Thrombosis In Myeloproliferative Neoplasms Research Articles

The CHIP-clinic as the catalyst of preventive medicine

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVD) and is a precursor stage to the BCR-ABL negative chronic myeloproliferative neoplasms (MPNs). These diseases are acquired stem cell neoplasms, arising due to mutations in the hematopoietic stem cell. The most prevalent is the JAK2V617F (JAK2) mutation, which potently generates reactive oxygen species (ROS), and accordingly contributes greatly to the chronic inflammatory state and the increased risk of thrombosis in MPNs. The MPNs are largely underdiagnosed blood cancers with a long pre-diagnostic phase of several years, when the elevated blood cell counts are considered reactive to smoking, blood clots, infections or chronic inflammatory diseases. Since the JAK2 mutation as CHIP-JAK2 associates with an increased risk of CVD and an increased risk of hematological and non-hematological cancers there is an urgent need to explore and validate the JAK2 mutation as a novel risk factor for CVD and to establish CHIP-clinics, which in an interdisciplinary collaboration between experts from several disciplines, and ensure timely diagnosis of the undiagnosed MPN patient and associated comorbidities. We envisage studies of the JAK2 mutation in large CVD cohorts to deliver the “Proof of Concept” for the JAK2 mutation to be implemented as a novel, highly important risk factor for CVD. These novel preventive strategies are considered to have the potential of reducing morbidity and mortality in a large population of citizens and patients, carrying the thrombosis- and CVD-promoting JAK2 mutation.

Association between JAK2V617F variable allele frequency and risk of thrombotic events in patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are a group of chronic disorders of the bone marrow characterised by the overproduction of clonal myeloid stem cells. The most common driver mutation found in MPNs is a point mutation on exon 14 of the JAK2 gene, JAK2V617F. Various studies have suggested that measuring the variable allele frequency (VAF) of JAK2V617F may provide useful insight regarding diagnosis, treatment, risks and outcomes in MPN patients. In particular, JAK2V617F has been associated with increased risk of thrombotic events, a leading cause of mortality in MPNs. The aim of this study was to determine if JAK2V617F VAF was associated with clinical outcomes in patients with MPN. JAK2V617F VAF was determined by quantitative PCR (qPCR) in a cohort of 159 newly diagnosed MPN patients, and the association of JAK2V617F VAF and risk of thrombosis was examined in this cohort. We observed a significantly higher JAK2V617F VAF in PV and PMF versus ET. A significant association was observed between JAK2V617F VAF and risk of thrombotic events. When patients were stratified by thrombotic events prior to and post diagnosis, an association with JAK2V617F VAF was only observed with post diagnosis thrombotic events. Of note, these associations were not observed when looking at each MPN subtype in isolation. We have shown that a higher JAK2V617F VAF is associated with thrombotic events post MPN diagnosis. JAK2V617F VAF may therefore provide a valuable prognostic indicator for risk of thrombosis in MPNs.

Management of thrombosis in myeloproliferative neoplasms

Management of thrombosis in myeloproliferative neoplasms

Anticoagulation for splanchnic vein thrombosis in myeloproliferative neoplasms: a systematic review and meta-analysis

BackgroundOptimal anticoagulation management in patients with myeloproliferative neoplasms (MPN) experiencing splanchnic vein thrombosis (SpVT) requires balancing risks of bleeding and recurrent thrombosis. ObjectivesWe conducted a systematic review and meta-analysis to assess the incidence of bleeding and thrombosis recurrence in patients with MPN-SpVT. MethodsWe included retrospective or prospective studies in English with ≥10 adult patients with MPN-SpVT. Outcomes included recurrent venous thrombosis (SpVT and non-SpVT), arterial thrombosis, and major bleeding. Pooled rates per 100 patient years with 95% CIs were calculated by DerSimonian–Laird method using random-effects model. ResultsOut of 4624 studies screened, 9 studies with a total of 443 patients were included in the meta-analysis with median follow-up of 3.5 years. In the 364 patients with MPN-SpVT treated with anticoagulation, pooled event rate for major bleeding was 2.8 (95% CI, 1.5-5.1; I2 = 95%), for recurrent venous thrombosis was 1.4 (95% CI, 0.8–2.2; I2 = 72%), and for arterial thrombosis was 1.4 (95% CI, 0.6-3.3; I2 = 92%) per 100 patient years. Among 79 patients (n = 4 studies) who did not receive anticoagulation, pooled event rate for major bleeding was 3.2 (95% CI, 0.7-12.7; I2 = 97%), for recurrent venous thrombosis 3.5 (95% CI, 1.8-6.4; I2 = 88%), and for arterial thrombosis rate 1.6 (95% CI, 0.4-6.6; I2 = 95%) per 100 patient years. ConclusionPatients with MPN-SpVT treated with anticoagulation have significant risks for both major bleeding and thrombosis recurrence. Further studies are necessary to determine the optimal anticoagulation approach in patients with MPN-SpVT.

Predictive significance of high neutrophil ratio for thrombosis in myeloproliferative neoplasms: JSH-MPN-R18 subanalysis.

