Real-time PCR of CD146 mRNA in peripheral blood enables the relative quantification of circulating endothelial cells and is an indicator of angiogenesis

Angiogenesis is a fundamental process in tumour growth and metastatic dissemination. Possible surrogate markers for tumour angiogenesis are the amounts of circulating endothelial cells (CEC) in peripheral blood and the plasma concentration of vascular endothelial growth factor ( VEGF). We tested the suitability of real-time PCR for CD146, an endothelial cell-specific antigen, to quantify CEC numbers in comparison to a flow cytometry quantification. Real-time PCR of CD146 mRNA showed high sensitivity and linearity for the quantification of cultivated primary endothelial cells added in different amounts to blood samples. Circulating endothelial cell numbers were quantified in peripheral blood samples of breast cancer patients and healthy controls by four-colour flow cytometry analysis and CD146 real-time PCR, and VEGF plasma concentrations were measured by ELISA. The amounts of CEC detected with both methods correlated significantly and CEC numbers were significantly increased in newly diagnosed breast cancer patients compared to healthy controls. Vascular endothelial growth factor concentrations correlated significantly with CEC numbers, but there was no significant difference in VEGF levels between breast cancer patients and healthy controls indicating that VEGF plasma levels cannot be used as surrogate marker for tumour angiogenesis. Taken together, the quantification of CEC by CD146 real-time PCR showed equivalent results to the flow cytometry analysis. Thus, CD146 real-time PCR may be an easy and reliable approach to quantify CEC in peripheral blood samples and could facilitate the integration of CEC measurements in clinical studies exploring the efficacy of antiangiogenic therapies.