HE4 Overexpression by Ovarian Cancer Promotes a Suppressive Tumor Immune Microenvironment and Enhanced Tumor and Macrophage PD-L1 Expression.

Ovarian cancer is a highly fatal malignancy characterized by early chemotherapy responsiveness but the eventual development of resistance. Immune targeting therapies are changing treatment paradigms for numerous cancer types but have had minimal success in ovarian cancer. Through retrospective patient sample analysis, we have determined that high human epididymis protein 4 (HE4) production correlates with multiple markers of immune suppression in ovarian cancer, including lower CD8+ T cell infiltration, higher PD-L1 expression, and an increase in the peripheral monocyte to lymphocyte ratio. To further understand the impact that HE4 has on the immune microenvironment in ovarian cancer, we injected rats with syngeneic HE4 high- and low-expressing cancer cells and analyzed the differences in their tumor and ascites immune milieu. We found that high tumoral HE4 expression promotes an ascites cytokine profile that is rich in myeloid-recruiting and differentiation factors, with an influx of M2 macrophages and increased arginase 1 production. Additionally, CTL activation is significantly reduced in the ascites fluid, and there is a trend toward lower CTL infiltration of the tumor, whereas NK cell recruitment to the ascites and tumor is also reduced. PD-L1 expression by tumor cells and macrophages is increased by HE4 through a novel posttranscriptional mechanism. Our data have identified HE4 as a mediator of tumor-immune suppression in ovarian cancer, highlighting this molecule as a potential therapeutic target for the treatment of this devastating disease.

Quality of life after pancreatic surgery - A systematic review.

BACKGROUND: Surgery for patients with pancreatic cancer carries a high risk of major post-operative complications and only marginally improves overall survival. This review aims to assess the impact of surgical resection on health-related quality of life (HRQOL) of pancreatic cancer patients. METHODS: A systematic review of the literature was performed according to the PRISMA guidelines. All studies assessing QOL using validated questionnaires in pancreatic cancer patients undergoing surgical resection were included. RESULTS: Twenty-two studies were assessed. Patients reported a decrease in physical, social and global scales within the first 3 months after surgery. These values showed improvement and were comparable to baseline values by 6 months. Recovery in emotional functioning towards baseline figures was demonstrated in the first 3 months post-operatively. Symptom scales including pain, fatigue and diarrhoea deteriorated after surgery, but reverted to baseline after 3-6 months. CONCLUSIONS: Surgical resection for pancreatic cancer has short-term negative impact on QOL. In the longer term, this will improve and eventually recover to baseline values after 6 months. Knowledge on the impact of surgery on QOL of pancreatic cancer patients is necessary to facilitate decision-making and tailoring of surgical techniques to the individual patient.

Genetically Determined Risk of Depression and Functional Outcome After Ischemic Stroke.

Background and Purpose- Psychosocial factors can have implications for ischemic stroke risk and recovery. This study investigated the effect of genetically determined risk of depression on these outcomes using the Mendelian randomization (MR) framework. Methods- Genetic instruments for risk of depression were identified in a discovery genome-wide association study of 246 363 cases and 561 190 controls and further replicated in a separate population of 474 574 cases and 1 032 579 controls. Corresponding genetic association estimates for risk of ischemic stroke were taken from 60 341 cases and 454 450 controls, with those for functional outcome 3 months after ischemic stroke taken from an analysis of 6021 patients. Following statistical power calculation, inverse-variance weighted MR was performed to pool estimates across different instruments. The Cochran Q heterogeneity test, weighted median MR, and MR pleiotropy residual sum and outlier were used to explore possible bias relating to inclusion of pleiotropic variants. Results- There was no MR evidence for an effect of genetically determined risk of depression on ischemic stroke risk. Although suffering low statistical power, the main inverse-variance weighted MR analysis was suggestive of a detrimental effect of genetically determined risk of depression on functional outcome after ischemic stroke (odds ratio of poor outcome [modified Rankin Scale, ≥3] per 1-SD increase in genetically determined risk of depression, 1.81; 95% CI, 0.98-3.35; P=0.06). There was no evidence of heterogeneity between MR estimates produced by different instruments (Q P=0.26). Comparable MR estimates were obtained with weighted median MR (odds ratio, 2.57; 95% CI, 1.05-6.25; P=0.04) and MR pleiotropy residual sum and outlier (odds ratio, 1.81; 95% CI, 0.95-3.46; P=0.08). Conclusions- We found no MR evidence of genetically determined risk of depression affecting ischemic stroke risk but did find consistent MR evidence suggestive of a possible effect on functional outcome after ischemic stroke. Given the widespread prevalence of depression-related morbidity, these findings could have implications for prognostication and personalized rehabilitation after stroke.

