Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas

Abstract

Brain tumors are the most common solid tumors of childhood, and pilocytic astrocytomas (PA) are the most common central nervous system tumor in 5 to 19 year olds. Little is known about the genetic alterations underlying their development. Here, we describe a tandem duplication of approximately 2 Mb at 7q34 occurring in 66% of PAs. This rearrangement, which was not observed in a series of 244 higher-grade astrocytomas, results in an in-frame fusion gene incorporating the kinase domain of the BRAF oncogene. We further show that the resulting fusion protein has constitutive BRAF kinase activity and is able to transform NIH3T3 cells. This is the first report of BRAF activation through rearrangement as a frequent feature in a sporadic tumor. The frequency and specificity of this change underline its potential both as a therapeutic target and as a diagnostic tool.

Figure 1. Identification of copy number gain at 7q34 in pilocytic astrocytomas

A, A representative 1Mb array plot from PA2 showing gain of clone RP5-886O8 at 7q34. B, A chromosome 7 tiling-path array plot from the same tumor, showing gain of approximately 2Mb spanning 22 clones between RP11-355D18 and RP11-543P6. C & D, A custom oligonucleotide array covering the ends of the region of gain, showing a change in copy number within the KIAA1549 and BRAF genes.

Figure 2. Tandem duplication at 7q34 produces a fusion gene between KIAA1549 and BRAF

A, A schematic diagram of the tandem duplication event observed at 7q34. Clones flanking the region of gain and the 1Mb clone from the region are indicated. Fluorescence in-situ hybridization analysis confirmed a tandem duplication (See Supplementary Fig. S1). B, RT-PCR analysis with primers in KIAA1549 exon 15 (PC4645) and BRAF exon 11 (PC4644) showing the three different mRNA fusion junctions observed (top) and a control locus in wild-type BRAF exons (bottom, expected product 214bp). PA25 does not harbour the 7q34 gain. PCR products were electrophoresed on a 1.5% agarose gel and visualised on a UV-transilluminator (UVP Ltd, Cambridge, UK). L; 100bp DNA size ladder (Invitrogen), NB; Normal brain cDNA (Ambion, Austin, TX), −ve: no template control. C, A schematic of the mRNA/proteins formed by the three different fusion products, and their frequency in our series. All three retain intact open reading frames. KD; BRAF kinase domain. D, A sequence trace confirming a fusion between KIAA1549 exon 16 and BRAF exon 9.

Figure 3. The KIAA1549:BRAF fusion gene shows constitutive kinase activity and transforms NIH3T3 cells

A, An in vitro BRAF kinase activity assay showing the fusion proteins to have constitutive kinase activity at a level similar to or higher than that of mutant BRAF. WT; wild-type BRAF, V600E; BRAF^V600E, l-KB: long-form KIAA1549-BRAF (K^Ex16B^Ex9) fusion, s-KB: short-form KIAA1549-BRAF (K^Ex16B^Ex9) fusion. Bars show the fold increase in activity over wild-type, averaged over triplicate assays, with standard deviation indicated by error bars. Two independent transfections gave similar results. B, NIH3T3 cells transduced with pBABE-puro vector alone (EV), HRas^V12 (Ras^V12), wild-type BRAF (WT), BRAF^V600E, or short-form KIAA1549-BRAF (K^Ex16B^Ex9) fusion (s-KB) were grown in soft agarose. Active RAS, mutant BRAF and the fusion protein all display anchorage-independent growth. Photos shown are representative fields taken at 11 days after plating.