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APOBEC3 enzymes are part of the innate immune system and they are important in retroviral defense. The number of mutations in ovarian cancer increases with rising levels of APOBEC3B mRNA. We could confirm that APOBEC3B mRNA is upregulated in ovarian cancer cell lines and in ovarian cancer tissue. We evaluated APOBEC3B expression in histologically defined subtypes of ovarian cancer to identify its influence on overall survival (OS) and progression-free survival (PFS). Tissue microarrays from 219 patients with high-grade serous (HGSC), 61 with low-grade serous (LGSC), 62 with endometrioid (EC) and 55 with clear cell (CCC) ovarian carcinoma were stained using an antibody against APOBEC3B. Real-time quantitative PCR was performed to detect APOBEC3B mRNA levels in 274 cases of HGSC, in 11 cases of LGSC, in 47 cases of EC and in 29 cases of CCC. Tumor-infiltrating lymphocytes (TILs) have been evaluated in a previous project. APOBEC3B staining was cytoplasmic as well as nuclear and both were positively correlated (P<0.001). In HGSC a trend was detectable for positive cytoplasmic staining as favorable regarding OS (P=0.283) and PFS (P=0.137). High levels of APOBEC3B mRNA were associated with prolonged PFS in HGSC in univariate analyses (P=0.043) and multivariate analyses (HR 0.55; 95%CI 0.35-0.88; P=0.012). APOBEC3B cytoplasmic staining and APOBEC3B mRNA were positively correlated with TILs. APOBEC3B in HGSC is related to an active immune infiltrate. However, there is no evidence for APOBEC3B as a clinically relevant prognostic biomarker.

High-grade serous ovarian carcinoma remains one of the most lethal malignancies in women. For histopathologic differentiation from mesothelioma cytokeratin, 5/6 immunohistochemistry is widely used. Another preferred marker for differential diagnosis to mesothelioma is estrogen receptor α (ER-α). In this study, we determined the rate of cytokeratin 5/6-positive cells in primary high-grade serous carcinoma. A cohort of 215 patients with high-grade serous ovarian carcinoma was evaluated immunohistochemically for the protein expression of cytokeratin 5/6. Most tumors demonstrated at least partly positive for cytokeratin 5/6 (n=148; 68.3%), showing different staining patterns from scattered stained cells to a diffuse staining, at times with a distinctive tumor-stroma border motif. Sixty-seven (31%) were entirely negative. No correlation of cytokeratin immunoreactivity score (IRS) with conventional staging parameters could be demonstrated. From the different IRS values for cytokeratin 5/6, IRS=12 (n=6; 2.9%) seemed to indicate a worse prognosis, albeit not statistically significant. An association with ER-α expression could not be detected but the combination of cytokeratin 5/6 IRS=12 and ER-α negativity resulted in a significant negative prognostic marker (overall survival: P=.003 and progression-free survival: P<.0001). We substantiate cytokeratin 5/6 protein expression as a frequent feature of high-grade serous ovarian carcinoma with various staining patterns, an important fact for the routine differential diagnosis with mesothelioma. Furthermore, cytokeratin 5/6 in combination with ER-α proved to be a negative prognostic marker, wherefore we suggest further investigation of its biological significance and possible manifestation of a basal differentiation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

HER3 is a member of the epidermal growth factor family and was predominantly described as a negative prognostic factor in various solid tumors as well as in ovarian cancer. In this study, we investigated HER3 on protein and mRNA expression in histologically defined subtypes of ovarian cancer looking for an influence on patient's survival. Altogether, we examined HER3 in ovarian high-grade serous (HGSC, n = 320), low-grade serous (LGSC, n = 55), endometrioid (EC, n = 33), and clear cell (CCC, n = 48) carcinomas using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR (qRT-PCR). Univariate and multivariate analyses were performed to explore the association between HER3 and overall survival (OS) as well as progression-free survival (PFS). In HGSC, high HER3 mRNA expression was a favorable prognostic factor for PFS (P = 0.008) and OS (P = 0.052), while for high HER3 protein expression, a trend towards better survival was seen (OS P = 0.064; PFS P = 0.099). A subgroup of HGSC with negative HER3 staining and negative HER3 mRNA levels showed most unfavorable OS and PFS (P = 0.002 and P = 0.004, respectively). Using the multivariate Cox regression model, HER3 was predictive for prolonged PFS (HR, 0.48; 95% CI, 0.26-0.88; P = 0.018). All in all, we cannot confirm the reported negative prognostic impact of HER3 expression in high-grade serous ovarian carcinoma and moreover find a rather positive prognostic implication of HER3 in this major ovarian cancer histological subtype.

With the approval of olaparib as monotherapy treatment in platinum-sensitive, relapsed high-grade serous ovarian cancer by the European Medical Agency (EMA), comprehensive genotyping of BRCA1 and BRCA2 in tumor tissue has become a mandatory pre-therapeutic test. This requires significant advances in routine tumor test methodologies due to the large size of both genes and the lack of mutational hot spots. Classical focused screening approaches, like Sanger sequencing, do not allow for a sensitive, rapid, and economic analysis of tumor tissue. Next-generation sequencing (NGS) approaches employing targeted panels for BRCA1/2 to interrogate formalin-fixed and paraffin-embedded tumor samples from either surgical resection or biopsy specimens can overcome these limitations. Although focused NGS methods have been implemented by few centers in routine molecular diagnostics for the analysis of some druggable oncogenic mutations, the reliable diagnostic testing of the entire coding regions of BRCA1 and BRCA2 was a new challenge requiring extensive technological improvement and quality management. Here, we describe the implementation and results of the first round robin trial for BRCA1/2 mutation testing in tumor tissue that was conducted in central Europe on May 2015, shortly after the approval and prior to the official release of olaparib. The high success rate of 81 % (21/26 test centers) demonstrates that BRCA1/2 multicenter mutation testing is well feasible in FFPE tumor tissue, extending to other tumor entities beyond ovarian cancer. The high number of test centers passing the trial demonstrates the success of the concerted efforts by German, Swiss, and Austrian pathology centers to ensure quality-controlled NGS-based testing and proves the potential of this technology in routine molecular pathology. On the basis of our results, we provide recommendations for predictive testing of tumor tissue for BRCA1/2 to clinical decision making in ovarian cancer patients.

