JAK2V617F-negative Myeloproliferative Neoplasms Research Articles

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Meth- ods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

Vascular Endothelial Dysfunction in Myeloproliferative Neoplasms and Gene Mutations.

Essential thrombocythemia (ET) and polycythemia vera (PV), are common Philadelphia-negative myeloproliferative neoplasms (MPN). Patients with MPN have a high rate of cardiovascular complications and often have acquired JAK2V617F and CALR genetic mutations. In this study, we aimed to analyze vascular endothelial function in patients with MPN.We evaluated 27 outpatients, including 10 patients diagnosed with MPN, flow-mediated dilatation (FMD), and nitroglycerin-mediated dilation (NMD), between September 2014 and August 2016. We measured serum adiponectin, which protects vascular endothelial function, and serum asymmetric dimethyl arginine (ADMA), which inhibits the production of adiponectin. The presence or absence of JAK2V617F and CALR mutations was evaluated in patients with MPN.Venous thrombosis was observed more frequently in patients with MPN than in those without. Seven MPN patients were diagnosed with PV, and 3 MPN patients were diagnosed with ET. JAK2V617F and CALR mutations were found in 5 and 3 MPN patients, respectively. FMD was significantly lower in JAK2V617F-positive MPN patients than in JAK2V617F-negative MPN patients, although NMD, adiponectin, and ADMA were similar in both groups. Adiponectin levels were higher and ADMA levels were lower in CALR-positive MPN patients than in CALR-negative MPN patients. There was no difference in FMD and NMD prevalence between the 2 groups. Furthermore, we had 3 representative MPN patients who were complicated with coronary spasm, possibly caused by MPN-related endothelial dysfunction.We found that patients with MPN presented with endothelial dysfunction, which was related to the presence of genetic mutations and was sometimes associated with cardiovascular disease.

Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

Objective:The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. Methods:The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F–negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. Results:28.3% of JAK2 V617F–negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. Conclusion:Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

Variation of STAT3 Gene in Myleproliferative Neoplasms and Its Significance

To detect the mutation and single nucleotide polymorphisms of STAT3 gene in the patients with myeloproliferative neoplasms (MPN), and to analyze the correlation between STAT3 gene and the subtypes of MPN. A total of 147 patients with MPN were selected, including 28 patients with polycythaemia vera (PV), 46 patients with essential thrombocythemia (ET), 10 patients with primary myelofibrosis (PMF), and 63 patients with chronic myeloid leukemia (CML); and 88 healthy persons were used as normal control. DNA of all cases was extracted from bone marrow or peripheral blood, and JAK2V617F gene mutation was detected by allele-specific PCR, then 23 exons of STAT3 gene were amplified by PCR. Mutation and single nucleotide polymorphism of Rs2293152 of STAT3 gene were identified by DNA sequencing. STAT3 gene mutation was found in 8 patients with CML. The mutation rate was 12.7%. the missense mutation（S629T）as found in 3 cases, the synonymous mutation was found in 5 cases (Q469Q 3 cases, G618G 2 cases). One case had mutations at the both sites of S629T and G618G. No mutation of STAT3 gene was found in the normal control group. Rs2293152: detection showed that the G allele of CML group was significantly higher than that of normal control, PV, ET and PMF group (P<0.01), suggesting that the patients with Rs2293152 G allele were more likely to develop CML. The C allele of PV, ET and PMF group was significantly higher than that of CML group (P<0.05), suggesting that the patients with Rs2293152 C allele were more likely to develop PV, ET and PMF. The G allele fiequency of JAK2V617F-negative group was significantly lower than that of the normal control and JAK2V617F positive group (P<0.01), suggesting that the Rs2293152 G allele may be a factor protecting against JAK2V617F mutation. In MPN patients, STAT3 gene is unstable and prone to mutation. The different alleles of the Rs2293152 locus of the STAT3 gene are relates with different subtypes and JAK2V617F-negative MPN.

