JAK2V617F-positive Polycythemia Vera Research Articles

Inflammation Mediated By TNFα Disrupts the Autophagic Flux and Oxidative Phosphorylation of Megakaryocytes and Platelets through the Downregulation of STX17

Background. JAK2 V617F-positive polycythemia vera (PV) is the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Elevated levels of TNFα, and a significantly higher risk of thrombosis and bleeding (thrombohemorrhage) are hallmarks of JAK2 V617F PV. Platelets from patients with PV are characterized by hyperreactivity (fast activation of the αIIbβ3 integrin), higher P-selectin exposure (thromboinflammation), and phosphatidylserine (procoagulant potential). We have also found that in addition to these phenotypes, platelets from PV patients have dysfunctional autophagy promoting mitochondrial dysfunction, accumulation of dysmorphic mitochondria, and decreased clot retraction. Therefore, we hypothesized that inflammation mediated by TNFα (like in some cancers) dysregulates autophagy in JAK2 V617F PV by reprogramming (immunometabolism) metabolism of MKs and platelets. Methods. Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were treated with chloroquine (CQ) to analyze the autophagic flux by western blot and electron microscopy. Platelet bioenergetics was analyzed using the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Thrombus formation was measured by microfluidics using T-TAS and ATLAS equipment. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Platelets for clot retraction were treated with CQ or EACC. The MEG-01 cell line was treated with TNFα to analyze its impact on the autophagic flux, mitochondrial metabolism, and transcription (RNAseq). Murine studies using platelets from wild type (WT), DARE (transgenic mice with constitutive expression of TNFα or a neutralizing antibody against TNFα were performed. Results. Platelets from patients with PV have significantly impaired autophagic flux (p<0.01), mitochondrial dysfunction (p<0.002), and accumulation of dysmorphic mitochondria (p<0.007). Platelets from PV patients have significantly lower levels of STX17 (p<0.008), a key regulatory molecule of autophagosome and lysosome fusion (Figure 1). The inhibition of STX17 from healthy donor platelets decreased clot stability (p<0.004). Moreover, platelets from chronically (DARE) and acutely inflamed mice (WT mice treated with TNFα) exhibited the same autophagic defect (p<0.03 and 0.02, respectively) secondary to the downregulation of STX17 (p<0.01 and 0.02, respectively). In vitro experiments showed that megakaryocytes downregulate the transcription and translation STX17 (p<0.006 and p<0.03, respectively) in response to TNFα. Finally, in vivo treatment of inflamed mice with a neutralizing anti-TNFa antibody restored the expression of STX17 (p<0.0001) and the autophagic flux (p<0.002). Conclusion. Using several in vitro and in vivo approaches, we demonstrated for the first time the immunometabolic effect of TNFα in platelets and megakaryocytes. TNFα is a key regulator of the autophagic flux in platelets inducing platelet hyperreactivity and decreasing clot stability. Our findings may explain why patients with JAK2 V617F PV are at increased risk for both thrombo-hemorrhagic events and suggest that targeting autophagy might lower this risk.

Dysregulated Autophagy in Platelets from JAK2V617F-Positive Polycythemia Vera and Essential Thrombocythemia Underlies Bioenergetic Failure As a Possible Mechanism for Thrombohemorrhagic Complications

Background: JAK2V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET) are the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). ET/PV patients have high levels of inflammation and are at increased risk of thrombosis. Platelets from patients with ET/PV are characterized by hyperreactivity (low activation threshold), contributing to the increased incidence of thrombosis in the MPNs. We previously described that platelets from PV patients accumulate dysmorphic mitochondria. Autophagy is a housekeeping and an inducible cellular process that eliminates damaged proteins and organelles (such as mitochondria and endoplasmic reticulum), providing biosynthetic and bioenergetic substrates for the cell. Dysregulated autophagy has been described in cancer, aging, and neurodegenerative, cardiac, and muscular diseases. We hypothesized that impairment of the platelet autophagic flux in ET/PV Jak2V617F alters platelet metabolism and function at the early and late stages of platelet activation. Methods: Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were incubated for two hours with chloroquine (CQ) [50 and 100 µM] to inhibit autophagy. CQ blocks the late stage of the autophagy process; therefore, functional autophagy was assessed by quantifying the accumulation of autophagic-related proteins by western blot. Oxidative stress (4-hydroxynonenal protein adducts) was measured by western blot. Platelet bioenergetics was studied by the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Results: Platelets from healthy patients showed basal and inducible autophagy characterized by the accumulation of autophagic markers such as LC3II (p<0.0024), GABARAP, the autophagic cargo receptor p62 and the mitochondrial marker TOM20 (p<0.021). However, ET/PV platelets showed dysfunctional basal and inducible autophagy. Moreover, PV platelets and platelets treated with CQ had higher levels of ROS-damaged proteins (p<0.0001) compared to healthy platelets. Platelets from PV patients exhibited hyperreactivity by flow cytometry (PAC-1 binding, p<0.05) and under flow conditions (p<0.03). Moreover, platelets from ET/PV patients showed marked bioenergetic failure characterized by increased mitochondrial depolarization, higher intracellular calcium, depressed mitochondrial respiration, and lower levels of ADP and ATP. Pharmacological inhibition of autophagy of platelets from healthy individuals with CQ recapitulated the bioenergetic and functional profiles of platelets from patients with ET/PV. Bioenergetically, autophagy inhibition resulted in depressed basal (p<0.03) and ATP-linked respiration (p<0.01). Functionally, pharmacological inhibition of the autophagic flux led to significantly decreased clot retraction, as observed with the platelets from patients with ET/PV. Conclusion: Our results suggest that impairment of autophagy in platelets from ET/PV patients might exert a negative bioenergetic effect on platelet function, promoting platelet hyperreactivity and dysfunctional clot retraction, which would likely favor the formation of unstable clots. Our findings may explain why patients with Jak2V617F ET and PV are at increased risk for both thrombosis and hemorrhage and suggest that targeting autophagy might lower this risk. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Autosomal Dominant JAK2R715T Mutation Causes Polycythemia Vera in 3 Generations

