Normal Fallopian Tube Samples Research Articles

Prediction of Ovarian Cancer with Deep Machine Learning and Alternative Splicing

Objective: Early detection of ovarian cancer could lead to improved survival rates, however no method has reliably been able to predict ovarian cancer. The aim of this study is to determine if processing alternative splicing data from high grade serous ovarian cancer patients using machine learning analytics will discriminate high grade serous ovarian cancer from normal fallopian tube samples. The ultimate goal would be to have a model that can predict high grade serous ovarian cancer with a blood test. Methods: This is a case-control study of patients with confirmed high grade serous ovarian cancer and those undergoing salpingectomy for benign indications. RNA-sequencing was performed on all samples. RNA-sequence data was then put into Deep-learning augmented RNA-seq analysis of transcript splicing software suite. Deep-learning augmented RNA-seq analysis of transcript splicing created a model of differential alternative splicing aimed to discriminate between high grade serous ovarian cancer and normal fallopian tube. DEXSeq analysis was used to determine exon-based expression. Initial results with both analytics were then modelled with multivariate lasso regression to create prediction models (performance determined by area under the curve and 95% CI). Models created were the validated using The Cancer Genome Atlas data sets. Results: One hundred and twelve high grade serous ovarian cancer and 12 benign samples were successfully sequenced. Deep-learning augmented RNA-sequencing analysis of transcript splicing identified 998 unique differentially expressed exons between high grade serous ovarian cancer and controls. Multivariate lasso regression analysis identified several exons that predicted high grade serous ovarian cancer with high performance. Specifically, ENSG00000182512:E001 from gene GLRX5 was highly predictive of high grade serous ovarian cancer with an area under the curve of 100%. Conclusions: Application of machine learning analytics to exon differential expression, most likely due to alternative splicing, predicted high grade serous ovarian cancer with high performance. These results were validated in an independent dataset of cases and controls. Differential exon expression from cell-free RNA potentially could be used for early diagnosis of high grade serous ovarian cancer.

Expression Profiling in Ovarian Cancer Reveals Coordinated Regulation of BRCA1/2 and Homologous Recombination Genes.

Predictive biomarkers are crucial in clarifying the best strategy to use poly(ADP-ribose) polymerase inhibitors (PARPi) for the greatest benefit to ovarian cancer patients. PARPi are specifically lethal to cancer cells that cannot repair DNA damage by homologous recombination (HR), and HR deficiency is frequently associated with BRCA1/2 mutations. Genetic tests for BRCA1/2 mutations are currently used in the clinic, but results can be inconclusive due to the high prevalence of rare DNA sequence variants of unknown significance. Most tests also fail to detect epigenetic modifications and mutations located deep within introns that may alter the mRNA. The aim of this study was to investigate whether quantitation of BRCA1/2 mRNAs in ovarian cancer can provide information beyond the DNA tests. Using the nCounter assay from NanoString Technologies, we analyzed RNA isolated from 38 ovarian cancer specimens and 11 normal fallopian tube samples. We found that BRCA1/2 expression was highly variable among tumors. We further observed that tumors with lower levels of BRCA1/2 mRNA showed downregulated expression of 12 additional HR genes. Analysis of 299 ovarian cancer samples from The Cancer Genome Atlas (TCGA) confirmed the coordinated expression of BRCA1/2 and HR genes. To facilitate the routine analysis of BRCA1/2 mRNA in the clinical setting, we developed a targeted droplet digital PCR approach that can be used with FFPE samples. In conclusion, this study underscores the potential clinical benefit of measuring mRNA levels in tumors when BRCA1/2 DNA tests are negative or inconclusive.

