Translating New science in Myeloproliferative Neoplasms Into Clinical practice Ayalew Tefferi, MD

1. Translating New science in Myeloproliferative Neoplasms Into Clinical practice Ayalew Tefferi, MD Professor of Medicine, Mayo Clinic, Rochester, MN, USA

2. 2008 WHO classification of chronic myeloid neoplasms Monocytosis Granulocytosis Thrombocytosis Eosinophilia Mastocytosis MPN MDS Erythrocytosis Dyserythropoiesis Dysgranulopoiesis Cytopenia Dysmegakaryopoiesis MDS/MPN Tefferi et al. Cancer in press

3. 2008 WHO classification of Chronic myeloid neoplasms other than MDS MPNs with eosinophilia and PDGFR or FGFR1 rearrangements MPNs MDS/MPN • Chronic myelomonocytic leukemia • Juvenile myelomonocytic leukemia • Atypical CML, BCR-ABL-negative • “MDS/MPN, unclassifiable” • Polycythemia vera • Essential thrombocythemia • Primary myelofibrosis • Chronic myelogenous leukemia • Mast cell disease • “Chronic eosinophilic leukemia, • not otherwise classified” • Chronic neutrophilic leukemia • “MPN, unclassifiable” • PDGFRA-rearranged • PDGFRB-rearranged • FGFR1-rearranged

4. MPNs are often associated with tyrosine kinase mutations Modified from Gotlib et al. Best Practice and Research Clinical Hematology 2006;19:535 KIT D816V Dasatinib

5. Semi-molecular classification of myeloproliferative neoplasms Clinicopathologically assigned Molecularly defined CMLABL PDGFRA-rearranged MPN Clinicopathologically assigned PDGFRB-rearranged MPN PVJAK2 SMKIT ETJAK2/MPL CMML FGFR1-rearranged MPN PMFJAK2/MPL Molecularly characterized MPNs with therapeutic validation CNL JMMLRAS/PTPN11/NF1 CEL Molecularly assigned MPNs without therapeutic validation HES MPNs with recurrent genetic markers Tefferi & Vardiman. Curr Opin Hematol 2007;14:115

6. 9p24 William Dameshek, 1900-1969 BJH111 (4), 1023-1034, 2000 ABL1 9q34.1 Green et al. Lancet. 2005;365:1054 Vainchenker et al. Nature 2005;434:1144 Skoda et al. N Engl J Med. 2005;352:1779 Gilliland et al. Cancer Cell. 2005;7:387 JAK2-V617F

7. JAK2 mutations PV 97% V617F and 3% JAK2 exon 12 mutations ET 60% V617F and 4% MPL515 mutations PMF 60% V617F and 8% MPL 515 mutations NOT FOUND IN SECONDARY POLYCYTHEMIA NOT FOUND IN REACTIVE THROMBOCYTOSIS NOT FOUND IN LYMPHOMA OR METASTATIC CANCER Levine et al. Nat Rev Cancer. 2007;7:673

8. JAK2V617F and JAK2 exon 12 mutation induce a PV-like phenotype in mice MPLW515L induces a PMF-like phenotype in mice Pikman et al. PLoS Med. 2006;3:e270. Scott et al. N Engl J Med. 2007;356:459 Wernig et al. Blood 2006;107:4274

9. Initial clonogenic event Pardanani et al. Stem Cells 2007;25:2358 Nussenzveig et al. Exp Hematol. 2007;35:32 Campbell et al. Blood 2006;108:3548 JAK2V617F Skoda et al. Blood in press Allele dose Pikman et al. PLoS Med. 2006;3:e270 Pardanani et al. Blood 2006;108:3472 Pardanani et al. Blood 2008;111:2785 Germline genetic variation Other interacting mutations MPLW515L/K PV PMF ET

10. Clonal Origination and Evolution in myeloproliferative neoplasms First mutation Clonal myelopoiesis without disease phenotype Disease-causing mutation Stem cell pool Committed myeloidprogenitor Phenotype patterning events Committed lymphoidprogenitor Disease-transforming events Polyclonal stem cell compartment Differentiatedcells Acute leukemia

11. Clinical relevance of V617F • Use as a diagnostic marker • Genotype-phenotype correlations • Therapeutic implications • Prognostic relevance • The prospect of targeted therapy

12. When PV is suspected Peripheral blood mutation screening for JAK2V617F & Serum erythropoietin measurement V617F (+) & Epo ↓ V617F (+) but Epo normal or ↑ V617F (-) but Epo ↓ V617F (-) & Epo normal or ↑ PV highly likely PV likely PV possible PV unlikely JAK2 exon 12 mutation screening BM biopsy Tefferi and Vardiman Leukemia 2008;22:14

