Disclosures for Ayalew Tefferi

Disclosures for Ayalew Tefferi Principal investigator role Employee Consultant Major Stockholder Speakers Bureau Scientific Advisory Board Janssen, Geron, Celgene, Sanofi-Aventis, Gilead Sciences, Incyte None None None None None Presentation includes discussion of the following off-label use of a drug or medical device: Hydroxyurea, Interferon-alpha, Busulfan, Thalidomide, Lenalidomide, Pomalidomide, Ruxolitinib, Androgen preparations, Erythropoiesis stimulating agents

Emerging Issues of Myeloproliferative Neoplasms Research 2016 Ayalew Tefferi, MD Mayo Clinic, Rochester, MN

Chronic Myeloid Malignancies Myelodysplastic Syndromes Myelodysplastic/ Myeloproliferative overlap Myeloproliferative Neoplasms Myeloid/Lymphoid neoplasm with eosinophilia and PDGFR/FGFR1 mutation MORPHOLOGY Absence of cytosis Dyserythropoiesis Dysgranulopoiesis Monocytosis Granulocytosis Thrombocytosis Erythrocytosis Eosinophilia Mastocytosis Presence of PDGFRA/B or FGFR1 mutation MUTATIONS MDS -TET2 20% RARS -SF3B1 80-90% Tefferi and Pardanani. JAMA Oncology 2015 (modified) CMML -TET2 40-60% -SRSF2 30-50% -ASXL1 40% Dyserythropoiesis RARS-T Dysgranulopoiesis -SF3B1 80-90% -JAK2V617F 50% acml -SETBP1 30% MDS/MPN-U Monocytosis CML -BCR-ABL1 100% PV -JAK2 99% ET -JAK2/CALR/MPL 85% PMF -JAK2/CALR/MPL 90% CNL -CSF3R 80-100% -SETBP1 30% SM -KITD816V 80-100% CEL MPN-U PDGFRA rearranged -FIP1L1-PDGFRA 100% PDGFRB rearrange -PDGFRB mutation 100% FGFR1 rearranged -FGFR1 mutation 100%

Objectives 1. New developments in laboratory investigation 2. WHO revisions in diagnostic criteria for PV, ET and PMF 3. Mutations and prognosis 4. Revised risk stratification in essential thrombocythemia 5. New developments in treatment

The calreticulin protein CALR located on 19p13.2 9 exons Mendlovic F. Nature Education 2010 Exon 9 indel mutations-1bp reading frameshift Mutational frequencies 20-30% in ET or PMF Specific to JAK2-unmutated ET and PMF Mutually exclusive of JAK2 and MPL mutations Klampfl et al. Nangalia et al. NEJM 2013

Chachoua et al. Blood First Edition Araki et al. Blood First edition Retroviral mouse transplant model for mutant CALR Marty et al. Blood First Edition CALR mutants specifically activate TpoR and the extracellular domain, but not the Tpo-binding site is essential in this regard Oncogenic signaling requires JAK2 Tpo-independent megakaryocyte proliferation in CALR-mutated patients requires TpoR and JAK2

Diagnosis

2016 Proposed Revised WHO Diagnostic Criteria (Barbui et al. Blood Cancer Journal (2015) 5, e337; doi:10.1038/bcj.2015.64 Published online 14 August 2015) Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis Prefibrotic Primary Myelofibrosis Major 1 Hemoglobin (Hgb) 1 Platelet count 450 x 10 9 /L 1 Megakaryocyte proliferation and atypia*** Megakaryocyte proliferation and atypia*** criteria >16.5 g/dl (men) >16 g/dl (women) or Hematocrit >49% (men) >48% (women) and grade 2 reticulin/collagen fibrosis ***megakaryocytes with aberrant nuclear/cytoplasmic ratio and hyperchromatic and irregularly folded nuclei and dense clustering and grade 1 reticulin/collagen fibrosis, Increased cellularity, granulocytic Proliferation and decreased erythropoiesis 2 BM trilineage myeloproliferation 2 BM megakaryocyte proliferation 2 Not meeting WHO criteria for Not meeting WHO criteria for with pleomorphic megakaryocytes with large and mature morphology other myeloid neoplasm other myeloid neoplasm 3 Not meeting WHO criteria for 3 Presence of JAK2, CALR or MPL mutation Presence of JAK2, CALR or MPL mutation 3 Presence of JAK2 mutation other myeloid neoplasms or or 4 Presence of JAK2, CALR or MPL mutation presence of another clonal marker or absence of evidence for reactive presence of another clonal marker or absence of evidence for reactive bone marrow fibrosis bone marrow fibrosis Minor 1. Subnormal serum Epo level 1. Presence of a clonal marker 1 1. Anemia 1. Anemia criteria or absence of evidence for reactive 2. Leukocytosis 2. Leukocytosis thrombocytosis 3. Palpable splenomegaly 3. Palpable splenomegaly 4. Increased LDH 4. Increased LDH 5. Leukoerythroblastosis (diagnosis requires meeting all three major criteria or the first two major criteria and one minor criterion diagnosis requires meeting all 4 major criteria or first three major criteria and one minor criterion diagnosis requires meeting all 3 major criteria and at least one minor criterion diagnosis requires meeting all 3 major criteria and at least one minor criterion

