Detection and Characterization of Circulating Tumor Cells in Early Breast Cancer

Abstract

Background: Detection of circulating tumor cells (CTCs) has demonstrated prognostic significance in metastatic breast cancer. This is less studied in early breast cancer due to the rarity of such cells in early disease and challenges in CTC detection, but shows strong clinical value as well. The purpose of this study was to collect CTCs in early breast cancer patients by use of an enhanced immunomagnetic enrichment method (MINDEC), detect and characterize them by multi-marker quantitative PCR (qPCR), and compare the results with clinicopathological data. Patients and Methods: CTCs were analyzed in 170 peripheral blood samples from 133 earlystage breast cancer patients. Blood samples from 30 healthy female volunteers were analyzed by the same methods as the patient group. CTC detection and characterization was performed using the MINDEC negative enrichment method (multi-marker depletion of leukocytes) followed by multi-marker qPCR. The multi-marker panel was selected based on previous literature, differential expression by serial analysis of gene expression (SAGE) data, and analysis of cell lines, breast tumor samples, and healthy controls. CTC status and clinicopathological factors were analyzed for statistical associations. The markers selected were EPCAM, ERBB2, KRT8, KRT19, SCGB2A2, SNAI1, SNAI2, TWIST1, and two novel markers, LUM and CCDC80. Results: Circulating tumor cells were detected in at least one blood sample in 35 of 133 (26.3%) of the patients and in 37 of 170 (21.8%) total samples. Of the CTC-positive patients, 7 (20%) were positive for more than one marker, 9 (24.3%) expressed only epithelial markers, 22 (59.5%) expressed only EMT markers, and 6 (16.2%) expressed both. Of the 35 CTC-positive patients, LUM was detected in 12 (34.3%) and CCDC80 detected in 10 (28.6%). CTC-status and individual markers were not significantly associated with any clinicopathological features. Conclusions: Detection and characterization of CTCs by the presented approach was feasible and revealed heterogeneous gene expression in CTC fractions from early breast cancer patients, with over 60% expressing EMT markers alone or with epithelial markers. Two novel extracellular matrix (ECM) markers (CCDC80 and LUM ) were selected for the panel and had the highest detection rates of all markers. Our detection rate of CTCs was similar to that observed with other methods in early-stage breast cancer, while allowing for expanded analysis of CTC characteristics. The clinical significance of these findings remains to be seen and will await further data on the clinical outcome for these patients.