Assessment of EGFR mutation status using cell-free DNA from bronchoalveolar lavage fluid

Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.