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Library of Congress Cataloging-in-Publication Data
Innovative leukemia and lymphoma therapy / edited by Gertjan J. L.
Kaspers . . . . [et al.].
p. ; cm. (Basic and clinical oncology ; 35)
Includes bibliographical references and index.
ISBN-13: 978-0-8493-5083-2 (hardcover : alk. paper)
ISBN-10: 0-8493-5083-2 (hardcover : alk. paper) 1. Leukemia
Treatment. 2. LymphomasTreatment. I. Kaspers, G. J. L., 1963- II. Series.
[DNLM: 1. Leukemiatherapy. 2. Lymphomatherapy. 3. Therapies,
Investigational. W1 BA813W v.35 2008 / WH 250 I58 2008]
RC643.I46 2008
616.99
0
41906dc22
2008006553
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Foreword
The outcome of therapy for leukemia and malignant lymphoma has improved
over the years, mainly in younger patients. Yet, there is no question that the
challenges in the area of developmental therapeutics have remained formidable.
These challenges relate to the patients who, from the start of treatment, fail to
respond to the currently available therapies or combinations of drugs. The
outlook of these primarily refractory patients is invariably dismal. Many of
the responder patients attaining an initial complete remission, unfortunately, will
finally present with relapse of disease. The relapses among the leukemias and
high-grade lymphomas usually occur early on, i.e., within the first two years.
Both groups, initial nonresponders and secondary failures, pose the notorious
difficulty of resistance to conventional therapy. These facts provide an overall
notion. Acquired somatic genetic abnormalities of the neoplasms provide keys to
the nature of the disease and offer important predictors of treatment failure. They
allow to pinpoint individual disease-specific features and distinguish variable
disease risks as well as identify those patients with the highest probability of
failure. The unmet therapeutic need is, by all standards, greatest among the large
population of older patients with hematological cancer in whom response rates
are comparatively low, relapse rates are high, and comorbidities prohibit the use
of classical chemotherapeutic agents at effective dose levels.
Scientists are on the way to discovering new drugs with different modes of
action that can overcome the limitations of todays selection of drugs. Numerous
new drugs are currently in early clinical development with the aim of circum-
venting the clinical bottleneck of chemotherapy resistance. In the coming years,
several of these compounds are expected to settle as members of the standard
armamentarium of drugs available to the patient with a hematological tumor.
New drugs may be designed with the deliberate objective of affecting a known
molecular lesion or signaling pathway in the cancer cell, thus critically inhibiting
iii
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tumor cell survival. These therapeutic compounds may tackle distinct, molecu-
larly defined subtypes of leukemia or lymphoma, and one would anticipate that
their greater specificity will allow for application with enhanced efficacy and
reduced toxicity.
Currently, we are witnessing the development of diagnostic technologies
that directly impact decision making in the clinical management of patients with
hematological malignancies. These technologies relate, on the one hand, to more
precise tissue diagnosis and involve innovative genomic, proteomic, and immu-
nological techniques. On the other hand, they involve improved in vivo imaging
methods, enabling a better and more sensitive visualization of neoplastic
deposits in the body. These techniques, when appropriately validated for clinical
use, will enable the distinction of prognostic disease subcategories and allow for
a specific diagnosis according quantitative, sensitive, and objective parameters.
This type of information will guide therapeutic decisions at the outset of
treatment. It will also provide substantial insights that will be useful in
monitoring treatment effects throughout the therapeutic management of patients
and redirect treatment choice. An ambitious diagnostic approach makes sense if
there is a choice for the physician among a broader scale of available therapeutic
options. One of the major objectives of todays molecular diagnostics relates to
the identification of new druggable targets for pharma developments.
Innovative Leukemia and Lymphoma Therapy appropriately and critically
deals with each of the issues and challenges as regards developmental thera-
peutics. The book highlights current, clinically relevant diagnostic strategies for
high-throughput diagnosis and disease response monitoring. The book covers, in
a series of individual chapters, a collection of overviews that highlight clinically
relevant novel therapeutic strategies in concise reviews. It also provides updates
on therapeutic compounds with new mechanisms of action that currently raise
intense interest and are in active development. This book comes as a timely
resource of information that furnishes a state-of-the-art and comprehensive
compendium, which will be of value to the interested clinician, researcher,
and student.
Bob Lowenberg
Erasmus University Medical Center
Rotterdam, The Netherlands
iv Foreword
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Preface
The treatment of leukemia and lymphoma is rapidly developing from conven-
tional chemotherapy toward a more tailored and targeted, innovative therapy.
However, conventional therapy is making progress as well. Targeted treatment
with increased efficacy and less side effects is becoming more and more a
reality, facilitated by fascinating developments such as oncogenomic studies and
sophisticated drug engineering. Knowledge on determinants of chemosensitivity
is also rapidly increasing. Together with pretreatment individualized tumor
response testing and with improved monitoring of treatment response by min-
imal residual disease measurements, treatment will indeed become more tailored
and individualized.
This book gives a complete and up-to-date overview of exciting new
treatment modalities in leukemia and lymphoma that have been introduced in the
clinic or will be introduced in the near future. Well-known international experts
summarize clinical studies on drugs such as tyrosine kinase inhibitors, mono-
clonal antibodies, proteasome inhibitors, farnesyl transferase inhibitors, hypo-
methylating agents, histone deacetylase inhibitors, mTOR targeting agents,
Notch pathway inhibitors, and inhibitors of cyclin-dependent kinases. The first
few chapters deal with methodological issues such as gene expression profiling
to detect new drug targets, individualized tumor response testing aiming at
selecting effective drugs, minimal residual monitoring to adapt treatment based
on actual treatment response, and statistical issues concerning clinical studies in
small subgroups of patients, while some discuss modulation of drug resistance
and improvements in allogeneic bone marrow transplantation. Other chapters
summarize targeting regulators of apoptosis, radioimmunotherapy, immunotherapy
by vaccination, gene-directed therapy, and anti-angiogenesis approaches. The
chapters provide a concise summary of the treatment rationale, of the pathways
that are involved, and of relevant preclinical research, whenever relevant.
v
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We recommend this well-illustrated, comprehensive book to students,
scientists, and clinicians with a special interest in innovative therapy who are
involved not only in research and/or treatment of leukemia and lymphoma in
particular, but in other malignancies as well.
G. J. L. Kaspers
Bertrand Coiffier
Michael C. Heinrich
Elihu Estey
vi Preface
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Contents
Foreword Bob Lowenberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1. Gene Expression Profiling to Detect New Treatment Targets
in Leukemia and Lymphoma: A Future Perspective . . . . . . . . . 1
Torsten Haferlach, Wolfgang Kern, and Alexander Kohlmann
2. Individualized Tumor Response Testing in Leukemia
and Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Andrew G. Bosanquet, Peter Nygren, and Larry M. Weisenthal
3. Minimal Residual Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Jacques J. M. van Dongen, Tomasz Szczepa nski,
and Vincent H. J. van der Velden
4. New Methods for Clinical Trials: AML as an Example . . . . . . 85
Elihu Estey
5. Monoclonal Antibody Mediated Treatment in Acute Myeloid
Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Ch. Michel Zwaan and Marry M. van den Heuvel-Eibrink
6. Monoclonal Antibodies in the Treatment of Malignant
Lymphomas and Chronic Lymphocytic Leukemia . . . . . . . . 125
Bertrand Coiffier
vii
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7. Radioimmunotherapy of Hematological Malignancies . . . . . . 149
Tim Illidge and James Hainsworth
8. Differentiation Induction in Acute Promyelocytic Leukemia . . . . 185
Adi Gidron and Martin S. Tallman
9. DNA Methylation and Epigenetics: New Developments
in Biology and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Jesus Duque, Michael L ubbert, and Mark Kirschbaum
10. The Emerging Role of Histone Deacetylase Inhibitors
in the Treatment of Lymphoma . . . . . . . . . . . . . . . . . . . . . . 233
Matko Kalac and Owen A. OConnor
11. Antileukemic Treatment Targeted at Apoptosis Regulators . . . 257
Simone Fulda and Klaus-Michael Debatin
12. Angiogenesis in Hematological Malignancies . . . . . . . . . . . . . 283
Alida C. Weidenaar, Hendrik J. M. de Jonge, Arja ter Elst,
and Evelina S. J. M. de Bont
13. Nucleic Acid-Based, mRNA-Targeted Therapeutics
for Hematologic Malignancies . . . . . . . . . . . . . . . . . . . . . . . . 311
Alan M. Gewirtz
14. Active Specific Immunization by the Use of Leukemic Dendritic
Cell Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Ilse Houtenbos, Gert J. Ossenkoppele,
and Arjan A. van de Loosdrecht
15. CDK Inhibitors in Leukemia and Lymphoma . . . . . . . . . . . . 353
Yun Dai and Steven Grant
16. FLT3: A Receptor Tyrosine Kinase Target in Adult
and Pediatric AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Mark Levis, Patrick Brown, and Donald Small
17. Treatment of Chronic Myeloid Leukemia with Bcr-Abl
Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Michael J. Mauro and Michael C. Heinrich
18. Tyrosine Kinase Inhibitors: Targets Other Than FLT3,
BCR-ABL, and c-KIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Suzanne R. Hayman and Judith E. Karp
viii Contents
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19. Tyrosine Phosphatases as New Treatment Targets
in Acute Myeloid Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . 449
I. Hubeek, K. Hoorweg, J. Cloos, and G. J. L. Kaspers
20. Proteasome and Protease Inhibitors . . . . . . . . . . . . . . . . . . . 469
N. E. Franke, J. Vink, J. Cloos, and G. J. L. Kaspers
21. Farnesyltransferase Inhibitors: Current and Prospective
Development for Hematologic Malignancies . . . . . . . . . . . . . 491
Judith E. Karp
22. Targeting Notch Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Jennifer ONeil and A. Thomas Look
23. mTOR Targeting Agents for the Treatment of Lymphoma
and Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Andrea E. Wahner Hendrickson, Thomas E. Witzig,
and Scott H. Kaufmann
24. Allogeneic Hematopoietic Cell Transplantation After
Nonmyeloablative Conditioning . . . . . . . . . . . . . . . . . . . . . . 539
Frederic Baron, Frederick R. Appelbaum, and Brenda M. Sandmaier
25. Modulation of Classical Multidrug Resistance and
Drug Resistance in General . . . . . . . . . . . . . . . . . . . . . . . . . 563
Branimir I. Sikic
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Contents ix
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Contributors
Frederick R. Appelbaum Fred Hutchinson Cancer Research Center and The
University of Washington, Seattle, Washington, U.S.A.
Frederic Baron Fred Hutchinson Cancer Research Center, Seattle, Washington,
U.S.A.
Andrew G. Bosanquet Bath Cancer Research, Royal United Hospital, Bath, U.K.
Patrick Brown Sidney Kimmel Comprehensive Cancer Center at Johns
Hopkins, Baltimore, Maryland, U.S.A.
J. Cloos Department of Pediatric Oncology/Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Bertrand Coiffier Hematology Department, Hospices Civils de Lyon and
Claude Bernard University, Pierre-Benite, France
Yun Dai Department of Medicine, Virginia Commonwealth University and
Massey Cancer Center, Richmond, Virginia, U.S.A.
Evelina S. J. M. de Bont Department of Pediatric Oncology/Hematology,
University Medical Center Groningen, University of Groningen, Groningen,
The Netherlands
Hendrik J. M. de Jonge Department of Pediatric Oncology/Hematology,
University Medical Center Groningen, University of Groningen, Groningen,
The Netherlands
Klaus-Michael Debatin University Childrens Hospital, Ulm, Germany
Jesus Duque Department of Hematology/Oncology, University Medical
Center Freiburg, Freiburg, Germany
xi
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Elihu Estey Division of Hematology, University of Washington Medical
Center, Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
N. E. Franke Department of Pediatric Oncology/Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Simone Fulda University Childrens Hospital, Ulm, Germany
Alan M. Gewirtz Division of Hematology/Oncology, Department of Medicine
& Abramson Family Cancer Research Institute, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania, U.S.A.
Adi Gidron Division of Hematology/Oncology, Department of Medicine,
Northwestern University Feinberg School of Medicine and The Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, U.S.A.
Steven Grant Department of Medicine, Biochemistry, and Pharmacology,
Virginia Commonwealth University and Massey Cancer Center, Richmond,
Virginia, U.S.A.
Torsten Haferlach Munich Leukemia Laboratory, Munich, Germany
James Hainsworth Paterson Institute of Cancer Research, School of
Medicine, University of Manchester, Manchester, U.K.
Suzanne R. Hayman Division of Hematology, Department of Medicine,
Mayo Clinic, Rochester, Minnesota, U.S.A.
Michael C. Heinrich Center for Hematologic Malignancies and Departments
of Medicine and Cell and Developmental Biology, Oregon Cancer Institute,
Oregon Health & Science University and Portland VA Medical Center, Oregon
Health & Science University, Portland, Oregon, U.S.A.
K. Hoorweg Department of Pediatric Oncology/Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Ilse Houtenbos Department of Hematology, VU University Medical Center,
Amsterdam, The Netherlands
I. Hubeek Department of Pediatric Oncology/Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Tim Illidge Paterson Institute of Cancer Research, School of Medicine,
University of Manchester, Manchester, U.K.
Matko Kalac Herbert Irving Comprehensive Cancer Center, The New York
Presbyterian Hospital, Columbia University, New York, New York, U.S.A.
Judith E. Karp Division of Hematologic Malignancies, Johns Hopkins Sidney
Kimmel Comprehensive Cancer Center, Baltimore, Maryland, U.S.A.
xii Contributors
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G. J. L. Kaspers Department of Pediatric Oncology/Hematology, VU
University Medical Center, Amsterdam, The Netherlands
Scott H. Kaufmann Department of Molecular Pharmacology and
Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, U.S.A.
Wolfgang Kern Munich Leukemia Laboratory, Munich, Germany
Mark Kirschbaum Division of Hematology and Hematopoietic Cell
Transplantation, City of Hope Comprehensive Cancer Center, Duarte,
California, U.S.A.
Alexander Kohlmann Roche Molecular Systems, Pleasanton, California, U.S.A.
Michael Lubbert Department of Hematology/Oncology, University Medical
Center Freiburg, Freiburg, Germany
Mark Levis Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,
Baltimore, Maryland, U.S.A.
A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer
Institute, Boston, Massachusetts, U.S.A.
Michael J. Mauro Center for Hematologic Malignancies, Oregon Cancer
Institute, Oregon Health & Science University, Portland, Oregon, U.S.A.
Peter Nygren Department of Oncology, Radiology, and Clinical Immunology,
University Hospital, Uppsala, Sweden
Owen A. OConnor Herbert Irving Comprehensive Cancer Center, The
New York Presbyterian Hospital, Columbia University, New York, New York,
U.S.A.
Jennifer ONeil Department of Pediatric Oncology, Dana-Farber Cancer
Institute, Boston, Massachusetts, U.S.A.
Gert J. Ossenkoppele Department of Hematology, VU University Medical
Center, Amsterdam, The Netherlands
Brenda M. Sandmaier Fred Hutchinson Cancer Research Center and The
University of Washington, Seattle, Washington, U.S.A.
Branimir I. Sikic Oncology Division, Department of Medicine, Stanford
University School of Medicine, Stanford, California, U.S.A.
Donald Small Sidney Kimmel Comprehensive Cancer Center at Johns
Hopkins, Baltimore, Maryland, U.S.A.
Tomasz Szczepanski Department of Immunology, Erasmus MC, University
Medical Center Rotterdam, Rotterdam, The Netherlands, and Department of
Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze,
Poland
Contributors xiii
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Martin S. Tallman Division of Hematology/Oncology, Department of
Medicine, Northwestern University Feinberg School of Medicine and The
Robert H. Lurie Comprehensive Cancer Center of Northwestern University,
Chicago, Illinois, U.S.A.
Arja ter Elst Department of Pediatric Oncology/Hematology,
University Medical Center Groningen, University of Groningen, Groningen,
The Netherlands
Marry M. van den Heuvel-Eibrink Department of Pediatric Oncology/
Hematology, Erasmus MC/Sophia Childrens Hospital, Rotterdam, The
Netherlands
Arjan A. van de Loosdrecht Department of Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Vincent H. J. van der Velden Department of Immunology, Erasmus MC,
University Medical Center Rotterdam, Rotterdam, The Netherlands
Jacques J. M. van Dongen Department of Immunology, Erasmus MC,
University Medical Center Rotterdam, Rotterdam, The Netherlands
J. Vink Department of Pediatric Oncology/Hematology, VU University
Medical Center, Amsterdam, The Netherlands
Andrea E. Wahner Hendrickson Department of Medicine, Mayo Clinic,
Rochester, Minnesota, U.S.A.
Alida C. Weidenaar Department of Pediatric Oncology/Hematology,
University Medical Center Groningen, University of Groningen, Groningen,
The Netherlands
Larry M. Weisenthal Weisenthal Cancer Group, Huntington Beach,
California, U.S.A.
Thomas E. Witzig Department of Medicine, Mayo Clinic, Rochester,
Minnesota, U.S.A.
Ch. Michel Zwaan Department of Pediatric Oncology/Hematology, Erasmus
MC/Sophia Childrens Hospital, Rotterdam, The Netherlands
xiv Contributors
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BASIC AND CLINICAL ONCOLOGY
Series Editor
Bruce D. Cheson
Professor of Medicine and Oncology
Head of Hematology
Georgetown University
Lombardi Comprehensive Cancer Center
Washington, D.C.
1. Chronic Lymphocytic Leukemia: Scientific Advances and Clinical
Developments, edited by Bruce D. Cheson
2. Therapeutic Applications of Interleukin-2, edited by Michael B.
Atkins and James W. Mier
3. Cancer of the Prostate, edited by Sakti Das and E. David Crawford
4. Retinoids in Oncology, edited by Waun Ki Hong and Reuben Lotan
5. Filgrastim (r-metHuG-CSF) in Clinical Practice, edited by George
Morstyn and T. Michael Dexter
6. Cancer Prevention and Control, edited by Peter Greenwald,
Barnett S. Kramer, and Douglas L. Weed
7. Handbook of Supportive Care in Cancer, edited by Jean Klastersky,
Stephen C. Schimpff, and Hans-Jorg Senn
8. Paclitaxel in Cancer Treatment, edited by William P. McGuire
and Eric K. Rowinsky
9. Principles of Antineoplastic Drug Development and Pharmacology,
edited by Richard L. Schilsky, G

erard A. Milano, and Mark J. Ratain


10. Gene Therapy in Cancer, edited by Malcolm K. Brenner and Robert
C. Moen
11. Expert Consultations in Gynecological Cancers, edited by Maurie
Markman and Jerome L. Belinson
12. Nucleoside Analogs in Cancer Therapy, edited by Bruce D. Cheson,
Michael J. Keating, and William Plunkett
13. Drug Resistance in Oncology, edited by Samuel D. Bernal
14. Medical Management of Hematological Malignant Diseases,
edited by Emil J Freireich and Hagop M. Kantarjian
15. Monoclonal Antibody-Based Therapy of Cancer, edited by Michael
L. Grossbard
16. Medical Management of Chronic Myelogenous Leukemia, edited
by Moshe Talpaz and Hagop M. Kantarjian
[pradeepr][D:/informa_Publishing/DK0832_Kaspers_112039/z_production/z_3B2_3D_files/978-0-
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17. Expert Consultations in Breast Cancer: Critical Pathways and
Clinical Decision Making, edited by William N. Hait, David A. August,
and Bruce G. Haffty
18. Cancer Screening: Theory and Practice, edited by Barnett S.
Kramer, John K. Gohagan, and Philip C. Prorok
19. Supportive Care in Cancer: A Handbook for Oncologists: Second
Edition, Revised and Expanded, edited by Jean Klastersky, Stephen
C. Schimpff, and Hans-Jorg Senn
20. Integrated Cancer Management: Surgery, Medical Oncology, and
Radiation Oncology, edited by Michael H. Torosian
21. AIDS-Related Cancers and Their Treatment, edited by Ellen
G. Feigal, Alexandra M. Levine, and Robert J. Biggar
22. Allogeneic Immunotherapy for Malignant Diseases, edited by John
Barrett and Yin-Zheng Jiang
23. Cancer in the Elderly, edited by Carrie P. Hunter, Karen A. Johnson,
and Hyman B. Muss
24. Tumor Angiogenesis and Microcirculation, edited by Emile E. Voest
and Patricia A. DAmore
25. Controversies in Lung Cancer: A Multidisciplinary Approach, edited
by Benjamin Movsas, Corey J. Langer, and Melvyn Goldberg
26. Chronic Lymphoid Leukemias: Second Edition, Revised and
Expanded, edited by Bruce D. Cheson
27. The Myelodysplastic Syndromes: Pathology and Clinical Manage-
ment, edited by John M. Bennett
28. Chemotherapy for Gynecological Neoplasms: Current Therapy and
Novel Approaches, edited by Roberto Angioli, Pierluigi Benedetti
Panici, John J. Kavanagh, Sergio Pecorelli, and Manuel Penalver
29. Infections in Cancer Patients, edited by John N. Greene
30. Endocrine Therapy for Breast Cancer, edited by James N. Ingle and
Mitchell Dowsett
31. Anemia of Chronic Disease, edited by Guenter Weiss, Victor
R. Gordeuk, and Chaim Hershko
32. Cancer Risk Assessment, edited by Peter G. Shields
33. Thrombocytopenia, edited by Keith R. McCrae
34. Treatment and Management of Cancer in the Elderly, edited by
Hyman B. Muss, Carrie P. Hunter, and Karen A. Johnson
35. Innovative Leukemia and Lymphoma Therapy, edited by G. J. L.
Kaspers, Bertrand Coiffier, Michael C. Heinrich, and Elihu Estey
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1
Gene Expression Profiling to Detect
New Treatment Targets in Leukemia
and Lymphoma: A Future Perspective
Torsten Haferlach and Wolfgang Kern
Munich Leukemia Laboratory, Munich, Germany
Alexander Kohlmann
Roche Molecular Systems, Pleasanton, California, U.S.A.
INTRODUCTION
The standard methods for establishing the diagnosis and prognosis of acute
leukemias and lymphomas are cytomorphology and cytochemistry in combina-
tion with multiparameter immunophenotyping. However, cytogenetics, fluores-
cence in situ hybridization (FISH), and polymerase chain reaction (PCR)-based
assays add important information with respect to biologically defined and
prognostically relevant subgroups. Together, a combination of different methods
allows a comprehensive diagnosis with relevant clearly defined subentities. It also
leads to a better understanding of the respective clinical course of defined disease
subtypes and to a more or less disease-specific therapeutic approach. However, not
all patients achieve complete remission during treatment, and many of those who
do, later develop relapse and treatment-resistant disease. To overcome these
problems, the microarray technology, which quantifies gene expression intensities
of thousands of genes in a single analysis, holds the potential to become an essential
tool for a strictly molecularly defined classification of leukemias and lymphomas.
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It may therefore be used at first as a novel routine method for diagnostic approaches
in the near future (1). But even more importantly, it will also reveal newgenetic and
therapeutically relevant markers and should guide the search for new targets. Gene
expression profiling will also improve patient selection to test therapeutic
hypothesis most efficiently and may help define dose and schedule determina-
tion. This chapter outlines the major steps for gene expression profiling analyses
to approach these different goals by starting at a better diagnostic character-
ization of leukemias and lymphomas hopefully ending up with new targets for
individual treatment of the respective patients.
MICROARRAYS AND THE ERA OF FUNCTIONAL GENOMICS
Both biology and medicine are undergoing a revolution that is based on the
accelerating determination of DNA sequences, including the completion of whole
genomes of a growing number of organisms (2). In parallel to the sequencing efforts,
a wide range of technologies with tremendous potential has been achieved that can
take advantage of the vast quantity of genetic information being now available. The
field of functional genomics seeks to devise and apply these technologies, such as
microarrays, to analyze the full complement of genes and proteins encoded by an
organism to understand the functions of genes and proteins (3) (Fig. 1).
Figure 1 Different types of microarray platforms. Microarray platforms vary according to
the solid support used (such as glass slides or silicon wafers), the surface modifications with
various substrates, the type and length of DNA fragments on the array (such as cDNA or
oligonucleotides), whether the gene fragments are presynthesized and deposited, or synthe-
sized in situ, the machinery used to place the fragments on the array (such as ink-jet printing,
spotting, mask, or micromirror-based in situ synthesis), and the method of sample preparation.
Currently, combinations of these variables are used to generate two main types of microarrays:
spotted glass slide arrays (right) and in situ synthesized DNA-oligonucleotide arrays (left).
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Glass Slide Microarrays
Glass slide microarrays were first produced in Patrick Browns laboratory at
Stanford University (4). In glass slide microarray studies, ribonucleic acid
(RNA) species from the test sample and from the reference sample are studied
pairwise as an equivalent mixture in which the control RNA is the reference for
expressing the gene transcript levels in the target sample (Fig. 1). Various direct
and indirect labeling methods for the sample have been developed (5). The
majority of expression analysis labeling protocols is based on the reverse tran-
scription of mRNA, either from highly purified poly(A) mRNA or total RNA
extracts and often include amplification steps. In most protocols, one sample is
labeled with the Cy3 (green) fluorochrome, the other with Cy5 (red). The labeled
cRNA molecules hybridize to the corresponding cDNA or long oligonucleotides,
of which the exact position on the array is known. The binding of the target to
the probe is detected by scanning the array, typically using either a scanning
confocal laser or a charge coupled device (CCD) camera-based reader. After
scanning, software calculations provide the ratios between green and red fluo-
rescence for each spot, corresponding to the relative abundance of mRNA from a
particular gene in the target sample versus the reference sample.
However, the technical difficulties in the reproducible production of glass
slide microarrays should not be underestimated (5). Much of this variation is
introduced systematically during the spotting of the DNA onto the slide surface,
and many of the initial cDNA clone sets were compromised by contamination
with T1 phage, multiple clones in individual wells, and incorrect sequence
assignment. Thus, given the lack of a gold standard for the production of glass
slide microarrays using current technologies, there is a high degree of variation
in the quality of data derived from glass slide microarray experiments. This poor
reproducibility not only adds to the cost of a given study but also leads to data
sets that are difficult to interpret.
MICROARRAYS AS AN INNOVATIVE TECHNIQUE
TO DETECT NEW TARGETS
For several reasons many investigations using microarrays for biological
approaches today are performed on the whole genome Affymetrix U133 set
(HG-U133A and HG-U133B or the HG-U133 2.0 plus array; Affymetrix, Santa
Clara, California, U.S.). A detailed up-to-date description on sequences and
probe selection rules is available as technical note from the manufacturer (www
.affymetrix.com).
Affymetrix HG-U133A and HG-U133B Microarrays
The U133 two-array set provides comprehensive coverage of well-substantiated
genes in the human genome. It can be used to analyze the expression level of
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39,000 transcripts and variants, including greater than 33,000 human genes. The
two arrays comprise more than 45,000 probe sets and 1,000,000 distinct oligo-
nucleotide features. The sequences from which these probe sets were derived
were selected from GenBank, dbEST, and RefSeq. The sequence clusters were
created from the UniGene database (Build 133, April 20, 2001) and then refined
by analysis and comparison with a number of other publicly available databases,
including the Washington University EST trace repository and the University of
California, Santa Cruz, Golden-Path human genome database (April 2001
release). In addition, an advanced understanding of probe uniqueness and
hybridization characteristics allowed an improved selection of probes based on
predicted behavior. The U133 chip design uses a multiple linear regression
model that was derived from a thermodynamic model of nucleic acid duplex
formation. This model predicts probe binding affinity and linearity of signal
changes in response to varying target concentrations. The two arrays are man-
ufactured as standard format arrays with a feature size of 18 mm and use 11 probe
pairs per sequence. The oligonucleotide length is 25 mer.
Human Genome U133 Plus 2.0 Array
In addition to all the sequences represented on the HG-U133A and HG-U133B
two-array set, the HG-U133 Plus 2.0 microarray also covers 9921 new probe sets
representing approximately 6500 new genes. These gene sequences were
selected from GenBank, dbEST, and RefSeq. Sequence clusters were created
from the UniGene database (Build 159, January 25, 2003) and refined by
analysis and comparison with a number of other publicly available databases,
including the Washington University EST trace repository and the NCBI human
genome assembly Build 31 (www.affymetrix.com). Thus, in using this com-
prehensive whole human genome expression array, an extensive coverage of the
human genome is reached. HG-U133 Plus 2.0 microarrays are manufactured as
standard format arrays with more than 54,000 probe sets of a feature size of
11 mm and use 11 probe pairs per sequence. The oligonucleotide length is 25 mer.
MICROARRAY DATA ANALYSIS
A wide range of approaches is available for gleaning insights from the data
obtained from transcriptional profiling. Data analyses are performed by two
different approaches, i.e., the supervised approach and the unsupervised
approach (Fig. 2). Unsupervised analyses are used to test the hypothesis whether
specific characteristics, e.g., genetic aberrations, are also reflected at the level of
gene expression signatures. Supervised analyses identify a minimal set of genes
that could be used to stratify those patients after a training of classification
engines (68). The gene lists from supervised analyses can also be further
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interpreted in terms of underlying biology. For all gene expression profiles,
master data tables have to be maintained. In these tables, rows represent all genes
for which data have been collected and columns represent microarray experi-
ments from individual patients. Each cell represents the measured fluorescence
intensity from the corresponding target probe set on the microarray. Before
analyzing the data, it is a routine procedure to normalize the data. This pro-
cedure is a mandatory step in the data-mining process to appropriately compare
the measured gene expression levels. U133 set microarray signal intensity
Figure 2 Overview about a common workflow to analyze microarray data. After
preparation of corresponding data sets from the main master table, the data are analyzed
either unsupervised or supervised. Unsupervised analyses are performed by hierarchical
clustering or principal component analysis. In the supervised analyses, differentially
expressed genes can be identified by various methods and selected for further inter-
pretations, e.g., visualization by hierarchical clustering, principal component analysis,
plotting as bar graphs, or generation of biological networks. In addition, differentially
expressed genes can be selected for classification tasks where several different machine-
learning approaches have to be applied.
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values can be normalized by scaling the raw data intensities to a common target
intensity using a recommended mask file.
Some Examples of Software to Identify Genes of Interest
Several software packages are used for principal data acquisition (GCOS),
storage (MicroDB), and analysis (DMT). The following tables give only some
examples to approach data. Individual gene expression profiles can also further
be prepared as Microsoft Excel tables.
Software Source Internet
GCOS Affymetrix, Inc. www.affymetrix.com/support/
MicroDB Affymetrix, Inc. www.affymetrix.com/support/
DMT Affymetrix, Inc. www.affymetrix.com/support/
The following packages can be applied for the identification of differ-
entially expressed genes and classification:
Software Source Internet
SAM Stanford University www-stat.stanford.edu/~tibs/SAM/
index.html
Bioconductor Open source www.bioconductor.org
q-Value University of
Washington
faculty.washington.edu/~jstorey/qvalue/
LIBSVM National Taiwan
University
www.csie.ntu.edu.tw/~cjlin/libsvm/
SAM is available as Microsoft Excel Add-in (9). Bioconductor is an open
source and open development software project for the analysis and comprehension
of genomic data. Bioconductor packages provide statistical and graphical
methodologies for analyzing genomic data. LIBSVM (Version 2.6) is a software
solution for SVM-based classification. The q-value software takes a list of p-values
resulting from the simultaneous testing of many hypotheses and estimates their
q-values (10). In addition, further third party software packages can be used for
statistical analyses and data visualization.
Software Source Internet
SPSS SPSS, Inc. www.spss.com/
Pathways Analysis Ingenuity Systems www.ingenuity.com
GeneMaths Applied Maths, Inc. www.applied-maths.com
Genomics Suits Partek, Inc. www.partek.com/
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Tools for Pathway Analyses to Detect New Targets and Correlations
The identification of diagnostic, prognostic, or therapeutic markers in leukemia
and lymphoma following microarray experiments and their biostatistical read outs
have to then focus on the discovery of important pathways in these tumors. Several
programs exist in order to identify pathways involved. These include Pathway
Assist (http://www.ariadnegenomics.com/products/pathway.html), DAVID (http://
apps1.niaid.nih.gov/david/), and Ingenuity (http://www.ingenuity.com/). As one
example, Ingenuity enables researchers to model, analyze, and understand complex
biological systems foundational to human health and disease. This includes
pathways analysis software and knowledge databases for biologists and bio-
statisticians and enterprise knowledge management infrastructure. Today, Ingenuity
is a useful knowledge base of biological networks with curated relationships between
proteins, genes, complexes, cells, tissues, drugs, and diseases.
Increasingly, also bioinformaticians are interested in developing analytical
tools that help scientists interpret experimental data especially in the context of
pathways and biological systems. These analytical tools have broad application
throughout research and development, from validating targets by uncovering
disease-related pathways to predicting pathways perturbed by therapeutic com-
pounds. As one example in Ingenuity, a broad genome-wide coverage of over
25,900 mammalian genes (11,100 human, 5500 rat, and 9300 mouse) can be
found and millions of pathway interactions extracted from literature are managed
interactively and web based.
At a basic level, an understanding of functions and pathways associated with
genes identified within an early-stage candidate region may assist in prioritizing
portions of this region for further investigation, e.g., targeted association using
higher densities of single nucleotide polymorphisms (SNPs). This type of approach
may even assist in identifying which genes to resequence in an attempt to identify
further SNPs for association studies. This is achievable now with the ability to
upload, for example, Affymetrix SNP identifiers directly into pathway software
such as Ingenuity. Future developments may increase the mapping coverage of
SNPs beyond the simple 1:1 gene to SNP mapping available today.
Beyond this, future functionality may even allow for the correlation
between multiple regions of the genome identified at a functional level and
findings of a genetic association study that identifies multiple, low scoring
regions. Previously, these may not have warranted further investigation based
solely on association scores. However, functional, process, pathway, or disease
annotations may implicate multiple regions as being relevant to a particular
phenotype by virtue of their compound effect. Evidence is already emerging
from the HapMap project that there are significant SNPs that are genetically
indistinguishable across large regions of individual chromosomes or even dif-
ferent chromosomes. It is anticipated that further development of software and
pathway analyses tools to approach the huge sets of data generated in microarray
experiments will lead to deeper insights.
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DETECTION OF NEW TARGETS IN LEUKEMIA AND LYMPHOMA
As has been outlined before, gene expression profiling has been extensively used
for tumor classification (8,1115) and is on the way to add important information
to predict response to therapy as well as for outcome in leukemia and lymphoma
patients. As these data are not in the focus of this article, they will only be cited,
if they add information also for new target detection.
Furthermore, there are only limited efforts yet to incorporate microarrays
into clinical trials in hematology and oncology because of several reasons: (1)
prospective sample acquisition parallel to the gold standard diagnostic proce-
dures is needed, (2) standardized equipment and software has to be used, (3)
experienced scientists and technicians with respect to microarray analyses have
to be involved, and (4) funding is mostly lacking and would be best if academic
institutions and industry combine efforts. Other factors like intra-laboratory and
inter-laboratory comparability have also to be taken into account.
This leads to the following relation according to Weeraratna (16): More
than 9000 references are available that concern microarrays, but only around
20 are clinical trials, and less than 10 of these pertain to cancer. As currently no
single prospective trial has been conducted to our knowledge to address the use
of microarrays within a clinical trial in leukemia and lymphoma, we only can
rely on information that was published in papers referring to diagnostic or
prognostic questions. On the basis of their findings, some preliminary statements
can also be made for the use of gene expression profiling to define new targets
and drugs in leukemia and lymphoma (17). The following chapters will comment
on these aspects and will be subdivided disease specifically.
Detection of New Targets in Lymphoma
Alizadeh et al. (13) defined distinct subtypes of diffuse large B-cell lymphoma
(DLBCL) by specific gene expression signatures. Although this paper mostly
focuses on newly defined biological subgroups of DLBCL, different prognosis
was also detected. This again leads to the detection of genes that are responsible
not only for a better and novel subclassification but also transfer into striking
differences in prognosis if patients are treated uniformly. Thus, the authors
concluded that a respective gene expression pattern and the IPI score for NHL in
combination will guide therapeutic decisions including bone marrow trans-
plantation as one option for high-risk patients. Furthermore, expression profiling
may also help to detect homogeneous groups of patients to improve the likeli-
hood of observing treatment efficacy in specific disease entities. This study was
the first to show that the two DLBCL subgroups differentially expressed entire
transcriptional modules composed of hundreds of genes. Polo et al. identified
a discrete subset of DLBCL that are reliant on Bcl6 signaling and uniquely
sensitive to Bcl6 inhibitors (18). Therefore, successful new therapeutics may be
aimed at the upstream signal-transducing molecules and further investigations
are needed.
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Microarrays have also been used to study the targets of c-Myc, a tran-
scription factor that plays a role in Burkitts lymphoma as c-Myc is involved in
the chromosomal translocation t(8;14). In this study genomic targets including
genes involved in cell cycle, cytoskeletal organization, cell growth, and adhesion
were identified (19). However, these structures have to be tested again as drug
targets after having been detected by gene expression profiling.
Detection of New Targets in Acute Myeloid Leukemia
Yagi et al. (20) analyzed 54 pediatric acute myeloid leukemia (AML) using
Affymetrix U95A arrays and focused on the reproducibility of some FAB sub-
types and especially on gene patterns to predict outcome. After unsupervised
clustering, they were able to differentiate patients with t(8;21) from those with
inv(16) and from those demonstrating an AML M4/5 or AML M7 phenotype or
immunophenotype by specific gene expression signatures. Within this unsu-
pervised analysis, no specific profile was found that correlated to the prognosis
of the patients. Since the inclusion of further cases with other FAB subtypes and
cytogenetic abnormalities (no karyotype was available in 9 of 54 cases) resulted
in an increased heterogeneity, the authors restricted their further analyses to the
genetically and morphologically better-defined subentities. For further calcula-
tion, data were analyzed and supervised with respect to outcome and prognosis.
A subset of 35 genes that were independent from the morphology or karyotype of
the patients was selected; some of them are associated with the regulation of the
cell cycle or with apoptosis. By hierarchical cluster analysis, patients could be
classified into high-risk and low-risk groups with highly significant differences
in event-free survival (EFS) ( p < 0.001).
Another approach was described by Qian et al. (21) in therapy-related AML
and myeloid cell lines focussing on CD34-positive selected cells. They were the
first ones to define a specific pattern of gene expression for t-AML in comparison
with other AML subtypes. The most discriminating genes were found to be
involved in arrested differentiation of early progenitor cells. A higher expression
of cell cycle control genes such as CCNA2, CCNE2, and CDC2 and genes for cell
cycle checkpoints such as BUB1 or growth (Myc) were found. Furthermore,
downregulation of transcription factors involved in early hematopoiesis (TAL1,
GATA1, EKLF) and overexpression of FLT3 was detected. The authors con-
cluded that these genes may be further investigated for new targets and drugs in
this very unfavourable subtype of AML.
As a further hallmark in AML, Bullinger et al. analyzed 65 peripheral
blood and 54 bone marrow samples in patients with AML (12). On the basis of
6283 most variably expressed genes they were able to reproduce cytogenetically
defined AML subgroups and, in addition, to define two different groups with
highly differing prognosis on the basis of gene expression profiles. While both
groups mainly included AML cases with normal karyotypes without differences
in many prognostic parameters, it is noteworthy that the group with the poorer
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prognosis included more patients with monosomy 7, complex aberrant
karyotypes, and length mutations of FLT3, while the group with the better
prognosis included more patients with inv(16). Thus, the observed differences in
the prognosis between both groups may be largely due to imbalances in profiles
of established prognostic factors rather than due to the identification of a newly
characterized biological subgroup of AML. Genes as published by Bullinger
et al. should be tested in independent cohorts of AML patients to further support
their prognostic power, and further investigations are again warranted for the
definition of such genes as new targets in AML treatment.
Similar results have been reported by Valk et al. (11) who discovered
16 groups of AML featuring distinct gene expression profiles on the basis of
microarray analysis, which, in addition, showed significant differences in clin-
ical course. However, while many of the identified groups were characterized by
specific cytogenetic aberrations known to be highly predictive of outcome, none
of the groups were restricted to cases without cytogenetic abnormalities. Thus,
the task remains to identify markers capable of discriminating prognostically
different cases out of the heterogeneous group of AML with normal karyotype
and to use these for target testing.
An improvement in this direction has been reported by Kern et al. who
analyzed gene expression profiles in 205 patients with AML and normal karyotype
(22). In order to identify genetically defined subgroups, an unsupervised principal
component analysis revealed 79% of cases clustering together, while a subgroup
comprising 21% of cases formed another cluster. Importantly, the analysis of
known genetic markers, including the presence of length mutations and point
mutations of FLT3, partial tandem duplications of MLL, or mutations of CEBPA,
NRAS, or CKIT, did not reveal differences between both groups. Significant
differences were found, however, in their phenotypes with more monocytic fea-
tures in the smaller group. Analysis of differentially regulated genetic pathways
revealed CD14, WT1, MYCN, HCK, and SPTBN1 as discriminating genes.
Stressing the potential impact of this analysis on the clinical management of AML,
these two groups significantly differed in the EFS. Thus, it was demonstrated here
also that within the group of AML with normal karyotype highly needed novel
molecular markers with prognostic impact can be identified by using gene
expression profiling. Some of the discriminating structures defined here may also
be used for future targets in specific AML subtypes.
However, regarding the biological heterogeneity of AML in general and of
AML with normal karyotype in particular, it is anticipated that further large-
scale studies in the context of clinical trials are needed to fully characterize and
validate novel and clinically relevant subgroups in AML and by doing so to
define new targets for individual treatment. A recent example is the study of
Bullinger et al. who further subclassified 93 patients with core binding factor
(CBF) leukemias (AML1-ETO and CBFB-MYH11) in different risk groups (23).
Another structure identified by gene expression profiling is the ubiquitin-
activating enzyme E1-like (UBE1) gene that is induced by all-trans retinoic acid
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(ATRA) in NB4 cells (24). Detailed investigation revealed that ATRA activates
the UBE1 promoter and the overexpression of UBE1 therefore triggers the
degradation of promyelocytic leukemia-retinoic acid receptor alpha (PML-
RARa) and leads to apoptosis in acute promyelocytic leukemia (APL) cells (25).
Clinical studies with UBE1 in leukemia are however missing.
Andersson et al. in a recent study compared (26) the molecular signatures
in childhood acute leukemias and their correlations to expression patterns in
normal hematopoietic subpopulations. 87 B-lineage acute lymphoblastic leukemia
(ALL), 11 T-cell ALL, 23 AML, and 6 normal bone marrows, as well as 10
normal hematopoietic subpopulations of different lineages and maturations were
ascertained by 27K cDNA microarrays. Not surprisingly, segregation according
to lineage and primary genetic changes was achieved. However, several genes
were identified that were preferentially expressed by the leukemic cells and not
by their normal counterparts. These genes suggest an ectopic activation and are
likely to reflect regulatory networks that may provide attractive targets for future
directed therapies. However, although this study clearly points to the right
direction, targets that were defined in this study have to be tested in an inde-
pendent cohort of patients before they may be used for drug design. This again
demonstrates that even if a variety of markers can be defined by gene expression
signatures in addition to the diagnostic pattern of a specific leukemia subtype,
the use of such information to find therapeutic structures or even targets is still
limited, which emphasizes the need for better support of translational research
and drug development in the future.
A possible approach to use expression profiling in a high-throughput
screening was published by Stegmaier et al. (27). They used HL-60 cells in
384-well culture plates and cultivated them with uniform concentrations of
1739 compounds to induce differentiation. By including different gene expression
signatures of AML-versus-monocyte and AML-versus-neutrophil distinctions as
measured by DNA microarrays, data were complemented by reverse transcription-
polymerase chain reaction (RT-PCR) and matrix assisted laser desorption/
ionization time-of-flight (MALDI-TOF). Because of this approach, finally eight
compounds were identified that reliably induced the differentiation signature. As a
result, a modest number of genes were sufficient to capture a complex cellular
response. However, the authors concluded that further investigations are needed to
identify the optimal gene signature. This work points to a possible scenario for the
identification of new targets and drugs by gene expression profiling. However, it
again demonstrates the complex problem to combine different highly sophisticated
methods in a high-throughput investigation to define at the end drugs to be tested
in a clinical trial.
Detection of New Targets in ALL
For sure, one of the most important questions posed by the use of gene expression
profiling is the identification of new targets for the further development of highly
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specific antileukemic drugs. One striking example is based on the results from
Armstrong et al. who found mutations and high-level expression in the FLT3
tyrosine kinase receptor gene in MLL-rearranged ALLs (28,29). FLT3 is known as
a tyrosine kinase receptor that is frequently activated by mutations in patients with
AML (30) but is rarely activated in ALL. However, by gene expression profiling it
was demonstrated that FLT3 was the gene most strongly associated with the
presence of MLL gene rearrangements in ALL. This leads to the idea (28,31,32) to
further investigate the potential role of this oncogene in the pathogenesis of MLL
tumors per se. Mutational analyses of FLT3 in MLL generearranged leukemias
clearly showed the presence of activating mutations in the activation loop of this
tyrosine kinase receptor in 5 of 30 cases studied. This was further validated by
treating leukemia cells with PKC412, a specific inhibitor of the FLT3 tyrosine
kinase. It was shown both in vivo and in vitro that PKC412 has differential cytotoxic
effects on MLL rearranged leukemia cells harbouring FLT3 activation (28).
Furthermore, it was demonstrated that also in ALL with hyperdiploid
cytogenetics, the FLT3 receptor is frequently expressed at a higher level. This
again reinforces the value of gene expression profiling as a powerful approach
for the identification of novel drugs also in ALL (3234), which should motivate
an urgent translation into clinical trials including high-risk patients.
Another approach in ALL to use gene expression for further insights in
biology of the disease was described by Zaza et al. (35). After intravenous
administration of thioguanine nucleotide (TGN), the TGN concentration was
determined in the leukemic blasts of 82 children with newly diagnosed ALL.
After analyses of 9600 genes, they identified 60 probes that were significantly
associated with TGN accumulation if patients were treated with mercaptopurine
(MP) alone and another 75 genes in patients treated with a combination of
metotrexate (MTX) and MP. There was no overlap between these two sets of
genes. The investigation was performed in parallel in vivo and in vitro and gene
expression profiling led to new insights into the genomic basis of interpatient
differences with respect to different treatment options. Through gene expression
profiling, clear correlations between a specific drugs level in vivo and increased
expression of specific genes were detected. It was even visible that expression
profiles correlated to mono or combined treatment modalities. Prospective
studies are needed to test these results.
Another outstanding investigation was conducted by Holleman et al. (36)
who identified a set of differentially expressed genes in B-lineage ALL being
sensitive or resistant to several drugs such as prednisolone (33 genes), vincristine
(40 genes), asparaginase (35 genes), and daunorubicin (20 genes). A score of genes
combined to define overall sensitivity or resistance to all four drugs was tested in a
multivariate analysis and predicted outcome of 173 children investigated
( p 0.027). Although these genes do not per se define new targets of treatment,
gene expression profiling clearly demonstrated in a prospective setting which
treatment may or may not be successful. This may serve as an example for the
application of gene expression profiling to improve treatment and to define targets
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and drugs against these targets in ALL. The authors further point to the aspect that
it may be important to determine whether specific modulation of proteins encoded
by genes that were found may describe treatment response best. These proteins may
also point to previously unrecognized potential targets and new agents to augment
the efficacy of current chemotherapy for ALL.
Brown et al. (37) investigated FLT3 inhibition by the selective inhibitor
CEP-701 in ALL. In this study eight ALL cell lines and primary ALL cells from
39 patients were evaluated and a high potency for this drug especially in ALL cells
overexpressing FLT3, i.e., MLL rearranged cases, as well as ALLs with hyper-
diploid karyotypes was identified. Seven of seven sensitive samples examined by
immunoblotting demonstrated constitutively phosphorylated FLT3 that was
potently inhibited by CEP-701, whereas zero out of six resistant samples expressed
constitutively phosphorylated FLT3. The authors concluded that the compound
CEP-701, a potent and selective FLT3 inhibitor, effectively suppresses FLT3-
driven leukemic cell survival and clinical testing of this compound as a novel
molecularly targeted agent for treatment of ALL is warranted.
However, in most cases the candidate targets identified in expression
studies (28) using relapse or treatment outcome as endpoints of their observation
and independent verification is missing (38). Therefore, conflicting results are
largely due to differences in treatment and biology of enrolled patients. The gap
between gene expression profiling to characterize biological entities in leukemia
and lymphoma and the targets to be tested is still not closed, and translation from
data management to drug design is still missing. However, the characterization
of molecular mutations and of pathway alterations in the leukemias proceeds
with high velocity as can be demonstrated by the recent study of Mullighan et al.
who revealed the PAX gene as the most frequent target of molecular mutation in
ALL and showed that direct disruption of pathways controlling B-cell devel-
opment and differentiation contribute to B-progenitor ALL pathogenesis (39).
This is just one more example of the recent progress in the identification of new
molecular targets in ALL.
Detection of New Targets in Chronic Myeloid Leukemia
McLean et al. (40) intended to define specific gene expression profiles in chronic
myeloid leukemia (CML) patients all treated with imatinib. In correlation to
cytogenetic response data, the expression pattern of a subset of 55 out of more
than 12,000 genes was identified that best predicted response to therapy. The
sensitivity to predict the individual response was 93.4%; however, the specificity
was only 58.3%. The authors further found that many of the genes identified
appeared to be strongly related to BCR-ABL transformation mechanisms. Thus,
these genes may need further investigation as potential new drug targets in CML.
Diaz-Blanco et al. described several novel transcriptional changes in pri-
mary CD34 positive CML cells in comparison with normal CD34-positive cells
including an upregulation of components of the TGFB signaling pathway or
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candidate genes such as the leptin receptor (LEPR), thrombin receptor (PAR1),
or the neuroepithelial cell transforming gene 1 (NET1) (41). It was further
possible to define differentially regulated candidate genes discriminating chronic
from blast phase of CML such as PRAME (preferentially expressed antigen of
melanoma) (42) or CAMPATH (CD52) (43) or deregulation of pathways, e.g.,
the WNTB catenin signaling system (42). These studies thus might be helpful for
definition of new novel stem or progentior cell-associated targets and of
mechanisms being responsible for the higher malignant transformation of CML
(41,42).
Detection of New Targets in Chronic Lymphocytic Leukemia
One highlight to establish the use of gene expression profiling to define new
targets in leukemia was the detection of ZAP70 to be expressed in a large
proportion of chronic lymphocytic leukemia (CLL) (14). As the expression of
ZAP70 was high in IgVH-unmutated cases of CLL, this gene was further cor-
related to distinction within CLL cases with respect to prognosis. This finding
also led to the investigation of the ZAP70 antigen expression by antibodies in
CLL using multiparameter immunophenotyping (44). Recently, it was demon-
strated that ZAP70 can also be successfully screened by a quantitative RT-PCR
method (45). After definition of CLL signature genes, the protein products of
these genes may represent such new targets for monoclonal antibodies or for
vaccine approaches. Another aspect detected in this investigation was the fact
that B-cell activation genes were upregulated in Ig-unmutated patients. Thus,
pathways downstream of the B-cell receptor may contribute to aggressive clinical
cases. It may be beneficial to target these signaling pathways.
However, again, gene expression profiling so far was helpful in finding
new epitopes in strict correlation to a specific disease or even subgroups within
such diseases, but targeted drugs are still under investigation.
Future Investigations to Diagnostic and Therapeutic Use
of Gene Expression Profiling: The MILE Study
The (microarray innovations in leukemia) MILE study is a cooperation of the
European Leukemia Network (ELN, work package 13) together with Roche
Molecular Systems. This innovative study was designed to test microarrays in
parallel to gold standard diagnostics in 4000 patients with leukemia in 11 different
sites (7 from ELN, 3 in United States, 1 in Singapore). At least 18 different classes
of leukemia shall prospectively be defined for diagnostic use in the MILE study
by their respective gene expression signatures. The ELN work package 13 is
per se the head of these activities.
In order to set up a clearly defined study with comparable sample quality,
as a first step, a prephase was conducted to harmonize laboratory workflows.
This prephase included tests of similar aliquots of two cell lines and three
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leukemia samplesAML, CML, and CLL. A first interim analysis was able to
demonstrate a very high inter- and intra-laboratory reproducibility (46). Figure 3
is an example of the data generated.
Stage I of the study now includes 2000 samples of leukemia all analyzed in
parallel with gold standard methods. After clarification of discrepant results
between gene expression analyses and gold standard report forms, the most
discriminating genes will be used to design a specific custom microarray for the
diagnosis of leukemias. This new microarray will then be tested prospectively in
stage II of the study by including another set of 2000 leukemia samples.
It is further intended to use a subset of this data to address further questions
like response to specific treatment as many patients are enrolled in prospective
clinical trials. Only studies like this may define new targets for treatment, because
information will be available on diagnosis, prognostic parameters, treatment, and
response as well as ultimately for treatment outcome. The power of gene expression
profiling may help in approaching such data sets from different perspectives and
may therefore be used to address several questions in parallel.
SUMMARY AND FUTURE TRENDS
As new drugs are classically tested in clinical trials, this may be an interesting
scenario for further use of microarrays. In many early clinical phase I/II studies
response rate is low and many patients have received some other treatment
before. However, if one is coupling clinical trials with gene expression profiling,
the investigators may enhance their information, as the identification of specific
gene expression profiles may correlate to drug response or resistance of the
individual patient. Products of such differentially expressed genes represent at
least plausible targets for inhibitors that may reverse the drug-resistance
Figure 3 Example for inter-laboratory reproducibility in MILE study: Center 7 versus all
other nine centers was calculated, all genes (38,000) were included in calculation.
Gene Expression Profiling 15
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phenotype. Thus, these markers may be prospectively used to identify those
patients who are likely to respond to the new agent. In follow-up studies far
fewer patients would then be required to prove efficacy (38,4750).
Today, we are on the way to design and to use specifically developed
microarrays with thousands of genes for the subclassification of leukemias and
lymphomas. The ongoing MILE study is one example of an international
approach to use gene expression profiling for the first time in a routine diag-
nostic setting. Also, a lymphoma microarray is already investigated for diagnosis
(51). Importantly, this information always includes information about a patients
prognosis, as clearly defined biological entities in leukemia and lymphoma lead
to disease-specific treatment and data are therefore also related to prognosis and
outcome. Some examples for this thesis are APL to be treated with ATRA or
arsenic trioxide, or BCR-ABL-positive leukemias that can be specifically treated
with imatinib or other tyrosine kinase inhibitors. Other examples are the use of
CD33-targeted treatment in AML with gemtuzumab ozogamicin, or anti-CD20
and anti-CD52 antibody-related treatment in lymphomas. In addition, several
studies were able to define a subset of genes that are not linked to a diagnostic
profile but can be also used for outcome prediction. These studies can even
demonstrate different marker genes that predict response to specific drugs.
So far, one has to accept that much less is known about the use of gene
expression profiling in finding new targets in leukemia and lymphoma. One nice
example may be the detection of ZAP70 in CLL that not only predicts the IgVH
status of the disease but can also be used as an antibody target to discriminate
patients at diagnosis. However, new treatment opportunities have not been
developed for this gene so far. Of course this does not mean that gene expression
profiling will never add information for new targets. By identifying new players
and pathways for resistance to therapy, DNA repair, and apoptosis, microarrays
open up new avenues for any targeted therapy that had not even existed a few
years ago. There is no evidence for any other technique today with so much
power for specific and less toxic treatment for cancer patients in the future.
However, the exact definition of the difference between a normal and a cancer
cell in all details is essentially required for the solution. The goal must be to
diagnose and stratify patients according to their disease-specific gene expression
profile before treatment starts and to treat individually with drugs specific for
such clearly defined biological entities. This does not mean that these drugs will
be individually defined for each patient but for a newly defined disease not based
only on morphology or cytogenetic parameters.
Models for the development of new targets in leukemia and lymphoma
should be adapted to large-scale clinical trials and have to focus in detail on new
medications tested. Thus, strong links between academic and industry initiatives
are urgently needed (52,53) to be the driving force behind the science. As cancer
pathways such as Ras, Src, or Myc are known and can be linked to several
tumors, their interaction and involvement can be studied by gene expression
profiling best. Therefore, not only single genes being over- or underexpressed
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but altered pathways in leukemia and lymphoma also may lead to new targets in
the near future.
CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS
Following its fast integration in hematological research we can expect gene
expression profiling to be included in clinical procedures already in the very near
future.
First, it might soon support the classification and risk stratification of
hematological malignancies as it provides a high degree of correlation with other
diagnostic methods such as flow cytometry (54,55) or PCR (56) and shows a
high diagnostic accuracy and reproducibility (7,57). The robustness of the
method is a further argument for its applicability in the clinical field (58).
Moreover, gene expression profiling is able to further subclassify distinct entities
such as chronic myelomonocytic leukemia, which could not be previously
subdivided by classical techniques (59). Although it is improbable that the new
technique will substitute all established methods such as cytomorphology,
cytogenetics, or PCR in the next years, it has to be expected that gene expression
profiling will become part of the diagnostic panel of hematological malignancies
and will be increasingly correlated with other methods or support those in case of
difficult differential diagnoses or decisions.
Second, a further step in the near future might be the inclusion in minimal
residual disease strategies. In combination with real-time PCR gene expression
profiling is able to serve for the definition of molecular markers, which can be
monitored during follow-up of the disease. This might be exemplified in AML in
the WT1 and PRAME genes (60).
Third, gene expression profiling will probably find its way in individu-
alized treatment planning as specific gene expression signatures are associated
with poor chemotherapy response and with drug resistance. These processes are,
e.g., mediated by a transcriptional program active in hematopoietic stem and
progenitor cells as was demonstrated in AML (61) and being associated with
nucleotide metabolism, apoptosis, and oxygen species metabolism (62). The
finding of such signatures therefore might be an indication for immediate
planning of allogeneic stem cell transplantation. However, such application of
gene expression profiling for the definition of chemosensitivity for individu-
alized treatment planning will probably have to be prepared somewhat longer
than the above mentioned indications and will probably be only part of research
studies rather than of routine strategies in the near future.
REFERENCES
1. Haferlach T, Kohlmann A, Kern W, et al. Gene expression profiling as a tool for the
diagnosis of acute leukemias. Semin Hematol 2003; 40:281295.
2. Wheeler DL, Church DM, Edgar R, et al. Database resources of the National Center
for Biotechnology Information: update. Nucleic Acids Res 2004; 32:D35D40.
Gene Expression Profiling 17
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0001_O.3d] [3/4/08/11:54:15] [1
22]
3. Fields S, Kohara Y, Lockhart DJ. Functional genomics. Proc Natl Acad Sci U S A
1999; 96:88258826.
4. Schena M, Shalon D, Davis RW, et al. Quantitative monitoring of gene expression
patterns with a complementary DNA microarray. Science 1995; 270:467470.
5. Holloway AJ, van Laar RK, Tothill RW, et al. Options availablefrom start to finish
for obtaining data from DNA microarrays II. Nat Genet 2002; 32(suppl):481489.
6. Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class
discovery and class prediction by gene expression monitoring. Science 1999; 286:
531537.
7. Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction
of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling.
Cancer Cell 2002; 1:133143.
8. Haferlach T, Kohlmann A, Schnittger S, et al. A global approach to the diagnosis of
leukemia using gene expression profiling. Blood 2005; 106:11891198.
9. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the
ionizing radiation response. Proc Natl Acad Sci U S A 2001; 98:51165121.
10. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl
Acad Sci U S A 2003; 100:94409445.
11. Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically useful gene-expression
profiles in acute myeloid leukemia. N Engl J Med 2004; 350:16171628.
12. 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:
16051616.
13. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell
lymphoma identified by gene expression profiling. Nature 2000; 403:503511.
14. Rosenwald A, Alizadeh AA, Widhopf G, et al. Relation of gene expression
phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic
leukemia. J Exp Med 2001; 194:16391647.
15. Tschoep K, Kohlmann A, Schlemmer M, et al. Gene expression profiling in sarcomas.
Crit Rev Oncol Hematol 2007; 63:111124.
16. Weeraratna AT. Discovering causes and cures for cancer from gene expression
analysis. Ageing Res Rev 2005; 4:548563.
17. Hanash SM, Madoz-Gurpide J, Misek DE. Identification of novel targets for cancer
therapy using expression proteomics. Leukemia 2002; 16:478485.
18. Polo JM, Juszczynski P, Monti S, et al. Transcriptional signature with differential
expression of BCL6 target genes accurately identifies BCL6-dependent diffuse large
B cell lymphomas. Proc Natl Acad Sci U S A 2007; 104:32073212.
19. Coller HA, Grandori C, Tamayo P, et al. Expression analysis with oligonucleotide
microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling,
and adhesion. Proc Natl Acad Sci U S A 2000; 97:32603265.
20. Yagi T, Morimoto A, Eguchi M, et al. Identification of a gene expression signature
associated with pediatric AML prognosis. Blood 2003; 102:18491856.
21. Qian Z, Fernald AA, Godley LA, et al. Expression profiling of CD34 hematopoietic
stem/progenitor cells reveals distinct subtypes of therapy-related acute myeloid
leukemia. Proc Natl Acad Sci U S A 2002; 99:1492514930.
22. Kern W, Schoch C, Kohlmann A, et al. Identification of biologically distinct and
clinically relevant subentities in patients with acute myeloid leukemia and normal
karyotypes by use of gene expression profiling. Blood 2004; 104:60A.
18 Haferlach et al.
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0001_O.3d] [3/4/08/11:54:15] [1
22]
23. Bullinger L, Rucker FG, Kurz S, et al. Gene-expression profiling identifies distinct
subclasses of core binding factor acute myeloid leukemia. Blood 2007; 110:
12911300.
24. Tamayo P, Slonim D, Mesirov J, et al. Interpreting patterns of gene expression with
self-organizing maps: methods and application to hematopoietic differentiation. Proc
Natl Acad Sci U S A 1999; 96:29072912.
25. Kitareewan S, Pitha-Rowe I, Sekula D, et al. UBE1L is a retinoid target that triggers
PML/RARalpha degradation and apoptosis in acute promyelocytic leukemia. Proc
Natl Acad Sci U S A 2002; 99:38063811.
26. Andersson A, Olofsson T, Lindgren D, et al. Molecular signatures in childhood acute
leukemia and their correlations to expression patterns in normal hematopoietic
subpopulations. Proc Natl Acad Sci U S A 2005; 102:1906919074.
27. Stegmaier K, Ross KN, Colavito SA, et al. Gene expression-based high-throughput
screening(GE-HTS) and application to leukemia differentiation. Nat Genet 2004;
36:257263.
28. Armstrong SA, Kung AL, Mabon ME, et al. Inhibition of FLT3 in MLL. Validation
of a therapeutic target identified by gene expression based classification. Cancer Cell
2003; 3:173183.
29. Armstrong SA, Staunton JE, Silverman LB, et al. MLL translocations specify a
distinct gene expression profile that distinguishes a unique leukemia. Nat Genet
2002; 30:4147.
30. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003
patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and
prognosis in the AMLCG study and usefulness as a marker for the detection of
minimal residual disease. Blood 2002; 100:5966.
31. Kohlmann A, Schoch C, Dugas M, et al. New insights into MLL gene rearranged
acute leukemias using gene expression profiling: shared pathways, lineage com-
mitment, and partner genes. Leukemia 2005; 19:953964.
32. Ferrando AA, Look AT. DNA microarrays in the diagnosis and management of acute
lymphoblastic leukemia. Int J Hematol 2004; 80:395400.
33. Armstrong SA, Mabon ME, Silverman LB, et al. FLT3 mutations in childhood acute
lymphoblastic leukemia. Blood 2004; 103:35443546.
34. Taketani T, Taki T, Sugita K, et al. FLT3 mutations in the activation loop of tyrosine
kinase domain are frequently found in infant ALL with MLL rearrangements and
pediatric ALL with hyperdiploidy. Blood 2004; 103:10851088.
35. Zaza G, Cheok M, Yang W, et al. Gene expression and thioguanine nucleotide
disposition in acute lymphoblastic leukemia after in vivo mercaptopurine treatment.
Blood 2005; 106:17781785.
36. Holleman A, Cheok MH, den Boer ML, et al. Gene-expression patterns in drug-
resistant acute lymphoblastic leukemia cells and response to treatment. N Engl J Med
2004; 351:533542.
37. Brown P, Levis M, Shurtleff S, et al. FLT3 inhibition selectively kills childhood
acute lymphoblastic leukemia cells with high levels of FLT3 expression. Blood
2005; 105:812820.
38. Cheok MH, Lugthart S, Evans WE. Pharmacogenomics of acute leukemia. Annu Rev
Pharmacol Toxicol 2006; 46:317353.
39. Mullighan CG, Goorha S, Radtke I, et al. Genome-wide analysis of genetic alterations
in acute lymphoblastic leukaemia. Nature 2007; 446:758764.
Gene Expression Profiling 19
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0001_O.3d] [3/4/08/11:54:15] [1
22]
40. McLean LA, Gathmann I, Capdeville R, et al. Pharmacogenomic analysis of cyto-
genetic response in chronic myeloid leukemia patients treated with imatinib. Clin
Cancer Res 2004; 10:155165.