Polycythemia vera (PV) has high hemoglobin (Hb) concentration caused by clonal proliferation of myeloid cells from a pluripotent stem cell (Prchal, N Engl J Med. 1974). Acquired somatic mutations of JAK2 gene, JAK2V617F and rarely exon 12, are present in virtually all PV patients (Ugo, Nature 2005, Scott N Engl J Med. 2007). The thromboses are the major causes of PV-related morbidity and mortality. A 38-year female suffered an ischemic stroke in 2017 at age 32 with negative thrombophilia work-up. She had elevated hemoglobin (Hb) 18 g/dL with intermittent elevation of leukocytes and normal platelets with borderline low erythropoietin (EPO) levels. JAK2V617F and exon 12 mutation analysis were negative. Her marrow was interpreted as compatible with PV and normal cytogenetics. Her hematologist treated her with phlebotomies. She was referred to us for an additional evaluation and management. Myeloid next generation sequencing (NGS) revealed a previously undescribed mutation JAK2R715T in the pseudokinase domain (JH2) with allelic burden ~50%. Some of her erythroid progenitors (BFU-Es) grew without extrinsic EPO, a hallmark of PV (Prchal, N Engl J Med. 1974). Phlebotomies were stopped; in two months, her Hb had risen to 17.4 g/dL and hematocrit had changed to 48.5% with normal leukocytes and platelets. We obtained JAK2 inhibitor fedratinib on compassionate basis from the manufacturer. On fedratinib, her Hb normalized to 15.2 g/dL with 42.8 % hematocrit. Her iron deficiency was corrected without increase of her Hb. The patient's germline mutation was confirmed by DNA sequencing of her nails. We encouraged her to have her only son and parents tested by NGS. The same JAK2R715T mutation was found in her 10-year-old son's blood sample. He also has elevated Hb at 16.2 g/dL (normal for age 11.5-15.5 g/dL), normal leukocytes and platelets were 400 K/µl. Her asymptomatic 63-year-old mother’ Hb was 17 g/dL, hematocrit 50.5%, and sequencing of her JAK2 also revealed JAK2R715T at ~50%. To assess how the R715T mutation may affect kinase activation, the JH1-JH2 domains of JAK2R715T and JAK2WT were modelled using AlphaFold 2.0 (Jumper, Nature 2021). The overall structure of JAK2R715T JH2 was more rigid than that of JAK2WT, in agreement with DynaMut in silico prediction (PMID: 29718330). -Helix C is shown in dark blue, Y570 and T555 in yellow, and ATP and T715 in red. Detailed comparison of JAK2R715T with JAK2WT JH2 (see Figure) indicated that the rigidification of the structure involved notably residues of the JH2-JH2 trans-dimerization domain centered around -Helix C. This has been suggested to mediate direct JH2-JH2 contacts (Wilmes, Science 2020) and a lengthening of the ß-sheet region surrounding the inhibitory phosphorylation site Tyr570 (Echarri, Mol Cell Biol. 2004). The R715 is in direct vicinity of the ATP binding pocket and promotes a closed lobe conformation through interaction with Thr555 of the nucleotide-binding loop (Bandaranayake, Nat Struct Mol Biol. 2012). We suggest that loss of this interaction may result in a more opened lobe conformation and disturbing of ATP binding to JH2, which regulates JAK2 kinase activity (Hammarén, Proc Natl Acad Sci USA 2015). To evaluate the effect of JAK2R715T in erythroid progenitor cells, the patient's erythroid progenitors derived from peripheral mononuclear cells were grown for 21 days with and without JAK inhibitor (ruxolitinib) following the previously published in vitro expansion protocol (Bruchova, Blood Cells Mol Dis, 2009). Compared to PVs and healthy controls, the patient cells showed increased proliferation. JAK inhibitor ruxolitinib treatment of patient cells induced decreased proliferation and differentiation compared to untreated cells. Assessment of STAT5 phosphorylation on patient's blood cells confirmed increased JAK2 activity to levels even higher than this of patients with confirmed JAK2V617F positive PV. X-chromosome based clonality studies of both females are ongoing. The EPO-independent colonies of her BFU-E is being evaluated for possible acquired conversion to homozygosity by uniparental disomy that is present in PV (Kralovics, Experimental Hematology, 2002). We conclude that this is the first example of dominantly inherited PV phenotype in 3 generations due to a novel germline JAK2R715T gain-of function mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical features and outcomes of JAK2 V617F-positive polycythemia vera and essential thrombocythemia according to the JAK2 V617F allele burden.