Bone Marrow Morphology in Myeloproliferative Neoplasms – Relevance in the Molecular Era

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders. The mutation BCR-ABL is sine qua non for diagnosis of chronic myeloid leukemia and JAK2 V617F is present in majority of cases of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Recent 2016 classification consigns importance to BM morphology as mutations are not universal. Identification of specific features would aid diagnosis of JAK2V617F negative MPN. This study aims to assess utility of peripheral blood and bone marrow morphology in myeloproliferative neoplasms in correlation with BCR-ABL and JAK2V617F. Methods: A retrospective 7 years study of peripheral blood, bone marrow aspirate (BMA) and bone marrow biopsy (BMB) of 44 cases of myeloproliferative neoplasms with BCR-ABL and JAK2 V617F mutation analysis. Results: Forty four cases of MPNs including 24 (54.5%) cases of CML, 7 (15.9% ) cases of PV, 5 (11.3%) cases of MF, 3 (6.8%) cases of ET, and 5 (11.3%) cases of MPN-U. MPV and LDH were significantly higher in MF in comparison to PV and ET. Higher haemoglobin (Hb) and platelets were seen in PV and ET. Dwarf MKs with hypolobated, cloud like nuclei in CML (fig 1), large MKs with staghorn nuclei were seen in ET. normal sized MKs with cloud like hypolobated forms in PV(fig2) and cloud like atypical dysmorphic nuclei with dense clustering were seen in MF. Conclusion: Hb, mean platelet volume (MPV), and LDH in conjunction with MK morphology aids in the diagnosis of MPNs. These findings will enable accurate diagnosis in JAK2 negative MPNs and benefit low resource center.

Generation, Characterization and Genetic Modification of Human iPSCs Containing Calr, MPL and JAK2 Mutations Found in MPN Patients

Introduction: Myeloproliferative neoplasms (MPNs) that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are associated with multiple somatic and myeloid-specific mutations. A major challenge to the study of these diseases has been the lack of model systems that can accurately represent their complexity; the clonal heterogeneity within the hematopoietic stem/progenitor cells (HSPC) population is confounding and it is technically challenging to isolate and expand patient HSPC clones in vitro. To overcome this hurdle, we have utilized induced pluripotent stem cell (iPSC) technology to generate disease and clone-specific iPSC lines that preserve the genetic identity of HSPC clones with the JAK2-V617F mutation (Ye et al., 2009 in Blood). Recent advances in MPN genetics have identified CALR and MPL mutations in the majority of JAK2-V617F negative MPN. We therefore created additional panels of iPSCs from patients carrying CALR and MPL mutations in addition to our JAK2-mutated iPSCs. In addition, we used CRISPR/Cas9 technology to study genetic lesions and their contribution the phenotypes by using iPSC in vitro hematopoietic differentiation assays (Ye, et al., 2015 in Molecular Therapy).Methods: We generated iPSC lines from MPN patients with JAK2V617F, CALR or MPL mutations using peripheral blood CD34+ cells by transient expression of reprogramming factors in plasmid vectors. We characterized the pluripotency and karyotype of the derived iPSC lines, and determined if they contained the same somatic mutation as found in blood samples of each MPN patient. Then the derived iPSC cell lines were differentiated to CD34+ HSPCs, erythroid or megakaryocytic cell lineages using defined in vitro culture systems. Erythroid differentiation was determined by flow cytometric analysis of CD235a (Glycophrin A)-positive and CD45-negative cells. Megakaryocytic differentiation was determined by the levels of CD41+CD42+ cells.Results: In vitro differentiation assays have shown increased erythroid differentiation of HSPCs derived from patient-specific iPSCs that carry the JAK2V617F mutation, recapitulating a major PV disease feature. Editing a PV-derived JAK2V617F/V617F iPSC line to JAK2V617F/WT abrogated endogenous erythroid colony (EEC) formation, erythroid expansion and altered erythropoietin dose-response curve characteristic of the parent line. Conversely, gene editing a JAK2V617F/WT to a JAK2V617F/V617F iPSC line from a different PV patient conferred EEC formation and expanded erythropoiesis. In addition, data generated from these isogenic iPSCs suggested the contribution of additional genetic lesions to MPN disease phenotypes in JAK2-V617F positive patients. We also generated iPSC lines from MPN patients with the CALR type II (+5 bp) mutation and the rare MPL-V501L mutation. We found that the iPSC line containing a heterozygous MPL-V501L mutation (derived from an ET patient) appeared normal with iPSC typical morphology and phenotypes (expressing OCT4 and TRA-1-60 pluripotency markers) as the wildtype and JAK2-V617F iPSCs. We also generated four iPSC lines from a MF patient: all the four clones contained the homozygous CALR type II (+5 bp) mutation as found in the blood cells of the MF patient. Two iPSC clones we examined had a normal karyoptype. However, the iPSC lines showed signs of increased spontaneous differentiation when being cultured under the standard culture condition, based on cell morphology and reduction of OCT4 & TRA-1-60 expression. We are currently testing the potential of these iPSC lines containing the CALR and MPLmutations in hematopoietic differentiation, focusing on erythrocytic and megakaryocytic differentiation activities.Conclusions: we believe that by comparative analysis of isogenic iPSCs containing the common and rare MPN driver mutations, both common and specific pathways essential for MPNs can be determined. Precise genome editing or surgery of these mutations by engineered targeting may also provide a novel approach to eliminate the malignant MPN clones.This project is supported in part by the MPN Research Foundation and LLS. DisclosuresNo relevant conflicts of interest to declare.

