口咽癌无浆细胞人乳头瘤病毒-16 DNA的高度多重检测。

IF 4 3区 医学 Q2 VIROLOGY Journal of Clinical Virology Pub Date : 2025-02-01 DOI:10.1016/j.jcv.2025.105760
Payton Clark , Natalya Karasik , Shauna R. Campbell , Neil M. Woody , Jamie A. Ku , Natalie Silver , Danielle Bottalico , Brandon L. Prendes , Eric D. Lamarre , Joseph Scharpf , Tamara A. Sussman , Jessica L. Geiger , Hannah Wang , Timothy A. Chan , Shlomo A. Koyfman , Jacob A. Miller
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引用次数: 0

摘要

背景:无浆细胞人乳头瘤病毒DNA (cfHPVDNA)是口咽癌的生物标志物。现有的诊断方法可能受到纵向监测灵敏度不足或成本高/复杂程度的限制。目的:我们假设通过高多重qPCR方法可以实现敏感和特异性的血浆cfHPVDNA检测。研究设计:我们设计并验证了一种单管一步基因型特异性qPCR检测方法,用于检测人血浆中cfHPV16DNA,该方法使用了跨越18个基因型的bbb8000个基因组。扩增子对cfHPVDNA片段大小进行优化。结果:cfHPV16DNA qPCR扩增子覆盖了16%的HPV16基因组。扩增子在3,944个基因组中保守的中位数为99.0%。95%检测下限为0.35个基因组拷贝/反应,空白下限为0。使用cfHPVDNA片段的硅模拟,与单个扩增子相比,多路复用的灵敏度提高了10倍,这与实验观察结果非常吻合。对HPV18进行了类似的实验。在36例头颈部粘膜癌患者中(HPV阳性26例,HPV阴性12例),与组织HPV状态和NavDx数字PCR的一致性为100%。检测预处理标本中cfHPVDNA亚基因组浓度。单扩增观察到假阴性,但这种多路扩增方法没有观察到假阴性。在17例治疗后地标标本中,复发的PPV为100%,NPV为100%。结论:该方法对血浆cfHPVDNA检测和复发预后具有特异性。亚基因组敏感性与计算机模拟结果非常一致。对于纵向测试,这种格式可能比dPCR或NGS更容易获得。
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Highly-multiplex detection of plasma cell-free human papillomavirus-16 DNA in oropharyngeal carcinoma

Background

Plasma cell-free Human Papillomavirus DNA (cfHPVDNA) is a biomarker for oropharyngeal carcinoma. Existing diagnostics may be limited by inadequate sensitivity or high cost/complexity for longitudinal monitoring.

Objectives

We hypothesized that sensitive and specific plasma cfHPVDNA detection may be achieved via a highly-multiplex qPCR method.

Study Design

We designed and validated a single-tube one-step genotype-specific qPCR assay for detection of cfHPV16DNA in human plasma using >8,000 genomes spanning 18 genotypes. Amplicons were optimized for cfHPVDNA fragment size.

Results

The cfHPV16DNA qPCR amplicons spanned 16 % of the HPV16 genome. Amplicons were conserved in a median of 99.0 % of 3,944 genomes in silico. The 95 % lower limit of detection was 0.35 genome copies/reaction and the limit of blank was 0. Multiplexing achieved a tenfold improvement in sensitivity compared with single amplicons using in silico simulations of cfHPVDNA fragmentation, which was in close agreement with experimental observations. An assay was replicated for HPV18 with similar observations.
Among 36 patients with head/neck mucosal carcinomas (26 HPV-positive, 12 HPV-negative), there was 100 % concordance with tissue HPV status and with NavDx digital PCR. Pre-treatment specimens with sub-genomic cfHPVDNA concentration were detected. False negatives were observed with single amplicons but not with this multiplexed method. Among 17 patients with post-treatment landmark specimens, there was 100 % PPV and 100 % NPV for recurrence.

Conclusions

This assay is specific for plasma cfHPVDNA detection and prognostic for recurrence. Sub-genomic sensitivity was in close agreement with in silico simulations. The format might be more accessible than dPCR or NGS for longitudinal testing.
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来源期刊
Journal of Clinical Virology
Journal of Clinical Virology 医学-病毒学
CiteScore
22.70
自引率
1.10%
发文量
149
审稿时长
24 days
期刊介绍: The Journal of Clinical Virology, an esteemed international publication, serves as the official journal for both the Pan American Society for Clinical Virology and The European Society for Clinical Virology. Dedicated to advancing the understanding of human virology in clinical settings, the Journal of Clinical Virology focuses on disseminating research papers and reviews pertaining to the clinical aspects of virology. Its scope encompasses articles discussing diagnostic methodologies and virus-induced clinical conditions, with an emphasis on practicality and relevance to clinical practice. The journal publishes on topics that include: • new diagnostic technologies • nucleic acid amplification and serologic testing • targeted and metagenomic next-generation sequencing • emerging pandemic viral threats • respiratory viruses • transplant viruses • chronic viral infections • cancer-associated viruses • gastrointestinal viruses • central nervous system viruses • one health (excludes animal health)
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