Quantitative detection of circulating epithelial cells by Q-RT-PCR

Abstract

It has been shown that the quantity of circulating tumor cells (CTCs) in breast cancer patients is an independent predictor of survival and treatment response. Real time quantitative reverse transcriptase PCR (Q-RT-PCR) is a sensitive technique for detection of CTCs. Our aim was to investigate whether the technique can be used also to quantitate these CTCs. We tested cytokeratin 19 (CK19), maspin, mammaglobin, GAPDH and RPL19 genes for their level of expression and linearity of amplification in serial dilutions of RNA extracted from the MDA-MB-231, UACC-812, T47D and HS578T breast cancer cell lines. To simulate CTCs, serial dilutions of cultured T47D and HS578T cells were added to peripheral blood from healthy volunteers. The samples were subjected to enrichment, RNA extraction and Q-RT-PCR. CK19 was reliably expressed in all four cell lines with a linear relationship between the quantity of added cells and the amount of CK19 RNA. The lower limit of reliable detection was 5 cells per sample, which corresponds to a concentration of 0.7 cell/ml in 7.5 ml of blood or would translate to a lower CTC concentration in a larger volume of blood. This technique may prove useful for high throughput comparative quantification of CTCs in individual patients during treatment and subsequent follow up for research and clinical management purposes.