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Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.

BACKGROUND: Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. METHODS: Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. RESULTS: We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls (p<0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites (p=0.04) and lymph node metastases (p=0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free (p=0.02) and overall survival (p=0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis (p=0.04, HR=2.35, CI(95%)=1.03-5.34). CONCLUSIONS: Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC. [ABSTRACT FROM AUTHOR]

Background: High-grade serous ovarian carcinoma (HGSOC) is generally associated with a very dismal prognosis. Nevertheless, patients with similar clinicopathological characteristics can have markedly different clinical outcomes. Our aim was the identification of novel molecular determinants influencing survival., Methods: Gene expression profiles of extreme HGSOC survivors (training set) were obtained by microarray. Differentially expressed genes (DEGs) and enriched signalling pathways were determined. A prognostic signature was generated and validated on curatedOvarianData database through a meta-analysis approach. The best prognostic biomarker from the signature was confirmed by RT-qPCR and by immunohistochemistry on an independent validation set. Cox regression model was chosen for survival analysis., Results: Eighty DEGs and the extracellular matrix-receptor (ECM-receptor) interaction pathway were associated to extreme survival. A 10-gene prognostic signature able to correctly classify patients with 98% of accuracy was identified. By an 'in-silico' meta-analysis, overexpression of FXYD domain-containing ion transport regulator 5 (FXYD5), also known as dysadherin, was confirmed in HGSOC short-term survivors compared to long-term ones. Its prognostic and predictive power was then successfully validated, both at mRNA and protein level, first on training than on validation sample set., Conclusion: We demonstrated the possible involvement of FXYD5 and ECM-receptor interaction signal pathway in HCSOC survival and prognosis.

Background: The existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance., Methods: We isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR., Results: Spheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival., Conclusions: This investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.

Background: Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro., Methods: Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT-qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients' prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR., Results: A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response., Conclusions: FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

MicroRNAs (miRNAs) belong to a family of small non-coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high-grade serous ovarian carcinoma (HGS-OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS-OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR-92a-3p, let-7a-5p, SNORD61, SNORD72, SNORD68, miR-103a-3p, miR-423-3p, miR-191-5p, miR-16-5p) were analysed on a total of 75 HGS-OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS-OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR-191-5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS-OvCa and normal controls, including the first time both the normal tissues supposed to be HGS-OvCa progenitors. In addition, we recommend miR-191-5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS-OvCa tissues., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t -test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.

Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.

Background: The aim of the present study was to investigate within ovarian carcinoma and normal ovarian biopsies the gene expression of multiple secretoglobin family members relative to mammaglobin B, which we previously reported as a promising novel ovarian carcinoma prognostic marker., Methods: Using quantitative real-time Reverse Transcription PCR we tested 53 ovarian carcinoma and 30 normal ovaries for the expression of 8 genes belonging to the secretoglobin family: mammaglobin A, lipophilin A, lipophilin B, uteroglobin, HIN-1, UGRP-1, RYD5 and IIS. Next, we decided to expand the LipB gene expression analysis to a further 48 ovarian carcinoma samples, for a total of 101 tumor tissues of various histologies and to study its protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tumors and normal ovaries. Finally, we correlated lipophilin B gene and protein expression to conventional patient clinico-pathological features and outcome., Results: We found significant mammaglobin A, lipophilin A, lipophilin B and RYD5 gene overexpression in ovarian carcinomas compared to normal ovaries. Lipophilin B mRNA showed a higher presence in tumors (75.4%) compared to normal ovaries (16.6%) and the most significant correlation with mammaglobin B mRNA (rs =0.77, p < 0.001). By immunohistochemical analysis, we showed higher lipophilin B expression in the cytoplasm of tumor cells compared to normal ovaries (p < 0.001). Moreover, lipophilin B gene overexpression was significantly associated with serous histology (serous vs clear cell p = 0.027; serous vs undifferentiated p = 0.007) and lower tumor grade (p = 0.02). Lower LipB mRNA levels (low versus high tertiles) were associated to a shorter progression-free (p = 0.03, HR = 2.2) and disease-free survival (p = 0.02, HR = 2.5) by univariate survival analysis and, importantly, they remain an independent prognostic marker for decreased disease-free (p = 0.001, HR = 3.9) and progression-free survival (p = 0.004, HR = 2.8) in multivariate Cox regression analysis., Conclusions: The present study represents the first quantitative evaluation of secretoglobin gene expression in normal and neoplastic ovarian tissues. Our results demonstrate lipophilin B gene and protein upregulation in ovarian carcinoma compared to normal ovary. Moreover, lipophilin B gene overexpression correlates with a less aggressive tumor phenotype and represents a novel ovarian carcinoma prognostic factor.

