Epidermal Growth Factor Receptor Mutation Status in Cell-Free DNA Supernatant of Bronchial Washings and Brushings

BACKGROUND: The aim of the current study was to examine whether it would be possible to detect epidermal growthw factor receptor (EGFR) mutations in cytology cell-free DNA (ccfDNA) from the supernatant fluids of bronchial cytology samples. METHODS: This study investigated cell damage via immunostaining with a cleaved caspase 3 antibody and the quantity of cell-free DNA in supernatant fluid from 2 cancer cell lines, and the EGFR mutation status was evaluated via polymerase chain reaction (PCR) analysis. EGFR mutations were also evaluated via PCR analysis in 74 clinical samples of ccfDNA from bronchial washing samples with physiological saline or from bronchial brushing liquid-based cytology samples with CytoRich Red. RESULTS: The quantity and fragmentation of cell-free DNA in the supernatant fluid and the cell damage and cleaved caspase 3 expression in the sediment gradually increased in a time-dependent manner in the cell lines. In the 74 clinical samples, the quantity of ccfDNA extracted from the supernatant was adequate to perform the PCR assay, whereas the quality of ccfDNA in physiological saline was often decreased. The detection of EGFR mutations with ccfDNA showed a sensitivity of 88.0%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 89.7%, and an accuracy of 94.1% in samples with malignant or atypical cells. CONCLUSIONS: These results suggest that activating EGFR mutations can be detected with ccfDNA extracted from the supernatant fluid of liquid-based samples via a PCR assay. This could be a rapid and sensitive method for achieving a parallel diagnosis by both morphology and DNA analysis in non-small cell lung cancer patients. Cancer (Cancer Cytopathol) 2015;123:620-8. (C) 2015 American Cancer Society.