1. Overview of Myelodysplastic Syndromes with Focus on Iron Overload Norbert Gattermann, MD, PhD Professor Department of Haematology, Oncology, and Clinical Immunology Heinrich-Heine-University Düsseldorf, Germany

2. Myelodysplastic Syndromes (MDS)–Age at Diagnosis Patients in the Düsseldorf Registry 700 600 500 400 300 200 100 0 7060504030208090 Düsseldorf MDS Registry. Graphic courtesy of Dr. U. Germing. Age (years) Number of Patients N = 3125

3. Age Group (Y)All PatientsMaleFemale <300.40.50.2 30–390.40.30.5 40–491.31.21.4 50–592.83.52.2 60–698.711.46.2 70–7924.538.817.2 80–8931.353.623.6 ≥9015.928.213.1 All ages4.95.54.4 MDS–Incidence by Age and Gender a Patients in the Düsseldorf Registry a Cases per 100,000 population/year. Düsseldorf MDS Registry. Graphic courtesy of Dr. U. Germing.

4. Oncogene Activation Growth Advantage Ineffective Haematopoiesis Functional Impairment, Decreased Life Span Excessive Apoptosis Bone Marrow Peripheral Blood MDS Pathophysiology Clonal Expansion Plus Ineffective Haematopoiesis Graphic courtesy of Dr. N. Gattermann.

5. Apoptosis in MDS Intrinsic and Extrinsic Causes? Primary Defect (Intrinsic) Excessive Apoptosis Activated Macrophages Pro-apoptotic Cytokines  Graphic courtesy of Dr. N. Gattermann.

6. High Apoptotic Activity Impaired Differentiation Early MDS (Low risk) Advanced MDS (High risk) Impaired Production of Blood Cells Graphic courtesy of Dr. N. Gattermann.

7. Oncogene Activation Growth Advantage Normal Haematopoiesis Clonal Haematopoiesis Clonal Evolution LeukaemiaMDS MDS Pathophysiology Clonal Evolution Toward Acute Myelogenous Leukaemia Graphic courtesy of Dr. N. Gattermann.

8. Incidence of Chromosomal Anomalies a in Patients with MDS and an Abnormal Karyotype 0 5 10 15 20 25 30 del(5q) -7/del(7q) +8 -18/del(18q) del(20q) -5 -Y -17/del(17p) +21 inv/t(3q) -13/del(13q) -21 t(5q) +11 +1/+1q del(12p) del(11q) t(7q) +Mar Frequency (%) Haase D, et al. Blood. 2007;110:4385-4395. Graphic courtesy of Dr. D. Haase. n = 1080 b a Isolated or in combination with 1 additional anomaly or complex anomalies. b Total data set, 2124; cytogenic analysis in 2072.

9. Cytogenetic Anomalies—Only Part of the MDS Story Useful for making the diagnosis Useful for prognostication Useful for treatment decisions Identified in about 50% of patients 1 Not specific for MDS Explanation for excessive apoptosis? Explanation for genomic instability? Explanation for dysplastic features? 1. Haase D, et al. Blood. 2007;110:4385-4395.

10. ErythropoiesisGranulopoiesisThrombopoiesis Blood Anisocytosis Poikilocytosis Macrocytosis Basophilic stippling Dimorphic blood film Pappenheimer bodies Nucleated red cells Pseudo-Pelger anomaly Hypersegmented nuclei Nuclei of bizarre shape Chromatin clumping Hypogranularity Monocytosis Blast cells, Auer rods Thrombocytopenia Giant platelets Agranular platelets Micromegakaryocytes Bone marrow Megaloblastic cells Nuclear fragmentation Internuclear bridges Multinuclearity Vacuolization Positive PAS staining Ring sideroblasts Increased blast cells Hypogranular Promyelocytes Hypogranular myelocytes Partial MPO defect Increase of promonocytes Lack of mature neutrophils Abnormal neutrophils Larger megakaryocytes with nonlobed nuclei Reduction of MEG Mononuclear MEG Multinucleated MEG MEG with botryoid nuclei MDS–Dysplastic Features in Blood and Bone Marrow Graphic courtesy of Dr. N. Gattermann. Abbreviations: MEG, megakaryocyte; MPO, myeloperoxidase; PAS, Periodic Acid-Schiff.