41. Diaz-Blanco E, Bruns I, Neumann F, et al. Molecular signature of CD34() hema-
topoietic stem and progenitor cells of patients with CML in chronic phase. Leukemia
2007; 21:494504.
42. Radich JP, Dai H, Mao M, et al. Gene expression changes associated with pro-
gression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S A 2006;
103:27942799.
43. Zheng C, Li L, Haak M, et al. Gene expression profiling of CD34 cells identifies a
molecular signature of chronic myeloid leukemia blast crisis. Leukemia 2006; 20:
10281034.
44. Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for
immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl
J Med 2003; 348:17641775.
45. Dicker F, Schnittger S, Kern W, et al. Complex aberrant karyotypes and unbalanced
translocations in CLL correlate with an unmutated IgVH status: a study on 133
patients studied with chromosome banding analysis, interphase FISH, IgVH mutation
status, ZAP-70 RNA exrpession and immunophenotyping. Blood 2005; 106:825a.
46. Haferlach T, Kohlmann A, Basso G, et al. A multi-center and multi-national program
to assess the clinical accuracy of the molecular subclassification of leukemia by gene
expression profiling. Blood 2005; 106:224a.
47. Cheok MH, Yang W, Pui CH, et al. Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells. Nat Genet 2003; 34:8590.
48. Golub TR. Mining the genome for combination therapies. Nat Med 2003; 9:510511.
49. Evans WE, Guy RK. Gene expression as a drug discovery tool. Nat Genet 2004;
36:214215.
50. Corchero J, Fernandez-Salguero PM. Improving cancer therapeutics by molecular
profiling. Curr Drug Metab 2005; 6:553568.
51. Staudt LM. Molecular diagnosis of the hematologic cancers. N Engl J Med 2003;
348:17771785.
52. Altman RB, Flockhart DA, Sherry ST, et al. Indexing pharmacogenetic knowledge
on the World Wide Web. Pharmacogenetics 2003; 13:35.
53. Downward J. Cancer biology: signatures guide drug choice. Nature 2006; 439:
274275.
54. Kern W, Kohlmann A, Schoch C, et al. Comparison of mRNA abundance quantified
by gene expression profiling and percentage of positive cells using immunopheno-
typing for diagnostic antigens in acute and chronic leukemias. Cancer 2006; 107:
24012407.
55. Basso G, Case C, DellOrto MC. Diagnosis and genetic subtypes of leukemia com-
bining gene expression and flow cytometry. Blood Cells Mol Dis 2007; 39:164168.
56. Sala-Torra O, Gundacker HM, Stirewalt DL, et al. Connective tissue growth factor
(CTGF) expression and outcome in adult patients with acute lymphoblastic leukemia.
Blood 2007; 109:30803083.
57. Kohlmann A, Schoch C, Schnittger S, et al. Molecular characterization of acute
leukemias by use of microarray technology. Genes Chromosomes Cancer 2003; 37:
396405.
20 Haferlach et al.
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0001_O.3d] [3/4/08/11:54:15] [1
22]
58. Kohlmann A, Schoch C, Dugas M, et al. Pattern robustness of diagnostic gene
expression signatures in leukemia. Genes Chromosomes Cancer 2005; 42:299307.
59. Gelsi-Boyer V, Cervera N, Bertucci F, et al. Gene expression profiling separates
chronic myelomonocytic leukemia in two molecular subtypes. Leukemia 2007; 21:
23592362.
60. Steinbach D, Schramm A, Eggert A, et al. Identification of a set of seven genes for
the monitoring of minimal residual disease in pediatric acute myeloid leukemia. Clin
Cancer Res 2006; 12:24342441.
61. Heuser M, Wingen LU, Steinemann D, et al. Gene-expression profiles and their
association with drug resistance in adult acute myeloid leukemia. Haematologica
2005; 90:14841492.
62. Eisele L, Klein-Hitpass L, Chatzimanolis N, et al. Differential expression of drug-
resistance-related genes between sensitive and resistant blasts in acute myeloid
leukemia. Acta Haematol 2007; 117:815.
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22]
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[2344]
2
Individualized Tumor Response Testing
in Leukemia and Lymphoma
Andrew G. Bosanquet
Bath Cancer Research, Royal United Hospital, Bath, U.K.
Peter Nygren
Department of Oncology, Radiology, and Clinical Immunology,
University Hospital, Uppsala, Sweden
Larry M. Weisenthal
Weisenthal Cancer Group, Huntington Beach, California, U.S.A.
INTRODUCTION
Individualized tumor response testing (ITRT) has a long history, with a number
of different technologies and many different tumor types tested. Almost all
technologies used for hematological malignancies are identical in their logic and
similar in their execution. The concepts underlying cell death assays are rela-
tively simple, even though the technical features and data interpretation can be
complex. The logic is that if the drug kills tumor cells from an individual patient
in a test tube, then it is more likely to be effective when administered directly
to a patient. Conversely, a drug that does not kill the patients cells, even at
concentrations significantly higher than can be achieved in the patient, is
unlikely to be effective. Considerable work based on these assays has been
reported during the past 25 years, and recently an ad hoc group of 50 scientists
from 10 countries agreed on the term individualized tumor response for these
23
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tests, describing them as the effect of anticancer treatments on whole living
tumor cells freshly removed from cancer patients and not including tests with
subcellular fractions, animals or cell lines (1).
We present results for hematological neoplasms, but note that analogous
results have been published for a variety of solid tumors in substantial numbers
of patients (2).
TOTAL CELL KILL/CELL DEATH ASSAYS
There is a clear divide between the two main technologies used in this work: an
ITRT endpoint can be based either on reduction of cell proliferation or on cell
death (36). Historically, the cell proliferation endpoint received great attention
as a result of studies by Salmon, Von Hoff, and others during the late 1970s and
early 1980s (7,8). These studies occurred during the heyday of the oncogene dis-
covery period in cancer research, when oncogene products were frequently found to
be associated with cell growth and when cancer was most prominently considered to
be a disease of disordered cell growth. In contrast, the concept of apoptosis had yet
to become widely recognized. Also unrecognized were the concepts that cancer may
be a disease of disordered apoptosis/cell death and that the mechanisms of action of
most, if not all, available anticancer drugs are mediated through apoptosis. When
problems with cell proliferation assays emerged (9,10), there was little enthusiasm
for studying cell death as an alternative endpoint (11). These factors explain many
abandoning research into ITRT during the 1980s.
As opposed to measuring cell proliferation, there is a family of assays
based on the concept of total cell kill or, in other words, cell death occurring in
the entire population of tumor cells (36).
The basic technology concepts are straightforward. Cells are isolated from
a fresh specimen obtained from a viable neoplasm. These cells are cultured in the
continuous presence or absence of a drug, most often for three to seven days. At
the end of the culture period, a measurement is made of cell injury, which
correlates directly with cell death, almost always by apoptosis (1214).
Although there are methods for specifically measuring apoptosis per se,
there are practical difficulties in applying these methods to mixed (and some-
times clumpy) preparations of tumor cells and normal cells. Thus, more general
measurements of cell death have been applied. One of these measurements is the
delayed loss of cell membrane integrity, which has been found to be a useful
surrogate for apoptosis. This is measured by differential staining in the Differ-
ential Staining Cytotoxicity (DiSC) assay method, which allows selective drug
effects against tumor cells to be recognized in a mixed population of tumor and
normal cells (6,15). More recently the Tumor Response to Antineoplastic
Compounds (TRAC) assay was described as a streamlined version of the DiSC
assay (16). Other cell death endpoints include loss of mitochondrial Krebs cycle
activity, as measured in the Methylthiazol Tetrazolium (MTT) assay (17), loss of
cellular adenosine triphosphate (ATP), as measured in the ATP assay (18), and
24 Bosanquet et al.
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loss of cytosolic esterase activity and cell membrane integrity, as measured by
the Fluorometric Microculture Cytotoxicity Assay (FMCA) and similar assays
(1921). Most recently, other methods including assays to measure apoptosis
more specifically have been described, although it remains to be seen if these
will offer any real advantages over the other measurements of cell death (2226).
These four endpoints produce valid and reliable measurements of cell
death. They also correlate well with each other on direct comparisons of the
different methods (17,19,20,2729). For instance, Weisenthal and associates have
performed direct correlations between the DiSC and MTT assays in approximately
5,500 fresh human tumor specimens, testing an average of 15 drugs per specimen
at two different concentrations. Although these endpoints agree with each other
in most solid tumors (overall correlation coefficient 0.85), we consider that
the MTT assay is more problematic in hematological neoplasms. For example,
correlations between treatment outcomes and assay results have been more
consistent in acute nonlymphocytic leukemia (ANLL) with the DiSC assay
endpoint (3032) than with the MTT endpoint (22,33,34).
Additionally, there is a clear relationship between prior treatment status
and assay results for anthracyclines in the case of the DiSC assay (relapsed
patients having blast cells that are clearly more resistant than those in previously
untreated patients, Table 1), which was not evident when ANLL was tested with
the MTT assay (35). The absolute magnitudes of drug effects (cell kill) are
substantially greater when scored in the DiSC assay than in the MTT assay in the
case of ANLL (Table 1). Finally, the correlation coefficient between DiSC and
MTT assays was weaker in the case of ANLL (median r 0.75), than in other
classes of neoplasms that Weisenthal had tested (median r 0.85).
There are at least two explanations for the greater drug effects detected in
the DiSC endpoint.
Firstly, the DiSC assay is a more specific endpoint for drug effects on blast
cells (as opposed to drug effects on blast cells plus the normal cells frequently
present in ANLL specimens).
Table 1 In Vitro Activity of Anthracyclines in ANLL As a Function of Prior Treatment
Status and Individualized Tumor Response Testing Endpoint
Drug/assay
Number
untreated
Number
treated
Cell fraction
surviving
(untreated)
Cell fraction
surviving
(relapsed) P
Doxorubicin/DiSC 12 16 0.11 0.33 0.020
Doxorubicin/MTT 12 16 0.34 0.42 0.428
Idarubicin/DiSC 10 16 0.06 0.25 0.0015
Idarubicin/MTT 10 16 0.35 0.45 0.180
Abbreviations: ANLL, acute nonlymphocytic leukemia; DiSC, differential staining cytotoxicity;
MTT, methylthiazol tetrazolium.
Source: From Ref. 35.
Individualized Tumor Response Testing in Leukemia and Lymphoma 25
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Secondly, it takes longer for cells to lose the ability to produce a signal in
the MTT assay than it does for them to be scored as dead in the DiSC assay
[e.g. (36)]. It is possible that the MTT assay would be more useful in ANLL (i)
were it applied only in cases in which there was a pure (>90%) population of
blast cells at the end of the assay, and/or (ii) were the duration of the cell culture
(and drug exposure) extended beyond the typical 96-hour period of these assays.
With regard to the first of these latter possibilities, it is notable that Hongo, et al.
(who contributed a disproportionate share of weak clinical correlations in Table 2)
did not attempt to determine the percentage of blast cells at the time the MTT
endpoint was measured (34).
A final point of emphasis is that it is important to rigorously standardize
assay conditions, including precisely controlling the duration of drug exposure
and cell culture. Thus, the DiSC assay and similar tests have some advantages
over the other short-term assays.
COMPLETED STUDIES OF CORRELATION BETWEEN CELL
DEATH ASSAY RESULTS AND CHEMOTHERAPY RESPONSE
As with other laboratory tests, the determination of the efficacy of ITRT is based
on comparisons of laboratory results with patient response (commonly referred
to as clinical correlations). The hypothesis to be tested with clinical corre-
lations is a simple onethat above-average drug effects in the assays correlate
with above-average drug effects in the patient, as measured by both response
rates and patient survival.
Table 2 and Figure 1 show that, with respect to response, the above
hypothesis has been confirmed to be true in all published studies. At each point
in the distribution of overall response rates, patients with test results in the
sensitive range were more likely to respond than the total patient population
as a whole. Conversely, patients with test results in the resistant range were
less likely to respond than the patient population as a whole. On average, patients
with assays in the test sensitive range were threefold more likely to respond than
patients with assays in the test resistant range (see the Overall relative risk
column in Table 2).
Considering this evidence as a whole, can it be inferred with confidence
that the cell death measured in the assays correlates with tumor cell death
measured in the patient? Comparing the chronic lymphocytic leukemia (CLL)
and acute lymphoblastic leukemia (ALL) data with the more limited but also
consistent data in non-Hodgkins lymphoma (NHL), a powerful case is made to
support the clinical relevance of this testing in human lymphatic neoplasms.
Considering the ANLL data in the context of the lymphatic neoplasm data, a
powerful case is made to support the clinical relevance of this testing in hem-
atological neoplasms in general.
The body of literature supporting cell death assays in lymphatic neoplasms
dates to studies in CLL published by Schrek in the 1960s (37,38). Schrek
26 Bosanquet et al.
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[2344]
measured the in vitro cell death effects of drugs, heat, and radiation on CLL cells
by means of phase contrast microscopy. He measured what we would today
recognize as apoptosis and undoubtedly being precisely congruent with the DiSC
assay. Radiation effects in vitro were strongly correlated with clinical outcome
(37,38).
In the late 1970s, Durkin compared in vitro drug effects in NHL and CLL
by means of trypan blue dye exclusion with clinical drug effects and reported
good correlations in a small study (39). The DiSC assay was developed inde-
pendently as an improved variation of the trypan blue test in which suspension
cultures of cells were first exposed to trypan blue, cytocentrifuged onto micro-
scope slides, and counterstained with either Haematoxylin/Eosin or Wright/
Giemsa (to identify the non-trypan blue-stained cells with respect to whether
these surviving cells were tumor cells or normal cells). With further improvement
(substitution of fast green stain for trypan blue and the addition of acetaldehyde-fixed
duck erythrocytes as an internal standard to aid in scoring the Cytospin slides),
clinical correlations in CLL and other neoplasms were first reported in an abstract
form and at meetings in the United States and Europe in 1981.
The first publications of clinical correlations with the DiSC assay, in 1983
and 1984, included studies of the activity of glucocorticoids and standard
cytotoxic agents, which were correlated with prior therapy and with clinical
outcome in ALL and CLL (3,15,40). In 1986, these were followed by a study
showing the clinical relevance of the DiSC assay in CLL, ALL, and NHL using
several clinical endpoints: (i) Correlations with known disease-specific activity
profiles, (ii) individual patient correlations with clinical response, (iii) greater
resistance of specimens from previously treated patients versus previously
untreated patients, and (iv) a shift to significantly greater drug resistance in
metachronous assays in the presence of intervening chemotherapy, but no shift in
the absence of intervening chemotherapy (41). These early findings were sub-
sequently independently confirmed by other investigators in more comprehen-
sive studies (19,28,4251). Additionally, studies in pediatric ALL reported that
resistance to dexamethasone in the DiSC assay predicted for poor survival (52),
findings also independently confirmed.
By the late 1980s, a number of other scientists were investigating the DiSC
assay and related cell death assays. These began with a head-to-head comparison
of the DiSC assay with the MTT assay in established cell lines by the National
Cancer Institute (NCI) lung cancer group (17). These studies established the
comparability of these endpoints in homogeneous cell populations.
A group at the VU University Medical Center of Amsterdam carried out a
head-to-head comparison of the DiSC endpoint with the MTT endpoint in ALL
(29). This group showed that the endpoints were comparable in specimens in
which the proportion of leukemia cells (relative to normal cells in the specimen)
was greater than 80% (29,53). They found the MTT endpoint to be less labor
intensive. They used the same general conditions originally described for the
DiSC assay (including a 96-hour continuous drug exposure, followed by
Individualized Tumor Response Testing in Leukemia and Lymphoma 27
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[2344]
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28 Bosanquet et al.
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duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
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comparisons between drug exposed and control cultures with the cell death
endpoint). These Dutch authors continue to publish an extensive, elegant, and
ongoing series of rigorous studies. They have established that the assay results
correlate with and predict both response and survival in ALL and that the assay
results are only two factors (the other being minimal residual disease), which
independently predict for survival in pediatric ALL (43,44,46,5457). They have
also extended this work to ANLL (22,35,45,58). Taken in the context of the
entire literature, these studies in pediatric ALL provide further support for
the validity of complementary studies in CLL.
Other investigators have also shown strong correlations between cell death
assay results and the clinical outcome (response and/or survival) in pediatric
ALL (19,49,50,52,55,59), adult ALL and ANLL (20,28,3032,6067), CLL
Figure 1 Correlations between ITRT results and clinical response. The data points are
the results of each of 33 individual studies that had at least 10 patients per data subset (see
Table 2). The data are plotted in order of the increasing response rate in the total patient
cohort studied (x-axis). The crosses (all of which lie on the x y line) represent the
response rates of patients in each study if the assay was of no value or not performed. The
squares represent the response rates for patients with assay results classified as in vitro
sensitive (&) versus resistant (&). The triangles show the weighted mean of all sensitive
results (~, n 1020)) and all resistant results (~, n 419) from Table 2. The greater the
vertical distance between the sensitive and resistant results for an individual study, the
more accurate the test results.
30 Bosanquet et al.
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(42,51,6871), and adult NHL (15,27,41,48). These studies included further
confirmation of the comparability between DiSC and MTT endpoints in assays
on clinical specimens. Larsson and Nygren also introduced the fluorescein
diacetate cell death endpoint (FMCA), which, like the DiSC endpoint, measures
cell membrane integrity and which correlates well with the DiSC endpoint in
homogeneous cell populations (19,27).
In 1991, Bosanquet, et al. published in Lancet a relatively large number of
correlations between clinical response and DiSC assay results, chiefly in CLL
(42). He showed, furthermore, highly significant correlations between assay
results and patient survival. This paper also confirmed the relevance of the
EDR (extreme drug resistance) endpoint, which is defined as an assay result
greater than 1 SD more resistant than the median of comparison assays. He later
described a paradoxical shift toward increased methylprednisolone sensitivity in
previously treated CLL and used the DiSC assay to identify high-dose methyl-
prednisolone as an effective treatment for otherwise refractory CLL (72,73).
These studies with the DiSC and MTT assays are further supported by studies
with the FMCA. Fluorescein diacetate is a lipid soluble material that readily
penetrates cell membranes. Viable cells contain cytosolic esterases that cleaves
the dye to nonlipid soluble fluorescein, which is concentrated in cells con-
taining a functionally intact membrane. Thus, the assay is conceptually similar to
the DiSC assay, which measures the ability of cells with functionally intact
membranes to exclude nonlipid soluble dyes. Delayed loss of this membrane
integrity is a marker of apoptotic cell death (74).
Investigators at Uppsala University in Sweden began work in the
1980s by comparing the DiSC and FMCA assays and establishing their com-
parability (19,27,48). They proceeded to publish a series of studies showing
correlations between assay results and treatment outcomes in NHL and ANLL
(19,20,47,48,61,62,75), confirming the specificity of the EDR endpoint in pre-
dicting clinical nonresponse (62), and confirming and extending earlier reports on
the capability of the cell death endpoint to identify the general disease-specific
activity patterns of a diverse spectrum of drugs (76).
Within the past several years, additional studies have provided strong
support for the clinical relevance of the information provided by cell death
assays in hematological neoplasms.
Table 2 shows response correlations for ALL, CLL, NHL, and ANLL and
may be summarized as follows:
l
ALL: n 304 published correlations between assay results and response
l
81% overall response rate for patients studied
l
91% response rate for patients treated with ITR testsensitive (ITR)
drugs; relative risk of failure to achieve remission 0.90 [95% confi-
dence interval (CI) 0.840.96, P 0.002]
l
49% response rate for patients treated with ITR testresistant (ITR)
drugs; relative risk 1.65 [95% CI 1.292.11, P < 0.0001]
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l
CLL: n 720
l
69% overall response rate
l
83% response rate with ITR drugs; relative risk 0.83 [0.780.89;
P < 0.0001]
l
19% response rate with ITR drugs; relative risk 3.66 [2.65.1;
P < 0.0001]
l
NHL: n 85
l
54% overall response rate
l
73% response rate with ITR drugs; relative risk 0.74 [0.580.95;
P 0.025]
l
12% response rate with ITR drugs; relative risk 4.69 [1.5913.8;
P < 0.0001]
l
ANLL: n 621
l
72% overall remission rate
l
88% response rate with ITR drugs; relative risk 0.82 [0.770.90;
P < 0.0001]
l
36% response rate with ITR drugs; relative risk 2.01 [1.652.45;
P < 0.0001]
Thus, there is a long, extensive, and consistent body of evidence supporting
the clinical relevance of cell death assays in human hematological neoplasms.
Gene Expression Profiling
Over the last few years, gene expression profiling has been suggested as the best
or only way of determining ex vivo drug sensitivity (77), and there has been
some recent progress for the concept of prediction of cytotoxic drug activity in
individual patients with solid tumors based on genomic signatures (80). How-
ever, due to almost all patients being treated with combination chemotherapy,
without ITRT, there are calibration difficulties with this methodology. Thus, in
one of the best papers on the subject, originating from work with childhood ALL
(78), the supervised cluster analysis was based on in vitro drug sensitivity
(79). This editorial then continued by erroneously suggesting that ITRT was
more cumbersome than gene expression profiling (79), whilst ITRT is
actually integrating all the gene expression into one convenient test. As a result,
and because it is as near real time and real life as is possible for a laboratory test,
we believe that ITRT may be most clinically relevant to the patient (Fig. 2).
COMPLETED STUDIES OF PATIENT SURVIVAL
In 1999, in a study of 243 CLL patients (70), Bosanquet identified 66 patients
who received fludarabine within a year of the performance of the DiSC assay
51 fludarabine testsensitive patients had a 69% response rate (80% for untreated
32 Bosanquet et al.
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patients; 64% for previously treated patients), while 15 fludarabine testresistant
patients had a 7% response rate (25% for previously untreated; 0% for previously
treated). None of these 15 fludarabine-resistant patients treated with fludarabine
survived 17 months; median survival was 7.9 months. In contrast, the fludarabine-
sensitive patients treated with fludarabine had an 80% chance of surviving beyond
17 months, a median survival of 41.7 months, and a 43% chance of surviving
beyond four years. Patients (n 42) with DiSC assay resistance to fludarabine but
treated with regimens other than fludarabine had a median survival of 16.3 months
and 10% survived beyond four years. The relative risk of death for patients with
DiSC assay resistance to fludarabine treated with fludarabine versus those treated
with a non-fludarabine regimen was 2.9. On multivariate analysis, fludarabine test
resistance was a more important determinant of survival in patients treated with
fludarabine than was any other clinical characteristic, including gender, Binet
stage, prior chemotherapy, and patient age. In a separate analysis, DiSC assay
directed therapy of CLL was calculated to be cost effective at only $2500 per
quality life-year saved (81).
Other investigators, as noted, have reported that assay results are
important predictors of patient survival in pediatric ALL and ANLL (33,49,
5658,82).
Similar studies from a number of different groups have published corre-
lations between ITRT results and survival in adult ANLL (32,33,63). Correla-
tions between DiSC assay results and patient survival in ANLL were first
published by a Swedish group in 1989 (31). These results were confirmed and
extended by a group at the University of Cologne (32), in a follow-up to their
earlier report of strong correlations between DiSC assay results and clinical
remission of adult ANLL a decade earlier (63). In their recent studies, the DiSC
assay results 100% accurately predicted clinical outcome and identified a group
of patients with a 100% early death rate, when treated with conventional
induction therapy (32,63). These studies are very analogous to the above-cited
work identifying a group of CLL patients in whom conventional treatment is
uniformly inactive (70).
Figure 2 Clinical relevance of various endpoints.
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The University of Cologne group followed up with a presentation at the
American Society of Hematology meeting in December, 1999, in which multi-
variate analysis showed DiSC assay results to be the strongest factor predicting
for clinical outcome (both complete remission and long-term patient survival) in
adult ANLL, using a novel and sophisticated method for calculating sensitive/
resistant cutoff boundaries (32). Additionally, a Danish group reported studies
correlating MTT assay results with both overall and relapse-free survival in 85
adult ANLL patients (33). Assay results remained significantly correlated with
survival on multivariate analysis. This work on ANLL is analogous and com-
plementary to the studies by the Dutch (Amsterdam) group in pediatric ALL,
discussed above.
ONGOING CLINICAL STUDIES
A major international clinical trial on ITRT in CLL is ongoing. The first random-
ization has closed and comparisons of ITRT by TRAC assay (83,84) and patient
response (85) have been published. The second randomization (at nonresponse or
relapse after first treatment) is between ITRT-directed therapy and protocol-guided
treatment. This second randomization continues to accrue patients and will deter-
mine to what extent ITRT can improve response and survival of CLL patients at
first relapse.
EXPERT OPINION: CURRENT USE OF ITRT IN CLINICAL ONCOLOGY
The American Society of Clinical Oncology (ASCO) working group recom-
mended against the use of ITRT in oncology practice, stating that Oncologists
should make chemotherapy treatment recommendations on the basis of published
reports of clinical trials. . . . (86). In a published objection to this recommen-
dation, Castro wrote: Paradoxically, as the number of possible treatment options
supported by completed randomized clinical trials increases, the scientific liter-
ature becomes increasingly vague in guiding physicians . . . moreover, clinicians
are confronted on nearly a daily basis by decisions that have not been addressed
by randomized trial evaluation (87).
The data in Table 3 support Castros point of view. These data are taken
from the United States NCI Web site, in which the so-called state-of-the-art,
standard therapy options are reviewed. It can be readily seen that 50 years of
prospective, randomized trials have failed to identify clear-cut best standard
therapies, even in the setting of first-line treatment (Table 3).
In each class of adult hematological neoplasms, there are a variety of
choices, similar in some respects but with key differences. One conservative
application of the assays would be to identify the most active of the otherwise
equally acceptable regimens. Another would be to eliminate the most inactive of
the regimens and choose from among the rest on the basis of other clinical
factors, including cost. In the setting of relapsed, refractory disease, ITRT
34 Bosanquet et al.
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provides a mechanism for choosing from an even larger number of potential
choices, many of which will not be tested in prospective, randomized trials for
years to come.
Beyond the potential advantage to the patient is the progress, which would
be fostered with regard to improving methodology, to make it available for
future applications to handle the explosive growth of new, expensive, potentially
toxic, and only partially effective drugs.
Castro further argued (87): Until the trialist approach has delivered
curative results with a high success rate, the clinical autonomy to integrate
promising insights and methods, including [ITRT], remains an essential com-
ponent of patient advocacy.
The members of the ASCO working group who formulated the ASCO
recommendations concerning ITRT agreed with Castro, stating: It is certainly
each practitioners prerogative to order [ITRT] . . . However, it is important to
specify to the patient what the treatment would be in the absence of the assay and
to be clear about if and how the information will be used to inform treatment
decision making(88).
Table 3 Equally Acceptable Standard Chemotherapy Options, According to the
United States National Cancer Institute
ALL (adult) AML CLL NHL (indolent)
NHL
(aggressive) Myeloma
D/V/P C R/F R R/Ctx/Dox/
V/P
Dex or P
D/V/P/A C/D R/F/Ctx R/F Ctx/Dox/V/P Ctx/P
D/V/P/A/Ctx C/D/T F/Ctx R/Ctx/V/P D/Ctx/V/B/P Melph/P
M/E Ctx/V/P R/Ctx/Dox/V/P Ctx/M/V/P Bortezomib
C/I Ctx/Dox/V/P R/F/M Mtx/B/Dox/
Ctx/V/Dex
V/Carm/
Melph/Ctx/P
F/Chl Chl (P or Dex) P/Dox/Ctx/E/
C/B/V/Mtx
V/Melph/
Ctx/P/C/
Carm/Dox/P
R/Ctx/F/M Thalidomide
Cda
Ctx (P or Dex)
F
Ctx/V/P
C/V/P/Procarb
Ctx/Dox/V/P
F/M/Dex
Abbreviations: A, Asparaginase; B, Bleomycin; C, Cytarabine; Carm, Carmustine; Cda, Cladribine;
Ctx, Cyclophosphamide; Chl, Chlorambucil; D, Daunorubicin; Dex, Dexamethasone; Dox, Doxor-
ubicin; E, Etoposide; F, Fludarabine; I, Idarubicin; M, Mitoxantrone; Melph, Melphalan; Mtx,
methotrexate; P, Prednisone; Procarb, Procarbazine; R, Rituximab; T, Tioguanine; V, Vincristine.
Individualized Tumor Response Testing in Leukemia and Lymphoma 35
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The authors of this chapter agree completely with the above quoted view-
points, as expressed by Castro and as modified by the ASCO working group.
CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS
Progress in the treatment of cancer has been surprisingly modest in light of the
rapid progress in tumor biology, although several new targeted drugs have
been introduced over the last few years. Most of them have so far been devel-
oped for use in solid tumors, but new drugs have also emerged for hematological
malignancies, for instance imatinib for chronic myeloid leukemia (CML), ri-
tuximab for lymphomas and bortezomib for myeloma. With the exception of the
new treatment situation in CML, there is little reason to believe that established
chemotherapy will not continue to form the basis of medical treatment of
hematological as well as solid tumors in the next five years. The new targeted
drugs mostly need to be combined with active chemotherapy to provide any
benefit and, thus, the need for predictive tests for individualized therapy selec-
tion has increased. Disappointingly, the introduction of the new drugs has not
been accompanied by specific predictive tests allowing for a rational and eco-
nomical use of the drugs. On the other hand, preliminary data indicate that
ITRTs also adequately reflect the clinical activity of, for instance, various
tyrosine kinase inhibitors. Given also the technical and conceptual advantages of
ITRTs together with their performance and the quite modest efficacy of therapy
prediction based on analysis of genome expression as published so far, there is
reason for a renewal in the interest for ITRTs for future optimized use of medical
treatment of malignant disease.
Thus, the current and potential role of ITRT in the management of patients
with hematological neoplasms remains controversial, although, for some years,
ITRT has been approved for reimbursement by Medicare in the United States.
Specifically excluded from consideration by the two editorial reviews published
in the Journal of Clinical Oncology (86,89) were studies which related only
to the performance characteristics of ITRTpredictive accuracy, sensitivity, and
specificity. The only studies considered were the very few which attempted to
show if treatment outcomes could be improved through the use of ITRT. These
criteria were surprising, as there are virtually no published studies with any other
laboratory test in which patients were randomized to treatment with and without
test information. The traditional (and heretofore only) criteria on which other
laboratory, clinical, and radiographic tests have been judged are the performance
characteristics (predictive accuracy, sensitivity, and specificity) and perceived
utility in the judgment of the clinician who orders the tests.
Only when these assays are widely performed and used and routinely
included as an integral part of clinical trials will these already promising tech-
nologies be improved and only then will their role in patient management
become better defined. But this is true for all complex laboratory technologies. A
good example is immunohistochemical staining for batteries of cell antigens, the
36 Bosanquet et al.
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use of which has never been shown by means of randomized controlled clinical
trials to improve treatment outcomes.
By raising the bar of acceptance to levels unprecedented for a laboratory
test, in essence a tariff has been erected to protect the paradigm of the best
empiric treatment for the average patient, as identified in the all-too-commonly-
nonproductive clinical trials. This tariff also discourages discovery of new,
effective drug regimens through the use of ITRT to guide drug selection.
With greater use of these assays in hemato-oncology and the ever-
increasing list of licensed drugs, it is very likely that the activity of new drugs
and new regimens would be identified at a much earlier time than with the
current system relying exclusively on usually empiric phase II trials (90,91).
If test results are used to assist in the selection of a regimen chosen from a
series of otherwise reasonable alternatives, then patients will never be harmed by
using the test result and best available evidence strongly indicates they will often
be helped.
In conclusion, there is a 45-year history of highly positive studies in
hematological neoplasms showing consistent, strong correlations between the
results of cell death assays and clinical outcomes (both initial response and long-
term patient survival). A similarly convincing body of evidence in solid tumors
(2) suggests these technologies are relevant for most, if not all, tumor types.
Thus, there is strong scientific rationale and good documentation for these tests
in a collectively large and diverse literature about hematological neoplasms for
the clinical relevance of the information provided by the tests. Their use, par-
ticularly where equally effective treatment options are possible, could improve
the rationale of treatment choice as well as probability of response and survival.
REFERENCES
1. Bosanquet AG, Kaspers GJ, Larsson R, et al. Individualized tumor response (ITR)
profiling for drug selection in tailored therapy: meta-analysis of 1929 cases of leu-
kemia and lymphoma. Blood 2007; 108:1017A (abstr).
2. Weisenthal LM, Nygren P. Current status of cell culture drug resistance testing
(CCDRT). Human Tumor Assay Journal 2002. Available at: http://www.weisenthal.
org/oncol_t.htm. Accessed February 2008.
3. Weisenthal LM, Shoemaker RH, Marsden JA, et al. In vitro chemosensitivity assay
based on the concept of total tumor cell kill. Recent Results Cancer Res 1984;
94:161173.
4. Weisenthal LM, Lippman ME. Clonogenic and nonclonogenic in vitro chemo-
sensitivity assays. Cancer Treat Rep 1985; 69:615632.
5. Weisenthal LM. Cell culture assays for hematologic neoplasms based on the concept
of total tumor cell kill. In: Kaspers GJL, Pieters R, Twentyman PR, et al., eds. Drug
Resistance in Leukemia and Lymphoma: The Clinical Value of Laboratory Studies.
Chur, Switzerland: Harwood Academic Publishers, 1993:415432.
6. Weisenthal LM, Kern, DH. Prediction of drug resistance in cancer chemotherapy: the
Kern and DiSC assays. Oncology (U.S.A.) 1992; 5:93103.
Individualized Tumor Response Testing in Leukemia and Lymphoma 37
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
7. Salmon SE, Hamburger AW, Soehnlen B, et al. Quantitation of differential sensitivity
of human-tumor stem cells to anticancer drugs. N Engl J Med 1978; 298:13211327.
8. Von Hoff DD, Casper J, Bradley E, et al. Association between human tumor colony-
forming assay results and response to an individual patients tumor to chemotherapy.
Am J Med 1981; 70:10271032.
9. Selby P, Buick RN, Tannock I. A critical appraisal of the human tumor stem cell
assay. New Engl J Med 1983; 308:129134.
10. Lieber MM, Kovach JS. Soft agar colony formation assay for chemotherapeutic
sensitivity of human solid tumors. Mayo Clin Proc 1982; 57:527528.
11. Nagourney RA. Ex vivo programmed cell death and the prediction of response to
chemotherapy. Curr Treat Options Oncol 2006; 7:103110.
12. Hickman JA. Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 1992;
11:121139.
13. Zunino F, Perego P, Pilotti S, et al. Role of apoptotic response in cellular resistance
to cytotoxic agents. Pharmacol Ther 1997; 76:177185.
14. Jaffrezou JP, Bettaieb A, Levade T, et al. Antitumor agent-induced apoptosis in
myeloid leukemia cells: a controlled suicide. Leuk Lymph 1998; 29:453463.
15. Weisenthal LM, Marsden JA, Dill PL, et al. A novel dye exclusion method for testing
in vitro chemosensitivity of human tumors. Cancer Res 1983; 43:749757.
16. Bosanquet AG, Durant J. Ex vivo drug sensitivity test by TRAC assay (Tumour
Response to Anti-neoplastic Compounds assay): a fourth-generation test based on the
DiSC assay. Br J Haematol 2005, 129(suppl 1):61.
17. Carmichael J, DeGraff WG, Gazdar AF, et al. Evaluation of a tetrazolium-based
semiautomatic colorimetric assay: assessment of chemosensitivity testing. Cancer
Res 1987, 47, 936942.
18. Kangas L, Gronroos M, Nieminen AL. Bioluminescence of cellular ATP: a new
method for evaluating cytotoxic agents in vitro. Med Biol 1984, 62, 338343.
19. Nygren P, Kristensen J, Jonsson B, et al. Feasibility of the fluorometric microculture
cytotoxicity assay (FMCA) for cytotoxic drug sensitivity testing of tumor cells from
patients with acute lymphoblastic leukemia. Leukemia 1992, 6, 11211128.
20. Larsson R, Nygren P, Ekberg M, et al. Chemotherapeutic drug sensitivity testing of
human leukemia cells in vitro using a semiautomated fluorometric assay. Leukemia
1990, 4, 567571.
21. Rotman B, Teplitz C, Dickinson K, et al. Individual human tumors in short-term
micro-organ cultures: Chemosensitivity testing by fluorescent cytoprinting. In Vitro
Cell Dev Biol 1988, 24, 11371138.
22. Zwaan CM, Kaspers GJ, Pieters R, et al. Cellular drug resistance in childhood acute
myeloid leukemia is related to chromosomal abnormalities. Blood 2002, 100, 3352
3360.
23. Staib P, Tiehen J, Strunk T, et al. Determination of caspase-3 activation fails to
predict chemosensitivity in primary acute myeloid leukemia blasts. BMC Cancer
2005, 5, 60 (8 pp).
24. Savasen S, Buck S, Ozdemir O, et al. Evaluation of cytotoxicity by flow cytometric
drug sensitivity assay in childhood T-cell acute lymphoblastic leukemia. Leuk
Lymph 2005; 46: 833840.
25. Nerenberg M, Kariv I, McNeeley P, et al. Use of optophoresis as an in vitro predictor
of cell response to chemotherapy for chronic lymphocytic leukemia. Leuk Lymph
2006; 47: 21942202.
38 Bosanquet et al.
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
26. Zhong Y, Bakke AC, Fan G, et al. Drug resistance in B-cell chronic lymphocytic
leukemia: predictable by in vitro evaluation with a multiparameter flow cytometric
cytotoxicity assay. Cytometry B Clin Cytom 2007; 72:189195.
27. Nygren P, Hagberg H, Glimelius B, et al. In vitro drug sensitivity testing of tumor
cells from patients with non-Hodgkins lymphoma using the fluorometric micro-
culture cytotoxicity assay. Ann Oncol 1994; 5(suppl 1):S127S131.
28. Kirkpatrick DL, Duke M, Goh TS. Chemosensitivity testing of fresh human leukemia
cells using both a dye exclusion assay and a tetrazolium dye (MTT) assay. Leuk Res
1990; 14:459466.
29. Pieters R, Huismans DR, Leyva A, et al. Comparison of the rapid automated MTT-
assay with a dye exclusion assay for chemosensitivity testing in childhood leukae-
mia. Br J Cancer 1989; 59:217220.
30. Lathan B, von Tettau M, Verpoort K, et al. Pretherapeutic drug testing in acute
leukemias for prediction of individual prognosis. Haematol Blood Transfus 1990;
33:295298.
31. Tidefelt U, Sundman-Engberg B, Rhedin AS et al. In vitro drug testing in patients
with acute leukemia with incubations mimicking in vivo intracellular drug concen-
trations. Eur J Haematol 1989; 43:374384.
32. Staib P, Staltmeier E, Neurohr K, et al. Prediction of individual response to che-
motherapy in patients with acute myeloid leukaemia using the chemosensitivity
index Ci. Br J Haematol 2005; 128:783791.
33. Norgaard JM, Langkjer ST, Palshof T, et al. Pretreatment leukaemia cell drug
resistance is correlated to clinical outcome in acute myeloid leukaemia. Eur J
Haematol 2001; 66:160167.
34. Yamada S, Hongo T, Okada S, et al. Clinical relevance of in vitro chemoresistance in
childhood acute myeloid leukemia. Leukemia 2001; 15:18921897.
35. Kaaijk P, Kaspers GJ, Van Wering ER, et al. Cell proliferation is related to in vitro
drug resistance in childhood acute leukaemia. Br J Cancer 2003; 88:775781.
36. Maehara Y, Anai H, Tamada R, et al. The ATP assay is more sensitive than the
succinate dehydrogenase inhibition test for predicting cell viability. Eur J Cancer
Clin Oncol 1987; 23:273276.
37. Schrek R. Differences between responsive and intractable chronic lymphocytic
leukemia. Med Hypotheses 1990; 31:8182.
38. Schrek R, Leithold SL, Friedman IA, et al. Clinical evaluation of an in vitro test for
radiosensitivity of leukemic lymphocytes. Blood 1962; 20:432442.
39. Durkin WJ, Ghanta VK, Balch CM, et al. A methodological approach to the pre-
diction of anticancer drug effect in humans. Cancer Res 1979; 39:402407.
40. Bosanquet AG, Bird MC, Price WJ, et al. An assessment of a short-term tumour
chemosensitivity assay in chronic lymphocytic leukaemia. Br J Cancer 1983;
47:781789.
41. Weisenthal LM, Dill PL, Finklestein JZ, et al. Laboratory detection of primary and
acquired drug resistance in human lymphatic neoplasms. Cancer Treat Rep 1986;
70:12831295.
42. Bosanquet AG. Correlations between therapeutic response of leukaemias and in-vitro
drug-sensitivity assay. Lancet 1991; 337:711716.
43. Pieters R, Loonen AH, Huismans DR, et al. In vitro drug sensitivity of cells from
children with leukemia using the MTT assay with improved culture conditions.
Blood 1990; 76:23272336.
Individualized Tumor Response Testing in Leukemia and Lymphoma 39
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
44. Pieters R, Kaspers GJ, Klumper E, et al. Clinical relevance of in vitro drug resistance
testing in childhood acute lymphoblastic leukemia: the state of the art. Med Pediatr
Oncol 1994; 22:299308.
45. Kaspers GJ, Kardos G, Pieters R, et al. Different cellular drug resistance profiles in
childhood lymphoblastic and non-lymphoblastic leukemia: a preliminary report.
Leukemia 1994; 8:12241229.
46. Klumper E, Pieters R, Veerman AJ, et al. In vitro cellular drug resistance in
children with relapsed/refractory acute lymphoblastic leukemia. Blood 1995; 86:
38613868.
47. Larsson R, Kristensen J, Sandberg C, et al. Laboratory determination of chemo-
therapeutic drug resistance in tumor cells from patients with leukemia, using a fluo-
rometric microculture cytotoxicity assay (FMCA). Int J Cancer 1992; 50:177185.
48. Larsson R, Jonsson B, Kristensen J, et al. Drug sensitivity testing of tumor cells from
patients with acute leukemia and non-Hodgkins lymphoma using a fluorometric
microculture cytotoxicity assay. In: Kaspers GJL, Pieters R, Twentyman PR, et al.,
eds. Drug Resistance in Leukemia and Lymphoma: The Clinical Value of Laboratory
Studies. Chur, Switzerland: Harwood Academic Publishers, 1993:399407.
49. Hongo T, Fujii Y, Igarashi Y. An in vitro chemosensitivity test for the screening of
anti-cancer drugs in childhood leukemia. Cancer 1990; 65:12631272.
50. Hongo T, Fujii Y. In vitro chemosensitivity of lymphoblasts at relapse in childhood
leukemia using the MTT assay. Int J Hematol 1991; 54:219230.
51. Silber R, Degar B, Costin D, et al. Chemosensitivity of lymphocytes from patients
with B-cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and camp-
tothecin analogs. Blood 1994; 84:34403446.
52. Nagourney RA, Weisenthal LM. Dexamethasone-induced cell death in primary
cultures of childhood ALL predict survival: a prospective trial with 13-year follow-up.
Leukemia 1995; 9:531 (abstr).
53. Kaspers GJL, Pieters R, Van Zantwijk CH, et al. In vitro drug sensitivity of normal
peripheral blood lymphocytes and childhood leukaemic cells from bone marrow and
peripheral blood. Br J Cancer 1991; 64:469474.
54. Pieters R, Huismans DR, Loonen AH, et al. Relation of cellular drug resistance to
long-term clinical outcome in childhood acute lymphoblastic leukaemia. Lancet
1991; 338:399403.
55. Styczynski J, Pieters R, Huismans DR, et al. In vitro drug resistance profiles of adult
versus childhood acute lymphoblastic leukaemia. Br J Haematol 2000; 110:813818.
56. Kaspers GJ, Veerman AJ, Pieters R, et al. In vitro cellular drug resistance and prognosis in
newly diagnosed childhood acute lymphoblastic leukemia. Blood 1997; 90:27232729.
57. den Boer ML, Harms DO, Pieters R, et al. Patient stratification based on prednisolone-
vincristine-asparaginase resistance profiles in children with acute lymphoblastic
leukemia. J Clin Oncol 2003; 21:32623268.
58. Klumper E, Ossenkoppele GJ, Pieters R, et al. In vitro resistance to cytosine ara-
binoside, not to daunorubicin, is associated with the risk of relapse in de novo acute
myeloid leukaemia. Br J Haematol 1996; 93:903910.
59. Hongo T, Fujii Y, Yajima S. In vitro chemosensitivity of childhood leukemic cells
and the clinical value of assay directed chemotherapy. In: Kaspers GJL, Pieters R,
Twentyman PR, et al., eds. Drug Resistance in Leukemia and Lymphoma: The
Clinical Value of Laboratory Studies. Chur, Switzerland: Harwood Academic Pub-
lishers, 1993:313319.
40 Bosanquet et al.
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
60. Santini V, Bernabei PA, Silvestro L, et al. In vitro chemosensitivity testing of leukemic
cells: prediction of response to chemotherapy in patients with acute non-lymphocytic
leukemia. Hematol Oncol 1989; 7:287293.
61. Kristensen J, Jonsson B, Sundstrom C, et al. In vitro analysis of drug resistance in tumor
cells from patients with acute myelocytic leukemia. Med Oncol Tumor Pharmacother
1992; 9:6574, 157 (erratum).
62. Larsson R, Nygren P. Prediction of individual patient response to chemotherapy by
the fluorometric microculture cytotoxicity assay (FMCA) using drug specific cut-off
limits and a Bayesian model. Anticancer Res 1993; 13:18251829.
63. Staib P, Lathan B, Schinkothe T, et al. Prognosis in Adult AML is precisely predicted
by the DiSC-assay using the chemosensitivity-index C1. Adv Exp Med Biol 1999;
457:437444.
64. Sargent JM, Taylor CG. Appraisal of the MTT assay as a rapid test of chemo-
sensitivity in acute myeloid leukaemia. Br J Cancer 1989; 60:206210.
65. Hwang WS, Chen LM, Huang SH, et al. Prediction of chemotherapy response in
human leukemia using in vitro chemosensitivity test. Leuk Res 1993; 17:685688.
66. Mollgard L, Tidefelt U, Sundman-Engberg B, et al. In vitro chemosensitivity testing
in acute non lymphocytic leukemia using the bioluminescence ATP assay. Leuk Res
2000; 24:445452.
67. Langkjer ST, Norgaard JM. Use of the MTT-assay for evaluation of chemosensitivity
in adult acute myeloid leukemia. In: Kaspers GJ, Pieters R, Twentyman PR, et al.,
eds. Drug Resistance in Leukemia and Lymphoma: The Clinical Value of Laboratory
Studies. Chur, Switzerland: Harwood Academic Publishers, 1993:279291.
68. Bird MC, Bosanquet AG, Forskitt S et al. Long-term comparison of results of a drug
sensitivity assay in vitro with patient response in lymphatic neoplasms. Cancer 1988;
61:11041109.
69. Bosanquet AG, Bell PB. Enhanced ex vivo drug sensitivity testing of chronic lympho-
cytic leukaemia using refined DiSC assay methodology. Leuk Res 1996; 20:143153.
70. Bosanquet AG, Johnson SA, Richards SM. Prognosis for fludarabine therapy of
chronic lymphocytic leukaemia based on ex vivo drug response by DiSC assay. Br J
Haematol 1999; 106:7177.
71. Morabito F, Stelitano C, Callea I, et al. In vitro drug-induced cytotoxicity predicts
clinical response to fludarabine in b-cell chronic lymphocytic leukaemia. Br J
Haematol 1998; 102:528531.
72. Bosanquet AG, McCann SR, Crotty GM, et al. Methylprednisolone in advanced
chronic lymphocytic leukaemia: rationale for, and effectiveness of treatment sug-
gested by DiSC assay. Acta Haematologica 1995; 93:7379.
73. Thornton PD, Matutes E, Bosanquet AG, et al. High dose methylprednisolone can
induce remissions in CLL patients with p53 abnormalities. Ann Hematol 2003;
82:759765.
74. Catchpoole DR, Stewart BW. Etoposide-induced cytotoxicity in two human T-cell
leukemic lines: delayed loss of membrane permeability rather than DNA fragmen-
tation as an indicator of programmed cell death. Cancer Res 1993; 53:42874296.
75. Larsson R, Nygren P. Laboratory prediction of clinical chemotherapeutic drug
resistance: a working model exemplified by acute leukaemia. Eur J Cancer 1993;
29A:12081212.
76. Jonsson E, Dhar S, Jonsson B, et al. Differential activity of topotecan, irinotecan and SN-
38 in fresh human tumour cells but not in cell lines. Eur J Cancer 2000; 36:21202127.
Individualized Tumor Response Testing in Leukemia and Lymphoma 41
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
77. Quintieri L, Fantin M, Vizler C. Identification of molecular determinants of tumor
sensitivity and resistance to anticancer drugs. Adv Exp Med Biol 2007; 593:95104.
78. Holleman A, Cheok MH, den Boer ML, et al. Gene-expression patterns in drug-
resistant acute lymphoblastic leukemia cells and response to treatment. N Engl J Med
2004; 351:533542.
79. Winick NJ, Carroll WL, Hunger SP. Childhood leukemia: new advances and chal-
lenges. N Engl J Med 2004; 351:601603.
80. Potti A, Dressman HK, Bild A, et al. Genomic signatures to guide the use of che-
motherapeutics. Nat Med 2006; 12:12941300.
81. Mason JM, Drummond MF, Bosanquet AG, et al. The DiSC assay: a cost-effective
guide to treatment for chronic lymphocytic leukemia? Int J Technol Assess Health
Care 1999; 15:173184.
82. Styczynski J, Wysocki M. Is the in vitro drug resistance profile the strongest
prognostic factor in childhood acute lymphoblastic leukemia? J Clin Oncol 2004;
22:963964.
83. Bosanquet AG, Raper SL, Durant J, et al. Comparison of ex vivo drug sensitivity by
TRAC assay and patient response in the UK LRF CLL4 trial. Haematologica/
Hematol J 2006: 91(suppl 1):100 (abstr).
84. Bosanquet AG, Raper SL, Durant J, et al. Drug sensitivity by TRAC (DiSC) assay as
a prognostic factor for patient response in untreated CLL: results from the UK LRF
CLL4 trial. Blood 2006; 108:94a (abstr).
85. Catovsky D, Richards S, Matutes E, et al. Assessment of fludarabine plus cyclo-
phosphamide for patients with chronic lymphocytic leukemia (the LRF CLL4 Trial):
a randomised controlled trial. Lancet 2007; 370:230239.
86. Schrag D, Garewal HS, Burstein HJ, et al. American Society of Clinical Oncology
technology assessment: chemotherapy sensitivity and resistance assays. J Clin Oncol
2004; 22:36313638.
87. Castro M. Resisting a fundamentalist policy. J Clin Oncol 2005; 23:36453646.
88. Schrag D, Samson DJ, Seidenfeld J, et al. In reply. J Clin Oncol 2005; 23:36463648.
89. Samson DJ, Seidenfeld J, Ziegler K, et al. Chemotherapy sensitivity and resistance
assays: a systematic review. J Clin Oncol 2004; 22:36183630.
90. Weisenthal LM. Antineoplastic drug screening belongs in the laboratory, not in the
clinic (editorial). J Natl Cancer Inst 1992; 84:466469.
91. Bosanquet AG, Burlton AR, Bell PB, et al. Ex vivo cytotoxic drug evaluation by
DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP.
Br J Cancer 1997; 76:511518.
92. Beksac M, Kansu E, Kars A, et al. A rapid drug sensitivity assay for neoplasmatic
cells. Med Oncol Tumor Pharmacother 1988; 5:253257.
93. Kaspers GJL, Pieters R, Van Zantwijk CH, et al. Prednisolone resistance in child-
hood acute lymphoblastic leukemia: vitro-vivo correlations and cross-resistance to
other drugs. Blood 1998; 92:259266.
94. Santini V, Bernabei PA, Dal Pozzo O, et al. Acute myeloid leukemia (AML) sen-
sitivity to antiblastics is predictable by INT assay. In: Kaspers GJ, Pieters R,
Twentyman PR, et al., eds. Drug Resistance in Leukemia and Lymphoma: The
Clinical Value of Laboratory Studies. Chur, Switzerland: Harwood Academic Pub-
lishers, 1993:365368.
95. Stute N, Kohler T, Lehmann L. Drug resistance testing in acute myeloid leukemia.
Adv Exp Med Biol 1999; 457:445452.
42 Bosanquet et al.
[sanjeev][6x9-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0002_O.3d] [7/4/08/15:32:42]
[2344]
96. Bosanquet AG, Copplestone JA, Johnson SA, et al. Response to cladribine in pre-
viously treated patients with chronic lymphocytic leukemia identified by ex vivo assess-
ment of drug sensitivity in the DiSC assay. Br J Haematol 1999; 106:474476.
97. Bosanquet AG. The DiSC assay: 10 years and 2000 tests further on. In: Kaspers GJL,
Pieters R, Twentyman PR, et al. eds. Drug Resistance in Leukemia and Lymphoma:
The Clinical Value of Laboratory Studies. Chur, Switzerland: Harwood Academic
Publishers, 1993:373383.
98. Leone LA, Meitner PA, Myers TJ, et al. Predictive value of the fluorescent cytoprint
assay (FCA): a retrospective correlation study of in vitro chemosensitivity and
individual responses to chemotherapy. Cancer Invest 1991; 9:491503.
99. Strauss SJ, Maharaj L, Hoare S, et al. Bortezomib therapy in patients with relapsed or
refractory lymphoma: potential correlation of in vitro sensitivity and tumor necrosis
factor alpha response with clinical activity. J Clin Oncol 2006; 24:21052112.
Individualized Tumor Response Testing in Leukemia and Lymphoma 43
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3
Minimal Residual Disease
Jacques J. M. van Dongen
Department of Immunology, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
Tomasz Szczepan ski
Department of Immunology, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands, and Department of Pediatric
Hematology and Oncology, Medical University of Silesia, Zabrze, Poland
Vincent H. J. van der Velden
Department of Immunology, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
MINIMAL RESIDUAL DISEASE
Current cytotoxic treatment protocols induce complete remission (CR) in most
acute leukemia patients [both acute lymphoblastic leukemia (ALL) and acute
myeloid leukemia (AML)], in some patients with chronic lymphocytic leukemia
(CLL), and in most non-Hodgkins lymphoma (NHL) and chronic myeloid
leukemia (CML) patients. Introduction of allogeneic and autologous hema-
topoietic stem cell transplantation (HSCT) in treatment protocols has further
increased the remission rates in ALL, AML, CML, and NHL. Nevertheless,
many of these patients ultimately relapse. Apparently, the treatment protocols
are not capable of killing all clonogenic malignant cells in these patients, even
though they reached CR according to cytomorphological criteria. The detection
limit of cytomorphological techniques is not lower than 1% to 5% of malignant
cells, implying that these techniques can provide only superficial information
45
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about the effectiveness of the treatment. More sensitive techniques are required
for the detection of low frequencies of malignant cells during and after treatment,
i.e., detection of minimal residual disease (MRD). MRD techniques should reach
sensitivities of at least 10
3
(one malignant cell within thousand normal cells),
but sensitivities of 10
4
to 10
6
are preferred. Such sensitivities allow true
MRD detection and thereby evaluation of the effectiveness of the total treatment
and assessment of the contribution of each treatment phase.
TECHNIQUES AND TARGETS FOR MRD MONITORING
For the detection of MRD, at present at least three methods are sufficiently
sensitive (10
3
), quantitative, and broadly applicable: flow cytometric immuno-
phenotyping, polymerase chain reaction (PCR)-based detection of junctional
regions of rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes
(mostly in lymphoid malignancies), and PCR-based detection of breakpoint
fusion regions of chromosome aberrations (Table 1).
Table 1 Applicability of MRD Techniques in Leukemias and Lymphomas
a
Flow cytometric
immunophenotyping PCR analysis
Aberrant
immunophenotypes
(10
3
10
4
)
Junctional regions
of Ig/TCR genes
(10
3
10
6
)
Chromosome
aberrations
(10
4
10
6
)
Precursor-B-ALL
children 7090% *95% 4045%
adults 7080% *95% 4045%
T-ALL
children *95% >95% 2530%
adults *95% >90% 1015%
Chronic B-cell
leukemias
>95% >95%
Chronic T-cell
leukemias
*95%
B-NHL 7080%
b
2530%
T-NHL 2025%
c
*95% 1015%
AML (non-M3) 6090% 1015% 1530%
APL NR NR >95%
CML >95%
a
The percentages indicate the applicability of the MRD techniques per category of hematopoietic
malignancies; J.J.M. van Dongen, unpublished results.
b
Somatic mutations hamper primer annealing in a part of the patients with B-NHL or B-CLL.
c
Based on T-ALL-like immunophenotype in lymphoblastic T-NHL and NPM-ALK expression in
*50% of large cell anaplastic lymphomas of T-cell lineage.
NR No reports on detailed studies.
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MRD Monitoring by Flow Cytometric Immunophenotyping
Principles
Hematological malignancies can be regarded as malignant counterparts of cells
in immature (acute leukemia) or mature (CLL, CML, NHL) stages of hemato-
poiesis. Although the immunophenotype of the malignant cells is often comparable
to their normal counterparts, tumor-associated immunophenotypes can be observed.
Such tumor-associated immunophenotypes can be identified in the vast major-
ity of acute leukemias, while they are less common in mature hematological
malignancies. Asynchronous antigen expression refers to the coexpression of
two or more antigens that are not present at the same time during normal dif-
ferentiation. Cross-lineage antigen expression represents the expression of
typical myeloid antigens on lymphoid cells or vice versa and the presence of
B-lineage antigens on T-lineage cells or vice versa. Ectopic antigen expression
refers to the presence of particular antigens on cells outside their normal
breeding sites or homing areas or to the expression of antigens that are normally
only expressed on nonhematopoietic cells.
MRD Monitoring in Acute Leukemias
Investigation of normal bone marrow (BM) B-cell precursors enabled establishment
of templates for normal B-cell development. Malignant precursor-B lymphoblasts
frequently display aberrant immunophenotypic features and thereby fall into empty
spaces outside the normal B-lineage pathways (Fig. 1) (1,2). Flow cytometric
investigations based on three- or four-color stainings showed the presence of
leukemia-associated phenotypes in 70% to 95% of precursor-B-ALL patients (3,4).
It should be noted that the detection of small numbers of precursor-B-ALL
cells in regenerating BM after chemotherapy or after HSCT can be hampered by
high frequencies of normal, regenerating precursor-B-cells (up to 50%) (5,6). The
extent of B-cell regeneration in BM differs per treatment protocol, per phase of
treatment, and seems to be dependent on the intensity of the preceding treatment (6).
Since nearly all T-ALL express terminal deoxynucleotidyl transferase (TdT)
as well as the pan-T-cell antigens CD2, cytoplasmic CD3 (CyCD3), CD5, and
CD7, the ectopic TdT expression allows MRD detection in 90% of T-ALL. Flow
cytometric analysis based on cross-lineage myeloid antigen expression, asyn-
chronous antigen expression (e.g., CD34-positive/CD3-positive), and antigen
overexpression (e.g., CD99 or CD7) can also be used for MRD detection in T-
ALL (7,8). Similar to precursor-B-ALL, multiparameter flow cytometry in
T-ALL reveals empty spaces outside normal T-cell development pathways.
Together the various leukemia-associated immunophenotypes can be employed
for MRD detection in virtually all T-ALL and lymphoblastic T-NHL.
In AML, tumor-associated immunophenotypes can be observed in about
70% to 85% of patients (913). However, it should be noted that the immuno-
phenotype of the AML cells may be heterogeneous and that several subpopulations
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can be present (14). For reliable MRD analysis, all leukemic subpopulations should
be monitored.
The detection limit of current flow cytometric MRD methods varies
between 0.1% and 0.01% for most precursor-B-ALL and AML, while in virtually
all T-ALL a detection limit of 0.01% can be reached (7,1517). It can be expected
that the recent introduction of flow cytometers with six to eight colors will further
improve the applicability and sensitivity of MRD detection in ALL and AML.
Furthermore, on the basis of a comparison of gene expression profiles of normal
and leukemic cells, new and widely applicable markers for MRD studies in acute
leukemias are being identified. The applicability of this approach has already been
proven for CD58, which is now one of the most useful markers to study MRD in
precursor-B-ALL (18).
MRD Monitoring in Mature B- and T-Cell Malignancies
In B-CLL, quantitative differences in the levels of antigen expression as compared
with normal B-cells can be observed in the vast majority of patients. This approach,
analogous to detection of empty spaces outside the normal B-cell development in
ALL, can potentially reach sensitivities of 10
4
to 10
5
(1921). Especially the
combination of CD19/CD5/CD20/CD79b or CD43 antibodies seems to be infor-
mative for MRD detection in PB of B-CLL patients. For MRD monitoring in BM, at
least one of the combinations CD19/CD5/CD20/CD79b, CD19/CD5/CD38/CD79b,
CD19/CD5/CD38/CD20 was found sufficiently sensitive and specific (19). A com-
bination of CD81/CD22/CD19/CD5 is particularly useful for MRD detection in
patients treated with CD20 antibody (rituximab) (22).
In B-NHL, the sensitivity of immunophenotypic MRD analysis is often
hampered by the lack of a tumor-associated immunophenotype and the presence
of normal B-cells with a comparable immunophenotype. Nevertheless, by the
use of markers that are normally only expressed on a subpopulation of B-cells,
sensitivities of 10
3
can be achieved. Examples include the CD103 antigen on
hairy cell leukemias and the CD5 antigen on mantle cell lymphoma (MCL) cells
(23). Furthermore, protein products from particular chromosome aberrations may
also be used as additional markers in flow cytometric analysis. BCL2/B-cell
antigen/Ig light chain stainings may be employed for MRD detection in patients
with follicular lymphomas (FL), since BCL2 overexpression is observed in this
type of B-NHL with t(14;18) (24). Similarly, the overexpression of CyclinD1 in
MCL with t(11;14) or of MYC in Burkitts lymphomas with t(2;8), t(8;14), or
t(8;22) may be employed for MRD detection of these types of B-NHL (25).
The vast majority of chronic T-cell leukemias and T-NHL belong to the
TCRab lineage, whereas only a minor fraction expresses TCRgd. Antibodies
against the protein products of V gene segments of TCR beta (TCRB) V gene
families, recognizing 60% to 70% of normal and malignant T-cells with TCRb
chain expression, can be used for detecting malignant cells (26). Also, Vg and Vd
antibodies might be useful for detecting malignant (clonal) TCRgd

T-cells,
although the presence of normal TCRgd

T-lymphocytes will interfere with these


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applications (27,28). This especially concerns Vg9/Vd2

phenotypes, because
most normal TCRgd

T-lymphocytes have this TCR phenotype (29). The appli-


cation of Vb, Vg, and Vd antibodies in well-chosen multicolor stainings can result
in sensitivity levels of approximately 10
2
. Such sensitivities do not allow true
MRD detection but may enable monitoring of T-cell leukemia patients during
treatment or predicting the possible outgrowth of a dominant subclone in case of
oligoclonal T-cell proliferations.
Detection of lower MRD levels (*10
3
) in chronic T-cell leukemia or
T-NHL is only possible upon analysis of tumor-associated immunophenotypes
(e.g., lack of CD2 expression) or translocation-specific fusion proteins such as
the NPM-ALK fusion protein in anaplastic large cell lymphoma (ALCL) with
t(2;5) (30).
Immunophenotypic Shifts
A potential pitfall of immunophenotypic MRD detection in hematological
malignancies is the occurrence of immunophenotypic shifts during the course of
the disease. Differences in immunological marker expression are particularly
frequent in acute leukemias and may concern up to 90% of patients (3133).
However, at least one leukemia-specific marker combination is retained by
leukemic cells at relapse in at least 80% of patients (31,34). In order to limit the
risk of false-negative results, at least two marker combinations per patient should
be used for immunophenotypic MRD monitoring. Furthermore, since in AML
shifts toward a more immature phenotype of the myeloblasts, consistent with
clonal evolution of a leukemic stem cell, are frequently observed (33), antibody
panels used for MRD monitoring in AML should preferably not be restricted to
the immunophenotype detected at presentation but should also include markers
of lineage immaturity.
Immunophenotypic shifts may also occur during the early phase of treat-
ment. Such immunophenotypic shifts have been reported in ALL and may be a
direct result of the effect of the drugs on the expression level of various antigens
or may be related to drug-induced cell kill (35,36).
MRD Monitoring by PCR Analysis of Junctional Regions
Principles
During early B- and T-cell differentiation the germline V, (D), and J gene seg-
ments of the Ig and TCR gene complexes rearrange, and each lymphocyte thereby
obtains a specific combination of V-(D-)J segments that codes for the varia-
ble domains of Ig or TCR molecules. The random insertion and deletion of
nucleotides at the junction sites of V, (D), and J gene segments make the
junctional regions of Ig and TCR genes fingerprint-like sequences, which
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are different in each lymphocyte and thus also in each lymphoid malignancy.
Therefore, junctional regions can be used as tumor-specific targets for MRD-
PCR analysis. Such targets can be identified (e.g., by PCR heteroduplex
analysis or GeneScan analysis) at initial diagnosis in more than 95% of lym-
phoid malignancies and in approximately 10% of AML (37,38). Subsequently,
the precise nucleotide sequence of the junctional regions can be determined.
