Normal Ovarian Samples Research Articles

Abstract 4800: Quantitative detection of discriminatory proteins in tissue and serum of ovarian cancer patients by LC-MSE

Abstract Purpose: Using label-free UPLC-MSE quantification methods to identifying proteins as well as determining their abundance in serum and microdissected ovarian cancer tissue. Experimental procedures: Serum of six patients with a serous adenocarcinoma of the ovary (stage IIIB or higher) was collected before treatment. Matched control patients with a serous cystadenoma of the ovary were chosen as a control group. In addition, homogeneous regions of cells that exhibited uniform histology were isolated from cancer tissue by laser capture microdissection (LCM), normal ovarian epithelial cell samples and stroma samples were taken from matched controls. We subsequently employed label-free ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC-MSE) to identify proteins and determine their absolute concentrations in serum and tissue lysates. Moderated t-tests were used to identify differentially expressed proteins between patient- and control-group. p-values were adjusted for multiple-testing using Benjamini-Hochberg false discovery rates and considered differentially expressed when <0.05. Summary of the data: In the serum specimens, 13 differentially expressed proteins were identified by LC-MSE profiling. Proteins with different concentrations in patients versus control sera include abundant serum proteins such as apolipoprotein AI and transferrin, both exhibited a lower concentration in serum of cancer patients. Differential expression was also observed for apolipoproteins (APOA IV, APOA II and APOC III) and other proteins that have not been associated with ovarian cancer previously such as C9 and Afamin. In the tissue-lysates we identified 535 Proteins of which 332 could be reliably quantified. Of these 56 were differentially expressed proteins of which 12 had non-overlapping standard deviations. Literature searches confirmed a link between ovarian cancer and 6 of these proteins, suggesting a possible role in disease progression. Of special interest was the identification of prohibitin (PHB) and cofilin-1 (CFL1). Immunohistochemical detection of CFL1 in formalin-fixed paraffin-embedded tissues of our patient group showed heterogeneous expression in 50% of the serous ovarian carcinomas, whereas all the benign lesions were negative. These results again demonstrate the validity of the protein quantitation results obtained by MSE. Interestingly, no overlap was found between the discriminatory proteins in serum specimens and corresponding tissue lysates, illustrating the challenge to identify a single biomarker in the circulation reflecting ovary cancer. Conclusions: Our study revealed several protein changes in serum and tissue of patients suffering from serous adenocarcinoma of the ovary. Further investigation of these proteins is warranted to establish whether they could provide new insights into disease etiology and act as potential new markers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4800. doi:1538-7445.AM2012-4800

Increased Expression of Dachshund Homolog 1 in Ovarian Cancer as a Predictor for Poor Outcome

This study aimed to determine the functional relationship between the levels of dachshund homolog 1 (DACH1) expression and different subtypes of ovarian cancer and to investigate the possible prognostic value of DACH1 in ovarian cancer. Immunohistochemical staining was deployed to determine the protein levels of DACH1. Staining was performed on patient samples, for whom the detailed follow-up data have been acquired during the last 10 years. Normal, benign, borderline, cancer, and metastatic ovarian cancer samples were included in this study. The results of our study show that DACH1 protein levels increase with the invasiveness of the ovarian cancer. As the cancer progresses from benign and borderline to metastatic, DACH1 protein expression increases as well. Moreover, with the increase in expression, the subcellular distribution of DACH1 changes from nucleus in normal tissue to cytoplasm in cancer. Finally, DACH1 expression levels were compared with estrogen receptor α (ERα) levels, and the results showed that overall DACH1 levels were higher, whereas also DACH1 exhibited increased cytoplasmic expression in ERα-positive ovarian cancer samples. These results indicate that DACH1 is highly expressed in metastatic ovarian cancer compared with that of normal, benign, and borderline ovarian tissues and that it could play an important role in cancer growth.