Aims: Wilms tumor protein 1 (WT1) expression is used in gynecological pathology as a diagnostic marker of serous differentiation, and is frequently co-expressed with ER-α. Early phase studies on WT1 vaccine in gynecological cancers are ongoing. In this study we aimed to determine the prognostic value of WT1 in high-grade serous ovarian carcinoma., Methods: WT1 protein expression was determined by immunohistochemistry in a cohort of 207 primary high-grade serous ovarian carcinomas. WT1 mRNA expression was evaluated in a cohort of 1137 ovarian carcinomas from publically available gene expression datasets., Results: High WT1 expression was a significant positive prognostic factor in primary high-grade serous ovarian carcinoma regarding overall survival (OS, p=0.008) and progression free survival (PFS, p=0.015), which was independent of age, stage, and residual tumor (OS: p=0.024, PFS: p=0.047). The prognostic significance of immunohistochemical WT1 expression could be reproduced in an independent cohort of 72 patients. On the mRNA level the prognostic significance was validated in silico in publically available gene expression datasets including TCGA data (OS: p=0.002, PFS: p=0.011). WT1 expression was significantly linked to ER-α expression (p=0.001), and tumors that co-expressed both markers (WT1+/ER-α+) had a longer survival time than tumors of all other marker combinations (OS: p=0.002, PFS: p=0.013)., Conclusion: We present WT1 as a robust prognostic marker in high-grade serous ovarian carcinoma, which adds prognostic information to ER-α. This should be kept in mind when WT1 is used as a biomarker in the context of WT1-targeting therapies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ovarian cancer is a heterogeneous disease of low prevalence, but poor survival. Early diagnosis is critical for survival, but it is often challenging because the symptoms of ovarian cancer are subtle and become apparent only during advanced stages of the disease. Therefore, the identification of robust biomarkers of early disease is a clinical priority. Metabolomic profiling is an emerging diagnostic tool enabling the detection of biomarkers reflecting alterations in tumor metabolism, a hallmark of cancer. In this study, we performed metabolomic profiling of serum and tumor tissue from 158 patients with high-grade serous ovarian cancer (HGSOC) and 100 control patients with benign or non-neoplastic lesions. We report metabolites of hydroxybutyric acid (HBA) as novel diagnostic and prognostic biomarkers associated with tumor burden and patient survival. The accumulation of HBA metabolites caused by HGSOC was also associated with reduced expression of succinic semialdehyde dehydrogenase (encoded by ALDH5A1), and with the presence of an epithelial-to-mesenchymal transition gene signature, implying a role for these metabolic alterations in cancer cell migration and invasion. In conclusion, our findings represent the first comprehensive metabolomics analysis in HGSOC and propose a new set of metabolites as biomarkers of disease with diagnostic and prognostic capabilities., (©2015 American Association for Cancer Research.)

Aims: Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma., Methods: PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset., Results: PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively)., Conclusions: Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity.

Background/aim: 3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates the cellular effects of various growth factors. Increased PDK1 expression is present in various cancers, suggesting that PDK1 may be a critical oncogene in cancer progression. However, only limited data exist on PDK1 expression in ovarian serous cancer., Materials and Methods: We used tissue microarrays to analyze PDK1 expression in 253 primary ovarian serous carcinoma samples., Results: A statistically significant negative correlation between PDK1 expression and tumor grade was found. In the high-grade group of ovarian serous carcinomas (n=189), there was a statistically significant difference in overall survival between cases with positive and negative PDK1 expression (p=0.035); positive cases showed longer overall survival. Multivariate analysis confirmed that slight PDK1 expression was an independent indicator for prolonged overall survival (HR=0.51, 95% CI=0.28-0.92, p=0.025)., Conclusion: PDK1 appears to be a prognostic marker and a possible therapeutic target in ovarian serous carcinoma., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Cyclin A1 is essential for male gametopoiesis. In acute myeloid leukemia, it acts as a leukemia-associated antigen. Cyclin A1 expression has been reported in several epithelial malignancies, including testicular, endometrial, and epithelial ovarian cancer (EOC). We analyzed Cyclin A1 expression in EOC and its correlation with clinical features to evaluate Cyclin A1 as a T-cell target in EOC., Methods: Cyclin A1 mRNA expression in EOC and healthy tissues was quantified by microarray analysis and quantitative real-time PCR (qRT-PCR). Protein expression in clinical samples was assessed by immunohistochemistry (IHC) and was correlated to clinical features., Results: Cyclin A1 protein was homogeneously expressed in 43 of 62 grade 3 tumor samples and in 1 of 10 grade 2 specimens (p < 0.001). Survival analysis showed longer time to progression (TTP) among patients with at least moderate Cyclin A1 expression (univariate: p = 0.018, multivariate: p = 0.035). FIGO stage, grading, age, macroscopic residual tumor after debulking, and peritoneal carcinomatosis / distant metastasis had no impact on TTP or overall survival (OS)., Conclusion: Cyclin A1 is highly expressed in most EOCs. The mechanism behind the prolonged TTP in patients with high Cyclin A1 expression warrants further investigation. The frequent, selectively high expression of Cyclin A1 in EOC makes it a promising target for T-cell therapies.

Neuroendocrine differentiation in high-grade serous ovarian carcinomas has only rarely been described. However, in our consultancy experience, we have been pointed at a case of neuroendocrine relapse in a patient with high-grade serous ovarian carcinoma where retrospectively, a minor neuroendocrine component in the primary tumor could be detected. Hypothesizing that immunohistochemical evidence of neuroendocrine differentiation might be more frequent in ovarian carcinoma than suspected by morphology, we immunophenotyped the tissue microarrays (TMAs) of a cohort of 178 high-grade serous carcinomas for chromogranin and synaptophysin expression. Synaptophysin expression was found in 12 (6.7 %) out of 172 patients, and chromogranin A expression was seen in 36 (20.7 %) out of 174 patients. Kaplan-Meier analysis revealed that carcinomas with synaptophysin expression of >20 % of positive cells (n = 4) had a significantly shorter survival time than those with 0-20 % of positive cells (p < 0.0001). Synaptophysin expression remained a significant prognostic factor in multivariate analysis (HR = 10.82, 95 % confidence interval 3.10-37.71, p < 0.0001), independently of age, FIGO stage, and residual tumor after surgery. A trend toward shorter survival was seen in patients with tumors that expressed chromogranin, irrespective of the amount of positive cells (p = 0.173). A neuroendocrine differentiation is important to keep in mind when a neuroendocrine tumor of unknown primary is detected in regional or temporal connection with an ovarian carcinoma. A minor neuroendocrine component in ovarian high-grade serous carcinomas might imply a dismal prognosis.

The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor κB pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P=0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of κB α (IκBα) (P=0.001) as well as nuclear and cytoplasmic phospho-IκBα (p-IκBα) expression (P=0.039 and P=0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P=0.002), and in multivariate analysis [hazard ratio=0.35 (95% confidence interval, 0.14-0.84, P=0.019)]. ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor κB pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor κB, particularly with regard to response to chemotherapy, would be desirable.

In models of triple-negative breast cancer (TNBC), it has recently been shown that the epidermal growth factor receptor (EGFR) pathway is up-regulated in the aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cell fraction. Because high-grade serous ovarian carcinoma (HGSC) reveals strong molecular similarities to TNBC, we aimed to investigate the potential link between ALDH1 and EGFR in this entity. Expression of ALDH1 was investigated in 131 primary HGSCs using immunohistochemistry. Expression data were correlated with EGFR expression as well as with clinicopathologic parameters and survival. Forty-two carcinomas (32.1%) were positive for ALDH1 protein expression. Data on EGFR expression were available for 112 tumors. In these cases ALDH1 was significantly linked to EGFR expression (P < .0001). ALDH1 positivity was a significant negative prognostic factor for overall survival both in univariate (P = .010) and in multivariate survival analyses (P = .041). Tumors that were positive for both ALDH1 and EGFR had an exceptionally bad prognosis as compared with carcinomas with 1 or both markers negative in univariate analysis (P < .0001) and in the multivariate setting (P = .004). Our study suggests that similar to TNBC, there is a link between ALDH1 and EGFR expression in HGSC. Double positivity for both markers identifies a subgroup of highly aggressive, poor-prognosis cancers for which alternative treatment options-potentially EGFR-targeting drugs-should be evaluated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ovarian high-grade serous carcinoma (HGSC, type 2 ovarian carcinoma) is a poor prognosis cancer with limited therapeutic options. We aimed to investigate the expression pattern and prognostic potential of the metastasis-promoting protein anterior gradient 2 (AGR2) in primary HGSC. Immunohistochemistry was applied to a cohort of 124 primary HGSCs using tissue microarrays. Additionally, in 48 type 1 carcinomas (low-grade serous (LGSC), endometrioid (EC), clear cell (CCC), and mucinous carcinoma (MC)), AGR2 expression was investigated in an exploratory approach. A strong expression of AGR2 was seen in 15 HGSCs (12.1 %) and was significantly linked to shortened overall survival (OS, p = 0.011) and also for progression-free survival (PFS, p = 0.001) in the setting of adjuvant platinum-based chemotherapy (CTX). Multivariate survival analysis including age, stage, and residual tumor after surgery revealed that AGR2 expression was an independent prognostic marker for OS (p = 0.001) and PFS (p = 0.001) in HGSC. In type 1 carcinomas, AGR2 was significantly increased as compared to HGSC (p = 0.001) and was seen in subsets of all histological types, low-grade serous LGSC, EC, CCC, and MC. In particular, strong diffuse staining was seen in LGSC and MC. There was no association between AGR2 and estrogen receptor expression in ovarian type 1 or type 2 carcinomas. AGR2 expression identifies highly aggressive HGSC with a compromised prognosis for which novel therapeutic options are needed. Our data strongly support the further evaluation of AGR2 as a therapeutic target and a potential marker for response to platinum-based CTX in this tumor entity.

