Esomeprazole and aspirin in Barrett's oesophagus (AspECT): a randomised factorial trial.

BACKGROUND: Oesophageal adenocarcinoma is the sixth most common cause of cancer death worldwide and Barrett's oesophagus is the biggest risk factor. We aimed to evaluate the efficacy of high-dose esomeprazole proton-pump inhibitor (PPI) and aspirin for improving outcomes in patients with Barrett's oesophagus. METHODS: The Aspirin and Esomeprazole Chemoprevention in Barrett's metaplasia Trial had a 2 × 2 factorial design and was done at 84 centres in the UK and one in Canada. Patients with Barrett's oesophagus of 1 cm or more were randomised 1:1:1:1 using a computer-generated schedule held in a central trials unit to receive high-dose (40 mg twice-daily) or low-dose (20 mg once-daily) PPI, with or without aspirin (300 mg per day in the UK, 325 mg per day in Canada) for at least 8 years, in an unblinded manner. Reporting pathologists were masked to treatment allocation. The primary composite endpoint was time to all-cause mortality, oesophageal adenocarcinoma, or high-grade dysplasia, which was analysed with accelerated failure time modelling adjusted for minimisation factors (age, Barrett's oesophagus length, intestinal metaplasia) in all patients in the intention-to-treat population. This trial is registered with EudraCT, number 2004-003836-77. FINDINGS: Between March 10, 2005, and March 1, 2009, 2557 patients were recruited. 705 patients were assigned to low-dose PPI and no aspirin, 704 to high-dose PPI and no aspirin, 571 to low-dose PPI and aspirin, and 577 to high-dose PPI and aspirin. Median follow-up and treatment duration was 8·9 years (IQR 8·2-9·8), and we collected 20 095 follow-up years and 99·9% of planned data. 313 primary events occurred. High-dose PPI (139 events in 1270 patients) was superior to low-dose PPI (174 events in 1265 patients; time ratio [TR] 1·27, 95% CI 1·01-1·58, p=0·038). Aspirin (127 events in 1138 patients) was not significantly better than no aspirin (154 events in 1142 patients; TR 1·24, 0·98-1·57, p=0·068). If patients using non-steroidal anti-inflammatory drugs were censored at the time of first use, aspirin was significantly better than no aspirin (TR 1·29, 1·01-1·66, p=0·043; n=2236). Combining high-dose PPI with aspirin had the strongest effect compared with low-dose PPI without aspirin (TR 1·59, 1·14-2·23, p=0·0068). The numbers needed to treat were 34 for PPI and 43 for aspirin. Only 28 (1%) participants reported study-treatment-related serious adverse events. INTERPRETATION: High-dose PPI and aspirin chemoprevention therapy, especially in combination, significantly and safely improved outcomes in patients with Barrett's oesophagus. FUNDING: Cancer Research UK, AstraZeneca, Wellcome Trust, and Health Technology Assessment.

The correlation between reading and mathematics ability at age twelve has a substantial genetic component.

Dissecting how genetic and environmental influences impact on learning is helpful for maximizing numeracy and literacy. Here we show, using twin and genome-wide analysis, that there is a substantial genetic component to children's ability in reading and mathematics, and estimate that around one half of the observed correlation in these traits is due to shared genetic effects (so-called Generalist Genes). Thus, our results highlight the potential role of the learning environment in contributing to differences in a child's cognitive abilities at age twelve.

Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo.

