JAK2V617F Mutant Allele Burden Research Articles

Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR.

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph-MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly β2-microglobulin (β2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.

Myeloproliferative Neoplasm Symptom Assessment Total Symptom Score (MPN-SAF TSS) in Chronic Myeloproliferative Neoplasms, Related with Genetic Burden, and Thrombosis.

Myeloproliferative Neoplasm Symptom Assessment Total Symptom Score (MPN-SAF TSS) is a surrogate marker for symptom evaluation in Chronic Myeloproliferative Neoplasms. There is not enough data to show the relationship between MPN-SAF TSS, JAK2 mutation allele burden, and thrombosis. In this retrospective analysis, we aimed to determine the genetic burdens, clinical features, and relationship with MPN-SAF TSS in MPN patients. One hundred thirty JAK2V617F positive MPN were included in our study. We have calculated MPN-SAF TSS and compared it with clinical features. Patients with higher JAK2V617F mutation allele burden had higher MPN-SAF TSS (p-value 0,008). Patients with thrombosis had higher MPN-SAF TSS scores than patients without thrombosis (p-value 0.003). The mean MPN-SAF TSS was higher in primary myelofibrosis (PMF) patients compared to PV and ET patients. Thrombosis was associated with increased symptom severity in several domains, including fatigue, abdominal discomfort, inactivity, night sweats, pruritus, weight loss, and early satiety. Additionally, an increase in JAK2 allele burden was observed with higher symptom scores. The MPN-SAF TSS proved to be a reliable tool for assessing symptom burden in Turkish MPN patients. Furthermore, a significant association between thrombosis occurrence and symptom severity suggests that thrombotic events may contribute to symptom development. Notably, increasing JAK2 allele burden was correlated with more severe symptoms, highlighting its potential role in predicting disease burden. This study emphasizes the importance of symptom assessment in MPN patients and supports the incorporation of MPN-SAF TSS in routine clinical practice to enhance patient care and management.

Comparison of Clinical and Molecular Features Between Patients With Essential Thrombocythemia and Early/Prefibrotic Primary Myelofibrosis Presenting With Thrombocytosis in Taiwan.

The clinical presentations of essential thrombocythemia (ET) may be quite similar to early/prefibrotic primary myelofibrosis (pre-PMF), especially in pre-PMF presenting with thrombocytosis (pre-PMF-T), but may be associated with a different outcome. It is very important to distinguish these two entities. The aim of this study was to address the clinical and prognostic relevance of distinguishing pre-PMF-T from ET. All patients, including 258 with ET and 105 with pre-PMF-T, received JAK2V617F, MPL (exon 10), and CALR (exon 9) mutation analysis and allele burden measurement for JAK2V617F and CALR mutants. Patients with pre-PMF-T had an older age and higher leukocyte and platelet counts but lower hemoglobin levels than patients with ET. Patients with pre-PMF-T had a shorter overall, leukemia-free, and thrombosis-free survival compared with patients with ET. Patients with ET had a higher rate of cerebral ischemic stroke, whereas patients with pre-PMF-T tended to have splanchnic vein thrombosis. The frequencies of JAK2V617F, CALR, and MPL mutations and CALR allele burden were no different, but JAK2V617F allele burden was significantly higher in pre-PMF-T. Patients with pre-PMF-T with the JAK2V617F mutation had an inferior overall survival and thrombosis-free survival, whereas the status of driver gene mutations did not influence the outcomes of patients with ET. ET and pre-PMF-T were two distinct disease entities and exhibited different clinical phenotype, genotype, and outcomes.

JAK2 V617F Mutation and Associated Chromosomal Alterations Involving JAK2 in Pre-HCT Blood Samples and Transplant Outcomes in Myelofibrosis Subtypes

