia. Tumor lysis syndrome The biochemical abnormalities associated with tumor lysis syndrome include hyperuricemia, azotemia, hyperkalemia, hyperphosphatemia, and hypocalcemia (Fig. 29.1). Hyperkalemia and hypocalcemia can cause cardiac arrhythmia or cardiac arrest; hyperuricemia and hyperphosphatemia can cause renal insufficiency, which in turn leads to oliguria, fluid overload, pulmonary edema, respiratory failure, hypoxia, cerebral edema, and death. In addition, hyperphosphatemia exacerbates hypocalcemia and can cause ectopic calcification at many sites, including the kidneys. Any child with newly diagnosed acute leukemia should be considered at risk of acute tumor lysis syndrome, especially those with an elevated WBC count, a high serum LDH level, a mediastinal mass or obstructive uropathy, or a rate of cytolysis predicted to be high on the basis of tumor type: Burkitt lymphoma, T-cell leukemia, or monoblastic leukemia. Serum electrolytes and biochemical markers of kidney and liver function should be carefully monitored. Patients in a dehydrated state, with hyperuricemia, hyperphosphatemia, renal failure, and elevated

Acute complications

Cell Lysis with Release of Intracellular Contents

Vasoactive Substances

Lactate Phosphate Potassium

Uric Acid Vasoconstriction

Decreased Tissue Perfusion Hyperkalemia

Capillary Leak Syndrome

Metabolic Acidosis

Hypotension

Intrarenal Precipitation

Ca Phosphate Precipitation

Kidney Dysfunction Decreased Preload

Pulmonary Edema

Hypocalcemia

Oliguria Anuria Fluid Overload

Heart Failure

Respiratory Failure

Cardiac Arrhythmia

Cardiac Arrest

Fig. 29.1 Pathophysiology of tumor lysis syndrome. Apoptosis and necrosis of leukemic cells releases intracellular contents, including vasoactive substances, purines that are metabolized to uric acid, phosphate, potassium, and lactate. All organs can be affected by these substances, but effects on the kidneys, lungs, and heart produce the most frequent and life-threatening clinical consequences.

serum LDH levels, are at the highest risk of life-threatening complications. Fever, infection, and hypoxia can further complicate the initial course. Patients at high risk of tumor lysis syndrome are best treated by a multidisciplinary team that includes a hematologist-oncologist, an intensivist, a nephrologist, a nurse, and a pharmacist. Failure to maintain strict control of the clinical course can have disastrous consequences because life-threatening complications may develop in a matter of hours. Uric acid metabolism An increased serum uric acid level is a common finding among patients with tumor lysis syndrome, with or without renal insufficiency.35,69 Uric acid, the end product of purine metabolism in humans (Fig. 29.2), is completely filtered into the glomerular space, 99% reabsorbed in the proximal tubule, and then actively secreted in the distal tubule. Daily excretion averages 500 mg. Uric acid is nearly com-

pletely ionized at physiologic pH and is found in blood as water-soluble urate. Because uric acid has a pKa of 5.35, the compound is nonionized and poorly soluble at the urinary pH of 5.0 found at the level of the distal tubules and collecting ducts. In these distal nephron segments, the uric acid concentration increases progressively as the filtrate reaches the collecting ducts, and the concentration is even higher in the presence of hyperuricemia. Uric acid is formed when xanthine oxidase metabolizes xanthine and hypoxanthine, which are also excreted by the kidney. Xanthine, hypoxanthine, and uric acid have different solubilities in urine, and crystallization can occur when the concentration of each compound exceeds its solubility, which are pH dependent (Fig. 29.3). Uric acid is much more soluble in alkaline conditions, xanthine and hypoxanthine are slightly more soluble at higher pH, and calcium phosphate is more soluble in acidic conditions; thus, calcium phosphate may crystalize in alkaline urine.70–74

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Fig. 29.3 Solubilities of uric acid, uric acid metabolites (xanthine, hypoxanthine), allantoin and calcium phosphate over a range of physiologic pH values. Alkalinization of urine dramatically increases the solubility of uric acid, but decreases that of calcium phosphate, so bicarbonate should be used with caution when serum (and therefore urine) phosphate is elevated. Solubilities depend on many factors in addition to pH, and published values differ. The interested reader should refer to source studies in the chemistry and thermodynamics literature for further details.70–76

Fig. 29.2 Pathway of purine catabolism and sites of action of agents that reduce the uric acid level (allopurinol and urate oxidase). Allopurinol prevents uric acid formation, but does not remove existing uric acid. It also causes accumulation of hypoxanthine and xanthine which, like uric acid, can crystallize in the kidneys. Urate oxidase breaks down uric acid to form water-soluble allantoin, which is 5 to 10 times more soluble than uric acid.

Hyperuricemia Patients with acute leukemia commonly excrete more uric acid than normal, even when serum urate levels are normal; there is no good correlation between serum uric acid levels and the amount of uric acid excreted.75 Histopathologic data from human autopsies and experimental animals demonstrate uric acid deposits within the luminal space of the distal tubules and collecting ducts (Fig. 29.4). Deposition of uric acid crystals leads to a peritubular granulomatous reaction and necrosis of the tubular epithelium. In some cases, uric acid stones are formed and create ureteral or pelvic obstruction, but this condition is less common among patients with acute leukemia than among patients with diseases that produce chronic hyperuricemia.76 Several factors modulate crystallization of uric acid within the luminal space: the concentration of the filtrate, the pH of the distal tubular fluid (Fig. 29.3), the tubular flow rates,

and hemoconcentration in medullary blood vessels. Dehydration and acidosis are associated with increased uric acid crystallization. Tumor lysis syndrome can produce nausea, vomiting, lethargy, agitation, somnolence, lumbar pain, hypertension, cloudy urine, and joint pain.

Xanthine nephropathy Xanthine nephropathy, elevated plasma xanthine levels, and increased urinary xanthine levels have been observed in patients with Burkitt lymphoma and leukemia who were receiving allopurinol for the prevention of uric acid nephropathy.77,78 Allopurinol inhibits the activity of xanthine oxidase and thus blocks the conversion of oxypurines (hypoxanthine and xanthine) to uric acid (Fig. 29.2). This condition leads to the accumulation of xanthine and hypoxanthine, which are excreted in the urine. Because xanthine is very insoluble (Fig. 29.3), its accumulation may lead to crystallization, and stone formation can occur.79 Furthermore, during massive tumor lysis, some uric acid is excreted in urine even in the presence of allopurinol because the massive release of purines during this process overwhelms the ability of allopurinol to completely block uric acid synthesis.80 Thus, patients with massive tumor lysis syndrome who are receiving this agent are at risk of uric acid and xanthine nephropathy despite adequate urine alkalinization. Because allopurinol and its metabolites are excreted only by the kidneys, this compound should be given at a reduced dosage in cases of renal insufficiency.

Acute complications

Hyperkalemia Hyperkalemia is an ominous sign during the initial treatment of leukemia and should be considered an emergency, because it can cause cardiac arrhythmia and sudden death. Serum potassium levels usually begin to rise within a few hours after the initation of chemotherapy. Hyperkalemia occurs before hyperphosphatemia, probably because the function of the energy-dependent sodium/potassium ATPase system is reduced before complete leukemia cytolysis occurs. Because potassium is an intracellular ion, massive destruction of tumor cells can release enough potassium to overwhelm excretory mechanisms. Cardiac complications due to increased serum potassium levels can be aggravated by acidosis and hypocalcemia. Acidosis also shifts intracellular potassium to the extracellular compartment and interferes with reuptake and reutilization of potassium by normal cells. Spurious elevations in serum potassium concentrations can result from cytolysis in vitro after sampling as the result of an elevated leukemic cell count.81 Calcium and phosphorus homeostasis Hyperphosphatemia with or without hypocalcemia occurs frequently during the first few days of induction chemotherapy. Within 12 to 24 hours after initiation of treatment, the blood load of phosphate is at its highest because of the cytolysis of leukemic cells, which are rich in organic and inorganic phosphorus compounds, and because of the shift of intracellular phosphate into the extracellular space in response to metabolic acidosis.82,83 Calcium and phosphorus homeostasis depends on the action of parathyroid hormone (PTH), which promotes phosphorus reabsorption by the renal tubules, and synthesis of 1,25-dihydroxycholecalciferol (vitamin D3 ). Elevation of serum phosphate levels inhibits PTH secretion, thereby decreasing renal phosphate reabsorption. In one study of childhood ALL, tubular reabsorption of phosphate decreased to 20% to 70% of the baseline, while phosphate clearance increased to 3 to 24 times the baseline during chemotherapy.84 Hyperphosphatemia and hypocalcemia Hyperphosphatemia has replaced hyperuricemia as the most common abnormality associated with renal failure among patients undergoing contemporary remission induction therapy.82–86 Hypocalcemia, which usually follows hyperphosphatemia, results from precipitation of calcium phosphate in tissue.84,85 In cases of severe hyperphosphatemia, despite a compensatory decrease in blood calcium levels, the calcium × phosphorus product can exceed 80 (mg/dL)2 , a level associated with cal-

Fig. 29.4 Linear streaks of precipitated uric acid (arrows) in the renal medulla of a 4-year-old boy who died of massive tumor lysis syndrome. (See color plate 29.4 for full-color reproduction.)

Fig. 29.5 Soft tissue calcification of the dorsum of the distal forearm. This 15-year-old boy with acute lymphoblastic leukemia and an initial white blood cell count of 283,000/mm3 developed tumor lysis syndrome with hyperphosphatemia and hypocalcemia. Multiple doses of intravenous calcium carbonate were administered via a peripheral intravenous catheter in the dorsum of the right hand to treat hypocalcemia. Several weeks later firm subcutaneous nodules were noted in the hand, wrist, and distal forearm. They were radiographically confirmed to be ectopic calcification, despite the absence of clinically evident extravasation at any time and a calcium × phosphate product that never exceeded 60 (mg/dL)2 .

cium phosphate precipitation in soft tissues (Fig. 29.5). In addition, administration of bicarbonate to alkalinize the urine increases the urinary precipitation of calcium phosphate, which is less soluble at an alkaline pH. Other factors contributing to hypocalcemia are decreased

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production of 1,25-dihydroxycholecalciferol by the kidneys and decreased levels of PTH with reduced calcium mobilization from the bones.88,89

Renal dysfunction Acute renal failure among children with acute leukemia and hyperuricemia has been attributed to intraluminal precipitation of uric acid, phosphates, xanthine, or some combination of these compounds and to the luminal obstruction caused by this precipitation.35,66,90,91 When hyperphosphatemia occurs alone or in combination with hyperuricemia, the most likely explanation for renal failure is renal intraluminal precipitation of calcium phosphate salts and uric acid. These deposits cause a renal interstitial inflammatory response, which in turn results in tubular atrophy and a spectrum of nephropathies ranging from tubular dysfunction to renal failure. However, the rapid improvement in renal function with proper hydration argues against glomerular or epithelial tubule damage. Vascular constriction or obstruction may also contribute to decreased filtration and renal insufficiency. Although patients with leukemia and hyperuricemia have an abnormal glomerular filtration rate, there is no absolute correlation between serum uric acid concentrations and the degree of renal insufficiency.

Management of tumor lysis syndrome Hyperhydration Specific measures for the prevention and management of tumor lysis syndrome are outlined in Table 29.2. Initial supportive care includes expansion of the intravascular volume, because many patients are dehydrated at the time of diagnosis because of low oral fluid intake, fever, or vomiting. Hyperhydration with a slightly hypotonic solution dilutes intravascular solutes such as urates and phosphates, increases renal blood flow and glomerular filtration, and flushes precipitated solutes from the renal tubules. Adverse effects of hyperhydration include hyponatremia, interstitial pulmonary edema, ascites, and cerebral edema. Fluid overload can cause respiratory distress, seizures, coma, and apnea. Therefore, fluid input and output should be monitored closely. A Foley catheter may be necessary in some cases of tumor lysis syndrome. Central venous monitoring should also be considered. Maintaining adequate urine output often requires the administration of mannitol, furosemide, or both, but diuretics should not be used until the patient has adequate intravascular volume (normovolemic or hypervolemic). Urine output should be kept above 100 mL/m2 per hour in patients receiving 3000

mL/m2 per day of fluid, allowing a maximum daily fluid retention of no more than 600 mL/m2 . Urine alkalinization and allopurinol Urine alkalinization facilitates excretion of urate and hypoxanthine but not of xanthine. Usually, the urine pH is kept above 6.5. Although this measure is important if uric acid nephropathy is to be avoided, overzealous urine alkalinization can lead to precipitation of calcium phosphate within the renal tubules and other tissues. When serum bicarbonate levels reach 30 mEq/L, bicarbonate should be discontinued; if necessary, acetazolamide can be given to increase urine pH. In addition to forced fluid intake and urine alkalinization, patients with hyperuricemia usually receive allopurinol. Allopurinol inhibits the activity of xanthine oxidase, the enzyme responsible for the conversion of hypoxanthine to xanthine and of xanthine to uric acid (Fig. 29.2). Allopurinol is metabolized to oxypurinol (alloxanthine), which also inhibits the activity of xanthine oxidase. Oxypurinol reaches its plasma concentration peak after 4.5 hours, and its plasma half-life is 15 hours. At St. Jude Children’s Research Hospital, we have largely abandoned the practice of urine alkalinization because it improves uric acid excretion only marginally, does not promote xanthine excretion, and significantly impairs phosphate excretion. Furthermore, hyperuricemia can be treated very effectively with recombinant urate oxidase (see below). Urate oxidase In the presence of massive tumor lysis, allopurinol may be insufficient to prevent hyperuricemia. Recombinant urate oxidase (rasburicase; Fasfurtec, Elitek) converts uric acid to allantoin (which is much more soluble than uric acid, Fig. 29.3) and offers an alternative treatment. This product is produced by a genetically modified Saccharomyces cerevisiae yeast in which the cDNA was cloned from a strain of Aspergillus flavus.92 It lowers plasma uric acid more rapidly and effectively than allopurinol.93 Most patients treated with rasburicase have had improved or stabilized renal function during rasburicase treatment, even in the face of chemotherapy-induced tumor lysis. Except for occasional instances of hemolytic anemia and methemoglobinemia in patients with glucose-6-phosphate-dehydrogenase deficiency (due to production of hydrogen peroxide as a byproduct of the reaction in which rasburicase converts uric acid into allantoin), rasburicase has been welltolerated, with a low frequency of adverse events.49,93–96 Hyperkalemia Preventing hyperkalemia in children with newly diagnosed leukemia is as important as adequate treatment.

Acute complications

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Table 29.2 Prevention and management of tumor lysis syndrome Goal

Intervention

Caution

Preservation of renal function

If the patient is hypotensive or severely dehydrated, begin fluid therapy with boluses of 20 mL/kg of normal saline over a period of 30 to 60 min until the patient is normovolemic

Aggressive hydration that accompanies treatment of hyperuricemia and hyperphosphatemia maximizes the chance of renal preservation during tumor lysis syndrome, presumably by flushing crystals from the renal tubules Patients may require dextrose-free fluids and 0.5 N saline if hyperglycemia or hyponatremia develop. Potassium should be avoided

Prevention and treatment of hyperuricemia

Prevention and treatment of hyperkalemia

Prevention and treatment of hyperphosphatemia and hypocalcemia

Intravenous fluid therapy with 5% dextrose in 0.25 N saline at a dosage of 3 to 5 L/m2 per day (2 to 4 times the rate of daily maintenance intravenous fluids) If hydration is adequate but urine output is <2 mL/kg per hour, add diuretics: mannitol at 0.5 g/kg for children younger than 9 years (15 g/m2 for those older) every 6 hours; or furosemide at 1 to 2 mg/kg every 6 hours to achieve the desired urine output Consider dopamine at a dose of 1 to 3 g/kg per min if poor renal perfusion is suspected Urine alkalinization with 20 to 40 mEq NaHCO3 per liter of intravenous fluid with intravenous boluses of 1.0 g/m2 NaHCO3 every 6 hours as needed to maintain urine pH between 6.5 and 7.0. Consider acetazolamide 150 mg/m2 every 6 to 8 hours (maximum, 1 g/day) if serum bicarbonate level is ≥30 mEq/L but the urine is not alkaline Allopurinol 100 to 500 mg/m2 per day (maximum, 800 mg/day) in three divided doses Recombinant urate oxidase (Rasburicase, 0.1 to 0.2 mg/kg) IV given over 30 min every 12 to 24 hours until the uric acid level normalizes

Hold intake of potassium in fluids and food. Kayexalate 1 to 2 g/kg per day given orally every 6 hours or as a retention enema in 20% sorbitol. If electrocardiographic changes are present, treat immediately with bicarbonate 0.5 mEq/kg by intravenous push, 10% calcium gluconate 0.5 mL/kg IV over 10 min, and 10% glucose 0.5 g/kg IV with 0.3 units of regular insulin per gram of glucose. Furosemide should be added only if the patient is well hydrated Aluminum hydroxide (50–150 mg/kg per day) or calcium carbonate (for patients with low serum calcium concentrations) in divided doses orally every 6 hours. Patients with symptomatic hypocalcemia or hyperkalemia should receive 10% calcium gluconate at a dosage of 0.5 mL/kg IV over 10 min

Do not treat with diuretics until the patient is adequately hydrated

Avoid nephrotoxins (hyperosmolar contrast agents, nonsteroidal anti-inflammatory drugs, aminoglycosides) Avoid overalkalinization of urine, which leads to intrarenal calcium phosphate precipitation. Decrease or discontinue bicarbonate if the serum level is ≥30 mEq/L, if urine pH is >7.3, if serum uric acid level normalizes, if serum phosphate level begins to rise, or if the patient is receiving urate oxidase The dose of allopurinol should be reduced in the presence of renal insufficiency Because of the risk of allergic reactions, epinephrine, hydrocortisone, and diphenhydramine should be readily available before administration of the first dose of urate oxidase in patients with a history of significant atopy or asthma. Known glucose-6-phosphate dehydrogenase deficiency is a contraindication to the use of urate oxidase, and clinicians should be alert to the possibility of hemolysis and methemoglobinemia. Do NOT treat hypokalemia unless it is causing symptoms (cramps, congestive heart failure, arrhythmia), because tumor lysis will increase the serum potassium level. If potassium supplementation is required to treat life-threatening hypokalemia, it should be administered judiciously. Arrange for urgent hemodialysis if hyperkalemia with electrocardiographic changes does not respond to these measures or if life-threatening hyperkalemia recurs after a transient response Avoid overalkalinization of urine, which leads to decreased phosphorus excretion and intrarenal calcium-phosphate precipitation. Patients treated with urate oxidase do not require urine alkalinization

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Patients at risk for tumor lysis syndrome should not receive potassium-containing intravenous fluids and their dietary potassium intake should be restricted. Symptomatic hyperkalemia or a serum potassium level near 6 mEq/L are indications for the use of potassium exchange resins such as Kayexalate, at a dosage of 0.25 to 0.5 g/kg every 6 hours (orally or as a retention enema in 20% sorbitol solution). Continuous cardiac monitoring and frequent serum potassium measurements are both indicated. If associated electrocardiographic abnormalities occur or if the serum potassium level exceeds 6.5 mEq/L, patients should immediately receive cardioprotective agents: NaHCO3 at a dose of 0.5 mEq/kg over 10 to 15 minutes, followed by a 10% calcium gluconate solution at a dose of 0.5 mL/kg over of 5 to 10 minutes. These agents should be administered separately to avoid precipitation. In addition, 0.5 g/kg of intravenous 10% glucose with 0.3 units of regular insulin per gram of glucose may be given over 1 hour to rapidly lower serum potassium. These measures shift potassium into the intracellular compartment, but are temporary in effect and should be used until body potassium can be reduced by other means, such as Kayexalate, forced diuresis with hyperhydration and furosemide, or dialysis. Hyperphosphatemia and hypocalcemia Hyperphosphatemia can be controlled by administering aluminum hydroxide or calcium carbonate orally or through a nasogastric tube. The choice between these agents depends on the serum calcium level. In the presence of hyperphosphatemia and a relatively high serum calcium level, administration of calcium may increase the precipitation of calcium phosphates in tissues if total calcium × phosphorus product is >80 (mg/dL)2 , and this increase may lead to nephrocalcinosis and extraskeletal calcification. In such cases, aluminum hydroxide, despite its unpleasant taste, is the preferred treatment. Calciumfree and aluminum-free phosphate binders, such as sevelamer hydrochloride,97,98 are now available and, in our limited experience, are quite effective. Asymptomatic patients with hypocalcemia are treated with oral calcium carbonate, but symptomatic patients should be treated with intravenous 10% calcium gluconate at a dose of 0.5 mL/kg administered over 5 to 10 minutes. If intravenously administered calcium solutions extravasate, or if the local concentration of calcium × phosphate exceeds 80 (mg/dL)2 , soft-tissue calcification can occur (Fig. 29.5). Renal failure Occasionally, even patients with well-managed leukemia have acute renal failure, manifested by oliguria, fluid overload, hyperkalemia, and other metabolic abnormalities.

Peritoneal dialysis, intermittent hemodialysis, and continuous arteriovenous or veno-venous hemofiltration have been used to treat these patients.99,100 Data comparing the different dialysis techniques are not available, but intermittent hemodialysis or continuous hemofiltration is preferred over peritoneal dialysis because the latter can exacerbate pre-existing diaphragm “splinting” and compromise respiration. The most common indication for dialysis is severe oliguria or anuria, with hypertension or fluid overload,66 and most patients have hyperphosphatemia as well. The duration of oliguria before the start of dialysis has been correlated with the length of oliguria after dialysis,101 a finding suggesting that dialysis is best initiated at the onset of significant oliguria or anuria. The timing of dialysis with respect to chemotherapy has not been clearly established. Stapleton et al.35 suggested that chemotherapy can be initiated soon after a dialysis treatment has been completed. Electrolytes are then monitored every 2 hours, and dialysis is repeated if necessary, usually 2 to 12 hours after the beginning of chemotherapy. Rarely, respiratory failure can complicate the course of patients with tumor lysis syndrome and renal failure (Fig. 29.1).102 Hypercalcemia Although common in adults with cancer,103 hypercalcemia (serum calcium concentration ≥11.5 mg/dL or 2.9 mmol/L) is rare in children with leukemia.104 Only 11 (0.6%) of 2816 patients with acute leukemia treated at St. Jude Children’s Research Hospital had hypercalcemia at the time of diagnosis: five with B-cell precursor ALL, three with mature B-cell ALL, one with T-cell ALL, one with unclassified ALL, and one with AML.105 Clinical manifestations of hypercalcemia include fatigue, anorexia, nausea, polyuria (nocturia), polydipsia, vomiting, abdominal and back pain, and constipation. Polyuria, vomiting, and anorexia lead to dehydration, which in turn leads to renal failure and acidosis. Severe hypercalcemia can cause neurologic abnormalities, including seizures, stupor, and coma, and even sudden death from cardiac arrhythmia.106–108 Because hypercalcemia resolves during the first few days of chemotherapy, chronic manifestations such as soft tissue and vascular calcifications, renal stones, and keratopathy are rarely seen in children with leukemia. Hypercalcemia is treated by aggressive hydration followed by forced diuresis. In the absence of severe organ dysfunction, children with mild asymptomatic hypercalcemia can be treated with fluid replacement (0.9% NaCl at an hourly rate of 20 mL/kg, if a 5% total body water loss is assumed). Thereafter, fluid replacement follows the recommendations listed in Table 29.2. If symptomatic hypercalcemia persists even after hydration and urine output

Acute complications

Fig. 29.6 (A) The anatomy of superior vena cava (SVC) syndrome. Complete or partial occlusion of the SVC decreases blood flow and predisposes to thrombus formation and intraluminal obstruction. Acute obstruction causes venous engorgement of the head and neck (see Fig. 29.7). (B) Central airway compression syndrome due to a mediastinal mass. The cross-sectional area of the trachea is 80% less than normal, a degree of compression associated with high risk of complications. Successful treatment requires removal of external compression by using chemotherapy, and treatment of the thrombus with heparin and in severe cases, catheter-directed thrombolytics. Without anticoagulant therapy, the thrombus can propagate from the SVC into the subclavian and jugular veins, as seen in the inset.

are adequate, furosemide, at a dose of 2 mg/kg, should be administered intravenously every 2 to 4 hours to promote urinary calcium excretion. The combination of 0.9% saline and furosemide usually decreases calcium concentrations by 2 to 3 mg/dL within the first 24 hours,109 a time period that should be sufficient for the initiation of effective antileukemic treatment. Calcitonin, bisphosphates, gallium nitrate, and plicamycin have been occasionally used in children with malignancy-associated hypercalcemia but are rarely, if ever, needed for children with leukemia since hypercalcemia in these patients is rapidly corrected after the initiation of remission induction chemotherapy that includes a glucocorticoid.

Compression of mediastinal structures Enlargement of hilar lymph nodes and the thymus is common among patients with T-cell ALL (50% to 60% of patients)54,110 but rare among those with AML.56 A chest

radiograph is mandatory for any patient with newly diagnosed leukemia or lymphoma. Children who have a large mediastinal mass at the time of diagnosis constitute a challenge that should be managed in the hospital by a multidisciplinary team consisting of a pediatric oncologist, an intensivist, a radiation oncologist, an anesthesiologist, a respiratory therapist, and a pathologist. Notable exceptions are patients with Hodgkin lymphoma, in whom even large mediastinal masses often cause no symptoms.

Central airway compression syndrome Approximately 50% of children with a mediastinal mass have compression of the trachea, a main bronchus, or both, but only 10% have severe symptomatic airway compression (central airway compression syndrome). Total airway occlusion can occur during the induction of general anesthesia, tracheal intubation or extubation, movement to a supine or flexed position (for lumbar puncture), and conscious sedation (Fig. 29.6).111,112 Therefore, severe central

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airway compression should be considered a medical emergency. A particularly ominous sign is the patient’s inability to lie supine because of exacerbation of dyspnea.113 Symptoms of airway compression include nonproductive cough, dyspnea on exertion, orthopnea, fatigue, and chest pain or discomfort. Signs of airway compression include cyanosis, stridor, decreased breath sounds, and wheezing. In addition to the anterior mediastinal mass and prominent hilar lymph nodes, chest radiographs usually show a posteriorly deviated trachea, atelectasis, and pleural effusion. All patients with suspected leukemia should have a chest radiograph as soon as the diagnosis is suspected. If a widened mediastinum is seen, a computed tomography (CT) scan of the chest can identify the degree of tracheal compression. Reduction of 50% or more in the tracheal cross-sectional area is associated with increased risk of complications with anesthesia.114 –116 Maximal inspiratory and expiratory flow-volume curves obtained with the patient in supine and sitting positions have been used to indicate the degree of impairment and can help to distinguish fixed from variable major intrathoracic airway lesions.112,115–118 For patients with central airway compression, the diagnosis of leukemia should be established by using the least invasive technique, because anesthesia carries a risk of fatal complications.112,119,120 Superior vena cava syndrome Among patients with leukemia, the superior vena cava is particularly susceptible to compression because it is surrounded by lymphoid tissue, lies against relatively rigid anatomic structures, and has a delicate vessel wall and low intraluminal pressure (Fig. 29.6). However, symptomatic compression and obstruction of the superior vena cava resulting in severely reduced venous return from the head, neck, and upper extremities (superior vena cava syndrome; SVCS) occurs in fewer than 5% of children with mediastinal involvement.121 The severity of clinical manifestations of SVCS depends on how rapidly the obstruction arises and whether there has been sufficient time for new collateral vessels to develop. Collateral veins drain to the inferior vena cava via the azygous vein when the obstruction is superiorly located, or via the hemiazygous or chest wall veins when the obstruction is below the azygous confluence. Children with severe SVCS experience dizziness, headaches, changes in facial color, and facial swelling. Physical examination is remarkable for facial and periorbital edema, cyanosis, plethora, neck and chest vein distention, papilledema, edema of the upper extremities, and increased pulsus paradoxus (Fig. 29.7). Development of collateral veins relieves the signs of SVCS. Chest radiographs reveal pleural and pericardial effusions in addi-

Fig. 29.7 Superior vena cava syndrome with venous engorgement of the neck and arms and development of collateral blood vessels in the trunk of a 10-year-old boy with T-cell ALL and a mediastinal mass. (See color plate 29.7 for full-color reproduction.)

tion to the superior mediastinal mass. CT imaging demonstrates the degree of displacement of normal anatomy, the extent of effusions, and the magnitude of central airway compression, but if the patient has dyspnea when supine, this procedure should be performed with the patient unanesthetized and in the prone position so that safety can be maximized. Doppler ultrasonography and echocardiography may be indicated for evaluating flow through the great vessels and cardiac function. Treatment of SVCS includes anticoagulation with unfractionated or low-molecular-weight heparin to prevent propagation of

Acute complications

the thrombus, catheter-directed thrombolysis to relieve obstruction if symptoms are severe,122 and, most important, relief of external SVC compression by prompt, effective treatment of the leukemia. Superior vena cava syndrome per se does not represent a medical emergency in children with a mediastinal mass; however, most pediatric patients with SVCS have concomitant central airway compression, which is responsible for rare, but tragic, complications. Furthermore, extension or embolism of the thrombus can compromise cardiac or pulmonary function, sometimes with fatal results.

Coagulopathies Incidence and risk factors Bleeding and thrombosis are important causes of morbidity and mortality during the first weeks of therapy among children with leukemia.61,123–129 Most patients are thrombocytopenic at the time of diagnosis, and many present with petechiae, bruising, or active bleeding. Laboratory evidence of coagulopathy without clinical manifestations is also common. Most coagulation defects can be successfully managed with blood product support, and they can be ultimately corrected by effective treatment of the leukemia. In a retrospective study of 1000 consecutive children with newly diagnosed acute leukemia, 25 of 805 with ALL (3.1%) and 27 of 195 with AML (14%) had laboratory evidence of coagulopathy before the start of treatment,61 and only 2.0% of patients with ALL and 9.7% of those with AML experienced clinically important bleeding. Two episodes of fatal hemorrhage occurred, both among patients with AML. Hence, extensive laboratory testing for coagulopathy is not warranted for patients with ALL, but all patients with promyelocytic leukemia should undergo coagulation screening at the time of diagnosis and as clinically indicated thereafter. Laboratory evaluation Laboratory evidence of coagulopathy varies depending on the subtype of leukemia. The most common abnormalities noted among children with ALL are thrombocytopenia and elevation of plasma fibrinogen and other clotting factors, including Factors V, VIII, IX, and XI.61,130,131 Among patients with AML, thrombocytopenia is accompanied by decreased plasma fibrinogen concentrations; prolonged prothrombin (PT), activated partial thromboplastin (aPTT), or thrombin time; and elevated levels of plasma fibrinogen or fibrin degradation products.61,125,130,131 Among patients with chronic myeloid leukemia, thrombocytosis and primary platelet dysfunction are frequent.132 Sensitive and specific methods have been developed for

the diagnosis of hypercoagulable states. The fibrinogen survival test has been used as an indirect marker of activation of the clotting system. Similarly, measurement of proteins of the fibrinolytic system (plasminogen, plasmin/antiplasmin complexes, plasminogen activators and inhibitors) and of natural clotting factor inhibitors (antithrombin III, protein C, protein S) can provide evidence of a hypercoagulable state. Other assays have been used to detect fibrinogen or fibrin degradation products (D-dimers, fibrinopeptide A), peptides released by procoagulants during activation (prothrombin fragment F1+2 , protein C activation peptide, Factor X activation peptide), and proteinase/inhibitor complexes (thrombin/antithrombin III complex) in patients with cancer. However, to date, these studies have not contributed substantially to the overall quality of coagulopathy management in pediatric leukemia. Coagulopathy in acute promyelocytic leukemia The leukemia subtype most commonly associated with potentially life-threatening coagulopathy, acute promyelocytic leukemia (APL),133–135 provides a paradigm for the mechanisms of hemorrhagic diathesis in leukemia. Most patients with APL have an increased tendency for bleeding and have laboratory evidence of coagulopathy, including thrombocytopenia, hypofibrinogenemia, and prolonged PT, aPTT, and thrombin times (see Chapter 19). 133–136 Because APL cells contain procoagulants that have been shown to activate Factor VII and Factor X, the release of procoagulants by senescent or chemotherapy-affected cells has been suggested as a mechanism of disseminated intravascular coagulation (DIC) in these patients; however, the fact that heparin does not effectively block procoagulant activity in APL suggests that other mechanisms account for the coagulopathy inherent in this disease.137 Primary or secondary fibrinolysis has also been suggested as a component of the bleeding diathesis that accompanies APL.138,139 APL cells have plasminogen activators in quantities sufficient to generate plasmin,140 and reduced plasminogen activator inhibitor activity has been demonstrated in such cells.141 These abnormalities cause rapid fibrinogen degradation that contributes to the hemorrhagic diathesis. Over the past decade, exciting progress in the treatment of coagulopathy among patients with APL has come from observations that all-trans retinoic acid (ATRA) and arsenic trioxide induce rapid improvement in clinical and biochemical coagulation abnormalities.142–146 In fact, in one study of APL treated with ATRA, no deaths were attributed to hemorrhage, and the signs of coagulopathy resolved after a mean of 4.2 days.147 Studies evaluating

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changes in coagulation abnormalities before and after the administration of ATRA have suggested that ATRA downregulates the production of prothrombotic products by leukemic cells.148 Hence, prompt treatment with ATRA and aggressive use of blood products are sufficient for most patients with APL.143–145,149,150 Heparin or antifibrinolytic agents should be reserved for patients who do not respond to these measures. With the advent of ATRA treatment, hemorrhagic complications have been reduced drastically.133,135,144,151–155 However, patients receiving this agent can experience retinoic acid syndrome, which has replaced hemorrhage as an important cause of morbidity and mortality.144,156,157 Because a similar condition can occur with use of arsenic trioxide,142 the syndrome is more accurately described as a “promyelocyte differentiation syndrome.” This syndrome and its treatment are described in Chapter 19. Coagulopathy in other childhood leukemias In types of AML other than APL, the pathogenesis of coagulopathy is unclear. The condition usually occurs in the context of hyperleukocytosis, infection, hypoxia, and hypotension. Thus, the use of heparin or antifibrinolytic agents in these AML subtypes is even more controversial and rarely, if ever, indicated. For these patients, reduction of the leukemic cell burden; transfusion of platelet concentrates, cryoprecipitate, and fresh frozen plasma; and vigorous treatment of concurrent conditions such as infection, hypoxia, and hypotension should precede any consideration of heparin or antifibrinolytic therapy. The patients may also benefit from a gradual reduction of the leukemic cell burden by treatment with a single effective agent, such as 2chlorodeoxyadenosine, cytarabine, or hydroxyurea, for the first few days. Thrombotic coagulopathy When blood vessel endothelium remains intact, blood fluidity is maintained through a fine balance between activation and inactivation of several proteinases.158–160 When this balance is disrupted and compensatory mechanisms are overwhelmed, localized intravascular coagulation produces thrombosis, and disseminated intravascular coagulation leads to coagulopathy and hemorrhage.158 The coagulation cascade can be activated in several ways among patients with newly diagnosed leukemia. First, intact leukemic cells can express a transmembrane protein that forms a complex with clotting Factor VII, which then produces a tissue factor–like activity that initiates the coagulation cascade.161–164 Second, leukemic cells can release a cysteine protease (cancer procoagulant) that directly activates Factor X even in the absence of Factor VII.134,150 ,165,166

Although tissue factor activity and cancer procoagulants are considered the two most important factors contributing to hypercoagulability in leukemia, other processes may play an important role as well. For example, endothelial cell integrity can be directly disrupted by leukemic cells, and this disruption can lead to platelet aggregation (Fig. 29.8). Inflammatory cytokines such as interleukin1, tumor necrosis factor, and vascular permeability factor indirectly activate the coagulation system by increasing the expression of tissue factor by endothelial cells and monocytes.167–169 Annexins, which have anticoagulant and phospholipase-A2 inhibitory activity, may also play a role in the pathogenesis of coagulopathy, and the increased release of plasminogen activators by either endothelial or leukemic cells can enhance fibrinolysis.170 Decreased synthesis of natural plasminogen activator inhibitors can further aggravate the hyperfibrinolytic state.141 In patients with newly diagnosed leukemia, studies do not support the hypothesis that deficiencies of antithrombin III, plasminogen, protein C, or protein S are important in the development of thrombosis;126,171,172 instead, they demonstrate a qualitative abnormality of von Willebrand Factor among patients with this complication.126,173,174 Increased endogenous thrombin generation may also contribute to thrombotic complications,174 though mucins from adenocarcinomas have been shown to cause thrombinindependent platelet aggregation by direct interaction with P-selectin on platelets and L-selectin on leukocytes.176 In addition to the thrombotic risk at diagnosis, thrombosis also commonly complicates the treatment course for patients with acute leukemia, especially children with ALL treated with glucocorticoids and L-asparaginase.124,177–181

Leukostasis syndrome Incidence and risk factors Patients with leukemia and hyperleukocytosis can develop leukostasis syndrome: progressive neurologic or respiratory symptoms or signs caused by small blood vessel infiltration and occlusion by leukemic blast cells (Table 29.1; Fig. 29.8).182–187 Death occurs in as many as 20% of patients with severe leukostasis as a result of intracranial bleeding or pulmonary insufficiency.185,188–191 Postmortem examination reveals widespread aggregates of leukemic blast cells and thrombi occluding small veins in the lungs, brain, and other organs. The pathophysiology of leukostasis is complex, and several factors contribute (Fig. 29.8).185 Although hyperleukocytosis is an important risk factor, an elevated leukocyte count alone is a poor predictor of leukostasis. In myeloid leukemias particularly, histopathologic evidence of leukostasis has been found in patients with a broad

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Fig. 29.8 Pathophysiology of hyperviscosity and leukostasis. Leukostasis sets off a chain of events that leads ultimately to pulmonary and cerebral damage.

range of presenting leukocyte counts.185,192–194 In our study of acute leukemia in children with leukocyte counts that exceed 100 × 109 /L, symptomatic pulmonary leukostasis occurred in 6 of 73 patients with myeloid leukemia and in none of 161 patients with lymphoid leukemia.195,196 Postmortem examination of lung specimens from three of the six affected patients revealed extensive leukemic infiltration of alveoli and pulmonary parenchyma. Moreover, in this study, intracerebral hemorrhage developed in 19% of patients with myeloid leukemia and in 2.5% of those with lymphoid leukemia. Conversely, metabolic complications associated with tumor lysis syndrome occurred more frequently among patients with lymphoid rather than myeloid leukemias.195

Symptoms and evaluation Leukostasis can affect any organ but most commonly damages the lungs and CNS (Fig. 29.8). Symptoms and signs include shortness of breath, tachypnea, dyspnea on exertion, hypoxia, confusion, somnolence, delirium, and coma (Table 29.3).185 Interpretation of laboratory data from patients with hyperleukocytosis must take into account the fact that leukemic cells are metabolically active in vitro, and thus results can be spurious. Discrepant values for arterial oxygen tension have been noted in patients with hyperleukocytosis.197 Moreover, normal oxygen saturation measurements by cutaneous oximetry have been observed in patients with abnormal arterial oxygen saturation, a finding suggesting oxygen consumption by leukocytes. 197

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Table 29.3 Symptoms and signs of leukostasis Organ system

Clinical manifestation

Bleeding Ocular

Oral, nasal, gastrointestinal, rectal, and uterine Papilledema, retinal hemorrhage and exudates, retinal detachment, and retinal vein thrombosis Headache, vertigo, tinnitus, hearing loss, ataxia, diplopia, paresthesias, mental status changes, syncope, seizures, chorea, somnolence, and coma Congestive heart failure, angina, and arrhythmias Tachypnea, dyspnea, pulmonary infiltrates, hypoxia, and respiratory failure

Neurologic

Cardiovascular Pulmonary

Importantly, the “oxygen steal” that occurs in vitro continues despite immediate placement of samples in ice. Spurious findings of high serum potassium concentration can also be observed in patients with hyperleukocytosis if excessive cytolysis occurs in vitro. The differential diagnosis of pulmonary leukostasis includes pneumonia, pneumonitis, pulmonary edema, pulmonary hemorrhage, and pulmonary embolism, all of which occur with greater frequency among children with newly diagnosed leukemia who have elevated WBC counts, sometimes with fatal results.196 A ventilation-perfusion lung scan can sometimes aid in diagnosis.184,198 Management The management of hyperleukocytosis in patients with ALL is controversial.189,194,195,199–203 In some centers, leukapheresis or exchange transfusion (for small children) is used to reduce the leukemic cell burden in patients with leukocyte counts exceeding 200 × 109 /L. Although these procedures effectively reduce the leukemic cell burden, their short- and long-term benefits have not been documented in controlled trials. Cranial irradiation for patients with CNS symptoms and renal irradiation for those with renal insufficiency have not proved effective and are no longer used. Coagulopathy may be present in patients with hyperleukocytosis, especially those with T-cell ALL; however, it generally resolves rapidly with chemotherapy and does not require specific treatment. Management of patients with AML and hyperleukocytosis represents an extraordinary challenge. Although tumor lysis syndrome and metabolic abnormalities are less common with AML than with ALL, early death due to leukostasis occurs much more often among patients with AML, possibly because of the increased rate of concomitant clinical problems such as

DIC, respiratory failure, and infection.189,195,204–206 We use leukapheresis more frequently to treat patients with AML than to treat patients with other types of leukemia because leukostasis is relatively frequent in patients with AML and there is no linear correlation between the leukocyte count and the risk of clinical events (stroke, pneumopathy).194 However, the efficacy of leukapheresis in this setting has not been proved. Leukostasis syndrome in monoblastic leukemia Patients with monoblastic leukemia (M5 AML) exhibit unique clinical features of the disease.64,189,207–214 They tend to be younger than 2 years and to have hyperleukocytosis, extramedullary organ involvement, coagulopathy, renal tubular dysfunction, leukostasis, and a very high incidence of early death.189 These clinical characteristics are explained in part by the unique properties of the leukemic monoblasts. Compared with other myeloid blast cells, monoblasts have increased adhesiveness, motility, invasiveness, and tissue life,210,211,215 features that facilitate tissue penetration and injury. In addition, the monoblasts are metabolically active, producing several cytokines and lysozymes213,216 that can worsen the adverse interactions between leukemic cells and organs such as lungs, brain, and kidneys.217 Patients with monoblastic leukemia commonly have very high fever and hypergammaglobulinemia, findings suggestive of increased cytokine production, as well as hypokalemia, presumably because of lysozymeinduced renal tubular dysfunction. Among 294 patients enrolled in the BFM group’s AML 78 and AML 83 studies, 30 died of hemorrhage or leukostasis (or both) either before therapy (11 patients) or during the first 2 weeks of therapy (19 patients). Although only 17% of the cohort had monoblastic leukemia, 91% of the deaths before treatment and 42% of the deaths during the first 2 weeks of induction therapy occurred in this group.189 In our AML 91 study, which accrued 90 patients, there was only one early death: this patient, who had M5 AML, died before chemotherapy could be given.196 It is possible that the low mortality rate of our patients with M5 AML and hyperleukocytosis is in part due to initial therapy with a 5-day course of 2-chlorodeoxyadenosine,218 which is especially effective against monoblastic leukemia.196 We believe that monoblastic leukemia with hyperleukocytosis should be treated as a medical emergency. In addition to supportive care that includes leukapheresis, correction of coagulopathy, and management of metabolic disturbances, the goal of chemotherapy should be a slow reduction of the initial leukemic cell burden. Patients with acute myelomonocytic leukemias and inv(16) also have an increased incidence complications at diagnosis, including pulmonary

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leukostasis and bronchiolitis obliterans with organizing pneumonia (BOOP).219 No specific etiology for these pulmonary complications was identified by the investigators.

Hyperviscosity syndrome Symptoms and pathophysiology Symptoms of hyperviscosity include visual disturbance, shortness of breath, bleeding, headache, vertigo, tinnitus, somnolence, delirium, and coma (Fig. 29.8).220–222 Because many patients with true hyperviscosity syndromes (e.g. polycythemia vera, Waldenstr¨om macroglobulinemia) have neurologic and respiratory symptoms similar to those found in patients with hyperleukocytic leukemias, changes in blood viscosity have been regarded as a component of leukostasis. However, with some exceptions,223–226 whole-blood viscosity is not elevated in hyperleukocytic leukemias, a finding suggesting that other events play a role in the pathophysiology of leukostasis. In this regard, leukostasis is a function of the number, deformability, size, surface markers, and tissue invasiveness of the leukemic cells and the volume fraction (leukocrit) that these cells represent.185,189,227 Lymphoblasts have mean cell volumes of 250 to 300 m3 , and myeloblasts have mean cell volumes of 350 to 450 m3 ; thus, when leukocyte counts are similar, a higher leukocrit is expected with myeloid leukemia than with lymphoid leukemia. Moreover, myeloblasts are less pliable and more tissue-invasive than lymphoblasts. Adaptive mechanisms To some extent, adaptive mechanisms mitigate the effect of hyperleukocytosis on blood viscosity. For example, in chronic leukemias there is a strong inverse correlation between the erythrocrit and the leukocrit. As the leukocrit rises, the erythrocrit falls, keeping blood viscosity within normal limits.226,227 In addition, local vascular regulatory mechanisms develop to preserve cerebral blood flow in response to increased blood viscosity. Brown and Marshall228 found that cerebral blood flow was greater than predicted in six of seven patients with hyperleukocytic leukemia and whole-blood hyperviscosity. Their findings suggest that compensatory mechanisms such as vasodilation might have mitigated the potential deleterious effects of hyperviscosity. Clinical management From both rheological and clinical perspectives, it is possible that the development of leukostasis syndrome depends mainly on events at the level of the microcirculation (Fig. 29.8).192,226 According to this hypothesis, leukemic blast cells reduce blood flow through small vessels. As the leuko-

cyte count increases, decreased blood flow and increased competition between red and white cells for access to the microvessels further reduce oxygen transport to tissues. Moreover, leukemic cells invade tissues and consume oxygen, thus resulting in focal occlusion of blood vessels by leukocyte aggregates and predisposing the patient to tissue ischemia. Furthermore, decreased blood flow dramatically increases viscosity and may cause symptomatic hyperviscosity in local capillary beds with consequent focal deficits that in some cases improve after appropriate therapy.220–222,229–232 Prophylactic transfusion of red blood cells should be avoided, because any elevation in hematocrit is accompanied by an increase in blood viscosity, and adequate hydration should be maintained. Although optimal management of microcirculatory hyperviscosity has not been defined, systemic hyperviscosity should be treated on an emergent basis with leukapheresis.222,231,233

Neurologic complications Clinically important involvement of the central or peripheral nervous system at the time of diagnosis of leukemia is rare but can cause devastating consequences in the absence of prompt emergency treatment. The signs and symptoms of these rare complications – weakness, back pain, and changes in mental status – may be incorrectly interpreted, and this false interpretation may lead to delayed diagnosis and irreversible damage to specific CNS functions. If such delays are to be prevented, a thorough neurologic examination must be included in the initial evaluation of patients with acute leukemia. Incidence of CNS leukemia Leptomeningeal leukemia, intracerebral myeloblastoma, epidural cord compression, or cranial or spinal nerve involvement are rare in acute leukemia, occurring in 3% to 5% of patients with ALL234 or the M1, M2, M6, or M7 subtype of AML,235,236 and in an even smaller fraction of M3 AML patients.237 However, they are found in about 30% of patients with M4 or M5 AML, especially in those with inv(16).238 Rarely, myeloid cell masses occur in the CNS in the absence of detectable bone marrow disease. In ALL, there is a correlation between leukemic cell immunophenotype and CNS leukemia. Patients with mature B-cell (Burkitt) leukemia (Chapter 18) or T-cell leukemia (Chapter 16) have the greatest risk of CNS involvement. Pathophysiology of CNS leukemia Although the pathogenesis of CNS leukemia is not completely understood, leukemic cells can gain access to the CNS in a variety of ways. In an animal model, leukemic

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cells extended from the skull marrow into the subarachnoid space via the bridging veins.239 Postmortem studies of children with ALL have suggested that leukemic cells enter the CNS by invading vein walls in the arachnoid membrane. The choroid plexus can also be the site of invasion in certain cases, thus facilitating the spread of leukemic cells into the cerebrospinal fluid. Some have speculated that certain molecules in the leukemic cell membrane, such as CD2, CD54, or CD22, may adhere to receptors on the endothelial cells of brain capillaries, thus enabling the cells to enter the CNS.240 Direct infiltration of the leptomeninges via bony lesions of the skull can occur as well.240,241 In B-cell leukemia, malignant cells can grow along nerve roots and invade the subarachnoid space through the neural foramina. Epidural spinal cord compression usually results from direct extension of disease from vertebral bodies. In cases of leukemic cell tumors (granulocytic sarcomas), aggregated tumor cells enter the extradural space through the intervertebral foramina. Finally, leukemic cells may be introduced iatrogenically at the time of the diagnostic lumbar puncture, especially when the puncture is traumatic.242 Treatment of CNS leukemia Most patients with CNS leukemic involvement do not have clinical manifestations and can be successfully treated by intensive intrathecal and systemic chemotherapy (see Chapters 16 and 19). Because of the numerous side effects associated with radiation therapy, most leukemia therapists limit the use of this modality. In fact, we have eliminated radiation therapy even in patients with CNS leukemia, with encouraging preliminary results.

Spinal cord compression Incidence and symptoms Epidural spinal cord compression occurs in approximately 0.4% of patients with newly diagnosed acute leukemia.60,243,244 The spinal cord can be compromised at any site, but in children the thoracic vertebral segment is most often affected. The pathophysiology of neurologic spinal cord dysfunction is not completely understood, but symptoms probably result from disruption of the vascular system, leading to vasogenic cord edema. Vertebral collapse resulting from leukemic invasion of the vertebral bodies can also compromise the spinal cord in rare cases. Most patients complain of progressive pain at the level of the epidural lesion, radicular pain, or both, and these complaints can be misinterpreted as pleurisy if thoracic vertebrae are involved. Motor abnormalities, including weakness or paralysis of the upper and lower extremities, are the second most common initial manifestation. Deep ten-

don reflexes are hyperactive, and the plantar (Babinsky) response is extensor. Sensory changes (including numbness, tingling, and paresthesia) occur in approximately 50% of patients. Autonomic dysfunction usually occurs later in the course of spinal cord compression, but when the cauda equina is the site of compression, neurogenic bladder dysfunction can be the presenting sign. Diagnosis and treatment Spinal cord compression is a medical emergency and should be evaluated promptly. Plain films and CT scans are not sensitive enough to identify spinal cord compression; myelography and MR imaging, on the other hand, are equally effective in making the diagnosis. Magnetic resonance imaging, which is usually the procedure of choice, allows visual evaluation of the spinal cord parenchyma and reveals the presence and extent of paraspinal involvement. In patients with newly diagnosed leukemia, intensive systemic chemotherapy plus high-dose corticosteroids should be used. Intravenous dexamethasone, the corticosteroid of choice, is effective in reducing spinal cord edema and the lymphoid tumor mass. Local radiotherapy is generally not necessary for these patients because newly diagnosed leukemia is chemosensitive. Laminectomy is rarely necessary and is reserved for diagnostic clarification when patients have extramedullary myeloid cell tumors without bone marrow involvement.

Intracerebral myeloblastoma Extramedullary myeloid cell tumors (granulocytic sarcoma, myeloblastoma, and chloroma) occur in 2% to 30% of patients with AML, depending on the leukemic cells’ genotype. Patients whose myeloid leukemia cells harbor the t(8;21) or rearrangements of chromosome 16 are at increased risk of extramedullary myeloid cell tumors. Approximately 15% of these tumors are intracranial (orbit, meninges, or brain; Fig. 29.9).57,238,245 These lesions may arise within the brain or the cerebellar parenchyma, or they may be associated with the dura mater, in which case the lesions can be indistinguishable from meningioma. Typically, the lesions are located adjacent to the ventricles or on the cisternal or sulcal meningeal surfaces. On CT scans, the lesions typically appear as isodense or hyperdense regions that are enhanced brightly with contrast.246 On T1 - and T2 -weighted MR images, myeloid tumors appear as isodense or hyperintense regions that are enhanced markedly with gadopentetate dimeglumine.247–249 Patients who have myeloblastoma without bone marrow involvement present a diagnostic quandary. The differential diagnosis includes non-Hodgkin lymphoma and metastatic

Acute complications

On-therapy complications Thrombosis In 1865, Trousseau noted an increased incidence of coagulopathy among patients with cancer.253 Virchow described a triad of abnormalities that contribute to a hypercoagulable state: abnormal blood flow (turbulence or stasis), abnormal blood constituents (coagulation factors or platelets), and vessel wall abnormalities (endothelial dysfunction or damage).254 Pediatric patients with leukemia may be affected by any or all parts of this prothrombotic triad, and possibly by direct stimulation of platelet aggregation via P-selectin on the platelet surface.176

Fig. 29.9 Intraorbital chloroma. On the soft-tissue window of this computed tomography scan, a mass is seen (arrow). It was the first sign of relapse in a 2-year-old child with T-cell acute lymphoblastic leukemia receiving maintenance chemotherapy.

neoplasms. Interpretation of the tissue biopsy samples can also be difficult, as reflected by the high percentage of incorrect pathologic diagnoses that are based solely on hematoxylin and eosin staining.250,251 Hence, a high degree of clinical suspicion and extensive immunohistochemical testing are indicated in cases of “atypical” primary histiocytic intracerebral lymphoma. The treatment of intracerebral myeloid tumors is controversial. While many patients are treated with combination chemotherapy plus whole-brain irradiation,252 cranial irradiation may not improve survival rates. Additionally, intensive systemic cytarabine-containing regimens with intrathecally administered methotrexate are effective in producing resolution of the intracerebral myeloid deposits. We recommend that such patients be evaluated in conjunction with a radiation oncologist. If the lesion does not shrink with chemotherapy or if the mass impinges on a critical area, radiotherapy should be started promptly.

Incidence and risk factors in childhood ALL Symptomatic thrombotic complications can develop among children with all types of leukemia,180,255–258 but they occur most frequently during ALL remission induction with prednisone, vincristine, and Lasparaginase.126–129,173,259 The BFM group found that genetic predisposition played an important role in the development of thrombosis among patients with childhood ALL.177,178 Of 288 patients treated on the ALL-BFM 90/95 study and screened for prothrombotic mutations, clinically evident central venous sinus thrombosis developed in 17 (5.9%); this condition was much more common among children with an inherited prothrombotic mutation than among those without such mutations (P < 0.0001).177 However, this finding has yet to be confirmed by other investigators. Malnick and coworkers260 identified inherited prothrombotic conditions in patients with AML, but the contribution of these conditions to the risk of thrombosis remains unknown, and even among children with ALL the association between prothrombotic conditions and clinically detectable thrombosis has not been observed in all studies.124 By contrast, the presence of an indwelling central venous catheter124,261–263 and concurrent treatment with L-asparaginase and glucocorticoids are undisputed risk factors.124,174,179–181,264,265 Clinical features in childhood ALL Sites of thrombosis include the CNS, the subclavian veins or SVC in proximity to an indwelling central venous catheter, and the lower extremities.124,177,178,266 Children with cerebral venous sinus thrombosis can exhibit symptoms such as severe headache, irritability, altered mental status, seizure, or some combination of these symptoms. The onset of these symptoms is an indication for MR or CT imaging of the brain. Magnetic resonance imaging is preferable because it can reveal abnormalities that are sometimes missed by CT scanning. Occasionally, the

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Fig. 29.10 Saggital vein thrombosis. On a sagittal T1-weighted MR image without contrast, a hyperintense signal (arrow) identified venous thrombosis in the superior sagittal sinus of a 13-year-old adolescent with B-precursor-cell ALL.

onset may be subacute, with a gradual increase in symptoms over a period of days or weeks. Cerebral thrombosis must be distinguished from transient ischemia associated with vincristine neuropathy and acute hypertension. Transient ischemic lesions are located in the watershed areas between the major cerebral arteries and are generally reversible.267 Sagittal sinus thrombosis, on the other hand, has characteristic findings and can be diagnosed readily by MR or CT imaging (Fig. 29.10). Thrombosis in the internal jugular veins, subclavian veins, or SVC produces swelling, pain, and sometimes inflammation of the affected extremity (Fig. 29.7).

Treatment The treatment of thrombosis, whether associated with ALL or AML, is controversial and depends on the site(s) affected. We treat patients who have symptomatic thrombosis with unfractionated or low-molecular-weight heparin to prevent propagation of the thrombus, with platelet transfusion to maintain the platelet count above 50 × 109 /L during

heparin treatment, and with catheter-directed thrombolysis in selected cases.122 In one study, the administration of fresh frozen plasma had no demonstrable benefit on the plasma levels of coagulation factors.268 If the antithrombin III level is low, we administer recombinant antithrombin III, and if deficiency of other anticoagulant proteins is suspected, we administer fresh frozen plasma. However, the benefit of this treatment approach has yet to be proved. Reinduction treatment with prednisone, vincristine, and L-asparaginase for relapsed leukemia in patients who had prior thrombotic complications has been associated with a high rate of recurrent thrombosis.266 For these patients, we recommend that prednisone and L-asparaginase be given separately and that prophylaxis with low-molecular-weight heparin be considered.

Gastrointestinal complications Mucositis – incidence and risk factors Mucositis, or inflammation of mucosal surfaces, is a common complication of chemotherapy. Anatomic locations

Acute complications

affected include the mouth (stomatitis), esophagus, stomach, duodenum, and colon. Inflammation of the cecum in neutropenic patients (typhlitis) is a special case and has been reviewed elsewhere.269,270 It should be noted that mucositis can occur in the terminal ileum or any part of the colon in patients who are not neutropenic with a clinical presentation identical to that of typhlitis, but such inflammation is more properly termed ileitis or colitis. Mucositis severity is defined as grade 3 or 4 if treated with intravenous fluids or parenteral/enteral nutrition, respectively. In trials of therapy for childhood AML, 11% to 47% of children develop mucositis,271–273 and 2% to 52% in trials of ALL.274–277 The incidence and severity depend on the treatment regimen employed, and those containing high-dose methotrexate and high-dose cytarabine are especially damaging to mucosae.275,277–280 Rask and colleagues275,276 documented a 52% incidence of stomatitis in children treated with methotrexate (5000 to 8000 mg/m2 ) given as a 24hour infusion and found that higher methotrexate plasma concentrations and delayed clearance were risk factors. In another study of high-dose methotrexate in children with ALL, mucositis occurred in 26% of cycles in patients who received a median dose of 1500 mg/m2 , but when the supportive care regimen was modified by increasing the rate of intravenous fluid administration, increasing the amount of bicarbonate given, and documenting a urine pH of 6.5 or higher prior to initiating the methotrexate infusion, the rate of mucositis decreased to 11% per cycle.281 This study highlights the importance of meticulous supportive care, which sometimes can affect patient outcomes more than the dose or schedule of chemotherapy. By contrast, in CCG1881, a treatment regimen for low-risk ALL that used neither high-dose methotrexate nor high-dose cytarabine, the cumulative incidence of stomatitis during the duration of therapy was only 2% in one treatment arm and 4% in the other.274 Mucositis – prevention and treatment Many methods have been studied for the prevention of chemotherapy-induced mucositis. Studies have demonstrated modest efficacy of oral medications, including chlorhexidine,282 transforming growth factor beta,283 granulocyte-monocyte colony stimulating factor,284 iseganan,285 pilocarpine,286 or a special oral care regimen.287 However, other studies have not confirmed their beneficial effects,288–290 and meta-analysis suggests that only ice chips during chemotherapy administration have a modest beneficial effect.291,292 Treatment of mucositis is supportive and consists of meticulous mouth hygiene, maintenance of fluid intake with enteral or parenteral supplementation, and analgesia. If, however, an infectious

pathogen (e.g. herpes simplex or Candida) is isolated, specific therapy is warranted. In this regard, patients with a history of stomatitis caused by herpes should be treated while awaiting culture results, and prophylactic therapy considered to prevent recurrence. Hepatotoxicity Cancer chemotherapy293–295 and a variety of other medications296,297 prescribed during leukemia therapy can cause hepatitis, cholestasis, and hepatic dysfunction. In a group of children treated for AML with cytarabine, etoposide, and daunorubicin (or idarubicin), 24% developed elevated bilirubin, serum glutamic oxaloacetic transaminase (SGOT), and/or alkaline phosphatase,273 a rate similar to that observed in other studies that employed similar treatment.271 Toxicity limited to changes in biochemical measurements of liver inflammation or function is rarely severe and frequently has no clinical consequence. In this regard, children with ALL who receive high-dose methotrexate experience a predictable rise in hepatic transaminases298 ; however, patients rarely have symptoms, and this transient hepatic inflammation usually does not lead to lasting liver dysfunction.299 Treatment of hepatotoxicity is supportive. Special attention should be paid to those with increased conjugated bilirubin, as they require dose reduction or delay of chemotherapy. Pancreatitis Pancreatitis is broadly defined as any inflammation of the pancreas. Mild forms may present with only edema of the pancreas; whereas, severe cases include hemorrhage and necrosis.300,301 Symptoms and signs of pancreatitis in children with ALL include abdominal pain and tenderness (100% of cases), anorexia (100%), nausea/vomiting (95%), fever (63%), gastrointestinal bleeding due to coagulopathy (42%), jaundice (26%), and oliguria (21%).302 In children with leukemia, the most common cause of pancreatitis is therapy with L-asparaginase,300,302,303 but other drugs (glucocorticoid, ATRA, cytarabine, mercaptopurine)304,305 and hypercalcemia have also been implicated.306 Hypertriglyceridemia, also a known risk factor for pancreatitis, can be caused by L-asparaginase,307 which is thought to be toxic to the pancreas because this organ utilizes extracellular Lasparagine and its cells have a high proliferative rate.308 Fortunately, pancreatitis is rare in patients with leukemia, occurring in 0% to 3% of children treated for ALL in large cooperative group studies277,302,309,310 and 5% of 134 adults treated for AML.306 Treatment is supportive, and includes withdrawal of the offending agent (usually L-asparaginase); administration of intravenous fluids and anti-emetics; careful monitoring

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of glucose, electrolytes, and coagulation parameters; and vigilance for complications. Fasting and nasogastric suction have been advocated, but two small studies showed a trend toward improved outcomes when enteral nutrition was used, and a recent review of this issue concluded that published studies were not sufficient to support either strategy over the other.311–314 Complications after acute pancreatitis are common, and include pancreatic pseudocyst, abscess, chronic or recurrent pancreatitis, and pancreatic insufficiency.301,315 The prognosis depends on the severity of the presentation, and adverse prognostic signs. In adults, “Ranson’s Criteria” for a poor prognosis include (at diagnosis) a WBC count of >16 × 109 /L, glucose >200 mg/dL, lactate dehydrogenase >350 units/L, SGOT (AST) >250 units/L, or (within 48 hours of onset) a 10% drop in hematocrit, elevation in blood urea nitrogen >5 mg/dL, serum calcium <8 mg/dL, arterial oxygen tension <60 mmHg, base deficit >4 mEq/L, or fluid retention >6 L.316 Other prognostic factors include age, serum albumin, highest serum creatinine value within 60 to 72 hours from hospital admission, need for mechanical ventilation, and chronic health status.317,318 None of the prognostic models proposed for adults have been validated in pediatric patients.

Neurologic complications The incidence and severity of treatment-related neurologic complications depends on the treatment regimen employed and patient susceptibility. For example, patients whose therapy includes a glucocorticoid may develop myopathy, insomnia, behavioral changes, psychosis, hiccup, tremor, or other neurologic manifestations; these symptoms are rarely caused by other agents. In this section, we focus on the neurologic complications that result from therapy specifically directed to the CNS, a prerequisite for successful treatment or prevention of CNS leukemia. Prophylaxis for CNS leukemia includes intrathecal chemotherapy (methotrexate, cytarabine, glucocorticoid, or a combination of drugs) with or without cranial radiation.319–322 The use of high-dose methotrexate and dexamethasone, which have activity in the CNS, may also contribute to disease control.323 Prophylactic CNS regimens may cause acute and delayed neurologic sequelae, including seizures, paresthesias, paresis, ataxia, debilitating headaches, cognitive dysfunction, and leukoencephalopathy.324–330 Seizures and leukoencephalopathy are discussed below; cognitive and neuropsychiatric dysfunction are discussed in Chapter 35. Seizures Seizures soon after diagnosis of acute leukemia may be caused by CNS leukemia, intracerebral hemorrhage, cere-

bral leukostasis, cerebral edema, thrombosis, metabolic disturbances, or drug-related acute neurotoxic reactions. By contrast, seizures that occur after the patient has attained remission are most often caused by hyponatremia due to SIADH, CNS thrombosis related to L-asparaginase use, or leukoencephalopathy.331–334 Constipation and hypertension have also been reported to predispose children with ALL to seizures, possibly because of a transient increase in intracranial pressure and ischemia while straining to defecate.267 Seizures occur in 4% to 22% of children treated for ALL and up to 5% of those treated for AML; both the incidence and timing of seizure onset depend on the treatment regimen.271,325,333,335 Absence, partial, generalized, and Lennox-Gastaut seizures have all been reported.271,332,336 Treatment must address the underlying cause of seizure: chemotherapy for CNS leukemia, correction of metabolic disturbances, relief of elevated intracranial pressure, surgical drainage of intracerebral hemorrhage, or anticoagulation for CNS thrombosis. Anticonvulsants that induce hepatic cytochrome P-450 enzymes, which metabolize chemotherapy, should be avoided, since they worsen prognosis.337,338 In this regard, Khan and colleagues339 documented seizure control in 74% of 50 children whose new-onset seizures were treated with gabapentin, an anticonvulsant that does not induce hepatic enzymes. At some point, patients in whom the underlying cause of seizure resolves should discontinue anticonvulsant therapy, since in many cases seizure will not recur. Leukoencephalopathy The term leukoencephalopathy denotes white matter damage in the CNS. It is identified by hyperintense signal on T2 -weighted MR imaging of the brain (Fig. 29.11).340 The US National Cancer Institute grades the severity of the radiological findings associated with leukoencephalopathy according to the degree of increase in the subarachnoid space, the degree of ventriculomegaly, and the extensiveness of T2 -weighted MR hyperintensities of cerebral white matter. Such MR findings do not always correlate with the clinical severity or neurologic prognosis.340 Symptoms of leukoencephalopathy can begin abruptly or gradually, and include cognitive deficits, personality changes, slurred or absent speech, altered mental status, seizures, focal neurologic deficits, spasticity, posturing, or coma.324,336,341–344 It has been associated with cranial radiation, intrathecal chemotherapy, and high-dose systemic methotrexate.336,342–345 High-dose cytarabine has also been reported to cause leukoencephalopathy.346 The incidence of leukoencephalopathy depends on the treatment administered and the aggressiveness of the

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Fig. 29.11 Leukoencephalopathy. These magnetic resonance images of a 3.5-year-old girl with acute lymphoblastic leukemia show normal T2 -weighted, fluid attenuation inversion recovery (FLAIR), and color-mapped images at the end of remission induction therapy (upper left, middle, and right panels, respectively). Marked white matter changes (arrows) are seen after four cycles of high-dose methotrexate (lower panels). The patient had no symptoms and a normal neuropsychological evaluation at the time of these studies. She is now 7 years old and has mild deficits in reading comprehension and mathematics, but functions at grade level and has no neurologic deficits. The color-mapped images were generated with digital imaging processing software.340 Yellow denotes normal gray matter; blue denotes cerebrospinal fluid; green denotes normal white matter; and red denotes abnormal white matter. (Courtesy of Dr. Gene Reddick; see color plate 29.11 for full-color reproduction.)

neurologic and radiologic screening program used to detect the disorder. The Children’s Cancer Group documented only three cases (1%) of symptomatic leukoencephalopathy in 301 children with ALL treated with cranial irradiation (18 or 24 Gy) and intrathecal methotrexate without high-dose methotrexate, but there were no screening procedures in the protocol, so that neurologic symptoms may have been underreported.277 The Berlin-Frankfurt¨ Munster (BFM) group studied 118 patients who had been treated on the ALL-BFM 81,347 ALL-BFM 83,348 or COALL349 studies with head CT or MR imaging at a mean of 7.2 years after the completion of therapy.324 The rates of abnormal scans were 56% and 61% in the two groups of children treated with cranial irradiation, but only 38% in those treated with intrathecal chemotherapy but no irradiation. Abnormalities included high- and low-density white matter

changes, gray matter changes, widening of the ventricles and sulci, and calcifications. The natural history of leukoencephalopathy is highly variable: some patients develop progressive neurologic dysfunction, while others have no symptoms and the MR imaging findings resolve. Reddick et al.340 documented improvement in MR findings in two patients with ALL, whose leukoencephalopathy peaked at 44 weeks from diagnosis (after repeated administration of high-dose methotrexate) and had improved at the time of re-evaluation at 132 weeks (with no further highdose methotrexate administered in the interval). They also documented a correlation between the amount of normal white matter and the degree of neurocognitive deficits in children with brain tumors treated with cranial radiation.350

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Leukoencephalopathy in childhood leukemia must be distinguished from the syndrome variously called reversible posterior leukoencephalopathy, reversible occipitoparietal encephalopathy, or hypertensive encephalopathy.351 This syndrome consists of headache, confusion, seizure, and visual disturbance associated with transient characteristic neuroimaging abnormalities in the posterior part of the brain.352,353 It is thought to result from a failure of the autoregulatory capabilities of the cerebral vessels, with consequent brain hyperperfusion and vasogenic edema.351,353–355 Reversible posterior leukoencephalopathy is often associated with hypertension, but a variety of drugs and medical conditions, including cancer, have also been associated with the syndrome.342,354,356–364 In fact, there have been reports of the syndrome after intrathecal chemotherapy,342,362,365,366 and it may not always be possible to distinguish reversible posterior leukoencephalopathy caused by dysregulated cerebral vasculature from leukoencephalopathy caused by intrathecal chemotherapy, CNS irradiation, or systemic methotrexate. In this regard, 10% of children with ALL develop hypertension during remission induction,367 and most take medications that could place them at risk for reversible posterior leukoencephalopathy. In both cases, the treatment is supportive: anticonvulsants, antihypertensives, rehabilitation, and avoidance of the precipitating agent.

Osteonecrosis Epidemiology Osteonecrosis (ON), or avascular necrosis of bone, is a recognized complication of treatment for leukemia and lymphoma whose pathophysiology is poorly understood. It occurs in the absence of associated trauma or infection and imposes substantial early morbidity with long-term functional consequences. The reported incidence of ON associated with steroid-containing leukemia treatment regimens ranges from 1% to 25%.368–373 This variation in reported incidence reflects the intensity and type of chemotherapy, the index of suspicion of clinicians, and the diagnostic methods used to detect the necrosis. For example, among 551 children with ALL or AML admitted to the University Children’s Hospital in M¨ unster and treated according to a BFM-like protocol from 1971 to 1985, six (1.1%) were diagnosed with symptomatic ON.372 In another study by Canadian investigators,374 5 of 113 children (4.4%) with high-risk ALL, who received treatment similar to the BFM group, developed symptomatic ON. Corticosteroids included induction with 28 days of prednisone at a dose of 60 mg/m2 per day, reinduction with 28 days of dexamethasone at a dose of 10 mg/m2 per day, and maintenance with prednisone at a dose of 40 mg/m2 per day for

5 days each month for 3 years. Of interest, none of the 115 children treated on the low-risk protocol developed symptomatic ON. The low- and high-risk protocols employed the same prednisone schedule and dosage in the induction and maintenance phases, but reinduction treatment with dexamethasone was not used in the low-risk patients. The Children’s Cancer Group (CCG) reported 111 cases of ON among 1409 children (9.3% incidence) treated on the CCG-1882 protocol.375 This protocol used prednisone during induction and maintenance and dexamethasone during delayed intensification. Patients 10 years of age or older had a 14% incidence of ON compared with 0.9% in younger patients (P < 0.001), while patients randomized to receive two 21-day dexamethasone courses had a 23% incidence compared with 16% in those who received only one course (P = 0.27). In the 10-year or older group, girls had an incidence of 17% and boys 12%, despite significantly lower cumulative glucocorticoid doses (2600 mg/m2 less prednisone) in girls, whose maintenance therapy lasted 1 year less than in boys. Ethnicity also affected the incidence of ON: 17% of whites, 3.3% of blacks, and 6.7% of children of other ethnicities developed this complication. These differences in ON incidence suggest that the risk of ON depends on a combination of patient factors (age, race, gender, pubertal stage, and genetics) and treatment factors (type, dose, and schedule of corticosteroid). At St. Jude Children’s Research Hospital, we used MR imaging of the hips and knees to screen patients treated for ALL or NHL with intensive prednisone.376 Of the 110 evaluable participants, 17 (15%) had ON. As in the CCG study,377 an age of 10 years or older was a significant risk factor for ON, with an incidence of 8% in patients younger than 10 compared with 31% in older children (P = 0.004). Six of the 17 patients with ON (35%) had no symptoms, a proportion similar to that of Ojala et al.378 , who found that six of nine patients identified by MR screening had no symptoms. Treatment and prognosis The management of asymptomatic ON is controversial, but when symptoms are present, treatment includes cessation of steroid therapy, symptomatic relief with analgesics, decreased weight-bearing, and surgical procedures such as core decompression and joint resurfacing. If the joint deteriorates further, replacement with a prosthetic joint is required, but this is problematic for growing children. In general, ON of the hips that involves the articular surface carries a poor prognosis, with a high probability of progressive joint deterioration and morbidity especially when more than 30% of the surface is affected. Symptomatic ON can lead to progressive irreversible weight-bearing joint destruction and ultimately joint replacement.379–383 In adults, anatomic and functional deterioration leads to a

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surgical procedure within 3 years of the diagnosis of symptomatic ON of the hip in 50% of cases384–387 and the knee in 18%.388 The best information on the natural history of ON in children with ALL comes from the large review of symptomatic patients by Mattano et al.389 Of 1409 children treated for ALL, 111 were diagnosed with symptomatic ON. After 1 to 7 years of follow-up, 84% had chronic pain and/or immobility, 24% had undergone orthopedic surgical procedures, and another 15% were considered candidates for surgery in the future. This morbidity occurred despite stopping of all glucocorticoid therapy at diagnosis of ON in over half the patients. Since established ON often leads to progressive joint deterioration, prevention and early recognition of this complication is essential. Attempts have been made by COG investigators and us to reduce the incidence of ON by interrupted (rather than continuous) use of glucocorticoid treatment. Preliminary results show a reduction in the frequency and severity of this complication, but it is uncertain whether antileukemic efficacy will be compromised.390

Endocrine complications Endocrinopathy is a common complication of leukemia and its treatment.391 Endocrine conditions that can develop include the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), diabetes mellitus, hypoglycemia, corticosteroid insufficiency, and hypercalcemia. Obesity also occurs with greater frequency in survivors of ALL, especially those treated with cranial irradiation.392,393 Growth velocity decreases during therapy, but survivors generally achieve normal adult height. Osteopenia can be present at the time of leukemia diagnosis or can develop during therapy. Growth and osteopenia are discussed in Chapter 30.

Hyponatremia and SIADH Hyponatremia most often results from administration of excess hypotonic intravenous fluids, but it can also be caused by SIADH, characterized by sustained release of excessive antidiuretic hormone (ADH) from the posterior pituitary gland. This hormone causes renal water retention and consequent serum hypoosmolarity, and is a known complication of treatment with vincristine, commonly used to treat ALL,394–397 and many other drugs. The condition typically develops a week after administration of a dose of vincristine and can occur even after a patient has tolerated multiple prior doses. SIADH often recurs if no adjustment is made to the vincristine dosage or schedule. Azole antifungal drugs, such as itraconazole, fluconazole, ketoconazole, and voriconazole inhibit the activity of

the cytochrome P-450 enzymes that metabolize vincristine and potentiate its toxicity.37,38,398–400 Adrenocortical insufficiency can lead to secondary SIADH.401–403 Lethargy, weakness, headache, oliguria, and weight gain are early symptoms of SIADH, which can progress to confusion, seizures, coma, and death.404,405 However, hyponatremia is often detected by a routine chemistry profile, at a time when early intervention can prevent the onset of symptoms. Mild hyponatremia occurs frequently when hyperhydration is given with chemotherapy, and SIADH must be distinguished from dilutional hyponatremia by evaluation of urine output, urine sodium, and urine osmolality. The diagnosis of SIADH is confirmed by elevated urine sodium concentration and osmolality in the presence of low serum sodium concentration and osmolality.

Treatment of SIADH SIADH is treated with fluid restriction; close monitoring of urine output, serum sodium concentration, and neurologic status; and discontinuation of vincristine therapy until the acute episode has resolved.406 Furosemide should be given if symptoms or signs of fluid overload are present. All medications should be evaluated, especially azole antifungals, selective serotonin reuptake inhibitors, tricyclic antidepressants, antipsychotics, anticonvulsants, thiazide diuretics, and azithromycin. Hospitalization is indicated for patients with significant oliguria, hyponatremia, or neurologic symptoms. Hyponatremic seizures require emergent intervention, including anticonvulsants and, if necessary, 3% sodium chloride solution intravenously (3–5 mL per kg of body weight given over 2 to 3 hours).406 If such aggressive therapy is necessary, the patient should be monitored in the intensive care unit, and serum sodium should be measured every 1 to 2 hours. In general, correction of hyponatremia should be no faster than 12 mEq/dL of sodium per 24 hours to prevent the rapid shift of water out of the brain and thereby minimize the risk of central pontine myelinolysis, a severe neurological disorder characterized by mutism, dysarthria, spastic quadriparesis, and pseudobulbar palsy.407,408 Patients who experience SIADH as a side effect of vincristine therapy should undergo prophylactic fluid restriction with future doses to prevent recurrence.395

Hyperglycemia – epidemiology Hyperglycemia occurs commonly among children treated with corticosteroids, which increase insulin resistance, and L-asparaginase, which reduces the production of insulin and possibly insulin receptors.409,410 Treatment with either glucocorticoids or L-asparaginase causes hyperglycemia in about 1% of patients410–412 ; the combination of these agents causes hyperglycemia in 4% to 10% of cases.412,413 In

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a study of 176 children with B-precursor ALL, Belgaumi and colleagues367 documented hyperglycemia during remission induction with L-asparaginase and prednisone 40 mg/m2 per day in 12% of patients, compared with 20% when dexamethasone 6 mg/m2 per day was substituted for prednisone (P = 0.20). Hyperglycemia occurred during remission induction in 41 (9.7%) of 421 pediatric patients who received prednisone and L-asparaginase for ALL.413 Risk factors for hyperglycemia included age of 10 years or more, obesity, Down syndrome, and a family history of diabetes mellitus. The incidence of hyperglycemia also depends on the type, dose, and schedule of L-asparaginase used,414,415 and in one study was more common in children treated with dexamethasone 6 mg/m2 per day (5%) than with prednisone 40 mg/m2 per day (1.5%).309

Hyperglycemia – symptoms, treatment, and prevention Children with chemotherapy-induced hyperglycemia usually exhibit symptoms such as polyuria, polydipsia, polyphagia, glucosuria, or some combination of these conditions on routine urinalysis. If these early signs are not heeded, dehydration, hypotension, somnolence, and coma can occur. Virtually all patients with therapy-induced hyperglycemia recover when L-asparaginase and glucocorticoids are discontinued, and they suffer no long-term adverse effects. Treatment includes monitoring of serum and urine glucose concentrations, diet modification, and increased exercise. Insulin therapy is needed only if clinically important hyperglycemia persists despite these measures and should be used with caution to avoid iatrogenic hypoglycemia. In our study, 29 of 41 patients with ALL, whose hyperglycemia developed during remission induction therapy with prednisone and L-asparaginase, required insulin therapy for an average of 11.5 days.413 The goal of therapy is to avoid extreme hyperglycemia. Strict glucose control is not necessary, and insulin requirements vary greatly depending on the timing of administration of steroids and L-asparaginase, and whether or not the patient is ambulatory. Treatment of hyperglycemia should begin with aggressive hydration and a low dose of regular insulin. If the glucose remains elevated, additional doses of regular insulin can be given to achieve adequate control while avoiding hypoglycemia. The best treatment is prevention. In this regard, the Dana-Farber Cancer Institute Consortium416,417 and the German Cooperative Study Group for treatment of ALL (COALL)418 avoid concurrent use of glucocorticoids and L-asparaginase to reduce the risk of hyperglycemia and thrombotic complications; these groups have maintained excellent treatment results for ALL.

Hypoglycemia The most common cause of hypoglycemia among patients with pediatric ALL is overtreatment of hyperglycemia with insulin, but the condition has also been observed in children receiving mercaptopurine.419,420 Halonen et al.420 reported hypoglycemia in 16 (54%) of 35 children after a 16-hour fast during continuation therapy that included oral methotrexate and mercaptopurine. Fasting blood glucose levels improved among all patients after completion of therapy and normalized completely in 67%. Ziino et al.419 found that 6 (6.9%) of 86 children treated with mercaptopurine and thioguanine experienced 18 episodes of symptomatic hypoglycemia during therapy. Affected patients were 3 to 5 years old, and hypoglycemia was successfully prevented in five of six patients by encouraging large evening meals and early breakfasts. Adrenal insufficiency may also cause hypoglycemia, and should be considered in children treated recently with glucocorticoids.421 Corticosteroid insufficiency Clinically important adrenocortical insufficiency during therapy for acute leukemia is uncommon after the 4 to 6 weeks of conventional prednisone or prednisolonecontaining remission induction regimens for ALL. However, the incidence depends on the dose, duration, and schedule of glucocorticoid therapy.391,422–426 Patients treated with dexamethasone at 6 mg/m2 per day for 28 consecutive days can have adrenal insufficiency lasting more than 4 weeks after cessation of treatment,426 and 4 of 10 children had adrenal insufficiency that persisted for 2.5 to 4 months after completion of treatment with prednisolone 60 mg/m2 per day for 35 consecutive days.427 Adrenal insufficiency usually manifests during times of metabolic stress, such as infection, chemotherapy-induced toxicity, or surgery.428 Under these circumstances, clinicians should be vigilant for symptoms of adrenal insufficiency: malaise, fatigue, weakness, anorexia, nausea, vomiting, weight loss, abdominal pain, diarrhea, hypothermia or hyperthermia, hypotension, altered mental status, and coma. Hyponatremia, hypoglycemia, hyperkalemia, metabolic acidosis, and prerenal azotemia may occur.423,428,429 Specific tests of hypothalamic-pituitary-adrenal axis function are discussed elsewhere.430 Empiric treatment should be initiated whenever the degree of hemodynamic or neurologic compromise is out of proportion to the severity of the inciting illness, because untreated adrenal insufficiency in patients undergoing severe infection, surgery, or other physiologic stress can be fatal.429,431,432 In such cases, an intravenous hydrocortisone bolus of 10 to 25 mg/m2 should be followed by 30 mg/m2 per day (moderate stress) or 100 mg/m2 per day (severe stress) in three or

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four divided doses.433 Because recovery of hypothalamicpituitary-adrenal axis function may occur within days425,426 or may require months,434 maintenance therapy with oral corticosteroids must be individualized.435,436 Supportive care medications, such as megestrol acetate, that potentially suppress adrenal function, should be used with caution in children who have received glucocorticoid therapy.437

Summary Acute complications include “early” complications that occur within 2 weeks of diagnosis and are caused by the leukemia itself and “on-therapy” complications that occur after the first 2 weeks of therapy and reflect the toxicity of leukemia therapy. The most common serious early complications are tumor lysis syndrome, central airway compression, coagulopathy, hyperviscosity/leukostasis, and neurologic dysfunction; whereas the most common on-therapy complications are thrombosis, endocrine dysfunction, gastrointestinal disorders, osteonecrosis, and neurologic dysfunction. Early recognition and prompt treatment of these problems can reduce early morbidity and mortality.

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41 Kalwinsky, D. K., Raimondi, S. C., Bunin, N. J., et al. Clinical and biological characteristics of acute lymphocytic leukemia in children with Down syndrome. Am J Med Genet Suppl, 1990; 7: 267–71. 42 Tamminga, R. Y., Dolsma, W. V., Leeuw, J. A., & Kampinga, H. H. Chemo- and radiosensitivity testing in a patient with ataxia telangiectasia and Hodgkin disease. Pediatr Hematol Oncol, 2002; 19: 163–71. 43 Chen, R. L., Wang, P. J., Hsu, Y. H., Chang, P. Y., & Fang, J. S. Severe lung fibrosis after chemotherapy in a child with ataxia-telangiectasia. J Pediatr Hematol Oncol, 2002; 24: 77–9. 44 Heller, P., Best, W. R., Nelson, R. B., & Becktel, J. Clinical implications of sickle-cell trait and glucose-6-phosphate dehydrogenase deficiency in hospitalized black male patients. N Engl J Med, 1979; 300: 1001–5. 45 Valaes, T. Severe neonatal jaundice associated with glucose6-phosphate dehydrogenase deficiency: pathogenesis and global epidemiology. Acta Paediatr Suppl, 1994; 394: 58–76. 46 Tanphaichitr, V. S., Mahasandana, C., Suvatte, V., et al. Prevalence of hemoglobin E, alpha-thalassemia and glucose-6phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok. Southeast Asian J Trop Med Public Health, 1995; 26(Suppl. 1): 271–4. 47 Mehta, A., Mason, P. J., & Vulliamy, T. J. Glucose-6-phosphate dehydrogenase deficiency. Baillieres Best Pract Res Clin Haematol, 2000; 13: 21–38. 48 Sklar, G. E. Hemolysis as a potential complication of acetaminophen overdose in a patient with glucose-6phosphate dehydrogenase deficiency. Pharmacotherapy, 2002; 22: 656–8. 49 Pui, C. H. Rasburicase: a potent uricolytic agent. Expert Opin Pharmacother, 2002; 3: 433–52. 50 Relling, M. V., Hancock, M. L., Rivera, G. K., et al. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J Natl Cancer Inst, 1999; 91: 2001–8. 51 McLeod, H. L., Coulthard, S., Thomas, A. E., et al. Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia. Br J Haematol, 1999; 105: 696–700. 52 McLeod, H. L., Krynetski, E. Y., Relling, M. V., & Evans, W. E. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia, 2000; 14: 567–72. 53 Lantz, B., Adolfsson, J., Lagerlof, B., & Reizenstein, P. Causes of death in leukemia and lymphoma with modern treatment. Acta Haematol, 1980; 63: 61–7. 54 Attarbaschi, A., Mann, G., Dworzak, M., et al. Mediastinal mass in childhood T-cell acute lymphoblastic leukemia: significance and therapy response. Med Pediatr Oncol, 2002; 39: 558–65. 55 Varma, S., Varma, N., Dhar, S., et al. Cytodiagnosis of granulocytic sarcoma presenting as superior vena cava syndrome in

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384 Bradway, J. K. & Morrey, B. F. The natural history of the silent hip in bilateral atraumatic osteonecrosis. J Arthroplasty, 1993; 8: 383–7. 385 Jacobs, B. Epidemiology of traumatic and nontraumatic osteonecrosis. Clin Orthop, 1978; 130: 51–67. 386 Ohzono, K., Saito, M., Sugano, N., Takaoka, K., & Ono, K. The fate of nontraumatic avascular necrosis of the femoral head. A radiologic classification to formulate prognosis. Clin Orthop, 1992; 277: 73–8. 387 Bozic, K. J., Zurakowski, D., & Thornhill, T. S. Survivorship analysis of hips treated with core decompression for nontraumatic osteonecrosis of the femoral head. J Bone Joint Surg (Am), 1999; 81: 200–9. 388 Houpt, J. B., Pritzker, K. P., Alpert, B., Greyson, N. D., & Gross, A. E. Natural history of spontaneous osteonecrosis of the knee (SONK): a review. Semin Arthritis Rheum, 1983; 13: 212–27. 389 Mattano, L. A., Jr., Sather, H. N., Trigg, M. E., & Nachman, J. B. Osteonecrosis as a complication of treating acute lymphoblastic leukemia in children: a report from the Children’s Cancer Group. J Clin Oncol, 2000; 18: 3262–72. 390 Mattano, L. A., Jr., Sather, H., La, M. K., Nachman, J. B., & Seibel, N. L. Modified dexamethasone reduces the incidence of treatment-related osteonecrosis in children and adolescents with higher risk acute lymphoblastic leukemia: A report of CCG-1961. Blood, 2003; 102: 221a. 391 Howard, S. C. & Pui, C. H. Endocrine complications in pediatric patients with acute lymphoblastic leukemia. Blood Rev, 2002; 16: 225–43. 392 Oeffinger, K. C., Mertens, A. C., Sklar, C. A., et al. Obesity in adult survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. J Clin Oncol, 2003; 21: 1359–65. 393 Mayer, E. I., Reuter, M., Dopfer, R. E., & Ranke, M. B. Energy expenditure, energy intake and prevalence of obesity after therapy for acute lymphoblastic leukemia during childhood. Horm Res, 2000; 53: 193–9. 394 Nicholson, R. G. & Feldman, W. Hyponatremia in association with vincristine therapy. Can Med Assoc J, 1972; 106: 356–7. 395 Stuart, M. J., Cuaso, C., Miller, M., & Oski, F. A. Syndrome of recurrent increased secretion of antidiuretic hormone following multiple doses of vincristine. Blood, 1975; 45: 315–20. 396 Robertson, G. L., Bhoopalam, N., & Zelkowitz, L. J. Vincristine neurotoxicity and abnormal secretion of antidiuretic hormone. Arch Intern Med, 1973; 132: 717–20. 397 Kebaili, K., Bertrand, Y., Foray, P., et al. A rare cause of hyponatremia during introductory treatment of acute lymphoblastic leukemia in an infant: inappropriate secretion of atrial natriuretic factor? Arch Pediatr, 1994; 1: 898–902. 398 Sathiapalan, R. K., Al-Nasser, A., El-Solh, H., Al-Mohsen, I., & Al-Jumaah, S. Vincristine-itraconazole interaction: cause for increasing concern. J Pediatr Hematol Oncol, 2002; 24: 591. 399 Kamaluddin, M., McNally, P., Breatnach, F., et al. Potentiation of vincristine toxicity by itraconazole in children with lymphoid malignancies. Acta Paediatr, 2001; 90: 1204–7.

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400 Bohme, A., Ganser, A., & Hoelzer, D. Aggravation of vincristineinduced neurotoxicity by itraconazole in the treatment of adult ALL. Ann Hematol, 1995; 71: 311–12. 401 Demura, R. The role of antidiuretic hormone in hyponatremia in adrenal insufficiency – is the guideline for the diagnosis of syndrome of inappropriate secretion of the antidiuretic hormone appropriate? Intern Med, 1999; 38: 382–3. 402 Kamoi, K., Tamura, T., Tanaka, K., Ishibashi, M., & Yamaji, T. Hyponatremia and osmoregulation of thirst and vasopressin secretion in patients with adrenal insufficiency. J Clin Endocrinol Metab, 1993; 77: 1584–8. 403 Spital, A. Hyponatremia in adrenal insufficiency: review of pathogenetic mechanisms. South Med J, 1982; 75: 581–5. 404 Krutisch, G. & Valentin, A. Comatose state due to severe hyponatremia in a patient with the syndrome of inappropriate antidiuretic hormone secretion. Intensive Care Med, 2001; 27: 944. 405 Kloster, R., Borresen, H. C., & Hoff-Olsen, P. Sudden death in two patients with epilepsy and the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Seizure, 1998; 7: 419–20. 406 Haycock, G. B. The syndrome of inappropriate secretion of antidiuretic hormone. Pediatr Nephrol, 1995; 9: 375–81. 407 Omari, A., Kormas, N., & Field, M. Delayed onset of central pontine myelinolysis despite appropriate correction of hyponatraemia. Intern Med J, 2002; 32: 273–4. 408 Lampl, C. & Yazdi, K. Central pontine myelinolysis. Eur Neurol, 2002; 47: 3–10. 409 Carpentieri, U. & Balch, M. T. Hyperglycemia associated with the therapeutic use of L-asparaginase: possible role of insulin receptors. J Pediatr, 1978; 93: 775–8. 410 Baillargeon, J., Langevin, A. M., Mullins, J., et al. Transient hyperglycemia in Hispanic children with acute lymphoblastic leukemia. Pediatr Blood Cancer, 2005; Feb. 7 [Epub ahead of print] PMID: 15700246. 411 Cetin, M., Yetgin, S., Kara, A., et al. Hyperglycemia, ketoacidosis and other complications of L-asparaginase in children with acute lymphoblastic leukemia. J Med, 1994; 25(3–4): 219–29. 412 Iyer, R. S., Rao, S. R., Pai, S., Advani, S. H., & Magrath, I. T. Lasparaginase related hyperglycemia. Indian J Cancer, 1993; 30: 72–6. 413 Pui, C. H., Burghen, G. A., Bowman, W. P., & Aur, R. J. Risk factors for hyperglycemia in children with leukemia receiving L-asparaginase and prednisone. J Pediatr, 1981; 99: 46–50. 414 Ridgway, D., Neerhout, R. C., & Bleyer, A. Attenuation of asparaginase-induced hyperglycemia after substitution of the Erwinia carotovora for the Escherichia coli enzyme preparation. Cancer, 1989; 63: 561–3. 415 Lavine, R. L., & DiCinto, D. M. L-Asparaginase diabetes mellitus in rabbits: differing effects of two different schedules of Lasparaginase administration. Horm Metab Res, 1984; 16(Suppl. 1): 92–6.

416 Silverman, L. B., Gelber, R. D., Dalton, V. K., Asselin, B. L., Barr, R. D., Clavell, L. A., et al. Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91–01. Blood, 2001; 97: 1211–18. 417 Silverman, L. B., Declerck, L., Gelber, R. D., et al. Results of Dana-Farber Cancer Institute Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1981–1995). Leukemia, 2000; 14: 2247–56. 418 Harms, D. O. & Janka-Schaub, G. E. Co-operative study group for childhood acute lymphoblastic leukemia (COALL): longterm follow-up of trials 82, 85, 89 and 92. Leukemia, 2000; 14: 2234–9. 419 Ziino, O., Russo, D., Orlando, M. A., et al. Symptomatic hypoglycemia in children receiving oral purine analogues for treatment of childhood acute lymphoblastic leukemia. Med Pediatr Oncol, 2002; 39: 32–4. 420 Halonen, P., Salo, M. K. & Makipernaa, A. Fasting hypoglycemia is common during maintenance therapy for childhood acute lymphoblastic leukemia. J Pediatr, 2001; 138: 428–31. 421 Artavia-Loria, E., Chaussain, J. L., Bougneres, P. F. & Job, J. C. Frequency of hypoglycemia in children with adrenal insufficiency. Acta Endocrinol Suppl (Copenh), 1986; 279: 275–8. 422 Giona, F., Annino, L., Donato, P., & Ermini, M. Gonadal, adrenal, androgen and thyroid functions in adults treated for acute lymphoblastic leukemia. Haematologica, 1994; 79: 141–7. 423 Krasner, A. S. Glucocorticoid-induced adrenal insufficiency. JAMA, 1999; 282: 671–6. 424 Rosmond, R., Chagnon, Y. C., Holm, G., et al. A glucocorticoid receptor gene marker is associated with abdominal obesity, leptin, and dysregulation of the hypothalamic-pituitaryadrenal axis. Obes Res, 2000; 8: 211–18. 425 Kuperman, H., Damiani, D., Chrousos, G. P., et al. Evaluation of the hypothalamic-pituitary-adrenal axis in children with leukemia before and after 6 weeks of high-dose glucocorticoid therapy. J Clin Endocrinol Metab, 2001; 86: 2993–6. 426 Felner, E. I., Thompson, M. T., Ratliff, A. F., White, P. C., & Dickson, B. A. Time course of recovery of adrenal function in children treated for leukemia. J Pediatr, 2000; 137: 21–4. 427 Petersen, K. B., Muller, J., Rasmussen, M., & Schmiegelow, K. Impaired adrenal function after glucocorticoid therapy in children with acute lymphoblastic leukemia. Med Pediatr Oncol, 2003; 41: 110–4. 428 Cooper, M. S. & Stewart, P. M. Corticosteroid insufficiency in acutely ill patients. N Engl J Med, 2003; 348: 727–34. 429 August, G. P. Treatment of adrenocortical insufficiency. Pediatr Rev, 1997; 18: 59–62. 430 Agwu, J. C., Spoudeas, H., Hindmarsh, P. C., Pringle, P. J., & Brook, C. G. Tests of adrenal insufficiency. Arch Dis Child, 1999; 80: 330–3. 431 Zaloga, G. P. Sepsis-induced adrenal deficiency syndrome. Crit Care Med, 2001; 29: 688–90.

