Comprehensive analysis of next generation sequencing and ARMS-PCR for detecting EGFR exon 20 insertion (ex20ins) mutations in Chinese non-small cell lung cancer patients

Introduction

Lung cancer is a leading cause of cancer incidence and mortality with an estimated 1.2 million diagnosis and 1.8 million deaths per year, globally (1). Non-small cell lung cancer (NSCLC) accounts for 85–90% of the lung cancer cases (2,3). Approximately 30% of NSCLC tumors harbor a mutation in the EGFR gene (4), with geographical variation in rates, the frequency of epidermal growth factor receptor (EGFR) genomic alterations was identified as 50.1% in Chinese NSCLC populations and significantly higher than the Western population, which was about 15% (5,6), and predominantly found in female, non-smoking, adenocarcinoma patients (7). EGFR tyrosine kinase inhibitors (TKIs) have led to better management and a standard of care for patients with recurrent or metastatic NSCLC that harbor EGFR activating mutations, such as L858R in exon 21 and in-frame deletions in exon 19 (ex19del), which are considered as classical mutations and account for almost 85% of observed EGFR mutations in NSCLC (8-10). With the wide application of next generation sequencing (NGS) in clinical practice, more and more rare mutations of EGFR have been discovered, of which EGFR exon 20 insertion (ex20ins) mutations constitute about 4–12% of all EGFR mutation types, and hence, are the third most common type of EGFR mutations in NSCLC, following ex19del and L858R point mutation (11-13). EGFR ex20ins mutations are reported to occur either toward the C terminal of the C-helix or in the loop that immediately follows it, and they lead to a prominent shift of the C-helix and P-loop into the drug-binding pocket, resulting in significant steric hindrance and limiting its binding to the traditional EGFR-TKIs (14,15). Thus, contrary to classical EGFR mutations that are sensitive to EGFR-TKIs, EGFR ex20ins mutations are associated with poor responses to approved first and second EGFR-TKIs (14). Further, NSCLC patients with EGFR ex20ins are known to have poorer prognosis compared to patients with other sensitizing EGFR mutations leading to unmet clinical need of newer specific therapeutic options (16). Fortunately, the US Food and Drug Administration (FDA) has approved mobocertinib (EGFR-TKI) and the EGFR-MET bispecific antibody amivantamab, as targeted treatments for adult patients with locally advanced or metastatic NSCLC with EGFR ex20ins in 2021 (17,18). In several clinical trials, the two targeted drugs were associated with clinically meaningful benefit in patients with previously treated EGFR ex20ins-positive NSCLC, with a manageable safety profile (19,20). Given its importance for diagnosis and treatment, accurate detection of EGFR ex20ins mutations is particularly important.

Several molecular diagnostic methods are currently used for the identification of EGFR ex20ins in NSCLC patients, including amplification refractory mutation system (ARMS), droplet digital polymerase chain reaction (ddPCR), sanger sequencing and NGS. Among them, ARMS and NGS are the most common methods recommended to detect EGFR mutations in domestic and foreign Clinical Practice Guidelines. ARMS is known to explore only a small number of certain hot spot EGFR mutations, which usually just contain several EGFR ex20ins variants. Therascreen (21,22) and Cobas EGFR v2 (23) are the two FDA approved PCR methods used for the assessment of EGFR mutations. Therascreen can detect three types of ex20ins, and Cobas EGFR v2 can detect five types of ex20ins. However, based on NGS data, so far, studies have identified at least 85 unique ex20ins variants in the Chinese population and 102 variants in the US population, with A767_V769dup and S768_D770dup being the most common ones (24,25). Therefore, the National Comprehensive Cancer Network (NCCN) and European Society of Medical Oncology (ESMO) guidelines currently recommend NGS to assess oncogenic drivers, including EGFR ex20ins in NSCLC patients (26,27). However, the efficacy of ARMS and NGS in detecting EGFR ex20ins mutations is unclear in clinical practice.

