Immune Response In Ovarian Cancer Research Articles

Construction of a PANoptosis-related Prognostic Signature for Predicting Prognosis, Tumor Microenvironment, and Immune Response in Ovarian Cancer.

The PANoptosis pathway is a recently identified mechanism of cellular death that involves the interaction and synchronization among cellular pyroptosis, apoptosis, and necrosis. More and more evidence suggests that PANoptosis is involved in the development and treatment of cancer. However, a comprehensive understanding of the influence of PANoptosis genes on prognostic value, tumor microenvironment characteristics, and therapeutic outcomes in patients with ovarian cancer (OC) remains incomplete. The present work was designed to devise a PANoptosis signature for OC prognosis and explore its potential molecular function. For this study, we obtained RNA sequencing and clinical data for ovarian cancer from the Cancer Genome Atlas (TCGA) and the GSE32062 cohort. Somatic variants of PANoptosis-related genes (PRGs) in OC were analyzed using GSCA. TCGA-OC and GSE32062 were used to construct training and validation cohorts for the model. Differential expression and correlation analyses were performed following the screening of genes with prognostic ability using univariate Cox analysis. Least Absolute Shrinkage and Selection Operator (LASSO) regression was performed to construct PRG signature based on genes that were differentially expressed and correlated with prognosis. CIBERSORT and ESTIMATE were used to analyze the relationship between the PRGs signature and immune infiltration. TIDE was used to analyze the relationship between the PRG signature and immune checkpoint genes. OncoPredict was used to analyze the relationship between the PRG signature and the drug sensitivity. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of PRGs in OC. The PRG signature was constructed using three prognostic genes (AIM2, APAF1, and ZBP1) in both TCGA-OC. The results showed that the PRGs signature had an AUC of 0.521, 0.546, and 0.598 in TCGA-OC and 0.620, 0.586, and 0.579 in GSE32062 to predict to predict OS at 1-, 3-, and 5-year intervals. Furthermore, a higher PRG signature risk score was significantly associated with shorter OS (HR = 1.693, 95% CI: 1.303 - 2.202, p = 8.34 × 10^-5 in TCGA-OC and HR = 1.63, 95% CI: 1.13 - 2.35, p = 0.009 in GSE32062). The risk score was identified as the independent prognostic factor for OC. Patients categorized according to their risk score exhibited notable variations in immune status, response to immunotherapy, and sensitivity to drugs. AIM2, APAF1, and ZBP1 were significantly aberrantly expressed in OC cell lines. The PRG signature has the potential to serve as a prognostic predictor for OC and to provide new insights into OC treatment.

Abstract B006: P53 transcriptional regulation of transposable elements and the downstream immune response in ovarian cancer

Abstract B006: P53 transcriptional regulation of transposable elements and the downstream immune response in ovarian cancer

NLRC5 overexpression in ovarian tumors remodels the tumor microenvironment and increases T-cell reactivity toward autologous tumor-associated antigens.

Epithelial ovarian cancer (OC) stands as one of the deadliest gynecologic malignancies, urgently necessitating novel therapeutic strategies. Approximately 60% of ovarian tumors exhibit reduced expression of major histocompatibility complex class I (MHC I), intensifying immune evasion mechanisms and rendering immunotherapies ineffective. NOD-like receptor CARD domain containing 5 (NLRC5) transcriptionally regulates MHC I genes and many antigen presentation machinery components. We therefore explored the therapeutic potential of NLRC5 in OC. We generated OC cells overexpressing NLRC5 to rescue MHC I expression and antigen presentation and then assessed their capability to respond to PD-L1 blockade and an infected cell vaccine. Analysis of microarray datasets revealed a correlation between elevated NLRC5 expression and extended survival in OC patients; however, NLRC5 was scarcely detected in the OC tumor microenvironment. OC cells overexpressing NLRC5 exhibited slower tumor growth and resulted in higher recruitment of leukocytes in the TME with lower CD4/CD8 T-cell ratios and increased activation of T cells. Immune cells from peripheral blood, spleen, and ascites from these mice displayed heightened activation and interferon-gamma production when exposed to autologous tumor-associated antigens. Finally, as a proof of concept, NLRC5 overexpression within an infected cell vaccine platform enhanced responses and prolonged survival in comparison with control groups when challenged with parental tumors. These findings provide a compelling rationale for utilizing NLRC5 overexpression in "cold" tumor models to enhance tumor susceptibility to T-cell recognition and elimination by boosting the presentation of endogenous tumor antigens. This approach holds promise for improving antitumoral immune responses in OC.