Abstract 70: A role for the chromatin remodeling protein CHD3 in ovarian cancer therapy response

Abstract Carboplatin and cisplatin are chemotherapeutic agents that are used extensively for treating epithelial ovarian cancer. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Epigenetic silencing, particularly at promoter regions of genes regulates important cell function and has been associated with all stages of tumor formation and progression and may contribute to therapy response. We analyzed the epigenome of 50 primary ovarian tumors and 12 normal ovarian samples using an array based method previously developed in our lab and associated Affymetrix U133 expression data. We then identified gene candidates that segregate patients based on platinum sensitivity and patient survival. These candidates were then pooled into a genome-wide RNAi-based screen where we validated a gene encoding a chromatin remodeling protein, CHD3, a member of the Mi-2 NuRD complex, and show that it is linked to chemoresistance. CHD3 is silenced through an epigenetic mechanism in both ovarian cancer cell lines and primary ovarian tumors. When ovarian cancer cell lines that are transcriptionally silenced for CHD3 are challenged with carboplatin they display a striking slow growth phenotype as well as increased resistance to the chemotherapy drugs carboplatin and cisplatin. Taken together, we provide the first evidence for a role for CHD3 as an important mediator of chemoresistance in ovarian cancer. Furthermore, CHD3 might represent a response predictor and potential therapeutic target for predicting chemoresistance in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 70. doi:10.1158/1538-7445.AM2011-70

Immunohistochemical evidence for the over-expression of Glutathione peroxidase 3 in clear cell type ovarian adenocarcinoma

Glutathione peroxidase 3 (GPX3) is a member of glutathione peroxidase family, exerting one of the most important cellular defense mechanisms against stress signals, including oxidative damage. In this study, the expression of GPX3 mRNA and protein was analyzed for ovarian cancer tissues to test its applicability as a biomarker that can distinguish the four major histologic types of epithelial ovarian cancer. A public microarray dataset containing 99 ovarian cancer and 4 normal ovary samples was downloaded, and GPX3 mRNA expression was analyzed. The expression of GPX3 protein was measured by immunohistochemical staining in 40 epithelial ovarian cancer tissues, 10 for each of the serous, endometrioid, mucinous, and clear cell type. Histoscores were made from the immunohistostaining, and analysis of variance (ANOVA) was performed to quantitate the differences in protein level. Analysis of genomic dataset confirms a GPX3 overexpression in clear cell type ovarian adenocarcinoma compared with normal ovary and 3 other subtypes of epithelial ovarian cancer at mRNA level. GPX3 also shows the highest average antibody staining intensities in clear cell type ovarian adenocarcinomas over the other 3 types in immunostaining on tissue arrays. This is the first validation of GPX3 as a clear cell type-specific biomarker in ovarian cancer patients' tissues by immunostaining. GPX3 may serve as an important molecular marker for the diagnosis and molecular understanding of clear cell carcinoma of the ovary.

Expression of the Wnt antagonist DKK3 is frequently suppressed in sporadic epithelial ovarian cancer

The Wnt pathway plays an important role in embryonic development, and defects in this pathway have been implicated in the tumorigenesis. The Dickkopf 3 (DKK3) is a putative Wnt signaling inhibitor that is frequently inactivated in human cancers. However, the expression of DKK3 in ovarian cancer remains unknown. We investigated the expression of DKK3 in silico using the Digital Differential Display. DKK3 mRNA expression was also analyzed by real-time RT-PCR in ovarian carcinomas and normal ovarian tissues. DKK3 protein expression was determined by immunohistochemistry in the same ovarian carcinomas and normal ovarian tissues. A significantly reduced expression of DKK3 (P < 0.05) was found after comparison of normal ovary- and tumor-derived libraries in the Cancer Genome Anatomy Project (CGAP). DKK3 mRNA expression was reduced in 63% (35 of 56) of tumors compared with normal ovarian samples (P < 0.02). Analysis of 13 matched pairs of ovarian carcinomas and adjacent normal tissues showed significant transcriptional downregulation of DKK3 (>twofold) in 9 paired carcinomas (69%). Loss or weak membranous expression of DKK3 protein was observed in 66% of ovarian cancers (37 of 56) including all tumors with low transcriptional level of DKK3 gene analyzed by real-time PCR. To our best knowledge, this is the first time to demonstrate altered expression of DKK3 in ovarian cancer. The latter could be a relevant mechanism for the activation of the Wnt pathway in the carcinogenesis of ovarian cancer but additional studies are required to elucidate the function of DKK3 silencing in ovarian carcinogenesis.

