An estimated 1500 people in Sweden will annually be diagnosed with head and neck cancer (HNC). Five year survival is approximately 69%. Long-term sequelae are common and in particular nutritional problems and fatigue. Radiotherapy (RT) is the cornerstone of treatment, either as single modality treatment or combined modality treatment. RT can induce immune responses at the site of tumor. It has been demonstrated that RT can lead to a strong systemic immune response . We have previously shown that an increase of conventional measures of systemic immune response to RT varied significantly across individuals. We predict that local immune response plays a major role in the antitumor effect. We also predict that a strong systemic immune response contributes to malnutrition and influence on survival. And malnutrition may lead to a worse response to RT.

The overall aim of this multicenter observational longitudinal study is to prospectively identify immunological and metabolic variables that affect the outcome of HNC patients. We will systematically investigate the local and systemic immune response induced by RT as well as explore alterations in metabolite composition induced by disease and treatment through global metabolite profiling. A platform for studies on immuno-metabolic changes in HNC patients has been established in the Uppsala-Orebro and Northern regions. Approximately 370 patients per year are eligible. Findings in this study can have implications on the development of personalized therapy in patients with HNC. The long-term benefit of the study will be the identification of measures for improved patient surveillance in order to improve the general and nutritional outcomes.

The incidence of head and neck cancer (HNC) has increased in Sweden during the last decades. Radiotherapy (RT) is a cornerstone of treatment either as single or combined modality treatment. There is emerging awareness that RT affects the immune system. For HNC patients treated with RT nutritional problems and fatigue are the most common long-term adverse effects. About 60% have advanced stage of disease at time of diagnosis. Recurrence as well as early death within two years is common.

Our hypothesis: there is a complex association between immune response, metabolic factors and survival.

The aim of this multicenter patient-centered observational longitudinal study is to prospectively identify immunological and nutritional/metabolic variables that affect the outcome of HNC patients. We will systematically investigate the local and systemic immune response induced by RT as well as explore alterations in metabolite composition induced by disease and treatment through global metabolite profiling (metabolomics). This highly biologically relevant data may be used to search for specific biomarkers of malnutrition, markers which would be of prognostic and/or diagnostic value. Moreover, analytically reliable data allows for a more comprehensive mechanistic understanding, as such, a particular focus lies in ensuring a high data quality. We have established a platform for a study on immuno-metabolic changes in HNC patients in the Uppsala-Orebro and Northern regions. Findings in this large-scale observational study can have implications on the development of personalized therapy in patients with HNC.

Survey of the field. There is restricted information on the importance of local and systemic immune response to RT in HNC patients. RT has systemic effects in addition to its local effects on tumors and surrounding tissue. Pro-inflammatory cytokines are activated by RT and have been associated with increased symptom burden in cancer treatment. Interleukin-6 (IL-6) is a potent inflammatory cytokine with diverse functions. In lung cancer mice xenograft studies irradiation has shown to induce an increase in IL-6 levels and migration of macrophages to the tumor tissue. An association between C-reactive protein (CRP) and IL-6 in plasma has been demonstrated in esophageal adenocarcinoma patients. Relapse-free patients are reported to have an increased proportion of Natural killer cells (NK-cells) in peripheral blood long-term after treatment. We have earlier followed a cohort of HNC patients during RT with repeated blood samples. high-sensitivity C-reactive protein (hCRP) increased during RT, and patients with the most pronounced weight loss had the greatest hCRP increase. Metabolic factors are reported to affect HNC patients in many ways. Severe malnutrition before treatment of HNC is reported to lead to increased mortality. In a secondary study in patients with oropharyngeal cancer (n = 357) we found that a high BMI gives significantly better 5-year survival than a low BMI. However, it is difficult to evaluate the results in the majority of nutritional studies in HNC patients because most prospective studies have included relatively few patients and many of the results obtained in retrospective studies have been conflicting. Patient's metabolic profile might work as a nutritional marker and be distinctly linked to nutritional status. The blood metabolome can be examined in detail in cancer patients to better identify different metabolic pathways. The nutritional status is strongly connected to the immune status. It is e.g. reported that L-arginine is crucial for the function of T-cell. As the immune system at the site of the tumor can inhibit cancer growth the local anti-tumorigenic immune status is important. Thus the nutritional status predicts the immune status and malnutrition is connected to a weaker immune response. It is unclear how the nutritional status is connected with the systemic immune status.

Research questions

1. The correlation between weight change and pro-inflammatory cytokines.
2. The correlation between weight change and immune response in tumour microenvironment.
3. Change of blood metabolome.
4. Change of Bioelectrical impedance analysis (BIA) measurements.
5. Change of fatty acid profile in blood.
6. Patient-reported outcome measure (PROM)
7. Patient-reported stress levels.
8. Return to work.
9. PROM and relation to treatment and overall survival.

Variables and measures All patients will be treated in accordance with local and national guidelines. Nutrient based local guidelines ensure that the patients receive nutritional support when needed. Baseline measurements will be taken before treatment starts, and follow-ups will take place at 4 and 7 weeks after start of treatment and at 3, 6, 12, and 24 months post treatment.

A Web-Based Patient Case Report Form (CRF) has been developed for a reliable, secure and easier data collection including the following variables:

Cancer-free survival, cancer-related death as well as death not related to cancer disease, interval between treatment and recurrence.

Explanatory variables:

1. Body Composition measured by BIA and validated against body composition measured by Dual-energy X-ray absorptiometry (DXA).
2. Characterization of immune profile in tumor tissue. Surgical biopsy of the tumor at the endoscopy for histopathological diagnosis and after RT when possible. Two methods will be applied to analyse the biopsies: 1) Multiplex tissue staining using the PerkinElmar Opal system which provides a quantitative and spatial composition of the immune cell infiltrates. 2) The RNA based Immunobiology Panel, which allows the measurement of the gene expression of hundreds of immune markers.
3. Immune profile in serum: analyzed with a multiplex assay using the Science for Life Laboratory (SciLife) platform.
4. Fatty acid profiling in serum with gas chromatography techniques.
5. Metabolomic parameters assessed with a multi-platform approach based on nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS) connected to the ultra-performance liquid chromatography (UPLC).
6. Body weight (kg) estimated 6 months before start of treatment, body weight registered at start of treatment and at all the follow-ups.

Questionnaires:

Nutritional follow-up with Patient Reported Outcome Measure Head and Neck Symptom Checklist (PROM - HNSC) (14). Quality of life (QoL) HNC specific questionnaire European Organization for Research and Treatment Quality of Life instrument (EORTC QLQ-H & N 35) (15). Hospital Anxiety and Depression Scale (HADS). Stress with questionnaire Perceived Stress Questionnaire (PSQ) (16).

Study design A prospective observational longitudinal study to elucidate all the research questions.

Estimated sample size About 1500 HNC patients are diagnosed annually in Sweden. The number of patients in this study before the exclusion criteria is approximately 370 patients/year.

Material: Patient selection - population, sample This multicenter study is a collaboration between the Uppsala-Orebro region and the Northern region. The patients are recruited at three university hospitals: Some of the patients are after treatment followed-up at their County Hospital. Inclusion criteria: curative treatment of patients with newly diagnosed HNC, and a World Health Organization (WHO) performance status of 0-2.

Inclusion Criteria:

Patients with newly diagnosed head and neck cancer

Exclusion Criteria:

Previous treatment for malignant disease except for skin cancer, severe alcohol abuse, psychiatric disorder, inability to understand Swedish

Patients diagnosed with head and neck cancer at the following three university hospitals:

Uppsala University Hospital, Orebro University Hospital, Umea University Hospital