Thus, in this study, we systematically analyzed and compared EGFR mutations in 3,606 Chinese NSCLC patients detected by NGS and 1,785 patients by ARMS. Further, we predicted the false negative rate of EGFR ex20ins mutation detection using different PCR kits in the real world based on the EGFR ex20ins variants detected by NGS. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-848/rc).

Methods

Patients and samples

A total of 3,606 patients diagnosed with primary or metastatic NSCLC in Fudan University Shanghai Cancer Center during July 2017 and June 2022 were submitted for detection of EGFR gene mutations by NGS using a capture-based targeted sequencing panel (Burning Rock Biotech, Guangzhou, China), including all exons in 68 genes (Table S1). And a total of 1,785 NSCLC patients recruited from March 2019 to June 2022 were detected using ADx-ARMS EGFR kit (Amoy Diagnostics CO., ltd., Xiamen, China), which can detect 18 variants of EGFR ex20ins mutations (Table 1). All test materials were formalin-fixed paraffin-embedded (FFPE) tumor samples, including surgical specimens, fine-needle aspirate and cytology specimens. All the samples were evaluated by an experienced pathologist prior to testing, and only samples with tumor cell content higher than 10% were further tested and analyzed. Clinicopathologic features, including patient age, gender, smoking history, pathological types and clinical stage, were obtained from the medical record, pathology report, and/or discharge summary. About 90% of the patients received the NGS and ARMS EGFR detection were treatment naïve. The study basically shown the original EGFR ex20ins mutation rates. Ethical approval was obtained from the Research Ethics Committee of Fudan University Shanghai Cancer Center (No. 050432-4-2108*). Informed consent was waived due to the retrospective nature of the study. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

DNA-based NGS

Total genomic DNA was extracted from tissue samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. Genomic DNA was profiled by using a capture-based targeted sequencing panel (Burning Rock Biotech), including all exons in 68 genes. The concentration of the DNA samples was measured with the Qubit dsDNA assay (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was then sheered to 300 bp using a Covaris S220 Focused ultrasonicator (Covaris, Woburn, Massachusetts, USA), followed by hybridization with the capture probe baits, hybrid selection with magnetic beads, and PCR amplification. QIAxcel Advanced automated nucleic acid analysis system (Qiagen) was then used to assess the quality and size range. Available indexed samples were then sequenced on a Nextseq 550 System (Illumina, San Diego, CA, USA) with paired-end reads. The sequencing data were mapped to the human genome (hg19) using Burrows-Wheeler Aligner version 0.7.10. Local alignment optimization, variant calling, and annotation were performed using the Genome Analysis Toolkit version 3.2 and VarScan version 2.4.3.

ARMS

Total DNA was extracted from three to four sections of 5µm thick FFPE tissues using a FFPE DNA kit (Qiagen GmbH, Hilden, Germany). The EGFR mutation was readily detected using the ARMS commercial regent (Amoy) according to the manufacturer’s protocol. The following PCR procedure was used: An initial denaturation at 95 °C for 5 min, followed by 95 °C for 25 sec, 64 °C for 20 sec and 72 °C for 20 sec to ensure the specificity, and 31 cycles of 93 °C for 25 sec, 60 °C for 35 sec and 72 °C for 20 sec to perform the data collection.

Statistical analyses

Fisher’s exact test was used to compare the gender, age, and smoking history of EGFR mutation and EGFR ex20ins groups. Chi-squared test for trend was used to analyze the relationship between EGFR mutations and pathological type and clinical stage. P<0.05 was defined as significant in the analysis.

