Abstract 2507: Relationship between cell surface expression level of EGF-R and cetuximab-mediated ADCC activity in human colorectal cancer cells

Abstract

Abstract Background: Cetuximab is a monoclonal antibody approved in the patients with metastatic colorectal cancer expressing epidermal growth factor receptor (EGF-R). It is considered that the proposed mechanism of action includes antibody-dependent cell mediated cytotoxicity (ADCC) by triggering FcαR on natural killer cells. However, it is unclear whether ADCC correlates with EGF-R expression and/or the mutational status of downstream effectors such as K-RAS, B-RAF, PI3K, and so on in colorectal cancer. Aim: The aim of this study is to investigate whether ADCC activities correlate with cell surface expression levels of EGF-R, and/or mutational status of K-RAS, B-RAF, and PI3K in human colorectal cancer cell lines. Methods: Five human colorectal cancer cell lines (HT29, HCT8, HCT116, DLD-1, SW480) and A431 (positive control) with different K-RAS, B-RAF, and PI3K mutational status were evaluated for: (i) cell surface expression level of EGF-R, (ii) cetuximab mediated ADCC activity. Cell surface expression of EGF-R was quantified using a flow cytometric analysis. Human peripheral blood mononuclear cells (hPBMC) were prepared from healthy donors and were isolated from heparinised peripheral blood on a Ficoll gradient. Cell lines were used to establish the assay condition as follows: (i) Cell line was incubated with cetuximab at various concentrations from 0 to 100 μg/ml as comparable experiments, (ii) hPBMC were added as effector cells at different effector:target (E:T) ratio, (iii) The cells were used as targets in a standard lactate dehydrogenase release assay to measure ADCC activity (Cytotox 96: Promega Corporation, WI, USA) as per the manufacturer's instruction. The correlation between the cell surface expression levels of EGF-R and ADCC activities were calculated using univariate regression analysis. Results: Cell surface expression levels of EGF-R in the HCT8, DLD-1, SW480, and A431 cell lines were high, whereas those of HT29 and HCT116 were low. The highest ADCC activity was detected in each cell line with the cetuximab concentration of 100 μg/ml at an E:T ratio of 20:1. A strong correlation was observed between the cell surface expression level of EGF-R and ADCC activity (correlation coefficient: 0.930 (P < 0.001)). There was no significant correlation between mutational status of K-RAS, B-RAF, and PI3K and ADCC activity in colorectal cancer cell lines. Conclusions: It was suggested that cetuximab mediated ADCC activity was correlated with cell surface expression level of EGF-R, but not with mutational status of K-RAS, B-RAF, and PI3K in human colorectal cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2507. doi:1538-7445.AM2012-2507