Thrombosis In Myeloproliferative Neoplasms Research Articles

Genetic Markers of Hereditary Thrombophilia and Risk of Thrombotic Complications in Patients with Polycythemia Vera

Background. Thrombotic complications are one of the main problems of polycythemia vera (PV) treatment. They significantly impair the quality of life of these patients and may lead to the lethal outcome. A thrombotic event often precedes the diagnosis of this hematological disease. The pathogenesis of thrombosis in myeloproliferative neoplasms, PV, in particular, is a complex one. Prescription of antiaggregants in the absence of thrombosis and anticoagulants after a thrombotic event requires special attention and development of corresponding recommendations. The prescription of anticoagulants is impossible without taking into account the risks of hemorrhagic complications, which are also typical for myeloproliferative neoplasms. Aim. Assessment of the impact of hereditary thrombophilia genetic markers on the risk of thrombotic complications in patients with PV. Methods. The study examined 116 patients with PV, who were screened for markers of hereditary thrombophilia: factor V (G1691A, FV Leiden), prothrombin, methylene-tetrahydrofolate reductase (MTHFR), fibrinogen (F/), plasminogen activator inhibitor (PA/-1), and platelet fibrinogen receptor type ///A (GP///A). The incidence of these markers and their role in thrombosis in such patients was investigated. Results. The study provided data on the incidence of hereditary thrombophilia markers in patients with PV. Statistically significant differences in the incidence of these markers and homocysteine level were found between patients with thrombosis and without them. Conclusion. The information about the hereditary thrombophilia markers presence may be useful for the prescription of adequate antiaggregant and anticoagulant therapy for PV patients. Further research in this field is justified and it will probably demonstrate the relevance of hereditary thrombophilia markers as prognostic factors for thrombotic complications risk assessment.

Splanchnic vein thrombosis in myeloproliferative neoplasms: pathophysiology and molecular mechanisms of disease.

Myeloproliferative neoplasms (MPNs) are the most common underlying prothrombotic disorder found in patients with splanchnic vein thrombosis (SVT). Clinical risk factors for MPN-associated SVTs include younger age, female sex, concomitant hypercoagulable disorders, and the JAK2 V617F mutation. These risk factors are distinct from those associated with arterial or deep venous thrombosis (DVT) in MPN patients, suggesting disparate disease mechanisms. The pathophysiology of SVT is thought to derive from local interactions between activated blood cells and the unique splanchnic endothelial environment. Other mutations commonly found in MPNs, including CALR and MPL, are rare in MPN-associated SVT. The purpose of this article is to review the clinical and molecular risk factors for MPN-associated SVT, with particular focus on the possible mechanisms of SVT formation in MPN patients.

Incidence of Early Thrombosis in Myeloproliferative Neoplasms (MPN): A Prospective Analysis from the Gruppo Laziale of Ph-Negative MPN

Background Thrombotic episodes are the major complication in the follow-up of Philadelphia negative Myeloproliferative Neoplasms (MPN), with high morbidity and mortality, as reported in several retrospective studies. At present, however, few prospective data are available on the early incidence of these complications.Methods To address this issue, we report on 1087 patients [M/F 508/579, median age 67.6 years, interquartile range (IQR) 55.2 - 75.9] with newly diagnosed MPN enrolled in the prospective database of our regional cooperative group since January 2011. Of them, 571 (52.5%) had Essential Thrombocythemia (ET), 303 (27.9%) Polycythemia Vera (PV) and 213 (19.6%) Primary Myelofibrosis (PMF). The main clinical features at diagnosis of the whole cohort and according to the different MPNs are reported in the Table 1.Results On the whole, 22 episodes of thrombotic complications were reported in 1087 patients (2.0%) at a median interval from diagnosis of 18.2 months (IQR 7.4 - 29.7): in particular, 15 (68.1%) were arterial (8 cerebral, 2 coronaric, 4 in the lower limbs, 1 splancnic) and 7 (31.9%) venous (5 in the lower limbs and 2 in the upper limbs). As to the incidence of early thrombosis in the different MPNs, they were 13/571 (2.2%) in ET patients, 5/303 (1.6%) in PV patients and 4/213 (1.8%) in PMF patients (p=0.810): median time from diagnosis to thrombotic event was also similar in the 3 MPNs (p=0.311). The 4-year cumulative Thrombosis-Free Survival (TFS) of the whole cohort was 97.3% (95%CI 96.0 - 98.6): there was no difference among the 3 MPNs as to 4-year TFS [96.7% (95%CI 94.8 - 98.6) in ET, 97.8% (95%CI 95.9 - 99.7) in PV and 98.7% (95%CI 96.9 - 100) in PMF, respectively, p=0.668). Several clinical features at diagnosis (age, gender, Hb levels, WBC and PLT counts, spleen enlargement, JAK-2 V617F mutation and previous thrombotic events) were evaluated for a role in predicting thrombotic events: only age (p=0.009) and previous thrombotic events (p=0.009) were significant.Conclusions The incidence of early thrombosis seems low in the first 4 years after diagnosis of MPN based on our prospective database, without any difference among ET, PV and PMF: it is worth of note that only age and previous thrombotic events had a predictive role, thus confirming many retrospective reported data and reinforcing the prognostic value of old scoring system for thrombotic risk in MPN. [Display omitted] DisclosuresBreccia:Ariad: Honoraria; Pfizer: Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria. Latagliata:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria.