Bacterial, archaea and viral transcripts (BAVT) expression in endometrial cancer

<h3>Objectives:</h3> Microbes are found virtually everywhere, including the human body. High-throughput sequencing of microbial genome, also known as metagenomic sequencing, may help to understand interactions between human and microbial processes. In this pilot study we aimed to determine differences of bacterial, archaea and viral expression between normal fallopian tube samples and ovarian and endometrial cancer samples. Then, we assessed these differentially expressed transcripts in endometrial and ovarian cancer from TCGA data. <h3>Methods:</h3> RNA was extracted and sequenced from 62 endometrial cancers, 104 high-grade serous ovarian cancers and 12 normal tubes specimens from our Biobank repository. Sequencing was performed using the Illumina HiSeq4000 platform that sequences 150 bp paired-end transcripts. Reads were processed with Centrifuge, a novel algorithm that classifies metagenomic sequences. We built our own index to differentiate between bacterial, archaea, viral and human transcripts. Resulting number of transcripts were normalized, log transformed and compared between tubal epithelium and ovarian and endometrial cancers (t-test). Lasso regression was used to identify significant independent transcripts between samples. TCGA ovarian and endometrial samples were processed as previously, and expression of significant probes were evaluated. <h3>Results:</h3> Bacterial, archaea and viral transcripts (BAVT) were 4.7% and 3,1% of all transcriptome in tubal and ovarian cancer respectively. 266 transcripts were differentially expressed between normal tubes and ovarian and endometrial cancer, with a p<0.005. 11 bacteria and 1 virus were independently significant in the multivariate regression model. These microbes were mapped close to genome regulatory areas. In general, there were more BVAT in normal tube samples. The amount of BAVT decreased in endometrial cancer and even more in ovarian cancer, supporting this regulatory role of BAVT. BAVTs in TCGA were 0.8% of all transcriptome. <h3>Conclusions:</h3> BAVT expression is different between normal tissues and cancers in the gynecologic tract. Cancer amount of BAVT seems to decrease in malignant tissues, supporting a regulatory role of BAVT. Further studies are needed to determine the significance of BAVT amount and change of expression.

Abstract PO032: Bacterial, archaea and viral transcripts (BAVT) expression in endometrial cancer

Abstract Objectives: Microbes are found virtually everywhere, including the human body. High-throughput sequencing of microbial genome, also known as metagenomic sequencing, may help to understand interactions between human and microbial processes. In this pilot study we aimed to determine differences of bacterial, archaea and viral expression between normal fallopian tube samples and ovarian and endometrial cancer samples. Then, we assessed these differentially expressed transcripts in endometrial and ovarian cancer from TCGA data. Methods: RNA was extracted and sequenced from 62 endometrial cancers, 104 high-grade serous ovarian cancers and 12 normal tubes specimens from our Biobank repository. Sequencing was performed using the Illumina HiSeq4000 platform that sequences 150 bp paired-end transcripts. Reads were processed with Centrifuge, a novel algorithm that classifies metagenomic sequences. We built our own index to differentiate between bacterial, archaea, viral and human transcripts. Resulting number of transcripts were normalized, log transformed and compared between tubal epithelium and ovarian and endometrial cancers (t-test). Lasso regression was used to identify significant independent transcripts between samples. TCGA ovarian and endometrial samples were processed as previously, and expression of significant probes were evaluated. Results: Bacterial, archaea and viral transcripts (BAVT) were 4.7% and 3,1% of all transcriptome in tubal and ovarian cancer respectively. 266 transcripts were differentially expressed between normal tubes and ovarian and endometrial cancer, with a p&amp;lt;0.005. 11 bacteria and 1 virus were independently significant in the multivariate regression model. These microbes were mapped close to genome regulatory areas. In general, there were more BVAT in normal tube samples. The amount of BAVT decreased in endometrial cancer and even more in ovarian cancer, supporting this regulatory role of BAVT. BAVTs in TCGA were 0.8% of all transcriptome. Conclusions: BAVT expression is different between normal tissues and cancers in the gynecologic tract. Cancer amount of BAVT seems to decrease in malignant tissues, supporting a regulatory role of BAVT. Further studies are needed to determine the significance of BAVT amount and change of expression. Citation Format: Silvana Pedra Nobre, Nicholas Cardillo, David Bender, Michael Goodheart, Eric Devor, Jesus Gonzalez Bosquet. Bacterial, archaea and viral transcripts (BAVT) expression in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO032.

Identification of Novel lncRNAs in Ovarian Cancer and Their Impact on Overall Survival.

