Tumor cell quantification and normalized multi-gene expression analysis of micrometastatic cells in blood and bone marrow of cancer patients: introduction of the embedded tumor cell calibrator technique.

Abstract #5029 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 process control, relative tumor cell enumeration and quantitative gene expression analysis of circulating tumor cells (CTC) in breast cancer patients.
 Methods: Samples from every patient were devided in native probes and matched calibrator probes containing either 2, 4 or 8 breast carcinoma tumor cells (ETC). The high affinity antibodies BM7 (MUC-1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment in 10 mL of peripheral EDTA-blood of patients with primary and metastatic breast cancer. Mononuclear cells from bone marrow of patients with primary breast cancer were analysed in parallel by standardized cytospin immunocytology techniques (2x10 6 cells) and by the immunobead enrichment technique (1x10 7 cells). Separated cells were lysed and used for mRNA isolation and c-DNA synthesis. A real-time quantitative RT-PCR approach for the tumor identifier cytokeratin 19 (CK19) and mammaglobin 1 (MG1) and the surrogate markers HER-2, survivin (Sur), prostate-specific ets factor (PSE), CXCR4, Nanog, CD276 and FGFR2 was established using primers and FAM-labeled TaqMan probes selected with the UniversalProbeLibrary system (Roche AG, Basel, CH).
 Results: Positivity rate of ETC controlled realtime RT-PCR on the basis of CK19 and MG1 was 6.9% in 205 patients with primary breast cancer and 61.1% in patients with metastatic disease. Clonal selection or conversion from a HER2 negative (histology) to a HER2 positive phenotype was determined by qRT-PCR in 21 from 73 (28.8%) patients. 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. Tumor cell detection rate in bone marrow on the basis of
 1x10 7 cells was 10.5% (11/104) while cytology at the level of 2x10 6 cells resulted in a positivity rate of only 1% (1/104).
 Conclusion: For the first time we describe embedded tumor cells (ETC) as internal calibrators for accurate process control and normalization of the complex techniques of the immunobead separation and the quantitative RT-PCR technique. Tumor cell quantification was performed with the tumor identifiers CK19 and MG1, while corresponding expression levels of surrogate markers were determined on the basis of spiked calibrator cells. The ETC technique improved monitoring and gene expression analysis of disseminated tumor cells in peripheral blood and bone marrow significantly.
 Characterization of clinically relevant markers from the networks of apoptosis (Sur, PSE), tumor related/induced angiogenesis (CD276), growth factor receptors (HER-2, FGFR2) and cytostatica resistence (ABCG2) may improve early detection of metastasis, monitoring of treatment regimes and prediction of (new) therapeutic targets. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5029.