BackgroundJAK2 mutation status is a well-known risk factor for thrombosis in patients with myeloproliferative neoplasms. However, the clinical usefulness of JAK2 V617F allele burden is under investigation.MethodsWe retrospectively evaluated the impact of the JAK2 V617F allele burden on clinical characteristics and outcomes of JAK2 V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET). The JAK2 V617F allele burden was measured using sequencing.ResultsAltogether, 127 patients with JAK2 V617F mutation (PV, N=61; ET, N=66) were included in this study. JAK2 V617F allele burdens were positively correlated with white blood cell counts, hemoglobin values, lactate dehydrogenase levels, and platelet counts. The median values of JAK2 V617F allele burden in patients with PV and ET were 58% and 30%, respectively. A JAK2 V617F allele burden of ≥30%, older age, and a higher hemoglobin level were risk factors for thrombotic events in ET. In patients with PV, older age was the only thrombotic risk factor. The 8-year probabilities of overall survival (OS) were 82.9% in all patients. A high JAK2 V617F allele burden (≥58%) was associated with poor OS in patients with PV. For the patients with ET, the difference in 8-year OS based on the JAK2 V617F allele burden was not significant.ConclusionThe JAK2 V617F allele burden was correlated with hematologic parameters and clinical outcomes. Assessing the JAK2 V617F allele burden can be helpful in predicting the thrombotic risk and disease course in patients with JAK2 V617F-positive PV and ET.

Open Label Phase I Study of Single Agent Oral RG7388 (idasanutlin) in Patients with Polycythemia Vera and Essential Thrombocythemia

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are associated with an increased risk of thrombosis, progression to myelofibrosis (MF) and significant symptom burden. While hydroxyurea, interferon, and JAK2 inhibitors are used to treat these myeloproliferative neoplasms (MPNs), hematopoietic stem cell-depleting therapies may result in an alternative mechanism to alter disease course and improve outcome. MDM2, a negative regulator of P53, is overexpressed in CD34+ MPN cells harboring wild type P53. It is the target of idasanutlin (IDA), an oral MDM2 antagonist that results in up-regulation of P53 and down-stream activators of apoptosis. Preclinical data from our laboratory group supports the clinical evaluation of IDA in patients (pts) with ET/PV (Lu et al Blood. 2014;124(5):771-9). We evaluated the safety and tolerability of IDA in a phase I dose escalation trial of pts with JAK2V617F-positive ET or PV [NCT02407080].Methods: 13 pts (1 withdrew prior to treatment) resistant/intolerant to hydroxyurea and/or interferon therapy and without prior JAK2 inhibitor therapy were enrolled in a single institution phase I trial of oral IDA at two dose levels of 100 mg and 150 mg daily for five consecutive days and repeated every 28 day cycles. The first cycle consisted of 56 days for the evaluation of dose limiting toxicities (DLT). A DLT was defined as any non-hematologic AE of grade 3+ or a hematologic AE of grade 2+ thrombocytopenia, or grade 3+ neutropenia or anemia. The absence of DLT in the initial cohort of 100 mg daily in 3 evaluable pts allowed for dose escalation in a new cohort to 150 mg and after a total of 6 pts were treated at this level an additional 3 pts were treated at 100mg to complete the intended study. Pts that did not attain at least a partial response (PR) by modified European LeukemiaNet (ELN) criteria after cycle 6 and met eligibility were able to continue receiving IDA in combination with pegylated interferon-α (PEG) at 45ug weekly. Correlative studies included baseline mutational profiling, MDM2 protein levels, changes in serum MIC-1 levels and JAK2V617F variant allele frequency (VAF) with therapy.Results: 12 pts (6 at 100 mg; 6 at 150 mg) were treated and their baseline characteristics are shown in Table 1. Pts received a median of 7 cycles (range, 1-12) over a median of 33 weeks (range, 8-107) on study. Three pts discontinued treatment due to pt decision (n=2) and physician decision (n=1). A DLT was not identified for either dose level during cycle 1, and no hematologic treatment emergent adverse events (TEAE) were seen at any time point. Table 2 shows the non-hematologic TEAEs that occurred in at least 2 pts regardless of attribution. Grade 3 fatigue (n=1) and grade 3 headache (n=1) were the only significant TEAE noted at any time on study (both in 100mg cohort). The overall response rate by modified ELN criteria by cycle 7 for the 9 evaluable pts was 7/9 (78%). 7 of 10 (70%) evaluable pts achieved a ≥50% improvement in total symptom score (TSS) from baseline. There were no thrombotic or major hemorrhagic episodes, or progression to MF. Bone marrow pathologic response was assessed in 2 pts after 5 cycles. One case showed histological improvement, with normalization of overall marrow cellularity and megakaryocyte number, and less pronounced megakaryocytic atypia. The median JAK2V617F VAF at baseline, 7 cycles, and 9 cycles of therapy was 45% (n=12), 12% (n=6) and 13% (n=6), respectively. One pt was eligible to receive combination therapy and achieved phlebotomy freedom, normalization of palpable spleen (baseline 18cm), leukocyte count (baseline 44 x109/L), and PV-related symptoms by cycle 8. This pt also attained a 20% reduction in JAK2V617F VAF.The percentage of mononuclear cells expressing MDM2 was dramatically increased as compared to normal controls (p=0.01) (Figure 1). Serum levels of MIC-1 was used to assess pharmacodynamic effects of IDA therapy. Serum MIC-1 levels were elevated (~ 5 fold) after 5 days of administration of IDA in 85% of the pts (Figure 2), indicating activation of P53 by the low doses of IDA administered. Persistent elevation in MIC-1 levels was observed on day 15 in 6 of the 12 pts treated.Conclusion: IDA is well tolerated and demonstrates a clear signal of clinical activity in pts with therapy refractory PV. Correlatives support on-target activity. Mature follow up on the entire cohort will be presented. A multicenter phase II trial of IDA at a dose of 150 mg is underway [Display omitted] DisclosuresMascarenhas:CTI Biopharma: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Novartis: Other: DSMB member , Research Funding; Merck: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding.