Contribution of m5C RNA Modification-Related Genes to Prognosis and Immunotherapy Prediction in Patients with Ovarian Cancer.

5-Methylcytosine (m5C) RNA modification is closely implicated in the occurrence of a variety of cancers. Here, we established a novel prognostic signature for ovarian cancer (OC) patients based on m5C RNA modification-related genes and explored the correlation between these genes with the tumor immune microenvironment. Methylated-RNA immunoprecipitation sequencing helped us to identify candidate genes related to m5C RNA modification at first. Based on TCGA database, we screened the differentially expressed candidate genes related to the prognosis and constructed a prognostic model using LASSO Cox regression analyses. Notably, the accuracy of the model was evaluated by Kaplan-Meier analysis and receiver operator characteristic curves. Independent prognostic risk factors were investigated by Cox proportional hazard model. Furthermore, we also analyzed the biological functions and pathways involved in the signature. Finally, the immune response of the model was visualized in great detail. Totally, 2,493 candidate genes proved to be involved in m5C modification of RNA for OC. We developed a signature with prognostic value consisting of six m5C RNA modification-related genes. Specially, samples have been split into two cohorts with low- and high-risk scores according to the model, in which the low-risk OC patients exhibited dramatically better overall survival time than those with high-risk scores. Besides, not only was this model a prognostic factor independent of other clinical characteristics but it predicted the intensity of the immune response in OC. Significantly, the accuracy and availability of the signature were verified by ICGC database. Our study bridged the gap between m5C RNA modification and the prognosis of OC and was expected to provide an effective breakthrough for immunotherapy in OC patients.

Abstract 5880: The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer

Abstract Introduction: Sterol-O acyltransferase 1 (SOAT1) functions by converting cholesterol and acyl-CoA into cholesterol ester and coA-SH. High SOAT1 expression with its mediated tumorigenesis has been shown in multiple cancers. Studies showed that SOAT1 inhibition by its inhibitors augmented the outcome of immunotherapies, in terms of the immune checkpoint blockades, cancer vaccines, and CAR-T cells, by boosting the function of T cells. However, in order to have a better understanding of the role played by SOAT1 in the tumor microenvironment of ovarian cancer (OC) and optimize strategies of combination immunotherapy, it’s necessary to detect the role of SOAT1 in OC cells on T cell-mediated immune response. Methods: SOAT1 knockdown was manipulated by siRNAs transfection. SOAT1 was inhibited by avasimibe and OC cells were treated in different doses of avasimibe. qPCR was performed to detect gene expressions in ovarian cancer cells. T cells, isolated and activated from healthy donors, were cocultured with SOAT1-silenced and avasimibe pre-treated OC cells and their tumor-conditioned medium (TCM). Flow cytometry analysis was used to measure cytotoxic secretory molecules of human T cells, like IFN-r, TNF-a, and Granzyme B. Results: The downregulation of intracellular cytotoxic cytokines was shown in the T cells cocultured with SOAT1-silenced and avasimibe-pretreated OC cells or treated by their TCM, thereby impairing the cytotoxic capacity of T cells, compared to the corresponding control group. qPCR results showed that several immunosuppressive cytokines, like IL-6, IL-8, TGF- were upregulated in the SOAT1-silenced or avasimibe-treated OC cells. Conclusion: SOAT1 inhibition in ovarian tumor cells impairs CD8+ T cell cytotoxic function, to some extent, via the upregulation of immunosuppressive-related cytokines. Citation Format: Jiangnan He, Michelle K.Y. Siu, Runying Long, Ruiqian Zhang, Mingo M.H. Yung, Hextan Y. S. Ngan, Karen K.L. Chan. The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5880.

PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling

BackgroundIncreased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian...

NR2F1 Regulates TGF-β1-Mediated Epithelial-Mesenchymal Transition Affecting Platinum Sensitivity and Immune Response in Ovarian Cancer.

