Abstract PR10: A decade in the making: Screening ctDNA detection in HPV-associated HNSCC
Authors: Hirayama Shun , Zhao Brian Y. , Naegele Saskia , Efthymiou Vasileios , Mendel Julia , Fisch Adam , Lawrence Michael S. , Adalsteinsson Viktor , Iafrate Anthony J. , Das Dipon , Faden Daniel L. 
Published: September 2023

Introduction HPV-associated head and neck squamous cell carcinomas (HPV+HNSCC) release circulating tumor DNA (ctDNA), including ctHPVDNA, into the blood, which is detectable at the time of diagnosis in nearly all patients with HPV+HNSCC using droplet digital PCR (ddPCR)-based approaches. We have previously demonstrated proof of principle that using these approaches, ctHPVDNA is detectable in a minority of patients up to 3 years prior to diagnosis, suggesting HPV+HNSCC may be reliably detected many years prior to clinical detection when using more sensitive approaches. Here, we applied a custom whole genome hybrid-capture-based next generation sequencing (NGS) assay, termed HPVseek, to plasma samples collected years prior to HPV+HNSCC diagnosis to test this hypothesis. Method Plasma samples from 28 patients collected 1-11 years prior to HPV+HNSCC diagnosis were obtained from the Mass General Brigham Biobank, along with 1:1 age and gender matched controls. Cell free DNA was extracted and run on HPVseek and our existing ddPCR assay. Pre-existing cut-points for a positive result were applied. In cases where tissue blocks were available from the time of diagnosis, DNA was extracted and underwent HPVseek for confirmatory molecular fingerprinting of the virus. Result 19 of 28 patients (68%) had detectable ctHPVDNA with lead times ranging from 1.0 to 7.5 years. Of the 11 samples obtained between 1-3 years before diagnosis, 11(100%) were ctHPVDNA positive. All controls were negative. HPV genotype distribution in positive samples was: 16 (n=16), 35 (1), 56 (1), and 59 (1) in blood with genotypes matching clinical genotype testing in 27/28 cases. 11/28 (39%) were positive on ddPCR, all of which were also positive on NGS. Molecular fingerprinting of viral genomes revealed unique viral genomes in each case and matching viral genomes within paired blood and tissue blocks, ruling out contamination and demonstrating the same inciting virus pre-diagnosis to diagnosis. Seven patients had diagnostic and post-diagnostic blood samples available as part of our ongoing prospective ctHPVDNA studies, allowing continuous tracking of cancer status along a time of up to 12 years, providing a global picture of HPV+HNSCC. Conclusion Here we demonstrate that HPV+HNSCC is detectable in the blood as ctDNA &gt;seven years prior to diagnosis, and was detectable in 100% of cases up to three years prior to diagnosis, using an ultrasensitive custom ctDNA NGS assay. Findings were orthogonally validated using molecular fingerprinting in tissue and blood. This study has broad implications for the field of liquid biopsy cancer screening, demonstrating perhaps the longest lead times ever reported for ctDNA detection prior to cancer clinical diagnosis and suggesting HPV+HNSCC could be screened for, detected, and potentially treated years before clinical cancer detection.

Citation Format: Shun Hirayama, Brian Y. Zhao, Saskia Naegele, Vasileios Efthymiou, Julia Mendel, Adam Fisch, Michael S. Lawrence, Viktor Adalsteinsson, Anthony J. Iafrate, Dipon Das, Daniel L. Faden. A decade in the making: Screening ctDNA detection in HPV-associated HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR10.