Overt Myeloproliferative Neoplasms Research Articles

Thrombotic, Cardiovascular, and Microvascular Complications of Myeloproliferative Neoplasms and Clonal Hematopoiesis (CHIP): A Narrative Review.

The most common causes of morbidity and mortality in the myeloproliferative neoplasms (MPNs), with the exception of myelofibrosis, are venous and arterial thrombosis, as well as more recently discovered cardiovascular disease (CVD). Clonal hematopoiesis of indeterminate potential (CHIP) is the subclinical finding in an individual of somatic mutations that are also found in clinically overt MPNs and other myeloid malignancies. The prevalence of "silent" CHIP increases with age. CHIP can transform into a clinically overt MPN at an estimated rate of 0.5 to 1% per year. It is likely, therefore, but not proven, that many, if not all, MPN patients had antecedent CHIP, possibly for many years. Moreover, both individuals with asymptomatic CHIP, as well as clinically diagnosed patients with MPN, can develop thrombotic complications. An unexpected and remarkable discovery during the last few years is that even CHIP (as well as MPNs) are significant, independent risk factors for CVD. This review discusses up-to-date information on the types of thrombotic and cardiovascular complications that are found in CHIP and MPN patients. A systemic inflammatory state (that is often subclinical) is most likely to be a major mediator of adverse reciprocal bone marrow-cardiovascular interplay that may fuel the development of progression of MPNs, including its thrombotic and vascular complications, as well as the worsening of cardiovascular disease, possibly in a "vicious cycle". Translating this to clinical practice for hematologists and oncologists who treat MPN patients, attention should now be paid to ensuring that cardiovascular risk factors are controlled and minimized, either by the patient's cardiologist or primary care physician or by the hematologist/oncologist herself or himself. This review is intended to cover the clinical aspects of thrombosis and cardiovascular complications in the MPN, accompanied by pathobiological comments.

The Prevalence of JAK2 Mutation in High-Altitude Patients with Unprovoked Thrombosis and Thrombosis at Unusual Sites

Introduction Thrombosis, both arterial and venous, is a major source of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Thrombosis can be the initial presentation of MPN and sometimes can predate its diagnosis. Most MPN-related thrombosis occurs on usual sites such as the cerebral and coronary arteries, and lower limbs deep venous thrombosis and pulmonary embolism. Rare thromboses can also occur with MPN, such as splanchnic and cerebral dural vein thromboses, with the former having a stronger correlation. Stroke in young people (under 50) and venous thrombosis at unusual sites (cerebral, splanchnic, renal and upper limbs) represent special scenarios for which workup includes exploring unusual etiologies, including inherited and acquired hypercoagulable states. JAK2 mutation is invariably present in polycythemia vera (PV), occurs in a significant proportion of other classical MPNs, and is directly associated with risk of thrombosis due to associated leukocytosis, platelet activation, and altered vascular endothelial function. JAK2-mutated MPNs are associated with higher thrombosis risk compared to JAK2 unmutated MPNs, and greater allele burden corresponds to greater risk. The association of JAK2 mutation with hepatic and splanchnic vein thrombosis is well established. There are evidence-based guidelines for testing in idiopathic splanchnic vein thrombosis; however, its association with other unusual site thrombosis is unclear. Clinicians may request JAK2 mutation testing in individuals with unprovoked VTE and VTE at unusual sites to uncover the underlying etiology. This is despite a low diagnostic yield, particularly in people with normal CBC. Cerebral sinus thrombosis, for example, occurs in 1% of MPNs, and an underlying MPN accounts for only 3.8% of CVT. Most documented MPNs presenting with thrombosis had abnormal CBC at presentation, either thrombocytosis or erythrocytosis. The aim of this analysis is to explore whether JAK2 mutation without overt MPN contributes to the unusual site venous thrombosis and stroke in young people in a high altitude population. Method: We collected lab records for all JAK2 requests made over three years (2020-2022) in two referral hospitals serving a population residing at an altitude of 2,270-3,000 meters above sea level. Requests that listed thrombosis as an indication were analyzed, and labs and imaging related to the corresponding requests were reviewed. Results: Among 208 JAK2 tests, 40 (19.2%) were requested during the evaluation of thrombotic events. Patients were relatively young, with a median age of 41, and the gender breakdown was 17 (42.7%) males and 23 females. About half (23, 57.5%) of the cohort had normal CBC. Those with abnormal CBC at presentation included 3 patients with leukocytosis, 10 with thrombocytosis, and 10 with erythrocytosis. Among males, 2 had an ischemic stroke, 5 had cerebral sinus thrombosis, 5 had splanchnic vein thrombosis, 4 had unprovoked DVT/PEs, and 1 had renal vein thrombosis. Among females, 4 experienced ischemic strokes, 3 had cerebral venous thrombosis, 10 had splanchnic vein thrombosis, 5 had unprovoked DVT/PEs, and 2 had renal vein thrombosis. Only 2 patients (5%) were positive for JAK2 mutations: a 17-year-old boy who presented with portal vein thrombosis and was diagnosed with polycythemia vera and a 31-year-old lady who presented with hepatic vein thrombosis and normal CBC. Conclusion: JAK2 mutation was rare in our high-altitude cohort with unprovoked DVT/PE/thrombosis at unusual sites. Outside the splanchnic venous system and with normal CBC, the diagnostic yield of JAK2 mutation testing is low. Hence, such testing might not be indicated.