This sequence information allows the design of junctional region-specific
oligonucleotides [either allele specific oligonucleotide (ASO) probes or ASO
primers], which can be used to detect malignant cells among normal lymphoid
cells during follow-up of patients. At present, real-time quantitative PCR (RQ-
PCR) analysis is the most frequently used approach for MRD monitoring in
hematological malignancies (Fig. 2) (39).
MRD Monitoring in Acute Leukemias
By applying appropriate primer sets, Ig/TCR gene rearrangements can be
detected in virtually all precursor-B-ALL and T-ALL patients. The number and
type of Ig/TCR rearrangements is however dependent on the age of the patient
and the presence of fusion gene transcripts, like TEL-AML1 and MLL-AF4
(4044). In order to limit the risk of false-negative MRD results due to clonal
evolution phenomena (e.g., ongoing rearrangements, loss of subclones), prefer-
ably two MRD-PCR targets with sufficient sensitivity (10
4
) should be used
for each ALL patient (4547).
MRD Monitoring by PCR Analysis of Junctional Regions
in Mature B- and T-Cell Malignancies
For MRD studies in B-CLL and B-NHL patients, Ig heavy (IGH) chain gene
rearrangements are frequently used, because they are present in virtually all
lymphoma patients (37). Also Ig kappa (IGK) light chain and Ig lambda (IGL)
light chain gene rearrangements can be applied as MRD-PCR target in lym-
phoma patients (48,49). A limitation of Ig gene rearrangements as MRD-PCR
target is the occurrence of somatic hypermutations in part of B-CLL patients and
in many B-NHL patients, especially FL and postfollicular B-NHL. This does not
seriously hamper initial diagnostics because currently available multiplex PCR
assays allow identification of clonal IGH, IGK, and/or IGL gene rearrangements
in more than 95% of mature B-cell malignancies (37,50). However, when the
somatic mutation process is active like in FL, this might result in the formation
of subclones, which are no longer recognized by the applied primer-probe set.
Since IGK-Kde and DH-JH rearrangements are not prone to somatic hyper-
mutations, theoretically they are preferred targets for MRD analysis. Clonal
evolution phenomena are rare in mature B-cell malignancies, and consequently
this does not hamper MRD monitoring in lymphomas.
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TCR gamma (TCRG) gene rearrangements are found in virtually all mature
T-lineage malignancies, whereas TCRB gene rearrangements can be detected in
all malignancies belonging to the TCRab lineage (51). Thus, MRD studies in
mature T-cell malignancies can generally use junctional regions of rearranged
TCRG and TCRB genes as PCR targets, whereas TCR delta (TCRD) gene
rearrangements are less often available (52). In mature T-cell malignancies, TCR
genes are not affected by somatic mutations and are not susceptible to ongoing
or secondary rearrangements. Consequently, one MRD-PCR target should be
sufficient for reliable monitoring of mature T-cell malignancies during and after
treatment.
RQ-PCR Analysis of Ig/TCR Gene Rearrangements
Several primer-probe sets for RQ-PCR-based detection of tumor-specific IGH
gene rearrangements have been described, particularly for application in ALL
patients (reviewed by van der Velden et al.) (39). In principle, these sets can also
be applied for MRD studies in mature B-cell malignancies, although the pres-
ence of somatic hypermutations might particularly hamper efficient annealing of
germline VH primers and probes. Application of 3
0
-minor groove binding (MGB)
probes allows design in small areas of the VH gene segments that are less
susceptible to somatic hypermutations (53). Also, for IGK, IGL, TCRG, TCRD,
and TCRB, several germline primer-probe sets have been designed (39).
In order to determine the sensitivity of the RQ-PCR assay, serial dilutions
of the diagnostic sample are generally used (Fig. 2). For defining the sensitivity,
several criteria (including reproducibility of the measurement, the difference
Figure 2 RQ-PCR assay for detection of MRD using IGK-Kde gene rearrangement as a
patient-specific target. (A) Schematic representation of an IGK-Kde rearrangement. The
position and sequences of the primers used for target identification at diagnosis are indi-
cated. (B) Sequences are given of the germline TaqMan
1
probe and the germline Kde
reverse primer used for RQ-PCR analysis during follow-up of patients. All sequences are
given from 5
0
to 3
0
. For each patient, a patient-specific forward primer is designed. (C)
RQ-PCR analysis of the Vk-Kde rearrangement in a precursor-B-ALL patient. Tenfold
dilutions of the diagnostic sample in normal MNC DNA were analyzed at an annealing
temperature of 608C; a quantitative range of 10
4
was reached. Normal MNC DNA did not
show amplification in any of the four wells tested. (D) Applicability of RQ-PCR analysis
of IGK-Kde rearrangements for MRD detection in follow-up samples of two patients with
precursor-B-ALL an MRD high-risk patient, 5257, with high MRD levels (10
3
) at the
early time points and a low-risk patient, 5397, with undetectable MRD already at the end of
induction treatment. RQ-PCR analysis (black diamonds) was compared with classical dot
blot analysis (open squares). Abbreviations: RQ-PCR, real-time quantitative polymerase
chain reaction; MRD, minimal residual disease; ALL, acute lymphoblastic leukemia;
MNC, mononuclear cells.
<
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between specific and nonspecific amplification, slope, and correlation coefficient
of the standard curve) should be taken into account (39). In order to compare
data between different studies and/or different laboratories, it is essential to have
uniform guidelines for RQ-PCR data interpretation (54). For Ig/TCR-based
MRD data in ALL, such guidelines have recently been developed within the
European Study Group on MRD detection in ALL (ESG-MRD-ALL; a consortium
of 30 international laboratories, coordinated by JJM van Dongen and VHJ van der
Velden) (55). These guidelines should be evaluated for use in monitoring of other
hematological malignancies as well.
The sensitivity of MRD-PCR analysis of junctional regions is dependent
on the type of rearrangement, on the size of the junctional region, and on the
background of normal lymphoid cells with comparable Ig/TCR gene rear-
rangements (56). One should be aware that the background of normal lymphoid
cells is not constant, but can differ per treatment phase. For example, high fre-
quencies of normal T-cells can be detected in postinduction follow-up samples, and
substantial expansions of normal precursor-B-cells can be detected in regenerating
BM after cessation of therapy (5,6,57).
To check the quantity and amplifiability of the DNA samples, a control
gene RQ-PCR should always be used.
MRD Monitoring by PCR Analysis of Chromosome Aberrations
Principles
In several hematological malignancies, chromosome aberrations can be detected
and may be used as MRD-PCR target. This includes breakpoint regions of fusion
genes, fusion gene transcripts, and aberrantly expressed genes (39). An advan-
tage of using chromosome aberrations as tumor-specific PCR targets for MRD
detection is their stability during the disease course. However, many hematological
malignancies do not have specific chromosome aberrations, which can be detected
by PCR. Nevertheless, new techniques for rapid and efficient screening of relatively
large breakpoint regions, such as long-distance PCR and long-distance inverse PCR,
should render such genomic breakpoint fusion sites into more feasible MRD-PCR
targets (58).
MRD Monitoring in Acute Leukemias
In about 40% of precursor-B-ALL patients and in about 15% of T-ALL patients,
fusion gene transcripts can be detected. These particularly concern MLL-AF4,
BCR-ABL, TEL-AML1, E2A-PBX1, CALM-AF10, and SIL-TAL1. In AML,
CBFB-MYH11 and AML1-ETO can be found in 10% to 25% of patients, the
frequency decreasing with age (59). PML-RARA transcripts can be detected in
virtually all acute promyelocytic leukemia (APL) patients. Primer-probe sets for
the detection of these fusion gene transcripts have been developed within the
Europe Against Cancer program (60,61).
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MRD Monitoring in CML
The BCR-ABL p210 fusion gene transcript can be detected in over 95% of
patients with CML and thereby is an excellent MRD-PCR target in this group of
patients. Moreover, both BM and PB can be applied for clinically relevant MRD
monitoring in CML (62). Several efforts to standardize the methodology of BCR-
ABL detection have been undertaken or are currently in progress (60,63,64).
MRD Monitoring by PCR Analysis of Chromosome Aberrations
in Mature B- and T-Cell Malignancies
In approximately 30% of B-NHL patients, chromosome aberrations can be
employed as tumor-specific MRD-PCR targets in which the PCR primers are
chosen at opposite sides of the breakpoint fusion region (65). One of the most
widely studied chromosomal translocations is t(14;18), involving the BCL2 and
IGH genes, which occurs in 80% of FL patients, 20% of DLBCL patients, and
which is detectable by standard PCR procedures in 60% to 70% of cases with
t(14;18). The t(11;14) is characteristic for most MCL and involves the BCL1 and
IGH genes. In 30% to 40% of MCL patients, the breakpoints are clustered within
a restricted area [the major translocation cluster (MTC) region], allowing easy
identification at the DNA level by standard PCR analysis. In the vast majority of
Burkitts lymphoma patients, chromosomal aberrations involving one of the Ig
genes in combination with the MYC gene, e.g., t(8;14), t(2;8), and t(8;22), can be
found. In all the above-mentioned B-NHL types the breakpoints generally occur
outside coding regions, implying that these translocations are not amenable to
reverse transcriptase PCR (RT-PCR) analysis for MRD detection, but should be
studied at the DNA level.
In some lymphomas, aberrantly expressed genes can theoretically be used
for MRD detection, although transcripts in normal cells may limit the sensitivity.
Examples include the expression of CCND1 transcripts in MCL with t(11;14),
and overexpression of MYC in Burkitts lymphomas with t(2;8), t(8;14), or
t(8;22). Although RQ-PCR assays for such transcripts have been reported, they
have not yet been used for MRD detection.
In T-NHL only a few well-defined translocations are known so far. This
concerns the NPM-ALK fusion gene that is observed in ALCL with t(2;5), and
that can be used for RT-PCR analysis and potentially in some cases for PCR
analysis at the DNA level as well (66).
RQ-PCR Analysis of Chromosome Aberrations
Depending on the type of chromosome aberration, detection limits of 10
3
to
10
6
can generally be reached. Because of the high sensitivity of PCR techni-
ques, cross-contamination of RT-PCR products between patient samples is a
major pitfall in RT-PCR-mediated MRD studies, resulting in up to 20% of false-
positive results (60). Such cross-contamination is difficult to recognize since
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leukemia-specific fusion gene transcript-derived PCR products and wild-type
transcript-derived PCR products are not patient specific. This is in contrast to
PCR products obtained from genomic breakpoint fusion regions, such as in
t(14;18) and TAL1 deletions, which can be identified by use of patient-specific
oligonucleotide probes.
For quantification of fusion gene transcript, generally plasmid standard
curves are used. For patient-specific fusion genes, the standard curve is usually
prepared from serial dilutions of the diagnostic sample. To correct for the
amount and quality of the RNA, the use of a control gene is essential (39,61).
CLINICAL RELEVANCE OF MRD MONITORING
IN LEUKEMIAS AND LYMPHOMAS
Clinical Relevance of MRD Monitoring in ALL
Clinical Value of MRD Detection During Frontline
Treatment of Childhood ALL
The most significant application of MRD monitoring in childhood ALL is the
evaluation of the initial response to chemotherapy, since numerous studies have
demonstrated that low levels or absence of MRD after completion of induction
therapy predicts excellent outcome (67,68) (Fig. 3). The level of MRD-PCR
positivity after induction therapy is independent of other clinically relevant risk
factors (e.g., age, blast count at diagnosis, immunophenotype at diagnosis,
presence of chromosome aberrations, response to prednizone, and classical
clinical risk group assignment) and is the most powerful prognostic factor.
Depending on the treatment protocol, the sensitivity of the MRD tech-
nique, and the timing of the follow-up BM samples, MRD negativity is asso-
ciated with overall relapse rates of only 2% to 10% (15,17,6972). Moreover,
Figure 3 (A) Hypothetical graph showing the kinetics of leukemic cell decrease and
regrowth in several ALL patients during and after treatment with the I-BFM-SG treatment
protocol. MRD curves represent individual patients of the three MRD-based risk groups
two patients with slow MRD clearance (high-risk group), two patients with moderate MRD
clearance (intermediate-risk group), and one patient with rapid MRD clearance (low-risk
group) (69). The detection limit of cytomorphologic techniques as well as the detection
limit of flow cytometric immunophenotyping and PCR techniques are indicated: I,
induction treatment; C, consolidation treatment; and II, reinduction treatment. (B) 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 and before consolidation
treatment (69). (C) EFS plot of patients divided according to the MRD prior to hema-
topoietic stem cell transplantation. The five-year EFS and number of patients for each
group are shown at the end of each curve (based on the report of the Pre-BMT MRD Study
Group) (82). Abbreviations: EFS, event-free survival; MRD, minimal residual disease.
>
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sensitive MRD detection during the induction phase seems capable of identi-
fying 20% to 50% of childhood ALL patients with a very rapid leukemia
clearance and long-term relapse-free survival (73,74). On the other hand, several
studies proved that high MRD levels at the end of induction treatment are
associated with high relapse rates of 70% to 100% (15,17,69,71).
MRD analysis at a single time point gives highly significant prognostic
information, but a single time point is not sufficiently precise to define MRD-
based low-risk and high-risk groups (15,17,6971). Depending on the MRD study,
the end-of-induction MRD status either identifies only patients at low risk of
relapse (69,70) or more frequently identifies exclusively high-risk patients (3,71).
In contrast, combined information on MRD at the end of induction treatment and
before consolidation treatment is significantly superior to single time point mea-
surement, which was first demonstrated by the International BFM Study Group
(I-BFM-SG) (69). Such combined MRD information distinguishes patients at low
risk with MRD negativity at both time points (5-year relapse rate of 2%); from
patients at high risk with an intermediate (10
3
) or high (10
2
) degree of MRD
at both time points (5-year relapse rate of 80%), and the remaining patients at
intermediate risk (5-year relapse rate of 22%) (Fig. 3) (69).
The group of MRD-based high-risk patients is larger than any previously
identified high-risk group (*15%) and has an unprecedentedly high five-year
relapse rate of 80%. In ongoing frontline protocols with MRD-based interven-
tion, the MRD-based high-risk group is subjected to further intensification of
treatment protocols, including HSCT during first remission or novel treatment
modalities, e.g., imatinib in t(9;22)-positive cases. On the other hand, the
MRD-based low-risk patients make up a group of a substantial size (*45%),
comparable to the frequency of survivors of childhood ALL before treatment
intensification was introduced. Therefore, low-intensity standard-risk protocols
may be sufficiently effective to cure such patients. MRD-based risk-group dis-
tribution is even more striking in T-ALL: with fewer (*25%) low-risk patients
with virtually no relapses, more (*25%) high-risk patients uniformly relapsing,
and approximately 50% intermediate-risk patients with 25% relapses (72).
Most ALL patients on conventional frontline chemotherapy protocols
reach MRD negativity at some point during the treatment, while approximately
10% of patients remain continuously MRD positive until the end of treatment.
These patients are usually at high or intermediate risk of relapse on the basis of
MRD monitoring. Future MRD-based protocols should demonstrate whether
continuous MRD monitoring in MRD-based high- and intermediate-risk patients
could be also used for treatment intervention.
Clinical Value of MRD Detection After Relapse of ALL
After first relapse MRD monitoring has strong predictive value by assessing early
treatment response after second induction treatment, although reported studies
involved small groups of patients and need to be confirmed (75,76). In the BFM
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ALL-REZ treatment protocol, patients with MRD levels less than 10
3
at day 36
had a probability of relapse-free survival of 86%, whereas MRD levels more than
or equal to 10
3
were uniformly predictive of dismal outcome (probability of
relapse-free survival of 0%) (75). Similarly, the study from St. Judes Childrens
Research Hospital showed that MRD investigation at day 36 of their protocol
could largely distinguish patients with relatively good prognosis (MRD 10
4
,
2-year incidence of relapse of 28%) and bad prognosis (MRD > 10
-4
, 2-year
incidence of relapse of 70%). Many current ALL relapse treatment protocols
include MRD measurements, which can be particularly useful for patients with late
(off-therapy) relapses for optimal qualification for and timing of HSCT (76).
Clinical Value of MRD Detection Before and/or After Stem Cell
Transplantation in Childhood ALL
Several studies have demonstrated that MRD monitoring is highly significant for
ALL patients undergoing HSCT (7781). Multicenter data combined by the Pre-
BMT MRD Study Group showed that the level of MRD prior to allogeneic
HSCT identifies a group of patients with a high risk of relapse after trans-
plantation (Fig. 3) (82). The five-year event-free survival (EFS) of the group
with negative-, low-, and high-level positive-MRD approximated 75%, 40%, and
20%, respectively (82).
MRD-PCR positivity in ALL patients after HSCT is also suggestive of
impending relapse (83). MRD was shown to occur in post-HSCT samples in 88%
of patients who subsequently relapsed, while only 22% of patients in long-term
CR showed MRD at any time after HSCT, mostly at low levels (83).
Therefore, the treatment of patients with a high MRD burden before HSCT
or persistent MRD positivity after HSCT should be modified (e.g., further
cytoreduction before HSCT, intensified conditioning, and/or early post-HSCT
immunotherapy to induce controlled graft-versus-leukemia effects) in order
to improve their generally poor outcome (7780,82).
Clinical Value of MRD Detection in Adult ALL
Adult ALL is more frequently characterized by high-risk features with greater
drug resistance, poorer tolerance of and compliance with treatment as compared
with childhood ALL (84) Also the frequencies of MRD positivity and the MRD
levels in adult patients are significantly higher than in comparably treated
children (85,86) Therefore, MRD information might be particularly important
for standard-risk ALL patient without known factors predictive of resistant
disease, which was clearly demonstrated by the study of the German Multicenter
Study Group for Adult ALL (87). Similar to studies in childhood ALL, MRD-
based measurement of early treatment response in adult ALL resulted in a very
precise new risk group definition: low-risk group with MRD less than or equal to
10
4
at day 11 and day 24 (10%, 3-year relapse rate of 0%), high-risk group with
MRD more than or equal to 10
4
or higher until week 16 (23%, 3-year relapse
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rate of 94%), and intermediate-risk group comprising the remaining patients
(3-year relapse rate of 47%) (87). Moreover, among patients who reached MRD
negativity, a conversion to MRD positivity (especially within the quantitative
range) during the follow-up was associated with significantly increased risk of
relapse (88) Another prospective study by Vidriales et al. has shown that MRD
detection remains highly relevant for the entire adult ALL group. Patients with
low/undetectable MRD early during induction treatment have excellent survival
rates, while high MRD positivity at the end of induction treatment was asso-
ciated with dismal outcomes (89). Introduction of imatinib into therapy of
t(9;22)-positive adult ALL brought new promises on improving treatment out-
come in this otherwise highly resistant disease (90). Therefore, MRD monitoring
becomes of high clinical value to analyze the effectiveness of different combi-
nations of chemotherapy, imatinib, and HSCT in t(9;22)-positive adult ALL
(91,92).
Clinical Relevance of MRD Monitoring in APL
The most extensive MRD studies in AML concerned the RT-PCR monitoring of
PML-RARA fusion transcripts in APL patients with t(15;17). The results from
several retrospective as well as prospective RT-PCR studies in APL patients
showed several distinct molecular characteristics of this AML subtype (93,94),
leading to the first successful treatment intervention protocol based on MRD
information (95). It was known for many years that treatment with all-
trans-retinoic acid (ATRA) alone is insufficient to eliminate all leukemic cells
(96) Rapid loss of MRD positivity during the first three months of ATRA and
cytotoxic treatment was associated with good outcome, whereas continuous
positivity was predictive of relapse (97). Nevertheless, MRD status at the end of
induction treatment is clinically insufficient to predict the patients subsequent
outcome as shown by several large prospective studies (98100). With modern
treatment protocols, combining ATRA with consolidation chemotherapy, PCR
negativity is achieved at the end of treatment in virtually all patients. A small
subset (*5%) of patients with refractory APL are RT-PCR positive even at the
end of treatment consolidation (9799), while the vast majority of relapsing
patients (2030% of total APL patients) is MRD negative at the end of con-
solidation treatment (98,101,102). To obtain clinically relevant information,
continuous prospective MRD monitoring is required during the first 6 to
12 months after consolidation treatment for early identification of patients at
increased risk of relapse (103). This is particularly important for patients with
high-risk features at presentation such as hyperleukocytosis, while the need
for continuous monitoring of patients with low initial white blood counts (i.e.,
<10 10
9
/L) is now questionable (94). Reappearance of detectable MRD
during the maintenance treatment usually precedes hematological relapse at a
median time of two to three months (101,102). This information led to the
definition of molecular relapse in APL, which is manifested by conversion from
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RT-PCR negativity to positivity in two successive BM samplings during follow-
up (101). Lo Coco et al. (95) demonstrated that patients treated at the time point
of molecular relapse have much better two-year EFS as compared with patients
treated with the same salvage therapy (ATRA, chemotherapy and subsequent
autologous HSCT in MRD-negative cases) at the time of hematological relapse
(92% vs. 44%). Allogeneic HSCT remains a valuable treatment option for
patients after hematological and/or molecular relapse. Graft-versus-leukemia
effect can even overcome molecularly persistent leukemia after intensive
second-line chemotherapy (104). For patients ineligible for allogeneic HSCT,
autologous HSCT can be considered, provided that MRD-negative grafts could
be obtained. Autologous HSCT with MRD-negative grafts might result in long-
term clinical remission in the majority of patients, while MRD-positive autol-
ogous HSCT grafts carry increased risk of subsequent relapse of disease
(94,105). Other treatment options for patients with molecular relapse of APL
include chemotherapy regimens containing gemtuzumab ozogamicin or arsenic
trioxide, which in selected cases might lead to prolonged clinical and molecular
remission (106,107).
Clinical Relevance of MRD Monitoring in Non-M3 AML
The only technique of MRD monitoring available for the vast majority of AML
patients is multiparameter immunophenotyping. Initial flow cytometric studies
already indicated that persistence and/or increase of cells with leukemia-
associated immunophenotypes precede hematological relapse (108). Similar to
what was found for ALL patients, several studies showed strong prognostic
significance of MRD detection for assessing the (early) response to chemo-
therapy in AML (12,109111) In the study by San Miguel et al. (110), the risk
for relapse was significantly increased in adult AML patients bearing equal or
more than 5 10
3
residual cells at the end of induction treatment (67%
incidence of relapse), while only 20% of the cases with less than 5 10
3
residual cells relapsed. At the end of intensification treatment, the threshold
value of 2 10
3
residual cells also identified two distinct groups with relapse
rates of 69% versus 36%. Multivariate analysis showed that this type of MRD
information was independent of the other known prognostic factors like cell
counts at diagnosis, age, or multidrug resistance (110). Extension of these
analyses on 126 patients resulted in an even more refined classification with four
risk groups: very low-risk group (MRD level <10
4
, 8 patients, no relapses),
low-risk group (MRD level 10
4
10
3
, 37 patients, 3-year cumulative relapse
rates of 14%), intermediate-risk group (MRD level <10
3
10
2
, 64 patients,
3-year cumulative relapse rates of 50%), and high-risk group (MRD >10
2
,
17 patients, 3-year relapse rate of 84%) (111). Similarly, Feller and colleagues
clearly showed the prognostic significance of MRD measured continuously after
the first three chemotherapy blocks and before HSCT. High MRD levels after
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each chemotherapy cycle (at a cutoff level of 1% after the first, 0.14% after the
second, and 0.11% after the third cycle) and in peripheral blood stem cell (PB-SC)
products (cutoff > 0.13%) were associated with significantly increased relative risk
of relapse (9). In contrast, two other prospective studies could not demonstrate
significant prognostic value of MRD determined after induction treatment (12,13).
Instead, MRD levels or log difference diagnosis-to-checkpoint after consolida-
tion treatment in adult AML were significantly related with outcome. While
approximately 80% of patients with high MRD levels after consolidation treatment
relapsed, only 18% to 25% of MRD low/negative suffered from disease recurrence;
patients with low/undetectable MRD had also significantly better overall outcome
(12,13). For many AML patients, HSCT is applied as treatment consolidation. The
preliminary results of MRD monitoring in AML patients after HSCT showed an
unequivocal association between the finding of cells with abnormal phenotype and
subsequent relapse. Multiparameter flow cytometry was also shown to be an
effective tool for discrimination between normal blasts transiently present in PB
after HSCT and leukemic blasts heralding medullar relapse (112). Furthermore, in
AML patients subjected to autologous HSCT, detection of leukemia-specific phe-
notypes in harvested BM was associated with treatment failure due to relapse (113).
Accordingly, MRD levels more than or equal to 10
3
in autologous PB-SC harvests
were found to be associated with AML relapse posttransplantation at a median time
of six months (114).
Two studies summarized the utility of MRD detection for pediatric AML
patients (11,115). In one study, the MRD levels at the end of induction treatment
were of clinical significance (11), while the second report emphasized the
prognostic significance of detecting more than 0.5% at any time point after
successful induction treatment (115). Currently, it is clear that detection of MRD
is also of high clinical value for AML patients, but the meaning of MRD differs
per treatment protocol and/or age group and depends on the laboratory approach
and the timing of follow-up sampling. Therefore, clinical significance of MRD
detection in pediatric AML should be further refined by future prospective
studies and standardized per treatment protocol.
The clinical value of the RT-PCR MRD studies in AML patients with
either t(8;21) or inv(16) is less certain. These chromosome aberrations charac-
terize a relatively small subset of AML patients (1020%) with a fairly good
prognosis. Moreover, several RT-PCR studies indicate that AML1-ETO or
CBFB-MYH11 fusion transcripts remain detectable in BM and PB of patients in
long-term remission, while other reports showed disappearance of fusion tran-
scripts in survivors of leukemia in sustained CR (116119). Preliminary results
from several quantitative RT-PCR and/or RQ-PCR studies indicate that gradual
reduction to very low fusion mRNA levels or to PCR-negativity throughout the
disease course is associated with durable clinical remission. In contrast, persis-
tently high MRD levels during treatment are associated with subsequent
hematological relapse (120).
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Clinical Relevance of MRD Monitoring in CML
Clinical Relevance of MRD Monitoring During
Frontline Treatment of CML
Introduction of imatinib has completely changed the treatment strategies in CML
and the role of MRD monitoring in this malignancy (121,122). More than 75% of
patients with newly diagnosed CML reach complete hematological and cytoge-
netic response on imatinib treatment (123). In contrast, only 25% to 30% of
patients were experiencing complete or partial cytogenetic remission with frontline
therapy based on IFN-a (62). Moreover, the degree of tumor reduction is sig-
nificantly higher in patients treated with imatinib as compared with patients on
IFN-a therapy. The International Randomized Study of Interferon and STI571
(IRIS) study demonstrated that after the first year of treatment, at least three-log
reduction of tumor load was achieved in 39% of all patients treated with imatinib,
but in only 2% of those given IFN-a and cytarabine (124). The probability of
progression-free survival for patients with such excellent molecular response to
imatinib was 100%at 60 months (125). Another study identified the BCR-ABL/ABL
ratio less than 0.1% as the MRD threshold associated with continuous remission on
imatinib treatment (126). Sequential MRD monitoring in patients demonstrating
complete response either to imatinib or to IFN-a therapy showed gradual decline
of MRD levels over time with imatinib superior to IFN-a/cytarabine in terms of
the speed and degree of molecular responses (126128). However, the subgroup
of patients in long-term CR who converted into sustained PCR-negativity is
very small (126,127,129). Most patients remain persistently RT-PCR positive
and in patients treated with imatinib, residual BCR-ABL-positive cells are even
more frequent in the CD34-positive stem cell compartment (126,130). These CML
stem cells have clonogenic capacities, as demonstrated by in vitro experiments
(130). MRD levels increasing at least twofold during imatinib treatment are
indicative of the emergence of a resistant CML clone, which is most frequently
characterized by acquired ABL kinase point mutation (131).
Clinical Relevance of MRD Monitoring After Stem
Cell Transplantation in CML
Allogeneic HSCT following initial cytoreductive phase can cure selected CML
patients. Current indications for such treatment include patients age, disease
phase, the degree of histocompatibility between the donor and the recipient, and
initial response to imatinib (122). Most CML patients are RT-PCR positive in the
first six to nine months after allogeneic HSCT, indicating that the conditioning
regimens before HSCT cannot eradicate all leukemic cells (132,133). Sustained
PCR negativity within one year after HSCT is associated with cure, while
patients with PCR positivity after one year or more post HSCT have significantly
greater risk of relapse than patients with PCR negativity (132). With serial
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quantitative PCR analyses, it is possible to identify the group of high-risk
patients that shows increasing MRD levels several months prior to hematological
or cytogenetic relapse (132,134). Patients who remain in remission generally
have decreasing or persistently low MRD levels, with some patients being BCR-
ABL mRNA positive even 10 years after allogeneic HSCT (135).
Quantitative MRD studies in CML enabled the definition of molecular
relapse after allogeneic HSCT, which is equivalent to rising or persistently high
MRD levels (BCR-ABL/ABL ratio of >0.02%) in two consecutive specimens
more than four months after HSCT (136). When donor lymphocyte infusions
(DLIs) are administered at the phase of molecular relapse, the outcome after
immunotherapy is more favorable (137,138). In some responders, such early
treatment results in conversion into sustained PCR negativity (137). Recently, it
was demonstrated that imatinib could be an alternative to DLI for the treatment
of molecular relapse of CML after HSCT (139). Interestingly, 40% of patients
remained in continuous molecular remission after imatinib discontinuation (139).
Clinical Relevance of MRD Monitoring in CLL
Clinical Relevance of MRD Monitoring in Chronic B-Cell Leukemias
B-CLL has a highly variable clinical course and shows heterogeneity in prog-
nosis. Many patients show a rather indolent disease without requiring treatment,
whereas others present with more aggressive forms that often lead to early death.
In the last few years, more insight has emerged into the biological prognostic
factors that determine differences in the disease course. The currently most
relevant parameters associated with unfavorable outcome include cytogenetic
aberrations (17p deletions, 11q aberrations), unmutated IGH VH segments, and
increased CD38 and ZAP70 expression (140).
Together with the increased knowledge on prognostic factors, therapy
results have further improved over the years. Moreover, the introduction of
newer treatment modalities, such as autologous and allogeneic HSCT and
especially therapy with antibodies such as rituximab or alemtuzumab (CD52) or
chemo-immunotherapy (combination of chemotherapy and antibodies), has
resulted in better responses in a significant proportion of B-CLL patients
(141,142). In fact, this has shifted therapeutic goals from palliation to cure, at
least in subsets of patients. With eradication of MRD becoming a realistic goal in
B-CLL, MRD detection has now become a relevant issue (142).
To measure MRD in B-CLL, both qualitative and quantitative approaches
have been applied. Qualitative MRD studies show variable sensitivities and
generally fail to show increased progression-free survival. Quantitative
approaches show higher sensitivities and seem more predictive (19,21). B ottcher
et al. have shown that four-color flow cytometry and RQ-PCR with ASO primers
have largely comparable sensitivities (around 10
4
) (21).
Measurement of MRD is not relevant in conventionally treated B-CLL
patients, because patients still show a relatively high tumor burden, even in CR. In
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contrast, several studies have demonstrated a role for MRD status evaluation in
clinical trials employing newer treatment modalities (142,143). Combination che-
motherapy (e.g., fludarabine and cyclophosphamide) has been shown to result in
MRD negativity in previously untreated patients or relapsed/refractory patients
(144). Furthermore, it has also been demonstrated that achieving an MRD-negative
response after alemtuzumab was the best predictor of survival in relapsed or
refractory B-CLL. Chemoimmunotherapy (e.g., fludarabine, with or without
cyclophosphamide, and rituximab, or fludarabine and alemtuzumab) also leads to
high overall response rates, including MRD-negative patients. Other studies showed
that molecular remission (MRD negativity) could be achieved upon alemtuzumab
consolidation therapy and after autologous and allogeneic HSCT (144,145).
Despite the molecular remissions that can be achieved in the above studies,
it remains to be shown that MRD negativity also correlates with improved and
prolonged survival rates in all these therapeutic approaches. In addition, MRD
detection has entered B-CLL treatment protocols only recently and is often
limited to some B-CLL patient groups. Two important actions are therefore
essential in the coming years. One is the further standardization of sensitive
methods for MRD detection in multicenter laboratory networks. The other is the
implementation of standardized MRD detection in multicenter clinical trials to
prove its clinical application (142). Several initiatives in these directions have
already been taken. One interesting trial in this respect could be the Nordic/
HOVON-68 trial in which fludarabine and cyclophosphamide will be compared
with fludarabine, cyclophosphamide, and alemtuzumab in biologically defined
[mutation status and fluorescence in situ hybridization (FISH) aberrations] high-
risk B-CLL patients. Included in the response criteria of this trial is MRD
detection via ASO-PCR and multiparameter flow cytometry in patients in CR.
Results from this and similar trials should help to reveal the true value of MRD
detection in clinical management in B-CLL patients as well as define the most
sensitive and practical approach for routine MRD analysis.
Clinical Relevance of MRD Monitoring in Chronic T-Cell Leukemias
In chronic leukemias of the T-cell lineage, such as T-cell large granular lym-
phocyte (T-LGL) leukemia, MRD evaluation has never been a goal so far; firstly,
because these diseases are mostly very indolent and secondly, as chronic T-cell
leukemias are relatively rare, large clinical studies with accurate follow-up
analysis have simply not been performed. Recently, a clinical trial for stand-
ardized treatment of T-LGL leukemias has been initiated in Germany in which
methotrexate and fludarabine are being evaluated. In this study, MRD detection
via TCRB/TCRG ASO RQ-PCR and four-color flow cytometry will be imple-
mented and be used to assess the treatment response. Eventually, such clinical
studies should reveal whether there is a true value for MRD detection as prog-
nostic factor or for evaluating treatment response in a relatively indolent disease
as chronic T-cell leukemia.
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Clinical Relevance of MRD Monitoring in NHL
In NHL patients, it is generally not possible to monitor MRD at the original site
of disease. However, in a proportion of lymphomas (particularly high and
intermediate-grade lymphomas) malignant cells can clearly be detected in BM
and PB at diagnosis (Table 2). Immunophenotypic and molecular studies can on
one hand contribute to better recognition of minimal BM infiltration at diagnosis,
undetectable with cytomorphological analyses (Fig. 4). On the other hand, the
presence of MRD in BM and to a lesser extent in PB might be applied as a
surrogate marker for treatment effectiveness (Table 2) (146).
Detection of BM Involvement During Initial Staging of NHL
Immunophenotypic and molecular detection of BM and/or PB involvement has
not yet been routinely implemented into clinical staging of NHL. Nevertheless,
the presence of aberrant clonal cells was demonstrated in BM of most children
Figure 4 An example of BM staging in a patient with NHL using flow cytometric
immunophenotyping. CD19-positive B-cells were gated and the expression of CD38,
SmIgk, and SmIgl were evaluated. In (A) a normal bone marrow sample is shown, with an
approximately equal distribution of SmIgk and SmIgl in the mature B-cells (black). In (B)
a bone marrow sample of a patient with B-NHL is shown; clearly an aberrant SmIgl-
positive B-cell population can be detected. The cells that are strongly positive for CD38
and SmIg-negative are precursor-B-cells (light gray); the cells that are very strongly
positive for CD38 are plasma cells (dark gray).
66 van Dongen et al.
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[4584]
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Minimal Residual Disease 67
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with T-LBL (147). BM involvement detected by BCL2-IGH PCR analysis is a
constant feature, not only of advanced stage FL with t(14;18) but also in patients
with localized stages (148). Using long-distance PCR for t(8;14), BM involve-
ment was found in more than one-third of samples from children with Burkitts
lymphoma, mainly patients without morphological BM involvement but with
advanced stage disease (149).
Immunophenotypic and/or molecular staging might have prognostic sig-
nificance. This was demonstrated for DLBCL, where it was possible to identify a
subset of patients with negative BM histology and positive Ig PCR results
characterized by significantly lower CR rate and significantly poorer overall
survival as compared with patients, in whom both BM histology and PCR results
were negative (150). Further prospective studies should reveal whether detection
of submicroscopic BM involvement with sensitivities of 10
3
to 10
5
would
improve prediction of clinical outcome in lymphoma patients. If so, patients with
detectable high MRD levels in BM at diagnosis might require more intensive
treatment.
Clinical Relevance of MRD Monitoring in FL Patients
The vast majority of clinical studies concentrated on FL with t(14;18) using the
BCL2-IGH fusion gene as DNA target for PCR-based MRD analysis (23,151,152).
Most FL patients harbor lymphoma cells in BM or PB at initial presentation (148).
Moreover, the lower the tumor load in BM at diagnosis (as quantitatively assessed
by RQ-PCR), the higher is the chance for the achievement of a complete clinical
and molecular response (153). For patients treated with conventional chemotherapy,
some studies could show a significant association of MRD-negativity during the
cytotoxic treatment with longer relapse-free survival, with BM being more infor-
mative for MRD monitoring than PB (153156). In contrast, several reports could
not find an obvious correlation between the presence or absence of t(14;18)-positive
cells in the circulation and relapse-free survival (157,158). More recently, the
combination of chemotherapy and treatment with rituximab was shown to produce
durable clinical remission in a subgroup of FL patients accompanied by PCR-
negativity in BM and/or PB (153,159,160). Patients who achieved sustained
molecular remission had significantly better clinical outcome at three-year follow-
up as compared with persistently MRD-positive patients or those who converted
from negativity to MRD-PCR positivity (153,160).
With recently developed high-dose sequential chemotherapy, it is possible to
harvest MRD-PCR-negative autologous BM grafts in most FL patients and MRD-
PCR-negative autologous PB grafts in more than half of the patients (161,162).
Such in vivo purging is even more effective after addition of rituximab as it was
shown that t(14;18)-positive B-cells can be effectively cleared from PB and/or BM
in a subset of patients treated with rituximab as single frontline treatment (163).
Preliminary data suggest that the combination of high-dose chemotherapy and
rituximab can yield MRD-PCR-negative autografts in virtually all patients (164).
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Initial data suggested that patients transplanted with MRD-PCR-negative
autologous grafts showed significantly longer disease-free survival in compari-
son with those whose BM contained residual clonal lymphoma cells after
purging (165). Recent studies also showed that only a small fraction of patients
who received MRD-PCR-negative autologous grafts collected after high-dose
sequential chemotherapy relapsed, while more than half of the patients who were
treated with MRD-positive grafts relapsed (161,162). In contrary, several other
studies could not demonstrate a significant correlation between FL outcome and
PCR status of the reinfused BM (166,167).
RQ-PCR data indicate that a small subset of patients in continuous clinical
remission (after high-dose chemotherapy supported by autologous HSCT)
become MRD-negative (166), while in most patients BCL2-IGH fusion genes
can be persistently found with stable levels within one order of magnitude (166).
Sequential MRD monitoring in a group of patients with advanced stage FL,
treated with high-dose sequential chemotherapy and autografting, showed that
virtually all patients with all follow-up samples PCR negative remained in CR
(168). In contrast, most patients who were persistently MRD positive relapsed.
These combined MRD studies indicate that intensified high-dose sequen-
tial chemotherapy followed by MRD-negative autologous HSCT is a promising
treatment modality in FL patients. Multicenter clinical studies using well-
standardized MRD-PCR techniques are required to establish the quantitative
criteria for molecular remission in FL and the potential applicability of MRD
information for clinical decisionmaking.
Another treatment option for patients with FL might be allogeneic HSCT.
Monitoring of the number of BCL2-IGH-positive cells in BM/PB after allogeneic
HSCT significantly reflects the clinical remission status. This information might
be used to assess the graft-versus-lymphoma effect of allogeneic HSCT and
subsequent DLIs in relapsing patients (169). Recently, allogeneic HSCT with a
reduced-intensity conditioning regimen including rituximab was shown to be
effective in a subset of FL patients. Recent data suggest that such treatment might
result in long-term clinical remission and sustained BCL2-IGH negativity (170).
Clinical Relevance of MRD Monitoring in MCL Patients
Using IGH gene rearrangements and BCL1-IGH fusion genes as DNA targets,
MCL patients were found to be continuously MRD-positive in BM and/or PB
during chemotherapy (171,172). Conventional induction therapy with CHOP-like
regimens does not significantly reduce the tumor load in BM and/or PB when
compared with pretreatment MRD levels (172,173). With more intensive treat-
ment, including a combination of the rituximab and conventional chemotherapy,
approximately one-third of MCL patients can reach an MRD-PCR-negative status
(174). However, in the majority of cases, this conversion to PCR negativity is
transient and this molecular remission is not associated with better progression-
free survival.
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Until recently, it was virtually impossible to harvest autologous MRD-
negative BM or PB-SC grafts in MCL (168,171173). Some mobilization regi-
mens before PB-SC even resulted in increased PB contamination with tumor cells
(172). Also the purging procedure in MCL was generally unsuccessful
(171,172,175). Reinfusion of MRD-positive grafts was frequently associated with
the relapse of MCL (171). Recently, significant progress has been achieved by
in vivo purging of PB CD34-positive autografts using a combination of high-
dose chemotherapy followed by immunotherapy with Rituximab (176). With such
a regimen, it was possible to obtain optimal amounts of PCR-MRD-negative
PB-SC in virtually all MCL patients (176,177) On the other hand, with intensive
conditioning treatment and pretransplant rituximab immunotherapy, MCL cells
conferred with the graft might have no major prognostic impact (173).
Recent data suggested that high-dose chemoradiotherapy followed by
autologous PB-HSCT might result in molecular remission in a subset of MCL
patients (168,173). MRD-PCR negativity after standard debulking chemotherapy
followed by high-dose chemotherapy, rituximab and autografting is strongly
predictive of durable clinical remission (173,177). Allogeneic HSCT represents
another effective treatment regimen for patients with advanced MCL and pre-
liminary data show conversion to MRD negativity after HSCT, which is related
to long-term hematological remission (171,178).
CONCLUSIONSCLINICAL PERSPECTIVES FOR
THE NEXT FIVE YEARS
Despite the complex methodology and the relatively high costs, MRD mon-
itoring is becoming an essential part of modern treatment protocols in different
leukemia and lymphoma categories (Table 3). Many ALL frontline treatment
protocols now use MRD diagnostics during the first three months for treatment
stratification, i.e., for recognition of patients at high risk of relapse who should
receive intensified treatment and for recognition of low-risk patients, who should
be rescued with standard-intensity or reduced chemotherapy. In addition, MRD
information is used for optimal timing of allogeneic HSCT in ALL, with the aim
to reach low or undetectable MRD status with pretransplant chemotherapy.
Within the next five years, it will become clear whether MRD diagnostics can
improve the overall clinical outcome in ALL patients. Hopefully, the treatment
reduction in the large group of MRD-based low-risk patients will not affect the
low relapse rate but will reduce the medical and psychological sequelae.
In current CML and APL treatment protocols, MRD is continuously
measured over a clinically relevant disease-specific time span. Such MRD
monitoring in CML and APL enables identification of patients at high risk of
relapse already at an early stage when the tumor load is relatively low (molecular
relapse) and when restarting of treatment is more effective. Within the next five
years, MRD monitoring will be routinely used to confirm remission in the vast
majority of CML and APL patients. For refractory or relapsing patients, MRD
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monitoring should be helpful to evaluate the effectiveness of alternative treatment
approaches, e.g., testing novel tyrosine kinase inhibitors in CML or combination
of ATRA, chemotherapy, and arsenic trioxide in APL.
The strategy of continuous MRD monitoring for therapy titration might
also be applicable in other subtypes of AML, CLLs, and different subtypes of
NHL. However, large-scale studies are required to fully define the disease-specific
MRD applications or MRD windows for clinically reliable MRD monitoring in
AML. Within next five years, improved strategies of MRD monitoring based on
multiparameter (eight-color) flow cytometric immunophenotyping should be
developed for AML patients and introduced into prospective clinical trials. Such
application of MRD diagnostics should become feasible in high-risk CLL patients
and NHL subsets with frequent BM involvement such as Burkitts lymphoma,
T-LBL, FL, and/or MCL.
Furthermore, sensitive MRD techniques will be more extensively applied
for other specific diagnostics aims, such as detection of minimal central nervous
system involvement in ALL, early diagnosis of ocular lymphomas, early diag-
nosis of leukemia/lymphoma in patients with unexplained cytopenias, improved
staging of lymphomas, and for assessing the effectiveness of newly available
cytotoxic or immunotherapeutic drugs.
Table 3 Prognostic Value and Clinical Applicability of MRD Detection in Leukemias
and Lymphomas
Type of MRD application
Disease
category
Early response
to frontline
treatment
Continuous
monitoring for
therapy titration
MRD
assessment
before HSCT
MRD
assessment after
HSCT
ALL
Chronic B-cell
leukemias
*
B-NHL *
Chronic T-cell
leukemias
*
T-NHL *
APL
AML (excl. APL)
CML
, value of MRD detection proven in large prospective studies
, potentially clinically relevant (e.g., in a subset of patients) but not yet proven by large pro-
spective studies
, MRD results are statistically significant but their clinical implication is not yet established
*, only relevant for patients treated with more aggressive protocols and/or including CD20 antibody
, MRD detection has no additional value as compared with conventional cytomorphological
techniques.
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Finally, it should be noted that accurate and sensitive MRD diagnostics is a
technically demanding and complex form of diagnostics that requires extensive
knowledge and experience as well as regular internal and external quality con-
trol. This implies that standardization, internationally accepted guidelines for
data analysis, and regular quality control rounds are essential for providing MRD
results that are comparable between different MRD laboratories and different
treatment protocols.
REFERENCES
1. Lucio P, Parreira A, van den Beemd MW, et al. Flow cytometric analysis of normal
B cell differentiation: a frame of reference for the detection of minimal residual
disease in precursor-B- ALL. Leukemia 1999; 13:419427.
2. Weir EG, Cowan K, LeBeau P, et al. A limited antibody panel can distinguish
B-precursor acute lymphoblastic leukemia from normal B precursors with four color
flow cytometry: implications for residual disease detection. Leukemia 1999; 13:558567.
3. Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal
residual disease in children with acute lymphoblastic leukaemia. Lancet 1998;
351:550554.
4. Ciudad J, San Miguel JF, Lopez-Berges MC, et al. Prognostic value of immuno-
phenotypic detection of minimal residual disease in acute lymphoblastic leukemia.
J Clin Oncol 1998; 16:37743781.
5. van Wering ER, van der Linden-Schrever BE, Szczepanski T, et al. Regenerating
normal B-cell precursors during and after treatment of acute lymphoblastic
leukaemia: implications for monitoring of minimal residual disease. Br J Haematol
2000; 110:139146.
6. van Lochem EG, Wiegers YM, van den Beemd R, et al. Regeneration pattern of
precursor-B-cells in bone marrow of acute lymphoblastic leukemia patients depends
on the type of preceding chemotherapy. Leukemia 2000; 14:688695.
7. Porwit-MacDonald A, Bjorklund E, Lucio P, et al. BIOMED-1 concerted action
report: flow cytometric characterization of CD7 cell subsets in normal bone
marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic
leukemia (T-ALL). Leukemia 2000; 14:816825.
8. Dworzak MN, Froschl G, Printz D, et al. CD99 expression in T-lineage ALL:
implications for flow cytometric detection of minimal residual disease. Leukemia
2004; 18:703708.
9. Feller N, van der Pol MA, van Stijn A, et al. MRD parameters using immuno-
phenotypic detection methods are highly reliable in predicting survival in acute
myeloid leukaemia. Leukemia 2004; 18:13801390.
10. Langebrake C, Creutzig U, Dworzak M, et al. Residual disease monitoring in
childhood acute myeloid leukemia by multiparameter flow cytometry: the MRD-
AML-BFM Study Group. J Clin Oncol 2006; 24:36863692.
11. Coustan-Smith E, Ribeiro RC, Rubnitz JE, et al. Clinical significance of residual
disease during treatment in childhood acute myeloid leukaemia. Br J Haematol
2003; 123:243252.
12. Kern W, Voskova D, Schoch C, et al. Determination of relapse risk based on
assessment of minimal residual disease during complete remission by multiparameter
72 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;
104:30783085.
13. 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;
96:39483952.
14. Macedo A, Orfao A, Gonzalez M, et al. Immunological detection of blast cell sub-
populations in acute myeloblastic leukemia at diagnosis: implications for minimal
residual disease studies. Leukemia 1995; 9:993998.
15. Coustan-Smith E, Sancho J, Hancock ML, et al. Clinical importance of minimal residual
disease in childhood acute lymphoblastic leukemia. Blood 2000; 96:26912696.
16. Lucio P, Gaipa G, van Lochem EG, et al. BIOMED-1 concerted action report: flow
cytometric immunophenotyping of precursor B-ALL with standardized triple-
stainings. BIOMED-1 Concerted action investigation of minimal residual disease in
acute leukemia: International Standardization and Clinical Evaluation. Leukemia
2001; 15:11851192.
17. Dworzak MN, Froschl G, Printz D, et al. Prognostic significance and modalities of flow
cytometric minimal residual disease detection in childhood acute lymphoblastic
leukemia. Blood 2002; 99:19521958.
18. Chen JS, Coustan-Smith E, Suzuki T, et al. Identification of novel markers for
monitoring minimal residual disease in acute lymphoblastic leukemia. Blood 2001;
97:21152120.
19. Rawstron AC, Kennedy B, Evans PA, et al. Quantitation of minimal disease levels in
chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the
prediction of outcome and can be used to optimize therapy. Blood 2001; 98:2935.
20. Esteve J, Villamor N, Colomer D, et al. Stem cell transplantation for chronic
lymphocytic leukemia: different outcome after autologous and allogeneic transplantation
and correlation with minimal residual disease status. Leukemia 2001; 15:445451.
21. B ottcher S, Ritgen M, Pott C, et al. Comparative analysis of minimal residual disease
detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH
PCR in CLL after allogeneic and autologous stem cell transplantation. Leukemia
2004; 18:16371645.
22. Rawstron AC, de Tute R, Jack AS, et al. Flow cytometric protein expression profiling
as a systematic approach for developing disease-specific assays: identification of a
chronic lymphocytic leukaemia-specific assay for use in rituximab-containing regimens.
Leukemia 2006; 20:21022110.
23. Szczepanski T, van Dongen JJM. Detection of minimal residual disease. In: Henderson
ES, Lister TA, Greaves MF, eds. Leukemia. Philadelphia, PA: WB Saunders, 2002:
249283.
24. Hermine O, Haioun C, Lepage E, et al. Prognostic significance of bcl-2 protein
expression in aggressive non-Hodgkins lymphoma. Blood 1996; 87:265272.
25. De Boer CJ, Schuuring E, Dreef E, et al. Cyclin D1 protein analysis in the diagnosis
of mantle cell lymphoma. Blood 1995; 86:27152723.
26. Langerak AW, van den Beemd R, Wolvers-Tettero ILM, et al. Molecular and flow
cytometric analysis of the Vbeta repertoire for clonality assessment in mature
TCRalphabeta T-cell proliferations. Blood 2001; 98:165173.
27. Langerak AW, Wolvers-Tettero ILM, van den Beemd MWM, et al. Immunophe-
notypic and immunogenotypic characteristics of TCR T cell acute lymphoblastic
leukemia. Leukemia 1999; 13:206214.
Minimal Residual Disease 73
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
28. Sandberg Y, Almeida J, Gonzalez M, et al. Clonal TCRab Large Granular
Lymphocyte proliferations reflect the spectrum of normal TCRab T-cells in
peripheral blood. Leukemia 2006; 20:505513.
29. Borst J, Wicherink A, van Dongen JJM, et al. Non-random expression of T cell
receptor gamma and delta variable gene segments in functional T lymphocyte
clones from human peripheral blood. Eur J Immunol 1989; 19:15591568.
30. Pulford K, Lamant L, Morris SW, et al. Detection of anaplastic lymphoma kinase
(ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and
neoplastic cells with the monoclonal antibody ALK1. Blood 1997; 89:13941404.
31. Macedo A, San Miguel JF, Vidriales MB, et al. Phenotypic changes in acute
myeloid leukaemia: implications in the detection of minimal residual disease. J Clin
Pathol 1996; 49:1518.
32. van Wering ER, Beishuizen A, Roeffen ET, et al. Immunophenotypic changes
between diagnosis and relapse in childhood acute lymphoblastic leukemia. Leukemia
1995; 9:15231533.
33. Langebrake C, Brinkmann I, Teigler-Schlegel A, et al. Immunophenotypic differ-
ences between diagnosis and relapse in childhood AML: implications for MRD
monitoring. Cytometry B Clin Cytom 2005; 63:19.
34. Campana D, CoustanSmith E. Detection of minimal residual disease in acute
leukemia by flow cytometry. Cytometry 1999; 38:139152.
35. Gaipa G, Basso G, Maglia O, et al. Drug-induced immunophenotypic modulation in
childhood ALL: implications for minimal residual disease detection. Leukemia
2005; 19:4956.
36. van der Sluijs-Gelling AJ, van der Velden VHJ, Roeffen ETJM, et al. Immuno-
phenotypic modulation in childhood precursor-B-ALL can be mimicked in vitro and
is related to the induction of cell death. Leukemia 2005; 19:18451847.
37. van Dongen JJM, Langerak AW, Bruggemann M, et al. Design and standardization
of PCR primers and protocols for detection of clonal immunoglobulin and T-cell
receptor gene recombinations in suspect lymphoproliferations: Report of the
BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17:22572317.
38. Boeckx N, Willemse MJ, 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; 16:368375.
39. van der Velden VHJ, Hochhaus A, Cazzaniga G, et al. Detection of minimal
residual disease in hematologic malignancies by real-time quantitative PCR: prin-
ciples, approaches, and laboratory aspects. Leukemia 2003; 17:10131034.
40. Brumpt C, Delabessae E, Beldjord K, et al. The incidence of clonal T-cell receptor
rearrangements in B-cell precursor acute lymphoblastic leukemia varies with age
and genotype. Blood 2000; 96:22542261.
41. van der Velden VHJ, Szczepanski T, Wijkhuijs JM, et al. Age-related patterns of
immunoglobulin and T-cell receptor gene rearrangements in precursor-B-ALL:
implications for detection of minimal residual disease. Leukemia 2003; 17:18341844.
42. Hubner S, Cazzaniga G, Flohr T, et al. High incidence and unique features of
antigen receptor gene rearrangements in TEL-AML1-positive leukemias. Leukemia
2004; 18:8491.
43. Jansen MW, Corral L, van der Velden VHJ, et al. Immunobiological diversity in
infant acute lymphoblastic leukemia is related to the occurrence and type of MLL
gene rearrangement. Leukemia 2007; 21,633641.
74 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
44. van der Velden VHJ, de Bie M, van Wering ER, et al. Immunoglobulin light chain
gene rearrangements in precursor-B-acute lymphoblastic leukemia: characteristics
and applicability for the detection of minimal residual disease. Haematologica
2006; 91:679682.
45. Szczepanski T, van der Velden VHJ, Raff T, et al. Comparative analysis of T-cell
receptor gene rearrangements at diagnosis and relapse of T-cell acute lymphoblastic
leukemia (T-ALL) shows high stability of clonal markers for monitoring of minimal
residual disease and reveals the occurrence of second T-ALL. Leukemia 2003;
17:21492156.
46. Szczepanski T, Willemse MJ, 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; 99:23152323.
47. Li A, Zhou J, Zuckerman D, et al. Sequence analysis of clonal immunoglobulin and
T-cell receptor gene rearrangements in children with acute lymphoblastic leukemia
at diagnosis and at relapse: implications for pathogenesis and for the clinical utility
of PCR-based methods of minimal residual disease detection. Blood 2003;
102:45204526.
48. Bertoni F, Zucca E, Genini D, et al. Immunoglobulin light chain kappa deletion
rearrangement as a marker of clonality in mantle cell lymphoma. Leuk Lymphoma
1999; 36:147150.
49. Hoogeveen-Westerveld M, Hupkes PE, Doekharan D, et al. Rearranged immunoglo-
bulin light chain genes as minimal residual disease markers in intermediate- and
high-grade malignant B cell non-Hodgkins lymphoma. Leukemia 1998; 12:
18101816.
50. Evans PA, Pott C, Groenen PJ, et al. Significantly improved PCR-based clonality
testing in B-cell malignancies by use of multiple immunoglobulin gene targets.
Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 2007;
21:207214.
51. Langerak AW, Wolvers-Tettero ILM, van Dongen JJM. Detection of T cell receptor
beta (TCRB) gene rearrangement patterns in T cell malignancies by Southern blot
analysis. Leukemia 1999; 13:965974.
52. Bruggemann M, White H, Gaulard P, et al. Powerful strategy for polymerase chain
reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2
Concerted Action BHM4 CT98-3936. Leukemia 2007; 21:215221.
53. Uchiyama M, Maesawa C, Yashima-Abo A, et al. Short consensus probes with
3
0
-minor groove binder of the immunoglobulin heavy-chain gene for real-time
quantitative PCR in B-cell non-Hodgkin lymphomas. Lab Invest 2004; 84:932936.
54. van der Velden VHJ, Panzer-Gr umayer ER, Cazzaniga G, et al. Optimization of
PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic
leukemia in a multi-center setting. Leukemia 2007; 21:706713.
55. van der Velden VHJ, Cazzaniga G, Schrauder A, et al. Analysis of minimal residual
disease by Ig/TCR gene rearrangements: Guidelines for interpretation of real-time
quantitative PCR data. Leukemia 2007; 21:604611.
56. van der Velden VHJ, Wijkhuijs JM, Jacobs DCH, et al. T cell receptor gamma gene
rearrangements as targets for detection of minimal residual disease in acute lym-
phoblastic leukemia by real-time quantitative PCR analysis. Leukemia 2002;
16:13721380.
Minimal Residual Disease 75
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
57. van Wering ER, van der Linden-Schrever BEM, van der Velden VHJ, et al. T
lymphocytes in bone marrow samples of children with acute lymphoblastic leukemia
during and after chemotherapy might hamper PCR-based minimal residual disease
studies. Leukemia 2001; 15:10311033.
58. Burmeister T, Marschalek R, Schneider B, et al. Monitoring minimal residual
disease by quantification of genomic chromosomal breakpoint sequences in acute
leukemias with MLL aberrations. Leukemia 2006; 20:451457.
59. van der Velden VHJ, Schoch C, Langerak AW, et al. Low frequency of reverse
transcription polymerase chain reaction-detectable chromosome aberrations in
relapsed acute myeloid leukaemia: implications for detection of minimal residual
disease. Br J Haematol 2001; 113:10821083.
60. Gabert J, Beillard E, van der Velden VHJ, et al. Standardization and quality control
studies of real-time quantitative reverse transcriptase polymerase chain reaction of
fusion gene transcripts for residual disease detection in leukemia: a Europe Against
Cancer program. Leukemia 2003; 17:23182357.
61. Beillard E, Pallisgaard N, van der Velden VH, et al. Evaluation of candidate control
genes for diagnosis and residual disease detection in leukemic patients using real-
time quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR): a
Europe against cancer program. Leukemia 2003; 17:24742486.
62. Hochhaus A, Lin F, Reiter A, et al. Quantification of residual disease in chronic
myelogenous leukemia patients on interferon-alpha therapy by competitive polymerase
chain reaction. Blood 1996; 87:15491555.
63. Branford S, Cross NC, Hochhaus A, et al. Rationale for the recommendations for
harmonizing current methodology for detecting BCR-ABL transcripts in patients
with chronic myeloid leukaemia. Leukemia 2006; 20:19251930.
64. Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to
treatment with tyrosine kinase inhibitors: review and recommendations for harmo-
nizing current methodology for detecting BCR-ABL transcripts and kinase domain
mutations and for expressing results. Blood 2006; 108:2837.
65. Gribben JG. Monitoring disease in lymphoma and CLL patients using molecular
techniques. Best Pract Res Clin Haematol 2002; 15:179195.
66. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a
nucleolar protein gene, NPM, in non-Hodgkins lymphoma. Science 1994; 263:
12811284.
67. Foroni L, Harrison CJ, Hoffbrand AV, et al. Investigation of minimal residual disease
in childhood and adult acute lymphoblastic leukaemia by molecular analysis. Br J
Haematol 1999; 105:724.
68. Szczepanski T, Flohr T, van der Velden VHJ, et al. Molecular monitoring of
residual disease using antigen receptor genes in childhood acute lymphoblastic
leukaemia. Best Pract Res Clin Haematol 2002; 15:3757.
69. van Dongen JJM, Seriu T, Panzer-Grumayer ER, et al. Prognostic value of minimal
residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998; 352:
17311738.
70. Nyvold C, Madsen HO, Ryder LP, 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; 99:12531258.
76 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
71. Cave H, van der Werff ten Bosch J, Suciu S, et al. Clinical significance of minimal
residual disease in childhood acute lymphoblastic leukemia. N Engl J Med 1998;
339:591598.
72. Willemse MJ, 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; 99:43864393.
73. Panzer-Grumayer ER, Schneider M, Panzer S, et al. Rapid molecular response
during early induction chemotherapy predicts a good outcome in childhood acute
lymphoblastic leukemia. Blood 2000; 95:790794.
74. Coustan-Smith E, Sancho J, Behm FG, et al. Prognostic importance of measuring
early clearance of leukemic cells by flow cytometry in childhood acute lympho-
blastic leukemia. Blood 2002; 100:5258.
75. Eckert C, Biondi A, Seeger K, et al. Prognostic value of minimal residual disease in
relapsed childhood acute lymphoblastic leukaemia. Lancet 2001; 358:12391241.
76. 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; 18:499504.
77. Knechtli CJ, Goulden NJ, Hancock JP, 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; 92:40724079.
78. van der Velden VHJ, Joosten SA, Willemse MJ, 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;
15:14851487.
79. Uzunel M, Mattsson J, Jaksch M, et al. The significance of graft-versus-host disease
and pretransplantation minimal residual disease status to outcome after allogeneic
stem cell transplantation in patients with acute lymphoblastic leukemia. Blood
2001; 98:19821984.
80. 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; 16:16681672.
81. Sanchez J, Serrano J, Gomez P, et al. Clinical value of immunological monitoring of
minimal residual disease in acute lymphoblastic leukaemia after allogeneic trans-
plantation. Br J Haematol 2002; 116:686694.
82. Krejci O, van der Velden VHJ, Bader P, et al. Level of minimal residual disease
prior to haematopoietic stem cell transplantation predicts prognosis in paediatric
patients with acute lymphoblastic leukaemia: a report of the Pre-BMT MRD Study
Group. Bone Marrow Transplant 2003; 32:849851.
83. Knechtli CJ, Goulden NJ, Hancock JP, 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; 102:860871.
84. Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med
2006; 354:166178.
85. Brisco MJ, Hughes E, Neoh SH, et al. Relationship between minimal residual disease
and outcome in adult acute lymphoblastic leukemia. Blood 1996; 87: 52515256.
Minimal Residual Disease 77
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
86. Foroni L, Coyle LA, Papaioannou M, et al. Molecular detection of minimal residual
disease in adult and childhood acute lymphoblastic leukaemia reveals differences in
treatment response. Leukemia 1997; 11:17321741.
87. Bruggemann M, Raff T, Flohr T, et al. Clinical significance of minimal residual
disease quantification in adult patients with standard-risk acute lymphoblastic
leukemia. Blood 2006; 107:11161123.
88. Raff T, Gokbuget N, Luschen S, et al. Molecular relapse in adult standard-risk ALL
patients detected by prospective MRD monitoring during and after maintenance
treatment: data from the GMALL 06/99 and 07/03 trials. Blood 2007; 109:910915.
89. Vidriales MB, Perez JJ, Lopez-Berges MC, et al. Minimal residual disease in
adolescent (older than 14 years) and adult acute lymphoblastic leukemias: early
immunophenotypic evaluation has high clinical value. Blood 2003; 101:46954700.
90. Yanada M, Takeuchi J, Sugiura I, et al. High complete remission rate and promising
outcome by combination of imatinib and chemotherapy for newly diagnosed BCR-
ABL-positive acute lymphoblastic leukemia: a phase II study by the Japan Adult
Leukemia Study Group. J Clin Oncol 2006; 24:460466.
91. Scheuring UJ, 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; 101:8590.
92. Wassmann B, Pfeifer H, Stadler M, et al. Early molecular response to post-
transplantation imatinib determines outcome in MRD Philadelphia-positive acute
lymphoblastic leukemia (Ph ALL). Blood 2005; 106:458463.
93. Grimwade D, Lo Coco F. Acute promyelocytic leukemia: a model for the role of
molecular diagnosis and residual disease monitoring in directing treatment approach
in acute myeloid leukemia. Leukemia 2002; 16:19591973.
94. Sanz MA, Lo Coco F. Standard practice and controversial issues in front-line
therapy of acute promyelocytic leukemia. Haematologica 2005; 90:840845.
95. Lo Coco F, Diverio D, Avvisati G, et al. Therapy of molecular relapse in acute
promyelocytic leukemia. Blood 1999; 94:22252229.
96. Miller WH Jr., Kakizuka A, Frankel SR, 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; 89:26942698.
97. Fukutani H, Naoe T, Ohno R, et al. Prognostic significance of the RT-PCR assay
of PML-RARA transcripts in acute promyelocytic leukemia. Leukemia 1995; 9:
588593.
98. 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
dellAdulto and Associazione Italiana di Ematologia ed Oncologia Pediatrica
Cooperative Groups. Blood 1997; 90:10141021.
