Forrest plot comparing PACC and non-PACC mutants can be found in Fig. 8h). Is this guidance up to date? 2a, Extended Data Fig. have no non-financial competing interests. Similarly, C797S mutations without T790M conferred resistance to Ex20ins-active inhibitors, but not first- or second-generation TKIs unless T790M was present (Fig. PubMed 6ac). We validated these findings without T790M mutations (Extended Data Fig. 6 Exon 20 loopinsertions are a distinct class of. This file contains Supplementary Tables 17. Dots are representative of each tumor, and bars are representative of average SEM for each group. receives consulting/advisory fees from Amgen, Navire Pharma, Intellisphere LLC; research funding to the institution from Amgen, Mirati Therapeutics, Boehringer Ingelheim, Merck&Co, Novartis, Pfizer; speakers fees from Bristol-Myers Squibb and RV Mais Promocao Eventos LTDS; other support from AstraZeneca Pharmaceuticals; travel expenses from Tango Therapeutics and reports past stock ownership of Moderna Inc and BioNTech SE. Soria, J. C. et al. The delta of the two rho values is shown as an overlapped grey bar. Tumours were validated for EGFR mutations by DNA fingerprinting and quantitative PCR as described46. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. 1c). c, Heat map with unsupervised hierarchical clustering of log(mutant/WT) ratios from Ba/F3 cells expressing indicated mutations after drug treatment. 1 and Extended Data Fig. PubMed 10, 1635 (2020). 77, 27122721 (2017). 10c); however, the structure-based approach identified nearly twice as many individuals who benefited from afatinib treatment. Sequist, L. V. et al. 4c, d), and structurefunction-based groups remained more predictive of mutation and drug sensitivity than exon-based groups (P=0.0034 and P<0.0001, respectively) (Extended Data Fig. d, Kaplan-Meier plot of TTF of patients with NSCLC harboring non-PACC atypical EGFR mutations (N= 56) treated with 1st- (N=25), 2nd- (N=13), or 3rd-gen (N=18) EGFR TKIs. Thress, K. S. et al. Similarly, acquired PACC mutations co-occurring with primary classical EGFR mutations retained sensitivity to second-generation TKIs while acquiring resistance to third-generation TKIs in an allele-specific manner (Fig. However, second-generation TKIs were less affected by shifts in the hinge region of the receptor and were predicted to maintain the orientation of the acrylamide group (Extended Data Fig. e21118 Background: Leptomeningeal metastasis (LM) is a debilitating condition associated with advanced non-small cell lung cancer (NSCLC). eh, Kaplan-Meier plots of TTF of patients atypical EGFR mutations stratified by EGFR TKI class for exons 18 (N=40) (e), 19 (N=19) (f), 20 (N=24) (g), and 21 (N=26) (h). R.M. Mutation order was assigned arbitrarily; groups were assigned on the basis of predicted mutational impact. Correspondence to For overlapping studies, only the largest dataset was used, and all known EGFR mutations were tabulated. Rafael Rosell, Andrs Felipe Cardona, Mariacarmela Santarpia, Margaret R. Smith, Yuezhu Wang, Fei Xing, Hannah L. Tumbrink, Alena Heimsoeth & Martin L. Sos, Alex Martinez-Marti, Enriqueta Felip, Miriam Sans, Guilherme Harada, Soo-Ryum Yang, Alexander Drilon, Marcel Wiesweg, Stefan Kasper, Martin Schuler, Nature Treatment groups included vehicle control (0.5% methylcellulose, 0.05% Tween-80 in dH2O), 100mgkg1 erlotinib, 20mgkg1 afatinib, 2.5mgkg1 poziotinib, 5mgkg1 osimertinib, and 25mgkg1 osimertinib. 4e). PubMed Central Background: Epidermal growth factor receptor gene (EGFR) exon 20 insertion (ex20-ins) mutations are an uncommon and heterogeneous group of non-small cell lung cancers (NSCLCs), resistant to conventional EGFR tyrosine kinase inhibitors (TKIs). Oncol. In b, d, data are representative of HR95%confidence interval. 7c). Google Scholar. PubMed are shareholders/full time employees of Guardant Health and report no non-financial interests. & Sievert, C. heatmaply: an R package for creating interactive cluster heatmaps for online publishing. Transl. Statistical differences in KaplanMeier plots, HR and Pvalues were generated using GraphPad Prism software and the MantelCox log-rank method. Guardant360 is a CLIA-certified, CAP/NYSDOH accredited comprehensive cfDNA NGS test that reports on SNVs, indels, fusions and SNVs in up to 73 genes. To determine whether structure function groups or exon groups were better predictor of drug sensitivity, we performed recursive-partitioning analyses to construct a decision tree for each drug using structure function group and mutation data on exons 18, 19, 20, and 21 as predictors. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. John V. Heymach. Using mutational mapping there were four distinct groups: (1) no obvious effect on the drug binding pocket (similar to L858R); (2) a mutation in the hydrophobic core (similar to T790M); (3) a large inward shift of both the C-helix and P-loop (similar to exon 20 insertions); and (4) a slight inward shift of the C-helix and/or P-loop due to direct changes to the either the C-helix and/or P-loop or indirectly through alterations of the -pleated sheets that are predicted to effect the position either the C-helix and/or P-loop. Prevalent hotspots for atypical mutations were the P-loop (L718V726, 13.6%) and the C-terminal loop of the C-helix (A767G779, 29.4%, Fig. and HolmSidaks multiple comparisons test. Clinical parameters were extracted from the respective databases. 