High platelet count and high probability of CALR detection in myeloproliferative neoplasms

Calreticulin (CALR) has been recently detected in BCR-ABL1 and JAK2V617F negative myeloproliferative neoplasms (MPNs). In this study, we used PCR-PAGE and DNA sequencing methods for detection of CALR mutations and analysis of laboratory findings in MPN patients. BCR-ABL1, JAK2V617F, andMPL515 were assessed using RT-PCR and ARMS-PCR methods in 103 suspected MPN patients. CALR was assessed in patients negative for BCR-ABL1 and JAK2V617Fpatients as well as six cell lines (Jurkat, U937, NB4, KCL22, THP1, and K562) using PCR-PAGE and DNA sequencing. Finally, laboratory data of patients were obtained. BCR-ABL1 and JAK2V617F were detected in 39 (37.86 %) and 34 (33 %) MPN patients, respectively. Two patients had combined BCR-ABL1/JAK2V617F mutation. MPL 515 was negative in all the patients. CALR mutation was found in six male JAK2V617F-negative MPN patients (28.6 %, p = 0.07). Platelet count was 1049.5 × 109/L (p = 0.01) in these patients. All patients with type 1 and 2 CALR mutations had platelet counts lower and higher than 1000 × 109/L, respectively. CALR-positive patients had lower WBC count versus JAK2V617F-positive patients. Finally, CALR mutations were negative in all the cell lines. PCR-PAGE detected mutations in a shorter time with lower cost. JAK2V617F may be detected in BCR-ABL1-positive patients and is not specific for these patients. CALR is useful for molecular diagnosis of JAK2V617F- and MPL-negative MPN patients. PCR-PAGE may be a rapid, cost-effective, and simple method, especially in developing countries. CALR mutations are detected in male patients, suggesting different incidences of CALR mutations in male/female populations in different nations and races.

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms.

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2(V617F)-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10(-10)) and rs2201862 (MECOM; meta-analysis P=1.96 × 10(-9)). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2(V617F)-positive cases. rs9376092 has a stronger effect in JAK2(V617F)-negative cases with CALR and/or MPL mutations (Breslow-Day P=4.5 × 10(-7)), whereas in JAK2(V617F)-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ(2) P=7.3 × 10(-7)). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype.

Integrated Genetic Profiling Of JAK2 Wildtype Chronic-Phase Myeloproliferative Neoplasms

