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Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) does not carry the 3 driver mutations (Triple negative (TN)) displaying, along with lower haemoglobin level and platelet number, significantly worse survival-rates. The malignant hemopoietic stem/progenitor cells compartment of TN MF pts has never been characterized. Here, we compared the in vitro effects of crucial factors of the inflammatory microenvironment on the functional behaviour of TN circulating CD34+ cells vs the JAK2(V617F) mutated counterparts. Methods: Peripheral blood was collected from 9 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) (n=5) mutated and TN (n=4). Circulating CD34+ cells from pts were enumerated by flow cytometry. Immunomagnetically isolated CD34+ cells were in vitro treated with selected inflammatory factors (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Interleukin-6) for 12/24 hours and migration/survival ability was investigated. The plasma concentration of these cytokines was evaluated by ELISA. Gene expression profiling analysis (GEP) was performed in CD34+ cells from 3 TN vs 3 JAK2(V617F) mutated pts (Human Transcriptome array 2.0, Affymetrix) and gene set enrichment analysis was conducted by GSEA. Results: GEP analysis showed a total of 165 genes differentially expressed (121 downmodulated and 44 upregulated in CD34+ cells from TN- vs JAK2(V617F) mutated pts). Specifically, we found that the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN-CD34+ cells. Consistent with the GEP data, the in vitro survival of untreated TN-CD34+ cells was significantly decreased as compared with the JAK2(V617F) mutated counterparts. GEP analysis identified also a network of genes involved in cell adhesion, proliferation and inflammation which were mainly down-regulated in TN-CD34+ cells. Specifically, GSEA highlighted an enrichment of cell adhesion, migration and chemotaxis signatures in TN-CD34+ cells. Accordingly, we found that the absolute number of circulating CD34+ and CD34+ CD184+ cells was significantly increased in TN pts as compared with the JAK2(V617F) mutated counterparts (p

Background: Microparticles (MPs) are small vesicles (0.1-1 micron) deriving from plasma membrane budding during homeostasis and cell activation. MPs express antigens and contain constituents from cell of origin and are increased in conditions that are characterized by high cell turnover or death, particularly inflammatory, autoimmune and neoplastic diseases. Myelofibrosis (MF) is a clonal neoplasia of the hematopoietic stem/progenitor cells characterized by distinctive abnormalities in megakaryocyte (MKC) development and platelet (PLT) activation. Mutations in 3 genes (JAK2, CALR, MPL) and chronic inflammation are the main pathogenetic drivers of MF. Ruxolitinib (RUX), a JAK1/2 inhibitor, suppresses both clonal myeloproliferation and release of proinflammatory cytokines, reducing splenomegaly and constitutional symptoms in around 50% of patients (pts). We hypothesized that MPs, as mediators of inflammation, could be overexpressed in MF and possibly predict responses to RUX. Aims: This study aims to: 1) enumerate circulating MK and PLT-derived MPs of MF pts; 2) evaluate the effect of RUX on MPs production by PLT and MK; 3) investigate whether circulating MK and PLT- MPs may be a biomarker of response to RUX. Methods: EDTA-anticoagulated peripheral blood from healthy donors (HD, n=10) and JAK2V617F positive MF pts (n=12) at intermediate-2/high IPSS risk was collected at baseline and 3 and 6 months after RUX therapy and immediately centrifuged. PLT (CD61+CD62P+) and MK (CD61+CD62P-)-derived MPs were analysed in PLT poor plasma samples by flow cytometry (CytoFLEX, Flow Cytometer-Beckman Coulter). The instrument was calibrated with MEGAMIX Beads (Beckman Coulter) with various diameters to cover the MPs (0.5 and 0.9 μm). Results: At 3 and 6 months, 5 out of 12 pts achieved a spleen response (R) according to 2013-IWG-MRT criteria. At baseline, the mean percentage of MK-derived MPs was significantly decreased (29±6 vs 72±5; p

The efficacy and safety of thrombopoietin receptor agonists (TRAs) in older patients with primary immune thrombocytopenia (ITP) are unknown. We investigated TRA response and switch, thrombotic/hemorrhagic risk, and sustained responses off-treatment (SROTs) in 384 patients with ITP aged ≥60 years. After 3 months, 82.5% and 74.3% of eltrombopag- and romiplostim-treated patients, respectively, achieved a response; 66.7% maintained the response (median follow-up, 2.7 years). Eighty-five (22.2%) patients switched to the alternative TRA; although no cross-toxicity was observed, 83.3% of resistant patients had a response after the switch. Thirty-four major thromboses (3 fatal) and 14 major hemorrhages (none fatal) occurred in 18 and 10 patients, respectively, while on TRAs and were associated with thrombosis history (subdistribution hazard ratio, 2.04, P = .05) and platelet count

