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Objectives: The chemokine CCL22 is recognized for recruiting immunosuppressive regulatory T-cells (Treg) that contribute to disease progression in various tumor entities helping them to evade the host immune response. Our study aims to identify the expressing cell types and to evaluate the prognostic significance of CCL22 secretion and its association with Treg invasion in endometrial cancer (EC), an immunogenic cancer. Methods: Specimens from 275 patients with EC and 28 healthy controls were screened immunohistochemically for CCL22. Immunofluorescence double-staining for CCL22 and different immune cell markers was performed. In vitro regulation of CCL22-expression was examined in EC cell lines (Ishikawa+, RL95–2) and human PBMCs in coculture settings via qPCR and ELISA. Results: Elevated CCL22 staining in tumor cells and CCL22-positive M1-macrophages in tumordistant areas were significantly associated with increased overall survival (OS). Conversely, high, secretory-appearing staining in the peritumoral and intratumoral stroma correlated with reduced OS. Although the analysis of the in vitro coculture model of epithelial tumor- and immune cells revealed PBMCs as the primary source of CCL22, we could confirm expression of the chemokine also in the EC epithelial cells. Conclusion: Our study suggests that CCL22 in EC is associated with OS, dependent on its location and the cell type producing it. Intracellular upregulation and extracellular secretion must be considered separately when investigating CCL22 expressing cell types in EC. These results may provide evidence for CCL22-mediated Treg recruitment in EC as a potential future therapeutic target.

Gestational diabetes mellitus (GDM) is a common condition during pregnancy. The prevalence of GDM is continuously increasing worldwide. Due to accessible diagnostic methods and a clear understanding of risk factors, GDM can be effectively diagnosed and managed. Galectins may influence immunomodulatory and inflammatory processes. This study examines the expression of galectin-7 in the placentas of women with gestational diabetes (GDM), compares it to its expression in healthy pregnancies, and evaluates the associated clinical outcomes. The placentas of 40 healthy women and 40 GDM placentas were included in the cohort. The expression level of galecin-7 was measured in the syncytiotrophoblast (SCT) and in the decidua of the placenta by immunohistochemistry and double immunofluorescence staining. The evaluation was performed by an immunoreactivity score (IRS). The study results show an increased expression of galectin-7 in the SCT and the decidua of GDM placentas as compared to the placentas of the control group. Elevated levels of galectin-7 were observed in both the nucleus and the cytoplasm. This study investigated the hypothesis that galectins are involved in pathophysiological processes of gestational diabetes. Statistical analysis of gene expression patterns confirmed that galectin-7 is indeed upregulated in GDM placentas. Further studies are needed to show the correlation of galectin-7 and the development and maintenance of gestational diabetes mellitus.

Galectins are known to play an important role in immunoregulatory processes and autoimmune diseases. Galectin-10 is a cytoplasmic protein of human eosinophils and is involved in various eosinophilic diseases. Since increased galectin expression is already detected in the placentas of mothers with gestational diabetes mellitus (GDM), this study focuses on the specific role of galectin-10 and hints at consequences for the diagnosis and therapeutic options of GDM. It is hypothesized that the difference in galectin-10 expression will raise the pathophysiological understanding of gestational diabetes. The study population consists of 80 women: 40 healthy mothers and 40 women suffering from gestational diabetes mellitus. The expression of galectin-10 was analyzed in the syncytiotrophoblast (SCT) and the decidua of the placenta via immunohistochemistry and immunofluorescence double staining. The immunoreactivity score (IRS) was used for evaluation. The results in this study were significant for an overexpression of galectin-10 in GDM placentas compared with the control group. The syncytiotrophoblast showed overexpression in the nucleus and the cytoplasm, whereas expression of galectin-10 in the decidua was significant in the cytoplasm only. This study identified the expression changes in galectin-10 in placental tissue between healthy and GDM mothers and intensified the understanding of gestational diabetes. Assuming that gestational diabetes mellitus is involved in inflammatory processes, galectin-10 might play a role in the development and maintenance of GDM. Further investigation is required to strengthen these findings.

Endometrial cancer (EC) is a common gynecological cancer worldwide. Treatment has been improved in recent years; however, in advanced stages, therapeutic options are still limited. The expression of galectins is increased in several tumor types and that they are involved in important cell processes. Large studies on endometrial cancer are still pending; Specimens of 225 patients with EC were immunohistochemically stained with antibodies for Gal-8 and Gal-9. Expression was correlated with histopathological variables. The cytosolic expression of both galectins is associated with grading and survival. Cytosolic Galectin-8 expression is a positive prognostic factor for overall survival (OS) and progression-free survival (PFS), while nuclear Gal-8 expression correlates only to OS. The cytosolic presence of Galectin-9 is correlated with a better prognosis regarding OS. Our results suggest that expression of both galectins is associated with OS and PFS in EC. Further studies are needed to understand the underlying molecular mechanisms.

Heat Shock Protein 90 (HSP90), a major molecular chaperone, plays a crucial role in cell function by folding and stabilizing proteins and maintaining proteostasis. This study aimed to elucidate the prognostic impact of HSP90 in cervical cancer. We analyzed HSP90 expression using immunohistochemistry in cervical cancer tissue microarrays from 250 patients. This study investigated correlations between HSP90 expression levels and key clinical outcomes, including overall survival (OS), progression-free survival (PFS), and FIGO classification. The statistical analyses employed included the Kruskal–Wallis-H test, log-rank (Mantel–Cox), and Cox regression. Our findings indicate that high nuclear HSP90 expression is associated with improved OS, while high cytoplasmic HSP90 expression correlates with better PFS and a lower FIGO classification in cervical squamous cell carcinoma patients. These results suggest that HSP90 could serve as a positive prognostic factor in patients diagnosed with cervical squamous cell carcinoma, underlining its potential as a biomarker for patient prognosis and as a target for therapeutic strategies.

