BRAF alterations in primary glial and glioneuronal neoplasms of the central nervous system with identification of 2 novel KIAA1549:BRAF fusion variants

Abstract

Recent studies highlight the importance of BRAF alterations resulting in mitogen activated protein kinase (MAK/ERK) pathway activation in low-grade CNS tumors. We studied 106 low-grade CNS neoplasms in a cohort of primarily pediatric patients to identify the prevalence and clinicopathologic significance of these alterations. Polymerase chain reaction testing identified KIAA1549:BRAF fusions in 51 (48%) tumors overall, including 42 (60%) pilocytic astrocytomas, 4 (17%) unclassifiable low-grade gliomas, 4 (36%) low-grade glioneuronal/neuroepithelial tumors, 0 (of 5) pleomorphic xanthoastrocytomas, 0 (of 4) diffuse astrocytomas (World Health Organization grade II), and 1 (of 3, 33%) pilomyxoid astrocytoma. KIAA1549:BRAF gene fusions confirmed by sequencing included the previously reported ones involving exons 1-16/9-18 (49%), 1-15/9-18 (35%), and 1-16/11-18 (8%) and 2 fusions with novel breakpoints: 1-15/11-18 (6%) and 1-17/10-18 (1%). DNA sequencing identified BRAF mutations in 8% of tumors. BRAF mutations were absent. KIAA1549:BRAF fusions were significantly more frequent in infratentorial (57%) and optic pathway (59%) tumors versus supratentorial (19%) tumors (p = 0.001). We did not identify significantly improved progression-free survival in tumors with fusions. In summary, KIAA1549:BRAF fusions predominate in pilocytic astrocytomas but are also present in some low-grade unclassifiable gliomas and glioneuronal tumors. The prognostic and therapeutic significance of this alteration is unclear and merits further study.

FIGURE 1

KIAA1549:BRAF is the most frequent genetic alteration in low-grade gliomas/glioneuronal tumors. KIAA1549:BRAF fusions were the most common genetic alteration in the 106 tumors examined. Pie chart represents the numbers of cases with the indicated specific alteration.

FIGURE 2

KIAA1549:BRAF distribution varies by location and histologic subtype. (A) KIAA1549:BRAF fusions were relatively more frequent in tumors located in the optic pathways (path) and infratentorial locations compared with the supratentorial compartment. (B) KIAA1549:BRAF fusions were most frequent in PA versus tumors with other histologic features. DA, diffuse astrocytoma, WHO grade II; LGG, low-grade glioma, indeterminate type; LGGN/NE, low-grade glioneuronal or neuroepithelial tumor; PA, pilocytic astrocytoma; PMA, pilomyxoid astrocytoma; PXA, pleomorphic xanthoastrocytoma.

FIGURE 3

Novel KIAA1549:BRAF fusion breakpoints. Polymerase chain reaction (PCR) identified 2 novel KIAA1549:BRAF fusion products with sizes corresponding to 210 nucleotide base pairs (bp) (A) and 800 nucleotide base pairs (bp) (B). Sanger sequencing confirmed novel breakpoints involving KIAA1549 exons 1–15/BRAF exons 11–18 and KIAA1549 exons 1–17/BRAF exons 10–18, respectively (A, B, bottom panel).

FIGURE 4

Relative distribution of KIAA1549:BRAF fusion subtypes. The distribution of all KIAA1549:BRAF fusions types identified by percentage (left). The specific KIAA1549 and BRAF exons involved in the different fusion types are labeled within the schematic boxes. Actual tumor numbers with each specific fusion type are indicated in the pie chart (right).

FIGURE 5

Expression levels of KIAA1549:BRAF fusion are similar or slightly lower than endogenous KIAA1549. Quantitative RT-PCR analysis demonstrated KIAA1549:BRAF fusion and KIAA1549 levels to be similar, but lower than endogenous BRAF. mRNA expression levels are expressed as −ΔC_t values (higher levels in the direction of the arrow), with matched Β-actin levels used as internal control. The PCR product sizes (bp) representing the 5 different KIAA1549:BRAF fusion variants are indicated in the x axis.

FIGURE 6

Extent of resection and age are significantly associated with progression-free survival (PFS) in low-grade glioma/glioneuronal tumors. (A–D) Kaplan Meier curves illustrate PFS comparisons by extent of surgical resection (gross total resection [GTR], red; subtotal resection [STR], blue) (A), age at surgical resection (>5 years, blue; ≤5 years, red) (B), KIAA1549:BRAF fused (blue) versus nonfused tumors in the whole group (C), and the clinically relevant group described by Hawkins et al (19) (D). The p values were obtained by log-rank analysis.