Summary Background There is no evidence that phlebotomy alone is sufficient to steadily maintain haematocrit on target level in low-risk patients with polycythaemia vera. This study aimed to compare the efficacy and safety of ropeginterferon alfa-2b on top of the standard phlebotomy regimen with phlebotomy alone. Methods In 2017, we launched the Low-PV study, a multicentre, open-label, two-arm, parallel-group, investigator-initiated, phase 2 randomised trial with a group-sequential adaptive design. The study involved 21 haematological centres across Italy. Participants were recruited in a consecutive order. Participants enrolled in the study were patients, aged 18–60 years, with a diagnosis of polycythaemia vera according to 2008–16 WHO criteria. Eligible patients were randomly allocated (1:1) to receive either phlebotomy and low-dose aspirin (standard group) or ropeginterferon alfa-2b on top of the standard treatment (experimental group). Randomisation sequence was generated using five blocks of variable sizes proportional to elements of Pascal's triangle. Allocation was stratified by age and time from diagnosis. No masking was done. Patients randomly allocated to the standard group were treated with phlebotomy (300 mL for each phlebotomy to maintain the haematocrit values of lower than 45%) and low-dose aspirin (100 mg daily), if not contraindicated. Patients randomly allocated to the experimental group received ropeginterferon alfa-2b subcutaneously every 2 weeks in a fixed dose of 100 μg on top of the phlebotomy-only regimen. The primary endpoint was treatment response, defined as maintenance of the median haematocrit values of 45% or lower without progressive disease during a 12-month period. Analyses were done by intention-to-treat principle. The study was powered assuming a higher percentage of responders in the experimental group (75%) than in the standard group (50%). Here we report results from the second planned interim analysis when 50 patients had been recruited to each group. The trial is ongoing, and registered with ClinicalTrials.gov , NCT03003325 . Findings Between Feb 2, 2017, and March 13, 2020, 146 patients were screened, and 127 patients were randomly assigned to the standard group (n=63) or the experimental group (n=64). The median follow-up period was 12·1 months (IQR 12·0–12·6). For the second pre-planned interim analysis, a higher response rate in the experimental group was seen (42 [84%] of 50 patients) than in the standard group (30 [60%] of 50 patients; absolute difference 24%, 95% CI 7–41%, p=0·0075). The observed z value (2·6001) crossed the critical bound of efficacy (2·5262), and the stagewise adjusted p value early showed superiority of experimental treatment. Thus, the data safety monitoring board decided to stop patient accrual for overwhelming efficacy and to continue the follow-up, as per protocol, for 2 years. Under the safety profile, no statistically significant difference between groups in frequency of adverse events of grade 3 or higher was observed; the most frequently reported adverse events were neutropenia (four [8%] of 50 patients) in the experimental group and skin symptoms (two [4%] of 50 patients) in the standard group. No grade 4 or 5 adverse events occurred. Interpretation Supplementing phlebotomy with ropeginterferon alfa-2b seems to be safe and effective in steadily maintaining haematocrit values on target in low-risk patients with polycythaemia vera. Findings from the current study might have implications for changing the current management of low-risk patients with polycythaemia vera. Funding AOP Orphan Pharmaceuticals, Associazione Italiana per la Ricerca sul Cancro

Background. The treatment of choice in steroid-resistant ITP is still controversial, due to the recent advent of new drugs (anti-CD20 and thrombopoietin mimetics) which have encouraged a generalized tendency to delay splenectomy. Consequently, the importance to define the efficacy and safety of splenectomy in the long-term is substantial. Patients and Methods. We retrospectively analyzed the data of 233 ITP patients who underwent splenectomy between 1959 and 2001, in 6 different European hematological Institutions and have now a minimum follow-up of 10 years from surgery. Results. Of the 233 patients, 206 (88%) achieved a response (87% complete). Sixty-eight out of 206 (33%) responsive patients relapsed, mostly (75%) within 4 years from first response. In 92 patients (39.5%), further treatment was required after splenectomy, which was effective in 76 cases (83%). In 138 patients (59%) response was maintained, free of any treatment, at last contact. No significant association between baseline characteristics and likelihood of stable response was found. Overall, 73 (31%) and 58 (25%) patients experienced at least one infectious or hemorrhagic complication. A stable response to splenectomy was associated with a lower rate of infectious (p=0.004) and hemorrhages (p

Previous observational studies suggest that rituximab may be useful in the treatment of primary immune thrombocytopenia (ITP). This randomized trial investigated rituximab efficacy in previously untreated adult ITP patients with a platelet count of 20 × 109/L or less. One hundred three patients were randomly assigned to receive 40 mg/d dexamethasone for 4 days with or without 375 mg/m2 rituximab weekly for 4 weeks. Patients who were refractory to dexamethasone alone received salvage therapy with dexamethasone plus rituximab. Sustained response (ie, platelet count ≥ 50 × 109/L at month 6 after treatment initiation), evaluable in 101 patients, was greater in patients treated with dexamethasone plus rituximab (n = 49) than in those treated with dexamethasone alone (n = 52; 63% vs 36%, P = .004, 95% confidence interval [95% CI], 0.079-0.455). Patients in the experimental arm showed increased incidences of grade 3 to 4 adverse events (10% vs 2%, P = .082, 95% CI, −0.010 to 0.175), but incidences of serious adverse events were similar in both arms (6% vs 2%, P = .284, 95% CI, −0.035 to 0.119). Dexamethasone plus rituximab was an effective salvage therapy in 56% of patients refractory to dexamethasone. The combination of dexamethasone and rituximab improved platelet counts compared with dexamethasone alone. Thus, combination therapy may represent an effective treatment option before splenectomy. This study is registered at http://clinicaltrials.gov as NCT00770562.

In 816 patients with 2016 World Health Organization-defined polycythemia vera (PV) enrolled in a multicenter retrospective study, we investigated the predictive value of Charlson comorbidity index (CCI) and body mass index (BMI) on thrombosis, progression to post-PV myelofibrosis (PPV-MF) and survival. Patients were subgrouped according to CCI=0 (58.1%, no comorbidities) or CCI≥1 (41.9%) and according to normal/underweight (BMI&nbsp

