Evidence for Clonality in Circulating Endothelial Progenitor Cells in Multiple Myeloma.

A governing role for neoangiogenesis in the progression of multiple myeloma (MM) is indicated by the finding in patients of increased bone marrow microvascular density that is positively correlated with disease activity and is attenuated by treatment with thalidomide. Bone marrow angiogenesis in MM is largely driven by autocrine and paracrine effects of vascular endothelial growth factor (VEGF) via VEGF receptor-2 (KDR). Elucidation of the role of neovascularization in MM growth and dissemination is likely to result in identification of better therapeutic targets than those currently available as well as provide insight into the pathogenesis of MM. Circulating endothelial cells (CECs) contribute to angiogenesis and comprise mature ECs and endothelial progenitor cells (EPCs). The present study sought to characterize CECs and their relation to disease severity in MM. CECs, identified as CD34⁺/CD146⁺/CD105⁺/CD11⁻ cells, were 6-fold higher in patients compared to controls, and correlated positively with serum M protein and β₂-microglobulin (P<.001 for both). In addition, circulating EPCs displayed late colony formation/outgrowth and capillary-like network formation on Matrigel. Effective thalidomide treatment inhibited these characteristics (P<.001). Co-expression of KDR and early vascular antigen CD133 characterized EPCs in MM, and KDR mRNA elevations correlated positively with M protein levels (P<.01). To evaluate the clonality of circulating EPCs, X-chromosome inactivation patterns were quantitated in female patients by human androgen receptor gene (HUMARA) assay. Radioactive (α-[³³P] -dCTP) polymerase chain reaction amplification of DNA extracted from peripheral blood mononuclear cells showed that X-chromosome inactivation status was informative in 9 of the 11 MM patients. Distribution of the X-inactivation profiles according to age did not show a shift towards the skewed range in older patients or controls. DNA obtained from confluent EPCs outgrown from 4 informative patients showed an extremely skewed pattern of X-chromosome inactivation, with allele ratios of 90/10% in one patient and 97/3% in another patient, whereas a random pattern of X-chromosome inactivation was observed in two other patients displaying clonal restriction (allele ratios of 55/45% and 54/46%). Collectively, these data underscore the angiogenic aspect of MM and suggest that angioblast-like EPCs are a pathogenic biomarker and a rational treatment target in MM. Furthermore, the clonality of EPCs in MM suggests that the pathogenesis of MM may be tightly related to vascular endothelial cells both at the functional/angiogenic and at the genetic and ontogenic levels.