Extracellular Vesicle Analysis Allows for Identification of Invasive IPMN.

BACKGROUND AND AIMS: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. METHODS: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). RESULTS: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-riskstigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). CONCLUSIONS: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

Evaluation of serum MUC5AC in combination with CA19-9 for the diagnosis of pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) is a highly aggressive tumor with a poor prognosis that lacks specific diagnostic markers. Mucin 5AC (MUC5AC) is a member of the mucin family, a heterogeneous group of high molecular weight, heavily glycosylated proteins that could be either membrane-bound or secreted. This multi-central study is to evaluate the performance of serum MUC5AC in combination with carbohydrate antigen 19-9 (CA19-9) for the diagnosis of PC in Asian. METHODS: Sixty-one patients with PC (comprised of early pancreatic cancer [n = 30] and late pancreatic cancer [n = 31] patients), 29 benign control, 35 choledocholithiasis, 25 chronic pancreatitis, and 34 healthy controls, were recruited from two hospitals. Serum levels of MUC5AC were evaluated by commercial ELISA kits. CA19-9 was measured by chemiluminescence immunoassay. The cutoff value of MUC5AC was determined based on optimal sensitivity and specificity. RESULTS: Serum MUC5AC in patients with PC (210.1 [100.5-423.8] ng/mL) presented higher levels than those in controls. The combined biomarker panel (MUC5AC and CA19-9) presented better performance and improved specificity to differentiate PC from controls (AUC 0.894; 95% CI (0.844-0.943), sensitivity 0.738, specificity 0.886) than CA19-9 (p = 0.043) or MUC5AC alone (p = 0.010); however, the latter two had no difference (p = 0.824). CONCLUSIONS: Serum MUC5AC is a potential biomarker for PC. The combination with CA19-9 presents improved specificity and better performance.

Antagonistic Effects of N-acetylcysteine on Mitogen-activated Protein Kinase Pathway Activation, Oxidative Stress and Inflammatory Responses in Rats with PM2.5 Induced Lung Injuries.

Objective To evaluate the antagonistic effects of N-acetylcysteine (NAC) on mitogen-activated protein kinases (MAPK) pathway activation, oxidative stress and inflammatory responses in rats with lung injury induced by fine particulate matter (PM2.5).Methods Forty eight male Wistar rats were randomly divided into six groups: blank control group (C1), water drip control group (C2), PM2.5 exposed group (P), low-dose NAC treated and PM2.5 exposed group (L), middle-dose NAC treated and PM2.5 exposed group (M), and high-dose NAC treated and PM2.5 exposed group (H). PM2.5 suspension (7.5 mg/kg) was administered tracheally once a week for four times. NAC of 125 mg/kg, 250 mg/kg and 500 mg/kg was delivered intragastrically to L, M and H group respectively by gavage (10 ml/kg) for six days before PM2.5 exposure. The histopathological changes and human mucin 5 subtype AC (MUC5AC) content in lung tissue of rats were evaluated. We investigated IL-6 in serum and bronchoalveolar lavage fluid (BALF) by Enzyme-linked immunosorbent assay (ELISA), MUC5AC in lung tissue homogenate by ELISA, glutathione peroxidase (GSH-PX) in serum and BALF by spectrophotometry, and the expression of p-ERK1/2, p-JNK1/2 and p-p38 proteins by Western blot. All the measurements were analyzed and compared statistically.Results Lung tissue of rats exposed to PM2.5 showed histological destruction and increased mucus secretion of bronchial epithelial cells. Rats receiving NAC treatment showed less histological destruction and mucus secretion. Of P, L, M and H group, MUC5AC in lung tissue, IL-6 in serum and BALF were higher than controls (C1 and C2) (all P<0.05), with the highest levels found in the P group and a decreasing trend with increase of NAC dose. The activity of GSH-PX in serum and BALF of PM2.5 exposed rats (P, L, M and H) was lower than that of controls (all P<0.05), with higher activities found in NAC treated rats (L, M, and H), and an increasing trend with increase of NAC dose. The expressions of p-ERK1/2, p-JNK1/2 and p-p38 proteins in PM2.5 exposed lung tissue (P, L, M and H) was higher than controls (all P<0.05), with decreased levels and dose dependent downregulation found in NAC treated rats.Conclusion NAC can antagonize major MAPK pathway activation, lung oxidative stress and inflammatory injury induced by PM2.5 in rats.

