在人类发展指数较高的国家,人乳头瘤病毒驱动的口鼻咽癌(HPV-OPC)的发病率正在增加。从3至4mL血浆中分离的HPV无细胞DNA(cfDNA)已成功用于治疗监测。HPV-cfDNA的一个高度讨论的应用是HPV-OPC的早期检测。这需要灵敏和特异性的cfDNA检测,因为cfDNA水平可能非常低。为了研究诊断前HPV-cfDNA的预测能力,来自血浆量有限的流行病学队列的存档样本是重要来源.为了建立低血浆体积的cfDNA检测工作流程,我们比较了cfDNA纯化方法[MagNAPure96(MP96)和QIAampccfDNA/RNA]和数字PCR系统(BioradQX200和QIAGENQIAcuityOne)。最终的测定验证包括来自具有确定的HPV状态的口鼻咽癌(OPC)患者的65个低容量血浆样品,其储存2-9年。与QIAamp相比,MP96的cfDNA分离效率提高了28%。两种数字PCR系统均显示出相当的分析灵敏度(HPV16和HPV33为6-17个拷贝),但QIAcuity在同一检测中检测到两种类型。在验证集中,该试验对HPV16和HPV33的敏感性为80%(n=28/35),特异性为97%(n=29/30).在血浆浓度≥750微升的样品中,灵敏度为85%(n=17/20),而在血浆<750微升的样品中,73%(n=11/15)。尽管随着血浆体积的减少,灵敏度预计会下降,即使在小量样本中,该检测方法也是灵敏且高度特异的,因此适用于在小量档案材料中探索HPV-cfDNA作为早期HPV-OPC检测标志物的研究.与HPV阴性OPC相比,IMPORTANCEHPV-OPC具有良好的预后。然而,大多数肿瘤是在局部扩散后诊断的,因此需要强化治疗。这可能导致持久的发病率,对生活质量有很大影响。降低治疗相关发病率的一种潜在方法是癌症的早期检测。HPVcfDNA已成功用于治疗监测,并且还在HPV-OPC患者的诊断前样品中检测到。这些诊断前样本通常只能从生物样本库中获得,通常只有少量的血浆。因此,我们开发了一个针对小批量归档样本优化的工作流程。使用这种方法,尽管样品有限,但可以实现高灵敏度,使其适合进行进一步大规模的HPV-cfDNA作为HPV-OPC的预后生物标志物的生物样本库研究。
The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-
OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-
OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (
OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-
OPC has a favorable prognosis compared to HPV-negative
OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.