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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: BRAF fusions; Hi-C; Juvenile Pilocytic Astrocytoma; Linked-reads; RNA sequencing; Single-cell RNA sequencing.

Fig. 1

Diagrams representing normal BRAF protein and fusions characterized in this study.

Fig. 2

Linked-reads data supporting BRAF fusions created by (a) a tandem duplication resulting in the canonical KIAA1549-BRAF fusion in JPA_2, (b) an interchromosomal translocation creating a TOP2B-BRAF fusion detected in JPA_3, and (c) a structural variant leading to the PTPRZ1-BRAF fusion detected in JPA_1, all visualized in Loupe. d Circos plots for linked-read datasets showing CNV calls made by TitanCNA (outer circle) and manually validated somatic SVs detected by at least two linked-read callers (inner circle). Copy number abnormalities are colour coded; structural variants are indicated by lines. e Circos plot of chromosome 7 in JPA_2 showing the canonical fusion, a catastrophic chromosomal event on chromosome 7 in JPA_1 and circos plot of chromosomes 3 and 7 with an interchromosomal fusion in JPA_3. Circos plots for linked-read datasets showing CNV calls made by TitanCNA (outer circle) and manually validated somatic SVs detected by at least two callers (inner circle). Copy number abnormalities are colour coded; BRAF fusions are shown in yellow and all other SVs in grey

Fig. 3

(a) UMAP clustering of JPAs based on the compartment scores along chromosome 7 (50 kb bins) shows two clusters separating JPAs and normal adult astrocytes from other tumor types and normal controls for other brain regions. b Correlation matrix showing hierarchical clustering by compartment score along chromosome 7 (50 kb bins) shows two clusters separating JPAs and normal astrocytes from other tumor types and normal controls for other brain regions. c Compartment score over KIAA1549 and BRAF showing that both genes are in open chromatin regions in JPAs (regardless of fusion) but not in normal adult astrocytes

Fig. 4

(a) Virtual 4C showing that the peak in the interaction between BRAF and KIAA1549 is exclusive to samples with the canonical fusion (50 kb resolution). Dotted lines indicate the genomic position were a peak would represent interaction of this region with BRAF. b Virtual 4C showing that the peak in the interaction between BRAF and KIAA1549 is exclusive to samples with the canonical fusion and that the samples with a novel fusion have a peak in the interaction between BRAF and the other fusion partner which is exclusive to those samples (500 kb resolution). Dotted lines indicate the genomic position were a peak would represent interaction of this region with BRAF