JAK2V617F Is a Risk Factor for TIA/Stroke in Young Patients.

The objective of this study was to assess the risk of arterial thrombosis in patients who harbor the JAK2V617F allele burden ≥1% detected during workup for myeloproliferative neoplasms (MPNs). We conducted a large cross-sectional analysis consisted of 5,220 patients who were tested for JAK2V617F and 1,047,258 people matched in age from health care insurance provider, taking into account age, sex, hypertension, diabetes, atrial fibrillation. Compared with noncarriers, mutation carriers were older, less likely to be current or past smokers and had lower body mass index. There was no significant difference between the groups regarding myocardial infarction and peripheral vascular disease. However, JAK2V617F ≥1% at age 34 to 54 years was associated with eightfold more likely to have transient ischemic attack (TIA)/stroke history unrelated to hypertension, diabetes, or atrial fibrillation. Association of JAK2V617F with TIA/stroke was also observed in the older age group, albeit a weaker association and not statistically significant. Prevalence of TIA/stroke was higher in patients with JAK2V617F negative, with odds ratio of 3.93 when compared with the general population after confounder adjustments. Further research is warranted to verify the relation between allele burden of JAK2V617F mutation and TIA/stroke and the role of JAK2V617F per se as a risk factor for arterial thrombosis in the absence of overt MPN. Also, consideration should be paid to the screened group with JAK2V617F negative due to the high incidence of TIA/stroke among them in comparison to the general population.

Treatment of Portal, Mesenteric, and Splenic Vein Thrombosis with Rivaroxaban: A Pilot, Prospective Cohort Study

Introduction: Anticoagulation plays a crucial role in the treatment of splanchnic vein thrombosis (SVT), including thrombosis of the portal (PVT), mesenteric (MVT) and splenic (SpVT) veins. Rivaroxaban is a potential alternative to heparins and vitamin K antagonists (VKA) in these patients, but data to support its use are scant. Several recent guidelines highlighted the limited evidence available for the use of the direct oral anticoagulants in these patients. In addition, despite anticoagulation, SVT patients carry high risk of recurrent venous thromboembolic events.The aim of this study was to evaluate the safety and efficacy of rivaroxaban for the acute-phase treatment of SVT (first 3 months of treatment).Methods: RIVASVT-100 (NCT02627053) was a prospective cohort study of adult patients with a first episode of symptomatic, objectively diagnosed PVT, MVT or SpVT. Exclusion criteria were known liver cirrhosis, Budd-Chiari syndrome, previous or ongoing variceal bleeding, portal cavernoma, thrombocytopenia, severe renal failure, life expectancy <3 months, concomitant interfering medications, treatment with therapeutic dose of heparins for >7 days, ongoing VKA treatment.Patients received rivaroxaban 15 mg twice daily for 3 weeks, followed by 20 mg once daily for a total of 3 months. Afterwards, the decision to continue any available anticoagulant drug was left to the discretion of the attending physicians. Follow-up was performed at 3 weeks, 2 months, 3 months and 6 months.Primary outcome was the occurrence of ISTH-defined major bleeding events during the 3 months of active treatment and up to 2 days after the end of study treatment. Secondary outcomes included death, clinically relevant non-major bleeding (CRNMB), recurrent SVT or symptomatic venous thromboembolism in other sites. We here report the results of the 3-month follow-up.Results: Between June 2015 and March 2021, 103 patients were enrolled from 18 participating centres. After excluding 3 patients who did not meet the criteria for eligibility, 100 patients were included in the analysis. Mean (SD) age was 54.4 (± 15.5) years; 64% were males. Overall, 74% of patients had PVT, 61% MVT and 48% SpVT; 53% of SVT occurred in multiple sites. The most common risk factors were abdominal inflammation/infection (26%), followed by solid cancer (9%), overt myeloproliferative neoplasms (9%) and oestrogen hormonal therapy (9%), while 43% of cases were unprovoked. The JAK2 V617F mutation was detected in 13 out of 50 tested patients (26%).Rivaroxaban was the sole anticoagulant agent used in 21% of patients, whereas the remaining received a combination of anticoagulants, which included low molecular weight heparin, unfractionated heparin or fondaparinux for a median of 5.0 days before transitioning to rivaroxaban.Three patients were lost to follow-up but known to be alive at the end of the study. At 3-month follow-up, 1 (1.0%) patient died due to a non-SVT related cause. Two patients (2.06%; 95% CI, 0.57-7.21) had major bleeding events (both gastrointestinal), while 3 patients (3.09%) had CRNMB. There were 2 recurrent SVT (2.06%) during rivaroxaban treatment, one of these occurred in a patient with metastatic solid cancer. The 6-month follow-up for the last enrolled patient is ongoing.Conclusions: Rivaroxaban appears to be safe and effective for the acute-phase treatment of SVT in non-cirrhotic patients. DisclosuresBeyer-Westendorf: Bayer: Other: Personal fees; Daiichi Sankyo: Other: Personal fees; Pfizer: Other: Personal fees; Portola-Alexion: Other: Personal fees. Carrier: BMS: Honoraria, Research Funding; Leo Pharma: Honoraria, Research Funding; Bayer: Honoraria; Pfizer: Honoraria, Research Funding; Servier: Honoraria; Sanofi: Honoraria. Bertoletti: BMS: Honoraria, Other: Personal Fees; Pfizer: Honoraria, Other: Personal fees; Aspen: Other: Personal Fees; Bayer: Other: Personal Fees; Leo Pharma: Other: Personal Fee. Di Nisio: Bayer, Daiichi Sankyo, BMS-Pfizer, Leo Pharma, and Sanofi: Other: Personal fees. Ageno: Bayer: Research Funding; Bayer, Portola, Janssen, Aspen, Norgine, Sanofi.: Other: Advisory Board. OffLabel Disclosure:The use of rivaroxaban in splanchnic vein thrombosis is off label in most countries.

