Fisiopatologia, diagnosi e classificazione della mielofibrosi

1. Fisiopatologia, diagnosi e classificazione della mielofibrosi Barosi Giovanni Laboratorio di Epidemiologia Clinica e Centro per lo Studio della Mielofibrosi. Fondazione IRCCS Policlinico S. Matteo, Pavia Catania, 7 Novembre 2008

2. Key Questions in Primary Myelofibrosis • Biological key questions • What genetic event preceding or concomitant to JAK2 V617F mutation contributes to the development of PMF? • Mechanism for constitutive mobilization of CD34+ cells in PMF • Mechanism for constitutive mobilization of endothelial progenitor cells and angiogenesis in PMF • Role of TGF beta in myelofibrosis and myeloproliferation of PMF • Operational (Clinical) key questions • JAK2 mutation load and prognosis • Definition of resistance to HU in PMF • Therapeutic key questions • Efficacy of molecularly targeted therapies

3. Toward a model of malignancy for primary myelofibrosis

4. Models of myeloproliferative neoplasms UPD: Uniparental disomy (inheritance of both homologues of a pair of chromosomes from one parent only).

5. Evidence that a preceding genetic event is necessary for developing the MPN disease • In certain MPN patients, the clonality of expanded myeloid progenitors is found to be larger than JAK2 V617F clone (JAK2 V617F being a late genetic event). Kralovics, R. et al. Blood 2006;108:1377-1380

6. Evidence that a preceding genetic event is necessary for developing the MPN disease • Familial MPNs prove genetic predisposition to acquire different JAK2 mutations Rumi et al, Cancer 2006

7. Evidence that a preceding genetic event is necessary for developing the MPN disease • Acute myeloid leukemia cases developed in JAK2 V617F positive patients can occur with leukemic blasts not harboring JAK2 V617F Pasqualucci et al, Leukemia 2008

8. Pasqualucci et al, Leukemia 2008

9. Other JAK2-independent molecular events influence the phenotype of PMF • Epigenetic alterations • SOCS • CXCR4 • miRNA

10. Socs 3 promoter methylation was detected in 32% of patients with PMF (Fourouclas et al, Haematologica, 2008) LIGAND CYTOKINE RECEPTOR Jak p Phosphorylation by JAKs of Stats NUCLEAR MEMBRANE Socs: Suppressor of Cytokine Signaling Stat (-) p Socs - CIS (cytokine inducible SH2 domain protein) - Socs 1-7 Stat p Gene transcription Socs

11. CXCR4 is down-regulated in PMF and is associated with the phenotype of the disease (Barosi et al, Vannucchi et al)

12. CD34+ cells p=.00001 p=.0007 p=.0001 p=.0001 Ctr PV ET PMF postPV-MF Micro-RNA is differentially expressed in MPNs and is associated with the phenotype of the disease (Guglielmelli et al, 2008) • Studio del profilo di espressione di miRNA in CD34+ di MPD • miR-16 risultava significativamente aumentato • Non mutazioni nei geni • La iper-espressione di miR16 induceva il differenziamento eritroide • La inibizione di miR16-2 risultava essere la più efficace rispetto a mir16-1 • Non significativa correlazione con JAK2 • La inibizione di miR16-2 abrogava la formazione di EEC • Anomalie nella espressione di miRNA possono contribuire al fenotipo delle MPD

13. Endothelial progenitor cells

14. Endothelial Progenitor Cells and Tumor Progression From Seandel et al. Cancer Cell, 2008

15. Methods of Detection of Endothelial Progenitor Cells Hirschi, K. K. et al. Arterioscler Thromb Vasc Biol 2008;28:1584-1595

16. Study Population

17. Clonality of ECFCs in CMPD ECFCs do not belong to the malignant clone

18. ECFCS in Myelofibrosis • ECFCs are not associated with: • Hemoglobin level • Disease duration • Spleen size • JAk2 mutational status or JAK2 allele burden

19. ECFCs in MF: Association with Young Age R=-0.258; P=0.02

20. ECFCs in MF: Association with High Platelet Count P=NS

21. ECFCs in MF: Association with Normal-Low WBC count R=-0.17; P=0.05

22. ECFCs in MF: Association with Normal-Low CD34+ count R=-0.32; P=0.01

23. ECFCs in MF: Association with a Diagnosis of Prefibrotic Myelofibrosis

24. Conclusion • With the current assay method, ECFCs in peripheral blood are measurable in a limited number of normal subjects and patients with CMPDs • The highest frequency of ECFCs is measured in patients with Myelofibrosis • High frequency of ECFCs is associated with young age, high number of platelet count, normal or low number of WBC, normal CD34+ cells and a diagnosis of prefibrotic myelofibrosis

