Screening and Identification of Biomarkers in Ascites Related to Intrinsic Chemoresistance of Serous Epithelial Ovarian Cancers

Objective

The ability to predict responses to chemotherapy for serous epithelial ovarian cancer (EOC) would be valuable since intrinsically chemoresistant EOC patients (persistent or recurrent disease within 6 months) gain little benefit from standard chemotherapy. The aim of this study was to screen and identify distinctive biomarkers in ascites of serous EOC associated with intrinsic chemoresistance.

Methods

Protein samples from ascites of 12 chemosensitive and 7 intrinsically chemoresistant serous EOC patients were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Furthermore, the identified proteins were validated by ELISA in ascites samples from 19 chemosensitive and 9 intrinsically chemoresistant EOC patients.

Results

The number of spots detected in all 2-D DIGE gels ranged from 1523–1711 using DeCyder software analysis. Thirty-four spots were differentially expressed based on the criteria of an average ratio of more than 1.5 and a student t-test P value <0.05. After MALDI-TOF/TOF MS analysis, 11 differentially expressed proteins, including 3 up-regulated and 8 down-regulated proteins, in ascites of chemoresistant tumors were successfully identified. Of the four selected proteins (ceruloplasmin, apoliprotein A-IV, transthyretin and haptoglobin) in ascites tested by ELISA, only ceruloplasmin was present at significantly different levels between the chemoresistant and chemosensitive ascites samples with average concentrations of 192.2 µg/ml and 157.5 µg/ml, respectively (P = 0.001).

Conclusion

The significantly up-regulated level of ceruloplasmin in the ascites fluid of intrinsic chemoresistant serous EOC patients suggests its potential as a prognostic biomarker for responses to chemotherapy. This finding prompts further investigation with a larger study in order to validate the clinical utility of ceruloplasmin.     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Deciphering the ovarian cancer ascites fluid peptidome

Background

Conventional proteomic approaches have thus far been unable to identify novel serum biomarkers for ovarian cancer that are more sensitive and specific than the current clinically used marker, CA-125. Because endogenous peptides are smaller and may enter the circulation more easily than proteins, a focus on the low-molecular-weight region may reveal novel biomarkers with enhanced sensitivity and specificity. In this study, we deciphered the peptidome of ascites fluid from 3 ovarian cancer patients and 3 benign individuals (ascites fluid from patients with liver cirrhosis).

Results

Following ultrafiltration of the ascites fluids to remove larger proteins, each filtrate was subjected to solid phase extraction and fractionated using strong cation exchange chromatography. The resultant fractions were analyzed using an Orbitrap mass spectrometer. We identified over 2000 unique endogenous peptides derived from 259 proteins. We then catalogued over 777 peptides that were found only in ovarian cancer ascites. Our list of peptides found in ovarian cancer specimens includes fragments derived from the proteins vitronectin, transketolase and haptoglobin.

Conclusions

Peptidomics may uncover previously undiscovered disease-specific endogenous peptides that warrant further investigation as biomarkers for ovarian cancer.     

Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer

Background

Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens.

Methodology and Principal Findings

We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-γ ELISPOT and MHC class I pentamer staining.

Conclusion and Significance

We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.     

Serum Protein Profile at Remission Can Accurately Assess Therapeutic Outcomes and Survival for Serous Ovarian Cancer

Background

Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer.

Experimental Design

Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis.

Results

Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69–0.86) and more than 10 three-marker combinations have excellent AUC values (0.91–0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10⁻³).

Conclusion

We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.     

Subtype specific elevated expression of hyaluronidase-1 (HYAL-1) in epithelial ovarian cancer

Background

Epithelial 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 Findings

We 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/Significance

This 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.     

Microarray-based oncogenic pathway profiling in advanced serous papillary ovarian carcinoma

Introduction

The identification of specific targets for treatment of ovarian cancer patients remains a challenge. The objective of this study is the analysis of oncogenic pathways in ovarian cancer and their relation with clinical outcome.

Methodology

A meta-analysis of 6 gene expression datasets was done for oncogenic pathway activation scores: AKT, β-Catenin, BRCA, E2F1, EGFR, ER, HER2, INFα, INFγ, MYC, p53, p63, PI3K, PR, RAS, SRC, STAT3, TNFα, and TGFβ and VEGF-A. Advanced serous papillary tumours from uniformly treated patients were selected (N = 464) to find differences independent from stage-, histology- and treatment biases. Survival and correlations with documented prognostic signatures (wound healing response signature WHR/genomic grade index GGI/invasiveness gene signature IGS) were analysed.

Results

The GGI, WHR, IGS score were unexpectedly increased in chemosensitive versus chemoresistant patients. PR and RAS activation score were associated with survival outcome (p = 0.002;p = 0.004). Increased activations of β-Catenin (p = 0.0009), E2F1 (p = 0.005), PI3K (p = 0.003) and p63 (p = 0.05) were associated with more favourable clinical outcome and were consistently correlated with three prognostic gene signatures.

