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

Genes ◽  
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.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5400-5400
Author(s):  
Tatiana V Makarik ◽  
Adhamjon O Abdullaev ◽  
Sergei M. Kulikov ◽  
Elena E Nikulina ◽  
Svetlana A Treglazova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cell Clone ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Allele Burden ◽  
Chronic Myeloproliferative Neoplasms ◽  
V617f Mutation ◽  
Calr Mutations

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.

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

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1021 ◽  
Author(s):  
Emir Hadzijusufovic ◽  
Alexandra Keller ◽  
Daniela Berger ◽  
Georg Greiner ◽  
Bettina Wingelhofer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Janus Kinase ◽  
Signaling Cascades ◽  
Nuclear Compartment ◽  
Downstream Signaling ◽  
Direct Targeting ◽  
Calr Mutations

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.

A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5215-5215
Author(s):  
Munazza Rashid ◽  
Rifat Zubair Ahmed ◽  
Shariq Ahmed ◽  
Muhammad Nadeem ◽  
Nuzhat Ahmed ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Diagnostic Algorithm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Common Mutation ◽  
Hypochondriac Region ◽  
Exon 9 ◽  
Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms

Blood ◽  
2014 ◽  
Vol 123 (24) ◽  
pp. 3714-3719 ◽  
Author(s):  
Mario Cazzola ◽  
Robert Kralovics
Keyword(s):  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Janus Kinase ◽  
Driver Mutations ◽  
Mutation Status ◽  
Genomic Landscape ◽  
Exon 10 ◽  
Stat Pathway

Abstract Our understanding of the genetic basis of myeloproliferative neoplasms began in 2005, when the JAK2 (V617F) mutation was identified in polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2 exon 12 and MPL exon 10 mutations were then detected in subsets of patients, and subclonal driver mutations in other genes were found to be associated with disease progression. Recently, somatic mutations in the gene CALR, encoding calreticulin, have been found in most patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. The JAK-STAT pathway appears to be activated in all myeloproliferative neoplasms, regardless of founding driver mutations. These latter, however, have different effects on clinical course and outcomes. Thus, evaluation of JAK2, MPL, and CALR mutation status is important not only for diagnosis but also for prognostication. These genetic data should now also be considered in designing clinical trials.

scholarly journals Bone marrow megakaryocytic activation predicts fibrotic evolution of Philadelphia-negative myeloproliferative neoplasms.

Haematologica ◽  
2020 ◽  
pp. 0-0
Author(s):  
Mattia Schino ◽  
Vincenzo Fiorentino ◽  
Elena Rossi ◽  
Silvia Betti ◽  
Monica Di Cecca ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Rapid Evolution ◽  
Progression Free Survival ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Serum Levels ◽  
Chronic Myeloproliferative Neoplasms ◽  
Calr Mutations ◽  
Wbc Count

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPNs’ prognosis needs to be personalized. We identified a new morphological parameter, defined as Megakaryocytic Activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) clusters formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPNs cohorts of patients with polycythemia (PV) (n=64) and non-PV patients [including essential thrombocythemia (ET), and early/prefibrotic primary myelofibrosis (PMF)] (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell (WBC) count, and LDH serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<.0001). Interestingly, M-ACT was not found in the subgroup of ET patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPNs and can represent a new morphologic parameter with a predictive value for progression of MPNs.

Frequency and Prognosis of JAK2 V617F, Calr, MPL and ASXL1 Mutations in Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5562-5562
Author(s):  
Marc Sorigue ◽  
Marta Cabezón ◽  
Olga Garcia ◽  
Patricia Velez ◽  
Silvia Marce ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Prognostic Impact ◽  
Wild Type ◽  
Exon 9 ◽  
V617f Mutation ◽  
Calr Mutations ◽  
Exon 10