Background Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. Methods Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. Results The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. Conclusions Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Background Previous reports highlighted the potential short and mid-term therapeutic activity and safety of rituximab in adult patients with autoimmune thrombocytopenias. Objectives Primary objectives of this study were to evaluate the long-term efficacy and toxicity profile. Patients and methods From October 1999 to April 2005, 37 adults patients, median age 54 years, with active and symptomatic autoimmune thrombocytopenias (30 idiopathic thrombocytopenic purpura, 1 idiopathic thrombocytopenia and neutropenia, 4 thrombocytopenia and concomitant undifferentiated connective tissue disease, 2 thrombocytopenia and concomitant B-cell lymphoprolipherative disorders) that had relapsed or were refractory to at least a full course of steroid therapy received weekly infusions of rituximab 375 mg/m2 for 4 weeks. The median interval from diagnosis to rituximab was 34 months (1–264 months) and the platelets median count was 11 x 109/L. The following parameters of efficacy and toxicity were considered: rate of complete and partial response, time to initial and maximum response, relapse rate, relapse free survival, treatment free survival, short and long-term toxicity. The possible prognostic influence of some clinical and laboratory parameters on the patients outcome were also analyzed. Results Complete and partial response (platelets count ≥ 100 x 109/L and ≥ 50 x 109/L) were 20/37 (54%) and 7/37 (19%), respectively. In most of the patients the time to initial and maximum response was prompt, already before the second administration of rituximab. The median period of observation from treatment was 22 months (1–48 months). Nine out 27 responding patients relapsed; 18/37 patients (49%) remained relapse free and 20/37 (54%) did not necessitated further therapy. A shorter interval period from the time of diagnosis and rituximab administration (≤ vs > 36 months) was associated with a lower relapse free survival (p= 0.03). During the period of rituximab administration, 2 patients experienced short term toxicity with one case of serum sickness syndrome; no infectious or other significant long term toxic complications were documented.. Conclusion Rituximab administration may allow to achieve long-lasting remission in nearly 50% of patients suffering from autoimmune thrombocytopenia with good toxic profile. The possibility to achieved long lasting response appeared related with an earlier timing of administration.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

BACKGROUND: After ruxolitinib discontinuation, the outcome of patients with myelofibrosis (MF) is poor with scarce therapeutic possibilities. METHODS: The authors performed a subanalysis of an observational, retrospective study (RUX-MF) that included 703 MF patients treated with ruxolitinib to investigate 1) the frequency and reasons for ruxolitinib rechallenge, 2) its therapeutic effects, and 3) its impact on overall survival. RESULTS: A total of 219 patients (31.2%) discontinued ruxolitinib for ≥14 days and survived for ≥30 days. In 60 patients (27.4%), ruxolitinib was rechallenged for ≥14 days (RUX-again patients), whereas 159 patients (72.6%) discontinued it permanently (RUX-stop patients). The baseline characteristics of the 2 cohorts were comparable, but discontinuation due to a lack/loss of spleen response was lower in RUX-again patients (P =.004). In comparison with the disease status at the first ruxolitinib stop, at its restart, there was a significant increase in patients with large splenomegaly (P 10 mg twice daily predicted better spleen (P =.05) and symptom improvements (P =.02), the reasons for/duration of ruxolitinib discontinuation and the use of other therapies before rechallenge were not associated with rechallenge efficacy. At 1 and 2 years, 33.3% and 48.3% of RUX-again patients, respectively, had permanently discontinued ruxolitinib. The median overall survival was 27.9 months, and it was significantly longer for RUX-again patients (P =.004). CONCLUSIONS: Ruxolitinib rechallenge was mainly used in intolerant patients; there were clinical improvements and a possible survival advantage in many cases, but there was a substantial rate of permanent discontinuation. Ruxolitinib rechallenge should be balanced against newer therapeutic possibilities.

Recently, treatment of immune‐mediated thrombotic thrombocytopenic purpura (ITTP) has changed with the advent of caplacizumab in clinical practice. The International Working Group (IWG) has recently integrated the ADAMTS‐13 activity/autoantibody monitoring in consensus outcome definitions. We report three ITTP cases during the coronavirus disease 2019 pandemic, that received a systematic evaluation of ADAMTS‐13 activity and autoantibodies. We describe how the introduction of caplacizumab and ADAMTS‐13 monitoring could change the management of ITTP patients and discuss whether therapeutic choices should be based on the clinical response alone. ADAMTS‐13 activity/antibodies were assessed every 5 days. Responses were evaluated according to updated IWG outcome definitions. These kinetics, rather than clinical remission, guided the therapy, allowing early and safe caplacizumab discontinuation and sensible administration of rituximab. Caplacizumab was cautiously discontinued after achieving ADAMTS‐13 complete remission. These cases illustrate that prospective ADAMTS‐13 evaluation and use of updated IWG definitions may improve real‐life patients’ management in the caplacizumab era.

The current COVID-19 pandemic requires revisiting our current approach to major blood disorders, including ITP (Immune Thrombocytopenia), stirring up the production of several disease-specific practical guidelines. This report describes an updated version of consensus-based practical guidelines on the management of ITP, adapted to the Italian health system and social context. It highlights the role of the hematologist in offering guidance for choosing differentiated approaches in relation to specific circumstances and is intended to provide them with a useful tool for sharing the decision-making process with their patients. Probably, the greatest risk to avoid for a patient with suspected, ongoing or relapsed ITP - that is not severe enough to place him or her at risk for major bleeding - is to be infected in non-hospital and hospital healthcare settings. This risk must be carefully considered when adapting the diagnostic and therapeutic approach. More in detail, the document first addresses the appropriate management for COVID-19 negative patients with newly diagnosed ITP or who experience a relapse of previous ITP, according to first and second lines of treatment and then the management of COVID-19 positive patients according to their severity, from paucisymptomatic to those requiring admission to Intensive Cure Units (ICU). The pros and cons of the different treatments required to correct platelet count are discussed, as are some specific situations, including chronic ITP, splenectomy, thromboembolic complication and anti COVID-19 vaccination.

Up to 30% immune thrombocytopenia (ITP) patients achieve a sustained remission off-treatment (SROT) after discontinuation of thrombopoietin receptor agonists (TPO-RAs). Factors predictive of response are lacking. Patients aged ≥18 years with newly diagnosed or persistent ITP were treated with eltrombopag for 24 weeks. Primary end-point was SROT: the proportion of responders that were able to taper and discontinue eltrombopag maintaining the response during a period of observation (PO) of six months. Secondary end-points included the association between some immunological parameters (TPO serum levels, cytokines and lymphocyte subsets) and response. Fifty-one patients were evaluable. Primary end-point was achieved in 13/51 (25%) treated patients and 13/34 (38%) patients who started the tapering. Baseline TPO levels were not associated with response at week 24 nor with SROT. Higher baseline levels of IL-10, IL-4, TNF-α and osteopontin were negative factors predictive of response (P = 0·001, 0·008, 0·02 and 0·03 respectively). This study confirms that SROT is feasible for a proportion of ITP patients treated with eltrombopag. Some biological parameters were predictive of response.

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No abstract available

The impact of ruxolitinib therapy on evolution to blast phase (BP) in patients with myelofibrosis (MF) is still uncertain. In 589 MF patients treated with ruxolitinib, we investigated incidence and risk factors for BP and we described outcome according to disease characteristics and treatment strategy. After a median follow-up from ruxolitinib start of 3 years (range 0.1-7.6), 65 (11%) patients transformed to BP during (93.8%) or after treatment. BP incidence rate was 3.7 per 100 patient-years, comparably in primary and secondary MF (PMF/SMF) but significantly lower in intermediate-1 risk patients (2.3 vs 5.6 per 100 patient-years in intermediate-2/high-risk patients, P

Myelofibrosis (MF) is characterized by chronic inflammation and hyper-activation of the JAK-STAT pathway. Infections are one of the main causes of morbidity/mortality. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, may further increase the infectious risk. Monocytes are critical players in inflammation/immunity through cytokine production and release of bioactive extracellular vesicles. However, the functional behavior of MF monocytes, particularly during RUX therapy, is still unclear. In this study, we found that monocytes from JAK2V617F-mutated MF patients show an altered expression of chemokine (CCR2, CXCR3, CCR5) and cytokine (TNF-α-R, IL10-R, IL1β-R, IL6-R) receptors. Furthermore, their ability to produce and secrete free and extracellular vesicles-linked cytokines (IL1β, TNF-α, IL6, IL10) under lipopolysaccharides (LPS) stimulation is severely impaired. Interestingly, monocytes from RUX-treated patients show normal level of chemokine, IL10, IL1β, and IL6 receptors together with a restored ability to produce intracellular and to secrete extracellular vesicles-linked cytokines after LPS stimulation. Conversely, RUX therapy does not normalize TNF-R1/2 receptors expression and the LPS-driven secretion of free pro/anti-inflammatory cytokines. Accordingly, upon LPS stimulation, in vitro RUX treatment of monocytes from MF patients increases their secretion of extracellular vesicles-linked cytokines but inhibits the secretion of free pro/anti-inflammatory cytokines. In conclusion, we demonstrated that in MF the infection-driven response of circulating monocytes is defective. Importantly, RUX promotes their infection-driven cytokine production suggesting that infections following RUX therapy may not be due to monocyte failure. These findings contribute to better interpreting the immune vulnerability of MF and to envisaging strategies to improve the infection-driven immune response.

Ruxolitinib (RUX), the first JAK1/JAK2 inhibitor approved for myelofibrosis (MF) therapy, has recently been associated with the occurrence of second primary malignancies (SPMs), mainly lymphomas and non-melanoma skin cancers (NMSCs). We analyzed the incidence, risk factors and outcome of SPMs in 700 MF patients treated with RUX in a real-world context. Median follow-up from starting RUX was 2·9years. Overall, 80 (11·4%) patients developed 87 SPMs after RUX start. NMSCs were the most common SPMs (50·6% of the cases). Multivariate analysis demonstrated that male sex [hazard ratio (HR): 2·37, 95% confidence interval (95%CI): 1·22–4·60, P=0·01] and thrombocytosis>400×109/l at RUX start (HR:1·98, 95%CI: 1·10–4·60, P=0·02) were associated with increased risk for SPMs. Risk factors for NMSC alone were male sex (HR: 3·14, 95%CI: 1·24–7·92, P=0·02) and duration of hydroxycarbamide and RUX therapy>5years (HR: 3·20, 95%CI: 1·17–8·75, P=0·02 and HR: 2·93, 95%CI: 1·39–6·17, P=0·005 respectively). In SPMs excluding NMSCs, male sex (HR: 2·41, 95%CI: 1·11–5·25, P=0·03), platelet>400×109/l (HR: 3·30, 95%CI: 1·67–6·50, P=0·001) and previous arterial thromboses (HR: 3·47, 95%CI: 1·48–8·14, P=0·004) were shown to be associated with higher risk of SPMs. While it is reassuring that no aggressive lymphoma was documented, active skin surveillance is recommended in all patients and particularly after prolonged hydroxycaramide therapy; oncological screening should be triggered by thrombocytosis and arterial thrombosis, particularly in males.