Long non-coding RNA’s (lncRNA) are RNA sequences that do not encode proteins and are greater than 200 nucleotides in length. They regulate complex cellular mechanisms and have been associated with prognosis in various types of cancer. We aimed to identify lncRNA sequences that are associated with high grade serous ovarian cancer (HGSC) and assess their impact on overall survival. RNA was extracted from 112 HGSC patients and 12 normal fallopian tube samples from our Biobank tissue repository. RNA was sequenced and the Ultrafast and Comprehensive lncRNA detection and quantification pipeline (UClncR) was used for the identification of lncRNA sequences. Univariate logistic and multivariate lasso regression analyses identified lncRNA that was associated with HGSC. Univariate and multivariate Cox proportional hazard ratios were used to evaluate independent predictors of survival. 1943 of 16,325 investigated lncRNA’s were differentially expressed in HGSC as compared to controls (p < 0.001). Nine of these demonstrated association with cancer after multivariate lasso regression. Our multivariate analysis of survival identified four lncRNA’s associated with survival in HGSC. Three out of these four were found to be independently significant after accounting for all clinical covariates. Lastly, seven lncRNAs were independently associated with initial response to chemotherapy; four portended a worse response, while three were associated with improved response. More research is needed, but there is potential for these lncRNAs to be used as biomarkers of HGSC or predictors of treatment outcome in the future.

Mining Featured Biomarkers Linked with Epithelial Ovarian CancerBased on Bioinformatics.

Epithelial ovarian cancer (EOC) is the18th most common cancer worldwide and the 8th most common in women. The aim of this study was to diagnose the potential importance of, as well as novel genes linked with, EOC and to provide valid biological information for further research. The gene expression profiles of E-MTAB-3706 which contained four high-grade ovarian epithelial cancer samples, four normal fallopian tube samples and four normal ovarian epithelium samples were downloaded from the ArrayExpress database. Pathway enrichment and Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) were performed, and protein-protein interaction (PPI) network, microRNA-target gene regulatory network and TFs (transcription factors) -target gene regulatory network for up- and down-regulated were analyzed using Cytoscape. In total, 552 DEGs were found, including 276 up-regulated and 276 down-regulated DEGs. Pathway enrichment analysis demonstrated that most DEGs were significantly enriched in chemical carcinogenesis, urea cycle, cell adhesion molecules and creatine biosynthesis. GO enrichment analysis showed that most DEGs were significantly enriched in translation, nucleosome, extracellular matrix organization and extracellular matrix. From protein-protein interaction network (PPI) analysis, modules, microRNA-target gene regulatory network and TFs-target gene regulatory network for up- and down-regulated, and the top hub genes such as E2F4, SRPK2, A2M, CDH1, MAP1LC3A, UCHL1, HLA-C (major histocompatibility complex, class I, C), VAT1, ECM1 and SNRPN (small nuclear ribonucleoprotein polypeptide N) were associated in pathogenesis of EOC. The high expression levels of the hub genes such as CEBPD (CCAAT enhancer binding protein delta) and MID2 in stages 3 and 4 were validated in the TCGA (The Cancer Genome Atlas) database. CEBPD andMID2 were associated with the worst overall survival rates in EOC. In conclusion, the current study diagnosed DEGs between normal and EOC samples, which could improve our understanding of the molecular mechanisms in the progression of EOC. These new key biomarkers might be used as therapeutic targets for EOC.

Networks of E-cadherin, β1 integrin, and focal adhesion kinase in the pathogenesis of tubal pregnancy

E-cadherin, β1 integrin, and focal adhesion kinase (FAK) are reported to involved in eutopic implantation by mediating cell adhesion. However, less is documented about their roles in ectopic implantation. This study was undertaken to evaluate the roles and networks of E-cadherin, β1 integrin, and FAK in tubal pregnancy. A total of 31 Fallopian tube specimens were obtained from tubal pregnant women. Immunohistochemistry and western blot were used to analyze the distributions and levels of E-cadherin, β1 integrin and phosphorylated-FAK (Pho-FAK) in the Fallopian tube epithelium. Normal Fallopian tube samples derived from non-pregnant women with benign genital diseases were used for comparison. E-cadherin presented in the cytomembrane of tubal epithelial cells and β1 integrin mainly expressed in the cytoplasm. A lowest-level of E-cadherin was detected in the implantation site (0.63 ± 0.29) when compared with the non-implantation site (0.95 ± 0.37) and the controls (0.89 ± 0.33) (P < 0.05). β1 integrin, as well as Pho-FAK in the implantation site (0.81 ± 0.35; 0.72 ± 0.24), showed a higher-level than that in the non-implantation site (0.59 ± 0.26; 0.48 ± 0.27) or the control group (0.38 ± 0.19; 0.36 ± 0.25) (p < .05). The decreased E-cadherin and increased β1 integrin are implicated in tubal pregnancy. The involvement of β1 integrin maybe depends on β1 integrin/FAK signaling.

Abstract 4587: ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

Abstract Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear. Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples. Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival. Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients. Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.