Introduction: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm that develops either de novo (primary myelofibrosis; PMF) or secondary to polycythemia vera (post-PV) or essential thrombocythemia (post-ET). JAK2 V617F is the most common somatic driver mutation in MF known to associate with disease progression. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment modality for MF. There are limited data regarding the prognostic utility of JAK2 V617F mutation and mosaic chromosomal alterations (mCAs) spanning JAK2 in MF patients undergoing HCT. Methods: The study included 924 patients undergoing HCT for myelofibrosis (634 PMF, 135 post-PV, and 155 post-ET) between 2000-2016. Pre-HCT peripheral blood samples, clinical and post-HCT data were provided by the Center for International Blood and Marrow Transplant Research (CIBMTR) registry and biorepository. We used PacBio Single Molecule Real-Time (SMRT) sequencing to detect the JAK2 V617F mutation, and Illumina Infinium Global Screening Array-24v1-0 to identify mCAs; this analysis focused on mCAs involving JAK2 (9p24.1). We used the Kaplan-Meier estimator to calculate the probability of overall survival (OS), and the cumulative incidence estimator for relapse/disease progression incidence and non-relapse mortality (NRM), where each was treated as a competing event for the other. Cox proportional hazard models were used for multivariable analyses. Analyses were completed separately by MF disease subtype. Follow up started at date of HCT. Results:JAK2 V617F mutation was detected in 562 patients (60.8%) with noted expected differences by disease subtype (57.6% of patients with PMF, 97% of post-PV, and 42.6% of post-ET). Almost all patients with mCAs involving JAK2 region were found to be JAK2 V617F mutation-positive (366 out of 374, 97.9%). In JAK2 V617F positive cases, the frequencies of mCA-positive patients per MF subtypes were as follow: 58.08% of PMF, 89.3% of post-PV, and 56.06% of post-ET. The mean allele burden for JAK2 V617F mutation was 57%, 78%, and 60% in PMF, post-PV, and post-ET, respectively. The probabilities of 3-year post-HCT OS were 55% (95% CI=51-58) for PMF, 59% (95% CI=51-67) for post-PV and 59% (51-66) for post-ET. The corresponding 3-year post-HCT cumulative incidences for relapse were 31% (95% CI=28-35), 31% (95% CI=24-40), and 33% (95% CI=26-41); and for NRM were 29% (95% CI=26-33), 25% (95% CI=18-32), and 23% (95% CI=17-30), for PMF, post-PV, and post-ET, respectively. In PMF, JAK2V617F mutation, JAK2 mCAs, or mutation allele burden were not associated with any of the evaluated HCT outcomes (p>0.05 for all). In post-PV, all four patients without JAK2V617F mutation died in the first 12 months after transplantation. No statistically significant associations between HCT outcomes and JAK2V617F allele burden was noted (p>0.05). For post-ET, patients with high mutation allele burden (³60%) had three-fold excess risk of NRM compared with mutation-negative patients (hazard ratio [HR]=2.9, 95% CI=1.21-6.93, p=0.01). No risk difference was noted between post-ET patients with low allele burden and risk of NRM (HR=0.89, 95% CI=0.34-2.31). No statistically significant association between JAK2V617F mutation status and risk of relapse for high or low mutation allele burden as compared with mutation-negative post ET patients was observed (HR=1.42, p=0.30 in patients with allele burden<60%, and HR= 1.54, p=0.26 for patients with allele burden³60%). JAK2V617F mutation status in post-ET patients was not associated with post HCT OS (HR=1.31, 95% CI=0.78-2.2, p=0.31). Conclusion: The effect of JAK2V617F mutation on post-HCT outcome differs by myelofibrosis subtypes. HCT outcomes in primary and post-PV myelofibrosis were not associated with JAK2 alterations, but high JAK2V617F mutation allele burden in post-ET was associated with elevated risk of non-relapse mortality but did not translate to a statistically significant survival disadvantage. Our findings suggest that allogeneic HCT for myelofibrosis may alleviate the known negative effect of JAK2V617F mutation in those patients, particularly for primary myelofibrosis and post-PV. It is essential to consider evaluating these findings within the context of other MF driver genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prevalence of the JAK2V617F Mutation in Patients with Ischemic Stroke

Introduction: Patients with the Philadelphia negative myeloproliferative neoplasms (MPN) have an increased risk of thromboembolic events, ischemic cerebrovascular events in particular. The somatic mutation JAK2V617F is associated with MPN with a prevalence of 96% in polycythemia vera (PV), 55% in essential thrombocythemia (ET), and 65% in primary myelofibrosis (PMF). The JAK2V617F mutation is found to be an independent risk factor for thrombosis in patients with MPN. However, whether the JAK2V617F mutation is a risk factor for arterial thrombosis in the absence of an MPN diagnosis is still uncertain. The sensitivity of the assay used for detection is crucial when assessing the presence of the JAK2V617F mutation. A higher sensitivity enables detection of lower allele burdens. This is illustrated by the varying prevalences (0.1-3.1%) in background population studies. In the Danish population study GESUS of approximately 20.000 individuals, the JAK2V617F mutation was detected by droplet digital PCR (ddPCR) with an assay sensitivity of 0.009%. The GESUS study found a JAK2V617F prevalence of 3.1%. Similarly, prevalence studies of JAK2V617F mutation in stroke have varying detection limits from 0.01-5% mutant alleles. The vast majority of these studies are retrospective studies in younger subpopulations of stroke patients. We aimed to investigate the prevalence of the JAK2V617F mutation in a prospective design of consecutively enrolled unselected patients with ischemic stroke or transient ischemic attack by the highly sensitive ddPCR. Method: We recruited 90 consecutive patients with ischemic stroke or transient ischemic attack during admission at the Department of Neurology, Zealand University Hospital, Denmark. A blood sample was taken within 7 days of the onset of symptoms and screened for the JAK2V617F mutation by ddPCR. Patients unable to give informed consent were excluded from the study. Results: Out of 90 patients (mean age 71, range 46-92; female 37%) with ischemic stroke or transient ischemic attack, the JAK2V617F mutation was found in 11 patients (12.2%). Allele burdens ranged from 0.0021 to 24. All 11 patients suffered an ischemic stroke. Six out of 11 patients with the JAK2V617F mutation had elevated blood cell levels i.e. erythrocytosis, thrombocytosis, and/or leukocytosis at the time of recruitment. Only three patients had continuously elevated blood cell levels after 1 month. Patient no. 6 with a JAK2V617F mutation allele burden of 34% had blood cell levels meeting the diagnostic criteria for MPN and was diagnosed with ET. Patient no. 2 had a JAK2V617F allele burden of 14% and increased levels of hemoglobin and platelet count, which both normalized spontaneously after 1 month. The patient was referred for a hematological workup and was found to have mild splenomegaly and a bone marrow biopsy showing myeloproliferative features. In light of the normal blood cell parameters, patient no. 2 was diagnosed with MPN-U. The patients with a JAK2V617F mutation are presented in table 1. Additional data will be presented at ASH. Conclusion and implications: The present study reveals a JAK2V617F mutation prevalence (12.2%) in a stroke population that is substantially higher compared to the background population (GESUS) in the same region of Denmark, using the same assay. The GESUS study showed a JAK2V617F mutationprevalence of 3.1% and for individuals above age 60 a prevalence of 4.1%. Given the fact that the JAK2V617F mutation is present in patients with and without cytosis, our results highly suggest that the JAK2V617F mutation per se is a thrombogenic factor in ischemic stroke. This calls for an exploration of the pathophysiological mechanisms and potential treatments to prevent future thrombotic events in individuals with and without cytosis. Table legend: Clinical and paraclinical characteristics of ischemic stroke patients with the JAK2V617F mutation. IS, ischemic stroke; TIA, transient ischemic attack; HTN, hypertension; HLP, hyperlipidemia; AF, atrial fibrillation; IHD, ischemic heart disease; DM2, diabetes mellitus type 2; Hb, hemoglobin; PLT, platelet count; WBC, white blood cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Micheliolide Exerts Therapeutic Effect in Myeloproliferative Neoplasms through Binding to STAT3 and STAT5 Proteins and Inhibiting Their Activities

