Molecular testing on bronchial washings for the diagnosis and predictive assessment of lung cancer

Cyto-pathological analyses of bronchial washings (BWs) collected during fibreoptic bronchoscopy are often inconclusive for lung cancer diagnosis. To address this issue, we assessed the suitability of conducting molecular analyses on BWs, with the aim to improve the diagnosis and outcome prediction of lung cancer. The methylation status of RASSF1A, CDH1, DLC1, and PRPH was analysed in BW samples from 91 lung cancer patients and 31 controls, using a novel two-colour droplet digital methylation-specific PCR technique. Mutations in ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 and gene fusions of ALK, RET, and ROS1 were also investigated, using next-generation sequencing (NGS) on 73 lung cancer patients and 14 tumour-free individuals. Our four-gene methylation panel had significant diagnostic power, with 97% sensitivity and 74% specificity (relative risk, 7.3; odds ratio, 6.1; 95% confidence interval, 12.7-127). In contrast, gene mutation analysis had a remarkable value for predictive, but not for diagnostic, purposes. Actionable mutations in EGFR, HER2, and ROS1 as well as in other cancer genes (KRAS, PIK3CA, and TP53) were detected. Concordance with gene mutations uncovered in tumour biopsies was higher than 90%. In addition, bronchial-washing analyses permitted complete patient coverage and the detection of additional actionable mutations. In conclusion, bronchial washings are a useful material on which to perform molecular tests based on gene panels: aberrant gene methylation and mutation analyses could be performed as approaches accompanying current diagnostic and predictive assays during the initial workup phase. This study establishes the grounds for further prospective investigation.