Sensitive and Specific Droplet Digital PCR Assays for Circulating Tumor HPV DNA: Development, Validation, and Clinical Application in HPV-Associated Cancers.

Background: Human papillomavirus (HPV) has emerged as a significant contributor to cancer incidence globally, particularly in the context of oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). This study aimed to develop and validate droplet digital PCR (ddPCR) assays for the detection of circulating tumor HPV DNA (ctHPV-DNA) in plasma, focusing on high-risk HPV genotypes associated with these cancers.

Methods: ddPCR assays for HPV16, 18, 33, 35, 56, and 59 were developed and tested using gBlocks, HPV cell-free DNA, fragmented tumor HPV+ DNA, and plasma samples from patients with HPV+ OPSCC (n = 110) and HNCUP (n = 9).

Results: Assays demonstrated robust technical sensitivity across all tested HPV genotypes. Clinical application of the assays on a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPV-DNA levels. Analyses revealed correlations between ctHPV-DNA levels and TNM stage and tumor viral load. The association between ctHPV-DNA and tumor viral load persisted even after adjusting for TNM stage. At posttreatment, 72.5% of samples had reached undetectable ctHPV-DNA levels. Having detectable ctHPV-DNA posttreatment was associated with a higher ctHPV-DNA level at diagnosis and higher viral load at diagnosis.

Conclusion: The findings underscore the potential of ctHPV-DNA as a biomarker for monitoring HPV+ cancers and offer insights into tumor dynamics. Implementation of these assays in clinical practice could enhance no-invasive treatment monitoring and recurrence detection in HPV-associated cancers.

Clinical trials: NCT05904327.