Prognostication in MF: From CBC to cytogenetics to molecular markers Alessandro M. Vannucchi University of Florence, Italy

Survival is Significantly Shortened in PMF Median survival: 4.6 versus 6.5 y Cervantes F et al. JCO 2012; 24:2891-7.

Why do we Need Accurate Prognostic Scores? Long term remissions with the potential of being cured have been described only in patients undergoing allogeneic HSCT However, HSCT approach to MF has several limitations, currently: Disease related Procedure related Median age is 65 y (but changing…) Donor availability Age-related comorbidities Only one prospective trial (+1 not yet fully reported) Fragile patients (splenomegaly, cytopenias, previous therapies…) High, and highly variable, TRM Rapid evolution to AML Choice of fully ablative versus RIC Uncertainties about whom and when Poor information about long-term effects

Risk Stratification in PMF Variable IPSS DIPSS Age >65 y  Constitutional symptoms Hemoglobin <10 g/dL Leukocyte count >25x109/L Circulating blasts > 1% Platelet count <100x109/L RBC transfusion need Unfavorable karyotype +8,-7/7q-,i(17q),inv(3), -5/5q-,12p-, 11q23 rearr. Cervantes et al, Blood 2009;113:2895-901 Passamonti et al, Blood 2010; 115:1703-8 Gangat N et al, J Clin Oncol 2011; 29:392-7

International Prognostic Scoring System-IPSS Points  Median survival (mo) Low 135 Int-1 1 95 Int-2 2 48 High >3 27 Low Int-1 Int-2 High Cervantes F et al. Blood 2009;113:2895-901

Impact of Anemia on Disease Progression Disease-related anemia* has been associated with worse prognosis in all risk scoring systems (Lille, IPSS and derivatives) RBC transfusional dependency is included in the DIPSS-plus score * NOT treatment-related Passamonti F.et al, Blood 2010; 115:1703-8

Dynamic IPSS (DIPSS) Points Median survival (mo) Low Not reach. Int-1 Not reach. Int-1 1-2 170 Int-2 3-4 48 High 5-6 18 Passamonti F et al. Blood 2010;115:1703-8

Survival by Cytogenetic Category in PMF At diagnosis Beyond initial diagnosis Survival by cytogenetic category. (A) Patients evaluated at diagnosis. (B) Patients evaluated beyond initial diagnosis. CPX indicates complex (≥ 3) abnormalities. CPX= complex (>3 abnormalities) Tam C S et al. Blood 2009;113:4171-4178

"Unfavorable" Karyotype in PMF: Effect on OS - Complex karyo, - Sole or 2 abnormalities including: Trisomy 8 -7/del(7q) Del(5q) Inv(3) isochromosome 17q/17p- 12p- 11q23 abnormality Unfavorable Karyo M 2.0 yr 5-yr survival: 8% Favorable Karyo M 5.2 yr 5-yr survival: 51% Caramazza D et al. Leukemia 2011; 25:82-88

"Unfavorable" Karyotype in PMF: Effect on LFS 5-yr AML transformation rate: 7% Unfavorable Karyo 5-yr AML transformation rate: 46% Caramazza D et al. Leukemia 2011; 25:82-88

Risk Stratification in PMF Variable IPSS DIPSS DIPSS-plus Age >65 y  Constitutional symptoms Hemoglobin <10 g/dL Leukocyte count >25x109/L Circulating blasts > 1% Platelet count <100x109/L RBC transfusion need Unfavorable karyotype +8,-7/7q-,i(17q),inv(3), -5/5q-,12p-, 11q23 rearr. Cervantes et al, Blood 2009;113:2895-901 Passamonti et al, Blood 2010; 115:1703-8 Gangat N et al, J Clin Oncol 2011; 29:392-7

Dynamic IPSS-plus (DIPSS-plus) Survival Months ∆ Low risk (0 adverse points) n=66; median survival ~ 185 months ▲ Intermediate-1 risk (1 adverse point) n=174; median survival ~ 78 months ○ Intermediate-2 risk (2 or 3 adverse points) n=360; median survival ~ 35 months High risk (4 or more adverse points) n=193; median survival ~ 16 months Low risk (0 variable) M 185 mo Int-1 risk (1 variable) M 78 mo Int-2 risk (2-3 variables) M 35 mo High risk (>4 variables) M 16 mo Gangat et al. J Clin Oncol 2011;29:392-7

Adverse Impact of Monosomal Karyotype Normal karyo M 50 mo Monosomal karyo M 6 mo Sole +8 M 20 mo Complex karyo No monosomal M 24 mo Vaidya R et al. Blood 2011;117:5612-15

