Detection Of JAK2 V617F Mutation Research Articles

The clinical value of plasma CFDNA JAK2 V617F mutation detection in myeloproliferative neoplasms

The clinical value of plasma CFDNA JAK2 V617F mutation detection in myeloproliferative neoplasms

Features of immune mediated diseases in JAK2 (V617F)-positive myeloproliferative neoplasms and the potential therapeutic role of JAK inhibitors

ObjectiveThe Janus Kinase (JAK) 2 (V617F) mutation is the most frequently detected in myeloproliferative neoplasms (MPN). JAK2(V617F) mutation displays a pro-inflammatory phenotype that may be associated to a higher risk of immune mediated diseases (IMIDs), thromboembolic complications or other cancers. We aimed to evaluate the prevalence and main features of both rheumatic and non-rheumatic IMIDs in a cohort of MPNs patients with JAK2 (V617F) mutation. MethodsStudy of all patients diagnosed with MPNs and JAK2 (V617F) mutation at a tertiary hospital in Northern Spain from 2004 to 2022. We focused on patients with rheumatic IMIDs to assess the time from IMIDs diagnosis to the detection of JAK2V617F mutation, the clinical course and severity of the disease, potential thrombotic complications, malignancies and therapeutic response. Results130 patients (73 men/57 women; mean age, 70.1 ± 14.5 years) were identified. Fifty-four (41.5 %) patients were diagnosed with at least one IMID. The prevalence of rheumatic IMIDs was 7.7 % (n = 10), including rheumatoid arthritis (n = 4), polymyalgia rheumatica (n = 3), Sjögren syndrome (n = 1), antiphospholipid syndrome (n = 1) and autoinflammatory syndrome with WDR1 mutation (n = 1). Thrombotic complications were observed in 4 of these 10 patients. The clinical course of the rheumatic IMID was mild in most cases and responded to conventional immunosuppressive therapy. One patient was successfully treated with Baricitinib, a JAK1/JAK2 inhibitor. ConclusionsA high prevalence of rheumatic IMIDs is observed in patients with MPNs and JAK2 (V617F) mutation. JAK inhibitors might be a targeted therapy option in these patients.

JAK2 V617F Mutation in Patients with Thrombosis

Background: Thrombosis is a major disorder with serious complications. The JAK2V617F mutation results in constitutive phosphorylation of JAK2 and activation of the cellular proliferation cascade.
 The present study aimed to clarify the prevalence of JAK2V617F mutation in patients with thrombosis.
 Subjects and Methods: This case-control study was conducted on 60 subjects subdivided into 2 groups: 30 healthy subjects as a control group (Group I) and 30 patients diagnosed with thrombosis (Group II). All subjects were subjected to full history taking, and clinical examination and investigated for Molecular detection of JAK2V617F mutation, FV (Factor V Leiden) – PTH (Prothrombin), and MTHFR (Methylene tetrahydro-folate reductase).
 Results: There was a statistically significant difference among the studied groups as regards the JAK2V617F mutation (P value < 0.05, odds ratio = 41.216, 95% C.I. 95% = 2.302 - 738.034, relative risk = 25, 95% C.I. 95% = 1.547– 404.012). This mutation was more significantly associated with venous thrombosis than arterial thrombosis (P = < 0.05).
 Conclusion: This study showed a significant association between JAK2V617F mutation and thrombosis with more significant evidence in the venous than the arterial thrombosis.

Clinical, Molecular, and Histopathological Correlates in a Series of 24 Consecutive Cases of Suspected Myeloproliferative Neoplasms with a JAK2V617F Variant Allele Frequency ≤1%

