JAK2V617F Mutational Status Research Articles

Correlation of Somatic Mutations and Clinical Characteristics at Presentation in Patients with Essential Thrombocytemia

BackgroundMost patients with essential thromocythemia (ET) have somatic mutations in janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL) genes. The clinical correlates at the presentation of patients with different mutations has not been established as yet.Materials and methodsWe evaluated the clinical, laboratory, and molecular features of 150 consecutive patients (93 females/57 males; average age 59,0+/- 14,2 years) with ET who were diagnosed at the University Clinic for Hematology in Skopje based on the revised WHO criteria. DNA from all patients was isolated from peripheral blood obtained at diagnosis using Qiagene DNA exctraction kit. The presence of JAK2V617F mutation was evaluated with fluorescent allele specific PCR/capillary electrophoresis. The CALR exon 9 insertions or deletions were initially detected with a fluorescent PCR/capillary electrophoresis and characterized with Sanger sequencing. The presence of mutations in exon 10 of the MPL gene was analyzed with direct sequencing of PCR amplified fragments. Informant consent was obtained from all participants. Statistical analysis was performed by Wilcoxon rank- sum test.ResultsThe basic hematological parameters at diagnosis for all patients were as follows; Plt = 962.1x109 ±351,2 x109/L; Hb = 126,8 ±39,2 g/L; Le = 10,2x103 ±4,2 x103/µL. No correlation was found between any of these parameters and age at diagnosis or sex of the patients. JAK2V617F, CALR and MPL mutations were found in 89 (59.3%), 42 (28%) and 2 (1.3%) patients, respectively, while 17 (11.4%) patients did not have a mutation in the 3 evaluated genes (triple negative). Twelve different mutations were found in CALR gene, the most common being 52-bp deletion (c.1092_1143del) and 5-bp insertions (c.1154_1155insTTGTC) which were present in 11 (26,2%) and 20 (47,6%) patients, respectively. Ten different mutations, including 6 insertions (c.1088insTTGTC; c.1088delinsTTTGTC; c.1113_1123delinsTTGT; c.1154delinsTATGTC; c.1126_1131delinsTGCGT; c.1154_1155insGTGTC) and 4 deletions (c.1092_1137del; c.1096_1129del; c.1089_1141del; c.1100_1145del), were detected in the other 11 patients with CALR mutation. The JAK2V617F mutation and triple negative status were more prevalent in female patients (67% and 68% respectively), while no sex preference was detected in patients with the CALR mutation. The average age of patients with JAK2V617F, CALR insertions, CALR deletions and triple negative status were 60,7±13,9, 54,5±12,9, 59,0±15,1 and 58,8+/-14,8 years, respectively. Patients with CALR mutation had a higher platelet count at the time of diagnosis (1134x109 ±372,9 x 109 /L) compared to both patients with mutated JAK2 (952 x109 ±335,6 x109/L) (P=0.047) and to triple negative patients (mean 886 x109/L±303,2 x109/L) (P=0.024). Patients who carried CALR insertions had higher platelet counts (1222,9 ±396,8 x109/L) than patients with CALR deletions (mean 956,8 ±287,8 x109/L ) (P=0.0405). White blood cells counts were the highest in patients with JAK2V617F mutation (mean 11.1±4,5x103/µL) compared to both CALR positive (mean 8,8±2,7x103/µL) and triple negative patients (mean 7,9±2,2x103/µL) (P=0.0049). No difference in Hb levels were present between patients with various mutations.ConclussionET patients with CALR insertions present with higher platelet counts and at an earlier age than patients with JAK2V617F, CALR deletions or triple negative patients, which might translate to differences in prognosis in these groups of patients. Higher incidence of JAK2V617F mutation and triple negative status in female patients point to a common mechanism for predisposition to these conditions. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value of JAK2V617F, Calr and MPL Mutational Status on Outcome and Thrombotic Risk in a Retrospective Cohort of 138 Patients with Essential Thrombocythemia

Background: Life expectancy in Essential Thrombocythemia (ET) patients is superimposed to normal population. The main causes of death are thrombotic events and evolution into myelofibrotic phase or secondary myelodisplasia/acute leukemia. Approximately 50% to 65% of patients with ET carry activating mutations in the Janus kinase 2 gene (JAK2), and an additional 5% in the thrombopoietin receptor gene (MPL) whereas 10 to 20% of patients have mutated calreticulin gene (CALR). Non-mutated JAK2, CALR and MPL ET (triple negative-TN) are about 10%.Patients and methods : In this study we analysed the prognostic value of JAK2V617F, CALR and MPL mutational status on outcome and thrombotic risk in a retrospective cohort of 138 ET patients defined according to WHO criteria, diagnosed from 1974 to 2013 in a single Italian centre (Turin). JAK2V617F mutation was assessed by Quantitative Real–Time PCR on DNA from peripheral blood or bone marrow samples. JAK2 negative cases were then analyzed for CALR mutations by Gene Scan Analysis in combination with direct sequencing or MPL W515L/K by Allelic Discrimination Real-Time PCR assay. Overall survival (OS), cumulative incidence of myelofibrotic transformations (CI-MT) and thrombosis (CI-TB) were calculated from the date of diagnosis. The between-group comparison for OS was performed with the log-rank test, whereas for CI-MT and CI-TB we using the Gray’s test considering death as competing event and adjusting for presence of cardiovascular risk factors .Results: Among 138 ET patients, 103 (74.6%) carried the JAK2V617F mutation, 3 (2.2%) carried activating mutations of MPL exon 10, 16 (11.6%) carried mutations of CALR exon 9, and only 16 patients (11.6%) had none of these markers (TN).An high incidence of elevated haemoglobin levels and/or haematocrit (males >16.5 g/dl or 49% and females >16.0 g/dl or 48%) was significantly associated with JAK2 positivity [13 pts JAK2+ (14.77%) vs 0 pts JAK2- (Fisher test p=0.019)]. Similarly, the CI-TB, analysed with a competing-risk approach, was higher in patients with a JAK2 mutation [(5-year CI-TB: 23.7% JAK2, 0% CALR, 0% MPL, 12.5% TN; P<0.001)].With a median follow-up of 48 months (IQR: 27-78; range 1-269), we observed 4 (3%) deaths, 9 (7%) myelofibrotic transformations and none leukemic evolution.The overall survival at 5 years was 98.4% (95%CI: 89.1-99.8) and the cumulative incidence of myelofibrotic transformations was 2.1% (95%IC: 1.0-8.6). The CI-MT was significantly higher in MPL-positive patients [5-year CI-MT: 0% JAK2, 0% CALR, 33.3% MPL, 8.3% TN; (P=0.006)]. Not significant difference on OS was found [5-year OS: 100% JAK2, 100% CALR, 100% MPL, 85.7% TN; (P=0.66)]. We did not include leukemia transformation, because of lack of event.Conclusion: In our retrospective cohort of ET patients, we found a significant correlation between JAK2 positivity and high risk of thrombosis. We also found a significant incidence of elevated haemoglobin levels and/or haematocrit in JAK2-positive patients. These data support the correlation between JAK2 positivity and cumulative risk of transformation to Polycythemia Vera. The correlation between MPL mutational status and evolution to myelofibrosis found in our study needs to be confirmed in a larger series of patients. DisclosuresNo relevant conflicts of interest to declare.