“Very-High Risk” Patients: >80% Mortality Very-High risk variables At 2 Years Very-High risk variables monosomal karyotype inv(3)/i(17q) or any 2 of the following: PB blasts >9% WBC >40x109/L other unfavorable karyotype Low (3%) Int-1 (11%) Int-2 (26%) High (53%) Very High (82%) N=884 pts at Mayo. 564 deaths, corresponding to 64%; 60 cases of AML l8% total population) The two strongest individual variables were MoKary and inv(3)/I(17q). The other predicted >80% mortality when present in at least two. The Very-High risk category included 52 pts vs 298 of high-risk. Of these 42 have been assigned to the HR and 10 to the Int-2 risk accoridng to DIPPSplus Leukemia risk was 31% vs 7%. Tefferi A et al. Blood 2011; 118:4595-8

Novel Prognostic Variables Somatic Mutations Germline Characteristics Cytokines Other biomarkers (FLC, hepcidin & ferritin levels)

JAK2 V617F Mutation and Prognosis in PMF WT V617F JAK2 V617F JAK2 wt 89 (31.1%) 68 (34.5%) 103.4 mo (77.5-129.3) 137.6 mo (48.5-226.8) N=483 Guglielmelli P et al, ASH2012

No Impact of JAK2V617F on Leukemia Risk WT V617F P=0.839 HR: 1.05 (95% CI, 0.7-1.7) (Competitive risk analysis) Guglielmelli P et al, ASH2012

Blast transformation: Kaplan-Meier analysis 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 et al. PlosOne 2013, In press

Mutation Complexity in PMF 382 (79.1%) of patients presented at least one somatic mutation 154 pts (32.5%) had >2 mutations 31 pts (6.4%) had >3 mutations Guglielmelli P et al, ASH2012

Mutations Associated with Reduced Overall Survival in Multivariate Analysis EZH2 ASXL1 WT WT Mut Mut P= 0.0008 P< 0.0001 SRSF2 Hazard Risk (95% CI range) P EZH2 1.91 (1.08-3.36) 0.025 ASXL1 2.21 (1.57-3.11) <0.0001 SRSF2 2.60 (1.63-41.6) WT Mut P< 0.0001 Guglielmelli P et al, ASH2012

Mutations Associated with Leukemia in Multivariate Analysis EZH2 ASXL1 WT Mut Mut WT P=.003 HR=1.98 (95%CI: 0.88-4.46) P<.0001 HR=2.5 (95%CI: 1.51-4.13) SRSF2 IDH1/2 Mut Mut WT WT P=.007 HR= 2.73 (95%CI: 1.34-5.55) P<.0001 HR= 2.66(95%CI: 1.10-6.47) * Competitive Risk Analysis Guglielmelli P et al, ASH2012

A "Molecularly High-Risk" Status Associates with Reduced Overall Survival Low Risk High Risk P<0.0001 EZH2 ASXL1 SRSF2 IDH1/2 HR= 2.29 (95%CI: 1.65-3.19) In the “molecularly high-risk” category, overall survival was 81 months (range: 61.9-99.5) compared with 148 months (range: 52.5-243.2) in the “molecularly low-risk” category (P<0.0001). Mutivariate analysis. Guglielmelli P et al, ASH2012

A "Molecularly High-Risk" Status Associates With Leukemia Transformation EZH2 ASXL1 SRSF2 IDH1/2 Low Risk P<0.0001 HR 2.96 (95%CI:1.85-4.76) In the “molecularly high-risk” category, leukemia-free survival was 129 months (range: 90-168) compared with 323 months (range: 194-452) in the “molecularly low-risk” category (P<0.0001) – Competitive risk analysis Guglielmelli P et al, ASH2012

The "Molecularly High-Risk" Status Contributes to Refined IPSS Categorization IPSS (LOW-INT1) IPSS (INT2-HIGH) P= 0.017 P= 0.002 Low Risk Low Risk High Risk High Risk Molecular Risk category Median Overal Survival, months (range) LOW 264.2 (184.7-343.6) HIGH 125.6 (77.4-173.8) Molecular Risk category Median Overal Survival, months (range) LOW 71.5 (59.2-23.7) HIGH 32.4 (26.6-38.3) Guglielmelli P et al, ASH2012

Hazard Risk (95%CI,range) The "Molecularly High-Risk" Status Predicts for Leukemia Risk within IPSS Categories IPSS (LOW-INT1) IPSS (INT2-HIGH) P= 0.001 P= 0.01 High Risk’ High Risk Low Risk Low Risk IPSS category Hazard Risk (95%CI,range) LOW-INT1 2.28 (1.20-4.36) INT2-HIGH 3.22 (1.52-6.83) Guglielmelli P et al, ASH2012

