FusionPlex Dx (FP Dx) is a 2-part multimodal next-generation sequencing (NGS)-based in vitro diagnostic kitted device that detects structural alterations and oncogenic isoforms in RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples from solid tumors using proprietary Anchored Multiplex PCR (AMP™) chemistry. FP Dx is approved as a companion diagnostic for five indications: METex14 skipping events, ALK, RET, ROS1 gene fusions to guide therapy in non-small cell lung cancer and NTRK1/2/3 gene fusions for all solid tumors. The assay also provides comprehensive genomic profiling for patients with solid tumors. This study summarizes the analytical performance of FP Dx.
FP Dx instructions for use were followed for all studies. Total nucleic acid was extracted from 1132 unique clinical FFPE specimens with positive or negative RNA alterations derived from ≥40 unique tumor types and 4 commercial reference FFPE samples generating a total of 3279 libraries. Libraries were sequenced on Illumina MiSeqDx and NextSeq 550Dx platforms. Pre-defined in-process control metrics were used for monitoring library preparation and sequencing, and only libraries meeting these metrics were included in the analysis (97.65%). Data were processed via Illumina sequencing software, R Analysis Suite Statistical Software, and Invitae Solid Tumor IVD Analysis.
The limit of detection of FP Dx was measured to be 22 reads for structural alteration (ALK, RET, and ROS1 gene fusions), 26 – 35 reads for METex14 skipping events, and 31 reads for NTRK gene fusions. The accuracy of FP Dx was evaluated by comparison to Illumina TST-170 NGS, Oncomine Focus NGS, and Illumina TSO Comp assays. Concordance agreement results were >95%, demonstrating the accuracy of FP Dx. Overall agreement reproducibility results for NTRK, METex14, ALK, RET, and ROS1 alterations were ≥99.15%, and repeatability results were ≥92.50%, demonstrating consistent precision of FP Dx.
Collectively, these data support FP Dx’s ability to detect RNA structural alterations. The unique capabilities of the Assay chemistry and software allow for exhaustive characterization of Assay sensitivity for all targeted alterations, even without prior knowledge of gene fusion partners or breakpoints.
Invitae Corporation.
Invitae Corporation.
V. Haddad, N. Hohos, M. Stueve, A. Timm, A. Rao, H. Slocum, J. Lee: Other, Institutional, Full or part-time Employment: Invitae.