Abstract 840: Development and validation of a real-time multiplex PCR assay for the simultaneous quantification of CK-19, MAGE-A3, HER-2 and PBGD in circulating tumor cells of breast cancer patients
Authors: Strati Areti D. , Markou Athina N. , Malamos Nikos , Georgoulias Vasilis , Lianidou Evi 
Published: February 2011

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. The aim of our work was to develop a quantitative real-time multiplex PCR assay for CK-19, MAGE-A3, HER-2 and PBGD and validate its performance in CTCs of early and metastatic breast cancer patients.
Materials and Methods: A tetraplex quantitative real time assay for CK-19, MAGE-A3, HER-2 and PBGD was developed based on the in-silico design of primers and four hybridization probe pairs with different fluorescence emission spectra. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. Specificity and sensitivity experiments were performed using the SKBR-3 cancer cell line as a positive control. The method was applied in 73 patients with early breast cancer before the administration of adjuvant chemotherapy, 27 patients with verified metastasis and 17 female healthy volunteers. Peripheral blood (20 mL in EDTA) was obtained and after density gradient centrifugation, immunomagnetic Ber-EP4 coated capture beads were used to enrich for epithelial cells, keeping for each sample two fractions: the CTC and corresponding PBMC fraction. Messenger RNA was isolated from enriched epithelial cells using oligo (dT)25 coated magnetic beads. After cDNA synthesis the expression of CK-19, MAGE-A3, HER-2 and PBGD was tested, in both fractions.
Results: The analytical performance of the method was evaluated in SKBR-3 tumor cell line in respect to analytical sensitivity and specificity. Cross reaction studies, performed for each gene target in the presence of all other targets have shown a very high specificity for each analyte. RNA quality in all samples was evaluated by PBGD gene expression. We found 30/73 (41%) patients with early breast cancer positive for CK-19, 14/73 (19,2%) for MAGE-A3 and 11/73 positive for HER-2 (15,1%). In patients with verified metastasis we found 14/27 patients positive for CK-19 (51.8%) 4/27 for MAGE-A3 (14,8%), 5/27 patients positive for HER-2 (18,5%). In healthy individuals we found 1/17 positive for CK-19 (5,9%) while no samples were found positive for MAGE-A3 and HER-2 (0%).
Conclusions: We report for the first time a highly specific, reproducible and sensitive quantitative multiplex real-time PCR assay for the simultaneous detection of CK-19, MAGE-A3, HER-2 and PBGD. The expression of these genes in CTCs will be further examined in a larger number of patients and results will be correlated with their clinical outcome.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 840.