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Abstract
Objective Barrett’s oesophagus is a premalignant condition that occurs in the context of gastro-oesophageal reflux. However, most Barrett’s cases are undiagnosed because of reliance on endoscopy. We have developed a non-endoscopic tool: the Cytosponge, which when combined with trefoil factor 3 immunohistochemistry, can diagnose Barrett’s oesophagus. We investigated whether a quantitative methylation test that is not reliant on histopathological analysis could be used to diagnose Barrett’s oesophagus.
Design Differentially methylated genes between Barrett’s and normal squamous oesophageal biopsies were identified from whole methylome data and confirmed using MethyLight PCR in biopsy samples of squamous oesophagus, gastric cardia and Barrett’s oesophagus. Selected genes were then tested on Cytosponge BEST2 trial samples comprising a pilot cohort (n=20 cases, n=10 controls) and a validation cohort (n=149 cases, n=129 controls).
Results Eighteen genes were differentially methylated in patients with Barrett’soesophagus compared with squamous controls. Hypermethylation of TFPI2, TWIST1, ZNF345 and ZNF569 was confirmed in Barrett’s biopsies compared with biopsies from squamous oesophagus and gastric cardia (p<0.05). When tested in Cytosponge samples, these four genes were hypermethylated in patients with Barrett’s oesophagus compared with patients with reflux symptoms (p<0.001). The optimum biomarker to diagnose Barrett’s oesophagus was TFPI2 with a sensitivity and specificity of 82.2% and 95.7%, respectively.
Conclusion TFPI2, TWIST1, ZNF345 and ZNF569methylation have promise as diagnostic biomarkers for Barrett’s oesophagus when used in combination with a simple and cost effective non-endoscopic cell collection device.
- hypermethylation
- non-endoscopic cell sampling
- biomarker
- barrett’s
- reflux
- cytosponge
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Footnotes
Contributors RCF conceived and supervised the overall study. RCF, HC and CR-I conceived and designed the experiments. HC analysed the data. OM, NG-D and NB performed the experiments. SM was involved in data collection. ID-B coordinated the clinical study. MO performed the pathology review. HC and RCF wrote the manuscript. All authors approved the final version of the manuscript.
Funding The BEST2 study was funded by Cancer Research UK (C14478/A12088). RCF receives core funding from the Medical Research Council. The study received infrastructure support from the Cambridge Human Research Tissue Bank, which is supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre and the Experimental Cancer Medicine Centre.
Competing interests Since this study was conducted, the Cytosponge™-TFF3 technology has been licensed to Covidien GI solutions (now owned by Medtronic) by the Medical Research Council. Rebecca Fitzgerald and Maria O’Donovan are named inventors on patents pertaining to the Cytosponge™. Covidien Solutions and Medtronic have not been privy to this manuscript or the data therein. All other authors declare no conflicts of interest.
Patient consent Obtained.
Ethics approval East of England – Cambridge central research ethics committee (No: 10/H0308/71).
Provenance and peer review Not commissioned; externally peer reviewed.