title: BARHL2 Methylation Using Gastric Wash DNA or Gastric Juice Exosomal DNA is a Useful Marker For Early Detection of Gastric Cancer in an H. pylori-Independent Manner.
abstract: OBJECTIVE
  The main purpose of this study was to develop a methylation analysis pipeline by using gastric wash-derived DNA and/or gastric juice-derived exosomal DNA (exoDNA), and to evaluate its suitability for the early detection of gastric cancer (GC) in clinical settings.
  
  
  METHODS
  We analyzed alterations of BarH-like 2 homeobox protein (BARHL2) in GC cell lines and tissues, as well as in DNA obtained from 128 gastric washes and 30 gastric juice-derived exosomes. GC cell lines were transfected with plasmids encoding BARHL2 and subjected to proliferation, colony formation, and gene expression analyses.
  
  
  RESULTS
  High levels of BARHL2 methylation were detected in three of seven GC cell lines; consistent with this, these cell lines expressed low levels of BARHL2. Treatment of these cell lines with 5-aza-2'-deoxycytidine restored BARHL2 expression. Levels of BARHL2 methylation in 18 normal and 14 atrophic gastritis samples were low irrespective of Helicobacter pylori infection. High levels of BARHL2 methylation were observed in gastric wash-derived DNA obtained from early GC patients before endoscopic resection (ER), but methylation was significantly lower after curative ER. Analysis using gastric juice-derived exoDNA samples revealed that BARHL2 methylation yielded an area under the curve of 0.923 with 90% sensitivity and 100% specificity with respect to discriminating GC patients from non-GC controls. BARHL2 nuclear immunoreactivity was found in all normal gastric epithelial cells and in cells from patients with gastritis and adenoma. In contrast, loss of BARHL2 expression was observed in the vast majority of the GC tissues. Finally, transfection of BARHL2 into MKN7 and MKN45 cell lines significantly inhibited their proliferation and ability to form colonies.
  
  
  CONCLUSIONS
  Methylation analysis of BARHL2 using gastric wash-derived DNA and/or gastric juice-derived exoDNA could be useful for early detection of GC in clinical settings.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.|Department of Internal Medicine, Kawasaki Rinko General Hospital, Kawasaki, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Oikawa
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Morita
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Tadateru
authors.initials: T
authors.lastname: Maehata
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yasuda
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Itoh