Aims: In vitro and epidemiological studies indicate an essential role for progesterone in the aetiology and progression of ovarian carcinoma. The aim of this study was to examine the prognostic role of progesterone receptor (PR) protein and mRNA expression., Methods and Results: PR expression was examined by immunohistochemistry (n=143) and kinetic reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed and paraffin-embedded tissue (n=55). PR mRNA and protein expression correlated (P<0.0001). PR mRNA was a positive predictor for overall and progression-free survival (P=0.0005 and P<0.0001, respectively). Protein expression was also prognostic (P=0.015 and P=0.0011, respectively), whereas only PR mRNA retained its prognostic value on multivariate analysis (P=0.04). PR mRNA was still a positive prognostic marker among oestrogen receptor 1 (ESR1) mRNA-positive tumours (P=0.0007) and survival was best in patients with PR- and ESR1-positive phenotypes (P=0.0155 and P=0.0016, respectively)., Conclusion: Expression of PR and ESR1 defines a subgroup of ovarian carcinomas with a favourable prognosis. PR and ESR1 mRNA expression analysis is a sensitive, quantitative and easy-to-perform high-throughput analytical tool for the identification of this subgroup and could be predictive in clinical trials focused on patients with potential benefit from hormonal treatment., (© 2011 Blackwell Publishing Limited.)

Estrogen as a potential factor of ovarian carcinogenesis, acts via two nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), but the cellular signal pathways involved are not completely clear so far. In this study we have described the expression of ERα, detected by immunocytochemistry in 11 ovarian carcinoma cell lines and by immunohistochemistry in 43 Federation Internationale des Gyneacologistes et Obstetristes stage III ovarian carcinoma specimens prepared before and after treatment with cisplatin-based schemes. For cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma, analysis of cisplatin sensitivity in 11 ovarian carcinoma cell line was also performed. The strong nuclear ERα expression was only shown in the single A2780P cell line. Expression of ERα in tissue specimens did not reveal any correlations between histopathological parameters (histologic type and grading). We demonstrated a significant association with ERα expression in specimens from primary laparotomies (PL) and cause-specific survival. In the cases terminated by death of the patient, overall immunoreactivity score of ERα expression at PL was significantly lower than in surviving patients. In addition, Kaplan-Meier analysis revealed significantly shorter overall survival time and progression-free time in cases with lower immunoreactivity score of ERα expression at PL. Our findings support the hypothesis that aberrant hormone activity, by way of altered receptor expression, might be an important factor in the malignant transformation of ovarian cancer.

Background: In ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting., Methods: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated EGFR expression in ten ovarian cancer cell lines and incubated cancer cells with Leptomycin B, a CRM1 specific inhibitor., Results: We observed a membranous and cytoplasmic EGFR expression in 36.4% and 64% of ovarian carcinomas, respectively. Membranous EGFR was an independent prognostic factor for poor overall survival in ovarian cancer patients (HR 2.7, CI 1.1-6.4, p = 0.02) which was also found in the serous subtype (HR 4.6, CI 1.6-13.4, p = 0.004). We further observed a significant association of EGFR with COX-2 and nuclear CRM1 expression (chi-square test for trends, p = 0.006 and p = 0.013, respectively). In addition, combined membranous EGFR/COX-2 expression was significantly related to unfavorable overall survival (HR 7.2, CI 2.3-22.1, p = 0.001).In cell culture, we observed a suppression of EGFR protein levels after exposure to Leptomycin B in OVCAR-3 and SKOV-3 cells., Conclusions: Our results suggest that the EGFR/COX-2/CRM1 interaction might be involved in progression of ovarian cancer and patient prognosis. Hence, it is an interesting anti-cancer target for a combination therapy. Further studies will also be needed to investigate whether EGFR is also predictive for benefit from EGFR targeted therapies.

AMP-activated protein kinase (AMPK) plays a central role in regulating energy metabolism in cells. AMPK activation results in down-regulation of anabolic pathways (e.g., fatty acid biosynthesis) and switches on catabolic processes such as glucose uptake, glycolysis or fatty acid oxidation. Recent studies in cell culture models have shown that the growth of tumor cell lines was inhibited by AMPK activation, but the expression of AMPK in human ovarian tumors has not been reported so far. In this study we investigated AMPK expression in a cohort of 70 ovarian carcinomas, 14 borderline tumors and 5 normal ovaries and linked the protein expression data to Gas chromatography/ time of flight mass spectrometry (GC/TOF-MS) based metabolomics. We observed a significantly higher expression in ovarian carcinomas compared to borderline tumors and normal ovaries (p=0.038). Decreased AMPK expression correlated significantly with higher tumor grade (p=0.009) and was of adverse prognosis in patients with advanced tumor stages (p=0.016) as well as in patients with serous ovarian carcinomas (p=0.037). GC/TOF-MS based metabolomics revealed a significantly higher concentration of glucose in AMPK-negative carcinomas (p=0.022) as well as overexpression of other metabolites from carbohydrate metabolism. Our results indicate a role for AMPK in progression of ovarian tumors and point towards a prognostic impact of AMPK expression for patient overall survival. Furthermore, our data suggest a deregulation of the AMPK-dependent energy metabolism in human ovarian carcinomas. In future clinical studies, activation of AMPK in ovarian carcinoma patients with advanced tumor stages might be an interesting therapeutic approach.

Intracellular signaling governed by serine/threonine kinases comprises the molecular interface between cell surface receptors and the nuclear transcriptional machinery. The protein kinase C (PKC) family members are involved in the control of many signaling processes directing cell proliferation, motility, and survival. Here, we examined a role of different PKC isoenzymes in protein phosphatase 2A (PP2A) and HRSL3 tumor suppressor-dependent cell death induction in the ovarian carcinoma cell line OVCAR-3. Phosphorylation and activity of PKC isoenzymes were measured in response to PP2A or phosphoinositide 3-kinase inhibition or HRSL3 overexpression. These experiments indicated a regulation of PKC, epsilon, zeta, and iota through PP2A and/or HRSL3, but not of PKCalpha and beta. Using isoform-specific peptide inhibitors and overexpression approaches, we verified a contribution to PP2A- and HRLS3-dependent apoptosis only for PKCzeta, suggesting a proapoptotic function of this kinase. We observed a significant proportion of human ovarian carcinomas expressing high levels of PKCzeta, which correlated with poor prognosis. Primary ovarian carcinoma cells isolated from patients also responded to okadaic acid treatment with increased phosphorylation of PKCzeta and apoptosis induction. Thus, our data indicate a contribution of PKCzeta in survival control in ovarian carcinoma cells and suggest that upregulation or activation of tyrosine kinase receptors in this tumor might impinge onto apoptosis control through the negative regulation of the atypical PKCzeta.

