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

Myeloproliferative Neoplasms 2017 Ayalew Tefferi, MD Mayo Clinic, Rochester, MN

Objectives 1. 2016 WHO revision of classification and diagnostic criteria 2. Practical diagnostic algorithm 3. Survival and prognosis 4. Treatment 5. Eosinophilic disorders 6. Systemic mastocytosis

Chronic Myeloid Neoplasms Myelodysplastic Syndromes (MDS) Myelodysplastic/ Myeloproliferative Overlap (MDS/MPN) Myeloproliferative Neoplasms (MPN) 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 or PCM1-JAK2 mutation MUTATIONS MDS with single lineage dysplasia MDS with multilineage dysplasia MDS with ring sideroblasts (MDS-RS) MDS-RS with single lineage dysplasia MDS-RS with multilineage dysplasia MDS with excess blasts MDS with isolated del(5q) MDS unclassifiable Provisional: Refractory cytopenia of childhood Tefferi and Pardanani. JAMA Oncology 2015 (modified) Chronic myelomonocytic leukemia (CMML) -TET2 40-60% -SRSF2 30-50% -ASXL1 40% Juvenile myelomonocytic leukemia (JMML) -genetic abnormality 90% Dyserythropoiesis Dysgranulopoiesis - RAS/MAPK pathway mutation 60% (PTPN11, KRAS, and NRAS) -CBL mutation 15% -Germline NF1 mutations 15% MDS/MPN-RS with thrombocytosis -SF3B1 80-90% -JAK2V617F 50% Atypical chronic myeloid leukemia (acml) -SETBP1 30% MDS/MPN-U Monocytosis CML -BCR-ABL1 100% PV -JAK2 99% ET -JAK2/CALR/MPL 85% JAK2 60% CALR 20% MPL 5% Triple-negative 15% PMF Same as ET CNL -CSF3R 80-100% CEL MPN-U PDGFRA rearranged -FIP1L1-PDGFRA 100% PDGFRB rearrange -PDGFRB mutation 100% FGFR1 rearranged -FGFR1 mutation 100% PCM1-JAK2 rearranged -PCM1-JAK2 100%

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

Diagnostic approach in routine clinical practice

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.

Essential thrombocythemia 1. Karyotype (i.e. cytogenetics) 7% abnormal at diagnosis (Trisomy 9 most frequent) Limited prognostic value 2. Driver mutational status JAK2 60% CALR 22% MPL 3% Triple negative 15% 3. Presence or absence of other mutations Prevalence of mutations/variants other than JAK2/CALR/MPL = 53% Driver mutational status did not affect prevalence Most frequent were ASXL1 andtet2 41%, 8% and 4% harbored 1, 2 or 3 such mutations 6 genes were identified as being affected by adverse mutations/variants SF3B1, SH2B3, EZH2, TP53, U2AF1, IDH2 (15% affected) Eur J Haematol. 2009;83:17 Blood 2014 124:2507 Blood Advances 2016;1:21

Am J Hematol. 2016;91:503 Overall survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Am J Hematol. 2016;91:503 Myelofibrosis-free survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Am J Hematol. 2016;91:503 Thrombosis-free survival in 495 patients with essential thrombocythemia stratified by driver mutational status

Prognostic relevance of ET mutations/variants other than JAK2/CALR/MPL Prevalence = 53% Driver mutational status did not affect prevalence Most frequent were ASXL1 andtet2 41%, 8% and 4% harbored 1, 2 or 3 mutations 6 genes were identified as being affected by adverse mutations/variants SF3B1, SH2B3, EZH2, TP53, U2AF1, IDH2 (15% affected) Mayo patients ET Italian patients Blood Advances 2016;1:21

Polycythemia Vera 1. Karyotype (i.e. cytogenetics) 19% abnormal at diagnosis (20% of abnormal karyotype were unfavorable) Most frequent were +9, 20q-, -Y and +8 Prognostically relevant for overall, leukemia-free and myelofibrosis-free survival 2. Driver mutational status JAK2 99% 3. Presence or absence of other mutations Prevalence of mutations/variants other than JAK2/CALR/MPL = 53% Most frequent were ASXL1 andtet2 30%, 20% and 3% harbored 1, 2 or 3 such mutations 3 genes were identified as being affected by adverse mutations/variants ASXL1, SRSF2, IDH2 ASH 2016 Blood 2014 124:2507 Blood Advances 2016;1:21

