Nearly one-third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial treatment with platinum-based therapy. Genomic and clinical characterization of these patients may lead to potential alternative therapies. Here, the objective is to classify non-responders into subsets using clinical and molecular features. Using patients from The Cancer Genome Atlas (TCGA) dataset with platinum-resistant or platinum-refractory HGSC, we performed a genome-wide unsupervised cluster analysis that integrated clinical data, gene copy number variations, gene somatic mutations, and DNA promoter methylation. Pathway enrichment analysis was performed for each cluster to identify the targetable processes. Following the unsupervised cluster analysis, three distinct clusters of non-responders emerged. Cluster 1 had overrepresentation of the stage IV disease and suboptimal debulking, under-expression of miRNAs and mRNAs, hypomethylated DNA, "loss of function"

BACKGROUND: Aberrantly expressed mucin glycoproteins (MUC) play important roles in pancreatic ductal adenocarcinoma (PDAC), yet their use as a diagnostic aid in fine-needle aspiration biopsy (FNAB) is poorly documented. The aim of this study was to investigate the rationale and feasibility of mucin (MUC1, MUC2, MUC3, MUC4, MUC5AC, and MUC6) expression profiling by RT-PCR for diagnostic applications in cytology.
METHODS: Mucin expression was examined by RT-PCR and immunohistochemistry in specimens resected from patients with pancreatic (n = 101), ampullary (n = 23), and common bile duct (n = 10) cancers and 33 with chronic pancreatitis. Furthermore, mucin profiling by RT-PCR was prospectively compared in surgical and biopsy specimens of 40 patients with pancreatic solid tumours qualified for FNAB prior to surgery.
RESULTS: A logistic regression model to distinguish PDAC from chronic pancreatitis using RT-PCR profiling included MUC3, MUC5AC, and MUC6. The same set of mucins differentiated ampullary and bile duct cancers from chronic pancreatitis. AUCs for the ROC curves derived from the two models were 0.95 (95%CI 0.87-0.99) and 0.92 (95%CI 0.81-0.98), respectively. The corresponding positive likelihood ratios were 6.02 and 5.97, while the negative likelihood ratios were 0.10 and 0.12. AUCs of ROC curves obtained by RT-PCR and immunohistochemistry demonstrated that both analytical methods were comparable. Surgical and cytological samples showed significantly correlated values of ΔCt for individual mucins with the overall Pearson's correlation coefficient r = 0.841 (P = 0.001).
CONCLUSIONS: Mucin expression profiling of pancreatic cancer with RT-PCR is feasible and may be a valuable help in discriminating malignant lesions from chronic pancreatitis in FNAB cytology.

BACKGROUND: Cancer genes tend to be highly mutated under positive selection. Better understanding the recurrently mutated genes (RMGs) in cancer is critical for explicating the mechanisms of tumorigenesis and providing vital clues for therapy. Although some studies have investigated functional impacts of RMGs in specific cancer types, a comprehensive analysis of RMGs and their mutational impacts across cancers is still needed.
METHODS: We obtained data from The Cancer Genome Atlas (TCGA) and calculated mutation rate of each gene in 31 cancer types. Functional analysis was performed to identify the important signaling pathways and enriched protein types of RMGs. In order to evaluate functional impacts of RMGs, differential expression, survival, and pairwise mutation patterns analyses were performed.
RESULTS: Totally, we identified 897 RMGs and 624 of them were specifically mutant in only a single cancer type. Functional analysis demonstrated that these RMGs were enriched in hydrolases, cytoskeletal protein, and pathways like MAPK, cell cycle, PI3K-Akt, ECM receptor interaction, and energy metabolism. The differentially expressed genes potentially affected by the same common RMG showed a relatively low overlap across different cancer types. For the 19 Mucin (MUC) family genes, nine of them were RMGs and four of them (MUC17, MUC5B, MUC4, and MUC16) were common RMGs shared in 8 to 17 cancer types. The results showed that recurrent mutations in MUC genes were significantly associated with better survival prognosis. Only a small part of RMGs was differentially expressed due to their own mutations and most of them were downregulated. In addition, pairwise mutation pattern analysis revealed the high frequency of co-occurred mutations among RMGs in STAD.
CONCLUSION: Through the functional analysis of RMGs, we found that six signaling pathways were disrupted in most cancer types and that energy metabolism was abnormal in tumors. The results also revealed a strong correlation between recurrently mutated genes from MUC family and human survival. In addition, gene expression and survival prognosis were associated with different mutation types of RMGs.

