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

Inclusion criteria

• Internal Training and Validation Cohorts
• BTC patients
• 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).
• Age 18-80 years (inclusive).
• Able to provide required blood samples.
• Pathologically confirmed biliary tract carcinoma (TNM stage I-IV).
• Stable vital signs; ECOG performance status 0-1.
• 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)
• Voluntary participation with signed informed consent (or by LAR).
• Age 18-80 years (inclusive).
• Able to provide required blood samples.
• Pathologically confirmed gastrointestinal malignancies other than BTC, including hepatocellular carcinoma, gastric cancer, colorectal cancer, and pancreatic cancer (TNM stage I-IV).
• Stable vital signs; ECOG performance status 0-1.
• Non-cancer participants (benign biliary disease)
• Able to provide written informed consent.
• Able to provide required blood samples.
• Age 18-80 years (inclusive).
• 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
• Voluntary participation with signed informed consent (or by LAR).
• 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.
• Age 18-80 years (inclusive).
• Able to provide required blood samples.
• Stable vital signs; ECOG performance status 0-1.
• 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
• Able to provide written informed consent.
• Able to provide required blood samples.
• Age 18-80 years (inclusive).

Exclusion criteria

• Training and Validation Cohorts
• Cancer patients
• Pregnant or breastfeeding women.
• History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
• Blood transfusion within 7 days prior to blood collection.
• History of curative cancer treatment within 3 years prior to blood collection.
• Use of anti-tumor drugs within 30 days prior to blood collection.
• Known bleeding disorders.
• Known autoimmune diseases.
• Concurrent other malignancies or multiple primary tumors.
• Non-cancer participants
• Pregnant or breastfeeding women.
• History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
• Blood transfusion within 7 days prior to blood collection.
• History of any malignant tumor.
• Known bleeding disorders.
• Known autoimmune diseases.
• Clinically significant abnormalities on routine examination (excluding hepatitis, hepatic cysts, or benign pulmonary nodules).
• External Validation Cohorts
• Cancer patients
• Pregnant or breastfeeding women.
• History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
• Blood transfusion within 7 days prior to blood collection.
• History of or ongoing curative cancer treatment within 3 years prior to blood collection.
• Use of anti-tumor drugs within 30 days prior to blood collection.
• Known bleeding disorders or autoimmune diseases.
• Concurrent other malignancies (including multiple primaries) or known cancer susceptibility gene carriers.
• Pathology confirmed benign disease after biopsy/surgery.
• Failure to confirm malignancy by pathology or imaging within 42 days after blood collection, or unclear lesion site/evidence.
• 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
• Pregnant or breastfeeding women.
• History of organ transplantation or prior allogeneic bone marrow/stem cell transplantation.
• Blood transfusion within 7 days prior to blood collection.
• History of any malignant tumor.
• Known bleeding disorders or autoimmune diseases.
• Clinically significant abnormalities on health examination (excluding hepatitis, hepatic cysts, or benign pulmonary nodules).