Neoadjuvant chemotherapy induces phenotypic mast cell changes in high grade serous ovarian cancer.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy in which patients have still yet to respond meaningfully to clinically available immunotherapies. Hence, novel immune targets are urgently needed. Our past work has identified that mast cells are significantly upregulated at the mRNA level in HGSOC patient tumors following neoadjuvant chemotherapy (NACT) exposure. Therefore, in this current investigation we sought to characterize intratumoral mast cell phenotypic changes as a result of NACT exposure and determine how these adaptations are associated with patient clinical outcomes. METHODS: Hematologic immunohistochemistry was employed to determine mast cell levels in 36 matched pre- and post-NACT HGSOC patient tumors. Fluorescent Immunohistochemistry was utilized to identify Tryptase+(carboxypeptidase A3 (CPA3) + mast cells as well as histamine levels in 29 and 20, respectively, matched pre- and post-NACT HGSOC patient tumors. Finally, human immortalized mast cells, LUVA were stimulated with carboplatin and paclitaxel and genomic changes were analyzed by quantitative PCR. RESULTS: Hematologic labeled intratumoral mast cells were significantly upregulated in the intraepithelial and stromal regions of the tumor, post-NACT. Lower levels of pre-NACT mast cells were significantly associated with an improved progression-free survival (PFS). Histamine, a marker of mast cell degranulation was similarly upregulated in post-NACT exposed tumors. Through the characterization of mast cell specific proteases Tryptase and CPA3, it was found that Tryptase+/ CPA3 + mast cells were significantly upregulated both in the intraepithelial and stromal compartments of the tumor, while Tryptase + cells were significantly upregulated in the stromal regions of the tumor. Lower post-NACT treated levels with Tryptase+/ CPA3 + cells were significantly associated with improved overall survival (OS) and PFS while higher Tryptase + mast cells were associated with improved OS. Finally, following chemotherapy exposure mast cell activating factors AREG and CCL2 were significantly upregulated while TGFB1, an inhibitor of mast cell activation was downregulated in LUVA cells. CONCLUSIONS: Enhanced mast cell numbers, as well as activation and degranulation are a consequence of NACT exposure. Post-NACT mast cells displayed differing associations with survival outcomes that was dependent upon granule classification. Ultimately, mast cells represent a clinically relevant putative HGSOC immune target.

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.

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.

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.

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.

The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes.

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.

Inhibition of DUSP6 sensitizes ovarian cancer cells to chemotherapeutic agents via regulation of ERK signaling response genes.

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.

Septin-2 is overexpressed in epithelial ovarian cancer and mediates proliferation via regulation of cellular metabolic proteins.

Epithelial Ovarian Cancer (EOC) is associated with dismal survival rates due to the fact that patients are frequently diagnosed at an advanced stage and eventually become resistant to traditional chemotherapeutics. Hence, there is a crucial need for new and innovative therapies. Septin-2, a member of the septin family of GTP binding proteins, has been characterized in EOC for the first time and represents a potential future target. Septin-2 was found to be overexpressed in serous and clear cell human patient tissue compared to benign disease. Stable septin-2 knockdown clones developed in an ovarian cancer cell line exhibited a significant decrease in proliferation rates. Comparative label-free proteomic analysis of septin-2 knockdown cells revealed differential protein expression of pathways associated with the TCA cycle, acetyl CoA, proteasome and spliceosome. Further validation of target proteins indicated that septin-2 plays a predominant role in post-transcriptional and translational modifications as well as cellular metabolism, and suggested the potential novel role of septin-2 in promoting EOC tumorigenesis through these mechanisms.

Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6.

