Abstract 5195: Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer
Authors: Zhang Qiang , Zhang Youbin , Flaum Lisa , Gerratana Lorenzo , Gradishar William , Platanias Leonidas , Cristofanilli Massimo 
Published: August 2018

Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease.

Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics.

Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had &gt;5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P&lt;0.01). The highest expression of HER-2 was found in Group 3 (66.67% samples are positive, and 120 positive cells/each sample). This value was significantly higher than Group 2 (27.27% samples are positive, 24.33 positive cells/each sample) and Group 1 (20.37% samples are positive, 6.18 positive cells/each sample) (P&lt;0.001). The results indicated that high level of CTCs clusters strongly associated with over expression of HER2 in CTCs.

Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (&gt; 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic.

Citation Format: Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5195.