scholarly journals JAK2V617F mutational status and allele burden have little influence on clinical phenotype and prognosis in patients with post-polycythemia vera and post-essential thrombocythemia myelofibrosis

Haematologica ◽  
2009 ◽  
Vol 94 (1) ◽  
pp. 144-146 ◽  
Author(s):  
P. Guglielmelli ◽  
G. Barosi ◽  
L. Pieri ◽  
E. Antonioli ◽  
A. Bosi ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Clinical Phenotype ◽  
Allele Burden ◽  
Mutational Status

High Rates of Molecular Response After Long-Term Follow-up of Patients with Advanced Essential Thrombocythemia (ET) or Polycythemia Vera (PV) Treated with Pegylated Interferon-ALFA-2A (PEG-IFN-α-2A; PEGASYS)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 461-461
Author(s):  
Alfonso Quintás-Cardama ◽  
Ross Levine ◽  
Taghi Manshouri ◽  
Outi Kilpivaara ◽  
Hagop M. Kantarjian ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Covalent Attachment ◽  
Molecular Response ◽  
Molecular Responses ◽  
Allele Burden ◽  
Mutational Status ◽  
Starting Dose ◽  
Jak2v617f Allele Burden

Abstract Abstract 461 Background: The use of IFN-α in polycythemia vera (PV) and essential thrombocythemia (ET) has been hampered by poor tolerance and inconvenient dosing schedules. The covalent attachment of polyethylene glycol to IFN-α renders a molecule with prolonged serum half-life, which can be administered weekly. Objectives: We conducted a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) in 84 patients (pts) with high-risk PV (n=44) or ET (n=40). We performed high throughput mutational analysis of JAK2, MPL, TET2, and ASXL1 in all pts. Patients and Therapy: Median age was 51 years (range, 18–79), time from diagnosis to PEG-IFN-α-2a 51 months (range, 0–355), and number of prior therapies was 1 (range, 0–6), including hydroxyurea (HU; n=47), anagrelide (AG; n=26), IFN-α (n=12: 5 oral and 7 sc), imatinib (n=7), and dasatinib (n=1). PEG-IFN-α-2a was initial therapy in 16 (19%) pts (7 PV) that refused HU. JAK2V617F was detected in 19/40 (48%) ET and in 42/44 (95%) PV pts. Nine (11%) pts had abnormal cytogenetics. Initial PEG-IFN-α-2a starting dose was 450 mcg/wk, but that was modified to the current starting dose of 90 mcg/wk. Results: After a median follow-up of 40 months (range, 8–62), 66/83 (80%) assessable pts have responded. Median time to response was 4 weeks (range, 0.5–26). Complete response (CR) was achieved by 62 (75%) pts (for ET: platelets <440×109/L, in the absence of thromboembolic events; for PV: Hb <15 g/dL, no phlebotomy, disappearance of splenomegaly) whereas 4 (5%) pts (2 PV, 2 ET) had a partial response ([PR]; no phlebotomy, off HU and AG, still palpable spleen). Of 5 pts with abnormal karyotype at study entry who were evaluable for response, 2 reverted to diploid