Which mutations matter in myelofibrosis? Alessandro M. Vannucchi Laboratorio Congiunto MMPC Department of Experimental and Clinical Medicine University of Florence, Italy

PMF-associated Mutations: Current Status JAK2 V617F MPL W515 Unmutated CALR Klampfl T et al. NEJM 2013; 369:2379-90; Nangalia J et al. NEJM 2013;369:2391-405.

JAK2 V617F Mutation and Prognosis in PMF Overall Survival Blast Transformation P=0.198 WT V617F WT V617F P=0.839 HR: 1.05 (95% CI, 0.7-1.7) A JAK2 V617F mutated genotype did not impact significantly on prognosis compared with JAK2 wild type (but might be different if we consider CALR mutated and triple-negative patients separate) N=483 Vannucchi AM et al, Leukemia 2013;27:1861-9

JAK2 V617F Allele Burden and Blast Transformation IPSS prognostic score 4.33 (1.5-12.6) 0.007 JAK2 V617F wt or Homo vs. Hetero 2.43 (1.44-4) 0.02 Barosi G. et al. PlosOne 2013; 3:e59791

Prognostic Impact of CALR Mutations in PMF Median 8.2 years P<0.0001 MPL mut Median 4.3 years Triple negative Median 2.5 years JAK2 mut Median 4.3 years CALR mutated patients have better prognosis than other mutation categories Klampfl T et al. NEJM 2013;369:2379-90; Tefferi A et al. Leukemia. 2014 Feb 5, online.

CALR mutations :Type 1 & Type 2 Klampfl T et al. NEJM 2013; 369:2379-90; Tefferi A et al., Leukemia 2014; Feb 26 Epub

CALR Type 1 vs Type 2 Mutations in PMF Type 1 CALR mut Median 10.3 years Median 3.1 years JAK2 V617F mut Median 4.0 years P<0.0001 Tefferi A et al., Leukemia 2014; Feb 26 Epub

Mutation Landscape* in PMF 79.1% had at least one mutation 154 pts (32.5%) had > 2 mutations 31 pts (6.4%) had > 3 mutations * Partial view Vannucchi AM et al, Leukemia 2013;27(9):1861-9

High Molecular Risk Prognostic Category harboring >1 mutation in any one of ASXL1, EZH2, SRSF2, IDH1/2 Overall Survival Blast Transformation A HMR status is associated with reduced OS and increased risk of blast transformation in PMF patients independent of IPSS/DIPPS-plus Vannucchi AM et al, Leukemia 2013;27:1861-9

HMR: How many patients would be reclassified? IPSS Risk Categories ASXL1 N. (%) EZH2 SRSF2 N.(%) IDHs N (%) Of HMR patients LOW 24/162 (14.8%) 6/165 (3.6%) 7/151 (4.6%) 2/157 (1.3%) 35/166 (21.1%) INT- 1 28/142 (19.7%) 6/143 (4.2%) 6/136 (4.4%) 6/142 34 /146 (23.4%) INT- 2 23/100 (23.0%) 4/99 (4.0%) 9/97 (9.3%) 2/96 (2.1%) 31 /104 (29.8%) HIGH 27/65 (41.5%) 8/66 (12.1%) 16/63 (25.4%) 1/60 (1.7%) 39/68 (57.3%)

Patients Distribution by Number of HMR Mutated Genes (no mut, 1 mut, > 2mut) LMR LMR Test Cohort N= 537 Validation Cohort N= 292 Refers to mutations in any one of ASXL1, EZH2, SRSF2, IDH1 & 2 Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

Overall Survival by Number of Mutated Genes P < 0.0001 No HMR mutation Median 12.2 years N=370 N=127 1 mutation Median 7.0 years HR 1.8 (1.3-2.5) N=40 >2 mutations Median 2.6 years HR 3.8 (2.6-5.7) In a Cox multivariable analysis adjusted for IPSS, the HR for OS for 2 or more mutations was 2.4 (95% CI=1.6-3.6) Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

The Impact of the Number of HMR Mutated Genes on Survival of IPSS-Stratified Patients LOW + INT-1 INT-2 + HIGH No mutation Median 20.2 years No mutation Median 5.7 years 1 mutation Median 4.4 years HR 1.4 (0.9-2.1) >2 mutations Median 2.2 years HR 2.2 (1.3-4.0) >2 mutations Median 3.2 years HR 4.0 (2.3-7.1) 1 mutation Median 11.3 years HR 1.4 (0.9-2.4) Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

Leukemia Free Survival by Number of HMR Mutated Genes P < 0.0001 No mutation Median 26.7 years 1 mutation Median 11.1years HR 2.6 (1.6-4.1) >2 mutations Median 6.6years HR 6.2 (3.5-10.7) N=370 N=127 N=40 The negative impact of harboring >2 mutated genes on the time to progression to blast transformation was maintained in the lower (HR 1.8; 95% CI 0.8-4.4; P=0.061) and higher (HR 2.5; 95% CI 1.1-5.8, P=0.02) IPSS risk categories. Guglielmelli P, et al. Leukemia 2014

Prognostic Relevance of the Number of Mutated Genes Lundberg P et al. Blood 2013; Jan 29

CALR and ASXL1 Mutations-Based Prognostic Risk Stratification Mayo Clinic, n=277 Florence, n=293 Overall survival Tefferi A et al, Leukemia 2014, Feb 5,online.

