1. Good Samaritan Medical Center, West Palm Beach, FL Guest Speaker Michael Savona, MD Associate Professor of Medicine Director, Hematology Early Therapeutics Program Vanderbilt-Ingram Cancer Center, Nashville, TN

2. Vardiman JW. Blood. 2009;114(5):937-51.

3. PREVALENCE OF MPNs PER 100,000 INDIVIDUALS Mehta J. Leuk Lymphoma. 2014;55(3):595-600 Age-Adjusted P Prevalence Per 100,000 Individuals Primary MF + Post PV MF + Post ET MF PV ET

4. 2013 Sep;27(9):1874-81 WHAT DOES A PV PATIENT LOOK LIKE? PRESENTING SIGNS AND SYMPTOMS Geyer H. Hematology Am Soc Hematol Educ Program. 2014;2014(1):277-286.

5. 2013 Sep;27(9):1874-81 Tefferi A. Leukemia. 2013;27(9):1874-81. WHAT DOES A PV PATIENT LOOK LIKE? CLINICAL FINDINGS

6. 2013 Sep;27(9):1874-81 OVERALL SURVIVAL AFTER DIAGNOSIS Tefferi A. Leukemia. 2013;27(9):1874-81. Median Survival = 18.9 years Observed Expected 100 0 80 60 40 20 P= 0.104 0 20 10 5 15 Years Number at Risk 1545 84 491 973 229

7. PART 1: ESTABLISHING A DIAGNOSIS

8. OUR PATIENT: CALVIN 59-year-old with left hand clumsiness and difficulty speaking Review of symptoms notable for fatigue Family notes recent reddish complexion Medical history: diet-controlled hypercholesterolemia No medications or drug allergies No hematological problems in the family Non-smoker, casual ETOH

9. CASE PRESENTATION: EXAM AND INITIAL DATA BP 176/91; oxygen saturation 95% Plethoric, ruddy appearance Regular heart rate and rhythm (S1S2, no M/R/G) Palpable spleen tip Neurological examination nonfocal

10. FURTHER WORKUP Complete blood count: –WBC 14.9 x 10 9 /L –Hgb 18.8 g/dl –Platelets 485 x 10 9 /L JAK2 V617F mutated Epo level: 1.9 mU/mL

11. QUESTION A.Oxygen saturation: 95% B.Platelet count: 485 x 10 9 L C.Epo level: 1.9 mU/mL D.WBC: 14.9 x 10 9 /L Which of Calvin’s characteristics are included within the WHO’s criteria for diagnosis of PV?

12. HISTORICAL PERSPECTIVE 1892: Vaquez disease 40-year-old with “cyanosis,” vertigo, dizziness, palpitations, and erythrocytosis; post-mortem exam showed marked hepatosplenomegaly 1903: A new clinical entity Osler publishes case series: “Chronic cyanosis, polycythemia and an enlarged spleen” Osler W. Br Med J. 1904;1(2246):121-122.

13. DIFFERENTIAL DIAGNOSIS: EXCLUDE OTHER CAUSES OF “POLYCYTHEMIA” Congenital –EPO-receptor mutation Acquired –Polycythemia vera Primary Polycythemia Secondary Polycythemia Congenital –VHL mutation, high affinity Hb, methemoglobinemia Acquired –Hypoxia –Inappropriate EPO production *Tumors “Relative” –Dehydration, diuretic use, etc. *Tumors include cerebellar hemangioblastoma, uterine leiomyoma, pheochromocytoma, renal cell carcinoma, hepatocellular cancer, meningioma, parathyroid adenoma

14. WILLIAM DAMESHEK, 1951: SPECULATIONS ON THE MYELOPROLIFERATIVE SYNDROMES Recognizes overlapping features shared by PV and the other “MPD” Leukocytosis Thrombocytosis Splenomegaly Marrow Fibrosis Dameshek W. Blood. 1951;6(4):372-5. “…Manifestations of proliferative activity of the bone marrow cells, perhaps due to a hitherto undiscovered stimulus”

15. DAMESHEK’S “MYELOSTIMULATORY” FACTOR Stein B. JAMA. 2010;303(24):2513-8. Wild-type JAK2 JAK2 mutated PV JAK2 V617F mutations present in ~95% of PV cases JAK2 Exon 12 mutations present in ~2-3% of PV cases *Typically isolated erythrocytosis Similar rate of MF, AML, thrombosis *Passamonti F. Blood. 2011; 117(10):2813-6.

