Ulcer associated cell lineage glands expressing trefoil peptide genes are induced by chronic ulceration in ileal pouch mucosa

BACKGROUND: Chronic ulcerative conditions in the gastrointestinal tract result in the appearance of the ulcer associated cell lineage (UACL). The glands of this new cell lineage secrete epidermal growth factor, transforming growth factor alpha, and the trefoil factor family (TFF) peptides, which are known to participate in repair processes. Pouchitis is the most frequent complication of ileal pouch-anal anastomosis. AIM: Our aim was to determine whether the mucosal ulceration present in pouchitis can induce the development of UACL glands. METHODS: Biopsies from ileal pouches with pouchitis (n=10), healthy pouches (n=5), and normal terminal ileum (n=5) were studied. Expression of TFF mRNA was assessed by in situ hybridisation. TFF1 and TFF2 proteins were localised by immunochemistry. RESULTS: UACL glands containing TFF1 and TFF2 were observed in six patients with pouchitis. In some glands, there was TFF3 mRNA as has been reported for Crohn's UACL. None of the biopsies from ileal reservoirs without pouchitis showed UACL glands (p<0.05). Neither TFF1 nor TFF2 expression was detected in ileal reservoirs without pouchitis. CONCLUSION: UACL glands arise de novo in ileal pouch mucosa of patients with pouchitis and express all three TFF peptide genes. Chronic inflammation alone, present in healthy pouches, is not enough to stimulate the growth of the UACL, and additional stimuli consequent on ulceration may be needed.     

Mucin gene expression in intestinal epithelial cells in Crohn's disease

BACKGROUND: Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease of unknown origin. It is characterised by chronic mucosal ulcerations which affect any part of the intestine but most commonly are found in the ileum and proximal colon. AIMS: Studies were undertaken to provide information regarding cell specific expression of mucin genes in the ileum of patients with CD. PATIENTS AND METHODS: Expression of mucin genes was analysed in the ileal mucosa of patients with CD and controls by in situ hybridisation and immunohistochemistry. RESULTS: In healthy ileal mucosa, patients with CD showed a pattern identical to normal controls with main expression of MUC2 and MUC3, lesser expression of MUC1 and MUC4, and no expression of MUC5AC, MUC5B, MUC6, or MUC7. In the involved mucosa, the pattern was somewhat comparable although heterogeneous to that observed in healthy ileal mucosa. Importantly, a particular mucin gene expression pattern was observed in ileal mucosa close to the ulcer margins in ulcer associated cell lineage, with the appearance of MUC5AC and MUC6 mRNAs and peptides, which are normally restricted to the stomach (MUC5AC and MUC6) and duodenum (MUC6), and disappearance of MUC2. CONCLUSIONS: Our results suggest that gel forming mucins (more particularly MUC5AC and MUC6) may have a role in epithelial wound healing after mucosal injury in inflammatory bowel diseases in addition to mucosal protection.     

Abnormalities in mucin gene expression in Crohn's disease

Alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation in Crohn's disease. To investigate the hypothesis of a mucin gene defect in Crohn's disease, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with Crohn's disease and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with Crohn's disease and (ii) between healthy mucosa of patients with Crohn's disease and controls. Expression of the different mucin genes was heterogeneous among controls and patients with Crohn's disease, except for MUC6 in controls. Nevertheless, MUC1 mRNA expression was significantly decreased in the involved ileal mucosa of patients with Crohn's disease when compared to the healthy mucosa (p = 0.02). Moreover, the expression levels of MUC3, MUC4, and MUC5B were significantly lower in both healthy and involved ileal mucosa of patients with Crohn's disease compared to controls (p < or = 0.05). The decrease of expression levels of some mucin genes (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD.     