JAK2V617F positive polycythemia vera with paroxysmal nocturnal hemoglobinuria and visceral thromboses: a case report and review of the literature

BackgroundPolycythemia vera (PV) is characterized by red cell mass expansion in the peripheral blood and can be complicated with thrombosis, bleeding, evolution to acute myeloid leukemia (AML) or a fibrotic phase. Paroxysmal nocturnal hemoglobinuria (PNH) in an acquired clonal haematopoietic stem cell disorder associated with chronic intravascular hemolysis, venous thrombosis, defective hematopoiesis, frequent episodes of infection and, rarely, leukemic transformation. Herein, we report an interesting case of a patient with coexistence of PNH clones and a JAK2V617F positive PV, with unusual thromboses without hemolysis.Case presentationA 51-year-old woman presented with increased levels of hematocrit, multiple liver, spleen, and left kidney infarctions and ascites; further investigation revealed a JAK2V617F-positive polycythemia vera and the presence of a significant PNH population (more than 90% CD55– CD59– cells among both granulocytes and red blood cells). Interestingly, the patient has experienced severe thrombotic events without any signs or symptoms of hemolysis.ConclusionsThis case raises questions over uncharted aspects of the PNH etiopathogenesis and its potential association with myeloproliferative neoplasms (MPN) and highlights the difficulty of diagnosing and managing patients with more than one potentially thrombophilic conditions, especially with established and severe thromboses.

Frequency of Zygosity in Jak-2 Positive Patients with Polycythemia Vera-Pakistan's Perspective.

Background:Estimation of JAK2V617F mutational load in Polycythemia Vera (PV) helps to determine the severity of the disease phenotype, the risk of thrombotic events, progression to post-PV myelofibrosis and survival. Amplification Refractory Mutation Screening (ARMS) PCR or Allele Specific (AS) PCR is a simple easy method with a reasonable sensitivity for screening of zygosity.The purpose of this study was to see the frequency of disease burden and phenotypic characteristics in Pakistani patients diagnosed with JAK2V617F mutation positive PV. Materials and Methods:A cross-sectional study using non probability consecutive sampling was conducted at Hematology Department, Liaquat National Hospital Karachi from October 2018 to July 2019.Adult newly diagnosed JAK2V617F positive PV patients of either gender were included. Patients’ demographics, clinical characteristics and baseline CBC were noted. JAK2V617F zygosity was qualitatively analyzed by ARMS-PCR technique. Age and gender were stratified to see to see the result of qualitative and quantitative effect modifiers on these patients using Chi Square and fisher exact test as appropriate while mean comparison was done by independent t-test and one way ANOVAtest. P value of ≤0.05 was considered as significant. Results:Fifty one patients were included in the study with an average age of 59.60±14.29years.90.2% of patients had hypertension.All patients tested positive for heterozygous state. Significant association of gender was found with smoking (p=0.001) while age was significantly linked with hypertension (p-0.033). Conclusion: JAK2V617F positive PV patients are mainly heterozygous males showing significant association with smoking and hypertension. ARMS-PCR is a robust technique to determine zygosity which can be used for screening purposes.

TET2 rs1548483 SNP Associating with Susceptibility to Molecularly Annotated Polycythemia Vera and Primary Myelofibrosis.

Background: The complexity of myeloproliferative neoplasms (MPNs) cannot be characterized by acquired somatic mutations alone. Individual genetic background is thought to contribute to the development of MPNs. The aim of our study was to assess the association between the TET2 rs1548483 single nucleotide polymorphism (SNP) and the susceptibility to polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) or chronic myeloid leukemia (CML). Methods: We evaluated the TET2 rs1548483 SNP through real-time PCR in 1601 MPN patients out of which 431 with PV, 688 with TE, 233 with PMF, 249 with CML and 197 controls. We included only patients with a molecularly proven driver mutation, such as JAK2 V617F, CALR or BCR-ABL1. Results: Significant association between TET2 rs154843 variant allele and JAK2 V617F-positive PV and PMF (OR = 1.70; 95% CI: 1.01–2.91; p-value = 0.046, and OR = 2.04; 95% CI: 1.10–3.77; p-value = 0.024, respectively), and type 2 CALR-positive PMF (OR = 2.98; 95% CI: 1.12–7.93; p-value = 0.035) was noted. Conclusions: The TET2 rs1548483 SNP is associated with the susceptibility to molecularly annotated PV and PMF.

