Exploiting the Full Potential of Novel Agents Targeting EGFR Exon 20 Insertions in Advanced NSCLC: Next-Generation Sequencing Outperforms Polymerase Chain Reaction–Based Testing

EGFR mutations emerged in the past two decades as one of the major therapeutic targets in advanced/metastatic NSCLC and more recently also in early stage disease. This molecularly defined subgroup of patients with NSCLC, which accounts for approximately 15% to 20% of all nonsquamous cases, is highly heterogeneous owing to the existence of several different variants with distinct biological properties and drug sensitivity to EGFR tyrosine kinase inhibitors (TKIs) that were historically divided in two subgroups—classical mutations (LREA exon 19 deletions and p.L858R exon 21 mutation) and uncommon mutations, which included all the other mutations that can occur in exons 18 to 21 of the EGFR gene.1Russo A. Franchina T. Ricciardi G.R.R. et al.A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): old successes and future perspectives.Oncotarget. 2015; 6: 26814-26825Crossref PubMed Scopus (131) Google Scholar Among the “uncommon mutations,” EGFR exon 20 insertions (EGFR ex20ins) have recently gained great interest owing to the approval of two selective inhibitors, amivantamab and mobocertinib. These mutations are short genetic in-frame insertions and 3- to 21-bp duplications (corresponding to 1–7 amino acids) that can occur within amino acids 762 and 774 and identify a highly heterogeneous subgroup of EGFR mutations with up to 122 different variants reported to date.2Friedlaender A. Subbiah V. Russo A. et al.EGFR and HER2 exon 20 insertions in solid tumours: from biology to treatment.Nat Rev Clin Oncol. 2022; 19: 51-69Crossref PubMed Scopus (60) Google Scholar On the basis of their location, EGFR ex20ins can be classified as near loop (AA 767–772; approximately 70%), far loop (AA 773–775; approximately 30%), and the helical mutants (AA 762–766; approximately 3%–6%).3Viteri S. Minchom A. Bazhenova L. et al.Frequency, underdiagnosis, and heterogeneity of epidermal growth factor receptor exon 20 insertion mutations using real-world genomic datasets.Mol Oncol. 2023; 17: 230-237Crossref PubMed Scopus (0) Google Scholar These mutations were traditionally considered resistant to all the available EGFR TKIs, including the third-generation inhibitor osimertinib, with very few exceptions, as mutations between codons 762 and 768 or those containing a glycine at position 770, and were preferably treated with platinum-based chemotherapy. In the past few years, we have witnessed a paradigm shift in molecular testing in advanced NSCLC moving from single-gene testing to next-generation sequencing (NGS), owing to the constant grow of the list of targetable oncogenes, making the use of multigene testing more cost effective.4Pennell N.A. Mutebi A. Zhou Z.Y. et al.Economic impact of next-generation sequencing versus single-gene testing to detect genomic alterations in metastatic non-small-cell lung cancer using a decision analytic model.JCO Precis Oncol. 2019; 3: 1-9Crossref PubMed Scopus (93) Google Scholar This major change has also affected liquid biopsy, in which plasma NGS is rapidly replacing polymerase chain reaction (PCR)–based technologies for cell-free DNA analysis, expanding its use beyond EGFR mutational status.5Rolfo C. Mack P. Scagliotti G.V. et al.Liquid biopsy for advanced NSCLC: a consensus statement from the International Association for the Study of Lung Cancer.J Thorac Oncol. 2021; 16: 1647-1662Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar NGS-based assays are associated with several advantages. The increasing use of NGS in both tissue and plasma leads to higher detection of low-frequency genetic aberrations, such as EGFR ex20ins, which were missed by conventional PCR-based approaches for EGFR testing. With the increasing use of NGS-based approaches, the incidence of EGFR ex20ins is destined to grow in the next few years. In addition, NGS can identify novel variants or very rare mutations that are not included in the limited set of EGFR ex20ins coverage of most of the commercially available PCR-based assays. Last, NGS-based approaches can identify the presence of co-mutations, which are increasingly associated with poorer prognosis, including TP53 mutations in EGFR ex20ins NSCLCs.6Christopoulos P. Kluck K. Kirchner M. et al.The impact of TP53 co-mutations and immunologic microenvironment on outcome of lung cancer with EGFR exon 20 insertions.Eur J Cancer. 