Background: Polycythemia Vera (PV) is a Ph1-negative myeloproliferative neoplasm characterized by vascular thrombosis and poor survival. The polycythemic thrombogenesis is associated with erythrocytosis and red cell adhesiveness angiopathy. Recent studies reported a JAK/STAT-mediated reduction of the regulatory T cells (Tregs) (CD4+, CD25high, CD127low, FoxP3+) and loss of self-tolerance. Aims: We investigated Tregs, anti-endothelial cell antibodies (AECA), and endothelial, platelet and coagulation activation, in Polycythemia Vera (PV) and thrombosis. Methods: We enrolled 60WHO-defined PV patients (30 men, 30 women; mean age 45±10 years) without cardiovascular risk factors, autoimmune disease or thrombotic history. Of PV patients, 40/60 had thromosis includingmyocardial infarction (MI) (10/60) according to the WHO critera, deep vein thrombosis (20/60) and pulmonary embolism (10/60) on lower-limb ultrasonography and computed tomography angiography, respectively. All patients were evaluated for JAK2V617F allele burden, Tregs, AECA,Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1), Intercellular Adhesion Molecule-1 (ICAM-1),prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). JAK2V617F allele burden were analyzed by Polymerase chain reaction, Tregs were measured by flow cytometry,AECA, ELAM-1 and ICAM-1 by ELISA, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA.Complete blood hemostasis was studied by PFA-100 on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30). Results: The patients with thrombosis had JAK2V616F allele burden higher (> 50%) compared to patients without thrombosis (< 50Kroll%), lower Tregs (1,5±0,5% vs 3.5±1%),higher AECA (200±20% vs 110±10%), ELAM-1 (80±10 ng/ml vs 45±5 ng/ml), and ICAM (170 ng/mL±10 vs 110±10 ng/mL), longer PT (30±10 s vs 20±2 s) and PTT (60±10 s vs 38±5 s), lower Fib (90±20 mg/dl vs 120±20 mg/dl), higher DD (650±100 mg/l vs 300±50 mg/l) and shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (40±10 s vs 55±20 s) and C/EPI n.v. 84-160 s (35±5 s vs 60±10 s). The patients with thrombosis had shorter CT (INTEM 35±20 s vs 70±20 s, EXTEM 20±10 s vs 30±5 s), shorter CFT (INTEM 15±10 s vs 25±5 s EXTEM 18±10 s vs 28±5 s), longer MCF (INTEM 130±10 mm vs 90±10 mm, EXTEM 120±10 mm vs 82±10 mm), and lower LY-30 (INTEM 0.9% vs 15%, EXTEM 0.8% vs 15%). A positive correlation there was between Tregs and AECA, ELAM-1 and ICAM-1 and thrombosis. Summary/Conclusion: These findings shed new light on thrombotic pathogenesis in patients with PV.

Background:The essential thrombocythemia (ET) is a myeloid neoplasm characterized by platelet hyperreactivity and thrombosis. The daily low‐dose aspirin (ASA) is a cornerstone in the prevention of the thrombotic events. In the ET an accelerated platelet turnover translates in a renewal of the drug target shortening the duration of cyclooxygenase (COX‐1) inhibition and may dictate new dosing strategies particularly in ASA “low‐responders” patients.Aims:Therefore, we evaluated platelet count, β‐thromboglobulin (β‐TG) and platelet factor 4 (PF4), as markers of platelet activation, the platelet function activity (PFA), as indicator of ASA platelet sensitivity, the clotting time (CT), clot formation time (CFT) and maximum clot formation/firmness (MCF), as indicators of aspirinated platelet contribution to clot firmness.MethodsWe studied 60 patients (20 men, 40 women; mean age 51 years, range 32‐70) with ET according to WHO criteria. The mean duration of disease was 11 years. All patients were on ASA 100 mg once daily. Of the 60 patients, 45 were on anagrelide hydrochloride (daily dose 1.5 mg) (10 men, 35 women), 15 were on hydroxyurea (daily dose 2 mg) (10 men 5 women). None had inherited or acquired thrombotic risk factors. Sixty subjects served as controls. Platelets were measured by automated analyzer. β‐TG and PF4 were determined by ELISA. ASA platelet sensitivity and CT, CFT and MCF were measured by Platelet Function Analyzer (PFA‐100) and by ROTEM delta, respectively.Results:The mean platelet count was 455 ± 200 × 109/L. All patients had normal β‐TG and PF4 (12 ± 5 IU/ml and 4 ± 1 IU/ml), prolonged C/EPI closure time (T, unit: s, n.v. 84‐160 s) (249 ± 40 s), normal CT (CT, unit: s. n.v. 100‐240 s) (110 ± 20 s), normal CFT (CFT, unit: s, n.v. 30‐110 s) (45 ± 5 s) and normal MCF (MCF, unit: mm, n.v. 50‐72 mm) (61 ± 2 mm).Summary/Conclusion:These findings suggest that in ET patients the daily low‐dose ASA represents an optimal dosing strategy.

