Introduction

To address the rapid rise in cases of oropharyngeal cancer (OPC) linked to human papillomavirus (HPV), effective biomarkers and innovative screening techniques are essential tools to facilitate early detection within well-defined high-risk populations. Among the most promising biomarkers are circulating tumor HPV DNA (ctHPV), antibodies (Abs) targeting HPV16 early (E) antigens, and persistent infection with oral HPV16 (oHPV16). In particular, HPV16 E Abs and ctHPV exhibit strong sensitivity and specificity, differentiating between HPV-related OPC and non-cancerous controls with sensitivities of 85% and 89% and specificities of 99% and 97%, respectively. Furthermore, the persistence of oHPV16 infection could play a role in assessing individual risk. This study aimed to assess the prevalence of these three biomarkers in a cohort of middle-aged men—a group with particularly high OPC rates—and to analyze their level of agreement.

Methods

Participants enrolled in the HPV-related Oropharyngeal and Uncommon Cancers Screening Trial of Men (HOUSTON) and Throat and Other HPV-Related Cancers in Men: Identifying Them Early (TRINITY; RP200025) study were included in the analysis. The purpose of these studies is to assess novel screening strategies and biomarkers for HPV-related cancers in middle-aged men (aged 50-64). Initial findings from the HOUSTON study, published previously, found the prevalence of HPV16 Abs to be 1.2% and oHPV16  to be 8% within this cohort.

Biomarker Assays.

Plasma ctHPV: digital droplet polymerase chain reaction (ddPCR) assay (NavDx, Naveris, Waltham, MA).

Plasma HPV16 Abs: novel RAPID ELISA.

Oral rinse oHPV16: cobas HPV Test (Roche Diagnostics, Indianapolis, IN).

Statistical Analysis.

Frequency distributions were tabulated for each biomarker and combinations of biomarkers. The kappa-statistic was used to gauge the level of agreement between the biomarkers. P < 0.05 was considered statitically significant, and all tests were two-sided. Stata 16.0 software was used for all analyses.

Results

A total of 880 male participants were enrolled in the HOUSTON and TRINITY trials between April 2017 and July 2023 (HOUSTON, N = 553 and TRINITY, N = 327). For the 640 samples with results available for all three biomarkers, 587 (91.7%) were negative for all three, 48 (7.5%) were positive for one (19 ctHPV-/Ab-/oHPV16+, 3 ctHPV-/Ab+/oHPV16-, 26 ctHPV+/Ab-/oHPV16-), 4 (0.6%) were positive for two (1 ctHPV-/Ab+/oHPV16+, 3 ctHPV+/Ab-/oHPV16+), and 1 (0.2%) was positive for all three biomarkers. ctHPV levels were significantly elevated in men positive for both HPV16 Abs and oHPV16 (20% vs. 0.6% for positive vs. negative Abs and 4.2% vs. 0.7% for positive vs. negative oHPV16).

Although the percent agreement between ctHPV and Abs or oHPV16 were notably high (95% and 92%, respectively), there was only a slight, although statisically significant, level of agreement (Κ ≤ 0.1; p < 0.05). The weighted average kappa value for all three biomarkers combined also indicated slight agreement (K = 0.076, p = 0.001).

Notably, the participant who tested positive for all three markers was subsequently diagnosed with stage II (T1N1) HPV16-positive/Epstein-Barr-negative nasopharyngeal cancer four months after study enrollment. Another individual who was negative for ctHPV but positive for HPV16 antibodies received a diagnosis of anal low-grade squamous intraepithelial lesion and consistently tested positive for high-risk HPV at the right tonsil/base of the tongue.

Conclusion

Prevalence was low for all three biomarkers within a middle-aged male population. The concordance among the three biomarkers was minimal, yet noteworthy. These findings imply that using these markers in tandem could potentially enhance the sensitivity and specificity for early detection of HPV-related cancers. Further extensive investigations are necessary to establish the most effective use of these markers within a screening framework.