11. WHO Classification of MDS Patients in the Düsseldorf Registry %MDSBlood Bone Marrow 3.75q– syndrome Anaemia Blasts <5% Platelets normal to increased Megakaryocytes normal to increased Blasts <5% No Auer rods Isolated del(5q) 8.8RA Anaemia Blasts ≤1% Dyserythropoiesis only Blasts <5% Ringed sideroblasts <15% 7.8RARS Anaemia Blasts ≤1% Dyserythropoiesis only Blasts <5% Ringed sideroblasts <15% 31.0RCMD Cytopaenia of ≥2 lineages Blasts ≤1% Monocytes <1000/µL >10% of cells per lineage dysplastic No Auer rods Blasts <5% Ringed sideroblasts <15% 15.4RCMD-RS Cytopaenia of ≥2 lineages Blasts ≤1% Monocytes <1000/µL >10% of cells per lineage dysplastic No Auer rods Blasts <5% Ringed sideroblasts ≥15% 15.0RAEB I Cytopaenia of ≥2 lineages Peripheral blood blasts ≤5% Monocytes <1000/µL Unilinear or multilinear dysplasia No Auer rods Blasts 5%–9% 18.3RAEB II Cytopaenia of ≥2 lineages Peripheral blood blasts ≤19% ± Auer rods Unilinear or multilinear dysplasia ± Auer rods Blasts 10%–19% Düsseldorf MDS Registry. Graphic courtesy of Dr. U. Germing.

12. Cumulative Survival of MDS Patients According to WHO Type Germing U, et al. Leuk Res. 2000;24:983-992. Graphic reflects updated data and is courtesy of Dr. U. Germing. Abbreviations: RA, refractory anaemia; RAEB, refractory anaemia with excess blasts; RARS, refractory anaemia with ring sideroblasts; RCMD, refractory cytopaenia with multilineage dysplasia; RSCMD, refractory sideroblastic cytopaenia with multilineage dysplasia.

13. Score Prognostic variable 00.51.01.52.0 Medullary blasts (%) <55–10––11–2021–30 KaryotypeGood a Intermediate b Poor c –– Cytopaenia0/12/3–– a Good: normal, -Y, del(5q), del(20q). b Intermediate: other abnormalities not seen in “good” or “poor.” c Poor: complex (≥3 abnormalities) or chromosome 7 anomalies. Low risk 0points Intermediate-1 0.5–1.0points Intermediate-2 1.5–2.0points High risk ≥2.5points International Prognostic Scoring System (IPSS) With permission from Greenberg P, et al. Blood. 1997;89:2079-2088.

14. 360 336 312 288 264 240 216 192 168 144 120 96 72 48 24 0 Cumulative Survival 1.0.8.6.4.2 0.0 Risk Survival Group (months) 31% Low 71 39% Int-131 20% Int-215 10% High 8 Months The IPSS Applied to Patients in the Düsseldorf MDS Registry Low Int 1 Int 2 High Düsseldorf MDS Registry. Graphic courtesy of Dr. U. Germing. n = 550

15. Score Prognostic Variable 0123 WHO categoryRA, RARS, 5q– RCMD, RCMD- RS RAEB-1RAEB-2 KaryotypeGood a Intermediate b Poor c –– Transfusion requirement YesRegular–– a Good: normal, -Y, del(5q), del(20q). b Intermediate: other abnormalities not seen in “good” or “poor.” c Poor: complex (≥3 abnormalities) or chromosome 7 anomalies. WHO Classification-Based Prognostic Scoring System (WPSS) Very low risk 0points Low risk 1points Intermediate 2points High risk3–4points Very high risk5–6points With permission from Malcovati L, et al. Blood. 2005;106:abstract 788.

16. Overall Survival (P <.001) Risk of AML Evolution (P <.001) WPSS at Diagnosis Applied to Patients in the Düsseldorf MDS Registry Malcovati L, et al. J Clin Oncol. 2007;25:3503-3510. Graphic courtesy of Dr. U. Germing.

17. MDS–Treatment Options Best supportive care, including iron chelation Growth factors Immunosuppressive treatment Differentiation induction Farnesyltransferase inhibitors Thalidomide/lenalidomide Arsenic trioxide Low-dose chemotherapy Epigenetic treatment Intensive chemotherapy Allogeneic stem cell transplantation Low Risk High Risk Prognostic Group

18. 1 unit PRC 200–250 mg 1–2 mg Daily Losses Iron Imbalance in Transfusion-Dependent Patients Abbreviation: PRC, packed red cells. Approximately 80% of MDS patients have a haemoglobin <10 g/dL at diagnosis 1 and the majority become transfusion-dependent 1. Sanz GF, et al. Blood. 1989;74:395-408. Graphic courtesy of Dr. N. Gattermann.

