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

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Science Translational Medicine  17 Jan 2018:
Vol. 10, Issue 424, eaao5848
DOI: 10.1126/scitranslmed.aao5848
  • Fig. 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 (NGS) 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 seven CpGs in biopsies of normal squamous (N Sq) mucosa, Barrett’s esophagus (BE), and esophageal adenocarcinoma (EAC) and in esophageal cell lines (CL).

  • Fig. 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, nondysplastic BE; HGD, BE with high-grade dysplasia. Fraction of methylated reads in each sample is indicated on the y axis. P < 0.001 for one-way analysis of variance (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. Fraction of methylated reads in each sample is indicated on the y axis. P < 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.

  • Fig. 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 and 108 cases. (B) mVIM, n = 59 controls and 107 cases. (C and D) Validation samples. (C) mCCNA1, n = 28 controls and 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 >.”

  • Fig. 4 DNA methylation in the proximal squamous esophagus of smokers versus nonsmokers.

    (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 nonsmokers were computed using the Mann-Whitney rank sum test.

  • Fig. 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.

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

    (A) mCCNA1, n = 36 controls and 50 cases. (B) mVIM, n = 36 controls and 50 cases. 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 >.”

  • Table 1 mVIM and mCCNA1 performance in the combined set of all distal esophagus brushings.

    VIM and CCNA1 gene methylation was assayed in DNA samples from cytology brushings of the distal esophagus from the following: unaffected controls brushed at the gastroesophageal junction (control GEJ); cases of NDBE, further subclassified as short-segment BE (SSBE) of 1 to 3 cm or long-segment BE (LSBE) of ≥3 cm; BE with low-grade dysplasia (LGD); BE with HGD; EAC (including junctional cancer of the esophagus). Samples were scored as VIM-methylated for mVIM >1.05% and as CCNA1-methylated for mCCNA1 >3.12% [using receiver operating characteristic (ROC) defined cutpoints from Fig. 3, A and B]. Cases were positive for the panel of mCCNA1 plus mVIM if either marker tested positive. Controls were negative for the panel when both mCCNA1 and mVIM were negative. Controls with one negative marker and one marker with assay failure were excluded. Entries indicate percent sensitivity or specificity (%) and total number of individuals tested (n).

    mVIMmCCNA1Either mVIM or mCCNA1
    %n%n%n
    Specificity control GEJ93.08696.68990.584
    Sensitivity all cases91.122490.122394.8229
    Sensitivity all NDBE91.57179.76991.772
    Sensitivity SSBE87.13176.73087.131
    Sensitivity LSBE95.04082.13995.141
    Sensitivity all
    dysplastic BE
    91.15694.55596.557
    Sensitivity LGD93.93390.63294.134
    Sensitivity HGD87.023100.023100.023
    Sensitivity EAC90.79794.99996.0100
  • Table 2 mVIM and mCCNA1 performance in esophageal balloon samples.

    VIM and CCNA1 gene methylation was assayed in DNA samples from non-endoscopic balloon sampling of the distal esophagus from unaffected controls [individuals with gastroesophageal reflux disease (GERD), erosive esophagitis, or no pathology detected during endoscopy]; cases of NDBE, further subclassified as SSBE of 1 to 3 cm or LSBE of ≥3 cm; BE with LGD; BE with HGD; EAC (including junctional cancer of the esophagus). Samples were scored as VIM-methylated for mVIM >1.0% and as CCNA1-methylated for mCCNA1 >1.0% (using ROC-defined cutpoints from Fig. 6). Samples were positive for the panel of mCCNA1 plus mVIM if either marker tested positive. Entries indicate percent sensitivity or specificity (%) and total number of individuals tested (n). We note that only four HGDs were studied, and in this small sample size, differences in the rate of mVIM and mCCNA1 detection of HGD versus detection of NBDE or EAC are not statistically significant (P > 0.088 for any between group comparisons).

    mVIMmCCNA1Either mVIM or mCCNA1
    %n%n%n
    Specificity unaffected
    controls
    91.736100.03691.736
    Sensitivity all cases80.05072.05088.050
    Sensitivity all NDBE80.63171.03190.331
    Sensitivity SSBE69.21353.81384.613
    Sensitivity LSBE88.91883.31894.418
    Sensitivity all
    dysplastic BE
    72.71172.71181.811
    Sensitivity LGD83.36100.06100.06
    Sensitivity HGD50.0450.0450.04
    Sensitivity EAC87.5875.0887.58
  • Table 3 mVIM and mCCNA1 detection in FFPE biopsies of upper gastrointestinal tract pathologies.