Is There a Role for Basophils in Cancer?

Basophils were identified in human peripheral blood by Paul Ehrlich over 140 years ago. Human basophils represent <1% of peripheral blood leukocytes. During the last decades, basophils have been described also in mice, guinea pigs, rabbits, and monkeys. There are many similarities, but also several immunological differences between human and mouse basophils. There are currently several strains of mice with profound constitutive or inducible basophil deficiency useful to prove that these cells have specific roles in vivo. However, none of these mice are solely and completely devoid of all basophils. Therefore, the relevance of these findings to humans remains to be established. It has been known for some time that basophils have the propensity to migrate into the site of inflammation. Recent observations indicate that tissue resident basophils contribute to lung development and locally promote M2 polarization of macrophages. Moreover, there is increasing evidence that lung-resident basophils exhibit a specific phenotype, different from circulating basophils. Activated human and mouse basophils synthesize restricted and distinct profiles of cytokines. Human basophils produce several canonical (e.g., VEGFs, angiopoietin 1) and non-canonical (i.e., cysteinyl leukotriene C4) angiogenic factors. Activated human and mouse basophils release extracellular DNA traps that may have multiple effects in cancer. Hyperresponsiveness of basophils has been demonstrated in patients with JAK2V617F-positive polycythemia vera. Basophils are present in the immune landscape of human lung adenocarcinoma and pancreatic cancer and can promote inflammation-driven skin tumor growth. The few studies conducted thus far using different models of basophil-deficient mice have provided informative results on the roles of these cells in tumorigenesis. Much more remains to be discovered before we unravel the hitherto mysterious roles of basophils in human and experimental cancers.

SH2B3 (LNK) rs3184504 polymorphism is correlated with JAK2 V617F-positive myeloproliferative neoplasms

Abstract Background: Pathogenesis and phenotypic diversity in myeloproliferative neoplasms (MPN) cannot be fully explained by the currently known acquired mutations alone. Some susceptible germline variants of different genes have been proved to be associated with the development of these diseases. The goal of our study was to evaluate the association between the rs3184504 polymorphism of SH2B3 (LNK) gene (p.R262W, c.784T&gt;C) and the risk of developing the four typical MPN - polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and chronic myeloid leukemia (CML). Material and methods: We investigated the SH2B3 rs3184504 T&gt;C polymorphism by real-time PCR in 1901 MPN patients (575 with PV, 798 with ET, 251 with PMF, and 277 with CML), all of them harboring one of the specific driver mutations - JAK2 V617F or CALR in case of PV, ET and PMF, or BCR-ABL1 in case of CML, and 359 controls. Results: Overall, the TT homozygous genotype was significantly associated with BCR-ABL1-negative MPN (OR = 1.34; 95% CI = 1.03-1.74; crude p-value = 0.02; adjusted p-value = 0.04). The most significant association was seen in case of PV (OR = 1.54; 95% CI = 1.14-2.06; crude p-value = 0.004; adjusted p-value = 0.024). Also, SH2B3 rs3184504 correlated significantly with JAK2 V617F-positive MPN (OR = 1.36; 95% CI = 1.04-1.77; crude p-value = 0.02; adjusted p-value = 0.08), but not with those CALR-positive. ET (regardless of molecular subtype) and CML were not correlated with SH2B3 rs3184504. Conclusions: The SH2B3 rs3184504 polymorphism is associated with risk of MPN development, especially PV. This effect is restricted to JAK2 V617F-positive PV and PMF only.

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes.

Inflammatory and oncogenic signaling, both known to challenge genome stability, are key drivers of BCR-ABL-positive chronic myeloid leukemia (CML) and JAK2 V617F-positive chronic myeloproliferative neoplasms (MPNs). Despite similarities in chronic inflammation and oncogene signaling, major differences in disease course exist. Although BCR-ABL has robust transformation potential, JAK2 V617F-positive polycythemia vera (PV) is characterized by a long and stable latent phase. These differences reflect increased genomic instability of BCR-ABL-positive CML, compared to genome-stable PV with rare cytogenetic abnormalities. Recent studies have implicated BCR-ABL in the development of a "mutator" phenotype fueled by high oxidative damage, deficiencies of DNA repair, and defective ATR-Chk1-dependent genome surveillance, providing a fertile ground for variants compromising the ATM-Chk2-p53 axis protecting chronic phase CML from blast crisis. Conversely, PV cells possess multiple JAK2 V617F-dependent protective mechanisms, which ameliorate replication stress, inflammation-mediated oxidative stress and stress-activated protein kinase signaling, all through up-regulation of RECQL5 helicase, reactive oxygen species buffering system, and DUSP1 actions. These attenuators of genome instability then protect myeloproliferative progenitors from DNA damage and create a barrier preventing cellular stress-associated myelofibrosis. Therefore, a better understanding of BCR-ABL and JAK2 V617F roles in the DNA damage response and disease pathophysiology can help to identify potential dependencies exploitable for therapeutic interventions.