4a) and then compared the median rho value of each correlation for both groups. Statistical differences were determined by ordinary one-way ANOVA with post-hoc Tukeys multiple comparisons test to determined differences between groups. Although all T790M-like mutants had at least one mutation in the hydrophobic core, there were two distinct subgroups of T790M-like mutantsthird-generation TKI sensitive (T790M-like-3S) and third-generation TKI resistant (T790M-like-3R) (Extended Data Fig. h, In silico modeling of EGFR Ex19del G796S (purple) with osimertinib in the reactive (blue) and predicted (orange) conformations demonstrate destabilization of TKI-protein interactions in the hinge region (yellow), displacing the reactive group (arrow). c, Comparison of osimertinib bound to wild-type EGFR (PDB 4ZAU, green) or EGFR G719S (PDB 2ITN, purple) demonstrates destabilization of TKI-protein interactions. Yun, C. H. et al. Bars are representative of average mutant/WT IC50 values SEM for each class of EGFR TKI and all cell lines. To determine the mutant/WT ratio, IC50 values for each drug and cell line were calculated and then compared to the average IC50 values of Ba/F3 cells expressing WT EGFR (+10ng/ml EGF to maintain viability). e, In silico modeling of EGFR G719S (purple) with poziotinib (blue) shows no predicted changes in poziotinib binding or TKI-protein interactions. All patients were treated with a second-generation TKI and experienced clinical benefits of stable disease and tumour shrinkage (Extended Data Fig. and J.V.H. The original draft of the manuscript was completed by J.P.R., J.B.C. This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program and the MD Anderson Cancer Center Support Grant P30 CA016672 including animal housing and care in the Research Animal Support Facility (RASF). MD Anderson Molecular Diagnostics Laboratory is a tissue molecular profiling method for NGS-based analysis to detect mutations in hotspot regions of 50 genes, and in April 2016, it was expanded to analyse 134 unique genes for the detection of somatic mutations in coding sequences of 128 genes and selected copy number variations (amplifications) in 49 genes. Finlay, M. R. et al. Structurefunction-based groups had a higher variable importance than exon-based groups, suggesting that structurefunction-based groups were more predictive of which mutational groups would be most sensitive to a particular drug compared with exon-based groups (P<0.0001) (Fig. Raw bioluminescence values were normalized to DMSO control-treated cells, and values were plotted in GraphPad Prism. Biotechnol. Data collection for Moffitt Cancer Center (MCC) patients was performed under the protocol (MCC 19161), which was formally reviewed and granted approval by MCC in accordance with the Declaration of Helsinki and the 21st Century Cures Act. While a separate classification for each individual mutation could provide more precision than the groups described here, validating the clinical activities of different drugs for each mutation is not feasible. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Response to afatinib and duration of afatinib treatment was tabulated from 803 patients in the Uncommon EGFR Database (www.uncommonegfrmutations.com). a, b, Heat maps supervised clustering by exon-based (a) or structure-function-based (b) groups of log (Mutant/WT) ratios from Ba/F3 cells expressing indicated mutations after 72h of indicated drug treatment. EGFR mutations were determined from formalin-fixed paraffin-embedded tumours or digital-droplet PCR for blood samples by CLIA-certified methods as previously described18,37. Tumor characteristics associated with engraftment of patient-derived non-small cell lung cancer xenografts in immunocompromised mice. 6d). Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. 8d, e). H.T. 15, 803815 (2020). d, KaplanMeier plot of time to treatment failure (TTF) (time from TKI commencement until radiologic progression, discontinuation, or death) of patients with NSCLC tumours containing classical (n=245 patients) or atypical (n=109 patients) EGFR mutations after EGFR TKI treatment. Tsigelny, I. F. et al. EGFR exon 18 mutations in lung cancer: molecular predictors of augmented sensitivity to afatinib or neratinib as compared with first- or third-generation TKIs. Holm-Sidaks multiple comparisons test was used to determine differences between groups. 5), and that structurefunction-based groups can predict drug sensitivity and patient outcomes better than exon-based groups. 16, 830838 (2015). Kosaka, T. et al. 5eg). 1 The prognosis of lung cancer detected at an advanced disease stage is considerably poor. b, Percentage of atypical EGFR mutations observed in patients with NSCLC (n=7,199 mutations). To determine the frequency of individual EGFR variants reported across the MD Anderson GEMINI database, cBioPortal, Foundation Medicine and the Guardant Health database, each database was analysed separately, and the average of all databases was determined. f, Dot plot of variable importance calculated as sum of the goodness of split for each split in the classification and regression trees (CART). p-values were determined by one-way ANOVA with unequal SD as determined by Brown-Forsythe test to determine differences in SD. Drug screens were performed in technical triplicate on each plate and either duplicate or triplicate biological replicates. Oncol. In all patients, a PACC mutation was identified upon biopsy at progression (Extended Data Fig. After 72h, 11l of Cell Titer Glo (Promega) was added to each well. Blue arrow indicates resolved pleural effusion. 