A Cell-free DNA Methylation Blood-Based Test for Biliary Tract Cancers Screening

A Cell-free DNA Methylation Liquid Biopsy for Diagnosis and Management of Biliary Tract Cancers

Biliary tract carcinoma (BTC), including gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma, ranks sixth in incidence among gastrointestinal malignancies and tenth in cancer-related mortality worldwide. Due to the lack of specific early symptoms, high malignancy, and frequent recurrence and metastasis, the rate of curative resection is only about 16.5%, and the overall 5-year survival rate is less than 5%. Early and accurate detection is therefore critical for improving patient outcomes. Circulating tumor DNA (ctDNA), a fraction of circulating free DNA (cfDNA), carries genetic and epigenetic information from tumor cells and can be detected even at the early stages of cancer development. Among various liquid biopsy biomarkers, ctDNA methylation shows particular advantages in sensitivity and specificity for early cancer detection and monitoring. This study aims to evaluate the application of cfDNA methylation liquid biopsy in the diagnosis and management of BTC.

Study Overview

• Status: Recruiting
• Conditions: Hilar Cholangiocarcinoma; Gall Bladder Cancer; Extrahepatic Cholangiocarcinoma; ctDNA; Intrahepatic Cholangiocarcinoma (Icc); Billiary Track Cancer

Detailed Description

Biliary tract carcinoma (BTC), encompassing gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma, is an aggressive malignancy with poor prognosis. Globally, it ranks sixth in incidence among gastrointestinal cancers and tenth in cancer-related mortality. BTC is characterized by the absence of specific early symptoms, high degree of malignancy, and a strong tendency for recurrence and metastasis. The curative resection rate remains around 16.5%, and the overall 5-year survival rate is less than 5%.

Biliary tract inflammation (such as cholangitis, acute cholecystitis, sclerosing cholangitis, and autoimmune cholangitis) can lead to abnormal CA19-9 elevation. In addition, IgG4-related sclerosing cholangitis often affects elderly patients and may mimic hilar cholangiocarcinoma, creating diagnostic challenges. Imaging alone often lacks accuracy in differentiating benign from malignant biliary lesions, resulting in misdiagnosis and inappropriate clinical decision-making. The number of patients with incidentally detected BTC during surgery is rapidly increasing, and reoperations impose substantial trauma and socioeconomic burden.

Circulating tumor DNA (ctDNA) refers to DNA fragments released into the bloodstream from tumor cells through apoptosis, necrosis, or secretion. ctDNA carries genomic alterations (point mutations, indels, CNVs, fusions), epigenetic modifications (DNA methylation [5mC], hydroxymethylation [5hmC]), and structural features (fragment length, fragmentation patterns). Circulating free DNA (cfDNA) represents the total extracellular DNA in plasma or serum, of which ctDNA accounts for less than 1%. Accumulating evidence shows that ctDNA is detectable in the early stages of tumorigenesis, highlighting its clinical potential in early detection, diagnosis, treatment guidance, and post-treatment monitoring.

The choice of biomarker type is key to liquid biopsy applications, and ctDNA methylation offers distinct advantages. Liquid biopsy analytes include circulating tumor cells (CTCs), ctDNA, exosomes, and microRNAs (miRNAs), each with unique detection characteristics. ctDNA, directly derived from tumor cells, provides relatively high sensitivity for early detection and is currently the most widely used target in clinical practice. Research on ctDNA has focused mainly on methylation, somatic mutations, and copy number variations. Among these, ctDNA methylation balances both signal abundance and signal strength, offering clear advantages over other approaches. Methylation analysis methods include restriction enzyme digestion, affinity enrichment, and bisulfite conversion. Among them, bisulfite-based approaches are the most established and widely used.

Therefore, the application of cfDNA methylation liquid biopsy in BTC for early screening, differential diagnosis, prognostic monitoring, and therapeutic guidance holds great significance for improving diagnosis, treatment, and patient outcomes.

• Study Type: Observational
• Enrollment (Estimated): 1800

Contacts and Locations

• Study Contact: Name: Yingbin Liu, PhD; Phone Number: +86 13918803900; Email: laoniulyb@163.com

Study Locations

• China
  • Shanghai Municipality
    • Shanghai, Shanghai Municipality, China, 200127
      • Recruiting
      • Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai
      • Contact: Yingbin Liu, PhD; Phone Number: +86 13918803900; Email: laoniulyb@163.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

The study population comprises individuals meeting inclusion criteria and not meeting any exclusion criteria.

Description

Inclusion Criteria Internal Training and Validation Cohorts

• BTC patients

  1. Willing to voluntarily participate and able to comply with study procedures; if unable to read or sign, informed consent must be signed by a legally authorized representative (LAR).
  2. Age 18-80 years (inclusive).
  3. Able to provide required blood samples.
  4. Pathologically confirmed biliary tract carcinoma (TNM stage I-IV).
  5. Stable vital signs; ECOG performance status 0-1.
  6. Adequate organ function: AST/ALT ≤ 5 × ULN; Child-Pugh class A or B; WBC > 3 × 10⁹/L; ANC ≥ 1.5 × 10⁹/L; Platelets ≥ 75 × 10⁹/L; Hemoglobin ≥ 90 g/L; Creatinine clearance ≥ 60 mL/min; Total bilirubin ≤ 3 × ULN.