Aims: Functional studies have demonstrated that nuclear factor (NF)-kappaB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF-kappaB pathway in human ovarian cancer in vivo., Methods and Results: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho-IkappaBalpha (P = 0.002 and P = 0.05, respectively), and IkappaBalpha mRNA expression (P = 0.032). In contrast, phospho-p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho-IkappaBalpha (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IkappaBalpha and phosphorylated nuclear and cytoplasmic IkappaBalpha expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho-IkappaBalpha expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR-3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001)., Conclusions: Total and phosphorylated IkappaBalpha protein expression might serve as markers for NF-kappaB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.

Background: Tamoxifen, used in breast cancer treatment, competitively inhibits estrogen receptor (ER) and also demonstrates direct antiproliferative effect on cancer cells even in ER lacking cancer tissue. However its molecular mechanism of action is still unclear, Material and Methods: We exposed on tamoxifen 11 ovarian cancer cell lines, including well-documented platinum-sensitive and platinum-resistant ones, and studied tamoxifen-, cisplatin-sensitivity and expression of ERalpha and beta., Results: We observed: no correlation between TAM-sensitivity and ERalpha and ERbeta expressions, no correlation between TAM influence on cisplatin-sensitivity and ERalpha and ERbeta expressions, increase of ERbeta expression after TAM-exposure in 3 cell lines; decrease in the 1 line, no TAM-exposure influence on ERalpha expression and increase of 1050 for cisplatin after TAM-exposure in 5 (45%) cell lines. These results show ovarian cancer cells being affected by TAM have different platinum sensitivity, Conclusions: Our data suggests that ovarian cancer cells platinum-sensitivity are not linked with ER expressions. We claim the necessity of seeking some TAM predicting factors, using DNA microarrays.

MicroRNA (miRNA) deregulation is a hallmark of human cancer. However, the mechanisms underlying miRNA alteration and the specific role of proteins involved in miRNA processing remains to be elucidated. Dicer is a key enzyme in the miRNA processing pathway that is essential for the production of mature miRNAs from their precursors. We tested the hypothesis that Dicer has biological and clinical relevance in ovarian cancer, using a range of methods including in vitro manipulation of Dicer expression. We observed down-regulation of Dicer in a subgroup of ovarian carcinomas, and found that decreased Dicer expression correlates significantly with reduced patient survival in serous cancers and advanced disease stages. Moreover, microarray and functional analysis suggest that reduced Dicer expression is connected with a global down-regulation of the microRNAome and with gene expression changes, particularly reduced expression of oestrogen receptor (ER) mRNA and protein in tumour tissue and in cell culture. Our data suggest a common mechanism for miRNAs changes by alterations in the basic machinery controlling miRNA biogenesis, of which Dicer is a central enzyme. These alterations of miRNA processing are of prognostic value and may play a role in the molecular pathogenesis of ovarian carcinoma and, possibly, other tumours. Knowledge of these molecular pathways may help toward new targeted therapeutic approaches for ovarian cancer., (Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Epidemiological and cell culture studies indicate that ovarian carcinoma growth is dependent on estrogen stimulation. However, possibly due to the lack of a reliable biomarker that helps to select patients according to prognostically relevant estrogen receptor (ER) levels, clinical trials using anti-estrogenic therapeutics in ovarian carcinoma have had inconsistent results. Therefore, we tested if ER expression analysis by a quantitative method might be useful in this regard in formalin-fixed paraffin-embedded (FFPE) tissue. In a study group of 114 primary ovarian carcinomas expression of estrogen receptor 1 (ESR1) mRNA was analyzed using a new method for RNA extraction from FFPE tissue that is based on magnetic beads, followed by kinetic PCR. The prognostic impact of ESR1 mRNA expression was investigated and compared to ERalpha protein expression as determined by immunohistochemistry. In univariate survival analysis the expression level of ESR1 mRNA was a significant positive prognostic factor for patient survival (hazard ratio (HR) 0.230 (confidence interval (CI) 0.102-0.516), P=0.002). ERalpha protein expression was correlated to ESR1 mRNA expression (P=0.0001); however, ERalpha protein expression did not provide statistically significant prognostic information. In multivariate analysis, ESR1 mRNA expression emerged as a prognostic factor, independent of stage, grade, residual tumor mass, age, and ERalpha protein expression (HR 0.227 (CI 0.078-0.656), P=0.006). Our results indicate that the determination of ESR1 levels by kinetic PCR may be superior to immunohistochemical methods in assessment of biologically relevant levels of ER expression in ovarian carcinoma, and is feasible in routinely used FFPE tissue.

Vascular endothelial growth factor C (VEGF-C) is a well described chemotactic and growth factor for lymphatic endothelial cells. Its inhibition leads to suppression of lymphatic and distant metastases in mouse models. In ovarian cancer, the relationship between VEGF-C expression and tumor behavior has not yet been determined by a quantitative method in vivo. Therefore, we used a new technique of RNA extraction from formalin-fixed paraffin-embedded tissue samples and determined the expression levels of VEGF-C mRNA in a study group of 97 ovarian cancer patients. Expression levels were correlated with clinicopathological features and patient survival. High VEGF-C expression was associated with worse overall (p = 0.0393) and progression-free (p = 0.0155) patient survival. In the subgroups of serous tumors and high-grade tumors, VEGF-C mRNA was still a negative indicator for patient survival (p = 0.0190 and 0.0311, respectively). A trend was observed among patients with high clinical stage (p = 0.0634). In multivariate survival analysis VEGF-C mRNA retained its prognostic influence on progression-free survival (p = 0.006, HR = 0.319 with a 95% confidence interval of 0.142-0.720). High VEGF-C expression was further associated with an increased residual tumor mass after primary cytoreductive surgery. We found no correlation of VEGF-C expression with tumor grade, FIGO stage, lymph node, or distant metastases. Our study demonstrates that high VEGF-C expression is associated with aggressive tumor behavior in ovarian cancer. mRNA extracted from paraffin-embedded tumor samples is suitable for VEGF-C gene expression studies.

Oestrogen receptors (ESRs) regulate the growth and differentiation of normal ovarian epithelia. However, to date their role as biomarkers in the clinical setting of ovarian cancer remains unclear. In view of potential endocrine treatment options, we tested the role of ESR1 mRNA expression in ovarian cancer in the context of a neo-adjuvant chemotherapy trial. Study participants had epithelial ovarian or peritoneal carcinoma unsuitable for optimal upfront surgery and were treated with neo-adjuvant platinum-based chemotherapy before surgery. RNA was isolated from frozen tumour biopsies before treatment. RNA expression of ESR1 was determined by microarray and reverse transcriptase kinetic PCR technologies. The prognostic value of ESR1 was tested using univariate and multivariate Cox proportional hazards models, Kaplan-Meier survival statistics and the log-rank test. ESR1 positively correlates with proliferation markers and histopathological grading. ESR1 was a significant predictor of survival as a continuous variable in the univariate Cox regression analysis. In multivariate analysis, elevated baseline ESR1 mRNA levels predicted prolonged progression-free survival (P=0.041) and overall survival (P=0.01) after neo-adjuvant chemotherapy, independently of pathological grade and age. We conclude that pretreatment ESR1 mRNA is associated with tumour growth and is a strong prognostic factor in ovarian cancer, independent of the strongest clinical parameters used in clinical routine. We suggest that ESR1 mRNA status should be considered in order to minimize possible confounding effects in ovarian cancer clinical trials, and that early treatment with anti-hormonal agents based on reliable hormone receptor status determination is worth investigating.