2022; 170: 106-118Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Therefore, it is not surprising that in the two registrative trials of amivantamab and mobocertinib, NGS-based approaches were used for EGFR ex20ins detection for biomarker analyses (patients were enrolled on the basis of local testing). Indeed, amivantamab was co-developed with two companion diagnostics, Guardant360 CDx (74-gene plasma NGS assay) and Oncomine Dx Target Test (46-gene tissue NGS assay), for EGFR ex20ins detection in the plasma and tissue, respectively.7Park K. Haura E.B. Leighl N.B. et al.Amivantamab in EGFR Exon 20 insertion-mutated non-small-cell lung cancer progressing on platinum chemotherapy: initial results from the CHRYSALIS phase I study.J Clin Oncol. 2021; 39: 3391-3402Crossref PubMed Google Scholar In the EXCLAIM trial, EGFR ex20ins were detected in the tissue with the Oncomine Dx Target Test or with the TruSight Tumor 170 Assay, a NGS assay that simultaneously analyzes DNA and RNA covering 170 genes.8Zhou C. Ramalingam S.S. Kim T.M. et al.Treatment outcomes and safety of mobocertinib in platinum-pretreated patients with EGFR exon 20 insertion-positive metastatic non-small cell lung cancer: a phase 1/2 open-label nonrandomized clinical trial.JAMA Oncol. 2021; 7e214761Crossref Scopus (94) Google Scholar Nevertheless, PCR-based assays are still largely used in clinical practice owing to the low turnaround time, relatively low costs, and large availability of this technology. Hence, in clinical practice, the use of NGS is still underutilized with more than 50% of the patients still receiving single-gene testing.9Robert N.J. Espirito J.L. Chen L. et al.Biomarker testing and tissue journey among patients with metastatic non-small cell lung cancer receiving first-line therapy in the US Oncology Network.Lung Cancer. 2022; 166: 197-204Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar The differential impact of NGS and PCR-based assays on EGFR ex20ins detection has been recently analyzed in two large retrospective analyses comparing the performance of these two technologies. Viteri et al.3Viteri S. Minchom A. Bazhenova L. et al.Frequency, underdiagnosis, and heterogeneity of epidermal growth factor receptor exon 20 insertion mutations using real-world genomic datasets.Mol Oncol. 2023; 17: 230-237Crossref PubMed Scopus (0) Google Scholar evaluated the real-world frequency of EGFR ex20ins using three large U.S. genomic NGS data sets with both tissue (GENIE and FoundationInsights) and plasma (GuardantINFORM) data with a reported frequency of approximately 7% to 8% of all lung adenocarcinomas. Interestingly, applying the coverage of EGFR ex20ins from two widely used commercially available PCR-based assays (EGFR RGQ PCR Kit version 2 and Cobas EGFR version 2), they revealed that approximately 50% of all EGFR ex20ins identified by NGS would have been missed using these tests. In this issue, Ou et al.10Ou S.-H.I Hong J.L. Christopoulos P. et al.Distribution and detectability of EGFR exon 20 insertion variants in non-small cell lung cancer.J Thorac Oncol. 2023; 18: 744-754Abstract Full Text Full Text PDF Scopus (0) Google Scholar reported the results of a large retrospective analysis (n = 636 patients, 104 unique variants), including patients with NSCLC harboring an EGFR ex20ins from a global phase 1/2 clinical trial (EXCLAIM), and real-world data from five data sets (a chart review study from Germany, the LC-SCRUM-Japan, GENIE, and U.S. COTA). The aim of this study was to evaluate the capability of six commercially available PCR-based assays versus NGS to identify EGFR ex20ins in patients with NSCLC, on the basis of the manufacturer-provided coverage information. Collectively, standard PCR kits would have missed more than 40% of patients harboring an EGFR ex20ins as compared with NGS, as these assays are designed to detect only the most prevalent EGFR mutations, with only one to seven EGFR ex20ins variants identified.10Ou S.-H.I Hong J.L. Christopoulos P. et al.Distribution and detectability of EGFR exon 20 insertion variants in non-small cell lung cancer.J Thorac Oncol. 