Thrombosis in myeloproliferative neoplasms (MPNs) is an important clinical problem, and risk-stratified management is essential. To identify the clinical characteristics of thrombosis in patients with MPNs, a nationwide multi-institutional retrospective analysis (JSH-MPN-R18) was conducted. The aim of the present study was to perform a sub-analysis of JSH-MPN-R18 findings to clarify the predictive parameters for thrombosis among complete blood count (CBC) results. Among the patients enrolled in JSH-MPN-R18, those with essential thrombocythemia (ET; n = 1152) and polycythemia vera (PV; n = 456) were investigated. We analyzed and compared CBC parameters between patients with and those without any thrombotic events using Welch's T-test. Statistical analyses were performed using the R statistical software. Thrombotic events were observed in 74 patients with ET. In multivariate analysis, only the neutrophil ratio was slightly but significantly higher for ET patients with thrombosis than for those without (p < 0.05). Of note, the absolute neutrophil count (aNeu) was considered a useful predictive tool for thrombosis among patients classified as low-risk according to the revised International Prognostic Score of Thrombosis for Essential Thrombocythemia. Among PV patients, those with thrombosis showed significantly higher hematocrit and aNeu than did those without thrombosis. As a thrombosis-associated factor, the neutrophil ratio was slightly but significantly elevated in patients with ET. This myeloid skew might reflect a higher value of JAK2 V617F allelic frequency in patients with ET with thrombosis; this was not clarified in JSH-MPN-R18. Further accumulation of evidence, including genetic information for JAK2 and other passenger mutations, is warranted.

Antiphospholipid antibodies and the risk of thrombosis in myeloproliferative neoplasms.

The morbidity and mortality of BCR-ABL-negative myeloproliferative neoplasia (MPN) patients is highly dependent on thrombosis that may be affected by antiphospholipid antibodies (aPLA) and lupus anticoagulant. Our aim was to evaluate the association of the aPLA together with platelet receptor glycoprotein (GP) Ia/IIa c.807C>T CT/TT genotypes and thrombotic complications in patients with MPNs. The study included 108 patients with BCR-ABL-negative MPN with data of previous thrombosis. Two different screening and one confirmatory test for the lupus anticoagulant were performed. Thrombotic complications were present in 59 (54.6%) subjects. aPLA were more frequently found in MPN patients with thrombosis vs no thrombosis (25.4 and 6.1%; p = 0.007). MPN patients with arterial thrombosis were more frequently positive for aPLA vs no arterial thrombosis (38.8 and 11.9%; p = 0.001). aPLA were more frequently found in patients with cerebrovascular events vs other arterial thrombotic complications or no thrombosis, respectively (39.3, 6.1, and 12.9%; p < 0.001). MPN patients with thrombosis were more frequently positive with aPLA and had platelet receptor GP Ia/IIa c.807C>T CT/TT genotypes compared to MPN patients without thrombosis (18.6 and 2.0%; p = 0.006). aPLA alone or with coexistence with platelet receptor GP Ia/IIa c.807C>T CT/TT polymorphism could be associated with thrombotic complications in patients with MPN.

Splanchnic Vein Thrombosis in Myeloproliferative Neoplasms: Treatment Considerations and Unmet Needs

Patients who develop splanchnic vein thrombosis (SVT) in the setting of a myeloproliferative neoplasm (MPN) are at risk for complications including portal hypertension, bleeding, thrombosis, and death. Prompt multidisciplinary treatment is thus necessary to prevent long-term sequelae. However, optimal management strategies are not well established due to a paucity of data. In this review, we very briefly discuss the epidemiology, pathophysiology, and prognosis of MPN-SVT and then more comprehensively explore treatment considerations of MPN-SVT, including anticoagulation, endovascular/surgical intervention, and cytoreductive therapy. We will also highlight current gaps in our knowledge of MPN-SVT and conclude by suggesting future directions to optimize the treatment of MPN-SVT and improve outcomes.

Global Hemostatic Assays for Risk Stratification of Thrombosis in Myeloproliferative Neoplasms (MPN): A Systematic Review and Meta-Analysis

