Characterization of Philadelphia-negative Chronic Myeloproliferative Neoplasms: identification of novel biomarkers by Next Generation Sequencing and study of interactions between hematopoietic stem cell and the inflammatory cell micro-environment

Myeloproliferative Neoplasms (MPN) are a heterogeneous group of hematological malignancies, consisting in clonal disorders of the hematopoietic stem/progenitor cell (HSC/PC). Molecular alterations and inflammatory microenvironment represent the two main etiopathogenic factors of MPN. The aim of this study was the molecular characterization of MPN patients and the study of interactions between HSC/PC and the inflammatory cell micro-environment. We investigated young (<40 years at diagnosis) ET and early-PMF patients, and patients with a JAK2V617F allele burden (AB) <3%, demonstrating that its determination is relevant both at diagnosis and during follow up. Indeed, it allows to prove the presence of a clonal hematopoiesis and, also, to predict clinical outcome. Of note, an AB≥0,8% always corresponds to an overt MPN phenotype. In this context, coordinating a network of 19 Italian laboratories, we identified the ipsogen JAK2 MutaQuant kit as the most sensitive and efficient assay for the quantification of samples with different mutation loads (in particular those with AB≤1%). With regard to the role of inflammatory microenvironment in the pathogenesis of MF, IL-1β and TIMP-1 seemed to confer a survival advantage to MF-derived HSPCs, enhancing their proliferation and in vitro migration, as well as their clonogenic ability. Finally, in this study we tested three different gene panels for mutations detection, obtaining promising results in terms of coverage analysis (more than 95% of target regions with depth greater than 500X) and identifying gene variants with very low mutation load (<1%) in all patients. In conclusion, this study set the basis for the standardization of molecular techniques for the determination of JAK2V617F AB, and for the validation of a robust NGS approach to be translated into a diagnostic setting. Moreover, IL-1β and TIMP-1 emerged as novel promoting factors of the in vitro maintenance of MF-derived HSPC, which may be exploited as potential targets of therapy.