Epithelial Ovarian Cancer Tissue Samples Research Articles

Potential molecular signatures in epithelial ovarian cancer by genome wide expression profiling

Ovarian cancer is the deadliest of all gynecologic cancers because of its late diagnosis and poor treatment outcomes. This study aimed to identify potential molecular signatures associated with biological processes that are implicated in epithelial ovarian cancer (EOC). Expression profiling was carried out on 16 fresh frozen EOC and normal ovarian tissue samples using the Illumina Whole Genome DASL assay (cDNA-mediated annealing, selection, extension and ligation). The differentially expressed genes were analyzed using the GeneSpring GX11.5 and Pathway Studio 8.0 software. The microarray results were validated using the immunohistochemistry analyses. Unpaired t-test identified 652 (270 up- and 382 downregulated) significant differentially expressed genes (P < 0.001 and fold change ≥2.0). Hierarchical clustering analysis displayed a distinct separation of cancer and normal samples. Gene set enrichment analysis identified alterations in the expression of genes associated with cancer development and progression. Positive immunostaining of claudin-7, ephrin receptor A1 and Forkhead Box M1 in EOC was consistent with the upregulation of these genes in the microarray result. However, the positive immunostaining of fibroblast growth factor-7 in cancer tissues was not in accordance with the downregulation of this gene in the microarray result. These results identify significant genes and their related biological processes which may contribute to the better understanding of development and progression of epithelial ovarian cancer.

An insulin-like growth factor-II intronic variant affects local DNA conformation and ovarian cancer survival

Insulin-like growth factor-II (IGF-II) may be a prognostic marker in ovarian cancer, and its intronic single nucleotide polymorphism (SNP) rs4320932 has been associated with risk of the disease. We determined whether rs4320932 is associated with IGF-II expression and patient survival in ovarian cancer, and explored whether the SNP variation affects DNA conformation both in the absence of and presence of carboplatin. IGF-II genotype (rs4320932) and phenotype were analyzed in 212 primary invasive epithelial ovarian cancer tissue samples with Taqman® SNP genotyping assays, quantitative reverse transcription-polymerase chain reaction and commercial enzyme-linked immunosorbent assay. DNA conformation was evaluated by circular dichroism (CD) spectra. Kaplan-Meier survival curves and Cox proportional hazard regression models were used to analyze the SNP associations with patient survival. The C allele of rs4320932, previously associated with decreased risk of ovarian cancer development, was here associated with significantly elevated risks of relapse (Ptrend = 0.0002) and death (Ptrend = 0.0006), remaining significant in multivariate analyses. The adjusted hazard ratios were 3.05 (95% confidence interval [CI]: 1.47-6.37) for relapse and 3.28 (95% CI: 1.64-6.57) for death, respectively. The variant was also significantly associated with chemotherapy response, but not with other clinicopathologic variables or with IGF-II expression. DNA with genotypes TT and CC had distinct CD spectra in both the absence of and presence of carboplatin. These findings suggest that the intronic SNP rs4320932 affects patient survival and chemotherapy response via alteration of DNA conformation, but not through regulation of IGF-II expression. This novel finding may have implications in individualized medicine for the design of specific molecules targeting DNA of specific conformations.

Subtype Specific Elevated Expression of Hyaluronidase-1 (HYAL-1) in Epithelial Ovarian Cancer

BackgroundEpithelial ovarian cancer (EOC) is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 3p in serous EOC pathogenesis. Our objective was to evaluate the expression of HYAL1, located at chromosome 3p21.3, in these EOC subtypes, and to investigate its correlation with the expression of steroid hormone receptors.Methodology/Principal FindingsWe determined the mRNA expression of HYAL1, estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in EOC tumor samples and cell lines using quantitative RT-PCR. We also examined the expression of these genes in a publicly available microarray dataset. HYAL-1 enzyme activity was measured in EOC cell lines and in plasma samples from patients. We found that HYAL1 mRNA expression was elevated in clear cell and mucinous EOC tissue samples, but not in serous and endometrioid samples, normal ovaries or benign tumors. Similar results were obtained by two different techniques and with tissue sample cohorts from two independent institutions. Concordantly, HYAL1 mRNA levels and enzymatic activity were elevated only in EOC cell lines derived from clear cell and mucinous subtypes. We also showed that HYAL1 mRNA was inversely correlated to that of ERα specifically in clear cell and mucinous EOCs. Additionally, ectopic expression of ERα in a clear cell EOC cell line (ER- and PR-negative) induced 50% reduction of HYAL1 mRNA expression, supporting a role of ERα in HYAL1 gene regulation. Significantly, HYAL-1 activity was also high in the plasma of patients with these EOC subtypes.Conclusions/SignificanceThis is the first report showing high HYAL-1 levels in EOC and demonstrating HYAL1 gene repression by ERα. Our results identify Hyaluronidase-1 as a potential target/biomarker for clear cell and mucinous EOCs and especially in tumors with low ERα levels.

