Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.     

Neurological disorders in essential thrombocythemia

Patients with essential thrombocythemia often complain of various subjective neurological symptoms. This prospective study aims to assess their incidence and response to therapy. Among 37 consecutive patients with essential thrombocythemia, 11 presented with neurological symptoms. Among them 4 had thrombotic events, 7 complained of transient or fluctuating subjective symptoms, and one had both. Brain magnetic resonance imagery failed to detect any substratum in patients with subjective symptoms. JAK2V617F mutation was found in 9 of 11 patients with neurological symptoms versus 14 of 26 patients without symptoms. Ten patients received low-dose aspirin for these symptoms: complete resolution was observed in 3, improvement with persisting episodes in 2, and resistance to aspirin in 2 patients, in whom addition of cytoreductive therapy became necessary to resolve those disabling symptoms. In this prospective cohort, 30% of patients with essential thrombocythemia presented neurological symptoms. Aspirin was fully efficient in only 30% of cases. JAK2V617F mutation could be a risk factor for such symptoms.     

Hydroxyurea (HU) is effective in reducing JAK2V617F mutated clone size in the peripheral blood of essential thrombocythemia (ET) and polycythemia vera (PV) patients

Ph-negative chronic myeloproliferative disorders (Ph^negcMPD) are treated according to the estimated vascular risk. The recent discovery of V617F point mutation of the JAK2 kinase, which frequently occurs in these diseases, has not changed their management so far. However, emerging data tend to support a prothrombotic role for the mutation, along with a better response of JAK2V617F mutated patients to hydroxyurea treatment. Our data further support this notion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

Background

The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes.

Design and Methods

We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63⁺ basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor.

Results

We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63⁺ basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63⁺ basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden.

Conclusions

These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus.     

Different involvement of the megakaryocytic lineage by the JAK2V617F mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis

Atypical megakaryocytes provide the histomorphological hallmark of all Philadelphia-chromosome negative chronic myeloproliferative disorder (Ph⁻ CMPD) subtypes and have not been studied so far for the JAK2^V617F mutation. The mutant gene dosage was determined in isolated megakaryocytes from 68 cases of JAK2⁺/Ph⁻ CMPD by a pyrosequencing assay. Megakaryocytes from essential thrombocythemia (ET) showed significantly lower levels of mutated JAK2 alleles compared to patients with chronic idiopathic myelofibrosis (cIMF) with manifest fibrosis and polycythemia vera (PV) but not to prefibrotic cIMF. Solely, ET JAK2V617F in megakaryocytes is associated with a PV-like phenotype, and at least in one patient, the JAK2 mutation was exclusively acquired within the megakaryocytic lineage. The overt differences between prefibrotic and fibrotic cIMF suggested a causative role of the gene dosage of mutant JAK2 in fibrotic progression. Megakaryocyte analysis of a follow-up of eight individual cases with sequential biopsies, however, showed that progression to homozygosity of V617F mutated JAK2 and onset of manifest fibrosis appeared to be independent events. We conclude that megakaryocytes might be the predominant or even the exclusive lineage that acquires the JAK2^V617F mutation in ET and that the JAK2^V617F evolution to higher gene dosages represents a dynamic and complex process substantially involving megakaryocytes.     

JAK2 V617F突变阳性原发性血小板增多症转化为纯红细胞白血病1例报告并文献复习

原发性血小板增多症(essential thrombocythemia,ET)是一种主要涉及巨核系的克隆性骨髓增殖性肿瘤(myeloproliferative neoplasms,MPN)。在30%~50%ET患者中检测出JAK2 V617F基因突变[1]。ET年发病率很低,为0.21/105~2.27/105[2]。ET诊断后15年内发生白血病转化的发生率为2%[3]。纯红细胞白血病是一类临床上很罕见的骨髓红系恶性增殖性疾病。     

Decrease in JAK2 V617F allele burden is not a prerequisite to clinical response in patients with polycythemia vera

Background

Although reduction in the JAK2^V617F allele burden (%V617F) has been suggested as a criterion for defining disease response to cytoreductive therapy in polycythemia vera, its value as a response monitor is unclear. The purpose of this study is to determine whether a reduction in %V617F in polycythemia vera is a prerequisite to achieving hematologic remission in response to cytoreductive therapy.

Design and Methods

We compared the clinical and hematologic responses to change in %V617F (molecular response) in 73 patients with polycythemia vera treated with either interferon (rIFNα-2b: 28, Peg-rIFNα-2a: 18) or non-interferon drugs (n=27), which included hydroxyurea (n=8), imatinib (n=12), dasatinib (n=5), busulfan (n=1), and radioactive phosphorus (n=1). Hematologic response evaluation employed Polycythemia Vera Study Group criteria, and molecular response evaluation, European Leukemia Net criteria.

Results

Of the 46 treated with interferon, 41 (89.1%) had a hematologic response, whereas only 7 (15.2%) had a partial molecular response. Of the 27 who received non-interferon treatments, 16 (59.3%) had a hematologic response, but only 2 (7.4%) had a molecular response. Median duration of follow up was 2.8 years. Statistical agreement between hematologic response and molecular response was poor in all treatment groups.

Conclusions

Generally, hematologic response was not accompanied by molecular response. Therefore, a quantitative change in %V617F is not required for clinical response in patients with polycythemia vera.     

