Molecular characterization of driver and somatic subclonal variants in patients with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal disorder of the hematopoietic stem cells due to an acquired mutation causing a hyper-sensibility or independence on a normal cytokine regulation and, consequently, an overproduction of blood cells. According to the recently revised World Health Organization (WHO) classification of myeloid neoplasms, MPNs include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and prefibrotic myelofibrosis (prePMF). In MPNs all the three myeloid lineages are usually involved, but one lineage is predominantly affected in each entity: PV is mainly characterized by the deregulation of the erythroid lineage associated with a variable hyperplasia, while ET is caused by an excessive platelets production and a megakaryocytic hyperplasia. PreMF and PMF are associated with the deregulation of the megakaryocytic and granulocytic lineage with the presence of bone marrow fibrosis. A somatic mutation in JAK2, CALR or MPL genes are found in the great majority of patients with MPNs. These driver mutations constitutively activate of the JAK-STAT pathway, resulting in increased phosphorylation of its substrates and leading to increased cytokine responsiveness of myeloid cells. MPN may progress to more aggressive disease, including post–PV MF, post–ET MF, acute myeloid leukemia, and blast-phase disease. Progression of the disease is typically associated with the acquisition of somatic mutations in genes responsible for subclonal evolution. These additional variants affect the functionality of different pathways, including DNA methylation and regulation of chromatin structure, transcriptional regulators, signaling pathway and splicing factors. The clinical diversity of MPNs seems to be mainly related to the combination of driver and subclonal variants and their order of acquisition. At any rate, the precise role of each mutation and its impact on MPN phenotype are not yet completely understood. In the era of precision medicine, the identification and characterization of a molecular target will allow to optimize the therapeutic choice in the individual patient. This work aimed to define the molecular characterization of driver and somatic subclonal variants in patients with MPNs. Our study included 147 patients diagnosed with PMF and preMF at the UOC Ematologia, Fondazione IRCCS Policlinico San Matteo and University of Pavia, between 1995 and 2015. DNA sequence variants were studied through a NGS approach using the Illumina Nextera Rapid Capture Custom Enrichment Kit and HiSeq2500 platform. The panel targeted the coding sequence of 81 genes known to be involved in myeloid neoplasms, with a cumulative target size of 235.9 kb. Overall, 179 additional somatic variants were found. Compared to preMF, PMF showed a larger proportion of patients with at least 1 variant (78% vs 45%, P<.001), a higher number of variants per patient (1.74 vs 0.74, P<.001), and a huger involvement of high molecular risk genes (70% vs 39%, P=.027). On the other hand, in preMF the molecular signature of a specific myelodisplastic syndrome (RARS-T) was found, with SF3B1 and DNMT3A being the most frequently mutated genes and DNA methylation and splicing the most affected pathways. Moreover, a significant difference in the timing of acquisition of additional somatic variants with respect of the driver mutation was observed, with CALR mutation being the first genetic event, while the JAK2 mutation occurring before or after other variants. In conclusion, our data suggested that not only clinical criteria but also distinct mutation patterns might differentiate PMF from preMF. Moreover, a continuum between preMF and RARS-T may be possible. This thesis is the initial part of a genotype-phenotype study aimed to correlate driver mutations, subclonal variants and clinical data in a cohort of 500 patients to define a possible diagnostic and prognostic role of these variants in MPNs.