Ph-negative MPN Research Articles

Presence of JAK2V617F Mutation in Endothelial Cells from Budd-Chiari Syndrome Patients Is Not Correlated with Ph-Negative Myeloproliferative Neoplasm

IntroductionThe role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation.Objectives1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs; 2. To determine the mutational landscape of granulocytes in patients with BCS who harbored the JAK2V617F mutation but did not have the clinical diagnosis of a Ph-negative MPN.MethodsWe identified 10 patients from our institution who had a diagnosis of BCS and harbored the JAK2V617F mutation in granulocytes. Three patients died from hepatic failure before they could be evaluated by bone marrow biopsy, so 7 patients remain for the analysis. All patients were investigated for the presence of Ph-negative MPNs with bone marrow trephine biopsy. ECs assays were performed according to the method of Hill. Briefly, Ficoll-Paque density gradient–isolated mononuclear cells were plated on fibronectin coated 6-well dishes with EndoCult medium (Stem Cell Technologies) for 48 hours, when non adherent cells were recovered and re-plated in a new dish at 106/mL concentration. After an additional 5 days, adherent cells were plucked and analyzed by flow cytometry. The ECs population was sorted using a FACS Aria BD Biosciences sorter according to the following phenotype: CD45-PerCP-negative, CD31-FITC-positve, VEGFR2-PE-positive, CD34-PECy7-positive, CD133-APC-negative. The presence of the JAK2V617F mutation was investigated by allele-specific PCR. Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 3 patients with JAK2V617F-positive BCS without a clinical diagnosis of Ph-negative MPN was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University), followed by in-house filters to reduce false positive calls.ResultsWe were able to obtain CECs from all 7 patients. The purity of the CECs populations obtained was over 96% in all cases. Among the 7 patients with BCS, five did not have any clinical feature of a Ph-negative MPN, with a normal bone marrow biopsy. Results are summarized in the table. The JAK2V617F mutation was positive in the CECs from 5 cases, including 3 patients who only had BCS. In one patient with BCS solely the reaction did not work, and in another the JAK2 was wild-type in the ECs. The mutation was positive in CECs from both patients with myelofibrosis and BCS. Three patients with BCS solely were evaluated by whole exome sequencing. The only known pathogenic abnormality found was the JAK2V617F mutation, albeit at a low allele fraction (5%, 6% and 12.6%).ConclusionThe presence of the JAK2V617F mutation in CECs from patients with BCS who did and did not have a diagnosis of Ph-negative MPN suggest that the mutation plays an important role in the development of vascular complications in these patients. Further studies with a larger number of patients are needed to precisely define the importance of CECs in the pathogenesis of MPNs. The sole presence of the JAK2V617F mutation in circulating granulocytes at a very low allele fraction in patients with BCS without Ph-negative MPNs suggest that these patients have a pre-malignant clone that would probably remain undiagnosed had it been not for the development of hepatic venous thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance Of ASXL1 and IDH Mutations inPhiladelphia Negative Myeloproliferative Neoplasms

