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A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers-Analytical validation and performance evaluation with clinical samples.

ABSTRACT: Introduction: A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types. Methods: The assay uses hybrid-capture probes capable of profiling DNA aberrations from 572 cancer-related genes and 91 RNA fusion partners. The panel can detect clinically-tractable biomarkers such as microsatellite instability (MSI) and tumor mutation burden (TMB). Results: Analytical evaluation demonstrated 100% specificity and 99.9% sensitivity within a ≥5% VAF limit of detection (LoD) for SNV/Indels. RNA-based fusion features an LoD of ≥5 copies per nanogram input when evaluated against commercial references. Excellent linearity and concordance were observed when benchmarking against orthogonal methods in identifying MSI status, TMB scores and RNA fusions. Actionable genetic alterations were identified in 65% of the clinical samples. Conclusion: These results demonstrate a molecular diagnostic assay that accurately detects genomic alterations and complex biomarkers. The data also supports an excellent performance of this assay for making critical diagnoses and well-informed therapeutic decisions in Asian prevalent cancers.

SUBMITTER: Ng CC

PROVIDER: S-EPMC9532579 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers-Analytical validation and performance evaluation with clinical samples.

Ng Cedric Chuan-Young CC Lim Sandy S Lim Abner Herbert AH Md Nasir Nur Diyana ND Zhang Jingxian J Rajasegaran Vikneswari V Lee Jing Yi JY Kok Jessica Sook Ting JST Thike Aye Aye AA Lim Johnathan Xiande JX Weng Ruifen R Yee Sidney S Choudhury Yukti Y Chan Jason Yongsheng JY Tan Puay Hoon PH Tan Min-Han MH Teh Bin Tean BT

Frontiers in molecular biosciences 20220921

<b>Introduction:</b> A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types. <b>Methods:</b> The assay uses hybrid-capture probes capable of profiling ...[more]

PMID: 36213130

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Project description:Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

Project description:Background Antithrombin deficiency is a rare but severe disorder leading to high risk of thrombosis. The current clinical care pathway relies on activity tests, which only provide overall functional information on the in vitro activity of antithrombin. However, antithrombin exists in many different forms, also known as proteoforms, with varying clinical phenotypes. Precision diagnostics, facilitated by mass spectrometry, provides a strategy to improve patient diagnostics by molecular characterization. Objectives To develop and analytically validate a mass spectrometry–based test for molecular characterization of antithrombin. Methods The test was analytically validated based on predefined analytical performance specifications. The validation covered imprecision, carryover, linearity, stability, analytical specificity, a provisional reference interval, and an explorative method comparison. Results The test passed the predefined analytical performance specifications with a mean within-laboratory imprecision of 5.9%, linearity between 0.08 and 2.58 μmol/L, and a provisional reference interval of 1.07 to 1.49 μmol/L. When measuring samples with a suspected quantitative deficiency, the test showed a good correlation with a commercial activity test (Pearson r = 0.88). Conclusion The test passed the validation, and we now envision the use of the test for exploration of the clinical relevance of specific antithrombin proteoforms. Puzzling cases of antithrombin deficiency, for instance, due to ambiguous activity results or an atypical clinical presentation, can be investigated by the LC-MRM mass spectrometry test serving as an add-on to the activity test and providing a molecular diagnosis. Clinical studies are planned to investigate the potential of the test to improve antithrombin diagnostics. Furthermore, the molecular information gained using the test may aid in establishing better risk stratification and a basis for personalized medicine. Graphical abstract Essentials • Current diagnostic tests for antithrombin (AT) deficiency only provide limited information.• Mass spectrometry (MS) is a technique that allows molecular characterization of AT.• An MS-based test for AT was developed, and full analytical validation was performed.• The test may serve as an add-on to current tests and a research tool to investigate the clinical picture of specific AT forms.

Dataset Information

A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers-Analytical validation and performance evaluation with clinical samples.

Publications

A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers-Analytical validation and performance evaluation with clinical samples.

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