Results

Clinical characteristics of NSCLC patients with EGFR mutations detected by NGS and ARMS

Among the 3,606 NSCLC patients detected by NGS, 2,077 (57.6%, 2,077/3,606) patients had EGFR mutations. Ninety-five patients had EGFR ex20ins mutations, contributing to 2.6% of all NSCLC cases (95/3,606) and 4.6% of EGFR-mutant tumors (95/2,077). The 3,606 NSCLC patients comprised 1,636 men and 1,970 women, at a median age of 74, ranged from 22–86 years old. The 2,077 patients with EGFR mutations comprised 724 men and 1,353 women, at a median age of 61, ranged from 22–86 years old. The 95 EGFR ex20ins patients included 33 men and 62 women. The median age was 57 years old, with a range of 22–79 years old. Among the patients with EGFR mutation, only 15.9% (330/2,077) patients were ≤50 years old, while the proportion was as high as 46.3% (44/95) in patients with EGFR ex20ins, the difference was statistically significant (P<0.001). Most of the NSCLC patients with EGFR mutations and/or EGFR ex20ins were non-smokers and adenocarcinoma with earlier clinical stage (I and II). There was a significant difference in clinical stages between EGFR mutation and EGFR ex20ins groups (P=0.05), and EGFR ex20ins mutations were more commonly identified in clinical stage I and II. A total of 1,785 NSCLC patients detected EGFR mutations by ARMS assay, most of them were in clinical stage III or IV. Eight hundred and sixty-four (48.4%, 864/1,785) patients had EGFR mutations, and only 20 patients had EGFR ex20ins, contributing to 1.1% of all NSCLC cases (20/1,785) and 2.3% of EGFR-mutant tumors (20/864). Similar with the NGS detection group, women, non-smokers, >50 years old and adenocarcinoma patients accounted for the majority of patients with EGFR mutations. However, no statistical significance for EGFR ex20ins was found among the different clinical stage groups (Table 2).

EGFR mutations and ex20ins in NSCLC patients detected by NGS

A total of 2,077 EGFR mutations, including 1,850 unique and 227 complex variants, were identified by NGS, accounting for 57.6% of the 3,606 NSCLC patients. In 1,850 EGFR unique mutations, exon 18 mutations (n=44, 2.4%), exon 19 mutations (n=759, 41.0%), exon 20 mutations (n=111, 6.0%; ex20ins, n=95, 5.1%), exon 21 mutations (n=886, 47.9%) and other exon mutations (n=50, 2.7%) were identified. In 227 EGFR complex variants, which indicating two to four EGFR mutations occurred simultaneously, exon 20 mutations accounted for 41.9% (95/227), most of them were T790M combined with L858R or ex19del, no complex variants with EGFR ex20ins were found (Figure 1).

Figure 1 Frequency and distribution of 2077 EGFR mutations in 3,606 NSCLC detected by NGS. NGS, next generation sequencing; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer.

Among the total of 206 EGFR exon 20 mutation patients, 95 unique EGFR ex20ins mutations, 16 unique exon20 other mutations and 95 complex exon20 other mutations were identified (Figure 2). Of the 95 EGFR ex20ins mutations, 34 unique ex20ins variants were identified by NGS (Table 1). The most frequent variant of EGFR ex20ins was A767_V769dup (29.5%, 28/95), followed by S768_D770dup (15.8%, 15/95), N771_H773dup (7.4%, 7/95) and P772_H773dup (7.4%, 7/95). These four types of mutations each accounted for over 5% of all EGFR ex20ins mutations. Additionally, 25 types of EGFR ex20ins variants were present in only one patient each, and interestingly, eight of which has never been reported according to Catalogue of Somatic Mutations in Cancer (https://cancer.sanger.ac.uk/cosmic) and cBioPortal databases (http://www.cbioportal.org/). EGFR ex20ins variants can be divided into two types: near-loop (A767-P772) and far-loop (H773-C775), based on the site of EGFR ex20ins helical region. Twenty-six kinds of EGFR ex20ins near-loop variants were found in 83 patients, while only eight kinds of EGFR ex20ins far-loop variants were found in 12 patients. EGFR ex20ins mutations detected by NGS are summarized in Figure 2 and Table 1.