IntroductionThe enzymes Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) und 3 (RIPK3) as well as the protein Mixed lineage kinase domain like pseudokinase (pMLKL) play a role in the signaling cascade of necroptosis. This is a form of programmed cell death which is caspase-independent. High-risk human papilloma virus infection can inhibit necroptosis. Thereby, a persistent infection and consequently the development of cervical cancer can be triggered. Aim of this study was the analysis of the expression of RIPK1, RIPK3 and pMLKL in cervical cancer tissue and the evaluation of its prognostic value on overall survival, progression-free survival and additional clinical parameters.MethodsThe expression of RIPK1, RIPK3, and pMLKL in cervical cancer tissue microarrays of n = 250 patients was analyzed immunohistochemically. Further, the effect of C2 ceramide on several cervical cancer cell lines (CaSki, HeLa, SiHa) was examined. C2 ceramide is a biologically active short-chain ceramide that induces necroptosis in human luteal granulosa cells.ResultsSignificantly longer overall survival and progression-free survival rates could be detected in cervical cancer patients expressing nuclear RIPK1 or RIPK3 alone or simultaneously (RIPK1 and RIPK3). Cell viability and proliferation was reduced through C2 ceramide stimulation of cervical cancer cells. Simultaneous stimulation of C2 ceramide and the pan-caspase inhibitor Z-VAD-fmk, or the RIPK1-inhibitor necrostatin-1, partly reversed the negative effect of C2 ceramide on cell viability. This observation could imply that caspase-dependent and -independent forms of cell death, including necroptosis, can occur. AnnexinV-FITC apoptosis staining induced a significant increase in apoptotic cells in CaSki and SiHa cells. The stimulation of CaSki cells with C2 ceramide led to a significant percentual increase in necrotic/intermediate (dying) cells after stimulation with C2 ceramide. In addition, after stimulation with C2 ceramide, CaSki and HeLa cells live cell imaging showed morphological changes which are common for necroptosis.DiscussionIn conclusion, RIPK1 and RIPK3 are independent positive predictors for overall survival and progression-free survival in cervical cancer patients. C2 ceramide can reduce cell viability and proliferation in cervical cancer cells by inducing most likely both apoptosis and necroptosis.

Abstract In recurrent epithelial ovarian cancer (EOC) most patients develop platinum-resistance. On molecular level the NRF2 pathway, a cellular defense mechanism against reactive oxygen species, is induced. In this study, we investigate AKR1C1/2, target of NRF2, in a well-established EOC collective by immunohistochemistry and in a panel of ovarian cancer cell lines including platinum-resistant clones. The therapeutic effect of carboplatin and MPA as monotherapy or in combination was assessed by functional assays, using OV90 and OV90cp cells. Molecular mechanisms of action of MPA were investigated by NRF2 silencing and AKR activity measurements. Immunohistochemical analysis revealed that AKR1C1/2 is a key player in the development of chemoresistance and an independent indicator for short PFS (23.5 vs. 49.6 months, p = 0.013). Inhibition of AKR1C1/2 by MPA led to a concentration- and time-dependent decline of OV90 viability and to an increased response to CP in vitro. By NRF2 silencing, however, the effects of MPA treatment were reduced. Concludingly, our data suggest that a combination therapy of carboplatin and MPA might be a promising therapeutic approach to increase response rates of EOC patients, which should be explored in clinical context.

Abstract Background For young borderline ovarian tumor (BOT) patients, preservation of the uterus was incorporated as an accepted option into treatment guidelines. For the endometrioid subtype (eBOT) however, adequate histological evaluation is challenging and might be associated with synchronous endometrial disorders or misinterpreted as spread from uterine primaries. Case presentation We report the cases of two young patients with eBOT who underwent treatment according to current guidelines. In both cases, unexpected findings of invasive uterine carcinomas were established in final histopathological evaluation. Conclusions This constellation highlights the challenging diagnostic workup of BOT and underlines that uterine curettage is indispensable for eBOT to exclude uterine primary tumors when fertility preservation is planned. Accordingly, we suggest to include this procedure into recommendations for diagnostic workup and to state the potential risk in treatment guidelines.

