Myelodysplastic Syndromes (Hematologic Malignancies)

H. J. Deeg ´ D. T. Bowen ´ S. D. Gore ´ T. Haferlach ´ M. M. Le Beau ´ C. Niemeyer

Hematologic Malignancies: Myelodysplastic Syndromes

H. J. Deeg ´ D. T. Bowen ´ S. D. Gore ´ T. Haferlach ´ M. M. Le Beau ´ C. Niemeyer

Hematologic Malignancies: Myelodysplastic Syndromes With 27 Figures and 25 Tables

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H. J. Deeg, M.D. Fred Hutchinson Cancer Research Center Seattle, USA D. T. Bowen, M.D. Ninewells Hospital & Medical School Dundee, DD1 9SY, Scotland, UK S. D. Gore, M.D. Johns Hopkins Oncology Center 600 North Wolfe Street, 2-109 Baltimore, MD 21287-8963, USA

M. M. Le Beau, Ph.D. University of Chicago Section of Hematology/Oncology 5841 S. Maryland, MC2115, Chicago, IL 60637, USA C. Niemeyer, M.D. Division of Pediatric Hematology and Oncology Department of Pediatrics and Adolescent Medicine, University Hospital of Freiburg Mathildenstrasse 1, 79106 Freiburg, Germany

T. Haferlach, M.D. MML Munich Leukemia Laboratory Max-Lebsche-Platz 31, 81377 Munich, Germany

ISBN-10 3-540-26188-5 Springer Berlin Heidelberg New York ISBN-13 978-3-540-26188-9 Springer Berlin Heidelberg New York Library of Congress Control Number: 2005933137 A catalog record for this book is available from Library of Congress. Bibliographic information published by Die Deutsche Bibliothek. Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springeronline.com ° Springer Berlin Heidelberg 2006 Printed in Germany The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about the application of operative techniques and medications contained in this book. In every individual case the user must check such information by consulting the relevant literature. Editor: Dr. Ute Heilmann, Heidelberg, Germany Desk editor: Meike Stoech, Heidelberg, Germany Production: LE-TEX Jelonek, Schmidt & Væckler GbR, Leipzig, Germany Typesetting: K + V Fotosatz GmbH, Beerfelden, Germany Cover design: Erich Kirchner, Heidelberg, Germany 21/3150/YL ± 5 4 3 2 1 0 ± Printed on acid-free paper

Preface

The group of diseases collectively referred to as myelodysplastic syndromes (MDS) has recently attracted considerable attention. Firstly, laboratory investigations have begun to shed light on the pathophysiology (if not etiology) of MDS, and considerable progress has been made in dissecting molecular pathways that contribute to the clinical manifestations of the disease. Secondly, several agents have been developed that appear to be of therapeutic benefit, at least for subgroups of patients. Thirdly, the development of novel hematopoietic cell transplant strategies has permitted to carry out successful transplants in a progressively increasing proportion of patients. Lastly, as the population ages, the number of patients diagnosed with MDS is growing. Several books dealing with various aspects of MDS have been published over the last few years. In the present volume we summarize the basic essentials of MDS and, in a very practice-oriented way, outline current approaches to the optimum management of patients with MDS. This should render the book useful to a broad spectrum of students and practioners in this field. We appreciate Springer's support for the project. H. Joachim Deeg

Contents

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Clinical Presentation . . . . . . . . . Bart Scott, H. Joachim Deeg

1

2

Differential Diagnosis . . . . . . . . Philip Nivatpumin, Steven D. Gore

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3

Etiology and Epidemiology of MDS . . . . . . . . . . . . . . . . . . . David T. Bowen

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4

Molecular Biology of Myelodysplasia . . . . . . . . . . . . . Philip Nivatpumin, Steven D. Gore

23

5

Classification and Staging of Myelodysplastic Syndromes . Torsten Haferlach, Wolfgang Kern

39

6

Cytogenetic Diagnosis of Myelodysplastic Syndromes . Harold J. Olney, Michelle M. Le Beau

55

Myelodysplastic Syndrome in Children . . . . . . . . . . . . . . . . Charlotte Niemeyer

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8

Management of Patients with Myelodysplastic Syndromes: Introductory Concepts . . . . . . . David T. Bowen

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Supportive Care . . . . . . . . . . . . David T. Bowen

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Hematopoietic Growth Factors . David T. Bowen

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Biologically Based Treatment . . Philip Nivatpumin, Steven D. Gore

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Hemopoietic Cell Transplantation Bart Scott, H. Joachim Deeg

123

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Second Malignancies . . . . . . . . H. Joachim Deeg

135

Subject Index . . . . . . . . . . . . . . . . . .

137

Contributors

David T. Bowen, M. D. Ninewells Hospital & Medical School, Dundee, DD1 9SY, Scotland, UK H. Joachim Deeg, M. D. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, P.O. Box 19024, Seattle, WA 98109-1024, USA Steven D. Gore, M. D. Johns Hopkins Oncology Center, 600 North Wolfe Street, 2-109, Baltimore, MD 21287-8963, USA Torsten Haferlach, M. D. MML Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany Wolfgang Kern, M. D. Medical Hospital and Health Center III, Clinical Center of the University of MunichGrosshadern, Marchioninistrasse 15, 81377 Munich, Germany

Michelle M. LeBeau, Ph. D. University of Chicago, Section of Hematology/ Oncology, 5841 S. Maryland, MC2115, Chicago, IL 60637, USA Charlotte M. Niemeyer, M. D. Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University Hospital of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany Philip Nivatpumin, M. D. Johns Hopkins Oncology Center, 600 North Wolfe Street, 2-109, Baltimore, MD 21287-8963, USA Harold J. Olney, M. D. Universit de Montral, CHUM HÖpital Notre-Dame, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1 Bart Scott, M. D. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, P.O. Box 19024, Seattle, WA 98109-1024, USA

Abbreviations

Ara-C BU CD CMML CR CSP CY EBMT FAB FAI FHCRC FM G-CSF GVHD HCT HLA I.V. IBMTR IPSS MCV MDS MRI MTX NK NMA PBPC PIG-A PNH RA RAEB RAEBt RARS RCMD

cytosine arabinoside busulfan cluster of differentiation chronic myelomonocytic leukemia complete remission cyclosporine cyclophosphamide European Blood and Marrow Transplant Group French-American-British (classification) fludarabine/cytosine arabinoside/idarubicin Fred Hutchinson Cancer Research Center fludarabine/melphalan granulocyte-colony stimulating factor graft-versus-host disease hematopoietic cell transplantation human leukocyte antigen intravenous(ly) International Bone Marrow Transplant Registry International Prognostic Scoring System mean cellular volume myelodysplastic syndrome magnetic resonance imaging methotrexate natural killer cells nonmyeloablative peripheral blood progenitor cells gene responsible for one step in the biosynthesis of the proteoglycan anchor of glycophosphatidylinositol-linked cell surface antigens paroxysmal nocturnal hemoglobinuria refractory anemia RA with excess blasts RAEB in transformation RA with ring sideroblasts refractory cytopenia with multilineage dysplasia

XII

Abbreviations

RCMD-RS RFS RIC tAML TBI THY TRM WHO

refractory cytopenia with ring sideroblasts relapse-free survival reduced-intensity conditioning acute myeloid leukemia transformed from MDS total body irradiation thymoglobulin transplant-related mortality World Health Organization

Clinical Presentation Bart Scott, H. Joachim Deeg

Contents 1.1 Symptoms upon Presentation . . . . . . .

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1.2 Past Medical History . . . . . . . . . . . . . .

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1.3 Family History . . . . . . . . . . . . . . . . . .

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1.4 Physical Examination . . . . . . . . . . . . .

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1.5 Laboratory Studies . . . . . 1.5.1 Blood . . . . . . . . . . . 1.5.2 Bone Marrow . . . . . . 1.5.3 Chromosome Analysis 1.5.4 Differential Diagnosis

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References . . . . . . . . . . . . . . . . . . . . . . . . .

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1.1 Symptoms upon Presentation

In keeping with the broad spectrum of disorders covered by the term myelodysplastic syndrome (MDS), the clinical presentation of patients varies widely (Greenberg 2000). The patients' symptoms are a direct result of cytopenias and cell function abnormalities. Most patients present with fatigue, a lack of energy, some with shortness of breath and dyspnea upon exertion, related to anemia. As their overall performance level declines, they may not be able to accomplish their jobs or tasks any longer. Other patients are noted incidentally to have a low hematocrit (or other cytopenias) on the occasion of a ªroutineº check-up. Some patients will notice petechiae or bruises, most frequently over the lower legs, and again others will complain of recurrent infec-

tions (10%; most of bacterial etiology) or may be noted by their dentist to have a root abscess that does not heal.

1.2 Past Medical History

The preceding medical history is generally not revealing. However, some patients will report that years ago they were told that they had ªanemia,º and occasional patients may have had a low platelet count ªall their lifeº (see Chapter 2, Differential Diagnosis). In addition, some patients give a history of autoimmune/inflammatory disorders, including vasculitis, polyneuropathy, a lupus-like syndrome, thyroiditis, arthritis and serositis (Hamblin 2002).

1.3 Family History

The family history is generally not contributory. Familial cases of MDS appear to be rare.

1.4 Physical Examination

Physical examination may be rather unremarkable. However, if patients are severely anemic they will be pale and show tachycardia. There may be petechiae, particularly in dependent parts, and bruises. Lymphadenopathy is an unusual finding, and clinically recognizable splenomegaly is infrequent. If serositis is a feature, there may be corresponding pulmonary (pleural), cardiac (pericardial), abdominal (peritoneal) and joint (synovial) findings.

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Chapter 1 ´ Clinical Presentation

1.5 Laboratory Studies 1.5.1 Blood

A low hematocrit/hemoglobin is the most frequent abnormality, and in about 30% of patients is the only cytopenia. The anemia is typically macrocytic, and the mean cellular volume (MCV) may be > 110 fl, particularly in patients who turn out to have a ª5q± syndromeº; B12 and folate deficiencies must be excluded. The reticulocyte count is typically low. There may be anisocytosis, poikilocytosis, acanthocytosis. Nucleated red blood cells may be seen, as well as basophilia and Howell-Jolly bodies. About two thirds of patients are neutropenia. Granulocytes tend to be hypogranular and hyposegmented with abnormal chromatin condensation. Cells with Pelger-HuŸt morphology may be present. The relative and absolute monocyte count may be elevated. Defects in adhesion, migration, phagocytosis and bacterial killing have been described. Cells respond poorly to hemopoietic growth factors. The peroxidase reaction may be negative. Natural killer cells (NK) function may also be abnormal. In patients with chronic myelomonocytic leukemia (CMML) (now generally classified under myeloproliferative disorders) the monocyte count is elevated (³ 1 ´ 109/liter). Patients with the proliferative variant have WBC > 12 ´ 109/liter. Hepatosplenomegaly may be present. Thrombocytopenia of various degrees is also present in about two thirds of patients; in maybe 5% of patients it is the only peripheral blood cytopenia. Cases of an Evans-like syndrome (with thrombocytopenia and hemolytic anemia) have been observed. Platelet function is often abnormal, as reflected in prolonged bleeding time and impaired aggregation. In patients with a 5q± syndrome, platelet counts are typically high (Fig. 1.1). A proportion of patients show evidence of hemolysis, and in some series, as many as 20% of patients have been reported to have a PIG-A mutation, a positive Ham test, abnormal or lacking expression of PIG-A anchored surface proteins, and increased sensitivity to complement-mediated lysis, consistent with (paroxysmal nocturnal hemoglobinuria) PNH (see Chapter 2). The proportion of patients with a documented PNH clone has been much lower in most series. Recent data from a Japanese study, on the other hand, suggest an even higher

Fig. 1.1 a±c. Illustration of morphologic findings in a patient with 5q± syndrome (courtesy of D. Myerson, M.D., Ph.D., Fred Hutchinson Cancer Research Center, Seattle, WA). a Peripheral blood smear showing Pelger-HuŸt appearance of granulocytes. b Bone marrow with typical megakaryocyte changes. c Megakaryocyte abnormalities (higher magnification)

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1.5 ´ Laboratory Studies

frequency of PNH positivity if sufficiently sensitive flow studies are applied (Ishiyama et al. 2003). Abnormalities in iron metabolism have been described, including iron absorption, and ferritin levels may be elevated. There are also reports of disturbed hemoglobin chain synthesis, an increase in fetal hemoglobin, Hgb H inclusions, abnormal erythrocyte enzymes and surface antigens. Erythropoietin levels in peripheral blood are generally high (except in patients with renal failure), although they may be low (< 200 units), even in patients with marked anemia, in about 15±20% of patients (Hellstrom-Lindberg 1995).

1.5.2 Bone Marrow

Bone marrow findings are described in detail in Chapters 2 and 5. Aspirates and biopsies typically reveal a hypercellular marrow. However, the marrow may be normocellular, and in as many as 20% of cases may be hypocellular (considering a 30±40% cellularity as the lower limit of normal), leading to potential difficulties with the differential diagnosis from aplastic anemia. It is also important to consider that there is a decline of overall marrow cellularity with age, and a lower cut-off for normal at 20% has been suggested for patients in their 70s and older (Hartsock et al. 1965). Generally, however, a limit of 40% is used. Since the marrow pattern may be quite heterogeneous (for reasons that are not well understood), multiple biopsies or a magnetic resonance imaging (MRI) scan may be required to be confident about the assessment. Microscopic examination of the marrow may reveal single- or multi-lineage dysplasia. There may be marked dyserythropoiesis including multinuclear fragments and bizarre nuclear shapes, mitosis, asynchrony between cytoplasm and chromatin, basophilia of cytoplasm, or Howell-Jolly bodies. Erythroid precursors may show prominent megaloblastic changes. Frequently ring sideroblasts are present (five or more siderotic granules are considered pathologic; if at least one third of the nuclear circumference is covered, the term ringed sideroblast is applied). To qualify as refractory anemia with ring sideroblasts (RARS) more than 15% ringed sideroblasts must be present. If more than 50% erythroid precursors are present (with more than 30% of the myeloid lineage being blasts), the criteria for erythroleukemia are met. Nuclear-cytoplasmatic asynchrony is also observed in

3

early myeloid cells, with granular cytoplasm, a reticulated nucleus, prominent nucleolus and perinuclear Golgi zone. The proportion of type I myeloblasts determines the morphologic subtype of MDS. Morphologic abnormalities in megakaryocytes include micromegakaryocytes, mononuclear forms, multiple small nuclei connected by strands of nuclear material, dysmorphic features and hypogranularity. In patients with a 5q± syndrome megakaryocytic abnormalities (usually small with single eccentric nuclei) may be prominent. Multiparameter flow cytometry of marrow cells may show a broad array of immunophenotypic aberrancies, apparently on all cell lineages, but best characterized in myeloid and monocytoid cells (reviewed in Benesch et al. 2004; Stetler-Stevenson et al. 2001). Immunophenotypic abnormalities may include the expression of new surface receptors, adhesion molecules or apoptosis-related surface markers. In addition, lineage infidelity, asynchronous antigen expression, differences in antigen density and homogeneity of expression may also be observed (Terstappen et al. 1992). These abnormalities may affect only subpopulations of cells that coexist with normal precursors or normal maturing cells. The proportion of CD34+ cells may be significantly increased in refractory anemia with excess blasts in transformation (RAEBt) compared with normal cells, whereas CD66+++ cells may be significantly decreased in patients with RAEB compared with patients with refractory anemia (RA) or normal marrow. CD33 intensity tends to be higher in diseases with more immature cells. One study used hierarchical clustering to assess for similarities and differences between patient groups (Maynadie et al. 2002). Eight clusters were identified based on intensity relationships between CD16, CD34, CD36, CD38, CD17 and HLA-DR on blasts. The eight groups exhibited differences in International Prognostic Scoring System (IPSS) scores, cytogenetic risk factors, and percentage of blasts. Clustering mean intensities for the granulocytes showed increased mean intensity expression of CD38, CD13, CD33 in patients with more advanced MDS stages (RAEB, RAEBT), as might be expected from a shift to the left reflecting increases in myeloblasts and immature myeloid cells. CD34+ cells in peripheral blood are decreased in RA patients but increased in RAEB and RAEBT patients as compared with normal. Patients with lower grades of MDS are more like normal in expression of CD114 on the CD34+ cells than patients with more advanced stages of disease (based on the French-American-British Clas-

4

Chapter 1 ´ Clinical Presentation

sification (FAB) classification). One report suggests that patients with CD90 expression on CD34+ cells are more likely to progress to leukemic transformation (Inaba et al. 1998). Markers reflecting immaturity of myeloid cells such as CD7 and CD117 tend to be more frequently expressed in advanced stages of MDS and acute myeloid leukemia transformed from MDS (tAML) (Ogata et al. 2002). Markers for maturity of myeloid cells (CD10 and CD15) appear to be more prevalent in early stages of MDS. Expression of CD7 on marrow cells, as detected in advanced stages of MDS, was found to be independently associated with a transformation-free survival (Ogata et al. 2002). A recently developed numerical scoring system based on phenotypic and scatter characteristics of MDS marrow cells suggests a strong correlation with IPSS scores and, in an initial analysis, was significantly correlated with post-transplant outcomes (Wells et al. 2003).

1.5.3 Chromosome Analysis

Chromosome abnormalities are described in detail in Chapter 6. Approximately 40±50% of patients with de novo, and as many as 90% of patients with secondary/treatment-related MDS show clonal cytogenetic abnormalities. The frequency may be even higher when molecular tools are used. It is important that a sample of marrow be sent for cytogenetic analysis in all patients suspected of having a diagnosis of MDS. Characteristic cytogenetic changes can confirm the diagnosis of MDS, and it also offers critical information regarding prognosis (IPSS). It is also becoming apparent that cytogenetic changes may affect treatment decisions.

1.5.4 Differential Diagnosis

The differential diagnosis is discussed in detail in Chapter 2. It is important to emphasize that in many instances MDS is a diagnosis of exclusion. Therefore, if there is any doubt about the diagnosis, the patient should be re-evaluated after an observation period of 2±3 months to substantiate (or exclude) the diagnosis of MDS.

References Benesch M, Deeg HJ, Wells D, Loken M (2004) Flow cytometry for diagnosis and assessment of prognosis in patients with myelodysplastic syndromes. Hematology 9:171±177 Greenberg PL (2000) Myelodysplastic syndrome. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE, McGlave P (eds) Hematology: basic principles and practice. Churchill Livingstone, New York, pp 1106±1129 Hamblin TJ (2002) Immunology of the myelodysplastic syndromes. In: Bennett JM (ed) The myelodysplastic syndromes: pathobiology and clinical management. Marcel Dekker, Inc., New York, pp 65±87 Hartsock RJ, Smith EB, Petty CS (1965) Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest: a study made from 177 cases of sudden death examined by necropsy. Am J Clin Pathol 43:326±331 Hellstrom-Lindberg E (1995) Efficacy of erythropoietin in the myelodysplastic syndromes: a meta-analysis of 205 patients from 17 studies. Br J Haematol 89:67±71 Inaba T, Shimazaki C, Sumikuma T, Shimura K, Takahashi R, Hirai H, Ashihara E, Sudo Y, Murakami S, Haruyama H, Fujita N, Yoshimura M, Nakagawa M (1998) Flow cytometric analysis of Thy-1 expression in myelodysplastic syndrome. Int J Hematol 68:403±410 Ishiyama K, Chuhjo T, Wang H, Yachie A, Omine M, Nakao S (2003) Polyclonal hematopoiesis maintained in patients with bone marrow failure harboring a minor population of paroxysmal nocturnal hemoglobinuria-type cells. Blood 102:1211±1216 Maynadie M, Picard F, Husson B, Chatelain B, Cornet Y, Le Roux G, Campos L, Dromelet A, Lepelley P, Jouault H, Imbert M, Rosenwadj M, Verge V, Bissieres P, Raphael M, Bene MC, Feuillard J, The Groupe d'Etude Immunologique des Leucemies (2002) Immunophenotypic clustering of myelodysplastic syndromes. Blood 100:2349± 2356 Ogata K, Nakamura K, Yokose N, Tamura H, Tachibana M, Taniguchi O, Iwakiri R, Hayashi T, Sakamaki H, Murai Y, Tohyama K, Tomoyasu S, Nonaka Y, Mori M, Dan K, Yoshida Y (2002) Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome. Blood 100:3887±3896 Stetler-Stevenson M, Arthur DC, Jabbour N, Xie XY, Molldrem J, Barrett AJ, Venzon D, Rick ME (2001) Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome. Blood 98:979±987 Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T, Hiddemann W, Wormann B (1992) Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia 6:70±80 Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, Deeg HJ (2003) Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hemopoietic stem cell transplantation. Blood 102:394±403

Differential Diagnosis Philip Nivatpumin, Steven Gore

Contents 2.1 Introduction . . . . . . . . . . . . . . . . . . . .

5

2.2 Nutritional Disorders . . . . . . . . . . . . .

6

2.3 Congenital . . . . . . . . . . . . . . . . . . . . . 2.3.1 Fanconi Anemia . . . . . . . . . . . . . . 2.3.2 Congenital Dyserythropoietic Anemia 2.3.3 Hereditary Sideroblastic Anemia . . .

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2.4 Toxic Disorders . . . . . . . . . . . . . . . . . .

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2.5 Infectious Disorders . . . . . . . . . . . . . .

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2.6 Other Hematopoietic Disorders . . . . 2.6.1 Idiopathic Aplastic Anemia . . . . . 2.6.2 Paroxysmal Nocturnal Hemoglobinuria . . . . . . . . . . . . . 2.6.3 Myeloproliferative/Myelodysplastic Disorders . . . . . . . . . . . . . . . . . . 2.6.4 Large Granular Lymphocytic Leukemia . . . . . . . . . . . . . . . . . .

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2.7 Summary . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . .

11

Introduction The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by two main features: ineffective hematopoiesis and a variable risk of transformation to acute myeloid leukemia (AML) (Steensma and Tefferi 2003).

The term ªmyelodysplastic syndromeº itself emerged in the 1970s amidst controversies surrounding the various presentations of this disorder; subsequent attempts have repeatedly been made to further specify a more precise classification (Anonymous 1976; Greenberg et al. 2000). Although advances in the genomic profiling of various malignancies promise improvement in a classification system for MDS as well, our current schemes still rely on a mixture of clinical, morphologic and cytogenetic features (Alizadeh et al. 2001; Culligan and Jacobs 1992; Miyazato et al. 2001). Given the lack of a pathologic ªgold standard,º various minimal criteria have been proposed for the diagnosis of MDS (Culligan and Jacobs 1992; Gardais 2000; Greenberg et al. 2000; Tricot 1992). They rely on morphologic evidence of dysplasia, peripheral cytopenias, karyotypic abnormalities and the presence of increased bone marrow blasts. However, those findings may be nonspecific and related to other pathologic processes, including nutritional, toxic, infectious and other clonal hematopoietic conditions. Bone marrow specimens from normal individuals have been reported to demonstrate mildly dysplastic hematopoiesis (Bain 1996; Champion et al. 1997). The diagnosis of MDS should be considered in any patient with unexplained cytopenia(s) or monocytosis. A careful examination of the peripheral blood smear and bone marrow aspirate is essential to identify characteristic morphologic changes in any or all hematopoietic lineages. In some cases, only the peripheral blood smear shows significant evidence of dysplasia. The bone marrow in MDS is typically hypercellular with dysplasia in one or more lineages (Rios et al. 1990). However, a distinct subset of MDS that has a hypocellu-

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Chapter 2 ´ Differential Diagnosis

lar marrow with features mimicking aplastic anemia, pure red cell aplasia, and other bone marrow failure states is well recognized (Garcia-Suarez et al. 1998). As stated, however, these features may be present in a variety of other conditions. A thorough workup of other causes of bone marrow conditions with pathologic features similar to MDS is mandatory, particularly in the absence of confirmatory clonal cytogenetic abnormalities.

2.2 Nutritional Disorders

Nutritional megaloblastic anemias have been described for over 100 years. The hallmark megaloblast results from impaired DNA synthesis as a result of vitamin B12 (cobalamin) or folate deficiency. Animal products (meat and dairy) are the sole dietary source of cobalamin in humans. It takes years to develop deficiency of cobalamin (Green and Kinsella 1995). Antibodies to intrinsic factor (pernicious anemia) are a common cause in the elderly, and other causes of intestinal malabsorption (e.g., sprue, bacterial overgrowth, etc.) account for remaining cases. Folate is found in animal products and leafy green vegetables. Because folate deficiency may develop within months, decreased dietary consumption accompanied with alcohol abuse is a common etiology. Macrocytic anemia is the most common presentation of vitamin B12 or folate deficiency. Bone marrow findings show erythroid hyperplasia with an abnormal morphology, as well as megaloblastoid granulopoiesis and megakaryocytopoiesis. These features may mimic either AML or MDS (Green and Kinsella 1995). MDS may present with macrocytosis, reduced reticulocytosis and pancytopenia, making it indistinguishable from these nutritional deficiencies. In addition to other clinical features (e.g., cognitive changes, decreased proprioception, glossitis), evaluation of the peripheral blood smear is often helpful to distinguish MDS from megaloblastic anemia. While reduced neutrophil lobulation (i.e., pseudo-Pelger-HuŸt abnormality) and hypogranular neutrophils are more characteristic of the peripheral blood smear of MDS, all of the ªtypicalº changes in megaloblastoid anemias may be seen in MDS. Hypersegmentation, increased neutrophil lobulation, and megaloblastoid changes have all been reported in MDS. Low serum B12 levels and elevated methylmalonyl CoA levels suggest the diagnosis of megaloblastic anemia in vitamin B12 deficiency. Reduced red blood cell

folate and an elevated homocysteine level are seen in folate deficiency. Bone marrow cytogenetics and flow cytometry studies are normal in B12 and folate deficiency, although one must be aware that MDS and nutritional deficiencies may coexist in some patients (Drabick et al. 2001). Treatment with intramuscular vitamin B12 or oral folate will result in improvement of anemia and resolution of hypersegmentation within several weeks. However, neurologic sequelae recover only over the course of months and, in some cases, may be irreversible. A ªtherapeutic trialº of B12 is not recommended in the absence of documented vitamin deficiency.

2.3 Congenital 2.3.1 Fanconi Anemia

In younger patients, congenital causes of bone marrow failure must be considered in the differential diagnosis of MDS. Although most of these disorders present very early in childhood and are associated with other characteristic distinguishing clinical features (e.g., anatomic abnormalities), there have been well-described reports of patients with Fanconi anemia (FA) presenting with atypical features later in adolescence and even early adulthood (Butturini et al. 1994; Cavenagh et al. 1996). Fanconi anemia is an autosomal recessive disease characterized by congenital abnormalities, ineffective hematopoiesis, and an increased risk of developing acute leukemias, myelodysplastic syndrome, and certain solid tumors (Tischkowitz and Hodgson 2003). A disorder of impaired DNA repair, FA has a heterogeneous clinical presentation with most patients, presenting in childhood with a combination of skeletal, gastrointestinal, renal, hematologic, and cardiac defects. Patients with FA may present with aplastic anemia, MDS, or acute leukemia. Macrocytosis is often the first hematologic abnormality, followed by thrombocytopenia and neutropenia, and, eventually, pancytopenia. In one study of 388 FA patients, the risks of developing hematologic abnormalities and death from hematologic causes were 98% and 81%, respectively (Butturini et al. 1994). In the presence of typical features, the diagnosis is easily made by the chromosome breakage test. In younger patients presenting with a hypocellular bone marrow and cytopenias, FA should be considered. Treatment is supportive in mild cases. The most definitive

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2.3 ´ Congenital

treatment is hematopoietic stem cell transplantation with 2-year survival rates as high as 60±70% with human leukocyte antigen (HLA)-identical sibling donors (Gluckman et al. 1995).

2.3.2 Congenital Dyserythropoietic Anemia

Congenital dyserythropoietic anemia (CDA) is a rare group of hereditary disorders characterized by ineffective erythropoiesis and distinct morphologic abnormalities in bone marrow erythroblasts (Heimpel 2004). The range of genetic heterogeneity and precise molecular defects is beyond the scope of discussion in this chapter. However, some key features are worth noting in the differential diagnosis with MDS. Three features are often present: ineffective erythropoiesis, hyperbilirubinemia, and a distinct morphologic appearance of erythroblasts that is easily recognized by trained individuals. The rarity of this disease often delays diagnosis, with estimates of up to 60% of cases diagnosed in adulthood, despite multiple prior evaluations for laboratory abnormalities (Greiner et al. 1992; Heimpel 2004). The peripheral blood smear may show anisocytosis, poikilocytosis, basophilic stippling, and occasional erythroblasts. The leading morphologic abnormality is the presence of binucleate erythroblasts in 10±50% of bone marrow erythroblasts. Bone marrow cellularity is often increased. Indirect hyperbilirubinemia is often present, and serum haptoglobin is low. Acid lysis testing may be helpful in some subtypes, but diagnosis often relies on expert morphologic analysis, high clinical suspicion, and genetic testing (Wickramasinghe 1998, 2000). Treatment is dependent on specific subtypes and may include interferon and splenectomy. Prevention of iron overload significantly improves outcome.

2.3.3 Hereditary Sideroblastic Anemia

Hereditary sideroblastic anemias (HSA) are a heterogeneous group of disorders and may have X-linked or autosomal inheritance or may be associated with sporadic congenital defects. Clinically, patients generally present with a mild anemia that is stable for many years. Mild to moderate hepatosplenomegaly is common. With time, patients develop symptoms and signs of chronic iron overload, reflecting the underlying pathophysiology of increased iron absorption due to ineffective

7

erythropoiesis and impaired heme biosynthesis (Bottomley 2000; Koc and Harris 1998). Iron overload may become manifest clinically with cirrhosis, cardiac disease, impaired glucose tolerance and diminished libido. In children, growth retardation may occur. A number of molecular defects have been identified in the pathophysiology of the hereditary sideroblastic anemias, including defects involving erythroid aminolevulinate synthase (ALAS2), ferrochelatase, cytochrome oxidase, thiamine transporter-1 and a variety of mitochondrial proteins (Alcindor and Bridges 2002). In many patients, the cause of sideroblastic anemia is unknown. However, the common mechanism involves impaired heme biosynthesis and an accumulation of mitochondrial iron in erythroblasts. The complete blood count typically shows a mild anemia. White blood cells and platelets are usually normal. Erythrocyte hypochromia is a consistent finding. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are all low and usually parallel the degree of anemia. Iron overload is reflected in the iron studies. Serum ferritin and transferrin saturation are increased, and serum transferrin levels are decreased. In X-linked sideroblastic anemia, free erythrocyte protoporphyrin is low. Bone marrow biopsy shows increased reticuloendothelial iron in bone marrow macrophages. Normoblastic erythroid hyperplasia with ringed sideroblasts is characteristic. Cytogenetic studies are normal unless a concomitant myelodysplasia is present. Depending on the inheritance pattern, various molecular tests may be used to evaluate for the underlying cause. Mutations in the ALAS2 gene are common in X-linked sideroblastic anemia. Mutations in the ABC7 transporter gene and SLC19A2 gene are found in X-linked sideroblastic anemia with ataxia and thiamine-responsive megaloblastic anemia syndrome (Alcindor and Bridges 2002; Bottomley 2000). A variety of other specific genetic tests may be conducted depending on clinical suspicion. There is no definitive treatment. Bone marrow transplantation has been attempted with some success (Gonzalez et al. 2000; Urban et al. 1992). X-linked sideroblastic anemia is responsive to pyridoxine (vitamin B6) treatment in up to two thirds of patients. In contrast, true acquired refractory anemia with ringed sideroblasts is rarely pyridoxine-responsive (May and Bishop 1998). Treatment of iron overload is critical to

8

Chapter 2 ´ Differential Diagnosis

improving survival. Therapeutic phlebotomy and iron chelation are both important mainstays of therapy.

2.4 Toxic Disorders

A variety of toxic exposures may result in bone marrow dysplastic changes that resemble myelodysplasia. A detailed clinical history and physical examination is the best way to elucidate these alternative diagnoses. Alcohol is a frequent cause of macrocytosis and anemia, independent of serum folate levels. Heavy use of alcohol has been reported to cause vacuolization of erythroid and granulocytic precursors (Girard et al. 1987). With severe alcohol abuse, bone marrow hypoplasia and pancytopenia may result. Changes are reversible with cessation of alcohol intake. Acute poisoning with heavy metals (e.g., lead, mercury, cadmium, gold, arsenic, etc.) is often suggested by a history of exposure. Chronic exposure, however, may have subtle clinical findings and may present with bone marrow changes, including dysplasia, hypoplasia and aplastic anemia. The diagnosis is made by a careful occupational and exposure history, clinical examination, and a heavy metal screen of the peripheral blood. Many medications can cause alterations in bone marrow pathology. Certain antibiotics, anticonvulsants, nonsteroidal anti-inflammatory drugs and antithyroid medications may all cause a clinical scenario similar to aplastic anemia. Dysplastic changes in the bone marrow aspirates of patients recovering from cancer chemotherapy and radiation therapy may be indistinguishable from MDS.

2.5 Infectious Disorders

Viruses are well-described causes of hematologic abnormalities. Cytopenias are common. Bone marrow changes include features ranging from dysplasia to outright aplasia. Hematologic abnormalities are well described in HIV-positive individuals. Macrocytosis is common either as a direct effect of the virus or a side effect of antibiotic or antiretroviral therapy. A comprehensive review of 216 bone marrow samples from HIV-positive individuals was reported in 1991 (Karcher and Frost 1991). Megaloblastic hematopoiesis and myelodysplasia were seen in 38% and 69% of specimens blindly reviewed, respectively. HIV testing should be

considered in patients with unexplained hematologic and bone marrow abnormalities. Many other viruses have been associated with aplastic bone marrow changes including parvovirus B19 and hepatitis (Brown et al. 1997; Kurtzman and Young 1989). Parvovirus B19 is a common viral infection causing erythema infections in children and an influenza-like illness in adults. In immunosuppressed patients or patients with high red blood cell turnover (e.g., hemolytic anemia), parvovirus infection may result in a pure red cell aplasia and rarely aplastic anemia. Bone marrow biopsy and aspirate may show classic giant pronormoblasts. The diagnosis is made by serology and polymerase chain reaction. Non-A, non-B, and non-C hepatitis have been reported in association with bone marrow failure and may be considered in the differential diagnosis. Virtually any other severe infection may cause significant bone marrow abnormalities, but the clinical scenario is usually helpful in establishing the underlying diagnosis.

2.6 Other Hematopoietic Disorders 2.6.1 Idiopathic Aplastic Anemia

A variety of other primary hematologic conditions may have similar features to MDS. Bone marrow specimens may display hypo- or hypercellularity. Idiopathic aplastic anemia (AA) can be difficult to distinguish from hypocellular MDS. Up to 20% of MDS patients have bone marrow cellularity that is less than 25% or less than expected based on age (Nand and Godwin 1988; Tuzuner et al. 1995). We have already discussed earlier many other causes of bone marrow failure and bone marrow hypoplasia/aplasia that may resemble hypocellular MDS. The etiology of ªidiopathicº AA is thought to be autoimmune in nature in most patients. This is bolstered by clinical observations of improvement with immunosuppressive therapy (Young 2002). Bone marrow immunostaining for CD34 positivity may distinguish aplastic anemia from MDS (Orazi et al. 1997; Scopes et al. 1994). Numerous studies have demonstrated that CD34+ bone marrow cells are reduced in specimens from patients with AA in comparison with patients with MDS. This is consistent with both the presumed mechanism of autoimmune destruction of progenitor cells in AA and the malignant clonal nature of MDS. Immuno-

a

2.6 ´ Other Hematopoietic Disorders

staining for CD34 combined with cytogenetic analysis help in distinguishing AA and hypocellular MDS. The clinical responsiveness of a subset of MDS to immunosuppressive therapy suggests an overlap with both aplastic anemia and paroxysmal nocturnal hemoglobinuria (see below). Ultimately, the ªdiagnosisº given to these disorders is of less importance than the underlying mechanism of bone marrow failure, which may allow appropriate selection of effective therapy.

9

Paroxysmal nocturnal hemoglobinuria (PNH) is another clonal hematopoietic disorder to consider in the differential diagnosis. It is characterized by a defect in the glycosylphosphatidylinositol (GPI)-anchor due to mutations in the PIG-A gene (Rosse 1997). The loss of many proteins bound to the GPI-anchor on the surface of hematopoietic cells is thought to lead to the clinical manifestations of hemolysis, venous thrombosis, and bone marrow failure. Furthermore, given its clonal nature, it has also been described in the setting of MDS, myeloproliferative disorders, and the progression to AML (Longo et al. 1994; Nakahata et al. 1993). The diagnosis of PNH should be considered in any patient presenting with cytopenias and a hypocellular bone marrow. Classically diagnosed by the sucrose lysis and Ham's test, it may now be identified by the absence of GPI-linked proteins, CD55 and CD59, on the surface of peripheral blood cells by using monoclonal antibodies and flow cytometry. Flow cytometric quantification of GPI-anchor binding using fluorescent-labeled inactive toxin aerolysin (FLAER) appears to be the most sensitive method for PNH diagnosis (Brodsky et al. 2000). Treatments include supportive care, immunosuppression, and hematopoietic stem cell transplantation.

proliferative disorder. The World Health Organization (WHO) recognized this in their recent recommendation that CMML, atypical chronic myelogenous leukemia (CML), and juvenile myelomonocytic leukemia (JMML) be removed from their prior CML and MDS categories and placed in a separate category of ªMDS/MPDº (Harris et al. 1999). Patients present with dysplastic bone marrow features and increased white blood cell counts. Karyotypic or molecular analysis to detect the Philadelphia chromosome or the BCR/ABL fusion gene easily identifies typical CML. However, some patients display features of both CML and CMML, yet lack the Philadelphia chromosome or the BCR/ABL fusion gene (Bennett et al. 1994). These patients with ªatypical CMLº show marrow dysplastic changes similar to MDS yet may have progressive leukocytosis and organomegaly reminiscent of myeloproliferative disorders. These patients have a significantly worse prognosis than typical CML patients, with a reported median survival of 24 months (Onida et al. 2002). Bone marrow fibrosis is another pathologic entity that is nonspecific and may cloud the diagnosis of MDS. Although mild to moderate degrees of bone marrow fibrosis have been reported in up to 50% of patients with MDS, diffuse fibrosis is rare (Steensma et al. 2001; Sultan et al. 1981). MDS with myelofibrosis may be distinguished from myelofibrosis with myeloid metaplasia (MMM), often on clinical grounds. Patients with the former are often pancytopenic with trilineage dysplasia and have atypical megakaryocytic proliferation. MMM typically has splenomegaly and extramedullary hematopoiesis. Furthermore, MMM is often more indolent. While the distinction with MMM may be easier, it may be difficult to distinguish MDS with myelofibrosis from acute myelofibrosis, the accelerated phase of chronic myelogenous leukemia, and acute megakaryocytic leukemia.

2.6.3 Myeloproliferative/Myelodysplastic

2.6.4 Large Granular Lymphocytic Leukemia

There can be overlap between some clinical and pathologic features of MDS and certain myeloproliferative disorders. Indeed, chronic myelomonocytic leukemia (CMML) was previously included in the French-American-British (FAB) classification of MDS due to the dysplastic features seen on bone marrow biopsy. However, in many ways, CMML shows classic features of a myelo-

Large granular lymphocytic (LGL) leukemia may also present with pancytopenia and bone marrow hypoplasia. It is a form of indolent non-Hodgkin lymphoma, characterized by a clonal proliferation of T cells or natural killer cells. The diagnosis is often suggested by findings of chronic neutropenia, a modest absolute lymphocytosis, and the presence of morphologically typical cells in the peripheral blood. Anemia and thrombocyto-

2.6.2 Paroxysmal Nocturnal Hemoglobinuria

Disorders

10

Chapter 2 ´ Differential Diagnosis

Table 2.1. Differential diagnosis of myelodysplasia Diagnoses

Clinical features

Smear/pathology

Testing

Vitamin B12 deficiency

Altered mental status, paresthesias, Glossitis, decreased proprioception, Elderly, history of malabsorption

Hypersegmented neutrophils, macrocytosis, hypercellular marrow

Serum B12 low, MMA high, elevated LDH and bilirubin

Folate deficiency

Elderly, history of alcohol use, or depression No neurologic symptoms

Similar to B12 deficiency

Elevated homocysteine level, decreased RBC folate

Fanconi anemia

Autosomal recessive, presents in childhood or early adulthood, other family members. Involved, other hematologic or solid malignancy. Usually with anatomic defects

Macrocytosis, cytopenias, hypocellular marrow

Chromosomal breakage test

Congenital dyserythropoietic anemia

Hyperbilirubinemia, hemolysis, tiredness, multiple prior evaluations without diagnosis

Anisocytosis, poikilocytosis, basophilic stippling, circulating erythroblasts, bone. Marrow with binucleate erythroblasts

Acid lysis test Direct genetic testing

Hereditary sideroblastic anemia

Low MCV, low MCH, low MCHC, elevated ferritin, low transferrin, iron overload, Pyridoxine responsive in some cases

Hypochromic anemia, increased marrow iron, ringed sideroblasts

Free erythrocyte protoporphyrin. Gene mutation analysis

Heavy metal exposure

Occupational history, mental status changes GI symptoms

Pancytopenia and hypocellular bone marrow

Heavy metal screen. TTesting of coworkers/ family

HIV

Risk factors, opportunistic infections. Virtually any other organ system may be affected

Any cytopenia may be seen, macrocytosis autoimmune thrombocytopenia, bone marrow with megaloblastosis and dysplasia

HIV testing; CD4 count; viral load

Parvovirus B19

Influenza-like illness, rash, arthralgias/arthritis. Erythema infectiosum in children, immunosuppressed and patients with hemolytic anemia most prone

Typically anemia, may resemble pure red cell aplasia, bone marrow with giant pronormoblasts

Parvovirus IgM/IgG titers PCR for Parovirus B19

Idiopathic aplastic anemia

Present with symptoms related to cytopenias

Pancytopenia, hypocellular bone marrow

Rule out other causes CD34+ percentage of marrow Precursors (usually low in AA)

Paroxysmal nocturnal hemoglobinuria

Thrombosis, Budd-Chiari presentation, hemolysis

May present with any cytopenia or pancytopenia. Bone marrow ranges from normal to similar to aplastic anemia; may progress to MDS or AML. May arise from prior treatment of AA

CD55, CD59 of peripheral blood

a

References

11

(continued) Diagnoses

Clinical features

Smear/pathology

Testing

Atypical CML

Splenomegaly, extramedullary infiltrates. Poor response to therapy, progression to AML

Leukocytosis, absence of basophilia, bone. Marrow hypercellular with erythroid dysplasia

BCR-ABL negative

Large granular lymphocytic leukemia

Associated malignancy or connective tissue disorder, especially rheumatoid arthritis

Neutropenia most common but other cytopenias. Bone marrow typically full of lymphoid infiltrates, but may be pancytopenic in nearly one fourth of cases

Peripheral blood or bone marrow flow cytometry for T cell and NK markers PCR for T cell antigen receptor genes

penia are not uncommon. Macrocytosis has been reported in over 20% of cases (Dhodapkar et al. 1994). However, in some patients these peripheral blood findings may be subtle, and patients may present with pancytopenia alone. In a Mayo clinic series of 203 patients with T-cell LGL leukemia, 14% presented with pancytopenia at initial presentation, and nine patients fulfilled criteria for aplastic anemia (Dhodapkar et al. 1994; Go et al. 2000). LGL leukemia has been described in association with rheumatoid arthritis and other hematologic disorders, including monoclonal gammopathy of undetermined significance and multiple myeloma (Loughran et al. 1988). Evaluation of the peripheral blood smear often shows the presence of LGLs. LGLs are large, typically contain azurophilic granules, and have an abundant cytoplasm and a round nucleus. Bone marrow pathology in the majority of cases shows clonal lymphocytic infiltrates. A minority of patients presents with a hypocellular bone marrow resembling aplastic anemia or hypocellular MDS. Diagnosis is confirmed by demonstration of a clonal population of T cells or natural killer (NK) cells either in the bone marrow or peripheral blood. Prognosis is generally excellent, with some reports of median survivals in excess of 10 years (Dhodapkar et al. 1994). Treatment consists of immunosuppressive therapy or low-dose cytotoxic therapy and is usually reserved for symptomatic patients with progressive disease (Loughran 1993).

2.7 Summary

The differential diagnosis of MDS is broad (Table 2.1). Many conditions resemble myelodysplasia in one or more features. In the absence of cytogenetic abnormal-

Table 2.2. Helpful tests to distinguish the diagnosis off MDS from other diagnoses 1. Complete blood count and differential 2. Peripheral blood smear 3. Bone marrow aspirate and biopsy 4. Bone marrow cytogenetics 5. Peripheral blood flow cytometry for CD55, CD59, FLAER and lymphocyte markers 6. Bone marrow staining for CD34+ cell percentage 7. Vitamin B12 level, red blood cell folate, methylmalonyl CoA and homocysteine level 8. HIV antibody testing 9. Heavy metal screen 10. Chromosome breakage testing

ities, a thorough evaluation of congenital, nutritional, toxic, infectious, and other clonal disorders is necessary. Based on the results of initial laboratory testing and careful evaluation of the peripheral blood smear and bone marrow, a focused number of additional tests often yield the diagnosis (Table 2.2).

References Alcindor T, Bridges KR (2002) Sideroblastic anaemias (Review). Br J Haematol 116:733±743 Alizadeh AA, Ross DT, Perou CM, van de Rijn M (2001) Towards a novel classification of human malignancies based on gene expression patterns (Review). J Pathol 195:41±52 Anonymous (1976) Is preleukemic states an adequate designation? Blood Cells 2:725±726

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Chapter 2 ´ Differential Diagnosis

Bain B (1996) The bone marrow aspirate of healthy subjects. Br J Haematol 94:206±209 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick H, Sultan C, Cox C (1994) The chronic myeloid leukaemias: guidelines for distinguishing chronic granulocytic, atypical chronic myeloid, and chronic myelomonocytic leukaemia. Proposals by the FrenchAmerican-British Cooperative Leukaemia Group. Br J Haematol 87:746±754 Bottomley SS (2000) Iron overload in sideroblastic and other non-thalassemic anemias. In: Barton JC, Edwards CQ (eds) Hemochromatosis. Genetics, pathophysiology, diagnosis and treatment. Cambridge University Press, Cambridge, UK, pp 442±452 Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT, Borowitz MJ (2000) Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 114:459±466 Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS (1997) Hepatitisassociated aplastic anemia. N Engl J Med 336:1059±1064 Butturini A, Gale RP, Verlander PC, Aldler-Bricner B, Gillio AP, Auerbach AD (1994) Hematologic abnormalities in Fanconi anemia: an international Fanconi anemia registry study. Blood 84:1650±1655 Cavenagh JD, Richardson DS, Gibson RA, Mathew CG, Newland AC (1996) Fanconi's anaemia presenting as acute myeloid leukaemia in adulthood. Br J Haematol 94:126±128 Champion KM, Gilbert JG, Asimakopoulos FA, Hinshelwood S, Green AR (1997) Clonal haemopoiesis in normal elderly women: implications for the myeloproliferative disorders and myelodysplastic syndromes. Br J Haematol 97:920±926 Dhodapkar MV, Li CY, Lust JA, Tefferi A, Phyliky RL (1994) Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a Tcell clonopathy of undetermined significance? Blood 84:1620± 1627 Drabick JJ, Davis BJ, Byrd JC (2001) Concurrent pernicious anemia and myelodysplastic syndrome. Ann Hematol 80:243±245 Garcia-Suarez J, Pascual T, Munoz MA, Herrero B, Pardo A (1998) Myelodysplastic syndrome with erythroid hypoplasia/aplasia: a case report and review of the literature (Review). Am J Hematol 58:319±325 Gardais J (2000) Dyshaemopoiesis in adults: a practical classification for diagnosis and management (Review). Leuk Res 24:641±651 Girard DE, Kumar KL, McAfee JH (1987) Hematologic effects of acute and chronic alcohol abuse (Review). Hematology-Oncology Clinics of North America 1:321±334 Gluckman E, Auerbach AD, Horowitz MM, Sobocinski KA, Ash RC, Bortin MM, Butturini A, Camitta BM, Champlin RE, Friedrich W, Good RA, Gordon-Smith EC, Harris RE, Klein JP, Ortega JJ, Pasquini R, Ramsay NK, Speck B, Vowels MR, Zhang M-J, Gale RP (1995) Bone marrow transplantation for Fanconi anemia. Blood 86:2856±2862 Go RS, Tefferi A, Li CY, Lust JA, Phyliky RL (2000) Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia. Blood 96:3644±3646 Gonzalez MI, Caballero D, Vazquez L, Canizo C, Hernandez R, Lopez C, Izarra A, Arroyo JL, Gonzalez M, Garcia R, San Miguel JF (2000) Allogeneic peripheral stem cell transplantation in a case of hereditary sideroblastic anaemia. Br J Haematol 109:658±660 Green R, Kinsella LJ (1995) Current concepts in the diagnosis of cobalamin deficiency (Review). Neurology 45:14351±440

Greenberg P, Anderson J, de Witte T, Estey E, Fenaux P, Gupta P, Hamblin T, Hellstrom-Lindberg E, List A, Mufti G, Neuwirtova R, Ohyashiki K, Oscier D, Sanz G, Sanz M, Willman C (2000) Problematic WHO reclassification of myelodysplastic syndromes. J Clin Oncol 18:3447±3452 Greiner TC, Burns CP, Dick FR, Henry KM, Mahmood I (1992) Congenital dyserythropoietic anemia type II diagnosed in a 69-year-old patient with iron overload. Am J Clin Pathol 98:522±525 Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, Lister TA, Bloomfield CD (1999) World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting, Airlie House, Virginia, November 1997. J Clin Oncol 17:3835± 3849 Heimpel H (2004) Congenital dyserythropoietic anemias: epidemiology, clinical significance, and progress in understanding their pathogenesis (Review). Ann Hematol 83:613±621 Karcher DS, Frost AR (1991) The bone marrow in human immunodeficiency virus (HIV)-related disease. Morphology and clinical correlation. Am J Clin Pathol 95:63±71 Koc S, Harris JW (1998) Sideroblastic anemias: variations on imprecision in diagnostic criteria, proposal for an extended classification of sideroblastic anemias (Review). Am J Hematol 57:16 Kurtzman G, Young N (1989) Viruses and bone marrow failure (Review). Baillieres Clin Haematol 2:51±67 Longo L, Bessler M, Beris P, Swirsky D, Luzzatto L (1994) Myelodysplasia in a patient with pre-existing paroxysmal nocturnal haemoglobinuria: a clonal disease originating from within a clonal disease. Br J Haematol 87:401±403 Loughran TP Jr (1993) Clonal diseases of large granular lymphocytes (Review). Blood 82:1±14 Loughran TP Jr, Starkebaum G, Kidd P, Neiman P (1988) Clonal proliferation of large granular lymphocytes in rheumatoid arthritis. Arthritis Rheum 31:31±36 May A, Bishop DF (1998) The molecular biology and pyridoxine responsiveness of X-linked sideroblastic anaemia (Review). Haematologica 83:56±70 Miyazato A, Ueno S, Ohmine K, Ueda M, Yoshida K, Yamashita Y, Kaneko T, Mori M, Kirito K, Toshima M, Nakamura Y, Saito K, Kano Y, Furusawa S, Ozawa K, Mano H (2001) Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 98:422±427 Nakahata J, Takahashi M, Fuse I, Nakamori Y, Nomoto N, Saitoh H, Tatewaki W, Imanari A, Takeshige T, Koike T (1993) Paroxysmal nocturnal hemoglobinuria with myelofibrosis: progression to acute myeloblastic leukemia. Leukemia & Lymphoma 12:137±142 Nand S, Godwin JE (1988) Hypoplastic myelodysplastic syndrome. Cancer 62:958±964 Onida F, Ball G, Kantarjian HM, Smith TL, Glassman A, Albitar M, Scappini B, Rios MB, Keating MJ, Beran M (2002) Characteristics and outcome of patients with Philadelphia chromosome negative, bcr/abl negative chronic myelogenous leukemia. Cancer 95:1673±1684 Orazi A, Albitar M, Heerema NA, Haskins S, Neiman RS (1997) Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anemia by CD34 and PCNA immunostaining of bone marrow biopsy specimens. Am J Clin Pathol 107:268±274

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Rios A, Canizo MC, Sanz MA, Vallespi T, Sanz G, Torrabadella M, Gomis F, Ruiz C, San Miguel JF (1990) Bone marrow biopsy in myelodysplastic syndromes: morphological characteristics and contribution to the study of prognostic factors. Br J Haematol 75:26±33 Rosse WF (1997) Paroxysmal nocturnal hemoglobinuria as a molecular disease (Review). Medicine 76:63±93 Scopes J, Bagnara M, Gordon-Smith EC, Ball SE, Gibson FM (1994) Haemopoietic progenitor cells are reduced in aplastic anaemia. Br J Haematol 86:427±430 Steensma DP, Tefferi A (2003) The myelodysplastic syndrome(s): a perspective and review highlighting current controversies (Review) [erratum appears in Leuk Res 29:117]. Leuk Res 27:95±120 Steensma DP, Hanson CA, Letendre L, Tefferi A (2001) Myelodysplasia with fibrosis: a distinct entity? (Review). Leuk Res 25:829±838 Sultan C, Sigaux F, Imbert M, Reyes F (1981) Acute myelodysplasia with myelofibrosis: a report of eight cases. Br J Haematol 49:11±16

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Tischkowitz MD, Hodgson SV (2003) Fanconi anaemia (Review). J Med Genet 40:1±10 Tricot GJ (1992) Minimal diagnostic criteria for the myelodysplastic syndrome in clinical practice. Leuk Res 16:56 Tuzuner N, Cox C, Rowe JM, Watrous D, Bennett JM (1995) Hypocellular myelodysplastic syndromes (MDS): new proposals. Br J Haematol 91:612±617 Urban C, Binder B, Hauer C, Lanzer G (1992) Congenital sideroblastic anemia successfully treated by allogeneic bone marrow transplantation. Bone Marrow Transplant 10:373±375 Wickramasinghe SN (1998) Congenital dyserythropoietic anaemias: clinical features, haematological morphology and new biochemical data (Review). Blood Rev 12:178±200 Wickramasinghe SN (2000) Congenital dyserythropoietic anemias (Review). Curr Opin Hematol 7:71±78 Young NS (2002) Acquired aplastic anemia (Review). Ann Intern Med 136:534±546

Etiology and Epidemiology of MDS David T. Bowen

Contents 3.1 Introduction . . . . . . . . . . . . . . . . . . 3.1.1 MDS Is a Heterogeneous Disease 3.1.2 Demographic Study . . . . . . . . . 3.1.3 Latency of Onset . . . . . . . . . . .

..

.. .. ..

15 15 15 16

3.2 Established Causative Factors for MDS: Cytotoxic Chemotherapeutic Drugs . .

16

3.3 Probable Causative Factors for MDS . . 3.3.1 Ionizing Radiation . . . . . . . . . . . . . 3.3.2 Benzene . . . . . . . . . . . . . . . . . . . .

17 17 17

3.4 Miscellaneous Potential Causative Factors: Case-Control Studies . . . . . . .

18

3.5 Aging and MDS . . . . . . . . . . . . . . . . .

20

3.6 Is There a Genetic Predisposition to MDS? . . . . . . . . . . . . . . . . . . . . . . .

20

3.7 Conclusion . . . . . . . . . . . . . . . . . . . . .

21

References . . . . . . . . . . . . . . . . . . . . . . . . .

21

3.1 Introduction

Despite at least two decades of epidemiological research, the causative factors leading to the development of myelodysplastic syndrome (MDS) remain largely unknown. The study of the causes of these relatively rare diseases is proving difficult for several reasons: 1. MDS comprises a spectrum of heterogeneous disorders.

