Abstract P4-01-16: 4-marker positive selection system for improved circulating tumor cell analysis of metastatic triple-negative breast cancer
Authors: Hancock Bradley A. , Zhong Yuan , Chang Chun-Li , Chen Yu-Hsiang , Solzak Jeffrey P. , Bales Casey L. , Nelson Emily M. , Savran Cagri , Radovich Milan 
Published: March 2020

Abstract
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 analysis is 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 20 healthy individuals with no history of cancer and 10 individuals with mTNBC. All blood samples were collected in CellSave tubes and were assessed using 4-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 4 markers, including TROP2, N-Cadherin, EGFR, and EpCAM, was assessed across eleven TNBC cell lines. All cell lines were positive for at least 1 of 4 markers in the panel. EpCAM-negative cell lines (4/11) were positive for N-Cadherin and/or EGFR. Unique to these cell lines, 4-marker IE was significantly more efficient than EpCAM alone (p=0.0006). The recruitment goals for the study have been reached. 39/40 (98%) of blood samples yieded successful analyses. 0 positive cells were detected from 18/20 healthy subjects, and 1 positive cell was detected in the remaining 2. 2 or more positive CTCs were detected in 4/10 (40%) participants with mTNBC when utilizing the experimental method, and 3/10 (30%) with EpCAM-directed detection. Futher, in 4 of 5 (80%) of cases in which CTCs were detected using either method, the 4-marker panel detected more cells compared to the EpCAM-based approach.
Discussion: Capturing the heterogeneity of CTCs in mTNBC by supplementing EpCAM-based analysis with additional markers is likely a meritorious approach. In this study gains in sensitivity were achieved while maintaining sufficient specificity using a multi-marker based positive-selection microfluidic device. Downstream studies focused on larger cohorts of patients are warranted.
Citation Format: Bradley A. Hancock, Yuan Zhong, Chun-Li Chang, Yu-Hsiang Chen, Jeffrey P. Solzak, Casey L. Bales, Emily M. Nelson, Cagri Savran, Milan Radovich. 4-marker positive selection system for improved circulating tumor cell analysis of metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-16.