Abstract 1466: Establishment of a multimarker gene panel for the characterization of circulating tumor cells in metastatic breast cancer.
Authors: Bredemeier Maren , Aktas Bahriye , Kimmig Rainer , Kasimir-Bauer Sabine 
Published: August 2014

Abstract
Background: Improved treatment strategies for metastatic breast cancer (MBC) patients are urgently needed. Since metastatic tissue may be difficult to obtain for repeated analysis, circulating tumor cells (CTC) would be an ideal surrogate tissue to identify prognostic and predictive factors that will help to select the optimal therapeutic strategy for each individual patient. Assuming that the population of CTC contains epithelial-like, EMT (Epithelial-Mesenchymal-Transition)-like and stem cell-like cells, we established a multi-marker qPCR for the characterization of these cells.Materials and Methods: Establishment of a 16 gene qPCR panel was performed using various epithelial cancer cell lines for the markers: EpCAM, MUC1 (epithelial); PI3K, PTEN, TWIST, mTOR, KRAS, AKT2 (EMT); ALDH1, CD44, CD24L4 (stem cell); ER, PR, HER2 and EGFR (receptors) and CD45 as a leucocyte control. The prostate cancer cell line LNCAP, expressing most of these genes, was used for spiking experiments. 10 ml blood of eight healthy donors (HDs), five HDs spiked with 10 LNCAP cells and 25 MBC patients were selected for CTC using AdnaTest BreastCancerSelect (AdnaGen AG) resulting in cDNA1. Subsequently, the same sample (with removed target cells) was processed again using the same procedure resulting in cDNA2. cDNA1 and cDNA2 were gene specifically preamplified using TaqMan PreAmp Master Mix according to in house designed assays. qPCR was performed using Bio-Rad SYBR Green Mix. If the CD45 deltaCt was &gt; zero, deltaCt value of a given gene was calculated as the difference between Ct (cDNA2) and Ct(cDNA1). A gene with a deltaCt &gt; zero was considered positive.Results: When HDs were tested for all genes, no false positive findings were observed except for AKT2, CD24L4, CD44 (n=3 cases). All of the genes except for TWIST, ER and PR could be positively detected in samples spiked with 10 LNCAP cells. In patient samples, at least one of all studied markers was detected in 21/25 (84%) of the patients. The distribution of the markers across all patients was highly variable. However, PI3K expression was observed most frequently (n=8/21 patients), followed by the expression of CD44 (n=6/21 patients), HER2 (n=5/21 patients) and TWIST, KRAS, mTOR and EpCAM (4/21 patients), respectively. No expression was observed for MUC1, PR and CD45. In general, EMT- and stem cell-like CTC were predominantly detected. HER2 positive and epithelial-like CTC as well as CTC expressing ER, PR and EGFR were observed less frequently. Interestingly, some patients expressed only one CTC-subtype.Conclusion: Multi-gene expression profiling improves the characterization of CTC of an individual patient. Furthermore, it was possible to classify individual patient samples into CTC subtypes. Despite these promising preliminary findings, the method has to be further optimized and needs to be verified in a larger patient population.
Citation Format: Maren Bredemeier, Bahriye Aktas, Rainer Kimmig, Sabine Kasimir-Bauer. Establishment of a multimarker gene panel for the characterization of circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1466. doi:10.1158/1538-7445.AM2013-1466