Targeted Sequencing of Sorted Peripheral Blood Cells Reveals Novel Germline and Somatic Variants in the Polymorphonuclear and Mononuclear Cells of Patients with Essential Thrombocythemia, Polycythemia Vera and Primary Myelofibrosis

Abstract Background The past decade has witnessed a significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). A large number of genes have now been implicated in the pathogenesis of MPN but their relative importance, the mechanisms by which they cause different cell types to predominate and their implications for prognosis remain unknown. Aim The aim of this study was to perform targeted next generation sequencing on different cell types directed at a panel of mutations shown to be associated with MPN in order to identify driver mutations and novel variants, to determine their relative importance to pathogenesis and to predict survival in patients with MPN. Methods We performed targeted sequencing on normal controls (NC) and patients with MPN from 3 different races (Malay, Chinese and Indian) in Malaysia who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria for MPN. Patients from 3 tertiary-level hospitals were recruited prospectively over 3 years and informed consents were obtained. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMNs), mononuclear cells (MNCs) and T cells. DNA was extracted from the different cell population and high-throughput sequencing was performed using an Ion Torrent PGM and AmpliSeq targeting platform, with a panel of 26 MPN-related genes. Results Twenty-nine patients (11 ET, 11 PV, 7 PMF) and 3 NC were recruited into the study. Ten of 29 (34.5%) and 9 of 29 (31%) patients were Malay and Chinese men respectively. Twenty-four of 29 (82.8%) patients harbored the JAK2V617F mutation which was detected in 7 (63.6%) ET, 11 (100%) PV and 6 (85.7%) PMF patients. The allelic frequency of JAK2V617F mutation was highest in PMF. 11 of the 24 JAK2V617F positive patients (2 ET, 5 PV and 4 PMF) also harbored potentially pathogenic variants involving the ASXL1, CALR, CBL, CHEK2, DNMT3A, TET2, U2AF1, RUNX1 , SF3B1 and TP53 genes. In patients who were found to have both JAK2V617F mutation and a TET2 variant, the allelic frequencies of the TET2 variantswere significantly lower than the JAK2V617F mutations. Conversely, in 1 ET patient with high TET2 allelic frequency, JAK2V617F mutation was not detected. Several novel variants involving the CHEK2, DNMT3A, RUNX1, SF3B1 and TET2 genes were identified in the study. All the novel variants were somatic (detected predominantly in the PMNs) except CHEK2 which was detected in all 3 cell types (PMNs, MNCs and T cells) at approximately 50% allele frequency. A similar spectrum of mutations was found in PMNs and MNCs, usually at a lower frequency in MNCs. Somatic mutations were absent or detected at very low frequencies in T cells. One of the 7 JAK2V617F positive ET patients harbored 4 somatic variants in CHEK2, SF3B1, TET2 and TP53 genes, with the highest allelic frequencies in the PMNs for 2 variants and MNCs for the other 2 variants. Three of the 4 JAK2V617F negative ET patients had CALR, MPL exon 10, SH2B3 and TET2 variants. One of the 3 ET patients with CALR and TET2 variant presented with a very high platelet count of 2251 x 109/L. One PV patient who harbored both JAK2V617F and CALR mutations presented with high haemoglobin and white cell counts. The CALR mutation in this patient was thought to be germline as it was detected in all cell types at approximately 50% allelic frequencies. The 2 PV patients who harbored both JAK2V617F and DNMT3A mutations presented with more severe phenotype with leukocytosis, splenomegaly of >15cm, constitutional and vasomotor symptoms requiring treatment with Hydroxycarbamide. The 3 PMF patients with JAK2V617F mutation in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 variants also presented with severe clinical phenotypes, 2 of whom died within 2 years from diagnosis. Conclusion JAK2V617F is the commonest somatic mutation detected in MPN patients in Malaysia and is predominantly found in the PMNs. Nearly half of the JAK2V617F positive patients also harbor other potentially pathogenic variants. Several novel somatic and one germline variants were found in this study. Patients who harbor the JAK2V617F in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 putatively pathogenic variants were found to have more severe clinical phenotypes with very poor prognosis. Further studies are required to explore cell type differences, prognosis and clonal evolution in MPN. Disclosures Aitman: Illumina: Honoraria.