Elevated mRNA Expression of FGF2 and TGFB1 Genes in Myelofibrosis and Essential Thrombocythemia Patients

Background: Myelofibrosis (MF) and essential thrombocythemia (ET) are clonal hematopoietic stem cell malignancies classified as myeloproliferative neoplasms (MPNs). FGF2 and TGF-β were associated with pathophysiology and progression of these diseases. Although FGF2 and TGF-β roles in angiogenesis and fibrosis are known, their mRNA expression in leukocytes of MPNs patients was not yet evaluated. Furthermore, the detailed mechanism of TGF-β signaling in the pathophysiology of these diseases remains unclear. In regular signaling process, Smads are a group of proteins critical for transmitting signals from TGF-β superfamily of the cell surface to the nucleus. Thus, Smads signaling plays an important role in the regulation of hematopoiesis and it could modulate TGF-β action in MPNs.Aims: The aim of this study was to investigate if there are differences in mRNA expression of FGF2, TGFB1 and SMADS 1 to 7 in MPNs patients and healthy controls and if it is related to JAK2 mutation status and allele burden.Methods: Fifteen primary myelofibrosis (PMF), 15 myelofibrosis post essential thrombocytemia (MPET), and 22 essential trombocythemia (ET) patients diagnosed according to the World Health Organization criteria (2008) were selected from the Hematology Department of the Federal University of de Sao Paulo and from the Hematology Division of the Catholic University of Sao Paulo. Other group with healthy subjects was also included. The control group was matched by age and gender with MPNs patients: 30 for PMF, 17 for MPET, and 34 for ET. Expressions of FGF2, TGFB1 and SMADs 1 to 7 mRNA were evaluated in total leukocytes by Real Time PCR, using Taqman assays. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by Real Time PCR, using Taqman MGB probes.Results: No difference was found between frequencies of JAK2V617F mutated subjects (P=0.520) and in allele burden percentage (P=0.415) in MPNs groups. Allele burden was also not associated with mRNA levels of studied genes in each group of MPNs, and none of the patients presented MPL W515K/L mutations. MF and ET patients had higher TGFB1 and FGF2 mRNA levels than its controls (P<0.05), while MPET patients had higher FGF2 levels than controls (P< 0.001). Both MPET and ET patients had higher SMAD6 expression than its controls (P<0.05), whereas ET patients also presented higher SMAD1 levels than controls (P=0.01). The SMADs expressions were similar in MF and their controls.Conclusions: Our findings suggest that FGF mRNA is overexpressed in leukocytes of MF, MPET and ET patients. It seems that higher TGFB1 mRNA levels do not affect the molecular signaling by SMADs in MF patients, although SMAD6 and SMAD1 mRNA levels in ET and MPET patients might be upregulated.Financing: FAPESP 2012/ 12957-5 DisclosuresNo relevant conflicts of interest to declare.

Calreticulin Mutation Is Associated with Milder Disease in Patients with Post Essential Thrombocythemia Myelofibrosis (PET-MF) Compared with JAK2V617F Mutation: A Study from the AGIMM Group