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30 Late complications after leukemia therapy Melissa M. Hudson

Introduction Because cure rates for children with acute lymphoblastic leukemia (ALL) have improved over the last three decades, information concerning late treatment sequelae has become increasingly important. Earlier reports described long-term complications after relatively homogeneous, less intensive chemotherapy given with cranial or craniospinal irradiation for central nervous system (CNS) preventive therapy. Recognition of new prognostic clinical and biological features has permitted risk-directed treatment that now cures at least 70% of children with ALL. Continued surveillance of the survivor population will elucidate the sequelae of these modern, intensive therapies. Similarly, the long-term survival of children with acute myeloid leukemia (AML) has improved substantially in the last decade with the use of more intensive chemotherapy regimens and allogeneic bone marrow transplantation. Today, approximately 40% of children with AML are cured of their disease. The increasing numbers of long-term survivors of AML mandate the evaluation of late treatment sequelae and their effect on morbidity and mortality. This chapter presents a systematic review of late treatment complications of childhood acute leukemia. My purpose is to assist the reader in identifying treatment and patient characteristics that predict the risk for adverse sequelae. Once these risk features are recognized, appropriate surveillance studies can be undertaken, and the potential effects of various therapy-related complications can be addressed during the design of future treatment regimens.

Endocrine toxicity Growth and pubertal development Survivors of childhood leukemia are at risk for impaired growth and short stature as adults due to intensive combination chemotherapy and radiation injury to the growth centers in the spine and long bones and various endocrine organs. The adverse effect of CNS radiation therapy on linear growth in children treated for leukemia has long been recognized, but its pathophysiology is unknown. Interference with hepatic synthesis of the essential insulinlike growth factor (IGF-1) and inhibition of amino acid uptake by cartilage have been hypothesized as possible mechanisms of chemotherapy-induced growth failure. 1 Additional factors that may compromise growth include the severity of the disease, the intensity and duration of chemotherapy, corticosteroid therapy, infections, and poor nutrition. 2–6 Sklar et al. 5 reported significant decrements in height standard deviation scores (SDS) in patients who did not receive cranial radiation. The greatest decrements in this measure occurred during treatment, but further decreases were observed during the period after completion of therapy and until final height was achieved. Dana-Farber investigators observed a statistically significant decrease in height z scores in a cohort of 618 children treated with either chemotherapy alone, or standard or twice-daily factionated cranial radiation, although only a fraction of this cohort (n = 93) was monitored until attainment of adult height. 7 Similar to findings of other investigators, young age at the time of diagnosis was the most influential

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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factor affecting long-term height. 7–9 In a cohort with more prolonged follow-up, chemotherapy was a risk factor for reduced final height only when administered in combination with cranial irradiation. 10 As relatively few studies describe final height outcomes in children treated with chemotherapy alone, further follow-up of contemporarily treated ALL survivors is needed to evaluate this issue. Cranial irradiation is clearly associated with the greatest loss in height in ALL survivors. Height loss from this modality may occur as the result of growth hormone deficiency, premature sexual development or an abnormally rapid progression through puberty, and hypothyroidism. The severity and frequency of radiation injury to the hypothalamic-pituitary axis are correlated with the total dose, fraction size, and patient age at the time of treatment. Blunted spontaneous basal pulsatile secretion of growth hormone has been consistently observed in children with ALL treated with 24 Gy cranial irradiation and intrathecal methotrexate. 11 However, individual growth patterns have correlated poorly with results from provocative growth hormone testing during and after completion of therapy. 2,12,13 Although investigators have compared the effects of 24 Gy versus 18 Gy cranial irradiation on growth, results conflict and are limited by follow-up times that are insufficient to determine if these radiation doses correlate with differences in the adult stature of ALL survivors. Uruena et al. 14 found that the long-term reduction in height SDS was similar for groups receiving 18 Gy or 24 Gy cranial radiation therapy. Cicognani et al. 15 observed less growth retardation in children treated with 18 Gy, but they were followed for only 5 years from diagnosis. Sklar et al. 5 observed the greatest loss in height SDS in children treated with 24 Gy cranial irradiation; these patients were followed until they attained adult height. However, significant decrements in height SDS were also observed in those who received no cranial radiation or 18 Gy cranial radiation. Young girls treated with cranial radiation were particularly vulnerable to height loss because of premature or early puberty combined with growth hormone deficiency. Stubberfield et al. 16 showed that during the first 5 years after diagnosis, the growth patterns of children whose treatment for ALL included 24 Gy cranial radiation were similar to those of patients who received 18 Gy. However, with further follow-up, a greater loss of height was seen for children who received 24 Gy. 16 This difference was correlated with a reduction in growth hormone secretion that was more marked in patients treated with 24 Gy. The results of these and other studies indicate that although cranial radiation therapy is the major factor inducing growth impairment, other variables such as age, gender, individual sensitivity to radiation therapy and dose fractionation, and intensity and

Fig. 30.1 Partial alopecia in a 24-year-old survivor of ALL who received a total cranial radiation dose of 48 Gy: 24 Gy for preventive CNS therapy and 24 Gy for treatment of CNS leukemia that developed 1 year after the completion of therapy.

duration of chemotherapy also affect final height. 17 Cranial irradiation can also result in a small cranium (especially when it is delivered at a young age) and partial alopecia (Fig. 30.1). Craniospinal irradiation is now reserved for patients with CNS relapse. Direct irradiation of the spine results in irreversible growth failure, primarily from destruction of the growth centers. The younger the child at the time of treatment, the more severe the reduction in spinal growth and sitting height, with disproportionate final height. 18–21 The full impact of radiation on spinal growth may not become apparent until the patient completes puberty, a time when the spine normally grows rapidly. True precocious puberty, early puberty, and menarche, as well as normally timed onset of puberty with rapid progression have been observed in children treated with cranial radiation therapy for CNS tumors. The effects of lowerdose cranial radiation therapy in ALL treatment regimens is not as well characterized, but early puberty in combination with reduced growth rates may contribute significantly to reduced final height in some survivors. Early puberty

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Fig. 30.2 (A) Growth grid of a 19-year-old survivor of ALL who received 24 -Gy cranial radiation as preventive CNS therapy, illustrating precocious puberty and rapid tempo that resulted in adult short stature. (B) Growth grid of 16-year-old survivor of ALL who received 24 Gy cranial radiation as CNS therapy, illustrating a reduction in growth velocity 5 years after completion of therapy and improvement after initiation of growth hormone therapy.

has been described in boys surviving ALL, but at the same frequency as in the normal population. 22,23 By contrast, multiple investigators have observed that girls whose ALL treatment included 18 Gy or 24 Gy cranial radiation therapy show loss in height because of early puberty or menarche (Fig. 30.2A). 14,16,17,22,23 In some studies, reduction in the amplitude 4,17,23–25 and duration 25 of the pubertal growth spurt exacerbated height loss. Several studies demonstrate a positive correlation between young age at the time of radiation therapy and the age at onset of puberty. 5,22 Monitoring for early puberty with its associated premature epiphyseal fusion and diminished growth spurt may allow timely

interventions such as growth hormone and gonadotropinreleasing hormone agonist therapies that reduce late-onset growth impairment (Fig. 30.2B).

Thyroid dysfunction Primary hypothyroidism is a well-known complication of head and neck radiation therapy when treatment exceeds 20 Gy. However, central or primary hypothyroidism after cranial or craniospinal radiation for childhood ALL is uncommon, although subclinical thyroid dysfunction may contribute to poor growth, particularly in children with

Late complications after leukemia therapy

concomitant growth hormone deficiency. 26–29 Elevated thyroid stimulating hormone (TSH) levels coupled with low or low-normal thyroxine levels suggest primary hypothyroidism. A low-normal free thyroxine level accompanied by a TSH surge and abnormal thyrotropin-releasing hormone tests more reliably identify children with the subtle growth abnormalities that result from central hypothyroidism. 30 Mild hypothyroidism may become more obvious after therapy with growth hormone is initiated because of increased turnover of thyroxine metabolites. 31 A recent study evaluating hypothalamic-pituitary-thyroid function in long-term ALL survivors (median follow-up time, 8 years) demonstrated normal thyroid function in survivors maintaining first continuous remission. These results suggest that adverse effects on the hypothalamic-pituitary-thyroid axis are uncommon after prophylactic cranial radiation doses of 24 Gy or lower. 32 Chemotherapy appears to contribute little, if any, excess risk for damage to the thyroid axis. 33

Growth after bone marrow transplantation Severe growth impairment after bone marrow transplantation is common in children with acute leukemia. Growth failure is attributed to an interplay of multiple factors including patient characteristics (such as age and gender), treatment variables (such as prior cranial radiation therapy and type of preparative regimen), and post-treatment complications, primarily graft-versus-host disease. In most of the groups, conditioning regimens that include total body irradiation (TBI) appear to result in the greatest height loss. 34–38 Several studies have indicated that growth impairment occurs earlier and more frequently in patients treated with both TBI and prophylactic cranial radiation therapy before transplantation. 35,39–43 Limited data are available on final height outcomes after allogeneic hematopoietic stem cell transplantation for pediatric malignancies. 38,44,45 The largest multicenter study evaluating final height achievement to date confirmed that irradiation is the major factor predisposing to longterm growth impairment; male and younger patients had the greatest loss in height SDS. 38 Previous cranial irradiation and single-dose TBI produced the greatest negative effect on final height, but fractionation of TBI significantly reduced height loss. 38 Patients conditioned with busulfan and cyclophosphamide had less interference with growth, as observed in other cohorts of transplant survivors. 38,43,46,47 Growth impairment following TBI appears to result primarily from direct radiation-induced damage to the epiphyses in the legs and spine 19,48 and is related to the total dose delivered and the fraction size. 49,50 Children

who receive TBI as a single exposure are more likely to develop growth failure than are those treated with fractionated schedules. 34,36–38,41,51,52 A variable but high incidence of growth hormone deficiency has been observed after TBI. 34,37,41,43,53 The poor correlation between results from growth hormone tests and subsequent growth suggests that other factors, such as radiation-induced skeletal dysplasia, poor nutrition, and hypothyroidism, 54 may contribute to impaired growth after transplantation. Intensive myeloablative chemotherapy, such as high-dose cyclophosphamide and busulfan without cranial radiation, can also result in growth retardation. 5 Other treatment complications including chronic graft-versus-host disease requiring corticosteroid therapy, and renal insufficiency may exacerbate the effects of growth hormone deficiency.

Growth hormone therapy Data regarding the effect of growth hormone treatment on growth and final height in children with acute leukemia are limited. Several studies of growth in children after bone marrow transplantation have indicated improvement in and normalization of growth velocity in most subjects given growth hormone therapy, although follow-up has been short 36,37,43,55 ; virtually all of the patients treated at St. Jude Children’s Research Hospital have shown an improvement in growth velocity (Fig. 30.2B). 55 For optimal therapeutic benefit, it is imperative that replacement growth hormone therapy should be started well before the patient matures sexually. The addition of a gonadotropin-releasing agonist in children with early puberty may also improve final height. 56 The use of growth hormone in childhood cancer survivors has raised concerns about the development or progression of acute leukemia. Several investigators have described the hormone’s in vitro mitogenic properties. 57,58 Earlier studies demonstrating an increased rate of leukemia in cohorts of growth hormone recipients were confounded by the presence of other risks factors for leukemia 59,60 ; more recent analyses do not support an excess risk of leukemia following growth hormone therapy. 61 To date, there is no evidence to support an increased risk of relapse in children with leukemia who receive growth hormone. 55,62–64 However, a recent report from the Childhood Cancer Survivor Study 63 demonstrated an excess risk of second neoplasms in survivors who received growth hormone replacement therapy that was primarily attributable to leukemia survivors who developed osteosarcoma. Due to the small number of events, the authors urge caution in the interpretation of their data.

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Fig. 30.3 (A) Obesity and short stature in a 22-year-old survivor who received her primary therapy with 24 Gy cranial radiation therapy for ALL at 2 years of age and an additional 24 Gy for CNS relapse 5 years later. (B) Growth grid demonstrating early puberty with rapid tempo, short stature, and morbid obesity after completion of growth.

Obesity after ALL therapy Obesity is a commonly reported treatment sequela in ALL survivors and has been observed in 35% to 57% of longterm survivors (Fig. 30.3). 7,8,65–72 Recent studies indicate a significant association between young age at diagnosis and an increased prevalence of obesity. 7,69 In some reports, obesity has been more frequent in female survivors, 66,69,73 but gender-associated differences in weight gain have not been apparent in other groups (Fig. 30.4). 67,68,72 Excessive weight gain is especially prevalent during the first year after cessation of therapy. 65,71,72 Reilly et al. 71 demonstrated that obesity in ALL patients was present as both an early and a late effect that developed even when cranial radiation

was not used as part of therapy. In their longitudinal study of body mass index (BMI) changes in ALL patients, they observed a seven-fold increase in obesity from diagnosis to 1 year after completion of therapy, as well as large increases in BMI standard deviation scores for the cohort during and after therapy. 71 Whereas many investigations have implicated cranial radiation-induced growth hormone deficiency as a causative factor, 8,65,66,69,72,73 this relationship has not been consistently observed in other cohort studies. 7,71 Some reports suggest that corticosteroid therapy, particularly with dexamethasone, may be an important factor in the development of obesity. 67,68 Treatmentinduced pituitary dysfunction resulting in growth hormone deficiency or leptin insensitivity has been proposed

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Fig. 30.4 Scatterplot of unadjusted body mass index (BMI) by age at diagnosis (in years) of ALL for females treated with ≥ 20 -Gy cranial radiotherapy. The odds ratio (OR) for obesity is greatest among females diagnosed at 0 to 4 years of age and treated with cranial radiation doses of ≥ 20 Gy (OR, 3.81; 95% CI, 2.34–5.99; P < 0.001). (Reprinted, with permission, from Oeffinger et al.69 )

as a potential pathophysiological mechanism predisposing to obesity in children with ALL. 74 This hypothesis is supported by studies demonstrating significantly reduced absolute lean body mass 74,75 and increased leptin concentrations in long-term survivors of childhood ALL compared with age- and sex-matched controls. 74 In these cases, growth hormone therapy may provide beneficial effects directly via its lipolytic properties 76 or indirectly through improved linear growth. Further studies are necessary to elucidate the mechanisms of obesity because of its potential for morbidity and mortality related to cardiovascular disease, insulin resistance, diabetes mellitus, and other health risks during adulthood.

Gonadal function Except for those given direct testicular radiation therapy, prophylactically or for relapsed disease, most boys treated for ALL progress through puberty. Early studies suggested that some Leydig cell function may be preserved, particu-

larly when radiation doses did not exceed 24 Gy. 77,78 The dose of 24 Gy given for leukemic relapse in the testes is associated with germ cell depletion and Leydig cell insufficiency. These patients may have some pubic hair (resulting from low levels of androgens secreted by adrenal gland), but they have very little penile development. Testosterone replacement therapy is required in these young men to assure normal pubertal progression. 79–81 Gonadal damage after chemotherapy for acute leukemia has been primarily associated with cyclophosphamide, used in intensive treatment regimens for high-risk patients. 25 Alkylating agent chemotherapy typically causes infertility by germ cell depletion, but Leydig cell function is preserved. Early impressions that the prepubertal testis may be less susceptible to damage by alkylating agent chemotherapy have not been confirmed by numerous studies of childhood cancer survivors, 82–87 which reported dose-related gonadal damage in males treated before, during, and after puberty. Standard regimens with antimetabolite chemotherapy are not known to impair testicular function. 88

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Leydig cell dysfunction is suggested by serum elevations of luteinizing hormone (LH), which regulates testosterone biosynthesis. Serum elevations of follicle-stimulating hormone (FSH), the mediator of spermatogenesis in the Sertoli cells, suggests germ cell depletion. Recent studies indicate that inhibin B, a regulatory protein produced in the Sertoli cells, appears to be valuable in assessing the integrity of the seminferous tubles and potential for spermatogenesis. 89,90 Information regarding pubertal development in female survivors of ALL has been conflicting and limited to results on small study populations. The currently available data indicate that girls tend to maintain ovarian function after conventional therapy for childhood leukemia. 91–93 In one of the largest cohort studies, which evaluated menarche in 188 long-term survivors of ALL, menarche was normally timed in more than 90% of subjects. 93 Girls treated with cranial radiation are at increased risk of premature puberty, which in combination with growth hormone deficiency, may result in a loss of final height. 17,24,25, 93 Precocious and early puberty may be more common in children irradiated before the age of 8 years. Elevated gonadotropin hormone levels suggestive of gonadal dysfunction have not correlated with pubertal development in studies of girls treated with cranial or craniospinal radiation; pubertal progress was early or normal in one study 25 but delayed and followed by ovarian failure in some cases in another. 94 Therefore, young girls often need to be monitored until they are 10 to 12 years of age to establish the status of ovarian function. However, women may be at increased risk for premature menopause after childhood leukemia if they received large doses of alkylating agent chemotherapy. 95 This observation correlates with early pathologic studies of childhood cancer patients that showed inhibited follicular development and reduced oocyte numbers. 96

Gonadal function after bone marrow transplantation The risk of gonadal dysfunction following transplant conditioning is related to the age at treatment, the cumulative dose of alkylator chemotherapy, the use of TBI, and history of previous alkylator therapy or cranial radiation. In general, the ovaries of prepubertal and adolescent girls are more resistant to alkylator-induced damage compared to adult ovaries because of their greater complement of follicles. 97 Ovarian function may remain normal after conditioning with high-dose cyclophosphamide before and after pubertal onset, 35,98 but premature ovarian failure may subsequently develop. 95 The risk of ovarian failure is very high after conditioning with cyclophosphamide and busulfan regardless of pubertal status. 46,99,100 Approximately 50% of girls transplanted before puberty

retain sufficient ovarian function to enter puberty and menstruate after TBI conditioning. 35,101 However, premature ovarian failure is universal in girls older than 10 years of age who received similar conditioning regimens. 98,101 Arrested or delayed puberty was associated with elevated levels of FSH and LH and low estradiol levels. Pubertal development in these girls is possible with hormone supplementation. In young men conditioned with high-dose cyclophosphamide, Leydig cell function and testosterone production is usually preserved. 46,99 However, germ cell damage is common in males treated with high-dose alkylators during or after puberty, typically manifesting as testicular atrophy and elevations of serum FSH. 35,102,103 After TBI conditioning, most males retain their ablity to produce testosterone regardless of their age at irradiation. Prepubertal boys usually have normal pubertal progression following TBI. 101 In older boys, elevations of serum LH suggests Leydig cell injury, but most have testosterone levels that are appropriate for age. Conversely, germ cell dysfunction develops in nearly all males treated with TBI. 104 Ovarian and testicular function has been reported to recover years after bone marrow transplantation in some children with hematologic malignancies. 104

Pregnancy outcomes Investigations of pregnancy outcomes are limited by the relatively brief period of follow-up of offspring born to childhood leukemia survivors. The concern about germ cell chromosomal mutagenesis from alkylating agent and radiation therapy has not been substantiated in several reports, 104–114 and offspring born to survivors of childhood cancer do not appear to be at increased risk of congenital malformations. 104–109,115 The exception to this is survivors of leukemia whose treatment included hematopoietic stem cell transplantation. Sanders et al. 104 observed an increased frequency of spontaneous abortion among women with aplastic anemia and hematologic malignancies whose treatment included hematopoietic stem cell transplantation and TBI. All women in their cohort treated with high-dose alkylators, with or without TBI, had an increased incidence of preterm labor and delivery and low-birth-weight babies. These complications were not observed in pregnancies among the partners of male transplant patients, whose offspring did not appear to have an excess of congenital malformations. Longer follow-up of larger groups is necessary to determine if these conclusions will hold true in childhood leukemia survivors treated with more intensive chemotherapeutic regimens.

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Neurologic sequelae Neurocognitive sequelae in children treated for ALL results primarily from the deleterious effects of CNS therapy. Numerous studies of children with acute leukemia conducted over the past 20 years have indicated that CNS treatment with radiation therapy and/or chemotherapy frequently results in neurocognitive deficits. 116–133 Factors predictive of increased risk of CNS injury are younger age at the time of CNS prophylaxis, 117,121,126,127,133–135 female gender, 116,123,132 higher radiation dose, 121,126 steroid treatment with dexamethasone, 116 and history of CNS relapse. 136,137 Contemporary treatment relies largely on intrathecal therapy for subclinical CNS disease and reserves cranial irradiation for only the subset of children at high risk of CNS relapse. 138,139 (A more extensive discussion of neurocognitive sequelae is presented in Chapter 35.) Pathologically, the late sequelae of radiation therapy are manifest by mineralizing microangiopathy and dystrophic calcification in the basal ganglia and periventricular regions. 140 Intrathecal and systemic chemotherapy can also cause some degree of brain atrophy and spinal cord dysfunction. 141,142 Leukoencephalopathy has been primarily attributed to injury of white matter from chronic methotrexate toxicity. Cranial radiation therapy may exacerbate leukoencephalopathy by compromising the blood–brain barrier and permitting increased concentrations of drug into the CNS. Numerous reports have described diagnostic imaging abnormalities in the CNS in children with ALL; the frequency of these changes ranges from 0% to 79%. 132,143–147 In several studies of longitudinal neuroimaging, CNS abnormalities were transient in some cases. 127,148 Younger age at diagnosis and higher doses of cranial radiation have been linked to higher frequencies of leukoencephalopathy 146 and intracerebral calcifications. 132 Many studies have shown poor correlation among diagnostic imaging abnormalities of the CNS, clinical symptoms, and neuropsychological deficiencies. 127,128,143 However, other reports have correlated morphologic abnormalities with specific neuropsychological deficits. 129,132,149 Iuvone et al. 132 correlated white matter abnormalities and intracerebral calcifications with poor visual integration skills in a cohort of ALL survivors who had maintained continuous remission for at least 4 years. Intracerebral calcifications were also correlated with the number of intrathecal methotrexate injections, low total intelligence quotient (IQ) and performance IQ scores, and significant impairment in attention. 132 Hertzberg et al. 129 observed similar relationships between leukoencephalopathy and performance and arithmetic IQ scores. While most investigations are limited by small study

populations, their findings support the need for periodic neuropsychological evaluation to ensure timely intervention for treatment-induced cognitive deficits. Until larger long-term studies correlating neuropsychological and academic evaluations become available, the clinical significance of any imaging changes should be estimated with caution. 120 Neurosensory treatment sequelae should be considered in childhood survivors of acute leukemia. Hearing loss related to high cumulative doses of aminoglycoside antibiotics can be significant enough to require amplification. 120,150,151 Audiologic screening should be considered in children with frequent admissions for fever and neutropenia who received prolonged antimicrobial treatment. Cataracts are a well-known complication of chronic steroid therapy and ionizing radiation in children with leukemia. 152–154 Posterior subcapsular cataracts were detected in 43 (52%) of 82 children 32 months after completing therapy for acute leukemia. 153 Although cataract formation was common, significant vision loss requiring lens extraction was not observed. Cataract formation is very common following TBI conditioning for hematopoietic stem cell transplantation; fractionation and low daily fractional dose administration significantly reduces the risk of this complication. 152,154 In a study of hematopoietic stem cell transplant recipients, 80% of patients given a single exposure of 10 Gy TBI a median of 6 years previously 154 developed posterior subcapsular cataracts, and nearly all required cataract repair. By contrast, among the patients who received fractionated TBI, 20% developed cataracts by 5 years, and only 20% of these have required lens extraction. Children with leukemia should have baseline ophthalmologic evaluation before institution of therapy and periodic follow-up evaluations after cranial radiation therapy and completion of therapy. Children with cataracts should have regular ophthalmologic evaluations to assess visual acuity.

Cardiopulmonary toxicity The restriction of cumulative anthracycline chemotherapy dosages to less than 550 mg/m2 in pediatric leukemia protocols has made congestive heart failure from acute cardiomyopathy a relatively rare occurrence during therapy. 155 However, several studies indicate an unanticipated high incidence of cardiac abnormalities in survivors of ALL that is characterized by left ventricular wall thinning and elevated cardiomyopathy. 156–158 Doxorubicininduced loss of cardiac myocytes has been implicated as the cause of the inadequate left ventricular mass that leads to subsequent clinically significant heart disease. In one

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study, up to one-half of patients who had received doxorubicin at a median cumulative dosage of 360 mg/m2 developed cardiomyopathy, manifested by increased left ventricular afterload or decreased contractility. 156 Children younger than 4 years at treatment, those receiving high cumulative doses of anthracyclines, and female patients are at increased risk of cardiac damage. 156,159 Using fractional shortening as the primary measure of left ventricular dysfunction, Kremer et al. 160 correlated the risk of subclinical cardiotoxicity with cumulative anthracycline dose reported in six methodologically sound studies of childhood cancer survivors. The frequency of subclinical cardiotoxicity was 15.5% to 27.8% for survivors treated with a cumulative dose of more than 300 mg/m2 compared with 0% to 15.2% in children receiving cumulative doses below 300 mg/m2 . Administration of anthracycline at doses above 45 mg/m2 within one week was associated with an increased frequency of clinical heart failure. 160 Recent investigation of ALL survivors demonstrate no or a very low frequency of echocardiographic abnormalities when doses were limited to 240 mg/m2 or lower. 161–164 Survivors with asymptomatic cardiac abnormalities may develop progressive left ventricular dysfunction manifest as late-onset congestive heart failure, symptomatic arrhythmias, or sudden death that occurs independently of congestive heart failure during therapy. 156,165–168 Other investigators have noted the reappearance of cardiac dysfunction in children who had shown at least partial resolution of cardiomyopathies developed during therapy. 158 Precipitating factors in cardiac decompensation after doxorubicin therapy are child birth, 165,169 viral infections, 170 isometric exercise, 158 and alcohol and cocaine ingestion. 158 Of concern, the prevalence and severity of abnormal cardiac growth and mechanics increased with prolonged follow-up. To reduce the cardiotoxicities of anthracyclines, efforts are being made to develop cardioprotective agents with improved therapeutic indices. 171 Contrary to previous assumptions, administration of anthracyclines by continuous infusion does not offer a cardioprotective advantage over bolus administration. 172,173 Studies of pulmonary function in childhood leukemia survivors are limited. A recent report indicated normal pulmonary capacity and subtle abnormalities of cardiac function in a small cohort of children who had completed therapy 1 to 7 years earlier. 174 Anthropometric assessments and body mass calculations revealed a significantly increased percentage of body fat and reduced exercise capacity in these survivors compared with age-matched healthy controls. The authors cautioned that continuing obesity and sedentary lifestyle might increase the risk of future heart disease. In another study, mild restrictive lung

disease and impaired exercise tolerance were observed in ALL survivors treated on United Kingdom ALL studies. 175 Factors predictive for restrictive lung disease were craniospinal radiation therapy, cyclophosphamide chemotherapy, and history of pneumonitis during treatment. Abnormalities of pulmonary diffusion in these patients correlated with anthracycline administration, craniospinal irradiation, and hematopoietic stem cell transplantation. Impaired diffusion and both restrictive and obstructive ventilatory defects have been observed in other studies of patients who had received hematopoietic stem cell transplantation. 175–178 Severe, persistent pulmonary defects have been reported in children who received TBI conditioning, but more recent investigations indicate a greater frequency of mild restrictive deficits than infectious or inflammatory pulmonary conditions. 177,179,180 Transplant recipients should be counseled to remain nonsmokers and have prompt evaluation for respiratory illnesses to reduce the risk of further pulmonary compromise.

Dental abnormalities The effects of cancer therapy on dental and craniofacial development have been reported in both the medical and dental literature. Developmental defects of tooth enamel and roots observed in ALL survivors include foreshortening and blunting of roots, incomplete calcification, premature closure of apices, tooth agenesis, arrested tooth development, microdontia, and enamel dysplasia (Fig. 30.5). 181–187 The severity of dental abnormalities is related to the age of the child at treatment and the use of cranial radiation therapy. Children treated before 5 years of age show the most severe dental defects, suggesting that immature teeth are more vulnerable to developmental disturbances than mature teeth. Radiation scatter from cranial radiation therapy results in disturbances of development of enamel and roots in the majority of ALL survivors studied, 182,184,186 and children younger than 5 years who received 24 Gy were most severely affected. Chemotherapy alone administered during the developmental stages of teeth increases the number of enamel defects. 181,182,184 Abnormalities of dentofacial development have also been observed after cranial radiation therapy and include deficient mandibular development not amenable to correction by conventional orthodontic therapy because of coexisting malformation of mandibular teeth roots. 183 Despite the prevalence of these abnormalities, one study suggests that ALL survivors have no greater risk of developing dental caries than does the normal population. 184 However, children treated with 24 Gy cranial radiation before 5 years of age appear to have

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Fig. 30.5 Panoramic radiograph of a child treated at a young age for ALL, demonstrating severe root stunting of all over-retained primary second molars, both primary maxillary canines, and the right mandibular primary canine, as well as the absence (hypodontia) of all permanent bicuspids.

an excess of periodontal disease compared to that in children given CNS preventive therapy by other methods.

Skeletal abnormalities after acute leukemia Cranial radiation therapy and high cumulative doses of steroids, methotrexate, and cyclophosphamide have all been associated with bony demineralization in pediatric leukemia survivors (Fig. 30.6). 188–197 Gilsanz et al. 188 observed significantly lower vertebral bone density in children after completion of ALL therapy (mean, 42 months) compared to that in nonleukemic control subjects. The decrease was entirely accounted for by the subset of patients who had undergone cranial radiation therapy and was unrelated to age at time of therapy or time after completion of therapy. These authors speculated that growth hormone levels affected bone density, as vertebral bone loss was more common in children with reduced skeletal growth. The adverse effect of cranial radiation on bone mineral density was subsequently reported in other studies of ALL survivors. 190,193,195,196 However, bone mineral deficits were not observed in patients treated with 18 Gy cranial radiation, who are more likely to maintain normal hypothalamic-pituitary function. 190 Several recent investigations noted more frequent and greater bone mineral decrements in male survivors, leading to the speculation of

subclinical hypogonadism in addition to growth hormone deficiency. 190,196 Lower bone mineral density z scores have also been observed in survivors treated with higher doses of antimetabolite therapy. 190 It is important to remember that osteopenia and osteoporosis may also occur in ALL survivors who do not receive cranial radiation; thus, treatment with corticosteroids and metabolites alone may be enough to predispose some survviors to bone demineralization. Therefore, any ALL survivor treated with corticosteroids, antimetabolites, or cranial radiation should be screened for bone mineral deficits so that appropriate interventions can be undertaken to reduce the risk of fractures and other complications related to osteopenia. Avascular necrosis (AVN), a well-recognized complication of corticosteroid therapy, has been observed with increasing frequency in recent trials of potent glucocorticoids (e.g. dexamethasone) at high cumulative doses. 198–202 Several centers describe patients with multifocal presentations during therapy that have significance for long-term follow-up because of the potential necessity of joint replacement. 198,199,202 In the largest study to date evaluating risk factors for AVN, the 3-year cumulative incidence of AVN was 9.3% ± 0.9% in 1409 children with ALL treated with intensive chemotherapy including multiple prolonged courses of corticosteroids. 203 The incidence was significantly higher for older children (≥10 years: 14.2% ± 1.3% versus <10 years: 0.9% ± 0.4%; P < 0.0001). 203 Other

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Fig. 30.6 Distribution of bone mineral density (BMD) standardized z scores for childhood leukemia patients. The number on each bar indicates the percentage of studied cases. The median BMD z score of the study population (0.78 SD below mean) lies between 1 and 2 SD below the expected median, which is significantly below that of the normal population (P <0.0001). Among the 13% with BMD z scores more than 2 SD below the mean, 9% were in the osteopenic range and 4% were in the osteoporotic range. (Reprinted, with permission, from Kaste et al.190 )

factors predicting a higher risk of AVN included female sex (age range, 10–20 years), white race, and more prolonged treatment with dexamethasone. 203 Dexamethasone use in antileukemia regimens, compared to prednisone, has also been associated with a higher risk of fractures. 204 After leukemic relapse has been excluded, avascular necrosis should be considered in leukemia survivors who present with persistent bone and joint pain. Magnetic resonance imaging is the diagnostic imaging modality of choice (Fig. 30.7), as plain radiographs may not show abnormalities during early phases of the disease.

Other organ toxicity Several additional toxicities are associated with antineoplastic agents commonly used in leukemia protocols. Therapy with cyclophosphamide only has been associated with a 10% incidence of hemorrhagic cystitis; several cases have occurred decades after chemotherapy. 205,206 Urinary bladder telangiectasia, fibrosis, and carcinoma are potential sequelae that need to be considered in long-term survivors who experience persistent hematuria and dysfunctional voiding after cyclophosphamide therapy. Elevations of liver function studies indicative of hepatocellular damage occur in many children, especially

during antimetabolite therapy with methotrexate and 6mercaptopurine, but most of these changes normalize during follow-up after completion of therapy. 207,208 Fibrosis, cirrhotic changes, and hepatoma were previously reported as complications of prolonged use of these agents, 209–215 but many of these cases were undoubtedly due to hepatitis C infection, which was not recognized until recently. Parenterally acquired hepatitis C infection should be considered in any patient with persistently elevated alanine aminotransferase levels and in any survivor of childhood leukemia who was transfused before 1990 (when blood product screening was implemented). Children who are negative according to the results of first-generation enzyme-linked immunosorbent assays (ELISA) should be retested with the more sensitive second-generation (available since 1992) or soon-to-be available third-generation ELISA tests. Confirmatory detection of hepatitis C RNA by polymerase chain reaction (PCR) testing indicates current infection. Young children who are seropositive for hepatitis C have shown incomplete serologic expression and fluctuating low levels of viremia that may complicate diagnosis. 216 Seropositive patients may have elevated alanine aminotransferase levels during treatment that normalize after therapy despite persistent viremia. 217 Because the degree of hepatocellular injury in hepatitis C seropositive children best correlates with viremia as detected by

Late complications after leukemia therapy

Fig. 30.7 (A) AP Plain radiograph of the right hip of a 17-year-old ALL survivor showing no convincing bony abnormalities or evidence of avascular necrosis. (B) Sagittal T1 -weighted magnetic resonance image, without contrast, of the same patient showing extensive bone marrow abnormalities consistent with bone marrow infarcts of the right iliac bone and ischium, as well as early avascular necrosis of the femoral head.

RT-PCR and not with serum alanine aminotransferase levels, 218 one should not dismiss the likelihood of hepatitis C infection in leukemia survivors with seemingly normal liver function studies. However, persistent elevations of serum alanine aminotransferase have a high degree of correlation of chronic liver injury, as indicated in one study of children with leukemia in first remission with chronic liver disease after cessation of chemotherapy who were hepatitis C seropositive. 219 The natural history of chronic hepatitis C infection in childhood leukemia survivors and the consequences of hepatotoxic chemotherapy to the risk and severity of chronic liver disease have been the focus of several investigations. 220–228 The reported prevalence of circulating viral RNA ranges from 6.6% to 49%. Earlier studies of chronically infected survivors suggested a slower progression of hepatic fibrosis. However, a recent report describing outcomes of a cohort with more prolonged follow-up (median, 19 years) demonstrated rates of fibrosis and end-stage liver disease that were similar to

those observed in larger adult cohorts with transfusionassociated liver disease. 228 A few investigations provide information about childhood cancer survivors’ response and tolerance to antiviral therapy, which appears to be comparable to that of chronically infected adults. 228–230 Reports of decompensated cirrhosis and hepatocellular carcinoma in childhood cancer survivors with chronic hepatitis C infection indicate that this population is at increased risk of liver-related morbidity and mortality. 220,228

Immunodeficiency in leukemia survivors Short-term abnormalities of immune function have been observed in children after completion of ALL therapy. 231–233 Defects in humoral and cellular immunity in disease-free children generally resolve in 6 months to 1 year. 234–244 However, some studies of long-term ALL survivors treated with intensive contemporary therapy have described isolated

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chemotherapy on memory B cells or plasma cell quantity or function. Their data suggest that reimmunization may be indicated for some high-risk ALL survivors following completion of contemporary intensive treatment regimens, although a revaccination policy has not been proposed. These findings notwithstanding, we recommend the discontinuation of trimethoprim-sulfamethoxazole prophylaxis for Pneumocystis carinii pneumonia 2 months after completion of therapy (after 6 months in patients who had received very intensive chemotherapy, as indicated for B-cell ALL) and the resumption of immunization after 6 months. Clinicians should also consider evaluating for antibodies against common vaccination antigens in intensively treated children to determine the need for revaccination. Progressive loss of protective antibody titers to viral and bacterial pathogens is well-established in recipients of autologous 248 or allogeneic bone marrow transplants, for whom routine reimmunization is recommended. 244,249–252

Second malignancies

Fig. 30.8 Cumulative incidence of second neoplasms (A) or any adverse event (B) among ≥10-year event-free survivors of childhood ALL according to whether they received cranial or craniospinal irradiation. The data are means and SEs. (Reprinted, with permission, from Pui et al.261 )

immune defects that persist for as long as 5 years after treatment cessation. 241,243,245 In several investigations, young age was correlated with more profound deficits in immune function. 243,246 Smith et al. 247 detected no protective antibodies to one or more vaccines previously administered for common childhood bacterial or viral diseases in children who were 2 to 5 years following completion ¨ of Berlin-Frankfurt-Munster therapy. Similarly, Nilsson et al. 243 observed that a high proportion of children treated intensively for ALL lost antibodies against common vaccination antigens. In 43 patients studied at a median of 5 years in continuous first remission, immune titers against measles and rubella were detected in only 26 (60%) and 31 (72%), respectively. 243 Despite revaccination, a small number of these patients failed to achieve protective levels of virus-specific antibioties. The authors speculate that this phenomenon may be related to the detrimental effects of

In general, reports of childhood cancer survivors indicate a lower frequency of second malignant neoplasms after acute leukemia than after treatment for solid tumors. 253–260 Investigators participating in the Childhood Cancer Survivors Study estimated a 20-year cumulative risk of 2.05% in the 4581 long-term survivors of leukemia registered in the multi-institutional retrospective cohort study. By standardized incidence ratios, this reflected an almost fourfold (95% CI, 1.3–7.6) and eightfold (95% CI, 3.6–15) excess risk of cancer for survivors of ALL and AML, respectively, compared with expected results for a comparable ageand sex-matched population. 257 CNS tumors and thyroid cancer were the most frequently observed histologies in survivors of acute leukemia; a younger age at diagnosis was a risk factor for both of these second malignancies. Several other large cooperative group studies revealed a comparably low incidence of second neoplasms in ALL survivors and similar distributions of second cancer histology. Neglia et al. 256 identified 43 second cancers in a retrospective review of 9720 children treated on Children’s Cancer Study Group (CCG) protocols from 1970 to 1988. Second cancers most commonly comprised CNS tumors (n = 24) and hematopoietic malignancies (n = 10), followed by other solid tumors, including carcinomas of the thyroid and parotid gland. These authors observed a sevenfold excess of all cancers compared with the incidence in the age-adjusted general population, and the estimated cumulative risk of developing a second cancer was 2.5% by 15 years after diagnosis. The higher risk of second cancers

Late complications after leukemia therapy

Fig. 30.10 Axial T2 -weighted magnetic resonance image demonstrating an astrocytoma with vasogenic edema in the left frontal lobe of a 10-year-old survivor of ALL who received 24 Gy to the cranium for prevention of CNS leukemia at 3 years of age.

Fig. 30.9 Axial contrast-enhanced T1 -weighted magnetic resonance image demonstrating an intensely enhancing occipital en plaque meningioma in a 31-year-old survivor of ALL who received 24 Gy to the cranium for CNS leukemia at 8 years of age.

was further increased in children 5 years of age or younger at the diagnosis of ALL. No association between cyclophosphamide or anthracycline chemotherapy and the risk of second malignancy could be determined. In a follow-up study of 8831 ALL survivors enrolled on CCG protocols between 1983 and 1995, Bhatia et al. 258 reported a 10-year cumulative incidence of 1.18% (95% CI, 0.8–1.5%) of any second cancer following treatment for ALL, which represented a sevenfold increased risk compared with that in the general population. Excess risks were observed for AML (standardized incidence ratio, or SIR, 8.3), non-Hodgkin lymphoma (SIR, 33.4), thyroid cancer (SIR, 13.3), brain tumors (SIR 10.1) and soft-tissue sarcoma (SIR, 9.1). Female sex (relative risk, RR, 1.8), craniospinal radiation (RR, 1.6), and relapse (RR, 3.5) were independently associated with an increased risk of developing a second cancer. The frequency and distribution of second cancers may change with more prolonged follow-up of adult survivors of child-

hood leukemia. This prediction was suggested by a recent study of ALL survivors in continuous remission 10 or more years from diagnosis. For survivors in the cohort treated with cranial or craniospinal radiation, the 10-year cumulative incidence of second malignancy was 5.8% ± 1.1%, but rose to 23.0% ± 3.8% by 20 years (Fig. 30.8). 261 Notably, a greater proportion of the excess neoplasms developing after 10 years from diagnosis were either benign tumors or low-grade malignancies, such as meningioma, basal cell carcinoma, thyroid cancer and parotid gland carcinoma. Tumors of the CNS are the most common second solid tumor in survivors of ALL (Figs. 30.9 and 30.10). 256–258,262,263 The most common histologies are high-grade gliomas, including glioblastomas and malignant astrocytomas, followed by peripheral neuroectodermal tumors, ependymomas, and meningiomas. 256–258 High-grade glioma confers a dismal prognosis, whereas meningiomas are highly curable. Hypotheses regarding the etiology of brain tumors include an inherent predisposition to cancer in children with ALL, 264 CNS therapy, or an interaction of these factors. The most commonly reported risk factors for brain tumor development include young age at diagnosis (especially 5 years or younger) 256,257 and cranial irradiation. 258,261 Most secondary CNS tumors occur

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after radiation therapy, with a range in median latency from 7 to 11 years. 256,258,263,265–274 However, St. Jude investigators observed an unusually high incidence of brain tumors with a briefer latency in onset (6 years) in ALL survivors with genetic defects in thiopurine metabolism who received cranial radiation and intensive systemic antimetabolite therapy. 274 The 8-year cumulative incidence of brain tumors among children with a defective versus a wild-type thiopurine methlytransferase phenotype was 42.9% (SE 20.6) versus 8.3% (SE 4.7; P = 0.0077). Thus, underlying genetic factors in concert with treatment variables may contribute to an excess risk of second malignancy in childhood leukemia survivors. The risk of secondary AML after ALL has been negligible in most treatment regimens, 256–260,275 with the exception of those incorporating epipodophyllotoxins. 276–280 In contrast to alkylating agent-induced AML, cases of AML after epipodophyllotoxin chemotherapy are characterized by a relatively brief latency period (1 to 6 years; median, 33 months), the absence of a myelodysplastic phase, monoblastic or myelomonoblastic morphology, and chromosomal 11q23 rearrangements that involve the MLL gene. 276,278,280–283 Frequent administration of an epipodophyllotoxin (i.e. weekly or twice weekly) at a relatively high dose, coadministration with certain agents (e.g. alkylating agents, L-asparaginase), and, to a lesser extent, a high cumulative dosage are associated with an increased risk of secondary AML; the short-term use of G-CSF may also increase the risk of this complication. 279,283,284 Although complete remissions can be readily induced in patients with epipodophyllotoxin-induced AML, longterm outcome is dismal, even with hematopoietic stem cell transplantation. 285 Therefore, this class of agents should be reserved for patients with high-risk leukemia and used judiciously.

Summary The development of curative therapy for childhood leukemias over the past three decades has resulted in the recognition of late treatment complications that can affect the patient’s quality of life and increase the risk of early mortality. Cancer and its treatment can produce chronic physical, psychologic, or neurocognitive sequelae that may adversely affect the health and socioeconomic status of the long-term childhood cancer survivor. 261,286 As late effects are treatment-specific, they can be anticipated and identified by careful monitoring of survivors after completion of therapy. Long-term monitoring is especially important because sequelae may not become apparent until

many years after therapy. Knowledge of potential treatment sequelae and appropriate screening measures that permit their early detection have the potential to greatly improve the health-related quality of life of childhood leukemia survivors. Furthermore, counseling about the contribution of health practices that exacerbate the risk of treatment complications can further reduce morbidity and mortality in childhood leukemia survivors.

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244 Ljungman, P., Lewensohn-Fuchs, I., Hammarstrom, V., et al. Long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation. Blood, 1994; 84: 657– 63. 245 Feldman, S., Gigliotti, F., Bockhold, C., & Naegele, R. Measles and rubella antibody status in previously vaccinated children with cancer. Med Pediatr Oncol, 1988; 16: 308–11. 246 Caver, T. E., Slobod, K. S., Flynn, P. M., et al. Profound abnormality of the B/T lymphocyte ratio during chemotherapy for pediatric acute lymphoblastic leukemia. Leukemia, 1998; 12: 619–22. 247 Smith, S., Schiffman, G., Karayalcin, G., & Bonagura, V. Immunodeficiency in long-term survivors of acute lymphoblastic leukemia treated with Berlin-Frankfurt-Munster therapy. J Pediatr, 1995; 127: 68–75. 248 Pauksen, K., Duraj, V., Ljungman, P., et al. Immunity to and immunization against measles, rubella and mumps in patients after autologous bone marrow transplantation. Bone Marrow Transplant, 1992; 9: 427–32. 249 Engelhard, D., Handsher, R., Naparstek, E., et al. Immune response to polio vaccination in bone marrow transplant recipients. Bone Marrow Transplant, 1991; 8: 295–300. 250 Ljungman, P., Wiklund-Hammarsten, M., Duraj, V., et al. Response to tetanus toxoid immunization after allogeneic bone marrow transplantation. J Infect Dis, 1990; 162: 496–500. 251 Ljungman, P., Fridell, E., Lonnqvist, B., et al. Efficacy and safety of vaccination of marrow transplant recipients with a live attenuated measles, mumps, and rubella vaccine. J Infect Dis, 1989; 159: 610–5. 252 Ljungman, P., Cordonnier, C., de Bock, R., et al. Immunisations after bone marrow transplantation: results of a European survey and recommendations from the infectious diseases working party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant, 1995; 15: 455–60. 253 Meadows, A. T., Baum, E., Fossati-Bellani, F., et al. Second malignant neoplasms in children: an update from the Late Effects Study Group. J Clin Oncol, 1985; 3: 532–8. 254 Zarrabi, M. H., Rosner, F., & Grunwald, H. W. Second neoplasms in acute lymphoblastic leukemia. Cancer, 1983; 52: 1712–19. 255 Nygaard, R., Garwicz, S., Haldorsen, T., et al. Second malignant neoplasms in patients treated for childhood leukemia. A population-based cohort study from the Nordic countries. The Nordic Society of Pediatric Oncology and Hematology (NOPHO). Acta Paediatr Scand, 1991; 80: 1220–8. 256 Neglia, J. P., Meadows, A. T., Robison, L. L., et al. Second neoplasms after acute lymphoblastic leukemia in childhood. N Engl J Med, 1991; 325: 1330–6. 257 Neglia, J. P., Friedman, D. L., Yasui, Y., et al. Second malignant neoplasms in five-year survivors of childhood cancer: childhood cancer survivor study. J Natl Cancer Inst, 2001; 93: 618– 29. 258 Bhatia, S., Sather, H. N., Pabustan, O. B., et al. Low incidence of second neoplasms among children diagnosed with

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acute lymphoblastic leukemia after 1983. Blood, 2002; 99: 4257–64. Kimball Dalton, V. M., Gelber, R. D., Li, F., et al. Second malignancies in patients treated for childhood acute lymphoblastic leukemia. J Clin Oncol, 1998; 16: 2848–53. Loning, L., Zimmermann, M., Reiter, A., et al. Secondary neoplasms subsequent to Berlin-Frankfurt-Munster therapy of acute lymphoblastic leukemia in childhood: significantly lower risk without cranial radiotherapy. Blood, 2000; 95: 2770– 5. Pui, C. H., Cheng, C., Leung, W., et al. Extended followup of long-term survivors of childhood acute lymphoblastic leukemia. N Engl J Med, 2003; 349: 640–9. Hawkins, M. M., Draper, G. J., & Kingston, J. E. Incidence of second primary tumours among childhood cancer survivors. Br J Cancer, 1987; 56: 339–47. Iyer, R. S., Soman, C. S., Nair, C. N., et al. Brain tumors following cure of acute lymphoblastic leukemia. Leuk Lymphoma, 1994; 13: 183–6. Farwell, J. & Flannery, J. T. Cancer in relatives of children with central-nervous-system neoplasms. N Engl J Med, 1984; 311: 749–53. Walter, A. W., Hancock, M. L., Pui, C. H., et al. Secondary brain tumors in children treated for acute lymphoblastic leukemia at St Jude Children’s Research Hospital. J Clin Oncol, 1998; 16: 3761–7. Cavin, L. W., Dalrymple, G. V., McGuire, E. L., Maners, A. W., & Broadwater, J. R. CNS tumor induction by radiotherapy: a report of four new cases and estimate of dose required. Int J Radiat Oncol Biol Phys, 1990; 18: 399–406. Rimm, I. J., Li, F. C., Tarbell, N. J., Winston, K. R., & Sallan, S. E. Brain tumors after cranial irradiation for childhood acute lymphoblastic leukemia. A 13-year experience from the DanaFarber Cancer Institute and the Children’s Hospital. Cancer, 1987; 59: 1506–8. Fontana, M., Stanton, C., Pompili, A., et al. Late multifocal gliomas in adolescents previously treated for acute lymphoblastic leukemia. Cancer, 1987; 60: 1510–8. McWhirter, W. R., Pearn, J. H., Smith, H., & O’Regan, P. Cerebral astrocytoma as a complication of acute lymphoblastic leukaemia. Med J Aust, 1986; 145: 96–7. Malone, M., Lumley, H., & Erdohazi, M. Astrocytoma as a second malignancy in patients with acute lymphoblastic leukemia. Cancer, 1986; 57: 1979–85. Judge, M. R., Eden, O. B., & O’Neill, P. Cerebral glioma after cranial prophylaxis for acute lymphoblastic leukaemia. Br Med J, 1984; 289: 1038–9. Salvati, M., Artico, M., Caruso, R., et al. A report on radiationinduced gliomas. Cancer, 1991; 67: 392–7.

273 Tiberin, P., Maor, E., Zaizov, R., et al. Brain sarcoma of meningeal origin after cranial irradiation in childhood acute lymphocytic leukemia. Case report. J Neurosurg, 1984; 61: 772– 6. 274 Relling, M. V., Rubnitz, J. E., Rivera, G. K., et al. High incidence of secondary brain tumours after radiotherapy and antimetabolites. Lancet, 1999; 354: 34–9. 275 Kreissman, S. G., Gelber, R. D., Cohen, H. J., et al. Incidence of secondary acute myelogenous leukemia after treatment of childhood acute lymphoblastic leukemia. Cancer, 1992; 70: 2208–13. 276 Pui, C. H., Ribeiro, R. C., Hancock, M. L., et al. Acute myeloid leukemia in children treated with epipodophyllotoxins for acute lymphoblastic leukemia. N Engl J Med, 1991; 325: 1682– 7. 277 Pui, C. H., Relling, M. V., Rivera, G. K., et al. Epipodophyllotoxinrelated acute myeloid leukemia: a study of 35 cases. Leukemia, 1995; 9: 1990–6. 278 Pui, C. H., Behm, F. G., Raimondi, S. C., et al. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med, 1989; 321: 136–42. 279 Pui, C. H., Relling, M. V., Behm, F. G., et al. L-asparaginase may potentiate the leukemogenic effect of the epipodophyllotoxins. Leukemia, 1995; 9: 1680–4. 280 Winick, N., Buchanan, G. R., & Kamen, B. A. Secondary acute myeloid leukemia in Hispanic children. J Clin Oncol, 1993; 11: 1433. 281 Albain, K. S., Le Beau, M. M., Ullirsch, R., & Schumacher, H. Implication of prior treatment with drug combinations including inhibitors of topoisomerase II in therapy-related monocytic leukemia with a 9;11 translocation. Genes Chromosomes Cancer, 1990; 2: 53–8. 282 Whitlock, J. A., Greer, J. P., & Lukens, J. N. Epipodophyllotoxinrelated leukemia. Identification of a new subset of secondary leukemia. Cancer, 1991; 68: 600–4. 283 Smith, M. A., Rubinstein, L., Anderson, J. R., et al. Secondary leukemia or myelodysplastic syndrome after treatment with epipodophyllotoxins. J Clin Oncol, 1999; 17: 569–77. 284 Relling, M. V., Boyett, J. M., Blanco, J. G., et al. Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment. Blood, 2003; 101: 3862– 7. 285 Sandler, E. S., Friedman, D. J., Mustafa, M. M., et al. Treatment of children with epipodophyllotoxin-induced secondary acute myeloid leukemia. Cancer, 1997; 79: 1049–54. 286 Hudson, M. M., Mertens, A. C., Yasui, Y., et al. Health status of adult long-term survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. JAMA, 2003; 290: 1583– 92.

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31 Therapy-related leukemias Carolyn A. Felix

Introduction The estimated frequency of second cancers in longterm survivors of primary cancers is about 7%.1 Although leukemias comprise only a small fraction of all second cancers, they are the major second cancers resulting from chemotherapy. The two broad classes of cytotoxic drugs associated with leukemia are alkylating agents and DNA topoisomerase II inhibitors.2–4 This chapter will focus on the features that distinguish alkylating agent-related leukemias and DNA topoisomerase II inhibitor-related leukemias as the two major forms of leukemia attributed to chemotherapy. Chemotherapy has been implicated in acute myelogenous leukemia (AML) of virtually all morphologic subtypes, myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (CML) (reviewed in Felix4 ). By contrast, therapeutic radiation primarily induces solid tumors,5–11 but also has been linked to an increased risk of leukemia.12,13 There are many more reports of chemotherapy-related leukemias in adults than in children14,15 ; however, leukemia has become an increasingly important complication of cytotoxic chemotherapy in children because they are more likely than adults to survive their primary cancers.2,16

Epidemiology of treatment-related leukemias Alkylating agent-related leukemias: incidence and risk A minority of patients (40% in one large series) who develop therapy-related leukemia have received chemotherapy alone,15 and there is often overlap between alkylating agents and DNA topoisomerase II inhibitors in the

chemotherapy regimen.15 The first observations of alkylating agent-related leukemia and MDS were reported in about 1970.17 Therapy-related MDS and AML can occur after administration of virtually all of the many different alkylating agents.15,18 Depending on the antileukemic agents, the regimens and the primary diseases, the cumulative risks of AML after alkylating agent treatment in adults followed for 4 to 11 years have ranged from 2% to more than 20%.19 Dose-intensive pediatric sarcoma regimens have been associated with a similar high incidence,20 although increasing age at the start of treatment is a major determinant of risk.21 The peak incidence of alkylating agentrelated leukemia after Hodgkin disease occurs at 6 years from the first exposure; however, the leukemia can occur from 2 to 12 years after the first treatment, after which there is a plateau in the cumulative probability of this complication.21 Cumulative dose is a primary determinant of risk for alkylating agent-related leukemia but the different alkylating agents are not equally leukemogenic.22 Therefore, Meadows et al.22 developed a predictive alkylating agent score that also incorporates the number of different alkylating agents administered and the duration of the treatment. The risk of leukemia in pediatric patients with primary Hodgkin disease increases with the alkylating agent score.23 The risk of secondary leukemia after childhood Hodgkin disease was greater with the MOPP regimen containing mechlorethamine than with regimens containing cyclophosphamide.24 Melphalan is a more potent leukemogen than cyclophosphamide25 and was 10 times more leukemogenic than cyclophosphamide (relative risk, 31.4 versus 3.1) in a study of breast cancer treatment in adults.26 Combining alkylating agents with radiation therapy increases the risk of leukemia

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 31.1 Alkylating agents used in anticancer treatment28–30 Bifunctional Nitrogen mustards Mechlorethamine Mechlorethamine analogs Melphalan Chlorambucil Cyclophosphamide Ifosfamide Azridines Thiotepa Alkyl alkane sulfonates Busulfan Nitrosoureas BCNU (carmustine) Nonclassic Procarbazine Dacarbazine Temazolamide Hexamethylmelamine Platinum analogs Cisplatin Carboplatin

over radiation therapy alone.21,26 In adults with primary breast cancer, moreover, adjuvant radiotherapy plus chemotherapy with 5-fluorouracil, doxorubicin and cyclophosphamide was associated with a higher risk of leukemia than the same chemotherapy alone (2.5% versus 0.5%), and the leukemias had typical features of alkylator agent-related MDS and AML.27 Alkylating agents form covalent bonds between alkyl groups (i.e. saturated carbon atoms) in the drugs and cellular biomolecules.28 Classic alkylating agents, nonclassic alkylating agents and platinum analogs are the types of alkylating agents used in anticancer treatment (Table 31.1).28–30 The oxygen and nitrogen atoms of the purines and pyrimidines in DNA are targets for alkylation. The classic bifunctional alkylating agents are nitrogen mustard and its analogs,28,31 which form covalent bonds with bases in the DNA and can cause interstrand crosslinks. This formation of interstrand DNA crosslinks defines the classic bifunctional alkylators and is the mechanism whereby they are cytotoxic.28 The resultant O6 -methylguanine residues also are carcinogenic.28,31 In addition, the N-7 position of guanine is a preferred target for alkylation, and various alkylating agents can bridge two guanine molecules at their N-7 positions.28 Nonclassic alkylating agents lack the feature of bifunctionality, but they contain N-methyl groups and can form

covalent bonds with DNA upon activation.29 Examples of nonclassic alkylating agents are procarbazine, dacarbazine, temazolamide and hexamethylmelamine.29 The transition metal platinum atom of the platinum analogs cisplatin and carboplatin forms strong covalent bonds with DNA.30 Cisplatin forms intrastrand N7 -alkyl adducts on adjacent deoxyguanosines or deoxyguanosine and deoxyadenosine.30–32 Monoadducts and interstrand crosslinks also can be formed but are less frequent types of DNA lesions with these agents.30–32 Although cisplatin has been in clinical use since 1969, it was not until more than two decades later that the platinum analogs cisplatin and carboplatin were linked with significantly increased, dose-dependent risks of leukemia. In children, there are reports of leukemias developing after chemotherapy that included platinum analogs and whose features of monosomies of chromosomes 5 and 7 were characteristic of alkylating agent-related leukemias.33–35 Platinum analogs are typically used with other leukemogenic drugs; in fact, it has been suggested that cisplatin is leukemogenic when administered with the DNA topoisomerase II inhibitors doxorubicin or etoposide.32,36 Cases of AML with translocations involving chromosome band 11q23 or 21q21, which are recurrent translocations in DNA topoisomerase II inhibitor-related leukemias, were reported after treatment of germ-cell tumors with cisplatin and etoposide.37–39 AML with the t(8;21) translocation was observed after osteosarcoma treatment with cisplatin and the DNA topoisomerase II inhibitor doxorubicin.36 However, most data on the platinum analog-leukemia association are from adults with primary gonadal tumors.40,41 There was a four-fold increased risk of leukemia in a casecontrol study of women with ovarian cancer who received cisplatin or carboplatin,40 and platinum-based testicular cancer treatment has been associated with a significant, dose-dependent increased leukemia risk.41 In patients with Hodgkin disease, splenectomy has been associated with an increased risk of alkylating agent-related AML.42,43 Autologous stem cell transplantation is associated with a significant risk of treatment-related AML, which increases with increasing age, cumulative alkylating agent dose, duration of prior chemotherapy, prior radiation therapy, use of radiation therapy in the conditioning regimen for autologous stem cell transplantation, and repeated transplantations.44–48 Most often the posttransplant leukemias are characterized by complex numerical and structural karyotypic abnormalities, including loss of chromosomes 5 and 7, the hallmark features of alkylating agent-related AML.47,49,50 The risk is higher after autologous transplantation of peripheral blood stem cells

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Table 31.2 Genetic predisposition to chemotherapy-related leukemia Alkylating agent-related Germline mutations in tumor suppressor genes Genetic variation in genes encoding DNA repair proteins Genetic variation in xenobiotic metabolizing enzymes

NF-1 TP53 XRCC1 hMSH2 CYP3A4 GSTT1 GSTP1 NQO1

Other

DNA topoisomerase II inhibitor-related

Unspecified secondary leukemia

CYP3A4 TPMT

CYP2D CYP2C19 CYP2D6 GSTM1

Trinucleotide repeat polymorphism in MLL

harvested after chemotherapy and cytokines than after bone marrow transplantation without priming.44,46,47 Leukemias developing after autologous stem cell transplantation generally occur early, often within 12 months of transplant, suggesting that the prior chemotherapy and events preceding transplantation are the primary determinants of risk.47 That the same cytogenetic abnormalities were detectable by FISH before conditioning with high-dose chemotherapy in 9 of 12 cases of posttransplantation MDS indicates that at least the initial damage in these cases resulted from the prior conventional chemotherapy and was unrelated to the preparative highdose chemotherapy used for transplantation.51 The question of whether post-transplant AML/MDS arises from the infused autograft or from residual disease in the patient has also been raised.46 The chromosomal aberrations in the post-transplant AML/MDS have not been detectable by FISH in the peripheral blood-derived autografts used for transplantation.52 Alternatively, the preparative high-dose chemotherapy and total-body irradiation used for transplantation may contribute to the risk.47

Alkylating agent-related leukemias: genetic predisposition Individuals with germline mutations in certain tumor suppressor genes are genetically predisposed to the form of leukemia that follows alkylating agent treatment. For example, germline mutations in the NF-1 tumor suppressor gene, which result in deregulation of the Ras signal transduction pathway, are associated with an increased risk of treatment-related monosomy 7-positive MDS after alkylating agent treatment (Table 31.2).53–56 Cyclophosphamide exposure leads to the development of myeloid leukemia in heterozygous Nf1 knockout mice, indicating that loss of the NF-1 gene product is a genetic predispos-

ing trait for this form of leukemia in a relevant animal model.57 Germline mutations in the conserved region of the TP53 tumor suppressor gene, the gene product of which is a central player in the cellular DNA damage response pathway, have been associated with leukemias following alkylating agent treatment,34,35,58 also indicating that certain individuals are genetically predisposed. Germline TP53 mutations may occur without a positive family history of the Li-Fraumeni syndrome58 and are detected in 23% of young children with rhabdomyosarcoma who lack a cancer family history,59 suggesting that this may be a highrisk population. Such mutations have also been detected in young children with treatment-related MDS and AML with chromosome 5 and 7 deletions after multimodality treatment for rhabdomyosarcoma.58,60 Germline TP53 mutations are present in about 3% of children with osteosarcoma without first-degree relatives with cancer.34,61 The occurrence of secondary AML with abnormalities of chromosomes 5, 7 and 17 suggests that patients with osteosarcoma with germline TP53 mutations also are at increased risk of alkylator-induced AML.34,35 The complex numerical and structural karyotypic abnormalities in alkylating agent-related leukemias associated with germline TP53 mutations, typically including loss of chromosomes 5, 7 and 17p, are consistent with the genomic instability that accompanies loss of wild-type TP53.34,35,58 The leukemias may also be characterized by segmental jumping translocations, in which multiple, intact, amplified copies of various oncogenes such as ABL or MLL are dispersed throughout the genome and extrachromosomally.60,62,63 Germline TP53 mutations appear to contribute to this form of genomic instability when DNA-damaging agents are administered.60,64,65 The recognition and processing of alkylating agentinduced DNA damage involves several DNA repair

Therapy-related leukemias

proteins, whose genetic variants can modulate the risk. The XRCC1 gene product is involved in base-excision repair and repair of single-strand breaks via interactions with other proteins in the cellular DNA repair machinery, including DNA polymerase , DNA ligase III and PARP.66 The XRCC1 gene contains an exon 10 polymorphism at codon 399 that results in an Arg-to-Gln amino acid substitution in the conserved PARP-binding domain of the protein.66 The proportion of patients homozygous for the wild-type XRCC1 genotype at codon 399 was higher in adults with treatmentrelated AML than in a control population without cancer, suggesting that the reduced DNA repair capacity associated with the variant allele may be protective against leukemogenesis66 ; however, the chemotherapy exposures were not specified.66 Another DNA repair protein variant that modulates the risk of alkylating agent-related leukemia was recently described. The observation of microsatellite instability in a large proportion of adult cases of treatmentrelated AML and MDS (94%)67 prompted investigation of an association between the intron splice acceptor Tto-C polymorphism at position 6 relative to exon 13 in the hMSH2 mismatch repair gene and leukemia following alkylating agent treatment.68 The polymorphic hMSH2 allele was significantly over-represented in cases of AML following exposures to agents that alkylate at O6 -guanine (cyclophosphamide, procarbazine, dacarbazine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) and was associated with microsatellite instability, possibly suggesting that the polymorphism predisposes to alkylating agent-related AML by inducing mismatch repair mutations.68 Genetic variations in enzymes involved in the metabolism of xenobiotic substances, including anticancer drugs, are other predisposing factors in the development of treatment-related leukemia. There are distinct phase I and phase II drug metabolism pathways whose genetic variation is relevant to individual susceptibility to cancer in general and to leukemia arising from exposure to genotoxic drugs in particular.69–71 Phase I metabolism by cytochrome P-450 (CYP) enzymes converts several anticancer drugs to reactive, electrophilic, watersoluble intermediates that can damage DNA,70,71 while phase II metabolism by the glutathione-S-transferases (GSTs), N-acetyltransferases (NATs), epoxide hydrolases and sulfotransferases results in inactivation and detoxification.69–72 CYP3A, the most abundant component of the CYP system in human liver, metabolizes several anticancer drugs, including the alkylators cyclophosphamide and ifosphamide, the epipodophyllotoxins etoposide and tenipo-

side, and vinblastine and vindesine.73,74 The nifedipinespecific response element (NFSE) in the 5 promoter region of the CYP3A4 gene contains an A-to-G transition polymorphism (CYP3A4-V) that has been examined as a risk factor for treatment-related leukemia.75 Although the majority of cases studied had chromosomal translocations of band 11q23, typical of DNA topoisomerase II inhibitor-related leukemias, several treatment-related leukemias without translocations also were examined.75 A significant deficit of the CYP3A4-V promoter polymorphism was observed for all cases of treatment-related leukemias compared to de novo cases, suggesting that the relationship of the CYP3A4 genotype with alkylating agent-induced leukemia should be studied further.75 The results also suggested that the wild-type CYP3A4 promoter genotype (CYP3A4-W) was significantly associated with epipodophyllotoxin-induced leukemogenesis,75 as elaborated on below. The A-to-G transition polymorphism in the NFSE of the CYP3A4 promoter, later named the CYP3A4*1B allele, was found to occur in individuals with CYP3AP1*1, CYP3A5*1 and other CYP3A genotypes,76 indicating the need to further evaluate not only the role of the CYP3A4*1B allele, but also the role of the other CYP3A genotypes in leukemia risk. Additional CYP polymorphisms occur at the CYP2D6 and CYP2C19 loci, slow metabolizer variants of which are associated with an increased risk of developing secondary leukemia.77 Although the risk was highest for leukemias with chromosomal abnormalities, these genotype–leukemia associations did not track with abnormalities specific for either alkylating agent-related or DNA topoisomerase II inhibitorrelated cases.77 There are many polymorphisms in the genes encoding GSTs.72 An increased risk of MDS has been reported in adults with the GSTT1 null genotype who lack the protein.78 Although the study was not focused on treatment-related MDS, these results predicted that genetic variation in the GSTT1 carcinogen detoxification pathway would prove to be a predisposing factor in alkylating agent-related MDS. In a Japanese population, it later was confirmed that individuals with the GSTT1 null genotype are at increased risk for treatment-related MDS.79 In a large British study the frequencies of GSTM1 and GSTT1 null genotypes were increased in both de novo and treatment-related AML compared with the control population; however, the polymorphism in the GSTP1 gene resulting in Val-for-Ile substitution at amino acid residue 105, the active site for binding hydrophobic electrophiles, was associated with susceptibility to treatment-related AML specifically.80 Furthermore, the leukemias were often characterized by complex karyotypes and loss of chromosomes 5q and 7q, and there was exposure to alkylating agents in the majority of patients

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where chemotherapy exposures had been documented.80 This polymorphism is of interest because reactive metabolites of several alkylating agents including ifosfamide, busulfan and chlorambucil are GSTP1 substrates.80 The enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) detoxifies simple quinones and their derivatives and protects cells against oxidative stress.81–84 The Cto-T polymorphism at position 609 in the NQO1 gene produces a proline-to-serine substitution at amino acid 187, which destabilizes and inactivates the enzyme.85 Individuals who are homozygous for the variant allele are completely lacking in NQO1 activity and heterozygotes have low-intermediate NQO1 activity.86 The association of this polymorphism with benzene poisoning in Chinese adults87 led to investigations of NQO1 C609T in leukemia susceptibility. The NQO1 C609T polymorphism is significantly over-represented in treatment-related leukemias, especially those with chromosome 5 and/or 7 abnormalities88 and the association has been validated.89

DNA topoisomerase II inhibitor-related-leukemias: incidence and risk By the late 1980s, coincident with the introduction of epipodophyllotoxins into clinical usage, a second distinct type of treatment-related leukemia was recognized in adults and children who previously had been treated for various primary leukemias and solid tumors.37,38,90–109 In 1985, Secker-Walker et al.110 observed ALL with the t(4;11) translocation as a late effect of neuroblastoma therapy with doxorubicin and teniposide.110 In 1987, Ratain et al.111 implicated etoposide in combination chemotherapy for non-small cell lung cancer in the subsequent development of leukemia with monoblastic features.111 DNA topoisomerase II mediates changes in DNA topology from relaxed to supercoiled states by transiently cleaving and religating DNA. The enzyme creates fourbase 5 overhangs by introducing coordinated staggered nicks on opposite strands of the DNA double helix when each enzyme subunit forms a covalent phosphodiester bond between its active-site tyrosine residue and the 5 phosphate terminus created in the scission. The resultant cleavage intermediate is called the cleavage complex.112 Agents that target DNA topoisomerase II act via either or both of two broad mechanisms: the first mechanism involves conversion of DNA topoisomerase II into a cellular toxin via stabilization of the normally transient cleavage complex, either by decreasing the reverse rate of religation or increasing the forward rate of scission, both of which have the overall effect of increasing cleavage.112,113 Several chemotherapeutic DNA topoisomerase II “inhibitors” disrupt the cleavage-religation equilibrium in this manner and

Table 31.3 DNA topoisomerase II “poisons” used in anticancer treatment Nonintercalating Epipodophyllotoxins Etoposide Teniposide (not currently in use) Intercalating Anthracyclines Daunorubicin Doxorubicin 4-Epi-doxorubicin Anthracinedione Mitoxantrone Dactinomycin

are also called DNA topoisomerase II “poisons” because the resultant DNA breaks can initiate apoptosis and also can promote illegitimate DNA recombination.113 Etoposide and doxorubicin are drugs that decrease religation.113 The second broad mechanism of action of compounds that target DNA topoisomerase II involves true catalytic inhibition of enzymatic function and activity of the enzyme.114,115 The DNA topoisomerase II-targeted anticancer drugs that have been associated with leukemia have active as DNA topoisomerase II “poisons,” although some have mixed effects as poisons and catalytic inhibition of this enzyme. There are two epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), both natural plant alkaloids and nonintercalative DNA topoisomerase II poisons,31,113 and both have been implicated in leukemia treatment complications (Table 31.3). Nonetheless, etoposide remains among the most widely used, highly efficacious anticancer drugs.112,113 Teniposide is no longer in clinical use but was previously used for pediatric ALL,90,98 pediatric solid tumors116 and lung cancer in adults.117–121 The treatment protocols associated with the highest 6-year cumulative risks of treatment-related AML, 12.4% and 12.3%, specified teniposide on weekly and twice-weekly schedules.90 Various intercalating agents that disrupt the cleavage-religation reaction of DNA topoisomerase II also have been implicated in leukemogenesis, including the anthracyclines daunorubicin, doxorubicin and 4-epi-doxorubicin, the anthracendione mitoxantrone,99,110,122–132 and dactinomycin,126,133 which also interferes with topoisomerase I.134 A recent rising incidence of therapy-related acute promyelocytic leukemia (APL) has been described in Europe and parallels increased use of the DNA topoisomerase II poison mitoxantrone as well as anthracyclines for breast cancer treatment.135 The use of high-dose and dose-intensive epirubicin-containing regimens, which is standard therapy for breast cancer

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in Canada, also carries a significantly increased risk of leukemia as a treatment complication.136 Agents targeting DNA topoisomerase II are administered with additional leukemogenic drugs, and there may be common risk factors for the primary cancers and the secondary leukemias.137 Hence, despite over 150 case reports137 and many cohort studies suggesting that etoposide is leukemogenic,38,39,90,93,99,105,109,111,138–142 the International Agency for Research on Cancer (IARC) concluded that there is “limited evidence” for the carcinogenicity of etoposide alone and that etoposide “probably” is carcinogenic in humans.137 On the basis of cohort studies on patients with gonadal tumors,38,39,105,109,141,142 the IARC concluded that there is “sufficient” evidence in humans for the carcinogenicity of etoposide in combination with cisplatin and bleomycin.137 In contrast to alkylating agent-related leukemias, the median latency from exposure to epipodophyllotoxinrelated AML is 24 to 30 months.2,143 However, there are cases with cytogenetic and molecular aberrations of epipodophyllotoxin-related AML where the latency has been up to 10 years, suggesting that the period of risk is variable and may be quite long.144,145 The overall incidence of leukemia following epipodophyllotoxin-containing regimens is about 2% to 3%.146 The NCI Cancer Therapy Evaluation Program monitored the occurrence of leukemia in 12 clinical trials in which low (<1500 mg/m2 ), moderate (1500–2999 mg/m2 ) or high (≥3000 mg/m2 ) total doses of epipodophyllotoxin were used. Respective calculated cumulative 6-year rates of 3.2%, 0.7%, and 2.2% indicated the lack of a dose–response effect, in contrast to alkylating agent-induced leukemia, suggesting that factors other than cumulative dose are of primary importance in predicting risk in the context of multiagent regimens.146 However, in a recent case-control study of treatment-related leukemia after pediatric solid tumors, the cumulative dose of etoposide and anthracyclines both were correlated with leukemia risk, although higher etoposide total doses, in excess of 6 g/m2 , were administered in many of the cases.147 Schedule may be an important factor in determining the risk.146,147 Intermittent weekly or twice-weekly epipodophyllotoxin schedules for primary ALL, used more commonly in the past, were associated with incidences as high as 5.9% and 12%.90,93 Schedule alterations and substitution of other agents for epipodophyllotoxins reduced but did not eliminate the risk of secondary leukemia following primary ALL.93,148,149 Etoposide administration on a semicontinuous (3 days/week for 3 weeks in a row out of 4 weeks) or a continuous (21 consecutive days out of 28) schedule may be associated with a higher risk, but the schedule effect could not be separated from the effect of dose because the majority of these patients also received in excess of

6 g/m2 of etoposide.147 One cohort study of patients with primary ALL suggested that L-asparaginase administration during the week preceding epipodophyllotoxin was associated with an increased risk and that L-asparaginase potentiates the leukemogenicity of epipodophyllotoxins.150 Twoyear cumulative risks of treatment-related AML were 5.4% and 1.1%, respectively, in patients who received an additional 16 to 19 doses of L-asparaginase compared with patients who received epipodophyllotoxin on the same schedule without the additional L-asparaginase.150 Methotrexate and mercaptopurine administration preceding epipodophyllotoxin-containing regimens may increase the risk of leukemia.93,139,148 There may be a disproportionate incidence of this form of secondary AML among Hispanic patients.93,151,152 AML also occurs after Langerhans’ cell histiocytosis (LCH) therapy with epipodophyllotoxins; because of the independent association between LCH and malignancies, the possibility of a contribution of the primary disease to risk has been raised.153–156 The incidence following treatment according to pediatric solid tumor protocols generally has been lower than with certain ALL regimens.91,157 However, an excess risk of this form of leukemia has been reported in patients with primary Hodgkin disease or osteosarcoma.147 More intensive solid tumor regimens are associated with a greater risk.128,129 It has also been suggested that in pediatric sarcoma regimens, it is the combined modality therapy of intercalating DNA topoisomerase II inhibitors with alkylating agents and irradiation that results in an increased risk.157 Leukemia is a complication of autologous stem cell transplantation, as described above, with a higher risk in recipients of autologous peripheral blood stem cells than in recipients of autologous bone marrow.44,46,47 In patients with lymphomas, a 12.3-fold increased risk of treatment-related leukemia was observed when the autologous stem cells used for transplantation were harvested after priming with etoposide; the leukemias had chromosomal translocations of bands 11q23 and 21q22 typical of leukemias associated with DNA topoisomerase II poisons.158 G-CSF administration was recently evaluated as a risk factor for secondary leukemia after primary ALL because G-CSF can induce growth of leukemia cells in vitro.159 Among pediatric patients randomized to receive G-CSF or placebo on the St. Jude’s Total XIIIA protocol, G-CSF was not independently associated with an increased risk when the patients were stratified for radiation therapy. However, when patients on two consecutive protocols were examined altogether and children receiving radiation were excluded, the incidence of treatment-related leukemia was higher in patients who received G-CSF, and the majority of leukemias were characterized by translocations of chromosome band 11q23, suggesting that G-CSF may increase the

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risk of DNA topoisomerase II inhibitor-related leukemia when these agents are administered as part of an intensive antileukemia regimen.159 Recently, pediatric and adult cases of MDS with hallmark cytogenetic features of alkylating agent-related leukemias, including complete or partial deletions of chromosomes 5 and 7, have emerged after the treatment of primary APL with all-trans-retinoic acid (ATRA) and DNA topoisomerase II inhibitors such as anthracyclines, mitoxantrone or etoposide.160–164 The incidence of MDS/AML after APL in one series was 6.5%.164 These cases have raised questions as to whether DNA topoisomerase II inhibitors can cause MDS similar to that induced by alkylators or, alternatively, whether the cases were not treatment-related at all but represented relapse of leukemia with an unrelated clone.160– 165 Although t(15;17) and inv(16) are among the characteristic chromosomal aberrations in leukemias following chemotherapy with DNA topoisomerase II inhibitors, these abnormalities sometimes are observed after therapy with radiation only,14,166 suggesting that heterogeneous exposures create a risk for treatment-related leukemias with balanced translocations.

DNA topoisomerase II inhibitor-related-leukemias: genetic predisposition Molecular cancer epidemiology has provided sparse information on specific polymorphisms or germline mutations that confer genetic susceptibility or mutagen sensitivity in this form of leukemia. CYP3A has several antineoplastic substrates as described above.73 CYP3A4 converts epipodophyllotoxin to a catechol metabolite that is readily oxidized to a quinone (Table 31.2). 167– 169 The polymorphism in the NFSE of the CYP3A4 promoter170 has been evaluated as a host factor that modulates leukemogenic drug effects in pediatric patients exposed to one or more anticancer drugs metabolized by CYP3A.75 The CYP3A4 wild-type genotype increased, and CYP3A4 heterozygous or homozygous variant genotypes decreased the risk of epipodophyllotoxinrelated leukemias with MLL translocations.75 This CYP3A4 promoter genotype association was validated in a study of Israeli adults with treatment-related leukemia with MLL translocations171 ; however, the same association was not observed in a population of pediatric patients who developed treatment-related AML following primary ALL therapy with etoposide- or teniposide-containing regimens.172 Newer data suggest that the wild-type (AA) genotype at the nifedipine specific element of the CYP3A4 promoter, which is associated with an increased risk of epipodophyllotoxin-

related AML, is associated with lower etoposide clearance in Caucasians.173 It is plausible that the CYP3A4 genotype confers susceptibility to this form of leukemia because the epipodophyllotoxins and their metabolites are genotoxins, 174– 176 the significance of which is described in the following section (‘Molecular pathogenesis’). Although etoposide catechol, the major metabolite of etoposide, is detectable in the plasma of patients receiving chemotherapy with etoposide, 177– 179 there are few studies of etoposide catechol disposition.177,178,180,181 In adults receiving etoposide at myeloablative 1-hour bolus doses of 600 mg/m2 per day for 4 days in a hematopoietic stem cell transplantation regimen, the catechol metabolite was observed to increase from the first day to the last day of treatment.177 Sequential pharmacokinetic studies also showed that etoposide catechol but not the parent drug increases when etoposide is administered as conventional, nonmyeloablative, multiple-day bolus doses in pediatric patients.181 These studies indicate that patients receiving multiple-day bolus doses of etoposide are exposed to higher concentrations of the genotoxic catechol metabolite with successive days of treatment. Although etoposide catechol is formed primarily through CYP3A4 metabolism,169 little is known about the relative importance of CYP3A5 in etoposide metabolism; it is conceivable that CYP3A5 induction76 could contribute to increased catechol formation. Ifosfamide induces CYP3A4 in primary hepatocytes maintained in culture182 and concurrent ifosfamide administration may contribute to increased etoposide catechol formation when these drugs are used in combination.181 Prednisone also induces etoposide clearance and affects catechol formation.173 Consistent with observations that the metabolism of most chemotherapeutic agents is polygenetically determined and that genetic polymorphisms can account for large differences in their pharmacokinetics, 69– 71 etoposide disposition is further affected by polymorphisms in the MDR1, CYP3A5, UGT1A1 (UDP-glucuronyltransferase 1A1) and VDR (vitamin D receptor) genes.173 The relationship of polymorphisms in these genes to the development of treatment-related leukemia has not yet been examined. There are also racial differences in the effects of specific genotypes on etoposide disposition.173 Woo et al.183 concluded that GSTT1 and GSTM1 null genotypes were not predisposing factors to epipodophyllotoxin-related leukemia following primary ALL,183 which contrasts with the importance of inactivating GST genotypes affecting phase II detoxification pathways in the genetic predisposition to leukemias following alkylating agent treatment (cf. previous section, “Alkylating agent-related leukemias: genetic predisposition”).

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Thiopurines are widely used for the therapy of ALL and, in some regimens, these agents are used in combination with etoposide.184 Thiopurine-S-methyltransferase (TPMT), which catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, is the principal metabolic inactivation pathway for the thiopurines.185 The TPMT gene is polymorphic; approximately 1 in 300 individuals is TPMT-deficient, and 11% of the population are heterozygotes with intermediate TPMT activity.185 TPMTdeficient and heterozygous individuals accumulate higher cellular levels of the active metabolites of thioguanine and experience more hematopoietic toxicities from the thiopurines.184 In a study of patients with primary ALL, a trend toward lower TPMT activity was reported among patients who developed treatment-related AML following a regimen specifying etoposide in induction and in maintenance, and the treatment-related leukemias were typical of epipodophyllotoxin-exposed cases.178 There are three polymorphic alleles – (GAA)4, (GAA)5, and (GAA)6 – at a trinucleotide (GAA) repeat tract within an Alu repeat sequence in intron 6 in the MLL breakpoint cluster region (bcr).186 Adult patients with DNA topoisomerase II inhibitor-related AML are almost exclusively (GAA)4/5 heterozygotes, which is different from the population distribution of the three alleles in normal individuals.186 The functional significance of this polymorphism in leukemia predisposition remains to be determined.

Molecular pathogenesis The vast majority (85%–90%) of cases of treatment-related leukemia and MDS have chromosomal aberrations that also occur in de novo leukemia and MDS, although the chromosomal and molecular aberrations in the treatmentrelated cases reflect prior therapy with distinct classes of cytotoxic drugs.187

Cytogenetic and molecular genetic changes in leukemias following alkylating agent treatment Complete or partial deletions of chromosomes 5 and 7 and complex, unbalanced numerical and structural cytogenetic abnormalities are the archetypal cytogenetic features of leukemias ascribed to alkylating agent treatment.19 Deletions of putative tumor suppressor genes at chromosomes 5q and 7q are believed to underlie the molecular pathogenesis of alkylating agent- related leukemias. Since similar aberrations occur in de novo MDS/AML, knowledge on potential regions of involvement at chromosomes 5q and 7q derives from de novo and treatment-related cases, but the specific genes in these regions that are important in leukemia pathogenesis continue to remain elusive.