Figure 2 Frequency of EGFR exon 20 insertions detected by NGS. NGS, next generation sequencing; EGFR, epidermal growth factor receptor.

EGFR mutations and ex20ins in NSCLC patients detected by ARMS

A total of 864 EGFR mutations, including 825 unique and 39 complex variants, were identified by ARMS, accounting for 48.4% of the 1,785 NSCLC patients. In 825 EGFR unique mutations, exon 18 mutation (n=23, 2.8%), exon 19 mutation (n=358, 43.4%), exon 20 mutation (n=27, 3.3%) and exon 21 mutation (n=417, 50.5%) were demonstrated. In 39 EGFR complex mutations, exon 20 accounted for 87.2% (34/39) (Figure 3). Similar with NGS group, most patients were T790M combined with L858R or ex19del.

Figure 3 Frequency and distribution of 864 EGFR mutations and 61 EGFR exon 20 mutation in 1,785 NSCLC detected by ARMS. ARMS, amplification refractory mutation system; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer.

Among the 61 EGFR exon 20 mutation patients, 19 unique EGFR ex20ins mutations, eight unique exon20 other mutations and one complex EGFR ex20ins mutations co-occurred with EGFR exon21 mutation, 33 complex exon20 other mutations were identified (Figure 3). In total, 20 patients with EGFR ex20ins were identified by ARMS, contributing 1.1% of all NSCLC cases (20/1,785) and 2.3% of EGFR-mutant cases (20/864), respectively (Table 2).

The comparison of frequencies of EGFR ex20ins in NSCLC patients detected by NGS and ARMS-PCR

Base on the above results, it is obvious that the positive rate of EGFR ex20ins mutations detected by ARMS method was significantly lower than that by NGS method, and the differences were statistically significant (P<0.01). In order to explore the potential causes, we carefully checked the design principle of ARMS method and found that it can just detect only 18 known EGFR ex20ins variants as shown in Table 1. Compared with 34 unique EGFR ex20ins variants in our study by NGS, only seven variants could be detected by ADx-ARMS EGFR kit mutation used in this study, accounting for 42.1% (40/95) of all EGFR ex20ins mutations by NGS. It meant that if ARMS was used to detect the 3,606 Chinese NSCLC patients tested by NGS, the false negative rate of EGFR ex20ins mutations reached 57.9%.

In addition, the ability to detect EGFR ex20ins mutations in two FDA-approved PCR kits, Therascreen and Cobas EGFR v2, was also evaluated. As shown in Table 1, Therascreen can detect three variants of ex20ins, and Cobas EGFR v2 can detect five types of ex20ins. Unfortunately, only one and two out of our 34 unique EGFR ex20ins variants could be detected, accounting for 1.1% (1/95) and 30.5% (29/95) of all EGFR ex20ins mutations based on our NGS data, respectively. It meant that if these two tests were used in Chinese NSCLC patients, the false negative rate of EGFR ex20ins mutations could be ranged about 69.5% to 98.9%.

Discussion

In NSCLC patients, the EGFR oncogenic driver mutations occur mainly in exons 18, 19, 20 and 21, which encode the tyrosine kinase domain. The in-frame insertion within exon 20 is the third most frequent class of EGFR mutations, accounting for 4–12% of EGFR mutations (14,28,29). Patients with EGFR ex20ins fundamentally have a shorter survival time compared with those who have common EGFR mutations due to the general lack of sensitivity to EGFR TKIs (11). Novel EGFR TKIs targeting EGFR ex20ins are emerging. Recently, amivantamab (JNJ-372) have been approved by the US FDA for the treatment of advanced or metastatic NSCLC patients with EGFR ex20ins mutations (30). Mobocertinib (TAK-788) has been granted priority review by the FDA for the same indication as amivantamab. In a phase 1/2 study, mobocertinib demonstrated antitumor activity in patients with NSCLC with diverse EGFR ex20ins variants with a safety profile consistent with other EGFR inhibitors in the first two parts of a three-part phase I/II study (ClinicalTrials. ID: NCT02716116) (31). In another study, mobocertinib also showed antitumor activity in patients with EGFR ex20ins-positive NSCLC (32,33). Although the sensitivity of most EGFR ex20ins to EGFR TKIs was generally lower, EGFR-A763_Y764ins FQEA is a unique EGFR ex20ins mutation which is sensitizing to clinically available EGFR TKIs (34). Thus, EGFR ex20ins are shown to have different sensitivity to different EGFR TKIs. Accurate detection of EGFR ex20ins is important for the treatment of NSCLC patients. Therefore, the latest NCCN guidelines recommend explicitly reporting specific variant types of EGFR ex20ins in the test reports, better guiding the choice of treatment options (27).