Simple Summary Patients with polycythemia vera, a myeloproliferative neoplasm, are at increased risk of thrombosis and progression to myelofibrosis. However, no disease-specific risk factors have been identified so far. Extracellular vesicles, released from a broad variety of cells, are receiving increasing attention for their effects on cell-to-cell communication. In addition, they play a role in cancer and thrombosis. Interestingly, circulating microbial components/microbes have been recently indicated as potential modifiers of inflammation and coagulation. Here, we identified a signature of thrombosis history and marrow fibrosis by analyzing the phenotype and the microbial DNA cargo of the circulating extracellular vesicles after isolation from the plasma of patients with polycythemia vera. These data may support the role of extracellular vesicles as liquid biomarkers of aggressive disease, thus contributing to refining the prognosis of polycythemia vera. Abstract Polycythemia vera is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. However, no disease-specific risk factors have been identified so far. Circulating extracellular vesicles (EVs) are mostly of megakaryocyte (MK-EVs) and platelet (PLT-EVs) origin and, along with phosphatidylethanolamine (PE)-EVs, play a role in cancer and thrombosis. Interestingly, circulating microbial components/microbes have been recently indicated as potential modifiers of inflammation and coagulation. Here, we investigated phenotype and microbial DNA cargo of EVs after isolation from the plasma of 38 patients with polycythemia vera. Increased proportion of MK-EVs and reduced proportion of PLT-EVs identify patients with thrombosis history. Interestingly, EVs from patients with thrombosis history were depleted in Staphylococcus DNA but enriched in DNA from Actinobacteria members as well as Anaerococcus. In addition, patients with thrombosis history had also lower levels of lipopolysaccharide-associated EVs. In regard to fibrosis, along with increased proportion of PE-EVs, the EVs of patients with marrow fibrosis were enriched in DNA from Collinsella and Flavobacterium. Here, we identified a polycythemia-vera-specific host/microbial EV-based signature associated to thrombosis history and marrow fibrosis. These data may contribute to refining PV prognosis and to identifying novel druggable targets.

The role of circulating microvesicles (MVs) in Myelofibrosis (MF) and Essential Thrombocythemia (ET) is far to be defined. Here we found that 1) circulating megakaryocyte-MVs were reduced in MF and ET while platelet-MVs were increased; 2) the proportion of circulating megakaryocyte- and platelet-MVs was associated with disease severity in MF; 3) ruxolitinib normalized the profile of circulating megakaryocyte- and platelet-MVs in spleen responders MF patients only. Of note, a cut-off value of 19.95% of circulating megakaryocyte-MVs predicts ruxolitinib spleen response. In light of these findings, circulating megakaryocyte/platelet-MVs may have a tissue specific diagnostic and prognostic role in MF.

Sequential use of the TPO-RAs romiplostim and eltrombopag in ITP patients failing either agent was retrospectively evaluated to assess efficacy and impact of clinical characteristics on outcome. Patients were grouped into 5 categories: efficacy issues: 1st TPO-RA failure; loss of response; non-efficacy issues: platelet fluctuations; patient's preference; adverse event development. Either one TPO-RA sequence was analyzed at 3 month and at last follow-up. 106/546 patients on TPO-RA underwent switch and 65% achieved, regained or maintained a short- term response independent of switch sequence, gender or age; lower response rates were associated with lines of previous therapy; disease duration lowers probability to respond. Clinically, patients switched for efficacy issue did not differ from those switched for non-efficacy issues. Response was achieved/regained in 57.8% of patients switched for efficacy issues, the lowest response rates were observed in non-responders to 1st TPO-RA; 80% of patients switched for non-efficacy issues maintained a response. Platelet fluctuation resolved in 44.4%. Of the 49 patients evaluable for long-term outcome, 27 were in response on therapy; 16 discontinued the TPO-RA for reasons other than efficacy, while only 6 were non responders. We confirm the efficacy of TPO-RA switch; once achieved, response to the 2nd TPO-RA seems durable.

Recently, the myelofibrosis secondary to PV and ET prognostic model (MYSEC-PM) was introduced to assess prognosis in myelofibrosis (MF) secondary to polycythemia vera and essential thrombocythemia (post-PV and post-ET MF), replacing the International Prognostic Scoring System (IPSS) and/or Dynamic IPSS (DIPSS) that was applied for primary MF (PMF). In a cohort of 421 ruxolitinib (RUX)-treated patients (post-PV and post-ET MF: 44.2%), we evaluated the following: (1) disease phenotype, responses, and toxicity to RUX; and (2) performance of the MYSEC-PM in post-PV or post-ET ME. While the IPSS failed to correctly stratify post-PV or post-ET MF patients at diagnosis, the MYSEC-PM identified 4 risk categories projected at significantly different survival probability (P < .001). Additionally, the MYSEC-PM maintained a prognostic value in post-PV and post-ET MF also when used over time, at RUX start. Notably, the MYSEC-PM reclassified 41.8% and 13.6% of patients into a lower and higher risk category, respectively. Finally, patients at intermediate-1 risk had significantly higher spleen responses and lower hematological toxicities compared to higher risk patients. Compared to PMF, post-PV and post-ET MF presented a more hyperproliferative disease, with higher leukocyte and/or platelet count and hemoglobin levels both at diagnosis and at RUX start. Despite comparable response rates, post-PV and post-ET MF had lower rate of RUX-induced anemia and thrombocytopenia at 3 and 6 months. The study validates MYSEC-PM in post-PV and post-ET MF prognostication. Post-PV or post-ET MF represents a separate entity compared to PMF in terms of clinical manifestations and toxicity to RUX. (C) 2018 Elsevier Inc. All rights reserved.

Objectives To evaluate the efficacy of a salvage treatment with rituximab (RTX) in adults with primary immune thrombocytopenia (ITP), in terms of short-term response and long-term response (LTR, i.e., probability to achieve and maintain response) and to identify biological and clinical predictors of response. Methods We retrospectively evaluated the outcome of patients with primary ITP treated with standard dosage RTX (375 mg/m2 × 4) as salvage therapy in five Italian centers. One hundred and three patients, median age of 46 yr, were included. The median period of observation was 59 months. Results Response (R) and complete response (CR) were documented in 57 (55%) and 37 (36%) patients, respectively. Patients younger than 40 yr had a higher probability to achieve CR (P = 0.025). Younger women (age < 40 yr) had a significantly higher probability to achieve R and CR (P = 0.039 and P = 0.009, respectively). The estimated LTR rate was 36% and 31% after 48 and 72 months, respectively; female sex (P = 0.033) and younger age (P = 0.021) were associated with better LTR. Younger women had the highest LTR rate (P = 0.006). Response duration was associated with the obtainment of CR after RTX (CR vs. partial response, P = 0.002). Conclusions The effect of RTX salvage treatment appears higher in younger women, with LTR rate possibly approaching that of splenectomy.