Simple SummaryIn ovarian cancer (OC), platinum-based therapy remains the front-line therapy, but drug resistance is common. In the current study, we analyzed two Gene Expression Omnibus (GEO) datasets to identify the responsible genes involved in the mechanism of platinum resistance. Thirteen co-upregulated genes and one co-downregulated gene were obtained. Among them, NR2F1 revealed the highest correlation with a poor prognosis. Mechanism research revealed that NR2F1 promotes epithelial-mesenchymal transition (EMT) through TGFβ-1 signaling, participating in platinum resistance. We also found that NR2F1 was positively correlated with the infiltration of immunosuppressive cancer-associated fibroblasts (CAFs). Higher expression of NR2F1 was correlated with a poorer response to immune check blockades including anti-PD-L1. In the future, by analyzing the expression status of NR2F1 and the effect of platinum and immunotherapy in the clinical setting, it is expected that NR2F1 will be established as an effective drug selection marker, guiding treatment selection in OC patients.The mechanism underlying platinum resistance in ovarian cancer (OC) remains unclear. We used bioinformatic analyses to screen differentially expressed genes responsible for platinum resistance and explore NR2F1′s correlation with prognostic implication and OC staging. Moreover, Gene-set enrichment analysis (GSEA) and Gene Ontology (GO) analyses were used for pathway analysis. Epithelial-mesenchymal transition (EMT) properties, invasion, and migration capacities were analyzed by biochemical methods. The association between NR2F1 and cancer-associated fibroblast (CAF) infiltration and immunotherapeutic responses were also researched. A total of 13 co-upregulated genes and one co-downregulated gene were obtained. Among them, NR2F1 revealed the highest correlation with a poor prognosis and positively correlated with OC staging. GSEA and GO analysis suggested the induction of EMT via TGFβ-1 might be a possible mechanism that NR2F1 participates in resistance. In vitro experiments showed that NR2F1 knockdown did not affect cell proliferation, but suppressed cell invasion and migration with or without cisplatin treatment through the EMT pathway. We also found that NR2F1 could regulate TGF-β1 signaling, and treating with TGF-β1 could reverse these effects. Additionally, NR2F1 was predominantly associated with immunosuppressive CAF infiltration, which might cause a poor response to immune check blockades. In conclusion, NR2F1 regulates TGF-β1-mediated EMT affecting platinum sensitivity and immune response in OC patients.

Abstract 1280: Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) are becoming the standard of care for ovarian cancer. The greatest benefit has been observed in patients who have mutations in BRCA1/2 or related genes in the homologous recombination (HR) DNA repair pathway. Nonetheless, responses to PARPi were not durable, indicating that strategies to prevent recurrence and overcome resistance, such as combinations with immune checkpoint inhibitors (ICIs), are of high priority. However, most studies on PARPi have focused on the tumor itself and synthetic lethality. The immunological effect, particularly the effect on adaptive immune response, has been overlooked. PARP is expressed in almost all cells, and it regulates the transcription of various genes through poly(ADP-ribosyl)ation. Therefore, we hypothesized that PARPi affect the composition and functions of T cells in the maintenance therapy setting, and consequently reconstitute the anti-tumor immune response in ovarian cancer. Here, we collected serial peripheral blood mononuclear cells (PBMCs) from PARPi-treated patients and analyzed their dynamic immunologic changes using muticolor flow cytometry. The PBMCs were collected during PARPi maintenance therapy, and the time points included pre-treatment as well as 1 month and 3 months after the initiation of treatment. Olaparib or niraparib was used as maintenance therapy. First, the percentages of CD4, CD8, and FoxP3+regulatory T cells (Treg cells) were compared at each time point and by PARPi type. Interestingly, the proportion of Treg cells increased at 1 and 3 months after initiation, especially in the niraparib group. However, there were no significant changes in the proportion of CD4 and CD8 T cells. Second, we analyzed immune checkpoints and properties in each T cell population. We found that the PD-1 expression on Treg cells decreased after 1 and 3 months. Although the percentage of Treg cells increased, proliferative Ki-67+Treg cells decreased after 1 and 3 months, especially in the niraparib group. Third, intracellular staining assay was performed to measure functional changes of T cells during PARPi treatment. IFN-γ and/or TNF-α producing CD8 and CD4 T cells decreased after 1 month compared to pre-treatment. Finally, proliferative Ki-67+CD8 T cells and PD-1+CD8 T cells decreased after 72 hours of in vitro olaparib treatment compared to the control group. In conclusion, long-term PAPRi treatment may affect Treg cells and CD8 T cells. However, strategies to overcome negative effects of PARPi and their detailed mechanisms need to be further investigated. Additional data in this regard will be presented at the meeting. Citation Format: Junsik Park, Miran Lee, Yup Kim, Yoo-Na Kim, Yong-Jae Lee, Jung-Yun Lee. Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1280.

Immune Subtypes Characterization Identifies Clinical Prognosis, Tumor Microenvironment Infiltration, and Immune Response in Ovarian Cancer.