Practical algorithm for diagnosis of myeloproliferative neoplasm Tefferi and Pardanani; JAMA Oncology 2015 Polycythemia vera suspected Essential thrombocythemia suspected Primary myelofibrosis suspected lood mutation screening JAK2V617F+ If negative Blood mutation screening JAK2V617F+ If negative Bone marrow biopsy with mutation screening and cytogenetics JAK2 exon 12+ If negative Diagnosis likely Bone marrow examination advised to confirm diagnosis CALR+ If negative Bone marrow examination required to confirm diagnosis and distinguish ET from prefibrotic PMF Subnormal serum erythropoietin level MPL+ If negative Diagnosis unlikely If JAK2 unmutated and serum erythropoietin level normal or increased Triple-negative Diagnosis considered If bone marrow morphology is consistent with PMF and 1. JAK2, CALR or MPL mutated or 2. trisomy 9 or del(13q) present or 3. Other myeloid malignancies are excluded

Survival and Prognosis

Tefferi et al. Blood 2014 Comparison of survival in 826 Mayo Clinic patients with essential thrombocythemia vs polycythemia vera vs primary myelofibrosis.

Tefferi et al. Blood 2015 Comparison of survival in 389 young patients with essential thrombocythemia vs polycythemia vera vs primary myelofibrosis.

Elala et al. Submitted Overall survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Elala et al. Submitted Myelofibrosis-free survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Elala et al. Submitted Thrombosis-free survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Targeted deep sequencing in 183 patients with essential thrombocythemia RED: Sequence variants previously associated with a hematologic malignancy and shown to be somatic PINK: Sequence variants previously associated with a hematologic malignancy and with 1% minor allele frequency in current databases for single nucleotide polymorphisms BLUE: Sequence variants with 1% minor allele frequency in current databases for single nucleotide polymorphisms 1. Prevalence of mutations/variants other than JAK2/CALR/MPL = 53% 2. Driver mutational status did not affect prevalence 3. Most frequent were ASXL1 andtet2 4. 41%, 8% and 4% harbored 1, 2 or 3 such mutations 5. 6 genes were identified as being affected by adverse mutations/variants Tefferi et al. Submitted

Targeted deep sequencing in 133 patients with polycythemia vera RED: Sequence variants previously associated with a hematologic malignancy and shown to be somatic PINK: Sequence variants previously associated with a hematologic malignancy and with 1% minor allele frequency in current databases for single nucleotide polymorphisms BLUE: Sequence variants with 1% minor allele frequency in current databases for single nucleotide polymorphisms 1. Prevalence of mutations/variants other than JAK2/CALR/MPL = 53% 2. Most frequent were ASXL1 andtet2 3. 30%, 20% and 3% harbored 1, 2 or 3 such mutations 4. 3 genes were identified as being affected by adverse mutations/variants Tefferi et al. Submitted

PV ET Tefferi et al. Submitted

Myelofibrosis-free survival in 132 Italian patients with polycythemia vera seen at the University of Florence, Italy, and stratified by the presence or absence of adverse sequence variants P<.0001 w/o adverse variants N=91 Median not reached with adverse variants N=41 Median 11.2 (7.5-14.9) yrs HR 9.5, 95% CI 3.9-23.4 Tefferi et al. Submitted

Leukemia-free survival Leukemia-free survival in 183 patients with essential thrombocythemia stratified by the presence or absence of adverse sequence variants/mutation 1 No adverse variants N=156 Leukemic transformations = 2 (1.3%).8.6.4 With adverse variants N=27 Leukemic transformations = 4 (14.8%).2 0 P<0.0001 0 5 10 15 20 25 30 35 40 Tefferi et al. Submitted Years