CALR and ASXL1 Mutations-Based Risk Stratification According to IPPS Categories IPSS LOW + INT1 IPSS INT2 + HIGH CALR- ASXL1+ Median 6.03 years HR 18.7 (2.3-153.9) Median 3.1 years HR 3.9 (1.1-13.4) CALR- ASXL1- CALR+ ASXL1+ Median n.r. HR 5.4 (0.7-40.4) Median 3.7 years HR 3.0 (0.9-9.8) CALR+ ASXL1- P<0.001 P=0.07 CALR/ASXL1 mutations-based prognostic model was DIPSS-plus independent (p<0.0001) Tefferi A et al, Leukemia 2014, Feb 5,online.

Patients’ Mutation Profile in COMFORT-II Trial 150 pts (90.4%) presented with at least one mutation There was no difference in the frequency of individual mutations between ruxolitinib and BAT arms Overall, 46 patients in the ruxo arm (38.3%) and 20 patients (43.5%) in the BAT arm were classified as "high molecular risk" (HMR) Mutations in % JAK2 75.5 ASXL1 32.5 TET2 10.7 MPL 7.7 EZH2 7.2 CBL 4.4 SRSF2 3.0 KRAS 1.4 SH2B3 1.3 IDH1 0.7 SOCS1 SOCS2 IDH2 41.8 39.3 6.0 3 9.6 1 2 3 >4 0 No. mutations BAT, best available therapy Guglielmelli P, et al. Blood 2014

HMR LMR Spleen volume reduction of >35% from baseline 48 weeks 24 weeks Mean spleen volume % change from baseline -23.5 -30.6 -29.0 -29.9 Proportion of patients (%) Spleen Response by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II HMR status did not impact on symptomatic improvement HMR status did not increase the risk of developing anemia or thrombocytopenia under ruxolitinib treatment Guglielmelli P, et al. Blood 2014, Jan 23

Symptomatic Improvement by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II HMR LMR Proportion of patients (%) 48 weeks 24 weeks None of the individual mutations with a frequency >2% correlated with symptomatic improvement Guglielmelli P, et al. Blood 2014, Jan 23

Hematologic Toxicity by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II HMR LMR Proportion of patients (%) Anemia Thrombocythopenia Mutations of genes of the JAK/STAT signalling pathway (JAK2, MPL, SH3B2, CBL, SOCSs) did not correlate with development of anemia or thrombocytopenia at 48 wk of treatment Guglielmelli P, et al. Blood 2014, Jan 23

Survival Estimates in Patients Stratified by Treatment and Molecular Score Ruxolitinib arm§ BAT arm§ HMR LMR 0.79 0.85 0.58 0.71 In multivariate analysis of overall survival by treatment and molecular risk, the HR for treatment (ruxolitinib vs BAT) was 0.57 (95%CI= 0.30-1.08) and for LMR vs HMR the HR was 0.62 (95%CI=0.33-1.16) §Median follow up= 151 weeks; Kaplan Meier estimates at 144 weeks Guglielmelli P, et al. Blood 2014, Jan 23

Therapeutic Efficacy of JAK2 Inhibitor Fedratinib in CALR Mutated PMF Patients Passamonti F et al, NEJM 2014; 370:1168-9

Which Mutations Matter in Myelofibrosis (take home message) A mutated CALR status delineates a better outcome in PMF patients compared with JAK2/MPL mutated and “triple negative” A IPSS/DIPSS-plus independent “High-Molecular Risk” (HMR) condition is defined by the presence of any one mutated gene among EZH2, ASXL1, SRSF2 and IDH1/2 ASXL1 and CALR contribute to refine prognostic stratification The number of (HMR-) mutated genes is an additional determinant of survival Clinical response to JAK2 inhibitors is independent of HMR category (ruxolitinib) and CALR mutations (fedratinib)

Which Mutations Matter in Myelofibrosis (work to do for the future) How to integrate all these information in an unified molecular/clinical scoring system

Acknowledgments Nick Cross, Amy Jones Salisbury & Southampton, UK Tony Green, Jyoti Nangalia Cambridge, UK Robert Kralovics, Thorsten Klampfl CERMM, Wien, Austria Ayalew Tefferi, Animesh Pardanani Mayo Clinic, Rochester, US William Vainchenker, Jean Luc Villeval Inst G. Roussy, Paris, France Niccolò Bartalucci Costanza Bogani Laura Calabresi Tiziana Fanelli Rajmonda Fjerza Ilaria M. Farina Paola Guglielmelli Carmela Mannarelli Serena Martinelli Lucia Merli Annalisa Pacilli Alessandro Pancrazzi Chiara Paoli Lisa Pieri Giada Rotunno Gianni Barosi Mario Cazzola Rossella Manfredini Alessandro Rambaldi, Tiziano Barbui Daniela Cilloni Elisabetta Dejana