Abstract A019: Analytical and clinical validation of HPV-SEQ, an NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA

Abstract Human papillomavirus (HPV) infection is the primary causative factor driving the rising incidence of oropharyngeal squamous cell carcinoma (OPSCC). High-risk HPV16 and HPV18 strains are responsible for the majority of HPV related OPSCC cases, with ∼90% and 3-5% respectively. As HPV positivity is associated with improved treatment response and prognosis, correct determination of HPV status is critical for risk stratification and treatment management. While diagnostic tumor biopsy remains a useful tool for HPV profiling, the drawbacks of this approach include pain and discomfort caused by its invasive nature, as well as inability to repeat multiple serial biopsies over time. Noninvasive detection of circulating tumor (ct)HPV-DNA in plasma has emerged as an attractive investigative strategy for early disease diagnosis, real-time response monitoring, and cancer surveillance. However, to date, there is no standard or routine liquid biopsy-based HPV testing for patients with OPSCC. The accuracy of HPV detection in plasma depends on the metrics of the specific method used. Comparison of the most common approaches for detection of ctHPV-DNA shows that next-generation sequencing (NGS)-based assays are superior to that of qPCR and ddPCR, especially in patients with low disease burden. Furthermore, as the majority of current liquid biopsy tests are designed to detect viral E6 and E7 genes, it may comprise a potential challenge for detection of patient-derived ctHPV-DNA among individuals treated with therapeutic vaccines expressing non-oncogenic E6/E7 constructs, actively evaluated in clinical trial settings. To this end, we have developed HPV-SEQ, a novel NGS method designed to achieve maximum sensitivity for HPV16/18 L1 gene in DNA libraries prepared from plasma of OPSCC patients. Here we describe the thorough analytical characterization and clinical validation studies of this lab-developed test conducted and certified under clinical laboratory improvement amendments regulations. HPV-SEQ assay were assessed using both contrived samples and biological specimens by measuring a set of performance attributes including limits of blank, limits of detection, limits of quantitation, accuracy and precision. The assay has demonstrated a robust quantitative detection across a wide ctHPV-DNA range, with low level of background signal (<0.05 copies/sample) in healthy donors and qualitative cut-off of >1.39 copies for both HPV strains (ensuring sufficient differentiation between negative and low positive samples), as well as outstanding sensitivity and specificity, approaching 100%. Given the rapid decrease in sequencing cost and effective integration of sequencing data in clinical care, NGS is likely to displace other HPV detection tests over the next few years. As such, our well-validated, ultra-sensitive, plasma-based HPV profiling method with optimal analytical performance may represent a significant advancement in risk stratification, treatment management, and surveillance for patients with OCSCC and other HPV- driven malignancies. Citation Format: Samaneh Eickelschulte, Anna Starus, David H Murray, Anja K Keyser, Oliver Schauer, Johannes Fredebohm, Frederick Jones, Alexander T Pearson, Everett E Vokes, Ari J Rosenberg, Nishant Agrawal, Evgeny Izumchenko. Analytical and clinical validation of HPV-SEQ, an NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A019.