Abstract 1392: Development of highly multiplexed, whole process reference materials for monitoring oncology RNA fusions
Authors: Huang Catherine E. , Konigshofer Yves , Nguyen Lequan , Anekella Bharathi 
Published: July 2016

Abstract
Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Next-generation sequencing methods are increasingly used to detect these fusion RNAs in FFPE material. However, quality control materials for these assays are lacking, and validation materials for rare fusions are frequently not available. We developed a novel FFPE Fusion RNA Reference Material to fill this unmet need.
Methods: Highly multiplexed RNAs were designed which contain multiple fusion targets observed predominantly in solid tumors including ALK, RET, ROS1 FGFR3, and NTRK1 fusions as well as PAX-PPARG fusion and ETV6-NTRK3 fusion. These RNAs were transcribed in vitro and contained a 5’ guanosine cap and a poly-adenosine tail to improve intracellular stability. The RNA was introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). After recovery from transfection, the cells were collected and fixed in formalin. Digital PCR with TaqMan chemistry was used to determine the average number of synthetic RNA transcripts per cell. These transfected cells were then mixed with non-transfected GM24385 cells to achieve a consistent “low positive”, formulation. The cell mixture was embedded in a paraffin block and 10 micron sections were produced. Quality control was performed by extracting RNA and testing using the ArcherDx FusionPlex™ Lung Thyroid Panel and the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel.
Results: All twelve (12) fusions present in the prototype were detected as high confidence calls on the ArcherDx Lung Thyroid panel. Whereas embedded cell lines (with genomic mutations) give extremely high positive results (typical results are thousands of reads across the fusion junction), Seraseq™ FFPE Fusion RNA Reference material gave low positive results, similar to patient samples. The reads spanning the fusion junction ranged from 38 to 396. The Ion AmpliSeq™ RNA Fusion Lung Cancer panel assays only 6 of the fusions in the Seraseq reference material due to their assay design; however, all assayed fusions were appropriately detected.
Conclusions: Highly multiplexed reference materials in FFPE format are needed for quality control of NGS-based detection of oncology RNA fusions. Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for twelve fusions observed in a variety of solid tumors, both common and rare. It generates low positive results on two leading assays, and this is important to truly challenge the assay system and verify performance at levels expected for patient samples.
Citation Format: Catherine E. Huang, Yves Konigshofer, Lequan Nguyen, Bharathi Anekella. Development of highly multiplexed, whole process reference materials for monitoring oncology RNA fusions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1392.