The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia

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.

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

Genes ◽

10.3390/genes12040559 ◽

2021 ◽

Vol 12 (4) ◽

pp. 559

Author(s):

Tatiana V. Makarik ◽

Adhamjon O. Abdullaev ◽

Elena E. Nikulina ◽

Svetlana A. Treglazova ◽

Elena E. Stepanova ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Jak2 V617f ◽

Janus Kinase ◽

Allele Burden ◽

Chronic Myeloproliferative Neoplasms ◽

Exon 9 ◽

Calr Mutations ◽

Exon 10 ◽

Proliferative Signal

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.

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Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽

10.1182/blood.v118.21.4687.4687 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4687-4687

Author(s):

Yue Xu ◽

Changxin Yin ◽

Han He ◽

Lingling Shu ◽

Fuqun Wu ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Western Countries ◽

Arms Pcr ◽

V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

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Isolation, Expansion and Characterization of BM-Mesenchimal Cells (MSCs) in Patients Affected By Chronic Myeloproliferative neoplasm (MPN)

Blood ◽

10.1182/blood.v124.21.5142.5142 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5142-5142

Author(s):

Patrizia Chiusolo ◽

Francesca Annunziata ◽

Elena Rossi ◽

Silvia Betti ◽

Silvia Bellesi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Gene Mutations ◽

Jak2 V617f ◽

Cell Types ◽

Erythropoietin Receptor ◽

Jak2 V617f Mutation ◽

Hematopoietic Stem ◽

V617f Mutation

Abstract Introduction MSCs constitute a pivotal cell type capable of shaping both the architecture of the microenvironment and modulating communication between the various cell types through effects on the extracellular matrix (ECM) and by secretion of various growth factors and cytokines. MSCs and hematopoietic stem cells are thought to share the same mesenchymal origin. Some data confirm that MSCs express a functional erythropoietin receptor and JAK2-transduction pathway, but their role in the development and evolution of MPN is still not well known so our aim was the isolation, expansion and characterization of MSCs in patients affected by MPN. Some data indicates that BM-MSCs of patients affected by MPN do not carry the JAK2-V617F mutation. We studied 20 patients affected by MPN with the following characteristics: M/F 12/8, median age 53years, 8 affected by PV, 8 by ET and 4 by PMF. 15 patients were positive for JAK2 V617F mutation, 1 pts for cMPL and 2 were CARL mutations carriers. Methods: MSC were isolated by bone marrow fraction by gradient separation on Lympholyte cell separation media and expanded in culture with a specific medium (MesenCult) in plastic-adherent cultures up to the second passage. DNA was extracted from MSC using QIAmp DNA Mini kit and the study of recurrent alterations (JAK2, MPL and CARL gene mutations was performed). Results: MCS were expanded in 14 on 21 patients. Flow citometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive). The molecular analysis of JAKV617F, cMPL and CARL mutations resulted negative in all analyzed samples both in patients carriers of mutations and in wild type ones. Conclusions: We conclude that common mutations markers of MPN neoplasm are absent in the mesenchymal compartment of bone marrows of patients affected by MPN and are restricted to the neoplastic clone. This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) Disclosures No relevant conflicts of interest to declare.

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

Annals of Hematology ◽

10.1007/s00277-016-2623-0 ◽

2016 ◽

Vol 95 (5) ◽

pp. 739-744 ◽

Cited By ~ 23

Author(s):

Miguel Waterhouse ◽

Marie Follo ◽

Dietmar Pfeifer ◽

Nikolas von Bubnoff ◽

Justus Duyster ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Jak2 V617f Mutation ◽

Chronic Myeloproliferative Neoplasms ◽

V617f Mutation ◽

Accurate Quantification

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Prevalence of the JAK2 V617F Mutation in Patients with Peripheral Arterial Disease

Blood ◽

10.1182/blood.v124.21.3165.3165 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3165-3165

Author(s):

Elena Kinz ◽

Klaus Gasser ◽

Axel Muendlein ◽

Andreas Leiherer ◽

Michael Steurer ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Arterial Disease ◽