Thrombosis in Myeloproliferative Neoplasms Is Linked to Increased Neutrophil Extracellular Trap (NET) Formation

▪Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders that are associated with an elevated risk for thrombosis. The reason for this association is incompletely understood. While elevated white blood cell (WBC) count is a risk factor for thrombosis in some models, the pathogenic pathways linking WBC abnormalities to thrombosis in MPNs are not clear.Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA, histones and serine proteases that contain and kill pathogens. There is a growing body of evidence linking NETs to thrombosis in various disease states, including cancer (Demers et al, PNAS 2012). We explored the contribution of NETs to the pro-thrombotic phenotype of MPNs.Methods: NET formation was assessed in neutrophils isolated from patients with MPNs and age-matched controls by using previously published immunofluorescence (IF) methods. c-KIT enriched marrow cells from Vav-Cre/Jak2V617F Knock-In (KI) mice were grafted into lethally irradiated wild-type (WT) recipients leading to constitutive heterozygous expression of the mutation in hematopoietic cells. Inferior vena cava (IVC) stenosis model was used to assess thrombotic tendency in mice. Mice were treated with ruxolitinib by oral gavage at a dose of 90 mg/kg BID for 3 days prior to thrombosis and NETosis experiments. NET formation was stimulated by ionomycin (4 µM).Results: Neutrophils from patients with MPNs (n=14) demonstrated significantly higher NET formation as compared to those from age-matched controls (n=10; p=0.003). This was true across specific MPN diagnoses and driver mutations (JAK2, CALR, MPL). Ex-vivo treatment of neutrophils with ruxolitinib (at 300nM for 2.5 hours) resulted in abrogation of NETosis as assessed by IF studies and was comparable to the effect achieved with the addition of compounds known to inhibit NET formation such as specific PAD4 and NADPH inhibitors; no difference was observed in Annexin V-mediated apoptosis between ruxolitinib treated and untreated neutrophils suggesting that ruxolitinib effect was not confounded by alternative apoptosis pathways.We next evaluated NETosis and its effect on thrombosis in MPN mouse models driven by Jak2 V617F. Neutrophils isolated from Vav-Cre/Jak2V617F demonstrated increased NET production ex-vivo as compared to WT littermate controls. Vav-Cre/Jak2V617F KI mice were grafted into lethally irradiated WT and assessed for their prothrombotic phenotype. VavCre/Jak2V617F KI demonstrated significantly higher thrombus formation at 2 hours using the IVC stenosis model (45% versus 0% for Jak2V617F KI (n=11) and Jak2WT (n=8), respectively; p=0.04 by Fisher's exact test). This was associated with significantly higher levels of free dsDNA in the plasma of KI stenosis mice as compared to WT stenosis controls (p=0.03 by Mann-Whitney). Furthermore, IHC and IF studies of lung tissue demonstrated that Vav-Cre/Jak2V617F KI have widespread thrombosis as compared to controls and IF studies demonstrated higher content of citrullinated histone H3 in the lung suggesting increased NETosis at that site. Treatment of Vav-Cre/Jak2V617F KI mice with ruxolitinib significantly abrogated thrombus formation (45% versus 0% for untreated (n=11) and treated (n=8) mice, respectively; p=0.04 by Fisher's exact test).We noted that expression of PAD4, an essential driver of NETosis, is elevated in MPN patients (Rampal Blood 2014). We found that PAD4 content was higher in protein lysates of neutrophils isolated from MPN patients as compared to controls. Moreover, abnormal nuclear localization was observed in patients. These observations may support a role for PAD4 in the excess NETosis observed in MPNs.Conclusion: We found that neutrophils from MPN patients and Jak2 V617F KI mice are sensitized to NET formation that may be mediated by increased PAD4 expression and nuclear localization. In mice, the increased ex-vivo NET formation is associated with an increased thrombosis rate and an abundance of NET related products in thrombus and tissue. Treatment with ruxolitinib abrogates NET formation in humans and significantly reduces thrombus formation in MPN mice models.These findings provide a pathogenic link between the genetic drivers of MPNs and thrombosis in these diseases. Pharmacologic inhibition of JAK-STAT pathway to reduce NETosis and thrombosis should be further investigated. DisclosuresDeAngelo:Novartis: Consultancy; Ariad: Consultancy; Celgene: Consultancy; Baxter: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy.