25. Hypothesis: EPCs mark an early phase of the disease (pre-angiogenic and pre-metastatic phase) • No hemopoietic progenitor cells mobilization (no myeloid metaplasia) • EPCs respond to angiogenic stimuli (hypoxia?) and migrate to extramedullary sites • Hemopoietic progenitor cells mobilization (myeloid metaplasia) • Intense angiogenesis Angiogenic switch

26. WHO (2008) criteria for primary myelofibrosis (PMF): diagnosis requires meeting all 3 major and 2 minor criteria • Major criteria • Presence of magakaryocyte proliferation and atypia*, usually accompanied by either reticulin and/or collagen fibrosis, or in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (i.e. prefibrotic cellular-phase disease) • Not meeting WHO criteria for polycythemia vera, BCR-ABL1+ chronic myelogenous leukemia, myelodysplastic syndrome, or other myeloid neoplasms • Demonstration of JAK2 V6127F or other clonal marker (e.g. MPL W515K/L), or in the absence of a clonal marker, no evidence that bone marrow fibrosis or other changes are secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathies • Minor criteria • Leukoerythroblastosis • Increased in serum lactate dehydrogenase level • Anemia • Splenomegaly ____________________________________________________________________________ • * Small to large megakaryocytes with an aberrant nuclear /cytoplasmic ratio and hyperchromatic, bulbous, or irregularly folded nuclei and dense clustering

27. The Italian Diagnostic Criteria of MMM NECESSARY CRITERIA A. Diffuse bone marrow fibrosis B. Absence of Philadelphia chromosome or BCR-ABL rearrangement in peripheral blood cells OPTIONAL CRITERIA 1. Splenomegaly of any grade 2. Anisopoikilocytosis with tear-drop erythrocytes 3. Presence of circulating immature myeloid cells 4. Presence of circulating erythroblasts 5. Presence of clusters of megakaryoblasts and anomalous megakaryocytes in bone marrow sections 6. Myeloid metaplasia DIAGNOSIS OF MMM:The two necessary criteria plus any other 2 optional criteria when splenomegaly is present, or any other 4 optional criteria when splenomegaly is absent Barosi et al. BJH 1999

28. Prefibrotic or Early Fibrotic Myelofibrosis • Female • Age: 34 year old • Splenomegaly: 2 cm from the costal margin • Hb = 13.4 g/dL • WBC = 7.5 x109/L • Ptl count = 465 x109/L • Tear drop cells: +/- • Immature myeloid cells in PB: 2% • Bone marrow fibrosis absent • LDH = 679 • CD34=11.4 x106/L

29. New Therapeutic Targets in Myelofibrosis • Non-JAK2 Inhibitors • JAK2 Inhibitors • Class I (JAK2 selective) • Class II (non JAK2 selective)

30. Non-JAK2 Inhibitors - Published Studies

31. Non-JAK2 Inhibitors - Published results

32. Lessons From Non-JAK2 Inhibiting Molecular Targets in Myelofibrosis • Targeting non disease-specific molecular pathways results in null or moderate response • Preclinical analysis of therapeutic agents may not predict clinical acitivity • -Bortezomib has activity in thrombopoietin-driven murine model of myelofibrosis (Wagner-Ballon, 2007), but no activity in humans. • Clinical activity is not correlated with the documented biological acitivty • - No difference in hypomethylation with 5-AZA between responders and non-responders (Quintas-Cardama, 2008)

33. New non-JAK2 Inhibitors Targets in Myelofibrosis – Ongoing and Pending Trials

34. JAK2 Inhibitors in Myelofibrosis - Clinical Trials

35. The First Results with a JAK2 Inhibitor in Myelofibrosis- INCB18424 • Rationale: INCB18424 is a small molecule that inhibits specifically JAK2 • Design: Phase I/II trial in patients with PMF and post PV/ET MF (both JAK2 V617F and wild type). Dose escalating from 25 mg PO BID to 50 mg PO BID • Response: • Six patients had a marked reduction in splenomegaly and symptomatic improvement • Serial JAK2 testing had gradual reduction in the percentage of PCR product with the V617F mutation • Toxicity: No significant toxicity Verstovsek et al, ASH 2007

36. Inhibitors of JAK2 Signal Transduction • Toxicities related to inhibition of JAKs other than JAK2 • First results highly encouraging • Most appropriate candidates • Optimum dose and schedule

37. RIMM- Italian Registry of Myelofibrosis. IRCCS Policlinico S. Matteo Foundation GIMEMA. Working Party on CMPD T. Barbui, Bergamo A.M. Vannucchi, Florence Coordination and Clinical studies G. Barosi M. Marchetti E. Gattoni C. Azzan Cytology R. Invernizzi A. Pecci FACS M. Massa R. Campanelli G. Viarengo Cell coltures V. Rosti E. Bonetti Clonality and JAK2 G. Bergamaschi L.Villani Pathology U. Magrini MPD-RC R. Hoffman J. Spivak J. Prchal A.R. Migliaccio L. Silverman R. Marchioli T. Barbui A.M. Vannucchi