JAK2V617F-positive Essential Thrombocythemia Patients Research Articles

Efficacy and safety of peginterferon-α2b for treatment of myeloproliterative neoplasms

To evaluate the clinical efficacy and adverse reactions of peginterferon-α2b for treatment of chronic myeloproliferative neoplasms (MPN). We retrospectively analyzed the data of 107 patients with MPN, including 95 with essential thrombocythemia (ET) and 12 with polycythemia vera (PV), who all received peginterferon-α2b treatment for at least 12 months. The clnical and follow-up data of the patients were analyzed to evaluate the efficacy and adverse reactions of the treatment. After receiving peginterferon- α2b treatment, both ET and PV patients achieved high hematological remission rates, and the total remission rates did not differ significantly between the two groups (86% vs 78%, P>0.05). In the overall patients, the spleen index decreased by 13.5% (95%CI: 8.5%-18.5%) after the treatment. The patients with hematological remission showed a significantly greater reduction of the total symptom score than those without hematological remission (P < 0.01). The median percentage of JAK2V617F allele load of PV patients decreased from 67.23% (49.6%-84.86%) at baseline to 19.7% (0.57%-74.6%) after the treatment, and that of JAK2V617F-positive ET patients decreased from 48.97% (0.45%-74.24%) at baseline to 22.1% (0.33%-65.42%) after the treatment. Mild adverse reactions (grade 1-2) were observed in both ET and PV groups without significant differences between them. The overall incidence of thrombotic events during the treatment was 2.8% in these patients, and no serious adverse reactions were observed. For patients with chronic myelodysplasia, peginterferon-α2b treatment can achieve a high peripheral blood cell remission rate and maintain a long-term stable state with good effect in relieving symptoms such as splenomegaly. Peginterferon- α2b treatment caused only mild adverse reactions, which can be tolerated by most of the patients.

Comparison of the effects between MPL and JAK2V617F on thrombosis and peripheral blood cell counts in patients with essential thrombocythemia: a meta-analysis

To assess the effects between MPL and JAK2V617F on the thrombosis risk and peripheral blood cell counts in patients with essential thrombocythemia (ET), we identified eligible studies from PubMed, Embase, and the Cochrane Library. Seven studies were ultimately included in this meta-analysis. All studies reported the peripheral blood cell counts of ET patients, and three of them reported the eligible thrombotic events. In comparing the effect of MPL versus JAK2V617F on thrombosis, 1257 ET patients (73 MPL + and 1184 JAK2V617F +) were included. MPL-positive (MPL +) ET patients had a higher risk of thrombosis than JAK2V617F-positive (JAK2V617F +) ET patients [RR = 1.80 (1.08–3.01), P = 0.025]. And 3453 ET patients (138 MPL + and 3315 JAK2V617F +) were included in the comparison of peripheral blood cell counts. Platelet counts of MPL + ET patients were higher than that of JAK2V617F + ET patients [WMD = 81.18 (31.77–130.60), P = 0.001]. MPL + ET patients had lower hemoglobin [WMD = − 11.66 (− 14.32 to − 9.00), P = 0.000] and white blood cell counts [WMD = − 1.01 (− 1.47 to − 0.56), P = 0.000] than JAK2V617F + ET patients. These findings indicate that the MPL mutation is a high-risk factor for thrombosis in ET patients, and it may be rational to include MPL mutation in the revised IPSET as a criterion for thrombosis prediction scores. And given the differences in peripheral blood, it is necessary to further study whether MPL + ET patients differ from JAK2V617F + ET patients in bleeding and survival.

Overexpression of PD-L1 Correlates with JAK2-V617F Mutational Burden and Is Associated with Chromosome 9p Uniparental Disomy in MPN

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P&amp;lt;.01) or HD (P&amp;lt;.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p&amp;lt;.005) and the same was observed in PMF (p&amp;lt;.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P&amp;lt;.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P&amp;lt;.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p&amp;lt;.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p&amp;lt;.001 and P&amp;lt;.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P&amp;lt;.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Clinical Characteristics and Cardioavscular Events in Patients with Esential Thrombocythemia with

