5035 Background: Penile squamous cell carcinoma (PSCC) is a rare, aggressive malignancy with limited treatment options and poor outcomes. Although immunotherapy has shown activity in a subset of patients, the spatial immune mechanisms underlying disease progression and treatment resistance remain unknown. We hypothesized that clinical behavior in PSCC is driven by spatial organization of the tumor microenvironment (TME), rather than immune cell abundance alone. Methods: We conducted the first and largest longitudinal, multi-omics study of PSCC, integrating spatial transcriptomics (NanoString CosMx 6K panel) and single-cell RNA sequencing across 81 patients spanning all disease stages. Spatial profiling was performed on a tissue microarray of 35 primary tumors from 31 patients, capturing >390,000 spatially resolved cells. Patients were stratified by progression status, HPV, stage, and chemotherapy response (n=12) per RECIST 1.1. Spatial architecture was quantified using neighborhood Enrichment Analyses, Spatial cell–cell proximity and Ripley K AUC analyses. Results: Two distinct and clinically divergent TME architectures were identified. Tumors with favorable clinical behavior exhibited organized, antigen-driven immune surveillance. HPV-positive (n=18) and non-progressed tumors (n=17) showed coordinated neighborhoods involving myeloid cells, dendritic cells, T cells, and endothelium. Proliferative tumor programs were spatially proximal to DCs, and effector CD8 T cells maintained close coupling to tumor states, with preserved CD8–DC interactions. In contrast, HPV-negative (n=17), progressed (n=18), and chemotherapy non-responders (n=4) converged on a dysregulated inflammatory state characterized by B cell–macrophage and CAF-associated neighborhoods, EMT–hypoxia tumor programs, and immune–stromal self-clustering. These tumors showed reduced tumor-directed immune engagement and loss of organized immune domains. Notably, chemotherapy non-responders were not immune-depleted but instead exhibited dense innate and antigen-presenting cell infiltrates that were spatially confined to stromal and vascular compartments, with limited tumor-directed immune engagement. Conclusions: This study establishes the first spatially resolved atlas of PSCC, integrating spatial and single-cell multi-omics to uncover distinct immune and stromal architectures linked to progression and therapy resistance. Distinct TME architectures define progression and treatment resistance, supporting spatial biomarkers for patient stratification and therapeutic optimization.
Hatoum et al. (Wed,) studied this question.