cytogenetics. JAK2V617F to total JAK2 ratio was determined by quantitative pyrosequencing assay in all 84 pts prior to PEG-IFN-α-2a. Sixty-one (73%) pts carried the JAK2V617F mutation, which was quantitated at least once on therapy in 54 (64%). Overall, 29 (54%) had >20% reduction in JAK2V617F allele burden, including 10 (19%) in whom the mutation became undetectable (complete molecular response [CMR]) and 15 (28%) who had a >50% reduction (partial molecular response). Molecular responses have not yet reached a plateau among pts with PV. We also analyzed pts for mutations in exon 12 of JAK2, MPL, and the tumor suppressors TET2 and ASXL1 to determine their impact on response to PEG-IFN-α-2a. No pts carried JAK2 exon 12 mutations. One JAK2V617F−negative pt with ET had a MPLW515L mutation, achieved CHR but did not achieve a molecular response. Full length resequencing of all exons of TET2 and ASXL1 genes identified somatic TET2 mutations in 9/71 (13%) and somatic ASXL1 mutations in 3/71 (4%) pts; we identified TET2 (3 JAK2V617F− ET, 2 JAK2V617F+ ET, 3 JAK2V617F+ PV, 1 JAK2V617F− PV) and ASXL1 (1 pt with ET JAK2V617F+, ET JAK2V617F−, and PV JAK2V617F+) mutations in PV and ET pts who were JAK2V617F–positive and negative. TET2 or ASXL1 mutational status did not impact the likelihood of achievement of JAK2 molecular responses, and there was no difference in JAK2V617F allele burden with PEG-IFN-α-2a according to TET2 or ASXL1 mutational status. One pt with baseline mutations in JAK2, TET2, and ASXL1 became JAK2V617F–negative on PEG-IFN-α-2a. Most pts had grade 1–2 toxicities but at doses ≤90 mcg/wk, grade 3–4 toxicity was infrequent. Twenty-five (30%) patients were taken off study after a median of 9 months (range, 3–36) but only 13 (15%) of them due to therapy-related toxicity: g3 neutropenia, anorexia, depression (n=3), ischemic retinopathy, g2 fatigue (n=5), dyspnea, g2 neuropathy. The remaining 59 pts are currently receiving 450 mcg/wk (n=1), 360 mcg/wk (n=1), 240 mcg/wk (n=1), 180 mcg/wk (n=2), 135 mcg/wk (n=3), 90 mcg every 1 (n=8), 2 (n=12), 3 (n=2), or 4 wks (n=1), 45 mcg every 1 (n=9), 2 (n=5), 3 (n=6), or 4 wks (n=8). Conclusion: PEG-IFN-α-2a is remarkably active and acceptably safe in advanced, previously treated PV and ET. Clinical responses are frequently accompanied by significant reduction of JAK2V617F allele burden, which becomes undetectable in a proportion of them suggesting selective targeting of the JAK2V617F clones. Quantitative analysis of ASXL1 and TET2 mutational allele burden during PEG-IFN-α-2a therapy to determine clonal evolution, and methylcellulose-based clonogenic assays in pts who achieved CMR to assess for the presence of erythropoietin independent colony formation are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