Abstract 3980: Quantitative detection of discriminatory proteins in tissue and serum of ovarian cancer patients by LC-MSE

Abstract Purpose To search for potential protein markers or activated pathways which play a role in ovarian cancer. Experimental procedures Serum of six patients with a serous adenocarcinoma of the ovary (stage IIIB or higher) was collected before treatment. Matched control patients with a serous cystadenoma of the ovary were chosen as a control group. In addition, homogeneous regions of cells that exhibited uniform histology were isolated from cancer tissue in three of these patients by laser capture microdissection (LCM), normal ovarian epithelial cell samples were taken from their matched controls. Subsequently we employed label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify proteins and determine their absolute concentrations in serum specimens and tissue lysates. Moderated t-tests were used to identify differentially expressed proteins between patient- and control-group, p-values were adjusted for multiple-testing using Benjamini-Hochberg false discovery rates. Proteins were considered to be differentially expressed if the adjusted p-values were &amp;lt;0.05. Summary of the data In the serum specimens, 13 differentially expressed proteins were identified by LC-MSE profiling. Proteins with different concentrations in patients versus control sera include abundant serum proteins such as apolipoprotein AI and transferrin, that both exhibited a lower concentration in serum of cancer patients. Differential expression was also observed for apolipoproteins (APOA IV, APOA II and APOC III) and other proteins that have not been associated with ovarian cancer previously such as C9 and Afamin. In the tissue lysates, 17 differentially expressed proteins were identified. Amounts of collagen found were decreased in cancer tissue. Furthermore, proteins associated with the respiratory chain and cell migration were found in increased amounts in the cancer tissue lysates. Interestingly, no overlap was found between the discriminatory proteins in serum specimens and corresponding tissue lysates, illustrating the challenge to identify a single biomarker in the circulation reflecting ovary cancer. Conclusions Our study revealed several protein changes in serum and the ovary of patients suffering from serous adenocarcinoma of the ovary (stage IIIB or higher). Further investigation of these proteins is warranted to establish whether they could provide new insights into the etiology of the disease and act as potential new disease markers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3980.

Abstract 4891: GULP1, a potential tumor suppressor gene in ovarian tumors and its utility as a biomarker

Abstract Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. GULP1, a molecule not previously related to cancer, is a cytoplasmic adaptor protein with a phosphotyrosine binding domain that plays a role in one of two partially redundant pathways that lead to the engulfment and clearance of apoptotic cells, according to several genetic studies performed in Caenorhabditis elegans. Performing a pharmacologic unmasking technique we observed that this molecule is downregulated in ovarian tumor tissues when compared to normal ovary. Ovarian cancer is the leading cause of death among gynaecological cancers worldwide, this due to the fact that women are diagnosed with advanced stage disease and because of the lack of truly sensitive/specific screening techniques and unavailability of individualized therapy. We performed an expression microarray on 15 ovarian tumor samples, 10 ovarian normal surface epithelium, 3 ovarian cancer cell lines and 3 normal cell lines. All cells have been treated with a demethylating agent which comprehensively uncovers genes silenced by promoter hypermethylation. The selection of the best candidates was based upon the differential expression between normal and tumor samples (or cell lines), them being downregulated in cancer when compared to normal, and finally the re-activation after the treatment with the demethylating agent. One of these promising genes was GULP1, which expression pattern was then assessed by Reverse Transcriptase PCR and Western Blot and compared to its methylation status in the same six ovarian cell lines and showed correlation between absence of expression and presence of methylation (or the opposite situation). We developed quantitative fluorogenic methylation specific PCR (QMSP) for GULP1 and profiled 437 ovarian tumor samples, 17 borderline tumors, 19 cystadenoma samples and 13 normal ovarian samples, finding 34.7% (151/437), 11.7% (2/17), 10.5% (2/19) and 0% (0/13) of methylation frequency, respectively, (establishing an empiric cutoff). Using fisher's exact test, we observe a significant increase in methylation when comparing tumors with cystadenomas and normals (p=0.0439 and p=0.0131, respectively). Late stage tumors also showed a higher frequency of methylation versus early stage (p=0.004). So far, we have demonstrated reduced log growth rates after transiently overexpressing GULP1 in one ovarian cell line (IGROVCP). To assess its potential as a TSG, we have ongoing experiments to assess its biological function as ectopic overexpression of this gene in ovarian cancer cell lines as well as creating knock downs using shRNAs. These preliminary data indicate that GULP1 may be an ovarian cancer biomarker. Further studies on the biology of this gene are needed to evaluate its potential as a TSG and its role in ovarian cancer carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4891.

