Determination of Sensitivity of RT-PCR for Cyokeratin 19 and Detection of Micrometastasis from Bone Marrow of Breast Cancer Patients

ABSTRACT

BACKGROUND: The detection of micrometastatic cells in patients with breast cancer may aid in determining of prognosis and in developing new therapeutic approaches. In this study, we evaluate an assay to identify breast cancer cells in the bone marrow of patients with breast cancer by using reverse-transcriptase polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (CK-19) transcripts. METHODS: A CK-19 specific-nested RT-PCR assay was developed and optimized by using limited dilutions of an MCF-7 breast-cancer cell line mixed with normal bone-marrow specimens. The optimized assay was then used to examine bone-marrow samples obtained from 60 patients with breast cancer. The specificity was assessed by examining 20 negative controls using malignant hematologic disease. RESULTS: In the sensitivity calibration system, CK-19 expressing tumor cells were detected in the mixture of 10 MCF-7 cells in 107 normal bone-marrow cells. All 20 neagtive control samples failed to amplify. Bone marrow samples from 10 of 60 patients (16.7%) with breast cancer scored positive, indicating micrometastasis of the bone marrow. Seven of the 37 samples from patients whose axillary lymph nodes were negative based on conventional histopathological studies were positive when the CK-19 RT-PCR method was used. CONCLUSION: RT-PCR for CK-19 is a sensitive, specific, and rapid method for detecting micrometastatic mammary carcinoma cells in the bone marrow of patients with breast cancer. It could be helpful in diagnosing and monitoring metastastic breast cancer and detecting of micrometastasis. This method should be evaluated using a larger number of patients for long-term follow-up.