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

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

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

Meccanismi molecolari: mutazioni di JAK2 e MPL Stat5 SOCS1 SOCS3 PI3K AKT mTOR FOXO Ras Raf MEK ERK Activation of genes (proliferation/survival/differentiation) EPOR MPL G/GM-CSFR IL3R IL5R GP130 JAK2 V617F Exon12 W515L/K Mpl

Only the MPL W515 mutations promote a G(1)/S-phase transition Chaligne, R. Leukemia 2008

Chronic Ph1-neg myeloproliferative neoplasms PV ET PMF JAK2 V617F ≈99 % ≈60% MPL W515L/K 8%* 8% JAK2 exon 12 ≈50% * nd * Referred to V617F-negative

A PV, rather than an ET phenotype, is reproduced in JAK2 V617F mice BM cells showed hypersensitivity of CFU-E to EPO, with 25-30% of CFU-E being EPO-independent. The number of BFU-E was much lower in these mice, suggesting that V617F acts in the most-differentiated erythroid progenitors to promote EPO_independent maturation. THE FACT THAT CENTRAL ERYTHROID FEATURE SOF PV ARE RECAPITULATED BY V617F ARGUES THAT IT IS THE PRIMARY AND DIRECT CAUSE OF PV. The effects on leukocytes were more variable and strain-dependent. No increase in plt count despite evidence of proviral integration in Mks, but there was a marked defect in plt function James C, Nature 2005;Wernig G, Blood 2006; Lacout C, Blood 2006; Zaleskas V, PLoS ONE 2006

Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice Transgenic line (FF1-Vav) with an expression of JAK2-V617F lower than the endogenous wild-type JAK2 developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia. Transgenic JAK2-V617F/MxCre mice had expression of JAK2-V617F approximately equal to wild-type JAK2 and developed a PV-like phenotype with increased hemoglobin, thrombocytosis, and neutrophilia. Higher levels of JAK2-V617F in mouse bone marrow by retroviral transduction caused a PV-like phenotype without thrombocytosis. Tiedt, R. et al. Blood 2008;111:3931-3940

JAK2 V617F allele burden in MPNs 20 40 60 80 100 JAK2 V617F allele burden (%) PV ET PMF postPV / ET MF Antonioli E, Haematologica 2007

Identification of disease subtypes in ET based on JAK2 V617F mutation JAK2V617F pos ET resembles PV: - increased erythropoiesis - higher leukocyte count - more venous thromboses - more frequent PV “transformation” - lower ferritin (p < 0.01) - lower MCV (p < 0.0001) - lower serum Epo (p < 0.0001) JAK2V617F neg ET - higher platelet counts Campbell et al Lancet 2005& Blood 2005; Antonioli et al Leukemia 2005;

Induction of thrombocytosis and myelofibrosis by MPLW515L MPLWT MPLW515L

Clinical correlates of MPL W515 mutations in ET mutant wild-type P value Median age, years 58 (22-84) 52 (8-97) 0.08 White blood cell count (x109/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 (x109/L) 960 ± 327 860 ± 309 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%) 994 ET pts 3.0% 418 V617Fneg 8.1% 12 (40%) W515K 18 (60%) W515L In 8 pts both MPLW515 and JAK2V617F AM Vannucchi et al., Blood, 2008

Essential Thrombocythemia 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 JAK2V617F or other clonal marker or no evidence of reactive thrombocytosis Minor Diagnostic combinations All four criteria must be met *, Tefferi A, 2007

ST ET PMF PV RARS-T 5q- CML 100% 60% 1- 4% 40% 60% 5- 8% 40% 60% 1- 4% 40% PMF 60% 5- 8% 40% PV 95% RARS-T 50% 50% 5q-  5% 95% CML 100% Vannucchi AM, ASH2007

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

Thrombosis-free survival in MPN Crude-analysis Years 1 2 3 4 5 6 7 8 9 10 Probability A GIMEMA retrospective study on 1,224 patients 67 397 339 275 235 206 168 139 120 95 82 PV Pts at risk 140 637 549 479 421 365 316 262 220 191 169 ET 28 190 123 94 78 68 60 48 40 36 30 MF Years Risultati di uno studio retropse4ttivo su oltre 1000 pz con MPD, multicentrioc, italiano, che è una fotografia dello stato attuale con le terapie attuali Vannucchi AM, Blood 2007 & unpubl.

