Exploring the potential of mucin 13 (MUC13) as a biomarker for carcinomas and other diseases.

BACKGROUND: Mucin 13 (MUC13) is a cell surface glycoprotein aberrantly expressed in a variety of epithelial carcinomas. Thus far, the role of MUC13 in various diseases remains elusive. To the best of our knowledge, this is the first study to examine the potential of MUC13 as a serum biomarker in a variety of carcinomas and other conditions. METHODS: We developed a recombinant MUC13 protein, mouse monoclonal antibodies and enzyme immunoassay (ELISA) for MUC13. We used this assay to measure MUC13 levels in the supernatants of cancer cell lines and a large cohort of serum samples from healthy and diseased individuals. RESULTS: MUC13 is secreted from cancer cell lines, with highest levels found in ovarian cancer cell lines. MUC13 levels in human sera were significantly increased in patients with renal failure and 20%-30% of patients with ovarian, liver, lung and other cancers. MUC13 was also elevated in 70% of patients with active cutaneous melanoma, but not uveal melanoma. Furthermore, we identified significant MUC13 elevations in the serum of patients with vasculitis (ANCA-positive) autoantibodies, but not in those with inflammatory bowel disease. CONCLUSIONS: Serum MUC13 is frequently elevated not only in a variety of malignant cases but also in some benign pathologies, thus appearing to be a non-specific disease biomarker. Nonetheless, serum MUC13 is clearly highly elevated in some carcinoma patients, and its relationship with tumor progression in this context warrant further research. Future studies that examine the correlation between serum MUC13 levels to stage of cancer could elucidate prognostic potential.

Mucins and Cytokeratins as Serum Tumor Markers in Breast Cancer.

Structural and functional characteristics of mucins and cytokeratins are shortly described. Thereafter, those commonly used in breast cancer as serum tumor markers are considered. First CA15.3, MCA, CA549, CA27.29 mucins and CYFRA21.1, TPA, TPS cytokeratins alone or in association have been examined in different stages and conditions. Then their usefulness in monitoring disease-free breast cancer patients is evaluated. The central role of the established cut-off and critical change, the "early" treatment of recurrent disease and the potential benefit in survival are other issues that have been highlighted and discussed. The successive sections and subsections deal with the monitoring of advanced disease. In them, the current recommendations and the principal findings on using the above mentioned mucins and cytokeratins have been reported. A computer program for interpreting consecutive measurements of serum tumor markers also has been illustrated. The final part of the chapter is devoted to mucins and cytokeratins as markers of circulating and disseminated tumor cells and their usefulness for prognosis.

Serum tumor markers in bile duct cancer--a review.

CONTEXT: Bile duct cancer (BDC) is a disease with a very grave prognosis, often diagnosed too late. OBJECTIVE: The aim of this review is to evaluate available literature on tumor markers in serum from patients with BDC. METHODS: Using the search words "serum markers", "bile duct cancer", "cholangiocarcinoma", "biomarker" and "tumor marker", a search was carried out. RESULTS: Seventy-five studies were included in the review. CONCLUSION: CA19-9 is by far the most studied and most promising diagnostic and/or prognostic marker in BDC. But also the different mucins are interesting as new markers of BDC in serum.

Challenging the limits of detection of sialylated Thomsen-Friedenreich antigens by in-gel deglycosylation and nano-LC-MALDI-TOF-MS.

The identification of sialylated Thomsen-Friedenreich antigens in proteins poses much interest in the context of cancer research. MALDI-TOF-MS is a powerful technique for this purpose; still it shows considerable low sensitivity for sialylated molecules due to in-source and metastable decomposition. Herein, we report a target-driven strategy to identify these antigens in minute amounts of glycoproteins isolated in polyacrylamide gels. The glycans were recovered from gel spots by reductive ß-elimination, permethylated and analyzed by nano-LC-MALDI-TOF-MS. A computational algorithm was developed to filter spectral noise and enhance/isolate the signals of interest. Sialylated antigens were identified in minute amounts of fetuin (0.1 µg) and plasminogen (1.0 µg) by this approach.MS assignments were further validated by enzymatic methods. This methodology allowed a fivefold decrease in the current LOD of fetuin sialylated O-glycans by MALDI-TOF-MS.

An improved lectin-based method for the detection of mucin-type O-glycans in biological samples.

