Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically 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. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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

Diagnostics ◽

10.3390/diagnostics10030143 ◽

2020 ◽

Vol 10 (3) ◽

pp. 143

Author(s):

Francesco La Rocca ◽

Vitina Grieco ◽

Vitalba Ruggieri ◽

Emanuela Zifarone ◽

Oreste Villani ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Digital Pcr ◽

Jak2 V617f Mutation ◽

Real Time Quantitative Pcr ◽

Mutational Status ◽

V617f Mutation ◽

Detection And Quantification

JAK2 V617F 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.

Download Full-text

JAK2 V617F Mutation Status of 232 Patients Diagnosed With Chronic Myeloproliferative Neoplasms

Clinical Lymphoma Myeloma & Leukemia ◽

10.1016/j.clml.2014.02.013 ◽

2014 ◽

Vol 14 (6) ◽

pp. 525-533 ◽

Cited By ~ 7

Author(s):

Kadriye Bahriye Payzin ◽

Kaan Savasoglu ◽

Inci Alacacioglu ◽

Fusun Ozdemirkiran ◽

Belgin Berber Mutlu ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Mutation Status ◽

Chronic Myeloproliferative Neoplasms ◽

V617f Mutation

Download Full-text

Relationship between JAK2-V617F mutation and hematologic parameters in Philadelphia-negative chronic myeloproliferative neoplasms

Turkish Journal of Biochemistry ◽

10.1515/tjb-2020-0267 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Murat Aksit ◽

Giray Bozkaya ◽

Nuriye Uzuncan ◽

Sibel Bilgili ◽

Can Ozlu ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Essential Thrombocytosis ◽

Mutation Status ◽

Chronic Myeloproliferative Neoplasms ◽

V617f Mutation ◽

Mutation Age

AbstractObjectivesWe aimed to investigate the prevalence of JAK2-V617F mutation and its association with hematologic parameters in polycythemia vera(PV), essential thrombocytosis(ET) and primary myelofibrosis(PMF) patients who have been tested for the mutation.MethodsWe retrospectively reviewed the records of 168 patients (82 males and 86 females) who were tested for JAK2-V617F mutation upon request of Hematology Clinic. JAK2-V617F mutation status, white blood cell (WBC) counts, platelet (PLT) counts, hemoglobin (Hb), hematocrit (Hct) levels and demographics of the patients were recorded.ResultsJAK2-V617F mutation was detected in 55.9% of the 168 patients. The mutation was observed in 58.2% of PV cases, in 54.4% of ET and in 54.5% of PMF cases. All patients were divided into two groups: mutation positive and negative. Age, WBC and PLT levels were significantly higher in mutation positive group (p<0.05). Age, WBC, Hb, Hct and PLT counts in PV cases with JAK2-V617F mutation, age and WBC counts in PMF cases with JAK2-V617F mutation were found to be significantly higher compared to mutation negative patients (p<0.05).ConclusionJAK2-V617F mutation is a very important parameter in diagnostic and prognostic evaluation. Thus, every patient suspected of having a myeloproliferative neoplasm should be screened for JAK2-V617F mutation.

Download Full-text

CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms

Biosensors ◽

10.3390/bios11080247 ◽

2021 ◽

Vol 11 (8) ◽

pp. 247

Author(s):

Miaomiao Chen ◽

Chunhua Zhang ◽

Zhiqing Hu ◽

Zhuo Li ◽

Menglin Li ◽

...

Keyword(s):

Detection System ◽

Myeloproliferative Neoplasms ◽

Cost Effective ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Minimum Detectable Concentration ◽

Lateral Flow Strip ◽

Whole Process ◽

Detectable Concentration ◽

V617f Mutation

The JAK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the JAK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.

Download Full-text

Splanchnic venous thrombosis in JAK2 V617F mutation positive myeloproliferative neoplasms – long term follow-up of a regional case series

Thrombosis Journal ◽

10.1186/s12959-018-0187-z ◽

2018 ◽

Vol 16 (1) ◽

Cited By ~ 3

Author(s):

Graeme Greenfield ◽

Mary Frances McMullin

Keyword(s):

Venous Thrombosis ◽

Myeloproliferative Neoplasms ◽

Case Series ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Long Term Follow Up ◽

V617f Mutation

Download Full-text

Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0054 ◽

2019 ◽

Vol 44 (4) ◽

pp. 492-498

Author(s):

Gonca Gulbay ◽

Elif Yesilada ◽

Mehmet Ali Erkurt ◽

Harika Gozukara Bag ◽

Irfan Kuku ◽

...

Keyword(s):

Blood Cell ◽

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Hematological Parameters ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

Download Full-text