BackgroundThe Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) are clonal hematopoietic disorders. JAK2V617F mutations are observed in approximately 90-95% of PV cases, but only 40-50% of ET and PMF cases. Although JAK2 exon 12 and LNK mutations are observed in the majority of JAK2V617F-negative PV patients, candidate gene and exome sequencing studies to date have not identified activating oncogenes in the majority of JAK2V617F-negative ET/PMF patients. Thus, further genetic investigations are needed to define the mutational architecture of these JAK2 wildtype MPNs in order to gain insight into the biology of these diseases, the clinical implications of genetic events that do occur, and the elucidation of potential therapeutic targets. AimsTo characterize the spectrum of genetic alterations in JAK2 wildtype chronic-phase myeloproliferative neoplasms. MethodsWe identified 32 patients with a confirmed diagnosis of an MPN (per 2008 WHO criteria), including MF, PV and ET, who were negative for JAK2V617F using a CLIA-certified allele specific assay for the JAK2 disease allele. Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. ResultsHigh coverage sequencing allowed us to identify JAK2V617F mutations in two patients (allele burden 3-5%) that were below the limit of detection of the CLIA assay. The most common mutations observed in JAK2V617F-negative MPN were in ASXL1 (22% of patients) and in TET2 (9%). Taken together, mutations in known epigenetic modifiers (ASXL1, TET2, DNMT3A, EZH2, MLL) were observed in 43% of samples, including a MLL-PTD mutation in one patient with PMF. We identified mutations in spliceosome components (SRSF2, U2AF1), in a subset of patients, consistent with previous reports, and in each case mutations in spliceosome components were mutually exclusive.We identified mutations in the JAK-STAT pathway (MPL, TYK2) and the RAS pathway components (KRAS, NF1) in 9% of this patient cohort, suggesting that there are alternate disease alleles that activate signaling in JAK2V617F-negative MPN. RNA-sequencing identified a ETV6-ABL1 fusion in one patient, and we identified amplification of PIK3CA in one patient in our cohort; these data suggest fusion genes and amplifications activate signaling in a subset of patients with JAK2V617F-negative MPN. We also identified novel mutations in MPN patients which have not been reported to date, including mutations in DNA repair genes (ATM and BRCA) in 25% of cases and mutations in the Notch signaling pathway (NOTCH1-4) in 31% of cases. The functional implications of these novel mutations remain to be elucidated.In univariate analysis, ASXL1 mutations were found to associate with impaired overall survival (Figure 1, p=0.049). These findings are consistent with data demonstrating an impaired survival in patients with MDS and PMF, and suggest that ASXL1 mutations represent an important biomarker for adverse outcome in JAK2V617F-negative MPN. [Display omitted] ConclusionsThese data demonstrate that the mutational spectra of JAK2V617F-negative MPN includes genes implicated in epigenetic regulation, novel mutations which activate gene signaling, and fusion genes/copy number alterations which provide a novel mechanism of oncogenic activation not previously reported in MPN. ASXL1 mutations occur frequently in JAK2 wildtype Philadelphia-Chromosome negative MPNs, and are associated with impaired overall survival. Collectively, these findings support the importance of ASXL1 mutations in predicting outcome in JAK2V617F-negative MPN, demonstrate that mutations in signaling effectors and in epigenetic regulators are common in MPN, and illustrate the genetic heterogeneity of JAK2V617F-negative MPN. Disclosures:Rampal:Foundation Medicine: Consultancy. Levine:Foundation Medicine, Inc: Consultancy.

High-Resolution Melting (HRM) Curve Analysis Is a Reliable and Relevant Tool In JAK2V617F-Negative Myeloproliferative Neoplasms