19. Erythroblasts Blood losses (eg, menstrual) Normal daily iron uptake ~1 mg Transferrin Other cells in the body Daily losses ~1 mg Urine, faeces, nails, hair, skin Duodenum Ineffective erythropoiesis Unrestrained intestinal iron absorption Suppressed hepcidin expression in the liver Increased GDF15 in the serum Macrophages (0.5–1.5 g) Haemoglobin (1.7–2.4 g) Liver Consequence of Ineffective Erythropoiesis–Increased Intestinal Iron Uptake Graphic courtesy of Dr. N. Gattermann.

20. MDS–Serum Ferritin at Diagnosis RARARSRAEBRAEB-TCMML 0 2000 4000 6000 8000 Serum Ferritin (ng/mL) 1290 1500 1800 1749 2000 2283 1590 1830 2000 2690 1309 20001980 2284 2500 2555 3839 4176 3526 7794 2980 6980 2136 2500 5000 Data on file. Düsseldorf MDS Registry. Graphic courtesy of Dr. N. Gattermann. Abbreviations: RA, refractory anaemia; RAEB, RA with excess blasts; RA with excess blasts in transformation; RARS, RA with ring sideroblasts; CMML, chronic myelomonocytic leukaemia. N = 650

21. Most Important Cause of Iron Overload in MDS–Transfusion Therapy Medium transfusion dependence –2 units/month –24 units/year –About 100 units/4 years Heavy transfusion dependence –4 units/month –48 units/year –About 100 units/2 years 200–250 mg iron 100 units = ≥20 g iron Total body iron in normal person 3–4 g

22. Impact of Transfusion Dependency on Nontransplantation Candidates Cazzola M, et al. N Engl J Med. 2005;352:536-538. Copyright © 2005. Massachusetts Medical Society. All rights reserved Cumulative probability of survival among patients given a diagnosis of MDS in Pavia, Italy from 1992–2002 Transfusion-Independent Transfusion-Dependent Survival Time (months) Cumulative Proportion Surviving 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 020406080100120140 N = 374 P =.005

23. Transfusion Dependency—A Risk Factor Independent of Cytogenetic Risk Group Not transfusion-dependent Transfusion-dependent a Only isolated del 5q. 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 020406080100120 Good a Months Intermediate 20406080100120 Poor 020 406080100120 Cumulative Proportion Surviving 0 With permission from Malcovati L, et al. J Clin Oncol. 2005;23:7594-7603. Cumulative Proportion Surviving

24. Malcovati L, et al. J Clin Oncol. 2005;23:7594-7603. Graphic courtesy of Dr. N. Gattermann. Survival of Patients with MDS According to Transfusion Dependency

25. Overall Survival in Patients with MDS by RBC Transfusion Dependency No RBC Transfusion Dependency RBC Transfusion Dependency P <.0001 With permission from Sanz G, et al. Abstract 640. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. N = 2241 Probability

26. With permission from Sanz G, et al. Abstract 640. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. Leukaemia-Free Survival in Patients with MDS by RBC Transfusion Dependency

27. Prognostic Impact of Development of Iron Overload is Independent of WPSS Score Overall Survival Leukaemia-Free Survival Variable a HRP-value Iron overload4.34<.001 WPSS1.60<.001 Variable a HRP-value WPSS2.24<.001 Iron overload2.13<.001 a Multivariate analyses including WPSS and development of iron overload (time-dependent) (n = 580). Cases with <3 serum ferritin measurements were excluded. With permission from Sanz G, et al. Abstract 640. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California.

28. Overall Survival in Patients with MDS by Serum Ferritin Level With permission from Sanz G, et al. Abstract 640. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. n = 762 Probability

29. With permission from Sanz G, et al. Abstract 640. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. n = 761 Leukaemia-Free Survival in Patients with MDS by Serum Ferritin Level Probability

30. Complications of Iron Overload Heart –Heart failure, arrhythmias Liver –Fibrosis, cirrhosis, cancer Endocrine tissues –Diabetes mellitus

31. Development of Diabetes During 3-Year Follow-Up 100% 80% 60% 40% 20% 0% 43% 33% 48% 32% 33% MDS Patients Overall Medicare Population Overall Medicare Population MDS Patients: Received RBC Transfusions MDS Patients: No RBC Transfusions P =.0001P =.02 Goldberg SL, et al. Abstract 636. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. Graphic courtesy of Dr. N. Gattermann.