Hypoxia Inducible Factors' Transcriptional Activity in Polycythemia Vera

Polycythemia vera (PV), a myeloproliferative neoplasms (MPN), is characterized by high red cell mass due to excessive erythrocytosis and by clonal proliferation of myeloid cells. The major PV complication, increased risk of thrombosis, is also seen in Chuvash erythrocytosis (CE), a disorder of upregulated hypoxia sensing. Hypoxia inducible factor (HIF)-1 and HIF-2 are the principle regulators of the hypoxic response by regulating the transcription of hypoxia responsive genes. We previously showed that MPN patients have upregulation of some HIF target genes in granulocytes and platelets along with increased glucose uptake (Sana, et al., Blood 2013 122:1604). Further, stem cell factor and thrombopoietin signaling increase HIF-1 activity (Kirito et al, Blood 2005); however, the role of HIFs in PV is unknown. We evaluated HIF transcriptional activity in PV by unbiased RNA sequencing of JAK2V617F positive PV granulocytes (22 patients and 10 controls) and platelets (24 patients and 4 controls). Differential gene expression was determined by DEseq2 and data stratified by p&lt;0.05 and Log2fold change&gt;1. BaseSpace Correlation Engine (Illumina Inc.) was used to identify genes regulated by HIF. Fifty-seven HIF target genes were dysregulated in PV granulocytes. There were 44 upregulated genes involved in apoptosis, metabolism, cellular proliferation, intracellular signal transduction, and ion transport. Thirteen genes were downregulated in PV, all associated with Ras and Rap1 signaling. Thirty-five genes were correlated with JAK2V617F allele burden; 27 genes were positively correlated while 8 genes were inversely correlated. SOCS2, FLT1, PDLIM1, ALDH1A2, NPR3, and BATF3 were regulated by both STAT5 and HIF suggesting that HIFs and the JAK-STAT pathway may cross-talk. CAV1, PLOD2, NRIP3, NGFRAP1, and UBXN10 inversely correlated with the white blood cell counts. Eighty-seven HIF target genes were differentially expressed in PV platelets. Seventy-two genes were upregulated and 15 genes were downregulated. Upregulated genes were associated with apoptosis, proliferation, and metabolism. Unlike in PV granulocytes, some of these upregulated genes were associated with immune responses including the TNF and NF-kB signaling pathways; NF-kB and HIF-1 co-stimulate each other. Twenty genes positively correlated with JAK2V617F allele burden but BCL2 and PNOC inversely correlated. There were 13 genes regulated by both STAT5 and HIF. DOT1L, GYG1, HOXA1, PRELID1, and RPIA transcript levels inversely correlated with the platelet counts. In both granulocytes and platelets, 17 genes were dysregulated. CAV1, CHAD, and SOCS2 are associated with negative regulation of receptor signaling via JAK-STAT. CHAD was downregulated while CAV1 and SOCS2 were upregulated in both granulocytes and platelets. ALDH1A2, CAV1, KCNMA1, and MMP9 genes were upregulated and associated with apoptosis. We also observed an increased transcript of inflammatory genes in PV. PV granulocytes had 82 upregulated and 11 downregulated inflammatory genes. Among these upregulated genes, MGLL, PROK2, and MAP2K6 were also HIF target genes. In platelets, 34 genes were dysregulated (31 genes were upregulated and 3 genes were downregulated). It is of interest that 2 prothrombotic genes are reported to be regulated by HIFs, tissue factor encoded by F3 (Giannarelli, et al., Circulation 2014) and protein S (encoded by PROS1). We found F3 transcript to be upregulated also in PV and CE granulocytes. PROS1 was reported to be negatively regulated by HIF-1 as detected by decreased protein S in mouse plasma and by decreased PROS1mouse liver transcript and human cell line HepG2 (Pilli, et al., Blood 2018). We found PROS1 to be also expressed in human granulocytes and platelets; however, it was overexpressed in PV and CP granulocytes but not in platelets. We demonstrate here high HIF transcriptional activity in granulocytes and platelets of PV; some of these HIF-regulated genes are also associated with immune response genes. Dysregulated HIF target genes were involved in apoptosis and cell proliferation pathways. Most HIF target gene transcription correlated with JAK2V617F allele burden, platelet counts, and white blood cell counts. These results suggest that HIF might be a therapeutic target of PV. Of drugs in clinical use, digoxin is a potent HIF-1 inhibitor (Zhang, et al., PNAS 2008); its potential role in PV and CE is being explored. Disclosures Gordeuk: Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding; Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding.

Synergistic Effect of Imatinib and Ruxolitinib in a Patient with JAK2V617F positive Myelofibrosis and Concomitant BCR-ABL1 positive Chronic Myeloid Leukemia

