A new nested primer pair improves the specificity of CK-19 mRNA detection by RT-PCR in occult breast cancer cells

Reverse transcription polymerase chain reaction (RT-PCR) of cytokeratin-19 (CK-19) has been widely used to detect small numbers of circulating malignant epithelial cells in the bone marrow or the peripheral blood of patients with breast cancer. However, a high percentage of false positive results has been recorded and conflicting reports question the clinical relevance of this technical approach. We demonstrate that the use of a new nested primer pair for CK-19 RT-PCR avoids false positive results without affecting the sensitivity of the assay. Our experiments were carried out using MCF-7 cells alone or mixed with peripheral blood mononuclear cells (PBMNC) of healthy donors. The results were also validated in a large series of healthy donors and in a preliminary study on a limited number of patients with breast cancer, thus suggesting that our assay is feasible for application in the clinical evaluation of occult malignant epithelial cells.