10eh). Shortly, cells were plated in 384-well plates (Greiner Bio-One) at 2,0003,000 cells per well in technical triplicate. HER2-positive breast cancer is a breast cancer that tests positive for a protein called human epidermal growth factor receptor 2 (HER2). 1b). CAS Epidermal growth factor receptor binds to at least seven different ligands. Cho, J. H. et al. 21, 53055313 (2015). J.S. A Phase II, Open-label, Single-arm, Multi-centre Study to Evaluate the Safety and Efficacy of Osimertinib With Amivantamab as First-line Treatment in Participants With Epidermal Growth Factor Receptor Mutation-Positive, Locally Advanced or Metastatic Non-small Cell Lung Cancer (OSTARA) Estimated Study Start Date : June 30, 2023 and M.B.N. There are different types of EGFR mutations. Code for this analysis can be found at https://github.com/MD-Anderson-Bioinformatics/EGFR-Structure-Function-Nature-Manuscript. The binding of a ligand to epidermal growth factor receptor allows the receptor to attach to another nearby epidermal growth factor receptor protein (dimerize), turning on (activating) the receptor complex. HR and Pvalues were determined using GraphPad Prism software and two-sided MantelCox log-rank tests. The structure function group variable was involved in the first and second splits in all of the 18 regression trees of drug sensitivity. a, b, Schematic below CT images shows timeline of patient treatments and outcomes. Statistical differences between near- and far-loop mutants was determined by two-sided unpaired t-test with unequal variance, a, Heat map with unsupervised hierarchical clustering of log (mutant/WT) ratios from Ba/F3 cells expressing indicated mutations after 72h of indicated drug treatment. EGFR is a cell surface protein that binds to epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation leading to cell proliferation. Mice were humanely euthanized if tumour sizes exceeded the maximum size. J. Deletions also including insertions were allowed and still considered classical exon 19 deletions. Further, G719S displays an inward shift of the C-helix compared to the WT crystal structure. Atypical EGFR mutations with US Food and Drug Administration (FDA)-approved treatments are EGFR S768I, L861Q and G719X, for which afatinib was deemed effective on the basis of retrospective studies13,14,15, and the EGFR/MET bispecific antibody amivantamab for exon 20 insertions (Ex20ins)16. EGFR mutations associated with acquired drug resistance, as described by the literature, are highlighted in red. Administration, U. S. F. a. D. (ed FDA) (2021). Concurrent use of aspirin with osimertinib is associated with improved survival in advanced EGFR-mutant non-small cell lung cancer. 3a) and structurefunction groups (Extended Data Fig. Classical EGFR mutations are L858R, T790M and various Ex19dels (Methods). In contrast, poziotinib (blue) is less effected by Q718 and is still positioned to react with C797, even in the context of L718Q mutations. Clinical trial design and treatment of patients with atypical EGFR mutations often rely on mutated-exon location to predict treatment, although heterogeneity in drug sensitivity across a single exon has been observed1,8,17. 3b) showed distinct differences, suggesting that structurefunction-based groups better defined groups of mutations by drug sensitivity than exon-based classification. p-value was determined using a paired two-sided t-test, and n = 18 drugs. 2a), T790M-like mutations in the hydrophobic core (Extended Data Fig. Squares are representative of the median of n=3 replicates. There were 333 patients with NSCLC identified in the MD Anderson GEMINI database who had tumours expressing atypical mutations. Methods 14, 323348 (2009). We also thank I. G. Munoz for critical review and editorial assistance. 7b). NS, not significant. ch, HRs and p-values were calculated using two-sided Mantel-Cox, Log-Rank tests. All atypical, n=109; all atypical without Ex20ins, n=97; exon 18, n=29; exon 19, n=22; exon 20, n=41; exon 21, n=18. Yoshikawa, S. et al. Globally, lung cancer is the leading cause of cancer-related deaths among men and women and is expected to have killed around 132 000 people in the United States in 2021. Further information on research design is available in theNature Research Reporting Summary linked to this paper. To determine the frequency of atypical mutations in the MD Anderson GEMINI and Foundation Medicine databases, atypical mutations were identified as described above and total number of known EGFR mutations across all patients was tabulated. JTO Clin. 1 are provided at https://github.com/MD-Anderson-Bioinformatics/EGFR-Structure-Function-Nature-Manuscript. 