开发一种高性能多探针液滴数字 PCR assay，用于从血浆中高灵敏度检测人乳头瘤病毒循环肿瘤 DNA。

Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma.

机构信息

Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, USA.

Department of Otolaryngology- Head and Neck Surgery, University of Michigan, USA.

出版信息

Oral Oncol. 2023 Aug;143:106436. doi: 10.1016/j.oraloncology.2023.106436. Epub 2023 Jun 1.

DOI:10.1016/j.oraloncology.2023.106436

PMID:37269557

Abstract

OBJECTIVES

To develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC).

MATERIALS AND METHODS

Plasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome.

RESULTS

The new assay termed 'ctDNA HPV16 Assessment using Multiple Probes' (CHAMP- 16) yielded significantly higher HPV16 counts compared to our previously validated 'Single-Probe' (SP) assay and a commercially available NavDx® assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal ∼ 20 months prior to the conventional SP assay.

CONCLUSION

Increased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Critically, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

摘要

目的

开发一种高性能的微滴式数字 PCR（ddPCR）检测方法，用于增强对 HPV 相关口咽鳞状细胞癌（HPV+OPSCC）患者血浆中循环肿瘤 DNA（ctDNA）中 HPV 的检测。

材料与方法

收集 HPV+OPSCC 患者的血浆样本。我们开发了一种高性能的 ddPCR 检测方法，该方法旨在同时靶向 HPV16 基因组的九个区域。

结果

新的检测方法称为“使用多重探针的 ctDNA HPV16 评估”（CHAMP-16）与我们之前验证的“单探针”（SP）检测方法和商业可用的 NavDx®检测方法相比，HPV16 计数显著更高。分析验证表明，CHAMP-16 检测方法的检测限（LoD）为每个反应 4.1 个拷贝，相当于 HPV16 的<1 个基因组当量（GE）。当用 SP 检测方法对 21 例早期 HPV+OPSCC 患者的血浆 ctDNA 和已知的 HPV16 ctDNA 进行检测时，所有患者在两种检测方法中均为 HPV16 ctDNA 阳性，并且 CHAMP-16 检测方法的 HPV16 信号平均高出 6.6 倍。最后，在对一位复发性疾病患者的样本进行的纵向分析中，CHAMP-16 检测方法在常规 SP 检测方法之前约 20 个月检测到 HPV16 ctDNA 信号。

结论

使用 CHAMP-16 检测方法检测到 HPV16 信号的增加表明，与常规 ddPCR 检测方法相比，HPV16+OPSCC 患者的复发检测可能显著更早。至关重要的是，这种多探针方法保持了 ddPCR 相对于下一代测序（NGS）方法的成本效益优势，支持了该检测方法在大规模人群筛查和常规治疗后监测中的成本效益。