Pro-Coagulant and Pro-Inflammatory Effect of Interferon Alpha in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Besides clinical factors (previous history of thrombosis, age > 60 years, cardiovascular risk factors), procoagulant phenotype, proadhesive properties of the endothelium and secretion of inflammatory cytokines, contribute to the pathogenesis of thrombosis in MPN. Pegylated interferon alpha (IFN) has obviously a great therapeutic efficacy on hematopoietic cell proliferation but its other biological effects particularly on hemostasis and inflammation have not been determined.The aim of the study was to determine whether IFN impacts the biological profile including endothelial and platelet markers compared to MPN patients treated by hydroxyurea (HU) and non-treated (NT) patients.Patients with polycythemia vera (PV) or essential thrombocythemia (ET) treated with IFN or HU or without any cytostatic drug were included. Platelet membrane glycoproteins and platelet activation were measured by flow cytometry. We evaluated shear-induced platelet aggregation (SIPA) at 4000 sec-1 that mimics stenotic arteries. We measured coagulation proteins by their activity and endothelial parameters (von Willebrand factor (VWF) antigen and activity, soluble thrombomodulin). Thrombin generation was measured in platelet poor plasma and platelet rich plasma. Platelet accumulation on immobilized collagen was measured using whole blood perfused at 1500 sec-1 in flow chambers. Statistical analysis was performed by ANOVA to compare the 3 groups of patients (p<0.05) and post-test Newman-Keuls to compare groups side-by-side (p<0.01).Eighty-eight patients were included: 28 treated by IFN (20 PV and 8 ET), 38 treated by HU (27 PV and 11 ET) and 22 NT (6 PV and 16 ET). All patients were treated with aspirin 75 or 100 mg/day. Prior history of thrombosis occurred in 25.7% of ET and in 31.5% of PV. During the follow up (> 6months), only 1 arterial and 2 venous thrombosis were observed.Hematological parameters and JAK2 V617F status was in the expected range in ET and PV patients.Surprisingly, we observed some significant effect of IFN on 1) platelets with a decreased GPVI expression compared to HU- and non-treated patients and increased levels of SIPA at 4000 sec-1. 2) endothelial cells with significantly increased levels of Willebrand antigen and activity. 3) coagulation parameters with decreased protein S activity and increased levels of factor VIII:C and fibrinogen (table 1). All the other parameters tested were similar in the 3 groups of patients.We had the opportunity to test 10 patients at least 6 months after IFN discontinuation. IFN was stopped for side effects in 6 patients, for complete molecular response in 2 and for normalization of hematological parameters in 2 others. GPVI expression, SIPA, VWF activity and antigen, protein S activity, FVIIIC and fibrinogen returned to levels similar to those of non-treated patients and were significantly different from the levels in IFN-treated patients (table 2). These results indicate that IFN was responsible for these biological modifications.In conclusion, these unexpected data showing an increase of procoagulant and inflammatory markers in IFN-treated MPN patients could be considered as a part of IFN biological activity. Though minimal, these alterations could require more intensive preventive anticoagulation than aspirin in patients with heterozygous FV Leiden or FII G20210A mutation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Splanchnic vein thrombosis in myeloproliferative neoplasms: risk factors for recurrences in a cohort of 181 patients.

We retrospectively studied 181 patients with polycythaemia vera (n=67), essential thrombocythaemia (n=67) or primary myelofibrosis (n=47), who presented a first episode of splanchnic vein thrombosis (SVT). Budd–Chiari syndrome (BCS) and portal vein thrombosis were diagnosed in 31 (17.1%) and 109 (60.3%) patients, respectively; isolated thrombosis of the mesenteric or splenic veins was detected in 18 and 23 cases, respectively. After this index event, the patients were followed for 735 patient years (pt-years) and experienced 31 recurrences corresponding to an incidence rate of 4.2 per 100 pt-years. Factors associated with a significantly higher risk of recurrence were BCS (hazard ratio (HR): 3.03), history of previous thrombosis (HR: 3.62), splenomegaly (HR: 2.66) and leukocytosis (HR: 2.8). Vitamin K-antagonists (VKA) were prescribed in 85% of patients and the recurrence rate was 3.9 per 100 pt-years, whereas in the small fraction (15%) not receiving VKA more recurrences (7.2 per 100 pt-years) were reported. Intracranial and extracranial major bleeding was recorded mainly in patients on VKA and the corresponding rate was 2.0 per 100 pt-years. In conclusion, despite anticoagulation treatment, the recurrence rate after SVT in myeloproliferative neoplasms is high and suggests the exploration of new avenues of secondary prophylaxis with new antithrombotic drugs and JAK-2 inhibitors.

