Elena I. Braicu

Serum Glycome Profiling: A Biomarker for Diagnosis of Ovarian Cancer

During the development of cancer, changes in cellular glycosylation are observed, indicating that alterations of the glycome occur in extracellular fluids as well as in serum and could therefore serve as tumor biomarkers. In the case of epithelial ovarian cancer (EOC), common tumor markers such as CA125 are known to have poor specificity; therefore, better biomarkers are needed. The aim of this work was to identify new potential glycan biomarkers in EOC-patients. N-Glycans were cleaved from serum glycoproteins from 63 preoperative primary EOC-patients along with 33 age-matched healthy women, permethylated, and analyzed using MALDI-TOF mass spectrometry. A value named GLYCOV was calculated from the relative areas of the 11 N-glycan biomarkers revealed by SPSS statistical analyses, namely four high-mannose and seven complex-type fucosylated N-glycans. GLYCOV diagnosed primary EOC with a sensitivity of 97% and a specificity of 98.4% whereas CA-125 showed a sensitivity of 97% and a specificity of 88.9%. Our study is the first one to compare glycan values with the established tumor marker CA125 and to give better results. Therefore, the N-glycome could potentially be used as a biomarker.

The N-terminally truncated p53 isoform Δ40p53 influences prognosis in mucinous ovarian cancer.

Objective The tumor suppressor p53 generates the N-terminally truncated isoforms &Dgr;40p53 and &Dgr;133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer. Methods &Dgr;40p53, &Dgr;133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase–quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, &Dgr;40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status. Results In endometrioid cancer specimens, &Dgr;133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated &Dgr;40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high &Dgr;40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094–0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193–1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53β and &Dgr;40p53&agr; in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters. Conclusions We show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of &Dgr;40p53 in patients with mucinous ovarian cancer.

Prognostic impact of AMP-activated protein kinase expression in ovarian carcinoma: Correlation of protein expression and GC/TOF-MS-based metabolomics

AMP-activated protein kinase (AMPK) plays a central role in regulating energy metabolism in cells. AMPK activation results in down-regulation of anabolic pathways (e.g., fatty acid biosynthesis) and switches on catabolic processes such as glucose uptake, glycolysis or fatty acid oxidation. Recent studies in cell culture models have shown that the growth of tumor cell lines was inhibited by AMPK activation, but the expression of AMPK in human ovarian tumors has not been reported so far. In this study we investigated AMPK expression in a cohort of 70 ovarian carcinomas, 14 borderline tumors and 5 normal ovaries and linked the protein expression data to Gas chromatography/ time of flight mass spectrometry (GC/TOF-MS) based metabolomics. We observed a significantly higher expression in ovarian carcinomas compared to borderline tumors and normal ovaries (p=0.038). Decreased AMPK expression correlated significantly with higher tumor grade (p=0.009) and was of adverse prognosis in patients with advanced tumor stages (p=0.016) as well as in patients with serous ovarian carcinomas (p=0.037). GC/TOF-MS based metabolomics revealed a significantly higher concentration of glucose in AMPK-negative carcinomas (p=0.022) as well as overexpression of other metabolites from carbohydrate metabolism. Our results indicate a role for AMPK in progression of ovarian tumors and point towards a prognostic impact of AMPK expression for patient overall survival. Furthermore, our data suggest a deregulation of the AMPK-dependent energy metabolism in human ovarian carcinomas. In future clinical studies, activation of AMPK in ovarian carcinoma patients with advanced tumor stages might be an interesting therapeutic approach.

An intracellular targeted antibody detects EGFR as an independent prognostic factor in ovarian carcinomas

BackgroundIn ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting. Methods: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated EGFR expression in ten ovarian cancer cell lines and incubated cancer cells with Leptomycin B, a CRM1 specific inhibitor.ResultsWe observed a membranous and cytoplasmic EGFR expression in 36.4% and 64% of ovarian carcinomas, respectively. Membranous EGFR was an independent prognostic factor for poor overall survival in ovarian cancer patients (HR 2.7, CI 1.1-6.4, p = 0.02) which was also found in the serous subtype (HR 4.6, CI 1.6-13.4, p = 0.004). We further observed a significant association of EGFR with COX-2 and nuclear CRM1 expression (chi-square test for trends, p = 0.006 and p = 0.013, respectively). In addition, combined membranous EGFR/COX-2 expression was significantly related to unfavorable overall survival (HR 7.2, CI 2.3-22.1, p = 0.001).In cell culture, we observed a suppression of EGFR protein levels after exposure to Leptomycin B in OVCAR-3 and SKOV-3 cells.ConclusionsOur results suggest that the EGFR/COX-2/CRM1 interaction might be involved in progression of ovarian cancer and patient prognosis. Hence, it is an interesting anti-cancer target for a combination therapy. Further studies will also be needed to investigate whether EGFR is also predictive for benefit from EGFR targeted therapies.

