Janus Kinase 2 V617F Mutation Research Articles

Abstract 7295: A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR

Abstract The Janus kinase 2 (JAK2) gene is located on chromosome 9p24.1 and encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells. In fact, the JAK2 mutation at amino acid 617 (Valine 617 to Phenylalanine, V617F) is the most frequently detected mutation in myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). This dominant gain-of-function V617F mutation results in constitutive tyrosine phosphorylation activity, leading to uncontrolled blood cell production, including increased numbers of erythrocytes, neutrophils, and platelets. To detect the JAK2 V617F mutation in DNA from patient blood samples, we developed a unique real-time PCR assay using our proprietary XNA technology that enables the assay to selectively amplify the mutant sequence by using a synthetic DNA analog XNA (Xenonucleic Acid). The JAK2 V617F mutation detection assay is designed to detect a valine-to-phenylalanine mutation at amino acid 617 (V617F). PCR primers, TaqMan probes, and XNA were designed for the JAK2 V617F mutation, along with primers and probes for an internal control gene in the duplex setting. The analytical performance of the assay was verified and validated using a variety of control samples. The results revealed that the XNA completely suppressed the amplification of wildtype sequence while only the V617F mutant sequence was amplified. The JAK2 V617F mutation detection assay is highly reproducible with intra- and inter-assay coefficients of variation of less than 10%. Results for the assay’s analytical sensitivity indicated that V617F could be detected with about 0.25% mutant allele frequency in 10 ng DNA input. In addition, no significant cross-amplification between V617F and V617I was observed. Thus, the JAK2 V617F mutation detection assay is specific for the V617F mutation. The JAK2 V617F mutation detection assay, using XNA-based technology, provides high sensitivity and specificity to detect the JAK2 V617F mutation. Thus, this assay would be a useful tool for clinical decision-making in determining the presence of the JAK2 V617F mutation in DNA from patient blood samples. Citation Format: Shuo Shen, Robert Brown, Daniel Kim, Larry Pastor, Andrew Y. Fu, Pushpinder Kaur, Alisha Babu, Wei Liu, Aiguo Zhang, Hiromi Tanaka, Michael Y. Sha. A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7295.

JAK2 V617F Mutation and Large Cerebral Artery Disease in Patients with Myeloproliferative Neoplasms.

The aim of the present study was to clarify the association between the Janus kinase 2 (JAK2) V617F mutation and large cerebral artery disease (LCAD) in patients with myeloproliferative neoplasms (MPNs). We retrospectively analysed patients diagnosed with MPNs between June 1992 and June 2022 who underwent brain magnetic resonance imaging. LCAD was defined as extracranial or intracranial large artery stenosis (≥ 50%) or occlusion on magnetic resonance angiography. A total of 86 patients (47 males; median age, 69 years old) were enrolled in this study. JAK2 V617F mutation was detected in 63 (73.3%) patients and LCAD in 35 (40.7%) patients. Univariate analysis showed that history of ischaemic stroke (LCAD, 62.9% vs. non-LCAD, 11.8%; P＜0.001), JAK2 V617F mutation (91.4% vs. 60.8%, P=0.002), and age ≥ 60 years (85.7% vs. 60.8%, P=0.016) were significantly associated with LCAD. Multiple logistic regression analysis showed that, in addition to ischaemic stroke, age ≥ 60 years and diabetes mellitus, JAK2 V617F mutation (odds ratio 29.2, 95% confidence interval 1.2-709.8, P=0.038) was independently associated with LCAD. LCAD was frequently observed in the intracranial carotid (14/35, 40.0%) and middle cerebral (13/35, 37.1%) arteries. This study revealed a significant association between the JAK2 V617F mutation and LCAD in patients with MPNs. This suggests that the JAK2 V617F mutation may promote cerebrovascular atherosclerosis and could be very important in determining therapeutic strategies for patients with not only JAK2 V617F-mutated MPNs but also LCAD-related stroke.