Ovarian carcinoma has the highest mortality rate among gynaecological malignancies. In this project, we investigated the hypothesis that molecular markers are able to predict outcome of ovarian cancer independently of classical clinical predictors, and that these molecular markers can be validated using independent data sets. We applied a semi-supervised method for prediction of patient survival. Microarrays from a cohort of 80 ovarian carcinomas (TOC cohort) were used for the development of a predictive model, which was then evaluated in an entirely independent cohort of 118 carcinomas (Duke cohort). A 300-gene ovarian prognostic index (OPI) was generated and validated in a leave-one-out approach in the TOC cohort (Kaplan-Meier analysis, p = 0.0087). In a second validation step, the prognostic power of the OPI was confirmed in an independent data set (Duke cohort, p = 0.0063). In multivariate analysis, the OPI was independent of the post-operative residual tumour, the main clinico-pathological prognostic parameter with an adjusted hazard ratio of 6.4 (TOC cohort, CI 1.8-23.5, p = 0.0049) and 1.9 (Duke cohort, CI 1.2-3.0, p = 0.0068). We constructed a combined score of molecular data (OPI) and clinical parameters (residual tumour), which was able to define patient groups with highly significant differences in survival. The integrated analysis of gene expression data as well as residual tumour can be used for optimized assessment of the prognosis of platinum-taxol-treated ovarian cancer. As traditional treatment options are limited, this analysis may be able to optimize clinical management and to identify those patients who would be candidates for new therapeutic strategies.

Aims: Multidrug resistance is a major impediment in chemotherapeutic treatment of ovarian carcinoma patients. The aim of this study was to investigate the expression of multidrug resistance-associated protein 1 (MRP1) and to assess the possible associations with clinicopathological variables and patient outcome in primary ovarian carcinoma., Methods and Results: Tumour specimens from 129 patients were obtained before chemotherapy and analysed by immunohistochemistry on tissue microarrays, and by real-time reverse transcriptase-polymerase chain reaction on RNA extracted from formalin-fixed paraffin-embedded tissue specimens using a new technique. Significantly increased MRP1 protein expression was observed in high-grade tumours (P = 0.005) and advanced International Federation of Gynaecology and Obstetrics stages (P = 0.036). On univariate Kaplan-Meier analysis, patients with higher expression of MRP1 protein had significantly decreased overall survival (P = 0.006). On multivariate Cox regression analysis, MRP1 protein expression retained its significance as an independent negative prognostic marker for overall survival (hazard ratio = 6.52, P = 0.003). Furthermore, MRP1 expression correlated with topoisomerase IIalpha expression both at mRNA and protein level (P < 0.001 and P = 0.023, respectively)., Conclusion: In summary, in patients with primary ovarian cancer, overexpression of MRP1 is an adverse marker for patient outcome and cancer aggressiveness. Our data provide a translational basis for further clinical studies on the predictive value of MRP1 expression for response to chemotherapy.

The insulin-like growth factor-II mRNA-binding protein IMP3 plays an important role in embryogenesis and recent reports suggest an involvement in tumorigenesis. Although IMP3 expression has been well studied in mouse and human fetal and adult gonads, its role in ovarian cancer is unknown. We investigated the expression of IMP3 at protein and mRNA levels in a cohort of primary ovarian carcinomas and in 11 ovarian cancer cell lines. Western blot analysis revealed an expression of IMP3 in all ovarian cancer cell lines and immunohistochemistry demonstrated a positive cytoplasmic staining in 32 of 68 carcinomas (47%). In contrast, epithelium of borderline tumors, as well as, benign ovarian lesions and normal ovaries exhibited only weak or no IMP3 expression. In univariate Kaplan-Meier analysis, IMP3 protein expression was significantly associated with better overall survival (P=0.048). To confirm these findings, we further determined IMP3 mRNA expression in 43 ovarian cancer specimens by real time quantitative reverse transcription-polymerase chain reaction. A significant correlation between protein and mRNA levels (r=0.414, P=0.006), as well as a correlation of IMP3 mRNA expression with patient overall survival (P=0.044), was observed. Our results demonstrate that IMP3 is expressed in a subpopulation of ovarian cancer and a marker of good prognosis.

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas.

Topoisomerase IIalpha (Top IIalpha) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIalpha has recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIalpha gene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles' separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIalpha mRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIalpha expression and clincopathological variables as well as patient outcome. Elevated Top IIalpha mRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan-Meier analysis, patients with higher expression of Top IIalpha nuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIalpha in a subset of samples. Cytoplasmic expression of Top IIalpha was associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)-a nuclear export protein (P=0.008). Our study suggests that Top IIalpha overexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIalpha expression in ovarian carcinoma, which might help to assess the patients' risk profile, and the planning of an individualized therapy.

Ovarian carcinoma patients have an extremely poor prognosis; therefore, new molecular therapeutic approaches are urgently needed. The mTOR pathway, which may be targeted by substances such as Rapamycin or RAD001, is emerging as a promising target for anticancer therapy. So far, the expression and prognostic impact of mTOR signalling elements have not been completely studied in ovarian tumors. We analyzed p-mTOR, p-4E-BP1 and p-eIF-4E in 107 human ovarian lesions and observed an overexpression of p-mTOR (47%) and p-eIF-4E (56%) protein in primary ovarian carcinomas as compared to borderline tumors. Phospho-mTOR expression was significantly related to p-eIF-4E (p< or =0.001) and serous histological type (p=0.03). Increased p-4E-BP1 (31%) was associated with poor differentiation (p=0.04) and higher mitotic rate (p=0.004). In univariate analysis, increased expression of p-mTOR and p-eIF-4E was significantly associated with better overall survival (p=0.003, p=0.029). To connect the expression data with mechanistic studies, a set of 10 ovarian cancer cell lines was used. Expression of p-mTOR was increased in all cancer cell lines as compared to ovarian surface epithelial (HOSE) cells. Rapamycin treatment revealed a reduction of p-mTOR and p-4E-BP1 but increased p-AKT levels. We show for the first time an association of p-mTOR and p-eIF-4E with better overall survival for ovarian cancer patients. The combined results of our in vivo and cell culture studies suggest that a subpopulation of these patients may benefit from mTOR inhibition. The design of future clinical trials should incorporate biomarker testing to determine predictive markers for response to mTOR inhibitors.

