Ian Perry

Barrett's metaplasia

The rate of oesophageal adenocarcinoma is increasing in the western world and has a poor prognosis mainly because individuals present at a late stage. Attempts to intervene at an early stage of tumour progression have not proven cost effective, although lesions identified during surveillance programmes have a better prognosis. As a consequence, there has been renewed interest in strategies that might prevent the precursor lesion Barretts oesophagus. Furthermore, there is an improved understanding of genetic and environmental interactions necessary for the clonal expansion and propagation of metaplastic premalignant lesions. Clearly, three mechanisms promote cancer progression--inheritance of germ-line mutations or polymorphisms, sporadic mutagenesis, and local epigenetic alterations. Locally produced cytokines and bile acids in the refluxate create a microenvironment that sets the scene for metaplastic transformation of the oesophageal epithelium, mainly by directly affecting metaplastic stem cells.

Detection of Intestinal Metaplasia in Barrett's Esophagus: An Observational Comparator Study Suggests the Need for a Minimum of Eight Biopsies

OBJECTIVES:Intestinal metaplasia (IM) and dysplasia in Barretts esophagus are recognized surrogates for esophageal adenocarcinoma risk. While few would argue with the “hunt for dysplasia,” there is a divide regarding the usefulness of the histological confirmation of intestinal metaplasia in endoscopically apparent long segment Barretts esophagus. We aimed to assess the frequency of intestinal metaplasia in 125 consecutive patients with columnar-lined esophagus and to determine the optimal biopsy protocol to detect intestinal metaplasia.METHODS:Two-hundred ninety-six endoscopies were performed over a 4-yr period in Barretts esophagus segments of mean length 4 cm (range 1–11 cm) at a single center and the resulting biopsies were analyzed retrospectively. Biopsies were all processed with routine hematoxylin and eosin (H&E) staining, and a subset (N = 92) was subject to alcian blue/periodic-acid Schiff staining.RESULTS:Using H&E staining, we found that the optimum number of biopsies to diagnose intestinal metaplasia was 8 per endoscopy, mean 67.9% endoscopies having intestinal metaplasia. In contrast, if only four were taken the yield was 34.7% with intestinal metaplasia. Unless more than 16 biopsies were taken (100% yield of intestinal metaplasia), no additional significant detection was achieved. Using additional alcian blue/periodic-acid Schiff staining only had a marginal benefit, with 5.4% of new cases of intestinal metaplasia being identified. There is a proximal cephalo-caudal gradient of intestinal metaplasia, especially with increased chronological age, but doing repeat endoscopies on patients did not increase the detection of intestinal metaplasia.CONCLUSIONS:The data suggest that at least 8 random biopsies is the minimum to be taken and analyzed with conventional H&E staining to diagnose benign intestinal metaplasia. Taking more biopsies did not statistically increase the diagnosis of intestinal metaplasia except when greater than 16 were taken when 100% yield was obtained.

Tumour necrosis factor-α in Barrett's oesophagus: a potential novel mechanism of action

Barretts metaplasia (BM) is an early lesion in the progression from oesophageal inflammation through dysplasia to the development of Barretts adenocarcinoma (BA). Previous work indicates that BM and BA are associated with reduced E-cadherin expression and increased cytoplasmic/nuclear pools of its associated protein β-catenin. β-catenin participates in Wnt signalling and activates oncogene transcription by complexing with T-cells factors (TCF). One such oncogene is c-myc. We have previously shown that TNF-α can down-regulate E-cadherin expression. Here, we assess TNF-α expression in Barretts metaplasia and examine if TNF-α can promote β-catenin mediated transcription of oncogenes in a gastrointestinal model system. Employing immunohistochemistry and Western blot analysis of oesophageal tissue, epithelial expression of TNF-α increases with progression along the metaplasia–dysplasia–carcinoma sequence (P<0.001). β-catenin mediated transcription was then assessed in TNF-α stimulated cell lines using the TOPFLASH reporter system whilst c-myc expression was assessed by real time PCR. In a columnar intestinal cell model, TNF-α induces c-myc expression which is induced via β-catenin mediated transcription (P<0.05). This β-catenin mediated transcription is independent of NF-κB activation. Thus, TNF-α is up-regulated in the progression of Barretts oesophagus and β-catenin mediated transcription of c-myc is a novel pathway whereby elevated levels of TNF-α may lead to oncogene transcription and altered biology in gastrointestinal epithelia and metaplasia.