EphA5 protein, a potential marker for distinguishing histological grade and prognosis in ovarian serous carcinoma.

BackgroundOvarian serous carcinoma (OSC) is the most common ovarian epithelial malignancy. Disregulation of Eph/ephrin signaling has been implicated in oncogenesis and tumor progression. EphA5 receptor is one of large families of Eph tyrosine kinase receptor and is documented in the development of nervous system. Till now, there is no published data about the role of EphA5 in ovarian epithelial neoplasmas.MethodsThis study aims to investigate the expression of EphA5 protein in ovarian serous carcinoma, and its relationship to clinical pathological characteristics. Sixty-one cases of ovarian serous carcinoma, 24 cases of benign ovarian serous tumors, 42 cases of serous borderline tumors and 20 cases of normal fallopian tubes were examined using immunohistochemical staining. The relationship between EphA5 expression and pathological parameters was analyzed. Kaplan-Meier survival function was used to analyze prognosis of patients.ResultsImmunostaining analysis demonstrated that the EphA5 protein was highly expressed in 100% (20/20) of normal fallopian tube samples, 100% (24/24) of benign epithelial ovarian tumors, 76% (32/42) of ovarian serous borderline tumors, and 31% (19/61) of ovarian serous carcinomas. Loss of EphA5expression was associated with tumor grade (P < 0.001) and FIGO stage (P = 0.005). The survival analysis showed that patients with negative or weak expression of EphA5 protein had a poor outcome than those with positive expression (P = 0.004).ConclusionsOur results show that EphA5 may be a potential biomarker for distinguishing high-and low-grade ovarian serous carcinoma and a potential prognostic marker.

Expression of EphB6 in ovarian serous carcinoma is associated with grade, TNM stage and survival

BackgroundOvarian serous carcinoma (OSC) is the most common ovarian epithelial malignancy. Despite new medical and surgical advances, the overall 5-year survival for OSC remains poor. There is an important need...

Abstract B02: DNA promoter methylation of HIST1H2BB as a potential early detection biomarker to reduce ovarian cancer disparities in the United States

Abstract Background: Ovarian cancer disproportionately affects African American women in the United States (US) at all stages of the disease, from presentation through treatment, and ultimately leads to increased mortality and decreased survival, compared to non-Hispanic White women. Till recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high-grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Purpose of study: Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium. Using the Infinium HumanMethylation 450 Array, that permits to quantitatively assess over 450,000 methylation sites, within and outside of CpG islands and more than 20,000 genes per sample, at single-nucleotide resolution, we have obtained the genome-wide methylation profile of HGSC and fallopian tubal epithelium. We conducted a pilot study by comparing the global methylation status in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Results: Our results identified the HIST1H2BB promoter as the most differentially methylated region when comparing HGSC with normal fallopian tube samples. Furthermore, we also found that HIST1H2BB is differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Conclusions: Taken together, these results support the potential use of HIST1H2BB promoter methylation as a diagnostic and prognostic biomarker for ovarian cancer. Our findings could lead to the development of a biomarker for the diagnosis and/or prognosis of ovarian cancer. An early detection biomarker for ovarian cancer could contribute to the reduction and eventual elimination of ovarian cancer health disparities in the US. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter methylation of HIST1H2BB as a potential early detection biomarker to reduce ovarian cancer disparities in the United States. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B02.

Abstract AS15: DNA promoter hypermethylation of HIST1H2BB as a diagnostic and prognostic biomarker for ovarian cancer

Abstract Background: Until recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Purpose of study: Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium. Using the Infinium HumanMethylation 450 Array, that permits to quantitatively assess over 450,000 methylation sites, within and outside of CpG islands and more than 20,000 genes per sample, at single-nucleotide resolution, we have obtained the genome-wide methylation profile of HGSC and fallopian tubal epithelium. We conducted a pilot study by comparing the global methylation in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Results: Our results identified the HIST1H2BB promoter as the most differentially methylated region when comparing HGSC with normal fallopian tube samples. Furthermore, we also found that HIST1H2BB is differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Conclusions: Taken together, these results support the potential of HIST1H2BB promoter hypermethylation as a diagnostic and prognostic biomarker. Our findings could lead to the development of a biomarker for the diagnosis and/or prognosis of ovarian cancer and could contribute to the current understanding of the mechanisms of progression in ovarian cancer. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter hypermethylation of HIST1H2BB as a diagnostic and prognostic biomarker for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS15.

