Low JAK2V617F Allele Burden Research Articles

Impact of JAK2 allele burden on MF outcome in the era of ruxolitinib.

6514 Background: Myelodepleted myelofibrosis (MF), characterized bycytopenias, lower JAK2V617F allele burden (JAK2%) and shorter benefit from JAKi ruxolitinib (RUX), has worse survival (OS) compared to myeloproliferative MF. Lower JAK2% and inferior OS is more typical for primary MF (PMF) vs MF from essential thrombocythemia or polycythemia vera (PPV/PET-MF). We sought to investigate the impact of JAK2% (</≥ 50%), cytopenias and the use of RUX in outcome of PMF/PPV-PET-MF patients from our center. Methods: 601 medical charts of JAK2 mutated patients with MF (known JAK2%) were retrospectively reviewed. We divided patients based on the absence (-) or presence (+) of cytopenias (hemoglobin < 10 g/dL or platelets < 100 x10^9/L) and leukocytosis (WBC ≥ 25 x10^9/L) into: Gr1 = (-)/(-) [absence of both]; Gr2 = (-)/(+) [proliferative]; Gr3 = (+)/(-) [cytopenic]; Gr4 = (+)/(+) [cytopenic and proliferative]) and evaluated OS per JAK2 </≥ 50% and PMF vs PPV/PET-MF. We assessed the tolerance of RUX ≥3 years. We used descriptive statistics, Kaplan-Meier curve with log-rank test and regression analysis for demographics, estimation of OS and its comparison. OS was censored at the time of stem cell transplantation. Results: Median age of the entire cohort was 75 years (64% males). Patients with JAK2 <50%, more likely PMF, had more anemia and thrombocytopenia, higher blasts, less leukocytosis, and smaller splenomegaly. JAK2 </≥ 50% did not discriminate OS of the entire group, PMF or PPV/PET-MF (median OS of ~ 45 months for each; only PPV/PET-MF with 64 months). OS of Gr1-4 according to JAK2 </≥ 50% and PMF/ PPV/PET-MF and combined OS of patients with comparable outcome (A-D) are shown in the table. The final groups with distinct outcomes included: A+B) non-cytopenic PET/PPV-MF and PMF irrespectively of proliferation and JAK2% - with one exception of non-cytopenic, proliferative PMF with JAK2 ≥50% - had median OS of 70 months (range, 54-86); D) cytopenic and proliferative PMF with any JAK2% and PPV/PET-MF with JAK2 < 50% of median OS 19 months (range, 13-25), and C) the rest of the cytopenic patients and non-cytopenic, proliferative PMF with JAK2 ≥50% with a median OS of 36 months (range, 32-42). Among the 3 final groups, similar proportion of patients were exposed to RUX during their follow-up: 36% (A+B), 31% (B) and 28% (C), respectively (p =0.13). The most patients in (A+B) group were able to tolerate RUX for ≥3 years (p =0.036). All patients had improved OS with RUX (data to be presented). Conclusions: Cytopenias and/or proliferation, rather than JAK2 </≥50%, define the outcome of PMF and PPV/PET-MF patients. [Table: see text]

HMR Mutations Drive Poor Prognosis in Myelofibrosis Patients with Lower JAK2V617F Allele Burden but Not in Those with Higher Allele Burden: Results of a Multicenter Study

JAK2V617F is the most common driver mutation in myelofibrosis (MF), followed by CALR and MPL alterations. Clinical variables included in the International Prognostic Scoring System (IPSS), Dynamic IPSS (DIPSS), and DIPSS plus, as well as JAK2V617F allele burden are all well-established prognostic factors influencing the survival of MF patients. In addition, ASXL1, EZH2, SRSF2, IDH and U2AF1 mutations are classified as high-molecular risk (HMR) mutations in this context because of their close association with disease progression and leukemic transformation. However, the relationship between JAK2V617F allele burden and HMR mutations remains unclear. To address this, we recruited 122 MF patients (discovery cohort) diagnosed at the National Taiwan University Hospital (Taipei, Taiwan) who had available mutation profile determined by next-generation sequencing. For external validation, 82 MF patients from the Christie Hospital (Manchester, U.K.) and 51 MF patients from the Cleveland Clinic (Ohio, U.S.A.) and Vilnius University Hospital Santaros Klinikos (Vilnius, Lithuania) were enrolled. The median age of the discovery cohort was 61 years. JAK2V617F was the most common driver mutation (64.8%), followed by CALR (18%) and MPL mutations (9%). ASXL1 mutation was the most common (37%) genetic alteration other than driver mutations, followed by TET2 (16%) and EZH2 mutations (12%). Overall, 55 patients (45%) had at least one HMR mutation ( ASXL1, EZH2, SRSF2, IDH and U2AF1). With a median follow-up of 28.2 months, the median overall survival (OS) of the discovery cohort was not reached (NR) and was well risk-stratified by current prognostication systems, including IPSS, DIPSS plus, MIPSS70, and GIPSS. Notably, patients with at least one HMR mutation had significantly shorter leukemia-free survival (LFS) and OS than those without (both p<0.001). Next, we divided the 79 JAK2-mutated patients into lower- and higher-allele burden groups ( JAK2low and JAK2high) using the median (75%), as the cutoff. Concurring with previous reports ( Tefferi et al, Leukemia 2008; and Guglielmelli et al, Blood 2009), JAK2low patients had significantly inferior OS than JAK2high patients (median, 53 months vs NR, p=0.037). Among all JAK2-mutated patients, those with HMR mutations had a significantly shorter OS than those without (50 months vs NR, p<0.001). When considering HMR mutations and JAK2V617F allele burden together, the presence of HMR alterations conferred worse LFS and OS in JAK2low but not in JAK2high patients (HMR vs without HMR among JAK2low patients, p<0.001 and p<0.001; and JAK2high, p=0.822 and p=0.699, respectively). By and large, patients with concurrent HMR mutations and lower JAK2V617F allele burden had a distinctively inferior OS than those with other constellations of genomic lesions ( p<0.001, Figure 1A). This group of patients also had the shortest survival among the total cohort including both JAK2-mutated and wild patients ( p<0.001). The discriminating power of integrating HMR/ JAK2 allele burden for LFS and OS remained valid in the UK cohort (HMR vs without HMR: JAK2low, p=0.013 and p=0.012; and JAK2high, p=0.337 and p=0.600, respectively) and the US/Lithuania cohort (HMR vs without HMR: JAK2low, p=0.016 and p=0.013; and JAK2high, p=0.643 and p=0.446, respectively). Co-occurrence tests in the discovery and validation cohorts revealed that there was no shared mutational co-occurrence pattern among the 3 cohorts, excluding the potential confounding influence of specific concurrent mutations. More strikingly, time-dependent ROC curve analyses suggested that integrating JAK2 allele burden with HMR mutations could be complementary to current risk stratification systems in predicting LFS and OS of MF patients (Figure 1B) across 3 cohorts. Finally, HMR/ JAK2low was identified as an independent adverse prognostic factor by multivariable analysis, irrespective of age, risk stratification systems, and other mutations. In summary, we observe that HMR mutations accompanied by a lower mutant JAK2V617F allele burden identified a distinct MF population with significantly reduced survival and high risk of leukemic transformation, with independent prognostic relevance extrapolated to and validated in two independent cohorts. Experimental studies are ongoing to ascertain the underlying mechanistic basis and resolve the clonal architecture of this distinct MF genomic grouping.

