This minisymposium was originally published in 2018. The information provided in this minisymposium was accurate and correct at the time of initial program release. Any changes in terminology since the time of initial publication may not be reflected in this minisymposium.
BRAF (v-raf murine sarcoma viral oncogene homolog B1 or B-Raf) is a RAS-regulated serine/threonine kinase and activator of the ERK/MAPK pathway, which promotes cellular proliferation and survival.
BRAF was first implicated in a broad range of human cancers in 2002 with the identification of a BRAF somatic missense mutation within the kinase domain in exon 15. Originally designated as a V599E mutation, the valine to glutamate substitution was later confirmed to be at codon 600 (V600E) of the full coding sequence. The V600E mutation disrupts the N-terminal auto-inhibitory mechanism and converts BRAF into a gain-of-function active form allowing constitutive activation of the MAPK pathway.
BRAF mutations tend to occur with high frequency in cancers known to harbor RAS mutations, indicating that ERK/MAPK pathway activation is common in these cancers and that mutations at various levels of the pathway may cause similar pathway activation. RAS and BRAF V600E mutations are mutually exclusive in most of these cancers. The V600E mutation is the most common BRAF alteration in the CNS and is most frequent in pleomorphic xanthoastrocytoma (PXA), ganglioglioma, and papillary craniopharyngioma; although it has been implicated in a large variety of primary CNS neoplasms including other localized gliomas and diffuse gliomas (see Table). BRAF V600E mutations have also been detected in some CNS histiocytoses. Other much less common BRAF mutations have also been described in CNS neoplasms.
The second most common BRAF alterations in CNS tumors are gene fusions resulting from duplication/rearrangement and manifesting as copy number abnormalities. BRAF duplications predominantly occur in pilocytic astrocytomas of the cerebellum and midline (hypothalamus and optic chiasm) but may occur in pilocytic astrocytomas from other sites and other low-grade pediatric neoplasms as well (see Table). In 2008, a chromosome 7q34 gain was detected in pilocytic astrocytomas and characterized as BRAF duplication with a tandem insertion in the KIAA1549 gene, creating a novel fusion oncogene. The most common fusion is between KIAA1549-exon 16 and BRAF-exon 9, but other less common fusion variants have been also identified. Such fusions result in oncoproteins that activate the MAPK signaling pathway, which appears to be the key signaling pathway in the development of pilocytic astrocytoma.
In general, the BRAF V600E mutation and the KIAA1549-BRAF fusion are mutually exclusive.
Table: BRAF alterations in CNS tumors.
|BRAF V600E Mutation|
|Pleomorphic xanthoastrocytoma (PXA) with and without anaplasia|
|Dysembryoplastic neuroepithelial tumour (DNT)|
|Pilocytic astrocytoma (PA)|
|Subependymal giant cell astrocytoma (SEGA)|
|Pilocytic astrocytoma (PA)|
|Diffuse leptomeningeal glioneural tumor (DLGNT)|
Initially, direct (Sanger) sequencing was considered the gold standard for BRAF mutation detection. However, other DNA-based assays with higher sensitivity were later developed including pyrosequencing, allele-specific quantitative PCR, high-resolution melting analysis, and next-generation sequencing (NGS). The most significant potential barrier to reliable molecular analysis is the quality of DNA preservation, which can be negatively impacted by formalin fixation and tissue processing. The other common barrier to molecular analysis is DNA quantity, both in absolute terms with regards to tumor sample volume and in relative terms due to contamination with DNA from nonneoplastic cells. Two commercially-available monoclonal antibodies (clone VE1 and clone V600) are suitable to detect BRAF V600E mutant protein in formalin-fixed, paraffin-embedded tissue by IHC. Clone VE1 is most widely utilized and has high sensitivity and specificity, according to studies. Concordance rates with molecular methods are strong but variable. IHC provides a potentially faster, cheaper, and more readily-available alternative to molecular assays in formalin-fixed, paraffin-embedded material. IHC has advantages over molecular studies, particularly as a convenient screening tool and when tissue is not sufficient for molecular analysis. However, IHC is not without limitations as nonspecific staining may pose a potential pitfall, especially with small samples, which could lead to false-positive results, and weak staining can lead to false-negative interpretations.
The preferred method to identify all BRAF fusion variants is by RNA-based sequencing, including by NGS. Fluorescence in situ hybridization (FISH) analysis, which demonstrates tandem duplication at chromosome 7q34, is an indirect, valid way to identify the presence of a KIAA1549-BRAF fusion.
