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This file contains supplementary methods and figures describing tumour severity scoring and tumour area measurements in all three tumour model. Additionally, histological representation of cleaved caspase 3 and BrdU staining and western blots showing activation of MAPK and PI3K signalling pathways in tumour models.

The objective of tailoring medicines for cancer patients according to the molecular profile of their disease holds great promise for the improvement of cancer therapy. Nevertheless, this approach has been limited, in part, due to the lack of predictive and informative preclinical studies. Herein, we describe an assessment of the therapeutic potential of targeting PI3K/mTOR and MAPK signaling in genetically defined mouse models of colorectal cancer mirroring disease subtypes targeted for novel therapy in the FOCUS4 trial. Our studies demonstrate that dual PI3K/mTOR inhibition is highly effective in invasive adenocarcinoma models characterized by combinatorial mutations in Apc and Pten; Apc and Kras; and Apc, Pten and Kras. MEK inhibition was effective in the combinatorial Apc and Kras setting, but had no impact in either Apc Pten mutants or in Apc Pten Kras triple mutants. Furthermore, we describe the importance of scheduling for combination studies and show that although no additional benefit is gained in Apc Pten mice, combination of PI3K/mTOR and MAPK inhibition leads to an additive benefit in survival in Apc Kras mice and a synergistic increase in survival in Apc Pten Kras mice. This is the first study using robust colorectal cancer genetically engineered mouse models to support the validity of PI3K/mTOR and MEK inhibitors as tailored therapies for colorectal cancer and highlight the potential importance of drug scheduling in the clinic. Mol Cancer Ther; 14(10); 2175–86. ©2015 AACR.

Pre- and Post-validation protein altering somatic variants identified by whole exome sequencing.

Purpose: Duodenal polyposis and cancer are important causes of morbidity and mortality in familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). This study aimed to comprehensively characterize somatic genetic changes in FAP and MAP duodenal adenomas to better understand duodenal tumorigenesis in these disorders.Experimental Design: Sixty-nine adenomas were biopsied during endoscopy in 16 FAP and 10 MAP patients with duodenal polyposis. Ten FAP and 10 MAP adenomas and matched blood DNA samples were exome sequenced, 42 further adenomas underwent targeted sequencing, and 47 were studied by array comparative genomic hybridization. Findings in FAP and MAP duodenal adenomas were compared with each other and to the reported mutational landscape in FAP and MAP colorectal adenomas.Results: MAP duodenal adenomas had significantly more protein-changing somatic mutations (P = 0.018), truncating mutations (P = 0.006), and copy number variants (P = 0.005) than FAP duodenal adenomas, even though MAP patients had lower Spigelman stage duodenal polyposis. Fifteen genes were significantly recurrently mutated. Targeted sequencing of APC, KRAS, PTCHD2, and PLCL1 identified further mutations in each of these genes in additional duodenal adenomas. In contrast to MAP and FAP colorectal adenomas, neither exome nor targeted sequencing identified WTX mutations (P = 0.0017).Conclusions: The mutational landscapes in FAP and MAP duodenal adenomas overlapped with, but had significant differences to those reported in colorectal adenomas. The significantly higher burden of somatic mutations in MAP than FAP duodenal adenomas despite lower Spigelman stage disease could increase cancer risk in the context of apparently less severe benign disease. Clin Cancer Res; 23(21); 6721–32. ©2017 AACR.

Summary of the 62 recurrently mutated genes in the exome dataset (supplementary table 2) and the number of mutations identified in each gene. Total numbers of variants in each gene take into account any mutations identified in duplicate adenomas.

Database of somatic APC mutations reported in FAP and MAP duodenal and colorectal adenomas. NA, Not available. NI, Not identified

Histogram showing number of validated protein changing somatic variants identified per adenoma.

Supplementary methods

Summary of all validated variants identified by exome sequencing. Patterned shading indicates duplicate adenomas.