A Comparison of Bone Marrow Morphology and Peripheral Blood Findings in Low and High Level JAK2 V617F Allele Burden.

Cases with low level JAK2 V617F mutations are increasingly detected; however, the clinical interpretation of the low allele JAK2 burden may be challenging. The aim of this study is to analyze and compare the bone marrow morphology and peripheral blood findings in the low level JAK2 V617F allele burden (≤15% of JAK2) and high JAK2 V617F mutation burden patients (>15% JAK2). In total, 122 JAK2 V617F positive cases with concomitant bone marrow biopsies and peripheral blood findings were re-evaluated (62 low and 60 high level JAK2 V617F positive). Within the low burden group, normal looking megakaryocytes (p = 0.0005) were more frequently found, compared with those with no atypia (p = 0.0003), their number was more frequently not increased (p = 0.009), and they did not form clusters (p = 0.001). We found statistically significant difference in the number of platelet (p = 0.0003) and hematocrit levels (p = 0.032) when comparing the JAK2 V617F <3% and ≥3% mutation burden. In the high-level burden, the megakaryocytes were more frequently atypical (p = 0.054), and more frequently formed clusters (p = 0.053) with nuclei with maturation defects (p ≤ 0.0001). In conclusion, the JAK2 V617F mutation burden is reflected by morphological changes in the bone marrow and careful follow up of each and every patient with a low JAK2 V617F positivity is mandatory.

P1026: COMPARATIVE GENOMIC PROFILING OF MYELOPROLIFERATIVE NEOPLASMS PRESENTING WITH AND WITHOUT SPLANCHNIC VEIN THROMBOSIS

Background: Splanchnic vein thrombosis (SVT) is the initial manifestation in 5% of myeloproliferative neoplasms (MPN). MPN patients presenting with SVT are usually younger, have less abnormal blood counts and lower JAK2V617F allele burden than other MPN patients. However, it is unknown whether they have a different genomic background. Aims: To describe the clinical and genomic profile of MPN presenting with SVT and to compare it with a matched group of MPN without SVT. Methods: Samples of 190 MPN patients were selected: 64 MPN presenting with SVT (SVT+) and 126 MPN without SVT (SVT-). SVT+ and SVT- were matched according to sex, age, MPN clinical subtype and driver mutation. Next Generation Sequencing was performed using Myeloid Solution by Sophia Genetics, which includes 30 genes recurrently mutated in MPN. Variants were selected using Sophia DDM software and were subsequently classified into 5 categories: 5 (definitely pathogenic), 4 (likely pathogenic), 3 (uncertain), 2 (likely benign), and 1 (benign). Only variants included into 4 and 5 categories were taken into account. Patients were classified according to the genomic classification proposed by Grinfeld and colleagues. Results: One hundred and three patients were women (54.2%) and 87 patients were men (45.8%), without significant differences between SVT+ and SVT- groups. Median age was 44 years (range: 15-85) and 50 years (range: 15-87) in SVT+ and SVT-, respectively (p not significant). Regarding MPN clinical subtype in SVT+ group, 39 (60.9%), 15 (23.4%) and 10 (15.6%) corresponded to Polycythemia Vera (PV), Essential Thrombocythemia (ET) and MPN unclassifiable, respectively. In the SVT- group, 75 (59.5%) and 51 (40.5%) corresponded to PV and ET, respectively. Genomic classification in the SVT+ and SVT- groups is shown in Table 1. The proportion of patients with high molecular risk (TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 mutation) was 14.1% and 26.2% for SVT+ and SVT-, respectively (p=0.06). Median number of pathogenic mutations was 1 (range: 1-4) in both SVT+ and SVT- groups. Pathogenic mutations in TET2, DNMT3A and ASXL1 were present in 10%, 3.7% and 3.7%, respectively, without significant differences between both groups. With a mean follow-up of 10.1 years for cases and 9.7 years for controls, 27 patients died (13 SVT+ and 14 SVT-). Median survival was 24 years and not reached for SVT+ and SVT-, respectively (p=0.1). On multivariate analysis, SVT+ group showed a higher risk of death (HR: 2.4, 95%CI: 1.1-5.5, p=0.03) after correction by age, sex and genomic classification. Transformation to myelofibrosis and acute leukemia was low and similar in both groups: 4.7% vs. 5.6% for myelofibrosis in SVT+ and SVT-, respectively (p not significant); and 0% vs. 2.4% for acute leukemia (p not significant). On multivariate analysis, patients with TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 showed a higher risk of disease progression to myelofibrosis or acute leukemia (HR: 14.5, 95%CI: 3.1-68.7, p=0.001) regardless of the presence of SVT at diagnosis, age and sex. Image:Summary/Conclusion: MPN presenting with SVT and MPN-matched controls showed a low molecular complexity. Genomic classification was helpful in identifying a small proportion of MPN patients presenting with SVT who are at high risk of disease progression. SVT at MPN diagnosis is associated with a higher risk of death independent of sex, age and genomic classification.

