Carriers Of JAK2V617F Mutation Research Articles

Thrombotic incidents in patients with myelofibrosis suggest to be independent of JAK2 V617f mutational status.

Myelofibrosis (MF) belongs to a group of conditions known as Philadelphia-negative myeloproliferative neoplasms (MPN). Bleeding or various vascular complications could be the main causes of morbidity and mortality in patients with MF. MPNrelated thrombosis is a multifactorial process and in the case of myelofibrosis, little is known. The risk factors for thrombotic complications in MF have been rarely assessed. The purpose of this study was to investigate the incidence of thrombotic events in MF and the role of JAK2 V617F mutation as a risk factor for thrombotic incidents in patients with MF. In our study of 37 patients, 35% had thrombotic events in the past. All patients were admitted to the Clinic of Hematology, St Ivan Rilski University Hospital, Sofia, Bulgaria between 2016 and 2019 and diagnosed based on the WHO criteria of 2016. The majority of patients (23, 62%) proved positive for JAK2 (Janus kinase) V617F mutation carrying one (16, 70%) or two (7, 30%) mutated alleles. Thirteen of the patients (35%) had a thrombotic event in the past and 9 of them (69%) were carriers of JAK2 V617F mutation. Fourteen patients of those without thrombotic history (24, 58%) were also carriers of JAK2 V617F mutation. As a whole, we did not find a statistically significant difference between JAK2 V617F mutation and the frequency of thrombotic events. Rendering an account to the possible life-threatening complications, treatment decisions should be undertaken upon possible antithrombotic prevention in MF.

Analysis of gene mutation characteristics in patients with chronic neutrophilic leukaemia

ABSTRACTObjective: This study aims to investigate the gene mutation characteristics of chronic neutrophilic leukaemia (CNL).Method: This study retrospectively analyses the molecular biological characteristics, laboratory characteristics and clinical data of four patients with CNL that were admitted in the second Hospital of Shanxi Medical University from May 2014 to October 2016. On the basis of the molecular biological data of 22 patients with CNL and 4 patients with CNL, we further analysed the characteristics of CNL molecular mutation.Results: Two out of the four patients with CNL were carriers of colony-stimulating factor 3 receptor (CSF3R) mutation, among which two were carriers of CSF3R T618I mutation combined with ASXL1 mutation and SETBP1 mutation, and two were only carriers of JAK2 V617F mutation. According to the molecular biological data of 22 patients with CNL, 20 patients were positive for CSF3R mutation. Two patients were positive for JAK2 V617F mutation. A total of 10 patients were positive for SETBP1 mutation which was correlated with the CSF3R T618I gene mutation (P = 0.03). A total of 13 patients were positive for ASXL1 mutation. No patients carried mutations in ASXL2 and MPL genes.Conclusion and Discussion: CSF3R mutation is the main tumorigenic mutation in CNL, in which CSF3R T618I mutation is the main mutation, and an extremely small number of CNL patients may be caused by JAK2 V617F mutation. SETBP1 and ASXL1 are the most common concomitant mutations in CNL with CSF3R mutation, and SETBP1 and CSF3R T618Imutations may have a certain correlation.

PO-19 - Platelet (PLT) adhesion under flow condition in essential thrombocythemia (ET) and polycythemia vera (PV) is variably influenced according to patient mutational status

The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels. To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV. 86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded. PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only. ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients. Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).

Diagnosis of latent polycythemia vera: A clinician's opinion

to identify the clinical features of latent polycythemia vera (PV) as an independent nosological entity. The investigation enrolled 81 patients (50 with extensive (manifest) PV and 31 with latent PV) who had visited the Outpatient Department, Hematology Research Center, Ministry of Health of Russia, in 2014 to October 2015. The gender distribution of the patients was statistically comparable in the analyzed groups. The patients with manifest PV were slightly older than those with latent PV: the median age in the compared groups was 56 and 44 years, respectively. Red blood cell counts, hemoglobin concentrations, and packed cell volume were higher in the patients with manifest PV. Blood platelet counts were higher in the latent PV group. There were no differences in the number of white blood cells in the compared groups. All the patients were JAK2 V617F mutation carriers. The JAK2 allele load was significantly higher in the manifest PV group than in the latent PV group. The compared patient groups differed in the rate of thromboses in the history or at diagnosis. In the patients with latent PV, thromboses were detected in 38% of cases versus 16% in those with manifest PV. In latent PV, there were mainly venous thromboses; abdominal vascular thromboses were diagnosed with a high frequency. Arterial thromboses were revealed in only 2 cases. Chronic myeloproliferative disease that is characterized by the JAK2 V617F mutation, borderline hemoglobin counts, and morphological features of a bone marrow trephine biopsy specimen, which are specific for PV, is an independent PV variant, namely: latent PV.