JAK2 (V617F)-Positive Essential Thrombocythemia and Polycythemia Vera Are Different Expressions Of a Genotypic/Phenotypic Continuum

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1592-1592
Author(s):  
Elisa Rumi ◽  
Daniela Pietra ◽  
Chiara Elena ◽  
Ilaria Casetti ◽  
Emanuela Sant 'Antonio ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Median Time ◽  
Essential Thrombocythemia ◽  
Cumulative Incidence ◽  
Mutant Allele ◽  
Clinical Phenotype ◽  
Jak2 V617f ◽  
Wild Type ◽  
Allele Burden ◽  
Wbc Count

Abstract Background About 95% of patients with polycythemia vera (PV) and 60-70% of those with essential thrombocythemia (ET) carry the unique JAK2 (V617F) mutation. Previous observations suggest that JAK2 (V617F)-positive ET and PV form a biological continuum, in which the degree of erythrocytosis is determined by physiological and genetic factors. Aims In this work, we studied the natural history of JAK2 (V617F)-positive ET and PV with the aim of establishing whether the two disorders indeed represent different phenotypic expression of a genotypic/phenotypic continuum. Methods We identified 1269 patients diagnosed with ET or PV at our Division between 1980 and 2012, for whom at least one DNA sample was available. The JAK2 (V617F) mutation was assessed using allele-specific quantitative PCR. As patients carrying JAK2 exon 12 or MPL mutations were excluded, the final study population included 1214 patients, 719 of whom with ET (463 JAK2 mutated, 256 JAK2 wild-type) and 495 with PV. Results I: presenting features The clinical phenotype of ET patients at diagnosis differed according to JAK2 mutational status. JAK2 mutated ET presented with older age at diagnosis, higher hemoglobin (Hb) level and white blood cell (WBC) count, lower platelet (PLT) count and erythropoietin level compared to JAK2 wild-type ET (Wilcoxon rank-sum test: P<.001 in all comparisons). The median V617F allele burden was significantly lower in JAK2 mutated ET than in PV (18.4% vs 43.4%, P<.001). A mutant allele burden greater than 50% was observed in 2% of patients with JAK2 mutated ET and 41.5% of those with PV (Fisher exact test: P<.001). In both JAK2 mutated ET and PV, the mutant allele burden was directly related to WBC count and Hb level. Results II: PV evolution Evolution to PV was observed in 53 JAK2 mutated ET patients (incidence 95% CI: 1.4-2.4 per 100 p-years) vs none of the 256 JAK2 wild-type ET (incidence 95% CI: 0-0.2 per 100 p-years), resulting in a significantly different occurrence. The median time to PV evolution was 54 months (range 3.5-220). The cumulative incidence of PV evolution in JAK2 mutated ET patients at 15 years was 28.8% (95% CI: 20.7-37.3; Figure 1). PV evolution was significantly associated with higher JAK2 allele burden at diagnosis (Cox regression HR=1.04, P<.001). Based on the hypothesis that PV patients might have had a silent “pre-PV phase”, we did an ad hoc search for any complete blood count (CBC) collected before diagnosis. Among PV patients, 177 (36%) had a previous CBC, collected at a median time of 22 months (range 1-305) before PV diagnosis. A normal CBC was observed in 15% of patients; the remaining subjects showed thrombocytosis (≥450 x 109/L) and/or leukocytosis (≥10 x 109/L) and/or erythrocytosis. The median time to PV onset was significantly shorter in patients showing at least one CBC abnormality than in those with normal CBC (24 vs 48 months, P=.011). Results III: clinical course The median follow-up was 5.1 years (range, 0-32 years). JAK2-mutated ET and PV did not differ in terms of cumulative incidence of thrombosis (25.3% vs 33.7% at 15 years, P=.35; Figure 2A) and had similar overall survival (OS) (90.3% vs 82.6% at 15 years, P=.29; Figure 2B). Conversely, JAK2 wild-type ET showed a better OS in comparison with both JAK2 mutated ET (P=.028) and PV (P=.004) and a lower incidence of thrombosis (12.7% at 15 years) than JAK2 mutated ET (P=.002) and PV (P<.001). A similar cumulative incidence of disease progression (leukemia and myelofibrosis) was observed in JAK2 mutated (11.7% at 15 years) and JAK2 wild-type ET (12.1% at 15 years), whereas a higher cumulative incidence was observed in PV (26% at 15 years; P=.011 and P=.007 when compared with JAK2 mutated ET and JAK2 wild-type ET respectively). Conclusions This study supports the hypothesis that JAK2 mutated ET and PV are different expressions of a genotypic/phenotypic continuum, in which the mutant allele burden contributes to determine the clinical phenotype. The risk of progression from JAK2 mutated ET to PV is about 2% per year. This work was supported by grant #1005 from Associazione Italiana per la Ricerca sul Cancro (AIRC) “Special Program Molecular Clinical Oncology 5x1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative - http:www-progettoagimm.it). Disclosures: No relevant conflicts of interest to declare.

scholarly journals JAK2V617F allele burden and thrombosis: A direct comparison in essential thrombocythemia and polycythemia vera

2009 ◽  
Vol 37 (9) ◽  
pp. 1016-1021 ◽  
Author(s):  
Alessandra Carobbio ◽  
Guido Finazzi ◽  
Elisabetta Antonioli ◽  
Paola Guglielmelli ◽  
Alessandro M. Vannucchi ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Allele Burden ◽  
Jak2v617f Allele Burden

JAK2 Mutations at Exon 12 and 14 in Polycythemia Vera and Idiopathic Erythrocytosis: Incidence and Correlation with Clinical Characteristics.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2532-2532
Author(s):  
Carlos Besses ◽  
Luz Martínez-Avilés ◽  
Alberto Alvarez-Larrán ◽  
Francisco Cervantes ◽  
Juan Carlos Hernández-Boluda ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Cell Sorting ◽  
Clinical Characteristics ◽  
Clinical Phenotype ◽  
Cell Lineage ◽  
Jak2 V617f ◽  
Free Survival ◽  
Mutational Status ◽  
Cell Subpopulations ◽  
Idiopathic Erythrocytosis