Pacritinib demonstrates spleen volume reduction in patients with myelofibrosis independent of JAK2V617F allele burden

AbstractMyelofibrosis (MF) has heterogeneous clinical manifestations, with some patients exhibiting a myelodepletive phenotype characterized by cytopenias and an absent or low JAK2V617F allele burden. Ruxolitinib may be less effective in these patients. We assessed the efficacy of pacritinib, a JAK2/IRAK1 inhibitor, in MF patients with low JAK2V617F allele burden. In this post hoc analysis of the PERSIST-1 and -2 trials, patients with MF randomized to pacritinib or best available therapy (BAT) were stratified by JAK2V617F allele burden quartile for spleen response of ≥35% and improvement in total symptom score of ≥50%. Five hundred thirty-six patients were included. Patients with lower JAK2V617F allele burden had smaller baseline spleens and lower hemoglobin and platelet counts as compared with higher allele burden patients. Among pacritinib-treated patients, spleen responses were observed across all JAK2V617F allele burden quartiles and in JAK2V617F− disease. No spleen responses were observed among BAT-treated patients with allele burden ≤50% or JAK2V617F− disease. The intention-to-treat response rate was significantly higher on the pacritinib arm for JAK2V617F− disease (23.0% vs 0%; P = .033), and for the lowest allele burden quartiles (0%-25%: 20.9% vs 0%, P < .001; 25%-50%: 15.4% vs 0%, P = .020). There were significantly more symptom responders with pacritinib vs BAT in the 0% to 25% and 25% to 50% cohorts. Pacritinib treatment led to superior spleen and symptom burden reduction compared with BAT in patients with absent or low JAK2V617F allele burden, suggesting that pacritinib may be uniquely suited for patients with myelodepletive MF.

MPN-376: The Clinical Impact of the Burden of JAK2V617F Mutated Allele in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Context: Several studies reported the impact of JAK2V617F allele burden on phenotypic properties and clinical course in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). We aimed to analyze the association of mutated JAK2V617F allele burden with clinical phenotype and laboratory characteristics in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Objective: A total of 202 consecutive Philadelphia-negative MPN patients, 118 with ET and 84 with PMF, were included in this study. Methods: We used a real-time, semi-quantitative polymerase chain reaction (PCR) with JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France) to screen JAK2V617 mutations and mutant allele burden. Results: According to the JAK2V617F status, ET patients were stratified into three groups: JAK2V617F mutation-negative (n=54) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=59) and upper quartile (n=5) ranges. ET patients with upper quartile JAK2V617F allele burden had significantly lower Hgb levels (p=0.001), lower Htc levels (p=0.001), and higher LDH levels (p=0.008) as compared to the other two groups. Also, ET patients with upper quartile JAK2V617F allele burden had significantly increased spleen size and higher bleeding complications (p=0.034; 0.002; respectively). We observed significant association between ET patients with high JAK2V617F allele burden and higher death rates (p=0.007). There was a trend towards higher incidence of venous thrombosis in ET patients with upper quartile JAK2V617F allele burden (p=0.057). According to JAK2V617F status, PMF patients were stratified into three groups: JAK2V617F mutation-negative (n=25) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=41) and upper quartile (n=18) ranges. Comparison across all three groups revealed significant associations between upper quartile allele burden and higher leukocyte counts (p=0.001). Also, PMF patients with upper quartile JAK2V617F allele burden had significantly increased spleen size compared to the other two groups (p=0.018). We observed significant association between PMF patients with low JAK2V617F allele burden and higher incidence of venous thrombosis (p=0.035). Conclusion: Our study reported that presence of higher V617F allele burden in ET is associated with defined haematological and clinical markers indicative of a more aggressive behavior while, in PMF, low allele burden was found to be associated with higher incidence of venous thrombosis.

The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity &gt;= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

PF687 RISK OF THROMBOSIS IN PATIENTS WITH POLYCYTHEMIA VERA AND ESSENTIAL THROMBOCYTHEMIA ACCORDING TO THE JAK2 V617F ALLELE BURDEN

Background: Thrombosis is one of the major causes of morbidity and mortality in patients with chronic phase myeloproliferative neoplasms (MPN) comprising polycythemia vera (PV) and essential thrombocythemia (ET). The JAK2 V617F mutation is the most common driver mutation in BCR-ABL1 negative MPNs and the presence of JAK2 V617F is an independent risk factor of thrombotic events in patients with ET. Aims: In this study, we analyzed whether the JAK2 V617F allele burden is associated with the incidence of thrombosis in JAK2 V617F-positive patients with PV or ET. Methods: This was a retrospective study that assessed the impact of JAK2 V617F allele burden on clinical and laboratory characteristics in JAK2 V617F-positive patients with PV or ET. Patients with a diagnosis of PV or ET according to the WHO criteria at Daegu Catholic University Hospital between 2008 and 2018 were included in this study. JAK2 V617F allele burden were measured by pyrosequencing. Results: A total of 127 patents, median age 68 years, with PV (n = 61) or ET (n = 66) were included in our analyses. The JAK2 V617F allele burden in the entire population of patients was 45.0 ± 25.9%. A higher V617F allele burden was associated with higher white blood cell counts, higher hemoglobin values, lower platelet counts and higher LDH level (p < 0.001, p < 0.001, p = 0.001, and p = 0.001, respectively). There was a statistically significant difference in the JAK2 V617F allele burden between PV and ET (p < 0.001). The median V617F allele burden in PV patients was 58%, and ET was 30%. Thrombotic events were observed in 27.8% and 51.5% in patients with PV and ET, respectively. Multivariate logistic regression analysis revealed that older age (odds ratio, 1.07; 95% CI 1.01–1.18) was associated with higher incidence of thrombosis in patients with PV. In patients with ET, multivariate logistic regression analysis revealed that older age (odds ratio, 1.36; 95% CI 1.14–1.72), higher hemoglobin (odds ratio, 1.26; 95% CI 1.09–1.54), and V617F allele burden≥30% (odds ratio, 1.03; 95% CI 1.00–1.08) was elucidated as risk factors for thrombotic complications. Summary/Conclusion: JAK2 V617F allele burden can represent the disease phenotype. The incidence of thrombotic complications in ET patients with high JAK2 V617F allele burden is significantly higher than patients with low JAK2 V617F allele burden. There was no significant difference of the rate of thrombosis in PV patients according to the JAK2 V617F allele burden.

