Embedded tumor cell calibrators (ETC) for normalized quantitative multi-gene expression analysis of micrometastatic cells in blood and bone marrow of cancer patients

Abstract

1065 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: Samples from every patient were devided in native probes and matched calibrator probes spiked with either 2, 4 or 8 breast carcinoma tumor cells. The high affinity antibodies BM7 (MUC-1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment of 10 mL peripheral EDTA-blood of patients with primary breast cancer and metastatic disease. Separated cells were used for mRNA isolation and c-DNA synthesis. A real-time quantitative RT-PCR approach for the markers cytokeratin 19 (CK19), survivin (Sur), mammaglobin 1 (MG1) and prostate-specific ets factor (PSE), HER-2, CD276 and FGFR2 was established using primers and FAM-labeled TaqMan probes selected with the Universal-Probe-Library system (Roche AG, Basel, CH). Results: Positivity rate of ETC controlled realtime RT-PCR on the basis of CK19 and MG1 in blood was 6.9% in 205 patients with primary breast cancer and 61.1% in patients with metastatic disease while 15/104 (14.4%) bone marrow samples were postive. Clonal selection or conversion from a HER2 negative (histology) to a HER2 positive phenotype was determined by qRT-PCR in 17 from 61 (27.8%) patients. During an 18-month 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. Conclusions: For the first time we describe embedded tumor cells (ETC) as internal calibrators for accurate process control and normalization of the complex immunobead quantitative RT-PCR technique. Tumor cell quantification was performed with the tumor identifier CK19 while corresponding expression levels of HER2 and the tumor-related/induced angiogenesis factor CD276 were determined on the basis of spiked calibrator cells. ETC should improve monitoring and gene expression analysis of disseminated tumor cells in bone marrow and circulating tumor cells during therapy. No significant financial relationships to disclose.