It was hypothesized that analysis of global gene expression in ovarian carcinoma can identify dysregulated genes that can serve as molecular markers and provide further insight into carcinogenesis and provide the basis for development of new diagnostic tools as well as new targeted therapy protocols. By applying bioinformatics tools for screening of biomedical databases, a gene expression profile databank, specific for ovarian carcinoma, was constructed with utilizable data sets published in 28 studies that applied different array technology platforms. The data sets were divided into four compartments: (i) genes associated with carcinogenesis: in 14 studies, 1881 genes were extracted, 75 genes were identified in more than one study, and only 4 genes (PRKCBP1, SPON1, TACSTD1, and PTPRM) were identified in three studies. (ii) Genes associated with histologic subtypes: in four studies, 463 genes could be identified, but none of them was identified in more than a single study. (iii) Genes associated with therapy response: in seven studies, 606 genes were identified from which 38 were differentially regulated in at least two studies, 3 genes (TMSB4X, GRN, and TJP1) in three studies, and 1 gene (IFITM1) in four studies. (iv) Genes associated with prognosis and progression: 254 genes were found in seven studies. From these genes, merely three were identified in at least two different studies. This snapshot of available gene expression data not only provides independently described potential diagnostic and therapeutic targets for ovarian carcinoma but also emphasizes the drawbacks of the current state of global gene expression analyses in ovarian cancer.

Histone deacetylase (HDAC) inhibitors are an emerging class of targeted cancer therapeutics, and little is known about HDAC expression in gynecologic malignancies. Therefore, we tested the hypothesis whether high-level expression of class 1 HDACs (HDAC1, 2, and 3) is associated with clinically distinct subsets of ovarian and endometrial carcinomas. Expression was assessed by immunohistochemistry in a population-based cohort of 465 ovarian and 149 endometrial carcinomas and correlated with clinicopathologic parameters. Each of the HDACs was expressed at high levels in most ovarian (HDAC1, 61%; HDAC2, 93%; HDAC3, 84%) and endometrial (HDAC1, 61%; HDAC2, 95%; HDAC3, 83%) carcinomas. Further, 55% and 56% of ovarian and endometrial carcinomas, respectively, expressed all three HDACs at high levels. Such cases were less common among endometrioid subtypes of ovarian and endometrial carcinomas (36% and 52% positive cases, respectively) compared with high-grade serous subtypes (64 and 69%, respectively, P < .001). High-level expression of all three HDACs is associated with a poor prognosis in ovarian endometrioid carcinomas (hazard ratio, 6.7; 95% confidence interval, 1.9-23.3). The independent prognostic information and the overall high rate of expression for class I HDACs suggest that these targets should be explored as predictive factors in ovarian and endometrial carcinomas prospectively.

Decreased expression of p16 may result from hypermethylation of the promoter or from deletion of the gene. It can lead to intensified proliferation of neoplastic cells and to cytostatic drug resistance. The study was aimed at the examination of prognostic value of p16 expression in relation to Ki67 and caspase-3 in ovarian cancers using immunohistochemistry. The immunohistochemical studies were performed on 73 paraffin-embedded samples of ovarian cancers from 43 patients and samples from 6 healthy ovaries. We have used monoclonal antibodies against p16. ABC method and DAB were used for antigens visualisation. The intensity of the immunohistochemical reactions was appraised using the semi-quantitative IRS scale. In healthy ovaries we have shown strong reaction in the nuclei of surface epithelium. In the case of studied ovarian cancers, the reaction of a nuclear and cytoplasmic localization was obtained. The mean overall immunoreactivity score of nuclear p16 expression amounted to 5.30+/-3.44 SD in primary laparotomy material and 6.61+/-4.34 SD in secondary cytoreduction material. Statistical analysis demonstrated that lower p16 expression was typical of the younger patients and the patients who died. Kaplan-Meier's analysis proved that lower expression of p16 was characteristic of cases with shorter overall survival. In the present study we have demonstrated that lowered p16 expression represented an unfavourable prognostic index in ovarian cancer. Lowered p16 expression was also typical for chemotherapy-resistant ceases (cases of lower caspase-3 and higher Ki67 at secondary cytoreduction expression).

Background: The human nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) mediates the nuclear export of proteins and messenger RNAs and, thus, is an important regulator of subcellular distribution of key molecules. Whereas cell-biologic studies have suggested a fundamental role for CRM1 in the regulation of mitosis, the expression of this protein in human tumor tissue has not been investigated to date., Methods: In this study, the expression of CRM1 was analyzed in a cohort of 88 ovarian tumors and 12 ovarian cell lines for the first time to the authors' knowledge., Results: Immunohistochemistry revealed increased nuclear (52.7%) and cytoplasmic (56.8%) expression of CRM1 in 74 carcinomas compared with the expression revealed in borderline tumors and benign lesions. Similarly, CRM1 expression was increased in ovarian cancer cell lines compared with human ovarian surface epithelial cells. Cytoplasmic CRM1 expression was related significantly to advanced tumor stage (P= .043), poorly differentiated carcinomas (P= .011), and higher mitotic rate (P= .008). Nuclear CRM1 was associated significantly with cyclooxygenase-2 (COX-2) expression (P= .002) and poor overall survival (P= .01). Because it was demonstrated previously that blocking of CRM1 by leptomycin B (LMB) contributes to the inhibition of nuclear export, the authors used a set of mechanistic assays to study the effects of CRM1 inhibition in cancer cells. Treatment of OVCAR-3 cells with LMB revealed a significant reduction of cell proliferation and increased apoptosis as well as suppressed interleukin-1beta-induced COX-2 expression., Conclusions: The current results indicated that CRM1 is expressed in a subpopulation of ovarian carcinomas with aggressive behavior and is related to poor patient outcome. A correlation also was demonstrated between CRM1 and COX-2 expression in ovarian cancer tissue. Furthermore, the treatment of ovarian cancer cells with LMB revealed a reduction in COX-2 expression. Therefore, the authors suggest that CRM1 may be an interesting biomarker for the assessment of patient prognosis and a molecular target for anticancer treatment.

Elevated metallothionein (MT) expression in ovarian cancers treated with cisplatin-based schemes represents an unfavorable prognostic index. MT expression is significantly higher in tumor samples obtained after chemotherapy. The present study aimed at examining MT expression in ovarian carcinoma cells sensitive (A2780) or resistant (A2780RCIS) against platinum drug treatment as well as examining effects of exposure to cisplatin on MT expression. Subcellular expression of MT was evaluated also in samples originating from 73 ovarian tumors. Cisplatin-resistant A2780RCIS cells were exposed to increasing cisplatin concentrations, and the subcellular expression of MT was determined by immunocytochemistry. The studies demonstrated that cisplatin-resistant A2780RCIS cells exposed to cisplatin typically manifested a nuclear MT expression. The study demonstrated also that exposure to cisplatin was paralleled by growing MT expression in cell nuclei. The nuclear expression of MT was also found to be specific for ovarian cancers of poor clinical outcome. No relationship could be demonstrated between cytoplasmic expression of MT and clinical variables. Nuclear MT expression is induced by cisplatin and seems to protect DNA in the cells from toxic effects of the drug.

The protein kinase AKT is involved in several signaling pathways that are important for tumor development and progression, suggesting that AKT might be an interesting target for a molecular tumor therapy. In this study, we investigated the AKT expression in ovarian carcinomas and the role of the AKT isoforms to ovarian cancer cell proliferation. We observed an increased AKT expression in 58% of the primary ovarian carcinomas as compared to normal ovaries by immunohistochemistry. AKT expression was significantly associated with positive lymph node status (P=0.002) and advanced FIGO stage (P=0.009). In western blot analysis, total AKT was expressed in all ovarian cancer cell lines and HOSE cells, while phosphorylated AKT was only observed in OVCAR-3 and SKOV-3 cells. The isoforms AKT1 and AKT2 were expressed at the mRNA level in all cell lines, while no relevant AKT3 mRNA levels were detected by conventional and quantitative RT-PCR. To determine the effects on cell proliferation, we used the unselective PI3K-inhibitor LY294002 as well as RNA interference to selectively inhibit the AKT isoforms. Treatment with LY294002 and the AKT2 siRNA reduced proliferation of OVCAR-3 cells. Our results show that AKT is expressed in a subpopulation of advanced ovarian carcinomas suggesting a role for this protein in the progression of this entity. Deactivation of AKT, especially AKT2 can result in reduction of cell growth. Accordingly, AKT is an interesting target for therapeutic intervention in ovarian cancer.

