Epitope mapping of epidermal growth factor receptor (EGFR) in lung cancer using immunohistochemistry: Fine tuning the protocols

Abstract

21162 Background: Until now, immunohistochemistry was not able to become a reliable diagnostic approach for EGFR targeted therapies. The golden standard of the determination of EGFR protein expression in paraffin embedded cancer tissues is the EGFRpharmDXtm kit. Methods: Here we show data based on analysis of 110 lung adenocarcinomas, that the recommended protocol may not be optimal for ideal performance of the immunodetection, since microwave retrieval, extended primary antibody-incubation time and replacement of the developer reagent converted four EGFR-negative tumor into EGFR protein positive out of eight lung adenocarcinoma cases. Protocol modification improved the performance of another widely used EGFR-kit, Ventana's CONFIRM, where replacement of the protease antigen retrieval with microwave cooking converted several EGFR-negative tumors to strongly positive. Meanwhile both EGFR-kits detect EGFR expression (juxtamembrane domain) but do not provide information on the expression of epitopes critical from the point of view of targeted therapy. Results: We have developed two protocols, which can detect the ligand-binding (AB-10, BioMarkers) and C-terminal (AB- 335, Biogenex) cytoplasmic domains of the EGFR protein in paraffin embedded lung cancer tissues. We have shown, based on the analysis of more than 110 lung adenocarcinoma tissues, that the ligand binding domain of EGFR is rarely expressed while the C-terminal domain is ubiquitously expressed in EGFR-PharmDX and CONFIRM-positive cancers. The biological activity of EGFR can be characterized either by autophosphorylation of the receptor or by detection of divers phosphorylated downstream signaling components. We have found that unlike p1086 (detected by a Zymed antibody), the p1173 site of EGFR (identified by a rabbit monoclonal of Epitomics) can be detected 27/110 paraffin embedded lung adenocarcinomas. Conclusions: Using the tested antibody panel we can reliably determine the EGFR protein expression in paraffin embedded (lung)cancer tissues. This work was supported by Ministry of Education (NKFP1a-0024–05). No significant financial relationships to disclose.