Introduction:JAK2V617Fmutation is present in majority of polycythemia vera (PV) and in 50-60% of patients with primary myelofibrosis (MF). JAK1/2 inhibitor ruxolitinib (RUX) is approved for treatment of both PV and intermediate/high risk MF. Two independent randomized studies showed the benefit of RUX in MF with significant reduction in spleen volume and improvement in constitutional symptoms compared to best supportive care. Clonal evolution may occur in PV and MF upon acquisition of new mutations and transformation to acute myeloid leukemia. Rarely, patients with JAK2V617Fpositive PV or MF have also concomitant BCR-ABL1 fusion gene with phenotype of chronic myelogenous leukemia (CML). Imatinib, a BCR-ABL1 inhibitor, induces long-term cytogenetic and molecular remissions in CML. Single agent imatinib has shown no clinical benefit in MF patients despite in vitro efficacy (Gaikwad et al Exp. Hematol. 2007). We describe here a patient with JAK2V617F positive post-PV MF who later developed concomitant BCR-ABL+ CML. Splenomegaly was initially resolved by RUX but returned coincidently with the diagnosis of CML and was again normalized by imatinib alone. Massive splenomegaly reappeared along with elevated hematocrit on imatinib and was resolved by the addition of low dose RUX.Case report: A 71-year old woman initially presented in September 2011 with isolated erythrocytosis and splenomegaly. A diagnosis of PV was established based on the presence of the JAK2V617Fmutation and hypercellular marrow. Bone marrow karyotype was normal (Table 1). The patient was randomized to pegylated interferon arm in the MPD Consortium study. A complete hematologic response was achieved after nearly 2 years on maximal dose 180 mcg weekly. In early 2015, she developed constitutional symptoms, and progressive splenomegaly. Progression to post-PV MF was confirmed on bone marrow evaluation. The patient was taken off study and started on RUX 20 mg BID. She developed severe anemia 4 months later requiring dose reduction to 10 mg BID. The anemia markedly improved, while JAK2V617F allele burden remained high at 88% with normal cytogenetics. In December 2017, the patient developed anemia, fatigue and rapid regrowth of spleen. Marrow karyotype showed Philadelphia (Ph) chromosome in 25% of the cells analyzed, PCR was positive for BCR-ABL1 p210, and JAK2V617F allele burden was reduced to 56.4%. In January 2018, she started imatinib 400 mg daily and RUX was discontinued due to toxicity concerns. In April 2018, her hematocrit increased and massive splenomegaly returned. Restart of initial RUX 20 mg BID was followed by severe pancytopenia which normalized when RUX was reduced to 5 mg BID with continuing imatinib regimen. After 8 weeks, splenomegaly completely resolved (Figure 1). A complete cytogenetic response and major molecular response were achieved after 3 months and 6 months of combination therapy respectively.Methods: Mononuclear cells from peripheral blood collected in June 2018 were plated in methylcellulose without erythropoietin or other cytokines. After 14 days, colonies were plucked, with ½ of each colony used for JAK2V617F allele-specific PCR and ½ prepared as cytospins for BCR-ABL fluorescent in situ hybridization (FISH) with the Vysis LSI BCR/ABL Dual Color, Dual Fusion Translocation Probe from Abbott Laboratories.Results: Analysis of 3-6 EPO-independent colonies of each CFU-E, G, M, GM, GEMM, BFU-E showed no BCR-ABL1 positive cells, while a single CFU-GEMM colony was positive and also homozygous for JAK2V617F. Frozen marrow cells from the time of the original diagnosis are being analyzed.Conclusion: The recurrence of splenomegaly in this patient following single agent RUX indicates emergence of novel BCR-ABL1+ clone. While single agent imatinib temporarily normalized the splenomegaly and leukocytosis for several months, MF phenotype reoccurred. Splenomegaly resolved completely in 8 weeks with the addition of very low dose RUX to standard dose imatinib, providing the first clinical observation of in vivo synergism of RUX and imatinib. We are investigating serial samples during her course, which will be presented at the meeting. The analysis of a single clone demonstrate that BCR-ABL1 mutation did not occur in previously normal dormant stem cell but in JAK2V617F positive progenitors and represent in this pts a novel subclone of previous PV/MF stem cell. DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro.We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. DisclosuresBrümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

A Novel Acquired t(2;4)(q36.1;q24) with a Concurrent Submicroscopic del(4)(q23q24) in An Adult with Polycythemia Vera

Background: Polycythemia vera (PV) is a clonal myeloid stem cell disease characterized by a growth-factor independent erythroid proliferation with an inherent tendency to transform into overt acute myeloid malignancy. Approximately 95% of the PV patients harbor the JAK2V617F mutation while less than 35% of the patients harbor cytogenetic abnormalities at the time of diagnosis. Methods and Results: Here we present a JAK2V617F positive PV patient where G-banding revealed an apparently balanced t(2;4)(q35;q21), which was confirmed by 24-color karyotyping. Oligonucleotide array-based Comparative Genomic Hybridization (aCGH) analysis revealed an interstitial 5.4 Mb large deletion at 4q23q24. Locus-specific fluorescent in situ hybridization (FISH) analyses confirmed the mono-allelic 4q deletion and that it was located on der(4)t(2;4). Additional locus-specific bacterial artificial chromosome (BAC) probes and mBanding refined the breakpoint on chromosome 2. With these methods the karyotype was revised to 46,XX,t(2;4)(q36.1;q24)[18]/46,XX[7]. Conclusions: This is the first report on a PV patient associated with an acquired novel t(2;4)(q36.1;q24) and a concurrent submicroscopic deletion del(4)(q23q24). The study also underscores the benefit of combined usage of FISH and oligo-based aCGH analysis in characterizing chromosomal abnormalities. The present findings provide additional clues to unravel important molecular pathways in PV to obtain the full spectrum of acquired chromosomal and genomic aberrations, which eventually may improve treatment options.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report

