This clinical study investigates whether the composition of the fecal microbiome and selected bacterial metabolites are associated with the efficacy and acute toxicity of radiotherapy in patients with locally advanced head and neck cancer treated with radical (chemo)radiotherapy. Increasing evidence suggests that gut bacteria and their metabolites may influence treatment response and side effects of cancer therapies, including radiotherapy.

Participants will receive standard radical radiotherapy with or without concomitant chemotherapy according to clinical guidelines. During the course of treatment, participants will provide two stool samples and two blood samples. Stool samples will be collected at home and delivered to the hospital on the day of radiotherapy. Blood samples (7 ml) will be collected during routine clinical blood draws. Samples will be analyzed to determine fecal microbiome composition using 16S rRNA sequencing and quantification of butyrate-producing bacterial genes (buk and but), as well as concentrations of short-chain fatty acids and tryptophan metabolites using targeted metabolomics.

Clinical outcomes will include treatment response assessed by CT or PET-CT imaging 3 months after completion of therapy, acute toxicity (CTCAE v5.0), and follow-up survival outcomes. The study aims to identify microbiome-related biomarkers that may contribute to improved personalization of supportive interventions in the future.

Head and neck cancer represents approximately 4-5% of all cancers worldwide and is associated with relatively poor survival outcomes. Most patients present with locoregionally advanced disease, and despite aggressive multimodal treatment including radiotherapy, recurrence occurs in up to half of patients within two years. Acute toxicities of radiotherapy, particularly radiomucositis and radiodermatitis, occur in more than 90% of patients and may lead to interruptions of treatment, negatively affecting outcomes.

Recent evidence indicates that the gut microbiome (fecal microbiota) and its metabolites may influence the efficacy and toxicity of cancer treatments. Studies have demonstrated associations between gut microbiota composition and response to immunotherapy and chemotherapy, and preclinical data suggest that specific bacterial metabolites, including short-chain fatty acids and tryptophan metabolites, may affect radiotherapy-related toxicity and survival. Emerging clinical studies have also shown associations between baseline gut microbiome diversity and survival or response to chemoradiotherapy in several cancer types. However, data regarding patients with head and neck cancer remain limited.

This prospective clinical study aims to evaluate the association between fecal microbiome composition and selected bacterial metabolites with treatment response and acute toxicity in patients with locoregionally advanced head and neck cancer undergoing radical radiotherapy with or without concomitant chemotherapy at the Institute of Oncology Ljubljana.

Participants will be treated according to standard institutional clinical practice and international guidelines. Biological samples will be collected at two time points during treatment: (1) from the start of radiotherapy until 7 days after initiation, but before administration of the first chemotherapy cycle (if applicable), and (2) during the last 7 days of radiotherapy. At each time point, participants will provide a stool sample (10-20 ml) collected at home and stored at 4°C until delivery to the hospital. Additionally, a 7 ml blood sample will be collected during routine clinical blood draws. Samples will be processed and stored at -80°C.

Microbiome analysis will be performed using sequencing of the V3-V4 regions of the 16S rRNA gene. Key bacterial genes involved in butyrate production (buk and but) will be quantified using PCR. Targeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) will be performed to quantify short-chain fatty acids and tryptophan metabolites in stool and serum samples.

Clinical and demographic data will be collected for each participant, including dietary intake during the 24 hours prior to sampling, radiotherapy dose-volume parameters, acute and late toxicity assessed using CTCAE v5.0, and response to treatment assessed by CT or PET-CT imaging 3 months after completion of (chemo)radiotherapy according to RECIST 1.1 criteria. Participants will be followed for recurrence-free survival and overall survival for two years.

The primary outcome is treatment response, defined as complete response versus incomplete response at the 3-month imaging evaluation. Secondary outcomes include overall survival, recurrence-free survival at two years, and acute treatment-related toxicity. Statistical analyses will include assessment of alpha and beta diversity, PERMANOVA analysis, and logistic regression models to evaluate associations between microbiome characteristics, metabolite concentrations, and clinical outcomes.

The study is exploratory and designed as a signal-finding study. Based on comparable published studies and feasibility considerations, approximately 60 patients will be enrolled.

Inclusion Criteria:

• Adult patients (≥18 years)
• Carcinoma of the oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, or carcinoma metastases in cervical lymph nodes of unknown primary origin
• Overall stage III-IV according to the 8th TNM classification
• Planned radical radiotherapy with or without concomitant chemotherapy

Exclusion Criteria:

• Planned induction chemotherapy
• Failure to complete radical (chemo)radiotherapy treatment