AimsAny reference laboratory testing non-small cell lung cancer samples for predictive biomarkers needs to develop and validate a wide range of different molecular techniques, each with a specific time requirement and application. Updated international guidelines suggest that next generation sequencing (NGS) to be the initial procedure. However, in a non-negligible subset of cases, library generation may fail or amplicon coverage may be insufficient. In these NGS ‘invalid’ cases, the Idylla system may represent a viable option for rapid epidermal growth factor receptor (EGFR) genotyping.MethodsThis retrospective study included 68 archival DNA samples previously processed by Ion Torrent NGS assay. Out of these, 43 cases, including 24 EGFRmutant samples, had a valid NGS result, whereas 25/68 (37%) were invalid. All samples were retested by directly pipetting the DNA inside the EGFRIdylla assay cartridge.ResultsIn all 43 cases with a valid NGS result, Idylla confirmed the EGFRmutational status. In particular, 24/24 (100%) of EGFRmutant samples as detected by NGS were confirmed by Idylla. Moreover, a large portion of cases (20/25; 80%) whose assessment by NGS was invalid were adequately processed by Idylla. Noteworthy, in 4/25 (16%) of cases, Idylla detected actionable EGFRmutations.ConclusionsIdylla assay could be very useful to quickly process cases for which NGS does not allow genotyping.