ABSTRACT #430 Prognostic Interactions Between SRSF2, ASXL1, and IDH Mutations in Primary Myelofibrosis and Determination of Added Value to Cytogenetic Risk Stratification and DIPSS-Plus Terra L Lasho, MT, (ASCP)1*, Naseema Gangat, MD1*, Christy Finke, BS1*, Rebecca R. Laborde, PhD1*, Curtis A Hanson, MD2*, Rhett P Ketterling, MD3*, Ryan A Knudson3*, Animesh Pardanani, MBBS, PhD1 and Ayalew Tefferi, MD1

The A3669G Polymorhism of Glucocorticoid Receptor Contributes to Blast Transformation in PMF The A3669G allele is a susceptibility allele for PMF (HR 1.6-1.8) The G/G allele associated with a «more-myeloproliferative» phenotype OS* BT* 0.47 per 100 pt-yr N=274 The A3669G allele in the untranslated region of human GR stabizes the mRNA of the dominant-negative Beta isoform of the GR, and contributes to development of erythrocytosis in PV where it is represented at highre frequency than in normal subjects. The variant has been associated with an excess proliferative signaling in hematopoietic cells. This study involved 499 PMF GG associated with higher WBC, larger spleen, higher CD34+ in PB (only the latter was JAK2V617F independent in multivariate) The association with BT remained significant after correction with know risk factors for AML (age, sex, WBC, IPSS, JAK2 homozygosity :HR 3.3, P=0.006) N=21 77.6mo vs 298mo; P=0.049 13.6 per 100 pt-yr 76.7mo vs 261mo; P=0.018 remained significant in multivariate *, restricted to JAK2V617Fpos pts, n=295 Barosi G et al, Blood 2012; 120:3112-7

Abnormally Increased IL-8 and IL2R Plasma Levels Are Prognostically Detrimental Int-1 (n=27) All (n=127) Survival data of (A) all 127 patients and (B) 90 treatment-naive patients with primary myelofibrosis stratified by the presence or absence of interleukin 8 (IL-8) and IL-2R levels that exceed three standard deviations above the normal mean. Survival data of (A) 27 intermediate-1–risk patients and (B) 70 intermediate-2–risk patients with primary myelofibrosis stratified by the presence or absence of interleukin 8 (IL-8) and IL-2R levels that exceed three standard deviations above the normal mean. Risk categorization is according to the Dynamic International Prognostic Scoring System plus model.5 Int-2 (n=70) Treatment naive (n=90) Tefferi A et al. JCO 2011;29:1356-1363

Plasma Free Light Chain (FLC) Levels Predict Survival in PMF FCL FCL +/- IL-8 and/or IL-2R < 3.78 mg/dL Both normal Both abnormal > 3.78 mg/dL Either abnormal Both abnormal Overall survival Kaplan-Meier survival curves of patients with primary myelofibrosis stratified by plasma free light chain (FLC) concentration above or below the receiver operating characteristic analysis–derived cutoff (3.78 mg/dL). (A) Patients with primary myelofibrosis with FLC more than 3.78 mg/dL had significantly inferior survival compared with those with an FLC level ≤ 3.78 mg/dL (log-rank P < .001). This association with inferior overall survival was sustained on multivariate analysis that considered other standard prognostic variables. (B) Patients with Dynamic International Prognostic Scoring System (DIPSS) -plus high and intermediate-2 risk stratified by DIPSS-plus and the FLC threshold of 3.78 mg/dL. (C) Composite risk model based on total FLC more than 3.78 mg/dL and interleukin-2 receptor (IL-2R) and/or IL-8 levels more than three standard deviations: both normal, either abnormal, and both abnormal. This refined risk model was predictive for survival independent of DIPSS-plus: either abnormal (hazard ratio, 2.1; P = .04) and both abnormal (hazard ratio, 3.7; P <= .001). Levels above or below the ROC cutoff (3.78 mg/dL) Pardanani A et al. JCO 2012;30:1087-94

Conclusions Prognostic scores in OMF are needed for therapeutic choices At present, they are mainly reserved for HSCT decision Most used scores are based on clinical and hematologic variables Cytogenetics has been show to add to clinical scores Molecular characterization may help refine clinical scores, be cost-effective and overcome technical limitations of conventional cytogenetics

Acknowledgments Section of Hematology, University of Florence Paola Guglielmelli Flavia Biamonte Tiziana Fanelli Ambra Spolverini Maria Chiara Susini Giada Rotunno Alessandro Pancrazzi Lisa Pieri Contributors Mario Cazzola - Pavia Gianni Barosi - Pavia Francisco Cervantes - Barcelona Andrea Reiter - Mannheim Andrew Duncombe - Southampton Katerine Zoe - Athens Nick Cross - Salisbury