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Normal homeostasis of adult intestinal epithelium and repair following tissue damage is maintained by a balance of stem and differentiated cells, many of which are still only poorly characterised. Enteroendocrine cells of the gut are a small population of differentiated, secretory cells that are critical for integrating nutrient sensing with metabolic responses, dispersed amongst other epithelial cells. Recent evidence suggests that sub-sets of secretory enteroendocrine cells can act as reserve stem cells. Given the link between cells with stem-like properties and cancer, it is important that we identify factors that might provide a bridge between the two. Here, we identify a sub-set of chromogranin A-positive enteroendocrine cells that are positive for the developmental and cancer-associated transcription factor Brachyury in normal human small intestinal and colonic crypts. Whilst chromogranin A-positive enteroendocrine cells are also Brachyury-positive in colorectal tumours, expression of Brachyury becomes more diffuse in these samples, suggesting a more widespread function in cancer. The finding of the developmental transcription factor Brachyury in normal adult human intestinal crypts may extend the functional complexity of enteroendocrine cells and serves as a platform for assessment of the molecular processes of intestinal homeostasis that underpins our understanding of human health, cancer and aging., The authors would like to thank the following organisations for supporting this work: J. Jezkova, R.J. McFarlane & J.A. Wakeman are supported by Cancer Research Wales; J.S. Williams is supported by Coleg Cymraeg Cenedlaethol; J. Sammut and R.J. McFarlane are supported by NWCR (grant no. CR950); S. Gollins is supported by National Institute for Social Care and Health Research Academic Health Science Collaboration (NISCHR AHSC) Clinical Research Fellow. F.Pinto is supported by Fundação para a Ciência e Tecnologia (FCT; grant no. SFRH/BD/81369/2011). R.M. Reis is recipient of a Brazilian National Counsel of Technological and Scientific Development (CNPq) scholarship. Geraint Williams is supported by the Wales Gene Park. are supported by NWCR (grant no. CR950); S. Gollins is supported by National Institute for Social Care and Health Research Academic Health Science Collaboration (NISCHR AHSC) Clinical Research Fellow. F.Pinto is supported by Fundação para a Ciência e Tecnologia (FCT; grant no. SFRH/BD/81369/2011). R.M. Reis is recipient of a Brazilian National Counsel of Technological and Scientific Development (CNPq) scholarship. Geraint Williams is supported by the Wales Gene Park., info:eu-repo/semantics/publishedVersion

Familial adenomatous polyposis (FAP) and MUTYH‐associated polyposis (MAP) are inherited disorders associated with multiple colorectal adenomas that lead to a very high risk of colorectal cancer. The somatic mutations that drive adenoma development in these conditions have not been investigated comprehensively. In this study we performed analysis of paired colorectal adenoma and normal tissue DNA from individuals with FAP or MAP, sequencing 14 adenoma whole exomes (eight MAP, six FAP), 55 adenoma targeted exomes (33 MAP, 22 FAP) and germline DNA from each patient, and a further 63 adenomas by capillary sequencing (41 FAP, 22 MAP). With these data we examined the profile of mutated genes, the mutational signatures and the somatic mutation rates, observing significant diversity in the constellations of mutated driver genes in different adenomas, and loss‐of‐function mutations in WTX (9%; p

The objective of tailoring medicines for cancer patients according to the molecular profile of their disease holds great promise for the improvement of cancer therapy. Nevertheless, this approach has been limited, in part, due to the lack of predictive and informative preclinical studies. Herein, we describe an assessment of the therapeutic potential of targeting PI3K/mTOR and MAPK signaling in genetically defined mouse models of colorectal cancer mirroring disease subtypes targeted for novel therapy in the FOCUS4 trial. Our studies demonstrate that dual PI3K/mTOR inhibition is highly effective in invasive adenocarcinoma models characterized by combinatorial mutations in Apc and Pten; Apc and Kras; and Apc, Pten and Kras. MEK inhibition was effective in the combinatorial Apc and Kras setting, but had no impact in either Apc Pten mutants or in Apc Pten Kras triple mutants. Furthermore, we describe the importance of scheduling for combination studies and show that although no additional benefit is gained in Apc Pten mice, combination of PI3K/mTOR and MAPK inhibition leads to an additive benefit in survival in Apc Kras mice and a synergistic increase in survival in Apc Pten Kras mice. This is the first study using robust colorectal cancer genetically engineered mouse models to support the validity of PI3K/mTOR and MEK inhibitors as tailored therapies for colorectal cancer and highlight the potential importance of drug scheduling in the clinic. Mol Cancer Ther; 14(10); 2175–86. ©2015 AACR.