Background: JAK2V617F is detected in >95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F variant allele frequency (VAF) is associated with different clinical characteristics and outcome. Highly sensitive molecular assays led to detect JAK2V617F mutation also in healthy subjects, associated with a higher risk of coronary heart disease (N Engl J Med. 2017;377(2):111-121) in the context of CHIP. To date, comprehensive data on suspected MPN patients with a JAK2V617F VAF≤1% are lacking. Aim and methods: The aim was to analyze clinical, molecular, and histopathological correlates in a series of suspected MPN patients (pts) with a JAK2V617F VAF≤1% (from 2017 to 2022) at our Center. JAK2V617F VAF was measured in granulocytes by highly sensitive RTQ-PCR. Conventional karyotype and target next-generation sequencing (NGS) analysis were performed (complete data will be presented at the meeting). Bone marrow (BM) biopsies were reviewed by two independent expert pathologists, R.S. and U.G., the latter included as an author in the International Consensus Classification (Blood 2022; 2022015850). Results: 21 males and 3 females (M/F=7:1), with a median age of 58 y (range, 22-81) were included. Median JAK2V617F VAF was 0.2% (0.1-1). In the suspicion of MPN, complete diagnostic work-up was carried out in pts presenting with erythrocytosis (n=15), thrombocytosis (n=4), history of atypical thrombosis (n=3), splenomegaly (n=1) and leukocytosis (n=1), (Fig.1). In pts with erythrocytosis, median (range) Hb and Hct values were 17 g/dL (15.8-18.5) and 51.5% (50.4-54.7), whereas leukocytes (6.6 x 109/L, 5-11) and platelet (258 x 109/L, 172-440) were within normal range. EPO level (10 mUI/mL; 1.8-12.5) was subnormal in 3 pts. Abnormal karyotype was documented in one case (45,X,-Y[7]/46,XY[13]). Additional mutations were documented in 3 pts: HBB T124N, SH2B3 S186I and cKIT D816V. In pts with thrombocytosis, platelet range was 460-1450 x 109/L; Hb, Hct and leucocytes values were normal. MPL W515K and CALR L367Tfs*46 additional mutations were in one and two pts, respectively. Considering pts with atypical thrombosis, one had a history of arterial events while the others experienced cerebral venous sinus and portal vein thrombosis. Thrombophilia screenings were negative. Karyotype was normal in two pts, whereas one had 45,X,-Y[3]/46,XY[20]. Additional CBL p.D460dup was detected in one case. The subjects investigated for splenomegaly/leukocytosis had normal karyotype, no additional mutations. Morphological features consistent with PV were documented in 4 pts (Fig. 2, A-B). In one of these cases, multifocal dense infiltrates of CD117+/CD25+ mast cells were detected, fulfilling the diagnostic criteria for systemic mastocytosis with associated hematological neoplasm. In the other cases, microscopic examination showed age-adjusted normal or focally increased cellularity, regular or mildly expanded erythroid lineage with no/only minor increase in granulocytic lineage and a higher number of megakaryocytes with PV-like features (i.e. polymorphic forms without atypia and no dense cluster formation) (Fig. 1, C-D). Neither reticulin fibrosis nor excess blasts were detected and these pts had a final diagnosis of MPN unclassifiable (MPN-U), with a PV-like morphology. In pts with thrombocytosis histology was consistent with ET in three cases and overt-PMF in one. Among ET, one pts harbored MPLW515K with grade 1 reticulin fibrosis (Fig. 2, E-F), whereas the overt PMF had CALR type 1 additional mutation. BM biopsies of pts presenting with atypical thrombosis, leukocytosis and mild splenomegaly showed non-specific MPN-like features (including focally increased cellularity, mild expansion of erythroid or granulocytic lineages, higher number of megakaryocytes with a TE-like or a PV-like morphology) and were diagnosed as MPN-U. Conclusions: This study highlights the importance of using highly sensitive assays to detect JAK2V617F, as well as the need to search for CALR and MPL mutations, particularly in pts with thrombocytosis. Moreover, in pts presenting with erythrocytosis and JAK2V617F positivity with VAF≤1%, histopathological diagnostic clues to MPN may be subtle as in the early phase of the disease, emphasizing the need to integrate histopathology with clinical and genetic data in order to address patients to proper clinical management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

79. Implications of fortuitous detection of JAK2 V617F mutations with solid tumor clinical sequencing

79. Implications of fortuitous detection of JAK2 V617F mutations with solid tumor clinical sequencing

Analysis of Clinical Characteristics of JAK2 V617F and BCR-ABL Double-Mutant Myeloproliferative Neoplasms