Abstract Background An abundance of tumor-associated macrophages has been shown to be an independent prognostic factor for a poor prognosis of human breast cancer (BC). Adipose tissue accounts for the largest proportion of the breast and has also been identified as an independent indicator of poor survival in BC. This study aims to elucidate if the influence of adipose tissue in BC might be mediated by macrophages. The roles of macrophages in the breast tumor-stroma (breast tumor stroma macrophages, BTSM) and macrophages in the surrounding adipose tissue (breast adipose tissue macrophages, BATM) were explored separately. Methods Two hundred ninety-eight BC tissue samples were analyzed immunohistochemically. The number of macrophages was detected by CD68+ staining. The quantity of BATMs and BTSMs was correlated to clinical and pathological parameters as well as to disease-free survival (DFS) and overall survival (OS). Results The amounts of BATMs and BTSMs strongly correlated with each other (r = 0.5, p = 2.98E−15). The quantity of BTSMs, but not of BATMs, was significantly associated with the BC molecular subtype (p = 0.000011), and all triple-negative BC tumors contained high amounts of BTSMs. BATMs were negatively associated with DFS (p = 0.0332). Both BATMs (p = 0.000401) and BTSMs (p = 0.021) were negatively associated with OS in the Kaplan-Meier analysis, but only BATMs remained an independent factor in the multivariate Cox-regression analysis (HR = 4.464, p = 0.004). Combining prostaglandin E2 receptor 3 (EP3)-expression and the quantity of BATMs, a subgroup with an extremely poor prognosis could be identified (median OS 2.31 years in the “high BATMs/low EP3” subgroup compared to 11.42 years in the most favorable “low BATMs/high EP3” subgroup, p = 0.000002). Conclusion Our findings suggest that BTSMs and BATMs seem to be involved differently in BC. Breast adipose tissue might contribute to the aggressiveness of BC via BATMs, which were independently associated with BC survival. BATMs’ role and occurrence might be functionally dependent on EP3, as a combination of both factors was strongly associated with survival. Targeting BATMs—eventually in combination with targeting the EP3-pathway—might be promising for future therapies.

Targeting the tumor-associated carbonic anhydrase XII (CA XII) is considered a promising strategy to improve cancer treatment. As such progress is highly demanded for ovarian carcinomas, the present study aimed to provide deeper information about their CA XII expression profile. A large collection of tissue specimens was stained immunohistochemically with a specific anti-CA XII antibody to evaluate the expression in neoplastic and non-neoplastic epithelial ovarian cells. In addition, flow cytometry was used to measure CA XII expression on tumor cells from malignant ascites fluid. Binding of the antibody revealed a significant CA XII expression in most ovarian carcinoma tissue samples and ascites-derived ovarian carcinoma cells. Moreover, CA XII was expressed at higher levels in ovarian carcinomas as compared to borderline ovarian tumors and non-neoplastic ovarian epithelia. Within the carcinoma tissues, high expression of CA XII was associated with higher tumor grading and a trend towards shorter overall survival. Our results indicate that CA XII plays a crucial role for the malignancy of ovarian carcinoma cells and emphasize the potential of CA XII as a diagnostic marker and therapeutic target in the management of ovarian carcinomas.

Macrophages are dynamic cells susceptible to the local microenvironment which includes tumor-associated macrophages (TAMs) in cancers. TAMs are a collection of heterogeneous macrophages, including M1 and M2 subtypes, shaped by various activation modes and labeled with various markers in different tumors. CCL22+-infiltrating cells are thought to be significantly associated with the prognosis of cervical cancer patients. Moreover, CCL22 is an established marker of M2a macrophages. Although the phenotypic identification of M1 and M2 macrophages is well established in mice and human macrophages cultured in a medium with fetal calf serum (FCS), fewer studies have focused on M2 subtypes. In addition, the question of whether CCL22 affects polarization of M2a macrophages remains unanswered. This study constructed a co-culture system to shape TAMs in vitro. We found that CCL22 was mainly secreted by TAMs but not cervical cancer cell lines. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1, M2a, M2b, and M2c macrophages using LPS plus IFNr, IL-4, LPS plus IL1β, and IL-10, respectively. Using flowcytometry, we found CD80++ was the marker of M1 and M2b, CD206++ was the marker of M2a, and CD163++ was the marker of M2c, compared with M0 macrophages. By regulating CCL22, we found that the mean fluorescence intensity (MFI) of CD206 in TAMs was significantly affected compared to the control group. Therefore, CCL22 could polarize TAMs of cervical cancer toward M2a macrophages. In conclusion, our study revealed that CCL22 could be a therapeutic target for cervical cancer, which might be because of its role in regulating macrophage polarization.

Abstract Background Regulatory T cells (Treg) suppress cytotoxic T cell anti-tumoral immune responses and thereby promote tumor progression. Prevention of intratumoral Treg accumulation by inhibition of their migration to the tumor microenvironment is a promising therapeutic strategy. The aim of this study was to identify the role of the two major Treg-attracting chemokines CCL1 and CCL22 in human breast cancer. Methods One hundred ninety-nine tissue samples of patients with invasive breast cancer were stained for CCL1 and CCL22 by immunohistochemistry. Chemokine expression and tumor infiltration by regulatory T cells, determined by expression of the transcription factor FoxP3, were quantified and their correlation to clinical features was statistically analyzed. Results Both CCL1 and CCL22 were expressed in most breast cancer tissues. CCL1 was significantly over-expressed in invasive breast cancer as compared to normal breast tissue. CCL1, but surprisingly not CCL22, showed a significant correlation with the number of tumor-infiltrating FoxP3+ Treg (p

Background:: Enolase-1, primarily known for its role in glucose metabolism, is overexpressed in various cancer entities. In contrast its alternative spliced nuclear isoform MBP-1 acts as a tumor suppressor. The aim of this study is to analyze the prognostic impact of Enolase-1/ MBP-1 and its functional significance in epithelial ovarian cancer (EOC). Methods:: By immunohistochemistry, Enolase-1 staining was examined in 156 EOC samples. Evaluation of Enolase-1 staining was conducted in the nucleus and the cytoplasm using the semi-quantitative immunoreactive score. Expression levels were correlated with clinical and pathological parameters as well as with overall survival to assess for prognostic impact. Results:: Cytoplasmic and nuclear Enolase-1 expression did not show a significant difference between the histological subtypes (p = 0.1). High nuclear Enolase-1/ MBP-1 staining negativly correlated with the tumor grading (p

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies.

