Abstract 4150: CK-19, MAGE-A3, HER-2 and TWIST-1 expression by RT-qPCR in CTCs of primary breast cancer patients after positive immune-magnetic selection using anti-EpCAM and anti-MUC1 magnetic beads
Authors: Strati Areti , Markou Athina , Hoffmann Oliver , Kimmig Rainer , Lianidou Evi S. , Kazimir-Bauer Sabine 
Published: August 2014

Abstract
Background: Circulating tumor cells (CTCs) are suggested as potential surrogate markers for minimal residual disease, the precursor of metastatic disease. Furthermore, to be able to disseminate and metastasize, CTCs must be able to perform epithelial-mesenchymal transition (EMT). The aim of our study was to evaluate the sensitivity of RT-qPCR for the detection and characterization of CTCs analyzing the expression of CK-19, MAGE-A3, HER-2 and TWIST-1 as a transcription factor for EMT in blood of early breast cancer patients after positive immunomagnetic selection using a combination of anti-EpCAM and anti-MUC1 magnetic beads.
Patients and Methods: Two × 5 ml blood samples were collected before surgery and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads (AdnaTest Breast Cancer Select, AdnaGen AG, Langenhagen, Germany). CK-19, MAGE-A3, HER-2 and PBGD transcripts were simultaneously quantified by multiplex RT-qPCR while TWIST-1 transcript by single RT-qPCR in the LightCycler 2.0 (Roche Diagnostics). All primers and probes were designed to match the assay conditions, such as amplicon sizes, and melting temperatures. Before proceeding to patients’ samples, the RT-qPCR assay was evaluated in respect to sensitivity, specificity, and reproducibilty. To ensure that amplifiable material was present in all specimens and to avoid false-negative results, real-time amplification of the reference gene porphobilinogen deaminase (PBGD) was performed for all samples. A total number of 436 samples were first evaluated for RNA quality by PBGD expression and finally 293 samples that were of good RNA quality were further processed.
Results: CK-19 was detected in 117/293 (39.9%), HER-2 in 35/293 (12.0%), MAGE-A3 in 25/293 (8.5%), and TWIST-1 in 16/293 (5.5 %) of all samples, respectively. One gene was detected in 135/293 (46.1%) of the patients, two genes in 23/293 (7.8%) of the patients, three genes in 3/293 (1.0%) patients and four genes in 0/293 (0 %) of the patients, respectively. In total, 161/293 (54.9%) patients were found positive for at least one gene expression, while in 132/293 (45.05%) patients we didn't detect CTCs at all.
Discussion: Our study has revealed a high sensitivity when using a combination of immunomagnetic separation and RT-qPCR, as well as a remarkable heterogeneity of gene expression for these genes between early breast cancer patients. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.
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 4150. doi:10.1158/1538-7445.AM2011-4150