The JAK1/2 inhibitor ruxolitinib is a cornerstone of management for some subsets of BCR-ABL negative myeloproliferative neoplasms (MPN). While many patients benefit with reduced splenomegaly and symptoms, some patients respond suboptimally to ruxolitinib, and even patients with a good initial response often develop resistance to ruxolitinib after several years of therapy. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, in the JAK2V617F positive MPN entities. We firstly evaluated the effect of MCL treatment on cell growth and survival in human MPN cell lines expressing the JAK2V617F mutation. MCL alone reduced cell viability and induced apoptosis of UKE1 and SET2 cells in a dose-dependent manner, and obviously improved the effect of ruxolitinib as monotherapy. Importantly, treatment with MCL also induced significant apoptosis of UKE1 cells which were selected for resistance against ruxolitinib. We further explored the therapeutic potential of MCL in vivo using a Jak2V617F knock-in (Jak2VF) mouse model, and found that DMAMCL, an orally available derivative of MCL, significantly increased ruxolotinib efficacy in Jak2VF mice, manifested as amplified hematological and spleen responses. What's more, DMAMCL, whereas not ruxolitinib, contributed to reduce the in vivo Jak2V617F mutant allele burden reflected by the percentage of mutant hematopoietic cells in peripheral blood and bone marrow. To determine the molecular mechanisms by which MCL exerts efficacy in MPN, we firstly performed western blot (WB) anlyses to evaluate the effect of MCL on hyper-activated JAK-STAT signaling, which is the cornerstone of MPN pathogenesis. We observed a dose-dependent inhibition of STAT3 and STAT5 phosphorylation caused by MCL treatment in UKE1 and SET2 cells (Figure 1A), and as expected, combination MCL with ruxolitinib provided an enhanced inhibitory effect. By next performing the affinity pull-down experiment, we confirmed that MCL suppressed the activation of STAT3 and STAT5 mainly through directly binding to the proteins (Figure 1B). We further conducted RNA-sequencing analysis in UKE1 cells treated with MCL and/or ruxolitinib to gain insights into mechanisms involved in MCL effect. Gene set enrichment analysis (GSEA) showed that genes related to TNF-α-NF-κB signaling and inflammatory response pathway were significantly downregulated upon treatment with MCL, that were verified by WB analyses and real-time quantitative PCR. Additionally, GSEA also revealed significant upregulation of genes related to reactive oxygen species (ROS) and unfolded protein response (UPR) pathway, respectively. Both flow cytometry and fluorescent microscopy imaging confirmed the ROS accumulation by MCL treatment. We also determined the activation of PERK branch of UPR pathway caused by MCL, as assessed by increased phosphorylation of eIF2α, and upregulation of pro-apoptotic genes, including ATF4, CHOP and GADD34. Finally, by addition of antioxidant agent, the activation of UPR pathway and increase of apoptosis in UKE1 cells induced by MCL were significantly rescued, indicating that MCL exerts cytotoxic effects mainly through triggering the oxidative stress in MPN cells. In summary, our results demonstrate that MCL is an effective agent with multiple biological activities in MPN, especially inhibition of STAT3/5 activity via directly binding to the proteins, and may be a promising drug combined with ruxolitinib to benefit patients who have suboptimal response or acquire resistance to ruxolitinib. Figure 1. MCL inhibits STAT3 and STAT5 phosphorylation through binding to the proteins directly. (A) Western blot analyses are shown for phosphorylated-STAT3 (p-STAT3), STAT3, phosphorylated-STAT5 (p-STAT5) and STAT5 levels in total cell extracts from UKE1 and SET2 cells treated with MCL (5, 10, 20μM) for 3 hours. (B) The biotin-conjugated MCL (biotin-MCL) and its inactive analogue S-MCL (biotin-S-MCL) were prepared as the positive- and negative- probe respectively. UKE1 and SET2 cell lysates were incubated with 20μM biotin-MCL or biotin-S-MCL for 1 hour at room temperature, then the mixtures were separately pulled down with streptavidin-coated agarose beads, and STAT3/STAT5 were detected with WB analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Using a Humanized Animal Model to Determine the Minimum JAK2V617F Mutant Allele Burden Required to Generate MPN Pathologies