Background: The purpose of this study was to assess the diagnostic and prognostic impact of preoperative serum determination of human epididymis protein 4 (sHE4), and to investigate its potential correlation with clinicopathological features and survival endpoints in endometrial cancer patients., Methods: Preoperative serum samples from 193 endometrial cancer patients and 125 women with normal endometrium were measured for sHE4 and serum CA125 (sCA125) concentrations by quantitative chemiluminescent microparticle immunoassays on the automated Architect instrument., Results: sHE4 concentrations were significantly higher in endometrial cancer patients regardless of tumour stage and grade compared with normal controls. Setting the specificity at 95 % , the sensitivities in detecting endometrial cancer patients were 66 % for HE4, 33 % for CA125 and 64 % for the combination of the two markers. High concentrations of both HE4 and CA125 significantly correlated with all clinicopathological features characterising a more aggressive tumour phenotype.In multivariate analysis, only high preoperative sHE4 concentrations, but not sCA125, were independent prognostic factors for shorter Overall Survival, Disease-Free Survival and Progression-Free Survival., Conclusions: HE4 is more sensitive and specifi c than CA125in distinguishing endometrial cancer patients from women with normal endometrium, regardless of tumour stage and grade. sHE4 appears to be associated with a more aggressive tumour variant and it could be clinically useful, in identifying high-risk endometrial cancer patients, for a tailored surgical and postoperative therapy. HE4 significant correlation with decreased Overall Survival, Disease Free Survival and Progression Free Survival suggests its potential role as a novel prognostic marker for endometrial cancer.

Background: Endometrial cancer is the most common gynecologic malignancy in developed countries. Trop-2 is a glycoprotein involved in cellular signal transduction and is differentially overexpressed relative to normal tissue in a variety of human adenocarcinomas, including endometrioid endometrial carcinomas (EEC). Trop-2 overexpression has been proposed as a marker for biologically aggressive tumor phenotypes., Methods: Trop-2 protein expression was quantified using tissue microarrays consisting of formalin-fixed paraffin-embedded specimens from 118 patients who underwent surgical staging from 2001-9 by laparotomy for EEC. Clinicopathologic characteristics including age, stage, grade, lymphovascular space invasion, and medical comorbidities were correlated with immunostaining score. Univariate and multivariate analyses were performed for overall survival, disease-free survival, and progression-free survival in relation to clinical parameters and Trop-2 protein expression., Results: Clinical outcome data were available for 103 patients. Strong Trop-2 immunostaining was significantly associated with higher tumor grade (p=0.02) and cervical involvement (p<0.01). Univariate analyses showed a significant association with reduced disease-free survival (DFS) (p=0.01), and a trend towards significance for overall and progression-free survival (p=0.06 and p=0.05, respectively). Multivariate analyses revealed Trop-2 overexpression and advanced FIGO stage to be independent prognostic factors for poor DFS (p=0.04 and p <0.001, respectively)., Conclusions: Trop-2 protein overexpression is significantly associated with higher tumor grade and serves as an independent prognostic factor for DFS in endometrioid endometrial cancer.

Background: The aim of this work was to analyze the diagnostic and prognostic value of serum human epididymis protein 4 (HE4) and Risk for Ovarian Malignancy Algorithm (ROMA) in epithelial ovarian cancer (EOC)., Methods: Preoperative serum samples of 419 women (140 healthy controls, 131 ovarian benign cysts, 34 endometriosis, and 114 EOC) were tested for CA125 and HE4 using fully automated methods (Abbott ARCHITECT) and validated cutoff values., Results: For the discrimination of benign masses from EOC, in premenopausal women, the sensitivity and specificity were 92.3% and 59.4% for CA125, 84.6% and 94.2% for HE4, and 84.6% and 81.2% for ROMA, whereas in postmenopausal women, the sensitivity and specificity were 94.3% and 82.3% for CA125, 78.2% and 99.0% for HE4, and 93.1% and 84.4% for ROMA. In patients with EOC, elevated CA125, HE4, and ROMA levels were associated with advanced Federation of Gynaecologists and Obstetricians (FIGO) stage, suboptimally debulking, ascites, positive cytology, lymph node involvement, and advanced age (all P ≤ 0.05). Elevated HE4 and ROMA (both P ≤ 0.01), but not CA125 (P = 0.0579), were associated with undifferentiated tumors. In multivariable analysis, elevated HE4 and ROMA (all P ≤ 0.05) were independent prognostic factors for shorter overall, disease-free, and progression-free survival., Conclusions and Impact: This study underlines the high specificity of HE4 in discriminating endometriosis and ovarian benign cysts from EOC and the high sensitivity of CA125 in detecting EOC. We showed HE4 and ROMA as independent prognostic factors. Multicenter studies are needed to draw firm conclusions about the applicability of HE4 and ROMA in clinical practice.

