Sample collection

Tumour specimens were collected from 10 patients enrolled between 2014 and 2018 in the German Society for Pediatric Oncology and Hematology (GPOH) NB2004 trial or NB registry 2016, and treated at the Charité University Medicine Berlin (Germany) according to the trial/registry protocols. All patients and/or their guardians gave written consent for the use of biosamples and clinical data for research in accordance with the local ethics review board of the medical faculty, University of Cologne as the trial sponsor of the NB2004 and the NB registry 2016 (https://clinicaltrials.gov/{"type":"clinical-trial","attrs":{"text":"NCT03042429","term_id":"NCT03042429"}}NCT03042429). Samples were collected by open surgical biopsy either at diagnosis, at tumour resection after 4–6 cycles of chemotherapy (according to neuroblastoma therapy regimen) or at diagnosis of relapse. Fresh samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C. Portions of tumour material were formalin-fixed and paraffin-embedded (FFPE) in parallel for diagnostics and preservation in the pathology unit. Two to 30 biopsies were taken from geographically separate areas of the tumour body with a minimal distance of 10 mm from each single tumour. From these biopsies, only tumour regions with high tumour cell content (>60% for WES and RNA sequencing, 10% for targeted re-sequencing) were included. A pathologist confirmed the diagnosis and assigned tumour regions for macrodissection with a high content of vital tumour cells on sequential hematoxylin and eosin-stained sections. Peripheral blood collected from each patient was used as a matched germline control for tumour samples. DNA was prepared using the Qiagen DNA Mini kit according to the manufacturer’s instructions.