Expression and Serum Levels of Mucin 5AC (MUC5AC) as a Biomarker for Cholangiocarcinoma: a Meta-analysis.

AIM: The potential of biomarkers in detecting early cholangiocarcinoma (CCA) is facilitated by examining CCA-associated proteins from primary studies. One such protein is mucin 5AC (MUC5AC) but inconsistency of reported associations between its expression/serum levels and CCA prompts a meta-analysis to obtain more precise estimates. METHODS: A literature search yielded 17 included articles where multiple data in some raised the number of studies to 22. We calculated pooled odds ratios (OR) and 95% confidence intervals from negative and positive readings of MUC5AC levels. Data were subgrouped by ethnicity, detection method, sample source, and cancer type. RESULTS: Outcome in the overall analysis was non-significant but those in the subgroups were. Thus, significant associations (P < 0.001) indicating high MUC5AC levels were found in three subgroups: (i) Thai (OR 8.32) and (ii) serum (OR 4.52). Heterogeneity of these two outcomes (I2 = 90-93%) was erased with outlier treatment (I2 = 0%) which also modulated the pooled effects (OR 2.48-2.59). (iii) Immunoblot (OR 2.61) had low initial heterogeneity (I2 = 2%). Robustness and significant tests for interaction (Pinteraction = 0.01-0.02) improved MUC5AC associations with CCA in the Thai population. CONCLUSIONS: Our pooled effect findings target the biomarker potential of MUC5AC to the Thai population.

Cerebral ischemia/reperfusion injury induces airway mucus hypersecretion in rats and activates IL­13­associated inflammatory mechanisms.

The majority of patients that suffer a stroke have excessive sputum, which accelerates the development of pulmonary complications. However, it is unclear whether cerebral ischemia and reperfusion (I/R) injury induces mucus hypersecretion, and the potential role of inflammation remains unknown. In the present study, the reversible middle cerebral artery occlusion model was applied in rats to induce cerebral I/R injury. The rats were grouped according to the duration of reperfusion (6, 12, 24, 48 and 72 h). Neurological dysfunction was evaluated by Longa scoring and lung dry­to­wet weight (dw/ww) ratios were determined to reflect the degree of mucus secretion. Inflammatory factor interleukin­13 (IL­13) and tumor necrosis factor­α (TNF­α) levels in serum and bronchoalveolar lavage fluid (BALF) were determined by enzyme­linked immunosorbent assay. Pulmonary levels of mucin 5AC (MUC5AC) and key molecules involved in nuclear factor­κB (NF­κB) signaling were determined by western blotting and immunohistochemistry. Rats with cerebral I/R had impaired neurological function, which was associated with the length of reperfusion time. In addition, the dw/ww lung ratio decreased and the pulmonary expression of MUC5AC increased with the increase in severity of neurological dysfunction, indicating that cerebral I/R may induce mucus hypersecretion in a reperfusion time­dependent manner. IL­13 and TNF­α levels in serum and BALF, as well as the nuclear translocation of NF­κB p65 in pulmonary tissues, significantly increased following cerebral I/R, which suggests that the activation of IL­13 and NF­κB inflammatory pathways may be involved. The present study concluded that cerebral I/R injury may induce airway mucus hypersecretion by activating IL­13 and NF­κB inflammatory pathways.

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing.

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 µL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

Assessment of bile and serum mucin5AC in cholangiocarcinoma: diagnostic performance and biologic significance.