Survival Survival data on 438 patients with primary myelofibrosis stratified by mutational status: Mayo-Florence patients 1.8.6.4 JAK2 mutated N=265 Median 5.7 years CALR mutated N=93 Median 16 years.2 Triple negative N=53 Median 2.3 years MPL mutated N=27 Median 9.9 years P<0.0001 0 0 5 10 15 20 25 30 Tefferi et al. Blood 2014 Years

Survival Survival data on 440 Mayo Clinic patients with JAK2 or CALR mutated primary myelofibrosis 1 JAK2V617F vs type 1/type 1-like, p<0.0001; HR 2.7, 95% CI 1.9-3.7 JAK2V617F vs type 2/type 2-like, p=0.84; HR 1.1, 95% CI 0.6-1.8 Type 2/type 2-like vs type 1/type 1-like, p=0.003; HR 2.5, 95% CI 1.4-4.5.8.6 Type-1/type 1-like CALR mutated N=110 Median 13.7 years.4.2 Type-2/type 2-like CALR mutated N=21 Median 3.3 3.5 years 0 JAK2-mutated N=309 Median 4 years 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Tefferi et al. Blood 2014 Years

Survival data on 325 Florence (Italy) patients with JAK2 or CALR mutated primary myelofibrosis Guglielmelli et al. Blood Cancer Journal (2015) 5, e360; doi:10.1038/bcj.2015.90

Survival Survival in 722 Mayo Clinic patients with primary myelofibrosis stratified by driver mutational status 1.8.6 CALR type 1/type 1-like N=115 Median 10.3 years P<0.0001.4 Triple-negative N=65 Median 3.1 years.2 CALR type 2/type 2-like N=24 Median 3.5 years MPL-mutated N=41 Median 6 years 0 JAK2-mutated N=477 Median 3.8 years 0 5 10 15 20 25 30 Tefferi et al. ASH 2015 Years

Targeted deep sequencing in 182 patients with primary myelofibrosis A) B) RED: Sequence variants previously associated with a hematologic malignancy and shown to be somatic PINK: Sequence variants previously associated with a hematologic malignancy and with 1% minor allele frequency in current databases for single nucleotide polymorphisms BLUE: Sequence variants with 1% minor allele frequency in current databases for single nucleotide polymorphisms 1. Prevalence of mutations/variants other than JAK2/CALR/MPL = 81% 2. Prevalence was 83% in JAK2, 73% CALR, 91% MPL and 82% triple-negative (p=0.43) 3. Most frequent were ASXL1 36%, TET2 18%, SRSF2 18%, U2AF1 16% 4. 35%, 26%, 10% and 9% harbored 1, 2, 3 or 4 such mutations 5. 7 genes were identified as being affected by adverse mutations/variants Tefferi et al. Submitted

Survival Survival in 182 patients with primary myelofibrosis stratified by the presence or absence of adverse or non-adverse sequence variants/mutation 1 Adverse sequence variants/mutations: 7 genes were identified as being involved with adverse mutations/variants.8.6 No sequence variants/mutations N=35 Median survival not reached.4 Non-adverse variants/mutations N=45 Median survival = 6.8 years HR 2.5 (95% CI 1.1-5.4).2 0 P<0.0001 Adverse variants/mutations N=102 Median survival = 3.6 years HR 5.1 (95% CI 2.6-10.2) Tefferi et al. Submitted 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Years

Survival Survival in 182 patients with primary myelofibrosis stratified by the number of adverse sequence variants/mutation Adverse sequence variants/mutations: 7 genes were identified as being involved with adverse mutations/variants 1.8 P<0.0001.6 No adverse variants/mutations N=80 Median survival = 8.5 years.4.2 0 Three or more adverse variants/mutations N=9 Median survival = 0.65 years HR 18.8 (95% CI 8.5-42.1) One or two adverse variants/mutations N=93 Median survival = 4 years HR 2.7 (95% CI 1.8-4.2) Tefferi et al. Submitted 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Years

Survival Overall survival in 369 patients with primary myelofibrosis stratified by genetics-based prognostic scoring system (GPSS) 1 GPSS risk categories High Score 5 Intermediate-2 Score 3 or 4 Intermediate-1 Score 1 or 2 Low Score 0 GPSS variables.8.6 Low N=31 Median not reached Karyotype Very high risk = 3 High risk = 1 Mutations Triple-negative = 2 JAK2 = 2 MPL = 2 Type 2/type 2-like CALR = 2 ASXL1 = 1 SRSF2 = 1 Age >60 = 2.4.2 0 High N=115 Median 2.2 yrs Intermediate-2 N=133 Median 5 yrs Intermediate-1 N=90 Median 9 yrs 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Karyotype Very high risk Monosomal Inv(3) i(17q) -7/7q- 11q abns 12p- High risk Complex not MK Two abns 5q- +8 Other trisomies Other sole abns Years Tefferi et al. ASH 2014