Background and Aims\ud \ud Duodenal polyposis and cancer have become a key issue for patients with familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). Almost all patients with FAP will develop duodenal adenomas, with 5% developing cancer. The incidence of duodenal adenomas in MAP appears to be lower than in FAP but the limited available data suggest a comparable increase in the relative risk and lifetime risk of duodenal cancer. Current surveillance recommendations, however, are the same for FAP and MAP, using the Spigelman score--incorporating polyp number, size, dysplasia, and histology--for risk stratification and determination of surveillance intervals. Previous studies have demonstrated a benefit of enhanced detection rates of adenomas by use of chromoendoscopy both in sporadic colorectal disease and in groups at high risk of colorectal cancer. We aimed to assess the effect of chromoendoscopy on duodenal adenoma detection, to determine the impact on Spigelman stage and to compare this in individuals with known pathogenic mutations in order to determine the difference in duodenal involvement between MAP and FAP.\ud \ud \ud Methods\ud \ud A prospective study examined the impact of chromoendoscopy on the assessment of the duodenum in 51 consecutive patients with MAP and FAP in 2 academic centers in the United Kingdom (University Hospital Llandough, Cardiff and St Mark's Hospital, London) from 2011 to 2014.\ud \ud \ud Results\ud \ud Enhanced adenoma detection of 3 times the number of adenomas after chromoendoscopy was demonstrated in both MAP (p=0.013) and FAP (p=0.002), but did not affect adenoma size. In both conditions, there was a significant increase in Spigelman stage after chromoendoscopy compared with endoscopy without dye spray. Spigelman scores and overall adenoma detection was significantly lower in MAP compared with FAP.\ud \ud \ud Conclusions\ud \ud Chromoendoscopy improved the diagnostic yield of adenomas in MAP and FAP 3-fold, and in both MAP and FAP this resulted in a clinically significant upstaging in Spigelman score. Further studies are required to determine the impact of improved adenoma detection on the management and outcome of duodenal polyposis.

Carcinomas of the biliary tract are aggressive malignancies in humans. Loss of the tumour suppressor PTEN has previously been associated with cholangiocarcinoma development in a murine model. Activation of KRAS is reported in up to one-third of human cholangiocarcinomas and 50% of gall bladder carcinomas. In this study we aimed to test the potential interaction between PTEN and KRAS mutation in biliary tract malignancy. We used an inducible Cre–LoxP-based approach to coordinately delete PTEN and activate KRAS within the adult mouse biliary epithelium. We found that activation of KRAS alone has little effect upon biliary epithelium. Loss of PTEN alone results in the development of low-grade neoplastic lesions, following long latency and at low incidence. Combination of both mutations causes rapid development of biliary epithelial proliferative lesions, which progress through dysplasia to invasive carcinoma. We conclude that activation of the PI3′K pathway following loss of PTEN is sufficient to drive slow development of low-grade biliary lesions in mice. In contrast, mutational activation of KRAS does not result in a similar phenotype, despite a prediction that this should activate both the RAF–MEK–ERK and PI3′-kinase pathways. However, mutation of both genes results in rapid tumourigenesis, arguing that PTEN normally functions as a ‘brake’ on the PI3′-kinase pathway, limiting the influence of KRAS activation. Mutation of both genes creates a ‘permissive’ environment, allowing the full effects of both mutations to be manifested. These data reveal an in vivo synergy between these mutations and provides a new mouse model of biliary tract malignancy

OBJECTIVES: To determine the differences between in-situ, prefixation and postfixation colorectal polyp measurements, their clinical impact upon determining adenoma surveillance intervals, and to compare postfixation measurements using three different devices. PATIENTS AND METHODS: A prospective study of 107 colorectal polyps resected from 65 consecutive patients (45 men, 20 women) undergoing colonoscopy as part of the Bowel Cancer Screening Programme was undertaken. The polyps were measured in situ, prefixation (study gold standard) and using three measurement devices (ruler, callipers and magnifying lens) postfixation in formalin. RESULTS: Prefixation ruler measurements were significantly higher than in-situ (P=0.02) and postfixation ruler measurements (P=0.04). No significant difference was observed between in-situ and postfixation ruler measurements (P=0.36), although in-situ measurements were more variable. In-situ measurements also generated more variation in surveillance intervals than postfixation measurements (9.3 vs. 5.6%). No significant difference was seen between measurements obtained by the three different devices postfixation (P=0.89). CONCLUSION: This study provides evidence supporting the use of postfixation polyp size measurements as advised by recent European pathology colorectal cancer screening recommendations. In the absence of a clinically significant difference between measurement devices, we advise the ruler to be used as a standard for postfixation measurements because of its widespread availability.

To assess the interobserver agreement in the reporting of colorectal polyps among histopathologists participating in the Welsh Bowel Cancer Screening (BCS) programme.Twelve benign polyps representative of BCS cases were identified from pathology files and reported by 28 BCS histopathologists using proforma sheets. The level of agreement between the participants and a gold standard was determined using kappa (κ) statistics. A moderate level of agreement was achieved in the reporting of polyp type [κ = 0.45; 95% confidence interval (CI) 0.34-0.59] and adenomatous lesions were distinguished from non-adenomatous lesions in 96% of cases. Substantial agreement was obtained in distinguishing low- and high-grade dysplasias (κ = 0.67; 95% CI 0.50-0.86), but there was only fair agreement in reporting excision margin status (κ = 0.24; 95% CI 0.07-0.43) with frequent use of the 'uncertain' category. Significant issues included categorizing serrated lesions, recognizing focal high-grade dysplasia and epithelial misplacement, and apparent overdiagnosis of villous change in adenomas.Interobserver variability in some aspects of reporting colorectal polyps by BCS pathologists is suboptimal, with a potential impact upon patient management and the efficient running of the screening service. Approaches to addressing this are discussed.

