Abstract 992: Normalized EGFR activation assessment in FFPE tissue sections using quantitative immunofluorescence
Authors: Mansfield James , Wetzel Randy , Hoyt Clifford 
Published: June 2012

Abstract
A wide variety of normal and neoplastic tissues express EGFR, and its overexpression has been detected in a number of human tumors including breast, lung, head and neck, glioblastoma multiforme, and colorectal carcinomas, to name a few. In addition, there are now several EGFR inhibitors- Herceptin (Trastuzumab), Erbitux (IMC-C225, cetuximab), Tarceva (OSI-774, erlotinib), Iressa (ZD 1839), Maztuzumab (EMD 72000) – which exhibit anti-cancer activity and are being used in clinical practice for the tumors of breast, colon, head and neck, lung, and others, although the precise mechanism by which EGFR inhibitors exert their anti-cancer effect remains unknown. However, because EGFR has not proven to be a useful prognostic/predictive marker of clinical response to EGFR-targeted therapies, other prognostic/predictive markers have been proposed, including the activated form of EGFR (pEGFR). Previously, EGFR phosphorylation was found to be associated with poor prognosis in non-small-cell lung cancer patients and was suggested to be an important predictor of clinical outcome.
To address the need to assess EGFR activation is a range of FFPE tissue sections, we have developed a method for measuring the normalized activation of EGFR, which incorporates a multilabel immunofluorescence reagent kit for EGFR, pEGFR and pERK, a multispectral imaging approach to provide quantitative imaging data and an automated morphologic image analysis method which returns “per-cell” EGFR activation, determined by normalizing pEGFR and pERK fluorescence intensity levels with total EGFR signal on a per cell basis. Cells analyzed are from particular morphologic regions (typically the tumor) detected automatically with pattern-recognition-based image analysis algorithms. This methodology was applied to tumor xenografts generated from four lung cancer cell lines (H1703, H1975, H3255, and Kyse450) each exhibiting different levels of activation.
Developing a multiplexed assessment methodology requires three components to come together to be effective: 1) a multilablel staining protocols that provide specific, balanced and robust signals; 2) a multispectral imaging methodology that is capable of removing the interference of tissue autofluorescence and residual cross-talk between the fluorophores; and 3) an image analysis methodology that is able to determine the per-cell intensity values of each fluorophore from cells of interest in the tumor.
Signals agreed measured in tissue sections of xenografts agree with expected relative expression as compared to cell lines in culture, with one exception which expressed stronger than expected. Data from cells grown in culture and from xenografts will be presented. This is the first demonstration of normalized EGFR activation assessment using multicolour immunofluorescence labelling in FFPE sections.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2011-992