IntroductionThe JAK2V617F mutation is observed in more than 95% of patients with Polycythemia Vera (PV), and 50–60% of patients with Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Refractory Anaemia with ringed Sideroblasts with marked Thrombocytosis (RARS-T). As a consequence, for JAK2V617F-negative cases it is worth looking for other mutations in the JAK2 and MPL genes as they are an important diagnostic aid for the myeloproliferative neoplasms (MPN). For example, half of JAK2V617F-negative PV patients carry mutations in the exon 12 of JAK2. To date, 40 different JAK2 exon 12 mutants have been reported. All are located between aa536 and aa547 and include deletions, insertions, and duplications. In the same way, nearly 5% and 10% of JAK2V617F-negative ET and PMF carry mutations in exon 10 of MPL, mainly affecting codons S505 and W515. Techniques such as allele-specific polymerase chain reaction (AS-PCR) are inappropriate for screening and sequencing, including Next Generation Sequencing (NGS), is time consuming as a first screening. High-resolution melting (HRM) curve analysis allows a rapid and whole exon mutation scanning approach associated with excellent sensitivity. The goal of our study was to evaluate the relevance of HRM in JAK2V617F-negative MPN and RARS-T in 3 French hospitals. MethodsHRM is based on the analysis of fusion curves of a short amplification product of PCR. This procedure, which derives PCR in real time and uses a fluorescent agent intercalated DNA, makes it possible to obtain a good resolution and to detect different types of mutations. The analyses were carried out on DNA from purified blood granulocytes. Diagnosis of PV, ET, PMF and RARS-T was established according to the 2008 WHO criteria. All patients were tested for their JAK2V617F status. To evaluate the efficiency of HRM in a routine diagnostic setting, we investigated 1997 patients with suspicion for a MPN from 3 French university hospitals. Samples with abnormal HRM curves were sequenced in order to confirm and characterize the mutations. ResultsFor 37/881 (4.2%) JAK2V617F-negative IE and PV patients, the HRM curve raised suspicion of the presence of a mutation in JAK2 exon 12, all confirmed by sequencing: the most frequent mutations were H538Q K539L (n=6), R541 E543 delins K (n=6), N542 E543 del (n=5), F537–I546 dup 10_F547L (n=4). For 2 additional PV cases, an abnormal HRM curve was obtained for JAK2 exon 14. Sequencing analysis revealed a V617F mutation with additional mutation of base 1831 (1831T>G), changing leucine 611 for a valine (L611V). For the second PV patient, a silent mutation corresponding to a substitution of a cytosine by a thymine in position 1848 of exon 14 of JAK2 was observed (C616), also associated with a V617F mutation. In both cases the JAK2V617F mutation had not been detected because the primers of the JAK2V617F AS-qPCR assay covered the sequence coding for L611 and C616. In addition, mutations in JAK2 exons 13 and 15 were tested on 33 PV-suspected samples: only one PV with a JAK2 exon 15 mutation was noted. Regarding the detection of mutations in exon 10 of MPL, an abnormal HRM curve was noted for 59/1116 (5.3%) MPN suspected. The mutations found were W515L (31 ET, 4 PMF), W515K (8 ET, 3 PMF), W515A (2 ET), W515R (1 ET, 1 PMF), W515S (1 ET), S505N (4 ET, 2 PMF, 1 RARS-T), V501A (1 ET). ConclusionIn ET and PMF, the HRM approach offers the advantage of quickly and reliably assessing patients for W515 and S505 mutations. In JAK2V617F-negative PV, due to the large number of possible mutations, HRM is a highly potent screening technique and allows a rapid scan of the whole exon 12, leading secondarily to sequence only a small number of HRM positive cases. Moreover, in 2 PV, HRM analysis of JAK2 exon 14 allowed to detect a V617F mutation hidden by a second mutation that inhibited primer hybridization and subsequent DNA amplification in the routine AS-qPCR assay. In summary, HRM is a highly relevant technique, cheaper than NGS, for routine molecular diagnosis of JAK2V617F-negative MPN, which typically carry a large variety of mutations, expressed at low levels. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Rapid Molecular Diagnosis Of JAK2V617F Negative MPN By Targeted Deep Sequencing Using The Ion Torrent PGM

Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells, increased risk of thrombotic complications and slow progression to myelofibrosis or, less often, leukemia. Activation of the JAK-STAT signaling pathway is a common underlying feature of these diseases and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The remaining 40% of cases of MF and ET harbor a broad range of mutations in many genes including those involved in cytokine receptor signaling, other components or the JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of thrombocytosis, many cases of chronic ‘ET' may not be clonal hematological neoplasms but reactive conditions.We have developed a rapid deep sequencing pipeline to detect mutations in 65 genes which have been implicated in MPN through previous reports of human mutations, mouse models of MPN, or other known components of hematopoietic cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway.In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN. Disclosures:Perkins:Novartis Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Controlling Thrombopoietin Receptor Dimerization, Orientation and Activation Via Tryptophan 515