Prognostic relevance of PV mutations/variants other than JAK2/CALR/MPL Prevalence = 53% Most frequent were ASXL1 andtet2 30%, 20% and 3% harbored 1, 2 or 3 such mutations 3 genes were identified as being affected by adverse mutations/variants ASXL1, SRSF2, IDH2 PV Blood Advances 2016;1:21

Primary myelofibrosis Br J Haematol. 2015;169:71 Blood. 2011;118:4595 Blood 2014 124:2507 Blood Advances 2016;1:105 1. Karyotype (i.e. cytogenetics) 43% abnormal at diagnosis (unfavorable 15%) Most frequent were 20q-, 13q-, +8. +9 and 1q+ Prognostically very relevant 2. Driver mutational status JAK2 60% CALR 24% MPL 6% Triple negative 10% 3. Presence or absence of other mutations Prevalence of mutations/variants other than JAK2/CALR/MPL = 81% Driver mutational status did not affect prevalence Most frequent were ASXL1 36%, TET2 18%, SRSF2 18%, U2AF1 16% 35%, 26%, 10% and 9% harbored 1, 2, 3 or 4 such mutations 7 genes were identified as being affected by adverse mutations/variants ASXL1, SRSF2, CBL, KIT, RUNX1, SH2B3 and CEBPA (56% affected)

Survival data in 903 patients with primary myelofibrosis stratified by karyotype 1.8.6 Confidence Intervals for DX-last f/u new Censor Variable: Censor survival tw o Model: Proportional Hazards Exp(Coef) 95% Low er 95% Upper BLOOD Cyto risk categories: High 4.355 3.311 5.727 BLOOD Cyto risk categories: Intermediat... 1.932 1.578 2.366 Confidence BLOOD Cyto Intervals risk categories: for DX-last Intermediat... f/u new 1.297 1.031 1.630 Censor Variable: Censor survival tw o Model: Proportional Hazards vs. low risk Row exclusion: 2015 PMF master database.svd vs. intermediate-1 risk Exp(Coef) 95% Low er 95% Upper BLOOD Cyto risk categories: High 3.348 2.406 4.659 BLOOD Cyto risk categories: Intermediat... 1.503 1.149 1.968 vs. intermediate-2 risk Survival BLOOD Cyto risk categories: High Exp(Coef) 95% Low er 95% Upper 2.146 1.581 2.914.4 Low risk Normal Sole 13q- Sole +9 Sole loss of Y chromosome N=548.2 High risk Monosomal Inv(3)/i(17q) -7/7q- 11q- 12p- N=67 0 Tefferi et al. ASH 2014 0 5 10 15 20 25 30 Years Int-2 risk Non-monosomal complex Two or more non-high risk abnormalities Sole +8 or other autosomal trisomies Sole 5q- Other sole abnormalities not included in low or int-1 risk categories N=65 Int-1 risk Sole 20q- Sole 1q duplications Sole translocation Sole extra sex chromosome N=124

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

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 ASXL1, SRSF2, CBL, KIT, RUNX1, SH2B3 and CEBPA (56% affected).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) 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Years Blood Advances 2016;1:105