Abstract is missing (Short communication)

There are several open questions to be answered regarding the pathophysiology of the development of preeclampsia (PE). Numerous factors are involved in its genesis, such as defective placentation, vascular impairment, and an altered immune response. The activation of the adaptive and innate immune system represents an immunologic, particularity during PE. Proinflammatory cytokines are predominantly produced, whereas immune regulatory and immune suppressive factors are diminished in PE. In the present study, we focused on the recruitment of regulatory T cells (Tregs) which are key players in processes mediating immune tolerance. To identify Tregs in the decidua, an immunohistochemical staining of FoxP3 of 32 PE and 34 control placentas was performed. A clearly reduced number of FoxP3-positive cells in the decidua of preeclamptic women could be shown in our analysis (p = 0.036). Furthermore, CCL22, a well-known Treg chemoattractant, was immunohistochemically evaluated. Interestingly, CCL22 expression was increased at the maternal-fetal interface in PE-affected pregnancies (psyncytiotrophoblast = 0.035, pdecidua = 0.004). Therefore, the hypothesis that Tregs undergo apoptosis at the materno-fetal interface during PE was generated, and verified by FoxP3/TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining. Galectin-2 (Gal-2), a member of the family of carbohydrate-binding proteins, which is known to be downregulated during PE, seems to play a pivotal role in T cell apoptosis. By performing a cell culture experiment with isolated Tregs, we could identify Gal-2 as a factor that seems to prevent the apoptosis of Tregs. Our findings point to a cascade of apoptosis of Tregs at the materno-fetal interface during PE. Gal-2 might be a potential therapeutic target in PE to regulate immune tolerance.

Background: Since the most well-known function of thyroid hormone receptors (TRs) relies on their ability to act as ligand-activated transcription factors, their subcellular localization has been recognized to be relevant for their biological meaning. The current study aimed to determine the prevalence and subcellular distribution of TR beta and TR beta-1 in ovarian cancer (OC). Methods: Tissue was collected from 153 patients that had undergone surgery due to OC at the Department of Obstetrics and Gynaecology of the Ludwig-Maximilians-University Munich. Immunohistochemistry detecting TR beta and TR beta-1 was performed. Staining signals were quantified and tested for association with clinico-pathological parameters including overall survival (OS). Results: The subcellular distribution of TR beta and TR beta-1 differed among histologic subtypes, grade and FIGO stage. TR beta positivity was strongly linked to shortened overall survival (p < 0.001). Strikingly, this shortened OS was mainly attributed to those cases showing complete (p = 0.005) or incomplete shift of TR beta to the cytoplasm (p < 0.001). Significance was lost in multivariate testing. Conclusions: Cytoplasmatic localization of TR beta was associated with reduced OS, at least in univariate analysis. Since TRs have long been supposed to mainly function via the regulation of gene transcription in the nucleus, cytoplasmatic shifting might be interpreted as a regulator of their activity.

Abstract Background In various cancers, overexpression of cyclooxygenase (COX)-2 and elevated prostaglandin (PG) E2 synthesis have been associated with tumor development and progression. The potential of COX-2 inhibitors in cancer prevention and treatment has been shown repeatedly; however, their clinical use is limited due to toxicity. PGE2 signals via EP receptors 1–4, whose functions are analyzed in current research in search for targeted anti-PG therapies. EP2 and EP4 rather promote tumorigenesis, while the role of EP3, especially in breast cancer, is not yet clear and both pro- and anti-tumorigenic effects have been described. Our study evaluates EP3 receptor expression in sporadic breast cancer and its association with clinicopathological parameters, progression-free and overall survival. Methods Two hundred eighty-nine sporadic breast cancer samples without primary distant metastasis were immunohistochemically analyzed for EP3 receptor expression. Tissue was stained with primary anti-EP3-antibodies. Immunoreactivity was quantified by the immunoreactivity-score (IRS); samples with an IRS ≥ 2 scored as EP3 positive. Chi-squared and Mann-Whitney-U test were used for comparison of data; Kaplan-Meier estimates and Cox-regression were used for survival analyses. Results EP3 receptor was expressed in 205 of 289 samples analyzed (70.9%). EP3 receptor expression was not associated with clinicopathological parameters (e. g. tumor size, hormone receptors, lymph node status). Kaplan-Meier estimates showed a significant association of EP3 positivity with improved progression-free survival (p = 0.002) and improved overall survival (p = 0.001) after up to 10 years. Cox regression analysis confirmed EP3 positivity as a significant prognostic factor even when other known prognosticators were accounted for. Conclusions In sporadic breast cancer, EP3 receptor expression is not significantly associated with clinicopathological parameters but is a significant prognostic factor for improved progression-free and overall survival. However, the functional aspects of EP3 receptor in breast cancer and the way how EP3 may oppose the pro-tumorigenic effects of PGE2 elevation and COX-2 overexpression are not fully understood so far. Further studies aiming at identification of the factors regulated by EP3 are necessary to evaluate the possibility of targeting EP3 in future anti-tumor therapy in breast cancer.

