Abstract P3-01-20: Proof-of-concept of a 4-marker system for improved CTC analysis of metastatic triple-negative breast cancer
Authors: Hancock BA , Chang C-L , Zhong Y , Solzak JP , Chen Y-H , Savran C , Radovich M 
Published: May 2019

Abstract
Introduction: Circulating tumor cells (CTCs) represent a potent opportunity to glean important information about in vivo breast cancer biology in a non-invasive fashion. A current limitation to CTC analysis is the inability of positive-selection systems to capture EpCAM-low/negative CTCs, a phenotype that is enriched in the CTCs of metastatic triple negative breast cancers (mTNBCs). This proof-of-concept study aims to increase CTC capture for downstream molecular analysis though the inclusion of additional markers specifically relevant to TNBC.
Methods: For inclusion in the analysis, marker candidates were: (1) sufficiently characterized in TNBC, (2) exclusively surface markers to avoid permeabilization, (3) not reported on leukocytes if a cluster of differentiation (CD) nomenclature was associated, and (4) targetable with commercially available antibodies. Cell lines were purchased as part of the TNBC Panel 3 (ATCC) and were characterized as positive or negative across the selected markers by flow cytometry. Capture efficiency of cell lines from culture medium was conducted using antibodies conjugated to magnetic beads in concert with an immunomagnetic detection system developed by the Savran Research Group at Purdue University. Comparisons to EpCAM-only based detection in capture efficiency experiments were completed using Student's t-test. EDTA-anticoagulated blood drawn from normal subjects was assessed for CTCs using the 4-marker capture and parallel 4-marker fluorescent cross-stain. This study was approved by the Institutional Review Board at Indiana University.
Results: We assessed surface expression of 4 markers (TROP2, N-Cadherin, EGFR, EpCAM) across 11 TNBC cell lines using flow cytometry. 100% of cell lines were positive for at least 1 of 4 markers in the panel. 7 of 11 cell lines were characterized by EpCAM positivity. The remaining 4 EpCAM-negative cell lines were positive for N-Cadherin, EGFR, or both in the absence of EpCAM. Immunomagnetic capture experiments performed across the 4 EpCAM-negative cell lines revealed a significant increase in capture efficiency yielded by the 4-marker panel as compared to EpCAM-only capture (p=0.0006). Capture efficiency for EpCAM-positive cell lines with the 4-marker panel was equivalent to EpCAM-only. The 4-marker panel was highly specific as CTC assessment of blood samples collected from 5 normal women without cancer yielded 0 positive cells.
Discussion: Compared to EpCAM-only based capture, the 4-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, while still maintaining sufficient specificity. The primary limitation of our study is that in vivo characterization of the system is incomplete. To address this, a clinical protocol for the performance assessment of this method compared to EpCAM-based detection in patients with mTNBC has been initiated at Indiana University and will begin enrolling patients in July 2018.
Citation Format: Hancock BA, Chang C-L, Zhong Y, Solzak JP, Chen Y-H, Savran C, Radovich M. Proof-of-concept of a 4-marker system for improved CTC analysis of metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-20.