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

Fangfang Xie, Xiaoxuan Zheng, Xiaowei Mao, Ruiying Zhao, Junyi Ye, Yujun Zhang, Jiayuan Sun
Annals of Thoracic Surgery 2019 March 15

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 (NSCLC) were collected via EBUS-TBNA and formalin-fixed paraffin-embedded. Three representative genes, EGFR, ALK and ROS1, were examined by amplification refractory mutation system polymerase chain reaction (ARMS-PCR), immunohistochemistry and quantitative reverse transcription PCR (RT-qPCR). 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 patients diagnosed with advanced NSCLC, 77 successfully performed all assays. Forty-one mutations in EGFR, ALK and ROS1 were detected in both conventional methods and NGS, representing a 100% concordance. In the contrast, four EGFR mutations detected by NGS were not covered in the targeted regions of ARMS-PCR, leading to a negative call in these patients. Altogether, NGS detected 12 additional variants including 6 KRAS mutations, 1 BRAF mutation, 1 RET fusion, 1 MET amplification concurrent with EGFR L858R, 1 KRAS amplification together with EGFR 19del and 1 ERBB2 amplification. The mean number of needle passes per lymph node was 5.2 in samples successfully applied all assays.

CONCLUSIONS: NGS assay can be successfully conducted with limited tissue samples obtained from EBUS-TBNA. Compared to conventional methods, NGS assay provides more comprehensive information on genetic alterations in tumor, which greatly assists therapeutic decision-making for advanced lung cancer.

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