Development and validation of an RNA sequencing panel for gene fusions in soft tissue sarcoma

Abstract

Gene fusions are one of the most common genomic alterations in soft tissue sarcomas (STS), which contain more than 70 subtypes. In this study, a custom‐designed RNA sequencing panel including 67 genes was developed and validated to identify gene fusions in STS. In total, 92 STS samples were analyzed using the RNA panel and 95.7% (88/92) successfully passed all the quality control parameters. Fusion transcripts were detected in 60.2% (53/88) of samples, including three novel fusions (MEG3–PLAG1, SH3BP1–NTRK1, and RPSAP52–HMGA2). The panel demonstrated excellent analytic accuracy, with 93.9% sensitivity and 100% specificity. The intra‐assay, inter‐assay, and personnel consistencies were all 100.0% in four samples and three replicates. In addition, different variants of ESWR1–FLI, COL1A1–PDGFB, NAB2–STAT6, and SS18–SSX were also identified in the corresponding subtypes of STS. In combination with histological and molecular diagnosis, 14.8% (13/88) patients finally changed preliminary histology‐based classification. Collectively, this RNA panel developed in our study shows excellent performance on RNA from formalin‐fixed, paraffin‐embedded samples and can complement DNA‐based assay, thereby facilitating precise diagnosis and novel fusion detection.

Improving fusion call confidence and reliability through an optimized process in low quality RNA from formalin-fixed, paraffin-embedded samples

“…Performance characteristics of V1 and V2 probe through the RNA fusion panel.The RNA fusion panel were evaluated primarily for performance characteristics, including sequencing performance, and fusion calling capacity by detecting gene fusions using 14 FFPE samples from sarcoma patients. All the samples were positive for gene fusion using an additional minor RNA panel with V1 probe, previously being run in our laboratory(Hu et al 2022), which corresponded with the results from the RNA fusion panel. The QC cutoffs of pre-libraries were 250-450 bp for base-pair sizing and 22.7 ng/µL for concentration, while 2 ng/µL was the minimal concentration for nal libraries.…”

“…Detailed sensitivity, speci city, reproducibility, and repeatability were also discussed. Evaluation of the primary performance characteristics of this assay in the rst 14 cases demonstrated that the assay is more inclusive than the minor RNA fusion panel previously run in our laboratory (Hu et al 2022). In summary, this study demonstrates that the RNA fusion panel is an excellent tool for detecting real gene fusion in solid tumors, and using tiling probes covering the fusion transcripts junction can improve the detection rate and reliability of gene fusions in low-quality RNA samples.…”

“…As mentioned in the "Introduction," the exclusive RNA fusion panel assay was developed as an improvement to our then current assay for fusion detection, the minor RNA panel which was the ability to identify novel fusions across 67 genes in sarcoma (Hu et al 2022). It did primarily suffer from the need for high RNA quality (DV 200 ≥ 40%).…”

“…The analysis procedure was similar to that described previously (Hu et al 2022). Some quality control (QC) parameters of the procedure should be noted, including the total number of reads, Q30, mapping ratio, target ratio, insert size, and mRNA − ratio.…”

Recurrent and novel fusions detected by targeted RNA sequencing as part of the diagnostic workflow of soft tissue and bone tumours

“…Similarly, reverse transcriptase PCR (RT‐PCR) requires upfront knowledge of fusion partners for primer design, making it challenging for the discovery of genes with multiple possible gene fusion partners and variable splicing. As opposed to whole transcriptome or whole genome approaches, targeted RNA‐seq currently remains more feasible and cost‐effective in a diagnostic setting [ 10 , 11 , 12 ].…”