BackgroundThe role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome.Case presentationA female presented with a history of JAK2V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35–45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells.ConclusionsThis is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

The Real-World Treatment of Polycythemia Vera: Adherence to Treatment Guidelines and Outcomes

Background The aim of therapy for polycythemia vera (PV), a myeloproliferative neoplasm, is to reduce cardiovascular risk without increasing the risk of bleeding or hematological transformation. Current consensus guidelines suggest low dose aspirin and phlebotomy for patients at low-risk for thrombosis ( Methods We performed a retrospective cohort study of newly diagnosed JAK2V617F positive PV patients over the age of 18 between January 1, 2005 and January 1, 2017 in Hamilton, Ontario, Canada. The primary outcome was all-cause mortality. Secondary outcomes were non-fatal cardiovascular events, major bleeding, and hematological progression. Statistical analysis was performed in STATA using Cox proportional hazard models, and Poisson regression. Results A total of 112 patients (58.0% female) met inclusion criteria. The mean (SD) age at diagnosis was 70 (14) years. Median follow-up was 3.38 years (Range = 0.04-11.89). At diagnosis, 82.1% of patients were classified as high risk for thrombosis. At diagnosis cardiovascular risk factors were present in 69.6% of patients including previous thrombotic events in 27.5%. Symptoms of PV (e.g. pruritus, vasomotor, fatigue) were found in 52.7% of patients at diagnosis. Patients received treatment with phlebotomy (90.2%), aspirin (87.5%), and hydroxyurea (74.1%). Monotherapy was used in 6 (24%) of the non-guideline patients and was not found in the guideline group. Anticoagulants were used in 17.2% of patients. Overall, only 77.7% of patients received treatment that was consistent with guidelines regarding aspirin and hydroxyurea use. A total of 22 (19.6%) of patients died during follow-up; increasing white blood cell count at diagnosis, increasing age at diagnosis, history of diabetes and history of congestive heart failure were associated with higher risk of death (Table 1). Patients who were not treated according to the guideline recommendations at any point during follow-up had an increased risk of death (HR, 2.88; 95% CI, 1.03-8.05; P Conclusion This contemporary North American PV patient cohort study showed that the majority of newly diagnosed PV patients were older adults at high risk of thrombosis consistent with previous studies. The presence of cardiovascular comorbidities at diagnosis appeared to increase the risk of death. About three-quarters of patients in our cohort received treatment according to consensus guideline recommendations. Although the reasons for discordance with guidelines are uncertain, those who did not receive recommended therapy appeared to have an increased risk of death. The results of this study should be interpreted with caution due to the possibility of confounding by other factors that influence mortality. Future research regarding the barriers and facilitators of PV treatment guideline use, and the optimal strategy for assessment and management of cardiovascular risk factors may provide opportunities to improve care and patient outcomes. Disclosures Siegal: Novartis: Other: Focus Group; Bayer: Other: Focus group; BMS-pfizer: Other: Presentation; Servier: Other: Focus Group; Portola Pharmaceuticals: Other: Presentation. Hillis: Celgene: Other: Personal fees; Novartis Oncology: Research Funding; Bristol-Myers Squibb: Other: Personal Fees.

前多血期に主幹動脈閉塞を伴う脳梗塞を発症し，数年後にcrescendo TIAを呈したJAK2V617F 変異遺伝子陽性真性多血症の1 例

前多血期に主幹動脈閉塞を伴う脳梗塞を発症し，数年後にcrescendo TIAを呈したJAK2V617F 変異遺伝子陽性真性多血症の1 例

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

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

Analysis of DNA methyltransferase 3A gene mutations in patients with Philadelphia-negative myeloproliferative neoplasms

Context: Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) from a group of disorders characterized by dysregulated JAK-STAT functionality, abnormal hematopoiesis, as well as increased production of proliferative cytokines. In addition to JAK2V617F mutation, additional gene alterations that are involved in epigenetic mechanisms, particularly de novo DNA methyltransferase 3A (DNMT3A), have been described in Ph-negative MPN biology. Aims: The aim of this study is to evaluate the H/C/S/P mutations in codon R882 of DNMT3A gene among patients with Ph-negative MPNs. Subjects and Methods: This study was conducted on 64 newly diagnosed patients with PV, ET, and PMF who referred to Shafa Hospital, Ahvaz, Iran. In the beginning, 5 mL whole blood was drawn from each patient, and the DNMT3A R882 codon mutations were investigated following the isolation of peripheral blood mononuclear cells by DNA amplification protocol using polymerase chain reaction and DNA sequencing techniques. Results: The R882H G > A mutation, which results in an amino acid substitution at position 882 of DNMT3A gene from arginine (R) to histidine (H), was observed in two patients (3.1%) with JAK2V617F positive PV and JAK2V617F negative PMFs. Conclusions: Based on the results, DNMT3A-R882 mutations occur at a low frequency in patients with Ph-negative MPNs. To the best of our knowledge, this is the first study to specifically estimate the prevalence of DNMT3A mutations among Ph-negative MPN patients living in the Middle East. It is recommended to investigate these mutants as a secondary defect along with common major complications in such patients.