Platelet Adhesion Under Flow Condition in Patients with Essential Thrombocythemia (ET) and Polycythemia Vera (PV): Analysis According to the Mutational Status

Background: ET and PV are two myeloproliferative neoplasms characterized by a high incidence of both arterial and venous thrombosis, and microcirculatory disturbances. So far, no studies have been performed to characterize ET and PV patient platelet (PLT) adhesion properties in flow conditions, an index of thrombus formation capacity in vitro (Platelets 2012; 23(3): 229-42).Aim: In this study, in a cohort of patients with ET or PV, we wanted to evaluate the PLT adhesiveness in vitro by a dynamic method and to estimate whether this property is influenced by any of the three somatic mutations, i.e. JAK2 V617F, Calreticulin (CalR) and MPL, typical of these diseases, of which, the JAK2 V617F mutation has a significant association with thrombosis occurrence, while CalR and MPL mutations have not. Further, the relations of PLT adhesion to main hematological parameters and to cytoreductive hydroxyurea (HU) therapy were also considered.Patients and Methods: Sixty-nine patients, i.e. 47 ET (20 M/27 F; mean age 60 years, range 33-86) and 22 PV (13 M/9 F; mean age 65 years, range 46-80), and 26 healthy controls (13 M/13 F; 44 years, range 27-61) were included into the study. For the adhesion assay, peripheral venous whole blood was drawn in sodium citrate, recalcified in the presence of heparin, and perfused over a collagen-coated surface for 4 min at a shear rate of 1,000 s-1. PLTs were then stained with an anti-CD62P (P-selectin)-FITC antibody as a PLT activation index, and annexin V-AlexaFluor647 to detect PLT procoagulant phosphatidylserine expression. After staining, images of adherent platelets in random fields were taken using phase contrast and fluorescence imaging with the EVOS® fluorescence microscope (Life Technologies). Results are expressed as the mean±SEM of the percentage of area covered by all PLTs (% coverage), or as the % of adherent PLTs positive for either P-selectin or phosphatidylserine. Main hematological parameters (i.e. PLT count, WBC count, RBC count, Hb, Hct, MCV, MPV), therapeutic regimens, and mutational status (JAK2 V617F, CalR, or MPL) were also recorded. Statistical analysis was performed by SPSS® (IBM) software package.Results: PLT adhesion was significantly (p<0.01) greater in both ET (44.6±2% coverage) and PV patients (45.6±2.7%) compared to controls (36±2.1%). The analysis of PLT adhesion according to the mutational status shows that, in ET patients, PLT adhesion was greatest in JAK2 V617F mutation carriers (n=22), followed by triple negative (n=7), and CalR-positive (n=15). The only 3 MPL-positive ET patients had the lowest PLT adhesion values. In PV patients, those with >50% JAK2 V617F allele burden carriers (n=8; 52.5±2% coverage) presented a significantly higher PLT adhesion than those with <50% allele burden (n=14; 42.4±3.9, p<0.05). Notably, in both ET and PV patients, while the overall PLT adhesion was significantly increased, the percentage expression of P-selectin by adherent PLTs (~ 75%) was not statistically different from that of control subjects. Differently, the expression of procoagulant phosphatidylserine on adherent PLTs was reduced in both ET (p<0.001) and PV patients (p=n.s.) compared to controls. Among hematological parameters, in ET patients, a significant (p<0.001) correlation was found between PLT adhesion with PLT count, but not with WBC count, RBC count, Hb, Hct, MCV, or MPV. As regard to treatments, a significant reduction in PLT adhesion was associated with cytoreductive treatment with HU (p<0.05) in patients with PV, but not with ET.Conclusions: ET and PV platelets show a greater thrombus formation capacity in vitro in a dynamic flow condition, as measured by an increase in adhesion to collagen. This capacity is significantly augmented according to the JAK2 V617F mutational allele burden and decreased by HU therapy. A prospective study of the PLT thrombus formation potential in a dynamic model is ongoing to evaluate the predictive value of this parameter on the thrombotic risk of ET and PV patients.Project funded by "AIRC IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research (A.I.R.C.)". DisclosuresNo relevant conflicts of interest to declare.

