Purpose: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. Experimental design: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. Results: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. Conclusion: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

目的：本研究比较两种单独的液体活检来源（无细胞（cf）DNA / RNA和细胞外囊泡（EV）相关的DNA / RNA（EV-DNA / RNA））的检测灵敏度，以鉴定循环中的人乳头瘤病毒（口咽鳞状细胞癌（OPCSCC）患者血浆中的HPV）DNA / RNA。我们还报告了对治疗产生反应的HPV-DNA水平的纵向变化。实验设计：进行了一项前瞻性研究，其中包括22例局部晚期疾病患者和6例转移性OPCSCC患者。23例患者通过p16免疫组化定义了HPV相关的OPCSCC。使用数字液滴PCR定量测定血浆来源的cf-DNA / RNA和EV-DNA / RNA的循环HPV-DNA和HPV-RNA的水平。结果：与EV-DNA相比，在cf-DNA中检测到循环HPV-DNA的敏感性更高，分别为91％和42％（p  = <0.001）。同样，与EV-RNA相比，cf-RNA中循环肿瘤HPV-RNA的检测灵敏度更高，分别为83％和50％（p = 0.0019）。在局部晚期队列中，治愈性治疗后100％（n = 16）的HPV-OPCSCC患者的cf-DNA循环HPV-DNA水平降低，其中81％的患者显示完全清除率达到无法检测的水平。但是，在转移性HPV-OPCSCC患者（n = 4）中，HPV-DNA水平与治疗反应无​​关。结论：我们的研究表明，尽管可以在EV相关DNA / RNA中检测到HPV-DNA / RNA，但cf-DNA / RNA是更敏感的液体活检介质。由于在我们的小群患者中发现循环中的HPV-DNA水平仅与局部晚期的治疗反应相关，而与转移环境无关，因此，使用HPV-DNA作为监测治疗反应的动态生物标记物需要进一步评估。