Color multiplex polymerase chain reaction for quantitative analysis of epidermal growth factor receptor genes in colorectal adenocarcinoma

Abstract

Background and objectives: Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane cell surface receptor, which belongs to the c-erbB family of tyrosine kinases. Chimeric anti-EGFR monoclonal antibody is an investigational therapy for advanced adenocarcinoma of the colon. Anti-EGFR is believed to be most effective against those neoplasms with elevated EGFR levels. Possible mechanisms for over expression of EGFR include gene amplification, transcriptional upregulation, or decreased degredation of the EGFR protein.

Methods: We analyzed a series of 36 specimens of colonic adenocarcinoma with known levels of EGFR protein expression for amplification of the gene sequence of EGFR. Carcinomas were considered positive for EGFR expression when greater than 10% of the neoplastic cells stained at a level of 2+ or 3+. Foci of adenocarcinoma were microdissected from paraffin sections and quantitative real-time PCR (polymerase chain reaction) performed using a thermal cycler with real time fluorescence capability (Light cycler(TM), Roche Diagnostics, Indianapolis, IN). A relative quantitation assay comparing the EGFR gene to the control albumin gene was performed by 2-color multiplexing.

Results: Usable data on gene amplification status were obtained in 31 of the 36 samples. The average EGFR/albumin gene copy number ratio for the 31 samples of colon adenocarcinoma in which PCR results were obtained was 1.13 +/- 0.55 with a range of 0.26-2.29. The average EGFR/albumin gene copy number ratio obtained for 16 normal DNA leukocyte samples used to establish the efficiency curves was 1.03 +/- 0.31 with a range of 0.49-1.51.

Conclusions: EGFR protein is overexpressed in a significant percentage of colonic adenocarcinomas. As with Her-2/neu overexpression, overexpression of EGFR serves as a basis for specific antibody therapy in a subset of carcinomas. Unlike Her-2/neu, where most overexpression is secondary to gene amplification, overexpression of EGFR appears to be unrelated to gene amplification.