Background: Mutations in the gene calreticulin (CALR) were recently discovered in 60-80% of patients (pts) with primary myelofibrosis (PMF) and essential thrombocythemia (ET) who were un-mutated for JAK2V617F and MPLW515. CALR mutated PMF pts had better overall survival (OS) compared with JAK2V617F or MPLW515 mutated while in ET CALR mutations were associated with lower incidence of thrombosis although the effect on survival was not significant. Conversely, there is no information concerning the impact of CALRmutation on disease phenotype and prognosis in post-essential thrombocythemia myelofibrosis (PET-MF).Aims: The aim of the study was to assess whether CALR mutational status and/or allele burden had clinical and/or prognostic relevance in PET-MF compared with JAK2, MPLmutated or triple-negative (TN) pts.Methods: ET and PET-MF were diagnosed by 2008 WHO and IWG-MRT criteria respectively; all pts provided an informed consent. Genotyping for CALR, JAK2V617F and MPLW515 was performed in granulocytes using allele specific RTQ-PCR (JAK2, MPL), capillary electrophoresis and direct sequencing (CALR, MPL). The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression.Results: A series of 147 PET-MF pts from 4 Italian centres was collected. Pts median age was 63y. Median follow up from PET diagnosis was 3.2y (0.07-18.8y) and the median time from ET to PET diagnosis was 11.6y (0.9-30.6y). Death occurred in 38 pts (26%) and 14 pts (9.5%) developed acute leukemia (AML). The median OS in the entire series calculated from PET-MF diagnosis was 10.9y (7.1-14.7y). Frequency of mutations was: CALR 16%, JAK2V617F 77%; MPLW515 4.3%; TN 2.8%. The frequency of CALR mutations in PET-MF patients was superimposable to that observed in a control group of 576 ET patients from our Institution (15.5%) and slightly lower compared with other series (20-25%). Type of CALRmutations was: 59.6% type 1, 23.1%, type 2, 17.3% others, significantly different (P=0.023) from ET: 46% type 1, 38% type 2, 16% others. Median CALR allele burden in PET-MF was 56% (20%-100%) with no significant differences in the CALR mutation subtypes (57.5% in type 1, 47.5% in type 2 and 45.0% in others); however, the median mutant allele burden of CALR-mutated PET-MF patients was significantly higher than in ET patients (33%, range 2%-52%; n=100) (P<0.03) suggesting a role for mutated allele accumulation in evolution to PET-MF. Similarly, the median V617F allele burden in JAK2 mutated patients was 50.5% (range 5-100%) significantly greater than the value (24%; range, 1-87%) (P=0.02) in ET pts, confirming previous data that evolution to PET-MF is associated with accumulation of mutated JAK2allele. We then compared hematological and clinical characteristics of the patients who were categorized according to their JAK2V617F, MPLW515 and CALR mutation status. There was no statistically significant difference among the unique patient mutational groups regarding age, hemoglobin, leukocyte and platelet count, peripheral blasts, LDH, circulating CD34+ cells, abnormal karyotype, grade of bone marrow fibrosis and cellularity, and pruritus. However, JAK2+ pts showed an increased rate of large (>10 cm) splenomegaly (28.6% vs 14% in CALR+, 7.1 in MPL+ and 25% in TN pts; P=0.02) and constitutional symptoms (50% vs 18.8% in CALR+, 45% in MPL+ and 12.5% in TN pts; P=0.002). The interval from ET to PET-MF was significantly longer in CALR+ pts (14.5y) compared with JAK2+ (10.2y) and TN patients (11.0y; P=0.04 for both) and similar to MPL+ (14y). There was a reduced rate of death (13.5%) in CALR+ compared with JAK2+ (30.6%), MPL+ (21.4%) and TN (66.7%) pts (P=0.005), although Kaplan Meier estimates did not reach a statistically significant difference. Finally, there were less AML transformation in CALR+ pts (1.9%) compared with JAK2+ (13.9%), MPL+(7.1%) and TN (22.2%) (P=0.04).Conclusion: These results show that CALR mutation is associated with delayed transformation of ET to PET-MF, a milder disease in terms of splenomegaly and symptom burden and a reduced risk of death compared with JAK2V617F PET-MF pts and more in general with MPL mutated and TN pts.In addition, similar to findings in primary MF and unlike in ET, PET-MF is characterized by prevalence of type 1 CALR mutations. DisclosuresNo relevant conflicts of interest to declare.

GATA1 Is Overexpressed in Patients with Essential Thrombocythemia and It Is Normalized By Anagrelide Treatment

GATA1 is the founding member of the GATA transcription factor family and is essential for cell maturation and differentiation within the erythroid and megakaryocytic lineages. GATA1 is a pleotropic transcription factor, whose expression is essential in these lineages; its function depends upon its ability to bind to both DNA and protein partners. Disturbance of either of these functions causes severe hematopoietic dysfunction and can result in blood disorders, such as thrombocytopenia, anaemia and even leukaemia. Ectopic expression of GATA1 in the murine myeloid cell line M1 induces c-Mpl appearance and megakaryocyte (MK) differentiation. The same phenomenon has been showed to occur in hematopoietic stem cells; in that forced increased expression of GATA1 blocked self-renewal and induced the exclusive generation of MegE lineages. Several studies, have suggested a connection between GATA1 and myeloproliferative neoplasia (MPN). We previously reported that high GATA1 transcript levels are found in the bone marrow of patients with ET and PV, independent of JAK2V617F or CALR mutation status. However, over expression of GATA1 is not seen in other MPNs.Anagrelide (ANA) has been proven to be an effective drug in reducing platelet count and thrombotic risk in management of ET and PMF patients. However, the mechanism by which this drug induces this effect is still unclear. Recently Erusalimsky and colleges have reported that ANA results in down-modulation of GATA1 and its co-factor FOG1 in MK during in vitro differentiation.In this study, we analysed the expression of GATA1 in peripheral blood (PB) samples from 38 patients with ET and compared the levels of expression before and after treatment with common cytoreductive agents such as hydroxyurea (HU) and ANA.We confirmed the data obtained in BM, with a significant up-regulation of GATA1 in ET compared to controls. When we measured the expression of GATA1 before and during treatment with ANA, there was a significant reduction of the GATA1 expression at 3 and 6 months of therapy, concomitant with a reduction in platelets (PLT) counts. Interestingly, this was not equally observed in patients treated with HU, where despite reduction in PLT counts, the GATA1 levels rose. Furthermore, when we analysed patients on combination treatment ANA + HU, GATA1 expression reduced only when patients were taking ANA. When ANA treatment was stopped and the patient continued only on HU, GATA1 levels rose again. This data suggests a direct effect of ANA on GATA1 expression.Although, no direct correlation between GATA1 expression and CALR mutations was found. However, a significant up regulation of CALR mRNA was observed independently from the CALR mutational status.GATA1 may represent a generic molecular marker in ET and a possible additional diagnostic criteria in thrombocytosis. GATA1 overexpression is independent from JAK2 mutations, and responds specifically to ANA therapy suggesting a role in monitoring therapy response. DisclosuresNo relevant conflicts of interest to declare.

