European Focus on MPN and MDS 2014 Leukemic Transformation in Myelofibrosis (MF): Molecular Pathogenesis and Clinical Implications Frank Stegelmann, MD Prague, 2 May 2014 Department of Internal Medicine III University Hospital of Ulm, Germany

Common Features of Classic MPN JAK2mut ~60% ET PV JAK2mut ~95% Hemorrhage Thrombosis Secondary AML (sAML) JAK2mut ~60% PMF SMF Primary / Secondary MF Levine et al., ASH 2006 (modified)

Background and Introduction Myelofibrosis (MF) is characterized by constitutional symptoms and poor prognosis; median survival ranges between 2-11 years Leukemic transformation occurs in ~20% of pts; it represents the most common cause of death in MF In MF and sAML, various genetic alterations were identified in recent years that involve cytokine signaling, RNA splicing, and epigenetic regulation Though many questions are still unanswered, novel pathogenic and clinical aspects of MF will be summarized in the next minutes, with the focus on their role in leukemic transformation

Genetic Evolution of MPN MPN in chronic phase Blast Phase >20% Blasts in PB/BM Tefferi and Vainchenker, J Clin Oncol 2011

Models of Leukemic Transformation 1. Accumulation of genetic events during the course of the disease cause sAML over time 2. MPN / sAML originates as a separate disease from a common founder clone 3. Separate founder clones are responsible for the development of MPN / sAML as separate diseases Rampal et al., Curr Opin Hematol 2013

Clinical Risk Factors for Leukemic Transformation DIPSS = Dynamic International Prognostic Scoring System DIPSS DIPSS-Plus 95% CI Hazard Ratio p 15% of pts Gangat et al., J Clin Oncol 2011

Clinical Risk Factors for Leukemic Transformation Frequency of Leukemic Transformation in PMF: 7% (n=52) Low risk: PLT >100/nL and No unfavorable karyotype High risk: PLT <100/nL or Unfavorable karyotype Unfavorable karyotype: Trisomy 8, -5/5q, -7/7q, 12p-, inv(3), Isochromosome(17q), 11q23 rearrangement, Complex karyotype (≥ 3 aberrations) 5-year Risk: 6% (low) vs. 18% (high) 10-year Risk: 12% (low) vs. 31% (high) Gangat et al., J Clin Oncol 2011

Frequent Mutations in PMF TET2 (10%) JAK-STAT activation Epigenetic dysregulation ASXL1 (22%) Spliceosome mutation SRSF2 (8%) MPL (5%) CBL (4%) DNMT3A (6%) EZH2 (5%) JAK2 (59%) IDH1/2 (3%) Vannucchi et al., Leukemia 2013

Impact of Mutations on Leukemic Transformation `Prognostic Detrimental Mutations´ EZH2 SRSF2 mut mut mut ASXL1 IDH1/2 mut Vannucchi et al., Leukemia 2013

Impact of Combinations of Detrimental Mutations ASXL1 +/- IDH1/2 +/- SRSF2 +/- EZH2 mutation: High Molecular Risk 69% 24% 7% Guglielmelli et al., Leukemia 2014

Novel Marker in MPN: Mutations of CALR Klampfl et al., N Engl J Med 2013 Screening for JAK2mut + CALRmut + MPLmut  Clonality in ~95% of MF

`Triple Negativity´: Adverse Outcome in PMF One Mutation (JAK2 or MPL or CALR) vs. Triple Negative (9%) Tefferi et al., Leukemia 2014

CALRunmut + ASXL1mut: Adverse Outcome in PMF Mutation Combinations of ASXL1 and CALR 17% 22% 61% Tefferi et al., Leukemia 2014

Genetic Complexity of sAML Milosevic and Kralovics, Int J Hematol 2013

Dismal Outcome by Conventional Treatment Regimen Survival Data Best Supportive Care 1.5 – 2.5 months Low-Intensity Treatment (Ara-C, 5-Aza, JAK Inhibitors etc.) 2.9 – 8.0 months Intensive Chemotherapy +/- Allogeneic Transplantation 3.9 – 47 months Mesa et al., Blood 2005 (91 cases) Tam et al., Blood 2008 (74 cases) Thepot et al., Blood 2010 (54 cases) Noor et al., Leuk Res 2011 (112 cases) Kennedy et al., Blood 2013 (75 cases)

Summary and Outlook Significant advances have been achieved in recent years regarding the molecular understanding of MF; biological and clinical impact of novel markers are under investigation Some favorable and unfavorable genotypes (e.g. ASXL1+/CALR-) have already been identified that are of prognostic relevance These data will likely be integrated into risk scores to improve prognostication of the individual risk of the patients in MF However, the enormous genetic complexity of MF and sAML is a challenge for translation of molecular data into clinical practise; patient-tailored, targeted treatment approaches will be necessary to overcome dismal outcome of sAML