Introduction The clinical characteristics, treatment, cardiovascular events (CVE) and evolution of patients diagnosed with JAK2 V617F positive essential thrombocythemia (ET) with low allele burden (LAB) are scarcely studied. Its presence in people without a confirmed diagnosis of malignant hemopathy is called clonal hematopoiesis of uncertain significance (CHIP) and confers higher risk of developing CVE. The objective of this study was to compare the clinical characteristics and CVE of a series of JAK2 V617F-positive ET patients with &lt;10% (LAB) vs. ≥10% allele burden (HAB), from the GEMFIN (Grupo Español de Enfermedades Mieloproliferativas Crónicas Filadelfia Negativas) Group. Methods From the database of the GEMFIN group, 410 ET patients were JAK2 V617F positive, 89 (21.7 %) with LAB and 321 (78.3%) with HAB. The clinical characteristics, treatment (cytoreduction, antiagregation, anticoagulation, JAK inhibitor), CVE (before, at and after diagnosis) and evolution to myelofibrosis (MF) or acute myeloid leukemia (AML) of these two groups of patients were compared. Results LAB and HAB groups did not significantly differ regarding the main clinical characteristics (i.e cardiovascular risk factors [CVRF] and International Prognostic Score for Thrombosis in Essential Thrombocythemia [IPSET] score) except for the median platelet count: LAB 636 x109/L [436- 2500] vs HAB 687 x109/L[440-1980L], p=0.035). CVE after diagnosis of ET were more frequent in patients with HAB (41/137, 30%) than in patients with LAB (5/48, 10%), p=0.007. Only one LAB patient with CVE had JAK2 allele burden &gt;5%. Treatments received by both groups were not significantly different. None of the patients from both groups progressed to AML, whereas 1/48 vs. 6/137 of patients evolved to MF. Median follow-up of patients with LAB and HAB was 3.4 years [0.1-17.7] and 4.3 years [0.1-27.8], respectively (Table 1). Conclusions In these series of ET patients from the GEMFIN group, patients with LAB had significantly lower median platelet count at diagnosis and less CVE after diagnosis than patients with HAB, although CVRF and IPSET scores and treatment approach were similar. The clinical behavior of LAB patients may resemble that of individuals with CHIP. The therapeutic algorithm of ET patients with LAB may be somehow different than that of patients with HAB and therefore, might be revised. Disclosures Bellosillo: Astra-Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biocartis: Honoraria; Merck-Serono: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman â€"La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; ThermoFisher: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; BMS: Honoraria. Hernandez Boluda:Incyte: Other: Travel expenses paid. Pérez:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Sequential analysis of 18 genes in polycythemia vera and essential thrombocythemia reveals an association between mutational status and clinical outcome.

Philadelphia-negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular-based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing-based 18-gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3-year follow-up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression. © 2017 Wiley Periodicals, Inc.

MiR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia.

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Targeted Sequencing of Sorted Peripheral Blood Cells Reveals Novel Germline and Somatic Variants in the Polymorphonuclear and Mononuclear Cells of Patients with Essential Thrombocythemia, Polycythemia Vera and Primary Myelofibrosis

