The A3669G Polymorphism of GR Is a Host Genetic Modifier Associated with Polycythemia Vera and Primary Myelofibrosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3067-3067
Author(s):  
Lilian Varricchio ◽  
Elena Masselli ◽  
Giovanni Migliaccio ◽  
Carolyn Whitsett ◽  
Ronald Hoffman ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Genetic Modifier ◽  
Primary Myelofibrosis ◽  
Jak2v617f Mutation ◽  
Single Mutation ◽  
Allele Burden ◽  
Host Genetic ◽  
Jak2v617f Allele Burden

Abstract Abstract 3067 The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) which are all associated with a gain-of-function mutation (JAK2V617F) of the JAK2 gene. There is ongoing discussion on the mechanism by which this single mutation may cause diseases with three different phenotypes. The consensus is that while the JAK2V617F may increase proliferation of hematopoietic cells in general, the lineage affected is determined by host genetic modifiers still to be identified. The glucocorticoid receptor (GR) is an important regulator of erythropoiesis in vivo and in vitro. It exerts its activity by synergistic transcriptional and membrane-associated mechanisms. Upon activation, GR forms a transcriptional complex with phosphorylated STAT-5 (P-STAT-5) which binds and alters the expression of a subset of the erythropoietin (EPO)- and stem cell factor (SCF)-target genes. Physical interaction between GR and the EPO receptor (EPO-R) on the cell membrane suppresses the ability of both receptors to phosphorylate STAT-5 when activated together. Insufficient GR/P-STAT-5 transcriptional activity has been suggested to result in delayed erythroid maturation and to induce eythrocytosis in patients exposed to excessive glucocorticoids. We had previously determined that erythroid cells (EBs) generated ex vivo from peripheral blood mononuclear cells of patients with PV and those obtained from normal donors (ND) stimulated with dexamethasone have similar biological (increased proliferation with delayed maturation) and biochemical (great levels of NFE-2, WT-1 and GATA2 and low levels of GATA1 and β-globin expression) properties (Varricchio et al,Blood 2009;114:3899a). This similarity is counterintuitive because STAT-5 phosphorylation was reduced in normal EBs exposed to dexamethasone and EPO while in EBs from PV patients it was constitutive due to the presence of the JAK2V617F mutation. In addition to the canonical GRα signalling isoform, cells may express the dominant negative GRβ isoform which binds poorly to glucocorticoids because its unique structure impairs ligand binding and determines nuclear retention. Expression of GRβ is regulated by the presence of the A3669G SNP in exon 9 which specifically stabilizes GRβ mRNA. The presence of this polymorphism has been associated with glucocorticoid resistance but its effects on erythropoiesis are unknown. To clarify the similarities between the biological properties of EBs expanded ex vivo from ND in the presence of dexamethasone and those obtained from PV patients, we determined the levels of expression (at the mRNA and protein level) of GRα and GRβ in EBs expanded ex vivo from 16 ND and 16 PV patients. While EBs from ND expressed GRα only, those expanded from PV patients expressed both GRα and GRβ. Therefore, in spite of constitutive STAT-5 phosphorylation, in EBs from PV patients formation of transcriptionally active GR/P-STAT-5 complexes is reduced by expression of GRβ. To clarify the mechanism underlying expression of GRβ in EBs obtained from PV patients, the frequency of the A3669G polymorphism was determined by specific PCR followed by sequencing of the amplified products using DNA obtained from the mononuclear cells of ND (7) and patients with PV (10). DNA from patients with PMF (10) and ET (14) was analysed for comparison. The polymorphism was detected in 60% of patients with PV but not in ND, indicating that GRβ mRNA is particularly stable in EBs from PV patients. In addition, the polymorphism was observed in 50% of patients with PMF but was not detected in patients with ET. A trend toward higher JAK2V617F allele burden was observed in A3669G positive PV patients (69.8±23.0) compared to A3669G negative PV patients (37.3±22.6) and ET patients (28.13±27.9) while the JAK2V617F allele burden in patients with PMF with or without the A3669G SNP was the same (51.2±37.5 vs 42.8±17.9). These data suggest that in addition to mutations of EPO-R and Von Hippel-Lindau gene, erythrocytosis may be induced by excess GR activation by glucocorticoids or, in association with the JAK2V617F mutation, by the presence of the A3669G SNP which favours GRβ expression. Moreover, the A3669G SNP may represent a host genetic modifier and a potential diagnostic tool to predict development of erythrocytosis in patients with MPN and GRβ may represent a potential therapeutic target for PV. 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.

