Spatial immune programs associated with nodal status differ between lymphoid follicular regions and peritumoral tonsillar tissue in HPV-positive oropharyngeal cancer

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Introduction: In human papillomavirus (HPV)-positive oropharyngeal cancer (OPC), lymph node metastasis is common, yet its biological and prognostic implications remain incompletely understood. Less is known about the spatial organization of immune programs within tumor-associated secondary lymphoid structures. Methods: Immune programs were analyzed by spatially distinguishing lymphoid follicular regions (LFRs) from tumor-free peritumoral tonsillar tissues (PTTs) in palatine tonsil-derived HPV-positive OPC. LFRs were analyzed using spatial transcriptomics and PTTs using bulk transcriptomics. Previously generated GeoMx Digital Spatial Profiler and bulk microarray datasets were reanalyzed to investigate compartment-specific immune programs. Immune pathway activity was assessed using single-sample gene set enrichment analysis according to nodal status. Results: Compartment-specific immune programs associated with nodal status were identified in both LFRs and PTTs. In LFRs, nodal-positive cases showed enrichment of stem-like, pre-exhausted, and T-cell activity programs accompanied by inflammatory and interferon-associated signaling. In contrast, nodal-negative PTTs exhibited enrichment of stem-like, pre-exhausted, and T-cell activity programs together with IL2-STAT5 and TGF-β signaling, without overt inflammatory activation. These findings indicate that identical immune pathways exhibit distinct biological associations depending on their spatial localization. Conclusion: Immune programs associated with nodal metastasis in HPV-positive OPC vary according to spatial immune context. Distinguishing LFRs from tumor-free PTTs provides a framework for interpreting immune programs beyond bulk tissue analysis and highlights the importance of spatial immune organization in shaping biological interpretation. Because the spatial transcriptomic analyses were performed in a limited number of patients, these findings should be considered exploratory and require validation in larger independent cohorts.