Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA
Background: Liquid biopsy is an important non-invasive method of sampling the molecular profile of tumors for patients to access personalized oncology therapeutics but can be challenging. NGS-based methods require high sample quality, high sequencing depth and associated cost, with complex workflows...
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| Language: | English |
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Elsevier
2025-06-01
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| Series: | The Journal of Liquid Biopsy |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2950195425000141 |
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| author | Ryan Thomas Evans Katherine Elizabeth Knudsen Elizabeth Gillon-Zhang Julia Natalie Brown Candace King Mary Beth Rossi Cory Kiser James Alexander Schaffernoth Amanda Shull Green Ana-Luisa Silva Kristine von Bargen Justyna Malgorzata Mordaka Rebecca Natalie Palmer Alessandro Tomassini Alejandra Collazos Simonetta Andreazza Iyelola Turner Chau Ha Ho Dilyara Nugent Jinsy Jose Christina Xyrafaki Prarthna Barot Magdalena Stolarek-Januszkiewicz Sam Abujudeh Eleanor Ruth Gray Jeffrey Gregg Wendy Jo Levin Barnaby William Balmforth Kelly Pitts Shari Brown |
| author_facet | Ryan Thomas Evans Katherine Elizabeth Knudsen Elizabeth Gillon-Zhang Julia Natalie Brown Candace King Mary Beth Rossi Cory Kiser James Alexander Schaffernoth Amanda Shull Green Ana-Luisa Silva Kristine von Bargen Justyna Malgorzata Mordaka Rebecca Natalie Palmer Alessandro Tomassini Alejandra Collazos Simonetta Andreazza Iyelola Turner Chau Ha Ho Dilyara Nugent Jinsy Jose Christina Xyrafaki Prarthna Barot Magdalena Stolarek-Januszkiewicz Sam Abujudeh Eleanor Ruth Gray Jeffrey Gregg Wendy Jo Levin Barnaby William Balmforth Kelly Pitts Shari Brown |
| author_sort | Ryan Thomas Evans |
| collection | DOAJ |
| description | Background: Liquid biopsy is an important non-invasive method of sampling the molecular profile of tumors for patients to access personalized oncology therapeutics but can be challenging. NGS-based methods require high sample quality, high sequencing depth and associated cost, with complex workflows, while PCR assays are limited in variant coverage. Aspyre Clinical Test for Lung® (Blood) is a simplified genomic profiling assay for NSCLC that targets 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, gene fusions and exon skipping events from plasma-derived cfDNA and cfRNA simultaneously. Method: Sensitivity, specificity, analytical accuracy and analytical precision at standard input levels (20 ng cfDNA and 42 ng cfRNA) were tested using a combination of contrived samples and extracts from clinical samples taken from both healthy volunteers and patients with NSCLC. The effects of potential interfering substances on assay performance were tested. Assay sensitivity and specificity were also assessed at lower sample input levels (5 ng cfDNA and 6 ng cfRNA). Results: At standard input levels, median limits of detection were ≤0.25 % variant allele fraction for single nucleotide variants, ≤0.4 % variant allele fraction for insertions or deletions, ≤6 copies for gene fusions, and ≤100 copies MET exon 14 skipping events. The specificity from variant-free samples was 100 %. Tests of analytical accuracy yielded 100 % NPA and 94 % PPA between Aspyre Clinical Test for Lung (Blood) and either results from orthogonal NGS testing or expected outcomes of contrived samples. Results were 100 % replicable across multiple operators, reagent lots, days and equipment. At low input levels, median limits of detection were ≤0.8 % for single nucleotide variants and insertions/deletions, 6 copies for gene fusions and 100 copies for MET exon 14 skipping, with a false-positive rate of 0 %. Conclusions: We present validation studies of Aspyre Clinical Test for Lung (Blood) using contrived and clinical samples. The technology is simple and fast, yet highly sensitive, specific, robust and reproducible with a turnaround time of two days. Aspyre Clinical Test for Lung (Blood) facilitates access to cost-effective, rapid, actionable molecular profiling of plasma for patients with NSCLC. |
| format | Article |
| id | doaj-art-4d40f691a1b1471e9ecb5ac22bef4672 |
| institution | Kabale University |
| issn | 2950-1954 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | The Journal of Liquid Biopsy |