BACKGROUND: Recent studies have showed the efficacy of mucin5AC (MUC5AC) as a diagnostic and prognostic serum biomarker in biliary tract tumors. The aim of the present investigation was to improve the current knowledge on the biologic relevance of MUC5AC in malignant and benign biliary disorders by comparing its diagnostic performance in both bile and serum samples of patients with cholangiocarcinoma (CCA) or benign biliary disorders. METHODS: A quantitative determination of MUC5AC by enzyme-linked immunosorbent assay was performed in bile and serum specimens from 26 patients with extrahepatic CCA and 20 subjects with benign biliary disorders (10 with biliary stones and 10 with cholangitis). Verification analysis was made by immunoblot. RESULTS: MUC5AC of serum and biliary origin contributed to different extent to total levels of MUC5AC in the different groups of patients. In particular, the transition toward a greater degree of injury of bile duct epithelium was accompanied by a greater amount of MUC5AC in serum than in bile. The diagnostic performance of MUC5AC expressed as serum/bile ratio showed excellent diagnostic performance for differentiating CCA from cholangitis (area under the curve [AUC], 0.94; 95% CI, 0.86-1.00; P < .0001), CCA from biliary stones (AUC, 0.99; 95% CI, 0.98-1.00; P < .0001), as well as cholangitis from biliary stones (AUC, 0.93; 95% CI, 0.82-1.00; P = .001). CONCLUSION: These findings provide new insight into the biologic importance of MUC5AC in biliary disorders and suggest that combined assessment of MUC5AC in bile and serum with expression of data in terms of serum to bile ratio may improve the diagnostic performance of MUC5AC quantification in serum alone.

A novel serum marker for biliary tract cancer: diagnostic and prognostic values of quantitative evaluation of serum mucin 5AC (MUC5AC).

BACKGROUND AND AIMS: Mucin 5AC (MUC5AC) is a glycoprotein found in different epithelial cancers, including biliary tract cancer (BTC). The aims of this study were to investigate the role of MUC5AC as serum marker for BTC and its prognostic value after operation with curative intent. PATIENTS AND METHOD: From January 2007 to July 2012, a quantitative assessment of serum MUC5AC was performed with enzyme-linked immunoassay in a total of 88 subjects. Clinical and biochemical data (including CEA and Ca 19-9) of 49 patients with BTC were compared with a control population that included 23 patients with benign biliary disease (BBD) and 16 healthy control subjects (HCS). RESULTS: Serum MUC5AC was greater in BTC patients (mean 17.93 ± 10.39 ng/mL) compared with BBD (mean 5.95 ± 5.39 ng/mL; P < .01) and HCS (mean 2.74 ± 1.35 ng/mL) (P < .01). Multivariate analysis showed that MUC5AC was related with the presence of BTC compared with Ca 19-9 and CEA: P < .01, P = .080, and P = .463, respectively. In the BTC group, serum MUC5AC ≥ 14 ng/mL was associated with lymph-node metastasis (P = .050) and American Joint Committee on Cancer and International Union for Cancer Control stage IVb disease (P = .047). Moreover, in patients who underwent operation with curative intent, serum MUC5AC ≥ 14 ng/mL was related to a worse prognosis compared with patients with lesser levels, with 3-year survival rates of 21.5% and 59.3%, respectively (P = .039). CONCLUSION: MUC5AC could be proposed as new serum marker for BTC. Moreover, the quantitative assessment of serum MUC5AC could be related to tumor stage and long-term survival in patients with BTC undergoing operation with curative intent.

CA-S27: a novel Lewis a associated carbohydrate epitope is diagnostic and prognostic for cholangiocarcinoma.

Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen-S27 (CA-S27) monoclonal antibody (mAb) was established using pooled CCA tissue-extract as immunogen. The epitope recognized by CA-S27-mAb was a new Lewis-a (Le(a)) associated modification of MUC5AC mucin. A Soybean agglutinin/CA-S27-mAb sandwich ELISA to determine CA-S27 in serum was successfully developed. High level of CA-S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro-intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA-S27 was dramatically reduced after tumor removal, indicating tumor origin of CA-S27. Patients with high serum CA-S27 had significantly shorter survivals than those with low serum CA-S27 regardless of serum MUC5AC levels. Fucosyltransferase-III (FUT3) was shown to be a regulator of CA-S27 expression. Suppression of CA-S27 expression with siRNA-FUT3 or neutralization with CA-S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA-S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA-S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells.

Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges.

BACKGROUND: Recent developments in LC-MS have turned it into a viable and valid alternative to ligand-binding assays. Large molecule bioanalysis by LC-MS is generally performed by tryptic digestion, purification and detection of one or more small signature peptides. High-resolution MS instruments offer quantification of intact small proteins or peptides and are able to increase the selectivity, while maintaining sensitivity. RESULTS: Unlike multiple reaction monitoring assays, several factors affecting data processing were presented and the optimal parameters to consider during quantification method building were also demonstrated. MUC5AC-13 (MW 1709.8 Da), human hepcidin/LEAP-1 (MW 2797.4 Da), porcine calcitonin (MW 3604 Da) and chicken lysozyme (MW 14.3 kDa) were selected as model compounds and the possibility of intact peptide and small protein quantification, without tryptic digestion, was demonstrated. CONCLUSION: Selectivity and sensitivity were improved using different scan modes, such as TOF-MS and TOF-MS/MS.

A novel carbohydrate antigen expression during development of Opisthorchis viverrini- associated cholangiocarcinoma in golden hamster: a potential marker for early diagnosis.

Poor prognosis of cholangiocarcinoma (CCA) is primarily due to delayed diagnosis because of the lack of appropriate tumor marker(s) to detect cancer development at an early stage. We have recently established a S121 monoclonal antibody (mAb) which recognizes an unidentified glycan epitope on MUC5AC, designated as CCA-associated carbohydrate antigen (CCA-CA). This antigen is expressed in human CCA cells but not in normal biliary epithelia. Detection of CCA-CA effectively distinguished CCA patients' sera from normal control sera with high specificity and sensitivity. In the present study, we examined a time profile of the expression of CCA-CA by immunohistochemical methods in the liver tissues of Opisthorchis viverrini (Ov)-associated CCA in a hamster model. Hamsters were divided into four groups; non-treated, Ov infected, NDMA (N-nitrosodimethamine) treated and Ov+NDMA treated groups, and animals from each group were euthanized at 1, 3 and 6 months post-treatment. CCA-CA was not detected in normal biliary cells of non-treated hamsters throughout the course of experiment. CCA-CA became detectable in the cytoplasm and apical surface of biliary cells of the NDMA and Ov+NDMA groups at early stage (1 month) of tumor development and increased with tumor progression. In contrast, CCA-CA was detected as nuclear staining at the 1 month post Ov infection and declined thereafter. These results suggest the possibility of CCA-CA as an early marker for CCA.

A novel serum carbohydrate marker on mucin 5AC: values for diagnostic and prognostic indicators for cholangiocarcinoma.

BACKGROUND: The incidence of cholangiocarcinoma (CCA) is increasing globally. Currently, there is no powerful marker for the diagnosis of CCA, which has led to late diagnosis and poor patient outcome. This study was designed to establish a new monoclonal antibody (MoAb) for detecting a serum marker associated with CCA. METHODS: Pooled CCA tissue extracts were immunized to germinal center associated nuclear protein (GANP)-transgenic mice. The antibody-producing hybridomas were prepared and initially screened by using an indirect enzyme-linked immunosorbent assay (ELISA). A positive clone that reacted strongly with CCA serum or tumor tissue extract and failed to react with normal human serum and liver extract was selected. RESULTS: An S121 immunoglobulin M MoAb that recognized a novel glycan epitope was obtained. Immunohistochemistry of CCA tissues revealed that the MoAb reacted strongly with hyperplastic/dysplastic and neoplastic bile ducts but not with normal bile ducts. In addition, experiments demonstrated that mucin 5AC (MUC5AC) is a core glycoprotein for the S121 epitope. A sandwich ELISA using soybean agglutinin and an S121 MoAb was developed for detecting S121 reactive antigen in patient sera. The level of serum S121 from patients with CCA was reduced significantly after tumor removal, indicating the tumor origin of this antigen. The test was able to distinguish patients with CCA from healthy individuals, active Opisthorchis viverrini-infected individuals and patients with various gastrointestinal cancers, hepatoma, and benign hepatobiliary diseases with 87.63% sensitivity, 89.58% specificity, an 80.95% positive predictive value, and a 93.47% negative predictive value. Moreover, high serum S121 levels were related to a poor patient outcome. CONCLUSIONS: The sugar antigen recognized by S121 MoAb is a new serum marker for the diagnosis and prognosis of CCA.