2. Few comprehensive patient registries exist to accurately determine the demographics of subtypes of MDS (e.g. age/sex distribution). 3. Determining the latency of onset of de novo MDS is not possible without large cross-sectional population studies.

3.1.1 MDS Is a Heterogeneous Disease

The diagnostic process involves categorizing an individual's disease into one of five French-American-British (FAB) groups or one of six World Health Organization (WHO) subgroups (the latter excluding sub-types of chronic myelomonocytic leukemia (CMML), and the FAB-defined refractory anemia with excess blasts in transformation (RAEB-t)). It seems likely that morphologically or molecularly distinct diseases (albeit with overlapping features) will have different causes.

3.1.2 Demographic Study

MDS is a rare disease, with an estimated incidence of 4 per 100,000 per year. The disease becomes more common with increasing age, such that the incidence rises to > 30 per 100,000 per year for people over 70 years of age (Germing et al. 2004; Williamson et al. 1994). Males are more commonly affected than females, although there is evidence that this is not so for refractory anemia with ring sideroblasts (RARS) (Germing et al. 2004). Recent data (Germing et al. 2004) do not support the widely held view that the incidence of MDS is increasing (Reizenstein and Dabrowski 1991). Neverthe-

16

Chapter 3 ´ Etiology and Epidemiology of MDS

less, an aging population will produce an increase in the absolute number of patients diagnosed as will more interventional investigation of older patients (Aul et al. 1998). MDS in children is more infrequent still, and has characteristics different from those in adults. Examples of these differences include the spectrum of FAB/WHO types (RARS and 5q± syndrome are almost never seen in childhood), and of chromosome abnormalities (a higher proportion of children have abnormalities of chromosome 7; see Chapter 7). 3.1.3 Latency of Onset

For patients with de novo MDS, the latency time of the disease is unknown. From a biological angle, there will be at least two phases of disease development, namely: 1) the time from the first damage in the bone marrow to the appearance of changes in the blood counts; and then 2) the time from the first blood count abnormality to the presentation with clinically relevant disease (usually symptoms of anemia) (Fig. 3.1). Both are impossible to study systematically at present. Physiological changes in hematological parameters with aging such as a reduction in hemoglobin concentration will need to be distinguished from those attributed to early MDS (Nilsson-Ehle et al. 2000). For cases of MDS developing after exposure to an agent known or presumed to cause MDS, the latency period varies. This may be from 1±41 years for different radiation exposures (Moloney 1987), 1±10 years for alkylator cytotoxic drugs (Mauritzson et al. 2002; Peder-

Fig. 3.1. Biological stages in the development of MDS

sen-Bjergaard et al. 2002 a), and even more difficult to assess for benzene (up to 30 years?) (Voytek and Thorslund 1991). MDS may also evolve from related disorders such as aplastic anemia, following immunosuppressive therapy, with a latency of up to 10 years (Kojima et al. 2002; Maciejewski et al. 2002; Soci 1996). 3.2 Established Causative Factors for MDS:

Cytotoxic Chemotherapeutic Drugs

Therapy-related MDS and AML (t-MDS/AML) are wellrecognized, though rare complications following cytotoxic drug therapy for malignant and some non-malignant (mainly autoimmune) diseases. It is estimated that prior exposure to chemotherapy increases the risk of developing MDS at least 100-fold (Pedersen-Bjergaard et al. 2002 b). Several classes of cytotoxic drugs are implicated, but alkylators most frequently cause an MDS phase. The increased risk following autologous stem cell transplantation may be more a function of prior exposure to relevant cytotoxic therapy than to the pathology of transplantation, and the cumulative incidence of tMDS/AML may be no higher than for conventional chemotherapy alone (Metayer et al. 2003; Pedersen-Bjergaard et al. 1997). One of the major challenges in the treatment of highly curable diseases such as Hodgkin lymphoma is now to reduce the risk of late complications, and early indications are that newer therapies will prove less likely to produce t-MDS/AML. Unfortunately, effective new agents for treating lymphoid malignancies such as fludarabine and radioimmunoconjugates may also be associated with an increased leukemogenic risk (Armitage et al. 2003). At least two subgroups of alkylator-induced t-MDS/ AML have been identified, with distinct cytogenetic, molecular and gene expression profiles (Pedersen-Bjergaard et al. 2002 b; Qian et al. 2002). One group consists of patients with abnormalities of chromosome 7 and normal chromosome 5, in whom mutations in the RAS oncogene are common. In the second group chromosome 5 abnormalities predominate, with or without abnormalities of chromosome 7, often associated with a complex karyotype and mutation of the p53 tumor suppressor gene (see Chapter 6). The second group has a poorer clinical outcome. The latency of onset from start of previous therapy to the development of t-MDS/AML following exposure to alkylating agent chemotherapy

a

3.3 ´ Probable Causative Factors for MDS

was 63 months (range 7±173) in a recent large series. However, t-MDS/AML constitute < 10% of all cases of adult MDS, and the study of t-MDS/AML as a model for the causes of de novo MDS is problematic. Many cases of t-MDS cannot be easily classified due to bone marrow fibrosis. RARS and CMML are relatively under-represented, and the chromosome abnormalities in t-MDS/AML, while qualitatively similar, are proportionately different from those of the de novo diseases (Mauritzson et al. 2002). Survival of patients with tMDS is also poorer than for de novo MDS. Nevertheless, the cytogenetic and molecular abnormalities identified in t-MDS (Pedersen-Bjergaard et al. 2002 a) (predominantly deletions involving chromosomes 5, 7, 17, 12 and 3) are similar to those in poor-risk de novo MDS, and detailed characterization of the pathways involved may lead to a better understanding of potential causative mechanisms in this subgroup of de novo MDS patients. 3.3 Probable Causative Factors for MDS 3.3.1 Ionizing Radiation

MDS cases are reported in cohorts of individuals exposed to radiation, for treatment of diseases such as ankylosing spondylitis (Brown and Doll 1965), or following exposure to the A-bomb in Hiroshima and Nagasaki (Matsuo et al. 1988). Some of these cases occurred up to 40 years after exposure and thus the precise association

Table 3.1. Potential causative agents in the etiology off MDS Definite Cytotoxic chemotherapeutic drugs, particularly alkylating agents Probable Benzene Ionizing radiation Tobacco smoke Possible Hair dye Pesticides Solvents

17

between the development of MDS and exposure to radiation is impossible to quantify. Mauritzson et al. (2002) also identified a longer latency period for the development of t-MDS/AML following radiotherapy exposure (median 207 months) compared with alkylating agent chemotherapy Ô radiotherapy (median 63 months). The incidence of t-MDS/AML does not appear to be increased following local radiotherapy for lymphoma, but is probably increased following total body irradiation (TBI) exposure at autologous stem cell transplant (Armitage et al. 2003). Similarly, weak associations between radiation exposure and MDS are identified in some (but not all) case-control epidemiology studies (vide infra).

3.3.2 Benzene

Early observations on the link between high concentration benzene exposure in Turkish shoe workers and leukemia/bone marrow failure identified a preleukemic pancytopenic phase in 13 of 51 patients (Aksoy et al. 1972). In a later follow up of this cohort (n = 44), approximately half of the pancytopenic patients had probable aplastic anemia, and the remainder had normo- or hypercellular marrows (Aksoy and Erdem 1978). A recent prospective cohort study has suggested a high incidence of morphological dysplasia in subjects exposed to benzene and a high rate of subsequently developing MDS or acute myeloid leukemia (Travis et al. 1994). The same cohort of benzene-exposed workers (74,828 subjects) showed a significantly elevated relative risk for the development of MDS (95%-confidence interval, 1.7±?) compared with a non-exposed cohort (35,805 subjects) (Yin et al. 1996). Legislation now ensures that exposure to high concentrations of benzene in the workplace or the environment must not occur. Thus, the main sources of exposure to low concentrations of benzene in daily life are tobacco smoke and petrol. Exposure to high concentrations of benzene clearly causes bone marrow toxicity, usually aplasia, some of which will progress to MDS or AML (Aksoy et al. 1972; Rothman et al. 1997). There are in vitro biological data to support a role for relevant cytotoxicity of benzene on hematopoietic cells. Incubation of human hematopoietic cells with benzene metabolites in vitro produces a variety of cytogenetic abnormalities also found in MDS and AML (Smith et al. 2000). Strong epidemiolog-

18

Chapter 3 ´ Etiology and Epidemiology of MDS

ical evidence definitively incriminating benzene as a cause of MDS is, however, still lacking.

3.4 Miscellaneous Potential Causative Factors:

Case-Control Studies

Attempts to study the environmental/occupational etiology of MDS have focused mainly on case-control studies. These are usually questionnaire-based studies, requesting information about the work and recreational background of MDS patients, compared with a ªcontrolº group of individuals who do not have MDS. Many of these studies are supported by occupational hygienists with knowledge of likely exposures within each line of work. While these efforts are commendable, and the best that can be achieved at present, there are many limitations to such studies. These include: 1) ªrecall biasª, relying on the patient's memory for accuracy; 2) considering MDS as one disease in order to produce a sample

size with sufficient power to answer the specific questions posed (something which has been done for the purposes of most of these studies); and 3) relying upon subgroup analysis to draw conclusions (all studies to date have done this). While each individual published case-control study has identified a number of occupations and substances that may be risk factors for MDS, there is little consistency between these studies. Many of the ªassociationsº are likely to represent statistical chance or very weak relative risks. Several modest size case-control studies of MDS patients and appropriate control subjects have now been reported. Most have involved a self-completed or assisted questionnaire exploring potential occupational, recreational and environmental factors associated with an increased odds ratio for MDS patients compared with controls. In the largest and most detailed study, 400 cases and controls (individually matched) were compared (West et al. 1995). Exposure histories including intensity assessment were obtained for 70 chemicals,

Table 3.2. Environmental agents most consistently associated with MDS in larger case-control studies (>100 cases) expressed as Odds ratio (95% confidence intervals where quoted in original references) Study

Tobacco smoking

Hair-dye use

Alcohol

Pesticides

Solvents

West et al. 1995

1.16 (0.8±1.6)

2.38 (1.0±5.9) (hydrogen peroxide exposure)

NA

1.0 (agrochemicals)

0.9 (combined paints, solvents, glues)

Mele et al. 1994

P < 0.05 (trend test for increased pack years)

1.5

NA

NA

NA

Ido et al. 1996

1.8 (0.8±3.9)

1.77 (0.9±3.5)

P < 0.02 (trend test for increasing consumption)

NA

1.5 (0.9±2.6) (organic only)

Pasqualetti et al. 1997

2.33 (1.1±5.1)

NA

NA

NA

NA

Rigolin et al. 1998

NA

NA

NA

2.1 (1.3±3.6)

7.1 (2.4±21)

Nagata et al. 1999

0.8 (0.4±1.6) (former)

1.99 (1.2±3.4)

1.01 (0.4±2.7) (former)

NA

1.99 (0.97±4.1) (organic only)

0.9 (0.5±1.8) (current)

0.8 (0.5±1.4) (current)

Bjork et al. 2000

1.8 (1.2±2.7)

NA

NA

NA

NA

Nisse et al. 2001

1.74 (1.1±2.7)

1.1 (0.4±3.1)

NA

3.2 (1.1±11.2)

2.6 (1.6±5.4)

NA not available

a

3.4 ´ Miscellaneous Potential Causative Factors: Case-Control Studies

and other hazards or radiation. An increased or possibly increased odds ratio was found for MDS patients exposed to radiation, halogenated organics and metals. Elevated odds ratios at higher exposure thresholds were found for copper, arc welding fumes and hydrogen peroxide, with borderline associations for degreasing agents, nickel, exhaust gases and radio transmissions. Pesticides were etiologically implicated in a smaller study (Goldberg et al. 1990), while a further study implicated plant and machine operation, and exposure to exhaust fumes, stone dust, cereal dust, fertilizers as well as petrol and diesel derivatives (Nisse et al. 1995). The data for exposure to commonly studied environmental toxins are summarized in Table 3.2. Tobacco smoking may represent the greatest source of benzene exposure for the population at large, with a 10-fold increase in benzene inhalation in smokers compared with non-smokers (Brownson et al. 1993). Cigarette smoke contains several thousand chemicals and in addition to benzene, many of these are known or suspected human carcinogens

19

(e.g., ethylbenzene, octane and radioactive lead-210) (Pasqualetti et al. 1997). Other factors with an elevated odds ratio for MDS that have emerged from case-control studies include alcohol excess (including a possible dose effect) (Ido et al. 1996) and childlessness (West et al. 1995), although this could not be confirmed in another smaller study (Nisse et al. 1995). Each of these studies is limited by insufficient numbers of cases and controls to identify significant odds ratios with high statistical power. It is clear that an association between exposure to an agent and the development of MDS is a long way from establishing cause and effect. Alternative explanations must always be considered, such as hair-dye use commonly associated with grey hair and the possibility that it is grey hair and not hair-dye chemicals that are associated with the development of MDS. Two studies have attempted to correlate cytogenetic abnormalities with exposure to environmental toxins (Table 3.3) (Rigolin et al. 1998; West et al. 2000). Both

Table 3.3. Cytogenetic abnormalities and their association with exposure to environmental toxins in MDS patients versus controls Study

MDS patients (number)

Controls (number)

Exposed vs. non-exposed

Exposure to specific agents vs. cytogenetic abnormalities

Rigolin et al. 1998

178 (cytogenetics known = 134)

178

P < 0.001 (trend test through IPSS good, intermediate to poor risk cytogenetic categories; poor risk category had highest ratio of exposed:non-exposed * cases)

NA

West et al. 2000

214 (cytogenetics known)

400

Cytogenetics abnormal: OR = 2 (0.8±5.9)

Chromosomes 5: inorganic gases/fumes, OR = 8 ** (1.1±356) and OR = 4.3 *** (1.3±13.6)

Cytogenetics normal: OR = 1 (0.6±1.8)

Chromosome 7: inorganic gases/fumes, OR = 5.1** (0.6±237) and OR = 8.4*** (1.7±42)

186 (cytogenetics unknown)

Chromosome 8: radiation, OR = 6.0** (0.7±28) and OR = 1.7*** (0.6±5.5) OR odds ratio, NA not available *** Exposure studied only to pesticides and organic solvents *** Paired comparison: chromosome abnormalities with normal *** Unpaired comparison: chromosome abnormalities with normal

20

Chapter 3 ´ Etiology and Epidemiology of MDS

studies suggest that a history of exposure to environmental toxins is more common in patients with an abnormal karyotype. Weak associations were also found for exposure to selected toxins and specific karyotypic abnormalities (e.g., higher incidence of exposure to inorganic gases and fumes in patients with abnormalities of chromosomes 5 and 7), although numbers were small and confidence intervals large (West et al. 2000). 3.5 Aging and MDS

The incidence of MDS increases with age. This observation has been interpreted in two ways: the disease must result from a progressive accumulation of a lifetime's exposure to a toxic agent, or the aged bone marrow ªstemº cell is easier to damage than its younger counterpart. Considerable evidence for genetic traits as determinants of stem cell function, including longevity, has emerged from the study of inbred mouse strains (Van Zant and Liang 2003). Although human progenitor numbers do not convincingly decrease with increasing age, their replating efficiency does decrease, suggesting a reduction in self-renewal (Marley et al. 1999). The process of ªagingº is not well defined, though much blame is heaped upon ªfree radicalsº (Kirkwood and Austad 2000), defense against which deteriorates with age. Mitochondrial DNA mutation would be potentially both caused by and result in increased intracellular oxidative stress. Although specific mutations in enzymes associated with electron transport have been identified in

Fig. 3.2. Age-related changes in hematopoietic stem cells (modified from Van Zant 2003)

MDS patients (Gattermann 2004) and appear to be associated with clonal expansion (Gattermann et al. 2004), the pathogenetic role and, indeed, the pathological relevance of these mutations remains debatable (Shin et al. 2003). It remains unclear in what way those changes are relevant to diseases of older age such as MDS (Fig. 3.2).

3.6 Is There a Genetic Predisposition to MDS?

The vast majority of MDS patients presenting in adulthood have no relatives with the disease, and no obvious inherited disease with a tendency for the development of MDS. In 30% of children with MDS, other abnormalities are present, and some of these are part of well-recognized syndromes including Fanconi anemia or Bloom Syndrome. Fanconi anemia may represent the best natural model for the mechanistic study of MDS, with 30% of patients developing MDS/AML by the age of 40 years (Kutler et al. 2003), and the spectrum of karyotypic abnormalities is similar to that seen with de novo MDS. Families with several members affected by MDS are described, but are exceptionally rare (Lucas et al. 1989). Although still very rare, familial MDS may be more likely in the family of a child with monosomy 7 (Luna-Fineman et al. 1995). An increased risk for autoimmune disease has been correlated with specific Human Leukocyte Antigen (HLA) subtypes. The response of selected MDS patients to immunosuppressive therapy catalyzed the search for an HLA restriction in MDS. Although an increased prevalence of HLA-DR2(15) has been reported in MDS (Saunthararajah et al. 2002), this finding has not been consistent (Deeg et al. 2004; Gowans et al. 2002). While the mechanisms underlying familial MDS are likely to involve abnormalities of high-penetrance genes, the emerging science of the study of interactions between variants in low-penetrance genes is now producing results. Polymorphic variants in genes encoding enzymes that metabolize xenobiotic environmental toxins are now widely studied as potential predisposing factors for cancer. No systematic study has yet been reported for MDS patients, although a study of a common variant in the NADP(H) quinone oxidoreductase 1 gene (NQO1) in patients developing hematopoietic toxicity after benzene exposure has suggested that this genetic variant may predispose to the development of MDS in this setting (Rothman et al. 1997). As for many of these

a

References

studies, the number of cases in this study was too small to draw confident conclusions.

3.7 Conclusion

Despite more than a decade of dedicated effort from epidemiologists, clinicians and scientists, the cause of MDS remains largely unknown. It is inevitable that the different subtypes of MDS will have different causes. Patients with RARS appear to have a different gender distribution and are under-represented in t-MDS; the cause of this disease is likely to be different from patients with RAEB, for example. We must use the limited high-quality demographic data to develop hypotheses, and test these in a combination of clinical and molecular epidemiology studies, which by definition will need to involve very large patient numbers.

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Goldberg H, Lusk E, Moore J, Nowell PC, Besa EC (1990) Survey of exposure to genotoxic agents in primary myelodysplastic syndrome: correlation with chromosome patterns and data on patients without hematological disease. Cancer Res 50:6876±6881 Gowans D, O'Sullivan A, Rollinson S, Roddam P, Groves M, Fegan C, Morgan G, Bowen D (2002) Allele and haplotype frequency at human leucocyte antigen class I/II and immunomodulatory cytokine loci in patients with myelodysplasia and acute myeloid leukaemia: in search of an autoimmune aetiology. Br J Haematol 117:541±545 Ido M, Nagata C, Kawakami N, Shimizu H, Yoshida Y, Nomura T, Mizoguchi H (1996) A case-control study of myelodysplastic syndromes among Japanese men and women. Leuk Res 20:727±731 Kirkwood TB, Austad SN (2000) Why do we age? (Review). Nature 408:233±238 Kojima S, Ohara A, Tsuchida M, Kudoh T, Hanada R, Okimoto Y, Kaneko T, Takano T, Ikuta K, Tsukimoto I (2002) Risk factors for evolution of acquired aplastic anemia into myelodysplastic syndrome and acute myeloid leukemia after immunosuppressive therapy in children. Blood 100:786±790 Kutler DI, Singh B, Satagopan J, Batish SD, Berwick M, Giampietro PF, Hanenberg H, Auerbach AD (2003) A 20-year perspective on the International Fanconi Anemia Registry (IFAR). Blood 101:1249± 1256 Lucas GS, West RR, Jacobs A (1989) Familial myelodysplasia. BMJ 299:551 Luna-Fineman S, Shannon KM, Lange BJ (1995) Childhood monosomy 7: epidemiology, biology, and mechanistic implications (Review). Blood 85:1985±1999 Maciejewski JP, Risitano A, Sloand EM, Nunez O, Young NS (2002) Distinct clinical outcomes for cytogenetic abnormalities evolving from aplastic anemia. Blood 99:3129±3135 Marley SB, Lewis JL, Davidson RJ, Roberts IA, Dokal I, Goldman JM, Gordon MY (1999) Evidence for a continuous decline in haemopoietic cell function from birth: application to evaluating bone marrow failure in children. Br J Haematol 106:162±166 Matsuo T, Tomonaga M, Bennett JM, Kuriyama K, Imanaka F, Kuramoto A, Kamada N, Ichimaru M, Finch SC, Pisciotta AV (1988) Reclassification of leukemia among A-bomb survivors in Nagasaki using French-American-British (FAB) classification for acute leukemia. Jpn J Clin Oncol 18:91±96 Mauritzson N, Albin M, Rylander L, Billstrom R, Ahlgren T, Mikoczy Z, Bjork J, Stromberg U, Nilsson PG, Mitelman F, Hagmar L, Johansson B (2002) Pooled analysis of clinical and cytogenetic features in treatment-related and de novo adult acute myeloid leukemia and myelodysplastic syndromes based on a consecutive series of 761 patients analyzed 1976±1993 and on 5098 unselected cases reported in the literature 1974±2001. Leukemia 16:2366±2378 Mele A, Szklo M, Visani G, Stazi MA, Castelli G, Pasquini P, Mandelli F (1994) Hair dye use and other risk factors for leukemia and preleukemia: a case-control study. Italian Leukemia Study Group. Am J Epidemiol 139:609±619 Metayer C, Curtis RE, Vose J, Sobocinski KA, Horowitz MM, Bhatia S, Fay JW, Freytes CO, Goldstein SC, Herzig RH, Keating A, Miller CB, Nevill TJ, Pecora AL, Rizzo JD, Williams SF, Li CY, Travis LB, Weisdorf DJ (2003) Myelodysplastic syndrome and acute myeloid leukemia after autotransplantation for lymphoma: a multicenter case-control study. Blood 101:2015±2023 Moloney WC (1987) Radiogenic leukemia revisited. Blood 70:905±908

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Nagata C, Shimizu H, Hirashima K, Kakishita E, Fujimura K, Niho Y, Karasawa M, Oguma S, Yoshida Y, Mizoguchi H (1999) Hair dye use and occupational exposure to organic solvents as risk factors for myelodysplastic syndrome. Leuk Res 23:57±62 Nilsson-Ehle H, Jagenburg R, Landahl S, Svanborg A (2000) Blood haemoglobin declines in the elderly: implications for reference intervals from age 70 to 88. Eur J Haematol 65:297±305 Nisse C, Lorthois C, Dorp V, Eloy E, Haguenoer JM, Fenaux P (1995) Exposure to occupational and environmental factors in myelodysplastic syndromes. Preliminary results of a case-control study. Leukemia 9:693±699 Nisse C, Haguenoer JM, Grandbastien B, Preudhomme C, Fontaine B, Brillet JM, Lejeune R, Fenaux P (2001) Occupational and environmental risk factors of the myelodysplastic syndromes in the North of France. Br J Haematol 112:927±935 Pasqualetti P, Festuccia V, Acitelli P, Collacciani A, Giusti A, Casale R (1997) Tobacco smoking and risk of haematological malignancies in adults: a case-control study. Br J Haematol 97:659±662 Pedersen-Bjergaard J, Pedersen M, Myhre J, Geisler C (1997) High risk of therapy-related leukemia after BEAM chemotherapy and autologous stem cell transplantation for previously treated lymphomas is mainly related to primary chemotherapy and not to the BEAM-transplantation procedure. Leukemia 11:16541±660 Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C (2002 a) Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood 99:1909±1912 Pedersen-Bjergaard J, Christiansen DH, Andersen MK, Skovby F (2002 b) Causality of myelodysplasia and acute myeloid leukemia and their genetic abnormalities (Review). Leukemia 16:2177±2184 Qian Z, Fernald AA, Godley LA, Larson RA, Le Beau MM (2002) Expression profiling of CD34+ hematopoietic stem/ progenitor cells reveals distinct subtypes of therapy-related acute myeloid leukemia. Proc Natl Acad Sci USA 99:14925±14930 Reizenstein P, Dabrowski L (1991) Increasing prevalence of the myelodysplastic syndrome: an international Delphi study. Anticancer Res 11:1069±1070 Rigolin GM, Cuneo A, Roberti MG, Bardi A, Bigoni R, Piva N, Minotto C, Agostini P, De Angeli C, Del Senno L, Spanedda R, Castoldi G (1998) Exposure to myelotoxic agents and myelodysplasia: case-control study and correlation with clinicobiological findings. Br J Haematol 103:189±197 Rothman N, Smith MT, Hayes RB, Traver RD, Hoener B, Campleman S, Li GL, Dosemeci M, Linet M, Zhang L, Xi L, Wacholder S, Lu W, Meyer KB, Titenko-Holland N, Stewart JT, Yin S, Ross D (1997) Benzene

poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C±>T mutation and rapid fractional excretion of chlorzoxazone. Cancer Res 57:2839±2842 Saunthararajah Y, Nakamura R, Nam JM, Robyn J, Loberiza F, Maciejewski JP, Simonis T, Molldrem J, Young NS, Barrett AJ (2002) HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome. Blood 100:1570±1574 Shin MG, Kajigaya S, Levin BC, Young NS (2003) Mitochondrial DNA mutations in patients with myelodysplastic syndromes. Blood 101:3118±3125 Smith MT, Zhang L, Jeng M, Wang Y, Guo W, Duramad P, Hubbard AE, Hofstadler G, Holland NT (2000) Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells. Carcinogenesis 21:1485±1490 Soci G (1996) Could aplastic anaemia be considered a pre-pre-leukaemic disorder? (Review). Eur J Haematol Supplementum 60:60±63 Travis LB, Li CY, Zhang ZN, Li DG, Yin SN, Chow WH, Li GL, Dosemeci M, Blot W, Fraumeni JFJ (1994) Hematopoietic malignancies and related disorders among benzene-exposed workers in China (Review). Leukemia & Lymphoma 14:91±102 Van Zant G (2003) Genetic control of stem cells: implications for aging (Review). Int J Hematol 77:29±36 Van Zant G, Liang Y (2003) The role of stem cells in aging (Review). Exp Hematol 31:659±672 Voytek PE, Thorslund TW (1991) Benzene risk assessment: status of quantifying the leukemogenic risk associated with the low-dose inhalation of benzene (Review). Risk Anal 11:355±357 West RR, Stafford DA, Farrow A, Jacobs A (1995) Occupational and environmental exposures and myelodysplasia: a case-control study. Leuk Res 19:127±139 West RR, Stafford DA, White AD, Bowen DT, Padua RA (2000) Cytogenetic abnormalities in the myelodysplastic syndromes and occupational or environmental exposure. Blood 95:2093±2097 Williamson PJ, Kruger AR, Reynolds PJ, Hamblin TJ, Oscier DG (1994) Establishing the incidence of myelodysplastic syndrome. Br J Haematol 87:743±745 Yin SN, Hayes RB, Linet MS, Li GL, Dosemeci M, Travis LB, Zhang ZN, Li DG, Chow WH, Wacholder S, Blot WJ (1996) An expanded cohort study of cancer among benzene-exposed workers in China. Benzene Study Group. Environmental Health Perspectives 104[Suppl 6]:1339±1341

Molecular Biology of Myelodysplasia Philip Nivatpumin, Steven Gore

Contents

4.2.9

24 24 25 25 25

Molecular Abnormalities Unrelated to Cytogenetic Abnormalities . . . 4.2.9.1 TP53 . . . . . . . . . . . . . . . . 4.2.9.2 FLT3 . . . . . . . . . . . . . . . . 4.2.9.3 AML1 . . . . . . . . . . . . . . . 4.2.9.4 Other Mutations . . . . . . . 4.2.10 Gene Expression Profiling . . . . . .

31 31 31 32 32 32

4.3 Summary . . . . . . . . . . . . . . . . . . . . . .

32

References . . . . . . . . . . . . . . . . . . . . . . . . .

33

26 26 27 28 28 28

4.1 Introduction

4.1 Introduction . . . . . . . . . . . . . . . . . . . .

23

4.2 Pathogenesis . . . . . . . . . . . . . . . . . . . 4.2.1 Susceptibility to Genomic Injury/ Genomic Instability . . . . . . . . . . . 4.2.2 Epigenetic Modifications . . . . . . . 4.2.3 Abnormalities in Signaling Pathways 4.2.3.1 EPO/Growth Factor Signaling 4.2.3.2 RAS/MAPK . . . . . . . . . . . . 4.2.3.3 Vascular Endothelial Growth Factor (VEGF) . . . . . . . . . . 4.2.4 Apoptosis . . . . . . . . . . . . . . . . . 4.2.5 Immune Dysregulation . . . . . . . . 4.2.6 Bone Marrow Microenvironment . . 4.2.6.1 Cytokine Milieu . . . . . . . . . 4.2.6.2 Neo-angiogenesis . . . . . . . 4.2.7 Molecular Abnormalities Identified by Cytogenetic Features . . . . . . . 4.2.7.1 Chromosome 5 Deletions . 4.2.7.2 Chromosome 7 Deletions . 4.2.7.3 Chromosome 20 . . . . . . . . 4.2.7.4 Chromosome 17 . . . . . . . . 4.2.7.5 Trisomy 8 . . . . . . . . . . . . . 4.2.7.6 Other Less Frequent Chromosomal Deletions . . . . . . 4.2.8 Translocations . . . . . . . . . . . . . . 4.2.8.1 TEL(ETV6) Fusion . . . . . . . 4.2.8.2 Nucleoporin Abnormality . . 4.2.8.3 MLL . . . . . . . . . . . . . . . . . 4.2.8.4 EVI-1 . . . . . . . . . . . . . . . . 4.2.8.5 NPM . . . . . . . . . . . . . . . .

24

29 29 29 30 30 30 30 30 30 30 31 31 31

The myelodysplastic syndromes (MDS) are a heterogeneous group of monoclonal disorders characterized by ineffective hematopoiesis and an increased risk of transformation to acute leukemia. Current classification systems (e.g., French-American-British (FAB), World Health Organization (WHO)) are based on morphological features of blood and bone marrow elements as well as cytogenetic abnormalities (Bennett et al. 1982; Harris et al. 1999). The natural history of MDS is highly variable. This likely reflects the myriad cytogenetic, genetic and epigenetic alterations that are associated with MDS. It has generally been suggested that MDS arises from a hematopoietic stem cell that has suffered irreversible DNA damage. Further events result in dominance of this damaged clone. Immunologic responses may occur that promote progenitor survival and eventual clonal dominance. In bone marrows from early-stage MDS patients, impaired differentiation and increased apoptosis pre-

24

Chapter 4 ´ Molecular Biology of Myelodysplasia

dominate, but as disease progression occurs, increased proliferation and accumulation of immature cells result. This chapter will summarize some of the observations of the numerous biologic mechanisms implicated in the pathophysiology of MDS, including intrinsic progenitor abnormalities, epigenetic changes, abnormal apoptosis machinery, immunologic influences, abnormal signal transduction pathways and the role of the bone marrow microenvironment.

4.2 Pathogenesis 4.2.1 Susceptibility to Genomic Injury/Genomic

Instability

MDS is thought to arise from the somatic mutation of a hematopoietic progenitor cell. Confirmation of the clonality has been bolstered by analysis of cytogenetic and X-chromosome inactivation studies (Abrahamson et al. 1991; Delforge 2003) of patients with MDS. However, scientific evidence has grown in support of the concept that the cytogenetic abnormalities seen so frequently in MDS may be acquired during disease progression, rather than reflecting the initial inciting clonal event (Delforge 2003; Nilsson et al. 2002). Whether a primary or secondary event, genomic instability, as evidenced by karyotypic changes common in MDS, is thought to play an important role in disease pathogenesis. Cytogenetic abnormalities in MDS result from the accumulation of genomic damage, failure to repair such damage, or both. Although the etiology of most cases of MDS is unknown, exposure to genotoxic agents such as benzene, radiation, or prior treatment with chemotherapeutic agents is known to increase the risk of developing MDS (Nisse et al. 2001). Other environmental agents that may increase the risk include smoking, heavy metals, pesticides, fertilizers, petroleum products, and organic chemicals (Garfinkel and Boffetta 1990; Rigolin et al. 1998; West et al. 1995) (see Chapter 3). The relationship between Fanconi anemia (FA) and childhood MDS point to increased susceptibility to genomic damage as an important cause of childhood MDS. Fanconi anemia is an autosomal recessive disorder of chromosomal instability. Patients are characterized by congenital abnormalities, ineffective hematopoiesis, and a high risk of developing MDS, acute leukemia, and solid tumors. FA proteins interact with other DNA damage repair proteins such as ATM and BRCA1 and

BRCA2 (Tischkowitz and Dokal 2004). Repair of DNA double-strand breaks is often inaccurate in the pre-leukemic Bloom's syndrome and in FA (Gaymes et al. 2002; Langland et al. 2002). Impaired response to oxidative stress has also been implicated in the pathophysiology of MDS. Defects in both glutathione transferase theta 1 (GSTT1) and NADPH quinone oxyreductase (NQO1) have been associated with an increased risk of myelodysplastic syndrome (Chen et al. 1996; Farquhar and Bowen 2003; Rothman et al. 1997). Glutathione is both an antioxidant and a cofactor for many antioxidant enzymes that are important in the metabolism of various toxins and carcinogens. NQO1 is another gene involved in antioxidant mechanisms. Mutations in this gene have been reported in association with benzene-induced bone marrow damage. Another possible mechanism underlying genomic instability involves telomere dynamics and the enzyme telomerase. Telomere erosion may result in chromosome end fusion and subsequent chromosome instability. Shortened telomere length has been reported to be associated with poor prognosis in patients with MDS (Engelhardt et al. 2004; Ohyashiki et al. 1994).

4.2.2 Epigenetic Modifications

While genetic alterations are critical in the pathogenesis of MDS, epigenetic changes also contribute significantly to the disease phenotype. A modern definition of epigenetics refers to ªmodifications in gene expression that are brought about by heritable, but potentially reversible changes in chromatin structure and/or DNA methylationº (Henikoff and Matzke 1997). Epigenetic changes include methylation of cytosine residues followed by a guanine base (DNA methylation) and post-translational modifications of histones that lead to alteration in chromatin structure at specific gene loci, which in turn determine the transcriptional output of the gene. These changes to histones, collectively referred to as the histone code, include lysine acetylation, lysine methylation, ubiquitination, phosphorylation, and sumoylation. These histone modifications are recognized by specific proteins that recruit transcriptional activators and corepressors, establishing a higher order of chromatin structure (Fischle et al. 2003; Hake et al. 2004). Methylation of CpG dinucleotides concentrated in the promoter regions of some genes (so-called `CpG islands') results in the functional inactivation of those

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genes without alteration in the primary sequence. Both hypomethylation and hypermethylation of the genome have been observed in hematological malignancies. Hypomethylation of the MDR1 gene has been described in AML samples and was shown to correlate with increased expression as measured by reverse transcription polymerase chain reaction (RT-PCR), possibly contributing to multidrug resistance in these patients (Attwood et al. 2002; Nakayama et al. 1998). Hypomethylation of c-myc and myeloperoxidase have also been described in samples from patients with primary AML and AML arising from MDS (Tsukamoto et al. 1992). While mutations in cell cycle control genes such as p15, p16, and p19 have rarely been described in MDS, hypermethylation of p15 is common. Hypermethylation of the p15INK4B gene promoter has been observed in 30± 50% of MDS cases and has been shown to correlate with the percentage of bone marrow blasts (Quesnel et al. 1998; Uchida et al. 1997). p15INK4B is a cyclin-dependent kinase inhibitor that is critical in regulating the G1 phase of the cell cycle. Its activation is downstream of the TGF-b/SMAD b pathway. This suggests that one mechanism of proliferation of leukemic cells is escape of regulation of G1 phase of cell cycle. Further evidence for the importance of this event in MDS pathogenesis derives from the observation that the degree of methylation correlates with the risk of evolution to AML and clinical prognosis (Quesnel et al. 1998). Other genes frequently methylated and silenced in myeloid malignancies include E-Cadherin, RARb R , and SOCS-1 (Esteller and Herman 2002; Herman and Baylin 2003). The clinical activity of the DNA methyltransferase inhibitors 5-azacitidine (Silverman 2004) and 2'deoxy-5-azacitidine (Wijermans et al. 1997, 2000) in MDS suggests that the methylation status of a subset of genes is likely to contribute significantly to the biological and clinical behavior of MDS. However, attempts to correlate the clinical activity of these agents with reversal of p15 methylation have demonstrated that methylation reversal may not be required for clinical response (Daskalakis et al. 2002; Issa et al. 2004; Lubbert 2003). Transcriptional silencing of methylated genes is mediated at least in part through the establishment of repressive chromatin conformation through the recruitment of histone deacetylases (Cameron et al. 1999). This has led to the strategy of combined DNA methyltransferase and histone deacetylase inhibition for the treatment of MDS.

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4.2.3 Abnormalities in Signaling Pathways

4.2.3.1 EPO/Growth Factor Signaling The ineffective hematopoiesis that is characteristic of MDS has led to the investigation of pathways involved in transducing signals from erythropoietin (EPO) and other growth factors. When bone marrow cells from MDS patients are cultured in colony forming assays in the presence of EPO, erythroid colony formation is reduced in comparison to normal controls (Backx et al. 1993; Mayani et al. 1989). EPO signaling involves a complex cascade of events beginning with the binding of EPO to the erythropoietin receptor (EPO-R). Upon binding EPO, the Janus kinase, JAK2, is activated (Tanner et al. 1995; Witthuhn et al. 1993). JAK2 activation leads to downstream tyrosine phosphorylation of a number of proteins. Activation of signal transducer and activator of transcription 5 (STAT5), a downstream protein of JAK2 that is thought to be important in EPO signaling, is impaired in myelodysplastic syndrome (Hoefsloot et al. 1997). This observation, combined with the other reports of normal presence of EPO-R in MDS patients, indicates that alteration of the EPO signaling pathway may have an important role in MDS (Backx et al. 1996). Alterations in other growth factor pathways have also been reported in MDS patient samples. GM-CSF and G-CSF priming of reactive oxygen species (ROS) production in neutrophils of patients with MDS is impaired (Fuhler et al. 2003). Thrombopoietin signaling has been investigated for its role in the dysmegakaryocytopoiesis seen in MDS, but its role is unclear (Hofmann et al. 2000; Kalina et al. 2000).

4.2.3.2 RAS/MAPK RAS is a critical component in the signaling cascade resulting in cellular proliferation in response to a variety of extracellular signals including growth factors. It acts as a molecular relay switch that is downstream of receptor tyrosine kinases and upstream of a cascade of mitogen activated protein kinases (MAPK) that result in activation of nuclear transcription factors. RAS mutations have been identified in up to 30% of human leukemias and up to 15% of patients with MDS (Paquette et al. 1993). N-ras mutations are associated with poor prognosis in MDS patients and increased rate of transformation to AML (Paquette et al. 1993). RAS activation is

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Chapter 4 ´ Molecular Biology of Myelodysplasia

mediated by the hydrolysis of GTP. RAS has intrinsic GTP-binding activity and GTP-hydrolyzing activity. This RAS/GTP complex activates target proteins such as RAF, leading to subsequent downstream signaling. Point mutations that interfere with the GTP hydrolyzing activity of RAS result in constitutive signaling and activation of downstream components, resulting in cell proliferation. An N-ras mutation has been associated with an increased risk of progression to AML (Padua et al. 1998). Preliminary clinical studies suggest that the farnesyl transferase inhibitors (FTI), tipifarnib and lonafarnib, are active in MDS; however, it is not clear that the clinical activity is related to the impact of the FTIs on RAS molecular signaling (Kantarjian et al. 2002; Kurzrock et al. 2004).

4.2.3.3 Vascular Endothelial Growth Factor (VEGF) Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. Angiogenesis is increasingly thought to have an important role in MDS (see Angiogenesis below). VEGF is regulated by multiple signals, including hypoxia inducible factor-1 (HIF-1) and Ras (Estey 2004). Myelomonocytic precursors of patients with MDS and AML overexpress both VEGF and its high affinity receptor, Flt-1 (Bellamy et al. 2001). Inhibition of VEGF reduces leukemia colony formation in clinical samples from MDS patients (Broxmeyer et al. 1995). The importance of VEGF has led to the clinical investigation of VEGF receptor antibodies and VEGF tyrosine kinase inhibitors for the treatment of MDS. 4.2.4 Apoptosis

Apoptosis is an ordered cellular process that regulates cell population size in a variety of conditions. First described by Wyllie and colleagues (Wyllie et al. 1980), apoptosis is an energy-dependent process characterized morphologically by cytoplasmic and nuclear condensation, fragmentation of nuclei into ªapoptotic bodiesº, preservation of plasma membrane integrity and phagocytosis of cellular debris by macrophages in the absence of an inflammatory response (Greenberg 1998; Walker et al. 1988; Wyllie 1985). This death mechanism is crucial in maintaining a precise number of cells in a given organism. Alterations in apoptosis have been implicated in a variety of medical disorders including myelodyspla-

sia. A variety of stimuli or insults serve as initiators of the apoptotic pathway. These include chemotherapy drugs, ultraviolet and gamma irradiation, chemical exposure, viral infection, steroid hormones, and various cytokines (e.g., TNF-a, Fas ligand, TGF-b) (Greenberg 1998). A comprehensive review of all the pathways involved in apoptosis is outside the scope of this chapter. However, the process of apoptosis may be conceptually divided into extrinsic and intrinsic pathways. Extrinsic triggers include death ligands (e.g., Fas ligand, TNF-a, TNF-related apoptosis-inducing ligand [TRAIL]) which bind to cell surface receptors and activate downstream signal transduction pathways. Intrinsic signals that activate the apoptotic pathway result from cellular stress, including exposure to radiation, chemicals or infectious processes. Removal of cellular survival signals (e.g., growth factors) may also trigger intrinsic activation of cell death. Both extrinsic and intrinsic apoptotic pathways culminate in the activation of a family of cytosolic aspartate-specific cysteine proteases (caspases). Caspases are the final effector molecules of apoptosis, responsible for the cleavage of both cytosolic and nuclear proteins that result in the stereotypic destruction of the cell. Extrinsic activation of apoptosis is mediated by the binding of death ligands (e.g., Fas ligand, TNF-a, TNFrelated apoptosis-inducing ligand [TRAIL]) to cell surface transmembrane receptors (Parker and Mufti 2004; Zang et al. 2001). For example, cytotoxic lymphocytes can initiate apoptosis by expressing Fas ligand, which binds to the Fas receptor on the surface of target cells. This binding results in a conformational change in the cytoplasmic death domain (DD) (Chinnaiyan et al. 1995; Nagata and Golstein 1995). The altered death domain then recruits intracellular adaptor molecules such as FADD (Fas-associated protein with a DD), which aggregate procaspase-8 molecules. These procaspase-8 molecules, which cleave and activate one another, in turn activate downstream caspases to induce apoptosis. Cellular stress and damage may initiate intrinsic aggregation and activation of procaspases. In the best-understood pathway, mitochondria are stimulated to release cytochrome c into the cytosol where it forms a complex with apaf-1 (apoptotic protease-activating factor-1), procaspase-9 and dATP (Li et al. 1997; Liu et al. 1996). This complex then triggers downstream effector caspases. The Bcl-2 family of intracellular proteins includes many of the most important regulators of apoptotic

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4.2 ´ Pathogenesis

pathways. Some proapoptotic members of this family such as Bad act by binding and inactivating death inhibiting members of the family (Parker and Mufti 2004). Other proapoptotic molecules such as Bax and Bak stimulate cytochrome c release from the mitochondrion. They form homo- or heterodimers that create membrane pores/ion channels that facilitate the release of cytochrome c and other apoptogenic proteins (Desagher et al. 1999; Hsu et al. 1997; Schendel et al. 1997; Zha et al. 1996). Some members of this family such as Bcl-2 and Bcl-XL inhibit apoptosis by blocking release of cytochrome c. They may directly bind and sequester cytochrome c and Apaf-1 or interact with Bax or Bak, thereby inhibiting pore formation (Eskes et al. 2000; Hu et al. 1998; Oltvai et al. 1993; Yang et al. 1997). There are numerous other mechanisms of positive and negative regulation of apoptosis that are outside the scope of this chapter. They may involve a variety of oncogenes and tumor-suppressor genes, including p53 and c-myc. Evidence for alterations in intramedullary apoptosis in early MDS was first suggested through morphological examination of bone marrow hematopoietic cells. Increased apoptosis of bone marrow progenitors was postulated to account for the clinical observation in early MDS of peripheral blood cytopenias in the presence of a hypercellular bone marrow. It was further postulated that decreased apoptosis may explain the later clinical disease progression and accumulation of immature progenitor cells. Increased apoptosis in MDS has been shown by morphology, immunohistochemistry, flow cytometry and the molecular detection of activated apoptosis-related proteins (Parker et al. 2000). Raza and colleagues were the first to show increased apoptosis in patients with early MDS (Raza et al. 1995 a, b). They used in situ end labeling (ISEL) of DNA strand breaks to detect apoptosis. ISEL labeling was elevated in patients with early MDS and low in AML and late MDS samples. Using nick-end labeling (TUNEL) techniques, various groups have demonstrated a higher percentage of labeled cells in the bone marrows of MDS patients than in healthy controls (Hellstrom-Lindberg et al. 1997; Lepelley et al. 1996). Upregulation of Fas in MDS bone marrow samples has also been reported (Bouscary et al. 1997; Gersuk et al. 1998; Kitagawa et al. 1998). Flow cytometry studies have been utilized to better characterize which specific MDS marrow cells are involved in apoptosis. Using flow cytometric detection of annex-

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in V on the surface of apoptotic cells, Parker and colleagues (1998) demonstrated increased apoptosis in CD34+ cells from early MDS patients compared with late MDS patients. Li et al. (2004) showed that apoptosis occurred predominantly (but not exclusively) in non-clonal cells as determined by concurrent FISH in patients with a suitable clonal marker. Rajapaksa and colleagues (1996) analyzed CD34+ and CD34± subfractions of bone marrow from MDS patients and evaluated the sub-diploid (sub-G1) DNA peak after staining with propidium iodide. They observed that the proportion of CD34+ cells with sub-G1 DNA (apoptotic) was increased in comparison to normal bone marrow and bone marrow from AML patients (Rajapaksa et al. 1996). Bcl-2 and cMyc oncoprotein levels were also evaluated. C-Myc:Bcl-2 oncoprotein ratios were highest in early MDS samples and lower in late MDS and AML samples. The ratio of the pro-apoptotic BAX to anti-apoptotic BCL-2 was increased in early-stage MDS but decreased in more advanced disease (Hellstrom-Lindberg et al. 1997; Parker and Mufti 2001; Parker et al. 2000). This observation supports the hypothesis that the relative balance between cell-death and cell-survival signals is associated with the increased apoptosis observed in MDS progenitors. The cause of abnormal apoptosis in MDS is unknown. Both intrinsic cellular defects and extrinsic factors are being investigated in ongoing research. Alterations in the immune-mediated signals, cytokine release, and other aspects of the bone marrow microenvironment have been implicated. Alterations in apoptosis appear to be a central feature of MDS. Increased apoptosis is frequently observed in the early stages of MDS. Further insight into the intrinsic and extrinsic factors that affect apoptosis is a central area of research and hold significant promise for the development of clinical therapies.

4.2.5 Immune Dysregulation

There is growing evidence that immune dysregulation plays a role in MDS pathophysiology. The relationship between MDS and autoimmunity stimulated the investigation into the role of the immune system in MDS. The incidence of autoimmune disorders appears to be increased in patients with MDS (Saif et al. 2002). Autologous cytotoxic T lymphocytes have been observed to exert inhibitory effects on MDS myelopoiesis in vitro.

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Chapter 4 ´ Molecular Biology of Myelodysplasia

Moreover, the features of MDS may overlap with aplastic anemia (AA) and large granular lymphocyte (LGL) disease, two diseases thought to be related to autoreactive T lymphocytes (Barrett et al. 2000; Kanchan and Loughran 2003). Clinical studies indicated activity of antithymocyte globulin (ATG) and cyclosporine in the treatment of select groups of patients with MDS (Jonasova et al. 1998; Killick et al. 2003; Molldrem et al. 2002). Given the added observation that tumor necrosis factor alpha (TNFa) mRNA and protein levels are elevated in both bone marrow and plasma samples of patients with MDS, recent clinical trials have evaluated the efficacy of treatment with immunosuppression and anti-TNF therapy (Deeg et al. 2004; Kitagawa et al. 1997; Maciejewski et al. 1995; Molnar et al. 2000; Rosenfeld and Bedell 2002). Single agent ATG has resulted in complete hematologic responses in up to 10±15% of patients (Killick et al. 2003). Predictors of response to immunosuppressive therapy include younger age, presence of a paroxysmal nocturnal hemoglobinuria (PNH) clone, human leukocyte antigen (HLA) DR15, hypocellularity, and a normal karyotype (Saunthararajah et al. 2002). Responses to ATG have been associated with disappearance of T-cell clones that demonstrate V beta clonality and which suppress hematopoiesis ex vivo (Kochenderfer et al. 2002). Deeg et al. treated fourteen transfusion-requiring patients with MDS with the combination of ATG and the soluble TNF receptor protein etanercept (Deeg et al. 2004). Forty-six percent of the patients responded, with five patients achieving periods of red blood cell and platelet independence that exceeded 2 years. These impressive results lend further evidence to the premise that immunomodulation may be effective in select patients with MDS. Fundamental questions remain unanswered about the precise mechanisms underlying autoimmunity in MDS. The hypothesis that T lymphocytes attack specific antigens on MDS clonal progenitors remains unproven. Likewise, it is unclear why some patients respond to immunosuppression and others do not. Important future investigations will include confirmation of the efficacy of immunosuppressive and anti-TNF therapies in phase III clinical trials and in identifying subsets of patients who will most benefit from these therapies.

4.2.6 Bone Marrow Microenvironment

4.2.6.1 Cytokine Milieu The observation of increased bone marrow apoptosis in patients with early MDS stimulated the evaluation of the bone marrow microenvironment as a mediator of MDS pathophysiology. Relative deficiency or overproduction of numerous cytokines, including interleukin 1b (IL1b), IL-6, IL-8, stem cell factor, erythropoietin, transforming growth factor beta (TGF-b), GM-CSF, and TNF-a have been measured in the bone marrow and serum of patients with MDS with unclear and sometimes conflicting results (Bowen et al. 1993; Fontenay-Roupie et al. 1999; Maurer et al. 1993; Verhoef et al. 1992). Of all of these cytokines, increased TNF-a has been consistently associated with elevated Fas antigen expression on CD34+ cells. Fas is a membrane protein that can initiate apoptotic signals in response to crosslinking by the Fas ligand (FasL) (Gersuk et al. 1998). The resultant downstream activation of caspases, which are the critical proteases that result in apoptotic cell death, indicate the importance of elevated TNF-a levels in promoting apoptosis in MDS (Mundle et al. 1999). The source of increased TNF-a is likely marrow macrophages and Tlymphocytes.

4.2.6.2 Neo-angiogenesis Another aspect of the bone marrow microenvironment that has emerged as an essential factor in the pathogenesis of MDS is neo-angiogenesis. Angiogenesis plays a critical role in tumor growth and metastasis (Folkman 1995). Increased microvessel density has been demonstrated in the bone marrow of patients with hematologic malignancies, including MDS (Alexandrakis et al. 2004, 2005; Padro et al. 2000; Pruneri et al. 1999). Neovascularization is mediated by a variety of angiogenic molecules that are released by both tumor cells and normal host cells. Abnormal elevation of several angiogenic cytokines and growth factors in AML samples has been reported. Important molecules include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin, TNF-a, and TGF-b (Albitar 2001; Alexandrakis et al. 2005; Faderl and Kantarjian 2004). Soluble VEGF receptor has been reported as a prognostic factor in both AML and MDS patients (Hu et al. 2004).

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4.2 ´ Pathogenesis

4.2.7 Molecular Abnormalities Identified

by Cytogenetic Features

Cytogenetic abnormalities occur in up to 70% of patients with primary MDS and up to 90% of patients with therapy-related MDS (Delforge 2003). A well-described difference between primary and secondary MDS is the complexity of abnormal karyotypes. Chromosomal deletions are common in MDS as opposed to the balanced translocations that are seen in AML. Over the last decade, intensive investigation has focused on identifying potential tumor suppressor genes in the regions of genetic loss in MDS. Here, we will review some of the insights based on particular chromosomal abnormalities.

4.2.7.1 Chromosome 5 Deletions Approximately 20% of patients with MDS have abnormalities of chromosome 5 (Third MIC Cooperative Study Group 1988). These abnormalities include interstitial deletions of the long arm (5q-), monosomy, and unbalanced translocations. A separate clinical entity of 5q± syndrome has been described in multiple studies in the literature and is now recognized as a separate entity in the WHO classification of MDS (Boultwood et al. 1994; Mathew et al. 1993). It is characterized by refractory macrocytic anemia with dyserythropoiesis, a striking female to male ratio of 3:1, normal or high platelet counts, 5q± as the sole cytogenetic abnormality, and a low propensity for transformation to AML. In other cases, abnormalities on chromosome 5 have been seen in familial cases of MDS and in therapy-related MDS (Grimwade et al. 1993; Mhawech and Saleem 2001). The most critical region of deletion is presumed to lie between 5q31 and 5q33. Numerous hematopoietic growth factors are encoded on the long arm of chromosome 5, and loss of these genes is presumed to play a role in MDS pathogenesis. The genes for IL-3, IL-4, IL-5, interferon regulator factor 1 (IRF-1), M-CSF, GMCSF, and the receptor for M-CSF are localized on the long arm of chromosome 5. These cytokines are critical in the proliferation of granulocytes. However, it is unclear how deletion of any one of these growth factor genes alone would result in a 5q± syndrome phenotype. IRF-1, a transcription activator of interferon 1 genes, results in a gene product that is anti-oncogenic and growth inhibitory (Harada et al. 1993). Although deletion of IRF-1 in one or both alleles by an accelerated exon skipping mechanism has been described in some

29

cases of MDS with 5q± abnormalities, specific mutations in this gene have not been described in MDS. Other insights into important genes involved in MDS result from the analysis of genes involved in chromosomal translocations on chromosome 5. t(3;5)(q25.1; q34) results in a chimeric protein, NPM-MLF1, that is associated with MDS prior to transformation to AML (Yoneda-Kato et al. 1996). t(5;11)(q31;q23) has been shown to result in a fusion between the MLL and GRAF genes in a child with JMML (Borkhardt et al. 2000). Homologs of the human GRAF gene have putative tumor suppressor properties and may indicate the importance of this gene in deletions of chromosome 5q. The unique clinical activity of CC-5013 in patients with abnormalities of chromosome 5 (List et al. 2005) may afford a unique reagent with which to probe this subset of patients for specific disease-defining molecular abnormalities.

4.2.7.2 Chromosome 7 Deletions Partial or complete deletion of chromosome 7 is a common finding in MDS and AML. It is seen in a variety of settings and is generally associated with poor prognosis. Approximately 10% of 7q± deletions are seen in the setting of de novo MDS. The remainder of 7q± deletions are seen in cases of MDS arising after environmental or chemotherapeutic exposure and in cases related to familial genetic disorders (e.g., Fanconi anemia, neurofibromatosis 1 [NF1], congenital neutropenia) (Mhawech and Saleem 2001). Analysis from patients with juvenile myelomonocytic leukemia, which often have monosomy 7, has shown that approximately 30% have NF1 gene mutations (Shannon et al. 1994). NF1 functions as a tumor suppressor gene, encoding a GTPase activation protein acts as a negative regulator of RAS activity (Martin et al. 1990). RAS activation occurs in a significant proportion of adult patients with MDS. Gene mutations of RAS or inactivation of the NF1 gene are thought to play an important role in the progression of MDS with monosomy 7 (Stephenson et al. 1995). The region at 7q22.1 has been suggested as a critical breakpoint in myeloid malignancies (Johnson et al. 1996). Genes of interest that have been mapped to chromosome 7q include erythropoietin, plasmin activator b, asparagine synthase gene inhibitor, T-cell receptor b and PIK3CG.

