Abstract 4699: Oncogenic fusion genes resulting from 7q34 deletions constitute a novel mechanism of MAPK pathway activation in pilocytic astrocytoma
Authors: Cin Huriye , Meyer Claus , Herr Ricarda , Janzarik Wibke G. , Lambert Sally , Jones David TW , Benner Axel , Witt Hendrik , Remke Marc , Bender Sebastian , Falkenstein Fabian , Anh Ton Nu Van , Olbrich Heike , von Deimling Andreas , Kulozik Andreas E. , Gnekow Astrid , Scheurlen Wolfram , Witt Olaf , Omran Heymut , Collins V Peter , Brummer Tilman , Marschalek Rolf , Lichter Peter , Korshunov Andrey , Pfister Stefan M. 
Published: June 2012

Pilocytic astrocytoma (PA) is the most common brain tumor in children. Underlying genetic driver aberrations can currently be determined for 75-80% of cases. In particular, we and others have recently shown that tandem duplication at 7q34, resulting in BRAF fusion genes and constitutive activation of the MAPK signaling pathway, is a hallmark genetic lesion in PA development. Alternative mechanisms of MAPK activation include BRAF and KRAS point mutations, RAF1 fusions, and Neurofibromatosis-associated NF1 mutations.

In order to examine more precisely the spectrum of alterations in PA, we screened 79 tumor samples for RAF fusion genes and mutations in KRAS, NRAS, PTPN11, BRAF and RAF1. We used multiplex and long-distance inverse (LDI) PCR to identify BRAF and RAF1 fusion genes and direct sequencing for detailed breakpoint mapping.

Strikingly, LDI-PCR revealed a novel BRAF fusion gene with an uncharacterized gene, FAM131B, as a partner. Array-based comparative genomic hybridization (aCGH), revealed an interstitial deletion of ∼2.5 Mb as a novel mechanism forming the FAM131B-BRAF fusion. As with the more common duplication, this deletion removes the N-terminal auto-inhibitory domain of BRAF kinase, resulting in constitutive kinase activity. Functional characterization of the novel fusion gene demonstrated constitutive MEK phosphorylation potential and transforming activity in NIH 3T3 cells. The same fusion gene was also identified in one PA in an additional series from Cambridge, UK (n=7, with no previously identified alteration). Furthermore, we have detected a larger deletion at 7q in one additional case from our series, in which the alternative fusion partner is currently being identified.

Overall, gene fusions targeting RAF kinases were identified in 68% (54/79) of PA. Detailed analysis of genomic DNA mapped 96% (52/54) 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 two novel SRGAP3-RAF1 fusion variants. BRAF, KRAS or NF1 mutations were observed as alternative mechanisms of MAPK activation in 9 tumors in which no RAF duplication was detected, as well as in two cases in our series which concomitantly harbored two or even three hits in the MAPK pathway.

In summary, we have identified a novel, recurrent BRAF fusion gene resulting in MAPK pathway activation in PA caused by a genomic deletion rather than amplification at 7q34, suggesting the possibility of further undiscovered fusion variants targeting RAF genes in this and other tumor types. Being a hallmark of PA tumorigenesis, these RAF fusion genes are expected to have clinical utility as both a specific marker for PA and a tumor-specific therapeutic target, which offers promise for applying novel treatment strategies in the near future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2011-4699