99. Burnett AK, 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; 93:41314143.
100. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracycline-
based consolidation results in high antileukemic efficacy and reduced toxicity in newly
78 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
diagnosed PML/RARalpha-positive acute promyelocytic leukemia. PETHEMA group.
Blood 1999; 94:30153021.
101. 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 GIMEMA-
AIEOP multicenter AIDA trial. GIMEMA-AIEOP Multicenter AIDA Trial.
Blood 1998; 92:784789.
102. Gallagher RE, Yeap BY, Bi W, et al. Quantitative real-time RT-PCR analysis of
PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic
significance in adult patients from intergroup protocol 0129. Blood 2003; 101:
25212528.
103. Lo Coco F, Diverio D, Falini B, et al. Genetic diagnosis and molecular monitoring
in the management of acute promyelocytic leukemia. Blood 1999; 94:1222.
104. Lo-Coco F, Romano A, Mengarelli A, et al. Allogeneic stem cell transplantation for
advanced acute promyelocytic leukemia: results in patients treated in second
molecular remission or with molecularly persistent disease. Leukemia 2003; 17:
19301933.
105. Meloni G, Diverio D, Vignetti M, et al. Autologous bone marrow transplantation for
acute promyelocytic leukemia in second remission: prognostic relevance of pre-
transplant minimal residual disease assessment by reverse-transcription polymerase
chain reaction of the PML/RAR alpha fusion gene. Blood 1997; 90:13211325.
106. Lazo G, Kantarjian H, Estey E, et al. Use of arsenic trioxide (As2O3) in the treatment
of patients with acute promyelocytic leukemia: the MD Anderson experience. Cancer
2003; 97:22182224.
107. Lo-Coco F, Cimino G, Breccia M, et al. Gemtuzumab ozogamicin (Mylotarg) as a
single agent for molecularly relapsed acute promyelocytic leukemia. Blood 2004;
104:19951999.
108. Drach J, Drach D, Glassl H, et al. Flow cytometric determination of atypical antigen
expression in acute leukemia for the study of minimal residual disease. Cytometry
1992; 13:893901.
109. Reading CL, Estey EH, Huh YO, et al. Expression of unusual immunophenotype
combinations in acute myelogenous leukemia. Blood 1993; 81:30833090.
110. San Miguel JF, 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; 90:24652470.
111. San Miguel JF, Vidriales MB, 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; 98:17461751.
112. Shulman HM, Wells D, Gooley T, et al. The biologic significance of rare peripheral
blasts after hematopoietic cell transplantation is predicted by multidimensional flow
cytometry. Am J Clin Pathol 1999; 112:513523.
113. Campana D. Applications of cytometry to study acute leukemia: in vitro determi-
nation of drug sensitivity and detection of minimal residual disease. Cytometry
(Communications in Clinical Cytometry) 1994; 18:6874.
114. 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; 13:12271234.
Minimal Residual Disease 79
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
115. Sievers EL, Lange BJ, Alonzo TA, et al. Immunophenotypic evidence of leukemia
after induction therapy predicts relapse: results from a prospective Childrens
Cancer Group study of 252 patients with acute myeloid leukemia. Blood 2003;
101:33983406.
116. Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21 translocation in patients
with acute myeloid leukemia type M2 in long-term remission. Blood 1993; 82:
712715.
117. Satake N, Maseki N, Kozu T, et al. Disappearance of AML1-MTG8(ETO) fusion
transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission.
Br J Haematol 1995; 91:892898.
118. Hebert J, Cayuela JM, Daniel MT, et al. Detection of minimal residual disease in
acute myelomonocytic leukemia with abnormal marrow eosinophils by nested
polymerase chain reaction with allele specific amplification. Blood 1994; 84:
22912296.
119. Tobal K, Johnson PR, Saunders MJ, et al. Detection of CBFB/MYH11 transcripts in
patients with inversion and other abnormalities of chromosome 16 at presentation
and remission. Br J Haematol 1995; 91:104108.
120. Krauter J, Wattjes MP, Nagel S, et al. Real-time RT-PCR for the detection and
quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients.
Br J Haematol 1999; 107:8085.
121. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of
the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;
344:10311037.
122. Goldman JM, Melo JV. Chronic myeloid leukemia: advances in biology and new
approaches to treatment. N Engl J Med 2003; 349:14511464.
123. OBrien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and
low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia.
N Engl J Med 2003; 348:9941004.
124. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to
imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid
leukemia. N Engl J Med 2003; 349:14231432.
125. Druker BJ, Guilhot F, OBrien SG, et al. Five-year follow-up of patients receiving
imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355:24082417.
126. Paschka P, Muller MC, Merx K, et al. Molecular monitoring of response to imatinib
(Glivec) in CML patients pretreated with interferon alpha. Low levels of residual
disease are associated with continuous remission. Leukemia 2003; 17:16871694.
127. Hochhaus A, Reiter A, Saussele S, et al. Molecular heterogeneity in complete
cytogenetic responders after interferon-alpha therapy for chronic myelogenous
leukemia: low levels of minimal residual disease are associated with continuing
remission. German CML Study Group and the UK MRC CML Study Group. Blood
2000; 95:6266.
128. Muller MC, Gattermann N, Lahaye T, et al. Dynamics of BCR-ABL mRNA
expression in first-line therapy of chronic myelogenous leukemia patients with
imatinib or interferon alpha/ara-C. Leukemia 2003; 17:23922400.
129. Kurzrock R, Estrov Z, Kantarjian H, et al. Conversion of interferon-induced, long-term
cytogenetic remissions in chronic myelogenous leukemia to polymerase chain reaction
negativity. J Clin Oncol 1998; 16:15261531.
80 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
130. Bhatia R, Holtz M, Niu N, et al. Persistence of malignant hematopoietic progenitors in
chronic myelogenous leukemia patients in complete cytogenetic remission following
imatinib mesylate treatment. Blood 2003; 101:47014707.
131. Branford S, Rudzki Z, Parkinson I, et al. Real-time quantitative PCR analysis can be
used as a primary screen to identify patients with CML treated with imatinib who
have BCR-ABL kinase domain mutations. Blood 2004; 104:29262932.
132. Cross NC, Hughes TP, Feng L, et al. Minimal residual disease after allogeneic bone
marrow transplantation for chronic myeloid leukaemia in first chronic phase: corre-
lations with acute graft-versus-host disease and relapse. Br J Haematol 1993; 84:
6774.
133. Hochhaus A, Weisser A, La Rosee P, et al. Detection and quantification of residual
disease in chronic myelogenous leukemia. Leukemia 2000; 14:9981005.
134. Lion T, Henn T, Gaiger A, et al. Early detection of relapse after bone marrow
transplantation in patients with chronic myelogenous leukaemia. Lancet 1993; 341:
275276.
135. van Rhee F, Lin F, Cross NC, et al. Detection of residual leukaemia more than 10 years
after allogeneic bone marrow transplantation for chronic myelogenous leukaemia. Bone
Marrow Transplant 1994; 14:609612.
136. Cross NC. Minimal residual disease in chronic myeloid leukaemia. Hematol Cell
Ther 1998; 40:224228.
137. van Rhee F, Lin F, Cullis JO, et al. Relapse of chronic myeloid leukemia after
allogeneic bone marrow transplant: the case for giving donor leukocyte transfusions
before the onset of hematologic relapse. Blood 1994; 83:33773383.
138. Raanani P, Dazzi F, Sohal J, et al. The rate and kinetics of molecular response to
donor leucocyte transfusions in chronic myeloid leukaemia patients treated for
relapse after allogeneic bone marrow transplantation. Br J Haematol 1997; 99:
945950.
139. Hess G, Bunjes D, Siegert W, et al. Sustained complete molecular remissions after
treatment with imatinib-mesylate in patients with failure after allogeneic stem cell
transplantation for chronic myelogenous leukemia: results of a prospective phase II
open-label multicenter study. J Clin Oncol 2005; 23:75837593.
140. Krober A, Seiler T, Benner A, et al. V(H) mutation status, CD38 expression level,
genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002;
100:14101416.
141. Shanafelt TD, Call TG. Current approach to diagnosis and management of chronic
lymphocytic leukemia. Mayo Clin Proc 2004; 79:388398.
142. Nabhan C, Coutre S, Hillmen P. Minimal residual disease in chronic lymphocytic
leukaemia: is it ready for primetime? Br J Haematol 2007; 136:379392.
143. Binet JL, Caligaris-Cappio F, Catovsky D, et al. Perspectives on the use of new
diagnostic tools in the treatment of chronic lymphocytic leukemia. Blood 2006;
107:859861.
144. Moreton P, Kennedy B, Lucas G, et al. Eradication of minimal residual disease in
B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with
prolonged survival. J Clin Oncol 2005; 23:29712979.
145. Montillo M, Tedeschi A, Miqueleiz S, et al. Alemtuzumab as consolidation after a
response to fludarabine is effective in purging residual disease in patients with
chronic lymphocytic leukemia. J Clin Oncol 2006; 24:23372342.
Minimal Residual Disease 81
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0003_O.3d] [10/4/08/10:9:53]
[4584]
146. van Dongen JJM, Langerak AW, Szczepanski T, et al. Molecular monitoring of
lymphoma. In: Canellos GP, Lister TA, Young BD, eds. The Lymphomas. Phila-
delphia, PA: Saunders Elsevier, 2006:83109.
147. van Dongen JJM, Hooijkaas H, Adriaansen HJ, et al. Detection of minimal residual
acute lymphoblastic leukemia by immunological marker analysis: possibilities and
limitations. In: Hagenbeek A, L owenberg B, eds. Minimal Residual Disease in Acute
Leukemia. Dordrecht, The Netherlands: M Nijhoff Publishers, 1986:113133.
148. Gribben JG, Freedman A, Woo SD, et al. All advanced stage non-Hodgkins
lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have
residual cells containing the bcl-2 rearrangement at evaluation and after treatment.
Blood 1991; 78:32753280.
149. Mussolin L, Basso K, Pillon M, et al. Prospective analysis of minimal bone marrow
infiltration in pediatric Burkitts lymphomas by long-distance polymerase chain
reaction for t(8;14) (q24;q32). Leukemia 2003; 17:585589.
150. Mitterbauer-Hohendanner G, Mannhalter C, Winkler K, et al. Prognostic significance
of molecular staging by PCR-amplification of immunoglobulin gene rearrangements
in diffuse large B-cell lymphoma (DLBCL). Leukemia 2004; 18:11021107.
151. Corradini P, Ladetto M, Pileri A, et al. Clinical relevance of minimal residual disease
monitoring in non-Hodgkins lymphomas: a critical reappraisal of molecular
strategies. Leukemia 1999; 13:16911695.
152. Buckstein R, Pennell N, Berinstein NL. What is remission in follicular lymphoma
and what is its relevance? Best Pract Res Clin Haematol 2005; 18:2756.
153. Rambaldi A, Carlotti E, Oldani E, et al. Quantitative PCR of bone marrow BCL2/
IgH cells at diagnosis predicts treatment response and long-term outcome in
follicular non-Hodgkin lymphoma. Blood 2005; 105:34283433.
154. Lopez-Guillermo A, Cabanillas F, McLaughlin P, et al. The clinical significance of
molecular response in indolent follicular lymphomas. Blood 1998; 91:29552960.
155. Gribben JG, Neuberg D, Barber M, et al. Detection of residual lymphoma cells by
polymerase chain reaction in peripheral blood is significantly less predictive for
relapse than detection in bone marrow. Blood 1994; 83:38003807.
156. Fernandez-Ruiz E, Cabrerizo M, Ortega M, et al. High molecular response rate and
clinical correlation in patients with follicular lymphoma treated with cyclophosphamide-
vincristine-prednisone plus interferon alpha 2b. Clin Cancer Res 2003; 9:24972503.
157. Lambrechts AC, Hupkes PE, Dorssers LC, et al. Clinical significance of t(14;18)-
positive cells in the circulation of patients with stage III or IV follicular non-
Hodgkins lymphoma during first remission. J Clin Oncol 1994; 12:15411546.
158. Mandigers CM, Meijerink JP, Mensink EJ, et al. Lack of correlation between
numbers of circulating t(14;18)-positive cells and response to first-line treatment in
follicular lymphoma. Blood 2001; 98:940944.
159. Czuczman MS, Grillo-Lopez AJ, White CA, et al. Treatment of patients with low-grade
B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody
and CHOP chemotherapy. J Clin Oncol 1999; 17:268276.
160. Rambaldi A, Lazzari M, Manzoni C, et al. Monitoring of minimal residual disease
after CHOP and rituximab in previously untreated patients with follicular lymphoma.
Blood 2002; 99:856862.
161. Corradini P, Astolfi M, Cherasco C, et al. Molecular monitoring of minimal residual
disease in follicular and mantle cell non-Hodgkins lymphomas treated with high-dose
82 van Dongen et al.
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
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[4584]
chemotherapy and peripheral blood progenitor cell autografting. Blood 1997; 89:
724731.
162. Ladetto M, Corradini P, Vallet S, et al. High rate of clinical and molecular remissions
in follicular lymphoma patients receiving high-dose sequential chemotherapy and
autografting at diagnosis: a multicenter, prospective study by the Gruppo Italiano
Trapianto Midollo Osseo (GITMO). Blood 2002; 100:15591565.
163. Colombat P, Salles G, Brousse N, et al. Rituximab (anti-CD20 monoclonal anti-
body) as single first-line therapy for patients with follicular lymphoma with a low
tumor burden: clinical and molecular evaluation. Blood 2001; 97:101106.
164. Galimberti S, Guerrini F, Morabito F, et al. Quantitative molecular evaluation in
autotransplant programs for follicular lymphoma: efficacy of in vivo purging by
Rituximab. Bone Marrow Transplant 2003; 32:5763.
165. Gribben JG, Freedman AS, 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; 325:15251533.
166. Hirt C, Dolken G. Quantitative detection of t(14;18)-positive cells in patients with
follicular lymphoma before and after autologous bone marrow transplantation. Bone
Marrow Transplant 2000; 25:419426.
167. Apostolidis J, Gupta RK, Grenzelias D, et al. High-dose therapy with autologous bone
marrow support as consolidation of remission in follicular lymphoma: long-term
clinical and molecular follow-up. J Clin Oncol 2000; 18:527536.
168. Corradini P, Ladetto M, Zallio F, et al. Long-term follow-up of indolent lymphoma
patients treated with high-dose sequential chemotherapy and autografting: evidence
that durable molecular and clinical remission frequently can be attained only in
follicular subtypes. J Clin Oncol 2004; 22:14601468.
169. Mandigers CM, Meijerink JP, Raemaekers JM, et al. Graft-versus-lymphoma effect
of donor leucocyte infusion shown by real-time quantitative PCR analysis of
t(14;18). Lancet 1998; 352:15221523.
170. Ho AY, Devereux S, Mufti GJ, et al. Reduced-intensity rituximab-BEAM-
CAMPATH allogeneic haematopoietic stem cell transplantation for follicular
lymphoma is feasible and induces durable molecular remissions. Bone Marrow
Transplant 2003; 31:551557.
171. Andersen NS, Donovan JW, Borus JS, et al. Failure of immunologic purging in
mantle cell lymphoma assessed by polymerase chain reaction detection of minimal
residual disease. Blood 1997; 90:42124221.
172. Jacquy C, Lambert F, Soree A, et al. Peripheral blood stem cell contamination in
mantle cell non-Hodgkin lymphoma: the case for purging? Bone Marrow Transplant
1999; 23:681686.
173. Pott C, Schrader C, Gesk S, et al. Quantitative assessment of molecular remission
following high-dose therapy with autologous stem cell transplantation predicts long
term remission in mantle cell lymphoma. Blood 2006; 107:22712278.
174. Howard OM, Gribben JG, Neuberg DS, et al. Rituximab and CHOP induction
therapy for newly diagnosed mantle-cell lymphoma: molecular complete responses
are not predictive of progression-free survival. J Clin Oncol 2002; 20:12881294.
175. Tarella C, Corradini P, Astolfi M, et al. Negative immunomagnetic ex vivo purging
combined with high-dose chemotherapy with peripheral blood progenitor cell
autograft in follicular lymphoma patients: evidence for long-term clinical and
molecular remissions. Leukemia 1999; 13:14561462.
Minimal Residual Disease 83
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[4584]
176. Magni M, Di Nicola M, Devizzi L, et al. Successful in vivo purging of CD34-
containing peripheral blood harvests in mantle cell and indolent lymphoma: evidence
for a role of both chemotherapy and rituximab infusion. Blood 2000; 96:864869.
177. Gianni AM, Magni M, Martelli M, et al. Long-term remission in mantle cell lym-
phoma following high-dose sequential chemotherapy and in vivo rituximab-purged
stem cell autografting (R-HDS regimen). Blood 2003; 102:749755.
178. Corradini P, Ladetto M, Astolfi M, et al. Clinical and molecular remission after
allogeneic blood cell transplantation in a patient with mantle-cell lymphoma. Br
J Haematol 1996; 94:376378.
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4
New Methods for Clinical Trials:
AML as an Example
Elihu Estey
Division of Hematology, University of Washington Medical Center,
Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
INTRODUCTION
This chapters fundamental tenet is that the conventional methodology used to
evaluate new drugs is incongruent with clinical reality; in particular, the former
frequently underestimates the complexity of the latter. I will point out that this situ-
ation is by no means inevitable. Indeed, a large number of papers in statistical liter-
ature have pointed out flaws in conventional designs and have proposed alternatives;
however, this literature has largely been ignored. By conventional methodology, I
refer to: (i) a phase 1 trial whose sole endpoint is toxicity that is quantified using
standard criteria and evaluated using a 33 design in order to determine a dose for
a subsequent phase 2 trial; (ii) a single-arm phase 2 trial formally concerned only
with a single measure of efficacy, followed by (iii) a large randomized phase 3 trial
intended to compare the new drug with standard treatment. Although the chapters
focus is acute myeloid leukemia (AML), I believe readers can generalize its points.
QUICKER PHASE 3 TRIALS
Because readers are perhaps most familiar with phase 3 trials, I will begin by
considering such a trial, which, in order to compare a new drug X with standard
37 therapy in older patients with untreated AML, typically enrolls 400 patients.
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The trial requires such a large number to detect a relatively small difference
between the two treatments with, at the most, a 5% false positive rate and a 10%
to 20% false negative rate corresponding to 80% to 90% power. For example,
a HOVON-MRC trial comparing standard induction therapy with or without
PSC833 considered it important to detect a minimum increase from9.5%to 18% in
a two-year event-free survival (EFS) probability (1), a CALGBstudy comparing the
same treatments wished to uncover a minimumimprovement in complete remission
(CR) rate from 50% to 65% (2), and an ECOG study comparing different anthra-
cylines was interested in a minimum increment in CR rate of 55 to 75% (3); given
the propensity to relapse, it seems fair to say that such improvements in CR rate
would result in gains in a two-year EFS probability similar to those of interest in the
HOVON-MRS study. I question whether such improvements, while statistically
significant given the aforementioned 5% false positive rate, are medically signifi-
cant in the minds of patients and treating physicians. The counter-argument is that
improvement in the treatment of AML will only come in small increments that must
not be missed. However, it is worth noting that dramatic advances have also
occurred, e.g., arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) for acute
promyelocytic leukemia (APL), 2 chlorodeoxyadenosine (2-CdA) for hairy cell
leukemia, and interferon and imatinib for chronic myelogenous leukemia (CML).
The cases of interferon for CML, 2 CdA, and ATO illustrate that a more sophisti-
cated understanding of disease pathogenesis than is currently possible for AML
need not underlie therapeutic advances. At any rate, it appears fair to ask whether
we would be better served by current phase 3 designs or by designs that by
focusing on larger differences require fewer patients and can thus be completed
more quickly (4).
The stipulation of a 5% significance (i.e., false positive) rate and a 10% to
20% false negative rate is also relevant in this regard. This formulation is used
for phase 3 trials in many different diseases that, however, are medically quite
different. Thus, while the desire to provide more protection against a false
positive result than a false negative one is appropriate in diseases for which
there is already reasonably effective treatment, the same desire appears less
appropriate in a disease such as AML for which treatment is less effective and,
accordingly in which replacement of a standard treatment by a (falsely positive)
new treatment is less consequential. Accordingly, I think that phase 3 studies
might be designed to provide the same protection say 10% to 15% against both
false positive and false negative conclusions. This proposal would also make
possible smaller sample sizes and allow more timely completion of studies.
ADAPTIVE RANDOMIZATION AND BAYESIAN INFERENCE
The word adaptive refers to the possibility, in light of current results, to
unbalance a 1:1 randomization scheme to favor a treatment arm that is out-
performing another arm (5). Although this possibility has intuitive appeal, it is
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rare for phase 3 studies to perform more than one or two interim examinations
of accumulating data. The reason is that, as detailed below, p-value-based
(frequentist) methods cannot maintain a 0.05 significance level at the
conclusion of the trial while supporting numerous interim analyses. In contrast,
Bayesian methodology permits (and encourages) such analyses. The Bayesian
paradigm is built around observed data and parameters, which I will denote by y.
An example of a parameter is the probability of CR. In the Bayesian paradigm,
parameters are random quantities, with probability distributions describing ones
uncertainty about them (68). The Bayesian paradigm begins with a prior distri-
bution, p(y), that characterizes ones uncertainty about y before conducting a trial
and observing the data. The next object is the likelihood, L(data | y), which
describes the probability of observing any specified data given any value of y; the
binomial distribution is an example of a likelihood for binary events such as CR.
The Bayesian paradigm combines the observed data with the prior to arrive at a
posterior distribution of y, which describes ones uncertainty about y after
observing the data. Specifically, Bayes theorem arrives at the posterior by mul-
tiplying the prior by the likelihood of observing the data given the parameter.
When making decisions or inferences on the basis of accruing data, Bayes the-
orem may be applied repeatedly, with the posterior at each stage becoming the
prior for the next stage. The probability distributions in this sequence become
increasingly informative about y as the data accumulate. This process, which is
known as Bayesian learning, (Fig. 1) is especially useful in sequential data
monitoring during a clinical trial. At each of the many times, the current posterior
probability distribution may be used to make a variety of decisions, including
modifying doses (as in a phase 1 trial), dropping an inferior treatment arm,
unbalancing a randomization in favor of a treatment or treatments that have rel-
atively superior performance, or terminating the trial early either because of the
superiority or futility of a treatment.
The Bayesian approach is properly contrasted with the conventional, fre-
quentist approach, which uses p-values as a basis for inferring strength of evi-
dence. The p-value is defined as the probability under the null hypothesis of
observing data as extreme, or more extreme, than that actually observed under a
predetermined experimental design. Many scientists erroneously believe that the
p-value is the probability that the null hypothesis is true, given the observed data.
In fact, this intuitively appealing quantity is a Bayesian posterior probability, not
a p-value. This is so because, given its definition, the calculation of a p-value
involves both observed and unobserved data and is heavily dependent on a
predetermined experimental design (9). An obvious logical flaw is that a given
data set could give rise to two or more different p-values, depending on
which design was intended. For observational data, where there is no experi-
mental design, the p-value of a given test may be a wide variety of different
quantities, depending on what sort of assumptions one makes about how the data
arose and the manner in which putative hypotheses are formulated. Indeed if the
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experimental design is not specified a p-value cannot be calculated. Once a
frequentist experimental design is specified, the investigator is permitted to look
at the accruing data only if and when an interim test is specified by the design. If
this constraint is violated, then the p-value must be adjusted upward to account
for the fact that a false positive decision might have been made. Many group
sequential designs have been described to deal with the issue of interim
analyses. Each designs statistical rules are constructed so that, accounting for
the interim analyses, the overall false positive probability (type I error) will be
maintained below a desired level, typically, p 0.05. In practice, this is done by
performing the interim tests at p-values much smaller than 0.05. However, the
same overall p-value can lead to different decisions depending on the particular
design employed. Importantly, the frequentist method punishes the investigator
for looking at data. Consider a trial designed to have an overall type I error 0.05.
If the investigator looks at the data at any unplanned time and performs a test at
each look, then it is possible that each of the planned 0.05 type I error has been
spent before the trial is completed. Thus, if new data become available there-
after, the frequentist approach makes no allowance for using it along with the
Figure 1 Bayesian learning. On the x-axis are various probabilities of CR following
receipt of a drug. On the y-axis are densities corresponding to the weight of the
evidence in favor of each probability. Each curve is characterized by a mean (the most
likely probability) and a dispersion that is proportional to the width of the curve. For
example, the left-most curve (the prior) indicates that 16% is the most likely CR rate,
but that some weight should be given to probabilities of 10% to 55%. Such a curve might
correspond to a prior probability as based on historical data. As patients are treated and a
CR rate greater than 16% is observed succeeding curves (posteriors) are shifted to the
right and become less disperse as more patients are treated. For each curve, it is simple to
compute the probability that the CR rate is greater than a given percentage with decisions
to stop or continue the study on the basis of this probability. An obvious issue is the choice
of the prior; in practice the effect of various priors on the posterior are examined.
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previous data! The same problem arises in a frequentist design for a trial in
which the final planned test yields p-value 0.051, but additional data are sub-
sequently obtained that strengthen the evidence in favor of a difference. The
frequentist approach does not permit these subsequent data to be used, since
they were not obtained as part of the planned experiment. In contrast, Bayesian
inference utilizes all of the available data, with inferences based on posterior
probabilities computed from observed data. In particular, Bayesian inference
does not involve unobserved data and is not affected by the experimental
design (9). That is, the data enter inferences only through the likelihood function.
Consequently, posterior probabilities, unlike p-values, can be used as an explicit
measure of support for a hypothesis.
An M.D. Anderson trial comparing idarubicin ara-C (IA), troxacita-
bine ara-C (TA), and troxacitabine idarubicin (TI) provides an example of
adaptive randomization (10). A maximum of 75 patients were to be randomized.
As each patient after the 16th one entered the trial, we calculated the Bayesian
posterior probability that the CR rate with either TA or TI was at least 10%
worse than the CR rate with IA; if this probability was greater than 85%, the
relevant arm closed, subject to the possibility of reopening depending on results
in subsequent patients receiving IA. Using this design, the TI arm closed after
each of the first five patients given TI did not achieve CR. The TA arm remained
open with patients adaptively randomized between it and IA in proportion to the
continuously updated CR rates with each regimen. Once the CR rate with TA
was 3 of 11 patients, this arm closed since by then the CR rate with IA was 10 of
18 patients.
An obvious question is the risk of a false negative using such a design.
This risk is quantified before the trial begins by examining the proposed designs
operating characteristics (OC). A designs OC consist of quantities that char-
acterize, under various hypothetical clinical scenarios, its average behavior (5),
e.g., sample size, probability of early termination for a given treatment if it is
truly ineffective and probability of selecting a given treatment if it is truly
superior, with truly here referring to the case where an infinite number of
patients are treated. As suggested by Gooley et al. (11) and Thall and Estey (12),
a practical way to choose design parameters in all but the simplest settings is to
first simulate the trial on the computer under several design parameterizations
and several reasonable clinical scenarios, and obtain the OC in each case. One
may then use this as a basis for choosing the design parameters to ensure that it
will have desirable OC, and in particular that the trial design is ethical.
Table 1 illustrates the OC for the IA versus TA versus TI trial described
earlier. In the scenario, where CR rates with TA and IA are 50% and 40%,
respectively, the design correctly selects TA in 80% of the 10,000 computer
simulations of the scenario. In these 80% IA and TI either drop out or have a
lower CR rate than TA, which is thus selected, i.e., the design has a power of
80%. Hence, the failure to discover the activity of TA (or TI) may have
reflected the inadequacies of these medicines rather than of the statistical design,
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whose power was comparable to that of a standard frequentist design. In contrast,
in the scenario where the IA has the highest CR rate, it is selected only 10% of
the time. Thus, the design has a 90% false positive rate, declaring the new
therapies TA or TI superior when they are not. If physicians wished a lower
false positive rate, the designs parameters would be changed; for example,
accrual to IA would stop only if the probability became greater than 95%
(rather than 85%) that the CR rate with IA was better (as opposed to > 10%
better) than those with TA or TI. Note however that these changes would also
reduce the probability of selecting TA when it is better. These considerations
illustrate the importance of interaction between the physician and the statistician,
an important feature of Bayesian designs with their emphasis of continual updating
of data such as CR rates.
Another potential disadvantage of adaptive randomized designs such as
those used for the IA versus TA versus TI trial is their failure to account for a
possible imbalance in prognostic covariates between the arms. This problem can
be ameliorated by the use of dynamic allocation when patients are random-
ized (13). Even if not, accounting for such covariates is possible, but would
obviously require entry of more patients before an arm might close; the number
is however still less than that used in conventional phase 3 trials.
Table 2 illustrates the consequences to patients of employing the adap-
tively randomized design used for the IA versus TA versus TI trial. The design
resulted in the administration of IA to 18 patients and of TA or TI to 16. In
contrast, use of a 1:1:1 randomization scheme throughout would have resulted in
seven fewer patients receiving the seemingly superior IA regimen than TA or TI.
Closing accrual to an arm after the entry of a relatively small number of patients
Table 1 Operating Characteristics of IA vs. TA vs. TI Trial
True probability CR
Probability arriving at
IA TA TI Correct conclusion correct conclusion
40% 50% 30% TA better 80%
40% 30% 30% IA better 10%
Abbreviations: IA, idarubicin ara-C; TA, troxacitabine ara-C; TI, troxacitabine idarubicin.
Table 2 Consequences of Adaptive Randomization
Pts entered with adaptive
randomization
Pts entered without adaptive
randomization
IA 18 11
TA or TI 16 22
Abbreviations: IA, idarubicin ara-C; TA, troxacitabine ara-C; TI, troxacitabine
idarubicin; pts, patients.
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could lead to failure to detect a small, perhaps biologically unique, subset of
patients who might respond to a treatment, even though the average patient is
highly unlikely to respond. It is unclear, however, whether any trial should
proceed with such a goal in mind.
NEED TO STUDY MORE TREATMENTS: SELECTION DESIGNS
Proposals for quicker phase 3 trials and adaptive randomization enable study of
many more drugs. Thus, for example, 200 patients might be randomized among
four or five, rather than two, treatments. This is possibly of value given the large
number of targeted therapies to be testeda number made all the more larger
by the recognized need to combine these with each other and with chemotherapy.
While in principle only a few promising treatments might be selected on the basis
of preclinical rationale, experience suggests this may not be possible. In addition
to the examples (2 CdA, ATO, IFN) of therapies for which the demand of a
convincing preclinical rationale might have prevented investigation of the
therapy, there are numerous examples of treatments that, despite seemingly sound
preclinical rationales, have been clinically ineffective. This has led to the proposal
of randomized selection designs (5) in which the above 200 patients would be
randomized among four or five treatments, with the treatment selected as best
subsequently investigated further. Selection designs are intended to choose the best
treatment, among those not dropped due to lack of efficacy regardless of the
degree of difference between the best and the second best treatment. This is very
different from a design in which the goal is to decide whether the best treatment
provides a specified degree of improvement over the others. Hypothesis-test-based
designs have the latter goal, and thus require a much larger sample size.
The OC of selection designs indicate that in the case of a trial randomizing
a maximum of 200 patients among four treatments, the probability of selecting a
treatment that is 20% superior to the other three is only about 60%. Thus, such
selection trials are often criticized as underpowered phase 2 studies. Note
however that the nominally high false negative rate of 40% must be compared
with what would obtain in the absence of the design. Assuming that is very
difficult to select the best treatment in the absence of clinical data such as would
emanate from a selection design, the effective false negative rate is 75% if one of
four new treatments is randomly selected for comparison with standard therapy
in a phase 3 study. Indeed, given the uncertainty inherent in selecting the best
arm, it might be said that the worse false negative result obtains if a drug is not
studied at all; the selection design is intended to prevent this possibility.
ARTIFICIALITY OF THE PHASE 2PHASE 3 DICHOTOMY
The phase 2 selection design randomizes patients thus flying in the face of the
concept of the phase 2 trial as single arm. However, in reality all phase 2 trials are
inherently comparative. In particular, patients are vitally interested in knowing
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which of the several new treatments they might receive is superiora question that
implies comparison. Stopping rules for single-arm phase 2 trials are based on his-
torical data with standard therapy further emphasizing the comparative nature of
phase 2 trials (14). However, using data fromsingle-armphase 2 trials as a basis for
comparison introduces bias in the form of trial-treatment confounding. The need to
avoid this problemleads to the use of randomization in phase 3. However, it appears
irrational to accept randomization in a phase 3 trial, but not in a phase 2 trial
intended to determine whether a new therapy should be investigated in phase 3.
Certainly the failure to randomize in phase 2 makes it impossible to use any phase 2
data in phase 3 comparisons. Even with randomization, however, decisions to move
fromphase 2 to phase 3 typically are based on early outcomes in phase 2 rather than
survival or disease-free survival (DFS) time. This requires the implicit assumption
that the early outcome is to some extent associated with improved survival or DFS.
To address these issues seamless phase 23 designs have been proposed (15).
Such a design randomizes between treatments, for example a standard S and an
experimental E, throughout and makes repeated interim decisions during the trial
based on both early response and survival time data. These decisions include
(i) stopping the trial and concluding that E is associated with longer survival than S,
(ii) stopping the trial because of futility, i.e., concluding that E and S are associated
with similar survival, (iii) continuing the trial, or (iv) expanding the phase 2 trial to
incorporate other centers, at which point the phase 3 trial begins. Accrual con-
tinues while the phase 3 trial is being organized, and the seamless nature of the
phase 23 transition allows for the use of all phase 2 data in all phase 3 decisions.
This is illustrated by a trial in which patients with stage 3 or 4 unresectable
nonsmall cell lung cancer receive docetaxel and radiation and are randomized
to receive or not receive an intratumor injection of an adenovirus containing wild
type p53 gene (Ad-p53). The hypothesis is that the transfected p53 will be
proapoptotic and also increase the tumors sensitivity to docetaxel and radiation
therapy. The data on each patient consist of (i) whether a response, defined as
local control (LC) of the tumor evaluated by fine-needle aspiration biopsy, is
observed at five months and (ii) the patients survival time, T. The overall
survival distribution is formulated as a mixture of three components: (i) the
survival distribution when LC is achieved the probability of achieving LC,
(ii) the survival distribution when LC is not achieved the probability that LC is
not achieved, and (iii) the survival distribution for patients who die in less than five
months, before LC is evaluated. The Ad-p53 effect on T may vary as a function of
the LC rates, the possibility that LC affects survival, and the possibility that there
is a direct Ad-p53 effect on T not mediated by LC. In particular, the model does
not assume that LC is a surrogate for T. The design specifies a maximum sample
size of 900 patients and maximum duration of 72 months. At each of up to
18 interim times during the trial, decisions are made on the basis of predictive
probability that survival in the Ad-p53 arm is greater, assuming either that
(i) accrual stops immediately but treated patients are followed for an additional
12 months or (ii) all 900 patients are accrued and followed until 72 months.
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This approach may be compared with a conventional frequentist design that
uses a log rank test to reject or accept the null hypothesis at up to four successive
times with an OBrienFleming test boundary while maintaining overall signifi-
cance level 0.05 and power 0.80 to detect a 25% increase in median survival time
from the null median of 15.5 months. A comparison of the two designs, assuming
the same maximum sample size (900) and maximum duration (72 months), is
summarized in Table 3. Six different hypotheses are assumed, the first three being
different types of null cases in which Ad-p53 does not improve survival,
whereas the last three are alternatives in which survival is longer in the Ad-p53
arm. The Bayesian design parameters were calibrated to maintain type I error less
than 0.05 and power more than 0.80. In all six cases, the Bayesian design has on
average a substantially smaller sample size, and a trial duration that is either
shorter than or the same as that of the conventional phase 3 design.
Although our example involves a lung cancer trial, this sort of seamless
phase 23 Bayesian design may easily be applied to trials of hematologic
malignancies where CR takes the part of LC.
MULTIPLE OUTCOMES
At the beginning of this chapter, I noted that commonly used statistical designs
pay insufficient regard to the complexities of medical practice. The failure to
explicitly recognize the inherently comparative nature of phase 2 trials or the
Table 3 Operating Characteristics of the Bayesian Phase IIIII Design and the
Conventional Designs Under the Six-Mixture Model-Based Hypotheses
Hypothesized Effects
Ad-p53
effect on
LC rate
LC effect
on survival
Ad-p53
direct
effect on
survival Design
Patients
(mean
number)
Mean
duration
(mo)
Probability
conclude
E>S
No No No Bayesian 425 20.4 0.03
conventional 842 28.1 0.05
Yes No No Bayesian 453 21.6 0.04
conventional 842 28.1 0.05
No Yes No Bayesian 452 21.7 0.03
conventional 854 28.5 0.05
Yes Yes No Bayesian 640 30.7 0.85
conventional 884 29.5 0.83
Yes Yes Yes Bayesian 525 23.2 0.97
conventional 861 28.7 >0.99
No No Yes Bayesian 576 29.2 0.56
conventional 873 29.1 0.79
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assumption of a relation between response and survival are examples, as is the
failure to provide for monitoring multiple outcomes in phase 2 (16). Specifically,
phase 2 trials focus on response and provide explicit stopping rules based on this
outcome. In contrast monitoring of toxicity is done informally, i.e., without
explicit stopping rules. The assumption is that toxicity rates are already known
from the phase 1 study. However, it has been pointed out that the number of
patients entered into typical phase 1 trials is often inadequate to estimate the
toxicity rate at any given dose (17). Furthermore, the patients treated on phase 2
trials may be quite different with regard to the likelihood of toxicity than those
entered on the corresponding phase 1 trial.
The following example illustrates the desirability of monitoring multiple
outcomes. We conducted a trial in which untreated AML patients under age 50
received double induction consisting of a course of idarubicin and high-dose
ara-C, followed by a second course of this combination 14 days after beginning the
first course, regardless of the status of the marrow at that time. While the objective
of intensifying therapy was to improve the 90-day CR rate, this approach also
carried the risk of increasing the 90-day mortality rate. Furthermore, the second
course was to be given only to patients judged eligible, i.e., recovered from
first course toxicity, by their attending physicians. Accordingly, it was possible that
the number of patients eligible for the second course would be sufficiently small that
the results would be of little practical significance. Our design formally
monitored three outcomes: the course 2 eligibility, and among eligible patients, the
CR rate and the death rate within 90 days after the start of course 1. The 90-day
window was selected after considering the anticipated accrual rate of three to four
patients per month, both because the risks of treatment failure or death within this
timeframe are high and for logistical reasons. To account for prognostic hetero-
geneity, the early stopping rules were applied separately in two subgroups:
patients with abnormalities of chromosomes 5 and/or 7 (5/7), and patients with
all other karyotypes. For illustration, we will focus attention on the latter sub-
group. It was decided that the requisite course 2 eligibility rate was greater than or
equal to 67%. On the basis of historical rates, a 4% increase in a 90-day mortality
rate (death), from 14% to 18%, was considered an acceptable trade-off for a
15% increase in the 90-day CR rate (success), from 67% to 82%. Death and
success were not complementary events because an eligible patient could be
alive but not in CR at day 90. The trial was to stop if, after evaluating each cohort
of five patients, the probability was too high that (i) the eligibility rate was less
than 67% or (ii) the death rate among eligible patients was increased by greater than
4%, or (iii) the probability of a 15% increase in the success rate among eligible
patients was too low. Criteria probabilities of 95%, 90%, and 5% for these events
quantified the terms too high and too low. These criteria led to three sets of
stopping boundaries, one for each rate being monitored. If early termination did not
occur, 50 patients would be entered, which would provide a 90% posterior credi-
bility interval for the 90-day success rate having limits within 0.12 of its mean,
assuming a mean success rate of 0.82 and mean eligibility rate of 0.68.
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This designs OC are summarized in Table 4. For example, under the
scenario where the true death rate is the unacceptably high value 33%, the
probability of early termination (PET) is 96% and 50% of the simulated trials
stopped after 15 patients were entered. For higher true death rates, the median
sample size becomes smaller. In contrast, if the minimum study goals were met,
the trial ran to completion in 79% of the simulated trials, with this percent
increasing for higher eligibility rate, higher success rate, or lower death rate. In
fact, the trial closed after 14 of the first 25 patients were ineligible although the
observed numbers of deaths (2/7) and success (5/7) within 90 days among
eligible patients were both acceptable.
Such multiple outcome designs allow the investigator to explicitly specify
a trade-off between efficacy and toxicity, which correspond to 90-day
success and 90-day death in the double induction trial. Different investigators
might have different trade-offs. For example, in the double induction trial, one
might consider a 4% increase in death rate acceptable only given a 25% increase
in success rate. In the scenario where the true death rate is 33% (row 3, Table 4),
on average, 5 of the median sample size of 15 would die compared to 2 of 15 in
the historical situation (14% death rate). If the investigator believes this is
unacceptable, the criterion probability could be lowered from 90% to 85%.
However, making it easier to stop the trial would also increase the PET if the true
success rate is the desired 82%. Our experience suggests that the need to specify
such trade-offs encourages dialogue between clinical investigators and statis-
ticians. Moreover, multiple outcome designs encourage the use of a wider range
of data in therapeutic decision-making.
CONCLUSION
Statistical methods for conducting clinical trials have remained essentially
static for the past 30 years. This phenomenon is difficult to explain in light of
the issues raised above and the profusion of papers in the statistical literature
describing new designs; I have referenced just a few of these here. It is difficult
to reconcile the eagerness with which physician or scientists have adopted new
Table 4 Operating Characteristics of the Double Induction Trial
Probability of
Achieved sample size percentiles
Clinical scenario early termination 25th 50th 75th
Same as historical rates 0.69 15 25 50
82% success rate and
18% death rate
0.21 50 50 50
67% success rate and
33% death rate
0.96 10 15 20
50% ineligibility rate 0.89 10 15 30
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molecular biologic techniques with their reluctance to adopt new statistical
techniques. Regardless of the reason for the contrast, I believe the con-
sequences of this reluctance are unfortunate from both academic and clinical
perspectives.
CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS
Hopefully, the next five years will see the adoption of many new methods for
conducting clinical trials. Phase 1 and phase 2 trials will be combined and will
monitor both efficacy and toxicity, consistent with patients primary reason for
entering phase 1 trials: to achieve a response, not no toxicity. Factors other
than dosage will be considered in assessing toxicity. For example, for purposes
of determining the dose for phase 2 trials, it is unrealistic to equate a 70-year-old
who has severe toxicity at a given dose with a 50-year-old who has the same
toxicity at the same dose. Yet this is what is done currently; pharmacoge-
nomics will also be considered as a determinant of toxicity.
The 33 design will be replaced with Bayesian methods, such as the
clinical reassessment method (CRM), which allow all information to be used,
rather than only the information obtained at the most recent dose.
Most importantly, given the large number of therapies to be investigated,
there will be a shift away from large randomized trials to smaller randomized
trials. The invariable need for the magic statistical significance level of 0.05
and equally magic power of 80% to 90% will be rethought. The question of
whether the optimal biological dose should be investigated rather than the more
traditional MTD will be explored, perhaps in separate arms of a randomized trial.
Using a single protocol, therapies that do poorly will be replaced by other therapies,
while if results are equivocal, more patients will be recruited into the study.
In general, there will then be a blurring of the artificial distinctions between
phases 1, 2, and 3.
The above will only occur given a willingness to forego habit and tradition.
However, the recent adoption by the MRC of the small, randomized trial, i.e., the
selection design described earlier in this chapter, is encouraging and will hopefully
provide a model for others.
REFERENCES
1. van der Holt B, Lowenberg B, Burnett A, et al. The value of the MDR1 reversal
agent PSC-833 in addition to daunorubicin and cytarabine in the treatment of elderly
patients with previously untreated acute myeloid leukemia in relation to MDR1 status
at diagnosis. Blood 2005; 106:26462654.
2. Baer M, George S, Dodge R, et al. Phase 3 study of the multidrug resistance
modulator PSC-833 in previously untreated patients 60 years of age and older with
acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood 2002;
100:12241232.
96 Estey
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3. Rowe J, Neuberg D, Friedenberg W, et al. A phase 3 study of three induction
regimens and of priming with GM-CSF in older adults with acute myeloid leukemia:
a trial by the Eastern Cooperative Oncology Group. Blood 2004; 103:479485.
4. Estey E. Clinical trials in AML of the elderly: should we change our methodology?
Leukemia 2004; 18:17721774.
5. Estey E, Thall P. New designs for phase 2 clinical trials. Blood 2003; 102:442448.
6. Berry D. Statistics: A Bayesian Perspective. Belmont, CA: Wadsworth Publishing
Company, 1996:124161.
7. Spiegelhalter D, Abrams K, Myles J. Bayesian Approaches to Clinical Trials and
Health-Care Evaluation. Chichester, UK: John Wiley & Sons, 2004.
8. Goodman, SN. Toward evidence-based medical statistics. 2: the Bayes factor. Ann
Intern Med 1999; 130:10051013.
9. Berger J, Berry D, Statistical analysis and the illusion of objectivity. American
Scientist 1988; 76:159165.
10. Giles F, Kantarjian H, Cortes J, et al. Adaptive randomized study of idarubicin and
cytarabine versus troxacitabine and cytarabine versus troxacitabine. J Clin Oncol
2003; 21:17221727.
11. Gooley T, Martin P, Fisher L, et al. Simulation as a design tool for phase I/II clinical
trials: an example from bone marrow transplantation. Control Clin Trials 1994; 15:
450462.
12. Thall PF, Estey E. A Bayesian strategy for screening cancer treatments prior to phase II
clinical evaluation. Stat Med 1993; 12:11971211.
13. Pocock SJ, Simon R. Sequential treatment assignment with balancing for prognostic
factors in the controlled clinical trial. Biometrics 1975; 31:102115.
14. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials
1989; 10:110.
15. Inoue LYT, Thall PF, Berry DA. Seamlessly expanding a randomized phase II trial
to phase III. Biometrics 2002; 58:823831.
16. Thall PF, Simon RM, Estey EH. New statistical strategy for monitoring safety and
efficacy in single-arm clinical trials. J Clin Oncol 1996; 14:296303.
17. Edler, L. Overview of phase I clinical trials. In: Crowley J, ed. Handbook of Statistics
in Clinical Oncology. New York, NY: Marcel Dekker, 2002:134.
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5
Monoclonal Antibody Mediated
Treatment in Acute Myeloid Leukemia
Ch. Michel Zwaan and Marry M. van den Heuvel-Eibrink
Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia
Childrens Hospital, Rotterdam, The Netherlands
GENERAL INTRODUCTION
Monoconal Antibodies
Thirty years have passed since Kohler and Milstein first described the possibility
of hybridizing murine cell lines, resulting in the expression and production of
specific murine antibodies (1,2). In 1984, they were awarded with the Nobel Prize
for their discovery.
Currently, more than 20 monoclonal antibody preparations are available
for clinical use for various diseases, including cancer and autoimmune diseases,
of which several are approved by the regulatory authorities, as summarized in
Table 1 (this list is neither meant to be exhaustive nor complete) (2). Monoclonal
antibody therapy is a type of immunotherapy and is referred to as passive
immunotherapy as the antibodies are not produced by the bodys own immune
system. Other examples are the use of cytokines such as interferones, inter-
leukines (ILs), or growth factors. This in contrast to cancer vaccines, which are
dependent on the host immune system to conquer the disease, are, therefore,
referred to as active immunotherapy. Another example of active immuno-
therapy in hematological malignancies is stem cell transplantation (SCT), aiming
99
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124]
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at inducing a graft-versus-leukemia effect, in addition to elimination of the
malignant stem cell by high-dose chemotherapy and/or irradiation.
Monoclonal antibodies have high specificity and are directed against a
single antigen. Ideally, they are directed against antigens that are present selec-
tively on tumor cells, which may result in limited side effects and organ toxicity.
Hence, broad tissue typing is required for the antigen of interest to study its
expression on normal human tissues. In addition, the expression should pref-
erably be strong and homogeneous throughout the tumor. Antibodies targeting
antigens that are also present in soluble form in the circulation may be less
effective because of rapid antibody clearance after infusion.
The use of murine antibodies in the clinical setting has not been very
successful, as they will be recognized by the human immune system as foreign
proteins, and generate an human anti-mouse antibody (HAMA) immune reaction
(3). This may result in inadequate exposure to the antibody due to diminished
stability in the circulation, as well as a severe allergic reaction in the recipient,
which interferes with repeated dosing. Murine antibodies also have less capacity
to recruit effector cells and complement to destruct cancer cells. They are referred
to as momabs, such as in ibritumomab, which is directed against the CD20
antigen and used in non-Hodgkins lymphoma. The ability to construct so-called
chimeric or humanized antibodies by genetic engineering has markedly
improved the possibilities to use monoclonal antibodies in the clinic (4). In chimeric
antibodies, the variable region is still from mouse origin, whereas in humanized
antibodies this is only the hypervariable region. Chimeric antibodies are approxi-
mately 60%to 95%human, whereas humanized antibodies are over 95%human (3).
In both instances the murine part is responsible for specificity and antigen
recognition. They are referred to as ximabs and zumabs, respectively, as in
rituximab (anti-CD20) and gemtuzumab (anti-CD33) or epratuzumab (anti-CD22).
Moreover, fully human antibodies are now available by using transgenic mice that
have been engineered to synthesize human antibodies. These antibodies usually
allow repeated dosing and do not result in severe allergic reactions.
Two different types of monoclonal antibodies can be distinguished, i.e.,
naked versus conjugated monoclonal antibodies:
1. Naked antibodies bind directly to antigens expressed on tumor cells, and
may either stimulate the immune system to destroy the cancer cell by
antibody-dependent cell mediated cytotoxicity (ADCC), complement
dependent cytotoxicity (CDC), or by induction of apoptosis. Examples are
rituximab, an anti-CD20 antibody used in the treatment of non-Hodgkins
lymphoma (5), and the anti-CD52 directed antibody alemtuzumab, which
may be used as part of the conditioning regimen in SCTs or to eliminate
T-cells from grafts (6). Rituximab was actually the first monoclonal antibody
registered (in 1997) by the Food and Drug Administration (FDA) for use
against cancer. Alternatively, monoclonal antibodies may exert their effect
as competitive antagonists, prohibiting a ligand to bind to a certain receptor,
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 101
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and thereby blocking the activity of the ligand, subsequently shutting down
the intracellular signaling cascades that would normally result from
ligand binding. The antiangiogenesis monoclonal antibody bevacizumab,
which interferes with vascular-endothelial growth factor receptor
signaling, is an example of the latter (7).
2. Conjugated monoclonal antibodies are antibodies linked to drugs, toxins or
radioactive compounds, and basically represent targeted delivery of the
conjugate to the tumor cell rather than inducing cell death by the afore-
mentioned mechanisms. An important issue to consider in developing such
conjugates is whether the conjugate will be internalized after binding to the
tumor cell. Examples are gemtuzumab ozogamicin (GO) (anti-CD33 linked
to calicheamicin) for the treatment of acute myeloid leukemia (AML) (8)
and radiolabeled antibodies directed against CD45 used in the conditioning
regimen of advanced leukemia patients (9).
These data show that the development of a monoclonal antibody represents
rigorous scientific effort and financial resources. Both the target and the antibody
need to be selected very carefully to ensure that the in vivo properties will allow
therapeutic efficacy. Hematological malignancies are ideally suited to treatment
with monoclonal antibodies because of the accessibility of malignant cells in the
blood, bone marrow, spleen, skin, and lymph nodes and the availability of targets
that are restricted to the hematopoietic system. Rapid and repeated evaluation of
target antigen expression is feasible using flowcytometry. In this chapter, we will
review the current status of targeted monoclonal antibody therapy in use or
development for the treatment of children and adults with AML, with the
exception of radiolabeled antibodies.
Acute Myeloid Leukemia
AML is a heterogeneous group of diseases and basically comprises all other than
lymphocyte-precursor derived acute leukemias. Traditionally, classification is
based on morphology according to the French-American-British (FAB) classi-
fication. Recently, a new classification for myeloid neoplasms has been intro-
duced by the WHO, which differentiates between AML with recurrent
cytogenetic abnormalities, AML with multilineage dysplasia, therapy-related
AML, and AML not otherwise specified (10). For stratification of patients in
clinical trials, cytogenetic abnormalities and early response to treatment are often
used, representing the underlying biology of the disease (11,12). Patients with
t(8;21), t(15;17), or inv(16)/t(16;16) are considered good risk by most collabo-
rative study groups, whereas abn(3q), monosomy 5 or 7 and deletion 5(q) or 7(q)
and complex abnormalities are considered poor risk by most groups (12). Cur-
rently, other molecular abnormalities, such as mutations in C/EBPa, or receptor
tyrosine kinases such as FLT3, KIT, and others may also be taken into account
(13). It is well known that the prognosis decreases with increasing age, which is
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partially due to an increasing frequency of unfavorable biology with increasing
age, but also represents unfavorable host characteristics (11). On the basis of
this, many study groups have separate protocols for children, younger adults, and
elderly people with AML.
Currently, induction regimens usually consist of intensive chemotherapy
comprised of cytarabine in combination with an anthracycline, with or without a
third drug such as etoposide or 6-thioguanine. Subsequently, the consolidation
phase consists of intensive courses of chemotherapy in children and younger
adults, with or without SCT, aiming at the eradication of minimal residual dis-
ease. Children and adults with acute promyelocytic leukemia [(APL) or AML
FAB M3] receive adapted treatment protocols incorporating all-trans retinoic
acid (ATRA), which results in much better treatment outcome than in non-M3
AML. Overall, 80% to 90% of children with AML reach complete remission
(CR) versus approximately 75% of younger adults and 50% of older adults
(11,13,14). This results in survival rates of approximately 50% to 70% for
children versus approximately 40% to 50% for younger adults and only 10% for
elderly AML patients (11,13,14). Relapse and nonresponse mainly contribute to
these high failure rates. Hence, outcome for most patients with AML is still
unsatisfactory in terms of antileukemic efficacy. Moreover, especially in chil-
dren, there is concern over the acute and long-term side effects associated with
the intensive chemotherapy (15). Therefore, there is a continuous interest in the
development of new antileukemic agents, preferably those that exert their action
without causing too many side effects.
CD33-DIRECTED MONOCLONAL ANTIBODIES IN ADULT AML
CD33 is expressed on the cell surface of malignant blast cells in 80% to 90% of
AML cases, but not on normal hematopoietic stem cells or nonhematopoietic
tissues. Antibody-based therapies for AML have, therefore, focused on CD33 as
a suitable target antigen. CD33 is also a useful target for conjugated antibodies,
as binding to CD33 results in internalization of the complex. The natural ligand
of CD33 and its function are currently not known (16).
Lintuzumab (HuM195)
At first, studies have been performed with lintuzumab (HuM195), which is a
naked antibody. This did not result in significant antileukemic activity in AML,
with the exception of molecular disease control in AML FAB M3 (APL), in
patients who were reverse transcriptase polymerase chain reaction (RT-PCR)
positive for the APL characteristic t(15;17) fusion transcript after induction
chemotherapy plus ATRA (17,18). These APL patients were treated with lin-
tuzumab twice weekly for three weeks. Half of the patients evaluable for con-
version of the RT-PCR of the fusion transcript became molecularly negative.
This is not surprising, as APL is characterized by relatively strong CD33
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 103
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expression. A recent randomized phase III study in relapsed/refractory AML
patients did not show a survival advantage in patients stratified to chemotherapy
plus lintuzumab (4 days of lintuzumab 12 mg/m
2
following induction chemo-
therapy were given, starting at day 6 after completion of induction chemotherapy
and repeated at day 1618) versus chemotherapy alone (pOS 36 vs. 28%, p 0.28)
(19). The addition of lintuzumab did not result in increased toxicity when compared
with the chemotherapy-only arm.
Gemtuzumab Ozogamicin
The most promising results have been obtained with GO, Mylotarg
1
, a humanized
anti-CD33 monoclonal antibody linked to calicheamicin, which is a potent
enidyne antileukemic antibiotic. Calicheamicin dissociates from the antibody-
calicheamicin complex after internalization, binds to the minor groove of
DNA, and results in DNA-double strand breaks (Fig. 1). The results of the
various phase I/II studies in adults are summarized in Table 2, and some are
discussed in more detail below.
Figure 1 Mechanism of action of gemtuzumab ozogamicin. (A) Gemtuzumab ozogamcin
consists of an anti-CD33 antibody linked to the antitumor antibiotic calicheamicin. (B) After
binding to CD33, the complex is internalized, after which the calicheamicin is released by
hydrolysis in lysosomes. (C) Free calicheamicin then translocates to the nucleus and cleaves
double-stranded DNA, resulting in apoptosis.
(text continues on page 112)
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Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 105
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124]
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[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
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[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
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duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
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[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
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[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
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.
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 111
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
The results of the adult phase I study in relapsed/refractory AML were
published in 1999 and showed that a postinfusion syndrome of rigor and chills
was the most frequent side effect (8). Two patients who were treated at the
9 mg/m
2
dose level had prolonged neutropenia and thrombocytopenia. Of the
40 patients who were treated, 20% showed a response (i.e., absence of leukemia
from bone marrow and peripheral blood). No antibodies against the antibody were
detected, but two patients developed antibodies against calicheamicin. The rec-
ommended dose for phase II studies was two times 9 mg/m
2
with a 14-day interval.
At this dose level, greater than 75% saturation of CD33 sites on peripheral blood
mononuclear cells was found.
Three phase II studies in adults with relapsed/refractory AML were started. It
has to be noted that inclusion in these three studies was restricted to a selected
population of adults with relapsed CD33-positive AML who had a first CR duration
of at least three months, and with a minimum of 80% of blasts staining positive for
CD33 at least four times above the background level. On the basis of the preliminary
data from142 patients enrolled in these studies, GO was given accelerated approval
by the FDA for patients with CD33-positive AML in first relapse who are 60 years
or older and who are not considered candidates for cytotoxic chemotherapy (20,21).
Administering two dosages of 9 mg/m
2
with a 14-day interval, toxicities were
similar as in the phase I study, but in addition hepatotoxicity was noted, including
elevated transaminases and hyperbilirubinaemia in 17% and 23%, respectively, as
well as one death due to liver failure. Of note, mucositis and severe neutropenic
infections were infrequent. CR was obtained in 16% of patients. However, another
subset of patients also showed remission but with insufficient platelet recovery, and
they were categorized as CR with insufficient platelet regeneration, but platelet
transfusion independence (CRp). The duration of response in these two subgroups
was similar, and hence the total response rate was considered to be 30% (21).
Recently, the final results of these phase II studies were published, now including a
total of 277 adults (22). The overall response rate was 26% and included 13% of
patients classified as CRand 13%as CRp, but nowa difference in median leukemia-
free survival time between these two patient categories of 6.4 versus 4.5 months was
noted. This suggest that the quality of remission in the CRp patients was less than in
patients with sufficient platelet recovery. Toxicities included grade 3 or 4 sepsis in
17% and grade 3 or 4 hyperbilirubinemia in 29%. Approximately 1% of patients
who did not undergo SCT (either before or after treatment with GO) also developed
hepatic veno-occlusive disease (VOD) following GO treatment.
Meanwhile more data became available on the hepatotoxicity of GO, again
showing that some patients developed clinical signs of VOD, which is thought to
be due to CD33 expression in hepatic sinusoids and perhaps better described as
sinusoidal obstruction syndrome (SOS) (23). Other factors involved may be
the liver leukemia load or circulating soluble CD33 levels. A high incidence of
VOD (in this particular single-center study as high as 64% of 14 patients) was
noted among patients who were transplanted following reinduction with GO,
mainly in patients who were transplanted shortly after GO treatment (24). This
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resulted in the recommendation to delay transplantation for at least 3.5 months
following treatment with GO. However, in another series VOD also occurred in
approximately 4% of patients without prior SCT history, as was also noted in the
phase II studies described above (25). Several case reports suggest that defib-
rotide may be useful in preventing or treating GO-induced VOD, but no larger
prospective or comparative studies have been performed (26,27).
Three studies have now been performed using single-agent GO as induction
therapy in older patients with newly diagnosed AML. The first study of Estey et al.
included 51 adults older than 65 years with either AML or myelodysplastic syn-
drome (MDS) (28). Patents were randomized to receive GOor GOplus interleukin-
11 (IL-11). CR rates were 8% and 36%, respectively. In this nonrandomized study,
the treatment results were compared with a historical cohort of patients receiving
cytarabine and idarubicin, and GO IL-11 was considered inferior to the standard
cytarabine/idarubicin schedule. The authors, therefore, argue against using GO as
standard regimen in elderly patients with AML. Another phase II study in 40 newly
diagnosed AML patients older than 60 years, who were considered not fit for
intensive chemotherapy, showed an overall response percentage (CR and CRp) of
17, although responses were mainly restricted to patients 61 to 75 years old (29).
Two doses of GO 9 mg/m
2
were given, and it was planned to give a third and fourth
dose of GO to responding patients. However, the second dose was only given to
57% of patients, either because of toxicity (n 9) or progressive disease (n 8).
Toxicity occurred mainly in patients older than 75 years of age, with 23%of patients
suffering from induction deaths, which led to the suggestion that a reduced dose
should be applied for this age group. Only one patient tolerated a third and fourth
dose of GO. The median survival in responding patients was 11.4 months. The third
study concerned 12 patients over 65 years of age (30). Three out of 12 patients were
in CR after two doses of GO at 9 mg/m
2
. Five patients had nonarrhythmia cardiac
adverse events, although it was not clear if they were attributable to treatment with
GO. Amadori et al. also mentioned 10%arrhythmias, 7%left ventricular dysfunction,
and 5% hypotension as grades 3 to 4 adverse events (29).
Most single-agent studies with GO have used the classical dosing
schedule of 9 mg/m
2
on day 1 and 14. However, based on in vitro data showing
rapid reexpression of CD33 after internalization of the CD33/GO complex, a
more fractionated dosing schedule may be more efficacious (31). These in vitro
data were recently translated to the clinic in a phase II study in which GO was
administered at 3 mg/m
2
on day 1, 4, and 7 in 57 patients with relapsed/refractory
AML (32). This schedule was very well tolerated, without hepatoxicity. The
response rate was 26% CR and 7% CRp.
Several studies have been reported on combination chemotherapy regi-
mens including GO, both in relapsed/refractory and in newly diagnosed patients.
Amadori et al. treated 57 newly diagnosed elderly AML patients with two
infusions of GO 9 mg/m
2
as induction therapy, which was followed subsequently
by conventional chemotherapy (33). Response to GO was observed in 35.1% of
patients, with an additional 10.5% of partial remissions. In total five patients
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suffered from VOD, with three cases during the initial GO treatment phase. One-
year survival was 34%. The value of this GO-containing regimen is currently
being compared in a prospective randomized study with a chemotherapy-only
arm. In another report, 59 patients with previously untreated AML or MDS were
treated with a single infusion of 6 mg/m
2
GO, combined with fludarabine,
cytarabine, and cyclosporine AMylotarg, fludarabine, cytarabine (ara-C), and
cyclosporine (MFAC) regimen, which induced CR in 46% of patients but also
resulted in considerable toxicity (34). Cyclosporine A was added as MDR1
reversal agent, as MDR1 has been reported to be involved in GO resistance (35).
This regimen was first tested in relapsed/refractory patients, and additionally as
postremission therapy in patients achieving CR after GO-containing induction
(36,37). Chevallier et al. have combined GO 9 mg/m
2
with cytarabine and
mitoxantrone in induction, in patients with relapsed/refractory AML, and report
a very promising response rate of 76% with acceptable toxicity, but the number
of patients was limited (38).
Kell et al. reported a feasibility study in newly diagnosed adult AML patients
in which GO was combined with several different standard AML induction regi-
mens (39). Several lessons were learned from this study: (i) the maximum dose of
GO in these combination was 3 mg/m
2
single infusion; (ii) this dose could not be
administered in consecutive courses because of hepatotoxocity and delayed hem-
atopoietic reconstitution, but it was possible to use GO in course 1 and 3; and
(iii) GO should not be combined with 6-thioguanine because of hepatotoxicity. A
very promising CR rate of 85% was noted, and a randomized phase III trial (study
AML15) based on this schedule has recently been completed in newly diagnosed
patients with AML in the MRC-group. The first results on the 1115 randomized
patients were reported by Burnett et al. at the ASH 2006 meeting, showing similar
remission induction rates of 85% both in the GO as well as in the non-GO arm.
However, there was a significant reduction in the relapse rate in patients included in
the GO-arm (37% vs. 52% at 3 years, p 0.01), resulting in improved disease-free
survival (51% vs. 40% at 3 years, p 0.008), although not yet in overall survival
(40). Considering toxicity, there was a significant increase in transaminase elevation
in the GO-arm but no difference in bilirubin elevation. Patients in the GO-arm
needed significantly more platelet transfusions (19 vs. 14; p < 0.0001), and more
days on IV antibiotics (20.6 vs. 18.6 days, p 0.001), although bone marrow
recovery was similar. GO did not increase the number of patients with death in CR,
after a median of 15 months of follow-up. Several other cooperative groups have
started similar prospective randomized studies.
Treatment of APL with GO seems promising, although the data are still
limited. Lo-Coco reported 16 patients with a molecular relapse of APL who were
salvaged by two (or more in 3 patients) infusions (6 mg/m
2
) of GO (41).
Approximately half of the responses were sustained for a median of 15 months.