Young Versus Old Age at Diagnosis Confers Distinct Genomic Profiles in Patients with Polycythemia Vera

Introduction: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by overproduction of mature erythrocytes and a propensity for transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). The defining genetic feature of PV is the JAK2 V617F mutation, present in 95-97% of patients, resulting in overactive JAK-STAT signaling. Secondary mutations have been identified which may modulate PV disease phenotype. Although typically diagnosed in older individuals, PV occurs in young patients (≤ 45 years old) as well. Compared with old patients (≥ 65 years old), a significant association with young PV and female gender, lower white blood cell count, lower JAK2 V617F allele burden, palpable splenomegaly, and splanchnic vein thrombosis has been reported. In the same study, the frequency of transformation to MF or AML was found to be similar, although the median time to transformation was shorter in old PV. The reasons for these age-related associations are not clearly understood. Since somatic mutations have been shown to accumulate with age, we hypothesized that the mutational profile of young PV patients may differ from that of old PV, which could contribute to the differences in clinical phenotypes observed.Methods: DNA from tumor (bulk peripheral blood mononuclear cells or granulocytes) and matched normal tissue (skin or sorted CD3+ T cells) was isolated from samples from 10 young (≤ 45 years old) and 11 old (≥ 65 years old) PV patients. Enhanced exome sequencing utilizing the standard IDT exome capture panel plus additional probes for genes known to be recurrently mutated in MPN and/or AML was performed. To determine order of mutation acquisition (and to rule out the possibility of biclonal disease) qPCR-based genotyping was performed on single cell-derived colonies from patients with ≥ 1 secondary mutation (in addition to JAK2 V617F). Plasma concentrations of 10 cytokines were assayed in 15 young and 23 old PV patient samples compared with age-matched healthy controls.Results: Comparative analysis of 7 young and 10 old patients (four samples were removed due to sample contamination/artifact issues) identified a total of 133 somatic mutations. A significantly higher overall mutational load was observed in the old group (mean 10.9 vs 3.4; p = 0.0025). A trend toward higher JAK2 V617F variant allele frequency (VAF) was observed in old versus young PV in the sequenced patients, and in a separate clinical cohort the JAK2 V617F VAF was significantly higher in old PV patients (p = 0.0067). Notably, putative secondary driver mutations were identified in 9/10 old PV patients (including ASXL1, TET2, and DNMT3A), whereas JAK2 appeared to be the only driver mutation present in each of the young PV patients. Secondary mutations were validated as being acquired before, after, or coincident with JAK2 V617F, with no specific predominant pattern observed. MPN-predisposing germline JAK2 46/1 haplotype occurrence did not differ with age, and additional germline mutations in MPN genes were not enriched in either cohort. Supranormal levels of multiple cytokines (e.g. IFNγ, IL-2Rα, IP-10, MIP-1α) were observed in both young and old PV, with a slight enhancement observed with increased age.Conclusions: These data indicate a striking age-based distinction in the near uniform absence (young) or presence (old) of secondary non-JAK2 mutations in PV patients. The finding that putative secondary driver mutations were acquired before JAK2 V617F in only a minority of old PV suggests that the overall increase of secondary mutations in this cohort may not merely be a function of aging, implying that these mutations do in fact contribute to PV development and/or progression. As transformation can be induced by accrual of disease-modifying mutations, the presence of secondary mutations in old PV patients is consistent with the reported shorter time to transformation in this age group, since young PV patients would likely need additional time to acquire secondary mutations. Abnormal inflammatory cytokine production was common to both young and old PV, suggesting this is not the predominant factor distinguishing these two cohorts. The contribution of additional non-genomic factors (e.g. epigenetic, hormonal) will be the subject of future study. DisclosuresOh:Janssen: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Research Funding; Gilead: Research Funding; Takeda: Research Funding.

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

AimsWe investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis...

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study.

Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%.Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (‘’Histology-based’’ diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (‘’Clinical-based’’ or ‘’Molecular-based’’ diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process.Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion.

Role of Non-Driver Mutations and JAK2V617F Allele Burden in Myelofibrotic and Acute Myeloid Transformation of Patients with Polycythemia Vera and Essential Thrombocythemia

