TRACE-BTC. Relation of Biomarkers and Patients Reported Quality of Life to Outcomes in Patients With Biliary Tract Cancer: a Real- World Cohort

This prospective, non-interventional observational study investigates the longitudinal dynamics of circulating tumor DNA (ctDNA), circulating biomarkers, and patient-reported outcomes in patients with biliary tract cancer (BTC) undergoing standard oncological treatment in routine clinical practice. The study is conducted across participating oncology centers and integrates translational laboratory analyses with structured clinical and patient-reported data collection.

# Study Design and Population

Participants are enrolled consecutively from the clinical population of patients diagnosed with BTC and assessed for curative-intent (including adjuvant or downstaging strategies) or palliative oncological treatment. Inclusion occurs within routine care pathways, and study participation does not influence treatment allocation, clinical decision-making, or follow-up schedules. All therapeutic interventions are delivered according to institutional standards and national guidelines.

# Longitudinal Sampling Framework

Peripheral blood samples are collected at predefined clinically relevant milestones across the treatment trajectory, including baseline, during systemic therapy, perioperative periods (if applicable), response evaluation, and follow-up. Sampling schedules are protocolized and may be reset if treatment strategy changes to ensure temporal alignment with clinical course. Each sampling allows collection of up to 40 mL of blood, with a maximum of 20 study-related samples per participant over the study period.

Specimens are processed using standardized pre-analytical workflows and stored at -80°C in a central research biobank. Temporary local storage at participating sites (maximum 5-7 days) is permitted prior to shipment to the central biobank under controlled conditions. All samples are pseudonymized using unique study identifiers.

# Translational Research Program

Retrospective batch analyses are performed on centrally stored plasma samples to ensure methodological consistency and reduce analytical variability. The translational laboratory program includes:

• Tumor-informed and tumor-uninformed ctDNA assays
• Detection of single nucleotide variants, indels, copy number alterations, and gene fusions
• Targeted methylation marker analyses
• Comparative analyses between plasma-derived biomarkers and archival tumor tissue
• Immunohistochemical characterization of tumor tissue when available
• Exploratory assessment of additional circulating biomarkers related to tumor burden, inflammation, and immune status

Whole-genome and whole-exome sequencing are not performed. All laboratory analyses follow validated, standardized workflows and are conducted at accredited academic laboratory facilities, including Aarhus University Hospital and collaborating molecular diagnostic laboratories.

# Patient-Reported Outcomes

Health-related quality of life (HRQoL) is assessed longitudinally using validated Danish-language instruments at predefined time points aligned with treatment and follow-up visits. Questionnaire burden is minimized while maintaining methodological robustness. PROM data are integrated with clinical and biomarker datasets to enable exploratory analyses of associations between biological markers and patient-reported outcomes.

• Data Collection and Management

Clinical data are extracted from electronic medical records and entered into a secure, centralized REDCap database hosted by Aarhus University. The database incorporates role-based access control, audit trails, predefined validation rules, and structured electronic case report forms. All data are pseudonymized, and no study data are recorded prior to written informed consent.

Data sources include routine clinical documentation, laboratory records, and PROM questionnaires. A predefined data dictionary ensures standardized variable definitions, harmonized terminology, and reproducibility across sites.

# Quality Assurance and Governance

The study is centrally coordinated and conducted in accordance with standardized operating procedures covering consent procedures, biospecimen handling, laboratory workflows, data entry, and protocol deviation management. Automated validation rules, logical consistency checks, and periodic internal data reviews are implemented to ensure data quality and completeness. Source data verification is supported by audit trails and controlled access to medical records for authorized personnel.

# Statistical and Analytical Approach

Analyses focus on the association between ctDNA dynamics, clinical variables, and outcomes. The statistical framework includes descriptive analyses, correlation analyses, survival modeling using Kaplan-Meier methods, and multivariable Cox regression to adjust for relevant clinical confounders. Comparative analyses between plasma-based biomarkers and tumor tissue findings will be performed where available. Integration of biomarker data with longitudinal PROM data will be exploratory.

Sample size targets are based on feasibility and predefined translational objectives for curative-intent monitoring, baseline ctDNA detection in advanced disease, and early ctDNA dynamics during treatment.

# Ethical Considerations and Safety

The study involves minimal risk, as the only study-specific procedure is additional peripheral blood sampling. Potential risks are limited to transient discomfort or minor bruising. Participation does not interfere with standard clinical care, and patients may withdraw consent at any time without consequences for treatment.

All biological materials and study data are handled in compliance with applicable data protection legislation and ethical regulations. Residual biological material may be stored for future ethically approved research if separate consent is provided; otherwise, excess material is destroyed after completion of study-specific analyses.

# Study Timeline

Patient inclusion is planned from 2026 through 2030, with longitudinal follow-up and final analyses expected to be completed in 2031. Results will be disseminated through peer-reviewed publications and scientific conferences irrespective of study outcomes.

Inclusion criteria

• Histopathologically verified biliary tract cancer (BTC) and/or Multidisciplinary Team (MDT) conference decision to define the patient as suffering from BTC.
• Eligible for curative, adjuvant, or palliative oncological treatment.
• Age ≥ 18 years.
• Written and oral consent.

Exclusion criteria

• Other malignant diseases within 5 years of BTC diagnosis, excluding early-stage non-melanoma skin cancer and carcinoma in situ of the cervix.
• Conditions that prohibit blood sampling.
• Known or suspected non-compliance.