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

Summary

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.

Eligibility

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).

Conditions8

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