The platelet-activating factor receptor (PAFR) and its ligand (PAF) are important inflammatory mediators that are overexpressed in ovarian cancer. The receptor is an important player in ovarian cancer development. In this study, we aimed to evaluate the prognostic value of PAFR in epithelial ovarian cancer (EOC) and the potential use of its antagonist, rupatadine, as an experimental treatment. Tissue microarrays of ovarian cancer patients, most markedly those with a non-mucinous subtype, immunohistochemically overexpressed PAFR. Elevated cytoplasmic PAFR expression was found to significantly and independently impair patients’ overall and recurrence-free survival (OS: median 83.48 vs. 155.03 months; p = 0.022; RFS: median 164.46 vs. 78.03 months; p = 0.015). In vitro, the serous ovarian cancer subtypes especially displayed an elevated PAFR gene and protein expression. siRNA knockdown of PAFR decreased cell proliferation significantly, thus confirming the receptor’s protumorigenic effect on ovarian cancer cells. The clinically approved PAFR antagonist rupatadine effectively inhibited in vitro cell proliferation and migration of ovarian cancer cells. PAFR is a prognostic marker in ovarian cancer patients and its inhibition through rupatadine may have important therapeutic implications in the therapy of ovarian cancer patients.

Trace amine-associated receptor 1 (TAAR1) is a Gαs- protein coupled receptor that plays an important role in the regulation of the immune system and neurotransmission in the CNS. In ovarian cancer cell lines, stimulation of TAAR1 via 3-iodothyronamine (T1AM) reduces cell viability and induces cell death and DNA damage. Aim of this study was to evaluate the prognostic value of TAAR1 on overall survival of ovarian carcinoma patients and the correlation of TAAR1 expression with clinical parameters. Ovarian cancer tissue of n = 156 patients who were diagnosed with epithelial ovarian cancer (serous, n = 110 (high-grade, n = 80; low-grade, n = 24; unknown, n = 6); clear cell, n = 12; endometrioid, n = 21; mucinous, n = 13), and who underwent surgery at the Department of Obstetrics and Gynecology, University Hospital of the Ludwig-Maximilians University Munich, Germany between 1990 and 2002, were analyzed. The tissue was stained immunohistochemically with anti-TAAR1 and evaluated with the semiquantitative immunoreactive score (IRS). TAAR1 expression was correlated with grading, FIGO and TNM-classification, and analyzed via the Spearman’s rank correlation coefficient. Further statistical analysis was obtained using nonparametric Kruskal-Wallis rank-sum test and Mann-Whitney-U-test. This study shows that high TAAR1 expression is a positive prognosticator for overall survival in ovarian cancer patients and is significantly enhanced in low-grade serous carcinomas compared to high-grade serous carcinomas. The influence of TAAR1 as a positive prognosticator on overall survival indicates a potential prognostic relevance of signal transduction of thyroid hormone derivatives in epithelial ovarian cancer. Further studies are required to evaluate TAAR1 and its role in the development of ovarian cancer.

Aberrantly activated Wnt/β-catenin signaling pathway, as well as platelet-activating factor (PAF), contribute to cancer progression and metastasis of many cancer entities. Nonetheless, the role of the degradation enzyme named platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in ovarian cancer etiology is still unclear. This study investigated the functional impact of platelet-activating factor acetylhydrolase on BRCA1 mutant ovarian cancer biology and its crosstalk with the Wnt signaling pathway. PAF-AH, pGSK3β, and β-catenin expressions were analyzed in 156 ovarian cancer specimens by immunohistochemistry. PAF-AH expression was investigated in ovarian cancer tissue, serum of BRCA1-mutated patients, and in vitro in four ovarian cancer cell lines. Functional assays were performed after PLA2G7 silencing. The association of PAF-AH and β-catenin was examined by immunocytochemistry. In an established ovarian carcinoma collective, we identified PAF-AH as an independent positive prognostic factor for overall survival (median 59.9 vs. 27.4 months; p = 0.016). PAF-AH correlated strongly with the Wnt signaling proteins pGSK3β (Y216; nuclear: cc = 0.494, p < 0.001; cytoplasmic: cc = 0.488, p < 0.001) and β-catenin (nuclear: cc = 0.267, p = 0.001; cytoplasmic: cc = 0.291, p < 0.001). In particular, high levels of PAF-AH were found in tumor tissue and in the serum of BRCA1 mutation carriers. By in vitro expression analysis, a relevant gene and protein expression of PLA2G7/PAF-AH was detected exclusively in the BRCA1-negative ovarian cancer cell line UWB1.289 (p < 0.05). Functional assays showed enhanced viability, proliferation, and motility of UWB1.289 cells when PLA2G7/PAF-AH was downregulated, which underlines its protective character. Interestingly, by siRNA knockdown of PLA2G7/PAF-AH, the immunocytochemistry staining pattern of β-catenin changed from a predominantly membranous expression to a nuclear one, suggesting a negative regulatory role of PAF-AH on the Wnt/β-catenin pathway. Our data provide evidence that PAF-AH is a positive prognostic factor with functional impact, which seems particularly relevant in BRCA1 mutant ovarian cancer. For the first time, we show that its protective character may be mediated by a negative regulation of the Wnt/β-catenin pathway. Further studies need to specify this effect. Potential use of PAF-AH as a biomarker for predicting the disease risk of BRCA1 mutation carriers and for the prognosis of patients with BRCA1-negative ovarian cancer should be explored.