16. JAK2 V617F ALLELE BURDEN CORRELATES Gender? Age? Disease Duration? JAK2 V617F allele burden Disease complications and evolution Pruritus Splenomegaly Myelofibrosis Thrombosis Clinical phenotype ThrombocytosisErythrocytosisLeukocytosis Adapted from Passamonti F. Haematologica. 2009; 94(1):7-10.

17. WHO CRITERIA 2008: POLYCYTHEMIA VERA Major Absolute erythrocytosis (>18.5 g/dL in men; >16.5 g/dL in women) JAK2 V617F mutation or similar (JAK2 exon 12) Minor Subnormal EPO level (<4 mU/mL) Bone marrow trilineage proliferation Endogenous erythroid colony growth Thiele J. Curr Hematol Malig Rep. 2009;4(1):33-40. *2 major and 1 minor, or 1 major and 2 minor required for diagnosis

18. “*AN ALTERNATIVE PROPOSAL” Hemoglobin/hematocrit is not a perfect surrogate for increase in red cell mass WHO criteria identified absolute erythrocytosis in only 35% and 63% of male and female PV patients Prospective, 5-year study showed 28/30 diagnosed with PV, based on ↑RCM, JAK2, and ≥1 minor criteria, but 8 (27%) and 18 (60%) did not meet Hct or Hgb criteria Red cell mass and plasma volume studies upgrade ~46% of JAK2-positive ET patients to PV Epo levels are neither sensitive nor specific, as they can be normal in PV and suppressed in ET EECF is neither widely available nor standardized for use *Spivak JL. Blood. 2008;112(2):231-9; Johansson PL. Br J Haematol. 2005;129(5):701-5; Silver J. Blood. 2013;122(11):1881-6; Alvarez-Larran A. Haematologica. 2012;97(11):1704-7; Cassinat B. Leukemia. 2008;22(2):452-3..

19. “MASKED” PV (mPV) One of Osler’s patients, Oxford 1916 397 patients with PV marrow morphology “Masked” (n=140) vs. overt PV (n=257) mPV typically male, with history of arterial thrombosis, and ↑platelets Similar vascular risk, but ↑rate of MF/AML, and ↓survival vs. overt PV mPV distinguished from ET by Hgb >16/16.5, and Hct 48/49% in M/F Plasma volume increase can mask PV, typically in cases of abdominal venous thrombosis with splenomegaly *Masked PV=Hgb values below WHO threshold Lamy T. Am J Med. 1997; 102(1):14-20; Spivak J. N Eng J Med. 2006; 355(7):737; Barbui T. Am J Hematol. 2014;89(1):52-4.

20. PROPOSED REVISIONS TO THE DIAGNOSTIC CRITERIA Tefferi A. Leukemia. 2014;28(7):1407-13. Major criteria: Hgb >16.5 g/dl (Hct >49%) in men; >16 g/dl (>48%) in women BM trilineage myeloproliferation with pleomorphic megakaryocytes Presence of JAK2 mutation Minor criteria: Subnormal Epo level Diagnosis would require all 3 major criteria or 2 major and 1 minor criteria

21. KEY TAKEAWAYS WHO criteria work well for many PV patients, but the Hgb threshold may be inadequate for some Be mindful of “masked” disease –JAK2 + ET, but generous Hct –JAK2 + with abdominal venous thrombosis –Pancytosis and/or splenomegaly but Hgb below threshold –Bone marrow morphology and RCM testing may better recognize PV in these settings Epo level imperfect, EEC testing not routinely available Updates have been proposed to WHO diagnostic criteria

22. PART 2: FRONTLINE TREATMENT OF PV

23. CASE REVIEW: CALVIN 59-year-old presents to the ER with clumsiness of the left hand and transient difficulties with his speech Fatigue is noted, along with ruddy complexion BP: 176/91; oxygen saturation 95% WBC: 14.9 x 10 9 /L Hgb: 18.8 g/dl Platelets: 485 x 10 9 /L JAK2 V617F positive Epo level: 1.9 mU/mL

24. QUESTION A.Phlebotomy (P) alone B.P + Aspirin (A) alone C.P + A + hydroxyurea D.P + A + ruxolitinib E.P + A + interferon alfa Which of the following would not be considered an appropriate first-line regimen for Calvin?

