Clonal Restriction in Endothelial Progenitor Cells in Patients with Multiple Myeloma.
Authors: Braunstein Marc , Özçelik Tayfun , Bagislar Sevgi , Vakil Varsha , Smith Eric L.P. , Batuman Olcay 
Published: April 2020

Abstract
In multiple myeloma (MM), neoplastic plasma cells and bone marrow microvascular cells have paracrine cross-regulatory interactions, and display abnormal features suggesting that these neoangiogenic cells are myeloma-related. Tumor neovascularization is closely linked to neoplastic growth. We have recently shown that circulating endothelial progenitor cells (EPCs) reflect disease severity as assessed by M protein and serum β2-microglobulin levels in MM. These observations linking circulating EPCs to disease activity in MM prompted us to study the genetic characteristics of EPCs using X-chromosome inactivation (XCI) analysis to determine their clonal restriction profile. In addition, we examined EPCs for the presence of genetic markers common to neoplastic MM cells by examining immunoglobulin heavy chain (IgH) rearrangement of germline DNA. Isolation and outgrowth of EPCs was done as previously described. Clonality within EPCs was evaluated by quantitation of XCI patterns in female patients by a human androgen receptor gene (AR) assay (HUMARA). DNA was prepared from EPCs, hair root, or PBMCs and digested by a methylase-sensitive restriction enzyme. The highly polymorphic X-linked human androgen receptor gene was amplified by PCR. In polyclonal populations, both alleles are seen as two distinct bands, while clonal populations yield a single band. Amplification of genomic DNA extracted from PBMCs showed that XCI status was informative in 7 of the 16 MM patients and in 7 of the 15 healthy controls. Next, EPCs and hair root cells from informative patients were studied for XCI patterns to determine clonality. DNA obtained from confluent EPCs outgrown from 7 of the 10 informative patients displayed extremely skewed XCI patterns (90% inactivation of one allele), while hair follicle cells in the same patients displayed a random pattern. These results suggest that EPCs share characteristics of clonal plasma cells in MM, and display clonal restriction that is lineage-specific. To further explore the genetic similarity between EPCs and MM cells, EPCs were assessed for clonotypic immunoglobulin heavy chain (IgH) VDJ sequences by PCR amplification of genomic DNA. In these experiments, DNA was extracted from bone marrow EPCs and used in a series of 7 PCR amplifications containing a specific VH forward primer and a consensus JH reverse primer. In EPCs from 2 patients, including one with skewed XCI, we found a single PCR product identical to the IgH VDJ rearrangement found in bone marrow containing 60% and 65 % plasma cells, respectively, suggesting that an identical IgH VDJ rearrangement was present in EPCs and plasma cells in the same patient. Controls for this experiment included HUVEC genomic DNA, which did not yield PCR products even when spiked with a positive control DNA (up to 50 %) extracted from the MM cell line U266, and PBMC genomic DNA from controls which yielded multiple bands produced from several of the VH primers. Our results show that EPCs in a population of MM patients display clonal restriction and genetic similarity to plasma cells. Thus, it is possible that neoplastic MM cells and EPCs are linked not only within a functional context by their paracrine cross-regulatory effects within bone marrow microenvironment, but also by their genetic ancestory. These conclusions await confirmation from ongoing molecular genetic comparisons of the two cell types.