1e, HR=1.6, P<0.0001) or when patients were stratified by mutation exon location (Fig. 4 Structure-function-based groupings are more predictive of drug and mutation sensitivity compared to exon-based groupings. For overlapping studies, the largest database was selected. We show that EGFR mutations, including atypical mutations, can be divided into four distinct subgroups based on structure and function (Fig. If you think of a mutation as a typo in the DNA, you can have missing or added words in the DNA, sometimes called deletions or insertions. Hierarchical clustering was determined by Euclidean distance between Mut/WT ratios. Article 57, 82498267 (2014). Nat. 20, 686702 (2018). Google Scholar. Tumors were measured three times per week and symbols are average of tumor volumes SEM. Klughammer, B. et al. Arrows indicate location of structural changes compared with wild-type EGFR. Vasconcelos, P. et al. Methods Sci. f, Tumor growth curves for PDXs harboring EGFR L858R E709K complex mutation treated with indicated inhibitors. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. 3b, Extended Data Fig. EGFR signaling cascade is a key regulator in cell proliferation, differentiation, division, survival, and cancer development. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7,8,9,10. Red arrow indicates lesion harboring L858R/L718V. A clinical challenge for physicians treating patients with EGFR-mutant cancers is to appropriately identify and match patient mutations with the best EGFR TKI. Patients that received prior chemotherapy or immunotherapy were included, but TTF was calculated for first EGFR TKI received. By contrast, TTF was not significantly different between classes of EGFR TKIs in patients with non-PACC mutations (Fig. Dots are representative of average of n=3 replicate mutant/WT IC50 values of individual cell lines with individual drugs and drugs of each class are grouped together. a, Dot plot of mutant/WT IC50 values of Ba/F3 cells expressing PACC mutations. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Article In a, b, classical-like, n=58; T790M-like, n=68; Ex20ins-L, n=76; PACC, n=156; exon 18, n=87; exon 19, n=19; exon 20, n=195; exon 21, n=63. c, KaplanMeier plot of TTF of patients with PACC mutations treated with first-, second- or third-generation EGFR TKIs. Dosing holidays were given if mouse body weight decreased by more than 10% or overall body weight dropped below 20 g. Maximum allowed tumour burden by approved IACUC protocol was a volume of 2,000mm3. discloses research support from Spectrum Pharmaceuticals, AstraZeneca, Takeda, Eli Lilly, Xcovery, Turning Point Therapeutics; advisory role for AstraZeneca, Eli Lilly and Turning Point Therapeutics; and accommodation expenses from Eli Lilly, and no non-financial competing interests. 2c), and mutations on the interior surface of the ATP-binding pocket or C-terminal end of the C-helix, which were predicted to be P-loop and C-helix compressing (PACC) (Fig. p-values were determined by one-way ANOVA with unequal SD as determined by Brown-Forsythe test to determine differences in SD. e, Dot plot of rho values from Spearman correlations of mutations vs exon-based group averages or structure-function based averages for each drug excluding T790M mutations. Patients were stratified by EGFR mutation, and EGFR mutations were manually curated as atypical or classical EGFR mutations. Other EGFR mutations in the kinase domain (exons 1821) have also been established as oncogenic drivers of NSCLC8. After sorting, EGFR-positive cells were maintained in RPMI containing 10%FBS, 1% penicillin-streptomycin, and 1ngml1 EGF to support cell viability. Uchibori, K. et al. Galili, T., OCallaghan, A., Sidi, J. 2b), insertions in the loop at the C-terminal end of the C-helix in exon 20 (Ex20ins-L; Extended Data Fig. Psychol. Data from Foundation Medicine and Guardant Health were provided under data use agreements; however, summarized data used in Fig. 2b). We applied the CART algorithm20,21 using the rpart R package. Commercial NGS platforms including FoundationOne and Guardant360 were used by both MD Anderson and Moffitt Cancer Center as described below. a, Percentage of patients with NSCLC containing classical and atypical EGFR mutations (n=11,619 patients). Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials. d, Dot plot of mutant/WT IC50 values of Ba/F3 cells expressing classical EGFR mutations (white bars) with or without PACC mutations (coloured bars). Thank you for visiting nature.com. Provided by the Springer Nature SharedIt content-sharing initiative, Journal of Experimental & Clinical Cancer Research (2023), Journal of Cancer Research and Clinical Oncology (2023). Russo, A. et al. For example, L718Q, S768I, and T854I correspond to exons 18, 20, and 21, respectively, but are all PACC mutations with similar structural effects on drug binding. In vivo, we also observed that NSCLC patient-derived xenografts (PDXs) containing G719A mutations were resistant to the third-generation TKI osimertinib, but most sensitive to the second-generation TKI poziotinib (Fig. As part of the MD Anderson Cancer Center Lung Cancer Moon Shots program, PDXs harbouring EGFR G719A and EGFR L858R/E709K were generated and maintained in accordance with Good Animal Practices and with approval from MD Anderson Cancer Center Institutional Animal Care and Use Committee on protocol number PA140276 as previously described46. Moffitt Trusight is a NGS Illumina sequencing platform with a panel of 170 genes. But contrary to other member of the ERBB family, HER2 does not directly bind ligand. All code used in this study can be found at https://github.com/MD-Anderson-Bioinformatics/EGFR-Structure-Function-Nature-Manuscript. 