To BRAF or not to BRAF: is that even a question anymore?

Abstract

Alterations in BRAF have been discovered in most pediatric low-grade gliomas. Because the field has moved quickly during the past few years, there is not yet widespread awareness about what B-Raf normally does, how the BRAF gene is modified in gliomas, why mutant proteins promote gliomagenesis, and what an abnormal BRAF result means for diagnosis, prognosis, and treatment. Depending on the data from ongoing clinical trials, however, BRAF mutation screening could quickly become mandatory for all pediatric gliomas and perhaps even a subset of adult gliomas. Herein, these topics and different methods of testing for BRAF fusions and V600E point mutations are reviewed.

Figure 1

MAPK signaling pathway and B-Raf derangements in gliomas. (A) Under normal circumstances Raf proteins require activation by Ras before activating MEK, which ultimately leads to promotion of cell division, survival, and in certain circumstances, differentiation. B-Raf is a more potent activator of MEK than C-Raf, which in turn is more potent than A-Raf. GF = growth factor; RTK = receptor tyrosine kinase. (B) When the N-terminal Ras-binding portion of B-Raf is lost in a BRAF:KIAA1549 (B–K) fusion, the mutant protein can activate MEK without first being activated by Ras, leading to tumor growth. A similar situation exists with SRGAP3:RAF1 fusion, wherein the kinase portion of C-Raf is joined to SRGAP3 (C–S). A V600E point mutation on B-Raf also has constitutive activity towards MEK. When the p16 checkpoint protein is intact, persistent over-activation of the MAPK pathway can lead to tumor senescence.

Figure 2

BRAF:KIAA1549 fusions and detection by fluorescence in situ hybridization (FISH). (A) The most common BRAF:KIAA1549 fusion in pediatric low-grade gliomas is between exons 1–16 of KIAA1549 and exons 9–18 of BRAF (16-9), seen in over 60% of tumors. 15-9 and 16-11 fusions are seen in approximately 25% and 15% of tumors, respectively. The common result in all of them is retention of the kinase portion of B-Raf. (B) Using a 3-probe FISH cocktail that encompasses the entire BRAF gene on 7q34, the classic BRAF fusion pattern is 2 large red signals plus a third smaller red signal near one of the larger signals, equating to 2 full copies of BRAF plus the kinase portion of a fusion gene. Green signals = centromeric enumeration probe for chromosome 7. (C) In tumors with high polysomy (frequently seen in pleomorphic xanthoastrocytomas and highly senescent pilocytic astrocytomas), reliable detection of a true BRAF fusion pattern is often difficult. (Probe signal size differences in panels B and C are due to post-image processing variables.)

Figure 3

Suggested method of incorporating BRAF testing in pediatric low-grade gliomas. When light microscopy suggests the possibility of a low-grade glioma, the presence of a BRAF fusion suggests either a pilocytic astrocytoma (PA) or its related tumor, pilomyxoid astrocytoma (PMA). Detection of B-Raf V600E, on the other hand, most represents a pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG), or a diffusely infiltrative astrocytoma (DA). Even a PA might still be in the differential depending on its histologic appearance.