Conclusions

Oncogenic pathway profiling of advanced serous ovarian tumours revealed that increased β-Catenin, E2F1, p63, PI3K, PR and RAS –pathway activation scores were significantly associated with favourable clinical outcome. WHR, GGI and IGS scores were unexpectedly increased in chemosensitive tumours. Earlier studies have shown that WHR, GGI and IGS are strongly associated with proliferation and that high-proliferative ovarian tumours are more chemosensitive. These findings may indicate opposite confounding of prognostic versus predictive factors when studying biomarkers in epithelial ovarian cancer.     

Measurement of Phospholipids May Improve Diagnostic Accuracy in Ovarian Cancer

Background

More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant.

Methodology/Principal Findings

We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates.

Results

The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%.

Conclusions/Significance

Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical presentation, to distinguish between women with ovarian cancer and those with benign conditions with shared symptoms and features.     

Integrative Proteomic Analysis of Serum and Peritoneal Fluids Helps Identify Proteins that Are Up-Regulated in Serum of Women with Ovarian Cancer

Background

We used intensive modern proteomics approaches to identify predictive proteins in ovary cancer. We identify up-regulated proteins in both serum and peritoneal fluid. To evaluate the overall performance of the approach we track the behavior of 20 validated markers across these experiments.

Methodology

Mass spectrometry based quantitative proteomics following extensive protein fractionation was used to compare serum of women with serous ovarian cancer to healthy women and women with benign ovarian tumors. Quantitation was achieved by isotopically labeling cysteine amino acids. Label-free mass spectrometry was used to compare peritoneal fluid taken from women with serous ovarian cancer and those with benign tumors. All data were integrated and annotated based on whether the proteins have been previously validated using antibody-based assays.

Findings

We selected 54 quantified serum proteins and 358 peritoneal fluid proteins whose case-control differences exceeded a predefined threshold. Seventeen proteins were quantified in both materials and 14 are extracellular. Of 19 validated markers that were identified all were found in cancer peritoneal fluid and a subset of 7 were quantified in serum, with one of these proteins, IGFBP1, newly validated here.

Conclusion

Proteome profiling applied to symptomatic ovarian cancer cases identifies a large number of up-regulated serum proteins, many of which are or have been confirmed by immunoassays. The number of currently known validated markers is highest in peritoneal fluid, but they make up a higher percentage of the proteins observed in both serum and peritoneal fluid, suggesting that the 10 additional markers in this group may be high quality candidates.     

Potential tumor biomarkers identified in ovarian cyst fluid by quantitative proteomic analysis,iTRAQ

Background

Epithelial-derived ovarian adenocarcinoma (EOC) is the most deadly gynecologic tumor, and the principle cause of the poor survival rate is diagnosis at a late stage. Screening and diagnostic biomarkers with acceptable specificity and sensitivity are lacking. Ovarian cyst fluid should harbor early ovarian cancer biomarkers because of its closeness to the tumor. We investigated ovarian cyst fluid as a source for discovering biomarkers for use in the diagnosis of EOC.

Results

Using quantitative mass spectrometry, iTRAQ MS, we identified 837 proteins in cyst fluid from benign, EOC stage I, and EOC stage III. Only patients of serous histology were included in the study. Comparing the benign (n = 5) with the malignant (n = 10) group, 87 of the proteins were significantly (p < 0.05) differentially expressed. Two proteins, serum amyloid A-4 (SAA4) and astacin-like metalloendopeptidase (ASTL), were selected for verification of the iTRAQ method and external validation with immunoblot in a larger cohort with mixed histology, in plasma (n = 68), and cyst fluid (n = 68). The protein selections were based on either high significance and high fold change or abundant appearance and several peptide recognitions in the sample sets (p = 0.04, FC = 1.95) and (p < 0.001, FC = 8.48) for SAA4 and ASTL respectively. Both were found to be significantly expressed (p < 0.05), but the methods did not correlate concerning ASTL.

Conclusions

Fluid from ovarian cysts connected directly to the primary tumor harbor many possible new tumor-specific biomarkers. We have identified 87 differentially expressed proteins and validated two candidates to verify the iTRAQ method. However several of the proteins are of interest for validation in a larger setting.     

Systematic Analysis of Immune Infiltrates in High-Grade Serous Ovarian Cancer Reveals CD20, FoxP3 and TIA-1 as Positive Prognostic Factors

Background

Tumor-infiltrating T cells are associated with survival in epithelial ovarian cancer (EOC), but their functional status is poorly understood, especially relative to the different risk categories and histological subtypes of EOC.

