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GENETICS & biology OF MYELOPROLIFERATIVE NEOPLASMS

GENETICS & biology OF MYELOPROLIFERATIVE NEOPLASMS. Jason Gotlib , MD, MS Associate Professor of Medicine (Hematology) Stanford Cancer Institute MPN Advocacy and Education International Patient Symposium, San Mateo, CA May 22, 2014. Discussion Points.

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GENETICS & biology OF MYELOPROLIFERATIVE NEOPLASMS

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  1. GENETICS & biology OF MYELOPROLIFERATIVE NEOPLASMS Jason Gotlib, MD, MS Associate Professor of Medicine (Hematology)Stanford Cancer Institute MPN Advocacy and Education International Patient Symposium, San Mateo, CA May 22, 2014

  2. Discussion Points • Refresher on the ‘established genetics’ & biology of MPNs: • JAK2 V617F, MPL • JAK-STAT signaling • Introduction to the ‘newer genetics’ • Calreticulin (CALR) • Other gene mutations outside of the JAK-STAT pathway • Relevance of mutations to diagnosis, prognosis and treatment of MPNs

  3. William Dameshek, 1951 Blood Editorial“Some Speculations on the Myeloproliferative Syndromes” “It is possible that these various conditions- ‘myeloproliferative disorders’- are all somewhat variable manifestations of proliferative activity of the bone marrow cells, perhaps due to a hitherto undiscovered stimulus.

  4. Normal Human Blood Development (Hematopoiesis) MPNs, and Stem Cells Normal MPN MPN Stem Cell MPN Progenitor Cell CD34+CD38+ Mature MPN Cells CD34- modified from Tan et al., 2006

  5. 2005: identification of jak2 v617F Lancet Nature Cancer Cell NEJM

  6. JAK2 V617F Mutation Frequency Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis 95-98% 50-60% 50-60% Exon 12 JAK2 ~2% JAK2 gene

  7. JAK-STAT Signaling EPO or TPO Receptor • A well characterized signaling pathway involved in normal hematopoiesis, inflammation, and immune function • Four members of JAK family • JAK1, JAK2, JAK3 and Tyk2 • They are tyrosine kinases • JAK2 specifically mediates growth factor signaling for red blood cells and for platelets Shuai, K. & Liu,B. (2003) Nature Reviews Immunology 3:900

  8. JAK2V617F JAK2 V617F Mutation EPO or TPO Receptor • Acquired • Arises in blood stem cells • Results in constitutively (i.e. always) • active JAK2 tyrosine kinase • Causes disease in mice (PV → MF) Shuai, K. & Liu,B. Nature Reviews Immunology 2003:3:900

  9. Mutations in other genes besides JAK2 cause activated JAK-STAT signaling in MPNs LNK (PV, ET, MF) <5% MPL 1-5% ET 5-10% PMF CBL ~6% (PMF) Oh and Gotlib, Exp Rev Hematol, 2010

  10. ASH 2013: CALR mutations in non-mutated JAK2 ET and MF patients ‘Triple negative’ 10% MPL Mutant 5% JAK2 exon 12 mutant ???? 50-60% JAK2V617F mutant 97% JAK2V617F mutant 30-40% CALR mutant 50-60% JAK2V617F mutant Essential Thrombocythemia Primary Myelofibrosis Polycythemia Vera Kralovics & Green labs, ASH 2013

  11. Mutations in the CALR gene all occur in one region (exon 9) Two most common mutations in the CALR gene: Type 1: 52-bp deletion Type 2: 5-bp insertion Referred to as ‘indels’ 5-bp insertion 52-bp deletion Klampfl et al, NEJM 2013

  12. Normal Functions of CALR in Cells CALR Functions CALR Mutation Activation of JAK-STAT signaling (but not previously known to be relevant to this pathway)

  13. Both JAK2- and CALR-mutated MPN patients show a gene expression signature associated with activated JAK-STAT signaling Rampalet al, Blood, 2014

  14. Mutations in genes outside of the JAK-STAT pathway in MPN patients JAK2 V617F JAK-STAT Pathway CBL JAK2 exon 12 MPL CALR LNK TET2 ASXL1 IDH1, IDH2 Outside of JAK-STAT Pathway DNMT3A EZH2 SRSF2

  15. Mutation Frequency in Chronic Phase and Post-MPN AML Mutated genes related to JAK-STAT signaling 5-20% frequency Mutated genes outside of the JAK-STAT pathway

  16. Average number of acquired mutations in: PV: 6.5 ET: 6.5 PMF: 13 Klampfl et al, NEJM 2013

  17. Mutations and Impact on Prognosis

  18. Prognostic Scoring Systems for Primary Myelofibrosis PROGNOSTIC FACTORS • Age >65 • Hb < 10 g/dL • WBC > 25,000/mm3 • Constitutional symptoms • Peripheral blood blasts >1% • RBC transfusion dependence • Platelet count < 100,000/mm3 • Unfavorable cytogenetics IPSS DIPSS Plus Cervantes et al, Blood, 2009 Gangatet al, J ClinOncol, 2011

  19. DIPSS Plus Gangatet al, J ClinOncol, 2011

  20. “High-Molecular Risk” Markers in PMF: ASXL1, EZH2, SRSF2, IDH1/2 Overall Survival Leukemia-free survival EZH2 Independent of IPSS or DIPSS-plus ASXL1 SRSF2 IDH1/2 Vannucchi et al. Leukemia 2012.