BACKGROUND: Human mutagenesis has a large stochastic component. Thus, large coding regions, especially cytoskeletal and extra-cellular matrix protein (CECMP) coding regions are particularly vulnerable to mutations. Recent results have verified a high level of somatic mutations in the CECMP coding regions in the cancer genome atlas (TCGA), and a relatively common occurrence of germline, deleterious mutations in the TCGA breast cancer dataset.
METHODS: The objective of this study was to determine the correlations of CECMP coding region, germline nucleotide variations with both overall survival (OS) and disease-free survival (DFS). TCGA, tumor and blood variant calling files (VCFs) were intersected to identify germline SNVs. SNVs were then annotated to determine potential consequences for amino acid (AA) residue biochemistry.
RESULTS: Germline SNVs were matched against somatic tumor SNVs (i.e., tumor mutations) over twenty TCGA datasets to identify 23 germline-somatic matched, deleterious AA substitutions in coding regions for FLG, TTN, MUC4, and MUC17.
CONCLUSIONS: The germline-somatic matched SNVs, in particular for MUC4, extensively implicated in cancer development, represented highly, statistically significant effects on OS and DFS survival rates. The above results contribute to the establishment of what is potentially a new class of inherited cancer-facilitating genes, namely dominant negative tumor suppressor proteins.

Colorectal cancer (CRC) accounts for about 8% of all new cancer cases diagnosed in the US. We used whole exome sequence data from triplet samples (colon carcinoma, colon adenoma, and normal tissue) from 18 individuals to assess gene mutation rates. Of the 2 204 genes that were mutated, APC, TTN, TP53, KRAS, OBSCN, SOX9, PCDH17, SIGLEC10, MYH6, and BRD9 were consistent with genes being an early driver of carcinogenesis, in that they were mutated in multiple adenomas and multiple carcinomas. Fifty-two genes were mutated in ≥12.5% of microsatellite stable (MSS) carcinomas but not in any of the adenomas, in line with the profile of a late driver event involved in tumor progression. Thirty-eight genes were sequenced in a larger independent set of 148 carcinoma/normal tissue pairs to obtain more precise mutation frequencies. Eight of the genes, APC, TP53, ATM, CSMD3, LRP1B, RYR2, BIRC6, and MUC17, contained mutations in >20% of the carcinomas. Interestingly, mutations in four genes in addition to APC that are associated with dysregulation of Wnt signaling, were all classified as early driver events. Most of the genes that are commonly associated with colon cancer, including APC, TP53, and KRAS, were all classified as being early driver genes being mutated in both adenomas and carcinomas. Classifying genes as potential early and late driver events points to candidate genes that may help dissect pathways involved in both tumor initiation and progression.

BACKGROUND: Mucins are heavily glycosylated glycoproteins, synthesized by mucosal surfaces and have an important role in healthy state and malignant diseases. Change in mucins synthesis or secretion may be primary event or secondary to inflammation or carcinogenesis.
AIM: The aim of this study is to assess the current knowledge about mucin expression in esophageal lesions, and to establish a role for different mucin expressions as prognostic markers.
METHOD: English Medical literature searches were conducted for "mucin" and "esophagus." Observational studies were included. Meta-analysis was performed using comprehensive meta-analysis software. Pooled odds ratios (OR) and 95% confidence intervals (CI) were calculated.
RESULTS: In the random-effect model, mucin expression was significantly higher in esophageal lesions than in normal esophageal mucosa with OR=5.456 (95% CI, 1.883-15.807, P=0.002). Measure of heterogeneity, demonstrated in the included studies, was high: Q=287.501, df (Q)=44.00, P<0.0001, I=84.696%. There is a gradient of mucin expression and complexity in esophageal premalignant to malignant lesions, lower in Barrett's mucosa with low grade dysplasia (LGD), increased in high grade dysplasia (HGD), and highest in esophageal adenocarcinoma (EAC). MUC2, MUC3, MUC5AC, and MUC6 expression was higher in EAC than HGD, and higher in HGD than in LGD mucosa. The opposite was found for MUC1 and MUC4.
CONCLUSION: Increased expression of certain mucin genes in esophageal mucosa may be further studied as a potential diagnostic tool, and this may add important information in the surveillance of Barrett's esophagus.

