Activated Epidermal Growth Factor Receptor: Studies in head and neck tumors and tumor cell lines after exposure to ligand and receptor tirosine-kinase inhibitors

J. Baselga, M.A. Cañadas, J. Codony, J.A. Hueto, A. Arcas, A. Lladó, X. Puig, M. Guix, G. Raspall, J. Albanell.

The epidermal growth factor receptor (EGFr) is frequently overexpressed in head and neck tumors. Therapeutic strategies directed at preventing or reversing activation of the EGFr are being studied in the clinic. In this setting, assays to determinate the level of EGRr activation prior and during therapy could be instrumental to find the optimal biological dose of anti-receptor therapies. We have studied in head and neck tumors and cell lines the presence of activated EGFr with an antibody (EGFr-act, Transduction Labs.) that specifically binds to the activated EGFr. Twenty one squamous head and neck frozen tumors were studied immunohistochemically with EFRr-act. And 8 (38%) of them showed a positive staining. The intensity of staining was mild in 2 tumors, moderate in 4, and strong in 2. These intensities paralleled the EGFRr staining with an antibody that reatcs with EGFRr independently of this activation. The specificity of EFRr-act. for activated EFRr was confirmed by western-blot analysis using both EGFr-act and an anti-phosphotyrosine antibody (4G10) on the same tumors. In parallel, the ability of EGFr-act. antibody to detect inhibition of EGFRr phosphorylation induced by an EGFr-tyrosine inhibitor (EGFr/TKI) was analyzed in human tumor cells. In EGFr overexpressing A431 and DiFi cancer cells, ligand-induced EGFr phosphorylation results in intense staining by EGFr-act. In A431 cells, treatment with the EGFr/TKI PD153035, at dose of 1 mM that results in inhibition of cell growth (Clin Cancer Res 1997;3 (11):2009), resulted in a complete loss of reactivity to EGFr-act., both by immunocytochemistry and western-blot analysis. In summary, EGFr is bassally activated in head and neck tumors and can be detected by immunohistochemistry and western blotting with the monoclonal antibody EGFr-act. In tumor cell lines the intensity of staining with EGFr-act. correlates with EGFr activation. Our findings suggest that it may be feasible to determinate the presence of activated EGFr and its inhibition by anti-EGFr therapies in head and neck tumors.

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Atlanta, USA, 15-18 Mayo 1999