Abstract 503: Molecular characterization of in vivo isolated EpCAM-positive circulating tumor cells in breast cancer
Authors: Strati Areti D. , Zavridou Martha , Kallergi Galateia , Politaki Eleni , Gorges Tobias , Kuske Andra , Bohnen Anna-Lena , Koutsodontis George , Psyrri Amanda , Lucke Klaus , Georgoulias Vasilis , Pantel Klaus , Lianidou Evi 
Published: July 2016

Abstract
Introduction: In the early stages of cancer, the chance to detect rare CTCs is increasing by increasing the sample volume. The aim of our study was to evaluate the diagnostic sensitivity of a novel clinical device for the in-vivo isolation of EpCAM-positive CTCs (CellCollectorTM, GILUPI, GmBH), by using highly sensitive RT-qPCR molecular assays.
Patients and methods: 29 breast cancer patients without overt metastases before the beginning of adjuvant chemotherapy (M0), 26 breast cancer patients with overt metastases before starting of therapy (M1) and 12/26 of them before the second cycle of therapy (M2), as well as 18 healthy donors participated in the study. After in-vivo isolation, total RNA was extracted from captured cells, lysed in Trizol, followed by cDNA synthesis. RT-qPCR was used for the molecular characterization of captured cells, for: CK-19, HER-2, TWIST1, VEGF, ER, PR, EGFR, CD44, CD24, and ALDH1, while B2M was used as a reference gene. Peripheral blood was also collected for CTC analysis by the FDA cleared CellSearchTM system. In addition, immunofluorescence staining of cytospins was performed and screened for CTCs using the ARIOL system, using ER, HER2, CK (8, 18, 19) and CD45 for CTC identification.
Results: Results are shown in Table 1. At least one gene was expressed in 10(34.5%) of M0, 15(57.7%) of M1 and 4(33.3%) of M2 patient groups, but in none of healthy donors 0/18(0%). CellSearchTM gave positive results in 5(17.2%) of M0, 10(38.5%) of M1 and 0(0%) of M2. Immunofluorescence (Ariol system) was positive for ER, HER2, CK (8, 18, 19) in 5/15(33.3%) M0, in 4/12(33.3%) M1 and in 1/7(14.3%) M2 groups. Table 1.Gene expression in CTCHealthy N = 18M0 N = 29M1 N = 26M2 N = 12CK-190 (0%)6(20.7%)6 (23.1%)2 (16.7%)HER20 (0%)2 (6.9%)0 (0%)0 (0%)ER0 (0%)2 (6.9%)0 (0%)0 (0%)PR0 (0%)0 (0%)0 (0%)0 (0%)EGFR0 (0%)0 (0%)0 (0%)0 (0%)TWIST10 (0%)1 (3.4%)0 (0%)2 (16.7%)VEGF0 (0%)3 (10.3%)5 (19.2%)1 (8.3%)CD44+/CD24−,0 (0%)4 (13.8%)3 (11.5%)1 (8.3%)ALDH1high/CD24−,0 (0%)2 (6.9%)8 (30.8%)1(8.3%)
Conclusions: In-vivo isolation of CTC is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems promising. Comparison studies with the CellSearch and immunofluorescence have shown poor agreement. These results should be validated in large patient cohorts, and in respect to the clinical outcome.
Citation Format: Areti D. Strati, Martha Zavridou, Galateia Kallergi, Eleni Politaki, Tobias Gorges, Andra Kuske, Anna-Lena Bohnen, George Koutsodontis, Amanda Psyrri, Klaus Lucke, Vasilis Georgoulias, Klaus Pantel, Evi Lianidou. Molecular characterization of in vivo isolated EpCAM-positive circulating tumor cells in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 503.