JAK2 Positive Patients Research Articles

Clinical and Disease Characteristics of Patients with Myelofibrosis and Essential Thrombocythemia Who Harbor a Calreticulin (CALR) Gene Mutation: Subanalysis of the MOST Study

Background: Calreticulin(CALR)gene mutations are the second most frequent driver of disease in myelofibrosis (MF) and essential thrombocythemia (ET). Compared with patients with JAK2 mutations, those with CALR mutations are considered to have lower-risk disease; however, prospective data on clinical characteristics and disease progression for patients with CALR mutations are limited. This study compared the disease characteristics and treatment patterns of patients with MF or ET who had CALR mutations vs those who had JAK2 mutations at enrollment in the Myelofibrosis and Essential Thrombocythemia Observational Study (MOST; NCT02953704). Methods: Patients enrolled in MOST were aged≥18 years with clinical diagnoses of MF or ET at clinical practices within the United States. Patients with MF included those with primary or secondary MF who were low risk (or intermediate-1 by age >65 years alone) per the Dynamic International Prognostic Scoring System (Passamonti F, et al. Blood. 2010;115:1703-1708). Patients in the ET cohort had a clinical diagnosis of ET and were ≥60 years or had history of thromboembolic events or were receiving ET-directed therapy at enrollment. Descriptive statistics were used to summarize the data. The median (range) follow-up time for CALR- and JAK2-positive patients was 52.8 (43-68) months and 56.6 (42-70) months, respectively. ET progression was defined as transformation to MF; MF progression was defined as worsening of objective clinical and laboratory assessments of disease. Results: A total of 881 patients ( CALR-positive, n=137 [16%]; JAK2-positive, n=750 [85%]; 6 patients had both CALR and JAK2 mutations) were included in this study. A summary of patient and disease characteristics are presented in Table 1 and Table 2. At enrollment, 25% and 75% of CALR-positive patients had a diagnosis of MF and ET, respectively, vs 16% and 84% of JAK2-positive patients. The majority of patients in both groups were women ( CALR-positive, 56%; JAK2-positive, 65%). CALR-positive patients were younger at enrollment and at diagnosis compared with JAK2-positive patients (median age at enrollment, 63.0 [24.0-89.0] vs 70.0 [26.0-93.0] years, P<0.001; median age at diagnosis, 59.3 [21.0-87.0] vs 66.3 [22.0-93.0] years, P<0.001). The majority of patients in this analysis (79% of CALR-positive, 90% of JAK2-positive) were receiving MPN-directed therapy at enrollment. The most common therapies in the CALR cohort were hydroxyurea (51%), interferon (12%), and anagrelide (8%); the common therapies among patients in the JAK2 cohort were hydroxyurea (66%), aspirin only (10%), and ruxolitinib (8%). Disease burden at enrollment was also assessed. The percentages of patients with white blood cell (WBC) count >10×10 9/L or platelet count >400×10 9/L were similar for both CALR- and JAK2-positive patients (13% vs 20% and 53% vs 51%, respectively). Percentages of patients with splenomegaly (SPM) were similar for CALR- vs JAK2-positive patients at enrollment (11.0% vs 13%). The mean MPN-SAF TSS was similar at baseline for CALR- and JAK2-positive patients (15.1 vs 16.5). The percentage of patients with MPN-SAF TSS >25 was 12% in the CALR-positive and 16% in JAK2-positive cohorts. Disease complications and progression were also assessed. Thrombotic and hemorrhagic events during the study period were infrequent in both CALR- and JAK2-positive patients (2% vs 4% and 1% vs 3%, respectively). Rates of disease progression were similar for CALR- and JAK2-positive patients, both for MF progression (50% vs 61%) and for ET progression to MF (2% vs 5%). Leukemic transformation occurred in 6 (4%) CALR-positive patients and 43 (6%) JAK2-positive patients. Deaths due to disease progression occurred in no CALR-positive patients and in 18 (2%) JAK2-positive patients. Conclusions: The results of this real-world analysis of the MOST study demonstrate that despite nearly 80% of CALR-positive patients receiving MPN-directed treatment, a substantial percentage of this group did not meet treatment goals, including control of symptom burden, WBC and platelet counts, and SPM. In addition, disease progression was observed in 50% of the patients with MF in the CALR-positive cohort. Further analysis of the MOST study may provide insight into optimal management of patients with CALR mutations.

Mutation Clearance of Driver Mutations after Transplantation for Myelofibrosis Predicts Outcome

Myelofibrosis is one of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by clonal myeloproliferation. Allogeneic hematopoietic stem-cell transplantation is the only potentially curative treatment for patients with myelofibrosis. With that, it can resolve fibrosis and lead to complete loss of the clonal burden. More than 90% of myelofibrosis patients harbor driver mutations in one of the 3 genes JAK2, CALR, and MPL, with JAK2 mutations being associated with worse outcomes. We herein report a study to determine whether the persistence of cells with MPN-associated driver mutations in the early period after allogeneic hematopoietic stem-cell transplantation was associated with outcomes. Patient samples for molecular analysis were prospectively collected from peripheral blood at 5 different time points: at start of conditioning prior to stem cell infusion, at days 30, 100, 180, and 1 year after transplantation. We applied quantitative PCR technology to detect individual driver mutations in JAK2, CALR, and MPL (with a high sensitivity of 0.01%). Simulation was applied to explain variances at different time points. Multivariable modelling was employed to determine independent effects. A total of 312 patients receiving first reduced-intensity conditioning transplantation were included. Distribution of driver mutation genotype at time of transplantation was as follows: 67% with JAK2, 19% with CALR, 4% with MPL, and 8% were triple-negative. Conditioning regimen comprised busulfan and fludarabine. Donor platform for transplantation was: matched related (18%), matched unrelated (58%), haploidentical (1%), and mismatched unrelated (23%). Disease risks at time of transplantation were low (1%), intermediate-1 (22%), intermediate-2 (55%), and high (22%). Median follow-up of the total cohort was 6 years. The median allele burden at time of transplantation was significantly (P<0.001) different between driver mutations: 20% (range, 0.1-100%) for JAK2, 44% (range, 3-54%) for CALR, 60% (range, 43-99%) for MPL. CALR and MPL showed earlier mutation clearance after transplantation compared with JAK2 ( Table). Mutation clearance was achieved at day 30 after transplantation in 46% with JAK2, 67% for CALR, and 30% for MPL (P=0.02). At day 100 after transplantation, 66% of JAK2, 75% of CALR, and 100% of MPL showed complete mutation clearance (P=0.09). Complete mutation clearance at day 30 after transplantation was predictive of post-transplantation relapse at 1 year (P=0.009), showing cumulative incidence of relapse of 6% versus 16%. Similar results were seen for complete mutation clearance at day 100 after transplantation, demonstrating cumulative incidence of relapse of 4% versus 24% (P<0.001). Mutation clearance at day 30 and 100 after transplantation explained 80% and 87% of variance of later time points. The effect of mutation clearance on post-transplant relapse was irrespective of driver mutation status ( Figure). 6-year disease-free survival according to mutation type was 52% for JAK2, 63% for CALR, and 80% for MPL (P=0.045). Mutation clearance at day 30 after transplantation was associated with better disease-free survival, irrespective of driver mutation status ( Figure). Moreover, day-30 complete mutation clearance predicted disease-free survival in JAK2, showing rates of 60% for negative versus 47% in JAK2 positive patients (P=0.05). Importantly, achievement of mutation clearance at day 30 after transplantation could also distinguish different risk groups in patients with onset better prognosis harboring CALR/MPL mutations. The 6-year disease-free survival was 76% for patients with mutation clearance versus 59% for CALR/MPL-positive patients(P=0.02) . Multivariate analysis (adjusting for donor type, diagnosis of primary or secondary myelofibrosis, allele burden at time of transplantation, and age) confirmed that patients who had mutation clearance at day 30 after transplantation had lower risk of relapse, better disease-free and overall survival. In conclusion, this is the first study to show that CALR and MPL are associated with earlier mutation clearance after transplantation compared with JAK2. Mutation clearance at day 30 and 100 after transplantation was the best predictor of relapse and disease-free survival, independent of driver mutation status.