Splanchnic Vein Thrombosis in Overt Myeloproliferative Neoplasms: The Mayo Clinic Experience with 107 Unselected Consecutive Cases

Background:Splanchnic vein thrombosis (SVT) is a characteristic feature of JAK2 mutation-enriched myeloproliferative neoplasms (MPN). MPN constitutes the most frequent cause of Budd-Chiari syndrome (BCS) and non-malignant, non-cirrhotic portal vein thrombosis (Blood. 2012;120:4921). Reported risk factors for recurrent SVT included BCS, thrombosis history, splenomegaly, leukocytosis and absence of warfarin therapy (Blood Cancer J. 2016;6:e493). The main objective of the current study was to share the experience from a single tertiary center.Methods:Study patients were selected from institutional databases, with additional cases identified through Advanced Cohort Explorer Tool. Conventional diagnostic criteria were utilized (Blood 2016;127:2391) Diagnosis of SVT in all cases was confirmed by imaging studies. Cases with latent MPN (MPN-U) or evidence of other cancer were excluded. All analyses considered clinical and laboratory parameters obtained at time of SVT.Results:i) Presenting features107 patients (median age 56 years; range 22-84; 62% females) were considered; specific MPN diagnosis was primary myelofibrosis (PMF) 36%, polycythemia vera (PV) 29%, essential thrombocythemia (ET) 29%, post-PV 6% and post-ET myelofibrosis (MF) 1%. Among evaluable cases, JAK2 mutation was detected in 85% and abnormal karyotype in 33%. At the time of SVT, 80% were not receiving aspirin (ASA) therapy and 4% had history of OCP use. Thrombosis history was recorded in 51% and included history of SVT in 38%. 39 (36%) patients had pre-SVT history of splenectomy, including 16 (15%) in the month before. Presentation included abdominal pain 62%, ascites 31% and GI bleed 15%.ii) Type of SVT and clinical correlatesSVT types included BCS ± other vessels (n=9; 6 PV, 2 ET and 1MF), portal ± splenic (n=73; 38 MF, 20 PV, 15 ET), portal + mesenteric ± splenic (n=23; 14 ET, 5 MF and 4 PV). Comparison of these three SVT categories disclosed significant associations with MPN sub-category (p<0.01; BCS with PV and “portal + mesenteric” with ET), abdominal pain (p=0.02; with BCS and “portal + mesenteric”) and ascites (p=0.007; with BCS).iii) Initial management of SVTInitial treatment included systemic anticoagulation (SA) only in 39% of the patients, SA + ASA 4%, SA + cytoreductive drug 21%, SA + ASA + cytoreductive drug 4%, and no SA 33%. In addition, trans-jugular intrahepatic portosystemic (TIPS) or other surgical shunting was undertaken in 13% and variceal banding in 14%.iv) Post-SVT survival, causes of death and prevalence of liver failureAt median post-SVT follow up of 2.6 years (range .02-17.5), 35 (33%) deaths and 5 (5%) leukemic transformations were noted; the corresponding percentages for PV/ET vs MF were 18% vs 53% and 1.6% vs 9%. Causes of death were unrelated to SVT in 17 instances, unknown in 6, liver failure in 5 (all MF), upper gastrointestinal bleed in 4 (3 MF 1 PV), AML in 2 MF cases and thrombosis in one ET patient.v) Risk factors for post-SVT survival and recurrent SVTAs expected, post-SVT survival was significantly shorter in patients with MF vs PV/ET (HR 4.6, 95% CI 2.2-9.6). In multivariable analysis, older age (p<0.01), MF diagnosis (vs PV/ET) (HR 4.0; 95% CI 1.9-8.4) and presentation with ascites (HR 2.7, 95% CI 1.3-5.8) were independently associated with shorter survival; the latter was also associated with post-SVT complications (p=0.01). When analysis was restricted to the 62 patients with PV/ET, median post-SVT survival was not reached and advanced age was the only predictor of shortened survival (p<0.01), while associations of BCS with PV and “portal + mesenteric” with ET were confirmed (p<0.01). Seven of 8 BCS cases occurred in patients younger than age 60 (p=0.07) and 7 of 8 BCS presented with ascites (p<0.01). The only predictor of SVT relapse was male sex (p<0.01). In general, post-SVT survival and recurrence rate were not affected by type of SVT or initial treatment strategy.Conclusions:Outcome in MPN-associated SVT mostly depends on the underlying MPN type, with limited influence from SVT-directed treatment strategies; importantly, there was no overt evidence of adverse effect, from SVT, on overall or leukemia-free survival in PV or ET; liver failure and GI bleed were relatively frequent causes of death in MF-associated SVT. Other noteworthy observations from the current study included association of BCS with PV, ascites and younger age; and recurrent SVT with male sex. DisclosuresNo relevant conflicts of interest to declare.

