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We compared light cycler reverse transcription polymerase chain reaction (LCRT-PCR) and next generation sequencing (NGS) to detect exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19)and Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) exon 2 mutations in endobronchial ultrasound-guided transbronchial needle aspirations (EBUS-TBNAs). LCRT-PCR additionally detected 2KRASexon 2 mutations and 3EGFRdelEx19mutations that were not detected using NGS. LCRT-PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance.First-line afatinib treatment prolongs overall survival in patients with metastatic non–small-cell lung cancer (NSCLC) harboring exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19) mutations. In contrast, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations are negative predictors for benefit from EGFR-targeting agents. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) allows for simultaneous interrogation for multiple mutations but has limitations (required tumor tissue amount, assay times). Reverse transcription polymerase chain reaction (RT-PCR) using light-Cycler technology (LCRT-PCR) can rapidly and sensitively detect somatic mutations from NSCLC patients. In the present study, we analyzed the feasibility of LCRT-PCR for rapid EGFRdelEx19 and KRAS exon 2 mutation detection in EBUS-TBNA samples and compared the LCRT-PCR and NGS results.A total of 48 EBUS-TBNA samples from 47 patients with a confirmed diagnosis of pulmonary adenocarcinoma were analyzed using LCRT-PCR (as previously described) and NGS (MiSeq; Illumina) using targeted resequencing and a customized multiplex PCR panel. The processing time was ˜1 week for the NGS and < 24 hours for the LCRT-PCR analyses.All (100%) EGFRdelEx19 and KRAS exon 2 mutations detected by NGS were detected by LCRT-PCR. In addition, LCRT-PCR detected 2 KRAS exon 2 mutations and 3 EGFRdelEx19 mutations that were not detected by NGS.LCRT-PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance (eg, EGFRdelEx19 and KRAS exon 2) in EBUS-TBNAs from NSCLC patients. It is of value as an initial assay for first-line treatment decisions.