Frequently used quantitative polymerase chain reaction–based methods overlook potential clinically relevant genetic alterations in epidermal growth factor receptor compared with next-generation sequencing: a retrospective clinical comparison of 1839 lung adenocarcinomas

Aims: The aim of the study was to investigate the advantage of implementing next-generation sequencing (NGS) compared with quantitative polymerase chain reaction (qPCR) when performing routine molecular diagnostics in adenocarcinomas of the lung. Methods: The study is a retrospective cross-sectional observational study of 1839 cytological and histological adenocarcinoma biopsies investigated for gene mutations from 2016 to 2018 at the Department of Pathology at Aarhus University Hospital. A total of 1169 samples were analyzed by qPCR for the presence of EGFR hotspot mutations from 2016 to 2017. A total of 670 samples were analyzed with NGS for the presence of EGFR mutations and other gene mutations in 2018. Results: The average frequency of EGFR mutations in the study population was 11.5%, with the highest frequency found in 2018, where NGS was implemented (10.8% in 2016, 11.5% in 2017, and 12.2% in 2018). Possible therapy resistance markers such as EGFR exon 20 mutations were found more commonly after NGS implementation, the difference being statistically significant (P = .015). In addition, NGS (2018) showed that 40.6% of the samples had KRAS mutations and 6.0% had BRAF mutations, mutations not commonly investigated in lung adenocarcinomas when qPCR is the method of choice. Among the EGFR-mutated samples analyzed with NGS, 13 contained a concurrent EGFR mutation, whereas three and two contained a concurrent KRAS and BRAF mutations, respectively. Conclusions: With the implementation in a clinical setting, NGS identifies more uncommon but potentially clinically important EGFR mutations, unique combinations of EGFR mutations, and concurrent mutations in KRAS and BRAF.