A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer

Abstract

Numerous variants of unknown significance (VUS) have been identified through large-scale cancer genome projects, although their functional relevance remains uninvestigated. We developed a mixed-all-nominated-mutants-in-one (MANO) method to evaluate the transforming potential and drug sensitivity of oncogene VUS in a high-throughput manner and applied this method to 101 nonsynonymous epidermal growth factor receptor (EGFR) mutants. We discovered a number of mutations conferring resistance to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib- and erlotinib-insensitive missense mutations within exon 19 and other gefitinib-resistant mutations, such as L833V, A839T, V851I, A871T, and G873E. -positive tumors (12.8%) harbored compound mutations primarily in the allele, which decreased the gefitinib sensitivity of these tumors. The MANO method further revealed that some EGFR mutants that are highly resistant to all types of TKIs are sensitive to cetuximab. Thus, these data support the importance of examining the clinical relevance of uncommon mutations within and of evaluating the functions of such mutations in combination. This method may become a foundation for the in vitro and in vivo assessment of variants of cancer-related genes and help customize cancer therapy for individual patients.

Colorectal adenocarcinoma‐derived EGFR mutants are oncogenic and sensitive to EGFR‐targeted monoclonal antibodies, cetuximab and panitumumab

“…Interestingly, with the exception of the previously well‐characterized L858R mutation (exon 21), most of the oncogenic kinase domain mutations found in CRC are located in exon 18, which are quite different from the distribution in lung adenocarcinoma (mainly exon 19 and 21 of kinase domain) or glioblastoma (extracellular domain) . Recently, a high‐throughput method for testing nonsynonymous EGFR mutants including R222C, S492R, P596L, E709K, G719S and L858R was reported and results are consistent with our study …”

“…22 Recently, a high-throughput method for testing nonsynonymous EGFR mutants including R222C, S492R, P596L, E709K, G719S and L858R was reported and results are consistent with our study. 23 Colon-cancer derived oncogenic EGFR mutants are sensitive to EGFR-targeted mAbs in vivo and in vitro Both cetuximab and panitumumab were able to inhibit the growth of the transformed Ba/F3 cells in a dose-dependent manner with an efficacy of less than 1 μg/mL of IC 50 with the exception of S492R expressing cells, which was suppressed by panitumumab, but resistant to cetuximab as previously reported ( Figs. 2a and 2b).…”

Implementation of “clinical sequencing” in cancer genome medicine in Japan

“…The clinical relevance of sequencing tests will also be enhanced by the development of therapies for gene alterations that are frequent but currently “undruggable”, such as deleterious mutations in the chromatin regulator genes . Functional annotation of mutations that are currently classified as variants of unknown significance will also facilitate clinical sequencing by prioritizing further actionable mutations …”

“…[27][28][29] Functional annotation of mutations that are currently classified as variants of unknown significance will also facilitate clinical sequencing by prioritizing further actionable mutations. 30,31 Of course, finding actionable mutations in a patient's tumor does not imply that the patient will respond to a therapeutic agent against that target. It is important to examine whether or not a treatment based on the gene profile of an individual patient can really improve the clinical course of his or her disease.…”

Revisited analysis of a SHIVA01 trial cohort using functional mutational analyses successfully predicted treatment outcome

“…Similar assays are reported in the literature enabling the measurements of variants' oncogenic activities and the efficacy of MTA on inhibiting signaling pathways. The mixed-all-nominated-mutants-inone (MANO) method recently reported allows evaluating in vitro the oncogenic activity and drug sensitivity of VUS in a high-throughput manner (Kohsaka et al, 2017). The application of this new method on 101 nonsynonymous EGFR variants allowed the discovery of a number of new mutations conferring resistance to EGFR tyrosine kinase inhibitors and pinpointed EGFR mutations that should rather be targeted by cetuximab.…”