13. When ET is suspected Peripheral blood mutation screening for JAK2V617F V617F (-) V617F (+) ET and PMF still possible & CML should be considered as well BM biopsy & cytogenetics ET, PV or PMF highly likely Use 2008 WHO criteria for specific diagnosis Consider FISH for BCR-ABL in the absence of the Ph chromosome but presence of dwarf megakaryocytes Tefferi and Vardiman Leukemia 2008;22:14

14. When PMF is suspected BM biopsy, reticulin stain, cytogenetic studies & mutation screening for JAK2V617F V617F (-) & normal cytogenetics Ph chromosome (+) V617F (+) or Del(13q) Other cytogenetic abnormalities CML PMF likely but use histology to exclude other myeloid neoplasm Could be PMF but also MDS or other myeloid neoplasm If megas dwarf consider FISH for BCR-ABL otherwise use histology for specific diagnosis Tefferi and Vardiman Leukemia 2008;22:14

15. Facts about laboratory testing for V617F • For the purposes of mutation detection, it really does not matter much if sample is from peripheral blood or bone marrow, granulocytes or whole blood, plasma or blood etc. • For the purposes of allele burden measurement, I recommend granulocytes for the sake of uniformity

16. Facts about laboratory testing for JAK2 exon 12 • For the purposes of mutation detection, one can start with peripheral blood first and then either granulocytes or bone marrow • Too many alleles to consider quantitative testing

17. Genotype-phenotype correlations • Positive vs. negative • V617F vs. Exon 12 JAK2 vs. MPLW515 • Relevance of allele burden

18. Mayo Clinic series of 176 ET patients examined for JAK2V617F by AS-PCR Kittur et al. Cancer 2007;:2279

19. Mayo Clinic series of 605 ET patients followed for median 7 years Tefferi et al. Blood. 2007;109:4105.

20. Clinical correlates of JAK2V617F in primary myelofibrosis

21. Does JAK2V617F presence in MF matter? Overall survival curves of V617F-positive and V617F-negative PMF patients (n=199) P=0.34 Survival V617F-negative; n=84 V617F-positive; n=115 Months Tefferi et al. Leukemia in press

22. Leukemia-free survival curves of V617F-positive and V617F-negative PMF patients (n=199) V617F-positive; n = 115 AML events = 7 V617F-negative; n = 84 AML events = 10 Survival P = 0.5 Months Tefferi et al. Leukemia in press

23. Overall survival curves of V617F-negative and V617F-positive PMF patients (n=129) stratified by mutant allele burden quartile ranges P = 0.0008 Upper quartile allele burden n = 19 Survival V617F-negative; n = 53 Lower quartile allele burden n = 19 Middle quartiles allele burden n = 38 Months Tefferi et al. Leukemia in press

24. Leukemia-free survival curves of V617F-negative and V617F-positive PMF patients (n=129) stratified by mutant allele burden quartile ranges P = 0.01 Middle quartiles allele burden n = 38 Upper quartile allele burden n = 19 Lower quartile allele burden n = 19 Leukemia-free Survival V617F-negative n = 53 Months Tefferi et al. Leukemia in press

25. ↑Hgb Pruritus ↑Fibrotic transformation ↑WBC ↓Plt Tefferi A, et al. Cancer. 2006;106:631 Vannucchi AM, et al. Blood. 2007;110:840 V617F allele burden “Homozygous” vs. “Heterozygous” PV V617F allele burden by qPCR ↑Hgb ↑WBC Pruritus ↑Spleen size Thrombosis Chemotherapy Tefferi A, et al. Leukemia. 2007;21:2074 Vannucchi AM, et al. Leukemia. 2007;21:1952 Dupont S, et al. Blood. 2007;110:1013

26. JAK2 exon 12 and MPL phenotypes • JAK2 exon 12 patients are younger and tend to present with with isolated erythrocytosis • MPLW515 ET or PMF patients are older and more anemic Scott et al. NEJM 2007;356:459 Vannucchi et al. ASH 2007; Guglielmelli et al. BJH 2007;137:244

27. Janus Kinases (JAK1, JAK2, JAK3, TYK2) are among over 90 tyrosine kinases in humans FERM Domain SH2-like Domain Rane et al. Oncogene 2000;19:5662

28. Anti-JAK2 drugs A B C JAK1 Flt3 90 25 Figure 1 The TargeGen compound Pardanani et al. Leukemia 2007;21:1658

29. Primary cell experiments A B Pardanani et al. Leukemia 2007;21:1658

30. TG101348 Given orally is effective in the treatment of PV in mouse models Gilliland group. Cancer Cell in press

31. ASH Abstract 2008A phase I/II study of TG101348, a JAK2-selective small molecule kinase inhibitor, in myelofibrosis Mayo, Dana Farber, UCSD, Stanford, MDACC, U of Michigan