Previous studies showed that the frequency of EGFR ex20ins mutations in Chinese NSCLC patients is about 2.21–2.27% (25,35,36). In this study, a total of 95 EGFR ex20ins mutations were identified in a subset of 3,606 NSCLC patients detected by NGS and 20 EGFR ex20ins were in 1,785 NSCLC patients by ARMS, with the frequency of EGFR ex20ins in Chinese NSCLC patients as 2.6% and 1.1%, respectively. The frequency of EGFR ex20ins of NGS detection in our results was slightly higher than that in previous studies and significantly higher than that by ARMS. The result was consistent with He et al. (37). They found the mutation rate of ex20ins by ARMS-PCR detection (1.3%) were statistically significantly lower than that of NGS detection (2.0%). In NGS patients, 46.3% (44/95) EGFR ex20ins patients were ≤50 years old and most of them were in clinical stages I and II. However, in ARMS patients, only 25.0% (5/20) EGFR ex20ins patients were ≤50 years old and most of them were in clinical stage III or IV.

The types of EGFR ex20ins mutations are numerous. Targeting EGFR ex20ins will be more complicated than targeting the classic EGFR mutations, L858R and ex19del, as the insertions are too diversified to support a one for-all solution. In the previous studies, 39–85 different molecular variants of EGFR ex20ins were identified in Chinese NSCLC patients with A767_V769dup and S768_D770dup being the most prevalent ones (38,39). Our study showed that 34 different subtypes of insertion variants were recorded in NGS, and the two most common EGFR ex20ins variants were consistent with previous studies, A767_V769dup (29.5%) and S768_D770dup (15.8%), comprised 45.3% of all EGFR ex20ins and can be the main targets in future drug development efforts. However, eight kinds of EGFR ex20ins variants were identified in our study, which were reported for the first time according to COSMIC and cBioPortal databases. These results suggested that accurate detection of EGFR ex20ins and subsequent individualized treatment are still full of challenges.