Quantitative RT-PCR studies showed that responding patients experienced a
dramatic decline (at least 2 logs) of the PML/RARalpha transcript after the first
GO dose. In a series from MD Anderson, GO was given to nine patients with
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untreated APL, in combination with ATRA and idarubicin in patients with high
white counts (42). After two induction courses, patients received eight mainte-
nance courses. GO was tolerated well and induced CR in 16/19 patients. Follow-
up was too short to determine whether these responses were durable. In a recent
study, GO (dose of 9 mg/m
2
) was given as consolidation in combination with
ATRA and arsenic trioxide, following arsenic trioxide reinduction, in patients
with relapsed APL, again showing good tolerability of this regimen (43).
CD33 DIRECTED MONOCLONAL ANTIBODIES IN PEDIATRIC AML
The results of the various studies in children are summarized in Table 3. The first
data reported on the use of GO in children concerned 15 patients with relapsed/
refractory AML who were treated on compassionate use basis with one to three
infusions of GO at a dosage of 4 to 9 mg/m
2
(44). Basically, efficacy and safety
mimicked the experience in adults. Durable responses were noted in two of the
eight patients who achieved a response, defined as no evidence of leukemia.
Brethon et al. published similar results in another compassionate use series (45).
The pediatric phase I study results were recently published by Arceci et al.
(46). They initially started at the 6-mg/m
2
-dose level, administering two infu-
sions with a 14-day interval, and subsequently escalated to 9 mg/m
2
. At this
level, 3 of 13 patients had grades 3 to 4 transaminase elevations, and one patient
developed VOD, after which dose de-escalation to the 6-mg/m
2
level was issued.
The last two patients received 7.5 mg/m
2
for two dosages, before the study was
closed. Overall, toxicities included grades 3 to 4 hyperbilirubinemia in 7% and
elevated hepatic transaminases in 21%; the incidence of grade 3 to 4 mucositis
(3%) or sepsis (24%) was relatively low. Eight of 29 patients achieved overall
remission (28%). Remissions were comparable in refractory (30%) and relapsed
(26%) patients.
Versluys et al. published a case series of another seven children treated
with GO prior to SCT as reinduction treatment (26). One to four doses of GO
9mg/m
2
were administered in these patients. After the second patient suffered
from severe VOD at subsequent SCT, routine defibrotide prophylaxis was given
to all patients, and none of them developed VOD.
Preliminary results from a phase II study with GO (2 doses of 7.5 mg/m
2
IV) in 20 patients who received homogenous pretreatment according to the
Relapsed AML 2001/01 protocol, but were either refractory to reinduction or
suffered from second relapse, showed a response rate of 40% (including both CR
and CRp) (47). The median survival of responders was longer than for non-
responders to GO (median 1.04 vs. 0.4 years, p 0.04). Transaminase and
bilirubin elevation was found in 5% of patients. One out of eight transplanted
children developed VOD.
A recent pilot study addressed the use of GO following reduced-intensity
stem cell transplantation (RIC-allo-SCT), in eight children with CD33 AML,
either in first or second CR (48). The first dose of GO was given after reconstitution
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116 Zwaan and van den Heuvel-Eibrink
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.
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of neutrophils and platelets at day 60-180 post RIC-allo-SCT. The second dose
was given after recovery of peripheral blood, usually around eight weeks following
the first dose. A dose escalation from 4.5 to 6.0 mg/m
2
appeared to be safe, and
further dose escalation is planned. Toxicity mainly consisted of bone marrow
suppression, infections, and transaminase elevations, but no VODs occurred. Fur-
ther studies are necessary to demonstrate whether GO is able to reduce the relapse
rate following RIC-allo-SCT and whether this approach is superior to myeloa-
blative allo-SCT.
Brethon et al. presented the first results of a combination of GO with
cytarabine, using a fractionated GO schedule; GO was given at 3 mg/m
2
/day on
day 1,4, and 7 plus cytarabine 100 mg/m
2
/day using continuous IV infusion for
seven days (49). Seventeen children received this course as induction regimen,
and six patients subsequently received a consolidation course with GO 3 mg/m
2
at day 1, plus cytarabine 100 mg/m
2
/day for seven days. Responses included
2 CRs, 4 CRps, and 4 PRs.
Currently, several pediatric studies are underway applying GO in several
phases of treatment. This includes the combination of reduced dosages of GO
(3 mg/m
2
) with induction and/or consolidation chemotherapy as well as testing
GO as single agent in postremission setting (following intensive AML treatment)
with the aim to eradicate minimal residual disease and reduce the relapse rate.
CD45-DIRECTED MONOCLONAL ANTIBODIES
CD45 is a tyrosine phosphatase and a common leucocyte antigen, expressed in
the membrane of all leucocytes, leukemic cells, and erythrocyte progenitors but
not outside the hematopoietic system (50). Different isoforms exist because of
alternative splicing. Its expression is more dense on lymphoid when compared
with myeloid cells, which explains why this antibody is used in conditioning
regimens of SCT to deplete the host from lymphocytes and reduce graft failure.
Antibody-bound CD45 tends to remain on the cell surface and does not inter-
nalize, which makes it an attractive target for radioimmunoconjugates, as the risk
of cleavage of the radioisotope and release in the circulation is limited. Several
studies on radiolabel-led anti-CD45 antibodies have been reported, but they are
outside the focus of this chapter. Experience with unconjugated anti-CD45
antibodies is limited. In a phase I study, patients who were to receive a SCT were
treated with the rat anti-CD45 antibodies YTH24 and YTH54. The MTD was
defined as 400 mg/kg/day for four days, with bronchospasm as dose-limiting
toxicity (50). This resulted in a decline in lymphocytes and granulocytes from
the peripheral blood (approximately 1 log reduction), as well as demonstrable
antileukemic efficacy (in the 3 patients with measurable disease before the
administration of the antibody). In a subsequent study in patients who could not
tolerate myeloablative conditioning, anti-CD45 was used in conjunction with
alemtuzumab, fludarabine, and total body irradiation, which resulted in successful
engraftment (51).
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OTHER ANTIBODIES
A phase I study was performed with an anti-GMCSF monoclonal antibody in eight
patients with relapsed AML, which was well tolerated but without significant
antileukemic activity (52).
FLT3 mutations have been detected in approximately 10% to 15% of
pediatric AML and 30% of adult AML cases. Apart from inhibitory small
molecules that interfere with signal transduction, monoclonal antibodies against
FLT3 have been developed (53). These antibodies interfere with ligand-mediated
autocrine FLT3 signaling and may induce antibody-dependent cellular cytotoxicity
(ADCC). Currently, these antibodies are in the preclinical phase of development,
and clinical studies have to be awaited.
RESISTANCE MECHANISMS
Similar to conventional chemotherapy, the use of monoclonal antibodies may results
in clinical resistance against these compounds (16). General mechanisms include
clonal evolution of clones that do not (or not high enough) express the selected target
antigen. In case of a high tumor load all the antibody may be trapped in the circu-
lation and hence not result in significant antileukemic efficacy, which has also been
described for GO (54). Especially when using naked antibodies, immune deficien-
cies, which may occur secondary to the disease itself or due to immunosuppressive
treatment, may impair with successful killing of tumor cells. Of particular relevance
for use of monoclonal antibodies in AML is whether the leukemic stem cell itself
expresses the target antigen of interest or whether this is only expressed by the more
mature bulk of cells (55). This issue is currently unresolved for GO and CD33,
although relapses are usually not characterized by CD33-negativity (16,56). For GO,
several other mechanisms have been reported, including MDR1 overexpression (35)
and differences in cellular calicheamicin sensitivity (57,58). Cell-line studies have
shown that GO-induced cytotoxicity was directly related to CD33 expression levels,
although several clinical studies have failed to relate CD33 expression to clinical
response to GO (59,60). In cell-line studies, apoptosis was not inhibited in the
presence of blocking anti-CD33 antibodies in case of continuous exposure to GO in
relatively high concentrations, whereas at lower concentrations apoptosis was
inhibited (61). This may be explained by other, CD33-independent uptake mecha-
nisms such as endocytosis. A more recent study, in patients undergoing treatment
with single-agent GO, showed that responders had higher median CD33 levels and
lower P-glycoprotein activity than nonresponders (62).
CLINICAL PERSPECTIVE FOR THE NEXT FIVE YEARS
The response rates to GO have not been promising enough to justify its use as a
single agent. Therefore, the current interest is to incorporate GO in modern
multiagent chemotherapy; however, only reduced dosages of GO can be used in
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this setting because of tolerability. Whether combination chemotherapy con-
taining GO will result in improved overall survival rates has to be awaited,
although the MRC AML15 study already showed a reduced relapse rate. It is
encouraging that the use of GO upfront did not lead to a significant increase in
death in CR in this study.
The current, conditional approval for GO by the FDA is restricted to
elderly AML patients in first relapse who are deemed unfit to receive more
intensive therapy only. In addition, there is currently no approval in Europe by
the European Agency for the Evaluation of Medicinal Product. This limited
approval status may be subject to change once more results become available.
GO may very well obtain an important role in the treatment of APL; for
instance, as salvage therapy in patients with detectable minimal residual disease
after consolidation chemotherapy or in molecular relapse, although larger pro-
spective studies are still lacking. Other options are to combine GO with ATRA
or arsenic trioxide, which might be an alternative to the use of regular chemo-
therapy in APL. GO is still an attractive drug for palliative treatment in AML,
given the lack of severe mucositis and alopecia and the possibility to administer
it in outpatient basis in most patients.
REFERENCES
1. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature 1975; 256:495497.
2. Margulies DH. Monoclonal antibodies: producing magic bullets by somatic cell
hybridization. J Immunol 2005; 174:24512452.
3. Harris M. Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol
2004; 5:292302.
4. Winter G, Harris WJ. Humanized antibodies. Trends Pharmacol Sci 1993; 14:139143.
5. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared
with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl
J Med 2002; 346:235242.
6. Gharib MI, Greenfield HM, Wynn RF, et al. Alemtuzumab (Campath IH) in con-
ditioning therapy with cyclophosphamide and total body irradiation in matched and
mismatched unrelated donor transplantation of children with acute lymphoblastic
leukaemia: a Report from 3 U.K. centres. Blood 2004; 104:593; (abstr).
7. Karp JE, Gojo I, Pili R, et al. Targeting vascular endothelial growth factor for
relapsed and refractory adult acute myelogenous leukemias: therapy with sequential
1-beta-d-arabinofuranosylcytosine, mitoxantrone, and bevacizumab. Clin Cancer Res
2004; 10:35773585.
8. Sievers EL, Appelbaum FR, Spielberger RT, et al. Selective ablation of acute
myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-
CD33 calicheamicin immunoconjugate. Blood 1999; 93:36783684.
9. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45
antibody plus cyclophosphamide and total body irradiation for advanced acute
leukemia and myelodysplastic syndrome. Blood 1999; 94:12371247.
120 Zwaan and van den Heuvel-Eibrink
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
10. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO)
classification of the myeloid neoplasms. Blood 2002; 100:22922302.
11. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia.
Blood 2005; 106:11541163.
12. Grimwade D, Moorman A, Hills R, et al. Impact of karyotype on treatment outcome
in acute myeloid leukemia. Ann Hematol 2004; 83(suppl 1):S45S48.
13. Lowenberg B. Strategies in the treatment of acute myeloid leukemia. Haematologica
2004; 89:10291032.
14. Creutzig U, Zimmermann M, Ritter J, et al. Treatment strategy and long-term results
in pediatric patients treated in four consecutive AMl-BFM trials. Leukemia 2005;
19:20302042.
15. Zwaan CM, Kaspers GJ. Possibilities for tailored and targeted therapy in paediatric
acute myeloid leukaemia. Br J Haematol 2004; 127:264279.
16. Linenberger ML. CD33-directed therapy with gemtuzumab ozogamicin in acute
myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms
of drug resistance. Leukemia 2005; 19:176182.
17. Jurcic JG, DeBlasio T, Dumont L, et al. Molecular remission induction with retinoic
acid and anti-CD33 monoclonal antibody HuM195 in acute promyelocytic leukemia.
Clin Cancer Res 2000; 6:372380.
18. Feldman E, Kalaycio M, Weiner G, et al. Treatment of relapsed or refractory acute
myeloid leukemia with humanized anti-CD33 monoclonal antibody HuM195.
Leukemia 2003; 17:314318.
19. Feldman EJ, Brandwein J, Stone R, et al. Phase III randomized multicenter study of a
humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with che-
motherapy, versus chemotherapy alone in patients with refractory or first-relapsed
acute myeloid leukemia. J Clin Oncol 2005; 23:41104116.
20. Bross PF, Beitz J, Chen G, et al. Approval summary: gemtuzumab ozogamicin in
relapsed acute myeloid leukemia. Clin Cancer Res, 2001; 7:14901496.
21. Sievers EL, Larson RA, Stadtmauer EA, et al. Efficacy and safety of gemtuzumab
ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse.
J Clin Oncol, 2001; 19:32443254.
22. Larson RA, Sievers EL, Stadtmauer EA, et al. Final report of the efficacy and safety
of gemtuzumab ozogamicin (Mylotarg) in patients with CD33-positive acute mye-
loid leukemia in first recurrence. Cancer 2005; 104:14421452.
23. Rajvanshi P, Shulman HM, Sievers EL, et al. Hepatic sinusoidal obstruction after
gemtuzumab ozogamicin (Mylotarg) therapy. Blood 2002; 99:23102314.
24. Wadleigh M, Richardson PG, Zahrieh D, et al. Prior gemtuzumab ozogamicin exposure
significantly increases the risk of veno-occlusive disease in patients who undergo
myeloablative allogeneic stem cell transplantation. Blood 2003; 102: 15781582.
25. Giles FJ, Kantarjian HM, Kornblau SM, et al. Mylotarg (gemtuzumab ozogamicin)
therapy is associated with hepatic venoocclusive disease in patients who have not
received stem cell transplantation. Cancer 2001; 92:406413.
26. Versluys B, Bhattacharaya R, Steward C, et al. Prophylaxis with defibrotide prevents
veno-occlusive disease in stem cell transplantation after gemtuzumab ozogamicin
exposure. Blood 2004; 103:1968; (letter).
27. Saviola A, Luppi M, Potenza L, et al. Late occurrence of hepatic veno-occlusive
disease following gemtuzumab ozogamicin: successful treatment with defibrotide.
Br J Haematol 2003; 123:752753.
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 121
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
28. Estey EH, Thall PF, Giles FJ, et al. Gemtuzumab ozogamicin with or without
interleukin 11 in patients 65 years of age or older with untreated acute myeloid
leukemia and high-risk myelodysplastic syndrome: comparison with idarubicin plus
continuous-infusion, high-dose cytosine arabinoside. Blood 2002; 99:43434349.
29. Amadori S, Suciu S, Stasi R, et al. Gemtuzumab ozogamicin (Mylotarg
1
) as single-
agent treatment for frail patients 61 years of age and older with acute myeloid
leukemia: final results of AML-15B, a phase 2 study of the European Organisation
for Research and Treatment of Cancer and Gruppo Italiano Malattie Ematologiche
dellAdulto Leukemia Groups. Leukemia 2005; 19:17681773.
30. Nabhan C, Rundhaugen LM, Riley MB, et al. Phase II pilot trial of gemtuzumab
ozogamicin (GO) as first line therapy in acute myeloid leukemia patients age 65 or
older. Leuk Res 2005; 29:5357.
31. van der Velden VH, Te Marvelde JG, Hoogeveen PG, et al. Targeting of the CD33-
calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in
vivo and in vitro saturation and internalization by leukemic and normal myeloid
cells. Blood 2001; 97:31973204.
32. Taksin AL, Legrand O, Raffoux E, et al. High efficacy and safety profile of fractionated
doses of Mylotarg as induction therapy in patients with relapsed acute myeloblastic
leukemia: a prospective study of the alfa group. Leukemia 2007; 21:6671.
33. Amadori S, Suciu S, Willemze R, et al. Sequential administration of gemtuzumab
ozogamicin and conventional chemotherapy as first line therapy in elderly patients
with acute myeloid leukemia: a phase II study (AML-15) of the EORTC and
GIMEMA leukemia groups. Haematologica 2004; 89:950956.
34. Tsimberidou A, Estey E, Cortes J, et al. Gemtuzumab, fludarabine, cytarabine, and
cyclosporine in patients with newly diagnosed acute myelogenous leukemia or high-
risk myelodysplastic syndromes. Cancer 2003; 97:14811487.
35. Linenberger ML, Hong T, Flowers D, et al. Multidrug-resistance phenotype and
clinical responses to gemtuzumab ozogamicin. Blood 2001; 98:988994.
36. Tsimberidou A, Cortes J, Thomas D, et al. Gemtuzumab ozogamicin, fludarabine,
cytarabine and cyclosporine combination regimen in patients with CD33 primary
resistant or relapsed acute myeloid leukemia. Leuk Res 2003; 27:893897.
37. Tsimberidou AM, Estey E, Cortes JE, et al. Mylotarg, fludarabine, cytarabine
(ara-C), and cyclosporine (MFAC) regimen as post-remission therapy in acute
myelogenous leukemia. Cancer Chemother Pharmacol 2003; 52:449452.
38. Chevallier P, Roland V, Mahe B, et al. Administration of mylotarg 4 days after
beginning of a chemotherapy including intermediate-dose aracytin and mitoxantrone
(MIDAM regimen) produces a high rate of complete hematologic remission in
patients with CD33 primary resistant or relapsed acute myeloid leukemia. Leuk
Res 2005; 29:10031007.
39. Kell WJ, Burnett AK, Chopra R, et al. A feasibility study of simultaneous admin-
istration of gemtuzumab ozogamicin with intensive chemotherapy in induction and
consolidation in younger patients with acute myeloid leukemia. Blood 2003;
102:42774283.
40. Burnett A, Kell WJ, Goldstone A, et al. The Addition of Gemtuzumab Ozogamicin
to Induction Chemotherapy for AML Improves Disease Free Survival without Extra
Toxicity: Preliminary Analysis of 1115 Patients in the MRC AML15 Trial. Blood
2006; 108:A8; (abstr).
122 Zwaan and van den Heuvel-Eibrink
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
41. Lo-Coco F, Cimino G, Breccia M, et al. Gemtuzumab ozogamicin (Mylotarg) as a
single agent for molecularly relapsed acute promyelocytic leukemia. Blood 2004;
104:19951999.
42. Estey EH, Giles FJ, Beran M, et al. Experience with gemtuzumab ozogamycin
(mylotarg) and all-trans retinoic acid in untreated acute promyelocytic leukemia.
Blood 2002; 99:42224224.
43. Aribi A, Kantarjian HM, Estey EH, et al. Combination therapy with arsenic trioxide,
all-trans retinoic acid, and gemtuzumab ozogamicin in recurrent acute promyelocytic
leukemia. Cancer 2007; 109:13551359.
44. Zwaan ChM, 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; 101:38683871.
45. Brethon B, Auvrignon A, Galambrun C, et al. Efficacy and tolerability of gemtuzumab
ozogamicin (anti-CD33 monoclonal antibody, CMA-676, Mylotarg) in children with
relapsed/refractory myeloid leukemia. BMC Cancer 2006; 6:172179.
46. Arceci RJ, Sande J, Lange B, et al. Safety and efficacy of gemtuzumab ozogamicin
(Mylotarg
1
)in pediatric patients with advanced CD33-positive acute myeloid
leukemia. Blood 2005; 106:11811188.
47. Zwaan CM, Reinhardt D, Zimmermann M, et al. A phase II study of single-agent
gemtuzumab ozogamicin in relapsed/refractory pediatric AML. J Clin Oncol 2005;
23:S806; (abstr).
48. Roman E, Cooney E, Harrison L, et al. Preliminary results of the safety of immu-
notherapy with gemtuzumab ozogamicin following reduced intensity allogeneic stem
cell transplant in children with CD33 acute myeloid leukemia. Clin Cancer Res
2005; 11:S7164S7170.
49. Brethon B, Yakouben K, Oudot C, et al. Efficacy of the combination of gemtuzumab-
ozogamicin (Mylotarg(R)) and cytarabine (GOCYT) in childhood myeloid leukemia: a
compassionate use based review in France. Blood 2006; 108:A570; (abstr).
50. Brenner MK, Wulf GG, Rill DR, et al. Complement-fixing CD45 monoclonal
antibodies to facilitate stem cell transplantation in mouse and man. Ann N Y Acad
Sci 2003; 996:8088.
51. Popat U, Carrum G, May R, et al. CD52 and CD45 monoclonal antibodies for
reduced intensity hemopoietic stem cell transplantation from HLA matched and one
antigen mismatched unrelated donors. Bone Marrow Transplant 2005; 35:11271132.
52. Bouabdallah R, Olive D, Meyer P, et al. Anti-GM-CSF monoclonal antibody therapy
for refractory acute leukemia. Leuk Lymphoma 1998; 30:539549.
53. Piloto O, Levis M, Huso D, et al. Inhibitory anti-FLT3 antibodies are capable of
mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment
of acute myelogenous leukemia blasts in nonobese diabetic/severe combined
immunodeficient mice. Cancer Res 2005; 65:15141522.
54. van Der Velden V, Boeckx N, Jedema I, et al. High CD33-antigen loads in peripheral
blood limit the efficacy of gemtuzumab ozogamicin (Mylotarg) treatment in acute
myeloid leukemia patients. Leukemia 2004; 18:983988.
55. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med 1997; 3:730737.
56. Bernstein ID. CD33 as a target for selective ablation of acute myeloid leukemia. Clin
Lymphoma 2002; (suppl 2):S9S11.
Monoclonal Antibody Mediated Treatment in Acute Myeloid Leukemia 123
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0005_O.3d] [3/4/08/12:47:6] [99
124]
57. Zwaan ChM, Reinhardt D, Jurgens H, et al. Gemtuzumab ozogamicin in pediatric
CD33-positive acute lymphoblastic leukemia: first clinical experiences and relation
with cellular sensitivity to single agent calicheamicin. Leukemia 2003; 17:468470.
58. Goemans BF, Kaspers GJL, Vijverberg SJH, et al. Large interindividual differences
in in-vitro calicheamicin sensitivity may underly gemtuzumab ozogamicin resistance
in acute myeloid leukemia. Blood 2005; 106:A35; (abstr).
59. Walter RB, Raden BW, Kamikura DM, et al. Influence of CD33 expression levels
and ITIM-dependent internalization on gemtuzumab ozogamicin-induced cytotoxicity.
Blood 2005; 105:12951302.
60. Jilani I, Estey E, Huh Y, et al. Differences in CD33 intensity between various
myeloid neoplasms. Am J Clin Pathol 2002; 118:560566.
61. Jedema I, Barge RM, van Der Velden V, et al. Internalization and cell cycle-dependent
killing of leukemic cells by Gemtuzumab Ozogamicin: rationale for efficacy in CD33-
negative malignancies with endocytic capacity. Leukemia 2004; 18:316325.
62. Walter RB, Gooley TA, van der Velden VH, et al. CD33 expression and P-glycoprotein-
mediated drug efflux inversely correlate and predict clinical outcome in patients with
acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy. Blood
2007; 109:41684170.
63. Piccaluga PP, Martinelli G, Rondoni M, et al. First experience with gemtuzumab
ozogamicin plus cytarabine as continuous infusion for elderly acute myeloid leu-
kaemia patients. Leuk Res 2004; 28:987990.
64. Alvarado Y, Tsimberidou A, Kantarjian H, et al. Pilot study of Mylotarg, idarubicin
and cytarabine combination regimen in patients with primary resistant or relapsed
acute myeloid leukemia. Cancer Chemother Pharmacol 2003; 51:8790.
65. Apostolidou E, Cortes J, Tsimberidou A, et al. Pilot study of gemtuzumab ozoga-
micin, liposomal daunorubicin, cytarabine and cyclosporine regimen in patients with
refractory acute myelogenous leukemia. Leuk Res 2003; 27:887891.
66. Cortes J, Tsimberidou AM, Alvarez R, et al. Mylotarg combined with topotecan and
cytarabine in patients with refractory acute myelogenous leukemia. Cancer Chemother
Pharmacol 2002; 50:497500.
67. Giles F, Garcia-Manero G, OBrien S, et al. Fatal hepatic veno-occlusive disease in a
phase I study of mylotarg and troxatyl in patients with refractory acute myeloid
leukemia or myelodysplastic syndrome. Acta Haematol 2002; 108:164167.
68. Lo-Coco F, Cimino G, Breccia M, et al. Gemtuzumab ozogamicin (Mylotarg) as a
single agent for molecularly relapsed acute promyelocytic leukemia. Blood 2004;
104:19951999.
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6
Monoclonal Antibodies in the Treatment
of Malignant Lymphomas and Chronic
Lymphocytic Leukemia
Bertrand Coiffier
Hematology Department, Hospices Civils de Lyon
and Claude Bernard University, Pierre-Benite, France
INTRODUCTION
Non-Hodgkins lymphoma (NHL) is a heterogeneous group of B- and T-cell
cancers, with a large variety of patterns of growth, clinical presentations, and
responses to treatment. Chronic lymphocytic leukemia (CLL) is a B-cell chronic
proliferation not very different from the small lymphocytic lymphoma, and both
diseases are often treated identically (1). The outcome depends on histological
subtype, tumor characteristics, host responses, and treatment. About 90% of
lymphomas have a B-cell phenotype, and for them recent therapeutic progress
came from the introduction of monoclonal antibodies (mAb) alone or in com-
bination with chemotherapy (24). The first antigen that has been targeted for
therapeutic purpose with success was the CD20 antigen, a trans-membrane
protein expressed by more than 99% of B-cell lymphomas. Rituximab was the
first mAb engineered to target the CD20 antigen and first approved mAb for
the treatment of lymphoma patients. Through the last 10 years, clinical trials
with rituximab have confirmed its efficacy in follicular lymphoma (FL) as well
as in aggressive lymphomas and its use has expanded significantly beyond the
initial indication of indolent B-cell lymphomas to virtually any CD20-positive
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lymphoma. The addition of rituximab to chemotherapy was the first real progress
in 10 years that has significantly prolonged the survival of patients with B-cell
lymphomas (4,5). In recent years, several other mAb targeting CD20 or other
lymphocyte antigens appeared, some of them associated with a toxin or a
radioisotope. However, most of the data generated today on mechanisms of
action or clinical efficacy have been for rituximab. Thus, rituximab will serve as
example in this review, and differences with other mAb will be outlined when
necessary.
MECHANISMS OF ACTION OF mAb
The mechanisms of action of mAb differ with the type of antibody, the antigen
they target, and their use: alone, in combination with chemotherapy, or con-
jugated to a toxin or a radionucleide. In case of a naked antibody, different
mechanisms have been identified (6). CD20 binding by rituximab is followed by
homotypic aggregation, rapid translocation of CD20 into specialized plasma
membrane microdomains known as rafts, and induction of apoptosis. Membrane
rafts concentrate Src family kinases and other signaling molecules (phospholi-
pases, caspases), and the anti-CD20-induced apoptotic signals are thought to
occur as a consequence of CD20 accumulation in rafts (7). Fas-induced apoptosis
occurs with the clustering of Fas molecules that leads to the formation of the
death-inducing signaling complex (DISC) and the downstream activation of the
death receptor pathway (8). The role of complement-dependent cytotoxicity
(CDC) is suggested by the consumption of complement observed after rituximab
administration, but in vitro CDC does not correlate with clinical response in
lymphomas (9,10). However, CDC seems to be the most important mechanism
of cell lysis in CLL patients (11). CDC is probably involved in the cytokine-
release syndrome and its toxicity (12). The importance of antibody-dependent
cellular cytotoxicity (ADCC) has been demonstrated in vivo when rituximab is
used alone (13). The Fc receptor (FcgR) of effector cells has two alleles and the
valine/valine (V/V) allele of FcgRIIIa which confers a higher affinity for
immunoglobulin G1 (IgG1) and rituximab is associated with an increased
responsiveness to rituximab (13,14). If the clinical relevance of the FcgRIIIa
receptor dimorphism was established in a number of studies with rituximab used
alone, it does not seem to play a major role when rituximab is used in combi-
nation with chemotherapy (15) even if one study showed an increased response
for patients with the V/V allele without difference for progression-free survival
(PFS) or overall survival (OS) (16). The immune mechanisms are probably valid
for other naked antigens but only those targeting the CD20 antigen may have the
direct action described here.
Finally, evidences that rituximab could synergize with chemotherapeutic
agents in B-cell killing were provided by Demidem (17). Subsequent inves-
tigations have confirmed synergy of rituximab with fludarabine, doxorubicin, and
other anticancer drugs (1820). In one hypothesis, this synergism is mediated, at
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least in part, via downregulation of interleukin-10 (IL-10) by rituximab, which in
turn causes downregulation of the antiapoptotic protein bcl2 and increased sen-
sitivity to apoptosis (21). Another mechanism involves the inhibition of the
activity of P-glycoprotein and, thus, the efflux of drugs like doxorubicin or vin-
cristine (22). In cell lines, the P-glycoprotein pump is translocated out of the lipids
rafts. Studies performed with cell lines as model systems revealed several
mechanisms that are involved in chemo/immunosensitization and the development
of resistance to rituximab. Rituximab has been shown to inhibit the p38 mitogen-
activated protein kinase, nuclear factor-kB (NF-kB), extracellular signal-regulated
kinase 1/2 (ERK 1/2) and AKT antiapoptotic survival pathways, all of which
result in upregulation of PTEN and Raf kinase inhibitor protein and in the
downregulation of antiapoptotic gene products (particularly Bcl-2, Bcl-xL, and
Mcl-1), and resulting in chemo/immunosensitization. Further, rituximab treatment
inhibits the overexpressed transcription repressor Yin Yang1 (YY1), which neg-
atively regulates Fas and DR5 expression, and its inhibition leads to sensitization
to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced
apoptosis (23).
If these mechanisms may have a role when mAb are combined with a radi-
onuclide, most of the antitumor effect resides in their capacity to deliver local
radiotherapy after the mAb is attached to tumor cells (24). The choice of the
antibody and therapeutic radioisotopes are critical for the success of radio-
immunotherapy (RIT). Several radiolabeled mAb have been studied in clinical trial
but only two, yttrium-90 (
90
Y or Y-90) ibritumomab tiuxetan and iodine-131 (
131
I or
I-131) tositumomab, have been registered for the treatment of lymphoma patients.
Both radiolabeled antibodies are mouse antibodies reacting with CD20 expressing
tumors. Y-90 is a pure b-emitter, with a half-life of 2.7 days (25). It is a link to the
antibody by a chelator (tiuxetan). The long pathlength of its b-particles is particu-
larly advantageous in tumor with heterogeneous or low distribution of the antigen
(26). I-131 is an a- and b-emitter that has a half-life of eight days. The path length of
its b-particles is relatively shorter than Y-90. Table 1 presents the differences
between Y-90 and I-131 radiolabeled antibodies.
Table 1 Characteristics of the Two Registered Radiolabeled MmAb
90
Y-ibritumomab
131
I-tositumomab
Linker Tiuxetan None
Isotope radiation decay Beta Beta and gamma
Half-life, days 2.7 8.0
Path length, mm 5.0 0.8
Energy, MeV 2.3 0.61
Non tumor distribution Bone Thyroid
Urine excretion Limited Substantial
Imaging Not possible Possible
Source: From Refs. 25, 106.
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An alternative approach to increase the activity of mAbs has been the
development of immunotoxin, a construct conjugating the antibody to cyto-
toxic plant, bacterial toxic proteins, or chemotherapy drugs (doxorubicin)
(27). The commonly used toxins, ricin or diphtheria toxin, are highly potent
natural products that disrupt protein synthesis. Unlike unconjugated mAb,
immunotoxins must be internalized after antigen binding to allow the toxin
access to the cytosol. Although the conjugation to mAbs confers some target
specificity, the toxin continues to mediate nonspecific toxicity to normal
tissues. Deglycosylated ricin A-chain has been used to eliminate such non-
specific toxicity.
MECHANISMS OF RESISTANCE
If multiple mechanisms of rituximab action have been reported, it remains
unclear which is or are most important in patients, and therefore it is difficult to
know the relative importance of potential mechanisms of resistance. This is true
for the other mAb too. Conceptual approaches of resistance mechanisms may be
resumed as was followed (28).
As far as events up to antigen binding are concerned, resistance to ritux-
imab may be secondary to low serum levels or rapid metabolism of the mAb;
development of human antimonoclonal antibodies (HAMA), most frequent with
nonhumanized antibodies than with rituximab, or human antichimeric antibodies
(HACA) (not yet demonstrated in patients); possibly different distribution within
malignant nodes, blood cells, marrow, and extranodal sites and responsible for
poor tumor penetration; high level of soluble antigen target (not yet demonstrated
for CD20 antigen); high tumor burden; and poor surface antigen expression.
Events that may induce resistance to rituximab after the antigen binding are
alteration of induced intracellular signals; reduction of direct apoptosis effect in
cases of elevated bcl-2 protein; inhibition of CDC by complement inhibitors; and
alteration of cell-mediated immunity. Gene microarray analysis has shown that
patients who failed to respond to rituximab have altered patterns of gene
expression, with an overexpression of genes important in cell-mediated immunity
(29). In vitro, long exposition to rituximab induced rituximab-resistant clones.
These clones exhibited constitutive hyperactivation of the nuclear factor-KB and
extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of
bcl-2 protein and bcl-2-related genes. These clones can be chemosensitized
following treatment with pharmacologic inhibitors like bortezomib (30).
In CLL patients, one particular mechanism of resistance has been
described where there is a high number of circulating B-cellsthe mono-
nuclear phagocytic system is rapidly saturated and rituximab-opsonized
cells are not cleared anymore. Then the complex rituximab-CD20 is shaved
from the cells that become CD20-negative or low and rituximab losses its
efficacy (31).
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SAFETY AND TOLERABILITY
The safety of mAb is mainly related to their origin and to the compound attached to
them. Radiolabeled mAb have a greater hematological toxicity than naked mAb
because of the effect of surrounding normal hematopoietic cells in bone marrow.
Immunotoxins also have greater toxicity because of the release of the toxin. Some
mAb such as alemtuzumab may have larger hematological toxicity because the
target antigen (CD52, in case of alemtuzumab) is not restricted to lymphoid cells.
The safety of rituximab is mainly related to infusion toxicity, a toxicity
most mAb have in common (32). These side effects are observed during the
infusion or in the first hours after drug infusion and particularly for the first
infusion. These include fever, chills, dizziness, nausea, pruritus, throat swelling,
cough, fatigue, hypotension, and transient bronchospasm in a majority of
patients. These symptoms are part of the cytokine-release syndrome. Their
intensity correlates with the number of circulating malignant cells at the time of
infusion. More severe infusional toxicity includes bronchospasm, angioedema,
and acute lung injury, which are often associated with high circulating cell
counts or pre-existing cardiac or pulmonary disease.
Another common toxicity is the rapid depletion of normal antigen-positive
B-lymphocytes from blood, bone marrow, and lymph nodes of the recipient, lasting
between six and nine months following the last administration of rituximab. In the
case of short rituximab treatment, this depletion does not compromise immunity:
Immunoglobulins do not decrease significantly, and patients do not have an
increased risk for infections during and after rituximab therapy (32,33); except for
some viruses like herpes virus, cytomegalovirus, or hepatitis B virus (HBV).
Maintenance treatment, particularly after autologous transplant, might be associated
with a decrease in immunoglobulins (34) and late toxicity (32).
Rare toxic events associated with rituximab comprised delayed neu-
tropenia and pulmonary reactions. Delayed neutropenia usually occurs in
patients treated with rituximab alone or in combination with chemotherapy. It
appears between one and six months after the last infusion, may be transient, is
rarely associated with infection, and resolves spontaneously in most of the cases
(35). The mechanisms are not fully understood. Pulmonary reactions are rare and
diverse, and usually related to rituximab because of the temporal relation (32).
RIT is associated with secondary myelodysplastic syndromes. Rituximab as
chemotherapy may induce a reactivation of hepatitis B in inactive HBV carriers.
Lamivudine or other antiviral treatment must be use prophylactically during
treatment and the following months to prevent this severe complication (36).
CLINICAL STUDIES
A few mAb have been registered for the treatment of lymphoma patients: rituximab
(Rituxan
1
or MabThera
1
),
90
Y-ibritumomab tiuxetan (Zevalin
1
),
131
I-tositumomab
(Bexxar
1
), and denileukin diftitox (OnTak
1
), the last two only in the United States.
However, a lot of other mAb are currently in preclinical, phase I, or phase II studies.
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Rituximab is certainly the mAb where the largest experience exists and the mAb
with several demonstrative randomized studies. We will focus on demonstrated
activity (phase III studies) and some phase II studies with promising results.
RITUXIMAB IN FOLLICULAR LYMPHOMA
Rituximab Alone in Relapse
When used alone, rituximab is usually given as four weekly injections of
375 mg/m
2
(37). The pivotal multicenter phase II study that included
166 patients treated with four infusions of rituximab showed an overall remission
rate of 48% [including 6% of complete response (CR)], and a median time to
progression (TTP) of 13 months (38). Elevated b2-microglobulin, elevated
lactate dehydrogenase (LDH), bulky disease, and age older than 60 years did not
appear to impact response, implying that patients regarded as having a poor
prognosis may respond to rituximab.
Patients relapsing after initial response to rituximab treatment may be
retreated with comparable response rates and adverse side effects, but, inter-
estingly, median time for progression might be longer than after first treatment
(39,40).
Whether prolonged treatment with rituximab or maintenance is able to
further improve response rates and prolong remission duration is of considerable
interest. Several arguments are in favor of this approach: the success of
re-treatment or the strong correlation between rituximab plasma levels and response
rates (41). A recent randomized trial showed that adding maintenance doses of
rituximab prolonged response duration (42); 202 patients with newly diagnosed
or refractory/relapsed FL were treated with rituximab. Patients responding, or
with stable disease, were randomized to no further treatment or prolonged rit-
uximab administration (375 mg/m
2
every two months for four times). With a
median follow-up of 35 months, the median event-free survival (EFS) was
prolonged in the treated group, 23 months versus 12 months in the control group.
However, patients relapsed within the six months after stopping rituximab
treatment. In another randomized study, Hainsworth showed that re-treatment at
relapse or prolonged treatment have the same benefit in terms of duration of
rituximab efficacy or time to chemotherapy (43).
Several questions remain without clear response: What is the optimal
prolonged treatment? What is the optimal duration maintenance? Which patients
benefit from prolonged treatment? And, finally, is prolonged treatment or
re-treatment at progression better in terms of survival or impact on transformation
rate?
Rituximab Alone in Untreated FL
Usually, patients with no adverse prognostic factors are not treated until they
develop such adverse parameters (44). However, because of its low-profile
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toxicity, its presumed low rate of secondary malignancy, and its lack of stem
cell toxicity, rituximab single agent was investigated in this setting (45): in a
series of 50 patients, a relative risk (RR) of 73% was obtained, with a CR of
26% ; 57% of the informative patients in CR reached a molecular remission.
However, even patients in CR and in molecular response did not seem to benefit
from this treatment because the median TTP was only two years, which is not
longer than that without treatment. A randomized study is currently underway in
the United Kingdom challenging this finding in these otherwise watch and
wait patients.
Rituximab alone was also studied in patients with a more aggressive
presentation, needing treatment at diagnosis, or after some follow-up without
treatment (46). The RR, just after four infusions, was comparable with the one
observed in relapsing patients (50% and <10% of CR). About 10% of patients had
progressive disease during the immediate posttreatment period and progression
occurred in less than 12 months among 50% of the responding patients.
Rituximab in Combination with Chemotherapy
In a phase II study, the combination of six cycles of CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) with rituximab given before, during,
and after chemotherapy in 40 patients with predominantly untreated FL
increased the RR [(55% CR, 40% partial response (PR)], with no added related
toxicity (47). Median TTP was 82 months.
Several randomized studies have now demonstrated that the addition of
rituximab to a standard chemotherapy regimen results in higher response rates
and longer TTP, EFS, and OS for patients treated with a combination of ritux-
imab plus chemotherapy (R-CHEMO) in first-line or in first-relapse patients
(Table 2). In first-line patients, four studies have reported a benefit in terms of
CR rates, PFS, and OS (4851). The first study randomized patients between
eight cycles of CVP (cyclophosphamide, vincristine, prednisone) and CVP
combined with rituximab (R-CVP) (48). At a median follow-up of 53 months,
patients treated with R-CVP had a highly significantly prolonged TTP (median
32 months vs. 15 months for CVP; P < 0.0001). Median time to treatment failure
(TTF) was 27 months in patients receiving R-CVP and 7 months in the CVP arm
(P < 0.0001). OS was longer for R-CVP than CVP (19% vs. 29% patients died,
P 0.03) (52). In the second study, patients were randomized between six
cycles of CHOP and CHOP combined with rituximab (R-CHOP) (51). In 428
patients, R-CHOP revealed a significantly higher RR (96% vs. 90%, P 0.011)
and a longer TTF (median not reached vs. 2.6 years, P < 0.0001).
In the French study, patients were randomized between CHVP (cyclo-
phosphamide, hydroxydaunomycin, vm 26, prednisone) plus interferon for
18 months and CHVP combined with rituximab (R-CHVP) plus interferon (53).
This analysis of all patients demonstrated a significant improvement of response
to therapy with R-CHVP plus interferon compared with CHVP plus interferon,
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both at six months [CR CRru 49% vs. 76%; PR 36% vs. 18%; respectively
(P < 0.0001)] and at 18 months [CR CRu 79% vs. 63%; PR 5% vs. 10%; res-
pectively (P 0.004)]. In the control arm, estimated 3.5 years EFS was 46% versus
67% with R-CHVP plus interferon (P < 0.0001). Even if the median follow-
up is only 3.5 years, this study showed a statistically significant OS advantage
for patients treated with rituximab (91% compared with 84% surviving at
3.5 years, P 0.029).
In relapsing patients, the FCM (fludarabine, cyclophosphamide, mitoxan-
trone) study showed that FCM combined with rituximab (R-FCM) is superior to
FCM alone for relapsing patients with follicular or mantle cell lymphoma (MCL)
(54). An update of this study showed that responding patients, treated either with
FCM or R-FCM had a prolonged PFS if they received a maintenance with
rituximab (55). The European Organisation for Research and Treatment of
Table 2 Randomized Studies Comparing Chemotherapy with the Combination of
Rituximab and Chemotherapy in Patients with Follicular Lymphoma
Setting
Response
rates
CR
rates EFS
Time to
progression OS
First-line patients
Marcus (48,52)
R-CVP 81% 41% 27 mo 32 mo Not different
CVP 57%
b
10%
b
7 mo
b
15 mo
b
Hiddemann (107)
R-CHOP 97% 20% 68 mo 50 mo Not analyzed
CHOP 90% 17% 21 mo
b
15 mo
b
Salles (50,53)
R-CHVP-Ifn
CHVP-Ifn
Not analyzed 79%
63%
b
Not reached Not reached Not analyzed
Herold (108)
R-MCP
MCP
85.5%
65.5%
b
42%
20%
b
Not reached
19 mo
b
Not reported Not reported
Relapsing patients
Forstpointer (54,55)
R-FCM
FCM
79%
58%
b
33%
13%
b
Not analyzed 16 mo
10 m
a
Not reached
24 m
b
Adjuvant rituximab
Hochster (57)
CVP?R
CVP
Not reported 30%
22%
a
Not reported 4.2 yr
1.5 yr
b
Trend in
favor of R
In parentheses are the references.
a
P < 0.05.
b
P < 0.01.
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Cancer (EORTC) study compared R-CHOP with CHOP alone in first or second
line patients not previously treated with doxorubicin (56). This last study is
particularly interesting because preliminary results showed a benefit of R-CHOP
over CHOP and also a benefit of rituximab maintenance after CHOP-only
induction. Rituximab maintenance yielded a median PFS from second ran-
domization of 51.5 months versus 15 months with observation [Hazard Ratio
(HR), 0.40; P < 0.001]. Improved PFS was found both after induction with
CHOP (HR, 0.30; P < 0.001) and R-CHOP (HR, 0.54; P 0.004). Rituximab
maintenance also improved OS from second randomization: 85% at three years
versus 77% with observation (HR, 0.52; P 011).
Finally, one study reported that maintenance with rituximab in patients
treated with chemotherapy increases CR rates and prolongs PFS (57). However, the
role of rituximab maintenance after a combination of rituximab plus chemotherapy
in first-line patients remains unclear and it is not currently recommended in CR
patients.
These different studies have implemented the use of combining rituximab
with chemotherapy as standard treatment in patients with FL who need to be
treated. Which of the chemotherapy regimens is better is not yet demonstrated
but the comparison of CR rates, EFS, PFS, and OS from the different studies
seems to show a larger benefit with the R-CHOP regimen. The comparison of
results obtained with R-CHOP to those reached with rituximab only in the same
type of patients equally favors the use of R-CHOP. However, these conclusions
need to be accepted with caution because no randomized study has compared
these different regimens.
RIT IN FOLLICULAR LYMPHOMA
Two mAb have been combined with a radionucleide and have been registered for
the treatment of patients with relapsing/refractory FL. RIT with Y-90 and I-131
labeled anti-CD20 antibodies (ibritumomab tiutexan and tositumomab) was
associated with a high response rate in relapsing/refractory patients. Zevalin was
not tested in untreated FL patients.
In the initial phase I/II study,
90
Y-ibritumomab was administered on
51 patients with relapsed and refractory CD20-positive B-cell NHL (58). The
overall response rate (ORR) for the 34 patients with indolent lymphoma was
82% (CR 26% and PR 56%). The estimate median TTP for the entire group was
12.9 plus months and the median duration of response was 11.7 plus months. The
major toxicity of
90
Y-ibritumomab was myelosuppression with thrombocyto-
penia being the most common.
90
Y-ibritumomab has been compared with
rituximab in a randomized controlled phase III study (59). The ORR was 80%
(CR/CRu 34% and PR 45%) for
90
Y-ibritumomab as compared with 56%
(CR/CRu 20% and PR 36%) for rituximab (P 0.002). The estimated TTP was
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12.6 months for
90
Y-ibritumomab and 10.2 months for rituximab (P 0.062). In
another study, Witzig treated with
90
Y-ibritumomab 54 patients with FL
refractory to rituximab (60). The ORR for the entire cohort was 74% (CR 14%
and PR 59%). The estimated TTP and response duration for responders were
8.7 and 6.4 months, respectively. Long-term responses are seen in 37% of the
patients with a median duration of response of 28 months in these good
responders (61).
131
I-tositumomab has been studied for more than 10 years. Vose et al. have
reported the final results of a multicenter phase II study with objectives to
evaluate the efficacy, dosimetry, methodology, and safety of
131
I-tositumomab
(62). Forty-seven patients with relapsed/refractory low-grade or transformed
NHL were treated with
131
I-tositumomab. The ORR for the entire group was
57% with 15 (32%) patients achieving CR. The ORR was similar in patients with
indolent (57%) or transformed lymphoma (60%). The median duration of
response was 8.2 months and 12.1 months, respectively, for each of these two
groups. The treatment was well tolerated and hematologic toxicity was the
principal adverse event. In the pivotal study, 60 patients with chemotherapy-
refractory indolent or transformed CD20-positive B-cell lymphoma (36 fol-
licular, 23 transformed, and 1 mantle cell) were treated with standard dose
131
I-tositumomab (63). The ORR was 65% (CR 20% and PR 45%). The median
duration of response was 6.5 months. Kaminski recently presented the results
of a phase II study evaluating
131
I-tositumomab alone in untreated patients with
FL (64). Of the 76 patients included, more than half did not have any criteria
associated with poor outcome and corresponded to patients who are usually not
treated. CR was observed in 75% of the patients, but only in 58% of those with a
large lymph node. Median PFS was 6.1 years for all patients, but less for patients
with criteria associated with poor outcome (details not given in the manuscript).
This study only showed that patients without large tumor may respond well to
131
I-tositumomab but it did not allow evaluating the role of this drug in patients
with FL.
Even though
131
I-tositumomab has shown interesting results in the phase II
study, duration of response is still limited. For this reason, some investigators are
beginning to evaluate
131
I-tositumomab in combination with other forms of
therapies. In a phase II study conducted by the Southwest Oncology Group
(SWOG) (65),
131
I-tositumomab was combined with CHOP for the treatment of
90 patients with untreated FL. Patients received six cycles of standard CHOP
followed by a consolidation dose of
131
I-tositumomab if PR was achieved. The
ORR after
131
I-tositumomab was 90% (CR/CRu 67% and PR 23%). More
interestingly, among patients assessable, 27 (57%) improved their level of
response after
131
I-tositumomab. The estimated two-year PFS and OS were 81%
and 97%, respectively (median follow-up 2.3 years). SWOG is currently con-
ducting a study comparing
131
I-tositumomab and rituximab in follicular patients
treated with CHOP as first treatment. Only such a study may evaluate the benefit
and toxicity of Bexxar in comparison with rituximab in first-line patients.
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A consensus meeting defined the optimal setting of RIT in the therapeutic
algorithm of patients with advanced stage of FL. According to the reviewed data,
RIT should preferably be used as consolidation after initial tumor debulking.
First-line RIT may be applied in patients not appropriate for chemotherapy
induction (66,67).
OTHER MONOCLONAL ANTIBODIES
Several MAb directed against CD20 (hA20, HuMax-CD20, ocrelizumab) or
other antigens (epratuzumab for CD22, apolizumab for HLA-DRB chain,
galiximab for CD80, SGN-40 for anti-CD40) are currently in phases I or II (68).
No definitive conclusion can be arrived at on their activity, toxicity, and benefit
compared with rituximab. The real interest of these new antibodies will have to
be demonstrated in randomized studies.
RITUXIMAB IN DIFFUSE LARGE B-CELL LYMPHOMA
The combination regimen R-CHOP, consisting of rituximab plus CHOP, is now
considered as the standard treatment for treating young and elderly patients with
diffuse large B-cell lymphoma (DLBCL) because of the superior activity dem-
onstrated in three randomized studies (Table 3). Results from the GELA study
have been recently updated with a five-year median follow-up and showed a
persisting advantage for patients treated with R-CHOP (Table 4) (69,70). In this
study, patients with DLBCL aged 60 to 80 years were treated either with eight
cycles of CHOP or eight cycles of R-CHOP. The difference observed between
the two arms was already statistically significant for EFS, PFS, and OS, with a
median follow-up of one year and improvement with follow-up.
The MInT study compared in 824 patients six cycles of R-CHOP-like
chemotherapy with CHOP-like chemotherapy in young patients with a low-risk
DLBCL (71). After a median time of follow-up of 34 months, chemotherapy
combined with rituximab (R-CHEMO) patients had a significantly longer TTF
(P < 0.00001), with estimated 2-year TTF rates of 60% (CHEMO) versus 76%
(R-CHEMO). Similarly, OS was significantly different (P < 0.001), with two-
year survival rates of 87% (CHEMO) and 94% (R-CHEMO), respectively. The
American study (ECOG/SWOG/CALGB study) was associated with a statistical
benefit in the primary endpoint TTF for R-CHOP versus CHOP alone (72).
However, the complicated design of this study makes conclusions difficult
compared with the two other studies. The interesting point of this study is the
second randomization looking at the effect of rituximab in patients who reached
a CR or a PR. If rituximab maintenance may decrease the progression rate in
patients treated with CHOP only, it has no effect on patients treated with
R-CHOP.
The same activity was shown in relapsing patients whether treated with
rituximab previously or not (73,74).
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Table 3 Randomized Studies Comparing CHOP with R-CHOP in Patients with DLBCL
Coiffier (69,70) Habermann (72) Pfreundschuh (71)
Setting
6080 yr old 6080 yr old <60 yr old
No stage I No stage I IPI 01
CHOP Maintenance CHOP or CHOP-like
Median follow-up 5 yr 2.7 yr 2 yr
CR rates
R-CHOP 75% 78% 85%
CHOP 63%
a
77% 65%
a
Early progression rates
R-CHOP 9% 15% 16%
CHOP 22%
a
17% 5%
a
Relapses
R-CHOP 34%
Not reported Not reported
CHOP 20%
a
Event-free survival 2-yr TTF
R-CHOP 3.8 yr 3.4 yr 81%
CHOP 1.1 yr
a
2.4 yr 58%
a
Progression-free survival
R-CHOP Not reached
Not reported Not reported
CHOP 1.0 yr
a
Overall survival 2-yr OS
R-CHOP Not reached
Not different
95%
CHOP 3.1 yr
a
85%
a
Note: In parentheses are the references.
a
P < 0.01
Abbreviations: DLBCL, diffuse large B-cell lymphoma; CR, complete response, IPI, International
Prognostic Index; TTF, time to treatment failure; OS, overall survival.
Table 4 5-Yr Survivals Observed in the GELA Study Comparing 8 Cycles of R-CHOP
and CHOP in Elderly Patients with DLBCL
R-CHOP CHOP P value
Median EFS 3.8 yr 1.1 yr 0.00002
5-yr EFS 47% 29%
Median PFS Not reached 1 yr <0.00001
5-yr PFS 54% 30%
Median OS Not reached 3.1 yr 0.0073
5-yr OS 58% 45%
Abbreviations: DLBCL, diffuse large B-cell lymphoma; EFS, event-free survival; PFS, progression-
free survival; OS, overall survival. Source: From Ref. 70.
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The experience of other mAb in DLBCL is quite restrained, but
90
Y-
ibritumomab tiuxetan has activity in rituximab-free relapsing patients (75).
mAb IN OTHER LYMPHOMAS
Small Lymphocytic Lymphoma
The efficacy of rituximab alone in this lymphoma is not very well known, with
few and discordant results. In a European study in relapsing patients, the efficacy
was low, with only a 10% RR (76). In untreated patients, in contrast, Hainsworth
found a 51% RR after four injections, with only 4% CR, and a median PFS of
18.6 months (77).
Marginal Zone Lymphoma
Most case reports have shown an efficacy of rituximab in these lymphomas.
Efficacy was demonstrated in relapsing mucosa-associated lymphoid tissue
(MALT) lymphoma (78). A current International Extranodal Lymphoma Study
Group (IELSG) trial randomizes chlorambucil versus chlorambucil plus rituximab
in new or relapsing patients with MALT lymphoma.
Mantle Cell Lymphoma
MCL has indolent lymphoma characteristics, but tends to pursue an aggressive
clinical course and is incurable with standard chemotherapy. An interim analysis
of a randomized trial comparing FCM with R-FCM has shown a striking
improvement in RR with rituximab (65% vs. 33%; CR 35% vs. 0%), with a trend
towards longer OS (54). Interestingly, about one-third of the patients achieved a
molecular remission. A maintenance with rituximab was scheduled for responding
patients and allowed to prolong the duration of response (55). Long-term remis-
sions have been reported with intensive chemotherapy and autologous stem cell
transplantation plus rituximab (see mAb and High Dose Therapy with Autologous
Stem Cell Transplantation.).
Chronic Lymphocytic Leukemia
Rituximab, given weekly as a single agent has low activity in relapsing patients
with CLL. A better activity has been observed in untreated patients (77). Dose
escalation, achieved by a thrice-weekly dosing schedule, (79) or higher weekly
doses, 500 to 2250 mg/m
2
(80) is necessary to reach significant clinical activity,
with an RR of 45% and 36%, respectively, as a single agent. The concurrent
administration of rituximab with fludarabine resulted in better results with an RR
rate of 90%, with 47% CR (81). Ongoing clinical studies are examining the use
of rituximab associated with fludarabine or pentostatin and cyclophosphamide,
which has shown great promise in a single-center phase II study (8284).
Monoclonal Antibodies 137
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Alemtuzumab is a humanized monoclonal antibody active against CD52.
Compared with CD20, CD52 is expressed at much higher density on the surface of
CLL cells. Activity of alemtuzumab in fludarabine-refractory CLL was established
in the pivotal trial conducted by Keating (85). In 93 patients, the ORR was 33%,
including 2% complete responders. The median time to response was six weeks and
the median time of therapy extended up to eight weeks. Median survival of all
patients was 16 months, but was 32 months for the responding patients. In heavily
pretreated patients with CLL, the ORR is approximately 35%, and in previously
untreated patients the ORR is greater than 80%, with a recent randomized study
suggesting it is superior to alkylator-based therapy (86). Importantly, alemtuzumab
is effective in patients with high-risk del(17p13.1) and del(11q22.3) CLL. Alem-
tuzumab combination studies with fludarabine and/or mAb such as rituximab have
demonstrated promising results. Alemtuzumab is also being studied in CLL patients
as consolidation therapy for treatment of minimal residual disease.
CLL cells are CD23 positive, and anti-CD23 (lumiximab) has clinical
activity in these patients (87).
Other Lymphomas
In posttransplant lymphoproliferative disease, several phase II have shown good
activity with rituximab alone or in combination with chemotherapy (88).
The only yet reported randomized study without benefit in the rituximab
arm was in patients with HIV-associated lymphoma: The response rate was not
statistically different in R-CHOP or CHOP arms (89). However, this study did
not have the power to show a significant difference, and the follow-up is
extremely short. In another phase II study, R-CHOP produced a CR rate of 77%
and a two-year survival rate of 75% without severe infectious complications (90).
In Europe, rituximab-containing chemotherapy is the standard for HIV-related
lymphomas (91).
Rituximab alone or combined with chemotherapy has activity in Wal-
denstrom macroglobulinemia (92).
Alemtuzumab have been reported active in cutaneous T-cell lymphomas
and peripheral T-cell lymphoma but activity was low and of short duration in
most of the cases (93,94).
The vast majority of the immunotoxin trials have been phase I studies
designated to determine the maximum tolerated dose. These trials have shown that
therapeutic serum levels may be achieved with tolerable toxicity. A relatively
uniform toxicity has been observed with vascular leak syndrome, hepatotoxicity,
and myalgia. The different trials have shown a low response rate of 10% to 25%
PRs without durable efficacy. The only available immunotoxin for patients is
OnTak for the treatment of cutaneous T-cell lymphoma; 30% of them responded
with a CR of 10% (95). The future of this therapy will depend on decreasing
toxicity, decreasing immune response against the construct, and increasing the
antitumor activity.
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Intrathecal rituximab was used in meningeal and CNS location of lymphomas.
It has no special toxicity and has activity for this difficult location (96).
mAb AND HIGH DOSE THERAPY WITH AUTOLOGOUS
STEM CELL TRANSPLANTATION
Rituximab has been used as an in vivo purging agent before and as maintenance
therapy after ASCT, in follicular and MCL (97,98) and in aggressive lymphoma
(99) in first-line or in relapse patients, with promising results. An ongoing
international trial in relapsed and refractory aggressive lymphoma randomizes
rituximab-DHAP (dexamethasone, aracytine, cisplatin) versus rituximab-ICE
(ifosfamide, carboplatin, etoposide) before ASCT and with a second random-
ization between rituximab maintenance and observation (CORAL study) (100).
Rituximab given after ASCT might have the interest to complete the remission
and to further decrease the relapse rate. However, this treatment may be asso-
ciated with more infections (101). It had been associated with severe decrease in
immunoglobulin levels (102) and more frequent neutropenia (35).
A few studies have looked at the potential use of radiolabeled mAb in the
context of HDT. As compared with external total body irradiation (TBI), radi-
olabeled monoclonal antibody could theoretically permit delivering higher dose
of radiation to the tumor while limiting radiation dose to normal tissues, thus
potentially reduce toxicity and treatmentrelated mortality (103). Press used
myeloablative doses of
131
I-tositumomab in combination with chemotherapy
(104). In this phase I/II study, 25 Gy was considered the maximum dose of
radiation that could be delivered to critical normal organs when combined with
cyclophosphamide (100 mg/kg) and etoposide (60 mg/kg). They observed an
objective response of 87% in a population of the patients with relapsed B-cell
lymphoma (73% indolent lymphoma). The estimated two years OS and PFS
were 83% and 68%. Ibritumomab tiuxetan followed by BEAM (Z-BEAM regimen)
has shown activity in patients with chemotherapy-refractory lymphomas, but its
advantage over BEAM alone is not yet demonstrated (105).
CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS
Rituximab has a large activity but not all patients respond. Defining at the molecular
level all mechanisms of action will allow defining the different mechanisms
involved in the resistance to this antibody. Thus, appropriate response may be
developed with the construct of new antibodies, defining new antigens, combination
of antibodies or antibodies plus chemotherapy or small molecules. The definitive
place of RIT must be defined.
Because of the large activity of rituximab, R-CHOP is now the standard for
treating patients with B-cell lymphoid malignancies. The role of the new anti-
bodies will have to be defined in comparison with R-CHOP in prospective
randomized studies. Because of the high ratio of efficacy/safety for rituximab,
Monoclonal Antibodies 139
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things will not be easy for the newcomers. Characteristics of patients not responding
well to R-CHOP will have to be found so that these new antibodies can be tested on
this poor-risk population.
T-cell malignancies do not have their antibodies yet, and different anti-
bodies will be tested. Other antigens will have to be found if the current ones do
not prove to be good.
Whatever the antigen and the antibody, mAb are now a standard part of the
treatment of lymphoma. Whether they will replace chemotherapy might be
considered, but this will not be the case for the next five years because the main
objective is to cure patients, and antibodies alone are not sufficient for that.
CONCLUSION
Rituximab was the first monoclonal antibody registered in the treatment of
lymphomas and it has allowed one of the major progresses for the treatment
of lymphoma patients. Alone, it is a very well-tolerated drug and has a great
activity in relapsing patients. However, it will hardly result in cure in this setting.
In combination with chemotherapy, rituximab allowed for the highest response
rates and longest EF and OS survivals ever described in FL and DLBCL. It has
activity, but it is less well demonstrated in other B-cell lymphomas. Other mAb
targeting CD20 or other antigens are on their way, but their activity is not yet
well defined compared with rituximab. RIT may add some specific activity, but
here too it is not well demonstrated. Antibodies conjugated with toxins are less
used at the moment.
REFERENCES
1. Jaffe ES, Harris NL, Stein H, et al., eds. World Health Organization Classification
of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid
Tissues. Lyon, France: IARC Press; 2001.
2. Coiffier B. Current strategies for the treatment of diffuse large B cell lymphoma.
Cur Op Hematol 2005; 12:259265 (review).
3. Coiffier B. First-line treatment of follicular lymphoma in the era of monoclonal
antibodies. Clin Adv Hematol Oncol 2005; 3:484491.
4. Coiffier B. Rituximab therapy in malignant lymphoma. Oncogene 2007; 26:
36033613.
5. Schulz H, Bohlius JF, Trelle S, et al. Immunochemotherapy with rituximab and
overall survival in patients with indolent or mantle cell lymphoma: a systematic
review and meta-analysis. J Natl Cancer Inst 2007; 99:706714.
6. Cartron G, Watier H, Golay J, et al. From the bench to the bedside: ways to improve
rituximab efficacy. Blood 2004; 104:26352642.
7. Janas E, Priest R, Wilde JI, et al. Rituxan (anti-CD20 antibody)-induced trans-
location of CD20 into lipid rafts is crucial for calcium influx and apoptosis. Clin
Exp Immunol 2005; 139:439446.
140 Coiffier
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
8. Stel AJ, Ten Cate B, Jacobs S, et al. Fas receptor clustering and involvement of
the death receptor pathway in rituximab-mediated apoptosis with concomitant
sensitization of lymphoma B cells to fas-induced apoptosis. J Immunol 2007; 178:
22872295.
9. Winkler U, Jensen M, Manzke O, et al. Cytokine-release syndrome in patients with
B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment
with an Anti-CD20 monoclonal antibody (Rituximab, IDEC-C2B8). Blood 1999;
94:22172224.
10. Weng WK, Levy R. Expression of complement inhibitors CD46, CD55, and CD59
on tumor cells does not predict clinical outcome after rituximab treatment in
follicular non-Hodgkin lymphoma. Blood 2001; 98:13521357.
11. Kennedy AD, Beum PV, Solga MD, et al. Rituximab infusion promotes rapid
complement depletion and acute CD20 loss in chronic lymphocytic leukemia.
J Immunol 2004; 172:32803288.
12. Bienvenu J, Chvetzoff R, Salles G, et al. Tumor necrosis factor alpha release is
a major biological event associated with rituximab treatment. Hematol J 2001; 2:
378384.
13. Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized anti-CD20
monoclonal antibody and polymorphism in IgG Fc receptor Fc gamma RIIIa gene.
Blood 2002; 99:754758.
14. Weng WK, Levy R. Two immunoglobulin G fragment C receptor polymorphisms
independently predict response to rituximab in patients with follicular lymphoma.
J Clin Oncol 2003; 21:39403947.
15. Boettcher S, Pott C, Ritgen M, et al. Evidence for Fcg receptor IIIA-independent
rituximab effector mechanisms in patients with follicular lymphoma treated with
combined immuno-chemotherapy. Blood 2004; 104:170a (abstr 589).
16. Kim DH, Jung HD, Kim JG, et al. FcGRIIIa gene polymorphisms may correlate
with response to frontline R-CHOP therapy for diffuse large B-cell lymphoma.
Blood 2006; 108:27202725.
17. Demidem A, Lam T, Alas S, et al. Chimeric anti-Cd20 (Idec-C2b8) monoclonal
antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic brugs.
Cancer Bioth Radiopharm 1997; 12:177186.
18. Alas S, Bonavida B, Emmanouilides C. Potentiation of fludarabine cytotoxicity on
non-Hodgkins lymphoma by pentoxifylline and Rituximab. Anticancer Res 2000;
20:29612966.