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Chapter 4 ´ Molecular Biology of Myelodysplasia

4.2.7.3 Chromosome 20 A chromosomal 20q deletion is seen in 10% of myeloproliferative disorders, particularly polycythemia vera, and in approximately 5% of primary MDS (Davis et al. 1984). Clinically, patients have a lower incidence of anemia and a relatively favorable prognosis. Only patients with 5q± syndrome have a more prolonged life expectancy. The crucial region deleted in MDS has been mapped to the region between D20S174 and D20S17 (Asimakopoulos et al. 1994; Roulston et al. 1993). Potential tumor-suppressor genes of interest in this region include phospholipase C d, adenosine deaminase, topoisomerase 1, hematopoietic cell kinase, growth hormone releasing factor, myeloid leukemia gene and SRC, the human homolog of Rous sarcoma virus.

4.2.7.4 Chromosome 17 Clinically, deletion of the short arm of chromosome 17 (17p±) is seen primarily in treatment-related MDS and is characterized by dysgranulopoiesis and pseudo-Pelger-HuŸt anomaly (Lai et al. 1995). The p53 gene is located at 17p13.1. P53 is a critical tumor suppressor gene that has significant roles in cell cycle control, DNA repair, and apoptosis. Loss of p53 has been documented in a variety of cancers, and it is likely that it plays a role in a subset of MDS cases.

4.2.7.5 Trisomy 8 Trisomy 8 occurs commonly in both acute and chronic leukemias. A curious observation has been the disappearance of trisomy 8 clones in the course of the disease. This phenomenon is independent of the percentage of blasts in the bone marrow or the clinical status of the disease (Iwabuchi et al. 1992; Matsuda et al. 1998). As such, it is unclear how significant trisomy 8 is in the pathogenesis of MDS, and therefore, it has not attracted as much attention as some of the other cytogenetic abnormalities.

4.2.7.6 Other Less Frequent Chromosomal Deletions Loss of portions of chromosomes 3, 11, 12, 13, and the Y chromosome have been described with varying frequency in the literature. Likewise, trisomies involving chromosomes 6, 13, and 21 have been documented.

Although critical regions on each chromosome have been identified and a number of candidate tumor suppressor genes have been identified, the molecular pathogenesis remains elusive.

4.2.8 Translocations

4.2.8.1 TEL(ETV6) Fusion First described by Srivastava et al. (1988) in patients with chronic myelomonocytic leukemia with eosinophilia, t(5;12)(q33;p13) is a recurrent chromosomal translocation that results in a fusion between the platelet-derived growth factor receptor-b (PDGRFR-b) gene on chromosome 5 and an ETS-like gene, TEL(ETV6) on chromosome 12 (Golub et al. 1994). It is now apparent that this translocation occurs in a broader group of myeloid malignancies with features of both myeloproliferative disorders (MPS) and MDS. ETS family members act as transcriptional activators, while PDGFR-b is a receptor tyrosine kinase that acts on multiple downstream targets including RAS. Oligomerization of the TEL/PDGRFR-b through the TEL HLH domain results in constitutive activation of the PDGRFR-b tyrosine kinase domain (Carroll et al. 1996). This constitutive activation results in cellular transformation. Demonstrated responses to the tyrosine kinase inhibitor, imatinib, in patients with chronic myeloproliferative diseases associated with TEL/PDGRFR-b lend further credence to the importance of this signaling pathway in the pathogenesis of subsets of MDS/MPS (Apperley et al. 2002). TEL gene fusion has also been reported in conjunction with ARNT, MN1, EVI-1, and ACS2 in MDS patients carrying a variety of translocations (Buijs et al. 1995; Raynaud et al. 1996; Salomon-Nguyen et al. 2000; Yagasaki et al. 1999).

4.2.8.2 Nucleoporin Abnormality Nucleoporins are molecules involved in the nuclear import and export of proteins and RNAs (Radu et al. 1995). The gene NUP98, in particular, has been identified as a fusion partner in patients with treatment-related AML or MDS with chromosomal translocations involving 11p15.5. Chimeric transcripts involving NUP98 combine the N-terminal GLFG repeats of NUP98 with the C-terminus of the partner gene (Ahuja et al. 1999; Arai et al. 1997; Nakamura et al. 1996; Nishiyama et al. 1999; Raza-

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4.2 ´ Pathogenesis

Egilmez et al. 1998; Slape and Aplan 2004). Up to 15 partnerships have been identified to date, clearly identifying NUP98 as a potentially important target in therapy-related leukemias and MDS. The mechanism of the formation of fusion and cellular transformation is an active field of investigation. The t(6;9)(p23;q34) translocation can occur in patients with both MDS and AML. This translocation results in a fusion protein between the DEK and CAN genes on chromosomes 6 and 9, respectively. CAN is structurally similar to a component of the nuclear pore complex, NUP214, which also facilitates nuclear import and export of RNA and protein (Kraemer et al. 1994).

4.2.8.3 MLL Chromosomal translocations involving 11q23 are well described in association with biphenotypic leukemias, monocytic leukemias, infant leukemias or secondary leukemias after treatment with topoisomerase inhibitors (Ayton and Cleary 2001; Ridge and Wiedemann 1994). The MLL gene involved in these translocations has homology to the trithorax gene of Drosophila, a homeotic regulator of pattern development in the fruit fly (Djabali et al. 1992). This suggests that MLL acts as a homeotic regulator of transcription. Although the most common 11q23 translocations are involved in AML as opposed to MDS, other less common MLL translocations are involved in primary or secondary MDS (Mitani et al. 1995; Taki et al. 1997). Although the conventional 11q23 translocations such as t(4;11)(q21;q23) and t(9;11)(q21;q23) have not been found in primary MDS, other translocations such as t(11;19)(q23;p13.1) and t(11;16)(q23;p13) have been reported. MLL gene tandem duplication has also been described in patients with MDS (Caligiuri et al. 1996).

4.2.8.4 EVI-1 Alterations in chromosome 3 involving 3q21 and 3q26 bands occur in up to 2% of patients with AML or MDS (Hirai 2003), conferring a uniformly poor prognosis. 3q21q26 syndrome has been described in patients with abnormal megakaryocytopoiesis, elevated platelet counts, and a poor prognosis (Jotterand Bellomo et al. 1992). Genes of interest within this region include transferrin, lactoferrin, transferrin receptor, melanotransferrin, CALLA/CD10 and ecotropic virus integration site 1

31

gene (EVI-1). The expression of the EVI-1 gene, located at 3q26, has generated the most interest in a variety of chromosomal abnormalities in MDS and AML. Inv(3) (q21q26) and t(3;3)(q21;q26) are classified with the 3q21q26 syndrome. The translocation t(1;3)(p36;q21) results in activation of MEL1, a homologous gene to EVI-1, under the control of ribophorin I, which is in the 3q21 region (Mochizuki et al. 2000). This is associated with trilineage dysplasia and dysmegakaryocytopoiesis. The translocation t(3;21)(q26;q22) generates an AML1/EVI-1 chimeric gene that is seen in therapy-related AML/ MDS and in the blast crisis of CML (Mitani et al. 1994). The pathobiology of EVI-1-mediated leukemogenesis is unclear, but may relate to downstream effects on TGF-b signaling (Izutsu et al. 2001). Buonamici et al. (2004) recently generated an EVI-1 murine model of myelodysplasia. These animals developed a fatal pancytopenia accompanied by a hypercellular bone marrow and dyserythropoiesis.

4.2.8.5 NPM Translocation t(3;5)(q25.1;q34) may occur in MDS and AML. It involves NPM on 5q34 and MLF1 on 3q25.1 (Yoneda-Kato et al. 1996). NPM has a role in transporting ribosomal nucleoproteins between the nucleolus and the cytoplasm. The fusion gene may affect cell growth by altering DNA replication, RNA processing or gene expression.

4.2.9 Molecular Abnormalities Unrelated

to Cytogenetic Abnormalities

4.2.9.1 TP53 Mutations in the tumor suppressor, TP53, are reported in up to 10% of MDS patients (Padua et al. 1998). Studies have shown that TP53 mutations occur in predominantly high-risk FAB subtypes. One study showed that mutations resulting in loss of heterozygosity of p53 were associated with therapy-related MDS and AML, deletion or loss of 5q, complex karyotype, and a uniform poor prognosis (Christiansen et al. 2001).

4.2.9.2 FLT3 Internal tandem duplication (ITD) of the FLT3 gene has been associated with adverse outcome in AML. FLT3 is a

32

Chapter 4 ´ Molecular Biology of Myelodysplasia

receptor-type tyrosine kinase that is involved in proliferation and differentiation in hematopoiesis. FLT3-ITD is found in up to 20% of AML and 5% of MDS (Shih et al. 2004; Yokota et al. 1997). It is associated with a high risk of transformation to AML and poor survival.

4.2.9.3 AML1 The AML1 (Runx1) gene encodes a heterodimeric transcription factor that is a critical regulator of hematopoiesis. Translocations involving this gene or its binding partners present in two forms, t (8; 21) and inv(16). Mutations in AML1 have been identified in therapy-related MDS following alkylating agents (Christiansen et al. 2004; Harada et al. 2003). Dominant negative effects of AML1 mutations on normal AML1 function are thought to be critical in pathogenesis (Imai et al. 2000). t (3; 21) is another MDS-associated translocation involving AML1. It has been described in MDS patients exposed to organic solvents, treatment-related MDS, and the blast crisis of CML (Mitani et al. 1994; Tasaka et al. 1992).

4.2.9.4 Other Mutations

diagnose and prognosticate various cancers, including myelodysplasia. 4.3 Summary

The pathogenesis of MDS is complex and remains elusive. Figure 4.1 shows a hypothetical model. The proposed models agree that a multistep process occurs through which a hematopoietic stem cell is mutated and attains a growth advantage. This may occur as a result of environmental damage or inherited predisposition. The mutated clone is associated with morphological dysplasia, impaired differentiation and hematopoiesis, and genomic instability. Cytokine secretion and apoptotic pathways are altered, and there may be impairment of immune responses. Presumably, in the early stages, increased production of proapoptotic cytokines leads to excessive apoptosis, correlating clinically with cytopenias and a cellular bone marrow. As the disease progresses, further genetic and epigenetic events occur, resulting in decreased apoptosis, clonal expansion, and progression to AML. There is considerable overlap between mechanisms, and MDS is likely a heterogeneous group of diseases with a similar clinical phenotype.

Other mutations have been described with RB, WT1, C/ EBPa and a variety of other genes with varying degrees of frequency. Their role in the pathogenesis of MDS is unclear.

4.2.10 Gene Expression Profiling

cDNA microarray technology has revolutionized molecular biology and medicine. For details in MDS, see Chapter 5. Using commercially available chip technology, investigators have investigated the expression of several thousands of genes in a variety of cancers, including diffuse large B cell lymphoma, follicular lymphoma, acute myeloid leukemia and myelodysplastic syndromes (Lossos et al. 2004; Pellagatti et al. 2004; Ross et al. 2004; Sigal et al. 2005). Identification of specific molecular ªsignaturesº in these and many other cancers holds promise for further advances in predicting prognosis and response to therapy. The role of these genome-wide analysis tools remains to be defined. They have the potential to completely transform the way we

Fig. 4.1. Hypothetical pathogenesis of MDS

a

References

Table 4.1. Examples of therapies currently under clinical evaluation for MDS and presumed their mechanism of action Agents

Mechanism of action

TLK199

Glutathione analog

Arsenic trioxide

Pro-apoptotic, antiangiogenesis, differentiation

Etanercept

Soluble TNF receptor

Pentoxifylline

Anti-TNF agent

Infliximab

Anti-TNF antibody

CEP701

FLT-3 inhibitor

Imatinib

Tyrosine kinase inhibitor

Tipifarnib

Farnesyl transferase inhibitor

Lonafarnib

Farnesyl transferase inhibitor

5-azacitidine

DNA methyltransferase inhibitor

2'-deoxy-5-azacitidine (decitabine)

DNA methyltransferase inhibitor

Sodium phenylbutyrate

Histone deacetylase inhibitor

MS-275

Histone deacetylase inhibitor

Valproic acid

Histone deacetylase inhibitor

Suberoylanilide hydroxamic acid (SAHA)

Histone deacetylase inhibitor

FK228 (Depsipeptide)

Histone deacetylase inhibitor

Thalidomide

Antiangiogenesis?, immunomodulatory

Lenalidomide

Antiangiogenesis?, immunomodulatory

PTK787

VEGF receptor kinase inhibitor

Bevacizumab

Anti-VEGF antibody

AG3340

Matrix metalloproteinase inhibitor

Bortezomib

Proteasome inhibitor

SCIO 469

MAP Kinase inhibitor

Clinical testing of a number of molecules that affect these myriad molecular mechanisms is currently being done (Table 4.1). Characterization of genomic expression patterns will inform both diagnosis and prognostication. Further insight into the molecular mechanisms of MDS will provide an avenue for more tailored and effective therapy in the future.

33

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Chapter 4 ´ Molecular Biology of Myelodysplasia

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a patient with refractory anaemia without haematological improvement. Eur J Haematol 60:260±261 Maurer AB, Ganser A, Buhl R, Seipelt G, Ottmann OG, Mentzel U, Geissler RG, Hoelzer D (1993) Restoration of impaired cytokine secretion from monocytes of patients with myelodysplastic syndromes after in vivo treatment with GM-CSF or IL-3. Leukemia 7:1728±1733 Mayani H, Baines P, Bowen DT, Jacobs A (1989) In vitro growth of myeloid and erythroid progenitor cells from myelodysplastic patients in response to recombinant human granulocyte-macrophage colony-stimulating factor. Leukemia 3:29±32 Mhawech P, Saleem A (2001) Myelodysplastic syndrome: review of the cytogenetic and molecular data (Review). Critical Reviews in Oncology-Hematology 40:229±238 Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M, Mano H, Yazaki Y, Ohki M, Hirai H (1994) Generation of the AML1-EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia. EMBO J 13:504±510 Mitani K, Kanda Y, Ogawa S, Tanaka T, Inazawa J, Yazaki Y, Hirai H (1995) Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation. Blood 85:2017± 2024 Mochizuki N, Shimizu S, Nagasawa T, Tanaka H, Taniwaki M, Yokota J, Morishita K (2000) A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells. Blood 96:3209±3214 Molldrem JJ, Leifer E, Bahceci E, Saunthararajah Y, Rivera M, Dunbar C, Liu J, Nakamura R, Young NS, Barrett AJ (2002) Antithymocyte globulin for treatment of the bone marrow failure associated with myelodysplastic syndrome [summary for patients in Ann Intern Med 2002 137:I-27]. Ann Intern Med 137:156±163 Molnar L, Berki T, Hussain A, Nemeth P, Losonczy H (2000) Detection of TNFalpha expression in the bone marrow and determination of TNFalpha production of peripheral blood mononuclear cells in myelodysplastic syndrome. Pathology Oncology Research 6:18± 23 Mundle SD, Reza S, Ali A, Mativi Y, Shetty V, Venugopal P, Gregory SA, Raza A (1999) Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes. Cancer Lett 140:201±207 Nagata S, Golstein P (1995) The Fas death factor. Science 267:14491456 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K, Chen SJ, Willman CL, Chen IM, Feinberg AP, Jenkins NA, Copeland NG, Shaughnessy JD (1996) Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 12:154±158 Nakayama M, Wada M, Harada T, Nagayama J, Kusaba H, Ohshima K, Kozuru M, Komatsu H, Ueda R, Kuwano M (1998) Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias. Blood 92:4296±4307 Nilsson L, Astrand-Grundstrom I, Anderson K, Arvidsson I, Hokland P, Bryder D, Kjeldsen L, Johansson B, Hellstrom-Lindberg E, Hast R, Jacobsen SE (2002) Involvement and functional impairment of the CD34+CD38± Thy-1+ hematopoietic stem cell pool in myelodysplastic syndromes with trisomy 8. Blood 100:259±267

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Raynaud SD, Baens M, Grosgeorge J, Rodgers K, Reid CD, Dainton M, Dyer M, Fuzibet JG, Gratecos N, Taillan B, Ayraud N, Marynen P (1996) Fluorescence in situ hybridization analysis of t(3; 12)(q26; p13): a recurring chromosomal abnormality involving the TEL gene (ETV6) in myelodysplastic syndromes. Blood 88:682±689 Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, Aplan PD (1998) NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer Res 58:4269±4273 Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R, Rifkin S, Iftikhar A, Shetty V, Parcharidou A, Loew J, Marcus B, Khan Z, Chaney C, Showel J, Gregory S, Preisler H (1995 a) Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 86:268±276 Raza A, Mundle S, Iftikhar A, Gregory S, Marcus B, Khan Z, Alvi S, Shetty V, Dameron S, Wright V (1995 b) Simultaneous assessment of cell kinetics and programmed cell death in bone marrow biopsies of myelodysplastics reveals extensive apoptosis as the probable basis for ineffective hematopoiesis. Am J Hematol 48:143±154 Ridge SA, Wiedemann LM (1994) Chromosome 11q23 abnormalities in leukaemia (Review). Leuk Lymphoma 14:11±17 Rigolin GM, Cuneo A, Roberti MG, Bardi A, Bigoni R, Piva N, Minotto C, Agostini P, De Angeli C, Del Senno L, Spanedda R, Castoldi G (1998) Exposure to myelotoxic agents and myelodysplasia: case-control study and correlation with clinicobiological findings. Br J Haematol 103:189±197 Rosenfeld C, Bedell C (2002) Pilot study of recombinant human soluble tumor necrosis factor receptor (TNFR:Fc) in patients with low risk myelodysplastic syndrome. Leuk Res 26:721±724 Ross ME, Mahfouz R, Onciu M, Liu HC, Zhou X, Song G, Shurtleff SA, Pounds S, Cheng C, Ma J, Ribeiro RC, Rubnitz JE, Girtman K, Williams WK, Raimondi SC, Liang DC, Shih LY, Pui CH, Downing JR (2004) Gene expression profiling of pediatric acute myelogenous leukemia. Blood 104:3679±3687 Rothman N, Smith MT, Hayes RB, Traver RD, Hoener B, Campleman S, Li GL, Dosemeci M, Linet M, Zhang L, Xi L, Wacholder S, Lu W, Meyer KB, Titenko-Holland N, Stewart JT, Yin S, Ross D (1997) Benzene poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C±>T mutation and rapid fractional excretion of chlorzoxazone. Cancer Res 57:2839±2842 Roulston D, Espinosa R, Stoffel M, Bell GI, Le Beau MM (1993) Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20. Blood 82:3424±3429 Saif MW, Hopkins JL, Gore SD (2002) Autoimmune phenomena in patients with myelodysplastic syndromes and chronic myelomonocytic leukemia (Review). Leuk Lymphoma 43:2083±2092 Salomon-Nguyen F, Della-Valle V, Mauchauffe M, Busson-Le CM, Ghysdael J, Berger R, Bernard OA (2000) The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion. Proc Natl Acad Sci USA 97:6757±6762 Saunthararajah Y, Nakamura R, Nam JM, Robyn J, Loberiza F, Maciejewski JP, Simonis T, Molldrem J, Young NS, Barrett AJ (2002) HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome. Blood 100:1570±1574 Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed JC (1997) Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA 94:5113±5118

38

Chapter 4 ´ Molecular Biology of Myelodysplasia

Shannon KM, O'Connell P, Martin GA, Paderanga D, Olson K, Dinndorf P, McCormick F (1994) Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N Engl J Med 330:597±601 Shih LY, Lin TL, Wang PN, Wu JH, Dunn P, Kuo MC, Huang CF (2004) Internal tandem duplication of fms-like tyrosine kinase 3 is associated with poor outcome in patients with myelodysplastic syndrome. Cancer 101:989±998 Sigal S, Ninette A, Rechavi G (2005) Microarray studies of prognostic stratification and transformation of follicular lymphomas. Bailliere's Best Practice in Clinical Haematology 18:143±156 Silverman LR (2004) DNA methyltransferase inhibitors in myelodysplastic syndrome. Bailliere's Best Practice in Clinical Haematology 17:585±594 Slape C, Aplan PD (2004) The role of NUP98 gene fusions in hematologic malignancy (Review). Leuk Lymphoma 45:1341±1350 Srivastava A, Boswell HS, Heerema NA, Nahreini P, Lauer RC, Antony AC, Hoffman R, Tricot GJ (1988) KRAS2 oncogene overexpression in myelodysplastic syndrome with translocation 5;12. Cancer Genet Cytogenet 35:61±71 Stephenson J, Lizhen H, Mufti GJ (1995) Possible co-existence of RAS activation and monosomy 7 in the leukaemic transformation of myelodysplastic syndromes. Leuk Res 19:741±748 Taki T, Sako M, Tsuchida M, Hayashi Y (1997) The t(11;16)(q23;p13) translocation in myelodysplastic syndrome fuses the MLL gene to the CBP gene. Blood 89:3945±3950 Tanner JW, Chen W, Young RL, Longmore GD, Shaw AS (1995) The conserved box 1 motif of cytokine receptors is required for association with JAK kinases. J Biol Chem 270:6523±6530 Tasaka T, Nagai M, Sasaki K, Murata M, Taoka T, Ikeda K, Tanaka T, Abe T, Takahara J, Irino S (1992) Translocation (3;21) (q26.2;q22.1) found in a patient with myelodysplastic syndrome and long-term exposure to organic solvents. Br J Haematol 82:473±475 Third MIC Cooperative Study Group (1988) Recommendations for a morphologic, immunologic, and cytogenetic (MIC) working classification of the primary and therapy-related myelodysplastic disorders. Report of the workshop held in Scottsdale, Arizona, USA, on February 23±25, 1987. Third MIC Cooperative Study Group. Cancer Genet Cytogenet 32:1±10 Tischkowitz M, Dokal I (2004) Fanconi anaemia and leukaemia clinical and molecular aspects (Review). Br J Haematol 126:176±191 Tsukamoto N, Morita K, Karasawa M, Omine M (1992) Methylation status of c-myc oncogene in leukemic cells: hypomethylation in acute leukemia derived from myelodysplastic syndromes. Exp Hematol 20:1061±1064 Uchida T, Kinoshita T, Nagai H, Nakahara Y, Saito H, Hotta T, Murate T (1997) Hypermethylation of the p15INK4B gene in myelodysplastic syndromes. Blood 90:1403±1409

Verhoef GE, De Schouwer P, Ceuppens JL, van Damme J, Goossens W, Boogaerts MA (1992) Measurement of serum cytokine levels in patients with myelodysplastic syndromes. Leukemia 6:1268±1272 Walker NI, Harmon BV, Gobe GC, Kerr JF (1988) Patterns of cell death (Review). Methods & Achievements in Experimental Pathology 13:18±54 West RR, Stafford DA, Farrow A, Jacobs A (1995) Occupational and environmental exposures and myelodysplasia: a case-control study. Leuk Res 19:127139 Wijermans P, Lçbbert M, Verhoef G, Bosly A, Ravoet C, Andre M, Ferrant A (2000) Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol 18:956±962 Wijermans PW, Krulder JW, Huijgens PC, Neve P (1997) Continuous infusion of low-dose 5-Aza-2'-deoxycytidine in elderly patients with high-risk myelodysplastic syndrome. Leukemia 11:1±5 Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN (1993) JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74:227±236 Wyllie AH (1985) The biology of cell death in tumours. Anticancer Res 5:131±136 Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251±306 Yagasaki F, Jinnai I, Yoshida S, Yokoyama Y, Matsuda A, Kusumoto S, Kobayashi H, Terasaki H, Ohyashiki K, Asou N, Murohashi I, Bessho M, Hirashima K (1999) Fusion of TEL/ETV6 to a novel ACS2 in myelodysplastic syndrome and acute myelogenous leukemia with t(5;12)(q31;p13). Genes Chromosomes Cancer 26:192±202 Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129±1132 Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T, Sonoda Y, Abe T, Kahsima K, Matsuo Y, Naoe T (1997) Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia 11:1605±1609 Yoneda-Kato N, Look AT, Kirstein MN, Valentine MB, Raimondi SC, Cohen KJ, Carroll AJ, Morris SW (1996) The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene 12:265±275 Zang DY, Goodwin RG, Loken MR, Bryant E, Deeg HJ (2001) Expression of tumor necrosis factor-related apoptosis-inducing ligand, Apo2L, and its receptors in myelodysplastic syndrome: effects on in vitro hemopoiesis. Blood 98:3058±3065 Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14±3-3 not BCL-X(L). Cell 87:619±628

of Myelodysplastic Syndromes Torsten Haferlach, Wolfgang Kern

Contents 5.1 Introduction . . . . . . . . . . . . . . . . . . . . 5.2 Diagnostic Procedures Needed for Staging and Classification . . . . . . . . . 5.2.1 Sample Collection and Preanalytic Procedures . . . . . . . . . . . . . . . . . 5.2.1.1 Cytomorphology and Cytochemistry . . . . . . . . 5.2.1.2 Multiparameter Flow Cytometry by FACS . . . . 5.2.1.3 Cytogenetics . . . . . . . . .

40

.

40

.

40

.

41

. .

42 42

5.3 Diagnostic Criteria . . . . . . . . . . . . . . . 5.3.1 Cytomorphology and Cytochemistry 5.3.1.1 Dysplasia in MDS: The FAB Criteria (1982) . . . 5.3.1.2 Dysplasia as Defined for AML by Goasguen et al. (1992) (also WHO 2001) . . 5.3.1.2.1 Criteria for Dysgranulopoiesis 5.3.1.2.2 Criteria for Dyserythropoiesis 5.3.1.2.3 Criteria for Dysmegakaryopoiesis . . . . 5.3.1.3 Ringed Sideroblasts According to FAB and to WHO Criteria . . . . . . . . . . . . . . 5.3.1.3.1 FAB Criteria . . . . . . . . . . . 5.3.1.3.2 WHO Criteria . . . . . . . . . . 5.3.1.4 Definition of Chronic Myelomonocytic Leukemia (CMML) . . . . . . . . . . . . . .

5.3.2

42 43 43 43 43 44 44 44 44 44 44

5.3.3 5.3.4 5.3.5

5.3.1.4.1 CMML: Proliferative and Non-proliferative Subtype, According to FAB Criteria (1994) (Bennett et al. 1994) 5.3.1.4.2 WHO Criteria for CMML (Dysplastic Type) (Jaffe et al. 2001) . . . . . . . . . . . . . . . Multiparameter Flow Cytometry (MFC) . . . . . . . . . . . . . . . . . . . . . 5.3.2.1 Granulocytes . . . . . . . . . 5.3.2.2 Monocytes . . . . . . . . . . . 5.3.2.3 Erythrocytes . . . . . . . . . . 5.3.2.4 ªBlastsº . . . . . . . . . . . . . 5.3.2.5 Additional Flow Cytometric Findings . . . . . . . . . . . . . Cytogenetics and FISH . . . . . . . . . Molecular Methods . . . . . . . . . . . Other Laboratory Features . . . . . .

5.4 Overview of Classification and Staging Systems . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Proposed Classification and Scoring Systems . . . . . . . . . . . . . . . . . . . 5.4.2 FAB Classification . . . . . . . . . . . . . 5.4.3 International Prognostic Scoring System (IPSS) (1997) . . . . . . . . . . . 5.4.4 WHO Classification (2001) . . . . . . . 5.5 Future Developments in Classification and Staging . . . . . . . . . . . . . . . . . . . . 5.5.1 Molecular Genetics . . . . . . . . . . . . 5.5.2 Microarrays for Classification and Staging in MDS? . . . . . . . . . . . . . 5.6 Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

44 44 44 44 45 45 45 46 46 47 47 47 47 47 47 48 50 50 50 51 51

40

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

5.1 Introduction

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematological disorders that are usually diagnosed based on findings in peripheral blood, and especially the bone marrow. MDS is characterized by ineffective hematopoiesis, showing dysplastic features in at least one lineage in the bone marrow. With the use of standard classification systems, e.g., the French-American-British (FAB) classification (Bennett et al. 1982), the new World Health Organization (WHO) proposal (Jaffe et al. 2001) or several other scoring systems such as the IPSS (Greenberg et al. 1997), the Dçsseldorf score (Aul et al. 1992) or the Bournemouth score (Mufti et al. 1985), staging and classification of MDS are readily achieved. However, various problems surface in daily routine. ªStandardº classification systems and problems will be discussed below. Some of the important issues include: 4 How to diagnose early stages of MDS and discriminate these from other non-malignant disorders 4 How to measure and reproducibly assess dysplasia, the hallmark of MDS staging and classification 4 How to reproducibly determine the proportion of blasts, an essential parameter for MDS classification and treatment decisions 4 How to incorporate biological markers, e.g., cytogenetics and molecular alterations, into prognostic staging and scoring systems 4 Researching other techniques to support or enhance our knowledge, and integrating those findings into staging and classification in the future 4 Determining whether MDS is different from AML and if current classification systems propagate artificial distinctions by adhering to rigid definitions Clearly, more than 20 years after development of the FAB classification many questions remain or have arisen since.

5.2 Diagnostic Procedures Needed

for Staging and Classification

During the past 20 years the diagnosis, classification, and staging of MDS have evolved from relying on cytomorphology alone to a comprehensive array of different methods that have improved our ability to establish the diagnosis and to arrive at treatment decisions. For state

of the art staging and classification an algorithm that combines cytomorphology and cytochemistry with immunophenotyping, accompanied by cytogenetics and molecular genetic methods has to be established in a laboratory setting (Haferlach and Schoch 2002). Generally, we start with peripheral blood smears and bone marrow cytomorphology, cytochemistry, and iron stains. Recent data suggest that multiparameter flow cytometry (FACS) should be included in the diagnostic work-up for MDS (Stetler-Stevenson et al. 2001; Wells et al. 2003). In addition, metaphase cytogenetics should be obtained in every case in which MDS is suspected. Where possible, the latter should be accompanied by FISH or 24-color FISH to confirm aberrant findings observed in metaphase karyotyping. As abnormalities of chromosomes 5 and 7, loss of a Y chromosome, or a complex aberrant karyotype play an important role in MDS classification according to IPSS (Greenberg et al. 1997) and WHO (Jaffe et al. 2001) (see below), these determinations are mandatory.

5.2.1 Sample Collection and Preanalytic

Procedures

Several prerequisites must be fulfilled for reproducible results: 4 Different methods rely on different sources of biologic materials. For example, cytomorphology is impaired by heparin, and good metaphase spreads cannot be expected if EDTA was added to the sample. 4 In all cases with cytopenia or suspected MDS blood and bone marrow samples should be obtained at the same time (Ludwig et al. 2005). 4 A trephine biopsy may be necessary, especially in cases with very hypocellular or inaspirable bone marrow (punctio sicca) and peripheral cytopenia (Cheson et al. 2003). In these circumstances peripheral blood should be analyzed, but smears for cytomorphology can also be produced from trephine cylinders. 4 Similarly, for cytogenetics (in case of a punctio sicca) a metaphase analysis can be done after culturing the trephine biopsy in appropriate medium and processing the medium (plus cells) for karyotyping. A comprehensive investigation at diagnosis requires 3± 5 ml EDTA anticoagulated bone marrow, 10 ml periph-

a

5.2 ´ Diagnostic Procedures Needed for Staging and Classification

eral blood in EDTA, 5±10 ml heparinized bone marrow, and 10±20 ml heparinized peripheral blood. Investigations can be performed with fewer cells; however, one should not jeopardize a comprehensive diagnostic workup by too limited a biologic specimen. The material should reach the laboratory within 24 h. It should be shipped at room temperature without adding cool packs or dry ice. With these precautions, a successful investigation is possible in more than 90% of cases, including metaphase cytogenetics (Bennett 2003). Similarly, measurements of protein expression by FACS or gene expression by PCR or microarrays are possible on these samples (Haferlach et al. 2003; Kern et al. 2003 b; Kohlmann et al. 2003; Schoch et al. 2002 a). This means, of course, that a central reference laboratory has to be available on a daily basis for optimal service (Haferlach and Schoch 2002).

41

Fig. 5.2. Pappenheim staining (´ 630) of a bone marrow smear. Typical aspects of a 5q± syndrome with megakaryocytes with round, non-segmented nuclei and relatively mature cytoplasm. No blasts are present

5.2.1.1 Cytomorphology and Cytochemistry For cytomorphology and cytochemistry at least five peripheral blood smears and five bone marrow smears should be available. After they have been air-dried without any further fixation, Pappenheim or May Grçnwald Giemsa (MGG) staining should be performed and accompanied by myeloperoxidase (MPO) (Figs. 5.1, 5.2 and 5.3) and non-specific esterase (NSE, Fig. 5.4) stainings in all cases (Cheson et al. 2003; Læffler et al. 2005; Theml 2004). These stains are necessary to satisfy the

Fig. 5.3. Myeloperoxidase reaction (MPO, ´ 630) on a bone marrow smear from a patient with RAEB-2. Most PMNs stain negative, demonstrating an MPO deficiency as a dysplastic feature in granulopoiesis

Fig. 5.1. Pappenheim staining (´ 630) of a bone marrow smear. In some cases the morphology is difficult and can lead to the diagnosis of MDS RAEB-2, CMML, or even AML M4. This difficulty underscores the need for clear definitions in classification and staging in MDS

cytomorphological needs: determination of percentage of blasts, degree of dysplasia in all three cell lineages, and myeloperoxidase deficiency in the NSE negative and positive cell compartments. In addition, an iron stain is essential for staging and classification in MDS (Fig. 5.5). Stainings such as periodic acid±Schiff reaction (PAS), acid phosphatase, or chloro-acetate esterase (CE) stains do not add important information (Læffler et al. 2005; Ludwig et al. 2005). Exceptions may be special cases, for instance the demonstration of glycogen in the erythroid lineage (PAS), or the use of CE in histological

42

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

Fig. 5.4. Non-specific esterase (NSE, ´ 630) on a bone marrow smear from a patient with CMML. The number of abnormal monocytes is high and would be underestimated if only MGG stains were used

viability of cells after 24 h. Whenever possible, bone marrow (rather than blood) should be analyzed. Similar to the cytomorphologic examination, MFC analysis includes separate evaluation of all cell lines. Therefore, it is essential to take advantage of CD45-gating, which allows for clear separation of granulocytes, monocytes, lymphocytes, blasts, and erythrocytes (Borowitz et al. 1993). Since the mainstay of flow cytometric evaluation of MDS samples is the detection of the aberrant expression of distinct antigens, which also includes the relation of the expression of different antigens to each other, MFC should use at least 4- or 5-fold staining. Care must be taken to analyze a sufficiently large number of events to allow for the assessment of aberrant antigen expression even in small subpopulations. Thus, to characterize a subpopulation comprising 4% of all cells, a total of at least 20,000 events should be analyzed. Furthermore, it is essential to compare flow cytometric findings in cases with suspected MDS with normal bone marrow controls as well as to non-malignant diagnoses sharing single clinical findings of MDS, i.e., isolated cytopenias (Wells et al. 2003).

5.2.1.3 Cytogenetics

Fig. 5.5. Iron staining (´ 630) in a case of RARS

sections where this stain represents the best method for the demonstration of neutrophilic/granulocytic lineage.

5.2.1.2 Multiparameter Flow Cytometry by FACS For multiparameter flow cytometry (MFC) cells can be processed after lysis or after Ficoll Hypaque gradient separation (Kern et al. 2003 a, b). Many laboratories use Ficoll in preparation for MFC if the workflow uses the same sample for MFC and molecular techniques. In general, heparin is preferred, in particular if samples are sent by overnight express, since EDTA reduces the

The importance of cytogenetics is addressed in detail in Chapter 6. A cytogenetic analysis in an essential component of the workup in each case of suspected or proven MDS. For fluorescence in situ hybridization (FISH) metaphases as well as interphase nuclei from cytomorphological smears of bone marrow or peripheral blood can be used. Probes for interphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24-color FISH, or comparative genomic hybridization (CGH) are usually hybridized in an overnight procedure and are available for analysis 24 h after the sample reached the laboratory (Schoch et al. 2002 b, 2003). Thus, a suspected 5q± syndrome can generally be proven in 24 h after biopsy using interphase FISH.

5.3 Diagnostic Criteria

The classification of MDS should follow the WHO proposal (Jaffe et al. 2001) published in 2001 and the IPSS (Greenberg et al. 1997), and will only rarely rely on the FAB or other proposed classification systems (Aul et al.

a

5.3 ´ Diagnostic Criteria

43

Table 5.1. Classifications and scoring systems for MDS Year

Parameters for subclassification Dys

Age

% blasts

Auer rods

RS

Hb

Plt

LDH

CG

CP

Subgroups

FAB (Bennett et al. 1982)

1982

+

0

+

+

+

(+) *

0

0

0

0

5

Mufti a (Mufti et al. 1985)

1985

0

0

+

0

0

+

+

0

0

(+) **

3

Sanz (Sanz et al. 1989)

1989

0

+

+

0

0

0

+

0

0

0

3

b

Aul (Aul et al. 1992)

1992

+

0

+

0

+

+

+

+

0

+

3

Morel c (Morel et al. 1993)

1993

0

0

+

0

0

0

+

0

+

0

3

IPSS (Greenberg et al. 1997)

1997

0

0

+

0

0

+

+

0

+

+

4

WHO (2001)

2001

+

0

+

+

+

+

+

0

+

+

10

Dys dysplasia, RS ringed sideroblasts, Plt platelets, CG cytogenetics, CP cytopenias (0±3), + needed for classification, 0 not taken into account for classification * in RA, ** neutrophils a

Mufti = Bournemouth score

b

Aul = Dçsseldorf score

c

Morel = Lille score

1992; Bennett et al. 1982; Mufti et al. 1985). Furthermore, some specific classification systems (see Table 5.1) (Morel et al. 1993) and specific terminological aspects for cytogenetics and FISH as defined by the ISCN nomenclature (International Standing Committee on Human Cytogenetic Nomenclature 1995) have to be considered.

5.3.1 Cytomorphology and Cytochemistry

The first step for the diagnosis of MDS is cytomorphology and cytochemistry. It is quick and cheap, and the results allow drawing up an optimal workflow for other, much more labor-intensive and expensive techniques. Cytomorphology can also discriminate MDS from AML or other diseases in most circumstances (Haferlach and Schoch 2002; Læffler et al. 2005; Ludwig et al. 2005). In most cases of MDS the classification based on cytomorphology is already sufficient to arrive at a treatment decision. Some aspects of cytomorphology deserve to be detailed here.

5.3.1.1 Dysplasia in MDS: The FAB Criteria (1982) In MDS, dysplasia is assigned to the respective lineage if at least 10% of cells show the following findings (note: in AML the threshold is 50%!) (Bennett et al. 1982):

4 Granulocytic dysplasia (Dys G): PMN agranular or hypogranular, basophilia in mature cells, PelgerHuŸt anomaly or hypersegmentation, MPO deficiency (Fig. 5.3) 4 Erythroid dysplasia (Dys E): ringed sideroblasts (> 15%) (Fig. 5.5), multinuclearity, nuclear fragments, two or more nuclear lobes, irregular cytoplasm 4 Megakaryocytic dysplasia (Dys M): micromegakaryocytes, large mononuclear forms, multiple small nuclei 4 For a diagnosis of MDS two or three lineages have to be dysplastic

5.3.1.2 Dysplasia as Defined for AML by Goasguen et al. (1992) (also WHO 2001) The threshold for MDS is 10% or more of cells demonstrating one or more features as outlined below; the threshold is 50% in AML (Goasguen et al. 1992). At least 500 bone marrow cells and 200 cells in the peripheral blood should be analyzed according to WHO standards (Jaffe et al. 2001).

5.3.1.2.1 Criteria for Dysgranulopoiesis 4 ³ 10% of PMN (at least 10) are agranular or hypogranular, or

44

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

4 Pseudo Pelger-HuŸt anomaly, or 4 Peroxidase deficiency 4 100 cells should be analyzed

5.3.1.2.2 Criteria for Dyserythropoiesis Of at least 25 erythroid precursors, ³ 10% show one of the following abnormalities: 4 Karyorrhexis 4 Megaloblastoid aspects 4 Multinuclearity 4 Nuclear fragments 5.3.1.2.3 Criteria for Dysmegakaryopoiesis Of megakaryocytes, ³ 10% show one of the following: 4 Micromegakaryocytes 4 Multiple separated nuclei 4 Large mononuclear forms 5.3.1.3 Ringed Sideroblasts According to FAB and to WHO Criteria As the presence of ringed sideroblasts defines specific subgroups of MDS according to both FAB (Bennett et al. 1982) and WHO (Jaffe et al. 2001) classifications, specific criteria for ringed sideroblasts have been established as follows.

5.3.1.3.1 FAB Criteria 4 ªRinged sideroblasts accounting for more than 15% of all nucleated cells in the bone marrowº 4 ªRinged sideroblasts have several siderotic granules on the nuclear membrane arranged in a collar around the nucleusº 5.3.1.3.2 WHO Criteria 4 In refractory anemia with ringed sideroblasts (RARS) ªringed sideroblasts account for more than 15% of red cell precursors in the bone marrowº 4 Ringed sideroblasts have ten or more iron granules encircling one third or more of the nucleus

5.3.1.4.1 CMML: Proliferative and Non-proliferative Subtype, According to FAB Criteria (1994) (Bennett et al. 1994) 4 Monocytosis of greater than 1,000/ll in blood (in both subgroups) 4 Proliferative subtype: WBC > 13,000/ll = myeloproliferative 4 Non-proliferative subtype: WBC £ 13,000/ll = MDS 5.3.1.4.2 WHO Criteria for CMML (Dysplastic Type) (Jaffe et al. 2001) 4 In peripheral blood monocytes are always > 1 ´ 109/l 4 Dysgranulopoiesis > 10% 4 Blasts and promonocytes < 20% of WBC in peripheral blood 4 Blasts and promonocytes < 20% in BM 4 NSE is strongly recommended for BM examination As outlined above, classification and staging on the basis of cytomorphology should rely on WHO criteria for international comparability of study results and consistent treatment decision making. 5.3.2 Multiparameter Flow Cytometry (MFC)

While cytomorphology has been the backbone for establishing the diagnosis of MDS for many decades, MFC has been used only in recent years to characterize MDS and to contribute to the diagnosis (Ogata et al. 2002; Stetler-Stevenson et al. 2001; Wells et al. 2003). Most importantly, MFC analysis should not be limited to the quantification and characterization of bone marrow blasts but must include the assessment of each subpopulation present in the bone marrow. Aberrant antigen expression patterns have been described specifically for each subpopulation and it is anticipated that even more details will be defined in the near future. The use of flow cytometric findings has not been limited to diagnostic purposes but includes also the potential to estimate the prognosis of a patient. The following sections provide published data on aberrant findings in the respective subpopulations (Table 5.2).

5.3.1.4 Definition of Chronic Myelomonocytic Leukemia (CMML)

5.3.2.1 Granulocytes

Since CMML is classified as MDS in only the FAB classification and not in the WHO classification, the defining criteria are given here:

The most prominent flow cytometric findings in granulocytes include the reduced side-scatter (SSC) signal (corresponding to hypogranularity; Fig. 5.6) and aber-

a

5.3 ´ Diagnostic Criteria

Table 5.2. Dysplastic features in MDS as analyzed by flow cytometry Blasts

Granulocytes

Monocytes

Erythrocytes

Homogenous expression of antigens

±

±

±

±

Abnormal CD11b/ CD16

Abnormal CD11b/ HLA-DR

±

±

Abnormal CD13/CD16

±

±

±

CD10±

±

±

CD11b+

CD11b±

CD11b±

±

CD13±

CD13±

CD13±

±

±

±

CD14±

±

CD15+

±

±

±

±

±

CD16-

±

CD19+

CD19+

CD19+

±

-

CD2+

CD2+

±

CD33±

CD33±

CD33±

±

CD34±

CD34+

CD34+

±

CD36+

CD36+

±

±

±

CD45±/(+)

CD45±/(+)

±

CD5+

CD5+

CD5+

±

CD56+

CD56+

CD56+

±

45

rant expression patterns of CD13 and CD16. In normal bone marrow granulocytes there is a typical convex distribution of granulocytes ranging from CD13+CD16± cells to CD13dimCD16dim and CD13+CD16+ cells. Aberrancies in MDS include a concave distribution with a lack of CD13dim cells, as well as a reduced expression of either CD13 or CD16. Other aberrancies are the lack or the reduced expression of the normally expressed antigens CD11b, CD33, CD64, CD66, and CD10. Antigens found aberrantly expressed on granulocytes in MDS include HLA-DR+ and CD34 as well as the lymphoid markers CD56+, CD2+, CD5+, CD7+, and CD19+. Furthermore, a reduced expression of CD45 may be present which can be easily detected in the CD45-SSCplot.

5.3.2.2 Monocytes Monocytes are readily identified in the CD45-SSC-plot since both CD45 and SSC signals are aberrant only in rare cases. Typical changes include an aberrant CD13CD16-expression, a lack of expression of CD11b, CD33, and CD14 as well as the aberrant expression of CD34, CD56, CD2, CD5, CD7, and CD19 (Fig. 5.7). Furthermore, HLA-DR may be expressed homogeneously, which is not normally seen in monocytes.

5.3.2.3 Erythrocytes

CD64+

CD64±

±

±

±

CD66(+)

CD66(+)

±

CD7+

CD7+

CD7+

±

±

CD71+

±

CD71±/ homogeneous

HLA-DR±

HLA-DR+

HLA-DR+ homogenous

±

Erythrocytes can be identified based on their low CD45SSC-signal and their expression of CD235a. The most prominent flow cytometric finding in erythrocytes in MDS is the aberrant expression of CD71 (Fig. 5.8). Thus, the vast majority of cells may be CD71-negative, which is seen only in a minority of normal erythrocytes. Furthermore, in CD71-positive cells the expression of CD71 frequently is homogeneous and strong, contrasting the heterogeneous expression ranging from strong to dim, which is found in normal erythrocytes.

Intensity of myeloid antigens

Intensity of myeloid antigens

±

±

5.3.2.4 ªBlastsº

SSC increased

SSC reduced

±

±

Based on CD45-SSC-gating bone marrow myeloblasts can be identified and accurately quantified. Furthermore, flow cytometrically detectable aberrancies can occur in the form of CD11b, which is normally expressed on mature cells only, and of the lymphoid and natural killer (NK)-cell markers CD19, CD7, CD5, and CD56,

46

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

Fig. 5.6 A, B. Flow cytometric findings in normal (A) and dysplastic granulocytes (B) in MDS marrow. Granulocytes are shown in red. Normal granulocytes have a strong SSC (side scatter) signal due to their granulation while hypogranulated granulocytes in MDS show reduced SSC signals

Fig. 5.7 A, B. Flow cytometric analysis of normal (A) and dysplastic monocytes (B) in MDS marrow. Bone marrow monocytes, shown in red, d show differences in CD56 expression. While normal monocytes are CD56-negative, dysplastic monocytes may display dim or strong CD56 signals

Fig. 5.8 A, B. Flow cytometric analysis of normal (A) and dysplastic erythrocytes (B) in MDS marrow. Normal erythrocytes show heterogeneous expression of CD71 with some cells being negative, while dysplastic erythrocytes may be CD71-negative with positive cells showing a homogeneous expression pattern

which are not normally expressed on myeloid blasts. Furthermore, the expression of HLA-DR which is regularly found on blasts may be absent, and there may be an abnormal intense expression of myeloid markers, although their expression per se is found in normal myeloid blasts.

5.3.2.5 Additional Flow Cytometric Findings In addition to the findings in the respective subpopulations described above, an abnormal ratio of myeloid cells to lymphocytes may correlate with abnormal myelopoiesis (Wells et al. 2003), and this parameter should,

therefore, also be analyzed in the flow cytometric workup of MDS.

5.3.3 Cytogenetics and FISH (See Chapter 6)

The first hierarchical step in the WHO classification of AML is based on cytogenetics and includes the balanced translocations t(15;17), inv(16), t(8;21), and 11q23 aberrations independently of the percentage of bone marrow myeloblasts. This is a very important step for a more biologically based approach in classification, and clearly separates these subentities from MDS. The same is true

a

5.4 ´ Overview of Classification and Staging Systems

when discriminating MDS with 5q± from all other MDS subtypes. The 5q± syndrome shows, by definition, less than 5% blasts and has been recognized as a unique entity for several reasons: age, gender, platelet counts, and prognosis (Van Den Berghe and Michaux 1997). As this entity is being discussed in more detail in another chapter, we will not further address the underlying biology and clinical aspects here. Other cytogenetic findings confer a significant prognostic impact and, thus, are needed to allow a risk-adapted treatment selection, although they are not yet included in diagnostic classification systems in MDS.

47

4 Platelet level 4 LDH Thresholds should be those defined in the IPSS. 5.4 Overview of Classification

and Staging Systems

Since the pivotal classification of MDS by the FAB group (Bennett et al. 1982) in comparison to acute leukemias (Bennett et al. 1976), several other staging and classification systems have been proposed for MDS. Table 5.1 summarizes features of some published staging systems.

5.3.4 Molecular Methods

Each method revealing new insights into the biologic background of MDS may generate disease-specific markers important for the management of the disease. Several new aspects have been added to the characterization of MDS by way of molecular biology. We will discuss this only briefly, as these findings have not yet found their way into new classification systems, but hopefully will in the near future. Abnormal gene expression includes: NRAS (found altered in 10% in MDS in our laboratory), MLL-PTD (5%), FLT3-ITD (2.5%), FLT3-TKD (1%), and CKIT (1%). Preliminary results point to a correlation between the mutations of these genes and the stage of MDS as well as the subtype according to FAB and WHO classification. Further studies are also warranted focusing an AML1 and CEBPA mutations, p15 methylation, and WT1 expression. The evaluation of these data should include correlations with findings of gene expression analyses using microarrays (see below). Some preliminary data have been published, using gene expression profiling in MDS that may alter current classifications and may allow for a refinement of treatment decisions.

5.3.5 Other Laboratory Features

FAB and WHO staging can be performed on the basis of cytomorphology alone, and its combination with cytogenetics, respectively. However, the IPSS grading and some other staging systems (see below) require a (limited) number of additional parameters: 4 Cytopenias (0±3) in general 4 Hemoglobin level

5.4.1 Proposed Classification

and Scoring Systems

Some scoring systems require a diagnosis of MDS (based on FAB definitions) before a score can be applied for the purpose of prognostication. Table 5.1 gives an overview of the parameters used in these staging and classification systems that may be helpful for future discussions. 5.4.2 FAB Classification

The definitions for FAB staging have been described. The different categories as defined by FAB in 1982 (Bennett et al. 1982) are summarized in Table 5.3. The FAB classification has enhanced our understanding of MDS more than any other classification so far, and has allowed for comparison of results with respect to epidemiology, treatment approaches, and prognostication. For more than 25 years, the FAB classification has served worldwide, and its importance cannot be overestimated. However, the inter-observer reproducibility is less than perfect (Argyle et al. 1989; Bennett and Begg 1981; Bennett et al. 1996; Browman et al. 1986; Castoldi et al. 1993; Chudgar and Khanduri 1992; Ost et al. 1983; Sales et al. 1991; Sultan et al. 1981). 5.4.3 International Prognostic Scoring System

(IPSS) (1997)

For the IPSS proposal, an international panel of experts combined their knowledge in analyzing 816 patient

48

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

Table 5.3. FAB classification (Bennett et al. 1982) Subtype

Blasts in PB

Blasts in BM

Ringed sideroblasts

Monocytes in PB

Auer rods

Refractory anemia (RA)

< 1%

< 5%

< 15% in BM

< 1,000/ll

No

Refractory anemia with ringed sideroblasts (RARS)

< 1%

< 5%

>15% in BM

< 1,000/ll

No

Refractory anemia with excess of blasts (RAEB)

< 5%

5±20%

< 15% in BM

< 1,000/ll

No

Refractory anemia with excess of blasts in transformation (RAEBt)

> 5%

20±30%

< 15% in BM

< 1,000/ll

Possible

Chronic myelomonocytic leukemia (CMML)

< 5%

0±20%

< 15% in BM

> 1,000/ll

no

4 Intermediate-2: score = 1.5±2 4 High: score ³ 2.5

Table 5.4. IPSS scores (Greenberg et al. 1997) Score value l Prognostic variable

0

0.5

1.0

1.5

2.0

BM blasts (%)

<5

5±10

±

11±20

21±30

Cytogenetics *

Good

Intermediate

Poor

±

±

Cytopenias

0/1

2/3

±

±

±

* good risk: normal, 5q±, 20q±, ±Y; poor risk: complex aberrant, ±7, 7q±; intermediate risk: all others

samples and outcome data (Greenberg et al. 1997). Multivariate analysis led to a score for prognostication, including the risk for progression to AML. The following parameters are included in the final score: 4 Cytopenias (0±3, e.g., Hb < 10 g/dl, neutrophils < 1,500/ll, platelets < 100,000/ll) 4 Bone marrow blasts (< 5%, 5±10%, 11±20%, 21±30%) 4 Cytogenetics (good risk: normal, 5q±, 20q±, ±Y; intermediate risk: neither good nor poor; poor risk: complex, ±7, 7q±) Based on these three parameters the IPSS score has been established as shown in Table 5.4. Application of this score allows for the separation of patients into four risk categories with significantly differing prognoses: 4 Low: score = 0 4 Intermediate-1: score = 0.5±1

The IPSS represents a major progress and provides, better than any other system, a basis to estimate prognosis and to guide treatment, which may range from a watchand-wait approach to allogeneic stem cell transplantation from an unrelated donor.

5.4.4 WHO Classification (2001)

The classification proposed by the WHO was published in 2001 (Jaffe et al. 2001). It should be used in staging and classification of MDS in future studies. It also can be applied in daily routine but should be accompanied by the IPSS score for clinical decision-making and prognostication. This is particularly important since the WHO system considers cytogenetics only for one category, the 5q± syndrome. As cytogenetics are very informative for prognostication in MDS (see IPSS score), it is mandatory to include the results of this text in staging and classification. As in the FAB classification, the WHO classification is mostly based on the percentage of myeloblasts in the bone marrow and peripheral blood, the type and degree of dysplasia, and the presence of ringed sideroblasts. Some new categories were introduced, which must be validated with regard to their unique biologic and clinical pattern in future studies. Further, some established categories such as refractory anemia with excess of blasts (RAEB) or CMML were subdivided with respect to the percentage of blasts in the bone marrow. An im-

a

5.4 ´ Overview of Classification and Staging Systems

portant and major change in comparison to the FAB classification has been the elimination of the former FAB-category RAEB in transformation (RAEBt), as all patients with 20% myeloblasts or more are to be classified as AML in the WHO system. RAEBt and AML appear to be very similar, and ªlumpingº seems a reasonable approach, although this has not been without controversy. The classification of MDS, especially with less than 5% bone marrow blasts, is the most difficult task for cytomorphologists in hematology today. Furthermore, the threshold for the definition of dysplasia (only 10%) is very different from that used in AML (50%). Thus, the MDS categories 5q± syndrome, refractory anemia (RA), refractory cytopenia with multilineage dysplasia (RCMD), and RARS according to the WHO classification are very difficult to diagnose unequivocally. In these cases it is recommended that the diagnosis be confirmed in a reference laboratory. In many cases with borderline features a repeated bone marrow examination should be performed after an interval of 2± 3 months before the final diagnosis of MDS is made. In all cases additional methods such as cytogenetics, FISH, and even MFC or molecular markers may support these sub-categories if they detect specific and unquestionable markers for MDS or can exclude this diagnosis. Several differential diagnoses have to be considered: nutritional or toxic factors such as alcohol; drugs, in-

49

cluding chemotherapy; arsenic intoxication; exposure to heavy metals; treatment of HIV; or virus infections. Other factors that may mimic MDS can be the administration of cytokines, parvovirus B19 infection, and especially deficiency in vitamin E, B12, or folic acid. Even congenital dyserythropoietic anemia, hairy cell leukemia, or paroxysmal nocturnal hemoglobinuria (PNH) have been misdiagnosed as MDS. This must lead to the conclusion that the classification of MDS is still difficult, needs highly experienced cytomorphologists and cytogeneticists, and should rely on the combination of several comprehensive techniques. In equivocal cases at least two different examinations at different time points and by different observers should be performed including a close follow-up of the patient's history. The percentage of blasts remains the major parameter in MDS classification, together with the degree of dysplastic cells in the bone marrow. This may be even more important now in the WHO proposal as RAEB has been subgrouped into RAEB-1 and RAEB-2 and CMML into CMML-1 and CMML-2 on the basis of bone marrow blasts. Conceivably, the percentage of blasts can in the future be replaced by other parameters and findings such as cytogenetics, FISH or MFC. Additional validation is required, and prospective, controlled studies, analyzed in a multivariate approach, should be conducted. Further details of the WHO classification for MDS are given in Table 5.5.

