Detection and genomic analysis of BRAF fusions in Juvenile Pilocytic Astrocytoma through the combination and integration of multi-omic data

Juvenile Pilocytic Astrocytomas (JPAs) are one of the most common pediatric brain tumors, and they are driven by aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway. RAF-fusions are the most common genetic alterations identified in JPAs, with the prototypical KIAA1549-BRAF fusion leading to loss of BRAF's auto-inhibitory domain and subsequent constitutive kinase activation. JPAs are highly vascular and show pervasive immune infiltration, which can lead to low tumor cell purity in clinical samples. This can result in gene fusions that are difficult to detect with conventional omics approaches including RNA-Seq. To this effect, we applied RNA-Seq as well as linked-read whole-genome sequencing and in situ Hi-C as new approaches to detect and characterize low-frequency gene fusions at the genomic, transcriptomic and spatial level. Integration of these datasets allowed the identification and detailed characterization of two novel BRAF fusion partners, PTPRZ1 and TOP2B, in addition to the canonical fusion with partner KIAA1549. Additionally, our Hi-C datasets enabled investigations of 3D genome architecture in JPAs which showed a high level of correlation in 3D compartment annotations between JPAs compared to other pediatric tumors, and high similarity to normal adult astrocytes. We detected interactions between BRAF and its fusion partners exclusively in tumor samples containing BRAF fusions. We demonstrate the power of integrating multi-omic datasets to identify low frequency fusions and characterize the JPA genome at high resolution. We suggest that linked-reads and Hi-C could be used in clinic for the detection and characterization of JPAs.