Glycated Hemoglobin (HbA1c) and Thrombotic Risk in Polycythemia Vera and Essential Thrombocythemia

BACKGROUND: Essential thrombocythemia (ET) and polycythemia vera (PV) are BCR::ABL1 negative myeloproliferative neoplasms (MPNs) characterized by overproduction of one or more mature myeloid cell lineages and high thrombotic risk. Diabetes mellitus (DM) is a rare cardiovascular (CV) comorbidity in MPNs and the optimal treatment target levels of glycated hemoglobin (HbA1c) in MPNs remain unknown. OBJECTIVE&METHODS: This multicenter study was conducted in Croatia and Serbia in the period between 01/1997 and 01/23 and investigated clinical correlations of HbA1c in ET and PV patients whose diagnoses were reassessed according to 2016 WHO criteria. We retrospectively collected data regarding diabetic ET and PV patients with available HbA1c at disease diagnosis, stratified them according to HbA1c levels, and compared their clinical outcomes to a control group of 93 non-diabetic ET and PV patients. Outcomes of interest were time to thrombosis (TTT; death as a censoring event) and overall survival (OS). Survival analyses were based on Kaplan-Meier method and performed with log-rank test and the Cox-regression analysis. The study was approved by Ethics Committees from all participating centers. RESULTS: We included 109 MPN patients (33 ET and 76 PV) with DM (all type II); median age was 69 years (range 37-86), 60 (55%) were females, 81 (74.3%) were JAK2- and 10 (9.2%) CALR-positive. Ninety-two (84.4%) patients were classified as high-risk (age>60 years or prior thrombosis), 68 (62.4%) received hydroxyurea (HU), 91 (83.5%) had arterial hypertension, 44 (40.4%) hyperlipidemia, and 36 (33%) were smokers. The median follow-up time was 38 months; 16 (14.7%) thrombotic (13 arterial and 3 venous) events and 25 deaths (22.9%) occured during this time. Median HbA1c in diabetic MPN patients was 6.8% (range 4.8-16.2%). There were no associations of HbA1c with disease phenotype, age, sex, mutational status, prior thrombosis, presence of individual CV risk factors, or any of the blood cell count components (p>0.050 for all analyses). ROC curve defined the optimal HbA1c cut-off level of ≥7.2% for the best discrimination of thrombotic events in MPN patients with DM. Diabetic MPN patients with low HbA1c (<7.2%) had similar TTT (median 182 months) as non-diabetic MPN patients (median 197 months) whereas those with higher HbA1c (≥7.2%) had significantly worse TTT (median 124 months; HR 3.76, p=0.012), as presented in Figure 1A. The detrimental effect of high HbA1c was evident in high-risk (HR 3.2, p=0.031) but not in low-risk diabetic MPN patients (p=0.115). Also, JAK2-negative diabetic MPN patients with higher HbA1c had inferior TTT when compared to their counterparts (HR 28.17, p<0.001), with no difference among JAK2-positive patients (Figure 1B). There were significant interactions between high HbA1c, thrombotic risk, and advanced age (p=0.026), higher leukocytes (p=0.028), higher erythrocytes (p=0.007), higher platelets (p=0.034), higher hematocrit (p=0.007) and higher hemoglobin (p=0.013), with older age and higher blood counts favoring higher thrombotic risk associated with high HbA1c. There was no difference in OS when comparing non-diabetic to diabetic MPN patients with respect to HbA1c (p=0.209). In the multivariate Cox regression model, high HbA1c (HR 5.04, p=0.013) remained as the only variable associated with inferior TTT among diabetic MPN patients when additionally controlling for age, sex, history of thrombosis, presence of JAK2 mutation, HU treatment, baseline leukocyte counts, and the presence of at least one other CV risk factor. DISCUSSION: This study demonstrated that diabetic MPN patients with low (<7.2%) HbA1c had similar TTT as non-diabetic patients suggesting that stringent control of DM in MPNs is extremely important; this effect was most evident in JAK2-negative diabetic MPN patients. Also, significant interactions with high trombotic risk were found between high HbA1c, older age, and different myeloid cell lineages, suggesting that uncontrolled DM acts synergistically with MPN clone and underlies CV development. However, no correlations between HbA1c and blood cell counts were detected, indicating that HbA1c measurements may not be accurrate in MPNs for DM diagnosis. The lack of association of high HbA1c with OS is probably related to optimal post-thrombotic management. Future studies are needed to elucidate optimal HbA1c values for the diagnosis and treatment of DM in MPNs.

