Essential Thrombocythemia Following Immune thrombocytopenia With JAK2 V617F Mutation: A Case Report

Immune Thrombocytopenia (ITP) is an autoimmune bleeding disorder. Tyrosine Kinase JAK2 (JAK2 V617F) mutation occurs in nearly 60% of Essential Thrombocythemia (ET) patients. Both diseases produce impaired platelet. We describe a case with ET following ITP. So far, only 3 reports described ET following ITP. We report the fourth patient with JAK2 V617F mutation at the onset of ITP presented 20 years ago that needed splenectomy. The association of these two diseases may recommend similar pathogenic mechanisms between Myeloproliferative Neoplasms (MPNs) and ITP that should be further explored.

Germline-Somatic Interactions in Myelofibrosis Susceptibility

Introduction: Myelofibrosis (MF) is a rare myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, progressive bone marrow failure, and increased risk of acute myeloid leukemia. While MF arises from somatic driver mutations in JAK2, MPL, and CALR, some MPN patients may have a heritable component. To comprehensively examine the genetic etiology of MF, we performed the first integrative analysis of SNP array genotyping (using Infinium Global Screening Array), targeted long-read sequencing (using PacBio SMRT sequencing) and telomere length (TL, using qPCR assay). Methods: Our study included 937 MF patients who received an allogeneic hematopoietic cell transplant (HCT) between 2000 and 2016 and had an available pre-HCT blood sample at the Center for International Blood and Marrow Transplant Research Repository. Somatic mosaic chromosomal alterations (mCAs, including deletions, duplications, or copy-neutral losses-of-heterozygosity (CNLOH)) were called with the Mosaic Chromosomal Alteration (MoChA) algorithm using raw genotyping intensity data. A genome-wide association study (GWAS) was restricted to include 827 MF patients of European ancestry and utilized 4,135 genetically-matched healthy controls. Results: GWAS identified six independent MF susceptibility loci at genome-wide significance (P< 5×10 -8); four of which replicate prior MPN susceptibility loci [9p24.1(JAK2), 5p15.33(TERT), 3q25.33(IFT80), and 4q24(TET2)] and two novel MF loci [6p21.35(HLA-DQB1-AS1) and 17p13.1(TP53)] (Figure 1). A transcriptome-wide association analysis using whole blood GTEx data highlighted the 9p24.1 locus with increased JAK2 expression associated with elevated risk of MF (P= 2.18×10 -19). A strong colocalization statistic further indicated shared genetic component between eQTL and this JAK2 locus (HyPrColoc Posterior Probability= 0.6) (Figure 2). Based on the strong signal identified at TERT (Figure 1), we investigated the relationship between MF risk and genetically-inferred telomere length using a panel of 19 germline variants previously found to be associated with telomere length. Of the 19 telomere-length associated variants investigated, 7 were found to be associated with MF risk (binomial P= 2.31×10 -5, linear trend P= 5.48×10 -4) (Figure 3). Both Mendelian randomization and genome-wide genetic correlation analyses further indicated that increased risk of MF was associated with longer inherited telomere length. Utilizing available clinical mutation data on a subset of 185 patients, MF cases carrying the germline risk haplotype of the 9p24.1(JAK2) susceptibility locus were observed to more frequently have the JAK2 V617F mutation (71% vs 59%; P= 0.02). Targeted PacBio long-read sequencing around JAK2 provided further evidence of linkage between the germline risk allele and the JAK2 V617F mutation. Detectable autosomal mCAs were also abundant in MF cases with 67.4% having at least one mCA (compared to ~3% in the general population) and 27.6% having an mCA spanning JAK2 (mostly CNLOH) (Figure 4). In addition, using a binomial test for biased allelic imbalance, a cis relationship was identified at 9p24.1 in which the MF risk haplotype was predominantly duplicated by CNLOH (binomial P=1.36×10 -9). Regional sequencing of JAK2 further confirmed duplication of JAK2 V617F by CNLOH. Finally, we observed an inverse association between autosomal mCAs and qPCR measured telomere length (OR= 0.22, 95% CI= 0.07-0.65, P= 6.40×10 -3). These results were consistent by mCA chromosomal region and copy number state. Conclusion: Our results suggest a molecular framework for the genetic etiology of MF in which both genetically-inferred telomere length and germline variation at JAK2 are associated with increased MF risk. The 9p24.1 risk haplotype predisposes to the acquisition of a somatic JAK2 V617F mutation in cis and subsequent duplication of JAK2 V617F by mCAs (usually CNLOH). This process leads to aberrant JAK2 activity and increased clonal proliferation, accelerating telomere length shortening and increasing genomic instability in patients with MF. Figure 1 Figure 1. Disclosures Gupta: AbbVie: Consultancy, Honoraria; Constellation Pharma: Consultancy, Honoraria; Roche: Consultancy; Pfizer: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Lee: Janssen: Other; Incyte: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Syndax: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Saber: Govt. COI: Other.

