Abstract 247: Twist pan-cancer synthetic RNA fusion control for assay development

Abstract

Abstract Fusion genes are structural variants that arise when the coding region of two previously independent genes become joined together; fusion genes can be a major driving cause of cancer development. Many gene fusion events have been classified as clinically-relevant and are targets for both diagnostic applications, such as TMPRSS2-ERG in prostate cancer, and therapeutic applications, such as CD74-ROS1 and EML4-ALK in non-small cell lung cancer. Given the potential clinical benefits of early detection, there is significant interest in developing highly sensitive and accurate diagnostic assays to detect cancer using these gene fusions as biomarkers. A significant barrier to the development of these diagnostic assays is the lack of a reliable and renewable sample source harboring gene fusions of interest for use in assay development and analytical validation. Here, we describe the development and performance of a new fusion reference material: the Twist Pan-cancer RNA Fusion Control, a highly-multiplexed positive control designed to be spiked into a selected RNA background or as a stand-alone positive control. The Twist Pan-cancer RNA Fusion Control provides a wide selection of 80+ common and curated cancer targets for analytical validation and assay development. The synthetic RNAs are designed centered around the known and documented fusion sites with 750 nt of RNA 5′- of and 3′- of the fusion junction, suitable for short read sequencing and qPCR experiments. Pooled synthetic fusion RNAs are quantified at known molar ratios, NGS QC’d for uniformity, and ddPCR’d to establish a highly precise pool concentration. Finally, in fusion detection NGS experiments, we observe over a 95% recall rate of fusion events. The Pan-cancer RNA Fusion Control is designed to serve as a spike-in or stand-alone positive control and specifically pairs well with a clinically-relevant target enrichment (TE) panel within the Twist Targeted Enrichment for Gene Expression solution. This control sample positive for RNA fusions can be applied in both qPCR and NGS workflows to validate panel/probe sets, establish limits of detection, and monitor ongoing assay performance. The Twist Pan-cancer RNA Fusion Control provides a valuable one-stop solution to assay developers seeking reference materials for a wide array of cancer-associated fusion transcripts. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Patrick Daniel Cherry, Jason Corwin, Kit Fuhrman, Jean Challacombe, Derek Murphy, Esteban Toro. Twist pan-cancer synthetic RNA fusion control for assay development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 247.