Table 5.5. The WHO classification proposal (2001) Entity

Dysplasia

Blasts in PB

Blasts in BM

5q± syndrome

mostly DysE

< 5%

< 5%

Ringed sideroblasts in BM < 15%

Cytogenetics

RA

mostly DysE

< 1%

< 5%

< 15%

various

RARS

mostly DysE

0%

< 5%

³ 15%

various

5q± only

RCMD

2±3 lineages

rare

< 5%

< 15%

various

RCMS-RS

2±3 lineages

rare

< 5%

³ 15%

various

RAEB-1

1±3 lineages

< 5%

5±9%

< 15%

various

RAEB-2

1±3 lineages

5±19%

10±19%

< 15%

various

CMML-1

1±3 lineages

< 5%

< 10%

< 15%

various

CMML-2

1±3 lineages

5±19%

10±19%

< 15%

various

MDS-U

1 lineage

0%

< 5%

< 15%

various

RA refractory anemia, RARS refractory anemia with ringed sideroblasts, RCMD refractory dysplasia with multilineage dysplasia, RCMS-RS refractory dysplasia with multilineage dysplasia and ringed sideroblasts, RAEB-1 refractory anemia with excess of blasts-1, RAEB-2 refractory anemia with excess of blasts-2, CMML-1 chronic myelomonocytic leukemia-1, CMML-2 chronic myelomonocytic leukemia-2, MDS-U MDS unclassified

50

Chapter 5 ´ Classification and Staging of Myelodysplastic Syndromes

The use of the WHO system in MDS in comparison to FAB and IPSS has been addressed in several reports over the last 3 years. In particular, in MDS cases with less than 5% bone marrow blasts, i.e., RA, RCMD, and 5q± syndrome, the WHO classification has been shown to provide significant prognostic information. In a series of 103 patients the median survival for RCMD cases was 27 months compared with 53 months and more than 102 months in cases with a 5q± syndrome and RA, respectively (Cermak et al. 2003). Importantly, RA and RCMD comprise similarly sized subgroups, making these differences in outcome clinically relevant. Furthermore, it has been reported that, within the subgroups of patients with less than 5% bone marrow blasts, the number of cell lineages involved has a prognostic impact. In a series of 64 patients, those with unilineage dysplasia had a significantly better survival than patients with multilineage dysplasia (median not reached vs. 29 months) (Howe et al. 2004). In a large series of 1,243 patients with MDS, similar differences were found between RA and RCMD with regard to median survival (69 vs. 33 months) (Germing et al. 2000). While in MDS subgroups with higher blast counts the similarities of the disease to AML may determine the clinical course, these data clearly suggest that in cases with low blast counts the WHO classification is an improvement over the former classifications and their clinical utility.

5.5 Future Developments in Classification

and Staging

Staging and classification in MDS are still a work in progress but are likely to be further improved in the near future based on new methods and insights. We expect an important step forward by taking advantage of MFC and molecular findings, in particular, gene expression profiling. How may these advances be accomplished, and how will they look?

5.5.1 Molecular Genetics

Beyond the scope of dysplastic features, blast percentage, and cytogenetic results, several new molecular markers have entered the scene but need to be assessed in regards to their impact compared with standard parameters currently in use.

5.5.2 Microarrays for Classification and Staging

in MDS?

Microarray analyses allow the assessment of the whole genome in one approach and, therefore, are anticipated to add significant information to current standard diagnostics. The studies performed so far have focused on differences among MDS, AML, and normal bone marrow with regard to gene expression signatures in the blast population as well as with regard to the total bone marrow. Miyazato et al. (2001) separated bone marrow cells positive for CD133 and compared MDS and AML signatures to each other within these populations. Differences were found in the expression of genes coding for growth factors and proteins involved in the redox regulation but also genes encoding membrane proteins. The Delta-like (Dlk) gene was expressed in the majority of 22 MDS cases but only rarely in 33 AML cases. These results were confirmed by PCR analyses; they may help in establishing a diagnostic signature for MDS but also point to differences in the pathogenesis between the two diseases. Hofmann et al. (2002) analyzed gene expression signatures in bone marrow samples from patients with low-risk (n = 7) and high-risk (n = 4) MDS as well as from healthy subjects (n = 4). Focusing on cells selected for CD34 positivity, they found differentially expressed genes, based on nine of which they discriminated all three groups. These findings were confirmed in additional samples with MDS (n = 8). The differentially expressed genes were also confirmed by PCR analysis. Besides these diagnostically relevant findings, based on their gene expression profiling analyses the authors suggested that low-risk MDS cases are characterized by the lack of defensive proteins, resulting in an increased susceptibility to cell damage while high-risk cases feature an expression of proliferation-associated genes. Analyses from our group (Haferlach et al. 2004) were guided by the fact that MDS and AML are distinguished by percentages of blasts in the bone marrow, the thresholds for which, however, are arbitrarily chosen and result only in a limited reproducibility in interlaboratory testings. Thus, other parameters have been assessed to discriminate these entities with respect to diagnosis and prognosis. In particular, identical karyotype aberrations have been observed between MDS and AML in the majority of cases, and these have a higher prognostic impact than blast percentages. We applied

a

References

gene expression profiling (HG-U133A+B microarrays, Affymetrix) in 70 MDS and 238 AML cases. In accordance with the WHO classification we excluded cases with balanced translocations, i.e., t(8;21), t(15;17), inv(16), or t(11q23), which were classified as AML irrespective of BM blast percentage. First, we aimed at identifying genes the expression of which correlated with the blast count. Among the top 50 genes the only one having a higher expression in cases with high blast counts was the FLT3 gene, while 12 other genes with a higher expression in cases with lower blast counts were identified (ARG1, CEACA1, LCN2, MMP9, STOM). Most of these latter genes are known to be expressed in mature granulocytes and are involved in differentiation and apoptosis. In a second step, we performed class prediction using support vector machines (SVM) to separate MDS and AML according to blast percentages as defined in the WHO classification. Using SVM and 10fold cross validation, the overall prediction accuracy was only 80%. Specifically, 230 of 238 AML cases were correctly assigned to the AML group, while eight cases were classified as MDS RAEB-2. However, none of the RA, 5q± syndrome, and RAEB-1 cases were correctly assigned to their groups but were either classified as AML or RAEB-2. Furthermore, only 16 of 38 RAEB-2 cases were correctly predicted, while the 20 remaining cases were assigned to the AML group. Thus, no clear gene expression patterns were identified which correlated with AML and MDS subtypes as defined by the WHO classification. Taking into account the common genetic background of MDS and AML, both entities were categorized in a third step according to cytogenetics and classified based on their respective gene expression profiles. In order to assess the impact of the common genetic background, the largest cytogenetically defined subgroups were compared to each other, i.e., AML and MDS with normal karyotype and with complex aberrant karyotype, respectively. Intriguingly, a correct classification of AML or MDS was found in 91%, and, even more impressive, classification into the correct cytogenetic groups was achieved in 95%. Consequently, all cases were divided into the two groups, complex aberrant karyotype (n = 60) and other or no aberrations (n = 248) irrespective of AML or MDS. A classification into these groups also yielded an accuracy of 93%. Overall, our data, thus, suggest that the biology of MDS or AML correlates with cytogenetics, and less well with the percentages of marrow blasts. These results under-

51

line the need for a revision of the current MDS and AML classification.

5.6 Conclusions

MDS classification and staging is difficult. The IPSS and the new WHO classification should be applied in parallel. Since parameters such as dysplasia and percentage of blasts are somewhat difficult to reproduce, future criteria for the evaluation of MDS should employ new markers from cytogenetics, MFC, and molecular methods, including gene expression profiling. This approach is needed because several characteristics of MDS represent dynamic processes, and if our staging and classification systems are rigidly based on blasts, we may reduce rather than increase our chances of understanding the underlying biology and of classifying and treating patients better in the future.

References Argyle JC, Benjamin DR, Lampkin B, Hammond D (1989) Acute nonlymphocytic leukemias of childhood. Inter-observer variability and problems in the use of the FAB classification. Cancer 63:295±301 Aul C, Gattermann N, Heyll A, Germing U, Derigs G, Schneider W (1992) Primary myelodysplastic syndromes: analysis of prognostic factors in 235 patients and proposals for an improved scoring system. Leukemia 6:52±59 Bennett JM (2003) Morphologic dysplasia in acute myeloid leukemia: importance of granulocytic dysplasia. (Comment). J Clin Oncol 21:3004±3005 Bennett JM, Begg CB (1981) Eastern Cooperative Oncology Group study of the cytochemistry of adult acute myeloid leukemia by correlation of subtypes with response and survival. Cancer Res 41:4833±4837 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C (1976) Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol 33:451±458 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C, The French-American-British (FAB) Co-Operative Group (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189±199 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick H, Sultan C, Cox C (1994) The chronic myeloid leukaemias: guidelines for distinguishing chronic granulocytic, atypical chronic myeloid, and chronic myelomonocytic leukaemia. Proposals by the FrenchAmerican-British Cooperative Leukaemia Group. Br J Haematol 87:746±754 Bennett JM, Cassileth PA, Paietta E, Rowe JM, Wiernik PH (1996) Morphologic classification of acute myeloid leukemia: concordance

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among Eastern Cooperative Oncology Group investigators: a comment. Leukemia 10:13±65 Borowitz MJ, Guenther KL, Shults KE, Stelzer GT (1993) Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in threecolor analysis. Am J Clin Pathol 100:534±540 Browman GP, Neame PB, Soamboonsrup P (1986) The contribution of cytochemistry and immunophenotyping to the reproducibility of the FAB classification in acute leukemia. Blood 68:900±905 Castoldi GL, Liso V, Fenu S, Vegna ML, Mandelli F (1993) Reproducibility of the morphological diagnostic criteria for acute myeloid leukemia: the GIMEMA group experience. Ann Hematol 66:171±174 Cermak J, Michalova K, Brezinova J, Zemanova Z (2003) A prognostic impact of separation of refractory cytopenia with multilineage dysplasia and 5q- syndrome from refractory anemia in primary myelodysplastic syndrome. Leuk Res 27:221±229 Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH, Schiffer CA, Doehner H, Tallman MS, Lister TA, Lo-Coco F, Willemze R, Biondi A, Hiddemann W, Larson RA, Lowenberg B, Sanz MA, Head DR, Ohno R, Bloomfield CD, LoCocco F, International Working Group for Diagnosis SoRCTOaRSfTTiAML (2003) Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol 21:4642±4649 Chudgar U, Khanduri U (1992) Intraobserver and interobserver reproducibility of the FAB classification in acute leukaemia. Indian J Pathol Microbiol 35:229±236 Germing U, Gattermann N, Strupp C, Aivado M, Aul C (2000) Validation of the WHO proposals for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients. Leuk Res 24:983±992 Goasguen JE, Matsuo T, Cox C, Bennett JM (1992) Evaluation of the dysmyelopoiesis in 336 patients with de novo acute myeloid leukemia: major importance of dysgranulopoiesis for remission and survival. Leukemia 6:520±525 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes (published erratum appears in Blood 1998; 91:1100). Blood 89:2079±2088 Haferlach T, Schoch C (2002) Modern techniques in leukemia diagnosis (Review) [German]. Internist 43:1190±1196 Haferlach T, Kohlmann A, Kern W, Hiddemann W, Schnittger S, Schoch C (2003) Gene expression profiling as a tool for the diagnosis of acute leukemias (Review). Semin Hematol 40:281±295 Haferlach T, Kern W, Kohlmann A, Dugas M, Merk S, Hiddemann W, Schnittger S, Schoch C (2004) MDS and AML are closely related diseases: gene expression patterns reveal clear similarities with respect to karyotypes and are less related to the bone marrow blast percentages [Abstract]. Blood 104:137 a Hofmann WK, de Vos S, Komor M, Hoelzer D, Wachsman W, Koeffler HP (2002) Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow. Blood 100:3553±3560 Howe RB, Porwit-MacDonald A, Wanat R, Tehranchi R, Hellstrom-Lindberg E (2004) The WHO classification of MDS does make a difference. Blood 103:3265±3270

International Standing Committee on Human Cytogenetic Nomenclature (1995) Mitelman F (ed) An international system for human cytogenetic nomenclature (1995): recommendations of the International Standing Committee on Human Cytogenetic Nomenclature, Memphis, Tennessee, USA, October 913, 1994. Karger, Farmington, CT Jaffe ES, Harris NL, Stein H, Vardiman JW (ed) (2001) World Health Organization classification of tumours. Pathology & genetics: tumours of hematopoietic and lymphoid tissues. IARCPress, Washington, DC Kern W, Danhauser-Riedl S, Ratei R, Schnittger S, Schoch C, Kolb HJ, Ludwig WD, Hiddemann W, Haferlach T (2003 a) Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow. Haematologica 88:646±653 Kern W, Kohlmann A, Wuchter C, Schnittger S, Schoch C, Mergenthaler S, Ratei R, Ludwig WD, Hiddemann W, Haferlach T (2003 b) Correlation of protein expression and gene expression in acute leukemia. Cytometry 55B:29±36 Kohlmann A, Schoch C, Schnittger S, Dugas M, Hiddemann W, Kern W, Haferlach T (2003) Molecular characterization of acute leukemias by use of microarray technology. Genes Chromosomes Cancer 37:396±405 Læffler H, Rastetter J, Haferlach T (2005) Atlas of clinical hematology. Springer, Berlin, Heidelberg, New York Ludwig W-D, Haferlach T, Schoch C (2005) Classification of acute leukemias. In: Pui C-H (ed) Treatment of acute leukemias: new directions for clinical research. Humana Press, Totowa, NJ, pp 3±42 Miyazato A, Ueno S, Ohmine K, Ueda M, Yoshida K, Yamashita Y, Kaneko T, Mori M, Kirito K, Toshima M, Nakamura Y, Saito K, Kano Y, Furusawa S, Ozawa K, Mano H (2001) Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 98:422±427 Morel P, Hebbar M, Lai J-L, Duhamel A, Preudhomme C, Wattel E, Bauters F, Fenaux P (1993) Cytogenetic analysis has strong independent prognostic value in de novo myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 cases. Leukemia 7:1315±1323 Mufti GJ, Stevens JR, Oscier DG, Hamblin TJ, Machin D (1985) Myelodysplastic syndromes: a scoring system with prognostic significance. Br J Haematol 59:425±433 Ogata K, Nakamura K, Yokose N, Tamura H, Tachibana M, Taniguchi O, Iwakiri R, Hayashi T, Sakamaki H, Murai Y, Tohyama K, Tomoyasu S, Nonaka Y, Mori M, Dan K, Yoshida Y (2002) Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome. Blood 100:3887±3896 Ost A, Lagerlof B, Sundstrom C, Lindstrom P, Gyllenhammer H, Engstedt L, Skoog L (1983) A study of the reproducibility of the diagnostic criteria for acute leukaemia. Scand J Haematol 31:257± 266 Sales CV, Rojo MJ, Chavez SG, Albisua GLM, Godinez R, Collazo JJ, Gaminio E, Rios D, Dominguez EME, Pizzuto CJ (1991) Agreement among observers in the classification of acute leukemias. [Spanish]. Revista de Investigacion Clinica 43:223±228 Sanz GF, Sanz MA, VallespÓ T, Caµizo MC, Torrabadella M, GarcÓa S, Irriguible D, San Miguel JF (1989) Two regression models and a scor-

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ing system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 74:395±408 Schoch C, Kohlmann A, Schnittger S, Brors B, Dugas M, Mergenthaler S, Kern W, Hiddemann W, Eils R, Haferlach T (2002 a) Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles. Proc Natl Acad Sci USA 99:10008±10013 Schoch C, Schnittger S, Kern W, Lengfelder E, Loffler H, Hiddemann W, Haferlach T (2002 b) Rapid diagnostic approach to PML-RARalphapositive acute promyelocytic leukemia. Hematol J 3:259±263 Schoch C, Schnittger S, Klaus M, Kern W, Hiddemann W, Haferlach T (2003) AML with 11q23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood 102: 2395±2402

53

Stetler-Stevenson M, Arthur DC, Jabbour N, Xie XY, Molldrem J, Barrett AJ, Venzon D, Rick ME (2001) Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome. Blood 98: 979±987 Sultan C, Deregnaucourt J, Ko YW, Imbert M, d'Agay MF, Gouault-Heilmann M, Brun B (1981) Distribution of 250 cases of acute myeloid leukaemia (AML) according to the FAB classification and response to therapy. Br J Haematol 47:545±551 Theml H (2004) Color atlas of hematology: practical microscopic and clinical diagnosis. Thieme Medical Publishers, Stuttgart Van Den Berghe H, Michaux L (1997) 5q-, twenty-five years later: a synopsis. (Review). Cancer Genet Cytogenet 94:1±7 Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, Deeg HJ (2003) Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hemopoietic stem cell transplantation. Blood 102:394±403

Cytogenetic Diagnosis of Myelodysplastic Syndromes Harold J. Olney, Michelle M. Le Beau

Contents 6.1 Introduction . . . . . . . . . . . . . . . . . . . .

56

6.2 Diagnosis . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Cytogenetic Analysis . . . . . . . . . . . 6.2.1.1 Specimen Collection . . . 6.2.1.2 Report and Interpretation of Results . . . . . . . . . . . 6.2.1.3 Applications of Conventional Cytogenetic Analysis 6.2.2 Fluorescence In Situ Hybridization (FISH) . . . . . . . . . . . . . . . . . . . . . 6.2.2.1 Background and Theory . 6.2.2.2 Types of Probes . . . . . . . 6.2.2.2.1 Centromere Probes . . . . 6.2.2.2.2 Chromosome Painting Probes . . . . . . . . . . . . . 6.2.2.2.3 Locus-Specific Probes . . . 6.2.2.3 FISH Strategies . . . . . . . 6.2.2.3.1 Dual-Color, Dual-Fusion Probes . . . . . . . . . . . . . 6.2.2.3.2 Dual-Color Break-Apart Probes . . . . . . . . . . . . . 6.2.2.4 Report and Interpretation 6.2.2.5 Applications of FISH . . . . 6.2.3 Advantages and Limitations of Cytogenetic and FISH Analysis . .

56 56 57

6.3 Recurring Abnormalities . . . . . . . . . . . 6.3.1 Cytogenetic Findings in MDS . . . . . 6.3.1.1 Normal Karyotype . . . . .

57 59 59 59 59 60 60 60 61 61 61 61 61 61 62 62 62

6.3.1.2 6.3.1.3 6.3.1.4

±Y . . . . . . . . . . . . . . . del(20q) . . . . . . . . . . . Loss of Chromosome 5 or del(5q) . . . . . . . . . . 6.3.1.5 The 5q± Syndrome . . . 6.3.1.6 +8 . . . . . . . . . . . . . . . 6.3.1.7 Loss of Chromosome 7 or del(7q) . . . . . . . . . . 6.3.1.8 The 17p± Syndrome . . 6.3.1.9 Translocations of 11q23 6.3.1.10 t(11;16) . . . . . . . . . . . 6.3.1.11 Complex Karyotypes . . 6.3.1.12 Rare Recurring Translocations . . . . . . 6.3.1.12.1 The Platelet-Derived Growth Factor Receptor Beta Translocations . . . 6.3.1.12.2 Translocations of 3q . . 6.3.2. Evolution of the Karyotype . . . . .

. .

62 63

. . .

63 63 63

. . . . .

64 65 65 65 66

.

66

. . .

66 67 67

6.4 The Genetics of the Myelodysplastic Syndromes . . . . . . . . . . . . . . . . . . . . 6.4.1 Molecular Models for Chromosome Abnormalities in MDS . . . . . . . . . . 6.4.2 Molecular Analysis of the del(5q) . . 6.4.3 Molecular Analysis of ±7/del(7q) . . 6.4.4 Molecular Analysis of the del(20q) . 6.4.5 Alterations in Gene Function . . . . . 6.4.6 Genetic Pathways Leading to MDS

68 68 69 69 69 73

References . . . . . . . . . . . . . . . . . . . . . . . . .

74

68

56

Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

6.1 Introduction

The cytogenetic evaluation of bone marrow samples from patients with a myelodysplastic syndrome (MDS) has become an integral part of clinical care. A cytogenetic analysis not only confirms the diagnosis but is invaluable in defining the prognosis and expected survival, as well as the risk for progression to an acute myeloid leukemia (AML). On a more fundamental level, cytogenetic analysis has been instrumental in establishing the clonality of these syndromes as well as providing hints at the pathobiology of these entities. Here we will review the most frequently encountered abnormalities, exploring their clinical and genetic features, as well as the techniques of cytogenetic analysis and their applications in MDS.

clonality in MDS is classic cytogenetic analysis. In fact, the World Health Organization (WHO) has included recurring cytogenetic abnormalities in the classification of several subtypes of MDS with distinct clinical presentations and natural histories as discussed below (Jaffe et al. 2001). Other techniques more suited for research rather than clinical application include the analysis of restriction fragment length polymorphisms, and mutated oncogenes or tumor suppressor genes, which have also confirmed the clonal nature of MDS (Weimar et al. 1994). Aberrant in vitro growth patterns of stem cells can be characteristic of MDS (Spitzer et al. 1979), yet this evaluation is restricted to laboratories with expertise in this technique and is not routinely available. Immunophenotyping protocols (Wells et al. 2003) and microarray techniques, including array comparative genomic hybridization (Walker et al. 2002) hold potential future clinical diagnostic and prognostic promise.

6.2 Diagnosis

The diagnosis of all hematological malignancies, including MDS, begins with the appropriate clinical evaluation combined with expert pathological and genetic analysis. An accurate diagnosis is crucial in management decisions. Dysplasia identified in bone marrow samples may be found in a number of benign and congenital conditions including nutritional disorders, toxic exposures and infectious states, as well in MDS and acute leukemias (Jaffe et al. 2001). In highly dysplastic cases of MDS, or when the blast count is elevated, the diagnosis of MDS is relatively straightforward and is characterized by typical laboratory findings discussed earlier in this volume. Given the varied pathological and clinical picture of MDS, however, more sophisticated testing can be useful in establishing the diagnosis. The key distinguishing feature of these syndromes is the clonal nature of the dysplasia. Initial work with X chromosome inactivation patterns in females, based on isozymes of the enzyme glucose-6-phosphate dehydrogenase, suggested that MDS was a clonal disorder (Prchal et al. 1978). This technique, however, is limited to females, and it can be difficult to interpret in cases with random imbalances in X inactivation (skewing) (Busque and Gilliland 1998). Amplification of a polymorphic short tandem repeat in the human androgen receptor gene (HUMARA) on the X chromosome with polymerase chain reaction techniques is an extension of this approach (Okamoto et al. 1998). The most widely available and standardized technique for identifying

6.2.1 Cytogenetic Analysis

The value of cytogenetic analysis in predicting survival and risk of leukemic transformation during a patient's clinical course has been well established (Jotterand and Parlier 1996; Morel et al. 1993; Sole et al. 2000; Toyama et al. 1993). Among the few independent variables identified that predict clinical outcomes in MDS, cytogenetic findings form the cornerstone of successful prognostic scoring systems (Table 6.1) (Greenberg et al. 1997). At the time of diagnosis, recurring chromosomal abnormalities are found in 40±70% of patients with primary MDS and in 95% of patients with therapy-related MDS (t-MDS) (Vallespi et al. 1998). The frequency of cytogenetic abnormalities increases with the severity of disease, as does the risk of leukemic transformation. Clonal chromosome abnormalities can be detected in marrow cells of 25% of patients with refractory anemia (RA), 10% of patients with refractory anemia with ringed sideroblasts (RARS), 50% of patients with refractory cytopenias with multilineage dysplasia (RCMD), 50±70% of patients with refractory anemia with excess blasts 1, 2 (RAEB-1,2), and 100% of patients with MDS with isolated del(5q) [5q±]. Most recurring cytogenetic abnormalities found in MDS are unbalanced, most commonly the result of the loss of a whole chromosome or a deletion of part of a chromosome, but unbalanced translocations and more complex derivative (rearranged) chromosomes

a

6.2 ´ Diagnosis

57

Table 6.1. Cytogenetic abnormalities of the international prognostic scoring system

Favorable risk

Cytogenetic abnormalities

25% AML progression

Median survival

Normal karyotype

5.6 years

3.8 years

Isolated del(5q) Isolated del(20q) Isolated ±Y Intermediate risk

Other abnormalities

1.6 years

2.4 years

Poor risk

±7/del(7q) complex karyotypes

0.9 years

0.8 years

6.2.1.1 Specimen Collection

Fig. 6.1. Karyotypic abnormalities in MDS

Fig. 6.2. Recurring chromosomal abnormalities in MDS

can be found (Figs. 6.1 and 6.2, Table 6.2). The most common cytogenetic abnormalities encountered in MDS are del(5q), ±7, and +8, which have been incorporated into the more robust prognostic scoring systems of MDS. Clones with unrelated abnormalities, one of which typically has a gain of chromosome 8, are seen at a greater frequency (~5% vs. ~1%) in patients with MDS than in patients with AML.

Detailed methods for the cytogenetic analysis of hematological malignant diseases have been described previously (Roulston and Le Beau 1997). Cytogenetic studies are performed on spontaneously dividing cells that are typically cultured for short periods (24±72 h). The dividing cells are collected by arresting them in metaphase using a spindle fiber inhibitor, e.g., Colcemid. A hypotonic solution is added to increase cell volume, which aids in chromosome spreading, and the cells are preserved using a methanol-acetic acid fixative. Slides are prepared by dropping the cell suspension onto microscope slides, which ruptures the cell membrane and spreads the chromosomes, followed by histological staining. Conventional cytogenetic studies can be performed on almost any tissue with actively dividing cells. Bone marrow is the tissue of choice for cytogenetic studies of MDS. Tissue should be collected in cell culture media with heparin to prevent clotting and antibiotics to prevent bacterial contamination, and transported to the laboratory immediately. Specimens should not be collected in tubes containing agents such as EDTA or Sodium Citrate, as these affect cell viability and preclude the analysis of cells undergoing mitosis.

6.2.1.2 Report and Interpretation of Results The International System of Human Cytogenetic Nomenclature (ISCN 1995) was developed as the result of several international conferences, and is used to describe chromosomal abnormalities in a consistent manner (Mitelman 1995). Using this system, abnormalities can be described using abbreviated terms for the rearrangement, followed by a numerical description of the chromosome(s), chromosome arm(s), and bands involved. A typical cytogenetic analysis includes the com-

58

Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Table 6.2. Recurring chromosomal abnormalities in myelodysplastic syndromes Frequency

Involved genes a

Consequence

+8

10%

±

±

±

±7 or del(7q)

10%

±

±

±

±5 or del(5q)

10%

±

±

±

del(20q)

5±8%

±

±

±

±Y

5%

±

±

±

i(17q)

3±5%

TP53

±

Loss of function

±13/del(13q)

3%

±

±

±

del(11q)

3%

±

±

±

del(12p)/t(12p)

3%

±

±

±

del(9q)

1±2%

±

±

±

idic(X)(q13)

1±2%

±

±

±

t(1;3)(p36.3;q21.2)

1%

±

t(2;11)(p21;q23)

1%

inv(3)(q21q26.2)

1%

t(6;9)(p23;q34)

1%

±7 or del(7q)

Disease

Chromosome abnormality

MDS

Unbalanced

Balanced

Therapy-related MDS

CMML

±

±

MLL

MLL fusion proteinaltered transcriptional regulation

RPN1

MDS1/EVI1

Fusion protein

DEK

NUP214

Fusion protein-nuclear pore protein

50%

±

±

±

±5 or del(5q)

42%

±

±

±

dic(5;17)(q11.1± 13;p11.1±13)

5%

±

TP53

Loss of function

der(1;7)(q10;p10)

3%

±

±

±

t(3;21)(q26.2;q22.1)

3%

EAP

RUNX1

RUNX1 fusion protein-altered transcriptional regulation

t(11;16)(q23;p13.3)/ t(11q23)

2%

MLL

CBP

MLL fusion protein-altered transcriptional regulation

t(5;12)(q33;p12)

25%

PDGFRB

ETV6/TEL

Fusion protein-altered signaling pathway

MDS myelodysplastic syndrome, CMML chronic myelomonocytic leukemia a

Genes are listed in order of citation in the karyotype, e.g., for the t(11;16), MLL is at 11q23 and CBP at 16p13.3

a

6.2 ´ Diagnosis

plete analysis of at least 20 banded metaphase cells. However, this may not always be possible and depends on the cellularity, mitotic index, and quality of the specimen. An analysis of less than 20 cells is still informative if a clonal abnormality is detected. The cytogenetic work-up should include the analysis of cells from more than one preparation, preferably two short-term cultures, e.g., 24- and 48-h cultures. Direct preparations, in which metaphase cells are prepared without prior culturing in vitro, have proven to be less suitable in that the percentage of cases with detectable abnormalities is substantially less than that observed with cultured samples. Genetic mutations accumulate during the progression of a normal cell to a malignant state. Therefore, multiple, related subpopulations of cells derived from a single progenitor may be present in any one specimen. A chromosomal abnormality is considered clonal if a structural abnormality or gain of a chromosome is identified in two or more cells. Chromosome loss can occur as a technical artifact during metaphase cell preparation; thus, a loss of a chromosome is considered to be clonal when it occurs in three or more cells. The number of cells observed in each clone is listed after the clone description in brackets [n]. The simplest clone is termed the ªstemlineº. The stemline is listed first, and related clones are listed in order of increasing complexity. To describe subclones, the term idem followed by any additional changes as compared to the stemline can be used. It is important to note that idem always refers to the abnormalities described in the stemline clone. This type of karyotypic progression is referred to as clonal evolution.

6.2.1.3 Applications of Conventional Cytogenetic Analysis Cytogenetic analysis should be requested for any patient with a suspected or confirmed MDS. The identification of recurring chromosomal abnormalities can aid in the diagnosis of the disorder. Moreover, cytogenetic abnormalities represent independent predictors of response to therapy and outcome. In addition, any abnormality noted at the time of diagnosis can be used as a biological marker to monitor the response to therapy or to detect residual disease in follow-up specimens. Subsequent specimens without this biological marker can be interpreted to represent a cytogenetic remission. Likewise, if the abnormal chromosome(s) is detected

59

in a follow-up specimen, it is indicative of residual disease, relapse, or in the case of karyotypic evolution, disease progression. In the future, cytogenetic results may be used to select risk-adapted therapies.

6.2.2 Fluorescence In Situ Hybridization (FISH)

6.2.2.1 Background and Theory In contrast to classical cytogenetic analysis, FISH can evaluate both metaphase and interphase cells in a rapid and efficient manner. The primary advantage of FISH is its simplified analysis permitting the evaluation of an increased number of cells greatly increasing the sensitivity. It can also be applied to histologically stained cells allowing a direct correlation of the genetic target's status within morphologically characterized cells. The technique does not, however, evaluate the entire chromosome complement but rather specific alterations based on probe selection. Not all recurring abnormalities of interest have probes suitable for clinical use and variation in the cytogenetic abnormality (with either complex rearrangement or differences in breakpoint) may occasionally not be detected with conventional probes. Fluorescence in situ hybridization is based on the same principle as Southern blot analysis, namely, the ability of single-stranded DNA to anneal to complementary DNA. In the case of FISH, the target DNA is the nuclear DNA of interphase cells, or the DNA of metaphase chromosomes affixed to microscope slides. The test probe is labeled through incorporation of a fluorescently tagged reporter nucleotide. The test probe anneals to its complementary sequences on fixed metaphase chromosomes or interphase nuclei, and is visualized by fluorescence microscopy as a brilliantly colored signal at the hybridization site. FISH can be performed using standard cytogenetic cell preparations, bone marrow or peripheral blood smears, or fixed and sectioned tissue.

6.2.2.2 Types of FISH Probes A variety of probes can be used to detect chromosomal abnormalities by FISH; a partial list of probes available for detection of the recurring abnormalities in MDS is given in Table 6.3. In general, these probes can be divided into three groups: (1) probes that identify a specif-

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Table 6.3. FISH probes to detect recurring chromosomal abnormalities in MDS Abnormality

Probe

Format a

Vendor b

t(11q23)

MLL

Two-color break-apart

Vysis, Ventana

Single color ±5/del(5q)

c

±7/del(7q)

a

EGR1/D5S23/D5S72

Two-color deletion

Vysis

CSF1R/D5S23/D5S72

Two-color deletion

Vysis

D7S522/CEP7

Two-color deletion

Vysis

D7S486/CEP7

Two-color deletion

Vysis

del(20q)

D20S108

Single color

Vysis

+8

CEP8

Single color

Vysis, Cytocell, Ltd

del(13q)

D13S25

Single color

Vysis

del(11q)

ATM

Single color

Vysis

±17/del(17p)

TP53

Single color

Vysis

Miscellaneous/ Transplants

CEPX/CEPY Subtelomeric (each arm)

Single color or two color Single color Two color

Vysis Vysis, Cytocell, Ltd

In two-color break-apart probes, DNA sequences from the 5' and 3' regions of a single gene are labeled and detected with red and green fluorochromes. In the germline configuration, a yellow fusion signal is observed, whereas individual red and green signals are observed when the sequences are separated as a result of a translocation

b

Vysis, Downers Grove, IL (http://www.vysis.com); Ventana Medical Systems, Tucson, AZ (http://www.ventanamed.com), Cytocell, Banbury, UK

c

The EGR1 gene at 5q31 is located within the commonly deleted segment of the del(5q) in AML, whereas the CSF1R gene is mapped to 5q33,

(http://www.cytocell.co.uk) and detects the del(5q) in the 5q± syndrome

ic chromosome structure, (2) probes that hybridize to multiple chromosomal sequences, and (3) probes that hybridize to unique DNA sequences (Gozzetti and Le Beau 2000).

6.2.2.2.1 Centromere Probes Examples of probes that hybridize to a specific chromosome structure include a- and b-satellite probes. These probes represent tandemly repeated DNA sequences that flank the centromeres of human chromosomes. In most instances, these sequences are distinct, such that an a-satellite probe derived from one chromosome will hybridize to that chromosome only. At present, specific probes are available for chromosomes 1±4, 6±12, 15±18, and 20, as well as the X and Y chromosomes. Hybridization of chromosome-specific repetitive sequence probes is particularly suitable for the detection of monosomy, trisomy and other aneuploidies, e.g., ±7 or +8 (Fig. 6.7) (Jenkins et al. 1992; Moyzis et al. 1987).

6.2.2.2.2 Chromosome Painting Probes Chromosome painting probes utilize cloned DNA libraries derived from whole, flow-sorted human chromosomes (Pinkel et al. 1988). After hybridization and detection, the result is the fluorescent staining or ªpaintingº of an entire chromosome. Chromosome-specific painting probes are available for each of the human chromosomes, and are particularly useful in characterizing marker chromosomes (a rearranged chromosome of unknown origin), or additional material of unknown origin translocated to other chromosomes. 6.2.2.2.3 Locus-Specific Probes Probes that hybridize to unique DNA sequences are usually genomic clones, which vary in size depending on the nature of the cloning vector. Probes in this group are particularly useful for detecting structural rearrangements. Using multi-color FISH, recurring translocations can be identified in interphase or metaphase cells by using genomic probes that are derived from

a

6.2 ´ Diagnosis

the breakpoints (Nederlof et al. 1990; Tkachuk et al. 1990). Similarly, recurring deletions can be detected using genomic probes located within the commonly deleted segment. For example, a locus-specific probe for the EGR1 gene at 5q31 detected with a green fluorochrome, and a locus-specific control probe for the short arm of chromosome 5 detected with a red fluorochrome can be used to identify ±5/del(5q) in MDS.

6.2.2.3 FISH Strategies 6.2.2.3.1 Dual-Color/Dual-Fusion Probes A variety of strategies have been developed for the detection of recurring translocations in interphase cells, each with variable sensitivity (Gozzetti and Le Beau 2000). The selection of a particular probe configuration may vary depending on the application. A common type of probe for detecting translocations with high sensitivity and specificity is a dual-fusion signal. In the example of the t(8;21), both probes include large regions encompassing the RUNX1 and ETO loci so that cells with the t(8;21) have one red and one green signal observed on the normal homologs, and yellow fusion signals observed on both the der(21) and der(8) homologs. This type of probe has a low rate of false positive cells, and the cut-off value for a positive result is ~1% (Dewald et al. 1998). The results of a recent study suggested that quantitative analysis of 6,000 nuclei could be used to detect MRD with a sensitivity of ~0.2% (Dewald et al. 1998). 6.2.2.3.2 Dual-Color Break-Apart Probes Two-color break-apart probes are designed such that DNA sequences from the 5' and 3' regions of a single gene are differentially labeled and detected with red and green fluorochromes. In the germline configuration, a yellow fusion signal is observed, whereas separate red and green signals are observed when the sequences are separated as a result of a translocation (Fig. 6.7). This probe configuration offers the advantage that knowledge of the partner gene is not necessary and, therefore, is most useful for loci that are translocated to multiple sites such as MLL. The sensitivity of this type of probe is exceedingly high (cut-off value of ~1.7%) with very high specificity.

61

6.2.2.4 Report and Interpretation When interpreting FISH results, it is important to first note the type of probe strategy used. This provides a general indication of the sensitivity of the specific test performed. Next, it is important to note the specific laboratory reference range for the probe used. The guidelines for clinical FISH analysis establish procedures for probe validation, assay validation, and assay analytical sensitivity (available through www.faseb.org/genetics/ acmg). Each laboratory must establish normal and abnormal reference ranges; without this information it is not possible to interpret FISH results accurately. FISH nomenclature, much like chromosome banding nomenclature, is outlined in the ISCN (1995) (Mitelman 1995).

6.2.2.5 Applications of FISH The applications of FISH in cancer diagnosis have been described in depth in several recent reviews (Gozzetti and Le Beau 2000; Kearney 1999). In many cases, FISH analysis provides increased sensitivity in that cytogenetic abnormalities have been detected in samples that appeared to be normal by morphological and conventional cytogenetic analysis. FISH is most powerful when the analysis is targeted toward those abnormalities that are known to be associated with a particular disease, or are known to occur in a particular patient's tumor. For example, cytogenetic analysis is often performed at the time of diagnosis to identify the chromosomal abnormalities in an individual patient's malignant cells. Thereafter, FISH can be used to detect residual disease or early relapse, and to assess the efficacy of therapeutic regimens.

6.2.3 Advantages and Limitations

of Cytogenetic and FISH Analysis

The advantages and limitations of cytogenetic and FISH analysis have been described in recent reviews (Gozzetti and Le Beau 2000). Perhaps the most critical parameters for consideration are the sensitivity and specificity of each method, and the speed with which each can be accomplished. Cytogenetic analysis has a relatively low sensitivity as compared with FISH (1:2±01:100 cells vs. 1 : 20± 1 : 1,000). Moreover, cytogenetic analysis requires highly skilled personnel, and is labor intensive; results are not available for 1±3 weeks in all but exceptional

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

cases. In contrast, material for FISH can be processed in 4±24 h, and the analysis of 1,000±2,000 cells can be accomplished in 15±45 min. Thus, FISH is a rapid technique that enables one to obtain information on the cytogenetic pattern of tumor cells in a time-frame sufficient for the data to be considered in treatment decisions. Both methods can be used to detect both numerical and structural chromosomal abnormalities. Cytogenetic analysis requires dividing cells from which metaphase chromosomes can be examined, whereas FISH may be applied to both metaphase cells and interphase cells, allowing for the accurate and informative analysis of those specimens for which no metaphase spreads could be isolated. The specificity of cytogenetic analysis is very high, and conventional cytogenetic analysis can detect the presence of virtually all chromosomal abnormalities with a single test. In contrast, the most notable limitation of FISH is that the detection of abnormalities is restricted to those loci tested, and to those probes that are currently available (Table 6.3). With the exception of several probes marketed by Vysis, Inc. (Abbott Laboratories), most of the commercially-available FISH probes have not yet received U.S. Food and Drug Administration approval for diagnostic use. This compels laboratories to continue to use both cytogenetic analysis and FISH methods to analyze the same clinical sample, rather than FISH alone. Other limitations of FISH relate to technical factors. First, the technique has been demonstrated to be highly sensitive for the detection of trisomy, but is less sensitive in detecting chromosome loss. The false monosomy rate (presence of only one signal in a diploid cell) can vary from 3±9% of all nucleated cells in preparations from bone marrow or peripheral blood; therefore, the application of FISH to detect monosomy in a small percentage of the cell population, e.g., MRD, is limited. Second, processing of bone marrow and peripheral blood cells is relatively simple; however, processing other tissues such as paraffin-embedded tissues or frozen sections from lymph node biopsies is substantially more difficult. Artifacts created by crushing and sectioning of tissue, and technical factors related to penetration of the probe into the cell nucleus can give misleading results. In these tissues, the percentage of cells showing false monosomy may be very high (> 50%).

6.3 Recurring Abnormalities

A handful of specific cytogenetic abnormalities, including the 5q± syndrome (Van den Berghe and Michaux 1997), the 17p± syndrome (Jary et al. 1997), and the isodicentric X chromosome (which is associated with RARS with a high likelihood of transformation to AML) (Dewald et al. 1982) are associated with morphologically and clinically distinct subsets of MDS (Table 6.2). Many findings, including loss or deletions of chromosomes 5 or 7, trisomy 8, and complex karyotypes, are common to both MDS and AML (Fig. 6.2). In rare cases, recurring balanced translocations have been reported. Abnormalities characteristic of acute leukemia without a prior myelodysplastic phase, such as the t(15;17), inv(16) and t(8;21), can be identified rarely in MDS (Rowley 1999). The t(9;22), diagnostic of chronic myelogenous leukemia and a subtype of acute lymphoblastic leukemia (ALL), has only rarely been reported in MDS (Smadja et al. 1989).

6.3.1 Cytogenetic Findings in MDS

6.3.1.1 Normal Karyotype In MDS, 30±60% of patients have a normal karyotype. This group of patients is almost certainly genetically heterogeneous where technical factors precluded the detection of chromosomally abnormal cells, or where leukemogenic alterations occur at the molecular level and are not detectable with standard cytogenetic methods. Nonetheless, despite this heterogeneity, these cases are a standard reference for comparison of outcomes. Patients with a normal karyotype fall within the favorable risk group. The median survival for these patients is estimated at 3.8 years, and the time to progression to AML in 25% of this cohort was 5.6 years (Greenberg et al. 1997).

6.3.1.2 ±Y The clinical and biological significance of the loss of the Y chromosome, ±Y, is unknown. Loss of the Y chromosome has been observed in a number of malignant diseases, but has also been reported to be a phenomenon associated with aging (Pierre and Hoagland 1972). The United Kingdom Cancer Cytogenetics Group undertook a comprehensive analysis of this abnormality in both

a

6.3 ´ Recurring Abnormalities

normal and neoplastic bone marrows (UKCCG 1992). A ±Y could be identified in 7.7% of patients without a hematologic malignant disease and in 10.7% of patients with MDS and, thus, was not reliable in documenting a malignant process. In a large series of 215 male patients, Wiktor et al. (2000) found that patients with a hematological disease had a significantly higher percentage of cells with a ±Y (52% vs. 37%, p = 0.036). In this series, the presence of ±Y in > 75% of metaphase cells accurately predicted a malignant hematological disease. A neutral or favorable prognosis for an isolated ±Y was noted by the authors. While loss of a Y chromosome may not be diagnostic of MDS, once the disease is identified by clinical and pathologic means, the International MDS Risk Analysis Workshop found that ±Y as the sole cytogenetic abnormality conferred a favorable outcome (Greenberg et al. 1997).

6.3.1.3 del(20q) A deletion of the long arm of chromosome 20, del(20q), is a common recurring abnormality in malignant myeloid disorders. The del(20q) is seen in approximately 5% of MDS cases and 7% of t-MDS cases (Vallespi et al. 1998). Clinical features characterizing MDS patients with a del(20q) include low-risk disease (usually RA), low rate of progression to AML, and prolonged survival (median of 45 months vs. 28 months for other MDS patients) (Wattel et al. 1993). Morphologically, the presence of a del(20q) is associated with prominent dysplasia in the erythroid and megakaryocytic lineages (Kurtin et al. 1996). The International MDS Risk Analysis Workshop noted that patients with a del(20q) observed in association with a complex karyotype identified a poor-risk group with a median survival for the entire poor-risk group of 9.6 months, whereas the prognosis for patients with an isolated del(20q) was favorable (Greenberg et al. 1997). These data suggest that the del(20q) in MDS may be associated with a favorable outcome when noted as the sole abnormality, but with a less favorable prognosis in the setting of a complex karyotype. This phenomenon is analogous to that observed for the del(5q) in MDS (discussed below).

6.3.1.4 Loss of Chromosome 5 or del(5q) In MDS or AML arising de novo, loss of a whole chromosome 5, or a deletion of its long arm, ±5/del(5q), are

63

observed in 10±20% of patients, whereas in t-MDS/tAML, it is identified in 40% of patients (Fig. 6.4) (Thirman and Larson 1996; Vallespi et al. 1998). A significant occupational exposure to potential carcinogens is present in many patients with AML or MDS de novo and either ±5/del(5q) or a ±7/del(7q) (discussed below), suggesting that abnormalities of chromosome 5 or 7 may be a marker of mutagen-induced malignant hematological disease (West et al. 2000). In primary MDS, abnormalities of chromosome 5 are observed in the 5q± syndrome (described below) or, more commonly, in RAEB 1, 2 of the WHO classification in association with a complex karyotype. Clinically, the patients with del(5q) coupled with other cytogenetic abnormalities have a poor prognosis with early progression to leukemia, resistance to treatment, and short survival. Abnormalities of 5q are associated with previous exposure to standard and high dose alkylating agent therapy, including use in immunosuppressive regimens (Aul et al. 1998; Larson et al. 1996; McCarthy et al. 1998; Pedersen-Bjergaard et al. 2000). A role for exposure to benzene (Hayes et al. 1997) as well as therapeutic ionizing radiation (Fenaux et al. 1989; Rowley and Olney 2002) as risks for MDS is emerging.

6.3.1.5 The 5q± Syndrome The 5q± syndrome represents a distinct clinical syndrome characterized by a del(5q) as the sole karyotypic abnormality (Boultwood et al. 1994; Van den Berghe and Michaux 1997). Unlike the male predominance in MDS in general, the 5q± syndrome has an over-representation of females (2 : 1). The initial laboratory findings are usually a macrocytic anemia with a normal or elevated platelet count. The diagnosis is usually RA (in two thirds), or RAEB (in one third). On bone marrow examination, abnormalities in the megakaryocytic lineage (particularly micromegakaryocytes) are prominent. These patients have a favorable outcome, in fact the best of any MDS subgroup, with low rates of leukemic transformation and a relatively long survival of several years duration (Boultwood et al. 1994; Greenberg et al. 1997).

6.3.1.6 +8 The incidence of a gain of chromosome 8 in MDS is ~10%. This abnormality is observed in all MDS subgroups varying with age, gender, and prior treatment

64

Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Fig. 6.3 A, B. Fluorescence in situ hybridization analysis of hematologic malignant diseases. Panels a and b illustrate images of metaphase and interphase cells following FISH; the cells are counterstained with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). A Hybridization of a directly labeled centromere-specific probe for chromosome 8 (CEP8 Spectrum Green, Vysis Inc.) to metaphase and interphase cells with trisomy 8 from a bone marrow aspirate of a

patient with AML. The chromosome 8 homologs are identified with arrows. B Hybridization of the MLL break-apart probe to metaphase and interphase cells with a t(11q23). In cells with a MLL translocation, a yellow fusion signal is observed for the germline configuration on the normal chromosome 11 homolog, a green signal is observed in the der(11) chromosome, and a red signal is observed on the partner chromosome

with cytotoxic agents or radiation (Greenberg et al. 1997; Morel et al. 1993; Paulsson et al. 2001; Vallespi et al. 1998). It can occur as both a constitutional and an acquired abnormality and can fluctuate throughout the disease course (Maserati et al. 2002; Mastrangelo et al. 1995; Matsuda et al. 1998). The significance of the gain of chromosome 8 in MDS patients is not fully characterized as a risk factor. The situation is complicated in that +8 is often associated with other recurring abnormalities known to have prognostic significance, e.g., del(5q) or ±7/del(7q), and may be seen in isolation as a separate clone unrelated to the primary clone in up to 5% of cases. The International MDS Risk Analysis Workshop ranked this abnormality in the intermediate risk group (Greenberg et al. 1997). Although only significant in univariate analysis, a large confirmatory study found that +8 as a sole abnormality had a worse behavior than expected for an intermediate IPSS risk group (Sole et al. 2000).

and Larson 1996). It can occur in three clinical situations (reviewed in Luna-Fineman et al. 1995): (1) de novo MDS and AML; (2) myeloid leukemia associated with constitutional predisposition; and (3) t-MDS/tAML. The similar clinical and biological features of the myeloid disorders associated with ±7/del(7q) suggest that the same gene(s) is altered in each of these contexts. The IPSS considers the ±7/del(7q) to be a poor prognosis cytogenetic finding (Greenberg et al. 1997). ªMonosomy 7 Syndromeº has been described in young children (see Chapter 7). It is characterized by a preponderance of males (~4 : 1), hepatosplenomegaly, leukocytosis, thrombocytopenia, and poor prognosis (Emanuel 1999; Martinez-Climent and Garcia-Conde 1999). Juvenile myelomonocytic leukemia (JMML, previously known as juvenile chronic myelogenous leukemia) shares many features with this entity, with ±7 observed either at diagnosis or as a new cytogenetic finding associated with disease acceleration on marrow examination (Luna-Fineman et al. 1995). An emerging paradigm is that ±7 cooperates with deregulated signaling via the RAS pathway in the pathogenesis of JMML. Activation of the RAS pathway occurs as a result of mutations in the NRAS or KRAS1 gene, inactivating mutations in the gene encoding NF1, a negative regulator of RAS proteins, or activating mutations in the gene encoding the PTPN11/SHP2 phosphatase, a positive regulator of RAS proteins. In constitutional disorders asso-

6.3.1.7 Loss of Chromosome 7 or del(7q) A ±7/del(7q) is observed as the sole abnormality in approximately 5% of adult patients with de novo MDS (Sole et al. 2000; Toyama et al. 1993), but in ~50% of children with de novo MDS (Kardos et al. 2003) and in ~55% of patients with t-MDS (Fig. 6.3, 6.4) (Thirman

a

6.3 ´ Recurring Abnormalities

65

normality of 17p; an inactivating mutation in the second allele on the remaining, morphologically normal chromosome 17 occurs in ~70% of cases (Lai et al. 1995; Wang et al. 1997). Sankar et al. (1998) mapped a CDS in leukemia and lymphoma patients to 17p13.3, suggesting the possible existence of a novel tumor suppressor gene distal to TP53. Fig. 6.4. Deletions of 5q and 7q in myeloid neoplasms. In this del(5q), breakpoints occur in q14 and q33 resulting in interstitial loss of the intervening chromosomal material. In this del(7q), breakpoints occur in q11.2 and q36. In both cases, the critical commonly deleted segments are lost. Normal chromosome 5 and 7 homologs are shown for comparison

ciated with a predisposition to myeloid neoplasms, including Fanconi anemia, neurofibromatosis type 1, and severe congenital neutropenia, a ±7/del(7q) is the most frequent bone marrow cytogenetic abnormality detected. As with ±5/del(5q), occupational or environmental exposure to mutagens including chemotherapy, radiotherapy, benzene exposure, and smoking (Bjork et al. 2000) as well as severe aplastic anemia (regularly treated with immunosuppressive agents alone) have been associated with ±7/del(7q).

6.3.1.8 The 17p± Syndrome Loss of the short arm of chromosome 17 (17p±) has been reported in up to 5% of patients with MDS. This loss can result from various abnormalities, including simple deletions, unbalanced translocations, dicentric rearrangements (particularly with chromosome 5), or less often ±17, or isochromosome formation (Johansson et al. 1993). The dic(5;17)(q11.1±13;p11.1±13) is a frequent recurring rearrangement (Lai et al. 1995; Wang et al. 1997). Approximately one third of these patients have t-MDS (Merlat et al. 1999), and most have complex karyotypes. The most common additional changes are ±7 or loss of 7q, and +8. Morphologically, the 17p± syndrome is associated with a characteristic form of dysgranulopoiesis combining pseudo-Pelger-HuŸt hypolobulation and the presence of small granules in granulocytes. Clinically, the disease is aggressive with resistance to treatment and short survival. The TP53 (p53) gene, an important tumor suppressor gene that functions in the cellular response to DNA damage, is located at 17p13.1. In these cases, one allele of TP53 is typically lost as a result of the ab-

6.3.1.9 Translocations of 11q23 The MLL (Mixed Lineage Leukemia) gene (also known as ALL1, HTRX, HRX) is involved in over 40 reciprocal translocations in acute leukemia (Rowley 2000). In a European workshop of 550 patients with 11q23 abnormalities, 28 cases (5.1%) presented with MDS, and five others with such an abnormality had evolved from tMDS to t-AML prior to cytogenetic analysis, for a total of 6% of all examined cases. One fourth of these cases were t-MDS (Bain et al. 1998). Other abnormalities, including complex karyotypes and a ±7/del(7q), frequently accompany the 11q23 abnormalities in both primary MDS and t-MDS. No association with FAB subgroup was identified, although RA was overrepresented, and RARS underrepresented as compared with most series of MDS patients. The median survival was short (19 months) with leukemic transformation in ~20% of cases. The classic association of prior exposure to topoisomerase II inhibitors in their 40 cases of t-MDS and t-AML was not confirmed in this workshop, but this may simply reflect the relatively small number (n = 23) of cases with full treatment details (Secker-Walker et al. 1998). Just under 12% of the 162 patients with 11q23 involvement included in an international workshop on MDS and leukemia following cytotoxic treatment presented with a t-MDS (Bloomfield et al. 2002; Rowley and Olney 2002). One third (6/19) of these patients had progression to an acute leukemia (five AML, one ALL). This study also did not find a clear association with FAB subtype. The most common translocations were t(9;11)(p22;q23) in six cases, t(11;19)(q23;p13.1) in three cases, and t(11;16)(q23;p13.3) in three cases.

6.3.1.10 t(11;16) The t(11;16)(q23;p13.3) occurs primarily in t-MDS, but some cases have presented as t-AML (Fig. 6.4) (Rowley et al. 1997). The t(11;16) is unique among at least 47 recurring translocations of MLL in myeloid malignancies

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Fig. 6.5. t(11;16)(q23;p13.3). In the t(11;16), breakpoints occur in 11q23 and 16p13.3, followed by a reciprocal exchange of chromosomal material. The 5' end of the MLL gene at 11q23 is fused to the 3' end of the CBP gene from 16p13.3 to form the MLL/CBP fusion gene on the der(11). Arrowheads indicate the breakpoints. Normal chromosome 11 and 16 homologs are shown for comparison

(with AML predominating), in that most patients have t-MDS. The MLL gene on chromosome 11 is fused with the CBP (CREB binding protein) gene on chromosome 16. The MLL protein is a histone methyltransferase that assembles in protein complexes that regulate gene transcription, e.g., HOX genes during embryonic development, via chromatin remodeling. CBP is an adapter protein involved in transcriptional control via histone acetylation, which mediates chromosome decondensation, thereby facilitating transcription. Both genes have multiple translocation partners in various hematological disorders; thus, elucidating their function will undoubtedly lead to significant progress in leukemia research.