Background & Aims: Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. Methods: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. Results: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G2/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. Conclusions: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC.

Background Preoperative (neoadjuvant) chemotherapy and radiotherapy are more effective than similar postoperative treatment for oesophageal, gastric, and rectal cancers, perhaps because of more effective micrometastasis eradication and reduced risk of incomplete excision and tumour cell shedding during surgery. The FOxTROT trial aims to investigate the feasibility, safety, and efficacy of preoperative chemotherapy for colon cancer. Methods In the pilot stage of this randomised controlled trial, 150 patients with radiologically staged locally advanced (T3 with >= 5 mm invasion beyond the muscularis propria or T4) tumours from 35 UK centres were randomly assigned (2:1) to preoperative (three cycles of OxMdG [oxaliplatin 85 mg/m(2), l-folinic acid 175 mg, fluorouracil 400 mg/m(2) bolus, then 2400 mg/m(2) by 46 h infusion] repeated at 2-weekly intervals followed by surgery and a further nine cycles of OxMdG) or standard postoperative chemotherapy (12 cycles of OxMdG). Patients with KRAS wild-type tumours were randomly assigned (1:1) to receive panitumumab (6 mg/kg; every 2 weeks with the first 6 weeks of chemotherapy) or not. Treatment allocation was through a central randomisation service using a minimised randomisation procedure including age, radiological T and N stage, site of tumour, and presence of defunctioning colostomy as stratification variables. Primary outcome measures of the pilot phase were feasibility, safety, and tolerance of preoperative therapy, and accuracy of radiological staging. Analysis was by intention to treat. This trial is registered, number ISRCTN 87163246. Findings 96% (95 of 99) of patients started and 89% (85 of 95) completed preoperative chemotherapy with grade 3-4 gastrointestinal toxicity in 7% (seven of 94) of patients. All 99 tumours in the preoperative group were resected, with no significant differences in postoperative morbidity between the preoperative and control groups: 14% (14 of 99) versus 12% (six of 51) had complications prolonging hospital stay (p=0.81). 98% (50 of 51) of postoperative chemotherapy patients had T3 or more advanced tumours confirmed at post-resection pathology compared with 91% (90 of 99) of patients following preoperative chemotherapy (p=0.10). Preoperative therapy resulted in significant downstaging of TNM5 compared with the postoperative group (p=0.04), including two pathological complete responses, apical node involvement (1% [one of 98] vs 20% [ten of 50], p

Background. There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented. Objective. To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. Methods. A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. Results. Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses. Conclusion. These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention.

Multiple rare nonsynonymous variants in APC predispose to colorectal adenomas. The mechanisms through which such variants act have been unclear, but it has been proposed that a specific ("just-right") level of beta-catenin signaling is required for colorectal tumorigenesis. This appears to be mediated by selection for APC genotypes that retain one, or rarely two, 20 amino acid beta-catenin downregulating repeats (20AARs). We investigated the mechanism through which the variant p.Glu1317Gln (c.3949GC) contributes to colorectal tumorigenesis. We compared the patterns of somatic APC mutations in tumors from patients with attenuated familial adenomatous polyposis (AFAP) who did, or did not, coinherit p.Glu1317Gln with their AFAP-causing APC mutations. Only 8.2% (4/49) of tumors carrying p.Glu1317Gln had somatic mutations predicted to result in mutant polypeptides retaining a single 20AAR, compared to 62.1% (36/58) of those which did not carry this variant (P=5.64 x 10(-9)). Furthermore, tumors with p.Glu1317Gln often carried somatic mutations that were unusually early or late (downstream of the second 20AAR) in the APC open reading frame. These data support a novel mechanism in which p.Glu1317Gln in combination with other weak mutant APC alleles (generating polypepetides with zero, two, or three 20AARs) can provide the necessary growth advantage for colorectal tumorigenesis.

Background The incidence of Crohn's disease rose rapidly in industralized countries over the past 50 years, but it is unclear whether the incidence is still rising or has reached a plateau. Aims To update the long-term incidence study of Crohn’s disease in Cardiff for 1996–2005, to investigate whether incidence is still rising and to study changes in disease characteristics over time. Method Crohn’s cases identified by retrospective analysis of hospital records as in previous studies in Cardiff. Results Two hundred and twelve cases were identified. Corrected incidence for this decade was 66 × 106 per year (95% confidence interval: 58–76), showing a continuing rise compared to previous decades. The proportion with colonic disease at presentation continues to rise (43%) with a corresponding fall in those with terminal ileal disease. There remains a strong female preponderance (F:M 1.6:1) as in previous studies. The incidence in children under age 16 continues to rise, and the median age at diagnosis has fallen slightly. Conclusion Crohn’s disease incidence continues to rise slowly in Cardiff with a continuing increase in those presenting with colonic disease, which is now the commonest disease pattern.