Abstract Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome. Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data. Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells. Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation. Table 1: JAK2 exon 12 and exon 14 mutations in blood cell subpopulations. Mutation Granulocytes Platelets CD14+ CD3+ CD19+ CD56+ * exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined Patient 1* K539L (AAA →TTA) + + − − − − Patient 2* K539L (AAA →CTA) + + + − − − Patient 3* H538_K539delinsL + + ND − − ND Patient 4* N542_E543del + + + − − − Patient 5# V617F (GTC →TTT) + + − − − ND C618R (TGT →CGT) + + − − − ND Patient 6# V617F (GTC →TTC) + + + − − ND C616C (TGT →TGC) + + + − − ND

miRNAs Expression Pattern in Essential Thrombocythemia and Polycythemia Vera Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1912-1912
Author(s):  
Aina Pons ◽  
Carlos Besses ◽  
Luz Martínez-Avilés ◽  
Alberto Alvarez-Larran ◽  
Raquel Longaron ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Expression Pattern ◽  
Essential Thrombocythemia ◽  
Mirna Expression ◽  
Fold Change ◽  
Differentially Expressed ◽  
Hematopoietic Stem ◽  
Mutational Status ◽  
Increased Risk ◽  
Normal Controls

Abstract Abstract 1912 Poster Board I-935 Background: Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are myeloproliferative neoplasms (MPN) arising from a multipotent hematopoietic stem cell characterized by an unregulated production of platelets, red cells and white cells alone or in combination, a tendency to clonal evolution and an increased risk of thrombohemorrhagic complications. MicroRNAs (miRNA) are negative regulators of genes involved in cellular proliferation, apoptosis and/or carcinogenesis. Aim: To analyze the expression pattern of miRNA between PV and ET patients and to find distinctive signatures in ET patients according to JAK2V617F and c-MPL mutational status. Material and Methods: Total RNA was extracted from peripheral blood granulocytes of 50 ET patients, 10 PV patients and 10 controls. Median age of patients was 57 years (range, 20-88); males 36%. The JAK2V617F mutation was present in 23 (46%) of 50 ET patients and in all PV patients. MPL mutations were present in 5 (18%) of 27 JAK2V617F negative cases (3 cases MPLW515L, 1 case MPLW515K and 1 case MPLS505N). miRNA expression was profiled in 384 miRNA via Taqman Low Density Array in ABI PRISM 7900. Expression data was normalized with RNU48 and relative quantification was calculated with the 2–σσCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by means of Significant Analysis of MicroArrays (SAM), Prediction Analysis of MicroArrays (PAM) and Class Comparison methods using BRB array tools version 3.7.0 and TIGR multiexperiment viewer version 4.3. Results: We found a general downregulation of miRNA in ET and PV patients respect to normal controls. A set of 29 miRNA allowed us to discriminate between ET and PV versus normal controls; three of these miRNA were up-regulated and 16 down-regulated in PV and ET vs. normal controls with a >2 fold change and p value <0.01. A distinctive signature of 79 miRNAs differentiated ET, PV and controls and a hierarchical clustering analysis defined miRNA expression profiles of the three particular groups. When we compared miRNA differentially expressed between PV and ET patients, we found nine miRNA, 4 up-regulated and 5 down-regulated in ET with respect to PV patients (p<0.01). Statistical comparisons between ET JAK2V617F-positive and ET JAK2V617F-negative cases showed a distinctive signature of 13 miRNA that allowed us to discriminate between the two groups. In addition, we also found in JAK2V617F cases 19 miRNA differentially expressed between MPL positive and MPL negative patients, with a >2 fold change and p<0.01. Finally, an increased expression of miR-142-5p correlated with JAK2V617F allele burden in ET patients Conclusions: Our study demonstrates that ET and PV can be defined by specific signatures of miRNA. In ET, some miRNA allow us to discriminate cases according to JAK2V617F mutational status and also between MPL-positive and MPL-negative JAK2V617F-negative patients. Research Funding: FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

JAK2V617F Complete Molecular Remission in Long-Term Follow-up of Patients with Polycythemia Vera and Essential Thrombocythemia Treated with Ruxolitinib

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3185-3185
Author(s):  
Lisa Pieri ◽  
Alessandro Pancrazzi ◽  
Annalisa Pacilli ◽  
Claudia Rabuzzi ◽  
Giada Rotunno ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Research Funding ◽  
Jak2v617f Mutation ◽  
Prolonged Treatment ◽  
Molecular Response ◽  
Molecular Remission ◽  
Allele Burden ◽  
Jak2v617f Allele Burden