Abstract INTRODUCTION: Several mutations have been described in patients with BCR-ABL1 –negative chronic myeloproliferative neoplasms, including primary myelofibrosis (PMF). The most frequent mutation is JAK2 V617F, followed by calreticulin exon 9 (CALR), MPL exon 10 and ASXL1 exon 12. Currently, less than 10% of patients lack molecular marker. CALR and ASXL1 mutations have been consistently found to have favorable and unfavorable prognostic implications, respectively. The aim of this study was to describe the frequency and prognostic impact of JAK2 V617F, CALR, MPL and ASXL1 mutations in patients diagnosed with PMF in 6 Spanish hospitals. METHODS: To detect the presence of JAK2 V617F mutation, an allele-specific PCR using TaqMan probes was used. Screening for insertions and deletions in CALR gene was performed with 6-FAM labeled primers spanning exon 9 and CALR mutations were described by Sanger sequencing. Sanger sequencing was also used to detect MPL exon 10 and ASXL1 exon 12 mutations. RESULTS: Sixty-eight patients were included in the study. All of them were screened for JAK2 and CALR mutations. Forty-five of them (66%) were positive for the JAK2 V617F mutation, while 11/68 (16%) were positive for CALR mutations. Of the 11 CALR mutations, 10 were JAK2 wild-type. MPL exon 10 mutation analysis was only performed in JAK2 wild-type patients and was positive in 4/23 patients (17%), and all of them were CALR wild-type. At the time of submission, ASXL1 exon 12 has been assessed in 18 patients (analysis in the rest of them is currently ongoing). ASXL1 mutations have been found in 3/18 (17%) patients, two of them also with a CALR mutation and the other one with the JAK2 V617F mutation. All three cases were indel mutations. Overall, no mutation was detected in 9/68 (13%) patients. JAK2 V617F, CALR and MPL mutations had no prognostic impact on overall survival. The effect of ASXL1 mutation on prognosis (with and without CALR mutation) will be assessed once all samples have been sequenced. CONCLUSION: JAK2 V617F, CALR and MPL mutations were found in our series of PMF patients in the same proportion found in larger series. ASXL1 has so far been found in a smaller percentage but the entire series of patients will need to be sequenced before reaching definitive conclusions. Studying these genes, only 13% of patients with PMF did not have a clonal marker. None of the studied mutations had prognostic significance. ACKNOWLEDGMENTS: The authors would like to thank Diana Dominguez for her excellent technical assistance and to the grant 2014 SGR225 (GRE), Generalitat de Catalunya. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Rapid, Sensitive and Specific Method for Quantifying Calr Mutant Allele Burden in Persons with Myeloproliferative Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5210-5210
Author(s):  
Jiao Zhou ◽  
Qiumei Yao ◽  
Robert Peter Gale ◽  
Jinlan Li ◽  
Lingdi Li ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Mutation Screening ◽  
Heterozygous Mutation ◽  
Specific Method ◽  
Wild Type ◽  
Allele Burden ◽  
Exon 9 ◽  
Calr Mutations ◽  
Calr Mutation

Abstract Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed. Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Disclosures No relevant conflicts of interest to declare.

scholarly journals Correlation analysis between JAK2, MPL, and CALR mutations in patients with myeloproliferative neoplasms of Chinese Uygur and Han nationality and their clinical characteristics

2018 ◽  
Vol 46 (11) ◽  
pp. 4650-4659
Author(s):  
Tao Lang ◽  
Yuling Nie ◽  
Zengsheng Wang ◽  
Qin Huang ◽  
Li An ◽  
...  
Keyword(s):  
Clinical Characteristics ◽  
Fusion Gene ◽  
Myeloproliferative Neoplasms ◽  
Pcr Amplification ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Chinese Patients ◽  
Patient Groups ◽  
Calr Mutations ◽  
The Relationship

Background Genetic factors play a role in the etiology of BCR-ABL-negative myeloproliferative neoplasms (MPNs). This study explored the relationship between mutations in the Janus kinase 2 gene ( JAK2), MPL, and the calreticulin gene ( CALR) in Uygur and Han Chinese patients with BCR-ABL fusion gene-negative MPN and corresponding clinical features. Methods A total of 492 BCR-ABL-negative MPN patients treated in our hospital from May 2013 to August 2016 were enrolled. Genomic DNA was extracted from peripheral blood and used for PCR amplification and DNA sequencing. Mutations including JAK2 V617F, MPL W515L/K, and those in JAK2 exon 12 and CALR were analyzed and compared with patient clinical characteristics. Results Of the 492 MPN patients, 169 were Uygur and 323 were Han. In these two patient groups, JAK2 mutations were detected in 39.64% and 52.63%, respectively, CALR mutations were detected in 10.06% and 20.43%, respectively, and MPL mutations were detected in 0.93% of Han patients. The age, white blood cell count, platelet levels, and hemoglobin levels in JAK2 in Han patients were higher than those in Uygur patients. Conclusion Han MPN patients harboring JAK2 mutations had higher level of age, WBC, PLT, and Hb than Uyghur patients with the same mutations.

Frequency and Molecular Characteristics of Calreticulin Gene (CALR) Mutations in Patients with JAK2-Negative Myeloproliferative Neoplasms

2014 ◽  
Vol 133 (2) ◽  
pp. 193-198 ◽  
Author(s):  
Marzena Wojtaszewska ◽  
Małgorzata Iwoła ◽  
Krzysztof Lewandowski
Keyword(s):  
Data Analysis ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Molecular Characteristics ◽  
New Mutation ◽  
Clinical Data Analysis ◽  
Leukocyte Counts ◽  
Exon 9 ◽  
Calr Mutations

In 2013, Nangalia et al. and Klampfl et al. found a recurrent and abundant mutation in the calreticulin gene (CALR), mutually exclusive with JAK2 and MPL alterations. At present, the data concerning the new mutation, i.e. its prevalence, allele burden and clinical significance, are scarce. We report the incidence and molecular characteristics of CALR mutations in a group of 184 Polish patients with myeloproliferative neoplasms (MPNs). Clinical data analysis revealed significant differences between JAK2 V617F-mutated and CALR-mutated groups. In essential thrombocythemia patients, hemoglobin levels and leukocyte counts were significantly higher in JAK2-positive than in CALR-positive patients (p = 0.023 and p = 0.017, respectively), but the CALR-positive patients had significantly higher platelet counts (p = 0.022). Patients harboring CALR mutations were also younger at the time of diagnosis (p = 0.039). In primary myelofibrosis patients, the degree of anemia was less severe in those who were CALR exon 9 mutation-positive than in those who were JAK2 V617F-positive (p = 0.048). © 2014 S. Karger AG, Basel

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