EGFR genetic heterogeneity of nonsmall cell lung cancers contributing to acquired gefitinib resistance.

Gefitinib is dramatically effective for nonsmall cell lung cancers (NSCLCs) with activating mutations of the epidermal growth factor receptor (EGFR) gene, but these tumors eventually develop drug resistance, attributable to a secondary T790M mutation or acquired MET amplification in some relapsed tumors. We analyzed EGFR mutations in matched pre- and post-therapeutic tumors of 6 gefitinib-responding lung cancers. With conventional PCR-based sequencing, classic mutations were detected in pretreatment samples of each case. The same mutations were readily confirmed in treated lesions of 4 cases, but were absent in those of Cases 1 and 2. Subsequent mutant-enriched peptide-nucleic-acid-mediated PCR clamping and subcloning assays detected the mutation in minor cells of treated lesions of Case 1, but still failed to detect a mutation in Case 2. We thus performed microdissection-based cell cluster mutation analysis of pretreatment tumors, and found that 3, including the first 2, concurrently contained tumor cells with either mutant or wild-type EGFR, although the latter was only a minor fraction. These findings suggest that some NSCLCs are genetically heterogeneous with regard to EGFR mutations; gefitinib-sensitive mutants decrease or vanish while wild clones selectively survive with gefitinib treatment. In addition, T790M was detected in a small fraction of treated lesions of 3 cases, and MET amplification was revealed in 3 treated tumors of Case 2. Thus, our results suggest that multiple mechanisms underlie acquired gefitinib resistance, and selection on a background of EGFR genetic heterogeneity also contributes to acquisition of resistance in a proportion of NSCLCs. (c) 2008 Wiley-Liss, Inc.