Abstract 2960: DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer

Abstract Till recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium, using the Infinium HumanMethylation 450 Array. We compared the methylation in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Our results identified the promoter regions of HIST1H2BB, MAGI2, and EPSTI1 to be differentially methylated when comparing HGSC with normal fallopian tube samples. Furthermore, these genes were differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Quantitative methylation specific PCR analysis confirmed our results of differential methylation among short-term and long-term survivors, and show that these genes are also methylated in ovarian cancer cell lines. Taken together, we have identified genes that could have potential as diagnostic and prognostic biomarkers. Our findings could lead to the development of a biomarker panel for the diagnosis and/or prognosis of ovarian cancer. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2015-2960

Low levels of IGFBP7 expression in high-grade serous ovarian carcinoma is associated with patient outcome.

BackgroundInsulin-like growth factor binding protein 7 (IGFBP7) has been suggested to act as a tumour suppressor gene in various human cancers, yet its role in epithelial ovarian cancer (EOC) has not yet been investigated. We previously observed that IGFBP7 was one of several genes found significantly upregulated in an EOC cell line model rendered non-tumourigenic as consequence of genetic manipulation. The aim of the present study was to investigate the role of IGFBP7 in high-grade serous ovarian carcinomas (HGSC), the most common type of EOC.MethodsWe analysed IGFBP7 gene expression in 11 normal ovarian surface epithelial cells (NOSE), 79 high-grade serous ovarian carcinomas (HGSC), and seven EOC cell lines using a custom gene expression array platform. IGFBP7 mRNA expression profiles were also extracted from publicly available databases. Protein expression was assessed by immunohistochemistry of 175 HGSC and 10 normal fallopian tube samples using tissue microarray and related to disease outcome. We used EOC cells to investigate possible mechanisms of gene inactivation and describe various in vitro growth effects of exposing EOC cell lines to human recombinant IGFBP7 protein and conditioned media.ResultsAll HGSCs exhibited IGFBP7 expression levels that were significantly (p = 0.001) lower than the mean of the expression value of NOSE samples and that of a whole ovary sample. IGFBP7 gene and protein expression were lower in tumourigenic EOC cell lines relative to a non-tumourigenic EOC cell line. None of the EOC cell lines harboured a somatic mutation in IGFBP7, although loss of heterozygosity (LOH) of the IGFBP7 locus and epigenetic methylation silencing of the IGFBP7 promoter was observed in two of the cell lines exhibiting loss of gene/protein expression. In vitro functional assays revealed an alteration of the EOC cell migration capacity. Protein expression analysis of HGSC samples revealed that the large majority of tumour cores (72.6%) showed low or absence of IGFBP7 staining and revealed a significant correlation between IGFBP7 protein expression and a prolonged overall survival (p = 0.044).ConclusionThe low levels of IGFPB7 in HGSC relative to normal tissues, and association with survival are consistent with a purported role in tumour suppressor pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1138-8) contains supplementary material, which is available to authorized users.

Differential Analysis of Ovarian and Endometrial Cancers Identifies a Methylator Phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.

Expressions of β3 Integrin and Fibronectin in Salpingitis

Objective To investigate the association between β3 integrin-fibronectin signaling and salpingitis. Methods Thirteen chronically inflamed tubal samples were included in the case group, while 29 normal fallopian tube samples were set as the controls. Immunohistochemistry was used to determine the distribution patterns and expressions of β3 integrin and fibronectin in the fallopian tube samples. Results β3 integrin was presented in the ciliated cells within the whole length of the normal fallopian tube, but no staining was detected in the tubal epithelium of salpingitis. A similar distribution pattern of fibronectin was revealed between the cases and the controls, with exclusive staining in the tubal mucosal matrix and the basement membranes of the tubal epithelium and mucosal vessels, whereas no staining was detected in the tubal epithelial cells. Fibronectin in the samples of salpingitis showed a remarkably stronger-staining than that of the normal controls (P Conclusion Salpingitis could reduce β3 integrin expression and raise fibronectin expression in the fallopian tube epithelium, concomitantly changed concentrations of β3 integrin and fibronectin seem to have impacts on the onset of salpingitis.