3e). ADS These data demonstrate that both primary and acquired PACC mutations are sensitive to second-generation TKIs, and structurefunction-based groupings could identify a novel class of mutations, PACC mutations, for which second-generation TKIs had higher selectivity and efficacy than third-generation drugs. As non-financial competing interests F.S. Wang, X. et al. Invest. In silico analysis of the interaction of osimertinib with PACC mutations G719S and L718Q predicted that changes in the orientation of the P-loop alter the position of TKI stabilization points tilting the indole ring of osimertinib away from the P-loop, destabilizing drug binding (Extended Data Fig. Robichaux, J. P. et al. Source data are provided with this paper. CAS As non-financial competing interests, J.V.H. 382, 4150 (2020). In addition, at Moffitt Cancer Center, there were 21 patients with NSCLC with tumours harbouring atypical EGFR mutations. Once tumours reached 2,000mm3, they were collected and re-implanted into the right flank of 6- to 8-week-old female NSG mice. Targeting EGFR exon 20 insertion mutations in non-small cell lung cancer, Coregulation of pathways in lung cancer patients with EGFR mutation: therapeutic opportunities, Genomic signatures define three subtypes of EGFR-mutant stage IIIII non-small-cell lung cancer with distinct adjuvant therapy outcomes, Prognostic Mutational Signatures of NSCLC Patients treated with chemotherapy, immunotherapy and chemoimmunotherapy, Comprehensive genomic profile of Chinese lung cancer patients and mutation characteristics of individuals resistant to icotinib/gefitinib, The next tier of EGFR resistance mutations in lung cancer, Genetic evolution to tyrosine kinase inhibitory therapy in patients with EGFR-mutated non-small-cell lung cancer, Impact of RAS mutation subtype on clinical outcomea cross-entity comparison of patients with advanced non-small cell lung cancer and colorectal cancer, https://github.com/MD-Anderson-Bioinformatics/EGFR-Structure-Function-Nature-Manuscript. To determine TTF after EGFR TKI treatment, patients with NSCLC harbouring an EGFR mutation in the tyrosine kinase domain (exons 1822) were identified in the MD Anderson GEMINI and Moffitt Cancer Center databases. Cancer Res. Objective response rate was reported in 529 patients. HER2 activation results from heterodimerization with another ERBB member or by homodimerization when HER2 concentration are high, for instance in cancer. The epidermal growth factor receptor (EGFR) belongs to the ERBB family of tyrosine kinase receptors. Dots are representative of average of n=3 replicate mutant/WT IC50 values of individual cell lines expressing exon 20 insertion mutations with individual drugs. Squares represent the median of n=3 replicates. To propagate tumours, 5- to 6-week-old female NSG mice (NOD.Cg-Prkdcscid IL2rgtmWjl/Szj) were purchased from Jackson Laboratories (005557). 6e). Abstract. The classification presented here provides a framework through which clinicians, informed by internet-based tools or companies providing NGS reports, could more effectively personalize EGFR TKI therapy. Med. Tamirat, M. Z., Koivu, M., Elenius, K. & Johnson, M. S. Structural characterization of EGFR exon 19 deletion mutation using molecular dynamics simulation. Lancet Oncol. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer. DNA from tumor cells is tested for mutations in this gene. Nat. Lung Cancer 108, 228231 (2017). Mutant to WT ratios for each drug were calculated by dividing the IC50 values of mutant cell lines by the average IC50 value of Ba/F3 cells expressing WT EGFR supplemented with 10ngml1 EGF for each drug. To test this hypothesis, we calculated the correlations of drug sensitivity and selectivity for each mutation to the predicted drug sensitivity by exon or structurefunction groups (Extended Data Fig. reports research funding and consulting fees from Bristol-Myers Squibb, AstraZeneca, Geneplus, OrigMed, Innovent, Merck, Johnson and Johnson, and no non-financial competing interests. Structure-based classification predicts drug response in, https://doi.org/10.1038/s41586-021-03898-1. and J.V.H. Extended Data Fig. Ou, S. I. et al. It is activated by various ligands in extracellular milieu and transmits the cellular response to mediate various cellular activities, including cell proliferation, cell survival, growth, and development. Le, X. et al. Epidermal growth factor receptor (EGFR) mutations are the second most common oncogenic driver event in non-small cell lung cancer (NSCLC). Mutations and assigned groups are in Supplementary Table 4. 1f). 7 Altmetric Metrics Abstract Purpose To assess whether the radiomic features of diffusion tensor imaging (DTI) and conventional postcontrast T1-weighted (T1C) images can differentiate the epidermal growth factor receptor (EGFR) mutation status in brain metastases from non-small cell lung cancer (NSCLC). For patients treated beyond progression, radiologic progression was recorded as the end point, and data cut-off was May 2021. Bars are representative of average mutant/WT ratio for all mutations and drugs in the indicated groups, dots are representative of average (n=3) mutant/WT ratio of individual mutations and drugs. Extended Data Fig. Bars are representative of average mutant/WT IC50 values SEM for each class of EGFR TKI and all classical-like cell lines. The study was conceptualized by J.P.R., X. d, Heat map with unsupervised hierarchical clustering of log (Mutant/WT) ratios from Ba/F3 cells expressing indicated exon 20 loop insertions after 72h of indicated