Background
Circulating tumour HPV DNA (ctHPVDNA) has emerged as a promising liquid biopsy biomarker for HPV-related cancers, with potential applications in both early detection and post-treatment surveillance. This may be particularly relevant for people living with HIV (PLHIV), who face elevated risks of HPV-related malignancies, as well as for patients with HPV-driven cancers where reliable biomarkers for recurrence monitoring are lacking.

Methods
We evaluated the performance of ctHPVDNA in two complementary clinical settings: pre-diagnostic detection of HPV-related cancers in PLHIV and longitudinal monitoring of recurrence in HPV-driven oropharyngeal cancer (OPC).
For early detection, we conducted a nested case–control study within the multinational EuroSIDA cohort (>24,500 PLHIV followed between 1994–2022). HPV-related cancer cases were matched 1:1 to cancer-free controls by country, sex, age, and calendar year. Serial plasma samples collected up to five years prior to cancer diagnosis, and one matched sample per control, were tested for ctHPVDNA (HPV16, HPV18/45, HPV33/52/58) using digital PCR, as well as HPV16-E6 antibodies. For recurrence monitoring, plasma from 38 HPV-driven and 20 HPV-negative OPC patients was collected at diagnosis and during follow-up, and ctHPVDNA was quantified. Tumor HPV status was confirmed using p16INK4A immunohistochemistry, HPV DNA PCR, and E6 serology.

Results
Among 101 HPV-related cancers identified in EuroSIDA (76 anal, 15 cervical, 3 penile, and 7 oropharyngeal cancers), ctHPVDNA detection increased closer to cancer diagnosis, from 15.0% at 4–5 years prior to diagnosis to 37.5% within one year. ctHPVDNA was detectable up to 14 years before cancer diagnosis in some cases, while detection among controls was rare (1/101; 1.0%), corresponding to 99% specificity. ctHPVDNA positivity was also associated with cancer site, lower CD4 counts (<400 cells/µL), and injection drug use.
In the OPC cohort, ctHPVDNA demonstrated high diagnostic performance at baseline, with 95% sensitivity (36/38) and 95% specificity (19/20). Among 24 HPV-driven OPC patients with follow-up samples (median follow-up 1.6 years), four experienced recurrence or persistent disease. In two patients, re-detection of ctHPVDNA after initial clearance preceded clinical diagnosis of recurrence by 4 and 8 months. ctHPVDNA positivity after therapy showed a positive predictive value of 75% for recurrence within one year.

Conclusions
ctHPVDNA shows promise as a minimally invasive biomarker across the HPV-related cancer continuum. It can be detected years before cancer diagnosis among PLHIV with high specificity and may also enable earlier identification of recurrence in HPV-driven OPC. With optimized sampling and analytical approaches, ctHPVDNA-based testing could support both early detection and re-occurence surveillance strategies for HPV-related cancers.

Longitudinal detection of ctHPVDNA in all serial pre-diagnostic plasma samples from ctHPVDNA-positive HPV-related cancer cases