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Updated: Sep 20, 2026
Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Published on: September 25, 2011
Color multiplex polymerase chain reaction for quantitative analysis of epidermal growth factor receptor genes in
Lester J Layfield1, Philip S Bernard, Neil S Goldstein
1Department of Pathology, University of Utah School of Medicine, and ARUP Laboratories, Inc., Salt Lake City, Utah 84132, USA. layfiel@aruplab.com
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.
Insights
Epidermal growth factor receptor (EGFR) protein is overexpressed in many colon cancers, but this is not due to gene amplification. This finding is crucial for developing targeted antibody therapies for colorectal adenocarcinoma.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) is a cell surface receptor implicated in cancer growth.
- Elevated EGFR levels are associated with advanced colorectal adenocarcinoma.
- Overexpression mechanisms include gene amplification, transcriptional upregulation, or decreased protein degradation.
Purpose of the Study:
- To investigate the relationship between EGFR gene amplification and EGFR protein overexpression in colonic adenocarcinomas.
- To determine if EGFR gene amplification is a common mechanism for EGFR overexpression in this cancer type.
Main Methods:
- Analyzed 36 colonic adenocarcinoma specimens for EGFR gene amplification using quantitative real-time PCR.
- Compared EGFR gene copy number to albumin gene copy number in tumor samples.
- Defined EGFR overexpression as >10% of neoplastic cells staining at 2+ or 3+ level.
Main Results:
- Gene amplification data were obtained for 31 of 36 samples.
- The average EGFR/albumin gene copy number ratio in tumors was 1.13 +/- 0.55.
- This ratio was similar to normal leukocyte samples, indicating no significant gene amplification.
Conclusions:
- EGFR protein is frequently overexpressed in colonic adenocarcinomas.
- EGFR overexpression can serve as a target for specific antibody therapies.
- Unlike Her-2/neu, EGFR overexpression in colorectal adenocarcinoma is generally not driven by gene amplification.