Molecular biomarkers of thrombosis in myeloproliferative neoplasms

JAK2 mutations define polycythemia vera (PV), CALR and MPL mutations are specific to JAK2 unmutated essential thrombocythemia (ET) and primary myelofibrosis (PMF). We overviewed the current knowledge on the relationship between these phenotypic driver mutations and thrombotic complications that are major cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPN) particularly PV and ET. The JAK2 mutation is found in 50-60% of patients with ET and PMF. The International Prognostic Score for Thrombosis in ET (IPSET-thrombosis) identified JAK2 mutation as an independent risk factor and a 3-tiered prognostic model was devised. IPSET-thrombosis model outperformed the 2-tiered conventional risk stratification that includes age and thrombotic history. PV is usually associated with a JAK2 mutation and studies looking at the role of JAK2V617F allele burden associated with thrombosis are so far inconclusive. In PMF, the rate of major thrombosis is around 2%pt-yr and JAK2 mutation emerged as an independent risk factor for these events. Calreticulin/MPL (CALR) is the second most frequent mutation and occurs in half of JAK2 and MPL wild-type patients with ET and PMF. Despite the fact that these mutations are associated with high platelet counts, the risk of thrombosis compared with JAK2 and MPL mutated cases is significantly lower. The role of MPL in the prediction of thrombosis is of difficult demonstration due to the low frequency in ET and PMF. Therefore, these epidemiologic studies pointed out the role of JAK2V617F mutation as a major contributory factor for the pathogenesis of thrombosis in MPN. Abnormalities of blood cells arising from the clonal proliferation of hematopoietic stem cells may explain the switch to a procoagulant phenotype.

Common Variation at 6q25.3 (TULP4) Influences Risk for Arterial Thrombosis in Myeloproliferative Neoplasms