Background: Arterial and venous thromboembolism are major complications of myeloproliferative neoplasms (MPN), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The pathogenesis of thrombosis in MPN is not fully understood. Global hemostatic assays, including thrombin generation assay (TGA), rotational thromboelastometry (ROTEM), and thromboelastography (TEG), have been evaluated as predictive markers of hypercoagulability in MPNs. However, validation of these and other laboratory surrogates is lacking. The objective of this systematic review is to summarize the literature and to examine the utility of global hemostatic assays as predictive markers for hypercoagulability and occurrence of thrombosis in MPNs. Methods: A systematic literature search was performed using MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews from inception to April 14, 2022, restricted to the English language. Criteria for eligible studies were studies that reported the results of TGA, ROTEM, and TEG in ET, PV, or PMF adult patients. Studies were required to have ≥10 subjects in each group. Two reviewers (A.T. and T.C.) independently searched the literature and extracted data from eligible studies. Disagreements were resolved by consensus or a third reviewer (C.K) when necessary. The methodological quality of included studies was appraised using the Newcastle - Ottawa Quality Assessment Scale (NOS) assessment tool. A meta-analysis was planned given sufficient data from included studies. The study protocol is available on PROSPERO (CRD42022325893). Results: A total of 815 records were retrieved from the literature search. After screening by title and abstract, 745 records were excluded. The remaining 70 references underwent full text review, 30 of which met eligibility criteria and were included in the analysis. These 30 studies (15 full-texts and 15 abstracts) collectively enrolled 926 controls, 700 ET, 556 PV, and 192 PMF patients. The number of studies that reported TGA, ROTEM, and TEG were 26, 3, and 3, respectively. The number of participants in each study ranged from 25 to 212 including the controls. Two studies originated from Oceania, 1 from North America, and 27 from Europe. JAK2 mutation was reported only in 15 studies and only two studies reported quantitative variant allele frequencies. There were 5 studies that did not have healthy controls. All of these studies were cross sectional analyses; no longitudinal prospective age/sex matched controlled studies have been published. Of the 26 TGA studies, comparison of TGA parameters were made between MPN vs. controls (15 studies), MPN with thrombosis vs. without thrombosis (3 studies), JAK2 positive vs. negative MPNs (11 studies), and MPN with vs. without treatment (6 studies). There is heterogeneity in the types of samples (platelet poor plasma or platelet rich plasma), trigger reagents (amounts of tissue factor, phospholipid, and thrombomodulin), and the automated platforms that were used. Significant differences in the endogenous thrombin potential (9 studies), peak height (10 studies), lag time (6 studies), and velocity index (3 studies) were found between MPN and controls. Thirteen studies reported rates of arterial and venous thrombotic outcomes in MPN patients, which ranged from 19 to 45%. Only 1 study found an association of TGA parameters and thrombosis. Two ROTEM studies showed ET having higher maximum clot firmness and shorter clot formation time compared to controls. Two TEG studies showed increased alpha angle between MPN and controls and between PV and controls. Risk of bias assessment with NOS showed summary scores ranging 1-3 for abstracts and 4-9 for full text studies. We were not able to perform meta-analysis due to insufficient data and the high heterogeneity in study objectives and laboratory methods. Conclusions: Studies were limited by small sample sizes and inconsistent results and do not comprehensively reflect the effects of treatment, duration of disease, or clonal status. The role of these global hemostatic assays in predicting thrombotic events remains inconclusive. Future studies are necessary to establish the usefulness and validity of these surrogate markers as predictors of thrombotic complications in the MPNs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Risk of Thrombosis in Myeloproliferative Neoplasms after Sars-Cov-2 Vaccination

Background SARS-CoV-2 infection has been associated with a poor prognosis in patients with Philadelphia-negative myeloproliferative neoplasms (MPN).(Passamonti et al. Lancet Haematol. 2020;7(10):e737-e745). Nevertheless, given the concerns about SARS-CoV-2 vaccine-associated thrombosis (Pavord et al. N Engl J Med 2021;385(18):1680-1689) and the well-known thrombotic risk associated with MPN (De Stefano et al. Leukemia 2016;30(10):2032-2038), patients had frequently vaccine-hesitancy and some of them even refused to be vaccinated. Methods To estimate the potential risk of thrombosis associated with SARS-CoV-2 vaccination in MPN, we evaluated 335 consecutive MPN patients vaccinated against SARS-CoV-2 with at least one dose. Proportional hazard Cox model for multiple failures was used to compare the risk of thrombotic complications according to vaccine exposure (treated as time-dependent covariate), and results of these models were reported in terms of Hazard Ratio (HR) for vaccine exposure vs non-exposure periods, together with its 95% Confidence Interval (95%CI). The exposure period was defined for each patient from the date of any SARS-CoV-2 vaccine dose till 30 days after; the non-exposure period included for each patient the six months before the first dose and the period beyond 30 days after each dose till last follow-up (see Figure 1A). To take into account time between diagnosis and enter date, left-truncation was applied. Results Starting from January 2021 we enrolled 335 MPN patients, including 99 affected with PV, 176 affected with ET, 49 affected with primary or secondary myelofibrosis and 11 affected with MPN unclassifiable. Median age was 55 years (range 15-88); 153 (46%) were males and 182 (54%) were females. Considering the mutational status, 252 patients (75.2%) were JAK2 V617F-mutated, 55 patients (16.4%) were CALR-mutated, 6 patients (1.8%) were MPL-mutated and 22 patients (6.6%) were triple negative. Seventy out of 335 patients (20.9%) were not receiving cytoreduction at the time of vaccination; the remaining 265 (79.1%) were under cytoreductive treatment (226 with hydroxyurea, 22 with ruxolitinib, 13 with interferon, 4 with busulfan). A previous history of thrombosis was reported in 100/335 (0.3%). Almost all patients received at least two doses of vaccination against SARS-CoV-2 (331/335, 98.8%); 75% of patients (224 of 335) received also the third dose. In most patients (320/335, 95.5%) the first two doses consisted of mRNA-based vaccines; in only 15 out of 335 (4.5%) the first two doses consisted of adenovirus-vectored vaccines. In all cases the third vaccination was performed with mRNA-based vaccines. Thrombotic complications included only major thrombosis. The risk of thrombosis in the exposure period did not significantly differ from the risk of thrombosis in the non-exposure period (HR=0.8; 95%CI 0.04-15.4, P 0.866), as shown in Figure 1B. No case of cerebral venous thrombosis was reported in the post-vaccine period. Conclusions Although these results need to be confirmed in larger studies, these data deliver an important message to the clinical community: SARS-CoV-2 vaccines do not increase the risk of thrombosis in MPN patients; thus, it is pivotal for health care professional to vaccinate the highest number of MPN patients, reassuring them regarding thrombotic complications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of the Platelet Proteome Reveals Insights into the Pro-Inflammatory and Pro-Thrombotic State Associated with the Philadelphia Chromosome-Negative Myeloproliferative Neoplasms

