Background:The detection of occult carcinoma cells in patientswith breast cancer has been shown to predict disease recurrence andmetastasis.
Materials and methods:To improve on molecular detection of breastcarcinoma cells in blood, we have developed a sensitive and quantitative assayusing real-time quantitative RT–PCR identifying transcripts of thecytokeratin-19 (CK19) gene.
Results:This real-time quantitative RT–PCR is sensitive,accurate and has a high reproducibility within a wide dynamic range, whichpermits simultaneous quantitative analysis of samples with varying inputconcentrations. Furthermore, the procedure offers several technical advantagesover classic quantitative PCR methods (competitive RT–PCR, Northernblotting) such as decreased likelihood of contamination due to absence ofpost-PCR manipulations, high sample throughput because of absence of post-PCRprocessing time (no agarose gel electrophoresis). In this pilot study, wedetected significantly elevated CK19 transcript levels in <10% ofthe volunteers, in · 30% of stage I–IIIa patientspreoperatively and in >70% of the and stage IV breast cancerpatients.
Conclusions:Analyses using this real time quantitativeRT–PCR for CK19 mRNA may prove to have clinical implications in theassessment of circulating tumour cells in peripheral blood, micrometastasesin bone marrow or lymph nodes in breast cancer patients. Application of thistechnique in a clinical population may improve diagnosis and monitoring ofmetastatic breast cancer and its validation is currently ongoing.