Though invasive mucinous adenocarcinoma of the lung (IMA) is pathologically distinctive, the molecular mechanism driving IMA is not well understood, which hampers efforts to identify therapeutic targets. Here, by analyzing gene expression profiles of human and mouse IMA, we identified a Mucinous Lung Tumor Signature of 143 genes, which was unexpectedly enriched in mucin-producing gastrointestinal, pancreatic, and breast cancers. The signature genes included transcription factors

Polymorphisms in microRNA (miRNA) binding sites may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and susceptibility to common diseases. Mucins have been identified as markers of adverse prognosis. We hypothesized that genetic variations in miRNA binding sites located in mucin genes may modulate signaling response and the maintenance of genomic stability ultimately affecting cancer susceptibility, efficacy of chemotherapy and survival. In this study, we analyzed the association of single nucleotide polymorphisms in predicted miRNA target sites (miRSNPs) of mucin genes with colorectal cancer (CRC) risk and clinical outcome. Thirteen miRSNPs in 9 genes were assessed in 1111 cases and 1469 controls. No strongly significant associations were observed in the case-control study. Patients carrying the CC genotype of rs886403 in MUC21 displayed a shorter survival and higher recurrence risk when compared with TT carriers [overall survival (OS): hazard ratios (HR) 1.69; 95% confidence intervals (CI) 1.13-2.46; P = 0.01 and event-free survival (EFS): HR 1.99; 95% CI 1.38-2.84; P = 0.0002, respectively]. The observed associations were more striking after stratification for tumor site (in patients with colon cancer, OS: HR 2.63; 95% CI 1.69-4.10; P < 0.0001 and EFS: HR 2.65; 95% CI 1.72-4.07; P < 0.0001). In contrast, rectal cancer cases carrying the CC genotype of rs4729655 in MUC17 displayed a longer survival (OS: HR 0.27; 95% CI 0.14-0.54; P = 0.0002) than those with the most common genotype. To our knowledge, this is the first study investigating miRSNPs potentially affecting miRNA binding to mucin genes and revealing their impact on CRC susceptibility or patient's survival.

AIMS: Mucin (MUC) glycoproteins are involved in various steps of the carcinogenesis and progression of human malignancies. The aim of this study was to verify whether semiquantitative evaluation of MUC staining by immunohistochemistry may help to differentiate pancreatic ductal cell adenocarcinoma (PDAC) from chronic pancreatitis and normal pancreas.
METHODS AND RESULTS: Mucin expression was examined by immunohistochemistry in surgical specimens resected from 101 patients with PDAC and 33 with chronic pancreatitis, and in 40 normal pancreatic tissue specimens. A quickscore (QS, range 0-300) was calculated by multiplying staining intensity by the percentage of positive cells. A diagnostic model was developed for MUC QS (MUC1, MUC2, MUC3, MUC4, MUC5AC, and MUC6), based on a receiver operating characteristic (ROC) curve and logistic regression analysis. Median QS values for MUC1 and MUC5AC were significantly higher for PDAC, whereas patients with non-malignant tissues had higher values for MUC3 and MUC6. The area under the curve for the ROC curve derived from the diagnostic model including MUC3, MUC5AC and MUC6 was 0.96 [95% confidence interval (CI) 0.91-0.98], with 85% sensitivity and 94% specificity. Median QS values for MUC2 were significantly higher in patients with less advanced tumours, whereas venous invasion was associated with a lower QS for MUC6. Moreover, multivariate survival analysis revealed that low MUC6 expression was a negative prognostic factor, with a hazard ratio of 1.73 (95% CI 1.07-2.81).
CONCLUSIONS: The three-MUC diagnostic model (MUC3, MUC5AC, and MUC6) showed an excellent ability to discriminate pancreatic cancer from non-malignant tissues, and yielded information that may prove useful for the development of clinical applications.

Gastric cancer is one of the most aggressive cancers and is the second leading cause of cancer death worldwide. Approximately 40% of global gastric cancer cases occur in China, with peritoneal metastasis being the prevalent form of recurrence and metastasis in advanced disease. Currently, there are limited clinical approaches for predicting and treatment of peritoneal metastasis, resulting in a 6-month average survival time. By comprehensive genome analysis will uncover the pathogenesis of peritoneal metastasis. Here we describe a comprehensive whole-genome and transcriptome sequencing analysis of one advanced gastric cancer case, including non-cancerous mucosa, primary cancer and matched peritoneal metastatic cancer. The peripheral blood is used as normal control. We identified 27 mutated genes, of which 19 genes are reported in COSMIC database (ZNF208, CRNN, ATXN3, DCTN1, RP1L1, PRB4, PRB1, MUC4, HS6ST3, MUC17, JAM2, ITGAD, IREB2, IQUB, CORO1B, CCDC121, AKAP2, ACAN and ACADL), and eight genes have not previously been described in gastric cancer (CCDC178, ARMC4, TUBB6, PLIN4, PKLR, PDZD2, DMBT1and DAB1).Additionally,GPX4 and MPND in 19q13.3-13.4 region, is characterized as a novel fusion-gene. This study disclosed novel biological markers and tumorigenic pathways that would predict gastric cancer occurring peritoneal metastasis.

