Aggiornamento Sindromi Mieloproliferative Croniche non LMC

1. Convegno Interregionale SIE Triveneto Verona, 29 maggio 2008 Aggiornamento Sindromi Mieloproliferative Croniche non LMC Francesco Rodeghiero Dipartimento di Terapie Cellulari ed Ematologia Ospedale San Bortolo - Vicenza

2. A brief history of Myeloproliferative Disorders • Second half of XIX century: CML (1845, Bennet), PMF* (1879, Henck), PV (1892, Vaquez), ET (1934, Epstein and Goedel) firstly described • 1951: Dameshek: concept of MPD as “…variable manifestations of proliferative activity of the bone marrow cells, perhaps due to a hitherto undiscovered stimulus” • 1960: Philadelphia chromosome(Nowell and Hungerford) • 1967: Clonal origin of MPD (Fialkow ) Creation of the PVSG • 1985: BCR-ABL fusion transcript (Shtivelman & al, Stam & al) • 2005: Mutation of JAK2 V617F in exon 14 (Levine et al; James et al; Kralovics et al, Baxter et al) William Dameshek (1900-1969) *new definition according to IWG- MRT (Mesa et al, 2007)

3. 2005: the JAK2 (V617F) mutation era (but at what level does the disease start?) Progenitor cells Terminally differentiated cells EPOR Red cells Stem cell Unknown receptor MPL Megakaryocytes G-CSFR Granulocytes

4. Role of JAK2 in Pathway Signaling The V617F gain-of-function mutation Wild-type JAK2 without ligand Wild-type JAK2 with specific ligand JAK2 with V617F mutation Ligand Receptor JAK2 P P P P JAK2 V617F P P JAK2 P P Valine-to-phenylalanine change at position 617 in the JH2 pseudo-kinase domain (exon 14) No signal RAS–MAPK STAT STAT PI3K RAS–MAPK PI3K (Kralovics et al, N Engl J Med 2005; 352:1779-90)

5. Frequency of JAK2 V617F mutation • 95% PV • 40-60% ET and PMF • Occasionaly in: - atypical MPD - MDS - CML - systemic mastocytosis - chronic neutrophilic leukemia - eosinophilic disorders Never seen in lymphoid tumors nor in healthy people

6. Mechanisms of 9pLOH Possible Roles of the JAK2 V617F mutation in Myeloproliferative Disease (Kralovics et al, N Engl J Med 2005; 352:1779-90)

7. Other somatic mutations in MPDs • 3% PV patients JAK2V617F neg present a mutation in exon 12 of the JAK2 • 5% PMF patients and 1% ET patients present mutations of the C-MPLgene (exon 10,W515L or W515K, S505N)

8. WHO criteria for PV 2001 criteria 2008 revised criteria (Tefferi et al, Blood 2007) • Major criteria 1. Hb>18.5 g/dL in men, 16.5 g/dL in women or increased red cell mass • JAK2 V617F or other similar mutations such as JAK2 exon 12 • Minor criteria • BM biopsy with panmyelosis • Low EPO level • EEC formation in vitro • A-criteria • Red cell mass>25% above mean value, or Hb>18,5 g/dL in men, 16,5 g/dL in women • No secondary erythrocytosis • Splenomegaly • Clonal genetic abnormality other than Ph’ • EEC formation in vitro • B-criteria • Thrombocytosis >400x 109/L • Leukocytosis >12 x109/L • BM biopsy with panmyelosis • Low EPO levels Dg: A1 + A2 and either another A or +2 B Dg: Both major critera + 1 minor or first major critera+ 2 minor criteria

9. WHO criteria for ET 2001 criteria 2008 revised criteria (Tefferi et al, Blood 2007) • Positive criteria • Sustained platelet count >600 x 109/L • BM with proliferation of the MK (enlarged,mature MK) • Criteria of exclusion • No evidence of PV, CML, CIMF, MDS • No reactive thrombocytosis • Sustained platelet count >450 x 109/L • BM biopsy with proliferation of the MK lineage (increased n. of enlarged, mature MK) • No evidence of PV, PMF, CML, MDS or other myeloid neoplasm • JAK2 V617F or other clonal marker, or in its absence, no reactive thrombocytosis Dg: all criteria Dg: all criteria