BackgroundThe past decade has witnessed a significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). A large number of genes have now been implicated in the pathogenesis of MPN but their relative importance, the mechanisms by which they cause different cell types to predominate and their implications for prognosis remain unknown.AimThe aim of this study was to perform targeted next generation sequencing on different cell types directed at a panel of mutations shown to be associated with MPN in order to identify driver mutations and novel variants, to determine their relative importance to pathogenesis and to predict survival in patients with MPN.MethodsWe performed targeted sequencing on normal controls (NC) and patients with MPN from 3 different races (Malay, Chinese and Indian) in Malaysia who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria for MPN. Patients from 3 tertiary-level hospitals were recruited prospectively over 3 years and informed consents were obtained. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMNs), mononuclear cells (MNCs) and T cells. DNA was extracted from the different cell population and high-throughput sequencing was performed using an Ion Torrent PGM and AmpliSeq targeting platform, with a panel of 26 MPN-related genes.ResultsTwenty-nine patients (11 ET, 11 PV, 7 PMF) and 3 NC were recruited into the study. Ten of 29 (34.5%) and 9 of 29 (31%) patients were Malay and Chinese men respectively. Twenty-four of 29 (82.8%) patients harbored the JAK2V617F mutation which was detected in 7 (63.6%) ET, 11 (100%) PV and 6 (85.7%) PMF patients. The allelic frequency of JAK2V617F mutation was highest in PMF. 11 of the 24 JAK2V617F positive patients (2 ET, 5 PV and 4 PMF) also harbored potentially pathogenic variants involving the ASXL1, CALR, CBL, CHEK2, DNMT3A, TET2, U2AF1, RUNX1 , SF3B1 and TP53 genes. In patients who were found to have both JAK2V617F mutation and a TET2 variant, the allelic frequencies of the TET2 variantswere significantly lower than the JAK2V617F mutations. Conversely, in 1 ET patient with high TET2 allelic frequency, JAK2V617F mutation was not detected. Several novel variants involving the CHEK2, DNMT3A, RUNX1, SF3B1 and TET2 genes were identified in the study. All the novel variants were somatic (detected predominantly in the PMNs) except CHEK2 which was detected in all 3 cell types (PMNs, MNCs and T cells) at approximately 50% allele frequency. A similar spectrum of mutations was found in PMNs and MNCs, usually at a lower frequency in MNCs. Somatic mutations were absent or detected at very low frequencies in T cells. One of the 7 JAK2V617F positive ET patients harbored 4 somatic variants in CHEK2, SF3B1, TET2 and TP53 genes, with the highest allelic frequencies in the PMNs for 2 variants and MNCs for the other 2 variants. Three of the 4 JAK2V617F negative ET patients had CALR, MPL exon 10, SH2B3 and TET2 variants. One of the 3 ET patients with CALR and TET2 variant presented with a very high platelet count of 2251 x 109/L. One PV patient who harbored both JAK2V617F and CALR mutations presented with high haemoglobin and white cell counts. The CALR mutation in this patient was thought to be germline as it was detected in all cell types at approximately 50% allelic frequencies. The 2 PV patients who harbored both JAK2V617F and DNMT3A mutations presented with more severe phenotype with leukocytosis, splenomegaly of >15cm, constitutional and vasomotor symptoms requiring treatment with Hydroxycarbamide. The 3 PMF patients with JAK2V617F mutation in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 variants also presented with severe clinical phenotypes, 2 of whom died within 2 years from diagnosis.ConclusionJAK2V617F is the commonest somatic mutation detected in MPN patients in Malaysia and is predominantly found in the PMNs. Nearly half of the JAK2V617F positive patients also harbor other potentially pathogenic variants. Several novel somatic and one germline variants were found in this study. Patients who harbor the JAK2V617F in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 putatively pathogenic variants were found to have more severe clinical phenotypes with very poor prognosis. Further studies are required to explore cell type differences, prognosis and clonal evolution in MPN. DisclosuresAitman:Illumina: Honoraria.

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET.Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients.Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future.

Clinical analysis of JAK2 V617F gene mutation in 120 patients with myeloproliferative neoplasms

Objective To investigate the JAK2 V617F gene mutations in myeloproliferative neoplasms (MPN) patients and its clinical significance. Methods 120 patients were detected bone marrow pathological conditions with bone marrow cytology and biopsy analysis,and monitored the Ph chromosome and bcr-abl fusion gene. Genomic DNA from bone marrow cells was extracted from 120 patients, JAK2 V617F gene mutations were tested by fluorescence quantitative PCR. Results All patients of each MPN type presented their typical characteristics. Ph chromosome and bcr-abl fusion gene were negative. The positive rate of JAK2 V617F gene mutations was 66.7 % (80/120), among them the positive rate in polycythemia vera (PV) was 72.7 % (16/22), the positive rate in essential thrombocythemia (ET) was 66.0 % (62/94), and among 4 patients primary myelofibrosis (PMF) JAK2 V617F was positive in 2 cases. In JAK2 V617F positive PV patients, the white blood cell count (P = 0.001) and platelet count (P = 0.01) were significantly higher than those in negative patients. In JAK2 V617F positive ET patients, the white blood cell count was significantly higher than that in negative patients (P = 0.006). In PMF patients, there was no significant difference (P> 0.05). Conclusion JAK2 V617F gene mutations testing is helpful to the diagnosis and definite diagnosis of bcr-abl negative MPN, which promote the early detection and treatment. Key words: Myeloproliferative neoplasms; JAK2 V617F gene mutation; Polycythemia vera; Essential thrombocythemia; Primary myelofibrosis

The impact of JAK2V617F mutation on different types of thrombosis risk in patients with essential thrombocythemia: a meta-analysis.

