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Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1-5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.

Background: Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations., Methods: 32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden., Results: The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative., Conclusion: The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Summary: Background: Essential thrombocythemia (ET) is classified as a chronic myeloproliferative neoplasm. JAK2 V617F mutation is found in about 50–60% patients with ET. We aim to determine the prevalence of JAK2 V617F mutation and its association with phenotype in adult Taiwanese patients with ET. Methods: In this combined retrospective and prospective study, adult ET patients, at least 18 years of age, were enrolled between November 2007 and September 2011. Genomic DNA was extracted from unsorted bone marrow and/or peripheral blood samples for the detection of JAK2 V617F mutation by allele-specific polymerase chain reaction. The clinical and laboratory characteristics of all patients at the time of diagnosis or referral were determined retrospectively by chart review. Results: A total of 82 patients were enrolled, and JAK2 V617F mutation was detected in 55 patients (67.1%). JAK2 V617F mutation was significantly more prevalent in old patients (36.4% vs. 14.8%, p =0.044), and associated with higher hemoglobin level (median 13.7 vs. 12.8g/dL p =0.012) and higher white blood cell count at diagnosis (12.1×103 vs. 8.8×103/μL p =0.015). ET patients with the mutation also tend to have lower platelet count (median 902×103 vs. 1078×103/μL p =0.051). In a binary logistic regression model, only higher hemoglobin concentration was significantly associated with JAK2 V617F mutational status (odds ratio 1.2 95% confidence interval 1.0–1.5; p =0.047). Conclusion: JAK2 V617F mutation in Taiwanese adult patients with ET has a high prevalence of 67.1% and is associated with old age, higher hemoglobin level, and higher leukocyte count. [Copyright &y& Elsevier]

Summary: In addition to the JAK2 V617F mutation, somatic mutation in DNMT3A has been described in BCL-ABL1-negative myeloproliferative neoplasms (MPNs). We have screened for DNMT3A exon 23 mutations in 130 adult Taiwanese patients with chronic phase myeloproliferative neoplasms. Only one somatic DNMT3A R882H mutation was identified in one JAK2 V617F mutation-positive essential thrombocythemia patient (1/91, 1%). Both mutations were detected in the CD34+-, CD19+-, peripheral blood mononuclear cell- and granulocyte-enriched fractions, but were not detected in the CD3+-enriched fraction by lineage analysis. Our findings suggest that DNMT3A mutation is not prevalent in MPNs, and further study is needed to clarify its role in the molecular pathogenesis of myeloproliferative neoplasms. [Copyright &y& Elsevier]