Mutational Analysis of Bile Cell-Free DNA in Primary Sclerosing Cholangitis: A Pilot Study

Overview

Journal Liver Int

Specialty Gastroenterology

Date 2025 Mar 3

PMID 40029142

Authors

Maria Arechederra

Emil Bik

Carla Rojo

Jasmin Elurbide

Maria Elizalde

Beata Kruk

Maciej Krasnodebski

Jan Pertkiewicz

Slawomir Koziel

Michal Grat

Joanna Raszeja-Wyszomirska

Maria Rullan

Gorka Alkorta-Aranburu

Daniel Oyon

Maite G Fernandez-Barrena

Lena S Candels

Andrzej Bialek

Lukasz Krupa

Kai M Schneider

Jesus Urman

Pavel Strnad

Christian Trautwein

Piotr Milkiewicz

Marcin Krawczyk

Matias A Avila

Carmen Berasain

Affiliations

Soon will be listed here.

Abstract

Background: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterised by inflammation and fibrosis of the bile ducts, conferring an increased risk of cholangiocarcinoma (CCA). However, detecting CCA early in PSC patients remains challenging due to the limited sensitivity of conventional diagnostic methods, including imaging or bile duct brush cytology during endoscopic retrograde cholangiopancreatography (ERCP). This study aims to evaluate the potential of bile cell-free DNA (cfDNA) mutational analysis, termed the Bilemut assay, as a tool for CCA detection in PSC patients.

Methods: Sixty-three PSC patients undergoing ERCP due to biliary strictures were prospectively recruited. Bile samples were collected, and cfDNA was extracted and analysed using the Oncomine Pan-Cancer Cell-Free assay. Twenty healthy liver donors were included for comparison. Samples with a mutant allele frequency (MAF) ≥ 0.1% were considered positive. Correlations between mutational status and clinical characteristics were assessed.

Results: cfDNA mutational analysis was successful in all bile samples. Mutations predominantly in KRAS, GNAS, and TP53 were detected in 36.5% (23/63) of PSC patients, compared to 10% (2/20) of healthy donors (p = 0.0269). The clinical characteristics of Bilemut-positive and -negative patients were comparable, though there was a trend towards a lower prevalence of inflammatory bowel disease in the Bilemut-positive group. Among PSC patients diagnosed with CCA during follow-up, 75% were Bilemut-positive, suggesting an association between mutational status and malignancy risk.

Conclusions: Mutational analysis of cfDNA obtained from bile collected from PSC patients undergoing ERCP is feasible. Implementing the Bilemut assay may help identify patients needing closer surveillance and further imaging studies.

References

Muraki R, Morita Y, Ida S, Kitajima R, Furuhashi S, Takeda M . Phosphatidylcholine in bile-derived small extracellular vesicles as a novel biomarker of cholangiocarcinoma. Cancer Med. 2023; 12(12):13007-13018. PMC: 10315768. DOI: 10.1002/cam4.5973. View

Saca D, Flamm S . Cholangiocarcinoma Surveillance Recommendations in Patients with Primary Sclerosing Cholangitis. Clin Liver Dis. 2023; 28(1):183-192. DOI: 10.1016/j.cld.2023.07.010. View

Oh J, Sung C . Comprehensive characteristics of somatic mutations in the normal tissues of patients with cancer and existence of somatic mutant clones linked to cancer development. J Med Genet. 2020; 58(7):433-441. DOI: 10.1136/jmedgenet-2020-106905. View

Weismuller T, Trivedi P, Bergquist A, Imam M, Lenzen H, Ponsioen C . Patient Age, Sex, and Inflammatory Bowel Disease Phenotype Associate With Course of Primary Sclerosing Cholangitis. Gastroenterology. 2017; 152(8):1975-1984.e8. PMC: 5546611. DOI: 10.1053/j.gastro.2017.02.038. View

Kanda M, Matthaei H, Wu J, Hong S, Yu J, Borges M . Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 2012; 142(4):730-733.e9. PMC: 3321090. DOI: 10.1053/j.gastro.2011.12.042. View

Shen N, Zhang D, Yin L, Qiu Y, Liu J, Yu W . Bile cell‑free DNA as a novel and powerful liquid biopsy for detecting somatic variants in biliary tract cancer. Oncol Rep. 2019; 42(2):549-560. PMC: 6610033. DOI: 10.3892/or.2019.7177. View

