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

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.

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).