PO.IM03.01 · 免疫学
肿瘤来源的游离 DNA 中病毒检测的分析验证
Analytical validation of viral detection in tumor-derived cell-free DNA
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：Epstein-Barr 病毒（EBV）和人乳头瘤病毒（HPV）是与特定恶性肿瘤相关的致癌病毒，但在临床实践中其在肿瘤中的检测常常未被评估。EBV 阳性胃癌患者可能从免疫治疗中获益，但病毒状态很少被常规评估。同样，HPV 相关恶性肿瘤在原发部位不明的病例中可能被遗漏。游离 DNA（cfDNA）分析提供了一种识别这些具有临床可操作性的病毒特征的非侵入性方法。在此，我们确立了 Guardant360 Liquid（Guardant Health，加州 Palo Alto）中 EBV 和 HPV 检测的分析性能，该检测是一款面向晚期癌症患者的全面的基于血液的分子分析检测。
方法：Guardant360 Liquid 使用靶向杂交捕获 cfDNA 检测靶向 EBV 和 14 种高危 HPV 基因型。我们评估了分析敏感性、特异性、准确性和精密度。准确性使用 75 份具有正交病毒检测结果的临床样本（43 份 HPV，33 份 EBV）以及 43 份具有 BD Onclarity HPV 检测（Becton Dickinson，新泽西州 Franklin Lakes）基因分型结果的样本进行评估。LoB（空白限）通过来自 30 名自报无癌供者样本的 120 次重复测定。LoD（检出限）通过 WHO HPV 参考标准品的系列稀释以及对临床样本和参考物质的计算机模拟降采样确立。精密度使用重复检测的临床样本进行评估。
结果：对于病毒检测状态，与正交方法相比，Guardant360 Liquid 显示出 93.48% 的阳性符合率（PPA）和 96.55% 的阴性符合率（NPA）。对于 HPV 基因分型，PPA 为 100%，NPA 为 97.39%。在无癌供者样本中，HPV 的假阳性率为 0%，EBV 为 0.83%。类别水平的 LoD 确立为每人类基因组 3.36 个病毒拷贝，检出率 ≥95%。精密度检测显示，在 1-5 倍 LoD 的所有重复中 PPA 为 100%。
结论：Guardant360 Liquid 在 cfDNA 中检测 EBV 和高危 HPV 基因型方面显示出高分析敏感性、特异性、准确性和可重复性。这一经过验证的非侵入性方法能够在晚期癌症患者中实现病毒检测，其潜在临床应用包括识别可能从免疫治疗中获益的 EBV 阳性胃癌患者，以及优化 HPV 阳性癌症的部位特异性治疗路径。这一能力将液体活检的临床效用从基因组分析扩展到包括具可操作性的病毒生物标志物检测。
查看英文原文 English abstract
Background: Epstein-Barr virus (EBV) and human papillomavirus (HPV) are oncogenic viruses linked to specific malignancies, yet their detection in tumors often goes unassessed in clinical practice. EBV-positive gastric cancer patients may benefit from immunotherapy, but viral status is rarely evaluated routinely. Similarly, HPV-associated malignancies can be missed in cases with unknown primary sites. Cell-free DNA (cfDNA) analysis offers a non-invasive approach to identify these clinically actionable viral signatures. Here, we established the analytical performance of EBV and HPV detection in Guardant360 Liquid (Guardant Health, Palo Alto, CA), a comprehensive blood-based molecular profiling assay for patients with advanced cancer.
Methods: Guardant360 Liquid targets EBV and 14 high-risk HPV genotypes using a targeted hybridization capture cfDNA assay. We evaluated analytical sensitivity, specificity, accuracy, and precision. Accuracy was assessed using 75 clinical samples with orthogonal viral testing results (43 HPV, 33 EBV) and 43 samples with BD Onclarity HPV Assay (Becton Dickinson, Franklin Lakes, NJ) genotyping results. LoB was determined using 120 replicates from 30 self reported cancer-free donor samples. LoD was established through serial titration of WHO HPV reference standards and in-silico down-sampling of clinical samples and reference materials. Precision was evaluated using clinical samples tested in replicate.
Results: For viral detection status, Guardant360 Liquid demonstrated a positive percent agreement (PPA) of 93.48% and a negative percent agreement (NPA) of 96.55% compared to orthogonal methods. For HPV genotyping, PPA was 100% and NPA was 97.39%. The false positive rate was 0% for HPV and 0.83% for EBV in cancer-free donor samples. The class-level LoD was established at 3.36 viral copies per human genome with ≥95% detection rate. Precision testing showed 100% PPA across all replicates at 1-5X LoD.
Conclusions: Guardant360 Liquid demonstrates high analytical sensitivity, specificity, accuracy, and reproducibility for detecting EBV and high-risk HPV genotypes in cfDNA. This validated, non-invasive approach enables viral detection in advanced cancer patients, with potential clinical applications including the identification of EBV-positive gastric cancer patients who may benefit from immunotherapy, and optimization of site-specific treatment pathways for HPV-positive cancers. This capability expands the clinical utility of liquid biopsy beyond genomic profiling to include actionable viral biomarker detection.
利益披露 Disclosure
H. Wang,
Guardant Health Employment, Stock.
L. Lawrence,
Guardant Health Employment, Stock.
J. Hutchins,
Guardant Health Employment, Stock.
T. Jiang,
Guardant Health Employment, Stock.