Mucins and mucin-type glycoproteins, collectively referred to as mucin-type O-glycans, are implicated in many important biological functions and pathological conditions, including malignancy. Presently, there is no reliable method to measure the total mucin-type O-glycans of a sample, which may contain one or more of these macromolecules of unknown structures. We report the development of an improved microassay that is based on the binding of lectins to the unique and constant GalNAc-Ser/Thr structural feature of mucin-type O-glycans. Since the sugar-amino acid linkage in the mucin-type O-glycans is invariably cryptic, we first chemically removed the heterogeneous peripheral and core saccharides of model glycoconjugates before examining for their interactions using an enzyme-linked lectin assay (ELLA). Desialylation of the model glycoconjugates led to maximal binding of the lectins but additional treatments such as Smith degradation did not result in increased binding. Of the lectins tested for their ability to probe the desialylated O-glycans, jacalin showed the highest sensitivity followed by champedak galactose binding (CGB) lectin and Vicia villosa agglutinin. Further improvement in the sensitivity of ELLA was achieved by using microtiter plates that were pre-coated with the CGB lectin, which increased the specificity of the assay to mucin-type O-glycans. Finally, the applicability of the developed sandwich ELLA to crude samples was demonstrated by estimating trace quantities of the mucin-type O-glycans in the human serum.

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II.

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galß1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.

[The diagnostic value of serum alveomucin test in diseases with lungs affection].

The study was organized to determine the diagnostic value of concentration of blood serum alveo-mucin as a marker of lesion of lung tissue in patients with community-acquired pneumonia and hemorrhagic fever with renal syndrome. The sampling included 86 patients with community-acquired pneumonia and 97 patients with hemorrhagic fever with renal syndrome. The patients with community-acquired pneumonia were divided in three groups according severity of disease course: slight form, medium-severe form and severe form of disease. The group of patients with medium-severe form of hemorrhagic fever with renal syndrome was divided on two groups depending on clinical roentgenologic pattern: with and without pathology of lungs. The high concentration of alveomucin at the day of hospitalization is typical for patients both with community-acquired pneumonia and with roentgenologically approved lungs affection in case of hemorrhagic fever with renal syndrome in comparison with patients with hemorrhagic fever with renal syndrome without affection of lung tissue (p=0.046). The concentration of alveomucin higher than 47.2 U/ml is established in 86% of patients with community-acquired pneumonia and affection of lung in case of hemorrhagic fever with renal syndrome and in 12% of patients with hemorrhagic fever with renal syndrome without affection of lungs (chi2=0.58 with Yeats equalization, p=0.44).

[The diagnostic significance of alveomucin in patients with hemorrhagic fever with renal syndrome].

The article considers the results of analysis of serum alveomucin levels in 97 patients with hemorrhagic fever with renal syndrome depending on the form and period of disease. It is demonstrated that in patients with hemorrhagic fever with renal syndrome and clinical radiologic symptoms of lung affection already in initial period of disease the level of serum alveomucin is reliably higher than in the control group and the group of patients with hemorrhagic fever with renal syndrome and without lung affection. The correlation analysis revealed the reverse dependence between the level of alveomucin and arterial blood saturation with oxygen in patients with hemorrhagic fever with renal syndrome and disease form of medium severity.

Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases.

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

Seromic profiling of colorectal cancer patients with novel glycopeptide microarray.

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures.

Carbohydrate markers of pancreatic cancer.

Pancreatic cancer is the fourth most common cause of death from cancer in the world and the sixth in Europe. Pancreatic cancer is more frequent in males than females. Worldwide, following diagnosis of pancreatic cancer, <2% of patients survive for 5 years, 8% survive for 2 years and <50% survive for only approx. 3 months. The biggest risk factor in pancreatic cancer is age, with a peak of morbidity at 65 years. Difficulty in the diagnosis of pancreatic cancer causes a delay in its detection. It is one of the most difficult cancers to diagnose and therefore to treat successfully. Additional detection of carbohydrate markers may offer a better diagnosis of pancreatic cancer. Carbohydrate markers of cancer may be produced by the cancer itself or by the body in response to cancer, whose presence in body fluids suggests the presence and growth of the cancer. The most widely used, and best-recognized, carbohydrate marker of pancreatic cancer is CA 19-9 [CA (carbohydrate antigen) 19-9]. However, the relatively non-specific nature of CA 19-9 limits its routine use in the early diagnosis of pancreatic cancer, but it may be useful in monitoring treatment of pancreatic cancer (e.g. the effectiveness of chemotherapy), as a complement to other diagnostic methods. Some other carbohydrate markers of pancreatic cancer may be considered, such as CEA (carcinoembryonic antigen), CA 50 and CA 242, and the mucins MUC1, MUC2 and MUC5AC, but enzymes involved in the processing of glycoconjugates could also be involved. Our preliminary research shows that the activity of lysosomal exoglycosidases, including HEX (N-acetyl-ß-D-hexosaminidase), GAL (ß-D-galactosidase), FUC (α-L-fucosidase) and MAN (α-D-mannosidase), in serum and urine may be used in the diagnosis of pancreatic cancer.