Determination of Driver Mutations in JAK2, MPL, and Calreticulin in Coronary Patients with Suspected Essential Thrombocythemia

Patients with essential thrombocythemia (ET) are at increased risk towards atherothrombotic complications. The JAK2 V617F mutation is found in at least half of the patients with ET and screening for JAK2 V617F mutational status has been included in the World Health Organization diagnostic criteria for ET. Further somatic driver mutations including mutations in exon 12 of the JAK2 gene, MPL mutations W515L/W515K, as well as mutations in exon 9 of the calreticulin (CALR) gene have been suggested as diagnostic markers for ET. Due to the close relation of ET to thrombotic events, early diagnosis of ET is of high clinical relevance, especially in patients with increased cardiovascular risk, like coronary patients. However, elevated blood cell counts are often ascribed to a reactive genesis within an acute coronary event. For this reason mostly no further clarification concerning myeloproliferative neoplasms is done.In the present study, therefore, we determined the prevalence of JAK2 V617F, JAK2 exon 12, MPL W515L, MPL W515K, and CALR exon 9 mutations in a cohort of patients undergoing coronary angiography for the evaluation of suspected or established stable coronary artery disease and suspected diagnosis of ET. We aimed at identifying those with occult ET proposing need for additional treatment.From a total of 1,589 coronary patients, 12 patients had increased platelets (>450 x10 9/L) predisposing them to ET. Mutation analysis among these patients showed three individuals with the JAK2 V617F mutation and one with an ins/del mutation in exon 9 of the CALR gene confirming suspected diagnosis of ET. Consequently, frequency of ET was 0.25% among our coronary patients. For comparison, in the United States between 38 and 57 ET cases per 100,000 age-adjusted inhabitants have been reported. This corresponds to an up to 7-fold accumulation of ET cases in coronary patients compared to the general population.We conclude that the prevalence of ET is increased in coronary patients compared to the general population. For this reason comprehensive mutation analysis should be considered in all cardiovascular risk patients with persistent elevation of thrombocytes, not only to identify patient subgroups at high risk but also to individualize therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

T Cell Mediated Suppression of Hematopoiesis in Myeloproliferative Neoplasm

Background:Increased inflammation is an established feature of MPN, but its precise role in disease pathogenesis has yet to be clearly defined. T cell mediated insult on hematopoietic stem cells can drive expansion of clones that have mutated in such a way as to escape these suppressive and/or apoptotic cues (this mechanism is clearly illustrated in aplastic anemia). To investigate whether T cell mediated suppression of hematopoiesis occurs in MPN we interrogated the T lymphocyte compartment of MPN patients, guided by abnormalities known to be present in aplastic anemia.Methods and Results:We first asked how selective depletion of T cells from peripheral blood mononuclear cells (MNC) would affect myeloid colony formation in MPN as compared to normal controls. MNC were depleted of T cells using CD3 magnetic beads or mock depleted and plated side by side in methylcellulose (+SCF, IL-3, Epo). Removal of CD3+ T cells from bulk MNC reduced colony formation in all 29 normal control samples tested, consistent with the ability of normal lymphocytes to enhance colony formation. In contrast, removal of CD3+ T cells from MPN MNC resulted in an increase in colony formation in 20 out of 51 MPN patient samples (containing an equal distribution of polycythemia vera, essential thrombocythemia, and myelofibrosis). From JAK2V617F-positive MPN patients, colonies were plucked and genotyped for JAK2V617F mutational status. The fraction of JAK2WT colonies increased with removal of T cells, demonstrating that JAK2WT progenitors are more sensitive to T cell mediated suppression as compared to their JAK2V617F counterparts. The observation that removal of T cells enhances myeloid colony forming ability of some MPN patients suggests that T mediated suppression of hematopoiesis may be occurring in MPN.Next, we tested whether MPN T cells have the ability to suppress colony formation of normal hematopoietic progenitors. CD3+ T cells from MPN (n=2) and normal controls (n=2) were added to normal control mobilized peripheral blood CD34+ cells (n=3). Each CD34+ sample was plated in methylcellulose by itself, with the addition of 100,000 MPN T cells/1.1ml or with the addition of 100,000 normal control T cells/1.1ml. For each CD34+ sample a unique MPN patient and normal control was used as the source of T cells. Addition of MPN T cells reduced colony formation in one of three CD34+ samples, whereas normal control T cells enhanced colony formation of all three CD34+ samples. In the remaining two CD34+ samples MPN T cells had an attenuated ability to enhance colony formation as compared to the normal control T cells.Conclusion:T-cell mediated suppression of myelopoiesis in vitro is demonstrable in MPN and may be a contributor to the anemia associated with myelofibrosis. Taken together, these data provide the rationale for further investigation into T mediated suppression of hematopoietic progenitors in MPN patients. DisclosuresNo relevant conflicts of interest to declare.

Primary Analysis Results from an Open-Label Phase II Study of INCB039110, a Selective JAK1 Inhibitor, in Patients with Myelofibrosis

Primary Analysis Results from an Open-Label Phase II Study of INCB039110, a Selective JAK1 Inhibitor, in Patients with Myelofibrosis

The JAK2 V617F mutational status and allele burden may be related with the risk of venous thromboembolic events in patients with Philadelphia-negative myeloproliferative neoplasms

IntroductionPatients with Philadelphia-negative myeloproliferative neoplasms (Ph- MPNs) are at increased risk of thromboembolic and hemorrhagic complications. The aim of the study was to determine the relationship between JAK2 V617F mutational status, JAK2 V617F allele burden and the risk of vascular complications occurrence. Materials and methodsAnalysis was performed in a cohort of 186 patients diagnosed with polycythemia vera (53), essential thrombocythemia (114), primary myelofibrosis (11), and unclassified MPN (8). The risk of vascular complications development was analyzed in 126 JAK2 V617F-positive patients with respect to allele burden assessed with allele-specific 'real-time' quantitative polymerase chain reaction (AS RQ-PCR). ResultsOverall prevalence of any vascular complications was 44.6%. Arterial thrombosis occurred in 20.4%, venous thromboembolism (VTE) in 11.3%, bleeding episodes in 24.7% of patients. Individuals harboring JAK2 V617F mutation, regardless of MPN type, were at higher risk of VTE (OR=5.15, 95%CI: 1.16-22.90, P=0.024), mainly deep vein thrombosis (DVT). JAK2 allele burden higher than 20% identified patients with 7.4-fold increased risk of VTE (95%CI: 1.6-33.7, P=0.004), but not of arterial thrombosis, neither of bleeding complications, and remained the only significant VTE risk factor in multivariate logistic regression. High allele burdens (over 50%) were strikingly associated with proximal DVT cases, but not with distal DVT. ConclusionsThe group of MPN patients with JAK2 V617F allele burden higher than 20% may benefit the most from vigilant monitoring and appropriate prophylaxis against vascular events. Inclusion of JAK2 V617F mutant allele burden in new risk stratifications seems to be justified and requires controlled prospective trials.