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

BACKGROUND: Mucins are implicated in survival in various cancers, but there have been no report addressed on survival in appendiceal carcinoma, an uncommon disease with different clinical and pathological features from those of other colon cancers. We aimed to investigate the clinical implications of expression of mucins in appendiceal carcinoma.
METHODS: Expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6, MUC16 and MUC17 in cancer tissue were examined by immunohistochemistry in 108 cases of surgically resected appendiceal carcinoma.
RESULTS: The following relationships of mucins with clinicopathologic factors were identified: MUC1 with positive lymphatic invasion (p = 0.036); MUC2 with histological type (mucinous carcinoma, p<0.001), superficial invasion depth (p = 0.007), negative venous invasion (p = 0.003), and curative resection (p = 0.019); MUC3 with non-curative resection (p = 0.017); MUC5AC with histological type (mucinous carcinoma, p = 0.002), negative lymphatic invasion (p = 0.021), and negative venous invasion (p = 0.022); and MUC16 with positive lymph node metastasis (p = 0.035), positive venous invasion (p<0.05), and non-curative resection (p = 0.035). A poor prognosis was related to positive lymph node metastasis (p = 0.04), positive lymphatic invasion (p = 0.02), positive venous invasion (p<0.001), non-curative resection (p<0.001), and positive expression of MUC3 (p = 0.004). In multivariate analysis, positive venous invasion (HR: 6.93, 95% CI: 1.93-24.96, p = 0.003), non-curative resection (HR: 10.19, 95% CI: 3.05-34.07, p<0.001) and positive MUC3 expression (HR: 3.37, 95% CI: 1.13-10.03, p = 0.03) were identified as significant independent prognostic factors in patients with appendiceal carcinoma.
CONCLUSIONS: Expression of MUC3 in appendiceal carcinoma is an independent factor for poor prognosis and a useful predictor of outcome in patients with appendiceal carcinoma after surgery.

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p = 0.026), venous invasion (p<0.001) and curative resection (p<0.001), in addition to expression of MUC1 (p = 0.042), MUC5AC (p = 0.007) and MUC16 (p<0.001). In subsequent multivariate analysis with curability as the covariate, lymph node metastasis, venous invasion, and MUC5AC and/or MUC16 expression were significantly related to the prognosis. Multivariate analysis in curative cases (n = 45) showed that SBC with MUC5AC and/or MUC16 expression had a significantly independent high hazard risk after adjusting for the effects of venous invasion (hazard ratio: 5.6, 95% confidence interval: 1.8-17). In conclusion, the study shows that a MUC5AC-positive and/or MUC16-positive status is useful as a predictor of a poor outcome in patients with SBC.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) may account for 20-30% of colon cancers. Although large SSA/Ps are generally recognized phenotypically, small (<1 cm) or dysplastic SSA/Ps are difficult to differentiate from hyperplastic or small adenomatous polyps by endoscopy and histopathology. Our aim was to define the comprehensive gene expression phenotype of SSA/Ps to better define this cancer precursor.
RESULTS: RNA sequencing was performed on 5' capped RNA from seven SSA/Ps collected from patients with the serrated polyposis syndrome (SPS) versus eight controls. Highly expressed genes were analyzed by qPCR in additional SSA/Ps, adenomas and controls. The cellular localization and level of gene products were examined by immunohistochemistry in syndromic and sporadic SSA/Ps, adenomatous and hyperplastic polyps and controls. We identified 1,294 differentially expressed annotated genes, with 106 increased ≥10-fold, in SSA/Ps compared to controls. Comparing these genes with an array dataset for adenomatous polyps identified 30 protein coding genes uniquely expressed ≥10-fold in SSA/Ps. Biological pathways altered in SSA/Ps included mucosal integrity, cell adhesion, and cell development. Marked increased expression of MUC17, the cell junction protein genes VSIG1 and GJB5, and the antiapoptotic gene REG4 were found in SSA/Ps, relative to controls and adenomas, were verified by qPCR analysis of additional SSA/Ps (n = 21) and adenomas (n = 10). Immunohistochemical staining of syndromic (n≥11) and sporadic SSA/Ps (n≥17), adenomatous (n≥13) and hyperplastic (n≥10) polyps plus controls (n≥16) identified unique expression patterns for VSIG1 and MUC17 in SSA/Ps.
CONCLUSION: A subset of genes and pathways are uniquely increased in SSA/Ps, compared to adenomatous polyps, thus supporting the concept that cancer develops by different pathways in these phenotypically distinct polyps with markedly different gene expression profiles. Immunostaining for a subset of these genes differentiates both syndromic and sporadic SSA/Ps from adenomatous and hyperplastic polyps.

