Abstract 465: Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples
Authors: Zhao Jingbo , Yang Shuang , Dong Hua 
Published: July 2021

Abstract
Background RNA sequencing (RNA-Seq) has been widely used for gene expression and fusion detection in fresh frozen samples for research use. In clinical studies, RNA was more commonly derived from FFPE samples, which may degrade or yield low due to preparation methods, storage conditions and time. We evaluate the feasibility, quality, and analytical performance of a targeted region RNA sequencing assay (we called as Master RNA Panel) on clinical FFPE tumor samples for gene fusion detection and expression quantification.
Methods Total RNA was extracted from FFPE tumor samples. cDNA synthesis and library preparation were used to prepare next-generation sequencing (NGS) libraries that were captured by a customized panel contains 31 gene fusion/splicing regions and 1813 gene expression regions. NGS libraries were then sequenced on Illumina NexSeq CN 500 or NovaSeq 6000 instrument. Sequencing data were analyzed and annotated with an in-house developed pipeline. A set of experimental and data quality control parameters were set up.
Results In both fresh tissue and FFPE samples, all known fusion sites (100%, 7/7) can be detected stably with the RNA input volume as low as 5 ng and 1 Gbp data, the gene expression of 5 ng RNA input volume shows a very high correlation with 100ng even under 1Gb data (R2≥ 0.95). Master RNA Panel can detect as low as 100 copies of the fusion gene with 50 ng RNA and the specificity was 100% based on a test of 10 fusion negative samples. The consistency of fusion gene detection by Master RNA Panel was 96.2% (51/53) compared with RNA-Seq in 44 clinical samples. To validate the accuracy of gene expression, we used both qPCR and RNA-Seq as comparison methods. Gene expression of 12 tumor-related genes (BAG1, BIRC5, CD68, ERBB2, ESR1, GRB7, GSTM1, MKI67, MMP11, MYBL2, PGR, SCUBE2) in 5 samples were profiled with qPCR assay and the correlation for each gene is greater than 0.8. 27 clinical samples were profiled with both Master RNA panel and RNA-Seq, the total correlation of gene expression between the two assay was also greater than 0.8. The precision verification results show that under the inspection of different operation dates, reagent batches and operators, the gene expression correlation between 5 replicates of 2 FFPE samples is greater than 0.99. The consistency of the fusion test for 5 replicates of 2 FFPE samples and 3 standards was 95.3% (81/85, &gt;=100 copies), indicating that the operation date, reagent batch and operator have no significant influence on the test results, and the test method has good precision.
Conclusions Master RNA Panel can help to identify both gene fusions and gene quantification in cancer patients with extremely low RNA input and high limit of detection (LoD), which is a good supplement for DNA based NGS assay, especially for gene expression quantification and fusion detection.
Citation Format: Jingbo Zhao, Shuang Yang, Hua Dong. Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples [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 465.