Diagnostic Performance of Red Blood Cell Indices, Serum Erythropoietin, and JAK2 Mutation Testing for the Evaluation of Polycythemia Vera at High Altitude

Introduction: Polycythemia vera (PV) is a clonal myeloproliferative neoplasm associated with mutation of Janus kinase 2 ( JAK2) in virtually all patients. Patients with PV typically present with constitutional symptoms and thrombotic complications related to myeloproliferation but can present with only mild or no symptoms. In 2016, the World Health Organization (WHO) diagnostic criteria for PV were revised to use lower hemoglobin and hematocrit cut-off levels in order to avoid missing masked polycythemia. Accordingly, a hemoglobin concentration of more than 16 g/dL (HCT>48%) for females and 16.5 g/dL (HCT>49%) for males can be considered for PV evaluation (previously >18.5 g/dL for men and >16.5 g/dL for women). In conjunction, concerns arise about the unnecessary evaluation of individuals with otherwise high-normal hemoglobin levels, more so for those living at high altitude. Altitude exposure, amongst others, decreases plasma volume to increase [Hb] which may accordingly muddle the diagnosis of PV at altitude. Nonetheless, Red Cell Mass (RCM) is seldom assessed in clinical practice. In addition, red blood cell count, related indices, and serum erythropoietin may also be affected by high altitude, and their diagnostic performance in the evaluation of PV at altitude has not been prospectively assessed. Method: We collected lab records for all JAK2 requests over three years (2020-2022) in two referral hospitals serving a population residing at an altitude of 2,270-3,000 meters above sea level. Indications for requests, as well as laboratory data and imaging, were gathered. Complete blood count (CBC) and other laboratory values were collected when ordered within one week of JAK2 testing. For CBC, EDTA anticoagulated samples were processed using Sysmex XN-3100. For JAK2 testing, RNA isolated from peripheral blood samples (using the MagNA Pure System) was reverse-transcribed, amplified, and sequenced for any mutations involving exons 12, 13, 14, or 15 using an ABI 3730 XL genetic analyzer. For serum EPO, frozen serum samples were outsourced to Bio Scientia Labs, Germany. Reference values for serum erythropoietin were lab generated at 4.3-29 mIU/ml and were not validated for our altitude. . Results:Of the 208 JAK2 tests performed during the reviewed period, 132 (63.4%) were for polycythemia (Table 1). The JAK2 V617F mutation was found in 22 (16.7%) patients, with 10 (45.5%) of them being men. No JAK2 mutation other than the exon 14 V617F substitution was found. JAK2-positive patients had a median age of 61 years, while JAK2-unmutated patients had a median age of 47 years. Overt polycythemia, according to the WHO 2016 classification, was identified in 15 (68.2%) , while the remainder had masked PV ( defined as increased RBCs with normal hemoglobin/ HCT). Patients with PV had significantly higher RBCs and lower MCV/MCH/MCHC compared to those with JAK2-unmutated polycythemia. They also exhibited significant elevation of RDW-CW and significant neutrophilia, basophilia, thrombocytosis, and splenomegaly. All PV patients with available serum EPO tests had low values (median 2 mIU/ml). Conclusion: At altitude, patients with PV show the same age and sex characteristics as those living at low elevations. Patients with PV have higher RBCs and lower MCV/MCH/MCHC compared to those with JAK-unmutated (secondary) polycythemia. RDW is significantly increased in patients with PV, reflecting an associated iron deficiency. Serum EPO performed well at altitude to suggest the presence of PV. The low rate of positive JAK2 mutation in our high-altitude cohort may reflect poor selection of subjects and over-workup of otherwise altitude-associated erythrocytosis. Isolated mild polycythemia at altitude remains common. Using hemoglobin/hematocrit alone to select patients for PV evaluation has a low positive predictive value and may be associated with unnecessary workup. RBC indices and RDW, along with an evaluation for iron status, might help select those more likely to have PV and could decrease unnecessary workups.

Enalaprilat and losartan decrease erythroid precursors frequency in cells from patients with polycythemia vera

ABSTRACTObjective:Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by the overproduction of red blood cells. First-line therapies are directed at lowering hematocrit levels. After the discovery of a mutation in the Janus kinase 2 (JAK2V617F), JAK2 inhibitors have been tested as second-line therapies. Despite these approaches, there is still the need for a major comprehension of the mechanisms involved in PV erythrocytosis and of more effective therapies. Angiotensin-converting enzyme (ACE) stimulates hematopoietic precursors proliferation and erythroid differentiation. We thus hypothesized that ACE inhibition could help in controlling erythrocytosis in PV.Methods:We assessed the clonogenic potential by colony-forming unit (CFU) assay of mononuclear cells isolated from PV JAK2 positive or JAK2 negative patients with erythrocytosis treated with enalaprilat or losartan.Results:Treatment with drugs led to a decrease of erythroid precursor frequency both in the presence and absence of JAK2 mutation, with a high extent in JAK2 positive cells and without affecting other types of precursors. No dose-dependent effect was observed.Conclusions:Our results demonstrate that ACE inhibition reduces erythroid precursor frequency, confirming the involvement of ACE in erythrocytosis despite the presence of JAK2 mutation and encouraging the hypothesis that ACE inhibitors and AT1R antagonists could help in directly managing erythrocytosis in PV.

Incidence of Thrombosis at Different Sites During the Follow-Up Period in Essential Thrombocythemia: A Systematic Review and Meta-Analysis.

Thrombotic events are the most frequent manifestations of essential thrombocythemia (ET). The objective of this study is to determine the incidence of thrombosis at different sites on follow-up in patients with ET. We searched PubMed, Web of Science, and The Cochrane Library databases and calculated the incidence of thrombosis by pooling and analyzing the extracted data using a random-effects model. A total of 70 studies (N = 25,805) were included in the analysis. The total and annual incidences of arterial thrombosis on follow-up were 13.4% and 2.0%, respectively. The total and annual incidences of the different manifestations of arterial thrombosis were as follows: stroke (5.3% and 0.8%), transient ischemic attack (5.1% and 1.2%), myocardial infarction (2.4% and 0.5%), unstable angina (0.9% and 0.2%), and peripheral arterial thrombosis (2.0% and 0.2%), respectively. In contrast, the total and annual incidences of arterial thrombosis in JAK2-positive patients were 18.4% and 2.7%, respectively. The total and annual incidences of arterial thrombosis in JAK2-negative patients were 5.9% and 0.8%, respectively. The total and annual incidences of venous thrombosis were 5.5% and 0.7%, respectively, and the incidences of the different manifestations of venous thrombosis at different sites were as follows: peripheral venous thrombosis (2.9% and 0.5%), superficial venous thrombosis (1.8% and 0.7%), deep venous thrombosis (1.6% and 0.3%), abdominal venous thrombosis (0.8% and 0.1%), pulmonary embolism (0.3% and 0.1%), and cerebral venous thrombosis (0.2% and 0%), respectively. The total and annual incidences of venous thrombosis in JAK2-positive patients were 7.4% and 1.2%, respectively. The total and annual incidences of venous thrombosis in JAK2-negative patients were 1.6% and 0.4%, respectively. The incidence of arterial thrombosis was higher than that of venous thrombosis in patients with ET. Arterial thrombosis manifested with cerebral arterial thrombosis, followed by cardiac thrombosis. Venous thrombosis events were mainly peripheral and superficial venous thrombosis. JAK2-positive patients have a higher incidence of arterial and venous thromboses than JAK2-negative patients, the sequence of thrombsis sites was similar to that of the overall patients.