25. TREATMENT GOALS FOR NEWLY-DIAGNOSED PV Reduce cardiovascular complications Improve disease-related symptoms Prevent transformation to myelofibrosis (15- 20%) and/or leukemia (3-5%) Improve overall survival (median 14 years) ?? Reduce/eliminate JAK2 allele burden ?? Reduce/eliminate other clones (eg, TET2) Tefferi A. Leukemia. 2013;27(9):1874-1881.

26. STANDARD FIRST-LINE TREATMENT FOR NEWLY-DIAGNOSED PV Phlebotomy Aspirin Hydroxyurea Interferon? Lifestyle modifications –Counseling about risk of thrombosis and hemorrhage –Smoking cessation, weight control, exercise –Management of HTN/DM/HL in susceptible patients –Avoidance of oral contraceptives –DVT prophylaxis (exercises, activity, enoxaparin sodium for long flights [controversial] or sedentary periods)

27. LOW-DOSE ASPIRIN IN PV: ECLAP STUDY Hypothesis: There is increased synthesis of platelet thromboxane in PV that can be suppressed by aspirin 100 mg daily 528 patients: 253 aspirin 100 mg daily, 265 placebo Inclusion Criteria: o No clear indication for, or contraindication to, aspirin o No significant comorbidities Primary endpoints: o Cumulative rates of nonfatal MI, stroke, or death from CV disease +/- PE or major venous thrombosis Landolfi R. N Engl J Med. 2004;350(2):114-124.

28. ECLAP RESULTS Probability of survival free of MI, stroke, death from CV disease, PE, or DVT Landolfi R. N Engl J Med. 2004;350(2):114-124. RR 0.40; [0.18,0.91]; P=0.03 Note: there were more smokers in the placebo group; HCT median was 46 during follow-up, with 25% of pts with HCT >48

29. CONCLUSIONS FROM ECLAP Aspirin significantly reduced primary endpoint by 60% –Significant decrease in combined endpoint of nonfatal MI, nonfatal stroke, PE, deep venous thrombosis, or death from any cause –Significant decrease in rates of thrombosis –No significant difference in rates of major adverse events –No significant reduction in overall and cardiovascular mortality –No significant increase in major bleeding events Landolfi R. N Engl J Med. 2004;350(2):114-124.

30. CARDIOVASCULAR EVENTS AND INTENSITY OF PV TREATMENT Cytoreductive Therapy in Polycythemia Vera –(CYTO-PV) study 365 adults w/JAK2+ PV, all treated with: –Low-dose aspirin –Phlebotomy (250-500 cc QOD until target HCT) and/or –Hydroxyurea (0.5-1 g daily until plt <400K) Hydroxyurea (HU) recommended for: –Patients at high risk for thrombosis (age >65 years, previous thrombosis) or –Progressive thrombocytosis or splenomegaly Marchioli R. N Engl J Med. 2013;368(1):22-33.

31. CYTO-PV STUDY TARGETS Two study arms o More intensive Tx (target hct = <45%) o Less intensive Tx (target hct = 45 to 50%) Primary end point: Time until death from CV complications or major thrombotic events Planned target was 1,000 patients, but slow enrollment and competing trials prevented completion Marchioli R. N Engl J Med. 2013;368(1):22-33.

32. CV EVENTS AND INTENSITY OF TREATMENT Marchioli R. N Engl J Med. 2013;368(1):22-33. 1.1 per 100 person-years in the low-hematocrit group 4.4 per 100 person-years in the high-hematocrit group

33. WHAT ABOUT INTERFERON (IFN) IN NEWLY-DIAGNOSED PV? IFN controls counts and can produce hematologic remission Progressively decreases JAK2 mutated clones and can induce molecular remission No evidence of leukemogenicity Pegylated formulation generally well-tolerated, but still associated with important side effects (flu-like symptoms, depression, autoimmune manifestations, ocular toxicity, etc.) Kiladjian JJ. Leukemia. 2008;22(11):1990-8; Kiladjian JJ. Blood. 2008;112(8):3065-72.