Inherent tendency for thrombosis is a major complication in myeloproliferative neoplasms (MPN). The molecular basis of thrombosis in MPN is not well understood, however, genetic factors have been proposed to play a role. To agnostically investigate the role of common germline variation in MPN thrombophilia, we performed genome-wide association studies (GWAS) in MPN patient cohorts characterized for arterial thrombosis (AT) events. In our discovery cohort (n=383) from Vienna, Austria, 18% of patients have suffered from AT after MPN diagnosis, and 33% of patients showed records for AT events at any time. Of this discovery cohort, a subset of patients (n=302) selected independently from thrombotic status was genotyped on the Affymetrix Genome-Wide SNP 6.0 array platform. After assessment of case-control setup and genotyping quality as standardly implemented in GWAS, confidently genotyped single nucleotide polymorphisms (SNPs) were tested for allelic association with occurrence of AT after MPN diagnosis. We observed an association signal beyond genome-wide statistical significance (P <5x10-8) at chromosome 6q25.3, tagged by rs6455579 and six additional closely related tag-SNPs. The 6q25.3 risk haplotype is common with a minor allele frequency (MAF) of ~8% in the Vienna MPN cohort. The minor allele rs6455579_C correlated with increased risk for AT. We next genotyped the full discovery cohort from Vienna (n=383) and a replication cohort from Pavia, Italy (n=505) using qPCR-based SNP genotyping for rs6455579 and computed odds ratios (ORs) to estimate the effect sizes of the association. In the Vienna cohort, rs6455579 genotype was significantly associated with both "AT after diagnosis" (P =1.90x10-8, ORhet=6.93) and "AT at any time" (P =6.96x10-4, ORhet=3.07). In the Pavia cohort, an association with "AT after diagnosis" could not be observed (P =0.14), however, the association of rs6455579 with "AT at any time" could be reproduced at formal statistical significance (P =7.92x10-3, ORhet=2.55). Moreover, Kaplan-Meier statistics on the Pavia cohort revealed a significant difference in AT-free survival after diagnosis upon rs6455579 genotype (log-rank-test P =0.009; rs6455579 homozygous major vs. heterozygous), underpinning the relevance of disease duration for the impact of the haplotype on MPN-related AT. To gain insight into the physiological mechanisms behind the 6q25.3 risk haplotype, we next evaluated the discovery cohort for correlations of the haplotype with clinical parameters other than AT. We could observe a significant trend for increased white blood cell (WBC) count in CALR mutated (n=90, P =2.50x10-3) but not in JAK2 mutated (n=182, P =0.52) ET and PMF patients carrying the risk allele. CALR mutated patients have been previously reported to exhibit significantly prolonged thrombosis-free survival as compared to JAK2 mutated patients. Thus, our observation indicates that the 6q25.3 germline risk haplotype might impact on WBC count most strongly in a subgroup of patients considered low risk for thrombosis based on the somatic mutational status. The 6q25.3 core haplotype (R2 >0.3) spans ~300 kilobases, covering intergenic sequence as well as promoter and 5' exons of the TULP4 gene. To test for the possibility of a rare coding variant in TULP4 or other more distant genes underlying the association through long-range linkage disequilibrium, we used genotype imputation in conjunction with the 1000 genomes reference panel to infer genotypes on all untyped variants (MAF>1%) in a 5 megabase region centered on the core haplotype. We did not detect any coding variants reflecting the association, and the tag-SNPs from the initial GWAS remained the most strongly associated variants. Causative non-coding genetic variation identified in GWAS is thought to exert its function through differential regulation of specific target genes. Therefore, we evaluated a potential influence of the 6q25.3 risk haplotype on TULP4 gene expression in peripheral blood (The Cancer Genome Atlas (TCGA) LAML dataset, RNA-Seq on 173 acute myeloid leukemia patients). Indeed we detected significantly decreased TULP4 expression in risk haplotype carriers (P =0.029), providing indirect evidence for reduced TULP4 transcript levels impacting on elevated risk for AT in MPN. Further studies will be required to functionally assess the potential role of TULP4 in MPN-related AT. DisclosuresGisslinger:Novartis: Honoraria, Research Funding, Speakers Bureau; Geron: Consultancy; Sanofi Aventis: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Incidence and Localization of Vascular Events in a Well Characterized Cohort of 477 Patients with Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are hematopoietic disorders characterized by bone marrow and blood myeloid cells proliferation. MPNs are secondary to the acquisition of clonal mutation. Polycythemia vera (PV) cases are mutated in JAK2V617F in 97% and JAK2 exon12 in 3%. Essential Thrombocythemia/ Primary Myelofibrosis (ET/PMF) cases are JAK2V617F mutated in 50-60%, CALR mutated in 20-30% and MPL mutated in 5-10%. Thromboses are the most frequent mid term complications in MPNs, and in most series the ratio arterial/venous thrombosis is 2/3 vs 1/3. The patients carrying CALR mutations have been recently reported as presenting a lower incidence of thrombosis, perhaps due to a lower leukocytes count and hemoglobin level.We report a cohort of 628 MPNs (ET and PV) patients followed in a single institution. JAK2, CALR and MPL mutational status is available to date in 477 cases. PV cases were divided into PV and masked PV (mPV; normal Hb/Ht level but increased red cell mass and high platelets count). Among the 477 patients, we identified 158 pts (33.1%) with PV, 57 pts (11.9%) with mPV, and 262 pts (54.9%) with ET. All PV and mPV cases were JAK2 mutated. In the ET group, 184 pts (70.2%) carried the JAK2 V617F mutation, 40 pts (15.3%) a CALR mutation, 5 pts (1.9%) a MPL mutation, and 33/262 pts (12.6%) were triple negative (JAK2V617F, CALR, MPL negative). All vascular events have been collected and the subtype (arterial or venous) and sites of thrombosis have been registered. Only the thrombotic events which occurred in the 2 years preceding the diagnosis of MPN were considered to be related to the MPN.We observed a similar incidence of thrombosis before and after diagnosis of MPN (22%, n=105 vs 21%, n=100). Among the 105 patients with prior thrombosis, 119 thrombotic events were recorded; during the follow-up, the 100 pts have presented 128 events of thrombosis. About one third of these 'post-diagnosis’ episodes were thrombotic recurrences. There were no significant differences in term of incidence of thrombosis before and after diagnosis according to the different phenotypes (PV 37 pts / 22% both; ET 19.1%, n=50 vs 19.8%, n=52) except for mPV: 18 pts (31.6%) with prior thrombosis vs 11 patients (19.3%) who presented a thrombosis after. This remained true among ET patients even when deciphering by the mutational status: JAK2V617F positive cases (24.5%, n=45 vs 23.9%, n=44), CALR positive (7.5%, n=3 vs 10%, n=4), MPL positive or triple negative cases (6.1%, n=2 vs 9.1%, n=3). We observed only 9 episodes of thrombosis in CALR mutated ET patients: 7 arterial events (3 cases of transient ischemic attack, 2 cases of ischemic stroke, 2 cases of acute myocardial infarction and 1 mesenteric ischemia) and 2 cases of venous thrombosis (1 deep venous thrombosis and 1 cerebral venous thrombosis).The frequency of CALR mutations was lower in our cohort than described in the literature, but allowed us to confirm the overall lower incidence of thrombosis in that subgroup of ET. In addition to previously published data we showed that venous thrombosis were extremely rare and that the majority of vascular events were arterial in CALR mutated patients who presented a thrombosis. Cerebral and myocardial infarctions were predominant. In parallel, we have performed a systematic CALR mutational analysis on 394 pts presenting a first unprovoked venous thrombosis without identify any mutant.Here we confirmed the very high incidence of thrombosis in MPNs, especially in JAK2 mutated cases, and showed that the frequency of vascular events remains very high even after the diagnosis of the MPN, demonstrating the lack of effective prevention of first episode or recurrence in this high-risk population. Future therapeutic strategies should better take into account all risk factors including the mutational status and the type of thrombotic events. DisclosuresNo relevant conflicts of interest to declare.

