20033 Background: The purpose of this study was detection and expression profiling of circulating tumor cells (CTC) in breast cancer patients. Methods: Two separate probes of 5 mL peripheral EDTA-blood from patients with primary breast cancer (n=167) and metastatic disease (n=111) were used for immunomagnetic tumor cell selection. Targets for preanalytical enrichment were the antigens EpCAM and MUC-1. Separated cells were lysed and used for mRNA isolation and c-DNA synthesis. The breast carcinoma-associated transcripts EpCAM, MUC-1, HER-2, claudin7, cytokeratin 19, mammaglobin 1, prostate-specific ets factor (PSE) and survivin were amplified by three separate multiplex RT-PCR reactions. Amplicons were analysed by capillary electrophoresis with the Agilent Bioanalyzer 2100. Specificity of the RT-PCR was confirmed by examination of blood of healthy donors. Results: Sensitivity for every single transcript was adjusted to 2 tumor cells per 5 ml blood. Tumor-associated transcripts were detected in 31 of of 167 (18.5%) patients with primary breast cancer and in 46 of 111 (41%) patients with metastatic disease. The marker with the highest incidence in both groups was MUC-1, with a positivity rate of 81%. Tumor-associated transcripts were heterogenouosly expressed, however multiple markers were identified in more than 50% of the positive samples. Conclusion: Using a combination of preanalytical immunomagnetic tumor cell enrichment followed by a multigen RT-PCR approach we describe a sensitive detection system for breast carcinoma cells. In this study a panel of 8 genes overexpressed at high levels in metastatic breast cancer was selected for the identification of disseminated tumor cells in the peripheral blood of breast cancer patients. HER-2, survivin as a unique member of the inhibitor of apotosis protein family, as well as PSE identified in circulating breast cancer cells may serve as prognostic indicators of tumor progression and could represent valid targets for new individualized therapeutic interventions. No significant financial relationships to disclose.
Detection of Circulating Tumor Cells in Breast Cancer Patients Using Cytokeratin-19 Real-Time RT-PCR