Evaluation of Clinical , Laboratory and Genetic Parameters in Patients with Primary Myelofibrosis in Nineveh province

Background :Primary myelofibrosis is a Philadelphia-negative myeloproliferative neoplasm(MPN) with predominant proliferation of megakaryocytes and granulocytes in the bone marrow characterized by initial proliferative phase, followed by a reactive deposition of fibrous connective tissue in the bone marrow. Objectives : To evaluate the clinical and laboratory data in primary myellofibrosis and to correlate the genetic disorders with clinical and other laboratory parameters. Setting : This case series study conducted at Ibn-Sina Teaching Hospital/Outpatients Hematology Department from November 2019 to April 2020. Patients and method : Total fifteen patients with primary myelofibrosis included in this case series (retrospective and prospective) study. The records of old cases were reviewed from patients for clinical history, clinical examination, previous blood counts, bone marrow study and genetic study. For new cases clinical history and physical examinations were noted, ten mls of venous blood were aspirated from patients (new cases) by clean venipuncture and delivered into sterile EDTA and gel tubes for doing complete blood pictures, genetic study (JAK2) and biochemical tests. Data were collected and analyzed by using SPSS software version 24. Result : The males were (60%) of patients and females were (40%) with male to female ratio was 1.5:1, their age ranged from 43-75 years with a mean of 59.3 years. The most common complains were fatigue (86.7%), fullness in the abdomen (73.3%) and constitutional symptoms (66.7-80%). All patients(100%) had splenomegaly at presentation. The total mean Hb was 8.2 g/dl with a range of 4.7-12.5 g/dl. Leukocytosis had been observed in (40%) of patients, while thrombocytosis was found in (26.6%) of patients. Compared in hematological parameters in PMF patients with JAK2 Positive and JAK2 Negative, it was found that MPV significantly increased in JAK2 positive patients with p-value 0.03 . Conclusion : old age groups (55-64 years) were commonly affected by primary myelofibrosis and males were predominance over females. The most common complains were fatigue and constitutional symptoms, while splenomegaly was the most common clinical sign. The mean platelets volume (MPV) significantly increased in JAK2 positive patients with p-value 0.03.

THE INFLUENCE OF THE JAK2V617F MUTATION ON CLINICAL, LABORATORIAL, AND MORPHOLOGICAL VARIABLES IN BCR-ABL NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

Investigate the correlation between the presence of the JAK2V617F mutation and laboratorial, morphological and clinical findings. This is a retrospective, observational, single-center cohort-study involving adults with classic BCR-ABL negative MPN and myelofibrosis secondary to them (n = 119), in a Brazilian universitary hospital. It were evaluated hematological parameters and nonspecific inflammatory biomarkers such as LDH, CRP, ESR, and ferritin, in peripheral blood, as well as the occurrence of thrombo-hemorrhagic events or death, by consulting medical records. Patients diagnosed with ET (n = 75), and JAK2V617F -positive (n = 69; 92,0%) had significantly higher values of marrow cellularity, hemoglobin, hematocrit, neutrophil to lymphocyte ratio (N/L ratio), ferritin, also higher RBC and WBC counts. Besides a higher occurrence of thrombotic and/or hemorrhagic events (p = 0.006). A positive correlation (Spearman) was observed between the JAK2V617F mutation and thrombo-hemorrhagic events, bone marrow cellularity, RBC count, hemoglobin concentration, hematocrit, WBC count, N/L ratio, and ferritin dosage. Patients with ET positive for JAK2V617F mutation showed a “PV-like” profile with higher values of hemoglobin, hematocrit, hematimetry, LDH, and higher medullary cellularity in addition to, lower platelet values and higher incidence of thrombotic events, as suggested by previous studies. After all, JAK2V617F status is incorporated into the International Prognostic Score for Thrombosis in ET (IPSET). Besides demonstrating the clinical, epidemiological, laboratory profile, and mutational status in Brazilian patients in a university hospital, our study points to the need to reinforce the orientation about certain exams. It is essential to evaluate not only the incorporation of ESR in the routine investigation of patients in whom there is suspicion of classic MPN BCR-ABL negative, especially PV, but also the inclusion of CRP dosage in the prognostic evaluation of patients with ET and PV. There is a correlation between the JAK2V617F mutation and higher values of RBC count, WBC count, N/L ratio, and ferritin, in addition to greater cellularity in the bone marrow. A “PV-like” profile was also observed in JAK2-positive TE patients.

Mutational Landscape of Patients Referred for Elevated Hemoglobin Level.

Background: Since the identification of JAK2 V617F and exon 12 mutations as driver mutations in polycythemia vera (PV) in 2005, molecular testing of these mutations for patients with erythrocytosis has become a routine clinical practice. However, the incidence of myeloid mutations other than the common JAK2 V617F mutation in unselected patients referred for elevated hemoglobin is not well studied. This study aimed to characterize the mutational landscape in a real-world population of patients referred for erythrocytosis using a targeted next-generation sequencing (NGS)-based assay. Method: A total of 529 patients (hemoglobin levels >160 g/L in females or >165 g/L in males) were assessed between January 2018 and May 2021 for genetic variants using the Oncomine Myeloid Research Assay (ThermoFisher Scientific, Waltham, MA, USA) targeting 40 key genes with diagnostic and prognostic implications in hematological conditions (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Results: JAK2 mutations were detected in 10.9% (58/529) of patients, with 57 patients positive for JAK2 V617F, while one patient had a JAK2 exon 12 mutation. Additional mutations were detected in 34.5% (20/58) of JAK2-positive patients: TET2 (11; 19%), DNMT3A (2;3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%), and ZRSR2 (1; 1.7%). Diagnosis of PV was suspected in 2 JAK2-negative patients based on the 2016 World Health Organization (WHO) diagnostic criteria. Notably, one patient carried mutations in the SRSF2 and TET2 genes, and the other patient carried mutations in the SRSF2, IDH2, and ASXL1 genes. Three JAK2-negative patients with elevated hemoglobin who tested positive for BCR/ABL1 fusion were diagnosed with chronic myeloid leukemia (CML) and excluded from further analysis. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and mutations were found in 6% (28/466) of these cases. Conclusion: Mutations other than JAK2 mutations were frequently identified in patients referred for erythrocytosis, with mutations in the TET2, DNMT3A, and ASXL1 genes being detected in 34.5% of JAK2-positive PV patients. The presence of additional mutations, such as ASXL1 mutations, in this population has implications for prognosis. Both the incidence and mutation type identified in patients with secondary erythrocytosis likely reflects incidental, age-associated clonal hematopoiesis of indeterminate potential (CHIP).

