Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera

Abstract Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca2+]c increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

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JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia

Blood ◽

10.1182/blood.v112.11.5228.5228 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5228-5228

Author(s):

Kohtaro Toyama ◽

Norifumi Tsukamoto ◽

Akio Saito ◽

Hirotaka Nakahashi ◽

Yoko Hashimoto ◽

...

Keyword(s):

T Cells ◽

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Mononuclear Cells ◽

Rna Extraction ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Mutation Status ◽

V617f Mutation

Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p < 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

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Evaluation Of The JAK2-V617F Gene Mutation In Egyptian Patients With Essential Thrombocythemia and Polycythemia Vera

Blood ◽

10.1182/blood.v122.21.5254.5254 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5254-5254

Author(s):

Maha Ibrahim El Zaafarany ◽

A. Hasan Abdel-Ghaffar ◽

Tawfik R. Elkhodary ◽

Dalia A. Salem ◽

Eman A. Soliman ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Hematological Parameters ◽

Mutation Screening ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Egyptian Patients ◽

V617f Mutation

Abstract An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80–90 % for patients with polycythemia vera (PV) and 40–70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Egyptian patients with MPD and compared these findings with published studies from other geographic regions and previous studies in EGYPT. A total of 56 patients were studied; of which, 32 were diagnosed with PV and 24 with ET. The mutation status of JAK2 was determined using allele-specific oligonucleotide (ASO) PCR assay. We found that 53% of the PV group and 79% of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed; patient age, levels of WBCs, levels of hemoglobin, levels of platelets and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). The JAK2-V617F mutation is frequently detected in the Egyptian patients with MPD, and especially in patients with ET. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD especially ET. Disclosures: No relevant conflicts of interest to declare.

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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.

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Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽

10.1182/blood-2005-09-3826 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3676-3682 ◽

Cited By ~ 164

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Daniela Pietra ◽

Matteo G. Della Porta ◽

Emanuela Boveri ◽

...

Keyword(s):

Polycythemia Vera ◽

Gene Dosage ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Disease Category ◽

Mutation Status ◽

Cell Counts ◽

Cd34 Cells ◽

V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

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Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

Blood ◽

10.1182/blood.v106.11.2578.2578 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2578-2578

Author(s):

Daniela Pietra ◽

Alessandra Balduini ◽

Carmela Marseglia ◽

Matteo G. Della Porta ◽

Luca Malcovati ◽

...

Keyword(s):

Mrna Expression ◽

Polycythemia Vera ◽

Protein Level ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Serum Samples ◽

Close Relationship ◽

V617f Mutation

Abstract A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation’s precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P<0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P<0.02) and of erythroid progenitors (P<0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

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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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The Red Cell Mass (RCM) Value In Polycythaemia Vera Disease In The JAK2 V617F Era

Blood ◽

10.1182/blood.v122.21.5249.5249 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5249-5249 ◽

Cited By ~ 1

Author(s):

Hassan A. Al-Jafar ◽

Leena M Aytoglu ◽

Issa Loutfi ◽

Iman Al-Shemmari ◽

Salem H Alshemmari

Keyword(s):

Polycythemia Vera ◽

Cell Mass ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Polycythaemia Vera ◽

Red Cell ◽

Red Cell Mass ◽

V617f Mutation ◽

Idiopathic Erythrocytosis

Abstract Introduction In Polycythaemia Vera (PV), the RBC lineage is involved with increased haemoglobin, RBC count and haematocrit. WHO diagnostic criteria for PV are JAK2 V617F mutation and elevated red cell mass (RCM) > 25% of mean normal value. In addition, tests of marrow hypercellularity, blood erythropoietin and colony formation, are minor criteria. However, the diagnostic role of RCM test is still controversial and requires clarification. In this work, PV patients who had both an RCM study and JAK2 V617F mutation test, and routine laboratory tests, are evaluated to check if RCM was essential in the diagnostic work up for PV. Methods Over 2 years, 75 patients with abnormal haematocrit (men ≥ 0.50, women ≥ 0.45) had RCM and JAK2 V617F mutation tests (except JAK2 exon 12 mutation). All subjects consented to the study approved by the ethics committee. RCM was done by Cr-51 RBC radiolabeling method (no prior venesection at least 1 month). Statistical analysis involved descriptive statistics and chi-square test. Results There were 71 males and 4 females, mean age 46 y (range 17-75 y). Increased RCM was found in 41/75 (55%). Positive JAK2 V617F was found in 13/75 patients (17%), who also had RCM above the mean normal predicted value, however, when the WHO RCM criteria were applied, only 7/13 (54%) could be considered as having “truly” increased RCM. In the patient group with negative JAK2 V617F test, 12/28 (43%) had RCM results as per WHO criteria. There was no statistical association between presence of JAK2 V617F and the RCM values. Conclusion In patients with negative JAK2 V617F but with high clinical suspicion for PV and all other causes of secondary and idiopathic erythrocytosis excluded, an increase in RCM would support the diagnosis of PV (about 10 % PV cases). In patients with JAK2 positive mutation and high haematocrit but RCM below the WHO cut-off level, an increased RCM would still count to confirm the diagnosis as the current standard level seems too stringent. References James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature 2005; 434(7037): 1144-8. Kralovics R, Passamonti F, Buser AS, Soon-Siong T, Tiedt R, Passweg JR, et al. A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders. Merck Manual of Diagnosis and Therapy. 16th Edition, 1992 McMullin MF, Bareford D, Campbell P, Green AR, Claire Harrison C, Hunt B, Oscier D, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis. British Journal of Haematology 2005; 130(2): 174-95. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med. 2007;356:459-468. Pardanani A, Lasho TL, Finke C, et al. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera. Leukemia. 2007;21:1960-1963. Pancrazzi A, Guglielmelli P, Ponziani V, et al. A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction. J Mol Diagn. 2008;10:435-441. Disclosures: No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v126.23.2808.2808 ◽

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.

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