NGS has the advantages of high flux and diverse detection types in detecting gene mutations. However, it also meets the problem of high cost in time and economy. From nuclein extraction to report diagnosis, it will take up about 14 days and the high expense of machine and reagent make it hard to popularize in the primary hospitals in China. Although ESMO 2022 suggested that NGS assay should be prioritized for EGFR ex20ins analysis to allow broader detection and characterization, ARMS is still one of the most common methods to detect EGFR gene mutations in Chinese NSCLC patients because of its simple operation, faster reports, reliable and cost-effectiveness. However, it has several limitations to detect EGFR ex20ins mutations. First, the reason for ARMS with the high sensitivity is its peculiar primer design, but it’s also the limitation. The primers make ARMS applies only in the detection of known mutations. The currently approved kits in China only cover up to 18 specific somatic insertion mutations in exon 20 of the EGFR oncogene (Table 1), while more than 100 EGFR ex20ins mutation types are currently identified and more novel previously unidentified mutations are being discovered. In our study, the second most frequent variant of EGFR ex20ins detected by NGS, S768_D770dup, was not included in the ADx-ARMS-EGFR kit. Based on the results of the NGS, the frequency of the 18 mutations detected by the ARMS kit were not high, the primers are designed based on the Western population, it may lead to EGFR mutations missed in the Chinese population. Second, EGFR ex20ins were shown to have different sensitivity to different EGFR TKIs. For example, one study found that poziotinib sensitivity was highly dependent on the insertion location, with near-loop insertions (amino acids A767 to P772) being more sensitive than far-loop insertions, an observation confirmed clinically with objective response rates (ORRs) of 46% and 0% observed in near versus far-loop, respectively (40). In this study, we found eight kinds of EGFR ex20ins far-loop variants and 26 kinds of near-loop variants by NGS. Thus, a structure-function-based approach may improve the prediction of drug sensitivity to targeted therapies for EGFR ex20ins mutations (41). The NCCN guidelines recommend reporting specific variant types of EGFR ex20ins in test reports (42). However, the ARMS testing could not report the specific variant type. Third, the ARMS detection kit currently does not cover the newly discovered rare lung cancer targets, such as FGFR2 fusion, NGR1 fusion, etc. Considering the advantages and disadvantages of NGS and ARMS, National Health Service (NHS) England guidance that describes a salvage testing pathway for patients with advanced lung cancer who would not survive to see the potential beneficial repercussions of NGS-based mutation detection. It allows local testing to continue by rapid PCR methods in a context of genomic testing in centralized laboratory hubs (43). Therefore, the commercial allele-specific ARMS testing solutions cannot fully meet clinical needs for EGFR ex20ins detection, and need iterative upgrading.

Conclusions

More and more variants of EGFR ex20ins mutations have been identified, and NGS has obvious advantages and is strongly recommended for the detection of EGFR ex20ins mutations. Using the existing PCR-based detection kit to detect EGFR ex20ins mutations has a high false negative rate. Considering the fast and cost-effective ARMS detection method, it suggests that the primers design should be updated according to the characteristics of EGFR ex20ins mutations in Chinese NSCLC patients.