SUMMARY Forty-five patients taking long-term indomethacin and with endoscopic erosions or superficial ulcers in the oesophagus, stomach or duodenal bulb were randomized in a double-blind study to continue with standard indomethacin or receive a ‘delayed-release’ formulation. This consisted of microgranules of indomethacin coated with an acrylic resin, Eudragit L, which releases drug in the small intestine. Endoscopy was repeated after 8 weeks and biopsies taken on both occasions. Changes in endoscopic lesions and histological appearances were similar in both groups, although mucosal erythema was less in those given Eudragit L indomethacin. Systemic, rather than topical, effects of indomethacin may therefore be responsible for persistence of gastro-duodenal mucosal lesions in these patients. There is reason to question the clinical practice of using expensive, delayed-release preparations.

This review provides an update on the pathogenesis and histopathological diagnosis of endocrine tumours of the gastrointestinal tract, concentrating on three different varieties whose careful assessment by pathologists is of particular clinical significance. These are the four types of enterochromaffin-like cell tumour of the gastric corpus, the periampullary somatostatin-containing D-cell tumour of the duodenum, and the frequently chromogranin A-negative L-cell tumour of the appendix and large intestine. In addition, the value of pathological factors in predicting the behaviour of gastrointestinal endocrine tumours and selecting therapy is discussed, and the crucial role of the pathologist in the multidisciplinary team management of these neoplasms is emphasized.

Appendicitis is the most commonly performed emergency abdominal surgery. The appendix can also be the site of a variety of neoplasms and unusual inflammatory conditions. A retrospective review was performed to determine the pathological diagnoses in appendicectomy specimens. This study is a retrospective analysis of 2660 appendicectomies performed from 1997 to 2003. The reports were analyzed for the following parameters: age-related incidence of acute appendicitis, seasonal variation in presentation, perforation rate, rate of negative and incidental appendicectomy, and the incidence of other pathologies encountered. Of the 2660 appendicectomy specimens, acute appendicitis was seen in 1718 patients (64.58%), with a peak in patients in their second decade (35.09% of cases of acute appendicitis). The perforation rate was 13.9% and was significantly higher in patients aged 70 years or more (P < 0.001). The negative appendicectomy rate was 28.8%, and was significantly higher in female patients (P < 0.001) and in the 11–30 year age group (P < 0.001). Other pathologies include carcinoid (0.52%), adenocarcinoma (0.39%), and mucinous cystadenoma (0.60%). The high rate of negative appendicectomy among female patients and the increased incidence of perforation in elderly patients reinforce the validity of the judicious use of laparoscopy in these populations. There are still a number of unusual histologies found in appendicectomy specimens supporting the continued use of routine histology.

BACKGROUND & AIMS: Ulcerative colitis (UC) is largely a disease of nonsmokers in which transdermal nicotine improves the symptoms but often causes adverse events (AEs). Nicotine enemas cause fewer AEs and were used as supplemental treatment for active UC. METHODS: We treated 104 patients with active UC with either 6-mg nicotine enemas or placebo enemas for 6 weeks in a randomized double-blind study. Patients continued their oral therapy, if any, for UC: 68 patients were taking mesalamine, 15 patients were taking prednisolone, and 12 patients were taking thiopurines during the study. Clinical, sigmoidoscopic, and histologic assessments were made at baseline and at the end of the study and symptoms were recorded daily on a diary card. The primary end point was induction of clinical remission and clinical improvement also was measured by the UC disease activity index. After the study, patients then used nicotine enemas daily for 4 weeks and sigmoidoscopy with a biopsy examination was repeated. AEs and salivary cotinine levels were monitored throughout the study. RESULTS: Clinical remission was achieved in 14 of 52 (27%) patients on active treatment and 14 of 43 (33%) patients on placebo (P = .55). The UC disease activity index improved by 1.45 points in the active group and by 1.65 points for those on placebo (P = .88). Only 1 patient discontinued treatment because of an AE (abdominal pain). In the 47 patients taking mesalamine only, active treatment conferred benefit that was not statistically significant; disease remission occurred in 9 of 25 patients on active therapy and 4 of 21 patients on placebo (P = .20). CONCLUSIONS: Six-milligram nicotine enemas were well tolerated but were not found to be efficacious for active UC.