19. Alas S, Bonavida B. Rituximab inactivates signal transducer and activation of
transcription 3 (STAT3) activity in B-non-Hodgkins lymphoma through inhibition
of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2
and sensitization to cytotoxic drugs. Cancer Res 2001; 61:51375144.
20. Ghetie MA, Bright H, Vitetta ES. Homodimers but not monomers of Rituxan
(chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize
with a chemotherapeutic agent and an immunotoxin. Blood 2001; 97:13921398.
21. Vega MI, Huerta-Yepaz S, Garban H, et al. Rituximab inhibits p38 MAPK activity
in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug
resistance. Oncogene 2004; 23:35303540.
22. Ghetie MA, Crank M, Kufert S, et al. Rituximab but not other anti-CD20 antibodies
reverses multidrug resistance in 2 B lymphoma cell lines, blocks the activity of
Monoclonal Antibodies 141
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
P-glycoprotein (P-gp), and induces P-gp to translocate out of lipid rafts. J Immunother
2006; 29:536544.
23. Bonavida B. Rituximab-induced inhibition of antiapoptotic cell survival pathways:
implications in chemo/immunoresistance, rituximab unresponsiveness, prognostic
and novel therapeutic interventions. Oncogene 2007; 26:36293636.
24. Lemieux B, Coiffier B. Radio-immunotherapy in low-grade non-Hodgkins lymphoma.
Best Prac Res Clin Haematol 2005; 18:8195.
25. Cheson BD. Radioimmunotherapy of non-Hodgkin lymphomas. Blood 2003; 101:
391398.
26. Juweid ME. Radioimmunotherapy of B-Cell Non-Hodgkins lymphoma: from
clinical trials to clinical practice. J Nucl Med 2002; 43:15071529.
27. Rosenblum M. Immunotoxins and toxin constructs in the treatment of leukemia and
lymphoma. Adv Pharmacol 2004; 51:209228.
28. Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and
resistance. Oncogene 2003; 22:73597368.
29. Bohen SP, Troyanskaya OG, Alter O, et al. Variation in gene expression patterns in
follicular lymphoma and the response to rituximab. Proc Nat Acad Sci U S A 2003;
100:19261930.
30. Jazirehi AR, Vega MI, Bonavida B. Development of rituximab-resistant lymphoma
clones with altered cell signaling and cross-resistance to chemotherapy. Cancer Res
2007; 67:12701281.
31. Williams ME, Densmore JJ, Pawluczkowycz AW, et al. Thrice-weekly low-dose
rituximab decreases CD20 loss via shaving and promotes enhanced targeting in
chronic lymphocytic leukemia. J Immunol 2006; 177:74357443.
32. Kimby E. Tolerability and safety of rituximab (MabThera). Cancer Treat Rev 2005;
31:456473.
33. Rafailidis PI, Kakisi OK, Vardakas K, et al. Infectious complications of monoclonal
antibodies used in cancer therapy: a systematic review of the evidence from
randomized controlled trials. Cancer 2007; 109:21822189.
34. Lim SH, Zhang Y, Wang Z, et al. Maintenance rituximab after autologous stem cell
transplant for high-risk B-cell lymphoma induces prolonged and severe hypo-
gammaglobulinemia. Blood 2004; p. 395a.
35. Lemieux B, Tartas S, Traulle C, et al. Rituximab-related late-onset neutropenia after
autologous stem cell transplantation for aggressive non-Hodgkins lymphoma. Bone
Marrow Transplant 2004; 33:921923.
36. Coiffier B. Hepatitis B virus reactivation in patients receiving chemotherapy for
cancer treatment: role of Lamivudine prophylaxis. Cancer Invest 2006; 24:548552.
37. Maloney DG. Preclinical and phase I and II trials of rituximab. Sem Oncol 1999;
26:7478.
38. McLaughlin P, Grillolopez AJ, Link BK, et al. Rituximab chimeric anti-CD20
monoclonal antibody therapy for relapsed indolent lymphoma: half of patients
respond to a four-dose treatment program. J Clin Oncol 1998; 16:28252833.
39. Davis TA, Grillo-Lopez AJ, White CA, et al. Rituximab anti-CD20 monoclonal
antibody therapy in non-Hodgkins lymphoma: safety and efficacy of re-treatment.
J Clin Oncol 2000; 18:31353143.
40. Lemieux B, Bouafia F, Thieblemont C, et al. Second treatment with rituximab in
B-cell non-Hodgkins lymphoma: efficacy and toxicity on 41 patients treated at
CHU-Lyon Sud. Hematol J 2004; 5:467471.
142 Coiffier
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
41. Berinstein NL, Grillolopez AJ, White CA, et al. Association of serum rituximab
(IDEC-C2B8) concentration and anti-tumor response in the treatment of
recurrent low-grade or follicular non-Hodgkins lymphoma. Ann Oncol 1998; 9:
9951001.
42. Ghielmini M, Schmitz SF, Cogliatti SB, et al. Prolonged treatment with rituximab in
patients with follicular lymphoma significantly increases event-free survival and
response duration compared with the standard weekly 4 schedule. Blood 2004;
103:44164423.
43. Hainsworth JD, Litchy S, Shaffer DW, et al. Maximizing therapeutic benefit of
rituximab: maintenance therapy versus re-treatment at progression in patients with
indolent non-Hodgkins lymphoma: a randomized phase II trial of the Minnie Pearl
Cancer Research Network. J Clin Oncol 2005; 23:10881095.
44. Ardeshna KM, Smith P, Norton A, et al. Long-term effect of a watch and wait
policy versus immediate systemic treatment for asymptomatic advanced-stage
non-Hodgkin lymphoma: a randomised controlled trial. Lancet 2003; 362:
516522.
45. Colombat P, Salles G, Brousse N, et al. Rituximab (anti-CD20 monoclonal antibody)
as single first-line therapy for patients with follicular lymphoma with a low tumor
burden: clinical and molecular evaluation. Blood 2001; 97:101106.
46. Hainsworth JD, Litchy S, Burris HA, et al. Rituximab as first-line and maintenance
therapy for patients with indolent non-Hodgkins lymphoma. J Clin Oncol 2002; 20:
42614267.
47. Czuczman MS, Weaver R, Alkuzweny B, et al. Prolonged clinical and molecular
remission in patients with low-grade or follicular non-Hodgkins lymphoma treated
with rituximab plus CHOP chemotherapy: 9-year follow-up. J Clin Oncol 2004; 22:
47114716.
48. Marcus R, Imrie K, Belch A, et al. CVP chemotherapy plus rituximab compared with
CVP as first-line treatment for advanced follicular lymphoma. Blood 2005; 105:
14171423.
49. Herold M, Pasold R, Srock S, et al. Results of a prospective randomized open label
phase III study comparing rituximab plus mitoxantrone, chlorambucil, prednisolone
chemotherapy (R-MCP) versus MCP alone in untreated advanced indolent non-
Hodgkins lymphoma and mantle cell lymphoma. Blood (ASH meeting abstracts)
2004; 104:169a (abstr 584).
50. Salles G, Foussard C, Mounier N, et al. Rituximab added to CHVPIFN improves
the outcome of follicular lymphoma patients: first analysis of the GELA-GOELAMS
FL-2000 randomized trial in 359 patients. Blood (ASH meeting abstracts) 2004;
104:49a50a (abstr 160).
51. Hiddemann W, Kneba M, Dreyling M, et al. Frontline therapy with rituximab added
to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone
(CHOP) significantly improves the outcome for patients with advanced-stage fol-
licular lymphoma compared with therapy with CHOP alone: results of a prospective
randomized study of the German Low-Grade Lymphoma Study Group. Blood 2005;
106:37253732.
52. Marcus R, Solal-Celigny P, Imrie K, et al. MabThera (rituximab) plus cyclo-
phosphamide, vincristine and prednisone (CVP) chemotherapy improves survival in
previously untreated patients with advanced follicular non-Hodgkins lymphoma
(NHL). Blood 2006; 108:146a (abstr 481).
Monoclonal Antibodies 143
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
53. Foussard C, Mounier N, van Hoof A, et al. Update of the FL2000 randomized trial
combining rituximab to CHVP-Interferon in follicular lymphoma (FL) patients
(pts). Proc Am Soc Clin Oncol 2006; 24:424s (abstr 7508).
54. Forstpointner R, Dreyling M, Repp R, et al. The addition of rituximab to a com-
bination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly
increases the response rate and prolongs survival as compared to FCM alone in
patients with relapsed and refractory follicular and mantle cell lymphomas: results
of a prospective randomized study of the German low grade lymphoma study group
(GLSG). Blood 2004; 104:30643071.
55. Forstpointner R, Unterhalt M, Dreyling M, et al. Maintenance therapy with ritux-
imab leads to a significant prolongation of response duration after salvage therapy
with a combination of rituximab, fludarabine, cyclophosphamide and mitoxantrone
(R-FCM) in patients with relapsed and refractory follicular and mantle cell lymphomas:
results of a prospective randomized study of the German low grade lymphoma study
group (GLSG). Blood 2006; 108:40034008.
56. van Oers M, Klasa R, Marcus RE, et al. Rituximab maintenance improves clinical
outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both
with and without rituximab during induction: results of a prospective randomized
phase 3 intergroup trial. Blood 2006; 108:32953301.
57. Hochster HS, Weller E, Ryan T, et al. Results of E1496: A phase III trial of CVP
with or without maintenance with rituximab in advanced indolent lymphoma. Proc
Am Soc Clin Oncol 2003; 22:6502 (abstr).
58. Witzig TE, White CA, Wiseman GA, et al. Phase I/II trial of IDEC-Y2B8 radio-
immunotherapy for treatment of relapsed or refractory CD20 B-cell non-Hodgkins
lymphoma. J Clin Oncol 1999; 17:37933803.
59. Witzig TE, Gordon LI, Cabanillas F, et al. Randomized controlled trial of Yttrium-
90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immuno-
therapy for patients with relapsed or refractory low-grade, follicular, or transformed
B-cell non-Hodgkins lymphoma. J Clin Oncol 2002; 20:24532463.
60. Witzig TE, Flinn IW, Gordon LI, et al. Treatment with ibritumomab tiuxetan
radioimmunotherapy in patients with rituximab-refractory follicular non-Hodgkins
lymphoma. J Clin Oncol 2002; 20:32623269.
61. Witzig TE, Molina A, Gordon LI, et al. Long-term responses in patients with
recurring or refractory B-cell non-Hodgkin lymphoma treated with yttrium 90
ibritumomab tiuxetan. Cancer 2007; 109:18041810.
62. Vose JM, Wahl RL, Saleh M, et al. Multicenter phase II study of iodine-131
tositumomab for chemotherapy-relapsed/refractory low-grade and transformed low-
grade B-cell non-Hodgkins lymphomas. J Clin Oncol 2000; 18:13161323.
63. Kaminski MS, Zelenetz AD, Press OW, et al. Pivotal study of iodine I 131 tosi-
tumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell
non-Hodgkins lymphoma. Journal of clinical oncology 2001; 19:39183928.
64. Kaminski MS, Tuck M, Estes J, et al. 131I-tositumomab therapy as initial treatment
for follicular lymphoma. N Engl J Med 2005; 352:441449.
65. Press OW, Unger JM, Braziel RM, et al. A Phase 2 trial of CHOP chemotherapy
followed by tositumomab/iodine I 131 tositumomab for previously untreated fol-
licular non-Hodgkin ymphoma: Southwest Oncology Group Protocol S9911. Blood
2003; 102:16061612.
144 Coiffier
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
66. Davies AJ. Radioimmunotherapy for B-cell lymphoma: Y90 ibritumomab tiuxetan
and I(131) tositumomab. Oncogene 2007; 26:36143628.
67. Dreyling M, Trumper L, von Schilling C, et al. Results of a national consensus
workshop: therapeutic algorithm in patients with follicular lymphoma: role of
radioimmunotherapy. Ann Hematol 2007; 86:8187.
68. Wedgwood A, Younes A. Targeting lymphoma cells and their microenvironment
with novel antibodies. Clin Lymphoma Myeloma 2006; 7(suppl 1):S33S40.
69. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared
with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl
J Med 2002; 346:235242.
70. Feugier P, Van Hoof A, Sebban C, et al. Long-term results of the R-CHOP study in
the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the
Groupe dEtude des Lymphomes de lAdulte. J Clin Oncol 2005; 23:41174126.
71. Pfreundschuh M, Trumper L, Osterborg A, et al. CHOP-like chemotherapy plus
rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis
diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera
International Trial (MInT) Group. Lancet Oncol 2006; 7:379391.
72. Habermann TM, Weller EA, Morrison VA, et al. Rituximab-CHOP versus CHOP
alone or with maintenance rituximab in older patients with diffuse large B-cell
lymphoma. J Clin Oncol 2006; 24:31213127.
73. Sieniawski M, Staak O, Glossmann JP, et al. Rituximab added to an intensified
salvage chemotherapy program followed by autologous stem cell transplantation
improved the outcome in relapsed and refractory aggressive non-Hodgkin lymphoma.
Ann Hematol 2007; 86:107115.
74. El Gnaoui T, Dupuis J, Belhadj K, et al. Rituximab, gemcitabine and oxaliplatin: an
effective salvage regimen for patients with relapsed or refractory B-cell lymphoma
not candidates for high-dose therapy. Ann Oncol 2007; 18:13631368.
75. Morschhauser F, Depil S, Jourdan E, et al. Phase II study of gemcitabine-
dexamethasone with or without cisplatin in relapsed or refractory mantle cell
lymphoma. Ann Oncol 2007; 18:370375.
76. Foran JM, Rohatiner AZS, Cunningham D, et al. European phase II study of rituximab
(chimeric anti-CD20 monoclonal antibody) for patients with newly diagnosed mantle-
cell lymphoma and previously treated mantle-cell lymphoma, immunocytoma, and
small B-cell lymphocytic lymphoma. J Clin Oncol 2000; 18:317324.
77. Hainsworth JD, Litchy S, Barton JH, et al. Single-agent rituximab as first-line and
maintenance treatment for patients with chronic lymphocytic leukemia or small
lymphocytic lymphoma: a phase II trial of the Minnie Pearl Cancer Research
Network. J Clin Oncol 2003; 21:17461751.
78. Conconi A, Martinelli G, Thieblemont C, et al. Clinical activity of rituximab
in extranodal marginal zone B-cell lymphoma of MALT type. Blood 2003; 102:
27412745.
79. Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing
schedule in B-Cell chronic lymphocytic leukemia and small lymphocytic lymphoma
demonstrates clinical activity and acceptable toxicity. J Clin Oncol 2001; 19:
21532164.
80. OBrien SM, Kantarjian H, Thomas DA, et al. Rituximab dose-escalation trial in
chronic lymphocytic leukemia. J Clin Oncol 2001; 19:21652170.
Monoclonal Antibodies 145
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
81. Byrd JC, Peterson BL, Morrison VA, et al. Randomized phase 2 study of fludar-
abine with concurrent versus sequential treatment with rituximab in symptomatic,
untreated patients with B-cell chronic lymphocytic leukemia: results from Cancer
and Leukemia Group B 9712 (CALGB 9712). Blood 2003; 101:614.
82. Keating MJ, OBrien S, Albitar M, et al. Early results of a chemoimmunotherapy
regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for
chronic lymphocytic leukemia. J Clin Oncol 2005; 23:40794088.
83. Wierda W, OBrien S, Wen S, et al. Chemoimmunotherapy with fludarabine,
cyclophosphamide, and rituximab for relapsed and refractory chronic lymphocytic
leukemia. J Clin Oncol 2005; 23:40704078.
84. Kay NE, Geyer SM, Call TG, et al. Combination chemoimmunotherapy with
pentostatin, cyclophosphamide, and rituximab shows significant clinical activity
with low accompanying toxicity in previously untreated B chronic lymphocytic
leukemia. Blood 2007; 109:405411.
85. Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (Campath-1H)
in patients who have failed fludarabine: results of a large international study. Blood
2002; 99:35543561.
86. Alinari L, Lapalombella R, Andritsos L, et al. Alemtuzumab (Campath-1H) in the
treatment of chronic lymphocytic leukemia. Oncogene 2007; 26:36443653.
87. Byrd JC, OBrien S, Flinn IW, et al. Phase 1 study of lumiliximab with detailed
pharmacokinetic and pharmacodynamic measurements in patients with relapsed or
refractory chronic lymphocytic leukemia. Clin Cancer Res 2007; 13:44484455.
88. Choquet S, Oertel S, Leblond V, et al. Rituximab in the management of post-
transplantation lymphoproliferative disorder after solid organ transplantation:
proceed with caution. Ann Hematol 2007; 86:599607.
89. Kaplan LD, Scadden DT, for the AIDS Malignancies Consortium. No benefit from
rituximab in a randomized phase III trial of CHOP with or without rituximab for
patients with HIV-associated non-Hodgkins lymphoma: AIDS malignancies
consortium study 010. Proc Am Soc Clin Oncol 2004; 22:564 (abstr 2268).
90. Boue F, Gabarre J, Gisselbrecht C, et al. Phase II trial of CHOP plus rituximab in
patients with HIV-associated non-Hodgkins lymphoma. J Clin Oncol 2006;
24:41234128.
91. Mounier N, Spina M, Gisselbrecht C. Modern management of non-Hodgkin
lymphoma in HIV-infected patients. Br J Haematol 2007; 136:685698.
92. Dimopoulos MA, Anagnostopoulos A, Kyrtsonis MC, et al. Primary treatment of
Waldenstrom macroglobulinemia with dexamethasone, rituximab, and cyclo-
phosphamide. J Clin Oncol 2007; 25:33443349.
93. Lundin J, Hagberg H, Repp R, et al. Phase 2 study of alemtuzumab (anti-CD52
monoclonal antibody) in patients with advanced mycosis fungoides/Sezary
syndrome. Blood 2003; 101:42674272.
94. Enblad G, Hagberg H, Erlanson M, et al. A pilot study of alemtuzumab (anti-CD52
monoclonal antibody) therapy for patients with relapsed or chemotherapy-refractory
peripheral T-cell lymphomas. Phase II trial of subcutaneous anti-CD52 monoclonal
antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell
chronic lymphocytic leukemia (B-CLL). Blood 2004; 103:29202924.
95. Olsen E, Duvic M, Frankel A, et al. Pivotal phase III trial of two dose levels of
denileukin diftitox for the treatment of cutaneous T-cell lymphoma. J Clin Oncol
2001; 19:376388.
146 Coiffier
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0006_O.3d] [3/4/08/12:51:14]
[125148]
96. Rubenstein JL, Fridlyand J, Abrey L, et al. Phase I study of intraventricular
administration of rituximab in patients with recurrent CNS and intraocular lymphoma.
J Clin Oncol 2007; 25:13501356.
97. Gianni AM, Magni M, Martelli M, et al. Long-term remission in mantle cell
lymphoma following high-dose sequential chemotherapy and in vivo rituximab-
purged stem cell autografting (R-HDS regimen). Blood 2003; 102:749755.
98. Belhadj K, Delfau-Larue MH, Elgnaoui T, et al. Efficiency of in vivo purging with
rituximab prior to autologous peripheral blood progenitor cell transplantation in
B-cell non-Hodgkins lymphoma: a single institution study. Ann Oncol 2004; 15:
504510.
99. Horwitz SM, Horning SJ. Rituximab in stem cell transplantation for aggressive
lymphoma. Curr Hematol Rep 2004; 3:227229.
100. Hagberg H, Gisselbrecht C. Randomised phase III study of R-ICE versus R-DHAP
in relapsed patients with CD20 diffuse large B-cell lymphoma (DLBCL) followed
by high-dose therapy and a second randomisation to maintenance treatment with
rituximab or not: an update of the CORAL study. Ann Oncol 2006; 17(suppl 4):
IV31IV32.
101. Neumann F, Harmsen S, Martin S, et al. Rituximab long-term maintenance therapy
after autologous stem cell transplantation in patients with B-cell non-Hodgkins
lymphoma. Ann Hematol 2006; 85:530534.
102. Lim SH, Zhang Y, Wang Z, et al. Maintenance rituximab after autologous stem cell
transplant for high-risk B-cell lymphoma induces prolonged and severe hypo-
gammaglobulinemia. Bone Marrow Transplant 2005; 35:207208.
103. Press OW, Eary JF, Appelbaum FR, et al. Phase II trial of
131
I-B1 (anti-CD20)
antibody therapy with autologous stem cell transplantation for relapsed B cell
lymphomas. Lancet 1995; 346:336340.
104. Press OW, Eary JF, Gooley T, et al. A phase I/II trial of iodine-131-tositumomab
(anti CD-20), etoposide, cyclophosphamide, and autologous stem cell transplantation
for relapsed B-cell lymphomas. Blood 2000; 96:29342942.
105. Shimoni A, Zwas ST, Oksman Y, et al. Yttrium-90-ibritumomab tiuxetan (Zevalin)
combined with high-dose BEAM chemotherapy and autologous stem cell trans-
plantation for chemo-refractory aggressive non-Hodgkins lymphoma. Exp Hematol
2007; 35:534540.
106. Dillman RO. Radiolabeled anti-CD20 monoclonal antibodies for the treatment of
B-cell lymphoma. J Clin Oncol 2002; 20:35453557.
107. Hiddemann W, Dreyling MH, Forstpointner R, et al. Combined immuno-chemo-
therapy (R-CHOP) significantly improves time to treatment failure in first line
therapy of follicular lymphoma: results of a prospective randomized trial of the
German Low Grade Lymphoma Study Group (GLSG). Blood 2003; 102:104a
(abstr 352).
108. Herold M, Haas A, Srock S, et al. Rituximab added to first-line mitoxantrone,
chlorambucil, and prednisolone chemotherapy followed by interferon maintenance
prolongs survival in patients with advanced follicular lymphoma: an East German
Study Group Hematology and Oncology Study. J Clin Oncol 2007; 25:19861992.
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7
Radioimmunotherapy of Hematological
Malignancies
Tim Illidge and James Hainsworth
Paterson Institute of Cancer Research, School of Medicine,
University of Manchester, Manchester, U.K.
GENERAL INTRODUCTIONTHE PRINCIPLES
OF RADIOIMMUNOTHERAPY
The use of monoclonal antibody (mAb) in routine clinical practice is now well
established and has arguably led to some of the most significant improvements in
outcome for patients in hematological malignancies as well as in a wide range of
other malignancies including breast and bowel cancer (13). Although the single
agent activity of most mAb has been modest when used in combination with
other antitumor therapies, an additive or synergistic effect has been seen with
both chemotherapy and radiotherapy (2). The impressive increases in clinical
response rates seen with the combination of mAb and combination chemother-
apy has led to not only highly impressive increases in response rates, relapse-free
survival (RFS) but even overall survival (3).
Radioimmunotherapy (RIT) is the administration of mAb or mAb-derived
constructs, which are chemically conjugated to therapeutic radioisotopes tar-
geted to tumor. Initially, mAbs were regarded simply as direct carriers for the
radioisotope, which deliver systemically targeted cytotoxic radiation to areas of
disease with relative sparing of normal tissue. It is, however, becoming clearer
that the mAb effector mechanisms may also play an important additional role in
killing lymphoma cells. The nature of RIT determines that its efficacy depends
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on a number of factors, including the properties of the targeted antigen (spe-
cificity, density, availability, shedding, and heterogeneity of expression), the
tumor (degree of vascularization, blood flow, and permeability), the mAb
(specificity, immunoreactivity, stability, and affinity), and the properties of
chosen radioisotope (emission characteristics, half-life, and availability) (4).
A wide variety of different mAbs, delivery schedules, radioisotopes, and doses
of radioactivity have been used in RIT and have resulted in impressive durable
partial and complete responses (PRs and CRs) in the treatment of non-Hodgkins
lymphoma (NHL) (5). Two RIT drugs namely iodine-131 (
131
I)-tositumomab and
yttrium-90 (
90
Y)-ibritumomab have been approved by the U.S. Food and Drug
Administration (FDA) and
90
Y-ibritumomab tiuxetan is approved by the European
Union (EU). The use of RIT in leukemias is less well developed but the emerging
data looks highly encouraging that RIT may play a useful role in the conditioning
regimens for a wide variety of hematological malignancies as part of transplantation
strategies. This chapter focuses on the current clinical indications of radio-
immunoconjugates in hematological malignancy and provides an overview of the
clinical trials and ongoing studies. Finally a perspective will be given of how the use
of RIT in lymphoma and leukemia might develop in the next five years.
BRIEF DESCRIPTION OF PATHWAY(S) INVOLVED, ESPECIALLY
PARTS RELEVANT TO THE TREATMENT
Antigen Targeting
The use of radiation therapy in the treatment of hematological malignancy has
been well established and is highly effective if the disease is localized, as both
lymphomas and leukemias are exquisitely sensitive to cell death by radiation.
The systemic nature of the majority of lymphomas and leukemias, however,
makes localized irradiation inappropriate for most patients. Therefore, the sys-
temic delivery of RIT is a logical strategy given that these disseminated diseases
are highly radiosensitive. The effective delivery of RIT requires the selection of
a suitable tumor antigen target.
Tumor-specific antigens would be the ideal targets for RIT, but such a
degree of specificity is unusual. In practice, tumor-associated antigens, expressed
abundantly on tumor cells as well as some normal tissues, represent the majority
of potential targets. As most NHL are of B-cell origin, the pan-B-cell antigens
such as human leukocyte antigen (HLA-DR), CD19, CD20, CD22, CD37, CD52,
and MHC II have been extensively evaluated as targets for RIT (1,3,69). Table 1
shows the antigen characteristics that are considered ideal for RIT.
From these initial investigations, the CD20 antigen emerged as having
many of the characteristics thought to be important for a good tumor target and
therefore targeting this antigen has dominated clinical RIT of lymphoma (10).
The CD20 antigen is a transmembrane phosphoprotein that is expressed on
mature B lymphocytes and to a lower degree on pre-B lymphocytes at a higher
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density. The antigen is also expressed on greater than 90% of B-cell NHL. The
CD20 complex does not internalize or shed from the cell surface and initiates
signal transduction that triggers apoptosis through a caspase-dependent pathway
(11). CD20 is highly expressed on the majority of B-cell lymphomas but is not
expressed on stem cells or plasma cells and consequently following radiolabeled
anti-CD20 mAb RIT, the B-cell pool is replenished over the next few months.
Although most clinical RIT work has been targeted against the CD20 antigen,
other B-cell antigens such as CD22 are still being actively investigated (12).
As regards RIT in leukemia, CD45 and CD33 have been the most exten-
sively investigated antigen targets. The CD45 antigen is the most broadly
expressed of the known hematopoietic antigens. CD45 is a tyrosine phosphatase
that is expressed in different isoforms ranging in molecular mass from 180 to
220 kD. CD45 is found on nearly all leukocytes, including lymphoid and
myeloid precursors. Greater than 90% of acute myeloid leukemia (AML) biopsy
samples and most acute lymphoblastic leukemia (ALL) biopsy samples show
CD45 antigen expression, where cell surface antigen expression averages
200,000 copies per cell. Importantly in the context of RIT, the antigen does not
internalize after mAb binding (13).
CD33 is a 67-kDa type 1 transmembrane protein whose expression is
restricted to early multilineage hematopoietic progenitors, myelomonocytic
precursors, and more mature myeloid cells. CD33 is absent on normal pluri-
potent hematopoietic stem cells, though 85% to 90% of adult and pediatric cases
of AML express CD33 (14). Therefore, CD33 has gained clinical importance as
a suitable tumor-associated antigen and target for mAb-based AML therapies.
The CD66 antigen is expressed at approximately 200,000 molecules per cell on
normal myelopoietic cells from the promyelocyte onward but not on AML blasts.
Fortunately for RIT, the CD66 molecule is neither internalized nor shed and aberrant
expression is found on a significant fraction of CD10-positive ALL blasts (15).
Radioisotopes Used in RIT
The physical characteristics considered important for a radioisotope in RIT
include half-life, type of radioactive emissions (a, b, or g), and ionization path
Table 1 Table of Characteristics Considered Ideal in an Antigen Target for RIT
The characteristics of an ideal target antigen
Tumor cell specific
Highly expressed on tumor cells
No tendency to mutation
Not secreted or shed
Not rapidly modulated on antibody binding
Critical for target cell survival
Not expressed on critical or nonrenewable host cells
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length. Particle energy and mean path length in tissue are important determinants
of therapeutic efficacy. The emission profile of the radioisotope not only
determines its suitability for therapy, but also the toxicological profile of the
radiopharmaceutical.
The majority of clinical trials to date have used either
131
I or
90
Y because
of their favorable emission characteristics, availability, and well-documented
radiochemistry that permit reliable and stable attachment to mAbs.
131
I has the
advantage of a long history of successful use in the management of thyroid
cancer and a well-documented safety profile. It is readily available, inexpensive,
easily conjugated, and emits both b-particles with a path length of 0.8 mm and
penetrating b-emissions. The g-photons enable uncomplicated imaging using a
gamma camera for dosimetry purposes but also result in a significant nontargeted
normal tissue radiation dose as well as radiation protection issues for visitors and
medical/nursing staff.
90
Y offers a number of theoretical advantages over
131
I, although the
radioisotopes have not been directly compared by labeling the same mAbs
described in this chapter with either isotope.
90
Y is a pure b-emitter that pro-
duces higher energy radiation (2.3 MeV vs. 0.6 MeV) at a longer path length
than
131
I (5.3 mm vs. 0.8 mm). Radiolysis induces cellular damage in both the
targeted lymphoma cells and neighboring cells. The increased path length would
be expected to enhance the cross fire effect and could, therefore, be poten-
tially advantageous in treating bulky, poorly vascularized tumors with hetero-
geneous antigen expression (4). This longer path length is likely, however, to
increase the normal tissue dose when targeting microscopic disease for which the
shorter b-particle path length of
131
I may be preferable.
The physical half-life of
90
Y is 64 hours and decays to a stable (non-
radioactive) form of zirconium (
90
Zr). The physical half-life of 64 hours
approximates to the biological half-life of murine mAbs and the absence of
penetrating g-emissions enables delivery as an outpatient (16). Additionally, if a
cell internalizes
90
Y, it is likely to be retained within the cell (12). In contrast,
once
131
I-conjugates are internalized by a cell, there is rapid dehalogenation of
the free iodide and subsequent excretion of the iodinated products out of the cell,
reducing desired tumor absorbed radiation dose and increasing normal tissue
radiation exposure (17). The major disadvantages of
90
Y relate to its greater
expense, relatively limited availability, and requirement for chelation radio-
chemistry making radiolabeling a more complex procedure.
90
Y does not emit
g-photons and, therefore, there is a need to use indium-111 as a surrogate to
obtain images for biodistribution and dosimetry studies. Rhenium-186 (
186
Re)
and copper-67 (
67
Cu) are both b-emitters and have physical and chemical
properties that make them attractive alternatives to either
131
I or
90
Y. Never-
theless, their current limited availability has meant that these radioisotopes have
received limited clinical use (18).
a-Emitters produce a helium nucleus particle of very high energy but with
a very short path length. The high linear energy transfer (LET) radiation of
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a-emitters may be lethal to cells with a single collision; however, the very short
path length means that the isotope must be adjacent to, or internalized by, the
cell to be effective and is likely to have little or no cross-fire effect. The suit-
ability of a-emitters, therefore, appears limited to readily accessible tumors such
as leukemia cells confined to the blood or bone marrow. The short half-life of
a-emitters [e.g., astatine-211 (
211
At)7 hours or bismuth-213 (
213
Bi)
45 minutes] complicates the radiopharmaceutical preparation, meaning that such
radioisotopes are likely to require generation on the same site as delivery in the
clinic. Despite this logistical hurdle, early clinical data in the treatment of leu-
kemia appear extremely promising (19,20).
In practice, the choice of the optimal radioisotope for RIT remains con-
troversial, with proponents advocating the relative merits of
131
I,
90
Y,
186
Re,
67
Cu, and a-emitters such as
211
At (16). Comparative studies are difficult to
conduct and scientifically robust randomized human trials have not been per-
formed. The ideal properties of a radioisotope for RIT remain unclear and it is
likely that the optimal radioisotope for a particular situation will depend upon the
bulk and type of tumor being targeted. An important area of potential future
research will be to define the optimal radioisotope, or cocktail of isotopes,
required for different tumor sizes.
BRIEF REVIEW OF RELEVANT TRANSLATIONAL RESEARCH
Factors Affecting the Therapeutic Efficacy of RIT in Lymphomas
Although RIT has emerged as a highly effective treatment for NHL, the
underlying mechanisms of action and in particular the interaction of tumor
irradiation and mAb signaling in RIT are still poorly understood (16,21). There
remain a number of important questions where further work is required to
address important issues required to optimize RIT delivery and where further
translational research may further inform our current knowledge. These areas
include
1. The optimal predose of cold mAb
2. The relative contribution of targeted radiation and mAb effector mecha-
nisms to the overall response and the mechanisms involved in the durable
responses seen in some patients
3. Defining whether a tumor radiation dose response exists in RIT for lym-
phomas and leukemias
Predosing of mAb in RIT
There are several factors that may theoretically limit lymphoma targeting of
radiolabeled pan-B-cell mAb in RIT. These include the complex formation of
administered mAb with free-circulating target antigen, the cross reactivity with
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antigen-positive circulating lymphoma cells, normal B cells in the blood or
spleen or nonlymphoid tissues, and finally the nonantigenic binding of mAb such
as binding by Fc arm of a mAb. Poor tumor targeting of a radioimmunoconjugate
leads to lower tumor to normal tissue radiation-dose ratios resulting in poten-
tially reduced therapeutic efficacy.
In order to improve the biodistribution of radiolabeled mAb in RIT, it has
become the established practice to give a predose of cold or unlabeled anti-
CD20 mAb prior to the therapeutic dose of the anti-CD20 radio-
immunoconjugate (22). The predose is considered to prolong the circulating
half-life of the radiolabeled mAb, block non-specific binding sites (e.g.,
circulating and splenic B cells), and result in increased tumor retention of the
labeled mAb. Buchsbaum et al. investigated whether a predose of anti-B1
improved the delivery of a subsequent radiolabeled mAb to tumor using in vivo
preclinical human xenograft models (23). The anti-B1 (anti-CD20) pan-B-cell
mAb that is reactive with human B-cell lymphomas but is not reactive with host
mouse B cells was used. A predose of unlabeled anti-B1 was found to sig-
nificantly increase the tumor-uptake of the subsequent radiolabeled anti-B1
although this improvement in tumor targeting appeared to plateau at the highest
predoses of unlabeled anti-B1.
Relative Contribution of Antibody Effector Mechanisms
and Targeted Radiation to Therapy
RIT has emerged as an effective treatment for lymphoma; however, the under-
lying mechanisms are poorly understood. By using different syngeneic murine
B-cell lymphoma models, the relative contributions of mAb and targeted radi-
ation to the clearance of tumor in vivo have been investigated (24). There is now
substantial evidence that mAbs can form an active component of RIT and that
mAb effector mechanisms may be important in the clearance of tumor in vivo.
Although the exact in vivo mechanisms of tumor killing by anti-CD20
mAb remain incompletely understood, preclinical data have suggested that the
action of rituximab may include mAb-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), and the direct induction of apoptosis
through cell surface mediated downstream signal transduction (25). Cragg and
Glennie reported that a panel of anti-CD20 mAbs (rituximab, 1F5, and anti-B1)
acts through distinctively different mechanisms in the therapy of two lymphoma
xenograft models (25). Rituximab and 1F5 redistribute CD20 into membrane
rafts and are bound efficiently by the complement component C1q and deposit
C3b resulting in CDC, which forms the major therapeutic effect of these two
mAbs. In contrast, complement depletion had no effect on the potent therapeutic
activity of anti-B1 (tositumomab), a mAb that does not redistribute CD20 into
membrane rafts, bind C1q, or cause efficient CDC. F(ab)2 fragments of anti-B1
(tositumomab), but not 1F5, were observed to be able to provide substantial
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immunotherapy, indicating that non-Fc-dependent mechanisms are involved in
the tositumomab action. In accordance with this, tositumomab was shown to
induce much higher levels of apoptosis than rituximab and 1F5, suggesting that
while complement is important for the action of rituximab and 1F5, this is not
the case for tositumomab, which more likely functions through its ability to
induce downstream signal transduction that results in apoptosis.
The relative importance of mAb effector mechanisms and targeted radia-
tion are difficult to measure and it is practically impossible to dissect the action
of the two components in clinical RIT. However, preclinical studies using well-
defined syngeneic animal models have helped to further clarify the relative
contributions of mAb effector mechanisms and targeted radiation. The ability of
some anti-B-cell mAbs to improve survival with targeted radiotherapy appeared
to correlate with their ability to initiate intracellular signal transduction.
Together these data illustrate that using one mAb to target radiation to tumor and
a second to induce cell signaling may be an effective new strategy in lymphoma
RIT.
Defining Whether a Tumor Radiation Dose Response Exists
in RIT for Lymphomas and Leukemias
There is currently little preclinical data that demonstrate whether a radiation dose
response exists. Using syngeneic B-cell lymphoma models, Du et al. have
demonstrated that with mAbs used solely as vectors to deliver radiation to tumor,
there does appear to be a radiation dose response (24).
To date, despite the high response rates seen in lymphoma RIT, clinical
dosimetry studies have thus far failed to show a consistent dose-response rela-
tionship (4). More recently, the Michigan group has concluded that there could
be a radiation dose response at least for
131
I-tositumomab (22,26); however, their
conclusions differ to others (27,28). Postema, for example, argues that none of
the RIT dosing methods use tumor dosimetry to determine the dose administered
to patients because the myelotoxicity of radiolabeled mAb will limit the incre-
ments of radioactivity dose but not the tumor absorbed dose (27). More recently,
Goldenberg and colleagues commented that because RIT has two potentially
therapeutic arms, namely radiation and mAb mechanisms, poor radiation tar-
geting does not exclude a good therapeutic response from the mAb (28).
Preclinical Leukemia Modeling
The group from Memorial Sloan-Kettering Cancer Center initially described the
preclinical administration of
213
Bi-HuM195 (anti-CD33) constructs (29). The
213
Bi-HuM195 construct was rapidly internalized into the cell in a time-depen-
dent manner ranging from 50% at 1 hour to 65% at 24 hours.
205
Bi/
206
Bi-labeled
constructs (bismuth isotopes with a longer physical half-life) were stable for
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at least two days in vitro in the presence of human serum at 378C. After injection
into mice, there was no observed uptake or loss of bismuth to tissues not
expressing the CD33 antigen, including the kidney which has a high affinity for
free bismuth. Mice were treated by intraperitoneal injection of
213
Bi-HuM195.
Doses ranging from 18.5 to 740 MBq/kg showed no toxicity, but at the higher
dose of 2590 MBq/kg, two-thirds of the mice died within two weeks and a third
of the mice showed significant reductions in white blood cell counts. Leukemia
cell killing in vitro with
213
Bi-HuM195 showed dose- and specific activity
dependent killing of CD33 positive HL60 leukemic cells; approximately 50%
killing was observed when two bismuth atoms (50 fM radiolabeled mAb) were
initially bound onto the target cell surface. The authors concluded that the
physical and biochemical properties are suitable for initial RIT studies in
humans.
The Seattle group has adopted a different targeting strategy and has tested
myeloablative doses of
131
I-anti-CD45 mAb prior to autologous stem cell
transplantation (ASCT) (30). This strategy was developed to reduce the overall
systemic radiation dose from total body irradiation (TBI) and to increase the
targeted radiation dose delivered to hematopoietic tissues with the goal of
decreasing relapse rates without increasing toxicity.
To determine whether the
131
I-anti-CD45 mAb was able to provide suf-
ficient immunosuppression for efficient transplantation across allogeneic bar-
riers, T-cell-depleted BALB.c marrow was transplanted into H2-compatible
B6-Ly5
a
mice after
131
I-30F11 (rat anti-murine CD45) mAb with or without
varying dose levels of TBI. Groups of five or six recipient mice per
131
I- or
TBI-dose level per experiment were given tail vein injections of 100 mg of
131
I-
30F11 mAb four days before marrow infusion, with or without TBI on day 0.
Engraftment, defined as 50% or greater donor B cells at three months post-
transplant, was determined by two-color flow cytometric analysis of peripheral
blood granulocytes, T cells, and B cells using mAbs specific for donor and host
CD45 allotypes and for CD3. Donor engraftment of 80% or more recipient
mice was achieved with either 8 Gy of TBI or 27.75 MBq (0.75 mCi) of
131
I-
30F11 mAb, with the radioimmunoconjugate delivering an estimated 26 Gy to
bone marrow. Subsequent experiments determined the dose of TBI alone or
TBI plus 27.75 MBq (0.75 mCi) of
131
I-30F11 mAb necessary for engraftment
in recipient mice that had been presensitized to donor antigens before trans-
plant, a setting requiring more stringent immunosuppression. Engraftment was
seen in 80% or more of presensitized recipients surviving after TBI (14 to
16 Gy or 12 to 14 Gy) and 27.75 MBq (0.75 mCi) of
131
I-30F11 mAb. How-
ever, only 28 of 69 (41%) presensitized mice receiving 10 to 16 Gy of TBI
alone survived. The authors suggest that targeted radiation delivered by
131
I-
anti-CD45 mAb provides sufficient immunosuppression to replace an appre-
ciable portion of the TBI dose used in matched sibling hematopoietic stem cell
transplant.
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REVIEW OF COMPLETED, ONGOING,
AND PLANNED CLINICAL STUDIES
Nonmyeloablative RIT in Lymphomas
Clinical RIT trials in NHL differ in terms of eligibility criteria, mAb and
radioisotope used, dose, number of treatments, doses of unlabeled mAb pre-
infused or coinfused, and the biodistribution or dosimetry estimations required
for administration of a therapeutic dose of radiolabeled mAb. Nevertheless,
virtually all clinical studies performed to date have shown high response rates in
for RIT in NHL and have been well reviewed (4,3135).
DeNardo et al. initially pioneered RIT for NHL with
131
I-anti-HLA-DR
mAb (Lym-1) (7). The efficacy of escalating fractionated doses of
131
I-Lym-1
ranging from 1480 to 3700 MBq/m
2
(40100 mCi/m
2
) resulted in an overall
response rate (ORR) of 52% in 21 treatment courses administered to 20
patients, with seven patients (33%) achieving CR, and four patients (19%)
achieving PR (7).
Goldenberg et al. used a
131
I-LL2 (anti-CD22) mAb to treat a variety of B-
cell lymphomas. In one of their trials, 4 out of 17 patients achieved objective
remission including 1 CR (36). In another trial,
90
Y-LL2 was administered to
seven patients with B-cell lymphomas, two of whom achieved PR (Table 2) (36).
Impressive responses have been observed in all of the clinical trials using
90
Y-ibritumomab tiuxetan and
131
I-tositumomab in relapsed B-cell lymphomas.
Although both
131
I-tositumomab and
90
Y-ibritumomab tiuxetan bind to the same
CD20 antigen, tositumomab binds to a unique epitope of CD20 (37). The
radioisotopes also have important differences in their emission characteristics.
Table 3 compares the main characteristics of
131
I-tositumomab and
90
Y-
ibritumomab tiuxetan.
Table 2 Results of a Randomized Controlled Trial of
90
Y-Ibritumomab Tiuxetan Vs.
Rituximab in Relapsed or Refractory Low-Grade or Transformed Follicular B-Cell NHL
Phase I/II
(n 51)
Phase II
(n 30)
Phase III
(n 73)
Overall response (%) 73 83 80
Median DR (mo) 11.7 11.5 13.9
CR, Cru (%) 29 47 34
Median DR (mo) 28 23 23
Ongoing CR, Cru (%) 19 14 32
Median DR (mo) 62.1 41.2 42.2
Range 60 to 66 40 to 42 33 to 48
Source: From Refs. 43,79.
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90
Y-ibritumomab tiuxetan consists of a monoclonal IgG1K anti-CD20
mAb, the murine parent immunoglobulin of rituximab, covalently attached to a
metal chelator molecule (tiuxetan, an isothiocyanatobenzyl derivative of the
polyaminocarboxylic acid DTPA), which stabilizes the mAb-isotope complex
for delivery to the lymphoma site (38). Biological half-life elimination of
90
Y-
ibritumomab tiuxetan is 30 hours. More than 90% of the b-radiation is absorbed
within a 5-mm proximity (corresponding to a diameter of 100 to 200 cells) of the
radiation source. This facilitates highly targeted delivery of radiation without
the need for patient isolation or shielding (16). The tiuxetan chelator molecule
provides a stable link between the mAb and the radioisotope, and, therefore, free
isotope clearance rates are minimal and predictable with 7.3% 3.2% of
the radiolabeled activity being excreted in the urine over seven days (39).
Consequently
90
Y-ibritumomab tiuxetan may be administered on an outpatient
basis. Figure 1 outlines the
90
Y-ibritumomab tiuxetan therapeutic regimen.
Four clinical trials, including three phase I/II and one randomized study
formed the basis of the FDA submission for
90
Y-ibritumomab tiuxetan. The
initial phase I/II study demonstrated that the dose limiting toxicity was myelo-
toxicity (39). The maximum-tolerated dose (MTD) was identified as 15 MBq/kg
(0.4 mCi/kg) to a maximum of 1184 MBq (32 mCi) for patients with a baseline
platelet count of greater than or equal to 150 10
9
/L and 11.1 MBq/kg
Table 3 Characteristics of
131
I-Tositumomab (Bexxar) and
90
Y-Ibritumomab Tiuxetan
(Zevalin)
131
I-tositumomab
90
Y-ibritumomab tiuxetan
U.S. trade name Bexxar Zevalin
Monoclonal antibody tositumomab (anti-B1)-murine ibritumomab (2B8)-murine
Chelation Simple More complex
Isotope
131
I
90
Y
Isotope emissions g and b b only
b-energy 0.606 MeV 2.293 MeV
b-particle path length 0.8 mm 5.3 mm
Isotope half-life 8 days 2.6 days
g-energy 0.364 MeV None
Radiation protection
measures
4- to 6-day inpatient stay in
shielded room
Outpatient
Isotope excretion Renal (variable) Limited
Normal tissue uptake Thyroid (preblocked with KI) Bone
Predose (unlabeled mAb) tositumomab (450 mg/patient) rituximab (250 mg/m
2
) 2
Dose 75 cGy whole body dose 15 MBq/kg (0.4 mCi/kg)
Dosimetric dose obligatory Dosimetric dose not required
Dose reduction for
thrombocytopenia
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(0.3 mCi/kg) for patients with baseline platelet counts of less than 150 10
9
/L
but greater than or equal to 100 10
9
/L. In this study, a high ORR for the intent-
to-treat population (n 51) was seen at 67% (26% CR; 41% PR); for low-grade
disease (n 34), 82% (26% CR; 56% PR); for intermediate-grade disease
(n 14), 43%.
A phase II study of patients with mild thrombocytopenia (baseline platelet
count of 100 10
9
/L to 150 10
9
/L) was conducted using the reduced dose of
11.1 MBq/kg (0.3 mCi/kg). The ORR was 83% (37% CR, 6.7% CR uncon-
firmed, and 40% PR). KaplanMeier estimated median time to progression
(TTP) was 9.4 months (range, 1.724.6). In responders, KaplanMeier estimated
median TTP was 12.6 months (range, 4.924.6). Toxicity was primarily hema-
tologic, transient, and reversible. The incidence of grade 4 neutropenia, throm-
bocytopenia, and anemia was 33%, 13%, and 3%, respectively. The conclusions
from this study were that reduced-dose ibritumomab tiuxetan is safe and well
tolerated and has significant clinical activity in this patient population (40).
A further single arm phase II study of
90
Y-ibritumomab tiuxetan was
undertaken to examine the efficacy of
90
Y-ibritumomab tiuxetan in a group with
rituximab refractory disease (41). Fifty-four heavily pretreated patients with
follicular lymphoma (FL) were recruited who were refractory to or progressed
after rituximab. The trial showed an ORR of 74% and a CR rate of 15%, despite
a median of four prior therapies and 73% of patients having bulky disease
(5 cm diameter). KaplanMeier-estimated DR was 6.4 months, with a TTP of
6.8 months in all patients and 8.7 months in responders. The randomized-
controlled trial has been described earlier and compared
90
Y-ibritumomab
tiuxetan with rituximab in relapsed or refractory low-grade B-cell NHL. This
confirmed that
90
Y-ibritumomab tiuxetan results in superior ORR and CR rates
to those seen with the naked mAb, rituximab.
A randomized phase III trial including 143 patients with relapsed or
refractory low grade, follicular, or transformed NHL compared efficacy of a
single dose of 15 MBq/kg (0.4 mCi/kg)
90
Y-ibritumomab tiuxetan with ritux-
imab (375 mg/m
2
once weekly for 4 weeks) (42). Response rates were sig-
nificantly higher in the
90
Y-ibritumomab tiuxetan arm with an ORR of 80%
Figure 1 Treatment regimen for
90
Y-ibritumomab tiuxetan. Intergroup European first-
line follicular lymphoma phase III study. Chemotherapy was left to the discretion of the
treating physician, e.g., chlorambucil, CHOP, CVP, fludarabine, CVP-R. **n 410
(accrual completed on January 2005).
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versus 56% (p 0.002) and a CR rate of 30% versus 16% (p 0.004). Subgroup
analysis revealed a superior benefit for patients with follicular histology with an
ORR of 86% versus 55% (p < 0.001) and a significantly increased (p < 0.04)
time to treatment progression for this subgroup. The overall TTP was, however,
not different in both treatment groups, but patients treated with
90
Y-ibritumomab
tiuxetan showed a trend toward longer median DR (14.2 months vs. 12.1 months)
and more often achieved responses lasting longer than six months duration
(64% vs. 47%).
A recent retrospective analysis suggests that treatment with
90
Y-
ibritumomab tiuxetan is associated with higher response rates and longer DR when
used earlier in the therapy schedule (43). An integrated analysis of 211 patients
treated in clinical trials compared efficacy and safety of
90
Y-ibritumomab tiuxetan
in patients with one prior therapy (n 63) to patients who received greater than or
equal to two prior therapies (n 148). Patients receiving
90
Y-ibritumomab tiuxetan
as second-line therapy had greater ORR (86% vs. 72%; p 0.051) and CR/CRu
(CR unconfirmed) rates (49% vs. 28%; p 0.004) and a significantly longer
median time to disease progression (TTP) (12.6 months vs. 7.9 months; p 0.038).
In CR/CRu patients, the median TTP (23.9 months vs. 15.6 months; p 0.0442)
and median DR (22.8 months vs. 14.6 months; p 0.0429) were both significantly
increased in patients with only one prior therapy (n 53).
A large European intergroup study of
90
Y-ibritumomab tiuxetan therapy of
previously untreated FL has now completed accrual with over 400 patients
recruited. Patients were treated initially with chemotherapy (the physicians
choice) and then randomized to
90
Y-ibritumomab tiuxetan or no further treat-
ment. The data from this study may provide data in determining whether
90
Y-
ibritumomab tiuxetan has a potential beneficial role after primary chemotherapy.
There was, however, relatively small numbers of patients treated in the latter part
of the study who received rituximab in combination with chemotherapy regi-
mens, which is now a widely adopted approach. Therefore, the additional role of
90
Y-ibritumomab tiuxetan RIT after full course rituximab, chemotherapy com-
binations will probably not be answered by this study.
Shipley and colleagues performed an evaluation of the efficacy of short-
duration R-CHOP followed by 15 MBq (0.4 mCi)
90
Y-ibritumomab tiuxetan as a
first-line treatment of patients with FL (44). The study was a multicenter phase II
trial that recruited 42 patients in which 39 completed the entire planned therapy.
Of the 40 patients that received chemotherapy, 12 (30%) were found to have a
CR/Cru and 28 (70%) showed a PR. After
90
Y-ibritumomab tiuxetan RIT, 26
patients (66%) showed a CR/Cru, 12 patients (31%) showed a PR and 1 patient
(3%) showed disease progression. The group showed that treatment with
90
Y-
ibritumomab tiuxetan after chemotherapy/rituximab increased the CR rate from
30% to 66%. Actuarial progression-free survival (PFS) after one year was 98%
(38 patients at risk) and after two years was 85% (15 patients at risk).
Clinical responses have also been observed for transformed follicular and
relapsed diffuse large B-cell lymphoma (DLBCL) when treated with
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90
Y-ibritumomab tiuxetan. Within an initial phase I/II study reported a response
rate of 58% with a 33% CR rate in a group of just 12 patients that had relapsed
following two previous chemotherapy regimens that included CHOP (42). A
prospective, single-arm, open-label, nonrandomized, multicenter phase II trial
was, therefore, undertaken to evaluate the efficacy and safety of
90
Y-
ibritumomab tiuxetan in patients over 60 years of age with relapsed or primary
refractory DLBCL not suitable for ASCT. Patients were divided into two groups,
with firstly those previously treated with chemotherapy alone (group A, n 76)
and secondly those previously treated with chemotherapy and rituximab (group B,
n 28) (45). All patients received a single dose of 15 MBq/kg (0.4 mCi/kg)
of
90
Y-ibritumomab tiuxetan up to a maximum dose of 1184 MBq/kg (32 mCi).
In total, 103 patients could be evaluated for treatment efficacy and 104 for
safety. An ORR of 44% was observed in the entire study population. In Group A,
the ORR was over 50%. In Group B, where 37% of patients were refractory to
rituximab-CHOP, the ORR was 19%. Adverse events (AEs), with the exception
of hematological AEs, were generally mild (grade 1/2) and the incidence of
severe infection was low, with only 7% of patients hospitalized for infection
during the study. The results of this study were encouraging and clinical trials are
now underway in the United States or at an advanced stage of development in the
EU to integrate
90
Y-ibritumomab tiuxetan into a front-line treatment for DLBC
alongside rituximab-chemotherapy schedules.
Tositumomab was the first mAb to be produced against a B-cell antigen
(46). The
131
I-tositumomab regimen is completed within one to two weeks and
consists of a tracer dose of the radioimmunoconjugate followed by the thera-
peutic dose 7 to 14 days later. Each infusion of
131
I-tositumomab is preceded
by an infusion of a predose of 450 mg cold or unlabeled tositumomab and
the therapeutic regimen is outlined in Figure 2. Whole body gamma camera
imaging is performed three times over the week following the trace labeled
infusion to calculate the whole body half-time and the dose required for the
therapeutic infusion to deliver a 65 to 75 cGy whole body dose (WBD) (usually
3700 to 5550 MBq (100150 mCi) (47). Dose adjustments to 65 cGy were
made for a baseline platelet count of 100,000/mm
3
to less than 150,000/mm
3
and for obesity.
Figure 2 Treatment regimen for
131
I-tositumomab.
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Kaminski and colleagues initially conducted a series of trials at the Uni-
versity of Michigan using the
131
I-tositumomab, for the treatment of relapsed FL
(48,49). In a pivotal study, 60 extensively pretreated patients were given a single
administration of
131
I-tositumomab (6). Disease responses were compared to
their previous responses to chemotherapy for follicular or transformed FL. A PR
or CR was observed in 39 patients (65%) after iodine
131
I-tositumomab, com-
pared with 17 patients (28%) after their last qualifying chemotherapy (LQC)
(p < 0.001).
131
I-tositumomab therapy was shown to provide greatly superior
RFS compared to the LQC.
Recently an integrated efficacy analysis of the five clinical trials presented
to the U.S. FDA for registration purposes of the
131
I-tositumomab regimen in
patients with relapsed or refractory low-grade, follicular, or transformed low-
grade NHL (50). This integrated analysis included 250 patients and response
rates in the five trials ranged from 47% to 68% with CR rates between 20% to
38%. With a median follow-up of 5.3 years, the five-year PFS was 17%. Of the
250 patients, 81 (32%) had a TTP of more than or equal to one year (termed
durable response population). For the durable response population, 44% had not
progressed at more than or equal to 2.5 to more than or equal to 9.5 years and had
a median DR of 45.8 months. The median duration of CR was not reached. The
durable response population had many poor prognostic characteristics, including
bone marrow involvement (41%), bulky disease greater than or equal to 5 cm
(49%), and transformed histology (23%). Forty-three percent of the patients had
been treated with more than four prior therapies and 36% had not responded to
their most recent therapy. The authors concluded that the
131
I-tositumomab
therapeutic regimen produces high response rates in patients with relapsed or
refractory low-grade, follicular, and transformed low-grade NHL, with a sizable
subgroup of patients achieving long-term durable responses.
Impressive response rates have also been seen in patients that were
refractory to rituximab, which were subsequently treated with
131
I-tositumomab.
Horning and colleagues used
131
I-tositumomab to treat 40 patients with low-
grade NHL, 72% of which had received four or more previous lines of therapy
and 60% of which had failed to respond to rituximab (51). An ORR of 68% with
a CR rate of 30% was noted and a median DR of 14.7 months reported. Of the 12
complete responders, 9 remained in CR at the time of presentation with a range
of 12 to 26 months. More recently, an analysis including 230 patients treated
with
131
I-tositumomab was made. Independently assessed durable CRs were
noted with similar frequency in patients with rituximab-refractory disease (28%)
and rituximab na

ive patients all of whom had chemotherapy refractory disease


(23%). With a median follow-up of 4.6 years, 75% of patients with durable CR
continue in complete remission (52).
Kaminski et al. have shown highly promising results in the front-line
treatment of previously untreated low-grade FL using
131
I-tositumomab (32). An
encouraging ORR of 95% was seen with 75% achieving CR. PCR (polymerase
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chain reaction) was used to detect rearrangement of the BCL2 gene, which
revealed molecular responses in 80% of assessable patients who had a clinical
CR. The most recent update included 76 patients with a median follow-up of
5.1 years. The actuarial five-year PFS for all patients was 59%, with a median
PFS of 6.1 years. Hematological toxicity was moderate, with no patient requiring
transfusion or granulocyte colony stimulating factor (G-CSF) (32,53); though 48
out of 76 (63%) patients developed detectable human anti-mouse antibody
(HAMA) responses after a single course of treatment with
131
I-tositumomab.
More recently Press and colleagues conducted a phase II trial in untreated
FL that consisted of six CHOP cycles followed up four to eight weeks later by
131
I-tositumomab (54). A cohort of 90 previously untreated eligible patients with
advanced-stage FL tolerated the treatment well. Reversible myelosuppression
was the main AE and was more severe during CHOP chemotherapy than after
RIT. The ORR over the entire treatment regimen was 90%, including 67% CR
and 23% PR. Of the 47 fully evaluable patients who achieved less than a CR with
CHOP, 27 (57%) improved their remission status after predosed tositumomab
and
131
I-tositumomab. With a median follow-up of 2.3 years, the two-year PFS
was estimated to be 81%, with a two-year overall survival of 97%. Having
established the feasibility and the efficacy of this approach, a randomized phase
III trial is currently being undertaken to compare this approach of chemotherapy
followed by RIT against immunochemotherapy with six cycles of CHOP-R.
CONTRIBUTION OF TARGETED RADIATION TO CLINICAL RESPONSE
The contribution of targeted radiation to the overall responses seen in RIT has
been addressed with two randomized studies comparing the radio-
immunoconjugates
90
Y-ibritumomab tiuxetan and
131
I-tositumomab with the
unlabeled mAbs (31,42). Both studies have shown greatly superior clinical
responses of RIT over the unlabeled mAb. The
90
Y-ibritumomab tiuxetan versus
rituximab is described above and the second study compared treatment outcomes
for unlabeled tositumomab (predose) and
131
I-tositumomab to an equivalent total
dose of unlabeled tositumomab involved 78 patients with refractory/relapsed
low-grade NHL (31). The investigators reported an ORR of 55% versus 19%
(p 0.002) with a CR 33% versus 8% (p 0.012) in
131
I-tositumomab versus
unlabeled tositumomab groups, respectively. The median duration of the ORR
was not reached for
131
I-tositumomab versus 28.1 months for unlabeled tositu-
momab. The median duration of CR was not reached in either arm, and the
median TTP was 6.3 months versus 5.5 months (p 0.031), respectively.
Although hematological toxicity was more severe and nonhematological AEs
were more frequent after
131
I-tositumomab than after tositumomab alone, there
were no serious infectious or bleeding complications. The frequency of devel-
oping HAMA was similar in the two arms of 27% (
131
I-tositumomab group)
versus 19% (tositumomab-alone group), respectively. This study demonstrated
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that although unlabeled tositumomab showed single agent activity, the conju-
gation of
131
I to tositumomab significantly enhanced the therapeutic
efficacy (31).
Toxicity and Safety of RIT
Adverse Effects of
90
Y-Ibritumomab Tiuxetan
Myelosuppression was found to be the dose-limiting toxicity of RIT for both
90
Y-ibritumomab tiuxetan and
131
I-tositumomab. An analysis of all patients
treated in
90
Y-ibritumomab tiuxetan trials (n 261) indicated that 28% will
experience grade 4 neutropenia and 8% will experience grade 4 thrombocyto-
penia (55). No significant differences in the incidence of hematological and
nonhematological grade 3 to 4 AEs were observed in patients 65 years or older
as compared with younger patients. However, despite concerns about the
potential of an increased risk of radiation-induced secondary hematological
malignancies, the observed rate of secondary myelodysplastic syndromes
(MDS)/AML was less than 1% (5/348), which is comparable with a similar
patient population treated with alkylating agents (55).
Safety data from the four clinical trials were reviewed retrospectively in an
integrated analysis encompassing 349 patients of whom 345 patients (99%)
completing
90
Y-ibritumomab (55). Although 80% of patients reported non-
hematological AEs, those were generally mild to moderate in severity with
asthenia, nausea, and chills being the most common events that were considered
to have probable or possible association related to the treatment. Only 11%
(39 patients) of all patients experienced grade 3 to 4 nonhematological toxicity.
For
90
Y-ibritumomab tiuxetan grades 1 to 2 and 3 to 4 thrombocytopenia
occurred in 37% and 63% of patients, respectively. Of the patients with grade 3
to 4 thrombocytopenia, 87% recovered to greater than or equal to 50 10
9
/L by
week 12 following therapy. Neutropenia grade 3 to 4 was observed in 30% of
patients, with 90% recovering to greater than 1.0 10
9
/L within 12 weeks
posttreatment. For patients, who received G-CSF support, the median duration of
neutropeania was reduced from 27 to 19 days. Grades 3 and 4 anemia developed
in 13% and 4% of patients, respectively. Of all patients, 22% required platelet
transfusions and 20% required red blood cell transfusions.
90
Y-ibritumomab
tiuxetan is dosed according to the patients body weight and baseline platelet
counts. For patients with platelet counts greater than or equal to 150,000/mm
3
,
5 MBq/kg (0.135 mCi/kg) body weight is given up to a maximum permissible
dose of 1200 MBq (32.4 mCi). For patients with platelet counts of 100 to 149
10
9
/L,
90
Y-ibritumomab tiuxetan is dosed at 11 MBq/kg (0.297 mCi/kg), up to a
maximum allowable dose of 1200 MBq (32.4 mCi).
The incidence of severe thrombocytopenia and neutropenia correlated
significantly with degree of bone marrow involvement and platelet counts at
baseline, underscoring the importance to exclude patients with greater than or
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equal to 25% bone marrow infiltration and inadequate bone marrow reserve.
Patients who had more than two prior chemotherapies were twice as likely to
develop grade 4 thrombocytopenia, whereas number of prior chemotherapies did
not correlate with longer median duration of neutropenia, thrombocytopenia, and
anemia.
Total number of B cells and levels of IgM declined after treatment but
recovered after six to nine months. The median T-cell counts and levels of IgG
and IgA remained stable following treatment with
90
Y-ibritumomab tiuxetan.
Most importantly, treatment with
90
Y-ibritumomab tiuxetan was not associated
with an excess rate of infections. In fact, incidence of infectious complications
was low with upper respiratory and urinary tract infections occurring in 5% and
7% of patients, respectively. Only 8% of all patients received antibiotic therapy
during the treatment period.
Adverse Effects of
131
I-Tositumomab RIT
Short-term nonhematological AEs with
131
I-tositumomab administration are
generally mild and include typically fatigue, nausea, fever, vomiting, pruritus,
and rash, which usually respond well to antihistamines. Follow-up examinations
include weekly blood and platelet counts following RIT until hematological
recovery occurs.
131
I-tositumomab is susceptible to dehalogenation and, therefore, a pre-
administration of cold iodine is given starting 72 hours prior to the dosimetric
dose and again at 14 days after the therapeutic dose in order to block the thyroid
from radioactive iodine uptake. Despite these attempts to prevent thyroid uptake
of
131
I, hypothyroidism appears one of the most consistent long-term adverse
effects after
131
I-mAb treatment. Hypothyroidism can, however, be easily
managed with thyroid stimulating hormone (TSH) replacement once detected
and screening is an important component of follow-up. Zelenetz reviewed the
multicenter RIT trials using
131
I-tositumomab in NHL patients and reported that
elevated TSH was observed in 5 out of 59 patients in the phase I study (56).
However, after a myeloablative dose of
131
I-tositumomab, elevated TSH was
observed in 59% of patients (57). HAMA reactions appear to be substantially
lower in previously treated NHL patients compared with the rates experienced in
solid tumor RIT (4). The incidence of HAMA was observed to be approximately
10% after
131
I-tositumomab treatment.
