Sialic Acid Linkage Analysis Refines the Diagnosis of Ovarian Cancer.

Epithelial ovarian cancer (EOC) is a rather rare but lethal disease that is usually diagnosed at an advanced stage; this is due to a lack of early diagnostic markers. At the time being, less than a quarter of patients are diagnosed when the tumor has not metastasized yet. In previous work, we demonstrated that antennarity, fucosylation, and sialylation increased in EOC patients and built a glycan-based score that was able to diagnose EOC better than CA125, the routine diagnostic marker, does. To date, little attention had been paid to the sialic acid linkages of N-glycans in the context of blood biomarker research. In this work, the sialic acid linkages of the serum glycome of ovarian cancer patients were investigated for the first time by MALDI-TOF-MS. To this end, we released N-glycans, derivatized sialic acids solely in a linkage-specific way and measured glycome profiles by MALDI-TOF mass spectrometry. A statistically significant decrease was observed between late stage patients and controls or early stage patients for high-mannose, hybrid-type, complex-type asialylated, bi, tri- and tetraantennary sialylated structures. A significant decrease of monosialylated monoantennary N-glycan structures was observed in early and late stage EOC when compared to healthy controls. Statistically significant increases were observed in early and late stage patients compared to controls for tri, tetraantennary fucosylated structures, afucosylated, and fucosylated triantennary structures taken as α-2,3-linked/α-2,6-linked sialic acid ratio. Moreover, all afucosylated and fucosylated structures taken as α-2,3-linked/α-2,6-linked sialic acid ratio and the α-2,3-linked/α-2,6-linked sialic acid ratio of all sialylated structures were increased significantly for early and late stage EOC patients when compared to healthy controls. Finally, ROC curves were built for the most significant glycan combinations and we were able to show that the serum glycome sialic acid ratio could enhance ovarian cancer diagnosis as sialic acid linkage modulations arise even in early stage ovarian cancer.

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. In the positive acute-phase proteins, haptoglobin, α1-antitrypsin, and α1-antichymotrypsin, an increase of antennarity and Lewis(X) motif could be measured in EOC patients on tri- and/or tetraantennary N-glycans. Tetraantennary N-glycans containing three Lewis(X) epitopes and triantennary N-glycans containing a ß(1-6) branch and a Lewis(X) epitope were only present in EOC patients. We also showed for the first time that the core-fucosylated biantennary digalactosylated N-glycan of α1-acid glycoprotein is a potential biomarker for EOC. To conclude, core-fucosylated biantennary N-glycans on α1-acid glycoprotein as well as higher antennarity and increased amounts of Lewis(X) motif on haptoglobin, α1-antitrypsin, and α1-antichymotrypsin are promising biomarkers for EOC. Nevertheless, their specificity and selectivity for the early detection of EOC should be evaluated in a larger study.

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

Alterations 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 Lewis(x) 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.

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.

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). IgGN-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.