Insights into the Molecular Genetics of Myeloproliferative Neoplasms

2012 ◽  
pp. 411-418
Author(s):  
Huong (Marie) Nguyen ◽  
Jason Gotlib
Keyword(s):  
Molecular Genetics ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Population Level ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Common Finding ◽  
Control Of Gene Expression ◽  
Disease Evolution ◽  
Molecular Abnormalities

Overview: The molecular biology of the BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) has witnessed unprecedented advances since the discovery of the acquired JAK2 V617F mutation in 2005. Despite the high prevalence of JAK2 V617F in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), and the common finding of dysregulated JAK-STAT signaling in these disorders, it is now appreciated that MPN pathogenesis can reflect the acquisition of multiple genetic mutations that alter several biologic pathways, including epigenetic control of gene expression. Although certain gene mutations are identified at higher frequencies with disease evolution to the blast phase, MPN initiation and progression are not explained by a single, temporal pattern of clonal changes. A complex interplay between acquired molecular abnormalities and host genetic background, in addition to the type and allelic burden of mutations, contributes to the phenotypic heterogeneity of MPNs. At the population level, an inherited predisposition to developing MPNs is linked to a relatively common JAK2-associated haplotype (referred to as ‘46/1’), but it exhibits a relatively low penetrance. This review details the current state of knowledge of the molecular genetics of the classic MPNs PV, ET, and PMF and discusses the clinical implications of these findings.

scholarly journals The Genetic Basis of Primary Myelofibrosis and Its Clinical Relevance

2020 ◽  
Vol 21 (23) ◽  
pp. 8885
Author(s):  
Elisa Rumi ◽  
Chiara Trotti ◽  
Daniele Vanni ◽  
Ilaria Carola Casetti ◽  
Daniela Pietra ◽  
...  
Keyword(s):  
Molecular Genetics ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Scoring Systems ◽  
Dynamic Structure ◽  
Primary Myelofibrosis ◽  
Driver Gene ◽  
Recent Effort ◽  
Biological Interactions ◽  
Hematopoietic Stem

Among classical BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) is the most aggressive subtype from a clinical standpoint, posing a great challenge to clinicians. Whilst the biological consequences of the three MPN driver gene mutations (JAK2, CALR, and MPL) have been well described, recent data has shed light on the complex and dynamic structure of PMF, that involves competing disease subclones, sequentially acquired genomic events, mostly in genes that are recurrently mutated in several myeloid neoplasms and in clonal hematopoiesis, and biological interactions between clonal hematopoietic stem cells and abnormal bone marrow niches. These observations may contribute to explain the wide heterogeneity in patients’ clinical presentation and prognosis, and support the recent effort to include molecular information in prognostic scoring systems used for therapeutic decision-making, leading to promising clinical translation. In this review, we aim to address the topic of PMF molecular genetics, focusing on four questions: (1) what is the role of mutations on disease pathogenesis? (2) what is their impact on patients’ clinical phenotype? (3) how do we integrate gene mutations in the risk stratification process? (4) how do we take advantage of molecular genetics when it comes to treatment decisions?

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

Blood ◽  
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.

Endothelial progenitor cells are clonal and exhibit the JAK2V617F mutation in a subset of thrombotic patients with Ph-negative myeloproliferative neoplasms

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2700-2707 ◽  
Author(s):  
Luciana Teofili ◽  
Maurizio Martini ◽  
Maria Grazia Iachininoto ◽  
Sara Capodimonti ◽  
Eugenia Rosa Nuzzolo ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Signal Transducer ◽  
Cell Compartment ◽  
Molecular Abnormalities ◽  
Endothelial Colony Forming Cells ◽  
Molecular Abnormality ◽  
Thrombotic Complications ◽  
Human Androgen Receptor