The differences of hemogram, myelogram, and driver gene mutations in classic myeloproliferative neoplasms

The aim of this study was to explore and compare routine blood features and pathological characteristics of bone marrow tissues in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis, prefibrotic stage (prePMF) and overt fibrotic stage (overtPMF), and the correlation between common driver gene mutations and clinical manifestations of myeloproliferative neoplasms (MPN). Methods: We analyzed 259 MPN patients treated at Tongji Hospital of Huazhong University of Science and Technology from January 2016 to December 2020. Results: Among ET, PV, prePMF, and overtPMF, the median leukocyte counts of PV and prePMF were significantly higher than those of ET. The average hemoglobin level of overtPMF was significantly lower than that of ET, PV, and prePMF. ET and prePMF had higher platelet counts than PV and overtPMF, whereas ET had the lowest platelet distribution width. Regarding hematopoietic tissues in the bone marrow, enlarged megakaryocytes were easily found in ET, PV, and prePMF, whereas the average diameter of megakaryocytes in prePMF was smaller than in ET, and PV showed various sizes of megakaryocytes. An increased M/E ratio and dilation of sinus were seen more frequently in PMF. Additionally, JAK2-positive patients tended to have significantly higher leukocyte counts than CALR-positive patients in ET and PMF.

Abstract 399: JAK2 Clonal Hematopoiesis Associates With Pulmonary Hypertension Severity

Pulmonary hypertension (PH) is a heterogenous condition characterized by high pulmonary vascular resistance and elevated arterial pressures. Clonal hematopoiesis of indeterminate potential (CHIP) occurs when a hematopoietic stem cell acquires a somatic mutation that confers a proliferative advantage. CHIP is associated with a range of cardiovascular diseases. Recent work in a murine model of PH demonstrated that JAK2 CHIP causes PH via neutrophil mediated vascular remodeling. Little is known about the association between CHIP and pulmonary pressures in humans. We hypothesized that CHIP would be associated with higher pulmonary pressure and a higher prevalence of clinical PH in patients referred for echocardiography. We evaluated patients in the Vanderbilt BioVU biobank for the presence or absence of several CHIP mutations (DNMT3A, TET2, ASXL1, PPM1D, and JAK2) and correlated this finding with standard echocardiography parameters extracted from the electronic health record. PH was defined as an estimated pulmonary artery systolic pressure (ePASP) >=40mmHg. We analyzed 12,007 patients who had a reported ePASP after the date of sequencing. Of those, 49% (5,883 of 12,007) were male, with a mean age of 55+/-19.2 years. CHIP mutations were present in 477 (3.9%). The mean ePASP was 34.5+/-13.9mmHg and 32% (3,842 of 12,007) had an ePASP >= 40mmHg. The ePASP was higher among JAK2 positive patients (42.3[39.4-36.5]mmHg vs. 34.6[34.4-34.2]mmHg; p = 0.0002). This association persisted after adjusting for age, race, and sex, (Beta coefficient 4.1[1.8-6.5]mmHg, p = 0.0005). We found no association between ePASP and other CHIP mutations. The presence of a JAK2 mutation was associated with a higher overall prevalence of PH (1.5% [180 of 12,007] vs. 0.9% [108 of 12,007]; p = 0.007). In a large echocardiographic referral population, JAK2 CHIP mutations are associated with higher ePASP and a higher prevalence of PH. Treatment implications and the association between CHIP mutations and clinical outcomes in patients with PH warrants further study.

Comparison of Clinical Data and Treatment Responses of Patients with Essential Thrombocythemia Using Anagrelide by JAK2 Gene Mutation Status

Introduction: Essential Thrombocytemia (ET) is a clonal stem cell disease that manifests itself with proliferation in the megakaryocytic lineage in the bone marrow, with clinical presentations ranging from asymptomatic to bleeding and thrombosis spectrum.
 In medical treatment, aspirin and/or monitoring are recommended for low-risk patients, while cytoreductive therapy is recommended for high-risk patients. Cytoreductive therapy is often used in patients with a very high platelet count (>1,000,000). The first choice in cytoreductive treatment is Hydroxyurea, and Anagrelid treatment is preferred in the young patient group and patients with hydroxyurea resistance/intolerance.
 This study aimed to evaluate the effects of the JAK2 mutation, which is associated with high risk, in the patient group receiving anagrelide therapy.
 Method: The files of patients diagnosed with ET according to 2016 WHO criteria and followed up under Anagrelide therapy in our center between January 2002 and December 2021 were reviewed retrospectively. In addition to the demographic data of the patients, diagnostic tests, bone marrow evaluations, JAK2 mutation status, and laboratory data were noted. Patients were divided into two groups according to JAK2 mutation positivity and negativity. The obtained data were compared between the two groups.
 Results: Thirty-three patients (male/female: 20/13) treated with Anagrelide for the diagnosis of ET were included in the study. It was observed that 14 (42%) of the patients were positive for JAK2 mutation. There was no significant difference between the groups regarding age at diagnosis, gender, duration of anagrelide use, and bone marrow fibrosis degrees. When the laboratory tests were compared at the time of diagnosis, the WBC count was significantly higher in the JAK2 positive group; other series were similar. When the last control laboratory data of the patients were compared, WBC, neutrophil, and hemoglobin levels were observed to be significantly higher in JAK2 positive patients, while LDH levels were significantly lower.
 Conclusion: It was observed that JAK2 mutation positivity, which is associated with high risk in ET risk staging, did not negatively affect anagrelide treatment response. In ET patients, leukocytosis (>11,000/mm3) has been identified as a risk factor for the whole lifespan. It was observed that the WBC counts of the patients who were positive for JAK2 were significantly higher at the time of diagnosis and during the treatment process. Since the LDH level after treatment is higher in patients with positive JAK2 mutation, it has been evaluated that JAK2 mutation may play a role in resistance to cytoreductive therapy.

ANALYSIS OF CALRETICULIN (CALR) MUTATION IN MYELOFIBROSIS PATIENTS IN KHYBER PAKHTUNKHWA

Objective: To analyze the prevalence of CALR and JAK2 mutation co-occurrence in MF patients in KPK.
 Methods: This cross-sectional study included MF patients (n = 50) enrolled in the Hematology/Oncology department of Hayatabad Medical Complex (HMC), IRNUM (Institute of Radiotherapy and Nuclear Medicine), Peshawar and Blood Diseases Clinic, Peshawar. Non-probability convenience sampling technique was used. All Patients with JAK2 V617F positive and negative primary or secondary myelofibrosis were included. After taking blood samples, DNA was extracted manually and analysed for JAK2 mutations using conventional PCR. Sanger sequencing technique was implied to analyse the samples for CALR mutations. Data were recorded & analysed statistically.
 Results: Among the 50 patients of myelofibrosis under study, 48(96%) were harboring JAK2 mutations while it was not present in 2 (4%) patients. The typical CALR mutations was not identified in the current study. However, 02 specific genetic variants were identified in 30 patients i.e., a single nucleotide polymorphism (c.1381 G > T) in the 3? UTR and a novel insertion-deletion variant (c. 1099 CTT > AC). Seven patients had the Indel frameshift variant (p.Leu 367 Thr Fx 63), whereas 23 had SNPs. In JAK-2 positive patients (n=28), 06 had the Indel variant, and 22 had the SNPs. Two patients were JAK2 negative, one with SNP and the other with a frameshift mutation.
 Conclusion: Majority of the MF patients had a novel frameshift variant (p. Leu367Thr Fx63) (n = 07) and an SNP in the 3? UTR region (G > T) (n = 23) in CALR gene.