BRAF as a Diagnostic Marker
The detection of a BRAF mutation has diagnostic implications in specific CNS tumors. BRAF V600E mutation distinguishes between papillary craniopharyngioma (96% are mutated) and adamantinomatous craniopharyngioma. The latter lack BRAF V600E mutations and instead harbor CTNNB1 mutations. The presence of the BRAF V600E mutation can be particularly useful as an ancillary diagnostic tool when handling localized pediatric low-grade glial neoplasms such as PXA (65% to 75% frequency), ganglioglioma (25% to 60%), dysembryoplastic neuroepithelial tumor (DNT) (20% to 25%), and pilocytic astrocytoma (7% to 10% of extracerebellar locations). Furthermore, detection of the mutation can aid in distinguishing ganglioglioma from cortical infiltration by a diffuse glioma. While most diffuse gliomas lack BRAF alterations, epithelioid glioblastomas have been shown to harbor BRAF V600E mutations. This and the fact that epithelioid glioblastomas share morphologic similarities to anaplastic PXAs have led to speculation that the two entities may be related. Future studies are likely to resolve this ambiguity, but for the time being, the distinction between the two remains highly subjective. BRAF V600E mutations also rarely occur in diffuse midline gliomas with H3F3A K27M mutations. Although most cutaneous melanomas demonstrate the BRAF V600E mutation, primary melanocytic neoplasms of the CNS characteristically lack BRAF mutations and instead show a high frequency of GNAQ and GNA11 mutations.
A clear majority of posterior fossa and midline pilocytic astrocytomas (>70%) show BRAF fusions making the identification of the KIAA1549-BRAF fusion a useful diagnostic biomarker.
BRAF as a Prognostic Marker
Thus far, the literature shows inconsistent trends as far as the relationship between BRAF status and outcome. Some studies suggest improved outcome in fusion-positive compared to fusion-negative pediatric low-grade gliomas, while others are less conclusive.
In a small cohort of pilocytic astrocytomas, the presence of the BRAF V600E mutation is significantly associated with infiltrative growth pattern and tumor progression.
Overall, BRAF V600E mutations appear to be more common in supratentorial neoplasms, while posterior fossa tumors tend to have higher frequency of BRAF fusions. One study demonstrated a significantly higher percentage of fusions in supratentorial midline tumors compared to more lateral hemispheric lesions.
BRAF as a Potential Therapeutic Target
Several small molecule inhibitors targeting the MAPK pathway, either directly through BRAF inhibition or downstream through inhibition of MEK, are available and include sorafenib, everolimus, and vemurafenib. However, unlike in metastatic melanoma, the benefit of targeting BRAF in pediatric low-grade gliomas is not well-established. As seen in melanomas, targeting one pathway may lead to resistance through upregulation of other pathways. Preclinical studies have suggested that combination therapy may be more successful. A recent study showed dramatic response of BRAF V600E–mutant papillary craniopharyngioma to targeted therapy. Neither of the antibodies used in IHC represent “companion diagnostics.”
Take Home Points
- BRAF alterations, acting through the MAPK pathway, are fairly frequent in CNS neoplasms and tend to occur predominantly in low-grade pediatric gliomas, papillary craniopharyngioma, and occasional diffuse gliomas.
- BRAF V600E mutations tend to predominate in supratentorial lesions (PXA, ganglioglioma, DNT, SEGA, and epithelioid GBM) while BRAF rearrangements/duplications occur with higher frequency in infratentorial tumors (eg, cerebellar pilocytic astrocytoma).
- IHC (particularly via the VE1 clone) offers a convenient method to screen for BRAF V600E mutations with relatively high sensitivity and specificity and is especially useful for samples with limited tissue.
- FISH is commonly used to identify the presence of the KIAA1549-BRAF fusion, although RNA-based assays are preferred.
- BRAF alterations can serve as critical diagnostic, prognostic, and predictive markers and may even be used for targeted chemotherapy.
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- Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949-54.
- Myung JK, Cho H, Park CK, et al. Analysis of the BRAF(V600E) mutation in central nervous system tumors. Transl Oncol. 2012;5(6):430-36.
- Penman CL, Faulkner C, Lowis SP, Kurian KM. Current understanding of BRAF alterations in diagnosis, prognosis, and therapeutic targeting in pediatric low-grade gliomas. Front Oncol. 2015;5:54.
- Ritterhouse LL, Barletta JA. BRAF V600E mutation-specific antibody: A review. Semin Diagn Pathol. 2015;32(5):400-8.
- Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011;121(3):397-405.