Defining the sensitivity landscape of EGFR variants to tyrosine kinase inhibitors

Open AccessPublished:November 05, 2022DOI:


      Tyrosine kinase inhibitor (TKI) is a standard treatment for patients with NSCLC harboring constitutively active epidermal growth factor receptor (EGFR) mutations. However, most rare EGFR mutations lack treatment regimens except for the well-studied ones. We constructed two EGFR variant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in the EGFR mutation hotspot (exons 18–21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line. A total of 3914 and 70,475 variants were detected in the constructed EGFR Substitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries. Of the 3914 Sub-Del variants, 221 proliferated fast in the control assay and were sensitive to EGFR-TKIs. For the 70,475 Ins variants, insertions at amino acid positions 770–774 were highly enriched in all 5 TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency. We present a comprehensive reference for the sensitivity of EGFR variants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs.


      Tyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non–small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor (EGFR) mutations are detected. However, except for the well-studied EGFR mutations, most EGFR mutations lack treatment regimens.

      Translational Significance

      The results demonstrated that patients with rare EGFR mutations were most likely to benefit from osimertinib therapy compared to afatinib, erlotinib, gefitinib, or icotinib therapy. This study provides a case of deep mutational scanning that simultaneously assayed substitution, deletion, and insertion variants. This approach is applicable for other oncogenes and targeted drugs.



      AA (Amino acid), Afa (Afatinib), ATCC (American Type Culture Collection), CIViC (Clinical Interpretation of Variants in Cancer), COSMIC (Catalogue of Somatic Mutations in Cancer), DMS (Deep mutational scanning), DMSO (Dimethyl sulfoxide), EGFR (Epidermal growth factor receptor), EGFR-TKI (EGFR-tyrosine kinase inhibitor), Erl (Erlotinib), ES (Enrichment Score), FACS (Fluorescent-activated cells sorting), FDR (False discovery rate), gDNA (Genomic DNA), Gef (Gefitinib), Ico (Icotinib), Ins (Insertion), MANO (Mixed-all-nominated-mutants-in-one), MITE (Mutagenesis by Integrated TilEs), NEB (New England Biolabs), NSCLC (Non–small-cell lung carcinoma), Osi (Osimertinib), sgRNA (Single guide RNA), STR (Short tandem repeat), Sub-Del (Substitution-Deletion), TKI (Tyrosine kinase inhibitor), VUS (Variants of unknown significance)
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