Abstract 1462: Development of a multiplexed RT-PCR-coupled liquid bead array assay for the molecular characterization of CTCs.
Authors: Parisi Cleo , Markou Athina , Strati Areti , Lianidou Evi 
Published: August 2014

Abstract
Introduction. The presence of circulating tumor cells (CTCs) in patients’ bloodstream is responsible for hematogenous metastases and as a “liquid biopsy” these cells should be considered as a useful tool for the detection and the characterization of micrometastasis. Molecular characterization of CTCs is really helpful for diagnostic and therapeutic purposes. We recently developed a liquid bead array hybridization assay for studying the expression of 6 genes in CTCs in breast cancer (Markou et al, Clin Chem 2011). The aim of the present study was to expand the potential of this assay by adding more genes of clinical interest in the genes panel.
Materials and Methods. We designed and evaluated a multiplexed PCR-coupled liquid bead array for studying simultaneously the expression of 14 genes. We firstly designed in silico primers and capture probes for: a) breast cancer stem cell markers: ALDH1, CD24, CD44, b) EMT markers: SNAIL, TWIST-1 and VIM, c) breast cancer markers: ERBB2, mammaglobin A, MAGEA3, PTEN, d) epithelial markers: CK-19, e) housekeeping genes: PBGD, HPRT1, and PDCD4 as a gene related with apoptosis. The assay is based on the following steps: total RNA isolation, cDNA synthesis, multiplex PCR for 14 genes, biotinylation of multiplex PCR products, hybridization of the biotinylated multiplex PCR products to xMAP microspheres, addition of streptavidin-phycoerythrin and measurement in the Luminex® 100 analyzer. Every single step was optimized in terms of analytical specificity, repeatability and reproducibility using the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3. To evaluate the performance of this assay we used cancer cell lines originated from the following tissues: a. human colon (COLO-205), b. human cervix (HeLa) and c. human mammary gland (T-47D).
Results. The total duration of the assay, including hands on work, is approximately 6h. The assay is highly specific for each gene in complex multiplexed formats. Intra-assay CVs (n=3) ranged from 0.2% to 15.3% and inter-assay CVs (n=3) ranged from 1.0% to 18.9%. The developed assay was successfully applied for studying gene expression in COLO-205, HeLa, MCF-7, MDA-MB-231, SK-BR-3 and T-47D cancer cell lines.
Conclusions. The previously developed liquid bead array has been successfully expanded including 8 additional gene targets of clinical interest. The developed assay could be applied for the molecular characterization of CTCs, since the expression of 14 genes can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis. The developed assay will be further extended to include additional gene targets.
Citation Format: Cleo Parisi, Athina Markou, Areti Strati, Evi Lianidou. Development of a multiplexed RT-PCR-coupled liquid bead array assay for the molecular characterization of CTCs. [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 1462. doi:10.1158/1538-7445.AM2013-1462