Histone H3 lysine 4 trimethylation (H3K4me3) is one of the most recognized epigenetic regulators of transcriptional activity representing, an epigenetic modification of Histone H3. Previous reports have suggested that the broad H3K4me3 domain can be considered as an epigenetic signature for tumor-suppressor genes in human cells. G-protein-coupled estrogen receptor (GPER), a new membrane-bound estrogen receptor, acts as an inhibitor on cell growth via epigenetic regulation in breast and ovarian cancer cells. This study was conducted to evaluate the relationship of GPER and H3K4me3 in ovarian cancer tissue samples as well as in two different cell lines (Caov3 and Caov4). Silencing of GPER by a specific siRNA and two selective regulators with agonistic (G1) and antagonistic (G15) activity were applied for consecutive in vitro studies to investigate their impacts on tumor cell growth and the changes in phosphorylated ERK1/2 (p-ERK1/2) and H3K4me3. We found a positive correlation between GPER and H3K4me3 expression in ovarian cancer patients. Patients overexpressing GPER as well as H3K4me3 had significantly improved overall survival. Increased H3K4me3 and p-ERK1/2 levels and attenuated cell proliferation and migration were observed in Caov3 and Caov4 cells via activation of GPER by G1. Conversely, antagonizing GPER activity by G15 resulted in opposite effects in the Caov4 cell line. In conclusion, interaction of GPER and H3K4me3 appears to be of prognostic significance for ovarian cancer patients. The results of the in vitro analyses confirm the biological rationale for their interplay and identify GPER agonists, such as G1, as a potential therapeutic approach for future investigations.

Vulvar cancer incidence numbers have been rising steadily over the past decades. Especially the number of young patients with vulvar cancer increased recently. Therefore, the need to identify new prognostic factors for vulvar carcinoma is more apparent. Cyclooxygenase-2 (COX-2) has long been an object of scientific interest in the context of carcinogenesis. This enzyme is involved in prostaglandin synthesis and the latter binds to nuclear receptors like PPARγ. Therefore, the aim of this study was to investigate COX-2- and PPARγ- expression in tissues of vulvar carcinomas and to analyze their relevance as prognostic factors. The cytoplasmatic expression of COX-2 as well as PPARγ is associated with a significantly reduced survival, whereas nuclear expression of PPARγ results in a better survival. Especially the combined expression of both COX-2 and PPARγ in the cytoplasm is an independent negative prognosticator for vulvar cancer patients.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are involved in various immune cell-mediated diseases. Their role in cancer is poorly investigated, and research focusses on Siglec-expression on immune cells interacting with tumor cells. This study evaluates the role of Siglec-8 in breast cancer (BC). Siglec-8 expression was analyzed immunohistochemically on 235 primary BC cases and was correlated with clinical and pathological parameters and outcome. Cell culture experiments were performed with various BC cell lines. Siglec-8 was expressed in 215 BC cases and expression was lowest in triple-negative BC. It correlated with estrogen receptor-status, grading and the prognostic factors galectin (Gal)-7 and tumor-associated mucin-1 (TA-MUC1). However, Gal-7 and TA-MUC1 were only prognosticators for clinical outcome in the cohort expressing high (Immunoreactivity score IRS > 3) Siglec-8 levels but not in the low-expressing cohort. Siglec-8 knockdown led to a significantly reduced Gal-7 expression in MCF7 cells. All BC cell lines expressed low Siglec-8-levels, that could be elevated in MCF7 by Peroxisome proliferator-activated receptor (PPARγ)-stimulation. This study demonstrates that Siglec-8 is expressed in BC cells and correlates with known clinical and prognostic parameters. It is probably associated with Gal-7 and TA-MUC1 and might be regulated via PPARγ. Further analyses focusing on functional associations will clarify Siglec-8’s eligibility as a possible therapeutic target.

So far, studies about targeted therapies and predictive biomarkers for vulva carcinomas are rare. The leucine zipper downregulated in cancer 1 gene (LDOC1) has been identified in various carcinomas as a tumor-relevant protein influencing patients’ survival and prognosis. Due to the lack of information about LDOC1 and its exact functionality, this study focuses on the expression of LDOC1 in vulvar carcinoma cells and its surrounding immune cells as well as its correlation to clinicopathological characteristics and prognosis. Additionally, a possible regulation of LDOC1 in vulvar cancer cell lines via the NF-κB signaling pathway was analyzed. Vulvar carcinoma sections of 157 patients were immunohistochemically stained and examined regarding LDOC1 expression by using the immunoreactive score (IRS). To characterize LDOC1-positively stained immune cell subpopulations, immunofluorescence double staining was performed. The effect of the NF-κB inhibitor C-DIM 12 (3,3′-[(4-chlorophenyl)methylene]bis[1 H-indole]) on vulvar cancer cell lines A431 and SW 954 was measured according to MTT and BrdU assays. Baseline expression levels of LDOC1 in the vulvar cancer cell lines A431 and SW 954 was analyzed by real-time PCR. LDOC1 was expressed by about 90% of the cancer cells in the cytoplasm and about half of the cells in the nucleus. Cytoplasmatic expression of LDOC1 was associated with decreased ten-year overall survival of the patient, whereas nuclear staining showed a negative association with disease-free survival. Infiltrating immune cells were mainly macrophages followed by regulatory T cells. Incubation with C-DIM 12 decreased the cell viability and proliferation of vulvar cancer cell line A431, but not of cell line SW 954. LDOC1 expression on mRNA level was twice as high in the cell line A431 compared to the cell line SW 954. Overexpression of LDOC1 was associated with unfavorable overall and disease-free survival. Tumor growth could be inhibited by C-DIM 12 in vitro if the expressed LDOC1 level was high enough.

