Abstract 2786: EGFR expression and phosphorylation in HNSCC predict response to EGFR inhibition but cell lines are not representative for the clinical situation
Authors: Ahsen Oliver von , Khaznadar Sami S. , Khan Martin 
Published: July 2017

Abstract
Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. Survival rates have not been improved for decades and conventional therapy is effective in only 50% of the patients. Based on broad expression in up to 80-90% of the HNSCC cases, epidermal growth factor receptor emerged as drug target but clinical efficacy of EGFR inhibitors in HNSCC is very limited. We therefore reinvestigated the EGFR expression levels necessary for response in cell lines and clinical samples.
Methods Standard procedures were used for IHC. The antibody clone D38B1 was used in 1:900 dilution for 2h at RT. Stainings were performed using the DAKO Envision system. EGFR expression and phosphorylation of Tyrosine-1173 were analysed by MSD (Mesoscale Discovery) in lysates from fresh frozen tumor or exponentially growing cells. For proliferation assays, 2000 cells per well were grown for 24 h before addition of inhibitors. Cell culture was continued for 72 h before testing viability using the CellTiter-Glo® Assay.
Results The majority (11/13) of HNSCC cell lines responded to the EGFR inhibitor Erlotinib. EGFR was highly expressed and phosphorylated in the Erlotinib responsive cell lines. Resistant cell lines displayed low level EGFR expression and phosphorylation. However, EGFR expression and phosphorylation in treatment naive clinical samples were significantly below the levels found in responding cell lines. In clinical samples EGFR was not overexpressed on the cellular level. Based on these findings, a clinical response to Erlotinib in HNSCC would not be expected.
Conclusion The prognostic value of EGFR expression has been used to argue for EGFR as a relevant target in HNSCC. Although most reviews claim that EGFR is overexpressed in HNSCC, clear data supporting this position are missing. Early studies tested the RNA levels and found the EGFR expression in tumors higher compared to control tissues. Studies using IHC assessed the association of EGFR expression with disease progression, but no comparison to expression in normal mucosa was described. Overexpression was based on percentage of positive cells not on the intensity of expression. We show similar levels of EGFR expression in growing keratinocytes and tumor cells. The often described overexpression only originated from a larger number of EGFR positive cells, not on overexpression on the cellular level. The high expression and functional relevance of EGFR in cell lines proves that EGFR activity is required for survival in cell culture. Our findings lead to the conclusion that this is not representative of the clinical situation. Definition of a response threshold for EGFR expression and clinical verification of this expression level is mandatory for the successful use of a predictive biomarker.
Citation Format: Oliver von Ahsen, Sami S. Khaznadar, Martin Khan. EGFR expression and phosphorylation in HNSCC predict response to EGFR inhibition but cell lines are not representative for the clinical situation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2786. doi:10.1158/1538-7445.AM2017-2786