Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett's esophagus

Abstract

We report a biomarker-based non-endoscopic method for detecting Barrett's esophagus (BE) based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma. Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve of 0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 min. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE.

Conflict of interest statement

Competing interests: Drs. Chak, Willis, and Markowitz have awarded patents on the use of methylated Vimentin for detection of Barrett’s esophagus and other GI cancers and have also pending patent on a balloon based device for non-endoscopic sampling of the esophagus. Drs. Chak, Willis, Markowitz, LaFramboise, de la Cruz Cabrera, and Moinova have pending patents on methylated CCNA1. Patent rights have been assigned to Case Western Reserve University and are managed under institutional conflict of interest policies. Awarded patents include: U.S. Patent 9580754, Methods and Compositions for Detecting Gastrointestinal and Other Cancers; U.S. Patent 8415100, Methods and Compositions for Detecting Gastrointestinal and Other Cancers; U.S. Patent 8221977, Methods and Compositions for Detecting Colon Cancers. Pending patents include: PCT/US2014/070060, Device for collecting a biological sample; PCT/US2010/030084, Digital quantification of DNA methylation; PCT/US2015/068131, Methods and compositions for detecting esophageal neoplasias and metaplasias; PCT/US2017/040708, Methods and compositions for detecting esophageal neoplasias and/or metaplasias in the esophagus. S.D.M. has consulting relationships with Rodeo Therapeutics, Jannsen Pharmaceuticals, and GlaxoSmithKline. A.C. has consulting relationships with U.S. Endoscopy and Coldplay Therapeutics. N.J.S. consults for Shire, Ambu, and Boston Scientific and has research funding from Medtronic, C2 Therapeutics, CSA Medical, EndoStim, CDx Medical, and Interpace Diagnostics. P.G.I. consults for Medtronic and has research funding from Exact Sciences, Intromedic, and C2 Therapeutics. M.I.C. consults for Pentax Medical Corporation and Cook Medical and has research funding from C2 Therapeutics, Inc. J.D. consults for US Endoscopy and CSA Medical and has research funding from C2 Therapeutics. No other financial conflicts of interest pertain to the authors of this paper.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1. CCNA1 region methylation in esophageal neoplasia

(A) Location of the differentially methylated region in the CCNA1 promoter on chromosome 13. The patch of CpGs found to be differentially methylated in reduced representation bisulfite sequencing (RRBS) and the amplicon assayed by next-generation sequencing are indicated above the map of the CpG island (green), with structures of CCNA1 RefSeq transcripts indicated below. (B) Average methylation of CpGs in the CCNA1 RRBS-defined patch of 7 CpGs in: biopsies of normal squamous mucosa (N Sq), Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), and in esophageal cell lines (CL).

Figure 2. NGS bisulfite sequencing assay of DNA methylation in esophageal biopsies

(A) CCNA1 locus methylation (mCCNA1) in esophageal neoplasia and control patients. N Sq (normal squamous biopsies); NDBE (non-dysplastic Barrett’s esophagus); HGD (Barrett’s esophagus with high-grade dysplasia); EAC (esophageal adenocarcinoma). Fraction of methylated reads in each sample is indicated on the Y-axis. P-value <0.001 for one way ANOVA comparison, and p<0.001 for post-hoc Student-Newman-Keuls test of N Sq versus BE, HGD, and EAC. (B) VIM locus methylation (mVIM) in esophageal neoplasia and control patients. N Sq (normal squamous biopsies); NDBE (non-dysplastic Barrett’s esophagus); HGD (Barrett’s esophagus with high-grade dysplasia); EAC (esophageal adenocarcinoma). Fraction of methylated reads in each sample is indicated on the Y-axis. P-value <0.001 for one way ANOVA comparison, and p<0.001 for post-hoc Student-Newman-Keuls test of N Sq vs BE, HGD, and EAC.

Figure 3. ROC curves of mCCNA1 and mVIM assayed in esophageal cytology brushings from control normal-appearing GE junctions versus BE and EAC cases

(A) and (B) Training samples. (A) mCCNA1, n= 61 controls, 108 cases. (B) mVIM, n= 59 controls and 107 cases. (C) and (D) Validation samples. (C) mCCNA1, n= 28 controls, 115 cases. (D) mVIM, n= 27 controls and 117 cases. Area under the curve (AUC) and the sensitivity and specificity of the assays at the indicated cutpoint are listed for each graph, with the cutpoint value of percent methylation that defines a positive test denoted by “At >”.

Figure 4. DNA methylation in the proximal squamous esophagus of smokers versus non-smokers

(A) mCCNA1, *p=0.0094; (B) mVIM, *p=0.0155. Patients were classified as smokers if they had any history of ever smoking. P values for differences between smokers and non-smokers were computed using the Mann-Whitney rank sum test.

Figure 5. Non-endoscopic balloon device

(A) Device capsule and catheter in comparison to a vitamin pill and a dime. (B) Capsule containing inverted balloon in configuration for swallowing. (C) Capsule with inflated balloon in configuration for esophageal sampling. (D) Capsule containing inverted balloon in configuration for device and biospecimen retrieval.

Figure 6. ROC curves of mCCNA1 and mVIM assayed on esophageal balloon samplings of the distal esophagus

(A) mCCNA1, n=36 controls, 50 cases. (B) mVIM, n=36 controls, 50 cases. Area under the curve (AUC), and the sensitivity and specificity of the assays at the indicated cutpoints are listed for each graph, with the cutpoint value of percent methylation that defines a positive test denoted by “At >”.