Introduction: Hypercoagulability, thrombosis and microvascular dysfunction are hallmarks of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). Elevated peripheral blood cell counts appear to contribute to thrombotic risk but the precise underlying mechanisms remain to be fully elucidated. The interplay between haemostatic and pro-inflammatory pathway activity ('thrombo-inflammation') has emerged in recent years as a source of hypercoagulability in MPN. Platelets are recognised as being mediators of thrombo-inflammation in other diseases and may also be effectors of pro-inflammatory/pro-coagulant activity in MPN. We hypothesized that the platelet proteome is altered in MPN and that its characterisation would reveal insights into the pathophysiology of the disease and the associated thrombotic risk. Aim: To determine if differences exist in the pattern of platelet protein expression in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) in contrast to healthy donors. Methods: 62 patients (ET, n=38; PV, n=24) and 9 healthy volunteers were recruited at Papa Giovanni XXIII Hospital, Bergamo, Italy and the Mater Misericordiae University hospital, Dublin, Ireland. Platelets were isolated from platelet rich plasma, washed in Krebs Ringer buffer and resuspended at 1x 109 platelets/mL in phosphate buffered saline. Whole platelets were lysed in RIPA buffer and differential proteomic signatures established using label-free quantification (LFQ) mass spectrometry (MS), where platelet lysate proteins were double digested using the commercially available PreOmics kit and analysed in a Bruker TimsTOF mass spectrometer connected to a EvoSep liquid chromatography system. Identified peptides were searched against a human FASTA using MaxQuant. For statistical analysis, proteins identified in a minimum of 70% of samples in at least one group were included. Differences in protein expression were determined using an unpaired t-test with a false discovery rate of 5% and a minimal fold change of 0.1 within the Perseus software; p- values below 0.05 were considered significant. Results: 2,180 proteins from platelet lysates were quantified across all patient and control samples. In MPN platelet lysates, 49 proteins were found to be differentially expressed in comparison to controls (p< 0.05) (Figure 1), including increased expression of markers of platelet activity such as vesicle-associated membrane protein 7 (VAMP-7; regulator of granule exocytosis and actin cytoskeleton activity) and CD109 (a GPI-linked glycoprotein expressed by activated platelets). Bioinformatical analysis revealed a cohort of mitochondrial, cytoskeletal & ribosomal proteins as well as potential effectors of thrombopoiesis and thrombo-inflammation which were significantly upregulated in the MPN cohort. Strikingly, protein disulfide-isomerase (PDI, a member of the thioredoxin superfamily of redox proteins) was increased in MPN lysates. PDI has been shown to contribute to thrombosis, platelet activation and platelet-neutrophil interactions in vivo; plasma levels of PDI have been shown to be increased in MPN and are associated with thrombosis risk. Conclusion: The platelet proteome is altered in MPN and is suggestive of an activated platelet phenotype with over-expression of proteins with known pro-coagulant activity. To our knowledge the over expression of PDI in MPN platelets has not previously been described. The identification of differentially expressed proteins, such as PDI, provides mechanistic insights into the pathophysiology underlying the substantial burden of arterial and venous thrombosis in MPN and may also assist in the identification of novel therapeutic targets. Additional proteomic analysis of platelet releasate and plasma extracellular vesicles is currently ongoing and may provide additional pathophysiological insights. Figure 1: Volcano plot of MPN versus control platelet lysate proteomes (x-axis, t-test difference between the mean log2 of the LFQ values; y-axis, the negative log transformed p-value) representing the proteins significantly altered in MPN. Black hyperbolic curves show the threshold for statistical significance. 36 proteins were found to be increased in MPN platelet lysates (including CD109, PDIA4 and VAMP-7) in comparison to control lysates (red) while 13 proteins were decreased in MPN platelet lysates (blue). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Site‐specific venous thrombosis in essential thrombocythemia: Impact on subsequent vascular events and survival

Site‐specific venous thrombosis in essential thrombocythemia: Impact on subsequent vascular events and survival

Assessment of relation between JAK2 gene and thrombosis in myeloproliferative neoplasms

Background Thrombotic complications are the most considerable etiology causing morbidity and mortality in patients with philadelphia (Ph) negative myeloproliferative neoplasms (MPN). There are many studies evaluating the association of JAK2 mutation and risk of thrombosis in MPN with inconclusive results. We also investigated the relation between JAK2 mutation in all Ph negative MPN and thrombosis.&#x0D; Material and Methods Thrombotic events and demographic features of 177 patients with Ph negative MPN were evaluated retrospectively.&#x0D; Results JAK2 V617 F mutation was detected in 57% of patients with essential thrombocythemia (ET), %90.3 of pateints with polycythemia vera (PV), 100% of pateints with primary myelofibrosis (PMF). Thrombotic complications occured more frequently with JAK2 mutation in all MPN patients than without (p=0.014). In JAK 2 mutation positive groups, the median age, thrombosis risk scores and leucocyte values are higher, splenomegaly and arterial and/or venous thrombosis are detected more frequently (p

