Abstract 3371: Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma
Authors: Baysal Hasan , De Pauw Ines , Zaryouh Hannah , Peeters Marc , Vermorken Jan Baptist , Smits Evelien , Lardon Filip , Jacobs Julie , Wouters An 
Published: September 2020

Abstract
The epidermal growth factor receptor (EGFR)-targeted therapies and immunotherapies are now at the forefront for treatment of head and neck squamous cell carcinoma (HNSCC). However, resistance to cetuximab, an anti-EGFR monoclonal antibody (mAb), is a major clinical problem, limiting the clinical benefit of treatment. Besides inhibition of downstream signalling, cetuximab can also induce antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to investigate the role of baseline EGFR expression and EGFR internalization following treatment with cetuximab as possible mechanisms that may explain differences in cetuximab-induced ADCC between HNSCC cell lines.
Ten HNSCC cell lines, comprising cetuximab sensitive as well as intrinsically and acquired resistant cell lines, differing in HPV-status, were used. ADCC was assessed using the xCELLigence RTCA system, by co-culturing HNSCC cells with natural killer cells from healthy volunteers (E:T; 5:1). Baseline EGFR expression was flow cytometrically determined, following staining with an EGFR PE-conjugated antibody or a PE-conjugated isotype control. Experiments were performed under normoxic and hypoxic (1% O2) conditions. To determine EGFR internalization, cetuximab and the corresponding isotype control were stained with FabFluor-pH reagent prior to incubation with HNSCC cells. Phase-contrast and red fluorescence kinetics were quantified using the IncuCyte Zoom.
We demonstrate that cetuximab-induced ADCC was highest in intrinsically cetuximab resistant cell lines compared to the acquired resistant and cetuximab sensitive cell lines. Under hypoxic conditions, a reduced ADCC was observed in the cetuximab sensitive and acquired resistant HNSCC cell lines but not in the intrinsically resistant cell lines. Baseline EGFR was expressed in a cell line-dependent manner and was not correlated with cetuximab sensitivity or HPV-status. Moreover, there was no correlation between baseline EGFR expression and the amount of cetuximab-induced ADCC. Importantly, the intrinsically cetuximab resistant cells demonstrated the highest level of EGFR internalization compared to the acquired resistant and cetuximab sensitive cell lines. Correlation analysis showed that the degree of EGFR internalization positively corelated with cetuximab-induced ADCC.
In conclusion, we showed that induction of ADCC is maintained, independent of EGFR expression. Furthermore, internalization of EGFR might play a role in cetuximab resistance and ADCC-induction. Future research further unravelling this mechanism will provide a better understanding of mAb-based anti-EGFR therapies in the future.
Citation Format: Hasan Baysal, Ines De Pauw, Hannah Zaryouh, Marc Peeters, Jan Baptist Vermorken, Evelien Smits, Filip Lardon, Julie Jacobs, An Wouters. Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3371.