Key Points• Activating mutations outside exon 10 of MPL were identified in 10% (7 of 69) of triplenegative cases of ET and PMF.• JAK2-V625F and JAK2-F556V were identified in 2 triple-negative cases of ET and were shown to activate JAK-STAT5 signaling.Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. (Blood. 2016;127(3):325-332) IntroductionEssential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV) are the classical BCR-ABL-negative myeloproliferative neoplasms (MPNs). They are chronic diseases, characterized by clonal expansion of differentiated myeloid cells driven by somatic mutations. Most of the cases are sporadic, however, familial clustering has also been observed. In 95% of PV cases and 50% to 60% of ET and PMF cases the disease is driven by an acquired mutation in the JAK2 gene V617F.1-4 JAK2-V617F leads to the constitutive activation of the Janus kinase 2 (JAK2) and subsequently the JAK-signal transducer and activator of transcription (STAT) signaling pathway. The remaining cases of PV carry mutations in exon 12 of JAK2. Although JAK2 mutations in MPNs are acquired, in recent years several families with hereditary thrombocytosis (HT) have been described to have germline JAK2 mutations. [5][6][7] The second most commonly mutated gene in ET and PMF is CALR encoding calreticulin. Mutations in exon 9 of CALR have been described in 25% and 35% of patients with ET and PMF, respectively. 8,9 There are o...

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