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases driven by somatic mutations acquired in hematopoietic stem cells (HSCs) and characterized by the deviant, unregulated proliferation of one or more myeloid lineages. Chronic MPN can present as polycythemia vera (PV; excess erythrocytes), essential thrombocythemia (ET; excess platelets), or myelofibrosis (MF; bone marrow fibrosis). More than 300,000 patients are currently living with an MPN diagnosis in the United States. The JAK2V617F variant is an acquired somatic mutation recurrently found in MPN patients and is used as a diagnostic marker along with increased hematocrit, increased hemoglobin, and adverse thrombotic events. In MPN patients, the frequency of JAK2V617F mutant cells varies widely but is known to induce an MPN phenotype with both high (>50%) and very low (<5%) variant allelic frequency (VAF). The reason for this is currently unknown. Most JAK2-mutant animal models model a VAF which is much higher than the lower end of detectable VAFs found in MPN patients. This results in a very limited understanding of what the minimal mutant cell burden required for disease manifestation is. The understanding of genotype/phenotype relationships in patients with low JAK2V617F VAF chronic MPN is paramount to our understanding of this disease and its hematological progression. To this end, we have designed a clinically relevant, humanized mouse model wherein we employ CRISPR and homology directed repair (HDR) via a single stranded oligo donor nucleotide sequence (ssODN) to knock-in the JAK2V617Fmutation at the endogenous locus of human cells at low VAF. In addition, a synonymous JAK2V617V mutation was introduced via CRISPR/ssODN in parallel cells to serve as a control tracked by VAF alongside the VAF of JAK2V617F in their corresponding cohorts. The respective ssODNs were nucleofected into either umbilical cord blood (UCB) derived CD34+ cells or healthy adult bone marrow (BM) derived CD34+ cells to investigate the possibility that developmental age is a variable in low JAK2V617F VAF inducing a clinical MPN phenotype. After nucleofection, 20,000 CD34+ cells were intra-tibially transplanted into sub-lethally irradiated (250 rads) NSGS mice. The low cell input was utilized to limit engraftment and initial disease burden of mutant cells. Effect was measured by mutant allele burden by droplet-digital PCR (ddPCR) analysis, flow cytometry at selected timepoints to track human cell engraftment, spleen weight, and complete blood count (CBC) analysis including hematocrit, hemoglobin, white blood cells, and platelets. At 4 months post-transplant, peripheral blood hCD45+ engraftment across all cohorts averaged less than 3%. Average VAF of circulating hCD45+ cells increased in both JAK2V617F cohorts 5 months post-transplant compared to day 0 post nucleofection. Notably, overall JAK2V617F VAF in each sample remained <5% when adjusted to include both human and mouse derived peripheral blood cells. Remarkably, both hematocrit (p= .037 in UCB cohorts and p=.0096 in BM cohorts; Figure 1A) and hemoglobin (p=.046 in UCB cohorts and p=.0026 in BM cohorts; Figure 1B) were significantly elevated in both JAK2V617F cohorts compared to their respective JAK2V617V controls, while white blood cell and platelet counts remained unchanged within normal ranges. In addition, spleen weights were significantly increased in both JAK2V617F cohorts compared to their respective controls. These data demonstrate a successful clinical model of MPN which induces a classical MPN phenotype where a low VAF of JAK2V617F corresponds with a significant increase of hematocrit, hemoglobin, and spleen weight in a humanized mouse model compared to relevant controls. In addition, we demonstrate that while engraftment due to low hCD34+ cell input remains low after 4 months, the VAF of JAK2V617F within the hCD45+ sub- population increases. This successfully enables the overall VAF contributing to the MPN phenotype to remain at clinically relevant lower levels compared to other models with much higher VAF contributions. We believe this model to be of high clinical relevance into the investigation of how low JAK2V617F burden relates to disease development and progression in MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IRF4 and IRF8 expression are associated with clinical phenotype and clinico-hematological response to hydroxyurea in essential thrombocythemia.

The morbidity and mortality of myeloproliferative neoplasms (MPNs) are primarily caused by arterial and venous complications, progression to myelofibrosis, and transformation to acute leukemia. However, identifying molecular-based biomarkers for risk stratification of patients with MPNs remains a challenge. We have previously shown that interferon regulatory factor-8 (IRF8) and IRF4 serve as tumor suppressors in myeloid cells. In this study, we evaluated the expression of IRF4 and IRF8 and the JAK2V617F mutant allele burden in patients with MPNs. Patients with decreased IRF4 expression were correlated with a more developed MPN phenotype in myelofibrosis (MF) and secondary AML (sAML) transformed from MPNs versus essential thrombocythemia (ET). Negative correlations between the JAK2V617F allele burden and the expression of IRF8 (P < 0.05) and IRF4 (P < 0.001) and between white blood cell (WBC) count and IRF4 expression (P < 0.05) were found in ET patients. IRF8 expression was negatively correlated with the JAK2V617F allele burden (P < 0.05) in polycythemia vera patients. Complete response (CR), partial response (PR), and no response (NR) were observed in 67.5%,10%, and 22.5% of ET patients treated with hydroxyurea (HU), respectively, in 12 months. At 3 months, patients in the CR group showed high IRF4 and IRF8 expression compared with patients in the PR and NR groups. In the 12-month therapy period, low IRF4 and IRF8 expression were independently associated with the unfavorable response to HU and high WBC count. Our data indicate that the expression of IRF4 and IRF8 was associated with the MPN phenotype, which may serve as biomarkers for the response to HU in ET.