The Role of JAK2 Mutation in Thrombotic Complications of Chronic Myeloproliferative Neoplasms

Patients diagnosed with myeloproliferative neoplasms (MPNs) often develop thrombotic events as an onset of symptoms or in evolution. The pathogenesis of thrombosis in patients with MPN is multifactorial. There are multiple prognostic score systems, but the presence of JAK2V617F (JAK2) mutation is an independent and strong thrombosis risk factor. Patients with MPN and JAK mutational status usually associate thrombocytosis, increased immature circulating platelets, and leukocytosis, with increased expression of CD62P and CD14, increased levels of circulating microparticles and leuko-platelet microaggregates, and altered endothelial function. This review aims to discuss different factors contributing to the increased thrombotic risk in association with JAK2 mutational status. Also, recent reports incriminate this mutation to have a possible role in spontaneous loss of pregnancy.

JAK2V617F mutation status and allele burden in classical Ph-negative myeloproliferative neoplasms in Japan

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, is frequently detected in classical myeloproliferative neoplasms (MPNs). In the present study, we determined the JAK2V617F allele burden in Japanese MPN patients using alternately binding probe competitive-polymerase chain reaction, a highly quantitative method recently developed by our group. Although we observed strong similarities in terms of epidemiological parameters associated with the JAK2V617F allele burden between our cohort and others, we found a higher JAK2V617F allele burden in Japanese polycythemia vera (PV) patients and lower frequencies of thrombosis in Japanese MPN patients compared with previous reports. In addition, despite the presence of high red blood cell counts, some patients bearing the JAK2V617F mutation were not diagnosed as PV, as their hemoglobin values were lower than the WHO PV criterion. In these patients, the JAK2V617F allele burden was strikingly similar to that in PV patients fulfilling the 2008 WHO criteria, suggesting that these patients can be classified as PV. Although isotopic measurement of red cell mass (RCM) is required for definitive diagnosis of PV, our data suggest that precise measurement of the JAK2V617F allele burden may improve the diagnosis of PV when RCM has not been determined.

IRS2 Associates With JAK2 and May Be Involved In Cell Proliferation Pathways In Chronic Myeloproliferative Neoplasms

BackgroundPolycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are BCR-ABL1 negative Chronic Myeloproliferative Neoplasms (MPN) characterized by increased myeloid proliferation, with predominant erythroid, megakaryocytic and megakaryocytic/granulocytic expansion, respectively. The finding of a recurrent mutation in the gene of the tyrosine-kinase Janus kinase 2 (JAK2 V617F) in these diseases has raised the hypothesis that this could be the main cause of their development. However, the evidence that MPN patients have a very similar response to JAK2 inhibitors regardless of JAK2 mutation status, and the knowledge that many receptors and substrates may lead to the activation of JAK/STAT, Ras/Raf/MAP kinases and PI3K/Akt/mTOR pathways, indicate the need to investigate other crucial proteins involved in the physiopathology of these diseases. Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF-IR, EPO-R and TPO-R. Previous studies performed on non-hematological cell lines have shown the association of IRS2 with JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAP kinases pathways, giving rise to the hypothesis that IRS2 could participate in the activation of crucial signaling pathways in MPN through direct interaction with JAK2 or through alternative mechanisms. AimsTo identify the JAK2/IRS2 protein interaction and to study the effects of pharmacological JAK1/2 inhibition (Ruxolitinib) over IRS2 phosphorylation in leukemia cell lines harboring or not the JAK2 V617F mutation; to characterize IRS2 expression in CD34+ cells from patients with MPN and its correlation with clinical data including JAK2 mutation status. MethodsLeukemia cell lines carrying JAK2 V617F mutation (HEL) or not (HL60) were used for immunoprecipitation and immunobloting with IRS2 and JAK2 antibodies. Cells treated or not with JAK1/2 inhibitor Ruxolitinib were also submitted to immunoprecipitation and immunobloting with IRS2 and anti-phosphotyrosine antibodies. Peripheral blood mononuclear cells from 28 healthy donors and 97 patients with MPN (PV=28, ET=38, PMF=31) were included, and CD34+ cells were submitted to quantitative PCR (q-PCR). Relative expression of IRS2 was correlated with clinical data and with JAK2 V617F mutation status. ResultsImmunoprecipitation analysis showed that IRS2 associates with JAK2 in leukemia cell lines harboring (HEL) or not (HL60) the JAK2 V617F mutation. Furthermore, treatment of HEL cell line with the JAK1/2 selective inhibitor Ruxolitinib resulted in decreased IRS2 tyrosine phosphorylation. IRS2 mRNA expression in CD34+ cells were significantly higher in patients with ET when compared to healthy donors (1.70 [0.42-10.60] versus 0.87 [0.01-11.22], p=0.03). There was no difference in IRS2 mRNA expression in PV or PMF patients when compared to healthy donors. Furthermore, significantly higher levels of IRS2 mRNA expression were observed in patients harboring JAK2 V617F mutation when compared to the wild type JAK2 for ET (2.37 [0.96-10.60], n=14 versus 1.54 [0.42-1.54], n=22; p=0.01); and for PMF (2.27 [0.003-10.59], n=20 versus 0.60 [0.02-2.42], n=11; p=0.02). Although there was also a significant difference in IRS2 mRNA expression in mutated versus non mutated JAK2 in PV (p=0.02), the number of non mutated samples was low (n=2). ConclusionsOur data indicate that IRS2 is a binding partner of JAK2 in myeloproliferative neoplasms and suggest that this protein association may be involved in cell proliferation in these diseases. The higher IRS2 expression in mutated samples (JAK2 V617F) might be associated with the constitutive activation of JAK2 in these samples. Disclosures:No relevant conflicts of interest to declare.