Background: Two commercial immunoassays for HE4 have been compared and the diagnostic accuracy of HE4, CA 125 and the combinatory ROMA algorithm for epithelial ovarian cancer (EOC) has been evaluated., Methods: HE4 and CA125 were measured on sera obtained from 259 women (73 healthy, 90 with benign ovarian or adnexal diseases, 96 with EOC). The ARCHITECT CMIA HE4 assay was compared with the Fujirebio EIA HE4, and the risk for EOC by the combinatory ROMA algorithm (HE4+CA 125) was assessed with both HE4 assays., Results: The CMIA HE4 assay showed a good linearity (r>0.9998) and precision (interassay and total CVs <4%). The correlation with EIA HE4 was linear (r=0.994), with an average bias of 0.4%. By ROC curve analysis, the sensitivity for EOC at a fixed specificity of 90%, 95% and 99% was 89.6%, 84.4% and 79.2% by CMIA HE4, 84.4%, 83.3% and 79.2% by EIA HE4, 86.5%, 76.0% and 59.4% by CMIA CA125. The accuracy of the ROMA algorithm determined by CMIA or EIA HE4 was very similar (AUC 87.1% vs. 87.6%; p=n.s.) and greater in menopause., Conclusions: The two HE4 assays showed a good correlation and similar clinical value, with a greater precision for CMIA. HE4 was more specific and accurate than CA125, supporting its use in addition to clinical and imaging criteria for the discrimination of benign from malignant ovarian lesions. The ROMA algorithm showed a good accuracy for discriminating women at high risk for EOC., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: To date, no good marker for screening or disease monitoring of endometrial cancer (EC) is available. The aims of this study were to investigate HE4 gene, protein expression and serum HE4 (sHE4) levels in a panel of ECs and normal endometria (NEs) and to correlate sHE4 with patient clinicopathological characteristics and prognosis., Methods: Using quantitative real-time PCR we tested 46 ECs and 20 NEs for HE4 gene expression. Protein expression was analysed by immunohistochemistry on tissue microarrays in 153 ECs and 33 NEs. Pre-operative serum samples from 138 EC and 76 NE patients were analysed with HE4-EIA assay. Association between sHE4 and patient clinicopathological characteristics or outcome was evaluated., Results: Protein and HE4 gene were significantly upregulated in EC tissues and sera, compared with controls. High sHE4 levels were significantly associated with worse EC clinical characteristics. By univariate survival analysis, high sHE4 levels significantly correlated with decreased overall survival, progression-free survival and disease-free survival, retaining their independent prognostic value on the poorly differentiated EC cohort., Conclusion: We demonstrate, for the first time, that high sHE4 levels correlates with an aggressive EC phenotype and may constitute an independent prognostic factor for poorly differentiated-ECs. Determination of sHE4 could be clinically useful in identifying high-risk EC patients for a more aggressive adjuvant therapy.

Background: Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients., Methods: MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome., Results: MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08-0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17-0.99, p = 0.048) had an independent prognostic value for disease-free survival., Conclusion: This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.

Objective: The purpose of this study was to develop and characterize a human antibody in a single-chain antibody fragment format (scFv) that is directed specifically against claudin-3 (CLDN3)., Study Design: The synthetic ETH-2 Gold human antibody phage display library was used to select scFv specific against CLDN3. scFv binding properties were analyzed by surface plasmon resonance; specificity was confirmed with enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry on a panel of ovarian and uterine serous carcinoma cell lines., Results: Surface plasmon resonance studies indicated scFv H6 to be the clone with the highest affinity against CLDN3 (K(D) of 23.60 nmol/L). scFv H6 efficiently stained CLDN3-expressing cells and recognized its epitope in enzyme-linked immunosorbent assay that was performed with uterine serous papillary carcinoma native protein extract, which suggested that a conformational epitope is recognized by this antibody. Cell surface immunofluorescence with laser scanning confocal microscopy confirmed the specific binding to the native membrane CLDN3., Conclusion: scFv H6 may represent a novel antitumor agent against chemotherapy-resistant ovarian and serous papillary carcinomas and other human malignancies that overexpress CLDN3.

