Abstract 7785: Spatial profiling reveals epigenetic and immunotherapy-driven remodeling of the tumor and lymph node microenvironment in HPV-associated HNSCC

Abstract Human papillomavirus (HPV) leads to most oropharynx cancers in the United States, with increasing incidence, significant morbidity from therapy and mortality from metastatic recurrence. HPV+ tumors exhibit a higher cure rate with surgery or chemoradiation but display lower sensitivity to immune checkpoint inhibitors (ICI). Tumor-draining lymph nodes (LNs), crucial for antigen presentation and T-cell priming in the cancer-immunity cycle, remain less well studied with respect to the effects of epigenomic and ICI agents on the LN microenvironment (LNME). HPV-positive tumors exhibit a distinct hypermethylation pattern compared with HPV-negative tumors, promoting immune evasion and providing rationale for an ongoing window-of-opportunity trial testing the epigenomic agent 5-azacytidine (5-AzaC) in combination with the ICI nivolumab. In this trial, patients with resectable HPV+ head and neck squamous cell carcinoma (HNSCC) are randomized 2:2:1 to nivolumab, nivolumab plus 5-AzaC, or 5-AzaC alone prior to standard-of-care resection and pathology-directed adjuvant therapy. We assessed the impact of trial therapies on immune infiltration and cell states in the TME and LNME using quantitative multiplex immunofluorescence on tumor and draining LN tissue donated by a subset of trial participants (n=14 patients) as well as untreated controls. We constructed a tissue microarray (TMA) comprising tumor resection tissue and involved LN tissue sampled with four-fold redundancy. Using qIF panels to assay protein expression of CD3, CD4, CD8, CD20, Ki67, GZMB, we investigated immune cell characteristics and activation. Initial analyses reveal the impacts of 5-AzaC vs nivolumab vs 5-AzaC+Nivo on tumor architecture and cell states in HNSCC tumor tissues. Trial therapies display effects on T cell infiltration, as hypothesized, with combination therapy leading to significant increases in CD3+ T cells, including both CD4+ and CD8+ subsets (p<0.05). We next performed spatial gene expression profiling to identify the impacts of trial therapies on architecture and cell states in tumor and LN. Included in our gene panel were HPV probes to mark cells bearing viral transformation. Initial analyses of 462,376 cells assayed in situ demonstrate the presence of varied immune populations, including macrophages, tumor infiltrating CD8 T cells, CD4 T cells, in tumor and LNs, confirming the utility of spatial approaches to correlate gene expression profiles with clinical drug response parameters. In depth immunologic analyses of patient tissues reveal potent effects of trial therapies on the LNME in HPV-associated HNSCC and suggest that epigenomic alteration may prime the TME for more effective immune responses. Detailed evaluation of the LNME may nominate novel biomarkers of immunotherapy response and identify future druggable targets within this critical anatomic site. Citation Format: Takeshi Ito, Alexander Ladenheim, Hans Zhao, Ernest Hidalgo Cedeno, Benjamin Judson, Saral Mehra, Ansley Roche, Zafar Sayed, Avanti Verma, Natalia Isaeva, Wendell Yarbrough, Aarti Bhatia, Barbara Burtness, Michael Chiorazzi. Spatial profiling reveals epigenetic and immunotherapy-driven remodeling of the tumor and lymph node microenvironment in HPV-associated HNSCC [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 7785.