Abstract A115: Mitochondrial analysis and epithelial ovarian carcinoma

Abstract Survival rates of ovarian cancer patients are low, largely due to late diagnosis. Early detection plays a major role towards improving outcomes in ovarian cancer, however few candidate biomarkers have shown efficacy for the detection of preclinical disease. Human mitochondrial gene mutations have increasingly been associated with various cancers. The mitochondrial genome is highly susceptible to oxidative damage and may accumulate damage over a long period of time. Consequently, variations in (mtDNA) may potentially play a role as a modifying risk factor in the development of age-related diseases such as ovarian cancer. The goal of this study is to examine correlations between mitochondrial gene alterations and three subtypes of ovarian tumor progression. Mitochondrial DNA fragments were obtained from 120 epithelial ovarian cancer tissue samples (40 serous; 40 endometrioid and 40 mucinous ovarian tumors respectively). The entire mitochondrial genome was PCR amplified using 9 overlapping primers sets and analyzed using both high resolution restriction digest and PCR-based sequencing techniques. Additionally, the levels of carbonyl proteins in cytosolic fraction of tissues were assessed by standard colorimetric technique, using 2-4-dinitrophenylhydrazine (DNPH) to derivatize protein, and measure the absorbance of the DNP-hydrazones at 370 nm. In this study we revealed the presence thirty-nine polymorphisms of which 28 were unreported. The observed mutations with notably frequencies (41-93%) among these samples were at np C7028T, C7256T, G7520A, T8548G, T8588C, A8860G, C9488G, C9500T, T9540C, C9857T, and T9951C. Furthermore, six unreported point mutations with frequencies of 14-41 % were observed at np G7520A, T8548G, C9488G, C9500T, C9857 and T9951C. A combined mutation of G7520A and C7256T was frequent at 45% in endometrioid stage III only. Interestingly, variants C7020T (56%) and at np A8860G (92%) were evenly distributed in all three studied ovarian tumor subtype and stages. Variants T9540C and C7520T showed significant higher frequency in African American samples compare to Caucasian samples and these may be used for further investigation for cancer disparity study. A number of the observed sequence variants were germ-lines with variants found in these mitochondrial genes/regions; D-Loop, 12S rRNA-tRNAphe, tRNAval, COX I, tRNAser, tRNAasp, COX II, tRNAlys, ATPase 6, ATPase 8, COX III, ND2, and ND3 genes among the three subtypes of ovarian cancer. Our results showed the elevation of the ratio of protein carbonyl/total protein in all three types of serous polyp as compare to their surrounding tissue. The level of the ratio of protein carbonyls/total protein decreased as a result of serous ovarian tumor progression. Our study suggests that certain mitochondrial DNA sequence variants and protein carbonyl (CO) groups, oxidative stress may play a potential role in etiological differences that may exist between the pathogenicity of subtypes and stages of benign and invasive epithelial ovarian tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A115.

Enhanced expression of Annexin A4 in clear cell carcinoma of the ovary and its association with chemoresistance to carboplatin

Clear cell carcinoma (CCC) of the ovary is known to be highly resistant to platinum-based chemotherapy. The purpose of our study was to identify a candidate protein that is associated with chemoresistance of CCC and to investigate the specific mechanism of chemoresistance conferred by the identified protein. Enhanced expression of Annexin A4 (Anx A4) was identified in ovarian CCC cells using 2-D differential gel electrophoresis (2D-DIGE) and mass spectrometry. Anx A4 levels were elevated in CCC cells compared with non-CCC cells as determined by real-time RT-PCR and Western blot analysis. Immunohistochemical analysis of Anx A4 was performed in 126 epithelial ovarian cancer tissue samples and demonstrated significantly elevated levels of Anx A4 protein levels in ovarian CCC tumors compared with ovarian serous and endometrioid tumors (p < 0.01). Anx A4-transfected ovarian non-CCC cells were more resistant to carboplatin (IC50 = 42 microM) compared with control cells (IC50 = 23 microM) as determined by modified MTT assay. Intracellular platinum levels were significantly lower in Anx A4-transfected cells compared with control cells after carboplatin treatment (p = 0.0020) and after an additional 360 min of carboplatin-free incubation (p = 0.0004), as measured by atomic absorption spectrophotometry. Expression of Anx A4 is elevated in ovarian CCC tumors and is associated with chemoresistance in cultured ovarian cancer cells. These results demonstrate that Anx A4 confers chemoresistance in ovarian cancer cells in part by enhancing drug efflux. Thus, Anx A4 may represent a novel therapeutic target of chemoresistance in patients with ovarian CCC.

Correlation of the survival of ovarian cancer patients with mRNA expression of the 60-kD heat-shock protein HSP-60.

Ovarian carcinomas express the 60-kD heat-shock protein HSP-60 at widely varying levels in different tumors. The aim of this study was to determine whether there was a relationship between expression of HSP-60 and survival in patients with ovarian carcinoma. Total RNA and DNA were prepared from 51 epithelial ovarian cancer tissue samples. The expression and structure of the HSP-60 gene were examined by Northern and Southern blot analyses using the carboxyl-terminal portion of this gene as a probe (0.89 kilobases [kb]). HSP-60 expression was correlated with overall survival by the Kaplan-Meier method. The 2.3-kb HSP-60 message was detected in all samples, but there was marked variation from tumor to tumor. Patients were classified into two groups on the basis of HSP-60 expression: group 1 (n = 25) included patients with low expression, and group 2 (n = 26) consisted of patients with high expression. There were no significant differences between the groups with respect to age, cell type, pathologic grade, clinical stage, and previous treatment. After a median follow-up period of 17 months, Kaplan-Meier plots demonstrated a much better survival for group 1 (median, 46.8 months; 41% at 4 years) than group 2 (median, 22.1 months; 16% at 3.9 years), a difference that was highly significant by the Mantel-Haenszel test (P = .00183). Southern blot analysis of these samples showed no amplification or rearrangement of the gene. The level of HSP-60 mRNA expression is a valuable prognostic factor in epithelial ovarian cancer. Variation in the level of expression is not due to amplification of this gene.