Claudin-7 (CLDN-7) is a tight junction protein recently found highly differentially expressed in ovarian carcinoma. To evaluate its potential as a novel biomarker, in this study, we quantified and compared claudin-7 expression at messenger RNA and protein level in 110 patients harboring various histologic types of epithelial ovarian carcinomas (EOC). CLDN-7 transcript was found significantly overexpressed in both primary and metastatic EOCs compared to normal human ovarian surface epithelium cell lines (fold change = 111.4, P < 0.001) by reverse transcription-polymerase chain reaction. At the protein level, CLDN-7 expression was found significantly higher in tumors of primary and metastatic origin when compared to normal ovaries (P < 0.001), regardless of the histologic type, the grade of differentiation, and the pathologic stage of the disease (P = 0.12). Moreover, a strong immunoreactivity for CLDN-7 was detected in EOC cells present in ascites fluids, whereas ascites-derived inflammatory cells, histiocytes, and reactive mesothelial cells were negative. Finally, immunohistochemical expression of CLDN-7 was observed in several human normal epithelial control tissues analyzed. CLDN-7 is significantly overexpressed in all main histologic types of EOC and in single neoplastic cells disseminated in peritoneal cavity and pleural effusions, suggesting its potential role as novel diagnostic marker in ovarian cancer. Despite widespread expression of CLDN-7 in several human normal tissues, the high density of CLDN-7 molecules, their membranous localization on EOC cells, and their lack of expression on the celomic epithelium in the peritoneal cavity suggest that this target could be potentially suitable for antibody-mediated localized therapies of ovarian adenocarcinoma.

This study identifies the genetic fingerprint of poorly differentiated endometrioid endometrial carcinomas (G3-EEC) and analyses the potential utility of trefoil factor 3 (TFF3) as novel serum marker in G3-EEC. Affymetrix microarrays were used to identify the gene expression patterns of 19 snap-frozen G3-EEC and 15 normal endometrium (NE) biopsies. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry were used to validate TFF3 expression. Finally, TFF3 serum levels were determined by ELISA in 25 G3-EEC patients, 42 healthy controls, and in 13 endometrial hyperplasia patients. Hierarchical cluster analysis showed TFF3 as the top differentially expressed gene between 363 upregulated genes in G3-EEC, when compared with NE. Trefoil factor 3 gene expression levels analysed by qRT-PCR significantly correlated with Affymetrix results (P<0.001; rs=0.85). By immunohistochemistry, TFF3 protein was significatively more expressed in EEC compared with NE (P<0.01), with cytoplasmatic positivity in 79% G3-EEC and 18% NE. Patients harbouring G3-EECs had significantly higher TFF3 serum concentration by ELISA when compared with healthy patients (P<0.001) or patients harbouring endometrial hyperplasia (P=0.012). In conclusion, TFF3 is highly expressed at gene and protein level in G3-EEC. Further investigations on a wider set of samples are warranted to validate TFF3 as a novel serum marker for early detection and/or monitoring of G3-EEC patients.

Mammaglobin B (MGB-2) is an uteroglobin gene family member recently found highly differentially expressed in ovarian cancer by gene expression profiling. To evaluate its potential as a novel endometrial cancer biomarker, in this study we quantified and compared MGB-2 expression at messenger RNA and protein levels in endometrial tumors (endometrioid endometrial cancer [EEC]) with different grades of differentiation. MGB-2 expression was evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) in fresh frozen biopsies and paraffin-embedded tissues derived from a total of 70 patients including 50 primary EEC and 20 normal endometria (NECs). High levels of MGB-2 gene expression were detected in 10 of 11 EEC G1 cases (91%), 16 of 17 EEC G2 cases (94%), and 6 of 22 EEC G3 cases (27%) by real-time PCR. In contrast, normal endometrial cells expressed low to negligible levels of MGB-2 by real-time PCR (P = 0.002 EEC vs NEC). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (P < 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were also statistically significant (G2 vs G3 P < 0.001, G1 vs G3 P = 0.016). MGB-2 protein expression was detected in 31 (86%) of 36 EEC and 0 of 5 atrophic NEC controls, while seven of eight (88%) of the proliferative/secretory/hyperplastic NECs focally expressed MGB-2 by IHC. MGB-2 is highly expressed in EEC, particularly in well- and moderately differentiated tumors, and may represent a novel molecular marker for EEC.