Diagnostic Performance of Red Blood Cell Indices, Serum Erythropoietin, and JAK2 Mutation Testing for the Evaluation of Polycythemia Vera at High Altitude

Introduction: Polycythemia vera (PV) is a clonal myeloproliferative neoplasm associated with mutation of Janus kinase 2 ( JAK2) in virtually all patients. Patients with PV typically present with constitutional symptoms and thrombotic complications related to myeloproliferation but can present with only mild or no symptoms. In 2016, the World Health Organization (WHO) diagnostic criteria for PV were revised to use lower hemoglobin and hematocrit cut-off levels in order to avoid missing masked polycythemia. Accordingly, a hemoglobin concentration of more than 16 g/dL (HCT>48%) for females and 16.5 g/dL (HCT>49%) for males can be considered for PV evaluation (previously >18.5 g/dL for men and >16.5 g/dL for women). In conjunction, concerns arise about the unnecessary evaluation of individuals with otherwise high-normal hemoglobin levels, more so for those living at high altitude. Altitude exposure, amongst others, decreases plasma volume to increase [Hb] which may accordingly muddle the diagnosis of PV at altitude. Nonetheless, Red Cell Mass (RCM) is seldom assessed in clinical practice. In addition, red blood cell count, related indices, and serum erythropoietin may also be affected by high altitude, and their diagnostic performance in the evaluation of PV at altitude has not been prospectively assessed. Method: We collected lab records for all JAK2 requests 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. Indications for requests, as well as laboratory data and imaging, were gathered. Complete blood count (CBC) and other laboratory values were collected when ordered within one week of JAK2 testing. For CBC, EDTA anticoagulated samples were processed using Sysmex XN-3100. For JAK2 testing, RNA isolated from peripheral blood samples (using the MagNA Pure System) was reverse-transcribed, amplified, and sequenced for any mutations involving exons 12, 13, 14, or 15 using an ABI 3730 XL genetic analyzer. For serum EPO, frozen serum samples were outsourced to Bio Scientia Labs, Germany. Reference values for serum erythropoietin were lab generated at 4.3-29 mIU/ml and were not validated for our altitude. . Results:Of the 208 JAK2 tests performed during the reviewed period, 132 (63.4%) were for polycythemia (Table 1). The JAK2 V617F mutation was found in 22 (16.7%) patients, with 10 (45.5%) of them being men. No JAK2 mutation other than the exon 14 V617F substitution was found. JAK2-positive patients had a median age of 61 years, while JAK2-unmutated patients had a median age of 47 years. Overt polycythemia, according to the WHO 2016 classification, was identified in 15 (68.2%) , while the remainder had masked PV ( defined as increased RBCs with normal hemoglobin/ HCT). Patients with PV had significantly higher RBCs and lower MCV/MCH/MCHC compared to those with JAK2-unmutated polycythemia. They also exhibited significant elevation of RDW-CW and significant neutrophilia, basophilia, thrombocytosis, and splenomegaly. All PV patients with available serum EPO tests had low values (median 2 mIU/ml). Conclusion: At altitude, patients with PV show the same age and sex characteristics as those living at low elevations. Patients with PV have higher RBCs and lower MCV/MCH/MCHC compared to those with JAK-unmutated (secondary) polycythemia. RDW is significantly increased in patients with PV, reflecting an associated iron deficiency. Serum EPO performed well at altitude to suggest the presence of PV. The low rate of positive JAK2 mutation in our high-altitude cohort may reflect poor selection of subjects and over-workup of otherwise altitude-associated erythrocytosis. Isolated mild polycythemia at altitude remains common. Using hemoglobin/hematocrit alone to select patients for PV evaluation has a low positive predictive value and may be associated with unnecessary workup. RBC indices and RDW, along with an evaluation for iron status, might help select those more likely to have PV and could decrease unnecessary workups.