Background:The evidence that currently used cytoreductive agents may affect the risk of second cancer (SC) remains uncertain because patients with myeloproliferative neoplasms (MPN) may have an intrinsic propensity to develop new malignancies.Aims:We assessed the influence of exposure to cytoreductive drugs on the occurrence of SC in a large multicenter international nested case‐control study.Methods:Cases (n = 647) were polycythemia vera (PV, n = 216), essential thrombocythemia (ET, n = 317) and myelofibrosis (MF, n = 114) patients with SC, and controls (n = 1,234) were MPN patients who did not present or develop SC during a comparable observation period. For each case, up to 3 controls who were SC‐free at the index date (date of SC occurrence in cases) were matched by each center for sex, age at MPN diagnosis (± 5 years), date of MPN diagnosis (± 6 years) and MPN disease duration (± 3 years).Results:Cases The most frequent category of SC was represented by carcinoma (n = 426, 65.8%) with a trend towards an higher frequency in patients with ET and PV compared to MF (p = 0.079). In MF patients, carcinoma occurred closer to MPN diagnosis (p = 0.062). Hematological SC (HSC), non‐melanoma skin cancer (NMSC) and melanoma were 62, 127 and 32, respectively. In 25.8% of MF patients, HSC were synchronous with MPN diagnosis, and indolent non‐Hodgkin lymphoma and CLL were prevalent (10 out of 16 patients).Drug exposure Hydroxyurea: Overall, the percentage of cases treated with hydroxyurea (HU) was similar to that of controls (p = 0.793) both in monotherapy as well as in combination with other drugs (p = 0.310). In a cancer‐specific stratified multivariable model, HU showed a two‐fold higher risk of NMSC irrespective of line of treatment (OR = 2.28, 95% CI 1.15–4.51).Pipobroman: Cases with SC were significantly more exposed to this drug than controls (p = 0.018). The independent role of this drug in this association was found in multivariable analysis (OR = 2.10, 95% CI 1.09–4.06) particularly for NMSC (OR = 3.74, 95% CI 1.00 ‐ 14.01)Ruxolitinib: In 17 patients, this drug was given in first‐line monotherapy (n = 16 MF and n = 1 ET) and the exposure was greater in cases than in controls (p = 0.033) The association of ruxolitinib with SC was significantly higher in patients with NMSC whose risk was almost 4‐fold higher than in non‐exposed (OR = 3.87, 95% CI 1.18–12.75).IFN, busulfan, anagrelide: The proportion of patients treated with these drugs did not differ in comparison with controls. No association of these drugs with the risk of overall SC was observed in multivariable model.Impact of SC on MPN‐related outcomesAfter the occurrence of SC, 647 cases were followed with a median of 3.0 years (interquartile range: 1.1–5.3). Major thrombosis was recorded in 10.5%, corresponding 3.1% pts/year. Major bleeding, evolution in acute leukemia/myelofibrosis were documented in 5.3% and 5.7%, respectively.Summary/Conclusion:This nested case‐control study performed in rare diseases such as MPN, represents the most efficient way, among retrospective studies, to identify the association between drug exposure and outcomes. Further strength of this survey is the large number of patients recruited and followed up in a network of 30 European hematological centers.

The optimal duration of treatment with vitamin K antagonists (VKA) after venous thromboembolism (VTE) in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) is uncertain. To tackle this issue, we retrospectively studied 206 patients with MPN-related VTE (deep venous thrombosis of the legs and/or pulmonary embolism). After this index event, we recorded over 695 pt-years 45 recurrences, venous in 36 cases, with an incidence rate (IR) of 6.5 per 100 pt-years (95% confidence interval (CI): 4.9-8.6). One hundred fifty-five patients received VKA; the IR of recurrent thrombosis per 100 pt-years was 4.7 (95% CI: 2.8-7.3) on VKA and 8.9 (95% CI: 5.7-13.2) off VKA (P=0.03). In patients receiving VKA, the IR of recurrent thrombosis per 100 pt-years was 5.3 (95% CI: 3.2-8.4) among 108 patients on long-term VKA and 12.8 (95% CI: 7.3-20.7) after discontinuation among the 47 who ceased treatment (P=0.008), with a doubled risk of recurrence after stopping VKA (hazard ratio: 2.21, 95% CI: 1.19-5.30). The IR of major bleeding per 100 pt-years was 2.4 (95%: CI: 1.1-4.5) on VKA and 0.7 (95% CI: 0.08-2.5) off VKA (P=0.08). In conclusion, in MPN patients with VTE recurrent thrombosis is significantly reduced by VKA and caution should be adopted in discontinuation; however, the incidence of recurrence on treatment remains high, calling for clinical trials aimed to improve prophylaxis in this setting.