34. TREATMENT OF PV WITH PIPOBROMAN Kiladjian JJ. J Clin Oncol. 2011;29(29):3907-13. Kiladjian et al (2011) 285 patients (all <65 years old) Two study arms: o HU 25 mg/kg/d, followed by maintenance (10-15 mg/kg/d) o Pipobroman 1.25 mg/kg/d, followed by maintenance (0.4-0.7 mg/kg/d)

35. OVERALL SURVIVAL DATA Kiladjian JJ. J Clin Oncol. 2011;29(29):3907-13.

36. TREATMENT-RELATED RISK FACTORS FOR TRANSFORMATION TO AML AND MDS Bjorkholm M. J Clin Oncol. 2011;29(17):2410-5. TreatmentOdds Ratio95% CI None1.0Reference Radioactive phosphorus (P 32 )1.50.8 to 2.8 Alkylating agent only0.90.4 to 2.1 HU only1.20.6 to 2.4 Mixed treatment (2 or 3)2.91.4 to 5.9

37. RISK OF TRANSFORMATION TO AML/MDS RELATIVE TO CUMULATIVE DOSE Bjorkholm M. J Clin Oncol. 2011;29(17):2410-5. TreatmentOdds Ratio95% CI HU, g 1-499 500-999 ≥1,000 1.5 1.4 1.3 0.6 to 2.4 0.6 to 3.4 0.5 to 3.3 Radioactive phosphorus (P 32 ), MBq 1-499 500-999 ≥1,000 1.5 1.1 4.6 0.6 to 3.3 0.5 to 2.2 2.1 to 9.,8 Alkylating agents, g 1-499 500-999 ≥1,000 1.1 1.7 3.4 0.5 to 2.3 0.6 to 5.0 1.1 to 10.6

38. KEY TAKEAWAYS Aspirin, phlebotomy and hydroxyurea remain the mainstays of therapy for PV Reduction of HCT to at least <45% is associated with significant clinical benefit Hydroxyurea is not associated with a significant independent risk for leukemia, but the issue is still of concern with long-term use and/or higher doses Pegylated interferon can also be considered for newly diagnosed PV The role of ruxolitinib in newly diagnosed PV is under investigation

39. PART 3: INADEQUATE RESPONSE TO HYDROXYUREA

40. OUR PATIENT: JOSEPH 71-year-old diagnosed with PV 3 years ago on the basis of erythrocytosis, thrombocytosis, and JAK2-V617F mutation in the setting of an unprovoked deep vein thrombosis Managed successful for 3 years with low-dose aspirin, phlebotomy (goal HCT≤45%), and hydroxyurea (500 g twice daily) Presents today with a non healing leg ulcer, fatigue, pruritus, and occasional night sweats Patient still requires occasional phlebotomy

41. HISTORY Past medical history –Squamous Cell Carcinoma (SCC) –Multiple actinic keratosis Surgical history – Mohs for SCC (2013) – Right inguinal hernia repair (2010) Medications – Aspirin 81 mg/day – Hydroxyurea 500 mg 2x/day – Atorvastatin 20 mg/day Social history –Former Smoker (quit 2008) –Retired attorney –Divorced 1986 –Remarried 2001 Family history –Mother had essential thrombocythemia –Father with myocardial infarction at age 68

42. PHYSICAL EXAM HEENT: Within normal limits CV/Lungs: Within normal limits Spleen: 3 cm below left costal margin Skin: Scar on forehead from Mohs surgery, numerous actinic keratoses

43. LABS Complete Blood Count –Hemoglobin: 16.7 g/dL –Hematocrit: 51% –Leukocytes: 16.4 x 10 9 /L –Platelets: 640 x 10 9 /L Peripheral smear –Leukocytosis, thrombocytosis, no nucleated red blood cells, no blasts Ferritin decreased, TIBC increased Chemistries: Within normal limits

44. QUESTION A.Complete response B.Partial remission C.Intolerant of hydroxyurea D.Resistant to hydroxyurea E.Evidence of progression to post-PV MF What would be the most accurate description of Joseph’s clinical status with respect to PV?