10. WHO criteria for PMF 2001 criteria 2008 revised criteria (Tefferi et al, Blood 2007) • Prefibrotic primary myelofibrosis • No or mild splenomegaly or hepatomegaly • Mild anemia, leukocytosis, thrombocytosis • No or mild leukoerythroblastosis, poikilocytosis, few dacryocytes • BM: hypercellularity, MK and neutrophilic proliferation, MK atypia,minimal or absent reticulin fibrosis • Fibrotic primary myelofibrosis • Moderate splenomegaly or hepatomegaly • Moderate anemia, WBC and plts decreased to elevate • Leukoerythroblastosis, poikilocytosis, dacryocytosis • BM: reticulin fibrosis, decreased cellularity, prominent MK proliferation and atypia, osteosclerosis • Major criteria • MK proliferation and atypia, with or without reticulin but increased BM cellularity/granulocytic proliferation • 2. No evidence of PV, CML, MDS or other myeloid neoplasm • JAK2 V617F or other clonal marker or in its absence, no evidence of secondary fibrosis • Minor criteria • Leukoerythroblastosis • Increase in serum LDH • Anemia • Palpable splenomegaly Dg: All major criteria + 2 minor criteria

11. Major criticisms of revised WHO diagnostic criteria (Spivak and Silver, Blood 2008) • Hb or Ht alone cannot substitute for red cell mass and plasma volume determinations • Since JAK2 causes a ‘PV-like’ phenotype in ET pts, red cell mass and plasma volume must be required in isolated trombocytosis. • Since JAK2 is the leading factor for classification, EPO and EEC assay (never standardized) are redundant • Marrow morphology reflects a particular stage in MPS evolution, so it is a non specific moving target with respect to phenotype • No differences between pts labeled as ‘prefibrotic MF’ or ‘true ET’ in clinical and laboratory features, JAK2 status, survival, thrombosis and hemorrhage. • Histologic criteria in WHO classification are unreliable (Wilkins et al, Blood 2008)

12. Influence of JAK2 on clinical phenotype of ET and PV ET JAK2: resembles PV increased risk of VT transformation to PV greater sensitivity to HU ET JAK2 homo: higher WBC count but comparable plt count more conspicuous spleen enlargment PV JAK2 homo: aquagenic pruritus more frequent highest degree of spleen enlargment higher rate of evolution into MF require cytostatic approach

13. Risk factors for thrombosis and hemorrhage in PV and ET PV: thrombotic and hemorrhagic events at diagnosis ET: thrombotic and hemorrhagic events at diagnosis

14. ECLAP, 2003: 1638 PV, prospective GISP 1995: 1213 PV, retrospective OLD thrombotic risk factors in PV • Age Vascular complication in pts age>65yrs: 5% patients-year, HR 2 (CI 1.2-3.3; p < 0.006) Age < 40 1.8 events/100 pts y Age > 70 5.1 events/100 pts y • Previous thrombosis Vascular complications in pts with a previous thrombotic events: 4.93%pts-year, HR: 1.95 (CI 1.3-3.0, p = 0.0017) 17.3% thrombosis without history vs 24.6% with history; p= 0.001

15. OLD thrombotic risk factors in ET • Age and previous thrombosis • Carobbio et al 2007, retrospective, 439 pts, FU 6.2 y. HR age > 60 and /or history of thrombosis : 2.3 (CI 1.3-3.9, p= 0.004) • Wolanskyj et al 2006, retrospective, 322 pts, FU 13.6 y RR age > 60: 1.51 (CI 1.05-2.18, p= 0.03), RR for previous vascular complications: 1.73 (1.07-2.81;p=0.03) • High platelet count • Van Genderen PJ, Michiels JJ, 1994: a very high plt count (>1500×109/L) major predictor of bleeding rather than thrombotic complications (due to acquired von Willebrand disease)

16. “NEW” thrombotic risk factors in PV White blood cell count(ECLAP, Blood 2007) Prospective, 1638 pts, F.U. 3 y; WBC > 15x109/L vs 10.1 to15x109/L baseline; HR adjusted for CHT and ASA Major thrombosis (169) HR 1.71 (1.10-2.65) p=0.017 AT (121) HR 1.67 (0.98-2.84) p ns VTE (51) HR 1.81 (0.84-3.89) p ns IMA (41) HR 2.84 (1.25-6.46) p=0.013 Stroke/TIA (54) HR 0.93 (0.37-2.34) p ns PAT (21) HR 1.72 (0.46-6.46) p ns WBC>15x109/L(p=0,001) is also an adverse prognostic factor for leukemic transformation(Gangat et al, BJH 2007)

17. “NEW” thrombotic risk factors in ET WBC count(Carobbio et al, Blood 2007) Retrospective, 439 pts, fu 6.2 y; WBC > 8.7x 109/L vs< 8.7x109/L baseline; RR multivariable analysis Major thrombosis during F/U RR 2.3 (1.4-3.9) p=0.001 Risk factors at dx HR CI Low risk + low WBC 1 - Low risk + high WBC 3.1 1.4-7.1 High risk* + low WBC 2.5 1.0-6.0 High risk* + high WBC 5 2.1-11.9 * age 60 yrs or older and/or previous thrombotic event