Niche-Targeted Clinical Grade Immunohistochemistry Reveals Heterogeneous Bone Marrow Neuropathy and Novel Stromal Patterns in Primary Myelofibrosis Patients

Regulation of the hematopoietic stem cell (HSC) niche includes elements of the nervous system, immune system as well as the vasculature and associated stroma. Evidence in murine models demonstrate microenvironmental influence on HSC and hematopoiesis however correlative clinical and translational data remain scarce. The sympathetic nervous system may contribute to the pathogenesis of myeloproliferative neoplasms (MPNs) mechanistically through effects on nestin+ mesenchymal stromal/stem cells. Temporally, bone marrow neuropathy may even precede overt MPN by promoting aberrant premalignant HSC expansion, at least in genetically engineered MPN mouse models. We hypothesize that clinical characterization of the bone marrow niche via conventional immunohistochemistry (IHC) on routine diagnostic trephine biopsies is feasible and may yield novel stromal architectural data supplementing classification and/or prognostic stratification of BCR-ABL1 negative MPN. Herein, we describe our initial results on normal bone marrows and patients with primary myelofibrosis (PMF) with a focus on the pan-neuronal marker (PGP9.5) and the stromal marker smooth muscle actin (SMA). Results: Bone marrow neuropathy, as assessed by loss of punctate or linear perivascular PGP9.5 reactivity around arterioles is highly variable at initial PMF diagnosis. This suggests that in PMF, bone marrow neuropathy may not be universal and if present may only occur in a subset of patients or affect only focal regions of the marrow. SMA staining reproducibly yielded 3 patterns of reactivity in PMF: paratrabecular, perivascular and interstitial. While perivascular SMA staining was observed in all cases of PMF, the interstitial SMA pattern exhibited both inter-patient and intra-patient variability. Intriguingly, SMA was capable of highlighting “hot” and “cold” spots in PMF marrows otherwise appearing homogeneous by conventional reticulin staining. These SMA patterns are presently being investigated in the context of clinical information and molecular profiling to further assess their prognostic relevance. From longitudinal biopsies, we show resolution of the PMF interstitial SMA pattern as early as 2 months post allo-hematopoietic stem cell transplant, far earlier than any reduction in reticulin fibrosis which suggests that SMA signal may have important biological implications - including as a biomarker of early response to JAK inhibition, superior to standard reticulin/collagen assessment. In summary, visualization of the bone marrow niche by routine clinical grade IHC is feasible and reveals stromal heterogeneity within WHO-defined PMF of potential interest for classification refinement, prognostication and therapeutic monitoring. DisclosuresTsui:Novartis: Honoraria. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding.

Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides – 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) – as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6–21%, p < 0.001) and 6% (95%CI: 0.4–11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12–2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49–110%, p < 0.001) and 105% (95%CI: 80–133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

Low prevalence of JAK2 V617F mutation in patients with thrombosis and normal blood counts: a retrospective impact study.

To determine the prevalence of the V617F Janus Kinase 2 (JAK2) mutation in patients with thrombosis without other biological signs of underlying myeloproliferative neoplasm (MPN) and identify associated risk factors for thrombosis. Over a 10-year period, datawere collected from patients with thrombotic events andwho had also been screened for the V617F JAK2 mutation. Patients with signs of underlying MPN, such as haematocrit levels ≥ 50% and/or platelet counts ≥ 450 × 109/L and/or splanchnic thrombosis were excluded from the study. Of 340 patients fulfilling inclusion criteria, JAK2 mutation was found in 9 (2.65%), the allele burden being at least 2% in 4 (1.1%). Upon follow-up, MPN was diagnosed in the latter 4. Univariate analysis of the whole cohort showed that age (54 ± 15 vs. 64 ± 13, p = 0.027), platelet count (317 ± 111 vs. 255 ± 75, p = 0.017), C-reactive protein level > 5mg/L (OR 7.29, p = 0.014), and splenomegaly (OR 54.5, p = 0.0002) were significantly associated with JAK2 mutation. There was also a trend for an increased risk of cerebral venous thrombosis (OR 6.54, p = 0.064). Logistic regression confirmed a significant association between splenomegaly and JAK2 mutation (OR 43.15 [95%CI, 3.05-610.95], p = 0.0054). The V617F JAK2 mutation is rarely found in patients with thrombotic events without overt MPN. Splenomegaly, however, is a statistically and clinically relevant indicator of a potential JAK2 mutation in patients with non-splanchnic thrombotic events. Such patients should require further assessment and a close follow-up.

