Whole exome sequencing identifies novel MPL and JAK2 mutations in triple negative myeloproliferative neoplasms.

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, exon 10 of MPL or JAK2-V617F in >90% of the cases, while 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 5/8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3/8 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 and a germline MPL-V285E mutation, as well as a germline JAK2-G571S variant. Sequencing of entire coding region of MPL and JAK2 was performed in additional 62 and 49 triple negative cases of ET or PMF, respectively. We detected new somatic (T119I, S204F, E230G, Y591D) and one germline (R321W) MPL mutation in 5/62 cases. All the mutations were gain-of-function mutations when analyzed in functional assays. JAK2 variants were identified in 5/57 triple negative cases and 3 of them were germline. 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