BACKGROUND: Barrett's oesophagus is a common premalignant lesion caused partly by acid reflux. Although the requisite therapy, proton pump inhibitors (PPIs), have been implicated in the progression of Barrett's oesophagus in animal models, harmful effects of prolonged PPI therapy in Barrett's oesophagus is both inconclusive and controversial. We therefore aimed to test the role of PPI-induced hypergastrinaemia in vitro and see whether any biological parameters were useful surrogates of long-term therapy in man. METHODS: We undertook detailed serological and tissue assessment of gastrin and CCK(2) receptors in 90 patients randomised to different doses of PPI therapy during a detailed 2-year follow-up. We also undertook a comprehensive study of cell models to study the consequential biological effects of gastrin on the mucosa. RESULTS: Gastrin and its cognate receptor CCK(2)R were expressed highest in the stomach, then less in Barrett's oesophagus and least in squamous oesophagus (SqE) (n=20 paired t-test, p<0.01). Analysis of the change in Barrett's oesophagus segment length change in 70 patients who were randomised to high or low PPI dose showed no difference over 2 years (n=70 t-test, p=0.8). Prolonged PPI use did, however, increase the serum gastrin, (36 pg/ml+/-57 pg/ml to 103 pg/ml+/-94 pg/ml (paired t test, p<0.05)). In vitro gastrin also induced changes in OE33(E)(cckr) Barrett's oesophagus cells, but not OE21(E)(cckr) squamous cells, transfected with CCK(2)R; migration was induced by 1 ng/ml of gastrin but proliferation only increased with 100 ng/ml (paired t-test, p<0.01) and both were abolished by antagonists. CONCLUSION: While the short-term effects of gastrin enhance epithelial restitution in Barrett's oesophagus (but not squamous mucosa) there is no clinical evidence that Barrett's oesophagus length expands over time. This study, which is the largest and longest term randomised controlled trial of gastrin biology in Barrett's oesophagus, is further proof of the clinical safety of PPI therapy.

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.

Ectopic expression of P-cadherin correlates with promoter hypomethylation early in colorectal carcinogenesis and enhanced intestinal crypt fission in vivo.

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

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.

Met receptor signaling: a key effector in esophageal adenocarcinoma.

PURPOSE: The incidence of esophageal adenocarcinoma is rising, and survival rates remain poor. The hepatocyte growth factor (HGF) receptor Met has been detected in esophageal cancer. The perturbation of cadherin/catenin complexes has also been shown. We sought to investigate a link among Met expression, cadherin/catenin biology, and cell growth. We assessed the prognostic significance of Met expression in esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Met and HGF expression in esophageal tissues were assessed using immunohistochemistry and ELISA. Met-positive cell lines (OE33 and SEG1) and a Met-negative cell line (TE7) were incubated with HGF. Real-time reverse transcription-PCR and Western blotting were used to assess levels of E-cadherin expression. Nuclear TCF/beta-catenin signaling was assessed following reporter construct transfection. Agar colony formation was used to assess anchorage-independent growth. A panel of 72 resected esophageal adenocarcinomas were assessed for Met expression by immunohistochemistry and correlated to survival data. RESULTS: An increased expression of Met was seen along the metaplasia- adenocarcinoma sequence. Met-positive cells showed reductions in E-cadherin mRNA (37% and 69%) and protein expression following stimulation with HGF (P < 0.01). OE33 and SEG-1 showed up to a 2-fold increase in the levels of beta-catenin nuclear signaling (P < 0.01). TE7 only responded when transfected to express Met; E-cadherin expression decreased by 64% (P < 0.01). HGF stimulation led to increased agar colony formation (P < 0.01). Patients with Met-positive tumors showed lower 6-month survival rates after surgical resection than those with Met-negative tumors (P < 0.05). CONCLUSIONS: Met activation induces changes consistent with early invasion, such as down-regulation of E-cadherin, increased nuclear TCF/beta-catenin signaling, and anchorage-independent growth. This is supported by ex vivo data associating Met with reduced short-term survival. Inhibitors of Met may be effective treatment for esophageal adenocarcinoma.

Mechanisms of disease: from stem cells to colorectal cancer.

Over the past decade, the advances in our understanding of stem cell biology and the role of stem cells in diseases, such as colorectal cancer, have been remarkable. In particular, discoveries related to the control of stem cell proliferation and how dysregulation of proliferation leads to oncogenesis have been foremost. For intestinal stem cells, the WNT family of growth factors, and events such as the regulation of the nuclear localization of beta-catenin, seem to be central to normal homeostasis, and mutations in the components of these pathways seem to lead to the development of colorectal cancer. A paradigm of abnormal stem cell biology is illustrated by patients with familial adenomatous polyposis, who have mutations in the adenomatous polyposis coli gene. The wild-type protein encoded by this gene is important for the prevention of mass beta-catenin accumulation in the nucleus and the subsequent overtranscription of cell cycle proteins. This review discusses the basic mechanisms behind stem cell regulation in the gut and follows their role in the natural history of tumor progression.

