PURPOSE

Recognition that the epidermal growth factor receptor (EGFR) was a therapeutic target in non-small cell lung cancer (NSCLC) and other cancers led to development of the small-molecule receptor tyrosine kinase inhibitors gefitinib and erlotinib. Clinical trials established that EGFR tyrosine kinase inhibitors produced objective responses in a minority of NSCLC patients. We examined the sensitivity of 23 NSCLC lines with wild-type or mutated EGFR to gefitinib to determine genes/proteins related to sensitivity, including EGFR and HER2 cell surface expression, phosphorylated EGFR expression, EGFR gene copy number, and EGFR mutational status. Downstream cell cycle and signaling events were compared with growth-inhibitory effects.

EXPERIMENTAL DESIGN

We determined gefitinib sensitivity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, EGFR expression by fluorescence-activated cell sorting and immunohistochemistry, phosphorylated EGFR by Western blotting, EGFR gene copy number by fluorescence in situ hybridization, and EGFR mutation by sequencing. The cellular effects of gefitinib on cell cycle were determined by flow cytometry and the molecular effects of gefitinib EGFR inhibition on downstream signal proteins by Western blotting. Gefitinib in vivo effects were evaluated in athymic nude mice bearing sensitive and resistant NSCLC xenografts.

RESULTS

There was a significant correlation between EGFR gene copy number, EGFR gene mutations, and gefitinib sensitivity. EGFR protein was necessary but not sufficient for predicting sensitivity. Gefitinib-sensitive lines showed a G(1) cell cycle arrest and inactivation of downstream signaling proteins; resistant cell lines had no changes. The in vivo effects mirrored the in vitro effects.

CONCLUSIONS

This panel of NSCLC lines characterized for gefitinib response was used to identify predictive molecular markers of response to gefitinib. Several of these have subsequently been shown to identify NSCLC patients likely to benefit from gefitinib therapy.