Abstract Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by the presence of JAK2V617F mutation in >95% and 60% of patients (pts), respectively. This mutation usually affects one allele in ET while most PV pts are homozygous due to mitotic recombination. Acquisition of the JAK2V617F mutation is strongly associated with the germline 46/1 predisposition haplotype. Ruxolitinib is a JAK1/JAK2 inhibitor recently approved for myelofibrosis (MF) and under investigation in PV and ET pts intolerant or resistant to hydroxyurea. We enrolled 24 pts, 11 with PV and 13 with ET, in the phase II INCB18424-256 trial that overall included 34 PV and 39 ET pts. 21/24 pts were still on treatment at 5 years (yr), of which 19 JAK2V617F mutated. Results of the PV cohort have been reported recently (Verstovsek et al. Cancer, 2014): with a median follow up of 35 months (mo), the JAK2V617F allele burden decreased by a mean of 8%, 14%, and 22%, respectively, after 12, 24 and 36 mo. The proportion of pts who achieved a reduction ≥50 % at any time during the 1st yr, 2nd yr, and 3rd yr were 5.9%, 14.7%, and 23.5%, respectively, but no patients achieved a complete remission. In our series of pts we evaluated the JAK2V617F allele burden by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. We also analysed by next generation sequencing (NGS; Ion Torrent platform) a series of MPN-associated mutations including TET2, ASXL1, IDH1/2, LNK, CBL, SRSF2, EZH2 and MPL at baseline and at 5 yr of treatment in ruxolitinib treated pts who achieved a >25% JAK2V617F allele burden reduction at 5 yr (n=13/19). JAK2V617F allele burden decreased by a mean of 7%, 11%, and 19% at 12, 24 and 36 mo, and decreased further by a mean of 28% after 60 mo. Three (1 PV, 2 ET) of 19 pts (16%) achieved a 50% or greater allele burden reduction after 2 yr; no additional pts achieved this degree of allele burden reduction even in prolonged follow up. These 3 pts further improved their molecular response to a complete molecular response (CMR) after 5 yr of treatment. Their mean JAK2V617F allele burden was 46.6% at baseline, 28.3%, 16.3%, 8.7% and 0% after 1 yr, 2 yr, 3 yr and 5 yr, respectively. The JAK2 CMR was confirmed in at least one independent sample at 3 mo after first discovery. At this last timepoint, the PV pt was in complete haematological remission according to ELN criteria, the 2 ET pts were in partial remission due to platelet count still >400x109/L: 422x109/L and 812x109/L, respectively. BM histopathology in the 2 ET pts at 5 yr, while they were in CMR, showed still evidence of megakaryocyte hyperplasia. In the PV pt, histopathology at 5 yr is pending; evaluation at 3 yr, a time when she was in complete hematologic remission and JAK allele burden had decreased from 69 to 8%, showed normalization of cellularity, megakaryocyte and myeloid lineage compared to baseline but still slight erythroid hyperplasia. All 3 pts had normal karyotype at baseline that remained unchanged thereafter. CMR for JAK2V617F was confirmed by NGS. The 2 ET pts achieving CMR did not show any additional mutations, while the PV pts presented a TET2 Y867H mutation with an allele burden of 48.9% and 52%, respectively at baseline and 5 yr. No recurrent mutations in genes other than JAK2 were found in all other examined cases at baseline or at 5 yr. In 3 informative pts, we also analysed the proportion of JAK2V617F homozygous, heterozygous and wild type clones by the method of Hasan et al (Leukemia 2013) based on allelic discrimination of 46/1 haplotype and JAK2. We found that JAK2V617F/V617F clones were reduced by a mean of 95.5%, JAK2V617F/WT showed an uneven trend with a mean reduction of 45.54% while JAK2WT/WT conversely increased (mean 61.43%) at 5 yr, suggesting that in a subset a patients who present significant reduction of VF allele burden ruxolitinib may preferentially target the homozygous clones. Until now, complete molecular remission in PV pts has been described only in patients treated with interferon. Our data suggest that a subset of pts who present a rapid and sustained reduction of the JAK2V617F allele burden under ruxolitinib may eventually reach a condition of CMR with prolonged treatment. However, similar to findings with interferon, mutations establishing clonality, such as in TET2, may still persist in patients who eventually show the disappearance of JAK2V617F mutated subclones. Disclosures Verstovsek: Incyte: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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 &gt;12G/L or immature granulocytes &gt;2% or erythroblasts &gt;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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