Enhanced amphiregulin exposure promotes modulation of the high grade serous ovarian cancer tumor immune microenvironment.

High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG's role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion (CXCL8, CXCL1, CXCL2) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of IFNy and IL-2. In vivo rAREG treatment promoted significant reductions in circulating levels of IL-2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8+ T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines OVCAR8 and PEA2. Overall, this study demonstrates AREG's ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

Fetal Ovarian Reserve: the Dynamic Changes in Ubiquitin C-Terminal Hydrolase L1.

The regulation of protein turnover by the unique deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCHL1) is only seen in oocytes, spermatogonia, and neurons. Our objective was to investigate variation in expression of UCHL1 across fetal maturation of oocytes that result in lifelong ovarian reserve. We performed a retrospective cohort study of 25 fetal autopsy specimens from 21 to 36 weeks. This was an IRB-approved protocol with parental permission for use of tissues for research purposes. Tissues were stained for expression of the oocyte-specific protein UCHL1, and expression levels were evaluated using quantitative immunofluorescence across gestational ages after correction for the area and background absorbance. Corrected total cell fluorescence (CTCF) for expression of UCHL1 within human oocytes was compared across fetal gestational ages and oocyte size. Trends were analyzed using a locally weighted scatterplot smoothing algorithm. Local expression of UCHL1 increases in oocytes across ovarian development reaching a plateau at 27 weeks with the maintenance of elevated levels through 36 weeks gestational age. This maturation trend is also evidenced by the increase in protein expression as oocyte area increases (r = 0.5530, p ≤ 0.001) with the largest rise occurring as oocytes are enveloped into primordial follicles. The increase in expression as oocytes transition from oogonia into oocytes in primordial follicles and beyond may be part of the preparation of both oocytes and the surrounding somatic cells for the long-term maintenance of the ovarian reserve.

Multiplex serum immune profiling reveals circulating LAG-3 is associated with improved patient survival in high grade serous ovarian cancer.

OBJECTIVE: High grade serous ovarian cancer (HGSOC) exhibits low response rates to clinically available immunotherapies. Nevertheless, emerging research has demonstrated that certain immune factors are predictive for HGSOC patient clinical outcomes, with our own groups previous work demonstrating that intratumoral levels of the immune checkpoint receptor LAG-3 is associated with improved patient survival. In this current study we sought to uncover non-invasive circulating immune prognostic and predictive signatures in HGSOC. METHODS: A multiplex approach was employed that examined circulating levels of immune checkpoint receptors LAG-3 and PD-1 along with 48 common cytokine and chemokines in a cohort of 75 HGSOC treatment naïve patient serum samples. RESULTS: Elevated serum LAG-3 was significantly associated with improved progression-free survival (PFS) and overall survival (OS) in HGSOC, while circulating PD-1 levels were largely unrelated with patient clinical outcomes. Cytokine and chemokine analysis revealed lower IL-15 expression correlated with improved PFS and OS, while increased IL-1α, IL-1Ra, IL-6, IL8 and VEGF were significantly associated with preoperative CA-125 levels. ROC analysis demonstrated that serum LAG-3 levels exhibited consistent reasonable predictability as a single agent. CONCLUSIONS: Serum-derived LAG-3 was identified out of a diverse array of chemokine and cytokines as the immune-based factor most significantly associated with improved HGSOC survival. These findings suggest that LAG-3 could be implemented as a non-invasive patient predictive marker for improved HGSOC clinical outcomes.

Adaptive transcriptomic and immune infiltrate responses in the tumor immune microenvironment following neoadjuvant chemotherapy in high grade serous ovarian cancer reveal novel prognostic associations and activation of pro-tumorigenic pathways.

The high rate of ovarian cancer recurrence and chemoresistance necessitates further research into how chemotherapy affects the tumor immune microenvironment (TIME). While studies have shown that immune infiltrate increases following neoadjuvant (NACT) chemotherapy, there lacks a comprehensive understanding of chemotherapy-induced effects on immunotranscriptomics and cancer-related pathways and their relationship with immune infiltrate and patient responses. In this study, we performed NanoString nCounter® PanCancer IO360 analysis of 31 high grade serous ovarian cancer (HGSOC) patients with matched pre-treatment biopsy and post-NACT tumor. We observed increases in pro-tumorigenic and immunoregulatory pathways and immune infiltrate following NACT, with striking increases in a cohort of genes centered on the transcription factors ATF3 and EGR1. Using quantitative PCR, we analyzed several of the top upregulated genes in HGSOC cell lines, noting that two of them, ATF3 and AREG, were consistently upregulated with chemotherapy exposure and significantly increased in platinum resistant cells compared to their sensitive counterparts. Furthermore, we observed that pre-NACT immune infiltrate and pathway scores were not strikingly related to platinum free interval (PFI), but post-NACT immune infiltrate, pathway scores, and gene expression were. Finally, we found that higher levels of a cohort of proliferative and DNA damage-related genes was related to shorter PFI. This study underscores the complex alterations in the ovarian TIME following chemotherapy exposure and begins to untangle how immunologic factors are involved in mediating chemotherapy response, which will allow for the future development of novel immunologic therapies to combat chemoresistance.

Intratumoral expression analysis reveals that OX40 and TIM-3 are prominently expressed and have variable associations with clinical outcomes in high grade serous ovarian cancer.