AbstractAbstract 3449 Background and Aims.The thrombopoietin receptor (TpoR, c-Mpl) plays a dual role in hematopoiesis. Beside the fact that it is one of the main regulators of megacaryopoiesis and platelet formation, TpoR is also expressed in HSCs and maintains them quiescent. TpoR signals as a homodimer after ligand binding in a 1:2 (ligand to receptor) ratio. Similar to other single pass membrane receptor, it contains an extracellular ligand binding domain, one helical transmembrane domain (TMD) and a cytoplasmic part important for JAKs binding and to mediate signaling. Unlike other cytokine receptors, TpoR contains a unique amphipathic motif (RW515QFP) at the junction between the TMD and the cytoplasmic domain. Mutations within this motif, namely W515K or W515L, lead to activation of the receptor and are associated with JAK2 V617F negative myeloproliferative neoplasms, specifically essential thrombocytopenia and primary myelofibrosis. We asked i) how a tryptophan at position 515 prevents autonomous activation of TpoR and ii) exactly how mutants at W515 impact on TpoR function and lead to oncogenesis. Methods.A combination of site-directed mutagenesis, biochemical and signaling assays, and spectroscopy analysis were employed. We made several specific mutations at position 515 to see if Trp515 is regulatory because of it size and/or aromatic character. We then tested the effect of mutations at adjacent positions in the wild type or mutated W515 constructs. Data were recorded from transiently transfected g2A cells and the results were confirmed in stably transduced BaF3 cells using dual luciferase and growth assays in the presence or absence of ligand. Adoptive transfer in lethally irradiated mice was used for assessing in vivo effects of TpoR mutants.The oligomerization status of the full length TpoR and some of our mutants was tested by employing the Gaussia princeps luciferase complementation assay, where Gaussia princeps luciferase fragments were fused in frame to the carboxyl terminus of TpoR or TpoR mutants. Recombination of the luciferase fragments results in a positive signal and reveals dimerization. Deuterium magic angle spinning (MAS) NMR spectroscopy and analytical ultracentrifugation were used to assess dimerization of the region encompassing the TMD. The tilt angle of this segment was assessed by FTIR spectroscopy. Results.Tryptophan seems to be absolutely required at position 515 to maintain the receptor inactive in absence of ligand. We identified secondary mutations (to tryptophan) that can prevent W515L/K/A activation of TpoR. The doubly mutated receptors behave like wild type receptors exhibiting no activation in absence of TPO and a normal response to TPO. These data suggest that W515 mutants may be specifically targeted for therapy. Mutations at W515 induced an increase in full length TpoR dimerization, and promoted dimerization of the segment comprising the TMD. However, our secondary site mutations showed differences between the mechanisms by which K or L versus A activate at W515. Overall, W515 impairs TpoR dimerization in a conformation productive for JAK2 activation. Measurements of the dichroic ratio by FTIR indicated that W515 increases the tilt angle of the TMD, thus reducing the interface required for TMD dimerization. We further explored the role of W515 in receptor activation by using a model where coiled coils are used to induce distinct dimeric conformations when fused to the TMD-cytosolic domains. W515 was essential for imparting differential signaling to the different dimers, both in cell lines and in vivo, where phenotypes in reconstituted mice no longer differ between receptors that have different dimeric orientations. Conclusions.Tryptophan 515 prevents TpoR dimerization in a productive orientation for JAK2 activation. This effect is reversible when secondary site mutations to tryptophan are introduced at certain positions around W515. Dimerization of TMD induced by ligand rotates W515 so that it is displaced from the headgroup region of the membrane to the dimer interface. This is crucial for imparting specific signaling as a function of dimeric orientation. Our results indicate that the region around W515 is a major switch for receptor function and could be exploited therapeutically. Disclosures:Constantinescu:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees.

JAK2 V617FMutation Prevalence in Myeloproliferative Neoplasms in Pernambuco, Brazil

The JAK2 V617F mutation is associated with three myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). It generates an unregulated clonal hematopoietic progenitor and leads to abnormal increased proliferation of one or more myeloid lineages. Subjects bearing this mutation may present more frequently with complications such as thrombosis and bleeding, and no specific treatment has yet been developed for BCR-ABL-negative JAK2 V617F-negative MPNs. To determine the prevalence of JAK2 V617F in MPNs in Pernambuco, Brazil, and to compare it with previous studies. 144 blood samples were collected at the Hospital of Hematology of the HEMOPE Foundation and were genotyped by polymerase chain reaction-restriction fragment length polymorphism with BsaXI enzymatic digestion. 88% (46/52) of the patients with PV, 47% (39/81) with ET, and 77% (8/11) with PMF were positive for JAK2 V617F, while more than 35% of the individuals were JAK2 V617F-negative, confirming a high prevalence of this abnormality in MPNs, more frequently with a low mutated allele burden, similar to what has been reported in other Western countries, despite differences among methods used to detect this mutation. Screening for JAK2 V617F may allow specific management of these diseases with JAK2 inhibitors in the future and highlights the need for further studies on the pathogenesis of BCR-ABL-negative JAK2 V617F-negative MPNs.

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

BackgroundPolycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population.MethodsWe sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression.ResultsA novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count.ConclusionsOur results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN.