Uterine serous papillary carcinoma (USPC) is a rare and highly malignant form of endometrial cancer (EC) characterized by early metastasis, chemoresistance, and high mortality rate. Little is known about USPC tumorigenesis even if recently a HER-2/neu role has been suggested in its development and progression. The aim of the present study was to evaluate HER-2 expression by immunohistochemistry (IHC) in 12 USPC formalin-fixed, paraffin-embedded (FFPE) samples. Moreover, we looked at the correlation between HER-2 protein expression and HER-2/neu gene amplification by fluorescence in situ hybridization (FISH), other than HER-2/neu messenger RNA expression by quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR). Finally, these results have been compared with commonly evaluated clinical features in EC patients, in order to define the potential prognostic value of HER-2/neu overexpression in USPCs. A high expression of HER-2 protein by IHC was noted in 2 of 12 patients (16.6%), and the same cases showed specific HER-2/neu gene amplification by FISH. All the samples investigated displayed a perfect concordance between IHC and FISH data. Five (41.6%) of 12 tumors demonstrated polysomy of chromosome 17 and, focusing on the 2 USPCs that showed HER-2/neu overexpression, one of them (50%) was polysomic for chromosome 17. All the other USPC cases (58.4%) showed to be disomic for chromosome 17. Quantitative RT real-time PCR performed on complementary DNA obtained from all FFPE USPC samples showed a complete correlation with FISH and IHC data. Moreover, HER-2/neu overexpression was associated with a poorer overall survival and a very low relapse-free survival time, thus being considered a candidate marker of worse overall prognosis in USPC. The use of trastuzumab (Herceptin), a monoclonal antibody directed against HER-2/neu, for the therapy of patients with HER-2/neu-positive USPCs should be further investigated in clinical trials.

Objective: Mammaglobin B is a uteroglobin gene family member recently found highly differentially expressed in serous papillary ovarian cancer by gene expression profiling. In order to evaluate its potential as a novel ovarian cancer biomarker, in this study we quantified and compared Mammaglobin B expression in various histologic types of epithelial ovarian carcinomas (EOC)., Methods: Mammaglobin B expression was evaluated by real-time PCR and/or immunohistochemistry in fresh-frozen biopsies and paraffin-embedded tissues derived from a total of 137 patients including 69 primary EOC with different histologies, 28 serous papillary omental metastasis, 8 borderline tumors, 26 benign cystadenomas and 14 normal ovaries., Results: High levels of Mammaglobin B gene expression were detected in 100% (68 out of 68) of the ovarian cancer biopsies tested by real-time PCR. In contrast, normal human ovarian surface epithelium (HOSE) expressed negligible levels of Mammaglobin B mRNA (EOC versus HOSE, p<0.01). Although Mammaglobin B gene expression levels were higher in endometrioid, mucinous and undifferentiated tumors when compared to serous papillary tumors, clear cell tumors and those with mixed histology, these differences were not statistically significant. In agreement with real-time PCR results, EOC were found to express significantly higher levels of Mammaglobin B protein when compared to normal ovaries and benign cystadenomas (p<0.01). However, only 29 out of 68 (42%) of the EOC samples found positive for Mammaglobin B by real-time PCR showed immunoreactivity by IHC., Conclusions: Mammaglobin B gene is highly expressed in EOC and may represent a novel molecular marker for multiple histological types of ovarian cancer. Additional studies to evaluate the clinical utility of Mammaglobin B as a diagnostic and/or therapeutic target in ovarian cancer are warranted.

