Abstract
BackgroundIn multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization.MethodsA total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH).ResultsIn 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI.ConclusionOur results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.
Highlights
In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression
Clonality in endothelial progenitor cells (EPCs) from MM patients was determined by X-chromosome inactivation (XCI) patterns using a highly polymorphic CAG repeat in the androgen receptor (AR) gene, a well-established method to assess clonality of tumor cells [14] based on random epigenetic inactivation of one of the two X chromosomes inherited from either parent [15]
Confluent primary EPCs outgrown from patients' peripheral blood mononuclear cells (PBMCs) or bone marrow (BM) were the source of genomic DNA used in our experiments
Summary
In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Experimental evidence shows that in MM, EPCs both in circulation and in the BM express angioblastic features–a high capacity for vasculogenesis and a pro-angiogenic gene expression pattern–suggesting that these cells resemble embryonic angioblasts, and supporting the thesis that BM EPCs help tumor neovascularization [8,11,13]. These observations prompted us to explore whether EPCs in MM display neoplastic features such as clonal restriction. Clonality in EPCs from MM patients was determined by X-chromosome inactivation (XCI) patterns using a highly polymorphic CAG repeat in the androgen receptor (AR) gene, a well-established method to assess clonality of tumor cells [14] based on random epigenetic inactivation of one of the two X chromosomes inherited from either parent [15]
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