Abstract 4620: The Secretory Leukocyte Peptidase Inhibitor (SLPI) is differentially expressed in normal ovary and ovarian tumor tissues

Abstract Background: The prognosis for epithelial ovarian cancer (EOC) is significantly improved if detected and treated while contained in the ovaries. However, the majority of ovarian cancer cases are diagnosed after the cancer has spread beyond the ovaries. Serum-based biomarker screening offers the potential to significantly increase the detection of cancer at earlier stages and therefore improving patient prognoses. CA125 (MUC16) is a serum biomarker that is elevated in the majority of ovarian cancer cases, but is limited in clinical utility due to low sensitivity for the detection of early stage disease. One potential complementary biomarker is Secretory Leukocyte Peptidase Inhibitor (SLPI). Objective: In order to assess the potential utility of SLPI as a biomarker for ovarian cancer detection, we have analyzed the expression of both SLPI mRNA and protein in a panel of 27 normal ovary tissue specimens and 61 epithelial ovarian tumors. Methods: SLPI mRNA expression analysis was performed with RNA isolated from fresh frozen tissue specimens using real-time RT-PCR. Median mRNA expression values for the normal and cancerous specimens were compared using the Mann-Whitney U test. Protein expression levels were characterized by immunoblotting whole cell lysates prepared from the same tissues. Results: SLPI transcript levels were found to be significantly up-regulated in the ovarian tumor specimens relative to the levels in normal ovary tissues (two-tailed p&amp;lt;0.0001). Eighty-seven percent (53/61) of the tumor specimens exhibited at least a 10-fold increase in SLPI mRNA as compared to the average expression measured in the 27 normal ovarian tissues. SLPI mRNA expression was increased at least 100-fold in 52% of the tumors. Furthermore, SLPI transcript levels differed significantly between normal ovarian tissue and each of the four major histological subtypes of epithelial ovarian cancer: serous (p&amp;lt;0.0001); endometrioid (p&amp;lt;0.0001), clear cell (p&amp;lt;0.0001) and mucinous (p&amp;lt;0.0001). Tumor specimens were compared to normal ovary samples on the same immunoblot, with equivalent loading of total protein. Overall, 82% (50/61) of the tumor specimens exhibited a qualitative increase in SLPI protein expression relative to the levels observed in normal ovarian tissues. Overexpression of SLPI protein was observed in all four major histological subtypes of EOC, with 79.3% (23/29) of serous and 69.2% (9/13) of mucinous samples demonstrating increased SLPI protein expression. One hundred percent of both the endometrioid and clear cell subtypes (9/9 and 8/8, respectively) also stained positive for SLPI protein. Immunoblot results were in concordance with the mRNA expression (cut-off of greater than 10-fold) for 85.2% (52/61) of the samples. Conclusion: These mRNA and protein expression data support further investigation into the potential effectiveness of SLPI as a biomarker for detection of EOC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4620.

FAU regulates carboplatin resistance in ovarian cancer

The development of chemotherapy resistance by cancer cells is complex, using different mechanisms and pathways. The gene FAU (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified through functional expression cloning and previous data have shown that overexpression enhances apoptosis in several cell types. We demonstrate that the expression of FAU was reduced in the A2780cis (cisplatin resistant subclone of A2780) cell line compared with the A2780 ovarian cancer cell line, and was directly related to the cell line's sensitivity to carboplatin. Downregulation of FAU in the A2780 cell line by transfection with two predesigned short-interfering RNAs (siRNAs) to FAU resulted in a significant increase in resistance to carboplatin-induced cell death. Downregulation resulted in increased cell viability and reduced apoptosis after 72 hr of drug treatment compared with the negative controls (Kruskal-Wallis P = 0.0002). Transfection of the A2780cis cell line with the pcDNA3 plasmid containing FAU was associated with increased sensitivity to carboplatin-induced apoptosis, with decreased cell viability and increased apoptosis (Mann Whitney P < 0.0001). The expression of FAU was examined by quantitative real-time reverse transcriptase polymerase chain reaction in normal and malignant ovarian tissue. A significant reduction in the expression of FAU was seen in the malignant compared with normal ovarian samples (Kruskal-Wallis P = 0.0261). These data support a role for FAU in the regulation of platinum-resistance in ovarian cancer. Further research is needed into the apoptotic pathway containing FAU to investigate the potential for targeted therapies to increase or restore the platinum sensitivity of ovarian cancer.