No abstract is available for this article.

Background Neutropenic patients with persistent fever despite antibiotic therapy are managed with empirical or pre-emptive antifungal therapy. The aim of the present study was to evaluate the current clinical use and efficacy of these two approaches in patients with high risk hematologic conditions. Design and Methods An electronic medical record system, the "Hema e-Chart", was designed and implemented to collect information prospectively on infectious complications, particularly on invasive fungal diseases, in patients with hematologic malignancies treated with chemotherapy and/or autologous or allogenic hemopoietic stem cell transplantation. The patients were enrolled from Hematology units distributed widely across Italy. Results Three hundred and ninety-seven adults with hematologic malignancies treated with chemotherapy with persistent fever and suspected invasive fungal disease were evaluable for the study (190 treated had been treated with empirical antifungal therapy and 207 with pre-emptive antifungal therapy). There was a significantly lower incidence of proven/probable invasive fungal diseases in patients treated with empirical antifungal therapy (n=14, 7.4%) than in patients treated with pre-emptive therapy (n=49, 23.7%) (P

OBJECTIVE: A novel mechanism of platelet destruction involving the CD47/ signal regulatory protein-α (SIRPα) system has recently been suggested in a mouse model of immune thrombocytopenia (ITP). The CD47 molecule serves as ligand for SIRPα receptor and as receptor for thrombospondin acting as antagonistic to phagocyte activity and a regulator of apoptosis, respectively. In this study, we evaluated if the CD47/SIRPα axis may be involved in the apoptosis and clearance of platelets in human ITP. MATERIALS AND METHODS: Using flow cytometry, we characterized whether expression of CD47 on fresh and in vitro-aged platelets- and of SIRPα receptor on CD14-derived dendritic cells (DCs), macrophages, circulating DCs, and monocytes is reduced is ITP; whether the in vitro platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRPα in ITP; and whether platelets are more susceptible to the CD47-induced death signal in ITP. RESULTS: We demonstrated that low platelet count in ITP is not due to increased phagocytosis associated with decreased expression of CD47 on the platelet surface and, despite reduced SIRPα expression, blockage of SIRPα on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs. In contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages, and platelets are not healthy because they show increased apoptosis and are resistant to CD47-induced death signal. CONCLUSIONS: Our results demonstrate that the CD47 pathway in ITP patients abnormally modulates platelet homeostasis.

International audience; Established risk factors for thrombosis in essential thrombocythemia (ET) include age (≥60 years) and previous vascular events. Recently, also leukocytosis has been proposed in risk stratification of ET patients. We report a retrospective study on 532 ET patients followed for a median of 7.6 years. Sixty-four patients (12%) developed 95 thrombotic events during follow-up. Together with the high-risk condition, a white blood cell (WBC) value above 11 × 10/L, corresponding to the fourth percentile value, significantly correlated with a higher thrombotic risk ( = 0.033) by Cox proportional hazards. Moreover, the cumulative risk of thrombosis was significantly higher in high-risk patients with WBC >11 × 10/L. JAK2 V617F mutation did not correlate with thrombosis. Overall, 123 (23%) patients died. Three independent parameters were noted as prognostic factors for survival in multivariate analysis: age >60 years, leukocytosis >11 × 10/L, and hemoglobin level below normal values. Based on these parameters, three groups of risk were defined, with significantly different survivals. Baseline leukocytosis correlated with a higher thrombotic risk in high-risk patients and identified a cohort of patients with worse survival.

Prior thrombosis is a well-established risk factor for re-thrombosis in polycythemia vera and essential thrombocythemia but scarce data are available on the rate of re-thrombosis and the optimal strategy for prevention of recurrence.We retrospectively estimated the rate of recurrence in a multicenter cohort of 494 patients (poly-cythemia vera/essential thrombocythemia 235/259) with previous arterial (67.6%) or venous thrombosis (31%) or both (1.4%). First thrombosis was cerebrovascular disease in 191 cases, acute coronary syndrome in 106, peripheral arterial thrombosis in 44, and venous thromboembolism in 160. Microcirculatory events were not computed.Thrombosis recurred in 166 patients (33.6%), with an incidence of 7.6% patient-years. Sex, diagnosis (polycythemia vera or essential thrombocythemia), and presence of vascular risk factors did not predict recurrence, whereas age60 years did (multivariable hazard ratio [HR], 1.67; 95% confidence interval [CI] 1.19-2.32). Increased leukocyte count at the time of the first thrombosis was a risk factor for recurrence in patients60 years old (HR 3.55; 95% CI 1.02-12.25). Cytoreduction halved the risk in the overall cohort (HR 0.53; 95% CI 0.38-0.73) and the combination with antiplatelet agents or oral anticoagulants was more effective than administration of single drugs. Significant prevention of rethrombosis was independently achieved in patients with venous thromboembolism by both oral anticoagulants (HR 0.32; 95% CI 0.15-0.64) and antiplatelet agents (HR 0.42; 95% CI 0.22-0.77), in those with acute coronary syndrome by cytoreduction (HR 0.30; 95% CI 0.13-0.68), and in those with cerebrovascular disease by antiplatelet agents (HR 0.33; 95% CI 0.16-0.66). The overall incidence of major bleeding was 0.9% patient-years and rose to 2.8% in patients receiving both antiplatelet and anti-vitamin K agents.In patients with polycythemia vera and essential thrombocythemia, cytoreduction protects against recurrent thrombosis, particularly after acute coronary syndrome. The contemporary use of oral anticoagulants (after venous thromboembolism) or antiplatelet agents (after cerebrovascular disease or venous thromboembolism) further improves the protective effect. Such findings call for prospective studies aimed at investigating whether strategies tailored according to the type of first thrombosis could improve prevention of recurrences.