    mVIM and mCCNA1 were assayed in microdissected formalin-fixed paraffin-embedded (FFPE) biopsies that captured each of the histologies shown. IM, intestinal metaplasia. Samples were scored as methylated for mVIM >1.05% and mCCNA1 >3.12% (the cutpoints established in ROC analysis of esophageal brushings assayed for each marker). Samples were positive for the panel of mCCNA1 plus mVIM if either marker tested positive. Entries indicate percent positive samples (%) and total number of individuals tested (n).

    mCCNA1mVIMEither mVIM or mCCNA1
    %n%n%n
    BE (IM)752090219021
    GEJ/cardia with IM (<1-cm extent)67970108010
    Non-IM columnar metaplasia
    concurrent with BE
    11930103010
    GEJ/cardia without IM (including
    15 cases with chronic carditis)
    053054055
    Distal normal squamous
    esophagus from control
    patients without
    glandular metaplasia
    024024024
    Eosinophilic esophagitis012015015
    Gastric fundic mucosa without IM424024424
    Intestinal metaplasia of stomach147119229
    H. pylori gastritis without IM15138131513

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/424/eaao5848/DC1

    Fig. S1. Flowchart of study analyses.

    Fig. S2. Comparison of methylation of individual CpGs versus CpG patches in discriminating esophageal lesions.

    Fig. S3. Comparing VIM and CCNA1 expression versus methylation.

    Fig. S4. Morphology of touch preps from balloon brushings of three intact porcine esophagus samples from esophagogastric organ explants.

    Table S1. Location of 26 differentially methylated patches identified by RRBS comparison of normal esophageal squamous mucosa versus Barrett’s lesions.

    Table S2. Demographic characteristics of training and validation esophageal brushing populations.

    Table S3. mVIM and mCCNA1 performance in training and validation esophageal brushing samples.

    Table S4. Comparison of sensitivities of mVIM plus mCCNA1 versus mVIM or mCCNA1, all at equal specificities.

    Table S5. Influence of smoking on VIM and CCNA1 methylation in proximal versus distal esophagus.

    Table S6. Participant evaluation of the non-endoscopic balloon sampling of the esophagus.

    Table S7. Demographic characteristics of subjects in non-endoscopic balloon study.

    Table S8. Methylation in post-ablation subjects.

    Table S9. Post-examination questionnaire.

    Table S10. Bisulfite-specific methylation-independent PCR primer sequences.

    Table S11. Index tags for bisulfite-specific methylation-independent PCR primer sequences.

    References (3366)

  • Supplementary Material for:

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

    Helen R. Moinova, Thomas LaFramboise, James D. Lutterbaugh, Apoorva Krishna Chandar, John Dumot, Ashley Faulx, Wendy Brock, Omar De la Cruz Cabrera, Kishore Guda, Jill S. Barnholtz-Sloan, Prasad G. Iyer, Marcia I. Canto, Jean S. Wang, Nicholas J. Shaheen, Prashanti N. Thota, Joseph E. Willis,* Amitabh Chak,* Sanford D. Markowitz*

    *Corresponding author. Email: SXM10{at}cwru.edu (S.D.M.); josephe.willis{at}uhhospitals.org (J.E.W.); amitabh.chak{at}uhhospitals.org (A.C.)

    Published 17 January 2018, Sci. Transl. Med. 10, eaao5848 (2018)
    DOI: 10.1126/scitranslmed.aao5848

    This PDF file includes:

    • Fig. S1. Flowchart of study analyses.
    • Fig. S2. Comparison of methylation of individual CpGs versus CpG patches in discriminating esophageal lesions.
    • Fig. S3. Comparing VIM and CCNA1 expression versus methylation.
    • Fig. S4. Morphology of touch preps from balloon brushings of three intact porcine esophagus samples from esophagogastric organ explants.
    • Table S1. Location of 26 differentially methylated patches identified by RRBS comparison of normal esophageal squamous mucosa versus Barrett’s lesions.
    • Table S2. Demographic characteristics of training and validation esophageal brushing populations.
    • Table S3. mVIM and mCCNA1 performance in training and validation esophageal brushing samples.
    • Table S4. Comparison of sensitivities of mVIM plus mCCNA1 versus mVIM or mCCNA1, all at equal specificities.
    • Table S5. Influence of smoking on VIM and CCNA1 methylation in proximal versus distal esophagus.
    • Table S6. Participant evaluation of the non-endoscopic balloon sampling of the esophagus.
    • Table S7. Demographic characteristics of subjects in non-endoscopic balloon study.
    • Table S8. Methylation in post-ablation subjects.
    • Table S9. Post-examination questionnaire.
    • Table S10. Bisulfite-specific methylation-independent PCR primer sequences.
    • Table S11. Index tags for bisulfite-specific methylation-independent PCR primer sequences.
    • References (3366)

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