Abstract 1178: Identification of a novel BRAF fusion partner in pilocytic astrocytoma
Authors: Cin Huriye , Meyer Claus , Witt Hendrik , Remke Marc , Janzarik Wibke , Van Anh Ton Nu , Olbrich Heike , von Deimling Andreas , Kulozik Andreas E. , Marschalek Rolf , Witt Olaf , Omran Heymut , Lichter Peter , Korshunov Andrey , Pfister Stefan 
Published: February 2011

Pilocytic astrocytoma (PA) constitutes the most common brain tumor in children. Recently, we and others have demonstrated that tandem duplications at chromosome 7q34 targeting the BRAF locus define a hallmark genetic lesion in PA development, resulting in BRAF fusion genes and constitutive activation of the MAPK signaling pathway. NF1, KRAS and activating BRAF mutations as well as RAF1 tandem duplications were identified as alternative mechanisms mostly in tumors without BRAF duplication.

To identify BRAF and RAF1 fusion genes and to discover novel fusion partners, we screened a total of 62 PA by using multiplex and long-distance inverse (LDI) PCR. Direct genomic sequencing was performed for detailed breakpoint mapping and to detect activating mutations. All translocations identified by PCR-based methods were validated by fluorescence in situ hybridization.

Overall, gene fusions targeting RAF kinases occurred in 71% (44/62) of PA. Sequencing of cDNA of the retrieved fusion transcripts confirmed all previously reported variants of the KIAA1549-BRAF fusion gene. Further detailed analysis of genomic DNA mapped 95% (42/44) of the breakpoints to the same breakpoint cluster region in intron 8 of the BRAF gene. Moreover, we identified the first non-intronic breakpoint in exon 8 of BRAF and detected one fusion gene product which additionally displayed an internal rearrangement of the remaining BRAF fragment. Fusion of SRGAP3 to RAF1 in two cases and activating mutations of BRAF, KRAS or NF1 in eleven cases were observed as alternative mechanisms of MAPK activation in tumors in which no BRAF or RAF1 duplication was detected. Interestingly, LDI-PCR analysis revealed fusion of BRAF to the first intron of the yet uncharacterized gene, FAM131B. Notably, all fusion events replaced the N-terminal auto-inhibitory domain of the respective RAF kinase with segments of the complementary fusion partner and retained the complete, in-frame coding sequence for the kinase domain enabling constitutive activation of the RAF kinase protein.

In summary, we identified fusion events targeting RAF kinase genes to be the predominant cause for aberrant MAPK activation in PA, accompanied by activating mutations as a complementary mechanism. This study significantly extends our knowledge of the high frequency and striking similarity of rearrangements resulting in KIAA1549-BRAF or SRGAP3-RAF1 fusion genes by discovering hitherto unreported fusion variants. Most importantly, we identified a novel fusion oncogene between BRAF and the so far uncharacterized gene FAM131B, representing the first report of a BRAF fusion partner in PA other than KIAA1549. Taken together, our results strengthen the role of BRAF fusion genes as a hallmark of PA tumorigenesis and highlight the potential of RAF kinase fusion products as a specific marker for PA and a promising tumor-specific therapeutic target which may open the avenue for developing novel treatment strategies in the future.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1178.