The present study was designed to characterize the genomic abnormalities in a series of pediatric low-grade astrocytomas using the 550K Illumina Bead Chip. The high-density SNP arrays utilized in this study provide a comprehensive genome-wide analysis that includes both CNAs and LOH. A variety of CNAs were detected, the majority of which were shown to be present in the normal population, and are less likely to be disease-causing. Using these arrays, a non-random 7q34 duplication was identified in 20 of the 28 primary astrocytomas, implicating a gene contained within the duplicated region that may play a role in the initiation of pediatric low-grade astrocytomas.
Clinical follow-up was available for one-third of the patients, and there were no confirmed recurrences. There was no correlation between patient age, gender, tumor histology or tumor location with the duplication. The 7q34 duplication was found in tumors that were located in both the cerebellum and the cerebral hemispheres. The frequency of the duplication was higher among the JPAs (77%) than the diffuse fibrillary astrocytomas (50%); however, this may have been biased by the relatively low number of fibrillary tumors in this cohort. The tumor from the one patient with documented NF1 (98–278), did not have the duplication, but had a very large region of CN-LOH starting at region 17q11.2, proximal to the location of the NF1 gene, and extending to the telomere. As NF1 is postulated to function as a tumor suppressor, loss of one copy of the gene would be consistent with an inactivating event that could predispose this patient to the development of multiple different tumor types, including low-grade gliomas. Peripheral blood studies were not available to determine if in fact this region of LOH was also present in the germline. If the remaining allele was mutated, this might be sufficient for tumor development. Interestingly, case 06–247 had a much smaller region of CN-LOH in region 17q11.2 overlapping with the proximal start of the NF1 gene. This case had nine alterations detected by the SNP analysis including deletions and CN-LOH, as well as the 7q34 duplication. As this patient did not have documented NF1 prior to the diagnosis of her right temporal fibrillary astrocytoma, mutation studies of the NF1 gene would provide insight as to whether it may be involved.
Trisomy 7 is a frequent finding in malignant gliomas in both children and adults, and has been reported in a limited number of low-grade astrocytomas, suggesting that it may be a marker of tumor progression. Identification of a candidate gene related to this chromosome copy number increase has been challenging because of the difficulty in narrowing a small region of duplication. Two recently published studies using array-based comparative genomic hybridization (aCGH) have also identified duplications in the 7q34 region, suggesting that this is the most frequent abnormality in (pediatric) low-grade astrocytomas (6
). It is unclear at the present time, however, how this may be related to tumors with trisomy 7. Deshmukh et al
) used a dual-platform aCGH approach (Nimblegen 385K oligonucleotide-array and Affymetrix 550K SNP-array) to characterize 10 JPA specimens. They identified a 1 Mb amplified region of 7q34 proximal to BRAF
, and suggested that HIPK2
was the critical target of the duplication. We found no evidence to support HIPK2
as the critical target gene with the 7q34 duplication, although increased expression because of the increased copy number may contribute to the biology of this disease. In contrast, Pfister et al
evaluated 66 pediatric low-grade astrocytomas and identified a 0.97 Mb (represented by two adjacent BAC clones) 7q34 duplication in 45% of tumors that spanned the BRAF
). Silencing BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2 resulted in decreased proliferation of cultured tumor cells, providing functional evidence for BRAF-induced activation of the MAPK pathway. Although both of these studies validate our finding of a novel duplication in the 7q34 region in pediatric low-grade astrocytomas, utilization of the Illumina 550K SNP-array allowed us to more precisely define the 7q34 breakpoints in our tumor samples. The array results, in combination with the patterns of copy number gain demonstrated by FISH, suggested that the mechanism of the 7q34 duplication may be variable among tumors, and combined with the mutation analyses of BRAF
, indicate that several types of genomic alterations can result in activation of BRAF
A number of solid tumors, including adult malignant gliomas, have demonstrated mutations in the BRAF
coding sequence, specifically in exons 11 and 15 (2
). Direct sequence analysis of exons 11 and 15 in genomic DNA from all 28 tumors and Western blot analysis of a subset of 10 tumors demonstrated a V600E mutation and increased protein expression in only one fibrillary tumor. As the one tumor with a mutation did not have the CNA involving 7q34, a limited number of pediatric gangliogliomas and high-grade pediatric gliomas with or without trisomy 7 were also analyzed by direct sequencing of exons 11 and 15. The same V600E mutation was found in 3 of 11 gangliogliomas, and 0 of 3 anaplastic astrocytomas. These results, although limited in scope, suggest that BRAF
mutations may be present in a spectrum of gliomas in both children and adults. Spittle et al
) have recently shown that CNAs of BRAF
are present in melanoma, however, in their series, the tumors with gains of 7 also had BRAF
mutations. In contrast, Jeuken et al
) reported few BRAF
mutations in adult gliomas, whereas copy number gains (as determined by CGH) of chromosome 7 were present in up to one-third of tumors. Again, the specificity of this CNA as it relates to BRAF activation is not known.
Our identification of a novel KIAA1549-BRAF
fusion gene may help to explain one of the mechanisms by which BRAF activation leads to development of pediatric astrocytomas. Ciampi et al
) demonstrated a fusion between AKAP9
, transcribed in opposite orientations, as a result of a paracentric inversion of chromosome 7 in a thyroid tumor. The AKAP9-BRAF fusion product resulted in elevated kinase activity and transformation of NIH3T3 cells, providing evidence for in vivo
activation of an intracellular effector along the MAPK pathway. Such a fusion product would be consistent with the results from the present series of astrocytomas. The predicted KIAA1549-BRAF
fusion gene would retain the highly conserved C-terminal kinase domain but lack the N-terminal regulatory domains, and could result in constitutive BRAF kinase domain activity and aberrant activation of the MAPK pathway.
Further studies are required to characterize the genomic structure, expression and function of the KIAA1549-BRAF fusion protein, and determine the specificity of BRAF alterations for low-grade pediatric gliomas. Ultimately the identification of a BRAF activating event may serve as a biological marker to differentiate low-grade from high-grade tumors and determine whether this is a prognostic indicator that could be used in a prospective setting. Clinical correlation with the long-term outcomes of patients both with and without the 7q34 duplication, preferably in the context of a clinical trial, may help to elucidate the prognostic significance of this novel duplication, and the potential role of BRAF in tumor initiation or progression. These studies also support BRAF and the MAPK pathway as potential therapeutic targets in pediatric gliomas.