Rebecca Harrison

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.

Advancing donor liver age and rapid fibrosis progression following transplantation for hepatitis C

Background and aims: Cirrhosis with liver failure due to hepatitis C virus (HCV) infection is the most common indication for liver transplantation (LT). Reinfection of the transplanted liver by HCV is inevitable, and aggressive hepatitis with accelerated progression to graft cirrhosis may be observed. Of concern, recent reports suggest that the outcome of LT for HCV may have deteriorated in recent years. Determinants of rate of progression to cirrhosis in the immunocompetent non-transplant patient are well defined, and the most powerful determinant is patient age at the time of infection. Following LT for HCV, recipient age does not affect outcome of HCV reinfection. However, the impact of donor age on graft fibrosis progression rate following LT has not been examined. Methods: We have examined post-transplant biopsies to assess histological activity, including fibrosis stage (scored 0–6 units, 6 representing established cirrhosis), and to calculate fibrosis progression rates in 101 post-transplant specimens from 56 HCV infected LT patients. Univariate and multivariate analyses examined the impact of parameters including recipient and donor age and sex on fibrosis progression rate, and on predicted time to cirrhosis. Results: For the cohort, median fibrosis progression rate was 0.78 units/year, and median interval from transplantation to development of cirrhosis was 7.7 years. In multivariate analysis, donor age (not recipient age) was a powerful determinant (p=0.02) of fibrosis progression rate. When the liver donor was younger than 40 years, median progression rate was 0.6 units/year and interval to cirrhosis was 10 years. When the donor was aged 50 years or more, median progression rate was 2.7 units/year and interval to cirrhosis only 2.2 years. During the observation period there has been a significant increase in donor age (p=0.01) but date of transplantation per se is not a determinant of progression rate when included in multivariate analyses. Conclusions: Donor age has a major influence on graft outcome following transplantation for HCV. The changing organ donor profile will affect the long term results of LT for HCV. These observations have important implications for donor liver allocation.

Consensus Statements for Management of Barrett's Dysplasia and Early-Stage Esophageal Adenocarcinoma, Based on a Delphi Process

BACKGROUND & AIMS Esophageal adenocarcinoma (EA) is increasingly common among patients with Barretts esophagus (BE). We aimed to provide consensus recommendations based on the medical literature that clinicians could use to manage patients with BE and low-grade dysplasia, high-grade dysplasia (HGD), or early-stage EA. METHODS We performed an international, multidisciplinary, systematic, evidence-based review of different management strategies for patients with BE and dysplasia or early-stage EA. We used a Delphi process to develop consensus statements. The results of literature searches were screened using a unique, interactive, Web-based data-sifting platform; we used 11,904 papers to inform the choice of statements selected. An a priori threshold of 80% agreement was used to establish consensus for each statement. RESULTS Eighty-one of the 91 statements achieved consensus despite generally low quality of evidence, including 8 clinical statements: (1) specimens from endoscopic resection are better than biopsies for staging lesions, (2) it is important to carefully map the size of the dysplastic areas, (3) patients that receive ablative or surgical therapy require endoscopic follow-up, (4) high-resolution endoscopy is necessary for accurate diagnosis, (5) endoscopic therapy for HGD is preferred to surveillance, (6) endoscopic therapy for HGD is preferred to surgery, (7) the combination of endoscopic resection and radiofrequency ablation is the most effective therapy, and (8) after endoscopic removal of lesions from patients with HGD, all areas of BE should be ablated. CONCLUSIONS We developed a data-sifting platform and used the Delphi process to create evidence-based consensus statements for the management of patients with BE and early-stage EA. This approach identified important clinical features of the diseases and areas for future studies.

Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells.

BACKGROUND & AIMS How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. METHODS Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. RESULTS We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell-derived unit-monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. CONCLUSIONS These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops.

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.

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus

Objectives: Current models of clonal expansion in human Barrett’s oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett’s segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett’s metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. Methods: Individual crypts across Barrett’s biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. Results: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett’s dysplasia. Conclusions: By studying clonality at the crypt level we demonstrate that Barrett’s heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett’s metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands.

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.

Clonality, Founder Mutations, and Field Cancerization in Human Ulcerative Colitis–Associated Neoplasia

BACKGROUND & AIMS The clonality of colitis-associated neoplasia has not been fully determined. One previous report showed polyclonal origins with subsequent monoclonal outgrowth. We aimed to assess the clonality and mutation burden of individual crypts in colitis-associated neoplasias to try to identify gatekeeping founder mutations, and explore the clonality of synchronous lesions to look for field effects. METHODS Individual crypts (range, 8-21 crypts) were microdissected from across 17 lesions from 10 patients. Individual crypt adenomatous polyposis coli (APC), p53, K-RAS, and 17p loss of heterozygosity mutation burden was established using polymerase chain reaction and sequencing analysis. Serial sections underwent immunostaining for p53, beta-catenin, and image cytometry to detect aneuploidy. RESULTS In most lesions an oncogenic mutation could be identified in all crypts across the lesion showing monoclonality. This founder mutation was a p53 lesion in the majority of neoplasms but 4 tumors had an initiating K-RAS mutation. Some nondysplastic crypts surrounding areas of dysplasia were found to contain clonal p53 mutations and in one case 3 clonal tumors arose from a patch of nondysplastic crypts containing a K-RAS mutation. CONCLUSIONS This study used mutation burden analysis of individual crypts across colitis-associated neoplasms to show lesion monoclonality. This study confirmed p53 mutation as initiating mutation in the majority of lesions, but also identified K-RAS activation as an alternative gatekeeping mutation. Local and segmental field cancerization was found by showing pro-oncogenic mutations in nondysplastic crypts surrounding neoplasms, although field changes are unlikely to involve the entire colon because widely separated tumors were genetically distinct.

A Phase II Study of Gefitinib Monotherapy in Advanced Esophageal Adenocarcinoma: Evidence of Gene Expression, Cellular, and Clinical Response

Purpose: At presentation, most cases of adenocarcinoma of the esophagus (ACE) are inoperable. Although chemotherapy can prolong survival, patients eventually die as a result of refractory disease. Epidermal growth factor receptor (EGFR) is almost universally expressed in ACE and is a negative prognostic factor. Experimental Design: This open-label, two-center, noncomparative, two-part phase II trial assessed the EGFR tyrosine kinase inhibitor gefitinib (500 mg/d) in patients with advanced, inoperable ACE. The primary end point was tumor response. The effect of EGFR inhibition was also evaluated by gene expression analysis of tumor biopsies taken before gefitinib treatment and 28 days after. Results: Twenty-seven patients were recruited and evaluable for tumor response and safety. Three patients had a partial response and seven had stable disease, giving a disease control rate (partial response + stable disease) of 37%. Drug-related adverse events were generally mild: diarrhea in 19 (grade 3 in three) and rash in 19 (grade 3 in five) patients, and there were no grade 4 drug-related adverse events. Microarray experiments on tumor biopsies showed that gefitinib also down-regulated oncogenes associated with tumor progression. Ki67 (a marker of tumor growth) expression decreased in five of seven biopsies taken before and after treatment. Conclusion: Gefitinib (500 mg/d) is an active and generally well-tolerated treatment for ACE. Studies on endoscopic biopsies are feasible and indicate that gefitinib inhibits both gene expression and cellular biology at 500 mg/d, and these may provide surrogate end points for predictive biomarkers. Further trials of gefitinib are warranted, particularly as patient response seems to be durable and current second-line chemotherapy options have no proven ability to prolong life.