The impact of administration of the
131
I-tositumomab therapeutic regimen
on circulating CD20 positive cells was assessed in two clinical studies (32,49),
one conducted in chemotherapy naive patients and one in heavily pretreated
patients. The assessment of circulating lymphocytes did not distinguish normal
from malignant cells. Consequently, assessment of recovery of normal B-cell
function was not directly assessed. Lymphocyte recovery began at approximately
12 weeks following treatment. Among patients who had CD20 positive cell
counts recorded at baseline and at six months, 8 of 58 (14%) chemotherapy na

ive
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patients had CD20 positive cell counts below normal limits at six months and
6 of 19 (32%) heavily pretreated patients had CD20 positive cell counts below
normal limits at six months. There was no consistent effect of the
131
I-tositu-
momab therapeutic regimen on posttreatment serum IgG, IgA, or IgM levels.
Secondary malignancy following RIT has fortunately proved to be rare
(32,56). Recently, 1071 RIT-treated patients were assessed for treatment-
related MDS/AML (58). Among these patients, 995 with low-grade and
transformed low-grade NHL had been previously treated with a median of three
therapies (range, 1 to 13 therapies) prior to RIT. As part of their initial therapy
for FL, 76 patients received RIT. For the previously treated patients, the
median follow-up from the diagnosis of NHL and RIT was six years and two
years, respectively; for the patients who received RIT as their initial therapy,
the corresponding median follow-up times were 5.6 years and 4.6 years,
respectively. Of the 995 previously treated patients, 35 (3.5%) cases of
treatment-related MDS/AML were reported, and 13 cases were confirmed to
have developed MDS/AML following RIT. This incidence was found to be
consistent with that expected on the basis of patients prior exposure to che-
motherapy. With a median follow-up approaching five years, no case of
treatment-related MDS/AML has been reported in the 76 patients, receiving
131
I-tositumomab as their initial therapy (58).
Subsequent Therapy After RIT
Data are emerging to suggest that subsequent therapy can be administered after
131
I-tositumomab and
90
Y-ibritumomab tiuxetan (59,60). Chemotherapy or
ASCT after prior therapy with
90
Y-ibritumomab tiuxetan also appears feasible
and reasonably well tolerated. The toxicity with subsequent therapy seems
similar to that in patients not treated with
90
Y-ibritumomab tiuxetan.
RIT After Autologous Transplantation
Despite initial concerns, preliminary data suggest that patients after prior bone
marrow transplant or stem cell support can be safely treated as long as a sig-
nificant dose reduction is made. Initially for
90
Y-ibritumomab tiuxetan, a
reduced dose of 11.1 MBq/kg, was suggested; however, recent data has sug-
gested that a full dose of 15 MBq/kg can be tolerated, although the number of
patients in this study was only eight (61). For
131
I-tositumomab a WBD of 65
cGy is feasible (62). The delivery of either radioimmunoconjugate after high
dose chemotherapy and ASCT is currently outside of the licensed indication.
This should not be confused with the application of RIT as part of the con-
ditioning regimen used as part of ASCT, where there is increasingly compelling
clinical safety and efficacy data.
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Myeloablative RIT in NHL
Myeloablative RIT has the theoretical advantage of delivering higher doses of
radiation and overcoming the dose-limiting myelotoxicity with autologous stem
cell and bone marrow rescue (56,57). Press and colleagues in Seattle pioneered
the myeloablative approach, demonstrating the feasibility of using high activity
doses of
131
I-anti-B-cell mAbs (mB-1 (anti-CD37) or anti-B1 (anti-CD20) with
either ASCT or PBSCT (57). Initially the group assessed the biodistribution,
toxicity, and efficacy of high doses of radiolabeled anti-B1 in 43 patients with
B-cell lymphoma. The dose limiting toxicity was found to be cardiopulmonary
and the lung MTD to be 27 Gy. Patients with a favorable biodistribution were
eligible for RIT with
131
I-tositumomab according to a phase 1 dose-escalation
protocol. Twenty-four patients had a favorable biodistribution and 19 received
therapeutic infusions of 8.7 to 28.7 GBq (234777 mCi) of
131
I-mAbs (58
1168 mg) followed by ASCT, resulting in CRs in 16, PRs in 2, and an MR (2550%
tumor regression) in 1. Of these patients, nine have remained in continuous CR
for 3 to 53 months. Toxic effects included myelosuppression, nausea, infections,
and two episodes of cardiopulmonary toxicity that were moderate in patients
treated with doses of
131
I- mAbs that delivered less than 27.25 Gy to normal
organs. For patients with a favorable biodistribution (tumor burdens <500 cm
3
and without massive splenomegaly), the CR rate was 84% with a PR rate of 11%.
These remissions appeared durable and the median DR exceeded 11 months at
the time of publication. Finally, the anti-CD20 mAb (B1) was considered to be
superior to the anti-CD37 (mB-1) because favorable biodistribution was ach-
ieved with 2.5mg/kg, as compared with 10mg/kg for the anti-CD37.
In the subsequent study, Press et al. evaluated the combination of high-
dose
131
I-tositumomab, etoposide, and cyclophosphamide (Cy) in conjunction
with ASCT in 38 patients with NHL (26 low grade, 12 aggressive) (63). Of the
37 evaluable patients, 33 (89%) were alive and 29 (78%) were progression free
after a median follow-up of 1.5 years. Toxicities included grade 4 myelosup-
pression in all patients, grade 2 to 3 nausea in 26 (70%), pulmonary infiltrate in
four, and grade 3 veno-occlusive disease (VOD) in two patients. These results
confirmed the feasibility of delivering high-dose RIT in combination with high-
dose chemotherapy in an ASCT setting for NHL. More recently, the long-term
follow-up of the 29 patients treated in phase I and phase II trials was reported
(57). Twenty-four of the 29 patients are alive and 15 progression free after a
median follow-up of 37 months. At five years, overall survival and PFS are
projected at 68% and 42%, respectively. None of the surviving patients has
objective impairment of performance status or cardiac function. Late toxicities
have been uncommon, except for elevation of the TSH levels seen in 60% of the
subjects.
The Seattle transplant group performed a multivariable comparison of
125 consecutive patients with FL treated with either high-dose RIT using
131
I-tositomamb (n 27) or conventional high-dose therapy (C-HDT) (n 98)
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and ASCT
64
. The groups were similar, although more patients treated with
high-dose RIT had an elevated pretransplantation level of lactate dehydro-
genase (41% vs. 20%, p 0.03) and elevated international prognostic score
(41% vs. 19%, p 0.02). Patients treated with high-dose RIT received indi-
vidualized therapeutic doses of
131
I-tositumomab [median, 19.7 GBq (531 mCi)]
to deliver 17 to 31 Gy (median, 27 Gy) to critical organs. Patients treated with
C-HDT received TBI plus chemotherapy (70%) or chemotherapy alone (30%).
Patients treated with high-dose RIT experienced improved overall survival and
PFS versus patients treated with C-HDT. The estimated five-year overall sur-
vival and PFS were 67% and 48%, respectively for high-dose RIT and 53% and
29%, respectively for C-HDT. The 100-day treatment-related mortality was
3.7% in the high-dose RIT group and 11% in the C-HDT group. The probability
of secondary MDS/AML was estimated to be 0.076 at eight years in the high-
dose RIT group and 0.086 at seven years in the C-HDT group. This data suggests
that high-dose RIT may improve outcomes when compared with C-HDT in
patients with relapsed FL.
Nademanee et al. recently studied the feasibility of including high dose
90
Y-ibritumomab with high-dose etoposide (VP-16) 40 to 60 mg/kg (day 4)
and Cy 100 mg/kg (day 2) followed by ASCT (65). This phase I/II trial of
high-dose
90
Y-ibritumomab tiuxetan in combination with etoposide and Cy
was conducted in 31 patients with CD20 positive NHL. Patients underwent
dosimetry (day 21) with 5 mCi (185 MBq)
111
In-ibritumomab tiuxetan fol-
lowing 250 mg/m
2
rituximab followed a week later by
90
Y-ibritumomab
tiuxetan to deliver a target dose of 1000 cGy to highest normal organ. Bone
marrow biopsy was later performed (day 7) to estimate radiation dose, and
stem cells were reinfused when the radiation dose was estimated to be less
than 5 cGy. The median
90
Y-ibritumomab tiuxetan dose was 2649.2 MBq
(range, 1354.23885 MBq) (71.6 mCi; range, 36.6105 mCi). At a median
follow-up of 22 months, the two-year estimated overall survival and RFS
rates were 92% and 78%, respectively. The author concluded that high-dose
90
Y-ibritumomab tiuxetan can be combined safely with high-dose etoposide
and Cy without an increase in transplant-related toxicity or delayed
engraftment.
Other myeloablative approaches have investigated the biodistribution and
pharmacokinetics of
131
I-rituximab in 35 patients with NHL (66). After
administration of a 20 to 40 mg mAb dose labeled with 250 MBq of
131
I,
biodistribution was determined by gamma camera imaging. The half-life of the
labeled mAb was 88 hours compared with the murine tositumomab with a half-
life of 56 hours. Greater dose was delivered to the whole body and organs,
although tumor doses were found to increase over time as opposed to other
organs. The authors concluded that patient-specific dosimetry was required and
that lower activity doses were required than those delivered by tositumomab
because of
131
I-rituximabs longer half-life.
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Clinical RIT Trials in Leukemias
RIT studies in leukemia have focused on the CD33, CD45, and CD66 anti-
gens. CD33 is a cell surface glycoprotein found on myeloid leukemia cells.
HuM195 is a humanized anti-CD33 mAb that has been used in its native form
as well as in a radiolabeled form to treat leukemias. The development over the
last decade culminated in a recently reported randomized cohort of 50 adult
patients [median age of patients was 62 years (range, 2686)] with relapsed or
refractory AML, which were treated with unconjugated HuM195 (67). CD33
was detected in 95% of patients for whom immunophenotyping was available.
All patients received a dose of either 12 or 36 mg/m
2
of HuM195 by intra-
venous infusion over four hours on days 1 to 4 and 15 to 18. Those patients
with stable or responding disease received two additional cycles on days 29 to
32 and 43 to 46. Of the 50 patients, 24 were treated with HuM195 as a first
salvage therapy and 26 as a second or subsequent salvage therapy. The per-
centage of blast involvement in the marrow ranged from 5% to 30% in
20 patients with the remainder having blast counts greater than 30%. Only two
CRs and one PR were observed from all 49 evaluable patients. All three
responses were in patients treated at the 12 mg/m
2
dose level, which all had
baseline blast percentages of less than 30%. Reductions in blast counts ranging
from 30% to 74% were described in nine other patients. HuM195 as a single
agent was observed to have minimal but measurable, antileukemic activity in
patients with relapsed or refractory AML, though its activity is confined to
patients with low-burden disease. No significant differences in clinical efficacy
or toxicity were seen between the two dose levels of mAb. HuM195 was well
tolerated with the predominant toxicities observed being infusion-related fevers
and chills, which were primarily related to the first dose of mAb. The authors
concluded that significant clinical efficacy with unconjugated HuM195 may only
be realized in patients with minimal residual disease or when combined with
chemotherapy.
131
I-M195 and
131
I-HuM195 has also been combined with busulfan (Bu)/
Cy as conditioning for allogeneic BMT (68). RIT with
131
I-M195 or
131
I-
HuM195 was performed on 31 patients with relapsed/refractory AML (n 16),
accelerated/myeloblastic chronic myeloid leukemia (CML) (n 14), or
advanced MDS (n 1). Subjects received 4.5 to 16.2 GBq (122437 mCi) plus
Bu (16 mg/kg) and Cy (90120 mg/kg) followed by infusion of related-donor
bone marrow (27 first ASCT; 4 second ASCT). Hyperbilirubinemia was the most
common extramedullary toxicity, occurring in 69% of patients during the first
28 days after ASCT. Gamma camera imaging showed targeting of the radio-
isotope to the bone marrow, liver, and spleen, with absorbed radiation doses to
the marrow of 272 to 1470 cGy. The median survival was 4.9 months (range,
0.390 months). Following bone marrow transplantation, three patients with
relapsed AML remained at the time of publication in CR for greater than 59, 87,
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and 90 months. The authors demonstrated the feasibility of adding CD33-
targeted RIT to a standard ASCT preparative regimen, though randomized trials
are required to demonstrate the benefit of intensified conditioning with RIT.
Feldman et al. compared the efficacy of HuM195 immunotherapy in
combination plus induction chemotherapy with chemotherapy alone in a
randomized study of 191 adults with first relapsed or primary refractory AML
(duration of first response, 012 months) (67). The two groups were admin-
istered with 8-mg/m
2
mitoxantrone, 80-mg/m
2
etoposide, and 1-g/m
2
cytar-
abine daily for six days in combination with 12 mg/m
2
with or without
HuM195. Of the cohort treated with the combination treatment, 36% showed
a CR or CR without complete platelet recovery, whereas only 28% showed
similar results in patients treated with chemotherapy alone (p 0.28). The
overall median survival was 156 days and was not different in the two arms of
the study. Apart from mild mAb infusion-related toxicities (fever, chills, and
hypotension), no differences in chemotherapy-related AEs were observed
with the addition of HuM195 to induction chemotherapy. Though the addition
of HuM195 to salvage induction chemotherapy was safe, the authors dem-
onstrated that the combination treatment did not result in a statistically sig-
nificant improvement in ORR or survival in patients with refractory/relapsed
AML.
A phase I activity dose escalation study was performed in nine patients
using
213
Bi-HuM195 in patients with refractory and relapsed myeloid leuke-
mias and the data used to estimate pharmacokinetics and dosimetry (69). This
was the first trial using an a-emitter in human leukemia patients. Patient
imaging was initiated at the start of each injection of 0.6 to 1.6 GBq (16.2
43.2 mCi) of activity. A set of 40 images of the liver and spleen were
dynamically obtained and blood samples were collected until three hours
postinjection and counted in a gamma counter. The percentage injected dose in
the liver and spleen volumes increased rapidly over the first 10 to 15 minutes to
a constant value for the remaining hour of imaging, yielding a very rapid
uptake followed by a plateau in the mAb uptake curves. The absorbed dose
equivalent to liver and spleen volumes ranged from 2.4 to 11.2 Sv and 2.9 to
21.9 Sv, respectively. Marrow (or leukemia) mean dose ranged from 6.6 to
12.2 Sv. The study demonstrated that patient imaging of
213
Bi, an a-particle
emitter, labeled to HuM195 was possible and may be used to derive pharma-
cokinetics and dosimetry data.
213
Bi-HuM195 was also studied in 18 refractory AML or CML patients
and was shown to localize to the marrow, spleen, and liver (19). Five dose levels
ranging from 10.36 to 37.0 MBq/kg of
213
Bi-HuM195 were administered to
18 patients. All 17 evaluable patients developed myelosuppression, with a
median time to recovery of 22 days and almost all of the activity was observed to
rapidly localize and remain in areas of leukemic involvement, including the bone
marrow, liver, and spleen. The reduced whole body and increased target organ
doses (bone marrow, liver, spleen) produced absorbed dose ratios approximately
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1000 times higher for
213
Bi-HuM195 than those for b-emitting anti-CD33 mAb
constructs. Fourteen (93%) of the 15 evaluable patients had reductions in cir-
culating blasts, and 14 (78%) of 18 patients had reductions in the percentage of
bone marrow blasts. This study further demonstrates the safety, feasibility, and
therapeutic effects of
213
Bi-HuM195 in treating leukemia.
In the phase I trial, 20% of 31 patients were shown to have a long-term
survival rate for patients with AML (68). The same group labeled HuM195 with
90
Y and has started a clinical trial using RIT in combination with TBI (2 Gy) and
fludarabine in a reduced intensity-conditioning program. The results of this trial
are anticipated but have not been revealed to date.
There have also been very encouraging results using
131
I-BC8 mAb
(anti-CD45) in combination with Cy and TBI as a marrow transplant con-
ditioning regimen for acute leukemia (70). Twenty patients were treated with
a radiation dose estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver,
with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by
120 mg/kg Cy and 12 Gy TBI. Toxicity was not measurably greater than that
of Cy/TBI alone and the MTD was not reached. Results from a phase I study
of
131
I anti-CD45 combined with 120 mg/kg Cy and 12 Gy TBI in HLA-
matched related transplants for AML in first remission were recently reported
(70). A biodistribution dose of 0.5 mg/kg
131
I-BC8 (murine anti-CD45) mAb
was given to 44 patients with advanced MDS/ALL. The mean / SEM
estimated radiation absorbed dose delivered to bone marrow and spleen was
6.5 / 0.5 and 13.5 / 1.3 cGy/mCi of
131
I, respectively. Liver (2.8 /
0.2 cGy/mCi), lung (1.8 / 0.1 cGy/mCi), kidney (0.6 / 0.04 cGy/mCi),
and total body (0.4 / 0.02 cGy/mCi), all received lower doses. Thirty-seven
patients (84%) had favorable mAb biodistributions, with a higher estimated
radiation absorbed dose to marrow and spleen than to normal organs. Of these
patients, 34 received a therapeutic dose of
131
I-BC8 mAb labeled with 2.8 to
22.6 GBq (76612 mCi) of
131
I to deliver estimated radiation absorbed doses
to liver (normal organ receiving the highest dose) of 3.5 Gy (level 1) to
12.25 Gy (level 6) in addition to Cy and TBI. The MTD was level 5 (delivering
10.5 Gy to liver), with grade 3/4 mucositis in two of two patients treated at level
6. The authors concluded that
131
I -anti-CD45 mAb can safely deliver substantial
supplemental doses of radiation to bone marrow (approximately 24 Gy) and
spleen (approximately 50 Gy) when combined with conventional Cy/TBI.
Matthews et al. undertook several studies using
131
I-anti-CD45 mAb (70).
The phase I study demonstrated mAb safety and acceptable pharmacodynamics
after 34 refractory AML/ALL patients, which had been evaluated. The patients
received a 2.8 to 22.6 GBq (76612 mCi) therapeutic dose of
131
I-anti-CD45 to
bring about calculated radiation-absorbed doses to the liver of 3.5 Gy (level 1
Bearman criteria) to 12.25 Gy (level 6) in addition to Cy (120 mg/kg) and TBI
(12 Gy). The red-marrow dose from the labeled mAb was 24 Gy, the spleen dose
was 50 Gy, and the LFS was 29%.
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Following the success of the phase I study, two phase II studies have been
performed to evaluate the short-term toxicity of the anti-CD45 radio-
pharmaceutical (68). In the first trial, 17 patients with advanced AML were
treated with
131
I-anti-CD45, TBI (12 Gy), and Cy (120 mg/kg) to obtain a red-
marrow dose of 21 to 22 Gy. Results of the trial showed LFS in 42% of patients,
29% relapsed and 29% died of nonrelapsed mortality. In the second study, 46
AML patients in CR1 with either an intermediate or high-risk cytogenetic fea-
tures were treated with the same treatment as before but with Bu (16 mg/kg)
instead of Cy. Red-marrow doses were 11 Gy, although average LFS was 60%
with intermediate patient LFS rates being 68% and average high-risk patient LFS
being 40%. Almost inevitably, the high-risk patients had a higher relapse rate
than the intermediate patients.
A further phase I trial has been initiated by the same group using only 2 Gy
TBI in combination with fludarabine (71,72). Thus far, the mean red-marrow
dose achieved with this treatment has been 24 Gy and of the 11 patients
recruited, 8 are in remission at the time of publication.
188
Reis an attractive radioisotope for RIT as it has a 16.9-hour half-life,
and emits a high energy (2.2 MeV b-particle and a 155 keV g-photon) facilitating
therapy and imaging. Bunjes et al. used a
188
Re- anti-CD66 mAb to intensify the
conditioning regimen prior to stem cell transplantation to treat 36 patients with
high-risk AML and MDS (73). As a favorable dosimetry was observed in all
cases, RIT was administered and provided a mean of 15.3 Gy of additional
radiation to the marrow. The normal organ receiving the highest dose was the
kidney (mean 7.4 Gy), which resulted in late renal toxicity in 17% of patients.
RIT was followed by standard full-dose conditioning with TBI (12 Gy) or Bu
and high-dose Cy with or without thiotepa. Patients subsequently received a
T-cell-depleted allogeneic graft from an HLA-identical family donor (n 15) or an
alternative donor (n 17). mAb infusion-related toxicity due was minimal, and
no increase in mAb treatment-related mortality was observed. After 30, 100 days
and 18 months postadministration, mortalities were 3%, 6%, and 22%, respec-
tively. Of the 15 patients undergoing transplantation in first CR or second CR,
the relapse rate was 20% and 21 patients found not to be in remission at the time
of transplantation had a 30% relapse rate.
Waldmann et al. have investigated the use of anti-Tac mAb (anti-
interleukin-2 receptor) in adult T-cell leukemia (ATL) caused by the retrovirus
human T-cell lymphotropic virus-I (74).
90
Y-anti-Tac was administered to the
first nine patients as part of a phase I dose-escalation trial and to the second
group of nine as part of a phase II trial involving a uniform 370 MBq (10 mCi)
dose. Patients in whom a response was observed were able to receive up to eight
additional doses. At the 185 to 555 MBq (515 mCi) doses used, 9 of 16
evaluable patients had responses (7 PR and 2 CR). The observed responses
appear better than those with unmodified anti-Tac and severe toxicity was
largely limited to the hematopoietic system.
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Expert Opinion, Including a Table with up to Five Key Issues
131
I-Tositumomab (Bexxar
1
) Versus
90
Y-Ibritumomab Tiuxetan
(Zevalin
1
)
No comparative clinical trial has ever been performed between
90
Y-ibritumomab
tiuxetan and
131
I-tositumomab and is unlikely ever to be either. However, it
appears reasonable to surmise that the two drugs have very similar response rates
and response durations from the published results.
The integration of
90
Y-ibritumomab tiuxetan and
131
I-tositumomab into
routine clinical practice seems, therefore, more likely to depend on the cost and
convenience of each therapy rather than perceived differences in clinical effi-
cacy.
131
I-tositumomab remains licensed in the United States only and if an EU
license is eventually gained, the radiation protection issues with the necessity for
five to six days inpatient stay for patients (within the EU) receiving
131
I-
tositumomab may influence clinicians on health economic grounds in favor of
90
Y-ibritumomab tiuxetan. The removal of the dosimetric dose, within the EU,
further simplifies the delivery of
90
Y-ibritumomab tiuxetan making the whole
regimen very easy for the patient.
RIT Induces Durable Remissions
Perhaps the most impressive finding to emerge from these maturing data using
RIT is for FLs, in all of the studies is the remarkable DR enjoyed by some
patients. This durability of treatment response was initially seen in patients
treated with
131
I-tositumomab (50) has also been observed with
90
Y-ibritumo-
mab tiuxetan (75). In all of the RIT studies, around 70% of patients who achieve
a CR remain in remission for years (43,50,75). Further, some patients treated in
the early studies have now been in remission for more than five years after and
with a median follow-up of almost four years (Table 2). An analysis of long-term
responders underscores the potential of
90
Y-ibritumomab tiuxetan to achieve
durable remissions with observed median DR approaching two years and
responses greater than six years being observed in some patients (43) (Fig. 3).
An analysis of prognostic factors has confirmed that this remarkable
durability of response is unlikely to be accounted for by patient selection as most
of these durable remissions have been achieved in heavily chemotherapy pre-
treated and chemo-refractory patients with validated poor prognostic factors such
as extensive prior therapy (19 regimens), bulky disease, high LDH, and extra-
nodal disease. Only disease bulk correlated with the ORR (<5 cm) [89 patients
ORR 90% (p < 0.001)] (55) (Fig. 4).
What Is the Role for Dosimetry in RIT?
One of the fundamental potential advantages of RIT is the ability to deliver
higher targeted radiation dose to the tumor than to normal tissue and thus
Radioimmunotherapy of Hematological Malignancies 173
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Figure 3 Progression-free survival for integrated summary of efficacy population
(n 250).
Figure 4 Progression-free survival for patients within each of the five clinical studies
that comprise the integrated summary of efficacy population.
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enhance the specific tumor killing. The fact that radioisotopes emit ionizing
radiation not only enables them to be used in therapy but also their emissions to
be quantified using radiation dosimetry. However, because of the relative
complexity of the radiation dose estimation for RIT, the clinical importance of
dosimetry in the RIT of NHL currently remains divisive. While some inves-
tigators regard dosimetry as essential component of RIT practice, others disagree
that it is necessary (27,28,53,62). It is possible that both groups of advocates may
be correct and that the requirement for dosimetry could be dependent on the
radioisotopes and the mAb targeting used. As ytrrium-90 is a pure b-emitter,
indium-111 is used as a surrogate and chelated to ibritumomab tiuxetan to
facilitate gamma camera imaging for dosimetry. Following the rituximab infu-
sion, imaging was performed using a dose of 185 MBq (5 mCi) and 1.6-mg total
mAb dose, as part of the registration studies at 2 to 24 hours, at 48 to 72 hours
and at 90 to 120 hours. For
131
I-tositumomab, gamma camera imaging, as pre-
viously described, is an essential part of the regimen and required for the patient-
specific dosimetry.
There are at least two important issues with regard to dosimetry; namely,
dosimetry can predict tumor response, and secondly it can predict normal tissue
toxicity. To date, despite the high response rates seen in lymphoma RIT, clinical
dosimetry studies have thus far failed to show a consistent dose-response rela-
tionship, although some investigators have found a correlation (30). One of the
factors that is highly likely to complicate the analysis and may underlie the
controversy is the inability of dosimetry to measure the tumoricidal capability of
some mAb.
Two imaging scans following rituximab injection do, however, form part
of the approved schedule within the United States, despite the fact that the
dosimetric studies failed to demonstrate a consistent correlation between the
estimated bone marrow dose and toxicity. It appears that
90
Y-ibritumomab
tiuxetan can be safely prescribed according to body weight and platelet count
(55), and this lack of positive correlation between imaging and bone marrow
toxicity led to imaging studies not being required within the EU (76).
INTEGRATION INTO TREATMENT ALGORITHMS FOR NHL
The high response rates and durable remissions achieved with either
90
Y-ibritumomab or
131
I-tositumomab make single agent RIT an attractive
treatment option for many patients with relapsed FL. Furthermore the impressive
DRs, seen after achieving a CR are achieved with a treatment that is completed
within a week, is very well tolerated and that has minimal nonmyelotoxicity
toxicity and easily manageable myelotoxicity.
The introduction of RIT has some parallels with the introduction of rit-
uximab into clinical practice in the late 1990s. There is no doubting that RIT
drugs are highly active but considerable uncertainty remains as to when and how
best to integrate RIT into clinical practice even within the licensed indication of
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relapsed low grade (United States) or relapsed rituximab failure or refractory FL
(within EU). The treatment is well tolerated by older patients and RIT makes this
approach a strong recommendation for relapsed FL.
In the education session on follicular at the American Society of Hema-
tologists (2004), an algorithm was suggested for FL where RIT was recom-
mended upon relapse first relapse after a rituximab-chemotherapy combination
(Fig. 5) (77). Currently this seems a reasonable treatment approach to FL, as it
does not exclude transplantation options at a later date, especially if, as rec-
ommended, progenitor stem cell collection is performed at the time of the initial
remission. Although data is emerging to suggest that RIT can be given after
transplantation (see above), this is inevitably at lower doses. Further clinical
trials will need to be performed to further define the role of RIT in the treatment
of other NHL.
CLINICAL PERSPECTIVES FOR THE NEXT FIVE YEARS
Although the results for single agent RIT are encouraging and make RIT an
attractive treatment option for relapsed FL, the future is likely to involve
integrating RIT into chemotherapy treatment protocols in an attempt
to increase relapse-free and overall survival. The current challenge for clinical
investigators is to determine the optimal approach of integrating RIT into
chemotherapy schedules. The emerging data using both
131
I-tostumomab and
Figure 5 A proposed treatment algorithm for follicular lymphoma patients after relapse
first relapse following a rituximab-chemotherapy combination. Source: From Ref. 77.
176 Illidge and Hainsworth
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90
Y-ibritumomab tiuxetan as consolidation following shortened or full course
chemotherapy look extremely promising and suggest that the quality of
responses can be substantially increased by this type of approach (44,54,78).
Future randomized clinical trials will define whether this type of approach
offers similar or perhaps even superior RFS over rituximab-chemotherapy
regimens followed by maintenance rituximab. Future clinical studies will
indicate whether the ability of RIT to improve the quality of the response from
partial to CR will add to RFS and perhaps even overall survival. The initial
highly promising phase II studies for
131
I-tositumomab suggest that this RIT as
a single treatment option may be worth exploring further for some patients and
preliminary data for patients using
90
Y-ibritumomab, tiuxetan, and rituximab,
as a first-line treatment for FL suggest very high response rates and will require
further exploration.
Given the high single agent activity of
90
Y-ibritumomab tiuxetan in DLBC
and
131
I-tositumomab in aggressive lymphoma, there are a number of clinical
trials that are now underway to integrate either of these two RIT reagents into the
front-line treatment of DLBC. There are a number of U.S. phase II studies and a
large European intergroup study with a randomization to
90
Y-ibritumomab
tiuxetan or no further treatment after full course CHOP-R chemotherapy that are
underway. Over the next five years the clinical studies that are currently
underway are likely to further define whether RIT has a role in further improving
the results achieved with R-CHOP immunochemotherapy in DLBCL.
Given the selection of higher risk poorer prognosis in patients who fail
R-CHOP regimens, it is to be expected that the results after standard BEAM
(BCNU, etoposide, cytarabine, melphalan) conditioning will result in less
impressive overall survival than prior to the introduction of R-CHOP. There is,
therefore, an urgent requirement to improve the results and an intensification of
the conditioning regimen is required. An area that is, therefore, currently
being intensely investigated is the integration of RIT into the conditioning
regimens instead of TBI or with reduced dose TBI, followed by ASCT rescue.
The studies to date have confirmed that higher myeloablative doses can be safely
delivered with ASCT support. While Press and colleagues have demonstrated
that myeloablative doses of
131
I-tositmomab with and without high dose che-
motherapy result in highly impressive RFS, the difficulty has been in repro-
ducing these excellent results given the extremely high doses of
131
I that are
manipulated in the conjugation process and then subsequently administered.
A more practical approach, which will allow the participation of many more
transplant centers, is the use of standard or escalated doses of
90
Y-ibritumomab tiuxetan. The early results are encouraging and confirm the
feasibility of the addition of escalated doses of
90
Y-ibritumomab tuxetan with the
standard conditioning regimen of BEAM (65). Appropriately designed
randomized studies will confirm over the next few years whether the addition of
RIT to high dose chemotherapy will result in improvement in treatment outcome
for patients with relapsed lymphoma.
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90
Y-ibritumomab tiuxetan is also being investigated in the treatment of
other aggressive CD20 positive lymphomas such as mantle cell lymphoma.
Preliminary data in patients with relapsed or refractory mantle cell lymphoma
suggest therapeutic efficacy of
90
Y-ibritumomab tiuxetan with disease control
being achieved in around half of the patients treated. Further clinical studies are
underway using RIT as consolidation after rituximab chemotherapy regimens,
and these studies should help to establish whether the poor results in this disease
can be improved upon.
The use of RIT in leukemia is less well developed and no radio-
immunoconjugate has yet been approved for routine delivery. The exquisite
sensitivity of these malignancies to targeted radiation alongside the highly
impressive results achieved by the pioneers in this field suggests that this is
highly likely to be a productive area for future clinical research and lead to
tangible patient benefits. The huge progress made over the last decade with the
development of RIT in the treatment of hematological malignancies leads to
distinct optimism that further development of RIT over the next five years will
lead to significant improvements in clinical outcomes for patients.
REFERENCES
1. Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol
2005; 23:11471157.
2. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared
with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J
Med 2002; 346:235242.
3. Robak T. Monoclonal antibodies in the treatment of chronic lymphoid leukemias.
Leuk Lymphoma 2004; 45:205219.
4. Knox SJ, Meredith RF. Clinical radioimmunotherapy. Semin Radiat Oncol 2000;
10:7393.
5. Press OW, Leonard JP, Coiffier B, et al. Immunotherapy of Non-Hodgkins Lym-
phomas. Hematology (Am Soc Hematol Educ Program) 2001; 2001:221240.
6. Kaminski MS, Zelenetz AD, Press OW, et al. Pivotal study of iodine I 131 tositu-
momab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-
Hodgkins lymphomas. J Clin Oncol 2001; 19:39183928.
7. DeNardo GL, DeNardo SJ, Goldstein DS, et al. Maximum-tolerated dose, toxicity,
and efficacy of (131)I-Lym-1 antibody for fractionated radioimmunotherapy of non-
Hodgkins lymphoma. J Clin Oncol 1998; 16:32463256.
8. Illidge TM, Cragg MS, McBride HM, et al. The importance of antibody-specificity
in determining successful radioimmunotherapy of B-cell lymphoma. Blood 1999;
94:233243.
9. Press OW, Farr AG, Borroz KI, et al. Endocytosis and degradation of monoclonal
antibodies targeting human B-cell malignancies. Cancer Res 1989; 49:49064912.
10. Grossbard ML, Press OW, Appelbaum FR, et al. Monoclonal antibody-based
therapies of leukemia and lymphoma. Blood 1992; 80:863878.
178 Illidge and Hainsworth
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0007_O.3d] [14/3/08/18:2:25]
[149184]
11. Chan HT, Hughes D, French RR, et al. CD20-induced lymphoma cell death is
independent of both caspases and its redistribution into triton X-100 insoluble
membrane rafts. Cancer Res 2003; 63:54805489.
12. Sharkey RM, Behr TM, Mattes MJ, et al. Advantage of residualizing radiolabels for
an internalizing antibody against the B-cell lymphoma antigen, CD22. Cancer
Immunol Immunother 1997; 44:179188.
13. Andres TL, Kadin ME. Immunologic markers in the differential diagnosis of small
round cell tumors from lymphocytic lymphoma and leukemia. Am J Clin Pathol
1983; 79:546552.
14. Griffin JD, Linch D, Sabbath K, et al. A monoclonal antibody reactive with normal
and leukemic human myeloid progenitor cells. Leuk Res 1984; 8:521534.
15. Hanenberg H, Baumann M, Quentin I, et al. Expression of the CEA gene family
members NCA-50/90 and NCA-160 (CD66) in childhood acute lymphoblastic leu-
kemias (ALLs) and in cell lines of B-cell origin. Leukemia 1994; 8:21272133.
16. Press OW, Rasey J. Principles of radioimmunotherapy for hematologists and
oncologists. Semin Oncol 2000; 27:6273.
17. Press OW, Shan D, Howell-Clark J, et al. Comparative metabolism and retention of
iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells.
Cancer Res 1996; 56:21232129.
18. DeNardo GL, Kukis DL, Shen S, et al. 67Cu-versus 131I-labeled Lym-1 antibody:
comparative pharmacokinetics and dosimetry in patients with non-Hodgkins lym-
phoma. Clin Cancer Res 1999; 5:533541.
19. Jurcic JG, Larson SM, Sgouros G, et al. Targeted alpha particle immunotherapy for
myeloid leukemia. Blood 2002; 100:12331239.
20. McDevitt MR, Sgouros G, Finn RD, et al. Radioimmunotherapy with alpha-emitting
nuclides. Eur J Nucl Med 1998; 25:13411351.
21. Illidge TM, Johnson PW. The emerging role of radioimmunotherapy in haemato-
logical malignancies. Br J Haematol 2000; 108:679688.
22. Wahl RL. The clinical importance of dosimetry in radioimmunotherapy with tosi-
tumomab and iodine I 131 tositumomab. Semin Oncol 2003; 30:3138.
23. Buchsbaum DJ, Wahl RL, Glenn SD, et al. Improved delivery of radiolabeled anti-
B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled
anti-B1 monoclonal antibody. Cancer Res 1992; 52:637642.
24. Du Y, Honeychurch J, Cragg MS, et al. Antibody-induced intracellular signaling
works in combination with radiation to eradicate lymphoma in radioimmunotherapy.
Blood 2004; 103:14851494.
25. Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector mechanisms of
anti-CD20 reagents. Blood 2004; 103:27382743.
26. Koral KF, Kaminski MS, Wahl RL. Correlation of tumor radiation-absorbed dose
with response is easier to find in previously untreated patients. J Nucl Med 2003;
44:15411543; (author reply 1543).
27. Postema EJ. Dosimetry and radioimmunotherapy of non-Hodgkins lymphoma.
J Nucl Med 2004; 45:21262127; (author reply 2127).
28. Goldenberg DM, Sharkey RM. Radioimmunotherapy of non-Hodgkins lymphoma
revisited. J Nucl Med 2005; 46:383384.
29. Nikula TK, McDevitt MR, Finn RD, et al. Alpha-emitting bismuth cyclohexylbenzyl
DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacoki-
netics, bioactivity, toxicity and chemistry. J Nucl Med 1999; 40:166176.
Radioimmunotherapy of Hematological Malignancies 179
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0007_O.3d] [14/3/08/18:2:25]
[149184]
30. Ruffner KL, Martin PJ, Hussell S, et al. Immunosuppressive effects of (131)I-anti-
CD45 antibody in unsensitized and donor antigen-presensitized H2-matched, minor
antigen-mismatched murine transplant models. Cancer Res 2001; 61:51265131.
31. Davis TA, Kaminski MS, Leonard JP, et al. The radioisotope contributes sig-
nificantly to the activity of radioimmunotherapy. Clin Cancer Res 2004; 10:7792
7798.
32. Kaminski MS, Tuck M, Estes J, et al.
131
I-tositumomab therapy as initial treatment
for follicular lymphoma. N Engl J Med 2005; 352:441449.
33. Press OW. Radioimmunotherapy for non-Hodgkins lymphomas: a historical per-
spective. Semin Oncol 2003; 30:1021.
34. Sharkey RM, Goldenberg DM. Perspectives on cancer therapy with radiolabeled
monoclonal antibodies. J Nucl Med 2005; 46:S115S127.
35. Wilder RB, DeNardo GL, DeNardo SJ. Radioimmunotherapy: recent results and
future directions. J Clin Oncol 1996; 14:13831400.
36. Goldenberg DM, Horowitz JA, Sharkey RM, et al. Targeting, dosimetry, and radio-
immunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal
antibody. J Clin Oncol 1991; 9:548564.
37. Tedder TF, Engel P. CD20: a regulator of cell-cycle progression of B lymphocytes.
Immunol Today 1994; 15:450454.
38. Grillo-Lopez AJ. Zevalin: the first radioimmunotherapy approved for the treatment
of lymphoma. Expert Rev Anticancer Ther 2002; 2:485493.
39. Witzig TE, White CA, Wiseman GA, et al. Phase I/II trial of IDEC-Y2B8 radio-
immunotherapy for treatment of relapsed or refractory CD20() B-cell non-
Hodgkins lymphoma. J Clin Oncol 1999; 17:37933803.
40. Wiseman GA, Gordon LI, Multani PS, et al. Ibritumomab tiuxetan radio-
immunotherapy for patients with relapsed or refractory non-Hodgkin lymphoma and
mild thrombocytopenia: a phase II multicenter trial. Blood 2002; 99:43364342.
41. Witzig TE, Flinn IW, Gordon LI, et al. treatment with ibritumomab tiuxetan radi-
oimmunotherapy in patients with rituximab-refractory follicular non-Hodgkins
lymphoma. J Clin Oncol 2002; 20:32623269.
42. Witzig TE, Gordon LI, Cabanillas F, et al. Randomized controlled trial of yttrium-
90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immuno-
therapy for patients with relapsed or refractory low-grade, follicular, or transformed
B-cell non-Hodgkins lymphoma. J Clin Oncol 2002; 20:24532463.
43. Gordon LI, Molina A, Witzig T, et al. Durable responses after ibritumomab tiuxetan
radioimmunotherapy for CD20 B-cell lymphoma: long-term follow-up of a phase
1/2 study. Blood 2004; 103:44294431.
44. Shipley DL, Greco FA, Spigel DR, et al. Rituximab with short duration chemo-
therapy followed by
90
Y-ibritumomab tiuxetan as first line treatment for patients
with follicular lymphoma: update of a Minnie Pearl Cancer Research Network
Phase II Trial. J Clin Oncol 2005; (abstr 6577).
45. Morschhauser F, Illidge T, Huglo D, et al. Efficacy and safety of yttrium-90 ibri-
tumomab tiuxetan in patients with relapsed or refractory diffuse large B-cell lym-
phoma not appropriate for autologous stem-cell transplantation. Blood 2007; 110:
5458.
46. Nadler LM, Ritz J, Hardy R, et al. A unique cell surface antigen identifying lym-
phoid malignancies of B cell origin. J Clin Invest 1981; 67:134140.
180 Illidge and Hainsworth
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0007_O.3d] [14/3/08/18:2:25]
[149184]
47. Hohenstein MA, Augustine SC, Rutar F, et al. Establishing an institutional model for
the administration of tositumomab and iodine I 131 tositumomab. Semin Oncol
2003; 30:3949.
48. Kaminski MS, Zasadny KR, Francis IR, et al. Radioimmunotherapy of B-cell lym-
phoma with [131I]anti-B1 (anti-CD20) antibody. N Engl J Med 1993; 329:459465.
49. Kaminski MS, Zasadny KR, Francis IR, et al. Iodine-131-anti-B1 radio-
immunotherapy for B-cell lymphoma. J Clin Oncol 1996; 14:19741981.
50. Fisher RI, Kaminski MS, Wahl RL, et al. Tositumomab and iodine-131 tositumomab
produces durable complete remissions in a subset of heavily pretreated patients with
low-grade and transformed non-Hodgkins lymphomas. J Clin Oncol 2005; 23:7565
7573.
51. Horning SJ, Younes A, Jain V, et al. Efficacy and safety of tositumomab and iodine-
131 tositumomab (Bexxar) in B-cell lymphoma, progressive after rituximab. J Clin
Oncol 2005; 23:712719.
52. Coleman M, Kaminski MS, Susan JK, Andrew DZ, et al. The BEXXAR therapeutic
regimen (tositumomab and iodine I 131 tositumomab) produced durable complete
remissions in heavily pretreated patients with non-Hodgkins lymphoma (NHL),
rituximab-relapsed/refractory disease, and rituximab-naive disease. Proc Am Soc
Hem Blood 2003; 102(11):29a; (abstr 89).
53. Koral KF, Dewaraja Y, Li J, et al. Update on hybrid conjugate-view SPECT tumor
dosimetry and response in 131I-tositumomab therapy of previously untreated lym-
phoma patients. J Nucl Med 2003; 44:457464.
54. Press OW, Unger JM, Braziel RM, et al. A phase 2 trial of CHOP chemotherapy
followed by tositumomab/iodine I 131 tositumomab for previously untreated folli-
cular non-Hodgkin lymphoma: Southwest Oncology Group Protocol S9911. Blood
2003; 102:16061612.
55. Witzig TE, White CA, Gordon LI, et al. Safety of yttrium-90 ibritumomab tiuxetan
radioimmunotherapy for relapsed low-grade, follicular, or transformed non-
Hodgkins lymphoma. J Clin Oncol 2003; 21:12631270.
56. Zelenetz AD. Radioimmunotherapy for lymphoma. Curr Opin Oncol 1999; 11:
375380.
57. Liu SY, Eary JF, Petersdorf SH, et al. Follow-up of relapsed B-cell lymphoma
patients treated with iodine-131-labeled anti-CD20 antibody and autologous stem-
cell rescue. J Clin Oncol 1998; 16:32703278.
58. Bennett JM, Kaminski MS, Leonard JP, et al. Assessment of treatment-related
myelodysplastic syndromes and acute myeloid leukemia in patients with non-
Hodgkin lymphoma treated with tositumomab and iodine I131 tositumomab. Blood
2005; 105:45764582.
59. Ansell SM, Ristow KM, Habermann TM, et al. Subsequent chemotherapy regimens
are well tolerated after radioimmunotherapy with yttrium-90 ibritumomab tiuxetan
for non-Hodgkins lymphoma. J Clin Oncol 2002; 20:38853890.
60. Dosik AD, Coleman M, Kostakoglu L, et al. Subsequent therapy can be administered
after tositumomab and iodine I-131 tositumomab for non-Hodgkin lymphoma.
Cancer 2006; 106:616622.
61. Jacobs SA, Vidnovic N, Joyce J, et al. Full-dose 90Y ibritumomab tiuxetan therapy is
safe in patients with prior myeloablative chemotherapy. Clin Cancer Res 2005; 11:
S7146S7150.
Radioimmunotherapy of Hematological Malignancies 181
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0007_O.3d] [14/3/08/18:2:25]
[149184]
62. Wahl RL. Tositumomab and
131
I therapy in non-Hodgkins lymphoma. J Nucl Med
2005; 46:S128S140.
63. Press OW, Eary JF, Gooley T, et al. A phase I/II trial of iodine-131-tositumomab
(anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation
for relapsed B-cell lymphomas. Blood 2000; 96:29342942.
64. Gopal AK, Gooley TA, Maloney DG, et al. High-dose radioimmunotherapy versus
conventional high-dose therapy and autologous hematopoietic stem cell trans-
plantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort
analysis. Blood 2003; 102:23512357.
65. Nademanee A, Forman S, Molina A, et al. A phase 1/2 trial of high-dose yttrium-90-
ibritumomab tiuxetan in combination with high-dose etoposide and cyclo-
phosphamide followed by autologous stem cell transplantation in patients with poor-
risk or relapsed non-Hodgkin lymphoma. Blood 2005; 106:28962902.
66. Scheidhauer K, Wolf I, Baumgartl HJ, et al. Biodistribution and kinetics of (131)I-
labelled anti-CD20 MAB IDEC-C2B8 (rituximab) in relapsed non-Hodgkins lym-
phoma. Eur J Nucl Med Mol Imaging 2002; 29:12761282.
67. Feldman EJ, Brandwein J, Stone R, et al. Phase III randomized multicenter study of a
humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with che-
motherapy, versus chemotherapy alone in patients with refractory or first-relapsed
acute myeloid leukemia. J Clin Oncol 2005; 23:41104116.
68. Burke JM, Caron PC, Papadopoulos EB, et al. Cytoreduction with iodine-131-anti-
CD33 antibodies before bone marrow transplantation for advanced myeloid leuke-
mias. Bone Marrow Transplant 2003; 32:549556.
69. Sgouros G, Ballangrud AM, Jurcic JG, et al. Pharmacokinetics and dosimetry of an
alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with
leukemia. J Nucl Med 1999; 40:19351946.
70. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45
antibody plus cyclophosphamide and total body irradiation for advanced acute leu-
kemia and myelodysplastic syndrome. Blood 1999; 94:12371247.
71. Niederwieser D, Maris M, Shizuru JA, et al. Low-dose total body irradiation (TBI)
and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-
matched or mismatched unrelated donors and postgrafting immunosuppression with
cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chi-
merism and sustained remissions in patients with hematological diseases. Blood
2003; 101:16201629.
72. McSweeney PA, Niederwieser D, Shizuru JA, et al. Hematopoietic cell trans-
plantation in older patients with hematologic malignancies: replacing high-dose
cytotoxic therapy with graft-versus-tumor effects. Blood 2001; 97:33903400.
73. Bunjes D, Buchmann I, Duncker C, et al. Rhenium 188-labeled anti-CD66 (a, b, c, e)
monoclonal antibody to intensify the conditioning regimen prior to stem cell
transplantation for patients with high-risk acute myeloid leukemia or myelodys-
plastic syndrome: results of a phase I-II study. Blood 2001; 98:565572.
74. Waldmann TA, White JD, Carrasquillo JA, et al. Radioimmunotherapy of
interleukin-2R alpha-expressing adult T-cell leukemia with Yttrium-90-labeled
anti-Tac. Blood 1995; 87:40634075.
75. Gordon LI, Witzig T, Molina A, et al. Yttrium 90-labeled ibritumomab tiuxetan
radioimmunotherapy produces high response rates and durable remissions in patients
with previously treated B-cell lymphoma. Clin Lymphoma 2004; 5:98101.
182 Illidge and Hainsworth
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[149184]
76. Weigert O, Illidge T, Hiddemann W, et al. Recommendations for the use of yttrium-90
ibritumomab tiuxetan in malignant lymphoma. Cancer 2006; 107:686695.
77. Winter JN, Gascoyne RD, Van Besien K. Low-grade lymphoma. Hematology (Am
Soc Hematol Educ Program) 2004; 2004(1):203220.
78. Leonard JP, Coleman M, Kostakoglu L, et al. Abbreviated chemotherapy with flu-
darabine followed by tositumomab and iodine I 131 tositumomab for untreated
follicular lymphoma. J Clin Oncol 2005; 23:56965704.
79. Witzig TE, Emmanouilides C, Molina A, et al. Yttrium-90 ibritumomab tiuxetan
radioimmunotherapy (RIT) induces durable complete responses (CR/CRu) in
patients with relapsed or refractory B-cell non-Hodgkins lymphoma (NHL). Proc
Am Soc Clin Oncol 2003; 22:597 (abstr 2400).
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8
Differentiation Induction in Acute
Promyelocytic Leukemia
Adi Gidron and Martin S. Tallman
Division of Hematology/Oncology, Department of Medicine, Northwestern
University Feinberg School of Medicine and The Robert H. Lurie Comprehensive
Cancer Center of Northwestern University, Chicago, Illinois, U.S.A.
INTRODUCTION
Acute myeloid leukemia (AML) is a heterogeneous group of diseases typically
associated with an aggressive course and generally a poor overall survival (OS).
Despite advances in therapy over the past 20 years, long-term survival for patients
with AML remains approximately 30% (1). Acute promyelocytic leukemia (APL)
represents a distinctive subtype of AML. It accounts for approximately 10% to
15% of all patients with AML and is distinguished from other types by a younger
median age (40 vs. 68 years), a unique genetic abnormality, the t(15;17) trans-
location and the formation of the promyelocytic leukemia-retinoic acid receptor
alpha (PML-RARa) fusion transcript, and most importantly, by the high cure rate
achieved with differentiation therapy. Until the late 1980s and early 1990s, APL
was considered the most fatal subtype of AML primarily because of a severe
coagulopathy often leading to catastrophic hemorrhage early in the natural history
of the disease or early in the course of treatment. The discovery that the leukemic
promyelocytes from patients with APL were uniquely sensitive to all-trans reti-
noic acid (ATRA) and that the breakpoint on chromosome 17 that resulted in the
development of an abnormal PML-RARa fusion product eventually led to the
recognition that disruption of RARa gene product was the major cause of
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maturation arrest in APL (2,3). Currently, APL is associated with several variant
chromosomal abnormalities leading to different gene rearrangements. PML-
RARa is the product of t(15;17), NPM (nucleophosmin)-RARa is the product of
t(5;17)(q35;q21), and is NuMA (nuclear matrix associated)-RARa the product of
t(11;17)(q13;q21), all of which lead to a syndrome that is responsive to ATRA.
PLZF (promyelocytic leukemia zinc finger)-RARa, the product of t(11;17)(q23;
q21), is unresponsive to ATRA. Signal transducer and activator of transcription
5B (STAT5B) is the product of t(17;17)(q11;q21) (4). In addition to cytogenetic
variants, molecular variant of the PML-RARa transcript such as bcr1, bcr2, and
bcr3 have also been described (5). Advances in the understanding of APL have
led to a new strategy in anti-leukemia therapy. Rather than relying on intensive
chemotherapy, the treatment of APL focuses now on differentiation therapy to
induce remission. Such a strategy results in long-term survival rates of more than
80% (6). This chapter will address the unique biology of APL, the role of ATRA
in differentiation therapy and arsenic trioxide (ATO) in inducing apoptosis. Both
agents contribute to the remarkable cure rates in APL. In addition, novel strat-
egies such as FLT3 inhibitors, antiangiogenesis agents, monoclonal antibodies,
differentiation agents, and histone deacetylase inhibitors will be described.
THE PATHOGENESIS OF APL
Retinoic acid (RA) is important in embryonic development and in a variety of
cellular processes. The activity of RA is mediated by RAR that are part of the
nuclear receptor superfamily. Several RARs have been described including
RXR, RARa, RARb, and RARg. RA activates RARa to bind to RAR elements
(RARE) located in the promoter region of genes important for differentiation
such as those of RARa, RARb, and RARg, and RXR. A heterodimer of RARa
and RXR forms and in conjunction with other coactivator proteins binds to DNA
and stimulates transcription through two domains. The ligand-independent
domain (AF-1) forms on the N-terminal of the protein and works in a promoter
context-dependent manner (5,7,8). The ligand-dependent domain (AF-2) is
associated with the corepressors NCoR, SMRT, Sin3A, and histone deacetylase.
Ligand binding to the AF-2 domain releases these corepressors and allows for
transcription of the target genes (5,911).
It was recognized early that vitamin Adeficient mice and humans developed
defects in hematopoiesis (5,12,13). However, the role of RA in myeloid dif-
ferentiation was more clearly demonstrated in cell lines such as HL60 and in
cells from patients with APL in the 1980s. HL60 cells that had a dominant
negative mutant of RARa were resistant to ATRA and showed a maturation
block. When these cells were infected with a retrovirus expressing RARa,
RARb, and RARg, differentiation was restored. It was then discovered that RA
acts directly through RARa to induce myeloid differentiation (14,15). In APL,
the fusion protein PML-RARa retains both the DNA and the ligand-binding
domains and inhibits terminal differentiation through abnormal recruitment of
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corepressors and histone deacetylases and acts as a dominant-negative RARa
mutant (5).
The fusion proteins characteristic of APL not only change the activity of
RARa, but also affect the normal function of the partner protein, PML. PML is
linked to RARa in 98% of all APL patients. The PML gene is located on
chromosome 15q22 and the protein localizes to nuclear bodies (NBs) (16). The
PML NB plays a key role in tumor suppression by regulating apoptosis, growth
arrest, and cellular senescence (16). The PML-RARa fusion results in delocal-
izing PML from the NB to microspeckled nuclear structures and exerting a
dominant-negative effect. The treatment of APL cells with ATRA not only
induces differentiation by releasing coreceptors, the release of RXR, and
restoring RA signaling, but also restores the PML-NB and therefore promotes
growth arrest and apoptosis (5).
CLINICAL TRIALS OF ATRA IN APL
The Predifferentiation Era of APL
Until the 1990s, combination chemotherapy with an anthracycline and cytosine
arabinoside (ara-C) was the standard treatment for APL. While most patients
achieved complete remission (CR), there was a high rate of early death and an
OS of only 20% to 40% was expected at two years. In the LAP0389 study
conducted by the Italian cooperative oncology group, GIMEMA (Gruppo Italiano
Ematologiche dellAdulto), 257 patients were randomized to receive either
idarubicin alone or idarubicin and ara-C for induction, followed by consolidation
with standard chemotherapy and then either by maintenance therapy with
6-mercaptopurine (6-MP) and methotrexate or observation. CR was achieved in
66% to 76% of patients and early death was reported in 15.3% to 21.4%. The
estimated eight-year event-free survival (EFS) was 23% to 35% and OS was
36% to 45% in favor of the maintenance arm. Early death was attributed mainly
to bleeding from worsening coagulopathy and resistance to chemotherapy (17).
In other studies, similar results were obtained and while it was shown that a
higher dose of anthracycline was associated with better OS, early death from
bleeding remained high (1520%) (18).
Choice of Anthracycline
It is not clear if one anthracycline or the other is more effective for induction
in APL. Fenaux et al. prospectively randomized patients to either rubidizone
or amsacrine, each with ara-C, without observing a difference between the
anthracyclines (19). There was a suggestion in a retrospective analysis that
idarubicin was associated with an improved outcome, but no prospective
randomized trial compared idarubicin with other anthracyclines (20). Although
anthracyclines improve OS, early death from bleeding and high relapse rates
characterize the predifferentiation era of APL treatment.
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Sequential Studies of ATRA in the Treatment of APL
Early phase II studies conducted in the late 1980s and early 1990s show a high
CR rate with ATRA alone. In 1988, Huang et al. were among the first to report
the activity of ATRA in 24 patients (16 previously untreated and 8 with resistant
disease) with 45 to 100 mg/m
2
/day of ATRA. All patients achieved CR (2). In
1991, Chen et al. reported 50 (47 previously untreated) patients treated with 60
to 80 mg/day of ATRA and observed a 94% CR rate. However, 40% of patients
relapsed (median duration of CR was 14.7 months) (21). Although a high CR
rate was achieved with ATRA monotherapy and early death was reduced
compared with historical chemotherapy studies (likely by resolution of the
coagulopathy with ATRA), some patients developed a rapid increase in the
leukocyte (WBC) count, which was often accompanied by the development of
the RA syndrome (RAS). The manifestations of this unique syndrome include
fever, weight gain, pulmonary infiltrates, pleural and pericardial effusions, renal
failure, rash, and occasionally death. In the North American Intergroup study
(protocol I0129), ATRA was definitively shown to prolong OS in APL patients
who were treated with ATRA at induction as compared with patients treated with
chemotherapy alone (22). In this study, 346 patients were randomized to receive
induction with chemotherapy (daunorubicin and ara-C) or ATRA. OS at one,
two, and three years was significantly improved in the ATRA group (75%, 57%,
and 50% vs. 82%, 72%, and 67%, respectively) (22). The APL 91 study by the
European APL group showed similar results (23). Subsequently, the European
APL group compared concurrent ATRA plus chemotherapy with ATRA fol-
lowed by chemotherapy. This approach resulted in a two-year EFS of 84% for
the patients treated concurrently versus 77% for the patients treated sequentially
(24). The incidence of RAS was also reduced with the concurrent approach from
25% to 10 % (25,26). Long-term follow-up to the APL 91 study by the European
APL group (ATRA followed by chemotherapy vs. chemotherapy alone) found an
estimated EFS and relapse rate at four years of 63% and 31 % in the ATRA group,
as compared with 17% and 87% in the chemotherapy group. OS at four years was
76% in the ATRA group as compared with 49% in the chemotherapy group (27).
Similarly, the North American Intergroup protocol reported a five-year DFS and
OS of 69% in patients who received ATRA for induction, compared with 29% and
45%, respectively, for patients induced with daunorubicin and ara-C (28). A recent
update of the Spanish cooperative group PETHEMA experience reported DFS of
81% to 90% at three years in 384 patients treated with ATRA and idarubicin for
induction followed by consolidation with idarubicn and mitoxantrone. ATRA was
added in consolidation in all patients who did not have low-risk disease (29).
Maintenance Therapy
The importance of ATRA maintenance was demonstrated by two large random-
ized trials. In the North American Intergroup trial, patients who were randomized
to daunorubicin and ara-C (DA) induction and no maintenance following
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consolidation (no exposure to ATRA) had a disease-free survival (DFS) of only
16% compared with 47% in patients who had DA induction and ATRA main-
tenance following identical consolidation and 74% in those who received ATRA
for both induction and maintenance (28). However, two recent studies have
suggested that maintenance therapy with ATRA or chemotherapy may not
benefit patients who are molecularly negative after intensive consolidation. The
Japanese Adult Leukemia Study Group (JALSG) has reported the results of a
trial (APL97) in which patients who were reverse transcriptase polymerase chain
reaction (RT-PCR) negative following intensive consolidation were randomized
to intensive maintenance chemotherapy (no ATRA) or observation (30). No
benefit was observed in the group of patients who received maintenance
chemotherapy. In a second trial, conducted by the GIMEMA, 586 patients
negative for the PML-RARa fusion transcript after consolidation chemotherapy
were randomized to one of three maintenance regimens for two years (low-dose
chemotherapy, ATRA, both chemotherapy and ATRA) or observation (31). No
benefit was observed for any of the maintenance regimens. These data suggest
that earlier studies may not have included optimal consolidation.
Treatment of Older Adults with APL
ATRA is also effective in both elderly patients and in children. In a trial from
Spain, 104 patients aged 60 and older received ATRA and idarubicin followed
by three consolidation courses of an anthracycline or anthracenedione followed
by ATRA maintenance and low-dose chemotherapy. Eighty-four percent
achieved CR, the six-year cumulative incidence of relapse was 8.5%, and DFS was
79% (32). Similarly, the GIMEMA conducted a trial in which 134 patients
between the ages of 60 and 75 years received a similar regimen as administered
in the Spanish trial. The DFS and OS at three years were between 72% and 83%
(33). The outcome for patients older than 60 in the APL93 trial was not as
favorable when compared with younger adults, with a lower CR rate of 86%
versus 94%, and OS at four years was only 57.6% compared with 78% in
younger adults. The higher death rate was attributed to higher mortality during
consolidation in the elderly group (34). A reduction in the intensity of consoli-
dation in older adults may be a useful strategy.
Treatment of Children with APL
In a subgroup analysis of the APL93 trial carried out by the European APL
Group, 97% of children achieved CR and five-year EFS and OS were 71% and
90%, respectively. Overall, there was no difference in outcome between children
and adults (35). In an analysis of the GIMEMA-AIEOP protocol, OS and EFS
were of 89% and 76% at over 10 years (36). Both trials confirm that when treated
with similar ATRA-based regimens to those used in adults, children fare as well
as adults. Similarly, the PETHEMA group treated 66 children with ATRA
and idarubicin, followed by consolidation with idarubicin monotherapy and
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ATRA-based maintenance. The OS and DFS at five years were 87% and 82%,
respectively (37). Overall, ATRA treatment in children resulted in results similar
to those obtained in adults. Because of potential excessive toxicity of ATRA in
children, many investigators use a reduced dose of 25 mg/m
2
/day (38).
Liposomal Formulation of ATRA
Although ATRA is an extremely effective drug in APL, this agent has the
limitation of being available only in an oral formulation. This limitation excludes
patients unable to take pills, such as young children and intensive-care patients
on life support. However, the introduction of a liposomal formulation proved to
be equally effective as oral ATRA and has the added benefit of overcoming the
declining blood levels that occur with the oral formulation after several weeks of
use (39). At the present time, this formulation is not commercially available.
Extramedullary Disease in APL
Before the introduction of ATRA, extramedullary disease (EMD) in APL was
rarely reported. However, the occurrence of this phenomenon is increasingly
observed after treatment with ATRA. While the incidence of EMD appears to be
increased in patients receiving ATRA, a report by the GIMEMA compared the
risk of developing EMD in two consecutive studies, the LAP0389 and AIDA.
Patients receiving ATRA did not appear to have an overall increased risk of
EMD (40). However, an increased risk of central nervous system disease was
suggested. It is possible that the prolonged survival achieved by treatment with
ATRA may account for the perceived increased occurrence of EMD or possibly
lack of exposure to ara-C in many patients. Common sites of EMD are the skin
(particularly at sites where vascular disruption occurred), central nervous system,
external auditory canal, and testis (4143). Risk factors for developing EMD in
APL appear to be a hyperleukocytosis at diagnosis, microgranular variant of
APL, and the bcr3 type of the PML-RARa fusion transcript (41). Patients with
EMD at relapse may still be salvaged with differentiation therapy. Tsimberidou
and colleagues, reported treating two patients with CNS relapse with a combi-
nation of ATRA, arsenic trioxide, and gemtuzumab ozogamicin and both
achieved CR (42). Others reported successful treatment of EMD relapses with
ATRA containing regimens as well (44,45).
In summary, ATRA is the treatment of choice for all patients with newly
diagnosed APL. It is now routine practice to combine ATRA with chemotherapy
during induction. When the presenting WBC is more than 10,000/mL, ATRA can
be started alone for two to four days to ameliorate the coagulopathy before
initiating chemotherapy (46). It is also routine to administer concurrent ATRA
and chemotherapy in this setting, particularly if the coagulopathy is not severe.
Following remission induction, consolidation with chemotherapy and ATRA
then maintenance therapy is currently the standard of care. However, despite the
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high rate of CRs and cures, some patients, particularly those with high-risk
disease, still relapse and require salvage therapy.
ARSENIC TRIOXIDE
Arsenic Trioxide (ATO) is one of the oldest medicines known and was used both
in Chinese medicine and in ancient Greece as a remedy for various ailments. The
use of arsenic in hematological disease was noted in the 1880s by Sir William
Osler (47). However, the activity of arsenic in modern medicine was revisited in
the last two decades in studies originating in China (48).
Mechanism of Action
ATO has a dual mechanism of action in APL patients. It induces both differ-
entiation and apoptosis. As discussed previously, the chromosomal abnormality
t(15;17) results in the PML-RARa fusion protein. This fusion protein causes a
maturation block at the promyelocytic stage and disaggregation of PML onco-
genic domains, disrupting the NBs and resulting in the loss of tumor suppression
(49). At low concentrations, ATO induces the phosphorylation of SMRT/NCoR
through the MAPK (mitogen-activated protein kinase) pathway. The SMRT/
NCoR complex dissociates from the PML-RARa fusion protein and allows for
coactivators to bind and for transcription to occur, which in turn results in
differentiation. In addition, ATO leads to the degradation of the PML-RARa
fusion protein and restoration of the normal RARa-RXR dimer, resulting in
differentiation (4850). ATO also induces apoptosis. PML normally controls
apoptosis, cell proliferation, and senescence and is localized to the NB (16). The
PML-RARa fusion protein disrupts the normal PML localization and function in
a dominant-negative way. ATO induces MAPK-mediated phosphorylation of
PML leading to sumoylation and retargeting it back to the NB. Restoring normal
function eventually triggers growth arrest and apoptosis (48). An additional
mechanism by which ATO promotes apoptosis is the generation of reactive
oxygen species (ROS). In APL cells, ATO increases the production of NADPH
(nicotinamide adenine dinucleotide) and results in the increase of superoxide
production. ROS production can induce phosphorylation and activate Jun N-
terminal kinase (JNK), which in turn trigger apoptosis (48). In summary, ATO
degrades the PML-RARa fusion protein, leads to the dissociation of the cor-
epressors, and allows for differentiation to occur. At higher doses, ATO restores
the normal function of PML and promotes apoptosis.
