Lucence Diagnostics Pte Ltd, Singapore, Singapore.
Lucence Health Inc, Palo Alto, California, United States of America.
PLoS One. 2022 Apr 29;17(4):e0267389. doi: 10.1371/journal.pone.0267389. eCollection 2022.
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.
下一代循环肿瘤 DNA 测序为癌症诊断提供了一种很有前途的方法，通过简单的血液采集即可进行微创的连续检测，并实现广泛的基因组覆盖，从而最大限度地提高患者的治疗获益，这与传统的基于组织的诊断检测相辅相成。LiquidHALLMARK® 是一种基于扩增子的下一代测序检测方法，用于对血浆游离 DNA（cfDNA）进行基因组分析。该综合 80 基因panel 检测点突变、插入/缺失、拷贝数改变和基因融合，还可检测致癌病毒（Epstein-Barr 病毒（EBV）和乙型肝炎病毒（HBV））和微卫星不稳定性（MSI）。本文报告了该检测方法的分析和临床验证。使用参考遗传材料进行分析验证，结果显示该检测方法对 0.1%变异等位基因频率（VAF）的点突变和 95.83%插入/缺失的灵敏度为 99.38%，对 0.5%VAF 的基因融合的灵敏度为 91.67%。在非癌症样本中，观察到高特异性（每个碱基≥99.9999%）。还通过经验确定了拷贝数改变、EBV、HBV 和 MSI 的检测下限。对 355 个肺癌样本进行的 LiquidHALLMARK 和参考等位基因特异性聚合酶链反应（AS-PCR）方法的 EGFR 变异检测结果的正交比较显示，总体一致性为 93.80%，而对 50 个肺癌样本进行的 cobas® EGFR 突变检测 v2 的外部验证显示，总体一致性为 84.00%，VAF 大于 0.4%的 EGFR 变体的一致性为 100%。在 1592 例连续患者中应用 LiquidHALLMARK 检测，结果显示其具有较高的检出率（癌症样本中 74.8%的循环肿瘤 DNA（ctDNA）呈阳性）和广泛的可操作性（50.0%的癌症样本存在具有可操作性生物证据的改变）。在 ctDNA 阳性的肺癌中，72.5%至少有一种具有指南批准药物适应证的生物标志物。这些结果证实了 LiquidHALLMARK 检测方法具有较高的灵敏度、特异性、准确性和精密度，支持其在基于血液的基因组检测中的临床应用。