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To the editor: Since the discovery that mutations in the 5′ untranslated region (UTR) of ANKRD26 are responsible for an autosomal-dominant form of thrombocytopenia ( ANKRD26 -RT),[1][1] 21 affected families were reported.[2][2] A study analyzing this series of patients suggested that ANKRD26 -RT

Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V–positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.

Rituximab is active in chronic lymphocytic leukemia (CLL) and may interfere with autoantibodies production in some immune diseases. We report the results of rituximab treatment in 7 patients with CLL-associated symptomatic autoimmune diseases refractory to standard immunosuppressive therapies: warm antibody hemolytic anemia (AHA) 4 patients, cold agglutinin disease (CAD) 1, immune thrombocytopenia (IT) 1, axonal degenerating neuropathy (ADN) 1. Rituximab was given at the dose of 375 mg/m2 per week for 4 weeks. One patient with AHA and one with CAD achieved complete normalization of hemoglobin levels and laboratory signs of haemolysis, with response duration (RD) of 8+ and 38+ months, respectively. In the patient with IT, complete remission was reached after the first week of treatment and RD was 6 months. The patient with ADN achieved a marked neurological improvement after rituximab therapy, with RD of 12 months. Retreatment of both patients with IT and ADN was effective. Rituximab may be an alternative agent for the treatment CLL-associated autoimmune diseases.

Essential thrombocythemia (ET) is a chronic myelo-proliferative disorder typical of middle age. However, it has also been observed in children and young adults.1–2 Major thrombotic episodes and microvascular disturbances have been described in young ET patients, but the real risk for vascular complications has not been clearly established, and specific therapeutic approaches have been investigated.3–7 In this study, we retrospectively analyzed a cohort of 76 thrombocytemic patients, younger than 40 years at diagnosis, with the following aims: (1) to evaluate thrombotic and hemorrhagic complications; (2) to specify the treatment adopted; and (3) to report pregnancy outcomes.

BACKGROUND: A risk score for invasive mold disease (IMD) in patients with hematological malignancies could facilitate patient screening and improve the targeted use of antifungal prophylaxis. METHODS: We retrospectively analyzed 1,709 hospital admissions of 840 patients with hematological malignancies (2005-2008) to collect data on 17 epidemiological and treatment-related risk factors for IMD. Multivariate regression was used to develop a weighted risk score based on independent risk factors associated with proven or probable IMD, which was prospectively validated during 1,746 hospital admissions of 855 patients from 2009-2012. RESULTS: Of the 17 candidate variables analyzed, 11 correlated with IMD by univariate analysis, but only 4 risk factors (neutropenia, lymphocytopenia or lymphocyte dysfunction in allogeneic hematopoietic stem cell transplant recipients, malignancy status, and prior IMD) were retained in the final multivariate model, resulting in a weighted risk score 0-13. A risk score of < 6 discriminated patients with low (< 1%) versus higher incidence rates (> 5%) of IMD, with a negative predictive value (NPV) of 0.99, (95% CI 0.98-0.99). During 2009-2012, patients with a calculated risk score at admission of < 6 had significantly lower 90-day incidence rates of IMD compared to patients with scores > 6 (0.9% vs. 10.6%, P

Hydroxyurea (Hydroxycarbamide; HU) is commonly used for the long-term treatment of patients with Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs). It is considered a first-choice agent for the treatment of these disorders as underlined by the European Leukemia Net Consensus Conference [1], although it is formally approved for this indication in some countries only. The drug is reportedly well tolerated in the large majority of subjects, although systemic and/or localized toxicities have been reported. Consensus criteria for definition of “intolerance” to HU have been described; patients who develop intolerance are candidate for second-line therapy and, more recently, for investigational drugs. However, no epidemiologic information about the occurrence of the most clinically significant HU-associated adverse events is yet available. In this study, the authors report on a multicenter series of 3,411 patients who were treated with HU among which 184, accounting for 5% of total, developed significant drug-related toxicities. These data provide an estimate of the frequency and a detailed characterization of clinically significant HU-related toxicities; these information have relevance for the management of MPN patients who require second-line therapy after developing HU-related intolerance.

No abstract is available for this article.

