Introduction: Gene rearrangement/fusion play an important role in cancer diagnosis and treatment. Comparing with routine diagnostic methods, Sanger sequencing, fluorescence in situ hybridization (FISH) & immunohistochemistry (IHC), next-generation sequencing (NGS)-based panel can provide genetic information with robust sensitivity and precision. Moreover, panel-based RNAseq can detect novel fusions and only requires a small amount of RNA. We have developed a 450-gene DNA & RNA NGS panel, YuanSu S, for clinical sample detection. In this study, YuanSu S was applied to detect gene rearrangements/fusions and determine its limit of detection (LOD) and concordance with FISH/IHC, and difference between DNAseq & RNAseq.

Methods: A total of 184 formalin fixed and paraffin embedded (FFPE) sarcoma samples were detected by YuanSu S at OrigiMed, a laboratory certified by College of American Pathologists (CAP) & Clinical Laboratory Improvement Amendments (CLIA). Gene fusions were analyzed by OriFusion, an OrigiMed's proprietary algorithm. For LOD determination, gradient amounts of RNA or mixed cell line samples were processed for sequencing. To confirm its concordance with FISH/IHC, 36 sarcomas FFPE samples were examined by FISH (33/36) or IHC (3/36). A cohort of 148 tumor samples was used to analyze rearrangement/fusion detection capability.

Results: For RNAseq in YuanSu S, LOD was determined as 5 copies/1ng RNA input and was verified by ddPCR. Samples with rearrangement/fusion ≥ 5% detected by DNAseq were confirmed by ddPCR. For the interfering substance validation, results of samples treated with ethanol or necrotic tissue were consistent with untreated samples. Furthermore, two different Illumina platforms, Nextseq 500 & Novaseq 6000, showed no difference in sequencing abilities. 36 sarcoma FFPE samples were assessed by NGS and verified by FISH or IHC. All the 19 samples with FISH or IHC positive were detected the same rearrangements/fusions by YuanSu S, whereas the negative samples had no fusion detected. In the cohort of 148 samples, 426 rearrangements/fusions were detected by YuanSu S. 101 (23.7%) were detected both by DNAseq & RNAseq; 204 (47.9%) were determined by DNAseq only; while 121 (28.4%) were determined by RNAseq only. Rearrangements were detected by DNAseq in 97 (65.5%) samples, and 40 (41.2%) may benefit from FDA-approved drugs. Importantly, 51 (34.5%) samples had fusions detected only by RNAseq, and 33 (64.7%) may benefit from targeted therapies.

Conclusion: Our results confirmed the reliability and advantages of YuanSu S, a DNA plus RNA NGS panel. Experience from YuanSu S indicated that integrative DNA & RNA sequencing could increase sensitivity of fusion detection. Therefore, the combination of DNAseq & RNAseq is able to screen more patients carrying fusion targets who could benefit from corresponding inhibitors and play an important role in guiding treatment decisions.

Citation Format: Jiacheng Cai, Feng Li, Haiyan Wu, Weifeng Wang, Qiuyue Pan, Lin Zhang, Kai Wang. Validation & application of next-generation sequencing panel YuanSu S for gene rearrangements/fusions detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2283.