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Patients with ovarian cancer exhibit low response rates to anti-programmed cell death protein-1 (PD-1) based therapies, despite ovarian tumors demonstrating measurable immune responses. Therefore, the aim of the present study was to comparatively examine expression of notable immune co-stimulatory and co-inhibitory receptors in order identify the most abundant receptors that could potentially serve as therapeutic targets to enhance immunotherapy response in high grade serous ovarian cancer (HGSOC). The Cancer Genome Atlas (TCGA) was employed to compare levels of various HGSOC and pan-cancer cohorts. To confirm these findings at the protein level, immunofluorescence of select receptors was performed in 29 HGSOC patient tissue samples. TCGA and Kaplan Meier analysis was employed to determine the association of highly expressed immune receptors with clinical outcomes. TIM-3 and OX40 exhibited the highest expression in HGSOC at both the gene and protein level, with TIM-3 demonstrating highest levels on both CD8

Dual specificity phosphatase 6 (DUSP6) is a protein phosphatase that deactivates extracellular-signal-regulated kinase (ERK). Since the ovarian cancer biomarker human epididymis protein 4 (HE4) interacts with the ERK pathway, we sought to determine the relationship between DUSP6 and HE4 and elucidate DUSP6's role in epithelial ovarian cancer (EOC). Viability assays revealed a significant decrease in cell viability with pharmacological inhibition of DUSP6 using (E/Z)-BCI hydrochloride in ovarian cancer cells treated with carboplatin or paclitaxel, compared to treatment with either agent alone. Quantitative PCR was used to evaluate levels of ERK pathway response genes to BCI in combination with recombinant HE4 (rHE4), carboplatin, and paclitaxel. Expression of EGR1, a promoter of apoptosis, was higher in cells co-treated with BCI and paclitaxel or carboplatin than in cells treated with chemotherapeutic agents alone, while expression of the proto-oncogene c-JUN was decreased with co-treatment. The effect of BCI on the expression of these two genes opposed that of rHE4. Pathway focused quantitative PCR also revealed suppression of ERBB3 in cells co-treated with BCI plus carboplatin or paclitaxel. Finally, expression levels of DUSP6 in EOC tissue were evaluated by immunohistochemistry, revealing significantly increased levels of DUSP6 in serous EOC tissue compared to adjacent normal tissue. A positive correlation between HE4 and DUSP6 levels was determined by Spearman Rank correlation. In conclusion, DUSP6 inhibition sensitizes ovarian cancer cells to chemotherapeutic agents and alters gene expression of ERK response genes, suggesting that DUSP6 could plausibly function as a novel therapeutic target to reduce chemoresistance in EOC.

Epithelial ovarian cancer (EOC) is a highly lethal gynecologic malignancy arising from the fallopian tubes that has a high rate of chemoresistant recurrence and low five-year survival rate. The ovarian cancer biomarker HE4 is known to promote proliferation, metastasis, chemoresistance, and suppression of cytotoxic lymphocytes. In this study, we sought to examine the effects of HE4 on signaling within diverse cell types that compose the tumor microenvironment. HE4 was found to activate STAT3 signaling and promote upregulation of the pro-angiogenic STAT3 target genes IL8 and HIF1A in immune cells, ovarian cancer cells, and endothelial cells. Moreover, HE4 promoted increases in tube formation in an in vitro model of angiogenesis, which was also dependent upon STAT3 signaling. Clinically, HE4 and IL8 levels positively correlated in ovarian cancer patient tissue. Furthermore, HE4 serum levels correlated with microvascular density in EOC tissue and inversely correlated with cytotoxic T cell infiltration, suggesting that HE4 may cause deregulated blood vessel formation and suppress proper T cell trafficking in tumors. Collectively, this study shows for the first time that HE4 has the ability to affect signaling events and gene expression in multiple cell types of the tumor microenvironment, which could contribute to angiogenesis and altered immunogenic responses in ovarian cancer.

