Experimentally Deduced Criteria for Detection of Clinically Relevant Fusion 3′ Oncogenes from FFPE Bulk RNA Sequencing Data

Abstract

Drugs targeting receptor tyrosine kinase (RTK) oncogenic fusion proteins demonstrate impressive anti-cancer activities. The fusion presence in the cancer is the respective drug prescription biomarker, but their identification is challenging as both the breakpoint and the exact fusion partners are unknown. RNAseq offers the advantage of finding both fusion parts by screening sequencing reads. Paraffin (FFPE) tissue blocks are the most common way of storing cancer biomaterials in biobanks. However, finding RTK fusions in FFPE samples is challenging as RNA fragments are short and their artifact ligation may appear in sequencing libraries. Here, we annotated RNAseq reads of 764 experimental FFPE solid cancer samples, 96 leukemia samples, and 2 cell lines, and identified 36 putative clinically relevant RTK fusions with junctions corresponding to exon borders of the fusion partners. Where possible, putative fusions were validated by RT-PCR (confirmed for 10/25 fusions tested). For the confirmed 3′RTK fusions, we observed the following distinguishing features. Both moieties were in-frame, and the tyrosine kinase domain was preserved. RTK exon coverage by RNAseq reads upstream of the junction site were lower than downstream. Finally, most of the true fusions were present by more than one RNAseq read. This provides the basis for automatic annotation of 3′RTK fusions using FFPE RNAseq profiles.

Clinically relevant fusion oncogenes: detection and practical implications

“…Furthermore, specific features such as conservation of open reading frame in both fusion partners, presence of RTK domain, and finding of several non-duplicate transcript reads for a fusion were shown as the efficient criteria for discriminating true versus artifact fusion reads in FFPEderived RNA sequencing data. 235 Indeed, despite the absolute gene expression levels being not necessarily the same, very similar pathways were overrepresented within dysregulated genes obtained from the FFPE and freshly extracted RNA, 232,233 thus demonstrating consistency with IHC studies. 236,237 Fusion transcript detection by RNA-seq from FFPE samples was recently reviewed, focusing on the experimental variables; however, the difference in bioinformatics approaches of fusion transcript detention was not discussed.…”

“…Furthermore, specific features such as conservation of open reading frame in both fusion partners, presence of RTK domain, and finding of several non-duplicate transcript reads for a fusion were shown as the efficient criteria for discriminating true versus artifact fusion reads in FFPE-derived RNA sequencing data. 235 …”

Cancer fusion transcripts with human non-coding RNAs

“…As a result, CML outcomes have improved dramatically when imatinib and other tyrosine kinase-specific targeted drugs were clinically introduced ( 18 , 30 ). With the advancement of sequencing technologies, the detection of fusion genes and transcripts was greatly facilitated, and numerous fusions have been identified in various tumor types ( 31 – 33 ). Moreover, some fusion types appeared to be strongly linked with specific cancer types and are considered responsible for roughly 20% of global human cancer morbidity ( 5 , 34 ).…”