Determination of the JAK2 V617F mutation in thrombosis patients

Background: Janus kinase 2 (JAK2) gene mutation causes uncontrolled myeloproliferation independent of cytokines and abnormal formation of the endogenous erythroid colony. JAK2 mutations were frequently observed in myeloproliferative neoplasms (MPNs), especially in polycythemia vera (PV) and essential thrombocythemia (ET). MPNs represent a risk factor for the development of thrombosis that is a significant cause of morbidity and mortality in patients. Materials and methods: We aimed to study the correlation between JAK2 mutation and thrombosis. Thirty-nine patients with a clinical diagnosis of thrombocytosis and Budd-Chiari syndrome were collected to determine the JAK2 V617F mutation. Genomic DNA from all specimens was amplified and detected the presence of JAK2 V617F mutation by AS-PCR. Results: We demonstrated JAK2 V617F mutation in both patients with a clinical diagnosis of thrombocytosis and Budd-Chiari syndrome. We found that 11 of 37 (29.7%) thrombocytosis patients had a JAK2 V617F mutation. Moreover, one of two patients who represented as Budd-Chiari syndrome was positive for JAK2 V617F mutation. In addition, JAK2 V617F mutation was associated with thrombosis. However, further study in large series is needed to support this finding. Conclusion: Determination of the JAK2 V617F mutation may be helpful for screening latent or occult MPNs patients who have an occurrence of thrombosis to adjust the appropriate treatment for good patient outcomes.

FTIR- based serum structure analysis in molecular diagnostics of essential thrombocythemia disease.

Essential thrombocythemia (ET) reflects the transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis remains obscure. Nevertheless, tyrosine kinase, especially Janus kinase 2 (JAK2), has been implicated in myeloproliferative disorders other than chronic myeloid leukaemia. FTIR analysis was performed on the blood serum of 86 patients and 45 healthy volunteers as control with FTIR spectra-based machine learning methods and chemometrics. Thus, the study aimed to determine biomolecular changes and separation of ET and healthy control groups illustration by applying chemometrics and ML techniques to spectral data. The FTIR-based results showed that in ET disease with JAK2 mutation, there are alterations in functional groups associated with lipids, proteins and nucleic acids significantly. Moreover, in ET patients the lower amount of proteins with simultaneously higher amount of lipids was noted in comparison with the control one. Furthermore, the SVM-DA model showed 100% accuracy in calibration sets in both spectral regions and 100.0% and 96.43% accuracy in prediction sets for the 800-1800cm-1 and 2700-3000cm-1 spectral regions, respectively. While changes in the dynamic spectra showed that CH2 bending, amide II and CO vibrations could be used as a spectroscopy marker of ET. Finally, it was found a positive correlation between FTIR peaks and first bone marrow fibrosis degree, as well as the absence of JAK2 V617F mutation. The findings of this study contribute to a better understanding of the molecular pathogenesis of ET and identifying biomolecular changes and may have implications for early diagnosis and treatment of this disease.

Study of the Janus kinase 2 (JAK2) gene haplotype 46/1 association with driver mutations of chronic Ph-negative myeloproliferative neoplasms

Introduction. Haplotype JAK2 46/1 is associated with more frequent development of Ph-negative myeloproliferative neoplasms (MPN) and with an increased detection rate of the JAK2 V617F mutation. At the same time, the molecular mechanisms of such associations remain unclear. Previously, there were no studies of regional, age and gender aspects of the predictive value of carriage of the 46/1 JAK2 haplotype, which could assess this relationship in some observations.Aim — to analyze the degree of association between 46/1 haplotype and the V617F mutation of the JAK2 gene depending on the sex, age, and place of residence of patients examined for suspected MPN.Methods. The study included 949 DNA samples from patients with suspected MPN. Samples of 150 volunteers and blood donors were included in the control group. Haplotype 46/1 (rs10974944), V617F mutation in the JAK2 gene, mutations in the CALR gene (type 1: c.1092_1143del; L367fs*46, COSV57116546; type 2: c.1154_1155insTTGTC; K385fs*47, COSV57116551) and in the MPL gene (W515K, W515L) were determined for all samples using real-time polymerase chain reaction (PCR-RT).Results. The 46/1 JAK2 haplotype were shown to be associated with a clinically significant level (> 2 %) of the allelic burden of the JAK2 V617F mutation. The odds ratio of the risk of developing a V617F positive MPN when carrying this haplotype variant did not depend on the main place of residence of the patients and was found to be most pronounced in men under 50 years of age. The odds ratio of the risk did not depend on the age of the examined women.Conclusion. The association of 46/1 haplotype with the presence of other drivers of MPN mutations in the CALR or MPL genes was also statistically significant, which confirms the hypothesis of “favorable soil” rather than “hypermutability” of the JAK2 gene.