Interlaboratory Quality Control Round of MPL Mutation Detection in Fourteen European Laboratories: A MPN&MPNr-EuroNet Study,

Abstract 3859▪▪This icon denotes a clinically relevant abstractThe MPL gene is located on chromosome 1p34 and encodes the thrombopoietin receptor. It includes 12 exons and is a key factor for growth and survival of megacaryocytes. Acquired mutations in this gene activate the thrombopoietin receptor constitutively. MPL515 somatic mutations are stem cell-derived events that involve both myeloid and lymphoid progenitors. Two distinct exon 10 mutations are found in 15% of JAK2-V617F negative myeloproliferative neoplasms (MPN),), i.e. 3% of essential thrombocythemia (ET) and 10% of primary myelofibrosis (PMF).: W515L, W515K and the rare W515A variant. A hereditary mutation, S505N, is associated with familial thrombocytosis.MPL mutation detection is a helpful new tool to detect clonality in JAK2 V617F negative MPN and to establish the diagnosis of MPN. As many laboratories use very different methods and interpretations, standardization is highly warranted. Particularly the methodology is diverse and the results need to be comparable, requiring comprehensive testing. This quality control project was established within workgroup 2 of the MPN&MPNr-EuroNet network, (www.mpneuronet.eu, supported by the European program COST (CoOperation in Science and Technology)). The lab from the ‘Hopital H. Mondor AP-HP Paris’ provided 29 samples containing randomized concentrations (between 100% and 1%) of the four mutations MPL W515L, W515K, W515A and S505N. The plasmids used for this quality control experiment spanned exons 9, intron 9 and exon10 of the MPL with S505N, W515L, W515K and W515A mutants diluted first with wild-type plasmid gene and then diluted in human genomic DNA. Thirteen European laboratories tested these 29 samples, each using their own chosen methods (14 altogether). The following methods were used: Mutascreen W515L/K Kit (Ipsogen, France):(n=4), allelic discrimination real-time PCR (n=2), high resolution melting (HRM) (n=7) and sequencing (n=2, 1 Sanger, 1 pyrosequencing)).There were no false positive results in any of the labs. All labs using the Mutascreen W515L/K Kit detected all W515L and W515K mutations, from 100% mutated down to 1% mutated plasmids. The allelic discrimination assays which were also designed for W515L and W515K only, detected the mutations down to 2%. The HRM methods were all designed differently. All except one (which did not recognize S505N) detected all 4 mutations with a sensitivity, down to 5% mutated plasmids, with few exceptions detecting either lower or higher amounts. The Sanger sequencing and pyrosequencing assays had a detection limit of 5–10%, with the pyrosequencing assay not being designed for the S505N mutation.All participating labs detected the most frequent MPL mutations in MPN W515L and W515K, with many designs not including W515A and S505N. Achieved sensitivities differed between methods with cutoffs of 1% to 10% (1.5% for the Ipsogen kit). Most laboratories reported the results as either positive or negative. However, the percentages of mutated alleles reported by a few labs differed greatly from each other and from the original dilutions (range 2–50 times) In conclusion, these results show that the diverse methods for MPL mutation detection used by different European labs yielded comparable specificity with varying sensitivity. Smaller clones might be missed by the less sensitive methods, and quantification of mutated alleles should be interpreted very carefully until standardised reference material for MPL mutation testing will be available. More extensive interlaboratory testing including patient samples is needed to identify the most robust assays suitable for diagnostic mutation detection and particularly for quantification of mutated alleles. Disclosures:No relevant conflicts of interest to declare.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Surface Expression of Mpl: Aberrant Mpl Trafficking In Myeloproliferative Neoplasms Is Linked to Insufficient Expression of Wild Type Jak2