Acknowledgments

Funding: This research was financially supported by the Innovation Group Project of Shanghai Municipal Health Commission (Project No. 2019CXJQ03), the Shanghai Science and Technology Development Fund (Project No. 19MC1911000), the Shanghai Municipal Key Clinical Specialty (Project No. shslczdzk01301) and the Innovation Program of Shanghai Science and Technology Committee (Project No. 20Z11900300).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-848/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-848/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-848/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-848/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ethical approval was obtained from the Research Ethics Committee of Fudan University Shanghai Cancer Center (No. 050432-4-2108*). Informed consent was waived due to the retrospective nature of the study. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Thandra KC, Barsouk A, Saginala K, et al. Epidemiology of lung cancer. Contemp Oncol (Pozn) 2021;25:45-52. [Crossref] [PubMed]
2. Li Y, Yan B, He S. Advances and challenges in the treatment of lung cancer. Biomed Pharmacother 2023;169:115891. [Crossref] [PubMed]
3. Abdelaziz HM, Gaber M, Abd-Elwakil MM, et al. Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates. J Control Release 2018;269:374-92. [Crossref] [PubMed]
4. Zhang YL, Yuan JQ, Wang KF, et al. The prevalence of EGFR mutation in patients with non-small cell lung cancer: a systematic review and meta-analysis. Oncotarget 2016;7:78985-93. [Crossref] [PubMed]
5. Chan BA, Hughes BG. Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Transl Lung Cancer Res 2015;4:36-54. [PubMed]
6. Wen S, Dai L, Wang L, et al. Genomic Signature of Driver Genes Identified by Target Next-Generation Sequencing in Chinese Non-Small Cell Lung Cancer. Oncologist 2019;24:e1070-81. [Crossref] [PubMed]
7. Zhang T, Joubert P, Ansari-Pour N, et al. Genomic and evolutionary classification of lung cancer in never smokers. Nat Genet 2021;53:1348-59. [Crossref] [PubMed]
8. Soria JC, Ohe Y, Vansteenkiste J, et al. Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer. N Engl J Med 2018;378:113-25. [Crossref] [PubMed]
9. Reungwetwattana T, Nakagawa K, Cho BC, et al. CNS Response to Osimertinib Versus Standard Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients With Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer. J Clin Oncol 2018; Epub ahead of print. [Crossref] [PubMed]
10. Vyse S, Huang PH. Targeting EGFR exon 20 insertion mutations in non-small cell lung cancer. Signal Transduct Target Ther 2019;4:5. [Crossref] [PubMed]
11. Arcila ME, Nafa K, Chaft JE, et al. EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics. Mol Cancer Ther 2013;12:220-9. [Crossref] [PubMed]
12. Zappa C, Mousa SA. Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res 2016;5:288-300. [Crossref] [PubMed]
13. Burnett H, Emich H, Carroll C, et al. Epidemiological and clinical burden of EGFR Exon 20 insertion in advanced non-small cell lung cancer: A systematic literature review. PLoS One 2021;16:e0247620. [Crossref] [PubMed]
14. Yasuda H, Park E, Yun CH, et al. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer. Sci Transl Med 2013;5:216ra177. [Crossref] [PubMed]
15. Robichaux JP, Elamin YY, Tan Z, et al. Mechanisms and clinical activity of an EGFR and HER2 exon 20-selective kinase inhibitor in non-small cell lung cancer. Nat Med 2018;24:638-46. [Crossref] [PubMed]
16. Hirose T, Ikegami M, Endo M, et al. Extensive functional evaluation of exon 20 insertion mutations of EGFR. Lung Cancer 2021;152:135-42. [Crossref] [PubMed]
17. Markham A. Mobocertinib: First Approval. Drugs 2021;81:2069-74. [Crossref] [PubMed]
18. Syed YY. Amivantamab: First Approval. Drugs 2021;81:1349-53. [Crossref] [PubMed]
19. Zhang SS, Zhu VW. Spotlight on Mobocertinib (TAK-788) in NSCLC with EGFR Exon 20 Insertion Mutations. Lung Cancer (Auckl) 2021;12:61-5. [Crossref] [PubMed]
20. Zhou C, Ramalingam SS, Kim TM, 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;7(12):e214761. Correction appears in JAMA Oncol 2022;8:1359.
21. Vallée A, Le Loupp AG, Denis MG. Efficiency of the Therascreen® RGQ PCR kit for the detection of EGFR mutations in non-small cell lung carcinomas. Clin Chim Acta 2014;429:8-11. [Crossref] [PubMed]
