Fisiopatologia diagnosi e classificazione delle trombocitemie Alessandro M

Fisiopatologia, diagnosi e classificazione delle trombocitemie Alessandro M. Vannucchi UF di Ematologia Università degli Studi, Firenze

Kaushansky K. N Engl J Med 2006; 354: 2034 -2045

Classification of Thrombocytosis n Primary Thrombocytosis n n Inherited, familial Acquired, clonal thrombocytosis n n n Chronic myeloproliferative neoplasms Chronic myelogenous leukemia Myelodysplastic syndromes (5 q- and RARS-T) • Secondary (Reactive) Thrombocytosis Acute & transient Acute blood loss n Recovery from thombocytopenia n Acute inflammation or infection n Response to exercise n Schafer AJ, NEJM 2004 Sustained Iron deficiency n Hemolytic anemia n Chronic inflammatory or infectious disease n Cancer n Drug reactions n Life-long Splenectomy n Splenic agenesia n

n Fisiopatologia, diagnosi e classificazione delle trombocitosi primarie n Fisiopatologia della trombosi nella ET

Meccanismi molecolari: mutazioni di JAK 2 e MPL EPOR MPL G/GM-CSFR IL 3 R IL 5 R GP 130 W 515 L/K P JAK 2 V 617 F JAK 2 P Exon 12 Stat 5 P Mpl JAK 2 P Ras SOCS 1 SOCS 3 P Stat 5 P PI 3 K AKT P Raf SOCS 1 SOCS 3 MEK P P m. TOR FOXO P ERK Activation of genes (proliferation/survival/differentiation) P

Only the MPL W 515 mutations promote a G(1)/S-phase transition Chaligne, R. Leukemia 2008

Chronic Ph 1 -neg myeloproliferative neoplasms JAK 2 V 617 F PV ET PMF ≈99 % ≈60% 0 8%* 8% ≈50% * nd nd MPL W 515 L/K JAK 2 exon 12 * Referred to V 617 F-negative

A PV, rather than an ET phenotype, is reproduced in JAK 2 V 617 F mice James C, Nature 2005; Wernig G, Blood 2006; Lacout C, Blood 2006; Zaleskas V, PLo. S ONE 2006

Ratio of mutant JAK 2 -V 617 F to wild-type Jak 2 determines the MPD phenotypes in transgenic mice n Transgenic line (FF 1 -Vav) with an expression of JAK 2 -V 617 F lower than the endogenous wild-type JAK 2 developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia. n Transgenic JAK 2 -V 617 F/Mx. Cre mice had expression of JAK 2 -V 617 F approximately equal to wild-type JAK 2 and developed a PV-like phenotype with increased hemoglobin, thrombocytosis, and neutrophilia. n Higher levels of JAK 2 -V 617 F in mouse bone marrow by retroviral transduction caused a PV-like phenotype without thrombocytosis. Tiedt, R. et al. Blood 2008; 111: 3931 -3940

JAK 2 V 617 F allele burden in MPNs JAK 2 V 617 F allele burden (%) 100 80 60 40 20 0 PV Antonioli E, Haematologica 2007 ET PMF post. PV / ET MF

Identification of disease subtypes in ET based on JAK 2 V 617 F mutation JAK 2 V 617 F pos ET resembles PV: - increased erythropoiesis - lower ferritin (p < 0. 01) - lower MCV (p < 0. 0001) - lower serum Epo (p < 0. 0001) - higher leukocyte count - more venous thromboses - more frequent PV “transformation” JAK 2 V 617 F neg ET - higher platelet counts Campbell et al Lancet 2005& Blood 2005; Antonioli et al Leukemia 2005;

Induction of thrombocytosis and myelofibrosis by MPLW 515 L MPLWT MPLW 515 L

Clinical correlates of MPL W 515 mutations in ET MPL mutant MPL wild-type P value Median age, years 58 (22 -84) 52 (8 -97) 0. 08 White blood cell count (x 109/L) 8. 8 ± 3. 0 9. 3 ± 2. 7 0. 6 Hemoglobin (g/L) 134 ± 13 140 ± 11 0. 02 Hematocrit (%) 40. 6 ± 3. 8 42. 0 ± 4. 1 0. 04 Platelet count (x 109/L) 960 ± 327 860 ± 309 0. 04 Splenomegaly, no-(%) 6 (19%) 178 (21%) 0. 9 Major thromb, no-(%) 10 (32%) 172 (19%) 0. 1 Microvessel dis no-(%) 19 (61%) 276 (30%) 0. 0005 Major hemorrh no-(%) 3 (10%) 35 (3. 6%) 0. 1 994 ET pts 3. 0% 418 V 617 Fneg 8. 1% 12 (40%) W 515 K 18 (60%) W 515 L In 8 pts both MPLW 515 and JAK 2 V 617 F AM Vannucchi et al. , Blood, 2008

WHO 2008 DIAGNOSTIC CRITERIA Essential Thrombocythemia Major criteria 1. Sustained platelet count ≥ 450 x 109/L 2. Bone marrow biopsy showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged mature megakaryocytes. No significant increase or left shift of neutrophil granulopoiesis or erythropoiesis 3. Not meeting WHO criteria for CML, PV, PMF, MDS or other myeloid neoplasm 4. Demonstration of JAK 2 V 617 F or other clonal marker or no evidence of reactive thrombocytosis Minor criteria Diagnostic combinations *, Tefferi A, 2007 All four criteria must be met

PRIMARY THROMBOCYTOSIS BCR-ABL JAK 2 V 617 F MPLW 515 L/K ST 100% No molec. abnormality 100% ET 60% 1 - 4% 40% PMF 60% 5 - 8% 40% PV 95% RARS-T 50% 5 q- 5% 95% CML Vannucchi AM, ASH 2007

n Fisiopatologia, diagnosi e classificazione delle trombocitosi primarie n Fisiopatologia della trombosi nella ET

Thrombosis-free survival in MPN A GIMEMA retrospective study on 1, 224 patients Probability Crude-analysis 1 Pts at risk Years PV 397 339 2 3 4 5 6 7 8 9 10 275 235 206 168 139 120 95 82 67 ET 637 549 479 421 365 316 262 220 191 169 140 MF 190 123 94 78 68 60 48 40 36 30 28 Vannucchi AM, Blood 2007 & unpubl.