CLINICAL EFFICACY OF ATO IN APL
Initial Clinical Trials from China
Investigators in Harbin, China treated APL patients with the arsenic-containing
remedy Ailing 1 in the early 1970s (51). In initial studies, Ailing 1 was able to
induce CR in 27% of APL patients (52,53). A later study, which was conducted
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in the 1970s and 1980s and published in 1992, reported a 65% CR rate in
32 patients with APL with a 50% OS at five years, 19% survived at 10 years (54).
The active agent was found to be ATO and it was purified and administered to
72 patients with a CR rate of 73% in 30 previously untreated patients and 52% in
42 relapsed or refractory patients (55). The lack of myelosuppression and cross-
resistance with ATRA as well as the lack of development of hyperleukocytosis in
34 of 44 patients who achieved CR in this study were confirmed in subsequent
trials. In 1999, the results of 242 patients with newly diagnosed, relapsed, or
refractory APL treated with ATO at Harbin Medical University demonstrated a
CR rate of 75% (56).
Clinical Trials in the United States in Patients with Advanced APL
These pioneering results from China led to clinical trials in the United States.
Initially, two trials of ATO in relapsed and refractory patients were conducted. A
single institution pilot study involving 12 patients who previously received
ATRA and chemotherapy were treated with 0.06 to 0.2 mg/kg/day until leukemic
cells were no longer detected in the bone marrow. CR was obtained in 11 patients
(92%) and 8 patients (73%) became negative for PML-RARa fusion transcript by
RT-PCR (57). A follow-up multicenter trial of 40 patients with relapsed or
refractory APL after ATRA and anthracycline therapy, who received 0.15 mg/kg/
day ATO until the bone marrow cleared or for a maximum of 60 days, reported an
85% CR rate and 86% of assessable bone marrows (29/40) tested negative for
PML-RARa transcripts (58). While ATO was active in patients in first or greater
relapse, results were best for patients in first relapse (Fig. 1). Niu et al. reported a
CR rate of 85% also among 47 patients with relapsed disease (59). Other studies
confirmed these results as well (60). Taken together, these studies and others
indicate that in patients with relapsed APL, ATO therapy leads to a high CR rate,
Figure 1 Overall and relapse-free survival for relapsed and/or refractory APL treated
with arsenic trioxide.
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induces molecular remission in most patients, and is associated with infrequent
drug resistance and reversible mild adverse effects.
ATO in Patients with Newly Diagnosed APL
As the efficacy of ATO in relapsed and refractory disease became clear,
investigators began exploring its role in newly diagnosed patients. Investigators
at Harbin University in China pioneered this effort. The 88% CR rate reported by
Sun and colleagues in 124 previously untreated patients that received single
agent ATO was similar to that seen in relapsed patients. These observations, that
single agent ATO leads to a high CR rate, were confirmed by studies from India
and Iran (54,56,61,62). In the Iranian study, 94 newly diagnosed patients were
treated with 0.15 mg/kg daily dose until CR was obtained or for 60 days.
Consolidation was given at the same dose for 28 days. These investigators
reported an 86.3% CR and 94.5% and 87.6% one- and three-year OS, respec-
tively, for patients who achieved CR. In this trial, patients did not receive
chemotherapy. However, hyperleukocytosis occurred in 58.6% of patients and
the APL differentiation syndrome occurred in 20.7% of patients. Ten of twenty-
three patients with the APL differentiation syndrome died of this complication
(62). Additional data from India support these findings. In this trial, 72 patients
with newly diagnosed APL were treated with ATO as a single agent. At a median
follow up of 31 months, EFS and OS were reported as 70.2% and 81.3%,
respectively. Many of these patients, however, received chemotherapy in the
form of either an anthracycline (6 patients) or hydroxyurea (53 patients) for the
APL differentiation syndrome or hyperleukocytosis (63). Table 1 summarizes
the experience with ATO as a single agent for induction.
Preclinical evidence suggests ATO and ATRA have different mechanisms
of action and that, in vitro, they can be synergistic or antagonistic (49,64,65).
However, in vivo, these agents have an additive or synergistic effect when
administered concomitantly or sequentially, respectively (65,66,67). Results from
a clinical trial in which patients received ATO and ATRA for relapsed disease
showed that the agents could be given together without producing more toxicity,
but without apparent additive benefit (68). In a study from China, Shen et al.
randomly assigned 61 newly diagnosed patients with APL to receive ATRA,
Table 1 Induction with Single-Agent Arsenic Trioxide for Patients with Untreated APL
Study (Ref.) N CR (%) PCR-neg (%) Postremission therapy
Zhang (56) 124 88 NR Chemotherapy
Ghavamzadeh (62) 94 86 92 ATO 1
Mathews (63) 72 80 95 ATO 6
Abbreviations: CR, complete response; PCR, polymerase chain reaction; NR, not reacted;
ATO, arsenic trioxide.
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ATO, or the combination of both for induction therapy (69). After achieving CR,
all patients received three consecutive courses of consolidation therapy with
daunorubicin, ara-C and homoharringtonine followed by maintenance with the
induction drug plus methotrexate and 6-MP for five cycles. Although chemo-
therapy was added when hyperleukocytosis occurred (equal among groups), CR
rates were more than 90% in all groups and time to CR was faster (25.5 days) in
the combination group compared with 40.5 days and 31 days in the ATRA and
ATO groups, respectively. Furthermore, in the combination group, platelets
recovered faster and PML-RARa transcript copies (as measured by RT-PCR) at
CR were significantly lower (69). All patients in the combination group remained
in CR at 18 months. At the MD Anderson Cancer Center, Estey and colleagues
treated 32 patients with ATRA and ATO as induction therapy. Patients received
chemotherapy (typically gemtuzumab ozogamicin) only if they presented with a
WBC count of more than 10,000/mL or if they failed to achieve molecular CR
three months after achieving CR. Thirteen patients were high risk and received
chemotherapy during induction. An 88% CR rate (85% in high-risk group) was
reported. Only two patients had a molecular relapse at 6 and 12 months from CR
(70). These studies highlight the finding that administering ATRA and ATO
concomitantly is safe and results in a greater reduction in disease burden without
increased toxicity as compared with each agent alone. In addition, these data
indicate the synergy between the agents as suggested by preclinical studies.
Despite the synergy between ATRA and ATO, the two agents may not be suf-
ficient for induction. The rate of hyperleukocytosis and APL differentiation
syndrome was significant in patients who received ATO as single agent in the
Iranian study (62) and mortality was 9%, highlighting the possible need for a
cytotoxic agent. Nevertheless, the results from the trial by Mathews and col-
leagues are very promising and suggest that some patients at very low risk
(defined as WBC < 5 10
9
/L and platelet count > 20 10
9
/L) may be curable
without chemotherapy and with single agent ATO (Fig. 2). Gemtuzumab
Figure 2 KaplanMeier product limit estimate of (A) OS and of EFS (n 72). (B) Kaplan
Meier product limit estimate of DFS (n 62). Abbreviations: OS, overall survival;
EFS, event-free survival; DFS, disease-free survival.
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ozogamicin (Mylotarg
TM
), an anti-CD33 antibody that is chemically linked to the
potent cytotoxic antibiotic calicheamicin, is active in APL. In early trials, using
gemtuzumab ozogamicin in conjunction with ATRA was effective in controlling
leukocytosis (71).
OTHER NOVEL THERAPIES IN APL
High-risk patients are the group most likely to relapse, and new therapies are
needed. Several new approaches are now under clinical investigation such as
FLT3 inhibitors, monoclonal antibodies, antiangiogenesis agents, differentiation
agents, and histone deacetylase inhibitors (Table 2).
FLT3 Inhibitors
FLT3 is a tyrosine kinase receptor expressed in leukemic cells. FLT3 mutations
that occur as internal tandem duplication (ITD) are the most common genetic
abnormality in AML. In APL patients FLT3 mutations are found in approxi-
mately 20% to 40% of patients (72,73). The presence of FLT-3/ITD was asso-
ciated with high peripheral leukemic cell count at presentation and may play a
role in the progression of APL (72). Currently FLT3 inhibitors are under clinical
investigation, and it remains to be seen if they will provide a therapeutic option
in the treatment of APL.
Monoclonal Antibodies
Alemtuzumab is a humanized anti-CD52 monoclonal antibody with activity in a
number of lymphoproliferative diseases and in transplant. Li and colleagues
Table 2 Novel Agents with Potential Activity in APL
Agent (Refs.) Rationale
FLT3 inhibitors (72,73) FLT3 mutations present in 2040% of APL
patients
Alemtuzumab (74) ATRA increases CD52 expression
Antiangiogenesis agents (75,76) Increased vessels and VEGF in bone marrow
samples of APL patients
Curcumin (78,79) Inhibits NF-kB and induces differentiation
with ATRA
Phosphodiesterase Inhibitors (80) Increases cAMPpromotes differentiation
with ATRA
Histone deacetylase inhibitors (81) Overcomes ATRA resistance by releasing
nuclear co-repressor complex
Abbreviations: APL, acute promyelocytic leukemia; VEGF, vascular endothelial growth factor;
ATRA, all-trans retinoic acid; camp, cyclic adenosine monophosphate.
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demonstrated that ATRA induces the expression of CD52 in NB4 cells. Fur-
thermore, they demonstrated that CD52 expression only happened in leukemic
cells that expressed PML-RARa or in cells at the promyelocytic stage (74).
Currently, data on the clinical efficacy of alemtuzumab in APL are lacking.
Antiangiogenesis Strategies
Angiogenesis has a critical role in the pathogenesis of hematologic diseases. In
APL, angiogenesis in the bone marrow is increased and is mediated by the
vascular endothelial growth factor (VEGF). ATRA therapy inhibits VEGF
production and decreases microvessel density in the bone marrow (75,76).
Clinical trials are needed to establish the role of antiangiogenesis agents such as
thalidomide and lenolidomide and other VEGF inhibitors in the treatment of
APL. It is hypothesized that VEGF promotes APL cell survival through external
and internal autocrine loops (76). It also appears that ATO may have an anti-
angiogenesis effect as well (77).
Differentiation Agents
Agents that enhance the activity of ATRA are currently under investigation.
Curcumin, an extract from the spice turmeric, inhibits the activation of NF-kB
(78) and induces differentiation in the presence of ATRA. Curcumin was found
to enhance the activity of ATRA in cell lines resistant to ATRA. NB4-R1 cells,
which are resistant to ATRA, differentiated in the presence of ATRA and cur-
cumin but not in the presence of either agent alone. Clinical data are not
available at this time. However, these results suggest that curcumin may be a
new unconventional agent in the treatment of APL (79). Another compound that
may overcome ATRA resistance is cyclic adenosine monophosphate (cAMP).
RA-and cAMP-signaling pathways converge on RARa, and cAMP greatly
enhances the differentiation mediated by ATRA and ATO. Phosphodiesterase
inhibitors such as theophylline, which increase the levels of cAMP, may prove
useful in overcoming ATRA resistance (80).
Histone Deacetylase Inhibitors
The PML-RARa fusion protein leads to abnormal recruitment of histone
deacetylase, which in turn leads to a transcription block (5). Therefore, treatment
with an inhibitor of histone deacetylase may help reverse this transcription block.
One patient with refractory APL resistant to ATRA alone was treated with
sodium phenylbutyrate in addition to ATRA. Twenty-three days after initiation
of therapy, the patient achieved clinical and cytogenetic CR. A second course of
phenylbutyrate resulted in a molecular CR (81). This experience shows that
histone deacetylase inhibitors may prove beneficial in ATRA-refractory APL.
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TREATMENT RECOMMENDATIONS FOR APL AND
MONITORING OF MINIMAL RESIDUAL DISEASE
Induction Therapy
Given the proven efficacy of ATRA and ATO in the treatment of newly diag-
nosed and relapsed APL, new treatment strategies are evolving. The evolution of
therapy with ATRA-based regimens is outlined in Table 3. On the basis of the
results of the European APL Group trials (APL91, APL93), the North American
Intergroup (28), and others, induction therapy should generally include ATRA at
a dose of 45 mg/m
2
/day in divided doses and at least an anthracycline. Because of
the apparent increased retinoid toxicities in younger patients, in general patients
younger than 20 years, receive ATRA at a dose of 25 mg/m
2
/day. There is
insufficient data to recommend one anthracycline over the other. Idarubicin has
been use more often as a single agent without ara-C, and daunorubicin has been
used more often in combination with ara-C. ATRA is given until CR or up to
60 days. It is important to resist premature evaluation of the bone marrow before five
to six weeks as responses are not expected earlier. In fact, a day-14 bone marrow
evaluation, which is so routine after conventional induction chemotherapy for
other subtypes of AML, is not useful in APL and can be abandoned. ATRA
should be started as soon as the diagnosis is suspected, before genetic confir-
mation. If the presenting WBC is more than 10,000/mL, it is reasonable to treat
with ATRA and concurrent chemotherapy to minimize the coagulopathy (46).
Alternatively, if the WBC is low and the coagulopathy is severe, the adminis-
tration of ATRA for two to four days is a reasonable approach.
Table 3 Evolution of Therapy with ATRA-Based Regimens for Patients with Newly
Diagnosed APL
Regimen
Study (Ref.) N Induction Consolidation Maint DFS (%)
APL 91 (23) 54 ATRA
DNR Ara-C
DNR Ara-C No 63
Interg 0129 (22) 49 ATRA DNR Ara-C Yes 74
GIMEMA (31) 108 ATRA IDA IDA/Mitox
Ara-C
Yes 89
PETHEMA (29) 384 ATRA IDA IDA/Mitox
ATRA
Yes 90
Shanghai (69) 20 ATRA ATO Chemotherapy Yes 100
Interg C9710 500 ATRA
DNR Ara-C
DNR ATRA
ATO
Yes 77*
Iran (62) 94 ATO ATO ATO 63.7
Abbreviations: ATRA, all-trans retinoic acid; DNR, daunorubicin; IDA, idarubicin; Mitox, mitoxantrone;
ATO, arsenic trioxide; Interg, North American Intergroup.
*Event-free survival.
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Consolidation Therapy
Standard consolidation chemotherapy regimens include anthracyclines with the
goal of eradicating the leukemic clone. The optimal number of consolidation
courses is not well defined, but most clinicians administer two to three courses.
The role of ara-C has been questioned in this setting. Data presented by Sanz and
colleagues demonstrated equal efficacy between anthracycline-based consolida-
tion therapy with and without ara-C (82). Hence, it appears that ara-C may not
have a role in this phase of treatment when idarubicin is given, as the anthracycline
and ATRA is administered in consolidation. However, in a randomized trial
carried out by the European APL group, newly diagnosed patients were
randomized to either ATRA, DA, or ATRA and daunorubicin without ara-C (83).
The study was closed early because of an increase in relapses among the patients
not receiving ara-C. The North American Intergroup (protocol C9710) studied the
role of ATO in consolidation. The results suggested a benefit for early consoli-
dation with two cycles of ATO (84). Several other studies have suggested that the
administration of intermediate-dose or high-dose ara-C either in induction or in
consolidation may benefit patients with high-risk disease (85,86).
Maintenance Therapy
Randomized studies have demonstrated the superiority of ATRA containing
maintenance regimens over that containing no maintenance in reducing relapse
(24,27,28,87). Long-term DFS rate with ATRA-based maintenance was 74% to
90%. The question if chemotherapy such as methotrexate and 6-MP add to ATRA
maintenance alone is still under investigation. It is currently recommended to give
ATRA-based maintenance for at least one to two years following consolidation.
However, recent studies have suggested that patients rendered molecularly neg-
ative with intensive consolidation may not benefit from maintenance.
Relapsed Disease
ATO has excellent activity in relapsed and refractory disease and is considered
the treatment of choice for relapsed disease. ATO is given at a dose of 0.15 mg/
kg/day for five days a week for five weeks. ATRA as a single agent in relapsed
disease produces inferior results compared with ATO (88). Patients who have
refractory disease by morphology or persistent disease by molecular studies and
are otherwise suitable candidates should be referred for allogeneic hematopoietic
stem cell transplant. If a donor is not identified, patients should enroll in a
clinical trial.
Monitoring of Minimal Residual Disease
The PML-RARa fusion protein allows for monitoring of disease at the molecular
level. Achieving complete molecular remission on two or more consecutive
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RT-PCR assays is strongly linked to a decreased rate of relapse and prolonged
survival. Conversely, patients with two or more positive RT-PCR were likely to
relapse. A molecular relapse may precede clinical relapse by several months
(89). As burden of disease is substantially lower at the time of molecular relapse
as compared with morphologic relapse, there may be a role for the use of ATO at
the time of molecular relapse without waiting for morphologic relapse (90). The
suggested molecular testing should consist of at least two successive marrow
samples at the end of treatment, then every three months for the first two years of
CR, then every six months for the next two to three years (90). Such monitoring
may provide the most benefit for high-risk patients.
CONCLUSION
The treatment of APL has evolved rapidly over the last 15 years. Once a disease
with poor OS and often an abrupt catastrophic course, APL has become the most
curable of all the subtypes of AML. The unique biology of APL, in which an
aberrant fusion protein inhibits differentiation and apoptosis, lends itself to a
therapeutic strategy that overcomes the transcription block. ATRA and ATO
both target the fusion protein and restore differentiation and apoptosis by
degrading the PML-RARa fusion protein. Both agents induce a high rate of
remission as single agents. Synergy between ATRA and ATO may permit a
reduction or possibly elimination of chemotherapy in most patients. This
approach could be used in conjunction with gemtuzumab ozogamicin for
induction and potentially offer patients with a curative approach without the use
of standard intensive chemotherapy. The role of other novel agents such as FLT3
inhibitors, monoclonal antibodies, antiangiogenesis agents, differentiation
agents, and histone deacetylase inhibitors remains to be determined. Insights
gained in the molecular events that lead to APL may potentially provide
advances in the understanding of leukemogenesis in other AML subtypes. These
may lead to the development of other differentiation-inducing therapies or novel
strategies for other AML subtypes.
Clinical Perspective for the Next Five Years
Improvements in the outcome of patients with APL will continue to occur and
potential short-term and long-term toxicities are likely to diminish. Further
characterization of the molecular genetics and clinical features of APL, partic-
ularly high-risk patients, may allow more specifically directed therapy. A sig-
nificant proportion of high-risk patients appear to express the FLT3/ITD gene
mutation raising the possibility of treatment with a FLT3 inhibitor (91). It is clear
that therapy in the next five years will involve less, and perhaps minimal, or even
no cytotoxic chemotherapy since the combination of ATRA and ATO appear to
be extremely effective in inducing a molecular remission. ATO alone may be
very effective in low-risk patients. More potent synthetic retinoids such as
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tamibarotene (AM80), which has a low affinity for the cellular RA-binding
protein and does not bind to RARg, which may limit toxicities such as rash, may
prove to be another important advance (92,93).
REFERENCES
1. Yanada M, Suzuki M, Kawashima K, et al. Long-term outcomes for unselected
patients with acute myeloid leukemia categorized according to the World Health
Organization classification: a single-center experience. Eur J Haematol 2005; 74(5):
418423.
2. Huang ME, Ye YC, Chen SR, et al. Use of all-trans retinoic acid in the treatment of
acute promyelocytic leukemia. Blood 1988; 72(2):567572.
3. Alcalay M, Zangrilli D, Pandolfi PP, et al. Translocation breakpoint of acute pro-
myelocytic leukemia lies within the retinoic acid receptor alpha locus. Proc Natl
Acad Sci U S A 1991; 88(5):19771981.
4. Zelent A, Guidez F, Melnick A, et al. Translocations of the RARalpha gene in acute
promyelocytic leukemia. Oncogene 2001; 20(49):71867203.
5. Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and
their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999; 93(10):
31673215.
6. Lowenberg B, Griffin JD, Tallman MS. Acute myeloid leukemia and acute promye-
locytic leukemia. Hematology (Am Soc Hematol Educ Program) 2003 2003(1): 82101.
7. Nagpal S, Saunders M, Kastner P, et al. Promoter context- and response element-
dependent specificity of the transcriptional activation and modulating functions of
retinoic acid receptors. Cell 1992; 70(6):10071019.
8. Nagpal S, Friant S, Nakshatri H, et al. RARs and RXRs: evidence for two autono-
mous transactivation functions (AF-1 and AF-2) and heterodimerization in vivo.
Embo J 1993; 12(6):23492360.
9. Chen JD, Evans RM. A transcriptional co-repressor that interacts with nuclear
hormone receptors. Nature 1995; 377(6548):454457.
10. Horlein AJ, Naar AM, Heinzel T, et al. Ligand-independent repression by the thyroid
hormone receptor mediated by a nuclear receptor co-repressor. Nature 1995; 377
(6548):397404.
11. Pazin MJ, Kadonaga JT. Whats up and down with histone deacetylation and tran-
scription? Cell 1997; 89(3):325328.
12. Wolbach SB, Howe PR. Nutrition classics from J Exp Med 42: 75377, 1925. Tissue
changes following deprivation of fat-soluble A vitamin. Nutr Rev 1978; 36(1):1619.
13. Hodges RE, Sauberlich HE, Canham JE, et al. Hematopoietic studies in vitamin A
deficiency. Am J Clin Nutr 1978; 31(5):876885.
14. Collins SJ, Robertson KA, Mueller L. Retinoic acid-induced granulocytic differen-
tiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic
acid receptor (RAR-alpha). Mol Cell Biol 1990; 10(5):21542163.
15. Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human
promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci U S A
1980; 77(5):29362940.
16. Salomoni P, Pandolfi PP. The role of PML in tumor suppression. Cell 2002; 108(2):
165170.
200 Gidron and Tallman
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0008_O.3d] [14/3/08/18:4:43]
[185206]
17. Avvisati G, Tallman MS. All-trans retinoic acid in acute promyelocytic leukaemia.
Best Pract Res Clin Haematol 2003; 16(3):419432.
18. Head D, Kopecky KJ, Weick J, et al. Effect of aggressive daunomycin therapy on
survival in acute promyelocytic leukemia. Blood 1995; 86(5):17171728.
19. Fenaux P, Tertian G, Castaigne S, et al. A randomized trial of amsacrine and rubi-
dazone in 39 patients with acute promyelocytic leukemia. J Clin Oncol 1991; 9(9):
15561561.
20. Berman E, Little C, Kher U, et al. Prognostic analysis of patients with acute pro-
myelocytic leukemia. Blood, 1991(suppl):43a.
21. Chen ZX, Xue YQ, Zhang R, et al. A clinical and experimental study on all-trans
retinoic acid-treated acute promyelocytic leukemia patients. Blood 1991; 78(6):
14131419.
22. Tallman MS, Andersen JW, Schiffer CA, et al. All-trans-retinoic acid in acute
promyelocytic leukemia. N Engl J Med 1997;337(15):10211028.
23. Fenaux P, Le Deley MC, Castaigne S, et al. Effect of all transretinoic acid in newly
diagnosed acute promyelocytic leukemia: results of a multicenter randomized trial.
European APL 91 Group. Blood 1993; 82(11):32413249.
24. Fenaux P, Chastang C, Chevret S, et al. A randomized comparison of all transretinoic
acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role
of maintenance therapy in newly diagnosed acute promyelocytic leukemia. The
European APL Group. Blood 1999; 94(4):11921200.
25. Vahdat L, Maslak P, Miller WH Jr., et al. Early mortality and the retinoic acid
syndrome in acute promyelocytic leukemia: impact of leukocytosis, low-dose che-
motherapy, PMN/RAR-alpha isoform, and CD13 expression in patients treated with
all-trans retinoic acid. Blood 1994; 84(11):38433849.
26. Tallman MS, Andersen JW, Schiffer CA, et al. Clinical description of 44 patients
with acute promyelocytic leukemia who developed the retinoic acid syndrome.
Blood 2000; 95(1):9095.
27. Fenaux P, Chevret S, Guerci A, et al. Long-term follow-up confirms the benefit of
all-trans retinoic acid in acute promyelocytic leukemia. European APL group.
Leukemia 2000; 14(8):13711377.
28. Tallman MS, Andersen JW, Schiffer CA, et al. All-trans retinoic acid in acute
promyelocytic leukemia: long-term outcome and prognostic factor analysis from the
North American Intergroup protocol. Blood 2002; 100(13):42984302.
29. Sanz MA, Martin G, Gonzalez M, et al. Risk-adapted treatment of acute promye-
locytic leukemia with all-trans-retinoic acid and anthracycline monochemotherapy: a
multicenter study by the PETHEMA group. Blood 2004; 103(4):12371243.
30. Asou N, Kishimoto Y, Kiyoi H, et al. A randomized study with or without intensified
maintenance chemotherapy in patients with acute promyelocytic leukemia who have
become negative for PML-RARalpha transcript after consolidation therapy: the Japan
Adult Leukemia Study Group (JALSG) APL97 study. Blood 2007; 110(1):5966.
31. Avvisati G Petti M, Lo Coco F, et al. AIDA: the Italian way of treating acute
promyelocytic leukemia (APL). Blood 2003(suppl 1):142a.
32. Sanz MA, Vellenga E, Rayon C, et al. All-trans retinoic acid and anthracycline
monochemotherapy for the treatment of elderly patients with acute promyelocytic
leukemia. Blood 2004; 104(12):34903493.
33. Mandelli F, Latagliata R, Avvisati G, et al. Treatment of elderly patients (> or
60 years) with newly diagnosed acute promyelocytic leukemia: results of the Italian
Differentiation Induction in Acute Promyelocytic Leukemia 201
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0008_O.3d] [14/3/08/18:4:43]
[185206]
multicenter group GIMEMA with ATRA and idarubicin (AIDA) protocols.
Leukemia 2003; 17(6):10851090.
34. Ades L, Chevret S, de Botton S, et al. Outcome of acute promyelocytic leukemia
treated with all trans retinoic acid and chemotherapy in elderly patients: the Euro-
pean group experience. Leukemia 2005; 19(2):230233.
35. de Botton S, Coiteux V, Chevret S, et al. Outcome of childhood acute promyelocytic
leukemia with all-trans-retinoic acid and chemotherapy. J Clin Oncol 2004; 22(8):
14041412.
36. Testi AM, Biondi A, Lo Coco F, et al. GIMEMA-AIEOPAIDA protocol for the
treatment of newly diagnosed acute promyelocytic leukemia (APL) in children.
Blood 2005; 106(2):447453.
37. Ortega JJ, Madero L, Martin G, et al. Treatment with all-trans retinoic acid and
anthracycline monochemotherapy for children with acute promyelocytic leukemia: a
multicenter study by the PETHEMA Group. J Clin Oncol 2005; 23(30):76327640.
38. Castaigne S, Lefebvre P, Chomienne C, et al. Effectiveness and pharmacokinetics of
low-dose all-trans retinoic acid (25 mg/m2) in acute promyelocytic leukemia. Blood
1993; 82(12):35603563.
39. 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(1):7380.
40. Specchia G, Lo Coco F, Vignetti M, et al. Extramedullary involvement at relapse
in acute promyelocytic leukemia patients treated or not with all-trans retinoic acid:
a report by the Gruppo Italiano Malattie Ematologiche dellAdulto. J Clin Oncol
2001; 19(20):40234028.
41. Sanz MA, Larrea L, Sanz G, et al. Cutaneous promyelocytic sarcoma at sites of
vascular access and marrow aspiration. A characteristic localization of chloromas in
acute promyelocytic leukemia? Haematologica 2000; 85(7):758762.
42. Tsimberidou AM, Estey E, Whitman GJ, et al. Extramedullary relapse in a patient
with acute promyelocytic leukemia: successful treatment with arsenic trioxide, all-
trans retinoic acid and gemtuzumab ozogamicin therapies. Leuk Res 2004; 28(9):
991994.
43. Lee KW, Yi J, Yun T, et al. Extramedullary relapse confirmed by fluorescence in situ
hybridization study of an ear mass in acute promyelocytic leukemia. Int J Hematol
2004; 79(5):462464.
44. Lederman CA, Weisberger J, Seiter K, et al. Differentiation of extramedullary acute
promyelocytic leukemia by all-trans-retinoic acid. Leuk Lymphoma 1995; 18(12):
189193.
45. Patriarca F, Fili C, Geromin A, et al. Activity of all-trans-retinoic acid in a case of
central nervous system extramedullary relapse of acute promyelocytic leukemia. Eur
J Haematol 2002; 68(5):310313.
46. Tallman MS, Nabhan C, Feusner JH, et al. Acute promyelocytic leukemia: evolving
therapeutic strategies. Blood 2002; 99(3):759767.
47. Robb-Smith AH. Oslers influence on haematology. Blood Cells 1981; 7(3):513536.
48. Chou WC, Dang CV. Acute promyelocytic leukemia: recent advances in therapy and
molecular basis of response to arsenic therapies. Curr Opin Hematol 2005; 12(1):16.
49. Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide (As2O3) in the treatment of
acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects
on APL cells. Blood 1997; 89(9):33453353.
202 Gidron and Tallman
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0008_O.3d] [14/3/08/18:4:43]
[185206]
50. Grignani F, De Matteis S, Nervi C, et al. Fusion proteins of the retinoic acid receptor-
alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 1998; 391(6669):
815818.
51. Zhang TD. Treatment of acute granulocytic leukemia with Ai ling No. 1: clinical
analysis and experimental research. Zhong Xi Yi Jie He Za Zhi 1984; 4(1):1920.
52. Douer D, Tallman MS. Arsenic trioxide: new clinical experience with an old
medication in hematologic malignancies. J Clin Oncol 2005; 23(10):23962410.
53. Zhang T. Leukemia treatment in traditional Chinese medicine. Chin J Traditional
Chin Med 1985; 10:1314.
54. Sun HP, Ma L, Hu C, et al. Thirty-two cases of treating acute promyelocytic leu-
kemia by Ailing-1 (cancer-cure-1) therapy combined with syndrome differentitation
teatment of traditional Chinese medicine. Chin J Comb Trad Chin Med West Med
1992; 12:170171.
55. Zhang P, Wang S, Hu LH, et al. Seventy-two cases of acute promyelocytic leukemia
treated with arsenic trioxide. Chin J Hematol 1996; 17(2):5860.
56. Zhang P. The use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic
leukemia. J Biol Regul Homeost Agents 1999; 13(4):195200.
57. Soignet SL, Maslak P, Wang ZG, et al. Complete remission after treatment of acute
promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998; 339(19):13411348.
58. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study of arsenic
trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001; 19(18):38523860.
59. Niu C, Yan H, Yu T, et al. Studies on treatment of acute promyelocytic leukemia
with arsenic trioxide: remission induction, follow-up, and molecular monitoring in
11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood
1999; 94(10):33153324.
60. Lazo G, Kantarjian H, Estey E, et al. Use of arsenic trioxide (As2O3) in the treatment
of patients with acute promyelocytic leukemia: the MD Anderson experience. Cancer
2003; 97(9):22182224.
61. Mathews V, Balasubramanian P, Shaji RV, et al. Arsenic trioxide in the treatment of
newly diagnosed acute promyelocytic leukemia: a single center experience. Am J
Hematol 2002; 70(4):292299.
62. Ghavamzadeh A, Alimoghaddam K, Ghaffari SH, et al. Treatment of acute pro-
myelocytic leukemia with arsenic trioxide without ATRA and/or chemotherapy. Ann
Oncol 2006; 17(1):131134.
63. Mathews V, Biju G, Lakshmi KM, et al. Single agent arsenic trioxide in the treat-
ment of newly diagnosed acute promyelocytic leukemia: durable remissions with
minimal toxicity. Blood 2006; 107(7):26272632.
64. Gianni M, Koken MH, Chelbi-Alix MK, et al. Combined arsenic and retinoic acid
treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells.
Blood 1998; 91(11):43004310.
65. Jing Y, Wang L, Xia L, et al. Combined effect of all-trans retinoic acid and arsenic
trioxide in acute promyelocytic leukemia cells in vitro and in vivo. Blood 2001; 97(1):
264269.
66. Rego EM, He LZ, Warrell RP Jr., et al. Retinoic acid (RA) and As2O3 treatment in
transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature
of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha
oncoproteins. Proc Natl Acad Sci U S A 2000; 97(18):1017310178.
Differentiation Induction in Acute Promyelocytic Leukemia 203
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0008_O.3d] [14/3/08/18:4:43]
[185206]
67. Lallemand-Breitenbach V, Guillemin MC, Janin A, et al. Retinoic acid and arsenic
synergize to eradicate leukemic cells in a mouse model of acute promyelocytic
leukemia. J Exp Med 1999; 189(7):10431052.
68. Raffoux E, Rousselot P, Poupon J, et al. Combined treatment with arsenic trioxide
and all-trans-retinoic acid in patients with relapsed acute promyelocytic leukemia.
J Clin Oncol 2003; 21(12):23262334.
69. Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 combination yields a
high quality remission and survival in newly diagnosed acute promyelocytic leu-
kemia. Proc Natl Acad Sci U S A 2004; 101(15):53285335.
70. Estey E, Garcia-Manero G, Ferrajoli A, et al. Use of all-trans retinoic acid arsenic
trioxide as an alternative to chemotherapy for untreated APL. Blood 2006; 107(9):
34693473.
71. Estey EH, Thall PF, Giles FJ, et al. Gemtuzumab ozogamicin with or without
interleukin 11 in patients 65 years of age or older with untreated acute myeloid
leukemia and high-risk myelodysplastic syndrome: comparison with idarubicin plus
continuous-infusion, high-dose cytosine arabinoside. Blood 2002; 99(12):43434349.
72. Kiyoi H, Naoe T, Yokota S, et al. Internal tandem duplication of FLT3 associated
with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group of the
Ministry of Health and Welfare (Kohseisho). Leukemia 1997; 11(9):14471452.
73. Au WY, Fung A, Chim CS, et al. FLT-3 aberrations in acute promyelocytic leu-
kaemia: clinicopathological associations and prognostic impact. Br J Haematol 2004;
125(4):463469.
74. Li SW, Tang D, Ahrens KP, et al. All-trans-retinoic acid induces CD52 expression in
acute promyelocytic leukemia. Blood 2003; 101(5):19771980.
75. Kini AR, Peterson LA, Tallman MS, et al. Angiogenesis in acute promyelocytic
leukemia: induction by vascular endothelial growth factor and inhibition by all-trans
retinoic acid. Blood 2001; 97(12):39193924.
76. Kini AR, Roychowdhury S, Dziuma A, et al. Vascular endothelial growth factor
(VEGF) is a critical autocrine survival factor in acute promyelocytic leukemia
(APL). Blood 2005; 106(11):772a.
77. Alimoghaddam K, Shariftabrizi A, Tavangar M, et al. Anti-leukemic and anti-
angiogenesis efficacy of arsenic trioxide in new cases of acute promyelocytic leu-
kemia. Leuk Lymphoma 2006; 47(1):8188.
78. Weber WM, Hunsaker LA, Roybal CN, et al. Activation of NFkappaB is inhibited by
curcumin and related enones. Bioorg Med Chem 2006; 14(7):24502461.
79. Kini AR, Nagabhushan M, Tallman MS, et al. Curcumin enhances differentiation of
all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant acute promyelocytic
(APL) cells. Blood 2005; 106(11):192b.
80. Guillemin MC, Raffoux E, Vitoux D, et al. In vivo activation of cAMP signaling
induces growth arrest and differentiation in acute promyelocytic leukemia. J Exp
Med 2002; 196(10):13731380.
81. Warrell RP Jr., He LZ, Richon V, et al. Therapeutic targeting of transcription in
acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl
Cancer Inst 1998; 90(21):16211625.
82. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracycline-
based consolidation results in high antileukemic efficacy and reduced toxicity in
newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. PETHEMA
group. Blood 1999; 94(9):30153021.
204 Gidron and Tallman
[sanjeev][69-Standard][D:/informa_Publishing/DK0832_Kaspers_112039/z_pro-
duction/z_3B2_3D_files/978-0-8493-5083-2_CH0008_O.3d] [14/3/08/18:4:43]
[185206]
83. Ades L, Chevret S, Raffoux E, et al. Is cytarabine useful in the treatment of acute
promyelocytic leukemia? Results of a randomized trial from the European Acute
Promyelocytic Leukemia Group. J Clin Oncol 2006; 24(36):57035710.
84. Powell B. Effect of consolidation with arsenic trioxide (As203) on event-free sur-
vival (EFS) and overall survival (OS) among patients with newly diagnosed acute
promyelocytic leukemia (APL): North American Intergroup protocol C9710. J Clin
Oncol 2007; 25:2 (abstr).
85. Sanz MA, Lo Coco F, Martin G, et al. Definition of relapse risk and role of non-
anthracycline drugs for consolidation in patients with acute promyelocytic leukemia:
a joint study of the PETHEMA and GIMEMA cooperative groups. Blood 2000; 96(4):
12471253.
86. Lengfelder E, Saussele S, Haferlach T, et al. Treatment of newly diagnosed acute
promyelocytic leukemia: the impact of high dose ara-C. Blood 2003; 102:142a(abstr).
87. Lo Coco F, Avvisati G, Vignetti M, et al. Front-line treatment of acute promyelocytic
leukemia with AIDA induction followed by risk-adapted consolidation: Results of
the AIDA-2000 trial of the Italian GIMEMA group. Blood 2004; 104:115a(abstr).
88. Dombret H, Fenaux P, Soignet SL, et al. Established practice in the treatment of
patients with acute promyleocytic leukemia and the introduction of arsenic trioxide
as a novel therapy. Semin Hematol 2002; 39(2 suppl 1):813.
89. Jurcic JG, Nimer SD, Scheinberg DA, et al. Prognostic significance of minimal
residual disease detection and PML/RAR-alpha isoform type: long-term follow-up in
acute promyelocytic leukemia. Blood 2001; 98(9):26512656.
90. Tallman MS. Acute promyelocytic leukemia as a paradigm for targeted therapy.
Semin Hematol 2004; 41(2 suppl 4):2732.
91. Gale RE, Hills R, Pizzey AR, et al. Relationship between FLT3 mutation status,
biologic characteristics, and response to targeted therapy in acute promyelocytic
leukemia. Blood 2005; 106:37683776.
92. Hashimoto Y, Kagechica H, Kawachi E. Et al. Correlation of differentiation-
inducing activity of retinoids on human leukemia cell lines HL-60 and NB4. J Can
Res Clin Oncol 1995; 121:696698.
93. Tobita T, Takeshita A, Kitamura K, et al. Treatment with a new synthetic retinoid,
Am80, of acute pormyelocytic leukemia relapsed from complete remission induced
by all-trans retinoic acid. Blood 1997; 90:967973.
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9
DNA Methylation and Epigenetics: New
Developments in Biology and Treatment
Jesus Duque and Michael Lu bbert
Department of Hematology/Oncology, University Medical
Center Freiburg, Freiburg, Germany
Mark Kirschbaum
Division of Hematology and Hematopoietic Cell Transplantation,
City of Hope Comprehensive Cancer Center, Duarte, California, U.S.A.
INTRODUCTION
Epigenetic changes are defined as all meiotically and mitotically heritable changes
in gene expression that are not encoded in the DNA sequence itself. The field of
epigenetics is becoming increasingly prominent because it presents a key to
understanding differences between growing and senescent cells, tumor and normal
cells, and differentiated and aging cells, which can be exploited for treatment of
multiple conditions. Interwoven mechanisms, which regulate the gene expression,
like DNA methylation, histone modification, and RNA-associated silencing, are
recognized as players in a system of growing complexity.
While it appears that DNA methylation is the paramount signal, over time,
it has become clear that this is one step of a well-choreographed set of modu-
lations at the histone level. The centrality to life of epigenetic regulation, in
processes such as differentiation, apoptosis, and cell replication necessitates tight
regulation of the accessibility to transcription of DNA. Whole sections of DNA
may need to be made unavailable to transcription, while certain spans may need
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to be activated or silenced rapidly in response to conditions inside and out of the
cell, and this is coordinated between changes in methylation at the DNA level
and the spectrum of changes at the histone level (1). Our ability to treat cancer
with hypomethylating agents will be improved as we unravel the systems with
which it collaborates. For this reason, we will review the current biology and
treatment paradigms based on DNA methylation and its inhibition as well as
survey several important examples of epigenetic coordination between DNA
methylation and other histone modifications.
DNA Methylation in Normal Hematopoiesis and in Cancer
Three to six percent of all cytosines are methylated in the genome of mammalian
cells. The reaction of cytosine methylation consists of the incorporation of a
methyl group from the S-adenosyl-methionine in a previously unmethylated
cytosine and it is catalysed by DNA methyltransferases (DNMTs) (2). There are
three functional DNMTs: DNMT1, which maintains the pattern of methylation
during replication, maintenance methylation (3), DNMT3A, and DNMT3B, both
of which can add methyl groups to previously unmethylated templatesde novo
methylation (4). The methylated cytosines are mostly localized in CpG dinu-
cleotides. In the mammalian genome, CpG dinucleotides are not found ran-
domly, but in clusters of CpGs, termed CpG islands, which are often associated
with sites where the transcription of DNA into RNA is initiated (5). About half
of the genes have CpG islands in their promoters (6) (Fig. 1).
The normal physiological function of DNA methylation is multifaceted.
The repetitive genomic sequences and noncoding regions of the genome are
heavily methylated; DNA methylation plays a role in the protection of chro-
mosomal integrity (7). Genomic imprinting, to establish which parental allele
will be expressed, is also determined by DNA methylation (8), and the inacti-
vated X chromosomes in females are heavily methylated (9). An important
function of DNA methylation is to regulate the expression of tissue-specific
genes (10). In normal granulopoiesis, it was shown that MPO-expression is
tightly controlled by its methylation status at different stages of myelopoiesis
(11). C/EBPa, an important transcription factor of myelopoiesis, is infrequently
methylated in some myeloid leukemias, whereas hypermethylation of its pro-
moter is predominantly found in M2 subtype from FAB classification (12,13). In
normal erythropoiesis, the g chain of the fetal hemoglobin (HbF) is silenced by
DNA methylation after fetal development and replaced by the b chain, which
forms the adult hemoglobin A (HbA) (14). DNA methylation has been related to
the normal aging process (15). CpG islands in promoter regions of some genes,
which have been studied mostly in colon cells, appear to undergo increasing
methylation with age. These changes underscore the potential role of aging in the
genesis of colorectal cancer (16,17).
Aberrant DNA methylation is implicated in cancerogenesis in several ways.
In tumor cells there is an apparent paradoxglobal genomic hypomethylation, but
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with hypermethylation concentrated in the promoters of several genes (18). This
leads to a picture of overall dysregulation favoring tumor formation, with both
inappropriate silencing and activation. For example, global genomic hypo-
methylation can lead to gene activation of the so-called cancer testis antigens
(CTAs) like MAGE and GAGE family and NY-ESO1 genes (19), but can also
lead to chromosomal instability and further genomic aberrations, e.g., Wilms
tumors (20), and in the rare ICF (immunodeficiency, chromosome instability, and
facial anomalies) syndrome. This syndrome is caused by a germline mutation
in DNMT3B and subsequent genomic hypomethylation, though without a
predisposition to cancer (2123).
Figure 1 Reversibility of DNA methylation in tumor cells. (A) The distribution of DNA
methylation in normal cells is predominantly concentrated in the CpG dinucleotides of
transcriptional region of genes. The promoters of genes are hypomethylated, so the
transcription could be induced by the presence of transcription factors. (B) In contrast,
there is a redistribution of the DNA methylation pattern in tumor cells. The transcriptional
regions of genes are hypomethylated and the promoters of tumor suppressor genes are
hypermethylated, thus avoiding the transcription. (C) The demethylating drugs, like
5-azacytidine and decitabine, can relieve this situation in tumor cells. They are incor-
porated in the DNA during the S phase of the cell cycle. The DNMTs bind covalently to
these molecules, depleting the amount of active DNMTs in the cells. The consequence is
a global DNA hypomethylation and the transcription of genes, which were silenced
by DNA hypermethylation, could be restored. Abbreviation: DNMTs, DNA methyl-
transferases.
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Aberrant de novo methylation of CpG islands is a hallmark of human
cancers and is found early during carcinogenesis. The CpG islands in the pro-
moters of several tumor suppressor genes are hypermethylated and thus silenced
in different tumor types, e.g., p16
INK4A
in several tumor types (24), p15
INK4B
cyclin-dependent kinase inhibitor in acute myeloid leukemia (AML) (25),
RARb2 in colon, lung, head, and neck tumors (26,27), retinoblastoma (Rb) cell
cycle inhibitor in sporadic and hereditary Rb (28,29), and MLH1 DNA mismatch
repair gene in colon tumors (30). Tumors with especially frequent aberrant gene
methylation appear to constitute a specific disease entity labeled CpG island
methylation phenotype (CIMP) with a worse prognosis (31).
How does hypermethylation and silencing promote tumorogenesis? Dis-
ruption of the function of a tumor suppressor gene, as defined by Knudson (32),
requires a complete loss of function of both copies of the gene involved.
Abnormal promoter methylation can have the same effect as a coding-region
mutation in one copy of the gene and in this manner cooperates with somatic and
familial mutations in the cancerogenesis. Thus, silencing of p16 does not appear
in tumors in which the Rb gene is mutated; similarly, mutations in the VHL
gene in renal cancer are found in 60% of the cases, while the gene is hyper-
methylated in another 20%. Also, mutations and epigenetic silencing of the same
gene or different genes with similar functions seem to be mutually exclusive
(33). Loss of imprinting (LOI) is another mechanism whereby DNA methylation
contributes to genetic activation in some types of cancers. Biallelic expression,
caused by LOI of the gene IGF2 is a common feature in normal colonic mucosa
in patients with colorectal cancer, and the detection of LOI can be used as a
predictive marker of individual risk (34).
What underlies these epigenetic changes? An aberrant pattern of methyl-
ation in tumor cells may be caused by external factors (e.g., toxins, virus, or diet)
or by other genetic aberrations (point mutations or chimeric fusion proteins from
chromosomal translocations). Toxins like cadmium may inhibit DNMTs and
lead to an acute hypomethylation of the DNA followed by hypermethylation
after chronic exposure (35) and, like arsenic, can induce Ras hypomethylation in
methyl-deficient C57BL/6J mice (36). The latency of cervical cancer and lym-
phoma seems to be caused, at least in part, by hypermethylation of the promoter
of the human papillomavirus 16 (HPV16) genome (37) and EpsteinBarr virus,
respectively (38). A polymorphism of the MTHFR gene, which plays a role in
the S-adenosyl methionine synthesis, was associated with increased colorectal
cancer in a population-based study, where cancer incidence was lower in patients
with high dietary methionine (39). In animal models, methyl-deficiency diets
(folic acid, methionine, choline, vitamin B12 deficiency diet) lead to increased
DNA hypomethylation and development of hepatomas (40).
Point mutations in oncogenes like the MYC gene, which has been shown to
associate with DNMT in osteosarcoma cells, might also target DNA methylation
pattern in tumor cells. Furthermore, the MYC protein does not bind to its rec-
ognition sequence in the methylated condition of that site, suggesting that DNA
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methylation may affect the ability of MYC to bind multiple sites within the
genome, thus also affecting the chromatin structure (41). Epigenetic dysregu-
lation of gene expression could also be secondary to chromosomal trans-
locations. In human leukemias, the chimeric fusion protein PML/RARa, which
arises from the chromosomal translocation t(15;17) in APL, recruits histone
deacetylases (HDACs) and DNMTs to the promoters of RARa target genes,
leading to hypermethylation and silencing of the RARb2 tumor suppressor gene
(42). Another chimeric fusion protein, AML1/ETO resulting from the t(8;21) in
AML, recruits HDACs via the corepressor complex NCoR and m-Sin3a (43,44)
and DNMTs (45) to target genes, e.g., IL-3 gene, silencing their expression.
Although this may not be a specific mechanism of these diseases, as RARb2 is
found to be methylated in several tumors and not only in APL (46,47), it is
illustrative of a link between genetic and epigenetic aberrations in cancer.
DNA methylation is likely not the sole mediator of gene silencing but
seems to be critical in maintaining silencing in tumors; DNA hyper- and
hypomethylation in tumors are good markers of epigenetic dysregulation in
cancer. It is generally accepted that WT1 functions as a tumor suppressor gene
in Wilms tumor. However, WT1 gene is highly expressed in other tumors,
including colorectal, breast, leukemia, and desmoid, suggesting a function also
as an oncogene (48). The WT1 locus was found affected in Wilms tumors by
intragenic deletions and mutations in about 20% of patients and by LOH because
of promoter hypermethylation (49), confirming the revised two hits hypothesis in
the tumorogenesis. DNA methylation is causal in maintaining the silenced epi-
genetic state but reactivation of silenced tumor suppressor genes can be achieved
by demethylating agents and by siRNAs to inactivate DNMTs.
DNA Methylation and Histone Acetylation
Epigenetic changes such as DNA methylation need to be maintained and
transmitted to daughter cells, necessitating tight coordination among the various
enzymes charged with histone regulation (1), for this reason a brief review of
other histone related modifications is necessary to fully explicate the role of
DNA methylation changes. Over 60 types of histone modification are described
at this time, with a paradigm shift from the study of individual changes to that of
signaling based on interrelationships between modification, which is referred to
as the histone code (50). This implies that the modifications are read in terms
of the overall set of changes at the histone tail, beyond the effect of any single
modification alone, and at the organization level of nucleosomes.
The nucleosomes are structures composed of a core of histone proteins
around which the DNA binds. At activated genes, where the promoters of the genes
are hypomethylated, the nucleosomes are widely and irregularly spaced, promoting
the access to transcription factors to initiate transcription. In contrast, at inactivated
genes, the nucleosomes are tightly compacted and regularly spaced (33). This
configuration of nucleosomes is controlled by posttranslational modifications in
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amino acid residues of histone tails and by DNA-binding proteins like MBD2,
which interacts with the nucleosomal remodeling complex NuRD (51), and
MeCP2, which associates with the SWI/SNF chromatin-remodeling complex (52).
As a result, it has been increasingly recognized that the histones, rather
than merely having a structural function, are dynamic regulators of gene activity
through posttranslational modifications like acetylation, methylation, phos-
phorylation, and ubiquitylation. Acetylation and methylation of conserved lysine
residues of histone tail domains are known to be important regulators of gene
expression. The biology of histone acetylation will be described in more detail in
a separate chapter in this volume; here, a brief overview is presented with a focus
on interactions between DNA methylation and the other epigenetic processes.
The presence of acetyl groups in the histone alters the charge so that
chromatin binds less tightly to the phosphate backbone of DNA. Acetylation, in
general, activates, whereas deacetylation is suppressive; these changes, modu-
lated by histone acetyltransferases (HATs) and HDACs are rapidly reversible
and subject to multiple signals. There are numerous proteins with HAT activity,
worthy of note for their relationship to hematological malignancies. CREB
(cyclic AMP response element binding protein) and p300 are frequent partners in
leukemia and myelodysplastic syndrome (MDS)-related translocations, fusing
with acetyltransferases of the MYST family such as MOZ and MORF and the
H3K4 methyltransferase mixed lineage leukemia (MLL) (53). It is of interest
that in these cases, the fusion protein often contains two distinct active HAT
domains. Mutations within the HAT domains are also associated with leukemias
(54), confirming the impression that changes in acetylation represent important
moments in leukemogenesis.
Deacetylation is accomplished via a large group of enzymes comprising, at
this time, four groups (based on homology to yeast proteins) of HDACs, with the
class I HDACs, similar to Rpd3, being the dominant group of nuclear HDACs,
class II, similar to HdaI, having cytoplasmic activity as well in their role as protein
acetylases, the class III HDACs, the Sir2 homologs, having very different structure
and activity, now generally referred to as Sirtuins, and the class IV HDAC11. In
order to understand their manifold activities, it is important to recognize that HATs
and HDACs (as well as many of the other enzymes which act in an epigenetic
manner) tend to be recruited to large complexes through which they exert their
action, with the presence of chromodomains and bromodomains, domains that bind
methyl-lysine and acetyl-lysine, respectively (55). HDAC 1 and 2 complex with
mSin3A to form a repressor complex (56,57), and complex with NCoR and SMRT
to act as nuclear hormone receptor corepressors (58,59). Complexes seem to
facilitate the activity of other complexes, so that the various SWI/SNF chromatin-
remodeling complexes act to recruit HAT complexes leading to acetylation (6062)
and retinoblastoma-E2F complexes recruit HDACs as part of their repressive
activity (63). Interestingly, SWI/SNF complex activity has been shown to bring
about loss of DNA methylation as well at targets such as CD44 and E-cadherin
(64), introducing further levels of network-like complexity to the interplay between
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the various epigenetic enzymes. Thus, it becomes less surprising that treatment with
the hydroxamate HDAC inhibitor (HDACi) LBH589 can lead to decreased histone
methylation via the disruption of the polycomb repressor complex 2 (65) or that
valproic acid, a drug used for years as an antiepileptic agent and recently found to
have HDAC activity (66), can lead to DNA demethylation (67). This may help
explain the dramatic activity of HDACi, such as vorinostat and LBH589, as single
agents particularly in lymphoid malignancies, with single agent activity seen in
Hodgkins lymphoma, diffuse large B cell lymphoma, ALL, and indolent lymphomas
(6871, Kirschbaum, personal communication) (Fig 2).
Figure 2 Modulation of gene transcription by HDACis. (A) The aberrant recruitment of
HDACs to genes is a common hallmark in cancer, so the balance between HATs and
HDACs to control gene transcription is disrupted. The HDAC activity predominate,
causing hypoacetylation of histone tails around the transcriptional start site. The con-
formation of histones is closer, and the distance between nucleosomes is shorter. In this
way, the transcription factors are not able to initiate the transcription. (B) However, this
scenario could be reverted by HDACis, which inactivate transiently the aberrant activity
of HDACs. The HAT activity dominates, and the histone tails become hyperacetylated,
opening the structure of histones and spacing the nucleosomes. The transcription factor
can start the transcription of genes, which were blocked by HDACs. Abbreviations: TF,
transcription factor; HATs, histone acetylases; HDACs, histone deacetylases; HDACis,
histone deacetylase inhibitors; Ac, acetyl group.
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A very important discovery in the past years was the finding that DNMTs
(DNMT3A and DNMT3B), the enzymes which mediate DNA methylation, as
well as methyl-binding proteins (MECP2 and MBD2) were physically associated
with HDACs and histone methyltransferases (HMT), leading to a connection
between the three major epigenetic events: DNA methylation, histone modifica-
tion, and nucleosomal remodeling (7274). It is thought that the first event is DNA
methylation of the gene promoters, catalyzed by DNMTs. When a cluster of CpGs
is methylated, the methyl-binding proteins can recognize the methylated DNA and
interact directly with the DNA, thus repressing the transcription by limiting the
access to transcription factors. The attached HDACs and HMTs modify further the
histone tails by removing acetyl groups from lysine residues or adding methyl
groups to lysine residues, leading to a change in the histone tail into a closed
conformation. Generally, DNA methylation is associated with histone hypo-
acetylation of histones 3 (H3) and 4 (H4) and transcriptional repression, whereas
hyperacetylated histone marks transcriptionally active genes (75,76).
Interestingly, histone modifications are not only related to epigenetic
regulation of transcription, but also cooperate with other proteins to regulate
nuclear processes like DNA repair. Certain genes, with tumor suppressor prop-
erties such as p21
WAF1
, are silent at the transcriptional level in the absence of
DNA hypermethylation and the presence of H3 and H4 hypoacetylation (77).
Moreover, H3 acetylation is functionally linked to H3K4 trimethylation by the
MLL4 protein, which has a very high homology to MLL1 gene. H3 acetylation
determines the degree and the abundance of H3K4 methylation and this interaction
plays an important role in the epigenetic response to the HDACis valproate and
butyrate (78).
DNA Methylation and Histone Methylation
It is important on the one hand to distinguish between DNA methylation and
histone methylation, although, as we will demonstrate, there are multiple points
of contact between the two. Histone methylation occurs on lysine and arginine,
with arginine capable of receiving up to two methyl groups, and lysine up to
three; the relevant genes are activated or repressed depending on the configuration
of histone methylation. Most known mammalian lysine methylating proteins have
homologous catalytic domains, known as SET (SuVar39, Enhancer of Zeste and
Trithorax), and divide in to families depending upon lysine target. We will review
histone methylation with regard to DNA methylation activity, grouped by histone
methylation mark.
Histone methylation is a marker of both active and inactive genes. Trime-
thylation of lysine 4 in H3K4 is linked to activated genes (79,80). Methylation at
this site is associated with multiple other modifications such as acetylation by
acetyltransferases (81) and deacetylation by deacetylases, the latter activity per-
haps acting as a brake on genes actively transcribed by H3K4 methylation (82). On
the other hand, some H3K4 interactions are specific for unmethylated DNA. The
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MLL enzymes, which can polymethylate H3K4 (83,84), exist as multicomponent
complexes containing differing catalytic SET-related units (85). MLL1 in partic-
ular interacts with other modifiers such as the acetylases MOF and CBP (86), and
along with other MLL enzymes also recruits the homeobox family genes. The Hox
transcription factors play a role in embryonic development, as well as in angio-
genesis, in which HoxD3, HoxB3, and HoxA9 are essential regulators. MLL1 is
responsible for H3K4 trimethylation at the HoxA9 locus (86). It has recently been
shown that suppression of MLL will inhibit Hox-related proangiogenic activity (87).
A necessary component of the MLL complex that regulates Hox gene expression is
menin, which specifically associates with MLL proteins among SET1 homologs.
Menin is the protein encoded by Men1, which when mutated leads to multiple
neoplasms, particularly in endocrine tissue (88). Hox gene expression is dependent
upon the association of menin with MLL (89).
It thus comes as no surprise that mutations in MLL would be associated
with cancer; over 50 mutated fusion proteins as the result of chromosomal
translocations have been described, and this mutation is frequent in ALL of
infants (90). It is noteworthy that MLL-related transformation, with the resultant
disregulation of Hox proteins, is dependent on its association with menin,
implicating the methyltransferase activity as critical for leukemogenesis (91,92).
It is thus interesting that the repression domain of MLL is specific for
unmethylated CpG sequences (93).
H3K9 methylation, particularly trimethylation, is believed to be crucial to
formation of heterochromatin (80), particularly as this methylation creates a
binding site for the chromodomain (chromatin organization modifier) of the
repression related protein HP1. Trimethylation at H3K9 is achieved by Suv39h1/
Suv39h2 (94) which was the first HMT identified. This process is involved in
several tumor-related signaling pathways, such as suppression achieved by
Smads after TGF-b signaling (95), or suppression of cyclin E and E2F by Rb
protein, the latter losing this suppressive ability when mutated (96,97). Relevant
to our argument is that this repression requires the recruitment of HDACs (98).
Furthermore, it appears that this Suv39h-HP1-mediated mechanism is intimately
involved with DNMT3A and DNMT3B DNA methylating activity (99). It is
worth noting that mice with decreased levels of Suv39h1 showed a high inci-
dence of B-cell lymphomas (94). Of particular interest with regard to leukemia,
it has been shown that residues 380432 and 351381 in the RUNX1 tran-
scription factor, bind Suv39h1 as well as HDAC1 and 3 (100). RUNX1 is a
component of fusion proteins found frequently in AML, such as t(8;21), t(12;21),
and related to the activity inv(16); this relationship to H3K9 methylation may
explain the release of lysosomal repression in cells with these mutations upon
treatment with HDACi and hypomethylating agents (101). SETDB1, another
enzyme which di- and trimethylates histones at H3K9, also interacts directly
with DNMT3A and DNMT3B (102).
Another silencing histone methylation mark with relationship to DNA
methylation activity is at H3K27 (103). The complexes that bring about this
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methylation share an EZH2 enzyme, part of the polycomb group (PcG) of
proteins, which have an important role in silencing the Hox transcription factors,
which as described earlier, are critical modulators of development and angio-
genesis (104). Methylated H3K27 leads to further repression through binding of
other PcG proteins, as well as specific interaction with all three DNMT (76).
This EZH2-based relationship between H3K27 and DNA methylation appears to
be particularly marked in cancers as opposed to normal tissue (105). Thus it is
indicative of the complexity of attempting epigenetic therapy, that treatment of
leukemia cells with the hydroxamic acid HDACi LBH589 led to depletion of
EZH2 and decreased histone methylation of H3K27 (65).
Clinical Use of DNA Hypomethylating Agents
One of the most intriguing and clinically exploitable aspect of epigenetics as a
target for anticancer therapy is that epigenetic aberrations, described above, are
pharmacologically reversible, as opposed to purely genetic aberrations, which are
irreversible.
Inhibitors of DNA methylation have been shown to reactivate the
expression of genes that have undergone epigenetic silencing, particularly if this
silencing has occurred in a pathological situation. 5-azacytidine (Vidaza
1
) and
5-aza-2
0
deoxycytidine (Dacogen
1
, decitabine, DAC), which were initially
developed as cytotoxic antineoplasic agents when used at maximally tolerated
doses as with standard chemotherapeutic agents, were found to have the clinically
more important DNA demethylating activity when used at very low doses (106)
leading to the recognition of its clinical activity in MDS and AML (107). Both
compounds are cytosine analogs that inhibit DNMT, reverse methylation, and
reactivate genes. When used at these doses, these agents were shown to differ-
entiate cells in tissues cultures (106) and to induce gene re-expression. These
agents do not have direct demethylation activity, rather, 5-azacytidine and deci-
tabine have to be incorporated in the DNA in the S-phase of the cell cycle,
covalently binding DNMTs, and thus depleting the nucleus of their enymatic
activity (108,109). DNA replication in the absence of DNMTs leads to global and
gene-specific hypomethylation (110). In vitro and in vivo, these compounds are
able to demethylate the promoter of hypermethylated genes, as was shown for
p15
INK4B
promoter in MDS patients, leading to a reactivation of silenced genes
(25,111). At the cytotoxic doses of these compounds, cell death may be due to
DNA damage and apoptosis (108). While decitabine at cytotoxic doses demon-
strated significant antitumor activity in hematological malignancies in clinical
trials, severe and prolonged myelosuppression was frequently observed (112).
In vitro, these substances have shown to produce more hypomethylation at
lower doses than at high doses (113,114). In vivo, paradoxically, it is at the
hypomethylating doses where they produce significant antitumoral activity after a
long latency to response in MDS and AML patients (115117). Repeated courses
of 5-azacytidine and decitabine are required to achieve a clinical response, which
216 Duque et al.
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is understandable from the biology of the activity, which necessitates incorpora-
tion and subsequent depletion of DNMTs. Thus, the older trials in hematologic and
nonhematologic malignancies from the early 1980s are now understood to have
been designed with supotimal dosing plans and premature clinical evaluations
(e.g., after only two courses of therapy) (118). The current recognition of con-
tinued treatment is predicated upon epigenetic changes being reversible, with
aberrant promoter methylation and gene silencing return, once 5-azacytidine or
decitabine treatment is stopped (119121). Clinical responses in the therapy with
demethylating agents have been related to hypomethylation during the treatment
of the p15 promoter (25) with reactivation of expression of p15 gene (122).
Hypomethylation in bone marrow of patients treated with decitabine appeared
after karyotype normalization, suggesting clonal demethylation changes and
demethylation of normal cells (123). In contrast, shorter overall survival was
significantly correlated with baseline hypermethylation of multiple genes like p15
and E-cadherin in a multivariate analysis of a phase III study (124).
These studies provide proof of principle, that 5-azacytidine and decitabine
can induce responses through gene hypomethylation in AML and MDS but other
mechanisms of action should not be excluded. In vitro, expression of p21
WAF1
was induced by decitabine in cell lines in association with the release of HDAC1
and increased acetylated H3 at the unmethylated p21
WAF1
promoter (125). In
vivo, global changes of gene expression of primary AML and MDS cells were
analyzed using microarrays before and after decitabine treatment. 80 of 22,000
genes were induced, half of them had a CpG island in the promoter region, and
the changes in methylation status of promoters could only be validated in one
gene, coding for myeloperoxidase (126).
Both azacytadine and decitabine have been approved by the FDA for the
treatment of MDS, with registration studies for the treatment of AML ongoing.
In future, there will be considerable focus on developing compounds that directly
inhibit DNMTs. RG101 is one such lead compound currently in clinical devel-
opment (127).
Hypomethylating Agents in Combination with Histone
Deacetylation Inhibition
HDACis can induce differentiation, growth arrest and/or apoptosis in transformed
cells, and have antitumoral properties (128,129). Accumulation of acetylated
proteins, particularly histones, results in the induction of several epigenetically
silenced genes, some of them with tumor suppressor properties, like the cell cycle
kinase inhibitor p21 (77). The first drug of this type, suberoylanilide hydroxamic
acid (SAHA) has very recently been approved by the FDA for the treatment of
cutaneous T-cell lymphoma, and very active development programs are ongoing
for other new HDACis (130) (Table 1).
The link between DNA methylation and histone modification in cancer
cells has encouraged several investigators to combine DNMT inhibitors with
DNA Methylation and Epigenetics 217
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218 Duque et al.
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[207232]
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DNA Methylation and Epigenetics 219
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[207232]
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