Myeloproliferative neoplasms – a global view

Myeloproliferative neoplasms – a global view

Comparison of Clinical and Hematological Parameters of Janus Kinase 2, Calreticulin or Myeloproliferative Leukemia Virus Oncogene Mutant Essential Thrombocythemia and Triple-Negative Essential Thrombocythemia.

IntroductionEssential thrombocythemia (ET) is one of the chronic myeloproliferative neoplasms. While Janus kinase 2 (JAK2) V617F mutation is defined in more than half of the patients with ET, calreticulin (CALR) or myeloproliferative leukemia virus oncogene (MPL) mutations are encountered more rarely. The discovery of the JAK2 V617F mutation in 2005, followed by the recognition of MPL and CALR mutations, brought up the idea of subdividing ET according to the mutation status. Our aim in this study is to investigate whether genetic mutations detected in patients diagnosed with ET cause a different clinical phenotype compared to triple-negative ET.MethodsThis retrospective study was conducted by evaluating the patients who were followed up with the diagnosis of ET in the hematology clinic of two tertiary centers in Turkey between 2009 and 2021. Patients with negative JAK2, CALR, and MPL mutations and meeting the diagnostic criteria for ET were defined as triple-negative ET. The patients were divided into two groups as triple-negative ET and mutation-positive ET according to the presence of a mutation. It was investigated whether there was a difference between these two groups in terms of demographic, laboratory, and clinical characteristics.ResultsA total of 109 patients were included in the study. The mean age of these patients was 54 (18-91) years and 85 (78%) patients were females. A total of 48 patients (44.0%) had JAK2 mutation, six (5.5%) had CALR mutation, and one (0.9%) had MPL mutation. It was observed that there was a significant difference between the two groups in terms of gender, mean age, and hemoglobin value. While 87% of patients with triple-negative ET were females, this rate was 69% in patients with mutation-positive ET (p = 0.036). The mean age was 41.8 years in triple-negative ET and 67.1 years in patients with mutation-positive ET (p = 0.0001). While the mean hemoglobin value was 12.9 g/dl in patients with triple-negative ET, it was 14.4 g/dl in patients with mutation-positive ET (p = 0.0001).ConclusionIt has been observed that ET with JAK2, CALR, or MPL mutations may have different phenotypic features compared to triple-negative ET, resulting in a clinical condition consisting of older patients with a higher erythrocyte count.

Comparative Effectiveness of Fedratinib and Pacritinib for the Treatment of Myelofibrosis in Patients with Low Platelet Counts: A Simulated Treatment Comparison Study