The Evaluation of Cardiac Disorders and Systemic Hypertension in JAK2 V617F Positive Patients at a Multicenter Institution

INTRODUCTION: Myeloproliferative neoplasms (MPN) represent a significant risk for arterial and venous thrombosis. The JAK2 V617F mutation is an acquired mutation common in patients with polycythemia vera and essential thrombocythemia. Studies have shown that both cardiovascular disorders and JAK2 V617F mutation are independent risk factors for thrombosis in MPN. Additionally in JAK2 V617F transgenic mice, there is reported association of cardiac hypertrophy and myeloproliferative neoplasms. However, there is limited clinical data regarding which cardiovascular risk factors are associated with JAK2 V617F mutations. Our study evaluated cardiac disorders in patients with and without JAK2 V617F mutation, and compared arterial and venous thrombosis rates between the two groups.METHODS: We performed a retrospective case series study at our multiinstitutional center. Demographic information was collected of patients who were evaluated for JAK2 V617F mutation from 2005 to 2014. 34 patients were female and 35 patients were male. Mean age was 64 .Cardiac disorders were characterized as coronary artery disease (CAD), atrial fibrillation (Afib), left heart failure (LHF), myocardial infarction (MI), and dysrhythmia (DYS) other than Afib. In total, thirty-four patients were JAK2 V617F positive while thirty-five patients were JAK2 V617F negative. Data was analyzed by SPSS software version 11.5 . We used pearson chi square test to compare the incidence of cardiac disorders and systemic hypertension (HTN) between two groups. Two tailed p-value <0.05 was considered statistically significant.RESULTS: 34 patients were female and 35 patients were male. Mean age was 64 .Baseline characteristics including mean age and gender were not significantly different between two groups. In JAK2 V617F positive patients, there was a statistically significant higher incidence of systemic hypertension and atrial fibrillation. The remaining characteristics were not significant. Additionally, we found no difference in rates of arterial and venous thrombotic events between two groups. We did not find any association between JAK2 V617F mutation and deep vein thrombosis (p= 0.154), stroke (p=0.289), pulmonary embolisms (p=0.538), and peripheral arterial disease (p=0. 572).TableP- VALUEJAK2 V617F MUTATIONCADMILHFAFIBHTNDYS0.2160.5380.289*0.018**0.012*0.289CONCLUSION: We identified systemic hypertension (p=0.012) and atrial fibrillation (p=0.018) to be strongly associated with JAK2 V617F mutation. This association has not been reported so far. Due to the retrospective nature of this case series, we are unable to determine whether there is a causal relationship between the presence of JAK2 V617F mutation and systemic hypertension and/or Afib. Further investigation is necessary to determine whether there is a causal relationship between the presence of JAK2 V617F mutation and systemic hypertension and/or Afib. DisclosuresNo relevant conflicts of interest to declare.

Thrombosis in Myeloproliferative Neoplasms

Thrombotic events are very frequent and represent the main cause of morbidity and mortality in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), mainly polycythemia vera and essential thrombocythemia. Pathogenesis of blood clotting activation in these diseases is multifactorial, and it involves various abnormalities of platelets, erythrocytes, and leukocytes, as well as dysfunctions of endothelial cells. These include not only elevations in the counts of circulating blood cells, arising from the clonal proliferation of hematopoietic progenitor cells, but also modifications of several physiological/molecular properties. Patients with MPN can be stratified in "high-risk" or "low-risk" thrombotic categories according to the age and history of thrombosis. The most commonly used front-line drugs for the treatment of high-risk patients include hydroxyurea and interferon alpha, whereas in low-risk patients, primary antithrombotic prophylaxis with aspirin is used. Future research should be focused on the evaluation of the role of biomarkers in identifying MPN patients at higher risk of thrombosis, who may benefit from primary thromboprophylaxis. Finally, a better understanding of the molecular events leading to the progress of the hypercoagulable state in MPN patients may provide appropriate tools for the development of targeted therapies based on reversal of coagulopathy.

The differential diagnosis of splachnic vein thrombosis

It was with great interest that we read the review regarding splachnic vein thrombosis in myeloproliferative neoplasms (Sekhar et al, 2013). We were surprised by the fact that screening for two important diseases predisposing to a splachnic vein thrombosis (SVT), namely antiphospholipid syndrome (APS) and paroxysmal nocturnal haemoglobinuria (PNH), was not mentioned in the ‘algorithm for the management of portal vein thrombosis/Budd Chiari syndrome’, which does not refer exclusively to patients with myeloproliferative neoplasms. In this algorithm, F5 R506Q (Factor V Leiden, FVL) and F2 G20210A (prothrombin G 20210A) mutation screening is detailed. We are not clear why the authors do not mention other inherited thrombophilias, such as antithrombin, Protein C and Protein S deficiency alongside the two aforementioned as potential risk factors for SVT. However, as many guidelines recommend anticoagulation with a vitamin K antagonist after SVT, to prevent further thrombotic events (De Stefano & Martinelli, 2010), the rational for screening for inherited thrombophilias is poor because there are no studies showing that such practice changes clinical management. The British Society for Haematology guidelines on thrombophilia testing state that ‘testing for heritable thrombophilias after a first episode of intraabdominal vein thrombosis has uncertain predictive value for recurrence (C). Decisions regarding duration of anticoagulant therapy in relation to the results of testing are not evidence-based’ (Baglin et al, 2010).

