Page 43 - Molecular features of low-grade developmental brain tumours

SEGA IN TSC HAVE TSC1/TSC2 BIALLELIC INACTIVATION & NO BRAF MUTATIONS
were used for additional immunocytochemical staining, as previously reported 36,37. The following antibodies have been used: glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4000; monoclonal mouse; DAKO; 1:50), microtubule-associated protein (MAP2; mouse clone HM2; Sigma 1:100), anti-human leukocyte antigen (HLA)- DP, DQ, DR (mouse clone CR3/43; DAKO; 1:100), CD3 (mouse monoclonal, clone F7.2.38; DAKO; 1:200; T-lymphocytes), phospho-S6 ribosomal protein (Ser235/236; pS6, rabbit polyclonal, Cell Signaling Technology, Beverly, MA, USA; 1:50) and Ki67 (mouse clone MIB-1, DAKO, Glostrup, Denmark. 1:20) were used in the routine immunocytochemical analysis of tumour specimens to document the presence of a heterogeneous population of cells and the activation of the mTORC1 pathway. After washing in PBS, sections were stained with a polymer based peroxidase immunocytochemistry detection kit (BrightVision Peroxidase system, ImmunoVision, Brisbane, CA, USA). Signal was detected using the chromogen 3-amino-9-ethylcarbazole (AEC, Sigma-Aldrich, St. Louis, MO, USA).
Table 1. Summary of clinicopathological features in TSC patients with subependymal giant cell astrocytoma.
Parameter
Number
%
Age
≤18
>18
37 21
64 36
Sex
Male
Female
36 22
62 37
Tumour location
Lateral ventricle
Foramen of Monro Third ventricle
49 5 4
84 9 7
TSC-lesions SEN/Tubers
56
97
Tuberous Sclerosis Complex Definite
Possible
56 2
97 3
DNA extraction and BRAFV600E Mutation Analysis
DNA was extracted from both FFPE (n=44) and frozen (n=14) SEGA tumour samples. Since SEGA often display intratumoural hemorrhages, areas of representative tumour (identified on hematoxylin & eosin stained sections) were selected for cases in which hemorrhages, were observed within the FFPE SEGA tissue samples (n=44). Tumour DNA was extracted from 10-μm-thick paraffin sections using BiOstic FFPE Tissue DNA Isolation kit (MO BIO) according to the manufacturer’s instructions. From frozen tissue samples (N=14) DNA was recovered from the organic phase following QIAzol (Qiagen) extraction of RNA and was
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