Abstract Introduction: Characterizing the immune contexture within the tumor microenvironment (TME) can provide valuable biological insights that may impact treatment regimens and improve clinical outcomes. The advent of neoadjuvant chemotherapy has provided a unique opportunity to study matched, pre- and post- treatment sample pairs, but these samples are often obtained from different metastatic sites. Innate differences in the immune profiles across metastatic sites could therefore be a confounding factor when attempting to determine treatment-related effects. Furthermore, immune contexture can also vary by region within the TME, so results could vary depending on the regions of interest (ROIs) selected for analysis. Methods: 36 matched, advanced primary ovarian cancer samples were obtained pre- and post- treatment with neoadjuvant chemotherapy aimed at reducing tumor burden. The first biopsy was taken at the time of initial diagnosis, whereas the second biopsy was collected after 3-4 rounds of carboplatin paclitaxel chemotherapy. Each slide was processed using a 26-plex multiplex immunohistochemistry (mIHC) assay, which interrogates immune cell densities and functional states for a broad range of immune cell populations, including CD8+ T cells, Tregs, TH1s, B cells, dendritic cells (DCs), granulocytes, macrophages, and monocytes. Tumor and stroma ROIs were selected by a board-certified pathologist using H&E slides immediately adjacent to the mIHC samples, and cell densities were calculated separately for each of the two tissue compartments. Results: When comparing pre-treatment samples from the omentum (n = 8) with post-treatment samples from the omentum (n = 14), all of the statistically significant changes were found within the tumor compartment, with higher densities in the post-treatment samples for CD8+ T cells (p = 0.009), Tregs (p = 0.012), DCs (p = 0.04), and monocytes (p = 0.027). In contrast, when comparing post-treatment samples from the omentum (n = 14) and the ovary (n = 14), all of the significant differences were found within the stroma compartment, with higher densities in the omentum for CD8+ T cells (p = 0.002), Tregs (p = 0.043), TH1s (p = 0.003), and macrophages (p = 0.048). Conclusions: These results demonstrate that the site of metastasis can be a significant confounding factor when making statistical comparisons between variables such as timepoint and that differences in immune cell density before and after treatment may be more apparent in the tumor, while differences between tissue types may be more apparent in the stroma. Further research into the immune profiles of different metastatic sites is warranted, but our findings demonstrate the importance of considering both tissue type and ROI selection strategy when designing a study with the intent to detect differences between longitudinal patient samples. Citation Format: Benjamin Tate, Elias Pavlatos, Teresa Kim, Kiaya Wilson, Erik Cid, Jian Rao, Gottfried Konecny, Joanna Pucilowska. Differences in immune contexture across metastatic sites and tissue compartments in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5532.