Analytical and clinical performance validation of HPV-SEQ, a novel NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA

作者：Samaneh Eickelschulte, Anna Starus, David H. Murray, Kirsten A. Keyser, Oliver Schauer, Simon Guellert, Emily L. Gramiccioni, Johannes Fredebohm, Frederick S. Jones, Harry Quon, Carmen Kut, Yilin Cao, Carole Fakhry, Tanguy Y. Seiwert, Yuxuan Miao, Mark W. Lingen, Alexander T. Pearson, Everett E. Vokes, Ari J. Rosenberg, Nishant Agrawal, Evgeny Izumchenko · 发表于：Oral Oncology · 年份：2025 · DOI：10.1016/j.oraloncology.2025.107445 · 被引用次数：8 · 研究领域：Head and Neck Cancer Studies、Cancer Genomics and Diagnostics、Cervical Cancer and HPV Research

BACKGROUND: Human papillomavirus (HPV) is the primary causative driver of oropharyngeal squamous cell carcinoma (OPSCC). Accurate detection of HPV-DNA is critical for risk stratification and management of OPSCC. However, assays designed to detect HPV in primary tumors do not allow monitoring of HPV-DNA over time, whereas commercially available droplet digital PCR-based methods for assessment of circulating cell free (cf)HPV-DNA in plasma remain suboptimal, hindering adaptation into clinical practice. We have developed HPV-SEQ, a novel next-generation-sequencing (NGS) based method for detection and quantification of HPV16/18 DNA in plasma of patients with OPSCC. METHODS: The assay uses primers targeting the L1 gene of HPV16 and HPV18 viral genomes and strain specific calibrators at a defined concentration to determine the ratio of native HPV to a known standard, enabling accurate reporting of patient-derived HPV16/18 viral load in a sample. This study was conducted using two different patient populations in addition to healthy donors and contrived material. All experiments were performed to fulfill several applicable analytical, performance and validation guidelines. RESULTS: A thorough analytical characterization and clinical validation of this platform demonstrates that HPV-SEQ detects cfHPV-DNA with exceptional limit of quantification and high precision, providing a foundation for integrating this platform into clinical settings. CONCLUSIONS: This ultra-sensitive HPV profil...