Constitutively Activated and Hyper-Sensitive Basophils in Patients with Polycythemia Vera: Role of JAK2V617F Mutation and Correlation with Pruritus

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3714-3714 ◽  
Author(s):  
Alessandro M. Vannucchi ◽  
Lisa Pieri ◽  
Costanza Bogani ◽  
Paola Guglielmelli ◽  
Maria Zingariello ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Ex Vivo ◽  
Absolute Number ◽  
Jak2v617f Mutation ◽  
Fixed Dose ◽  
Median Frequency ◽  
Direct Role ◽  
Allele Burden

Abstract Aquagenic pruritus is a typical feature of polycythemia vera (PV) reported by up to 65% of patients (pts) at diagnosis (Mesa R, Cancer 2007). It can either ante date diagnosis or appear during the course of disease; it is poorly responsive to phlebotomy or myelosuppressive therapy, while treatment with interferon-alpha or selective serotonin reuptake inhibitors can be sometimes successful. Its pathophysiologic mechanisms are poorly known, and eosinophils, basophils or dermal mast cells have all been hypothesized to contribute. An association with JAK2V617F burden has been reported, since pruritus was significantly more common in pts with >50% mutated alleles (Tefferi A, Cancer 2006; Vannucchi AM, Blood 2007), and the risk of having it was 4.2-fold greater in pts in the highest mutated allele quartile as compared to the lowest one (Vannucchi AM, Leukemia 2007). Therefore, we sought to focus on basophils to underscore their possible involvement in the pathogenesis of pruritus and the role of V617F mutation. According to previous reports, among the 75 pts included in this study the V617F allele burden was significantly greater in those referring pruritus (71±18%) than in those who denied it (48±19%; P=.002). We found that the V617F allele burden measured in immunomagnetically purified basophils was comparable to granulocytes (r=0.9), while the content of JAK2 mRNA was significantly higher in PV basophils than in both PV granulocytes (P<.0001) and normal basophils (P=.02) and granulocytes (P=.01). This was not due to a preferential representation of mutated RNA since the proportion of wild-type and V617F-mutated JAK2 mRNA transcripts was consistent with quantitative genotyping. CD63 is a tetraspanin contained in the granules of resting basophils, which is expressed on outer membrane following their activation. We measured by flow cytometry the number of circulating CD63+ basophils in PV pts. We found that median frequency of CD63+ cells in the CD123+/HLA-DR− basophil gate was significantly greater in PV pts than in controls (13.0±10.5% versus 1.8±1.7%; P= .01), and their absolute number increased from 0.4±0.3×106/L in controls to 15.5±11.0 in PV pts (P=.003). Also the expression level of CD63, measured as the mean fluorescence intensity (MFI), was significantly greater in PV pts than in controls (1,405±856 vs 740±145.2; P=.04). PV pts suffering from pruritus had significantly greater percentage of activated basophils than those without: 18.8±10.6% vs 5.6±3.6% (P=.009). Finally, both the percentage and the absolute number of CD63+ basophils were greater in pts with >50% mutated allele compared to those with <50%: 15±9.5% vs 4.7±3.8 % (P= .02) and 21.2±10.8×106/L vs 7.1±6.7×106/L (P= .04), respectively, and they were linearly correlated with V617F burden (r=0.76, P<.01). Transmission electron microscopy analysis showed that, compared to healthy counterpart, PV basophils had reduced number of granules most of which were empty and displayed a disrupted membrane, while the cytoplasm presented abnormal electrondensity and vacuolization. To evaluate the role of JAK2V617F mutation in the activated status of circulating basophils, we analyzed their ex-vivo activation in response to the N-Formyl-Met-Leu-Phe (fMLP) peptide and IL-3 that is the most potent priming cytokine of basophils. Using progressive dilutions of fMLP in IL-3-primed cells, we found that the increase of CD63+ basophils was always significantly greater in PV pts than in controls, particularly in case of >50% V617F allele (P<.01); similarly, when basophils were challenged with varying amounts of IL3 (from 0.1 to 10 ng/mL) and a fixed dose of fMLP, the response of PV cells resulted significantly greater than control cells at all concentrations employed, overall pointing to an hypersensitivity of mutated cells. Finally, preincubation with the potent and selective JAK2 inhibitor AZD1480 (kindly provided by Astra Zeneca Ltd) largely prevented the ex-vivo activation of PV basophils which proved to be even more sensitive than normal cells. These data point to constitutively activated and hyper-reactive basophils as one mechanism contributing to pruritus in PV, and provide evidence for a direct role of JAK2V617F mutation. However, it is conceivable that additional circulating and/or dermal cells may be involved as well as in the pathogenesis of this agonizing symptom.

