Introduction: Circulating tumor cells (CTCs) represent a potent opportunity to glean important information about in vivo breast cancer biology using a non-invasive technique. However, EpCAM-based techniques are significantly limited by the presence of marker-negative CTCs, a phenotype that is enriched in the CTCs of metastatic triple-negative breast cancers (mTNBCs). This interim analysis is part of an exploratory study to investigate the utility of supplementing EpCAM with additional markers specifically relevant to mTNBC for more complete CTC enumeration.

Methods: Marker candidates used in this analysis were: (1) previously associated with TNBC, (2) exclusively surface markers to avoid permeabilization, (3) not associated with leukocytes or endothelial cells, and (4) targetable with commercially available antibodies. TNBC cell lines were characterized across the selected markers by flow cytometry. Immunomagnetic enrichment (IE) of cell lines from culture medium was conducted using antibodies conjugated to magnetic beads in concert with a rare cell detection system developed by the Savran Research Group at Purdue University. The recruitment goals for the study stand at twenty healthy individuals and twenty individuals with mTNBC. All blood samples were collected in CellSave tubes and were assessed using four-marker IE with a parallel monochromatic cross-stain. Anti-CD45 and Hoechst stain were incorporated into the cross-stain in standard fashion. Samples from individuals with mTNBC were also subjected to standard EpCAM-based CTC detection on the same device, which served as a pairwise baseline comparator.

Results: Surface expression of four markers, including TROP2, N-Cadherin, EGFR, and EpCAM, was assessed across eleven TNBC cell lines. All cell lines were positive for at least one of four markers in the panel. EpCAM-negative cell lines (4/11) were positive for N-Cadherin and/or EGFR. Unique to these cell lines, four-marker IE was significantly more efficient than EpCAM alone (p=0.0006). As of November 2018, ten normal and six mTNBC participants have been recruited. All blood samples were able to be analyzed. Healthy participants yielded 0 CTCs, with the exception of one participant, in which a single positive cell was found. CTCs were detected in 50% and 33% of participants with mTNBC when utilizing the experimental method vs. EpCAM-directed detection. Futher, in 100% (3/3) of cases in which CTCs were detected, the four-marker system detected more CTCs than the EpCAM-based comparator.

Discussion: Compared to EpCAM-based capture, the four-marker experimental system presented here has potential to enhance CTC analysis by more completely representing the heterogeneity of TNBC. This results in better overall capture efficiency and enumeration while still maintaining sufficient specificity. The parent analysis is expected to be completed in the summer of 2019.

Citation Format: Bradley Hancock, Chun-Li Chang, Yuan Zhong, Yu-Hsiang Chen, Jeffrey Solzak, Casey Bales, Emily Nelson, Robin Paul, Cagri Savran, Milan Radovich. 4-marker positive selection system for improved CTC analysis in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1354.