Chromosome 7 abnormalities are the most commonly observed changes in leukemias following alkylating agent treatment.187 Chromosome 7 aberrations are present in 50% of cases of treatment-related AML/MDS.188 More than 80% of cases of MDS and AML with 7q deletions have allelic loss of the entire region from chromosome 7q22 to 7q31. However, rare cases have been observed with allelic loss at 7q31 and 7q22 loci and retention of sequences between these loci or submicroscopic allele imbalance for a different distal locus,189 suggesting that multiple distinct critical chromosme 7q genes are involved in MDS and AML.189 The hDMP1 (cyclin D-binding Myb-like protein) gene is one example of a candidate tumor suppressor gene at chromosome band 7q21 frequently deleted in AML and MDS with 7q deletion.190 In another search for candidate tumor suppressor genes, the human Trx family member MLL5 also was discovered within a commonly deleted 2.5 Mb segment at chromosome band 7q.191 However, inactivating mutations and decreased MLL5 mRNA expression were not found, possibly suggesting inactivation of one MLL5 allele by haploinsufficiency as a potential mechanism whereby MLL5 may contribute to leukemogenesis.191 Alternatively, the 7q22 region may harbor other genes important to leukemogenesis.191 Kratz et al.192 also extensively characterized an estimated 2-Mb commonly deleted segment of chromosome 7q22.192 Although a missense mutation in the PIK3CG gene encoding the catalytic subunit p110 gamma of phosphoinositide 3-OH-kinase-gamma (PI3K gamma) at chromosome band 7q22 was identified in two cases, the same alteration was observed in unaffected parents and, infrequently, in control individuals, suggesting that this is unlikely to be the tumor suppressor gene associated with monosomy 7.193 The second most common cytogenetic abnormality in alkylating agent-related AML/MDS is −5/del(5q).187 FISH has revealed that the del(5q) is often a covert unbalanced translocation.187 There is marked variability in the breakpoints of the 5q deletion, but many genes that are important in hematopoiesis have been localized to the region 5q13–q33.188,194 The IRF-1 (interferon-regulatory factor-1) gene at chromosme band 5q31.1 between IL-5 and CDC25C and centromeric to IL-3 and GM-CSF, was consistently deleted at one or both alleles in 13 cases of leukemia or MDS with chromosome 5q31 aberrations, possibly suggesting that IRF-1 may be a critically deleted gene in leukemias with −5/del(5q) abnormalities.195 The EGR1 (early growth response 1) gene, FMS oncogene and the M-CSF1R gene are also at chromosome band 5q31.188 A commonly deleted segment of chromosome 5q31 has been localized to a 1.5 Mb interval.196 PURA is within the most commonly deleted segment in del(5)(q31).197 PURA

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and PURB at chromosome bands 5q31.1 and 7p13, respectively, encode functionally cooperative proteins in the Pur family. FISH has shown frequent simultaneous hemizygous PURA and PURB deletions in MDS and AML.197 The del(5)(q13q33) and del(5)(q13q35) are major subsets of deletions.198 Translocations or paracentric inversions involving 5q11 to 5q13 also are observed,198 and there has been considerable attention paid to more proximal regions of loss at chromosome 5q. A deleted region in a 2.0-Mb interval at 5q13.1 was identified in a subset of cases of MDS/AML with chromosome 5 anomalies.198 A fragile site involved in unbalanced translocations and interstitial deletions has been suggested within a locus in a less than 3-Mb region at chromosome band 5q13.3.199 The region of loss at chromosome band 5q13.3 was narrowed to approximately 1.5 Mb, and SSBP2, which encodes a sequence-specific single-stranded DNA binding protein with potential tumor suppressor activity, was identified in this region.200 Cosegregating deletions of chromosome 5q13.3 and the TP53 gene at chromosme 17p13 have suggested functional cooperation between loss of a putative tumor suppressor gene at chromosome 5q13.3 followed by loss of TP53.201 Ras family members regulate cellular proliferation and differentiation by cycling between active GTP-bound and inactive GDP-bound states. 202– 206 Many hematopoietic growth factors transduce signals from the cell surface to the nucleus through Ras family members.207,208 Accompanying mutations in RAS oncogenes occur in a significant proportion of cases of alkylating agent-related leukemia. 209– 211 Activating KRAS and NRAS mutations at codons 12, 13, or 61 deregulate this signal transduction pathway and are oncogenic.212 The overall incidence of KRAS and NRAS mutations in one series of cases of treatment-related AML and MDS with deletions of chromosomes 5 and 7 was 9%, but there was a higher incidence in treatment-related AML with monosomy 7 or del(7q) (19%).211 The finding of germline TP53 mutations in several cases of alkylating agent-related leukemias in children predicted that alkylating agent-related leukemia was a type of cancer where somatic TP53 mutations also would be found. Such mutations were observed in the leukemic cells in 21 of 77 unselected cases (27%) of treatment-related AML and treatment-related MDS in adults.213 Alkylating agent exposure was documented in 19 of the 21 cases with TP53 mutations, indicating that these mutations are frequent in AML and MDS following alkylating agent treatment.213 TP53 mutations also were found to be common in leukemia and MDS following multiagent ovarian cancer therapy, which often included platinum analogues.214 Although constitutional DNAs were not available, the mutations likely were not of germline origin because of the nature of the muta-

tions, the older ages of the patients and the fact that ovarian cancer is not a Li-Fraumeni syndrome tumor.214 A total of 11 TP53 mutations were detected in 9 of 17 cases studied, and the propensity for G-to-A transitions possibly suggested specific DNA damage in leukemias following platinum analogues.214 The phenomenon of MLL gene amplification in the form of segmental jumping translocations also was found to be a recurrent abnormality in treatment-related leukemias associated with TP53 mutations and alkylating agent exposure,215 although the role of the oncogene amplification in leukemia pathogenesis is unknown. The high frequency of TP53 mutations in leukemia and MDS following alkylator therapy contrasts with the lower reported frequencies in primary MDS.216,217 Hypermethylation resulting in inactivation of the negative cell-cycle regulatory genes p15(INK4B) and p16(INK4A) has been observed in a large proportion of cases of therapyrelated MDS and AML.218 p15 methylation, which occurs more often, is significantly associated with leukemias with −7/del(7q) abnormalities.218 These observed molecular aberrations led to a model subdividing alkylating agent-related leukemias based on involved genetic pathways and biologic features.187 The first subgroup includes cases with −7/del(7q) abnormalities with a normal chromosome 5. The −7/del(7q) abnormalities may be present only in subclones of the cells, and these leukemias may contain additional aberrations including t(3;21), RAS mutations and p15 promoter methylation but usually not TP53 mutations.187 The second subgroup includes cases with −5/del(5q), which, unlike the −7/del(7q) abnormalities, appear to represent primary alterations and not subclone evolution.187 Additional aberrations including −7/del(7q) abnormalities and unidentified marker chromosomes may occur, and TP53 mutations are very common in cases with −5/del(5q).187 Duplication or amplification of chromosome band 11q23, including the unrearranged MLL gene, occurs as a recurrent abnormality in this subgroup and is associated with TP53 mutations.187,215 The ability to distinguish treatment-related leukemias with −7/del(7q) abnormalities without chromosome 5 involvement and cases with −5/del(5q) on the basis of molecular genetic pathways187 was recently validated by gene expression profiling experiments comparing normal CD34+ hematopoietic stem cells with CD34+ hematopoietic stem cells from a small number of cases of treatment-related AML.219 A characteristic gene expression pattern was observed in the treatmentrelated leukemias with −5/del(5q) and complex karyotypes, which includes higher expression of several genes involved in cell cycle control, including cyclin

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A2 (CCNA2), cyclin E2 (CCNE2) and CDC2, the checkpoint gene BUB1, the cell growth gene MYC, and the MYC-regulated gene CDC28 protein kinase 2 (CKS2).219 Loss of expression of the IFN consensus sequencebinding protein (ICSBP) gene, the product of which regulates proliferation and differentiation in hematopoietic cells, was characteristic of treatment-related AML with the −5/del(5q).219 In addition, this subgroup was characterized by reduced expression of several chromosome 5 genes such as APC.219 The same study suggested a second subgroup of treatment-related AML with −7 or simple karyotypes without −5/del(5q) abnormalities characterized by downregulation of the TAL1, GATA1, and EKLF genes, which encode transcription factors that are involved in early hematopoiesis, and upregulation of FLT3 and BCL2.219 The AML-1 gene encodes the DNA-binding subunit of the core-binding factor  (CBF) transcription factor complex, which is critical for hematopoiesis. Somatically acquired point mutations in the AML1 (RUNX1, CBFA2) gene, which affect the DNA-binding potential of the AML1 gene product, have been identified in a large proportion (38%) of cases of therapy-related AML or MDS following alkylating agent treatment with or without local irradiation for various primary cancers.220 These data indicate that AML1 point mutations are induced by the treatment, but it is not yet known whether the AML1 point mutations represent primary or secondary alterations in the progression of this form of AML.220 The gene encoding DAP (death-associated protein)kinase, a calcium/calmodulin-regulated serine/threonine kinase that positively regulates apoptosis, has been found to be hypermethylated in 50% of cases of treatment-related AML and MDS following alkylating agent treatment.221 DAP-kinase hypermethylation is more common in alkylating agent-related leukemias than in DNA topoisomerase II inhibitor-related cases.221 Since the DAP-kinase hypermethylation is associated with loss of DAP-kinase gene expression, loss of DAP kinase function appears to be important in treatment-related MDS.221 Mitochondrial (mt) transfer RNA mutations and mtDNA mutations currently are emerging as new types of abnormalities that may be relevant to alkylating agent-related AML and MDS. A somatic G3242A mt transfer RNALeu(UUR) mutation recently was observed in the CD34+ cells in a case of myelodysplastic syndrome presenting as refractory anemia with excess blasts (RAEB).222 Although mt transfer RNA and mtDNA mutations have not yet been examined in treatment-related MDS and leukemia, they may result in ineffective hematopoiesis via mitochondrial respiratory chain dysfunction222 and trigger ROS-mediated damage to the genome.223 mtDNA mutations are also of interest

because treatment with a fludarabine/alkylator regimen has resulted in the acquisition of mtDNA mutations and higher levels of superoxide radical generation in the primary leukemia cells of patients undergoing chemotherapy for CLL.223

Cytogenetic and molecular genetic changes in leukemias following DNA topoisomerase II inhibitors Translocations are the primary molecular alterations in leukemias related to chemotherapy with DNA topoisomerase II inhibitors.187,224 Translocations of the 100-kb MLL gene at chromosome band 11q23 occur most often, 225– 229 but virtually all of the translocations observed in de novo AML including t(8;21) and its variants, 39,91,132,157,230– 237 t(15;17), 125,135,146,166,238– 241 inv(16) and 143,166,230,231,240,242 t(8;16),231 t(9;22)243 and heterot(16;16), geneous translocations of the NUP98 gene at chromosome band 11p15 244– 253 can also be found. A recent international workshop examining 511 cases of treatment-related leukemia with balanced translocations found translocations of the MLL gene at chromosome band 11q23 in 162 cases, translocations of the AML1 (CBFA2) gene at chromosome band 21q22 in 79 cases, inversions or translocations fusing the CBFB gene at chromosome band 16p13 to the MYH11 at chromosome band 16q22 in 48 cases, translocations of the PML and RARA genes at chromosome bands 15q22 and 17q12 in 41 cases, and rare and unique translocations in the remaining cases (Fig. 31.1)14 . MLL contains 36 exons and encodes a 431-kDa, 3969amino acid protein with various transcription factor structural motifs, a DNA methyltransferase (MT) domain, and regional amino acid with homology to Drosophila trithorax (trx) in the N-terminal speckled nuclear localization (SNL) motifs, the central zinc fingers comprising a plant homeodomain (PHD) and the SET domain at the C-terminus. 254– 259 Drosophila trx group (trxG) and Polycomb-group (PcG) proteins maintain expression or repression, respectively, of homeotic gene complexes for the execution of developmental programs during embryogenesis; trx maintains but does not initiate expression of HOX genes.260,261 MLL and BMI-1, mammalian homologs of trx and PcG proteins, are antagonistic regulators of HOX gene expression.262 MLL maintains HOX gene expression early during mammalian skeletal, craniofacial and neural development and hematopoiesis.260,263,264 The N-terminal AT hook motifs localize MLL to the nucleolus, nuclear matrix and mitotic chromosomal scaffolds, consistent with a role in epigenetic control of gene expression and chromatin regulation.265 The AT hook motifs promote p21 and p27 upregulation,

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Fig. 31.1 Distribution of balanced chromosomal translocations in 511 cases of treatment-related leukemia. (Adapted, with permission, from Rowley and Olney.14 )

cell cycle arrest and monocytic differentiation.266 The MT domain is part of a transcriptional repression region.266 The PHD mediates MLL homodimerization and binding to a nuclear cyclophilin, which modulates target gene expression.267 The SET domain interacts with the SWI/SNF chromatin remodeling complex, which activates transcription.268 Consistent with its role in epigenetic gene regulation, the C-terminal MLL SET domain has specific histone H3 lysine-4-specific methyltransferase activity that regulates HOX promoters.269 MLL exists in a large protein supercomplex that is involved in the remodeling, acetylation, deacetylation, and methylation of nucleosomes and histones.270 The MLL protein is proteolytically cleaved into N- and C-terminal fragments with transcriptional repression and transcriptional activation properties, which associate in an intramolecular complex.271 The proteolytic cleavage of MLL recently was shown to be mediated by Taspase1.272 The association of the MLL N- and C-terminal fragments is important for proper nuclear sublocalization of MLL.273 Further, MLL proteolytic cleavage by Taspase1 is essential for proper expression of HOX target genes.272 The de novo counterpart of treatment-related leukemias with MLL translocations is leukemia in infants. MLL translocations, which characterize approximately 80% of cases of infant ALL and approximately 80% of cases of FAB M4/M5 AML in infants and young children,274 disrupt an 8.3-kb breakpoint cluster region (bcr) between exons 5-11. Since MLL translocation breakpoints are in introns of the bcr and the breakpoints in the partner genes also are in introns, the fusion transcripts are in-frame 127,130,133,275– 277 ; MLL translocations are thought to cause leukemia by producing chimeric oncoproteins from the der(11) chro-

mosome, in which the amino terminus of MLL is joined to the carboxyl terminus of the partner protein.278 The fusion proteins retain the AT-hook and SNL motifs and MT domain of MLL but replace the PHD, transactivation domain, and C terminal SET domain with the C-terminus of the partner protein.278 The MLL proteolytic cleavage site is lacking from the fusion proteins, and the fusion proteins cannot interact with the MLL C-terminus.271 MLL translocations involve many partner genes that encode different types of proteins. 278– 280 Genomic breakpoint junction sequences or MLL chimeric transcripts involving more than 40 partner genes including MLL itself have been described and others are expected. Many MLL partner proteins have structural motifs of nuclear transcription factors (LAF-4, AF4, AF5
, AF5q31, AF6q21, AF9, AF10, MLL, AF17, ENL, AFX), 255,256,281– 289 proteins involved in transcriptional regulation (CBP, ELL, p300) 290– 293 or, in two cases, nuclear proteins of unknown function (LCX, AF15q14). 294– 296 Other MLL partner proteins are found in the cytoplasm (AF1p, AF1q, AF3p21, GMPS, LPP, GRAF, CDK6, FBP17, ABI-1, CBL, MPFYVE, GAS7, MSF, LASP1, EEN, hCDCrel, SEPTIN6), 130,144,145,277,297– 312 or at the cell membrane (AF6, CALM, LARG, GPHN, MYO1F), 313– 317 endoplasmic reticulum and Golgi apparatus (ALKALINE CERAMIDASE),317 microtubules (EBI),318 or ribosome (RPS3).319 The t(4;11), which fuses MLL and AF-4, accounts for about 70% of MLL translocations in ALL,274 but significantly more partner gene diversity occurs in AML. The partner genes of MLL in treatmentrelated leukemias often occur as partner genes in de novo cases.127,133,275,320 However, several partner genes were discovered in treatment-related cases including AF3p21,144 GMPS (guanosine 5  -monophosphate synthetase) at band 3q24,145 LPP at band 3q28,299 AF-6q21,284 CBP (CREBbinding protein) at band 16p13.3,290,291,321 GAS7 (growth arrest-specific 7) at band 17p13,130 MSF (MLL septinlike fusion) at band 17q25308 and the p300 gene at band 22q13,293 and some but not all of these MLL translocations have been found as recurring abnormalities in de novo cases. Treatment-related leukemias with a normal karyotype or karyotypic del(11)(q23) abnormalities may harbor cryptic MLL translocations130,320 or MLL tandem duplications.133 Until recently, all MLL translocations identified in patients were associated with the clinical diagnosis of leukemia; however, a novel fusion of MLL to the partner gene called MIFL (MLL Insufficient for Leukemia) from chromosome band 4p12 was discovered in the bone marrow of a patient treated for neuroblastoma in whom clinical leukemia has not occurred.322 Similarly a translocation fusing MLL to ARHGEF 17 from chromosome band 11q13 occurred after therapy of primary AML with t(8;21), and

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has not been associated with a leukemia phenotype in the patient.323 It remains to be determined whether any relevant secondary alteration will result in transformation to leukemia in either of these patients or whether the translocations are fundamentally different in their function than the others. The functional contribution of the partner protein in MLL fusion proteins to leukemogenesis is not well understood because of the plethora of partner proteins. Some of the partner genes are members of the same gene families. LAF-4, for example, is a gene family with three members (LAF-4, AF4, AF5q31) that fuse with MLL.278,283,324,325 Three SEPTIN family members, hCDCrel, MSF (AF-17q25), and SEPTIN6, are disrupted by MLL translocations.307,309– 312,326 AFX and AF6q21 encode forkhead domain proteins with site-specific DNA-binding and transcriptional regulation properties.284,289,327,328 However, there is no known unifying functional relationship between the many partner proteins. Much information on the function of MLL fusion proteins derives from murine models. The first murine model for MLL fusion protein leukemogenesis were Mll-AF9 heterozygous knock-in mice, which developed AML.329 Various retroviral transplantation murine models have used 5 -MLL-partner-3 constructs to establish that the der(11) protein products are leukemogenic, 278,330– 334 but whether MLL fusion proteins confer a gain of function or dominantnegative inhibition of normal MLL265,335 remains a topic of investigation. It has been suggested that transcriptional activation may be a key function encoded by MLL fusion partners.328,335,336 ENL and AF-9 contain a similar highly conserved C-terminal transcriptional transactivating region that is retained in the fusion proteins.337 Structure–function analysis of the MLL-ELL fusion protein has shown a domain with potent transcriptional activation properties.338 MLL-AFX requires two conserved transcriptional effector domains for leukemogenesis.327 One group has shown that MLL-ENL, MLL-AF9, and MLL-ELL fusion proteins associate with GADD34 and abrogate apoptosis mediated by GADD34, which binds SNF5/INI1 in the SNF/SWI complex, suggesting that these fusion proteins may confer a gain of function.339 The HOXA9 gene and the MEIS1 gene, which encodes a HOX coregulator, are overexpressed in MLL-rearranged leukemias,340 and these genes were shown to be the key targets in MLL-ENL-mediated cellular immortalization.341 Consistent with a gain of function mechanism, it has been suggested that MLL fusion proteins constitutively activate Hoxa9, which was found to be essential for leukemogenesis induced by the MLLENL fusion protein in a murine retroviral transplantation model.342 By contrast, Hoxa9 expression influenced the myeloid phenotype but not the incidence of leukemia and

was not required for Mll-AF9 leukemogenesis in a transgenic model. In addition, leukemic transformation by MLLGAS7 occurred in the absence of Hoxa9 and Hoxa7 in a different murine model, although the Hox genes influenced the phenotype, latency and penetrance of the leukemia that emerged.343 An alternative mechanism that might account for the contribution of cytoplasmic MLL partner proteins to leukemogenesis involves dimerization, since the coiledcoil domains of the cytoplasmic partner proteins AF1p and GAS7 proved necessary and sufficient for leukemic transformation.344 The roles of loss of function of MLL and/or the partner protein are unknown.278,335 It has been suggested that it is less likely that MLL fusion proteins are leukemogenic by the creation of MLL haploinsufficiency, dominant-negative inhibition of MLL or mutation of both MLL alleles.335 The presence and potential functional role of MLL fusion proteins predicted by in-frame 5 -partner-MLL-3 fusion transcripts from the other derivative chromosomes found in many cases remains unexplored.277 The latency time to leukemia in the murine models has suggested that secondary alterations may be important in addition to the MLL translocations.278,335 However, unlike in alkylator-induced leukemias, TP53 mutations are not observed in treatment-related leukemias associated with DNA topoisomerase II inhibitors,58 possibly suggesting that intact p53-dependent DNA repair may be required for the translocations to be formed.345 Overexpression of FLT3 and FLT3 mutations have emerged as important secondary alterations in leukemias with MLL translocations and MLL tandem duplications,340,346–348 although treatment-related cases have not been extensively examined. FLT3 internal tandem duplication (ITD) mutations also appear to be especially common in APL with the t(15;17), another recurrent translocation in treatmentrelated leukemia, but, again, mostly de novo cases were examined.349 In pediatric cases of treatment-related AML following primary neuroblastoma130 or primary ALL,350 the MLL translocation was absent in the marrow at primary cancer diagnosis but emerged during the treatment, suggesting that treatment with DNA topoisomerase II inhibitors caused and did not select for a pre-existing translocation. In MLL-rearranged infant leukemias the translocation is a somatic, in utero event and the latency is short (from some time in pregnancy to the time of leukemia diagnosis in the infant).307,351,352 However, de novo MLL-rearranged leukemias in older children generally are not traceable to birth,350 which is also consistent with a causative role of DNA topoisomerase II inhibitors in the translocations in treatment-related cases. Nonetheless, MLL translocations can be present early during anticancer treatment at

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Clinical DX NBL

Monocytosis/Cytopenias

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Fig. 31.2 Detection of a leukemia-associated translocation fusing MLL with GAS7 early during neuroblastoma chemotherapy. The MLL translocation was not detectable by PCR at diagnosis of neuroblastoma, but was detectable by clonotypic PCR analysis of the der(11) genomic breakpoint junction in all marrow specimens obtained at and after 6 weeks from the start of treatment (after two cycles of cyclophosphamide, doxorubicin and vincristine).130 AML was diagnosed 15.5 months after the translocation was detectable by PCR.130 (Adapted, with permission, from Megonigal et al.130 )

low cumulative doses of DNA topoisomerase II inhibitors (Fig. 31.2).130 There has been considerable attention to the DNA damage and repair mechanisms whereby DNA topoisomerase II inhibitors result in MLL translocations. The association of DNA topoisomerase II-targeted chemotherapy with leukemias has suggested that repair of drug-induced DNA topoisomerase II-mediated damage may result in translocations (reviewed in Felix3,354,355 and Rowley356 ). First, Southern blot analysis suggested that there was a biased distribution of MLL genomic breakpoints in treatmentrelated leukemias in adults 3 in the bcr and disruption of a scaffold attachment region containing a putative DNA topoisomerase II site,357 although the breakpoint junctions were not cloned. There is heterogeneity in the MLL translocation breakpoint distribution in the bcr in treatment-related leukemias in children, but a hotspot region in intron 8, 3 in the bcr, also has emerged from the molecular cloning of MLL genomic breakpoint junctions (Fig. 31.3).127,130,133,275– 277,290,358,359 In the treatmentrelated leukemias, where both genomic breakpoint junctions have been characterized (Fig. 31.3, bold arrows), the sequences reveal precise or near-precise interchromosomal DNA recombinations with gains or losses of no or, more often, a few bases.130,275–277,358,359 The relatively precise interchromosomal DNA recombinations in the treatmentrelated cases are consistent with the processing of fourbase, staggered double-stranded breaks from DNA topoi-

somerase II.276,277,359 In a case of treatment-related ALL with t(4;11)276 and in a case of treatment-related AML with t(9;11),359 the translocation breakpoints in MLL and in its partner gene were reciprocally cleaved by DNA topoisomerase II in an in vitro assay, and the functional, druginduced DNA topoisomerase II cleavage sites could be resolved to form the breakpoint junctions observed in the leukemias.276,359 CYP3A4 metabolizes etoposide by O-demethylation to etoposide catechol, which is readily oxidized via sequential one-electron oxidation steps to etoposide semiquinone and etoposide quinone.169,360 These etoposide metabolites as well as the etoposide parent drug are genotoxins that can stimulate DNA topoisomerase II cleavage.176,276,359 The quinone metabolite can damage DNA through alkylation.168,176,361–365 Redox cycling by etoposide catechol and etoposide quinone results in the formation of reactive oxygen species (ROS).362 Abasic sites in DNA from ROS can be more potent enhancers of DNA topoisomerase II cleavage than the etoposide parent drug.366,367 The catechol and quinone metabolites of etoposide, like the parent drug, increase the formation of DNA topoisomerase II cleavage complexes at or near the translocation breakpoints in MLL and its partner genes in treatment-related leukemias.176,276,359 These data, taken together, also favor the model in which the chromosomal breakage leading to MLL translocations in DNA topoisomerase II inhibitor-related leukemias

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6610; Domer 6784; 10/Atlas

6006; Daheron 6230; 5/Atlas

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6587-6792; t-24/Whitmarsh 6588-6589; t-120/Raffini 6591-6592; P4/Langer 6593; P6/Langer 6592-6594; P8/Langer 6593; P9/Langer 6589; P10/Langer

6588-6793; t-24/Whitmarsh 6594-6596; t-120/Raffini 6588-6590; P4/Langer 6588; P6/Langer 6596-6597; P8/Langer 6600; P9/Langer Fig. 31.3 Emergence of a translocation breakpoint hotspot in MLL intron 8, which is 3 in the genomic breakpoint cluster region (bcr), in treatment-related leukemia in children. A schematic of the MLL bcr is shown. Fine arrows indicate MLL translocation breakpoints on the der(11) chromosome (top) or on the other derivative chromosome (bottom) in cases of treatment-related leukemias where only one of the genomic breakpoint junctions has been cloned. Bold arrows (top and bottom) indicate MLL translocation breakpoints in cases of treatment-related leukemia in which both genomic breakpoint junctions have been cloned.127,130,133,275–277,290,299 Note precise or near-precise nature of the translocations in the latter cases (bold arrows). Nucleotide coordinates are indicated relative to the MLL (ALL–1) bcr genomic sequence (GenBank number, U04737). Translocation breakpoints in eight treatment-related leukemias have now been mapped within the hotspot region from positions 6587 to 6610 in intron 8, and the hotspot region contains several functional DNA topoisomerase II cleavage sites.359

is a consequence of DNA topoisomerase II cleavage.359 However, an alternative mechanism has been proposed whereby apoptotic nucleolytic cleavage causes the damage to MLL in these translocations.368–370 Microhomologies at or near the translocation breakpoint junctions130,275–277,307,359,371–374 are consistent with nonhomologous end-joining (NHEJ) as the mechanism of DNA repair in treatment-related and de novo leukemias with MLL translocations.276,277,359,373,375 Most DNA doublestrand breaks generated by mutagenic agents cannot be religated directly, and some limited processing, including

small deletions and/or DNA polymerization, is required before NHEJ can ensue,376 which is consistent with several small deletions130,277 and a templated single-base insertion276 that have been observed at various breakpoint junctions in treatment-related leukemias with MLL translocations. MLL tandem duplications were found to involve Alu repeat elements in 9 of 14 cases, suggesting, though not uniformly, Alu recombinations in this special form of MLL rearrangement.377 Recently it was shown that the intercalating DNA topoisomerase II poison mitoxantrone induces DNA

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topoisomerase II cleavage at a translocation breakpoint hotspot in the PML gene in treatment-related acute promyelocytic leukemia in cases where there was clinical exposure to mitoxantrone. This finding is also consistent with a model of the translocation mechanism in treatmentrelated APL, where the DNA damage leading to the t(15;17) is a consequence of DNA topoisomerase II cleavage.378

Clinical features of treatment-related leukemias Despite the multiagent and multimodality context in which chemotherapy is used, the therapy-related MDS and AML attributed to alkylating agents is recognizable as a distinctive clinical syndrome.15,18 Alkylating agent-related leukemias typically present with antecedent myelodysplasia, have a long latency and the archetypal cytogenetic abnormalities described at length above. Leukemias associated with DNA topoisomerase II inhibitors have heterogeneous presentations, which generally are characteristic of particular chromosomal translocations. Epipodophyllotoxin-related leukemias with MLL translocations usually are FAB-M4 (myelomonocytic) or FAB-M5 (monoblastic) variants but other AML subtypes, MDS, and ALL also may occur.93,246,379 The morphologic heterogeneity in presentations of MLL-rearranged leukemias is a reflection of the partner gene. Cases with t(11;16) fusing MLL with CBP (CREB binding protein) often present as MDS, similar to CMMoL.290,291,321,380 Cases with t(4;11) fusing MLL with AF-4 present as ALL.133,275–277,381 The archetypal presentation of AML with t(8;21) is that of FAB M2 AML,39,91,132,157,230–237 but variant translocations disrupting the AML1 (CBFA2) gene at chromosome band 21q22 have heterogeneous presentations of MDS and AML of other morphologic subtypes.236 The t(15;17) is associated with APL.125,135,146,166,230,238–241,382 Cases with inv(16) and t(16;16) present as FAB M4 myelomonocytic leukemia with eosinophilia.143,166,230,231,240,242 The t(8;16) typically is associated with FAB M4 morphology and prominent erythrophagocytosis.231 The presentations include CML, AML, or ALL when the translocation is t(9;22).243 Treatment-related leukemias may be associated with a preleukemic phase in the early stage of disease that is detected by routine laboratory testing in the follow-up of the first malignancy.187 The cytopenias and peripheral blood monocytosis, which can be interpreted as general chemotherapy effects, may be harbingers of leukemia.130 Many cases of treatment-related AML and MDS present with high percentages of marrow blasts enabling FAB classification.187 However, especially after autologous stem cell transplantation, the treatment-related AML and MDS may not present with high percentages of marrow blasts,

and the presentation includes refractory cytopenias and clonal chromosomal abnormalities.47 A study comparing the clinical presentations in 24 children with treatment-related MDS or AML with those of 960 patients with de novo disease suggested that patients with treatment-related MDS or AML were significantly older at diagnosis, had lower white blood cell counts, were more likely to have MDS and were less likely to have hepatomegaly, splenomegaly or hepatosplenomegaly, and that the leukemias were less likely to exhibit classic AML translocations than were the de novo cases.152

Treatment options for treatment-related leukemias The treatment options for treatment-related leukemias and MDS include a range of approaches or combinations of approaches that include cytotoxic chemotherapy, hematopoietic stem cell transplantation, differentiating agents, palliation/supportive care, and targeted therapeutics.14,125,149,187,229,383–395 Evaluation of the efficacy of these treatment options is confounded by the general lack of clinical trials or series of uniformly treated patients.152,396 Prior treatment using aggressive regimens for the primary malignancy is an important determinant of the ability to administer further intensive therapy in the leukemia treatment.152 In both adults and children with treatment-related AML and MDS, the use of intensive therapy is advantageous; nonetheless, it is well recognized that treatment-related leukemias respond less well to either chemotherapy or hematopoietic stem cell transplantation than do de novo cases. Early results on patients with Hodgkin disease who developed treatment-related AML suggested some successes with hematopoietic stem cell transplantation in younger patients transplanted in remission, but the deaths from complications were significant.397,398 One retrospective analysis found a mean complete remission rate among adults with treatment-related leukemia of 30.7% and a mean 2-year disease-free survival with allogeneic bone marrow transplantation of 19%.391 More recent studies have reported long-term disease-free survival rates with allogeneic stem cell transplantation for treatment-related AML/MDS of about 30%, depending on the regimen.399,400 The value of allogeneic transplantation for treatmentrelated AML extends to transplantation with an HLAmatched unrelated donor in patients less than 35 years old.401 A recent workshop retrospectively gathered outcome data on mostly adults with therapy-related AML and MDS.14 Although the regimens varied, there was an

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overall survival advantage in patients undergoing intensive therapy with hematopoietic stem cell transplantation (n = 67) compared with intensive therapy without hematopoietic stem cell transplantation (n = 289).14 The median survival was 15 months (95% CI, 10.3–30.9 months) and the 5-year survival 26.7% in the former group, compared with a median survival of 10 months (95% CI, 8.0–12.0 months) and a 5-year survival of 16.2% without transplantation.14 Chromosome 5 and/or 7 abnormalities were associated with a median survival of only 7 months (95% CI, 6.0–9.9 months) and overall survival rates at 1, 2, and 5 years of 28.5%, 12.5%, and 3.2%, respectively.14 In contrast, in cases without these abnormalities, the median survival was 10 months (95% CI, 8.0– 12.0 months) with overall survival rates at 1, 2, and 5 years of 42.6%, 42.6%, and 19.3%, respectively.14 Others have suggested that the genetic pathways distinguishing alkylating agent-related leukemias with −7/del(7q) without chromosome 5 abnormalities from those cases with −5/del(5q) and unbalanced chromosome 5 translocations may have an impact on prognosis and that, although the outcome is usually unfavorable, patients with treatment-related MDS with monosomy 7 as the sole abnormality without excess blasts may have relatively prolonged survival.187 The heterogeneity in outcome associated with treatment-related MDS with monosomy 7 or del(7q) extends further to a subset of children who achieve spontaneous hematologic and cytogenetic improvement without any therapy; however, the features predicting this clinical outcome are not well understood.402,403 Transient MDS with monosomy 7 has been described after regimens incorporating G-CSF in the primary cancer treatment, suggesting that management by observation may be indicated in this setting.390 In contrast, leukemias with chromosome 5 abnormalities and the associated molecular aberrations, especially TP53 loss of heterozygosity, are uniformly associated with an extremely poor prognosis.187 It also has been recognized that treatment-related leukemias with MLL translocations are associated with a grave prognosis with significantly shorter median survival than leukemias with 21q22 abnormalities, inv(16), or t(15;17).14 Gene expression profiling has shown that the presence of the t(8;21), inv(16), or t(15;17) drives the clustering of AML into unique, genetically defined, favorable prognostic subgroups.404,405 The more favorable responses to therapy in cases with t(8;21), inv(16), and t(15;17) are similar to de novo cases with the same translocations.125,231,242,398,406 Most cases of treatment-related MDS or AML in the pediatric population likewise prove resistant to the current therapeutic options.149,152,229 In two retrospective

pediatric reviews of epipodophyllotoxin-related AML, the long-term survival rates were 10% to 20%, and the few survivors generally were managed with allogeneic bone marrow transplantation.149,229 In one retrospective pediatric study of allogeneic bone marrow transplantation for treatment-related MDS or AML following primary ALL, the 3-year disease-free survival was 19%.407 The 2-year disease-free survival was 24% (95% CI, 5–53%) in another retrospective study of allogeneic bone marrow transplantation for treatment-related MDS or AML in children.408 In the CCG 2891 study, patients with treatment-related MDS or AML were randomly assigned to receive standardtiming or intensive-timing five-drug induction therapy with dexamathasone, cytarabine, thioguanine, etoposide, and daunorubicin – with or without G-CSF. The 50% induction rate in 24 children with treatment-related MDS or AML was significantly worse than the 72% induction rate in children with de novo disease.152 However, disease-free survival was similar (45% versus 53%) if induction was achieved.152 The overall long-term survival rates with standard-timing versus intensive-timing induction therapy were 0% and 32%, respectively, suggesting that intensive-timing induction can improve long-term survival.152 Intensive chemotherapy and hematopoietic stem cell transplantation are not options for elderly patients or patients heavily pretreated for their primary disease. Lowdose chemotherapy with melphalan occasionally has been used successfully and may prove an option in some cases.409 The use of low-dose cytarabine as a differentiating agent has been disappointing as an alternative in this population because of a significant frequency of toxic deaths from myelosuppression and the short overall survival (∼3 months) comparable to that with supportive care.410 Other approaches to MDS have used the differentiating agent arsenic trioxide or the combined differentiating agents all-trans-retinoic acid and erythropoietin, or G-CSF and erythropoietin,393–395 but efficacy in treatment-related cases remains to be determined. Hematopoietic stem cell transplantation with reduced intensity conditioning also merits further study.411,412 Durable remissions consistent with a graft-versusleukemia effect were recently reported in adults with de novo or treatment-related AML and MDS, using the strategy of reduced intensity conditioning with hematopoietic stem cell transplantation as consolidation therapy in first remission; results were especially promising for the three patients with treatment-related AML or MDS included in the series.412 Experience in pediatric de novo MDS suggests that allogeneic bone marrow transplantation can be performed without conditioning, at least in some cases.413

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Because of the poor treatment outcomes and the toxicities associated with intensive chemotherapy and hematopoietic stem cell transplantation in patients with treatment-related leukemias, molecularly targeted approaches are especially attractive. The development of targeted therapeutics is a major research focus, and preclinical and clinical investigations are being actively pursued. The first example of a targeted agent that has proven useful for at least a subset of patients with de novo and treatmentrelated leukemia is all-trans-retinoic acid, which has a role in therapy for APL.125 The tyrosine kinase inhibitor imatinib mesylate targeting BCR-ABL now is used routinely to effectively induce remission in newly diagnosed BCR-ABL (+) CML.?? This agent may enable reduction in the intensity of conditioning for hematopoietic stem cell transplantation?? and, although heretofore unstudied in treatment-related CML, it may prove highly relevant in heavily pretreated patients. Imatinib also has been tested in BCR-ABL (+) ALL.??,?? This kinase inhibitor targets other tyrosine kinases that are deregulated in leukemia and has resulted in complete remission in a case c-Kit(+) refractory secondary AML.??

Future directions Treatment-related leukemias are generally clinically aggressive, and reported long-term survival rate are poor. Preventative strategies and better treatment options are both needed. Progress in pharmacogenomics directed at clarification of host risk factors for treatment-related leukemia, and unraveling the DNA damage and repair mechanisms that underlie the chromosomal aberrations resulting from anticancer treatment with different classes of cytotoxic drugs together offer future hope for identifying at-risk individuals and arriving at safer regimens. In addition, further elucidation of the molecular basis of the various forms of treatment-related leukemia and their de novo counterparts is expected to inform new molecularly targeted therapies that are not only more efficacious but also less toxic. Progress in this area has established paradigms for the rapid translation of knowledge on leukemogenesis into preclinical testing and, ultimately, the clinical usage of molecularly targeted agents, examples of which were described above. Other promising preclinical data showing sensitivity to various tryosine kinase inhibitors suggest that FLT3 may prove an important therapeutic target in patients with MLL-rearranged leukemias with FLT3 mutations.348,?? The leukemia-specific fusion proteins from chromoso-

mal translocations disrupting various transcription factors offer “vistas” for novel, disease-specific drug design.?? Chromosomal translocations creating leukemia-specific fusion proteins via breakage within introns and the generation of unique in-frame fusion transcripts may afford opportunities for new targeting of mRNA with antisense, ribozymes or RNA interference, or other custom therapeutic agents.?? Several investigators have now shown that experimental downregulation of translation of the MLL fusion proteins MLL-AF-9, MLL-ENL, MLL-ELL, and MLLCBP with various antisense oligodeoxynucleotides is possible in the laboratory.??–?? The finding of hypermethylation of specific genes such as p15/p16218 and DAP-kinase221 in treatment-related MDS may support a role for demethylating agents such as 5-aza–2 -deoxycytidine in the therapy of treatment-related AML/MDS, and there are data indicating that this agent can result in clinical responses.?? Burgeoning proteomic approaches that complement genomics are expected to accelerate these efforts.

REFERENCES 1 Flannery, J. T., Boice, J. D., Jr., Devesa, S. S., et al. Cancer registration in Connecticut and the study of multiple primary cancers, 1935–1982. Natl Cancer Inst Monogr, 1985; 68: 13–24. 2 Smith, M. A., McCaffrey, R. P., & Karp, J. E. The secondary leukemias: challenges and research directions. JNCI, 1996; 88: 407–18. 3 Felix, C. A. Secondary leukemias induced by topoisomerase targeted drugs. Biochimica et Biophysica Acta, 1998; 1400: 233– 55. 4 Felix, C. Chemotherapy-related second cancers. In A. I. Neugut, A. T. Meadows, & E. Robinson, eds., Multiple Primary Cancers: Incidence, Etiology, Diagnosis and Prevention (Baltimore, MD: Williams & Wilkins, 1999), pp. 137–64. 5 Meadows, A. T., Baum, E., Fossati-Bellani, F., et al. Second malignant neoplasms in children: an update from the late effects study group. J Clin Oncol, 1985; 3: 532–8. 6 Kaldor, J. Second cancer following chemotherapy and radiotherapy. An epidemiological perspective. Acta Oncol, 1990; 29: 647–55. 7 Donaldson, S. S. & Hancock, S. L. Second cancers after Hodgkin’s disease in childhood. N Engl J Med, 1997; 334: 792–3. 8 Wolden, S. L., Lamborn, K. L., Cleary, S. F., Tate, D. J., & Donaldson, S. Second cancers following pediatric Hodgkin’s disease. J Clin Oncol, 1998; 16: 536–44. 9 Ron, E., Lubin, J. H., Shore, R. E., et al. Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res, 1995; 141: 259–77. 10 Travis, L. B., Curtis, R. E., Boice, J. D., Jr., et al. Second malignant neoplasms among long-term survivors of ovarian cancer. Cancer Res, 1996; 56: 1564–70.

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368 Betti, C. J., Villalobos, M. J., Diaz, M. O., & Vaughan, A. T. M. Apoptotic triggers initiate translocations within the MLL gene involving the nonhomologous end joining repair system. Cancer Res, 2001; 61: 4550–5. 369 Sim, S.-P. & Liu, L. F. Nucleolytic cleavage of the mixed lineage leukemia breakpoint cluster region during apoptosis. J Biol Chem, 2001; 276: 31 590–5. 370 Betti, C. J., Villalobos, M. J., Diaz, M. O., & Vaughan, A. T. Apoptotic stimuli initiate MLL-AF9 translocations that are transcribed in cells capable of division. Cancer Res, 2003; 63: 1377–81. 371 Super, H. G., Strissel, P. L., Sobulo, O. M., et al. Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia. Genes Chromosomes Cancer, 1997; 20: 185–95. 372 Felix, C. A., Kim, C. S., Megonigal, M. D., et al. Panhandle PCR amplifies genomic translocation breakpoint involving unknown partner gene. Blood, 1997; 90: 4679–86. 373 Gillert, E., Leis, T., Repp, R., et al. A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells. Oncogene, 1999; 18: 4663–71. 374 Felix, C. A., Hosler, M. R., Slater, D. J., et al. Duplicated regions of AF-4 intron 4 at t(4;11) translocation breakpoints. Molecular Diagnosis, 1999; 4: 269–83. 375 Whitmarsh, R., Saginario, C., Zhuo, Y., et al. Reciprocal DNA topoisomerase II cleavage events at 5 ;-TATTA-3 sequence in MLL and AF-9 create homologous single-stranded overhangs that anneal to form der(11) and der(9) genomic breakpoint junctions in treatment-related AML without further processing [abstract]. Blood, 2002; 100(Suppl. 1): 530–1a. 376 Jackson, S. P. Sensing and repairing DNA double-strand breaks. Carcinogenesis, 2002; 23: 687–96. 377 Wiedemann, L. M., MacGregor, A., & Caldas, C. Analysis of the region of the 5 end of the MLL gene involved in genomic duplication events. Br J Haematol, 1999; 105: 256–64. 378 Mistry, A. R., Felix, C. A., Whitmarsh, R. J., et al. DNA topoisomerase II in therapy-related acute promyelocytic leukemia. N Eng J Med, 2005; 352: 529–38. 379 Smith, M. A., Rubenstein, L., & Ungerleider, R. S. Therapyrelated acute myeloid leukemia following treatment with epipodophyllotoxins: estimating the risks. Med Pediatr Oncol, 1994; 23: 86–98. 380 Satake, N., Ishida, Y., Otoh, Y., et al. Novel MLL-CBP fusion transcript in therapy-related chronic myelomonocytic leukemia with a t(11;16)(q23;p13) chromosome translocation. Genes Chromosomes Cancer, 1997; 20: 60–3. 381 Hunger, S. P., Sklar, J., & Link, M. P. Acute lymphoblastic leukemia occurring as a second malignant neoplasm in childhood: report of three cases and review of the literature. J Clin Oncol, 1992; 10: 156–63. 382 Pedersen-Bjergaard, J. Acute promyelocytic leukemia with t(15;17) following inhibition of DNA topoisomerase II. Ann Oncol, 1995; 6: 751–3. 383 Preisler, H. D., Early, A. P., Raza, A., et al. Therapy of secondary acute nonlymphocytic leukemia with cytarabine. N Engl J Med, 1983; 308: 21–3.

384 Longmore, G., Guinan, E. C., Weinstein, H. J., et al. Bone marrow transplantation for myelodysplasia and secondary acute nonlymphoblastic leukemia. J Clin Oncol, 1990; 8: 1707–14. 385 Cortes, J., O’Brien, S., Kantarjian, H., et al. Abnormalities in the long arm of chromosome 11 (11q) in patients with de novo and secondary acute myelogenous leukemias and myelodysplastic syndromes. Leukemia, 1994; 8: 2174–8. 386 de Witte, T., Suciu, S., Peetermans, M., et al. Intensive chemotherapy for poor prognosis myelodysplasia (MDS) and secondary acute myeloid leukemia (sAML) following MDS of more than 6 months duration. A pilot study by the Leukemia Cooperative Group of the European Organisation for Research and Treatment in Cancer (EORTC-LCG). Leukemia, 1995; 9: 1805–11. 387 O’Donnell, M. R., Long, G. D., Parker, P. M., et al. Busulfan/cyclophosphamide as conditioning regimen for allogeneic bone marrow transplantation for myelodysplasia. J Clin Oncol, 1995; 13: 2973–9. 388 Gardin, C., Chaibi, P., de Revel, T., et al. Intensive chemotherapy with idarubicin, cytosine arabinoside, and granulocyte colony-stimulating factor (G-CSF) in patients with secondary and therapy-related acute myelogenous leukemia. Leukemia, 1997; 11: 16–21. 389 Anderson, J. E., Gooley, T. A., Schoch, G., et al. Stem cell transplantation for secondary acute myeoid leukemia: Evaluation of transplantation as initial therapy or following induction chemotherapy. Blood, 1997; 89: 2578–85. 390 Laver, J. H., Yusuf, U., Cantu, E. S., et al. Transient therapy-related myelodysplastic syndrome associated with monosomy 7 and 11q23 translocation. Leukemia, 1997; 11: 448–55. 391 Applebaum, F. R., Le Beau, M. M., & Willman, C. L. Secondary leukemia. In Hematology 1996. Education program of the American Society of Hematology (Washington, DC: American Society of Hematology, 1996), pp. 33–47. 392 Tohyama, K., Tsutani, H., Wano, Y., et al. Anti-leukemia chemotherapy of high-risk myelodysplastic syndromes. Oncologist, 1997; 2: 160–3. 393 List, A., Beran, M., DiPersio, J., et al. Opportunities for Trisenox (arsenic trioxide) in the treatment of myelodysplastic syndromes. Leukemia, 2003; 17: 1499–507. 394 Stasi, R., Brunetti, M., Terzoli, E., & Amadori, S. Sustained response to recombinant human erythropoietin and intermittent all-trans retinoic acid in patients with myelodysplastic syndromes. Blood, 2002; 99: 1578–84. 395 Negrin, R. S., Stein, R., Doherty, K., et al. Maintenance treatment of the anemia of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor and erythropoietIn evidence for in vivo synergy. Blood, 1996; 87: 4076–81. 396 Rowe, J. M. Therapy of secondary leukemia. Leukemia, 2002; 16: 748–50. 397 Geller, R. B., Vogelsang, G. B., Wingard, J. R., et al. Successful marrow transplantation for acute myelocytic leukemia following therapy for Hodgkin’s disease. J Clin Oncol, 1988; 6: 1558– 61.

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398 Kantarjian, H. M., Estey, E. H., & Keating, M. J. Treatment of therapy-related leukemia and myelodysplastic syndrome. Hematol Oncol Clin North Am, 1993; 7: 81–107. 399 Witherspoon, R. P. & Deeg, H. J. Allogeneic bone marrow transplantation for secondary leukemia or myelodysplasia. Haematologica, 1999; 84: 1085–7. 400 Yakoub-Agha, I., de La Salmoniere, P., Ribaud, P., et al. Allogeneic bone marrow transplantation for therapy-related myelodysplastic syndrome and acute myeloid leukemia: a long-term study of 70 patients-report of the French society of Bone Marrow Transplantation. J Clin Oncol, 2000; 18: 963–71. 401 Arnold, R., de Witte, T., Biezen, A. van, et al. Unrelated bone marrow transplantation in patients with myelodysplastic syndromes and secondary acute myeloid leukemia: an EBMT survey. European Blood and Marrow Transplantation Group. Bone Marrow Transplant, 1998; 21: 1213–16. 402 Mantadakis, E., Shannon, K. M., Singer, D. A., et al. Transient monosomy 7: a case series in children and review of the literature. Cancer, 1999; 85: 2655–61. 403 Leung, E. W., Woodman, R. C., Roland, B., et al. Transient myelodysplastic syndrome associated with isochromosome 7q abnormality. Pediatr Hematol Oncol, 2003; 20: 539–45. 404 Bullinger, L., Dohner, K., Bair, E., et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med, 2004; 350: 1605–16. 405 Valk, P. J., Verhaak, R. G., Beijen, M. A., et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med, 2004; 350: 1617–28. 406 Grimwade, D., Walker, H., Oliver, F., et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood, 1998; 92: 2322–33. 407 Hale, G. A., Heslop, H. E., Bowman, L. C., et al. Bone marrow transplantation for therapy-induced acute myeloid leukemia in children with previous lymphoid malignancies. Bone Marrow Transplant, 1999; 24: 735–9. 408 Leahey, A. M., Friedman, D. L., & Bunin, N. J. Bone marrow transplantation in pediatric patients with therapy-related myelodysplasia and leukemia. Bone Marrow Transplant, 1999; 23: 21–5. 409 Anargyrou, K., Vaiopoulos, G., Terpos, E., et al. Low dose melphalan is a treatment option in elderly patients with high risk myelodysplastic syndrome or secondary acute myeloblastic leukaemia. Haematologia (Budap), 2002; 32: 169–73. 410 Ballen, K. K. & Antin, J. H. Treatment of therapy-related acute myelogenous leukemia and myelodysplastic syndromes. Hematol Oncol Clin North Am, 1993; 7: 477–93. 411 Fung, H. C., Cohen, S., Rodriguez, R., et al. Reducedintensity allogeneic stem cell transplantation for patients whose prior autologous stem cell transplantation for hematologic malignancy failed. Biol Blood Marrow Transplant, 2003; 9: 649–56. 412 Taussig, D. C., Davies, A. J., Cavenagh, J. D., et al. Durable remissions of myelodysplastic syndrome and acute myeloid

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leukemia after reduced-intensity allografting. J Clin Oncol, 2003; 21: 3060–5. Hasle, H., Kerndrup, G., Yssing, M., et al. Intensive chemotherapy in childhood myelodysplastic syndrome. A comparison with results in acute myeloid leukemia. Leukemia, 1996; 10: 1269–73. Goldman, J. M. & Melo, J. V. Chronic myeloid leukemia – advances in biology and new approaches to treatment. N Eng J Med, 2003; 349: 1451–64. Scheuring, U. J., Pfeifer, H., Wassmann, B., et al. Early minimal residual disease (MRD) analysis during treatment of Philadelphia chromosome/Bcr-Abl-positive acute lymphoblastic leukemia with the Abl-tyrosine kinase inhibitor imatinib (STI571). Blood, 2003; 101: 85–90. Ottmann, O. G., Druker, B. J., Sawyers, C. L., et al. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood, 2002; 100: 1965–71. Kindler, T., Breitenbuecher, F., Marx, A., et al. Sustained complete hematologic remission after administration of the tyrosine kinase inhibitor imatinib mesylate in a patient with refractory, secondary AML. Blood, 2003; 101: 2960–2. Spiekermann, K., Dirschinger, R. J., Schwab, R., et al. The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3. Blood, 2003; 101: 1494–504. Karamouzis, M. V., Gorgoulis, V. G., & Papavassiliou, A. G. Transcription factors and neoplasia: vistas in novel drug design. Clin Cancer Res, 2002; 8: 949–61. Rabbitts, T. H. & Stocks, M. R. Chromosomal translocation products engender new intracellular therapeutic technologies. Nat Med, 2003; 9: 383–6. Kawagoe, H., Kawagoe, R., & Sano, K. Targeted downregulation of MLL-AF9 with antisense oligodeoxyribonucleotide reduces the expression of the HOXA7 and -A10 genes and induces apoptosis in a human leukemia cell line, THP-1. Leukemia, 2001; 15: 1743–9. Niitsu, N., Hayashi, Y., & Honma, Y. Downregulation of MLLCBP fusion gene expression is associated with differentiation of SN-1 cells with t(11;16)(q23;p13). Oncogene, 2001; 20: 375– 84. Johnstone, R. W., Gerber, M., Landewe, T., et al. Functional analysis of the leukemia protein ELL: evidence for a role in the regulation of cell growth and survival. Mol Cell Biol, 2001; 21: 1672–81. Akao, Y., Mizoguchi, H., Misiura, K., et al. Antisense oligodeoxyribonucleotide against the MLL-LTG19 chimeric transcript inhibits cell growth and induces apoptosis in cells of an infantile leukemia cell line carrying the t(11;19) chromosomal translocation. Cancer Res, 1998; 58: 3773–6. Daskalakis, M., Nguyen, T. T., Nguyen, C., et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2 -deoxycytidine (decitabine) treatment. Blood, 2002; 100: 2957–64.

32 Infectious disease complications in leukemia Jeremy A. Franklin and Patricia M. Flynn

Introduction Children with leukemia experience the common infections of childhood – such as upper respiratory tract infection, otitis media, and gastroenteritis – which are generally managed in the same manner as those occurring in the immunocompetent host. However, the immunocompromised status of many of these children leaves them susceptible to various opportunistic infections, which generally occur at times when the host defense mechanisms are at nadirs of efficiency. This chapter reviews the factors that contribute to infectious complications in patients with leukemia and suggests strategies for the prevention and management of these infections.

Defects in the host defense mechanisms associated with leukemia Numerous factors contribute to the decreased efficacy of the host defense mechanisms in patients with leukemia. The malignancy itself and the therapeutic modalities necessary for a successful outcome can affect the physical barriers to infection, impair immune system functioning, disrupt cytokine mediators, and alter the normal microbiological flora. A major defense against infection is the integrity of the mucosal membranes and the integument, which act as biologic barriers against potential pathogens. Breaches in these barriers provide ready access for endogenous microbiological flora and other opportunistic pathogens. Venipuncture, catheter entry sites, bone marrow aspirates, mucosal ulcerations, and mucosi-

tis are common defects in these important barriers encountered in patients with leukemia. A variety of deficiencies may occur in the immune system of patients with leukemia, including impaired humoral antibody responses, impaired cell-mediated immunity, quantitative and qualitative phagocytic defects, and the disruption of cytokine mediators. Much of the immune system dysfunction seen in patients with leukemia is actually due to therapy rather than the malignancy itself. For example, systemic corticosteroids alter both humoral and cellular immune responses, decreasing phagocytosis and neutrophil migration. Numerous chemotherapeutic agents have well-defined adverse effects on the host immune system. As examples, methotrexate, cyclophosphamide, and 6-mercaptopurine can cause leukopenia and impair humoral responses, while azathioprine affects the primary and secondary immune responses in addition to causing leukopenia. Irradiation impedes both cellular and humoral immunity. Alterations in the endogenous microbiological flora due to antibiotic therapy may allow one or more species to increase to levels that may result in an opportunistic infection. The single most important and readily available measurement of susceptibility to infection is the absolute neutrophil count. Neutropenia with counts of 500 cells/ L or less has become firmly established as a risk factor for infection, with the frequency and severity of the infection being inversely related to the absolute neutrophil count. In practice, patients with leukemia who have an infection often are categorized into two groups: those who are neutropenic and those who are non-neutropenic. The treatment phase and status of the primary disease (i.e. induction of remission,

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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remission, and relapse) should also be taken into account. The remission induction phase is a time of high vulnerability to infection and is generally comparable to periods of relapse with regard to the causes of infectious diseases. The high frequency of febrile episodes in neutropenic patients with leukemia has led to use of the term “febrile neutropenia” as a diagnostic entity. Although this label provides a practical approach to the management of such patients, it fails to recognize the causative factor. Because the management of febrile episodes in neutropenic patients is discussed in a subsequent section, consideration will be given here to those infections that may occur in patients with adequate neutrophil counts. Such patients are usually in remission and have absolute neutrophil counts of 1000 cells/ L or greater. However, it must be emphasized that any infection that occurs in a nonneutropenic patient may also occur in the neutropenic patient under some circumstances. The immunosuppressed patient who is not neutropenic may have impaired cell-mediated immunity. The considerable knowledge gained from the treatment of AIDS patients has been applied to the treatment of children with cancer. Especially useful is the number and percentage of CD4 Tlymphocytes as a measure of susceptibility to certain infections. For example, AIDS patients with CD4 cell counts of less than 200 cells/ L are at high risk for Pneumocystis jiroveci (previously Pneumocystis carinii) pneumonitis, and those with counts of less than 75 cells/ L are at high risk for Mycobacterium avium complex infections.1 Unfortunately, similar correlations have not been made in children with leukemia, but several factors at play in these patients, including the use of purine antagonists, corticosteroid use, cyclosporine use, irradiation, and malnutrition, may diminish T-lymphocyte activity.

Viral infections The viruses most often involved in the infections seen in children with leukemia are listed in Table 32.1. The most common viral infections in non-neutropenic leukemia patients are due to respiratory tract viruses; however, it is the herpesviruses that pose the greatest threat to these children. Cytomegalovirus (CMV) may cause pneumonia, retinitis, gastroenteritis, hepatosplenomegaly, and a heterophile-negative infectious mononucleosis-like syndrome; however, CMV rarely causes serious disease in leukemia patients who have not undergone bone marrow transplantation (BMT). Of 9029 autopsies of cancer patients at M.D. Anderson Cancer Center who had not received a transplant, only 20 showed histologic evidence

Table 32.1 Viruses most often infecting children with leukemia Respiratory tract viruses Adenovirus Parainfluenza viruses Influenza virus Respiratory syncytial virus Rhinovirus Herpesviruses Varicella-zoster virus Herpes simplex virus Cytomegalovirus Epstein–Barr virus Human herpesvirus 6 Molluscum contagiosum Hepatitis viruses A, B, and C

of CMV pneumonia, representing a frequency of 2.2 cases per 1000 autopsies.2 Human herpesvirus 6 (HHV-6) causes roseola (exanthem subitum) in infants and children, may cause febrile seizures, and has been reported to cause fatal pneumonitis and encephalitis in immunosuppressed patients.3 HHV-6 infection also occurs frequently in organ transplant patients but is not associated with clinical disease unless accompanied by concomitant CMV infection.4 In addition to the infectious mononucleosis syndrome experienced by normal persons infected with Epstein–Barr virus (EBV), malignant lymphoproliferative disease may occur in severely immunosuppressed patients, such as those undergoing BMT and those receiving cyclosporine.5 Varicella is the primary infection produced by varicellazoster virus (VZV), while herpes zoster represents an activation of the latent and persistent VZV infection. The clinical features of varicella and zoster in cancer patients are similar to those in otherwise normal individuals, with the exception that systemic involvement of deep organs is more frequent.6,7 Complications of varicella include pneumonitis,8 meningoencephalitis,8 hepatitis, nephritis, “atypical generalized zoster,”8 and secondary bacterial infections. Most fatalities are associated with varicella pneumonitis.

Bacterial infections Most bacterial infections that occur in non-neutropenic children with leukemia are the same as those encountered in healthy children. These include otitis media, pharyngitis, and pneumonitis due to Streptococcus pneumoniae, group A -hemolytic streptococcus, and Hemophilus influenzae;

Infectious disease complications in leukemia

impetigo and cellulitis due to Staphylococcus aureus and group A -hemolytic streptococcus; urinary tract infection due to Escherichia coli; and enteritis caused by Clostridium difficile, Salmonella spp., Yersinia spp., Shigella spp., enteropathogenic E. coli, and Campylobacter spp. These and other bacterial infections are treated in the same manner as those in otherwise normal children. Occasionally, the bacterial infections that are encountered in the neutropenic patient will occur in leukemia patients with normal neutrophil counts (see Tables 32.2 and 32.3 and the section on Infections in the neutropenic patient). It must be emphasized that any of the bacterial infections that affect the neutropenic patient could also occur in the nonneutropenic patient. At particular risk are children with indwelling central catheters, those with lesions that break the normal mucosal and skin barrier, and those with a severely compromised immune system for reasons other than inadequate neutrophil counts.

Fungal infections Most of the systemic fungal infections in leukemic patients occur during periods of profound neutropenia. These infections are usually caused by Candida and Aspergillus species; however, many less common fungi, such as Curvularia, Alternaria, Trichosporon asahii, and a multitude of others, have been implicated in invasive fungal disease in patients with hematologic malignancies. Infections due to Candida and Aspergillus in neutropenic patients are emphasized later in this chapter. Although non-neutropenic patients rarely experience systemic fungal infections, when such infections do occur the causative fungi are usually different from those found in neutropenic patients. Cryptococcus neoformans, Histoplasma capsulatum, and Coccidiodes immitis cause systemic mycoses in otherwise normal immunocompetent individuals as well as in non-neutropenic and neutropenic children with leukemia.

Protozoan infections Pneumocystis jiroveci, Cryptosporidium parvum, and Toxoplasma gondii are three protozoan organisms that cause little, if any, disease in normal persons but may cause severe illnesses in children with leukemia. (P. jiroveci has been classified as a fungus based on DNA sequence homologies; however, based on morphologic and biologic similarities, some experts classify P. jiroveci as a protozoa.) Impairment of cell-mediated immunity is the major risk factor for these infections.

807

Table 32.2 Causes of the most common serious bacterial infections and drugs for their treatment Bacteria

Drugsa

Bacteroides spp.

Clindamycin, metronidazole, chloramphenicol Metronidazole, clindamycin, penicillin Vancomycin, ceftriaxone Meropenem, aminoglycoside ± 3rdgeneration cephalosporin Ampicillin + aminoglycoside, vancomycin + aminoglycoside 2nd- or 3rd-generation cephalosporin, aminoglycoside, meropenem Ampicillin, metronidazole, clindamycin 2nd- or 3rd-generation cephalosporin, meropenem Ampicillin ± aminoglycoside, TMP/SMX TMP/SMX, amikacin, meropenem Penicillin, clindamycin, tetracycline Cefotaxime, ceftriaxone, meropenem Antipseudomonal -lactam + aminoglycoside, meropenem Ceftriaxone, cefotaxime, ampicillin Nafcillin, vancomycin Vancomycin, nafcillin

Clostridium spp. Corynebacterium spp. Enterobacter spp. Enterococcus spp. Escherichia coli Fusobacterium spp. Klebsiella spp. Listeria monocytogenes Nocardia asteroids Propionibacterium spp. Proteus spp. Pseudomonas aeruginosa Salmonella spp. Staphylococcus aureus Staphylococcus, coagulase negative Streptococcus pneumoniae Streptococcus pyogenes (group A) Viridans streptococci group

Penicillin, ceftriaxone, vancomycin Penicillin, clindomycin, macrolides Vancomycin, penicillin + aminoglycoside

a

Drug of choice underlined, unless antibiotic susceptibility tests indicate resistance.

Topographic classification of infections Patients often have infections that are localized to an organ or system, leading to pneumonia, meningitis, hepatitis, cellulitis, enteritis, urinary tract infection, or others. Localization is less likely in patients who are neutropenic than in those able to elicit an acute inflammatory response at the infected site.

Infections of the integument Patients with leukemia are at increased risk of cutaneous infections from bacteria, viruses, and fungi. In addition to the suppression of the host immune response as a result of chemotherapy, the intergrity of the integument as a primary defensive barrier against infection is often

Table 32.3 Most common bacterial infections in children with cancer Escherichia Klebsiella Enterobacter

Staphylococcus

Coag. neg.

Enterococcus

Clostridium Corynebacterium Viridans

P. aeruginosa

coli

spp.

spp.

aureus

Staph. spp. pneumoniae

spp.

monocytogenes

spp.

difficile

Bacteremia/sepsis

+

+

+

+

+

+

+

+

+

Ecthyma gangrenosum

+

Type of infection

Pneumonia

+

Meningitis

+

+

+

+

+

+

+

+

+

+

+

Endophthalmitis

+

+

Osteomyelitis

+

+

+

Urinary tract infection

+

+

+

+

+

+

+

+

+ +

+

Mastoiditis

+

+

Thrombophlebitis

+

+

+

+

+

+

Intra-abdominal

+

+

+

+

Otitis media

Endocarditis

Streptococcus Bacillus Listeria

+

+

+

Cellulitis

+

+

+ +

+

+

+

+

+

+

+

+

+

+

+

+ +

+ +

+ +

+

+ +

+

Gastroenteritis

+

+

Lymphadenitis Sinusitis

streptococci

+

infection Septic arthritis

spp.

+

+

Infectious disease complications in leukemia

B

C

E

F

A

D

Fig. 32.1 (A) Target lesions in Pseudomonas aeruginosa sepsis. (B) Ecthyma gangrenosum due to P. aeruginosa. (C) Cutaneous lesions associated with molluscum contagiosum. (D) Cutaneous lesions associated with disseminated candidiasis. (E) CT scan showing two areas of necrosis with an associated infiltrate due to pulmonary aspergillosis. (F) MRI of the brain showing large abscess due to rhinocerebral zygomycosis. (See color plates 32.1A–D for full-color reproduction.)

compromised in these patients by the presence of various foreign bodies, such as long-term venous access devices. Areas of cellulitis usually represent either bacterial or fungal infection. The extent of neutropenia and anemia may determine the features of cellulitis. With severe anemia (hemoglobin <7.0 g/dL), the infected tissue may lack erythema, whereas with severe neutropenia (neutrophil count

<200 cells/ L) induration may not be present due to the lack of an inflammatory response. Cellulitis occurs most frequently in the perineal area and at sites of skin entry for indwelling catheters, venipuncture, finger sticks, and bone marrow aspiration. Lesions due to Pseudomonas aeruginosa have a somewhat characteristic appearance due to the greenish discharge and black eschar formation known

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Table 32.4 Causes of the most common systemic mycoses and drugs for their treatment Fungus

Drugs

Aspergillus spp. Blastomyces dermatiditis Candida spp.

Voriconazole, amphotericin B, caspofungin Itraconazole, amphotericin B, voriconazole Azole compounds, amphotericin B, caspofungin, micafungin Amphotericin B, itraconazole, voriconazole, fluconazole Amphotericn B ± flucytosine, voriconazole Itraconazole, fluconazole, amphotericin B, voriconazole Amphotericin B, posaconazolea Itraconazole, voriconazole, posaconazole Voriconazole, itraconazole, posaconazole

Coccidiodes immitis Cryptococcus neoformans Histoplasma capsulatum Zygomycoses Pseduallescheria boydii Trichosporon beigelii a

Investigational.

as ecthyma gangrenosum (Figs. 32.1A and B). Diagnostic methods include needle aspiration for material to be cultured, stains, and biopsy with punch or surgical excision. Treatment is based on the causative agent, as suggested in Tables 32.2 and 32.4

Herpes simplex virus The usual clinical manifestations of herpes simplex virus (HSV) infections include vesicular or ulcerative lesions of the lips or genitalia, gingivostomatitis with fever, occasionally generalized vesicular skin lesions, and rarely systemic dissemination. Blackish eschars may sometimes form over ulcerative lesions. The diagnosis is made by isolation in tissue culture after 1 to 3 days, direct fluorescent antibody staining of vesicle scrapings, or enzyme immunoassay detection of viral antigens. Intravenous administration of acyclovir is indicated for systemic herpes simplex disease. Oral or intravenous acyclovir may be used for extensive cutaneous and mucosal lesions. The intravenous dose is 750 mg/m2 per day in three divided doses; the oral dose is 80 mg/kg per day in four divided doses. Oral valacyclovir9 and famciclovir10 may also be considered for cutaneous and mucosal lesions. Cidofovir may be considered in cases of drug-resistant HSV infection.8 Contact precautions are recommended.

pes zoster can be made on the basis of characteristic cutaneous lesions. In questionable cases, the organisms can be cultured from vesicular fluid or identified by immunofluorescent staining with monoclonal antibodies. The less sensitive Tzanck smear shows multinucleated giant cells containing intranuclear inclusions. Intravenous acyclovir has been the treatment of choice for VZV infections in leukemic patients.11 Usually, a dose of 500 mg/m2 every 8 hours is given intravenously for about 7 days. Experience with oral acyclovir for the treatment of varicella is limited, and studies are inadequate to recommend the formulation. However, oral acyclovir has been successfully used for the treatment of uncomplicated herpes-zoster virus infections.12 Oral administration of valacyclovir results in bioavailability three to five times greater than that of oral acyclovir,13 and its use has been successfully demonstrated in herpes zoster.14 Famciclovir has also been used effectively in the therapy of varicella-zoster.15 Cidofovir has been used in the therapy of drug-resistant VZV infections.16 Patients with varicella-zoster infections are highly contagious and must be kept in isolation.17 Aspirin should never be given because of its association with Reye syndrome in patients with varicella or influenza.18 Patients who are susceptible to varicella and become exposed to either varicella or zoster should be given varicella zoster immune globulin (VZIG) within 96 hours of exposure. Limited studies support the use of oral acyclovir for postexposure prophylaxis to varicella-susceptible patients,19,20 although these data are inadequate for firm recommendations. The greatest risk for infection comes from close household and classroom exposures.

Molluscum contagiosum Lesions of molluscum contagiosum vary from tiny nodular lesions to large confluent lesions (Fig. 32.1C). The typical lesion produced by this virus is a round, flesh-colored, smooth, umbilicated papule associated with many other lesions of the same type. In immunosuppressed cancer patients, the lesions are more extensive, generalized, and persistent than in the normal host. Diagnosis is often obvious from the typical lesion with a dimpled bit containing creamy material consisting of globular or nucleated Henderson-Paterson inclusion cells. A biopsy is diagnostic. No satisfactory treatment has been established. Topical cidofovir appears promising.21

Varicella-zoster virus Herpes zoster represents activation of latent VZV. Initial lesions are limited to a dermatomal distribution, but generalized skin lesions and deep organ involvement may occur in some cases. The diagnosis of varicella and her-

Infections of the bloodstream Infection of the bloodstream is not uncommon in patients with hematologic malignancies, particularly

Infectious disease complications in leukemia

during periods of neutropenia. In fact, 10% to 20% of cases of febrile neutropenia have a documented bacteremia.22 Because of the various predisposing factors discussed previously, microorganisms can more readily penetrate the integument or translocate across mucosal barriers or invade the bloodstream from other sites of infection. In addition, the increased use of central venous access devices has contributed to the increased incidence of bacteremia, particularly bloodstream infections related to gram-positive organisms and yeast-type fungi. Occult bacteremia in patients with hematologic malignancies is relatively common; however, neither clinical observation nor rapidly available laboratory tests have proven useful in distinguishing between patients with or without bacteremia. Bacteremia in the non-neutropenic leukemia patient is diagnosed and treated in much the same way as bacteremia in immunocompetent children. Empiric therapy for presumed bacteremia has traditionally been directed towards gram-negative bacteria due to the potentially high morbidity and mortality associated with these infections. The widespread use of thirdgeneration cephalosporins, most commonly ceftazidime, as empiric therapy has contributed to the emergence of resistance in some bacteria while the patient is receiving therapy. The fourth-generation cephalosporin cefepime is useful in preventing breakthrough infections due to these bacteria. Unless a specific risk factor for infection due to a gram-positive organism exists, empiric antibiotics directed at gram-positive organisms are not typically initiated. The incidence of bacteremias due to gram-positive organisms, such as Staphylococcus aureus, Streptococcus spp., and coagulase-negative staphylococci is increasing; however, infections due to these particular organisms tend to produce less morbidity and mortality than infections due to gram-negative organisms.22 Studies have shown no increased morbidity or mortality due to delayed initiation of gram-positive antimicrobial coverage in patients with occult bacteremia without an obvious risk factor for infection due to gram-positive organisms.22 Catheter-related bloodstream infections are an increasingly prevalent entity among patients with hematologic malignancies. Children with leukemia frequently have long-term tunneled central venous lines to ensure reliable venous access while minimizing the number of needle sticks they must receive. Catheter-related bloodstream infection is a complicated issue. Differentiating between bloodstream infections related or not related to central venous lines is a difficult task. Currently, cultures of approximately 75% to 85% of all catheter tips from central venous lines, removed due to suspected

catheter-related bloodstream infection, are negative.23 Numerous methods have been tried to help differentiate catheter-related from catheter-unrelated bloodstream infections, including quantitative comparative cultures,24–26 different times to positivity,27,28 cytocentrifugation and acridine orange staining of blood drawn from the catheter,25 and absolute counts of colony-forming units in blood cultures drawn from the catheter.23,24 The issue of diagnosis and treatment of catheter-related bloodstream infections was addressed in a consensus statement published by the Infectious Disease Society of America (IDSA) in May 2001.23 Efforts aimed at prevention of catheter-related bloodstream infections have included strict attention to aseptic technique by patients and caregivers,26,29 use of preservative-containing catheter flush solutions,26,30–32 use of antibiotic-containing flush solutions,26,33–35 and use of antimicrobial-impregnated catheters.36 Studies of these measures in children remain limited.

Infections of the central nervous system Meningitis Meningitis is surprisingly uncommon in children with leukemia in comparison with infection at other sites. A history of CNS surgery, the presence of a foreign body (such as an Omaya reservoir or a ventriculoperitoneal shunt), or a cerebrospinal fluid (CSF) leak are factors that increase the risk of CNS infection. Patients with neutropenia may present a diagnostic dilemma, as they lack the inflammatory response necessary to elicit signs of meningeal inflammation and white blood cells to produce a CSF pleocytosis. The white cells, protein, and glucose of the spinal fluid may be normal in neutropenic patients, even with advanced bacterial meningitis. Diagnosis requires cultivation of the organism from the cerebrospinal fluid, some causative organisms are listed in Table 32.3. Treatment is based on the causative agent, as summarized in Table 32.2. Coccidioides immitis causes disseminated disease in less than 1% of all cases.37 Meningitis due to Coccidioides is a serious sign of disseminated disease and may be fatal if left untreated. Diagnosis may be made by serology, complement fixation on CSF samples, culture (which is dangerous and should be performed only by highly trained personnel), and a DNA probe. In patients with meningitis, complement fixation on CSF samples may also serve to monitor response to therapy, as persistent or increasing titers indicate progressive disease and declining titers indicate improvement,

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with the caveat that low or undetectable titers in severely immunocompromised hosts can be unreliable and must be interpreted with caution. Therapy for CNS infection is fluconazole.38 For unresponsive infection, intravenous amphotericin B augmented with repeated instillation of amphotericin B into the CSF is indicated. Cryptococcus neoformans may cause many different clinical syndromes, including meningitis. Cryptococcus rarely causes extrapulmonary disease in children with leukemia; however, the most common extrapulmonary site in these children is the CNS.39 Cryptococcal meningitis often follows an indolent course and may present only as very subtle changes in personality. The diagnosis of cryptococcal meningitis can be made by isolating the organism from the CSF; however, since very few organisms are typically present in CSF samples, large quantities of fluid are required. Tests for the cryptococcal antigen on CSF are available, and the antigen is detected in approximately 90% of cases with cryptococcal meningitis.40 Treatment of cryptococcal meningitis should begin with amphotericin B plus flucytosine.40,41 If the patient is responding well, therapy can be changed after 2 weeks to fluconazole for 8 weeks or more. The relapse rate is approximately 50%.40

Encephalitis Encephalitis due to infection is rare in patients with leukemia. Such cases are usually viral in origin, arising primarily from herpes simplex virus (HSV), varicella-zoster virus (VZV), or rubeola.22 Cerebral toxoplasmosis and fungal or bacterial infection must also be considered in the diagnosis. In addition, encephalitis can arise from noninfectious causes, such as neoplastic disease, metabolic derangements, and toxic effects of chemotherapy. Serologies, CSF findings, and CT scans are often nonspecific, and a definitive diagnosis can often be made only by brain biopsy, which is rarely performed. PCR analysis performed on the CSF is a very useful adjunct in the diagnosis of encephalitis due to infections including HSV, VZV, and toxoplasmosis. Although PCR methods to detect infectious causes of encephalitis may not be available commercially, they are often established in governmental and private research labs. Empiric therapy is warranted for cases of encephalitis believed but not proven to have an infectious etiology. The clinical features of cerebral toxoplasmosis include the signs and symptoms of diffuse encephalopathy, meningomyelitis, or localized CNS lesions: fever, lethargy, headache, seizures, and focal neurologic signs. The demonstration of tachyzoites, or cysts of Toxoplasma gondii, in affected tissues or body fluids, the detection of T. gondii DNA by PCR,42 and serologic tests for antibody are help-

ful in establishing the diagnosis. The recommended treatment is a combination of pyrimethamine, 1.0 mg/kg per day (maximum, 25 mg), with double doses for the first 2 days of therapy, and sulfadiazine, 85 mg/kg per day, in two or three doses.43 Folinic acid, 5 mg every 3 days, should be used to prevent folate deficiency.43

Progressive multifocal leukoencephalopathy Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease seen in immunocompromised ˚ str¨om in patients. First described as a clinical entity by A 1958,44 PML presents as focal neurologic deficits (such as aphasia, cranial nerve deficits, and hemiparesis) rapidly progressing to cortical blindness, dementia, and coma.44 PML is due to the JC virus,45 a polyomavirus first isolated in 1971.44 Approximately, 60% to 80% of adults have antibodies to JC virus, which enters a latent phase after the initial infection (usually asymptomatic). The virus can reactivate in patients who are immunosuppressed and causes PML via virus-induced killing of oligodendrocytes.45 A definitive diagnosis of PML due to JC virus requires a brain biopsy with characteristic pathologic findings; in addition, the virus particles can be visualized by electron microscopy. When performed on CSF, PCR for JC virus DNA is positive in 60% to 100% of cases of PML; however, this diagnosis should not be made on the basis of PCR results alone in a patient without a clinical syndrome compatible with PML.44 CT and MRI can be used to help support the diagnosis of PML. Although lesions are present in the white matter, they often appear inadequate to explain the severity of clinical findings; indeed, the dissociation of clinical and radiologic findings suggests the diagnosis of PML.44 Various drugs have been evaluated for the therapy of PML, but none have proved satisfactory. Cidofovir is currently being investigated, but the early results are not encouraging.46,47

Infections of the respiratory tract The most common causes of pneumonia in leukemia patients not receiving bone marrow transplants include bacteria (Staphylococcus aureus, Streptococcus pneumoniae, viridans streptococci, Pseudomonas aeruginosa, E. coli, and Klebsiella spp.), viruses (respiratory syncytial virus, parainfluenza virus, influenza virus, adenoviruses, varicella-zoster virus, and cytomegalovirus), fungi (Candida spp., Aspergillus spp., Histoplama capsulatum, and Coccidioides immitis), and protozoa (Pneumocystis jiroveci). However, almost any of the organisms listed

Infectious disease complications in leukemia

in Tables 32.1–4 can produce pneumonia in patients with leukemia. The chest radiograph must be interpreted in the light of other clinical features of the illness. A few clinical associations may suggest but not prove the etiology. For example, lobar pneumonia with an acute onset in a patient who has not recently received antibiotics may be bacterial, while pneumonia developing in a neutropenic patient after several days of broad-spectrum antibiotics may be fungal. An acute onset of diffuse bilateral pneumonitis in a nonneutropenic patient who is not receiving antibiotic prophylaxis may be due to a respiratory virus (Table 32.1) or to Pneumocystis jirovecii. In endemic areas, non-neutropenic patients in remission who have prolonged pneumonitis unresponsive to antibiotics should be evaluated for histoplasmosis or coccidiomycosis. Diagnostic specimens should include: (1) blood for culture of bacteria and fungi, (2) sputum for culture and cytology, (3) bronchoalveolar lavage fluid for culture and cytology, and (4) lung biopsy specimens for culture and cytology. Cytologic studies include appropriate stains for bacteria (including Legionella and Mycobacterium spp.), fungi, chlamydia, and P. jirovecii. Direct antigen detection and culture for respiratory viruses is also indicated. Serologic tests are of little help for the initial diagnostic approach. The decision to use invasive techniques depends on the status of the patient, the urgency for a definitive diagnosis, and the capabilities for an invasive procedure (either bronchoalveolar lavage or biopsy) and processing of specimens. The respiratory tract viral infections that occur most commonly in leukemia patients are listed in Table 32.1. In a prospective study of children receiving anticancer therapy, 75 consecutive episodes of febrile infections in 32 children were evaluated with sensitive virologic techniques to detect respiratory viruses.48 Evidence for a respiratory viral infection was found in 28 (37%) of these cases. The frequency of infection by specific viruses in febrile cases was: rhinovirus, 17%; respiratory syncytial virus, 8%; parainfluenza virus, 6.7%; adenovirus, 5.3%; and influenza viruses, 5%. These respiratory viruses were found with equal frequency among neutropenic and non-neutropenic patients.

Adenovirus Adenoviruses typically produce respiratory infections with bronchiolitis and pneumonia.49 In the severely immunocompromised host, adenoviral infection can occur in other organ systems and manifest as hepatitis, nephritis,50 colitis, meningoencephalitis,51 carditis,52 and hemorrhagic cystitis;53 in addition, fatal disseminated adenoviral infection is well described and may involve multi-

ple organs.51,54,55 The diagnosis is made by isolation of the virus from infected sites (using conjunctival swabs, nasopharyngeal aspirates, stool, urine, blood, or spinal fluid) in human cell lines (e.g. HEK, HEP-Z, or HeLa cells). A cytopathic effect is seen by the tenth day of culture. Antigen can be directly detected with monoclonal antibodies using an enzyme-linked immunosorbant assay (ELISA) or by other methods. Adenovirus PCR studies are also available. A well-established drug treatment for adenoviral infections is lacking, but a few reports on intravenous ribavirin suggest some beneficial effects from this drug.55 Two studies have shown cidofovir to be efficacious in cases of severe adenoviral infection.56,57

Human parainfluenza viruses Parainfluenza viruses commonly cause bronchiolitis, croup, and pneumonia with epidemics occurring during the fall and early winter. For an accurate diagnosis, nasopharyngeal washes and aspirations are cultured for virus and examined by a direct antigen detection method. There is currently no treatment of proven efficacy. Aerosolized ribavirin has been tried in a few cases with variable success.58,59

Influenza virus Influenza virus may cause fatal infections in severely compromised patients; however, most influenza infections in leukemia patients are similar to those in otherwise normal children. In a study of 25 immunosuppressed patients infected with influenza A, only two had severe infections.60 Feldman et al. 61 described 20 children with cancer and influenza at St. Jude Children’s Research Hospital. The clinical manifestations of influenza were similar to those in otherwise normal children (fever, cough, myalgia), but the clinical course lasted twice as long (2 weeks) as in the general population. No deaths occurred, but three patients had secondary bacterial infections. One study showed the incidence of influenza in cancer patients to be twice that in the normal population.62 Complications of influenza include bacterial pneumonia, primary viral pneumonia, myositis, toxic shock syndrome, and Reye syndrome.63 The diagnosis is established by isolating of influenza A or B virus from nasopharyngeal swabs or washes, pharyngeal gargles, or bronchoalveolar lavage specimens, cultured with primary rhesus monkey kidney (PRMK) or Madin–Darby canine kidney (MDCK) cells. A shell vial technique allows detection of the virus in 72 hours. A new enzyme immunoassay membrane technique can be completed in 15 minutes and is undergoing further evaluation.63 Amantadine and

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rimantadine are approved for the treatment of influenza A infection,64 but should be given within the first 2 days of illness to ensure an adequate therapeutic effect. Thereafter, the drugs may be used prophylactically in exposed susceptible patients. Zanamivir and oseltamivir, which specifically target the neuraminidase activity of influenza, are also effective against influenza A and B.65 Oseltamivir has also been approved by the FDA for prophylaxis of influenza.64

Respiratory syncytial virus Severe respiratory syncytial virus (RSV)-induced disease with increased morbidity may occur in children with leukemia. Most patients have pronounced bronchiolitis or giant cell pneumonia, with prolonged virus shedding for periods of up to 3 months.66 In over half of the immuncompromised patients with RSV infection, the virus is acquired nosocomially. The usual incubation period is 2 to 8 days. RSV can be isolated in cultures of nasopharyngeal secretions within 3 to 8 days. Rapid tests for RSV antigen, including immunofluorescent and enzyme immunoassay techniques, will identify the virus in about 80% to 90% of cases.66 Ribavirin administered by small-particle aerosol spray is the treatment of choice for patients with serious pulmonary disease due to RSV. The drug is administered into an oxygen hood, tent, or mask as a ribavirin solution (20 mg/mL in water) for a period of 12 to 20 hours daily. Efficacy has not been well-established, particularly in children with cancer and RSV infection.67,68 RSV polyclonal immune globulin is commercially available and has been effective in the prevention of RSV disease in high-risk infants; however, there are few data supporting this preparation as a treatment for RSV infection in immunocompromised patients.69,70 The monoclonal antibody preparation palivizumab has been shown to be safe and efficacious in the prevention of RSV disease in high-risk infants71 ; but again, only scant evidence supports the use of palivizumab for RSV infection or prophylaxis in immunocompromised patients.70 Infected patients shedding RSV are highly contagious and should be isolated until they no longer excrete the virus.

Rhinovirus Clinical manifestations of rhinovirus infection include rhinitis, bronchitis, sinusitis, otitis media, and possibly lower respiratory tract disease. The diagnosis is established by culture of rhinovirus from nasal secretions. No specific treatment is available. Pneumocystis jiroveci (previously Pneumocystis carinii) The disease caused by P. jiroveci presents with an acute or subacute onset of tachypnea, dyspnea, cough, and fever. Bilateral diffuse alveolar disease is often but not always

evident on a chest radiograph. Arterial oxygen tension is decreased and the alveolar-arterial oxygen gradient is increased. Without prophylaxis, P. jiroveci pneumonitis occurs in about 20% of acute leukemia cases during therapy. The mortality rate is approximately 100% if the cases are not treated.72 Diagnosis requires the demonstration of P. jiroveci in lung parenchyma or secretions from the lower respiratory tract. Specimens may be obtained by bronchoalveolar lavage or biopsy. Sputum may occasionally contain organisms of diagnostic significance. GrocottGomori, Giemsa, toluidine blue, and fluorescein-labeled antibody stains identify the organisms. Twenty milligrams of trimethoprim with 100 mg of sulfamethoxazole per kg per day in four divided doses, orally or intravenously, is the treatment of choice.73 Alternative regimens include pentamidine isethionate,74 dapsone-trimethoprim,75 atovaquone,76 and trimetrexate with leocovorin.77 The treatment course is usually 21 days. If patients who recover are not given prophylaxis with trimethoprim-sulfamethoxazole, one-third will have a recurrence.78 P. jiroveci pneumonitis can be effectively prevented with 5 mg of trimethoprim and 20 mg of sulfamethoxazole per kg per day. Prophylaxis may be given daily79 or 3 days a week80 and should be administered during the course of immunosuppressive therapy. Histoplasma capsulatum Typically, the clinical features are fever and pulmonary infiltrates with hilar adenopathy. Disseminated infection may occur and usually involves liver, spleen, blood, and bone marrow but may affect any organ of the body. In a study of 30 cases of disseminated histoplasmosis in children with malignancy, 21 (70%) were in remission.81 At the time of diagnosis, 87% were febrile, 45% had experienced weight loss of more than 1 kg, 52% had cough, 25% had hepatosplenomegaly, and 42% had neutrophil counts of 1500/ L or less. A definitive diagnosis requires the isolation of H. capsulatum in culture from infected tissue. The radioimmunoassay for H. capsulatum antigen in urine is a useful aid in the diagnosis of disseminated histoplasmosis.82 Amphotericin B is used for lifethreatening cases, while itraconazole or voriconazole is effective for less severe cases. Guidelines regarding the treatment of histoplasmosis were published by the Infectious Disease Society of America in 2000.83 Coccidioides immitis C. immitis is typically acquired by the respiratory tract, and infection is usually clinically inapparent. Symptomatic disease due to this organism most often resembles influenza. Extrapulmonary disease is rare,37 usually developing as a result of trauma at the site of direct inoculation of the

Infectious disease complications in leukemia

organism. Diagnosis is by serology, skin testing in patients with clinical disease compatible with this organism, culture (which can be hazardous), and DNA probe analysis. Amphotericin B provides effective therapy for disseminated disease (without involvement of the CNS) or pulmonary disease in immunocompromised patients. Azole therapy is effective in mild to moderate cases or with meningitis. Cryptococcus neoformans C. neoformans may present with many different clinical features, such as meningitis, pneumonitis, cutaneous lesions, bone lesions, fever, and disseminated disease; however, only nine cases of extrapulmonary cryptococcosis were reported in children with leukemia during the 25-year period from 1966 to 1992.39 Diagnosis is established by isolation of C. neoformans in culture from infected tissue. Detection of cryptococcal antigen in CSF, blood, or urine offers a sensitive and specific diagnostic tool. Fluconazole has been effective in the treatment of pulmonary and disseminated cryptococcosis in adults, but its use in children has been limited. A course of at least 6 weeks is required for treatment.84,85 Life-threatening or disseminated disease is treated with amphotericin B plus oral flucytosine.

Infections of the orointestinal tract Oral mucositis Antileukemic drugs are frequently associated with toxic effects on mucous membranes. The drug-induced mucositis may be managed with good oral hygiene, pain control, and diets that ensure adequate nutrition. Distinguishing drug-induced mucositis from mucositis due to infectious agents or drug-induced mucositis that has become secondarily infected is an important and difficult task. Specimens should be obtained for special stains and cultures to detect bacteria, viruses, or fungi that require intervention with antimicrobial therapy. Superinfection of drug-induced mucositis by HSV, Candida, and bacteria of the endogenous oral flora is common. Since the etiologic agent cannot be identified from clinical manifestations, swabs of the lesions should be obtained and sent for appropriate staining and culture. Oral candidiasis is treated with nystatin suspension, amphotericin B, clotrimazole troches, or caspofungin.86 HSV gingivostomatitis may be treated with oral acyclovir if the patient is not neutropenic and can tolerate oral medication. In addition, valacyclovir9 and famciclovir10 have proven efficacy against herpes simplex virus and are increasingly being used as

first-line therapy for outpatient therapy in patients who are not neutropenic and are able to tolerate oral medications. Neutropenic patients and patients unable to tolerate oral therapy should receive intravenous acyclovir. Therapy for bacterial superinfection of drug-induced mucositis should be directed by culture results.

Esophagitis In the non-neutropenic patient, infectious esophagitis is rare. The infectious agents implicated in esophagitis in neutropenic leukemia patients are Candida, HSV, and bacteria. Definitive diagnosis requires an endoscopic procedure with biopsies. Due to the intrinsic difficulties in performing such diagnostic procedures, patients are frequently given a trial of empiric antifungal, antiviral, or antibacterial agents, as discussed previously.

Hepatitis Jaundice is a common sign of hepatitis from several microbial and nonmicrobial entities. Excluding drug-induced hepatitis, hepatotropic viruses represent the most common causes of hepatitis. The main hepatotropic viruses include hepatitis viruses A, B, and C, EBV, and CMV. Bacterial sepsis and disseminated fungal infections may affect the liver, but usually the hepatic component is not extensive. The diagnosis of viral hepatitis is based on serologic studies for the hepatitis viruses, EBV, and CMV. Only CMV can be cultured. Hepatitis A87 and hepatitis B88 are preventable by proper immunization. CMV may be effectively treated with ganciclovir, foscarnet, or cidofovir89 ; Chronic hepatitis B may be treated with lamivudine, adefovir, or interferon alfa-zb. Hepatitis C can be treated with pegylated interferon plus ribavirin.

Gastroenteritis It is often difficult to determine whether diarrhea is due to an infection or to chemotherapy. For patients receiving prolonged broad-spectrum antibiotics, pseudomembranous enterocolitis should be considered as well as mucosal candidiasis. The evaluation should include culture of the stool for bacteria and fungi, tests for Clostridium difficile toxin, rotavirus, Cryptosporidium parvum, Giardia lambia, and other parasites, as indicated by clinical circumstances. Treatment is supportive unless a causative organism is identified. Cryptosporidiosis is a disease that causes acute and chronic diarrhea. The diagnosis is established by clinical manifestations together with the demonstration of oocysts of Cryptosporidium parvum in feces. Acid-fast, Giemsa, auramine, and fluorescent monoclonal antibody stains

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may be useful. Nitazoxanide was approved by the FDA in December 2002 as the first available medication for the treatment of diarrheal disease due to Cryptosporidium parvum.90 Nitazoxanide effectively reduces the duration of diarrheal disease and has no significant adverse effects compared to placebo in a randomized controlled trial.90 Clostridium difficile pseudomembranous colitis is a frequent occurrence in leukemic patients. Leukemia patients are frequently exposed to prolonged courses of broadspectrum -lactam antibiotics, posing a major risk factor for this disease. Distinguishing C. difficile colitis from other causes of colitis is not possible on clinical grounds. Detection of the C. difficile toxin B in stool samples is the diagnostic test of choice.91 C. difficile colitis is effectively treated with oral metronidazole given for 10 to 14 days. Oral vancomycin should be reserved for patients who fail to respond to or cannot tolerate metronidazole. Approximately 15% of patients will relapse after “successful” treatment of C. difficile.91

Typhlitis (neutropenic colitis) Typhlitis is a diagnosis unique to the neutropenic patient. Inflammation of the cecum due to gram-negative bacteria of the gut flora may occur in severely neutropenic patients. The diagnosis of typhlitis has become increasingly more common due to the intensification of chemotherapy and an increased awareness of the problem. Patients are febrile and have right-sided or generalized abdominal pain; however, no clinical findings differentiate typhlitis from other abdominal diseases.92 CT scanning reveals distention of the cecum with circumferential thickening of the cecal wall.93 Ultrasound studies show thickening of the bowel wall and marked pseudopolypoid formation of the mucosa. Patients with bowel wall thickening of more than 10 mm on ultrasound examination have a significantly higher mortality rate than those with less pronounced thickening (≤10 mm).94 Recovery of neutrophil counts is an important factor influencing outcome and whether or not surgery may become necessary.95 Treatment includes broad-spectrum antibiotics, empiric antifungal therapy in certain cases, bowel rest, and, in the most severe cases, surgical resection. Rapid diagnosis and initiation of empiric therapy with appropriate broad-spectrum antimicrobials have helped reduce the need for surgical procedures.92,96

Urinary tract infections Urinary tract infections are relatively uncommon among all other infections experienced by children with leukemia.

Signs, symptoms, diagnosis, and treatment are similar to those of otherwise healthy children. However, it should be kept in mind that severely neutropenic patients may not have pyuria with infection.

Infections in the neutropenic patient The major infections in the neutropenic patient are caused by gram-positive cocci, gram-negative bacilli, Candida spp., and Aspergillus spp. However, it must be emphasized that all infections described earlier for non-neutropenic patients, as well as those affecting otherwise normal people, can also occur in neutropenic patients, and most of the myriad bacteria and fungi that colonize the mucosal surfaces and the skin have been implicated in infection of the severely immunocompromised host. Neutropenia represents an ominous prognosis in leukemia patients with an infection; therefore, the approach to management of the severely neutropenic patient is immediate aggressive therapy at the earliest sign of infection, usually manifested by fever. Thus, the entity of “fever and neutropenia” has become established as a functional diagnosis in the management of patients with leucopenia.

Management of the febrile neutropenic patient The following guidelines were recommended by the Infectious Diseases Society of America in 2002.97

Definitions Fever is defined as a single temperature of 38.3 ◦ C (101 ◦ F) or higher or one of 38.0 ◦ C (100.4 ◦ F) or more for at least 1 hour, and neutropenia as less than 500 neutrophils/ L or less than 1000 neutrophils with a predicted decline to 500 neutrophils/ L or lower levels.

Evaluation Patients should be examined carefully for the site of infection, keeping in mind that the lack of neutrophils impedes the acute inflammatory response. The most commonly infected sites are the perineum, catheter sites, breaks in the skin and mucosa, lungs, pharynx, middle ear, lower esophagus, periodontium, and sinuses. The minimal initial diagnostic tests should include bacterial and fungal cultures of peripheral blood and each lumen of any vascular catheters, cultures of lesions and diarrheal stools; chest

Infectious disease complications in leukemia

FEVER AND NEUTROPENIA

EVALUATE

IS VANCOMYCIN NEEDED?

NO INDICATIONS

INDICATIONS

Severe mucositis Quinolone prophylaxis Colonized w i t h Meth.-resistant S. aureus Pen.–Ceph.-resistant Streptococcus pneumoniae Obvious catheter-related infection

MONOTHERAPY

DUOTHERAPY

Cetazidime/cefepime OR Imipenem/meropenem

Aminoglycoside + Antipseudomonal β-lactam OR Imipenem/meropenem

Vanc. + ceftazidime/cefepime OR Vanc. + imipenem/meropenem ± Aminoglycoside

REASSESS AFTER 3 DAYS

Fig. 32.2 Guide to initial antibiotic management of the febrile neutropenic patient. Meth., methicillin; Pen., penicillin; Ceph., cephalosporin; Vanc., vancomycin.

radiograph; complete blood count; and measurements of serum transaminases, sodium, potassium, creatinine, and blood urea nitrogen, and other tests as indicated.

Initial antibiotic therapy At least 50% of patients with febrile neutropenia have an established or occult infection, and at least 20% of febrile patients with neutrophil counts of 100 cells/ L or lower have bacteremia; therefore, broad-spectrum, bactericidal antibiotics should be administered immediately in adequate intravenous dosages (Fig. 32.2). Many antibiotics and antibiotic combinations are highly effective in the treatment of febrile neutropenic episodes. One of three basic strategies may be used: single-drug

therapy, two-drug therapy without vancomycin, or vancomycin plus one or two additional antibiotics (Fig. 32.2). With the emergence of antibiotic-resistant strains of bacteria, an attempt is being made to limit the use of vancomycin. Accordingly, the first criterion of decision-making is whether or not the patient is likely to have an infection that is responsive only to vancomycin. If vancomycin is needed, it is given with ceftazidime or cefepime, with or without an aminoglycoside. If there are no indications for vancomycin, the choice is between monotherapy with ceftazidime/cefepime or imipenem/meropenem or twodrug therapy with an aminoglycoside (tobramycin, gentamicin, or amikacin) plus an antipseudomonal penicillin (piperacillin or ticarcillin), a cephalosporin (cefepime or ceftazidime), or a carbopenem (imipenem or meropenem).

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PERSISTENT FEVER DURING FIRST 3 DAYS OF TREATMENT.NO ETIOLOGY

REASSESS PATIENT ON DAYS 4–5

ADD AMPHOTERICIN B CONTINUE INITIAL ANTIBIOTICS

If no change in patient, consider stopping the vancomycin

CHANGE ANTIBIOTICS

If progressive disease, add vancomycin plus gram-negative bacillus coverage

±

CHANGE ANTIBIOTICS

If febrile through days 5 to 7 and resolution of neutropenia is not imminent, add empiric antifungal therapy with amphotericin B

Fig. 32.3 Guidelines for the management of patients with persistent fever of unknown origin after 3 days of treatment.

Recent randomized trials in selected low-risk pediatric patients have demonstrated the safety and efficacy of oral ciprofloxacin given alone or following a dose of parenteral antibiotics as initial therapy for febrile neutropenia in the outpatient setting.98–100 Although admission rates were as high as 20%, there were no serious complications or deaths in these patients.98–100 Careful selection of patients for this alternative is crucial to success.

Management of antibiotics during the first week of therapy If the causative organism is identified by culture, treatment should be modified for optimal coverage (see Table 32.2), although broad-spectrum coverage must be continued. If the infection is confined to an organ system or specific site, one may suspect a particular pathogen (Table 32.3), but clinical judgment is never sufficiently precise to allow narrow-spectrum antibiotic coverage. Unless broadspectrum coverage is maintained, secondary infections may arise. The most common bacterial infections and their causative organisms are listed in Table 32.3. Patients who are afebrile within 3 to 5 days of treatment, are considered at low risk for progressive, worsening infection, are clinically well, have negative cultures, and show hematologic evidence of imminent bone marrow recovery – as demonstrated by 2 or more days of an increasing absolute leukocyte count, ANC, absolute phagocyte count, and platelet count – may be considered for discharge.101–105 Regardless of their ANC and regardless of whether an etiology for the initial fever has been determined, they should

continue to receive an oral drug, such as cefixime (for children) or a quinolone plus amoxicillin-clavulanate (for adults), on an outpatient basis. If the patient is afebrile but considered at high risk at initial presentation, the initial antibiotic regimen should be continued intravenously. Factors that contribute to a high-risk status at presentation include a neutrophil count of 100/ L or less, evidence of skin or mucosal lesions, discernible disease (pneumonia, endocarditis, cellulitis, catheter site infection, etc.), chills, or hypotension on admission or during treatment. If fever persists through the first 3 days of treatment, reassessment of therapy should begin (Fig. 32.3). In addition to repeating the initial studies, one should consider a CT scan to evaluate the liver, spleen, kidneys, and lungs for evidence of new or progressive lesions. The scan will provide information on possible fungal lesions and will serve as a baseline for later evaluations. If the disease is progressive and worsening, and vancomycin has not been used, the drug should be started empirically. If vancomycin was included in the initial antibiotic regimen, it should be stopped if no organisms have been cultured that would suggest a need for this agent. A change to the alternative antibiotics mentioned earlier for the initial therapy may be tried, but if fever persists through days 5 to 7, antifungal therapy with amphotericin B should be instituted empirically. Treatment should be administered for the shortest time needed to eradicate the causative organism. Much depends on the resolution of fever and return of the neutrophil count to values greater than 500/ L. A general scheme regarding the duration of antibiotic therapy is provided in Figure 32.4. If no evidence of fungal infection is found by

Infectious disease complications in leukemia

DURATION OF ANTIBIOTIC THERAPY

AFEBRILE BY DAY 3–5

ANC ≥ 500 for 2 consecutive days Stop antibiotics 48 hours after afebrile and resolution of neutropenia

PERSISTENT FEVER

ANC < 500 by day 7

Low risk at presentation; clinically well

High risk at presentation
ANC < 100
Mucositis
Unstable vital signs

Stop antibiotics when afebrile for 5 to 7 days

Continue antibiotics

ANC ≥ 500

ANC < 500

Stop antibiotics 4 to 5 days after ANC ≥ 500

Continue for 2 weeks then reassess

Reassess

Stop if no infectious etiology isolated and patient is clinically stable

Fig. 32.4 Guidelines regarding the duration of antibiotic therapy when no infectious agent is isolated. ANC, absolute neutrophil count/ L.

cultures and CT scans, amphotericin B may be discontinued after 2 weeks. If a fungal infection is identified, a specific treatment course should be undertaken (see Table 32.4). Routine use of a granulocyte-stimulating factor (either G-CSF or GM-CSF) is not recommended because in most cases such treatment has little impact on the final outcome and is not cost effective.106 However, their use may be indicated in cases with a predictive worsening of the clinical course or an expected long delay in bone marrow recovery. Only G-CSF has been approved by the FDA for use in chemotherapy-induced neutropenia.106 Granulocyte transfusions are not routinely recommended; however, recent studies have shown that G-CSF-primed granulocyte transfusions achieve near normal neutrophil counts in recipients,107 that the transfused granulocytes function normally for up to 24 hours in the recipient,107 and that granulocyte transfusions may be beneficial in the treatment of severe infections in neutropenic patients.108 The use of antiviral therapy should be specific and directed by clinical or laboratory evidence suggesting a viral infection.

and Absidia spp.) cause infections in the neutropenic host. A variety of fungi, including Alternaria, Cladosporium, Cunninghamella, Curvularia, Exserohilum, Fusarium, Geotrichum, Hansenula, Malassezia, Penicillium, Phialophora, Pichia, Rhodotorula, Saccharomyces, and Trichosporon spp., have caused mycoses in the compromised host85,109 Traditionally, amphotericin B deoxycholate has been the drug of choice for most severe fungal infections in neutropenic patients; however, this conventional formulation of amphotericin B is extremely toxic and is associated with a large number of adverse effects in patients. In the mid-1990s, various lipid formulations of amphotericin B were devised in an attempt to find a less toxic alternative. Currently, three lipid formulations are available: amphotericin B colloidal dispersion (ABCD),110–112 amphotericin B lipid complex (ABLC),110,111,113 and liposomal amphotericin B110,111,114,115 ; each of which is as efficacious as conventional amphotericin B with substantially less toxicity.110–116 New azole agents and echinocandins offer alternatives to amphotericin B, but ongoing investigation is needed to define their use in pediatric patients with leukemia.

Fungal infections in neutropenic patients Most of the fungal infections in neutropenic patients are caused by Candida and Aspergillus spp. Less frequently the aseptate fungi of the order Mucorales (Rhizopus, Mucor,

Candidiasis Mild infections due to Candida species include oropharyngeal thrush, vaginitis, enteritis, and dermatitis. Moderately

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severe localized infections include esophagitis. Systemic candidiasis is a serious disease predominately involving the lungs, liver, spleen, brain, and kidneys. However, with systemic candidiasis, any organ may be affected. Cutaneous lesions may be seen in cases of disseminated disease (Fig. 32.1D). Among 109 children with cancer and systemic candidiasis studied antemortem and at autopsy, Candida colonization, fever, neutropenia, relapse of malignancy, therapy with antibiotics, and the use of immunosuppressive drugs were noted in about 90% of cases.117 Candida endocarditis was more frequent in patients with indwelling central venous catheters.117 In a review of reports of 1591 cases of systemic candidiasis infection, Wingard118 found that species other than Candida albicans accounted for 46% of all such infections in cancer patients. Specifically, C. tropicalis accounted for 25%, C. glabrata 8%, C. parapsilosis 7%, C. krusei 4%, and other Candida species for 2%. Patients with leukemia were more likely to have C. albicans or C. tropicalis than other Candida species.119 Definitive diagnosis requires culture of the organism from infected tissue, histologic demonstration of yeast, and pseudohyphal forms in tissue samples. Blood cultures are often sterile despite systemic candidiasis. Of 1032 blood cultures from patients with systemic candidiasis, only 17% yielded Candida spp.117 Serologic tests for antibody, antigen, metabolic products, and nucleotide sequences are currently not of sufficient specificity to serve as diagnostic tests. CT scans are useful in identifying characteristic hypodense circular lesions in liver, spleen, kidneys, and brain that warrant a presumptive diagnosis of a systemic mycosis (Fig. 32.5).120,121 Oral candidiasis may be treated with nystatin suspension, amphotericin B suspension, or clotrimazole troches. Fluconazole or systemic amphotericin B may be needed for severe cases (Table 32.4). Esophageal candidiasis is treated with fluconazole, amphotericin B, caspofungin or mica fungin,122,123 an echinocandin compound with a broad spectrum of antifungal activity and an excellent safety profile.86,124 Systemic candidiasis is treated with amphotericin B, with or without flucytosine. Lipid formulations of amphotericin B may be considered for patients who are intolerant or nonresponsive to conventional amphotericin B, but the cost is often prohibitive. Fluconazole alone is a useful alternative in some cases. Caspofungin is a potent alternative in therapy for systemic candidiasis.

