Objective The relevance of EGFR gene amplification in patients with EGFR-mutated advanced non-small cell lung cancer (NSCLC) has not been fully elucidated. Moreover, a limited number of studies have been published on methods to estimate gene amplifications based on routinely performed targeted next generation sequencing (NGS). In this study we aimed to determine whether PCR-based targeted NGS data on tumor tissue can be used to estimate presence of gene amplifications and explored the prognostic value of EGFR gene amplification in EGFR mutated NSCLC patients.

Materials and methods A total of 3,194 good quality targeted NGS data files were retrieved from 2014 to 2017. Among those, 1,729 NSCLC samples originated from 1,586 NSCLC patients of whom 134 had an EGFR mutation (8.2%). Clinical data were available for 66 of the patients. Raw sequencing data were re-analyzed using a custom designed pipeline. The presence of an amplification was based on the read depth of a given amplicon relative to a set of reference amplicons from the sample or relative to a set of normal control samples. Reference amplicons were selected based on low degree of variation in read depth amongst all tested samples. Amplifications were regarded significant when the ratio was ≥3 and the z score was ≥3.5. Technical validation was done by FISH and MLPA. Cox regression analysis on overall survival was done with each of the amplification parameters using SPSS.

Results No amplifications were detected for the ALK, KIT, NRAS, PDGFRA, GNAQ and MAP2K1 gene loci, whereas amplifications for BRAF, ESR1, GNA11, HRAS, KRAS, MET and PIK3CA were observed at a low frequency. Depending on the amplification analysis strategy (within sample or relative to normal controls), 19% and 13% of the EGFR mutated group had an EGFR amplification, respectively. In EGFR wild type patients, amplifications were detected in 5% and 4% of the patients using the two methods, respectively. The sensitivity and specificity of the NGS based estimation of amplifications for EGFR was 94% (14/15) and 97% (34/35) respectively for both data analysis approaches. Patients with concurrent EGFR mutations and amplifications (estimated within sample) treated with EGFR-TKI had a significantly worse overall survival compared with those without concurrent EGFR amplifications (ratio, p=0.047 and z score, p=0.015). Cox regression analysis indicated a borderline significant interaction between ratio and z score (p=0.052).

Conclusion Routinely obtained amplicon-based NGS data can be used to identify gene amplifications. The presence of EGFR amplifications in EGFR mutant patients is predictive of a worse overall survival.

Keywords: Lung adenocarcinoma, EGFR, survival, tyrosine kinase inhibitor

Citation Format: Pei Meng, Jiacong Wei, Miente Martijn Terpstra, Anke van Rijk, Menno Tamminga, Frank Scherpen, Arja ter Elst, Mohamed Z. Alimohamed, Lennart F. Johansson, T. Jeroen N. Hiltermann, Harry J. Groen, Klaas Kok, Anthonie J. van der Wekken, Anke van den Berg. Clinical value of EGFR gene amplifications detected using amplicon based targeted next generation sequencing data in lung adenocarcinoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4335.