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

scholarly journals Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 115 (15) ◽  
pp. 3109-3117 ◽  
Author(s):  
Alfonso Quintás-Cardama ◽  
Kris Vaddi ◽  
Phillip Liu ◽  
Taghi Manshouri ◽  
Jun Li ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Interleukin 6 ◽  
Myeloproliferative Neoplasms ◽  
Primary Cultures ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Experimental Models ◽  
Jak2v617f Mutation ◽  
Erythroid Progenitor ◽  
Therapeutic Implications

AbstractConstitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Cytogenetic Studies at Diagnosis in Polycythemia Vera: Clinical and JAK2V617F Allele Burden Correlates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2545-2545
Author(s):  
Naseema Gangat ◽  
Jacob J. Strand ◽  
Terra L. Lasho ◽  
Christy M. Finke ◽  
Ryan A. Knudson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
World Health ◽  
Chromosome 1 ◽  
Leukemic Transformation ◽  
Cytogenetic Abnormalities ◽  
Allele Burden ◽  
Significant Difference ◽  
Cytogenetic Studies ◽  
Jak2v617f Allele Burden

Abstract Background: Previous cytogenetic studies in polycythemia vera (PV) have included a relatively small number of patients (“n” ranging 10–64). In the current study (n=137), we describe cytogenetic findings at presentation and examine their relationship to clinical and laboratory features, including bone marrow JAK2V617F allele burden. Methods: The study consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom bone marrow biopsy and cytogenetic studies were performed at diagnosis. Results I: cytogenetic details At diagnosis: A total of 137 patients (median age, 64 years; 49% females) were studied at diagnosis and had adequate metaphases for interpretation. Cytogenetics were normal in 117 patients (85%) and displayed either a sole -Y abnormality in 5 patients (7% of the male patients), and other chromosomal abnormalities in 15 (11%). The latter included trisomy 8 in five patients, trisomy 9 in three patients, two patients each with del(13q), del(20q), and abnormalities of chromosome 1, and one patient each with del(3)(p13p21), dup(13)(q12q14), and del(11)(q21). At follow-up: Repeat cytogenetic studies while still in the chronic phase of the disease were performed in 19 patients at a median of 60 months (range, 8–198) from diagnosis. Of these, 4 had aquired new cytogenetic clones including 3 with normal cytogenetics at time of initial PV diagnosis. The new abnormalities included del(20q), del(5q), del(1p), chromosome 1 abnormality, and inv(3)(q21q26.2). At time of disease transformation: Leukemic transformation was documented in 3 patients of whom cytogenetic information at the time was available in 2 patients; both patients had normal results at time of initial PV diagnosis and complex cytogenetic abnormalities at time of leukemic transformation. In contrast, among 6 patients with available cytogenetic information at time of fibrotic transformation, the results were unchanged from those obtained at time of diagnosis in 5 patients. ii) Correlation between cytogenetics at diagnosis and JAK2V617F allele burden: Allele-specific, quantitative PCR analysis for JAK2V617F was performed in 71 patients using genomic DNA from archived bone marrow obtained at the time of the initial cytogenetic studies. JAK2V617F mutation was detected in 64 of the 71 (90%) patients; median mutant allele burden was 16% (range 3–80%) without significant difference among the different cytogenetic groups: normal vs. –Y vs. other cytogenetic abnormalities (p=0.72). iii) Clinical correlates and prognostic relevance of cytogenetic findings at diagnosis: Among several parameters studied for significant correlations with cytogenetic findings at diagnosis, an association was evident only for age (p=0.02); all –Y abnormalities (n=5) as well as 13 of the 15 (87%) other cytogenetic abnormalities occurred in patients ≥ 60 years of age. Stated another way, the incidence of abnormal cytogenetics (other than -Y) was 4% for patients younger than age 60 years and 15% otherwise. The presence of abnormal cytogenetics at diagnosis had no significant impact on either overall or leukemia-free survival. Conclusions: Abnormal cytogenetic findings at diagnosis are infrequent in PV, especially in patients below age 60 years. Furthermore, their clinical relevance is limited and there is not significant correlation with bone marrow JAK2V617F allele burden.