Abstract In this study we investigated whether neoplastic transformation occurring in Philadelphia (Ph)–negative myeloproliferative neoplasms (MPNs) could involve also the endothelial cell compartment. We evaluated the level of endothelial colony-forming cells (E-CFCs) in 42 patients (15 with polycythemia vera, 12 with essential thrombocythemia, and 15 with primary myelofibrosis). All patients had 1 molecular abnormality (JAK2V617F or MPLW515K mutations, SOCS gene hypermethylation, clonal pattern of growth) detectable in their granulocytes. The growth of colonies was obtained in 22 patients and, among them, patients with primary myelofibrosis exhibited the highest level of E-CFCs. We found that E-CFCs exhibited no molecular abnormalities in12 patients, had SOCS gene hypermethylation, were polyclonal at human androgen receptor analysis in 5 patients, and resulted in JAK2V617F mutated and clonal in 5 additional patients, all experiencing thrombotic complications. On the whole, patients with altered E-CFCs required antiproliferative therapy more frequently than patients with normal E-CFCs. Moreover JAK2V617F-positive E-CFCs showed signal transducer and activator of transcription 5 and 3 phosphorylation rates higher than E-CFCs isolated from healthy persons and patients with MPN without molecular abnormalities. Finally, JAK2V617F-positive E-CFCs exhibited a high proficiency to adhere to normal mononuclear cells. This study highlights a novel mechanism underlying the thrombophilia observed in MPN.

scholarly journals In-Frame Exon 9 CALR Deletions Co-Occur with Other Alterations in the JAK-STAT Pathway in Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4588-4588 ◽  
Author(s):  
Yongbao Wang ◽  
Albert K Ho ◽  
Qiulu Pan ◽  
Frederick Karl Racke ◽  
Dan Jones
Keyword(s):  
Mutation Analysis ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Point Mutations ◽  
Jak2 V617f ◽  
Illumina Miseq ◽  
Primary Myelofibrosis ◽  
Ensembl Database ◽  
Stat Pathway

Abstract Introduction: Mutations in the chaperone gene calreticulin (CALR) have been recently identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF), and are essentially mutually exclusive with JAK2 or MPL mutations. Normal and mutant CALR proteins may differentially affect the subcellular trafficking of JAK-STAT signaling components. CALR mutations previously reported in ET and PMF have been +1 frameshift (fs) mutations localized to exon (E) 9 that generate a novel C-terminal protein sequence with a shift from acidic to basic residues. CALR E9 in-frame (IF) deletions have been recently rarely reported as polymorphisms such as TMP_ESP_19_13054686_13054688 and TMP_ESP_19_13054650_13054658 (Ensembl database entries). We sought to determine the frequency and associated clinical features of CALR with E9 IF alterations in samples submitted for suspicion of a myeloproliferative neoplasm (sMPN). We also assessed whether CALR IF alterations are differentially associated with +1fs mutations or with JAK2 V617For other somatic mutations in MPN-associated genes. Materials and Methods: CALR mutation analysis of E9 was performed on genomic DNA extracted from blood, bone marrow (BM) aspirate or fixed BM biopsy sections using a Sanger sequencing assay with an analytic sensitivity of at least 15%. E9 IF cases were further assessed and mutations quantified by an Ion torrent sequencing panel assessing CALR, CSF3R, JAK2 and MPL, a second panel containing ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 and an Illumina MiSeq extended panel with 20 additional MPN-associated genes. These assays had a sensitivity of approximately 5%. JAK2 V617Fmutations were quantitated using a pyrosequencing assay with an analytic sensitivity of 1%. Results: We assessed CALR E9 mutation status in 733 sMPN samples that were negative for JAK2 V617F mutation. 148 (20.1%) had typical +1fs mutations (95 type 1 and variants, 53 type 2 and variants); 2 (0.3%) had point mutations (E381A and D7373M); 7 (1.0%) had IF deletions including E381_A382>A, D397_D400>D (n =4), D400_K401>D and E405_V409>V. All E9 IF deletions were present at ~50% of reads. Clinical diagnoses were cytopenia/BM fibrosis, ET, thrombocytosis/anemia, and sMPN unspecified. Mutation analysis for 27 additional MPN-associated genes revealed mutations in 5/7 (71.4%) IF deletion cases including in MPL (W515L,40%; D163Y,12%), CSF3R (A470T 46%), ASXL1 (D954fs*26, 45%) and ZRSR2 (S449_R450dup, 27%). No additional mutations were found in the 2 cases with non-synonymous CALR point mutations/SNPs. In a parallel set of 76 MPN samples that had JAK2 V617F at varying levels, we noted 1 E9 IF deletion (D397_D400>D) in a sMPN case with 21.6% JAK2 V617F, and a typical +1fs mutation (K385fs*47) in a case with low (4.2%) JAK2 V617F. All other JAK2 V617F cases had no E9 CALR alterations. Conclusions: CALR E9 in-frame deletions occur in up to 1% of sMPN samples and involve a variety of codons in the acidic domain. Therefore, sizing assays without DNA sequencing are not sufficient to unequivocally distinguish IF deletions from the characteristic +1 frameshift somatic mutations associated with ET and PMF. Given their level, these CALR IF deletions are likely germline sequence variants but are associated with a high frequency of somatic mutations in other MPN-associated genes but not with CALR +1fs mutations. Their co-occurrence with pathogenic somatic mutations in JAK2, MPL and CSF3R affecting the JAK-STAT pathway raises the possibility for a contributory role of altered CALR proteins produced by these E9 deletions in the pathogenesis of MPN. Disclosures Wang: Quest Diagnostics: Employment. Ho:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

