A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood

Abstract

Summary Background The detection of occult carcinoma cells in patients with breast cancer has been shown to predict disease recurrence and metastasis Materials and methods To improve on molecular detection of breast carcinoma cells in blood, we have developed a sensitive and quantitative assay using real-time quantitative RT–PCR identifying transcripts of the cytokeratin-19 (CK19) gene. Results This real-time quantitative RT–PCR is sensitive, accurate and has a high reproducibihty within a wide dynamic range, which permits simultaneous quantitative analysis of samples with varying input concentrations Furthermore, the procedure offers several technical advantages over classic quantitative PCR methods (competitive RT–PCR, Northern blotting) such as decreased likelihood of contamination due to absence of post-PCR manipulations, high sample throughput because of absence of post-PCR processing time (no agarose gel electrophoresis). In this pilot study, we detected significantly elevated CKI9 transcript levels in 70% of the and stage IV breast cancer patients. Conclusions Analyses using this real time quantitative RTPCR for CK.19 mRNA may prove to have clinical implications in the assessment of circulating tumour cells in peripheral blood, micrometastases in bone marrow or lymph nodes in breast cancer patients Application of this technique in a clinical population may improve diagnosis and monitoring of metastatic breast cancer and its validation is currently ongoing