title: 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.
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kawahara
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Fukumitsu
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Taira
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Abe
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Yorihiko
authors.initials: Y
authors.lastname: Takase
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Murata
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Azuma
authors.affiliation: Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Ishii
authors.affiliation: Department of Surgery, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Shinzo
authors.initials: S
authors.lastname: Takamori
authors.affiliation: Department of Pathology, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Akiba
authors.affiliation: Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Hoshino
authors.affiliation: Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Kage
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001980:Bronchi / Q000378:metabolism / Q000473:pathology*; D002474:Cell-Free System; D003581:Cytodiagnosis; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D011379:Prognosis
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: cytology cell-free DNA; epidermal growth factor receptor mutation; liquid-based cytology; lung cancer; supernatant fluid