CLONAL PROGRESSION IN THE LEUKEMIC TRANSFORMATION OF CHRONIC MYELOPROLIFERATIVE NEOPLASMS

Myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell progenitor. These diseases include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Evolution to acute myeloid leukemia occurs in 8-20% of MF and 10% of PV/ET with incidence after 10 years and with a dismal prognosis. The main aim of this project is to elucidate the molecular hierarchy of clonal cells during the chronic and acute phase and to identify additional mutations occurring at acute leukemia. In order to validate the predictive significance for leukemia of myeloid-associated mutations in MPN patients, we analysed granulocytes DNA of 215 pts with PV and 174 with ET and we identified ASXL1 and SRSF2 as prognostically significant in PV and U2AF1 and TP53 in ET with a worst impact in leukemia-free and MF-free survival. To study clonal heterogeneity and clonal progression in the leukemic transformation of MPN we performed whole exome (WES) and trascriptome (RNAseq) sequencing of 12 paired samples (chronic (CP)/blast phase(BP), using CD34 positive cells. In addition, a myeloid neoplasm panel was used for target NGS sequencing of paired samples plus an additional 23 samples collected from MPN pts at leukemic phase. The most frequent mutated genes detected by NGS at BP, other than driver mutations, were ASXL1 (51.4%), RUNX1 (37.1%), TET2 (17%), SRSF2 (16%), IDH1 (14%), TP53 (14%), NRAS (14%), FLT3 (14%), U2AF1 (11%) and KRAS (11%); three (8%) cases were mutated for EZH2 DNMT3A, CBL and PTPN11, 2 cases for IDH2, SH2B3, SF3B1 IKZF1, SETBP1 and ZRSR2, and 1 case for ABL1, ETV6, BRAF and ARID1A. Compared to the CP of the 12 paired samples, 10 pts (83%) acquired novel mutations, 3 of which acquired ≥3 variants. RUNX1 and ASXL1 were those with the highest number of acquisition (n=7 and 5, respectively). Conversely, loss of variants was observed only in ASXL1 (n=3). In order to confirm the differential expression of genes and to evaluate the reliability of the RNA Seq, a Gene Expression assay was performed by Real Time PCR and Droplet Digital PCR. The expression of SH2D1A, PRTG and CDKN2A were differentially expressed compared to the healthy controls; we observed an over expression of CDKN2A in BP respect of CP at limit of significance, so it could be considered as new predictive markers of leukemic progression, but we need to extend the number of patients in CP and BP. Our data indicate that BP is associated with significant changes in mutation complexity and RNA expression, overall affecting different intracellular pathways whose further characterization might help to identifying potential targets for therapy.