Background:Non-driver mutations and JAK2V617F allele burden have been involved in progression to myelofibrosis (MF) or acute myeloid leukemia (AML) in patients with polycythemia vera (PV) and essential thrombocythemia (ET). It is unknown if both mechanisms play a different role in disease transformation and if they are useful in routine clinical practice.Methods:JAK2V617F allele burden was monitored in 208 patients (PV n=106, ET n=102) for a median of 6.5 years (range: 1-13). Quantification of JAK2V617F allele burden was assessed on the first available sample and every year thereafter. The evolutionary pattern of JAK2V617F allele burden was categorized as persistently low (<50%), persistently high (>50%), progressive increase (> 25% from baseline) or unexplained decrease (not therapy related).Next generation sequencing (NGS) analysis of 51 myeloid-related genes was performed in 100 patients with a median molecular follow-up of 10 years including all cases with transformation to AML or MF. Detected mutations by NGS in the last sample were studied in first paired sample obtained in the chronic phase (median time from diagnosis: 1.6 years).Time to myelofibrosis and time to AML were calculated according to the presence of non-driver mutations or the JAK2V617F evolutionary pattern. Multivariate analysis was performed by Cox regression.Results:With a median follow-up of 13 years (range: 1-30) 32 patients died whereas 24 and 12 patients progressed to MF and AML, respectively. Median age at diagnosis was 63 years (range: 20-94), 115 were women (55%) and 173 (83%) received cytoreduction. A persistently low JAK2V617F allele burden was observed in 62% of patients whereas the remainder presented a persistently high (25%), a progressive increase (11%) or a non-therapy-related decrease of JAK2V617F allele burden (2%).Non-driver mutations were detected in last sample in 48% of patients. Median number of mutations was 1 (range: 1-5). Mutational frequencies were: TET2 12%, DNMT3A 12%, TP53 9%, ASXL1 7%, RUNX1 4%, SF3B1 4%, SRSF2 4%, IDH1/2 4%, SH2B3 3 % and <2% for EZH2, ZRSR2, SETBP1, FLT3, NPM, BCOR, CBL, PTPN11, KAT6A, NF1, U2AF1, GNAS, PHF6, KTM2A, JAK2, BCORL1, NOTCH1, MPL and PRPF40B.Mutations were detected in first sample in 28% of patients (58% of those with mutations in last sample). Frequencies of mutations in first sample were: TET2 10%, DNMT3A 5%, TP53 5%, ASXL1 4%, SRSF2 4%, IDH1/2 3%, and < 2% for SF3B1, SH2B3, KMT2A and ZRSR2. The evolutionary pattern of JAK2V617F allele burden was not associated with the presence of mutations in first or last sample.Twelve patients progressed to AML (post-PV n=7, post-ET n=5), nine of them presented mutations in first sample. AML transformation at 15 years was 27% and 6.8% for patients with and without additional mutations in first sample, respectively (p=0.001). Mutated genes associated with a higher probability of AML transformation were DNMT3A (p<0.0001), SRSF2 (p<0.0001) and IDH (p<0.0001). Other variables associated with a higher probability of AML were age > 65 years (p=0.012) and exposure to busulfan (p=0.003). Evolutionary JAK2V617F pattern was not associated with the probability of AML (p=0.667). In multivariate analysis, an increased risk of AML transformation was observed for patients with additional mutations in the chronic phase (HR: 6.3; 95%CI 1.6-24.7, p=0.008) after adjusting for initial diagnosis, age and exposure to busulfan.Twenty-four MF transformations were documented (post-PV n= 18, post-ET n=6). Presence of additional mutations was not associated with the probability of MF (p=0.189). Patients with persistently high or a progressive increase of the JAK2V617Fallele burden showed a higher probability of MF transformation (24% versus 1.5% at 10 years, p<0.0001) that persisted in multivariate analysis after correction for age and initial diagnosis (HR 13.9, 95%CI: 2.9-65.6, p=0.001).Conclusion:Non-driver mutations are involved in the progression of PV and ET to AML but not to MF. NGS could be useful for identifying patients with PV or ET at risk of AML transformation.Acknowledgment:This work was supported by grants from the Instituto de Salud Carlos III, Spanish Health Ministry, FISPI13/00557, FISPI1300393, RD012/0036/0004, 2014 SGR567. Alicia Senín received a grant from Sociedad Española de Hematología y Hemoterapia. DisclosuresNo relevant conflicts of interest to declare.

Masked polycythaemia vera: presenting features, response to treatment and clinical outcomes.

Masked polycythaemia vera (PV) has been proposed as a new entity with poorer outcome than overt PV. In this study, the initial clinical and laboratory characteristics, response to treatment and outcome of masked and overt PV were compared using red cell mass and haemoglobin or haematocrit levels for the distinction between both entities. Sixty-eight of 151 PV patients (45%) were classified as masked PV according to World Health Organisation diagnostic criteria, whereas 16 (11%) were classified as masked PV using the British Committee for Standards in Haematology (BCSH). In comparison with overt PV, a higher platelet count and a lower JAK2V617F allele burden at diagnosis were observed in masked PV. Patients with masked PV needed lower phlebotomies and responded faster to hydroxcarbamide than those with overt PV. Complete haematological response was more frequently achieved in masked than in overt PV (79% vs. 58%, P = 0.001). There were no significant differences in the duration of haematological response, the rate of resistance or intolerance to hydroxycarbamide and the probability of molecular response according to type of PV (masked vs. overt). Overall survival, rate of thrombosis and major bleeding, and probability of transformation was superimposable among patients with masked and overt PV.

JAK2, Calr and MPL Mutation Status Predicts the Survival Outcome of Patients with Primary Myelofibrosis

BackgroundPatients with primary myelofibrosis survive for a median of 5-7 years after diagnosis. However, survival outcomes markedly vary; some patients experience rapid progression and others survive for 10 years or more. The international prognostic scoring system (IPSS) stratifies patients into 4 risk groups on the basis of clinical features (age and constitutional symptoms) and blood counts (hemoglobin, white blood cells, and the presence of circulating blasts).Most patients with primary myelofibrosis harbor mutually exclusive somatic mutations in 1 of 3 genes. In 60% of patients, a substitution mutation is detected in the Janus Kinase 2 (JAK2) gene (JAK2V617F). In another 30% of patients, a frameshift mutation is identified in the Calreticulin (CALR) gene. In 5% of patients, a substitution mutation is found in the gene that codes for the thrombopoietin receptor (MPL). Although patients with mutated CALR generally have more indolent disease than patients with JAK2V617F, the prognostic significance of harboring JAK2V617F is confounded by the observation that low JAK2V617F allele burden is associated with aggressive disease and shortened overall survival (OS) duration.AimTo develop a prognostic model based on the mutation status of the JAK2, CALR, and MPL genes and to determine whether JAK2, CALR, and MPLmutation status provides additional prognostic value to the IPSS.Patients and methodsBone marrow samples were collected and processed from 344 patients with primary myelofibrosis at The University of Texas MD Anderson Cancer Center between 2000 and 2013 (157 months), at the initial visit. All samples were screened for JAK2V617F and for exon9 deletion or insertion mutations in CALR. We used quantitative PCR to quantify the levels of wild-type and mutated JAK2 and determine the JAK2V617F burden. Patients who did not have a mutation in either gene (n = 73) were also screened for an exon10 substitution mutation in MPL.ResultsOf the 344 patients screened for JAK2V617F and mutations in CALR, 226 (66%) had JAK2V617F and 43 (13%) had mutations in CALR. Of the 75 patients who did not have JAK2 or CALR mutations, 16 (21%) had mutated MPL. In 59 patients (17%), we did not identify mutations in any of the genes (“triple negative”). In the 226 patients who harbored the JAK2V617F mutation, a cut-point of 50% dichotomized patients into those with a high JAK2V617F burden (≥50%) and a favorable survival outcome (median OS: 80 months, 95% confidence interval [CI]: 51-109 months) and those with a low JAK2V617F burden and an adverse survival outcome (median OS: 50 months, 95% CI: 40-60 months; Figure 1). Clinically, patients with a high JAK2V617F burden had larger spleens than those with a low JAK2V617Fburden (P < .0001).We then classified patients according to mutation status into 5 groups: mutated CALR, mutated MPL, high JAK2V617F burden, low JAK2V617F burden, or triple negative) and used mutation status and the IPSS to fit a multivariate model to predict OS. In this model, the IPSS and mutation status independently predicted OS, and as implicated by the higher p-value of the Wald chi-square test, the mutation status was a superior estimate of OS. On the basis of this analysis, patients were stratified according to mutation status into 3 groups with different survival outcomes. Patients had a favorable outcome if they harbored CALR or MPL mutations or if they had a high JAK2V617F burden (n = 170, OS: 104 months, 95% CI: 86-122 months). Patients who were triple negative had an intermediate survival outcome (n = 59, OS: 56 months, 95% CI: 35-77 months). Patients with a low JAK2V617F burden had an adverse outcome (n = 115, OS: 45 months, 95% CI: 38-52 months; Figure 2). The risk of transformation to acute myeloid leukemia was similar across groups except that patients with a low JAK2V617Fburden had a lower risk of transformation (hazard ratio = 0.27, 95% CI: 0.1-0.8).ConclusionOn the basis of mutation status in JAK2, CALR, and MPL, we stratified patients into 3 groups with good (CALR or MPL mutation or high JAK2V617F burden), intermediate (triple negative), or adverse (low JAK2V617Fburden) survival outcomes. Our results indicate that prognostication based on mutation status is independent of and superior to the IPSS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rapid Clearance Of JAK2 V617F Allele Burden In Patient With Advanced Polycythemia Vera (PV) During Combination Therapy With Ruxolitinib and Peg-Interferon Alpha-2a