There is accumulating evidence to indicate that during the early years of renal replacement therapy, peritoneal dialysis (PD) provides an equivalent, if not superior, mode of dialysis to hemodialysis (HD) for a substantial number of patients [1, 2]. The benefit of PD, however, appears to be limited to the first three or four years, and the majority of patients switch therapy to HD because of technique failure. The causes of this treatment failure are multifactorial and include recurrent episodes of peritonitis, loss of residual renal function, and loss of peritoneal function [3]. The most common change in peritoneal function is a loss of ultrafiltration capacity, although a reduction in solute clearance is not an infrequent occurrence [4–6]. The causes of such functional changes are poorly understood but there is increasing evidence that they are related to changes in the structure of the membrane which correlates in most patients with the longevity of dialysis [7–10], although other factors clearly are influential. Structural changes include the loss of mesothelial cells, an increase in the thickness of the submesothelial compact zone, and a plethora of vascular changes, which range from classical small vessel atherosclerosis to venular changes. The etiology of these structural changes, however, remains speculative and includes the uremic process itself, recurrent infections, and the continuous exposure of the membrane to bioincompatible dialysis fluids [11]. Dialysis fluid has long been recognized as “bioincompatible” with the homeostasis of the peritoneal cavity [12–16]. A low pH, hyperosmolar fluid containing lactate as a buffering agent, and glucose degradation products (GDP) as a byproduct of production will inevitably have long-term negative pathophysiologic consequences [17, 18, 19]. By definition however, the fluid cannot be phys

The aetiology of inflammation in cardiac mucosa at the gastro-oesophageal junction (carditis) is unclear, although gastro-oesophageal reflux has been suggested.To correlate histological features of carditis with oesophageal acid exposure (gastro-oesophageal reflux) and proximal gastric bile exposure (duodenogastric reflux) in patients with symptoms of gastro-oesophageal reflux disease (GORD).Sixty-six patients with reflux symptoms underwent endoscopy with biopsy, oesophageal manometry, 24-h oesophageal pH testing and 24-h proximal gastric Bilitec 2000 testing. Inflammation in glandular mucosa was assessed using the updated Sydney System. Fifteen healthy volunteers underwent pH and Bilitec 2000 testing and served as controls.There was no correlation between either the presence or histological grade of carditis and oesophageal acid exposure or proximal gastric bilirubin exposure. Patients with reflux symptoms had as much duodenogastric reflux into the proximal stomach as did control subjects.We were unable to establish either gastro-oesophageal or duodenogastric reflux as the predominant cause of inflammation in cardiac mucosa.

Prolonged patient survival after surgical resection, is associated with a higher cytotoxic and memory T cell density within colorectal cancers (CRC). High endothelial venules (HEVs) are specialized blood vessels present in secondary lymphoid organs (SLO) that allow ingress of naïve and central memory T cells from the blood. It has been proposed that HEVs in tumors might serve as a similar route of entry for lymphocytes into the tumor and result in an improved prognosis. The present study aimed to characterize HEVs and their microenvironment in resected tumors from colorectal cancer patients (n = 62). We observed HEVs in association with lymphoid aggregates in 49 out of 62 patients. However, these HEV+ lymphoid aggregates were largely at the invasive margin of the tumor and although there was an association with lymphocytes and HEVs at the invasive margin (p = 0.002) there was only a very weak association with tumor infiltrating lymphocytes. Indeed, lymphoid aggregates were associated with more advanced disease (Dukes’ stage C) and did not indicate a favorable prognosis.

Background:\ud \ud Molecular characteristics of cancer vary between individuals. In future, most trials will require assessment of biomarkers to allocate patients into enriched populations in which targeted therapies are more likely to be effective. The MRC FOCUS3 trial is a feasibility study to assess key elements in the planning of such studies.\ud \ud \ud Patients and Methods:\ud \ud Patients with advanced colorectal cancer were registered from 24 centres between February 2010 and April 2011. With their consent, patients’ tumour samples were analysed for KRAS/BRAF oncogene mutation status and topoisomerase 1 (topo-1) immunohistochemistry. Patients were then classified into one of four molecular strata; within each strata patients were randomised to one of two hypothesis-driven experimental therapies or a common control arm (FOLFIRI chemotherapy). A 4-stage suite of patient information sheets (PISs) was developed to avoid patient overload.\ud \ud \ud Results:\ud \ud A total of 332 patients were registered, 244 randomised. Among randomised patients, biomarker results were provided within 10 working days (w.d.) in 71%, 15 w.d. in 91% and 20 w.d. in 99%. DNA mutation analysis was 100% concordant between two laboratories. Over 90% of participants reported excellent understanding of all aspects of the trial. In this randomised phase II setting, omission of irinotecan in the low topo-1 group was associated with increased response rate and addition of cetuximab in the KRAS, BRAF wild-type cohort was associated with longer progression-free survival.\ud \ud \ud Conclusions:\ud \ud Patient samples can be collected and analysed within workable time frames and with reproducible mutation results. Complex multi-arm designs are acceptable to patients with good PIS. Randomisation within each cohort provides outcome data that can inform clinical practice.