32. Development of Cardiac Complications During 3-Year Follow-Up 100% 80% 60% 40% 20% 0% 80% 42% 80% 69% 42% MDS Patients Overall Medicare Population Overall Medicare Population MDS Patients: Received RBC Transfusions MDS Patients: No RBC Transfusions P =.001P =.002 Goldberg SL, et al. Abstract 636. Presented at: 50th Annual American Society of Hematology Meeting, December 6-9, 2008, San Francisco, California. Graphic courtesy of Dr. N. Gattermann.

33. 131 transfused adult patients 101 leukaemias 30 other anaemias Relation of Cardiac Iron Deposits to Amount of Blood Transfused Buja LM, et al. Am J Med. 1971;51:209-221. Graphic courtesy of Dr. N. Gattermann. Patients with Cardiac Iron (%) Units of Blood Transfused

34. Increased LPI or “Free” Iron Cell Death Fibrosis Organelle Damage TGF- β 1 Hydroxyl Radical Generation Lipid Peroxidation Lysosomal Fragility Enzyme Leakage Collagen Synthesis Abbreviations: LPI, labile plasma iron; TGF, transforming growth factor. Consequences of Iron-Mediated Toxicity During Iron Overload With permission from Cohen AR, et al. In: Disorders of Hemoglobin. Steinberg MH, et al, eds. Cambridge University Press; 2001:979-1027.

35. Which Patients with MDS May Benefit from Chelation Therapy?

36. Cumulative Survival of MDS Patients According to WHO Type Germing U, et al. Leuk Res. 2000;24:983-992. Graphic reflects updated data and is courtesy of Dr. U. Germing. Abbreviations: RA, refractory anaemia; RAEB, refractory anaemia with excess blasts; RARS, refractory anaemia with ring sideroblasts; RCMD, refractory cytopaenia with multilineage dysplasia; RSCMD, refractory sideroblastic cytopaenia with multilineage dysplasia.

37. Survival of RCMD + RSCMD Patients According to IPSS (Düsseldorf MDS Registry) MDS type:RARARSRCMDRSCMD RAEBIRAEBII5q- n13114642024720021141 IPSS scoreLowInt-1Int-2High 36%50%13%1% Median survival (months) 6236127 Düsseldorf MDS Registry. Graphic courtesy of Dr. N. Gattermann.

38. Requirement for Iron Chelator Therapy in Patients with MDS (Crude Estimate) ~ 20% of MDS patients (RA + RARS) ~6 years ~ 15% of MDS patients (RCMD ± RS)5–6 years 36% low-risk ~ 20% of MDS patients(RCMD ± RS) 3 years 50% int-1 Median Survival Düsseldorf MDS Registry. Graphic courtesy of Dr. N. Gattermann.

39. Profile of patients who might benefit from the treatment of iron overload Gattermann N. Hematol Oncol Clin North Am. 2005;19(suppl 1):18-25. Iron Overload in MDS Consensus Meeting Nagasaki, Japan – May 11, 2005 Transfusion-dependent patients Low-risk MDS: IPSS low or Int-1 WHO-type RA and RARS and 5q- Candidates for allografting Patients with documented stable disease Ferritin levels >1000–2000 ng/mL or other evidence of significant tissue iron overload Absence of comorbidities severely limiting prognosis

40. The Relative Importance of Iron Overload in MDS Morbidity Clinical problems in MDS Concomitant diseases Complications of chronic anaemia Problems of aging Complications of marrow failure Leukaemic transformation Iron chelation Graphic courtesy of Dr. N. Gattermann.

41. Conclusions Iron overload in MDS starts before patients become transfusion-dependent, but its most important cause is chronic transfusion therapy Transfusion therapy is associated with decreased likelihood of survival –Iron overload represents an independent prognostic factor Cardiac iron overload, which is detectable after about 75 units of red blood cells, appears to contribute to cardiac complications in MDS patients Retrospective data suggest improved survival with iron chelation therapy –Patients with lower-risk MDS are most likely to benefit

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