Analytical performance assessment of ASPYRE-Lung: A new assay for rapid detection of actionable variants from tissue and plasma in NSCLC patients.

Todd William Kelley Biofidelity Ltd, Cambridge, United Kingdom info_outline Todd William Kelley, Ana-Luisa Silva, Rebecca Palmer, Ryan Evans, Iyelola Turner, Christina Xyrafaki, Justyna Mordaka, Kristine von Bargen, Chau Ha Ho, Nicola Potts, Eleanor Gray, Alejandra Collazos, Elizabeth Gillon-Zhang, Julia Brown, Candace King, Alessandro Tomassini, Aishling Cooke, Magdalena Stolarek-Januszkiewicz, Robert Osborne, Barnaby Balmforth

Authors Todd William Kelley Biofidelity Ltd, Cambridge, United Kingdom info_outline Todd William Kelley, Ana-Luisa Silva, Rebecca Palmer, Ryan Evans, Iyelola Turner, Christina Xyrafaki, Justyna Mordaka, Kristine von Bargen, Chau Ha Ho, Nicola Potts, Eleanor Gray, Alejandra Collazos, Elizabeth Gillon-Zhang, Julia Brown, Candace King, Alessandro Tomassini, Aishling Cooke, Magdalena Stolarek-Januszkiewicz, Robert Osborne, Barnaby Balmforth Organizations Biofidelity Ltd, Cambridge, United Kingdom Abstract Disclosures Research Funding Pharmaceutical/Biotech Company Biofidelity Ltd Background: We developed ASPYRE technology to make detection of genomic variants in oncology patients more accessible. Although ASPYRE is not traditional PCR, it can be performed using standard qPCR instruments on nucleic acid extracted from FFPE tissue or plasma and is therefore easily adoptable by clinical laboratories. Using ASPYRE, we designed a targeted panel called ASPYRE-Lung TM consisting of 114 variants in 11 genes ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3 ) to inform the clinical management of patients with NSCLC. We have previously published general assay descriptions of ASPYRE-Lung (Silva et al ., 2021, Scientific Reports and Gray et al ., 2022, BMC Medical Genomics ). Here we extend our prior studies with a detailed analytic performance assessment across all variants using precisely controlled synthetic samples. We determined assay-wide performance using samples with low variant allele fractions (VAFs) near the limit of detection (LoD) of the assay and compared results across 2 separate laboratory settings. Methods: Nine independent batches of reagents were split between 2 laboratories. Contrived test samples were prepared with known VAFs (DNA variants) or copy numbers (RNA variants). Each sample contained a single variant with VAFs of 0.1-0.5% for DNA variants, and 6-150 copies for RNA variants. Multiple replicates were performed at both testing sites using 20ng DNA/6ng RNA. Data was processed using an internally designed algorithm and analyzed using a machine learning classifier which determined the presence or absence of each individual variant. Results: Study data resulted in a total of 133,083 variant calls from 2,319 samples. For each variant at each VAF or copy number, the detection rate was calculated, and the results for each variant were used to calculate an LoD95 using probit regression. Target assay specifications and the assay-wide analytical performance data are shown in the table below. Conclusions: The data show that ASPYRE-Lung meets or exceeds target specifications for the detection of single nucleotide variants (SNVs), insertions/deletions (indels), and gene fusions. Estimates of LoD for key variants fall within target specifications, and assay-wide performance demonstrates highly sensitive detection of each class of variant. Performance was similar at the two laboratory sites further supporting the clinical potential of the assay. Moreover, the assay only requires 20ng DNA and as little as 6ng RNA, depending on sample type, so it is highly suitable for use with typical NSCLC patient samples such as needle core biopsies or plasma where available nucleic acid may be limited. This data supports further clinical development of ASPYRE-Lung. Class Criteria Target threshold Acceptable Threshold Result SNVs and Indels Median panel wide LoD95 0.45% 0.8% 0.31% Fusions Median panel wide LoD95 18 copies 48 copies 6.1 copies

Pre-clinical