JAK Inhibitors Are Not Associated With a Reduction In Novel Markers Of Thrombosis In Myeloproliferative Neoplasms

BackgroundThrombosis is a common and sometimes unpredictable complication of the myeloproliferative neoplasms (MPN). MPN management is often directed by perceived thrombotic risk. Numerous factors are predictive for thrombosis including haematocrit, age and prior thrombotic event. More recently leucocyte count and JAK2V617F allele burden have been described as potentially contributing to this thrombotic risk profile. In addition, both platelet activation status and tissue factor expression have been shown to be elevated in MPN patients compared to controls and postulated to predict for thrombosis. Cytoreduction with hydroxycarbamide has been shown to decrease thrombotic risk in this population. The introduction of JAK inhibitors (JAKi) has had a profound effect upon the therapeutic landscape for MPN, however the potential effect of these drugs upon thrombotic risk is unknown. We postulated that treatment with JAKi may affect key surrogate markers for thrombosis. Materials and MethodsPatients were recruited prior to the commencement of JAKi therapy, either with ruxolitinib or SAR302503. Baseline citrated samples were taken pre drug administration and subsequent samples were collected following a minimum of 1 month of JAKi treatment for flow cytometric analysis on a Beckman Coulter Navios instrument. Platelet-neutrophil aggregates were defined as CD15+/CD42b + and CD15+/CD62p+. Platelet-monocyte aggregates were defined as CD14+/CD42b+ and CD14/CD62p+. Platelet activation was defined by expression of CD63 and CD62p. Monocyte tissue factor expression was defined as co-expression of CD14+ and CD142+. Activated neutrophils were defined as CD15/CD63+. ResultsWe evaluated 15 MPN patients (13 myelofibrosis and 2 polycythaemia vera) pre and post JAKi therapy as well as 14 MPN patients not receiving JAKi (10 patients on hydroxycarbamide, 1 on interferon-α and 3 on no treatment). Among the 15 MPN patients receiving JAKi, the median age was 64 years and the median duration of disease was 6 years and 1 month, 9 patients were JAK2V617F positive, 1 was MPLW515L positive, and 3 had a prior history of thrombosis (2 deep venous thromboses, 1 arterial thrombosis). Four patients received SAR302503 and 11 patients received ruxolitinib. At the time of sample collection, 8 patients were receiving aspirin and 1 was receiving warfarin.We found no statistically significant difference in surrogate markers of thrombosis between MPN patients on conventional therapy and MPN patients selected to receive JAKi treatment. When comparing paired samples pre and post JAKi we found a decrease in platelet monocyte aggregates, neutrophil activation and monocyte tissue factor expression following JAKi therapy but these results did not reach statistical significance (see table). Of note, there was a significant increase in monocyte count with treatment (p=0.0078) but no significant change with respect to neutrophil, total leucocyte and platelet count. No thrombotic events occurred during the study period. [Display omitted] ConclusionsGiven the substantial interest in the role of JAKi in MPN, it will be of key importance to evaluate the effects of these agents upon risk of thrombosis, a surrogate biomarker for that could be a demonstrated effect upon laboratory markers. In this study we were unable to detect a statistically significant effect of JAKi upon novel markers for thrombosis although the overall trend was for a decrease in monocyte activation despite an increase in monocyte count. This may be due to a number of factors including small sample size and short interval (1 month) between testing but warrants further investigation to address this important question. Disclosures:Harrison:Novartis: Honoraria; Sanofi : Honoraria.

Are MPNs Vascular Diseases?

A high risk of arterial and venous thrombosis is the hallmark of chronic myeloproliferative neoplasms (MPNs), particularly polycythemia vera (PV) and essential thrombocythemia (ET). Clinical aspects, pathogenesis and management of thrombosis in MPN resemble those of other paradigmatic vascular diseases. The occurrence of venous thrombosis in atypical sites, such as the splanchnic district, and the involvement of plasmatic prothrombotic factors, including an acquired resistance to activated protein C, both link MPN to inherited thrombophilia. Anticoagulants are the drugs of choice for these complications. The pathogenic role of leukocytes and inflammation, and the high mortality rate from arterial occlusions are common features of MPN and atherosclerosis. The efficacy and safety of aspirin in reducing deaths and major thrombosis in PV have been demonstrated in a randomized clinical trial. Finally, the Virchow's triad of impaired blood cells, endothelium and blood flow is shared both by MPN and thrombosis in solid cancer. Phlebotomy and myelosuppressive agents are the current therapeutic options for correcting these abnormalities and reducing thrombosis in this special vascular disease represented by MPN.

