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DOI: 10.1055/s-0028-1089146
Normalized quantitative gene expression analysis of circulating tumor cells in primary and metastatic breast cancer patients – Embedded Tumor Cell Calibrator Technique
Background: The purpose of this study was the validation of a new preanalytical enrichment and molecular detection method using embedded tumor cell calibrators (ETC) for quantitative gene expression analysis of circulating tumor cells (CTC) in breast cancer patients.
Methods: The high affinity antibodies BM7 (MUC–1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment. Samples from every patient were devided in native probes and matched calibrator probes containing either 2 or 10 breast carcinoma tumor cells (ETC). A real-time quantitative RT-PCR approach was established using primers and FAM-labeled TaqMan probes of the UniversalProbeLibrary system (Roche AG, Basel, CH).
Results: Positivity rate of ETC controlled realtime RT-PCR on the basis of CK19 as tumor identifier was 6.9% in 105 patients with primary breast cancer and 61.1% in patients with metastatic disease. During a 18 months follow-up of 92 patients with primary breast cancer, multimarker positivity was determined in 10 patients, and in three of these patients early metastasis was clinically confirmed. In patients with metastatic disease CTCs expressed the tumor-identifier CK19 and MG1 together with various surrogate markers.
Conclusion: We describe embedded tumor cells as internal calibrators for accurate process control and normalization of tumor cell enrichment and quantitative RT-PCR approaches. Characterization of clinically relevant markers from the networks of apoptosis (Sur), angiogenesis (CD276), growth factor receptors (HER–2) and cytostatica resistence (ABCG2) may improve early detection of metastasis, monitoring of treatment regimes and prediction of (new) therapeutic targets.
immunomagnetic enrichment - real-time RT-PCRtum