BACKGROUND: Lung adenocarcinoma (LAC) is the most common type of lung cancer, accounting for 30-35% of all cases.
AIM: In this study we aim to predict potential genes and confirm pathways which are associated with LAC.
MATERIALS AND METHODS: By using the meta-analysis method, GSE10072 and GSE 2514 datasets were merged to find potential genes and pathways which are associated with LAC.
RESULTS: Our analysis indicated identified differentially expressed genes enriched in multicellular organismal metabolic process, gland development, and urogenital system development. Further, we predicted genes including EGF-like domain might be the potential target genes for further study, such as NGX6, MUC17, and Nel. In addition, a number of genes that associated with axon guidance, focal adhesion, and complement and coagulation cascades pathway might be also involved in LAC in a direct or indirectly manner.
CONCLUSIONS: Our analysis indicated identified differentially expressed genes enriched in multicellular organismal metabolic process, gland development, and urogenital system development We anticipate numerous advances in LAC research in the coming years based on our meta-analysis.

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3'), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

AIMS:   To analyse the expression of several mucins (MUC1, MUC2, MUC3, MUC5AC and MUC6), epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2 (HER2), thyroid transcription factor-1 (TTF-1), caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20), and the presence of mutations of EGFR, KRAS and HER2 in congenital pulmonary airway malformations (CPAM).
METHODS AND RESULTS:   Forty-one cases of CPAM and six pulmonary sequestrations were included. TTF-1 expression was observed in all cases but was not seen in mucinogenic growths in CPAM. CDX2 expression was completely negative. MUC1 expression was noted in 12 (29%) CPAM and in 33% sequestrations. MUC5AC was noted in only five cases (26%) by immunohistochemistry and was found in the mucinogenic proliferations of type 1 CPAM. No immunolabelling was noted for the other mucins. EGFR was expressed variably in almost all cases, while HER2 and CK20 was seen exclusively in the mucinogenic proliferations. All mucinous growths were characterized by KRAS mutations. No EGFR and HER2 gene alterations were identified.
CONCLUSIONS:   KRAS mutations and MUC5AC, CK20 and HER2 expression was seen in all mucinogenic proliferations, supporting the neoplastic nature of these cytologically bland growths. These findings emphasize the importance of complete surgical resection of such lesions.

This review focuses on the immunohistochemical expression of members of the MUC-type mucin family in salivary gland tumors and head and neck squamous cell carcinomas (HNSCC). Information is available on changes in the expression levels and distribution profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7 in tumors of the salivary glands; and of MUC1, MUC2 and MUC4 in HNSCC. In salivary gland tumors the expression patterns of MUC2, MUC3, MUC5AC and MUC6 appear to be very closely correlated with the histopathological tumor type indicating their potential use to improve diagnostic accuracy in salivary gland neoplasia. Some MUC-type mucins have emerged as valuable prognostic indicators in pleomorphic adenoma, mucoepidermoid carcinoma and HNSCC. Nine antibodies directed against different MUC1 antigens have thus far been examined in HNSCC of which monoclonal antibodies DF3, HMFG-1 and Ma695 have shown significant correlations with disease outcome. The importance of taking the specific anti-MUC antibody into consideration when comparing the results of different studies on MUC expression in salivary gland tumors and HNSCC is also highlighted in this review.