Acute‐phase glycoprotein N‐glycome of ovarian cancer patients analyzed by CE‐LIF

Epithelial ovarian cancer (EOC) is the most frequent cause of death from all gynecological malignancies because of its late diagnosis. As N‐glycosylation is modified in the course of ovarian cancer, it is a promising source of tumor biomarkers. In this work, serum glycoproteins, depleted from albumin and IgG, were separated by 2DE. Protein spots of acute‐phase proteins were identified by peptide mapping and their corresponding glycan moieties were released enzymatically, fluorescently labeled and analyzed by CE‐LIF.

The Serum Glycome to Discriminate between Early-Stage Epithelial Ovarian Cancer and Benign Ovarian Diseases

Epithelial ovarian cancer (EOC) is the sixth most common cause of cancer deaths in women because the diagnosis occurs mostly when the disease is in its late-stage. Current diagnostic methods of EOC show only a moderate sensitivity, especially at an early-stage of the disease; hence, novel biomarkers are needed to improve the diagnosis. We recently reported that serum glycome modifications observed in late-stage EOC patients by MALDI-TOF-MS could be combined as a glycan score named GLYCOV that was calculated from the relative areas of the 11 N-glycan structures that were significantly modulated. Here, we evaluated the ability of GLYCOV to recognize early-stage EOC in a cohort of 73 individuals comprised of 20 early-stage primary serous EOC, 20 benign ovarian diseases (BOD), and 33 age-matched healthy controls. GLYCOV was able to recognize stage I EOC whereas CA125 values were statistically significant only for stage II EOC patients. In addition, GLYCOV was more sensitive and specific compared to CA125 in distinguishing early-stage EOC from BOD patients, which is of high relevance to clinicians as it is difficult for them to diagnose malignancy prior to operation.

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery

AbstractAlterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewisx epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery. Graphical abstractᅟ

Vascular endothelial growth factor C mRNA expression is a prognostic factor in epithelial ovarian cancer as detected by kinetic RT-PCR in formalin-fixed paraffin-embedded tissue.

Vascular endothelial growth factor C (VEGF-C) is a well described chemotactic and growth factor for lymphatic endothelial cells. Its inhibition leads to suppression of lymphatic and distant metastases in mouse models. In ovarian cancer, the relationship between VEGF-C expression and tumor behavior has not yet been determined by a quantitative method in vivo. Therefore, we used a new technique of RNA extraction from formalin-fixed paraffin-embedded tissue samples and determined the expression levels of VEGF-C mRNA in a study group of 97 ovarian cancer patients. Expression levels were correlated with clinicopathological features and patient survival. High VEGF-C expression was associated with worse overall (p = 0.0393) and progression-free (p = 0.0155) patient survival. In the subgroups of serous tumors and high-grade tumors, VEGF-C mRNA was still a negative indicator for patient survival (p = 0.0190 and 0.0311, respectively). A trend was observed among patients with high clinical stage (p = 0.0634). In multivariate survival analysis VEGF-C mRNA retained its prognostic influence on progression-free survival (p = 0.006, HR = 0.319 with a 95% confidence interval of 0.142–0.720). High VEGF-C expression was further associated with an increased residual tumor mass after primary cytoreductive surgery. We found no correlation of VEGF-C expression with tumor grade, FIGO stage, lymph node, or distant metastases. Our study demonstrates that high VEGF-C expression is associated with aggressive tumor behavior in ovarian cancer. mRNA extracted from paraffin-embedded tumor samples is suitable for VEGF-C gene expression studies.

Sialic acid methylation refines capillary electrophoresis laser‐induced fluorescence analyses of immunoglobulin G N‐glycans of ovarian cancer patients

Alterations in IgG N‐glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE‐LIF of 8‐aminopyrene‐1,3,6‐trisulfonic acid labeled N‐glycans is a well‐established rapid method to characterize IgG N‐glycans that needs only low amounts of starting material. However, sialylated N‐glycans have short migration times due to their negative charge. As a result, some of them are not well resolved and co‐migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgG N‐glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co‐migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented, which allowed an accurate quantification of these N‐glycans. Finally, we investigated the IgG N‐glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods. With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.

Endo-β-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: application to biomarker discovery.

Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-β-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55 min instead of 21 h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis-laser-induced fluorescence (CE-LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker.