RAF gene fusions are specific to pilocytic astrocytoma in a broad paediatric brain tumour cohort

Abstract

Brain tumours are the most common solid tumour in children and are the primary cause of cancer-related death in children and young adults [4, 6]. The most prevalent childhood brain tumours are low-grade gliomas, specifically pilocytic astrocytomas (PAs, WHO Grade I) [1]. PAs are slow-growing tumours which are often cystic, and may occur sporadically or in association with the genetic disorder Neurofibromatosis type 1. Several recent studies including our own have identified novel KIAA1549–BRAF and SRGAP3–RAF1 gene fusions in the majority of PAs tested [3, 7, 8, 12]. In these fusions, the N-terminal autoinhibitory domains of the RAF proteins are replaced by those of KIAA1549 or SRGAP3, resulting in constitutive activation of the ERK/MAPK pathway. A recent study has suggested that the KIAA1549–BRAF fusion is more common in PAs originating in the cerebellum [5]. In low-grade glioma without RAF gene fusions there is increasing evidence for activation of the ERK/MAPK pathway through alternative mechanisms, such as point mutation of KRAS or BRAF [2, 11, 13]. Despite the high frequency of RAF gene fusions in PAs, they have not been investigated in other types of paediatric brain tumours. In this study, we screened a new cohort of 74 paediatric brain tumours, with a range of different pathologies, for all known KIAA1549–BRAF and SRGAP3–RAF1 fusion variants. Access to tumours and clinical data was in accordance with Local Research Ethics Committee (LREC) regulations: Great Ormond Street Hospital LREC reference number 05/Q0508/153. Tumours were classified by diagnostic criteria defined by the World Health Organization (WHO) [10]. Total RNA was extracted from fresh frozen tissue samples using the miRNeasy mini kit (Qiagen, Crawley, UK) and reverse transcribed using the SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA). KIAA1549–BRAF fusions were detected using previously described primers and techniques [3]. The primers used for detecting SRGAP3–RAF1 fusions were 50-TGG CAGTAACCTCATCACCA-30 (located in SRGAP3 exon 10) and 50-GGTTGGGTCGACAACCTTTA-30 (located in RAF1 exon 11). All fusions identified by PCR were confirmed by direct sequencing on a 3100 Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0693-y) contains supplementary material, which is available to authorized users.