BackgroundTreatment with pegylated interferon-alpha2 (IFN) is associated with complete hematologic remissions (CHR) in a large majority of patients with essential thrombocythemia (ET) and PV. In a subset of patients, IFN can induce major molecular remissions that can be sustained for up to 4 years after discontinuation of therapy. In hypercellular myelofibrosis (MF), IFN may also induce regression of bone marrow fibrosis. To this end, long-term IFN treatment with normalization of elevated blood counts is accompanied by a reduced risk of thrombosis and hemorrhage. When initiated at the early disease stage, IFN may have the greatest chance to induce “minimal residual disease” with a low JAK2 V617F allele burden. For these reasons, IFN is foreseen to be a cornerstone in the treatment of these neoplasms. Ruxolitinib (RUX) is a JAK1-2 inhibitor that is highly efficacious in alleviating hypermetabolic symptoms and enlarged spleens in patients with MF. Although the earliest reports suggested that RUX did not significantly lower JAK2 V617F mutational load, newer findings clearly show that in a subset of patients RUX may indeed profoundly decrease JAK2 V617F allele burden; these reductions could be even larger if treatment is instituted in early stages of MF or PV. Preliminary data show RUX to be highly efficacious in PV as well. With both drugs having potent antiproliferative and anti-inflammatory effects, combination therapy with IFN and RUX may potentially promote tumor regression in MPNs, as assessed by rapid induction of CHR, reduction in spleen size, and enhanced reduction of JAK2 V617F allele burden. Case reportA 55-year-old woman with PV diagnosed in the precursor stage of ET 12 years ago was referred for a second opinion due to intolerance of hydroxyurea (HU; monosymptomatic fever) and pegylated interferon-alpha2b (PEG-Intron; exanthema). The patient had suffered 2 episodes of transitory ischemic attack (TIA) since 2009, and therefore, permanent treatment with clopidogrel had been instituted. Several CT scans were normal. At the time of referral, the patient was receiving anagrelide and clopidogrel. The patient's hemoglobin was 11.9 g/dL, her leukocyte count was 21.9 x 109/L, and platelets were elevated at 526 x 109/L. CRP levels were normal. An abdominal ultrasound revealed a spleen length of 20 cm, causing intermittent pain and abdominal discomfort. Because of hypermetabolic symptoms, pronounced abdominal discomfort, and intolerance of PEG-Intron and HU, treatment with RUX was initiated at a dose of 20 mg twice daily. Within the first 3 days of RUX therapy, the patient experienced remarkable clinical improvement with resolution of severe fatigue, night sweats, abdominal pain, and pruritus, which had negatively impacted the patient's quality of life during the past 2 to 3 years. Four days after starting RUX, the patient experienced acute TIA-like symptoms with transient decrease of strength in the left arm and abnormal sensations in the left half of the tongue and neck. The symptoms were similar to those that the patient experienced during previous TIAs. The blood values disclosed a slight decrease in the leukocyte count (17.6 x 109/L) and an increase in the platelet count (573 x 109/L). A CT scan was normal. Consequently, RUX therapy was combined with PEG-IFN-alpha2a (Pegasys) at a very low dose of 45 µg every second week. After 1 month, a CHR was achieved (Figure 1), and after 2.5 months, an abdominal ultrasound showed a spleen length reduction from 20 to 13.8 cm. After 6 months, the spleen was no longer palpable. The patient had no need of phlebotomies during treatment with RUX and Pegasys. The combination therapy was exceedingly well tolerated without any side effects to either drug. Remarkably, despite an advanced PV stage, JAK2 V617F allele burden was rapidly lowered from 90% mutated alleles at referral to 59% at 6 months and 20% within 1 year (Figure 2). [Display omitted] ConclusionThis case report demonstrates for the first time that combination therapy with RUX and “low–low-dose” Pegasys is highly efficacious in rapidly reducing JAK2 V617F allele burden in concert with achieving CHR and resolution of splenomegaly. This observation warrants prospective trials of this combinatorial approach in patients with PV and hyperproliferative MF to assess whether combination therapy induces a more rapid decline in the JAK2 V617F allele burden than monotherapy with either drug. Disclosures:Hasselbalch:Novartis: Consultancy, Honoraria, Speakers Bureau. Off Label Use: Interferon-alpha2a is used routinely in Denmark for treatment of essential thrombocythemia polycythemia vera and in some myelofrbrosis patients , as well. Ruxolitinib is being used routinely in myelofibrosis patients on strict indications including hypermetabolic symptoms and abdominaldiscomfort due to large splenomegaly ln rare cases Ruxolitinib is being used in the transitional stage of PV towards myelofibrosis when other options are not available due to intolerance to eg. interferonlor hydroryurea.