DIAGNOSTIC VALUE OF JAK2 V617F MUTATIONAL SCREENING IN PATIENTS WITH BUDD-CHIARI SYNDROME

Background: The diagnosis of underlying myeloproliferative neoplasms (MPNs) is often problematic in patients with Budd Chiari syndrome (BCS). A clonal mutation in JAK2 tyrosine kinase (JAK2V617F) occurs in a high proportion of patients with MPNs and is of use in the characterization of occult MPNs in BCS. Aim of the work: Detection of JAK2 V617F mutation in patients with BCS and its value in detection of occult MPNs. Patients and Methods: This study was carried out on fifty seven newly diagnosed Budd Chiari syndrome patientswho were attending tropical department in Ain Shams University Hospitals during the period from July 2017 to July 2018.Detection of JAK2V617F mutation was done by real time polymerase chain reaction. Results: Out of the studied 57 BCS patients, JAK2 V617F mutation was detected in 12 patients (21.1%) {10 (83.3%) were heterozygous and 2(16.7%) were homozygous for mutation}, while 45 patients (78.9%) were negative.On comparing JAK2 V617F positive and negative groups, there was a highly statistically significant relation regarding MPNs diagnosis, where all JAK2 V617F positive patients were diagnosed as MPNs of whom 7 (58.3%) had overtpresentation and 5(41.7%) had occult presentation, while in JAK2 V617F negative patients only 2 were diagnosed overt MPNs (p=0.001). Conclusion: In conclusion the JAK2 V617F mutation is an acquired mutation that can be used for diagnosis of latent MPNs presenting with thrombotic events, thus it is recommended to include JAK2 V617F gene analysis in the research panel for BCS patients.

Should We Screen for Janus Kinase 2 V617F Mutation in Cerebral Venous Thrombosis?

Background: The presence of Janus Kinase 2 (JAK2) V617F mutation represents a major diagnostic criterion for detecting myeloproliferative neoplasms (MPN) and even in the absence of overt MPN, JAK2 V617F mutation is associated with splanchnic vein thrombosis. However, the actual prevalence and diagnostic value of the JAK2 V617F mutation in patients with cerebral venous thrombosis (CVT) are not known. The aims of this study were to assess the prevalence of JAK2 V617F mutation in a large group of consecutive CVT patients, to detect clinical, biological, and radiological features associated with the mutation, and to determine the long-term venous thrombosis recurrence rate in CVT patients with JAK2 mutation but without overt MPN in order to recommend the best preventive treatment. Methods: This was a prospective study conducted on consecutive patients with a first-ever radiologically confirmed CVT. JAK2 V617F mutation analysis was assessed in all the study subjects. JAK2 V617F-positive patients were followed up to detect new venous thrombotic events. Results: Of the 125 included subjects, 7 were found to have JAK2 V617F mutation (5.6%; 95% CI 2.3-11.2). Older age (p = 0.039) and higher platelet count (p = 0.004) were independently associated with JAK2 V617F positivity in patients without overt MPN. During a mean follow-up period of 59 (SD 46) months, 2 JAK2 V617F-positive patients presented with 4 new venous thromboembolic events. Conclusions: Screening for the JAK2 V617F mutation in CVT patients seems to be useful even in the absence of overt MPN and/or in the presence of other risk factors for CVT because of its relatively high prevalence and the risk of thrombosis recurrence.

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study.

Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%.Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (‘’Histology-based’’ diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (‘’Clinical-based’’ or ‘’Molecular-based’’ diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process.Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion.

Unusual Site Thrombosis: Focus on Myeloproliferative Neoplasms with Splanchnic or Cerebral Venous Thrombosis

This study retrospectively evaluated 144 consecutive patients with unusual site thrombosis who referred to our Thrombosis Center between 2000 and 2016. All patients were classified as having either splanchnic venous thrombosis (SVT; n=127) or cerebral venous thrombosis (CVT; n=17). On the presence and type of provoking risk factors, then patients were categorized into three groups: unprovoked, those with possibly resolved provoking factors (PR), and those with persistent provoking factors (PP). Among the identified risk factors regarded as PP, we focused on Myeloproliferative Neoplasms (MPN) and performed a clinical comparison between MPN patients with SVT (MPN-SVT) and those with CVT (MPN-SVT). The characteristics of our cohort well reflects the clinical heterogeneity of clinical features commonly found in the routine clinical practice of diagnosing unusual site thrombosis. One hundred and twenty seven SVT patients were included: 7 unprovoked SVT, 10 SVT with PR, 110 SVT with PP; seventeen patients showed CVT, 5 unprovoked, 6 CVT with PR and 6 CVT with PP. Major risk factor for SVT with PP was liver cirrhosis (71.6%), whereas for CVT were MPN (5 patients, 29.4%). MPN was present in 8 patients (6.2%) of SVT (MPN-SVT vs MPN-CVT, p=0.009). For MPN-SVT, 3 patients showed the morphological features of polycytemia vera (PV), 2 of essential thrombocytemia (ET), 1 of primary myelofibrosis (PMF) and 2 fell into the MPN unclassified category (U-MPN); distribution of MPN subtypes for CVT was as follows: 1 PV, 2 ET and 2 U-MPN. Molecular analysis identified the JAK2V617F mutation respectively in 75% patients with MPN-SVT and in 60% patients with MPN-CVT; bone marrow histological features supported the diagnosis of MPN in all cases. Median age at MPN-SVT diagnosis was 46 years (range 17-78) vs 43 years (range 31-84) for MPN-CVT. Coexisting PR and thrombophilic abnormalities were identified in 75% and 20% of MPN-SVT and MPN-CVT respectively, so MPN are per se a strong risk for thrombosis and the leading systemic cause of CVT. In four of five cases (80%), CVT antedate the clinical phenotype of an overt MPN, whereas SVT occurred at MPN diagnosis in five patients and during MPN follow-up in three patients (median 164 months, range 88-215). At diagnosis MPN-SVT tended to have significantly lower platelet and white blood cell counts and haemoglobin concentration than MPN-CVT (respectively p<0.001, p<0.001 and p=0.002). Similar proportion of MPN patients in the two groups received cytoreductive treatment (hydroxyurea and alpha interferon) and appropriate anticoagulant therapy that consisted of low molecular weight heparin (LMWH) followed by oral vitamin K antagonists (VKA). MPN-SVT and MPN-CVT experienced similar median duration of anticoagulation (26 months for MPN-SVT, range 1-62, and 26 months for MPN-CVT, range 4-168) and a good quality anticoagulation control (median time within therapeutic range of 71 vs 87% respectively for MPN-SVT and MPN-CVT). Seventy-five percent MPN-SVT and 80% of MPN-CVT remain on indefinite anticoagulation. All patients were alive at last follow-up and the results of imaging techniques showed resolution of thrombosis in 87.5% and 80% of MPN-SVT and MPN-CVT respectively; the probability of recanalization of the occluded vessels was 18 months and 4 months for MPN-SVT and MPN-CVT, respectively. Only in one patient with MPN-SVT, a major bleed occurred on-treatment while the incidence of recurrent thrombosis was the same for both MPN-SVT and MPN-CVT (0.02 per 100 patients-year). In conclusion, our study evaluated unselected populations with unusual site thrombosis that were followed in our Thrombosis Center, an anticoagulation clinic well experienced on pathogenic mechanisms and anticoagulant treatment. Our findings have practical implications and point out the role of MPN as a major contributory factor for the pathogenesis of CVT with PP, even in absence of overt myeloproliferative features and additional prothombotic factors. Regarding management of vascular complications, patients with MPN-SVT and MPN-CVT responded equally well and efficacy of anticoagulation was not affected by the site of thrombosis. DisclosuresOffidani:Celgene: Honoraria, Research Funding; Janssen: Honoraria.