Introduction: Myelofibrosis (MF) is a Philadelphia chromosome-negative myeloproliferative neoplasm with an estimated prevalence of 4-6/100,000 persons in the USA. Patients with MF experience aberrant hematopoiesis, bone marrow fibrosis, splenomegaly, and cytopenias, including thrombocytopenia, as well as diminished survival ranging from months to years, depending on risk status. Symptoms progressively worsen, underscoring the need for effective treatments across heterogeneous risk statuses and patient dispositions. Among intermediate- to high-risk patients, Janus kinase 2 (JAK2) inhibitors are the primary treatment for MF. Fedratinib was approved by the US Food and Drug Administration (FDA) based on the results of the JAKARTA trials (NCT01437787; NCT01523171), and a new drug application for pacritinib has been submitted to the FDA for the treatment of MF in patients with severe thrombocytopenia. A head-to-head trial of fedratinib and pacritinib for the treatment of MF has not been conducted. Moreover, there is a paucity of evidence evaluating these agents in patients with MF and thrombocytopenia (platelets < 100 × 10 9/L). Therefore, indirect treatment comparisons (ITCs) are required to assess the comparative efficacy of fedratinib and pacritinib in this population.Methods: A systematic literature review (SLR) was conducted to identify all clinical evidence in patients with MF and thrombocytopenia. Based on the SLR results, the JAKARTA, JAKARTA-2, and PERSIST-2 (NCT01773187) trials were identified as the studies to form the basis of the ITC. A pooled analysis data set was developed based on individual patient-level data from the fedratinib 400-mg arms of the JAKARTA and JAKARTA-2 trials including patients with platelets < 100 × 10 9/L. Published summary data from the pacritinib 200-mg arm of the PERSIST-2 trial served as the comparator. Simulated treatment comparisons (STCs) were used to compare spleen volume reduction (SVR) ≥ 35% while adjusting for mutually reported baseline patient characteristics such as age, sex, Dynamic International Prognostic Scoring System, Eastern Cooperative Oncology Group (ECOG) performance status (PS), JAK2 V617F mutation status, prior ruxolitinib exposure, and laboratory tests. Final adjustment models were selected based on fit statistics and the literature review. Indirect relative risk (RR) was estimated with 95% confidence intervals (CIs) using unanchored naive ITC (unadjusted) and STC (adjusted) methodologies.The PERSIST-2 trial included patients with baseline platelets < 50 × 10 9/L, but did not report median baseline counts. Therefore, a sensitivity analysis was conducted to evaluate the impact of differential median baseline platelet counts. In the sensitivity analysis, an outcome model was generated in the full pooled JAKARTA trial population irrespective of platelet count, and platelet count was forced into the multivariable adjustment model. Outcomes were simulated at 3 median baseline platelet counts (25, 50, and 75 × 10 9/L) and compared using similar methodology to the main analysis.Results: The main analysis suggests that fedratinib is associated with a greater proportion of SVR ≥ 35% than pacritinib (Table, A). According to the naive ITC, fedratinib was numerically favored over pacritinib in terms of SVR ≥ 35% (RR, 1.67 [95% CI, 0.94-2.97]). After adjustment for ECOG PS, JAK2 V6127F mutation status, and prior ruxolitinib exposure, fedratinib was statistically favored relative to pacritinib for SVR ≥ 35% (RR, 1.76 [95% CI, 1.00-3.10]).Results of the platelet-count-adjusted sensitivity analysis showed that a significant difference was observed in favor of fedratinib with respect to SVR ≥ 35% (Table, B). The resulting indirect RRs were similar regardless of simulated median baseline platelet count, which suggest that differential baseline platelet count has a minimal impact on the ITC results.Conclusion: This analysis used a population-adjusted ITC to assess the comparative efficacy of pacritinib and fedratinib in patients with MF and thrombocytopenia. Following population adjustment, fedratinib was associated with a greater proportion of patients achieving SVR ≥ 35% than pacritinib. In lieu of a head-to-head clinical trial, further real-world evidence studies should be conducted to assess the effectiveness of these treatments in the clinical setting. [Display omitted] DisclosuresDaniele: BMS/Celgene: Consultancy; Purple Squirrel Economics: Current Employment. Abraham: Bristol Myers Squibb: Current Employment. Kee: Bristol Myers Squibb: Current Employment. Tremblay: Cytel: Consultancy; Purple Squirrel Economics: Current Employment. Rose: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. McBride: BMS: Current Employment.

Silica-Coated Iron Oxide Nanoparticles for DNA Isolation for Molecular Genetic Studies in Hematology.