The pathogenesis of duodenal gastric metaplasia: the role of local goblet cell transformation

BACKGROUND AND AIMS: Gastric metaplasia is frequently seen in biopsies of the duodenal cap, particularly when inflamed or ulcerated. In its initial manifestation small patches of gastric foveolar cells appear near the tip of a villus. These cells contain periodic acid-Schiff (PAS) positive neutral mucins in contrast with the alcian blue (AB) positive acidic mucins within duodenal goblet cells. Previous investigations have suggested that these PAS positive cells originate either in Brunner's gland ducts or at the base of duodenal crypts and migrate in distinct streams to the upper villus. To investigate the origin of gastric metaplasia in superficial patches, we used the PAS/AB stain to distinguish between neutral and acidic mucins and in addition specific antibodies to immunolocalise foveolar cell mucin MUC5AC, the foveolar cell secretory product, gastric trefoil factor (TFF1), the mature goblet cell mucin MUC2, and MUC2 core antigen. RESULTS: Cells in focal patches of gastric metaplasia contained secretory granules of both gastric and goblet cell phenotypes. MUC5AC and TFF1 were present as expected in gastric foveolar cells but in addition, MUC2 core antigen, normally present only in the Golgi of intestinal goblet cells, was expressed in secretory granules. Goblet cells in the vicinity of metaplastic patches also expressed both gastric and intestinal antigens. MUC5AC/MUC2 containing goblet cells were most common near the villus tip but were also seen at the base of crypts. Where crypts and Brunner's gland ducts merged they were always seen on the crypt side of the junction. Goblet cells were the only cells to express gastric antigens in these areas. In advanced metaplastic lesions, dual phenotype goblet cells were less evident and fewer cells expressed intestinal mucin antigens. CONCLUSIONS: We suggest that goblet cells that express both intestinal and gastric antigens may represent local precursors of gastric metaplasia undergoing a transition to foveolar-like cells of mixed phenotype at the site of early metaplastic patches. As metaplasia becomes more widespread, a more pure gastric phenotype emerges. This progression is likely to be controlled by local inflammatory signals.     

Spatio-temporal expression of trefoil peptide following severe gastric ulceration in the rat implicates it in late-stage repair processes

BACKGROUND: The trefoil peptide (TFF1) is a member of a family of mucin-associated regulatory peptides that are widely distributed in gastrointestinal tissues and have been implicated in the maintenance of the gastric mucosa. The role of TFF1 in gastric mucosal repair was examined by analysis of the spatio-temporal expression of TFF1 following gastric ulceration in the rat. METHODS: Gastric ulcers were induced in rats by application of glacial acetic acid to the serosa of the fundus. At various time points post injury (0-28 days), macroscopic and microscopic examination of the gastric mucosa was performed. In addition, the spatio-temporal expression of TFF1 protein and proliferating cell nuclear antigen were identified by immunohistochemistry, TFF1 message by in situ hybridization, and acidic/neutral secreting mucins by Alcian blue-periodic acid-Schiff staining. RESULTS: In normal rat gastric tissue, TFF1 peptide and mRNA were expressed in mucosal cells of the superficial epithelium. Trefoil peptide and mRNA were significantly induced between 4 and 28 days post ulceration, with expression extending beyond the superficial epithelium and being localized to acidic mucin-producing cells deep within the repairing mucosa. CONCLUSIONS: Spatio-temporal expression of TFF1 mRNA and peptide following macroscopic repair implicates TFF1 as a potential mediator of late stage-repair processes. Whether this is through direct stimulation of cellular differentiation or the enhancement of mucosal protective properties through an interaction with gastric mucins remains to be elucidated.     

Intestinal Goblet Cells and Mucins in Health and Disease: Recent Insights and Progress

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 β (RELMβ), and Fc-γ 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.     