To assess the effect of JAK2V617F on different thrombotic risks in essential thrombocythemia (ET) patients, we identified eligible studies from several databases including Pubmed, Embase, and Cochrane Central Register of Controlled Trials (up to November 2014). Twenty-two studies of 2922 ET patients were included in exploring the relationship between JAK2V617F and the risk of thrombosis. Compared to JAK2V617F-negative ET patients, JAK2V617F-positive ET patients had higher odd risks (ORs) of arterial thrombosis [OR=2.59 (1.84-3.65)] and venous thrombosis [OR=2.10 (1.53-2.88)]. The JAK2V617F-positive group was also more prone to increased risk of microcirculatory disturbances [OR=1.50 (0.97-2.32)]. Moreover, JAK2V617F may indicate increased risk of either arterial [OR=1.71 (1.22-2.39)] or venous thrombosis [OR=2.90 (1.54-5.46)] before diagnosis of ET. During follow-up, JAK2V617F might not be related to arterial thrombosis [OR=1.90 (0.90-2.08)], but rather venous thrombosis [OR=1.95 (1.08-3.53)]. In conclusion, JAK2V617F increased the risk of arterial and venous thrombosis in ET patients, while understanding its role in microcirculatory disturbances will require further studies.

Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms

Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells.Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN.Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki.Results:CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001).The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05).Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004).Conclusion:CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients. DisclosuresNo relevant conflicts of interest to declare.

A 40-Microrna Signature Characterizes JAK2V617F-Negative Essential Thrombocythemia: Role Of Mir-221 and Mir-203 In The Regulation Of The JAK-STAT Pathway

BackgroundMicroRNAs (miRNAs) act as negative regulators of expression of genes that participate in cellular proliferation, apoptosis and/or carcinogenesis. MiRNAs have been shown to play a role in both solid and hematological tumors. Interestingly, several miRNA profiles have been differentially related to the mutational status of multiple tumors. A mutation in the JAK2 kinase (V617F) has been associated with essential thrombocythemia (ET) in around 40–50% of cases. However, the biological basis of ET in patients lacking the JAK2V617F mutation is still unknown. miRNAs may be crucial to the development of ET in these patients, yet their role in JAK2V617F-negative ET remains to be elucidated. AimTo characterize the expression pattern of miRNAs involved in JAK2V617F-negative ET and to identify the targets of these miRNAs. MethodsTotal RNA was extracted from isolated platelets from 19 ET patients (10 JAK2V617F-positive and 9 JAK2V617F-negative) and 10 healthy controls. The expression of 670 mature miRNAs was determined by TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT real time PCR system. miRNA expression data was analyzed by the 2-ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software v 2.13.To identify molecular pathways potentially altered by the expression of multiple miRNAs, we used Diana-mirPath, which performs an enrichment analysis of multiple miRNA target genes, comparing each set of miRNA targets to all known KEGG pathways. The mRNA expression of identified putative targets was analyzed by TaqMan gene expression assays (Applied Biosystems) and correlated with miRNA expression in order to select candidate genes for further target validation by Renilla-Luciferase assay and Western Blot. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in K562 together with 1μM of modified psicheck2 vector containing the 3'UTR region to be validated. Renilla luciferase levels were measured at 24h post-tranfection. The miRNAs identified in the Renilla-Luciferase assay were further confirmed by Western Blot. 100nM of pre-miR/pre-miR negative control were transfected in the HL60 cell line and protein levels were measured at 24h post-transfection. Anti-SOCS1 (ab119954) and Anti-SOCS3 (ab62584) primary antibody (abcam) were used. ResultsHierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic miRNA signature (p<0.0001). ET patients harbored a distinctive signature of 101 miRNAs, 99 of which were downregulated. Furthermore, a 40-miRNA signature was found to be differentially expressed between JAK2V617F-positive and JAK2V617F-negative ET patients. Diana-mirPath analysis showed that 8 of these 40 miRNAs had putative targets in the JAK-STAT pathway. The mRNA expression analysis of these genes showed an inverse correlation between the expression of three miRNAs and their target genes. An inverse correlation was found between SOCS1 and miR-221 (r2 = -0.719, p=0.001), SOCS3 and miR-221 (r2 = -0.644, p=0.005), SOCS3 and miR-203 (r2 = -0.447, p=0.072) and PTPN11 and miR-23a (r2 = -0.494, p=0.044). All three miRNAs were upregulated in JAK2V617F-negative ET patients in comparison with JAK2V617F-positive ET patients. The Renilla-Luciferase assay validated SOCS1 as a target of miR-221 (28.9% Renilla luciferase protein reduction, p=0.002) and SOCS3 as a target of miR-203 (19.6% Renilla luciferase protein reduction, p=0.04). No significant modifications were observed for PTPN11. SOCS1 and SOCS3 were further validated by Western Blot, with significant reduction of protein levels of SOCS1 (16%) and SOCS3 (19%) after increasing the levels of miR-221 and miR-203 respectivelly. ConclusionA 40-miRNA signature is differentially expressed in JAK2V617F-negative and JAK2V617F-positive ET patients. Two of these miRNAs, miR-221 and miR-203, target SOCS1 and SOCS3, which are involved in the JAK-STAT pathway. Further investigation is warranted to shed light on the role of these miRNAs in the development of JAK2V617F-negative ET. AcknowledgmentsThis study was supported in part by grants EC10-136, FISPI10/01807, RD09/0076/00036, RD12/0036/0010, 2009SGR929 and AECC Catalunya 2011 Disclosures:No relevant conflicts of interest to declare.

Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

AbstractPatients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca2+ mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F-positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin αIIbβ3 activation.

Genetic Predisposition to Molecular Response in Patients with Myeloproliferative Neoplasms Treated with Hydroxycarbamide

Abstract 1738 Introduction:Hydroxycarbamide (HC) is an antimetabolite used as first-line therapy in high risk essential thrombocythemia (ET) and polycythemia vera (PV). In treatment-naive patients, HC achieves complete hematological and partial molecular response in 80% and 50% of patients, respectively. Genetic factors involved in the pharmacokinetics of HC, as well as in the acquisition of the JAK2V617F mutation, could play a role in the variability among these patients in achieving a molecular response. Objective:To assess the influence of 46/1 JAK2 haplotype and urea transporters polymorphisms on the molecular response in JAK2V617F-positive PV and ET patients treated with HC. Methods:JAK2V617F allele burden was measured by quantitative PCR at diagnosis and every 6 months during follow-up in 53 PV patients and in 41 ET patients receiving HC therapy. Molecular response to HC treatment was defined according to European LeukemiaNet (ELN) criteria. SNPs rs12340895 and rs12343867 were used to determine 46/1 haplotype status and SNPs rs2298720 (SLC14A1), rs9960464 (SLC14A2), were used to genotype urea transporters. The rate of molecular response was compared according to 46/1 JAK2 haplotype status (negative, heterozygous, homozygous), and genotype of 2 urea transporters: SLC14A1 (GG, GA, AA) and SLC14A2 (GG, GA, AA). The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results:Overall, molecular response (CR or PR) was achieved in 48 patients (51%), corresponding to 46% and 57% in PV and ET patients, respectively (p=0.3). In ET patients, there were no significant differences in the proportion of patients achieving a molecular response according to 46/1 JAK2 haplotype or to the different transporter genotypes. PV patients with the GG genotype in transporter SLC14A2 obtained more frequently a molecular response than those with AA or GA genotype (71% versus 36%, p=0.03). No significant differences in molecular response were observed in PV patients according to transporter SLC14A1 and 46/1 JAK2 haplotype. Conclusion:46/1 JAK2 haplotype status did not influence molecular response in PV or ET patients treated with HC. Polymorphisms in urea transporters showed a minimal effect on the rate of molecular response in these patients. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiling Distinguishes Essential Thrombocythemia from Polycythemia Vera Patients and Identifies a Common Expressed Set of Genes in Relation to JAK2V617F Status

