Abstract 3825: Digital PCR-characterized, highly multiplexed, oncology RNA fusion reference materials: Performance on multiple NGS platforms
Authors: Huang Catherine , Konigshofer Yves , Nguyen Lequan , Vemula Rajeswari , Kamineni Praveena , Philkana Deepika , Jaiswal Ekta , Anekella Bharathi 
Published: July 2017

Abstract
Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Molecular diagnostics are rapidly being introduced for detection of fusion RNAs by highly multiplexed next-generation sequencing assays. However, reference materials to aid in the development, optimization and validation of these assays are lacking. We developed Seraseq FFPE Fusion RNA Reference Material to fill this unmet need and show that this material is compatible across a wide range of NGS assay platforms.
Methods: Biosynthetics were used for transcription of clinically actionable RNA fusions, including fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. Sixteen (16) in vitro transcribed RNAs were introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). The cells were collected, fixed in formalin, and total RNA was isolated. Digital PCR with TaqMan® chemistry was used to determine the target Fusion RNA copies per microliter. Use of fusion-specific digital PCR provides an orthogonal method of verifying transcript levels and serves as the “ground truth” for the abundance of each RNA. NGS testing of the purified RNA used the ArcherDx FusionPlex™ CTL Panel, QIAGEN QIAseq Targeted RNAscan Panel, and the Thermo Fisher Oncomine Focus Assay.
Results: All sixteen (16) fusions present in the prototype were detected as expected on each NGS platform. Results among the three different NGS platforms were generally concordant, although the reads across the fusion junctions did vary slightly among NGS assays and with digital PCR results. However, all methods indicated that the reference material gave low positive results, similar to a patient sample, and that the single reference material could serve as a positive control for detection of sixteen different fusions, which represent a variety of different solid tumor types.
Conclusions: Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for sixteen fusions observed in a variety of solid tumors, both common and rare. It provides a consistent, unlimited supply of QC materials particularly valuable for difficult to find rare fusions. The reference material generates low positive results on three leading assays, and this is important to truly challenge the assay system. This material is handled identically to a patient sample from extraction through analysis, and verifies performance at levels expected for patient samples.
Citation Format: Catherine Huang, Yves Konigshofer, Lequan Nguyen, Rajeswari Vemula, Praveena Kamineni, Deepika Philkana, Ekta Jaiswal, Bharathi Anekella. Digital PCR-characterized, highly multiplexed, oncology RNA fusion reference materials: Performance on multiple NGS platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2017-3825