6.3.1.11 Complex Karyotypes Complex karyotypes are variably defined, but generally involve the presence of ³ 3 chromosomal abnormalities. The majority of cases with complex karyotypes involve unbalanced chromosomal abnormalities leading to the loss of genetic material. Complex karyotypes are observed in ~20% of patients with primary MDS, and in as many as 90% of patients with t-MDS (Le Beau et al. 1986; Thirman and Larson 1996). Abnormalities involving chromosomes 5, 7, or both are identified in most cases with complex karyotypes. There is general agreement that a complex karyotype carries a poor prognosis (Greenberg et al. 1997; Hamblin and Oscier 1987).

6.3.1.12 Rare Recurring Translocations The identification of genes involved in recurring cytogenetic abnormalities has been extremely useful in gaining insights into their normal functions and their

role in leukemogenesis (Look 1997; Rowley 2000). The consequence of the recurring translocations is the deregulation of gene expression with increased production of a normal protein product, or the generation of a novel fusion gene and production of a fusion protein. To date, all of the recurring translocations cloned in malignant myeloid disorders result in fusion proteins. In MDS, several such translocations have been identified and examined by molecular analysis.

6.3.1.12.1 The Platelet-Derived Growth Factor Receptor Beta Translocations The t(5;12)(q33;p12) is observed in ~1% of patients with chronic myelomonocytic leukemia (CMML). In 1994, the molecular consequences of this translocation were elucidated. The gene encoding the beta chain of the platelet derived growth factor receptor (PDGFRB) is involved on chromosome 5. A novel ETS-like (Erythroblastosis Virus Transforming Sequence) transcription factor, TEL (translocated ETS in leukemia, also known as ETV6), is the gene affected on chromosome 12. The translocation creates a fusion gene; and the encoded fusion protein contains the 5' portion of TEL and the 3' portion of PDGFRB (Golub et al. 1994). Biochemical studies have revealed that the PDGFRB kinase activity is perturbed and contributes to the transformed phenotype. TEL encodes a transcriptional repressor, and is promiscuously involved in translocations with some 40 genes in hematologic malignancies (Rowley 2000). Interest has increased in identifying this translocation, which predicts for a response to imatinib mesylate, a selective inhibitor of the tyrosine kinase activity of the PDGFRB protein (Apperley et al. 2002). Similarly, PDGFRB participates in other rare translocations involving genes encoding the membrane associated protein HIP1 (Huntington interacting protein 1) in the t(5;7)(q33;q11.2) (Ross et al. 1998), the small GTPase RABPT5 (Rabaptin 5) in the t(5;17)(q33;p13) (Magnusson et al. 2001) and H4, a ubiquitous protein of unknown function in the t(5;10)(q33;q21) (Kulkarni et al. 2000) to produce CMML, and with CEV14 (clonal evolutionrelated gene on chromosome 14, also known as TRIP11, thyroid hormone receptor interactor 11) in the t(5;14)(q33;q32) in a case of AML (Abe et al. 1997). A unifying feature of these various translocations is the presence of eosinophilia.

a

6.3 ´ Recurring Abnormalities

6.3.1.12.2 Translocations of 3q The t(3;21)(q26.2;q22.1) has been linked to acute leukemia arising after cytotoxic therapy. This abnormality was first recognized in chronic myelogenous leukemia in blast crisis (Rubin et al. 1987) and later in t-MDS/tAML (Rubin et al. 1990). The EAP gene (Epstein-Barr small RNAs Associated Protein) at 3q26.2 encodes a highly expressed small nuclear protein associated with EBV small RNA (EBER1). EAP was found to be fused with the RUNX1 (Runt related transcription factor, also known as AML1) gene at 21q22, retaining the DNA binding sequences of EAP. P The fusion is out-of-frame; thus, the RUNX1 gene is truncated and loses its functional activity. Further work has identified two additional genes 400±750 kb centromeric to EAP, also at 3q26.2, namely MDS1/EVI1 (MDS associated sequences) and EVI1 (Ecotropic Virus Insertion site) (Nucifora et al. 1994). Both genes encode nuclear transcription factors containing DNA-binding zinc finger domains, which are identical other than an N-terminal extension of 12 amino acids in the MDS1/EVI1 protein, representing a splicing variant. Each gene has independent and tightly controlled expression during differentiation (Sitailo et al. 1999). The MDS1/EVI1 and EVI1 proteins have opposite functions. EVI1 inhibits G-CSF mediated differentiation and TGFb F 1 growth-inhibitory effects, whereas MDS1/ F 1 EVI1 has no effect on G-CSF and enhances TGFb growth-inhibition (Sitailo et al. 1999). RUNX1 fuses with MDS1/EVI1 in-frame, resulting in the loss of the first 12 amino acids, producing a novel EVI1 protein, and a phenotype of arrested differentiation, which leads to apoptosis in vitro (Sood et al. 1999). MDS1/EVI1 serves as a translocation partner with the ribosome binding protein RPN1 (ribophorin 1) (Martinelli et al. 2003) or the gene encoding GR6, a poorly characterized protein in fetal development (Pekarsky et al. 1997) in the inv(3)(q21q26.2) or the t(3;3)(q21;q26.2) associated with normal or increased platelet counts as well as TEL1 (Raynaud et al. 1996) (discussed above) in the t(3;12) (q26.2;p13). Common features of myeloid diseases associated with abnormalities of 3q are a previous history of cytotoxic exposure, prominent bone marrow dysplasia, and a poor prognosis. Abnormalities of chromosome 7 [±7/del(7q)] are observed in most cases with rearrangements of 3q. In an international workshop on therapy-related hematologic disease, inv(3)/t(3;3) abnormalities were the most frequent of the 3q abnormalities (Block et al. 2002).

67

6.3.2 Evolution of the Karyotype

Serial evaluations can be informative, particularly when there is a change in the clinical features of a patient. The identification of new abnormalities in the karyotype often coincides with a change in the behavior of the disease, usually to a more aggressive course, and may herald incipient leukemia. Cytogenetic evolution is the appearance of an abnormal clone where only normal cells have been seen previously, or the progression from the presence of a single clone (often with a simple karyotype) to multiple related, or occasionally unrelated, abnormal clones. The abnormal clones may evolve, acquiring additional abnormalities with disease progression, and typically resolve with remission of disease following treatment. In published series, most MDS patients die of bone marrow failure, close to half progress to acute leukemia, and a few die of intercurrent illness. The natural history of MDS is generally characterized by one of three clinical scenarios: (1) a gradual worsening of pancytopenia, where the marrow blast count is found to be increasing; (2) a relatively stable clinical course followed by an abrupt change with a clear leukemic transformation; and (3) a stable course over many years without significant change in the marrow blast counts when reevaluated (Hamblin and Oscier 1987). In the first group, the karyotype typically remains stable, and the progression to leukemia is based on the relatively arbitrary finding of greater than 20% blasts (30% in the FAB classification) in the marrow, making the transition to AML a relatively ill-defined event. In the second group, a change in the karyotype with the gain of secondary clones, and complex karyotypes, is typical. Both the karyotype and the disease tend to remain stable in the third group. Few series with sequential cytogenetic studies have been published, and most series are small with short follow-up periods (de Souza Fernandez et al. 2000; Geddes et al. 1990; Horiike et al. 1988). Nonetheless, karyotypic evolution in MDS is associated with transformation to acute leukemia in about 60% of cases, and reduced survival, particularly for those patients who evolve within a short period of time (less than 100 days) (Geddes et al. 1990).

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

6.4 The Genetics of the Myelodysplastic

Syndromes

6.4.1 Molecular Models for Chromosome

Abnormalities in MDS

As described earlier, many of the recurring chromosomal abnormalities in MDS lead to the loss of genetic material. Such loss is the hallmark of tumor suppressor genes, which normally function to control cell growth or cell death by regulating the cell cycle, the response to DNA damage, and apoptosis. A simple ªtwo-hitº model involving a single target tumor suppressor gene (Knudson's model) predicts that loss of function of both alleles must occur for the malignant phenotype to be expressed (Knudson 1971). Loss of gene function may occur by chromosomal deletion or loss, point mutations, or methylation of the regulatory elements of the gene (transcriptional silencing). A clinical example to illustrate this principle is the occurrence of MDS or AML following cytotoxic therapy (t-MDS and t-AML, respectively). A relatively long latency period following cytotoxic exposure precedes bone marrow dysfunction. This latency is compatible with a two-step mechanism in which a second mutation of a target gene must occur in a myeloid progenitor cell. Given two normal alleles at the tumor suppressor gene locus initially, one would be mutated as a result of therapy. Subsequent loss of the second allele in a bone marrow stem cell would permit leukemia development. Alternatively, because AML develops in only 5±15% of patients who are treated for a primary tumor, these individuals may have inherited a predisposing mutant allele; subsequent exposure to cytotoxic therapy may induce the second mutation, giving rise to leukemia. In these cases, characterization of the predisposing mutations will be important in identifying individuals who are at risk of developing t-AML, and in the selection of the appropriate therapy for the primary malignant disease. In an alternative model, loss of only a single copy of a gene may result in a reduction in the level of one or more critical gene products (haploinsufficiency). Several reports implicate haploinsufficiency of the TP53 and p27Kip1 genes in the pathogenesis of tumors in mice, where a substantial percentage of tumors developing in heterozygous mice retain a functional copy of TP53 or p27Kip1 (Fero et al. 1996; French et al. 2001). In humans, haploinsufficiency of the RUNX1 gene results in a familial platelet disorder with a predisposition

to AML (Michaud et al. 2002; Song et al. 1999). Importantly, the few leukemias available for analysis from affected family members appear to retain one normal RUNX1 allele. Support for this mechanism in sporadic cases of MDS and AML with point mutations in the RUNX1 gene is also emerging (Nakao et al. 2004). Despite intensive analysis, homozygous deletions have not been detected in myeloid leukemia cells characterized by deletions of 5q, 7q, or 20q in MDS and AML, an observation that is compatible with a haploinsufficiency model in which loss of one allele of the relevant gene (or genes) alters the cell's fate. Moreover the absence of inactivating mutations in the remaining allele of candidate genes located within the commonly deleted segments of these chromosomes lends further support for the haploinsufficiency model. 6.4.2 Molecular Analysis of the del(5q)

Several groups of investigators have defined a commonly deleted segment (CDS) on the long arm of chromosome 5 predicted to contain a myeloid tumor suppressor gene that is involved in the pathogenesis of MDS and AML (Fig. 6.5) (Fairman et al. 1995; Jaju et al. 1998; Horrigan et al. 2000; Lai et al. 2001; Zhao et al. 1997). By cytogenetic and fluorescence in situ hybridization (FISH) analysis, Le Beau and colleagues defined a 1.5-Mb CDS within 5q31 flanked by D5S479 and D5S500 (Zhao et al. 1997). The function of the genes within this

Fig. 6.6. Ideogram of the long arm of chromosome 5 showing chromosome markers and candidate genes within the commonly deleted segments (CDSs) as reported by various investigators. The proximal CDS in 5q31 was identified in MDS, AML and t-MDS/t-AML, whereas the distal CDS in 5q32±33 was identified in the 5q± syndrome

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6.4 ´ The Genetics of the Myelodysplastic Syndromes

CDS covers a spectrum of activities including regulation of mitosis and the G2 checkpoint, transcriptional and translational regulators, and cell surface receptors. Analysis of myeloid leukemia cells for inactivating mutations has eliminated 20 genes within the CDS, suggesting that a novel myeloid tumor suppressor gene is located in this interval, or that mechanisms such as haploinsufficiency may be involved in the pathogenesis of these disorders (Lai et al. 2001; Zhao et al. 1997). In the 5q± syndrome, molecular analysis of bone marrow cells suggests that a different region is involved. Boultwood and colleagues (2002) identified a 1.5-Mb CDS within 5q32 between D5S413 and GLRA1, which is also gene-rich. This region is distal to the CDS in 5q31 found in the patients with RAEB-1, RAEB-2 and AML with del(5q). Whether all patients with the 5q± syndrome have involvement of a gene in this distal region, and whether this gene plays a role in the pathogenesis of other subtypes of MDS or AML remains to be defined. In summary, the existing data suggest that there are two non-overlapping CDSs in 5q31 and 5q32. The proximal segment in 5q31 is likely to contain a tumor suppressor gene involved in the pathogenesis of both de novo and therapy-related MDS/AML. Band 5q32 is likely to contain a second myeloid tumor suppressor gene involved in the pathogenesis of the 5q± syndrome.

6.4.3 Molecular Analysis of ±7/del(7q)

As with the ±5/del(5q), the breakpoints and extent of the deletions of 7q in patients have been investigated to identify a CDS (Fischer et al. 1997; Johnson et al. 1996; Kere 1989; Le Beau et al. 1996; Liang et al. 1998; Tosi et al. 1999). Two distinct CDSs were identified by molecular analysis of 81 patients with de novo and therapy-related MDS/AML (Le Beau et al. 1996) In 65 patients, the CDS was within q22, whereas in 16 other patients, interstitial deletions of a more distal segment were detected with a CDS of q32±33. Using FISH analysis, an ~2-Mb CDS in 7q22 was defined, which is consistent with most published data (Dæhner et al. 1998; Fischer et al. 1997; Kere 1989; Lewis et al. 1996). Tosi et al. (1999) evaluated patients with 7q abnormalities and identified an interesting patient with a complex karyotype and a t(7;7) who had a deletion associated with the translocation breakpoint of 150 kb proximal to the CDS defined by Le Beau et al. (1996). A number of candidate genes have been identified and evaluated for mutations within the

69

CDS at 7q22, including genes encoding extracellular (or extracellular-like) proteins, replication and transcriptional control elements, a splicing factor kinase and a mitochondrial processing peptidase (Kratz et al. 2001); however, no inactivating mutations have been identified in the remaining allele. Data from cytogenetic, FISH, and loss of heterozygosity (LOH) studies performed in a number of laboratories paint a complex picture of 7q deletions in myeloid malignancies. There is general agreement that 7q22 is involved in the majority of cases. Defining a consistent CDS has been hampered by (1) the relatively low frequency of del(7q) compared with the complete loss of chromosome 7; (2) the use of different techniques to investigate marrow samples, e.g., FISH vs. LOH; (3) the wide clinical spectrum of myeloid disorders with alterations in chromosome 7, suggesting genetic heterogeneity; and (4) the existence of multiple and sometimes complex cytogenetic abnormalities in most cases.

6.4.4 Molecular Analysis of the del(20q)

The majority of deletions of 20q are large with loss of most of the long arm, although cytogenetic analysis of the deleted chromosome 20 homologs has revealed that the deletions are variable in size. By using FISH with a panel of probes from 20q, combined with LOH studies, investigators have identified an interstitial CDS of 4 Mb within 20q12 that is flanked by D20S206 proximally and D20S424 distally, containing a number of genes. Despite the availability of detailed physical maps and transcripts maps, the identity of a myeloid tumor suppressor gene on 20q is unknown (Bench et al. 2000; Wang et al. 2000). The functions of candidate genes within the CDS are diverse, and include transcription factors, components of signal transduction pathways, an RNA transcription modulator, and a regulator of apoptosis (Wang et al. 2000).

6.4.5 Alterations in Gene Function

A growing body of evidence suggests that mutations of multiple genes cooperate to mediate the pathogenesis and progression of MDS. A detailed review of these genes is beyond the scope of this chapter. Table 6.4 provides a partial list and overview of some of the salient features of genes implicated in MDS.

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Table 6.4. Genes altered in myelodysplastic syndromes Gene

Alteration

Associated features

Reference

BCL2

Overexpressed in all FAB subtypes

Encodes a protein product that suppresses apoptosis; No correlation with survival; Highest levels noted in higher risk entities where apoptosis is reduced

Hayes et al. 1997; Lepelley et al. 1995; Parker et al. 2000

CSF1R/ FMS

Mutated in 12±20%, increased with higher risk MDS

Encodes the macrophage colony-stimulating factor receptor with tyrosine kinase activity; Karyotype predominantly normal; Increased frequency of transformation to AML and poor survival

Padua et al. 1998; Ridge et al. 1990

FLT3

Internal tandem duplication (ITD) in ~10% of MDS and AML with trilineage dysplasia

Encodes a class III receptor tyrosine kinase playing a role in stem cell differentiation; ITD results in constitutive activation of protein; Associated with progression to AML and poor prognosis; Frequently observed with normal karyotype in AML

Horiike et al. 1997; Kiyoi et al. 1998

GCSFRG

Point mutations identified

Encodes the G-CSR receptor; Severe congenital neutropenia (SCN) patients with G-CSF receptor defects can progress to MDS and/or AML; Mutation alone is not sufficient for transformation; Progression to leukemia in SCN associated with loss of chromosome 7 and NRAS/KRAS1 mutations

Tidow et al. 1998

HLADR15 (DR2)

Overrepresentation in MDS of RA subtype (36% vs. 21% in normal blood donors)

T T-cell mediated autoimmune mechanism implicated in some forms of MDS; Correlated with response to immunosuppression of carefully defined MDS

Saunthararajah et al. 2002

KIT

Overexpressed; no mutations found

Encodes the stem cell factor receptor; May provide an autocrine growth pathway

Arland et al. 1994; Siitonen et al. 1994

MDR1

Expressed in ~60%

Encodes a transmembrane drug efflux pump; May be involved in resistance of MDS to drug therapy; Associated with monosomy 7

Zochbauer et al. 1994

MDM2

Overexpressed in ~70%

Encodes a protein product (murine double minute-2) which abrogates the function of the p53 tumor suppressor protein via ubiquitination and degradation of p53; Gene amplification not detected; Associated with unfavorable cytogenetic abnormalities; Shorter remission duration

Bueso-Ramos et al. 1995; Faderl et al. 2000

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6.4 ´ The Genetics of the Myelodysplastic Syndromes

71

Table 6.4 (continued) Gene

Alteration

Associated features

Reference

MPL

Overexpressed in ~45% of CMML, and ~40% of RAEB-1,2 patients; underexpressed (~50% of normal levels) in most MDS patients, especially RA

Encodes the thrombopoietin receptor; Higher expression in RAEB-1,2 associated with poor prognosis, increased progression to AML; Correlated with dysmegakaryocytopoiesis

Gouscary et al. 1995; Ogata and Tamura 2000

NF1

Loss and mutations identified, particularly in pediatric MDS/MPS

Encodes neurofibromin, a tumor suppressor gene product, that functions as a GTPase activating (GAP) protein to downregulate RAS function; High incidence of MDS and AML in children with neurofibromatosis type I; No structural alteration in homologous allele in adults with loss of one chromosome 17

Gallagher et al. 1997 b; Shannon et al. 1994

NRAS

Mutated in 20±40%; overexpressed in RA, RARS

Encodes a component of various cytokine signal transduction pathways; Activating mutations result in constitutive signaling; Associated with monocytic component; Increased risk of progression to AML; Overexpression may represent an early event in the multi-step process of transformation

Padua and West 2000

CDKN2B/ p INK4B p15

Decreased expression via gene silencing by DNA methylation in 68% of t-MDS/t-AML

Closely associated with deletion or loss of 7q

Christiansen et al. 2003

p15INK4B p

±

Independently associated with poor survival

PTPN11

Somatic missense mutations in 33% of JMML patients

A non-receptor tyrosine phosphatase that functions as a positive regulator of RAS proteins, mutations activate the phosphatase activity; Mutations of NRAS/KRAS1, NF1 and PTPN11 are mutually exclusive

Loh et al. 2003

Telomerase (including TERT, TR, and TP1)

Increased activity late in disease, particularly TERT

Enzyme complex responsible for chromosome telomere maintenance and replication; Variable levels of activity; Abnormal telomere maintenance may be an early indication of genetic instability; Telomeres shortened with disease progression

Counter et al. 1995; Li et al. 2000; Norrback and Roos 1997; Xu et al. 1998

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Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

Table 6.4 (continued) Gene

Alteration

Associated features

Reference

TP53

Mutated in 5±25%; higher frequency in t-MDS

Encodes G1, S, and G2 checkpoint protein product which monitors integrity of genome; arrests cell cycle in response to DNA damage; Loss of wild type allele; Associated with weak BCL2 expression; Observed as both early and late genetic event in MDS; Associated with rapid progression and poor outcome seen with loss of 17p or ±5/del(5q) suggesting pathogenic exposure to carcinogens; Significantly differentiates worse prognosis within each IPSS subgroup

Kita-Sasai et al. 2001; Misawa and Horiike 1996; Padua et al. 1998

WT1

Associated with overexpression

Overexpressed in 65% of bone marrow specimens and 78% of peripheral blood specimens compared with normal cells, including all RAEB and t-AML samples; Correlated with blast counts and cytogenetic abnormalities; Significantly correlated with IPSS score

Cilloni et al. 2003

The RAS family is the most extensively studied gene family in MDS. RAS proteins are a critical component of signaling pathways from cell-surface receptors to the nucleus, and result in the control of cellular proliferation, differentiation, and cell death. These proteins bind guanine nucleotides, with activation controlled by cycling between the guanosine triphosphate bound (active) and guanosine diphosphate bound (inactive) forms (Rebollo and Martinez 1999). Once activated by a cell surface receptor, RAS proteins induce a cascade of kinase activity, resulting in the transduction of the signals to the nucleus. The RAS signaling cascade is downstream of a number of activated cytokine receptors, including the FLT3, IL3, and GM-CSF receptors; thus, this signaling pathway plays a pivotal role in hematopoiesis. Mutant RAS proteins retain the active GTP-bound form, promoting constitutive activation. The most frequent mutation is a single base change at codon 12 of the protein, but codons 13 and 61 are also frequently mutated. Codons 12 and 13 are located within the pocket that binds GTP, and mutant proteins have decreased phosphatase activity reducing inactivation to the GDPbound form (Neubauer et al. 1994; Gallagher et al. 1997 a; Gallagher et al. 1997 b). Constitutively activating point mutations of NRAS have been detected at high fre-

quency in hematologic malignancies. In MDS, NRAS mutations have been detected in 10±40% of cases. These mutations are associated with a poor prognosis, with higher incidence of transformation to AML and shorter survival. Those patients with both abnormal karyotypes and NRAS mutations have the highest likelihood of transformation (Beaupre and Kurzrock 1999; de Souza Fernandez et al. 1998; Neubauer et al. 1994; Padua et al. 1998; Tien et al. 1994). Many therapeutic molecules entering clinical trials, including the farnesyl transferase inhibitors and imatinib, interrupt various steps in the RAS signaling pathways (Apperley et al. 2002; Kurzrock et al. 2003). One of the paradoxes associated with MDS is the presence of peripheral cytopenias, frequently involving all three lineages (granulocytic, erythroid, and megakaryocytic), with the presence of a hypercellular bone marrow where cells in both the peripheral blood and bone marrow exhibit varying degrees of dysmorphic features. Many genes are involved in the tightly regulated and complex process of apoptosis (programmed cell death), which plays an important role in maintaining normal homeostasis by removing immature and damaged cells. Although some of the findings are conflicting, there is consensus on a number of points. Mea-

a

6.4 ´ The Genetics of the Myelodysplastic Syndromes

surements of cell cycle kinetics demonstrate an increase in the proliferation of all hematopoietic cell lineages, particularly the myeloid cell lines (Parker et al. 2000; Raza et al. 1995). This proliferation is balanced by an increase in apoptosis in MDS. It is well documented that altered cytokine levels play a pivotal role in this process (Westwood and Mufti 2003). The pro-apoptotic tumor necrosis factor alpha (TNFa), transforming growth factor alpha (TGFa), interferon gamma (IFNc) and interleukin-1 beta (IL1b) are increased in MDS (Allampallam et al. 2002; Reza et al. 1999; Yoshida and Mufti 1999). They may function to suppress the growth of hematopoietic progenitors and induce expression of the FAS receptor which, when appropriately triggered, can initiate the apoptotic pathways. The prominent role of some cytokines has been examined in clinical studies. Strategies to neutralize TNFa by decreasing its production with pentoxifylline or thalidomide and with soluble TNFa receptors (to competitively bind the excess TNF) have resulted in clinical responses in a minority of MDS patients (Raza et al. 1996, 2001; Turk et al. 1996).

6.4.6 Genetic Pathways Leading to MDS

Extensive experimental evidence indicates that more than one mutation is required for the pathogenesis of hematological malignant diseases (Alcalay et al. 2001; Kelly et al. 2002). That is, expression of translocationspecific fusion genes or deregulated expression of oncogenes is required, but insufficient by itself to induce leukemia. Thus, an important aspect of leukemia biology is the elucidation of the spectrum of chromosomal abnormalities and molecular mutations that cooperate in the pathways leading to leukemogenesis (Pedersen-Bjergaard et al. 2002). There is growing evidence that a limited number of molecular pathways may be involved. Gilliland and colleagues have described an emerging paradigm in AML, namely, the cooperation between constitutively activated tyrosine kinase molecules such as FLT3 and transcription factor fusion proteins (Kelly et al. 2002). In this model, the activated tyrosine kinase confers a proliferative or anti-apoptotic activity, whereas the fusion protein impairs normal differentiation pathways, but has a limited effect on cellular proliferation. The existing evidence suggests that this model is also applicable to MDS. As described in the previous section, activating mutations of oncogenes, and inactivating mutations of tu-

73

mor suppressor genes have been identified in a number of genes in MDS and AML, and may occur in conjunction with recurring chromosomal abnormalities. For example, ±7/del(7q) has been associated with activating mutations of the RAS pathway (activating KRAS1, NRAS, or PTPN11 mutations, or inactivating mutations of NF1), as well as methylation silencing of the CDKN2B (p15INK4B) gene (Christiansen et al. 2003; Loh et al. 2003; Side et al. 2004). TP53 (p53) mutations are uncommon in this subgroup. In contrast, MDS associated with ±5/del(5q) is associated with TP53 mutations and a complex karyotype (Christiansen et al. 2001; Side et al. 2004). With respect to the t(11;16) observed in a small subset of t-MDS patients, overexpression of the FLT3 gene is characteristic of MLL-associated leukemias (Armstrong et al. 2002). Although our understanding of the association of chromosomal abnormalities with gene mutations in MDS is incomplete, several patterns of cooperating mutations have emerged, suggesting that there are multiple genetic pathways leading to MDS. Gene expression profiling of MDS and t-MDS has also provided support for the concept of distinct molecular and genetic subsets of MDS. Using expression profiling of CD34+ cells in t-MDS and t-AML, Qian et al. (2002) found that patients with a ±5/del(5q) have a higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), and loss of expression of the gene encoding interferon consensus sequence binding protein (ICSBP/ IRF8). A second subgroup of t-AML, including patients with ±7/del(7q) is characterized by down-regulation of transcription factors involved in early hematopoiesis (TAL1, GATA1, and EKLF), and overexpression of proteins involved in signaling pathways in myeloid cells (FLT3), and cell survival (BCL2). Similarly, expression profiling of CD34+ cells from patients with primary MDS revealed that the expression profile of 11 selected genes could accurately distinguish low-risk from highrisk MDS cases (Hofmann et al. 2002). Pellagatti et al. (2004) performed expression profiling analysis of peripheral blood neutrophils in patients with primary MDS, predominantly RA (including seven cases with the 5q± syndrome). In this report, differential expression of 71 genes distinguished patients with the 5q± syndrome from RA patients with a normal karyotype. The cyclic AMP-responsive element modulator (CREM), cylindromatosis tumor suppressor gene (CYLD), and RB1inducible coiled-coil 1 (RB1CC1) genes were expressed at higher levels in the 5q± syndrome group, whereas the

74

Chapter 6 ´ Cytogenetic Diagnosis of Myelodysplastic Syndromes

genes encoding the antioxidant protein 1 (ATOX1), and the SP1 transcriptional activation subunit 9 cofactor (CRSP9) genes were expressed at lower levels. When all patients were considered, those genes upregulated in the most patients included RAB20 (a small GTP-binding protein of the RAS superfamily), ZNF183 (a zinc finger protein), ARG1 (liver arginase) and ACPL (IL18 receptor accessory protein). The genes most commonly down-regulated were COX2 (cyclooxygenase 2), CD18, G-protein coupled receptor 105, FOS, IL7R, ACT2 (immune activation 2), and IFI56 (interferon-inducible 56). Establishing the molecular pathways involved in MDS may facilitate the identification of selectively expressed genes that can be exploited for the development of urgently needed targeted therapies. At present, there are a number of unanswered questions. For example, we do not yet know the full spectrum of genetic mutations in MDS within each pathway, nor do we know the order in which these mutations occur, and the prognostic significance associated with various cooperating mutations. Several possible models are outlined in Fig. 6.7. A variety of experimental evidence suggests that the recurring chromosomal abnormalities in MDS and AML are likely to be the initiating event. With respect to the recurring translocations, the rearrangement is likely to occur in a hematopoietic progenitor cell or, in some cases, in a committed myeloid progenitor cell. Leukemogenesis may entail a linear process in which the initiating mutation leads to a specific pat-

tern of stepwise, additional mutations that complete malignant transformation. In MDS, the process may vary somewhat in that the initiating mutations may occur in a hematopoietic stem cell. In the setting of a normal bone marrow microenvironment, the initiating mutation may result in clonal expansion coupled with emerging genetic instability (or the selection of cooperating mutations that lead to instability), and the development of a clonal population. Selective pressures created both by the microenvironment as well as the initiating events would lead to the acquisition of additional mutations necessary to complete malignant transformation. Alternatively, MDS may arise in the setting of an abnormal bone marrow microenvironment, resulting in the generation of multiple populations with varying initiating events. Some clonal populations may persist, whereas others may undergo cell death, and yet others may go on to acquire additional mutations necessary to complete malignant transformation. The latter model would account for the observation of unrelated cytogenetic clones in the bone marrow of MDS patients, as well as the observation of persistent dysplasia in MDS or AML patients following therapy. Emerging technologies such as the ability to culture stromal cell populations and proteomics and genomics technologies may facilitate the evaluation of these various models.

References

Fig. 6.7. Models for the genetic pathways leading to MDS. See text for a discussion of the alternative models. In the lower panel, the examples of the ±5/del(5q) and ±7/del(7q) are used to illustrate the models of MDS arising in the setting of a normal bone marrow environment vs. an abnormal bone marrow environment, respectively. It is possible that either abnormality can arise in both settings, and that each model may occur

Abe A, Emi N, Tanimoto M, Terasaki H, Marunouchi T, Saito H (1997) Fusion of the platelet-derived growth factor receptor beta to a novel gene CEV14 in acute myelogenous leukemia after clonal evolution. Blood 90:4271±4277 Alcalay M, Orleth A, Sebastiani C, Meani N, Chiaradonna F, Casciari C., Sciurpi MT, Gelmetti V, Riganelli D, Minucci S, Fagioli M, Pelicci PG (2001) Common themes in the pathogenesis of acute myeloid leukemia. Oncogene 20:5680±5694 Allampallam K, Shetty V, Mundle S, Dutt D, Kravitz G, Reddy PL, Alvi S, Galili N, Saberwal GS, Anthwal S, Shaikh MW, York A, Raza A (2002) Biological significance of proliferation, apoptosis, and monocyte/ macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome. International Journal of Hematology 75:289±297 Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ, Chase A, Chessells JM, Colombat M, Dearden CE, Dimitrijevic S, Mahon FX, Marin D, Nikolova Z, Olavarria E, Silberman S, Schultheis C, Cross NC, Goldman JM (2002) Response to imatinib mesylate in patients with chronic myeloproliferative disease with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med 347:481±487

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Toyama K, Ohyashiki K, Yoshida Y, Abe T, Asano S, Hirai H, Hirashima K, Hotta T, Kuramoto A, Kuriya S (1993) Clinical implications of chromosomal abnormalities in 401 patients with myelodysplastic syndromes: a multicentric study in Japan. Leukemia 7:499±508 Turk BE, Jiang H, Liu JO (1996) Binding of thalidomide to alpha 1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production. Proc Natl Acad Sci U S A 93:7552±7556 Vallespi T, Imbert M, Mecucci C, Preudhomme C, Fenaux P (1998) Diagnosis, classification, and cytogenetics of myelodysplastic syndromes. Haematologica 83:258±275 Van den Berghe H, Michaux L (1997) 5q±, twenty-five years later: a synopsis. Cancer Genet Cytogenet 94:1±7 Walker J, Flower D, Rigley K (2002) Microarrays in hematology. Current Opinion in Hematology 9:23±29 Wang P, Spielberger RT, Thangavelu M, Zhao N, Davis EM, Iannantuoni K, Larson RA, Le Beau MM (1997) dic(5;17): a recurring abnormality in malignant myeloid disorders associated with mutations of TP53. Genes Chromosomes Cancer 20:282±291 Wang PW, Eisenbart JD, Espinosa III R, Davis EM, Larson RA, Le Beau MM (2000) Refinement of the smallest commonly deleted segment of chromosome 20 in malignant myeloid diseases and development of a PAC-based physical and transcription map. Genomics 67:28±39 Wattel E, Lai JL, Hebbar M, Preudhomme C, Grahek D, Morel P, Bauters F, Fenaux P (1993) De novo myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 20: a subtype of MDS with distinct hematological and prognostic features? Leuk Res 17:921±926 Weimar IS, Bourhis JH, De Gast GC, Gerritsen WR (1994) Clonality in myelodysplastic syndromes. Leukaemia Lymphoma 13:215±221 Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, Deeg HJ (2003) Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood 102:394±403 West RR, Stafford DA, White AD, Bowen DT, Padua RA (2000) Cytogenetic abnormalities in the myelodysplastic syndromes and occupational or environmental exposure. Blood 95:2093±2097 Westwood NB, Mufti GJ (2003) Apoptosis in the myelodysplastic syndromes. Curr Hematol Rep 2:186±192 Wiktor A, Rybicki BA, Piao ZS, Shurafa M, Barthel B, Maeda K, Van Dyke DL (2000) Clinical significance of Y chromosome loss in hematologic disease. Genes Chromosomes Cancer 27:11±16 Xu D, Gruber A, Peterson C, Pisa P (1998) Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia. Br J Haematol 102:1367±1375 Yoshida Y, Mufti GJ (1999) Apoptosis and its significance in MDS: controversies revisited. Leuk Res 23:777±785 Zhao N, Stoffel A, Wang PW, Eisenbart JD, Espinosa R, 3rd, Larson RA, Le Beau MM (1997) Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1±1.5 Mb and preparation of a PAC-based physical map. Proc Natl Acad Sci U S A 94:6948±6953 Zochbauer S, Gsur A, Gotzl M, Wallner J, Lechner K, Pirker R (1994) MDR1 gene expression in myelodysplastic syndrome and in acute myeloid leukemia evolving from myelodysplastic syndrome. Anticancer Res 14:1293±1295

Myelodysplastic Syndrome in Children Charlotte M. Niemeyer

Contents 7.1 Introduction . . . . . . . . . . . . . . . . . . . .

81

7.2 Classification of Childhood MDS . . . . .

81

7.3 Cytogenetics . . . . . . . . . . . . . . . . . . . .

82

7.4 Primary MDS . . . . . . . . . . . . . . . . . . . 7.4.1 Primary MDS Without Increase in Blast Count (Refractory Cytopenia [RC]) . . 7.4.2 Primary MDS with Increased Blasts (RAEB and RAEBt) . . . . . . . . . . . . .

83 83 84

7.5 Secondary MDS . . . . . . . . . . . . . . . . . 7.5.1 MDS in Congenital Bone Marrow Failure . . . . . . . . . . . . . . . . . . . . . 7.5.2 MDS After Acquired Aplastic Anemia 7.5.3 Familial MDS . . . . . . . . . . . . . . . . 7.5.4 Therapy-related MDS . . . . . . . . . . .

85 85 86 86

Summary . . . . . . . . . . . . . . . . . . . . . . . . . .

86

References . . . . . . . . . . . . . . . . . . . . . . . . .

86

85

7.1 Introduction

Myelodysplastic syndrome (MDS) is much rarer in children than in adults, accounting for less than 5% of hematopoietic neoplasia in childhood. Population-based data suggest an annual incidence of 1±2/million (Hasle et al. 1995, 1999 b; Passmore et al. 2003). There are significant differences in presentation, underlying cytogenetic abnormalities, and classification between MDS in children and adults. The therapeutic aim in children with MDS is a

cure, and therapeutic efforts concentrate on hematopoietic stem cell transplantation (HSCT) rather than on novel therapeutics such as anti-angiogenic therapy, farnesyl transferase, or DNA methylation inhibitors.

7.2 Classification of Childhood MDS

MDS in childhood is a very heterogeneous group of disorders associated with a variety of different clinical conditions. This diversity hampered a generally accepted classification in the past. Although some investigators have argued that childhood MDS can be classified according to the French-American-British (FAB) nomenclature in the same subgroups as adult cases (Brandwein et al. 1990; Hasle et al. 1995, 1999 b; Passmore et al. 2003), others pointed out the system was rarely used in practice (Bader-Meunier et al. 1996; Luna-Fineman et al. 1999). In addition, an infantile monosomy 7 syndrome characterized by male predominance, hepatosplenomegaly, and leukocytosis had been included as a separate entity in the classification of childhood MDS (Passmore et al. 1995). Because complete loss of chromosome 7 occurs in all morphologic MDS subgroups (Hasle et al. 1999 a) and there is no evidence that monosomy 7 represents a discrete entity, it should no longer be referred to as the monosomy 7 syndrome (Hasle et al. 1999 a; Woods et al. 2002). Similar to the FAB classification, the recent World Health Organization (WHO) classification of neoplastic diseases of the hematopoietic and lymphoid tissues (Jaffe et al. 2001) is based on a review of adult cases. It recognizes juvenile myelomonocytic leukemia (JMML) as a distinct entity, but the subdivision of

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Chapter 7 ´ Myelodysplastic Syndrome in Children

Table 7.1. Classification of MDS and myelodysplastic// myeloproliferative disorders of childhood I. Myelodysplastic/myeloproliferative disease Juvenile myelomonocytic leukemia (JMML) Chronic myelomonocytic leukemia (CMML) (secondary only) BCR-ABL-negative chronic myeloid leukemia (Ph-CML) II. Down syndrome disease Transient abnormal myelopoiesis (TAM) Myeloid leukemia of Down syndrome III. Myelodysplastic syndrome (MDS) Refractory cytopenia (RC) (PB blasts < 2% and BM blasts < 5%) Refractory anemia with excess blasts (RAEB) (PB blasts 2±19% or BM blasts 5±19%) RAEB in transformation (RAEBt) (PB or BM blasts 20±29%)

MDS does not reflect the hematological and clinical picture of MDS in childhood. To account for the special features of MDS in children, an international consensus on a pediatric modification of the WHO classification has been proposed (Table 7.1) (Hasle et al. 2003). Myelodysplastic and myeloproliferative disorders in children are separated into three main groups: JMML, MDS, and Down syndrome. JMML is a unique disorder of infancy characterized by a hyperactive RAS signaling pathway due to molecular aberrations in the genes encoding for SHP-2, RAS, or neurofibromatosis type 1. MDS in Down syndrome in the first 5 years of life is not biologically different from AML in these patients. The unifying term ªmyeloid leukemia of Down Syndromeº is proposed for this disorder, and patients should be excluded from MDS series. Childhood MDS, arising de novo or secondary to a predisposing condition, is subdivided into refractory cytopenia (RC), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBt) (Hasle et al. 2003). The term ªRCº rather than ªrefractory anemia (RA)º was chosen because thrombocytopenia and neutropenia are more frequently observed than anemia (Kardos et al. 2003). In childhood MDS, RC with ringed sideroblasts is infrequently found, the unique 5q± syndrome has not been described, and the importance of multilineage dysplasia

in RA is unknown. Consequently, these adult subtypes were omitted from the pediatric WHO modification. For the definition of MDS, the WHO classification eliminated RAEBt (Jaffe et al. 2001). Because there are no data indicating whether a blast threshold of 20% is better than the traditional 30% to distinguish MDS from de novo AML in children, the International Consortium suggested retaining the subtype of RAEBt with 20±30% blasts in the bone marrow until more data are available. It should be emphasized, however, that the blast count is insufficient to differentiate de novo AML from MDS (see below). MDS after prior chemo- or radiation therapy, prior acquired aplastic anemia, in congenital bone marrow failure disorders, and in familial disease are classified as secondary MDS (Hasle et al. 2003). All other cases are named primary MDS, although it is reasonable to assume that most of these disorders are secondary to some yet unknown genetic predisposition. These presumed underlying genetic changes may also give rise to subtle phenotypic abnormalities observed in many children with primary MDS. The Toronto group suggested a descriptive CCC classification schema in which the first C stands for ªcategory of underlying disease,º the second C for ªcytology,º and the third C for ªcytogeneticsº (Mandel et al. 2002). Cytology is used to subdivide both RC and RAEB into three subgroups based upon level of dysplasia. The CCC classification emphasizes the importance of underlying pathogenetic factors because they may determine treatment preferences. However, due to an infinite number of possible subgroups, the CCC system will be difficult to use in clinical practice. Although there is no perfect system for classifying pediatric MDS, lumping as proposed in the modified WHO approach may be more appropriate for classification and clinical studies than splitting as proposed in the CCC system.

7.3 Cytogenetics

The frequency of an abnormal karyotype in hematopoietic cells varies among subtypes of MDS in childhood (Table 7.2). In advanced primary MDS, about 60% of children have chromosomal aberrations (Groupe Francais de Cytogenetique Hematologique 1997; Luna-Fineman et al. 1999). In contrast to AML, numerical abnormalities dominate; structural abnormalities are frequently part of a complex karyotype with nu-

a

7.4 ´ Primary MDS

83

Table 7.2. Results of cytogenetics of patients with primary MDS and with MDS secondary to chemo- or radiation therapy. Interim analysis of Study EWOG-MDS 98 Karyotype

Primary MDS (%) All (n = 199)

Complex (³ 3 abnormalities) Monosomy 7

RC (n = 105)

RAEB/RAEBt (n = 94)

MDS secondary to chemo- or radiation therapy (%) (n (n = 44)

6

3

9

36

24

17

32

23

Trisomy 8

4

3

5

0

Other abnormalities

9

5

14

23

57

72

40

18

Normal karyotype

meric aberrations. Monosomy 7 is the most common cytogenetic abnormality being identified in approximately 25% of cases. In the absence of standard banding cytogenetics, in situ hybridization (FISH) for identification in monosomy 7 is helpful (Ketterling et al. 2002), although the importance of small clones (< 30%) of monosomy 7 cells remains unknown. Constitutional trisomy 8 mosaicism may remain unrecognized and should be tested for when trisomy 8 is found in the bone marrow. Monosomy 7 is associated with a shorter time to progression in RC of childhood (Kardos et al. 2003). In advanced MDS, monosomy 7 as the sole cytogenetic aberration has not been an unfavorable feature in most studies (Hasle et al. 1999a; Woods et al. 2002), in contrast to findings in adults. Favorable cytogenetic aberrations, ±Y, 20q± and 5q±, have been reported in adults, but these aberrations are so infrequent in children that they are of no practical importance. AML-specific translocations, including t(8;21)(q22;q22), t(15;17)(q22;q12), or inv(16)(p13q22), may occur in cases of de novo AML with a low blast cell count. Their response to therapy is favorable, and they should not be considered MDS (Chan et al. 1997). In an ongoing prospective trial of the European Working Group of MDS in Childhood (EWOG-MDS) (Rogge and Niemeyer 2000), the distribution of karyotypes in advanced MDS shows normal cytogenetics in hematopoietic cells in 40% of patients, while 9% have three or more aberrations (complex karyotype) (Table 7.2). In contrast, only 18% of children with MDS secondary to chemo- or radiation therapy display a normal karyotype, while a complex karyotype is noted in 36%.

7.4 Primary MDS

In primary MDS the main diagnostic challenges are to differentiate low-grade MDS from aplastic anemia or congenital bone marrow failure syndromes, and highgrade MDS from AML.

7.4.1 Primary MDS Without Increase in Blast

Count (Refractory Cytopenia [RC])

MDS with less than 5% blasts in the bone marrow is particularly difficult to diagnose because dysplasia of hematopoietic cells occurs frequently in children with a variety of disease states. In the absence of a cytogenetic marker, the clinical course will have to be carefully evaluated before a diagnosis of RC can be established. Minimal diagnostic criteria for pediatric MDS have been proposed by an International Consortium (Hasle et al. 2003). At least two of the following criteria must be fulfilled: sustained unexplained cytopenia, at least bilineage morphologic dysplasia, acquired clonal cytogenetic abnormality, and increased myeloblasts (³ 5% in bone marrow) (Hasle et al. 2003). RC is the most common subtype of childhood MDS, accounting for about half of the cases (Passmore et al. 2003). As in adults, MDS without increase in blast count in children and adolescents can present with cytopenia and a hyperplastic bone marrow. However, in a retrospective study on RC in childhood, about half of the cases had decreased marrow cell content (Kardos et al. 2003). In the ongoing prospective EWOG-MDS trial (Rogge and Niemeyer 2000), the percentage of hypocellular RC was 76% (Fig. 7.1). Compared with normo- or

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Chapter 7 ´ Myelodysplastic Syndrome in Children

no information normal karyotype

Fig. 7.1. Cellularity and karyotype in 158 children with refractory cytopenia. Increased or normal cellularity for age was noted in 40 patients (24%) while 118 (76%) had decreased cellularity. Interim analysis of study EWOG-MDS 98 (November 2004)

Fig. 7.2. Cumulative incidence of progression to advanced MDS for patients with refractory cytopenia and either monosomy 7, trisomy 7 or normal karyotype at the time of diagnosis. Patients who underwent stem cell transplantation were censored at the time of transplantation (adjusted from [12])

hypercellular bone marrows, the percentage of patients with monosomy 7 was 11% in hypocellular marrows, while the percentage of children with normal karyotypes was 64% (Fig. 7.1). This observation, together with a low rate of leukemic transformation, raises the question whether some of the children with hypocellular RC and a normal karyotype have an unrecognized congenital disorder with dysplasia and marrow failure rather than acquired MDS. In addition, the separation of RC from acquired severe aplastic anemia (SAA) can

be difficult. Differences in cytogenetic composition or response to immunosuppressive therapy among MDS and SAA study populations may depend at least in part on patient selection. Karyotype is the most important factor for progression of RC to advanced MDS and shortened survival (Kardos et al. 2003) (Fig. 7.2). The median time to progression for children with RC and monosomy 7 is less than 2 years. In contrast to patients with monosomy 7, patients with trisomy 8 and other karyotypes may experience a long stable course of their disease. HSCT from a human leukocyte antigen (HLA)-compatible related or unrelated donor early in the course of their disease is the treatment of choice for patients with RC and monosomy 7 (Anderson et al. 1996; Kardos et al. 2003). For children with normal karyotypes or chromosomal abnormalities other than monosomy 7 and absence of transfusion dependence or neutropenia, a watch and wait strategy can be appropriate. If cytopenia necessitates treatment, current therapeutic options include HSCT with either myeloablative or reduced-intensity (Strahm et al. 2003) preparative therapies. Some patients will respond to immunosuppressive therapy with cyclosporine and anti-lymphocyte/thymocyte globulin (Peters et al. 2003). Whether immunosuppression can result in sustained responses in a substantial number of children with RC is currently unknown. Therapy with hematopoietic growth factors, differentiating agents, or hypomethylating agents is generally felt not to be indicated in children and adolescents with RA because none of these approaches has been shown to prolong survival.

7.4.2 Primary MDS with Increased Blasts

(RAEB and RAEBt)

There is consensus that the relationship between MDS and de novo AML is better defined by biological and clinical behavior than by myeloblast count. Consequently, myeloid disease with low blast count and cytogenetic abnormalities typically associated with de novo AML is classified as AML. Because monosomy 7 is the only chromosomal abnormality strongly suggestive of MDS, children presenting with a low blast count and other chromosomal aberrations or normal karyotype have to be observed closely before a diagnosis of MDS can be established. Disease with rapid increase in marrow blasts or organ infiltration should be considered de novo AML, which is by far the more common disorder.

a

Probability of Event-free Survival

7.5 ´ Secondary MDS

Fig. 7.3. Event-free survival of 88 patients with primary advanced MDS transplanted after a preparative regimen with busulfan 16 mg/ kg, cyclophosphamid 120 mg/kg and melphalan 140 mg/m2 from an HLA-identical/1 Ag mismatched family donor (MFD) (N = 38) or an HLA-compatible/1 Ag mismatched unrelated donors (UD) (N = 50) (Niemeyer et al. 2004)

To allow for the interface between MDS and de novo AML, RAEBt was retained in the pediatric classification until new data become available (Hasle et al. 2003). The most appropriate therapy for children with RAEB or RAEBt is unknown (Niemeyer et al. 2004; Webb et al. 2002; Woods et al. 2001). Although most investigators agree that HSCT can improve survival and is the treatment of choice, the importance of cytoreductive therapy prior to grafting remains controversial (Niemeyer et al. 2004; Webb et al. 2002). Data from the EWOG-MDS study indicate that intensive chemotherapy prior to HSCT will not improve survival (Niemeyer et al. 2004). In this study of advanced primary MDS, 88 patients with advanced MDS were given an unmanipulated graft after a preparative regimen with busulfan (16 mg/kg), cyclophosphamide (120 mg/kg), and melphalan (140 mg/m2). Thirtyeight patients were transplanted from HLA-identical/ 1 antigen mismatched family donors, while 50 children received transplants from HLA-compatible/1 Ag mismatched unrelated donors. The event-free survival at 5 years was 68% and 46% for HSCT from matched family and unrelated donors, respectively (Fig. 7.3). While the highest FAB type prior to SCT predicted relapse, with cumulative incidence rates increasing from RAEB to RAEBt and myelodysplasia-related AML, the use of intensive chemotherapy prior to SCT or blast percentage at SCT did not. Hopefully, well-controlled international clinical trials will resolve the issues on pre-HSCT remission induction therapy, optimal preparative regimen, and stem cell source in the future.

85

7.5 Secondary MDS 7.5.1 MDS in Congenital Bone Marrow Failure

Among the congenital bone marrow failure disorders, Fanconi anemia is the one most frequently evolving to hematopoietic neoplasia. MDS or AML develop in as many as 50% of patients with Fanconi anemia before the age of 40 years (Kutler et al. 2003). Because most patients with Fanconi anemia have a dysplastic bone marrow and the definition of clonality is problematic, it is difficult to diagnose MDS in Fanconi anemia in the absence of an increased blast count. Because the natural history and therapy differ between MDS in Fanconi anemia and other patients, therapy results should be reported separately. For patients with severe congenital neutropenia (Kostmann syndrome), an incidence of MDS/AML of about 13% after 8 years of granulocytecolony stimulating factor (G-CSF) treatment has been reported (Dale et al. 2003). In most Kostmann patients with MDS/AML, acquired mutations in the G-CSF receptor are noted, and partial or complete loss of chromosome 7 is found in more than half (Kalra et al. 1995; Tidow et al. 1997). MDS may occur in as many as one third of patients with Shwachman-Diamond syndrome (Smith 2002), but has less frequently been described in patients with Diamond-Blackfan anemia (Hansen and Martin 1982; Janov et al. 1996; Martin et al. 1981). It is noteworthy that not all bone marrow failure syndromes predispose to the development of MDS, e.g., patients with dyskeratosis congenita develop bone marrow failure (aplastic anemia) in 95% of the cases but MDS is rare (Dokal 2000). 7.5.2 MDS After Acquired Aplastic Anemia

MDS develops in 10±15% of children with aplastic anemia not treated with HSCT (Ascensao et al. 1976; Kojima et al. 2002; Locasciulli et al. 2001; Rosenfeld et al. 1985; Tichelli et al. 1988). Most cases of MDS in children are diagnosed within the first 3 years from presentation with aplastic anemia. The fast progression to MDS raises the question whether at least some of the patients had MDS from the beginning. One might also speculate that there is a biological overlap between aplastic anemia and hypoplastic RC.

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Chapter 7 ´ Myelodysplastic Syndrome in Children

7.5.3 Familial MDS

Familial occurrence of MDS, especially with ±7/7q-, has been reported in a number of cases (Anasetti et al. 1987; Hasle and Olsen 1997; Shannon et al. 1989; Singer et al. 1983; Storb et al. 1983; Thomas et al. 1984). Some families show discordance for ±7; therefore, it is uncertain whether ±7 per se increases the risk for familiar cases. The inherited predisposing locus in familial MDS or AML with ±7/7q- may not be located on chromosome 7 (Gao et al. 2000). Familial MDS does also occur without ±7/7q- (Press et al. 1986).

7.5.4 Therapy-related MDS

New intensive treatment protocols for various disorders may lead to an increased risk of therapy-related diseases (Barnard et al. 2002). Children with MDS secondary to chemo- or radiation therapy generally have a poor survival (Sasaki et al. 2001). Even if remission can be achieved with AML-type therapy, only very few patients remain disease-free, and only HSCT offer cure in about a third of the patients (Badger and Bernstein 1983; L'Esperance et al. 1975).

Summary The proposal of the pediatric modification of the WHO classification of MDS reflects our current state of knowledge. With additional information on the underlying biological processes concepts and therapies will change. In children with refractory cytopenia a watch and wait strategy or immunosuppressive therapy can be appropriate in the absence of monosomy 7. In all other cases, HSCT is the treatment of choice. With the possibility to cure at least half of the children with MDS, there is very little room for palliative therapy approaches.