Splanchnic Vein Thrombosis Associated With Myeloproliferative Neoplasms. A Study Of The IWG-MRT In 475 Subjects

▪Philadelphia-negative Myeloproliferative Neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis Primary (PMF) and secondary to PV and ET (PPV-, PET-MF); included are also some less characterized entities defined as unclassified MPN (U-MPN). Risk of arterial and venous thrombosis is increased in MPN patients, and thrombosis is one of most important causes of mortality and morbidity. The risk of venous thrombosis in unusual sites, such as splanchnic vessels (SVT), is particularly associated with MPN; SVT can lead to complications such as portal hypertension, esophageal and gastric varices, ascites and hepatic failure. A recent meta-analysis reported that a MPN is the underlying cause of portal vein thrombosis in 31.5% and of Budd Chiari syndrome in 40.9% of cases (Smalberg, 2012). A significant association of SVT with JAK2V617F mutated MPN was reported (Dentali, 2009) but study of other correlations has been hampered by heterogeneity of available patient cohorts comprising relatively small number of cases.We conducted a retrospective multicenter study collecting clinical and biological data of patients (pts) with SVT associated with MPN diagnosed according to WHO2008 criteria, aiming to describe patients' characteristics, trends and prognostic factors, and their potential implications for clinical practice. Data were collected from 15 international hematology centers in the framework of IWG-MRT.We collected 475 cases of pts with portal, splenic or mesenteric vein thrombosis (75.2%) or Budd Chiari syndrome (24.8%) associated with MPN. In 32% of cases, simultaneous involvement of portal (69.1% of total thrombosis), splenic (30.5%) and mesenteric (25.3%) veins occurred, and in 1.7% they were associated with Budd Chiari syndrome. Frequency of MPN subtype: 38.1% ET (n=181), 34.9% PV (n=166), 16.2% MF (n=77), 10.8% U-MPN (n=51). Median follow-up 87.9 mo (range 0.5-430); female 61.3% (n=292; P<0.0001 vs male); median age at MPN diagnosis (dg) 44.4 y (range 12-90), at SVT dg 44.9 y (range 17-85). In 229 cases (48%) MPN and SVT dg were coincident, while in 104 (22%) SVT occurred before MPN dg (median 40 mo, range 5-335) and in 129 (27%) during MPN follow up (median 79 mo, range 5-394). JAK2V617F mutational status is available for 361 pts: 99% PV, 84.7% ET, 88.1% PMF and 92.9% U-MPN pts were JAK2V617F positive, with a mean allele burden of 56±27.4%, 33.1±25.5%, 39.3±19.4% and 23.8±11.9%, respectively. Erythropoietin-independent colonies (EEC) were present at diagnosis in 80/110 evaluated cases (72.7%), 38/47 PV (84.4%), 32/45 ET (71.1%), 8/11 PMF (72.7%) and 2/7 U-MPN (28.6%). A concurrent thrombophilic state was found in 38.9% of cases. A 12.3% of pts experienced a recurrence of SVT after a median of 29 mo (range 1-378.3) and 35.8% developed thrombosis in other sites (17.7% arterial, 19.3% venous). Esophageal varices were found in 70.6% from which 31.9% bled.MF transformation occurred in 32/166 PV (19%) and in 23/181 ET (13%) pts, with median time to progression of 122.3 mo (range 5.4-377.3) and 125.1 mo (range 39.3-255.3), respectively. Evolution to acute leukemia (AL) occurred in 12 pts (2.7%), of which 2 PMF, 6 PV and 4 ET. In 3 PV and 1 ET pts a PPV and PET-MF transformation occurred before AL.After SVT, 77% of pts received anticoagulation, 23.5% antiaggregant therapy and 1.5% both; 68.8% received cytotoxic drugs, 11.4% of pts were treated with trans jugular porto-systemic shunt. No differences in survival were noted with these approaches. Beta blocker therapy was used in 48.5% of pts and correlated with improved survival (p=0.041)At last follow up 70/473 pts (14.8%) died; causes of death are evolution to AL (16.4%), other cancers (14.5%), disease progression without AL (12.7%), SVT (10.9%), hepatic failure and venous thrombosis other than SVT (9.1% each), heart failure and arterial thrombosis (7.3% each), hemorrhage (5.5%), renal failure and infection (3.6% each). After 10 y follow up 8/166 PV (5%), 14/181 ET (8%), 14/77 PMF (18%) and 1/51 U-MPN (1.96%) pts died (p<0.01). Survival was significantly affected by occurrence of thrombosis other than SVT (p<0.0001) but not recurrence in splanchnic vessels (p=0.068).This large study confirms the strong association between JAK2V617F-mutated MPN and SVT and identifies the category of U-MPN as the prognostically more favorable; thrombosis at sites outside the splanchnic vasculature remains the leading cause of death. Disclosures:No relevant conflicts of interest to declare.

