Abstract
Introduction
There is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures.
Materials and Methods
We performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients.
Results
In the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases.
To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo.
Discussion
Together with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS.
Disclosures
Schmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.
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