Protocol optimization of a targeted sequencing panel for genomic profiling of bronchoalveolar lavage fluid in lung cancer.

Introduction: We investigated a commercially available sequencing panel to study the effect of sequencing depth, variant calling strategy, and targeted sequencing region on identifying tumor-derived variants in cell-free bronchoalveolar lavage (cfBAL) DNA compared with plasma cfDNA.

Methods: Sequencing was performed at low or high coverage using two filtering algorithms to identify tumor variants on two panels targeting 77 and 197 genes respectively.

Results: One hundred and four sequencing files from 40 matched DNA samples of cfBAL, plasma, germline leukocytes, and archival tumor specimens in 10 patients with early-stage lung cancer were analyzed. By low-coverage sequencing, tumor-derived cfBAL variants were detected in 5/10 patients (50%) compared with 2/10 (20%) for plasma. High-coverage sequencing did not affect the number of tumor-derived variants detected in either biospecimen type. Accounting for germline mutations eliminated false-positive plasma calls regardless of coverage (0/10 patients with tumor-derived variants identified) and increased the number of cfBAL calls (5/10 patients with tumor-derived variants identified). These results were not affected by the number of targeted genes.