Clonal expansion in the human gut: mitochondrial DNA mutations show us the way.

The mechanisms of how DNA mutations are fixed within the human gastrointestinal tract and how they spread are poorly understood and are hotly debated. It has been well documented that human colonic crypts are clonal units; one epithelial stem cell within the crypt becoming dominant and taking over the crypts' entire stem cell population--so called monoclonal conversion. Studies have revealed that crypts can exist as families and develop into patches. The questions have been how do such patches in the human colon develop? Does this have implications on how DNA mutations spread? We have previously shown that mitochondrial DNA (mtDNA) mutations, which result in the deficiency of cytochrome c oxidase, are established within a single colonic crypt stem cell, resulting in a crypt with a mixed phenotype. Over time that mutated stem cell can take over the entire stem cell population resulting in a wholly-mutated crypt. We have furthered this research by showing that entirely cytochrome c oxidase-deficient crypts are able to divide by a process called crypt fission, to form two cytochrome c oxidase-deficient daughter crypts, each sharing the exact parental mtDNA mutation. Furthermore, patches of these crypts also possess a founder mtDNA mutation suggesting that fission repeats itself to form patches, which increase in size with age. Here, we hypothesize that this can be expanded into other areas of the gastrointestinal tract, especially the stomach, where there is a paucity of data regarding clonality and the spread of DNA mutations. We ask if these mutated crypts expand at a different rate to wild type ones. We also discuss the implications for the spread of potential carcinogenic mutations within the gut.

Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.

The understanding of the fixation of mutations within human tissues and their subsequent clonal expansion is a considerable problem, of which little is known. We have previously shown that nononcogenic mutations in the mitochondrial genome occur in one of a number of morphologically normal colonic crypt stem cells, the progeny of which later occupy the whole crypt. We propose that these wholly mutated crypts then clonally expand by crypt fission, where each crypt divides into two mutated daughter crypts. Here we show that (i) mutated crypts in the process of fission share the same mutated mitochondrial genotype not present in neighboring cytochrome c oxidase-positive crypts (the odds of this being a random event are >or=2.48 x 10(9):1); (ii) neighboring mutated crypts have the same genotype, which is different from adjacent cytochrome c oxidase-positive crypts; (iii) mutated crypts are clustered together throughout the colon; and (iv) patches of cytochrome c oxidase-deficient crypts increase in size with age. We thus demonstrate definitively that crypt fission is the mechanism by which mutations spread in the normal human colon. This has important implications for the biology of the normal adult human colon and possibly for the growth and spread of colorectal neoplasms.

Transient P-cadherin expression in radiation proctitis; a model of mucosal injury and repair.

Morphology at both cellular and glandular levels in the colon is dependent to an extent on cell-cell adhesion mediated by cadherin-catenin complexes. Alterations in the expression of E-cadherin, the cadherin normally present in colon, have been shown to be implicated in tissue remodelling within the gastrointestinal tract. Furthermore, it has previously been shown that P-cadherin, normally present only in stratified epithelia and placenta, is expressed in colitis and during neoplastic change in the colon. The morphological features of mucosal injury induced by pre-operative radiotherapy in the non-neoplastic rectal mucosa were studied in patients with rectal adenocarcinoma. Three characteristic phases of radiation proctitis were defined on histological grounds (acute injury, and early and late regenerative phases) essentially correlating with the time interval between radiotherapy and surgery; such features were mirrored by alterations in cadherin-catenin expression and localization in rectal crypts. On immunohistochemistry and western blotting, P-cadherin was highly expressed in the acute injury and early regenerative phases, with a decreased level of expression during late regeneration. E-cadherin and associated catenins were translocated from membrane to cytoplasm in degenerating crypts, with return of normal membranous expression in regenerating crypts. In conclusion, radiation-induced proctitis represents an in vivo model of mucosal injury and regeneration and provides a valid model in which to study events during epithelial injury and repair. Altered cadherin expression, in particular transient aberrant P-cadherin expression, is intimately associated with these processes.