Objective: The goal of this study is to comprehensively determine the most clinically relevant immune checkpoint receptor in epithelial ovarian cancer (EOC). Methods: Stage III, Grade III EOC formalin-fixed, paraffin-embedded (FFPE) tumors from ten patients were submitted to Cofactor Genomics to undergo RNA sequencing and machine learning analysis to determine immune cell content and levels of the ten most studied immune escape genes. Patient samples were stratified by high progression-free survival (PFS) of 65 months or greater (n=5) and low PFS of 7 months or less (n=5). All patient tumors submitted were from primary debulking surgery and were naïve to chemotherapy. Immunohistochemistry (IHC) was employed to validate findings. Results: The two immune escape genes ICOS and CTLA-4 were found to be the most predictive biomarkers differentiating short and long PFS. ICOS median transcripts per million (TPM) were 418 and 1,621 in patients with short and long PFS, respectively (p Conclusions: Immune modeling analysis revealed ICOS and CTLA-4 as the most predictive immune biomarkers for PFS in EOC. Interestingly, it was discovered that levels of both activating and inhibitory immune checkpoint receptors correlate to a higher percentage of immune cell populations, indicating overall immune content is important in predicting improved outcomes. Future directions include repeating this assay in a larger patient cohort to validate the predictive threshold determined for ICOS and CTLA-4. Citation Format: Nicole James, Matthew Oliver, Joyce Ou, Jenna Emerson, Katherine Miller, Erin Lips, Ashley Borgstadt, Paul DiSilvestro, Jennifer Ribeiro. Immune modeling analysis identifies ICOS and CTLA-4 as predictive biomarkers in serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B04.

Objective: To determine the effect of HE4 on expression of immune-related genes in CD8+ T cells and explore the role of HE4 in immunosuppression and angiogenesis in epithelial ovarian cancer (EOC). Methods: Tissue HE4 levels and T cell counts were determined by immunohistochemistry (IHC). Recombinant HE4 (rHE4)-mediated gene expression in CD8+ T cells was measured by qPCR array. Cytokine levels were determined using a human cytokine array. Cell viability of ovarian cancer cells co-cultured with pooled peripheral blood mononuclear cells (PBMC) in the presence or absence of rHE4 was determined using MTS cell viability reagent. P-values were determined by unpaired 2-tailed student t test. Activation of STAT3 was evaluated by Western blot, and the interaction of STAT3 and HE4 was queried by immunoprecipitation of STAT3. Finally, HE4 and IL8 levels were measured by IHC in a microarray of human EOC and normal adjacent tissue (NAT), and levels were correlated using Spearman Rank test. Results: Tissue HE4 levels correlated with CD8+ T cell counts in EOC. rHE4 treatment of CD8+ T cells upregulated expression of a panel of immunosuppressive and proangiogenic genes, including VEGF, HIF1A, STAT3, IDO1, FOXP3, and IL8. IL8 was most robustly upregulated (99-fold vs control). HE4 also resulted in alterations in cytokine levels in ovarian cancer cells co-cultured with PBMCs. OVCAR8 and SKOV3 ovarian cancer cell lines were treated with rHE4, and levels of phospho-STAT3—a known regulator of IL8—were measured. HE4 was found to activate STAT3 in a time-dependent manner, which occurred in the absence of a physical interaction between HE4 and STAT3. rHE4 treatment of OVCAR8 and SKOV3 cells co-cultured with PBMCs suppressed PBMC-mediated cytotoxicity. Finally, IL8 and HE4 mean levels were elevated in EOC tissue compared to NAT and significantly correlated (R=0.49332; p=0.00142). Conclusions: HE4 upregulates expression of genes involved in promoting a proangiogenic and immunosuppressive microenvironment in EOC, which may play a role in reduced cytotoxic ability of immune cells. Upregulation of IL8 may occur via HE4 regulation of STAT3 signaling. Future directions include testing the effect of HE4 on endothelial cell angiogenesis and determining if STAT3 and VEGF inhibitors modulate HE4 suppression of immune cell cytotoxicity. Citation Format: Nicole James, Jenna Emerson, Ashley Borgstadt, Lindsey Beffa, Anze Urh, Kyukwang Kim, Rakesh Singh, Richard Moore, Paul DiSilvestro, Jennifer Ribeiro. Genomic analysis of immunosuppressive and proangiogenic genes in recombinant HE4 treated immune cells and implications for T-cell cytotoxicity in ovarian cancer cell co-culture [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B33.