Treatment in essential thrombocythemia and polycythemia vera

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 2017

Additional management issues in PV and ET 1. What if you can t use hydroxyurea i. Interferon alpha (Qunitas-Cardama et al. Blood 2013; CHR 76% in PV, 77% in ET; CMR 18% in PV and 17% in ET) i. Busulfan (Alvarez-Larran et al. Ann Hematol 2014; CHR in HU-refractory PV or ET was 83%; Kuriakose et al. Haematologica 2013; CMR in 2 (33%) of 6 PV patients) i. Anagrelide (Not recommended because of its association with disease progression into myelofibrosis and increased thrombosis risk in ET phase-3 study; Harrison et al, NEJM 2005) i. Ruxolitinib (Vannucchi et al. NEJM 2015; randomized study in HU-refractory PV with ruxo vs standard therapy; 59% of patients on standard therapy received HU??? 21% of ruxo treated patients achieved both hematocrit control and 35% reduction in spleen volume; 60% hematocrit control; 49% symptoms control; CHR 24%; No CMR reported) 2. What about treatment during pregnancy? i. Low-risk ASA only ii. High-risk IFN alpha 3. What about treatment of pruritus?...paroxetine, IFN-alpha, UVB, ruxolitinib

Treatment in myelofibrosis

Survival data of 793 patients with primary myelofibrosis evaluated at time of their first Mayo Clinic referral and stratified by their Dynamic International Prognostic Scoring System (DIPSS-plus) that employs eight variables: Age >65 yrs; Hgb <10 g/dl; RBC transfusion-dependent; platelets <100 x 10(9)/L; WBC > 25 x 10(9)/L; 1% circulating blasts; Constitutional symptoms; karyotype prognostic scores (DIPSS-plus). 0 risk factors 1 risk factor 2 or 3 risk factors 4 or more risk factors Median 15.4 years 6.5 years 2.9 years 1.3 years Gangat N et al. JCO 2011;29:392-397 2011 by American Society of Clinical Oncology

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

Leukemia 2014

COMFORT-2 Ruxolitinib vs best available therapy (BAT) long-term follow-up Median f/u 4.3 years 27% ruxo-randomized patients completed 5-year treatment P=0.06 AML 5.5% with ruxo and 6.8% with BAT Skin cancer 17% with ruxo and 3% with BAT Harrison et al. Leukemia (2016) 30, 1701

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

Diagnostic Algorithm for Primary Eosinophilia 1st step Peripheral blood screening for FIP1L1-PDGFRA using FISH or RT-PCR Mutation present FIP1L1-PDGFRA associated clonal eosinophilia PDGFRB rearranged clonal eosinophilia 2nd step Bone marrow biopsy with cytogenetics 8p11 translocation present FGFR1 rearranged clonal eosinophilia CEL-NOS 3rd step Peripheral blood lymphocyte phenotyping and TCR gene rearrangement studies Abnormal or clonal lymphocytes present lymphocytic variant hypereosinophilia Tefferi et al. Mayo Clin Proc 85:158, 2010 All the above negative Idiopathic eosinophilia including HES

98 Mayo Clinic patients with WHO-defined HES/IH (Pardanani et al. Leukemia 2016;30:1924) Males (%) 54 (55%) Age (years), median (range) 53 (19 83) AEC (x10 9 /l), median (range) 3 (1 59) AEC 3 10 9 /l (%) 52 (53%) AEC 5 10 9 /l (%) 35 (36%) No. (%) of organs involved (including skin) None 15 (16%) One 58 (59%) Two 20 (20%) Three or more 5 (5%) No. (%) of organs involved (excluding skin) None 45 (46%) One 40 (41%) Two 10 (10%) Three or more 3 (3%) Cardiac involvement (%) 8 (8%) Palpable hepatosplenomegaly (%) 4 (4%) Pulmonary involvement (%) 27 (28%) Gastrointestinal involvement (%) 16 (16%) Hemoglobin (g/dl), median (range) 13.3 (7.9 16.3) Hemoglobin <LNL (%) 39 (40%) Hemoglobin <10 g/ dl (%) 11 (11%) Leukocyte count 10 9 / l, median (range) 9.4 (3.6 87) Leukocyte count >15 10 9 / l (%) 18 (18%) Platelet count 10 9 /l, median (range) 276 (79 631) Platelet count <150 10 9 /l (%) 4 (4%) AST >UNL (%) 5 (6%) (n=87) Total bilirubin >UNL (%) 4 (7%) (n=58) LDH >UNL (%) 22 (36%) (n=61) Tryptase >UNL (%) 14 (24%) (n=59) IL-5 >UNL (%) 15 (31%) (n=49) NGS revealed 11% harbored pathogenic mutation; TET2=3, ASXL1 =2, KIT=2, and IDH2, JAK2, SF3B1 and TP53=1 each. 15% harbored a variant of unknown significance; TET2=8, ASXL1=2, SETBP1=2, and CALR, CEBPA and CSF3R=1 each. NO DIFFERENCE IN MUTATED VS NON-MUTATED IN PHENOTYPE MUTATED PATIENTS HAD INFERIOR SURVIVAL IN UNIVARIATE ANALYSIS Risk factors for survival: Advanced age (2 points) Hgb <10 g/dl (one point) Cardiac involvement (one point) Hepatosplenomegaly (3 points) Low risk 0-1 points High risk 2 or more points