| spelling | doaj-art-4d40f691a1b1471e9ecb5ac22bef46722025-08-20T03:30:44ZengElsevierThe Journal of Liquid Biopsy2950-19542025-06-01810029810.1016/j.jlb.2025.100298Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNARyan Thomas Evans0Katherine Elizabeth Knudsen1Elizabeth Gillon-Zhang2Julia Natalie Brown3Candace King4Mary Beth Rossi5Cory Kiser6James Alexander Schaffernoth7Amanda Shull Green8Ana-Luisa Silva9Kristine von Bargen10Justyna Malgorzata Mordaka11Rebecca Natalie Palmer12Alessandro Tomassini13Alejandra Collazos14Simonetta Andreazza15Iyelola Turner16Chau Ha Ho17Dilyara Nugent18Jinsy Jose19Christina Xyrafaki20Prarthna Barot21Magdalena Stolarek-Januszkiewicz22Sam Abujudeh23Eleanor Ruth Gray24Jeffrey Gregg25Wendy Jo Levin26Barnaby William Balmforth27Kelly Pitts28Shari Brown29Biofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Inc., Morrisville, NC, USABiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Ltd., Cambridge, United KingdomBiofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABiofidelity Ltd., Cambridge, United Kingdom; Corresponding author. 330 Cambridge Science Park, Milton rd, Cambridge, CB4 0WN, United Kingdom.Biofidelity Inc., Morrisville, NC, USABiofidelity Inc., Morrisville, NC, USABackground: Liquid biopsy is an important non-invasive method of sampling the molecular profile of tumors for patients to access personalized oncology therapeutics but can be challenging. NGS-based methods require high sample quality, high sequencing depth and associated cost, with complex workflows, while PCR assays are limited in variant coverage. Aspyre Clinical Test for Lung® (Blood) is a simplified genomic profiling assay for NSCLC that targets 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, gene fusions and exon skipping events from plasma-derived cfDNA and cfRNA simultaneously. Method: Sensitivity, specificity, analytical accuracy and analytical precision at standard input levels (20 ng cfDNA and 42 ng cfRNA) were tested using a combination of contrived samples and extracts from clinical samples taken from both healthy volunteers and patients with NSCLC. The effects of potential interfering substances on assay performance were tested. Assay sensitivity and specificity were also assessed at lower sample input levels (5 ng cfDNA and 6 ng cfRNA). Results: At standard input levels, median limits of detection were ≤0.25 % variant allele fraction for single nucleotide variants, ≤0.4 % variant allele fraction for insertions or deletions, ≤6 copies for gene fusions, and ≤100 copies MET exon 14 skipping events. The specificity from variant-free samples was 100 %. Tests of analytical accuracy yielded 100 % NPA and 94 % PPA between Aspyre Clinical Test for Lung (Blood) and either results from orthogonal NGS testing or expected outcomes of contrived samples. Results were 100 % replicable across multiple operators, reagent lots, days and equipment. At low input levels, median limits of detection were ≤0.8 % for single nucleotide variants and insertions/deletions, 6 copies for gene fusions and 100 copies for MET exon 14 skipping, with a false-positive rate of 0 %. Conclusions: We present validation studies of Aspyre Clinical Test for Lung (Blood) using contrived and clinical samples. The technology is simple and fast, yet highly sensitive, specific, robust and reproducible with a turnaround time of two days. Aspyre Clinical Test for Lung (Blood) facilitates access to cost-effective, rapid, actionable molecular profiling of plasma for patients with NSCLC.http://www.sciencedirect.com/science/article/pii/S2950195425000141Analytical validationLiquid biopsyNSCLCMolecular profiling |
| spellingShingle | Ryan Thomas Evans Katherine Elizabeth Knudsen Elizabeth Gillon-Zhang Julia Natalie Brown Candace King Mary Beth Rossi Cory Kiser James Alexander Schaffernoth Amanda Shull Green Ana-Luisa Silva Kristine von Bargen Justyna Malgorzata Mordaka Rebecca Natalie Palmer Alessandro Tomassini Alejandra Collazos Simonetta Andreazza Iyelola Turner Chau Ha Ho Dilyara Nugent Jinsy Jose Christina Xyrafaki Prarthna Barot Magdalena Stolarek-Januszkiewicz Sam Abujudeh Eleanor Ruth Gray Jeffrey Gregg Wendy Jo Levin Barnaby William Balmforth Kelly Pitts Shari Brown Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA The Journal of Liquid Biopsy Analytical validation Liquid biopsy NSCLC Molecular profiling |
| title | Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA |
| title_full | Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA |
| title_fullStr | Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA |
| title_full_unstemmed | Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA |
| title_short | Analytical validation of Aspyre Clinical Test for Lung (Blood): A multiplexed PCR and pyrophosphorolysis-based assay for detecting actionable NSCLC variants in plasma cfDNA and cfRNA |
| title_sort | analytical validation of aspyre clinical test for lung blood a multiplexed pcr and pyrophosphorolysis based assay for detecting actionable nsclc variants in plasma cfdna and cfrna |
| topic | Analytical validation Liquid biopsy NSCLC Molecular profiling |
| url | http://www.sciencedirect.com/science/article/pii/S2950195425000141 |
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