IntroductionASXL1 gene mutations and IDH1/IDH2 mutations were described in Ph-negative MPN in 2009 and 2010, respectively. We aimed to study frequency of aferomentioned mutations and their clinical importance, prevelance and potential prognostic significance in Ph-negative MPN. MethodsStudy group consisted of 107 ET (58 females, 49 males) and 77 PMF (43 females, 34 males) patients diagnosed according to WHO 2008 criteria. Exon 12 of ASXL1 was amplified from genomic DNA and bidirectionally sequenced. High-resolution melting was performed for IDH1 mutations (R130) and IDH2 mutations (R140 and 172) followed by confirmatory bidirectional sequence analysis. To our knowledge, this is the largest cohort of ET studying ASXL1 mutations. ResultsFrequency of ASXL1 mutations (exon 12) was significantly higher in PMF (24.7%) compared to ET patients (8.4%; p=0.005). Our analysis identified 29 different mutations of ASXL1 in 28 out of a total 184 patients, (15.2%) (one PMF patient presented with two simultaneous mutations). Nonsense mutations, including frameshift mutations were detected in 19 patients (10.3%). Mutant IDH was detected in 2 ET (1 IDH1 R140Q, 1 IDH1 R132C) and 5 PMF patients (3 IDH1 R123C, 1 IDH1 R132S, 1 IDH2 R140Q). The ET patient with IDH2 mutation (R140Q) harbored JAK2V617F mutation with an allele burden of 5% while the other patient with IDH1 mutation (R132C) did not harbor JAK2V617F mutation. 3 of 5 IDH mutated PMF patients (1 IDH1 R132C, 1 IDH1 R132S and 1 R140Q) concurrently carried JAK2V617F mutation with an allel burden of 31-50%, 5-12.5% and 31-50%, respectively. Frequency of IDH mutation for ET and PMF patients was 1.9% and 6.5%, respectively (p=0.131). We observed shortened OS in ASXL1-mutated PMF patients as compared to ASXL1 wild-type PMF patients(mean 108 months; 95% CI: 62-153 and 202 months; 95% CI: 123-282, respectively; p=0.025). (Figure 1). Also, trend towards shorter OS was observed in presence of ASXL1 nonsense sequence variations with respect to other sequence variations (p=0.09). Multivariate survival analysis confirmed independent prognostic value of ASXL1 mutations (OR: 2.75; 95% CI:1.37-5.5; p=0.033) but not ASXL1 nonsense sequence variations (p=0.131). Although number of IDH mutated PMF patients was small, we observed significantly shorter LFS in IDH mutated patients as compared to IDH wild-type patients (p=0.024). [Display omitted] ConclusionIn our study group, 7% of JAK2V617F-negative ET and 26.3% of JAK2V617F-negative PMF patients showed ASXL1 mutations. Based on our findings, it may be deduced that analysis of ASXL1 genes provided an additional 7% increase in ET and a 26.3% increase in PMF patients with a detected molecular marker of clonality. In our relatively large cohort of PMF patients, ASXL1 and IDH mutations were associated with shortened OS and LFS, respectively. One of 5 IDH mutated PMF patients harboring IDH mutations showed leukemic transformation. Thus, it can be suggested that IDH mutations may play a role in prediction of leukemic transformation. ASXL1 mutated PMF patients did not show an increase in poor karyotype features. In PMF, death and bleeding complications were associated with combined ASXL1 and IDH mutations. Additional studies are needed to clearly assess impact of mutation combinations on disease course and severity. An important characteristic feature of IDH mutated PMF in this study was the paucity of abnormal or unfavorable karyotype. All IDH mutated PMF patients had normal karyotype. Thus, it seems that ASXL1 mutations and IDH mutations represent independent prognostic biomarkers in PMF. Our findings support previous observations that mutations affecting epigenetic regulation might be prognostically more relevant than those involving JAK-STAT signaling in PMF. Disclosures:No relevant conflicts of interest to declare.

JAK2 Inhibitors: Are They the Solution?

The discovery of the JAK2V617F mutation in patients with Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPN) started the era of targeted therapy for these diseases. Until now, patients had few treatment options available, which usually were restricted to hydroxyurea, interferon preparations, and chemotherapy in more aggressive cases. JAK2 inhibitors have been developed over the past 5 years, and the results of the first clinical trials with JAK2 inhibitors for patients with myelofibrosis were recently published. Current research results suggest that JAK2 inhibitors have a potential to decrease disease burden and its activity, as manifested by a decrease in splenomegaly and improvement in systemic disease-related symptoms, but they do not seem to be able to eradicate the malignant clone. However, JAK2 inhibitors help patients regardless of their mutation status, because patients without JAK2V617F mutation benefit to the same extent as patients with JAK2V617F mutation. A greater understanding of the pathophysiology of MPNs is needed before we can cure myelofibrosis with drug therapy. Currently, several new JAK2 inhibitors are in clinical trials for patients with myelofibrosis, and clinical trials for patients with polycythemia vera and essential thrombocythemia have also started. We review recent data on JAK2 inhibitors for the management of patients with Ph-negative MPNs.

Prevalence of MPL W515L/K Mutations in Taiwanese Patients With Philadelphia-negative Chronic Myeloproliferative Neoplasms

The discovery of Janus kinase 2 (JAK2)-V617F has provided important insight into the pathogenesis of Philadelphia-negative chronic myeloproliferative neoplasms (Ph-negative MPNs); however, the etiology of JAK2(V617F)-negative Ph-negative MPN remains unidentified. MPL(W515L) and MPL(W515K) (MPL(W515L/K)) are 2 gain-of-function mutations, which have been found in some Ph-negative MPN patients from Western countries. However, little is known about the incidence of these mutations in Taiwanese Ph-negative MPN patients. We determined the MPL sequence of DNA samples from 105 patients, including 88 patients with Ph-negative MPNs and 17 with myelodysplastic syndrome, using polymerase chain reaction amplification of the cytokine receptor MPL exon 10 sequence. All the patients were normal at codon 515 regardless of their JAK2 status. The MPL W515L/K mutations are rare in Taiwanese patients with Ph-negative MPNs.