Detection, quantitation, and genotyping of human papillomavirus circulating tumor DNA by droplet digital PCR.

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Journal of Clinical Microbiology Pub Date : 2025-09-10 Epub Date: 2025-08-19 DOI:10.1128/jcm.00585-25
Emily C Fernholz, David M Routman, Kathryn M Van Abel, Eric J Moore, Daniel J Ma, Danielle E Hunter, Kathleen R Bartemes, James S Lewis, Erik B Wendlandt, Matthew J Binnicker
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引用次数: 0

Abstract

Human papillomavirus (HPV) is comprised of >200 genotypes and has an ~8 kb, circular, double-stranded DNA genome. Transmission of HPV occurs through skin-to-skin contact and infection of squamous epithelial cells of cutaneous and mucosal surfaces. HPV genotypes are categorized as low- or high-risk (hrHPV) based on oncogenic potential. There are approximately 14 types of hrHPV that can cause several types of cancer, including HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). Detection of HPV(+)OPSCC is traditionally accomplished using p16 immunohistochemistry (IHC) and HPV-specific testing, either DNA or RNA in situ hybridization (ISH) staining or DNA-based PCR of suspected tumor biopsy tissue. More recently, platelet-poor plasma (PPP) samples from patients with HPV(+)OPSCC have proven useful for detection and quantitation of fragments of HPV circulating tumor DNA (ctDNA). ctDNA has been shown to be useful in determining treatment response and monitoring for disease recurrence. In this study, a novel droplet digital PCR assay (ddPCR) was developed and validated for the detection and quantitation of ctDNA from 5 hrHPV genotypes in PPP. Analytical sensitivity ranged from 7.71 to 19.45 fragments of HPV ctDNA per milliliter of PPP across five hrHPV genotypes. In patients with confirmed primary or recurrent HPV(+)OPSCC or HPV(-)OPSCC, testing of corresponding PPP samples (n = 32) by ddPCR demonstrated 90.63% (29/32) overall agreement with p16/HPV-ISH biopsy results. Compared with reference ddPCR assays performed at outside laboratories, our ddPCR assay yielded 90% (9/10) overall agreement. This assay may provide clinicians with a tool for monitoring HPV ctDNA prior to, during, and after treatment of an HPV-associated cancer.

Importance: At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.

Abstract Image

人乳头瘤病毒循环肿瘤DNA的微滴数字PCR检测、定量和基因分型。
人乳头瘤病毒(HPV)由约200个基因型组成,具有约8 kb的环状双链DNA基因组。HPV通过皮肤与皮肤的接触和皮肤和粘膜表面的鳞状上皮细胞的感染而传播。HPV基因型根据致癌潜力分为低危型和高危型(hrHPV)。大约有14种hrHPV可导致几种类型的癌症,包括HPV相关的口咽鳞状细胞癌(HPV(+)OPSCC)。HPV(+)OPSCC的检测传统上使用p16免疫组织化学(IHC)和HPV特异性检测,DNA或RNA原位杂交(ISH)染色或DNA-based PCR对可疑肿瘤活检组织进行检测。最近,来自HPV(+)OPSCC患者的血小板缺乏血浆(PPP)样本已被证明可用于HPV循环肿瘤DNA (ctDNA)片段的检测和定量。ctDNA已被证明在确定治疗反应和监测疾病复发方面是有用的。在这项研究中,我们开发了一种新的液滴数字PCR法(ddPCR),用于检测和定量PPP中5种hrHPV基因型的ctDNA。在五种hrHPV基因型中,每毫升PPP的分析灵敏度为7.71至19.45个HPV ctDNA片段。在确诊为原发性或复发性HPV(+)OPSCC或HPV(-)OPSCC的患者中,通过ddPCR检测相应的PPP样本(n = 32)显示90.63%(29/32)与p16/HPV- ish活检结果总体一致。与外部实验室进行的参考ddPCR分析相比,我们的ddPCR分析的总体一致性为90%(9/10)。这种检测方法可以为临床医生提供在HPV相关癌症治疗之前、期间和之后监测HPV ctDNA的工具。重要性:至少有14种人类乳头瘤病毒(HPV)基因型已被确定具有高致癌潜力。虽然HPV的分子诊断检测可广泛用于宫颈液体细胞学样本,但对其他类型样本的检测有限,包括液体活检样本,如血小板不足的血浆(PPP)。随着HPV相关口咽鳞状细胞癌(HPV(+)OPSCC)发病率的上升,实验室检测是患者诊断、管理和监测的重要组成部分。在这里,我们总结了用于PPP中HPV循环肿瘤DNA (ctDNA)检测和定量的多路、液滴数字PCR (ddPCR)方法的发展和分析性能验证。该检测可能为临床医生提供了一种工具,以解决与hpv相关的癌症患者的最小残留疾病。
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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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