CALR mutation analysis is not indicated in patients with splanchnic vein thrombosis without evidence of a myeloproliferative neoplasm: a micro-review

Th e recent discovery of exon 9 insertion and/or deletion mutations of the CALR gene in up to 80% of JAK2and MPL-unmutated essential thrombocythemia and primary myelofi brosis patients compels the incorporation of CALR mutational analysis into the molecular diagnostic algorithm for these myeloproliferative neoplasms (MPN). MPN are a major cause of splanchnic vein thrombosis (SVT) which encompasses Budd-Chiari syndrome, portal and mesenteric vein thrombosis. Up to 40% of SVT patients are diagnosed with an overt or latent MPN [1]. While the MPN-associated JAK2 V617F mutation is consistently reported in cohorts of SVT patients, several studies have investigated the role of CALR mutation analysis for MPN diagnosis in the presence of SVT with some debate existing [2-10]. Here, all reports published to date are summarized (Table 1). Briefl y, of 944 patients studied only eight (0.8%) had evidence of a CALR mutation and of whom seven already had a previous diagnosis of an MPN. MPN patients with CALR mutations have a signifi cantly lower overall risk of thrombosis than their counterparts harboring the JAK2 V617F and this is clearly the case with respect to SVT. While CALR mutated MPN patients may develop SVT, summarizing those published studies to date, routine investigation for these mutations appears not to be indicated in the diagnostic algorithm for SVT where no clinical or hematological features of an MPN are present. References

Thrombotic risk factors in Chinese nonmalignant and noncirrhotic patients with portal vein thrombosis: an observational study with a systematic review of the literature.

Until now, no data on the routine screening for thrombotic risk factors in Chinese nonmalignant and noncirrhotic patients with portal vein thrombosis (PVT) have been reported. A total of 141 nonmalignant and noncirrhotic patients with PVT who underwent screening tests for thrombotic risk factors between September 2009 and August 2012 were included in this study. The JAK2 V617F mutation was found in 35 of the 141 patients tested. Neither the JAK2 exon 12 mutation nor the MPL W515 L/K mutation was found in any of the 50 patients tested. Overt myeloproliferative neoplasms (MPNs) were diagnosed in 13 patients (polycythemia vera, n=1; essential thrombocythemia, n=9; idiopathic myelofibrosis, n=3). Latent MPNs were considered in 23 patients with the JAK2 V617F mutation but without any significant abnormalities, as determined through regular blood tests. Anticardiolipin IgG antibodies were positive in none of the 136 patients tested. Paroxysmal nocturnal hemoglobinuria was not found in any of the 141 patients tested. Neither the factor V G1691A mutation nor the factor II G20210A mutation was found in any of the 72 patients tested. The C677T mutation in 5,10-methylenetetrahydrofolate reductase (MTHFR) was found in 29 of the 38 patients tested. Hyperhomocysteinemia was detected in eight of the 39 patients tested. MPNs are an important thrombotic risk factor in Chinese patients with PVT. However, the extreme rarity of paroxysmal nocturnal hemoglobinuria, anticardiolipin IgG antibodies, and factor V G1691A and factor II G20210A mutations has precluded any support for the implementation of routine screening for these thrombotic factors in such patients. Additional case-control studies should confirm the role of the MTHFR C677T mutation and hyperhomocysteinemia in the pathogenesis of PVT.