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.

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

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.

scholarly journals Aetiology of Myeloproliferative Neoplasms

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1810
Author(s):  
Mary Frances McMullin ◽  
Lesley Ann Anderson
Keyword(s):  
Blood Donation ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Occupational Exposures ◽  
Population Level ◽  
Primary Myelofibrosis ◽  
Epidemiological Studies ◽  
Incidence Rates ◽  
Autoimmune Conditions ◽  
History Of

Myeloproliferative neoplasms (MPNs) have estimated annual incidence rates for polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis of 0.84, 1.03, and 0.47 per 100,000. Prevalence is much higher, particularly for PV and ET, as mortality rates are relatively low. Patients are often concerned about why they developed an MPN and epidemiological studies enable the identification of potential causative factors. Previous work in small heterogeneous studies has identified a variety of risk factors associated with MPNs including family history of MPN, autoimmune conditions, some occupational exposures, and blood donation. At a population level, germline predisposition factors in various populations have been associated with MPNs. The pilot MOSAICC (Myeloproliferative Neoplasm: An In-depth Case-Control) study is one of the largest epidemiological studies in MPN ever carried out to date. It demonstrated the most effective methods for carrying out a significant epidemiological study in this patient group including the best way of recruiting controls, as well as how to evaluate occupational and lifestyle exposures, evaluate symptoms, and collect biological samples. Significant results linked to MPNs in the pilot study of 106 patients included smoking, obesity, and childhood socioeconomic status. The methodology is now in place for a much larger ongoing MOSAICC study which should provide further insight into the potential causes of MPNs.

Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3074-3074
Author(s):  
Brady L Stein ◽  
Donna M Williams ◽  
Michael A McDevitt ◽  
Christine L. O'Keefe ◽  
Ophelia Rogers ◽  
...  
Keyword(s):  
Molecular Genetics ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Single Nucleotide Polymorphism Array ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

Sequential Analysis By Next Generation Sequencing of 18 Genes in Polycythemia Vera and Essential Thrombocythemia Reveals a Association Between Mutational Status and Clinical Outcome after 3-Year Follow-up

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2808-2808
Author(s):  
Damien Luque Paz ◽  
Aurelie Chauveau ◽  
Caroline Buors ◽  
Jean-Christophe Ianotto ◽  
Francoise Boyer ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Poor Prognostic Factor ◽  
Disease Evolution ◽  
Allele Burden ◽  
V617f Mutation