Recurrent Thrombosis in PV or ET  Time after first thrombosis (months) 5.6%pt-yr 3.4%pt-yr 2.2%pt-yr Cumulative probability (%) 49.9% ET (n=259) PV (n=235) n=494 A retrospective GIMEMA study on 494 pts with first thrombosis La probabilita’ cumulativa di ricorrenza era del 17.7% a 2 anni, 30.8% a 5 anni, de.l 49.9% a 10 anni De Stefano V, Haematologica 2007

Cardiovascular risk factors (*) Risk Stratification in ET Risk category Age >60 or history of thrombosis Cardiovascular risk factors (*) Low NO Intermediate YES High (**) ---- Although their contribution to thrombotic risk is controversial, it is apporpiate counselinmg patients as concerns style of life, particularly smoking, and eventually correcting associated abnormalities such as hypertension or diabetes (*) Hyperthension, Hypercholesterolemia, Diabetes, Smoking; Familial thrombophilia. Their contribution to thrombotic risk is controversial

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

Thrombocytosis and Thrombosis: an Uneven Relationship - 2 ECLAP cohort 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* (x109/L) Hazard Ratio (95% CI) p-value 650 to 1,000 More than 1,000 Total thrombosis 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) (0.3-0.8) P=0.01 3) +JAK2V617F adjusted (0.3-1.1) 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 (x109/L) HR (95% CI) P (*) >15.0 1.74 (1.1-2.7) 0.01 Carobbio A, Blood 2007 WBC (x109/L) HR (95% CI) P (*) <7.2 1 7.2-8.7 2.4 (1.1-5.6) 0.04 8.7-10.4 2.7 (1.2-6.2) 0.02 >10.4 3.0 (1.3-6.9) 0.008 Carobbio 439 pts: count at diagnosis precits for thrombosis in the follow-up; Wolanskyj 322 Unlike Carobbio, Tefferi et al bllod (2007, letter) found an association of leukocytosis with both Arterial and Venous events at diagnosis, BUT NOT in follow-up unlike Carobbio. However, he did not perform multivariable time-dependent analysis. (*) Multivariable analysis

Leukocytosis and “conventional” risk factors in ET Low-risk High-risk P=.003 Cumulative probability of thrombosis in the follow-up Carobbio A et al, Blood 2007; JCO 2008

Rate of CV events (% pts/year) Interaction of leukocytosis and thrombocytosis on thrombotic risk in ET WBC Platelet count Rate of CV events (% pts/year) JAK2V617F mutated (%) RR (P=) <11,000 >1,000 1.59 26 1 <1,000 2.26 56 1.92 (0.034) >11,000 2.88 61 2.38 (0.026) 2.95 77 2.43 (0.017) Carobbio A, Blood 2008

V617F Status and Thrombosis in ET Venous thrombosis Arterial thrombosis Thrombosis at presentation 2.09 [1.44, 3.05] 1.96 [1.43, 2.67] 1.88 [1.38, 2.56] Ziakas PD et al, Haematologica 2008

V617F Burden and Thrombosis in ET (95%CI) P WT 1 Hetero 1.49 (0.9-2.5) 0.13 Homo 3.97 (1.3-11.7) 0.013 * adjusted for age, sex, previous thrombosis and leukocytosis Vannucchi AM et al, Blood 2007

Interactions between JAK2 V617F genotype and inherited thrombophilia 132 ET pts, 45 pts (34%) with major thrombosis. 83 pts (62.8%) V617F+, 8 pts (6%) were Homo RR JAK2 V617F+ 2.1 (1.1-3.8) Homo 3.7 (1.8-7.2) Hetero 1.9 (1.0-3.5) Homo vs Hetero 1.9 (1.2-3.2) RR JAK2+ Thr’ph’+ vs WT Thr’ph’- 4.4 (2.2-8.8) JAK2+ Thr’ph’- 2.1 (1.1-4.0) vs JAK2+Thr’ph’- 2.1 (1.3-3.4) De Stefano V, EHA 2008

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