Background & Aims: The megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a rare disease of childhood that presents early with intestinal hypoperistalsis, hydronephrosis, and hydroureters. Transgenic mice that lack the a3 subunit containing nicotinic acetylcholine (nAChR) have a phenotype similar to that of MMIHS. Methods: We examined the expression of this subunit in control and MMIHS tissue derived from patients using in situ hybridization (ISH) and immunocytochemistry (ICC). Results: In controls, both techniques showed a wide distribution of a3 nAChRs present in ganglion cells, muscle, and epithelium. By contrast, most MMIHS tissue gave negative staining with ISH and variable results with ICC. Conclusions: These observations are consistent with a lack of a3 nAChRs contributing to the pathogenesis of MMIHS.

Objective To report the incidence of Crohn's disease in the city of Cardiff between 1991 and 1995, in relation to the data of the preceding 65 years. Methods The incidence of Crohn's disease was studied by collecting information from clinical records, the department of pathology database and a questionnaire sent to local family practitioners. Results Eighty-four new patients with Crohn's disease, and resident in Cardiff, were diagnosed between 1991 and 1995. The mean incidence for this quinquennium was 56 cases per 106 population per year (95% confidence interval, 44-68). There was a female predominance, particularly in young adults, with an overall male to female ratio of 0.47. Colorectal disease was the most common site of disease at the time of diagnosis. Conclusions In relation to the findings of our previous studies, the data suggest that the overall incidence of Crohn's disease is now stable, but that the proportion with colorectal disease continues to increase and there is a marked female preponderance in Crohn's disease presenting in young people.Eur J Gastroenterol Hepatol12:907-911

Endoscopic ultrasound of esophageal carcinoma is conventionally performed using the 13-mm fiberoptic/ultrasound echoendoscope. However, the large diameter results in an inability to negotiate the primary tumor in 25% of patients. The aim of this study was first to determine whether use of the 8-mm esophagoprobe would overcome this problem and second to evaluate the accuracy of the smaller diameter instrument. One hundred consecutive patients with esophageal cancer underwent initial gastroscopy. Based upon the degree of luminal stenosis, patients were staged with either the conventional echoendoscope (luminal diameter > or = 15 mm) or the esophagoprobe (luminal diameter < 15 mm). The primary tumor was successfully negotiated in all subjects (echoendoscope 30, esophagoprobe 70) so that T- and N-staging was accomplished in every patient. Esophageal dilatation was performed in 12 patients (12%). The procedure was well tolerated and there were no complications, in particular no patient suffered esophageal perforation. The accuracy of the esophagoprobe for T-staging was 90% (19 out of 20) and that for N-staging was 75% (15 out of 20). This was similar to the accuracy of staging with the conventional echoendoscope, 90% (9 out of 10) for T-stage and 90% (9 out of 10) for N-stage. The esophagoprobe can safely and accurately stage patients with esophageal carcinoma, including those with high-grade stenoses.

To evaluate the accuracy of thin-section magnetic resonance (MR) imaging (in-plane resolution, 0.6 x 0.6 mm) in the preoperative assessment of the depth of extramural tumor infiltration, which is a major prognostic indicator in rectal cancer.In a prospective study of 28 consecutive patients, preoperative MR imaging was performed. The tumor stage according to the TNM classification system and the measured depth of extramural tumor invasion in matched MR images and histopathologic slices were compared.Preoperative MR imaging correctly indicated the histopathologic tumor stage in all 25 patients in whom comparisons were possible. The difference between the depth of extramural tumor measured on preoperative MR images and corresponding measurements on histopathologic slices of the resection specimen ranged from -5.0 mm to +5.5 mm (mean difference, +0.13 mm; 95% CI: -2.72, +2.98 mm), indicating good agreement. The mesorectal fascia, and the relation of the tumor to it, could be visualized in every case. In all five patients with involvement of the circumferential excision margins of resection specimens, extensive extramural invasion was identified on preoperative MR images.Preoperative thin-section MR imaging accurately indicates the tumor stage of rectal cancer and depth of extramural tumor infiltration. It provides valuable information for identifying T3 tumors for preoperative adjuvant therapy in patients who are at high risk of failure of complete excision.