Introduction. The existence of the JAK2V617F mutation in a high proportion of Myeloproliferative Disorders (MPD) BCR-ABL-negative has provided important insight into the pathogenesis of these diseases. However, much of the molecular abnormalities associated to BCR-ABL-negative MPD remain unknown, specially in those which do not display JAK2V617F. In a previous study, we performed gene expression analysis by using the microarray technique in 20 essential thrombocythemia (ET) patients (44K whole human genome oligo microarrays, Agilent Technologies) and the results were confirmed in 40 ET patients by using TaqMan® Low Density Arrays Arrays (LDA, Applied Biosystems). In our previous experience the results showed different gene expression patterns in ET and a supervised clustering of the data identified genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients, and a characteristic gene expression profile for JAK2V617F-negative patients (Puigdecanet et al.,2008).Aim. Our aim was to confirm the ET gene expression profile in an extended group of patients and to explore the differences and similarities in polycythemia vera (PV) and reactive thrombocytosis (RT) patients by real-time quantitative RT-PCR (RQ-PCR) technique using the LDA platform. In addition, we wanted to analyze the relationship between gene expression data and JAK2V617F status.Patients and Methods. The following patients were included in the study: 58 ET (23 JAK2V617F-negative, 34 JAK2V617F in heterozygosity and one JAK2V617F in homozygosity) and 41 PV (7 JAK2V617F-negative, 25 JAK2V617F in heterozygosity and 9 JAK2V617F in homozygosity) patients, diagnosed according to the WHO criteria (2001) and who had never received cytoreductive treatment, and six patients with RT. Based on the previous results, we designed a new LDA platform containing 96 assays in duplicate, which included the most expressed genes in ET in relation to healthy controls and the most differentially expressed genes between JAK2V617F-negative and JAK2V617F-positive ET patients. The RQ-PCR analysis was performed in RNA from peripheral blood granulocytes and the relative gene expression quantification was achieved using GAPDH as the endogenous control and a pool of 10 healthy individuals as the calibrator.Results. ETvsPV: The majority of the genes studied presented significant higher expression in ET than in PV patients. Interestingly, FOSB was one of the most differentially expressed gene (FC= 8.3), and CISH and C13orf18 did not distinguish between the two groups.ET: We confirmed the differentially expression of the majority of the genes previously detected between JAK2V617F-negative and JAK2V617F-positive ET patients and we extended the set of genes. Among them, we highlight the differential expression of CISH, FOSB and C13orf18 genes (p<0.01).PV: Supervised analysis showed that CD44, BATF and CISH clearly distinguish JAK2V617F-negative PV patients from the JAK2V617F-positive.ET and PVvsRT: Some differentially expressed genes between ET and RT patients were detected, but the most significant gene was TNF (p<0.001), which presented a higher expression in RT (FC=5.9). The same difference was observed between PV and RT.Conclusions. We have detected a different gene expression pattern in ET and in PV patients. However, we also identified a set of genes which expression was related to JAK2V617F status, both in ET and PV patients. These findings would be interesting to identify other signal transduction pathways besides JAK-STAT involved in the pathogenesis of ET and PV.

The Relationship Between Wild Type and Mutant-Positive Platelets in Essential Thrombocythemia Patients with a JAK2 V617F Mutation

The myeloproliferative condition essential thrombocythemia (ET) is characterized by a persistent thrombocytosis in the absence of a recognizable cause. Approximately 50% of patients carry the acquired mutation JAK2 V617F, but it is unclear whether this is the initiating event in the overproduction of platelets or a secondary process arising in a situation where the thrombopoietic drive is already increased. In vitro studies have shown that mutant-positive erythroid cells have a proliferative advantage compared to wild-type (WT) cells. However, in JAK2 V617F-positive ET patients, the mutation is only found in a proportion of neutrophils, with the mutant level remaining stable over many years, and the JAK2 WT neutrophils are polyclonal by X-chromosome inactivation analysis. This may not be true of platelets as expansion of the mutant-positive cells may be restricted to the megakaryocytic lineage. We therefore quantified the mutant level in neutrophils and platelets purified from 10 JAK2 V617F-positive ET patients prior to the initiation of cytoreductive therapy using PCR with a fluorescently-labeled reverse primer and a mismatch forward primer that allowed discrimination between WT and JAK2 V617F alleles following AflIII digestion. There was no significant difference in the mutant levels determined using neutrophil DNA and RNA (median 15% [range, 11%–27%] and 21% [12%–31%] respectively). Mutant levels in platelet RNA were significantly higher than those in neutrophil RNA (median 27% [range, 20%–39%] versus 21% [12%–31%] respectively; P = 0.002), but still indicated that the JAK2 mutant was present in only a subpopulation of platelets. Assuming that all cells were heterozygous for the mutation, the data indicate that a median of only 54% (range 40%–78%) of the platelets were mutant-positive. We then calculated the absolute number of JAK2 WT and mutant-positive platelets for each patient from the quantified proportion of mutant alleles in platelet RNA and the total platelet count at the time of testing. The absolute number of JAK2 mutant-positive platelets in the patients varied between 263 and 798 × 109/L, and strongly correlated with the percentage of JAK2 mutant alleles (r2 = 0.81, P = 0.0004). The WT platelet count varied between 225 and 426 × 109/L, and there was a significant negative correlation between the absolute number of WT and mutant-positive platelets (r2 = 0.70, P = 0.002). However, when the absolute number of WT platelets was plotted against the total platelet count, there was no relationship between them (r2 = 0.003, P = 0.87). These data suggest that the negative feedback from the total platelet mass on normal (JAK2 WT) thrombopoiesis was incomplete; in no case was the WT platelet count below the lower limit of normal. This may relate to the observation that in ET, levels of thrombopoietin, the lineage-specific cytokine which drives platelet production, are often normal or even increased, unlike the situation in polycythemia vera where erythropoietin levels are reduced and normal red cell production is suppressed. Furthermore, extrapolation of the data from the WT platelet counts and JAK2 V617F mutant levels raises the possibility that when the mutation was first acquired and mutant levels were very low, the WT platelet count was at the upper limit of normality or elevated. This suggests that the JAK2 V617F mutation could have arisen on a background of increased thrombopoiesis, and was not the initiating event in the development of the disorder.

