Genome-Wide Cell-Free DNA Profiling Enables Early Detection of Head and Neck Cancers

INTRODUCTION: Over a billion people globally are impacted by risk factors for head and neck squamous cell carcinoma (HNSCC) - primarily smoking and smokeless tobacco, alcohol and human papillomavirus (HPV) - and may benefit from screening programs if they were available (five-year survival more than doubles for early-stage compared to metastatic disease across tumor sites but currently only 30% of cancers are diagnosed in early stages). We previously developed non-invasive blood tests for multiple cancer types using cell-free DNA (cfDNA) fragmentation profiles (DELFI) and repeat landscapes (ARTEMIS). These tests use cost-effective low-coverage whole-genome sequencing (WGS) to analyze millions of cfDNA fragments whose length, composition, and genome-wide distribution reflect cancer-related genetic and epigenetic changes. Here, we expand these analyses to another non-invasively sampled biofluid, saliva, and demonstrate their use for HNSCC detection. METHODS: We established a prospective single-center collection of blood and saliva samples from individuals with and without HNSCC (n=329). These include 68 healthy individuals without high-risk exposures, 62 healthy individuals who use chewing tobacco, 17 individuals with high-risk leukoplakia, and 182 individuals with cancer, including 39, 37, 27, and 79 with stages I-IV of HNSCC respectively. We extracted cfDNA from blood (all, n=329) and saliva (subset, n=96), performed low-coverage WGS, and split individuals into Discovery (n=244) and Validation (n=85) cohorts. We evaluated genome-wide fragmentation profiles (DELFI) and repeat landscapes (ARTEMIS) in blood and saliva. We cross-validated three ARTEMIS-DELFI classifiers using blood, saliva, or features from both biofluids when available in the Discovery Cohort, and evaluated the locked models in the held-out Validation Cohort. RESULTS: Genome-wide fragmentomes were concordant between blood and saliva samples from the same patients (median correlation coefficient r=0.76, IQR 0.64 - 0.87), and reflected cancer-related alterations including chromosomal changes such as 8q gain and 3p and 9p loss. In the Discovery Cohort, individuals with HNSCC were detected with high performance (AUC=0.87, 95% CI=0.83-0.91 and AUC=0.85, 95% CI=0.76-0.94 for plasma and saliva, respectively), and a combined model using features from both biofluids achieved an AUC of 0.89 (95% CI 0.82-0.97). In the Validation Cohort, at locked thresholds corresponding to 90% specificity, 65%, 71%, and 71% of cancers were detected by the blood, saliva and combined models, respectively. Across cohorts, sensitivity for Stage I tumors was >= 50% for all models. CONCLUSIONS: We demonstrated that cfDNA fragmentation profiles and repeat landscapes can be obtained from both blood and saliva. These approaches may enable accessible, high-performance screening for HNSCC in global high-risk populations. Citation Format: Akshaya Vijaya Annapragada, Shanaya Patel, Sarah Short, Keerti Boyapati, Shashi Koul, Hope Orjuela, Alice Eastman, Aditi Patel, Shreya Lotia, Vaishnavi Patel, Jason Zavras, Adrianna Bartolomucci, Dushyant Mandlik, Kaustubh Patel, Vivek Tanavde, David Sidransky, Mariana Brait, Rob Scharpf, Victor Velculescu, Jillian Phallen. Genome-wide cell-free DNA fragmentomes from blood and saliva enable early detection of head and neck cancers [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 1145.