[Graphic][1] Introduction . Availability of the 2 TPO-RAs Romiplostim (Rom) and Eltrombopag (El) offers an effective treatment option for primary ITP patients (pts). However, some pts are either not responsive or lose response - i.e. desired platelet (plt) count achieved but not sustained over time, or experience wide fluctuations in plt count with either TPO-RA. Adverse events may cause treatment discontinuation. Finally, ptOs preference may be an important issue considering the different route and timing of administration of the two agents. Availability of two TPO-RAs for clinical use, with different molecular structure and site of binding within the TPO receptor, makes it appealing to try switching with the aim of overcoming treatment limitations of either agent. The present survey offers insight into outcome of TPO-RA switching in a group of ITP pts treated at 8 Centers representative of the Italian territory. Patients. Charts of ITP pts on treatment who underwent TPO-RA switch were retrospectively reviewed. Results . Between Jan 2009 and Feb 2015, 57 of 249 pts on TPO-RA (22,9%) underwent switch: El ˆ Rom 26/57 (45.6%), Rom ˆEl 31/57 (54.4%). Median age at 1st TPO-RA administration was 55 yrs (range 16-81); M/F = 23/34. Median disease duration prior to 1st TPO: 58 mos (range 2-648). Median lines of previous therapy 3 (range 1-6; splenectomy: 23/57, 40.4%). Overall 42/57 pts (73,7%) had received maximum product dose as per prescribing information prior to switch. Table 1 summarizes reasons for TPO-RA switch and outcome. Overall, 32/57 pts (56.1%) achieved, regained or maintained a response upon switching. The majority of pts (39/57, 68.5%) were switched for efficacy issues, i.e. failure to respond to 1st TPO (27 pts) or response loss (12 pts); among these 39 pts, 48.7% responded to the 2nd TPO-RA. One pt lost response to Rom because of development of neutralizing antibodies; response was regained upon switching to El. In this subgroup of pts, disease duration and lines of previous therapy (but not splenectomy status) seem to have an impact on response to switching. Each one month increase in disease duration determines a 0.7% decrease in the odds of achieving a response (WaldOs test p=0.065). More than 2 lines of therapy determine a 72% decrease in the odds of achieving a response (WaldOs test p=0.077). Either TPO-RA switch sequence was equally effective in yielding response (FisherOs exact test p=0.752) and age at 1st TPO-RA had no impact on response. Of the 18 pts switched for reasons other than efficacy, 13 (72.2%) maintained a response on the 2nd TPO-RA: 5/6 switched for plt count instability (counts stabilized = 2/5 responding pts), 4/7 switched for ptOs preference, 4/5 switched for side effects. Four pts (1 plt count instability, 3 ptOs preference) underwent Odouble switchO (i.e. Rom ˆ El ˆ Rom): re-exposure to Rom was not associated with response loss. Discussion . Switching enables approximately 56% of pts to achieve, regain or maintain a plt response; switching for inefficacy yields lower response rates (48.7%) compared to switching for reasons other than efficacy (72.2%) Plt counts fluctuation stabilized in 40% of pts. Re-exposure to R in the 4 pts who underwent Odouble switchO was not associated with response loss, confirming absence of tachyphylaxis with this TPO-RA. Our results are in line with those reported by Khellaf (Haematologica 2013) and Gonzales-Porras (BJH 2014): TPO-RA switch can be a safe and appealing treatment option for ITP pts who experience suboptimal results with either agent. | REASON for SWITCHING | n (%) | NR (%) | R (%) | CR (%) | | | --------------------- | --------- | --------- | --------- | --------- | | | All | 57 (100) | 25 (43.9) | 14 (24.6) | 18 (31.5) | | | El->Rom | 26 (45.6) | 10 (38) | 8 (31) | 8 (31) | | | Rom->El | 31 (54.4) | 10 (48.4) | 6 (19.4) | 10 (32.3) | | | 1st TPO-RA failure | 27 (47.4) | 16 (59) | 4 (15) | 7 (26) | | | El->Rom | 15 | 7 (47) | 3 (20) | 5* (33) | | | Rom->El | 12 | 9 (75) | 1 (8) | 2 (17) | | | Loss of response | 12 (21.1) | 4 (33) | 2 (17) | 6 (50) | | | El->Rom | 4 | 2 (50) | 1 (25) | 1 (25) | | | Rom->El | 8 | 2 (25) | 1 (12.5) | 5 (62.5) | | | Plt count fluctuation | 6 (10.5) | 1 (17) | 3 (50) | 2 (33) | | | El->Rom | 2 | | 1 | 1 | | | Rom->El | 4 | 1 | 2 | 1 | | | PtOs preference | 7 (12.3) | 3 (42.9) | 2 (28.6) | 2 (28.6) | | | El->Rom | | | | | | | Rom->El | 7 | 3 | 2 | 2 | | | Adverse eventii | 5 (8.8) | 1 (20.0) | 3 (60.0) | 1 (20.0) | | | El->Rom | 5 | 1 | 3 | 1 | | | Rom->El | | | | | | * CR: complete response; R: response; NR: no response (Rodeghiero et al, Blood 2009) * *1 NR secondary to neutralizing antibodies development * ii1 hepatic enzyme increase, 1 CPK increase; 2 skin rash; 1 retinal thrombosis. Table 1. Disclosures De Stefano: Janssen Cilag: Research Funding; Shire: Speakers Bureau; Amgen: Speakers Bureau; Bruno Farmaceutici: Research Funding; Novartis: Research Funding, Speakers Bureau; Roche: Research Funding; Celgene: Speakers Bureau; GlaxoSmithKline: Speakers Bureau. [1]: /embed/inline-graphic-2.gif

Evidence suggests that the JAK2 V617F mutation is associated with an increased risk of first thrombosis in patients with essential thrombocythemia (ET). Whether this mutation is also a risk factor for recurrent thrombosis is currently unknown. To investigate the impact of the JAK2 V617F mutation on the risk of recurrent thrombosis in patients with ET, we carried out a multicentre retrospective cohort study. We recruited 143 patients with previous arterial (64.4%) or venous major thrombosis (34.8%) or both (0.8%); 98 of them (68.5%) carried the mutation. Thrombosis recurred in 43 of the patients (30%); overall, after adjustment for sex, age, presence of vascular risk factors, and treatment after the first thrombosis, the presence of the JAK2 mutation did not predict recurrence (multivariable hazard ratio, HR, 0.88, 95% CI 0.46-1.68). Indeed, the individuals homozygous for the JAK2 V617F (allele burden >50%) mutation had an increased risk of recurrence in comparison with wild-type patients (HR 6.15, 95% CI 1.51-24.92). In conclusion, a homozygous JAK2 V617F mutation is an independent risk factor for recurrent thrombosis in patients with ET. © Springer-Verlag 2009.