Evaluation of the prevalence and prospective clinical impact of the JAK2 V617F mutation in coronary patients

The JAK2 V617F mutation is not only found in the majority of patients with myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), but also has been reported in individuals without overt MPN. A close relation of the JAK2 V617F mutation to atherothrombotic events has been described, at least in patients with MPN. The prevalence of the JAK2 V617F mutation and its clinical impact in coronary patients is unknown. To address this issue, DNA samples from 1,589 subjects undergoing coronary angiography with up to 11 years of follow up were genotyped using allele-specific real-time PCR assays. Prevalence of the JAK2 V617F mutation was 1.32% (n = 21) in coronary patients. Two JAK2 V617F positive patients showed baseline platelet counts indicative for ET and a third patient developed ET during follow up, finally resulting in a percentage of 0.188% of ET cases. This corresponds to an up to fivefold accumulation of ET cases in coronary patients compared with the general population. Our study showed no impact of the JAK2 V617F mutation on future atherothrombotic events or overall survival (HR = 1.04 [0.33-3.27]; P = 0.949 and HR = 0.35 [0.05-2.46]; P = 0.288, respectively). Therefore, our data suggest that JAK2 V617F positive coronary patients are not at increased risk for future atherothrombotic complications. Routine mutation screening in coronary patients is, therefore, not warranted. However, number of ET cases appears to be accumulated in coronary patients. For this reason, we recommend JAK2 V617F testing only in coronary patients showing abnormal blood cell counts for further clarification.

Focal 18F-FDG uptake in bone marrow on PET/CT in a patient with JAK2 mutation without overt myeloproliferative neoplasm

A 58-year-old female diagnosed with early stage esophageal carcinoma in our hospital underwent endoscopic resection by endoscopic submucosal dissection (ESD) in April 2013. F-FDG PET/CT performed immediately prior to the ESD showed focal F-FDG accumulations in the vertebral body of Th8, and vertebral body and arch of L4 with SUV max of 3.98–4.42 (Fig. 1a–c). No masses or osteoclastic lesions were observed. MRI findings of the lesions showed low intensity on T1WI and high intensity on STIR image (Fig. 2a–d). To clarify the cause of the FFDG accumulation in the bone, we performed bone biopsy from the vertebral arch of L4. Histopathological findings revealed hypercellular marrow (80 % cellularity) and increases in number and size of megakaryocytes, most of which were in maturated form with hyperlobulated nuclei, which are usually found in cases with myeloproliferative neoplasm (MPN) (Fig. 3a, b). Reticulin fibrosis of marrow was observed minimally by Gitter staining (Fig. 3c), and collagen fibrosis was not observed. In contrast, laboratory workup for peripheral blood showed no abnormality: WBC 6,920/lL (stab 0.0 %, seg 61.0 %, lymph 30.0 %, mono 6.0 %, eosino 1.5 %, and baso 1.5 %), RBC 452 9 10/ lL, Hb 14.1 g/dL, Ht 40.6 %, PLT 33.3 9 10/lL, reticulocytes 1.2 %, LDH 229 U/L, VB12 351 mg/dL, and NAP score 223. Bone marrow biopsy subsequently performed from the left iliac bone showed normocellular marrow, but the number of megakaryocytes was also increased (Fig. 3d). Furthermore, JAK2 V617F mutation was detected in the bone marrow sample by real-time qualitative PCR with a sensitivity of more than 2 % of all alleles. G-banding showed normal diploid karyotype, and BCR–ABL translocation was not detected by FISH analysis. 5 months after the first visit, her laboratory data of peripheral blood was within the normal range, and FFDG PET/CT also showed similar F-FDG accumulation in the bone, without new lesions. We will continue to follow her progress carefully. The JAK2 V617F mutation is present in patients with Philadelphia-negative MPN, including over 90 % of polycythemia vera cases and about half of essential thrombocythemia and primary myelofibrosis cases [1]. The JAK2 V617F mutation may also be detected in healthy individuals without overt MPN [2]. Nielsen et al. [2] reported that the JAK2 V617F mutation was detected in 18 of 10,507 participants (0.2 %) in the general population, and three of these 18 individuals with the JAK2 V617F mutation developed overt myeloproliferative disorder during up to 17.6 years of follow-up. In the present case, focal F-FDG accumulation in bone marrow and histopathological findings, other than the finding of the left iliac bone marrow as positive for JAK2 V617F mutation, suggest that the patient is more likely to develop some form of overt MPN in the A. Fujimi (&) Y. Kanisawa Department of Hematology and Oncology, Oji General Hospital, 3-4-8 Wakakusa-cho, Tomakomai 053-8506, Japan e-mail: Akihito.fujimi@ojihosp.or.jp

Outcome of patients with splanchnic venous thrombosis presenting without overt MPN: A role for the JAK2 V617F mutation re-evaluation