Cumulative probability (%) Recurrent Thrombosis in PV or ET n=494 49. 9% (n=259) ET PV (n=235) 5. 6%pt-yr 3. 4%ptyr 2. 2%ptyr Time after first thrombosis (months) De Stefano V, Haematologica 2007

Risk Stratification in ET Age >60 Cardiovascular Risk category or risk history of thrombosis factors (*) Low NO NO Intermediate NO YES High (**) YES ---- (*) Hyperthension, Hypercholesterolemia, Diabetes, Smoking; Familial thrombophilia. Their contribution to thrombotic risk is controversial

Thrombocytosis and Thrombosis: an Uneven Relationship - 1 Clonal Thrombocytosis Secondary Thrombocytosis Digital or cerebrovascular ischemia Characteristic No Large vessel arterial or venous thrombosis Increased risk No Bleeding complications Increased risk No Finding Schafer AJ, NEJM 2004

Thrombocytosis and Thrombosis: an Uneven Relationship - 2 ECLAP cohort v In a series of 99 young (16 -59) ET pts, receiving or not prophylactic cyroreductive therapy, no difference in thrombosis (nor hemorrhages) was noted Berk PD et al, 1995; Di Nisio M et al, BJH 2006; Tefferi A et al, Blood 2006

Thrombocytosis and Thrombosis: an Uneven Relationship - 3 Platelet count* (x 109/L) Hazard Ratio (95% CI) pvalue 650 to 1, 000 More than 1, 000 1) Unadjusted 0. 7 (0. 4 -1. 1) 0. 5 (0. 3 -0. 9) P= 0. 03 2) Variables adjusted 0. 6 (0. 4 -1. 0) 0. 5 (0. 3 -0. 8) P=0. 01 3) +JAK 2 V 617 F adjusted 0. 6 (0. 4 -1. 1) 0. 6 (0. 3 -1. 1) Total thrombosis Carobbio A, Blood 2008

Thrombocytosis and Thrombosis: an Uneven Relationship - 4 MRC PT-1 Trial Harrison C et al, NEJM 2005

Leukocytosis and Thrombosis in ET Wolanskyi A, Mayo Clin Proc, 2006 WBC (x 109/L) HR (95% CI) P (*) >15. 0 1. 74 (1. 1 -2. 7) 0. 01 Carobbio A, Blood 2007 WBC (x 109/L) HR (95% CI) P (*) <7. 2 -8. 7 1 2. 4 (1. 1 -5. 6) 0. 04 8. 7 -10. 4 >10. 4 2. 7 (1. 2 -6. 2) 3. 0 (1. 3 -6. 9) 0. 02 0. 008 (*) Multivariable analysis

k -ri s gh Hi Lo w- ris k Leukocytosis and “conventional” risk factors in ET P=. 003 Carobbio A et al, Blood 2007; JCO 2008

Interaction of leukocytosis and thrombocytosis on thrombotic risk in ET WBC <11, 000 >11, 000 Carobbio A, Blood 2008 Platelet count Rate of CV events JAK 2 V 617 F (% pts/year) mutated (%) RR (P=) >1, 000 1. 59 26 1 <1, 000 2. 26 56 1. 92 (0. 034) <1, 000 2. 88 61 2. 38 (0. 026) >1, 000 2. 95 77 2. 43 (0. 017)

V 617 F Status and Thrombosis in ET Venous thrombosis 2. 09 [1. 44, 3. 05] Arterial thrombosis 1. 96 [1. 43, 2. 67] Thrombosis at presentation 1. 88 [1. 38, 2. 56] Ziakas PD et al, Haematologica 2008

V 617 F Burden and Thrombosis in ET HR (95%CI) WT Hetero * Homo adjusted for age, sex, previous thrombosis and leukocytosis Vannucchi AM et al, Blood 2007 1 1. 49 (0. 9 -2. 5) 3. 97 (1. 3 -11. 7) P 0. 13 0. 013

Interactions between JAK 2 V 617 F genotype and inherited thrombophilia n 132 ET pts, 45 pts (34%) with major thrombosis. n 83 pts (62. 8%) V 617 F+, 8 pts (6%) were Homo RR RR JAK 2 V 617 F+ 2. 1 (1. 1 -3. 8) JAK 2+ Thr’ph’+ vs WT Thr’ph’- 4. 4 (2. 2 -8. 8) Homo 3. 7 (1. 8 -7. 2) 2. 1 (1. 1 -4. 0) Hetero 1. 9 (1. 0 -3. 5) JAK 2+ Thr’ph’vs WT Thr’ph’JAK 2+ Thr’ph’+ vs JAK 2+Thr’ph’- 2. 1 (1. 3 -3. 4) Homo vs Hetero 1. 9 (1. 2 -3. 2) De Stefano V, EHA 2008

Acknowledgments n Tiziano Barbui Chairman of the GIMEMA-MPD WP and all the colleagues of the WP n Paola Guglielmelli, Elisabetta Antonioli, Lisa Pieri, Alberto Bosi Dip. Ematologia, Firenze