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Objective: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients., Methods: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration., Results: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates., Conclusion: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies., Competing Interests: Declaration of Competing Interest NSM has received a travel grant from NanoString technologies, unrelated to this work. DDLB has received research support grants from Roche-Genentech, AstraZeneca, and BeiGene (paid to institution); and personal consulting fees from Exo Therapeutics, that are outside the submitted work. MF has participated in Advisory Boards/Consulting from Astra Zeneca, Novartis, GSK, Takeda, Lilly, MSD Eisei and received honoraria/speakers' fees from AstraZeneca, GSK, ACT-Genomics; Research funding to institution: from AstraZeneca, Novartis, Beigene, all unrelated to this work. ADeF has received funding from AstraZeneca, unrelated this work. NS has received honoraria for invited participation in advisory boards hosted by Astra-Zeneca-MSD and Glaxo SmithKline, unrelated to this work. UM had stock ownership in Abcodia until October 2021, and UCL had a license agreement (2011–2021) with Abcodia not related to this work. BHN is a co-founder of Innovakine Therapeutics Inc., unrelated to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Chimeric antigen receptor (CAR)-T cell strategies ideally target a surface antigen that is exclusively and uniformly expressed by tumors; however, no such antigen is known for high-grade serous ovarian carcinoma (HGSC). A potential solution involves combinatorial antigen targeting with AND or OR logic-gating. Therefore, we investigated co-expression of CA125, Mesothelin (MSLN) and Folate Receptor alpha (FOLRA) on individual tumor cells in HGSC., Methods: RNA expression of CA125, MSLN, and FOLR1 was assessed using TCGA (HGSC) and GTEx (healthy tissues) databases. Antigen expression profiles and CD3+, CD8+ and CD20+ tumor-infiltrating lymphocyte (TIL) patterns were assessed in primary and recurrent HGSC by multiplex immunofluorescence and immunohistochemistry., Results: At the transcriptional level, each antigen was overexpressed in >90% of cases; however, MSLN and FOLR1 showed substantial expression in healthy tissues. At the protein level, CA125 was expressed by the highest proportion of cases and tumor cells per case, followed by MSLN and FOLRA. The most promising pairwise combination was CA125 and/or MSLN (OR gate), with 51.9% of cases containing ≥90% of tumor cells expressing one or both antigens. In contrast, only 5.8% of cases contained ≥90% of tumor cells co-expressing CA125 and MSLN (AND gate). Antigen expression patterns showed modest correlations with TIL. Recurrent tumors retained expression of all three antigens and showed increased TIL densities., Conclusions: An OR-gated CAR-T cell strategy against CA125 and MSLN would target the majority of tumor cells in most cases. Antigen expression and T-cell infiltration patterns are favorable for this strategy in primary and recurrent disease., Competing Interests: Declaration of Competing Interest MSB discloses involvement in clinical trials supported by the following companies: Merck, Marker Therapeutics, Transgene, Immune Design, Bristol-Myers Squib, Genentech, and Pharmacyclics. MM discloses involvement with the following companies: Celgene, Genentech, Kite Pharma, Morphosys, Nanostring, and Pfizer. BHN discloses involvement with Virogin Biotech, Akamara Therapeutics, IMV Technologies and Innovakine Therapuetics. None of these relationships influence or are influenced by the results presented in this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: We recently showed that tumors with an immunologically 'cold' phenotype are enriched for expression of stemness-associated genes and PVR/CD155, the ligand of the immunosuppressive molecule TIGIT. To explore the therapeutic implications of this finding, we investigated the relationship between PVR/CD155 expression, tumor-infiltrating lymphocytes (TIL), and prognosis in high-grade serous ovarian cancer (HGSC) and other cancers., Methods: Expression of CD155, TIGIT, PD-1, PD-L1, and other immune markers in HGSC was assessed by high-dimensional flow cytometry, multi-color histological imaging, and/or gene expression profiling. The prognostic significance of PVR/CD155 and CD274/PD-L1 expression was assessed bioinformatically in HGSC and 32 other cancers in The Cancer Genome Atlas., Results: T cells from HGSC frequently co-expressed TIGIT and PD-1, and the ratio of TIGIT to PD-1 expression increased markedly after in vitro expansion with a clinically relevant