Comparative evaluation of targeted RNA sequencing and RNA-exome sequencing for fusion detection in soft tissue sarcomas.

Abstract

e23524 Background: Soft tissue sarcomas are heterogeneous mesenchymal malignancies with overlapping histology, making diagnosis challenging. According to World Health Organization (WHO) classification of STS and bone sarcomas, >150 recurrent fusions have emerged as central diagnostic hallmarks for many sarcoma entities. RNA-based next-generation sequencing, particularly RNA-exome sequencing, enables sensitive detection of known and novel fusion transcripts with precise breakpoint characterization, and comprehensive molecular profiling even from limited tissue samples. The aim of this study was to assess the diagnostic utility of exome-capture RNA sequencing compared to targeted RNA next-generation sequencing for the detection of sarcoma-associated gene fusions, particularly in cases with challenging histology or negative results on targeted fusion panels. Methods: Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue using the MagMAX FFPE DNA/RNA Ultra Kit (Thermo Fisher Scientific). RNA-exome libraries were prepared with the NadPrep Total RNA to DNA – EZ DNA Library Preparation Kit (Nanodigmbio) and sequenced on the DNBSEQ-T7 platform (MGI Tech). Fusion transcript detection was performed using SeqPilot, complemented by bioinformatic tools STAR-Fusion and FusionInspector. In parallel, targeted RNA NGS libraries were generated using a custom sarcoma fusion panel, sequenced on the Ion GeneStudio S5 Prime System (Thermo Fisher Scientific) and analysed via the Torrent Suite software. Results: A total of 143 samples from patients with various histological subtypes of sarcoma were analyzed using a custom sarcoma panel (Ion AmpliSeq). Sarcoma-associated rearrangements were identified in 34% of cases (49/143). The most frequently detected alteration was the EWSR1::FLI1 fusion, observed in 7.7% of samples, a well-established molecular hallmark of Ewing sarcoma. Subsequently, 24 samples that were negative by the custom sarcoma panel were further analyzed using the RNA exome panel. In addition, in 17% (4/24) of cases, detection of TFE3::ASPSCR1 , HEY1::NCOA2 , FGFR1::WHSC1L1 , and EWSR1-NR4A3 fusions aided histological classification and confirmed the initial sarcoma diagnosis. Conclusions: RNA-exome sequencing provided additional diagnostic yield in panel-negative cases, enabling the detection of clinically informative fusions that supported or refined histological classification. These findings highlight the significant role of RNA-exome sequencing in the molecular diagnosis of STSs, particularly in diagnostically challenging cases, and support its integration into advanced diagnostic workflows. Nevertheless, prior review by sarcoma-expert pathologists remains essential to determine the appropriate indication for NGS testing.