Screening MPL515 mutations from 286 cases of essential thrombocythemia and primary myelofibrosis

Objective To establish a simple and sensitive method to detect MPL515 mutations in peripheral blood of ET and PMF patients, and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients. Methods Totally 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases.SYBR Green Ⅰ real-time PCR was used to detect JAK2V617F mutation.Taqman probe was designed to be specific for the three types of mutations (MPL515wt, MPLW515L and MPLW515K).Real-time PCR was used to detect MPL515 mutations.The results were confirmed by sequencing after T-A cloning. Results Among 261 ET patients, 119 cases (45.6%) were identified as JAK2V617F mutation carriers and 7 cases (2.7%) were detected to be MPL515 mutation carriers, including 5 cases with MPLW515L, 1 case with MPLW515K and 1 case with MPLW515L+K.Additionally 10 cases with JAK2V617F（40.0%） and 3 cases with MPL515 （12.0%） were screened out in 25 PMF patients, including 1 case with MPLW515L and 2 cases with MPLW515L+K.One ET patient was found to harbor concurrent JAK2V617F and MPL515 mutations. Conclusion JAK2V617F mutation is the major molecular marker of ET and PMF, meanwhile MPL515 mutation is important and useful complement.(Chin J Lab Med,2012,35:634-638) Key words: Thrombocytosis; Myelofibrosis; Receptors，thrombopoietin; Janus kinase 2; Mutation; Polymerase chain reaction

ADP-Induced Whole Blood Aggregometry and Platelet-Associated Thrombin Generation (TG) In Essential Thrombocythemia (ET) and Polycythemia Vera (PV) Patients

AbstractAbstract 1981Patients with ET and PV are characterized by an increased rate of thrombotic complications and by several abnormalities of platelets, more pronounced in JAK2V617F mutation carriers. Platelet function inhibitors are widely utilized for thromboprophylaxis in ET and PV patients. An increased reactivity to ADP of platelets from these patients has previously been shown by light transmission platelet aggregometry studies, which might explain the failure of aspirin to fully protect from thrombosis these patients, mainly those at high risk. While ADP activation pathway is critical for platelet aggregation, less is known about its role to the initiation of blood coagulation by platelets. In this study we wanted to evaluate in a group of patients with ET and PV, the platelet reactivity to ADP in terms of both proaggregating and procoagulant responses.ADP-induced platelet aggregation and platelet procoagulant potential (i.e. thrombin generation capacity) were measured by the whole blood impedance aggregometry (Multiplate analyser) and by the Calibrated Automated Thrombogram (CAT) assay, respectively. Whole blood platelet aggregation was induced by 6.5 μM ADP. Whereas thrombin generation (TG) was measured in platelet rich plasma (PRP: 150,000 platelets/μl) both in resting conditions and after stimulation by 1.6 μM and 8.3 μM ADP. Fifty patients with ET (58% V617F mutation carriers) and 40 patients with PV (95% V617F mutation carriers) were enrolled into the study.The results show that ET and PV patients presented with significantly higher ADP-induced whole blood platelet aggregation compared to control subjects (786±70 AU*min, and 777±54 AU*min, respectively, versus 465±70 AU*min, both p<0.05), the highest values being observed in ET and PV patients carriers of the JAK2V617F mutation (806±75 AU*min; p<0.001 vs controls). In resting conditions, TG in PRP from ET (133±38 nM) and PV (144±54 nM) patients was significantly greater compared to control PRP (103±21 nM; p<0.05). Similarly, in ADP-stimulated conditions, platelet TG was significantly higher (p<0.05) in ET and PV patients at both 1.6 μM ADP (140±32 nM and 154±59 nM; respectively) and 8.3 μM ADP (161±38 nM and 181±65 nM; respectively) compared to controls (1.6 μM ADP: 112±19 nM; 8.3 μM ADP: 132±26 nM). The analysis according to the JAK2 mutational status showed that platelets from JAK2V617F carriers induced significantly higher TG when stimulated by 8.3 μM ADP (185±47nM) compared to 1.6 μM ADP (146±45nM; p<0.001). Differently, this difference was not observed in JAK2 wild type patients. While in resting conditions patients on hydroxyurea (HU) generated less thrombin compared to non-HU treated patients, no effects of patients' therapy on TG (i.e. aspirin, HU, aspirin+HU) was observed in the ADP-stimulated conditions.In conclusion, for the first time we demonstrated that platelets from ET and PV patients are more reactive to ADP, not only in terms of increased platelet aggregation in a whole blood system, but also as an enhanced thrombin generation, particularly in those carriers of JAK2V617F mutation. These data support the hypothesis that the use of ADP receptor-inhibitors, in addition to aspirin, might be of help in the prevention of thrombosis in these conditions, by allowing a more complete inhibition of platelet functions. Disclosures:No relevant conflicts of interest to declare.

