MUTATIONAL ANALYSIS OF BONE MARROW MESENCHIMAL STROMAL CELLS IN MYELOID MALIGNANCIES

Growing evidence suggest the involvement of the microenvironment in the pathophysiology of hematopoietic malignancies. Mesenchimal stromal cells (MSC) support hematopoiesis through the production and secretion of cytokines, cell-cell interactions and immunomodulating properties. These cells are defined plastic-adherent growing cells, are known to express CD105, CD73 and CD90, lack common hematopoietic antigens and differentiate to osteoblasts, adipocytes and chondroblasts in vitro.1 Anomalies of multiple MSC features have been described in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). These include significantly reduced growth, proliferative and differentiating capacities, premature replicative senescence, abnormal expression of surface molecules and chemokines, and reduced ability to support hematopoietic stem and progenitor cell (HSPC) growth in long-term culture assays. The molecular basis of MSC dysfunction in MDS and AML are still under investigation. Previous studies have shown the occurrence of non-clonal chromosomal aberrations in bone marrow MSC isolated from patients with MDS and AML, which only very rarely correspond to the cytogenetic markers observed in the hematopoietic leukemic clone of the same individual. Somatic mutations of multiple genes have been described in myeloid malignancies and often concur to identify distinct leukemia subtypes or prognostic subgroups. Recently, deep sequencing approaches have identified new recurrent mutations of genes involved in epigenetic and spliceosome machineries in AML and MDS samples. We investigated the frequency of recurrent mutations of epigenetic and spliceosomal genes, of FLT3 and NMP1 genes in matched bone marrow hematopoietic cells and MSC isolated from 41 patients with myeloid malignancies. The study population included 9 de novo AML, 9 MDS, 7 chronic myeloproliferatve neoplasms (MPN), 3 secondary AML (sAML, 2 evolved from MDS, 1 from MPN), and 13 therapy-related myeloid neoplasm (7 t-AML and 6 t-MDS). Bone marrow mononuclear cells (BM-MNC) were isolated from all patients at the time of diagnosis by Ficoll gradient centrifugation. MSCs were expanded using Mesencult medium (Stem Cell Technologies, Voden Medical Instruments spa, Milan, Italy) in plastic-adherent cultures up to the second passage. Flow cytometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive) in more than 99% of MSC population. DNA was extracted from BM-MNC and MSC using QIAamp DNA Mini Kit (Qiagen srl, Milan, Italy). The following hot-spot mutations were studied on genomic DNA by Sanger sequencing (ABI PRISM 3100; Applied Biosystems/Life technologies, Milan,Italy): IDH1 R132, IDH2 R140 and R172, DNMT3A R882, U2AF1 S34 and R35, SF3B1 exons 13–14 and 15–16, and SRSF2 exon 1.11 In addition, FLT3 and NPM1 mutations were analyzed in patients with de novo or therapy-related AML. FLT3 Internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations were studied by RT-PCR and RFLP RTPCR, respectively, while NPM1 exon 12 mutations were detected by RT-PCR with high resolution melting curve analysis (HRMCA), followed by Sanger sequencing of positive cases. In BM-MNC, FLT3 ITD and FLT3 TKD mutations were found in 2 patient (one patient with de novo AML and one patient with a t-AML respectively), while NPM1 exon 12 mutation were 3 present in 6 AML patients (4 de novo, 3 COSM158604 and 1 COSM1319219; 1 sAML, COSM158604; 1 t-AML, COSM28937). IDH1 R132 mutations were found in one patient with de novo AML (R132C) and in two t-AML patients (one R132H and one R132L). One t-AML patient presented an IDH2 R140Q mutation, while no mutations were detected at the codon R172. Three DNMT3A R882 mutations were found in one de novo AML (R882H), one t-AML (R882H) and one sAML evolved from Polycythemia Vera (R882C). One U2AF1 S34Y mutation was found in a patient with in