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9.

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.

[Serum biomarkers in idiopathic pulmonary fibrosis]. / Biomarkery surowicze w samoistnym wlóknieniu pluc.

Idiopathic pulmonary fibrosis (IPF) is the most common of interstitial lung diseases and is characterised by a significant mortality rate. That is way both clinicists and patients are interested to identify factors that may influence outcome of disease. This factors are named biological markers or biomarkers. Their usefulness in diagnostic, monitoring and prognosis of interstitial pneumonia, and idiopathic pulmonary fibrosis was estimated in many researches. The most of them was concerned to serum biomarkers, such as surfactant proteins, mucin-connected proteins, Clara cells proteins, cytoceratines and cytokines.

[Clinical and diagnostic value of the determination of alveomucin in patients with community-acquired pneumonia].

The study was undertaken to define the clinical and diagnostic value of the determination of the serum levels of alveomucin in patients with community-acquired pneumonia and the correlation of this index with the etiological factors of the disease and its severity. The investigators used general clinical, laboratory, and instrumental studies and specific serodiagnostic assays of the pathogen and measured alveomucin levels on the principle of a double-site enzyme immunoassay. Examining the serum level of alveomucin in the examinees indicated that on admission day this index was 34% higher in the patients with community-acquired pneumonia than in the healthy individuals (52.59 +/- 2.8 and 39.22 +/- 2.8 U/ml, respectively). The mean value of this index was reduced to 49.88 +/- 2.29 U/ml) in the course of the disease. A certain association was found between the study index and the clinical characteristics of the disease: a direct correlation was established between the extent of lung injury and the level of mucin antigen. An assay of serum alveomucin depending on the severity of community-associated pneumonia revealed that on the first days of admission its values did not differ significantly from that in patients with different forms of the disease. However, there were statistically significant differences in this index according to the severity of the disease in its course. Analysis of serum alveomucin values in relation to the etiology of the disease also revealed certain regularities.

Squaraine dyes as fluorescent turn-on sensors for the detection of porcine gastric mucin: A spectroscopic and kinetic study.

Mucin-type glycoproteins are the principal components of mucus which cover all the mucosal surfaces of the human body. The mucus and mucins are essential mediators of the innate immune system, however in the last decades mucins have been identified even as an important class of cancer biomarkers. Luminogenic materials with fluorescence turn-on behavior are becoming promising materials because of their advantages of label free, relatively inexpensive and simple to use properties for biological detection and imaging. Squaraines are luminogens characterized by high fluorescence in organic media but poor emission in aqueous environments due to their tendency to self-aggregate. Herein we investigate the interaction between porcine gastric mucin (PGM) and several squaraines in aqueous media. While squaraine dyes showed low fluorescence intensity and quantum yield in water, as a result of the formation of aggregates, an enhancement of fluorescence up to 45-fold was achieved when PGM was added. PGM was detected in a linear range of 10-300 µg/mL with a limit of detection of 800 ng/mL. The assay was used to quantify mucin in diluted human serum samples and recoveries of 94.9-116.2% were achieved. To the best of our knowledge, this is the easiest and convenient method for mucin detection in the reported literature.

MUC4 and MUC5AC are highly specific tumour-associated mucins in biliary tract cancer.

