Sebastian Zeki

Characterization of Inflammatory Bowel Disease With Urinary Metabolic Profiling

OBJECTIVES:Distinguishing between the inflammatory bowel disease (IBD), Crohns disease (CD), and ulcerative colitis (UC) is important for both management and prognostic reasons. Discrimination using noninvasive techniques could be an adjunct to conventional diagnostics. Differences have been shown between the intestinal microbiota of CD and UC patients and controls; the gut bacteria influence specific urinary metabolites that are quantifiable using proton high-resolution nuclear magnetic resonance (NMR) spectroscopy. This study tested the hypothesis that such metabolites differ between IBD and control cohorts, and that using multivariate pattern-recognition analysis, the cohorts could be distinguished by urine NMR spectroscopy.METHODS:NMR spectra were acquired from urine samples of 206 Caucasian subjects (86 CD patients, 60 UC patients, and 60 healthy controls). Longitudinal samples were collected from 75 individuals. NMR resonances specific for metabolites influenced by the gut microbes were studied, including hippurate, formate, and 4-cresol sulfate. Multivariate analysis of all urinary metabolites involved principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA).RESULTS:Hippurate levels were lowest in CD patients and differed significantly between the three cohorts (P<0.0001). Formate levels were higher and 4-cresol sulfate levels lower in CD patients than in UC patients or controls (P=0.0005 and P=0.0002, respectively). PCA revealed clustering of the groups; PLS-DA modeling was able to distinguish the cohorts. These results were independent of medication and diet and were reproducible in the longitudinal cohort.CONCLUSIONS:Specific urinary metabolites related to gut microbial metabolism differ between CD patients, UC patients, and controls. The emerging technique of urinary metabolic profiling with multivariate analysis was able to distinguish these cohorts.

Stem cells and their implications for colorectal cancer

The colonic crypt is home to several multipotent stem cells. These stem cells reside in a niche at the base of the crypt, which controls their behavior and maintains the stem cells homeostasis through a variety of signaling pathways and interactions. Several attempts have been made to define markers that can identify colonic stem cells, the most useful of which is Lgr5, a Wnt target gene. Although the crypt base contains several stem cells, each colonic crypt comprises a single clone of cells. Investigators have attempted to reconcile these apparently contradictory observations by conducting research into stem cell division. The propagation of stem-cell-acquired mutations through a crypt results in a monocryptal adenoma that, through crypt fission, develops into a microadenoma. Some early adenomas become polyclonal through an as yet unknown mechanism. The discovery of subpopulations of cancer cells that can initiate tumors when implanted into mice has renewed interest in the existence of cancer stem cells, especially with regard to their implications for the use of chemotherapy. Various potential markers of cancer stem cells have been investigated, particularly CD133, but the cancer stem cell theory still has some limitations.

Risk Factors for Severe Disease in Adults with Falciparum Malaria

BACKGROUND Over a 16-year period, we conducted a clinical study of malaria acquired worldwide in adults from malaria-nonendemic countries, to determine risk factors for severe Plasmodium falciparum malaria. METHODS All patients with confirmed malaria who were managed by our unit from 1991 to 2006 were prospectively evaluated. Factors predicting disease severity according to (1) strict World Health Organization (WHO) criteria, (2) a composite measure of unfavorable outcome, and (3) length of hospital stay were identified by logistic and linear regression analyses. RESULTS We evaluated 676 episodes of malaria, 482 (71%) due to P. falciparum and 194 (29%) due to nonfalciparum parasites. Black patients had a significantly reduced risk of developing WHO-defined severe falciparum malaria, with Asian patients having odds of severe falciparum malaria that were 8.05-fold (95% confidence interval [CI], 2.93-22.1-fold) higher and white patients having odds that were 8.20-fold (95% CI, 2.94-22.9-fold) higher. Black patients also had a reduced risk of an unfavorable outcome and of a prolonged stay in the hospital, compared with the risks for white or Asian patients. Of 6 patients with falciparum malaria who died, none were black. In univariate analysis, patients with parasitemias of >or= 2% had odds of severe falciparum malaria 12-fold higher than those of patients with parasitemias of <2% (73% vs. 19%). Patients with a history of previous clinical malaria, regardless of ethnicity, had a significantly reduced risk of WHO-defined severe falciparum malaria (odds ratio, 0.35 [95% CI, 0.15-0.80]). CONCLUSIONS The findings of this study demonstrate that ethnicity and parasitemia are important independent risk factors for severe falciparum malaria in adults from malaria-nonendemic countries and that a history of previous clinical malaria significantly reduces the risk of WHO-defined severe falciparum malaria.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective Barretts oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barretts glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barretts glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barretts mirrors pyloric glands and is preserved in Barretts dysplasia. MUC2-positive goblet cells are localised above the neck in Barretts glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barretts glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barretts glands are clonal, indicating derivation from a single stem cell. Conclusions Barretts shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barretts oesophagus.

