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 intermediate-risk MDS, while a SF3B1 K666N mutation was detected in one patient with AML following a primary MDS. Six SRFS2 P95 mutations were found in two patients with de novo AML (P95L and P95R), three patients with MDS (P95L, P95H and P95 frameshift mutation) and one patient with MPN (P95H). All mutations described were heterozygous. Results of mutational analysis are reported in Figure 1. We found no mutations for any of the studied genes in the MSC compartment, both in carriers of mutations in the hematopoietic compartment and in wild-type patients. Exemplary sequencing reactions for all genes studied in matched bone marrow hematopoietic cells and MSC, are reported in Figure 2. The absence of NMP1 and FLT3 mutations in MSC from AML patients has been previously reported.5 So far, the prevalence of somatic mutations of epigenetic and spliceosomal genes in MSC compartment in patients with myeloid malignancies is not known. Mutations of DNMT3A and IDH1/2 have been described in 22% and 16% of AML patients and in 8% and 12% of MDS patients, respectively.7,12,13 These mutations have been associated to deregulated DNA methylation and poor prognosis.14 Abnormal DNA methylation has been advocated as responsible of the premature senescence and reduced growth of MSC in MDS. Specific patterns of DNA methylation in MSC have been reported in MDS subtypes, such as Refractory Anemia with Excess Blasts (RAEB) and Refractory Cytopenia with Multilineage Dysplasia (RCMD), compared to MSC form healthy donors.3 According to our results, abnormal methylation profiles described in MSC are unlikely to be related to mutations of epigenetic regulatory genes. Abnormal splicing patterns of multiple genes have been described in myeloid neoplasms due to mutations of spliceosome machinery genes.15 This phenomenon is considered to play a significant role in myeloid leukemogenesis due to selective missplicing of tumor-associated genes. The contribution of missplicing to MSC dysfunction in myeloid neoplasms is still a matter of investigation. The absence of recurrent spliceosome gene mutations in MSC contrasts with the hypothesis that these mutations may play a significant role. Finally, we conclude that common mutations of genes involved in epigenetic regulation and spliceosome machinery are absent in the mesenchimal compartment of leukemic bone marrows and are restricted only to the malignant hematopoietic clone. Further investigation is required to ascertain the role of methylation and missplicing in the microenvironment dysfunction observed in myeloid malignancies.