P2.03-17 EGFR T790M Mutation may not be Generated through Selection by EGFR-TKI from Randomly Occurring Mutations in Vitro Using ENU

Abstract

It is postulated that T790M resistant mutation is a result of selection by EGFR-TKI from clones that have randomly generated mutations. If so, it would be possible to detect the process of convergence from random mutations to T790M, depending on exposure time for EGFR-TKI. After exposure of Ba/F3 cells expressing EGFR Del19 to ENU (N-ethyl-N-nitrosourea) for 24 hours, they were cultured with various concentrations of 3 EGFR-TKIs at trough concentrations (Fig.). At 1, 6, 12, 24, and 48 hours, cell viability was evaluated and single cell cloning was performed. EGFR exons 18 to 21 were analyzed by Sanger sequencing. Percentages of viable cells exposed to TKI were approximately 80%, 50%, < 20%, and < 3% at 1, 6, 12, 48 hours, respectively. These ratios were almost similar among 3 TKIs. Of 50 clones that had survived TKI treatment, T790M was detected in 12 (24%) (Fig.). Notably, two were detected in cells exposed to erlotinib or afatinib only for one hour (Fig.). In addition, we found 2 G873E (c.2876G>A) mutations, known to be oncogenic, with the same TKI treatment. In contrast, 36 clones (72%) with TKI or additional 19 clones without TKIs that had been exposed to only ENU did not harbor any secondary EGFR mutations. It is unlikely that EGFR T790M mutation is generated through selection by EGFR-TKI from randomly occurring mutations. Rather, it appears that T790 is the preferred target of mutagenesis through yet an unknown mechanism which occurs after only one-hour treatment.