Major Hematological Response Is the Main Factor for Achieving a Major Molecular Response in JAK2V617F-Positive Essential Thrombocythemia and Polycythemia Vera Patients Treated with Hydroxyurea

Background. Hydroxyurea (HU) remains the cytoreductive drug of choice in high-risk patients with essential thrombocythemia (ET) and polycythemia vera (PV). There is scarce information about its effect upon the JAK2V617F allele burden in these patients.Aim. To analyze prospectively the JAK2V617F response to HU treatment in newly diagnosed patients with ET or PV and to investigate the clinical and/or biological variables associated to a good molecular response.Material and Methods. Sixty patients (25 PV and 35 ET) consecutively diagnosed in a single institution according to WHO criteria were included in the study. All PV patients were JAK2 V617F positive (14 homozygotes) whereas 21 (60%) ET patients were positive for the mutation (all in heterozigosity). Cytoreductive treatment was started according to standard guidelines with the mean HU daily dose being 894 ± 171 mg and 867±223 mg for PV and ET, respectively. Blood samples were taken immediately before starting treatment, at 3 months and then every 6 months. A mean number of 4.6 samples per patient were analyzed by quantitative real-time PCR assay after a median of 16 months (range: 3.5–57) of cytoreductive treatment. Determination of the relative change of the allelic ratio of JAK2V617F was calculated as follows: (% last V617F minus % first V617F) divided by % first V617F × 100. Major molecular response (MMR) was defined when pre-treatment V617F allele burden was reduced by > 50%. Major hematological response (MHR) was defined in PV when Hct <45 L/L (M), < 42 L/L (F), > 50% decrease in palpable splenomegaly, > 50% reduction in phlebotomies and normal platelet (< 400 × 109/L) and leukocyte (≤ 10 × 109/L) counts were achieved. MHR was defined in ET when normal platelet and leukocyte counts and > 50% reduction in palpable splenomegaly were obtained. The probability of major hematological and molecular response was calculated by the Kaplan-Meier method, followed by the log-rank test.Results. MHR was seen in 46 (77%) patients. Median time to MHR was 4 months with the probability of response at 6 and 12 months being 55% and 71% respectively in the whole series. MHR was faster and more frequently observed in ET patients than in PV (MHR probability at 6 months: 36% in PV and 69% in ET, p=0.009). Patients with a higher JAK2 allele burden showed a significantly lower probability of response (MHR at 6 months: 36% in patients with 50% of mutated JAK2 alleles versus 63% in patients with < 50% of mutated JAK2 alleles, p=0.01). MMR was observed in 15 out of 46 assessable patients (33%). Median time to MMR was 7.5 months (range 2–57) with the probability of response at 12 and 18 months being 21% and 30%, respectively, in the whole series. JAK2V617F mean decrease was −22.7±45.5% in PV and −31.8±39.1% in ET, respectively. Six out of 25 PV patients switched from homozygous to a heterozygous status. The probability of obtaining a MMR was significantly higher in patients achieving a MHR than in those who did not obtained a MHR (MMR at 18 months: 41% versus 0%, p=0.02). Age, gender, spleen size, leukocyte count, platelet count, serum LDH level and JAK2V617F allele burden prior to cytoreductive treatment were not associated with the likelihood of MMR. ET patients with an Htc 0.45 L/L at diagnosis obtained a MMR more frequently than those patients with Htc < 0.45 L/L (MMR probability at 18 months: 57% versus 16%, p=0.003).Conclusions. Major hematological response is the main factor predictive for attaining a major molecular response in JAK2V617F-positive PV and ET patients treated with HU.

Recurrent der(9;18) in essential thrombocythemia with JAK2 V617F is highly linked to myelofibrosis development

We studied JAK2 mutational status, in combination with cytogenetic analysis in 54 patients with essential thrombocythemia (ET), and attempted to obtain greater insight into the correlation between clinicohematologic features and genetic abnormalities. We found that six ET patients developed myelofibrosis and four of them had JAK2 V617F mutation. It is noteworthy that three of the four ET patients with JAK2 V617F had add(18)(p11). In contrast, the remaining two ET patients who developed myelofibrosis had neither JAK2 V617F nor add(18)(p11). Moreover, none of the ET patients with JAK2 V617F and chromosome changes other than add(18)(p11) developed myelofibrosis. The current results indicate that add(18)(p11), possibly due to der(9;18), may contribute a link to myelofibrosis in JAK2 V617F–positive ET patients, while those with wild-type JAK2 may use another pathway toward myelofibrosis.