Aspergillus The clinical features of Aspergillus infection include sinusitis, pneumonitis, hemoptysis, tracheobronchitis, brain abscess or infiltrate, cutaneous necrosis, and

Fig. 32.5 CT scan showing multiple hypodense circular lesions in liver and spleen due to systemic candidiasis.

ulceration.85,125,126 Isolation of the organism in cultures and a histologic finding of septate hyphae in affected biopsy or autopsy tissue provide a definitive diagnosis. CT scans of lungs showing nodular lesions with a “halo” of reduced density127 or a cavitary lesion with an intracavitary mass (half-moon sign) are highly suggestive of pulmonary aspergillosis (Fig. 32.1E). The diagnosis is confirmed by the finding of Aspergillus spp. in bronchoalveolar lavage specimen or on lung biopsy specimen. CT scans of sinuses showing a soft tissue mass within the sinus,128 mucosal thickening, fluid level, and bony invasion128 strongly suggest aspergillosis of the sinuses. Cultures of Aspergillus spp. from sinus fluid or tissue, together with a compatible CT scan, establish the diagnosis. A useful diagnostic guide for aspergillosis was developed from the work of Gerson and colleagues129 in neutropenic patients. These investigators reported that any five of the following 11 abnormalities in neutropenic patients indicated aspergillosis in 60% to 90% of cases, with a specificity in excess of 85%: fever at time of admission; neutropenia for more than 30 days; two or more febrile episodes without a source identified; fever for 14 days or more without an etiology; fever for longer than 18 days while antibiotics are being administered; rales; nasal signs of ulceration, eschar, epistaxis, or sinus tenderness; pleuritic pain; cavitary or nodular lesions in the lungs; new pulmonary infiltrate after 2 weeks in the hospital; and multilobar infiltrates. Intravenous amphotericin B in high doses (up to 1.5 mg/kg per day) is the traditional drug of choice. Amphotericin B lipid complex, liposomal amphotericin B, and amphotericin B colloidal dispersion have all been approved by the FDA for the treatment of aspergillosis in patients who are intolerant or unresponsive to conventional amphotericin B treatment. The echinocandin

Infectious disease complications in leukemia

compound caspofungin received original FDA approval for the treatment of Aspergillus infection in patients who failed therapy with conventional agents or in patients who were unable to tolerate conventional therapy.86 Recent studies have shown the utility of voriconazole as initial therapy for invasive aspergillosis. A noncomparative study of voriconazole as primary therapy demonstrated a response rate of 48% in patients with invasive aspergillosis.130 A more recent, comparative study suggested that voriconazole led to better responses and improved survival rates with fewer severe side effects in patients with invasive aspergillosis.131 It is possible that voriconazole will replace amphotericin B as the drug of choice for invasive aspergillosis. Some benefit may also come from the concomitant use of rifampin, flucytosine, and colony-stimulating factors (G-CSF or GMCSF), but proof of efficacy is lacking. Surgical intervention with excision of localized lesions may be indicated in some cases.126

Zygomycoses Zygomycoses may present with the clinical features of sinusitis, pulmonary infarct/infiltrate, or CNS lesions. Sinusitis may progress to involve the brain (rhinocerebral zygomycosis, Fig. 32.1F). Although uncommon, other deep organs and skin may be sites of infection. Isolation of aseptate fungi (Rhizopus, Mucor, and Absidia spp.) from infected tissue (biopsy/autopsy) or otherwise sterile body fluids is needed for diagnosis. Amphotericin B is the first drug of choice, although responses are often poor. Optimal therapy requires aggressive antifungal therapy coupled with the cessation of immunosuppression (if possible), and surgical resection of all infected tissue.132–134 Posaconazole, an investigational azole, appears very promising for the treatment of zygomycosis.

Recommendations for the prevention of infections Only three approaches are available to prevent infectious diseases: (1) isolation, (2) immunization, and (3) chemoprophylaxis.

Isolation Patients and parents must be educated regarding the principles of contagion and hygiene. The leukemic child should avoid contact with patients known or suspected to be infected with readily transmissible microbes. Such information is best presented to patients and parents by descriptions of clinical syndromes associated with these microbes

such as “common cold,” diarrhea, fever, or rash. Printed material that can be referenced at a later date is particularly useful. When hospitalized, patients with leukemia should be admitted to a clean private room. Most experts agree that “total protected environments” or “life-island” rooms are not necessary. However, severely immunocompromised patients are much more prone to nosocomial infections with Aspergillus; therefore, certain measures are indicated to reduce this risk. Studies have shown that in the nonepidemic setting there is a significant relationship between fungal contamination of the hospital environment and the rate of invasive nosocomial aspergillosis135,136 ; therefore, frequent sampling of the hospital environment to evaluate the level of fungal contamination is indicated. Two commonly implemented measures that have shown a significantly beneficial effect in reducing fungal contamination and subsequent nosocomial aspergillosis are highefficiency particulate air (HEPA) filtration and laminar airflow (LAF) rooms.136–139 Further details on nosocomial infections and their prevention are given elsewhere.140,141 Controversy exists over so-called neutropenic diets, which have long been part of the standard of care in many pediatric oncology hospitals. The concept of a neutropenic diet began to evolve in the 1960s in an attempt to minimize the risk of opportunistic infections in patients with hematologic malignancies.142 Such diets frequently restrict fresh fruits and juices, fresh vegetables, and raw eggs,143 yet, there are no data to support an independent effect of diet on reducing the number of opportunistic infections in patients with leukemia.142,144 In a recent survey of 156 institutions belonging to the Association of Community Cancer Centers, 78% reported placing patients with moderate or severe neutropenia on dietary restrictions aimed at reducing the risk of opportunistic infections in these patients.143 Although most institutions reported the use of restricted diets only during periods of neutropenia, 17% maintained them throughout the duration of chemotherapy.143

Immunization Although children with leukemia differ in their degree of immunocompetence, and in their responses to vaccines, the routine administration of childhood vaccinations is recommended with some exceptions and modifications. The following are recommendations of the Advisory Committee on Immunization Practices (ACIP).145 Live-bacterial and live-virus vaccines are contraindicated in immunocompromised children with hematologic malignancies. All children, especially those with leukemia, should receive the inactivated poliovirus vaccine rather

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Vaccine

Birth Hep B

1 month

2 month

4 month

6 month

12 month

15 month

18 month

24 month

only if mother HBsAg (−)

Hep B DTaP

DTaP

Haemophilus influenzae type b

Hib

Hib

Hib

Inactivated polio

IPV

IPV

DTaP Hib IPV

IPV

Hold until 3 months after completion of chemotherapy

Varicella

Hold until 3 months after completion of chemotherapy PCV

PCV

Td

DTaP

Measles, mumps, rubella

PCV

13 –18 years

Hep B

DTaP

Pneumococcal

11–12 years

Hepatitis B series

Hepatitis B Diphtheria, tetanus, pertussis

4–6 years

PCV

PCV

PPV

Vaccines below this line are for selected populations

Hepatitis A series

Hepatitis A

Influenza (should be given yearly)

Influenza

Range of recommended ages

Catch-up vaccination

Preadolescent assessment

Fig. 32.6 Recommended immunization schedule, modified for children with leukemia. DTaP, diphtheria, tetanus toxoids and acellular pertusssis; Td, tetanus and diphtheria toxoids; PCV, pneumococcal conjugate vaccine; PPV, pneumococcal polysaccharide vaccine; IPV, inactivated polio vaccine.

than the oral live poliovirus vaccine. The measles, mumps, and rubella (MMR) vaccine should not be given during periods of immunocompromise. Routine administration of varicella vaccine is not advised; however, under some special circumstances, it may be given to leukemic children.146 Figure 32.6 shows the modified immunization schedule for children with leukemia. Normal siblings and other household contacts of children with leukemia should not receive the oral poliovirus vaccine, but may receive the MMR and varicella vaccines. Of note, if a family member or other household contact receiving varicella vaccine develops a presumed vaccinerelated rash 7 to 25 days after vaccination, the immunocompromised patient should avoid direct contact with the affected household member for the duration of the rash. Most experts agree that VZIG is not indicated in this situation. Children who were vaccinated during or within 2 weeks before immunosuppressive therapy should be considered unvaccinated and require revaccination after the completion of chemotherapy.145 Patients in remission

whose chemotherapy has been terminated for at least 3 months may receive live-virus vaccines for infections to which they remain susceptible. A recent study suggests that chemotherapy for ALL can induce the loss of humoral immunity to measles, rubella, and other viral infections against which the patients were previously immunized,147 supporting reimmunization after the completion of chemotherapy.

Chemoprophylaxis Antibiotic, antiviral, and antifungal prophylaxis are not routinely recommended for patients with leukemia who are not undergoing allogeneic hematopoietic stem cell transplantation, except for the use of trimethoprimsulfamethoxazole to prevent Pneumocystis jiroveci pneumonitis. Antimicrobial prophylaxis is administered to prevent P. jiroveci pneumonia, which occurs in 15% to 20% of children with ALL who do not receive appropriate prophylaxis. The drug of choice is the combination of trimethoprim

Infectious disease complications in leukemia

and sulfamethoxazole, 150 mg and 750 mg/m2 per day, which may be given daily or on 3 consecutive days a week.79,80 Second-line drugs for this prophylaxis include atovaquone,76,148,149 dapsone,149,150 and aerosolized pentamidine.148,149 Although several studies have shown that the administration of antibiotics, such as trimethoprimsulfamethoxazole or quinolones, such as ofloxacin, will reduce the number of febrile episodes during periods of profound neutropenia, this practice is not recommended for routine use because of the potential for the development of antibiotic-resistant bacteria.97 Because of the dramatically increased incidence of sepsis secondary to viridans streptococci in neutropenic patients with hematologic malignancies, and the potential seriousness of this complication, even in cases treated with appropriate antimicrobial agents,151 some experts would recommend prophylaxis against viridans streptococci for patients during periods of neutropenia. At particular risk are profoundly neutropenic patients who have recently received high doses of cytarabine,151–154 which often causes severe mucositis; in addition, high-dose cytarabine appears to increase the risk of viridans streptococcal sepsis beyond its association with mucositis.151 Agents used as prophylaxis against viridans streptococci include penicillin, ampicillin, vancomycin, and fluoroquinolones.155 No consensus exists on prophylaxis against viridans streptococci. The potential benefits of prophylaxis must be weighed against the risk of selecting for resistant bacteria with an attendant increase in morbidity and mortality.152,155 Children exposed to active cases of influenza A virus infection can be given rimantadine or amantadine prophylactically. These drugs have no effect against influenza B virus. Zanamivir and oseltamivir are newer medications that specifically target the neuraminidase activity of influenza A and B. Both zanamivir and oseltamivir are efficacious either as therapy or as prophylaxis65 ; however, zanamivir has not been approved by the FDA for influenza prophylaxis. Patients with hematologic malignancies are susceptible to invasive mycoses, especially during periods of prolonged neutropenia. The diagnosis of invasive fungal disease can be problematic, and the available treatments for established invasive fungal disease often yield poor response rates. Thus, the concept of primary antifungal prophylaxis is appealing and has been under investigation for many years. Agents used for prophylaxis have included fluconazole, itraconazole, micafungin, and amphotericin B. A large study that reviewed 50 previous studies with more than 9000 total patients concluded that evidence to support routine primary antifungal prophylaxis in patients

not undergoing allogeneic stem cell transplantation is poor; however there was no evidence against the use of antifungal prophylaxis.156 Antifungal medications such as the echinocandins, the recently developed triazoles such as voriconazole and posaconazole, and terbinafine have not been evaluated sufficiently as antifungal prophylactic agents to make recommendations regarding their use in leukemia patients.

Future directions Children with leukemia remain at risk for infections that occur commonly in children as well as those that result from their immunosuppression. Changes in chemotherapy regimens are likely to result in changes in the types and severity of infections that are observed. Continuing research to define these risks, describe new infections, diagnostic tests, and treatment and prophylaxis strategies are critical to providing the best supportive care. The emergence of antibiotic-resistant organisms presents one of the greatest challenges. Balancing the availability of prophylactic regimens for disease prevention against the risk of inducing resistant organisms is an ongoing challenge. For decades, amphotericin B has been the gold standard for treatment of systemic fungal infection. Newer lipid formulations of amphotericin B promise greater tolerability but at markedly increased cost. Determining when to use standard amphotericin B or a lipid preparation remains a question. Several new antifungal agents have recently become available or are in investigational trials. The impact that these agents may have on treatment and prophylaxis of fungal infections remains unknown. While it is tempting to use these agents, defining the benefits and limitations are still necessary. In addition, many clinicians opt for combination antifungal therapy for severe infections. To date there is no data to support this approach defining another key area for future research. Also, there remains great opportunity to develop diagnostic tests for pathogens we isolate from our patients. Molecular microbiology laboratories have rapidly improved our diagnostic ability and will continue to provide new tests that must be applied in a logical and costconscious fashion.

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34 Barriga, F. J., Varas, M., Potin, M., et al. Efficacy of a vancomycin solution to prevent bacteremia associated with an indwelling central venous catheter in neutropenic and non-neutropenic cancer patients. Med Pediatr Oncol, 1997; 28: 196–200. 35 Rackoff, W. R., Weiman, M., Jakobowski, D., et al. A randomized, controlled trial of the efficacy of a heparin and vancomycin solution in preventing central venous catheter infections in children. J Pediatr, 1995; 127: 147–51. 36 Schutze, G. E. Antimicrobial-impregnated central venous catheters. Pediatr Infect Dis J, 2002; 21: 63–4. 37 Rosenstein, N. E., Emery, K. W., Werner, S. B., et al. Risk factors for severe pulmonary and disseminated coccidioidomycosis: Kern County, California, 1995–1996. Clin Infect Dis, 2001; 32: 708–15. 38 Saitoh, A., Homans, J., & Kovac, A. Fluconazole treatment of coccidioidal meningitis in children: two case reports and a review of the literature. Pediatr Infect Dis J, 2000; 19: 1204–8. 39 Leggiadro, R. J., Barrett, F. F., & Hughes, W. T. Extrapulmonary cryptococcosis in immunosuppressed infants and children. Pediatr Infect Dis J, 1992; 11: 43–7. 40 Perfect, J. R., & Casadevall, A. Cryptococcosis. Infect Dis Clin North Am, 2002; 16: 837–74. 41 Cunha, B. A. Central nervous system infections in the compromised host: a diagnostic approach. Infect Dis Clin North Am, 2001; 15: 567–90. 42 Sugane, K., Takamoto, M., Nakayama, K., et al. Diagnosis of Toxoplasma meningoencephalitis in a non-AIDS patient using PCR. J Infect, 2001; 42: 159–60. 43 Abrams, E. J. Opportunistic infections and other clinical manifestations of HIV disease in children. Pediatr Clin North Am, 2000; 47: 79–108. 44 Demeter, L M. JC, BK, and other polyomaviruses; progressive leukoencphalopathy. In G. L. Mandell, J. E. Bennett, & R. Dolin, eds., Principles and Practice of Infectious Diseases, 5th edn. (New York: Churchill Livingstone, 2000), pp. 1645–51. 45 Richardson-Burns, S. M., Kleinschmidt-DeMasters, B. K., DeBiasi, R. L., et al. Progressive multifocal leukoencephalopathy and apoptosis of infected oligodendrocytes in the central nervous system of patients with and without AIDS. Arch Neurol, 2002; 59: 1930–6. 46 Osorio, S., De La Camara, R., Golbano, N., et al. Progressive multifocal leukoencephalopathy after stem cell transplantation, unsuccessfully treated with cidofovir. Bone Marrow Transplant, 2002; 30: 963–6. 47 Marra, C. M., Rajicic, N., Barker, D. E., et al. A pilot study of cidofovir for progressive multifocal leukoencephalopathy in AIDS. AIDS, 2002; 16: 1791–7. 48 Arola, M., Ruuskanen, O., Ziegler, T., & Salmi, T. T. Respiratory virus infections during anticancer treatment in children. Pediatr Infect Dis J, 1995; 14: 690–4. 49 Wulffraat, N., Geelan, S., Dijken, P. van, et al. Recovery from adenovirus pneumonia in a severe combined immunodeficiency patient treated with intravenous ribavirin. Transplantation, 1995; 59: 927.

50 Liles, W. C., Cushing, H., Holt, S., et al. Severe adenoviral nephritis following bone marrow transplantation. Bone Marrow Transplant, 1993; 12: 409–12. 51 Davis, D., Henslee, P. J., & Markesberry, W. R. Fatal adenovirus meningoencephilitis in a bone marrow patient. Ann Neurol, 1988; 23: 385–9. 52 Shenep, J. L., Srinivas, R. V., Jenkins, J. J., et al. A young woman with lymphoma and endocarditis. Lancet, 1995; 346: 1532. 53 Murphy, G. F., Wood, D. P., Jr, McRoberts, J. W., et al. Adenovirus associated hemorrhagic cystitis treated with intravenous ribavirin. J Urol, 1993; 149: 565–6. 54 Wreghitt, T. G., Gray, J. J., Ward, K. N., et al. Disseminated adenovirus infection after liver transplantation and its possible treatment with ganciclovir [letter]. J Infect, 1989; 19: 88–9. 55 McCarthy, A. J., Bergin, M., DeSilva, L. M., et al. Intravenous ribavirin therapy for disseminated adenovirus infection. Pediatr Infect Dis J, 1995; 14: 1003–4. 56 Carter, B. A., Karpen, S. J., Quiros-Tejeira, R. E., et al. Intravenous cidofovir therapy for disseminated adenovirus in a pediatric liver transplant recipient. Transplantation, 2002; 74: 1050–2. 57 Leather, H. L. & Wingard, J. R. Infection following hematopoietic stem cell transplantation. Infect Dis Clin North Am, 2001; 15: 483–520. 58 Sidwell, R. W., Khare, G. P., Allen, L. B., et al. In vitro and in vivo effect of 1-beta-D-ribofuranosyl-1,2,4-triazole-3carboxamide (ribavirin) on types 1, 2 and 3 parainfluenza virus infections. Chemotherapy, 1975; 21: 205–20. 59 Wright, R. B., Pomerantz, W. J., & Luria, J. W. New approaches to respiratory infections in children. Bronchiolitis and croup. Emerg Med Clin North Am, 2002; 20: 93–114. 60 Junkman, P., Anderson, J., Ashcan, J., et al. Influenza A in immunocompromised patients. Clin Infect Dis, 1993; 17: 244– 7. 61 Feldman, S., Webster, R. G., & Sung, M. Influenza in children and young adults with cancer. Cancer, 1977; 39: 350–3. 62 Kemp, A., Hall, C. B., MacDonald, N. E., et al. Influenza in children with cancer. Pediatrics, 1989; 115: 33–9. 63 Ryan-Poirier, K. Influenza virus infection in children. Adv Pediatr Infect Dis, 1995; 10: 125–56. 64 Englund, J. A. Antiviral therapy of influenza. Semin Pediatr Infect Dis, 2002; 13: 120–8. 65 Prober, C. G. Antiviral therapy for influenza virus infections. Semin Pediatr Infect Dis, 2002; 13: 31–9. 66 Ogre, P. L. & Patel, J. Respiratory syncytial virus infection and the immunocompromised host. Pediatr Infect Dis J, 1988; 7: 246–8. 67 Tsutsumi, H., Sone, S., Yoto, S., et al. Respiratory syncytial virus bronchiolitis in a girl undergoing chemotherapy for active lymphoblastic leukemia: an immunologic study of local secretions. Pediatr Infect Dis J, 1996; 15: 635–6. 68 Hall, C., Powell, K. R., MacDonald, N. E., et al. Respiratory syncytial virus infection in children with compromised immune function. N Engl J Med, 1986; 315: 77–81.

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87 Bell, B. P. Hepatitis A vaccine. Semin Pediatr Infect Dis, 2002; 13: 165–73. 88 Coates, T., Wilson, R., Patrick, G., et al. Hepatitis B vaccines: assessment of the seroprotective efficacy of two recombinant DNA vaccines. Clin Ther, 2001; 23: 392–403. 89 Kimberlin, D. W. Antiviral therapy for cytomegalovirus infections in pediatric patients. Semin Pediatr Infect Dis, 2002; 13: 22–30. 90 Amadi, B., Mwiya, M., Watuka, A., et al. Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet, 2002; 360: 1375–80. 91 Mylonakis, E., Ryan, E. T., & Calderwood, S. B. Clostridium difficile – associated diarrhea: a review. Arch Intern Med, 2001; 161: 525–33. 92 Otaibi, A. A., Barker, C., Anderson, R., et al. Neutropenic enterocolitis (typhlitis) after pediatric bone marrow transplant. J Pediatr Surg, 2002; 37: 770–2. 93 Horton, K. M., Corl, F. M., & Fishman, E. K. CT evaluation of the colon: inflammatory disease. Radiographics, 2000; 20: 399– 418. 94 Cartoni, C., Dragoni, F., Micozzi, A., et al. Neutropenic enterocolitis in patients with acute leukemia: prognostic significance of bowel wall thickening detected by ultrasonography. J Clin Oncol, 2001; 19: 756–61. 95 Pastore, D., Specchia, G., Mele, G., et al. Typhlitis complicating induction therapy in adult acute myeloid leukemia. Leuk Lymphoma, 2002; 43: 911–14. 96 Sclatter, M., Snyder, K., & Freyer, D. Successful nonoperative management of typhlitis in pediatric oncology patients. J Pediatr Surg, 2002; 37: 1151–5. 97 Hughes, W. T., Armstrong, D., & Bodey, G. P. Guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis, 2002; 34: 730–51. 98 Mullen, C. A., Petropoulos, D., Roberts, V. W., et al. Outpatient treatment of fever and neutropenia for low risk pediatric cancer patients. Cancer, 1999; 86: 126–34. 99 Petrilli, A. S., Dantas, L. S., Campos, M. C., et al. Oral ciprofloxacin versus intravenous ceftriaxone administered in an outpatient setting for fever and neutropenia in low-risk pediatric oncology patients: randomized prospective trial. Med Pediatr Oncol, 2000; 34: 87–91. 100 Paganini, H., Rodriguez-Brieshcke, T., Zubizarreta, P., et al. Oral ciprofloxacin in the management of children with cancer with lower risk febrile neutropenia. Cancer, 2001; 91: 1563– 7. 101 Aquino, V. M., Tkaczewski, I., & Buchanan, G. R. Early discharge of low-risk febrile neutropenic children and adolescents with cancer. Clin Infect Dis, 1997; 25: 74–8. 102 Aquino, V. M., Buchanan, G. R., Tkaczewski, I., et al. Safety of early hospital discharge of selected febrile children and adolescents with cancer with prolonged neutropenia. Med Pediatr Oncol, 1997; 28: 191–5. 103 Bash, R. O., Katz, J. A., Cash, J. V., et al. Safety and cost effectiveness of early hospital discharge of lower risk children with

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119 Flynn, P. M., Marina, N. M., Rivera, G. K., et al. Candida tropicalis infection in children with leukemia. Leuk Lymphoma, 1993; 10: 369–76. 120 Flynn, P. M., Shenep, J. L., Crawford, R., et al. Use of abdominal computed tomography for identifying disseminated fungal infection in pediatric cancer patients. Clin Infect Dis, 1995; 20: 964–70. 121 Bartley, D. L., Hughes, W. T., Parvey, L. S., et al. Computed tomography of hepatic and splenic fungal abscesses in leukemic children. Pediatr Infect Dis, 1982; 1: 317–21. 122 Villanueva, A., Arathoon, E. G., Gotuzzo, E., et al. A randomized double-blind study of caspofungin versus amphotericin for the treatment of candidal esophagitis. Clin Infect Dis, 2001; 33: 1529–35. 123 Villanueva, A., Gotuzzo, E., Arathoon, E. G., et al. A randomized double-blind study of caspofungin versus fluconazole for the treatment of esophageal candidiasis. Am J Med, 2002; 113: 294– 9. 124 Abruzzo, G. K., Gill, C. J., Flattery, A. M., et al. Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrob Agents Chemother, 2000; 44: 2310–8. 125 Pagano, L., Ricci, P., Montillo, M., et al. Localization of aspergillosis to the central nervous system among patients with acute leukemia: report of 14 cases. Clin Infect Dis, 1996; 23: 628–30. 126 Denning, D. W. & Stevens, D. A. Antifungal and surgical treatment of invasive aspergillosis: review of 2,121 cases. Rev Infect Dis, 1990; 12: 1147–201. 127 Hauggaard, A., Ellis, M., & Ekelund, L. Early chest radiography and CT in the diagnosis, management and outcome of invasive pulmonary aspergillosis. Acta Radiol, 2002; 43: 292–8. 128 Dahniya, M. H., Makkar, R., Grexa, E., et al. Appearances of paranasal fungal sinusitis on computed tomography. Br J Radiol, 1998; 71: 340–4. 129 Gerson, S. L., Talbot, G. H., Hurwitz, S., et al. Prolonged granulocytopenia: the major risk factor for invasive pulmonary aspergillosis in patients with acute leukemia. Ann Intern Med, 1984; 10: 345–51. 130 Denning, D. W., Ribaud, P., Milipied, N., et al. Efficacy and safety of voriconazole in the treatment of acute invasive aspergillosis. Clin Infect Dis, 2002; 34: 563–71. 131 Herbrecht, R., Denning, D. W., Patterson, T. F., et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med, 2002; 347: 408–15. 132 Lee, F. Y., Mossad, S. B., & Adal, K. A. Pulmonary mucormycosis: the last 30 years. Arch Intern Med, 1999; 159: 1301–9. 133 Jimenez, C., Lumbreras, C., Aguado, J. M., et al. Successful treatment of mucor infection after liver or pancreas-kidney transplantation. Transplantation, 2002; 73: 476–80. 134 Ryan, M., Yeo, S., Maguire, A., et al. Rhinocerebral zygomycosis in childhood acute lymphoblastic leukaemia. Eur J Pediatr, 2001; 160: 235–8. 135 Alberti, C., Bouakline, A., Ribaud, P., et al. Relationship between environmental fungal contamination and the

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33 Hematologic supportive care Fariba Navid and Victor M. Santana

Introduction The great strides that have been made in curing children of acute leukemia can be attributed in part to improvements in supportive care during periods of morbidity due to therapeutic interventions. Such care is a major component of the total patient management strategy and includes nutritional support, prophylaxis against life-threatening infections, empiric use of antibiotics during periods of neutropenia, blood component support, adequate venous access, and, most recently, the use of hematopoietic growth factors to ameliorate hematologic complications. In this chapter, we review the current status of blood component support, intravenous catheter placement, and supportive therapy with granulocyte colony-stimulating factor and other cytokines. Although the focus is on experience with childhood leukemias, some examples are drawn from experience with solid tumor patients, particularly in situations where limited data are available from leukemia studies.

Blood component support Since 1828, when Blundell1 initiated the use of blood transfusion to counteract postpartum hemorrhages, the demand for blood component products in the United States has increased exponentially. Each year, approximately 12 million units of blood are transfused in this country, with surgery, motor vehicle accidents, and complications of cancer accounting for the majority of this usage. Cancer patients receive blood component support because of deficient hemoglobin levels and platelet counts caused by the suppression of blood cell progenitors or by bone marrow aplasia due to tumor cell infiltration.

The use of red cell and plasma products has remained constant over the last decade, while platelet usage has steadily increased. This rise is in part due to the evergrowing need to support patients during marrow-ablative chemotherapy and the lack of clinically effective cytokines to augment the production of platelets. In spite of advances in supportive care for cancer-related complications, the use of blood component therapy is still associated with substantial risks. These include alloimmunization, transmission of viral and bacterial infections, graft-versus-host disease, transfusion-related acute lung injury, and substantial financial costs. Therefore, adherence to established guidelines for the use of blood component therapy, designed to ensure that patients receive this support only when it is absolutely necessary, has become an important aspect of clinical management. A summary of available blood components and their indications is given in Table 33.1.

Packed red cell transfusions The primary indication for red blood cell transfusion is an inadequate oxygen-carrying capacity in blood. Anemia is the most common (but not the only) cause of this deficiency. Although anemia most often results from the effects of the malignancy itself or from its treatment, other causes such as hemorrhage and hemolysis must be considered. The need for red cell transfusion in patients with childhood leukemia is greatest during the first 3 months of treatment.2,3 Accurate measurement of the hemoglobin concentration or hematocrit level is essential to determining the need for red cell transfusion. A hemoglobin concentration that absolutely indicates the need for red cell transfusion in childhood cancer patients has not been defined.

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 33.1 Summary of blood componentsa Component

Major indication

Action

Contraindication

Special precautions

Hazardsa

Rate of infusion

Whole blood

Symptomatic anemia with large volume deficit

Condition responsive to specific components

For massive blood loss, as fast as patient can tolerate

Symptomatic anemia

Pharmacologically treatable anemia, coagulation deficiency

Must be ABO-identical; labile coagulation factors deteriorate within 24 hours after collection Must be ABO-compatible

Infectious diseases; septic, toxic, allergic, or febrile reactions; circulatory overload; GVHD

Red blood cells, red blood cells with adenine-saline added Red blood cells with or without leukocyte reduction

Restoration of oxygen-carrying capacity, restoration of blood volume Restoration of oxygen-carrying capacity

As fast as patient can tolerate, but less than 4 hours

Symptomatic anemia, febrile reactions from leukocyte antibodies

Restoration of oxygen-carrying capacity

Pharmacologically treatable anemia, coagulation deficiency

Must be ABO-compatible

Infectious diseases; hemolytic, septic/toxic, allergic, or febrile reactions; GVHD Infectious disease; hemolytic, septic, toxic, or allergic reactions; GVHD

Deficit of labile and stable plasma coagulation factors, TTP Deficit of stable coagulation factors

Source of labile and nonlabile plasma factors

Condition responsive to volume replacement

Must be ABO-compatible

Infectious disease, allergic reactions, circulatory overhead

Source of nonlabile factors

Deficit of labile coagulation factors or volume replacement Deficit of any plasma protein other than those enriched in cryoprecipitated antihemophilic factor Plasma coagulation deficits and some conditions with rapid platelet destruction (ITP)

Should be ABO-compatible

Infectious diseases, allergic reactions

Less than 4 hours

Frequent repeat doses may be necessary

Infectious diseases, allergic reactions

Less than 4 hours

Should not use some microaggregate filters (check manufacturer’s instructions) Must be ABO-compatible; avoid depth-type microaggregate filters

Infectious diseases; septic, toxic, allergic, or febrile reactions; GVHD

Less than 4 hours

Infectious diseases, allergic febrile reactions, GVHD

One unit over 2–4 hours (closely observe for reaction)

Fresh frozen plasma

Liquid plasma, plasma, and thawed plasma Cryoprecipitated antihemophilic factor Platelets, platelets with pheresisc

Granulocytes, pheresis

Hemophilia A,d von Willebrand’s disease, hypofibrinogenemia, factor XIII deficiency Bleeding due to thrombocytopenia or platelet function abnormality Neutropenia with infection

Provides factor VIII, fibrinogen vWF, factor XIII Improves hemostasis

Provides granulocytes

Infection responsive to antibiotics

As fast as patient can tolerate but less than 4 hours (longer in patients with severe anemia) Less than 4 hours

Abbreviations: GVHD, graft-versus-host disease; TTP, thrombotic thrombocytopenic purpura; ITP, idiopathic thrombocytopenic purpura. Adapted from circular of information for the use of human blood and blood components (American Red Cross 1751, August 2000). b For all cellular components, there is a risk that the recipient will become alloimmunized. c Red blood cells and platelets may be processed in a manner that yields leukocyte-reduced components, for which the main indications are prevention of febrile, nonhemolytic transfusion reactions and prevention of leukocyte alloimmunization. Risks are the same as those for standard components, except for the reduced risk of febrile reactions. d When virus-inactivated concentrates are not available. a

Hematologic supportive care

Traditionally, physicians have tried to maintain hemoglobin levels above 10 g/dL in children with leukemia; more recently, they have followed a more conservative guideline, approximately 7 to 8 g/dL, if there are no other clinical conditions that would require higher hemoglobin levels. This change in strategy was prompted largely by the desire to reduce the indiscriminate use of packed red cells. In addition to the hemoglobin concentration, one should consider the patient’s cardiopulmonary status and physical condition, length of the anemia, and the possibility of correcting the hemoglobin deficiency by other means when deciding if a transfusion is indicated. Administration of packed red blood cells (PRBC) is almost universally preferred over whole blood replacement in the treatment of anemia; whole blood should be reserved for clinical emergencies characterized by hypovolemia and signs of circulatory insufficiency. Children generally need the oxygen-carrying capacity of red cells and receive little or no benefit from the extra fluid or protein in the plasma component of whole blood. In fact, the extra fluid volume can be hazardous, potentially contributing to circulatory overload. Washed red blood cells or frozen red blood cells should be considered for frequently transfused patients who are at risk for the development of febrile or urticarial transfusion reactions. Individually bagged aliquots of packed red blood cell units (“quad packs”) are useful for infants and small children, who require repeated transfusion of less than a full unit of cells.4 Infusion of red blood cells can elicit a variety of adverse reactions (Table 33.2), including acute hemolytic reactions due to incompatibility of major erythrocyte antigens and antibodies; delayed transfusion reactions (3–10 days post-transfusion) as a result of non-ABO antigenantibody incompatibility; allergic reactions due to antibodies to plasma proteins; febrile reactions due to antibodies to leukocytes or plasma proteins; hypervolemic reactions; sepsis as a result of contaminated blood; infectious complications; and graft-versus-host disease as a result of transfused lymphocytes in severely immunocompromised cancer patients. A single blood donation contains from 450 to 500 mL of blood with a minimum hematocrit of 38%. This “whole blood” is usually stored for 21 days at 1 ◦ C to 6 ◦ C in a sterile container with anticoagulant solution. If the container is opened for any reason, the unit becomes unsuitable for transfusion after 24 hours. After plasma removal, the resulting red blood cell component has a hematocrit of 65% to 80% and a volume of 300 to 350 mL. Red blood cells stored with additive solutions have a shelf-life of 42 days at 1 ◦ C to 6 ◦ C. Each unit of whole blood or red blood cells contains enough hemoglobin to raise the hemoglobin

concentration in an average-sized adult by approximately 1 g/dL (3% increase in the hematocrit). The ABO group of all red-cell-containing components must be compatible with ABO antibodies in the recipient’s plasma. Except in cases of emergency, serologic compatibility must be established between the recipient and donor (usually by crossmatching) before any red cell-containing components are transfused. The volume of PRBC required by a particular patient can be calculated by the following formula: Volume of PRBC (mL) = patient’s weight (in kg) × patient’s estimated blood volume (mL/kg) × (desired hematocrit – observed hematocrit)/hematocrit of PRBC The estimated blood volume in children varies with age, and the hematocrit also varies with each unit of PRBC. However, with the formula above, the standard volume to transfuse should be approximately 10 to 20 mL/kg. To avoid hypervolemia in patients with chronic anemia, one should either consider lower volumes of transfusion (5 mL/kg) or prolonged infusion times (>4 hours). The primary reasons for low post-transfusion increments in hemoglobin or hematocrit include alloimmunization, continued blood loss, autoimmune hemolytic anemia, hypersplenism, or incorrect calculation of the packed red cell volume. Graft-versus-host disease is an infrequent but potentially severe complication in severely immunosuppressed childhood cancer patients with pancytopenia. Every unit of packed red cells contains a small volume of immunocompetent lymphocytes that, in an immunosuppressed host, can induce graft-versus-host disease. Patients at highest risk for this condition are children with severe combined immune deficiency, Wiskott–Aldrich syndrome, immunosuppression due to bone marrow transplantation, and leukemia or lymphoma treated with aggressive chemotherapy. Newborns receiving transfusions, either by the intrauterine route or postpartum, are also at risk for this complication. Irradiation of blood products is the most effective and economical way of preventing graft-versushost disease in high-risk patients. The recommended dose of radiation, approximately 1500 cGy, inactivates immunocompetent lymphocytes without damaging other components of the transfusion product.

Platelet transfusions The use of platelet products has increased dramatically over the last decade in the United States, largely because of

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Table 33.2 Noninfectious transfusion reactions Febrile Leukoagglutinins in patient destroy transfused leukocytes ≥1 ◦ C (2 ◦ F) in temperature rise Chill common, but alone is not a transfusion reaction Fever may be sign of septic, hemolytic, or vascular reaction Prophylaxis/treatment: acetaminophen Poses no threat to patient, although continued transfusion may not be advisable Allergic IgE-medicated reaction to substances in transfused product Usually limited to urticarial rash Occasional wheezing and angioedema Symptoms similar to those in vascular reactions Prophylaxis/treatment: diphenhydramine; if severe, corticosteroids or epinephrine Vascular Caused by leukocyte products secreted during storage of blood components, especially platelets (interleukins-1, -6 and -8, as well as TNF
, histamine, and serotonin) May be severe or life-threatening Signs/symptoms: feeling of doom, fever, generalized pruritus, flushing, erythema, gastrointestinal symptoms, urticaria, angioedema, dyspnea, wheezing, bronchospasm, hypotension, tachycardia, respiratory/cardiac arrest, shock, and pulmonary hemorrhage Prophylaxis: leukoreduction Treatment: supportive, depending on manifestations Hemolytic, acute Immune-mediated destruction of erythrocytes Administrative error usual cause (e.g. sample mislabeled for type and cross-match) May be fatal or result in permanent renal damage Signs/symptoms: first signs are typically fever, tachycardia, chills, dyspnea, feeling of doom, chest or back pain, abnormal bleeding, DIC, shock, instability of blood pressure in surgery; first clues, hypotension, DIC, hemoglobinuria Prophylaxis: close attention to administrative procedures Treatment: stop transfusion, maintain blood pressure and urine flow, correct hemostasis May involve other causes of hemolysis, such as intraoperative cell saver, simultaneous transfusion of lysing solution, freezing or overheating of erythrocyte products Hemolytic, chronic Anamnestic antibody response in previously alloimmunized patient Onset 2–14 days after transfusion Signs/symptoms: unexplained fever, positive direct Coomb’s test, falling hematocrit, increased bilirubin and LDH Treatment: usually none required Septic Bacterial contamination of blood unit, typically cryophilic bacteria that metabolize citrate in erythrocytes (Yersinia enterocolitica), but also a variety of organisms in platelets and thawed plasma products ≥2 ◦ C (3–4 ◦ F) rise in temperature, severe chills, hypotension or shock Treatment: stop transfusion, give broad-spectrum antibiotics, maintain blood pressure Anaphylactic IgA deficiency in patient Severe dyspnea, pulmonary or laryngeal edema, severe bronchospasm, laryngospasm Treatment: corticosteroids, epinephrine Other noninfectious complications Acute noncardiogenic pulmonary edema Acute increased permeability of pulmonary microcirculation, with massive pulmonary edema Likely cause: complement activation and secondary cytokine release induced by transfused granulocyte antibiotics Treatment: corticosteroids, epinephrine HLA and other antigen alloimmunizations Circulatory overload Graft-versus-host disease Hypothermia with massive transfusion (>15 mL/kg per hour in children) Metabolic complications with massive transfusion Usually concomitant with liver or kidney failure Citrate toxicity Hyper- or hypokalemia Acidosis/alkalosis Immunomodulation Abbreviations: TNF, tumor necrosis factor; DIC, disseminated intravascular coagulation; LDH, lactate dehydrogenase; HLA, human leukocyte antigen.

Hematologic supportive care

their wider use in cancer patients and transplant recipients. Platelet support is of critical importance in patients undergoing bone marrow transplantation, who have long periods of thrombocytopenia. Thrombocytopenia in children with cancer most often develops as a result of decreased platelet production by the bone marrow secondary to tumor progression, myelosuppressive chemotherapy, or both. Other factors include hypersplenism, sepsis, disseminated intravascular coagulation and bleeding; antiplatelet antibodies can produce thrombocytopenia by decreasing platelet survival. Platelet transfusion therapy reduces the morbidity and mortality from hemorrhage, but is much less efficacious if increased platelet consumption or destruction markedly shortens platelet survival. The number of platelets required to produce or maintain adequate hemostasis has been extensively studied. Analysis of the temporal relationship between bleeding time and platelet count has demonstrated that bleeding time is not significantly increased until the platelet count falls below 100,000/mm3 . Thereafter, bleeding time increases in direct proportion to the decreased platelet number. In studies conducted at the National Cancer Institute in the early 1960s, Gaydas et al.5 demonstrated that moderateto-severe hemorrhage did not increase in incidence until the platelet count decreased below 20,000/mm3 . Since this study, there has been considerable discussion regarding the use of prophylactic platelet transfusion for a threshold platelet count. In 2001, the American Society of Clinical Oncology, published a set of clinical practice guidelines for platelet transfusions in cancer patients.6 The expert panel that formulated these guidelines recommends, based on several retrospective and prospective adult studies, that platelet transfusions be given to adult patients with leukemia who have a platelet count of less than or equal to 10,000/mm3 . However, in children with ALL during remission induction, platelet transfusion is rarely indicated, because they are hypercoagulable as a result of treatment with glucocorticoids and L-asparaginase. After remission induction, platelet transfusion is rarely necessary since thrombocytopenia is generally mild. Similar to the practice with adults, prophylactic platelet transfusions are given to pediatric patients with AML when the platelet count falls below 10,000/mm3 . In all instances, prophylactic transfusions should be administered at a higher threshold (e.g. 20,000/mm3 ) to patients with fever, sepsis, previous bleeds, hyperleukocytosis, severe mucositis, coagulopathy, and falling platelet counts.7–11 Careful attention to oral hygiene and suppression of menses, if relevant, should also be aggressively pursued.

In general, a threshold platelet count of 50,000/mm3 is used for minor procedures and 75,000 to 100,000/mm3 for patients undergoing a major invasive surgical procedure, such as laparotomy or craniotomy. Whether platelet transfusions are necessary before minimally invasive procedures, such as bone marrow aspiration or lumbar puncture in an asymptomatic patient, is less clear. To answer this question, at least with respect to the risks associated with thrombocytopenia and lumbar punctures, Howard et al.12 retrospectively reviewed the records of newly diagnosed patients with ALL undergoing induction and consolidation therapy at St. Jude Children’s Research Hospital. The results of that study demonstrate that lumbar puncture can safely be performed in children with ALL if the platelet count is 10,000/mm3 or higher. Although thrombocytopenic patients do not suffer neurologic complications of lumbar puncture, their risk for blood contamination of the cerebrospinal fluid (CSF) does increase at low platelet counts.13 Blood contamination of CSF, or “traumatic lumbar puncture,” in newly diagnosed children with circulating leukemic blasts confers a worse prognosis, presumably by introducing leukemia into the CSF.14 Based on these observations, the authors recommend that in the setting of a diagnostic lumbar puncture or suspicion of circulating leukemic cells or bacteremia, a platelet count greater than 100,000/mm3 is desirable. For a routine lumbar puncture to facilitate intrathecal chemotherapy, a platelet count of 10,000/mm3 is adequate. A unit of platelets is defined as a concentrate of platelets separated from a single unit of whole blood and suspended in a small amount of the original plasma. The typical unit contains 0.5 to 1×1011 platelets suspended in 40 to 70 mL of plasma. On average, 4 to 8 units from different donors are pooled for a transfusion product. These are generally referred to as random donor platelets. Alternatively, multiple platelet units may be processed from the same donor via an apheresis machine, which continually processes and centrifuges blood. The red cells and plasma are returned to the donor and the platelet-rich fraction is collected. This “single-donor unit” has the same number of platelets as found in the 4 to 8 pooled multidonor units. Commonly, this product is referred to as an apheresis-platelet product. Apheresis platelets have several advantages (e.g. reduced antigen exposure and lower risk of infection) that make them preferable when multiple transfusion are anticipated, or when patients are refractory to platelets from unmatched donors. Donor platelets should be stored with gentle, continuous agitation at 20 ◦ C to 24 ◦ C for no more than 5 days. The number of platelet concentrates to be administered depends on the clinical situation. A unit of platelets is generally sufficient to produce a rise of approximately 10,000 to

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15,000 platelets/mm3 per square meter of body surface area in a child or 5000 to 10,000 platelets/mm3 per square meter in an adult. Thus, 0.5 to 1 unit of platelets is commonly given to children for every 10 kg of body weight. Larger doses may be required if the disease is complicated by fever, sepsis, or both. Platelet concentrates should be both Rh- and ABOtype specific. As with packed red cells and whole blood, platelets should be irradiated to prevent graft-versus-host disease. In a large, multi-institutional, randomized, blinded trial in adult patients with AML, the Trial to Reduce Alloimmunization to Platelets (TRAP), the frequency of immunemediated platelet refractoriness with antiplatelet antibodies was 13% in the control group of patients who received unmodified, random donor platelets, as compared with 3% and 4% in patients who received filtered, leukocytereduced platelets from random (P = 0.004) or single donors (P = 0.01), respectively.15 Hence, for patients expected to require multiple platelet transfusions, the use of leukocytedepleted blood products should be considered in order to prevent alloimmunization. Platelet responses may be more precisely determined by calculating a corrected count increment (CCI), which gives the increase in platelet count adjusted for the number of platelets infused and the size of the recipient. The formula for calculating the CCI is given below: observed platelet count increment CCI =

× body surface area (m2 ) number of platelets transfused (×1011 )

The observed platelet count increment is the difference between the platelet count 1 hour or 10 minutes16 posttransfusion and the pretransfusion level. The expected increment in a stable patient who has received fresh or properly stored platelets should be 6000 to 8000/mm3 per unit of platelets transfused, with a CCI of 10,000 to 20,000. CCI values can be useful in quantifying platelet transfusion responsiveness. Values above 10,000 are considered to indicate good responses, whereas those below 7500 to 5000 on two consecutive ABO-compatible transfusions, stored less than 72 hours, are considered to indicate platelet refractoriness. There are many reasons for a reduced CCI. It may represent a simple indirect measure of alloimmunization, but HLA antibodies are only one of many factors that influence the CCI. In a study that used multilinear regression analysis, major factors influencing the CCI were prior splenectomy, bone marrow transplantation, disseminated intravascular coagulation, use of amphotericin B, splenomegaly, and HLA antibody grade.17 In addition, the length and condition of platelet storage may have a profound impact on the survival of platelets. Although platelets can be stored for

Poor corrected count increment after platelet transfusion Consider alloimmunization (lymphocytoxicity testing)

−

+ • Use HLA-matched

•

Rule out drug effects, fever, DIC

•

Administer ABO – matched platelets or pheresed, fresh products

platelets

Fig. 33.1 Strategy for evaluating the response to platelet transfusion. DIC, disseminated intravascular coagulation.

up to 5 days, their function and viability decrease with storage time, even when conditions are optimal. Under normal circumstances, the life-span of the transfused platelets is 9.5 days, but in thrombocytopenic patients, it ranges from only 4 to 5 days, and in the presence of autoantibodies it can be less than 24 hours. Fresher platelet products can make an essential difference in the incremental platelet number in patients with alloimmunization, splenomegaly, or sepsis. A useful schema for evaluating the response to platelet transfusion is presented in Figure 33.1. Approximately 50% to 60% of patients with plateletrefractory alloimmunization will respond to HLA-matched platelets.18 For patients who do not have an adequate response, cross-matching techniques can be used to identify suitable donors.19 In patients that are actively bleeding, the use of large numbers of pooled random donor platelets may be beneficial.20 Other approaches, including the use of corticosteroids, high-dose intravenous immunoglobulin, epsilon aminocaproic acid and desmopressin have been evaluated in a limited number of patients, with variable results.21–23

Fresh frozen plasma Fresh frozen plasma (FFP) consists of the fluid portion of blood that is separated and frozen at −18 ◦ C or below within 8 hours after the collection of whole blood in anticoagulant solution [citrate phosphate dextrose (CPD), citrate phosphate double dextrose (CP2D), or citrate phosphate dextrose adenine solution (CPDA-1)]. Plasma collected in acid citrate dextrose or 2% to 3% sodium citrate must be frozen within 6 hours. The volume of this component generally ranges from 180 to 300 mL. The plasma proteins in FFP include all coagulation factors in concentrations of

Hematologic supportive care

1 IU/mL of plasma. Hence, a unit of FFP contains about 200 IU of each coagulation factor. The amount of coagulation factor activity in one unit of FFP equals approximately 7% of the coagulation factor activity in a 70-kg patient. FFP frozen at −18 ◦ C becomes outdated at 1 year; however, when stored at −65 ◦ C, it can be held for as long as 7 years. The indications for FFP are limited and include the following24,25 :

1. Requirement for replacement of plasma coagulation factors in preoperative patients when specific corrective factors are not available. 2. Massive transfusion requirement in patients with abnormal coagulation. 3. Bleeding in patients receiving warfarin who will undergo an invasive procedure before vitamin K administration can reverse the warfarin effect. 4. Plasma exchange procedures for a diagnosis of thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome. Because of potential infectious risks, FFP should not be used when coagulopathy can be corrected more effectively with specific therapy, such as vitamin K, cryoprecipitated AHF, or Factor VIII:C concentrates, or when blood volume can be safely and adequately replaced with other volume expanders, such as 0.9% sodium chloride, Ringer’s lactate, 5% albumin, or plasma protein fraction. Complications of FFP administration are uncommon; however, antibodies in the plasma may react with the recipient’s red cells, causing a positive direct antiglobulin test. In rare instances, noncardiogenic pulmonary edema (transfusion-related acute lung injury) may develop due to antibodies in donor plasma that react with the recipient’s leukocytes. One approach to limiting FFP-related complications is to ensure that the plasma is ABO-compatible with the recipient’s red cells. The volume of FFP transfused depends on the clinical situation and the size of the patient, with laboratory assays of coagulation function serving as a useful ancillary guide. Plasma must be thawed in a water bath at 30 ◦ C to 37 ◦ C or in an FDA-approved microwave device, and then infused immediately or stored at 1 ◦ C to 6 ◦ C for no more than 5 days. Thawed plasma contains all of the stable proteins found in FFP, including reduced amounts of Factor VIII and Factor V. The reduction in Factor VIII:C is clinically significant.

Granulocyte transfusions Granulocyte infusions, as a means to enhance host defenses, have been of interest for more than 60 years. However, progress was felt to be limited by inadequate yields of functional granulocytes from donors. Recently, this obsta-

cle has been circumvented by the administering of granulocyte colony-stimulating factor, G-CSF, with or without corticosteroids to donors 12 hours before leukopharesis.26 This method can result in granulocyte yields that are sufficient to allow for large numbers of neutrophils to be transfused into neutropenic patients with life-threatening infections unresponsive to conventional therapies. The benefit of this approach remains to be proven in large randomized trials. Of note, although some data are emerging on the longterm effects of G-CSF administration in healthy donors,27 the practice of using G-CSF to mobilize white blood cells in healthy donors is controversial from an ethical standpoint. The concern is that the long-term risks of treating normal donors with G-CSF is not known. Hence, some believe that this practice should be considered experimental and subject to review by institutional review boards.28,29 Granulocyte transfusions are usually prepared by intermittent or continuous centrifugation methods. Because granulocyte function declines rapidly with storage, the cells should be transfused as soon as possible after preparation. The cells should be irradiated, ABO-compatible and Rh-compatible. Granulocyte transfusions should not be filtered before use. Alloimmunization, febrile reactions, fluid overload, graft-versus-host disease, and pulmonary complications are potential adverse effects of granulocyte transfusion. The optimal dose, frequency, and duration of granulocyte transfusions have not been established.

Transfusion-associated infections Over the past 15 years, concerns regarding the transmission of infectious diseases through the blood supply have shaped our transfusion practices. A three-tiered system for donor screening is the mainstay for the prevention of transfusion-associated infections. These include: (1) a voluntary self-exclusion of donors at risk for various infections based on a history of exposure; (2) confidential selfexclusion of donors (the donor requests that the blood collected not be used for transfusion); and (3) testing for evidence of infection in donor units. Table 33.3 lists the viral agents currently screened for by US blood banks on all blood products, the estimated risk of infection per unit of blood transfused, and current screening procedures. In addition to these viral agents, each unit of blood donated is also tested for evidence of syphilis. Directed donations are generally discouraged, as this strategy may pressure high-risk individuals to donate under peer pressure. In addition, direct donation from family members may sensitize the recipient to HLA antigens, thus precluding bone marrow transplantation. Potential transfusion-transmitted

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Hepatitis B virus

Table 33.3 Transfusion-associated viral infections

Agent

Estimated risk of Prevalence in infection per unit donors (%)92 transfused32

Hepatitis B

0.2

1:31,000–1:147,000

Hepatitis C

0.4

1:1,600,000

HIV-1

0.015

1:1,900,000

HIV-2

Essentially 0 in US 0.046

<1:1,000,000

HTLV-1/2

Current tests EIA for HBsAg, EIA for anti-HBc EIA for anti-HCV, RIBA for confirmation, NAT EIA for anti-HIV, Western blot for p24 antigen confirmation and NAT EIA for anti-HIV-2

1:256,000–1:2,000,000 EIA for anti-HTLV-1,2

Abbreviations: HBsAg, Hepatitis B surface antigen; HBc, hepatitis B core antigen; EIA, enzyme immunoassay; RIBA, recombinant immunoblot assay; NAT, nucleic acid amplification testing.

infectious agents and their impact on the cancer population, if pertinent, are described below.

Cytomegalovirus Cytomegalovirus (CMV) remains one of the most common viruses found in the general population. As many as 80% of blood donors have CMV antibodies with the prevalence increasing with donor age. However, only about 1% of donors are able to transmit the virus. CMV may be present in the white cell-containing components from donors previously infected with the virus, which can persist lifelong in the presence of protective serum antibodies. Among previously negative patients who have received multiple transfusions, 9% to 58% will become CMV-positive. The importance of CMV-induced disease in the general population of pediatric oncology patients is unclear, but transmission of the virus by transfusion may be of concern in severely immunocompromised patients, including low-birth-weight premature infants born to CMVseronegative mothers, AIDS patients, and bone marrow transplant recipients. Approximately one-half to one-third of all previously CMV-negative marrow transplant recipients will become CMV-positive after receiving unscreened blood transfusions.30,31 The risk of CMV transmission in this patient population has been markedly reduced by transfusing components from CMV-seronegative donors and/or by leukocyte filtration of blood into CMV-negative bone marrow transplant candidates and recipients.

Accounting for 10% to 15% of post-transfusion hepatitis cases in the era prior to routine screening, hepatitis B virus (HBV) now has an estimated donor prevalence in the range of 0.1% to 2.0%. The risk of HBV transmission is calculated to be between 1:31,000 to 1:147,000 units transfused, based on the current seroprevalence rate and the sensitivity of current assays.32 Possible reasons for “breakthrough” infections from screened units include the 1- to 6-week “window” between viral infection and antibody response in donors, true false-negative testing, and the possibility of a low-titer carrier state in which the virus can be transmitted but is present in too low a concentration to allow detection. Because of overlapping risk factors, measures taken to reduce the risk of HIV infection have also led to decreases in the incidence of HBV positivity among blood donors. The risk of HBV infection can also be reduced by prior vaccination of persons who would be expected to have large transfusion requirements.

Hepatitis C virus Hepatitis C virus (HCV) was responsible for the majority (>90%) of non-A, non-B hepatitis cases associated with transfusions in the 1970s and 1980s. Such infections are of major concern, since approximately one-half of the patients with non-A, non-B hepatitis will progress to chronic liver disease, which may be fatal. The institution of HCV screening in donors using enzyme immunoassays (EIA) to detect antibodies to HCV has reduced the incidence of post-transfusion HCV detection from 1% to 4% per unit to approximately 0.03% per unit. The incidence has been further reduced by nucleic acid amplification testing (NAT) for HCV RNA. In 1999, NAT was implemented as a donor screening assay for HIV and HCV under the FDA Investigational New Drug and recently approved by the FDA for donor testing.33 Unlike other screening assays that rely on the detection of antibodies generated by the infected individual, NAT detects genetic material from the infectious agent, allowing earlier detection of an infected donor. In over 25 million donations screened with NAT, eight HIV-positive and 113 HCV-positive units were detected that tested negative by other methods.32

Human immunodeficiency viruses HIV-1 accounts for most of the public concern over risks associated with blood transfusions. About 50% of persons seroconverting from blood transfusions are expected to develop acquired immunodeficiency syndrome (AIDS) within 7 years. The combination of donor self-selection and

Hematologic supportive care

HIV antibody testing is estimated to be 99.9% effective in eliminating HIV-positive donors. The risk of HIV positivity in transfused blood appears to be decreasing yearly, and the most recent estimate of the risk of HIV infection from a single unit of blood product is approximately 1 in 2 million units transfused. As indicated in the above discussion of HCV, the significant decrease in the risk of acquiring HIV from a blood transfusion can be attributed to the recent approval by the FDA of nucleic acid amplification testing. HIV-2 currently presents virtually no risk to transfusion recipients in the United States, as its distribution is limited mainly to western Africa. Routine screening for anti-HIV-1 antibodies will also detect over 75% of HIV-2infected units. Since 1992, there has been a national policy mandating specific screening of blood for HIV-2.

platelets is reported to be between 1 and 200 per 100,000 units.37,38 Gram-positive and gram-negative bacteria have been implicated in these fatalities. Transfusion-transmitted bacterial infection of red cells is rare. The estimated frequency of contaminated red cells is 1 per 0.5 to 1 million units.36 The organism most often implicated in bacterial sepsis associated with red cell transfusion is Yersinia enterocolitica, followed by Serratia and Pseudomonas species. In contrast to contaminated platelet transfusions, transfusion of contaminated red cell units is associated with high fever and chills during or immediately after infusion. A greater than 50% mortality rate has been reported with contaminated red cell transfusions.

Human T-cell leukemia viruses

Hepatitis A virus and hepatitis E virus pose only a minuscule transfusion risk, owing to the very short viremic phase, the low concentration in blood during the viremic phase, and the absence of a prolonged carrier state. Hepatitis D virus (HDV) can cause significant disease; however, it is usually found in the presence of HBV and is therefore eliminated when donors are screened for the latter virus. Nearly all donors are positive for Epstein–Barr virus, which may be responsible for rare cases of a post-transfusion infectious mononucleosis-like illness. The seroconversion rate following transfusion is approximately 1:200. Parvovirus B-19 is the agent responsible for erythema infectiosum, a contagious febrile illness of childhood, and has been implicated in the development of severe anemia in immunocompromised patients or those with chronic hemolytic anemias. Thirty to sixty percent of adults have antibodies to this virus. It has been estimated that 1 in 10,000 to 1 in 20,000 donors may be viremic with parvovirus B-19, although it is unclear whether transfusions are responsible for disease transmission. An impending threat to the cancer population is the recent spread of the West Nile virus (WNV) to the United States. This virus primarily infects birds and mosquitoes, with horses and humans serving as incidental hosts. Since the virus is transmitted from mosquitoes to humans, the incidence of disease peaks in the late summer and early fall. It is estimated that one out of five infected individuals will have a mild febrile illness and that 1 out of 150 will develop meningitis or encephalitis.39 The risk for severe disease is thought to be higher in the elderly and the immunosuppressed. During the 2002 WNV epidemic in the United States, 23 patients were confirmed to have acquired WNV infection through transfusion.40 This observation led to a program of nation-wide screening of all blood donations using a recently developed NAT assay for this virus. More than 800 viremic blood donations have been identified

Human T-cell leukemia virus-1 (HTLV-1) is associated with the development of adult T-cell leukemia or HTLVassociated tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). These diseases develop in a small minority of infected persons after an incubation period of years to decades. HTLV-2 has not been conclusively implicated in the development of any human disease, although there are recent reports suggesting its involvement in a TSP/HAM-like illness. HTLV shares many of the same routes of transmission as HIV-1, including sexual contact, intravenous drug use, transfusion and breast milk; however, it is considerably less infectious.34 Serologic testing for HTLV-1/2 is part of the routine screening of donor units in the United States.

Bacterial contamination Although the focus of public attention has been on virally transmitted agents in the blood supply, the most commonly transfused infectious agents are bacteria, specifically through contaminated platelets. The risk of receiving contaminated platelets may be 50- to 250-fold higher than the combined risk of transfusion-related infection per unit associated with HIV, HCV, HBV, and HTLV-1/2.35 Platelets are highly susceptible to bacterial contamination because they are stored at 20–24 ◦ C for as long as 5 days. The estimated incidence of contaminated platelets is approximately 1 in 2000 to 12,000 units.26,36 Contamination of platelets is thought to mainly occur during the blood donation process. The clinical presentation of a patient transfused with contaminated platelets is variable – depending on the extent of the contamination and the virulence of the bacteria. The signs and symptoms are often delayed and often go unrecognized as being related to the transfusion of platelets.35 The risk of death from contaminated

Other infectious agents

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since this screening program was implemented, with six cases of transfusion-acquired WNV reported during the same time period, indicating that there is still a risk of transfusion-associated transmission.41 Creutzfeldt–Jakob disease (CJD) and a new variant CJD (nvCJD) pose a theoretical threat to the blood supply. CJD is a human transmissible spongiform encephalopathy believed to be caused by a prion protein. CJD has been described in persons who received contaminated pituitary growth hormone, corneas, or dura mater from human cadavers or who were operated on with contaminated neurosurgical instruments. nvCJD has recently been described in the United Kingdom with features distinctly different from CJD. The cause of this new human transmissible spongiform encephalopathy appears to be the same agent responsible for an outbreak of bovine spongiform encephalopathy (BSE) among cattle in the UK.42 Although there have been no confirmed reports of either CJD or nvCJD transmission by blood or blood products, some studies in experimental animal models suggest that both CJD and nvCJD can be transmitted through blood transfusion.43,44 Based on these observations, and due to the relatively new discovery of these agents, certain precautions have been put into effect regarding blood donations in the United States. Sporozoan parasites, the causative agents in malaria, are detected in approximately 0.25 cases per million units of blood collected in this country. In addition, rare cases of Chagas disease and babesiosis have been linked to transfusions.

General guidelines for blood product use The indications for and techniques of blood product support vary from institution to institution. Table 33.4 summarizes the use of blood products at St. Jude Children’s Research Hospital.

Blood substitutes In theory, the wide availability of blood substitutes should have a dramatic impact on transfusion practices. They could be used as adjuncts to current human products and could reduce the risk of transfusion-associated infections or other complications. At present, preclinical development of blood substitutes has focused on strategies for improving the oxygen-carrying capacity of blood. Two major approaches have been undertaken: one relies on synthetic oxygen-carrying compounds, known as perfluorocarbons (PFCs), while the other is a hemoglobinbased strategy that takes advantage of the blood’s natural

Table 33.4 Guidelines for the use of blood products at St. Jude Children’s Research Hospital Irradiate all cellular products with 25 Gy/unit Purpose: prevent GVHD mediated by transfused lymphocytes Reduce leukocytes in all cellular products to <106 /unit during pheresis or by filtration (preferably before storage) Purpose: ↓HLA alloimmunization ↓CMV transmission ↓Non-hemolytic transfusion reactions Prevention of immunomodulation by transferred leukocytes Use CMV sero-negative products for selected patients Purpose: prevention of CMV transmission in immunosuppressed patients (primarily allogeneic BMT candidates) Use single-donor pheresis platelets whenever possible Purpose: ↓exposure to potential transfusion-transmitted infectious agents Abbreviations: GVHD, graft-versus-host disease; BMT, bone marrow transplantation; CMV, cytomegalovirus.

ability to capture and transport gases. A recent review by Stowell45 summarizes the current status of these materials as blood substitutes. Research on other substitutes is under way but so far has not yielded any products that are being tested in clinical trials. The future of blood substitutes in supportive therapy for childhood leukemia patients remains in doubt. However, increased societal concern over infectious disease transmission through blood products, respect for religious and ethical concerns, and the introduction of modern cancer therapy to children in developing countries lacking adequate blood-banking infrastructure are compelling reasons to continue the development of substitutes. Before these strategies become feasible in cancer patients, it will be necessary to address the safety issues posed by administering very large volumes of hemoglobin-based substitutes (in the range of 50 to 100 g), as well as the potentially serious long-term effects of these supportive therapies. Several of the agents that have been studied show some short-term toxicity – including hypertension and renal damage, tachycardia, and gastrointestinal pain. Finally, studies of bovinebased substitutes must address the danger of transmission of bovine spongiform encephalopathy and perhaps other, as yet unidentified, diseases.

Venous access support Optimal delivery of intravenous chemotherapy to leukemia patients requires stable venous access. Central venous catheters meet this need by preventing damage to

Hematologic supportive care

peripheral veins, ensuring safe administration of chemotherapy and supportive care, and providing relief from psychological trauma due to multiple venous punctures. There are two major groups of venous devices presently in use in pediatric leukemia patients. The first includes totally implanted devices such as the Port-A-Cath, Infus-A-Port, or Mediport, which are placed intracorporeally under the skin. The second group are the tunneled, cuffed silicone catheters such as Hickman or Broviac catheters, which are tunneled subcutaneously to a skin exit site. Selection of a venous access device depends on numerous variables, such as: the planned length of the patient’s treatment; the complexity of the treatment course; the use of vesicants; the status of the patient’s peripheral veins; the frequency of blood sampling, nutritional support, and fluid administration; and the ability of the patient and family to care for the catheter. More complex treatment regimens or the need for parenteral nutrition with chemotherapy would suggest the use of a double-lumen central catheter. Patients receiving intensive chemotherapy for acute myeloid leukemia or B-cell acute lymphoid leukemia, or hematopoietic stem cell transplantation, typically need double-lumen catheters. Small children may require single-lumen external catheters due to size constraints. Given a choice, teenagers often prefer implanted ports, which may give better freedom of activity and are better concealed.

External tunneled catheters The first external catheters were introduced by Broviac et al. in 197346 and by Hickman et al. in 1974.47 These silastic catheters are placed by use of a venous cut-down approach (external, internal jugular or cephalic vein) or a percutaneous subclavian approach. All Hickman–Broviac catheters have a radio-opaque strip for visualization by radiography or fluoroscopy. The optimal location for placement is the superior vena cava at or above the junction of the right atrium. Catheters of this type are inserted beneath the skin for several inches from exit site to vein. A Dacron cuff proximal to the exit site secures the catheter and minimizes the risk of infection. The external portion of the catheter is connected to a heparin lock. Another type of external catheter is the Groshong catheter, which has a three-way valve at the tip that opens for aspiration or infusion and remains closed when not in use. Unlike their Hickman–Broviac counterparts, Groshong catheters do not require heparin flushes to maintain patency.

Implanted venous access devices Various types of catheters that are totally implanted under the skin, (e.g. Port-A-Cath, Infus-A-Port, and Mediport) are

now available. The ports are inserted in the infraclavicular area (the subclavian or jugular vein), or lower thoracic area, the abdominal wall (femoral vein), or a peripheral upper extremity vein (the basilic or cephalic vein). The catheter port is a stainless steel or titanium plastic chamber with a rubber top and a long thin catheter connected to the side. Both the port and the catheter are implanted completely under the skin and sutured in place. Single- and doublelumen ports, both accessed with a special Huber needle, are available.

Nontunneled venous access devices Several alternative catheters have been used successfully to gain venous access. The PICC line (peripherally inserted central catheter) is a catheter made of silicone or a polymer, which is usually inserted in the basilic or cephalic vein and advanced to the superior vena cava for tip placement. All PICC lines must be placed with a strict sterile technique, requiring the skills of physicians or nurses who are experienced in intravenous therapy and have specialized education on PICC insertion.

Complications associated with central venous catheters Catheter placement can produce numerous complications, including pneumothorax, hemothorax, hydrothorax, and injury to the carotid artery or brachial nerve plexus. Air embolism is an immediate concern and can occur even during catheter maintenance when the tube is being manipulated. The greatest frequency of complications is seen with external tunneled catheters, with relatively few difficulties arising from use of implanted ports.48

Occlusions Catheter occlusion may be partial, allowing fluids to be infused but not withdrawn, or it may be complete, so that neither infusion nor withdrawal is possible. The basis for occlusions is not always obvious, but may entail clot formation at the end of the catheter, fibrin sheath formation around the catheter with slippage over the catheter tip, or build-up of blood products or precipitate within the catheter itself. This complication is seen more often with heavier silicone catheters than with lighter, more flexible ones. The incidence of occlusion ranges from 15% to 19% and occlusion generally occurs at 15 to 65 days postinsertion.48 Previously, fibrinolytic therapy with urokinase or streptokinase was used at many institutions to treat suspected

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clot formation resulting in catheter lumen occlusions. However, in January 1999 the Food and Drug Administration (FDA) discontinued the use of urokinase in the United States due to quality control issues by the manufacturer. Furthermore, in December 1999, the FDA released a warning letter that streptokinase was associated with an unacceptable level of allergic reactions and was not to be used to restore patency to central venous catheters. An alternative approach to catheter occlusion has been the use of another fibrinolytic agent, recombinant tissue plasminogen activator (rt-PA). Several studies have recently been published demonstrating the efficacy and safety of rt-PA for catheter occlusion in children.49–52 The recommended dose of rt-PA and the dwell time in the catheter differ in these studies. At St. Jude Children’s Research Hospital, the dose of rt-PA (1 mg/mL) is based on the internal volume of the central venous catheter. If blood cannot be aspirated from the catheter after a one hour incubation of rt-PA in the lumen of the occluded catheter, the rt-PA is left in for another hour. Catheter occlusions presumed to be due to mineral or drug precipitates can be cleared with 0.1 N hydrochloric acid (0.2–0.5 mL). Lipid deposits can usually be cleared with up to 3 mL of a solution of 70% ethanol in water.30,53

Venous thrombus formation Thrombus formation in catheters can result from many different mechanisms, including damage to the vessel wall from the catheter tip, leading to platelet aggregation and formation of mural thrombi initially and then large veno-occlusive thrombi that enter the vena cava or right atrium. Large-bore catheters and left-sided catheterization are more likely to induce thrombus formation than are other procedures, and external catheters are more thrombogenic than implanted ports. Sclerosing chemotherapy, venous compression by mediastinal tumors, and bacterial colonization can aggravate thrombus formation. Signs and symptoms are usually subtle and may include mild pain in the chest wall, neck, or scapular area. Physical findings are more evident after complete occlusion and include regional swelling and the development of collateral circulation. The diagnosis is confirmed by venography. Treatment includes thrombolytic agents (discussed above) and the use of anticoagulants (standard heparin, low molecular weight heparin, warfarin); surgical removal is rarely needed. Anticoagulants prevent further propagation of a thrombus and helps in the formation of collateral veins. Direct infusion of thrombolytics through the device is most effective in treating a catheter tip thrombi, while mural thrombi require infusions into nearby veins.

Catheter-related infections All types of catheters can promote life-threatening infections in patients with absolute neutrophil counts below 500/mm3 .54 These complications may arise locally (e.g. at the exit site, tunnel, or port pocket) or may be systemic due to intraluminal or extraluminal colonization by bacteria, with or without thrombus formation. Gram-negative bacteria, especially Pseudomonas aeruginosa and E. coli, were once the predominant organisms associated with catheterrelated infections in febrile neutropenic patients. Today, they account for only about 30% of infections, while grampositive aerobes from the skin (e.g. Staphylococcus aureus, S. epidermidis, and Streptococcus species) are identified in about 50% of cases. Candida species account for 5% to 7% of the total infections. Coagulase-negative cocci have high pathogenic potential when introduced through catheter insertion or other procedures that violate intact skin. Catheter infection rates range from 2.7% to 60%. Contributing factors include the lack of immunocompetency, the type of device used, the development of extraluminal thrombosis, and care of the catheter. Neutropenia is associated with about 70% of catheter-related infectious episodes. Uncontrolled bacteremia is a contraindication for catheter insertion. In early studies, multilumen catheters were thought to promote infection more often than single-lumen devices, ostensibly because of the increased number of manipulations they required, but this statement has been refuted in more recent analyses.55 Patients requiring parenteral nutrition and chemotherapy are more prone to infections than those requiring only chemotherapy. Exit-site infections arise in tissue at the point where the catheter exits the skin, whereas tunnel infections occur along the subcutaneous tract extending proximally from the skin exit wound to the site of entry in the central vein. The former are indicated by local erythema, pain, or exudate at the exit site, while the latter are associated with erythema and tenderness along the subcutaneous catheter tract. Systemic infection is usually suspected when the patient presents with fever and chills, which may be evidence of mural thrombus or fibrin sheath at the catheter tip. Patients suspected of having catheter-related infection should have blood culture drawn from all lumens as well as a peripheral vein. Diagnosis of catheter-related sepsis is made when 15 or more colonies of a single organism are apparent in cultures or if the colony count in the catheterblood sample is 10 times more than in the peripheral blood. However, because most institutions do not perform quantitative blood cultures, any positive culture from a catheter

Hematologic supportive care

should be presumptive evidence of catheter-related bacteremia/sepsis. Importantly, one can minimize the risk of catheterrelated bacteremia by “locking” the catheter with vancomycin and heparin.56 A physical and antimicrobial barrier can be created by use of a device called a “vita cuff” (Vita, San Carlos, CA), which is constructed of biodegradable collagen impregnated with silver ions. Indeed, extraluminal surface colonization was substantially reduced in randomized studies of the vita cuff.57 The optimal dressing for the exit site and the frequency of dressing change are still controversial. Indications for catheter removal include persistent bacteremia or clinical symptoms beyond 48 to 72 hours despite appropriate antibiotic treatment; signs and symptoms of hypotension and chills following each catheter flush; infections of the progressive exit site, insertion site, or tunnel tract despite antibiotic treatment; candidiasis or atypical mycobacterial infections; and a catheter that is nonfunctional or no longer required for treatment.

Extravasation Extravasation can occur with use of a vesicant or nonvesicant-type agent.58 The patient may experience severe pain and irritation that requires removal of the catheter. Pain is usually in the area of the subcutaneous tunnel, port pocket, or clavicular area at the time of infusion. Erythema may be present or develop later on. Mechanisms of extravasation include backtracking due to occlusion of the catheter tip. This complication is more likely with percutaneous catheters but can occur with any catheter. Extravasation may also reflect catheter damage or separation during insertion or incorrect locking. The catheter sometimes is pinched off between the clavicle and the first rib if it is inserted into the median subclavian vein. The distal portion of the catheter may fracture due to constant friction, so that it creates an embolus in the right heart or pulmonary artery. Embolization can be asymptomatic or can lead to cardiac arrhythmia, chest pain, and a swishing sound in the ipsilateral ear with catheter flushing. Needle dislodgement from a port, due to use of the incorrect needle or inadequate stabilization of the needle in the port, can cause extravasation in as many as 50% of patients. Hence, care should be taken to ensure that the needle is properly taped, and that the port is placed away from the shoulder to minimize displacement.

Malposition Malpositioning of the catheter may result from: (1) faulty placement into a smaller tributary of the superior vena

cava; (2) migration of the catheter tip into another vein, such as the jugular system; or (3) perforation of superior vena cava or endomyocardium. With catheter-tip migration, patients may be asymptomatic or experience pain with infusion or audible catheter flush in the ipsilateral ear. Thus, catheter placement must be checked by fluoroscopic examination using a lateral view. Perforation of the superior vena cava is a possible complication of silastic and polyurethane catheter placement. The patient experiences sudden onset of chest pain and shortness of breath and may manifest mediastinal widening, pleural and pericardial effusion, or cardiac tamponade. Intimal injury of the blood vessel results from cannulation of the left subclavian vein and curving of the catheter tip.

Hematopoietic growth factors The myelosuppressive effects of chemotherapy and irradiation are clinically important because they compromise the immune system and increase the risk of serious or life-threatening infection and hemorrhage due to thrombocytopenia. Hematopoietic growth factors, or cytokines, are proteins that stimulate bone marrow cells to expand and differentiate along specific pathways, resulting in the maintenance of blood cell populations that are vital to normal immune function. Over the last decade, innovations in recombinant DNA technology have led to the development of cytokines with significant impacts on clinical practice. The application of these cytokines to pediatric patients was recently the subject of two reviews.59,60 Granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF) are perhaps the most thoroughly studied cytokines in the childhood leukemias and will be the major focus of discussion here. Cytokines to stimulate red cell production (erythropoietin) and platelet production (thrombopoietin) are not routinely used in pediatric cancer patients; however, their efficacy and safety in pediatric cancer patients have recently been the subject of a number of studies that will briefly be reviewed here.

Granulocyte-stimulating factors Although multiple factors determine the risk of infection in patients treated with myelosuppressive agents, the best predictor is the neutrophil count. Thus, there has been great interest in the development and use of hematopoietic growth factors to ameliorate or prevent neutropenia in children with leukemia. Two of these cytokines, G-CSF and GM-CSF, stimulate myelopoiesis

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directly, although GM-CSF stimulates multiple cell lineages at intermediate stages of development while G-CSF acts in a relatively lineage-restricted manner in later stages of myeloid differentiation.61,62 Although initially thought to stimulate myelomonocytic and megakaryocytic elements, GM-CSF produces its greatest effects on granulocytes and monocytes. Whether or not these cytokines are beneficial depends on the risk of neutropenia due to myelosuppressive therapy in individual patients. In principle, treatment with growth factors should decrease the duration of neutropenia, thereby reducing the need for hospitalization and antibiotic therapy, as well as the risk of bacterial and fungal infections. Experience with GM-CSF to prevent infectious complications of chemotherapy-induced myelosuppression in children with leukemia has been limited. In contrast, the prophylactic use of G-CSF after myelosuppressive chemotherapy decreased the depth and/or duration of neutropenia in several randomized clinical trials in pediatric patients with ALL.63–66 The difficulty in making absolute recommendations regarding the use of G-CSF in children with leukemia is best exemplified by two pivotal trials of this cytokine in children with ALL conducted by investigators at St. Jude Children’s Research Hospital and by members of the Berlin¨ Frankfurt-Munster (BFM) study group.63,64 In the St. Jude trial, 164 patients with ALL (age range, 2 months to 17 years) were randomized to receive placebo or G-CSF (10 g/kg of body weight per day subcutaneously), beginning 1 day after completion of remission induction therapy and continuing until the neutrophil count was at least 1000/ L for 2 days. Among the 148 evaluable patients, G-CSF treatment did not significantly alter the major endpoints of the study: rate of hospitalization for febrile neutropenia (58% in the G-CSF group versus 68% in the placebo group), event-free survival probability at 3 years (83% in both groups), and the number of severe infections (five in the G-CSF group versus six in the placebo group). The median total costs of supportive care were similar in the two groups ($8768 for G-CSF and $8616 for the placebo). The authors concluded that while G-CSF therapy did in fact produce some short-term clinical benefits in children who had received intensive induction therapy for ALL, including shorter hospital stays and fewer infections overall, its use could not be justified on the basis of more stringent efficacy criteria. Additionally, the cost savings associated with the use of G-CSF were not sufficient to offset the cost of the growth factor. European investigators performed a prospective, randomized, open-label study in children with high-risk ALL in hematologic remission. G-CSF or placebo was given after each of nine cycles of intensive chemotherapy at 5 g/kg per day subcutaneously, starting on the day after com-

pletion of chemotherapy and ending on day 20 of each treatment course. In the 34 patients studied, the average incidence of febrile neutropenia per cycle was significantly reduced in the G-CSF group, from 40% to 17%. The median total duration of severe neutropenia over all treatment cycles was likewise significantly reduced in the G-CSF group (6.2 days per patient versus 20.3 days in the control group), as were the average incidence of neutropenia per treatment cycle and the number of days of neutropenia per patient. This comparison of randomized clinical trials (Table 33.5) illustrates well the difficulties of deciding whether or not to administer G-CSF to children with ALL. With treatment plans that specify only a single cycle of intensive chemotherapy, as in the St. Jude study, it appears that the growth factor will not produce major clinical benefits. If, on the other hand, remission induction-type therapy is repeated frequently, as in the BFM trial, G-CSF may indeed be beneficial. Unfortunately, it is still not possible to predict the outcome of G-CSF therapy on the basis of clinical and laboratory risk factors. Thus, the decision to use G-CSF in individual cases is ultimately a matter of clinical judgment. Clinical studies have also demonstrated beneficial effects on neutrophil counts from G-CSF and GM-CSF in patients with myelodysplastic syndromes.67 A major concern is that, in this setting, the growth factors may accelerate the progression of cases to acute myeloid leukemia (AML), so that physicians must carefully weigh this hazard against potential gains from such therapy. Routine administration of G-CSF after induction chemotherapy for AML still cannot be recommended. Although such treatment may eventually prove harmless, the unknown potential for leukemic cell stimulation and the current lack of adequate studies indicating definite therapeutic benefits in children with AML are sufficiently dissuasive. There have also been reports of the use of these agents to enhance the cycling of leukemic cells prior to remission induction therapy and to accelerate recovery from neutropenia before scheduled treatment.68 Although sound in principle, this strategy has the potential to stimulate an underlying clone of leukemic cells. The use of colony-stimulating factors is contraindicated in patients with massively enlarged spleens, as stimulation of hematopoiesis in extramedullary sites may predispose the patient to splenic infarction. Caution should also be exercised in treating patient with colony-stimulating factors in combination with chest irradiation. Irradiation of the thoracic duct can lead to depletion of peripheral stem cells and lymphocytes, causing prolonged and profound neutropenia when given in regimens that include

Hematologic supportive care

Table 33.5 Comparison of definitive clinical trials of G-CSF in children with ALL Feature

St. Jude study63

BFM study64

Study design

Randomized, double-blind, placebo-controlled

Randomized, open-label, placebo-controlled

No. of evaluable patients

148 (all risk categories)

34 (high-risk)

Treatment period evaluated

Postremission induction (one cycle)

Postintensification (nine cycles)

G-CSF dosage ( g/kg per day)

10

5

Major endpoints (G-CSF versus placebo) Percent patients with febrile neutropenia No. with severe infections

58 versus 69 (P = 0.23) 5 versus 6 (NS)

17 versus 40 (P = 0.007) 10 versus 15 (NS)

Secondary endpoints (G-CSF versus placebo) Percent days ANC <500/ L No. of documented infections overall Median duration of antibiotic use (days)

5.3 versus 12.7 (P = 0.007) 12 versus 27 (P = 0.009) 6 versus 9 (NS)

17.4 versus 61.6 (P < 0.001) 10 versus 21 (P = 0.04) 18.2 versus 32.2 (P = 0.02)

Other Shorter durations of fever Therapy administered on schedule Favorable cost:benefit ratio

No Yes No

Yes Yes Not assessed

Abbreviations: ANC, absolute neutrophil count; NS, not significant.

colony-stimulating factors. The potential for enhanced toxicity when giving G-CSF with intensive chemotherapy and radiation also warrants caution. Although the shortterm side effects of G-CSF have been well described, the long-term effects of this agent in cancer patients have not been closely studied. Recently, Relling et al.69 showed an increased risk of secondary AML in patients who received G-CSF in the context of epipodophyllotoxin-based therapy. The optimal G-CSF dosage has not been established; however, most investigators treating leukemia patients use between 5 and 10 g/kg per dose once daily.63,64,70 Optimal dosages of GM-CSF are likely to be the same as those recommended for G-CSF; however, it will be important to substantiate this impression by comparing the effects of these cytokines in particular clinical situations to determine the doses that lead to a desired clinical outcome. Either colony-stimulating factor can be administered subcutaneously or intravenously. The subcutaneous route is preferred because less drug is needed to produce a given hematologic effect, and the lower peak concentrations achieved by the subcutaneous route lower the risk of serious toxicity. Serious bleeding at injection sites in thrombocytopenic patients is generally not a major concern, and subcutaneous administration is more likely to maximize the cost: benefit ratio. There are certain settings in which intravenous administration may be more appropriate than subcutaneous, for example, in the treatment of patients in septic shock or children with indwelling central lines,

for whom subcutaneous dosing could induce considerable psychological trauma. Current recommendations specify that a course of GCSF should begin at least 24 hours after completion of chemotherapy because of the possibility that earlier initiation of the cytokine might induce rapid progenitor cycling that could heighten chemosensitivity. Lending credence to this concern is preliminary information from a trial in which patients receiving G-CSF therapy concurrently with 5-fluorouracil/leucovorin appear to have had more profound neutropenia than was seen in historical controls or in patients not treated simultaneously with G-CSF and chemotherapy.71 Patients receiving concurrent topotecan and G-CSF for several days also appeared to have more serious neutropenia than those beginning G-CSF after completion of the cytotoxic agent.72 In an effort to reduce the cost of supportive care, several studies in adult cancer patients and one in pediatric cancer patients suggest that waiting to start G-CSF several days after the completion of chemotherapy does not compromise neutropenia recovery.73–76 Whether these findings will alter the current practice of administering G-CSF 24 hours after the end of chemotherapy remains to be determined. The duration of G-CSF therapy has also been a matter of some uncertainty. The package insert advocates continuing the cytokine until the absolute neutrophil count (ANC) has reached at least 10,000/mm3 on one determination. The major concern has been that stopping G-CSF at a

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lower count (e.g. ANC ≥ 1500/mm3 ) might allow the granulocyte count to decline to neutropenic levels, rendering patients susceptible to further infection or delaying the start of a new chemotherapy cycle. However, the basis for this concern is unclear in light of data indicating that tissue neutrophil levels are generally replete prior to the restoration of circulating neutrophil numbers.77 The usual convention is to withhold subsequent chemotherapy for at least 24 to 48 hours after cessation of G-CSF therapy. In the absence of firm guidelines, and in view of the positive results obtained when G-CSF is administered during the intervals between weekly chemotherapy, we think it prudent to allow a wash-out period between administration of G-CSF and chemotherapy. Clinicians should be aware of the fact that immediately after discontinuation of growth factor, there may be a rapid decrease in the ANC, possibly as much as 50%. Therefore, in light of this expected decline, one must allow the ANC to reach a relatively high level before the growth factor is withdrawn. Decisions to use G-CSF in support of cytotoxic therapy cannot be based on conventional risk-benefit assessments. Rather, the prescribing physicians must consider the more ambiguous issue of whether they are obtaining good therapeutic value for individual patients at an acceptable cost. The issue of cost is accentuated when one realizes that GCSF prophylaxis does not materially affect acute mortality. Current antibiotic therapy is generally so effective that the chance of infectious death cannot be discernibly reduced by G-CSF administration. Thus, the immediate benefit of G-CSF must be weighed against the more elusive endpoints of treatment cost and quality of life. Clearly, better guidelines are needed for the use of G-CSF in patients with febrile neutropenia. In the meantime, judicious administration of G-CSF to patients with a critical need for neutrophils (for example, during a serious uncontrolled infection) is difficult to challenge. These and other concerns are addressed by the recommendations of the American Society of Clinical Oncology, which were developed to aid practicing oncologists in the proper use of colony-stimulating factors.78–80 With similar objectives in mind, a European panel of experts published a set of guidelines specifically for the use of colony-stimulting factors in children.81 Recently, the Food and Drug Administration approved a new sustained-duration formulation of recombinant human G-CSF, pegfilgrastim (Neulasta TM, Amgen, Inc., CA), for the management of chemotherapy-induced neutropenia in patients with a body weight of greater than 45 kg. Pegfilgrastim was developed by attaching a polyethylene glycol moiety to the G-CSF protein, resulting in a molecule that is minimally cleared by the kidneys and has a longer half-life than the unmodified protein.82 Consequently, this

formulation can be administered as a single subcutaneous injection per cycle of chemotherapy. Several randomized, active-control adult studies have demonstrated that a single dose of pegfilgrastim per cycle is as safe and effective as daily injections of standard G-CSF.83–85 The safety and efficacy of pegfilgrastim is currently being investigated in children with cancer.

Erythropoietin Erythropoietin is a glycoprotein hormone, produced primarily in the kidney, that regulates red cell production in humans. The two major stimuli for the induction of erythropoietin production are hypoxia and a decrease in hemoglobin. Erythropoietin acts on hematopoietic progenitors in the bone marrow and induces them to mature into red cells. Extensive laboratory work has shown the necessity for erythropoietin in induction of erythroid production and hemoglobin synthesis. Recombinant DNA technology has permitted production of human erythropoietin in large amounts for use in clinical studies, and it has been shown to be effective in amelioration of anemia associated with renal disease and rheumatoid arthritis, induced by zidovudine treatment of HIV patients, or occurring in adult cancer patients. Large randomized, doubleblind, placebo-controlled studies and large communitybased trials in adult cancer patients have shown that erythropoietin increases hemoglobin levels, reduces transfusion requirements, and improves quality of life.86–89 However, in children with cancer, a limited number of studies, most involving small numbers of patients, are available on the efficacy, safety, optimal dose and duration of erythropoietin. These studies are summarized in Table 33.6. Three large multicenter, randomized studies in pediatric cancer patients are under way. Of note, the requirement for exogenous erythropoietin may be greater in adults than in children, based on comparisons of erythropoietin levels and serum soluble transferrin receptors (sTfR) at diagnosis and during chemotherapy. In contrast to adults, pediatric cancer patients have an adequate erythropoietin response to anemia at diagnosis and after exposure to chemotherapy, although their sTfR levels are decreased, suggesting a defect in erythropoiesis is due to a lack of erythropoietic progenitors.90 Most experts agree that the use of erythropoietin in childhood cancer patients requires further investigation. New information from the current multicenter trials should help establish guidelines for erythropoietin administration, especially in patients whose social, cultural, or religious beliefs preclude the use of red cell transfusions to alleviate anemia.

Table 33.6 Published clinical trials testing erythropoietin in children with cancer Reference

Wagner et al.93

Porter et al.94

Bolonaki et al.95

Varan et al.96

Kronberger et al.97

Leon et al.98

Patient population

Neuroblastoma

Sarcoma

Leukemia/ lymphoma/solid

Solid tumor

Solid tumor

Solid tumor

tumor No. of patients

38

24

15

34

37

25

Study design

Randomized, open-label

Randomized, double-blind

Nonrandomized,

Randomized

Nonrandomized,

Nonrandomized,

placebo-controlled Dose and schedule

200 U/kg SC daily if Hb <

Placebo versus 150 U/kg

open-label, intrapatient

open-label, historical

control

controls

150 U/kg three times per

150 U/kg three times

150 U/kg if Hb between

open-label, historical controls 150 U/kg five times per week

10 g/dL, three times a

three times per week IV or

week SC × 8 weeks (if no

per week SC for 8

12–16 g/dL and 300 U/kg

for 12 weeks ± iron

week

SC × 16 weeks (↑50 U/kg

response, ↑ to 250 U/kg

weeks

if Hb < 12 g/dL, three

supplementation

if Hb ≥ 10 g/dL

until no transfusions

then 400 U/kg) + iron

times per week SC or IV

required or max. 300

supplementation

for 28 weeks

U/kg) + iron supplementation Study endpoints ↑ Hb/Hct (Hb in EPO

NS

NA

NS

Yes (10.21 g/dL versus

NS

8.41 g/dL, P = 0.027)

group versus Hb

Yes (12.4 g/dL versus 9.6 g/dL, P < 0.001)

controls, P value) ↓ Transfusion requirements (EPO group versus

No

Yes

Yes

Yes

Yes

Yes

(161 mL/kg versus 106.6

(23 mL/kg versus 80 mL/kg,

(5 units versus 15 units of

(1 pt. versus 8 pts.,

(23 pts. versus 33 pts.,

(4 pts. versus 24 pts.,

mL/kg, P = 0.005)

P = 0.02)

PRBC, P < 0.05)

P = 0.008)

P = 0.007)

controls, P value) Abbreviations: NA, not assessed; EPO, erythropoietin; SC, subcutaneous; IV, intravenous; Hb, hemoglobin; NS, not significant; PRBC, packed red blood cells.

P < 0.001)

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Thrombopoietic growth factors Platelet transfusion therapy is currently the only treatment for severe thrombocytopenia. As discussed earlier, platelet transfusions temporarily control severe thrombocytopenia but are associated with several risks, including resistance and alloimmunization, infection, and transfusion reactions. Unfortunately, the successful clinical use of cytokines to stimulate the production of erythrocytes and neutrophils and other hematopoietic cells has not been equaled with platelets, although several interleukins (IL-1, IL-3, IL-6, IL11) and thrombopoietin (TPO) have been shown to promote thrombopoietic activity. One disadvantage to the use of interleukins is that their pleiotropic effects often results in unwanted side effects. On the other hand, TPO (also known as c-Mpl ligand) is a relatively lineage-specific cytokine that stimulates megakaryocyte growth and maturation in vitro and is a potent thrombopoietic growth factor in vivo. In contrast to the granulocyte colony-stimulating factors, TPO acts on early megakaryocytes. Two recombinant forms of human thrombopoietin, rhTPO and PEGrHuMGDF (pegylated form), have been evaluated in several adult clinical trials. Because of the detection of neutralizing antibodies to endogenous TPO in a few patients who received PEG-rHuMGDF, the development of this formulation was suspended by the pharmaceutical industry. Both rhTPO and PEG-rHuMGDF have been shown to enhance platelet recovery in patients who receive nonmyeloablative chemotherapy, but the effect was not clinically relevant in adult patients who receive myeloablative chemotherapy for acute leukemias and stem cell transplantation.91 At present, the practical value of thrombopoietic cytokines remains to be established. Because chemotherapy-induced thrombocytopenia does not carry the same high risks and costs as neutropenia, the future role of these agents will likely be limited. Preliminary studies indicate that one area of promise for these growth factors may be in enhancing the production of platelets in apheresis donors and increasing the yield of CD34+ cells during stem cell mobilization.

Conclusions and future directions Advances in supportive care measures for children with leukemia have improved treatment- and disease-related morbidity and mortality in this patient population. Evidence-based guidelines have been established for prophylactic platelet transfusions and the use of granulocytestimulating factors. The risk, both infectious and noninfectious, associated with blood transfusions remains a

significant problem.92 Strategies to lessen the exposure to blood products using growth factors, i.e. erythropoietin and thrombopoietin, and blood substitutes, have had limited success and require further investigation. Thus, blood transfusions should continue to be used judiciously. In the light of the improved survival of patients with childhood leukemia, the risk and benefit of current supportive care measures, as well as those developed in the future, should be evaluated in terms of both acute as well as long term effects.

REFERENCES 1 Blundell, J. Successful case of transfusion. Lancet, 1828; 1: 431. 2 Skillings, J. R., Sridhar, F. G., Wong, C., & Paddock, L. The frequency of red cell transfusion for anemia in patients receiving chemotherapy. A retrospective cohort study. Am J Clin Oncol, 1993; 16: 22–5. 3 Simone, J. V. Use of fresh blood components during intensive combination therapy of childhood leukemia. Cancer, 1971; 28: 562–5. 4 Warkentin, P. Transfusion therapy for the pediatric oncology patient. In D. Kasprisin & N. Luban, eds., Pediatric Transfusion Medicine, vol. 2. (Boca Raton, FL: CRC Press, 1987). 5 Gaydas, L., Freireich, E., & Mantel, N. The quantitative relation between platelet count and hemorrhage in patients with acute leukemia. N Engl J Med, 1963; 266: 905–9. 6 Schiffer, C. A., Anderson, K. C., Bennett, C. L., et al. Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol, 2001; 19: 1519–38. 7 Gmur, J., Burger, J., Schanz, U., Fehr, J., & Schaffner, A. Safety of stringent prophylactic platelet transfusion policy for patients with acute leukaemia. Lancet, 1991; 338: 1223–6. 8 Heckman, K. D., Weiner, G. J., Davis, C. S., et al. Randomized study of prophylactic platelet transfusion threshold during induction therapy for adult acute leukemia: 10,000/microL versus 20,000/microL. J Clin Oncol, 1997; 15: 1143–9. 9 Wandt, H., Frank, M., Ehninger, G., et al. Safety and cost effectiveness of a 10 × 10(9)/L trigger for prophylactic platelet transfusions compared with the traditional 20 × 10(9)/L trigger: a prospective comparative trial in 105 patients with acute myeloid leukemia. Blood, 1998; 91: 3601–6. 10 Rebulla, P., Finazzi, G., Marangoni, F., et al. The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. N Engl J Med, 1997; 337: 1870–5. 11 Friedmann, A. M., Sengul, H., Lehmann, H., Schwartz, C., & Goodman, S. Do basic laboratory tests or clinical observations predict bleeding in thrombocytopenic oncology patients? A reevaluation of prophylactic platelet transfusions. Transfus Med Rev, 2002; 16: 34–45.

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12 Howard, S. C., Gajjar, A., Ribeiro, R. C., et al. Safety of lumbar puncture for children with acute lymphoblastic leukemia and thrombocytopenia. JAMA, 2000; 284: 2222–4. 13 Howard, S. C., Gajjar, A. J., Cheng, C., et al. Risk factors for traumatic and bloody lumbar puncture in children with acute lymphoblastic leukemia. JAMA, 2002; 288: 2001–7. 14 Gajjar, A., Harrison, P. L., Sandlund, J. T., et al. Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukemia. Blood, 2000; 96: 3381–4. 15 Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med, 1997; 337: 1861–9. 16 O’Connell, B., Lee, E. J., & Schiffer, C. A. The value of 10-minute posttransfusion platelet counts. Transfusion, 1988; 28: 66–7. 17 Bishop, J. F., McGrath, K., Wolf, M. M., et al. Clinical factors influencing the efficacy of pooled platelet transfusions. Blood, 1988; 71: 383–7. 18 Engelfriet, C. P., Reesink, H. W., Aster, R. H., et al. Management of alloimmunized, refractory patients in need of platelet transfusions. Vox Sang, 1997; 73: 191–8. 19 Rebulla, P. Refractoriness to platelet transfusion. Curr Opin Hematol, 2002; 9: 516–20. 20 Nagasawa, T., Kim, B. K., & Baldini, M. G. Temporary suppression of circulating antiplatelet alloantibodies by the massive infusion of fresh, stored, or lyophilized platelets. Transfusion, 1978; 18: 429–35. 21 Kobrinsky, N. L. & Tulloch, H. Treatment of refractory thrombocytopenic bleeding with 1-desamino-8-D-arginine vasopressin (desmopressin). J Pediatr, 1988; 112: 993–6. 22 Kickler, T., Braine, H. G., Piantadosi, S., et al. A randomized, placebo-controlled trial of intravenous gammaglobulin in alloimmunized thrombocytopenic patients. Blood, 1990; 75: 313–16. 23 Zeigler, Z. R., Shadduck, R. K., Rosenfeld, C. S., et al. High-dose intravenous gamma globulin improves responses to singledonor platelets in patients refractory to platelet transfusion. Blood, 1987; 70: 1433–6. 24 Consensus conference. Fresh-frozen plasma. Indications and risks. JAMA, 1985; 253: 551–3. 25 National Blood Resource Education Program. Indications for the Use of Red Blood Cells, Platelets, and Fresh Frozen Plasma. NIH Publication 89–2974. (Rockville, MD: National Institutes of Health, 1989). 26 Dale, D. C. The discovery, development and clinical applications of granulocyte colony-stimulating factor. Trans Am Clin Climatol Assoc, 1998; 109: 27–36, 36–8. 27 Cavallaro, A. M., Lilleby, K., Majolino, I., et al. Three to six year follow-up of normal donors who received recombinant human granulocyte colony-stimulating factor. Bone Marrow Transplant, 2000; 25: 85–9. 28 Volk, E. E., Domen, R. E., & Smith, M. L. An examination of ethical issues raised in the pretreatment of normal volunteer granulocyte donors with granulocyte colony-stimulating factor. Arch Pathol Lab Med, 1999; 123: 508–13.

