title: A non-endoscopic device to sample the oesophageal microbiota: a case-control study.
abstract: The strongest risk factor for oesophageal adenocarcinoma is reflux disease, and the rising incidence of this coincides with the eradication of Helicobacter pylori, both of which might alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett's carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota.
  
  
  
  In this case-control study, 16S rRNA gene amplicon sequencing was done on 210 oesophageal samples from 86 patients representing the Barrett's oesophagus progression sequence (normal squamous controls [n=20], non-dysplastic [n=24] and dysplastic Barrett's oesophagus [n=23], and oesophageal adenocarcinoma [n=19]), relevant negative controls, and replicates on the Illumina MiSeq platform. Samples were taken from patients enrolled in the BEST2 study at five UK hospitals and the OCCAMS study at six UK hospitals. We compared fresh frozen tissue, fresh frozen endoscopic brushings, and the Cytosponge device for microbial DNA yield (qPCR), diversity, and community composition.
  
  
  
  There was decreased microbial diversity in oesophageal adenocarcinoma tissue compared with tissue from healthy control patients as measured by the observed operational taxonomic unit (OTU) richness (p=0·0012), Chao estimated total richness (p=0·0004), and Shannon diversity index (p=0·0075). Lactobacillus fermentum was enriched in oesophageal adenocarcinoma (p=0·028), and lactic acid bacteria dominated the microenvironment in seven (47%) of 15 cases of oesophageal adenocarcinoma. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-times higher quantities of microbial DNA than did endoscopic brushes or biopsies using quantitative PCR (p<0·0001). The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga, and Dialister. The Cytosponge detected decreased microbial diversity in patients with high-grade dysplasia in comparison to control patients, as measured by the observed OTU richness (p=0·0147), Chao estimated total richness (p=0·023), and Shannon diversity index (p=0·0085).
  
  
  
  Alterations in microbial communities occur in the lower oesophagus in Barrett's carcinogenesis, which can be detected at the pre-invasive stage of high-grade dysplasia with the novel Cytosponge device. Our findings are potentially applicable to early disease detection, and future test development should focus on longitudinal sampling of the microbiota to monitor for changes in microbial diversity in a larger cohort of patients.
  
  
  
  Cancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).
authors.affiliation: Medical Research Centre Cancer Unit, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK.
authors.forename: Daffolyn R Fels
authors.initials: DRF
authors.lastname: Elliott
authors.affiliation: Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, UK; Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Walker
authors.affiliation: Department of Histopathology, Cambridge University Hospital NHS Trust, Cambridge, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: O'Donovan
authors.affiliation: Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Medical Research Centre Cancer Unit, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK. Electronic address: RCF29@MRC-CU.cam.ac.uk.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Fitzgerald
mesh_terms: D000230:Adenocarcinoma / Q000382:microbiology* / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001471:Barrett Esophagus / Q000382:microbiology* / Q000473:pathology; D001706:Biopsy; D016022:Case-Control Studies; D003581:Cytodiagnosis / Q000379:methods*; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000382:microbiology* / Q000473:pathology; D004945:Esophagoscopy; D004947:Esophagus / Q000382:microbiology*; D005260:Female; D006801:Humans; D008297:Male; D064307:Microbiota; D008875:Middle Aged; D018570:Risk Assessment / Q000379:methods*; D017422:Sequence Analysis, DNA