• Other gastrointestinal malignancies (to exclude BTC non-specific signals)

  1. Voluntary participation with signed informed consent (or by LAR).
  2. Age 18-80 years (inclusive).
  3. Able to provide required blood samples.
  4. Pathologically confirmed gastrointestinal malignancies other than BTC, including hepatocellular carcinoma, gastric cancer, colorectal cancer, and pancreatic cancer (TNM stage I-IV).
  5. Stable vital signs; ECOG performance status 0-1.

• Non-cancer participants (benign biliary disease)

  1. Able to provide written informed consent.
  2. Able to provide required blood samples.
  3. Age 18-80 years (inclusive).
  4. Pathologically or clinically diagnosed benign biliary diseases, including cholecystitis, cholelithiasis, choledocholithiasis, adenomyomatosis, gallbladder polyps, xanthogranulomatous cholecystitis, or primary sclerosing cholangitis.

External Validation Cohorts

• BTC patients

  1. Voluntary participation with signed informed consent (or by LAR).
  2. Imaging findings of malignant biliary stricture or mass, or serum CA19-9 > 100 U/mL, highly suspicious for BTC, with planned surgery or biopsy for pathological confirmation.
  3. Age 18-80 years (inclusive).
  4. Able to provide required blood samples.
  5. Stable vital signs; ECOG performance status 0-1.
  6. Adequate organ function: AST/ALT ≤ 5 × ULN; Child-Pugh class A or B; WBC > 3 × 10⁹/L; ANC ≥ 1.5 × 10⁹/L; Platelets ≥ 75 × 10⁹/L; Hemoglobin ≥ 90 g/L; Creatinine clearance ≥ 60 mL/min; Total bilirubin ≤ 3 × ULN.

• Healthy volunteers

  1. Able to provide written informed consent.
  2. Able to provide required blood samples.
  3. Age 18-80 years (inclusive).

Exclusion Criteria Training and Validation Cohorts

• Cancer patients

  1. Pregnant or breastfeeding women.
  2. History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
  3. Blood transfusion within 7 days prior to blood collection.
  4. History of curative cancer treatment within 3 years prior to blood collection.
  5. Use of anti-tumor drugs within 30 days prior to blood collection.
  6. Known bleeding disorders.
  7. Known autoimmune diseases.
  8. Concurrent other malignancies or multiple primary tumors.

• Non-cancer participants

  1. Pregnant or breastfeeding women.
  2. History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
  3. Blood transfusion within 7 days prior to blood collection.
  4. History of any malignant tumor.
  5. Known bleeding disorders.
  6. Known autoimmune diseases.
  7. Clinically significant abnormalities on routine examination (excluding hepatitis, hepatic cysts, or benign pulmonary nodules).

External Validation Cohorts

• Cancer patients

  1. Pregnant or breastfeeding women.
  2. History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
  3. Blood transfusion within 7 days prior to blood collection.
  4. History of or ongoing curative cancer treatment within 3 years prior to blood collection.
  5. Use of anti-tumor drugs within 30 days prior to blood collection.
  6. Known bleeding disorders or autoimmune diseases.
  7. Concurrent other malignancies (including multiple primaries) or known cancer susceptibility gene carriers.
  8. Pathology confirmed benign disease after biopsy/surgery.
  9. Failure to confirm malignancy by pathology or imaging within 42 days after blood collection, or unclear lesion site/evidence.
  10. Special exclusion criteria:
• Pathology confirmed precancerous lesions.
• Any local/regional or systemic anti-tumor therapy (including surgery, radiotherapy, targeted therapy, or immunotherapy) prior to blood collection.

• Healthy volunteers

  1. Pregnant or breastfeeding women.
  2. History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
  3. Blood transfusion within 7 days prior to blood collection.
  4. History of any malignant tumor.
  5. Known bleeding disorders or autoimmune diseases.
  6. Clinically significant abnormalities on health examination (excluding hepatitis, hepatic cysts, or benign pulmonary nodules).

