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

Objectives: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.

A new bioluminescent reporter system to study the biodistribution of systematically injected tumor-derived bioluminescent extracellular vesicles in mice

“…Western blot analysis confirmed the presence of EV marker proteins like ALIX and CD63. The presence of these markers is consistent with observations of purified EV samples, all EV preparations were free from contaminating cell organelles as indicated by the absence of markers of Golgi apparatus or endoplasmic reticulum [ 53 , 54 ]. TEM analysis of the isolated EVs revealed the presence of vesicles with a characteristic round shape and a clear-cut lipid bilayer, showing good evidence of intact EVs.…”

Identification of DNA mutations in gastric washes from gastric adenocarcinoma patients: Possible implications for liquid biopsies and patient follow‐up

“…As GW fluids are in intimate contact with the gastric mucosa (normal or neoplastic), they represent a possible source of valuable biomarkers, including ctDNA . However, very few studies have focused on the use of GWs as a source of tumor markers and most were limited to the identification of proteins, tumor cells, extracellular vesicles, microRNAs and lncRNAs or cytological markers . To our knowledge, the results presented here are the first to demonstrate tumor‐derived mutations in the DNA from the GW of GAC patients using next‐generation DNA sequencing (NGS).…”

“…22 However, very few studies have focused on the use of GWs as a source of tumor markers and most were limited to the identification of proteins, tumor cells, extracellular vesicles, microRNAs and lncRNAs or cytological markers. [22][23][24][25][26][27][28] To our knowledge, the results presented here are the first to demonstrate tumorderived mutations in the DNA from the GW of GAC patients using next-generation DNA sequencing (NGS). Our results highlight the potential of GW as a relevant source for detecting GAC mutations and its potential utility for tracking tumor dynamics, patient prognosis and treatment efficiency.…”

The roles of extracellular vesicles in gastric cancer development, microenvironment, anti-cancer drug resistance, and therapy

“…In 30 gastric juicederived exosomes, BarH-like 2 homeobox protein (BARHL2) showed high levels of methylation. Interestingly, BARHL2 methylation generated an area under the curve of 0.923 with 90% sensitivity and 100% specificity concerning recognizing GC patients from healthy controls when analysis of gastric juice-derived exosome DNA samples [33]. All these results suggested that methylation analysis of BARHL2 using gastric juice-secreted exosome DNA could be beneficial for early diagnosis of GC in clinical settings.…”