Single Cell Analysis Of JAK2V617F Positive MPN Stem/Progenitor Cells In Chronic Phase and Leukemic Transformation

IntroductionEssential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of leukemic transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior. We therefore investigated JAK2V617Fallele burden and genotype in the stem/progenitor populations of MPN patients in chronic and leukemic phases. MethodsWe studied 39 JAK2V617Fpositive MPN patients evaluated between 2005 and 2013, including 8 patients at leukemic transformation. CD34+ cells isolated from peripheral blood were flow sorted based on CD34, CD38, and the stem cell marker aldehyde dehydrogenase (ALDH). JAK2V617F allele percentages were calculated using an allele specific real time PCR assay. Cells were sorted into 96 well plates and single cell JAK2V617Fgenotypes were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted fractions when applicable. ResultsIn all MPN cases, the JAK2V617F mutation was detected in the CD34+CD38-ALDHhigh fraction – the same population in which the normal hematopoietic stem cell resides. Quantitative JAK2V617F allele burdens in this fraction were highest in MF > PV > ET. Single cell JAK2V617F genotyping revealed a higher proportion of JAK2V617F-/- cells in ET and PV than in MF, but JAK2V617F-/- cells were detectable in the CD34+CD38-ALDHhigh fraction of all cases. In most cases of PV and MF, this fraction contained a mixture of JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ cells. Additional chronic phase lesions (including mutations of ASXL1 &TET2) were also found in the CD34+CD38-ALDHhighfraction.Two patterns of leukemic transformation were observed. The first pattern (in 7/8 patients) was identical to that of de novo AML (Gerber JM, Blood 2012), with emergence of a unique CD34+CD38-ALDHint fraction, which was clonal by JAK2V617F genotype and contained leukemia-specific lesions (e.g., 5q deletion). In contrast, the residual CD34+CD38-ALDHhigh population lacked the leukemic abnormalities and was oligoclonal with respect to JAK2V617F. In 3 of these AML cases, the CD34+CD38-ALDHint fraction was JAK2V617F-/-, while the JAK2V617F mutation remained detectable in the CD34+CD38-ALDHhigh fraction. Single cell genotyping performed during the leukemic phase of a PV patient revealed only JAK2V617F-/- CD34+CD38-ALDHint cells but identified JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ CD34+CD38-ALDHhigh cells; JAK2V617F levels were barely detectable in the progenitors and neutrophils during this leukemic phase. Upon achievement of complete remission from AML, high JAK2V617F allele burdens were then found in the progenitors and neutrophils, as well as in the CD34+CD38-ALDHhigh fraction. A second pattern of leukemic transformation was seen in one patient, in whom no CD34+CD38-ALDHint population was present. The CD34+CD38-ALDHhigh population was expanded in this case and harbored JAK2V617F+/+ positive cells with the leukemia-specific lesion. ConclusionsUnlike CML, in which the BCR/ABL oncogene is typically present in the majority of CD34+CD38-ALDHhigh cells at diagnosis (Gerber JM, Am J Hematol 2011), the JAK2V617F mutation was present in only a minority of CD34+CD38-ALDHhigh cells in JAK2V617F positive ET and PV. Moreover JAK2V617F-/- cells were detected even in longstanding, advanced phase PV and MF. Lower JAK2V617F clonal burdens in the primitive CD34+CD38-ALDHhigh compartment as compared to neutrophils in most cases of MPN suggest that the JAK2V617F mutation does not confer a significant advantage at the stem cell level and that other genetic lesions may drive expansion of this population. JAK2V617F negative leukemias occur in about 35% of PV patients, apparently arising from the residual JAK2V617F negative CD34+CD38-ALDHhigh reservoir. We conclude that primitive stem/progenitor cells are mosaic with regard to JAK2V617F mutation status in the majority of MPN patients. Furthermore, acquisition of JAK2V617F, development of JAK2V617F homozygosity, and accrual of other acquired lesions in chronic phase MPN all occur in the primitive CD34+CD38-ALDHhigh compartment. Lesions specific to post MPN AML segregate to a distinct CD34+CD38-ALDHint population. Disclosures:Jones:Cytomedix: Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination., Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination. Patents & Royalties.

Ruxolitinib for Myelofibrosis–An Update of Its Clinical Effects

Myelofibrosis (MF), a Philadelphia chromosome-negative myeloproliferative neoplasm, is characterized by progressive bone marrow fibrosis and ineffective hematopoiesis. Clinical hallmarks include splenomegaly, anemia, and debilitating symptoms. In 2 randomized phase III studies, the Janus kinase (JAK) 1/JAK2 inhibitor ruxolitinib significantly improved splenomegaly and disease-related symptoms compared with placebo (Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment [COMFORT-I]) or best available therapy (COMFORT-II) in patients with intermediate-2 or high-risk MF. Although ruxolitinib therapy was associated with dose-dependent anemia and thrombocytopenia, these adverse events rarely led to treatment discontinuation. This update of the clinical effects of ruxolitinib in patients with MF was based on original articles and meeting abstracts published after the primary publication of the COMFORT trials in March 2012. Long-term follow-up data from the COMFORT trials and clinical experience with ruxolitinib in unselected patient populations suggest that improvement of splenomegaly and symptoms is durable. Patients benefit from ruxolitinib therapy across subgroups defined by age, MF type, risk category, performance status, JAK2 V617F mutation status, extent of splenomegaly, or presence of cytopenias. In COMFORT-I, platelet counts stabilized with dose adjustments, and hemoglobin levels gradually recovered to slightly below baseline after the first 8 to 12 weeks of therapy. After initial increases, the need for red blood cell transfusions decreased to a level similar to that found in the placebo group. The 2-year follow-up data from the COMFORT trials suggest that patients with intermediate-2 or high-risk MF receiving ruxolitinib therapy may have improved survival compared with those receiving no (placebo) or traditional therapy.