Splanchnic vein thrombosis in myeloproliferative neoplasms

Splanchnic vein thrombosis (SVT) is one of the most important complications of myeloproliferative neoplasms (MPN). Although MPN are common causes of SVT, the pathophysiological mechanisms underlying this predisposition, their epidemiology and natural history are not fully understood. Studies have concentrated on the generalized prothrombotic environment generated by MPN and their relationship with abnormal blood counts, thereby furthering our knowledge of arterial and venous thrombosis in this population. In contrast, there are few studies that have specifically addressed SVT in the context of MPN. Recent research has demonstrated in patients with MPN the existence of factors increasing the risk of SVT such as the presence of the JAK2 V617F mutation and its 46/1 haplotype. Features unique to the circulating blood cells, splanchnic vasculature and surrounding micro-environment in patients with MPN have been described. There are also abnormalities in local haemodynamics, haemostatic molecules, the spleen, and splanchnic endothelial and endothelial progenitor cells. This review considers these important advances and discusses the contribution of individual anomalies that lead to the development of SVT in both the pre-neoplastic and overt stage of MPN. Clinical issues relating to epidemiology, recurrence and survival in these patients have also been reviewed and their results discussed.

Thrombosis in Myeloproliferative Neoplasms With JAK2V617F Mutation

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders and are characterized by advanced proliferation and survival advantage. These abnormalities are considered to derive from the point mutation in exon 14 of the Janus kinase 2 genes (JAK2 V617F). The thrombosis rate and the high prevalence of JAK2V617F in patients with MPN suggest that there is an association between the 2 in MPN. Apart from the mutation, other variables are documented to cause endothelial dysfunction and involve in thrombotic tendency. Endothelial progenitor cells differentiated from hematopoietic stem cell in patients with JAK2V617F MPN play an indispensable role in initiating and modulating neoangiogenesis. Although a risk-oriented therapeutic approach has been applied to MPN treatments, the further study on pathogenesis of MPN may provide more novel preventions and therapies for MPN.

JAK2V617F Mutation Is Not Associated with Thrombosis in Behcet Syndrome

The Janus kinase 2(V617F) (JAK2 (V617F)) mutation is an acquired genetic defect that is considered to enhance thrombosis in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Thrombosis is also a well-defined component of Behcet syndrome (BS). The aim of this study was to determine the frequency of JAK2 ( V617F ) mutation in BS-associated thrombosis. A total of 152 patients with BS (62 with thrombosis and 90 without thrombosis) were enrolled. An additional 186 patients with MPNs and 107 healthy blood donors were included to serve as diseased and healthy controls, respectively. None of the patients with BS and healthy controls carried the JAK2 (V617F) mutation, whereas 67% of patients with MPNs were positive for JAK2 ( V617F ). The frequency of thrombosis in patients with MPNs was not statistically different between carriers and non-carriers of JAK2 ( V617F ) mutation. Our data suggest that JAK2 (V617F) is not directly related to thrombosis in MPNs and in other thrombotic entities, such as BS.

Venous thromboembolism in patients with essential thrombocythemia and polycythemia vera

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs), which generally follow a benign and indolent clinical course. However, venous thromboses are common and constitute the main cause of morbidity and mortality. The discovery of the JAK2V617F mutation and other biomarkers has advanced our understanding of these diseases. There is a strong association between the presence of the JAK2V617F mutation and the development of thrombosis in ET. If venous thrombosis presents with unusual manifestations, the diagnosis of a MPN, such as PV or ET, should be part of the differentials. Treatment of venous thrombosis in MPN follows the same principle as in other patients with venous thrombosis, but careful attention to primary and secondary prophylaxis in addition to heparin-induced thrombocytopenia should be given. Cytoreductive therapy is indicated in high-risk subgroups of PV and ET patients, and alternative therapeutic agents have different effects on risk of venous thrombosis. New therapeutic approaches are emerging, and JAK2 inhibitors, histone deacetylase inhibitors and next-generation anticoagulants are in various stages of clinical development for the treatment of MPN, but their exact role in thrombosis prevention and treatment remains unclear.

How to manage thrombosis in myeloproliferative neoplasms

In this article, an updated overview of risk factors and management of vascular complications in myeloproliferative neoplasms (MPN) is presented. The rate of thrombosis in patients with polycythemia vera, essential thrombocythemia, and primary myelofibrosis is elevated and a risk-oriented therapeutic approach is recommended to avoid inappropriate exposure to cytotoxic drugs on one side or suboptimal treatment on the other. Established thrombosis risk factors are age and previous vascular events but novel disease-related determinants such as leukocytosis and JAK2V617F mutational status and/or mutational burden are now under active investigation. The theory that elevated platelet count increases thrombosis risk in these diseases is now challenged. Recent recommendations by the European Leukemia Net suggest that MPN patients should be stratified and treated according to conventional risk factors (age and previous events) and that baseline leukocytosis and JAK2V617F mutation should be considered once more information is available and when they have been eventually validated in prospective studies.

Pathophysiology of thrombosis in myeloproliferative neoplasms

Life expectancy of patients with myeloproliferative neoplasms (MPNs) and particularly that of subjects with polycythemia vera (PV) and essential thrombocythemia (ET) has significantly increased over the last three decades, largely due to the use of cytoreductive treatments. Currently, polycythemia