Upregulation of the oncogene c-myc in Barrett’s adenocarcinoma: induction of c-myc by acidified bile acid in vitro

Background and aims: C-myc over expression is implicated in malignancy although to date this has not been studied in Barrett’s metaplasia. We sought to determine c-myc expression in the malignant progression of Barrett’s metaplasia and whether it may be induced by bile acids seen in gastro-oesophageal refluxate. Methods: C-myc protein and mRNA levels were assessed in 20 Barrett’s metaplasia and 20 oesophageal adenocarcinoma samples by western blotting and real time polymerase chain reaction. Levels of c-myc and proliferation were also assessed in cell lines OE21, OE33, SW-480, and TE-7 stimulated with pulses or continuous exposure to the bile acids deoxycholic acid and chenodeoxycholic acid. Results: C-myc protein was upregulated in 50% of Barrett’s metaplasia and 90% of oesophageal adenocarcinoma samples compared with squamous, gastric, and duodenal controls. C-myc immunolocalisation in Barrett’s metaplasia revealed discrete nuclear localisation, becoming more diffuse with progression from low to high grade dysplasia to adenocarcinoma. Both continual and pulsed bile acid induced c-myc at pH 4, with no effect at pH 7 or with acidified media alone. Pulsed bile acid treatment induced proliferation (p<0.05); in contrast, continuous exposure led to suppression of proliferation (p<0.05). Conclusions: We have shown upregulation of c-myc with malignant progression of Barrett’s metaplasia and suggest that acidified bile may be a novel agent responsible for induction of this oncogene.

Gastro-oesophageal cancer: death at the junction

The death rates from cancers of the oesophagus and gastro-oesophageal junction, adjusted for age, have risen steadily since the early 1970s (from 3 to 6 per 100 000 and from 1.5 to 3 per 100 000 population in the United Kingdom respectively).1 These figures are comparable to those in northern Europe and the United States. The incidence of Barretts adenocarcinoma in the United States has increased from 0.3 per 100 000 to 2.3 per 100 000 over the past three decades. Despite improvements in multimodality therapy, especially chemotherapy regimens of combined epirubicin, cisplatin, and fluorouracil combined with surgery, survival has not improved significantly, suggesting that alternative strategies for identifying and treating these conditions are needed. The incidence of intestinal metaplasia of both the oesophagus (Barretts oesophagus) and the gastro-oesophageal junction are also increasing. This metaplastic tissue is believed to have a premalignant potential, and Barretts oesophagus is related to bile and acid reflux disease.2 About 8% of patients undergoing routine endoscopy and 3% of …

Aberrant P-cadherin expression is a feature of clonal expansion in the gastrointestinal tract associated with repair and neoplasia.

The recognition of key roles for cadherins in the determination of epithelial cell phenotype, migration, differentiation, and tumour dissemination have stimulated much interest in this family of adhesion molecules. In the gastrointestinal tract, alteration of the expression of classical cadherins with aberrant P‐cadherin up‐regulation, associated with co‐expression or loss of E‐cadherin expression, is seen in neoplastic transformation of oral and oesophageal squamous mucosa and in lesions representing early neoplastic transformation of glandular mucosa, such as aberrant crypt foci and metaplastic and adenomatous polyps. This same phenotype is seen in enterocytes adjacent to foci of ulceration in the intestine in colitis, including inflammatory bowel disease, and in colitis‐associated dysplasia. In coeliac disease, reversible E‐cadherin down‐regulation correlates with the degree of villous atrophy, but in contrast with colitis, aberrant P‐cadherin expression is not a feature. Aberrant epithelial P‐cadherin expression is thus associated with a proliferative phenotype related to ulceration and neoplastic transformation in the gastrointestinal tract, which may confer a survival advantage on these cells, but the relative functional roles of P‐cadherin and E‐cadherin and the molecular mechanisms underlyingP‐cadherin/catenin interactions have yet to be elucidated. Copyright

Inflammation promotes Barrett's metaplasia and cancer: a unique role for TNFalpha.

Immune responses in the gastrointestinal tract directed against gut-derived pathogens are crucial in Ž . maintaining mucosal integrity Daig et al., 2000 . In most circumstances this process is self-limiting and characterized by an acute inflammatory response involving cytokine release and the recruitment of Ž . inflammatory cells Jung et al., 1995 . Clearance of the pathogen is subsequently followed by stem cell proliferation, mucosal remodelling and eventually Ž . healing McKay and Perdue, 1993 . Deregulation of these processes with aberrant cytokine production Ž . such as tumour necrosis factor TNF or persistence of pathogens such as Helicobacter pylori leads to chronic inflammation and in some circumstances may initiate a metaplasia dysplasia carcinoma Ž . sequence Prach et al., 1997; Jankowski et al., 1999 . One process that appears to be of key importance in the carcinogenesis of epithelial neoplasia is the Ž development of metaplasia Jankowski et al., 1999; . Spechler, 1999; Voutilainen et al., 1999 . In Barrett’s oesophagus, a premalignant lesion of the oesophagus, the native non-stratified squamous epithelium is replaced by a mucin-secreting columnar-lined intestinal-type epithelium, which is thought to be more resistant to continued duodeno-gastro-oesophageal Ž . reflux Jankowski et al., 2000 . A similar association between chronic inflammation and metaplasia is seen throughout the gut and includes Helicobacter pyloriassociated chronic atrophic gastritis with intestinal Ž . metaplasia Sung et al., 2000 , colonic metaplasia Ž . within ileal pouches Schouten, 1998 , and ulcerative Ž . colitis with metaplastic polyps Higaki et al., 1999 . These changes in epithelial morphology or metaplasia are hypothesized to reflect a phenotypic change at the level of the pluripotent stem cells in the mucosal crypts in response to stimulation with inŽ flammatory cytokines and growth factors Jankowski . Ž et al., 1999 . The biology of these stem cells Jankow. ski and Wright, 1992 and the mechanisms by which these changes are mediated has been an area of intense research with only limited data available, Ž implicating cytokines such as TNF Jankowski et . Ž . Ž al., 2000 , interleukin 1 IL-1 El Omar et al., . Ž . 2000 and transforming growth factor TGF Ž . Jankowski, 1993 . Epidemiological data have demonstrated a strong association between chronic inflammation with metaplasia such as Barrett’s oesophagus and Helicobacter pylori-associated atrophic gastritis and subŽ sequent progression to neoplasia Sipponen and Hy. varinen 1993; Prach et al., 1997 Barrett’s adenocarcinoma of the oesophagus has seen a rapid increase in incidence in the western world and is characterized by a uniformly poor prognosis, with a median survival time, following diagnosis, of less than one Ž . Ž year Powell and McConkey, 1992 . Chronic 20 . years , symptomatic gastro-oesophageal reflux and Barrett’s metaplasia are generally regarded to be the most important risk factors for developing

