Abstract 2946: Gene expression profiling in circulating cells (CTCs) of breast carcinoma patients - a tool for early metastasis detection and therapy individualization

Background: Tumor cell dissemination is an early process in breast cancer (BC) and circulating tumor cells (CTCs) are considered potential surrogate marker for the detection and characterization of minimal residual disease. Here we monitored hematogenous micrometastasis in BC patients by gene expression profiling of CTCs based on CTC-abundance in blood and expression of 35 oncomarkers on the mRNA level by multimarker quantitative PCR (qPCR). Characteristics of the early dissemination process and gene expression profile changes were studied by analyzing tumor tissue and CTCs of primary and metastatic BC patients. Methods: In primary BC five ml blood was collected before and after chemotherapy (n=87 patients). In metastatic BC 5 ml blood of 72 patients was studied either at the time of relapse of BC or at a documented progressive BC before receiving new therapy. All samples were analyzed for CTCs using the AdnaTest BreastCancer (AdnaGen AG, Langenhagen, Germany). CTCs were immunomagnetically enriched using the AdnaTest BreastCancerSelect followed by RNA isolation, subsequent gene expression analysis by reverse transcription and multiplex-PCR for the detection of EpCAM, MUC-1 and HER-2 transcripts. RNA from formalin fixed paraffin embedded (FFPE)-tumor tissue (n=46) was isolated with RecoverAll® (Ambion). After reverse transcription the cDNA was gene-specifically pre-amplified for multimarker qPCR analysis on the Biomark® (Fludigm, USA) microfluidic chip for 48 genes in each of 48 samples (2034 rxn in total). qPCR data were analyzed with GenEx ver. 5.0 (MultiD, SE) and correlated to available clinical data. Results: 286 CTC samples were analyzed in total. Blood sample was considered positive in the AdnaTest if at least one marker was measured above the cut-off level in the sample. CTCs were found in 29/87 (33,3%) of primary BC patients before chemotherapy and in 10/87 (11,5 %) patients after chemotherapeutical treatment. Among metastatic BC patients 48/72 (67%) were CTC positive. Gene expression analysis of the CTCs in metastatic BC patients and FFPE samples revealed 20 genes that were differentially expressed (p