PB2053: PRIMARY ARTERIAL HYPERTENSION IN MYELOPROLIFERATIVE NEOPLASMS: A SYSTEMATIC REVIEW

Background: Patients diagnosed with myeloproliferative neoplasms (MPNs) are prone to experience thrombotic events and often experience a high burden of cardiovascular comorbidities. Primary arterial hypertension (HTN) remains one of the most common comorbidities in the elderly. However, the impact of HTN on MPNs is unclear. Aims: Report on the occurrence of HTN in MPNs. Describe its epidemiology, influence on development of thrombosis, HTN treatment strategies. Methods: Systematic search in PubMed/Medline, Web of Science and SCOPUS, from the inception of these databases to retrieve relevant English-written articles. Eligibility criteria: 1. Confirmed MPNs diagnosis, 2. Confirmed HTN diagnosis, 3. Studies reported on the epidemiology of HTN in MPNs, impact of HTN on the development of thrombosis or on the therapeutic options used to treat HTN in MPNs. We excluded studies investigating secondary HTN, e.g., drug-induced HTN. Results: The systematic search resulted in 590 potentially relevant manuscripts. After removal of duplicates and screening of abstracts/titles (n=503), 87 manuscripts were selected for full-text review. After applying the eligibility criteria and full-text evaluation, 25 articles were excluded and the remaining 62 were entered into the qualitative/quantitative analysis. HTN emerged as the most common comorbidity in MPNs, affecting 41-63% of primary myelofibrosis (PMF), 40-64% of essential thrombocythemia (ET), 46-90% of polycythemia vera (PV) cases. In PV, the presence of HTN was associated with decreased serum free epinephrine and aldosterone levels, reduced retinal microperfusion, elevated kidney arteries resistance, JAK2-V617F mutation and high-risk PV. In PMF, it was linked with high uric acid concentrations and elevated risk of thrombosis (OR=1.96). PV subjects with HTN were non-dippers, suffered from systolic/diastolic dysfunction as assessed by cardiac ultrasound, displayed elevated cell-free hemoglobin and nitrite/nitrate ratio, and an increased risk of thrombosis (HR=1.77). HTN in ET was associated with overall thrombosis, especially arterial thrombosis (HR=1.91-3.8). The cardio-IPSET score was superior in predicting thrombosis in ET patients with HTN. Individuals with MPNs and HTN exhibited signs of carotid artery injury, end-organ damage, increased albumin-to-creatinine ratio, P-selectin concentrations and were prone to develop kidney dysfunction. Moreover, stroke was more common in MPNs with concomitant HTN (HR=4.24) and HTN was a predictor of ischemic stroke recurrence in MPNs. The risk of thrombosis in MPNs was higher if HTN was also associated with other cardiovascular risk factors, i.e., dyslipidemia, diabetes or smoking. The risk of cerebral venous sinus thrombosis was lower in MPNs with co-occurring HTN. In terms of treatment options for patients with HTN and MPNs, angiotensin-converting enzyme inhibitors (ACEI), e.g., lisinopril, and calcium-channel blockers (CCB - nifedipine for hypertensive emergencies) have been successfully used. The recommendation to avoid the use of diuretics in patients with elevated blood viscosity, e.g., PV, remains unaltered. Summary/Conclusion: Our findings suggest that HTN is the most common comorbidity in MPNs and is associated with development of thrombosis, end-organ damage, i.e., retinal and kidney dysfunction. Integration of HTN in the current scores employed to predict the development of thrombosis and stratify MPNs subjects in warranted. Screening for HTN in MPNs is urgently needed. Accurate management strategies based on the use ACEI and CCB need further exploration in MPNs with associated HTN.

JAK2V617F-Positive Endothelial Cells Induce Apoptosis and Release JAK2V617F-Positive Microparticles

Objective:Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) have a high propensity for thrombosis, which has been attributed to increased blood counts, endothelial cell (EC) dysfunction, and inflammation. The presence of the JAK2V617F mutation in the ECs of MPN patients has been confirmed, but the consequences of EC involvement by JAK2V617F in the pathogenesis of thrombosis are unclear. Endothelial microparticles (EMPs) released from ECs play an important role in endothelial dysfunction and also in the intercellular exchange of biological signals and information. Several studies have revealed that patients with JAK2V617F and a thrombosis history have increased numbers of MPs in their circulation.Materials and Methods:The current study utilized a lentiviral transduction model of JAK2 wild type (JAK2wt) or JAK2V617F encoding green fluorescent protein (GFP) into human umbilical vein ECs to determine the effect of JAK2V617F on ECs. EC infected with JAK2V617F, JAK2WT, and only-GFP were tested after two days of culture.Results:The proteins of ECs that potentially play a role in the development of thrombosis, including endothelial protein C receptor, thrombomodulin, and tissue factor, were detected by flow cytometry analysis with no statistical significance. Increased annexin-V uptake of JAK2V617F and JAK2wt ECs compared to GFP-alone ECs was detected. The EMP production in the supernatants of the EC culture was investigated. Genotyping of the EMPs revealed the presence of genomic DNA and RNA fragments in EMP cargos. JAK2V617F-positive DNA was detected in EMPs released from JAK2V617F-infected ECs and EMPs were shown to carry the genotype of the cell of origin.Conclusion:JAK2V617F-positive EMPs were shown for the first time in the literature. This novel research provides the first evidence that EMPs might regulate neighboring and distant cells via their cargo materials. Thus, the direct effect of JAK2V617F on ECs and their functions suggests that different mechanisms might play a role in the pathogenesis of thrombosis in MPNs.