Trefoil peptides

There is a growing body of evidence supporting the hypothesis that members of the trefoil peptide family are involved actively in maintaining the integrity of the gastrointestinal mucosa and facilitating its repair.To date, three trefoil peptides are known in man: pS2, ITF and SP. Each is a secretory peptide expressed in specific compartments throughout the gut, in patterns that appear generally to be conserved between mammalian species.Ulceration, whether due to common pathological processes or experimentally induced, results in altered local expression of trefoil peptides. In diverse chronic ulcerative conditions in man, glandular structures develop within the mucosa, derived from the UACL. These UACL glands express three trefoil peptides, EGF and lysozyme, all potentially able to contribute to the healing process. In fact local goblet and endocrine cell types may also be recruited to secrete pS2 into the local environment. In experimental ulcers, in rat stomach or intestinal resection margins, there is also accentuation of trefoil peptide expression at the margins and in the poorly differentiated mucous cells extending out presumably in attempts to restore epithelial integrity.Several trefoil peptides have been expressed as ‘recombinant’ proteins in bacterial, baculoviral or yeast systems, and these procedures have allowed some of the biological properties of these peptides to be determined.In vitro, rITF, hITF and hSP are motogens, able to promote migration of epithelial cells. In vivo, rITF and hSP are able to prevent much of the gastric damage effected by a single dose of indomethacin, when given systemically. There is synergy between EGF and rITF both in vitro and in vivo, which may allow the development of new peptide therapies for ulceration that will maximize repair and minimize cell proliferation.     

Mucin gene expression in Barrett's oesophagus: an in situ hybridisation and immunohistochemical study

BACKGROUND AND AIMS: Mucin genes are expressed in a site specific manner throughout the gastrointestinal tract. Little is known about the expression pattern in the oesophagus. In this study we have investigated MUC gene expression in both the normal oesophagus and specialised intestinal metaplasia (Barrett's oesophagus). PATIENTS: Archived paraffin embedded material from eight specimens of normal oesophagus, 18 Barrett's oesophagus, eight gastric metaplasia, six high grade dysplasia, and six cases of adenocarcinoma were examined for expression of the mucin genes MUC1-6. METHODS: Mucin mRNA was detected by in situ hybridisation using [(35)S] dATP labelled oligonucleotide probes. Mucin core protein was detected by immunohistochemistry. RESULTS: Normal oesophagus expressed MUC5B in the submucosal glands and MUC1 and MUC4 in the stratified squamous epithelium. Barrett's oesophagus strongly expressed MUC5AC and MUC3 in the superficial columnar epithelium, MUC2 in the goblet cells, and MUC6 in the glands. In high grade dysplasia and adenocarcinoma there was downregulation of MUC2, MUC3, MUC5AC, and MUC6, but upregulation of MUC1 and MUC4 in half of the specimens examined. CONCLUSIONS: Normal oesophagus and Barrett's oesophagus have a novel pattern of mucin gene expression. Barrett's oesophagus expressed the mucins associated with normal gastric epithelium and normal intestinal epithelium. While most mucin genes were downregulated in severely dysplastic and neoplastic tissues, there was upregulation of the membrane bound mucins MUC1 and MUC4. This may prove useful in detecting early signs of progression to adenocarcinoma of the oesophagus.     

Trefoil factor 2 in gland mucous cell mucin in the mucous gel covering normal or damaged gastric mucosa using the Mongolian gerbil model

OBJECTIVE: Trefoil factor 2 (TFF2) is localized in gastric gland mucous cells. The purpose of the study was to determine whether TFF2 and gastric mucin are localized in mucous cells and in the surface mucous gel layer (SMGL) of the normal gastric mucosa or in the mucoid cap adherent to gastric mucosal lesions in Mongolian gerbils. MATERIAL AND METHODS: Gastric mucosal lesions were induced in Mongolian gerbils using oral administration of Helicobacter pylori (H. pylori), subcutaneous administration of indomethacin, or oral administration of 30% ethanol. Tissue samples were fixed in Carnoy's solution for preservation of the SMGL, dehydrated, and embedded in paraffin. Histochemical staining for gastric mucins and immunostaining for TFF2 were performed. RESULTS: It was found that surface mucous cell mucin and gland mucous cell mucin were segregated in the SMGL covering the normal gastric mucosa, and the mucin of the mucoid cap covering the mucosal lesions was primarily gland mucous cell mucin. There was a co-localization of TFF2 in gland mucous cell mucin in gland mucous cells, the SMGL, and the mucoid cap. CONCLUSIONS: The co-localization of TFF2 in gland mucous cells and in the adherent mucus suggests a physical interaction between TFF2 and gland mucous cell mucin, and the participation of TFF2 trapped in the adherent mucus functions in mucosal defense, healing, and repair.     

