Abstract LB-C008: Replication stress coupled to cGAS–STING activation defines an innate immune–inflamed transcriptional state in head and neck squamous cell carcinoma that Is enriched for HPV-positive disease

Abstract Background Replication stress is a fundamental driver of genomic instability in cancer, reflecting chronic perturbations in DNA replication caused by oncogene activation, cell-cycle dysregulation, and impaired DNA damage response signaling. These processes shape tumor evolution and therapeutic vulnerability. In head and neck squamous cell carcinoma (HNSCC), the transcriptional consequences of replication stress states remain poorly defined. Methods Primary HNSCC tumors from TCGA-HNSC cohort with matched RNA sequencing, somatic mutation, copy number, and overall survival data were analyzed (N = 499). Replication stress was quantified using a multi-module transcriptomic signature spanning checkpoint signaling, fork stabilization, replication licensing, homologous recombination, Fanconi anemia pathways, and nucleotide metabolism. Innate immune activation was captured using a cGAS–STING axis signature (CGAS, TMEM173, TBK1, IRF3, IRF7, IFNB1, IFNA1, ISG15, MX1, OAS1). Tumors were stratified by median splits for replication stress and STING activity, yielding four groups. Immune activation was assessed using an interferon-γ response signature, cytolytic activity (GZMB, PRF1, GZMA), and PD-L1 expression (CD274). HPV status was obtained from TCGA PanCancer Atlas annotations. Associations were evaluated using Kruskal–Wallis tests, Mann–Whitney U tests, Fisher’s exact tests, and multivariable Cox regression adjusting for age, stage, and tumor mutational burden. Results RS-high/STING-high tumors comprised 25.9% of cases (129/499). Across RS/STING strata, there were highly significant differences in interferon-γ signaling (χ2 = 102.5, p < 2.2 × 10-16), cytolytic activity (χ2 = 73.4, p = 8.0 × 10-16), and PD-L1 expression (χ2 = 76.7, p < 2.2 × 10-16). RS-high/STING-high tumors were significantly enriched for HPV-positive disease (28/124 vs 44/363; OR 2.11, 95% CI 1.20–3.68; p = 0.0078). Importantly, within both HPV-positive and HPV-negative strata, RS-high/STING-high tumors retained significantly higher interferon-γ signaling, cytolytic activity, and PD-L1 expression compared with all other tumors (all p < 0.05), indicating that this immune-inflamed state is not simply a surrogate for viral etiology. In multivariable survival models (N = 474), RS-high/STING-high status was not independently prognostic (HR 1.34, 95% CI 0.96–1.87; p = 0.084),with no evidence of HPV-dependent effect modification (interaction HR 0.38, 95% CI 0.12–1.22; p = 0.11). HPV positivity showed a non-significant trend toward improved survival (HR 0.62, 95% CI 0.35–1.10; p = 0.10). Conclusions In HNSCC, high replication stress coupled with innate immune activation defines a distinct immune-inflamed transcriptional state that is enriched for HPV-positive tumors. These findings suggest that replication stress can demarcate tumors with coordinated interferon and cytolytic programs. Joint quantification of replication stress and innate immune activation may provide a rational biomarker framework for immune-priming strategies and rational combination therapies in HNSCC. Citation Format: Harold Nathan Tan, Michael Wotman, Luana Sousa, Khaled Sanber, Neal Akhave, George Blumenschein, Renata Ferrarotto, Maura Gillison, Faye Johnson, Timothy A. Yap. Replication stress coupled to cGAS–STING activation defines an innate immune–inflamed transcriptional state in head and neck squamous cell carcinoma that Is enriched for HPV-positive disease [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C008.