Abstract 4255: Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel

Abstract

Abstract Gene fusions and RNA splicing errors are important events frequently involved in tumorigenesis, and often represent clinically addressable targets for various cancers. The reliable detection of such mutations via NGS requires the development of cutting-edge technologies capable of overcoming many challenges. These include detecting fusion mutations regardless of the complexity of fusion structures, removing the length and quantity constraints of FFPE RNA, reducing ribosomal RNA interference, eliminating false negatives and positives, revealing the existing copies of mutations, and improving the reproducibility and ease of workflow. Here we describe the development and validation of a fast and user-friendly RNA-based fusion detection technology - CleanPlex® Fusion. Adding a universal primer at the 3' end of single-stranded cDNA via template switching allows us to eliminate the constraint of template length, and hence amplify damaged RNA fragments, such as FFPE RNA. The presence of a target-specific primer on one side enables the detection of any mutations on the RNA fragments, including novel ones. With a panel of 61 primers (covering most of fusion mutations of NSCLC) annealing to the 3' end portion of RNA, we demonstrated the high on-target rate, mapping rate, and low rRNA rate and primer-dimer rate. We validated the amplification of each amplicon by using fragmented total RNA. Additionally, we also made a dual-primer multiplex PCR panel covering the same set of fusion mutations as defined by the single-primer panel, confirmed its coverage of all fusion mutations using a synthetic pool of fusion targets, and detected all commercial reference fusion mutations targeted by the panel. We again demonstrated the detection of such reference fusion mutations using the single-primer mPCR panel, and demonstrated that the single-primer method even detected fusion mutations outside the boundary of the dual primers. Moreover, we showed that the detected fusion reads significantly correlate with copy numbers determined by digital PCR. Lastly, we validated detection of fusion mutations in cell lines and FFPE RNA. CleanPlex® Fusion therefore represent a robust and efficient NGS method for detecting fusion mutations. Citation Format: Chenyu LI. Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4255.