BRAF V600E Mutation As Initiating Event In The Causation Of Rat Gliomas Following Postnatal Exposure Of N-Ethyl-N-Nitrosourea (ENU)

Kaishi SATOMI, International Agency for Research on Cancer, Germany
SATOMI K. 2 , OH J. 2 , HUTTER B. 1 , BRORS B. 1 , DIESSL N. 3 , LIU H. 4 , WOLF S. 5 , WIESTLER O. 6 , KLEIHUES P. 7 , KOELSCH B. 8 , KINDLER-RÖHRBORN A. 8 , OHGAKI H. 2

1 Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany
2 Section of Molecular Pathology, International Agency for Research on Cancer, Lyon, France
3 Genomics and Proteomics Core Facility, High Throughput Sequencing, DKFZ, Heidelberg, Germany
4 Division of Molecular Neurogenetics, DKFZ, Heidelberg, Germany
5 Next Generation Sequencing Unit, DKFZ, Heidelberg, Germany
6 Helmholtz-Gemeinschaft Deutscher Forschungszentren, Berlin, Germany
7 Faculty of Medicine, University of Zurich, Zurich, Switzerland
8 Institute of Pathology, University Hospital Essen, Essen, Germany

Purpose
A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. While T:A -> A:T mutations in the transmembrane domain of the neu (c-erbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas was unknown. The objectives of this study were to identify driver mutations in ENU-induced rat gliomas.
Methods
We performed whole genome sequencing of gliomas that developed in 3 BDIV and 2 BDIX rats exposed to a single dose of ENU (80 mg / kg body weight) on postnatal day one. Somatic mutations in gliomas were obtained by direct comparison with normal DNA from the same strain of rats. Bioinformatics were carried out to identify common somatic alterations. Candidate alterations were validated by Sanger sequencing and further screened for in ENU-induced gliomas in 33 BDIV and 12 BDIX rats. Immunohistochemistry was carried out in 10 rat gliomas with Braf V600E mutation.
Results
T:A -> A:T and  T:A -> C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41-55% of all somatic single nucleotide mutations). Braf V600E (A -> T) mutations were detected in all 5 rat gliomas in bioinformatic analysis, and  were validated by Sanger sequencing. Further screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V600E mutation, while peritumoral brain tissue of either strain (n=16) had the wild-type sequence. Immunohistochemistry showed cytoplasmic expression of BRAF V600E in gliomas.
Conclusions
Braf V600E mutations, which are present in several human neoplasms, are the initiating event in the development of rat gliomas caused by a single dose of ENU.
Funding sources
This project was funded by the German Cancer Research Center (DKFZ) and the International Agency for Research on Cancer.