29 Anderlini, P., Korbling, M., Dale, D., et al. Allogeneic blood stem cell transplantation: considerations for donors. Blood, 1997; 90: 903–8. 30 Dodd, R. Infectious complications of blood transfusion. Hematol Oncol Ann, 1994; 2: 280–7. 31 Bowden, R. A., Slichter, S. J., Sayers, M., et al. A comparison of filtered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion-associated CMV infection after marrow transplant. Blood, 1995; 86: 3598– 603. 32 American Association of Blood Banks. Transfusion transmitted diseases. In M. Brecher, ed., Technical Manual, 50th, Anniversary AABB Edition, 1953–2003, 14th edn. (Bethesda, MD: American Association of Blood Banks, 2002). 33 Grant, P. R. & Busch, M. P. Nucleic acid amplification technology methods used in blood donor screening. Transfus Med, 2002; 12: 229–42. 34 Williams, A. E. & Sullivan, M. T. Transfusion-transmitted retrovirus infection. Hematol Oncol Clin North Am, 1995; 9: 115–36. 35 Reading, F. C. & Brecher, M. E. Transfusion-related bacterial sepsis. Curr Opin Hematol, 2001; 8: 380–6. 36 Goodnough, L. T., Brecher, M. E., Kanter, M. H., & AuBuchon, J. P. Transfusion medicine. First of two parts – blood transfusion. N Engl J Med, 1999; 340: 438–47. 37 Kuehnert, M. J., Roth, V. R., Haley, N. R., et al. Transfusiontransmitted bacterial infection in the United States, 1998 through 2000. Transfusion, 2001; 41: 1493–9. 38 Workshop on Safety and Efficacy of Methods in Reducing Pathogens in Cellular Products Used in Transfusion, Bethesda, Maryland, August 7, 2002. (Rockville, MD: FDA, Center for Biologics Evaluation and Research, 2002). 39 Petersen, L. R., Roehrig, J. T., & Hughes, J. M. West Nile virus encephalitis. N Engl J Med, 2002; 347: 1225–6. 40 Pealer, L. N., Marfin, A. A., Petersen, L. R., et al. Transmission of West Nile virus through blood transfusion in the United States in 2002. N Engl J Med, 2003; 349: 1236–45. 41 Centers for Disease Control and Prevention. Update: West Nile virus screening of blood donations and transfusion-associated transmission–United States, 2003. MMWR, 2004; 53: 281–4. 42 Brown, P., Will, R. G., Bradley, R., Asher, D. M., & Detwiler, L. Bovine spongiform encephalopathy and variant CreutzfeldtJakob disease: background, evolution, and current concerns. Emerg Infect Dis, 2001; 7: 6–16. 43 Hunter, N., Foster, J., Chong, A., et al. Transmission of prion diseases by blood transfusion. J Gen Virol, 2002; 83: 2897–905. 44 Chamberland, M. E., Alter, H. J., Busch, M. P., Nemo, G.,& Ricketts, M. Emerging infectious disease issues in blood safety. Emerg Infect Dis, 2001; 7(Suppl.): 552–3. 45 Stowell, C. P. Hemoglobin-based oxygen carriers. Curr Opin Hematol, 2002; 9: 537–43. 46 Broviac, J. W., Cole, J. J., & Scribner, B. B. A silicone rubber atrial catheter for prolonged parenteral alimentation. Surg Gynecol Obstet, 1973; 136: 602–6. 47 Hickman, R. O., Buckner, C. D., Clift, R. A., et al. A modified right atrial catheter for access to the venous system in

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chemotherapy does not affect stem and progenitor cell apheresis yield: a retrospective study of 65 cases. Transfusion, 1999; 39: 561–4. Wright, D. G., Meierovics, A. I., & Foxley, J. M. Assessing the delivery of neutrophils to tissues in neutropenia. Blood, 1986; 67: 1023–30. American Society of Clinical Oncology. Recommendations for the use of hematopoietic colony-stimulating factors: evidencebased, clinical practice guidelines. J Clin Oncol, 1994; 12: 2471– 508. American Society of Clinical Oncology. Update of recommendations for the use of hematopoietic colony-stimulating factors: evidence-based clinical practice guidelines. J Clin Oncol, 1996; 14: 1957–60. Ozer, H., Armitage, J. O., Bennett, C. L., et al. 2000 update of recommendations for the use of hematopoietic colony-stimulating factors: evidence-based, clinical practice guidelines. American Society of Clinical Oncology Growth Factors Expert Panel. J Clin Oncol, 2000; 18: 3558–85. Schaison, G., Eden, O. B., Henze, G., et al. Recommendations on the use of colony-stimulating factors in children: conclusions of a European panel. Eur J Pediatr, 1998; 157: 955–66. Morstyn, G., Foote, M. A., Walker, T., & Molineux, G. Filgrastim (r-metHuG-CSF) in the 21st century: SD/01. Acta Haematol, 2001; 105: 151–5. Vose, J. M., Crump, M., Lazarus, H., et al. Randomized, multicenter, open-label study of pegfilgrastim compared with daily filgrastim after chemotherapy for lymphoma. J Clin Oncol, 2003; 21: 514–19. Holmes, F. A., O’Shaughnessy, J. A., Vukelja, S., et al. Blinded, randomized, multicenter study to evaluate single administration pegfilgrastim once per cycle versus daily filgrastim as an adjunct to chemotherapy in patients with high-risk stage II or stage III/IV breast cancer. J Clin Oncol, 2002; 20: 727–31. Green, M. D., Koelbl, H., Baselga, J., et al. A randomized double-blind multicenter phase III study of fixed-dose singleadministration pegfilgrastim versus daily filgrastim in patients receiving myelosuppressive chemotherapy. Ann Oncol, 2003; 14: 29–35. Demetri, G. D., Kris, M., Wade, J., Degos, L., & Cella, D. Qualityof-life benefit in chemotherapy patients treated with epoetin alfa is independent of disease response or tumor type: results from a prospective community oncology study. Procrit Study Group. J Clin Oncol, 1998; 16: 3412–25. Abels, R. Erythropoietin for anaemia in cancer patients. Eur J Cancer, 1993; 29A(Suppl. 2):S2–8.

88 Glaspy, J., Bukowski, R., Steinberg, D., et al. Impact of therapy with epoetin alfa on clinical outcomes in patients with nonmyeloid malignancies during cancer chemotherapy in community oncology practice. Procrit Study Group. J Clin Oncol, 1997; 15: 1218–34. 89 Littlewood, T. J., Bajetta, E., Nortier, J. W., Vercammen, E., & Rapoport, B. Effects of epoetin alfa on hematologic parameters and quality of life in cancer patients receiving nonplatinum chemotherapy: results of a randomized, double-blind, placebocontrolled trial. J Clin Oncol, 2001; 19: 2865–74. 90 Corazza, F., Beguin, Y., Bergmann, P., et al. Anemia in children with cancer is associated with decreased erythropoietic activity and not with inadequate erythropoietin production. Blood, 1998; 92: 1793–8. 91 Kuter, D. J. & Begley, C. G. Recombinant human thrombopoietin: basic biology and evaluation of clinical studies. Blood, 2002; 100: 3457–69. 92 Glynn, S. A., Kleinman, S. H., Schreiber, G. B., et al. Trends in incidence and prevalence of major transfusion-transmissible viral infections in US blood donors, 1991 to 1996. Retrovirus Epidemiology Donor Study (REDS). JAMA, 2000; 284: 229–35. 93 Wagner, L. M., Billups, C. A., Furman, W. L., Rao, B. N., & Santana, V. M. Combined use of erythropoietin and granulocyte colony-stimulating factor does not decrease blood transfusion requirements during induction therapy for high-risk neuroblastoma: a randomized controlled trial. J Clin Oncol, 2004; 22: 1886–93. 94 Porter, J. C., Leahey, A., Polise, K., Bunin, G., & Manno, C. S. Recombinant human erythropoietin reduces the need for erythrocyte and platelet transfusions in pediatric patients with sarcoma: a randomized, double-blind, placebo-controlled trial. J Pediatr, 1996; 129: 656–60. 95 Bolonaki, I., Stiakaki, E., Lydaki, E., et al. Treatment with recombinant human erythropoietin in children with malignancies. Pediatr Hematol Oncol, 1996; 13: 111–21. 96 Varan, A., Buyukpamukcu, M., Kutluk, T., & Akyuz, C. Recombinant human erythropoietin treatment for chemotherapyrelated anemia in children. Pediatrics, 1999; 103: E16. 97 Kronberger, M., Fischmeister, G., Poetschger, U., Gadner, H., & Zoubek, A. Reduction in transfusion requirements with early epoetin alfa treatment in pediatric patients with solid tumors: a case-control study. Pediatr Hematol Oncol, 2002; 19: 95–105. 98 Leon, P., Jimenez, M., Barona, P., & Sierrasesumaga, L. Recombinant human erythropoietin for the treatment of anemia in children with solid malignant tumors. Med Pediatr Oncol, 1998; 30: 110–6.

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34 Pain management Alberto J. de Armendi and Doralina L. Anghelescu

Principles of pain management for children with cancer In identifying and treating pain in children with cancer, one must keep in mind the complexity of pain as a physiological, psychological, and social phenomenon. The etiology of pain in the cancer patient is diverse. Children with cancer experience pain associated with the disease, pain caused by procedures to establish a diagnosis and monitor the disease, and pain related to therapeutic interventions (postoperative pain or pain related to chemotherapy or radiotherapy). Because of the multitude and complexity of factors involved in the pathogenesis of pain in children with cancer, the diagnostic and therapeutic strategies for pain management need to be individualized to the particular needs of each patient. The treatment plan most often includes multiple pharmacologic and nonpharmacologic interventions delivered by a multidisciplinary team that includes pediatricians, oncologists, anesthesiologists, nurses, psychologists, child life specialists, and physical therapists. The principles of pain management for children with cancer are presented in Table 34.1.

Assessment of pain Before pain can be successfully managed or controlled, it must first be assessed using tools that are age appropriate and well suited to the clinical situation. In assessing pain, the health care provider should always assume that the child’s report of pain is valid. Self-report is the gold standard for assessment of pain. For infants, toddlers, or children

who cannot self-report, the FLACC (face, legs, activity, cry, consolability) Scale is recommended (Table 34.2) and has received clinical validation.1,2 Each component is scored separately, and these scores are summed to determine the FLACC score. The Faces Pain Scale (Fig. 34.1) is used for older children who can self-report.3 The Faces Pain Scale has been validated in children as young as 5 years.4 To assess the level of pain in patients older than 13 years, a numeric scale (0 to 10) is used. The point at which the patient would choose treatment to control his or her pain must be identified. Frequent reassessments are imperative; specifically, hourly assessments should be conducted after the analgesic has been given. If the pain is new or if it changes in severity or character, a full diagnostic work-up is warranted. This work-up is especially needed if the new or different pain is headache, backache, or neuropathic pain.

Classification of the type of pain Usually described as aching or throbbing, nociceptive pain is related to disease, surgery, or side effects of treatment (e.g. mucositis), and arises from bone, joint, muscle, skin or connective tissue. Neuropathic pain results from damage or inflammation of the nerve. Neuropathic pain is often described as burning, tingling, “pins-and-needles,” or a piercing sensation, and it may follow the anatomical distribution of the injured nerve. The cause of the nerve injury can be surgical interruption of nerves (e.g. phantom limb pain), chemotherapy-induced (e.g. vincristine), or radiotherapy-induced nerve damage.

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Table 34.1 Principles of pain management for children with cancer 1. 2. 3. 4. 5.

6. 7. 8.

Pain is a complex phenomenon. Multiple etiologies are involved in pain generation. Complex multimodal treatment plans are needed. The dynamic nature of the pain process requires frequent reassessment and flexibility in formulation of the therapeutic plan. Individual components of the “pain complex” should be targeted by specific pain intervention (anti-inflammatory drugs for inflammation-related pain, opioid receptor agonists for severe nociceptive pain, neuropathic pain-directed pharmacologic interventions for neuropathic pain: anticonvulsants, tricyclic antidepressants, and systemically administered local anesthetics). The side effects of analgesics should be aggressively prevented and treated. Escalate the level of invasiveness based on severity of pain and side-effect profile: oral regimen, parenteral regimen, neuraxial delivery of drug (intrathecal, epidural), neuroablation (chemical, surgical, thermal). Nonpharmacological interventions should be considered at each level of therapeutic intervention: physical therapy, psychological intervention.

Table 34.2 FLACC Score Scoring Categories

0

1 Occasional grimace or frown, withdrawn, disinterested Uneasy, restless, tense Squirming, shifting back and forth, tense

Frequent to constant quivering chin, clenched jaw Kicking or legs drawn up Arched, rigid or jerking

Cry (C)

No particular expression or smile Normal position or relaxed Lying quietly, normal position, moves easily No cry (awake or asleep)

Consolability (C)

Content, relaxed

Moans or whimpers; occasional complaint Reassured by occasional touching, hugging or being talked to; distractible

Crying steadily, screams or sobs, frequent complaints Difficult to console or comfort

Face (F) Legs (L) Activity (A)

2

Each of the five categories – Face (F), Legs (L), Activity (A), Cry (C), and Consolability (C) – is scored from 0 to 2, resulting in a total score range of 0 to 10.

Pharmacological management of pain These guidelines provide information about the initial doses of analgesics administered to opioid na¨ıve patients. Doses should be individualized on the basis of age, disease status, and previous or current opioid exposure. Medications to treat mild nociceptive pain are presented in Table 34.3. All of the drugs used to treat mild nociceptive pain are antipyretic; therefore, these drugs should be used with caution in patients which are neutropenic or who have recently undergone transplantation. An alternative for treating mild pain is codeine (0.5 mg/kg orally q 4 h). The analgesic effect of codeine is based on its metabolism into morphine. Ten percent of the Caucasian population cannot metabolize codeine into morphine; therefore, in this group codeine has no analgesic effects.5 Because naproxen (Naprosyn) and ibuprofen (Motrin) inhibit platelet function, these two drugs should be used with extreme caution

in patients with thrombocytopenia. Choline magnesium trisalicylate (Trilisate) has less antiplatelet effect than naproxen or ibuprofen. All nonsteroidal anti-inflammatory drugs (NSAIDs) should be used with extreme caution in patients with renal dysfunction, concomitant diuretic use, or hypovolemia, because these agents can precipitate renal failure. NSAIDs can affect the disposition of methotrexate by reducing its clearance; hence, NSAID use should be avoided by patients who are receiving high-dose methotrexate.6 Choline magnesium trisalicylate should not be used in patients with presumed or confirmed viral infections because of the association between Reye syndrome and salicylate use. Moderate nociceptive pain can be controlled by acetaminophen (Tylenol) or NSAIDs (see Table 34.3), or by opioids such as: codeine 0.5 to 1 mg/kg orally every 4 hours, oxycodone (Roxicodone) 0.1mg/kg orally every 4 hours, or morphine 0.15 mg/kg orally every 4 hours.

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0 No hurt

2 Hurts little bit

4 Hurts little more

6 Hurts even more

8 Hurts whole lot

10 Hurts worst

Fig. 34.1 Faces Pain Scale. Say to the child, “The first face has no pain. The last face has the worst pain. Point to the face that shows how you are feeling.”

Severe nociceptive pain can often be controlled by intermittent administration of morphine (0.3 mg/kg orally every 4 hours, or 0.1 mg/kg IV every 4 hours). When prolonged pain is expected (e.g. early mucositis), it may be more appropriate to start a continuous morphine infusion at 0.01–0.04 mg/kg per hour with an initial bolus of 0.05 mg/kg, and consider the use of patient-controlled analgesia (PCA). If the pain is not relieved by this approach, 50% of the above doses can be given every hour until the pain is controlled, and then full doses can be given every four hours to maintain a constant level of pain control. Alternatively, morphine 0.05 mg/kg IV boluses every 5 to10 minutes can be given with the physician at the bedside until the pain is controlled. Once controlled, a morphine PCA infusion may be started at a rate of 0.02 mg/kg per hour with boluses of 0.02 mg/kg every 15 to 20 minutes. Administration can be titrated to the desired effect. If unacceptable side effects (e.g. sedation, pruritus, myoclonus) occur with the use of morphine, hydromorphone (Dilaudid) or fentanyl (Sublimaze) can be used in equianalgesic doses according to the conversion table (Table 34.4). If tolerance develops to one analgesic, better pain control may be achieved by changing to another opioid; 100% equianalgesic dose is not necessary and may lead to unacceptable side effects or overdose. Due to incomplete tolerance, we start at 60% to 70% of the equianalgesic dose if we change opioid (i.e. morphine or fentanyl) or route of administration (intravenous to oral). Morphine conversion from IV to oral can be done using the following formula: Average hourly IV dose × 4 (hours) × 3 (IV to oral conversion factor) = approximate oral dose of immediate-release morphine to be given every 4 hours The conversion ratio of 1:3 from IV to oral is based on the bioavailability of oral morphine being 30%. When converting from IV to oral morphine, a clinical order should be provided for the as-needed hourly rescue dosing of immediaterelease morphine. The rescue dose should be equal to 50%

Table 34.3 Pharmacological management of mild nociceptive pain Medications

Dose

Acetaminophen (Tylenol) Naproxen (Naprosyn) Ibuprofen (Motrin) Choline magnesium trisalicylate (Trilisate) Ketorolaca

a

15 mg/kg orally q 4 h (maximum 1000 mg/dose or 4000 mg/day) 5 mg/kg orally q 12 h (maximum 200 mg/dose or 1000 mg/day) 5–10 mg/kg orally q 8 h (maximum 400 mg/dose or 1200 mg/day) 10–15 mg/kg orally q 12 h (maximum 1000 mg/dose) <60 kg body wt: 0.5 mg/kg IV q 6 h ≥60 kg body wt: 15–30 mg/kg IV q 8 h

Do not administer for more than 5 days.

Table 34.4 Opioid equianalgesic doses Equianalgesic dose (mg) Drug Morphine Hydromorphone Fentanyl Oxycodone

IM/IV

Orally

10 1.5 0.1–0.2 Not available

30 7.5 Not available 15–30

of the dose that is administered orally every 4 hours. If frequent rescue dosing is required, the scheduled dose should be adjusted accordingly in order to minimize the use of rescue dosing to only 3 to 4 times per day. A combined regimen of long-acting opioid and immediate-release opioid should provide adequate pain control for constant pain as well as intermittent breakthrough pain. Opioid weaning is commenced when the decrease in pain indicates a decrease in the need for opioids. When a patient is receiving opioid via PCA, the infusion should be stopped or its rate should be reduced while the patient is allowed to continue to self-administer boluses of opioid. If a decrease in pain occurs suddenly, the opioid dosage should be reduced by 20% to 30% every 1 to 2 days. Patients

Pain management

who have been treated with low-to-moderate doses of opioids for fewer than 5 days can be weaned from the drug within 3 or 4 days. The time required for weaning must be increased proportionately if opioids have been given for more than 5 days.7 The longer the patient has been receiving opioids, the longer it will take to wean in order to avoid withdrawal symptoms. One approach to weaning is to calculate the total amount of opioid received over a 24-hour period, and then reduce this amount by 20% on the first day of weaning. Subsequent daily dose reductions should be in the range of 10% to 20% per day as tolerated. Signs and symptoms of withdrawal include: sweating, diarrhea, jitteriness, piloerection, agitation, tachycardia, and stuffy nose. Careful monitoring of the signs and symptoms of withdrawal should occur, and the opioid dose should be adjusted if any signs or symptoms are observed.

Ondansetron should be used first, because in combination with opioids, it has no cumulative CNS depressant effects. Treatment of neuropathic pain is initiated with gabapentin (Neurontin) 5 mg/kg or 100 mg orally three times daily. The usual effective dose range is 1800 to 3600 mg per day. The dose can be gradually increased to a maximum daily dose of 3600 mg or 70 mg/kg per day.9 If the pain control is not adequate when the dose of gabapentin is at a maximum level, addition of amitriptyline (Elavil) 0.2 mg/kg PO every night can be beneficial. The dose of amitriptyline can be increased every 3 to 5 days until the maximum dosage (1 mg/kg every night) is reached. In addition to the use of gabapentin and amitriptyline, systemic local anesthetic agents can be considered (IV lidocaine to determine effectiveness, followed by oral regimen of mexiletine).10

Treatment of common side effects

Pain management for diagnostic and therapeutic procedures

Most patients on opioid drugs for more than 1 or 2 days should receive regular laxatives (bulk and stimulant, such as a senna combination or lactulose) to prevent opioidinduced constipation. Simple stool softeners are not sufficient. Patients should have a bowel movement every 2 to 3 days. As a general rule, senna and docusate (Senokot S) 1 tablet twice daily, or casanthranol and docusate (PeriColace) 1 capsule twice daily should be given for every 30 mg of oral morphine administered in a 12-hour period. Another approach to preventing constipation is to administer naloxone (Narcan) orally at a dosage of 3 mg three times daily, titrated up to a maximum of 12 mg three times daily depending on laxation and withdrawal symptoms. To prevent withdrawal, therapy should be started with low doses and patients should be carefully monitored during titration.8 Pruritus may be encountered when an opioid drug is used, particularly morphine. If pruritus should occur with the use of morphine, consider using an alternative opioid. Diphenhydramine (Benadryl) 0.5–1 mg/kg orally or IV may be used to effectively treat pruritus (maximum 150 mg/day). An alternative approach is to administer a low-dose IV infusion of naloxone (Narcan) 0.25 g/kg per minute up to1 g/kg per minute. If a patient who is receiving opioids experiences nausea and vomiting, it is first necessary to exclude a primary condition as the cause. If nausea and vomiting are secondary to opioid use, they may be treated with one of the following: ondansetron (Zofran) 0.15 mg/kg IV with a maximum dosage of 8 mg every 8 hours, promethazine (Phenergan) 0.25–0.5 mg/kg or diphenhydramine (Benadryl) 0.5–1 mg/kg every 6 hours IV as needed.

The value of adequate analgesia for pediatric patients who undergo painful procedures cannot be overemphasized. Inadequate analgesia for initial procedures (e.g. bone marrow aspiration or lumbar puncture) in young children may diminish the effect of adequate analgesia given for subsequent procedures.11 Many survivors of pediatric cancer and their parents experience severe symptoms of posttraumatic stress as late as 12 years after completion of successful treatment. Medical procedures such as bone marrow aspirations and lumbar punctures are frequently recalled as traumatic events.12 The psychological distress that patients and their parents experience in response to invasive procedures persists past the end of treatment for both children and parents.13,14 The components of the anesthetic intervention during painful procedures in pediatric oncology are: hypnosis (unconsciousness), amnesia, and analgesia. Adequate general anesthesia is defined as the prevention of both purposeful movement and autonomic signs of inadequate analgesia, achieved with hemodynamic stability, during noxious stimulation.15 Ensuring analgesia has the merit of suppressing the somatic motor, cardiovascular and hormonal responses to the noxious stimulation. The neuroendocrine stress responses to noxious stimulation during surgery include the release of adrenocorticotropic hormone (ACTH), growth hormone (GH), renin, prolactin, endorphins, and antidiuretic hormone (ADH). Catabolic hormones such as cortisol, catecholamines, glucagon and thyroxine are also secreted in increased amounts in response to noxious stimuli, whereas plasma concentrations of anabolic hormones such as insulin and testosterone are usually decreased.16 Increased levels of stress hormones are

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undesirable because they promote hemodynamic instability and increase metabolic catabolism.17 Most painful procedures in pediatric oncology are brief, intensely painful, and performed in the clinical setting. Therefore, the ideal anesthetic regimen for painful procedures should include the following characteristics: (1) availability in intravenous and/or oral forms; (2) rapid onset of action; (3) short duration; (4) maximization of the patient’s comfort and cooperation; and (5) safety.18 Rapid titration, fast emergence, and avoidance of side effects such as prolonged somnolence and nausea and vomiting are primary considerations for procedures performed in the outpatient setting. The use of general anesthesia during the performance of painful diagnostic procedures is the currently accepted standard of care at St. Jude Children’s Research Hospital. Historically, different methods of sedation and analgesia have been used at pediatric hospitals. A survey of the institutions associated with the Pediatric Oncology Group in 1985 found that 20% of the institutions never used premedications for children undergoing bone marrow aspiration procedures, 12% routinely used it, and 68% occasionally used it. When drugs were used, 40% reported use of a meperidine (Demerol)/promethazine (Phenergan)/chlorpromazine (Thorazine) “pediatric cocktail;” 20% used chloral hydrate; and 18% used diazepam (Valium).19 A more recent review (1995) of 27 institutions associated with the Pediatric Oncology Group found that 67% routinely used only local anesthesia, 22% used systemic premedication, and 11% used relaxation.20 The use of equimolecular oxygen and nitrous oxide mixture for procedure-related pain has been reportedly successful in children.21,22 The anesthetic intervention currently used at St. Jude includes a combination of topical analgesia and total intravenous anesthesia, propofol and fentanyl, offering the essential components of general anesthesia: hypnosis (unconsciousness), amnesia, and analgesia, while providing rapid titration to effect. The goals of this anesthetic intervention are to prevent somatic and hemodynamic responses to noxious stimuli, to permit maintenance of spontaneous ventilation, and to produce residual post-procedure analgesia with minimal side effects. Propofol is a sedative–hypnotic agent used for induction and maintenance of anesthesia. Its rapid onset is due to rapid distribution into vessel-rich organs based on the high lipophilicity. The rapidity of redistribution away from vessel-rich organs accounts for the brief pharmacodynamic action, hence the need for repeated boluses or continuous infusion to maintain a stable level of anesthesia

and sedation.23–25 Termination of propofol’s effect is determined by redistribution and rapid hepatic and extrahepatic (lung, kidney) clearance. Propofol determines a dosedependent depression of central nervous system function. A dose of 2.5–3 mg/kg is the recommended effective induction dose in children 3 to 12 years old. The incidence of transient apnea associated with this dose was found to be 21%.26 The onset of hypnosis is one arm-brain circulation time; the peak effect occurs 90 to 100 seconds after the drug administration. At subhypnotic doses propofol causes sedation and amnesia and has antiemetic activity.27 The ability to rapidly titrate its use and rapid recovery make propofol an ideal agent for anesthesia during painful procedures.28,29 Fentanyl is the opioid that is most commonly administered to supplement general anesthesia to pediatric patients. The advantage of using fentanyl during pediatric oncology procedures is that it provides analgesia with rapid onset and short duration of action. Fentanyl is highly liposoluble, is able to penetrate the blood–brain barrier, and can undergo rapid redistribution and hepatic clearance.30 The eutectic mixture of local anesthetics (EMLA) is a cream that consists of 2.5% lidocaine and 2.5% prilocaine in an oil-in-water emulsion. Lidocaine and prilocaine are amide-type local anesthetic agents. The local anesthetic action is the result of the stabilization of neuronal membranes by the inhibition of ionic fluxes required for initiation and conduction of impulses. The onset, depth and duration of dermal analgesia provided by EMLA depend primarily on the duration of application. To provide sufficient analgesia for clinical procedures, EMLA should be applied under an occlusive dressing for at least 1 hour. Satisfactory dermal analgesia is achieved 1 hour after application, maximal analgesia occurs 2 to 3 hours after application, and persists 1 to 2 hours after removal. When the use of EMLA was evaluated in double-blinded, placebo-controlled studies of pediatric patients with cancer, EMLA substantially reduced pain caused by venous puncture, 31–35 subcutaneous drug reservoir puncture, and lumbar punctures.33,36 An alternative to EMLA, ELA-Max, has been found to be as safe and as effective as EMLA with the advantage of a shorter time to onset (30 minutes versus 60 minutes).37

Neuraxial analgesia and neurolytic blocks Neuraxial-delivered opioids are used for pain control because analgesic efficacy and efficacy-to-side-effect ratio are better than those of opioids delivered by other routes.

Pain management

The equianalgesic ratio for morphine administered intravenously, epidurally, and intrathecally is 100:10:1. Opioids administered intrathecally are effective at doses 100to 600-fold lower than doses of opioids administered systemically; epidural opioids are effective at doses 10-fold lower than systemic doses.38,39 Lower doses delivered by the neuraxial route allow for the optimization of the sideeffect profile, and lower incidence of sedation, respiratory depression, itching, nausea, and myoclonus. The choice between epidural catheters and intrathecal catheters for cancer pain management is based on the patient’s life expectancy. Epidural catheters are more likely to dislodge, can develop catheter-tip fibrosis, and can produce patchy analgesia because of adhesions in the epidural space.38,40 Nevertheless, the use of tunneled epidural catheters for prolonged analgesia in children has provided excellent pain relief.41 Therefore, these catheters are recommended for short-term use (between 3 and 6 months), whereas intrathecal catheters can be used for prolonged periods of time. Similar infection rates associated with the use of long-term epidural and intrathecal catheters have been reported.42,43 Analgesia induced by neuraxial delivery of opioids such as morphine, hydromorphone, fentanyl, and sufentanil can be augmented by the addition of other pharmacologic agents, including local anesthetics, clonidine, and baclofen.38,44 Ketamine has been used as an additive to local anesthetics administered via the epidural route.45,46 Enhanced neuraxial analgesia is mediated by ketamine’s action on the N-methyl-D-aspartate receptor. Neurolytic blocks using phenol or alcohol can be considered for intractable localized pain, if the associated risk of loss of sensory or motor function is considered to be acceptable. Neuroablative techniques are reserved for patients with terminal disease and have to be performed under general anesthesia in children.47 Documentation of successful pain relief with a temporary block produced by a local anesthetic is mandatory before a neuroablative procedure with phenol or alcohol can be performed.

Nonpharmacological interventions for pain management Age-appropriate nonpharmacological interventions can also be used for the reduction of pain. These interventions work best when introduced early in the course of illness as a part of a multi-modal treatment approach. They should not be considered as a substitute for analgesics, but as a means to increase the effectiveness of the pharmacological management. A wide array of interventions is available and

effective in making pain more tolerable for some children, especially during medical procedures.48–50 Unfortunately, there are few studies that have determined the effectiveness of various techniques for children during painful procedures. Current practice continues to rely on clinical judgment to select the most effective strategies to help children cope with painful procedures. Generally, distraction is seen as a pain-reducing strategy, as it diverts attention from a noxious stimulus by passively redirecting the child’s attention or by actively involving the child in the performance of a distracting task.51 Other approaches are primarily sensory, including touch, massage, stroking, and rocking, which are almost automatic means for caregivers to comfort distressed children. Children older than age 5 years can usually participate in guided imagery. They have incredible imaginations and the ability to focus, thus “tuning out” their environment. Children should never feel threatened or made to feel ashamed of being unable to cooperate. Ideally, these strategies should be introduced by a skilled health-care provider who is able to instruct the parents how to be a future coach prior to the pain experience.

Clinical implications The challenge of pain management in children with cancer can be met only if the goal of pain control is associated with an acceptable side-effect profile and an improvement in function and quality of life. Appropriate approaches to achieve this balance include individualized treatment plans to address various pain entities and gradual, stepwise escalation of pain interventions from least invasive to most aggressive modalities.

REFERENCES 1 Merkel, S. I., Voepel-Lewis, T., Shayevitz, J. R., et al. The FLACC: a behavioral scale for scoring postoperative pain in young children. Pediatr Nurs, 1997; 23: 293–7. 2 Manworren, R. C. & Hynan, L. S. Clinical validation of FLACC: preverbal patient pain scale. Pediatr Nurs, 2003; 29: 140–6. 3 Wong, D. L., Hockenberry-Eaton, M., Winkelstein, M. L., et al. Whaley & Wong’s Nursing Care of Infants and Children, 6th edn. (St. Louis, MO: Mosby, 1999), p. 2040. 4 Hicks, C. L., Baeyer, C. L. von, Spafford, P. A., et al. The Faces Pain Scale-Revised: toward a common metric in pediatric pain measurement. Pain, 2001; 93: 173–83. 5 Golianu, B., Krane, E. J., Galloway, K. S., et al. Pediatric acute pain management. Pediatr Clin North Am, 2000; 47: 559–87.

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6 Litalien, C. & Jacqz-Aigrain, E. Risks and benefits of nonsteroidal anti-inflammatory drugs in children: a comparison with paracetamol. Paediatr Drugs, 2001; 3: 817–58. 7 Anand, K. J. & Ingraham, J. Pediatric Tolerance, dependence, and strategies for compassionate withdrawal of analgesics and anxiolytics in the pediatric ICU. Critical Care Nurse, 1996; 16: 87–93. 8 Meissner, W., Schmidt, U., Hartmann, M., et al. Oral naloxone reverses opioid-associated constipation. Pain, 2000; 84: 105–9. 9 Rusy, L. M., Troshynski, T. J., & Weisman, S. J. Gabapentin in phantom limb pain management in children and young adults. Report of seven cases. J Pain Symptom Manage, 2001; 21: 78–82. 10 Massey, G. V., Pedigo, S., Dunn, N. L., et al. Continuous lidocaine infusion for the relief of refractory malignant pain in a terminally ill pediatric cancer patient. J Pediatr Hematol Oncol, 2002; 24: 566–8. 11 Weisman, S. J., Bernstein, B., & Schechter, N. L. Consequences of inadequate analgesia during painful procedures in children. Arch Pediatr Adolesc Med, 1998; 152: 147–9. 12 Stuber, M. L., Christakis, D. A., Houskamp, B., et al. Posttrauma symptoms in childhood leukemia survivors and their parents. Psychosomatics, 1996; 37: 254–61. 13 Jacobsen, P. B., Manne, S. L., Gorfinkle, K., et al. Analysis of child and parent behavior during painful medical procedures. Health Psychol, 1990; 9: 559–76. 14 LeBaron, S. & Zeltzer, L. Assessment of acute pain and anxiety in children and adolescents by self-reports, observer reports, and a behavior checklist. J Consult Clin Psychol, 1984; 52: 729–38. 15 Stanski, D. R. & Shafer, S. L. Quantifying anesthetic drug interaction. Implications for drug dosing. Anesthesiology, 1995; 83: 1–5. 16 Oyama, T. & Wakayama, S. The endocrine responses to general anesthesia. Int Anesthesiol Clin, 1998; 26: 176–81. 17 Bailey, P. L., Egan, T. D., & Stanley, T. H. Intravenous opioid anesthetics. In R. D. Miller, ed., Anesthesia, 5th edn. (Philadelphia, PA: Churchill Livingstone, 2000), pp. 302–3. 18 Krane, E. Oncology procedures. In M. Yaster, E. J. Krane, R. F. Kaplan, C. J. Cot´e, & D. G. Lappe, eds., Pediatric Pain Management and Sedation Handbook (St Louis, MO: Mosby, 1997), pp. 481–8. 19 Hockenberry, M. J. & Bologna-Vaughan, S. B. Preparation for intrusive procedures using noninvasive techniques in children with cancer: state of the art versus new trends. Cancer Nurs, 1985; 8: 97–102. 20 Broome, M. E., Richtsmeier, A., Maikler, V., et al. Pediatric pain practices: a national survey of health professionals. J Pain Symptom Manage, 1996; 11: 312–20. 21 Pietrement, C., Salomon, R., Monceaux, F., et al. Analgesia with oxygen-nitrous oxide mixture during percutaneous renal biopsy in children. Arch Pediatr, 2001; 8: 145–9. 22 Annequin, D., Carbajal, R., Chauvin, P., et al. Fixed 50% nitrous oxide oxygen mixture for painful procedures: a French survey. Pediatrics, 2000; 105: E47.

23 Fulton, B. & Sorkin, E. M. Propofol. An overview of its pharmacology and a review of its clinical efficacy in intensive care sedation. Drugs, 1995; 50: 636–57. 24 Bryson, H. M., Fulton, B. R., & Faulds, D. Propofol. An update on its use in anaesthesia and conscious sedation. Drugs, 1995; 50: 513–59. 25 Smith, I., White, P. F., Nathanson, M., et al. Propofol: an update on its clinical use. Anesthesiology, 1994; 81: 1005– 43. 26 Hannallah, R. S., Baker, S. B., Casey, W., et al. Propofol: effective dose and induction characteristics in unpremedicated children. Anesthesiology, 1991; 74: 217–19. 27 Reves, J. G., Glass, P. S. A., & Lubarsky, D. A. Nonbarbiturate intravenous anesthetics. In R. D. Miller, ed. Anesthesia, 5th edn. (Philadelphia, PA: Churchill Livingstone, 2000), pp. 251–4. 28 Jayabose, S., Levendoglu-Tugal, O., Giamelli, J., et al. Intravenous anesthesia with propofol for painful procedures in children with cancer. J Pediatr Hematol Oncol, 2001; 23: 290–3. 29 Hertzog, J. H., Dalton, H. J., Anderson, B. D., et al. Prospective evaluation of propofol anesthesia in the pediatric intensive care unit for elective oncology procedures in ambulatory and hospitalized children. Pediatrics, 2000; 106: 742–7. 30 Cot´e, C. J., Lugo, R. A., & Ward, R. M. Pharmacokinetics and pharmacology of drugs in children. In C. J. Cote, I. D. Todres, N. G. Goudsouzian, & J. F. Ryan, eds., A Practice of Anesthesia for Infants and Children, 3rd edn. (Philadelphia, PA: W. B. Saunders, 2001), pp. 148–9. 31 Maunuksela, E. L. & Korpela, R. Double-blind evaluation of a lignocaine-prilocaine cream (EMLA) in children. Effect on the pain associated with venous cannulation. Br J Anaesth, 1986; 58: 1242–5. 32 Soliman, I. E., Broadman, L. M., Hannallah, R. S., et al. Comparison of the analgesic effects of EMLA (eutectic mixture of local anesthetics) to intradermal lidocaine infiltration prior to venous cannulation in unpremedicated children. Anesthesiology, 1988; 68: 804–6. 33 Halperin, D. L., Koren, G., Attias, D., et al. Topical skin anesthesia for venous, subcutaneous drug reservoir and lumbar punctures in children. Pediatrics, 1989; 84: 281–4. 34 Paut, O., Calmejane, C., Delorme, J., et al. EMLA versus nitrous oxide for venous cannulation in children. Anesth Analg, 2001; 93: 1590–3. 35 Cordoni, A. & Cordoni, L. E. Eutectic mixture of local anesthetics reduces pain during intravenous catheter insertion in the pediatric patient. Clin J Pain, 2001; 17: 115–18. 36 Juarez Gimenez, J. C., Oliveras, M., Hidalgo, E., et al. Anesthetic efficacy of eutectic prilocaine-lidocaine cream in pediatric oncology patients undergoing lumbar puncture. Ann Pharmacother, 1996; 30: 1235–7. 37 Eichenfield, L. F., Funk, A., Fallon-Friedlander, S., et al. A clinical study to evaluate the efficacy of ELA-Max (4% liposomal lidocaine) as compared with eutectic mixture of local anesthetics cream for pain reduction of venipuncture in children. Pediatrics, 2002; 109: 1093–6.

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38 Mercadante, S. Neuraxial techniques for cancer pain: an opinion about unresolved therapeutic dilemmas. Reg Anesth Pain Med, 1999; 24: 74–83. 39 Nichols, D. G., Yaster, M., Lynn, A. M., et al. Disposition and respiratory effects of intrathecal morphine in children. Anesthesiology, 1993; 79: 733–8. 40 Collins, J. J., Grier, H. E., Kinney, H. C., et al. Control of severe pain in children with terminal malignancy. J Pediatr, 1995; 126: 653–7. 41 Aram, L., Krane, E. J., Kozloski, L. J., et al. Tunneled epidural catheters for prolonged analgesia in pediatric patients. Anesth Analg, 2001; 92: 1432–8. 42 Byers, K., Axelrod, P., Michael, S., et al. Infections complicating tunneled intraspinal catheter systems used to treat chronic pain. Clin Infect Dis, 1995; 21: 403–8. 43 Du Pen, S. L., Peterson, D. G., Williams, A., et al. Infection during chronic epidural catheterization: diagnosis and treatment. Anesthesiology, 1990; 73: 905–9. 44 Galloway, K., Staats, P. S., & Bowers, D. C. Intrathecal analgesia for children with cancer via implanted infusion pumps. Med Pediatr Oncol, 2000; 34: 265–7.

45 Naguib, M., Sharif, A. M., Seraj, M., et al. Ketamine for caudal analgesia in children: comparison with caudal bupivacaine. Br J Anaesth, 1991; 67: 559–64. 46 Semple, D., Findlow, D., Aldridge, L. M., et al. The optimal dose of ketamine for caudal epidural blockade in children. Anaesthesia, 1996; 51: 1170–2. 47 Staats, P. S. & Kost-Byerly, S. Celiac plexus blockade in a 7-yearold child with neuroblastoma. J Pain Symptom Manage, 1995; 10: 321–4. 48 Chen, E., Joseph, M. H., & Zeltzer, L. K. Behavioral and cognitive interventions in the treatment of pain in children. Pediatr Clin North Am, 2000; 47: 513–25. 49 Kuppenheimer, W. G. & Brown, R. T. Painful procedures in pediatric cancer. A comparison of interventions. Clin Psychol Rev, 2002; 22: 753–86. 50 Christensen, J. & Fatchett, D. Promoting parental use of distraction and relaxation in pediatric oncology patients during invasive procedures. J Pediatr Oncol Nurs, 2002; 19: 127–32. 51 Kleiber, C. & Harper, D. C. Effects of distraction on children’s pain and distress during medical procedures: a meta-analysis. Nurs Res, 1999; 48: 44–9.

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35 Psychosocial issues Raymond K. Mulhern, Sean Phipps, and Vida L. Tyc

Introduction Serious challenges to the psychosocial adjustment of the patient, parents, and patient’s siblings are presented by the diagnosis of leukemia, its treatment and its clinical outcome. Many of these challenges, such as temporary shock and denial in the acute phase of diagnosis, are not specific to cancer and may be associated with other life-threatening conditions of childhood. Further challenges to adjustment, such as chronic learning problems associated with central nervous system (CNS) therapy, are more unique to childhood cancer and to leukemia in particular. The purpose of this chapter is to highlight psychosocial adjustment challenges to patients and their families that are associated with acute and subacute reactions following diagnosis and with late adverse events following the completion of therapy. We focus special attention on issues that tend to be most prevalent in, if not unique to, childhood leukemia. Overviews of psychosocial research issues relevant to childhood cancer in general are available elsewhere, a recent review deals specifically with concerns in leukemia.1–3 For issues not addressed in this chapter, the reader is referred to several excellent recent publications, including a review of terminal care.4

including painful procedures and chemotherapy-induced side effects. Standard methods, including patient support, patient education, and use of pharmacologic agents, have been employed with variable success in an attempt to decrease the distress associated with invasive procedures and treatment-induced symptoms. Several nonpharmacologic psychologic interventions are also available for managing the pediatric cancer patient’s treatment-related anxiety and distress.

Acute procedural pain and distress Pediatric cancer patients undergo frequent invasive procedures, including venipunctures, bone marrow aspirations (BMAs), and lumbar punctures (LPs), the latter two interventions representing the most distressing and feared aspect of the pediatric cancer patient’s treatments.5 Many patients describe these procedures as more dreaded than the disease itself.6 There is a large literature on nonpharmacologic methods to reduce children’s distress during painful medical procedures. The controlled research on psychological interventions conducted since 1990 is summarized elsewhere.7

Hypnosis and cognitive-behavioral interventions

Issues during treatment Nonpharmacologic approaches to pain and symptom management Children with cancer are repeatedly subjected to a number of medical stressors in the course of their treatment,

Earlier studies of the use of hypnotic techniques in pediatric patients demonstrated significant improvements over baseline levels in procedure-related distress in children as young as 6 years old.6,8,9 Procedures labeled as “hypnotic” involve intensely engaging the child in a fantasy experience with the assistance of suggestion, then reframing and

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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altering the sensory experiences.10 Although early research on hypnosis was methodologically limited,7 more recent studies have compared hypnotic interventions with cognitive strategies to determine the most effective method for reducing procedural distress. For example, Kuttner and colleagues11 compared the efficacy of a hypnotic “imaginative involvement” with the efficacy of behavioral distraction and attention-placebo control condition in reducing pain and distress during BMAs in children stratified by age. Two intervention sessions were given, each prior to a BMA procedure. For the first BMA, younger children showed significant reductions in distress with the hypnotic treatment; older children showed less anxiety and pain during distraction. By the second BMA, the younger children in the three treatment conditions showed equivalent reductions in distress scores. Although distraction required both intervention sessions for optimal effect, hypnosis had an all-ornone effect. These results, combined with earlier findings,9 indicate that hypnotic procedures, although effective, provide no substantially greater benefits than those achievable with cognitive strategies such as distraction and imagery. A series of studies have examined the efficacy of multicomponent cognitive-behavioral treatment (CBT) programs for reducing procedural distress and providing pain relief in children undergoing BMAs and LPs.12–15 These packaged interventions typically include filmed modeling, relaxation training/breathing exercises, emotive imagery, distraction, behavioral rehearsal, self-regulation, and tangible reinforcers to increase motivation and compliance. A more recent study demonstrated the utility of a novel distraction technique (wearing virtual reality glasses) as an adjunct to conventional care in managing pain associated with LPs in adolescents with cancer.16 Collectively, these studies indicate that CBT is a useful approach to the management of self-reported procedural pain and behavioral distress.12–15,17

A comparison of pharmacologic and psychologic interventions Jay et al.13 evaluated three separate interventions using a randomized crossover design: a CBT package, oral diazepam (Valium), and an attention control condition. Children receiving diazepam showed reductions in anticipatory distress, although distress during procedures was not affected. The CBT group had significantly lower behavioral distress ratings, self-reported pain ratings, and pulse rates in comparison to the diazepam and attention-control groups. The benefits of CBT, however, did not generalize to subsequent procedures. Furthermore, children did not appear to benefit from cognitive-behavioral strategies

unless there was a coach present during the painful procedure. Thus, the most effective component of behavioral approaches may be distraction.18 When CBT plus diazepam was compared with CBT alone, both groups had reduced observed behavioral distress and self-reported pain scores after the intervention. However, diazepam did not potentiate the efficacy of CBT in alleviating children’s distress during BMAs or LPs.14 In fact, the CBT plus diazepam group had only one-third the reduction in observed behavioral distress and self-reported pain compared with children in the CBT-alone group, suggesting that the use of some sedatives may hinder a child’s ability to learn and implement CBT. The efficacy of a CBT package has also been compared with that of general anesthesia (nitrous oxide and halothane) in alleviating distress in 18 pediatric cancer patients undergoing BMAs.19 The CBT intervention and short-acting mask anesthesia were delivered according to a repeated-measures counterbalanced design. Results of the study were surprisingly equivocal. No significant differences were found between the two groups of children in self-reported fear, pain, pulse, or anticipatory BMA anxiety. Children who received CBT exhibited more distress for the first minute after lying down on the treatment table. However, parents reported significantly more behavioral adjustment problems 24 hours after the procedure when their children had received anesthesia. More recent reports support the utility of combining CBT with pharmacologic approaches in reducing children’s distress during invasive procedures. In a controlled prospective study, Kazac and colleagues20,21 evaluated a combined pharmacologic and parent-centered psychologic intervention, compared with pharmacologic intervention only, in 92 children under 18 years of age with leukemia undergoing LPs and BMAs.20,21 For older children, the parent-based psychologic intervention consisted of guided imagery, relaxation, and hypnosis, while distraction and play-related techniques were employed with younger children. Medications administered in the pharmacologic intervention included EMLA cream (lidocaine 2.5% plus prilocaine 2.5%), ELA-Max cream (lidocaine 4%), lidocaine, midazolam and morphine sulfate according to the American Academy of Pediatrics Guidelines.22 A third arm of the study incorporated a cross-sectional control group of patients with leukemia in first remission prior to the prospective study. Results from this randomized trial showed that the combined psychologic and pharmacologic treatment was superior to the pharmacologic treatment alone on ratings of child distress by mothers and nurses at 6 and more than 12 months following diagnosis. Both the combined and pharmacologic-only groups demonstrated

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lower child distress than the cross-sectional control group at more than 12 months. These findings are important and suggest that psychologic intervention can further enhance the success of pharmacologic protocols, based on recommended standards of care, in reducing procedural distress. Increased attention has also been given to parentmediated interventions for patients undergoing aversive procedures. In one study, parents were taught to coach their child in the use of attentional distraction and paced breathing during venipunctures.23 Significant reductions were reported in observed child distress, parent-rated child distress, and parents’ ratings of their own distress. Similar results have been reported in other studies in which parents were used as coaches during painful procedures.24–26 Parents were no more likely to use distress-reducing interventions with their children during venipuncture when they received coaching from a health-care professional.26 The long-term maintenance of parent-mediated treatment effects has not been consistently documented. In summary, CBT strategies have been shown to be effective in reducing children’s acute procedural distress, yet little is known about the variables that predict individual patient responses to these interventions. A recent report claimed that children’s pain sensitivity, as measured by selfreport, moderated their response to a psychologic intervention aimed at reducing LP distress.27 Mean reductions of observed behavioral distress for BMAs range from 18% to 50% after intervention, but the residual distress exhibited by some children has been discouragingly high.12–14 Pharmacologic management, alone or combined with psychologic management, may be required for some patients for whom a single approach does not provide adequate amelioration of distress. Local anesthesia (EMLA cream or ELA-Max cream), frequently used in conjunction with systemic pharmacologic and CBT interventions, appears to be useful in providing some pain relief to the surface area where BMAs and LPs are conducted, but it is not sufficiently effective in reducing overall levels of pain and distress. To date, few well-controlled empirical studies that evaluate the efficacy of conscious sedation or short acting general anesthetic agents (i.e. Propofol) in reducing pain and distress in pediatric cancer patients undergoing painful procedures have been conducted.7 Ideally, children should be individually matched to the most appropriate intervention based on predictive factors such as age, coping style and preference.22 Specific recommendations made by Schecter et al.28 in their report of the Consensus Conference on Management of Childhood Cancer Pain include aggressive pharmacologic management (i.e. conscious sedation or general anesthesia) for the initial BMA and LP. For children under 5 years of age, conscious sedation or general

anesthesia is recommended for all subsequent procedures. For children older than 5 years of age, an individualized approach using behavioral and pharmacologic interventions alone or in combination is suggested.22

Psychologic interventions Klosky et al.29 recently evaluated the efficacy of an interactive educational intervention for pediatric cancer patients, aged 2 to 7 years, undergoing radiation therapy simulation. Children who received a Barney® intervention – involving filmed modeling, exposure to an interactive didactic Barney character, and passive auditory distraction – experienced lower levels of observed behavioral distress and heart rate than did children in a control environment. However, no differences in sedation rates were found. Older patients and those who exhibited behavioral distress were more likely to be sedated. This intervention approach was unique in that it incorporated well-established cognitivebehavioral strategies, delivered via automated, interactive technology, in the pediatric oncology setting. A cognitivebehavioral intervention was also found to produce a measurable, although small, reduction in MRI-related distress in an older group of pediatric patients, aged 6 to 18 years, with CNS cancer. The cognitive-behavioral package was built on strategies used for children undergoing invasive procedures,14 and included filmed modeling, breathing exercises, emotive imagery, behavioral rehearsal, and positive incentive. There were no significant differences between the intervention and control groups on child, parent, or staff ratings of MRI distress or observed behavioral distress. Yet, the cognitive-behavioral intervention was effective in reducing distress during the intravenous line insertion preceding the MRI on the basis of staff but not child or parent ratings of distress.30 Sedation rates were similar between the intervention and control groups, and child ratings of prior MRI distress was the only variable to significantly predict sedation. Intervention effects were small in this study, in part, because of obtained “floor effects” for distress ratings that reflected the sample’s repeated exposure and experience with the MRI procedure. In summary, multicomponent CBT interventions, similar to those that have been effective for reducing children’s acute procedural pain, but with slight modifications that account for the unique components of noninvasive procedures, may be equally effective for children undergoing radiation therapy and MRI.30,31 Of note in these studies is that observed behavioral distress did not consistently predict the need for sedation, despite the fact that medical staff often report reliance on observed behavioral distress as a criterion to identify patients that require sedation. Better

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criteria and guidelines that define subgroups of patients who are candidates for sedation or who may be most responsive to cognitive behavioral approaches are needed.

Chemotherapy-related distress Anticipatory nausea and vomiting Nausea and vomiting are common adverse side effects for pediatric patients receiving cancer chemotherapy. Although postchemotherapy nausea and vomiting (PNV) is more common, some patients develop anticipatory nausea and vomiting (ANV) before chemotherapy is given. ANV aversions are thought to be acquired through a classical conditioning process. According to this hypothesis, cues in the environment that are repeatedly paired with the infusion of emetogenic chemotherapy can elicit nausea and vomiting. The emetogenicity of the chemotherapy, the severity of the postchemotherapy symptoms, and the number of treatment sessions are important predictors of ANV, supporting the concept of ANV as a conditioned response.32,33 Patient variables, including high anxiety level, susceptibility to conditioned taste aversions, and increased autonomic reactivity, have also been associated with ANV.34–36 Tyc et al.37 recently found that pediatric cancer patients with expectations of severe postchemotherapy symptoms and those who were more distressed by nausea and vomiting were more likely to experience ANV symptoms, suggesting that cognitive factors may also influence ANV. Major advances have been made in the pharmacologic management of chemotherapy-induced nausea and vomiting with the introduction of serotonin (5-HT) antagonists (e.g. ondansetron), which have considerable antiemetic potency in children, with fewer side effects. Although these new antiemetic agents are quite effective in preventing acute nausea and vomiting, they are less effective in treating delayed nausea and vomiting.38 More intensive chemotherapy regimens and platinum drugs are also associated with lower rates of response to 5-HT antagonists.39 Thus, successful control of nausea and vomiting has not been achieved in all patients. Prior to the use of serotonin antagonists, ANV occurred in an estimated 20% to 30% of pediatric cancer patients, but the rates may be higher for specific chemotherapeutic regimens.35 A recent study conducted at our institution found that 59% of pediatric cancer patients receiving various chemotherapy regimens reported at least mild ANV symptoms despite the use of ondansetron; roughly 14% reported severe ANV symptoms.37 Those patients whose symptoms remain refractory to antiemetics may benefit from nonpharmacologic approaches.

Hypnosis and cognitive-behavioral interventions A comprehensive review of controlled research on psychological interventions for chemotherapy-associated distress (1990 to present) has recently been published.40 Similar psychologic interventions have been used to treat both postchemotherapy and ANV symptoms; in earlier studies, patients receiving hypnosis reported significant reductions in the severity and intensity of nausea and vomiting.41–43 These studies, however, were limited by small sample size and lack of appropriate control groups. In a more recent study that included a no-treatment control group,44 54 pediatric cancer patients were randomly assigned to receive either hypnosis, nonhypnotic cognitive distraction, or an attention-placebo condition. The greatest reduction in observed and self-reported ANV, PNV, and distress was seen in children treated with hypnosis. The cognitive distraction intervention appeared to have a minimal effect, whereas symptoms of children receiving no intervention progressively worsened. The severity of these side effects was not affected by the intervention. Symptom duration, therefore, may be more responsive to hypnotic intervention than is symptom severity. Jacknow et al.45 examined PNV and ANV symptoms and antiemetic usage in pediatric cancer patients who were randomly assigned to receive hypnosis or standard antiemetics. The hypnosis group was trained during their initial chemotherapy course and used antiemetics on a supplemental basis only, whereas the control group received a standard antiemetic regimen. There were no differences between the groups in reports of nausea and vomiting severity; however, patients undergoing hypnosis relied less on antiemetic treatment than did control subjects during the first two courses of chemotherapy. The hypnosis group also had reduced anticipatory symptoms as compared to the control group at 1 to 2 months postdiagnosis. Behavioral interventions, such as progressive muscle relaxation training, systematic desensitization, biofeedback, and cognitive distraction, have also been effective in reducing chemotherapy distress in children with cancer.33,46 External distractors (e.g. video games) reduced the frequency and severity of anticipatory and postchemotherapy side effects in pediatric oncology patients as long as distracting stimuli were available.47,48 Dahlquist and colleagues49 reported a 46% to 68% reduction in behavioral distress during chemotherapy venipunctures in three adolescent patients who received training in cue-controlled muscle relaxation, controlled breathing, pleasant imagery, and positive self-statements. Recently, Dahlquist et al.50 demonstrated the efficacy of a multisensory distraction intervention involving exposure to an electronic interactive toy with the voice of Mickey Mouse® for

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children aged 2 to 5 years who received repeated injections during chemotherapy. Compared with the wait-list control group, children in the intervention group demonstrated significantly lower levels of observed behavioral distress and were rated by parents and nurses as less anxious. These improvements were maintained over the 8-week period of distraction intervention. In summary, studies that compare behavioral interventions with no treatment/attention control conditions show that behavioral interventions are effective in reducing chemotherapy-related distress in pediatric oncology patients. A collective review of intervention studies to control ANV and PNV among pediatric cancer patients concludes that imagery is a well-established treatment, that active cognitive distraction with relaxation is probably an efficacious treatment, and that access to video games is a promising intervention based on the Chambless/Society for Pediatric Psychology criteria.51,52 Moreover, imagery, cognitive distraction with relaxation, and videogames may be equally effective in reducing nausea, although imagery may be more effective than the other treatment approaches in reducing vomiting, particularly its duration.44 Media and technology-driven interventions that employ developmentally appropriate interactive electronic toys as distractors may offer a potentially cost-effective alternative to traditional delivery of nonpharmacologic treatment packages, particularly in preschool children. The pharmacologic advances in nausea and vomiting control have certainly reduced the need for psychologic intervention for chemotherapy-related symptoms. However, behavioral strategies clearly provide an invaluable approach to managing distress associated with brief and repeated stressors such as finger sticks, venipuncture, and chemotherapy injections, for which pharmacologic management may be less appropriate.

Family adjustment The psychosocial impact of childhood leukemia cannot be captured solely through study of the patient in isolation. Rather, childhood leukemia affects every member of the family, and must be understood within that context. The early studies in this area focused on the direct, or linear impact of childhood cancer on family adjustment, although most researchers now favor a systems perspective which recognizes reciprocal influences within the family, i.e. how the coping, adaptation, and illness-related behavior of one family member can affect those of all other family members.53–57 In this section, we briefly review the literature relating to the linear impact of leukemia on the family, the relationship of global family functioning

to patient adjustment, the reciprocity of child and parent adjustment, the influence of leukemia on parenting behavior, and the consequent influence of parenting behavior on patient treatment-related distress. Studies of the linear impact of childhood cancer on the family have reported a broad range of outcomes, from significant psychopathology to very healthy adaptation. Some disparities can be attributed to the rapid changes in treatment regimens and improved prognoses over the years. For example, some of the first studies of family adaptation were reported at a time when childhood leukemia was almost invariably fatal, and these tended to highlight pathologic responses.58–61 As cure rates began to improve dramatically, some studies emphasized the relatively healthy adaptation of families of children with leukemia in comparison to control families,62–66 and others continued to show higher levels of emotional disturbance.67–70 Studies pertaining specifically to parental functioning show a similar pattern, with some reports of increased emotional distress and psychopathology,71–78 but at least an equal number reporting generally healthy parental adaptation.79–83 More recently there has been a focus on symptoms of post-traumatic stress disorder in families of children with cancer.76–78 Most studies have shown a moderate increase in such symptoms in the parents of children with cancer, but no significant increase in the child-patients themselves.76,77 Regarding the adaptation of siblings, research findings have also been variable, with some suggestion that having a seriously ill sibling may even be beneficial to psychologic development.54,84–87 These diverse and occasionally contradictory findings have contributed to a shift in focus, away from a deficit or psychopathology model in which children with cancer are compared with physically healthy children in search of differences. Rather, research has shifted to processes of coping and adaptation, and identification of factors that predispose to more or less healthy family outcomes. Models of family adaptation to a crisis such as childhood leukemia have been broadly incorporated into a systems perspective.88–93 In the Double ABCX model, a stress event (A) interacts with family resources (B), followed by the family’s appraisal of the event (C), leading to an outcome (X).88,89 The model has been expanded to include the dynamic aspects of adjustment to an ongoing stressor over time. A central concept in this model is that of “pile-up” of stresses and demands that occur as families cope with a continuing stressor. This model is illustrated in the longitudinal study by Kupst and colleagues of families coping with childhood leukemia.54,64,65,94,95 The “circumplex” model of family systems has also been very influential in research on childhood cancer.90 This

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model focuses on two dimensions, family adaptability and cohesion, and posits that moderate levels of cohesion and adaptability are optimal, thus predicting a curvilinear relationship between measures of cohesion or adaptability and family adjustment. This approach is illustrated by the research of Kazak and colleagues.54,55,63,79 Interestingly, most studies of families experiencing pediatric cancer have pointed to a linear rather than a curvilinear relationship, such that greater cohesion and adaptability are associated with better adjustment across all levels.54,55,96 Furthermore, families of children with cancer appear more likely to score at the extremes of cohesion and adaptability.54,97 A third model that has been influential in psychooncology research is the family life cycle model,91–93 which predicts that certain normal developmental challenges (e.g. having a new baby) will act to pull the family together (centripetal orientation), and that other developmental changes (e.g. older children leaving home) will act to pull the family apart (centrifugal forces). Serious illness in a family member, particularly a child, is thought to produce centripetal forces, which may either exacerbate or conflict with the family’s normative developmental orientation.93 This model is illustrated in a study by Rait et al.98 of family predictors of adjustment in adolescent cancer survivors. The family environment may predict individual adjustment in families of children with cancer. Barbarin et al.96 found that higher levels of family cohesion and support were associated with lower levels of parental stress and better marital functioning in parents of children with cancer. This finding has been replicated by Noll et al.80 Similarly, the family environment has also been shown to be a significant determinant of adjustment in the child-patient. For example, Kazak and Meadows63 found that higher levels of family adaptability were related to the patient’s report of self-concept, particularly regarding perceived social acceptance and scholastic competence. Phipps and Mulhern97 documented that family cohesion, expressiveness, and conflict were all significant predictors of adjustment in children undergoing bone marrow transplantation (BMT). Moreover, family cohesion and expressiveness were found to act as protective factors, promoting resilience to BMT-related stressors. Not surprisingly, a similar relationship between family environment factors and adjustment has also been reported for siblings of cancer patients.54,99 The cross-cultural robustness of these findings was also demonstrated in a study by Dolgin et al.100 The reciprocal relationship of parental distress to child adjustment is clearly established: higher levels of parental distress or the presence of parental psychopathology, are associated with greater adjustment difficulty in both childpatients and siblings.71,82,91,92 One of the most consistent

findings from the longitudinal family coping study of Kupst and colleagues64,65,94,95 is the strength of the relationship between parental adjustment and the child-patient’s adjustment to illness. This finding has led to the conclusion that adequacy of coping runs in families; hence, the effectiveness of the parents’ coping is directly related to that of the children’s. Brown et al.71 reported on the relationship of maternal psychopathology and adjustment outcome in a cross-sectional sample of children with leukemia. Despite the relatively good adjustment of the overall sample, the subset of children whose mothers met the DSM-IIIR criteria for psychopathology demonstrated significantly higher levels of emotional distress and behavioral problems than did those whose mothers showed no evidence of psychopathology. Moreover, these differences were seen whether measures were based on maternal report or child self-report, providing stronger evidence that these findings reflect real adjustment difficulties in this subset of children, rather than distortion in parental ratings due to the parent’s emotional state. Blotcky et al.101 explored the influence of family and parental function on the experience of hopelessness in children recently diagnosed with cancer. Parental emotional distress and the coping behaviors of both parents were strongly related to the child’s sense of hopelessness. Interestingly, the mother’s and father’s coping behavior scores were unrelated to each other, and each contributed independently to the child’s sense of optimism and hopefulness. These findings illustrate strong reciprocity occurring on a vertical level (parent–child) but not on a horizontal level (mother–father). Parental adjustment has also been related to emotional and behavioral problems in siblings of children with cancer.85 The presence of childhood leukemia may also lead to changes in parenting behavior, or in the child-rearing practices employed by parents in managing their children’s behavior and meeting their emotional needs. Overindulgence of the child-patient, breakdown of discipline and limits within the family, overprotectiveness, and excessive expectations of well siblings are examples of behavioral tendencies associated with increased parenting stress from the demands of childhood cancer.53,102 However, in one of the few studies to address this problem directly, Davies et al.102 found that parents of children with leukemia did not differ significantly from control parents of healthy children in their reports of child-rearing practices, with the exception of items relating to concern over the child’s health. Parenting behavior may influence the medically related fears of children and their adjustment to illness, treatment, or hospitalization. Regarding child anxiety and medically related fears, most studies have shown that parental disciplinary strategies that focus on the use of force or threat

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of punishment are associated with greater anxiety, in contrast to parental use of positive reinforcement, modeling, or reassurance, which promote lower anxiety.103–105 The child’s medical status is predictive of parental anxiety (i.e. sicker children have more anxious parents, who in turn are more likely to use force and less likely to use modeling or reassurance).104 These findings provide another clear example of reciprocal influences, insofar as characteristics of the child-patient (medical status) predict parental responses (discipline strategies) that are associated with differential outcomes for the child (fear/anxiety). A more recent study examined change in parental distress during the early months following the diagnosis of childhood cancer, and its effect on parenting behaviors.106 Although distress tended to decline over time, the pattern of change in distress was a significant predictor of parental control and responsiveness.106 Parenting practices may play a significant role in determining how children cope with specific aversive medical events. For example, in a study of ANV in pediatric cancer patients receiving chemotherapy, parental disciplinary styles predicted the development of conditioned aversions.35 Specifically, children whose parents relied on threat of punishment were more likely to develop ANV, whereas those whose parents relied on modeling and reassurance were less likely to show symptoms of ANV. Other parental behaviors and patterns of parent–child interactions during medical procedures have also been shown to significantly influence child tolerance and procedural coping.107–114 Given the centrality of family and parental functioning on child adjustment, recent intervention approaches have targeted the family or the parents as a means of improving outcomes for children with cancer.115–117 For example, the surviving cancer competently intervention program (SCCIP) is a family group intervention that combines both family therapy and cognitive-behavioral approaches with the ultimate goal of improving psychological functioning for the child-patient.115 Initial pilot work suggests that such an approach helps to decrease anxiety and symptoms of post-traumatic stress disorder, even in the absence of measurable changes in family function.115 An alternative approach is the targeting of intervention towards mothers of children with cancer. The rationale behind this approach is that maternal functioning is significantly related to the adjustment of both children with cancer and their siblings, so that improvement in maternal mental health would be expected to have multiple positive effects ‘downstream’ within the family.115 In two separate randomized trials, a program of problem-solving training for mothers of children with newly diagnosed cancer has been shown to

increase their problem solving skills and reduce negative affectivity during the crucial period of adjustment following the crisis of cancer diagnosis.115,117 The impact of the intervention on child adjustment has yet to be demonstrated, but represents an important question for future research.

Issues after treatment The study of late effects of cancer among children presupposes that the children are long-term survivors of their disease, if not permanently cured. Late effects are defined as occurring after the successful completion of medical therapy, usually 2 or more years after diagnosis. They are generally assumed to be chronic, if not progressive. This definition serves to separate late effects from the acute and time-limited effects of disease and treatment, such as chemotherapy-induced nausea and vomiting.

Neuropsychologic late effects Neuropsychologic late effects, as a subset of psychosocial late effects, are defined as pathologic changes in the child’s CNS secondary to cancer or its treatment that are manifested by stable changes in the child’s behavior. The most often studied behavioral correlates include intellectual, cognitive, and academic performance, beginning with early negative findings published by Soni et al.118 At best, even the more recent research designs are quasi-experimental because of limitations in controlling for essential features of the child’s disease and medical therapy. Pathophysiology Relevant sources of neuropathology for children with ALL include chemotherapy and cranial radiation therapy (CRT). Although acute and subacute radiation side effects such as anorexia, confusion, and somnolence are reversible within weeks of stopping treatment, late CNS effects may be irreversible if not progressive. The severity of somnolence does not necessarily predict later neuropsychologic decline.119 Radiation damage to the normal tissues of the CNS may manifest as cortical atrophy, vascular damage (mineralizing microangiopathy), or white matter destruction (leukoencephalopathy).120 Severity and areas of involvement are associated with the volume of brain irradiated, dose of radiation per fraction, and total dose of radiation received.121 However, these abnormalities, as detected on neuroimaging studies such as computed tomography (CT) or magnetic resonance imaging (MRI), are not highly correlated with neuropsychologic function, except in selected

Psychosocial issues

series of patients.122 At least some recent evidence indicates that calcifications of the basal ganglia and/or grey/white matter junctions predicts poor intellectual and memory functioning.123 Various forms of chemotherapy, administered intravenously or intrathecally, have been used for CNS treatment of leukemia in combination with CRT. Recently, efforts to eliminate the need for CRT, thereby averting the neurophysiologic and neuropsychological seguelae of radiation have prompted the use of more intensive CNS chemotherapy, most often with methotrexate. However, the well-documented potential neurotoxicity of methotrexate is especially problematic following CRT, apparently because of changes in the blood–brain barrier following irradiation.124 Meadows and Evans125 reported that of 13 patients previously treated for ALL, 10 demonstrated neurologic and psychologic deficits. Four of the 10 children with neurotoxicity had received methotrexate but not CRT. Global neuropsychological deficits The most commonly used psychological index of neurotoxicity has been the IQ score or pattern of IQ subtest scores of long-term survivors. A meta-analysis of 30 studies published before 1988 concluded that children receiving CRT, those who are younger at the time of CRT, and those who are farther from completion of their therapy with CRT will show the greatest intellectual deficits, averaging a 10-point decline.126 The results of more recent nonquantitative reviews have generally confirmed this finding.127 Retrospective studies have demonstrated significantly lower IQ in irradiated patients than in nonirradiated patients,123,128–131 although some studies have failed to find an effect.132,133 Moore et al.134 found that children receiving 18-Gy CRT had mean IQ scores approximately 9 points higher than did children receiving 24-Gy (103.5 versus 94.6). Simbert et al.135 found that children given 18- or 24-Gy CRT had lower IQ scores (94.9) than did those treated with chemotherapy alone (102.4) or healthy controls (101.7), and that those treated with 24-Gy scored lower than the other groups on several other measures of cognitive functioning. Similar findings have been independently reported by other investigators.136,137 Longitudinal studies have also documented significant declines in IQ from first to last observations among long-term survivors who had received CRT138–140 ; Ochs et al.140 reported similar declines among children who received chemotherapy alone, although other studies did not report a decline with time.141,142 In most studies, no correction was made for the use of different IQ tests as children became older.138–140 When this correction was made in one study, differences between treatment groups remained insignificant, but 22% to 30% of irradi-

ated and nonirradiated patients had clinically significant declines in IQ.143 The controversy as to whether treatment with low-dose CRT (e.g. <18 Gy) is more harmful than treatment with chemotherapy alone remains active. Raymond-Speden144 compared the post-treatment neuropsychological functioning of 20 children given 18-Gy CRT with that of 21 children treated with chemotherapy alone and two control groups: 21 children with asthma and 21 healthy controls. Full-Scale IQs were not significantly different between the two ALL groups, although both groups had lower IQ values than either of the two comparison groups. Only with regard to arithmetic achievement did the irradiated children perform worse than the non-irradiated children with ALL. Espy et al.145 conducted a prospective study of 30 children treated for ALL and analyzed the data using growth curve analysis. All children had been treated with chemotherapy alone. Overall, at 4 years post-diagnosis, the leukemia patients fell significantly behind their age-matched peers on tests of academic achievement and verbal fluency. No significant changes were noted in IQ values. Patients treated with intrathecal (IT) and intravenous (IV) MTX performed more poorly on tasks of visual-motor integration than those treated with IT therapy alone. Waber et al.146 recently reported on 61 survivors of ALL treated on a single Dana-Farber protocol using 18-Gy CRT. Measures of IQ and verbal and nonverbal memory did not differ from normal expectations using normative test values. However, the absence of control or comparison groups limits generalization of these findings. Kingma et al.147 followed 20 children with ALL, all diagnosed before the age of 7 years, for up to 7 years post-diagnosis. Patients were treated with chemotherapy alone, including IT and IV MTX. A strength of this study is that patients were compared to matched healthy controls from local schools. At the last evaluation, patients scored less well with regard to their Verbal IQ (mean change, −9 points) and a test of mental flexibility. No age or gender effects were noted. In a cross-sectional study of 121 long-term survivors of ALL, Langer et al.148 found that those receiving chemotherapy alone had a FullScale IQ approximately 9 points higher than those treated with CRT (≤18 Gy). Statistical interactions between age and gender were specific to particular outcomes with younger age at CRT associated with greater risk for impairment. Moleski149 has provided an extensive review of the cognitive effects of CNS chemotherapy among children treated for ALL, including methodologic issues. One of the conclusions is the need for matched healthy controls. Nevertheless, the conclusion was that the majority of studies do support the conclusion that CNS chemotherapy has some deleterious effect on intellectual development, attention,

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nonverbal memory, and academic achievement (especially mathematics). The effect of a child’s age at the time of CNS treatment is less ambiguous. Although some studies have found age-related effects128,139,141,150,151 and others have failed to identify such effects,129,130–133,139,142,143 all positive findings indicate that younger children are more vulnerable than older children. Simbert et al.135 found that young age was a risk factor for those receiving 18- or 24-Gy CRT but not chemotherapy alone. Similarly, Jankovic et al.152 found that patients given 18-Gy CRT had significantly lower IQs than those treated with chemotherapy alone; however, most of the difference was attributed to the very poor outcomes among children who received CRT when less than 3 years of age. Younger age at treatment may place females, but not males, at greater risk.137 Even among similarly irradiated children, differences in the amount and type of chemotherapy given may help to explain these discrepancies between studies.153 By using regression analyses on a combined group of children of various ages who had survived ALL or brain tumors, Silber et al.153 have recently developed a mathematical algorithm for predicting IQ loss when the dose of CRT and age at CRT are known. Mulhern et al.123 compared the functional and neuropsychological status of 26 long-term survivors of ALL diagnosed in the first 24 months of life with that of 26 children previously treated for Wilms tumor. Of the 26 children with ALL, CNS prophylaxis included no CRT in 6, 18-Gy CRT in 5, 20-Gy CRT in 7, and 24-Gy CRT in 5. Three additional children experienced CNS relapse and received total CRT doses of 24, 40, and 44-Gy. Children treated for ALL had significantly lower mean IQ scores (87 versus 96), poorer performance on four of six measures of visual and auditory memory, lower achievement in arithmetic skills, and a greater frequency of special educational interventions than those treated for Wilms tumor. IQ and memory performance in the ALL group were inversely correlated with total CRT dose, supporting the contemporary practice of avoiding prophylactic CRT in very young children who are not at risk for CNS relapse. A more recent report on the treatment of children 12 months or younger with chemotherapy alone has failed to find early evidence of developmental problems.154 Retrospective studies of the relationship between IQ changes and time post-treatment have had mixed findings.129,134,151 Schlieper et al.129 and Said et al.151 failed to find a significant correlation between time elapsed after treatment and IQ levels in their retrospective studies of childhood ALL survivors, although Schlieper et al.129 detected effects of CRT relative to controls. Moore et al.134 retrospectively evaluated 35 long-term survivors of ALL, all of whom had received CRT, and reported that Verbal

IQ (but not other IQ measures) was inversely correlated with time elapsed since CRT. Mulhern et al.123 also found a significant inverse correlation between the IQ levels of children treated for ALL in infancy and time elapsed since therapy. This relationship was not noted in a group of children treated for cancer without CNS therapy. At least two prospective studies have reported declines in intellectual function over time. Rubenstein et al.139 evaluated 24 children with ALL prior to CRT and again 1 year later and 4 to 5 years later. Statistically significant declines of approximately 6 to 7 points were observed in Verbal, Performance, and Full-Scale IQ. There has been interest recently in the possibly different vulnerabilities of male and female children to the effects of CNS treatment for ALL. Although the mechanisms are not yet clear, some evidence suggests that girls are more likely than boys to suffer deleterious effects. Robison et al.,150 using multiple regression analyses of 50 long-term survivors, identified female gender as a risk factor accounting for mean Verbal and Full-Scale IQ differences of almost 10 points as compared to males. Schlieper et al.129 compared the intellectual performance of male and female long-term survivors of ALL who had received CNS treatment with CRT or chemotherapy alone. Few differences were seen between the males and females who had received only chemotherapy. Among the children receiving CRT, however, females consistently scored significantly lower than males on Performance and Full-Scale IQ measures. Waber et al.137 compared the neuropsychological performances of 27 female and 24 males long-term survivors of ALL who were treated with chemotherapy and 24-Gy CRT. Males and females were closely matched for age at diagnosis and relevant treatment features. Although females had a mean IQ 8.5 points lower than that of males, this difference was not statistically significant. Interestingly, the proportion of females who were short and overweight for age was also significantly greater than that of males, implying that females are more vulnerable to neuroendocrine as well as cognitive impairment secondary to treatment of the CNS. Increasing concerns have been expressed regarding the deleterious effects of corticosteroids, especially dexamethasone, on neurocognitive development of children treated for ALL. Waber et al.155 compared the cognitive performance of 44 children previously treated with prednisone to 23 children treated with dexamethasone. Those treated with dexamethasone had a mean IQ 11 points lower that those treated with prednisone, although this difference is confounded by changes in IQ test version. Nevertheless, children receiving dexamethasone performed more poorly on several measures of academic achievement and visualspatial abilities.

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In a prospective evaluation of children treated for ALL over a period of almost 10 years, Mulhern et al.143 used regression analysis to identify factors associated with IQ declines as compared to pre-CNS treatment levels. Female gender accounted for a significant amount of the variance in Verbal IQ decline; this effect was most pronounced among children in the higher dose (24-Gy) CRT group. However, other investigators have failed to identify gender differences among survivors treated with or without CRT.134 A Children’s Cancer Group (CCG) study has reported the provocative finding that pre-CRT intrathecal methotrexate may actually help protect the CNS, accounting for a unique 25-point IQ difference among girls under 5 years of age at the time of therapy.156 In contrast, Waber et al.157 reported that girls receiving high-dose intrathecal methotrexate prior to 18-Gy CRT performed more poorly than those treated with only a single modality. Obviously, further research is indicated to clarify risks and underlying biologic vulnerability. Specific neuropsychological impairments Several investigators have moved from characterizing deficits merely in terms of patterns of IQ scores toward evaluation of more specific neuropsychological functions. An emerging general consensus holds that IQ declines are secondary to one or more central processing deficits involving short-term memory, speed of processing, visual-motor coordination, or sequencing abilities.127,158–160 Memory functions have been among the more frequently investigated deficits in this context. Copeland et al.130 compared long-term survivors of ALL who did or did not have CRT with a cancer control group that had no CNS therapy. The Verbal Selective Reminding Test and the Nonverbal Selective Reminding Test were included in the battery to assess memory functioning. Nonirradiated patients with ALL did not differ from controls on these measures, but the irradiated patients with ALL had lower nonverbal memory scores than did the other two groups. Because the irradiated patients also had significantly lower IQs, this difference may have been secondary to a generalized cognitive impairment. Among children treated with 24-Gy CRT, specific deficits in verbal memory and verbal learning have been described in the absence of comparable nonverbal deficits.161 These verbal learning problem were most pronounced among survivors with intracerebral calcifications as seen on CT. When combined with the results of the Copeland series,130 these results suggest that specific memory and attentional difficulties are biologically based. Interestingly, one recent study has documented cognitive processing problems and learning disabilities among ALL survivors treated with chemotherapy alone.162 Mulhern et al.163 observed significant deficits in

long-term recall of visually and orally presented verbal material (Learning Efficiency Test) and in recall and reproduction of geometric patterns (Target Test), as compared to test norms, in children treated for ALL with either high-dose chemotherapy without CRT or low-dose chemotherapy plus CRT. After the scores were adjusted for IQ, these deficits remained significant and there was no difference between treatment groups. More recent research has sought to further define the core symptomatology underlying learning problems among survivors of ALL. Lockwood et al.164 tested 56 longterm survivors of ALL who had been randomized to receive 18-Gy CRT or chemotherapy only for treatment of ALL. The focus of the patient assessment was attention, specifically, sensory selection, response selection, attentional capacity, and sustained attention. Patients receiving CRT performed more poorly on most measures composing the above factors with younger children receiving CRT with young age comprising an additional risk factor. Schatz et al.165 compared 27 long-term survivors of ALL, most of whom had been treated with CRT (15 with 18-Gy; 3 with 24-Gy) to 27 demographically matched controls on tests of IQ, working memory, and processing speed. Surprisingly few differences were found between the performance of the two ALL groups. However, more importantly, the authors presented preliminary validation for a model that explains IQ performance following CRT as a function of changes in processing speed and working memory. Studies such as these are important in that they more clearly direct the development of potential interventions for neurocognitive loss. Impact on educational attainment Most studies showing significant decreases in intellectual development as a result of treatment for ALL have also shown a generally higher risk of school failure, special educational needs and, sometimes, specific learning disabilities (Table 35.1). A study of 593 ALL survivors and 409 sibling controls from CCG protocols revealed a 3.6-fold increase in the risk of learning disability placement in school among former patients.166 The need to formally assess at-risk children and to inform parents and teachers of potential signs and symptoms of these learning problems cannot be overemphasized.167 Among the first complaints of parents are that the child appears to have mastered the material the night before a test but remembers little the next morning, and that increasing amounts of time are spent at home finishing work not completed at school. Subtle changes, such as problems with handwriting speed as a result of chemotherapy, may go unrecognized.168 Teachers, not understanding the neurobiological basis for the underlying learning problems, may believe the child to be poorly motivated or a “daydreamer.” The present standard

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Table 35.1 Risk for and types of neuropsychological deficits after treatment Risk factors Younger age at CNS therapy Increasing cranial radiation dose Relapse High dose methotrexate Female gender Increasing time elapsed from CNS therapy Abnormal MRI findings Deficit areas Global IQ decline “Slow learner” profile Mental retardation (rare) Specific cognitive processing deficits Attention Memory Processing speed Visual–motor coordination Academic achievement deficits Reading comprehension Written expression Mathematical computation

care for these children is a comprehensive psychoeducational evaluation supplemented with special measures to probe cognitive processing deficits commonly associated with cancer therapy. The results of the evaluation process then guide the type and intensity of special educational interventions.168 In summary, for children who receive CRT at or before the age of 2 years and for those who experience a CNS relapse (especially those who receive a second course of CRT), all available evidence points to an increased risk of chronic and pervasive neurological and neuropsychological deficits. Thus, the development of these children should be formally monitored by the treating institution. For older children who receive CRT and remain in remission, the risk of clinically significant intellectual or neuropsychological decline is equivocal. Memory functions may be affected independently of IQ changes. The contribution of other risk factors, such as female gender, are less well documented but are potentially important areas for future research.

Bone marrow transplantation The use of BMT in the treatment of pediatric leukemia has continued to grow over the past two decades, and improved survival rates due to this procedure have contributed to a rapidly growing long-term survivor population.169–171

Leukemia patients comprise more than 70% of pediatric BMT recipients.170 Research regarding the psychosocial consequences of BMT has begun to accumulate from both adult172,173 and pediatric174,175 populations, but progress has been slow and many issues remain unresolved. This discussion will focus on two areas of interest: the acute distress experienced during the transplant procedure; and late effects in long-term survivors, particularly those related to the CNS and neurocognitive sequelae. Although it is generally accepted that hematopoietic stem cell transplantation (HSCT), is a very demanding and stressful experience for children, the degree to which this procedure causes distress in children has not been widely studied empirically. Research on the psychosocial consequences of BMT has come predominantly from adult patients, with only a small number of studies involving pediatric samples.172,175 Studies addressing acute issues have frequently been limited by retrospective designs, with the few prospective studies involving very small samples, particularly in pediatric populations.176–179 A retrospective study of childhood HSCT patients suggests that children may experience milder levels of distress than those reported by adults.180 More recently, our group reported a prospective, longitudinal study of the acute responses of a large cohort of children undergoing HSCT.181,182 A total of 153 children were assessed at the time of admission for transplantation and then underwent weekly assessments through week +6, followed by monthly assessment through month +6. Data were obtained from reports by either parents or patients (if 5 years of age or older) using the BASES183,184 scales. The major findings of this study were: (1) children undergoing HSCT enter the hospital with an already heightened level of distress (defined by high levels of somatic symptoms and mood disturbance, and low levels of activity) that increases dramatically following conditioning, reaching a peak approximately 1 week post-transplantation; (2) this increased distress is transient, declining rapidly back to admission levels by week +4 to week +5, followed by a further decline to presumed basal levels by months 4 to 6; and (3) the trajectories of distress depicted by the reports of both parents and children are remarkably similar, each providing confirmatory support for the validity of the findings. This pattern differs from the more commonly depicted “trajectory to recovery” following transplantation, in which adult patients have been reported to show gains in functioning for up to 2 years post-HSCT, before leveling off.185 It may be that there are two separate trajectories, one reflecting acute processes and mood disturbances specific to the tranplant procedure, which recovers more quickly, and a second trajectory related to a more global quality of life,

Psychosocial issues

which shows a longer time to recovery. Nevertheless, our findings confirm a number of clinical impressions that had not been documented empirically, and point to the need for new interventions or more intensive approaches to supportive care aimed at reducing levels of distress during the acute phase of transplantation.186 The major late effects studied thus far in long-term survivors of HSCT involve endocrine dysfunction (affecting both growth and gonadal development), ophthalmologic abnormalities, and CNS abnormalities.169–171 Cataracts, once a common complication of early transplantconditioning regimens that included single-dose total body irradiation (TBI), have been significantly reduced since the advent of fractionated TBI.170 Endocrine problems that typically result from TBI in the pre-HSCT conditioning regimen include thyroid dysfunction, decreased growth velocity, growth hormone deficiency, delay or absence of sexual development, and gonadal failure.187 To date, no studies have been reported that relate pediatric patients’ attitudes and affective status post-HSCT to issues of fertility, sexuality, or other neuroendocrine outcomes. However, in the adult literature, sexuality and sexual functioning have received more attention. Overall, results indicate that sexual difficulties are reported by many adult HSCT recipients, and that at least 25% report diminished sexual interest, enjoyment, and/or activity, with women appearing to be at greater risk.188,189 Whether similar difficulties will be experienced by pediatric BMT recipients in adulthood remains a question for future research. Late effects in the CNS have been a considerable focus of research on pediatric HSCT survivors. Patients undergoing HSCT are at risk for a number of adverse CNS events during the early post-transplant period, including cerebral hemorrhage, infectious complications such as viral encephalitis, metabolic encephalopathy, and other encephalopathies of unknown cause.190–194 Although the mortality associated with these complications is quite high, the surviving children are likely to recover with significant neurological impairments. Estimates of the frequency of neurological complications in HSCT patients have ranged from 11% to as high as 70%, depending on the survey methods.191,193–195 One of the earliest reports of the late effects of HSCT on the CNS indicated a 7% incidence of leukoencephalopathy in patients, but only in the subgroup who had received both previous CNS therapy and TBI.196 More recent studies that have included diagnostic imaging have indicated MRI abnormalities in nearly two-thirds of survivors.194,197 The most common findings involve white matter lesions or mild cerebral atrophy. In general, abnormalities have not been associated with TBI, but relate to graft-versushost disease (GVHD), in particular, to the corticosteroids

and cyclosporine used in the treatment of GVHD.194,197,198 These studies have been limited primarily to adult patients. Beyond specific neurological complications, research has focused on the risk for global cognitive and academic deficits in pediatric survivors of HSCT. Survivors of hematopoietic stem cell transplantation are thought to be at risk for cognitive deficits as a result of their exposure to numerous potentially neurotoxic agents, including TBI, busulfan and other cytotoxic conditioning agents, together with high-dose steroids, cyclosporine, and other agents used for the prophylaxis and/or treatment of GVHD.199–203 The findings reported thus far on neurocognitive outcomes in pediatric HSCT have been somewhat contradictory, although a consensus is beginning to emerge. A few studies have indicated declines in cognitive or academic function following the procedure,204–206 but a somewhat larger number of studies have reported normal neurodevelopment, with no evidence of declines in cognitive function.207–212 Our interpretation is that many of the divergent findings in the literature may be related to age effects within and between cohorts, and that with due consideration of age effects, a relatively coherent picture of the neurocognitive late effects of HSCT can be drawn. This suggests that HSCT, even with TBI, poses a low-to-minimal risk for late cognitive and academic deficits in patients who are at least 6 years old at the time of transplantation. For patients age 5 and under, and particularly for those younger than 3 years of age, the risk of cognitive impairment is apparently increased, regardless of whether or not TBI is used in conditioning. Clearly, further research is necessary to confirm this interpretation.

Interventions for neurocognitive deficits Interventions can take the form of prevention of neurocognitive deficits, primarily by reducing toxicity associated with treatment, or remediation of deficits that cannot be avoided. Although modification of treatment approaches to ALL, especially the reduction or elimination of the use of CRT, have lessened the neurocognitive morbidities, many children continue to experience significant but more subtle problems. Only recently, however, have investigators begun to focus on interventions intended to treat residual neurocognitive problems directly. Generally, these efforts are quite preliminary at present but are characterized by two separate approaches. These include cognitive/behavioral interventions and the use of psychostimulants, with both approaches borrowing heavily from the existing literature on rehabilitation methods for childhood Attention Deficit Hyperactivity Disorder (ADHD) and traumatic brain injury.

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Butler and Copeland213 have published preliminary data on the efficacy of a cognitive/behavioral intervention for survivors of childhood cancer with attentional deficits. In its current form, the intervention consists of 20, twohour sessions with a therapist. The difficulty of the material increases with mastery. The content of the sessions is divided into components intended to improve organization and planning, goal setting, self-evaluation, problem solving, and strategy acquisition. The results from a multiinstitutional randomized clinical trial coordinated by Dr. Butler through Oregon Health Sciences Center will be forthcoming. Medication for attentional problems with methylphenidate, a drug with known effectiveness in ADHD, has yet-unknown potential for helping children surviving cancer with learning problems. The only placebocontrolled, randomized study of this agent published thus far did show promise on laboratory measures of attention214 ; however, the practical benefits in an academic setting are not known. Currently, a randomized clinical trial of methylphenidate is under way at St. Jude Children’s Research Hospital, Duke University Medical Center, and the Medical University of South Carolina. One of the future goals of these intervention studies will be to define which children respond better to each type of treatment.

Health promotion Increasing numbers of childhood cancer survivors are now reaching adulthood due to progress in the development of curative therapy. This population is quite vulnerable to chronic adverse effects of treatment that may diminish their quality of life and increase their risk of early death. Late treatment-related effects can differ in severity and are highly dependent on the specific cancer diagnosis, type of treatment received, and the age of the child during treatment. For example, young children treated with radiation therapy for brain tumors are at an increased risk for neurocognitive deficits, while adolescents undergoing chemotherapy and radiation for a malignancy may have reproductive and body image concerns.215 In addition, cumulative doses of certain chemotherapeutic agents may affect specific organs (e.g. anthracyclinerelated cardiomyopathy).216 Second malignancies may also result directly from therapy or from genetic determinants that gave rise to the initial cancer.217

Health behaviors Behavioral health habits can influence a young patient’s risk of treatment-specific toxic effects and the development

of subsequent cancers. Adaptive health habits may serve to minimize or even prevent some adverse late effects of cancer treatment, while maladaptive health behaviors may exacerbate the risks. For example, good oral hygiene may lessen the adverse periodontal affects that can result from cranial irradiation at an early age.218 Survivors of childhood leukemia are especially vulnerable to tobacco-related health problems because they have a sevenfold excess risk for second cancers219 as well as an increased risk for cardiac and pulmonary damage due to previous therapy.219,220 Adverse health behavior such as cigarette smoking can promote or exacerbate these predispositions.221 Few studies have examined health behavior among adolescents with cancer. Early studies indicated that a smaller proportion of adolescents with cancer engage in health-risking behaviors such as excessive alcohol use and marijuana use than do healthy adolescents. In the first comprehensive study of the health-related behaviors of childhood cancer survivors,222 our research group surveyed 40 young adult long-term survivors ranging in age from 18 to 29 years and 110 parents of long-term survivors aged 11 to 17 years. The survey included questions about the former patient’s frequency of alcohol and tobacco use, as well as diet, exercise, sleep, dental, and seat belt habits. The reported prevalence of tobacco and alcohol use was slightly less than 10% among those less than 18 years old. Among the young adults, 17.5% were currently smoking and 15% were using smokeless tobacco products (“dip” or “snuff”). Alcohol use was more frequent (72.5%), but problem drinking was infrequently reported. More than 25% of both samples reported brushing their teeth once a day or less, eating balanced meals infrequently, and wearing seat belts infrequently. Approximately 6.4% of the preadolescent/adolescent age group exercised less than 1 hour weekly, the definition of sedentary behavior according to the Centers for Disease Control. Similar results were obtained in a follow-up study of 46 pediatric cancer survivors, ages 10 to 18 years, who provided self-reports of their health behavioral practices.223 In this sample, younger patients and those from higher socioeconomic backgrounds more frequently engaged in healthy behaviors. More recently, Hudson and colleagues224 investigated the self-reported health behaviors of a larger sample of 266 adolescent cancer survivors and found that 94% abstained from tobacco, 64% practiced sun protection, 27% performed monthly self-examination, 40% ate nutritious diets, and 52% performed aerobic exercise. Fifty-seven percent indicated that they wanted to change their behavioral practices to be healthy. In the largest study examining tobacco use habits in cancer survivors over 18 years of age, Emmons et al.225 found that 28% reported ever smoking and

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17% were current smokers. Factors associated with risk of smoking initiation included older age at cancer diagnosis, lower household income, less education, not having had pulmonary-related cancer treatment and not having had brain radiation. Although survivors smoke at rates below that of the general population, these figures are unacceptably high and indicate a need for smoking prevention and cessation interventions with this population. Collectively, health behavior studies indicate that young cancer survivors report lifestyles that are at least as healthy as their peers.226,227 The prevalence of risky health behaviors is low across studies. However, the threshold for adverse health outcomes is lowered and the risks for chronic illness are consequently increased among longterm survivors. Also, the prevalence data reported for unhealthy habits are likely to be underestimates, due to the reluctance of patients and their parents to admit maladaptive health habits to their health care providers. The development of effective health interventions that educate patients and parents as to the importance of healthprotective behaviors and of methods to increase patients’ compliance with healthy behaviors, is certainly warranted.

