Abstract P1-04-11: Recovery rates of circulating tumor cells in breast cancer cell lines spiked into peripheral blood
Authors: Zhu W , Mineyev N , Ring A , Tripathy D , Punj V , Barsky LW , Lang JE 
Published: August 2014

Abstract
Background: Circulating tumor cells (CTCs) have been demonstrated to be prognostic in all stages of breast cancer. Immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) isolates enriched populations of CTCs derived from peripheral blood without the need for background subtraction of leukocytes. We previously reported IE/FACS as a strategy for gene expression profiling of CTCs. We hypothesized that variable expression of the epithelial cell adhesion marker (EpCAM) may bias IE/FACS recovery rates and composition.
Patients and Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). Unknown quantities of cell lines were spiked into PBS or peripheral blood (PB) from healthy female donors. IE/FACS was performed using EpCAM (MJ37) ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD45 PE-Cy7, and EpCAM (EBA-1) mAb conjugated to phycoerythrin. FACS sorting was performed using a FACS Aria II (BD Biosciences) and a gating strategy devised based on negative controls (n = 23). Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. Two-way ANOVA was used to analyze variation in recovery rates between groups (Prism GraphPad 6.0).
Results: Overall mean recovery rates for the 10 cell lines were 51.4% from PBS and 39.5% from PB. The specific cell type being analyzed was a more significant source of variation (p = 0.03) than was whether measurements were made from PBS or PB (p = 0.2). However, analysis by molecular subtype did not show differences between intrinsic groups (p = 0.23) nor did it show differences in recovery rates from PBS or PB (p = 0.26). The two inflammatory breast cancer cell lines (SUM 149 and SUM190) showed no differences in recovery rates compared to other cell lines (p = 0.41) nor in recovery rates from PBS versus PB (p = 0.75). Recovery rates for the 10 cell lines are shown in Table 1.
Recovery Rate Recovery from PBS (%)Recovery from PB (%)SKBR368.051MDA-MB-45361.270.6T47D28.911.4MCF794.024.4BT47497.147.6ZR75152.850.8SUM1496372.9SUM19048.358.3MDA-MB-2310.927.04Hs578T0.250.84Recovery rate of immunomagnetic enrichment/fluorescence activated cell sorting of cell lines spiked into saline (PBS) or peripheral blood (PB)
Conclusions: Significant variation occurred in recovery rates of spiked, sorted cells depending on cell line type. Negative control PB specimens from healthy individuals could successfully define a consistent gating strategy that captured zero CTCs from healthy individuals and permitted acquiring a portion of CTCs from each of the 10 cell lines. Further experiments will characterize the gene expression of the sorted cells compared to bulk RNA for each cell line. Better biomarkers are needed to improve upon the recovery rates of CTCs while minimizing selection bias. Future studies are required to determine if expression profiling of CTCs is an informative biomarker that may be applied clinically as a prognostic or predictive tool.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-11.