ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue

• Published in: Frontiers in oncology Vol. 14; p. 1420162
• Main Authors: Evans, Ryan T., Gillon-Zhang, Elizabeth, Brown, Julia N., Knudsen, Katherine E., King, Candace, Green, Amanda S., Silva, Ana-Luisa, Mordaka, Justyna M., Palmer, Rebecca N., Tomassini, Alessandro, Collazos, Alejandra, Xyrafaki, Christina, Turner, Iyelola, Ho, Chau Ha, Nugent, Dilyara, Jose, Jinsy, Andreazza, Simonetta, Potts, Nicola D., von Bargen, Kristine, Gray, Eleanor R., Stolarek-Januszkiewicz, Magdalena, Cooke, Aishling, Reddi, Honey V., Balmforth, Barnaby W., Osborne, Robert J.
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 25.09.2024
• Subjects: assay validation; molecular diagnosis; NSCLC; Oncology; precision oncology; pyrophosphorolysis; targeted panel; pyrophosphorolysis; assay validation; precision oncology; NSCLC; targeted panel; molecular diagnosis
• ISSN: 2234-943X; 2234-943X
• DOI: 10.3389/fonc.2024.1420162

Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes ( ) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.