Abstract Background: Epstein-Barr virus (EBV) and human papillomavirus (HPV) are oncogenic viruses linked to specific malignancies, yet their detection in tumors often goes unassessed in clinical practice. EBV-positive gastric cancer patients may benefit from immunotherapy, but viral status is rarely evaluated routinely. Similarly, HPV-associated malignancies can be missed in cases with unknown primary sites. Cell-free DNA (cfDNA) analysis offers a non-invasive approach to identify these clinically actionable viral signatures. Here, we established the analytical performance of EBV and HPV detection in Guardant360 Liquid (Guardant Health, Palo Alto, CA), a comprehensive blood-based molecular profiling assay for patients with advanced cancer. Methods: Guardant360 Liquid targets EBV and 14 high-risk HPV genotypes using a targeted hybridization capture cfDNA assay. We evaluated analytical sensitivity, specificity, accuracy, and precision. Accuracy was assessed using 75 clinical samples with orthogonal viral testing results (43 HPV, 33 EBV) and 43 samples with BD Onclarity HPV Assay (Becton Dickinson, Franklin Lakes, NJ) genotyping results. LoB was determined using 120 replicates from 30 self reported cancer-free donor samples. LoD was established through serial titration of WHO HPV reference standards and in-silico down-sampling of clinical samples and reference materials. Precision was evaluated using clinical samples tested in replicate. Results: For viral detection status, Guardant360 Liquid demonstrated a positive percent agreement (PPA) of 93.48% and a negative percent agreement (NPA) of 96.55% compared to orthogonal methods. For HPV genotyping, PPA was 100% and NPA was 97.39%. The false positive rate was 0% for HPV and 0.83% for EBV in cancer-free donor samples. The class-level LoD was established at 3.36 viral copies per human genome with ≥95% detection rate. Precision testing showed 100% PPA across all replicates at 1-5X LoD. Conclusions: Guardant360 Liquid demonstrates high analytical sensitivity, specificity, accuracy, and reproducibility for detecting EBV and high-risk HPV genotypes in cfDNA. This validated, non-invasive approach enables viral detection in advanced cancer patients, with potential clinical applications including the identification of EBV-positive gastric cancer patients who may benefit from immunotherapy, and optimization of site-specific treatment pathways for HPV-positive cancers. This capability expands the clinical utility of liquid biopsy beyond genomic profiling to include actionable viral biomarker detection. Citation Format: Hao Wang, Lauren Lawrence, Jamie Hutchins, Tingting Jiang. Analytical validation of viral detection in tumor-derived cell-free DNA abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 224.
Wang et al. (Fri,) studied this question.