Roles of vascular endothelial growth factor and platelet-derived growth factor in lymphangiogenesis in epithelial ovarian carcinoma.

To assess roles of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the mechanisms of lymphangiogenesis in epithelial ovarian carcinoma. (1) Expression of Prox1, a newly described lymphatic endothelial cell nucleus marker, VEGF-A, VEGF-C, VEGF-D and PDGF-A, PDGF-B, PDGF-C, PDGF-D were detected by RT-PCR in SKOV3 cell line and in 90 ovarian tissue samples, included 15 benign tumors, 10 borderline tumors, 45 malignant tumors and 20 normal ovarian samples. (2) Expression levels of Prox1, VEGF-A, -C, -D and PDGF-A, -B, -C, -D were detected in 90 ovarian tissue sample mentioned above by real-time quantitative PCR (RTQ-PCR). (1) Prox1 was expressed in ovarian samples mentioned above, while not detected in SKOV3 cell. VEGF-A, -C, -D and PDGF-A, -B, -C, -D were found in SKOV3 cell and various ovarian tissues. (2) Expression levels of Prox1 (2.2 +/- 1.3, P < 0.01), VEGF-A (3.5 +/- 1.5, P < 0.01), VEGF-C (19 +/- 14, P < 0.01), VEGF-D (3.0 +/- 1.8, P < 0.01) and PDGF-A (3.3 +/- 3.3, P < 0.05), PDGF-C (6.9 +/- 4.6, P < 0.01) in malignant group were found to be significantly higher than those in borderline group and benign group. (3) The expression levels of Prox1, VEGF-A and PDGF-A were significantly greater in samples from the patients with lymph node metastasis (Prox1: 3.0 +/- 1.4, VEGF-A: 4.1 +/- 1.7, PDGF-A: 4.9 +/- 4.1), peritoneum metastasis (Prox1: 2.8 +/- 0.9, VEGF-A: 4.0 +/- 1.8, PDGF-A: 4.5 +/- 4.0) and in stage III - IV (Prox1: 2.6 +/- 1.3, VEGF-A: 4.0 +/- 1.4, PDGF-A: 4.1 +/- 3.7) than those without lymph node metastasis, without peritoneum metastasis and in stage I - II. There was a significant increased in the degree of VEGF-C and VEGF-D expression in positive lymph node metastasis group (VEGF-C: 24 +/- 13, VEGF-D: 3.9 +/- 2.0) compared with negative group (P < 0.05). (4) There were significant positive correlations between the expression levels of Prox1 and VEGF-D (r = 0.62, P < 0.01), PDGF-C (r = 0.91, P < 0.01) or PDGF-D (r = 0.61, P < 0.01). VEGF-A, VEGF-C and PDGF-A may promote lymphatic metastasis in epithelial ovarian carcinoma through else mechanisms other than lymphangiogenesis. VEGF-D may facilitate lymphangiogenesis and lymph node metastasis in epithelial ovarian cancer. There is no significant correlation between the expression of PDGF-B and lymphangiogenesis and lymph node metastasis. PCGF-C and PDGF-D may motivate lymphangiogenesis, but could not participate in lymph node metastasis in ovarian carcinoma.

PAX2 expression in low malignant potential ovarian tumors and low-grade ovarian serous carcinomas