Infection with Helicobacter pylori Affects All Major Secretory Cell Populations in the Human Antrum

We have investigated how gastric H. pylori infection affects antrum secretory cell types by studying the expression of secretory proteins in antrum epithelium. Antrum biopsy specimens were prospectively collected from 102 individuals (49 H. pylori-infected). Immunohistochemistry was performed for secretory mucins (MUC5AC, MUC5B, MUC6), Trefoil factor family (TFF)-peptides (TFF1, TFF2), endocrine peptides (gastrin, chromogranin A), and proliferating cells (Ki-67). Protein expression was quantified morphometrically. H. pylori infection was significantly correlated to mucosal inflammation and to epithelial atrophy and proliferation. In H. pylori-infected patients the number of proliferating cells increased significantly, and the zone of proliferating cells shifted toward the surface epithelium of the antral glands. Infection was correlated with decreased MUC5AC, TFF1, and TFF2 expression and increased MUC6 and MUC5B expression. Endocrine cells expressing chromagranin A and gastrin shifted toward the surface epithelium of the antral glands in H. pylori-infected patients. H. pylori infection and concomitant inflammation induced increased epithelial proliferation and triggered coordinate deregulation of secretory cell populations in the antrum. In particular, infection led to a coordinated increase in cells expressing MUC6 and MUC5B at the expense of MUC5AC-producing cells.     

Dramatic diurnal variation in the concentration of the human trefoil peptide TFF2 in gastric juice

BACKGROUND: TFF2, a member of the trefoil factor family of proteins, is a glycosylated protein of 106 amino acids. It is secreted by gastric antral and pyloric glands and by Brunner's glands of the duodenum. TFF2 is found in high concentrations around sites of ulceration. It stimulates cell motility and is probably the principal cytoprotective trefoil peptide in the stomach. AIMS: To determine if production of TFF2 follows a circadian rhythm and to measure changes in secretion of TFF2 in response to food intake and during sleep. SUBJECTS: Young healthy adults were recruited. They were asymptomatic and were not receiving medication. The 24 hour regimen was designed to allow normal stimulation of gastric secretion in response to food intake and sleep. Gastric juice was collected two hourly via a nasogastric tube. METHODS: Glycosylated and non-glycosylated TFF2 proteins were measured by quantitative western transfer analysis. The results were analysed statistically using SPSS software. RESULTS: There was a dramatic diurnal variation in the concentration of TFF2. The mean concentration was lowest in the early evening (0.29 microg/ml), increased gradually during the evening, and then sharply during the night to reach 7.9 microg/ml. The ratio of glycosylated to non-glycosylated TFF2 varied and was higher during the night than in the afternoon. pH, total protein, and pepsin concentrations in gastric juice did not vary significantly over 24 hours. CONCLUSION: The data suggest that diurnal variations in TFF2 secretion occur independently of pepsin and gastric acid secretion. The concentration of glycosylated TFF2 in the gastric lumen falls in response to food intake. TFF2 secretion increases during inactivity and sleep. These results suggest that secretion of TFF2 in the stomach is highest during the night and that the cytoprotective effects of TFF2 on the gastric mucosa occur mainly during sleep.     