protocol. CD155 was commonly expressed on malignant epithelium in HGSC and showed a negative or non-significant association with TIL. In contrast, PD-L1 was predominantly expressed by tumor-associated macrophages and positively associated with TIL. These contrasts between CD155 and PD-L1 were seen across HGSC patients, across metastatic sites within individual patients, and even within individual tumor deposits. PVR/CD155 and CD274/PD-L1 exhibited divergent prognostic associations across diverse cancer types in TCGA, including HGSC., Conclusions: CD155 and PD-L1 exhibit contrasting expression patterns, TIL associations and prognostic significance, suggesting they represent non-redundant immunosuppressive mechanisms. The CD155/TIGIT pathway represents a compelling immunotherapeutic target for HGSC and for immunologically cold tumors in general., Competing Interests: Declaration of competing interest No potential conflicts of interest to disclose., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Objective: As a negative regulator of T cells, Programmed Death Ligand 1 (PD-L1) is both an indicator and inhibitor of anti-tumor immune responses, which has led to confusion about its prognostic significance. We investigated the primary source of PD-L1 expression in epithelial ovarian cancer and its relationship to tumor-infiltrating lymphocytes (TIL) and associated gene products., Methods: Tissue microarrays containing high-grade serous carcinomas (HGSC) and endometrioid, clear cell and mucinous ovarian cancers from optimally debulked patients were assessed by immunohistochemistry for expression of PD-L1 and other markers (CD68, CD3, CD8, PD-1, CD103, FoxP3 and CD25). The Cancer Genome Atlas was interrogated for associations between PD-L1 expression and immune-related transcriptional and genomic features of HGSC., Results: PD-L1 was primarily expressed by tumor-associated CD68(+) macrophages rather than tumor cells. PD-L1(+) cells frequently co-localized with CD8, CD4 and PD-1(+) TIL, CD25(+)FoxP3(+) Tregs, and other TIL subsets. PD-L1(+) cells were prognostically favorable in HGSC. Moreover, the presence of both PD-L1(+) cells and CD8 TIL was associated with better prognosis than CD8 TIL alone. PD-L1 gene expression was independent of BRCA status. At the transcriptional level, PD-L1 was associated with both cytolytic (granzyme B, T-bet and IFN-γ) and suppressive (PD-1, CTLA-4, LAG3 and IDO-1) gene products., Conclusions: PD-L1 is primarily expressed by macrophages in ovarian cancer and is strongly associated with both cytolytic and regulatory TIL subsets, resulting in a net positive association with survival. Tumors containing PD-L1(+) macrophages appear caught in an immunological stalemate that may require multi-pronged immunotherapy to alleviate., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Introduction: Tumor-infiltrating CD8(+) T cells are strongly associated with survival in high-grade serous ovarian cancer, but their functional phenotype remains poorly defined. The mucosal integrin CD103 (alpha(E)/beta(7)) facilitates the infiltration of T cells into epithelial tissues, including gut and lung mucosa, solid organ allografts, and various epithelial cancers. We reasoned that CD103 might also be expressed by tumor-reactive T cells in ovarian cancer., Methods: Flow cytometry was used to assess the frequency and phenotype of CD103-expressing T cells in primary ascites fluid from 13 patients with high-grade serous ovarian cancer and 2 patients with recurrent disease., Results: We report that a subset of patients with advanced serous ovarian cancer have profoundly elevated frequencies of CD103-expressing CD8(+) cells in ascites (between 20% and 70% of CD8(+) cells in ascites were CD103(+)) and that CD103 expression correlated with levels of TGF-beta in ascitic fluid. Conversely, CD103 was not expressed on CD4(+) cells, even in those patients with very high frequencies of CD8(+)CD103(+) cells. CD8(+)CD103(+) cells were antigen-experienced (CD45RA(-)CD45RO(+)CD62L(lo)CCR7(-)) and of an intermediate (EM2) effector memory phenotype (CD27(+)CD28(-)). TCR repertoire analysis indicated significant skewing between CD8(+)CD103(-) and CD8(+)CD103(+) T cell subsets, suggesting the two populations contain distinct antigenic specificities. Lastly, HLA pentamer analysis revealed that one patient in the cohort harbored a high frequency of CD8(+) T cells in ascites that were specific for the tumor antigen NY-ESO-1, and that approximately 75% of these NY-ESO-1 specific CD8(+) T cells were CD103(+)., Conclusions: CD103(+) may be a marker of activated and tumor-reactive CD8(+) T cells in high-grade serous ovarian cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)