Barrett’s Esophagus: Disregulation of Cell Cycling and Intercellular Adhesion in the Metaplasia-Dysplasia-Carcinoma Sequence

The incidence of both esophageal adenocarcinoma and Barrett’s esophagus, a premalignant condition predisposing to this cancer, is rising rapidly. There is growing evidence that both of these conditions are related to the reflux of acid and bile into the esophagus. This results in inflammation and cell damage which initiates a sequence of events termed the metaplasia-dysplasia-carcinoma sequence in which the squamous epithelium is replaced by columnar epithelium exhibiting increasing degrees of dysplasia and overt malignancy. This sequence of events is underpinned by changes in cell cycling, such as accumulation of p16 and p53 mutations and increased cyclin D1 activity. Progression along this pathway is characterized by changes in intercellular adhesion, in particular, loss of adenomatous polyposis coli, reduced cadherin expression and increased catenin phosphorylation resulting in its nuclear translocation. Herein, we detail these molecular defects and propose how they may interrelate in an ordered progression in the development of esophageal adenocarcinoma.

Barrett's mucosa: remodelling by the microenvironment

Barretts metaplasia and the associated adenocarcinoma are composed not only of epithelial cells, but also of inflammatory cells and endothelial cells in the lamina propria and around the tumour, respectively. The early incidence of vascular invasion and metastasis is a feature of Barretts adenocarcinomas. A paper in this issue of The Journal of Pathology shows that vascular endothelial growth factor (VEGF) is expressed in both metaplastic cells and endothelial cells of pre‐neoplastic Barretts epithelium early in the development of neoplasia. It is becoming clearer that the harmful insults of acid and bile reflux alter not only the epithelium, but also the lamina propria. Cytokines such as tumour necrosis factor alpha (TNFα) or transforming growth factor (TGFα) are implicated in the formation of early Barretts adenocarcinomas. From current knowledge it is possible to hypothesize that metaplastic cells, perhaps as a consequence of either TNFα or TGFα stimulation, secrete VEGF. VEGF can promote adjacent endothelial cell growth through phosphorylation of β‐catenin and vascular endothelial cadherin (VE‐cadherin) in endothelial cells. In this de novo microenvironment, angiogenesis is therefore accelerated, enhancing the chance of microvascular invasion. Copyright

Cadherin adhesion in the intestinal crypt regulates morphogenesis, mitogenesis, motogenesis, and metaplasia formation.

The topographical organisation of the epithelium lining mucous membranes has been an intense point of research. One of the fundamental biological issues underpinning this and associated issues relates to the role and regulation of epithelial adhesion molecules. Adhesion between individual cells allows an intact layer to be formed, which is selectively permeable. In addition, the orchestrated regulation of multiple adhesion molecules allows the gradual transition from basal secretory cells to apical absorptive cells in the crypt-villus gradient. Moreover, it is becoming clear that no one class of adhesion molecule can sufficiently govern crypt architecture; however, the main cell-cell adhesion molecules are the cadherins and the related desmosomal cadherins. These latter molecules interact with the catenins, which bind directly or indirectly with cytoskeletal molecules such as Rho and Rac. In addition, other complex glycoproteins, such as the carcinoembryonic antigens, might contribute to adhesion, although their mechanisms of function are distinctly different. Integrins on the basal aspect of the cells also signal important morphoregulatory signals as a result of their binding to the extracellular maxtrix. The disruption of these physiological processes also provides a necessary and, in some cases, sufficient molecular mechanism for cancer invasion and metastasis, such as occurs in E-cadherin mutation positive familial gastric cancer.