To analyze the disease types, clinical manifestations, efficacy and outcome of JAK2 V617F and BCR-ABL double-mutant myeloproliferative neoplasms (MPN), and provide a reference for the diagnosis, treatment and prognosis of MPN. The clinical characteristics, diagnosis, therapeutic efficacy and outcome of JAK2 V617F and BCR-ABL double-mutant MPN were analyzed comprehensitively by combining a clinical case diagnosed and treated in our hospital with literature cases from CNKI and PubMed databases. A total of 38 related literatures were retrieved from the two databases by searching "JAK2 V617F" and "BCR-ABL" as key words from 1990 to 2019, and 59 cases were involved. Among all the 60 cases, 41 were males (68.3%) with a median age of 61 (32-77) years old, while 19 were females (31.7%) with a median age of 58 (21-82) years old. The BCR-ABL fusion gene and JAK2 V617F mutation were found simultaneously in 21 cases (35%), 19 cases (31.7%) with JAK2 V617F mutation were found during the treatment of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). Ph+CML was detectable in 20 cases (33.3%) during the treatment of JAK2 V617F mutation positive MPN. Polycythemia vera (PV) was the most common MPN coexisting with CML (30%), followed by essential thrombocythemia (ET) (26.7%) and primary myelofibrosis (PMF) (21.7%). In addition, there were 13 cases (21.7%) not classified in the literature. Among the 60 cases, 35 CML patients were clearly staged, including 31 in the chronic phase, 3 in the accelerated phase, and 1 in the blast crisis phase. As for the subtypes of BCR-ABL fusion gene, there were 30 cases with clear classification, including 28 cases of p210, 1 case of p190 and 1 case of p230. As cases of BCR-ABL and JAK2 V617F double-mutant MPN are reported, simultaneous detection of JAK2 V617F mutation and BCR-ABL fusion gene in MPN patients is necessary to avoid misdiagnosis and missed diagnosis.

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

Myeloproliferative neoplasm (MPN) is a group of myeloid disorders which leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with BCR-ABL positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia Vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, formalin-fixed paraffin-embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. This study is a descriptive crosssectional study. Forty FFPE marrow specimens were retrieved from the years 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. JAK2-V617F mutation was positive in 13 out of 40 MPN cases. Level of agreement between HRM and AS-PCR was 97.5%. HRM is a rapid and powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

JAK2V617F mutation prevalence on Chilean adults suffering from primary mesenteric and portal venous thromboses.

Mesenteric and portal venous thromboses are rare diseases with high mortality rates and are strongly associated with hepaticcirrhosis, and abdominal inflammatory or tumoral processes, but in some cases can be the first sign of myeloproliferative neoplasm (MPN) or hereditary thrombophilia. JAK2V617F mutation detection is an important diagnostic tool for MPN patients. The aim of this study was to describe the JAK2V617F mutation prevalence on Chilean patients suffering from a primary splanchnic venous thrombosis (SVT), in order to assess how it relates to primary MVT and PVT in our specific population. A retrospective observational study was conducted in patients referred to the University of Chile Clinical Hospital with mesenteric and/or portal venous thrombosis diagnosis over a 7-year period. Patients with primary thrombosis underwent hereditary thrombophilia study and JAK2V617F mutation screening. A total of 123 patients had splanchnic venous thrombosis(mesenteric and/or portal) as their main discharge diagnosis. Sixty patients (49%) had primary mesenteric or portal venous thrombosis(no attributable secondary cause). Hereditary thrombophilia and MPN were diagnosed in 21.6% and 43.3% of SVT patients, respectively. Twenty SVT patients remained without an etiologic diagnosis. In MPN patients, almost all had the JAK2V617F mutation (92.3%). About 16% of patients who had positive JAK2V617F mutation did not meet diagnostic criteria for MPN. In this Chilean cohort, half of mesenteric or portal venous thrombosis showed no secondary cause. In this group, the main causes were MPN and hereditary thrombophilia. Nearly, all MPN patients had JAK2V617F mutation, but there was a group of patients having JAK2V617F mutation but did not meet MPN criteria.

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study.

JAK2V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR’s technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

A centrifugal microfluidic emulsifier integrated with oil storage structures for robust digital LAMP.

