Page 156 - Molecular features of low-grade developmental brain tumours

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post-transcriptional regulators of gene expression, involved in the pathogenesis of different neurological diseases and oncogenesis 25-28. In particular we investigated the expression and function of miR-519d and miR-4758, two miRNAs involved in the regulation of the PI3K/AKT3/ P21 pathway, that could be used to distinguish GGs from DNTs and other low and high grade gliomas.
Materials & Methods
Subjects
The majority of the cases included in this study were obtained from the archives of: the Academic Medical Center of Amsterdam and the University Medical Center Utrecht. A further DNT case was included from the University College London Institute of Neurology in London. SEGA tissue was obtained from the following institutes: University Hospital Erlangen, Children’s Memorial Health Institute in Warsaw, Hacettepe University in Ankara, and the University Hospital de Santa Maria (CHLN) in Lisbon. A total of 99 surgical tumour specimens were examined (Table 1; n=35, GNT (WHO grade I); n= 64 astrocytomas; SEGA (TSC1/TSC2 mutated) n=10; PA (WHO I) n=15; AII (IDH-1 mutant) n=10; AIII (IDH-1 mutant) n=14 and GB (IDH-1 WT) n=15). Eight surgical specimens contained sufficient amount of peritumoural tissue (normal-appearing cortex/white matter adjacent to the tumour; Table 1). All tumour cases were reviewed independently by eight neuropathologists, and the diagnosis was confirmed according to the revised WHO classification of tumours of the central nervous system 1,7; only entities with consensus agreement between the neuropathologists were included (CD34 positive GGs and CD34 negative DNTs, all IDH1 negative). Additional molecular diagnosis was performed on GNT samples with sufficient amounts of DNA to identify mutations in BRAF and FGFR1 genes. Control cortical specimens were obtained at autopsy from 17 patients. For the cortical specimens attention was taken to provide equal grey/white matter tissue components from the tissue. For control tissue, all autopsies were performed within 24 hours after death. Tissue was obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee and approved by the science committee of the UMC Utrecht Biobank.
Mutation analysis
GG samples were screened using sanger sequencing as described previously 29. Next generation sequencing (NGS) was performed on 10 GGs and 8 DNTs using a customised Ion AmpliSeqTM Neurology Panel (ThermoFisher Scientific, Waltham, Massachusetts, USA) for targeted multi-gene amplification, as previously reported 18. This panel consists of the following genes; AKT1, AKT3, ATRX, BRAF, CDK6, CIC, CTNNB1, DDX3X, DEPDC5, EZH2, FGFR1 (exon 12 and exon 14), FUBP1, H3F3A, HIST1H3b, HIST1H3c, IDH1, IDH2, KDM6A, mTOR, MYB, MYBL1, NPRL2, NPRL3, PIK3CA, PIK3R1, PIK3R2, PTCH1, PTEN, SMARCA4, SMARCB1, SMO, SUFU, TP53. FGFR1 mutations below 5%, duplications and fusion genes cannot be detected with this panel.