Objective: Because of the modest immunotherapeutic response among ovarian carcinoma (OC) patients, it is significant to evaluate antitumor immune response and develop more effective precision immunotherapeutic regimens. Here, this study aimed to determine diverse immune subtypes of OC. Methods: This study curated the expression profiles of prognostic immunologically relevant genes and conducted consensus clustering analyses for determining immune subtypes among OC patients in TCGA cohort. With Boruta algorithm, characteristic genes were screened for conducting an immune scoring system through principal component analysis algorithm. The single-sample gene set enrichment analysis and ESTIAMTE methods were adopted for quantifying the immune infiltrates and responses to chemotherapeutic agents were estimated with pRRophetic algorithm. Two immunotherapeutic cohorts were used for investigating the efficacy of immune score in predicting therapeutic benefits. Results: Two immune subtypes were conducted among 377 OC patients. Immune subtype 2 was characterized by worse clinical prognosis, more frequent genetic variations and mutations, enhanced immune infiltrates, and increased expression of MHC molecules and programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1). In total, 30 prognosis-relevant characteristic immune subtype–derived genes were identified for constructing the immune score of OC patients. High immune score was linked with more dismal prognosis, decreased immune infiltrations, and expression of MHC molecules. High immune score presented favorable sensitivity to doxorubicin and vinorelbine and reduced sensitivity to cisplatin. In addition, immune score possessed the potential in predicting benefits from anti–PD-1/PD-L1 therapy. Conclusion: Collectively, our findings propose two complex and diverse immune subtypes of OC. Quantitative assessment of immune subtypes in individual patients strengthens the understanding of tumor microenvironment features and promotes more effective immunotherapeutic regimens.

Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

BackgroundImmune checkpoint blockades (ICBs) therapy showed limited efficacy in ovarian cancer management. Increasing evidence indicated that conventional and targeted therapies could affect tumor-associated immune responses and increase the effectiveness of immunotherapy. However, the effects of Niraparib, one of the poly (ADP) ribose polymerase (PARP) inhibitors, on the immune response remains unclear. Delineating the crosstalk between cytotoxic anticancer agents and cancer-associated immunity may lead to more efficient combinatorial strategies.MethodsProgrammed death ligand 1 (PD-L1) expression in human ovarian cancer cells after PARP inhibitors treatment was examined by western blotting (WB) and flow cytometry. The expression of poly ADP-ribose polymerase (PARP1), PD-L1, and CD8 in human ovarian cancer tissues was detected by immunohistochemistry(IHC). The effect of Niraparib and PD-L1 blockade in ovarian cancer progression was investigated in vivo. The changes of immune cells and cytokines in vitro and in vivo were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Changes of cGAS/STING signal pathway after Niraparib treatment were determined by WB, ELISA.ResultsNiraparib upregulated membrane PD-L1 and total PD-L1 expression in ovarian cancer cells and had a synergistic effect with PD-L1 blockade in vivo. In clinical patient samples, Niraparib augmented cytotoxic CD8+T cell proportion and function. In vivo and vitro, Niraparib can also increase the proportion of T cells and combined with PD-L1 blockade could further enhance the effect. Besides, Niraparib activated the cGAS-STING pathway, increasing the levels of cytokines such as CCL5 and CXCL10, which played a vital role in augmenting the infiltration and activation of cytotoxic T cells.ConclusionsNiraparib could modulate the immune response via the activation of the cGAS/STING pathway, and combination with PD-L1 blockade could further enhance the effect. These results provide a sound theoretical basis for clinical treatment.

Loss of Adar1 increases T cell migration and prolongs survival in ovarian cancer

Abstract Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. OC is generally characterized by an immunosuppressive tumor microenvironment (TME) and less than 10% of patients respond to immune checkpoint blockade (ICB) therapy, though better OC prognosis is associated with high tumor-infiltrating CD8 T cells. DNA methyltransferase inhibitors (DNMTis) remove methylation and activate transcription of double-stranded (ds)RNA in OC. These dsRNAs are sensed in the cytoplasm and trigger the induction of type I IFN and also the transcription of interferon-stimulated genes (ISGs). Adenosine deaminase 1 (ADAR1) is an ISG that edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 can inhibit this type I IFN response. Our preliminary RNA-seq analysis from human OC cell lines indicates that RNA editing by ADAR1 is increased after DNMTi treatment. To test the hypothesis that Adar1 loss will amplify the DNMTi-induced IFN response in OC, we implanted mice with syngeneic ID8 murine OC Adar1 knockdown (KD) cells or control cells and treated with DNMTi. We show that DNMTi treatment reduces tumor burden and extends survival in this OC model. Loss of Adar1 significantly prolongs survival in this model, which is further improved with DNMTi treatment. DNMTi treatment of murine OC cells combined with Adar1 KD increases T cell migration in transwell migration assays and also enhances expression of chemokines CCL5, CCL2, and CXCL10. We are currently performing immunophenotyping analyses to interrogate the composition of immune cells in the TME in the mouse model of OC. These studies thus describe a role of Adar1 in the DNMTi-induced immune response in OC.

