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.

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.

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.