Microparticle-Associated Thrombin Generation and Procoagulant Activity Is Increased In Patients with Essential Thrombocythemia

AbstractAbstract 1985Essential Thrombocythemia (ET) is a chronic myeloproliferative neoplasm characterized by an increased thrombotic risk. Numerous quantitative and qualitative abnormalities of platelets and leukocytes, arising from the clonal proliferation of hematopoietic progenitor cells, have been described as responsible of the thrombophilic state in this disease. Recently, a high number of plasma microparticles (MP) of different cellular origin has been described in ET patients. MP are a key component of the hemostatic response and have been found increased in diseases at high thrombotic risk. To explore the contribution of plasma MP to the hypercoagulable state of patients with ET, in this study we aimed to characterize the MP functional procoagulant features. We used two different methods: the calibrated automated thrombogram (CAT), to determine the MP-associated thrombin generation (TG), and the P-PPL/1 assay (Stago R&D) to measure the MP-associated procoagulant activity (PCA). Both assays were performed in platelet free plasma (P-FP) obtained from 69 ET patients (24M/45F; 33 carriers of JAK2V617F mutation) and 67 control subjects (32M/35F). In a subgroup of 23 ET patients and 23 controls MP-associated TG and PCA were also determined in MP-free plasma (MP-FP). PFP was obtained by two serial centrifugations (4,000 rpm for 15 min, then 11,000 rpm for 10 min) and MP-FP by re-centrifuging P-FP at 14,000 rpm for 30 min. MP were isolated from the pellet. For the TG assay, 80 ul of P-FP or MP-FP and 20 ul of buffer were mixed, and TG started by adding CaCl2. The results were expressed as lag-time, peak, area under the curve (ETP), and time-to-peak (ttPeak). For P-PPL/1 assay, 100 ul of P-FP or MP-FP were mixed with 50 ul of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds.The results show that P-FP from ET patients generated significantly higher quantity of thrombin compared to controls, as demonstrated by the shorter lag-time (22.1±8.7 vs 29.5±14.7 min; p=0.004) and time to peak (26.6±8.4 vs 33.3±13.2 min; p=0.004) and the significantly greater peak (52.9±24.9 vs 38.24±20.9 nM; p=0.002) and ETP (765.6±206.4 vs 537.5±295 nM*min; p=0.001). Similarly, the MP-associated PCA was significantly increased in ET patients (79±11 sec) compared to controls (89±11 sec; p<0.05). This increase was due to the presence of MP, as no TG and little PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples to the original values of P-FP for both assays. TG was significantly (p<0.05) increased in the JAK2V617F mutation carriers (lag-time: 19.5±7.4 min; peak: 59.8±26.9 nM; ttpeak: 23.9± 4.9 min) compared to wild-type subjects (lag-time: 24.5±9.1 min, peak: 45.4±20.6 nM, ttpeak: 29.3±10.3 min), while no significant differences were found for PCA. Significant correlations were found between the PCA by PPL-assay and the different parameters of TG assay [lag-time (R2= 0.414), peak (R2= -0.542), ETP (R2= -0.514)].In conclusion, our results show that MP-associated TG capacity, as well as PCA, are increased in plasma from ET patients. The highest MP-associated TG was found in JAK-2 mutation carriers, who also are at higher risk for thrombosis compared to wild-type subjects. Our data provide evidence for a contribution of MP to the thrombophilic state of these patients and suggest to test MP associated TG and PCA in prospective studies to predict thrombosis in ET patients. Disclosures:No relevant conflicts of interest to declare.