While selective overexpression of serum clinical biomarker Human epididymis secretory protein 4 (HE4) is indicative of ovarian cancer tumorigenesis, much is still known about the mechanistic role of the HE4 gene or gene product. Here, we examine the role of the secretory glycoprotein HE4 in ovarian cancer immune evasion. Through modified subtractive hybridization analyses of human peripheral blood mononuclear cells (PBMCs), we have characterized gene targets of HE4 and established a preliminary mechanism of HE4-mediated immune failure in ovarian tumors. Dual specificity phosphatase 6 (DUSP6) emerged as the most upregulated gene in PBMCs upon in vitro exposure to HE4. DUSP6 was found to be upregulated in CD8+ cells and CD56+ cells. HE4 exposure reduced Erk1/2 phosphorylation specifically in these cell populations and the effect was erased by co-incubation with a DUSP6 inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI). In co-culture with PBMCs, HE4-silenced SKOV3 human ovarian cancer cells exhibited enhanced proliferation upon exposure to external HE4, while this effect was partially attenuated by adding BCI to the culture. Additionally, the reversal effects of BCI were erased in the co-culture with CD8+ / CD56+ cell deprived PBMCs. Taken together, these findings show that HE4 enhances tumorigenesis of ovarian cancer by compromising cytotoxic CD8+ and CD56+ cells through upregulation of self-produced DUSP6.

Beyond the Biomarker: Understanding the Diverse Roles of Human Epididymis Protein 4 in the Pathogenesis of Epithelial Ovarian Cancer.

Human epididymis protein 4 (HE4) is an important clinical biomarker used for the detection of epithelial ovarian cancer (EOC). While much is known about the predictive power of HE4 clinically, less has been reported regarding its molecular role in the progression of EOC. A deeper understanding of HE4's mechanistic functions may help contribute to the development of novel targeted therapies. Thus far, it has been difficult to recommend HE4 as a therapeutic target owing to the fact that its role in the progression of EOC has not been extensively evaluated. This review summarizes what is collectively known about HE4 signaling and how it functions to promote tumorigenesis, chemoresistance, and metastasis in EOC, with the goal of providing valuable insights that will have the potential to aide in the development of new HE4-targeted therapies.

Human Epididymis Protein 4 Promotes Events Associated with Metastatic Ovarian Cancer via Regulation of the Extracelluar Matrix.

Human epididymis protein 4 (HE4) has received much attention recently due to its diagnostic and prognostic abilities for epithelial ovarian cancer. Since its inclusion in the Risk of Ovarian Malignancy Algorithm (ROMA), studies have focused on its functional effects in ovarian cancer. Here, we aimed to investigate the role of HE4 in invasion, haptotaxis, and adhesion of ovarian cancer cells. Furthermore, we sought to gain an understanding of relevant transcriptional profiles and protein kinase signaling pathways mediated by this multifunctional protein. Exposure of OVCAR8 ovarian cancer cells to recombinant HE4 (rHE4) promoted invasion, haptotaxis toward a fibronectin substrate, and adhesion onto fibronectin. Overexpression of HE4 or treatment with rHE4 led to upregulation of several transcripts coding for extracellular matrix proteins, including SERPINB2, GREM1, LAMC2, and LAMB3. Gene ontology indicated an enrichment of terms related to extracellular matrix, cell migration, adhesion, growth, and kinase phosphorylation. LAMC2 and LAMB3 protein levels were constitutively elevated in cells overexpressing HE4 and were upregulated in a time-dependent manner in cells exposed to rHE4 in the media. Deposition of laminin-332, the heterotrimer comprising LAMC2 and LAMB3 proteins, was increased in OVCAR8 cells treated with rHE4 or conditioned media from HE4-overexpressing cells. Enzymatic activity of matriptase, a serine protease that cleaves laminin-332 and contributes to its pro-migratory functional activity, was enhanced by rHE4 treatment in vitro. Proteomic analysis revealed activation of focal adhesion kinase signaling in OVCAR8 cells treated with conditioned media from HE4-overexpressing cells. Focal adhesions were increased in cells treated with rHE4 in the presence of fibronectin. These results indicate a direct role for HE4 in mediating malignant properties of ovarian cancer cells and validate the need for HE4-targeted therapies that will suppress activation of oncogenic transcriptional activation and signaling cascades.