Assessment of the role of the Tumour microenvironment and the checkpoint TIGIT in Metastatic high grade serous ovarian cancer

Immune checkpoint inhibitors have yielded impressive results in melanoma, renal cell and non-small cell lung carcinoma, however results of large phase 3 trials in epithelial ovarian cancer (EOC) JAVELIN 100 and ImaGYN500 failed to meet their primary endpoints and overall response rates are relatively low (5.9–20.0%)..Attention therefore, has shifted to the identification of novel checkpoints. The T-cell immunoglobulin and ITIM domain (TIGIT) which regulates T cell-mediated and natural killer cell-mediated tumor recognition and thus represents a promising target for new immunotherapy interventions. Using an in silico approach we assessed the prognostic and predictive impact of TIGIT mRNA expression in high grade serous ovarian cancer (HGSOC). We sought to validate our findings in a cohort of eleven patients with matched primary ovarian and metastatic high grade serous ovarian cancer (HGSOC) tissue and used flow cytometry and immunohistochemistry (IHC). Methods: The Cancer Genome Atlas (TCGA) was analysed to ascertain the prognostic impact of TIGIT high mRNA expression. Differentially Expressed genes (DEGs) between patients with high and low TIGIT expression in the TCGA, stratified based on an unsupervised tree analysis, were calculated using EdgeR . Statistical significance was defined as a p value 10e-20 and Log2 fold change >2. Enriched pathways were identified using Gene Set Enrichment Analysis (GSEA) pathways using a False Discovery Rate (FDR) <0.25 as statistically significant. An ARACNe analysis was performed to identify the master regulator genes associated with TIGIT high expression. FACs and IHC for CD4 and CD8 were performed on 26 samples from women with HGSOC. Tissue samples, labelled with fluorescent antibodies against CD3, CD4, CD8, checkpoints TIGIT, PD1 and cytokine IFN-γ also were analysed with a FACS Fortessa (BD Biosciences). Results: An increased expression of mRNA TIGIT is associated with an improved overall survival in HGSOC (p=0.034). 975 DEGs were identified in the TIGIT high group, of which 240 were associated with TIGIT high expression. We employed a GSEA and ssGSEA that identified the pathways predominantly involved in complement activation and the humoral immune response as upregulated. Flow cytometry demonstrated a difference within the TME of the matched ovarian and metastatic samples. A reduction in the frequency of both CD4+ and CD8+ TILs on the metastatic samples was noted (primary median CD8+ TILs i 35.2%(± 10.08) metastatic median T cells 12%(±20.9) (p=0.06).In addition, TIGIT and CD155 were overexpressed on primary tissue in comparison to PD-L1 suggesting TIGIT axis as a potential target for immunotherapy. Within the metastatic tissue, we observed a significant reduction in the frequency of PD-1 and TIGIT expression on both CD4+ TILs (p=0.011, p = 0.007 respectively) and CD8+ TILs (p=0.017, p = 0.007) .Within IHC, the majority of primary ovarian samples (5/7, 71.4%) a higher density of CD8⁺ stromal TILs (sTILs) compared to intraepithelial (iTILs) (median sTILs 381 (SD±335) cells/mm² versus median iTILs 200 (±299) cells/mm² (p=0.8) suggesting an immune excluded environment. In 4 of 6 different metastatic sites, the density of CD8⁺ sTILs was higher than iTILs, demonstrating these tumours were also immune excluded. Within the five omental samples, the majority of the TILs were concentrated in the stroma (median sTILs 1271± 1089 cells/mm² , median iTILs 728±993 cells/mm² ). The two splenic samples were also immune excluded (median sTILs 728±197, median iTILs 369 ± 297cells/mm² ). T Traditionally ovarian cancer research has focused on primary tumour samples, however owing to its late presentation with widespread metastasis, we highlight the importance of studying metastatic samples. Our findings demonstrate an immune-suppressive environment within metastases that make this cancer difficult to tackle with single agent ICIs.