OBJECTIVES: Alterations in mucin (MUC) glycosylation and expression have been described in cancer. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) can provide material for molecular biology analysis. This study assessed the feasibility of evaluating MUC expression from material obtained by EUS-FNA and studied the profile of MUC expression in benign and malignant pancreatic lesions.
METHODS: A total of 90 patients with solid or cystic pancreatic lesions underwent FNA. The aspirated material was used for cytological analysis and RNA extraction to assess the expression pattern of MUCs by reverse transcription-PCR with primers specific for the MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes.
RESULTS: RNA extraction was successful in 81% of the biopsies. The prevalences of MUC1, MUC2, MUC4, and MUC7 in ductal adenocarcinoma were 57.7, 51.4, 18.9, and 73.0%, respectively. Fifty percent of benign lesions and neuroendocrine tumors (NETs), and 63% of intraductal papillary mucinous neoplasms (IPMNs) were positive for MUC1. Twenty-five percent of benign lesions, 86% of NETs, and 47% of IPMNs were positive for MUC2. Of NETs, 50% were positive for MUC1, and 14% were positive for MUC7. None of the benign lesions or NETs expressed MUC4. MUC7 expression was highly significant for adenocarcinoma (P=0.007) and borderline for IPMN (P=0.05). MUC7 was expressed in 37.5% of chronic pancreatitis cases.
CONCLUSIONS: RNA can be extracted from samples obtained under EUS-FNA. MUC7 could serve as a potential biological marker to identify malignant lesions, especially pancreatic adenocarcinoma.

MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been suggested that MUC17 expression is correlated with the malignancy potential of pancreatic ductal adenocarcinomas (PDACs). In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. Near the transcriptional start site, the DNA methylation level of MUC17-negative cancer cell lines (e.g. PANC1) was high, whereas that of MUC17-positive cells (e.g. AsPC-1) was low. Histone H3-K9 (H3-K9) modification status was also closely related to MUC17 expression. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC17 expression. Furthermore, the hypomethylation status was observed in patients with PDAC. This indicates that the hypomethylation status in the MUC17 promoter could be a novel epigenetic marker for the diagnosis of PDAC. In addition, the result of miRNA microarray analysis showed that five potential miRNA candidates existed. It is also possible that the MUC17 might be post-transcriptionally regulated by miRNA targeting to the 3'-untranslated region of its mRNA. These understandings of the epigenetic changes of MUC17 may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for cancer patients.

The mucus layer coating the gastrointestinal tract is the front line of innate host defense, largely because of the secretory products of intestinal goblet cells. Goblet cells synthesize secretory mucin glycoproteins (MUC2) and bioactive molecules such as epithelial membrane-bound mucins (MUC1, MUC3, MUC17), trefoil factor peptides (TFF), resistin-like molecule beta (RELMbeta), and Fc-gamma binding protein (Fcgbp). The MUC2 mucin protein forms trimers by disulfide bonding in cysteine-rich amino terminal von Willebrand factor (vWF) domains, coupled with crosslinking provided by TFF and Fcgbp proteins with MUC2 vWF domains, resulting in a highly viscous extracellular layer. Colonization by commensal intestinal microbiota is limited to an outer "loose" mucus layer, and interacts with the diverse oligosaccharides of mucin glycoproteins, whereas an "inner" adherent mucus layer is largely devoid of bacteria. Defective mucus layers resulting from lack of MUC2 mucin, mutated Muc2 mucin vWF domains, or from deletion of core mucin glycosyltransferase enzymes in mice result in increased bacterial adhesion to the surface epithelium, increased intestinal permeability, and enhanced susceptibility to colitis caused by dextran sodium sulfate. Changes in mucin gene expression and mucin glycan structures occur in cancers of the intestine, contributing to diverse biologic properties involved in the development and progression of cancer. Further research is needed on identification and functional significance of various components of mucus layers and the complex interactions among mucus layers, microbiota, epithelial cells, and the underlying innate and adaptive immunity. Further elucidation of the regulatory mechanisms involved in mucin changes in cancer and inflammation may lead to the development of novel therapeutic approaches.

Lymph node metastasis (LNM) is the most important prognostic factor in patients undergoing surgical resection of pancreatic ductal adenocarcinoma (PDAC). In this study, we aimed to identify molecular markers associated with LNM in PDAC using genome-wide expression profiling. In this study, laser microdissection and genome-wide transcriptional profiling were used to identify genes that were differentially expressed between PDAC cells with and without LNM obtained from 20 patients with PDAC. Immunohistochemical staining was used to confirm the clinical significance of these markers in an additional validation set of 43 patients. In the results, microarray profiling identified 46 genes that were differently expressed between PDAC with and without LNM with certain significance. Four of these biomarkers were validated by immunohistochemical staining for association with LNM in PDAC in an additional validation set of patients. In 63 patients with PDAC, significant LNM predictors in PDAC elucidated from multivariate analysis were low expression of activating enhancer binding protein 2 (AP2alpha) (P = 0.012) and high expression of mucin 17 (MUC17) (P = 0.0192). Furthermore, multivariate analysis revealed that AP2alpha-low expression and MUC17-high expression are independent prognostic factors for poor overall survival (P = 0.0012, 0.0001, respectively). In conclusion, AP2alpha and MUC17 were independent markers associated with LNM of PDAC. These two markers were also associated with survival in patients with resected PDAC. We demonstrate that AP2alpha and MUC17 may serve as potential prognostic molecular markers for LNM in patients with PDAC.

