Page 60 - Molecular features of low-grade developmental brain tumours

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on the molecular contribution of DNA methylation in SEGAs specifically 17,27,29. Therefore, in this study we aimed to identify distinct methylation patterns and pathways that might contribute to SEGA pathogenesis by performing a comprehensive analysis of genomic DNA methylation in SEGAs.
Materials & Methods
SEGA tumour specimens
A total of 55 SEGA specimens were obtained from the following sites: the Amsterdam University Medical Center (location AMC), the University Medical Center Utrecht, University Medical Center Groningen, Medical University of Vienna, Children’s Memorial Health Institute in Warsaw, Meyer Children’s Hospital in Florence, University Hospital Erlangen, University Hospital Münster, Hacettepe University in Ankara, the University Hospital of Santa Maria (CHLUN) in Lisbon, and the North Bristol NHS Trust as part of the UK Brain Archive Information Network (BRAIN UK: Ref.: 16/002). Fifty-one of the SEGA samples were obtained from patients that met the clinical diagnostic criteria for TSC. Histological diagnosis was confirmed following the current WHO classification guidelines by two independent neuropathologists 7. TSC1/TSC2 mutation analysis was performed in blood or tumour sample DNA (for samples with a sufficient amount of DNA) as part of routine clinical care or was detected using massively parallel sequencing as described previously (Table 1) 30. The following clinical data was collected from medical records: TSC1/TSC2 mutation status, gender, localization of the SEGA, size of the tumour, age at seizure onset, seizure frequency, drug management at time of surgery (including treatment with mTORC1 inhibitors), tumour recurrence/regrowth and presence of other TSC related malformations. Periventricular brain tissue was used as control and was obtained from autopsy controls without a history of TSC, epilepsy, brain tumour or other neurological manifestations. Specimens were obtained and used in accordance with the Declaration of Helsinki and this study was approved by the Medical Ethics Committees of each institution.
DNA extraction & 450k methylation analysis
DNA was extracted from FFPE SEGA tumour samples (n=42) and location matched controls (n=8). Representative tumour regions were identified on hematoxylin & eosin sections for cases in which hemorrhages were prominent and were macrodisected from unstained sections (10-μm-thick) to reach a tumour cell content of 70% or more. For controls a punch of 2 mm diameter and up to 3 mm depth was taken from the periventricular zone. DNA was extracted using BiOstic FFPE Tissue DNA Isolation kit (MO BIO) according to the manufacturer’s instructions. 200-300 ng of DNA per sample was analysed using Illumina Infinium HumanMethylation450 BeadChip (450k) arrays according to the manufacturer’s instructions at the Genomics and Proteomics Core Facility of the DKFZ (Heidelberg, Germany).
RNA isolation and RNA sequencing
Frozen tissue of 12 SEGAs was homogenized with Qiazol Lysis Reagent (Qiagen