Background. Telomere shortening, dysfunction, and fusion may facilitate the acquisition of large-scale genomic rearrangements, driving clonal evolution and tumor progression. The relative contribution that telomere dysfunction and/or APC mutation play in the chromosome instability that occurs during colorectal tumorigenesis is not clear. Methods. We used high-resolution telomere length and fusion analysis to analyze 85 adenomatous colorectal polyps obtained from 10 patients with familial adenomatous polyposis and a panel of 50 colorectal carcinomas with patient-matched normal colonic mucosa. Telomerase activity was determined using the telomeric repeat amplification protocol. Array-CGH was used to detect large-scale genomic rearrangements. Pearson correlation and Student t test were used, and all statistical tests were two-sided. Results. Despite the presence of telomerase activity, we observed apparent telomere shortening in colorectal polyps that correlated with large-scale genomic rearrangements (P < .0001) but was independent of polyp size and indistinguishable from that observed in colorectal carcinomas (P = .82). We also observed apparent lengthening of telomeres in both polyps and carcinomas. The extensive differences in mean telomere length of up to 4.6kb between patient-matched normal mucosa and polyps were too large to be accounted for by replicative telomere erosion alone. Telomere fusion events were detected in both polyps and carcinomas; the mutational spectrum accompanying fusion was consistent with alternative nonhomologous end joining. Conclusions. Telomere length distributions observed in colorectal polyps reflect the telomere length composition of the normal originating cells from which clonal growth was initiated. Originating cells containing both short telomeres and APC mutations may give rise to polyps that exhibit short telomeres and are prone to telomere dysfunction, driving genomic instability and progression to malignancy.

Chk1 is essential in maintaining genomic stability due to its role in cell cycle regulation. Several recent studies have indicated that the abrogation of checkpoints in tumourigenesis through the inhibition of Chk1 may be of therapeutic value. To further investigate the role of Chk1 in the mouse small intestine and its potential role as a therapy for colorectal cancer, we simultaneously deleted Chk1 and Apc in the mouse small intestine. We found that homozygous loss of Chk1 is not compatible with Wnt-driven proliferation and resulted in the suppression of Wnt-driven tumourigenesis in the mouse small intestine. In contrast, heterozygous loss of Chk1 in a Wnt-driven background resulted in an increase in DNA damage and apoptosis and accelerated both tumour development and progression.

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with ApcMin/+ and AhCre+Apcfl/fl mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in ApcMin/+ mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of β-catenin and increased levels of dephosphorylated β-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of β-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in ApcMin/+ mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1., Author Summary Colorectal cancer is the fourth leading cause of cancer related deaths worldwide, and a key genetic change associated with this disease is mutation of the gene APC. APC encodes a protein which plays a regulatory role in the Wnt signalling pathway. To better understand the mechanisms leading to colorectal cancer after APC loss, we have used a mouse model in which we deleted Apc in the bowel and which developed several characteristics of early stage cancers. Here, we show that after Apc loss, the expression of another gene, Cited1, is increased in mice and human colorectal tumours. To study the role of Cited1 in bowel cancer after loss of Apc, we generated mice mutant for Apc (Min) or mutant for Apc and Cited1 (MinCited1). We observed that MinCited1 mice developed fewer intestinal tumours and lived longer than Min mice suggesting that Cited1 is pro-tumourigenic. However, we also observed that Cited1 deficiency actually increased many of the aspects associated with loss of Apc, including deregulation of the Wnt pathway and cell death. To explain this apparent paradox, we propose a model whereby loss of Cited1, in the context of deregulated Wnt signalling, ‘over-stimulates’ the Wnt pathway, the net effect of which is to inhibit tumourigenesis.

BACKGROUND: Colorectal crypts are clonal units in which somatic mutation of marker genes in stem cells leads to crypt restricted phenotypic conversion initially involving part of the crypt, later the whole crypt. Studies in mice show that the time taken for the great majority of mutated crypts to be completely converted, the clonal stabilisation time, is four weeks in the colon and 21 weeks in the ileum. Differences in the clonal stabilisation time between tissues and species are thought to reflect differences in stem cell organisation and crypt kinetics. AIM: To study the clonal stabilisation time in the human colorectum. METHODS: Stem cell mutation can lead to crypt restricted loss of O-acetylation of sialomucins in subjects heterozygous for O-acetyltransferase gene activity. mPAS histochemistry was used to visualise and quantify crypts partially or wholly involved by the mutant phenotype in 21 informative cases who had undergone colectomy up to 34 years after radiotherapy. RESULTS: Radiotherapy was followed by a considerable increase in the discordant crypt frequency that remained significantly increased for many years. The proportion of discordant crypts showing partial involvement was initially high but fell to normal levels about 12 months after irradiation. CONCLUSIONS: Crypts wholly involved by a mutant phenotype are stable and persistent while partially involved crypts are transient. The clonal stabilisation time is approximately one year in the human colon compared with four weeks in the mouse. The most likely reason for this is a difference in the number of stem cells in a crypt stem cell niche, although differences in stem cell cycle time and crypt fission may also contribute. These findings are of relevance to colorectal gene therapy and carcinogenesis in stem cell systems.