Abstract
A two-step reverse-transcriptase-based polymerase chain reaction (PCR) with nested primer pairs was developed to amplify sensitive and specific cytokeratin-19 (CK-19) mRNA sequences from human breast cancer cells. No CK-19 pseudogene interference was seen. The larger DNA-derived amplification products could be clearly discriminated from mRNA-derived products. The CK-19 message was not amplified from bone marrow or blood of healthy volunteers and patients with haematological malignancies nor from myeloid and lymphoid cell lines. Breast cancer cells were diluted in buffy coat cells up to 10(-6) and CK-19 mRNA sought by PCR. The CK-19 message was detected in 14 of 26 blood samples and 14 of 24 marrow samples but in neither of two peripheral blood stem cell samples taken from 35 breast cancer patients. By sequence-analysis control of two of these samples and two cell lines, the amplified DNA fragments were confirmed to be homologous with the CK-19 sequence. The CK-19 message was further sought in matched blood/marrow samples taken from 13 untreated women in the same cohort at the time of diagnosis. In 3 of these, CK-19 RNA was detected in blood and marrow and, in 3 others, only in blood, but never in marrow alone. The results show that CK-19 assay by reverse transcriptase/PCR is a sensitive and specific technique for the detection of cancer cells in bone marrow and blood. It could be helpful in diagnosis and monitoring of metastatic breast cancer and detection of micrometastases. This should be evaluated on larger numbers of patients, with different clinical samples and epithelial malignancies.