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Niemeyer CM, Ptoszkova H, Ritter J, Slater R, Stary J, StollmannGibbels B, Testi AM, van Wering ER, Zimmermann M (1999a) Myelodysplastic syndrome, juvenile myelomonocytic leukemia, and acute myeloid leukemia associated with complete or partial monosomy 7. Leukemia 13:376±385 Hasle H, Wadsworth LD, Massing BG, McBride M, Schultz KR (1999b) A population-based study of childhood myelodysplastic syndrome in British Columbia, Canada. Br J Haematol 106:1027±1032 Hasle H, Niemeyer CM, Chessells JM, Baumann I, Bennett JM, Kerndrup G, Head DR (2003) A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases (Review). Leukemia 17:277±282 Jaffe ES, Harris NL, Stein H, Vardiman JW (ed) (2001) World Health Organization classification of tumours. Pathology & genetics: tumours of hematopoietic and lymphoid tissues. IARCPress, Washington, DC Janov AJ, Leong T, Nathan DG, Guinan EC (1996) Diamond-Blackfan anemia. Natural history and sequelae of treatment. Medicine 75:77±78 Kalra R, Dale D, Freedman M, Bonilla MA, Weinblatt M, Ganser A, Bowman P, Abish S, Priest J, Oseas RS (1995) Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia. Blood 86:4579±4586 Kardos G, Baumann I, Passmore SJ, Locatelli F, Hasle H, Schultz KR, Stary J, Schmitt-Graeff A, Fischer A, Harbott J, Chessells JM, Hann I, Fenu S, Rajnoldi AC, Kerndrup G, Van Wering E, Rogge T, Nollke P, Niemeyer CM (2003) Refractory anemia in childhood: a retrospective analysis of 67 patients with particular reference to monosomy 7. Blood 102:1997±2003 Ketterling RP, Wyatt WA, VanWier SA, Law M, Hodnefield JM, Hanson CA, Dewald GW (2002) Primary myelodysplastic syndrome with normal cytogenetics: utility of 'FISH panel testing' and M-FISH. Leuk Res 26:235±240 Kojima S, Ohara A, Tsuchida M, Kudoh T, Hanada R, Okimoto Y, Kaneko T, Takano T, Ikuta K, Tsukimoto I (2002) Risk factors for evolution of acquired aplastic anemia into myelodysplastic syndrome and acute myeloid leukemia after immunosuppressive therapy in children. Blood 100:786±790 Kutler DI, Singh B, Satagopan J, Batish SD, Berwick M, Giampietro PF, Hanenberg H, Auerbach AD (2003) A 20-year perspective on the International Fanconi Anemia Registry (IFAR). Blood 101:1249± 1256 L'Esperance P, Hansen JA, Jersild C, O'Reilly R, Good RA, Thomsen M, Nielsen LS, Svejgaard A, Dupont B (1975) Bone-marrow donor selection among unrelated four-locus identical individuals. Transplant Proc [Suppl 1]:823±831 Locasciulli A, Arcese W, Locatelli F, Di Bona E, Bacigalupo A (2001) Treatment of aplastic anaemia with granulocyte-colony stimulating factor and risk of malignancy. Lancet 357:43±44 Luna-Fineman S, Shannon KM, Atwater SK, Davis J, Masterson M, Ortega J, Sanders J, Steinherz P, Weinberg V, Lange BJ (1999) Myelodysplastic and myeloproliferative disorders of childhood: a study of 167 patients. Blood 93:459±466 Mandel K, Dror Y, Poon A, Freedman MH (2002) A practical, comprehensive classification for pediatric myelodysplastic syndromes: the CCC system [republished from J Pediatr Hematol Oncol 2002 24:343±352]. J Pediatr Hematol Oncol 24:596±605

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Martin PJ, Hansen JA, Siadak AW, Nowinski RC (1981) Monoclonal antibodies recognizing normal human T lymphocytes and malignant human B lymphocytes: a comparative study. J Immunol 127:1920± 1923 Niemeyer CM, Zecca M, Korthoff E, Duffner U, Zintl F, Ebell W, Stary J, Dilloo D, Peters C, Schmugge M, Sedlacek P, Messina C, van Heuvel M, Bergstraesser E, Trebo M, Noelke P, Locatelli F (2004) Allogeneic stem cell transplantation for children with advanced primary MDS: results from the EWOG-MDS Study Group employing a pre-transplant preparative regimen with busulfan, cyclophosphamide and melphalan (Abstract). Blood 104:632 a Passmore SJ, Hann IM, Stiller CA, Ramani P, Swansbury GJ, Gibbons B, Reeves BR, Chessells JM (1995) Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood 85:1742±1750 Passmore SJ, Chessells JM, Kempski H, Hann IM, Brownbill PA, Stiller CA (2003) Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia in the UK: a population-based study of incidence and survival. Br J Haematol 121:758±767 Peters AMJ, Baumann I, Strahm B, Gerecke A, Bergstraesser E, Burdach S, Dilloo D, Fenu S, Holter W, Klingebiel T, Koscielniak E, Welte K, Fischer A, Fuehrer M, Niemeyer CM (2003) Immunosuppressive therapy for children with refractory anemia (Abstract). Bone Marrow Transplant 31[Suppl 1]: S183, #P670 Press OW, Vitetta ES, Martin PJ (1986) A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins. Immunol Lett 14:37±41 Rogge T, Niemeyer CM (2000) Myelodysplastic syndromes in childhood. Onkologie 23:18±24 Rosenfeld M, Bowen-Pope DF, Singer JW, Ross R (1985) Responsiveness of the in vitro hematopoietic microenvironment to platelet-derived growth factor. Leuk Res 9:427±434 Sasaki H, Manabe A, Kojima S, Tsuchida M, Hayashi Y, Ikuta K, Okamura I, Koike K, Ohara A, Ishii E, Komada Y, Hibi S, Nakahata T, MDS Committee of the Japanese Society of Pediatric Hematology J (2001) Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan. Leukemia 15:1713±1720 Shannon KM, Turhan AG, Chang SS, Bowcock AM, Rogers PC, Carroll WL, Cowan MJ, Glader BE, Eaves CJ, Eaves AC (1989) Familial bone marrow monosomy 7. Evidence that the predisposing locus is not on the long arm of chromosome 7. J Clin Invest 84:984±989 Singer JW, Keating A, Ramberg R, McGuffin R, Sanders JE, Sale G, Fialkow PJ, Thomas ED (1983) Long-term stable hematopoietic chimerism following marrow transplantation for acute lymphoblastic leukemia: a case report with in vitro marrow culture studies. Blood 62:869±872 Smith OP (2002) Shwachman-Diamond syndrome (Review). Semin Hematol 39:95±102 Storb R, Deeg HJ, Thomas ED, Buckner CD, Clift RA, Flournoy N, Kennedy MS, Doney K, Appelbaum FR, Sanders JE, Stewart P, Shulman H, Sullivan KM, Witherspoon RP (1983) Preliminary results of prospective randomized trials comparing methotrexate and cyclosporine for prophylaxis of graft-vs.-host-disease after HLA-identical marrow transplantation. Transplant Proc 15:2620±2623 Strahm B, Greil J, Kremens B, Peters C, Stary J, Vormoor J, Zintl F, Rogge T, Locatelli F, Niemeyer CM (2003) A new conditioning regimen for patients with refractory anemia and congenital disorders (Abstract). Bone Marrow Transplant 31[Suppl 1]: S26, #198

88

Chapter 7 ´ Myelodysplastic Syndrome in Children

Thomas ED, Clift RA, Storb R (1984) Indications for marrow transplantation. Annu Rev Med 35:1±9 Tichelli A, Gratwohl A, Wursch A, Nissen C, Speck B (1988) Late haematological complications in severe aplastic anaemia. Br J Haematol 69:413±418 Tidow N, Pilz C, Teichmann B, Muller-Brechlin A, Germeshausen M, Kasper B, Rauprich P, Sykora KW, Welte K (1997) Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia. Blood 89:2369±2375 Webb DK, Passmore SJ, Hann IM, Harrison G, Wheatley K, Chessells JM (2002) Results of treatment of children with refractory anaemia

with excess blasts (RAEB) and RAEB in transformation (RAEBt) in Great Britain 1990-1999 (Review). Br J Haematol 117:33±39 Woods WG, Neudorf S, Gold S, Sanders J, Buckley JD, Barnard DR, Dusenbery K, DeSwarte J, Arthur DC, Lange BJ, Kobrinsky NL (2001) A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission: a report from the Children's Cancer Group. Blood 97:56±62 Woods WG, Barnard DR, Alonzo TA, Buckley JD, Kobrinsky N, Arthur DC, Sanders J, Neudorf S, Gold S, Lange BJ (2002) Prospective study of 90 children requiring treatment for juvenile myelomonocytic leukemia or myelodysplastic syndrome: a report from the Children's Cancer Group. J Clin Oncol 20:434±440

Management of Patients with Myelodysplastic Syndromes: Introductory Concepts David T. Bowen

Contents 8.1 Introduction . . . . . . . . . . . . . . . . . . . .

89

8.2 Disease Heterogeneity . . . . . . . . . . . .

89

8.3 Evaluating Clinical Trial Data: Response Criteria . . . . . . . . . . . . . . . . . . . . . . . .

90

8.4 Guidelines for the Diagnosis and Management of MDS . . . . . . . . . .

90

References . . . . . . . . . . . . . . . . . . . . . . . . .

93

8.1 Introduction

The management discussion presents sizable challenges for physician and myelodysplastic syndrome (MDS) patient alike. Firstly, a confident diagnosis is not always possible, particularly in patients with refractory anemia plus other confounding medical conditions (e.g., autoimmune disease, rheumatoid arthritis) where honesty requires discussion with the patients about the balance of probabilities. Secondly, assuming a confident diagnosis, a prognosis should be established to inform discussion about treatment options. The International Prognostic Scoring System (IPSS) score (Greenberg et al. 1997) and the World Health Organization (WHO) classification (Bennett 2000) provide valuable prognostic guidance, but only in broad terms. Thirdly, co-morbidity is common in this predominantly older patient population, restricting the use of more aggressive interventions such as chemotherapy and stem cell transplanta-

tion. Finally, the physician and patient must evaluate the efficacy of all interventions from the medical literature, with inevitable interpretive difficulties on both sides. 8.2 Disease Heterogeneity

The heterogeneity of the group of diseases collectively known as the myelodysplastic syndromes is obvious, but is often overlooked or over-simplified when considering the management recommendations for an individual patient. It is easy to comprehend the differences between diseases with discrepant morphological features such as refractory anemia with ringed sideroblasts (RARS) versus refractory anemia with excess blasts. What is less accessible is the considerable heterogeneity within individual disease categories, which is only starting to be disentangled with the progression from French-American-British (FAB) to WHO classification (witness the WHO distinction between RARS and refractory cytopenia with multilineage dysplasia RCMD ± RS). The WHO classification clearly has improved prognostic power in comparison with FAB (Germing et al. 2000) and is now a better complementary prognostic tool together with the IPSS score. While the IPSS score allows discussion of median survival times with patients, the ranges (confidence intervals) are not provided and clinical experience indicates that these are wide.

90

Chapter 8 ´ Management of Patients with Myelodysplastic Syndromes: Introductory Concepts

8.3 Evaluating Clinical Trial Data:

Response Criteria

The acceptance of new interventions into routine management is dependent upon a solid evidence base for its efficacy and safety. The demonstration of efficacy of an interventional agent will be judged by its ability to produce clinical or hematological improvement or both to an extent that is considered clinically meaningful by both physician and patient. The debate surrounding the clinical use of recombinant erythropoietin in the management of MDS is a good example. An International Working Group (IWG) has attempted to address this issue producing standardized response criteria for MDS patients treated with interventional therapy (Cheson et al. 2000). These criteria include responses that alter the natural history of MDS, cytogenetic response, quality of life, and hematologic improvement (HI) (Table 8.1). Also included are recommended study endpoints. These standardized response criteria are perhaps most useful for Hematologic Improvement, given that those criteria describing the alteration of natural history of the disease are similar to those in routine use for the therapy of acute leukemia, with a few notable exceptions including criteria for disease progression. Hematologic Improvement is now subdivided by lineage and defined in terms of quality (major/minor). Importantly, a minimum qualifying duration of response (at least 2 months) is also included. While considered by some to be somewhat cumbersome (Raza 2001), at least this approach attempts to dissect the nature of response, allowing the reader to distinguish responses that they would consider clinically meaningful (e.g., major erythroid response with transfusion independence) from those that may be less so (e.g., a minor platelet response in a patient with no clinical bleeding, incrementing from 25,000/mm3 to 50,000/mm3). The IWG group accept that these criteria are a useful template to inspire further work in this area (Cheson et al. 2001). Some important discussion points have emerged, including a lack of standardization in the reporting of baseline parameters, the time points at which response should be evaluated (Raza 2001), and the relevance of small increments in neutrophil counts (Steensma et al. 2001). The quality of response may need to be more stringent, given that at least in the context of therapy with hematopoietic growth factors, ªcomplete erythroid responsesº using a more stringent definition

than the IWG criteria for HI-E major, are considerably more durable and clinically desirable than ªpartial responsesº which would still be considered as HI-E major (Hellstrom-Lindberg et al. 2003). Finally the IWG criteria do not address the issue of potential detrimental effects of interventional therapy, an example of which is progressive thrombocytopenia in some patients treated with recombinant erythropoietin plus granulocyte-colony stimulating factor (Hellstrom-Lindberg et al. 1997), nor potential beneficial effects in patients with rare variants of MDS such as a patient with thrombocytosis whose platelet count is controlled on novel therapy such as thalidomide (personal observation).

8.4 Guidelines for the Diagnosis and

Management of MDS

Expert working groups from three countries have published guidelines to aid practicing physicians in the dayto-day management of MDS patients (Alessandrino et al. 2002; Bowen et al. 2003; Greenberg et al. 2004). Each guideline was developed using different methodology, from consensus statement (National Cancer Center Network [NCCN]) (Greenberg et al. 2004), through consensus plus comprehensive literature review (UK) (Bowen et al. 2003), to systematic review plus scenarios (Italian) (Alessandrino et al. 2002) (Table 8.2). It is reassuring that all guidelines have broadly drawn the same conclusions, which is largely a consequence of the limited high-quality evidence base for most interventions considered. Two guidelines (UK and NCCN) are driven by the IPSS category and presented as flow charts. In the absence of karyotypic information, the UK guideline recommends the Sanz score for prognostic determination (Sanz et al. 1989), based on the methodological rigor of the derivation of this score. The use of a prognostic model to inform discussion of appropriate management of MDS is, thus, endorsed by the guidelines and works effectively in practice. The UK guideline produced only four grade-A recommendations, defined as requiring at least one randomized controlled trial or meta-analysis. Two of these were positive: Hydroxyurea as the recommended treatment for proliferative chronic myelomonocytic leukemia (CMML), and EPO to be considered for selected patients with RA/RARS, while two were negative; low-dose cytosine arabinoside and 13-cis retinoic acid were not

a

8.4 ´ Guidelines for the Diagnosis and Management of MDS

91

Measurement of response/treatment effect in MDS Altering disease natural history 1. Complete remission (CR) Bone marrow evaluation: Repeat bone marrow showing less than 5% myeloblasts with normal maturation of all cell lines, with no evidence for dysplasia a. When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid cells, the percentage blasts should be based on the nonerythroid cells. Peripheral blood evaluation (absolute values must last at least 2 months) b: ± Hemoglobin greater than 11 g/dl (untransfused, patient not on erythropoietin) ± Neutrophils 1,500/mm3 or more (not on a myeloid growth factor) ± Platelets 100,000/mm3 or more (not on a thrombopoietic agent) ± Blasts % ± No dysplasia a 2. Partial remission (PR) (absolute value must last at least 2 months) All the complete remission (CR) criteria (if abnormal before treatment), except: bone marrow evaluation. Blasts decreased by 50% or more over pre-treatment, or a less-advanced MDS FAB classification than pre-treatment. Cellularity and morphology are not relevant. 3. Stable disease Failure to achieve at least a PR, but with no evidence of progression for at least 2 months. 4. Failure Death during treatment or disease progression characterized by worsening of cytopenias, increase in the percentage bone marrow blasts, or progression to an MDS FAB subtype more advanced than pre-treatment. 5. Relapse after CR or PR, one or more of the following: (a) Return to pre-treatment bone marrow blast percentage (b) Decrement of 50% or greater from maximum remission/response levels in granulocytes or platelets (c) Reduction in hemoglobin concentration by at least 2 g/dl or transfusion dependence c 6. Disease progression (a) For patients with less than 5% blasts, a 50% or more increase in blasts to more than 5% blasts (b) For patients with 5±10% blasts, a 50% or more increase to more than 10% blasts (c) For patients with 10±20% blasts, a 50% or more increase to more than 20% blasts (d) For patients with 20±30% blasts, a 50% or more increase to more than 30% blasts (e) One or more of the following: 50% or greater decrement from maximum remission/response levels in granulocytes or platelets, reduction in hemoglobin concentration by at least 2 g/dl or transfusion dependence c 7. Disease transformation Transformation to AML (30% or more blasts) 8. Survival and progression-free survival Cytogenetic response (Requires 20 analyzable metaphases using conventional cytogenetic techniques) Major: no detectable cytogenetic abnormality, if pre-existing abnormality was present Minor: 50% or more reduction in abnormal metaphases

92

Chapter 8 ´ Management of Patients with Myelodysplastic Syndromes: Introductory Concepts

(continued) Fluorescent in situ hybridization may be used as a supplement to follow a specifically defined cytogenetic abnormality Quality of life Measured by an instrument such as the FACT Questionnaire Clinically useful improvement in specific domains: Physical Functional Emotional Social Spiritual Hematologic improvement (HI) Improvements must last at least 2 months in the absence of ongoing cytotoxic therapy b Hematologic improvement should be described by the number of individual, positively affected cell lines (e.g., HI-E, HI-E + HI-N, HI-E + HI-P + HI-N) 1. Erythroid response (HI-E) Major response: for patients with pre-treatment hemoglobin less than 11 g/dl, greater than 2 g/dl, increase in hemoglobin; for RBC transfusion-dependent patients, transfusion independence Minor response: for patients with pre-treatment hemoglobin less than 11 g/dl, 1±2 g/dl increase in hemoglobin; for RBC transfusion-dependent patients, 50% decrease in transfusion requirements 2. Platelet response (HI-P) Major response: for patients with a pre-treatment platelet count less than 10,000/mm3, an absolute increase of 30,000/mm3 or more; for platelet transfusion-dependent patients, stabilization of platelet counts and platelet transfusion independence Minor response: for patients with a pre-treatment platelet count less than 10,000/mm3, a 50% or more increase in platelet count with a net increase greater than 10,000/mm3 but less than 30,000/mm3 3. Neutrophil response (HI-N) Major response: for absolute neutrophil count (ANC) less than 1,500/mm3 before therapy, at least a 100% increase, or an absolute increase of more than 500/mm3, whichever is the greater Minor response: for ANC less than 1,500/mm3 before therapy, ANC increase of at least 100%, but absolute increase less than 500/mm3 4. Progression/relapse after HI (one or more of the following): (a) 50% or greater decrement from maximum response levels in granulocytes or platelets (b) A reduction in hemoglobin concentration by at least 2 g/dl (c) Transfusion dependence c For a designated response (CR, PR, HI), all relevant response criteria must be noted on at least two successive determinations at least 1 week apart after an appropriate period following therapy (e.g., 1 month or longer) a

The presence of mild megaloblastoid changes may be permitted if they are thought to be consistent with treatment effect. However, persistence of pre-treatment abnormalities (e.g., pseudo-Pelger-HuŸt cells, ringed sideroblasts, dysplastic megakaryocytes) is not consistent with CR

b

In some circumstances, protocol therapy may require the initiation of further treatment (e.g., consolidation, maintenance) before the 2month period. Such patients can be included in the response category into which they fit at the time the therapy is started

c

In the absence of another explanation such as acute infection, gastrointestinal bleeding, hemolysis, and so on

From: Bruce D. Cheson. Report of an International Working Group to standardize response criteria. Blood 2000; 96: 3671±3674. Copyright American Society of Hematology, used with permission.

a

References

93

Table 8.2. (Methods used by three separate groups (US NCCN, UK BCSH, and Italian Society of Hematology) to derive guidelines for the management of MDS Consensus

ªSystematicº literature review

Systematic literature review including abstracts, proceedings, etc.

Scenario-based

US, NCCN

´

±

±

±

UK, BCSH

´

´

±

±

Italy, Italian Society of Hematology

´

´

´

´

´ = Method employed

Table 8.3. Discrepant recommendations for the management of MDS between the three guideline groups Azacytidine/decitabine

Antilymphocyte globulin/cyclosporin A

Autologous SCT

US

Consider in:

Consider for hypoplastic MDS and for PNH+/ HLA DR2

Not specifically recommended

UK

Not considered

Consider for hypoplastic MDS (and PNH clone)

In clinical research protocol: as per Italian study

Italy

Highly recommended for IPSS high not suitable for intensive therapy (esp. if abnormal karyotype)

Highly recommended for hypoplastic MDS or HLA DR2

Recommended for patients in CR after intensive chemotherapy but lacking an allogeneic donor

Low/INT-1: various INT-2/High: alternative to intensive therapy

recommended. The Italian guideline produced three grade-A recommendations, including that for EPO use in RA/RARS, but also immunosuppressive therapy for hypoplastic MDS (especially HLA DR2+), and AMLtype chemotherapy for patients who are not candidates for stem cell transplantation, who are < 55 years old with INT-2/High risk by IPSS, and ECOG performance score 0/1. These latter two recommendations would not have satisfied criteria for a grade A recommendation in the UK guideline. Noteworthy minor differences between the three guidelines included recommendations for autologous transplantation, demethylating therapy (5azacytidine/Decitabine) and immunosuppressive therapy (Table 8.3). No published randomized controlled trials are available to evaluate these interventions, although randomized international studies of both demethylating agents and autologous transplantation are ongoing.

References Alessandrino EP, Amadori S, Barosi G, Cazzola M, Grossi A, Liberato LN, Locatelli F, Marchetti M, Morra E, Rebulla P, Visani G, Tura S (2002) Evidence- and consensus-based practice guidelines for the therapy of primary myelodysplastic syndromes: a statement from the Italian Society of Hematology. Haematologica 87:1286±1306 Bennett JM (2000) World Health Organization classification of the acute leukemias and myelodysplastic syndrome. Int J Hematol 72:131±133 Bowen D, Culligan D, Jowitt S, Kelsey S, Mufti G, Oscier D, Parker J, UK MDS (2003) Guidelines for the diagnosis and therapy of adult myelodysplastic syndromes. Br J Haematol 120:187±200 Cheson BD, Bennett JM, Kantarjian H, Pinto A, Schiffer CA, Nimer SD, Læwenberg B, Beran M, de Witte TM, Stone RM, Mittelman M, Sanz GF, Wijermans PW, Gore S, Greenberg PL (2000) Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 96:3671±3674 Cheson BD, Bennett JM, Kantarjian H, Schiffer CA, Nimer SD, Lowenberg B, Stone RM, Mittelman M, Sanz GF, Wijermans PW, Greenberg PL (2001) Myelodysplastic syndromes standardized response criteria: further definition. Blood 98:19±85

94

Chapter 8 ´ Management of Patients with Myelodysplastic Syndromes: Introductory Concepts

Germing U, Gattermann N, Strupp C, Aivado M, Aul C (2000) Validation of the WHO proposals for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients. Leuk Res 24:983±1092 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes (published erratum appears in Blood 1998; 91:1100). Blood 89:2079±2088 Greenberg PL, Baer M, Bennett JM, Bloomfield CD, DeCastro CM, Deeg HJ, Devetten M, Emanuel PD, Erba HP, Estey E, Foran J, Gore SD, Millenson M, Navarro W, Nimer SD, O'Donnell MR, Saba HI, Spiers K, Stone R, Tallman MS (2004) Myelodysplastic syndromes. National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology v.1.2005: Hellstrom-Lindberg E, Negrin R, Stein R, Krantz S, Lindberg G, Vardiman J, Ost A, Greenberg P (1997) Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model. Br J Haematol 99:344±351

Hellstrom-Lindberg E, Gulbrandsen N, Lindberg G, Ahlgren T, Dahl IM, Dybedal I, Grimfors G, Hesse-Sundin E, Hjorth M, Kanter-Lewensohn L, Linder O, Luthman M, Lofvenberg E, Oberg G, Porwit-MacDonald A, Radlund A, Samuelsson J, Tangen JM, Winquist I, Wisloff F, Scandinavian MDS (2003) A validated decision model for treating the anaemia of myelodysplastic syndromes with erythropoietin + granulocyte colony-stimulating factor: significant effects on quality of life. Br J Haematol 120:1037±1046 Raza A (2001) Improve or abandon the standardized response criteria for myelodysplastic syndromes recommended by the International Working Group. Blood 98:251±252 Sanz GF, Sanz MA, VallespÓ T, Caµizo MC, Torrabadella M, GarcÓa S, Irriguible D, San Miguel JF (1989) Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 74:395±408 Steensma DP, Letendre L, Tefferi A (2001) Clarifications to the standard neutrophil response criteria for clinical trials in myelodysplastic syndromes are needed. Blood 97:3321±3322

Supportive Care David T. Bowen

Contents

survival is lacking and has not been systematically studied. It is paradoxical that supportive care, which is difficult to standardize, has remained the standard of care against which new therapeutic interventions are compared. Nevertheless, the goal of supportive care is to improve quality of life, and where possible, prolong survival.

9.1 Introduction . . . . . . . . . . . . . . . . . . . .

95

9.2 Co-morbidity and Causes of Death . . .

95

9.3 Quality of Life . . . . . . . . . . . . . . . . . . .

96

9.4 Red Blood Cell Transfusions . . . . . . . .

96

9.5 Iron Chelation Therapy . . . . . . . . . . . .

97

9.6 Thrombocytopenia and Bleeding . . . .

97

9.2 Co-morbidity and Causes of Death

9.7 Neutropenia and Infection . . . . . . . . .

98

References . . . . . . . . . . . . . . . . . . . . . . . . .

98

Transformation to acute myeloid leukemia (AML) and overall survival are the endpoints most widely reported for large cohort studies of unselected patients with MDS. AML transformation is easy to recognize and almost always contributes directly to the cause of death in the affected patients, predominantly in the high-risk MDS subtypes (refractory anemia with excess blasts (RAEB)-2 by the World Health Organization (WHO), INT-2/high-risk groups by International Prognostic Scoring System (IPSS)). However, causes of death among patients who do not transform to AML may be directly related to their MDS, including therapy-related complications such as iron overload, or alternatively to co-morbid conditions. Analysis of the largest cohort of MDS patients, including all categories, in whom the causes of death were reported, identified 28% of deaths due to AML transformation, 60% due to hemorrhage/ infection, 12% due to apparently MDS-unrelated causes, and 1% (3/216 deaths) due to secondary hemosiderosis (Table 9.1) (Sanz et al. 1989). In low-risk MDS, death due to unrelated causes occurred in up to 45% of patients with the low-risk WHO subtype refractory anemia with ringed sideroblasts (RARS), and in approxi-

9.1 Introduction

Although active therapeutic interventions are available for patients with myelodysplastic syndrome (MDS), many patients may be suitable for supportive care only. This is a consequence of both a lack of effective therapeutic strategies, and the relatively high co-morbidity in this predominantly older patient cohort. Supportive care usually refers to the replacement of deficient blood components (red blood cells and platelets) in symptomatic patients, treatment of infection, and iron chelation therapy for transfusional iron overload. These interventions will not alter the natural history of the disease, though there is some evidence that iron chelation therapy may have a disease-modifying effect. Highquality evidence for the effectiveness of supportive care in terms of outcome measures such as quality of life and

96

Chapter 9 ´ Supportive Care

Table 9.1. Causes of death in MDS patients Study

Subtype

Sanz et al. (1989)

All MDS

Matsuda et al.l (1999)

RA (high risk)

2 (22)

4 (44)

3 (33)

0

0

0

RA (int. risk)

6 (25)

8 (33)

5 (21)

3 (13)

0

2 (8)

RA (low risk)

1 (11)

4 (44)

2 (22)

0

2 (22)

0

PSA

0

9 (24)

0

6 (16)

11 (29)

12 (31)

38

RARS*

6 (8)

9 (12)

5 (7)

12 (16)

18 (25)

74

Germing et al.l (2000)

Hemorrhage (%)

Infection (%)

129 (45)

AML transformation (%)

Cardiac failure (%)

Unrelated to MDS (%)

Unknown (%)

Total

60 (21)

3 (1)

24 (8)

72 (25)

288

24 (32)

9 24 9

RA refractory anemia (FAB), PSA pure sideroblastic anemia (WHO = RARS) *

RARS now reclassified as RCMD-RS by WHO

mately 15±20% of patients with multilineage dysplasia (Germing et al. 2000; Matsuda et al. 1999; Mufti et al. 1985).

Table 9.2. Supportive care: options and indications Red cell transfusion Symptomatic anemia

9.3 Quality of Life

Parameters of physical quality of life (QOL) are impaired in MDS patients compared with healthy age and sex-matched subjects. These physical parameters include physical functioning and vitality (Short Form36 tool) and the physical component of fatigue (EuroQOL-5D Visual Analogue Scale instrument). Each of these physical parameters was correlated to hemoglobin concentrations in a cohort of regularly transfused MDS patients (Jansen et al. 2003). Mental components of the QOL instruments were not influenced by hemoglobin concentrations in this small study. In separate cohorts of patients with cancer-related anemia treated with recombinant Epo, a similar improvement in physical QOL parameters has been observed, correlating well with an improvement in hemoglobin concentration (Boogaerts et al. 2003; Fallowfield et al. 2002). Options and indications for supportive care are listed in Table 9.2.

9.4 Red Blood Cell Transfusions

Chronic anemia, though seldom life threatening, can produce significant morbidity and is, therefore, important in relation to QOL and to co-morbid conditions.

Platelet transfusion Chronic thrombocytopenia with clinical bleeding Planned surgical intervention Granulocyte-colony stimulating factor Infectious episodes Chronic neutropenia with recurrent infections Antibiotic therapy Infectious episodes Iron chelation therapy Patients with longer life expectancy (e.g., 5q± syndrome, WHO subtypes RA, RARS) Initiate after 25 units transfused (500 mg iron)

The influence of chronic anemia on cardiac complications in MDS patients remains unknown. Chronic anemia increases cardiac pre-load, which leads to left ventricular hypertrophy and an increased cardiac output. In well-chelated patients with b-thalassaemia, asymptomatic abnormalities of cardiac function have been documented, including both an increase in left ventricular mass, and reduced contractility (Bosi et al. 2003). Left ventricular ejection fraction was reduced in patients with greater iron load but was also abnormal in some patients who were well chelated.

a

9.6 ´ Thrombocytopenia and Bleeding

97

Red cell transfusions should be considered in any patient symptomatic of anemia, irrespective of hemoglobin concentration and after exclusion of other causes. Patients with MDS may, however, compensate well for their anemia and the hemoglobin trigger for the introduction of red cell transfusions will vary between individuals. The frequency of red cell transfusion is variable, and anecdotal evidence would suggest that the intertransfusion interval decreases with time. For patients with short transfusion intervals, bleeding and hemolysis must be considered, but the high transfusion requirement usually simply reflects profound erythroid failure (severe reticulocytopenia) with or without peripheral consumptive processes such as hypersplenism. Although most physicians and patients accept the ªupdownº cyclical symptoms of a conventional transfusion program, evidence from cancer patients treated with recombinant Epo would indicate the greatest incremental benefit in QOL to occur at hemoglobin concentrations > 12 g/dl (Cella 1997; Demetri et al. 1998). Most regularly transfused MDS patients rarely achieve this level, even transiently. The risks associated with red cell transfusions are considerable, and many may not have been recognized. Increasing recognition of transfusion-transmitted infection drives the search for alternative strategies for the management of anemia. Recent infectious agents considered or proven to be associated with red cell transfusions include hepatitis C, TT virus, and new variant Creutzfeldt-Jakob disease. Although these infections with long incubation times, if transmitted, appear of little relevance to the majority of MDS patients whose life expectancy is < 6 years, there are long-term transfused patients for whom the concern for infections is a major issue. Further, red cell alloimmunization is common and the frequency increases with increasing numbers of blood units transfused. Allosensitization may be of particular concern in patients who are candidates for hemopoietic cell transplantation. Of note, reactions to red cell transfusion are equally frequent in patients with and without red cell antibodies, presumably because of sensitization to antigens expressed on leukocytes (Fluit et al. 1990).

dotally, all Hematologists will recall patients with transfusional iron overload who have died of cardiac failure. For most of these patients, it is difficult to exclude at least a contribution from co-existent cardiac disease or the cardiac effects of chronic anemia. For practical purposes, the UK Guidelines for the Diagnosis and Management of MDS have recommended instituting iron chelation therapy for those transfusion-dependent patients who have a good prognosis (e.g., 5q± syndrome, WHO category RARS), commencing after approximately 25 units of packed red blood cells transfused (Bowen et al. 2003). Desferrioxamine remains the iron chelator of choice, usually administered subcutaneously by prolonged infusion or twice daily bolus. The support of an active multidisciplinary team is essential to maximize compliance with this cumbersome and invasive therapy. Desferrioxamine, given as a regular subcutaneous infusion, can reduce serum ferritin and liver iron concentration in MDS patients (Jensen et al. 1995). Addition of Vitamin C, 100±200 mg daily, taken about 1 h prior to desferrioxamine infusion, increases the proportional iron excretion but should not be started until approximately 4 weeks after desferrioxamine therapy is initiated. The use of twice daily subcutaneous bolus injections of desferrioxamine (Franchini et al. 2000) may be considered where infusions are not tolerated, but there is even less information about their potential value in MDS than for the prolonged infusions. Observational studies have also suggested that desferrioxamine therapy may be associated with improved marrow function, and reduced transfusion requirements (Haines and Wainscoat 1991; Jensen et al. 1996). The oral iron chelator Deferiprone is increasingly used, although large studies demonstrating efficacy and safety in MDS patients are lacking. The availability of new oral iron chelators will catalyze research and hopefully lead to a more rational approach to iron chelation in MDS patients (Nisbet-Brown et al. 2003). The main challenges are to clearly link transfusional iron overload to life-threatening complications such as cardiac failure, and to develop reliable methods for monitoring iron status in relevant end organs such as the liver and heart (Jensen et al. 2003).

9.5 Iron Chelation Therapy

9.6 Thrombocytopenia and Bleeding

There are no data on which to base recommendations for iron chelation therapy in patients with MDS. Anec-

Bleeding occurs usually coincident with severe thrombocytopenia due to bone marrow failure, but may rarely

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Chapter 9 ´ Supportive Care

also be a manifestation of MDS-associated functional defects of platelets. Prophylactic platelet transfusions are not indicated in asymptomatic thrombocytopenic patients and should be reserved to cover symptomatic bleeding or planned interventional procedures such as surgery or dental extraction. The efficacy of danazol in transiently improving platelet counts in MDS patients is controversial (Chabannon et al. 1994; Chan et al. 2002).

9.7 Neutropenia and Infection

Neutropenia is common in MDS patients, and infection is one of the major causes of death (Table 9.1). Most MDS patients treated with granulocyte-colony stimulating factor (G-CSF) increase their blood neutrophil counts, indicating reasonable marrow reserve. No randomized studies have yet demonstrated a clear benefit for routine use of G-CSF, although in one cohort study, patients with neutrophil counts maintained at >1.5 ´ 109/ L had fewer infections than those with lower values (Negrin et al. 1992). Prophylactic G-CSF therapy may have some role in patients with severe chronic neutropenia and recurrent infections. There is no evidence for prophylactic antibiotic therapy in this context.

References Boogaerts M, Coiffier B, Kainz C (2003) Impact of epoetin beta on quality of life in patients with malignant disease. Br J Cancer 88:988± 995 Bosi G, Crepaz R, Gamberini MR, Fortini M, Scarcia S, Bonsante E, Pitscheider W, Vaccari M (2003) Left ventricular remodelling, and systolic and diastolic function in young adults with beta thalassaemia major: a doppler echocardiographic assessment and correlation with haematological data. Heart (British Cardiac Society) 89:762±766 Bowen D, Culligan D, Jowitt S, Kelsey S, Mufti G, Oscier D, Parker J, UK MDS (2003) Guidelines for the diagnosis and therapy of adult myelodysplastic syndromes. Br J Haematol 120:187±200 Cella D (1997) The Functional Assessment of Cancer Therapy-Anemia (FACT-An) Scale: a new tool for the assessment of outcomes in cancer anemia and fatigue. Semin Hematol 34:13±19 Chabannon C, Molina L, Pegourie-Bandelier B, Bost M, Leger J, Hollard D (1994) A review of 76 patients with myelodysplastic syndromes treated with danazol (Abstract). Cancer 73:3073±3080 Chan G, DiVenuti G, Miller K (2002) Danazol for the treatment of thrombocytopenia in patients with myelodysplastic syndrome. Am J Hematol 71:166±171 Demetri GD, Kris M, Wade J, Degos L, Cella D (1998) Quality-of-life benefit in chemotherapy patients treated with epoetin alfa is inde-

pendent of disease response or tumor type: results from a prospective community oncology study. J Clin Oncol 16:3412±3425 Fallowfield L, Gagnon D, Zagari M, Cella D, Bresnahan B, Littlewood TJ, McNulty P, Gorzegno G, Freund M (2002) Multivariate regression analyses of data from a randomised, double-blind, placebo-controlled study confirm quality of life benefit of epoetin alfa in patients receiving non-platinum chemotherapy. Br J Cancer 87: 1341±1353 Fluit CR, Kunst VA, Drenthe-Schonk AM (1990) Incidence of red cell antibodies after multiple blood transfusion. Transfusion 30:532± 535 Franchini M, Gandini G, de Gironcoli M, Vassanelli A, Borgna-Pignatti C, Aprili G (2000) Safety and efficacy of subcutaneous bolus injection of deferoxamine in adult patients with iron overload. Blood 95:2776±2779 Germing U, Gattermann N, Aivado M, Hildebrandt B, Aul C (2000) Two types of acquired idiopathic sideroblastic anaemia (AISA): a timetested distinction. Br J Haematol 108:724±728 Haines ME, Wainscoat JS (1991) Relapsing sideroblastic anaemia. Br J Haematol 78:285±286 Jansen AJ, Essink-Bot ML, Beckers EA, Hop WC, Schipperus MR, Van Rhenen DJ (2003) Quality of life measurement in patients with transfusion-dependent myelodysplastic syndromes. Br J Haematol 121:270±274 Jensen PD, Jensen FT, Christensen T, Ellegaard J (1995) Evaluation of transfusional iron overload before and during iron chelation by magnetic resonance imaging of the liver and determination of serum ferritin in adult non-thalassaemic patients. Br J Haematol 89:880±889 Jensen PD, Heickendorff L, Pedersen B, Bendix-Hansen K, Jensen FT, Christensen T, Boesen AM, Ellegaard J (1996) The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload. Br J Haematol 94:288±299 Jensen PD, Jensen FT, Christensen T, Eiskjaer H, Baandrup U, Nielsen JL (2003) Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool. Blood 101:4632±4639 Matsuda A, Jinnai I, Yagasaki F, Kusumoto S, Murohashi I, Bessho M, Hirashima K, Honda S, Minamihisamatsu M, Fuchigami K, Matsuo T, Kuriyama K, Tomonaga M (1999) New system for assessing the prognosis of refractory anemia patients. Leukemia 13:1727±1734 Mufti GJ, Stevens JR, Oscier DG, Hamblin TJ, Machin D (1985) Myelodysplastic syndromes: a scoring system with prognostic significance. Br J Haematol 59:425±433 Negrin RS, Nagler A, Kobayashi Y, et al. (1992) Maintenance treatment of patients with myelodysplastic syndromes using recombinant human granulocyte colony stimulating factor. Blood 78:36±43 Nisbet-Brown E, Olivieri NF, Giardina PJ, Grady RW, Neufeld EJ, Sechaud R, Krebs-Brown AJ, Anderson JR, Alberti D, Sizer KC, Nathan DG (2003) Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet 361:1597±1602 Sanz GF, Sanz MA, VallespÓ T, Caµizo MC, Torrabadella M, GarcÓa S, Irriguible D, San Miguel JF (1989) Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 74:395±408

Hematopoietic Growth Factors David T. Bowen

Contents

10.3.1.5 Dosing Schedule . . . . . . 106 10.3.1.6 Mechanism of Action? . . 106 10.3.2 Erythroid Response to Other Growth Factors and Combination Therapy 106

10.1 Introduction . . . . . . . . . . . . . . . . . . . .

99

10.2 Mechanism of Anemia . . . . . . . . . . . . 10.2.1 Ineffective Erythropoiesis . . . . . . . 10.2.1.1 Clonogenic Erythroid Growth . . . . . . . . . . . . . 10.2.1.2 Erythroid Apoptosis . . . . 10.2.1.3 Incriminating the Mitochondria . . . . . . . . . . . . 10.2.2 Additional Contributors to Anemia in MDS . . . . . . . . . . . . . . . . . . . 10.2.3 Myelodysplasia Subtypes with Preferential Erythroid Lineage Involvement . . . . . . . . . . . . . . . . 10.2.3.1 Sideroblastic Anemia with Single-lineage Involvement . . . . . . . . . . 10.2.3.2 Refractory Anemia with Single-lineage Involvement 10.2.3.3 5q± Syndrome . . . . . . . .

100 100

References . . . . . . . . . . . . . . . . . . . . . . . . .

100 100

10.1 Introduction

10.3 Treatment of Anemia with Hematopoietic Growth Factors . . . . . . . . . . . . 10.3.1 Recombinant Erythropoietin Therapy . . . . . . . . . . . . . . . . . . . 10.3.1.1 Which Patients Will Respond? . . . . . . . . . . . . 10.3.1.2 How Prolonged a Therapeutic Trial? . . . . . . 10.3.1.3 How Durable Are Erythroid Responses to Growth Factors? . . . . . . . . . . . . . 10.3.1.4 Quality of Life . . . . . . . . .

101 101 101 101 101 102 102 102 102 105 105 105

106

The first report of recombinant hematopoietic growth factor therapy in myelodysplastic syndrome (MDS) patients was published in 1987, in a landmark study of eight patients with predominantly high-risk MDS treated with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (Vadhan-Raj et al. 1987). An increase in mature leukocytes (neutrophils, monocytes, eosinophils, and lymphocytes) was observed in all patients, with an erythroid response in three subjects. Since these early studies, the focus has shifted to stimulants of erythropoiesis with the availability of recombinant erythropoietins (EPO; alpha and beta), and recently the longer-acting derivative erythropoietin compounds. The currently available hematopoietic growth factors have no clear role in the routine management of neutropenia or thrombocytopenia in MDS patients, although low-dose granulocyte colonystimulating factor (G-CSF) therapy is sometimes advocated for patients with recurrent infections and severe neutropenia (Negrin et al. 1992). Despite the vast literature and multitude of clinical studies, the precise role of hematopoietic growth factors in the management of MDS patients remains to be clearly defined. Definitive clinical trials powered to demonstrate improved quality of life and improved survival are still required.

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Chapter 10 ´ Hematopoietic Growth Factors

The most prevalent clinical problem for patients with myelodysplastic syndromes is anemia. Eighty percent of patients are anemic at presentation (Sanz et al. 1989) and the majority require red cell transfusion at some stage of the disease. Although transfusion remains an appropriate intervention for many patients, increasing understanding of the optimal use of therapeutic agents, including the availability of several new drugs, will lead to an increasing proportion of patients treated with the intention of eliminating the transfusion need. 10.2 Mechanism of Anemia

As predicted from the clinical presentation, in vitro studies of clonogenic growth have confirmed erythropoiesis as the lineage expressing the most prominent defect (Backx et al. 1993; Sawada et al. 1995). While the anemia of MDS can often be multifactorial (Fig. 10.1), the most prominent pathological processes are ineffective and hypoproliferative erythropoiesis. Ineffective erythropoiesis is more commonly seen in refractory anemia (RA), refractory anemia with ring sideroblasts (RARS) and refractory anemia with excess blasts (RAEB)(usually < 10% blasts), while patients with >10% blasts have more hypoproliferative erythropoiesis (Cazzola et al. 1982).

10.2.1 Ineffective Erythropoiesis

Morphological abnormalities of erythroid precursors are one of the diagnostic hallmarks of MDS (Bennett et al. 1982). These include nuclear abnormalities such as megaloblastoid changes and nuclear irregularity. Bone marrow from patients with RARS tends to show less nuclear irregularity, an increase in proerythroblasts and the classical iron deposition in mitochondria demonstrated on staining with Perl's reagent. The morphological similarities with megaloblastic anemia suggest a fundamental defect in DNA synthesis as the pathological process underpinning ineffective erythropoiesis in MDS, although the nature of this defect remains elusive.

10.2.1.1 Clonogenic Erythroid Growth Committed erythroid progenitor growth is reduced in most patients (May et al. 1985). MDS bone marrow is relatively deficient in erythroid (compared with myeloid) clonogenicity as demonstrated both by replating of blast colonies (Backx et al. 1993) or by early CD34+ cell lineage commitment (Sawada et al. 1995). Erythroid progenitor growth in semi-solid culture can be partially augmented in vitro by a variety of survival-augmenting (anti-apoptotic) strategies. These include increased concentrations of early-acting hematopoietic growth factors such as stem cell factor (Backx et al. 1992) and granulocyte-colony stimulating factor (Schmidt-Mende et al. 2001), antioxidants (amifostine) (List et al. 1997), glucocorticoids (Koeffler et al. 1978) and caspase inhibitors (Hellstrom-Lindberg et al. 2001). Residual non-clonal erythroid progenitors can be identified in some patients (Asano et al. 1994), and may be preferentially stimulated by in vivo hematopoietic growth factor therapy (Rigolin et al. 2002).

10.2.1.2 Erythroid Apoptosis Fig. 10.1. Mechanisms of hematopoietic progenitor/precursor cell death in MDS. Extrinsic factors may include inhibitory cytokines (e.g., TNF-a, IFN-c and autoimmune attack (?HLA restricted). Intrinsic abnormalities include genetic (chromosome anomalies/gene mutations) or epigenetic phenomena (e.g., promoter methylation). Mitochondrial dependent and independent pathways are implicated. TNF-a tumor necrosis factor-a, IFN-c interferon-c, Th1 subset of T-helper lymphocytes, HLA human leucocyte antigen, RS ring sideroblasts

The paradox of a morphologically expanded bone marrow erythron with peripheral anemia is explained by an increased rate of intramedullary erythroid cell death, most likely by augmented apoptosis (Hellstrom-Lindberg et al. 1997 a; Lepelley et al. 1996; Raza et al. 1996). While augmented erythroid apoptosis has been demonstrated by a variety of techniques, conflicting data cloud the clarification of the pathological processes that induce this apoptosis.

a

10.2 ´ Mechanism of Anemia

Apoptosis and DNA fragmentation can clearly be demonstrated in the progenitor-enriched bone marrow CD34+ cells (Parker et al. 2000; Peddie et al. 1997) as well as in erythroid precursors (Matthes et al. 2000; Raza et al. 1995; Suarez et al. 2004). The final common effector pathway of apoptosis via caspase-3 is augmented in mononuclear cells (Boudard et al. 2000; Hellstrom-Lindberg et al. 2001) and erythroid cells (Hellstrom-Lindberg et al. 2001), predominantly in RA and RARS, and inhibition of caspase-3 promotes in vitro erythroid colony growth (Boudard et al. 2000). Caspase-3 can be activated through mitochondrial-dependent pathways (via Caspase-9), and also mitochondria-independent signals initiated via caspase-8. Erythroid precursors from MDS patients are more sensitive to Fasmediated cell death, but the evidence for release of a Fas-mediated erythroid block by in vitro inhibition of the Fas pathway is conflicting (Boudard et al. 2002; Claessens et al. 2002; Dror 2003; Hellstrom-Lindberg et al. 2001). Clarification of the role of ªdeath receptorº pathway proteins (including Fas, FADD) may further elucidate the emerging contribution of extrinsic inhibitory and autoimmune processes to ineffective erythropoiesis.

10.2.1.3 Incriminating the Mitochondria Several strands of evidence support a pathological role for mitochondrial defects in low-risk MDS. The pathological accumulation of ferritin in mitochondria is the basis for the diagnosis of the MDS subtypes with sideroblastic changes. This ferritin represents almost exclusively the recently described mitochondrial ferritin (Cazzola et al. 2003), which appears to preferentially accumulate in RARS and congenital X-linked sideroblastic anemia, although the pathological relevance has yet to be established. While iron accumulation occurs only in sideroblastic mitochondria, augmented release of cytochrome c, activation of caspase-9 (Tehranchi et al. 2003), and loss of mitochondrial membrane potential (Matthes et al. 2000; Michalopoulou et al. 2004) incriminate the mitochondrion as at least an intermediary, and perhaps the primary perpetrator of premature erythroid apoptosis in both RARS and RA. Mitochondrial DNA mutations at loci encoding critical mitochondrial genes have recently been described in MDS patients (Gattermann 2004; Gattermann et al. 2004; Shin et al. 2003), but these may equally represent expansion of a clone with a coincident random heteroplasmic mutation, or a pathologically irrelevant abnormality.

101

10.2.2 Additional Contributors to Anemia in MDS

Peripheral red cell destruction/loss may also produce anemia in MDS. Red cell loss may result from bleeding associated with thrombocytopenia or platelet functional defects. The red cell lifespan is shortened in some MDS patients, and this may be due to hemolysis (often with a positive direct antiglobulin test) (Sokol et al. 1989) or hypersplenism. Many diverse red cell abnormalities are also described in MDS, though their clinical significance is less clear (Chalevelakis et al. 1991; Higgs et al. 1983; Lintula 1986). Many patients with long-standing transfusion therapy show progressively increasing transfusion needs, possibly due to multiple mechanisms.

10.2.3 Myelodysplasia Subtypes with Preferential

Erythroid Lineage Involvement

10.2.3.1 Sideroblastic Anemia with Single-lineage Involvement RARS patients as defined by the French-American-British (FAB) classification (Bennett et al. 1982) can be divided into two groups on the basis of single lineage (erythroid) vs. multilineage dysplasia and degree of nonerythroid lineage cytopenias. This is now recognized in the World Health Organization (WHO) classification (Bennett 2000) with the single lineage subtype renamed RARS, and the multilineage subtype refractory cytopenia with multilineage dysplasia (RCMD)-RS. RARS has a better prognosis (77% vs. 56% 3-year survival) with no cases of leukemic transformation in a recently reported study (Germing et al. 2000 a). However, myeloid colony growth may also be severely reduced in typical RARS patients, indicating a stem cell origin of this form of MDS (Hellstrom-Lindberg et al. 2001).

10.2.3.2 Refractory Anemia with Single-lineage Involvement As for sideroblastic anemia the WHO classification recognizes two forms of the old FAB subtype refractory anemia (RA). The single-lineage subtype is renamed RA, and the multilineage involved subtype RCMD. The prognosis and AML transformation rates are better for RA than for RCMD, but the difference is not as marked as for sideroblastic anemia (Germing et al. 2000 b).

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10.2.3.3 5q± Syndrome The 5q± syndrome occurs predominantly in elderly women and is characterized by anemia, frequently thrombocytosis, and an isolated deletion of the long arm (or part thereof) of chromosome 5. Overall, 46% of patients have erythroid hypoplasia, with only 13% showing hyperplastic erythropoiesis (Giagounidis et al. 2004). The recent observation that the karyotypic defect is present in precursors of myeloid and lymphoid lineages as well as erythroid cells poses some fundamental mechanistic questions as to why the clinical picture predominates in the erythroid lineage (Nilsson et al. 2000). It should be noted that not all patients with a 5q± abnormality have a typical 5q± syndrome.

10.3 Treatment of Anemia with Hematopoietic

Growth Factors

The aim of interventional therapy for anemia is to improve quality-of-life benchmarked against either the baseline untreated state, or against best supportive care in the form of red cell transfusions. These interventional therapies have the potential to produce sustained increases in hemoglobin concentration and to thus avoid the up-and-down lifestyle accepted by so many regularly transfused patients and their physicians.

10.3.1 Recombinant Erythropoietin Therapy

The therapeutic efficacy of recombinant Erythropoietin (EPO) (Table 10.1), alone or combined with granulocyte colony-stimulating factor (G-CSF) (Table 10.2) in the treatment of anemia is now well established for selected patients with MDS. Cohort studies have clearly demonstrated responses, and two randomized studies have confirmed the superior erythroid response rates compared with either best supportive care plus placebo (versus EPO alone) (Italian Cooperative Study Group for rHuEpo in Myelodysplastic Syndromes 1998), or best supportive care alone (versus EPO + G-CSF) (Casadevall et al. 2004). EPO therapy is generally well tolerated with the most common side effects being flu-like symptoms and occasional splenic pain and enlargement. Thrombocytopenia can be accentuated in non-responders to EPO, but rarely with adverse clinical consequences (Hellstrom-Lindberg et al. 1997 b).

The role of EPO therapy remains to be precisely defined and several key questions remain unanswered:

10.3.1.1 Which Patients Will Respond? Early indications of response predictors to EPO therapy alone were identified in a meta-analysis covering trials of EPO for MDS patients up to 1994, and including 205 patients from 17 trials (Hellstrom-Lindberg 1995). Overall response rate was 16%, using 100% reduction of transfusion need as minimal response criteria. Factors predictive of response were non-RARS subtype, pre-treatment serum Erythropoietin concentration of less than 200 units/l, and absent need for red cell transfusion. Patients with RARS responded less well to Erythropoietin therapy alone with an overall response rate of 8% (Hellstrom-Lindberg 1995). The only double blind, randomized, placebo-controlled study of Erythropoietin treatment in MDS showed an overall benefit for Erythropoietin over placebo (p=0.007) in MDS patients with < 10% bone marrow myeloblasts. However, analysis of patient subgroups showed a significant effect of treatment only in non-transfused patients and in patients with RA. Again baseline serum Erythropoietin levels of less than 200 units/l predicted for response (Italian Cooperative Study Group for rHuEpo in Myelodysplastic Syndromes 1998). Taking these studies together, it is likely that RARS with transfusion need responds poorly to Erythropoietin as monotherapy. The synergistic therapeutic effect of G-CSF added to EPO has now been convincingly demonstrated (Hellstrom-Lindberg et al. 1998; Negrin et al. 1996; Remacha et al. 1999). This effect was most pronounced in patients with RARS, who have shown the best response rate to the combination (~50%). The combination therapy was well tolerated. Using pre-treatment Erythropoietin as a ternary variable (< 100, 100±500 or > 500 u/l) and RBC transfusion requirement (< 2 or ³ 2 units per month) as a binary variable, a predictive model for response was developed from the data of 98 patients treated in two multi-center studies (Hellstrom-Lindberg et al. 1997 b). Three groups were identified with predicted response rates of 74%, 23% and 7%. The model remained predictive of response in a prospective validation study, although response rates were predictably lower at 61% and 14% in the ªgoodº and ªintermediateº predictive groups, respectively (Hellstrom-Lindberg et al. 2003). It is important to emphasize that this predic-

a

10.3 ´ Treatment of Anemia with Hematopoietic Growth Factors

103

Table 10.1. Trials of erythropoietin alone in MDS Number of patients

Results

Comments

205 from 17 trials

16% overall response **

Higher response rates if:

Meta analyses Hellstrom-Lindberg (1995)

Serum EPO < 200 U/l Non-RARS FAB type Non-transfusion dependent Rodriguez et al. (1994)

115 from 10 studies

23.5% response

(abstract only; Spanish language)

Higher response for RAEB No relationship to EPO level or transfusion requirement

Post-meta analyses (larger studies) Terpos et al. (2002) Italian Cooperative Study Group for rHuEpo in Myelodysplastic Syndromes

281 87

(1998)

45% overall at 26 weeks (18% at 12 weeks) *

Prolonged therapy increased response rates especially for RARS

14/38 vs. 4/37 responders; ( =0.007) (p

Randomized double-blind placebo-controlled study of EPO in low-risk MDS

In favor of EPO *

Response assessed at 8 weeks Response predictors = FAB type RA, basal EPO level < 200 U/l, non-transfused

**

Stasi et al. (1997 a)

43

16.7% (CR+PR) *

±

Stasi et al. (1997 b)

25

4 CR, 5PR *

Responders had lower serum concentration of TNF-/ *

Di Raimondo et al. (1996)

12 with RA only, normal WBC and platelets and short duration of disease

7 (58.3%) CR, 2PR *

Highly selected, mild cases of RA

Rose et al. (1995)

116

28% *

Serum EPO < 100 Mu/ml predicted for response (54% of RA with low EPO responded)

Less stringent CR/PR criteria: for example CR = increase in Hb ³ 20 g/l and elimination of transfusion need; PR = increase in Hb of 10±20 g/l in non-transfused patients, or a reduction of transfusions by 50%

**

CR/PR criteria per Hellstrom-Lindberg: CR = Hb > 115 g/l and no transfusion need; PR = rise in Hb ³ 15 g/l in on-transfused patients, or elimination of transfusion need with stable Hb in previously transfused patients

tive model was derived for patients treated with a therapeutic trial of 12-week duration only. Relatively low serum EPO concentration has also been predictive of response in other studies (Terpos et al. 2002), although the above predictive model (EPO concentration plus transfusion need) was not helpful in a recently reported trial (Mantovani et al. 2000). The response criteria used for different studies have varied in their stringency, and those using the most stringent criteria (complete re-

sponse = achievement of Hb > 11.5 g/dl and transfusion independence; partial response = > 2 g/dl increment in Hb concentration and independence from transfusion) (Casadevall et al. 2004; Hellstrom-Lindberg et al. 2003) will be most likely to identify durable responses. Despite the availability of a validated predictive model for response prediction, 39% of patients with a high predictive score still fail to respond. Improved predictors of response or earlier indicators of therapeutic

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Chapter 10 ´ Hematopoietic Growth Factors

Table 10.2. Trials of erythropoietin PLUS G-CSF in MDS Number of patients

Erythroid response

Comments

Casadevall et al. (2004)

60 (randomized 1 : 1)

42% in EPO/G-CSF group **

No improvement in QOL in treatment group; only 3/30 patients still responding at 12 months

Hellstrom-Lindberg et al. (1997 b)

53

61%, good predictive group **

Prospective study validating predictive model

14%, intermediate predictive group Median response duration = 29 months (CR) vs. 5.5 months (PR) Mantovani et al. (2000)

33

61% erythroid response at 12 weeks *

Twelve of 17 responders at 12 weeks and 14/20 responders at 20 weeks had ªgood erythroid responseª, namely independent of red cell transfusion/sustained increase in Hb > 2 g/dl

80% erythroid response at 36 weeks

Thirteen responders maintained response on treatment for 2 years

Remacha et al. (1999)

32

Erythroid response in 50% (12 patients CR and 4 PR) *

±

Hellstrom-Lindberg et al. (1997 b)

Development of predictive model from (Hellstrom-Lindberg et al. 1998) and (Negrin et al. 1996)

36% response **

In multivariate analysis, serum EPO levels and initial transfusion need were significant predictors of response

Median duration of response = 11±24 months

Predictive score for response developed by log-likelihood and logistic

High predictive group = 74% probability of response

regression analyses.