Age-related differences in disease characteristics and clinical outcomes in polycythemia vera

The natural history and prognosis for young patients with polycythemia vera (PV) in the post-JAK2 V617F era are not well defined. Therefore, we retrospectively analyzed disease characteristics and clinical outcomes in 120 patients ≤ 45 years and 84 patients ≥ 65 years at diagnosis. Despite lower white blood counts (9.2 vs. 13.4 × 109/L, p = 0.004) and a lower JAK2 V617F allele burden (51% vs. 66%, p = 0.015), younger patients with PV had comparable rates of vascular complications compared to older patients (27% vs. 31%, p = 0.64). However, splanchnic vein thrombosis occurred more frequently in younger patients (13% vs. 2%, p = 0.0056). Myelofibrotic and leukemic transformation, the most serious complications of myeloproliferative neoplasms (MPN), occurred with similar frequencies in young versus older patients (15% vs. 10%, p = 0.29). Prevention or delay of these complications is currently the most urgent challenge in the care of younger patients with PV.

Clinical Implications for Patients with Low JAK2V617F Allele Burden

AbstractAbstract 1745In classical Philadelphia-negative myeloproliferative neoplasms (MPN), JAK2V617F is considered as a driver mutation when the threshold of 1% JAK2V617F/JAK2total allele burden is reached. However a lower ratio is sometimes detected with highly sensitive assays. We investigated the clinical significance of such minor clones by describing the characteristics of 27 patients with a low JAK2V617F allele burden ranging from 0.1% to 0.99%. Material and MethodsA commercially available quantitative ASO-PCR assay of 0.1% sensitivity (MutaQuant® kit, Ipsogen) was used. Two thousand five hundred consecutive blood samples were sent to our lab for JAK2V617F mutation between 2009 and 2012. Total blood DNA was extracted by an automated standardized procedure (Qiasymphony®, Qiagen). All samples were tested in duplicate. The 27 samples of our cohort were controlled using a second assay of 0.01% sensitivity (Larsen et al, BJH 2007). Thirty samples from healthy donors were also tested. High resolution melting curve (HRM) analysis of JAK2 exon 14 ruled out the possibility of an additional mutation hampering the annealing of a primer. Patients with a known classical MPN clinical phenotype were also tested for JAK2 exons 12–17 (entire pseudo-kinase domain) or for MPL exon 10 depending on the context.Results Laboratory FindingsAmong the 2500 samples, 735 (29.4%) were positive above 1%, 27 (1.1%) had low JAK2V617F allele burden ranging from 0.12 to 0.99%. The patient with the lowest ratio (0.12%) was not confirmed by the second assay and therefore was excluded from the study. This allowed the median to settle at 0.40%. No associated mutations were found in the JAK2 pseudo-kinase domain in patients with polycythemia vera (PV) and in MPL exon 10 in patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF). Healthy patients were all tested JAK2V617F negative. Clinical AspectsThe cohort included 19 men and 7 women ranging from 28 to 95 years of age (median 63 years old).Two patients had secondary acute myeloid leukaemia following JAK2V617F positive MPN indicating the presence of residual JAK2V617F cells and the negativity of the myeloblastic population.Thirteen patients (50%) had a classical MPN with a median ratio of 0.36%: 7 ET, 5 PV and 1 PMF according to WHO 2008 criteria. However a bone marrow biopsy was available for only two patients (1 ET, 1 PMF). None of them had received pegylated interferon alpha-2a. Four patients had a prior history of thrombosis: two strokes, one pulmonary embolism, two portal vein thrombosis (PVT). For one PV patient, a 6 months follow-up blood and bone marrow sample confirmed a low allele burden in the same range (0.4%) and in vitro Epo-independant erythroid colonies were observed.Five patients had other chronic myeloid neoplasms (two myelodysplastic/myeloproliferative neoplasms, one chronic eosinophilic leukaemia, one chronic myeloid leukaemia, one refractory anaemia with ring sideroblasts). Among these five, four had an abnormal karyotype. We did not observe any thrombotic event in these patients.We cannot conclude on hematological diagnosis for the last six patients: four patients were screened for JAK2 mutation because of PVT. One patient had chronic polycythemia in a context of alcohol and tobacco abuse. One patient had homozygous hemochromatosis with a normal haemoglobin level in spite of repeated phlebotomies. DiscussionIn this single centre study low JAK2V617F allele burden represented 1% of all samples sent for JAK2V617F study and 3.5% of JAK2V617F positive patients.Seventeen patients (65%) had classical MPN or splanchnic vein thrombosis. To our knowledge PV patients with such low JAK2V617F allele burden have not been reported in the absence of associated JAK2 pseudo-kinase domain mutation. A larger screen for cooperating mutations responsible for the PV phenotype is under process.In the context of other chronic myeloid neoplasms, the JAK2V617F mutation is thought to belong to a more complex clonal architecture mostly implicating chromatin remodeling genes. Here, the presence of a JAK2 mutation could argue in favour of clonal haematopoiesis.In conclusion the clinical phenotype of low JAK2V617F patients overlaps with classical JAK2V617F MPN. The technical implications might be challenging for molecular diagnostic platforms. More data are needed to further characterize these patients. Disclosures:No relevant conflicts of interest to declare.

