Abstract 408: Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC)
Authors: Zhang Qiang , Gerratana Lorezo , Shah Ami N. , Davis Andrew A. , Flaum Lisa , Zhang Youbin , Pestell Richard G. , Wehbe Firas , Behdad Amir , Platanias Leonidas , Gradishar William , Cristofanilli Massimo 
Published: August 2019

Abstract
Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs.
Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics.
Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs.
Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis.
Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) [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 408.