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

Prior treatment

• 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:
• 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).

Other

• Pathology confirmed precancerous lesions.

• 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).