DNA Methylation Markers for Detection of Cholangiocarcinoma: Discovery, Validation, and Clinical Testing in Biliary Brushings and Plasma

Ju Dong Yang, Hassan Ghoz, Mohammed M. Aboelsoud, William R. Taylor, Tracy C. Yab, Calise K. Berger, Xiaoming Cao, Patrick H. Foote, Nasra H. Giama, Emily G. Barr Fritcher, Douglas W. Mahoney, Catherine D. Moser, Thomas C. Smyrk, Benjamin R. Kipp, Gregory J. Gores, Lewis R. Roberts, John B. Kisiel

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Abstract

Cholangiocarcinoma (CCA) has poor prognosis due to late-stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced-representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin-fixed, paraffin-embedded CCA tumors and adjacent hepatobiliary control tissues using methylation-specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95-1.0). In the clinical pilot on plasma, a cross-validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81-0.95]) but not for primary sclerosing cholangitis–associated eCCA (AUC, 0.54 [0.35-0.73]). Conclusion: Next-generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

Original languageEnglish (US)
Pages (from-to)1448-1459
Number of pages12
JournalHepatology Communications
Volume5
Issue number8
DOIs
StatePublished - Aug 2021

Bibliographical note

Funding Information:
The authors dedicate this work to the memory of Dr. David A. Ahlquist (1951-2020). They thank Amanda Bedard for her administrative assistance and are grateful for the Genome Analysis Core (GAC) and co-directors Julie M. Cunningham, Ph.D., and Eric Wieben, Ph.D. GAC is supported, in part, by the Center for Individualized Medicine and the Mayo Clinic Comprehensive Cancer Center grant (National Cancer Institute P30CA15083).

Funding Information:
Potential conflict of interest: Ms. Berger owns intellectual property rights in Exact Sciences. Dr. Cao was employed by Exact Sciences. Mr. Foote owns intellectual property rights in Exact Sciences. Dr. Kisiel consults, received grants from, and owns intellectual property rights in Exact Sciences. Mr. Mahoney owns intellectual property rights in Exact Sciences. Dr. Roberts advises and received grants from Bayer. He consults for AstraZeneca, The Lynx Group, and MJH Life Sciences. He advises Exact Sciences, Envision Communications, Gilead, OED Therapeutics, GRAIL, and Genentech. He received grants from Ariad, Glycotest, RedHill, TARGET, BTG International, and Wako Diagnostics. Mr. Taylor owns intellectual property rights in Exact Sciences. Ms. Yab owns stock and intellectual property rights in Exact Sciences.

Funding Information:
The authors dedicate this work to the memory of Dr. David A. Ahlquist (1951‐2020). They thank Amanda Bedard for her administrative assistance and are grateful for the Genome Analysis Core (GAC) and co‐directors Julie M. Cunningham, Ph.D., and Eric Wieben, Ph.D. GAC is supported, in part, by the Center for Individualized Medicine and the Mayo Clinic Comprehensive Cancer Center grant (National Cancer Institute P30CA15083).

Funding Information:
Supported by the Jack and Maxine Zarrow Family Foundation, Multisite Gastrointestinal Cancer Detection by Stool DNA Methylation (CA214679), Paul Calabresi Program in Clinical‐Translational Research (CA90628), Carol M. Gatton endowment for Digestive Diseases Research, Eddie Gong and Dana Clay, The Cholangiocarcinoma Foundation, and Mayo Clinic SPORE in Hepatobiliary Cancer (P50 CA210964).

Publisher Copyright:
© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.

PubMed: MeSH publication types

  • Journal Article

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