Approach to HES or HES-like clonal eosinophilia Peripheral blood mutation screen for PDGFRA and PDGFRB mutations Positive Negative CEL or other myeloid malignancy T clone present HES Asymptomatic may not need therapy Imatinib 100 mg/day CSA MTX Cytoxan If treatment necessary PDGFRA-rearranged MPN PDGFRB-rearranged MPN Chronic therapy Prednisone for acute therapy Low-dose prednisone Hydroxyurea Interferon alpha Imatinib Mepolizumab Alemtuzumab

When should you suspect mastocytosis? Urticaria pigmentosa-like lesions Mast cell mediator symptoms Anaphylactoid symptoms/dizziness/headache Diarrhea Flushing/urticaria Osteopenia/unexplained fractures

Practical classification of mast cell disease 1 Cutaneous mastocytosis (skin-only disease) Both can manifest mast cell mediator release symptoms Hartmann. & Henz, Br J Derm 2001;144:682 2 Systemic mastocytosis (SM) i Indolent SM ii Aggressive SM (cytopenia, bone disease, organomegaly, etc.) Travis et al. Medicine 1988;67:345 Valent et al. Leukemia Research 2001;25:603 1. SM without associated 2 nd myeloid neoplasm 2. SM with associated 2 nd myeloid neoplasm 3. Mast cell leukemia

Survival for 342 systemic mastocytosis patients classified by disease type compared with the expected age and gender matched US Population s survival Expected US Survival compared to WHO classification Survival 0 20 40 60 80 100 ISM, (n=159) ASM, (n=41) AHD, (n=138) MCL, (n=4) Expected US Survival 0 10 20 30 Lim et al. Blood 2009;113:5727. Years from Dx 01Oct08

Mutation-augmented prognostic scoring system (MAPSS) in 94 patients with advanced mastocytosis ISM (n=44) KIT 73% TET2 7% No other mutations ASM (n=25) KIT 84% TET2 20% ASXL1 16% AHN (n=80) KIT 75% TET2 45% ASXL1 26% CBL 19% JAK2 DNMT3A U2AF1 RUNX1 SF3B1 Others Two or three risk factors or low platelet count Four or more risk factors One risk factor other than platelet count Risk factors: Platelet count <150; albumin <3.5; age >60; ASXL1/CBL mutated; Hgb <10 Pardanani et al. AJH 2016;91:888 Pardanani et al. BJH 2016;175:531

Treatment for Systemic Mastocytosis Indolent Aggressive Associated with MDS or CMML Mast cell leukemia H1 and H2 blockers Cromolyn Phototherapy Topical steroids Cladribine or IFN-α or Experimental therapy Cladribine or AML-like therapy or Experimental therapy followed by If this fails, OK to try IFN-α or cladribine Treat as MDS or CMML Transplant? J Clin Oncol. 2014 ;32:3264

Response and progression-free survival in 116 patients with advanced mastocytosis treated with oral midostaurin (PKC-412) 100 mg twice-daily Gotlib J et al. N Engl J Med 2016;374:2530

Allogeneic hematopoietic stem-cell transplantation (allohct or HCT) outcomes in 57 patients with advanced systemic mastocytosis (SM): 38 SM-AHNMD; 12 MCL and 7 aggressive SM. Celalettin Ustun et al. JCO 2014;32:3264