Gene expression profiling distinguishes JAK2V617F-negative from JAK2V617F-positive patients in essential thrombocythemia

To explore the gene expression signature in essential thrombocythemia (ET) patients in relation to JAK2V617F mutational status, expression profiling in circulating granulocytes was performed. Twenty ET were studied by microarray analysis and the results were confirmed by real-time quantitative RT-PCR in 40 ET patients, not receiving cytoreductive treatment. A heterogeneous molecular signature characterized by two main gene expression patterns was found: one with an upregulation of inflammatory genes related to neutrophil activation and thrombosis, and the other with significantly lower expression of these genes. Supervised clustering analysis showed 30 genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients. Among the JAK2V617F-negative, a set of 14 genes (CISH, C13orf18, CCL3, PIM1, MAFF, SOCS3, ID2, GADD45B, KLF5, TNF, LAMB3, HRH4, TAGAP and TRIB1) showed an abnormal expression pattern. In this group of patients, CISH, SOCS2, SOCS3 and PIM1 genes, all involved in JAK-STAT signalling pathway, presented a lower expression. A two-gene predictor model was built comprising FOSB and CISH genes, which were the best discriminators of JAK2V617F status. In conclusion, JAK2V617F-negative ET patients present a characteristic gene expression profile, different from JAK2V617F-positive patients. Other pathways, besides JAK-STAT, might be implicated in the pathophysiology of JAK2V617F-negative ET patients.

Frequency of the JAK2V617F Mutation in Platelets from Essential Thrombocythemia (ET) Patients.

The JAK2V617F mutation has been recently reported in patients with polycythemia vera (PV) and a proportion of patients with ET and myelofibrosis with myeloid metaplasia. This acquired point mutation constitutively activates JAK2 tyrosine kinase and is believed to underlie growth factor hypersensitivity displayed by hematopoietic progenitors in these disorders. Identification of this genetic abnormality may be useful for diagnostic purposes although its prognostic implication remains to be determined. The prevalence of this mutation in granulocytes from ET patients ranges from 23 to 57% depending on the detection technique used. ET is characterized by predominant involvement of the megakaryocytic lineage and increased platelet production. In order to evaluate whether the frequency of the V617FJAK2 mutation in platelets from ET patients differs from that previously reported in granulocytes, we amplified a fragment of JAK2 cDNA from leukocyte-depleted platelets by RT-PCR followed by digestion with BsaXI restriction endonuclease (PCR-RFLP). The JAK2V617F mutation abolishes a BsaXI restriction site present in the wild-type sequence and generates a different band pattern. In addition, allele-specific RT-PCR (ASPCR) was performed with a primer pair common to both wild-type and mutant alleles and a third primer complementary to the mutant allele. Forty four patients with ET diagnosed according to the PVSG criteria were included, median age 39 years old (14–81), 10 were men; 21 were studied before treatment, 10 during treatment with anagrelide or hydroxyurea and 13 both before and during treatment. Twenty-two (50%) patients were found to harbour the JAK2V617F mutation by both PCR-RFLP and ASPCR, 3 were homozygous. No change in genotype was found in repeated samples from patients studied both before and during treatment. Hemoblobin levels and the frequency of thrombosis were significantly higher in patients with the mutation respect to those without (141 ±14 g/L vs. 129 ± 11 g/L, p=0.009, and 41% vs. 4.5%, p=0.004, respectively). In addition, patients carrying the mutation were significantly older (51 vs. 33 years old, p=0.002) and had lower platelet counts at diagnosis (1.070 x 109/L vs. 1.500 x 109/L, p=0.02). Interestingly, 2 patients harbouring the mutation developed PV on follow-up. In conclusion, the prevalence of the JAK2V617F mutation in platelets from ET patients did not differ from that previously reported in granulocytes. The increase in the frequency of thrombosis found in patients carrying the mutation should be confirmed in a larger study. Besides, the finding of higher hemoglobin levels in this group of patients is of interest since JAK2V617F-positive ET patients have been reported to display PV markers such as endogenous erythroid colony growth and PRV-1 overexpression, suggesting they may have larger involvement of the erythroid lineage. Further studies will help clarify whether the risk of developing PV is higher in this subset of patients.