In this study, the investigators are examining the role of the immune system in pheochromocytoma and paraganglioma. The investigators aim to examine the differences in the immune system between people who have these tumors with and without a hereditary predisposition. The investigators also want to see how the immune system changes during the development of the tumor in people with a hereditary predisposition. Finally, the investigators will compare the data with a control group of people without these tumors. Ultimately, the investigators hope that the results will contribute to the discovery of new immune system-targeted medications for pheochromocytoma and paraganglioma.

Rationale: Pheochromocytoma and Paraganglioma (PPGL) represent rare, catecholamine-secreting tumours (1). Genetic or sporadic mutations, accounting for approximately 70% of cases, significantly contribute to PPGL development, and are categorized into three clusters based on tumour formation mechanisms (2,3). Current treatment options, particularly for advanced or metastatic disease, are limited (4). Understanding the immune system's role and the impact of genetics on tumour immunology in PPGL could unveil crucial insights for therapeutic advancements. Earlier studies emphasized the immunogenic nature of PPGL, highlighting the tumour microenvironment (TME) and circulatory factors as key components (5-7). However, these studies lack specific examination of genotypes' effect on the immune system. This study aims to address this gap in two parts, particularly focusing on differences between genetic clusters.

Objective: To examine the differences in the immune system in PPGL regarding genetics. Part I will examine immune cell composition and response in circulation. Part II will examine immune cell composition in TME.

Study design: Part I will be a partly cross-sectional and partly prospective cohort study. Part II will be a histological study of retrospectively and prospectively collected PPGL samples.

Study population: Part I will include 80 patients with PPGL, 80 carriers of germline mutations predisposing for PPGL, and 40 sex and age matched healthy volunteers. Part II will include histological samples of 80 patients with hereditary disease and 80 patients with sporadic disease.

Main study parameters/endpoints: The main study outcomes are inflammatory molecules and proteins produced by stimulated and unstimulated immune cells from circulation, immune cell composition in histological PPGL samples and in circulation, and their genetic determinants. Secondary outcomes will comprise of transcriptional and epigenetic signature of circulating immune cells, circulating immunomodulating metabolites, trained immunity, and clinical outcomes such as tumour metastasis, survival.

Nature and extent of the burden and risks associated with participation, benefit and group relatedness: For patients and mutation carriers, there is no direct benefit in participating in this study. However, by participating, they can contribute to the acquisition of scientific knowledge and the development of new therapeutic targets and novel disease management strategies. Such strategies might benefit patients and mutation carriers in the future if they potentially develop advanced disease. There are no risks associated with the study. There are no interventions other than those related to the regular patient care (venipuncture). Thus, this study is considered to impose a low burden on patients.

Inclusion Criteria:

Part I:

• Newly diagnosed patients with PPGL or newly diagnosed patients with (metastatic) PPGL recurrence.
• OR patients with mutations which predispose for the development of PPGL.
• Aged > 18 years.

Part II:

• Confirmed PPGL on pathology.
• Aged > 18 years.

Exclusion Criteria:

• Unable to provide informed consent.
• Active inflammatory or infectious comorbidities.
• Other malignancies which are under active treatment (except for basal cell carcinoma, other in situ carcinomas).
• Using medication interfering with the immune system
• Pregnancy or breastfeeding
• A self-reported alcohol consumption of >21 units per week

For part I, the study population will consist of patients who are presenting or are currently under follow-up at the Radboudumc endocrinology department for their PPGL. Eigthy patients will be included with hereditary or sporadic cases of PPGL and an equal number of asymptomatic carriers. The healthy control group will consist of 40 age and sex matched healthy volunteers.

Part II will include histological samples of 160 patients with PPGL, of which 80 hereditary and 80 sporadic. A large part of the population will overlap between part I and part II, since almost all patients with PPGL undergo surgery for their tumour. These samples will partly be retrospectively collected from the Radboudumc Urology Biobank, where biomaterials from previously treated patients with PPGLs are kept and stored or future scientific research (Human Subjects Review Board approval number: CWOM-9803-0060). The remaining samples will be collected prospectively from patients undergoing surgery for their PPGL.

Data will be made available by the corresponding author upon reasonable request.

One year after completion of the study.