The influence of JAK2 V617F mutational status and allele burden on the risk of vascular complications in patients with chronic myeloproliferative neoplasms

The influence of JAK2 V617F mutational status and allele burden on the risk of vascular complications in patients with chronic myeloproliferative neoplasms

Aberrant expression of signaling proteins in essential thrombocythemia

Dysregulated expression of signaling proteins may contribute to the pathophysiology of essential thrombocythemia (ET). This study aimed to characterize protein expression in ET and to correlate the dysregulated proteins with phenotypes and prognosis of ET patients. The expression of 128 proteins in peripheral blood neutrophils from 74 ET patients was assessed and compared with those from 29 healthy subjects and 35 polycythemia vera (PV) patients using protein pathway array. Fifteen proteins were differentially expressed between ET patients and normal controls. These dysregulated proteins were involved in the signaling pathways related with apoptosis and inflammation. Our results showed a significant overlap in protein expression between ET patients with JAK2V617F mutation and PV patients. In addition, nine proteins were associated with JAK2V617F mutation status in ET patients. Furthermore, estrogen receptor beta (ERβ) and Stat3 were independent risk factors for subsequent thrombosis during follow-up on multivariable analysis. Our study shows a broad dysregulation of signaling protein in ET patients, suggesting their roles in ET pathogenesis. The expression levels of ERβ and Stat3 could be promising predictors of subsequent thrombosis in ET patients.

Primary myelofibrosis: risk stratification by IPSS identifies patients with poor clinical outcome

OBJECTIVES:To evaluate whether risk scores used to classify patients with primary myelofibrosis and JAK-2 V617F mutation status can predict clinical outcome.METHODS:A review of clinical and laboratory data from 74 patients with primary myelofibrosis diagnosed between 1992 and 2011. The IPSS and Lille scores were calculated for risk stratification and correlated with overall survival.RESULTS:A V617F JAK2 mutation was detected in 32 cases (47%), with no significant correlation with overall survival. The patients were classified according to the scores: Lille - low, 53 (73.%); intermediate, 13 (18%); and high, 5 (7%); and IPSS – low, 15 (26%); intermediate-1, 23 (32%); intermediate-2, 19 (26%); and high, 15 (31%). Those patients presenting a higher risk according to the IPSS (high and intermediate-2) had a significantly shorter overall survival relative to the low risk groups (intermediate-1 and low) (p = 0.02).CONCLUSIONS:These results emphasize the importance of the IPSS prognostic score for risk assessment in predicting the clinical outcome of primary myelofibrosis patients.

Prognostic impact of JAK2V617F mutation in myelodysplatic syndromes: A matched case control study

While in RARS-T, JAK2V617F mutation is common and associated with good prognosis, the clinical and prognostic impact of this mutation in other MDS is unknown. We collected data from 132 non-RARS-T MDS with known JAK2V617F mutation status. JAK2V617F mutation was significantly correlated with lower progression to AML (p<.0011) and better overall survival (OS, p=.011). OS difference persisted after matching on age, sex, IPSS and % marrow blast (p=.031). Thus, in MDS other than RARS-T, JAK2V617F mutation may be associated with favorable outcome.

A Germline Variant in the TERT Gene Is a Novel Predisposition Allele Associated with Myeloproliferative Neoplasms

AbstractAbstract 707 Background:Myeloproliferative neoplasms (MPNs) are disorders that result in unregulated overproduction of one or more myeloid blood cell types by the bone marrow. Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) comprise the three classic MPNs. The somatic JAK2 V617F mutation is present in 95% of PV patients, and 50–60% of ET and PMF patients. Past work has identified a germline haplotype in JAK2 associated with risk of developing a V617F-positive MPN (Kilpivaara et al; Olcaydu et al; Jones et al, Nat. Genet., 2009). Methods:We recruited a web-based participatory cohort of individuals with MPNs to better understand the genetic basis of these conditions. Participation in this MPN research initiative included free genotyping, and participants provided informed consent and responses to surveys online, under a protocol approved by the external AAHRPP-accredited IRB, Ethical & Independent Review Services. We enrolled and collected saliva samples from more than 700 participants with the following self-reported diagnoses: systemic mastocytosis (n=142), ET (n=145), PV (n=137), PMF (n=50), chronic myelogenous leukemia (n=108), and 97 with overlapping diagnoses (e.g. ET or PV with PMF). Participants were genotyped using a derivative of the Illumina HumanOmniExpress BeadChip with additional custom content, including probes for the JAK2 V617F variant. We performed a genome-wide association study (GWAS) of classic MPN diagnoses (at least one of ET, PV, or MF), using 407 cases from this MPN research initiative and 94,037 controls drawn from the broader 23andMe research participant community. Association was assessed by logistic regression adjusted for age, gender, and 5 principal components to model ancestry. Results:We replicated the known association between germline variation in JAK2 and classic MPNs (rs12340895: odds ratio=2.5, P=7.4 × 10−35). We also identified a novel genome-wide-significant association in TERT, or telomerase reverse transcriptase (rs2853677: odds ratio=1.6 per G allele, P=6.3 × 10−11). The association is consistent with an additive model on the logistic scale (odds ratio=2.6 for 2 versus 0 G alleles). Another variant in TERT, rs2736100, has previously been associated with several non-hematological cancers and with red blood cell count, and our lead SNP is in linkage disequilibrium with TERT variant rs2736100 (r2=0.54). While no other loci reached the threshold of genome-wide significance (P<5.0 × 10−8), we saw a suggestive association with a germline missense variant (F858L) in ATM, or ataxia telangiectasia mutated gene (rs1800056: odds ratio=2.6 per C allele, P=1.4 × 10−5). This variant has also been reported to be associated with breast cancer and chronic lymphocytic leukemia.The rs2853677 TERT variant was associated with each of ET, PV, and PMF, with similar effect sizes. It was not associated with JAK2 V617F mutation status within the MPN cohort (odds ratio=1.0, P=1.0), while the germline JAK2 variant was preferentially associated with V617F-positive MPNs (odds ratio=2.1, P=3.0 × 10−6). The high-risk allele of rs2853677 was also predictive of V617F status among 23andMe participants who had not been recruited into the MPN community (odds ratio=1.6, P=0.0012). These results suggest that while variation in TERT facilitates either acquisition or proliferation of V617F-positive myeloid cells, it also predisposes to MPNs through a V617F-independent mechanism. Conclusion:We have identified a germline variant in TERT which is a new predisposition allele for classic MPNs. These results demonstrate the potential for web-based recruitment methods to contribute to genetic research for uncommon diseases. Disclosures:Hinds:23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other.