Abstract 969: Molecular characterization of circulating tumor cells in breast cancer by liquid bead array hybridization assay

Abstract

Abstract Introduction: The aim of our study was to develop and validate a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in Circulating Tumor Cells (CTCs), and application of this assay in peripheral blood samples of breast cancer patients. Patients and method: We designed a novel multiplexed PCR-coupled liquid bead array that consist of the following steps: a) in silico designed gene-specific primers and capture probes for CK-19, HER-2, Mammaglobin (hMAM), MAGE-A3, TWIST-1 and PBGD (as a control gene), b) RNA isolation from immunomagnetically enriched CTCs, c) multiplex PCR, d) sequence hybridization array, e) detection in the Luminex platform. We performed extensive optimization experiments using the SKBR3 and MDA-MB-231 cell lines as positive controls, to maximize analytical sensitivity and specificity and we further validated the performance of this array in respect to cross reactivity and intra and inter-precision. Finally, we applied the developed methodology in peripheral blood samples of 64 patients with operable breast cancer, 25 patients with verified metastasis and 17 healthy individuals. Results: The analytical performance of the developed array was evaluated in tumor cell lines in respect to analytical sensitivity and specificity. Cross reaction studies were performed for each gene target in the presence of all other targets. The assay is highly specific for each gene in complex multiplexed formats, since the discriminatory power of fluorescent bead sets is unique to specifically detect by sequence hybridization the presence of individual gene specific PCR products. The developed liquid bead-based array is highly sensitive, since it can detect the expression of each individual gene at one SKBR3 cell level. The validation of the array included within day and between-days precision studies. None of the genes tested was detected in the CTC fraction of healthy donors while in patients with verified metastasis, CK-19 was detected in 65%, HER-2 in 20%, MAGE-A3 in 30%, hMAM in 20% and TWIST-1 in 20%. In operable breast cancer patients, CK-19 was detected in 26.6%, HER-2 in 12.5%, TWIST-1 in 31.2%, MAGE-A3 in 18.7% while hMAM was detected in 10.9% of the patients. Conclusions: Our results show that individual gene expression can be readily measured in CTCs using a bead-based platform. This may form an efficient basis for a multiplex approach to measure multiple genes (up to 100) in the same sample, thus saving sample volume and reducing the total cost and time of analysis. This is the first time that the Luminex technology is used for gene expression studies in CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 969. doi:10.1158/1538-7445.AM2011-969