Int. predictive group = 23% probability of response Low predictive group = 7% probability of response Hellstrom-Lindberg et al. (1998)

50 in randomized study

Overall response rate was 38%**

In randomized study:

Response rates in the two arms were identical

Arm A = G-CSF for 4 weeks followed by combination for 10 weeks

Median survival = 26 months, leukemic transformation 28%

Arm B= EPO for 8 weeks followed by the combination for 10 weeks

Median duration of response in 20 long-term maintenance patients = 24 months

Response rates for RA, RARS, RAEB were 20%, 46%, 37%, respectively

a

10.3 ´ Treatment of Anemia with Hematopoietic Growth Factors

105

Table 10.2 (continued)

Negrin et al. (1996)

Number of patients

Erythroid response

Comments

55

53 (96%) had a neutrophil response

Response predicted by low serum EPO level, higher absolute basal reticulocyte counts and normal cytogenetics at study entry

44 patients evaluated for an erythroid response and 21(48%) had a response 81% of these responders maintained their response during an 8-week maintenance phase* Imamura et al. (1994)

**

10

No responses in erythroid or platelets following 10 weeks of treatment

One delayed erythroid response following cessation of treatment

80% had a neutrophil response *

Considerably higher doses of G-CSF (intravenous) than in other studies

Hellstrom-Lindberg et al. (1993)

22

8 (38%) showed a significant response in Hb **

Less-advanced pancytopenia, lower levels of serum EPO and ring sideroblasts predicted for response

Negrin et al. (1993)

24

10 (42%) had an erythroid response *

Low pre-treatment EPO levels only predictor for response

Less stringent CR/PR criteria: for example, CR = increase in Hb ³ 20 g/l and elimination of transfusion need; PR = increase in Hb of 10±20 g/l in non-transfused patients, or a reduction of transfusions by 50%

**

CR/PR criteria per Hellstrom-Lindberg: CR = Hb > 115 g/l and no transfusion need; PR = rise in Hb ³ 15 g/l in on-transfused patients, or elimination of transfusion need with stable Hb in previously transfused patients

response are clearly required. Early indications are that these may include relatively simple parameters such as those reflected in the WHO classification (Howe et al. 2004).

10.3.1.2 How Prolonged a Therapeutic Trial? Two recent studies with less stringent response criteria than the Scandinavian trials have demonstrated an increased response rate with prolonged growth factor treatment. Responses to therapy with EPO + G-CSF increased from 61% at 12 weeks to 80% at 36 weeks (Mantovani et al. 2000), while responses to EPO therapy alone increased from 18% at 12 weeks to 45% at 26 weeks in another cohort (Terpos et al. 2002). Both studies indicate that RARS (FAB classification) patients benefit most from these prolonged therapeutic trials, but the quality of response was not described.

10.3.1.3 How Durable Are Erythroid Responses to Growth Factors? Quality of response determines durability with a median response duration for complete responders (achievement of Hb >11.5 g/dl and transfusion independence) of 29 months versus 5.5 months for partial responders (> 2 g/dl increment in Hb concentration and independence from transfusion) (Hellstrom-Lindberg et al. 2003). Prolonged responses are reported with similar durability in some cohorts (Hast et al. 2001; Mantovani et al. 2000), but not others (Casadevall et al. 2004).

10.3.1.4 Quality of Life No randomized studies have been conducted with sufficient statistical power to demonstrate differences in quality of life (QOL). One small cohort study identified an increase in global QOL (EORTC QLQ-C30 instrument) in responding patients (Hellstrom-Lindberg et

106

Chapter 10 ´ Hematopoietic Growth Factors

al. 2003), while a small randomized study failed to show any difference in QOL (Functional Assessment of Cancer Therapy-anemia tool) between cohorts treated with EPO + G-CSF versus best supportive care (Casadevall et al. 2004).

10.3.1.5 Dosing Schedule The vast majority of published studies have used dosing schedules for EPO of approximately 50,000±70,000 units/week in 3±5 divided doses for a minimum of 6 weeks. Small studies have indicated equivalent efficacy of once weekly dosing of EPO for MDS patients, usually at a total dose of approximately 40,000 U/week (Garypidou et al. 2003), but larger studies are required to confirm this. Given the higher response rate of RARS patients to combination therapy, it is reasonable to initiate treatment with EPO plus G-CSF for this group. If G-CSF is used, it should be added at a dose to normalize (and at least double) the neutrophil count if it is less than 1.5 ´ 109/l or double the neutrophil count if it is more than 1.5 ´ 109/l. As for all other patients on EPO treatment, functional iron deficiency has to be considered, though this has not been systematically studied as a cause for non-response to EPO in MDS.

10.3.1.6 Mechanism of Action? A response to EPO + G-CSF therapy is morphologically associated with a reduction of bone marrow apoptosis, reduced (but more effective) bone marrow erythropoiesis and, in RARS, a reduced number of ring sideroblasts (Hellstrom-Lindberg et al. 1997 a). In vitro, EPO + G-CSF reduces mitochondria-mediated pro-apoptotic pathway activation in erythroid culture systems from both RARS and RA patients (Tehranchi et al. 2003). A preferential stimulation of non-clonal hematopoietic cells in patients responding to EPO alone has also recently been reported (Rigolin et al. 2002).

10.3.2 Erythroid Response to Other Growth

Factors and Combination Therapy

Several smaller cohort studies have indicated that response rates to the combination of EPO + granulocyte macrophage-colony stimulating factor (GM-CSF) are comparable to those with EPO + G-CSF (Economopou-

los et al. 1999; Hansen et al. 1993; Stasi et al. 1999). However, the only randomized study of GM-CSF + placebo vs. GM-CSF + EPO showed low response rates (< 10% in each arm) and little difference between the two arms (Thompson et al. 2000). GM-CSF has more side effects than G-CSF and there is little evidence to recommend GM-CSF therapy in combination with EPO. Small studies have also examined combinations of other agents (including growth factors) with EPO such as IL-3, 13cis retinoic acid, cyclosporin-A, All-trans retinoic acid, or vitamin D, but none appear superior to EPO alone or in combination with G-CSF.

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recombinant human granulocyte macrophage colony stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 317:1545±1552

Biologically Based Treatment Philip Nivatpumin, Steven D. Gore

Contents 11.1 Introduction . . . . . . . . . . . . . . . . . . . .

111

11.2 Therapies Targeting Epigenetic Changes 11.2.1 DNA Methyltransferase Inhibitors . 11.2.2 Histone Deacetylase Inhibitors . . . 11.2.3 Combinations of DNA Methyltransferase with Histone Deacetylase Inhibitors . . . . . . . . . . . . . . . . . .

112 112 113

11.3 Therapies Targeting Angiogenesis 11.3.1 Thalidomide . . . . . . . . . . . . . 11.3.2 Lenalidomide . . . . . . . . . . . . 11.3.3 Bevacizumab . . . . . . . . . . . . 11.3.4 SU11248 and PTK787 . . . . . . 11.3.5 Arsenic Trioxide . . . . . . . . . .

. . . . . .

114 114 114 114 115 115

..... ..... .....

.....

115 115 115 116

11.5 Therapies Targeting the Immune System . . . . . . . . . . . . . . . . . . . . . . . . 11.5.1 ATG . . . . . . . . . . . . . . . . . . . . . . 11.5.2 Cyclosporine . . . . . . . . . . . . . . .

116 116 116

11.6 Therapies Targeting Signaling 11.6.1 Tipifarnib . . . . . . . . . . . 11.6.2 Lonafarnib . . . . . . . . . . 11.6.3 Imatinib . . . . . . . . . . . . 11.6.4 PKC412 . . . . . . . . . . . .

.. . . . .

116 116 117 117 117

11.7 Therapies Targeting Aberrant Differentiation . . . . . . . . . . . . . . . . . .

117

11.4 Therapies Targeting Disordered Cytokine Milieu . . . . . . . . . . . . 11.4.1 Pentoxifylline . . . . . . . . . . 11.4.2 SCIO-469 . . . . . . . . . . . . . 11.4.3 Etanercept and Infliximab .

. . . .

. . . . .

. . . . .

. . . . . .

.. . . . .

. . . .

. . . . . .

. . . .

11.7.1 Retinoic Acid . . . . . . . . . . . . . . . 11.7.2 Vitamin D . . . . . . . . . . . . . . . . .

117 118

11.8 Miscellaneous Therapies . . . . . . . . . . 11.8.1 TLK199 . . . . . . . . . . . . . . . . . . .

118 118

11.9 Conclusion . . . . . . . . . . . . . . . . . . . . .

118

References . . . . . . . . . . . . . . . . . . . . . . . . .

118

113 11.1 Introduction

With improved insights into the cellular and molecular pathophysiology of myelodysplastic syndrome (MDS), new agents for its treatment have been developed. While these putative `targeted' therapies have been based on testable hypotheses developed from in vitro models, in many instances it has been difficult to validate the mechanism underlying clinical activity. Nevertheless, these studies have led to FDA approval of the first drug for the treatment of MDS. The availability of increasing numbers of active agents is leading to changes in the management of MDS. Patients with MDS must undergo a thorough analysis of prognostic factors, and examination for clinical parameters that may be predictive of response to specific therapies. Whether these therapies (other then hemopoietic cell transplantation) will alter the natural history of MDS requires further observation.

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Chapter 11 ´ Biologically Based Treatment

11.2 Therapies Targeting Epigenetic Changes 11.2.1 DNA Methyltransferase Inhibitors

The cytosine analogues 5-azacitidine (5AC) and 2'deoxy-5-azacitidine (decitabine) are both potent inhibitors of DNA methyltransferase 1, the enzyme primarily responsible for converting CpG dinucleotides to methyl-CpG dinucleotides in a replicating DNA strand. This mechanism enables the faithful replication of the CpG methylation pattern in the daughter DNA strand (Jones and Baylin 2002). 5-Azacitidine is converted to 2'-deoxy-5-azacitidine intracellularly; both are then phosphorylated and incorporated into DNA in lieu of cytosine residues. The azacytosine nucleoside is recognized by DNA methyltransferase and forms an irreversible adduct. As the DNA undergoes successive cycles of replication, methyltransferase is not available to reproduce the CpG methylation pattern; thus, with several cell divisions, the DNA is increasingly demethylated. As promoter methylation is associated with transcriptional silencing of the gene, reversal of gene methylation leads to re-expression of genes, at least in vitro. A similar mechanism is presumed to occur in response to the administration of DNA methyltransferase inhibitors in vivo. Initial development of the DNA methyltransferase inhibitor 5AC for the treatment of MDS was based on the observation that low doses of this nucleoside analogue could induce terminal differentiation in in vitro models including mouse erythroleukemia cells (MEL) (Creusot et al. 1982). Initial studies administered 5AC intravenously (Silverman et al. 1993), but due to its extremely short half-life, effective intravenous administration required continuous infusion. Subsequent trials utilized the daily subcutaneous administration of a reconstituted slurry (Silverman 2001; Silverman et al. 2002). Promising Phase II data led to a definitive Phase III trial conducted within the CALGB (Silverman 2001; Silverman et al. 2002). In this trial, patients with all French-American-British (FAB) classifications, including low- and high-risk MDS categories, were randomly assigned to treatment with 5AC, 75 mg/m2/day, administered subcutaneously daily for 7 consecutive days on a 28-day cycle or observation/supportive care. Assigned therapy was to continue for a minimum of four cycles. Patients whose disease progressed on the observation arm could cross over after 4 months or longer and receive 5AC. This trial showed significant hematologic responses to 5AC, including 21% complete (CR) and par-

tial (PR) remissions, and overall hematologic responses in 60% of patients, compared with 5% in the observation arm (and here apparently related to increasing neutrophils in the setting of progressive leukemia). Patients assigned to azacitidine experienced a median delay in progression to leukemia of about 8 months. A companion quality of life assessment study showed that treatment with 5AC was associated with improvement in several quality of life parameters, including improved fatigue, dyspnea, physical functioning, positive affect, and psychological distress (Kornblith et al. 2002). However, no survival advantage with azacitidine was observed in this trial, possibly due to the allowed crossover, such that the majority of patients eventually received azacitidine. Azacitidine responses were seen in patients with all FAB subtypes, including patients with AML evolved from MDS. Decitabine has been administered (intravenously) three times daily for 3 days with treatment cycles repeated every 6 weeks. Response rates were similar to those with 5AC; however, delayed cytopenias appeared to be more pronounced with this schedule of decitabine, particularly in the first cycle of therapy (Wijermans et al. 2000). Cytogenetic remissions occurred in 17% of patients in an intention-to-treat analysis (Lubbert et al. 2001). Results from a randomized trial of decitabine versus observation (similar to the above 5AC trial) in patients with International Prognostic Scoring System (IPSS) intermediate 1, 2 or high-risk MDS have been presented in abstract form. The median number of treatment cycles required to attain remission with decitabine was 3±4, and the trial showed hematologic responses in a large proportion of patients and again an increase in the time to disease progression or death (Saba et al. 2004). A recent dose-finding study of decitabine suggests that schedules of this nucleoside that deliver lower daily doses of drug for longer exposures may increase the response rate, consistent with the requirement for exposure to the azacytosine analogues for several cell divisions to effect reversal of DNA methylation (Issa 2003; Issa et al. 2004). Like decitabine, azacitidine therapy has been associated with cytogenetic responses; however, hematologic responses have also been observed in patients who continue to show their original clonal cytogenetic abnormalities. These trials suggest that DNA methyltransferase inhibitors not only ameliorate cytopenias but also delay

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11.2 ´ Therapies Targeting Epigenetic Changes

disease progression. Therefore, while supportive care and growth factors may remain reasonable treatment options, DNA methyltransferase inhibitors, by delaying disease progression, may represent attractive alternatives for some patients. Despite their important activity, the relationship between their clinical effects and their molecular properties as DNA methyltransferase inhibitors remains uncertain. In earlier studies of decitabine, decreased methylation of the p15INK4B promoter was demonstrated in selected patients, apparently along with re-expression of this cyclin-dependent kinase inhibitor as shown by an immunohistochemical technique (Daskalakis et al. 2002). Another study found no relationship between pre-treatment p15 promoter methylation density or change in p15 methylation density and clinical response (Issa et al. 2004). Global and gene-specific DNA methylation has not been systematically analyzed in any study of sufficient size to allow for a meaningfully powered statistical examination of a potential correlation between methylation, reversal of methylation, and clinical response. Similarly, no trials have adequately explored the issue of re-expression of methylated genes and clinical response. Treatment with DNA methyltransferase inhibitors is reasonably well tolerated; however, up to four cycles of therapy are required for most clinical responses to occur. Therapy with DNA methyltransferase inhibitors is often associated with worsening of cytopenias before improvement occurs; thus, a commitment should be made to a minimum of four cycles of therapy with either nucleoside analogue before deeming the treatment unsuccessful. The optimal duration of administration of the DNA methyltransferase inhibitors is similarly uncertain. In the randomized trial of 5AC, patients who achieved less than a complete response received ongoing therapy with the drug, while patients who achieved a complete response received only two additional cycles of drugs after documentation of response. In contrast, decitabine trials have in general administered a maximum of six cycles of drug. This difference in trial design may account for the difference in median response duration (15 months for 5AC, greater than 9 months for decitabine). Determination of the optimal dose schedule and duration of therapy for both drugs will require further trials.

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11.2.2 Histone Deacetylase Inhibitors

Initial interest in the use of histone deacetylase inhibitors to treat MDS derived from the observation that butyrate could induce differentiation in a variety of myeloid leukemia models (Leder and Leder 1975; Novogrodsky et al. 1983; Schroter et al. 1981). Clinical administration of butyrate led to induction of remission in a patient with AML (Novogrodsky et al. 1983), and administration of phenylbutyrate in association with all-trans retinoic acid (ATRA) led to remission in an ATRA-resistant relapse of acute promyelocytic leukemia (Warrell et al. 1998). The polar planar compound hexamethylene bisacetamide (HMBA) was associated with limited clinical activity in MDS (Andreeff et al. 1992). Second generation compounds based on the activity of HMBA to induce differentiation led to the synthesis of suberoylanilide hydroxamic acid (SAHA), another potent HDAC inhibitor (Richon et al. 1996). Administration of the short chain fatty acid HDAC inhibitor sodium phenylbutyrate (NaPB) as 7-day infusions every 14 or 28 days, and as a 21-day infusion repeated every 28 days (21 days on/7 days off) yielded modest lineage responses in patients with MDS and AML (Gore and Carducci 2000; Gore et al. 1997, 2001, 2002). Sustained plasma concentrations of 0.3±0.5 mM were achieved, comparable to concentrations associated with inhibition of histone deacetylase in vitro. The oral short chain fatty acid, valproic acid, in clinical use as a neuroleptic drug, has similar activity in vitro (Gottlicher 2004; Gottlicher et al. 2001), and has been shown to have clinical activity in selected patients with MDS and AML (Kuendgen et al. 2004). The more potent and specific HDAC inhibitors SAHA, FK228 (depsipeptide), MS-275, and LAQ824 have undergone phase I testing in MDS; however, clinical outcomes have not been published. The relationship between clinical response to these agents, inhibition of histone deacetylase or other protein deacetylases, and re-expression of silenced genes, has not been systematically investigated to date.

11.2.3 Combinations of DNA Methyltransferase

and Histone Deacetylase Inhibitors

The observation that transcriptional silencing of genes with hypermethylated CpG islands is mediated via recruitment of transcriptional corepressor complexes, in-

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cluding histone deacetylases, led to the in vitro demonstration that optimal re-expression of such genes required exposure to a DNA methyltransferase inhibitor prior to addition of an HDAC inhibitor (Cameron et al. 1999). This has led to clinical studies investigating the use of HDAC inhibitors in combination with DNA methyltransferase inhibitors for the treatment of MDS and other myeloid malignancies. Trials have been performed (or are underway) examining combinations of 5AC and NaPB, MS-275, and SAHA, and decitabine in combination with valproate, SAHA, and FK228. No results have been published to date.

11.3 Therapies Targeting Angiogenesis

Angiogenesis is a complex process that regulates the formation of blood vessels and plays an important role in both normal development and tumorigenesis (Folkman 1995). Increased microvessel density has been demonstrated in the bone marrow of patients with hematologic malignancies, including MDS (Alexandrakis et al. 2004, 2005; Padro et al. 2000; Pruneri et al. 1999). While neovascularization has been well documented in bone marrow biopsies from patients with MDS, it is not clear whether this represents a primary pathogenic process or, rather, a response to the disordered cytokine milieu in the MDS microenvironment. In vitro, antagonism of angiogenic factors using antibodies that neutralize vascular endothelial growth factor (VEGF) or interfere with its signal transduction leads to increased erythropoiesis from MDS progenitor cells (Bellamy et al. 2001). However, the mechanism underlying the effect of the best studied agents on angiogenesis in MDS remains unclear. Nonetheless, significant activity of these agents has been documented.

11.3.1 Thalidomide

Thalidomide was the first putative anti-angiogenesis agent studied in MDS. Highly active in the treatment of multiple myeloma, the relationship between its activity in myeloma and its anti-angiogenesis activity is unclear (Anderson 2003; Moehler et al. 2004). Thalidomide has been studied in four MDS trials. Three trials showed erythroid responses in 29/142 patients treated; the fourth trial targeted higher doses of thalidomide and responses were observed in only 4/69 patients

(Moreno-Aspitia et al. 2002; Musto 2004; Musto et al. 2002; Raza et al. 2001; Strupp et al. 2002). Thalidomide is poorly tolerated in this patient population due to its neuropathic and sedative effects. Responses are limited to the erythroid series and appear restricted to patients with low-risk MDS. A randomized trial of thalidomide versus placebo has been completed but results have not been released. 11.3.2 Lenalidomide

Lenalidomide is a thalidomide analog with reduced neurotoxicity and increased immunomodulatory activity. In a Phase I/II study of lenalidomide in low-risk MDS, major erythroid responses were observed in 24/ 43 patients (List et al. 2005). An especially impressive response rate was observed in patients whose karyotype included deletions at chromosome 5q31.1. Major cytogenetic responses developed in 10/12 patients with 5q deletions, including complete cytogenetic responses in 9/10. The molecular mechanism of lenalidomide is unclear. Confirmatory large Phase II trials of lenalidomide in MDS patients with and without 5q deletions have been completed and await reporting of the data. Should the unique sensitivity of MDS associated with interstitial deletions on chromosome 5q be confirmed in the Phase II trials, lenalidomide may represent a powerful tool for probing the molecular pathogenesis of that subset of MDS (Giagounidis et al. 2004). 11.3.3 Bevacizumab

Bevacizumab is a monoclonal antibody to VEGF that has been studied in both solid and hematologic malignancies. It was recently approved for use in metastatic colorectal cancer after a clinical trial demonstrated improved overall survival when given in combination with standard chemotherapy (Hurwitz et al. 2004). In patients with refractory and relapsed AML, a combination of cytarabine, mitoxantrone and bevacizumab was tested (Karp et al. 2004). The overall response rate was 48%, and 33% of patients had complete responses. Serum VEGF levels were decreased significantly in the majority of patients in comparison to pretreatment levels.

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11.4 ´ Therapies Targeting Disordered Cytokine Milieu

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11.3.4 SU11248 and PTK787

11.4.1 Pentoxifylline

SU11248 is a small molecule receptor tyrosine kinase (RTK) inhibitor that blocks VEGF-mediated cell growth (Glade-Bender et al. 2003). A Phase I trial of SU11248 in 15 patients with refractory or resistant AML showed six responses in ten evaluable patients (Fiedler et al. 2005). All patients with FLT3 mutations (n=4) had morphologic or partial responses, compared with two of ten evaluable patients with wild-type FLT3. Another Phase I trial in 29 AML patients showed responses in five of 29 patients (O'Farrell et al. 2003). PTK787 is another potent orally administered inhibitor with activity against all VEGF RTKs, PDGFR, c-kit and Flt-1 (Drevs 2003; Thomas et al. 2003). It is currently under evaluation by Cancer and Leukemia Group B (CALGB study 10105).

Pentoxifylline (PTX), a xanthine derivative known to interfere with the lipid-signaling pathway used by TNFa, a TGFb F and IL-1b, has been studied in combination with other agents in the treatment of MDS. Raza et al. administered a combination of pentoxifylline, ciprofloxacin and dexamethasone to 25 patients with MDS. Eighteen patients showed some hematologic response, nine of 18 showing an improvement in absolute neutrophil counts only, and nine of 18 showing multi-lineage responses. Raza et al. also administered pentoxifylline, ciprofloxacin and amifostine with and without dexamethasone to 35 patients with MDS (Raza et al. 2000). Fifteen patients did not respond until dexamethasone was added, and seven responded before. When examined by lineage, 19 of 22 patients showed improved neutrophil counts, 11 of 22 demonstrated more than 50% reduction in blood transfusion requirements, improved Hb levels, or both, and seven of 22 showed improvement in platelet counts. The studies of pentoxifylline have been difficult to assess due to the inclusion of steroids in most combinations; additionally, the response criteria used in these studies pre-dated the now-standard IWG criteria (Cheson et al. 2000).

11.3.5 Arsenic Trioxide

Arsenic trioxide, a potent agent for the treatment of relapsed acute promyelocytic leukemia, possesses a variety of molecular activities, including inhibition of angiogenesis (List et al. 2003; Sekeres 2005). This observation led to several Phase II studies of arsenic trioxide for the treatment of MDS that have not yet been published. In a trial combining arsenic trioxide with thalidomide in 28 patients with MDS, seven patients responded, including one complete hematologic and cytogenetic response and one with regression in spleen size (Raza et al. 2004). Two trilineage responses were seen in patients with inv (3)(q21q26.2).

11.4 Therapies Targeting Disordered Cytokine

Milieu

As with angiogenesis, it is not clear whether the disordered cytokine milieu in the microenvironment of MDS bone marrows represents a primary pathogenic event or a secondary phenomenon. Nonetheless, the dysregulated cytokines (especially increased TNFa and TGFb) can clearly serve as negative regulators of hematopoiesis, in particular erythropoiesis. Thus, targeting this cytokine dysregulation could be associated with amelioration of cytopenias in early stage MDS.

11.4.2 SCIO-469

SCIO-469 is an orally bioavailable inhibitor of p38a mitogen-activated protein kinase (MAPK). The mammalian MAPK cascades are information highways for the transmission of extracellular signals from the cell surface to the nucleus. In response to upstream signals, MAPKs phosphorylate their specific substrates at serine or threonine residues. These phosphorylation events can positively or negatively regulate substrates and, thus, the entire signaling cascade. MAPK signaling pathways have numerous roles, including modulation of gene expression, mitosis, proliferation, motility, metabolism, and apoptosis (Milella et al. 2003; Wada and Penninger 2004). The p38 MAPK cascade in particular appears to be important for the synthesis and secretion of IL-6, VEGF, and TNFa, a important components of the bone marrow microenvironment. SCIO-469 has demonstrated activity in preliminary models of both multiple myeloma and MDS. SCIO-469 treatment augmented cytotoxicity of the proteasome in-

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hibitor PS-341 against PS-341-resistant cell lines and patient multiple myeloma cells (Hideshima et al. 2004). 11.4.3 Etanercept and Infliximab

Tumor necrosis factor alpha (TNFa) mRNA and protein levels have been reported to be elevated in both bone marrow and blood plasma samples of patients with MDS, and recent clinical trials have evaluated the efficacy of treatment with immunosuppression and antiTNF therapy (Kitagawa et al. 1997; Maciejewski et al. 1995; Molnar et al. 2000; Rosenfeld and Bedell 2002). Pilot studies using the soluble TNFa receptor protein have shown its safety in patients with MDS (Deeg et al. 2002; Rosenfeld and Bedell 2002). Deeg et al. (2004) treated 14 transfusion-requiring patients with MDS with the combination of antithymocyte globulin (ATG) and the soluble TNF receptor protein etanercept. Forty-six percent of the patients responded with five patients achieving periods of red blood cell and platelet independence that exceeded 2 years. These results support the premise that immunosuppression is effective in select patients with MDS. Infliximab is a chimeric anti-TNFa monoclonal antibody. It binds both soluble and membrane-bound TNFa. a Stasi and Amadori (2002) reported one major and one minor erythroid response in two MDS patients treated with infliximab. Other data that have been presented in abstract form show some activity of infliximab in patients with MDS.

11.5.1 ATG

Single agent ATG has resulted in complete hematologic responses of up to 10±15% of patients with MDS (Killick et al. 2003; Molldrem et al. 2002). Predictors of response to immunosuppressive therapy include younger age, presence of a paroxysmal nocturnal hemoglobinuria (PNH) clone, HLA DR15, hypocellularity and a normal karyotype (Saunthararajah et al. 2002). Response to ATG has been associated with disappearance of T cell clones that demonstrate T cell receptor V beta clonality, and which suppress hematopoiesis ex vivo (Deeg et al. 2004).

11.5.2 Cyclosporine

Cyclosporine, a calcineurin inhibitor that is a potent immunosuppressive agent used in solid organ and bone marrow transplantation, has been studied in the treatment of MDS. Shimamoto et al. (2003) examined the efficacy of cyclosporine A (CsA) in 50 patients with myelodysplastic syndrome. Hematological improvement was observed in 30 (60%) patients, all with refractory anemia (RA). There were significantly more responders among patients with good risk karyotype or HLADRB1*1501 than among patients with intermediate/poor risk karyotypes or with other HLA-DRB1 types. This supports the notion that certain subsets of MDS have a more ªimmunologicº pathophysiology that is responsive to immune suppression.

11.5 Therapies Targeting the Immune System

11.6 Therapies Targeting Signaling

The incidence of autoimmune disorders is increased in some MDS populations (Saif et al. 2002). Autologous cytotoxic T-lymphocytes have been observed to exert an inhibitory effect on MDS myelopoiesis in vitro. The clinical features of subsets of MDS overlap with aplastic anemia (AA) and large granular lymphocyte (LGL) lymphoproliferative disorders, two diseases thought to be related to dysregulation of the immune system (Barrett et al. 2000). Clinical studies have shown activity of the immunosuppressives antithymocyte globulin and cyclosporine in the treatment of select groups of MDS patients.

11.6.1 Tipifarnib

Farnesyltransferase (FT) inhibitors have emerged as novel inhibitors of signaling for hematologic malignancies (Lancet and Karp 2003). They were originally developed to interfere with the farnesylation of Ras, a gene mutated in a wide array of cancers and in up to 20% of MDS patients, and have been the focus of intense investigation (Beaupre and Kurzrock 1999; Reuter et al. 2000). Two orally bioavailable compounds, tipifarnib and lonafarnib, are the most advanced to date in terms of clinical testing (Kurzrock et al. 2002). In an initial Phase I trial in patients with refractory and relapsed MDS and AML, tipifarnib produced clini-

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11.7 ´ Therapies Targeting Aberrant Differentiation

cal responses in 29% of patients, including two complete remissions (Karp et al. 2001). Interestingly, none of the patients enrolled in the study were found to have Ras mutations. Correlative studies showed inhibition of farnesyltransferase activity in the bone marrow cells of treated patients. Non-dose-limiting toxicities included nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. A Phase II study of tipifarnib in MDS patients reported responses in three of 28 treated patients (Kurzrock et al. 2004). The initial dose of 600 mg orally twice daily was not well tolerated; a lower dose of 300 mg orally twice daily was acceptable.

11.6.2 Lonafarnib

Abstracts presented in recent meetings of the American Society of Hematology have shown promising activity for lonafarnib in the treatment of MDS and AML patients. A Phase III randomized trial is being planned to investigate the clinical benefit and frequency of platelet response to lonafarnib in patients with chronic myelomonocytic leukemia (CMML) or advanced MDS with severe thrombocytopenia.

11.6.3 Imatinib

In a subset of patients with CMML, a reciprocal translocation occurs that places the platelet-derived growth factor receptor beta (PDGFRb R ) next to various fusion partners, resulting in constitutive activation of the tyrosine kinase function of PDGFRb R (Levitzki 2004). Imatinib therapy has had promising activity in a subset of patients with a PDGFRb R translocation (Apperley et al. 2002). Four patients with CMML and a PDGFRb R translocation treated with imatinib 400 mg orally daily sustained complete cytogenetic responses.

11.6.4 PKC412

Protein kinase C (PKC) plays an important role in signaling pathways that regulate cell structure and gene expression (Newton 2001). Downstream mediators such as MAPKs and phosphatidylinositol 3-kinase are important for growth and differentiation (Franklin and McCubrey 2000). PKC412, a potent kinase inhibitor that has activity against PKC and FLT-3, has been studied in he-

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matologic and solid malignancies both alone and in conjunction with other agents (George et al. 2004; Monnerat et al. 2004; Virchis et al. 2002). It is currently under evaluation in myelodysplastic syndromes.

11.7 Therapies Targeting Aberrant

Differentiation

With the success of all-trans retinoic acid (ATRA) in treating acute promyelocytic leukemia, there has been increased interest in targeting aberrant differentiation pathways in the treatment of AML and MDS. Retinoic acid derivatives such as ATRA, cis-retinoic acid and vitamin D have been studied alone and in combination with other agents.

11.7.1 Retinoic Acid

Single agent ATRA has shown limited activity in patients with MDS. Visani et al. (1995) treated ten patients with MDS with oral ATRA for 6 weeks. A rise in hemoglobin concentration > 1 g/dl was observed in 3/10 patients, while 5/10 patients showed an increase in granulocyte counts > 0.5 ´ 109/l without concomitant increase in the percentage of blast cells in the bone marrow. All the effects were transient and maximal responses were obtained by the fourth week of treatment. 13-cis-retinoic acid (13-CRA) has shown mild single agent activity in MDS (Ohno 1994). Koeffler et al. (1988) randomized 68 patients with MDS to receive a single daily dose of either 13-CRA or placebo. Treatment was continued up to 6 months. No significant difference was noted between the two treatment groups in response to test drug. Clark et al. (1987) evaluated the effect of 13-CRA in 70 patients with MDS and 5% or fewer bone marrow blasts. Among non-sideroblastic patients, 1-year survival was 77% in the treatment arm versus 36% in the control group. Hematologic responses were not characterized with the precision of modern International Working Group criteria. More recently, Bourantas et al. (1995) administered 13-CRA to 34 patients with MDS. Partial remission was achieved in four patients, one with RA, two with RA with excess blasts (RAEB) and one with CMML. Questions remain as to the optimal dosage, timing and combination of this agent in the treatment of MDS.

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Combination of differentiation agents with other agents such as chemotherapy or histone deacetylase inhibitors has also shown promising results. Ferrero et al. (2004) tested 13-cis retinoic acid + (OH)2 vitamin D3 + low-dose 6-thioguanine and cytarabine in 26 patients with AML and in four patients with MDS. The response rate was 50%, with 27% complete remissions. Kuendgen et al. (2004) treated 18 patients with MDS and AML with valproic acid (VPA), a histone deacetylase inhibitor. Five patients were treated up front with VPA and ATRA. There were no responses. Eight patients responded to VPA therapy alone. Four of five patients who relapsed were treated with VPA and ATRA, and two responded. Erythropoietin has been given in conjunction with ATRA with some clinical responses. In one study, 27 patients with low- or intermediate-risk MDS were enrolled in a 12-week study (Stasi et al. 2002). ATRA was administered orally at doses of 80 mg/m2 per day in two divided doses for 7 consecutive days every other week. Recombinant human EPO was given subcutaneously three times a week. Clinically significant erythroid responses with increases in hemoglobin levels of at least 1 g/dl or reduction of transfusion needs were seen in 13 (48%) patients, with four showing improved responses after dose escalation of EPO. 11.7.2 Vitamin D

Vitamin D3 analogs can act on the differentiation and maturity of different cell lines in vitro. Vitamin D has been studied in the treatment of both solid and hematologic cancers. Mellibovsky et al. (1998) studied the effects of vitamin D analogs in patients with MDS. All the patients were in a low- to intermediate-risk group. In the calcidiol-treated group, one patient responded, three were nonresponders, and one showed progression. In the calcitriol group, ten were responders (two with major response), and four were non-responders. Side effects were minimal. Studies of vitamin D analogs in conjunction with other agents in the treatment of MDS are underway.

11.8 Miscellaneous Therapies 11.8.1 TLK199

TLK199 is a novel liposomal glutathione derivative that is a peptidomimetic inhibitor of glutathione S-transferase P1-1 (GSTP1-1) (Ruscoe et al. 2001). GSTP1-1 is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues. It is thought to act as a negative growth regulator; inhibition of this pathway might promote cellular proliferation and differentiation. The results of TLK199 in the treatment of MDS patients have been reported in abstract form. Response rates appear to be in the 20±30% range in low-risk patients. 11.9 Conclusion

The recent FDA approval of azacitidine and the likely fast track review of lenalidomide indicate the emergence of a number of biologically based therapies for MDS. The market for new therapies is great, given that MDS is primarily a disease of older adults, and demographics in the United States are shifting towards an older population. Individually tailored combination therapies targeting multiple molecular mechanisms will be the norm in the future. Ideally, orally bioavailable, less toxic therapies will improve the availability and tolerability of treatments for older individuals. Rationally designed clinical studies are critical to sorting through the increasing number of agents available for testing. Emphasis must be placed on verifying the putative `targeted' molecular mechanisms of the novel agents. Understanding of the relevant operative mechanisms underlying clinical response will contribute to a better understanding of the biology of MDS and will facilitate the rational design of more effective drugs.

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ment of old/poor prognosis patients with acute myeloid leukemia or myelodysplastic syndrome. Haematologica 89:619±620 Fiedler W, Serve H, Dohner H, Schwittay M, Ottmann OG, O'Farrell AM, Bello CL, Allred R, Manning WC, Cherrington JM, Louie SG, Hong W, Brega NM, Massimini G, Scigalla P, Berdel WE, Hossfeld DK (2005) A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 105:986±993 Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease (Review). Nat Med 1:27±31 Franklin RA, McCubrey JA (2000) Kinases: positive and negative regulators of apoptosis (Review). Leukemia 14:2019±2034 George P, Bali P, Cohen P, Tao J, Guo F, Sigua C, Vishvanath A, Fiskus W, Scuto A, Annavarapu S, Moscinski L, Bhalla K (2004) Cotreatment with 17-allylamino-demethoxygeldanamycin and FLT-3 kinase inhibitor PKC412 is highly effective against human acute myelogenous leukemia cells with mutant FLT-3. Cancer Res 64:3645±3652 Giagounidis AA, Germing U, Wainscoat JS, Boultwood J, Aul C (2004) The 5q± syndrome. Hematology 9:271±277 Glade-Bender J, Kandel JJ, Yamashiro DJ (2003) VEGF blocking therapy in the treatment of cancer (Review). Expert Opinion On Biological Therapy 3:263±276 Gore SD, Carducci MA (2000) Modifying histones to tame cancer: clinical development of sodium phenylbutyrate and other histone deacetylase inhibitors (Review). Expert Opinion on Investigational Drugs 9:2923±2934 Gore SD, Samid D, Weng LJ (1997) Impact of the putative differentiating agents sodium phenylbutyrate and sodium phenylacetate on proliferation, differentiation, and apoptosis of primary neoplastic myeloid cells. Clin Cancer Res 3:1755±1762 Gore SD, Weng LJ, Zhai S, Figg WD, Donehower RC, Dover GJ, Grever M, Griffin CA, Grochow LB, Rowinsky EK, Zabalena Y, Hawkins AL, Burks K, Miller CB (2001) Impact of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia. Clin Cancer Res 7:2330±2339 Gore SD, Weng LJ, Figg WD, Zhai S, Donehower RC, Dover G, Grever MR, Griffin C, Grochow LB, Hawkins A, Burks K, Zabelena Y, Miller CB (2002) Impact of prolonged infusions of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia. Clin Cancer Res 8:963±970 Gottlicher M (2004) Valproic acid: an old drug newly discovered as inhibitor of histone deacetylases (Review). Ann Hematol 83[Suppl 1]:S91±S92 Gottlicher M, Minucci S, Zhu P, Kramer OH, Schimpf A, Giavara S, Sleeman JP, Lo CF, Nervi C, Pelicci PG, Heinzel T (2001) Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J 20:6969±6978 Hideshima T, Podar K, Chauhan D, Ishitsuka K, Mitsiades C, Tai YT, Hamasaki M, Raje N, Hideshima H, Schreiner G, Nguyen AN, Navas T, Munshi NC, Richardson PG, Higgins LS, Anderson KC (2004) p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells. Oncogene 23:8766±8776 Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335±2342

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Issa JP (2003) Decitabine (Review). Curr Opin Oncol 15:446±451 Issa J-P, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, Bayar E, Lyons J, Rosenfeld CS, Cortes J, Kantarjian HM (2004) Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103:1635±1640 Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer (Review). Nat Rev Genet 3:415±428 Karp JE, Lancet JE, Kaufmann SH, End DW, Wright JJ, Bol K, Horak I, Tidwell ML, Liesveld J, Kottke TJ, Ange D, Buddharaju L, Gojo I, Highsmith WE, Belly RT, Hohl RJ, Rybak ME, Thibault A, Rosenblatt J (2001) Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood 97:3361±3369 Karp JE, Gojo I, Pili R, Gocke CD, Greer J, Guo C, Qian D, Morris L, Tidwell M, Chen H, Zwiebel J (2004) 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 10:3577±3585 Killick SB, Mufti G, Cavenagh JD, Mijovic A, Peacock JL, Gordon-Smith EC, Bowen DT, Marsh JC (2003) A pilot study of antithymocyte globulin (ATG) in the treatment of patients with 'low-risk' myelodysplasia. Br J Haematol 120:679±684 Kitagawa M, Saito I, Kuwata T, Yoshida S, Yamaguchi S, Takahashi M, Tanizawa T, Kamiyama R, Hirokawa K (1997) Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes. Leukemia 11:2049±2054 Koeffler HP, Heitjan D, Mertelsmann R, Kolitz JE, Schulman P, Itri L, Gunter P, Besa E (1988) Randomized study of 13-cis retinoic acid v placebo in the myelodysplastic disorders. Blood 71:703±708 Kornblith AB, Herndon JE, Silverman LR, Demakos EP, Odchimar-Reissig R, Holland JF, Powell BL, DeCastro C, Ellerton J, Larson RA, Schiffer CA, Holland JC (2002) Impact of azacytidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol 20:2441±2452 Kuendgen A, Strupp C, Aivado M, Bernhardt A, Hildebrandt B, Haas R, Germing U, Gattermann N (2004) Treatment of myelodysplastic syndromes with valproic acid alone or in combination with alltrans retinoic acid. Blood 104:1266±1269 Kurzrock R, Cortes J, Kantarjian H (2002) Clinical development of farnesyltransferase inhibitors in leukemias and myelodysplastic syndrome (Review). Semin Hematol 39:20±24 Kurzrock R, Albitar M, Cortes JE, Estey EH, Faderl SH, Garcia-Manero G, Thomas DA, Giles FJ, Ryback ME, Thibault A, De Porre P, Kantarjian HM (2004) Phase II study of R115777, a farnesyl transferase inhibitor, in myelodysplastic syndrome. J Clin Oncol 22:1287±1292 Lancet JE, Karp JE (2003) Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy (Review). Blood 102:3880± 3889 Leder A, Leder P (1975) Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells. Cell 5:319±322 Levitzki A (2004) PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases (Review). Cytokine & Growth Factor Reviews 15:229±235

List A, Beran M, DiPersio J, Slack J, Vey N, Rosenfeld CS, Greenberg P (2003) Opportunities for Trisenox (arsenic trioxide) in the treatment of myelodysplastic syndromes. Leukemia 17:1499±1507 List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D, Rimsza L, Heaton R, Knight R, Zeldis JB (2005) Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 352:549±557 Lubbert M, Wijermans P, Kunzmann R, Verhoef G, Bosly A, Ravoet C, Andre M, Ferrant A (2001) Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. Br J Haematol 114:349±357 Maciejewski J, Selleri C, Anderson S, Young NS (1995) Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood 85:3183±3190 Mellibovsky L, Diez A, Perez-Vila E, Serrano S, Nacher M, Aubia J, Supervia A, Recker RR (1998) Vitamin D treatment in myelodysplastic syndromes. Br J Haematol 100:516±520 Milella M, Kornblau SM, Andreeff M (2003) The mitogen-activated protein kinase signaling module as a therapeutic target in hematologic malignancies (Review). Reviews in Clinical & Experimental Hematology 7:160±190 Moehler TM, Hillengass J, Goldschmidt H, Ho AD (2004) Antiangiogenic therapy in hematologic malignancies (Review). Current Pharmaceutical Design 10:1221±1234 Molldrem JJ, Leifer E, Bahceci E, Saunthararajah Y, Rivera M, Dunbar C, Liu J, Nakamura R, Young NS, Barrett AJ (2002) Antithymocyte globulin for treatment of the bone marrow failure associated with myelodysplastic syndrome [summary for patients in Ann Intern Med 2002 137:I-27]. Ann Intern Med 137:156±163 Molnar L, Berki T, Hussain A, Nemeth P, Losonczy H (2000) Detection of TNFalpha expression in the bone marrow and determination of TNFalpha production of peripheral blood mononuclear cells in myelodysplastic syndrome. Pathol Oncol Res 6:18±23 Monnerat C, Henriksson R, Le Chevalier T, Novello S, Berthaud P, Faivre S, Raymond E (2004) Phase I study of PKC412 (N-benzoyl-staurosporine), a novel oral protein kinase C inhibitor, combined with gemcitabine and cisplatin in patients with non-small-cell lung cancer. Ann Oncol 15:316±323 Moreno-Aspitia A, Geyer S, Li C-Y, Tefferi A, Witzig T, Niedrinhaus RD, Yukov AM, Morton R, Fitch T, Addo FE, Dakhil SR, Tschetter L, Colon-Otero G (2002) N998B: Multicenter phase II trial of thalidomide (Thal) in adult patients with myelodysplastic syndromes (MDS) [Abstract]. Blood 100:96 a Musto P (2004) Thalidomide therapy for myelodysplastic syndromes: current status and future perspectives (Review). Leuk Res 28: 325±332 Musto P, Falcone A, Sanpaolo G, Bisceglia M, Matera R, Carella AM (2002) Thalidomide abolishes transfusion-dependence in selected patients with myelodysplastic syndromes. Haematologica 87:884± 886 Newton AC (2001) Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions (Review). Chemical Reviews 101:2353±2364 Novogrodsky A, Dvir A, Ravid A, Shkolnik T, Stenzel KH, Rubin AL, Zaizov R (1983) Effect of polar organic compounds on leukemic cells.

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Schroter H, Gomez-Lira MM, Plank KH, Bode J (1981) The extent of histone acetylation induced by butyrate and the turnover of acetyl groups depend on the nature of the cell line. Eur J Biochem 120:21±28 Sekeres MA (2005) Arsenic trioxide as a treatment for myelodysplastic syndrome. Current Hematology Reports 4:59±63 Shimamoto T, Tohyama K, Okamoto T, Uchiyama T, Mori H, Tomonaga M, Asano Y, Niho Y, Teramura M, Mizoguchi H, Omine M, Ohyashiki K (2003) Cyclosporin A therapy for patients with myelodysplastic syndrome: multicenter pilot studies in Japan. Leuk Res 27:783±788 Silverman LR (2001) Targeting hypomethylation of DNA to achieve cellular differentiation in myelodysplastic syndromes (MDS) (Review). Oncologist 6[Suppl 5]:8±14 Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC, Odchimar-Reissig R, Stone RM, Nelson D, Powell BL, DeCastro CM, Ellerton J, Larson RA, Schiffer CA, Holland JF (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20:2429±2440 Silverman LR, Holland JF, Weinberg RS, Alter BP, Davis RB, Ellison RR, Demakos EP, Cornell CJJ, Carey RW, Schiffer C (1993) Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes. Leukemia 7[Suppl 1]:21±29 Stasi R, Amadori S (2002) Infliximab chimaeric anti-tumour necrosis factor alpha monoclonal antibody treatment for patients with myelodysplastic syndromes. Br J Haematol 116:334±337 Stasi R, Brunetti M, Terzoli E, Amadori S (2002) Sustained response to recombinant human erythropoietin and intermittent all-trans retinoic acid in patients with myelodysplastic syndromes. Blood 99:1578±1584 Strupp C, Germing U, Aivado M, Misgeld E, Haas R, Gattermann N (2002) Thalidomide for the treatment of patients with myelodysplastic syndromes. Leukemia 16:1±6 Thomas AL, Morgan B, Drevs J, Unger C, Wiedenmann B, Vanhoefer U, Laurent D, Dugan M, Steward WP (2003) Vascular endothelial growth factor receptor tyrosine kinase inhibitors: PTK787/ZK 222584 (Review). Semin Oncol 30:32±38 Virchis A, Ganeshaguru K, Hart S, Jones D, Fletcher L, Wright F, Wickremasinghe R, Man A, Csermak K, Meyer T, Fabbro D, Champain K, Yap A, Prentice HG, Mehta A (2002) A novel treatment approach for low grade lymphoproliferative disorders using PKC412 (CGP41251), an inhibitor of protein kinase C. Hematol J 3:131±136 Visani G, Tosi P, Manfroi S, Ottaviani E, Finelli C, Cenacchi A, Bendandi M, Tura S (1995) All-trans retinoic acid in the treatment of myelodysplastic syndromes. Leuk Lymphoma 19:277±280 Wada T, Penninger JM (2004) Mitogen-activated protein kinases in apoptosis regulation (Review). Oncogene 23:2838±2849 Warrell RPJ, He LZ, Richon V, Calleja E, Pandolfi PP (1998) Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst 90:1621± 1625 Wijermans P, Lçbbert M, Verhoef G, Bosly A, Ravoet C, Andre M, Ferrant A (2000) Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol 18:956±962

Hemopoietic Cell Transplantation Bart Scott, H. Joachim Deeg

Contents 12.1 Introduction . . . . . . . . . . . . . . . . . . . .

123

12.2 General Considerations for HCT . . . . .

124

12.3 Transplant Strategies . . . . . . . . . . . . . 12.3.1 Conventional (ªMyeloablativeº) HCT 12.3.1.1 ªLess Advancedº MDS . . . 12.3.1.2 ªAdvancedº MDS . . . . . . . 12.3.2 Reduced-Intensity Conditioning Regimens . . . . . . . . . . . . . . . . . . 12.3.3 Autologous HCT . . . . . . . . . . . . .

125 125 125 127

12.4 Special Considerations . . . . . . . . . . . . 12.4.1 MDS Is a Disease of ªOlderº Patients 12.4.2 Childhood MDS . . . . . . . . . . . . . 12.4.3 Other Parameters . . . . . . . . . . . . 12.4.4 Secondary MDS . . . . . . . . . . . . . 12.4.5 Post-transplant Relapse . . . . . . . .

129 129 130 130 130 131

12.5 Conclusions . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . .

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128 129

12.1 Introduction

Hemopoietic cell transplantation (HCT) provides effective therapy for various malignant and non-malignant disorders. The indications are relatively clear for some diseases, but are less well defined for others, including myelodysplastic syndrome (MDS). Firstly, MDS is predominantly a disease of older patients, and conventional transplant approaches have not been well tolerated in

that age group. Secondly, in many patients, MDS progresses slowly over many years with only little morbidity, rendering the decision as to the optimum time for transplantation difficult (Cutler et al. 2004). Thirdly, our understanding of the pathophysiology of MDS has improved, and several therapeutic compounds directed at exploiting biologic features of the disease have shown efficacy, at least transiently, as non-transplant therapy for MDS (Deeg et al. 2004 b; List et al. 2003; Molldrem et al. 2002; Raza et al. 2001; Silverman et al. 2002). Nevertheless, MDS are clonal stem cell disorders, and the only therapeutic modality with proven curative potential is HCT. The indications for HCT depend upon disease stage/risk, patient interest, patient age, donor availability, the promise of alternative modalities, and, in the end, the probability of success with a transplant. Details of disease classification are discussed elsewhere in this volume. According to the French-American-British (FAB) classification, MDS includes refractory anemia (RA; < 5% marrow blasts), RA with ringed sideroblasts (RARS; > 15% marrow ringed sideroblasts), RA with excess blasts (RAEB; 5±20% marrow blasts), RAEB in transformation (RAEBt; 21±30% marrow blasts), and chronic myelomonocytic leukemia (CMML) (Bennett et al. 1982). The World Health Organization (WHO) recently defined MDS subgroups more narrowly in a revised classification including RA, RARS, refractory cytopenia with multilineage dysplasia (RCMD), del 5q± syndrome, RAEB-1 (5± 10% marrow blasts) and RAEB-2 (11±20% marrow blasts), and unclassifiable MDS. Furthermore, the threshold for the diagnosis of AML was reduced to > 20% myeloblasts, effectively eliminating RAEBt as a diagnostic category (Vardiman et al. 2002). In addition, CMML was reclassified as a myeloproliferative disorder.

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Chapter 12 ´ Hemopoietic Cell Transplantation

The incorporation of cytogenetic findings and the number of cytopenias, in addition to the blast count, into a new risk scoring system termed International Prognostic Scoring System (IPSS) provides improved prognostic precision (Greenberg et al. 1997), not only for the natural history of the disease, but also for results with HCT (Deeg et al. 2002; Nevill et al. 1998). 12.2 General Considerations for HCT

Until quite recently very few patients above the age of 55 years were offered HCT from allogeneic donors (autologous ªtransplantsº using the patient's own cells have been performed up to a higher age ceiling). This policy was based on the observation that the severity and frequency of transplant-related morbidity and mortality (TRM) increased progressively with age. Those complications were related not only to the intensity of the transplant conditioning regimens, but also to graft-versus-host disease (GvHD), the most frequent complication after allogeneic transplantation. Therefore, since even in the most favorable group of patients overall mortality rates have been in the range of 25±30%, careful assessment of the indications for and timing of transplantation is needed. As discussed in Chapter 8, patients in IPSS risk groups low or intermediate-1 may have life expectancies in the range of 5±10 years with supportive care only or low-intensity therapy (Greenberg et al. 1997). However,

Fig. 12.1. Impact of acute GvHD on RFS. There were 151 patients with grades 0±I and 126 with grades II±IV acute GvHD (p = 0.01). This figure is adapted from research originally published in Castro-Malaspina et al. (2002)

reassessment in regard to transplantation is indicated in any patient with disease progression. For patients with intermediate-2 or high-risk MDS by IPSS, HCT is considered the treatment of choice if they are younger than 65 years old and have good performance status. In patients more than 65 years of age with adequate performance status, low-intensity (non-transplant) therapy might be preferable, unless these patients qualify for transplantation following non-myeloablative (NMA)/reduced intensity conditioning (RIC). It is of note that the IPSS, while originally derived from data on survival and leukemic transformation in non-transplanted patients, also impacts on survival after HCT. Among 251 patients transplanted at the Fred Hutchinson Cancer Research Center (FHCRC), the 5year relapse-free survival (RFS) was 60% with low and intermediate-1 risk, 36% for intermediate-2 risk, and 28% for patients with high-risk disease (Appelbaum and Anderson 1998). Similar results have been reported by Neville et al. (1998), who showed 7-year RFS for patients in the good-, intermediate-, and poor-risk cytogenetic subgroups (as determined by IPSS) to be 51%, 40% and 6%, respectively. The corresponding figures for actuarial relapse were 19%, 12% and 82%, respectively. There was no difference for NRM between the three groups. In addition to single or multi-organ failure, the major causes of NRM after allogeneic HCT are GvHD and associated complications, in particular, infections. Figure 12.1 illustrates the impact of acute GvHD on RFS among patients with MDS transplanted from unrelated donors (Castro-Malaspina et al. 2002). Taking into consideration the IPSS information and transplant results, Cutler et al. (2004), in an analysis involving patient data from multiple institutions, have suggested that patients in risk groups intermediate-2 and high, who a priori are transplant candidates, will have the best overall life expectancy if they proceed to transplantation without delay. Patients with low to intermediate-1 risk disease, on the other hand, may have the longest life expectancy if HCT is delayed, maybe by several years (Cutler et al. 2004) (Fig. 12.2). The role of intensive remission-induction and consolidation chemotherapy before HCT in patients with MDS has remained controversial. De Witte et al. (2001) reported on 184 patients who received 1 or 2 remission-induction courses followed by consolidation (in patients with complete remission [CR]; patients who did not achieve a CR with induction were advised

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12.3 ´ Transplant Strategies

125

12.3.1 Conventional (ªMyeloablativeª) HCT

12.3.1.1 ªLess Advancedº MDS

Fig. 12.2. Impact of IPSS risk category on likely benefit from HCT. While based on several assumptions, the data suggest that patients in risk groups intermediate-2 (int-2) and high, if candidates for HCT, should be transplanted without delay. Patients in the lower risk groups may benefit from delaying transplantation until there is evidence for disease acceleration. This figure was originally published in Cutler et al. (2004)

to undergo HCT as salvage therapy or receive therapy with high-dose cytosine-arabinoside [Ara-C]). Following consolidation, patients then proceeded to either allogeneic or autologous HCT depending on donor availability. Four-year overall survival in the entire cohort was 26%, and RFS was 29% (de Witte et al. 2001). Yakoub-Agha et al. (2000) have shown that patients who achieve remissions with pre-transplant chemotherapy have a substantially better outcome after HCT than patients who do not achieve a remission. However, patients who are given induction chemotherapy and fail to respond, have a lower probability of a successful post-transplant course than patients who were not treated pre-transplant (Scott et al. 2005). Controlled studies comparing HCT with and without prior chemotherapy are necessary to definitely answer the question as to the role of induction chemotherapy.