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+ and CD4+ T cell subsets. Pan-cancer analysis determined that TIM-3 and OX40 levels were similar to those in immunotherapy-responsive cancers, while PD-1 exhibited much lower expression in HGSOC. Finally, OX40 was most strongly associated with improved patient survival. Overall, the current study suggested that TIM-3 and OX40 are frequently expressed intratumoral immune receptors in HGSOC and thus represent promising immune targets. Furthermore, the present analysis strongly suggested that OX40 was significantly associated with a longer survival and could potentially be utilized as a prognostic factor for improved patient outcomes in HGSOC.

A bioinformatic analysis of WFDC2 (HE4) expression in high grade serous ovarian cancer reveals tumor-specific changes in metabolic and extracellular matrix gene expression.

Human epididymis protein-4 (HE4/WFDC2) has been well-studied as an ovarian cancer clinical biomarker. To improve our understanding of its functional role in high grade serous ovarian cancer, we determined transcriptomic differences between ovarian tumors with high- versus low-WFDC2 mRNA levels in The Cancer Genome Atlas dataset. High-WFDC2 transcript levels were significantly associated with reduced survival in stage III/IV serous ovarian cancer patients. Differential expression and correlation analyses revealed secretory leukocyte peptidase inhibitor (SLPI/WFDC4) as the gene most positively correlated with WFDC2, while A kinase anchor protein-12 was most negatively correlated. WFDC2 and SLPI were strongly correlated across many cancers. Gene ontology analysis revealed enrichment of oxidative phosphorylation in differentially expressed genes associated with high-WFDC2 levels, while extracellular matrix organization was enriched among genes associated with low-WFDC2 levels. Immune cell subsets found to be positively correlated with WFDC2 levels were B cells and plasmacytoid dendritic cells, while neutrophils and endothelial cells were negatively correlated with WFDC2. Results were compared with DepMap cell culture gene expression data. Gene ontology analysis of k-means clustering revealed that genes associated with low-WFDC2 were also enriched in extracellular matrix and adhesion categories, while high-WFDC2 genes were enriched in epithelial cell proliferation and peptidase activity. These results support previous findings regarding the effect of HE4/WFDC2 on ovarian cancer pathogenesis in cell lines and mouse models, while adding another layer of complexity to its potential functions in ovarian tumor tissue. Further experimental explorations of these findings in the context of the tumor microenvironment are merited.

The requirement of ubiquitin C-terminal hydrolase L1 in mouse ovarian development and fertility†.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Prognostic immunologic signatures in epithelial ovarian cancer.

Epithelial Ovarian Cancer (EOC) is a deadly gynecologic malignancy in which patients frequently develop recurrent disease following initial platinum-taxane chemotherapy. Analogous to many other cancer subtypes, EOC clinical trials have centered upon immunotherapeutic approaches, most notably programmed cell death 1 (PD-1) inhibitors. While response rates to these immunotherapies in EOC patients have been low, evidence suggests that ovarian tumors are immunogenic and that immune-related genomic profiles can serve as prognostic markers. This review will discuss recent advances in the development of immune-based prognostic signatures in EOC that predict patient clinical outcomes, as well as emphasize specific research areas that need to be addressed to drive this field forward.

Immune Modeling Analysis Reveals Immunologic Signatures Associated With Improved Outcomes in High Grade Serous Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy worldwide, as patients are typically diagnosed at a late stage and eventually develop chemoresistant disease following front-line platinum-taxane based therapy. Only modest results have been achieved with PD-1 based immunotherapy in ovarian cancer patients, despite the fact that immunological responses are observed in EOC patients. Therefore, the goal of this present study was to identify novel immune response genes and cell subsets significantly associated with improved high grade serous ovarian cancer (HGSOC) patient prognosis. A transcriptomic-based immune modeling analysis was employed to determine levels of 8 immune cell subsets, 10 immune escape genes, and 22 co-inhibitory/co-stimulatory molecules in 26 HGSOC tumors. Multidimensional immune profiling analysis revealed CTLA-4, LAG-3, and Tregs as predictive for improved progression-free survival (PFS). Furthermore, the co-stimulatory receptor ICOS was also found to be significantly increased in patients with a longer PFS and positively correlated with levels of CTLA-4, PD-1, and infiltration of immune cell subsets. Both ICOS and LAG-3 were found to be significantly associated with improved overall survival in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Finally, PVRL2 was identified as the most highly expressed transcript in our analysis, with immunohistochemistry results confirming its overexpression in HGSOC samples compared to normal/benign. Results were corroborated by parallel analyses of TCGA data. Overall, this multidimensional immune modeling analysis uncovers important prognostic immune factors that improve our understanding of the unique immune microenvironment of ovarian cancer.

The Perfect Combination: Enhancing Patient Response to PD-1-Based Therapies in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, with an overall 5-year survival of only 47%. As the development of novel targeted therapies is drastically necessary in order to improve patient survival, current EOC clinical trials have heavily focused on immunotherapeutic approaches, centered upon programmed cell death 1 (PD-1) inhibitors. While PD-1 monotherapies have only exhibited modest responses for patients, it has been theorized that in order to enhance EOC patient response to immunotherapy, combinatorial regimens must be investigated. In this review, unique challenges to EOC PD-1 response will be discussed, along with a comprehensive description of both preclinical and clinical studies evaluating PD-1-based combinatorial therapies. Promising aspects of PD-1-based combinatorial approaches are highlighted, while also discussing specific preclinical and clinical areas of research that need to be addressed, in order to optimize EOC patient immunotherapy response.