Gefitinib (Iressa) and erlotinib (Tarceva), which target the epidermal growth factor receptor (EGFR), are approved for treatment of patients with advanced non-small cell lung cancer (NSCLC). Patients whose tumors harbor mutations in the EGFR gene, including delE746-A750 in exon 19 and L858R in exon 21, may benefit in particular from gefitinib treatment. However, acquired resistance to gefitinib has been a serious clinical problem, and further optimization is needed for application of EGFR-targeted drugs in lung cancer patients. In this study, we established gefitinib-resistant NSCLC cells from PC-9 cell line, which harbors the delE746-A750 mutation, by exposing the cell line to gefitinib for over 7 months. Gefitinib-resistant PC-9/GEFs cell lines showed a marked downregulation of PTEN expression and increased Akt phosphorylation. In revertant, gefitinibsensitive clones (PC-9/Rev) derived from PC-9/GEF1-1 and PC-9/GEF2-1, PTEN expression, as well as sensitivity to gefitinib and erlotinib, was restored. Knockdown of PTEN expression using small interfering RNA specific for PTEN in PC-9 cells resulted in drug resistance to gefitinib and erlotinib. Nuclear translocation of the EGR1 transcription factor, which regulates PTEN expression, was shown to be suppressed in resistant clones and restored in their revertant clones. Reduced PTEN expression was also seen in tumor samples from a patient with gefitinib-refractory NSCLC. This study thus strongly suggests that loss of PTEN expression contributes to gefitinib and erlotinib resistance in NSCLC. Our findings reinforce the therapeutic importance of PTEN expression in the treatment of NSCLC with EGFR-targeted drugs. Cancer Res; 70(21); 8715-25. ©2010 AACR.

Purpose: Therapeutic responses of non-small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for ∼90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of ∼90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC.Experimental Design: We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing.Results: We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti-delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry.Conclusions: This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFRtargeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics.