Analytical And Clinical Validation Of An Epcam-Independent Assay For Ctc Detection In Peripheral Blood Of Early Breast Cancer Patients Based On Cytokeratin-19 (Ck-19) Rt-Qpcr

Introduction: The aim of our study was to evaluate the prognostic significance of our previously developed EpCAM independent RT-qPCR assay for CK-19 mRNA (Stathopoulou et al, Int J Cancer 2006) and validate its analytical and clinical performance in early breast cancer. Methods: Quality control on analytical sensitivity and specificity, linearity, intra- and inter-assay reproducibility, and stability of the external standard used for the preparation of the calibration curves was performed. Reproducibility of the assay between our labs was evaluated by analyzing 26 cDNAs. The prognostic significance of the assay in respect to DFI and OS was evaluated by analysing peripheral blood of 179 patients with stage I/II breast cancer postoperatively, before the administration of adjuvant chemotherapy. Results: The limit of detection (LOD) is 3 copies/reaction and limit of Quantitation (LOQ) 10 copies/reaction. The linear range of the calibration curve was 10-10 5 copies/reaction and the intra- and inter-assay reproducibility are shown below: The inter-lab reproducibility of the assay was evaluated by analyzing 26 cDNA samples in both labs and there was a 100% concordance. During the follow up period (8 years) 32/179 (17.9%) patients relapsed and 18/179 (10%) patients died from the disease. 45/179 (25.1%) samples were found positive for CK-19 and 134/179 (74.9%) negative. In the group of CK-19 positive patients 15/45(33.3%) relapsed and 9/45(20.0%) died while in the group of CK-19 negative patients 17(12.7%) patients relapsed and 9(6.7%) died. Kaplan-Meier analysis showed that CK-19 mRNA positivity was significantly associated with DFI (P = 0.014) and OS (P = 0.051). Conclusions: This EpCAM independent assay can be used for a high-throughput detection of CTCs in peripheral blood and has prognostic significance in early breast cancer. Citation Format: Areti Strati, Athina Markou, Aliki Stathopoulou, Stella Apostolaki, Dimitris Mavroudis, Vasilis Georgoulias, Evi S. Lianidou. Analytical and clinical validation of an EpCAM-independent assay for CTC detection in peripheral blood of early breast cancer patients based on Cytokeratin-19 (CK-19) RT-qPCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 373. doi:10.1158/1538-7445.AM2015-373