An obstacle in chemotherapy of ovarian cancer is the development of drug resistance. Taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) was found to be overexpressed in a paclitaxel-resistant ovarian carcinoma cell line. However, clinical impact of TRAG-3 in ovarian carcinoma has not been demonstrated previously. For demonstration of potential clinical impact of TRAG-3, immunohistochemistry was applied to determine TRAG-3 protein expression in specimens obtained from ovarian carcinoma patients (n=37) who received a paclitaxel-based chemotherapy at two different time points, initial laparotomy before chemotherapy, and secondary cytoreduction after chemotherapy. The TRAG-3-specific immunohistochemical staining was correlated with clinical outcome. In ovarian carcinoma specimens obtained at the initial laparotomy, an advantage in overall (P < 0.001) and progression-free (P = 0.003) survival for patients with weak TRAG-3 expression could be demonstrated. Tumor specimens excised at secondary cytoreduction procedure were not predictive for clinical outcome. In summary, TRAG-3 was found to be a prognostic factor for the prediction of clinical outcome after the application of paclitaxel-based chemotherapy.

Aims: NF-kappaB has been demonstrated to activate proliferative, inflammatory, and angiogenic processes in ovarian cancer cells in vitro. To add translational information on the situation in vivo, we determined the expression pattern of p65, an important subunit of the classic NF-kappaB pathway, in ovarian carcinoma tissue, and investigated in vivo and in vitro whether this pathway is implicated in the known overexpression of cyclooxygenase-2 (COX-2)., Methods: p65 siRNA, chemiluminescent NF-kappaB transcription factor assay, Taqman PCR, as well as immunoblotting were performed with OVCAR-3 ovarian cancer cells. 83 primary ovarian cancinomas as well as 17 cases of benign ovarian tissue were analyzed by p65 and COX-2 immunohistochemistry using a tissue microarray., Results: DNA-binding avtivity as well as COX-2 mRNA and protein expression were strongly inducible by IL-1beta treatment in OVCAR-3 cells, while p65 siRNA inhibited IL-1beta-dependent p65 activity (p = 0.037) as well as COX-2 expression on the mRNA (p < 0.03) and on the protein level. In human tumor tissue, p65 protein expression was significantly associated with COX-2 expression (p = 0.002) as well as tumor grading (p = 0.005). Furthermore, p65 expression was a significant prognostic indicator of a reduced patient survival both in univariate (p = 0.038) and in multivariate analysis (p = 0.014)., Conclusion: Our study indicates a deregulation of the classical NF-kappaB pathway in ovarian cancer, which results in the overexpression of the NF-kappaB target gene COX-2. Components of this pathway might constitute novel attractive targets for a specific therapy of advanced ovarian cancer.

Purpose: Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells., Experimental Design: For the subcellular detection of ABCC2, immunohistochemistry was done using 41 Federation Internationale des Gynaecologistes et Obstetristes stage III ovarian carcinoma specimens prepared before treatment with cisplatin-based schemes and 35 specimens from the same group after chemotherapy. Furthermore, 11 ovarian carcinoma cell lines as well as tissue microarrays consisting of various human tissues were analyzed., Results: Nuclear membranous localization of ABCC2 was associated with response to first-line chemotherapy at primary (P = 0.0013) and secondary surgery (P = 0.0060). Cases with relapse showed higher nuclear membrane expression at primary (P = 0.0003) and secondary surgery (P = 0.0024). Kaplan-Meier analyses showed that weak nuclear membrane ABCC2 expression before treatment was associated with significantly longer overall (P = 0.04) and progression-free survival (P = 0.001); following chemotherapy, it correlated with significantly longer progression-free survival (P = 0.038). Tissue microarrays confirmed nuclear membranous localization of ABCC2, in particular, in poorly differentiated cells. In ovarian carcinoma cells, it correlated with resistance against cisplatin, whereas localization in the cytoplasmic membrane did not., Conclusions: ABCC2 confers resistance to cisplatin of ovarian carcinoma in cell culture systems and in clinics when expressed in the nuclear membrane. Thus, ABCC2 localization can predict platinum therapy outcome. Furthermore, expression of ABCC2 in nuclear membranes in human tissues is specific for poorly differentiated cells including stem cells.

Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. We have used a metabolite profiling approach to test the hypothesis that quantitative signatures of primary metabolites can be used to characterize molecular changes in ovarian tumor tissues. Sixty-six invasive ovarian carcinomas and nine borderline tumors of the ovary were analyzed by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS) using a novel contamination-free injector system. After automated mass spectral deconvolution, 291 metabolites were detected, of which 114 (39.1%) were annotated as known compounds. By t test statistics with P < 0.01, 51 metabolites were significantly different between borderline tumors and carcinomas, with a false discovery rate of 7.8%, estimated with repeated permutation analysis. Principal component analysis (PCA) revealed four principal components that were significantly different between both groups, with the highest significance found for the second component (P = 0.00000009). PCA as well as additional supervised predictive models allowed a separation of 88% of the borderline tumors from the carcinomas. Our study shows for the first time that large-scale metabolic profiling using GC-TOF MS is suitable for analysis of fresh frozen human tumor samples, and that there is a consistent and significant change in primary metabolism of ovarian tumors, which can be detected using multivariate statistical approaches. We conclude that metabolomics is a promising high-throughput, automated approach in addition to functional genomics and proteomics for analyses of molecular changes in malignant tumors.

Background: The membrane cofactor protein CD46 represents a complement inhibitor, which protects autologous cells from complement - mediated cytotoxicity. CD46 may exhibit the potential to protect tumor cells from the immune responses of the host. The present study aimed to evaluate the prognostic significance of CD46 expression in ovarian cancers., Materials and Methods: The analyses were performed on 73 ovarian cancer samples. Immunohistochemical reactions were performed on paraffin sections of tumors using monoclonal antibodies directed against CD46. The immunohistochemical reactions and the clinical observations results were subjected to statistical analysis., Results: Expression of CD46 was demonstrated in 60% of primary laparotomy cases and in 70% secondary cytoreduction cases. Kaplan-Meier analysis showed that a significantly shorter revival-free time was linked to cases with CD46 expression at PL (p= 0.01)., Conclusion: Ovarian cancers manifest CD46 expression that is linked to a less favourable prognosis.

Aminopeptidase N/CD13 (EC 3.4.11.2) is suggested to play a role in cancer cells invasion, and its activity can be inhibited using specific inhibitors. CD13 inhibitors evoke apoptosis of CD13-positive cancer cells. However, expression of CD13 has not been described in specimens obtained from ovarian carcinomas. Thus, in the present study, the expression of CD13 and its significance was examined in samples of ovarian cancers. The analyses were performed on sections originating from 73 tumor samples (43 from primary laparotomies [PL] and 30 from secondary cytoreductions [SCRs]). Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD13. The analysis demonstrated no relationships between the expression of CD13 on one hand and clinical variables and pathologic variables of the patients on the other hand. Expression of CD13 was demonstrated to be significantly more pronounced in samples obtained in PLs as compared to samples from SCRs (P < 0.001). Thus, the data indicate that a potential treatment of ovarian carcinoma with CD13 inhibitors should be performed before chemotherapy or in parallel to first-lapse chemotherapy.