JAK2Exon 12 Mutant Allele Burden Study (JETMABS): A European Multicenter Study

BackgroundA subset of polycythemia vera (PV) patients harbor mutations in a discrete region of exon 12 in the JAK2 gene. So far more than 40 individual mutations have been described involving amino acids 533 to 547 including substitutions, deletions, and duplications. Several studies have indicated that a higher JAK2V617F mutant allele burden may be associated with a more extreme PV, but both the variability and rarity of the JAK2exon 12 mutations have limited the number of studies describing how they reflect disease development and influence thrombotic events. To investigate the impact of the JAK2exon 12 mutant allele burden on the disease phenotype and its utility as a prognostic marker, we established a European collaboration termed JAK2Exon 12 Mutated Allele Burden Study (JETMABS). ResultsEighty six samples from 66 individual JAK2exon 12 positive PV patients (median age 64 years, range 32-88 years) were collected from participating centers and mutant allele burden was determined by allele specific qPCR evaluating 11 different JAK2exon 12 mutations. Depending on the participating center the DNA was either purified from whole blood (n=36; mutated alleles, median 20%, range 1.0-99%) or from granulocytes (n=30; mutated alleles, median 21%, range 8.9-51%). These were divided into separate groups as purification of DNA from granulocytes appeared to result in a slightly higher estimated mutant allele burden, as expected. The vast majority of patients had a mutant allele burden below 50% regardless of the source of DNA (60 out of 64 patients). Both hemoglobin (Hb) (Spearman r=0.41, P<0.05) and hematocrit (Hct) (Spearman r=0.49, P<0.05) levels correlated significantly with the mutant allele burden in the samples purified from granulocytes. The pre-diagnosis thrombotic events did not correlate with the mutant allele burden, but the two cases with the highest mutant allele burdens (88% and 99%) were the only ones to experience thrombotic events post-diagnosis. Four patients developed non-hematological secondary cancers, two patients progressed to post-PV myelofibrosis, and one patient developed AML. ConclusionThe JAK2exon 12 mutant allele burden did not seem to influence the pre-thrombotic events in PV patients. The allele burden only exceeded 50% in a few cases, which was associated with post-thrombotic events. Although this does not seem to reflect the dynamics of the more aggressive JAK2V617F counterpart, these few patients may require a more intensive treatment strategy. Disclosures:No relevant conflicts of interest to declare.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality

AbstractWe studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

IL6 inhibition of inflammatory S100A8/9 proteins is NF-κB mediated in essential thrombocythemia.

This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34+ cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-κB, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-κB and inflammation signalling in MPN-derived CD34+ cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-κB and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. SIGNIFICANCE OF THE STUDY: We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-κB and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.

Germline Genetic Factors Affecting Interferon Alpha Therapy in Polycythemia Vera

Introduction: Interferon alpha (IFNa) based therapies can induce sustained hematological and molecular responses (HR/MR) in Polycythemia Vera (PV) and other Myeloproliferative Neoplasms (MPN), however, not all patients respond sufficiently. While previous studies suggest that disease driving somatic mutations and genomic aberrations do not predict response to IFNa, the role of common germline polymorphisms remains elusive. We addressed the effect of germline genetic factors on PV therapy with Ropeginterferon alfa-2b (Ropeg), a novel monopegylated IFNa. We performed genome-wide association studies (GWAS) as an unbiased approach, and additionally evaluated a previously reported influence of polymorphisms at the IFNL4 locus in a large PV patient cohort. Methods: Genomic DNA was isolated from whole blood of PV patients (n=115) on Ropeg therapy, who provided consent within the PROUD-PV (NCT01949805) and CONTI-PV (NCT02218047) study programs. Patients were genotyped for &gt;900k tagging single nucleotide polymorphisms (SNPs) across the genome on the Affymetrix SNP 6.0 array platform. Additional IFNL4 SNPs were typed using an amplicon-based next generation sequencing approach. Association analyses were performed using the PLINK toolset. JAK2-V617F mutant allele burden was quantified using a qPCR-based assay (ipsogen MutaQuant, Qiagen). MR was measured based on changes in mutant allele burden upon therapy as defined by the European LeukemiaNet (ELN) criteria. Results: To test for potential associations between germline genetic variation and response to IFNa therapy, we performed GWAS for MR data upon 12 months (M), 18M, 24M and 30M and 36M follow-up on Ropeg treatment. Genome-wide tagging SNPs were tested both for association in a case-control setup applying chi-square statistics as well as for quantitative trait association, using peripheral blood JAK2-V617F mutational burden changes from baseline as continuous variables. After Bonferroni correction for multiple testing, none of those analyses revealed a statistically significant association, suggesting the absence of strong germline predisposition factors for MR. Germline variation at the interferon-lambda (IFNL) locus was previously reported to strongly influence viral clearance during IFNa therapy of Hepatitis C (reviewed in Wack et al., Nat Immunol, 2015). While the same variants were recently reported to also affect HR during IFNa treatment in an MPN patient cohort (Lindgren et al., EJH, 2018), their potential impact on MR as surrogate marker for the size of the malignant MPN clone has not yet been evaluated. Upon testing for MR in our PV cohort, in a case-control setup we observed a statistically significant association only upon 36M follow-up (p=0.02; OR=2.51; 95%CI=[1.14-5.62]; Figure 1a). Notably, testing for change of JAK2-V617F mutational burden under Ropeg treatment as quantitative trait, we found the association to be present at formal statistical significance at all stages during follow-up (Figure 1a). For the Hepatitis C association, it is now widely acknowledged that a diplotype of two coding variants covers most of the causality, where rs368234815_TT disrupts the open reading frame (no IFNL4) while rs117648444_G results in the impaired IFNL4-S70 in contrast to the fully functional IFNL4-P70, the latter paradoxically impacting negatively on viral clearance (Terczynska-Dyla et al., Nat Commun, 2014). Similarly, in our PV cohort genotyping and phasing of this diplotype revealed a significant influence on MR in carriers of the fully functional IFNL4-P70 (p=0.01; Figure 1b; shown are patients at 36M follow-up (n=70)). Conclusions: The absence of a strong germline predisposition factor for Ropeg treatment response in our cohort implies that any PV patient may be eligible for Ropeg therapy independently of their genetic makeup. While the IFNL4 diplotype is of potential use for patient stratification, it remains to be investigated whether increase of treatment duration and/or dose adjustments can overcome the adverse effect of functional IFNL4 on Ropeg therapy. Longitudinal monitoring of JAK2-V617F mutational burden under Ropeg treatment in conjunction with determination of the IFNL4 germline genetic status may allow for optimizing patient management. Disclosures Krejcy: AOP Orphan Pharmaceuticals AG: Employment. Klade:AOP Orphan Pharmaceuticals AG: Employment. Zoerer:AOP Orphan Pharmaceuticals AG: Employment. Gisslinger:Janssen-Cilag: Honoraria; Roche Austria GmbH: Consultancy; Novartis Pharma GmbH: Consultancy, Honoraria, Research Funding; Myelopro GmbH: Consultancy; Celgene GmbH: Honoraria; Pharma Essentia: Other: Personal fees; AOP Orphan Pharmaceuticals: Consultancy, Honoraria, Research Funding. Kralovics:Novartis: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Other: Advisory board; Pharma Essentia: Honoraria; Qiagen: Honoraria; MyeloPro Diagnostics and Research: Equity Ownership.