drug treatment. Median TTF was calculated using the KaplanMeier method. Osimertinib for patients with non-small-cell lung cancer harboring uncommon EGFR mutations: a multicenter, open-label, phase II trial (KCSG-LU15-09). N. Engl. b, Dot plot of Spearmans rho values for correlations of mutations versus exon-based group averages or structurefunction-based averages for each drug. 2a, Extended Data Fig. Background Activating mutations in the epidermal growth factor receptor gene (EGFR) confer hypersensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in patients with advanced non . Forrest plot comparing HRs and p-values across exons can be found in Fig. Both protocols include informed consent for publication of deidentified data. Slider with three articles shown per slide. When treated with an EGFR TKI, patients with atypical EGFR mutations had a shorter TTF compared with patients with classical EGFR mutations (Fig. Front. Mutations in this gene are associated with lung cancer. g, Representative CT images from a patientharbouring E709K G719S complex mutation before and after four weeks of afatinib treatment. 10 Structure-function groups identify patients with greater benefit to 2, http://creativecommons.org/licenses/by/4.0/, Patient-derived cell-based pharmacogenomic assessment to unveil underlying resistance mechanisms and novel therapeutics for advanced lung cancer, Structure and dynamics of the EGFR/HER2 heterodimer, Treatment of advanced non-small cell lung cancer with driver mutations: current applications and future directions, Multiregional radiomics of brain metastasis can predict response to EGFR-TKI in metastatic NSCLC, Two germline mutations can serve as genetic susceptibility screening makers for a lung adenocarcinoma family. ; and editing was completed by J.P.R., R.S.K.V., J.B.C., X. Natl Acad. Patients were consented under the GEMINI protocol (PA13-0589) which was approved in accordance with the MD Anderson Institutional Review Board, or protocol MCC 19161, which was formally reviewed and granted approval by Moffitt Cancer Center in accordance with the Declaration of Helsinki and the 21st Century Cures Act for retrospective analysis of patient outcomes and treatment course for case studies of patients presented. We generated a panel of 76 cell lines expressing EGFR mutations spanning exons 1821 and screened these cell lines against 18 EGFR inhibitors representing first- (non-covalent), second (covalent) and third- (covalent, T790M targeting) generation and Ex20ins-active TKIs (Supplementary Table 1). [6] For each correlation, the mutation tested was removed from the average structure function and exon-based groups. Of these patients, 88 patients received at least one line of EGFR TKI treatment. Insertions in the C-helix (for example, an FQEA insertion at A763 (A763insFQEA)) were pan-sensitive to EGFR TKIs23,24, whereas those in the loop following the C-helix (A767C775) were not25,26, and the T790M mutation was sensitive to third- but not first- or second-generation TKIs. When we compared the selectivity ofEGFR TKIs for PACC mutations, we found that second-generation TKIs were significantly more selective for PACC mutations than any other TKIclass (Fig. Mice received drug 5 days per week, and mice were euthanized at day 28 to harvest tumors. Mutation variants in the EGFR gene can cause the . Le and J.V.H. 24, 61956203 (2018). Outcomes were recorded for patients for only first EGFR TKI. Oncogene 32, 2738 (2013). CAS 5, 216ra177 (2013). J.P.R. Mice received drug 5 days per week, and mice were euthanized at day 21 to harvest tumors. For the analysis of cBioPortal, all non-overlapping studies were selected and exported. g, Dot plot of percent change in tumor volume on day 28 of tumors described in f. Dots are representative of each tumor, and bars are representative of average SEM for each group. serves as scientific advisor for Rexanna Foundation and the EGFR resisters. Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations. 2a, Extended Data Fig. Among those patients, 67.1% had classical EGFR mutations (L858R and/or Ex19del with or without T790M); 30.8% had atypical EGFR mutations, including Ex20ins (9.1%), atypical mutations (12.6%), or a complex mutation including an atypical mutation (9.1%); and 2.2% had a classical mutation with T790M and an atypical mutation (Fig. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Classical activating mutations (exon 19 deletions and the L858R point mutation) comprise the vast majority of EGFR mutations and are well defined as strong predi a, KaplanMeier plot of DOT of patients with NSCLC tumours containing atypical EGFR mutations (n=358 patients) stratified by structure-based groups treated with afatinib. Google Scholar. Funding was provided by J.V.H., X. Fragments of NSCLC tumours expressing EGFR S768dupSVD, G719A or L858R/E709K were implanted into 6- to 8-week-old female NSG mice. Rep. 1, 100051 (2020). J. Med. An integrated TCGA pan-cancer clinical data resource to drive high-quality survival outcome analytics. Evidence-based recommendations on osimertinib (Tagrisso) for untreated locally advanced or metastatic epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer in adults. 