New Risk Stratification in Essential Thrombocythemia

Practice-relevant revision of IPSET-thrombosis based on 1019 patients with WHO-defined essential thrombocythemia Barbui et al. Blood Cancer Journal (2015) 5, e369; doi:10.1038/bcj.2015.94

Contemporary treatment algorithm in essential thrombocythemia (ET) and polycythemia vera (PV) (all patients with polycythemia vera require phlebotomy to a hematocrit target of <45%) Very low-risk disease No history of thrombosis Age 60 years JAK2-unmutated Low-risk disease No history of thrombosis Age 60 years JAK2 mutated Intermediate-risk disease Age >60 years No history of thrombosis JAK2 unmutated High-risk disease History of thrombosis or Age >60 years with JAK2 mutation Without CV risk factors With CV risk factors Arterial thrombosis history at any age Venous thrombosis history at any age Observation alone Once-daily aspirin Without CV risk factors Once-daily aspirin With CV risk factors Hydroxyurea + once-daily aspirin Hydroxyurea + once-daily aspirin Hydroxyurea + systemic anticoagulation Avoid aspirin in the presence of extreme thrombocytosis and acquired von Willebrand syndrome With CV risk factors age >60 years or JAK2-mutated or CV risk factors JAK2-mutated or CV risk factors Consider twice-daily aspirin Cytoreductive therapy might not be essential Consider twice-daily aspirin Consider once-daily aspirin Modified from Tefferi and Barbui AJH 2015

Treatment Myelofibrosis

Myelofibrosis Rx Algorithm Type 1 Type 1 Type 1 Type 1 Tefferi A. AJH 2014

Leukemia 2014

Survival Figure 1a 1.8 Ruxolitinib-treated, n=51.6 P=0.43.4.2 No ruxolitinib, n=410 0 0 20 40 60 80 100 120 140 Months Tefferi et al. NEJM 2011:365;15

Survival Figure 1 Survival in 542 Mayo Clinic patients with high or intermediate-2 risk myelofibrosis stratified by treatment with momelotinib 1.8 Momelotinib treated N=100 Median 3.2 years P=0.99.6.4.2 Momelotinib-naive N=442 Median 3.8 years 0 Tefferi et al. ASH 2015 0 2 4 6 8 10 12 14 16 18 Years

Pardanani et al. Leukemia (2015) 29, 741 744

Non-canonical mechanism of action Canonical mechanism of telomerase activity Telomeres Telomerase Imetelstat Regulation of transcription Mitochondrial RNA processing and ROS production

Status of All 33 Patients Treated with Imetelstat 9.4 mg/kg IV every 1 to 3 weeks Median age 67 years; primary PMF 55%; DIPSS-plus high 52% and int-2 48%; prior JAKi therapy 49%; Abnormal karyotype 55%; Tx-dependent 39%; Median palpable spleen 15 cm CR/PR = 7 (21.2%) patients Median time to response onset=3.5 months (1.4-7.2) Median response duration= 18/10 months in CR/PR All 4 CR patients achieved reversal of fibrosis 3 CR patients had molecular responses CR/PR 27% in JAK2-mutated vs 0% in unmutated CR/PR 32% in ASXL1-unmutated vs 0% in mutated CR 38% in SF3B1/U2AF1 mutated vs 4% otherwise CR/PR/CI = 12 (36%) patients Spleen response =35% Tx-independency = 31% LES response = 82% Efficacy Leukocytosis response = 80% Thrombocytosis response = 100% Toxicity ( treatment-related ) Grade 3 non-hematologic toxicity infrequent Grade 4 myelosuppression: 18.2% thrombocytopenia 12.1% neutropenia 30% grade-3 anemia Grade 1 or 2 LFT frequent Bili 12.1% ALP 21.2% Transaminases 27.3% 41 Tefferi et al. NEJM 2015

Pre and post imetelstat mutation screening in 2 CR patients Patient #1 U2AF1Q157P T>G Patient #2 Baseline Granulocyte Buccal 3 month Granulocyte 4 month Granulocyte 6 month Granulocyte 10 month Granulocyte 13 month Granulocyte 16 month Granulocyte Tefferi et al. NEJM 2015