Current health promotion interventions Clinical interventions and health education programs designed to prevent or modify maladaptive health behaviors and promote healthy habits in young cancer prevention in healthy populations and have used educational interventions that focus on the long-term health consequences of behavior. More recently developed programs have sought to provide training aimed at increasing protective health behaviors. These programs have targeted behaviors implicated in the development of cancer, such as tobacco use, dietary fat intake, and sun exposure,228–230 as well as cancer screening behaviors such as breast and testicular self-examination.231,232 Typically, these programs have been geared toward the healthy youngster through community and school-based interventions using various instructional modalities. However, information from existing programs and curricula may assist health care providers in developing health promotion programs tailored to the pediatric cancer care setting. In a recent controlled prospective trial, Hudson et al.224 evaluated the efficacy of an educational risk counseling intervention to promote protective health behaviors among childhood cancer survivors. Survivors attending a long-term follow-up clinic were randomized to receive standard follow-up care or standard care plus an educational intervention. The intervention consisted of breast or testicular self-examination (BSE or TSE) instruction

by a nurse using a breast or testicular model, targeted late effects screening based on clinical history and treatment exposures, clinical assessment and discussion of the patient’s written clinical summary, health behavior training, health goal commitment, and telephone follow-up. The intervention targeted tobacco use, sun protection, diet, exercise or self-examination. No significant differences between the intervention and standard care groups in health knowledge, health perceptions, and health behaviors were noted at the 12 month assessment relative to baseline. However, female survivors in the cohort demonstrated greater increase in knowledge, while survivors for whom self-examination was the health goal demonstrated more frequent practice of BSE/TSE. Using a similar approach, Hollen et al.233 found no significant reductions in the frequency of smoking, alcohol use, or illicit drug use in a convenience sample of adolescent cancer survivors, ages 13 to 21 years, who participated in a 5-hour decision-making and risk-reduction program focused on health behaviors, by comparison with adolescent survivors who did not receive the intervention. However, the shortterm effects of improved health-related decision-making and decreased motivation for alcohol use were observed for the intervention group. Results from the first randomized smoking prevention trial conducted with 103 preadolescent and adolescent cancer survivors were recently published by Tyc and colleagues.234 Compared to a standard care control group, survivors who received a single session tobacco risk counseling intervention demonstrated an increase in tobaccorelated knowledge and perceived vulnerability to tobaccorelated health risks, as well as a reduction in intentions to use tobacco at 12 months following the intervention. The intervention consisted of late effects risk counseling in addition to an educational video, goal setting, written physician feedback, smoking literature, and followup telephone counseling. A similar randomized clinical trial examining the efficacy of a behavioral risk counseling intervention to reduce the pediatric cancer patient’s exposure to environmental tobacco smoke (ETS), also known as secondhand smoke, is currently under way at our institution. Young cancer patients treated at our institution are regularly exposed to ETS in the home, typically from parental sources, despite their increased health risks associated with tobacco smoke secondary to treatment-related toxicities.235 One of the primary goals of this intervention study is to prospectively examine the relationship between ETS exposure, as measured by urine cotinine levels and parents’ report of the number of cigarettes to which their child is exposed, and the child’s short-term health outcomes and treatment-related side effects.

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The effectiveness of the interventions presented here may not have been highly salient in the cohorts described because of the moderate level of intervention that is typically provided to patients as routine standard care in the survivor clinic settings in which the studies were conducted. The limited intervention effects may also suggest that brief, broad-based risk counseling may not be sufficient to produce dramatic changes in knowledge, health perceptions, and health practices in this vulnerable patient group. More intensive interventions conducted over several sessions may be necessary to enhance the impact of health counseling programs. However, they should be balanced against the constraints of daily clinical practice in order to be maximally cost-effective. Specific recommendations for conducting health promotion interventions with young cancer survivors have been discussed in more detail elsewhere.236,237 Moreover, the inclusion of biologic markers to validate adolescents’ self-report of behavioral practices would be a useful addition to health promotion trials with high risk pediatric populations.

A combined model approach to health promotion Several theories have been proposed to explain health behaviors in adolescents and adults. Perception of health risk, also referred to as “perceived vulnerability,” is a central component of all current models of health-protective behavior.238 According to these models, persons who perceive their risk of a negative health outcome to be high are more likely to engage in some preventive action to reduce that risk. In the Health Belief Model, for example, perceived vulnerability, combined with perceived severity of the potential outcome, perceived benefits of the protective behavior, and personal costs of engaging in a particular selfprotective behavior, reflect one’s “health motivation” or readiness to undertake that behavior. Studies have demonstrated that informing young adults and healthy adolescents about their personal vulnerability to negative health outcomes can increase healthy preventive behaviors.239,240 We recently examined perceptions of vulnerability to health-related problems among cancer survivors at our institution.222 Specifically, preadolescent, adolescent, and young adult survivors and their parents were asked to rate the strength of their belief that remaining healthy is more important for the patient than for most other children or young adults. Respondents were asked to rate this belief on a 5-point scale ranging from “much less important” to “much more important.” More than 80% of parents and 60% of young adult survivors surveyed believed it was more important for the former patient to remain healthy than for most other people to do so. Somewhat surprisingly, 3 of

the 40 young adults responded that their health was less or much less important than that of most other people. Demographic factors such as the patient’s gender, socioeconomic level, and time elapsed since completion of therapy had no significant effect. The perceived importance of health protection was inversely related to the patient’s current age and to the total duration of therapy. In addition, the relationship between health protective beliefs and health protective behaviors was inconsistent, especially among younger patients. Later studies also support the finding that young cancer survivors perceive themselves to be vulnerable to health problems as a result of their treatment.223,234,241 Translation of the cancer survivors’ heightened health concerns into protective health behavioral practices, however, has not been consistently demonstrated. For example, perceived vulnerability was not found to be a significant predictor of future intentions to use tobacco among adolescent cancer survivors.241 Without comparison to healthy control groups, it is difficult to determine whether heightened concerns about health vulnerability are uniquely characteristic of young patients treated for cancer. Although adolescent cancer survivors’ personal susceptibility to negative health outcomes may serve to motivate good behavioral health habits, variables encompassed by other theoretical models (e.g. Social Learning Theory), may also play a role in promoting healthy behaviors in this patient group. For example, the utility of the Social Learning Theory framework in health education has been demonstrated for a number of health practices in a variety of populations.242–244 Its application, particularly when combined with the Health Belief Model, may help to clarify why survivors engage in particular health behaviors following the cancer experience.

Resources for caregivers Numerous resources that describe psychosocial issues for children with ALL are available to parents, teachers, and physicians. Issues related to planning for school re-entry after diagnosis of cancer, including preparation of the teachers and other students and parental rights, have recently been reviewed elsewhere, and these publications provide excellent resources for teachers and parents.245,246 Information about childhood ALL and psychosocial issues is available from several websites, including the Candlelighters Foundation (http://www.candlelighters.org/), a patient advocacy group with a strong network of parent support groups. This website includes a thorough summary of cognitive late effects and parental rights written in lay language. The American Cancer

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Society (http://www.cancer.org/docroot/home/index.asp) site includes information about insurance and nutritional aspects of care. The National Cancer Institute has a childhood cancer home page (http://www.nci.nih.gov/ cancerinfo/types/childhoodcancers) that includes extensive information about ALL for parents/patients as well as for health-care professionals; descriptions of current clinical trials and assistance in searching the research literature are at this site. The Childhood Leukemia Foundation (http://www.clf4kids.com/) offers summer camp scholarships to patients.

Conclusions The present standard of care in pediatric oncology includes the availability of psychosocial services for all patients and their families. Psychosocial research has significantly advanced our understanding of the behavioral and social sequelae of leukemia and its treatment encountered by patients and their families. Although the severity of these effects varies widely, many of their associated medical and nonmedical risk factors have been identified, allowing atrisk patients or families to be targeted for interventions. Such studies have also helped to quantify the psychosocial costs of cure, prompting increased efforts to reduce acute and late adverse effects (for example, by reducing the number of bone marrow aspirates and using cranial radiotherapy more selectively) while maintaining or improving cure rates. A smaller but equally important number of studies have attempted to translate descriptive findings into psychosocial interventions. These studies are labor-intensive and fraught with logistic problems in the cancer care setting. Nevertheless, psychosocial research will continue to evolve toward direct intervention strategies for improving the quality of life for patients and their families. In some areas, such as family coping and procedural distress, successful interventions are in use and have defined contemporary standards of care. In other areas, such as health promotion and neuropsychological rehabilitation, there is an urgent need for translation of descriptive data into studies that test the efficacy of psychosocial interventions.

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impairments of concentration, attention, and memory. Med Pediatr Oncol, 2002; 38: 320–8. Moleski, M. Neuropsychological, neuroanatomical, and neurophysiological consequences of CNS chemotherapy for acute lymphoblastic leukemia. Arch Clin Neuropsychol, 2000; 15: 603–30. Robison, L. L., Nesbit, M. E., Sather, H. N., et al. Factors associated with IQ scores in long-term survivors of childhood acute lymphoblastic leukemia. Am J Pediatr Hematol/Oncol, 1984; 6: 115–21. Said, J. A., Waters, B. G. H., Cousens, P., et al. Neuropsychological sequelae of central nervous system prophylaxis in survivors of childhood acute lymphoblastic leukemia. J Consult Clin Psychol, 1989; 57: 251–6. Jankovic, M., Brouwers, P., Valsecchi, M. G., et al. Association of 1800cGy with intellectual function in children with acute lymphoblastic leukaemia. Lancet, 1994; 344: 224–7. Silber, J. H., Radcliffe, J., Peckham, V., et al. Whole-brain irradiation and decline in intelligence: the influence of dose and age on IQ score. J Clin Oncol, 1992; 10: 1390–6. Kaleita, T. A., Reaman, G. H., MacLean, W. E., Sather, H. N., & Whitt, J. K. Neurodevelopmental outcome of infants with acute lymphoblastic leukemia. Cancer, 1999; 85: 1859–65. Waber, D., Carpentieri, S. C., Klar, N., et al. Cognitive sequelae in children treated for acute lymphoblastic leukemia with dexamethasone or prednisone. J Pediatr Hematol/Oncol, 2000; 22: 206–13. Balsom, W. R., Bleyer, W., Robison, L. L., et al. Intellectual function in long-term survivors of childhood acute lymphoblastic leukemia: protective effect of pre-irradiation methotrexate? A Children’s Cancer Study Group Study. Med Pediatr Oncol, 1991; 19: 486–92. Waber, D. P., Tarbell, N. J., Gariclough, D., et al. Cognitive sequelae of treatment in childhood acute lymphoblastic leukemia: cranial radiation requires an accomplice. J Clin Oncol, 1995; 13: 2490–6. Goff, J. R., Anderson, H. R., & Cooper, P. F. Distractibility and memory deficits in long-term survivors of acute lymphoblastic leukemia. Dev Behav Pediatr, 1980; 1: 158–63. Taylor, H. G., Albo, V. C., Phebus, C. K., et al. Postirradiation treatment outcomes for children with acute lymphocytic leukemia: clarification of risks. J Pediatr Psychol, 1987; 12: 395– 411. Cousens, P., Ungerer, J. A., Crawford, J. A., et al. Cognitive effects of childhood leukemia therapy: a case for four specific deficits. J Pediatr Psychol, 1991; 16: 475–88. Brouwers, P. & Poplack, D. Memory and learning sequelae in long-term survivors of acute lymphoblastic leukemia: association with attention deficits. Am J Pediatr Hematol Oncol, 1990; 12: 174–81. Brown, R. T., Madden-Swain, A., Pais, R., et al. Chemotherapy for acute lymphocytic leukemia: cognitive and academic sequelae. J Pediatr, 1992; 121: 885–9. Mulhern, R. K., Wasserman, A. L., Fairclough, D., et al. Memory function in disease-free survivors of childhood acute

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lymphocytic leukemia given CNS prophylaxis with or without 1,800cGy cranial irradiation. J Clin Oncol, 1988; 6: 315–20. Lockwood, K. A., Bell, T. S., & Colegrove, R. W. Long-term effects of cranial radiation therapy on attention functioning in survivors of childhood leukemia. J Pediatr Psychol, 1999; 24: 55– 66. Schatz, J., Kramer, J. H., Ablin, A., & Matthay, K. K. Processing speed, working memory, and IQ: a developmental model of cognitive deficits following cranial radiation therapy. Neuropsychology, 2000; 14: 189–200. Haupt, R., Fears, T. R., Robison, L. L., et al. Educational attainment in long-term survivors of childhood acute lymphoblastic leukemia. J Am Med Assoc, 1994; 272: 1427–32. Armstrong, F. D. & Horn, M. Educational issues in childhood cancer. School Psychol Q, 1995; 10: 292–304. Reinders-Messelink, H. A, Shoemaker, M. M., Snijders, T. A. B., et al. Analysis of handwriting of children during treatment for acute lymphoblastic leukemia. Med Pediatr Oncol, 2001; 37: 393–9. Treleaven, J. G. & Barrett, J. Introduction. In J. Barrett & J. G. Treleaven, eds., The Clinical Practice of Stem-Cell Transplantation, vol. 1 (Oxford, UK: Isis Medical Media, 1998), pp. 2–16. Sanders, J. E. Bone marrow transplantation in pediatric oncology. In P. A. Pizzo & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology, 3rd edn. (Philadelphia, PA: J. P. Lippincott, 1997), pp. 357–74. Wingard, J. R. Bone marrow to blood stem cells; past, present, future. In M. B. Whedon & D. Wujcik, eds., Blood and Marrow Stem Cell Transplantation (Boston, MA: Jones and Bartlett Publishers, 1997), pp. 3–24. Andrykowski, M. A. Psychiatric and psychosocial aspects of bone marrow transplantation. Psychosomatics, 1994; 35: 13– 24. Andrykowski, M. A. Psychosocial factors in bone marrow transplantation: a review and recommendations for research. Bone Marrow Transplant, 1994; 13: 357–75. Phipps, S. Bone marrow transplantation. In D. Bearison & R. Mulhern, eds., Pediatric Psychooncology: Psychological Perspectives on Children with Cancer (New York: Oxford University Press, 1994), pp. 143–70. Phipps, S. & Barclay, D. Psychosocial consequences of pediatric bone marrow transplantation. Int J Pediatr Hematol Oncol, 1996; 3: 171–82. Wettergren, L., Langius, A., Bjorkholm, M., & Bjorvell, H. Physical and psychosocial functioning in patients undergoing autologous transplantation – a prospective study. Bone Marrow Transplant, 1997; 20: 497–502. Hjermstad, M. J., Loge, J. H., Evensen, S. A., et al. The course of anxiety and depression during the first year after allogeneic or autologous stem cell transplantation. Bone Marrow Transplant, 1999; 24: 1219–28. Rodrigue, J. R., Graham-Pole, J., Kury, S., Kubar, W., & Hoffman, G. Behavioral distress, fear, and pain among children hospitalized for bone marrow transplantation. Clin Transplant, 1995; 9: 454–6.

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179 Gunter, M., Karle, M., Werning, A., & Klingbiel, T. Emotional adaptation of children undergoing bone marrow transplantation. Can J Psychiatry, 1999; 44: 77–81. 180 Parsons, S. K., Barlow, S. E., Levy, S. L., Supran, S. E., & Kaplan, S. H. Health-related quality of life in pediatric bone marrow transplant survivors: according to whom? Int J Cancer (Suppl.), 1999; 12: 46–51. 181 Phipps, S., Dunavant, M., Garvie, P., Lensing, S., & Rai, S. N. Acute health-related quality of life in children undergoing stem cell transplant. I. Descriptive outcomes. Bone Marrow Transplant, 2002; 29: 425–34. 182 Phipps, S., Dunavant, M., Lensing, S., & Rai, S. N. Acute health-related quality of life in children undergoing stem cell transplant. II. Medical and demographic determinants. Bone Marrow Transplant, 2002; 29: 435–42. 183 Phipps, S., Hinds, P. S., Channel, S., & Bell, G. L. Measurement of behavioral, affective and somatic responses to pediatric bone marrow transplantation. Development of the Bases scale. J Pediatr Oncol Nurs, 1994; 11: 109–17. 184 Phipps, S., Dunavant, M., Jayawardene, D., & Srivastava, D. K. Assessment of health related quality of life in acute inpatient settings: Use of the BASES scale in children undergoing bone marrow transplantation. Int J Cancer, 1999; 12: 18–24. 185 McQuellon, R. P., Russell, G. B., Rambo, T. D. et al. Quality of life and psychological distress of bone marrow transplant receipients: the ‘time trajectory’ to recovery over the first year. Bone Marrow Transplant, 1998; 21: 477–86. 186 Phipps, S. Reduction of distress associated with pediatric bone marrow transplant: complementary health promotion interventions. Pediatr Rehab, 2002; 5: 223–34. 187 Sanders, J. E. Late effects following marrow transplantation. In F. L. Johnson & C. Pochedly, eds., Bone Marrow Transplantation in Children (New York: Raven Press, 1990), pp. 471–96. 188 Mumma, G. H., Mashberg, D., & Lesko, L. M. Long-term psychosexual adjustment of acute leukemia survivors: impact of marrow transplantation versus conventional chemotherapy. Gen Hosp Psychiatr, 1992; 14: 43–55. 189 Wingard, J. R., Curbow, B., Baker, F., et al. Sexual satisfaction in survivors of bone marrow transplantation. Bone Marrow Transplant, 1992; 9: 185–90. 190 Garrick, R. Neurologic complications. In K. Atkinson, ed., Clinical Bone Marrow and Blood Stem Cell Transplantation, 2nd edn. (New York: Cambridge University Press, 2000), pp. 58–79. 191 Graus, F., Saiz, A., Sierra, J., et al. Neurologic complications of autologous and allogeneic bone marrow transplantation in patients with leukemia: a comparative study. Neurology, 1996; 46: 1004–9. 192 Marks, P. V. Neurological aspects of stem-cell transplantation. In J. Barrett & J. Trealeaven, eds., The Clinical Practice of Stem-Cell Transplantation, vol. 1 (Oxford, UK: ISIS, 1998), pp. 787–94. 193 Meyers, C. A., Weitzer, M., Byrne, K., et al. Evaluation of the neurobehavioral functioning of patients before, during and

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after bone marrow transplantation. J Clin Oncol, 1994; 12: 820–6. Padovan, C. S., Tarek, Y. A., Schleuning, M., et al. Neurological and neuroradiological findings in long-term survivors of allogeneic bone marrow transplantation. Ann Neurol, 1998; 43: 627–33. Patchell, R. A., White, C. L., Clark, A. W., Beschorner, W. E., & Santos, G. W. Neurologic complications of bone marrow transplantation. Neurology, 1985; 35: 300–6. Thompson, C. B., Sanders, J. E., Flournoy, N., et al. The risks of central nervous system relapse and leukoencephalopathy in patients receiving marrow transplants for acute leukemia. Blood, 1986; 67: 195–9. Coley, S. C., Jager, H. R., Szydlo, R. M., & Goldman, J. M. CT and MRI manifestations of central nervous system infection following allogeneic bone marrow transplantation. Clin Radiol, 1999; 54: 390–7. Pace, M. T., Slovis, T. L., Kelly, J. K., & Abella, S. D. Cyclosporin A toxicity: MRI appearance of the brain. Pediatr Radiol, 1995; 25: 180–3. Chou, R. H., Wong, G. B., Kramer, J. H., et al. Toxicities of totalbody irradiation for pediatric bone marrow transplantation. Int J Radiat Oncol, 1996; 34: 843–51. Miale, T. D., Sirithorn, S., & Ahmed, S. Efficacy and toxicity of radiation in preparative regimens for pediatric stem cell transplantation. I: clinical applications and therapeutic effects. Med Oncol, 1995; 12: 231–49. Peper, M., Schraube, P., Kimming, C., et al. Long-term cerebral side-effects of total body irradiation and quality of life. Recent Results Cancer Res, 1993; 130: 219–30. Reece, D. E., Frei-Lahr, D. A., Shepherd, J. D. et al. Neurologic complications in allogeneic bone marrow transplant patients receiving cyclosporin. Bone Marrow Transplant, 1991; 8: 393– 401. Padovan, C. S. Gerbitz, A., Sostak, P., et al. Cerebral involvement in graft-versus-host disease after murine bone marrow transplantation. Neurology, 2001; 56: 1106–8. Cool, V. A. Long-term neuropsychological risks in pediatric bone marrow transplant: what do we know? Bone Marrow Transplant, 1996; 18(Suppl. 3), S45–9. Kramer, J. H., Crittenden, M. R., DeSantes, K., & Cowan, M. J. Cognitive and adaptive behavior 1 and 3 years following bone marrow transplantation Bone Marrow Transplant, 1997; 19: 607–13. Smedler, A. C. & Bolme, P. Neuropsychological deficits in very young bone marrow transplant recipients. Acta Pediatr, 1995; 84: 429–33. Arvidson, J., Kihlgren, M., Hall, C., & Lonnerholm, G. Neuropsychological functioning after treatment for hematological malignancies in childhood, including autologous bone marrow transplantation. Pediatr Hematol Oncol, 1999; 16: 9–21. Llach, D. M., Campdepadros, P. M., Ceballos, B. N., et al. Secuelas neuropsicologicas a medio y largo plazo del trasplante de medula osea en pacientes con enfermedades

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heamtologicas [Long and medium-term neuropsychological sequelae of bone marrow transplantation in pediatric patients with hematological disease.] An Esp Pediatria, 2001; 54: 463–7. Phipps, S., Dunavant, M., Srivasatava, D. K., Bowman, L., & Mulhern, R. K. Cognitive and academic functioning in survivors of pediatric bone marrow transplantation. J Clin Oncol, 2000; 18: 1004–11. Pot-Mees, C. C. The Psychological Aspects of Bone Marrow Transplantation in Children. (The Netherlands: Eburon Delft, 1989). Simms, S., Kazak, A. E., Golumb, V., Goldwein, J., & Bunin, N. Cognitive, behavioral, and social outcome in survivors of childhood stem cell transplantation. J Pediatr Hematol Oncol, 2002; 24: 115–19. Kupst, M. J., Penati, B., Debban, B., et al. Cognitive and psychosocial functioning of pediatric hematopoietic stem cell transplant patients: a prospective longitudinal study. Bone Marrow Transplant, 2002; 30: 609–17. Butler, R. W. & Copeland, D. R. Attentional processes and their remediation in children treated for cancer: a literature review and the development of a therapeutic approach. J Int Neuropsychol Soc, 2002; 8: 115–24. Thompson, S. J., Leigh, L., Christensen, R., et al. Immediate neurocognitive effects of methylphenidate on learningimpaired survivors of childhood cancer. J Clin Oncol, 2001; 19: 1802–8. Konsler, G. K. & Jones, G. R. Transition issues for survivors of childhood cancer and their health care providers. Cancer Pract, 1993; 1: 319–24. Lipshultz, S. E., Colan, S. D., Gelbar, R. D., et al. Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in childhood. N Engl J Med, 1991; 324: 808–15. Meadows, A. T. Risk factors for second malignant neoplasms: report from the Late Effects Study Group. Bull Cancer, 1988; 75: 125–30. Neglia, J. P., Meadows, A. T., Robison, L. L., et al. Second neoplasms after acute lymphoblastic leukemia in childhood. N Engl J Med, 1991; 325: 1330–6. Lipshultz, S. E., Colan, S. D., Gelber, R. D., et al. Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in childhood. N Engl J Med, 1991; 324: 808–15. Shaw, N. J., Tweeddale, P. M., & Eden, O. B. Pulmonary function in childhood leukemia survivors. Med Pediatr Oncol, 1989; 17: 149–54. Tao, M. L., Guo, M. D., Weiss, R., et al. Smoking in adult survivors of childhood acute lymphoblastic leukema. J Natl Cancer Inst, 1998; 90: 219–25. Mulhern, R. K., Tyc, V. L., Phipps, S., et al. Health-related behaviors of survivors of childhood cancer. Med Pediatr Oncol, 1995; 25: 159–65. Tyc, V. L., Hadley, W., & Crockett, G. Prediction of health behaviors in pediatric cancer survivors. Med Pediatr Oncol, 2001; 37: 42–6.

224 Hudson, M. M., Tyc, V. L., Srivastava, D. K., et al. Multicomponent behavioral intervention to promote health protective behaviors in childhood cancer survivors: the Protect Study. Med Pediatr Oncol, 2002; 39: 2–11. 225 Emmons, K., Frederick, P. L., & Whitton, J. Predictors of smoking initiation and cessation among childhood cancer survivors: a report from the Childhood Cancer Survivor Study. J Clin Oncol, 2002; 20: 1608–16. 226 Adams, P. F., Schoenborn, C. A., Moss, A. J. et al. Health risk behaviors among our nation’s youth: United States, 1992. In Vital and Health Statistics. Series 10, No. 192. (Hyattesville, MD: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for Health Statistics, 1995), pp. 1–51. 227 Kann, L., Kinchen, S. A., Williams, B. I., et al. Youth Risk Behavior Surveillance: United States, 1997. (Silver Spring, MD: Division of Adolescent and School Health, National Center for Chronic Disease Prevention and Health Promotion, 1998), pp. 1–89. 228 Perry, C. L., Stone, E. J., Parcel, G. S., et al. School-based cardiovascular health promotion: the child and adolescent trial for cardiovascular health (CATCH). J School Health, 1990; 60: 406–13. 229 Perry, C. L., Kelder, S. H., Murray, D. M., et al. Communitywide smoking prevention: long-term outcomes of the Minnesota Heart Health Program. Am J Public Health, 1992; 82: 1210–6. 230 Lombard, D., Neubauer, T. E., & Canfield, D. Behavioral community intervention to reduce the risk of skin cancer. J Appl Behav Anal, 1991; 24: 677–86. 231 Lerman, C., Kash, K., & Stefanek, M. Younger women at increased risk for breast cancer: psychological well-being perceived risk, and surveillance behavior. N Can Inst Monogr, 1994; 16: 171–6. 232 Rosella, J. D. Testicular cancer health education: an integrative review. J Adv Nurs, 1994; 20: 666–71. 233 Hollen, P. J., Hobbie, W. L., & Finley, S. M. Testing the effects of a decision-making and risk reduction program for cancer-surviving adolescents. Oncol Nurs Forum, 1999; 26: 1475–86. 234 Tyc, V. L., Rai, S., Lensing, S., et al. An intervention to reduce intentions to use tobacco among pediatric cancer survivors. J Clin Oncol, 2003; 21: 1366–72. 235 Tyc, V. L., Hudson, M. M., Hadley, W., et al. Pediatric cancer patients and parents who smoke: counseling guidelines. Primary Care Cancer, 2001; 21: 9–16. 236 Tyc, V. L., Hudson, M. M., Hinds, P., et al. Tobacco use among pediatric cancer patients: recommendations for developing clinical smoking interventions. J Clin Oncol, 1997; 15: 2194– 204. 237 Tyc, V. L., Hudson, M. M., & Hinds, P. Health promotion interventions for adolescent cancer survivors. Cognitive Behavioral Prac, 1999; 6: 128–36. 238 Weinstein, N. D. Testing four competing theories of healthprotective behavior. Health Psychol, 1993; 12: 324–33.

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239 Blalock, S. J., McEvay Devellis, B, Afifi, R. A., et al. Risk perceptions and participation in colorectal cancer screening. Health Psychol, 1990; 87: 71–81. 240 Eiser, M., Zellman, G. L., & McAlister, A. L. A health belief model-social learning theory approach to adolescents’ fertility control: findings from a controlled field trial. Health Edu Q, 1992; 19: 249–62. 241 Tyc, V. L., Hadley, W., & Crockett, G. Brief report: predictors of intentions to use tobacco among adolescent survivors of cancer. J Pediatr Psychol, 2001; 26: 117–21. 242 Strecher, V. J., Devellis, F. R., Giorgino, K. B., et al. The role of self-efficacy in achieving health behavior change. Health Edu Q, 1986; 13: 73–91.

243 Strecher, V. J., Bauman, K. E., Goat, B., et al. The role of outcome and efficacy expectations in an intervention designed to reduce infants’ exposure to environmental tobacco smoke. Health Edu Res, 1993; 8: 137–143. 244 Bandura, A. Social Learning Theory (Englewood Cliffs, NJ: Prentice-Hall, 1977). 245 Leigh, L. & Miles, M. A. Educational issues for children with cancer. In P. A. Pizzo & D. G. Poplack, eds., Principles and Practice of Pediatric Oncology, 4th edn. (Philadelphia, PA: J. B. Lippincott, 2002), pp. 1463–75. 246 Woznick, L. A. & Goodheart, C. D. Living with Childhood Cancer: A Practical Guide (Washington, DC: APA Life Tools, 2002).

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36 Nursing care Pamela S. Hinds, Jami S. Gattuso, and Belinda N. Mandrell

Introduction In the treatment of children and adolescents with leukemia, nursing care complements and extends the efforts of other members of the health-care team. The focus of nursing care is on the long-term as well as immediate needs of patients and their families, and thus includes strategies to prevent or manage complications and sequelae of the disease and its treatment. Another integral aspect of nursing care is assisting individual patients and families in achieving their developmental goals during the period of treatment and follow-up. In this chapter, rather than describing the challenges of pediatric oncology nursing or detailing practice recommendations specific to the care of young patients with leukemia, we provide a framework within which these aspects can be addressed and apply this framework to several practice examples. This framework includes three levels of environment and seven possible treatment phases. The relationships between nursing strategies and both the level of environment and the phase of care are illustrated in examples of several major concerns that can arise in the treatment of childhood leukemia. For convenience, we have based our examples primarily on the treatment of childhood acute lymphoblastic leukemia (ALL), but the same principles apply to other leukemias in children. Whenever possible, we have focused on the results of nursing research and their application to practice.

An environmental model The nursing care of a child or adolescent with leukemia extends beyond a set of symptoms or specific treatment and

disease-related events. Further, the nurse’s ability to provide care and the patient’s response to that care are affected by environmental factors. Thus, an environmental model is both appropriate and necessary in evaluating the care needs of these patients. The model we have chosen (see Fig. 36.1) specifies three interactive levels of environment that directly influence nursing care and its outcomes. This model, derived in part from the work of two other groups,1,2 has proven useful in our own studies of the quality of life in children and adolescents with cancer, and of selected nursing procedures and their outcomes.3

The internal environment The internal environment represents the patient’s genetic, physical, and psychological characteristics. Typically, these factors indicate a patient’s levels of functioning and feelings toward self, which in turn influence the extent to which patients participate in their own care and respond to nursing care. For example, the extent to which a young patient grasps the nature and seriousness of the malignancy and the need for treatment directly influences his or her receptiveness to the information nurses provide regarding the disease and its treatment, the potential for infection, and the importance of a hopeful attitude. These responses in turn affect the outcome of nursing care.

The immediate environment The immediate environment represents the influence of family members and health-care providers on a patient’s responses to the care provided. Family influences include a supportive involvement in the child’s care and compliance

C Cambridge University Press 2006. Childhood Leukemias, ed. Ching-Hon Pui. Published by Cambridge University Press.


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Nursing care

Patient outcomes

Institutional environment Immediate environment (parent, healthcare providers)

NURSING CARE

Internal environment (patient)

Family outcomes

Community outcomes

Fig. 36.1 Environmental model of nursing care for children and youths diagnosed with leukemia, and for their families. The model reflects the nursing care process and its outcomes and the three levels of environment that directly influence nursing care strategies.

with the treatment requirements. Supportive involvement is more likely when the family is adapting to the cancer experience.3 High levels of parental distress have been significantly correlated with high levels of hopelessness in the child with leukemia.4 Hence, family coping is a focal concern for nurses. The second source of influence in the immediate environment – health-care providers – is reflected in the provider’s technical competence, knowledge of leukemia and its treatment and potential complications, and understanding of child development.5 Nursing care for the child with leukemia and the child’s family is provided by the staff nurse and the advanced practice nurse, who have overlapping and complementary roles. The staff nurse is a registered nurse who has acquired specialty knowledge primarily through job-related experiences and is primarily responsible for the safe administration of prescribed therapies and for determining and documenting the effects on patients of those therapies. The advanced practice nurse is a registered nurse with a minimum of a master’s degree in nursing who has acquired advanced, in-depth knowledge and preceptored clinical experience in pediatric oncology.6,7 The advanced practice nurse, who works closely with the child and the child’s family, could be a clinical nurse specialist, nurse practitioner, or nurse anesthetist. The clinical nurse specialist and the nurse practitioner may follow the child from the point of diagnostic procedures through treatment and into survival or end of life.

The institutional environment The institutional environment represents the influence of the health-care and social systems and of society as a whole

(e.g. financial contributions to research and care-related projects; guidelines for reimbursement). This level extends beyond the health-care setting to include the community and society at large. These dynamic and interactive levels of environment directly influence the quality of care provided by nurses and the outcomes of that care. For example, how well the patients understand the importance of care of the oral mucosa during treatment (internal environment), combined with how firmly the family (immediate environment) supports this need will directly influence the impact of a nurse’s efforts to teach patients about mouth care and to monitor the condition of the mucosa. In turn, nurses’ documentation of the extent and frequency of mucositis, and of its impact on patients’ well-being, could motivate a drug company or academic researcher (institutional environment) to create a new preventive or treatment agent. Ultimately, the three levels of environment and the nursing care efforts interact to determine whether and to what extent the patient experiences mucositis, and for how long. A framework that is strictly patient- or family-centered is not sufficient to adequately meet the short- and long-term health-care needs of children with leukemia.8

Nursing care strategies There are five essential strategies that pediatric oncology nurses use in caring for children with leukemia and their families: (1) patient and family education; (2) providing a supportive presence; (3) actively monitoring and anticipating; (4) being technically competent; and (5) serving as an advocate for patients and families. Each of these strategies is used to help patients recover from the leukemia, develop behaviors that will protect them from complications, and generally promote health and wellbeing. Each strategy is briefly described below, and examples incorporating the five strategies are subsequently included in the descriptions of the phases of care that follow.

Patient and family education This strategy is based on learning activities that are carefully created and strategically timed9,10 to reflect the acute and chronic nature of childhood leukemia; the symptoms most often seen in clinical settings (anemia, fatigue, petechiae, lethargy, bruising, and pain);11–17 the developmental and health status of the patient,18 parents, and family unit; the target symptom or behavior (e.g. monitoring body temperature); and the desired outcome (e.g. remaining infection free). It is important to balance disease and

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treatment-related education with health promotion and childhood development information.19 Examples of content areas and specific topics for patient and family education, by phase of care, are displayed in Table 36.1. The underlying assumptions are that the patient and family have a right to easily understood information about the disease and its treatment, and that such information can prevent or lessen physical and emotional complications and facilitate adherence to treatment regimens and adaptation to the cancer experience. Patients and families also have the right to, and need for, support from nurses and other health-care providers in order to use this information to their benefit. Nurses, other health-care professionals, and family members sometimes mistakenly assume that children and adolescents cannot understand illness-related information,20,21 thereby decreasing the likelihood that the young patient will be given this information. However, there is evidence that children (some younger than 5 years) with cancer develop an advanced understanding of the concepts of illness, suffering, and dying.2,22,23 In this regard, Kamps et al.23 found that parents of childhood cancer survivors would prefer that their child be present to hear treatmentand disease-related information, should the cancer recur. These parents also indicated that their child (even at ages as young as 4 years) should be allowed to decide whether or not to receive further treatment in the event of a recurrence. Essential information about treatment can in some situations be effectively shared by parents of children with leukemia with each other.24 Books that describe treatment and family coping with the cancer experience such as the one by Keene,25 a mother of a child who was treated for leukemia, can be recommended by nurses to parents who prefer learning from such sources.

Providing a supportive presence Conveying to patients and parents that they are not alone in this experience and that what is happening to them matters to others is an important nursing strategy. A supportive presence includes the ability to listen nonjudgmentally to expressions of distress, anger, fear, confusion, and suffering. It also means being able to provide active comfort, to demonstrate sensitivity to the family’s expressed and unexpressed needs, and to involve other knowledgeable persons in efforts to better assess and address these needs. Providing a supportive presence occasionally involves directly cautioning a parent or patient about their stated conclusions, impressions, or planned actions that may be based on incomplete or misinterpreted information and that could result in negative outcomes, such as ignoring a treatment guideline designed to prevent infection.

Nursing humor and honesty have been described by adolescents with leukemia as especially helpful in facilitating their efforts to be hopeful about their treatment outcomes. These patients have indicated that hope is important during all phases of care, including the end of life.26 Parents have reported finding much support from the opportunity to share their feelings with health-care providers, in the honesty of health care providers, and from health-care providers acknowledging the difficulties of having a child in treatment for leukemia.27 Supporting patient hopes is a strategy that can be taught to all levels of nurse providers.

Monitoring and anticipating This strategy is designed to prevent or rapidly identify complications of the disease and its treatment. To do this effectively, nurses must be watchful and must know the patient’s most vulnerable times, such as the expected nadirs in neutrophil counts. Nursing assessments can be planned carefully to determine the presence or absence of complications at times of predicted vulnerability. However, some complications are not predictable (e.g. the presence, intensity, and location of pain) and thus require routine nursing monitoring.24,28 The monitoring–anticipating strategy can be effectively combined with patient and family education when the nurse teaches patients and families how and (if possible) when to watch for specific symptoms.

Technical competence The ability to skillfully initiate and complete care procedures, whether they are part of planned care or emergency care, is a prerequisite of quality care. The nurse’s ability to convey this competence to patients, families, and coworkers is essential to ensuring that all participate actively in the care process, thereby diminishing the likelihood of adverse events.5 In fact, the parents of a childhood cancer patient wrote that “competency commensurate to one’s position” is an essential requirement for all health-care workers in pediatric oncology.29

Advocacy The final nursing strategy, advocacy, involves providing patients and families with accurate information, in a sensitive manner, so that they will know how to negotiate within a system of health care to meet their care needs. Advocacy can require speaking on behalf of the patient and family when they are unable to serve as their own advocates, or providing sufficient support to enable them to make their desires known.30 This strategy is based in part on the principle of

Table 36.1 Topics for inclusion in patient and family education efforts, by phase of care Phase of care

Content area

Diagnosis/induction

Consolidation/ intensification

Continuation chemotherapy

Bone marrow transplant

Disease process

Diagnosis defined, causes, incidence

Bone marrow functioning, response to treatment

Diagnostic procedures

BMA, LP Venipuncture Laboratory tests X-rays Sedation/conscious sedation

Treatment protocol

Meaning of Roadmap protocol/schema Informed consent

Treatment devices/ modalities

Surgery Antineoplastic agents Intrathecal therapy Venous access devices

Radiation therapy Antibiotics

Review of devices

Total-body irradiation High-dose chemotherapy

Symptom management

Pheresis fever Blood counts Neutropenia Infection prevention Pain control Blood products Alopecia

Nutrition Anorexia Constipation Diarrhea Nausea/vomiting Mucositis Skin care Pain control Fatigue

Rehabilitation

Dry mouth Pain Graft versus host disease

Psychosocial issues

Hopefulness Employment (parental)

Loneliness Impact on siblings Coping strategies Reaction of peers

Establishing routines Family relationships Sexuality Late effects Body image

Treatment setting/staff

Introduction of staff/roles Telephone contacts Maps Routines of care

Support groups Educational and supportive resources

Isolation Hopefulness Anxiety Irregular school attendance Introduction of staff/roles

Ablative therapy, engraftment of donor marrow Periodic BMA/ LP

Recurrence

Terminal care

Follow-up

Diagnosis defined, prognosis

Progressive disease symptoms described

Roadmap/schema Clinical trials/ experimental agents Best clinical management Informed consent Biologic agents

Palliative care

Height/weight History (e.g. school or social problems)

Pain control

Pain control Nutrition Anorexia Elimination Skin care Breathing changes Parental fatigue

Fertility Growth/development Insurability School success Late effects Continued access to staff

Monitoring Preparing Fears Grieving

Decision making Do not resuscitate status Grieving Emotional withdrawal

Physical exams Diagnostic imaging Laboratory tests

Type of transplant defined Preparative regimen Informed consent

Abbreviations: BMA, bone marrow aspiration; LP, lumbar puncture.

Completion of therapy

Health promotion Well-child care

Infusion devices

Introduction of hospice/home care staff and roles Telephone contacts Continued access to hospital staff

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Table 36.2 Strategies to help patients and families prevent and detect infection Internal environment (patient) Wash hands after bowel or bladder elimination Don’t share eating utensils or cups Avoid activities that can compromise skin integrity (i.e. shaving, rough play, sharp toys) Menstruating female patients: use sanitary napkins only Maintain adequate diet and fluid intake to avoid constipation Maintain personal hygiene (e.g. daily bath, shower, oral care, perineal care) Immediate environment Avoid activities that can compromise skin integrity (i.e., diaper pins, sharp toys) Avoid people with recent or current infections Inspect skin and mucous membranes for signs of infection Encourage adequate sleep and rest Cover bone marrow and lumbar puncture sites for 24 hours Know who and when to call about elevated temperature or signs of infection Teach parents (and patient, if possible) how to calculate the absolute neutrophil count Institutional environment Remind friends/classmates to cover mouth when sneezing or coughing Remind friends/classmates of importance of handwashing Provide literature that describes the risk of infection for patients with leukemia

respect for the autonomy of the child or adolescent with leukemia, and for his or her family and their ability to make informed choices when they have adequate information and assistance.

Phases of care Seven distinct phases have been identified in the care of children with ALL. Not all patients with ALL or with other types of leukemia will experience each of these phases, some of which are dependent on treatment response or risk features at the time of diagnosis, but each merits a brief discussion.

Diagnosis/remission induction The initial examination and laboratory analysis will include bone marrow aspiration or biopsy and lumbar puncture for diagnostic purposes. While this first set of procedures may be completed under emergency conditions, the nurse should ensure that the procedures are thoroughly

explained to the parent and patient and that adequate time is available to review the reasons for and the steps involved in these procedures. Beginning with confirmation of the suspected diagnosis, remission induction is a particularly stressful period. Treatment is initiated with a sense of urgency. The first objective is to reduce the leukemic cell burden as rapidly as possible without placing the patient at risk of severe complications.31 The nurse contributes to this objective through skilled administration of chemotherapy, use of supportive and technical approaches to manage symptoms, and coordination of the necessary clinical and related services. Infection is one of most common causes of morbidity and mortality during induction therapy,32 so that its prevention is a primary nursing objective. Nursing care strategies to prevent infection are especially important during periods of neutropenia, with patient and family education affording an effective means to achieve this end. These efforts focus on providing clear, specific guidelines for avoiding situations that could place the patient at risk, and on prompt reporting of symptoms. The parent and when possible, the patient need to be instructed by the nurse on how to calculate the absolute neutrophil count (ANC). The nurse needs to emphasize that the risk of infection is greater when the ANC is less than 500/mL. Table 36.2 presents examples of nursing strategies to prevent, monitor, and report symptoms of infection, within the environmental model of care. One particular strategy – teaching family members and, when possible, patients how to monitor body temperature – is an example that combines the five nursing care strategies. How to take a temperature is taught in the early phase of care, as temperature is a critical indicator of a patient’s response to infection. Learning the meaning of temperature as a symptom and how to safely and accurately monitor temperature represents a concrete way in which families can protect the patient. Thus, parents (and some patients) practice taking a temperature with the nurse and are expected to demonstrate a clear understanding of the temperature that represents fever and the steps that they must then take. Information on monitoring body temp is also provided in written form, which includes both pictorial and verbal descriptions (nursing care strategy of patient and family education). Family members are assured that the treating staff will respond promptly and competently and that the symptom will be taken seriously (nursing care strategy of supportive presence). Family members are reminded of critical times when monitoring temperature will be particularly necessary and are encouraged to learn the signs and symptoms or behaviors that their child has when experiencing a change in body temperature (nursing care strategy of monitoring and anticipating). Patients who

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are admitted to the care setting with fever are considered a priority admission, and the nurse moves quickly to initiate venous access and obtain blood cultures while simultaneously soliciting a detailed history of the preceding days and events and the pattern of temperature (nursing care strategy of technical competence). In addition, the nurse helps the family or patient secure any additional information needed from the treating team to interpret the fever episode (nursing care strategy of advocacy). Expert nursing care, combined with appropriate infection control measures, decreases the need for hospitalization and facilitates outpatient therapy. Surgical placement of venous access devices during remission induction therapy is more common now than in the past. Technical competence during this phase of therapy includes skilled manipulation of the device. Treatment requires repeated entry into these devices to infuse antibiotics, antineoplastic agents, antifungal agents, hydrating fluids or blood products, and to collect blood samples. If not performed carefully, each of these procedures can place the patient at risk for infection, hemorrhage, or embolus.12 The nurse’s ability to enter the device accurately and collect a necessary blood sample can lower the total number of entries required as well as the likelihood of complications. Our studies have shown that when nurses carefully follow the institutional policy for aseptic handling of venous access devices, entering the device does not represent a source of infection.33 We also found that a 3-mL discard volume is sufficient to yield accurate values for potassium, sodium, ionized calcium, glucose, hematocrit, and hemoglobin concentrations in blood obtained through a tunneled venous access device (P. Hinds, J. Gattuso, L. Walters, and L. Oakes, unpublished data). Occasionally, some patients will benefit from placement of a peripherally inserted central catheter (PICC), for example, if their venous access device is not functioning competently or if additional access is needed. A PICC will likely be a new device and experience for the patient and family, who have become familiar and comfortable with the venous access device and therefore will need a careful explanation of the construction, function, and placement of the line as well as an overview of potential complications and how to evaluate them. Practice time in flushing the line will be needed before the family feels confident about performing this task. Families and patients are urged to alert staff to any signs of potential difficulties with the PICC and are instructed about the actions they need to take should they suspect anything unusual (monitoring and anticipating). The nurse can skillfully insert and tape the PICC, assess its function for possible complications such as infection,

phlebitis, or clotting, and remove the PICC (technical competency). The nurse also provides verbal and written information to the family about the PICC in case their child is treated elsewhere during part of the care (advocacy). Finally, the nurse responds attentively to any anxiety the family or patient may express or convey about the PICC and offers to be available to repeat the information or demonstrations (supportive presence). Another nursing care objective during this initial phase is helping the patient and family adapt to the diagnosis of leukemia, to the planned treatment, and to the resulting alterations in lifestyle. Strategies that contribute to this goal include being available to respond to questions or offer information, assessing the patient’s and family’s responses to the information provided and to treatmentrelated experiences, clarifying and reinforcing previously provided information, providing additional written material, initiating referrals to other health-care workers for necessary supportive care, and offering to introduce the patient and parents to others who are or have been in similar situations. A useful strategy is to collect key treatment descriptions, patient-care materials, and educational handouts in a notebook to which the patient and family can refer throughout treatment. Table 36.3 lists the contents of a notebook that is used at our facility.

Consolidation/intensification Many treatment protocols for leukemia specify a consolidation/intensification phase, which begins immediately after remission is achieved. The primary goal is to decrease or eliminate minimal residual disease in all sites.31 Therapy during this phase is very intensive, resulting in neutropenia, fatigue, nausea and vomiting, hair loss, and other physical changes, including weight gain or loss. Also, changes in life schedule (inability to attend school or participate in other age-related activities) become quite noticeable to patients and can produce a profound sense of loneliness and of being different from friends and peers. Pain-related functional problems can also occur.34 A common cause of pain is mucositis, which can range from a limited irritation of the oral mucosa (Grade I) to severe ulceration, severe erythema, and bleeding that preclude swallowing, eating, or drinking (Grade III). Table 36.4 presents specific examples of preventive and corrective care actions, within the three-level environmental model. An additional example that combines the five nursing care strategies is preparing the patient and family for bone marrow aspiration and biopsy. Although the patient will usually have had both an aspiration and biopsy for

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Table 36.3 Contents of a patient and parent handbook 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24.

Patients bill of rights and responsibilities Support and services What is a protocol? Preventing infection Bacterial infection Opportunistic infection Viral infection HIV infection Glossary of terms Diagnosis Treatments Protocols How does a protocol affect treatment? Temperature conversion/taking your child’s temperature Activity and rest Diet and nutrition Immunization Medicines Exposure to sun Family relationships Hope Side effects of cancer treatment Digestive system side effects Mouth sores Diarrhea Constipation Bone marrow side effects Blood counts during therapy Red blood cells Platelets Pain Pain mission statement Body image changes Hair loss Excessive weight gain or loss Skin changes Other side effects of treatment Long-term effects of treatment

diagnostic purposes, they may have been done under emergency conditions. Additional nursing time is needed during this phase of care to review the reasons for these procedures, to explain the position the patient’s body will need to be in for the procedures (patient and family education), and to solicit patient preferences for handling procedurerelated pain. Written materials are also provided. The nurse develops a written profile regarding the patient’s preferences for the procedure so that others on the treating team may similarly practice and respect the patient’s preferences (advocacy). The nurse periodically updates the profile and confirms the preferences with the patient at the time of each

procedure. The nurse conveys an appreciation of the difficulties of the procedures and a willingness to be close by or to arrange for others preferred by the patient to be close by (supportive presence). When patients choose to have the procedures without sedation or with conscious sedation, the nurse emphasizes the need to remain in a certain physical position in order to avoid repeated needle insertions. Throughout the procedure, if the patient prefers, the nurse can explain what is about to happen and what sensations the patient may feel (monitoring and anticipation). Following the procedure, the nurse positions the patient for comfort and safety and monitors recovery to full alertness (technical competency). A treatment-related symptom commonly experienced during this phase of treatment is fatigue, described by children as a physical heaviness that makes participating in usual daily activities impossible.13 Adolescents describe it as an emotional, social, cognitive and physical experience that includes loss of energy, lowered ability to concentrate, and difficulty responding to social overtures from others.15 Fatigue intensifies towards the end of treatment. The nursing role includes asking patients about their tiredness, describing fatigue to the patient and family and urging them to monitor it for patterns of occurrence and intensity, and suggesting strategies for decreasing the fatigue. Strategies favored by patients include distraction that does not require their participation, schedules that take advantage of higher points of energy, and having protected rest or sleep periods. Strategies for parents include having information about fatigue to share with other family members or friends of the patient and ideas about planning family events around the patient’s times of higher energy. During this and earlier treatment phases, the need for transfusion of red cells or platelets can be an unsettling experience for the child and parent. Competence in transfusing blood products requires adequate preparation of the patient and family for the procedure, accurate infusion, and monitoring for immediate, short-term, or long-term outcomes. Allergic reactions, fluid volume overload, and electrolyte disturbances must be anticipated.35 Studies at our institution have shown that infusing platelets by electromechanical pump does not damage the quality of the platelets or affect patient outcomes.36 We have also found that infusing platelets at 10 mL/kg per hour – twice the previous standard rate – produced a similar medical outcome and substantially shortened the infusion time.37 This type of research-based improvement in nursing care helps to promote a patient’s and family’s ability to tolerate the treatment by reducing certain demands, such as time spent in the treatment facility for procedures.

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Table 36.4 Strategies to prevent and treat mucositis Internal environment (patient) No mucositis Practice brushing with a soft-bristle toothbrush and fluoride toothpaste Floss after meals and at bedtime Rinse mouth with 5 to 10 mL of Peridex or half-strength Cepacol for 30 seconds in the morning and in the evening Grade I mucositis Rinse mouth with alkaline-saline solution for 30 seconds every 2 hours while awake Clean teeth and gingivae twice daily with a soft brush dipped in Peroxyl, then rinse mouth Eat a bland diet Grade II mucositis Clean teeth and gingivae with soft brush dipped in diluted Peroxyl (1:1 dilution), then rinse with alkaline-saline solution Grade III mucositis Rinse for 30 seconds with Ulcer-Ease to anesthetize the mucosa Clean teeth and gingivae using a soft brush dipped in alkaline-saline solution Report pain intensity Attempt to eat high moisture foods such as jello, apple sauce, canned fruits Moisten lips every 2 to 4 hours if they are dry or chapped If lips are cracked or crusted, clean every 2–4 hours with saline-saturated gauze and then apply lanolin Provide information to patient about recovery period (7–14 days) Immediate environment (nurse) No mucositis Encourage brushing, gentle flossing, and rinsing after meals and at bedtime Explain symptoms to report (e.g. oral burning, red areas in oral cavity, lesions) Grade I Obtain cultures if white patches are present Review food appropriate for a bland diet and foods to avoid Grade II Apply Kanka solution directly to small isolated ulcers Use Ulcer-Ease solution (5–10 mL) for larger or multiple ulcers Encourage frequent rinsing Monitor Peroxyl use; encourage use every other day, or for no more than 3 consecutive days Grade III Encourage gentle mouth care, including frequent rinsing Assess pain intensity on a regular basis Review importance of high-moisture foods Explain expanded recovery period (7–14 days) to family Institutional environment (nurse) Share information on mucositis (grade, duration, pain intensity), and/or effective relief measures with other health-care professionals Develop intervention studies to determine most-effective prevention and treatment measures Coordinate care efforts to ensure adequate nutritional and fluid intake and effective pain management Develop standardized orders/guidelines for assessment and management

Continuation chemotherapy During the continuation phase of treatment, the goals are to maintain control of the leukemia, to prevent the development of drug-resistant cells,31 and to avert or minimize long-term complications. For patients with ALL who continue in complete remission, continuation therapy usually lasts 2.5 to 3 years. During this phase, patients and parents develop a routine of sorts, and a sense of normalcy emerges.

Insofar as possible, treatment is scheduled in accord with school- or other age-related commitments in order to help the patient reintegrate into the community.34 The period of continuation treatment is also a particularly good time to assess the patients’ and parents’ understanding of the vast amount of information about leukemia and its treatment to which they have been exposed. Nurses spend time reviewing information and inviting questions to make certain that the information has been assimilated. Of particular

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Table 36.5 Strategies to prevent, understand, monitor, and report pain Internal environment Teach patients 5 years or older to indicate location, presence, and intensity of pain <5 years or for patients unable to self-report: FLACC Scale by nurse report 5–13 years: Faces Pain Scale ≥14 years: 0–5 scale and verbal instructions to rate pain Voice concerns about pain medications Teach patient to describe perceptions about the causes and meanings of the pain (when age-appropriate) Immediate environment Believe that the patient is the real authority on his or her pain Assess pain regularly and frequently Recognize that pain is emotionally and physically debilitating Seek ways to decrease pain stimuli Administer analgesia (when appropriate) on a regular or continuous basis versus a PRN basis Document pain assessments, including effectiveness of attempted relief measures Address fears and myths about addiction or drug-seeking behavior Institutional environment Address fears and myths about possible addiction or drug-seeking behavior Document the effectiveness of pharmacologic and nonpharmacologic pain measures with different types of pain; share these findings Assist in developing and applying pain relief measures.

importance, they re-emphasize the need to adhere to the oral medication schedule for dexamethasone or prednisolone, prophylactic antibiotics, and 6-mercaptopurine. Careful instructions to patients and their parents regarding the need to avoid dairy products when swallowing and absorbing 6-mercaptopurine are especially important.

body irradiation. During and after this preparatory regimen, multiple agents must be infused and numerous procedures performed. Expert nursing care involves creating a schedule that will facilitate the completion of all of these aspects of treatment. Scheduling issues include decisions about when to infuse blood products, antibiotics, antifungal agents, and pain control medications in relation to other procedures and to respiratory and physical therapy. Technical competence during this phase of care includes the ability to accurately and painlessly complete venous punctures. Our own research indicates that coagulation indicators are more accurately sampled when collected from a venous puncture than from a venous access device.39 Transplant patients often have considerable anxiety about potential treatment complications. Nurses need to anticipate spoken and unspoken fears and be prepared to offer simple, straightforward explanations. Pain is a particular concern. Treatment-related pain can occur during any phase of leukemia therapy but is a particular problem for patients undergoing transplantation. Table 36.5 presents examples of strategies for preventing and addressing pain responses. Prerequisites of successful pain management are a thorough understanding of pain and of both pharmacologic and nonpharmacologic pain relief measures (technical competence); sensitivity to patients’ reports of pain and to behaviors that may indicate the presence of pain (supportive presence); and a commitment to providing the most effective possible pain relief to each patient (advocacy). To do this, the nurse needs a reliable and valid way to measure and monitor pain. Our studies of children with cancer26,40 led to the conclusion that the Faces Pain Scale (FPS) provides a reliable and valid measure of pain in patients 5 to 13 years old.

Completion of therapy/follow-up Bone marrow transplantation Patients whose disease characteristics are associated with a high risk of relapse may undergo allogeneic bone marrow transplantation, rather than continuation treatment, as part of their front-line therapy. Because certain aspects of pediatric bone marrow transplantation are still experimental, and the transplant period is considered to be an acute psychological stressor, transplantation constitutes a distinct phase of care.38 The primary treatment goals are to obliterate any possible remaining disease, reduce the body’s ability to recognize the infused marrow as foreign, and restore hematopoiesis by replacing the ablated marrow with healthy donor marrow. The intensive preparatory regimen comprises high-dose chemotherapy and total-

After treatment is complete, patients require regular evaluation. The primary medical focus is monitoring for disease recurrence and for complications related to leukemia or its treatment. By contrast, the primary nursing focus is to promote the practice of healthy behaviors and lifestyles. Although patients and families are relieved to have reached this point, they may also be uneasy, having come to view treatment as necessary to prevent the return of leukemia. Also, some patients and families develop an emotional dependency on the treatment setting.35,41 Supportive nursing strategies include explaining the likelihood of family members’ experiencing ambivalent feelings about completing treatment, and reminding the patient and family members that they may contact the treating facility at any

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time in the future with any kind of concern, and reassuring them that they will not be forgotten by the staff. To assist in monitoring for recurrence or late effects, nurses must carefully document the patient’s weight and height at each follow-up visit, and can also contribute by taking a careful history regarding intellectual development and academic performance.12 In addition, the nurse can anticipate patients’ unspoken fears regarding recurrence, and should be prepared to discuss these fears. The major nursing focus during follow-up care is to help patients and families concentrate on well-being and on health-promoting behaviors. These include diet, exercise, self-examination of breasts or testicles, use of sunscreens, and the avoidance of smoking and other high-risk behaviors. In an ongoing study at our institution, the majority of long-term survivors returning for their annual evaluation indicated a “need to improve” their health behaviors and had chosen to take an active approach, by selecting a health promotion goal to concentrate on during the next year. Members of the health-care team participate in this process by discussing the chosen goal, demonstrating the behavior, when appropriate, and checking on the patient’s progress by telephone 3 and 6 months after the annual examination. Our preliminary impressions are that adolescent survivors participate actively in this health promotion approach.42

Recurrence of leukemia Relapse from a state of remission can occur during continuation therapy or after the completion of therapy.43 The treatment for relapse depends upon the timing of the recurrence, the involved sites, and the classification of the leukemia. However, most patients and family members will not be familiar with the terms used to describe relapsed disease and will benefit from additional time with members of the treating team, including the nurse, learning the meaning of these terms. There have been extensive biological studies of cancer recurrence,44–46 but only limited research on the impact of the recurrence on pediatric patients and families.47,48 Treatment of relapsed disease typically involves drugs, terminology, and approaches that are unfamiliar to patients and families (e.g. those used in Phase I and Phase II studies). For these reasons, and because of the emotional distress associated with the return of disease, nurses must give particularly careful attention to all aspects of the informed consent process and patient’s and parent’s understanding of the process. In a recent study, St. Jude researchers identified the behaviors of nurses and other health-care providers that

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Table 36.6 Strategies to help patients and families with the dying process Internal environment Determine the patient’s preferences regarding pain control (when age-appropriate) and management of other symptoms Listen attentively to patient’s concerns, expressed fears, feelings Provide skilled administration of pain control medications, blood products, oxygen, or other treatments Teach family how to turn and position their child for comfort (age-appropriate) Immediate environment Determine the family’s views on pain control Advocate for effective pain control Share information on developmental stages and understanding of death Share information on the physiologic process of dying Prepare family for changes in patient’s appetite, fatigue level, and breathing Promote fastidious hygiene to minimize odors secondary to breakdown products Listen attentively to family member concerns, feelings, and fears Determine family’s preferences regarding inclusion of patient in end-of-life discussions Convey respect for family’s wishes Consider referral to hospice care if patient and family prefer Institutional environment Communicate significant clinical changes to members of health-care team Coordinate final care preferences, such as a “Do Not Resuscitate” letter for patients receiving home care Encourage health-care team members to contact family before and after the patient’s death Advocate for federal and other organizations to fund research related to end of life

either facilitated or impeded the parents’ ability to cope with a first recurrence of their child’s cancer.47,48 Nursing behaviors that facilitated the process included sharing information with parents (type, detail, and frequency of information provided at the level requested by parents), providing hope, demonstrating technical competence, and responding to the parents’ questioning of nursing care processes, actions, or judgments. Conveying support for the parents’ previous treatment-related decisions was considered a helpful nursing behavior whereas questioning previous and current decisions about treatment was viewed as an obstacle to parental coping.

Terminal care Terminal care issues are critically important for patients whose leukemia cannot be cured with available therapies. This phase is defined as the period when the patient’s death

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is imminent or likely to occur within 2 to 6 weeks. The primary nursing care goals are to help keep the child or adolescent physically comfortable and to help prepare the family members for the process (see Table 36.6). The nursing strategy is to describe honestly to the family the changes that are most likely to occur as the patient’s death nears. These changes may include a loss of appetite and an inability to eat or swallow food, constipation or other changes in elimination such as urine retention, decreased interaction with others and an apparent emotional withdrawal (even from close friends and relatives), respiratory changes, pain or bleeding.49–51 The nursing role may also include discussion with patients or family members about fears, desires, and impressions of what constitutes a peaceful or “good” death. In some instances, it may also be possible to discuss the emotional, physical, and behavioral responses to grief that family members may experience as the child’s death approaches, and after the death.11,52,53 Finally, the nurse can reassure the patient and family that they will not be abandoned during this phase of care.8

Conclusions Each time a nurse prepares to give care to a child or adolescent with leukemia, he or she must consider the care needs of the individual patient (the internal environment), the family members and members of the health care team (the immediate environment), and the larger community (the institutional environment). Nursing actions directed toward one level of environment will simultaneously or ultimately affect the other two levels. The nurse should carefully consider these potential effects when planning care. In addition, the nurse must plan for the particular needs at each of these levels in terms of phase of care (diagnosis/remission induction, intensification/consolidation, continuation, bone marrow transplantation, completion/follow-up, recurrence of leukemia, and terminal care). Certain care needs are common to most or all of these phases, but care approaches should be tailored to better match the most pressing concerns of each phase of care and each level of environment. The nurse can select one care strategy or a combination of strategies (e.g. patient and family education and serving as an advocate) to meet the needs of each level of environment and then the same or different strategies can be used to reinforce the initial care efforts. Finally, toward the shared goal of providing technically competent and sensitive nursing care, a concentrated research effort is needed to better define optimal nursing actions and procedures during each phase of care.

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20 Koocher, G. & O’Malley, J. The Damocles Syndrome: Psychosocial Consequences of Surviving Childhood Cancer (New York: McGraw-Hill, 1981). 21 Mann, J. R. Psychosocial aspects of leukemia and other cancers during childhood. In N. K. Aaronson & J. H. Beckmann, eds., The Quality of Life of Cancer Patients (New York: Raven Press, 1987), pp. 135–40. 22 Hinds, P. & Martin, J. Hopefulness and the self-sustaining process in adolescents with cancer. Nurs Res, 1988; 37: 336–40. 23 Kamps, W., Akkerboon, J., Kungma, A., et al. Experimental chemotherapy in children with cancer – a parents’ view. Pediatr Hematol Oncol, 1987; 4: 117–24. 24 Rahman, S. Leukemia. In Hockenberry-Eaton, M., ed., Essentials of Pediatric Oncology Nursing: A Core Curriculum (Glenview, IL: APON, 1998), pp. 15–20. 25 Keene, N. Childhood Leukemia: A Guide for Families, Friends and Caregivers (Sebastopol, CA: O’Reilly & Associates, Inc, 2002). 26 Hinds, P. & Martin, J. Hopefulness and the self-sustaining process in adolescents with cancer. Nurs Res, 1988; 37: 336–40. 27 McGrath, P. Beginning treatment for childhood acute lymphoblastic leukemia: insights from the parents’ perspective. Oncol Nurs Forum, 2002: 29: 988–96. 28 West, N., Oakes, L., Hinds, P., et al. Measuring pain in pediatric oncology ICU patients. J Ped Oncol Nurs, 1994; 11: 64–8. 29 Peterson, D. & Peterson, G. The new challenge in pediatric cancer care. In J. Van Eys, ed., The Truly Cured Child (Baltimore, MD: University Press, 1977), pp. 101–5. 30 Curley, M. The essence of pediatric critical care nursing. In M. A. Curley, J. B. Smith, & P. A. Moloney-Harmon, eds., Critical Care Nursing of Infants and Children, (Philadelphia, PA: W. B. Saunders, 1996), pp. 3–14. 31 Westlake, S. K. & Bertolone, K. L. Acute lymphoblastic leukemia. In C. R. Baggott, K. P. Kelly, D. Fochtman, & G. V. Foley, eds., Nursing Care of Children and Adolescents with Cancer, 3rd edn. (Philadelphia, PA: W. B. Saunders, 2002), pp. 466–90. 32 Ablin, A. R. Supportive Care of Children with Cancer: Current Therapy and Guidelines from the Children’s Cancer Group (Baltimore, MD: The Johns Hopkins University Press, 1993). 33 Hinds, P., Wentz, T., Hughes, W., et al. An investigation of the safety of the blood reinfusion step used with tunneled venous access devices in children with cancer. J Ped Oncol Nurs, 1991; 8: 159–64. 34 Spitzer, A. Primary nursing in childhood cancer as applied in Israel. Cancer Nurs, 1985; 8: 89–95. 35 Hockenberry, M. J. & Coody, D. K., eds., Pediatric Oncology and Hematology: Perspectives on Care (St. Louis, MO: Mosby, 1986). 36 Norville, R., Hinds, P., Wilimas, J., et al. The effects of infusion methods on platelet count, morphology, and corrected count increment in children with cancer: in vitro and in vivo studies. Oncol Nurs Forum, 1994; 21: 1669–73.

37 Norville, R., Hinds, P., Wilimas, J., et al. The effects of infusion rates on platelet outcomes and patient responses in children with cancer. Oncol Nurs Forum, 1997; 24: 179–93. 38 Nespoli, L., Verri, A. P., Locatelli, F., et al. The impact of paediatric bone marrow transplantation on quality of life. Qual Life Res, 1995; 4: 233–40. 39 Hinds, P., Quargnenti, A., Gattuso, J., et al. Comparing the results of coagulation tests on blood drawn by venipuncture and through tunneled venous access devices in pediatric patients with cancer. Oncol Nurs Forum, 2002; 29: E1–9. 40 Oakes, L., Hinds, P., Bhaskar, R., et al. Chest tube stripping in pediatric oncology patients: an experimental study. Am Crit Care, 1993; 2: 293–301. 41 Hockenberry, M. J. Crisis points in cancer. In M. J. Hockenberry & D. K. Coody, eds., Pediatric Oncology and Hematology: Perspectives on Care, (St. Louis, MO: Mosby, 1986), pp. 432–49. 42 Hudson, M. M., Tyc, V. L., Srivastava, D. K., et al. Multicomponent behavioral intervention to promote health protective behaviors in childhood cancer survivors. Med Pediatr Oncol, 2002; 39: 2–11. 43 National Cancer Institute. When Cancer Recurs: Meeting the Challenge Again, NIH Publication 90–2709. (Bethesda, MD: National Cancer Institute, 1990). 44 Green, D. M. The diagnosis and management of Wilms tumor. Pediatr Clin North Am, 1985; 32: 735–54. 45 Grier, H. E. & Weinstein, H. J.. Acute nonlymphocytic leukemia. Pediatr Clin North Am, 1985; 32: 653–67. 46 Poplack, D. G. Acute lymphoblastic leukemia in childhood. Pediatr Clin North Am, 1985; 32: 669–97. 47 Hinds, P., Birenbaum, L., Clarke-Steffen, L., et al. Coming to terms: parent’s response to a first cancer recurrence in their child. Nurs Res, 1996; 45: 148–53. 48 Hinds, P., Birenbaum, L., Pedrosa, A., & Pedrosa, M. Guidelines for the recurrence of pediatric cancer. Sem Oncol Nurs, 2002; 18 50–9. 49 49 Coyle, N. & Layman-Goldstein, M. Pain assessment and management in palliative care. In M. L. Matzo & D. L. Sherman, eds., Palliative Care Nursing: Quality Care to the End of Life (New York: Springer, 2001), pp. 362–486. 50 Wolfe, J., Grier, H. E., Klar, N., et al. Symptoms and suffering at the end of life in children with cancer. N Eng J Med, 2000; 342: 326–33. 51 Kane, J. R. & Himelstein, B. P. Palliative care in pediatrics. In A. M. Berger, R. K. Portenoy, & D. E. Weissman, eds., Principles and Practice of Palliative Care and Supportive Oncology, 2nd edn. (Philadelphia, PA: Lippincott, Williams & Wilkins, 2002), pp. 1044–59. 52 Bradlyn, A. S. & Patterson, K. Psychosocial management in pediatric oncology. In A. K. Ritchey, ed., Supportive Care Manual (St. Louis, MO: Pediatric Oncology Group, 1996). 53 Field, M. J. & Behrman, R. E., eds., When Children Die: Improving Palliative and End-of-life Care for Children and their Families, (Washington, DC: National Academies Press, 2003).

893

Index

A20 gene, 132 AAVs see adeno-associated viruses (AAVs) ABC (ATP-binding cassette) transporters, 523 ABCG2 gene, expression, 83 Abelson murine leukemia virus, 8 ABL gene, 8, 582, 681 activation, 272–4 deficiency, 274 ABL protein, tyrosine kinase activity, 273 Absidia spp., infections, 819 accessory proteins, 419–20 and cellular drug resistance, 419–20 acetaminophen, 851 acid phosphatase staining, 29 ACIP (Advisory Committee on Immunization Practices) (US), 821 acquired immunodeficiency syndrome (AIDS) see HIV/AIDS ACTH (adrenocorticotrophic hormone), 9 activins, 129 mediation, 129 acute, use of term, 21 acute bilineal leukemias see bilineal acute leukemias acute complications, 709–35 early, 709, 712–27 coagulopathies, 721–2 hyperviscosity syndrome, 725 incidence, 709 intracerebral myeloblastoma, 726–7 leukostasis syndrome, 722–5 mediastinal structural compression, 719–21 metabolic abnormalities, 712–19 neurologic, 725–6 spinal cord compression, 726 types of, 709 incidence, 710 late, 709 on-therapy, 727–35 endocrinal, 733–5 gastrointestinal, 728–30

894

Index

incidence, 709 neurologic, 730–2 osteonecrosis, 732–3 thrombosis, 727–8 types of, 709 risk factors, 709–12 leukemic, 711–12 patient, 709–11 treatment, 712 types of, 709 acute erythroblastic leukemia, 28, 176–7 acute granulocytic leukemia see acute myeloid leukemia (AML) acute leukemias with aberrant antigen expression, 179–83 advanced, 644–5 of ambiguous lineage, 36–7 aminopterin treatment, 8–9 B-precursor cell, 5 CD19 expression, 645 CD45 expression, 644–5 chemotherapy, 12 classification, 21, 29–39 future trends, 39–40 colony-forming cells in, 81 cytogenetics, 235–61 definition, 183 in developing countries, 625–36 treatment, 632–6 diagnosis, 23 future trends, 39–40 epidemiology, 48–59 etiology, 48–59 genetic factors, 50–1 immunophenotyping, 162 incidence, 48–9 management, 235 neuropsychological consequences, 14 risk factors, 6, 52, 363 and skeletal abnormalities, 759–60 subclassification, 4 subtypes, 24 total care, 14 undifferentiated, 37 see also bilineal acute leukemias acute lymphatic leukemia see acute lymphoblastic leukemia (ALL) acute lymphoblastic leukemia (ALL), 3–4, 7, 29–32, 439–61 abnormalities immunophenotype-specific, 248–51 with no immunophenotype association, 238–48 adrenocorticotrophic hormone treatment, 9 AF4-MLL fusion transcript, 32 age peaks, 57 and alcohol consumption, 55 antigen receptor gene rearrangements, 691–4 and aplastic marrows, 172

assessment, RT-PCR assays, 236 BCR-ABL fusion transcript, 32 and birth weight, 55–6 B-lineage, 688 and cancer risk, 362, 363 classification, 166–9 cytogenetics, 249 and Down syndrome, 365 IG/TCR gene rearrangements, 222–4 incidence, 223 oligoclonality, 223–4 see also B-cell acute lymphoblastic leukemia (B-ALL) cellular drug resistance, 415–16 genetic abnormalities, 417 chemotherapy, 10, 391, 639 chromosomal abnormalities numeric, 238–41 structural, 241–8 classification, 238 future trends, 39–40 genetic, 447–9 immunophenotypic, 31, 445–6 molecular (genetic), 31–2 morphologic, 29–30 clinical presentation, 441–3 clinical trials, 453 coagulopathies, 721 colony-forming cells, 81 cranial irradiation studies, 866 cure rates, 750 cures, 9 phases, 10 cyclophosphamide treatment, 403 cytogenetics, 238–51 cytoplasmic granules, 26 in developing countries, 632–4 diagnosis, 23, 28, 445–6 future trends, 39–40 issues, 150 diploidy, 240 and Down syndrome therapeutic outcomes, 366–7 treatment, 460–1 drug cytotoxicity assays, 414 E2A-PBX1 fusion transcript, 32 etiology, 272, 439 flow cytometry, 694–6 and folic acid deficiency, 7 and genetic syndromes, 50–1 glucocorticoid treatment, 402 granular, 30 hematopoietic stem cell transplantation, 459–60, 603–6 high-hyperlipoid, 31 high-risk, 238 and HLA-DR, 58

895

896

Index

acute lymphoblastic leukemia (ALL) (cont.) hyperdiploidy >50 chromosomes, 239–40 47–50 chromosomes, 240 hyperlipoid, 417 hypodiploidy, 31, 240–1 and hypoplastic marrows, 172 immunophenotyping, 160–2 incidence, 48–9 age-specific, 48 ethnic and racial differences, 48–9 geographic differences, 49 rates, 49 sex differences, 48 temporal trends, 49–50 intensification therapy, 456–7 L1, 25 L2, 25 L3, 25, 29–30 diagnosis, 30 laboratory findings, 443–5 lymphoblast origins, 4 mature B, 168–9 minimal residual disease assessment, 690–6 continuous monitoring, 693 detection, 694, 695 markers, 687–8 prognosis, 694–6 molecular genetic abnormalities, 690–1 molecular genetics, 272–85 clinical implications, 284–5 future trends, 285 molecular lesions, 284 mortality rates, treatment-related, 709 near-haploidy, 240 near-tetraploidy, 238–9 near-triploidy, 238–9 pathobiology, 439–40 pathophysiology, 439–40 theories, 439 Philadelphia chromosome in, 242–3, 603, 605 ploidy, 31 postnatal events, 440 prevalence, 21 prognosis, 439 prognostic factors, 449–51 proto-oncogene activation, 272 pseudodiploidy, 240 recurrent genetic changes, 241 reinduction therapy, 456–7 relapse, 449 remission induction therapy, 452–6 remission rates, 392, 455 risk classification schemes, 284, 450

risk factors, 440 risk groups, 449–51 subtypes, 10, 31, 446 characteristics, 448 ethnic and racial differences, 441, 447 supportive care, 451–2 survival rates, 473, 474, 603, 629 symptoms, 441–3 TEL-AML1 fusion transcript, 32 and thrombosis incidence, 727 risk factors, 727 symptoms, 727–8 treatment, 728 T-lineage, 169, 687–8 classification, 169, 224 cytogenetics, 251 IG/TCR gene rearrangements, 224 incidence, 222, 223 treatment outcomes, 456 see also T-cell acute lymphoblastic leukemia (T-ALL) translocations, 241, 272, 447 treatment CNS-directed therapy, 457–8 continuation, 458–9, 633 effects on educational attainment, 867–8 failure, 629, 630 future trends, 461 issues, 460–1 and obesity, 754–5 pharmacological effects, 450 principles, 452 protocols, 632–4 and relapse risk, 238 risk classification, 450, 452 total therapy approach, 451–60 twin studies, 439–40, 694 xenotransplantation studies, 76–7 see also B-cell acute lymphoblastic leukemia (B-ALL); common acute lymphoblastic leukemia (cALL); infant acute lymphoblastic leukemia; lymphoid antigen-positive acute lymphoblastic leukemia (Ly+ ALL); myeloid antigen-positive acute lymphoblastic leukemia (My+ ALL); relapsed acute lymphoblastic leukemia; T-cell acute lymphoblastic leukemia (T-ALL) acute lymphocytic leukemia see acute lymphoblastic leukemia (ALL) acute lymphoid leukemia see acute lymphoblastic leukemia (ALL) acute megakaryoblastic leukemia (AML M7), 5, 28, 48, 183, 253, 254 classification, 507–8 diagnosis, 35, 160, 172–3, 507 differential diagnosis, 556 and Down syndrome, 186, 366, 417–18, 499 therapeutic outcomes, 367 extramedullary involvement, 508

Index

immunophenotyping, 177–8 incidence, 507 laboratory findings, 507–8 molecular genetics, 323–5 pathogenesis, 324 prognosis, 522 subtypes, 28, 323–4 acute megakaryocytic leukemia see acute megakaryoblastic leukemia (AML M7) acute monoblastic/monocytic leukemia (AML M5), 3, 27, 48 cellular drug resistance, 417 complications, 712 diagnosis, 34 immunophenotyping, 175–6 and leukostasis syndrome, 724–5 acute monocytic leukemia see acute monoblastic/monocytic leukemia (AML M5) acute myeloblastic leukemia see acute monoblastic/monocytic leukemia (AML M5) acute myelocytic leukemia see acute myeloid leukemia (AML) acute myelogenous leukemia see acute myeloid leukemia (AML) acute myeloid leukemia (AML), 3–4, 499–526 adult vs. pediatric, 506 advanced, 644 and alcohol consumption, 55 allogeneic stem cell transplantation, 606–7 AML1-ETO fusion transcript, 35 assessment, RT-PCR assays, 236–7 benzene-induced, 7 and birth weight, 55–6 blast counts, 553 CBFB-MYH11 fusion transcript, 35 CD33 expression, 642–4 cellular drug resistance, 416 and central nervous system leukemia, 518–19 central nervous system treatment, 518–21 chemotherapy, 639 chromosomal abnormalities, 56 future research, 299 monosomy 7, 253 numeric, 253, 254 structural, 254–60 trisomy 8, 253 trisomy 21, 253–4 classification, 32–8, 48, 298, 502–3, 506–13 future trends, 39–40 molecular, 298–9 clinical presentation, 501–5 clinical trials, 514 coagulopathies, 721, 722 colony-forming cells, 81 common genetic lesions, 260 comorbidity, 503 complications, 524–5 and core-binding factor complex, 307–15

cure rates, 750 cytogenetics, 251–60 definition, 499 de novo, 298 in developing countries, 635–6 diagnosis, 23, 150, 501–5 algorithms, 33 differential, 30, 504–5 future trends, 39–40 diagnostic criteria, 502–3 and Down syndrome, 365, 508–10 therapeutic outcomes, 367 epidemiology, 499–500 etiology, 56–7, 299, 499–500 and familial myeloid disorders with monosomy 7, 375 flow cytometry, 697 FLT3 gene, internal tandem duplications, 681–2 Flt3 receptor mutations, 108 functional assays, 73 future trends, 499, 525–6 and genetic syndromes, 50–1 hematopoietic stem cell transplantation, 518, 606–7 IG/TCR gene rearrangements, 224 immunophenotyping, 160–2, 172–9 incidence, 48–9, 499–500 age factors, 500 ethnic and racial differences, 499 geographic differences, 625 rates, 49, 50 temporal trends, 49–50 induction therapy, 513 intensification, 513–17 infants, 506–7 inversions, 299 laboratory findings, 503–4 long-term culture-initiating cell assay studies, 78–9 minimal granulocytic differentiation, 26 minimal residual disease assessment, 688–9, 696–7 detection issues, 688 MLL gene abnormalities, 316–23 transformation mechanisms, 322–3 MLL gene-induced, genetic models, 322 MLL gene rearrangements, 36 molecular genetic abnormalities, 696–7 molecular genetic lesions, rare, 325 molecular genetics, 298–326 future trends, 325–6 molecular pathogenesis, 298–9 mortality rates, treatment-related, 709 murine studies, 275, 305 and myeloid tumors, 510 myeloperoxidase positivity, 26 pathogenesis, 500–1 pharmacogenomics, 521

897

898

Index

acute myeloid leukemia (AML) (cont.) polymerase chain reaction analysis, 683–4 postremission therapy, 517–18 prevalence, 21 prognostic factors, 521–3 protein alterations, 501 recurrent genetic changes, 252 remission rates, 10–11, 513, 540 risk factors, 499, 500 RUNX1 mutations, 314–15 and severe congenital neutropenia, 373–4 subtypes, 28, 502, 506–13 rare, 512–13 supportive care, 524–5 survival rates, 606, 750 relapse-free, 644 symptoms, 503 translocations, 35, 299 treatment, 513–25 and infection, 524 outcomes, 499 phases, 513 and tumor masses, 712 use of term, 172 vs. myelodysplastic syndrome, 556, 557 xenotransplantation studies, 76–7 young children, 506–7 see also acute megakaryoblastic leukemia (AML M7); acute monoblastic/monocytic leukemia (AML M5); acute myeloid leukemia with maturation (AML M2); acute myeloid leukemia with minimal evidence of myeloid differentiation (AML M0); acute myeloid leukemia with predominant erythroid differentiation (AML M6); acute myeloid leukemia without maturation (AML M1); acute myelomonocytic leukemia (AML M4); acute promyelocytic leukemia (APL); chronic myeloid leukemia (CML); congenital acute myeloid leukemia; familial platelet disorder/acute myelogenous leukemia (FPD/AML); infant acute myeloid leukemia; myeloid leukemia in Down syndrome; refractory acute myeloid leukemia; relapsed acute myeloid leukemia; treatment-related acute myeloid leukemia (t-AML) acute myeloid leukemia with associated multilineage dysplasia, diagnosis, 36 acute myeloid leukemia with maturation (AML M2), 26, 48, 254 diagnosis, 34 immunophenotyping, 173–4 acute myeloid leukemia with minimal evidence of myeloid differentiation (AML M0), 48 chromosomal abnormalities, 178–9 classification issues, 178 diagnosis, 33–4, 512 immunophenotyping, 178–9 acute myeloid leukemia with predominant erythroid differentiation (AML M6), 28

diagnosis, 34–5, 172–3 immunophenotyping, 176–7 acute myeloid leukemia without maturation (AML M1), 26, 48 diagnosis, 34 immunophenotyping, 173 acute myelomonocytic leukemia (AML M4), 27, 48 diagnosis, 34 immunophenotyping, 175 inversions, 35 M4Eo variant, 175 see also juvenile myelomonocytic leukemia (JMML) acute nonlymphoblastic leukemia see acute myeloid leukemia (AML) acute procedural pain, 858 acute promyelocytic leukemia (APL), 27, 48, 511–12 all-trans-retinoic acid treatment, 400 and bleeding, 524–5 characterization, 511 chromosomal abnormalities, 255, 299–300 classification, 511 clinical presentations, 512 coagulopathies, 721–2 consolidation chemotherapy, 520 diagnosis, 34, 512 differentiation therapy, 544 and extramedullary disease, 512 genetic bases, 511–12 Hispanics, 5, 48–9 HuM195 maintenance therapy, 643 hypergranular variant, 26 immunophenotyping, 174–5 incidence, 511 ethnic and racial differences, 499 geographic differences, 625 microgranular, 27 molecular genetics, 299–307 murine studies, 305 and obesity, 511, 512 prognosis, 522 RAR
gene, variant translocation effects, 305–7 secondary mutations, 315–16 survival rates, 696 symptoms, 512 transformations, molecular mechanisms, 303–5 treatment, 512, 518–21 acute undifferentiated leukemia (AUL) classification issues, 179 immunophenotyping, 179 acyclovir, 810, 815 in herpes zoster treatment, 810 introduction, 13 ADCC (antibody-dependent cellular cytotoxicity), 639–40 adeno-associated viruses (AAVs) properties, 663–4 structure, 663–4

Index

adenomatous polyposis coli (APC), 127 adenoviral infections, 612, 813 diagnosis, 813 adenoviruses, 813 properties, 662–3 adenyl cyclases, activation, 126–7 ADHD see attention deficit hyperactivity disorder (ADHD) adolescents health behaviors, 870–1 studies, 870–1 adrenocorticotrophic hormone (ACTH), in acute lymphoblastic leukemia treatment, 9 adult T-cell leukemia/lymphoma (ATLL), 187 early studies, 187 immunophenotyping, 187 incidence, 187 symptoms, 187 see also HTLV-1-associated leukemia/lymphoma Advisory Committee on Immunization Practices (ACIP) (US), 821 advocacy, and nursing care, 884–6 AF-1p gene, 321 AF2p21 gene, 784 AF4 gene, 278, 691 AF4-MLL fusion genes, 32, 246 AF-6 protein, 322 AF-6q21 gene, 784 AF-6q21 protein, 321 AF9 gene, 278, 321 affluence, and infection, 57 Africa, Burkitt lymphoma, 625 AF-X protein, 321 age and cellular drug resistance, 416 and chemotherapy, 711 and complication risk, 711 and cranial irradiation, 866 risk factors, 711 AGM (aortic-gonad-mesonephros) region, 71 agnogenic myeloid metaplasia see chronic idiopathic myelofibrosis (CIMF) AHOPCA (Association of Pediatric Oncologists of Central America), 635–6 AIDS see HIV/AIDS AIF (apoptosis-inducing factor), 342 Akt kinases, 142 functions, 346 roles, 346–7 AKT pathways, 346 and chemoresistance, 345–7 ALCL see anaplastic large cell lymphoma (ALCL) alcohol, in pain management, 855 alcohol consumption and childhood leukemias, 55, 56 health risks, 870–1 prevalence, 870

aldehyde dehydrogenase, expression, 82 alkaline phosphatase, 159, 160 ALK (anaplastic lymphoma kinase), 190 alkylating agent-related leukemias clinical features, 788 cytogenetic changes, 781–3 genetic predisposition, 776–8 incidence, 774–6 molecular genetic changes, 781–3 risk factors, 774–6 alkylating agents, 12 in chemotherapy, 774, 775 classic, 775 cytotoxicity, 400 DNA damage induction, 776–7 exposure, 56 leukemogenicity, 7, 774 nonclassic, 775 platinum analogues, 775 types of, 775 ALL1 gene see MLL gene ALL see acute lymphoblastic leukemia (ALL) allantoin, 716 alleles, polymorphic, 781 allele-specific oligonucleotide (ASO) probe method, 685, 687 alloantigens, 650 allogeneic stem cell transplantation in acute myeloid leukemia treatment, 606–7 in chronic myeloid leukemia treatment, 583–4 and event-free survival rates, 481 from HLA-matched related donors, 480–1, 558 from HLA-matched unrelated donors, 481 and HLA mismatch, 481 in juvenile myelomonocytic leukemia treatment, 581–2 in myelodysplastic syndrome treatment, 558–9 and survival rates, 480 vs. autologous stem cell transplantation, 543 alloimmunization prevention, 834 treatment, 834 allophycocyanate (APC), 150 allopurinol, 12 in tumor lysis syndrome management, 716 xanthine oxidase inhibition, 714, 716 all-trans-retinoic acid (ATRA), 255, 301, 304 in acute promyelocytic leukemia treatment, 400, 512, 519, 520, 544, 552, 696 in coagulopathy treatment, 721–2 in consolidation chemotherapy, 520–1 effects on PML-RAR
fusion gene, 304–5 leukemogenicity, 780 in pediatric patients, 525 pharmacology, 400 plasma concentrations, 400 toxicity, 525

899

900

Index

alopecia, partial, 751 alpha interferons, 11 alpha-2a-interferon, 607 alpha naphthyl acetate esterase (ANA), 27 applications, 28–9 alpha naphthyl butyrate esterase (ANB), 27 applications, 28–9 reactivity, 28 Alternaria spp., infections, 807 amantadine, 813–14, 823 American Academy of Pediatric Guidelines, 859 American Cancer Society, 872–3 American Society of Clinical Oncology, 833 recommendations, 844 amino acids, metabolism, 420–1 aminoglycoside antibiotics, 13, 817 and hearing loss, 757 aminopterin, in acute leukemia treatment, 8–9 amitriptyline, 853 AMKL see acute megakaryoblastic leukemia (AML M7) AML1-ETO fusion genes, 504 detection, 541–2, 697 transcript, 35 AML1 gene encoding, 783 haploinsufficiency, 500 mutations, 33, 501, 561 roles, 279 translocations, 783–8 see also CBFA2 gene; RUNX1 gene AML see acute myeloid leukemia (AML) AML M0 see acute myeloid leukemia with minimal evidence of myeloid differentiation (AML M0) AML M1 see acute myeloid leukemia without maturation (AML M1) AML M2 see acute myeloid leukemia with maturation (AML M2) AML M3 see acute promyelocytic leukemia (APL) AML M4 see acute myelomonocytic leukemia (AML M4) AML M5 see acute monoblastic/monocytic leukemia (AML M5) AML M6 see acute myeloid leukemia with predominant erythroid differentiation (AML M6) AML M7 see acute megakaryoblastic leukemia (AML M7) amphotericin B, 13–14, 815, 818, 819, 820–1 lipid formulations, 524 ANA see alpha naphthyl acetate esterase (ANA) analgesia neuraxial, 855 patient-controlled, 852 procedural, 853–4 anaplastic large cell lymphoma (ALCL) incidence, 190 leukemic phase, 190–1 symptoms, 190–1 anaplastic lymphoma kinase (ALK), expression, 190 ANB see alpha naphthyl butyrate esterase (ANB)

anemia, 23, 459 Diamond–Blackfan, 552 red blood cell transfusions, 829 see also aplastic anemia; Fanconi anemia (FA); refractory anemia (RA) anesthesia and cognitive-behavior therapy compared, 859 components, 853–4 general, 854, 860 local, 860 see also eutectic mixture of local anesthetics (EMLA) annexins, 722 Antennapedia gene, 275, 277, 317 anthracenediones, leukemogenicity, 778–9 anthracyclines, 423–4, 477–8 accumulation, 423 adverse reactions, 398 and apoptosis, 424 cardiotoxicity, 398, 758 mediation, 758 cellular drug resistance, 422 mechanisms, 423–4 in consolidation chemotherapy, 520 DNA damage, 424 free radical scavenger formation, 423–4 leukemic cell sensitivity, 519 leukemogenicity, 56, 778–9 pharmacology, 398 in relapsed acute myeloid leukemia treatment, 542–3 in remission induction therapy, 455 retention, 423 sequestration, 423 therapeutic mechanisms, 423 antiangiogenic drugs, 545 antiapoptotic proteins, expression, 85 antibiotic-resistant bacteria, 817, 823 antibiotic therapy in febrile neutropenia treatment, 817–18 guidelines, 819 antibodies applications, clinical, 639 monoclonal, 639, 640 radiolabeled, 640–2 unconjugated, 639–40 see also anti-CD19 antibodies; anti-CD33 antibodies; anti-CD45 antibodies antibody-dependent cellular cytotoxicity (ADCC), 639–40 antibody-targeted therapies, 639–46 categories, 639–42 future trends, 645–6 hematopoietic cell surface antigens, 642–5 anti-CD19 antibodies conjugated with blocked ricin, 645 with genistein, 645

Index

anti-CD33 antibodies conjugated, with calicheamicin, 643–4 radiolabeled, 641–2, 644 unconjugated, 640–1, 642–3 anti-CD45 antibodies, 641–2 radiolabeled, 644–5 anticipatory nausea and vomiting (ANV), 861 and parenting behavior, 864 pharmacotherapy, 861 treatment, 861 antifolate drugs, 14 development, 14 antifungal drugs, 524 antigen expression, 150–4 antigen presentation, 648–9 and cellular immunotherapy, 655 antigen-presenting cells (APCs), 652–3 antigen receptor genes, rearrangements, 691–4 antigens categorization, 649–50 differentiation, 649–50 recognition,by immune system, 210 tumor-specific, 650–1 see also hematopoietic cell surface antigens; human leukocyte antigens (HLAs) antileukemic drugs, 5 assays, 14–15 cellular drug resistance dose–response relationship, 391 effects on MLL gene, 440 efficacy, 414 mucositis induction, 815 therapeutic indices, 391 antimycin A, 344 antineoplastic agents apoptosis induction, 342 toxicity, 760 antipurine drugs, 14 ANV see anticipatory nausea and vomiting (ANV) ANZCCSG (Australian and New Zealand Children’s Cancer Study Group), 513 aortic-gonad-mesonephros (AGM) region, 71 AP-1, and cellular drug resistance, 420 AP1903, 669 Apaf-1 gene, 340 binding, 342 expression, 340–1 APC (adenomatous polyposis coli), 127 APC (allophycocyanate), 150 APC gene, mutations, 364 APCs (antigen-presenting cells), 652–3 apheresis-platelet products, 833 APL see acute promyelocytic leukemia (APL)

aplastic anemia acquired, 552 vs. refractory cytopenia, 555 aplastic marrows, and acute lymphoblastic leukemia, 172 apoptosis, 107, 639–40 and anthracyclines, 424 and caspases, 131–2, 340 and chemoresistance, 339–50 future research, 350 and chemotherapy, 339 E2A-HLF fusion gene-induced, 276 and FLIP protein, 132 induction, 130–1 inhibition, 107, 108, 342–3 lymphocytes, 275 mechanisms, 283–4 murine studies, 342–3 pathways, 339–41 abnormalities, 429–30 extrinsic, 340, 345 intrinsic, 340, 345 and radiotherapy, 339 regulatory mechanisms, 283, 339 and BCL2 gene family, 341–3 roles, 339–40 see also inhibitors of apoptosis proteins (IAPs) apoptosis-inducing factor (AIF), release, 342 apoptosome, 340 activation, 342 ara-C see cytarabine (ara-C) ARF gene, 251 expression, 350 inactivation, 349 ARF protein, 349 armadillo gene, 127 arsenic trioxide, 545 in acute promyelocytic leukemia treatment, 521, 544 in coagulopathy treatment, 721–2 arsenious oxide, 8 arthralgia, and leukemia, 441 AS (asparagine synthetase), 420–1 ASO (allele-specific oligonucleotide) probe method, 685, 687 asparaginase (ASP), 10, 420–1, 477–8 cellular drug resistance, 416, 421 genetic abnormalities, 417 mechanisms, 420–1 dosage, 454–5 drug–drug interactions, 402 forms of, 452–4 and hyperglycemia, 733–4 hypersensitivity, 401 leukemogenicity, 779 mechanisms, 401 occurrence, 400–1, 452–4 and pancreatitis, 729

901

902

Index

asparaginase (ASP) (cont.) pharmacokinetics, 400–1 pharmacology, 400–2 and protein synthesis, 421 in relapse treatment, 479 in remission induction therapy, 455 therapeutic mechanisms, 420 and thrombosis risk, 727 asparaginase synthetase (AS), metabolism, 420–1 asparagine, 10 plasma depletion, 401–2 aspergillosis, 13–14, 524, 820–1 diagnosis, 820 pulmonary, 809 symptoms, 820 treatment, 820–1 Aspergillus spp., 812–13 infections, 612, 807, 819, 820–1 risk factors, 821 Aspergillus flavus, 716 aspiration see bone marrow aspiration (BMA) Association of Community Cancer Centers (US), 821 Association of Pediatric Oncologists of Central America (AHOPCA), 635–6 astrocytomas, 763 AT see ataxia telangiectasia (AT) ataxia telangiectasia (AT), 51, 224, 375–8, 444 and cancer risk, 362, 363, 376 and cancers, 376 comorbidity, 376, 489 demography, 375–6 diagnosis, 376–7 incidence, 375–6 and leukemia, 377 pathogenesis, 377 phenotype, 376–7 prognosis, 377–8 symptoms, 376, 442–3 therapy, 377–8 ataxia-telangiectasia mutant (ATM), 349 ataxia telangiectasia-mutated (ATM) signaling pathway, 303 A-T gene, mutations, 376 AT hook motifs, 317 ATLL see adult T-cell leukemia/lymphoma (ATLL) ATM (ataxia-telangiectasia mutant), 349 ATM (ataxia telangiectasia-mutated) signaling pathway, 303 ATM gene, 376 mutations, 376, 377, 440 ATM protein kinase, 273–4 atomic bombs, 6, 52, 53 atovaquone, 452, 822–3 ATP assay, 415 ATP-binding cassette (ABC) transporters, prognostic significance, 523 ATRA see all-trans-retinoic acid (ATRA)

attention deficit hyperactivity disorder (ADHD), 869 cognitive-behavior therapy, 870 audiologic screening, 757 Auer rods, 25, 38 characteristics, 26 multiple, 34 staining, 27 AUL see acute undifferentiated leukemia (AUL) Australia, folate supplementation studies, 54 Australian Leukemia Study Group, 525 Australian and New Zealand Children’s Cancer Study Group (ANZCCSG), idarubicin studies, 513 Austrian-German-Italian Pediatric Registry, 543 autoimmune lymphoproliferative syndromes, 118 autologous bone marrow grafts, 694 autologous stem cell transplantation, 481 complications, 779 disadvantages, 481 gene marking for, 670–1 and treatment-related acute myeloid leukemia, 775–6 vs. allogeneic stem cell transplantation, 543 avascular necrosis (AVN), 761 glucocorticoid-induced, 759–60 avian leukemia oncogenes, 8 avidin substrates, binding, 159 AVN see avascular necrosis (AVN) Ayudame a Vivir (El Salvador), 634 azoles, 524 babesiosis, 838 bacille Calmette–Gu´erin (BCG), 648 in leukemia treatment, 14 Bacillus cereus, infection, 524 background radiation, and childhood leukemias, 53 bacteremia, 13 catheter-related, 840, 841 diagnosis, 811 incidence, 811 risk factors, 811 treatment, 811 bacteria antibiotic-resistant, 817, 823 gram-negative, 13, 811, 840 bacterial infections and cancers, 808 and leukemia, 806–7 transfusion-associated, 837 treatment, 807 BAD protein, 85, 142, 342 BAK protein, 85, 342 mutations, 343 regulatory mechanisms, 342 B-ALL see B-cell acute lymphoblastic leukemia (B-ALL) Bang, O., 5 Bannayan-Zonana syndrome, 346

