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 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

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.

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

2005: identification of jak2 v617F Lancet Nature Cancer Cell NEJM

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

JAK-STAT Signaling 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 EPO or TPO Receptor Shuai, K. & Liu,B. (2003) Nature Reviews Immunology 3:900

JAK2 V617F Mutation Acquired Arises in blood stem cells 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

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

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 30-40% CALR ???? 50-60% JAK2V617F mutant 97% JAK2V617F mutant Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis Kralovics & Green labs, ASH 2013

Referred to as ‘indels’ 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

(but not previously known to be relevant Normal Functions of CALR in Cells CALR Mutation Calcium Regulation in the cell Protein folding Cell adhesion Immune-mediated cell death Programmed cell removal Activation of JAK-STAT signaling (but not previously known to be relevant to this pathway) CALR Functions

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

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

Mutation Frequency in Chronic Phase and Post-MPN AML Gene Chronic Phase Blast Phase / AML JAK2 V617F PV: 98%; ET /PMF: 50-60% Exon 12 JAK2 PV: ~1-2% CALR ET/PMF: ~30-40% MPL ET: 1-5%; PMF: 5-10% LNK PV, ET, PMF: <5% ~10% CBL PMF: 6% TET2 PV: 7-16%, ET: 4-11%, PMF: 8-17% ASXL1 PV: 2-5%; ET: 5-8%; PMF: 7-17% 19% DNMT3A PV: 7%, ET: 3%, PMF: 7-15% 17% IDH 1/2 PMF: 4% 21% IKZF1 EZH2 5-13% of MPNs P53 27% SRSF2 Mutated genes related to JAK-STAT signaling 5-20% frequency Mutated genes outside of the JAK-STAT pathway

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

Mutations and Impact on Prognosis

IPSS Prognostic Scoring Systems for Primary Myelofibrosis DIPSS Plus 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 Gangat et al, J Clin Oncol, 2011

DIPSS Plus DIPSS Plus # Adverse Points Median Survival Low risk 185 months (15.4 yrs) Intermediate-1 risk 1 78 months (6.5 yrs) Intermediate-2 risk 2-3 35 months (2.9 yrs) High risk 4-6 16 months (1.3 yrs) Gangat et al, J Clin Oncol, 2011

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

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

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

Type 1 vs Type 2 CALR mutations may have different effects on prognosis Tefferi et al, Leukemia, 2014

Two Faces of ET Chao, Gotlib, Blood, 2014

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

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

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. Curr Opin Immunology 2010 Busque et al. Nat Genetics 2013

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

JAK2 V617F: 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 Wernig et al, Blood, 2006 Zaleskas et al, PLoS ONE, 2006

Predisposition genes identified in collaboration with 23andMe 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

Diagnosis and Treatment Genetic Mutations in Diagnosis and Treatment

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

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?

Do mutations in MPN affect response to therapy? Examined impact of mutations on outcome in MF patients treated on COMFORT-II trial. Frequency JAK2 75% ASXL1 33% TET2 11% MPL 7% EZH2 CBL 4% SRSF2 3% SH2B3 1% IDH1/2 73 patients best Available therapy ** ** = High molecular risk (HMR) category 219 patients 146 patients Ruxolitinib Survival at 114 weeks Best available tx Ruxolitinib Low Molecular Risk 71% pts alive 85% pts alive High Molecular Risk 58% pts alive 79% pts alive Guglielmelli et al, Blood, 2014

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