Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients

Abstract

Abstract Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients. Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL. Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients. Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2014-4018