In the present study we examined prognostic value of immunohistochemical estimation of topoisomerase 1A (TOP 1A) and HER-2/neu expression in ovarian cancers treated with platinum-based drugs but not with topotecan and the relation between expression of these proteins on the one hand and intensity of proliferation (Ki67) on the other. The analyses were performed on 73 samples of ovarian carcinoma originating from 43 first-look laparotomies (FLL) and, in 30 cases, from secondary cytoreductions (SCR)(after chemotherapy) from the same patients. In paraffin sections immunohistochemical reactions were performed using antibodies directed to HER-2/neu, TOP 1A and Ki67. Kaplan-Meier's analysis disclosed a shorter overall survival time in cases with augmented expression of TOP 1A at FLL and with higher expression of Ki67 at SCR. A shorter progression-free time was detected in cases with higher proportion of Ki67 positive cells at FLL. No relationship could be disclosed between HER-2/neu expression and the studied clinicopathological parameters. The studies confirmed high value of Ki67 estimation. The augmented expression of TOP 1A was demonstrated to represent an unfavourable prognostic factor. Thus, in cases with elevated expression of TOP 1A application of topotecan-based therapeutic schemes should be considered.

Immunohistochemical analysis of prognostic significance of COX-2 and P-gp expression in ovarian cancers was performed on samples originating from 73 tumors. COX-2-positive cases were shown to demonstrate higher expression of P-gp. The studies demonstrated also that, higher P-gp expression was typical for cases which responded poorly to chemotherapy and for cases with shorter progression-free time. Expression of COX-2 predisposed to a more rapid disease progression. The study documented a relationship between COX-2 and P-gp suggesting that COX-2 inhibitors might investigated in clinical trials as a treatment supplementary to chemotherapy of ovarian cancers.

High cytoplasmic expression of maspin was described in ovarian cancers of shorter survival rates. Until now, no relationship has been described between expression of maspin and sensitivity to cisplatin in ovarian cancers. This study aimed at examining the relationship between expression of maspin, detected by immunohistochemistry and clinical response to cisplatin in ovarian cancer cases as well as the in vitro sensitivity to cisplatin of 11 ovarian cancer cell lines. The analyzes were performed on 73 samples of ovarian cancer and on A2780P, A2780RCIS, CAOV-3, EFO 21, EFO 27, ES-2, Mdah 2774, OAW 42, OVCAR-3, PA-1, and SKOV-3 ovarian cancer cells. Cytoplasmic maspin expression in studied cells significantly correlated with cisplatin sensitivity. A significantly shorter overall survival and progression-free survival was associated with lower cytoplasmic maspin expression at first-look laparotomies and nuclear maspin expression and secondary cytoreductions. Higher nuclear maspin at first-look laparotomies expression was specific for cases of complete response. In the study, the elevated expression of maspin was shown to be typical for cisplatin-sensitive ovarian cancers.

Expression of CD24 represents a poorly recognized, unfavorable prognostic factor. Expression of the protein is supposed to facilitate extravasation of tumor cells. Our study aimed at examination of prognostic significance of CD24 estimation in samples obtained from primary surgeries (PS) and secondary cytoreductions (SCR) (after chemotherapy) in ovarian cancer patients. The analyses were performed on sections originating from 73 tumor samples. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD24. Kaplan-Meier's analysis showed that a significantly shorter overall survival time and progression-free time was demonstrated to characterize cases with cytoplasmic membranous expression of CD24 (CD24c-m) (P < 0.001). The calculations performed demonstrated also a significantly higher proportion of CD24c-m positive cases in sections from SCR as compared to that from PS (P= 0.04) and in cases of progressive disease as compared to complete response at PS and SCR (P= 0.002 and P= 0.05, respectively). Summing up, in this study, we have demonstrated a negative prognostic significance of a cytoplasmic membranous expression of CD24 in cases of ovarian cancer.

Actinomycosis in the pelvic region is an uncommon diagnosis. This infection is caused by Actinomyces israelii, a gram-positive anerobic saprophyte bacterium that is a normal inhabitant of the upper intestinal tract in humans. Pelvic actinomycosis is difficult to diagnose pre-operatively and is diagnosed, in most cases, accidentally. Actinomycosis can mimic pelvic and abdominal malignancies. A case report of a 35-year-old female patient with a fixed pelvic mass is presented and the diagnosis and treatment of pelvic actinomycotic inflammatory disease in relation to ovarian cancer are discussed. Clinicians should be aware of this rare infection to spare women potential morbidity from excessive surgical procedures.

Resistance to cis- or carboplatin represents the principal cause of therapeutic failures in ovarian carcinoma. The phenomenon of resistance to platinum-based drugs is partly related to expression of metallothionein (MT) and of glutathione S-transferase pi (GST-pi), but opinion on the subject is discordant. Documentation of a negative predictive effect of MT and GST-pi expression for the therapy employing platinum-based drugs would permit to select resistant cases in which other therapeutic approaches could be employed. The present study aimed at examining the relation between intensities of MT and GST-pi expressions in ovarian carcinomas and dynamics of the clinical course in the neoplastic disease in a group of cisplatin-treated patients. The analyses were performed on samples of ovarian carcinoma originating from 43 first-look laparotomies (FLLs) and, in 30 cases, from second-look laparotomies (SLL) from the same patients. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies to MT and GST-pi. The calculations showed that in cases with augmented expression of MT, mortality was higher. On the other hand, augmented expression of GST-pi predisposed to more frequent relapses, deaths and progression of the tumor. Kaplan-Meier analysis showed that a significantly shorter survival time was linked to cases of higher expression of MT at FLL and of higher expression of GST-pi at FLL, whereas a shorter progression-free time was manifested by cases with higher expression of GST-pi at FLL. The performed investigations indicate that augmented expressions of MT at FLL and GST-pi at FLL in ovarian cancer represent an unfavourable predictive factor in cisplatin-treated patients.

Lysophosphatidic acid acyltransferase beta (LPAAT-beta) is an enzyme involved in lipid biosynthesis whose role in tumour progression has been of emerging interest in the last few years. We investigated the expression of LPAAT-beta by reverse transcriptase-polymerase chain reaction and immunohistochemistry in 10 ovarian cell lines as well as in a cohort of 106 ovarian tumours and normal ovaries. Lysophosphatidic acid acyltransferase beta mRNA was found in all cell lines and ovarian tumours examined. Expression of LPAAT-beta protein was significantly increased in ovarian carcinomas compared to benign ovarian tissue (chi2 test P-value=0.001, Kruskal-Wallis test P-value <0.0001). Furthermore, LPAAT-beta expression was positively associated with higher tumour grade (P=0.044), higher mitotic index (P<0.0001) and tumour stage (P=0.032). Expression of LPAAT-beta was significantly linked to reduced overall survival time (P=0.024) as well as to shorter progression-free survival time (P=0.012) in patients younger than 60 years. Our study shows that LPAAT-beta is upregulated in ovarian cancer and is more prevalent in poorly differentiated tumours. In addition, LPAAT-beta expression is a predictor of a worse prognosis in patients younger than 60 years. Further studies are needed to investigate if LPAAT-beta may serve as a therapeutic target for certain subgroups of patients.