Aim: To develop magnetic nanoparticles (MNPs) based on iron oxide for DNA isolation from blood cells for quantitative molecular genetic analyses of the V617F mutation in the Januskinase 2 (JAK2) gene. Materials and Methods: MNPs were synthesized by the coprecipitation method and coated with tetraethyl orthosilicate (TEOS). The size and shape of the complexes were estimated using transmission electron microscopy. Twenty blood samples from patients with myeloproliferative disorders were used for DNA isolation with the MNPs. DNA quality and compatibility for molecular genetic studies of the JAK2 V617F mutation were investigated by gel electrophoresis and real-time polymerase chain reaction (RT-PCR). Results: The average amount of DNA isolated from 150 μL of whole blood was 75.2 ng when MNPs were used and 72.5 ng when standard silica sorbent was used. There was no DNA damage observed after interaction with MNPs. RT-PCR demonstrated similar values for the JAK2 V617F mutant DNA ratios in the samples after DNA isolation with MNPs and by standard sorption on silica. Conclusions: MNPs with silicate capsules of sufficient thickness were obtained and the undesirable damaging effect of iron oxides on nucleic acids during isolation from cells were eliminated. Designed MNPs allow obtaining intact DNA for molecular genetic studies using the example of the JAK2 V617F for study.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?

Background: A substitution of G to T at nucleotide 1849 in exon 14 of the Janus kinase2 (JAK2) gene is well recognized in myeloproliferative neoplastic disorders (MPNs). Based on WHO guidelines, detection of the mutation is very important to confirm the disease in suspected patients.&#x0D; Methods: Eighty-seven patients with different background diseases were tested for JAK2 V617F mutation by four different methods, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), polymerase chain reaction-high resolution melting (PCR-HRM), and two different commercial kits.&#x0D; Results: The mean age of patients was 53.38±17.43 years, 72.4% were males, and 37.6% were females. JAK2 mutation was detected in 16 patients (18.3%). Of those, 7 (43.75%) suffered from PV, 5 (31.25%) from ET, 3 (18.75%) from PMF, and 1 (6.15%) from unclassified neoplastic disorders. The frequency of JAK2 mutation was 71.4% (5/7) in PV, 80% (4/5) in ET, and 66.7% (2/3) in PMF patients. The sensi- tivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and GE of PCR-HRM for detection of the JAK2 mutation was 86.7%, 100%, 100%, 97.3%, and 97.7%, respectively. While the sensitivity, specificity, PPV, NPV, and GE of PCR-RFLP were 93.3%, 80.5%, 50%, 98.3%, and 82.7%, respectively. On the other hand, the sensitivity, specificity, PPV, NPV, and GE of ARMS assays were evaluated by about 80%, 96%, 100%, 96%, and 96.5%, respectively.&#x0D; Conclusion: This study showed that PCR-HRM was a more sensitive assay to detect the JAK2 V617F mutation than the other assays. So, it can be used as a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. PCR-RFLP must accompany it as a gold standard method for confirmation of the mutation of JAK2 V617F.

Somatically acquired mutations in primary myelofibrosis: A case report and meta-analysis.

Familial myeloproliferative disease (MPD) cases account for 7.6% of the global MPD cases. The present study reported 2 cases of primary myelofibrosis (PMF). The patients were two sisters; the older sister succumbed to the disease at the age of 37, whereas the younger sister maintained a stable disease status and gave birth to a son through in vitro fertilization. Genetic analysis of bone marrow DNA samples showed that both sisters carried a Janus kinase 2 (JAK2) V617F mutation, and the older sister also had a trisomy 8 chromosomal abnormality (47, XX, +8). A systematic literature search was also performed using PubMed, CNKI and Wanfang databases, to determine the association between JAK2 and PMF. Following comprehensive screening of the published literature, 19 studies were found to be eligible for the current meta-analysis. The results showed that JAK2 V617F was a risk factor of PMF, and no sex dimorphism was observed in JAK2 V617F mutation prevalence amongst all PMF cases. In addition, there was a lack of association between the JAK2 V617F mutation and PMF-related mortality.

Frequency and characteristics of the JAK2 V617F mutation in 23 cerebral venous sinus thrombosis patients with thrombocytosis.