32. Abstract #558INCB18424, an Oral, Selective JAK2 Inhibitor, Shows Significant Clinical Activity in a Phase I/II Study in Patients With Primary Myelofibrosis (PMF) and Post Polycythemia Vera/Essential Thrombocythemia Myelofibrosis (Post-PV/ET MF) Srdan Verstovsek, Hagop Kantarjian, Animesh Pardanani, Deborah Thomas, Jorge Cortes, Ruben Mesa, John Redman, Carl-Michael Staschen, Jordan Fridman, Kris Vaddi, and Ayalew Tefferi Leukemia Department, M.D. Anderson Cancer Center, Houston, Texas Mayo Clinic, Rochester, Minnesota Incyte Corporation, Wilmington, Delaware

33. Potent and selective ATP competitive JAK inhibitor • 100-fold selectivity against a broad panel of kinases • Excellent preclinical pharmacokinetic properties • High oral availability • Efficacious and well-tolerated in a JAK2V617F-driven animal model • Preclinical toxicology: • Findings restricted to myelosuppression and reduced lymphoid organ cellularity at high doses

34. Total 32 patients MTD 25 mg BID Toxicity ● ● ● ● ● ● ● ● • DLT = Thrombocytopenia grade 4 • Toxicity at MTD = Thrombocytopenia up to grade 3 • No other toxicities • Two SAEs with non-neutropenic fever and hypotension • Constitutional symptoms improved in all patients • Spleen size decreased to over 50% in the majority • Only 1 patient became transfusion-independent • Mutation status did not matter • Leukoerythroblastosis so far not affected • V617F allele burden so far not affected • Too early for bone marrow assessment ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Efficacy June 2007 July 2007 October 2007 November 2007 August 2007 September 2007 ● denotes 1 patient starting at 25 mg BID and ● at 50 mg BID

35. Hydroxyurea Interferon Transplant Clonal myeloproliferation JAK2 inhibition Altered cytokine milieu Altered stroma Extra- medullary hematopoiesis Prednisone Thalidomide Lenalidomide JAK2 and/or JAK1 inhibition Abnormal immunologic response Ineffective hematopoiesis Epo Androgens Tefferi and Gilliland, Cell Cycle 2007

36. Effect of INCB18424 on Angiogenic Factors & Pro-inflammatory Cytokines N = 5 N = 3 N = 5 Percent of Baseline N = 2 N = 2 IL-6 VEGF

37. SEMI- • Low CYP inhibition profile • High oral bioavailability • Myelosuppression seen • at highest dose levels Abstract # 553A Phase I Study of XL019, a Selective JAK2 Inhibitor, in Patients with Primary and Post-PV/ET Myelofibrosis S. Verstovsek, A. D. Pardanani, N. P. Shah, L. Sokol, M. Wadleigh, D. G. Gilliland, A. F. List, A. Tefferi, H. Kantarjian, G. M. Schwab, L. A. Bui, D. O. Clary MD Anderson Cancer Center, Mayo Clinic, University of California, San Francisco, H Lee Moffitt Cancer Center, Dana Farber Cancer Institute and Exelixis, Inc., San Francisco, CA, USA.

38. XL019-001: Dose escalation phase…this is why it is semi-hot • N= total 9, evaluable 6; spleen size reductions of 33% to 100% observed in all 5 subjects with mutations; no myelosuppression • Grade 1 adverse events: nausea, headaches, dysequilibrium, dizziness, chest discomfort, scintillating scotomas, fatigue, hypertension • Grade 2 adverse events: lightheadedness and decreased sensation in soles • 2 serious adverse events: confusion, peripheral neuropathy

39. NOT THAT Abstract # 3543Phase II Study of CEP701, an Orally Available JAK2 Inhibitor, in Patients with Primary Myelofibrosis and Post Polycythemia Vera/Essential Thrombocythemia Myelofibrosis Srdan Verstovsek, Ayalew Tefferi, Steven Kornblau, Deborah Thomas, Jorge Cortes, Farhad Ravandi-Kashani, Guillermo Garcia-Manero, and Hagop Kantarjian, M.D. Anderson Cancer Center, Houston, TX, Mayo Clinic College of Medicine, Rochester, MN. • Orally available high potency JAK2/FLT3 inhibitor; 80 mg PO BID • 22 patients with V617F-positive MF; median f/u 5 months (range 1-5) • 5 had spleen response and 1 became transfusion independent • Side effects included nausea, vomiting, diarrhea and thrombocytopenia

40. So who with PV or ET is eligible for JAK2 inhibitor clinical trials? • Low-risk patients without leukocytosis or anemia have a near-normal life expectancy with indolent clinical course and very low risk of thrombosis. NOT. • High-risk patients are currently well managed with hydroxyurea and aspirin. A major phase III trial is needed to show advantage over this inexpensive and relatively well tolerated drug. POSSIBLE. • High-risk patients who do not tolerate hydroxyurea or are refractory to it. Current alternative drugs include IFN, pipobroman (in Europe) or busulfan. • PROBABLE

41. So who with MF is eligible for JAK2 inhibitor clinical trials? • Low-risk patients. NOT in early phase trials. • Intermediate-risk patients. YES. • High-risk patients. YES.