Significant Impact of the Molecular Profile on the Prognosis of Patients with Myeloproliferative Neoplasms and Splanchnic Vein Thromboses

Introduction: Myeloproliferative neoplasms (MPN) are the most frequent underlying causes of splanchnic vein thromboses (SVT), including Budd-Chiari syndrome (BCS), portal, splenic and mesenteric vein thromboses. This subgroup of MPN patients with SVT (MPN/SVT) has been shown to have distinct clinical and molecular features like younger age and lower JAK2V617Fmutant allele burden (the most predominant driver mutation in MPN/SVT patients) and are often considered as early stages of MPN. However, there is no study that has investigated the contribution of NGS data to the risk stratification of these MPN/SVT patients. Main objective of this study was to evaluate the impact of the presence of additional mutations to the driver mutation in the long term outcome of MPN patients with SVT. Patients and methods: Over the past 10 years, a total of 286 patients with SVT have been referred to our center for diagnostic assessment of an underlying MPN. Among them, a diagnosis of MPN (WHO criteria) was finally made in 197. Full molecular analyses by NGS was available in 61 of these MPN/SVT patients. The molecular profiling of patients was performed using a Capture-based custom NGS panel provided by Sophia Genetics. This panel comprised the coding sequences of 36 genes involved in MPN. Bioinformatics analysis was performed by Sophia Genetics, with a detection limit of variants set at 1%. We compared (i) MPN/SVT patients to our local cohort of 1371 MPN patients without SVT with full NGS data available; (ii) MPN/SVT patients with and without additional mutation to the driver mutation. Results: Median follow-up of MPN/SVT patients was 11 years, 62% were females, 33% had BCS and 67% portal vein thrombosis (Table 1). All patients received anticoagulants and 58 (95%) a cytoreductive therapy after the diagnosis of MPN. The type of MPN was polycythemia vera in 69%, essential thrombocythemia in 25% and myelofibrosis in 6%. The driver mutation was JAK2V617F in 57 (93%), and CALR mutation in 4 patients, respectively, and no patient had MPL mutation. By NGS, an additional mutation was identified in 27 (44%) patients, while 34 patients had only the driver mutation. The most frequent additional mutations were in TET2 (23%), ASXL1 (8%), DNMT3A (7%), IDH1/2 (5%), and LNK (5%) genes. EZH2, SRSF2 and TP53 mutations were found in only 1 patient, respectively. When compared to NGS data from 1371 MPN patients, there was no significant difference in the frequency of each additional mutation, except a slightly lower incidence of ASXL1 mutations in MPN/SVT patients (8% vs 23% in MPN patients, p=0.07). Of note, MPN/SVT patients were younger at MPN diagnosis than the control MPN cohort (44 vs 61 years, p&amp;lt; 10-5). Clinical characteristics were not significantly different between MPN/SVT patients with and without additional mutations (Table 1), including age at SVT or at MPN diagnosis, type of MPN or type of SVT, use of cytoreductive therapy, and median follow-up (11 years in both groups). In contrast, MPN/SVT patients with additional mutations had a significantly higher median JAK2V617F mutant allele burden than those with only JAK2 mutation (34% vs 11%, p=0.003, Figure 1); and higher risk of hematological transformation to myelofibrosis or acute leukemia or death (30% vs 6%, p=0.017). Only few patients experienced recurrence of thrombosis during follow up, with no difference according to the mutational profile (3 (11%) and 6 (17%) in patients with and without additional mutations, respectively). Conclusion: This is to our knowledge the first study assessing the impact of molecular abnormalities in the long term outcome of MPN/SVT patients showing a possible prognostic role for NGS data in these patients. Our results suggest that MPN/SVT patients with only JAK2V617F mutation and a low mutant allele burden (below 50%) are at low risk of adverse hematological outcome. In contrast, NGS may identify a group of patients with additional mutations at very high long term risk of unfavorable hematological evolution (30% transformation or death after 11 years of median follow-up, that is unexpected in such a young MPN population). In this group, a disease modifying therapy like interferon or other targeted therapies should be proposed when possible to reduce this risk of transformation. Disclosures Kiladjian: Novartis: Honoraria, Research Funding; Celgene: Consultancy; AOP Orphan: Honoraria, Research Funding.