Centrifugal droplet-based microfluidic devices have been applied to biomedical analysis and diagnostics recently. However, in centrifugal droplet-based microfluidic devices, droplets are tightly packed (i.e., the oil film between neighbouring droplets is thin). Therefore, droplet coalescence usually occurs especially during thermal incubation process. To preserve individual droplets in the devices, we report a new design for monodisperse droplet generation and storage that exploits a centrifugal configuration for droplet emulsification and oil-storage structures (OSSs) for regulation of the thickness of oil film between neighbouring droplets. The centrifugal emulsifier was well designed to ensure uniform droplet generation. Meanwhile, the OSSs could store oil during centrifugal emulsification while release oil before thermal incubation, which "loosen" tightly packed droplets to prevent droplets from coalescing. In this paper, the working process of OSS was analysed, and its shape and size were optimized. Then, the optimized OSSs were integrated into a centrifugal emulsifier for droplet digital loop mediated isothermal amplification (ddLAMP) by which detection of JAK2 V617F mutation within myeloproliferative neoplasms with a dynamic range of 101 to 104 copies per μL was achieved. We anticipate that the simplicity and robustness of our system make it attractive as an inexpensive and easy-to-operate device for DNA amplification, particularly applicable in point-of-care settings.

Establishment of a system for rapid detection of JAK2 V617F mutation in myeloproliferative diseases

To develop a system for rapid detection of JAK2 V617F mutation among patients with myeloproliferative diseases. Specific primers and TagMan probes were designed for the mutant and wild type alleles based on the principle of real-time PCR. A complete system including the method for detection and product for quality control were established through the evaluation of sensitivity and accuracy of the method, double-blind trial, and preparation of negative and positive controls through site-directed mutagenesis and molecular cloning. A system for rapid detection of the JAK V617F mutation has been developed. Compared with Sanger sequencing, the sensitivity and specificity of the method have both reached 100%. Meanwhile, 1000 normal samples and 1 case with the JAK2 V617F mutation were detected, which gave a population rate of 1‰. The system was fast, accurate, cheap, high throughput, and easy to use. It can be utilized as a routine test. Although the JAK2 V617F mutation is rare in the population, it should be screened among myeloproliferative neoplasm patients.

Somatic mutation of the V617F JAK2 gene in patients of the cardiovascular diseases

The study included 930 venous blood samples. JAK2 V617F mutation was detected by using the allele - specific real time polymerase chain reaction. JAK2 V617F mutation was detected in 15 (1.6%) patients, but only two of them had blood cell count that could cause a hematological disease to be suspected. The inclusion of the JAK2 V617F mutation detection in the complex of laboratory tests of the cardiovascular patients can facilitate the timely identification of patients with increased thrombotic risk, as well as the timely diagnosis of myeloproliferative diseases.

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.

PF685 THE RELATIONSHIP BETWEEN TOTAL ANTIOXIDANT CAPACITY AND JAK2V617F MUTATION IN ESSENTIAL THROMBOCYTHEMIA

Background:Essential thrombocythemia (ET) is a BCR‐ABL1‐negative myeloproliferative neoplasm associated in 50–60% of cases with the presence of the JAK2V617F mutation. Several studies have revealed that oxidative stress induces a growth advantage to JAK2V617F‐ positive clones, but little is known about the antioxidant status of ET patients.Essential thrombocythemia (ET) is a BCR‐ABL1‐negative myeloproliferative neoplasm associated in 50–60% of cases with the presence of the JAK2V617F mutation. Several studies have revealed that oxidative stress induces a growth advantage to JAK2V617F‐ positive clones, but little is known about the antioxidant status of ET patients.Aims:To evaluate the total antioxidant capacity (TAC) in patients with ET and to investigate if there are any differences between JAK2V617F‐positive cases and JAK2V617F‐negative ET cases in terms of TAC.Methods:We evaluated patients diagnosed with ET according to the WHO criteria (2016 revised) vs. healthy subjects (control group). Patients were classified based on age, sex and JAK2V617F mutational status. Haematological and biochemical parameters, as well as bone marrow aspirate or biopsy, were analyzed. Detection of JAK2V617F mutation was performed by RT‐PCR. TAC was measured using a multi‐detection microplate reader FLUOstar Omega (reagents from Sigma‐Aldrich). Data analysis was conducted using the student T‐test and a p‐value ≤ 0.05 was considered statistically significant. The study was approved by the Ethics Committee of the University of Medicine and Pharmacy of Craiova, Romania, approval no. 79/23.02.2017. All subjects agreed to partake in the study and signed a written informed consent.Results:The study group involved 58 ET patients (mean age 59.72 ± 14.63 years, age range 22–82): 25 males (40.32%, mean age 55.77 ± 15.80 years, age range 22–82) and 37 females (59.68%, mean age 62.14 ± 13.30 years, age range 23–81). 36 patients (62.07%) were JAK2V617F‐positive and 22 patients (37.93%) were JAK2V617F‐negative. 7 patients (12.07%) had homozygous genotype and 29 patients (50.00%) had a heterozygous genotype. TAC was decreased in patients with ET compared with healthy subjects and in JAK2V617F‐positive vs. JAK2V617F‐negative cases (p < 0.05). We found no significant differences between sexes in terms of age (men = 55.77 ± 15.80 years vs. women = 62.14 ± 13.30 years, p = 0.1116) or TAC (men = 0.50 ± 0.04 mM/L vs. women = 0.49 ± 0.06 mM/L, p = 0.3043). Also, we did not record significant differences in terms of TAC between JAK2V617F homozygous genotype vs. JAK2V617F heterozygous genotype (0.48 ± 0.05 mM/L vs. 0.49 ± 0.06 mM/L, p = 0.1686).Summary/Conclusion:In patients diagnosed with ET, TAC is decreased compared to normal individuals. Also, the decrease in TAC is more expressed in JAK2V617F‐positive cases vs. JAK2V617F‐negative cases or controls. No significant differences in terms of TAC were found between sexes or JAK2V617F homozygous vs. heterozygous genotype.

Detection of JAK2 V617F Mutation among Donors with Erythrocytosis

Introduction: It is mandatory that every blood donor must have their haemoglobin (Hb) values measured before blood donation. High Hb may indicate an underlying hidden pathological condition. The aim of this study is to investigate the occurrence of the JAK2 V617F gene mutation in blood donors with erythrocytosis. Methods: A cross-sectional study was conducted over a nine-month period involving blood donors with high pre-donation Hb. A total of 45 blood donors with total white cell (TWC) > 12.0x 109/l, platelet > 450x109/ l and Hb > 18g/dL were subjected to JAK2 V617F gene mutation analysis. Samples were collected and analysed for haematological tests and detection of JAK2 V617F mutation. Results: From a total of 2238 blood donors, 175 blood donors had high haemoglobin value. Samples from forty-five of these donors were then analysed for JAK2 V617F using allele-specific polymerase chain reaction (PCR). The prevalence of blood donors with erythrocytosis was 7.8%. All samples were negative for the JAK2 V617F mutation. Conclusions: Erythrocytosis can be relative or absolute and the different causes can be distinguished on the basis of clinical signs and symptoms. An absence of the JAK2 V617F mutation cannot by itself excludes the diagnosis of polycyhaemia vera (PV) since erythrocytosis is the single clinical feature that sets PV apart from other types of myeloproliferative neoplasm (MPN). Further study is required for the detection of other gene mutations that activates the JAK-STAT signalling pathway that could be identified in JAK2 V617F-negative MPN patients. KEYWORDS: Blood donors, JAK 2 V617F mutation, erythrocytosis

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorder which lead to erythrocytosis, thrombocytosis and leukocytosis. MPN with BCR-ABL positive is chronic myeloid leukemia (CML) while BCR-ABL negative MPN includes polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, Formalin-Fixed Paraffin-Embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive cross-sectional study. Forty FFPE marrow specimens were retrieved from the year 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 3 out of 4 in PV, 0/2 in ET, 4/4 in PMF, 4/10 in non-classified MPN and 1/20 in CML. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay, which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

Performance evaluation of the automated SNPs analyzer for the detection of JAK2 V617F mutation

