Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR‐tyrosine kinase inhibitors

Abstract

The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations. (Cancer Sci 2013; 104: 584-589)

Determining EGFR-TKI sensitivity of G719X and other uncommon EGFR mutations in non-small cell lung cancer: Perplexity and solution

“…They found that the auto-phosphorylation level of G719S was lower, indicating that the oncogenicity of G719S was weaker than that of the other two common mutants (51,53). Subsequent studies further confirmed the conclusions using western blotting and immunofluorescence staining (51,53–57). …”

“…However, compared with L858R, G719S required a higher concentration of gefitinib (54). Their conclusions were further validated by subsequent studies using other techniques (53,57–59). In general, based on the studies mentioned above, G719X was found to have moderate oncogenicity and intermediate sensitivity to TKIs regarding kinase function.…”

“…To be more quantitative, the 50% inhibiting concentration (IC 50 ) of gefitinib in various mutants was further measured in series of studies. The IC50 of gefitinib in G719S mutants was between that of gefitinib in wild-type cells and Del19/L858R mutants, implying an intermediate sensitivity of G719S (16,52,54,55,57,63). …”

Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay

“…In contrast, the YFP‐EGFR‐ICD assay allows both the use of transiently transfected cells and quantitative measurement of EGFR activity on a single‐cell basis. We previously investigated the EGFR‐TKI sensitivity of EGFR harboring common or single uncommon mutations with this assay and found that the results reflected the clinical outcome . The increasing application of genomic sequencing to NSCLC patients is likely to reveal new combinations of EGFR mutations.…”

“…Constructs were confirmed by DNA sequencing. HEK293 cells (5.0 × 10 4 per well) were seeded in 24‐well plates, transfected for 30 hours with 500 ng of plasmid DNA with the use of Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA), and then exposed to erlotinib (Cayman Chemical, Ann Arbor, MI, USA) or afatinib (Selleck Chemicals, Houston, TX, USA) in culture medium at 37°C for 6 hours. The distribution of YFP‐EGFR‐ICD fluorescence was then examined with a BZ‐8100 fluorescence microscope (Keyence, Osaka, Japan).…”