BACKGROUND: The detection of mutated epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) with residual cell pellets derived from liquid-based cytology (LBC) samples (eg, endoscopic ultrasound-guided fine-needle aspiration) has been validated with allele-specific polymerase chain reaction. The aim of this study was to validate next-generation sequencing (NGS) technology for detecting gene mutations with residual cell pellets from LBC. METHODS: Archived DNA extracted from LBC samples of adenocarcinoma stored in PreservCyt with a known EGFR mutation status was retrieved. Genomic DNA was multiplex-amplified and enriched with Ion AmpliSeq Cancer Hotspot Panel v2 chemistry and the OneTouch 2 instrument; this was followed by semiconductor sequencing on the Ion Personal Genome Machine platform. The mutation hotspots of 6 NSCLC-related genes (BRAF, EGFR, ERBB2, KRAS, MET, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a [PIK3CA]) were analyzed with NextGENe and Torrent Suite bioinformatics tools. RESULTS: The commonly identified EGFR sequence changes, including 4 L858R mutations, 3 exon 19 deletions, and 1 exon 20 insertion, were in 100% concordance between the assay platforms. Less common NSCLC variants were also found in the mutation hotspots of ERBB2, KRAS, MET, and PIK3CA genes. CONCLUSIONS: NSCLC mutation analysis using NGS can be successfully performed on residual cell pellets derived from LBC samples. This approach allows the simultaneous examination of multiple mutation hotspots in a timely manner to improve patient care. (C) 2017 American Cancer Society.