Management of Myeloproliferative Neoplasms during Pregnancy: A Systematic Review

▪Introduction:Myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Pregnancy in the presence of MPN increases the risk of thrombosis, hemorrhagic events, pre-eclampsia, progression to myelofibrosis (MF) or acute myeloid leukemia (AML) leading to poor fetal outcome.Methods:We conducted a literature search on PubMed, Embase, Web of Science, and Clinicaltrials.gov using MeSH terms and key words for MPN, PV, and ET from date of inception to January 2021. We excluded studies reporting less than 10 patients and those not in English language.Results:ET studies:How et al. (121 pregnancy (P)/52 women (W)) reported Aspirin (ASA) was protective and was used in 51.2% P (n=62/107) with OR (odd's ratio) of 0.304 (p=0.048; 90% CI 0.102-0.795). Similar benefit of ASA was also reported by Gangat e t al.(2009) (63P/36W ; P loss 21% in 24 patients receiving ASA vs 75% in 12 patients not receiving ASA), Niittyvuopio et al. (40P/16W; live births (LB) in all 13 P treated with ASA or ASA with prior cytoreduction therapy vs 67% complications and 56% loss of P in patients not treated with ASA), Randi et al. (1999) (16P/13W; 7/13 LB occurred on ASA, and only one P was lost on ASA), Bangerter et al.( 17P/9W; 100% P treated with ASA resulted in LB vs 45% LB in P without any treatment, p=0.04), and Pagliaro et al. (15P/9W; ASA monotherapy (n=3/15P) or ASA+ subcutaneous heparin (n=7/15P) resulted in nine LB). Cincotta e t al. (30P/12W) reported 79% LB with ASA or IFN-α vs 38% LB without any treatment (p=0.06). Among the treatment cohort, 5/7 patients on ASA had a LB, and all 4/4 patients on IFN-α had a LB.However, Beressi et al. (31P/18W) reported 4/8 untreated and 10/19 ASA treated P were lost with no statistically significant benefit. Palandri et al. reported 24P/13W treated with IFN-α (n=1), IFN-α + ASA (n=2) and ASA alone (n=9). The LB rate was not influenced significantly with treatment at conception (treated vs. untreated: 8/12 vs. 7/12, P=0.99). Melillo e t al. (122P/92W) reported no benefit with ASA. IFN-α had positive outcomes with 95% LB than patients not receiving IFN-α with 71.6% LB (OR 0.10 (95%CI: 0.013-0.846, p=0.034)). JAK2 mutation was associated with poorer outcomes (OR 6.19; 95%CI: 1.17-32.61; p=0.038). Wright et al. (43P/20W) reported no statistically significant difference between no treatment (n=16/43), ASA alone (n=24/43), ASA+cytoreduction (n=2/43), and low molecular weight heparin (LMWH) (n=1/43) groups.Passamonti et al.( 96P/58W ) reported statistically significant worse outcomes in JAK2 negative patients, no statistically significant benefit in JAK2 positive patients (p=0.05) with ASA and higher complications with conception while on cytoreduction therapy (n=6/13 abortion, n=3/13 pre-eclampsia).ET + Other MPN studies:Gangat et al. (2020) reported (152W ET and 36W PV) that in 95P/55W with ET, 60% pregnancy loss occurred without ASA vs 27% with ASA (p=0.02). All 4/36W with PV who were treated with one or more of ASA, enoxaparin, and hydroxyurea delivered LB, and 50% developed venous thromboembolism. Sokolowska et al. reported 104P/60W with MPN (47 ET, 3 PV, and 10 PMF) treated with various combinations of cytoreduction therapy (IFN-α in 48P, apheresis in 1P and anticoagulation (LMWH+ASA in 10, LMWH alone in 19 and ASA alone in 24P) during the pregnancy with 72.1% LB. Lapoirie et al. stratified 27P/14W (ET 19P/9W, PV 8P/5W) with MPN into high risk (67%) and standard risk and found significant P loss in all high-risk P that did not receive ASA (n=2). Maternal complications occurred in 3 high-risk P that received ASA. 77% of P treated with ASA, LMWH, or both resulted in a LB. Alimam et al. (58W; ET 47, PV 5, PMF 5, MPN-unclassified 1) reported better than predicted outcomes in 88% of women receiving ASA during P, 38% receiving ASA + prophylactic LMWH and 3% receiving ASA + therapeutic LMWH resulting in 58 LB with a P loss rate of 1.7/100 (95% CI; 0.04-9.24).PV studies:Irene et al. (24P/15W) reported a 40% incidence of P loss without antithrombotic therapy vs 10.5 % P loss receiving ASA, LMWH, or both. Willie e t al. divided 32P/14W with PV into two groups (treatment group: 18 P, treated with ASA during P and LMWH for six weeks postpartum; non-treatment group: 14 P) with 72% vs 14% LB respectively.Conclusion:Use of ASA, LMWH and cytoreductive therapy is the existing practice for pregnancies with MPN. However, proper evidence via a RCT is an unmet need. [Display omitted] DisclosuresAnwer: Janssen pharmaceutical: Honoraria, Research Funding; BMS / Celgene: Honoraria, Research Funding; Allogene Therapeutics: Research Funding; GlaxoSmithKline: Research Funding.