Ovarian tumors of low malignant potential and low-grade ovarian serous carcinomas are thought to represent different stages on a tumorigenic continuum and to develop along pathways distinct from high-grade ovarian serous carcinoma. We performed gene expression profiling on three normal human ovarian surface epithelia samples, and 10 low-grade and 10 high-grade ovarian serous carcinomas. Analysis of gene expression profiles of these samples has identified 80 genes upregulated and 232 genes downregulated in low-grade ovarian serous carcinomas. PAX2 was found to be one of the most upregulated genes in low-grade ovarian serous carcinoma. The upregulation of PAX2 was validated by real-time quantitative RT-PCR, western blot and immunohistochemical analyses. Real-time RT-PCR showed a statistically significant difference in PAX2 mRNA expression (expressed as fold change in comparison to normal human ovarian surface epithelia) among ovarian tumors of low malignant potential (1837.38, N=8), low-grade (183.12, N=17), and high-grade (3.72, N=23) carcinoma samples (P=0.015). Western blot analysis revealed strong PAX2 expression in ovarian tumors of low malignant potential (67%, N=3) and low-grade carcinoma samples (50%, N=10) but no PAX2 protein expression in high-grade carcinomas (0%, N=10). Using immunohistochemistry, tumors of low malignant potential (59%, N=17) and low-grade carcinoma (63%, N=16) samples expressed significantly stronger nuclear staining than high-grade ovarian carcinoma samples (9.1%, N=263). Furthermore, consistent with earlier immunohistochemical findings, PAX2 expression was expressed in the epithelial cells of fallopian tubes but not in normal ovarian surface epithelial cells. Our findings further support the two-tiered hypothesis that tumors of low malignant potential and low-grade ovarian serous carcinoma are on a continuum and are distinct from high-grade ovarian carcinomas. In addition, the absence of PAX2 expression in normal ovarian epithelia but expression in fallopian tube fimbria and ciliated epithelial inclusions would suggest the potential development of tumors of low malignant potential and of low-grade ovarian serous carcinomas from secondary Müllerian structures.

Nuclear expression of Snail1 in borderline and malignant epithelial ovarian tumours is associated with tumour progression

BackgroundTranscription factor Snail1 has a central role in induction of epithelial-mesenchymal transition (EMT). The aim of the present study was to elucidate the expression of Snail1 protein during epithelial ovarian tumourigenesis and to study the association of Snail1 expression with clinicopathological factors and prognosis.MethodsEpithelial and stromal fibroblast-like fusiform cells of 14 normal ovarian samples, 21 benign, 24 borderline and 74 malignant epithelial ovarian tumours were studied for Snail1 protein using immunohistochemistry.ResultsNuclei of surface peritoneal cells of normal ovaries (n = 14) were regarded as negative for Snail1. Nuclear expression of Snail1 protein in epithelial ovarian tumours was increased during tumour progression from precursor lesions into carcinomas both in epithelial (p = 0.006) and stromal cells (p = 0.007). Nuclei of benign tumours (n = 21) were negative for Snail1. In borderline tumours (n = 24) occasional positive epithelial cells were found in 2 (8%) samples and in 3 (13%) samples stromal cells were focally positive for Snail1. In carcinomas (n = 74) focal Snail1 staining in epithelial cells was present in 17 (23%) tumours, and in stromal cells in 18 (24%) tumours. Nuclear expression of Snail1 in epithelial or stromal cells was not associated with clinicopathological factors or prognosis.ConclusionNuclear Snail1 expression seems to be related to tumour progression, and expression in borderline tumours indicates a role for Snail1 in early epithelial ovarian tumour development. Snail1 also appears to function more generally in tissue remodelling as positive staining was demonstrated in stromal cells.

Nuclear cyclin B1 is overexpressed in low-malignant-potential ovarian tumors but not in epithelial ovarian cancer

To investigate the role of cyclin G1 and cyclin B1 in ovarian tumorigenesis. We examined cyclin B1 and G1 expression in 58 epithelial ovarian cancer, 18 low-malignant-potential ovarian tumors, and 6 normal ovarian epithelium samples using immunohistochemistry. We also examined cyclin G1 and p53 expression in 7 epithelial ovarian cancer cell lines using Western blot analysis. Nuclear cyclin B1 expression was significantly higher in low-malignant-potential tumors than in normal ovarian epithelium. There was no difference in nuclear or cytoplasmic cyclin B1 or cyclin G1 expression between epithelial ovarian cancer and normal ovarian epithelium. Cyclin G1 and B1 expression was not associated with p53 expression or clinicopathologic features in patients with epithelial ovarian cancer or low-malignant-potential tumors. Our data demonstrated that nuclear cyclin B1 is overexpressed in low-malignant-potential tumors, which may contribute to the development of low-malignant-potential tumors. Cyclin B1 and G1 may not be suitable targets for epithelial ovarian cancer treatment.

Downregulation of connective tissue growth factor by three‐dimensional matrix enhances ovarian carcinoma cell invasion

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intraperitoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hr of 3D collagen culture) coupled with confirmatory real-time reverse-transcriptase polymerase chain reaction, multiple 3D cell culture matrices, Western blot, immunostaining, adhesion, migration and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion- mimicking conditions (3D Type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n = 41), but was present in 100% of normal ovarian epithelium samples (n = 7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using alpha6beta1 and alpha3beta1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion.

