Diagnostic power of DNA methylation markers suggestive of cholangiocarcinoma in ERCP-based brush cytology

      Background and Aims

      Accurate differentiation between cholangiocarcinoma (CCA) and benign biliary stricture is of paramount importance. Biliary brush cytology is a simple and safe diagnostic approach that provides relatively high specificity; however, sensitivity is limited. Previous reports indicated the aberrations of DNA methylation in CCA. This study aimed to investigate the diagnostic performance of the methylation index (MI) of HOXA1 and NEUROG1 gene promoters in CCA.

      Methods

      Patients with biliary stricture who underwent ERCP with brush cytology in Siriraj Hospital from September 2016 to December 2019 were prospectively enrolled. The MI of HOXA1 (MI_H) and MI of NEUROG1 (MI_N) were determined by quantitative methylation-specific polymerase chain reaction. The diagnostic power for CCA was tested for MI from both genes and serum carbohydrate antigen 19-9 (CA19-9).

      Results

      Sixty-seven patients were included in the study; 41 patients had a final diagnosis of CCA, and 26 patients were determined to have a benign biliary stricture. The results showed that both MI_H and MI_N had higher sensitivity and accuracy (95.1% and 82.3% and 90.2% and 89.5%, respectively) than brush cytology (61.5% and 78.1%) and CA19-9 (69.4% and 77.8%). The combination of brush cytology, both methylation markers, and CA19-9 increased the sensitivity and accuracy to 97.4% and 91.0%. Methylation markers were positive in 5 of 6 patients with confirmed CCA whose cytology and CA19-9 were negative.

      Conclusions

      DNA methylation increased the sensitivity for the diagnosis of CCA; therefore, the use of DNA methylation is promising for diagnosis of CCA in patients with biliary strictures. A future validation study is warranted to assess its role in clinical practice. (Clinical trial registration number: NCT 04568512.)

      Graphical abstract

      Abbreviations:

      ALP (alkaline phosphatase), CA19-9 (carbohydrate antigen 19-9), CCA (cholangiocarcinoma), FISH (fluorescence in situ hybridization), HOXA1 (Homeobox A1), MI (methylation index), NEUROG1 (Neurogenin 1), PCR (polymerase chain reaction)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Gastrointestinal Endoscopy
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Bowlus C.L.
        • Olson K.A.
        • Gershwin M.E.
        Evaluation of indeterminate biliary strictures.
        Nat Rev Gastroenterol Hepatol. 2016; 13: 28-37
        • Navaneethan U.
        • Hasan M.K.
        • Lourdusamy V.
        • et al.
        Single-operator cholangioscopy and targeted biopsies in the diagnosis of indeterminate biliary strictures: a systematic review.
        Gastrointest Endosc. 2015; 82: 608-614
        • Trikudanathan G.
        • Navaneethan U.
        • Njei B.
        • et al.
        Diagnostic yield of bile duct brushings for cholangiocarcinoma in primary sclerosing cholangitis: a systematic review and meta-analysis.
        Gastrointest Endosc. 2014; 79: 783-789
        • Navaneethan U.
        • Njei B.
        • Lourdusamy V.
        • et al.
        Comparative effectiveness of biliary brush cytology and intraductal biopsy for detection of malignant biliary strictures: a systematic review and meta-analysis.
        Gastrointest Endosc. 2015; 81: 168-176
        • Pan Y.
        • Liu G.
        • Zhou F.
        • et al.
        DNA methylation profiles in cancer diagnosis and therapeutics.
        Clin Exp Med. 2018; 18: 1-14
        • Henriksen S.D.
        • Madsen P.H.
        • Larsen A.C.
        • et al.
        Promoter hypermethylation in plasma-derived cell-free DNA as a prognostic marker for pancreatic adenocarcinoma staging.
        Int J Cancer. 2017; 141: 2489-2497
        • Pixberg C.F.
        • Raba K.
        • Müller F.
        • et al.
        Analysis of DNA methylation in single circulating tumor cells.
        Oncogene. 2017; 36: 3223-3231
        • Gkountela S.
        • Castro-Giner F.
        • Szczerba B.M.
        • et al.
        Circulating tumor cell clustering shapes DNA methylation to enable metastasis seeding.
        Cell. 2019; 176: 98-112
        • Larsen L.K.
        • Lind G.E.
        • Guldberg P.
        • et al.
        DNA-methylation-based detection of urological cancer in urine: overview of biomarkers and considerations on biomarker design, source of DNA, and detection technologies.
        Int J Mol Sci. 2019; 20: 2657
        • Zhao F.
        • Vesprini D.
        • Liu R.S.C.
        • et al.
        Combining urinary DNA methylation and cell-free microRNA biomarkers for improved monitoring of prostate cancer patients on active surveillance.
        Urol Oncol. 2019; 37: 297.e9-297.e17
        • Angsuwatcharakon P.
        • Rerknimitr R.
        • Kongkam P.
        • et al.
        Identification of pancreatic cancer in biliary obstruction patients by FRY site-specific methylation.
        Asian Pac J Cancer Prev. 2016; 17: 4487-4490
        • Prachayakul V.
        • Kanchanapermpoon J.
        • Thuwajit C.
        • et al.
        DNA methylation markers improve the sensitivity of endoscopic retrograde cholangiopancreatography-based brushing cytology in extrahepatic cholangiocarcinoma.
        Technol Cancer Res Treat. 2017; 16: 1252-1258
        • Nakaoka T.
        • Saito Y.
        • Saito H.
        Aberrant DNA methylation as a biomarker and a therapeutic target of cholangiocarcinoma.
        Int J Mol Sci. 2017; 18: 1111
        • Zhang C.
        • Zhang B.
        • Meng D.
        • et al.
        Comprehensive analysis of DNA methylation and gene expression profiles in cholangiocarcinoma.
        Cancer Cell Int. 2019; 19: 352
        • Kim B.H.
        • Cho N.Y.
        • Choi M.
        • et al.
        Methylation profiles of multiple CpG island loci in extrahepatic cholangiocarcinoma versus those of intrahepatic cholangiocarcinomas.
        Arch Pathol Lab Med. 2007; 131: 923-930
        • Patel A.H.
        • Harnois D.M.
        • Klee G.G.
        • et al.
        The utility of CA19-9 in the diagnoses of cholangiocarcinoma in patients without primary sclerosing cholangitis.
        Am J Gastroenterol. 2000; 95: 204-207
        • Amornyotin S.
        • Na-pomphet S.
        • Wongwathanyoo T.
        • et al.
        Anesthesia for endoscopic retrograde cholangiopancreatography (ERCP) from 1999--2003 in Siriraj Hospital: a retrospective study.
        J Med Assoc Thai. 2004; 87: 1491-1495
        • Boldorini R.
        • Paganotti A.
        • Andorno S.
        • et al.
        A multistep cytological approach for patients with jaundice and biliary strictures of indeterminate origin.
        J Clin Pathol. 2015; 68: 283-287
        • Brooks C.
        • Gausman V.
        • Kokoy-Mondragon C.
        • et al.
        Role of fluorescent in situ hybridization, cholangioscopic biopsies, and EUS-FNA in the evaluation of biliary strictures.
        Dig Dis Sci. 2018; 63: 636-644
        • Kipp B.R.
        • Stadheim L.M.
        • Halling S.A.
        • et al.
        A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures.
        Am J Gastroenterol. 2004; 99: 1675-1681
        • Navaneethan U.
        • Njei B.
        • Venkatesh P.G.
        • et al.
        Fluorescence in situ hybridization for diagnosis of cholangiocarcinoma in primary sclerosing cholangitis: a systematic review and meta-analysis.
        Gastrointest Endosc. 2014; 79: 943-950
        • Dudley J.C.
        • Zheng Z.
        • McDonald T.
        • et al.
        Next-generation sequencing and fluorescence in situ hybridization have comparable performance characteristics in the analysis of pancreaticobiliary brushings for malignancy.
        J Mol Diagn. 2016; 18: 124-130
        • Singhi A.D.
        • Nikiforova M.N.
        • Chennat J.
        • et al.
        Integrating next-generation sequencing to endoscopic retrograde cholangiopancreatography (ERCP)-obtained biliary specimens improves the detection and management of patients with malignant bile duct strictures.
        Gut. 2020; 69: 52-61
        • Cazacu I.M.
        • Semaan A.
        • Stephens B.
        • et al.
        Diagnostic value of digital droplet polymerase chain reaction and digital multiplexed detection of single nucleotide variants in pancreatic cytology specimens collected by EUS-guided FNA.
        Gastrointest Endosc. 2020; 12
        • Kobayashi M.
        • Ryozawa S.
        • Araki R.
        • et al.
        Investigation of factors affecting the sensitivity of bile duct brush cytology.
        Intern Med. 2019; 58: 329-335