Thrombosis in Myeloproliferative Neoplasms: A Single Center Experience of Using Whole Blood Platelet Aggregation Studies for Risk Assessment and Thromboprophylaxis

Thromboembolic complications are the most common causes of morbidity and mortality in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN); and prevention of these complications remains a significant clinical challenge. Effective thromboprophylaxis in MPN patients generally requires use of anti-platelet therapy, commonly aspirin; however, there are no standardized or universally accepted guidelines regarding the dose of aspirin. This study evaluates the usefulness of whole blood platelet aggregation (WBPA) studies to identify patients at risk for thrombosis and to achieve safe and effective long term thromboprophylaxis. One hundred and thirty-two consecutive patients were enrolled into this study. WBPA studies were performed at diagnosis in 125 patients to identify those with platelet hyperactivity (deemed to be at risk for thrombosis) and repeated 4 weeks after commencement of anti-platelet therapy to ascertain the efficacy. In patients with incomplete drug effect, treatment was revised and the study repeated until optimum effect was achieved. Results of the WBPA studies and anti-platelet therapy requirements were correlated with the underlying driver mutations and various international prognostic score of thrombosis for essential thrombocythemia (IPSET- Thrombosis) sub-groups. WBPA studies showed varying degrees of platelet hyper-activity in 115 patients. Based on these results, the patients were commenced on anti-platelet therapy comprising aspirin (dose ranging from 100mg twice or thrice weekly to 400mg daily) and clopidogrel (75mg daily) alone or in combination with aspirin or odorless garlic. None of the patients developed thrombosis during the follow up period ranging from 1-23 years (median 8yrs), while on the prescribed, individualized anti-platelet therapy. No significant differences were noted in terms of aspirin dose requirements between the JAK-2 positive and CALR or MPL positive patients, and, among the four IPSET-Thrombosis sub-groups. Patients with normal (9) or hypo (1) – activity were not given any anti-platelet therapy at diagnosis.ConclusionRoutine use of WBPA studies enables safe and effective risk-adapted thromboprophylaxis in MPN patients, irrespective of the underlying driver mutation and their risk predicted by the IPSET- thrombosis criteria.

The Risk Factors of Thrombosis in Patients with Philadelphia Chromosome-negative Myeloproliferative Neoplasms

To investigate the overview of thrombosis in myeloproliferative neoplasms(MPN) patients, and to explore the risk factors of thrombosis at diagnosis and during follow-up. The clinical data of 388 MPN patients treated in our hospital were collected. The patients were followed up by outpatient and phone. The risk factors of thrombosis were analyzed by statistical methods. Among 388 MPN patients, 161 patients (41.49%) showed thromboses at diagnosis or during follow-up. Among them, 92.55% were arterial thromboses, 146 cases (96.27%) were complicated with thromboses at diagnosis, and 36 cases (11.46%) showed newly thromboses or progression of previous thromboses among the 314 received full follow-up patients. Age (P＜0.001, HR:1.033, 95%CI:1.016-1.051), JAK2V617F mutation (P=0.037, HR:1.72, 95%CI: 1.033-2.862), hypertension (P＜0.001, HR:2.639, 95%CI:1.659-4.197) and hyperlipidemia (P＜0.001, HR:2.659, 95%CI:1.626-4.347) were the independent risk factors affecting thrombosis at diagnosis of the patients. During the follow-up, age (P=0.016, HR:1.032, 95%CI: 1.006-1.059) and previous thrombosis history (P=0.019, HR:2.194, 95%CI: 1.135-4.242) were the independent risk factors affecting the progression of thrombosis at different sites or on the basis of the previous thrombosis in the patients. Patients with advanced age, JAK2V617F mutation or complicated with hypertension and hyperlipidemia shows a higher risk of thrombosis at diagnosis, while the patients with advanced age or previous thrombosis history shows a higher risk of progression of thrombosis during the follow-up.

Correlation between leukocyte-platelet aggregates and thrombosis in myeloproliferative neoplasms.

The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P<.05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89ng/mL vs 286.39ng/mL, P=.034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR=1.561, 95% CI: 1.007-2.420, P=.046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR=1.975, 95%CI: 1.215-3.212, P=.006) for thrombosis occurrence. Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

Neutrophil-to-Lymphocyte Ratio (NLR) Is a Risk Factor for Venous Thrombosis in Polycythemia Vera