Alterations in epithelial mucin expression are associated with carcinogenesis, but there are few data in biliary tract cancer (BTC). In pancreatic malignancy, MUC4 is a diagnostic and prognostic tumour marker, whereas MUC5AC has been proposed as a sensitive serological marker for BTC. We assessed MUC4 and MUC5AC expression in (i) prospectively collected bile and serum specimens from 72 patients with biliary obstruction (39 BTC) by real-time reverse transcriptase-PCR (qPCR) and western blot analysis, and (ii) 79 archived biliary tissues (69 BTC) by immunohistochemistry. In bile, MUC4 protein was detected in 27% of BTC and 29% of primary sclerosing cholangitis (PSC) cases, but not in other benign and malignant biliary diseases (P<0.01 and P=0.06). qPCR revealed a 1.9-fold increased MUC4 mRNA expression in BTC patients' bile compared with benign disease. In archived tissues, MUC4 protein was detected in 37% of BTC but in none of the benign samples (P=0.03). In serum, MUC5AC was found exclusively in BTC and PSC sera (44% and 13%, respectively; P<0.001 for BTC vs non-BTC) and correlated negatively with BTC survival. Biliary MUC4 and serum MUC5AC are highly specific tumour-associated mucins that may be useful in the diagnosis and formulation of therapeutic strategies in BTC.

Multiple hepatic receptors cooperate to eliminate secretory mucins aberrantly entering the bloodstream: are circulating cancer mucins the "tip of the iceberg"?

Hollow organs lined by columnar epithelial cells normally secrete mucins and their proteolytic fragments vectorially into the lumen. These heterogeneously O-glycosylated molecules are known to aberrantly enter the bloodstream in the setting of epithelial carcinomas and possibly during injury or inflammation. We have recently shown that carcinoma mucin fragments can trigger the rapid formation of platelet-rich microthrombi in vivo. Thus, mechanisms to clear such aberrantly secreted mucins must exist. Indeed, we found that i.v. injected carcinoma mucin fragments had an approximately 1 minute half-life in mice, which was primarily due to rapid clearance by hepatic reticuloendothelial cells. Inhibition of known glycan-recognizing hepatic clearance receptors showed involvement of multiple partially overlapping clearance systems. Studies of genetically deficient mice and incomplete competition between different mucins confirmed this result. Thus, multiple hepatic clearance receptors cooperate to eliminate secretory mucins entering the circulation, limiting potential pathology. This may also explain why mucin-type clustered O-glycosylation is rare on plasma proteins. Notably, small subsets of injected carcinoma mucins remained unrecognized by clearance systems, had a much longer half-life, and carried highly sialylated O-glycans. Similar circulating mucins were found in tumor-bearing mice despite lack of saturation of hepatic clearance mechanisms. Thus, circulating cancer mucins currently used as clinical diagnostic markers likely represent only the clearance-resistant "tip of the iceberg." Such aberrantly circulating mucins could play pathologic roles not only in cancer but also during injury or inflammation of hollow organs and in liver disease.

Microparticle-associated tissue factor activity: a link between cancer and thrombosis?

BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

A new mucin antibody/enzyme-linked lectin-sandwich assay of serum MUC5AC mucin for the diagnosis of cholangiocarcinoma.

We have previously used agarose gel electrophoresis and immunoblotting to qualitatively measure serum MUC5AC mucin for diagnosing cholangiocarcinoma. In this study, we developed a quantitative determination of serum MUC5AC by sandwich ELISA using MUC5AC mucin monoclonal antibody and soybean agglutinin. A cut-off value of the absorbance 0.074 was obtained from a complete statistical Receiver Operating Characteristic curves with an area under the curve=0.8141. The assay could discriminate cholangiocarcinoma patients from the controls with 71% sensitivity and 90% specificity. The test is simple to perform, reproducible, and probably used for detecting cholangiocarcinoma in a high-risk group or suspected patients.

Overproduction of PGE2 in peripheral blood monocytes of gastrointestinal cancer patients with mucins in their bloodstream.

When monocytes from healthy donors were cultured in the presence of sera from patients with gastrointestinal cancer, PGE2 production from the monocytes was elevated. Serum proteins were fractionated on Sepharose 4B and the inducing activity was found in the excluded fractions. By excluding some mucins from the serum, the inducing activity was reduced effectively. The activity was also reduced by adding binding inhibitors to the scavenger receptor. These results suggest that peripheral blood monocytes in epithelial cancer patients may be continuously stimulated by mucins in the bloodstream through the scavenger receptor, resulting in overproduction of PGE2.