Index

Barnes, D. W. H., 11 Barney intervention, 860 basic helix-loop-helix/leucine zipper (bHLHZip) protein, 279 basophilia, 39 BAX gene expression, 284 mutations, 284 BAX protein, 85, 342, 344 mutations, 343 regulatory mechanisms, 342 BC8 antibodies B-cell acute lymphoblastic leukemia (B-ALL), 487–94 cellular drug resistance, 416–17 chemotherapy, 492 complications avoidance, 492 chromosomal abnormalities, 249 CNS prophylaxis and therapy, 492–3 cytogenetics, 488–9 diagnosis, 490–1 differential diagnosis, 487 epidemiology, 489 and Epstein–Barr virus, 490 IG/TCR gene rearrangements, 224 immunophenotyping, 446, 487–8 incidence, 487 geographic differences, 489–90 lymphoblast characterization, 488 management, initial, 492 mature, 168–9 molecular pathology, 488–9 MYC gene activation, 279–80 pathology, 487–8 prevalence, 221–2 prognosis, 449 proto-oncogene activation, 281–2 reinduction therapy, 494 relapse, 494 risk factors, 489 staging, 490–1 subtypes, 168–9 survival rates, 487, 494 symptoms, 490–1 transcription factors, dysregulation, 272 translocations, 39, 279, 488 treatment, 452, 491 emergency situations, 493–4 future trends, 494 outcomes, 493 use of term, 224 see also Burkitt lymphoma; precursor B-cell acute lymphoblastic leukemia (precursor-B-ALL) B-cell receptor (BCR), 138, 155 complexes, 154 expression, 155

B cells see B lymphocytes BCG see bacille Calmette–Gu´erin (BCG) BCL2/BAX ratios, 429–30 BCL2 gene discovery, 341 early studies, 341 expression, 284 homologues, 341 regulatory mechanisms, 341 roles, 344 in apoptosis, 283–4 BCL2 gene family, 342, 343, 346 alterations, 343–4 apoptosis inhibition, 342–3 definition, 341 drug targeting, 344 members, 341–2 overexpression, 343 roles, 343–4 in apoptosis regulation, 341–3 BCL2 homology domains, 341 BH1, 341–2 BH2, 341–2 BH3, 341–2, 344 BH4, 341–2 BCL2 protein, 85 BCL10 gene, 347 BCL-ABL fusion gene, 186, 242, 243 BCL-X gene, 668–9 BCL-XL protein, 85, 342 overexpression, 343 roles, 344 BCR see B-cell receptor (BCR) BCRA2 gene, mutations, 372 BCR-ABL fusion gene, 5, 8, 12, 186, 238, 650 activity, 8 in acute lymphoblastic leukemia, 272–5, 285 childhood, 274–5 age differences, 416 in chronic myeloid leukemia, 582 cytogenetic studies, 272–80 detection, 476 generation, 272 neutralization, 664 as relapse marker, 474 roles, 274, 344 targeting, 583 transcript, 32 see also Philadelphia chromosome BCR-ABL fusion protein, 12, 650 transformation, 274 BCR gene, 582 BCRP (breast-cancer resistance protein), 429 BCRP1 (ABCG2) gene, expression, 83

903

904

Index

benign lymphocytosis differential diagnosis, 172 mimicking lymphoblastic leukemia, 171–2 Benjamin Bloom Hospital (El Salvador), facilities, 634 Bennett, John Hughes (1812–75), 3 benzene leukemogenicity, 7 in vehicle exhaust, 54 benzene intoxication, 7 ¨ Berlin–Frankfurt–Munster (BFM) Consortium, 445, 450, 456 BFM-93 protocol, 635–6 chemotherapy regimens, 492 cranial irradiation studies, 518 idarubicin studies, 513 Relapse Study Group, 477–8 relapse trials, 478 ¨ see also International Berlin–Frankfurt–Munster Study Group (I-BFM-SG) ¨ BFM Consortium see Berlin–Frankfurt–Munster (BFM) Consortium BFU-Es see burst-forming units-erythroid (BFU-Es) BFU-Mks (burst-forming units-megakaryocyte), 80–1 BH3 mimetics, 344 bHLHZip (basic helix-loop-helix/leucine zipper) protein, 279 biclonal leukemias see bilineal acute leukemias BID protein, 342 BIK protein, 342 bilineal acute leukemias, 37 characterization, 512–13 diagnosis, 37 immunophenotyping, 183 treatment outcomes, 513 BIM protein, 85, 342 expression, 342 biofeedback, 861–2 bioflavonoids, leukemogenicity, 56 biological response modifiers, 11 biomarkers applications, 58 for leukemogenesis, 58–9 biopsy (bone marrow) see bone marrow biopsy biotin, avidin substrates, 159 biphenotypic leukemias, 179–83 diagnosis, 37, 181–3 Philadelphia chromosome in, 37 see also mixed-lineage leukemias birth order, and childhood leukemias, 55 birth weight, and childhood leukemias, 55–6 bismuth isotopes, in radiolabeled antibody treatment, 644 Bithorax gene, 277, 317 blast cell chromosomes abnormalities, 272 detection, 272 blast cells characteristics, 447

counts, 553, 556–7 cycling, 23 cytoplasmic vacuolization, 30 formation, chronic myeloid leukemia, 186 genetic alterations, 439 immunologic analysis, 31 karyotyping, 247 L1-type, 29 L2-type, 29 see also bone marrow blasts; erythroblasts; fibroblasts; lymphoblasts; megakaryoblasts; monoblasts; myeloblasts bleeding, and acute promyelocytic leukemia, 524–5 bleeding time, and platelet count, 833 B-lineage cells see B lymphocytes BLM gene mutations, 380 murine models, 380 blood banks, introduction, 12, 14 blood cells biosynthesis, 70 daily output, 70 persisting populations, 70 terminal differentiation, 70 types of, 69–70 see also red blood cells (RBCs); white blood cells (WBCs) blood component products, 830 demand for, 829 guidelines, 838 see also packed red blood cells (PRBCs); platelet products blood component support, 829–35 guidelines, 829 risk factors, 829 see also blood transfusions blood groups, identification, 12 bloodstream infections, 810–11 catheter-related, 811 blood substitutes development, 838 future trends, 838 guidelines, 838 oxygen transport, 838 blood transfusions early, 12 noninfectious reactions, 832 screening, 835–6 in supportive care, 451 see also granulocyte transfusions; platelet transfusions; red blood cell transfusions Bloom syndrome (BS), 51, 379–80 and cancers, 379 comorbidity, 379 demography, 379 etiology, 362–3 incidence, 379 pathogenesis, 380

Index

phenotype, 379–80 therapy, 380 Bloom Syndrome Registry, 379 Blundell, J., 12 B lymphocytes, 109 apoptosis, 155 differentiation, 214–16 germinal, 156 in Hodgkin disease, 112 immunoglobulins on, 210 immunophenotyping, 154–6 maturation, 154, 155 na¨ıve, 155 migration, 155, 212 precursors, 111–12 progenitors, 688 BM11 gene, 87 BM see bone marrow (BM) BMA see bone marrow aspiration (BMA) BMD see bone mineral density (BMD) BMI see body mass index (BMI) BMPs see bone morphogenetic proteins (BMPs) BMT see bone marrow transplantation (BMT) BO see bronchiolitis obliterans (BO) Bodey, G. P., 13 body height, treatment effects, 750–1 body mass control, 113 body mass index (BMI), 755 obesity studies, 754 body temperature, monitoring, 886–7 bone marrow (BM) abnormalities, 761 characteristics, in myelodysplastic syndrome, 553–4 congenital failure, 551–2 infiltration, 23 as relapse site, 540–1 sampling, 21–2 sites, 22 smear tests, 473 stem cell harvesting, 601 as stem cell source, 601 bone marrow aspiration (BMA), 21–2, 445 in AML diagnosis, 503–4 bone marrow sampling, 21–2 complications, 22 distress management, 858 cognitive-behavior therapy, 859, 860 hypnosis vs. distraction, 859 in JMML diagnosis, 574–5 preparation for, 887–8 procedures, 21–2 bone marrow biopsy, 21, 445 in AML diagnosis, 503–4 preparation for, 887–8

procedures, 22 specimen preparation, 23 bone marrow blasts, 23–5 expression profiles, 39 reduction, 402 bone marrow relapse and event-free survival rates, 481 late, remission rates, 478 postremission chemotherapy, 478 reinduction therapy, 477 remission rates, 477–8 bone marrow transplantation (BMT), 11, 494 in acute myeloid leukemia treatment, 518 allogeneic, 518, 606, 650 autologous, 606, 694 disadvantages, 518 and family adjustment, 863 and gonadal function, 756 and growth impairment, 753 and nursing care, 890 and ovarian function, 756 psychosocial issues, 868–9 see also European Group for Blood and Marrow Transplantation (EBMT); hematopoietic stem cell transplantation (HSCT) bone mineral density (BMD), 760 and cranial irradiation, 759–61 bone morphogenetic proteins (BMPs), 129 mediation, 129 BOOP see bronchiolitis obliterans-organizing pneumonia (BOOP) Borella, Luis, 4, 14 Borgomano, C., 7 Bortezomib, 348 box 1 motif, 135 box 2 motif, 135 BR140 protein, 321 brain tumors etiology, 763–4 incidence, 763–4 radiation-associated, 395 Brazil acute lymphoblastic leukemia, 629, 630 acute myeloid leukemia, 636 Burkitt lymphoma, 635 high-dose methotrexate guidelines, 636 pediatric cancers, treatment outcomes, 631 pediatric cancer units, 628 collaborative treatment programs, 633 BRCA1 protein, 299–300 breakpoint fusion genes, 680–1 breast-cancer resistance protein (BCRP), and cellular drug resistance, 429 breast feeding, and infection, 57 Brewster, H. F., 14 bronchiolitis obliterans (BO), 611 treatment, 612

905

906

Index

bronchiolitis obliterans-organizing pneumonia (BOOP), 611 CT scans, 611 treatment, 612 BS see Bloom syndrome (BS) Btk kinases, 138 mutations, 138 BUB1 gene, 782–3 Burchenal, J. H., 9, 9 Burkitt-like lymphoma, and Burkitt lymphoma compared, 487–8 Burkitt lymphoma, 5, 29, 30, 168, 224, 487–94 and Burkitt-like lymphoma compared, 487–8 chemotherapy, 492 complications avoidance, 492 classification, 487–8 CNS prophylaxis and therapy, 492–3 CT scans, 491 cytogenetics, 488–9 in developing countries, 634–5 diagnosis, 490–1 differential diagnosis, 487 epidemiology, 489 and Epstein–Barr virus, 490, 625 histological sections, 488 immunophenotype, 487–8 incidence, geographic differences, 489–90, 625 of jaw, 491 lymphoblast characterization, 488 management, initial, 492 molecular pathology, 488–9 murine models, 489 MYC gene translocations, 279 pathology, 487–8 proliferation rates, 488 reinduction therapy, 494 relapse, 494 risk factors, 489 staging, 490–1 subtypes, 489–90 supportive care, 635 survival rates, 494 symptoms, 490–1 translocations, 488 treatment, 491 emergency situations, 493–4 future trends, 494 outcomes, 493 use of term, 224, 488 see also B-cell acute lymphoblastic leukemia (B-ALL) burst-forming units-erythroid (BFU-Es), 80, 110 proliferative potential, 80 quantitation, 81 use of term, 80 burst-forming units-megakaryocyte (BFU-Mks), 80–1 busulfan, 583 and ovarian failure, 756

busulfan, cyclophosphamide and melphalan regimen, 558 busulfan/cyclophosphamide regimen, 602 CAE (chloroacetate esterase), 23 Caenorhabditis elegans ces-2 gene, 242, 276 SMADs, 129 CAE stain see chloroacetate esterase (CAE) stain calcium homeostasis, and tumor lysis syndrome, 715 CALGB (Cancer and Leukemia Group B), 517–18, 645 calicheamicin, 544, 643–4 see also gemtuzumab ozogamicin cALL see common acute lymphoblastic leukemia (cALL) CALLA (common acute lymphoblastic leukemia antigen), 150 CALM-AF10 fusion gene, 250, 257 Campath-1, 640 cAMP (cyclic AMP), 126–7 camptothecin, in relapsed acute myeloid leukemia treatment, 543 Campylobacter spp., infections, 806–7 cancer cells, p53 function loss, 348–9 Cancer and Leukemia Group B (CALGB), 517–18, 645 cancers acquired genetic alterations, 362 and ataxia telangiectasia, 376 and bacterial infections, 808 and Bloom syndrome, 379 and childhood leukemia treatment, 51 and cigarette smoke exposure, 55 differentiating-inducing therapy, 11–12 etiology, 59 pain management, 850–5 risk factors, 362, 363 second, 774 and tobacco smoking, 870 see also familial cancer syndromes; pediatric cancers cancer survivors, second malignancies, 762 cancer–testis antigens (CTAs), expression, 650 Candida spp., 812–13 infections, 807, 819–20 catheter-related, 840 Candida albicans, 820 Candida glabrata, 820 Candida krusei, 820 Candida parapsilosis, 820 Candida tropicalis, 820 candidiasis, 524, 809, 819–20 diagnosis, 820 etiology, 820 systemic, 820 treatment, 820 Candlelighters Foundation, 872 CAN gene, 325 Cannon, H. E., 14 cap gene, 663–4 Capizzi II regimen, studies, 517

Index

carbapenems, 524, 757 carboplatin, 775 carboxyfluorescein diacetate succinimidyl ester (CFSE), 82 CARD (caspase recruitment domain), 347 cardiopulmonary toxicity, 757–8 caregivers handbooks, 887, 888 resources, 872–3 case studies, early, 14 caspase recruitment domain (CARD), 347 caspases, 131–2, 340, 347 activation, 130 and apoptosis, 131–2, 340 biosynthesis, 131 caspase-1, 131 expression, 340–1 caspase-3, 132 cleavage, 340 roles, 340 caspase-5, expression, 340–1 caspase-6, roles, 340 caspase-7 expression, 340–1 roles, 340 caspase-8, 131–2 activation, 340 roles, 340 caspase-9, 341, 346 activation, 340, 342 expression, 340–1 roles, 340 caspase-10, expression, 340–1 caspase-11, 131 caspase-12, 131 effector, 341 executioner, 340 initiator, 340, 341 roles, 131, 340 Casper protein see FLIP protein caspofungin, 815, 820 Cas proteins, tyrosine phosphorylation, 139 cataracts, 757 and total-body irradiation, 869 CAT (cyclophosphamide/cytarabine/topotecan) regimen, 543 category, cytology and cytogenetics (CCC), 549 -catenin, 127 accumulation, 127–8 phosphorylation, 127 catheter occlusions, 839–40 treatment, 839–40 catheter-related infections, 840–1 bloodstream, 811 catheters epidural, 855 external tunneled, 839

intrathecal, 855 malpositioning, 841 see also central venous catheters CBF (core binding factor), 501 CBF1 (RBP-J) protein, 134 CBFA2-CBFA2T fusion gene, 259 CBFA2-ETO fusion gene, 254–5 CBFA2 gene abnormalities, 235, 259–60 and Down syndrome, 366 mutations, 254, 375 translocations, 783–8 see also AML1 gene; RUNX1 gene CBFB-MYH11 fusion gene, 255–6 transcript, 35 CBF gene, 307–8 expression, 308 CBF-MYH11 fusion gene, 697 CBF-MYH11 fusion protein, 311 expression, 310–11 functions, 310–11 CBF protein, 311 roles in hematopoiesis, 309–10 in leukemogenesis, 311–12 cbl-b gene, 143 CBP (CREB-binding protein), 259, 321 CBP gene, 784 CBP-MLL fusion gene, 259 CBP-MORF fusion gene, 259 CBP-MOZ fusion gene, 259 CBS see cystathionine B synthase (CBS) CBS gene, 367 CBT see cognitive-behavior therapy (CBT) c-cbl protein, 143 CCC (category, cytology and cytogenetics), 549 CCG see Children’s Cancer Group (CCG) (US) CCI see corrected count increment (CCI) CCNA2 gene, 782–3 CCNC gene, 248 CCNE2 gene, 782–3 CCR1 receptor, deficiency, 116 CCR2 receptor, deficiency, 116 CD3 expression, 164, 216 immunophenotyping, 164 CD3-activated cells, 652 CD4+ cells antigen presentation, 648, 649–50 chemoattractants, 119–20 cloning, 650 counts, 806 CD7 expression, 164 immunophenotyping, 164

907

908

Index

CD8+ cells, antigen presentation, 648, 649–50 CD13 expression, 157–8, 164 immunophenotyping, 164–5 CD14, expression, 158 CD15, expression, 158 CD19 biology, 645 expression, 163–4, 645 immunophenotyping, 163 as target, 657 see also anti-CD19 antibodies CD20 expression, 154 as suicide molecule, 669 as target, 657 CD22 expression, 154, 164 immunophenotyping, 163 CD25, 652 CD28-activated cells, 652 CD31, polymorphisms, 650 CD33 biology, 642 expression, 158, 544, 642–4 immunophenotyping, 164–5 targeting, 641–2 see also anti-CD33 antibodies CD34 expression, 82, 157–8, 159, 166, 601 immunophenotyping, 166 phenotype analysis, 82–3 CD34+ cells, 82, 782–3 CD36 expression, 159 localization, 158 CD38, expression, 83 CD40, characterization, 118 CD40L, characterization, 118 CD41a, expression, 159 CD42b, expression, 159 CD45 biology, 644 expression, 163, 644–5 flow cytometry, 160, 161, 162 immunophenotyping, 162–3 targeting, 641–2 see also anti-CD45 antibodies CD58, as marker, 689 CD61, expression, 159 CD64, 158 CD65, structure, 158 CD71, 159 CD79, expression, 215–16

CD79a, 155, 210 expression, 164 CD79b, 155, 210 CD79
, immunophenotyping, 163 CD95, and apoptosis, 424, 639–40 CD117, immunophenotyping, 165 CD179a, encoding, 155 CD179b, encoding, 155 CD235a, expression, 159 2-CdA (2-chlorodeoxyadenosine), 543 CDC2 gene, 782–3 CDC28 gene, 782–3 CDC (Communicable Disease Center) (US), 13 CDKIs (cyclin-dependent kinase inhibitors), 282–3 CDKN1B gene, 248 CDKs (cyclin-dependent kinases), 85–6 CD markers, expression, 154 CDR3 (complimentarity determining region 3), 214 CDs (Clusters of Differentiation), 150 CEBPA gene, mutations, 523 cefepime, 817 ceftazidime, 817 in bacteremia treatment, 811 CEL (chronic eosinophilic leukemia), 587 cell adhesion, 139 cell cycle, 282–3 schematics, 283 cell cycle progression, regulatory mechanisms, 85–6 cell death see apoptosis cells antigen-presenting, 652–3 CD3-activated, 652 malignant, 227–8 myeloid, 70 neoplastic, 666 precursor, 70 see also blast cells; blood cells; B lymphocytes; colony-forming cells (CFCs); dendritic cells (DCs); leukemic cells; long-term culture-initiating cells (LTC-ICs); lymphoblastoid cell lines (LCLs); natural killer (NK) cells; side population (SP) cells; stem cells; T lymphocytes; tumor cells cellular drug resistance age differences, 416 assays, 414–31 cross-class resistance, 429–30 future trends, 430–1 and genetic abnormalities, 417–18 molecular determinants, 414–31 predictive value, 415–16 risk factors, 416–18 immunophenotype, 416–17 cellular immunotherapy adoptive, 648–57 and antigen presentation, 655 clinical experience, 651–5

Index

future trends, 657 historical background, 648 improvements, 655 limitations, 655 targets, 649 cellular theory, of leukemia, 3 cellulitis, 809–10 central airway compression syndrome, 719–20 symptoms, 720 central nervous system (CNS) chemotherapy, 865 infections of, 811–12 neurocognitive deficits, 757 radiation damage, 864–5 radiation effects, 750 radiotherapeutic effects, 757 relapse, 541 status, classification, 445 treatment, in acute myeloid leukemia, 518–21 central nervous system (CNS) leukemia, 445 and acute myeloid leukemia, 518–19 diagnosis, 504–5 incidence, 725 pathophysiology, 725–6 relapse, 445, 457–8, 633–4 chemotherapy, 479 craniospinal irradiation vs. cranial irradiation, 479 diagnosis, 473 treatment, 478–9 treatment, 457–8, 726 central venous catheters advantages, 838–9 complications, 839–41 extravasation, 841 implanted, 839 infections, 840–1 malpositioning, 841 occlusions, 839–40 venous thrombus formation, 840 see also peripherally inserted central catheters (PICCs) centroblasts, 156 centrocytes, 156 cephalosporins, 757 in bacteremia treatment, 811 cerebral toxoplasmosis, 812 diagnosis, 812 symptoms, 812 treatment, 812 cerebrospinal fluid (CSF) analysis, 445 blood contamination risk, 833 cytarabine concentrations, 396–7 ces-1 gene, 276–7 ces-2 gene, 242 roles, 276

CFCs see colony-forming cells (CFCs) CFSE (carboxyfluorescein diacetate succinimidyl ester), 82 CFTR (cystic fibrosis transmembrane conductance regulator), 664 CFU-C (colony-forming units-culture), 79 CFU-Es see colony-forming units-erythroid (CFU-Es), 79 CFU-GEMM see colony-forming units-granulocyte, erythroid, megakarocytic, macrophage (CFU-GEMM) CFU-GMs see colony-forming units-granulocyte macrophage (CFU-GMs) CFU-Mks see colony-forming units-megakaryocyte (CFU-Mks) CFU-S see colony-forming unit-spleen (CFU-S) Chagas disease, 838 Chambless/Society for Pediatric Psychology criteria, 862 Charcot-Marie-Tooth hereditary neuropathy, 456 Chelyabinsk reactor accident (Russia), 53 chemical exposure and childhood leukemias, 54 and leukemia, 7 parental, 54 chemokine receptors, 106, 115 CCR2, 126 CCR3, 126 CCR4, 126 CCR5, 126 CXCR2, 126 CXCR3, 126 CXCR4, 126 CXCR5, 126 deficiency, 126 murine studies, 126 nomenclature, 126 roles, 126 signaling, 125, 126–7 diversity, 126 mechanisms, 126–7 structure, 126 chemokines, 115–16 biological activity, 116 classification, 116 deficiency, 116 family, 115 mediation, 126 nomenclature issues, 115–16 structure, 115 in tumor vaccines, 666
-chemokines, 116 -chemokines, 116  -chemokines, 116 -chemokines, 116 chemoprophylaxis, 822–3 chemoresistance and AKT pathways, 345–7 and apoptosis, 339–50 future research, 350 and death receptor pathways, 344–5

909

910

Index

chemoresistance (cont.) mechanisms, 339 and NF-B, 347–8 and p53 gene, 348–50 tumors, 339 see also drug resistance chemotherapy, 11, 12, 13–14 in acute leukemia treatment, 12 in acute lymphoblastic leukemia treatment, 10, 391, 639 in acute myeloid leukemia treatment, 639 age factors, 711 and apoptosis, 339 in B-cell acute lymphoblastic leukemia treatment, 492 in central nervous system leukemia relapse treatment, 479 and cognitive processing, 867 consolidation, 520 and craniofacial developmental defects, 758 developmental effects, 613 developments, 414 and distress, 861–2 hepatotoxicity, 729 in Hodgkin disease treatment, 639 and hyperphosphatemia, 715 intensive, 473, 580, 632–3 and learning disabilities, 867 and leukemia induction, 7, 774 and Leydig cell dysfunction, 755–6 low-dose, 580 and mucositis, 728–9 myelodysplastic syndrome, 558 and NF-B, 347–8 and nursing care, 889–90 and radiotherapy, 11 relapsed acute myeloid leukemia, 542–3 responsiveness, 391 risk factors, 639 translocation induction, 783 and treatment-related leukemias, 789, 774–90 vs. hematopoietic stem cell transplantation, 480 see also postremission chemotherapy chemotherapy-related distress, 861–2 cognitive-behavior therapy, 861–2 hypnosis, 861–2 Chernobyl reactor accident (Russia), 53 child adjustment and parental distress, 863 and parenting behavior, 863–4 Childhood Cancer Survivor Study, 753, 762 childhood hematologic malignancies future research, 380 heritable predispositions, 362–80 Childhood Leukemia Foundation, 873 childhood leukemias acute complications, 709–35 incidence, 710 risk factors, 710

age peaks, 57 and alcohol consumption, 55 and birth order, 55 and birth weight, 55–6 and chemical exposure, 54 chromosomal abnormalities, 58 classification, 3–4, 12, 21–40, 48 and coagulopathies, 722 cures discovery, 3 historical background, 9–12 cytogenetic abnormalities, 50 diagnosis, 21–40 and diet, 54 drug cytotoxicity assays, 415–18 drug responsiveness, 391 epidemiology, 48–59 future trends, 59 etiology, 5–8, 48–59 future research, 59 familial patterns, 51 and family adjustment, 862–4 and fetal loss, 55 fetal origins, 4, 5 genetic factors, 50–1, 58–9 and genetic syndromes, 50–1 immunogenotypes, 220–8 incidence, 48–50, 499–500 geographic differences, 625 studies, 53 and ionizing radiation, 51, 52–3 and lifestyle, 54–5 and maternal age, 55 and maternal reproductive history, 55–6 microsatellite instability in, 58 and neoplasms, 51 and nonionizing radiation, 53–4 and population mixing, 57–8 psychosocial issues, 858–73 and radiography, 6 risk factors, 52 assessment, 48 demographic, 51–4 environmental, 51–4, 58–9 susceptibility, 59 and tobacco smoking, 55 treatment, cancers in offspring, 51 trends, 48–50 and tumors, 51 unique populations, 56–8 and vitamin supplementation, 54 see also infant leukemias childhood monosomy 7 syndrome, 510, 548–9 diagnostic issues, 510 prognosis, 510 symptoms, 510

Index

Children’s Cancer Group (CCG) (US), 367, 447, 450 CCG-1882 protocol, 732 chemotherapy studies, 492, 580 flow cytometry studies, 697 induction therapy intensification studies, 517, 559 methotrexate studies, 867 osteonecrosis studies, 732 postremission therapy studies, 517 second malignancy studies, 762–3 studies, 9, 51, 456, 457 Children’s Oncology Group chemotherapy studies, 580 splenectomy studies, 580 children’s rights, 630 chimeric genes formation, 299 roles, 299 chimeric leukemias see mixed-lineage leukemias chimerism status, post-transplant, 602–3 China, mortality rates, under-five, 626–7 chloroacetate esterase (CAE), 23 chloroacetate esterase (CAE) stain applications, 29 limitations, 29 2-chlorodeoxyadenosine (2-CdA), in relapsed acute myeloid leukemia treatment, 543 chloromas see myeloid sarcomas chlorpromazine, 854 choline magnesium trisalicylate, 851 chondroitin protoglycan sulfate, 167 chromosomal abnormalities accumulation, 55 in acute myeloid leukemia, 56 in childhood leukemias, 58 classification, 284, 298–9 clonal, 238 cryptic, 260 definition, 238 and leukemia, 7 monosomy 7, 253 nomenclature, 238 nonrandom, 4, 7–8, 225 numeric, 238–41, 253, 254 secondary, 238 structural, 241–8, 254–60 common recurrent, 254–7 uncommon recurrent, 257–60 trisomy 8, 253 trisomy 21, 253–4 see also inversions; translocations chromosome 5, abnormalities, 258, 781 chromosome 7, abnormalities, 362–3, 781 chromosome 19, 242 chromosomes abnormalities, 272 detection, 272

derivative, 238 dicentric, 238, 240 marker, 238 metaphase, 239 see also blast cell chromosomes; Philadelphia chromosome chronic, use of term, 21 chronic cardiomyopathy, risk factors, 398 chronic eosinophilic leukemia (CEL), characterization, 587 chronic idiopathic myelofibrosis (CIMF), 587 characterization, 587 diagnosis, 587 disease course, 587 incidence, 587 treatment, 587 chronic leukemias classification, 21 diagnosis, 23 chronic lymphatic leukemia see chronic lymphocytic leukemia (CLL) chronic lymphocytic leukemia (CLL), 3 leukocyte counts, 23 prevalence, 21 xenotransplantation studies, 76–7 chronic monocytic leukemia see myelodysplastic syndrome (MDS) chronic myelogenous leukemia see chronic myeloid leukemia (CML) chronic myeloid leukemia (CML), 3, 582–4 abl proto-oncogene, 8 adult-type, hematopoietic stem cell transplantation, 607–8 allogeneic stem cell transplantation, 583–4 atypical, 571 and blast formation, 186 clinical findings, 582 deletions, 243 diagnosis, 39 disease course, 582–3 epidemiology, 582 genetics, 582 hematologic findings, 582 long-term culture-initiating cell assay studies, 78–9 Philadelphia chromosome, 4, 70 prevalence, 21 prognosis, 582–3 relapse, treatment, 584, 651 treatment, 583–4 palliative, 8 xenotransplantation studies, 76–7 see also acute myeloid leukemia (AML) chronic myelomonocytic leukemia (CMML) classification, 38–9, 549, 571 issues, 549 use of term, 572 see also juvenile myelomonocytic leukemia (JMML) cidofovir, 810, 812

911

912

Index

cigarette smoke benzene in, 7 exposure, 55, 871 see also tobacco smoking CIMF see chronic idiopathic myelofibrosis (CIMF) ciprofloxacin, 817–18 circumplex model, 862–3 cisplatin, leukogenicity, 775 citrate dextrose, 12 CJD (Creutzfeldt-Jakob disease), 838 c-kit, 107–8 deficiency, 108 roles, 108 c-kit gene, mutations, 588 CKS2 gene, 782–3 cladribine, in acute myeloid leukemia treatment, 513 CLL see chronic lymphocytic leukemia (CLL) clonal chromosomal aberrations, definition, 238 clonal cytopenia, use of term, 549 clonality detection, and IG/TCR gene rearrangements, 216–20 cloning, mammals, 88 Clostridium difficile infections, 806–7 tests, 815 Clostridium difficile colitis diagnosis, 816 treatment, 816 clotrimazole, 815, 820 Clusters of Differentiation (CDs), 150 CML see chronic myeloid leukemia (CML) CMML see chronic myelomonocytic leukemia (CMML) c-mpl gene, 110 CMV see cytomegalovirus (CMV) CMYC gene, 249, 251 overexpression, 342–3 translocation, 168 CNS leukemia see central nervous system (CNS) leukemia CNS prophylaxis and therapy, in B-cell acute lymphoblastic leukemia treatment, 492–3 coagulation function, 835 coagulopathies, 721–2 in acute promyelocytic leukemia, 721–2 in childhood leukemias, 722 and hyperleukocytosis, 724 incidence, 712, 721 laboratory evaluation, 721 risk factors, 721 thrombotic, 722 treatment, 721 Coccidioides immitis, 812–13, 814–15 infections, 807 and meningitis, 811 codeine, 851 effectiveness, 851

cognitive-behavior therapy (CBT), 858–9, 869 and anesthesia compared, 859 attention deficit hyperactivity disorder, 870 chemotherapy-related distress, 861–2 distress management, 860 effectiveness, 859, 860 multicomponent, 859, 860–1 with pharmacotherapy, 859–60 cognitive processing, and chemotherapy, 867 colony-forming cells (CFCs), 79 in acute leukemias, 81 assays, 78 applications, 79 frequencies, 79 high proliferative potential, 81 megakarypoietic, 80–1 pluripotent, 70, 81 see also erythroid colony-forming cells; granulopoietic colony-forming cells colony-forming units-culture (CFU-C), use of term, 79 colony-forming units-erythroid (CFU-Es), 80, 110 detection, 80 proliferative potential, 80 stimulation, 80 colony-forming units-granulocyte, erythroid, megakarocytic, macrophage (CFU-GEMM), 81 use of term, 81 colony-forming units-granulocyte macrophage (CFU-GMs), 79–80, 373 proliferation, 79 use of term, 79 colony-forming units-megakaryocyte (CFU-Mks), 80–1 quantitation, 81 use of term, 80 colony-forming unit-spleen (CFU-S), 74 properties, 74 seeding fraction, 74 self-renewal, 86 use of term, 74 colony-scoring analysis, 73 assumptions, 73 colony-stimulating factor-1 (CSF-1) biosynthesis, 111 characterization, 110, 111 receptors, 106, 107, 308 see also granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF) common acute lymphoblastic leukemia antigen (CALLA), 150 common acute lymphoblastic leukemia (cALL) age peaks, 57 future research, 58 infection studies, 57–8 Communicable Disease Center (CDC) (US), Pneumocystis pneumonia studies, 13

Index

community participation, in pediatric cancer units, 632 competitive repopulating units (CRUs), 74–7 assay, 75 procedures, 74 robustness, 75 specificity, 74 characterization, 78 detection, 75 frequencies, 76, 77 localization, 75 markers, 84 competitive transplantation methodology, 75 complications gastrointestinal, 728–30 pulmonary, 611–12 see also acute complications; endocrinal complications; infectious disease complications; late treatment complications; major transplant-related complications (MTCs); neurologic complications complimentarity determining region 3 (CDR3), 214 concurrent organ dysfunction, 711 congenital acute myeloid leukemia diagnosis, 506–7 prognosis, 507 spontaneous remission, 507 symptoms, 507 use of term, 506 congenital leukemias, not associated with Down syndrome, 183 congenital neutropenia and cancer risk, 362 and myeloid malignancies, 362–3 see also severe congenital neutropenia (SCN) Consensus Conference on Management of Childhood Cancer Pain, 860 consolidation chemotherapy, acute promyelocytic leukemia, 520 consolidation therapy see intensification therapy constipation, treatment, 853 Convention on the Rights of the Child, 630 Cooke, J. V., 6 core-binding factor (CBF), roles, in acute myeloid leukemia, 501 core-binding factor complex, and acute myeloid leukemia, 307–15 core-binding factor leukemia, secondary mutations, 315–16 corrected count increment (CCI) determination, 834 reduced, 834 corticosteroids bone marrow blast cell reductions, 402 effects on IQ, 866 insufficiency, 734–5 and obesity development, 754–5 cortisone, in leukemia treatment, 9 cotrimoxazole (sulfamethoxazole), 13, 822–3 Cowden disease, 346 Coxsackie virus, 14

c-Raf, 143 Craigie, David (1793–1866), 3 cranial irradiation, 457 age factors, 866 and bone mineral density, 759–61 and early puberty, 751–2 effects on body height, 751 and growth impairment, 752, 754 and intelligence quotient, 865–7 and leukoencephalopathy, 757 and memory function, 867 neuropsychologic effects, 864–5 sex differences, 866–7 studies, 518 vs. craniospinal irradiation, 479 craniofacial development, and chemotherapy, 758 craniospinal irradiation, vs. cranial irradiation, 479 CREB-binding protein (CBP), 259, 321 Creutzfeldt-Jakob disease (CJD), transmission, 838 Crk proteins, 139 Crohn’s disease, 114 CRT see cranial irradiation CRUs see competitive repopulating units (CRUs) cryptococcal meningitis diagnosis, 812 etiology, 812 treatment, 812 cryptococcosis, 815 Cryptococcus neoformans, 815 infections, 807 and meningitis, 812 cryptosporidiosis, treatment, 815–16 Cryptosporidium parvum, 815–16 infections, 807 CSF-1 see colony-stimulating factor-1 (CSF-1) CSF see cerebrospinal fluid (CSF) C-SMADs, 129 CTAs (cancer–testis antigens), 650 CTLs see cytotoxic T lymphocytes (CTLs) CTP (cytidine triphosphate), 429 Curie, Marie (1867–1934), 6 Curvularia spp., infections, 807 cyclic AMP, 126–7 cyclin-dependent kinase inhibitors (CDKIs), 282–3 cyclin-dependent kinases (CDKs), 85–6 cyclins, 85–6 cyclophosphamide, 602, 655 in acute lymphoblastic leukemia treatment, 403 in Burkitt lymphoma treatment, 634–5 gonadal damage, 755 in juvenile myelomonocytic leukemia treatment, 581 leukemogenicity, 775 in lymphoid leukemia treatment, 9 metabolism, 403 and ovarian failure, 756

913

914

Index

cyclophosphamide (cont.) pharmacology, 403 toxicity, 760 cyclophosphamide/cytarabine/topotecan (CAT) regimen, in relapsed acute myeloid leukemia treatment, 543 cyclosporine A, 610 CYP1A1, polymorphisms, 59 CYP2D6, polymorphisms, 59, 777 CYP3A, metabolism, 777 CYP3A4 gene polymorphisms, 403, 777 roles, 780 CYP3A4 protein, metabolism, 780 CYP3A5 gene, polymorphisms, 403, 780 CYP3A5 protein, metabolism, 780 cystathionine B synthase (CBS), 367 activity, 562 polymorphisms, 521 cysteines, 116 cystic fibrosis transmembrane conductance regulator (CFTR), 664 cytarabine (ara-C), 10–11, 32, 254, 427–9 in acute myeloid leukemia treatment, 513 adverse reactions, 823 applications, 396, 460 in Burkitt lymphoma treatment, 634–5 cellular drug resistance, 416, 428 mechanisms, 427 cerebrospinal fluid concentrations, 396–7 clinical resistance, 396 in consolidation chemotherapy, 520 cytotoxicity, 396 and Down syndrome, 397 drug–drug interactions, 397–8 metabolic activation, 427–9 myeloblast sensitivity, 562 pharmacology, 396–8 in relapsed acute myeloid leukemia treatment, 542 in relapse treatment, 479 retention, 396 therapeutic mechanisms, 427 see also high-dose cytarabine (HDAC) regimen cytidine triphosphate (CTP), synthesis, 429 cytochemical analysis, 22–9 specimen preparation, 22–3 cytochemical staining, 22 advantages, 27 cytochrome c, release, 342 cytochrome P-450, 59 cytogenetic abnormalities analysis, 448–9 in childhood leukemias, 50 in infant leukemias, 56 primary, 238 cytogenetics of acute leukemias, 235–61 conventional, 235

future trends, 260–1 interphase, 235 molecular, 235–6 myelodysplastic syndrome, 554 nomenclature, 238 cytokine receptors classification, 125 cloning, 125 roles, 134–5 and self-renewal, 87 signaling pathways, 120, 136, 139–42 structure, 135 superfamily, 108, 125, 134–7 classification, 135 cytokines applications, 524 biological activity, 107 common receptor chains, 111 effects on hematopoiesis, 107 and hematopoietic stem cell proliferation, 86, 109 interferon-related, 114–15 lymphoid lineages, 111–13 myeloid lineages, 108–11 nomenclature issues, 106, 115–16 novel, 119–20 regulatory mechanisms, 111 roles, 120, 125 in hematopoiesis, 126 in lymphoid development, 111–13 specificity, 107 structural characterization, 106 in tumor vaccines, 666 see also hematopoietic growth factors; interferons (IFNs); transforming growth factors (TGFs); tumor necrosis factors (TNFs) cytomegalovirus (CMV), 612 detection, 612 infections, 806 transfusion-associated, 836 prevalence, 836 transmission, 836 cytomorphology, diagnostic issues, 225 cytopenia, 23 clonal, 549 single-lineage, 553 see also refractory cytopenia (RC); thrombocytopenia cytoplasmic dyes, 82 cytoplasmic granules, 26 cytoplasmic protein tyrosine kinases, 135 identification, 135 cytoplasmic vacuolization, blast cells, 30 cytotoxic drugs, 543 sensitivity, modification, 661, 669–70 cytotoxicity antibody-dependent cellular, 639–40

Index

and Fas ligand, 118 see also drug cytotoxicity assays cytotoxic therapy, and Down syndrome, 366–7 cytotoxic T lymphocytes (CTLs) antigen-specific, applications, 652–4 generation issues, 655 and Hodgkin disease, 654 targets, 649–50 therapeutic applications disadvantages, 657 effectiveness, 655 risk factors, 656 transduction, 656 dactinomycin, leukemogenicity, 778–9 DAG see diacylglycerol (DAG) Dalldorf, Gilbert, 14 Dameshek, William (1900–69), 571 Dana-Farber Cancer Institute, 517 Consortium, 456 protocol, 454, 865 DAPK (death-associated protein kinase), 350, 783 dapsone, 822–3 DAT (daunorubicin/cytarabine/thioguanine regimen), 516 daunorubicin, 10–11 in acute myeloid leukemia treatment, 513 in acute promyelocytic leukemia treatment, 519–20 cellular drug resistance, 416, 423–4 dosage, 444 and idarubicin compared, 513 leukemogenicity, 56, 778–9 liposomal, 542–3 pharmacodynamics, 398 pharmacokinetics, 398 pharmacology, 398 sensitivity to, 632–3 therapeutic mechanisms, 423 daunorubicin/cytarabine/thioguanine (DAT) regimen, studies, 516 DCC gene, expression, 523 dCK (deoxycytidine kinase), 396 dCK gene, abnormalities, 427–8 DCs see dendritic cells (DCs) DCTER (dexamethasone/ cytarabine/thioguanine/etoposide/daunomycin) regimen, 517 DDs see death domains (DDs) DEA see dye exclusion assay (DEA) death-associated protein kinase (DAPK), 350, 783 death domains (DDs), 130 activation, 130 roles, 130–1 death-inducing signaling complex (DISC), 344, 345 death receptors DR4, 344

DR5, 344–5 pathways, and chemoresistance, 344–5 DEK-CAN, 258 DEK gene, 325 delayed intensification therapy see reinduction therapy deletions, definition, 238 Delore, P., 7 Delta notch ligands, 134 demographic factors, and childhood leukemias, 51–4 denaturing gradient gel electrophoresis (DGGE), 220 dendritic cells (DCs), 654 antigen presentation, 648 immunotherapy with, 654–5 Denmark, family studies, 7 dental abnormalities, 758–9 deoxycytidine kinase (dCK), 396 Dependovirus spp., 663 desensitization, systematic, 861–2 developed countries mortality rates, 627 under-five, 626 developing countries acute leukemias, 625–36 future trends, 636 acute leukemia treatment, 632–6 acute lymphoblastic leukemia, 632–4 acute myeloid leukemia, 635–6 Burkitt lymphoma, 634–5 cancers, 625–6 cultural development, 626–7 epidemiology, 625 health priorities, 625–6 infectious diseases, 625 lymphoma treatment, 632–6 malignant neoplasms, social burden, 625–6 mortality causes, 625 rates, 626–7 pediatric cancers, 625–6 survival rates, 627 pediatric cancer units establishment, 630–2 establishment requirements, 628 implementation barriers, 628–30 implementation rationale, 630 political development, 626–7 socioeconomic development, 626–7 development and hematopoietic stem cell transplantation, 613 see also pubertal development dexamethasone in acute lymphoblastic leukemia treatment, 402 cellular drug resistance, 416 in continuation treatment, 459 dosage, 402

915

916

Index

dexamethasone (cont.) effects on IQ, 866 and fracture risk, 760 and obesity development, 754–5 and osteonecrosis, 732 pharmacology, 402–3 in remission induction therapy, 455 see also prednisolone dexamethasone/cytarabine/thioguanine/etoposide/daunomycin (DCTER) regimen, studies, 517 dexrazoxane, in remission induction therapy, 455 dFDC see gemcitabine DFS see disease-free survival (DFS) DGGE (denaturing gradient gel electrophoresis), 220 DHFR gene, mutations, 426–7 diacylglycerol (DAG), 139 biosynthesis, 127 diagnostic clonality, IG/TCR gene analysis in, 220–8 dialysis, 718 Diamond–Blackfan anemia, 552 diarrhea, treatment, 815–16 diazepam, effectiveness, 859 dic(9;20)(p13;q11.2), 248 diepoxybutane testing, 372 diet and childhood leukemias, 54 neutropenic, 821 and topoisomerase II inhibition, 56–7 differential staining cytotoxicity (DiSC) assay see dye exclusion assay (DEA) differentiating-inducing therapy, 11–12 differentiation concept of, 106 lineages, 227 regulatory mechanisms, 106 see also hematopoietic cell differentiation differentiation antigens, 649–50 differentiation therapy, acute promyelocytic leukemia, 544 diffuse large B-cell lymphomas (DLBCLs), 347 2 ,2 -difluorodeoxycytidine (dFDC) see gemcitabine digital karyotyping, 235 dimorphic T/myeloid acute biphenotypic leukemia, 37 diphenhydramine, 853 diploidy, 240 see also hyperdiploidy; hypodiploidy DiSC assay see dye exclusion assay (DEA) DISC (death-inducing signaling complex), 344, 345 disease-free survival (DFS), 558 rates, 558–9 distraction cognitive, 861–2 effectiveness, 859 multisensory, 861–2 in pain management, 855, 859 and parent-mediated interventions, 860

distress cognitive-behavior therapy, 860 parental, 863 parent-mediated interventions, 860 procedural, 858 see also chemotherapy-related distress diversity and joining (DJ) regions, 154 DLBCLs (diffuse large B-cell lymphomas), 347 DLIs see donor lymphocyte infusions (DLIs) DNA amplification, 4, 679–80 DNA-based vaccines, 666–7 DNA damage, 56 alkylating agent-induced, 776–7 anthracyclines, 424 DNA index, 473–4 DNA integrase, 5 DNA microarray analysis, 39–40, 284, 449 DNA polymerase, 5 DNA probes, 4, 236 centromeric, 236 in fluorescence in situ hybridization, 236 painting, 236 telomeric, 236 unique-sequence, 236 DNA repair proteins, 777 DNA topoisomerase II, 424–5 DNA topoisomerase II inhibitor-related leukemias clinical features, 788 cytogenetic changes, 783–8 genetic predisposition, 776, 780–1 incidence, 778–80 molecular genetic changes, 783–8 risk factors, 778–80 DNA topoisomerase II inhibitors in chemotherapy, 774, 778 leukemogenesis, 774, 780 mechanisms, 778 DNRs (dominant-negative receptors), 669 DOCK180, 139 dominant-negative receptors (DNRs), 669 donor lymphocyte infusions (DLIs), 602–3 post-transplant, 607 donor matching and graft-versus-host disease, 600 for hematopoietic stem cell transplantation, 599–600 matched sibling donors, 601 see also matched family donors (MFDs); matched unrelated donors (MUDs) dorsal receptor complex, 128 dose–response relationship, antileukemic drugs, 391 Double ABCX model, 862 Dounreay (UK), childhood leukemia incidence, 53 Down syndrome, 5, 7, 50–1, 364–7 abnormal hematopoiesis, 509 and acute lymphoblastic leukemia

Index

therapeutic outcomes, 366–7 treatment, 460–1 and acute megakaryoblastic leukemia, 186, 366, 417–18, 499 therapeutic outcomes, 367 and acute myeloid leukemia, 365, 508–10 treatment outcomes, 367 and cancer risk, 362, 363 comorbidity, 365 risk factors, 365 and complication risk, 711 and cytarabine treatment, 397 demography, 364 gene mutations, 254 and immunologic abnormalities, 365 incidence, 364 and leukemia, 183–6, 363, 365 cellular drug resistance, 417–18 pathogenesis, 365–6 predisposing genes, 366 and myelodysplastic syndrome, 509–10, 551 pathogenesis, 365–6 phenotype, 364–5 and proliferative disorders, 363 therapy, 366–7 and transient myeloproliferative disorder, 559–60 incidence, 509 and transient myeloproliferative disorders, 183–6, 508–9 see also myeloid leukemia in Down syndrome doxorubicin, 775 and heart disease, 757–8 leukemogenicity, 56, 775, 778–9 pharmacodynamics, 398 pharmacokinetics, 398 pharmacology, 398 DQA1 alleles, 58 Drosophila spp. (flies) Antennapedia gene, 275, 277, 317 armadillo gene, 127 Bithorax gene, 277, 317 exd protein, 275 Frizzled receptors, 127 HOM proteins, 275 nervy gene, 312 notch gene, 282 notch receptors, 134 notch signaling pathway, 117 runt gene, 307 Serrate gene, 134 SMADs, 129 toll/IL-1 receptors, 128 toll receptor, 128 transcription factors, 272 trithorax gene, 244, 277, 317, 319, 783–4 Ultrabithorax gene, 275

drug cytotoxicity assays, 414–15 advantages, 418 in childhood leukemias, 415–18 developments, 414 disadvantages, 418 less-used, 415 drug-metabolizing enzymes, polymorphisms, 59 drug resistance, 9–10 in relapsed acute myeloid leukemia, 542 in relapse treatment, 476 see also cellular drug resistance; chemoresistance; multiple drug resistance (MDR) drug resistance proteins, 429 drug response antileukemic drugs, 391 and childhood leukemias, 391 interpatient variability, 391 drugs antiangiogenic, 545 antifungal, 524 antipurine, 14 cross-resistance, 429–30 sedative-hypnotic, 854 see also antifolate drugs; antileukemic drugs; cytotoxic drugs; nonsteroidal anti-inflammatory drugs (NSAIDs) Druker, B. J., 12 Duffy blood group, 116 duplications, 238 Dutch-Belgian Hemato-Oncology Cooperative Group, 517 Dutch Childhood Leukemia Study, 415 dye exclusion assay (DEA), 414 advantages, 414 applications, 415 disadvantages, 414 and methyl tetrazolium assay compared, 415 dysplasia definitions, 553 see also myelodysplasia E2A-FB1 fusion gene, 242 E2A-HLF fusion gene, 242, 276–7 apoptosis induction, 276 E2A-PBX1 fusion gene, 5, 167–8, 242, 275, 276, 440 detection, 276 expression, 276 roles, 275–6 transcript, 32 E2A proteins deficiency, 275 roles, 275 early puberty, and cranial irradiation, 751–2 Eastern Cooperative Oncology Group, 517 EBMT see European Group for Blood and Marrow Transplantation (EBMT)

917

918

Index

EBNA-1 antigen expression, 648–9, 654 roles, 490 EBNA-3B antigen, deletion, 653 EBNA (Epstein–Barr nuclear antigen) testing, 490 EBV-LPD see Epstein–Barr virus-associated lymphoproliferative disease (EBV-LPD) ECD (phycoerythrin-Texas red conjugate), 150 echinocandins, 524, 823 ECM (extracellular matrix), 139 ecthyma gangrenosum, 809 edema, vasogenic, 763 educational attainment, acute lymphoblastic leukemia treatment effects, 867–8 EFS see event-free survival (EFS) EGIL (European Group for the Immunological Characterization of Leukemias), 169, 181–2 egl1 gene, 276–7 EGR1 gene, 781 Egypt, T-cell acute lymphoblastic leukemia, 625 Ehrlich, Paul (1854–1915), 3 EKLF gene, 782–3 ELA2 gene, mutations, 373 ELA-Max cream, 859, 860 electricity power supplies, exposure studies, 53–4 electromagnetic radiation and childhood leukemia, 53–4 exposure, 52 electrophoresis, in polymerase chain reaction, 680 Elion, Gertrude Belle (1918–99), 9, 13 Elitek (rasburicase), 716 Ellerman, V., 5 ELL gene, 321 El Salvador pediatric cancer units, 628–9, 630 collaborative treatment programs, 632–4 treatment abandonment issues, 634 El Salvador-I protocol, 632–3 El Salvador-II protocol see Guatemala, Honduras, El Salvador-II (GHS-II) protocol Elspar, 452–4 embryogenesis, and hematopoiesis, 71 EMBT see European Bone Marrow Transplantation (EMBT) EMLA see eutectic mixture of local anesthetics (EMLA) encephalitis, 812 diagnosis, 812 etiology, 812 endocrinal complications, 733–5 and hematopoietic stem cell transplantation, 869 endocrine toxicity, 750–6 and gonadal function, 755–6 and growth, 750–2 and pregnancy outcomes, 756 and pubertal development, 750–2

ENL gene, 278, 321 ENT1 protein, expression, 428–9 environmental exposure, and leukemogenesis, 362, 363 environmental factors and childhood leukemias, 51–4 and genetic factors, 58–9 environmental model institutional level, 883 intermediate level, 882–3 internal level, 882 of nursing care, 882–3 environmental tobacco smoke (ETS), exposure, 871 enzyme deficiencies and complication risk, 711 ethnic and racial differences, 711 eosinophilia, 39 comorbidity, 587 etiology, 587 eosinophils, 116 eotaxin, 116 epipodophyllotoxins, 10, 424–5, 552 cellular drug resistance, 422 mechanisms, 424–5 leukemogenicity, 56, 256, 778 dosage factors, 779 multidrug resistance transmembrane transporters, 425 pharmacology, 398–400 and second malignancies, 764 therapeutic mechanisms, 424 topoisomerase II inhibition, 56 EPO see erythropoietin (EPO) Epstein–Barr nuclear antigen (EBNA) testing, 490 Epstein–Barr virus-associated lymphoproliferative disease (EBV-LPD), 613 cellular immunotherapy, 652–4 Epstein–Barr virus (EBV), 5, 648–9 and B-cell acute lymphoblastic leukemia, 490 and Burkitt lymphoma, 490, 625 infections, 806, 837 and lymphomas, 649 ERB-2 gene, expression, 668 Erwinia chrysamthemi asparaginase, 400–1, 454 dosage issues, 454–5 erythema infectiosum, 837 erythroblasts characteristics, 24, 26 leukemic, 3 erythrocytes see red blood cells (RBCs) erythroid colony-forming cells, 80 terminal differentiation, 70 erythroid lineages immunophenotyping, 158–9 maturation, 158–9 erythroleukemia, 3

Index

erythropoiesis, 159 mechanisms, 110 erythropoietin (EPO), 80, 134, 137, 844 applications, 80, 844 clinical trials, 845 deficiency, 584–5 receptors, 106, 135 expression, 109–10 roles, 109–10 nonredundant, 110 structure, 106, 109 Escherichia coli, 812–13 asparaginase, 400–1, 452–4 dosage issues, 454–5 infections, 806–7 catheter-related, 840 esophagitis, 815 essential thrombocytosis (ET), 80, 586 diagnosis, 586 disease course, 586 etiology, 586 incidence, 586 treatment, 586 esterase enzymes, staining, 28–9 ET see essential thrombocytosis (ET) Etanercept, 612 ETO gene, 312–13 etoposide, 10–11, 775 accumulation, 399 cellular drug resistance, mechanisms, 424–5 cytotoxicity, 400 demethylation, 399 leukemogenicity, 7, 56, 256, 778 issues, 779 metabolism, 786 pharmacokinetics, 398–9 pharmacology, 398–400 therapeutic mechanisms, 424 toxicity, 399 etoposide catechol, metabolism, 780 ETS (environmental tobacco smoke), 871 ETV6-CBFA2 fusion gene, 5, 246–7 detection, 247–8 occurrence, 247 prognostic significance, 247 see also TEL-AML1 fusion gene ETV6 gene abnormalities, 235, 246, 259 deletion, 248 rearrangements, 247, 248 see also TEL gene European Group for Blood and Marrow Transplantation (EBMT), 543, 558 Chronic Leukemia Registry, 584

European Group for the Immunological Characterization of Leukemias (EGIL), 169, 181–2 European Lymphoma Bone Marrow Transplant Registry, 494 European Working Group on Myelodysplastic Syndrome (EWOG-MDS), 553, 555–6, 607 studies, 557, 581 eutectic mixture of local anesthetics (EMLA), 854 creams, 859, 860 event-free survival (EFS), 239, 474, 475, 476, 629 and allogeneic stem cell transplantation, 481 myeloid leukemia in Down syndrome, 562 in pediatric cancer units, 628 and relapse category, 475 EVI1 gene, 257–8, 313, 671–3 EWOG-MDS see European Working Group on Myelodysplastic Syndrome (EWOG-MDS) exd protein, 275 exhaust gases, benzene in, 7 external tunneled catheters, 839 extracellular matrix (ECM), 139 extramedullary disease, and acute promyelocytic leukemia, 512 extramedullary myeloid tumors see myeloid sarcomas extramedullary relapses, 541 extravasation, 841 mechanisms, 841 E -Myc transgenic mice, 344, 347 genetic studies, 342–3 lymphomagenesis studies, 489 FA see Fanconi anemia (FA) FAB classification see French-American-British (FAB) classification Faber, S., 14 Face, Legs, Activity, Cry, Consolability (FLACC) Scale, 850, 851 Faces Pain Scale (FPS), 850, 852, 890 FADD protein, 130–1, 132, 344 roles, 131 up-regulation, 345 FAK see focal adhesion kinase (FAK) famciclovir, 810, 815 familial cancer syndromes genetic studies, 362 incidence, 362 mechanisms, genetic, 363 inheritance, 362 familial erythrocytosis, 585–6 familial (hereditary) thrombocytopenia, 586–7 familial monosomy 7, 51 familial myeloid disorders with monosomy 7, 374–5 demography, 374 pathogenesis, 374–5 phenotype, 374 risk factors, 500 therapy, 375 familial patterns, and childhood leukemias, 51

919

920

Index

familial platelet disorder/acute myelogenous leukemia (FPD/AML), 375 comorbidity, 375 demography, 375 incidence, 375 leukemogenesis, 363 pathogenesis, 375 phenotype, 375 therapy, 375 familial Wilms tumor, etiology, 362 family adjustment and bone marrow transplantation, 863 and childhood leukemias, 862–4 and family cohesion, 863 and family environment, 863 models, 862–3 circumplex, 862–3 Double ABCX, 862 family life cycle, 863 see also child adjustment family cohesion, and family adjustment, 863 family education content, 884, 885 and nursing care, 883–4 family environment, and family adjustment, 863 family life cycle model, 863 family studies, leukemia, 7 FANCD2 protein, 372 FANC gene, mutations, 375 Fanconi anemia (FA), 51, 371–3, 550, 553 and cancer risk, 362 characterization, 371 comorbidity, 371–2 and complication risk, 711 demographics, 371 diagnostic criteria, 372 hematopoietic stem cell transplantation, 372–3 incidence, 371 and leukemia, 372 and myelodysplastic syndrome, 551 and myeloid malignancies, 362–3 pathogenesis, 372 phenotype, 371–2 therapy, 372–3 Farber, Sidney (1903–73), 8–9, 14 farnesyl transferase inhibitors (FTIs), 544 synthesis, 578–9 Fas-associated Death Domain (FADD) protein see FADD protein Fasurtec (rasburicase), 716 Fas gene, mutations, 118 Fas ligand and cytotoxicity, 118 roles, 117 Fas receptors, 125 activation, 340

and chemoresistance, 344–5 regulatory mechanisms, 344 fatigue, treatment-induced, 888 Fc receptors, 125, 138 family, 137–8 signaling, 137–9 tyrosine phosphorylation, 138 FDA (Food and Drug Administration) (US), 13, 639, 643, 839–40, 844 febrile neutropenia antibiotic therapy, 817–18 management, 818–19 definitions, 816 evaluation, 816–17 management, 816–19 use of term, 806 Feline leukemia virus, 134 females, pubertal development, 756 fentanyl, 854 fetal liver kinase 1 (Flk1) deficiency, 108 expression, 107, 108 fetal loss, and childhood leukemias, 55 fever and leukemia, 441 management, 816–19 see also febrile neutropenia FFP see fresh frozen plasma (FFP) FGFR1 gene, 258 fibroblasts, 78, 119 sources, 78 fibrosis, 23 FIP1L1 gene, 588 FISH see fluorescence in situ hybridization (FISH) FITC (fluorescein isothiocyanate), 150 FK506-binding protein (FKBP), 656 modification, 669 FKHR protein, 321 FLACC (Face, Legs, Activity, Cry, Consolability) Scale, 850, 851 Flice-like inhibitory protein (FLIP) see FLIP protein flies notch signaling pathway, 117 see also Drosophila spp. (flies) FLIP protein, 344 and apoptosis, 132 Flk1 see fetal liver kinase 1 (Flk1) flow cytometry, 27, 31, 32–3 acute lymphoblastic leukemia, 694–6 acute myeloid leukemia, 697 MRD detection issues, 688 advantages, 690 disadvantages, 160, 690 four-color, 688 immunophenotypes, 687 in immunophenotyping, 150, 160

Index

limitations, 554–5, 690 lineage markers, 82 minimal residual disease detection, 687–90, 695 multiparameter, 81–2 advantages, 160 principles, 687 Flt1 (fms-like tyrosine kinase 1), 107, 108 Flt3 see fms-like tyrosine kinase 3 (Flt3) FLT3 gene, 255, 785 expression, 245 internal tandem duplications, 501, 522–3, 681–2 mutations, 315–16, 501, 522–3 overexpression, 449 prognostic significance, 523 fluconazole, 13–14, 820, 823 flucytosine, 820 fludarabine, 602, 655 in relapsed acute myeloid leukemia treatment, 543 fluorescein isothiocyanate (FITC), 150 fluorescence in situ hybridization (FISH), 32, 235, 236 applications, 236, 237–8, 239, 245, 278 myelodysplastic syndrome studies, 554 DNA probes, 236 multicolor karyotyping, 236 procedures, 236 see also multiplex-fluorescence in situ hybridization (M-FISH); spectral karyotyping (SKY) fluorescent probes, 236 fluorochromes, 150 fluorometric microculture cytotoxicity assay (FMCA), 415 fluoroscopes, 6 5-fluorouracil, leukemogenicity, 775 FMCA (fluorometric microculture cytotoxicity assay), 415 fms-like tyrosine kinase 1 (Flt1), expression, 107, 108 fms-like tyrosine kinase 3 (Flt3) biological activity, 108 expression, 278–9 inhibitors, 278–9 mutations, 108, 526 roles, 108 in acute myeloid leukemia, 501 focal adhesion kinase (FAK), 139 recruitment, 139 folate-related genes, polymorphisms, 440 folate supplementation, studies, 54 folic acid, 14 deficiency, and leukemia, 7 in leukemia treatment, 8–9 follicle-stimulating hormone (FSH), 756 folylpolyglutamate synthetase (FPGS), 391, 425 activity, 426 Food and Drug Administration (FDA) (US), 13, 639, 643, 839–40, 844 Ford, A. M., 7 formazan, 414

Fowler solution, 8 fowl leukemia, transmission, 5 FoxO proteins, 346 FPC scoring system, 557 FPD/AML see familial platelet disorder/acute myelogenous leukemia (FPD/AML) FPGS see folylpolyglutamate synthetase (FPGS) FPS (Faces Pain Scale), 850, 852, 890 Fractalkine, 116 free radical scavengers, formation, 423–4 French-American-British (FAB) classification, 23, 30, 38, 166, 473 acute lymphoblastic leukemia acute myeloid leukemia, 33, 502–3 issues, 178 myelodysplastic syndrome, 549 French Society of Pediatric Hematology and Immunology, postremission therapy studies, 517 French Society for Pediatric Oncology (SFOP), LMB-89 regimen, 492, 493 fresh frozen plasma (FFP), 834–5 administration, 835 complications, 835 components, 834–5 indications, 835 risk factors, 835 Friedreich, Nikolaus (1796–1862), 3 Frizzled receptors occurrence, 127 signaling, 125, 127–8 mechanisms, 128 proteins, 127–8 structure, 127 FSH (follicle-stimulating hormone), 756 FTIs see farnesyl transferase inhibitors (FTIs) fungal diseases, 823 fungal infections, 524 and leukemia, 807 and neutropenia, 819–21 FUS-ERG fusion gene, 259 fusion transcripts, 664 Fy gene, 116 Fyn kinases, 155 gabapentin, 853 GAS7 gene, 784 gastroenteritis, 815–16 diagnosis, 815 gastrointestinal complications, 728–30 GATA1 gene, 782–3 mutations, 254, 324–5, 509, 561 and Down syndrome, 366, 367 GC-GCRs see glucocorticoid-glucocorticoid receptors (GC-GCRs) GCRs see glucocorticoid receptors (GCRs) G-CSF see granulocyte colony-stimulating factor (G-CSF) GDP (guanosine diphosphate), 126

921

922

Index

GEF (guanine exchange factor), 142–3 gemcitabine, 525–6 in relapsed acute myeloid leukemia treatment, 543 gemtuzumab ozogamicin, 526, 544, 643–4 in acute promyelocytic leukemia treatment, 521 FDA approval, 639, 643 see also calicheamicin Genasense, 344 gene expression aberrant, 681–2 changes, 276, 280 during hematopoietic cell differentiation, 84 regulatory mechanisms, 87 gene expression profiling, 39–40, 449 MLL fusion genes, 278–9 gene knock-out studies, 87, 277 gene marking, 670–1 for autologous stem cell transplantation, 670–1 T lymphocytes, 671 new methodology, 671 genes antigen receptor, 691–4 in cancer therapy, 665 roles, in apoptosis, 284 see also chimeric genes; oncogenes; tumor suppressor genes GeneScan analysis, 220, 221, 222 detection limits, 227 procedures, 220 gene therapy, leukemia induction, 671–3 genetic abnormalities, and cellular drug resistance, 417–18 genetic disorders and complication risk, 711 and leukemogenesis, 711 use of term, 362 genetic factors and childhood leukemias, 50–1 and environmental factors, 58–9 genetic mutations frequencies, 58 secondary, 439 genetic risk evaluation, 284 genetics and acute lymphoblastic leukemia classification, 31–2 and leukemia, 7–8 see also cytogenetics; molecular genetics; pharmacogenetics genetic syndromes, and childhood leukemias, 50–1 genetic targeting, 11–12 gene transfer applications, 661–73 concept of, 661 drug-resistant, 669–70 disadvantages, 670 efficiency improvements, 666 future trends, 673 host modification, 667

methods, 661–73 strategies, 661 tumor cell modification, 664–7 gene transfer vectors, 661–4 applications, 672 properties, 671 synthetic, 664 genistein, 645 German AML Cooperative Group, HAM regimen studies, 517 German Registry, 50 GHS-II protocol see Guatemala, Honduras, El Salvador-II (GHS-II) protocol Giardia lambia, 815 GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto), 520–1 Gleevac see imatinib mesylate Gloor, W., 9, 14 glucocorticoid-glucocorticoid receptors (GC-GCRs), 419 mechanisms, 418 glucocorticoid receptors (GCRs) binding, 418 and cellular drug resistance, 418–19 polymorphisms, 418–19 splice variants, 418 glucocorticoids, 418–20 in acute lymphoblastic leukemia treatment, 402 cellular drug resistance, 415, 416 mechanisms, 418–20 in continuation treatment, 459 drug–drug interactions, 402–3 pharmacology, 402–3 target gene interactions, 420 therapeutic mechanisms, 418 and thrombosis risk, 727 glucocorticoids-efflux, via transmembrane transporters, 420 glucose-6-phosphate dehydrogenase deficiency, 711 studies, 439 glutathione S-transferases, 59 free radical scavenging, 424 glycogen synthase kinase-3 (GSK-3), 127, 346 inhibition, 127–8 roles, 127 GM-CSF see granulocyte-macrophage colony-stimulating factor (GM-CSF) GM-CSF gene, 308 activation, 576 GMPS gene, 784 Goldin, A., 9 gonadal function and bone marrow transplantation, 756 and endocrine toxicity, 755–6 Good, R. A., 7 G-proteins, subunits, 126, 127 graft failure, 608

Index

graft-versus-host disease (GvHD), 75, 480, 581, 608–10 acute, 608–9 and alloreactivity, 650, 651–2 chronic, 609–10 and donor matching, 600 and graft-versus-leukemia effect, 602 induction, 652 prevention, 601 prophylaxis, 558 protective effects, 543–4 and red blood cell transfusions, 831 symptoms, 609 treatment, 610 graft-versus-leukemia (GvL) effect, 480, 581 and alloreactivity, 650 and graft-versus-host disease, 602 roles, 602 gram-negative bacteria, 13, 811, 840 granulocyte colony-stimulating factor (G-CSF), 134 administration, 835, 843 duration, 843–4 post-chemotherapy, 843 applications, 601, 819 precautions, 842 therapeutic, 110–11, 452, 524, 841–4 chemotherapy sensitization studies, 517 contraindications, 842–3 cost issues, 844 dosage issues, 843 quality-of-life issues, 844 roles, 107, 110 in granulocyte production, 110, 111 secondary leukemia risk factors, 779–80 in severe congenital neutropenia treatment, 373, 374, 551–2 structure, 110 trials, 842, 843 granulocyte-macrophage colony-stimulating factor (GM-CSF) applications, 601 therapeutic, 524, 652, 819 biosynthesis, 576 dose–response curves, 576 hypersensitivity, 576 receptors, 135 roles, 109, 571, 578 signaling pathways, 109, 577 granulocytes generation, 79–80, 110, 111 neutrophilic, 69 granulocyte-stimulating factors, 841–4 granulocyte transfusions, 835 administration, 835 preparation, 835 granulocytic sarcomas see myeloid sarcomas granulocytopenia, 443

granulopoietic colony-forming cells, 79–80 terminal differentiation, 70 granzyme B, 340 Grb-2 protein, 143–4 Greater Delaware Valley Pediatric Tumor Registry (US), 50 Greaves, M. F., 6 GR gene, mutations, 402–3 Gro (melanoma growth stimulatory activity), 116 Gross, Ludwik (1904–99), 5, 6, 14 murine studies, 14 growth, and hematopoietic stem cell transplantation, 613 growth factors identification, 111 receptors, 106, 279 thrombopoietic, 846 see also hematopoietic growth factors; tumor growth factor
(TGF
); tumor growth factor  (TGF) growth hormone, receptors, 135 growth hormone therapy effects, 753 and leukemogenesis, 753 and neoplasms, 753 growth impairment and bone marrow transplantation, 753 and cranial irradiation, 752, 754 and endocrine toxicity, 750–2 Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA), consolidation chemotherapy studies, 520–1 GSK-3 see glycogen synthase kinase-3 (GSK-3) GSTM1 gene, 780 polymorphisms, 59, 777–8 GSTP1 gene, polymorphisms, 777–8 GSTT1 gene, 780 deletions, 521 expression, 400 polymorphisms, 59, 777–8 GTP (guanosine triphosphate), 126 guanine exchange factor (GEF), 142–3 guanosine diphosphate (GDP), 126 guanosine triphosphate (GTP), 126 Guatemala, Honduras, El Salvador-II (GHS-II) protocol, 633–4 applications, 634 continuation therapy, 633 GUS gene, 681 Guthrie blood spots, neonatal, 4, 5, 14–15 Guthrie, Robert, 14–15 GvHD see graft-versus-host disease (GvHD) GvL effect see graft-versus-leukemia (GvL) effect H&E (hematoxylin-eosin) stains, 23 HA1 antigen, 650–1, 652 HA2 antigen, 650–1 HA-14–1, 344 Haemophilus influenzae, 612 HAM (high-dose cytarabine and mitoxantrone) regimen, 517

923

924

Index

haploinsufficiency, and leukemogenesis, 363 haplotypes, 600 Hartenstein, V., 7 HAV (hepatitis A virus), 837 HbF see hemoglobin F (HbF) HBV see hepatitis B virus (HBV) 4-HC (hydroperoxycyclophosphamide), 670 HCV see hepatitis C virus (HCV) HDAC regimen see high-dose cytarabine (HDAC) regimen HDM2 gene, 349 hDMP1 gene, 781 HDMTX see high-dose methotrexate (HDMTX) HDV (hepatitis D virus), 837 health behaviors, 870–1 adaptive, 870 adolescents, 870–1 maladaptive, 870 Health Belief Model, 872 health education programs, 871–2 effectiveness, 871–2 see also family education; patient education health professionals behaviors, 891 in pediatric cancer units, 632 see also nurses health promotion, 870–2 combined model approach, 872 current interventions, 871–2 effectiveness, 871–2 and nursing care, 891 health risk, perceptions, 872 hearing loss, drug-induced, 757 heart disease, drug-induced, 757–8 hematogones, 171–2 hematologic malignancies, incidence, 551 hematologic supportive care, 829–46 blood components, 829–35 future trends, 846 venous access, 838–9 hematopoiesis abnormal, 509 anatomy, 69–88 changes during, 71–2 concept of, 69 cytokine effects, 107 developmental origins, 71–2 and embryogenesis, 71 genes in, 299 genetic disorders, 8 hierarchical model of, 69–70 localization, 71 notch signaling pathway, 117 and peripheral blood stem cells, 601 physiology, 69–88

regulatory mechanisms, 84–8, 106, 107 concepts, 84–5 extrinsic control, 85 intrinsic control, 85 signal transduction in, 125–44 in Shwachman–Diamond syndrome, 370 transcription factors, 87 hematopoietic cell differentiation, 69 asymmetric, 70 functional assays, 72 gene expression changes, 84 mechanisms, 71 murine, 83 stages, phenotypic markers, 81–4 hematopoietic cell surface antigens, 639 targeting studies, 642–5 hematopoietic growth factors, 106–20, 841–6 characterization, 106 mechanisms, 841 roles, 106 structure, 106 see also granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF) hematopoietic malignancies, prevalence, 220–1 hematopoietic neoplasms, classification, 33 hematopoietic stem cells (HSCs) assays, 70 characterization, 75 functional assays, 70, 72–4 colony-scoring analysis, 73 in vitro, 73 in vivo, 72–3 limitations, 73 limiting-dilution analysis, 73 reproducible conditions, 72 specificity, 73 gene marking, 670–1 hierarchy, 69 with in vitro repopulating activity, 132 with in vivo repopulating activity, 74–7 lineage restriction control, 87–8 lineage restriction events, 71 murine, 74 mutations, 69 pluripotent, 70 progressive development, 72 proliferation control, 85–6 proliferative potential, 74 quiescent phase, 107 regulatory mechanisms, 85, 107–8 research, 107 self-renewal, 76 staining, 82 survival, 107, 108

Index

transdifferentiation, 88 viability control, 85–6 hematopoietic stem cell transplantation (HSCT), 364, 599–614 in acute lymphoblastic leukemia treatment, 459–60, 603–6 in acute myeloid leukemia treatment, 518, 606–7 in adult-type chronic myeloid leukemia treatment, 607–8 in advanced acute leukemia treatment, 644–5 advantages, 480 complications infectious, 612 post-transplant, 610–13, 869 pulmonary non-infectious, 611–12 and development, 613 donor selection, 599–600 and endocrinal complications, 869 in Fanconi anemia treatment, 372–3 future trends, 614 graft failure, 608 and growth, 613 historical background, 599 immune reconstitution, 613 as immunotherapy, 602–3 indications, 603–8 in infant ALL, 460 and infertility, 613 in juvenile myelomonocytic leukemia treatment, 581–2 matching issues, 543 and minimal residual disease monitoring, 693–4 morbidity factors, 608–13 mortality factors, 608–13 in myelodysplastic syndrome treatment, 607 and neurocognitive deficits, 869 and neurologic impairments, 869 nonmyeloablative, 602 opportunistic pathogens, 612 post-engraftment phase II, 612 post-engraftment phase III, 612 post-transplant malignancies, 614 pre-engraftment phase I, 612 preparative regimens, 602 psychosocial issues, 868–9 relapse after, 544 in relapsed acute lymphoblastic leukemia treatment, 480–1 experimental approaches, 481 in relapsed acute myeloid leukemia treatment, 543 relapse risk, 602–3 second transplants, 608 in severe congenital neutropenia treatment, 374 and sexual dysfunction, 869 stem cells ex-vivo manipulations, 601 sources, 600–1 and survival rates, 480 in treatment-related acute myeloid leukemia treatment, 607 in treatment-related leukemia treatment, 789–90

in treatment-related myelodysplastic syndrome treatment, 607 vs. chemotherapy, 480 see also allogeneic stem cell transplantation; autologous stem cell transplantation; bone marrow transplantation (BMT) hematopoietic system formation, 309–10 reconstitution, 107 hematopoietic transplantation, 11, 12 risk factors, 14 hematosuppression, concept of, 11 hematoxylin-eosin (H&E) stains, 23 hemoglobin levels, 831 as marker, 159 hemoglobin F (HbF), 575 elevated levels, 572, 575 Hemophilus influenzae, infections, 806–7 hemopoietins, roles, 345 heparan sulfates, 78 heparin, in thrombosis treatment, 728 hepatic fibrosis, and transient myeloproliferative disorder, 367 hepatic insufficiency, and complication risk, 711 hepatitis, 815 hepatitis A virus (HAV), 837 hepatitis B virus (HBV) infections, transfusion-associated, 836 prevalence, 836 hepatitis C virus (HCV) infections, 760–1 transfusion-associated, 836 prevalence, 836 screening, 836 hepatitis D virus (HDV), risk factors, 837 hepatitis E virus (HEV), 837 hepatosplenic T-cell lymphoma, 188–90 hepatotoxicity, 729 hereditary thrombocytopenia, 586–7 heritable predispositions childhood hematologic malignancies, 362–80 genetic mechanisms, 363 leukemia, genetic mechanisms, 363 herpes simplex virus (HSV) and encephalitis, 812 infections, 810 diagnosis, 810 symptoms, 810 treatment, 810 herpes simplex virus I, 669 herpes simplex virus 1-thymidine kinase, 656 herpesviruses, 806 as gene transfer vectors, 664 human herpesvirus 6, 806 and leukemia, 5, 12–13 see also Epstein–Barr virus (EBV) Herpesvirus saimiri, 119

925

926

Index

herpesvirus thymidine kinase, 656 herpes zoster, 12–13, 806 diagnosis, 810 treatment, 810 HES see hypereosinophilic syndrome (HES) heteroduplex analysis, 220, 221, 222 advantages, 220 detection limits, 227 procedures, 220 HEV (hepatitis E virus), 837 HGPRT see hypoxanthine–guanine phosphoribosyltransferase (HGPRT) HHV-6 (human herpesvirus 6), 806 hierarchical models, of hematopoiesis, 69–70 high-dose cytarabine (HDAC) regimen, 396 in postremission therapy, 517–18 studies, 516 high-dose cytarabine and mitoxantrone (HAM) regimen, studies, 517 high-dose methotrexate (HDMTX), 392 guidelines, 636 high proliferative potential colony-forming cells (HPP-CFCs), use of term, 81 Hispanics acute promyelocytic leukemia, 5, 48–9, 625 secondary leukemias, 779 histochemical staining, 23 Histoplasma capsulatum, 812–13, 814 infections, 807 histoplasmosis diagnosis, 814 symptoms, 814 Hitchings, George H. (1905–98), 9 HIV/AIDS, 132 comorbidity, 489 proteins, toxicity, 662 transfusion-associated, 836–7 risk factors, 836–7 screening, 836–7 HLAs see human leukocyte antigens (HLAs) HLF gene, 276 HMG proteins, roles, 277 hMSH2 gene, 777 Hodgkin disease, 649 chemotherapy, 639 and cytotoxic T lymphocytes, 654 and interleukin 9, 112 and secondary leukemias risk, 774 treatment, 788–9 Hodgkin lymphomas, 347 HOM proteins, 275 Honduras El Salvador-II protocol, 634 treatment abandonment issues, 630

horseradish peroxidase, 159, 160 host-defense mechanisms defective, 612, 805–6 see also immune system host modification, 667 HOX11 gene, 40, 251 activation, 682 expression, 281, 323, 449, 783–4 roles, 281 HOX11L2 gene, 250 activation, 281, 682 expression, 281, 449 overexpression, 225 roles, 281 HoxA9 gene, 322, 785 HOXA gene, expression, 317–19 HOX gene, 306, 319 expression, 277, 319–20 family, 87, 785 regulatory mechanisms, 278–9 HOX proteins, 275–6 in leukemogenesis, 275 specificity, 275 HPP-CFCs (high proliferative potential colony-forming cells), 81 HPRT (hypoxanthine phosphoribosyltransferase), 393–4 HPRT gene, 58 HRX gene see MLL gene HSCs see hematopoietic stem cells (HSCs) HSCT see hematopoietic stem cell transplantation (HSCT) HS-tk gene, 656 HSV see herpes simplex virus (HSV) HSV-tk gene, 669 hTERT antigen, expression, 650 HTLV-1-associated leukemia/lymphoma, 150, 187 early studies, 187 see also adult T-cell leukemia/lymphoma (ATLL) HTLVs see human T-cell leukemia viruses (HTLVs) HTRX gene see MLL gene 5-HT (serotonin) agonists, 861 Huang, M. E., 11–12 Hughes, Walter T., 13 HuM195, 642–3 clinical trials, 642 maintenance therapy, 643 human hematopoietic stem cells, murine studies, 74–7 human herpesvirus 6 (HHV-6), infections, 806 human immunodeficiency virus (HIV) see HIV/AIDS human leukocyte antigens (HLAs), 11 and donor matching, 599 HLA-A2, 599–600 HLA-DR, 150 and childhood leukemias, 58 matched in allogeneic stem cell transplantation, 480–1 optimally, 600

Index

mismatched, 481, 600, 601 nomenclature, 599–600 specificities class I, 599–600 class II, 599–600 see also major histocompatibility complex (MHC) human T-cell leukemia viruses (HTLVs) HTLV-1, 837 HTLV-2, 837 transfusion-associated, 837 Hungerford, D. A., 7 HY antigen, 650 hybrid leukemias see mixed-lineage leukemias hydrocarbons exposure, 51–2 parental, 54 hydroperoxycyclophosphamide (4-HC), 670 hydroxyurea, 11, 583 hyperbilirubinemia, 444 hypercalcemia, 444, 718–19 treatment, 718–19 hyperdiploidy, 447 47–50 chromosomes, 240 >50 chromosomes, 239–40 cytogenetic issues, 239 etiology, 239–40 hypereosinophilia, 281–2, 443–4 hypereosinophilic syndrome (HES), 587–8 definition, 588 etiology, 588 treatment, 588 use of term, 587 hyperglycemia epidemiology, 733–4 prevention, 734 symptoms, 734 treatments, 734 hyperhydration, in tumor lysis syndrome, 716 hyperkalemia, and tumor lysis syndrome, 715, 716–18 hyperleukocytosis, 443 and coagulopathies, 724 and leukemia, 722 management, 724–5 see also leukostasis syndrome hypermethylation, 782 hyperphosphatemia and chemotherapy, 715 prevention, 451–2 and remission induction therapy, 715–16 and tumor lysis syndrome, 715–16, 718 hypertrophy, gingival, 503 hyperuricemia prevention, 451–2 treatment, 12 and tumor lysis syndrome, 714

hyperviscosity syndrome, 725 adaptive mechanisms, 725 clinical management, 725 pathophysiology, 723, 725 symptoms, 725 hypnosis, 858–9 for chemotherapy-related distress, 861–2 hypocalcemia, and tumor lysis syndrome, 715–16, 718 hypodiploidy, 31, 240–1, 447 low, 240–1 hypogammaglobulinanemia, 138 hypoglycemia, etiology, 734 hyponatremia, and syndrome of inappropriate antidiuretic hormone secretion, 733 hypoplastic marrows, and acute lymphoblastic leukemia, 172 hypopyon, and relapsed acute lymphoblastic leukemia, 442 hypothalamic-pituitary-thyroid axis, studies, 753 hypothyroidism, 752–3 hypoxanthine–guanine phosphoribosyltransferase (HGPRT), 58, 427 and cellular drug resistance, 427 hypoxanthine phosphoribosyltransferase (HPRT), 393–4 IAP gene, 420 IAPs see inhibitors of apoptosis proteins (IAPs) IARC (International Agency for Research on Cancer), 779 ¨ I-BFM-SG (International Berlin–Frankfurt–Munster Study Group), 691 ibuprofen, precautions, 851 ICE see interleukin 1 converting enzyme (ICE) ICSBP gene, 782–3 idarubicin in acute myeloid leukemia treatment, 513 in acute promyelocytic leukemia treatment, 519–20 idiopathic pneumonia syndrome (IPS), 611 treatment, 612 IDSA see Infectious Disease Society of America (IDSA) IFAR (International Fanconi Anemia Registry), 372 IFN
see interferon-
(IFN
) IFN (interferon-), gene, 115 IFN gene, 251 IFN see interferon- (IFN ) IFNs see interferons (IFNs) ifosfamide in acute lymphoblastic leukemia treatment, 403 metabolism, 403, 780 IgA (immunoglobulin A), 109 IgE (immunoglobulin E), 109, 112 IgG see immunoglobulin G (IgG) IgG4 antibody, 643 IG gene complexes, schematic, 211 IG genes, 272 rearrangements, 439–40, 683, 684 junctional regions, 682, 685, 686 translocations, 279

927

928

Index

IGG gene, 377 IGH gene, 214 rearrangements, 214–16, 222–3, 225, 239, 682, 683 biallelic, 214 detection, 685–6 secondary, 216 Southern blot analysis, 218, 226, 227 IGK gene deletions, 222–3 rearrangements, 214–16, 222–3, 225, 682 consecutive, 217 secondary, 214 IGL gene rearrangements, 214–16, 222–3, 682 secondary, 214 IgM (immunoglobulin M), 109, 155 IG/TCR gene rearrangements, 210–28 aberrant, 224–5 in B-lineage ALL, 222–4 clonality detection, 216–20 as clonality markers, 694 combinatorial diversity, 213 cross-lineage, in acute myeloid leukemia, 224 diagnostic applications, 226 during lymphoid differentiation, 217 frequencies, 223 junctional diversity, 214 in lymphoid differentiation, 214–16 in lymphoid leukemias, 210 lymphoid malignancy analysis, 226–7 oncogenic, 224–5 polymerase chain reaction amplification, 218–20 polymerase chain reaction analysis, 684–5 preferential, 214 processes, 210–16 secondary, 214, 216 Southern blot analysis, 216–18 in T-lineage ALL, 224 V(D)J coupling, 211–12, 219 IG/TCR genes analysis, applications, 220–8 molecular diversity, 213, 214 IgTCRs (immunoglobulin T-cell receptors), 667–8 IB proteins, 347 IL-1 see interleukin 1 (IL-1) IL-1ra (interleukin 1ra), 118 IL-1
see interleukin 1
(IL-1
) IL-1 see interleukin 1 (IL-1) IL-2 see interleukin 2 (IL-2) IL-3 see interleukin 3 (IL-3) IL-3 gene, 281–2, 308 IL-4 see interleukin 4 (IL-4) IL-5 see interleukin 5 (IL-5) IL-6 see interleukin 6 (IL-6) IL-7 see interleukin 7 (IL-7)

IL-8 see interleukin 8 (IL-8) IL-9 (interleukin 9), 112 IL-10 (interleukin 10), 115 IL-11 (interleukin 11), 114 IL-12 see interleukin 12 (IL-12) IL-13 see interleukin 13 (IL-13) IL-14 (interleukin 14), 141 IL-15 see interleukin 15 (IL-15) IL-16 (interleukin 16), 119–20 IL-17 see interleukin 17 (IL-17) IL-18 see interleukin 18 (IL-18) IL-19 (interleukin 19), 115 IL-20 (interleukin 20), 115 IL-21 (interleukin 21), 113 IL-22 (interleukin 22), 115 IL-24 (interleukin 24), 115 IL-26 (interleukin 26), 115 imagery, effectiveness, 862 imatinib mesylate, 243, 285 activity, 544 in chronic myeloid leukemia treatment, 583 issues, 583, 607–8 imipenem, 817 immune reconstitution after hematopoietic stem cell transplantation, 613 factors affecting, 613 immune system antigen recognition, 210 defects, 805–6 drug-induced, 805 modifying, 667 tumor cell recognition, 648–51 immunization infections, 821–2 see also alloimmunization immunoconjugates, 640–2 immunodeficiency, 761–2 immunogenotypes and cellular drug resistance, 416–17 of childhood leukemias, 220–8 immunoglobulin A (IgA), 109 immunoglobulin E (IgE), 109, 112 immunoglobulin G (IgG), 109, 112 and Down syndrome, 365 immunoglobulin M (IgM), 109, 155 immunoglobulins (Igs), 150 encoding, 210–11 genes, 210–11, 439 somatic hypermutations, 214 heavy-chain, 154, 210 light-chain, 155, 210 roles, 210 structure, 210 see also IG/TCR genes immunoglobulin T-cell receptors (IgTCRs), 667–8

Index

immunohistochemical assays advantages, 159–60 applications, 160 disadvantages, 160 immunohistochemical staining, 23, 160 immunohistochemistry, 150, 159–60 immunologic abnormalities, and Down syndrome, 365 immunologic testing see immunophenotyping immunophenotyping, 150–91 in acute lymphoblastic leukemia diagnosis, 445–6 advantages, 150 diagnostic issues, 225 early studies, 150 future trends, 191 lineage development, 150–4 methods, 159–66 myelodysplastic syndrome, 554–5 panels, 160–6 in relapse diagnosis, 473 immunoreceptor tyrosine-based activation motifs (ITAMs), 138 immunoreceptor tyrosine-based inhibitory motifs (ITIMs), 138 immunosuppression, chemotherapy-induced, 12–14 immunosuppressive therapy, myelodysplastic syndrome, 558 immunotherapy with dendritic cells, 654–5 hematopoietic stem cell transplantation as, 602–3 relapsed acute myeloid leukemia, 544–5 see also cellular immunotherapy immunotoxins, 640–2 India, T-cell acute lymphoblastic leukemia, 625 induction therapy acute myeloid leukemia, 513 and infections, 886 intensification, 513–17 studies, 516–17, 559 see also reinduction therapy; remission induction therapy infant acute lymphoblastic leukemia chemotherapy, 460 genetic abnormalities, 460 hematopoietic stem cell transplantation, 460 issues, 604–6 incidence, 460 treatment, 460 infant acute myeloid leukemia, 506–7 use of term, 506 infantile monosomy 7 syndrome see childhood monosomy 7 syndrome infant leukemias cytogenetic abnormalities, 56 etiology, 56–7 infant monosomy 7 syndrome see childhood monosomy 7 syndrome infants, acute myeloid leukemia, 506–7 infections and acute myeloid leukemia treatment, 524

and affluence, 57 and breast feeding, 57 of central nervous system, 811–12 classification, topographic, 807 defenses, 805 detection strategies, 886 early exposure, 57 future research, 58 immunization, 821–2 and induction therapy, 886 of integument, 807–10 isolation, 821 and leukemia, 5–6, 14, 57–8 evidence, 5–6 and neutropenia, 816 of orointestinal tract, 815–16 prevention, 821–3, 886 protozoan, 807 of respiratory tract, 812–15 serological studies, 57–8 susceptibility measurement, 805–6 of urinary tract, 816 see also adenoviral infections; bacterial infections; bloodstream infections; catheter-related infections; fungal infections; respiratory tract infections; transfusion-associated infections; viral infections infectious disease complications in leukemia, 805–23 future trends, 823 infectious diseases, developing countries, 625 Infectious Disease Society of America (IDSA), 811 guidelines, 814, 816 infertility, and hematopoietic stem cell transplantation, 613 influenza complications, 813 diagnosis, 813 symptoms, 813 treatment, 813–14, 823 influenza virus, 813–14 inherited cancer syndromes see familial cancer syndromes inhibin B, 756 inhibins, 129 mediation, 129 inhibitors of apoptosis proteins (IAPs), 420 overexpression, 347 INK4A gene, 251, 349 deletions, 240, 250 roles, 282–3 inositol-1,4,5-triphosphate, biosynthesis, 127 institutions, and nursing care, 883 insulin response substrate (IRS) proteins IRS-1, 143 IRS-2, 143 IRS-3, 143 roles, 143

929

930

Index

integrin receptors, 125 adapter/linker protein recruitment, 139 regulatory mechanisms, 139 roles, 139 signaling, 139 integument infections, 807–10 roles, 805 intelligence quotient (IQ) corticosteroid effects, 866 and cranial irradiation, 865–7 intensification therapy acute lymphoblastic leukemia, 456–7 continuous administration, 456–7 and nursing care, 887–8 see also postremission therapy; reinduction therapy intensified polychemotherapy, in relapsed acute lymphoblastic leukemia treatment, 473 interactive-educational interventions, 860 interferon-
(IFN
) in chronic myeloid leukemia treatment, 583 gene, 115 in juvenile myelomonocytic leukemia treatment, 580 interferon- (IFN), gene, 115 interferon- (IFN ) immunomodulatory activity, 115 roles, 115 interferon- inducing factor see interleukin 18 (IL-18) interferons (IFNs), 114–15 receptors, 135 roles, 114 type-I, 114–15 interleukin 1 (IL-1) receptors, 106, 128 regulation, 131 related factors, 118–19 interleukin 1R
(IL-1R
), roles, 118 interleukin 1
(IL-1
) biological activity, 118–19 biosynthesis, 118 roles, 119 interleukin 1 (IL-1) biological activity, 118–19 biosynthesis, 118, 131 roles, 119 interleukin 1 converting enzyme (ICE), 118 roles, 131 interleukin 1 receptor associated kinases (IRAKs), 128 roles, 128–9 interleukin 2 (IL-2), 111, 670 applications, therapeutic, 656, 668–9 receptors, 111, 135 in remission induction, 652 roles, in lymphoid development, 112 structure, 106, 112

interleukin 3 (IL-3) in vitro activity, 109 receptors, 135 roles, 108, 109 interleukin 4 (IL-4) deficiency, 112 and interleukin 13 compared, 112 properties, 112 roles, 107 in lymphoid development, 112 interleukin 5 (IL-5) receptors, 135 roles, 109 interleukin 6 (IL-6) biological activities, 113–14 deficiency, 114 family, 113–14 receptor chains, 113 structure, 110 and tumor cells, 114 interleukin 7 (IL-7) deficiency, 137 receptor chains, 113 receptors, 144 roles, in lymphoid development, 111–12 interleukin 8 (IL-8) binding, 116 nomenclature issues, 115–16 interleukin 9 (IL-9), roles, in lymphoid development, 112 interleukin 10 (IL-10), roles, 115 interleukin 11 (IL-11), roles, 114 interleukin 12 (IL-12), 141 cloning, 113 and interleukin 18 compared, 119 roles, 107, 113 interleukin 13 (IL-13) and interleukin 4 compared, 112 roles, 112 structure, 112 interleukin 14 (IL-14), 141 interleukin 15 (IL-15) biological activity, 112–13 receptors, 135 roles, 113 interleukin 16 (IL-16), roles, 119–20 interleukin 17 (IL-17) biological activity, 119 family, 119 nomenclature issues, 119 structure, 119 interleukin 18 (IL-18), 131 and interleukin 12 compared, 119 receptors, 128 roles, 119 interleukin 19 (IL-19), roles, 115

Index

interleukin 20 (IL-20), roles, 115 interleukin 21 (IL-21), roles, 113 interleukin 22 (IL-22), roles, 115 interleukin 24 (IL-24), roles, 115 interleukin 26 (IL-26), roles, 115 International Agency for Research on Cancer (IARC), 779 ¨ International Berlin–Frankfurt–Munster Study Group (I-BFM-SG), minimal residual disease studies, 691 International Bone Marrow Transplant Registry/Autologous Marrow Transplant Registry, 543 International Fanconi Anemia Registry (IFAR), 372 International Outreach Program (IOP), 631–2 collaborative treatment programs, acute lymphoblastic leukemia, 632–4 International Prognostic Scoring System (IPSS), 557 International Shwachman-Diamond Family Conferences, 371 International Standing Committee on Human Cytogenetic Nomenclature (ISCN), 238 International Workshops on Leukocyte Differentiation Antigens, 150 Internet resources, for caregivers, 872–3 interpatient variability definition, 391 drug response, 391 intracerebral myeloblastoma, 726–7 inv(16), 35, 562, 697, 783 inv(16)(p13q22), 310–12 inv(16)(p13.1q22), 255–6 inv(19)(p13.3q13.4), 242 inversions in acute myeloid leukemia, 299 definition, 238 inv(16), 35, 562, 697, 783 inv(16)(p13q22), 310–12 inv(16)(p13.1q22), 255–6 inv(19)(p13.3q13.4), 242 iodine isotopes, in radiolabeled antibody treatment, 644–5 ionizing radiation and childhood leukemias, 51, 52–3 and leukemia, 6, 12 IOP see International Outreach Program (IOP) IPS see idiopathic pneumonia syndrome (IPS) IPSS (International Prognostic Scoring System), 557 IQ see intelligence quotient (IQ) IRAKs see interleukin-1 receptor associated kinases (IRAKs) IRF-1 gene, 781 irradiation craniospinal, 479 early studies, 8 in testicular relapse treatment, 480 thymic, 6 see also cranial irradiation; total-body irradiation (TBI) IRS proteins see insulin response substrate (IRS) proteins ISCN (International Standing Committee on Human Cytogenetic Nomenclature), 238

isochromosomes, 238 Italians, acute promyelocytic leukemia, 625 ITAMs (immunoreceptor tyrosine-based activation motifs), 138 ITIMs (immunoreceptor tyrosine-based inhibitory motifs), 138 Itk kinases, 138 itraconazole, 823 Jagged notch ligands, 134 Jak kinases, 127 activation, 136–7 Jak1, 136, 137 Jak2, 135, 136, 137 Jak3, 136, 137 members, 135–6 roles, 137 tyrosine phosphorylation, 137 Jak-Stat signaling pathway, 140 Jamshidi needle, 22 Janus protein tyrosine kinases, 134–7 see also Jak kinases Japan, atomic bombs, 6, 52, 53 jaundice, 815 jaw, Burkitt lymphoma of, 491 jCML see juvenile myelomonocytic leukemia (JMML) J gene, 211, 218–19 JMML see juvenile myelomonocytic leukemia (JMML) JNK kinases, 128, 129 activation, 131 juvenile chronic myeloid leukemia (jCML) see juvenile myelomonocytic leukemia (JMML) juvenile myelomonocytic leukemia (JMML), 4, 363 age differences, 572 chemotherapy intensive, 580 low-dose, 580 chromosomal studies, 575 classification, 38–9, 549, 571–2 clinical presentation, 573 clonality, 575–6 comorbidity, 573 diagnosis, 38–9, 574, 575 diagnostic criteria, 572 differential diagnosis, 576 disease course, 579 early studies, 571–2 epidemiology, 572–3 genetic events, 579 genetic features, 577–9 hematologic features, 573–5 hematopoiesis, in cell culture studies, 576–7 hematopoietic stem cell transplantation, 581–2, 607 historical background, 571–2 incidence, 572 laboratory features, 573–5 and monosomy 7, 575

