Next-Generation Sequencing for Genotyping of Endobronchial Ultrasound- Guided Transbronchial Needle Aspiration Samples in Lung Cancer

Background. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can obtain a small amount of specimen. This study aims to evaluate the feasibility and robustness of using EBUS-EBNA samples to perform capture-based targeted next-generation sequencing (NGS). Methods. Tissue samples from patients with advanced non-small cell lung cancer were collected by EBUS-TBNA and were formalin-fixed paraffin-embedded. Three representative genes, EGFR, ALK, and ROS1, were examined by amplification refractory mutation system polymerase chain reaction, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction. The remaining samples were processed with NGS assay with a 56-gene panel. Classic driver mutations detected by NGS were verified by conventional methods. Results. Of the 85 samples from patients with advanced non-small cell lung cancer, 77 were performed successfully with all assays. Forty-one mutations in EGFR, ALK, and ROS1 were detected in both conventional methods and NGS, representing a 100% concordance. In contrast, four EGFR mutations detected by NGS were not covered in the targeted regions of amplification refractory mutation system polymerase chain reaction, leading to a negative call in these patients. Altogether, NGS detected 12 additional variants, including six KRAS mutations, one BRAF mutation, one RET fusion, one MET amplification concurrent with EGFR L858R, one KRAS amplification together with EGFR 19del, and one ERBB2 amplification. The mean number of needle passes per lymph node was 5.2 in samples successfully applied in all assays. Conclusions. NGS assay can be successfully conducted with limited tissue samples obtained from EBUS-TBNA. Compared with conventional methods, NGS assay provides more comprehensive information on genetic alterations in tumors, which greatly assists therapeutic decision making for advanced lung cancer. (C) 2019 by The Society of Thoracic Surgeons