Evaluating several methods of JAK2 V617F mutation-genotyping to predict the risk of getting polycythemia vera and other myeloproliferative neoplasm diseases

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are members of the Philadelphia negative chronic subgroup of Myeloproliferative neoplasm. Published studies showed that the mutation JAK2 V617F is mostly responsible for the diseases; therefore, an accurate, low-cost and rapid molecular method to identify this mutation is important in screening and early diagnostic of these diseases. Different methods for genotyping of JAK2 V617F have been proposed. In this study, we evaluated the quality and cost-effectiveness of three genotyping methods, i.e., PCR-ARMS, PCR-RFLP, Sanger sequencing, to determine the appropriate genotyping for JAK2 V617F and predicted in silico the effect of this mutation on the structure and function of Janus kinase 2 protein. Results showed that the Sanger sequencing and PCR-RFLP genotyping methods were more accurate than PCR-ARMS. PCR-RFLP was also more rapid and economical than the other methods. In silico studies also demonstrated that the JAK2 V617F mutation had a large effect on the activity of corresponding protein. These results provided the initial data for further studies on genetic screening and prediction of myeloproliferative neoplasm and other related diseases in the Vietnamese population.

CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms

The JAK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the JAK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.

The mortality outcomes and survival pattern of patients of Myeloproliferative Neoplasms in Malaysia

Background: The prognostication of myeloproliferative neoplasm (MPN) has always been challenging even in the advent of Janus kinase 2 (JAK2 V617F) molecular studies. The survival pattern of MPN in a developing country such as Malaysia is still undetermined.Materials and Methods: This was a retrospective study using information from 774 patients from the National MPN Registry conducted from the year 2009 to 2015 in Malaysia. Patients with the diagnosis of essential thrombocythaemia (ET), polycythaemia vera (PV), primary myelofibrosis (PMF) and unclassified MPN (MPN-U) were included. Survival data were traced until December 2018. Results: The cohort consisted of 42.0% ET, 41.0% PV, 8.9% PMF and 8.1% MPN-U, with 48.8% Malay, 39.1% Chinese, 7.1% Indian, 5.0% Others. The subtypes analysis revealed that male MPNs was more than female MPNs except in ET. The Chinese ethnicity was associated with the highest incidence of ET. The mortality rate was the highest in PMF followed by MPN-U then PV and ET (p<0.0001). Survival analysis revealed that the overall survival differed significantly according to characteristics such as sex, sub-types, JAK2 V617F mutation, bone marrow fibrosis, presence of splenomegaly, diabetes mellitus, hypertension, and bleeding manifestation. Cox regression analysis identified age, haemoglobin level, sex, and subtype as a significant risk factor for mortality outcome. Conclusion: Patients with ET had the slightly better OS while PMF had the worst OS. This is in conjunction with low haemoglobin, worsening bone marrow fibrosis, splenomegaly, diabetes mellitus, hypertension and bleeding. JAK2 V617F mutation was seemingly resulting in inferior overall survival especially in ET and PMF. The survival outcome of the MPN registry is instrumental for future policy development of effective healthcare in Malaysia.

Cerebellar venous thrombosis mimicking a cerebellar tumor due to polycythemia vera: a case report

Background Cerebral venous thrombosis (CVT) occurs rarely in the general population and is frequently associated with confused clinical findings and delayed diagnosis. Isolated cerebellar cortical vein thrombosis is a very rare phenomenon. Case presentation This report describes a case with CVT, which is manifested as space-occupying lesions of the cerebellar hemisphere and mimics a cerebellar tumor at the beginning. The diagnosis of CVT was finalized given the laboratory and brain biopsy findings. The etiology may be related to polycythemia vera with Janus Kinase 2 V617F mutation. Conclusion Isolated cerebellar vein thrombosis should be considered when swelling and enhancing cerebellar lesions are detected. Polycythemia vera, especially with a positive JAK2 V617F mutation, may be a rare risk factor for CVT.

