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Introduction. Circadian rhythm is involved in multiple biological activities and implicated in cancer development. However, the role of circadian rhythm in head and neck squamous cell carcinoma (HNSCC) has not been fully interpreted yet. Herein, the present study set out to explore the significance of circadian regulator genes (CRGs) in HNSCC. Materials and Methods. The molecular landscape and clinical significance of 13 CRGs in HNSCC were explored based on The Cancer Genome Atlas (TCGA). The biological functions of PER3, a key CRG, were validated by cellular experiments. The correlation of CRGs with microenvironment, pathway activities, and prognosis was determined by bioinformatic algorithms. A novel circadian score was introduced to evaluate the circadian modification pattern of each patient and further validated in an independent cohort from the Gene Expression Omnibus (GEO) dataset. Results. CRGs presented high heterogeneity in HNSCC at both genomic and transcriptomic levels. Specifically, PER3 indicated a better prognosis and inhibited HNSCC cell proliferation. Moreover, HNSCC tissues displayed three circadian regulator patterns with distinct clinical outcomes, transcriptomic characteristics, and microenvironment features. Circadian score was an independent risk factor and exhibited excellent predictive efficiency in both the training cohort from the TCGA database and the validation cohort from the GEO database. Conclusions. CRGs played an indispensable role in HNSCC development. An in-depth exploration of circadian rhythm would improve the understanding of HNSCC carcinogenesis and confer novel insights for future clinical practices.

Background: HPV-associated oropharyngeal cancer (HPV+OPSCC) is the most common HPV-associated cancer in the United States yet unlike cervical cancer lacks a screening test. HPV+OPSCCs are presumed to start developing 10-15 years prior to clinical diagnosis. Circulating tumor HPV DNA (ctHPVDNA) is a sensitive and specific biomarker for HPV+OPSCC. Taken together, blood-based screening for HPV+OPSCC may be feasible years prior to diagnosis., Methods: We developed an HPV whole genome sequencing assay, HPV-DeepSeek, with 99% sensitivity and specificity at clinical diagnosis. 28 plasma samples from HPV+OPSCC patients collected 1.3-10.8 years prior to diagnosis along with 1:1 age and gender-matched controls were run on HPV-DeepSeek and an HPV serology assay., Results: 22/28 (79%) of cases and 0/28 controls screened positive for HPV+OPSCC with 100% detection within four years of diagnosis and a maximum lead time of 7.8 years. We next applied a machine learning model classifying 27/28 cases (96%) with 100% detection within 10 years. Plasma-based PIK3CA gene mutations, viral genome integration events and HPV serology were used to orthogonally validate cancer detection with 68% (19/28) of the cohort having multiple cancer signals detected. Molecular fingerprinting of HPV genomes was performed across patients demonstrating that each viral genome was unique, ruling out contamination. In patients with tumor blocks from diagnosis (15/28), molecular fingerprinting was performed within patients confirming the same viral genome across time., Conclusions: We demonstrate accurate blood-based detection of HPV-associated cancers with lead times up to 10 years before clinical cancer diagnosis and in doing so, highlight the enormous potential of ctDNA-based cancer screening., Competing Interests: Daniel L. Faden has received research funding or in-kind funding from Bristol-Myers Squibb, Calico, Predicine, BostonGene and Neogenomics. He has received consulting fees from Merck, Noetic, Chrysalis Biomedical Advisors, Arcadia and Focus. None of these sources relate to the work in this manuscritpt.