45. Complete Remission Complete Remission Partial Remission Partial Remission Molecular Response Resolution of PV signs ≥10 pt.  MPN TSS Near normal counts No progressive disease or vascular event Bone marrow remission & ≤Gr 1 reticulin fibrosis Resolution of PV signs ≥10 pt.  MPN TSS Near normal counts No progressive disease or vascular event Bone marrow remission & ≤Gr 1 reticulin fibrosis Resolution of PV signs ≥10 pt.  MPN TSS Near normal counts No progressive disease or vascular event Resolution of PV signs ≥10 pt.  MPN TSS Near normal counts No progressive disease or vascular event Peripheral blood granulocytes CR – Eradicated mutation PR - ≥50%  allele burden, ≥20% allele burden at baseline Peripheral blood granulocytes CR – Eradicated mutation PR - ≥50%  allele burden, ≥20% allele burden at baseline Barosi G. Blood. 2013:121(23):4778-4781. RESPONSE CRITERIA FOR PV (≥12 WEEKS) Progressive Disease = Post- PV MF, MDS, or AML

46. Assessing MPN Burden WHO Diagnosis Does Not Tell Whole Story MPN Symptoms MF>PV>ET Multifactorial Some ET/PV > MF Cytoreductive rx frequently not effective MPN Symptoms MF>PV>ET Multifactorial Some ET/PV > MF Cytoreductive rx frequently not effective Vascular Events PV/ET > MF Counts matter Can be unrecognized Vascular Events PV/ET > MF Counts matter Can be unrecognized Progression PV/ET to MF PV/ET to AML MF to AML ? 2 nd MDS Progression PV/ET to MF PV/ET to AML MF to AML ? 2 nd MDS Cytopenias MF> ET/PV Anemia MF 75% TX Dep 25% TPN 30% Cytopenias MF> ET/PV Anemia MF 75% TX Dep 25% TPN 30% Splenomegaly MF> ET/PV Pain not always a function of size Splenomegaly MF> ET/PV Pain not always a function of size Baseline Health AGE/ Medicines Comorbidities Baseline Health AGE/ Medicines Comorbidities

47. PRIMARY COMMERCIALLY AVAILABLE MPN DRUGS ET PV MF Hydroxyurea Interferon/ Peg-INF Interferon/ Peg-INF Anagrelide Ruxolitinib +++ ++ +++ + + + + + + + + ++ +++ ++ + + EXPERIMENTAL – OFF LABEL Ruxolitinib Approved in PV December 4, 2014 “In patients having inadequate response to or intolerant of hydroxyurea” Ruxolitinib Approved in PV December 4, 2014 “In patients having inadequate response to or intolerant of hydroxyurea” + to +++ Strength of Evidence

48. WHAT DOES INTOLERANCE/RESISTANCE TO HYDROXYUREA IN PV MEAN? 1.Need for phlebotomy (HCT<45%) 2.PLT >400 x 10 9 /L and WBC >10 x 10 9 /L 3.Failure to reduce spleen by > 50% 4.No reduction of spleen symptoms 1.Cytopenias (any) –ANC <1.0 x 10 9 /L –Hemoglobin <100 g/l –Platelets <100 x 10 9 /L 2.Leg ulcers 3.GI toxicity 4.Fever 5.Mucocutaneous manifestations 6.Skin cancers RESISTANCE INTOLERANCE Please Note 1.After > 3 Months 2.At maximum tolerated dose or 2 g/day Please Note 1.After > 3 Months 2.At maximum tolerated dose or 2 g/day Please Note At lowest dose to achieve either a PR or CR Please Note At lowest dose to achieve either a PR or CR Barosi G et. al. Br J Haematol. 2009;148:961-3.

49. CASE CONTINUATION Joseph undergoes a repeat bone marrow aspirate and biopsy. Results: –1+ out of 3+ fibrosis, normal cytogenetics. –Diagnosis: –PV patient intolerant to hydroxyurea Ruxolitinib 10 mg twice daily is initiated

50. QUESTION A.Control of hematocrit B.Reduction in splenomegaly, if present C.Reduction in pruritus D.Prevention of vascular events E.All of the above Which of the following is among the goals of ruxolitinib therapy in PV?

