Abstract 2082: Multiplex immunofluorescence reveals compartment-specific immune cell correlations associated with age and immunotherapy response in ovarian carcinoma.

Abstract Background Ovarian carcinoma, an aggressive malignancy typically diagnosed at advanced stages, has shown limited response to immune checkpoint inhibitors. Increasing evidence suggests that age-related immune alterations and spatial heterogeneity within the tumor microenvironment influence immunotherapy outcomes. This study investigates whether immune cell organization across tissue compartments correlates with clinical response to immunotherapy. Method Twenty patients treated with immunotherapy were identified. FFPE tumor blocks were retrieved, and slides were stained using a multiplex immunofluorescence panel (DAPI, PD-L1, CD4, CD8, CD20, FoxP3, PanCK). Regions of interest were annotated by a pathologist and analyzed using supervised learning in inForm and PhenoChart. Immune cell phenotypes were quantified across tissue compartments, and correlation analyses were performed using RStudio, Excel, and GraphPad Prism. Result Older patients demonstrated significantly improved immunotherapy outcomes, including longer progression-free survival and duration of effective immunotherapy, confirming an age-associated advantage in therapeutic response. Multiplex immunofluorescence revealed distinct immune correlation networks between responders and non-responders, driven by tissue compartmentalization. Among immunotherapy responders, stromal regions showed a positive correlation between FoxP3+ and PanCK+ tumor cells, whereas tumoral regions exhibited a negative correlation between CD8+ cells and PanCK+ cells. Whole-tissue analysis revealed a strong correlation between FoxP3+ and CD8+ cells in responders. In contrast, non-responders displayed a different pattern of immune cell organization, characterized by strong correlations between CD4+ and CD8+ T cells in the stroma and between CD20+ B cells and PanCK+ cells in the tumor compartment. Spatial proximity heatmaps demonstrated that responders had higher local clustering of CD8+, CD4+, and CD20+ cells across multiple radii within both tumor and stromal regions, while non-responders exhibited limited spatial clustering consistent with “immunologically cold” tumors. Conclusion This study identifies distinct, compartment-specific immune cell relationships associated with immunotherapy response in ovarian carcinoma highlights age as a significant modifier of immune architecture. Responders, predominantly older patients, exhibited coordinated stromal FoxP3+ interactions and tumoral CD8+ engagement, whereas non-responders, predominantly younger patients, showed stromal CD4+/CD8+ clustering and tumoral CD20+ associations characteristic of cold immune phenotypes. These findings suggest that immune cell correlations, rather than absolute densities, may serve as informative spatial biomarkers for predicting immunotherapy response. Citation Format: Kathy Kwock, Okan Gültekin, Hanim I. Ozkizilkaya, Lynda D. Roman, William Dean Wallace, Tirzah Petta, Joseph W. Carlson. Multiplex immunofluorescence reveals compartment-specific immune cell correlations associated with age and immunotherapy response in ovarian carcinoma 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 2082.