Chemotherapy Induces Tumor-Associated Macrophages that Aid Adaptive Immune Responses in Ovarian Cancer.

Neoadjuvant chemotherapy (NACT) may stimulate anticancer adaptive immune responses in high-grade serous ovarian cancer (HGSOC), but little is known about effects on innate immunity. Using omental biopsies from HGSOC, and omental tumors from orthotopic mouse HGSOC models that replicate the human tumor microenvironment, we studied the impact of platinum-based NACT on tumor-associated macrophages (TAM). We found that chemotherapy reduces markers associated with alternative macrophage activation while increasing expression of proinflammatory pathways, with evidence of inflammasome activation. Further evidence of a shift in TAM functions came from macrophage depletion via CSF1R inhibitors (CSF1Ri) in the mouse models. Although macrophage depletion in established disease had no impact on tumor weight or survival, CSF1Ri treatment after chemotherapy significantly decreased disease-free and overall survival. This decrease in survival was accompanied by significant inhibition of adaptive immune response pathways in the tumors. We conclude that chemotherapy skews the TAM population in HSGOC toward an antitumor phenotype that may aid adaptive immune responses, and therapies that enhance or sustain this during remission may delay relapse.

Development and Validation of an Immune-Related Prognostic Signature for Ovarian Cancer Based on Weighted Gene Coexpression Network Analysis.

Background Ovarian cancer is one of the most lethal diseases of women. The prognosis of ovarian cancer patients was closely correlated with immune cell expression and immune responses. Therefore, it is important to identify a robust prognostic signature, which correlates not only with prognoses but also with immune responses in ovarian cancer, thus, providing immune-related patient therapies. Methods The weighted gene coexpression network analysis (WGCNA) was used to identify candidate genes correlated with ovarian cancer prognoses. Univariate and multivariate Cox regression analyses were used to construct the prognostic signature. The Kaplan-Meier method was used to predict survival, and the immune-related bioinformatics analysis was performed using the R software. The relationship between the signature and clinical parameters was analyzed with the GraphPad Prism 7 and SPSS software. Results Gene expression from The Cancer Genome Atlas dataset was used to perform the WGCNA analysis, and candidate prognostic-related genes in patients with ovarian cancer were identified. According to the Cox regression analysis, the prognostic signature was constructed, which divided patients into two groups. The high-risk group showed the least favorable prognosis. Three independent cohorts from the Gene Expression Omnibus (GEO) database were used for the validation studies. According to the immune analyses, the GEO database signatures were significantly correlated with the immune statuses of ovarian cancer patients. By analyzing the combination of the prognostic signature and total mutational burden (TMB), ovarian cancer patients were divided into four groups. In these groups, memory B cell, resting memory CD4 T cell, M2 macrophage, resting mast cell, and neutrophil were found with significant distinctions among these groups. Conclusions This novel signature predicted the prognosis of ovarian cancer patients precisely and independently and showed significant correlations with immune responses. Therefore, this prognostic signature could indicate targeted immunotherapies for ovarian cancer patients.

Abstract 3267: Identification of a novel biomarker response in a prospective clinical trial of immune checkpoint blockade in recurrent ovarian carcinoma