AB0825 JAK2 (V617F) MUTATION AND ASSOCIATION TO IMMUNE MEDIATED DISEASES. STUDY OF 130 PATIENTS FROM A SINGLE UNIVERSITY HOSPITAL

Background:Janus Kinases (JAK) are tirosin-kinases that can promote cytokine production in immune and hematopoietic cells. The JAK-2 (V617F) mutation is the most frequently detected mutation in myeloproliferative neoplasms (MPN) which include essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF) and undifferenciated MPN. JAK-2 (V617F) mutation displays a pro-inflammatory phenotype that may be associated to a higher risk of immune mediated diseases (IMID), thromboembolic complications or other cancers (1-3).Objectives:To evaluate the presence of a) IMID (rheumatic and non-rheumatic), b) cancer and, c) Deep vein thrombosis/ Venous thromboembolism (DVT/VTE) events in a cohort of patients with a positive JAK-2 (V617F) mutation.Methods:We studied all the patients diagnosed with a positive JAK-2 (V16F) mutation in a single University Hospital between January, 2004 and December, 2019.Results:The study included 130 patients (73 men/57 women; mean age, 70.1±14.5 years). They were diagnosed of ET (n=64, 49.2%), PV 46 (35.4%), undifferentiated MPN (n=12, 9.2%) and PMF (n=8, 6.1%). Of these patients, 54 (41.5 %) (44 non rheumatic and 10 rheumatic) were diagnosed with at least one IMID, 46 (35.4%) with other cancer different of MPN and 36 (27.7%) with DVT/VTE events. (Table 1/Figure 1).Conclusion:Due to its prevalence and potential complications, IMID should be taken into consideration when a patient is diagnosed with a positive JAK-2 (V617F) mutation.References:[1]Perner F, et al. Cells. 2019;8:854.[2]Xu Q, et al. Clin Rheumatol. 2020 Jul 16.[3]Hasselbalch HC, et al. 2020; 23;17(1):248.Table 1.Associated diseases in 130 patients with JAK2 (V617F) mutation. Data are n (%)Myeloproliferative neoplasms (MPN)130 (100) Essential thrombocythemia (ET)64 (49.2) Polycythemia vera (PV)46 (35.4) Undifferentiated MPN12 (9.2) Primary myelofibrosis (PMF)8 (6.5)Non-rheumatic IMID44 (33.8) Diabetes mellitus22 (50) Asthma10 (22.7) Psoriasis6 (13.6) Crohn disease2 (4.5) Autoimmune thyroiditis2 (4.5)Rheumatic IMID10 (7.7) Rheumatoid arthritis4 (40) Polymyalgia rheumatica3 (30) Sjögren disease1 (10) Antiphospholipid syndrome1 (10) Adult-onset Still’s disease1 (10)Malignancies different of MPN44 (33.8) Solid tumours // Hematologic malignancies // Skin cancer22 (50) // 13 (29.5) // 9 (20.4)Deep vein thrombosis/Venous thromboembolism (DVT/VTE) events35 (26.9)Figure 1.Associated diseases accordingly to the subtype of Myeloproliferative neoplasm. Data are n.ABBREVIATIONS: DVT/VTE: Deep vein thrombosis/ Venous thromboembolism. ET: Essential Thrombocythemia, IMID: Immune Mediated Diseases, PV: Polycythemia Vera; MPN: Myeloproliferative Neoplasms; PMF: Primary myelofibrosis; UMPN: Undifferenciated myeloproliferative neoplasms.Disclosure of Interests:Carmen Álvarez-Reguera: None declared, Lara Sanchez-Bilbao: None declared, Ana Batlle-López: None declared, Sara Fernández López: None declared, Miguel Á. González-Gay Speakers bureau: Abbvie, Pfizer, Roche, Sanofi and MSD., Grant/research support from: Abbvie, MSD, Janssen and Roche, Ricardo Blanco Speakers bureau: Abbvie, Pfizer, Roche, Bristol-Myers, Janssen, Lilly and MSD., Grant/research support from: Abbvie, MSD and Roche