ObjectiveTo analyse the frequency and characteristics of the Janus kinase 2 (JAK2) V617F mutation in patients with cerebral venous sinus thrombosis (CVST) with thrombocytosis.MethodsThe study enrolled CVST patients with thrombocytosis that had undergone JAK2 V617F mutation detection to determine the frequency of the JAK2 V617F mutation in this cohort. Correlations between patient demographics, whole blood cell counts, targeted sequencing results and JAK2 V617F mutation status were determined.ResultsA total of 23 patients were enrolled in the study: 11 (47.8%) with the JAK2 V617F mutation and 12 (52.2%) without the JAK2 V617F mutation. The mean platelet count was significantly higher in patients with the JAK2 V617F mutation than in patients without the mutation (478.1 ± 107.4 × 109/l versus 374.4 ± 54.1 × 109/l, respectively). There were no significant differences in age, sex, white blood cell count or haemoglobin level between the two groups. Other than single nucleotide polymorphisms, no hot-spot mutations associated with myeloid tumours other than the JAK2 V617F mutation were detected in four CVST patients that underwent targeted sequencing.ConclusionThe JAK2 V617F mutation was frequently detected in CVST patients with thrombocytosis and it was associated with higher platelet counts.

STAT5 is Expressed in CD34+/CD38- Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

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.

Unclassifiable non-CML classical myeloproliferative diseases with microcytosis: findings indicating diagnosis of polycythemia vera masked by iron deficiency

Background/aimPolycythemia Vera (PV) is a myeloproliferative disorder characterized by overproduction of morphologically normal red blood cells (RBCs), granulocytes, and platelets, a phenotype that is caused by a mutation (V617F) in Janus kinase 2 (JAK2). However, JAK2 V617F is also found in approximately 50% of patients with essential thrombocytosis and primary myelofibrosis, rendering its presence nonspecific as a diagnostic test. An increased red cell mass is a major criterion for the diagnosis of PV according to World Health Organization (WHO) 2016 criteria. High hemoglobin (Hgb) or Hematocrit (Hct) are universally used as indicators of an increased red cell mass for the diagnosis of PV. However, conditions such as iron deficiency (ID) with decreased mean cell volume may mask the diagnosis due to nonelevated Hct level. The aim of this study was to investigate the clinical characteristics of the patients with unclassifiable non-CML classical myeloproliferative disease with microcytosis (MPD/M) and nonelevated Hgb and Hct levels at diagnosis and to determine if some of these cases could be real PV cases masked due to ID-related microcytosis.Materials and methodsThere were 23 MPD/M cases among 208 non-CML classical MPD cases (11%). Among 22 patients who had adequate test results related to the cause of microcytosis, ID and beta-thalassemia trait (TT) were the apparent causes of microcytosis in 15 and 1 cases, respectively.ResultsClinicopathological correlations revealed consistently positive JAK2 V617F mutation status (20/20, 100%), frequently elevated RBC count (17/23, 73.9%), and PV-compatible bone marrow findings (10/12, 83.3%). These findings are compatible with PV instead of essential thrombocytopenia or primary myelofibrosis. In spite of frequent cytoreductive treatment, 3 patients developed increased Hgb/Htc levels during median 58.2 (279–63) months’ follow-up.ConclusionThese data show that the majority of MPD/M cases are PV patients masked due to ID-related microcytosis.

Escalated activation ofSTAT3 due to JAK2 V617F activatingmutation causesdownregulation ofPTEN via miRNA 21overexpression

Abstract Background Mutations in Janus kinase 2 (JAK2) genes are the genetic characteristic of BCR-ABL1-negative myeloproliferative neoplasms (MPN). JAK2 V617F mutation is frequently found in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). We aimed to determine the effect of JAK2 V617F on expression of phosphatase and tensin homolog (PTEN) and its regulator miR-21. Materials and Methods Seventy two (72) (MPN) patients were screened for JAK2 V617F burden using ASO-PCR. Out of 72 MPN patients, 58 were JAK2 V617F positive and 14 were JAK2 V617F negative. BCR/ABL1 fusion gene transcript screening by multiplex PCR was done to rule out Philadelphia positive MPN. We performed SYBR green based qRT-PCR assay to characterize relative STAT3, PTEN mRNA expression and miR21 expression in 72 MPN patients. Results The mRNA expression levels of STAT3 and PTEN and mi-21 expression in all MPN patients were evaluated and compared withJAK2 (V617F) and JAK2 (WT) MPN patients. We identified that STAT3 mRNA expression was significantly upregulated in patients with JAK2 (V617F) genotype (mean fold±SD=2.14 ±1.2) compared to patients with JAK2 (WT) genotype (mean fold±SD=1.23±0.95). Further, we observed a significantly increased miR 21 expression (mean fold±SD=2.18±0.44) in JAK2 (V617F) patients in comparison to JAK2 (WT) patients (mean fold±SD=1.70±0.49) (p = 0.0003). In addition, we observed that PTEN expression was substantially and significantly downregulated in JAK2 (V617F) patients (mean fold±SD= 1.73 ± 0.36) compared JAK2 (WT) patients (mean fold±SD=2.55 ± 0.25) (p Conclusion In summary, STAT3 activation caused by JAK (V617F) mutation in MPN patients causes overexpression of miR-21 which leads to downregulation of tumour supressor PTEN MPN patients. Thus, our suggests that JAK2 activated STAT3/miRNA-21 may play a valuable prognostic biomarker for MPN.

Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method.

BackgroundThe Janus kinase 2 (JAK2) has an important role in the intracellular signaling in normal and neoplastic cells. JAK2 mutation, called JAK2 V617F, is frequently found in Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess the analytical efficiency of high-resolution melting (HRM) method using reannealing-curve analysis in comparison with routine melting-curve analysis for JAK2 V617F mutation detection.MethodTwenty-three samples including one negative synthetic standard DNA, two 50% and 75% positive synthetic standard DNA samples, five wild-type samples and 15 samples positive for JAK2 V617F were examined by HRM. Melting and reannealing stages were performed, and then, raw and normalized curves were compared between the two stages.ResultsIn melting-curve analysis, the wild-type and mutant samples had different melting temperatures (75/53°C and 75/10°C, respectively). In normalized curves corresponding to reannealing method, mutant samples were better separated from the baseline than in melting method as well as for samples with different mutant DNA burden from each other. Furthermore, wild-type samples were more homogenous in the normalized curves corresponding to reannealing than in melting method. This means that patients with a low allelic burden may be wrongly interpreted as normal in the common melting method.ConclusionWe suggest the use of reannealing instead of the melting-curve analysis for the detection of sequence variations, especially for large-scale mutation and allele burden measurement in clinical settings. However, more evaluations with more sample size will better improve the benefits of reannealing-curve analysis in research and clinic.

V617F-independent upregulation of JAK2 gene expression in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is one of the most important immune-mediated disorders of the gastrointestinal tract. Besides, IBD is associated with numerous extraintestinal complications such as venous thromboembolism (VTE), an important risk factor for vascular complications, which results in the increased morbidity and mortality. The JAK2 (Janus kinase 2) V617F mutation is a well-known point mutation which is involved in the pathogenesis of IBD, and VTE. Therefore, the aims of this study were to evaluate expression of JAK2 and association of V617F mutation in JAK2 of Iranian patients with IBD. Two hundred and forty-six patients with IBD (209 UC and 37 CD) and 206 healthy controls were enrolled in this study. The genomic DNA and total RNA were extracted from peripheral blood mononuclear cells (PBMCs). Then, the JAK2 V617F mutation detection was performed using the restriction fragment length polymorphism (RFLP) method. In addition, the JAK2 mRNA expression was evaluated using a quantitative polymerase chain reaction (q-PCR) using the SYBR Green assay. There was no association of V61F mutation in patients with IBD with or without thrombosis compared with healthy control. However, the relative mRNA expression of JAK2 was significantly upregulated in patients with IBD in comparison with healthy control (P < 0.0001). In addition, the JAK2 mRNA expression was significantly decreased in patients with IBD having thrombosis compared with those without thrombosis ( P < 0.0001). Taken together our findings suggested that JAK2 V61F-independent upregulation of JAK2 mRNA expression in patients with IBD. Moreover, despite the absence of JAK2 V617F mutation in patients with IBD, the increased gene expression of JAK2 can be explained by another molecular mechanism such as regulation of gene expression at the transcriptional level which may play crucial roles in the pathogenesis of IBD.