Spectrum of Myeloid Mutations in Patients with Elevated Hemoglobin

▪Background: JAK2 V617F and exon 12 mutations are the characteristic driver mutations in polycythemia vera (PV), identified in more than 95% of patients. In addition, other genetic mutations have previously been described in JAK2-positive PV that appear to have prognostic significance (Tefferi et al., Blood 2016). The incidence of other driver mutations in unselected patients referred for elevated hemoglobin is less well studied. This study aims to characterize the genetic mutational landscape in a real-world population of patients referred for elevated hemoglobin using a targeted Next-Generation Sequencing (NGS)-based assay.Methods: We reviewed all patients referred for elevated hemoglobin levels (>160 g/L in females or >165 g/L in males) between 2018 and 2020 to hematology clinics at London Health Sciences Centre in Southwestern Ontario, Canada who underwent testing for genetic variants using the NGS-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA). This assay targets 40 key genes with diagnostic and prognostic implications in several myeloid malignancies (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Patient demographics, laboratory data and final diagnosis were extracted from the electronic medical record. For all patients with genetic mutations, clinical diagnosis was confirmed by three independent reviewers.Results: A total of 529 patients underwent genetic testing for elevated hemoglobin levels: 389 (73.5%) were males (mean age 58; range 18-95) and 140 (26.5%) were female (mean age 60; range 24-85). JAK2 mutations were detected in 10.9% (58/529) of patients and a diagnosis of PV was confirmed. The majority of JAK2-mutated PV patients (n=57) were positive for JAK2 V617F, while one patient had an exon 12 mutation. Additional single myeloid mutations were detected in 34.5% (20/58) of JAK2-positive patients and involved the following genes: TET2 (11; 19%), DNMT3A (2; 3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%) and ZRSR2 (1; 1.7%) (Figure 1A). JAK2 mutations were not detected in 89.0% (471/529) of our cohort. A diagnosis of PV was confirmed in 2 JAK2-negative patients based on clinical features and myeloid mutations were detected in both: SRSF2 and TET2 gene mutations in 1 patient and SRSF2, IDH2, ASXL1 gene mutations in the other patient. Three JAK2-negative patients tested positive for the BCR-ABL fusion and were diagnosed with chronic myeloid leukemia. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and myeloid mutations were found in 6% (28/466) of these cases. Mutations were detected in DNMT3A (12; 2.6%), TET2 (5; 1.1%), ASXL1 (5; 1.1%), TP53 (2; 0.4%), NF1 (2; 0.4%), KIT (1; 0.2%), U2AF1 (1; 0.2%) (Figure 1B). All patients with JAK2-negative secondary erythrocytosis had only one myeloid gene mutation detected.Conclusion: Additional myeloid mutations other than JAK2 mutations are frequently identified in patients referred for erythrocytosis, with the highest frequencies observed in the TET2, DNMT3A and ASXL1 genes. The spectrum of myeloid mutations and overall incidence in JAK2-negative patients with secondary erythrocytosis is similar to the reported incidence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) (Jaiswal et al., NEJM 2014), and suggests that these may represent incidental age-related mutations. By contrast, among the JAK2-positive patients, 34.5% had at least one additional myeloid mutation supporting a pathogenic role in these patients with myeloproliferative neoplasms. While concomitant myeloid mutations in patients with PV are well-described, further research is required to elucidate the significance of variants identified in JAK2-negative patients classified as secondary erythrocytosis in order to determine whether these mutations contribute to clinical phenotype or represent background CHIP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Prediction Rule to Guide JAK2 Testing in Patients with Suspected Polycythemia Vera

▪Background: The widespread availability of molecular testing for JAK2 mutations has facilitated the diagnosis of polycythemia vera (PV) but also raises the concern of test overutilization in patients referred for elevated hemoglobin. At our institution, we have observed increased molecular testing in these patients with declining rates of JAK2 mutation positivity, suggesting that a prediction rule could be useful to guide such testing. In this study, we report the derivation and validation of a simple rule using complete blood count (CBC) parameters to predict the likelihood of having a JAK2 mutation in patients referred for elevated hemoglobin.Methods: We examined all patients with elevated hemoglobin (≥160 g/L for women, or ≥165 g/L for men), who underwent JAK2 mutation testing using the Next-Generation Sequencing (NGS)-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA), between 2018 and 2021 at the London Health Sciences Centre in Ontario, Canada. We extracted data including age and sex as well as CBC parameters at the time of testing, including hemoglobin, hematocrit, erythrocytes, leukocytes, neutrophils, platelets and mean corpuscular volume. All CBCs were performed on a Sysmex XN Analyzer (Sysmex Corporation, Japan). In the derivation cohort, JAK2-positive and -negative groups were compared using Student's t-tests or c 2 tests, as appropriate. We dichotomized potentially significant continuous variables at an optimal cut-off point using receiving operating characteristic curves. Potentially significant predictors were evaluated using multiple variable stepwise logistic regression analysis with JAK2 positivity as the dependent variable. The model was evaluated using Hosmer-Lemeshow tests and pseudo-R2 measures. A dichotomous score was derived based on the presence or absence of significant variables and subsequently evaluated and internally validated using logistic regression and c 2 tests using non-parametric bootstrapping with 1000 samples. The model was subsequently validated in the second cohort.Results: The derivation cohort included 308 patients tested between January 9, 2018 and December 19, 2019, and the validation cohort included 223 patients tested between January 7, 2020 and May 12, 2021. The characteristics of both cohorts are shown in Table 1. The final model included platelets above the upper quintile (308 × 10 9/L) and erythrocytes above the upper quartile (6.17 × 10 12/L) and a score of one was assigned to patients with either of these characteristics. The odds ratio for JAK2 positivity in patients with a score of 1 was 14.6 (95% CI 5.5-38.8) compared to those with a score of 0. The model had a sensitivity of 87.8% and a negative predictive value of 97.4% in the derivation cohort, and of 100% for both in the validation cohort. The percentage of JAK2 positive patients in patients with a score of 1 was 28%. The percent of false negatives was 2.6% (95% CI 1.1-6.0) and 0 (95% CI 0-2.8) in the derivation and validation cohorts, respectively. The use of this rule to guide molecular testing would have resulted in approximately 60% fewer tests.Conclusion: We developed and validated a simple rule to predict the likelihood of JAK2 mutation positivity in patients with a hemoglobin of 160 or higher, based on CBC parameters with a high negative predictive value (Figure 1). If implemented, this prediction rule could result in a significant reduction in molecular testing avoiding 60% or approximately 100 tests per year at our institution. This approach would be particularly beneficial for broader health system management of hematological malignancies, facilitating the reallocation of resources to emerging higher-yield molecular diagnostic investigation (Kawata et al., BJH 2021). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Understanding the Secondary Transformation of JAK2 Myeloproliferative Neoplasms: Genomic Analysis