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram- bacteria from the apical side and the proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human beta-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-alpha. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-alpha levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-alpha induced significantly increased levels of IL-8 and TNF-alpha itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-gamma was selective for CEACAM1-L while TNF-alpha upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

AIMS: Oncocytic change in papillary neoplasms of the biliary tract is a very uncommon finding with little known about pathogenesis, immunophenotype and prognosis, especially in comparison to similar lesions in the pancreatic ductal system. We report four cases of oncocytic biliary intraductal papillary neoplasms (IPNs), highlighting the clinicopathological characteristics of these tumours, the immunohistochemical profile with regard to Wnt pathway proteins and mucin core protein (MUC) status, and compare these findings with the oncocytic variant of intraductal papillary mucinous neoplasm (IPMN) of the pancreas.
METHODS: Four cases of oncocytic IPN of the extrapancreatic, biliary tree (two with accompanying invasive carcinomas) were examined for mucin profiles and Wnt signalling proteins. The cases were stained for: beta-catenin, c-myc, glutathione synthase kinase (GSK), E-cadherin, cyclin D1, and adenomatous polyposis coli (APC), and MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B and MUC6, using standard immunohistochemistry.
RESULTS: The cases occurred in three males and one female, ranging in age from 59 to 81 years. The lesions caused obstructive symptoms related to the biliary tree as well as non-specific abdominal symptoms. Typically, cystic lesions were noted grossly. All four of the IPNs were composed of distinctive oncocytic cells. The invasive carcinomas accompanying two of the cases were also composed of oncocytes. None of the cases showed aberrant expression of the Wnt signalling proteins, although cyclin D1 was markedly over-expressed in all four cases. Three of four cases showed the following mucin profile: MUC3, MUC4, MUC5AC, MUC5B and MUC6 positive.
CONCLUSIONS: The Wnt pathway proteins (especially beta-catenin and E-cadherin) are expressed normally in oncocytic variants of intraductal papillary neoplasms of the biliary tree, and the mucin profile is similar to their counterparts in the pancreas.

BACKGROUND: MUC1 and MUC3 are from a large family of glycoproteins with an aberrant expression profile in various malignancies. Much interest has been focused on the role of these proteins in the development and progression of colorectal cancer; however, no previous studies have included the highly confounding variable of vascular invasion in their survival analysis. Using high throughput tissue microarray technology we assessed the prognostic value of MUC1 and MUC3 expression in the largest cohort of colorectal cancer patients to date. We propose that tumours lacking expression of MUC1 and MUC3 will be more likely to metastasise, due to previously observed loss of cell-cell adhesion, and this will therefore lead to more aggressive cancers with poorer prognosis.
METHODS: A tissue micro-array was prepared from tumour samples of 462 consecutive patients undergoing resection of a primary colorectal cancer. A comprehensive prospectively recorded data base with mean follow up of 75 months was collected and included common clinicopathological variables and disease specific survival. Immunohistochemical analysis of MUC1 and MUC3 expression was performed using antibodies NCL-MUC1 and 1143/B7 respectively, results were correlated with the variables within the database.
RESULTS: Positive expression of MUC1 and MUC3 was seen in 32% and 74% of tumours respectively. On univariate analysis no correlation was seen with either MUC1 or MUC3 and any of the clinicopathological variables including tumour grade and stage, vascular invasion and tumour type. Kaplan-Meier analysis demonstrated a significant reduction in disease specific survival with MUC1 positive tumours (p = 0.038), this was not seen with MUC3 (p = 0.552). On multivariate analysis, using Cox proportional hazards model, MUC1 expression was shown to be an independent marker of prognosis (HR 1.339, 95%CI 1.002-1.790, p = 0.048).
CONCLUSION: MUC1 expression in colorectal cancer is an independent marker of poor prognosis, even when vascular invasion is included in the analysis. These results support previous studies suggesting a role for MUC1 in colorectal cancer development possibly through its effects on cell adhesion.

