Non-small cell lung cancer (NSCLC) comprises approximately 80% to 85% of all lung cancers [1]. In Taiwan, lung cancer is the leading cause of cancer-related deaths, and most patients with lung cancer have stage III or IV disease at time of diagnosis [2]. Therefore, the NCCN Guidelines for NSCLC recommend biomarker testing for actionable oncogenic driver mutations [3]. Targeted therapy has proven to improve the quality of life of patients with specific somatic genomic alterations compared with traditional chemotherapy [3]. In East Asia and Taiwan, more than half of the patients with advanced lung adenocarcinoma have EGFR mutations [4], [5]. Moreover, exon 19 deletions and exon 21 L858R point mutations comprise the majority of these EGFR-mutated NSCLC cases, and lung adenocarcinomas with classic EGFR mutations are generally sensitive to all three generations of EGFR tyrosine kinase inhibitors (EGFR-TKIs) [5]. Owing to the high prevalence of EGFR mutations in Asia, expert consensus recommendations on biomarker testing in metastatic NSCLC in Asia emphasize the need for EGFR testing, including both common (exon 19 deletions and L858R substitutions) and uncommon EGFR mutations [4]. Although upfront next-generation sequencing (NGS) for patients with metastatic NSCLC has cost savings and shorter time-to-test results in the United States, it has been demonstrated that exclusionary testing for EGFR has the shortest time-to-result and is the most cost-saving method in East Asia, including Taiwan [6], [7], [8].

Different EGFR testing methods using formalin-fixed paraffin-embedded (FFPE) samples are available, including Sanger sequencing, PCR-based methods —such as the Roche cobas EGFR mutation test v2 (Roche Molecular Systems, Inc., CA, USA) or the Idylla EGFR mutation test (Biocartis, Mechelen, Belgium)—, and NGS. In Taiwan, Roche cobas EGFR mutation test v2 is the most commonly used method for detecting EGFR mutations in NSCLC. The Roche cobas EGFR mutation test v2 is a real-time polymerase chain reaction (RT-PCR) test that identifies 42 hot spot mutations in exons 18, 19, 20, and 21 of the EGFR gene. Similarly, the Idylla EGFR mutation test is an RT-PCR-based, fully automated in vitro diagnostic test for the qualitative detection of hot spot mutations in exons 18, 19, 20, and 21 of the EGFR gene. Additionally, the PlexBio IntelliPlex Lung Cancer Panel (LCP) (PlexBio; Taipei, Taiwan) is an enriching PCR assay that uses πCode MicroDisc technology to detect specific mutations. The IntelliPlex-LCP-DNA assay can detect 74 hot spot mutations in the KRAS, NRAS, PIK3CA, BRAF, EGFR, ERBB2, MEK1, and AKT1 genes, whereas the IntelliPlex-LCP-RNA assay can detect 28 fusions in the ALK, MET, NTRK1, RET, and ROS1 genes from FFPE specimens. AmoyDx Pan Lung Cancer (PLC) Panel (Amoy Diagnostics, Xiamen, China) is a multigene panel used as a companion diagnostic test for advanced stage lung cancer approved in Japan, China and in Europe. The AmoyDx PLC is designed to detect 167 hotspot mutations and fusions in 11 genes (EGFR, ALK fusion, ROS1 fusion, RET fusion, MET ex14 skipping mutations, ERBB2, BRAF, KRAS, NTRK1/2/3 fusion) by real-time PCR [9]. Amoy PLC was introduced as part of LC-SCRUM Asia genetic screening project with a higher success rate, shorter turnaround time, and higher detection rate than NGS panels like Oncomine Comprehensive Assay and Oncomine Precision Assay [9]. These four PCR-based tests (cobas, Idylla, PlexBio IntelliPlex LCP, AmoyDx PLC) are more sensitive than Sanger sequencing, with an approximate limit of detection (LOD) of 5% of the allele frequency.

Although the cobas EGFR test is a convenient and sensitive test that is widely used in Taiwan, it has some limitations. For example, it does not include the exon 20 C797S mutation and only detects limited types of exon 20 insertion mutations. A case report from Japan showed that the cobas test did not detect the L858R mutation in a patient with lung adenocarcinoma harboring the L858R-K860I doublet mutation [10]. In this study, we identified four cases of L858R doublet mutations and used them to evaluate the L858R detection ability of three four PCR-based EGFR tests: the cobas, Idylla EGFR test, IntelliPlex-LCP-DNA assay, and AmoyDx PLC panel.