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Background: Many patients with non-small cell lung cancer (NSCLC) lack access to highly effective approved targeted therapeutics due to multiple gaps in biomarker testing. Challenges in comprehensive molecular testing include complexities associated with the need to assess the presence of multiple variants, costs of running multiple sequential assays per sample, high assay quality control (QC) failure rates, clinical need for rapid turn-around time (TAT) to initiate therapy, and insufficient tissue samples. The ASPYRE-Lung NSCLC assay addresses gaps in multiplexed testing by simultaneously analyzing DNA and RNA, detecting 114 actionable genomic variants across 11 genes, consistent with current NSCLC treatment guidelines. This study was to assess the ease of adoption and performance of ASPYRE-Lung in third-party laboratories, comparing concordance across sites and with orthogonal methods., Methods: ASPYRE-Lung was established at two academic centers with multiple operators per site. Assay concordance was evaluated across three sites using 77 patient samples [61 derived from formalin-fixed paraffin-embedded (FFPE) tissue and 16 from cytology specimens]., Results: Reproducibility for all 77 samples yielded a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.99%. Concordance with next-generation sequencing (NGS)-based methods across all three sites was high with PPA of 97.2% and NPA of 99.96%., Conclusions: ASPYRE-Lung assay is a cost-effective, easy to adopt testing method requiring no specialized expertise or complicated bioinformatics, with the potential to inform genomic data on small tissue samples, thus enabling all patients with NSCLC to undergo biomarker testing in a timely manner and benefit from appropriate targeted therapies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-525/coif). All authors report that this work was supported by Biofidelity Ltd., UK. S.E.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. She served as technical consultant for potential investors at Biofidelity (Oct 2023 to Jan 2024). She received honoraria and travel expenses from ThermoFisher to present on NSCLC at a corporate workshop at AMP 2024. C.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. S.J.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.F.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. R.N.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.N.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. C.X. reports that Biofidelity provided all materials (reagents, consumables, travel expenses to laboratories) to complete the work. C.X. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.G.Z. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.G.Z. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. C.K. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.K. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. R.T.E. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.T.E. is an employee of Biofidelity Inc. and has financial interest including salary, stock and stock options. A.S.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.S.G. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. A.L.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.L.S. is an employee of Biofidelity Inc. or Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. M.S.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. M.S.J. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. K.v.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.v.B. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. I.T. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. I.T. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. C.H.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.H.H. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. A. Collazos reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Collazos is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. N.D.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. N.D.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. D.N. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. D.N. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. J.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.J. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.R.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.R.G. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.S. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. E.S. is a former employee of Adaptive Biotechnologies, which have no interests in the work presented herein. W.J.L. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. WJL. is a consultant of Biofidelity Inc. and has financial interest including consultancy fees and work-related travel to AACR 2024 where data from this work was presented. A. Cooke reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Cooke was an employee of Biofidelity Ltd. and has financial interest including salary and stock. B.W.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.W.B. reports that Biofidelity Ltd. has provided travel expenses for work-related travel to AACR 2024 where data from this work was presented. B.W.B. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. R.J.O. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.J.O. was an employee of Biofidelity Ltd., and has financial interest including salary, stock, and stock options. H.V.R. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. H.V.R. was a consultant of Biofidelity Inc. and had financial interest including consultancy fees. H.V.R. is a current employee of Belay Diagnostics, which have no interests in the work presented herein. V.M.V.D. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. The authors have no other conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Introduction: 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 ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3 ) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously., Methods: 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., Results: 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 MET 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., Conclusions: 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., Competing Interests: Authors RE, EG-Z, JB, KK, CK, AG, and BB are employed by the company Biofidelity Inc. Authors A-LS, JM, RP, AT, AC, CX, IT, CH, DN, JJ, SA, NP, KB, EG, MS-J, AC and RO are employees of Biofidelity Ltd, a privately held company. All authors may hold stock or stock options. Author HR was a consultant of Biofidelity Inc at the time of the study. Biofidelity has filed patent applications on aspects of this research WO2021130494A1. The author HR declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The authors declare that this study received funding from Biofidelity Ltd. The following had the following involvement in the study: study design, data collection, analysis, decision to publish, and preparation of the manuscript., (Copyright © 2024 Evans, Gillon-Zhang, Brown, Knudsen, King, Green, Silva, Mordaka, Palmer, Tomassini, Collazos, Xyrafaki, Turner, Ho, Nugent, Jose, Andreazza, Potts, von Bargen, Gray, Stolarek-Januszkiewicz, Cooke, Reddi, Balmforth and Osborne.)

Background: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed., Results: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run., Conclusion: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day., (© 2022. The Author(s).)

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)