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

Sep 21, 2026

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Human papillomavirus (HPV) contributes substantially to global cancer incidence, especially oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). The study developed and validated droplet digital PCR (ddPCR) assays to detect circulating tumor HPV DNA (ctHPV-DNA) in plasma, targeting high-risk HPV genotypes linked to these cancers. Assays for HPV16, 18, 33, 35, 56, and 59 showed robust technical sensitivity and enabled clinical assessment.

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Abstract

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Introduction

Molecular Diagnosis & Therapy (2024) 28:835–845 [https://doi.org/10.1007/s40291-024-00743-9](https://doi.org/10.1007/s40291-024-00743-9) Sensitive and Specific Droplet Digital PCR Assays for Circulating Tumor HPV DNA: Development, Validation, and Clinical Application in HPV‑Associated Cancers Alvida Qvick1 · Elin Andersson2 · Anna Oldaeus Almerén3 · Max Waenerlund4,5 · Bianca Stenmark1 · Christina Karlsson6 · Mats G. Karlsson1 · Gisela Helenius7 Accepted: 20 August 2024 / Published online: 26 September 2024 © The Author(s) 2024 Abstract 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 assayson a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPVDNA 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 . * Alvida Qvick[alvida.qvick@regionorebrolan.com](alvida.qvick@regionorebrolan.com) 1 Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro University, Örebro, Sweden 2 Clinical Research Center, Örebro University Hospital,Örebro, Sweden 3 Department of Otolaryngology, Faculty of Medicine and Health, Örebro University, Örebro, Sweden 4 Department of Otolaryngology, Sahlgrenska University Hospital, Göteborg, Sweden 5 Faculty of Medicine and Health, Örebro University, Örebro, Sweden 6 School of Health Sciences, Örebro University, Örebro, Sweden 7 ATMP Center, Skåne University Hospital, Lund, Sweden 1 Introduction Due to the significant increase in human papilloma virus (HPV)-induced cancers over recent decades, the role of DNA diagnostics has become of great importance. Almost 0.7 million new HPV cancer cases were diagnosed in 2018, equal to 10% of the total cancer incidence worldwide [1] . HPV, a DNA virus that infects squamous cells, is widely recognized for its potential to cause genital cancer through persistent infections. However, HPV+ head neck cancer, such as oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP), has become oneof the most frequent HPV driven cancer types worldwide. The prevalence varies by region, but HPV+ OPSCC has been reported to have a pooled global burden of 33%, with a higher prevalence in the Nordic countries [2] . High-risk HPV genotypes, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, are considered carcinogenic due to their expression of E6 and E7 oncoproteins, along with their ability to integrate genomic components into the human genome [3]. Consequently, circulating tumor DNA (ctDNA) released from HPV-induced tumors contains fragments of HPV DNA seque

FAQs

What is the study trying to achieve with droplet digital PCR?

Develop and validate ddPCR assays to detect circulating tumor HPV DNA (ctHPV-DNA) in plasma for HPV-associated cancers.

Which HPV genotypes were included in the assay development?

HPV16, 18, 33, 35, 56, and 59 were developed and tested in the ddPCR assays.

What clinical performance and biological associations were reported?

Clinical use showed high sensitivity (91.6%) and ctHPV-DNA levels varied widely; ctHPV-DNA correlated with TNM stage and tumor viral load, and undetectable ctHPV-DNA after treatment was common.

Sep 21, 2026

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