Multi-analyte blood-based screening early detection of HPV-associated oropharyngeal and anal cancer within the PLCO cohort

Background: Despite rising incidence, there are no early detection methods for HPV+ oropharyngeal (HPV+OPC) and anal (HPV+AC) cancers. HPV16 E6 antibody (E6 Ab) positivity is a promising early biomarker, appearing in most patients at diagnosis and years before symptoms, but the long interval between seroconversion and cancer limits clinical utility. Additional tumor-indicative biomarkers are needed. Circulating tumor HPV DNA (ctHPVDNA) is highly sensitive and specific for detecting HPV+ cancers at diagnosis. Ultrasensitive HPV whole genome sequencing (WGS) assays such as HPV-DeepSeek have shown strong performance for HPV+OPC early detection in a single institution cohort. Methods: ctHPVDNA testing was performed on serial pre-diagnostic plasma samples from 73 OPC and 19 AC cases and 183 matched PLCO controls with available E6 Ab results. Samples were blinded, underwent cfDNA extraction, and were analyzed using HPV-DeepSeek as previously described. Results: ctHPVDNA detection was strongly associated with future OPC and AC development (OR 20.52 [95% CI 4.80-87.59], P<0.0001; OR 33.63 [95% CI 7.05-Infinity], P<0.0001). Among E6 Ab positive cases (N=31, surrogate marker to HPV+), ctHPVDNA sensitivity was 61.3% (95% CI 42.2-78.2) for OPC and 71.4% (95% CI 29-96.3) for AC. Sensitivity was highest near diagnosis (100% for OPC within 2 years; 100% for AC within 5 years) and declined with longer lead time. ctHPVDNA read counts increased as the lead time to diagnosis decreased. Eighteen OPC and nine AC cases positive for ctHPVDNA had serial samples. 10 OPC (56%) were positive in all serial samples (median time between first positive ctHPVDNA test to diagnosis: 3.5 years) and 8 (44%) converted from ctHPVDNA negative to positive over follow-up (median time from conversion to diagnosis: 4.7 years, range 0.6 to 8.9 years). 2 AC (22%) were positive in all serial samples (median time between first positive ctHPVDNA test to diagnosis: 6.6 years) and 6 (67%) converted from ctHPVDNA negative to positive over follow-up (median time from conversion to diagnosis: 2.4 years, range 1.2 to 15.4 years). Conclusions: ctHPVDNA, measured using an ultrasensitive WGS assay, HPV-DeepSeek, was strongly associated with future HPV cancer risk. Among E6 Ab positive cases, ctHPVDNA achieved 100% sensitivity in the years immediately preceding diagnosis in both OPC and AC. Serial sampling showed increasing detection rates and read counts as diagnosis neared, supporting ctHPVDNA as a time-dependent marker of emerging disease. Combining E6 Ab testing with ultrasensitive ctHPVDNA detection provides complementary information for blood-based early detection of HPV+ cancers which should be tested in prospective early detection trials.