The Ep-CAM: A potential candidate for diagnosis in ovarian cancer

e16571 Background: The purpose of this study was to examine the expression of splice variants of the Ep-CAM gene in normal ovarian epithelial tissue and ovarian carcinoma and further to associate the expression of Ep-CAM with clinicopathologic characteristics if such an association exists. Methods: Ep-CAM expression was examined by semiquantitative PCR in 122 ovarian tumors (92 adenocarcinomas, 10 low malignant potential (LMP) tumors, and 10 adenomas) and 10 normal ovaries. Results: In carcinomas as well as LMP tumors and adenomas, Ep-CAM mRNA expression was significantly elevated compared to that in normal ovary samples. Ep-CAM mRNA expression level in carcinomas was significantly elevated compared to that in adenomas and Ep-CAM mRNA expression level in advanced clinical stage diseases was significantly higher than that in early stage diseases in ovarian carcinomas. With regard to histological type, Ep-CAM mRNA expression level in mucinous adenocarcinomas was significantly higher than those in the other tissue subtypes. Conclusions: These features imply that Ep-CAM expression may play an important role in ovarian cancer development and progression, and this may be useful as a diagnostic marker. No significant financial relationships to disclose.

Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas

BackgroundGene expression profiling of formalin-fixed, paraffin-embedded (FFPE) samples represents a valuable approach for advancing oncology diagnostics and enhancing retrospective clinical studies; however, at present, this methodology still requires optimization and thus has not been extensively used. Here, we utilized thorough quality control methods to assess RNA extracted from FFPE samples and then compared it to RNA extracted from matched fresh-frozen (FF) counterparts. We preformed genome-wide expression profiling of FF and FFPE ovarian serous adenocarcinoma sample pairs and compared their gene signatures to normal ovary samples.MethodsRNA from FFPE samples was extracted using two different methods, Ambion and Agencourt, and its quality was determined by profiling starting total RNA on Bioanalyzer and by amplifying increasing size fragments of beta actin (ACTB) and claudin 3 (CLDN3) by reverse-transcriptase polymerase chain reaction. Five matched FF and FFPE ovarian serous adenocarcinoma samples, as well as a set of normal ovary samples, were profiled using whole genome Agilent microarrays. Reproducibility of the FF and FFPE replicates was measured using Pearson correlation, whereas comparison between the FF and FFPE samples was done using a Z-score analysis.ResultsData analysis showed high reproducibility of expression within each FF and FFPE method, whereas matched FF and FFPE pairs demonstrated lower similarity, emphasizing an inherent difference between the two sample types. Z-score analysis of matched FF and FFPE samples revealed good concordance of top 100 differentially expressed genes with the highest correlation of 0.84. Genes characteristic of ovarian serous adenocarcinoma, including a well known marker CLDN3, as well as potentially some novel markers, were identified by comparing gene expression profiles of ovarian adenocarcinoma to those of normal ovary.ConclusionConclusively, we showed that systematic assessment of FFPE samples at the RNA level is essential for obtaining good quality gene expression microarray data. We also demonstrated that profiling of not only FF but also of FFPE samples can be successfully used to identify differentially expressed genes characteristic of ovarian carcinoma.

High-resolution analysis of copy number alterations and associated expression changes in ovarian tumors

BackgroundDNA copy number alterations are frequently observed in ovarian cancer, but it remains a challenge to identify the most relevant alterations and the specific causal genes in those regions.MethodsWe obtained high-resolution 500K SNP array data for 52 ovarian tumors and identified the most statistically significant minimal genomic regions with the most prevalent and highest-level copy number alterations (recurrent CNAs). Within a region of recurrent CNA, comparison of expression levels in tumors with a given CNA to tumors lacking that CNA and to whole normal ovary samples was used to select genes with CNA-specific expression patterns. A public expression array data set of laser capture micro-dissected (LCM) non-malignant fallopian tube epithelia and LCM ovarian serous adenocarcinoma was used to evaluate the effect of cell-type mixture biases.ResultsFourteen recurrent deletions were detected on chromosomes 4, 6, 9, 12, 13, 15, 16, 17, 18, 22 and most prevalently on X and 8. Copy number and expression data suggest several apoptosis mediators as candidate drivers of the 8p deletions. Sixteen recurrent gains were identified on chromosomes 1, 2, 3, 5, 8, 10, 12, 15, 17, 19, and 20, with the most prevalent gains localized to 8q and 3q. Within the 8q amplicon, PVT1, but not MYC, was strongly over-expressed relative to tumors lacking this CNA and showed over-expression relative to normal ovary. Likewise, the cell polarity regulators PRKCI and ECT2 were identified as putative drivers of two distinct amplicons on 3q. Co-occurrence analyses suggested potential synergistic or antagonistic relationships between recurrent CNAs. Genes within regions of recurrent CNA showed an enrichment of Cancer Census genes, particularly when filtered for CNA-specific expression.ConclusionThese analyses provide detailed views of ovarian cancer genomic changes and highlight the benefits of using multiple reference sample types for the evaluation of CNA-specific expression changes.

Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries

BackgroundMicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.MethodologyWe used massively parallel pyrosequencing (i.e., “454 sequencing”) to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.ConclusionsThis report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer.

Expression of gonadotrophin releasing hormone receptor I is a favorable prognostic factor in epithelial ovarian cancer

The majority of epithelial ovarian cancers originate in the ovarian surface epithelium. The ovarian surface epithelium is a hormonally responsive tissue, and hormones are thought to play a key role in the development of this type of cancer. Gonadotrophin releasing hormone II is one of 2 isoforms which are thought to act through gonadotrophin releasing hormone receptor I, and gonadotrophin releasing hormone II has been shown to cause growth inhibition of cultured ovarian surface epithelium. The aim of this study was to investigate the expression levels and prognostic significance of gonadotrophin releasing hormone II and the gonadotrophin releasing hormone receptor I in epithelial ovarian cancer. Gonadotrophin releasing hormone II and gonadotrophin releasing hormone receptor I messenger RNA expression was examined in 23 cancers and 7 normal ovarian surface epithelium samples by quantitative real time polymerase chain reaction. An ovarian cancer tissue microarray containing 139 cases was constructed and immunohistochemical analysis of gonadotrophin releasing hormone II and gonadotrophin releasing hormone receptor I protein expression was performed and correlated with clinical outcome data. Gonadotrophin releasing hormone II messenger RNA expression was lower in cancer samples compared to normal ovarian surface epithelium samples (P < .05). Gonadotrophin releasing hormone II protein expression correlated with histologic subtype (25% serous versus 45% nonserous, P < .05) but not with overall survival. Gonadotrophin releasing hormone receptor I messenger RNA expression was highest in serous tumors when compared to non serous (P < .05) and normal tissue (P < .001). Expression of the gonadotrophin releasing hormone receptor I protein was also found to correlate with patient survival (P < .05). We have demonstrated gonadotrophin releasing hormone II and its receptor, gonadotrophin releasing hormone receptor I, are present in clinical ovarian samples, and that gonadotrophin releasing hormone receptor I protein expression is a favorable prognostic factor, suggesting these proteins play an important role in the development of epithelial ovarian cancer.

CAMP response element–binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation

Approximately 90% of human ovarian tumors result from transformation of ovarian surface epithelial cells. It has been hypothesized that repeated destruction of the epithelial cells during ovulation, followed by proliferation and migration of epithelial cells to restore the ovarian surface, renders these cells susceptible to mutagenic events. One of the proteins found to promote ovarian surface epithelial cell survival and proliferation was the transcription factor, cAMP response element-binding protein (CREB). Thus, the objective of this study was to determine whether CREB was also highly expressed in tumor cells originating from the ovarian epithelium. Using an ovarian cancer tissue array, it was observed that approximately 54% of the epithelial-derived human ovarian tumors displayed moderate or high levels of CREB immunostaining, while none of the normal ovarian samples did. Comparison of CREB levels in a human ovarian tumor cell line to those of a normal ovarian epithelial cell line revealed elevated levels of CREB and phosphorylated CREB in the ovarian tumor cells. To determine whether CREB regulated proliferation and/or apoptosis in the ovarian tumor cell line, CREB expression was suppressed using RNA interference. Decreased CREB expression significantly reduced ovarian tumor cell proliferation, while there was no effect on apoptosis in these cells. Finally, we showed that CREB is highly expressed in an in vivo murine model of ovarian tumorigenesis. Therefore, CREB is frequently overexpressed in ovarian cancer where it appears to promote cell proliferation.