18. “NEW” thrombotic risk factors in ET WBC count Tefferi et al, Blood 2008: 605 pts, F/U 84 months “Accurate interpretation of the relationship between WBC count at diagnosis and subsequent thrombosis is counfounded by treatment. Prospective studies are needed to clarify the issue further”

19. “NEW” thrombotic risk factors in ET The role of JAK2 V617F • The mutation is an indipendent predictor of pregnancy complication; JAK2V617F pos women had an HR=2,02 of developing complication respect to JAK2V617F neg.(Passamonti et al,Blood 2007)

20. 25-30% PV and 2-4% ET patients have JAK2 in homozygous status Vannucchi et al, Blood 2007, 1035 pts, retrospective: • 118 HOMO patients (104 PV and 14 ET) were older, displayed higher WBC and Htc values at diagnosis, presented larger spleen, had more frequent evolution in MF, were more likely to receive chemotherapy for disease control than 587 hetero and 257 WT. • ET pts have higher risk of CV events

21. Thrombotic and Hemorrhagic Complications After Surgery In Patients With Essential Thrombocythemia And Polycythemia Vera(Ruggeri et al, Blood 2008) • Rate of thrombosis and major hemorrhagic episodes (around 7%) higher than that observed in clinical trials evaluating heparin prophylaxis and in surgical pts with cancer (1%) • No correlation between bleeding episodes and type of diagnosis, use of antithrombotic prophylaxis, type of surgery • Restrict antithrombotic prophylaxis with LMWH in PV pts undergoing major surgery • In ET pts with several risk factors use antiplatelet drugs

22. Risk factors in PMF • Cox regression analysis: HR of JAK2V617Fmutation for different outcomes (Barosi et al, Blood 2008)

23. JAK2 in PMF Heterozygotes: higher Hb level reduced transfusion requirement aquagenic pruritus higher platelet count Homozygotes: more enlarged pleen higher WBC count greater need of cytoreductive therapy The evolution hetero  homo (10 per 100 pts-yr) is a critical step in the progression of the disease and represents a major determinant for the disease evolution (Barosi et al, Blood 2008)

24. Dynamic prognostic model to predict survival in post-PV myelofibrosis(Passamonti et al, Blood 2008) In PV pts, the 15-year risk of evolution to MF is 6%; no risk factors identified so far 68 post PV MF pts (cohort of 647 PV) Prognostic factors at diagnosis: 6.6 yrs of survival for pts with Hb > 10 g/dL vs 1.9 years for pts with Hb < 10 g/dL (p <0.001) Time-dependent analysis: Hb < 10 g/dL; PLT < 100 x 109/L; WBC > 30 x 109/L have statistical significance on survival= HR of 4.2(95% CI:2,4-7,7; p<0,001) for pts who acquires 1, 2 or 3 of these risk factors at any time from the diagnosis of post-PV MF)

25. New mutations: JAK2 exon 12 • Pts JAK2 exon 12+ have isolated erythrocytosis, low Epo levels, JAK2 exon 14 WT, isolated erythroid hyperplasia in the BM and, in some case, EEC growth • Only one case described V617F pos and exon 12 pos • JAK2 exon12 mutations in PV(3%) and IE (27%) :simply different stages of the same erythrocytosic MPD? • According to WHO criteria, this disorder may be diagnosed as IE or PV. Progression to full-blown PV may occur during the clinical course. (Cazzola M, Haematologica 2007)

26. New mutations in c-mpl • PT-1 studies, retrospective, 776 ET pts: 4 different mutations in exon 10 c-mpl gene. • MPL mutations in 8.5% of WT ET pts, only one was also JAK2 pos • MPL mutate pts have lower Hb and higher plts levels at diagnosis, higher Epo levels, endogenous MK but not EEC growth and reduced BM erythroid and overall cellularity than JAK2 pts • MPL mutate pts were older with reduced BM cellularity but could not be identified as a discrete clinico-pathological subgroup vs WT • No differences in thrombosis, major hemorrhage, MF transformation or survival rates (Beer et al, Blood 2008)