pS2/TFF1 interacts directly with the VWFC cysteine-rich domains of mucins

BACKGROUND & AIMS: Trefoil factors (TFFs) are secreted gastrointestinal proteins that have been shown to protect and promote healing of the gastrointestinal tract. Moreover, pS2/TFF1 is essential for normal differentiation of the gastric mucosa because deficient mice develop antropyloric adenomas. To date, it is unclear how TFFs mediate their functions. METHODS: Using the yeast 2-hybrid system, we attempted to identify murine TFF1 interacting proteins by screening a stomach and duodenum complementary DNA (cDNA) expression library. RESULTS: Four positive clones were isolated. Sequence and expression studies showed that they corresponded to the murine counterpart of human cDNA sequences encoding carboxy-terminal fragments of mMuc2 (489 residues) and mMuc5AC (427, 430, and 894 residues) mucin proteins. Mutagenesis experiments showed that TFF1 interacts with the 2 mucins through binding with their VWFC1 and VWFC2 (von Willebrand factor C) cysteine-rich domains. CONCLUSIONS: These results show that the gastrointestinal protective effect of TFF1, and presumably of the other TFFs, is caused at least partially by their participation, via mucin binding, in the correct organization of the mucous layer that protects the apical side of the mucosa from deleterious luminal agents.     

Epithelial restitution and wound healing in inflammatory bowel disease

Inflammatory bowel disease is characterized by a chronic inflammation of the intestinal mucosa. The mucosal epithelium of the alimentary tract constitutes a key element of the mucosal barrier to a broad spectrum of deleterious substances present within the intestinal lumen including bacterial microorganisms, various dietary factors, gastrointestinal secretory products and drugs. In addition, this mucosal barrier can be disturbed in the course of various intestinal disorders including inflammatory bowel diseases. Fortunately, the integrity of the gastrointestinal surface epithelium is rapidly reestablished even after extensive destruction. Rapid resealing of the epithelial barrier following injuries is accomplished by a process termed epithelial restitution, followed by more delayed mechanisms of epithelial wound healing including increased epithelial cell proliferation and epithelial cell differentiation. Restitution of the intestinal surface epithelium is modulated by a range of highly divergent factors among them a broad spectrum of structurally distinct regulatory peptides, variously described as growth factors or cytokines. Several regulatory peptide factors act from the basolateral site of the epithelial surface and enhance epithelial cell restitution through TGF-13-dependent pathways. In contrast, members of the trefoil factor family （TFF peptides） appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-13-independent mechanism from the apical site of the intestinal epithelium. In addition, a number of other peptide molecules like extracellular matrix factors and blood clotting factors and also non- peptide molecules including phospholipids~ short-chain fatty acids （SCFA）, adenine nucleotides, trace elements and pharmacological agents modulate intestinal epithelial repair mechanisms. Repeated damage and injury of the intestinal surface are key features of various intestinal disorders including inflammatory bowel diseases and require constant repair of the epi     

The role of mucin in GERD and its complications

Acid, pepsin and other noxious material reach the esophageal mucosa and interact with the luminal aspect of the squamous epithelium. The first protective barrier to these potentially injurious substances is the mucus buffer layer that covers the mucosa. In healthy people, the esophagus has a protective surface adherent mucus gel barrier. Levels of mucin glycoprotein are considerably increased in response to acid and pepsin. A wide spectrum of mucin genes are expressed in normal esophageal mucosa, squamous cell carcinoma of the esophagus, Barrett epithelium and esophageal adenocarcinoma. The mucins MUC5AC and MUC6 are expressed to a similar degree in Barrett metaplasia and gastric mucosa, as is MUC2 in Barrett intestinal metaplasia and small bowel mucosa. Increased expression of MUC1 is associated with progression from dysplasia to adenocarcinoma of the esophagus. Thus, mucins have an important role in the defense of esophageal mucosa against the acid, pepsin and bile that are present in the refluxate. Changes in the expression of mucins occur in patients with GERD, and might lead to the development of new drugs.     