Myeloproliferative neoplasms (MPNs) comprise of a group of clonal stem cell disorders characterized by proliferation of red blood cells (RBC; polycythemia vera or PV), platelets (essential thrombocytosis or ET) or white blood cells (WBC; primary myelofibrosis or PMF). Biggest challenge with MPNs is the leukemic transformation that occurs in 1-2%, 4-5%, and 20-30% of patients over a 10-year period in ET, PV, and PMF, respectively. These secondary leukemia patients are associated with dismal prognosis, with median survival of 6 months. Mutations in JAK2 have been observed in the majority of MPN patients, and though JAK2 mutation seems to be the phenotypic driver of MPNs, recent evidence suggest clonality and mutational events antecedent to JAK2 are responsible for leukemic transformation. To better understand the impact of somatic mutations that contribute to transformation of MPN to leukemia, and the clonal architecture of these mutations, we conducted this study. The collection of bone marrow samples, saliva samples and clinical data of 48 clinically JAK2 positive MPN patients (i.e. JAK2 V617F PCR positive) was carried out at Seoul National University Hospital, and this study was approved by the institutional review board. Written informed consents were obtained from all patients in accordance with the Declaration of Helsinki. The bone marrow samples obtained at chronic MPN state were subjected to target sequencing. Target genes providing diagnostic or prognostic information in myeloid malignancies were selected, and the final gene panel consisted of 2228 amplicons covering 47 genes. Amplified targets were sequenced on the Illumina TruSeq Amplicon. The mean coverage depth was 1056X. For 3 patients with sequential paired samples at MPN diagnosis and at secondary transformation, whole genome sequencing (WGS) was performed using Illumina HiSeq2000. Fisher's exact test was used to compare the mutation frequency of single genes in different MPN subgroups, while Levene's test was used to for comparison of mutation frequency of gene groups. Two-sided t-test was used to evaluate the statistical significance. There were 18 ET patients (37.5%), 21 PV patients (43.8%) and 9 PMF patients (18.8%), and the median age at MPN diagnosis was 63 years for the total cohort. Not surprisingly, PMF was associated with most secondary transformations (P=0.030). About 89.6% of the patients carried somatic mutations other than JAK2. The most frequently observed mutations included TP53, PTCH1, ASXL1, TET2, CALR, EZH2, KMT2A, MPL and SETBP1. Interestingly, majority of the patients carrying ASXL1 were PV. PMF patients more frequently carried mutated splicing genes per patient (p=0.005), while PV patients seem to carry more mutated epigenetic regulator genes per patient, especially the chromatin modifiers (P=0.077). In addition, somatic mutations in TP53 and NF1 are associated with increased risk of secondary transformation and these mutations seem to be present already in the chronic MPN state at a low allelic burden. The WGS results showed the number of mutations at chronic MPN state versus at secondary transformation did not significantly differ, suggesting novel mutation rate is low in MPN and the secondary transformation is indeed driven by expansion of already existent small clones. Efforts to effectively incorporate next generation sequencing into MPN diagnosis and follow-up will be helpful for recognition of patients at high risk of secondary transformation, and can ultimately lead to better clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pulmonary hypertension as a risk factor in JAK2-positive polycythemia rubra vera

Introduction The occurrence of primary pulmonary arterial hypertension (PAH (in primary myeloid proliferation neoplasms seems quite high, up to 22% in polycythemia vera. Polycythemia rubra vera (PRV) is one of the myeloproliferative neoplasms. We aimed to study the incidence of PAH among patients with PRV. Patients and methods A prospective cross-section study was performed on 60 (PRV) patients with PRV confirmed by bone marrow and JAK2 positivity. Abdominal ultrasonography, transthoracic echocardiography, and computed tomography chest were done to estimate the pulmonary hypertension (HTN) and exclude other chest diseases. Results Among the studied 60 patients, we found 14 patients with pulmonary HTN (23.4%) who had significantly increased incidence of comorbidities than patients with normal pulmonary pressure (P=0.009). Moreover, there were statistically significant differences in the size of spleen by ultrasound between the two groups (P=0.008). Patients with pulmonary HTN had a higher hemoglobin level compared with those with normal pulmonary pressure (P=0.006). There was a significant positive correlation between pulmonary pressure and existing comorbidities (diabetes mellitus, HTN, or both) but no correlation with the size of the spleen, hemoglobin level, or white blood cells. Conclusion The prevalence of PAH in JAK2-positive patients with PRV is 76.6%, and there was significant relationship between hematological parameters (hemoglobin, white blood cells, lactate dehydrogenase, and urea level and PAH in JAK2-positive patients with PRV.

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.

JAK2 V617F Mutation Negative Polycythemia Diagnosed By IGF-1R, EEC Formation , and IRS2 over Expressions

We previously reported that by flow cytometry study of mononuclear cells (peripheral blood) of IGF-1 R (insulin growth factor -1 receptor) can help in differentiating polycythemia vera (PV) from secondary erythrocytosis (PLoS ONE 11(11): e016529), in patients negative for JAK2V617F mutation who requires frequent phlebotomy. EEC (Endogenous erythroid colony formation) formation which was shown to be related to IGF-1 signaling was employed as a minor criteria of PV prior to 2016 WHO criteria for the diagnosis of PV. IRS-2, an IGF-1 activation downstream signaling protein was found to be a docking protein, specific for erythropoietin-induced erythropoiesis. Current studies were designed to correlation between IGF-1R over-expression to EEC (Endogenous erythroid colony formation) formation and IRS-2 over expression in those patients who required frequent phlebotomy with elevated IGF-1R and JAK2 mutation negative patients. Materials and Methods: Patients: 8 patients withsecondary polycythemia (JAK2V617F mutation negative), and 12 MPN patients (6 ET, 3 PV and 3 MF) were studied. Of the 12 MPN patients, 11 were JAK2 mutation positive and 1 was CALR mutation positive. All 20 patients had elevated IGF-1 R. Methods: 1) IGF-1R expression: 106 cells blood mononuclear cells were incubated with 10 μL of PE-conjugated control or anti-IGF1R IgG (R&D Systems) for 30 minutes. Fluorescent intensity was detected by a flow cytometry. 2) EEC formation: Erythroid colony cultures using Methyl cellulose based medium without (MethoCult H4230) or with pre-inclusion of cytokines (MethoCult H4230, including erythropoitin) from StemCell Technologies Inc. were used. On day 10-12, colonies were scored for Burst forming unit-erythroid (BFU-e), Colony forming unit-erythroid (CFU-e), and Colony forming unit cells (CFU-c) according to the manufacturer's graphic guide. 3) IRS-2 Expression: RT-PCR Predesigned primers for IRS-2 and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR blood mononuclear cells was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of IRS-2 was compared with each internal control and the final patient to normal control ratio was the average of the three. Results: 7 out of 8 patients with secondary PV, were found to have EEC formation. 12 MPN patients had increased expression of IRS-2 (Fig 1). 2 patients with secondary PV, so far studied also were found to have increased expression of IRS-2. Conclusion: 1) In patients with secondary PV (JAK2 mutation negative and increased IGF-1 and requiring frequent phlebotomy0, 7 out of 8 (87%) were found to have EEC formation. This suggests that IGF-1 by Flow cytometry can replace EEC as a supplement to the diagnosis of PV. 2) In JAK2 or CALR mutation positive patients , IGF-1R over-expression were found to have to have IRS-2 over-expression , 2 patients with secondary PV so far studied were also found to have elevated IRS-2 signaling. This suggests that the etiology of erythrocytosis in these secondary PV is likely through IRS-2 pathway. Further studies on this is still in progress. Disclosures No relevant conflicts of interest to declare.