Gleeson D, Walmsley M, Trivedi P, Joshi D, Rea B . Surveillance for cholangiocarcinoma in patients with primary sclerosing cholangitis: Can we be more proactive?. Frontline Gastroenterol. 2023; 14(2):162-166. PMC: 9933607. DOI: 10.1136/flgastro-2022-102172. View

Molodecky N, Kareemi H, Parab R, Barkema H, Quan H, Myers R . Incidence of primary sclerosing cholangitis: a systematic review and meta-analysis. Hepatology. 2011; 53(5):1590-9. DOI: 10.1002/hep.24247. View

Palmela C, Peerani F, Castaneda D, Torres J, Itzkowitz S . Inflammatory Bowel Disease and Primary Sclerosing Cholangitis: A Review of the Phenotype and Associated Specific Features. Gut Liver. 2017; 12(1):17-29. PMC: 5753680. DOI: 10.5009/gnl16510. View

10.

Kinugasa H, Nouso K, Ako S, Dohi C, Matsushita H, Matsumoto K . Liquid biopsy of bile for the molecular diagnosis of gallbladder cancer. Cancer Biol Ther. 2018; 19(10):934-938. PMC: 6300345. DOI: 10.1080/15384047.2018.1456604. View

11.

Rey Rubiano A, Gonzalez-Teshima L, Arango L, Blanco-Avellaneda C, Carvajal Gutierrez J, Castano-Llano R . Clinical practice guideline on the use of single-operator cholangioscopy in the diagnosis of indeterminate biliary stricture and the treatment of difficult biliary stones. Surg Endosc. 2023; 38(2):499-510. PMC: 10830582. DOI: 10.1007/s00464-023-10569-x. View

12.

Singhi A, Nikiforova M, Chennat J, Papachristou G, Khalid A, Rabinovitz M . 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. 2019; 69(1):52-61. PMC: 6943248. DOI: 10.1136/gutjnl-2018-317817. View

13.

Karlsen T, Folseraas T, Thorburn D, Vesterhus M . Primary sclerosing cholangitis - a comprehensive review. J Hepatol. 2017; 67(6):1298-1323. DOI: 10.1016/j.jhep.2017.07.022. View

14.

Arechederra M, Rullan M, Amat I, Oyon D, Zabalza L, Elizalde M . Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures. Gut. 2021; 71(6):1141-1151. PMC: 9120390. DOI: 10.1136/gutjnl-2021-325178. View

15.

Gerges C, Beyna T, Tang R, Bahin F, Lau J, van Geenen E . Digital single-operator peroral cholangioscopy-guided biopsy sampling versus ERCP-guided brushing for indeterminate biliary strictures: a prospective, randomized, multicenter trial (with video). Gastrointest Endosc. 2019; 91(5):1105-1113. DOI: 10.1016/j.gie.2019.11.025. View

16.

Kano Y, Ishikawa T, Yamao K, Mizutani Y, Iida T, Uetsuki K . What is the appropriate method of pathological specimen collection for cholangiocarcinoma detection in primary sclerosing cholangitis?. J Gastroenterol. 2024; 59(7):621-628. DOI: 10.1007/s00535-024-02105-y. View

17.

Goeppert B, Folseraas T, Roessler S, Kloor M, Volckmar A, Endris V . Genomic Characterization of Cholangiocarcinoma in Primary Sclerosing Cholangitis Reveals Therapeutic Opportunities. Hepatology. 2020; 72(4):1253-1266. DOI: 10.1002/hep.31110. View

18.

Loi E, Zavattari C, Tommasi A, Moi L, Canale M, Po A . HOXD8 hypermethylation as a fully sensitive and specific biomarker for biliary tract cancer detectable in tissue and bile samples. Br J Cancer. 2022; 126(12):1783-1794. PMC: 9174245. DOI: 10.1038/s41416-022-01738-1. View

19.

Nagai K, Kuwatani M, Hirata K, Suda G, Hirata H, Takishin Y . Genetic Analyses of Cell-Free DNA in Pancreatic Juice or Bile for Diagnosing Pancreatic Duct and Biliary Tract Strictures. Diagnostics (Basel). 2022; 12(11). PMC: 9689036. DOI: 10.3390/diagnostics12112704. View

20.

Trikudanathan G, Navaneethan U, Njei B, Vargo J, Parsi M . Diagnostic yield of bile duct brushings for cholangiocarcinoma in primary sclerosing cholangitis: a systematic review and meta-analysis. Gastrointest Endosc. 2013; 79(5):783-9. DOI: 10.1016/j.gie.2013.09.015. View