  21. “High-Molecular Risk” Markers in PMF: 0, 1, or >2 mutations 0 0 1 1 >2 >2 Guglielmelliet al, Leukemia, 2014

  22. Impact of CALR Mutations on Outcomes in ET / PMF Klampfl et al, NEJM 2013

  23. Type 1 vs Type 2 CALR mutations may have different effects on prognosis Tefferiet al, Leukemia, 2014

  24. Two Faces of ET Chao, Gotlib, Blood, 2014

  25. How does one mutation cause 3 diseases? • (1) JAK2 Dependent Effects JAK2V617F homozygosity Polycythemia Vera • JAK2V617F heterozygosity Essential Thrombocytosis • (2) JAK2-Independent Effects • - Co-occurring mutations (3) Genetic background of the patient - Variations in the DNA that one is born with that may predispose to greater susceptibility to MPN later in life

  26. Are TET2 mutations the “pre-JAK2” mutation? JAK2 normal TET2 mutant colonies No JAK2 mutant TET2 normal colonies!! JAK2 mutant + TET2 mutant colonies JAK2 mutant + TET2 mutant CD34+ High % engraftment These data suggest that TET2 mutations preceded acquisition of JAK2 mutations in MPN patients. *Delhommeauet al NEJM 2009 Low % engraftment JAK2 mutant + TET2 normal CD34+

  27. TET2 mutations in normal elderly individuals with clonal blood formation A proportion of patients with clonal blood formation and no clinically apparent hematological disorder have TET2 mutations. In some cases, the acquisition of the TET2 mutation actually preceded development of JAK2 mutant MPN. . Beerman et al. CurrOpin Immunology 2010 Busque et al. Nat Genetics 2013

  28. How does one mutation cause 3 diseases? • (1) JAK2 Dependent Effects JAK2V617F homozygosity Polycythemia Vera • JAK2V617F heterozygosity Essential Thrombocytosis • (2) JAK2-Independent Effects • - Co-occurring mutations (3) Genetic background of the patient - Variations in the DNA that one is born with that may predispose to greater susceptibility to MPN later in life or type of MPN

  29. JAK2V617F: One Mutation, Three Diseases: Effect of genetic background: mice example • Balb/c mice: • High red blood cell count, high white blood cell count, and myelofibrosis • C57Bl/6 mice: • High red blood cell count, normal-mildly increased white blood cell count, and fibrosis only in the spleen (not marrow) Bumm , et al, Cancer Res, 2006 Lacout, et al, Blood, 2006 Werniget al, Blood, 2006 Zaleskaset al, PLoS ONE, 2006

  30. Model of MPN Development ? Environmental Factors Inherited Variations in DNA that Predispose To MPN Acquired MPN Mutations Chronic Phase MPN JAK2 V617F Blast Phase JAK2 46/1 LNK ASXL1 TERT EZH2 LNK TET2 TET2 MPL IDH 1/2 Predisposition genes identified in collaboration with 23andMe SRSF2 P53

  31. Genetic Mutations in Diagnosis and Treatment

  32. Summary: Role of JAK2 and other mutations in the diagnosis of MPNs • JAK2 V617F, MPL, or CALR mutations establish the presence of a primary bone marrow disorder, almost always an MPN, instead of a reactive condition (e.g. infection, inflammation) • However, the diagnosis of an MPN requires a combination of clinical, laboratory, histopathology, and mutation testing • The majority of patients with ET and MF with non-mutated JAK2 or MPL have CALR mutations

  33. Summary: Mutations and Treatment of MPNs In 2014: • Treatment decisions about PV, ET, or PMF are not based on JAK2 mutation status • IPSS/DIPSS-Plus are used to risk stratify patients into low, intermediate-1/2, and high risk groups • JAK inhibitors demonstrate activity in myelofibrosis patients with normal or mutant JAK2 • Gene panels are becoming available in labs to evaluate for mutations in 20+ genes • May be useful: 1) in triple negative patients 2) to assess for poor-risk molecular markers: ASXL1, EZH2, SRSF2, IDH 1/2  Poor risk markers: earlier referral to transplant for intermediate-1 risk patients?

  34. Do mutations in MPN affect response to therapy? • Examined impact of mutations on outcome in MF patients treated on COMFORT-II trial. 73 patients best Available therapy ** 219 patients 146 patients Ruxolitinib ** Survival at 114 weeks ** ** ** = High molecular risk (HMR) category Guglielmelliet al, Blood, 2014

  35. Acknowledgements Stanford Andrea Linder Jim Zehnder Cheryl Langford Jason Merker Cecelia Perkins Andy Fire Jenny Ma BiquanLuo Cristina Williams Krishna Roskin Wan-Jen Hong Mark Chao Colleagues Ruben Mesa Ross Levine Claire Harrison AnimeshPardanani AyalewTefferi 23andMe David Hinds and team Our Patients Stanford Division of Hematology MPN Advocacy and Education International Charles and Ann Johnson Foundation

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