Although investigation for JAK2 V617F mutation is recommended in patients presenting with splanchnic venous thrombosis (SVT), no specific clinical advice is given to SVT patients presenting without myeloproliferative neoplasms (MPN) and JAK2 V617F mutation. In MPN-free SVT patients, to investigate the clinical outcome, the clinical impact of re-evaluation for the JAK2 V617F mutation, and relationships with the occurrence and time to diagnosis of MPN. A cohort of non-cirrhotic SVT patients, enrolled at a single centre and prospectively analyzed. In 121 SVT patients prospectively followed from 1994 to 2012, a MPN was present in 28 (23.1%). Additional 13 patients (10.7%) showed only the JAK2 V617F mutation. During the follow-up, the JAK2 V617F mutation and/or MPN were identified in 8 patients (median time of development: 21 months, range 6-120), whereas 72 remained (MPN and JAK2 V617F)-free until the end of the observation. The mortality rate was higher among patients presenting with MPN and/or the JAK2 V617F mutation than in patients who developed later or remained disease-free (p=0.032). The thrombosis-free survival was lower in patients with (p=0.04) or developing later MPN and the JAK2 V617F mutation (p=0.005) than in patients (MPN and JAK2 V617F)-free. The incidence of bleeding was similar among groups. MPN with or without circulating positive clones for JAK2 V617F mutation can occur long after a SVT, identifying at risk patients for new thrombotic events. If confirmed in other studies, re-evaluation for JAK2 V617F mutation may be of help in early MPN detection and clinical management of SVT patients.

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

Author SummaryRecent descriptions of the existence of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumours. In this study, we focus on understanding the stem cell defect that results from a mutation in the JAK2 tyrosine kinase gene, which is present in the majority of patients with myeloproliferative neoplasms (MPNs), a group of clonal bone marrow diseases that are characterised by the overproduction of mature blood cells and increased frequency of leukaemia development. By using single-cell assays and mathematical modeling, followed by the individual assessment of daughter cells from single HSCs, we identify a distinct cellular mechanism that differentially affects stem cell and progenitor cell expansion. Specifically, we show that this single point mutation can negatively affect HSCs while leaving progenitor cell expansion intact. Characterising the mechanisms that link JAK2 mutations with clonal expansions that eventually lead to development of MPNs will inform our understanding of the earliest stages of tumour establishment and of the competition between subclones of proliferating progenitor/stem cells. These findings have direct relevance to all cancers of a suspected stem cell origin.

Thrombotic risk factors in Chinese Budd-Chiari syndrome patients

In Western countries, thrombotic risk factors for Budd-Chiari syndrome (BCS) are very common, including factor V Leiden mutation, prothrombin G20210A mutation, myeloproliferative neoplasms, paroxysmal nocturnal haemoglobinuria, etc. However, the data regarding thrombotic risk factors in Chinese BCS patients are extremely limited. An observational study was conducted to examine this issue. A total of 246 BCS patients who were consecutively admitted to our department between July 1999 and December 2011 were invited to be examined for thrombotic risk factors. Of these, 169 patients were enrolled. Neither factor V Leiden mutation nor prothrombin G20210A mutation was found in any of 136 patients tested. JAK2 V617F mutation was positive in four of 169 patients tested. Neither MPL W515L/K mutation nor JAK2 exon 12 mutation was found in any of 135 patients tested. Overt myeloproliferative neoplasms were diagnosed in five patients (polycythemia vera, n=3; essential thrombocythemia, n=1; idiopathic myelofibrosis, n=1). Two of them had positive JAK2 V617F mutation. Both CD55 and CD59 deficiencies were found in one of 166 patients tested. This patient had a previous history of paroxysmal nocturnal haemo-globinuria before BCS. Anticardiolipin IgG antibodies were positive or weakly positive in six of 166 patients tested. Hyperhomocysteinaemia was found in 64 of 128 patients tested. 5,10-methylenetetrahydrofolate reductase C677T mutation was found in 96 of 135 patients tested. In conclusion, factor V Leiden mutation, prothrombin G20210A mutation, myeloproliferative neoplasms, and paroxysmal nocturnal haemoglobinuria are very rare in Chinese BCS patients, suggesting that the etiological distribution of BCS might be different between Western countries and China.

The clinical significance of JAK2V617F mutation for Philadelphia-negative chronic myeloproliferative neoplasms in patients with splanchnic vein thrombosis

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF), collectively known as Philadelphia-negative (Ph-negative) chronic myeloproliferative neoplasms (MPNs), MPNs represent the most common causes of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT). The JAK2V617F mutation has been demonstrated in most of the Ph-negative chronic MPNs. The study objective was to assess the diagnostic value of JAK2V617F mutation in patients with SVT in a group of 68 patients with SVT (42 PVT,19 BCS, 7 combined PVT and BCS). By DNA-melting curve analysis, the JAK2V617F mutation was detected in 42.1 % of BCS, 38.1 % of PVT and 71.4 % of combined PVT and BCS groups. Thirteen of 15 (86.6 %) SVT patients with overt MPN and 16 of 53 (30.1 %) SVT patients without overt MPN (patients with either normal blood counts or cytopenias), including 6 of 16 with BCS (37.5 %), 7 of 33 with PVT (21.2 %) and 3 of 4 with combined BCS and PVT (75 %) possessed JAK2V617F mutation. A substantial proportion of patients with SVT were recognized as carriers of the JAK2V617F mutation despite the absence of overt signs of MPN. Receiver Operating Characteristic (ROC) curve analysis determined a platelet count of 190,000mm(3) (area under the curve; AUC=0.724, p=0.002) and a white blood cell (WBC) count of 8,150mm(3) (AUC=0.76, p=0.001) as the best cut-off values for the highest sensitivity and specificity ratios of the JAK2V617F mutation in patients with SVT. A significant positive correlation existed between the JAK2V617F mutational status of SVT patients and the WBC and platelet counts. Our results imply that JAK2V617F mutation screening should be an initial test for MPN in patients with SVT.