TERT and JAK2 polymorphisms define genetic predisposition to myeloproliferative neoplasms in Japanese patients.

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are often characterized by specific somatic mutations in any of the three genes: JAK2, CALR, or MPL. A single nucleotide polymorphism (SNP), rs2736100, in the reverse transcriptase gene (TERT) and a germline JAK2 46/1 haplotype have been associated with MPNs in North American and European patients. We examined 201 Japanese MPN patients, including 52 with PV, 131 with ET, and 18 with PMF, as well as 366 control individuals for TERT rs2736100 and JAK2 rs10974944, a tagging SNP of the 46/1 haplotype. Furthermore, correlations between the JAK2 V617F allele burden at diagnosis and TERT rs2736100 or JAK2 rs10974944 were evaluated using a digital PCR assay for accurate quantitation. The JAK2 46/1 haplotype, but not the TERT rs2736100 SNP, was correlated to the JAK2 V617F mutant allele burden in JAK2 V617F-positive MPN patients. In conclusion, we demonstrated that both TERT rs2736100_C and JAK2 46/1 haplotype are predisposing factors for MPNs in Japanese patients. While TERT rs2736100_C tended to have a more general, non-specific effect on all MPNs, the JAK2 46/1 haplotype was essentially predisposed to the JAK2 V617F-positive MPNs.

A Prospective Evaluation of Vitamin B1 (thiamine) Level in Myeloproliferative Neoplasms: Clinical Correlations and Impact of JAK2 Inhibitor Therapy

Background:Vitamin B1 (Thiamine) deficiency might result in Wernicke's encephalopathy (WE); the latter was reported in clinical studies with the JAK2 inhibitor fedratinib resulting in disruption of clinical development, despite efficacy in patients with myelofibrosis (MF) (JAMA Oncol, 2015, 5). Our objectives were i) to determine the incidence of thiamine deficiency in patients with myeloproliferative neoplasms (MPN), ii) assess clinical correlations of thiamine level and iii) determine the impact of JAK2 inhibitor therapy on thiamine level in MPN.Methods:After Institutional review board approval, patients referred with a suspected diagnosis of MPN were enrolled. All clinical and laboratory variables including treatment details were collected at the time of referral. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of thiamine diphosphate in whole blood was performed at the time of referral. Reference range for thiamine level in whole blood was 70-180 nmol/L. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all statistical analysis.Results:A total of 115 patients were prospectively enrolled and included 28 patients without MPN and 87 with MPN. 87 MPN patients (median age, 65 years, 52% males) included 32 with primary myelofibrosis (PMF), 17 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 11 with post PV MF, 8 with post ET MF, and 2 with MPN-U. The MPN and non-MPN patients were similar in their gender distribution, pattern of alcohol use, malnutrition and multivitamin use (p=0.24, 0.12, 0.54 and 0.78 respectively), although the former were older (p=0.09).Median thiamine level of the study patients was 167 nmol/L (range; 60-442 nmol/L) with only two (1.7%) female patients displaying level below the normal reference range. One belonged to the non-MPN group (thiamine level 68 nmol/l); a 26 year old female with erythroid leukemia in the absence of alcohol use or malnutrition; the second patient (thiamine level 60 nmol/l) was a 38 year old female with ET, CALR type 2 mutated on hydroxyurea, without a history of alcohol use or malnutrition. Overall, both MPN and non-MPN patients displayed similar thiamine level (p=0.89).Clinical correlations of thiamine level in MPNMedian thiamine level was similar amongst post ET MF, ET, PMF, PV, and post PV PMF patients (127, 162, 166, 190 and 209 nmol/L respectively, p=0.13). No significant effect on thiamine level was apparent for age, gender, hemoglobin, platelet count, dynamic international prognostic scoring system (DIPSS) score, unfavorable cytogenetics, palpable splenomegaly, or constitutional symptoms. Interestingly, thiamine level was significantly higher in JAK2 mutated cases (median 177 nmol/L vs 145 nmol/L in unmutated cases, p=0.03). Furthermore, when analysis was restricted to PMF patients, thiamine level positively correlated with JAK2V617F mutant allele burden (p=0.01) and leukocyte count > 10 x 109/L (p=0.03). JAK2 mutated PMF patients with leukocyte count > 10 x 109/L depicted significantly higher thiamine levels (median 313 nmol/L) compared to JAK2 mutated patients without leukocytosis (median 164 nmol/L) and JAK2 unmutated patients with or without leukocytosis (median 192 nmol/L and 143 nmol/L respectively)(p=0.004, Figure 1a).Impact of treatment on thiamine levelTreatment with hydroxyurea was documented in 35 patients with no significant impact on thiamine level (p=0.28). 27 patients were on JAK inhibitors (median duration at referral; 41 months); 12 patients on ruxolitinib and 15 MF patients on momelotinib as part of a clinical trial. Thiamine level was similar amongst JAK inhibitor treated vs untreated patients (median 166 vs 169.5 nmol/L respectively, p=0.72). Annual thiamine measurements were performed on 15 patients treated with momelotinib (median duration 52 months, range 18-60 months). Despite fluctuations in thiamine levels, only two patients were noted to have thiamine level below normal (65 and 69 nmol/L at year 3), which subsequently improved to 101 and 121 nmol/L by year 4, while still on treatment (Figure 1b).Conclusions:The current study confirms the rarity of subnormal thiamine levels in MPN and the limited impact of treatment on thiamine levels, including with JAK2 inhibitors. Our novel observation regarding the correlation of thiamine level with JAK2V617F mutant allele burden and leukocyte count requires further exploration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Longitudinal Mutational Analysis in Hydroxycarbamide-Resistant/Intolerant Essential Thrombocythemia Treated on the Majic-ET Study

