BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas

Abstract

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

“…In this regard, we found this amplification in 24 of 30 (80%) of cerebellar and 10 of 16 (62.5%) of brainstem/ hypothalamic/optic-pathway JPA, whereas only 1 of 7 of hemispheric JPA had this duplication, which did not include BRAF (Tables 1, 2a and b; Figure 2; Po0.001). We also sequenced BRAF in 52 samples and found point mutations affecting the hot spot codon 600 in exon 15 of BRAF, V600E (an activating mutation previously described in melanomas and other cancers (Wan et al, 2004)) only in 1 JPA and 1 grade II ganglioglioma (Tables 1, 2a and b; Supplementary Table 3) in keeping with previous studies (Jeuken et al, 2007;Jones et al, 2008;Pfister et al, 2008).…”

“…We also establish that its prevalence varies with the site of origin within the brain of the JPA, and is more frequent in cerebellar (24 of 30 -80%) followed by brainstem and optic pathway tumours (10 of 16 -62.5%), whereas it is rare in hemispheric JPA (1 of 7 -14%; Tables 1, 2a and b; Figures 1 and 2). Our analysis and the several validation steps we performed characterise this region with increased precision compared to the recently published concurrent studies, which missed either BRAF or HIPK2 (Deshmukh et al, 2008;Jones et al, 2008;Pfister et al, 2008) and confirm that 7q34 amplification includes both BRAF and HIPK2 in as many as 34 of 35 JPA samples. We identify additional genetic regions in JPA without 7q34 duplication ( Table 3) and show that most genes within these regions are modulators of the immune system.…”

“…Their silencing of HIPK2 in U87 (a glioblastoma (GBM) cell line) decreased the cells proliferation rate. Pfister et al (2008) identified duplication of 7q34 within 139186224 -140156951 in 30 of 66 (45.5%) paediatric LGA. This region included BRAF, a gene which was not in the genetic interval described in Deshmukh et al (2008).…”

“…Recently several consecutive papers described duplication of 7q34 in LGA including JPA (Bar et al, 2008;Deshmukh et al, 2008;Jones et al, 2008;Pfister et al, 2008;Sievert et al, 2008). However, the data reported are conflicting regarding the subgroup of LGA affected by this genetic event, the size of the duplication and its anatomical localisation within the brain.…”

Pilocytic astrocytomas of the optic nerve and their relation to pilocytic astrocytomas elsewhere in the central nervous system

“…[8][9][10][11][12][13] The biologic significance of BRAF duplication lies in the activation of the MAPK pathway, which can drive tumor proliferation. 9 In addition to KIAA1549:BRAF fusion product, other molecular alterations have been reported in pilocytic astrocytoma, including other BRAF fusion products, 14,15 rare BRAF V600E mutations, 16 BRAF insertions, 17 and KRAS mutations.…”