Abstract 608: Oncogenic role of cullin 4 E3 ubiquitin ligase (CRL4-CDT2) in epithelial ovarian cancer

Abstract Objective: Ubiquitination is a key posttranslational modification involved in the tight regulation of biological processes including cell-cycle regulation and DNA replication and repair. Our objective was to investigate the expression and function of CDT2, a substrate recognition factor for the CUL4 E3 ubiquitin ligase family (CRL4-CDT2) in ovarian cancer. Methods: CDT2 expression was evaluated using realtime RT-PCR in 26 serous ovarian cancers 11 normal ovaries, and 14 fallopian tube fimbria specimens and confirmed by Western blotting on a subset of the above. Immunohistochemistry was performed using tissue microarrays consisting of over 130 independent ovarian cancer samples of various histologies, as well as, normal fallopian tube samples using antibodies to CDT2 and p21. Each samples was represented at least in triplicate and scored semi-quantitatively. CDT2 knockdown was accomplished using siRNA and overexpression via retroviral transduction. Cell growth was evaluated using MTT assays. Statistical analysis were performed using either parametric (t test) as well as non-parametric (Mann-Whitney and Kruskal Wallis) methods as indicated. Comparison of p21 and CDT2 expression was performed using Spearman's correlation. Results: CDT2 mRNA expression was approximately 9 fold higher in serous ovarian cancers compared to normal ovary and fallopian tube fimbria samples (P&amp;lt;0.00001). Western blotting confirmed this differential expression at the protein level. Immunohistochemistry validated CDT2 overexpression in ovarian cancers and did not reveal any differences between serous and other histological subtypes. Immunohistochemical staining did not reveal any significant correlation between the expression of CDT2 and p21, a previously identified target of CRL4-CDT2. Knockdown of CDT2 expression resulted in 31% reduction in cell viability in high CDT2 expressing ES2 cells (P&amp;lt;0.0001) but had minimal effect in low CDT2 expressing SKOV3 cells. However, overexpression of CDT2 in SKOV3 cells did not influence growth of these cells. Knockdown of other component of CRL4-CDT2 ubiquitin ligase (Cul4A and DDB1) also reduced growth of ES2 cells. Finally CDT2 protein levels significantly increased upon in vitro transformation of fallopian tube epithelial cells (described elsewhere) and in subsequent mouse xenografts established from these cells. Conclusions: The results of this ongoing investigation are the first to suggest an oncogenic role for the CRL4-CDT2 ubiquitin ligase in ovarian cancer. The mechanism of action remains unknown but does not appear to involve p21 degradation. 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 608. doi:10.1158/1538-7445.AM2011-608

MicroRNA Profiling of BRCA1/2 Mutation-Carrying and Non-Mutation-Carrying High-Grade Serous Carcinomas of Ovary

BackgroundMicroRNAs (miRNA) are 20∼25 nucleotide non-coding RNAs that inhibit the translation of targeted mRNA, and they have been implicated in the development of human malignancies. High grade serous ovarian carcinomas, the most common and lethal subtype of ovarian cancer, can occur sporadically or in the setting of BRCA1/2 syndromes. Little is known regarding the miRNA expression profiles of high grade serous carcinoma in relation to BRCA1/2 status, and compared to normal tubal epithelium, the putative tissue of origin for high grade serous carcinomas.Methodology/Principal FindingsGlobal miRNA expression profiling was performed on a series of 33 high grade serous carcinomas, characterized with respect to BRCA1/2 status (mutation, epigenetic silencing with loss of expression or normal), and with clinical follow-up, together with 2 low grade serous carcinomas, 2 serous borderline tumors, and 3 normal fallopian tube samples, using miRNA microarrays (328 human miRNA). Unsupervised hierarchical clustering based on miRNA expression profiles showed no clear separation between the groups of carcinomas with different BRCA1/2 status. There were relatively few miRNAs that were differentially expressed between the genotypic subgroups. Comparison of 33 high grade serous carcinomas to 3 normal fallopian tube samples identified several dysregulated miRNAs (false discovery rate <5%), including miR-422b and miR-34c. Quantitative RT-PCR analysis performed on selected miRNAs confirmed the pattern of differential expression shown by microarray analysis. Prognostically, lower level miR-422b and miR-34c in high grade serous carcinomas were both associated with decreased disease-specific survival by Kaplan-Meier analysis (p<0.05).Conclusions/SignificanceHigh grade serous ovarian carcinomas with and without BRCA1/2 abnormalities demonstrate very similar miRNA expression profiles. High grade serous carcinomas as a group exhibit significant miRNA dysregulation in comparison to tubal epithelium and the levels of miR-34c and miR-422b appear to be prognostically important.