Comparison of clinicopathologic findings according to JAK2 V617F mutation in patients with essential thrombocythemia

The JAK2 V617F mutation is present in most patients with polycythemia vera, but in fewer patients with essential thrombocythemia (ET). We have assessed the frequency of this mutation in ET patients using amplification refractory mutation system PCR and determined the relationship of the mutation with disease phenotypes. Clinical-laboratory findings and histomorphological features were compared according to mutational status in 108 ET patients. The mutation was detected in 61 patients (56.5%) including one homozygous patient. Those with the mutation had significantly higher leukocyte (P = 0.003) and neutrophil (P = 0.007) counts. However, the incidences of thrombotic events and progression to advanced stages did not differ significantly between patients with and without the mutation. Thrombotic events were significantly correlated with older age (P = 0.025). Morphological analysis revealed that erythroid hypoplasia was exclusively found in the mutation-negative patients (P = 0.027). We could confirm previous findings of higher leukocyte count in ET patients with JAK2 mutation, but could not find any correlation with thrombotic events. Therefore, the detection of the mutation could characterize a subset of ET patients with distinct phenotype, despite its clinical significance being still undetermined.     

Neurological symptoms in essential thrombocythemia: impact of JAK2V617F mutation and response to therapy

Patients with essential thrombocythemia (ET) often suffer from neurological symptoms (NS) not ever resulting from previous thrombotic cerebral events (TCE). We reported NS occurred in 282 patients, in order to identify the factors influencing ET‐related NS in the absence of TCE, and the response to therapy. Overall, 116 of 282 patients (41%) presented NS; 101 of them (87%) reported subjective transient and fluctuating NS, without concurrent TCE, which we defined as ET‐related NS, by frequency: cephalalgia, chronic paresthesias, dizziness or hypotension, visual disturbances, and tinnitus. In univariate analysis, ET‐related NS resulted more frequently in young people (P = 0.017) and in females (P = 0.025). We found a higher prevalence of JAK2V617F mutation in ET‐related NS patients (P = 0.021). In multivariate analysis, gender (P = 0.024) and JAK2V617F mutation (P = 0.041) remained significantly associated with the development of ET‐related NS, with a risk of about four times higher for JAK2V617F‐mutated patients (OR = 3.75). Ninety‐seven of 101 patients with ET‐related NS received an antiplatelet (AP) agent at the time of NS, whereas only selected high‐risk ET‐related NS patients were treated with a cytoreductive drug, according to the published guidelines and similarly to patients without NS. We observed that only 32 of 97 (33%) patients with ET‐related NS achieved a complete response after AP treatment. Among the 65 non‐responder patients, 36 (55.4%) improved NS after the introduction of cytoreductive therapy; therefore, the addition of cytoreductive treatment should be considered in this setting.     

Influence of the JAK2 V617F mutation and inherited thrombophilia on the thrombotic risk among patients with essential thrombocythemia

It is uncertain whether the JAK2 V617F mutation increases the thrombotic risk in patients with essential thrombocythemia, and it is unknown whether inherited thrombophilia is an additive risk factor in mutated subjects. We studied 132 patients with essential thrombocythemia, 38 of them (29%) with a history of thrombosis. The JAK2 mutation was present in 83 (63%), and inherited thrombophilia in 7. The mutated patients <60 years had a relative risk (RR) for thrombosis at any time of 3.83 (95%CI 1.27–11.49) in comparison with wild-type patients; in those with both the mutation and thrombophilia the RR was 2.23 (95%CI 1.57–3.18) and 7.66 (95%CI 2.66–22.03) in comparison with mutated or wild-type patients without thrombophilia, respectively. During the follow-up, only the homozygotes for JAK2 V617F were more prone to thrombosis (RR 17.25, 95%CI 2.33–127.4). Among the patients >60 years, no increase in RR was associated with the JAK2 mutation. In conclusion, in the younger patients with ET the thrombotic risk is higher in the JAK2 V617F-mutated and is further increased by the presence of inherited thrombophilia.     

Modulation of JAK2 V617F allele burden dynamics by hydroxycarbamide in polycythaemia vera and essential thrombocythaemia patients

The definition of primary refractory acute myeloid leukaemia is the failure to achieve a response after one or two cycles of induction. Given that there are many different strategies involving different doses of cytarabine and anthracyclines, which may or may not be equivalent, and as this is an area of unmet need with the potential for the development of new agents and strategies, uniform criteria for response have been described that need to be adhered to. The outcome of patients with chemoresistant disease is poor with only a proportion of patients salvaged by allogeneic stem cell transplantation. Progress in supportive care strategies and donor identification has enabled more of these patients to undergo unrelated donor transplantation. Novel strategies and new agents directed at the biology of the disease and the mechanisms of resistance are needed.     

A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.     

Leucocytosis in polycythaemia vera predicts both inferior survival and leukaemic transformation

Leucocytosis (leucocyte count >15 x 10(9)/l) was recently associated with thrombosis in polycythaemia vera (PV). This study sought the prognostic relevance of leucocytosis for survival and leukaemic or fibrotic transformation. Amongst 459 patients with PV seen at our institution in recent years (median age, 60 years; 56% males), 146 deaths and 88 leukaemic (n = 34) or fibrotic (n = 54) transformations were documented. Arterial or venous thrombosis occurred in 14% and 9% of patients at diagnosis and in 25% and 15% during follow-up, respectively. Multivariate analysis identified the advanced age (P < 0.0001), leucocytosis (leucocyte count >/=15 x 10(9)/l; P = 0.0006) and arterial thrombosis at diagnosis (P = 0.01) as independent predictors of inferior survival. In the absence of the first two risk factors, median survival was projected at 272 months as opposed to 108 months in the presence of both risk factors (P < 0.0001). Leucocytosis was also identified as an independent predictor of both leukaemic transformation and venous thrombosis during follow-up. Time-to-event analysis did not disclose a significant association between single or multiple cytotoxic drug exposure and either leukaemic or fibrotic transformation. The current study highlighted the prognostic relevance of leucocytosis on various aspects of the disease in PV.