A whole-genome sequencing liquid biopsy designed to detect human papillomavirus (HPV) circulating tumor DNA (ctDNA) demonstrated high diagnostic accuracy across a broad range of HPV-associated cancers, according to data presented Thursday at the Association for Molecular Pathology (AMP) 2025 annual meeting in Boston. The assay, called HPV-DeepSeek, showed 98.7% sensitivity and 99.1% specificity in a cohort of more than 400 patients and controls, offering pathologists a potential tool for noninvasive detection years before cancer symptoms occur.
The findings were presented by Adam S. Fisch, MD, PhD, a pathologist at Massachusetts General Hospital and assistant professor of pathology at Harvard Medical School. Dr. Fisch said the assay’s performance was consistent across cancers arising in the oropharynx, anus, cervix, and other anatomic locations.
HPV-DeepSeek is a hybrid-capture, next-generation sequencing assay that targets 43 HPV genotypes, Dr. Fisch explained. Positive calls require at least 10 HPV-specific reads and at least 10% genome coverage—thresholds established through orthogonal comparison with known positives and known negatives. Dr. Fisch noted that these cutoffs “were shown to be a very robust” basis for classification in prior work involving oropharyngeal squamous cell carcinoma.
The current analysis included 224 patients with histologically confirmed HPV-associated squamous cell carcinoma, all sampled before treatment. Reflecting the demographics of oropharyngeal cancer, the HPV-positive group was predominantly male, with a median age of 61. The 212-person control group included individuals without cancer and those with cancers unrelated to HPV.
Most cases originated from the tonsil or oropharynx, due in part to referral patterns at Massachusetts Eye and Ear, the primary source of the patient cohort. Anal, cervical, and less common anatomic sites were also represented. HPV16 was the most frequent genotype detected, followed by HPV33 and HPV35, consistent with established genotype distributions.
Across the full cohort, nearly all true-positive cases exceeded both assay thresholds by substantial margins. The study identified 2 false positives and 3 false negatives. Median genome coverage in true-positive cases approached 100%, reflecting the assay’s depth and breadth of detection.
In addition to HPV identification, HPV-DeepSeek reports several optional features, including PIK3CA mutation status, viral integration patterns, and structural variation within the viral genome. Dr. Fisch presented multiple integration signatures observed in sequencing data, distinguishing among episomal HPV, episomal HPV with internal rearrangements, mixed episomal–integrated patterns, and clonally integrated HPV accompanied by viral gene deletions. Integration into human chromosomes 6, 9, and 17 appeared in several samples.
PIK3CA mutations were observed across multiple anatomic sites using a 4% variant allele frequency cutoff. Dr. Fisch said these additional data types may support future research into how genomic alterations and integration profiles influence tumor development.
Integration findings also aligned with known site-specific tendencies. Oropharyngeal carcinomas, typically associated with episomal HPV, showed fewer integration events. In contrast, all cervical carcinoma cases in the study exhibited integration patterns, echoing long-established observations in the field.
Dr. Fisch said the assay’s ability to reliably detect circulating HPV nucleic acids across diverse cancer types suggests potential utility beyond diagnosis, including treatment monitoring and residual disease assessment. However, he emphasized that the study presented at AMP focused on diagnostic performance in the pretreatment setting.
Although additional validation is required through prospective, longitudinal studies, Dr. Fisch emphasized the advantages of a whole-genome approach. Broader HPV genome coverage, he noted, enables confident determination of HPV presence, mutation status, and structural features not detectable in assays targeting smaller genomic regions.
In closing, Dr. Fisch said HPV-DeepSeek “is able to robustly detect circulating HPV nucleic acid in blood samples from different types of HPV-associated cancers,” while also providing information on PIK3CA mutations, integration events, and structural rearrangements.