The JAK2V617F tyrosine kinase mutation has no impact on overall survival and the risk of leukemic transformation in myelofibrosis

The prevalence of JAK2V617F tyrosine kinase mutation differs between various variants of myelofibrosis with the higher detection rate for patients with post-polycythemia vera myelofibrosis (post-PV MF; 91%) if compared to primary myelofibrosis (PMF; 45%) and post-essential thrombocythemia myelofibrosis (post-ET MF; 39%). The impact of V617F point mutation and its allele burden on overall survival (OS) and the risk of leukemic transformation (LT) has been the subject of several studies, but the results were ambiguous. Our study included 77 patients with the following variants: 42 patients with PMF (55%), 16 with post-ET MF (21%) and 19 with post-PV MF (24%). Median age at diagnosis for the entire cohort was 61 years (range 19-81), with 53% of female. A total of 42 patients were JAK2V617F positive, giving an overall frequency of 55%; the median allele burden was 22% (range 2-96%). The JAK2V617F point mutation was detected in 21 patients with PMF (50%), 14 with post-PV MF (88%) and 7 with post-ET MF (37%). Lower JAK2V617F allele burden was more frequently detected in PMF patients, whereas higher allele burden was predominantly seen in post-PV/ET MF group. There was no significant difference between V617F-positive and V617F-negative patients in terms of studied parameters in PMF as well as in post-PV/ET MF subgroup. No significant difference was also demonstrated when the above-mentioned subpopulations were analyzed according to JAK2V617F allele burden, except higher leukocyte count in post-PV/ET MF patients with higher allele burden (14.3×10(9)/L vs. 6.2×10(9)/L; p=.03). Median follow-ups for V617F-positive and V617F-negative patients were 16.6 months (range 3.6-206.4) and 36.4 months (range 2.5-142.1), respectively. The presence of JAK2V617F mutation did not affect OS and the risk of LT development.

Prognostic Impact of EZH2 and ASXL1 Mutation in Myelofibrosis

Abstract▪2811▪This icon denotes a clinically relevant abstractAmong chronic myeloproliferative neoplasms (MPN) myelofibrosis (MF) has the worst prognosis. The International Prognostic Score System-IPSS reliably discriminates four categories of patients (pts) with different survival (OS); however, the possibility to identify pts with very high-risk disease, based on intrinsic characteristics of the underlying disease, would be particularly useful for therapeutic choices that include early stem cell transplantation. Several studies have addressed the prognostic value of molecular abnormalities harbored by MF pts, but a part from a negative role of low JAK2V617F allele burden on survival independently reported by the Mayo Clinic and our own group, none of them impacted significantly on survival. Here we have genotyped at diagnosis 370 pts with primary MF (PMF) and 148 with post-polycythemia vera/essential thrombocythemia MF (PPV/PET-MF) for mutations of EZH2 and ASXL1. EZH2 is the catalytic component of polycomb repressive complex 2 involved in trimethylation of H3 lysine 27, while ASXL1 is a member of enhancer of trithorax and polycomb group required for repression of HOX genes through deubiquitination of histone H2A. All 20 EZH2 exons were screened by high-resolution melting followed by direct sequencing whereas direct sequencing was used for ASXL1 exon 12 genotype. Pts median FU was 39 mo (1–340); 128 pts died (25.9%), 81 (18.3%) because of leukemia (AL). Among PMF pts survival differed significantly according to the IPSS category and was also predicted by the lowest V617F quartile.EZH2 mutation. We found 22 PMF (6%), 1 PPV-MF (1.2%) and 6 PET-MF (9.4%) mutated pts, harboring 25 different mutations, 20 exonic (heterozygous in 83%), 5 intronic. Concurrent JAK2 V617F, MPL W515L/K, IDH1/2, TET2, CBL and ASXL1 mutations were found in 41.4%, 0%, 0%, 6.9%, 3.4% and 20.7% of EZH2 -mutated pts. In multivariate analysis EZH2 mutation was associated with leukocytosis (12.3±13.2 vs 18.7±11.5×10e9/L) in PMF pts, but no additional clinical/hematological correlation was discovered. EZH2 mut pts clustered preferentially in the IPSS high-risk category (52.6%; P=.002). More EZH2 mut than wt PMF pts died (51.9% vs 24.4%; P<.001); median OS was shorter in EZH2 mut pts (32 vs 137 mo, P=.001). Leukemia occurred in 31.8% of EZH2 mutated and 17.6% of wt pts; leukemia-free survival (LFS) was shorter in EZH2 mut PMF pts (25.7 vs 63.6 mo; P=.028).ASXL1 mutation. In a total number of 334 evaluated patients we found 38 different mutations in 49 pts (14.7%), 35 PMF (15%), 5 PPV-MF (8.6 %) and 9 PET-MF (21.4%). Mutations were frameshift, non sense and missense mutations, heterozygous in 46 of 49 cases. Co-expression of JAK2 V617F, MPL W515L/K and EZH2 mutation was found in 55.1%, 4.1% and 12.2%, respectively. Co-espression of ASXL1 and IDH1/2, TET2 or CBL was observed in two, one and one of the evaluable subjects, respectively. ASXL1 mutation was associated with leucocytosis (P=.006), a blast count >1% (43.8% vs 16.6%, P<.0001) and constitutional symptoms (P=.027). ASXL1 mutated pts clustered preferentially in the IPSS high-risk category (46.4%; P<.0001). OS was significantly shortened in ASXL1 mutated PMF patients (median= 87.6 mo vs 264; P=.004); leukemia occurred in 34.9% of ASXL1 mutated and 15.2% of wt pts (P=.002). LFS was shorter in ASXL1 mutated PMF patients [137 mo than wt 264 mo, (P=.01)].According to EZH2 and ASXL1 mutational status, median OS was significantly shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (34 vs 116 vs 198 vs 272 mo, respectively. P<.0001). On multivariate analysis, OS was predicted by IPSS score (P<.001), ASXL1 (P=.016) and EZH2 mutational status (P=.011). Leukemia-free survival (LFS) was shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (25 vs 153 vs 138 vs 264 mo; P=.002), but the low number of cases (n=6) prevented multivariate analysis.No impact of EZH2 or ASXL1 mutations on OS or LFS was seen in PPV/PET-MF.Overall, these results showed that mutations in genes involved in epigenetic gene regulation such as EZH2 and ASXL1 negatively impact on OS and LFS in pts with PMF, suggesting that screening for these mutations might be useful for risk stratification and treatment decisions. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria.