Epidermal growth factor receptor mutation status in cell‐free DNA supernatant of bronchial washings and brushings

Abstract

BACKGROUND:The aim of the current study was to examine whether it would be possible to detect epidermal growth 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. V C 2015 American Cancer Society.

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

“…As a result, a total of 74 lung cancer patients with paired samples between primary tumour and corresponding metastatic pleural effusion tumour with both cell sediment DNA and supernatant ccfDNA were enrolled in this study. EGFR mutations at the primary site in these lung patients had already been confirmed by TaqMan Mutation Detection assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA) or fluorescence resonance energy transfer‐based preferential homoduplex formation assay (F‐PHFA; Riken Genesis Co., Ltd. Tokyo, Japan) based on histological tissue samples taken at the time of diagnosis (Table ). Although cytological diagnosis (i.e.…”

“…Cell sediment from the samples was immersed in a solution of CytoRich Red (Becton Dickinson, Franklin Lakes, NJ, USA), and fixed for at least 30 min. Each liquid‐based cytology (LBC) smear was processed using the SurePath TM system according to the manufacturer's instructions as per our previous report . All LBC slides were then stained using the Papanicolaou method for cytological diagnosis.…”

“…Mutation of the EGFR gene was examined in exons 19 (delE746‐A750) and 21 (L858R) by TaqMan Mutation Detected assay or F‐PHFA assay according to the manufacturer's instructions. In brief, genomic DNA was purified from supernatant fluids and cell sediment of LBC smears in pleural effusion samples using a DNA extractor Master Pure™ kit (Wako, Osaka, Japan) according to the protocol described in our previous report . DNA quantity was assessed with a BioPhotometer (Eppendorf AG, Hamburg, Germany).…”

“…In addition, Che et al reported a newly developed molecular assay for the detection of cell‐free Mycobacterium tuberculosis DNA in pleural effusion. We have also reported that EGFR mutations can be detected from cytology cell‐free DNA (ccfDNA) in the supernatant fluids of liquid‐based cytology samples . In effusion cytology, the sediment materials available for EGFR mutation analysis are limited, because EGFR mutation analysis is often performed after immunostaining for cytological diagnosis, and it is known that EGFR mutation analysis yields false‐negative results when there are few cancer cells in the sediment.…”

Dysregulation and detection methods of EGFR in oral cancer. A narrative review.

“…Polymerase chain reaction (PCR) has been used to detect EGFR mutations in normal and neoplastic tissues. 3,17,31,35,49 In oral cancer specific tests to evaluate deletions, silent mutations or changes at amino acid level encoded in exons 18 to 21 have been used. 33 Bagan et al 26 studied the genomic amplification of EGFR in frozen tissue samples.…”