Epidermal growth factor receptor dynamics influences response to epidermal growth factor receptor targeted agents.

Analysis of gene expression of cancer cell lines exposed to erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), showed a marked increase in EGFR mRNA in resistant cell lines but not in susceptible ones. Because cetuximab induces EGFR down-regulation, we explored the hypothesis that treatment with cetuximab would interfere with erlotinib-induced EGFR up-regulation and result in antitumor effects. Exposure of the resistant biliary tract cancer cell line HuCCT1 but not the susceptible A431 epidermoid cell line to erlotinib induced EGFR mRNA and protein expression. Combined treatment with cetuximab blunted the erlotinib-induced EGFR up-regulation and resulted in inhibition of cell proliferation and apoptosis in the HuCCT1 cells. Blockage of erlotinib-induced EGFR synthesis in HuCCT1 cells by small interfering RNA resulted in identical antitumor effects as cetuximab, providing mechanistic specificity. In mice xenografted with A431, HuCCT1, and the pancreatic cancer cell line Panc430, maximal growth arrest and decrease in Ki67 proliferation index were documented with combined therapy, and EGFR down-regulation was observed in cetuximab-treated tumors. These results may indicate that resistance to EGFR kinase inhibition may be, at least in part, mediated by a highly dynamic feedback loop consisting of up-regulation of the EGFR upon exposure to EGFR kinase inhibitors. Abrogation of this response by small interfering RNA-mediated EGFR mRNA down-regulation and/or by cetuximab-mediated protein clearance induced tumor arrest across several cancer models with different EGFR expression levels, suggesting that resistance and sensitivity are dynamic events where proportional decrease in the target rather than absolute content dictates outcome.

An epidermal growth factor receptor intron 1 polymorphism mediates response to epidermal growth factor receptor inhibitors.

This study tested the hypothesis that the number of CA single sequence repeat (CA-SSR) in the intron 1 of the epidermal growth factor receptor (egfr) gene, which affects transcription efficiency of the gene, is associated with the response to EGFR inhibitors. To this end, we determined the number of CA dinucleotides in the intron 1 of the egfr gene in a panel of 12 head and neck cancer cell lines that lack egfr gene amplification and measured the expression of EGFR (mRNA and protein), as well as response to EGFR inhibition. Cells with lower number of CA dinucleotides in the CA-SSR had higher expression of the EGFR gene and protein and were more sensitive to the inhibitory effects of erlotinib, a small molecule inhibitor of the EGFR tyrosine-kinase. Phenotypic modification by silencing EGFR mRNA expression in a susceptible cell line induced resistance to the drug. The number of CA dinucleotide was equivalent in genomic and tumor DNA obtained from 30 patients with head and neck cancer. In a clinical study in colorectal cancer, subjects with lower number of CA dinucleotide frequently developed skin toxicity, a feature that is related to the antitumor activity of this class of drugs. These results suggest that polymorphic variations in the intron 1 of the egfr gene is associated with response to EGFR inhibitors and may provide an explanation as to why the development of skin toxicity is associated with a favorable outcome in patients treated with these agents.

Assessment of Epidermal Growth Factor Receptor (EGFR) signaling in paired colorectal cancer and normal colon tissue samples using computer-aided immunohistochemical analysis.

The Epidermal Growth Factor Receptor (EGFR) plays a role in multiple tumor cell processes and is targeted by several anticancer therapies. Although EGFR mutations may determine tumor susceptibility in a small proportion of patients, knowledge of the EGFR signaling pathway status in tumors may help guide further drug development and hypothesis-driven combination studies. We aimed to validate and apply a novel computer-aided immunohistochemical (IHC) technique to characterize the status of EGFR signaling in matched colorectal tumor and normal colon tissue samples. Tissue Microarrays (TMA)were made from both cancerous and normal colorectal tissue in 18 patients and stained with antibodies against EGFR, phospho-EGFR (pEGFR), Akt, pAkt, MAPK, and pMAPK. TMA's were quantitatively scored using the Automated Cellular Imaging System (ACIS II, Chromavision, Inc). ACIS was compared against cell line Western blotting, ELISA, and visual scoring (0-3+) by a pathologist. We found that ACIS analysis was highly reproducible and results were well correlated with other techniques. A post-scan "image microdissection" technique of analyzing heterogeneous human samples showed good correlation between paired human samples [Pearson correlation for tumors, 0.922 (p < .001)]. Cancer samples had markedly higher staining of pEGFR, Akt, pAkt, MAPK, and pMAPK. We conclude that ACIS IHC of human tissue samples is quantitative, reproducible, and correlates with Western blots and ELISA in cell line pellets as well as pathologist's scores of human samples. Colorectal tumors show higher staining of pEGFR and downstream effectors compared to matched normal colorectal tissues.