51. SIX WEEKS LATER Joseph reports marked decrease in fatigue, pruritus, and night sweats Spleen no longer palpable Leg ulcer is healing Hemoglobin 14.0 g/dL (without phlebotomy), leukocytes 11 x 10 9 /L, and platelets 390 x 10 9 /L

52. RUXOLITINIB (SINGLE AGENT) IN PV BAT Week 32 (Primary analysis) Week 80 n = 110 n = 112 Crossover to ruxolitinib Resistance to or intolerance of HU (modified ELN criteria) Phlebotomy requirement Splenomegaly Pre-randomization (Day -28 to Day -1) Hct 40%-45% Randomized (1:1) Extended Treatment Phase Ruxolitinib 10 mg BID Week 208 Compared to BAT, results showed that ruxolitinib led to: 1.Superior control of hematocrit 2.Superior reduction in splenomegaly 3.Superior reduction in PV-related symptoms 4.Trend for less thrombotic events Compared to BAT, results showed that ruxolitinib led to: 1.Superior control of hematocrit 2.Superior reduction in splenomegaly 3.Superior reduction in PV-related symptoms 4.Trend for less thrombotic events Vannucchi et al. EHA 2014. Abstract LB-2436.

53. RESPONSE TRIAL: CHANGE IN PV SYMPTOMS Median Percentage Changes From Baseline at Week 32 in Individual MPN-SAF Symptom Scores Improvement −120 −100 −80 −60 −40 −20 0 20 40 Mesa R. ASH 2014. Abstract 709.

54. TOXICITY PROFILES OF PV AGENTS DrugCommon Adverse Effects Hydroxyurea Stomatitis Leg ulcers Dry skin, acne Gastric pain, diarrhea Interferon alfa Neutropenia Elevated LFTs Fatigue Headache Ruxolitinib Headache Abdominal Pain Diarrhea Fatigue Dyspnea Najean Y. Blood. 1997;90(9):3370-7; Kiladjian JJ. Blood. 2008;112(8):3065-72; Mesa R. ASH 2014. Abstract 709. Kiladjian JJ

55. POSSIBLE ALGORITHM OF THERAPY OF PV IN 2015 Diagnosis of PV Front Line Cytoreduction HU, or HU vs. INF Clinical Trial Consider ruxolitinib, INF (if not previously received), or JAK2 clinical trial Assess Symptom Quartile by MPN 10 Q1:TSS <8 Q2:TSS 8-17 Q3:TSS 18-31 Q4:TSS ≥32 Decide on need for concurrent cytoreduction based on risk and symptoms YES Monitor for symptom burden, vascular events, progression Worsening symptom burden Vascular event, progression Phlebotomy intolerance Worsening symptom burden Vascular event, progression HU Resistance/ Intolerance Assess MPN Risk Score & Symptoms Control Hematocrit (<45%) Low-dose aspirin in appropriate patients NO

56. KEY CASE TAKEAWAYS Polycythemia vera is a heterogeneous disease A subset of patients have worsening symptoms, splenomegaly, and difficulty with hematocrit control Hydroxyurea is largely considered front-line cytoreductive therapy for high-risk PV patients, but some patients have an inadequate response based either on resistance or intolerance Ruxolitinib is FDA-approved for patients with PV who have an inadequate response or are intolerant of hydroxyurea

57. ACTIVITY POST-TEST

58. QUESTION A.Oxygen saturation >95% B.Platelet count: 485 x 10 9 L C.Epo level: 1.9 mU/mL D.WBC: 14.9 x 10 9 /L According to the WHO, which of the following would be included among the characteristics that qualify a patient for diagnosis of PV?

59. QUESTION A.Phlebotomy (P) alone B.P + Aspirin (A) alone C.P + A + hydroxyurea D.P + A + ruxolitinib E.P + A + interferon alfa Which of the following would not be considered an appropriate first-line regimen for the treatment of PV?

60. QUESTION A.Control of hematocrit B.Reduction in splenomegaly, if present C.Reduction in pruritus D.Prevention of vascular events E.All of the above Which of the following is among the goals of ruxolitinib therapy in PV?