Abstract The role of immune checkpoint inhibitors (ICPIs) in ovarian cancer continues to be refined. The presence of intratumoral T cells in ovarian tumors correlates with improved survival and progression, suggesting that immune modulation, such as by ICPIs, may be beneficial. However, a better understanding of the immune effects following ICPI use, particularly in those with a long progression free survival, is needed. Thus, we used imaging mass cytometry (IMC) to compare the tumor immune landscape of patients with ovarian cancer before and after a CTLA4 immune checkpoint inhibitor treatment to determine the association between alterations in immune landscapes and progression free survival. Fine needle biopsies were obtained from 8 patients with recurrent platinum-resistant ovarian carcinoma enrolled in a phase 2 randomized trial evaluating the efficacy of Tremelimumab and Durvalumab (CTLA4 and PDL1 checkpoint inhibitors, respectively) in recurrent ovarian cancer treatment. Each patient had a pre-treatment biopsy and on-treatment biopsy after 3 cycles of CTLA4 inhibitor therapy. Progression free time (PFS) was recorded with 3 patients with a long PFS (>180 days) and 5 with short PFS (<60 days). Formalin fixed paraffin embedded (FFPE) tissue sections were stained for IMC analysis via Fluidigm protocol with 34 metal-tagged antibodies to detect various cell and immune related markers. The IMC data was obtained using the Fluidigm Helios CyTOF instrument and Hyperion Imaging System laser ablation module. A novel image informatics pipeline through Matlab was used to assess the image intensity of each marker and cell location. Using the developed image informatics pipeline, cell types and locations were analyzed for 16 samples from 8 patients. In all patients, mean CD8+ T cell densities had an increased trend in on-treatment vs pre-treatment samples (205.6 v 129.9 cells/mm2, p=0.086). An analysis of cell location found that in all patients on-treatment, the mean number of CD8+ T cells adjacent to M2 macrophages increased significantly (0.09 v. 0.17 cells, p = 0.0460) as well as the mean number of CD8+ T cells adjacent to B7H4+ tumor cells (0.035 v. 0.01 cells, p = 0.046). Furthermore, patients with a long PFS had a significantly higher number of CD8+ cells neighboring B cells on-treatment than short PFS (0.12 v. 0.27 cells, p=0.028). Using IMC and novel image informatics pipeline, we found that patients with recurrent ovarian cancer treated with Tremelimumab with a long PFS mounted a significant immune response compared to patients with a short PFS, with a higher number of CD8+ T cells neighboring B cells after treatment. For all patients, the immune landscape altered after treatment. To our knowledge, this is the first known use of IMC to demonstrate immune response in ovarian cancer following ICPI therapy. Overall, IMC and image informatics pipeline are robust tools that simultaneously analyze multiple biomarkers and spatial location of cells to better assess the tumor immune microenvironment and cellular interactions. Citation Format: Han Cun, Emily M. Hinchcliff, Ying Zhu, Sammy Ferri-Borgogno, Rita Cheng, Jared K. Burks, Stephen T. Wong, Amir A. Jazaeri, Samuel C. Mok. Identification of a novel biomarker response in a prospective clinical trial of immune checkpoint blockade in recurrent ovarian carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3267.

Abstract B58: The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer

Abstract While therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, less than 10% of ovarian cancer (OC) patients have responded. Previous work from our group showed that DNA methyltransferase inhibitors (DNMTis) activate the interferon response in tumors through upregulation of repetitive elements (REs) including endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline several million years ago. DNMTis increase ERVs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill the tumors. Importantly, nearly all high-grade serous OCs harbor a mutation in the TP53 gene, encoding the tumor suppressor protein P53. p53 may transcriptionally repress ERVs to ensure genome stability; 30% of all p53 binding sites are in ERV-derived sequences. We treated seven human cancer cell lines, including four OC lines (3 TP53 wild-type, 4 TP53 mutant) with DNMTi and analyzed repetitive element expression (RNA-Seq), open chromatin (ATAC-Seq), and DNA methylation (MeDIP + MRE). Interestingly, RE upregulation was significantly higher in the P53 mutant cell lines. This was true for the SINE, LINE, and ERV groups of repetitive elements. The DNA methylation of these elements was not significantly different between TP53 wild-type and mutant cell lines, but the open chromatin peaks (ATAC-Seq) were significantly increased in the TP53 mutant cell lines. We thus hypothesize that wild-type TP53 may partner with DNA methylation to transcriptionally repress RE transcription. Treating TP53 wild-type cells with Nutlin-3A, which activates P53, represses transcription of some ERVs. When we overexpressed R175H or R273H mutant P53 constructs in the SKOV3 P53-null cell line and treated with DNMTi, we observed much higher upregulation of ERVs after DNMTi treatment than when we overexpressed the control wild type P53 vector. We predicted that higher ERVs would induce a higher interferon response and perhaps change the microenvironment in P53 mutant tumors. We used the ID8 MOSE mouse ovarian cancer model modified by the MacNeish laboratory to compare p53+/+ and p53-/- tumor response to DNMTi treatment. Characterization of the tumor microenvironment indicated a significantly higher frequency of lymphocytes, Treg cells, MDSCs, and myeloid cells in the p53-/- model in comparison to the p53 wild-type model. In current work, we are creating isogenic cell lines of human and mouse wild-type, mutant, and null p53 and comparing the response to DNMTi in vitro and in vivo. We will then determine whether P53 binding directly regulates repetitive elements transcriptionally, and more broadly, whether mutant P53 cells are more sensitive to DNMTi upregulation of repetitive elements and induction of an immune response. Results of this work will be particularly relevant for high-grade serous OC, which is driven by mutant P53 and is generally unresponsive to immunotherapies. Citation Format: Stephanie Gomez, James I. McDonald, Elisa Arthofer, Noor Diab, Aneil Srivastava, Paul Austin, Katherine B. Chiappinelli. The role of mutant P53 in repetitive element regulation and the immune response in 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 B58.