Objective To test and evaluate the JAK2 gene V617F mutation in patients with myeloproliferative tumors based on i-densy IS-5320 platform according to ISO15189 accreditation requirements. Methods Instrument performance verification. Selected from December 2014 to February 2017, 20 cases of JAK2 V617F mutation positive peripheral blood samples from Huashan Hospital of the Shanghai FuDan University Medical College and 20 cases of peripheral blood samples with negative JAK2V617F mutation.The Realtime PCR with TaqMan MGB probe was selected as the control method to verify and evaluate the accuracy of testing JAK2 V617F mutation on i-densy IS-5320 platform. Whole blood samples were used to evaluate the reproducibility, cross-contamination and anti-interference ability of this platform. The ability of mutation load was verified by detecting mixtures of human erythnoleukemia cells and colorectal cancer cell HCT116 with 12 different proportions. Results I-densy IS-5320 platform and TaqMan MGB probe real-time fluorescence quantitative PCR show the same result.The within-run reproducibility and between-run reproducibility are both 100%. There is no observed contamination. High bilirubin and high triglyceride blood samples have no obvious interference on mutation detection. The mutation ratio with a load as low as 0.25% could be tested stably by i-densy IS-5320 platform. The detecting peak of melting curve can reflect the ratio of JAK2 V617F mutation to some extent. Conclusions I-densy IS-5320 can detect the mutation of JAK2 V617F gene in the whole blood directly. It has high sensitivity, accuracy and stability, and is easy to operate, and also can reflect the mutation load of JAK2 V617F, which could meet the clinical requirements for the detection of mutations. (Chin J Lab Med, 2018, 41: 470-474) Key words: Myeloproliferative neoplasms; JAK2 V617F; High resolution melting curve analysis

Relationship between JAK2V617F mutation, allele burden and coagulation function in Ph-negative myeloproliferative neoplasms

ABSTRACTObjectives: Our aim was to explore the relationship between JAK2V617F mutation allele burden and hematological parameters especially in coagulation function in Chinese population.Methods: This study included 133 Ph-negative myeloproliferative neoplasms (MPNs) patients between 2013 and 2016. All the clinical and experimental data of patients were collected at the time of the diagnosis without any prior treatment, including blood parameters, coagulation function, splenomegaly, vascular events and chromosome karyotype. PCR and qPCR were used to detect JAK2V617F mutation and JAK2V617F mutation allele burden.Results: In polycythemia vera patients, a positive correlation between the allele burden of JAK2V617F mutation and PLT counts was found; in essential thrombocythemia (ET) patients, WBC counts, RBC counts, HB, and HCT were higher in mutated patients than in wild-type patients. Furthermore, PT-INR was higher in ET and PMF mutated patients. In addition, a positive correlation between the allele burden of JAK2V617F mutation and activated partial thromboplastin time (APTT) was observed in JAK2V617F mutated ET patients.Conclusions: Higher hematologic parameters including counts of WBC, RBC, and PLT are closely associated with JAK2V617F mutation and its burden in Ph-negative MPNs; importantly, PT-INR, APTT are also related to JAK2V617F mutation and allele burden. Thus, our data indicate that JAK2V617F mutation allele burden might not only represent the burden of MPN but also alter the coagulation function.

O1-13-3 - Rapid detection of JAK2 V617F mutation in myeloproliferative disorders

O1-13-3 - Rapid detection of JAK2 V617F mutation in myeloproliferative disorders

Assessment of interleukin-10 level and Janus kinase 2 V617F mutation incidence in patients with primary myelofibrosis

Background: Myelofibrosis (MF) is largely documented by an abnormal cytokine expression, which in turn could contribute to bone marrow fibrosis, angiogenesis, and constitutional symptoms. To gain additional pathogenetic insight regarding cytokine phenotype correlations in MF, this study estimated the level of interleukin-10 (IL-10) abnormality and Janus kinase (JAK2 V617F) mutation in primary MF. Objective: The objective of this study was to assess serum IL-10 level and it's relation with the presence of JAK2 V617F mutation in patients with MF. Materials and Methods: JAK2 V617F mutation detection was performed in 32 patients with MF using amplification refractory mutation screening-polymerase chain reaction. IL-10 level was estimated in 36 patients with MF using enzyme-linked immunosorbent assay technique compared to 27 healthy controls who were enrolled for comparison. Results: The study showed higher significant (P ≤ 0.002) increase of IL-10 in patients with MF with cutoff value ≥8.9510 pg/ml and area under the curve value of 0.749 (P Conclusion: JAK2 mutation and IL-10 as anti-inflammatory cytokines may play a role in the pathogenesis and hematological presentation of patients with PMF. High IL-10 level may predict good prognosis in patients with PMF.