Abstract A068: Dual multiplexing analysis of the tumor immune microenvironment of rare ovarian granulosa cell tumors identified predictors of recurrence and potential therapeutic targets
Authors: Hilliard Tyvette S. , Juncker-Jensen Anna 
Published: March 2024

Abstract
Ovarian granulosa cell tumors (GCT) are a rare neoplasm of the ovary representing 2-5% of all ovarian cancers. Although most women are diagnosed at an early stage, the slow pattern of progression combined with a high rate of recurrence leads to ~80% of women succumbing to the disease. Prevention of recurrence is of importance in GCT patients. Typically, recurrences occur within 5-10 years making reliable prognostic indicators or predictors of recurrence a challenge. Identification of the tumor immune profile could clarify how these factors facilitate the balance between pro-tumor and anti-tumor immunity and furthermore improve patient survival. As GCTs are rare tumors and tissue availability is very limited, we used a dual multiplexing approach in order to maximize the data output from a total of 14 FFPE tumor samples (6 primary tumors, and 8 recurrent tumors). With this approach we used a single 4μm section to detect 15 markers in an immunofluorescence multiplexing assay, and an adjacent 10μm section to analyze expression of 770 immuno-related cancer genes using the Nanostring nCounter assay PanCancer Immune Panel, with the aim to spatially profile immune cell subsets, angiogenic vessels, as well as markers differentially expressed between primary and recurrent tumors. On the protein level we confirmed previous findings that ovarian GCTs are “cold” tumors, with a very low density of T cell infiltration. Likewise, levels of CTLA-4, PD-1, and PD-L1 were very low in all GCTs. When we analyzed the presence of tumor associated macrophages (TAMs) in the tumor microenvironment (TME) however, we found a 113% increase in TAM density in recurrent tumors compared to primary tumors, corresponding to an increase in the macrophage score on the mRNA level. Furthermore, nearest neighbor analysis of the proximity between M2-type TAMs and angiogenic vessels in the TME, demonstrated M2 TAMS to be much closer associated with the vessels in recurrent tumors compared to primary tumors. We detected a differential gene expression for 66 genes in recurrent tumors compared to primary tumors. IDO1, a key regulator of immune tolerance, showed the highest differential with a significant 14-fold increase in recurrent tumors and thereby represents a therapeutic target within immuno-oncology beyond checkpoint blockade. Additionally, we found SPP1 (the gene coding for osteopontin) to have a 9-fold higher expression in recurrent versus primary GCT tumors, while VEGFA was found to have a 10-fold higher expression. As a pro-inflammatory cytokine osteopontin is thought to both facilitate the recruitment of TAMs and to stimulate angiogenesis by inducing VEGF expression in endothelial cells, suggesting that osteopontin could be a therapeutic target in patients with recurrent GCT. Taken together, these data support the utility of a dual multiplexing approach to immunoprofiling the TME of rare ovarian granulosa cell tumors to identify novel targets for further investigation.
Citation Format: Tyvette S. Hilliard, Anna Juncker-Jensen. Dual multiplexing analysis of the tumor immune microenvironment of rare ovarian granulosa cell tumors identified predictors of recurrence and potential therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A068.