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

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.

Use of RT-PCR for Cytokeratin 19 to Detect Micrometastases in Bone Marrow of Follow-up Patients with Breast Cancer after Surgery

PURPOSE: We have previously reported that the reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK 19) is a highly sensitive and specific method for detecting micrometas tases in the bone marrow of patients with primary breast cancer. Thus, we conducted this study to identify occult metastatic cells in the bone marrow of follow-up patients with breast cancer by using the RT-PCR for cytokeratin 19 after surgery. METHODS: We studied 42 patients with breast cancer who had no evidence of relapse, 3 patients who had a local relapse, and 7 patients who had distant metastases. Bone-marrow aspirates were taken from the single posterior iliac crest under local anesthesia anesthesia at a median of 45 months after surgery. RT-PCR for CK19 was done by using two 30-cycle rounds with nested primers. RESULTS: Occult metastases were found in 23% of the 52 breast-cancer patients. None of the bone-marrow aspirates showed any evidence of tumor involvement by using conventional cytology. RT- PCR was positive for CK 19 in 19% of the 42 patients without relapse, and in 40% of the 10 patients with local or distant relapse. CONCLUSION: RT-PCR for CK 19 is a sensitive method for detecting tumor cells in the bone marrow of follow-up patients with breast cancer after surgery. This assay may be useful in detecting metastastic disease, as well as in monitoring the effectiveness of systemic therapy. However, a large study with long-term follow-up is required in order to clarify its clinical significance and usefulness.