The Ovarian Cancer Tumor Immune Microenvironment (TIME) as Target for Therapy: A Focus on Innate Immunity Cells as Therapeutic Effectors.

Ovarian cancer (OvCA) accounts for one of the leading causes of death from gynecologic malignancy. Despite progress in therapy improvements in OvCA, most patients develop a recurrence after first-line treatments, dependent on the tumor and non-tumor complexity/heterogeneity of the neoplasm and its surrounding tumor microenvironment (TME). The TME has gained greater attention in the design of specific therapies within the new era of immunotherapy. It is now clear that the immune contexture in OvCA, here referred as tumor immune microenvironment (TIME), acts as a crucial orchestrator of OvCA progression, thus representing a necessary target for combined therapies. Currently, several advancements of antitumor immune responses in OvCA are based on the characterization of tumor-infiltrating lymphocytes, which have been shown to correlate with a significantly improved clinical outcome. Here, we reviewed the literature on selected TIME components of OvCA, such as macrophages, neutrophils, γδ T lymphocytes, and natural killer (NK) cells; these cells can have a role in either supporting or limiting OvCA, depending on the TIME stimuli. We also reviewed and discussed the major (immune)-therapeutic approaches currently employed to target and/or potentiate macrophages, neutrophils, γδ T lymphocytes, and NK cells in the OvCA context.

Prognostic significance of lymphocyte monocyte ratio in patients with ovarian cancer.

Background:This study aimed to systematically assess the prognostic value of lymphocyte monocyte ratio (LMR) in patients with ovarian cancer through performing a meta-analysis.Methods:Web of Science, PubMed, EMBASE, Cochrane Library, and China National Knowledge Infrastructure databases were searched for potentially eligible studies. The baseline characteristics and relevant data were extracted. Hazard ratios with 95% confidence intervals (CIs) were combined to assess the prognostic value of LMR in patients with ovarian cancer.Results:Nine studies enrolling 2809 patients were included. The pooled hazard ratios of lower LMR for overall survival and progression free survival in patients with ovarian cancer were 1.71 (95% CI, 1.40–2.09) and 1.68 (95% CI, 1.49–1.88), respectively. Subgroup analysis and sensitivity analysis were also performed. No significant publication bias was found.Conclusion:Our results suggested that lower LMR was associated with poorer overall survival and progression free survival in patients with ovarian cancer. The findings may assist prognosis evaluation and future research on therapies based on modulating host immune response in ovarian cancer.

Multisite Tumor Sampling Reveals Extensive Heterogeneity of Tumor and Host Immune Response in Ovarian Cancer.

Ovarian cancer (OVCA) is characterized by genomic/molecular intra-patient heterogeneity (IPH). Tissue histology and morphological features are surrogates of the underlying genomic/molecular contexture. We assessed the morphological IPH of OVCA tumor compartments and of lymphocytic infiltrates in multiple matched samples per patient. We examined 294 hematoxylin & eosin (H&E) OVCA tumor whole sections from 70 treatment-naïve patients who had undergone cytoreductive surgery. We assessed morphological subtypes as immunoreactive (IR), solid - proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per patient; stromal tumor-infiltrating lymphocyte (sTIL) density as average per sample; and, as maximal sTIL values (max-TILs) among all samples per patient, ovaries and implants. Among all 294 tumor sections, the most frequent primary morphological subtype was PG (n=150, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%). Subtype combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p<0.001), SD and MT in implants (p=0.023 and p<0.001, respectively). sTILs were higher in SD compared to non-SD (p=0.019) and lower in PG, respectively (p<0.001). sTIL density was higher in implants than in ovaries (p<0.001). Higher max-TILs were associated with stage IV disease (p=0.043), upper abdominal dissemination (p=0.024), endometrioid histology (p=0.013), and grade 3 tumors (p=0.021). Favorable prognosticators were higher max-TILs per patient (PFS, OS) and higher SD-load (PFS). Clinically relevant morphological and host immune-response IPH appear to be the norm in OVCA. This may complicate efforts to decipher sensitivity of the tumor to certain treatment modalities from a single pre-operative biopsy.