2023; 18: 744-754Abstract Full Text Full Text PDF Scopus (0) Google Scholar The results of these two studies further reinforce the notion that NGS-based tests should replace single-gene testing for tumor genotyping of advanced NSCLCs. The missed identification of EGFR ex20ins might result in poorer outcomes for these patients, precluding the access to effective targeted therapies, but also exposing them to potentially ineffective drugs, such as immune checkpoint inhibitors. This is a common problem with other clinically relevant oncogenic driver with a low prevalence, such as HER2 mutations, RET rearrangements, MET exon 14 skipping mutations, and NTRK 1 to 3 rearrangements, which are associated with high sensitivities to appropriate targeted agents but are often undertested in clinical practice. Increasing testing rates of these biomarkers is essential to fully translate in clinical practice the unprecedented results found in clinical trials with targeted therapies in these molecularly selected subgroups of NSCLCs (Fig. 1). In addition, most PCR-based tests do not discriminate the exact EGFR ex20ins variant. Nevertheless, the notion of the specific EGFR ex20ins variant can be useful in clinical practice, as these mutations are highly heterogeneous and sensitivity to some ex20ins-specific EGFR TKIs and older generation TKIs seems influenced by their location. For instance, far-loop mutants have been recently found to be less sensitive to poziotinib than near-loop mutants (overall response rate 0% and disease control rate 67% versus overall response rate 46% and disease control rate 84%, respectively).11Elamin Y.Y. Robichaux J.P. Carter B.W. et al.Poziotinib for EGFR exon 20-mutant NSCLC: clinical efficacy, resistance mechanisms, and impact of insertion location on drug sensitivity.Cancer Cell. 2022; 40: 754-767.e6Abstract Full Text Full Text PDF PubMed Google Scholar Furthermore, most helical mutations are EGFR TKI sensitive,2Friedlaender A. Subbiah V. Russo A. et al.EGFR and HER2 exon 20 insertions in solid tumours: from biology to treatment.Nat Rev Clin Oncol. 2022; 19: 51-69Crossref PubMed Scopus (60) Google Scholar and the use of former EGFR TKIs can be proposed. In contrast, activity of mobocertinib and amivantamab seems independent of mutation position.7Park K. Haura E.B. Leighl N.B. et al.Amivantamab in EGFR Exon 20 insertion-mutated non-small-cell lung cancer progressing on platinum chemotherapy: initial results from the CHRYSALIS phase I study.J Clin Oncol. 2021; 39: 3391-3402Crossref PubMed Google Scholar,8Zhou C. Ramalingam S.S. Kim T.M. et al.Treatment outcomes and safety of mobocertinib in platinum-pretreated patients with EGFR exon 20 insertion-positive metastatic non-small cell lung cancer: a phase 1/2 open-label nonrandomized clinical trial.JAMA Oncol. 2021; 7e214761Crossref Scopus (94) Google Scholar The identification of concomitant mutations (i.e., TP53 mutations, RB1 mutations), only possible with NGS, albeit currently useful exclusively for prognostic considerations, in the next future could support escalating and de-escalating strategies in these patients. Finally, the use of liquid biopsy, namely cell-free DNA NGS analysis, can further increase the testing rates for EGFR ex20ins and other uncommon oncogenic drivers in advanced NSCLC, which is historically characterized by tissue availability issues. The tissue and plasma should be identified as complementary tools for tumor genotyping, and optimal use of all the available resources can spread the identification of all the relevant biomarkers with clinically approved targeted therapies, not leaving behind any single patient who might benefit from these agents. In conclusion, the study of Ou et al.10Ou S.-H.I Hong J.L. Christopoulos P. et al.Distribution and detectability of EGFR exon 20 insertion variants in non-small cell lung cancer.J Thorac Oncol. 2023; 18: 744-754Abstract Full Text Full Text PDF Scopus (0) Google Scholar adds further evidence to the clinical utility of NGS in advanced NSCLC and should represent the definitive standard for tissue and plasma testing in this disease, progressively replacing single-gene testing strategies that should be limited to selected cases. Only adopting NGS on a large-scale basis we can finally reach the goal of delivering appropriate treatments to all the patients with specific oncogene drivers, finally entering in the era of personalized medicine for all the approved therapeutic targets, and not only for those more common. Christian Rolfo, Alessandro Russo: Conceptualization; Writing—original draft; Writing—review and editing; Supervision; Project administration. Distribution and Detectability of EGFR Exon 20 Insertion Variants in NSCLCJournal of Thoracic OncologyVol. 18Issue 6PreviewEGFR exon 20 insertion (ex20ins) mutations represent 5% to 10% of EGFR mutations in NSCLC. Identifying patients with EGFR ex20ins is challenging owing to the limited coverage of polymerase chain reaction (PCR) assays and the relatively recent use of next-generation sequencing (NGS). This study analyzes the spectrum of EGFR ex20ins variants in a large patient population from a global clinical trial and several real-world cohorts and the ability of PCR kits to identify these alterations. Full-Text PDF Open Access