Prognostic factors are of great interest in patients with endometrial cancer. One potential factor could be the protein MSX1, a transcription repressor, that has an inhibitory effect on the cell cycle. For this study, endometrioid endometrial carcinomas (n = 53), clear cell endometrial carcinomas (n = 6), endometrioid ovarian carcinomas (n = 19), and clear cell ovarian carcinomas (n = 11) were immunochemically stained for the protein MSX1 and evaluated using the immunoreactive score (IRS). A significant stronger expression of MSX1 was found in endometrioid endometrial carcinomas (p < 0.001), in grading 2 (moderate differentiation) (p = 0.001), and in tumor material of patients with no involvement of lymph nodes (p = 0.031). Correlations were found between MSX1 expression and the expression of β-Catenin, p21, p53, and the steroid receptors ERα, ERβ, PRα, and PRβ. A significant (p = 0.023) better survival for patients with an MSX1 expression in more than 10% of the tumor cells was observed for endometrioid endometrial carcinomas (21.3 years median survival (MSX1-positive) versus 17.3 years (MSX1-negative)). Although there is evidence that MSX1 expression correlates with improved long-term survival, further studies are necessary to evaluate if MSX1 can be used as a prognostic marker.

Posttranslational histone modification plays an important role in tumorigenesis. Histone modification is a dynamic response of chromatin to various signals, such as the exposure to calcitriol (1α,25(OH)2D3). Recent studies suggested that histone modification levels could be used to predict patient outcomes in various cancers. Our study evaluated the expression level of histone 3 lysine 4 trimethylation (H3K4me3) in a cohort of 156 epithelial ovarian cancer (EOC) cases by immunohistochemical staining and analyzed its correlation to patient prognosis. The influence of 1α,25(OH)2D3 on the proliferation of ovarian cancer cells was measured by BrdU proliferation assay in vitro. We could show that higher levels of H3K4me3 were correlated with improved overall survival (median overall survival (OS) not reached vs. 37.0 months, p = 0.047) and identified H3K4me3 as a potential prognostic factor for the present cohort. Ovarian cancer cell 1α,25(OH)2D3 treatment induced H3K4me3 protein expression and exhibited antiproliferative effects. By this, the study suggests a possible impact of H3K4me3 expression on EOC progression as well as its relation to calcitriol (1α,25(OH)2D3) treatment. These results may serve as an explanation on how 1α,25(OH)2D3 mediates its known antiproliferative effects. In addition, they further underline the potential benefit of 1α,25(OH)2D3 supplementation in context of ovarian cancer care.

AIM:The objective of this study was to evaluate the prognostic value of ARID1A, p53, p21, p16 and ß-Catenin in endometrioid and clear cell ovarian and endometrial carcinomas. MATERIALS AND METHODS:97 tumors were available for analysis of ARID1A, p53, p21, p16 and ß-Catenin with the techniques of tissue microarray and immunohistochemistry. 32 were ovarian carcinomas and 65 were endometrial carcinomas. RESULTS:Endometrioid ovarian carcinomas showed negative staining for ARID1A (a) and p21 (b), aberrant expression of p53 (c) and p16 (d) and ß-Catenin positive nuclear expression (e) respectively in 19% (a), 100% (b), 28.6% (c), 52.4% (d) and 4.8% (e) of all cases. In the group of clear cell ovarian carcinomas it was 63.6% (a), 100% (b), 81.8% (c), 54.5% (d) and 0% (e). For endometrioid uterine carcinomas it was 75.7% (a), 94.9% (b), 30.5% (c), 52.1% (d) and 6.8% (e) and for clear cell uterine carcinomas it was 8.6% (a), 100% (b), 50% (c), 100% (d) and 0% (e). Survival analysis showed that negative expression of ARID1A, p53 aberrant expression and ß-Catenin nuclear positive staining are independent negative prognosticators in both, clear cell and endometrioid carcinoma, regardless of ovarian or uterine origin. Cox-Regression analysis showed them again as negative prognostic factors. Furthermore, we found a significant correlation between ARID1A and ß-Catenin expression in endometrioid uterine tumors. CONCLUSION:The analyzed gynaecological carcinoma showed a distinct expression scheme of proteins that are associated with tumor suppression. We may conclude that ARID1A, p53 and ß-Catenin are the strongest prognostic factors by analyzing a subgroup of tumor suppressor genes in clear cell and endometrioid subtypes of ovarian and endometrial cancer and may be used along with traditional morphological and clinical characteristics for prognosis.

Anti-tumor efficacy of Gatipotuzumab, a therapeutic antibody targeting Tumor-Associated Mucin-1 (TA-MUC1), in relapsed ovarian cancer (OC) appeared to be rather heterogeneous. Whether adding a second anti-neoplastic drug may augment response towards Gatipotuzumab, has not been elucidated so far. Since it is known that anti-MUC1 antibodies may alter estrogen receptor activity in breast cancer, this potential interplay was investigated in OC. The correlation between TA-MUC1, estrogen receptors (ERs) and another 12 protein markers as well as their correlation with clinico-pathological parameters in 138 ovarian cancer cases was studied. Finally, Gatipotuzumab and 4-Hydroxy-TTamoxifen (4-OHT) as well as the combination of both was tested for its impact on cell viability in COV318, OV-90, OVCAR-3, and SKOV-3 cells. A strong positive correlation between TA-MUC1 and ERs was detected in OC tissue. Those cases missing ERs but staining positive for TA-MUC1 had significantly reduced overall survival. The combination of 4-OHT and Gatipotuzumab significantly reduced cell viability and was more effective than treatment with Gatipotuzumab alone. Co-stimulation with Gatipotuzumab enhanced the efficacy of 4-OHT in OVCAR-3 and SKOV-3. The data suggest an interplay of TA-MUC1 and ERs in OC. Whether the combination of Gatipotuzumab and TTamoxifen may enhance efficacy of either of the two drugs in vivo, or may even translate into a clinically relevant benefit over the respective monotherapies, remains to be investigated.

Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cytoprotective antioxidant processes. In this study, the prognostic potential of NRF2 and its interactions with the estrogen receptor α (ERα) in ovarian cancer cells was investigated. NRF2 and ERα protein expression in ovarian cancer tissue was analyzed as well as mRNA expression of NRF2 (NFE2L2) and ERα (ESR1) in four ovarian cancer and one benign cell line. NFE2L2 silencing was carried out to evaluate a potential interplay between NRF2 and ERα. Cytoplasmic NRF2 expression as inactive form had significantly higher expression in patients with low-grade histology (p = 0.03). In the serous cancer subtype, high cytoplasmic NRF2 expression (overall survival (OS), median 50.6 vs. 29.3 months; p = 0.04) and high ERα expression (OS, median 74.5 vs. 27.1 months; p = 0.002) was associated with longer overall survival as well as combined expression of both inactive cytoplasmic NRF2 and ERα in the whole cohort (median 74.5 vs. 37.7 months; p = 0.04). Cytoplasmic NRF2 expression showed a positive correlation with ERα expression (p = 0.004). NFE2L2 was found to be highly expressed in the ovarian cancer cell lines OVCAR3, UWB1.289, and TOV112D. Compared with the benign cell line HOSEpiC, ESR1 expression was reduced in all ovary cancer cell lines (all p < 0.001). Silencing of NFE2L2 induced a higher mRNA expression of ESR1 in the NFE2L2 downregulated cancer cell lines OVCAR3 (p = 0.003) and ES2 (p < 0.001), confirming genetic interactions of NRF2 and ERα. In this study, both inactive cytoplasmic NRF2 and high ERα expression were demonstrated to be associated with improved survival in ovarian cancer patients. Further understanding of interactions within the estradiol–ERα–NRF2 pathway could better predict the impact of endocrine therapy in ovarian cancer.

PPARγ belongs to the group of nuclear receptors which is expressed in the trophoblast and together with other factors is responsible for the maintenance of pregnancy. Apart from that PPARγ is also a main factor for macrophage polarization. The aim of this study was to investigate the combined expression pattern and frequency of PPARγ under physiological circumstances and in spontaneous and recurrent miscarriages in the trophoblast and in maternal macrophages of the decidua. Human placental tissues of the first trimester (15 physiologic pregnancies, 15 spontaneous abortion and 16 recurrent miscarriage placentas) were analyzed for expression of the nuclear receptor PPARγ. Expression changes were evaluated by immunohistochemistry and real time PCR (RT-PCR) in trophoblast and in maternal macrophages of the decidua. Maternal macrophages were identified by double immunofluorescence using cluster of differentiation 68 (CD68) as marker for macrophages and further characterized regarding their M1/M2 polarization status. The intermediate villous trophoblast revealed a significantly lower PPARγ expression in spontaneous and recurrent abortion. Maternal macrophages express PPARγ. Their number is significantly enhanced in the decidua of spontaneous miscarriages whereas in recurrent miscarriages maternal macrophages seem to express PPARγ only in very few cases. PPARγ is associated with an M2 polarization state that is common for decidual macrophages. The lack of PPARγ in recurrent miscarriage decidual macrophages seems to be associated with a specific inflammatory response against the fetus.

The evaluation of new prognostic factors that can be targeted in ovarian cancer diagnosis and therapy is of the utmost importance. Galectins are a family of carbohydrate binding proteins with various implications in cancer biology. In this study, the presence of galectin (Gal)-8 and -9 was investigated in 156 ovarian cancer samples using immunohistochemistry (IHC). Staining was evaluated using semi-quantitative immunoreactivity (IR) scores and correlated to clinical and pathological data. Different types of galectin expression were compared with respect to disease-free survival (DFS) and overall survival (OS). Gal-8 served as a new positive prognostic factor for the OS and DFS of ovarian cancer patients. Gal-9 expression determined the DFS and OS of ovarian cancer patients in two opposing ways—moderate Gal-9 expression was correlated with a reduced outcome as compared to Gal-9 negative cases, while patients with high Gal-9 expression showed the best outcome.

There is a tremendous need for developing new useful prognostic factors in ovarian cancer. Galectins are a family of carbohydrate binding proteins which have been suggested to serve as prognostic factors for various cancer types. In this study, the presence of Galectin-1, -3, and -7 was investigated in 156 ovarian cancer specimens by immunochemical staining. Staining was evaluated in the cytoplasm and nucleus of cancer cells as well as the peritumoral stroma using a semi quantitative score (Remmele (IR) score). Patients’ overall survival was compared between different groups of Galectin expression. Galectin (Gal)-1 and -3 staining was observed in the peritumoral stroma as well as the nucleus and cytoplasm of tumor cells, while Gal-7 was only present in the cytoplasm of tumor cells. Patients with Gal-1 expression in the cytoplasm or high Gal-1 expression in the peritumoral stroma showed reduced overall survival. Nuclear Gal-3 staining correlated with a better outcome. We observed a significantly reduced overall survival for cases with high Gal-7 expression and a better survival for Gal-7 negative cases, when compared to cases with low expression of Gal-7. We were able to show that both tumor and stroma staining of Gal-1 could serve as negative prognostic factors for ovarian cancer. We were able to confirm cytoplasmic Gal-7 as a negative prognostic factor. Gal-3 staining in the nucleus could be a new positive prognosticator for ovarian cancer.