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DOI: 10.1055/s-0028-1089154
Detection and characterization of micrometastatic cells with the embedded tumor cell calibrator technique – Normalized RT-PCR
Background: We present a new technique called embedded tumor cell calibrators (ETC) for tumor cell enumeration and gene expression analysis.
Material and Methods: Bone marrow and blood samples from normal donors and patients with breast carcinoma were devided in at least one native probe and several matched calibrator probes. Calibrator probes were spiked with either 2, 4, 8 or 16 breast carcinoma cells (embedded tumor cell calibrators, ETC). Native and matched ETC samples were processed side by side under identical conditions through the following steps: a) immunomagnetic tumor cell enrichment with high affinity antibodies BM7 (MUC–1) and VU1D9 (EpCAM), b) m-RNA isolation, c) c-DNA-synthesis d) real-time RT-PCR using primers and FAM-labeled TaqMan probes of the UniversalProbeLibrary system (Roche AG, Basel, CH).
Results: The new diagnostic tool was established in a modell systems with cytokeratin 19 (CK19) as tumor identifier and with HER–2 and CD276 as modell targets for gene expression analysis. Using 5ml whole blood (106 samples) and Ficoll separated bone marrow (72 samples,1×107 cells) we have established and validated conditions for tumor cell quantification in the range of 1 to 16 tumor cells. In 25 probes from patients with metastatic disease tumor cells were successfully quantified (mean 2.5 cells) and corresponding HER2 and CD276 expression ratios were normalized to the calibrator cells HD-ZE.
Discussion: We have designed a new technical approach which allowed simultaneous tumor cell quantification and relative gene expression analysis of rare tumor cells in bone marrow and blood. The technique can be adopted to other carcinoma types and further surrogate markers.
CTC - DTC - gene expression analysis