Detection of a Stem Cell Population with Hemangioblastic Characteristics in Primary Myelofibrosis (PMF),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3860-3860
Author(s):  
Ioanna N Trivai ◽  
Thomas Stuebig ◽  
Anita Badbaran ◽  
Ursula Gehling ◽  
Asterios Tsiftsoglou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Primary Myelofibrosis ◽  
Jak2v617f Mutation ◽  
Cell Subpopulation ◽  
Stem Cell Population ◽  
Allele Burden

Abstract Abstract 3860 Primary myelofibrosis (PMF) comprises a myeloproliferative neoplasia accompanied by imbalance of various tissues of the mesoderm, let alone the hematopoietic tissue. Involvement of multiple hematopoietic lineages during disease progression suggests the clonality of myelofibrosis that can be attributed to an initial stem cell defect at the very early stage of the stem cell hierarchy. Hematopoietic and endothelial phenotypes of circulating multipotent stem cells in patient peripheral blood, along with the increased microvascular density in the bone marrow, leads to the hypothesis that the critical event in PMF involves malignant transformation of a stem cell with hemangioblastic potential. Former studies have provided functional evidence that activated JAK2 signalling in primitive human hematopoietic cells is sufficient to drive key processes involved in the pathogenesis of the disease. In this study, the functionality and differentiation potential of circulating primitive JAK2V617F+ stem cells from primary myelofibrosis patients is assessed. Primitive stem cells were isolated from peripheral blood of 25 patients. All patients participating in the study were diagnosed with primary myelofibrosis, have been untreated, and were found positive for JAK2V617F mutation. Isolated stem cells were analysed for purity and assessed for the expression of markers characteristic for the hemangioblast phenotype (CD34, CD133, CD45, VEGFR2, VE-Cadherin, E-Cadherin, CD31) with flow cytometry. Genomic DNA was isolated from various stem cell populations to determine the mutational status by PCR. Our results indicate that long term repopulating stem cells circulating in peripheral blood bear the JAK2V617F mutation. Hemangioblast resembling populations within the isolated prime stem cells were also found positive for the mutation. Long term repopulating stem cells bearing different allele burden for JAK2V617F mutation from PMF patient peripheral blood were expanded for up to 4 months. Various colonies formed after seeding in semisolid media were characterised by morphological features (CFU-GEMM, CFU-GM, CFU-E, CFU-M, CFU-Endo) and expressing genes by quantitative PCR. Moreover, allele burden determination for various progenitors of both hematopoietic and endothelial lineages was performed. JAK2V617F allele burden varied within individual progeny phenotypes, indicating the acquisition of the mutation that boosts the outgrowth of the malignant clone within the hemangioblast compartment of the bone marrow. Endothelial and macrophage progenitors appear heterozygotic while all rest progenitors of various hematopoietic lineages can be either heterozygotic or homozygotic. This indicates high genomic instability of the JAK2V617F+ malignant clone as it is driven into hematopoietic differentiation. Our results indicate the existence of a malignant clone with hemangioblast phenotype in PMF which can differentiate into hematopoietic and/or endothelial progenitors in vitro. Our experiments shed light to the pathogenesis of PMF by characterising the potential of the defective stem cell subpopulation responsible for the disease. 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 &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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