Detection of circulating tumor cells in blood of metastatic breast cancer patients using a combination of cytokeratin and EpCAM antibodies

Abstract

Background Circulating tumor cells (CTCs) are detectable in peripheral blood of metastatic breast cancer patients (MBC). In this paper we evaluate a new CTC separation method based on a combination of anti-EpCAM- and anti-cytokeratin magnetic cell separation with the aim to improve CTC detection with low target antigen densities. Methods Blood samples of healthy donors spiked with breast cancer cell line HCC1937 were used to determine accuracy and precision of the method. 10 healthy subjects were examined to evaluate specificity. CTC counts in 59 patients with MBC were measured to evaluate the prognostic value on overall survival. Results Regression analysis of numbers of recovered vs. spiked HCC1937 cells yielded a coefficient of determination of R 2 = 0.957. The average percentage of cell recovery was 84%. The average within-run coefficient of variation for spiking of 185, 85 and 30 cells was 14%. For spiking of 10 cells the within-run CV was 30%. No CTCs were detected in blood of 10 healthy subjects examined. A standard threshold of 5 CTC/7.5 ml blood as a cut-off point between risk groups led to a highly significant prognostic marker (p < 0.001). To assess the prognostic value of medium CTC levels we additionally considered a low (CTC-L: 0 CTC), a medium (CTC-M: 1–4 CTC) and a high risk group (CTC-H: ≥5 CTC). The effect of this CTC-LMH marker on overall survival was significant as well (p < 0.001). A log-ratio test performed to compare the model with 3 vs. the model with 2 risk groups rejected the model with 2 risk groups (p = 0.026). For CTC as a count variable, we propose an offset reciprocal transformation 1/(1 + x) for overall survival prediction (p < 0.001). Conclusions We show that our CTC detection method is feasible and leads to accurate and reliable results. Our data suggest that a refined differentiation between patients with different CTC levels is reasonable.

Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

“…Some more methods for CTC detection are described in the literature for example by immunomagentic cell separation [38], by EPISPOT assays [39], by Flow Cytometry followed by Real-Time PCR [40] and by immunomagnetic cell enrichment also followed by Real-Time PCR [41]. These methods, although they might have a higher sensitivity and specificity, are much more laborious, expensive, and time consuming.…”

Gene Knockdown by EpCAM Aptamer–siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells

“…In one study, about 2/3 of TNBCs, presumably the basal-like subtype, stained strongly for EpCAM (25). The number of EpCAM þ circulating cells is linked to poor prognosis in breast cancer (32)(33)(34)(35)(36). An EpCAM antibody has been evaluated clinically for epithelial cancers, but had limited effectiveness on its own (37)(38)(39).…”

“…An EpCAM antibody has been evaluated clinically for epithelial cancers, but had limited effectiveness on its own (37)(38)(39). EpCAM expression identifies circulating tumor cells in an FDA-approved method for monitoring metastatic breast, colon, and prostate cancer treatment (32)(33)(34)(35)(36). Moreover, about 97% of human breast cancers and virtually 100% of other common epithelial cancers, including lung, colon, pancreas, and prostate, stain brightly for EpCAM (23), suggesting that the platform developed here could be adapted for RNAi-based therapy of common solid tumors.…”

2-NBDG Fluorescence Imaging of Hypermetabolic Circulating Tumor Cells in Mouse Xenograft model of Breast Cancer

“…Using the Cell Tack system based on enumeration of CTC positive for EpCAM, CTC could be detected in only 60 % of patients with metastatic breast cancer [5, 6, 32]. It was suggested to use a combination of cytokeratin and EpCAM antibodies for detection of circulating tumor cells in blood of metastatic breast cancer patients [33, 34]. In conjunction with use of 7-ADD [9] or other probes, quantification of 2-NBDG uptake may be used to differentiate viable, hyper-metabolic CTC from other blood cells or nonviable, apoptotic CTC by multi-color flow cytometry.…”