Methodology/Principal Findings

Tissue microarrays containing high-grade serous, endometrioid, mucinous and clear cell tumors were analyzed immunohistochemically for the presence of lymphocytes, dendritic cells, neutrophils, macrophages, MHC class I and II, and various markers of activation and inflammation. In high-grade serous tumors from optimally debulked patients, positive associations were seen between intraepithelial cells expressing CD3, CD4, CD8, CD45RO, CD25, TIA-1, Granzyme B, FoxP3, CD20, and CD68, as well as expression of MHC class I and II by tumor cells. Disease-specific survival was positively associated with the markers CD8, CD3, FoxP3, TIA-1, CD20, MHC class I and class II. In other histological subtypes, immune infiltrates were less prevalent, and the only markers associated with survival were MHC class II (positive association in endometrioid cases) and myeloperoxidase (negative association in clear cell cases).

Conclusions/Significance

Host immune responses to EOC vary widely according to histological subtype and the extent of residual disease. TIA-1, FoxP3 and CD20 emerge as new positive prognostic factors in high-grade serous EOC from optimally debulked patients.     

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors

Background

Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.

Methods

In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSⁿ). The LC-ESI-MSⁿ of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.

Results

The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC).

Conclusion

Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles.     

Identification of the chemokine CX3CL1 as a new regulator of malignant cell proliferation in epithelial ovarian cancer

Background

Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX₃CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX₃CL1 in EOC.

Results

Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX₃CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX₃CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX₃CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX₃CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX₃CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX₃CL1 products. Conversely, CX₃CL1 promoted through its binding to CX₃CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX₃CL1 and faster tumor growth.

Conclusion

Our findings highlight the previously unappreciated constitutive expression of CX₃CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.     

Oncologic Trogocytosis of an Original Stromal Cells Induces Chemoresistance of Ovarian Tumours

Background

The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.

Methodology/Principal Findings

We isolated an original type of stromal cells, referred to as “Hospicells” from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.

Conclusions/Significance

This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient''s tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.     

Integrated Analysis of Gene Expression Profiles Associated with Response of Platinum/Paclitaxel-Based Treatment in Epithelial Ovarian Cancer

Purpose

This study aims to explore gene expression signatures and serum biomarkers to predict intrinsic chemoresistance in epithelial ovarian cancer (EOC).

Patients and Methods

Gene expression profiling data of 322 high-grade EOC cases between 2009 and 2010 in The Cancer Genome Atlas project (TCGA) were used to develop and validate gene expression signatures that could discriminate different responses to first-line platinum/paclitaxel-based treatments. A gene regulation network was then built to further identify hub genes responsible for differential gene expression between the complete response (CR) group and the progressive disease (PD) group. Further, to find more robust serum biomarkers for clinical application, we integrated our gene signatures and gene signatures reported previously to identify secretory protein-encoding genes by searching the DAVID database. In the end, gene-drug interaction network was constructed by searching Comparative Toxicogenomics Database (CTD) and literature.

Results

A 349-gene predictive model and an 18-gene model independent of key clinical features with high accuracy were developed for prediction of chemoresistance in EOC. Among them, ten important hub genes and six critical signaling pathways were identified to have important implications in chemotherapeutic response. Further, ten potential serum biomarkers were identified for predicting chemoresistance in EOC. Finally, we suggested some drugs for individualized treatment.

Conclusion

We have developed the predictive models and serum biomarkers for platinum/paclitaxel response and established the new approach to discover potential serum biomarkers from gene expression profiles. The potential drugs that target hub genes are also suggested.     

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Background

Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/Principal Findings

A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/Significance

Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.     

Circulating antinuclear antibodies in patients with pelvic masses are associated with malignancy and decreased survival

Background

Circulating autoantibodies occur more frequently in cancer patients than in patients without cancer.

Methods and Findings

We examined sera from patients referred for pelvic mass symptoms to a tertiary university clinic. A total of 127 were diagnosed with epithelial ovarian cancer while 386 had a benign condition. A screen for IgG anti-nuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells confirmed a highly significant overrepresentation of ANA in the cancer group where 40% had detectable (i.e., a titer ≥160) ANA compared with less than 12% in the benign group. The overrepresentation of ANA in the cancer group persisted (p<0.0001) after matching the age-profile of the benign group with the ovarian cancer group. Only 19 out of 127 patients in the age-matched benign subgroup were positive for ANA corresponding to an 85% specificity at 40% sensitivity of ANA as the only marker for malignancy. No correlation of ANA positivity in either group with specific bands in immunoblots could be demonstrated even though immunoblot positivity was clearly increased in the malignant group (41% vs. 3%). The presence, strength, and type of ANA did not correlate with serum CA-125 values or with staging, and ANA outcome did not contribute with independent diagnostic information. However, survival was significantly shorter in ANA-positive compared with ANA-negative cancer patients and patients with CA-125 below the median CA-125 value in the cancer group had a significantly decreased survival when positive for ANA. ANA status made no difference in the group with CA-125 values above the median. Also, there was a significant correlation between speckled ANA-strength and histological tumor grade.

Conclusions

Circulating antibodies are a promising source for new biomarkers in cancer. Characterization of epitope specificities and measurements of consecutive samples will be important for further elucidating the role of ANA in evaluating ovarian cancer patients.