Abstract KN01: Dissecting antitumor immune response in ovarian cancer

Abstract In epithelial ovarian cancer (EOC), intratumoral or intraepithelial tumor infiltrating lymphocytes (TILs), i.e. T cells specifically infiltrating tumor islets, occur in approximately half of the patients and correlate with longer survival. As with many other solid tumors, despite promising results in mouse models, response of EOC to PD-1 blockade has been quite limited in the clinic, but the mechanisms underlying therapeutic failures in immunoreactive human tumors harboring TILs are not well understood. Our work has sought to answer the following key questions: a) whether ovarian TILs are tumor-specific; b) if so, what are the relevant antigens recognized by TILs; c) whether the PD-1/PD-L1 pathway is relevant; d) if so, what are the mechanisms underlying response to PD-1 blockade; and finally e) what mechanisms govern TIL recruitment and engraftment. The data presented will show that there is indeed a spontaneous and tumor-specific T-cell response in a proportion of EOC, accurately heralded by intraepithelial CD8+ TILs. Tumor-specific antigens will be discussed. Our work also shows that the PD-1/PD-L1 is a central immune checkpoint pathway in these tumors. However, only a fraction of such tumors with ieTILs respond to PD-1. We dissected the mechanisms underlying response to PD-1 blockade and therapeutic approaches to enhance response to PD-1 in human and mouse preclinical models. Therapeutic approaches to achieve effective immunotherapy in ovarian cancer will be discussed, including combination checkpoint blockade, adoptive T cell therapy and new rational combinations. Finally, successful T-cell engraftment in ovarian cancer requires tumor cell-derived CCL5 and is amplified by myeloid cell-secreted IFNg-inducible CXCL9, dependent on activation of tumor-infiltrating lymphocytes (TILs). Spontaneous, epigenetically-mediated loss or stable knockdown of ccl5 leads to CD8+ T-cell loss and immune desertification in the mouse, while forced ccl5 expression prevents loss of cxcl9 and TILs, and leads to attenuated tumor growth. Accordingly, CCL5-CXCL9 co-expression reveals immunoreactive tumors with prolonged survival and response to PD-1 blockade. The impact of homologous recombination and BRCA loss on the establishment of immunoreactive tumors will be also discussed. Citation Format: George Coukos. Dissecting antitumor immune response in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr KN01. doi:10.1158/1535-7163.TARG-19-KN01

Abstract 3199: Targeting LHRH-R to improve immune response in ovarian cancer

Abstract Background: While immune checkpoint inhibitors have revolutionized the treatment of multiple cancers, such therapies have had limited efficacy in ovarian cancer. We hypothesized that a lytic peptide could stimulate local immune response and enhance efficacy of checkpoint blockade. Here, we tested the effects of a synthetic lytic peptide targeting the luteinizing hormone receptor (LHRH-R), EP-100, on immune therapy in ovarian cancer models. Methods: We carried out series of in vitro (MTT assay, immunoblot analysis, cytokine array, and immune profiling assay) and in vivo (orthotopic mouse model) experiments to determine the biological effects of EP-100 monotherapy and its combination with checkpoint blockade, α-PD-L1. Results: We found that LHRH-R positive murine ovarian cancer cells (ID8, IG10, IF5, and 2C12) were sensitive to EP-100 and were specifically killed at low micromolar levels through LHRH-R. The in vivo syngeneic mouse models (ID8 and IG10) demonstrated that EP-100 reduced tumor volume, tumor weight, and ascites volume as a single agent. The greatest effects on tumor volume reduction and ascites volume were observed with the combination of EP-100 and α-PD-L1 antibody, indicating a potentially synergistic effect in vivo. Immune profiling of tumors showed that the population of CD8+ T cells, NK cells, dendritic cells, and macrophages were significantly increased in tumor and ascites treated with α-PD-L1, EP-100 and in the combination group. However, monocytic myeloid suppressor cells, B cells, and regulatory T cells were decreased in tumors treated with α-PD-L1, EP-100 and in the combination group. We found similar effects of EP-100 combination with α-PD-L1 antibody in ascites from each group. These data indicate that EP-100 promotes a tumor microenvironment that is favorable for immune response. In vitro cytokine arrays revealed that EP-100 induced IL-1α, IL-33, CCL20, VEGF, and LDLR secretion. Among them, we validated increasing Il-33 level following EP-100 treatment in vivo and in vitro and determined the specific biological role of CD8+ T cell activation with IL-33 gene silencing using siRNA and Cas9-CRISPR approaches. In addition, we found that CD8+ T cell expressed a low level of LHRH-R and had minimal direct effects from EP-100. Conclusion: These data demonstrate strong efficacy of EP-100 and checkpoint blockade therapy. Our results provide new directions to enhance the efficacy of immune therapies for ovarian cancer. Citation Format: Mark S. Kim, Shaolin Ma, Carola Leuschner, Sanghoon Lee, Robert L. Coleman, Anil K. Sood. Targeting LHRH-R to improve immune response in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3199.