Abstract 2820 Background:Mpl, the receptor for thrombopoietin (Tpo), activates Jak2. Mpl and Jak2 are expressed both on platelets and on hematopoietic progenitors, where they initiate critical cell survival and proliferation signals in response to Tpo. Thus, interfering with the “Mpl/Jak2 couple” is likely to alter myelopoiesis. Indeed mutations in JAK2 or MPL are characteristic of myeloproliferative neoplasms (MPN). However, platelet Mpl expression is frequently low in MPN, including MPN with no mutation of JAK2. Aim:To determine whether expression of wild type Jak2 (Jak2wt) is altered in MPN, and whether changes in expression of Jak2wt affect Mpl expression. Methods:Mpl and Jak2 protein expression was analysed by immunoblotting in platelet and cell lysates. Expression of JAK2 wt and JAK2 V617F was also studied at the mRNA level using quantitative RT-PCR. Transient transfections of HEL and UKE-1 cells were used to study the effects of Jak2wt on Mpl and of MPL on Jak2wt and Jak2V617F. We also studied the cellular localization of Jak2wt, Jak2V617F and Mpl, expressed as chimeric proteins fused to Visible Fluorescent Proteins (VFP, derivatives of GFP) or detected by immunofluorescence methods. Results:Following SDS-PAGE and western blotting of normal platelet proteins, Mpl was typically found in two isoforms: the long, mature 84 kDa isoform (Golgi-processed, glycosylated, functional, Tpo-binding form expressed at the cell surface) and the short, 74 kDa isoform (immature Mpl). Four subgroups of MPN patients were defined according to platelet Mpl patterns: pattern 1, mature and immature Mpl expressed at comparable levels, typical of healthy donors and found in 37% of MPN tested; pattern 2, mature Mpl only, typical of reactive states, associated with high JAK2 wt expression and observed in 15% of MPN; pattern 3: immature Mpl only; and pattern 4: no Mpl. Patterns 3 and 4 were observed in 48% of MPN and were associated with low JAK2 wt levels (<200 copies/100 ABL copies). JAK2 wt mRNA levels varied widely in MPN and Mpl pattern was correlated with levels of JAK2 wt, but not JAK2 V617F, mRNA. Blotting studies performed on cell lysates from cultured lines homozygous for JAK2V617F (HEL, UKE-1) detected mainly the 74 kDa immature form of Mpl.Transfection of JAK2wt in HEL and UKE-1 cells, alone or together with MPL, increased the yield of mature Mpl. Fluorescence studies of HEL cells revealed that both endogenous Mpl and the Mpl-VFP chimeric proteins were largely restricted to an intracellular pool, without significant co-localization with Jak2V617F. In contrast, significant levels of transfected Mpl-VFP reach the plasma membrane in K562 cells that express only Jak2wt; co-localization of Jak2 and Mpl in these cells was significant. Conclusion:JAK2 wt and Mpl levels are linked and both are frequently low in MPN, including JAK2 V617F-negative MPN. Co-localization of Mpl and Jak2V617F is poor whereas Mpl and Jak2wt are strongly co-localized, consistent with constitutive association and indicating a role for Jak2wt as chaperone along the exocytic or recycling pathways. Future work will seek to determine the intracellular location of aberrantly trafficked Mpl and establish if other mutations found in MPN are linked to defects in the expression or trafficking of Mpl. Disclosures:No relevant conflicts of interest to declare.

Novel multiplex bead-based assay with LNA-modified probes for detection of MPL exon 10 mutations

MPL exon 10 mutations were the second class of mutations shown to be associated with the pathogenesis of some Philadelphia chromosome – negative myeloproliferative neoplasms (MPNs). Recently, their identification gained wide recognition in the diagnostic work-up for suspected cases of JAK2 V617F negative MPNs. Various molecular approaches have been applied, yet universally accepted method is still lacking. We aimed at development and validation of a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the following MPL mutations: W515L/K/A/R. Testing on both artificial plasmid constructs and on clinical samples revealed that the method was comparable in terms of specificity to direct sequencing and had a much higher sensitivity of 1% mutant alleles. This method could be successfully implemented in the diagnostic work-up for MPNs. Furthermore, this system allows further multiplexing for single-tube identification of different mutations associated with MPNs.

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane–juxtamembrane region of MPL ( MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients.

The JAK2 46/1 haplotype in Budd-Chiari syndrome and portal vein thrombosis

AbstractThe germline JAK2 46/1 haplotype has been associated with the development of JAK2V617F-positive as well as JAK2V617F-negative myeloproliferative neoplasms (MPNs). In this study we examined the role of the 46/1 haplotype in the etiology and clinical presentation of patients with splanchnic vein thrombosis (SVT), in which MPNs are the most prominent underlying etiological factor. The single-nucleotide polymorphism rs12343867, which tags 46/1, was genotyped in 199 SVT patients. The 46/1 haplotype was overrepresented in JAK2V617F-positive SVT patients compared with controls (P < .01). Prevalence of the 46/1 haplotype in JAK2V617F-negative SVT patients did not differ from prevalence in the controls. However, JAK2V617F-negative SVT patients with a proven MPN also exhibited an increased frequency of the 46/1 haplotype (P = .06). Interestingly, 46/1 was associated with increased erythropoiesis in JAK2V617F-negative SVT patients. We conclude that the 46/1 haplotype is associated with the development of JAK2V617F-positive SVT. In addition, our findings in JAK2V617F-negative SVT patients indicate an important role for the 46/1 haplotype in the etiology and diagnosis of SVT-related MPNs, independent of JAK2V617F, that requires further exploration.