7 Drug repurposing can overcome T790M-like resistance mutations. J.V.H. Article Surgical samples were rinsed with serum-free RPMI supplemented with 1% penicillin-streptomycin then implanted into the right flank of 5-to6-week-old NSG female mice within 2h of resection. Clinical outcomes of patients with early-stage epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer who underwent reflex molecular testing. Dots are representative of each mutation; bars are representative of the average rho value standard deviation (SD). Cancer Cell 36, 444457.e447 (2019). To determine whether structure-based groups could identify which class of inhibitors would provide the most benefit to patients with atypical EGFR mutations, we performed retrospective analyses of TTF of patients with atypical EGFR mutations treated with EGFR TKIs in MD Anderson Cancer Center GEMINI and Moffitt Cancer Center databases, and TTF was determined for the firstEGFR TKI for which patientswere treated. The diversity and higher than previously appreciated prevalence of atypical EGFR mutations shown here highlights the necessity of comprehensive next-generation sequencing (NGS) for patients with NSCLC. Heterogeneous responses to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with uncommon EGFR mutations: new insights and future perspectives in this complex clinical scenario. Nature (Nature) We calculated variable importance as the sum of the goodness of split measures for each split. Therefore, structurefunction classification identified not only a larger subgroup of patients, but also a subgroup with greater benefit from second-generation TKIs than the exon-based classification. The results predicted the treatment efficacy of these combinations. Chem. Lancet Oncol. Frampton, G. M. et al. J. Cancer 125, 37383748 (2019). However, the frequency of EGFR gene mutation in TNBC is low and varies with ethnicity. Rep. 2, 100105 (2021). & Tutz, G. An introduction to recursive partitioning: rationale, application, and characteristics of classification and regression trees, bagging, and random forests. Residues important in receptor signaling and drug binding are highlighted. By contrast, second-generation TKIs do not interact with the P-loop of EGFR and maintain interaction points in the hydrophobic cleft (Extended Data Fig. Sci. Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6. PubMed Central Med. Liu, J. et al. T790M-like-3R mutants, complex mutations comprising T790M and a known drug-resistance mutation (that is, C797S30, L718X31 or L792H18,31), were resistant to classical EGFR TKIs but retained selectivity for select ALK and PKC inhibitors such as brigatinib or midostaurin (Extended Data Fig. Nature 597, 732737 (2021). These two factors make EGFR and its family members prime. Bioinformatics 34, 16001602 (2018). Pan-cancer landscape and analysis of ERBB2 mutations identifies poziotinib as a clinically active inhibitor and enhancer of T-DM1 activity. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Each decision rule was constrained in an internal node, and every internal node points to yes-or-no questions that result in a yes or no branch. Structurefunction-based groups identified that patients with PACC mutations (n=156) had a significantly longer DOT for afatinib than other structure-based groups (DOT: 17.1 months, P<0.0001) (Fig. Ramalingam, S. S. et al. L747_K754del-insATSPE, L747P and E746-A750del mutations occur in exon 19 but are T790M-like, PACC, and classical mutations, respectively, with distinct drug sensitivity and structural effects. receives research support from OneOme, is a consultant for Quest Diagnostics and 23andMe, and has served on an advisory committee for Novartis, and has provided educational content for the American College of Clinical Pharmacy, Florida Pharmacy Association and HorizonCME. Retrospectively, we identified three patients with NSCLC containing EGFR L858R mutations that received first-line osimertinib and subsequently developed an EGFR-dependent mechanism of resistance. Chin. Atypical EGFR mutations (n=7,199) occurred primarily in exons 18 (23.7%) and 20 (20.9% insertions and 19.2% point mutations; Fig. 2 Among patients with advanced lung adenocarcinomas, 51.4% in Asia and 15% to 22% outside Asia have epidermal growth factor receptor . 11, 545555 (2016). Targeted therapies. 8g). Studies showed that exon 20 mutations are heterogenous in their response to TKIs10,22. e, Bar plot of average mutant/WT ratio of Ba/F3 cells expressing exon 20 loop insertions separated by mutational groups for indicated drug classes. Targeted therapies are approved for patients with classical mutations and a small number of other mutations4,5,6. Le, Y.Y.E., J.K.H., A.B.S., J.E.G. Groups were assigned on the basis of structural predictions (Methods). Dots are representative of variable importance for each drug in the exon and structure-function-based groups as indicated and excluding T790M mutations. Kobayashi, Y. et al. Cancer Res. and J.B.C., and figures were prepared by J.P.R., S.H. The gene encoding the epidermal growth factor receptor (EGFR) tyrosine kinase is somatically mutated in a substantial fraction of patients with lung cancer. Heat maps and hierarchical clustering were generated by plotting the median log (Mut/WT) value for each cell line and each drug using R and the ComplexHeatmap package40 2.6.2 (R Foundation for Statistical Computing). Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. ), the Hallman Fund, the Stading Fund for EGFR inhibitor resistance, the Gil and Dody Weaver Foundation, the Hanlon Fund, the Richardson Fund for EGFR mutant lung cancer research, and ASCO CDA-57112 (Y.Y.E.). PubMed Central p-values were determined by one-way ANOVA with unequal variance as determined by Brown-Forsythe test to determine differences in variance. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Lung Cancer 149, 3340 (2020). Validating these findings in the cBioPortal database, patients with atypical EGFR mutations had a shorter progression free interval19 and overall survival, irrespective of treatment (Extended Data Fig. Plates were incubated for a minimum of 10min, and bioluminescence was determined using a FLUOstar OPTIMA plate reader (BMG LABTECH). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Cancer Res. X. PubMed Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. EGFR mutations associated with acquired drug resistance are highlighted in red. Atypical EGFR mutations are defined as non-classical, non-synonymous mutations. Error bars are representative of SEM for each bar. 6a). Overall survival with osimertinib in untreated, EGFR-mutated advanced NSCLC. c, Dot plot of variable importance calculated from CART. The delta of the two rho values is shown as an overlapped grey bar. Structural features of EGFR mutations with known drug sensitivity (that is, classical EGFR mutations41,42, T790M43,44,45 and exon 20 insertions22,25) were used as the basis for predicting the impact of mutations on drug sensitivity. However, even within Ex20ins-L mutations, some degree of heterogeneity in drug sensitivity was observed (Fig. Wu, Y. L. et al. https://doi.org/10.1038/s41586-021-03898-1, DOI: https://doi.org/10.1038/s41586-021-03898-1. ISSN 0028-0836 (print). Noncovalent wild-type-sparing inhibitors of EGFR T790M. of mutant/WT ratio for all mutations and drugs; dots show representative average mutant/WT of n=3 replicates. J.K.H. PubMedGoogle Scholar. Previous reports have shown that protein kinase C27 (PKC) and anaplastic lymphoma kinase28,29 (ALK) inhibitors exhibit off-target activity for EGFR mutations including T790M, and the non-covalent nature of these compounds predict that they retain activity in mutations that interrupt covalent binding. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Mice were randomized into four groups: vehicle (N=4), poziotinib 2.5mg/kg (N=5), osimertinib 5mg/kg (N=4), and osimertinib 25mg/kg (N=5) . [5] The epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). Public datasets used in this study include non-overlapping studies including NSCLC in cBioportal (www.cbioportal.org) and the Uncommon EGFR Database (www.uncommonegfrmutations.com). a, Dot plot of mutant/WT IC50 values of Ba/F3 cells expressing exon 20 loopinsertion mutations and treated with indicated classes of EGFR TKIs. a, Overlap of G719S (PDB 2ITN, green) and WT EGFR (PDB 2ITX, grey) crystal structures demonstrate arobust shift of F723 (red arrow) in the P-loop orienting the benzyl ring in a downward position condensing the P-loop in the drug binding pocket. T790M-like-3S mutants had high selectivity for third-generation TKIs and some Ex20ins-active inhibitors and moderate selectivity for ALK and PKC inhibitors (Extended Data Fig. The OlympiAD trial showed the poly-ADP ribose . In the meantime, to ensure continued support, we are displaying the site without styles 36, 22442250 (2018). Landscape of EGFR -dependent and -independent resistance mechanisms to osimertinib and continuation therapy post-progression in EGFR-mutant NSCLC. d, Forest plot of HRs calculated from KaplanMeier plots in c and Extended Data Fig. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structurefunction-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations. 13, e23e31 (2012). There were 11,619 patients in whom primary and/or co-occurring mutations were recorded on a per-patient basis (Extended Data Fig. Rosell, R. et al. Notably, a patient with a complex PACC mutation, E709K/G719S, saw clinical benefit and tumour shrinkage with afatinib treatment after progressing on osimertinib (Extended Data Fig. J. Med. Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor. 1d, e). J.P.R. Le, W.R., J.P.R., H.T., J.K.H., J.E.G., Y.Y.E., X. Liu, J.Z., F.S., S.V., R.M., A.B.S. and J.V.H., for the classification of EGFR mutations. Details of specific studies accessed can be found in the Reporting Summary. Dots are representative of variable importance for each drug; bars show median+95%confidence interval of variable importance for all drugs (n=18 drugs) (Supplementary Table 2). Oncol. Article J. Thorac. Using a panel of an additional 15 Ba/F3 cell lines expressing Ex20ins-L mutations spanning A767V774, we found that Ex20ins-L mutations could be subdivided into two subgroups: near- and far-loop Ex20ins (Extended Data Fig. These data support expanding testing of ALK and/or PKC inhibitors or development of novel non-covalent inhibitors for the broader group of T790M-like-3R mutations. and J.S. The order of co-occurring mutations was assigned arbitrarily. Article Using hierarchical clustering of in vitro selectivity over WT EGFR and mutational mapping of EGFR mutations, we observed four distinct subgroups of EGFR mutations: classical-like mutations that were distant from the ATP-binding pocket (Extended Data Fig. Source data for all figures can be found at https://github.com/MD-Anderson-Bioinformatics/EGFR-Structure-Function-Nature-Manuscript.

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