▪Background. The tendency towards thrombosis in myeloproliferative neoplasms (MPNs) is linked to the JAK2-mutant clone which leads to hypercellularity and functional interplay between abnormal erythrocytes, platelets, leukocytes and dysfunctional endothelium. The resulting cell activation that also involves stromal cells in their microenvironment, has been shown associated with chronic, systemic, subclinical pro-inflammatory state, and has been implicated in the pathogenesis of thrombosis in MPN. Neutrophil to lymphocyte ratio (NLR) is a novel inflammatory marker found to be associated with the severity and prognosis of cardiovascular diseases but there is little evidence for its prognostic significance in MPN. We investigated whether NLR could predict the onset of arterial and venous thrombosis in polycythemia vera (PV).Methods. Subjects of this study were 1508 patients included in the ECLAP trail with NLR evaluation available. In addition to standard statistical methods, we used proportional hazards additive models (GAM) as they can provide an excellent fit in the presence of nonlinear relationships, for the prediction of total, arterial and venous thrombosis as smooth functions with cubic splines of different blood parameters.Results. After a median follow-up of 2.76 years, 169 thrombotic events (10.3%) were objectively diagnosed and analyzed: 87 arterial (MI, Stroke, TIA, PAT) and 88 venous thrombosis (DVT±PE, superficial thrombophlebitis). •In univariate analysis, arterial thrombosis was associated with age (p=0.003) and previous thrombosis, especially if arterial (p<0.001), and with the presence of at least one cardiovascular risk factor (p=0.009). No association between baseline platelet and leukocyte counts and NLR with arterial events was found. Conversely, in venous thrombosis, beside age (p=0.039) and history of venous thrombosis (p<0.001), additional risk factors were low hemoglobin (p=0.039) and increasing values of NLR (p=0.002) resulting from a simultaneous increase of neutrophils (p=0.002) and to a decrease of absolute number lymphocytes (p=0.002).•To predict outcomes, we tested total leukocytes, neutrophils, lymphocytes, platelets and NLR by GAM to evaluate their trend in continuous scale of thrombotic risk. A significant association between the risk of venous thrombosis and the progressive lower counts of lymphocytes (p=0.002) emerged, leading to increase the NLR values (p=0.005). However, given the greater precision of the NLR estimates and the markedly wide confidence interval of lymphocyte counts estimates, NLR was used in multivariate model. Conversely, neither absolute values of neutrophils and lymphocytes nor NLR were found associated with arterial events.•In multivariate model, adjusted for age, gender and treatments at baseline, the risk of venous thrombosis was independently associated with previous venous events (HR=5.48, p ≤0.001) and NLR values ≥5 -the best cut-off resulted applying the Liu's method (HR=2.13, p=0.001). NLR effect for total venous thrombosis was not modified by excluding superficial venous thrombosis (HR=2.90, p=0.001) in a sensitivity analysis. Older age (i.e., ≥65 years, HR=1.88, p=0.005) and previous arterial thrombosis (HR=1.92, p=0.003) retained the prognostic statistical significance for arterial thrombosis.•Our learning cohort findings, indicating a correlation between NLR values and venous thrombosis, have been validated in two Italian independent external cohorts (Florence, n=282 and Rome, n=173) of contemporary PV patients (Figure 1).Conclusions. The NLR is an inexpensive and easily accessible test compared to other inflammatory markers. We found that NLR-alone is an independent predictor of venous thrombosis and could be included in a new scoring system. In addition to guiding the stratification of thrombotic risk, these data confirm that inflammation is a relevant target of antithrombotic therapy in PV. [Display omitted] DisclosuresVannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Globally Applicable “Triple AAA” Risk Model for Essential Thrombocythemia Based on Age, Absolute Neutrophil Count, and Absolute Lymphocyte Count

▪BackgroundThe detrimental effect of leukocytosis on survival in myeloproliferative neoplasms (MPN) has been well established for primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET ) (JCO. 2018;36:310; BJH. 2020;189:291) Previous studies have also implicated leukocytosis as a risk factor for leukemic transformation (Mayo Clin Proc. 2017;92:1118) and thrombosis in MPN (Blood Adv. 2019;3:1729). However, it is currently not clear as to which component(s) of white blood cells is responsible for these observations. In the current study, we sought to examine the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts, on overall (OS), leukemia-free (LFS), and myelofibrosis-free (MFFS) survival and in ET.MethodsStudy patients (n=349) were retrospectively recruited from the Mayo Clinic MPN database of 1,249 WHO-defined ET patients, evaluated over five decades (1967-2021), based on availability of information on ANC, ALC and AMC. Conventional criteria were used for diagnosis and definitions of major complications, including leukemic or fibrotic transformation (Blood 2016;127:2391). Conventional statistical methods were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for survival.Results349 patients (median age 57 years, range 18-89; females 61%) with ET were included in the study: 46% JAK2, 34% CALR, 16% triple-negative and 4% MPL mutated; IPSET risk category high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1-16.4) for hemoglobin, 8.2 x 10(9)/L (3.2-52) for leukocyte count, and 859 x 10(9)/L (451-3460) for platelet count; palpable splenomegaly was present in 48 (14%); median followup was 10 years (range 0-47), during which time 118 deaths, 52 fibrotic progressions, and 14 leukemic transformations were documented.Multivariable analysis identified older age (p<0.001), increased ANC (p<0.001), decreased ALC (p=0.03), and male sex (p=0.04), but not AMC (p=0.8), venous thrombosis (p=0.4), or arterial thrombosis (p=0.4), as independent risk factors for OS. ANC of ≥8 x 10(9)/L and ALC of <1.8 x 10(9)/L were determined as appropriate cut-off values by ROC analysis. Subsequent multivariable analysis using these cut-off values resulted in HR (95% CI) of 5.2 (3.4-7.9; p<0.001) for age >60 years, 3.1 (2.1-4.6; p<0.001) for ANC, and 2.0 (1.4-3.0; p<0.001) for ALC; male sex was no longer significant in this analysis (p=0.14). An operational HR-based risk score assigned 3 adverse risk points for older age (>60 years), 2 for increased ANC (≥8 x 10(9)/L) and 1 for ALC (<1.8 x 10(9)/L), resulting in an new Age Anc Alc (AAA; triple A) risk model for survival in ET with estimates of median survival ranging from 9.7 to 36.6 years (Figure 1). In addition, ALC <1.8 x 10(9)/L was associated with inferior LFS (p=0.06) and MFFS (p=0.07) while AMC as a continuous variable showed borderline significance for MFFS (p=0.18). In univariate analysis, JAK2V617F allele burden showed significant association with OS (p=0.02), LFS (p=0.05) and MFFS (p=0.001); however significance for OS was lost in mutivariable analysis that included ANC and ALC.An external validation cohort from the University of Florence (n=485) confirmed the independent survival risk contribution from age >60 years (p<0.001; HR 9.9, 95% CI 5.4-18.0), ANC ≥8 x 10(9)/L (p=0.02; 1.9, 1.1-3.5) and ALC <1.8 x 10(9)/L (p<0.001; 2.0, 1.3-3.0). The Triple A risk model was also effectively applied on the Florence validation cohort (Figure 1): median follow-up 8.4 years; 87 deaths; 43 fibrotic progression; 12 leukemic transformations. The association of ALC <1.8 x 10(9)/L (p=0.04) and AMC (p=0.002) with fibrotic transformation was also validated in the Florence cohort.Conclusions:The current study identifies increased ANC and decreased ALC as age-independent risk factors for survival in ET, thus allowing the development of a globally applicable simple to use “Triple A” risk model that is based on Age, ANC and ALC. Decreased ALC also predicted fibrotic and leukemic progression. Our observations suggest potential value for immune profiling as an additional prognostic tool in MPN. [Display omitted] DisclosuresSzuber: Novartis: Honoraria. Vannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Deciphering the Individual Contribution of Absolute Neutrophil, Lymphocyte and Monocyte Counts to Thrombosis Risk in Patients with Myeloproliferative Neoplasms