Abstract Introduction Myeloproliferative neoplasms (MPN) are molecularly characterized by driver mutations of JAK2, MPL or CALR. Other somatic mutations may occur in epigenetic modifiers or oncogenes. Some of them have been shown to confer a poor prognosis in primary myelofibrosis, but their impact is less known in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). In this study, we investigated the mutational profile using NGS technology in 50 JAK2 V617F positive cases of MPN (27 PV and 23 ET) collected at the time of diagnosis and after a 3 year follow-up (3y). Patients and Methods All patients were JAK2 V617F positive and already included in the prospective cohort JAKSUIVI. All exons of JAK2, MPL, LNK, CBL, NRAS, NF1, TET2, ASXL1, IDH1 and 2, DNMT3A, SUZ12, EZH2, SF3B1, SRSF2, TP53, IKZF1 and SETBP1 were covered by an AmpliseqTM custom design and sequenced on a PGM instrument (Life Technologies). CALR exon 9 mutations were screened using fragment analysis. Hotspots that mutated recurrently in MPN with no sequencing NGS coverage were screened by Sanger sequencing and HRM. A somatic validation was performed for some mutations using DNA derived from the nails. The increase of a mutation between diagnosis and follow-up has been defined as a relative increase of twenty percent of the allele burden. An aggravation of the disease at 3y was defined by the presence of at least one of the following criteria: leukocytosis >12G/L or immature granulocytes >2% or erythroblasts >1%; anemia or thrombocytopenia not related to treatment toxicity; development or progressive splenomegaly; thrombocytosis on cytoreductive therapy; inadequate control of the patient's condition using the treatment (defined by at least one treatment change for reasons other than an adverse event). Results As expected, the JAK2 V617F mutation was found in all patients with the use of NGS. In addition, we found 27 other mutations in 10 genes out of the 18 genes studied by NGS (mean 0.54 mutations per patient). Overall, 29 of 50 patients had only the JAK2 V617F mutation and no other mutation in any of the genes analysed. No CALR mutation was detected. Nine mutations that were not previously described in myeloid malignancies were found. The genes involved in the epigenetic regulation were those most frequently mutated: TET2, ASXL1, IDH1, IDH2 and DNMT3A. In particular, TET2 mutations were the most frequent and occurred in 20% of cases. There was no difference in the number or in the presence of mutations between PV and ET. At 3y, 4 mutations appeared in 4 patients and 15 out of 50 patients (9 PV and 6 ET) were affected by an allele burden increase of at least one mutation. At 3y, 24/50 patients suffered an aggravation of the disease as defined by the primary outcome criterion (16 PV and 8 ET). The presence of a mutation (JAK2 V617Fomitted) at the time of the diagnosis was significantly associated with the aggravation of the disease (p=0.025). Retaining only mutations with an allele burden greater than 20%, the association with disease aggravation is more significant (p=0.011). Moreover, a mutation of ASXL1, IDH1/2 or SRSF2, which is a poor prognostic factor in primary myelofibrosis, was found in 8 patients, all having presented an aggravation of their disease (p=0.001). Only 4 patients had more than one somatic mutation other than JAK2 V617F and all of them also had an aggravation at 3y (p=0.046). In this cohort, appearance of a mutation at 3y was not associated with the course of the disease. Conversely, the increase of allele burden of at least one mutation was associated with an aggravation (p=0.019). Discussion and conclusion Despite the short follow-up and the limited number of patients, this study suggests that the presence of additional mutations at the time of the diagnosis in PV and TE is correlated to a poorer disease evolution. The increase of mutation allele burden, which reflects clonal evolution, also seems to be associated with the course of the disease. These results argue for a clinical interest in large mutation screening by NGS at the time of the diagnosis and during follow-up in ET and PV. Disclosures Ugo: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: ASH travel.

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.

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