27. Familial MPD Hereditary predisposition to true MPD Inherited disorders • Mendelian transmission • High penetrance • Polyclonal hematopoiesis • Low penetrance • Clonal hematopoiesis • Frequent occurence of somatic mutations Erythrocitosis/PV • Mutation of EPOR • mutation of VHL • mutation of PHD2 • mutation of HIF-2A • high oxygen–affinity haemoglobin • Methaemoglobinemia • 2,3-bisphosphoglyceratedeficiency • In the same pedigree at least two relatives have a MPD (PV,ET or MF)→ prevalence:7,6% - autosomal dominant trait with decreased penetrance • Clinical presentation, complications, disease evolution not different from sporadic MPD • JAK2 mutations are acquired and occured as secondary genetic events - evidence of disease anticipation ET • Mutation of Tpo • Mutation in c-mpl

28. Treatment outline (Mesa, ASH 2007)

29. RCT in Essential Thrombocythemia

30. PT 1Results

31. ECLAP(Landolfi et al, NEJM 2005)

32. Stem cell transplantation in MFDupriez scoring system Adverse Prognostic Factors: Hb < 10 g/dL; WBC < 4000/µL or > 30.00/µL

33. Stem cell transplantation in high risk MFResults of ALLO-TMO * at 2.2 - 5 years; ° in pts < 45 years of age

35. Risk adapted treatment strategies § 1 RCT; * RCT ongoing (Ht < 45% vs 45 – 50%, GIMEMA)

36. Acknowledgments • Marco Ruggeri • Elena Albiero • Martina Bernardi • Silvia Finotto

39. Therapy • PVSG prospective trial, 431 PV pts,1987: phlebotomyalone/phlebotomy+chlorambucil/phlebotomy+radioactive phosphate. Phlebotomy is recommended in all pts with PV and should represent the only treatment in pts at low risk for vascular complications. • ECLAP Study, double-blind randomized trial, 518 PV pts, 2005: low dose aspirin (100 mg daily)/placebo. Low dose aspirin (75-100 mg daily) is recommended in all PV ptswithout history of major bleeding or gastric intolerance • PT-1 Study, randomized trial, 809 high risk ET pts, 2005: HU+low-dose aspirin/ anagrelide+low-dose aspirin. HU is very effective in preventing thrombosis and thus should be considered the drug of choice in high-risk patients. Progression to MF and transformation to AML are similar in the two groups • Silver RT, Cancer 2006,’long-term effects of treatment of PV with rINFα’(55 pts, 13 yrs therapy).Complete responses in the great majority of case but two important problems: its cost and incidence of side effects. It is now reserved to selected categories (pregnant women, very young subjects or those with intolerance to HU)

40. Therapy Landolfi, R. et al. Haematologica 2008;93:331-335

41. Erythrocitosis (I) • Primary (caused by an acquired or inherited mutation expressed within the erythroid progenitors) -Familiar: 1. mutation of EPOR 2. “Hereditary predisposition to PV” - Acquired: 95% JAK2V617F, 5% JAK2WT 3% JAK2 exon12 positive JAK2 exon12 negative ??

42. Erythrocitosis (II) • Secondary(caused by circulating plasma factors stimulating erythropoiesis) -Congenital/Familiar: 1. mutation of VHL 2. mutation of PHD2 3. mutation of HIF-2A 4. high oxygen–affinity haemoglobin 5. methaemoglobinemia 6. 2,3-bisphosphoglycerate deficiency - Acquired: 1.Hipoxia-driven 2. Oxygen-indipendent

43. Hypoxia-sensing pathway

45. Schematic representation of the erythropoietin receptor and the defect in the receptor underlying primary familial and congenital polycythemia. HCP, hematopoietic cell phosphatase.

46. The 2008 WHO classification for Myeloid Neoplasms • 1. Acute myeloid leukemia • 2. Myelodysplastic syndromes (MDS) • 3. Myeloproliferative neoplasms (MPN) 3.1 Chronic myelogenous leukemia 3.2 Polycythemia vera 3.3 Essential thrombocythemia 3.4 Primary myelofibrosis 3.5 Chronic neutrophilic leukemia 3.6 Chronic eosinophilic leukemia, not otherwise categorized 3.7 Hypereosinophilic syndrome 3.8 Mast cell disease 3.9 MPNs, unclassifiable • 4. MDS/MPN 4.1 Chronic myelomonocytic leukemia 4.2 Juvenile myelomonocytic leukemia 4.3 Atypical chronic myeloid leukemia 4.4 MDS/MPN, unclassifiable • 5. Myeloid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 5.1 Myeloid neoplasms associated with PDGFRA rearrangement 5.2 Myeloid neoplasms associated with PDGFRB rearrangement 5.3 Myeloid neoplasms associated with FGFR1 rearrangement (8p11 myeloproliferative syndrome)

47. Cascata fosforilativa nella via JAK-STAT a seguito della formazione del complesso ligando - recettore

49. Meccanismo con cui le mutazioni della Tpo possono causare un aumento della traduzione nella sintesi della proteina.