Role of mucins in inflammatory bowel disease: important lessons from experimental models

Inflammatory bowel disease (IBD) is characterized by a chronically inflamed mucosa of the gastrointestinal tract, caused by an underlying immune imbalance and triggered by luminal substances, including bacteria. Mucus forms a gel layer covering the gastrointestinal tract, acting as a semi-permeable barrier between the lumen and the epithelium. Mucins, the building blocks of the mucus gel, determine the thickness and properties of mucus. In IBD in humans, alterations in both membrane-bound and secretory mucins have been described involving genetic mutations in mucin genes, changes in mucin mRNA and protein levels, degree of glycosylation, sulphation, and degradation of mucins. As mucins are strategically positioned between the vulnerable mucosa and the bacterial contents of the bowel, changes in mucin structure and/or quantity probably influence their protective functions and therefore constitute possible aetiological factors in the pathogenesis of IBD. This hypothesis, however, is difficult to prove in humans. Animal models for IBD permit detailed analysis of those aspects of mucins necessary for protection against disease. These models revealed pertinent data as for how changes in mucins, in particular in MUC2, imposed by immunological or microbial factors, may contribute to the development and/or perpetuation of chronic IBD, and shed some light on possible strategies to counteract disease.     

Trefoil factor 2 in gland mucous cell mucin in the mucous gel covering normal or damaged gastric mucosa using the Mongolian gerbil model

Objective. Trefoil factor 2 (TFF2) is localized in gastric gland mucous cells. The purpose of the study was to determine whether TFF2 and gastric mucin are localized in mucous cells and in the surface mucous gel layer (SMGL) of the normal gastric mucosa or in the mucoid cap adherent to gastric mucosal lesions in Mongolian gerbils. Material and methods. Gastric mucosal lesions were induced in Mongolian gerbils using oral administration of Helicobacter pylori (H. pylori), subcutaneous administration of indomethacin, or oral administration of 30% ethanol. Tissue samples were fixed in Carnoy's solution for preservation of the SMGL, dehydrated, and embedded in paraffin. Histochemical staining for gastric mucins and immunostaining for TFF2 were performed. Results. It was found that surface mucous cell mucin and gland mucous cell mucin were segregated in the SMGL covering the normal gastric mucosa, and the mucin of the mucoid cap covering the mucosal lesions was primarily gland mucous cell mucin. There was a co-localization of TFF2 in gland mucous cell mucin in gland mucous cells, the SMGL, and the mucoid cap. Conclusions. The co-localization of TFF2 in gland mucous cells and in the adherent mucus suggests a physical interaction between TFF2 and gland mucous cell mucin, and the participation of TFF2 trapped in the adherent mucus functions in mucosal defense, healing, and repair.     

Enhanced expression of mucin 6 glycoprotein in cholangiocarcinoma tissue from patients in Thailand as a prognostic marker for survival

Background and Aim: Cholangiocarcinoma (CCA) is a mucin‐producing cancer that has poor prognosis. Mucin 6 (MUC6) is a mucin that is normally co‐expressed with the trefoil factor family‐2 (TFF2) trefoil peptide. Both MUC6 and TFF2 have been reported to be involved in the progression of many types of cancers. The aim of this study was to determine the expression of MUC6 and TFF2 in CCA tissues and associate these results with clinical data. Methods: MUC6 and TFF2 were detected in CCA tissues by immunohistochemistry. The correlations of MUC6 and TFF2 expressions with clinical data were analyzed. Results: We determined the significant co‐expression of both proteins in serial CCA tissues. The high expressions of MUC6 and TFF2 were demonstrated in 37% and 31% of patients, respectively. The expression levels decreased in the advanced stage of CCA when clinical metastasis was exhibited. The high expression of either protein showed a correlation with prolonged postoperative survival time, but only a high expression of MUC6 is significantly correlated with a 5‐year survival rate. A multivariate Cox regression analysis revealed that a low expression of MUC6, high expression of TFF2, age of patients >56 years, tumor size >5 cm, and poorly‐differentiated histological type were independent, poor prognostic indicators for CCA. Conclusion: MUC6 showed a good correlation with the survival of CCA patients. It may be of value to propose that MUC6 is a good prognostic marker for CCA management.