INTRODUCTION: Recent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells.
METHODS: Promoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays.
RESULTS: We confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas.
CONCLUSION: Our study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.

PURPOSE: To compare survival and histologic features of hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome) cases to well-matched sporadic colon cancers from the same patient population.
EXPERIMENTAL DESIGN: Between January 1995 and March 2002, a total of 5,138 consecutive patients underwent resection of primary colorectal adenocarcinoma in a single institution. According to the Amsterdam criteria, 56 HNPCC patients were matched to 147 sporadic colorectal cancer (SCRC) with no family history of cancer and with the same gender, tumor location, and age within 3 years. Immunohistochemical analyses were done for MUC1, MUC2, MUC3, and MUC5AC.
RESULTS: The HNPCC group had a marginally significantly better long-term outcome than the SCRC group (P = 0.058). The trend disappeared after adjustment by tumor-node-metastasis stage in a Cox model (P = 0.774). We noted a difference of >50% in the 5-year cancer-specific survival rates of HNPCC- and SCRC-mucinous groups (92% versus 31%, P = 0.0003). Interaction between mucin and HNPCC and its effects on survival were further confirmed by comparing the Cox models with and without interaction terms (hazard ratio, 0.1; P = 0.034 with adjusting stage). Patients with tumors showing dual expression of mucin and MUC1, which appeared in 11% of those with HNPCC and 50% of those with SCRC, had a lower 5-year cancer-specific survival rate than patients without (30% versus 60%; P = 0.004 by log-rank test; P = 0.039 with adjustment for tumor-node-metastasis stage).
CONCLUSIONS: These results suggest that mucin has an inverse effect on survival in patients with HNPCC and SCRC, which might be partly explained by a lower prevalence of MUC1 expression in the mucinous HNPCC group than in the SCRC groups.

With increasing interest on mucins as diagnostic and therapeutic targets in cancers and other diseases, it is becoming imperative to characterize novel mucins and investigate their biological significance. Here, we present the completed coding sequence and genomic organization of the previously published partial cDNA sequence of MUC17. Rapid amplification of cDNA ends with PCR, sequences from the Human Genome databases, and in vitro transcription/translational assays were used for these analyses. The MUC17 gene is located within a 39-kb DNA fragment between MUC12 and SERPINE1 on chromosome 7 in the region q22.1. The full-length coding sequence of MUC17 transcribes a 14.2-kb mRNA encompassing 13 exons. Alternate splicing generates two variants coding for a membrane-anchored and a secreted form. The canonical variable number of tandem repeats polymorphism of the central tandem repeat domain of the MUC genes is not significantly detected in the MUC17 gene. In addition, we show the overexpression of MUC17 by Western blot and immunohistochemical analyses in pancreatic tumor cell lines and tumor tissues compared with the normal pancreas. The expression of MUC17 is regulated by a 1,146-bp fragment upstream of MUC17 that contains VDR/RXR, GATA, NFkappaB, and Cdx-2 response elements.

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the "non-mammary" MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.

Cell surface mucins are complex glycoproteins expressed on the apical membrane surface of mucosal epithelial cells. In malignant epithelial cells they are thought to influence cell adhesion, and are clinical targets for tumor immunotherapy and serum tumor marker assays. We have compared expression of MUC1, MUC3, MUC4, MUC11, MUC12 and MUC13 mRNA in epithelial cancers and/or cell lines with non-malignant tissues. In non-malignant tissues, MUC3, 4, 11, 12 and 13 were expressed at highest levels in gastrointestinal tissues, whereas MUC1 was more widely distributed. Significant down-regulation of the MUC4, MUC12 and MUC13 genes was observed in colonic cancers compared with normal tissue, whereas MUC1 was upregulated. In rectal cancers, levels of all six mucin genes were not significantly different to those in normal rectal tissues. Both MUC1 and MUC4 were down-regulated in gastric cancers, whereas cancer and normal tissue levels were similar for MUC3, 11, 12 and 13. In esophageal cancers there was a general trend toward higher levels than in normal tissue for MUC1, 3, 12 and 13. In ovarian cancers MUC1 levels were very high, whereas only low levels of all other mucins were observed. We also report expression in renal cell carcinomas, bladder carcinomas and breast cancer cell lines. The reported expression profiles of the cell surface mucin gene family will help direct biological and clinical studies of these molecules in mucosal biology, and in malignant and inflammatory diseases of epithelial tissues.