Human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) represents a distinct clinical and molecular entity, characterized by its favorable prognosis and distinct immunological profiles compared to HPV independent OPSCC tumors. Elucidating the transcriptional and immune alterations underlying this association is essential for identifying biomarkers that may improve diagnostic accuracy, therapeutic decision-making, and patient stratification. In this study, we sought to identify a robust, differentially expressed master regulator gene and validate its utility as a biomarker for HPV-driven OPSCC in both tumor tissue and peripheral blood.
Methods:
An integrative transcriptomic analysis was conducted using publicly available RNA-seq datasets from HPV-associated and non-associated oropharyngeal cancer samples.  Differentially expressed genes (DEGs) were identified with DESeq2, and immune cell composition was estimated through CIBERSORT and validated using single-cell RNA sequencing (scRNA-seq) data and SingleR annotation. Functional enrichment analyses were conducted to identify biological pathways related to viral response and immune regulation. Experimental validation was performed in two complementary cohorts: (1) RNA from an independent set of FFPE HPV+ and HPV- OPSCC tumor tissues (n=20) via RT-qPCR, and (2) peripheral blood buffy coat samples from 10 HPV+ OPSCC and 10 HPV- oral cavity SCC patients. The latter was explored to assess its potential as a non-invasive, systemic biomarker of the HPV-associated tumor immune microenvironment. ASCL4 expression was assessed in both cohorts to confirm its tumor-specific upregulation and explore its detectability in blood. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control, and relative gene expression was calculated using the 2^−ΔΔCt method.
Results:
Transcriptomic and immune deconvolution analyses of HPV+ OPSCC revealed a distinct molecular profile characterized by enriched immune activation pathways and significantly increased tumor infiltration of B and NK cells. From this integrated analysis, the transcription factor ASCL4 emerged as a top differentially expressed gene. Interrogation of public cohorts confirmed that high ASCL4 expression was associated with improved overall survival, aligning with the favorable prognosis of HPV-driven disease. Notably, ASCL4 was also significantly overexpressed in the peripheral blood buffy coat of HPV+ OPSCC patients compared to HPV- controls (p=0.0019), supporting its potential as a novel, non-invasive systemic biomarker.
Conclusions:
We report, for the first time, the consistent upregulation of the transcription factor ASCL4 in HPV+ OPSCC. Our integrative profiling validates ASCL4 as a tumor-specific biomarker linked to a favorable prognosis and a robust immunogenic microenvironment. Its detection in peripheral blood warrants investigation as a non-invasive monitoring tool. These findings position ASCL4 as a compelling candidate for clinical translation in HPV-driven head and neck cancer, with potential applications in diagnosis and treatment stratification.