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

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 carcinom a cells in blood, we have developed a sensitive and quantitative assay usin g 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 reproducibility within a wide dynamic range, which permits simultane ous quantitative analysis of samples with varying input concentrations. Fur thermore, the procedure offers several technical advantages over classic qu antitative PCR methods (competitive RT-PCR, Northern blotting) such as decr eased 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 significantl y elevated CK19 transcript levels in < 10% of the volunteers, in . 30% of s tage I-IIIa patients preoperatively and in > 70% of the and stage IV breast cancer patients. Conclusions: Analyses using this real time quantitative RT-PCR for CK19 mRN A may prove to have clinical implications in the assessment of circulating tumour cells in peripheral blood, micrometastases in bone marrow or lymph n odes in breast cancer patients. Application of this technique in a clinical population may improve diagnosis and monitoring of metastatic breast cance r and its validation is currently ongoing.