931

932

Index

juvenile myelomonocytic leukemia (JMML) (cont.) murine models, 578 and neurofibromatosis type 1, 368, 369, 572–3, 578 and Noonan syndrome, 363, 369–70, 573 therapeutic outcomes, 370 pathophysiology, 571 progenitor cells, 576 factors affecting, 576 spontaneous growth, 576 prognosis, 579 relapse, 581 sex differences, 572 survival, 579, 581 symptoms, 573 treatment, 579–82 experimental, 580–2 twin studies, 572 use of term, 572 Kaplan-Meier plots, 474, 605 Kaposi sarcoma, incidence, geographic differences, 625 kappa-deleting element (Kde), 214–16, 217, 682, 693 rearrangements, 222–3 Karnofsky performance scores, 610 Karon, M., 14 karyotypes abnormal, 238 leukemic cells, 31 partial, 247 karyotyping digital, 235 translocation studies, 272 see also spectral karyotyping (SKY) Kayexalate, 718 KBF1 (RBP-J) protein, 134 KC (melanoma growth stimulatory activity), 116 Kde see kappa-deleting element (Kde) Kellett, C. E., 6, 14 ketamine, 855 killer cell immunoglobulin-like receptors (KIRs), 603 killer inhibitory receptors (KIRs), 655 Kinlen, L. J., 6 KIRs (killer cell immunoglobulin-like receptors), 603 KIRs (killer inhibitory receptors), 655 KIT gene, mutations, 316 Klebsiella spp., 812–13 Klinefelter syndrome, 551 Knudson model, 363–4, 375 Kostmann granulocytic leukemia, 51 Kostmann syndrome (KS), 373, 551–2 KRAS gene, mutations, 782 Krivit, W., 7, 9 KS (Kostmann syndrome), 373, 551–2

lactoferrin expression, 158 as marker, 158 LAF-4 genes, 785 Landsteiner, K., 12 Langerhans’ cell histiocytosis (LCH), 779 large granular lymphocyte leukemia (LGLL), 150, 188 natural killer cell, 188 T lymphocyte, 188 large granular lymphocytes (LGLs), 188 increased numbers, 188 late treatment complications after leukemia therapy, 750 cardiopulmonary toxicity, 757–8 dental abnormalities, 758–9 endocrine toxicity, 750–6 immunodeficiency, 761–2 neurologic sequelae, 757 organ toxicity, 760–1 pulmonary function, 758 second malignancies, 762 skeletal abnormalities, 759–60 LCH (Langerhans cell histiocytosis), 779 Lck kinases, 138 LCLs see lymphoblastoid cell lines (LCLs) L-DNR (liposomal daunorubicin), 542–3 lead, parental exposure, 54 learning disabilities, and chemotherapy, 867 Learning Efficiency Test, 867 Leder stain, 23 LEFs (lymphoid enhancer-binding factors), 127 lentiviruses, properties, 662 leptin, roles, 113 leukapheresis, 452 leukemia cutis, 504 leukemia-free survival (LFS), 603–4 leukemia inhibitory factor receptors (LIFRs), 113 LIFR, 136 leukemia research approaches, 14–15 early, 3 future, 58, 59 goals, 298 leukemia(s) acute erythroblastic, 28, 176–7 and ataxia telangiectasia, 377 and bacterial infections, 806–7 Bcl2 gene alterations, 343–4 biology, 712 B-lineage, and IG/TCR gene rearrangements, 222 case studies, 14 cell types, 3 cellular theory of, 3 chemoresistance and AKT pathways, 345–7

Index

and NF-B, 347–8 and p53 gene, 348–50 chemotherapy-induced, 7, 774 and chromosomal abnormalities, 7 classification, 3, 14, 21, 166–91 current, 4 genetic, 4–5 immunologic, 150–4 modern approaches, 21 World Health Organization, 5 curative therapy, 9–12 issues, 12 diagnosis, 21, 150 bone marrow sampling, 21–2 criteria, 5 establishment, 23–9 historical background, 3–5 morphologic, 23 technological advancements, 3–4 and Down syndrome, 183–6, 363, 365 cellular drug resistance, 417–18 pathogenesis, 365–6 etiology, 5–8 chemical causes, 7 genetic causes, 7–8 hematopoietic stem cell mutations, 69 infectious causes, 5–6, 14, 57–8 physical causes, 6 viral, 5 family studies, 7 and Fanconi anemia, 372 and folic acid deficiency, 7 gene therapy-induced, 671–3 genotyping, 4 heritable predispositions, genetic mechanisms, 363 history of, 3–15 lessons from, 14–15 and hyperleukocytosis, 722 incidence, 5 geographic differences, 625, 626 rates, 6 infectious disease complications, 805–23 future trends, 823 and ionizing radiation, 6, 12 late treatment complications, 750 and lymphomas, 3, 186–7 molecular characterization, 284 psychosocial impacts, 858 relapse, and nursing care, 891 smoldering, 694 and socioeconomic status, 6 splenic, 3 subclonal detection, 226 subtypes, 712 supportive therapy, 12–14

T-lineage, and IG/TCR gene rearrangements, 222 treatment, 8–14 goals, 712 late complications, 750 palliative, 8–9 psychosocial issues, 14 twin studies, 7, 439–40 types of, 3 use of term, 3 and viral infections, 806 xenotransplantation studies, 76–7 see also acute leukemias; acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); acute undifferentiated leukemia (AUL); alkylating agent-related leukemias; biphenotypic leukemias; central nervous system (CNS) leukemia; childhood leukemias; chronic leukemias; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myelomonocytic leukemia (CMML); DNA topoisomerase II inhibitor-related leukemias; infant leukemias; large granular lymphocyte leukemia (LGLL); lymphoid leukemia; mixed-lineage leukemias; myelogenous leukemia; precursor-NK leukemia/lymphoma; treatment-related leukemias Leukemia Study Group, 9 leukemia viruses Abelson murine, 8 evidence, 5–6 feline, 5 mammalian, 5, 14 murine, 5 see also human T-cell leukemia viruses (HTLVs) leukemic cells accumulation, 440 anaplastic lymphoid, 4 anthracycline sensitivity, 519 characterization, 473 chemotherapy sensitization studies, 517 division, 440 genetic analysis, 473–4 immunophenotyping, 4–5 karyotypes, 31 myeloid, 4 targets, 649–50 and tumor vaccines, 666 leukemic erythroblasts, 3 leukemic monoblasts, 3 leukemic myeloblasts, 3 leukemic retinopathy, 442 leukemogenesis biomarkers, 58–9 CBF protein in, 311–12 and chemotherapy, 774 and environmental exposure, 362, 363 and genetic disorders, 711 and growth hormone therapy, 753

933

934

Index

leukemogenesis (cont.) and haploinsufficiency, 363 mechanisms, 5, 8 proteins in, 500–1 risk factors, 774 leukocytes see white blood cells (WBCs) leukocytosis, 3, 23 inflammatory, 5 in juvenile myelomonocytic leukemia, 573–4 leukemic, 5 see also hyperleukocytosis leukoencephalopathy, 731–2 and cranial irradiation, 757 differential diagnosis, 731–2 incidence, 730–1 symptoms, 731 use of term, 730 leukostasis syndrome, 722–5 in acute monoblastic/monocytic leukemia, 724–5 and complication risk, 712 evaluation, 723–4 incidence, 722–3 management, 724–5 pathophysiology, 723 risk factors, 722–3 symptoms, 723–4 see also hyperleukocytosis Leunase, 452–4 Levy, M. A., 7 Leydig cell dysfunction and bone marrow transplantation, 756 and chemotherapy, 755–6 and radiotherapy, 755 LFS (leukemia-free survival), 603–4 LGLL see large granular lymphocyte leukemia (LGLL) LGLs see large granular lymphocytes (LGLs) LH (luteinizing hormone), 756 lidocaine, 859 lifestyle, and childhood leukemias, 54–5 Li-Fraumeni syndrome, 282 LIFRs see leukemia inhibitory factor receptors (LIFRs) ligand binding, 125, 126 limiting-dilution analysis, 73 assumptions, 73 lineage markers, 82 applications, 82 flow cytometry, 82 lineage restriction control, 87–8 events, 71 regulatory mechanisms, 87–8 lineages development, 150–4 differentiation, 227 lymphoid, 111–13

megakaryocytic, 158–9 see also monocytic lineages; myeloid lineages liposomal daunorubicin (L-DNR), in relapsed acute myeloid leukemia treatment, 542–3 liposome–DNA complexes, 664 liposomes, 664 Lissauer, H., 8, 14 Listeria monocytogenes (bacteria), 118 liver function, late treatment complications, 760–1 LMB-89 regimen, 492, 493, 635 modified, 635 LMO1 gene overexpression, 225 roles, 281 LMO2 gene, 671–3 overexpression, 225 roles, 281 LMP-1 antigen, expression, 654 LMP-2 antigen expression, 654 toxicity, 654 LOH see loss of heterozygosity (LOH) long-term culture-initiating cells (LTC-ICs), 77–9 assay development, 77 in leukemia studies, 78–9 principles, 78 characterization, 78 detection, 78 frequencies, 78 self-renewal, 86 loss of heterozygosity (LOH), 246 tumor studies, 364 Louis–Bar syndrome see ataxia telangiectasia (AT) Loutit, J. F., 11 LPP gene, 784 LTC-ICs see long-term culture-initiating cells (LTC-ICs) lumbar puncture distress management, 858, 860 indications, 833 luteinizing hormone (LH), and Leydig cell dysfunction, 756 LY294002, 142 Ly+ ALL see lymphoid antigen-positive acute lymphoblastic leukemia (Ly+ ALL) LYL gene, 250 LYL1 gene, 40, 281 expression, 281 lymphoblastic leukemias benign lymphocytosis mimicking, 171–2 see also acute lymphoblastic leukemia (ALL) lymphoblastoid cell lines (LCLs) antigen expression, 652–3 applications, 652–3 lymphoblasts, 171 characteristics, 24, 25, 488

Index

identification, 23–6 L3, 488, 491 leukemic, 443 staining, 27 thymic origin, 4 lymphocytes, 69, 171 apoptosis, 275 development, 70 differentiation, 107 see also B lymphocytes; large granular lymphocytes (LGLs); T lymphocytes lymphoid antigen-positive acute lymphoblastic leukemia (Ly+ ALL), 179–83 studies, 181 lymphoid development, cytokines in, 111–13 lymphoid differentiation, IG/TCR gene rearrangements, 214–16 lymphoid enhancer-binding factors (LEFs), 127 lymphoid leukemia, 3 cyclophosphamide treatment, 9 early studies, 7, 14 IG/TCR gene rearrangements, 210 vincristine treatment, 9 lymphoid lineages, 111–13 lymphoid malignancies analysis, via IG/TCR gene rearrangements, 226–7 incidence, geographic differences, 625 prevalence, 221 lymphomagenesis, murine models, 489 lymphomas Bcl2 gene alterations, 343–4 chemoresistance and AKT pathways, 345–7 and NF-B, 347–8 and p53 gene, 348–50 classification, 166–91 immunologic, 150–4 incidence, geographic differences, 625, 626 and leukemia, 3, 186–7 treatment, developing countries, 632–6 see also adult T-cell leukemia/lymphoma (ATLL); anaplastic large cell lymphoma (ALCL); Burkitt lymphoma; HTLV-1-associated leukemia/lymphoma; non-Hodgkin lymphoma (NHL); precursor-NK leukemia/lymphoma; T-cell lymphoblastic lymphomas (T-LBL) lymphopoiesis, mechanisms, 309–10 lymphoproliferations, monoclonal vs. polyclonal, 225–6 lymphotactin
, 116 lymphotactin , 116 Lyn kinases, 138, 155 M195 antibody, 644 Mck protein, 139 MacMahon, B., 7 macrocytosis, 553 macrophages, generation, 79–80

MACS (magnetically activated cell sorting), 601 MAD:MAX heterodimer, 280, 488 MAD protein, 280 MAGE proteins, 667 magnetically activated cell sorting (MACS), 601 major histocompatibility complex (MHC) antigens expression, 649 as targets, 650 and donor matching, 599 and murine leukemia, 58 protein binding, 648–9 see also human leukocyte antigens (HLAs) major transplant-related complications (MTCs), 610–11 risk factors, 611 malaria, 625, 838 Malawi, Burkitt lymphoma, 635 malignant cells, detection, 227–8 malignant neoplasms inherited, 362 social burden, in developing countries, 625–6 malpositioning, catheters, 841 MALT (mucosa-associated lymphoid tissue) lymphomas, 347, 489 mammals cloning, 88 notch receptors, 133–4 notch signaling pathway, 117 toll/IL-1 receptors, 128 MAP kinases see mitogen-activated protein (MAP) kinases marijuana exposure, 55 health risks, 870–1 matched family donors (MFDs), 558, 581 studies, 558 matched sibling donors (MSDs), stem cell sources, 601 matched unrelated donors (MUDs), 558, 581 stem cell sources, 601 studies, 558 maternal age, and childhood leukemias, 55 maternal reproductive history, and childhood leukemias, 55–6 MAX:MAX heterodimers, 488 MAX protein, 8, 280 May–Grunwald–Giemsa stain, 414 MCL-1 protein, overexpression, 343 Mdm2, 348 functions, 349 MDR1 gene, polymorphisms, 403, 521, 780 MDR see multiple drug resistance (MDR) MDR gene, 669–70 MDS see myelodysplastic syndrome (MDS) MDS-related AML, use of term, 553 measles, mumps, and rubella (MMR) vaccine, 822 mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) regimen, and secondary leukemia risk, 774 mediastinal structures, compression, 719–21

935

936

Index

Medical Research Council (MRC) (UK), 644, 709 Childhood Leukemia Working Group, 447 induction therapy intensification studies, 516–17 MidAC regimen studies, 517 megablastosis, folic acid treatment, 8–9 megakaryoblastic leukemia, differential diagnosis, 177 megakaryoblasts characteristics, 24, 26–7 staining, 29 megakaryocytes differentiation, 110 identification, 80–1 maturation, 158–9 proliferation, 110 megakaryocytic lineages, immunophenotyping, 158–9 megakaryopoiesis, 80 megakarypoietic colony-forming cells, 80–1 MEIS1 gene, 785 melanoma growth stimulatory activity (MGSA), 116 melphalan, 602 leukemogenicity, 774 membrane dyes, 82 membrane folate receptor (MFR), 425 membrane transporters, overexpression, 339 Memorial Sloan-Kettering Cancer Center (MSKCC) (US), 642 M195 antibody studies, 644 memory function, and cranial irradiation, 867 meningeal relapse, 10 meningiomas, 763 meningitis, 811–12 diagnosis, 811 etiology, 811 treatment, 811–12 see also cryptococcal meningitis meperidine, 854 6-mercaptopurine (6MP), 9, 10–11 in continuation treatment, 458 and hypoglycemia, 734 metabolism, 394, 427 pharmacokinetics, 393–4 pharmacology, 393–5 sensitivity to, 458 Merek disease virus, 5 meropenem, 817 metabolic abnormalities, and early complications, 712–19 metal fumes, parental exposure, 54 methionine synthase reductase, 367 methotrexate (MTX), 3–4, 9, 10, 425–7 administration routes, 458–9 in Burkitt lymphoma treatment, 634–5 cellular drug resistance, 426 mechanisms, 425–7 in continuation treatment, 458 IQ studies, 865 membrane transport, 425

neurotoxicity, 865 pharmacodynamics, 392–3 pharmacogenetics, 393 pharmacokinetics, 391 pharmacology, 391–3 polyglutamation, 425–6 protective effects, issues, 867 target enzymes, 426–7 therapeutic mechanisms, 425 toxicity, 393 see also high-dose methotrexate (HDMTX) methotrexate polyglutamates (MTXPGs), 391 5, 10-methylenetetrahydrofolate reductase, 7 methylenetetrahydrofolate reductase (MTHFR), 367 polymorphisms, 440 methylguanine methyltransferases (MGMTs), 670 methylmercaptopurine, biosynthesis, 393–4 methylphenidate, 870 methyl-thiazol-tetrazolium (MTT) assay, 414–15, 476 applications, 415 and dye exclusion assay compared, 415 metronidazole, 816 MFDs see matched family donors (MFDs) M-FISH see multiplex-fluorescence in situ hybridization (M-FISH) MFR (membrane folate receptor), 425 MGMT gene, 670 MGMTs (methylguanine methyltransferases), 670 MGSA (melanoma growth stimulatory activity), 116 MHC see major histocompatibility complex (MHC) mice ABL gene deficiency, 274 acute myeloid leukemia studies, 275 apoptosis studies, 342–3 BLM gene studies, 380 bone marrow, phenotype analysis, 82 CD40 deficiency, 118 chemokine deficiency, 116 chemokine receptor studies, 126 colony-stimulating factor-1 mutations, 111 competitive repopulating units, 75, 78 E2A protein deficiency, 275 hematopoiesis, 71 hematopoietic cell differentiation, 83 hematopoietic stem cells, 74 studies, 75 human hematopoietic stem cells repopulating activity, 76, 77 studies, 74–7 interleukin 1 deficiency, 119 interleukin 4 deficiency, 112 interleukin 6 deficiency, 114 interleukin 15 deficiency, 113 leukemia studies, 7, 11, 14, 322 megakaryocytes, 80–1

Index

MLL gene deficiency studies, 277, 319–20 Nf1 gene, 368–9, 776 RUNX1-ETO fusion protein studies, 312–13 Runx1 gene, 309–10 Runx2 gene, 310 severe combined immunodeficiency, 111 Stat1 studies, 141 Stat5 deficiency, 141–2 thrombocytopenia, 110 tumor necrosis factor receptor deficiency, 118 see also nonobese diabetic-scid/scid (NOD/SCID) mice; transgenic mice MIC (morphologic, immunologic, and cytogenetic) classification, 31, 33 microarrays applications, 689 DNA microarray analysis, 39–40, 284, 449 microsatellite DNA, 58 microsatellite instability (MSI), 58 in childhood leukemias, 58 MidAC (mitoxantrone/cytarabine) regimen, 517 midazolam, 859 MIF (MYC-inhibiting factor), 488 minimal residual disease (MRD), 679–98 acute lymphoblastic leukemia studies, 694, 690–6 acute myeloid leukemia studies, 696–7 assays, 541–2 detection advantages, 679 methods, 679 in peripheral blood, 696 via flow cytometry, 687–90, 695 via polymerase chain reaction, 679–86, 694 early studies, 679 future trends, 698 issues, 679 markers in ALL, 687–8 in AML, 688–9 identification, 689 measurement, 451, 523–4, 603–4, 690 monitoring, 235, 236 continuous, 693 and hematopoietic stem cell transplantation, 693–4 relapsed acute lymphoblastic leukemia, 693, 696 and relapse risk, 480 sensitivity, 690 MIP-1
, deficiency, 116 Mitelman Database of Chromosome Aberrations in Cancer, 238 mitochondrial membrane potential (MMP), reduction, 342 mitogen-activated protein (MAP) kinases, 127 Ras pathway, 143–4, 571 mitoxantrone, leukemogenicity, 778–9 mitoxantrone/cytarabine (MidAC) regimen, trials, 517

mixed-lineage leukemias characterization, 513 treatment, 461 treatment outcomes, 513 see also biphenotypic leukemias MKL-1 gene, 325 MLL5 gene, 781 MLL-ABI1 fusion gene, 257 MLL-AF4 fusion gene, 440 MLL-AF6 fusion gene, 257 MLL-AF9 fusion gene, 278 expression, 322 MLL-AF10 fusion gene, 257, 278, 321 MLL-CBP fusion gene, 256, 278, 321, 788 MLL-ELL fusion gene, 278, 785 MLL-ENL fusion gene, 40, 278, 321 expression, 322 transcript, 32 MLL-FK506 fusion protein, 322 MLL fusion genes, 277–9 gene expression profiling, 278–9 MLL fusion proteins, murine models, 785 MLL-GAS7 fusion gene, 786 expression, 322 MLL gene, 5, 7 abnormalities, 235, 277, 306–7, 316–23, 691 detection, 278 prognostic significance, 522 transformation mechanisms, 322–3 bioflavonoid-induced breaks, 56 deficiency, 277, 319–20 functions, 317–20 leukemia induction, genetic models, 322 in leukemogenesis, 277–8 partner genes, 784 rearrangements, 32, 167, 244–6, 284, 604–6 in acute lymphoblastic leukemia, 238, 243–6 in acute myeloid leukemia, 36, 56 age differences, 416, 417 detection, 245 drug-induced, 440 evaluation, 448 in infant ALL, 460 twin studies, 56 regulatory mechanisms, 317–19 tandem duplication, 254 translocations, 50, 56–7, 317, 783–7 leukemia-associated, 320–2, 783–8 MLL-LacZ fusion protein, 322 MLL-p300 fusion gene, 256 MLL protein, 319, 320 homology, 277 roles, 277, 783–4 structure, 277, 318 transformation mechanisms, 322–3

937

938

Index

MMP (mitochondrial membrane potential), 342 MMR (measles, mumps, and rubella) vaccine, 822 modal number (MN), definition, 238 molecular genetics of acute lymphoblastic leukemia, 272–85 of acute megakaryoblastic leukemia, 323–5 of acute myeloid leukemia, 298–326 of acute promyelocytic leukemia, 299–307 future trends, 285 research goals, 298 molecular lesions identification, 284 rare, 325 molluscum contagiosum, 809, 810 monoblasts characteristics, 24, 26 staining, 27 monoclonal antibodies, 23, 150 monocytes, 69 monocytic leukemia see myelodysplastic syndrome (MDS) monocytic lineages antigens, 157–8 immunophenotyping, 157–8 maturation, 157–8 monosomy 7, 240, 253 childhood, 572 familial, 51 and juvenile myelomonocytic leukemia, 575 and myelodysplastic syndrome, 551, 554 see also familial myeloid disorders with monosomy 7 monosomy 7 syndrome, 4 and myelodysplastic syndrome, 548–9, 552 see also childhood monosomy 7 syndrome MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) regimen, 774 morphine adverse reactions, 853 in pain management, 851–2 morphine sulfate, 859 morphologic analysis, 22–9 specimen preparation, 22–3 morphologic, immunologic, and cytogenetic (MIC) classification, 31, 33 mortality rates developed countries, 626, 627 developing countries, 626–7 see also under-five mortality rates (U5MRs) MOZ-CBP fusion protein, 321–2 MOZ gene, 258 6MP see 6-mercaptopurine (6MP) MPDs see myeloproliferative disorders (MPDs) mpl gene, 110 MPO see myeloperoxidase (MPO) MPO gene, expression, 178 MPO staining see myeloperoxidase (MPO) staining

MRC see Medical Research Council (MRC) (UK) MRD see minimal residual disease (MRD) MRD1 gene, overexpression, 542 MRP1 protein, 423 and cellular drug resistance, 420, 423, 429 expression, 424 MSDs see matched sibling donors (MSDs) MSF gene, 784 MSI see microsatellite instability (MSI) MSKCC see Memorial Sloan-Kettering Cancer Center (MSKCC) (US) MTCs see major transplant-related complications (MTCs) mtDNA, mutations, 783 MTG16-CBFA2 fusion gene, 256 MTG16 gene, 312, 313 MTGX gene, 312 MTHFR see methylenetetrahydrofolate reductase (MTHFR) mTOR protein, 346 MTT assay see methyl-thiazol-tetrazolium (MTT) assay MTX see methotrexate (MTX) MTXPGs (methotrexate polyglutamates), 391 Mucor spp., infections, 819 Mucorales (fungi), 819 mucormycosis, 13–14 mucosa-associated lymphoid tissue (MALT) lymphomas, 347, 489 mucosal membranes, integrity, 805 mucositis, 13 drug-induced, 815 incidence, 728–9 and pain, 887 prevention, 729, 889 risk factors, 728–9 treatment, 729, 815, 889 MUDs see matched unrelated donors (MUDs) multiple drug resistance (MDR) in relapsed acute myeloid leukemia, 542 relapse treatment issues, 476 multiplex-fluorescence in situ hybridization (M-FISH), 236 modifications, 236 Murphy, M. L., 9 mustard gas, leukemia treatment studies, 8 MVP/LRP protein, expression, 423 My+ ALL see myeloid antigen-positive acute lymphoblastic leukemia (My+ ALL) MYB gene, 87 neutralization, 664 MYC gene, 8, 420 activation, 279–80 dysregulation, 30, 488 mechanisms, 488–9 expression, 488 MYC-inhibiting factor (MIF), mutations, 488 MYC:MAX heterodimers, 280, 488 Mycobacterium avium, infections, risk factors, 806

Index

mycoses, 13–14 etiology, 810 invasive, 823 treatment, 810 MYC protein, roles, 280 MyD88 (Myeloid differentiation 88), 128 myeloablation, 11, 12 myeloblasts, 23 characteristics, 24, 25–6 cytarabine sensitivity, 562 identification, 23–6 staining, 27, 28–9 myelodysplasia benzene-induced, 7 classification, 31 incidence, 362–3 myelodysplastic syndrome (MDS), 4, 548–63 abnormalities, 38, 178–9, 551 age differences, 549, 550–1 allogeneic stem cell transplantation, 558–9 blast counts, 553, 556–7 bone marrow features, 553–4 characterization, 548 chemotherapy, 558 classification, 21, 38, 550 current approach, 549–50 historical background, 548–9 clinical features, 553–5 comorbidity, 551 and congenital bone marrow failure, 551–2 cytogenetics, 554 diagnosis, 38 diagnostic criteria, 555, 556 diagnostic work-up, 556 differential diagnosis, 557 disease course, 553, 557 and Down syndrome, 509–10, 551 drug-induced, 780 epidemiology, 550–2 etiology, 552–3 familial, 544, 552 and familial myeloid disorders with monosomy 7, 375 functional assays, 73 future trends, 562–3 geographic differences, 550–1 hematopoietic stem cell transplantation, 607 immunophenotyping, 554–5 immunosuppressive therapy, 558 incidence, 548, 550–1 laboratory features, 553–5 nomenclature issues, 548 and nonclonal disorders, 555 pathophysiology, 552–3 primary, 550 prognosis, 557

secondary, 550 etiology, 550 treatment, 559 and severe congenital neutropenia, 373–4 sex differences, 550–1 and Shwachman–Diamond syndrome, 370–1 subtypes, 38, 549, 550–1 symptoms, 553–5 treatment, 557–9 use of term, 548 vs. acute myeloid leukemia, 556, 557 xenotransplantation studies, 77 myelodysplastic syndrome (MDS), 548–9 see also childhood monosomy 7 syndrome; juvenile myelomonocytic leukemia (JMML); treatment-related myelodysplastic syndrome (t-MDS) myelogenous leukemia, 3 use of term, 3 see also chronic myeloid leukemia (CML); splenic leukemia myeloid antigen-positive acute lymphoblastic leukemia (My+ ALL), 179–83 studies, 181 myeloid-associated antigens, expression, 446 myeloid cells, development, 70 myeloid differentiation 88 (MyD88), 128 myeloid leukemia in Down syndrome, 560, 559–62 classification, 560 clinical features, 561–2 epidemiology, 561 event-free survival, 562 laboratory features, 561–2 pathobiology, 561 pathogenic model, 561 prognosis, 560, 562 symptoms, 561–2 treatment, 562 use of term, 560 myeloid lineages, 108–11 antigens, 157–8 immunophenotyping, 157–8 maturation, 157–8 see also acute myeloid leukemia (AML) myeloid malignancies, incidence, 362–3 myeloid neoplasia, 550 myeloid neoplasms, classification, 571 myeloid sarcomas, 29, 502 diagnosis, 37–8 intraorbital, 727 myeloid tumors and acute myeloid leukemia, 510 characteristics, 510 differential diagnosis, 511 incidence, 511 intracerebral, 726–7 treatment, 727

939

940

Index

myeloid tumors (cont.) localization, 510–11 magnetic resonance imaging, 511 treatment, 511 myeloperoxidase (MPO), 26, 308 detection, 165 expression, 158 immunophenotyping, 165 as marker, 158 in tumor cells, 510 myeloperoxidase (MPO) staining, 27 applications, 27–8 myeloproliferative disorders (MPDs), 70, 368–9 chronic, 571–88 classification, 549–50, 571, 572 functional assays, 73 risk factors, 363 transient, and Down syndrome, 183–6 use of term, 571 see also transient myeloproliferative disorder (TMD) myeloproliferative leukemia virus oncogene, 110 myelosclerosis with myeloid metaplasia see chronic idiopathic myelofibrosis (CIMF) MYH11 gene, 310–11 myleran, 11 Mylotarg see gemtuzumab ozogamicin naproxen, precautions, 851 narcotics, 12 National Cancer Institute (NCI) (US), 9, 730, 833 chemotherapy studies, 492 resources, 873 Working Formulation, 487 NAT (nucleic acid amplification testing), 836 natural killer (NK) cells, 113, 340, 655 alloreactivity, 604 antileukemic activity, 603 development, 170 precursors, 169–71 nausea treatment, 853 see also anticipatory nausea and vomiting (ANV); postchemotherapy nausea and vomiting (PNV) NBS see Nijmegen breakage syndrome (NBS) NBs (nuclear bodies), 301–3 NBS1 gene, 378 mutations, 378 NCI see National Cancer Institute (NCI) (US) near-haploidy, 240 near-tetraploidy, 238–9 near-triploidy, 238–9 neo gene, 670 neoplasms and childhood leukemias, 51 and growth hormone therapy, 753

hematopoietic, 33 myeloid, 571 second, 762 see also malignant neoplasms neoplastic cells, in tumor vaccines, 666 nervy gene, 312 Neumann, Ernst (1834–1918), 3 neuraxial analgesia, 855 neurocognitive deficits and central nervous system treatment, 757 and hematopoietic stem cell transplantation, 869 interventions, 869–70 post-treatment, 868 neurofibromatosis type 1 (NF1), 51, 367–9 and cancer risk, 362 comorbidity, 368 demography, 367–8 diagnostic criteria, 368 incidence, 367–8 and juvenile myelomonocytic leukemia, 368, 369, 572–3, 578 myeloid leukemia risk, 363 pathogenesis, 368, 369 phenotype, 368 and proliferative disorders, 363 roles, 577 therapy, 369 neurologic complications, 725–6, 730–2 and hematopoietic stem cell transplantation, 869 neurologic sequelae, 757 neurolytic blocks, 855 neuropathic pain, 850 treatment, 853 neuropsychologic late effects, 864–8 definition, 864 global, 865–7 pathophysiology, 864–5 specific impairments, 867 neurosensory deficits, 757 neutropenia, 13–14, 612, 886 and catheter-related infections, 840 and fungal infections, 819–21 and infections, 805–6, 816 treatment, 110–11, 452, 841–4 see also congenital neutropenia; febrile neutropenia neutropenic colitis see typhlitis neutropenic diet, 821 neutropenic patients, 805–6, 807 infections, 816 fungal, 819–21 management, 816–19 neutrophil counts, 805–6 neutrophil elastase gene, 308 neutrophilia, 23 neutrophils, 69 Nevada (US), nuclear weapons testing, 53

Index

NF1 see neurofibromatosis type 1 (NF1) Nf1-/- cells, 577–8, 579 NF1 gene functions, 368 mutations, 368, 369, 375, 572–3, 578, 579 germline, 776 mice, 368–9, 776 mutations, 578 NFKB2 gene, 347 NF-B, 125, 128 activation, 129, 130, 131 and cellular drug resistance, 420 and chemoresistance, 347–8 and chemotherapy, 347–8 discovery, 347 roles, 132, 347 in tumorigenesis, 347 sequestration, 347 suppression, 348 NGOs (non-governmental organizations), 632, 634 NHEJ (nonhomologous end-joining), 787 NHL see non-Hodgkin lymphoma (NHL) Nijmegen breakage syndrome (NBS), 224, 378–9 comorbidity, 378 demography, 378 incidence, 378 pathogenesis, 378 phenotype, 378 prognosis, 378–9 therapy, 378–9 nitazoxanide, 815–16 nitrogen mustard, leukemia treatment studies, 8 N-nitroso compounds, 54 nitrous oxide, 854 NK cells see natural killer (NK) cells nociceptive pain, 850 mild, treatment, 851, 852 moderate, treatment, 851 severe, treatment, 851–2 NOD/SCID mice see nonobese diabetic-scid/scid (NOD/SCID) mice NOD/SCID-2microglobulin-/- mice, hematopoietic stem cell studies, 76 NOD/SCID-nu/nu mice, 76 non-governmental organizations (NGOs), and pediatric cancer units, 632, 634 non-Hodgkin lymphoma (NHL), 29, 649 incidence geographic differences, 625 rates, 6 nonhomologous end-joining (NHEJ), 787 nonionizing radiation, and childhood leukemias, 53–4 non-neutropenic patients, 805–6, 807

nonobese diabetic-scid/scid (NOD/SCID) mice hematopoietic stem cell studies, 76 repopulating activity, 76 side population cells, 84 nonspecific esterase (NSE) stains, 27 nonsteroidal anti-inflammatory drugs (NSAIDs), 851 precautions, 851 Nonverbal Selective Reminding Test, 867 Noonan syndrome, 369–70 demographics, 369 incidence, 369 and juvenile myelomonocytic leukemia, 363, 369–70, 573 therapeutic outcomes, 370 pathogenesis, 369–70 phenotype, 369 and proliferative disorders, 363 therapy, 370 Nordic Society of Pediatric Hematology and Oncology (NOPHO), 562 ALL-92 protocol, 415 North American Intergroup, consolidation chemotherapy studies, 520 notch genes, 282 NOTCH1, 133, 134, 449 deficiency, 117 signaling, 134 notch2, 134 deficiency, 117 notch3, 134 notch4, 134 notch-ligands, roles, 78 notch receptors early studies, 133–4 ligands, 134 roles, 134 signaling, 125, 133–4 mechanisms, 134 structure, 134 notch signaling pathway, 117 discovery, 117 in hematopoiesis, 117 Nowell, P. C., 7 NOXA protein, 342 expression, 342 NPM-MLF1 fusion gene, 258 NPM-RAR
fusion gene, rearrangements, 511–12 NQO1 gene, polymorphisms, 778 NRAS gene, mutations, 782 NSAIDs see nonsteroidal anti-inflammatory drugs (NSAIDs) NSE (nonspecific esterase) stains, 27 nuclear bodies (NBs), 301–3 nuclear fallout and childhood leukemia, 53 nuclear weapons testing, 53 nuclear power industry, and childhood leukemia, 53

941

942

Index

nuclear weapons testing, nuclear fallout, 53 nucleic acid amplification testing (NAT), 836 nucleotide incorporation assay, 415 NUP98 gene, 325 NUP98-LEDGF fusion gene, 258–9 NUP98-NSD1 fusion gene, 258–9 NUP98-TOP1 fusion gene, 258–9 nurses behaviors, 891 roles, 883 nursing care, 882–92 and advocacy, 884–6 anticipating, 884 and bone marrow transplantation, 890 and chemotherapy, 889–90 and diagnosis, 886–7 environmental models, 882–3 and family education, 883–4 goals, 882 and intensification therapy, 887–8 and leukemia relapse, 891 monitoring, 884 and patient education, 883–4 patient handbooks, 887, 888 phases, 886–92 post-treatment, 890–1 recommendations, 892 remission induction therapy, 886–7 strategies, 883–6 and supportive care, 884 and technical competence, 884 terminal patients, 891–2 nursing staff, in pediatric cancer units, 632 nutritional status, and complication risk, 709–11 nystatin, 815 obesity and acute lymphoblastic leukemia treatment, 754–5 and acute promyelocytic leukemia, 511, 512 and complication risk, 711 occlusions, catheter, 839–40 oligonucleotides, antisense, 665 oliguria, 718 oll/IL-1 receptors, signal transduction mediation, mechanisms, 128 ON see osteonecrosis (ON) oncogenes chimeric, 272–9 formation, via aberrant regulation, 279–81 mutant, 664, 667 translocation-associated, 273 see also proto-oncogenes

oncogenesis genetic studies, 362 mechanisms, 439 oncoproteins, aberrant expression, 439 oncostatin M (OSM), roles, 114–15 ondansetron, 853, 861 opioids, 851 adverse reactions, treatment, 853 dosage, 852 neuraxial delivery, 854–5 weaning, 852–3 oral mucositis, 815 orchiectomy, in testicular relapse treatment, 480 Oregon Health Sciences Center (US), 870 organ dysfunction, concurrent, 711 organ toxicity, 760–1 orointestinal tract, infections, 815–16 oseltamivir, 813–14, 823 OSM (oncostatin M), 114–15 osteonecrosis (ON), 732–3 epidemiology, 732 incidence, 732 prognosis, 732–3 treatment, 732–3 osteoporosis, 111 osteosarcoma, 753 OTT-MAL fusion gene, 257 ovarian function and bone marrow transplantation, 756 and leukemia treatment, 756 oxazaphosphorines metabolism, 403 pharmacology, 403 oxycodone, 851 1p32, abnormalities, 250 8p11.2, abnormalities, 258 9p, abnormalities, 250–1 11p15.5, abnormalities, 258–9 12p, abnormalities, 246–8, 259 12p13 abnormalities, 235 see also ETV6 gene 16p13/CBP, abnormalities, 259 p14ARF gene, 282–3, 313 p15INK4A gene, 283 p15INK4B gene, inactivation, 450, 782 p16 gene, 58, 282, 417 p16INK4A gene, 282–3, 349, 417 inactivation, 349, 450, 782 p19ARF gene, 282–3, 313, 417 p21CIP1 gene, 350 p53 gene, 282 and chemoresistance, 348–50 expression, 282

Index

function loss, 348–9 inactivation, 450 mutations, 282, 349, 489, 782 germline, 776 roles, 282, 348, 476 p53 protein, mediated responses, 348 p63 gene, 349 roles, 349–50 p73 gene, 349 roles, 349–50 p85 gene, 138–9 p110 gene, 138–9 p300 gene, 784 packed red blood cells (PRBCs) administration, 831 storage, 831 transfusions, 829–31 volume determination, 831 pain acute procedural, 858 assessment, 850 classification, 850 and mucositis, 887 self-report, 850 see also neuropathic pain; nociceptive pain pain management, 850–5 adverse reactions, treatment, 853 clinical implications, 855 in diagnostic procedures, 853–4 nonpharmacological interventions, 855, 858–68 and nursing care, 890 pharmacological, 851–3 principles, 850, 851 strategies, 890 in therapeutic procedures, 853–4 painting probes, 236 palivizumab, 814 pancreatitis, 729–30 risk factors, 729 treatment, 729–30 pancytopenia, 553 parainfluenza viruses, 813 paraparesis, 443 parasites, sporozoan, 838 parathyroid hormone (PTH), activity, 715 parental distress, and child adjustment, 863 parental exposure, chemicals, 54 parenting behavior changes, 863–4 and child adjustment, 863–4 parent-mediated interventions, 859–60 parent participation, in pediatric cancer units, 628 parents alcohol consumption, 55 handbooks, 887, 888

post-traumatic stress disorder, 862 tobacco smoking, 55 parotid gland, enlargement, 443 Parvoviridae, 663 parvovirus B-19, 837 parvovirus infection, 459, 837 PAS (periodic-acid Schiff ) stain, 29 paternal preconception radiation exposure, 53 patient-controlled analgesia (PCA), 852 patient education content, 884, 885 and nursing care, 883–4 patients age factors, 711 concurrent organ dysfunction, 711 genetic conditions, 711 handbooks, 887, 888 nutritional status, 709–11 risk factors, 709–11 see also neutropenic patients; non-neutropenic patients paxillin, 139 PBMCs (peripheral blood mononuclear cells), 654 PBSCs see peripheral blood stem cells (PBSCs) PBSF/SDF-1 (pre-B-cell growth-stimulating factor/stromal cell-derived factor 1), 116 PC5 (phycoerythrin-cyanine 5), 150 PCA (patient-controlled analgesia), 852 PCR see polymerase chain reaction (PCR) PDGFRA gene, 588 PEBP2AB gene see CBFA2 gene pediatric cancers developing countries, 625–6 political status, 630 survival rates, 627 procedural analgesia, 853–4 treatment outcomes, 631 pediatric cancer units community participation, 632 databases, 632 developing countries establishment, 630–2 establishment requirements, 628 implementation barriers, 628–30 implementation rationale, 630 diagnostic facilities, 632 educational resources, 632 event-free survival rates, 628 health professionals, 632 nursing staff, 632 operating costs, 628–9 parent participation, 628 patient records, 632 twinning partnerships, 630–1 communications, 632

943

944

Index

Pediatric Oncology Group (POG) (US), 239, 240, 367, 445 anesthesia studies, 854 bone marrow relapse studies, 477–8 induction therapy intensification studies, 516 interferon-
studies, 580 myeloid leukemia studies, 562 relapse studies, 454–5 pegaspargase, 452–4 pegfilgrastim, 844 pelle, 128 penicillins, 757 pentamidine, 822–3 pentamidine isethionate, 13 PE (phycoerythrin), 150 perceived vulnerability, 872 studies, 872 PerCP (peridinium chlorophyll complex), 150 perfluorocarbons (PFCs), 838 perforin, 340 peridinium chlorophyll complex (PerCP), 150 periodic-acid Schiff (PAS) stain, applications, 29 peripheral blast cell count (PBC), as relapse prognosis factor, 475–6 peripheral blood hematological data, 574 minimal residual disease detection, 696 peripheral blood mononuclear cells (PBMCs), 654 peripheral blood stem cells (PBSCs) advantages, 607 harvesting, 601 and hematopoiesis, 601 peripherally inserted central catheters (PICCs), 839 placement skills, 887 PEST domain, 134 pesticides and childhood leukemias, 54 exposure, 51–2 PETHEMA group (Spain), 520 PFCP (primary familial and congenital polycythemia), 585–6 PFCs (perfluorocarbons), 838 Pgp encoding, 542 inhibition, 542 pharmacodynamics definition, 391 issues, 391–404 and treatment outcomes, 450 pharmacogenetics definition, 391 issues, 391–404 and treatment outcomes, 450 pharmacogenomics, acute myeloid leukemia, 521 pharmacokinetics definition, 391 issues, 391–404

pharmacological interventions, and psychological interventions compared, 859–60 phenol, in pain management, 855 phenotype analysis, 81–2 limitations, 82–4 murine bone marrow, 82 phenotype instability, 82–4 phenotypic markers, in hematopoietic cell differentiation studies, 81–4 phenylalanine, 15 phenylpyruvic oligophrenia, 14 pheochromocytomas, 368–9 Philadelphia chromosome, 7–8, 32, 238 in acute lymphoblastic leukemia, 242–3, 603, 605 in biphenotypic leukemias, 37 and cellular drug resistance, 417 in chronic myeloid leukemia, 582 cytogenetic studies, 272–80 detection, 476 discovery, 4, 70, 572 see also BCR-ABL fusion gene phosphatidylinositol-3-kinase activation, 142, 345 catalysis, 346 roles, 346–7 in signaling, 142 phosphoinositol-3-kinase activation, 133 inhibitors, 142 mediation, 142 phospholipase C (PLC), 139 activation, 127, 133, 138 family, 127 phosphorus, radioactive, 8 phosphorus homeostasis, and tumor lysis syndrome, 715 phosphorylation, protein tyrosine, 142–3 phosphotyrosine binding (PTB) domain, 133 phycoerythrin (PE), 150 phycoerythrin-cyanine 5 (PC5), 150 phycoerythrin-Texas red conjugate (ECD), 150 PICCs see peripherally inserted central catheters (PICCs) PIK3CG gene, 781 PKA (protein kinase A), 126–7 PKC see protein kinase C (PKC) PKH 26 (dye), 82 plakoglobin, 127 plasma collection, 834–5 see also fresh frozen plasma (FFP) Plasmodium falciparum (malarial parasite), 635 Plasmodium knowlesi (malarial parasite), resistance, 116 Plasmodium vivax (malarial parasite), resistance, 116 platelet count and bleeding time, 833 threshold, 833

Index

platelet products apheresis, 833 applications, 831–3 platelets bacterial contamination, 837 roles, 69–70 units, 833 random-donor, 833 single-donor, 833 platelet transfusions, 831–4, 846 administration, 833–4 advantages, 12 guidelines, 833 indications, 833 and nursing care, 888 response determination, 834 see also corrected count increment (CCI) PLC see phospholipase C (PLC) pleocytosis, 473 plexiform neurofibromas, 368 ploidy, 31, 284, 447 applications, 284 DNA analysis, 473–4 pluripotent colony-forming cells, 70, 81 PLZF protein, 306 expression, 305 PLZF-RARA fusion gene, 255 PLZF-RAR
fusion protein, 306 binding, 306 expression, 305–6 transcriptional activity, 307 PML see progressive multifocal leukoencephalopathy (PML) PML gene, 255, 299–300 functions, 301–3 roles, 302–3 PML nuclear bodies, 301, 302 PML oncogenic domains (PODs), 301 PML protein, 300, 303 roles, 299–300 structure, 300 PML-RARA fusion gene, 255 PML-RAR
fusion gene, 34, 300–1, 696 all-trans-retinoic acid effects, 304 expression, 305, 542 leukemia induction, 303 rearrangements, 511–12 regulatory mechanisms, 304 roles, 299–300, 303–4, 344 transcripts, 521, 524 translocation, 34 PML-RAR
fusion protein, 650 Pneumocystis carinii see Pneumocystis jiroveci Pneumocystis jiroveci, 10, 13, 762, 812–13, 814 classification issues, 807

infections, 612 risk factors, 806, 807 Pneumocystis jiroveci pneumonia prevention, 822–3 treatment, 452 Pneumocystis jiroveci pneumonitis, 822 diagnosis, 814 treatment, 814 Pneumocystis pneumonia drug therapy, 13 early studies, 13 pneumonia diagnosis, 812–13 etiology, 812–13 see also idiopathic pneumonia syndrome (IPS) PNV see postchemotherapy nausea and vomiting (PNV) PODs (PML oncogenic domains), 301 POG see Pediatric Oncology Group (POG) (US) poliomyelitis, paralytic, 6, 57 polycythemia vera (PV), 80, 584–5 complications, 585 diagnostic criteria, 585 etiology, 584–5 incidence, 585 risk factors, 585 survival rates, 585 symptoms, 585 treatment, 585 palliative, 8 Polycythemia Vera Study Group (PVSG), 585 polymerase chain reaction (PCR), 84 acute myeloid leukemia studies, 683–4 analysis, 219, 220, 222, 684 detection limits, 227 disadvantages, 219–20 sensitivity, 227–8 DNA sequence length, 680 of IG/TCR gene rearrangements amplification, 218–20, 685 analysis, 684–5 leukemia-specific targets, 680–2 minimal residual disease detection, 679–86, 694 sensitivity, 685 modified methods, 236–7 molecular targets, 681 precursor B-cell acute lymphoblastic leukemia studies, 682–3 principles, 679–80 product visualization, 680 quantitative competitive, 236 sensitivity increase, 680 T-cell acute lymphoblastic leukemia studies, 683 see also real-time quantitative polymerase chain reaction (RQ-PCR); real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-RT-PCR); reverse transcriptase-polymerase chain reaction (RT-PCR)

945

946

Index

polymorphisms and acute lymphoblastic leukemia risk, 440 and acute myeloid leukemia risk, 500 in drug-metabolizing enzymes, 59 polyubiquitination, 300 population mixing, and childhood leukemias, 57–8 posaconazole, 821, 823 postchemotherapy nausea and vomiting (PNV), 861 treatment, 861 postrelapse survival, 541 postremission chemotherapy, 478 protocols, 478 postremission therapy acute myeloid leukemia, 517–18 evaluation, 517 see also intensification therapy post-traumatic stress disorder (PTSD), in parents, 862 PR1 antigen, 652 PRAME (preferentially expressed antigen of melanoma), 682 PRBCs see packed red blood cells (PRBCs) pre-B-cell complexes, roles, 216 pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1), 116 pre-B-cell receptor (pre-BCR), 154–5 complexes, 154 expression, 155 precursor B-cell acute lymphoblastic leukemia (precursor-B-ALL), 31, 167–8 cellular drug resistance, 417 early, 166–7 gene expression profiling, 39–40 IGH gene studies, 226 IG/TCR gene rearrangements, 222–3, 224 polymerase chain reaction analysis, 682–3 prognosis, 449 transitional, 168 xenotransplantation studies, 76–7 see also common acute lymphoblastic leukemia (cALL) precursor cells, use of term, 70 precursor-NK leukemia/lymphoma, 169–71 classification issues, 169–70 diagnosis, 170 symptoms, 170 precursor T-cell acute lymphoblastic leukemia (precursor-T-ALL), 31 chromosomal abnormalities, 32 gene expression profiling, 39–40 markers, 164 prednisolone, 477–8 cellular drug resistance, 415–16 see also dexamethasone prednisone, 3–4 in acute lymphoblastic leukemia treatment, 402 cellular drug resistance, 415–16 dosage, 402

in leukemia treatment, 9 and osteonecrosis, 732 pharmacology, 402–3 preferentially expressed antigen of melanoma (PRAME), 682 pregnancy outcomes, and endocrine toxicity, 756 preleukemia, use of term, 548 pre-T1 cells, 156–7 pre-T cell complexes, expression, 216 pre-T-cell receptor (pre-TCR), 157 primary cytogenetic lesions, 238 primary familial and congenital polycythemia (PFCP), 585–6 priming, 84 proapoptotic proteins, expression, 85 probes fluorescent, 236 painting, 236 TaqMan, 685 see also DNA probes progenitor cells, 688 erythroid, 80 granulopoietic, 79 maturation, 154 megakaryopoietic, 80–1 proliferative potential, 78, 79 use of term, 70 progressive multifocal leukoencephalopathy (PML), 812 diagnosis, 812 etiology, 812 symptoms, 812 treatment, 812 progressive muscle relaxation training, 861–2 prolactin, 107 receptors, 109, 135 expression, 109–10 proliferative disorders risk factors, 363 see also myeloproliferative disorders (MPDs) promethazine, 853, 854 promyeolocytes, 34 pronormoblasts, 459 propofol, 854 Protein Design Labs, 642 protein kinase A (PKA), activation, 126–7 protein kinase C (PKC) activation, 139 overexpression, 423–4 proteins roles, in leukemogenesis, 500–1 virus-associated, 649 see also accessory proteins; insulin response substrate (IRS) proteins; Stat proteins; tumor necrosis factor receptor associated factor (TRAF) proteins protein synthesis, and asparaginase, 421

Index

protein tyrosine, phosphorylation, substrates, 142–3 protein tyrosine kinase receptors characterization, 132 inactivation, 133 mechanisms, 132–3 roles, 132 signaling, 132–3 substrate recruitment, 133 protein tyrosine kinases, 155 protein tyrosine phosphatase, 578 proto-oncogenes activation, 272, 281–2 mutations, 363 protozoan infections, and leukemia, 807 pruritus, treatment, 853 pseudodiploidy, 240 Pseudomonas spp., 837 Pseudomonas aeruginosa, 13, 809, 812–13 infections, 524, 809–10 catheter-related, 840 psychological interventions, 860–1 and pharmacological interventions compared, 859–60 psychosocial issues, 14, 858–73 in bone marrow transplantation, 868–9 caregiver resources, 872–3 during treatment, 858–68 health promotion, 870–2 post-treatment, 864 psychostimulants, 869 PTB (phosphotyrosine binding) domain, 133 PTEN gene, 346 PTEN signaling pathway, 346 PTH (parathyroid hormone), 715 PTPN11 gene, mutations, 363, 368, 369–70, 573, 578 PTSD (post-traumatic stress disorder), 862 pubertal development and endocrine toxicity, 750–2 females, 756 pulmonary complications, in hematopoietic stem cell transplantation, 611–12 pulmonary function, late treatment complications, 758 PUMA protein, 342 expression, 342 PURA gene, 781 PURB gene, 781 purging effects, 670 techniques, 670 purines catabolism, 714 metabolism, 9 PV see polycythemia vera (PV) PVSG (Polycythemia Vera Study Group), 585 PZLF-RAR
fusion gene, rearrangements, 511–12

3q21, abnormalities, 257–8 3q26, abnormalities, 257–8 5q abnormalities, 781 deletions, 781–3 5q-syndrome, classification, 549 6q, abnormalities, 248 7q abnormalities, 781 deletions, 782–3 7q22, abnormalities, 781 7q31, abnormalities, 781 7q35, abnormalities, 250 11q23 abnormalities, 235, 256–7, 277, 316, 320–1, 604–6 prognostic significance, 256, 522 as gene rearrangement site, 243–4 translocations, 244–5, 783 see also MLL gene 11q23/MLL gene rearrangement, 238, 243–6 14q11.2, abnormalities, 251 21q22 abnormalities, 235, 259–60, 561 see also CBFA2 gene 22q13/p300, abnormalities, 259 quantitative competitive polymerase chain reaction (QC-PCR), 236 quinones, 7 RA see refractory anemia (RA) Rac proteins, activation, 139 RAD18 protein, 299–300 radiation (ionizing) see ionizing radiation radiographic equipment, upgrading, 6 radiography, and childhood leukemias, 6 radiolabeled antibodies, 640–2 radiotherapy, 11 adverse reactions, 750 and apoptosis, 339 and chemotherapy, 11 and childhood leukemia, 52 effects on central nervous system, 757 and Leydig cell dysfunction, 755 neuropsychologic effects, 864–5 see also cranial irradiation radon, exposure, 53 RAEB see refractory anemia with an excess of blasts (RAEB) RAEB-T see refractory anemia with an excess of blasts in transformation (RAEB-T) RAF kinases, 579 RAG1 protein, 211, 299–300 binding, 212 RAG2 protein, 211 binding, 212 Rag2-/-  c-/- mice, 76 RANK receptor, 344

947

948

Index

Ranson’s criteria, 730 RARA gene, 255 RARA-PML fusion gene, 255 RAREs (retinoic acid response elements), 301 RARS see refractory anemia with ringed sideroblasts (RARS) RAR
gene, 299–300, 303 functions, 301 roles, 299 structure, 299 variant translocation effects, 305–7 RAR
-PLZF fusion protein, expression, 305–6 rasburicase, 716 see also urate oxidase Ras family, roles, 782 RAS gene, mutations, 544, 782 Ras pathways, 142, 143–4, 571 activation, 133 hyperactive signaling, 363 mechanisms, 143–4 targeting, 578–9 and Vav gene, 142–3 RAS peptides, 650 RAS proteins biosynthesis, 578 mutations, 316, 368, 369, 577 regulatory mechanisms, 577 roles, 577 RAS-RAF-MAP pathways, 571, 579 RBCC motif, 300 RBCs see red blood cells (RBCs) RBM15 gene, 325 RBM15-MKL1 fusion gene, 504 RBP-J protein, in notch receptor signaling, 134 RC see refractory cytopenia (RC) REAL (Revised European-American Lymphoma), 487 real-time quantitative polymerase chain reaction (RQ-PCR), 236, 686 advantages, 680–1 applications, 671 minimal residual disease quantification, 685, 687 sensitivity, 685–6 precautions, 681 real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-RT-PCR) advantages, 541–2 applications, 542 recombinant DNA analysis, 439 recombinant tissue plasminogen activator (rt-PA), in catheter occlusion treatment, 840 recombination signal sequences (RSSs), 211 red blood cells (RBCs) bacterial contamination, 837 development, 107 hematopoiesis, 71 maturation, 158–9

preservation, 12, 14 roles, 69 thiopurine methyltransferase activity, 394–5 see also packed red blood cells (PRBCs) red blood cell transfusions adverse reactions, 831 applications, 829 complications, 831 hemoglobin levels, 831 indications, 829 and nursing care, 888 packed, 829–31 reduced folate carrier (RFC), 425 Reed-Sternberg cells, 654 refractory, use of term, 549 refractory acute myeloid leukemia prognosis, 540 treatment, 540, 542–5 refractory anemia (RA), 607 classification, 549 see also refractory cytopenia (RC) refractory anemia with an excess of blasts (RAEB) classification, 549 diagnosis, 553 hematopoietic stem cell transplantation, 607 prognosis, 557 treatment outcomes, 555–6 refractory anemia with an excess of blasts in transformation (RAEB-T) chemotherapy, 558 classification, 549 hematopoietic stem cell transplantation, 607 treatment outcomes, 555–6 refractory anemia with ringed sideroblasts (RARS), 549, 607 classification, 549 refractory cytopenia (RC) classification, 549 hematologic data, 553 vs. aplastic anemia, 555 see also refractory anemia (RA) refractory cytopenia with multilineage dysplasia, classification, 549 registries, 50 reinduction therapy acute lymphoblastic leukemia, 456–7 acute myeloid leukemia, 513 bone marrow relapse, 477 multiagent, 494 principles, 456 relapsed acute lymphoblastic leukemia, 477 see also intensification therapy RelA protein, roles, 347 relapsed acute lymphoblastic leukemia, 473–82 bone marrow involvement, 694 diagnosis, 473–4 future issues, 481–2

Index

hematopoietic stem cell transplantation, 480–1, 603–4 experimental approaches, 481 and hypopyon, 442 immunophenotypes, 475 incidence, 473 intensified polychemotherapy, 473 isolated bone marrow relapse vs. combined bone marrow relapse, 474–5 vs. extramedullary relapse, 474 management issues, 473 minimal residual disease monitoring, 693, 696 prognostic factors, 474–6 classification, 475 reinduction therapy, 477 remission rates, 476–7 treatment, 476–81 outcomes, 477 resistance mechanisms, 476 relapsed acute myeloid leukemia, 540–5 central nervous system relapse, 541 chemotherapy, 542–3 definition, 540 diagnosis, 540–1 drug resistance, 542 extramedullary relapses, 541 hematologic relapse, 540–1 hematopoietic stem cell transplantation, 543 graft manipulation, 543–4 preparative regimens, 543–4 relapse after, 544 immunotherapy, 544–5 minimal residual disease assays, 541–2 multiple drug resistance, 542 multiple relapses, 541 prognosis, 540, 541 relapse sites, 540–1, 542 survival rates, 545 treatment, 540, 542–5 relaxation techniques, 861–2 REL gene, amplification, 347 Rel proteins, 347 remission induction therapy, 452–6 assessment, 691–3 goals, 452 and hyperphosphatemia, 715–16 intensification, 455 nursing care, 886–7 supportive care, 455–6 renal failure, and tumor lysis syndrome, 716, 718 renal insufficiency, and complication risk, 711 rep gene, 663–4 research see leukemia research respiratory syncytial virus (RSV), 814 diagnosis, 814

symptoms, 814 treatment, 814 respiratory tract infections, 812–15 incidence, 813 retinoblastoma epidemiology, 363–4 etiology, 362 13-cis-retinoic acid, in juvenile myelomonocytic leukemia treatment, 580 retinoic acid response elements (RAREs), 301 retinoic acid syndrome, 525 retinoid-X receptors (RXRs), 301 signaling, 303 retroviruses, 110, 663 and leukemia, 5, 662 murine, 662 properties, 662 structure, 661 reverse genetics, 364 reverse transcriptase-polymerase chain reaction (RT-PCR), 235, 236–8, 643 advantages, 236 applications, 237–8, 276, 278 in minimal residual disease detection, 523–4, 541–2 oligonucleotide probes, 236 precautions, 681 use of term, 680 see also real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-RT-PCR) Revised European-American Lymphoma (REAL), 487 RFC gene, polymorphisms, 425 RFC (reduced folate carrier), 425 RFC1 gene, polymorphisms, 425 RHD (runt homology domain), 307 rhinocerebral mucormycosis, 809 rhinovirus, infections, 814–15 Rhizopus spp., infections, 819 rhodopsin, 106 Rho proteins, regulatory mechanisms, 139 ribavirin, 814 ribozymes, applications, 665 ricin, anti-B4 blocked, 645 rights, children’s, 630 rimantadine, 813–14, 823 RING fingers, 299–300 RIP protein, 131 risk, classification schemes, 284, 452 risk counseling programs, 871 tobacco smoking, 871 risk evaluation, genetic, 284 rituximab, 639, 669 Rlk kinases, 138 RNA interference, mechanisms, 665 Robertson, O. H., 12, 14 Rockefeller Institute (US), 14

949

950

Index

roentgen rays, discovery, 8 Romanowsky staining, 22–3 Rous, P., 12 Rowley, J. D., 7 RQ-PCR see real-time quantitative polymerase chain reaction (RQ-PCR) RQ-RT-PCR see real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-RT-PCR) R-SMADs, 129, 130 phosphorylation, 130 RSSs (recombination signal sequences), 211 RSV see respiratory syncytial virus (RSV) rt-PA (recombinant tissue plasminogen activator), 840 RT-PCR see reverse transcriptase-polymerase chain reaction (RT-PCR) runt gene, 307 runt homology domain (RHD), 307 RUNX1/CBF transcription factor complex, 307 alterations, 316 function, 307–10 regulatory mechanisms, 310 roles, 307–8 in lymphopoiesis, 309–10 RUNX1-ETO fusion protein, 308, 312–13 expression, 314 murine studies, 312–13 roles, 312 RUNX1 gene, 307–8, 312–13 abnormalities, 306–7, 313–14 encoding, 307 mutations, roles, 314–15 promoters, 309 see also AML1 gene; CBFA2 gene Runx1 gene (murine), deletion, 309–10 RUNX1 protein, 307–8 binding, 308–9 roles, in hematopoiesis, 309–10 transactivational properties, 309 Runx2 gene (murine), 310 RUNX2 protein, 307–8 functions, 310 RUNX3 gene, expression, 310 RUNX3 protein, 307–8 functions, 310 RXR/RAR
heterodimers, 301 RXRs see retinoid-X receptors (RXRs) Saccharomyces cerevisiae (yeast), 716 SAGE (serial analysis of gene expression), 84 St. Jude AML-97 protocol, 636 St. Jude Children’s Research Hospital (SJCRH) (USA), 9–10, 13, 180, 278, 445, 457 anesthesia procedures, 854 antibody screening panels, 160–2 blood product use, 838

classification issues, 178 flow cytometry studies, 697 minimal residual disease studies, 687, 688 nursing care studies, 891 postremission therapy studies, 517 risk classification scheme, 284, 450 studies, 457 TPMT studies, 395 see also International Outreach Program (IOP) St. Jude Total XI protocol, 632–3 St. Jude Total XIIIB protocol, 633–4 Salmonella spp., infections, 806–7 SBB staining see Sudan black B (SBB) staining SBDS gene, mutations, 370 SCCIP (surviving cancer competently intervention program), 864 SCF see stem cell factor (SCF) Schistosoma haematobium (blood fluke), 635 SCID see severe combined immunodeficiency (SCID) SCN see severe congenital neutropenia (SCN) SCNIR (Severe Chronic Neutropenia International Registry), 371, 373 SCT see hematopoietic stem cell transplantation (HSCT) SDS see Shwachman–Diamond syndrome (SDS) second malignancies, 762 sedative-hypnotic drugs, 854 Seeger, D. R., 8–9 SEER Registry (US), 50 seizures, 730 incidence, 730 self-renewal divisions, 70 Sellafield (UK), childhood leukemia incidence, 53 Sen, L., 4 Senn, N., 8 sepsis, 823, 837 catheter-related, 840–1 SEPTIN genes, 785 serial analysis of gene expression (SAGE), 84 serine hydroxymethyltransferase, polymorphisms, 440 serine/threonine kinases, 106, 125 serotonin agonists, 861 serpentine receptors, 106, 125, 126 Serrate gene, 134 Serratia spp., 837 SET domain, 320 Severe Chronic Neutropenia International Registry (SCNIR), 371, 373 severe combined immunodeficiency (SCID), 137 mice, 111 X-linked, 662, 671–3 severe congenital neutropenia (SCN), 371, 373–4 and acute myeloid leukemia, 373–4 demographics, 373 incidence, 373 and myelodysplastic syndrome, 373–4 pathogenesis, 373–4

Index

phenotype, 373 subtypes, 373 symptoms, 373 treatment, 374, 551–2 use of term, 373 sexual dysfunction, and hematopoietic stem cell transplantation, 869 SFOP (French Society for Pediatric Oncology), 492, 493 SH2 (src homology 2), 133, 137 SHC protein, 143–4 sheep, hematopoietic stem cells, studies, 75 Shigella spp., infections, 806–7 SHP-1, 137 SHP-2, 363 and juvenile myelomonocytic leukemia, 578 mutations, 579 roles, 577 Shwachman–Diamond syndrome (SDS), 370–1 comorbidity, 370 demographics, 370 diagnostic criteria, 370 and hematopoiesis, 370 incidence, 370 and myelodysplastic syndrome, 370–1, 552 and myeloid malignancies, 362–3 pathogenesis, 370–1 phenotype, 370 prognosis, 371 therapy, 371 treatment, 371 Shwachman syndrome, 51 SIA see stroma-supported immunocytometric assay (SIA) SIADH see syndrome of inappropriate antidiuretic hormone secretion (SIADH) side population (SP) cells, 82 localization, 84 phenotype analysis, 83–4 Sierra Leone, mortality rates, under-five, 626 signaling pathways, 139–44 common, 125 cytokine receptors, 120 intracellular, 129–30 signal transducers and activators of transcription (Stats) activation, 140–1 dimerization, 140 discovery, 139–40 structure, 140 tyrosine phosphorylation, 140 see also Stat proteins signal transduction, 129 apoptotic pathways, 341 in hematopoiesis regulation, 125–44 toll/IL-1 receptor-mediated, 128–9

signal transduction inhibitors, 544 mechanisms, 544 simian virus 40 (SV40), 649 studies, 667 Simpson, C. L., 6 single-strand conformation polymorphism (SSCP), 220 sinusitis, 821 SJCRH see St. Jude Children’s Research Hospital (SJCRH) (USA) skeletal abnormalities, 759–60 skin, infections, 807–10 skin rashes, 459 photosensitive, 459 Skipper, H. E., 9 SKY see spectral karyotyping (SKY) SLUG gene, expression, 276–7 SM see systemic mastocytosis (SM) SMADs activation, 129 C-SMADs, 129 functional groups, 129–30 inhibitory, 129–30 in intracellular signaling, 129–30 mediation mechanisms, 129 use of term, 129 see also R-SMADs small cell tumors, 163 SMCY protein, 650 smear tests, bone marrow, 473 Smith, M. T., 7 smoking see tobacco smoking smoldering leukemia, 694 social learning theory, 872 socioeconomic status, and leukemia, 6 solid organ transplantation (SOT), 653 solvents, parental exposure, 54 Soni, S. S., 14 SOT (solid organ transplantation), 653 Southern blot analysis, 235 detection limits, 227 IGH gene, 218, 226, 227 IG/TCR gene rearrangements, 216–18 limitations, 225 procedures, 217–18 Soviet Union (former), reactor accidents, 53 SP cells see side population (SP) cells spectral karyotyping (SKY), 236, 237 applications, 239 limitations, 236 spinal cord compression, 726 diagnosis, 726 incidence, 726 symptoms, 726 treatment, 726 splenectomy, in juvenile myelomonocytic leukemia treatment, 580

951

952

Index

splenic leukemia, 3 use of term, 3 see also chronic myeloid leukemia (CML); myelogenous leukemia sporozoans, 838 src homology 2 (SH2), 133, 137 Src kinases, 155 SSBP2 gene, 782 SSCP (single-strand conformation polymorphism), 220 ST1571 see imatinib mesylate staining acid phosphatase, 29 hematopoietic stem cells, 82 histochemical, 23 immunohistochemical, 23, 160 Romanowsky, 22–3 see also chloroacetate esterase (CAE) stain; cytochemical staining; myeloperoxidase (MPO) staining; Sudan black B (SBB) staining Staphylococcus aureus, 812–13 infections, 806–7 catheter-related, 840 Staphylococcus epidermidis, infections, catheter-related, 840 Stat proteins, 127 signaling pathways, 139–42 Stat1, 140–1 murine studies, 141 Stat2, 140–1 Stat3, 140, 141 Stat4, 141 Stat5, 140, 141 Stat5a, 141–2 Stat5b, 141–2 Stat6, 141 Stats see signal transducers and activators of transcription (Stats) stem cell factor (SCF) applications, 107–8 properties, 107–8 receptors, 106 roles, 108 see also c-kit stem cells ex-vivo manipulation, 601 harvesting, 601 identification, 601 mobilization, 601 plasticity, 88 self-renewal control, 86–7 sources, 600–1 use of term, 70 see also hematopoietic stem cells (HSCs); peripheral blood stem cells (PBSCs)

stem cell transplantation (SCT) see hematopoietic stem cell transplantation (HSCT) steroids, 610 see also corticosteroids STI571 see imatinib mesylate Streptococcus spp., infections, catheter-related, 840 Streptococcus pneumoniae, 612, 812–13 infections, 806–7 Streptococcus viridans, infection, 524, 823 streptokinase, in catheter occlusion treatment, 839–40 stroma-supported immunocytometric assay (SIA), 415 disadvantages, 415 Sudan black B (SBB) staining, 27 applications, 28 Su(H) (Suppressor of Hairless) protein, 134 suicide genes, 669 sulfamethoxazole, 13, 822–3 Sullivan, M. P., 10 SUMO1 protein, 301–2 superior mediastinal syndrome, 444 superior vena cava syndrome (SVCS), 444, 720–1 anatomy, 719 diagnosis, 720 symptoms, 720 treatment, 720–1 supportive care acute lymphoblastic leukemia, 451–2 acute myeloid leukemia, 524–5 blood transfusions in, 451 Burkitt lymphoma, 635 developments, 829 and nursing care, 884 remission induction therapy, 455–6 see also hematologic supportive care Suppressor of Hairless (Su(H)) protein, 134 surviving cancer competently intervention program (SCCIP), 864 SV40 see simian virus 40 (SV40) SVCS see superior vena cava syndrome (SVCS) Sweden, mortality rates, under-five, 626 Swedish Family-Cancer Database, 55 Syk kinases, 138, 155 symptom management, nonpharmacological interventions, 858–68 syndrome of inappropriate antidiuretic hormone secretion (SIADH) and hyponatremia, 733 symptoms, 733 treatment, 733 systemic mastocytosis (SM), 588 diagnosis, 588 disease course, 588 symptoms, 588 tachyzoites, 812 TAL1:E2A complexes, 281

Index

TAL1 gene, 40, 250, 782–3 deletions, 240, 680 expression, 281 TAL1 transcription factor, overexpression, 225 TAL2 gene, 250 roles, 281 T-ALL see T-cell acute lymphoblastic leukemia (T-ALL) Talpaz, M., 11 TAM see transient myeloproliferative disorder (TMD) t-AML see treatment-related acute myeloid leukemia (t-AML) TAN1 gene, 282 TAP proteins see transporter-of-antigen processing (TAP) proteins Taq DNA polymerase, 680 TaqMan probes, 685 TaqMan technology, 685, 686 Target Test, 867 target therapies advantages, 639 see also antibody-targeted therapies Taspase1, 317–19 TBI see total-body irradiation (TBI) Tc2 cells, 652 T-cell acute lymphoblastic leukemia (T-ALL), 4, 29, 58, 76–7 anterior mediastinal masses, 445 and ataxia telangiectasia, 376 cellular drug resistance, 416–17 and genetic abnormalities, 417 chromosomal abnormalities, 32, 249–50 nonrandom, 225 classification, 32, 224 diploidy, 240 IG/TCR gene rearrangements, 224 immunophenotyping, 446 incidence, geographic differences, 625 minimal resistance disease studies, 679 polymerase chain reaction analysis, 683 prevalence, 222 prognosis, 449 proto-oncogene activation, 281–2 relapse, prognosis, 475 risk factors, 440 transcription factor genes, dysregulated expression, 281 transcription factors, 281 dysregulation, 272 translocations, 279, 281 treatment-induced, 671–3 see also precursor T-cell acute lymphoblastic leukemia (precursor-T-ALL) T-cell factors (TCFs), 125, 127 TCF-1, 128 T-cell lymphoblastic lymphomas (T-LBL), 222 classification, 224 T-cell receptor excision circle (TREC), 155, 212 T-cell receptors (TCRs), 138, 157, 308, 613, 657 artificial, 667–8

chimeric, 668 encoding, 211 genes, 211 roles, 210 types of, 211 see also IG/TCR genes T cells see T lymphocytes TCFs see T-cell factors (TCFs) TCL1 gene, 377 TCR genes, 250, 272, 377, 439 future research, 225 oligoclonality, 684–5 rearrangements, 214–16, 222, 223, 439–40, 683 aberrant, 224 cross-lineage, 682–3 junctional regions, 682, 685, 686 patterns, 683 secondary, 684 translocations, 279, 377 TCRA gene, 250, 365 rearrangements, 216 PCR analysis, 219 secondary, 214 schematic, 212 TCRB gene, 157, 250, 281, 282, 365 multiple junction sites, 214 rearrangements, 213, 216, 224, 225, 682–3 biallelic, 214 detection, 686 frequency, 224 mechanisms, 212 occurrence, 223 PCR analysis, 219 secondary, 214, 224 schematic, 212 TCR–CD3 complexes, expression, 216 TCRD gene, 157, 250 rearrangements, 216, 223, 224, 682–3 PCR analysis, 219 secondary, 214 schematic, 212 TCRG gene, 157, 214 rearrangements, 216, 223, 224, 682–3 biallelic, 214 detection, 686 PCR analysis, 219 secondary, 214, 224 schematic, 212 TCRs see T-cell receptors (TCRs) TCR
gene, 157 TCR
 proteins, 157 expression, 211 TCR proteins, 157 TDT see terminal deoxynucleotidyl transferase (TDT) technical competence, and nursing care, 884

953

954

Index

Tec kinases, 138 teeth, developmental defects, 758, 759 TEL-AML1 fusion gene, 5, 12, 50, 279, 474 age differences, 416 expression, 279, 280, 440 identification, 447–8 mediation, 279 as relapse marker, 476 roles, 279 transcript, 32 see also ETV6-CBFA2 fusion gene TEL gene, 58 rearrangements, 168, 247, 284 roles, 279 see also ETV6 gene TEL/RUNX1 fusion protein, 313 temozolomide, 525–6 temperature gradient gel electrophoresis (TGGE), 220 teniposide, 10 accumulation, 399 cellular drug resistance, mechanisms, 424–5 continuous-infusion, 399 demethylation, 399 dosage, 399 leukemogenicity, 7, 56, 256, 778 pharmacokinetics, 398–9 pharmacology, 398–400 systemic exposure, 399–400 therapeutic mechanisms, 424 toxicity, 399 terbinafine, 823 terminal care, 891–2 strategies, 891 terminal deoxynucleotidyl transferase (TDT), 150 detection, 165–6 expression, 679 immunophenotyping, 165–6 testicular leukemia, symptoms, 441–2 testicular relapse, 442 bilateral irradiation, 480 diagnosis, 473 minimal residual disease detection, 694 orchiectomy, 480 treatment, 479–80 tetrasomy 13, 254 tetrasomy 21, 253 and myeloid leukemia, 561–2 TGFs see transforming growth factors (TGFs) TGF
(tumor growth factor), 116 TGF see tumor growth factor  (TGF) TGGE (temperature gradient gel electrophoresis), 220 TGNs (6-thioguanine nucleotides), 393–5 Th2 cells, 652 therapeutic indices, antileukemic drugs, 391 therapeutic response, determinants, 450–1

therapy-related leukemias see treatment-related leukemias thioguanine, 9, 10–11 6-thioguanine cellular drug resistance, 416 metabolism, 394 6-thioguanine nucleotides (TGNs), pharmacology, 393–5 thiopurine methyltransferase (TPMT), 5, 393–4 activity, 394–5 and cellular drug resistance, 427 deficiency, 395, 458, 711 metabolism, 780–1 tumorigenicity, 395 thiopurines, 427 cellular drug resistance, 416 mechanisms, 427 inactivation, 780–1 therapeutic mechanisms, 427 thiotepa, 602 Thomas, E. D., 11 Three Mile Island accidents (US), 53 thrombocytes see platelets thrombocytopenia, 23, 375, 561 drug-induced, 12 hereditary, 586–7 immune, 444 mice, 110 pain management issues, 851 and platelet transfusions, 833, 846 thrombopoietic growth factors, 846 thrombopoietin (TPO), 81, 134 properties, 110 receptors, 109, 135 abnormalities, 586 regulatory mechanisms, 110 roles, 110 thrombosis, 727–8 and acute lymphoblastic leukemia incidence, 727 risk factors, 727 symptoms, 727–8 treatment, 728 sagittal vein, 728 thrombus formation, venous, 840 thymic cell leukemia, 14 thymic irradiation, neonatal, 6 thymic lymphoma, risks, 6 thymic stromal lymphopoietin (TSLP) receptors, 113 roles, 113 thymidylate synthase, polymorphisms, 440 thymidylate synthetase inhibition assay (TSIA), 415 thymomegaly, 14 thymus, T lymphocyte precursors, 156 thyroid carcinoma, risks, 6 thyroid dysfunction, 752–3

Index

TIF1 protein, 299–300 TILs (tumor-infiltrating lymphocytes), 655 time to relapse definition, 474 use of term, 474 TIRAP protein, 128 TK gene, 656, 669 TL see transient myeloproliferative disorder (TMD) T-LBL see T-cell lymphoblastic lymphomas (T-LBL) T-lineage cells see T lymphocytes Tlx genes, 281 T lymphocytes, 58, 114 activation, 668 requirements, 649 adoptively transferred, 651 allodepleted, 652 in cellular immunotherapy, 648 depletion, 601 differentiation, 113, 216 donor, unmanipulated, 651–2 expansion, 652 gene marking, 671 new methodology, 671 genetic modification, 656–7, 661, 668–9 gene transfer, 657 for drug sensitivity, 656 growth factors, 112–13 immature, 687–8 and immune reconstitution, 613 immunophenotyping, 156–7 in vivo expansion, 655–6 maturation, 156 precursors, 111–12, 156 T-cell receptors on, 210 transduction, 657 tumor antigen responses, 650–1 see also cytotoxic T lymphocytes (CTLs) TMD see transient myeloproliferative disorder (TMD) t-MDS see treatment-related myelodysplastic syndrome (t-MDS) TNFs see tumor necrosis factors (TNFs) tobacco smoking and childhood leukemias, 55, 56 during pregnancy, 51–2 environmental exposure, 871 maternal, 55 prevalence, 870 prevention programs, 871 and secondary cancers, 870 see also cigarette smoke tobramycin, 524 toll/IL-1 receptors in mammals, 128 signal transduction mediation, 128–9 tollip protein, 128 toll-like receptors, 128

toll receptor, use of term, 128 topoisomerase binding agents, leukemogenicity, 7 topoisomerase I, inhibitors, 543 topoisomerase II catalysis, 56 and cellular drug resistance, 424–5 inhibition, 56, 440 and diet, 56–7 topoisomerase II agents, cytotoxicity, 400 topoisomerase II inhibitor hypothesis, 56–7 topoisomerase II inhibitors, and childhood leukemia risk, 500 topotecan, in relapsed acute myeloid leukemia treatment, 543 total-body irradiation (TBI), 480, 543, 602 and cataracts, 869 developmental effects, 613 and growth impairment, 753 in juvenile myelomonocytic leukemia treatment, 581 Toxoplasma gondii, 812 infections, 807 toys, in distraction interventions, 861–2 TP53 gene, 349 mutations, 364 T-PLL (T-prolymphocytic leukemia), 376 TPMT see thiopurine methyltransferase (TPMT) TPMT gene, 780–1 TPO see thrombopoietin (TPO) TPO gene, mutations, 586–7 T-prolymphocytic leukemia (T-PLL), and ataxia telangiectasia, 376 TRADD protein, 130–1 roles, 131 TRAF proteins see tumor necrosis factor receptor associated factor (TRAF) proteins TRAIL receptors, 345 binding, 344 TRAIL-R1, 344 TRAIL-R2, 344 transcription factor genes, dysregulated expression, 281 transcription factors, 127 activation, 272 definition, 272 homology, 272 roles, 87 transdifferentiation, hematopoietic stem cells, 88 transforming growth factors (TGFs) discovery, 116 receptors, 106 transfusion-associated infections, 835–8 screening, 835–6 transgenic mice acute myeloid leukemia studies, 305 Burkitt lymphoma studies, 489 see also E -Myc transgenic mice transient abnormal myelopoiesis (TAM) see transient myeloproliferative disorder (TMD)

955

956

Index

transient leukemia see transient myeloproliferative disorder (TMD) transient myeloproliferative disorder (TMD), 184–6, 254, 560 complications, 509, 559–60 diagnosis, 509 and Down syndrome, 365, 508–9, 559–60 incidence, 509 therapeutic outcomes, 367 and hepatic fibrosis, 367 incidence, 324, 508 management, 509 pathobiology, 561 use of term, 559 translocations in acute lymphoblastic leukemia, 241, 447 in acute myeloid leukemia, 299 chemotherapy-induced, 783 definition, 238 effects on RAR
gene, 305–7 karyotyping, 272 leukemia-specific, 272 t(1;11)(q21;q23), 322 t(1;14)(p32;q13), 281 t(1;19), 440, 447, 690–1 t(1;19)(q23;p13), 275 t(1;19)(q23;p13.3), 239, 241–2 t(1;22)(p13;q13), 257, 325, 562 t(2;8)(p11;q24), 488 t(2;8)(p12;q24), 279–80 t(3;5)(q25;q34), 258 t(3;21), 313 t(4;11), 440, 690–1 t(5;11)(q35;p15.5), 258 t(5;14)(q35;q32), 250, 682 t(5;17), 305 t(6;9)(p23;q34), 258, 325 t(6;11)(q21;q23), 321 t(6;11)(q27;q23), 257–322 t(8;14), 39 t(8;14)(q11.2;q32), 249 t(8;14)(q24;q32), 279, 488 t(8;14)(q24.1;q11.2), 251 t(8;14)(q24.1;q32), 249 t(8;16), 783 t(8;16)(p11;p13.3), 321–2 t(8;21), 35, 562, 783 t(8;21)(q22;q22), 254–5, 312–13 t(8;22)(q24;q11), 279–80, 488 t(9;22), 474, 690–1, 783 t(9;22)(q34;q11), 272 t(9;22)(q34;q11.2), 239, 242–3 t(10;11)(p11.2;q23), 257 t(10;11)(p12;q13), 257 t(10;11)(p12;q23), 257, 321

t(10;14)(q24;q11.2), 251 t(11;14)(p15;q11.2), 251 t(11;16)(q23;p13.3), 256, 321 t(11;17)(q13;q21), 305 t(11;17)(q23;q21), 305 t(11;19)(q23;p13.3), 246 t(11;22)(q23;p13), 256, 321 t(12;21), 313, 417, 474, 690–1 t(12;21)(p13;q22), 246–7, 476 t(14;18)(q32;q21), 341 t(15;17), 299–301, 562, 783 t(15;17)(q22;q12–21), 255 t(15;17)(q22;q21), 299–300 t(16;16), 783 t(16;16)(p13;q22), 310–12 t(16;16)(p13.1;q22), 255–6 t(16;21)(p11.2;q22.2), 259 t(16;21)(q24;q22), 256, 259, 312, 313 t(17;19)(q22;p13), 276 t(17;19)(q22;p13.3), 242 in treatment-related leukemias, 784, 786 t(X;11)(q13;q23), 321 see also chromosomal abnormalities transmembrane transporters and glucocorticoids-efflux, 420 multidrug resistance, 425 and vinca alkaloid-efflux, 420 transphosphorylation, 136 transplant-related mortality (TRM), 558 rates, 558–9 transporter-of-antigen processing (TAP) proteins roles, 648 selectivity, 648–9 TRAP (Trial to Reduce Alloimmunization to Platelets), 834 traumatic lumbar puncture, 445 treatment abandonment issues, 630 reduction strategies, 634 treatment protocols, for acute lymphoblastic leukemia, 632–4 treatment-related acute myeloid leukemia (t-AML), 56, 525 alkylating agent-induced, 774 diagnosis, 36 hematopoietic stem cell transplantation, 607 incidence, 525 treatment, 789 treatment-related leukemias, 56, 774–90 chemotherapy, 774–90 clinical features, 788 epidemiology, 774–81 future trends, 790 hematopoietic stem cell transplantation, 789–90 molecular pathogenesis, 781–8

Index

prognosis, 789 translocations, 784 treatment, options, 788–90 treatment-related myelodysplastic syndrome (t-MDS), 525, 552 alkylating agent-induced, 774 hematopoietic stem cell transplantation, 607 incidence, 525 treatment, 789 TREC (T-cell receptor excision circle), 155, 212 tretinoin, 11–12 Trial to Reduce Alloimmunization to Platelets (TRAP), 834 triazoles, 823 Trichosporon asahii, infections, 807 Trif protein, 128 trimethoprim, 814, 822–3 early studies, 13 trimethoprim-sulfamethoxazole, 452, 762, 814, 822 TRIM (TRIpartite Motif), 300 trisomy 8, 240, 253 and myelodysplastic syndrome, 551 and myeloid leukemia, 561–2 trisomy 11, 254 trisomy 13, 254 trisomy 19, 254 trisomy 21, 240 acquired, 253–4 and Down syndrome, 365–6 and myelodysplastic syndrome, 551 and myeloid leukemia, 561 trisomy 21 syndrome see Down syndrome trisomy 22, 254 trithorax gene, 244, 277, 317, 319, 783–4 TRM see transplant-related mortality (TRM) troxacitabine, 525–6 truthfulness, in professional–child communication, 14 Trx gene family, 781 tryptan blue, 414 TSIA (thymidylate synthetase inhibition assay), 415 TSLP see thymic stromal lymphopoietin (TSLP) tube protein, 128 tuberin, 346
-tubulin, binding, 423 -tubulin, binding, 423 tumor ablation, mechanisms, 639–40 tumor antigens, 650–1 tumor burden, 711–12 tumor cells, 23 clonal chromosomal aberrations, 238 genetic modification, 661, 664–7 and interleukin 6, 114 in myeloid tumors, 510 recognition, by immune system, 648–51 transduction, 665–6 tumor growth factor
(TGF
), 116

tumor growth factor  (TGF), 81, 656 secretion, 669 superfamily, 116–17 members, 116–17 TGF1, 117 TGF2, 117 TGF3, 117 tumor growth factor  receptors, 106 activation pathways, 129 in intracellular signaling, 129–30 roles, 125, 129 signal transduction, 129 structure, 129 type I, 129 type II, 129 tumorigenesis mechanisms, 362 NF-B in, 347 tumor-infiltrating lymphocytes (TILs), 655 tumor lysis syndrome, 712–16 and calcium homeostasis, 715 comorbidity, 712 hyperhydration, 716 and hyperkalemia, 715, 716–18 and hyperphosphatemia, 715–16, 718 and hyperuricemia, 714 and hypocalcemia, 715–16, 718 management, 712–13, 716–18 pathophysiology, 713 and phosphorus homeostasis, 715 prevention, 717 and renal failure, 716, 718 risk factors, 712–13 urate oxidase treatment, 716 uric acid metabolism, 713, 715 urine alkalinization, 716 use of term, 712–13 and xanthine nephropathy, 714 tumor masses, 711–12 tumor necrosis factor receptor associated factor (TRAF) proteins characterization, 132 TRAF1, 131, 132 TRAF2, 131 deletion, 131, 132 TRAF3, deletion, 132 TRAF6, 128 deletion, 132 tumor necrosis factor receptors, 125 activation, 130 characterization, 130 and chemoresistance, 344–5 signaling, 130–2 tumor necrosis factors (TNFs) characterization, 117

957

958

Index

tumor necrosis factors (TNFs) (cont.) family, 106, 117–18 members, 117 nomenclature issues, 117 regulatory mechanisms, 344 roles, 118, 576 TNF, 118 tumors analysis, 364 chemoresistance, 339 and childhood leukemias, 51 correction, 664–6 deletion, 664–6 primary cytogenetic lesions, 238 recognition, requirements, 649 second malignancies, 763 see also brain tumors; myeloid tumors tumor suppressor genes, 282–3 identification, 364 inactivation, 282, 364 mutations, 363–4, 776 tumor suppressors, regulatory mechanisms tumor vaccines, 666 Turkey, chloromas, 625 Turk, W., 3 Turner, J. R., 12 Turner syndrome, 551 twinning model, 630–1 twin studies, 7, 51 acute lymphoblastic leukemia, 439–40, 694 juvenile myelomonocytic leukemia, 572 typhlitis, 816 diagnosis, 816 treatment, 816 tyrosine kinases, 106 tyrosine phosphorylation, 125–6, 133, 135 Fc receptors, 138 Jak kinases, 137 signal transducers and activators of transcription, 140 U5MRs see under-five mortality rates (U5MRs) UCB (umbilical cord blood), 601 UGT1A1 gene, polymorphisms, 780 UKCCG (United Kingdom Cancer Cytogenetics Group), 239 Ultrabithorax gene, 244 umbilical cord blood (UCB), as stem cell source, 601 unconjugated antibodies, 639–40 under-five mortality rates (U5MRs) developed countries, 626 developing countries, 626–7 reduced, 626–7, 631–2 use of term, 626 UNICEF (United Nations International Children’s Emergency Fund), 626

United Kingdom (UK) childhood leukemias incidence, 50, 53 infection studies, 57 Creutzfeldt-Jakob disease, 838 leukemia, cure rates, 10 United Kingdom Cancer Cytogenetics Group (UKCCG), 239 United Nations General Assembly, 630 United Nations International Children’s Emergency Fund (UNICEF), 626 United States(US) blood component products, 829 childhood leukemias, incidence, 48–9, 499–500 Creutzfeldt-Jakob disease, 838 infant leukemia studies, 56–7 leukemia, cure rates, 10 West Nile virus, 837–8 University of Toronto (Canada), 370 urate oxidase, 12, 451–2 in tumor lysis syndrome management, 716 uric acid serum levels, 444, 714 solubility, 714 uric acid metabolism, and tumor lysis syndrome, 713, 715 uridine triphosphate (UTP), 429 urinary tract, infections, 816 urine alkalinization, in tumor lysis syndrome, 716 urokinase, in catheter occlusion treatment, 839–40 Utah (US), nuclear weapons testing, 53 UTP (uridine triphosphate), 429 vaccines, 821–2 DNA-based, 666–7 tumor, 666 valacyclovir, 810, 815 vancomycin, 13, 524, 816, 817, 818 varicella, 12–13 complications, 806 diagnosis, 810 treatment, 810 varicella-zoster immune globulin (VZIG), 13 in varicella treatment, 810 varicella-zoster virus (VZV), 810 and encephalitis, 812 infections, 806 treatment, 810 vascular endothelial growth factor (VEGF) binding, 107, 108 expression, 523, 545 Vav gene, 142 roles, 142–3 v-cbl oncogene, 143

Index

V(D)J exon, 211 combinatorial diversity, 213 coupling, 211–12 and preferential rearrangements, 214 recombination mechanism, 212 somatic hypermutations, 214 VDR gene, polymorphisms, 780 VEGF see vascular endothelial growth factor (VEGF) VEG-F gene, 665 vehicle exhaust, exposure, 54 Velcade, 344 venous access devices implanted, 839 nontunneled, 839 placement skills, 887 selection criteria, 839 tunneled, 839 types of, 839 venous access support, 838–9 venous thrombus formation, 840 Verbal Selective Reminding Test, 867 Vernick, V., 14 V gene, 211, 218–19 segments, 214 Videbaek, A., 7 vinca alkaloid-efflux, via transmembrane transporters, 423 vinca alkaloids, 421–3 cellular drug resistance, 422 mechanisms, 423 microtubule interactions, 423 therapeutic mechanisms, 421–3 vincristine, 477–8 cellular drug resistance, 416 in continuation treatment, 459 dosage, 444 drug–drug interactions, 403 in lymphoid leukemia treatment, 9 neurotoxicity, 403 pharmacokinetics, 403 pharmacology, 403 viral infections and leukemia, 806 screening, 835–6 transfusion-associated, 835–7 Virchow, Rudolf Ludwig Karl (1821–1902), 3, 4, 12, 14, 727 case studies, 14 viridans streptococci, 823 virus-associated proteins, 649 viruses Coxsackie, 14 influenza, 813–14 Merek disease, 5 parainfluenza, 813 West Nile, 837–8

see also adeno-associated viruses (AAVs); adenoviruses; cytomegalovirus (CMV); Epstein–Barr virus (EBV); herpes simplex virus (HSV); herpesviruses; leukemia viruses; respiratory syncytial virus (RSV); retroviruses; varicella-zoster virus (VZV) vita cuffs, 841 vitamin supplementation, and childhood leukemias, 54 vomiting treatment, 853 see also anticipatory nausea and vomiting (ANV); postchemotherapy nausea and vomiting (PNV) voriconazole, 524, 823 VZIG see varicella-zoster immune globulin (VZIG) VZV see varicella-zoster virus (VZV) Wang, Zhen Yi, 11 Ward, G., 6 WBCs see white blood cells (WBCs) West Nile virus (WNV), transmission, 837–8 white blood cells (WBCs), 5, 12 counts, 23, 443 and acute promyelocytic leukemia prognosis, 522 in chronic myeloid leukemia, 582 in juvenile myelomonocytic leukemia, 573–4 and leukostasis syndrome, 712 roles, 69 see also leukemic cells; lymphocytes white blood disease see leukemia(s) WHO see World Health Organization (WHO) Wilms tumor, cranial irradiation studies, 866 Wilms tumor antigen-1 (WT-1), expression, 649–50 Wilms tumor gene see WT1 gene Wiskott-Aldrich syndrome, comorbidity, 489 Wnt proteins, 125 WNV (West Nile virus), 837–8 wood dust, maternal exposure, 54 World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissue, 487 human leukocyte antigen specificities, 599–600 leukemia classification, 5, 23, 31, 33, 38 algorithms, 33 bases, 298–9 hematopoietic neoplasms, 33 myelodysplastic syndrome classification, 549 World War I, mustard gas, 8 wortmannin, 142 Wright stain, 22–3 WRPY motif, 309 WSXWS motif, 134 WT1 gene, 282 expression, 681 xanthine nephropathy, and tumor lysis syndrome, 714 xanthine oxidase, inhibition, 714, 716

959

960

Index

xenobiotics, metabolism, 777 xenotransplantation hematopoietic stem cell studies, 74–7 leukemia studies, 76–7 X-linked lymphoproliferative (XLP) disease, comorbidity, 489 X-linked severe combined immunodeficiency (X-SCID), 662, 671–3 XLP (X-linked lymphoproliferative) disease, 489 X-RAR
fusion proteins, 305 XRCC1 gene, 776–7 X-SCID (X-linked severe combined immunodeficiency), 662, 671–3

Yersinia spp., infections, 806–7 Yersinia enterocolitica, 837 young children, acute myeloid leukemia, 506–7 yttrium isotopes, in radiolabeled antibody treatment, 644 Zamyl see HuM195 zanamivir, 813–14, 823 ZAP-70 kinases, 138 zinc finger genes, 132, 321 Zuelzer, W. W., 9 zygomycoses, 821