The best results with allogeneic HCT are achieved in patients with low myeloblast counts in the marrow, i.e., RA/RARS (or RCMD, RCRS), at the time of transplantation, and patients without high-risk clonal cytogenetic abnormalities (less advanced MDS) (Table 12.1). The European Group for Blood and Marrow Transplantation (EBMT) reported on 131 patients, most conditioned with total body irradiation (TBI)-based regimens (70%) and transplanted from HLA-identical siblings. Five-year RFS was 52%, and relapse incidence 13% for patients with RA/RARS (Runde et al. 1998). Among 510 patients with MDS transplanted from unrelated donors (National Marrow Donor Program) those conditioned with busulfan (BU) plus cyclophosphamide (CY) [BUCY] fared better than patients prepared with other, generally TBI-containing regimens (Castro-Malaspina et al. 2002). RFS and relapse rate in patients with RA were 40% and 5%, respectively (Castro-Malaspina et al. 2002). BUCY regimens have been used by several transplant teams (Nevill et al. 1998; O'Donnell et al. 1995); some have added cytosine arabinoside (Ratanatharathorn et al. 1993). Despite encouraging results, however, NRM due to infections, GvHD, and single or multi-organ toxicity was in the range of 30±54% (Castro-Malaspina et al. 2002; Nevill et al. 1998; Runde et al. 1998). The team at the FHCRC recently reported results achieved with a BUCY regimen in which the busulfan

Table 12.1. Transplant outcome in patients with MDS conditioned with a regimen of ªtargetedº busulfan and cyclophosphamide (Deeg et al. 2002) MDS risk group

12.3 Transplant Strategies

RFS

Relapse l

NRM

0.57

0.13

0.31

Low

0.80

0.00

0.20

Intermediate-1

0.64

0.06

0.30

All patients (n = 109)

We will discuss various transplant approaches. As results with transplants from unrelated donors who are matched with recipients on the basis of high-resolution human leukocyte antigen (HLA) typing are approaching those with HLA-identical sibling transplants, we will present those data together rather than strictly separating them.

Transplant outcome (proportion) a

IPSS

Intermediate-2

0.40

0.29

0.31

High

0.29

0.42

0.29

RFS relapse-free survival, NRM non-relapse mortality a

At 3 years after transplantation

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Chapter 12 ´ Hemopoietic Cell Transplantation

Fig. 12.3. Impact of pre-transplant IPSS risk on relapse (A) and RFS (B) after allogeneic HCT. int-1 intermediate-1; int-2 intermediate-2. This figure is adapted from research originally published in Deeg et al. (2002)

(BU) dose was adjusted to maintain steady state plasma levels of 800±900 ng/ml (targeted BUCY) (Deeg et al. 2002) (Fig. 12.3). The 3-year probability of RFS was 68% among 69 patients with RA/RARS transplanted from HLA-identical sibling donors, and 70% with unrelated donors. NRM among all patients combined was 12% at 100 days, and 31% at 3 years; relapse occurred in 5% of patients. Outcome tended to be superior in patients transplanted with granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) rather than marrow. A large retrospective survey of the EBMT group compared results with marrow and G-CSF-mobilized PBPC for allogeneic HCT from HLA-identical siblings (TBI- or chemotherapy-based conditioning regimens). Similar to the FHCRC data, the incidence of treatment failure in all MDS subgroups was lower with PBPC than with marrow (Guardiola et al. 2002) (Fig. 12.4). Only patients with RA and high-risk cytogenetics did not derive a net benefit from the use of PBPC. The incidences of acute GvHD were comparable, while chronic GvHD was more frequent with PBPC. Nevertheless, these studies showed excellent overall results with allogeneic HCT in less advanced MDS with up to 70% RFS. They suggest, furthermore, that the lack of a suitably matched related donor should not be cause to abandon plans for transplantation, and a search for an unrelated donor should be pursued.

Fig. 12.4. Relapse-free (A, C C) and overall survival (B, D) after HCT for MDS. All patients received transplants from HLA-identical siblings, using either bone marrow (Ðб) or peripheral blood (...........) as a source of stem cells. Panels A and B include patients in all IPSS groups, panels C and D only intermediate-2 and highrisk patients. This figure is adapted from research originally published in Guardiola et al. (2002)

a

12.3 ´ Transplant Strategies

12.3.1.2 ªAdvancedº MDS The incidence of post-transplant relapse increases with the proportion of marrow blasts present at the time of transplantation (advanced MDS [RAEB/RAEBt]) and with increasing IPSS scores, reflecting primarily the impact of high-risk karyotypes in addition to the proportion of myeloblasts (Castro-Malaspina et al. 2002; Deeg et al. 2002; Nevill et al. 1998; Sierra et al. 2002). Relapse rates in the range of 15±80% have been reported (Appelbaum et al. 1990; Castro-Malaspina et al. 2002; Deeg et al. 2002; Runde et al. 1998). Studies in the 1980s using CY and TBI containing regimens reported 30±40% RFS (Appelbaum et al. 1990). To determine if more intensive conditioning would improve results, 31 patients with RAEB, RAEBt, or CMML to be transplanted from related or unrelated donors at the FHCRC were prepared with a BUCY plus TBI regimen (Anderson et al. 1996). Compared with historical controls conditioned with CYTBI, relapse rates were lower (28% vs. 54%), but NRM was markedly increased (68% vs. 36%), and RFS at 3 years was not improved (23% vs. 30%). The EBMT study already cited also included 63 patients with RAEB/RAEBt, and 18 patients with acute myeloid leukemia transformed from MDS (tAML). The 5-year RFS was 34%, 19%, and 26% for patients with RAEB, RAEBt, and tAML, respectively (Runde et al. 1998). Most of the patients (70%) were prepared with TBI-containing conditioning regimens. Relapse occurred in 28 patients (at 1±33 months); RFS at 5 years was 34% for RAEB, 19% for RAEBt, and 26% for tAML. Younger age and shorter disease duration were associated with better outcome. Another EBMT trial included 105 patients (69 conditioned with TBI) who received HLA-matched unrelated donor transplants. RFS was 27%, 8%, and 27% for RAEB, RAEBt and tAML patients, respectively (Arnold et al. 1998). A recent report from the International Bone Marrow Transplant Registry (IBMTR) on 452 patients transplanted from HLAidentical siblings (44% conditioned with TBI regimens) between 1989 and 1997 showed a RFS of 40% at 3 years (Sierra et al. 2002). Corresponding figures for relapse incidence and NRM were 23% and 37%, respectively. The proportion of marrow blasts at transplantation was the strongest predictor for relapse and RFS, and younger age correlated with higher probability of survival. CY is not stem cell toxic but contributes to non-hemopoietic toxicity. Thus, in another trial, 60 patients

127

with RAEB, RAEBt, CMML, or tAML (20 related, 40 unrelated donors) were conditioned with BU plus TBI, aimed at reducing the relapse rate, and did not receive CY (Jurado et al. 2002). The Kaplan-Meier estimate of survival at 3 years was 26%, while the relapse incidence of 25% was comparable to that observed previously with a regimen combining BUTBI with CY (BUCYTBI) (Anderson et al. 1996). Overall NRM was 38% at 100 days. Particularly disappointing were results with unrelated donors. The data showed that CY was not required to achieve engraftment of donor cells, and suggested that high-dose TBI may not be the optimum modality for conditioning. Similar to the results in patients with less advanced MDS, the use of PBPC resulted in a lower incidence of treatment failure than the use of marrow in all patient groups with advanced MDS (Guardiola et al. 2002). Other trials have evaluated toxicity and efficacy of conditioning regimens that combine (targeted) BU with fludarabine (Flu) rather than CY (Bornhåuser et al. 2003). The transplant team in Calgary used a regimen of intravenous (i.v.) Flu, given over 5 days, and i.v. BU, 3.2 mg/kg, given once a day over 3 h on 4 consecutive days, combined with rabbit ATG (Thymoglobulin [THY]), 4.5 mg/kg, plus methotrexate (MTX) and cyclosporine (CSP). The study enrolled 70 patients with various diagnoses, including MDS, and patients were given marrow or PBPC from related or unrelated donors. There were two cases of graft failure (from unrelated donors), and the incidence of acute GvHD was 8%, and chronic GvHD 36%. The day 100 mortality was 2% with related, and 8% with unrelated donors. Projected RFS at 2 years was 74% for low-risk, and 65% for high-risk disease (Russell et al. 2002). A recently concluded FHCRC trial of targeted BUCY in patients with high-risk MDS also incorporated THY in a dose escalation design (Deeg et al. 2004 a). While the rates of GvHD were higher than in the Calgary study, they were lower than in concurrent trials not using THY. A trial in 96 patients at the M.D. Anderson Cancer Center in Houston including 22 patients with MDS also used a FluBU regimen (without THY) (de Lima et al. 2004). While results for patients with MDS are difficult to separate out, the incidence of acute GvHD (grades II±IV) was 15% for related and 68% for unrelated transplant recipients. These data suggest excellent tolerability of FluBU regimens, and additional trials are warranted.

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Chapter 12 ´ Hemopoietic Cell Transplantation

12.3.2 Reduced-Intensity Conditioning Regimens

As outlined above, a major drawback of HCT has been the high rate of TRM. The recent development of NMA or RIC transplant regimens (ªmini-transplantsº) has, therefore, met with considerable interest (Carella et al. 2000). The rationale is that a reduction in the intensity of cytotoxic conditioning regimens will be associated with lower toxicity and thereby reduce NRM. The post-transplant administration of immunosuppressive drugs (e.g., CSP plus mycophenolate mofetil or tacrolimus plus MTX) will facilitate donor cell engraftment and enhance the allogeneic (immunologic) effect of donor lymphocytes against the patients' cells (graft-versus-MDS effect) (McSweeney and Storb 1999). In view of the generally high incidence of NRM in older patients, such an approach is particularly attractive for the treatment of patients with MDS. RIC regimens are also of interest for patients with co-morbid medical conditions, and for patients who relapse after a conventional transplant, since conventional transplants have generally not been very successful because of severe toxicity, particularly in adult patients. Earlier transplant strategies generally developed in the direction of intensification of conditioning regimens to eradicate the underlying disease and prevent relapse. The use of NMA regimens represents a dramatic departure from that approach, counting heavily on immunologic effects of allogeneic donor cells against the patient's disease. The field is developing rapidly (Kroger et al. 2001, 2003; Parker et al. 2002; Sierra et al. 2002; Storb et al. 2000; Stuart et al. 2003; Taussig et al. 2003). It would be inappropriate, however, to simply contrast NMA with more conventional (ablative) regimens. All efforts are directed at achieving the best results with the least toxic regimens. Thus, as already described above, we are also witnessing progressive modifications of conventional (ablative) regimens. Finally, because of problems with sustained engraftment of donor cells and, in parallel, disease progression in the patient, we are observing ªre-intensificationº of NMA regimens, in other words, steps toward optimization of conditioning from both directions. Kroger et al. (2003) have used a regimen of fludarabine combined with a reduced dose of BU and showed a RFS of 38% at 3 years among 37 patients with MDS or tAML (transplanted from related (n = 19) or unrelated HLA-matched (n = 18) donors). The cumulative incidence of relapse was 32%. De Lima and colleagues

(2004) recently reported the M. D. Anderson experience with two regimens: Flu, 100 mg/m2, plus cytosine arabinoside, 4 mg/m2, and idarubicin, 36 mg/m2 (FAI), compared with Flu 100±150 mg/m2, plus melphalan, 140 mg/ m2 (FM). There were 26 patients with high-risk MDS included in this study of 94 patients. The FM regimen was significantly associated with a higher degree of donor cell engraftment, and a lower incidence of relapse (30% vs. 61%; p = 0.029) but also a higher incidence of TRM (p ( = 0.036). The 3-year survival rates were 30% and 35% for the FAI and the FM regimens, respectively. A recent update on NMA regimens by the Seattle consortium included 78 patients with MDS (45 related, 33 unrelated transplants; 46 were IPSS low/intermediate-1, and 32 intermediate-2/high or unknown) (Stuart et al. 2003). These patients were conditioned with Flu (3 ´ 30 mg/m2) plus 200 cGy of TBI. Graft failure occurred in 6% of patients, and 42% of patients relapsed. The NRM was 14% at day 100, and 25% at 1 year. Approximately 20% of patients were surviving at 3 years (25% with low-risk, and less than 10% with high-risk disease) (Stuart et al. 2003). Ho et al. (2004) reported on 62 patients (24 with HLA-identical siblings and 38 with unrelated donors) conditioned with RIC regimens (FluBU/campath). The day 100 NRM was 0% for siblings and 11% for patients transplanted from unrelated donors. The incidence of relapse ranged from 7 to 80% for IPSS risk groups intermediate-1 to high. There were 26 patients who received donor lymphocyte infusions at a median of 273 days after HCT. RFS ranged from 86% for IPSS low-risk patients to 40% among patients with high-risk disease. An increased relapse rate in patients with MDS prepared with RIC was also reported by Martino et al. (2003). It is important to note, of course, that patients prepared with RIC regimens tended to be older and often had co-morbid conditions. An ªintermediateº intensity regimen was used by Chan et al. (2003). These investigators combined 2 days of photopheresis with pentostatin, 4 mg/m2 for 2 consecutive days, and 3 ´ 200 cGy of TBI given over 2 days to prepare 18 patients (30±70 [median 54] years old) with MDS for transplantation. Sixteen patients achieved full donor chimerism, and all patients survived beyond day 100. The incidence of acute GvHD was 19%. With a median follow-up of 14 months, the 1 year RFS was 64%. It appears, therefore, that a standard regimen for allogeneic transplants in patients with MDS has yet to be

a

12.4 ´ Special Considerations

established. Phase III randomized studies comparing different regimens in well-defined patient populations should be helpful in achieving that goal.

12.3.3 Autologous HCT

Autologous HCT generally does not lead to GvHD and is associated with lower TRM than allogeneic transplants. It may hold promise in patients in whom a ªpureº population of normal hemopoietic stem cells is obtainable. The EBMT reported results in 79 patients with MDS, showing 2-year RFS of 28% after autologous HCT (de Witte et al. 1997). NRM was 39% in patients more than 40 years of age. These results were restricted, however, to patients who achieved complete remissions after induction chemotherapy. Wattel et al. prospectively assessed feasibility of autologous HCT (either with bone marrow or PBPC) after conditioning with BUCY in 24 of 39 patients who achieved complete remissions after induction chemotherapy. Among these, 50% were alive 8±55 months after transplantation (Wattel et al. 1999). De Witte and colleagues (2001) presented data on 184 patients with MDS/tAML who received induction chemotherapy. Among these, 56 had HLA-identical related donors available, and 128 did not. One hundred patients achieved remission and, with or without additional consolidation, 39 were transplanted with allogeneic and 61 with autologous cells. The rate of continuous complete remission was 33% for allogeneic, and 31% for autologous transplants. The 4-year RFS (expressed as proportion of the total cohort) was 25% for allogeneic, and 15% for autologous transplants (de Witte et al. 2001). The same authors recently reported results in patients with or without HLA-identical sibling donors on an intent to treat basis (Oosterveld et al. 2003). There were 159 patients who received remission induction and consolidation chemotherapy. Sixty-five patients had no donor available, and among these, 33 ultimately received autologous transplants. RFS was 23% for patients with, and 21% for patients without, a donor. Transplants from alternative donors did not significantly alter the survival of the group without a related donor. This intention to treat analysis failed to show a survival advantage for patients with HLA identical sibling donors compared to those without such a donor. The data indicate, however, that outcome with autologous transplantation is superior to that with chemotherapy alone (without a transplant).

129

12.4 Special Considerations 12.4.1 MDS Is a Disease of ªOlderº Patients

The median age of patients at the time of diagnosis of MDS is in the early 70s, an age when aggressive therapy is generally not well tolerated. Transplantation for these patients was, therefore, viewed with great skepticism. Results show, nevertheless, that many older patients do quite well, and the age ªceilingº for transplantation has been raised progressively. In an FHCRC trial, 50 patients with MDS, 55±66 years of age, were conditioned with either BU (7 mg/ kg)/CY plus fractionated TBI or targeted BU (prescribed dose 16 mg/kg)/CY (Deeg et al. 2000). Donors were HLA-identical siblings in 34, HLA-non-identical family members in four, identical twins in four, and unrelated volunteers in six patients. The RFS at 3 years was 53% for RA, 46% for RAEB, and 33% for RAEBt/tAML or CMML (Deeg et al. 2000). When only transplants from HLA-identical siblings were considered, RFS for patients with RA was 67%. Survival in all FAB categories was highest among patients conditioned with targeted BUCY. A recent analysis in a larger cohort of patients confirmed those results and showed no significant impact of age up to 66 years on transplant outcome (Deeg et al. 2002). Of course, in view of the concern about TRM, it was in this older patient population that NMA/RIC regimens were developed and are being exploited. The average age of patients in those studies has generally been a decade or more above that of patients treated with more conventional regimens such as targeted BU/CY. We recently analyzed data on 172 patients, more than 40 years of age with MDS/tAML, who were transplanted at the FHCRC. Patients receiving targeted BUCY were 40±65 (median 52) years old, compared with 40±73 (median 62) years for patients conditioned with a regimen of 200 cGy of TBI with or without the addition of Flu. Patients with RA/RARS fared slightly better with BUCY; however, no difference was observed among patients with more advanced MDS (Scott et al. 2004). The data suggest that various regimens can be used for older patients; however, toxicity is still a problem, and patients should be carefully selected.

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Chapter 12 ´ Hemopoietic Cell Transplantation

12.4.2 Childhood MDS

HCT in children with MDS is discussed in Chapter 7.

12.4.3 Other Parameters

There are additional disease parameters, which are not considered in currently used classification schemes, but may well be relevant for prognosis and treatment outcome. For example, we have shown that the severity of immunophenotypic aberrancies and scatter properties of myeloid and monocytic marrow cells pre-transplant correlates directly with the probability of post-transplant relapse, and inversely with RFS (Wells et al. 2003). Therefore, one may want to consider HCT in patients in whom sequential marrow aspirates show worsening of flow parameters, even if by established classification schemes the disease is ªstable.º Patients with MDS and associated marrow fibrosis tend to have more rapid disease progression than patients without fibrosis (Maschek et al. 1992). Preliminary transplant data suggest, however, that fibrosis does not have a negative impact on transplant outcome (B. Scott, unpublished). Thus, in patients with MDS and associated fibrosis one may want to consider HCT early in the disease course, even if by other criteria, such as IPSS classification, these patients are considered ªgood riskº. The impact of factors such as cellularity or neo-angiogenesis in the marrow remains to be determined.

12.4.4 Secondary MDS

Treatment-related (secondary) MDS occurs after therapy for various disorders (see Chapter 3). At 10 years after autologous HCT for Hodgkin disease and nonHodgkin lymphoma incidence figures of 1±20% have been reported (Friedberg et al. 1999; Micallef et al. 2000; Sobecks et al. 1999). The median time from primary disease to secondary MDS ranges from 2±9 years (Friedberg et al. 1999; Micallef et al. 2000). Abnormal, usually high-risk karyotypes (monosomy 7; complex abnormalities) are present in 80±90% of patients (Friedberg et al. 1999; Micallef et al. 2000; Sobecks et al. 1999). Exposure to irradiation and chemotherapy, as given for the patient's original disease, is thought to be the major cause, although a genetic predisposition cannot be excluded. As prior therapy (given for the

original disease) is expected to result in tissue damage, this is likely to predispose the patient to substantial morbidity and mortality with a transplant for secondary MDS. Among 552 patients who had received autologous HCT for non-Hodgkin lymphoma, Friedberg et al. (1999) observed 41 who developed MDS at a median of 47 months, for an actuarial incidence of 19.8% at 10 years. Thirteen patients underwent allogeneic HCT, and all died with a median survival of 1.8 months. These results are in agreement with an earlier report by the EBMT group, which showed a 5-year survival of 0% in patients with secondary MDS (de Witte 1999). A French group reported on 70 patients receiving allogeneic HCT (after various conditioning regimens) for therapy-related MDS and AML (Yakoub-Agha et al. 2000). Overall 54 patients died, 19 of relapse, 34 of NRM, and one of relapse of the primary disease. Age greater than 37 years, absence of complete remission at HCT, and intensive schedules for conditioning were associated with poor outcome. RFS, relapse incidence, and NRM rates at 2 years were 28%, 42%, and 49%, respectively (Yakoub-Agha et al. 2000). It is of note, however, that all these patients had been given pre-transplant induction chemotherapy, and patients who achieved remissions had a substantially higher chance of RFS than patients who were not in remission at transplantation. We analyzed results in 111 consecutive patients with secondary MDS transplanted at the FHCRC between 1971 and 1998 from either related or unrelated donors using the same conditioning regimens as employed concurrently for patients with de novo MDS (Witherspoon et al. 2001). The primary diagnoses included Hodgkin disease, non-Hodgkin lymphoma, carcinoma of the breast, aplastic anemia, multiple myeloma, polycythemia vera, and other malignancies or immunologic disorders. The 5-year RFS was 8% for patients prepared with TBI regimens, 19% for those given fixed-dose BUCY, and 30% for those prepared with targeted BUCY. The 5-year relapse rates were 40% for tAML, 40% for RAEBt, 26% for RAEB, and 0% for RA and RARS (Witherspoon et al. 2001). Thus, as with de novo MDS, disease stage was the most important risk factor for outcome, and the conditioning regimen had a major impact. Ballen et al. (1997) reported RFS of 14%, and NRM of 50% at 3 years for 18 patients with secondary MDS treated with HCT from matched related or unrelated donors after preparation with various conditioning regimens.

a

References

Thus, results obtained with allogeneic HCT for treatment-related MDS are currently not satisfactory, although transplantation may be the only viable option for many of these patients. Efforts must be directed firstly at the prevention of secondary MDS, and secondly at improved tolerability of transplant conditioning. Some preliminary studies with induction chemotherapy followed by RIC regimens have yielded encouraging results.

12.4.5 Post-transplant Relapse

Post-transplant relapse remains a problem in patients with a high myeloblast count or ªhigh-riskº cytogenetics or both. Reports on the efficacy of donor lymphocyte infusions in patients with MDS are still limited; however, there is evidence that this approach may be effective (Bader et al. 1999; Bethge et al. 2004; Castagna et al. 1998; Ho et al. 2004; Shiobara et al. 2000). A Japanese series noted complete remissions in five of 11 MDS patients (Shiobara et al. 2000). Similar results have been presented by Ho et al. (2004) and Depil et al. (2004). We have given donor lymphocytes to seven patients with MDS (five with RAEB and two with RA), and three (all with RAEB) achieved complete remissions. Two patients are alive, disease free, at more than 2 years (M. Flowers et al., unpublished observations). These observations are of interest, but firm conclusions cannot be drawn at this point. Some patients with relapse have undergone successful second transplants (Stuart et al. 2003). Conceivably, RIC transplants are effective in these patients, particularly if carried out before disease evolution.

12.5 Conclusions

HCT offers potentially curative therapy for patients with MDS. Patients with high-risk MDS (intermediate-2 or high by IPSS criteria) who have HLA-identical related or unrelated donors should be transplanted early in their disease course. Patients with less advanced MDS by FAB criteria (< 5% marrow blasts) but with high-risk IPSS cytogenetic findings or severe multilineage cytopenias according to IPSS, and transfusion dependence or severe neutropenia should also be considered for early transplantation. Patients with low-risk cytogenetic features and without severe cytopenias may do well for ex-

131

tended periods of time with more conservative management. HCT can be carried out successfully, even in the seventh decade of life. Overall, regimens without highdose TBI appear to be better tolerated than TBI containing regimens. The use of PBPC may offer an advantage over marrow cells, although at the price of a higher incidence of chronic GvHD. The place of RIC/NMA transplants, other than for patients of advanced age (older than 65 years), remains to be determined. Improved survival with transplants from unrelated volunteer donors, in part, reflects selection of donors on the basis of high resolution (allele-level) HLA typing. Autologous transplants are an option for patients without a suitable donor if a remission can be induced pretransplant. The role of pre-transplant induction chemotherapy is currently not clear. Future investigations will focus on identification of additional prognostic parameters allowing to predict prognosis as well as on determination of the optimal timing of HCT (Benesch et al. 2002; Cutler et al. 2004). Acknowledgements. This work was supported by PHS Grants CA87948, CA18029, and HL36444.

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Chapter 12 ´ Hemopoietic Cell Transplantation

diatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of increasing mixed chimerism: a single center experience of 12 children. Leukemia 13:2079±2086 Ballen KK, Gilliland DG, Guinan EC, Hsieh CC, Parsons SK, Rimm IJ, Ferrara JL, Bierer BE, Weinstein HJ, Antin JH (1997) Bone marrow transplantation for therapy-related myelodysplasia: comparison with primary myelodysplasia. Bone Marrow Transplant 20:737± 743 Benesch M, Wells DA, Leisenring W, Loken MR, Myerson D, Deeg HJ (2002) Prognostic significance of pretransplant multidimensional flow cytometric parameters for posttransplant survival and relapse in 111 patients with myelodysplastic syndrome (MDS) [Abstract]. Blood 100:97 a Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C, The French-American-British (FAB) Co-Operative Group (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189±199 Bethge WA, Hegenbart U, Stuart MJ, Storer BE, Maris MB, Flowers MED, Maloney DG, Chauncey T, Bruno B, Agura E, Forman SJ, Blume KG, Niederweiser D, Storb R, Sandmaier BM (2004) Adoptive immunotherapy with donor lymphocyte infusions after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Blood 103:790±795 Bornhåuser M, Storer B, Slattery JT, Appelbaum FR, Deeg HJ, Hansen J, Martin PJ, McDonald GB, Nichols G, Radich J, Woolfrey A, Jenke A, Schleyer E, Thiede C, Ehninger G, Anasetti C (2003) Conditioning with fludarabine and targeted busulfan for transplantation of allogeneic hematopoietic stem cells. Blood 102:820±826 Carella AM, Champlin R, Slavin S, McSweeney P, Storb R (2000) Miniallografts: ongoing trials in humans (Editorial). Bone Marrow Transplant 25:345±350 Castagna L, El Weshi A, Bourhis JH, Ribrag V, Naccache P, Vantelon JM, Brault P, Pico JL (1998) Successful donor lymphocyte infusion (DLI) in a patient with myelodysplastic syndrome (MDS) after failure of T-cell-depleted bone marrow transplantation (TD-BMT) (Letter). Br J Haematol 103:284±285 Castro-Malaspina H, Harris RE, Gajewski J, Ramsay N, Collins R, Dharan B, King R, Deeg HJ (2002) Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the National Marrow Donor Program. Blood 99:1943±1951 Chan GW, Foss FM, Klein AK, Sprague K, Miller KB (2003) Reduced-intensity transplantation for patients with myelodysplastic syndrome achieves durable remission with less graft-versus-host disease. Biol Blood Marrow Transplant 9:753±759 Cutler CS, Lee SJ, Greenberg P, Deeg HJ, Prez WS, Anasetti C, Bolwell BJ, Cairo MS, Gale RP, Klein JP, Lazarus HM, Liesveld JL, McCarthy PL, Milone GA, Rizzo JD, Schultz KR, Trigg ME, Keating A, Weisdorf DJ, Antin JH, Horowitz MM (2004) A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed transplantation for low-risk myelodysplasia is associated with improved outcome. Blood 104:579±585 de Lima M, Anagnostopoulos A, Munsell M, Shahjahan M, Ueno N, Ippoliti C, Andersson BS, Gajewski J, Couriel D, Cortes J, Donato M, Neumann J, Champlin R, Giralt S (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose

is relevant for long-term disease control after allogeneic hematopoietic stem cell transplantation. Blood 104:865±872 de Witte T (1999) Stem cell transplantation in myelodysplastic syndromes (Review). Forum 9:75±81 de Witte T, van Biezen A, Hermans J, Labopin M, Runde V, Or R, Meloni G, Mauri SB, Carella A, Apperley J, Gratwohl A, Laporte J-P (1997) Autologous bone marrow transplantation for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia following MDS. Blood 90:3853±3857 de Witte T, Suciu S, Verhoef G, Labar B, Archimbaud E, Aul C, Selleslag D, Ferrant A, Wijermans P, Mandelli F, Amadori S, Jehn U, Muus P, Boogaerts M, Zittoun R, Gratwohl A, Zwierzina H, Hagemeijer A, Willemze R (2001) Intensive chemotherapy followed by allogeneic or autologous stem cell transplantation for patients with myelodysplastic syndromes (MDSs) and acute myeloid leukemia following MDS. Blood 98:2326±2331 Deeg HJ, Shulman HM, Anderson JE, Bryant EM, Gooley TA, Slattery JT, Anasetti C, Fefer A, Storb R, Appelbaum FR (2000) Allogeneic and syngeneic marrow transplantation for myelodysplastic syndrome in patients 55 to 66 years of age. Blood 95:1188±1194 Deeg HJ, Storer B, Slattery JT, Anasetti C, Doney KC, Hansen JA, Kiem HP, Martin PJ, Petersdorf E, Radich JP, Sanders JE, Shulman HM, Warren EH, Witherspoon RP, Bryant EM, Chauncey TR, Getzendaner L, Storb R, Appelbaum FR (2002) Conditioning with targeted busulfan and cyclophosphamide for hemopoietic stem cell transplantation from related and unrelated donors in patients with myelodysplastic syndrome. Blood 100:1201±1207 Deeg HJ, Appelbaum FR, Storer B, Cassarella M, Scott B, McDonald G, Storb R (2004 a) Reduced incidence of acute and chronic graft-versus-host disease (GvHD) without increased relapse in patients with high-risk myeloid disorders given thymoglobulin (THY) as part of the transplant conditioning regimen: a dose finding study [Abstract]. Blood 104:56 a Deeg HJ, Jiang PYZ, Holmberg LA, Scott B, Petersdorf EW, Appelbaum FR (2004 b) Hematologic responses of patients with MDS to antithymocyte globulin plus etanercept correlate with improved flow scores of marrow cells. Leuk Res 28:1177±1180 Depil S, Deconinck E, Milpied N, Sutton L, Witz F, Jouet JP, Damaj G, Yakoub-Agha I (2004) Donor lymphocyte infusion to treat relapse after allogeneic bone marrow transplantation for myelodysplastic syndrome. Bone Marrow Transplant 33:531±534 Friedberg JW, Neuberg D, Stone RM, Alyea E, Jallow H, LaCasce A, Mauch PM, Gribben JG, Ritz J, Nadler LM, Soiffer RJ, Freedman AS (1999) Outcome in patients with myelodysplastic syndrome after autologous bone marrow transplantation for non-Hodgkin's lymphoma. J Clin Oncol 17:3128±3135 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes (erratum appears in Blood 1998 91:1100). Blood 89:2079±2088 Guardiola P, Runde V, Bacigalupo A, Ruutu T, Locatelli F, Boogaerts MA, Pagliuca A, Cornelissen JJ, Schouten HC, Carreras E, Finke J, van Biezen A, Brand R, Niederwieser D, Gluckman E, de Witte TM, Subcommittee for Myelodysplastic Syndromes of the Chronic Leukaemia Working Group of the European Blood and Marrow Transplantation Group (2002) Retrospective comparison of bone marrow and granulocyte colony-stimulating factor-mobilized peripheral

a

References

blood progenitor cells for allogeneic stem cell transplantation using HLA identical sibling donors in myelodysplastic syndromes. Blood 99:4370±4378 Ho AYL, Pagliuca A, Kenyon M, Parker JE, Mijovic A, Devereux S, Mufti GJ (2004) Reduced-intensity allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome and acute myeloid leukemia with multilineage dysplasia using fludarabine, busulphan and alemtuzumab (FBC) conditioning. Blood 104:1616±1623 Jurado M, Deeg HJ, Storer B, Anasetti C, Anderson JE, Bryant E, Chauncey T, Doney K, Flowers MED, Hansen J, Martin PJ, Nash RA, Petersdorf E, Radich J, Sale G, Sandmaier BM, Storb R, Wade J, Witherspoon R, Appelbaum FR (2002) Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome after conditioning with busulfan and fractionated total body irradiation is associated with low relapse rate but considerable nonrelapse mortality. Biol Blood Marrow Transplant 8:161±169 Kroger N, Schetelig J, Zabelina T, Kruger W, Renges H, Stute N, Schrum J, Kabisch H, Siegert W, Zander AR (2001) A fludarabine-based dose-reduced conditioning regimen followed by allogeneic stem cell transplantation from related or unrelated donors in patients with myelodysplastic syndrome. Bone Marrow Transplant 28:643± 647 Kroger N, Bornhauser M, Ehninger G, Schwerdtfeger R, Biersack H, Sayer HG, Wandt H, Schafer-Eckardt K, Beyer J, Kiehl M, Zander AR (2003) Allogeneic stem cell transplantation after a fludarabine/busulfan-based reduced-intensity conditioning in patients with myelodysplastic syndrome or secondary acute myeloid leukemia. Ann Hematol 82:336±342 List A, Beran M, DiPersio J, Slack J, Vey N, Rosenfeld CS, Greenberg P (2003) Opportunities for Trisenox (arsenic trioxide) in the treatment of myelodysplastic syndromes. Leukemia 17:1499±1507 Martino R, van Biezen A, Iacobelli S, Brand R, Niederwieser DW, de Witte TM (2003) Reduced-intensity conditioning (RIC) for allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical siblings in adults with myelodysplastic syndromes (MDS): a comparison with standard myeloablative conditioning: a study of the EBMT-Chronic Leukemia Working Party (EBMTCLWP) [Abstract]. Blood 102:184 a Maschek H, Georgii A, Kaloutsi V, Werner M, Bandecar K, Kressel M-G, Choritz H, Freund M, Hufnagl D (1992) Myelofibrosis in primary myelodysplastic syndromes: a retrospective study of 352 patients. Eur J Haematol 48:208±214 McSweeney PA, Storb R (1999) Mixed chimerism: preclinical studies and clinical applications (Review). Biol Blood Marrow Transplant 5:192±203 Micallef INM, Lillington DM, Apostolidis J, Amess JAL, Neat M, Matthews J, Clark T, Foran JM, Salam A, Lister A, Rohatiner AZS (2000) Therapy-related myelodysplasia and secondary acute myelogenous leukemia after high-dose therapy with autologous hematopoietic progenitor-cell support for lymphoid malignancies. J Clin Oncol 18:947±955 Molldrem JJ, Leifer E, Bahceci E, Saunthararajah Y, Rivera M, Dunbar C, Liu J, Nakamura R, Young NS, Barrett AJ (2002) Antithymocyte globulin for treatment of the bone marrow failure associated with myelodysplastic syndrome [summary for patients in Ann Intern Med 2002 137:I±27]. Ann Intern Med 137:156±163 Nevill TJ, Fung HC, Shepherd JD, Horsman DE, Nantel SH, Klingemann HG, Forrest DL, Toze CL, Sutherland HJ, Hogge DE, Naiman SC, Le

133

A, Brockington DA, Barnett MJ (1998) Cytogenetic abnormalities in primary myelodysplastic syndrome are highly predictive of outcome after allogeneic bone marrow transplantation. Blood 92:1910±1917 O'Donnell MR, Long GD, Parker PM, Niland J, Nademanee A, Amylon M, Chao N, Negrin RS, Schmidt GM, Slovak ML, Smith EP, Snyder DS, Stein AS, Traweek T, Blume KG, Forman SJ (1995) Busulfan/cyclophosphamide as conditioning regimen for allogeneic bone marrow transplantation for myelodysplasia. J Clin Oncol 13:2973± 2979 Oosterveld M, Suciu S, Verhoef G, Labar B, Belhabri A, Aul C, Selleslag D, Ferrant A, Wijermans P, Mandelli F, Amadori S, Jehn U, Muus P, Zittoun R, Hess U, Anak O, Beeldens F, Willemze R, de Witte T (2003) The presence of an HLA-identical sibling donor has no impact on outcome of patients with high-risk MDS or secondary AML (sAML) treated with intensive chemotherapy followed by transplantation: results of a prospective study of the EORTC, EBMT, SAKK and GIMEMA Leukemia Groups (EORTC study 06921). Leukemia 17:859± 868 Parker JE, Shafi T, Pagliuca A, Mijovic A, Devereux S, Potter M, Prentice HG, Garg M, Yin JA, Byrne J, Russell NH, Mufti GJ (2002) Allogeneic stem cell transplantation in the myelodysplastic syndromes: interim results of outcome following reduced-intensity conditioning compared with standard preparative regimens. Br J Haematol 119:144±154 Ratanatharathorn V, Karanes C, Uberti J, Lum LG, de Planque MM, Schultz KR, Cronin S, Dan ME, Mohamed A, Hussein M, Sensenbrenner LL (1993) Busulfan-based regimens and allogeneic bone marrow transplantation in patients with myelodysplastic syndromes. Blood 81:2194±2199 Raza A, Meyer P, Dutt D, Zorat F, Lisak L, Nascimben F, du RM, Kaspar C, Goldberg C, Loew J, Dar S, Gezer S, Venugopal P, Zeldis J (2001) Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes. Blood 98:958±965 Runde V, de Witte T, Arnold R, Gratwohl A, Hermans J, van Biezen A, Niederwieser D, Labopin M, Walter-Noel MP, Bacigalupo A, Jacobsen N, Ljungman P, Carreras E, Kolb HJ, Aul C, Apperley J (1998) Bone marrow transplantation from HLA-identical siblings as first-line treatment in patients with myelodysplastic syndromes: early transplantation is associated with improved outcome. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 21:255±261 Russell JA, Tran HT, Quinlan D, Chaudhry A, Duggan P, Brown C, Stewart D, Ruether JD, Morris D, Glick S, Gyonyor E, Andersson BS (2002) Once-daily intravenous busulfan given with fludarabine as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical outcomes. Biol Blood Marrow Transplant 8:468±476 Scott BL, Maris M, Sandmaier B, Storer B, Chauncey T, Maloney DG, Sorror M, Storb R, Deeg HJ (2004) Myeloablative versus nonmyeloablative hemopoietic cell transplantation (HCT) for patients with myelodysplasia (MDS) or AML with multilineage dysplasia following MDS (tAML) [Abstract]. Blood 104:638 a Scott BL, Storer B, Loken M, Storb R, Appelbaum FR, Deeg HJ (2005) Pretransplantation induction chemotherapy and posttransplantation relapse in patients with advanced myelodysplastic syndrome. Biol Blood Marrow Transplant 11:65±73

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Shiobara S, Nakao S, Ueda M, Yamazaki H, Takahashi S, Asano S, Yabe H, Kato S, Imoto S, Maruta A, Yoshida T, Gondo H, Morishima Y, Kodera Y (2000) Donor leukocyte infusion for Japanese patients with relapsed leukemia after allogeneic bone marrow transplantation: lower incidence of acute graft-versus-host disease and improved outcome. Bone Marrow Transplant 26:769±774 Sierra J, Prez WS, Rozman C, Carreras E, Klein JP, Rizzo JD, Davies SM, Lazarus HM, Bredeson CN, Marks DI, Canals C, Boogaerts MA, Goldman J, Champlin RE, Keating A, Weisdorf DJ, de Witte TM, Horowitz MM (2002) Bone marrow transplantation from HLAidentical siblings as treatment for myelodysplasia. Blood 100: 1997±2004 Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC, Odchimar-Reissig R, Stone RM, Nelson D, Powell BL, DeCastro CM, Ellerton J, Larson RA, Schiffer CA, Holland JF (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20:2429±2440 Sobecks RM, Le Beau MM, Anastasi J, Williams SF (1999) Myelodysplasia and acute leukemia following high-dose chemotherapy and autologous bone marrow or peripheral blood stem cell transplantation. Bone Marrow Transplant 23:1161±1165 Storb R, McSweeney PA, Sandmaier BM, Nash RA, Georges G, Maloney DG, Molina A, Chauncey T, Yu C, Zaucha JM, Blume KG, Shizuru J, Niederwieser D (2000) Allogeneic hematopoietic stem cell transplantation: from the nuclear age into the 21st century. Transplant Proc 32:2548±2549 Stuart MJ, Cao TM, Sandmaier BM, Hegenbart U, Maris M, Agura E, Chauncey TR, Maloney DG, Pulsipher MA, Wong R, Niederwieser DW, Blume KG, Storb RF (2003) Efficacy of non-myeloablative allogeneic transplant for patients with myelodysplastic syndrome

(MDS) and myeloproliferative disorders (MPD) (except chronic myelogenous leukemia) [Abstract]. Blood 102:185 a Taussig DC, Davies AJ, Cavenagh JD, Oakervee H, Syndercombe-Court D, Kelsey S, Amess JA, Rohatiner AZ, Lister TA, Barnett MJ (2003) Durable remissions of myelodysplastic syndrome and acute myeloid leukemia after reduced-intensity allografting. J Clin Oncol 21:3060±3065 Vardiman JW, Harris NL, Brunning RD (2002) The World Health Organization (WHO) classification of the myeloid neoplasms (Review). Blood 100:2292±2302 Wattel E, Solary E, Leleu X, Dreyfus F, Brion A, Jouet JP, Hoang-Ngoc L, Maloisel F, Guerci A, Rochant H, Gratecos N, Casassus P, Janvier M, Brice P, Lepelley P, Fenaux P (1999) A prospective study of autologous bone marrow or peripheral blood stem cell transplantation after intensive chemotherapy in myelodysplastic syndromes. Leukemia 13:524±529 Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, Deeg HJ (2003) Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hemopoietic stem cell transplantation. Blood 102:394±403 Witherspoon RP, Deeg HJ, Storer B, Anasetti C, Storb R, Appelbaum FR (2001) Hematopoietic stem-cell transplantation for treatment-related leukemia or myelodysplasia. J Clin Oncol 19:2134±2141 Yakoub-Agha I, de La Salmoni re P, Ribaud P, Sutton L, Wattel E, Kuentz M, Jouet JP, Marit G, Milpied N, Deconinck E, Gratecos N, Leporrier M, Chabbert I, Caillot D, Damaj G, Dauriac C, Dreyfus F, Franœois S, Molina L, Tanguy ML, Chevret S, Gluckman E (2000) Allogeneic bone marrow transplantation for therapy-related myelodysplastic syndrome and acute myeloid leukemia: a long-term study of 70 patients-report of the French Society of bone marrow transplantation. J Clin Oncol 18:963±971

Second Malignancies H. Joachim Deeg

Contents 13.1 Introduction . . . . . . . . . . . . . . . . . . . .

135

13.2 Synchronous Malignancies . . . . . . . . .

135

13.3 Metachronous Malignancies . . . . . . . . 13.3.1 Chemotherapy . . . . . . . . . . . . . . 13.3.2 Hemopoietic Cell Transplantation .

136 136 136

13.3 Conclusions . . . . . . . . . . . . . . . . . . . .

136

References . . . . . . . . . . . . . . . . . . . . . . . . .

136

Introduction Second malignancies, defined as new malignancies that develop as a consequence of therapy, are not a frequent complication in patients with myelodysplastic syndrome (MDS). For one, patients with less advanced or low-risk MDS are generally managed conservatively, and do not receive cytotoxic chemotherapy or irradiation, the two modalities most frequently associated with the induction of secondary malignancies. Secondly, patients with more advanced or high-risk MDS, who may receive cytotoxic therapy in the non-transplant setting, usually are not cured of MDS and have a short life expectancy. Thus, on the basis of the time course alone, these patients are unlikely to manifest second (treatment-related) malignancies. The situation is different for patients who are cured of MDS by hematopoietic cell transplantation (HCT). These patients received chemotherapy or irradiation

(or both) as conditioning for HCT and may have experienced graft-versus-host disease (GvHD), all factors that have been associated with new malignancies (Curtis et al. 1997, 1999; Flowers and Deeg 2004; Friedman et al. 2004). Some of these patients have now been followed for two decades, a risk period of sufficient length to allow for at least a preliminary assessment. To what extent genetic predisposition and polymorphism of genes, especially in xenobiotic pathways, are relevant for both the initial diagnosis of MDS, and the chances for a secondary malignancy remains to be determined. Of greater relevance appears to be the presentation of secondary MDS as a malignancy following therapy for other disorders (Metayer et al. 2003); these cases account for maybe 10±15% of all cases of MDS (see Chapter 3). 13.2 Synchronous Malignancies

Many reported series include cases where patients presented with ªmarrow failureº and were subsequently diagnosed as having MDS as well as another, generally lymphoid malignancy, including chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, hairy cell leukemia or other. These observations again raise the question of the role of underlying genetic factors, but no systematic studies addressing these possibilities are currently available. In addition, these observations raise the question of the effect of the location of the mutagenic lesion relative to the steps in stem cell differentiation on the clinical presentation.

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13.3 Metachronous Malignancies 13.3.1 Chemotherapy

Recent observations in patients with MDS treated with CC-5013 (Revlimid) show that some patients, in particular those with a 5q± syndrome, who achieve hemopoietic and even cytogenetic responses, may indeed develop new clonal cytogenetic abnormalities. The mechanism leading to those abnormalities and the clinical relevance are currently not clear. However, the observations are somewhat reminiscent of those in patients with chronic myeloid leukemia treated with imatinib. As with all clonal (non-constitutional) abnormalities, close monitoring appears to be indicated. 13.3.2 Hemopoietic Cell Transplantation

Numerous analyses of the development of post-transplant malignancies have been reported, although not specifically for patients with a primary diagnosis of MDS. Curtis et al. (1997) analyzed results in 19,229 patients (including 643 with MDS or myeloproliferative disorders), 97.2% of whom had received allogeneic HCT and who had been observed for 5±30 years. The ratio of observed/expected tumors was 2.7 (P < 0.001); among patients surviving beyond 10 years, the risk was 8.3 times that of the population at large. The cumulative incidence was 6.7% at 15 years. Elevated in particular were the risks for malignant melanoma, cancers of the buccal cavity, liver, brain, thyroid, bone, and connective tissue. Higher doses of total body irradiation (TBI) were associated with solid tumors, and male sex and chronic GvHD with squamous cell carcinoma of the oral cavity and skin. Patients were also at risk for post-transplant lymphoproliferative disorders. The risk was highest during the first 5 months after transplantation. The cumulative incidence was 1% (Curtis et al. 1999). The risk for early occurring lymphoproliferative disorders was strongly associated (P < 0.0001) with transplants from unrelated

or human leukocyte antigen (HLA) non-identical donors, T-cell depletion of donor marrow, the use of polyclonal or monoclonal anti-T-cell antibody preparations to prevent or treat acute GvHD, the development of acute GvHD, and the use of TBI in the conditioning regimen. Late onset lymphomas were associated with extensive chronic GvHD (P = 0.01).

13.3 Conclusions

Thus, second malignancies have not been a major problem in patients with MDS. The occurrence of new malignancies is most likely related to conditioning and transplant-related problems, but a contribution of genetic factors can, at present, neither be proven nor disproven.

References Curtis RE, Rowlings PA, Deeg HJ, Shriner DA, Soci G, Travis LB, Horowitz MM, Witherspoon RP, Hoover RN, Sobocinski KA, Fraumeni JF Jr, Boice JD Jr, Schoch HG, Sale GE, Storb R, Travis WD, Kolb H-J, Gale RP, Passweg JR (1997) Solid cancers after bone marrow transplantation. N Engl J Med 336:897±904 Curtis RE, Travis LB, Rowlings PA, Soci G, Kingma DW, Banks PM, Jaffe ES, Sale GE, Horowitz MM, Witherspoon RP, Shriner DA, Weisdorf DJ, Kolb H-J, Sullivan KM, Sobocinski KA, Gale RP, Hoover RN, Fraumeni JF Jr, Deeg HJ (1999) Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study. Blood 94:2208±2216 Flowers MED, Deeg HJ (2004) Delayed complications after hematopoietic cell transplantation. In: Blume KG, Forman SJ, Appelbaum FR (eds) Thomas' hematopoietic cell transplantation. Blackwell Publishing Ltd., Oxford, UK, pp 944±961 Friedman DL, Leisenring W, Schwartz JL, Deeg HJ (2004) Second malignant neoplasms following hematopoietic stem cell transplantation. Int J Hematol 79:229±234 Metayer C, Curtis RE, Vose J, Sobocinski KA, Horowitz MM, Bhatia S, Fay JW, Freytes CO, Goldstein SC, Herzig RH, Keating A, Miller CB, Nevill TJ, Pecora AL, Rizzo JD, Williams SF, Li CY, Travis LB, Weisdorf DJ (2003) Myelodysplastic syndrome and acute myeloid leukemia after autotransplantation for lymphoma: a multicenter case-control study. Blood 101:2015±2023

Subject Index

A alcohol 6, 7, 10 AML1 31, 32, 47 anemia ± congenital dyserythropoietic 49 ± growth factor 105, 106 ± mechanism 100, 101 angiogenesis 26, 28, 33, 115 antithymocyte globulin (ATG) 28, 116 aplastic anemia (AA) 6, 7, 8, 10, 11, 82, 116 apoptosis 24, 26, 27, 28, 30, 32 arsenic trioxide 33, 115 azacitidine 118 5-azacitidine 25, 33

B benzene 17, 18, 19 bevacizumab 33, 114 blasts 40, 48 bone marrow 3 Bournemouth score 40, 43

C causes of death 96, 97, 98 CCC classification 82 CD45-gating 42 CEBPA 47 chromosome 5 29, 30 chromosome 7 29 chromosome 17 30

chromosome 20 30 chronic myelomonocytic leukemia (CMML) 44, 48 ± dysplastic type 44 ± non-proliferative subtype 44 ± proliferative subtype 44 CMML-1 49 CMML-2 49 C-Kit 47 classification 40, 47 ± CCC 82 ± French-American-British (FAB) 40, 47, 48 ± WHO 48, 49, 50, 81 comparative genomic hybridization (CGH) 42 congenital dyserythropoietic anemia 49 cyclosporine 28, 116 cytochemistry 40, 43 cytogenetics 42, 46, 48 cytomorphology 40, 43 cytopenias 47, 48

D decitabine, 2'-deoxy-5-azacitidine 25, 33, 112 depsipeptide 33, 113 diagnosis 40 disorders, myeloproliferative 9, 30 DNA Methyltransferase 25, 33, 113 Down syndrome 82 Dçsseldorf score 40, 43 dyserythropoiesis 44 dysgranulopoiesis 43 dysmegakaryopoiesis 44 dysplasia 40, 43 ± erythroid 43

± granulocytic 43 ± megakaryocytic 43

E epigenetic alterations 23 epigenetic changes 24, 112 epigenetic events 32 erythropoietin (EPO) 25, 118 ± therapy 102 etanercept 28, 33, 116 etiology ± benzene 16, 17, 19, 20 ± chemotherapy 16, 17 ± radiation 16, 17, 19 EVI-1 30, 31

F Fanconi anemia (FA) 6, 10, 24, 29, 85 farnesyltransferase (FT) 26, 33, 116, 117 Ficoll Hypaque gradient separation 42 FK228 33, 113, 114 FLT3 31, 51, 115 FLT3-ITD 47 FLT3-TKD 47 fluorescence in situ hybridization (FISH) 40, 42, 46 folate 6, 10, 11 French-American-British (FAB) classification 40, 43, 47, 48, 82, 85

138

Subject Index

G gene expression 41, 51 ± profiling 50 ± signatures 50 genes, blast count 51 guidelines 89, 90, 93

H heavy metal 7, 10, 11, 24 hemopoietic cell transplantation (HCT) 131 ± autologous 129 hemoglobin level 47 hexamethylene bisacetamide (HMBA) 113 histone code 24 histone deacetylase 33, 113, 114,118 HIV 7, 10, 11 HLA-DR15 28, 116

I immunophenotyping 40 infliximab 33, 116 international prognostic scoring system (IPPS) 40, 43, 47, 48 iron chelation 96, 97 iron stains 40

J juvenile myelomonocytic leukemia (JMML) 81, 82

K karyotype ± complex aberrant, gene expression profiling 51 ± normal, gene expression profiling 51 Kostmann syndrome, see severe congenital neutropenia

L laboratory studies 2 large granular lymphocytic (LGL) 116 lenalidomide 33, 8 Lille score 43 lonafarnib 26, 33, 116, 117

P 9, 11, 28,

M malignancies, second 135 MAP-Kinase (MAPK) 25, 115, 117 May Grçnwald Giemsa (MGG) 41 microarrays 41, 50 mitochondria 100, 101, 106 MLL 31 MLL-PTD 47 molecular biology 47 molecular genetics 50 monosomy 7 82, 83, 84 ± infantile monosomy 7 syndrome 81 MS-275 33, 113, 114 multiparameter flow cytometry (MFC) 40, 42, 44 ± aberrant antigen expression 44 ± blasts 45 ± erythrocytes 45 ± granulocytes 44 ± monocytes 45 myelodysplastic syndrome (MDS) 81, 82 ± advanced 127 ± familial 86 ± in children 82 ± secondary 130 ± therapy-related 87 myelofibrosis 9 myeloperoxidase (MPO) 41 myeloproliferative disorders 9, 30

N neurofibromatosis type 1 82 neutropenia 96, 98 non-specific esterase (NSE) 41 NRAS 47 NUP98 30, 31

p15 INKK4B 25, 113 p15 methylation 47 p53 27, 30, 31 paroxysmal nocturnal hemoglobinuria (PNH) 9, 10, 28, 49 patients, older 129 pentoxifylline 115 Peroxidase 44 phenylbutyrate 33, 113 platelet level 47 platelet-derived growth factor receptor (PDGFR) 30, 115, 117 presentation 1 pseudo-Pelger-HuŸt cells 6, 30, 44 pyridoxine 7, 10

Q quality of life 96, 97

R RAEB-1 49 RAEB-2 49 RAEB in transformation (RAEBt) 49 RAS 25, 26, 29, 30, 116, 117 ± signaling pathway 82 reduced-intensity conditioning regimens 128 refractory anemia (RA) 49 refractory cytopenia (RC) 82, 83 refractory cytopenia with multilineage dysplasia (RCMD) 49, 50 relapse 131 response criteria 90, 92 retinoic acid (ATRA) 113, 117, 118

S severe congenital neutropenia 85 Shwachman-Diamond syndrome 87 sideroblasts, ringed 44 Src homology protein 2 (SHP-2 ) 82 staging 40 staging systems 47

a

Subject Index

113, 114 syndrome ± Down syndrome 82 ± Kostmann syndrome, see severe congenital neutropenia ± myelodysplastic (MDS) 81, 82 ± Shwachman-Diamond syndrome 87 5q-syndrome 42, 47, 48, 49, 50

T thalidomide 33, 114 thrombocytopenia 96, 97 ± management 97 tipifarnib 26, 33, 116, 117 transfusion ± platelets 98 ± red cells 96, 97 transplantation 123 trephine biopsy, inaspirable bone marrow 40 tumor necrosis factor alpha (TNF-a) 26, 27, 115, 116

139

V valproic acid (VAP) 33, 113, 118 vascular endothelial growth factor (VEGF) 33, 26, 28, 114, 115 vitamin B12 6, 10, 11

W World-Health Organisation (WHO) ± classification 48, 49, 50, 81 ± pediatric modification 82 WT1 47

40, 43