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort
control（Internal training and validation sets）; Healthy individuals Patients with pathologically confirmed benign biliary lesions Patients with other gastrointestinal malignancies
malignant（Internal training and validation sets）; patients with intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer
malignant (Independent validation set); patients with suspected biliary tract malignancies
control (Independent validation set); Healthy individuals

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Diagnostic performance of the ctDNA methylation model in biliary tract cancer	Evaluate the overall sensitivity, specificity, and accuracy of the ctDNA methylation liquid biopsy model in the diagnosis of biliary tract cancer (BTC).	Baseline

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Subtype-specific diagnostic performance	Sensitivity, specificity, and accuracy of the ctDNA methylation model in gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma.	Baseline
Stage-specific diagnostic performance	Sensitivity, specificity, and accuracy of the model across BTC stages I-IV (TNM staging).	Baseline
Differential diagnosis	Sensitivity, specificity, and accuracy of the ctDNA methylation model in distinguishing BTC from benign biliary lesions.	Baseline

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Progression-Free Survival (PFS)	To assess the association between ctDNA methylation and PFS in BTC patients.	From date of diagnosis until documented progression or death, whichever occurs first, up to 5 years.
Overall Survival (OS)	To assess the association between ctDNA methylation and OS in BTC patients.	From date of diagnosis until death from any cause, up to 5 years.
Recurrence monitoring performance	Sensitivity and specificity of ctDNA methylation for monitoring recurrence and progression of biliary tract malignancies.	Up to 5 years post-treatment

Collaborators and Investigators

• Sponsor: Yingbin Liu, MD, PhD, FACS
• Collaborators: Xijing Hospital; Sun Yat-sen University; West China Hospital; Qilu Hospital of Shandong University; First Affiliated Hospital Xi'an Jiaotong University; Hunan Provincial People's Hospital; Fudan University Shanghai Cancer Center ( FUSCC )
• Investigators: Principal Investigator: Yingbin Liu, PhD, Renji Hospital

Publications and helpful links

General Publications

• Chung DC, Gray DM 2nd, Singh H, Issaka RB, Raymond VM, Eagle C, Hu S, Chudova DI, Talasaz A, Greenson JK, Sinicrope FA, Gupta S, Grady WM. A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening. N Engl J Med. 2024 Mar 14;390(11):973-983. doi: 10.1056/NEJMoa2304714.
• Nagino M, Hirano S, Yoshitomi H, Aoki T, Uesaka K, Unno M, Ebata T, Konishi M, Sano K, Shimada K, Shimizu H, Higuchi R, Wakai T, Isayama H, Okusaka T, Tsuyuguchi T, Hirooka Y, Furuse J, Maguchi H, Suzuki K, Yamazaki H, Kijima H, Yanagisawa A, Yoshida M, Yokoyama Y, Mizuno T, Endo I. Clinical practice guidelines for the management of biliary tract cancers 2019: The 3rd English edition. J Hepatobiliary Pancreat Sci. 2021 Jan;28(1):26-54. doi: 10.1002/jhbp.870. Epub 2020 Dec 23.
• Yang M, Zhao Y, Li C, Weng X, Li Z, Guo W, Jia W, Feng F, Hu J, Sun H, Wang B, Li H, Li M, Wang T, Zhang W, Jiang X, Zhang Z, Liu F, Hu H, Wu X, Gu J, Yang G, Li G, Zhang H, Zhang T, Zang H, Zhou Y, He M, Yang L, Wang H, Chen T, Zhang J, Chen W, Wu W, Li M, Gong W, Lin X, Liu F, Liu Y, Liu Y. Multimodal integration of liquid biopsy and radiology for the noninvasive diagnosis of gallbladder cancer and benign disorders. Cancer Cell. 2025 Mar 10;43(3):398-412.e4. doi: 10.1016/j.ccell.2025.02.011.
• Vogel A, Bridgewater J, Edeline J, Kelley RK, Klumpen HJ, Malka D, Primrose JN, Rimassa L, Stenzinger A, Valle JW, Ducreux M; ESMO Guidelines Committee. Electronic address: clinicalguidelines@esmo.org. Biliary tract cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2023 Feb;34(2):127-140. doi: 10.1016/j.annonc.2022.10.506. Epub 2022 Nov 10. No abstract available.

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2020
• Primary Completion (Estimated): March 21, 2026
• Study Completion (Estimated): May 1, 2026

Study Registration Dates

• First Submitted: September 10, 2025
• First Submitted That Met QC Criteria: September 10, 2025
• First Posted (Estimated): September 16, 2025

Study Record Updates

• Last Update Posted (Estimated): September 16, 2025
• Last Update Submitted That Met QC Criteria: September 10, 2025
• Last Verified: September 1, 2025

More Information

Terms related to this study

• Keywords: Liquid biopsy; Early diagnosis; ctDNA methylation; Billiary Track Cancer
• Additional Relevant MeSH Terms: Pathologic Processes; Neoplasms by Site; Neoplasms; Neoplasms by Histologic Type; Digestive System Neoplasms; Digestive System Diseases; Biliary Tract Diseases; Neoplasms, Glandular and Epithelial; Adenocarcinoma; Carcinoma; Gallbladder Diseases; Biliary Tract Neoplasms; Pathological Conditions, Signs and Symptoms; Disease; Cholangiocarcinoma; Klatskin Tumor; Gallbladder Neoplasms; Cirrhosis, Familial, with Pulmonary Hypertension
• Other Study ID Numbers: BTC_ctDNAm_LYB_2025

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No