22. Hsiue EH, Lee JH, Lin CC, et al. Profile of the therascreen® EGFR RGQ PCR kit as a companion diagnostic for gefitinib in non-small cell lung cancer. Expert Rev Mol Diagn 2016;16:1251-7. [Crossref] [PubMed]
23. Malapelle U, Sirera R, Jantus-Lewintre E, et al. Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer. Expert Rev Mol Diagn 2017;17:209-15. [Crossref] [PubMed]
24. Bauml JM, Viteri S, Minchom A, et al. FP07.12 Underdiagnosis of EGFR Exon 20 Insertion Mutation Variants: Estimates from NGS-based Real-World Datasets. J Thorac Oncol 2021;16:S208-9. [Crossref]
25. Qin Y, Jian H, Tong X, et al. Variability of EGFR exon 20 insertions in 24 468 Chinese lung cancer patients and their divergent responses to EGFR inhibitors. Mol Oncol 2020;14:1695-704. [Crossref] [PubMed]
26. Mosele F, Remon J, Mateo J, et al. Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group. Ann Oncol 2020;31:1491-505. [Crossref] [PubMed]
27. Ettinger DS, Wood DE, Aisner DL, et al. Non-Small Cell Lung Cancer, Version 3.2022, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2022;20:497-530. [Crossref] [PubMed]
28. Lassalle S, Hofman V, Heeke S, et al. Targeted Assessment of the EGFR Status as Reflex Testing in Treatment-Naive Non-Squamous Cell Lung Carcinoma Patients: A Single Laboratory Experience (LPCE, Nice, France). Cancers (Basel) 2020;12:955. [Crossref] [PubMed]
29. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol 2012;13:e23-31. [Crossref] [PubMed]
30. Neijssen J, Cardoso RMF, Chevalier KM, et al. Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET. J Biol Chem 2021;296:100641. [Crossref] [PubMed]
31. Gonzalvez F, Vincent S, Baker TE, et al. Mobocertinib (TAK-788): A Targeted Inhibitor of EGFR Exon 20 Insertion Mutants in Non-Small Cell Lung Cancer. Cancer Discov 2021;11:1672-87. [Crossref] [PubMed]
32. Riely GJ, Neal JW, Camidge DR, et al. Activity and Safety of Mobocertinib (TAK-788) in Previously Treated Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations from a Phase I/II Trial. Cancer Discov 2021;11:1688-99. [Crossref] [PubMed]
33. Janne PA, Neal JW, Camidge DR, et al. Antitumor activity of TAK0788 in NSCLC with EGFR exon 20 insertions. J Clin Oncol 2019;37:9007. [Crossref]
34. Vasconcelos PENS, Gergis C, Viray H, et al. EGFR-A763_Y764insFQEA Is a Unique Exon 20 Insertion Mutation That Displays Sensitivity to Approved and In-Development Lung Cancer EGFR Tyrosine Kinase Inhibitors. JTO Clin Res Rep 2020;1:100051. [Crossref] [PubMed]
35. Song Y, Hu C, Xie Z, et al. Circulating tumor DNA clearance predicts prognosis across treatment regimen in a large real-world longitudinally monitored advanced non-small cell lung cancer cohort. Transl Lung Cancer Res 2020;9:269-79. [Crossref] [PubMed]
36. Liu Z, Wu L, Cao J, et al. Clinical characterization of ERBB2 exon 20 insertions and heterogeneity of outcomes responding to afatinib in Chinese lung cancer patients. Onco Targets Ther 2018;11:7323-31. [Crossref] [PubMed]
37. He C, Wei C, Wen J, et al. Comprehensive analysis of NGS and ARMS PCR for detecting EGFR mutations based on 4467 cases of NSCLC patients. J Cancer Res Clin Oncol 2022;148:321-30. [Crossref] [PubMed]
38. Yang G, Li J, Xu H, et al. EGFR exon 20 insertion mutations in Chinese advanced non-small cell lung cancer patients: Molecular heterogeneity and treatment outcome from nationwide real-world study. Lung Cancer 2020;145:186-94. [Crossref] [PubMed]
39. Xu CW, Wang WX, Wang D, et al. Pemetrexed-based chemotherapy for non-small-cell lung cancer patients with EGFR exon 20 insertion mutation: a multicenter study. Transl Lung Cancer Res 2020;9:1853-61. [Crossref] [PubMed]
40. Elamin YY, Robichaux JP, Carter BW, 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.e6. [Crossref] [PubMed]
41. Robichaux JP, Le X, Vijayan RSK, et al. Structure-based classification predicts drug response in EGFR-mutant NSCLC. Nature 2021;597:732-7. [Crossref] [PubMed]
42. Passaro A, Leighl N, Blackhall F, et al. ESMO expert consensus statements on the management of EGFR mutant non-small-cell lung cancer. Ann Oncol 2022;33:466-87. [Crossref] [PubMed]
43. Group NGSTW. Solid cancer genomics testing: the salvage pathway. London: NHS England and NHS Improvement; 2021. Available online: https://www.england.nhs.uk/publication/nhs-improvement-annual-report-and-accounts-2021-to-2022/