▪MAJIC-ET is a phase 2 study comparing ruxolitinib (RUX) to best available therapy (BAT) in hydroxycarbamide resistant/intolerant (HC-RES/INT) essential thrombocythaemia (ET). The presence of additional non-driver mutations at baseline, in particular TP53 and SF3B1, were predictive for increased risk of disease transformation in this cohort (EHA-4276). Now, we expand the mutation analysis up to 5 years to evaluate clonal evolution after prolonged treatment and correlate with clinical outcome.Driver mutation (JAK2/CALR/MPL) allele burdens were quantified using targeted next-generation sequencing and non-driver mutation analysis was performed using an ISO accredited Illumina TruSeq Custom Amplicon Panel, including 31 gene mutation hotspots & exons (~36,000 bp, 287 amplicons).Data was censored in January 2017 with 47.3% (n=52) randomised to BAT & 52.7% (n=58) to RUX. Overall, 49.1% were JAK2-mutated, 30% CALR-mutated, 4.5% MPL-mutated and 16.4% triple-negative (TN). At 12 months (m), molecular responses (MR) occurred rarely with complete MR (CMR) in 2 patients (pts) and partial MR (PMR) in 3 pts. MR were exclusively seen in RUX-treated pts. 54% (50/92) of those with driver mutations had a >12m allele burden analysis. Median sample time was 48m after trial entry (24 - 60m). There was no significant change in allele burden in the overall cohort. A new JAK2 V617F mutation (allele burden of 6%) was detected at 36m in a “TN” pt at baseline. Of the 5 pts achieving a molecular response at 12m, 2 of these subsequently lost their molecular response; a JAK2-mutated pt achieving CMR at 12m but at 52m had an allele burden of 4% (now PMR) and a CALR-mutated pt with PMR (allele burden 65 to 9%) at 12m, switched from RUX due to toxicity and subsequently lost their MR at 60m (allele burden of 44%), associated with clonal evolution (gain of a new ASXL1 mutation). New MRs occurred in only 2 pts; PMR in a MPL-mutated pt on pegylated-interferon and in a CALR-mutated pt on RUX. 4 pts with a baseline allele burden of <20% had a ≥50% reduction at latest time point (TP); interferon-treated (n=2) and RUX-treated (n=2).Analysis of non-driver mutations was performed on latest TP samples in 51 pts (46%) at a median of 40m after trial entry (6 - 60m). Additional non-driver mutations were present in 30% (33/110) of the cohort at baseline (EHA-4276). In 94% of pts with non-driver mutations at baseline, these were still detected at the latest TP (median 38m, 12 - 52m). There was a <10% allele burden change in 92%. However, a ≥10% increase in allele burden occurred in a TP53-mutated pt who transformed to acute leukaemia: allele burden at baseline of 9% to 39% at transformation. New non-driver mutations were frequently acquired; 9/51 pts (18%) with a total of 15 mutations detected; TET2 [5], ASXL1 [3], TP53 [3], SF3B1 [2], U2AF1 [1], SETBP1 [1]. Retrospective review of the paired baseline sample showed 93% of these mutations present at levels below the pre-specified threshold for variant calling. Mutations were equally acquired in JAK2- or CALR-mutated pts with no mutations acquired in MPL-mutated or TN pts. New mutations occurred on RUX (5/9, 56%) at a similar frequency to BAT (4/9, 44%). There was no association between acquisition of a subsequent new mutation and presence of non-driver mutation at baseline or haematological response at 12m. In transformed pts (12/110), 75% (9/12) had additional non-driver mutations at baseline. Of those with later samples for analysis (n=8) at the time of transformation, no new mutations were detected. New mutation acquisition was not associated with disease duration, age, gender or prior treatments and did not influence 2-year overall survival.We report for the first time a comprehensive longitudinal mutational analysis of HC-RES/INT ET patients within the context of a clinical trial. In this cohort, we found that MR was rare, both early and late in the course of treatment. Initial MR was often not sustained and may signify clonal evolution. As the presence of SF3B1 and TP53 mutations at trial entry predicted for disease progression, we anticipate that the new SF3B1 and TP53 mutations identified at later TP will also increase risk of subsequent transformation during longer follow-up. These data highlight the clinical utility of longitudinal molecular analysis of patients with HC-RES/INT ET. DisclosuresHarrison:Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; CTI BioPharma: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Speakers Bureau. Mead:ARIAD: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Cell Therapeutics: Consultancy; Celgene: Research Funding; Elstar: Research Funding; Evotek: Research Funding.

Alisertib (MLN8237), an Oral Selective Inhibitor of Aurora Kinase a, Has Clinical Activity and Restores GATA1 Expression in Patients with Myelofibrosis

Alisertib (MLN8237), an Oral Selective Inhibitor of Aurora Kinase a, Has Clinical Activity and Restores GATA1 Expression in Patients with Myelofibrosis

Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients

To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34+ cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV. From our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

Quantitative assessment of JAK2 V617F and CALR mutations in Philadelphia negative myeloproliferative neoplasms

BackgroundPhiladelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. Patients and methodsQuantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. ResultsTwelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). ConclusionOur study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.