Development of a serum biomarker assay that differentiates tumor-associated MUC5AC (NPC-1C ANTIGEN) from normal MUC5AC.

A serum ELISA using a monoclonal antibody that detects a MUC5AC-related antigen (NPC-1C antigen) expressed by pancreatic and colorectal cancer was developed. The NPC-1C antibody reacts with specific epitopes expressed by tumor-associated MUC5AC that does not appear on MUC5AC from normal tissues. Based on observations of a highly specific antibody, we tested the ELISA to differentiate serum from healthy blood donors compared to serum from patients with colorectal or pancreatic cancer. Additionally, patient tumor tissue was stained to examine the expression pattern of MUC5AC-related antigen in pancreatic and colorectal cancers. The results indicate the NPC-1C antibody ELISA distinguished serum of cancer patients from normal donors with very good sensitivity and specificity. Most patient's tumor biopsy exhibited NPC-1C antibody reactivity, indicating that tumor-associated MUC5AC antigen from tumor is shed into blood, where it can be detected by the NPC-1C antibody ELISA. This serum test provides a new tool to aid in the diagnosis of these cancers and immune monitoring of cancer treatment regimens.

Role of mucins in the skin during benign and malignant conditions.

Skin-related diseases comprise a major health challenge to the practicing physician, and constitute a significant psychological, social and financial burden to the society. Further, skin cancer, especially non-melanoma skin cancer is currently the leading type of malignancy in the Western world. Given the huge burden of skin diseases, there is growing emphasis on understanding their pathophysiology, and towards their early detection. Mucins are high-molecular weight O- and N-linked glycoproteins that have emerged in recent years as important molecules in maintaining health and in promoting or protecting against inflammation and cancer. They have also begun to emerge as highly specific diagnostic and prognostic markers and novel therapeutic targets in several malignant disorders. However, their role in cutaneous pathologies has remained largely obscured. The present review provides the expression patterns and proposed role of mucins in the healthy skin and various benign and malignant skin diseases. The review has immense clinical significance as the availability of highly specific reagents including monoclonal antibodies against mucins makes them extremely attractive targets for specific diagnosis and/or immunotherapy of benign and malignant cutaneous diseases.

Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers.

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.

Potential application of alternatively glycosylated serum MUC1 and MUC5AC in gastric cancer diagnosis.

Post-transcription modification of proteins can be altered during carcinogenesis. In this study, quantitative sandwich enzyme immunoassays were utilized to explore the clinical diagnostic value of the alternatively glycosylated MUC1 and MUC5AC. Four pairs of antibodies were selected to construct quantitative sandwich enzyme immunoassay. Serum mucin levels of 104 primary gastric cancer patients and 120 healthy individuals were measured using the four antibody pairs. The detection sensitivities of each antibody pair against gastric cancers were 42.31%, 25.00%, 38.46% and 30.77% respectively, with a specificity of 90.00%, significantly higher than widely used tumor markers CEA (21.15%) and CA19-9 (18.27%). When monitoring in parallel with all of the four antibody pairs, the detection sensitivity increased to 75.00%, with the same 90.00% specificity. Immunoblotting of the serum samples using the anti-mucin antibodies revealed highly variable glycosylation patterns among gastric cancer patients. In addition, real-time PCR indicated the elevated mRNA levels of MUC1 and/or MUC5AC in gastric cancers. The cancer-specific epitopes were also detected in other alimentary canal epithelium cancers such as colonic, nasopharyngeal and esophageal cancers, but with much lower sensitivities. Our results suggested that alternatively glycosylated MUC1 and MUC5AC could be of significant potential as effective tumor markers in gastric cancer diagnosis.