Jak2 V617f Mutation ◽

Allele Burden ◽

Cell Counts ◽

V617f Mutation ◽

Artery Disease ◽

Peripheral Arterial

Abstract Introduction: The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms and increases thrombotic risk. We previously showed that JAK2 V617F is also found in healthy subjects as well as in patients with coronary artery disease (0.6% and 1.3%, respectively). Peripheral arterial disease (PAD) is an important manifestation of diffuse atherosclerosis and PAD patients are at exceptionally high risk for cardiovascular events, showing a worse prognosis than that of patients with coronary artery disease Due to the close relation of the JAK2 V617F mutation to thrombotic events we hypothesized that this mutation may play an important role in the risk management of PAD patients. However, prevalence of JAK2 V617F or of occult myeloproliferative neoplasms is unknown in PAD patients. Methods: In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD. JAK2 mutational status from 997 age-matched healthy people was available from a previous study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with allele-specific quantitative real-time PCR. Results: From a total of 287 PAD patients samples, 9 (3.1%) were tested positive for JAK2 V617F mutation corresponding to a 5-fold, highly significant increase compared with healthy people (p<0.001). Mutant allele burden of JAK2 V617F positive samples was ranging between 0.2% and 96.2% (median=0.75%). Generally, our study showed no significant association of the JAK2 V617F mutation with abnormal blood cell counts. However, the patient with the highest mutant allele burden showed elevated hemoglobin values (> 18.5 g/dL) indicating polycythemia vera (PV). Conclusion: We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. For this reason mutation analysis should be considered in PAD patients with abnormal blood cell counts to identify occult myeloproliferative neoplasms and to adjust therapeutic treatment, possibly reducing the risk of future vascular complications. Disclosures No relevant conflicts of interest to declare.

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Comparative Analysis of Proinflammatory Cytokine Levels in Peripheral Blood and Bone Marrow of Patients with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-115600 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4995-4995

Author(s):

Pu Chen ◽

Boting Wu ◽

Xia Shao ◽

Chanjuan Liu ◽

Zhenglin Yu ◽

...

Keyword(s):

Bone Marrow ◽

Proinflammatory Cytokines ◽

Peripheral Blood ◽

Proinflammatory Cytokine ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Cytokine Levels ◽

V617f Mutation ◽

Calr Mutations

Abstract Background Myeloproliferative neoplasms (MPNs) are characterized by marker somatic mutations involving JAK2, MPL, and CALR, which lead to constitutive activation of tyrosine kinase signaling cascades, subsequently dysregulated proliferative of myeloid linages, and eventually myelofibrosis or leukemic transformation. Recently, it has been argued that proinflammatory processes are crucial to the pathogenesis and progression of MPNs. A number of proinflammatory cytokines including lipocalin, IL-1, IL-2R, IL-6, IL-8, IL-12, IL-15, and IP-10 have been found elevated in the peripheral blood (PB) of MPN patients. However, there has been limited data on the levels of proinflammatory cytokines in the bone marrow (BM) of MPN patients. The present study determined and compared 40 proinflammatory cytokine levels in the PB and BM plasma of MPN patients with unequivocal molecular background, thus intending to illustrate the proinflammatory features of BM microenvironment as well as to evaluate the credibility of PB cytokine profiles. Methods Newly diagnosed MPN patients (n=12, 8 had JAK2 V617F mutation, 4 had CALR mutations) were included in the present study. PB samples were taken within 48 hours of BM samples. Paired PB and BM plasma cytokine profiles were measured as well as 10 health control PB plasma samples in a single procedure by Quantibody Human Inflammatory Array 3 (RayBiotech, Norcross, GA) which permitted detection of 40 inflammation-associated cytokines. Results Among 12 MPN patients, 8 had JAK2 V617 mutation (6 males, median age 61.5 years), and 4 had CALR mutations (3 males, median age 53.5 years). Positive linear correlations between PB and BM levels were found in 12 proinflammatory cytokines including BLC (r=0.613, p=0.034), I-309 (r=0.872, p<0.001), IL-1α (r=0.666, p=0.018), IL-1β (r=0.929, p<0.001), IL-12p40 (r=0.642, p=0.024), IL-15 (r=0.608, p=0.036), M-CSF (r=0.906, p<0.001), MIG (r=0.596, p=0.041), MIP-1α (r=0.787, p=0.002), MIP-1δ (r=0.648, p=0.023), sTNFRI (r=0.827, p=0.001), and sTNFRII (r=0.644, p=0.024). Compared to health controls, BM levels of G-CSF, IL-2, IL-4, IL-6R, IL-7, IL-8, IL-10, IL-13, MIP-1β, PDGF-BB, RANTES, and TIMP-1 were markedly elevated (all p<0.01), among which IL-2, IL-4, and IL-7 levels were even higher in patients with CALR mutations than those with classic JAK2 V617F mutation (all p<0.05). Conclusions Optimal linear correlation could only be found in limited species of proinflammatory cytokines, especially I-309, IL-1β, M-CSF, and sTNFRI, between PB and BM plasma of MPN patients. Therefore, caution should be recommended during attempts to illustrate the status of inflammation in MPN patients by circulating cytokine markers. A stronger inflammatory component might exist in MPN patients with CALR mutations than those with classic JAK2 V617F mutation. Disclosures No relevant conflicts of interest to declare.

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Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience

Blood ◽

10.1182/blood.v124.21.5569.5569 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5569-5569

Author(s):

Amin Ben Lassoued ◽

Nathalie Beaufils ◽

Caroline Bonnefoy ◽

Jean Gabert

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Molecular Diagnostic ◽

Platelet Counts ◽

Study Population ◽

Calr Mutations

Abstract Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region. The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations. The study was approved by the local ethics committee. Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included. Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test. A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants. Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients. Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). Disclosures No relevant conflicts of interest to declare.

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