Purpose of the Study:

Bile duct cancers are rare and aggressive. About 250 new cases are diagnosed each year in Denmark. These cancers are difficult to detect early, so only about 20% of patients can have surgery when diagnosed. Even after surgery, the cancer often returns, and chemotherapy only slightly reduces the risk of relapse.

For patients who cannot have surgery, treatments such as chemotherapy (sometimes combined with immunotherapy) can relieve symptoms and extend life, but their effect is limited. A small number of patients have specific genetic changes in their cancer that can be treated with targeted medicines.

Currently, doctors cannot predict which patients will benefit from treatment. Standard monitoring methods like CT scans are expensive, inconvenient, and sometimes unreliable because bile ducts are hard to see clearly on scans.

Blood tests that detect cancer DNA in the blood (called circulating tumor DNA or ctDNA) and other biological markers may be a better way to monitor the disease and adjust treatment. These tests could help detect cancer recurrence earlier and determine whether treatment is working. Measuring patients' quality of life and symptoms over time may also help predict treatment benefit and evaluate effectiveness.

The goal of this study is to:

• Investigate how biomarkers, including ctDNA, can predict disease course, detect relapse, and monitor treatment response.
• Identify the best way to measure ctDNA in patients with bile duct cancer.
• Examine whether patients' own reports of quality of life and symptoms can help assess treatment effect and prognosis.

Study Design and Procedures:

This is a prospective cohort study focusing on blood biomarkers and patient-reported symptoms and quality of life.

Participants agree to provide blood samples:

• Before treatment
• During treatment
• During follow-up

Each sample involves up to 40 ml of blood, with a maximum of 20 samples per patient.

The blood will be analyzed for:

• ctDNA and genetic changes
• Cancer-related markers
• Inflammation markers
• Immune system markers

Tumor tissue samples will also be examined to compare blood and tissue results. Full genome or exome sequencing will not be performed. Samples will be stored in a research biobank.

For patients with incurable disease, quality of life and symptom burden will be monitored repeatedly using Danish questionnaires.

Participants:

The study will include:

• Up to 100 patients with potentially curable disease
• Up to 200 patients with incurable disease

To participate, patients must:

• Have confirmed bile duct cancer
• Be eligible for curative, additional (adjuvant), or palliative treatment
• Be over 18 years old
• Provide written and verbal consent

Patients cannot participate if they:

• Had another cancer within the past 5 years (except early skin cancer or very early cervical cancer)
• Cannot safely provide blood samples
• Are unable to cooperate with study procedures

Risks and Inconveniences:

Participants will have extra blood samples taken, usually during regular hospital visits. Possible side effects include mild soreness or small bruises at the needle site. The extra blood amount (40 ml per sample) is considered medically insignificant.

Participants will also spend time filling out questionnaires. The number and frequency of questions have been kept as low as possible while still providing meaningful data.

Financial Information:

Extra costs for blood sampling, laboratory analysis, and data collection will be covered by external research funding managed by Aarhus University Hospital.

The researchers have no financial interest in the project. Patients will not receive financial compensation for participating.

Recruitment and Consent:

Potential participants are identified during routine clinical care. During a planned meeting with a doctor, patients receive written and verbal information about the study, including its purpose, risks, advantages, and disadvantages.

The conversation takes place in a calm and private setting. Patients may bring a support person. They have time to ask questions and at least 24 hours to consider participation.

Patients can withdraw their consent at any time without affecting their treatment. Consent must be given before any study-related procedures begin.

Publication of Results:

The results - whether positive or negative - will be presented at national and international conferences and submitted to peer-reviewed scientific journals.

Ethical Considerations:

All participants receive standard medical treatment. The risks and disadvantages are limited, and participants are unlikely to benefit directly from the study. However, the research may improve how biomarkers and patient-reported outcomes are used to predict prognosis and treatment response, potentially leading to better treatment for future patients with bile duct cancer.

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.

Study participants will be selected from adult patients with histopathologically verified BTC-or those defined as having BTC by a Multidisciplinary Team (MDT) conference-who are candidates for curative (including downstaging and adjuvant) or palliative treatment and who have no contraindications to blood sampling. Patients are identified in routine clinical practice and referred for oncological management.

Patients will be recruited prospectively as they are seen in daily clinical care at participating oncology centers. Eligible individuals must be able to provide written and oral informed consent. The population therefore includes patients across the disease spectrum (localized, locally advanced, or metastatic BTC) undergoing standard-of-care treatments such as surgery, systemic therapy, interventional procedures, or surveillance, with no alteration of therapy due to study participation.

Individual participant data (IPD) will not be shared due to ethical, legal, and privacy considerations. The dataset contains sensitive clinical information, biomarker results (including ctDNA analyses), and patient-reported outcomes that could potentially allow re-identification of participants despite pseudonymization. Data collection and storage are governed by informed consent, data protection regulations, and regional legislation (e.g., GDPR), which restrict the sharing of identifiable or potentially identifiable health data beyond the approved research purposes. Additionally, the consent obtained specifies use of data within the scope of the study and related ethically approved research. Therefore, only aggregated and anonymized results will be reported in publications, and any future data sharing would require additional ethical approval and appropriate data-sharing agreements to ensure participant confidentiality and compliance with applicable regulations.