Objective: The purpose of this study was to identify genes that are highly differentially expressed in metastatic serous papillary ovarian tumors (MET) when compared with primary ovarian serous carcinomas (OSPC)., Study Design: An oligonucleotide microarray with probe sets complementary to >14,500 human genes was used to determine whether patterns of gene expression may differentiate OSPC from MET in 31 snap-frozen serous papillary ovarian carcinomas (ie, 14 primary OSPC and 17 omental metastasis [MET])., Results: Hierarchic cluster analysis of gene expression in OSPC and MET identified 156 genes that exhibited > 2-fold differences (P < .05) and that distinguished OSPC from MET. A number of invasion and metastasis predictive genes (including plasminogen activator; matrix metalloproteinase; matrix structural constituent genes encoding products with collagen, heparin, and hyaluronic acid binding activity; genes encoding receptors for insulin-like growth factors; vascular endothelial growth factor; endothelin type A; fibroblast growth factor; thrombospondin 1 and 2; type A and B integrins, and chemokines [stromal cell-derived factor 1 (CXCL12)]) were found among the 120 genes that were highly differentially overexpressed in MET, when compared with OSPC. Down-regulated genes in MET compared with OSPC included hepsin and testisin. Overexpression of CXCL12, matrix metalloproteinase, plasminogen activator, type A and B integrins, and hepsin genes was validated by quantitative real-time polymerase chain reaction in all samples. Finally, overexpression of CXCL12 in MET, when compared with OSPC, was validated at the protein level by immunohistochemistry., Conclusion: Gene expression profiling may differentiate metastatic ovarian cancer from primary OSPC. The identification of metastasis-associated genes may provide a foundation for the development of new type-specific diagnostic strategies and treatment for metastatic ovarian cancer.

Objective: To identify novel molecular biomarkers useful for the early diagnosis and therapy of ovarian cancer by gene expression profiling. To compare the genetic fingerprints of flash-frozen ovarian serous carcinomas to those of matched highly purified primary tumor cell cultures., Methods: Gene expression profiles of 19 flash-frozen ovarian serous papillary carcinoma (OSPC) were analyzed and compared to 15 controls (highly purified human ovarian surface epithelium short-term cultures, HOSE) using oligonucleotide microarrays complementary to >14,500 human genes. In addition, gene expression profiling of 5 highly purified primary OSPC cultured in vitro for less than 2 weeks was compared to flash-frozen ovarian carcinoma biopsies obtained from matched samples. Quantitative RT-PCR and IHC staining techniques were used to validate microarray data at RNA and protein levels for some of the differentially expressed genes., Results: Unsupervised analysis of gene expression data readily distinguished normal tissue from flash-frozen OSPC and identified 901 and 557 genes that exhibited >3-fold up-regulation or down-regulation, respectively, in OSPC when compared to HOSE. Mammaglobin 2, an ovarian secreted protein, was identified as the top differentially expressed gene in OSPC (19 out 19 OSPC versus 0 out of 15 HOSE) with over 827-fold up-regulation relative to HOSE. The claudin and kallikrein family of proteins including the clostridium perfringens enterotoxin receptors claudin 3 and 4, kallikreins 6, 7, 8, 10, 11 and the immunomodulatory molecule B7-H4 were found among the most highly overexpressed genes in OSPC when compared to HOSE. Genetic fingerprints of flash-frozen OSPC were found to have high correlation with those of purified primary OSPC short-term in vitro cultures with only 31 out of 8,637 genes (0.35%) differentially expressed between the two groups., Conclusions: Short-term in vitro culture of primary ovarian carcinomas may greatly increase the purity of ovarian tumor RNA available for gene expression profiling without causing major alteration in OSPC fingerprints. Mammaglobin 2, kallikreins 6, 7, 8, 10, 11, claudin 3 and 4 and B7-H4 gene expression products represent candidate biomarkers endowed with great potential for early screening and therapy of OSPC patients.

Human papillomaviruses (HPVs), particularly HPV-16/18, are linked to cervical cancer development. Full-length, recombinant HPV-16/18 E7 oncoproteins were used in a new streptavidin-biotin capture ELISA method to investigate anti-HPV E7 antibody prevalence in serum. Sera from 99 healthy women, 70 cervical cancer patients, and 30 patients with cervical pre-invasive neoplasia were analyzed. Anti-HPV-16/18 E7 positivity was found in 53% of cervical cancer patients, in 40% with cervical pre-invasive neoplasia, and in 8% of healthy women. Serum samples from 12 cervical cancer patients were obtained at different time intervals during the treatment. Eleven out of 12 showed a correspondence between HPV-E7 antibody levels (decreasing versus increasing) and the type of response (clinically complete or partial response versus progression or stable disease) at each serological evaluation. Five patients with recurrent HPV-16/18-positive cervical carcinoma were analyzed before and after vaccination with HPV-16/18 E7-pulsed autologous dendritic cells; anti-HPV-16/18 E7 positivity was found in 3 out of 5 women. In conclusion, this assay could potentially be used as an adjunctive tool to monitor the type of response to treatment and possibly to detect antibody induction in cervical cancer patients after vaccination, as a potential marker to evaluate its efficacy.