▪BackgroundIn addition to its influence on survival, leukocytosis in myeloproliferative neoplasms (MPN) has also been implicated as a risk factor for thrombosis, including venous thrombosis in PV (Blood Cancer J, 2017;7:662) and arterial thrombosis in ET (Blood. 2011;117:5857). In the current study, we sought to clarify the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts, for arterial and venous thrombotic events in essential thrombocythemia (ET) and polycythemia vera (PV).MethodsThe current study included 487 patients with ET (n=349) or PV (n=138), recruited from the Mayo Clinic MPN database, based on availability of information on ANC, ALC and AMC. Conventional criteria were used for diagnosis (Blood 2016;127:2391) and definitions of major vascular events (Blood Cancer J. 2018;8:25). Conventional statistical models were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for arterial or venous thrombosis.ResultsEssential thrombocythemia patients:349 patients (median age 57 years, range 18-89; females 61%) with ET were included in the study: 46% JAK2, 34% CALR, 16% triple-negative and 4% MPL mutated; IPSET risk category high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1-16.4) for hemoglobin, 8.2 x 10(9)/L (3.2-52) for leukocyte count, and 859 x 10(9)/L (451-3460) for platelet count; palpable splenomegaly was present in 48 (14%) patients and cardiovascular risk factors in 56%; median follow-up was 10 years (range 0-47). There were 38 (11%) documented venous events at diagnosis and 31 (9%) after diagnosis and 42 (12%) arterial events at diagnosis and 64 (18%) after diagnosis.Polycythemia vera patients:138 patients (median age 62 years, range 20-94; females 50%) with PV were included in the study; presenting median (range) values were 17.9 g/dL (16.1-24) for hemoglobin, 11.8 x 10(9)/L (2.7-65.8) for leukocyte count, and 434 x 10(9)/L (44-1679) for platelet count; 57% of patients presented with leukocytosis >11 x 10(9)/L; palpable splenomegaly was present in 37 (27%) patients; abnormal karyotype was documented in 17% of patients. Median follow-up was 11 years (range 0.03-36.7). There were 22 (16%) documented venous events at diagnosis and 21 (16%) after diagnosis and 28 (20%) arterial events at diagnosis and 15 (11%) after diagnosis.ANC/ALC/AMC associations with thrombosis in ET and PV:In both ET and PV, ANC/ALC/AMC did not correlate with arterial thrombosis at/prior to diagnosis (p>0.1 in all instances). By contrast, in both ET and PV, higher ANC (p=0.05 and 0.04, respectively) and higher AMC (p=0.005 and 0.07), but not ALC (p=0.6 and 0.7), were correlated with venous thrombosis at/prior to diagnosis.Arterial thrombosis-free survival was not affected by ANC/ALC/AMC in either ET or PV (p>0.1 in all instances). By contrast, venous thrombosis-free survival in both ET and PV was compromised by higher ANC (p=0.05 and 0.003, respectively), but not ALC or AMC. The significant association between higher ANC and inferior venous thrombosis-free survival in both ET (p=0.01) and PV (p=0.007) was sustained during multivariable analysis that included history of venous thrombosis, age and sex; the only other variable of significance was older age in ET (p<0.001). The significant association between ANC and venous thrombosis-free survival was also noted in an external cohort of PV patients from the University of Florence (n=576).Conclusions:The current study identifies ANC as having a direct correlation with venous thrombosis in both ET and PV, independent of currently known risk factors. An additional risk contribution from AMC was apparent for events occuring at or prior to but not after diagnosis. Taken together, these observations flag both neutrophils and monocytes as potential contributors to venous but not arterial thrombosis in MPN, at least not by themselves. Additional collaborative studies are ongoing in order to integrate and reconcile our observations in the context of neutrophil/lymphocyte ratio (Carobbio et al) and JAK2V617F allele burden (Loscocco et al), on venous thrombosis-free survival in PV, reported in concomitantly submitted ASH 2021 abstracts, especially in light of the relatively small number of events in the current study. DisclosuresBarbui: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Vannucchi: AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.