Clonal Selection and Persistence in Dysplastic Barrett's Esophagus and Intramucosal Cancers After Failed Radiofrequency Ablation

OBJECTIVES:Radiofrequency ablation (RFA) is used to successfully eliminate Barretts esophagus (BE)-related dysplasia or intramucosal carcinoma and aims to cause reversion to squamous epithelium. However, in 20% of cases RFA fails to return the epithelium to squamous phenotype. Follow-up studies show a similar dysplasia recurrence rate. We hypothesize that failed RFA is due to clonally mutated epithelial populations harbored in RFA-privileged sites and that RFA can select for the mutant clonal expansion.METHODS:A longitudinal case series of 19 patients with BE and high-grade dysplasia or intramucosal carcinoma were studied. DNA was extracted from individual Barretts glands, deep esophageal glands within mucosal resections and biopsy specimens before and after RFA. Mutations were identified by targeted sequencing of genes commonly mutated in Barretts adenocarcinoma.RESULTS:Five patients demonstrated persistent post-RFA pathology with persistent mutations, sometimes detected in deep esophageal glands or neighboring squamous epithelium after several rounds of RFA preceded by mucosal resection. Recurrence of pathology in three other patients was characterized by de novo mutations.CONCLUSIONS:Protumorigenic mutations can be found in post-ablation squamous mucosa as well as in mutant deep esophageal glands; both are associated with dysplasia recurrence. Following RFA, non-dysplastic Barretts epithelium can contain mutant clones that are found in a subsequent adenocarcinoma. Ablation may also drive the clonal expansion of pre-existing clones after a “bottleneck” created by the RFA. Overall, recurrence of dysplasia post RFA reflects the multicentric origins of Barretts clones and highlights the role of clonal selection in carcinogenesis.

Crypt dysplasia in Barrett's oesophagus shows clonal identity between crypt and surface cells.

Epithelial dysplasia is an important histological diagnosis signifying the presence of pre‐invasive disease, usually needing intervention. However, the specific genetic changes responsible for the induction of this phenotypic change are unknown. Moreover, recent reports indicate that the dysplastic phenotype may not be immutable: in basal crypt dysplasia (CD), unequivocal dysplastic changes are seen in the crypts in Barretts oesophagus and other pre‐invasive lesions in the gastrointestinal tract, but the upper crypts and surface epithelium associated with these dysplastic crypts show the definitive morphology of a differentiated epithelium. The genotypic relationship between CD and the differentiated surface epithelium is presently unclear. We obtained 17 examples of CD: the lower and upper crypts and surface epithelium were differentially laser‐microdissected from formalin‐fixed, paraffin‐embedded sections and mutations were sought in tumour suppressor genes frequently associated with progression in Barretts oesophagus. We found two patients who both showed a c. C238T mutation in the CDKN2A (CDKN2AInk4A) gene and where the precise microanatomical relationships could be discerned: this mutation was present in both the CD at the crypt base and in the upper crypt and surface epithelium. We conclude that, in CD, the dysplastic basal crypt epithelium and the upper crypt and surface epithelium show clonal CDKN2A mutations, thus showing definitively that the surface epithelium is derived from the dysplastic crypt epithelium: the dysplastic phenotype is therefore not fixed and can be reversed. The mechanism of this change is unclear but may be related to the possibility that dysplastic cells can, probably early in their progression, respond to differentiation signals. However, it is also clear that a heavy mutational burden can be borne by crypts in the gastrointestinal tract without the development of phenotypic dysplasia. We are evidently some way from understanding the plasticity and the genotypic correlates of the dysplastic phenotype. Copyright

The use of molecular markers in predicting dysplasia and guiding treatment

The ability to stratify patients based on the risk of progression to oesophageal adenocarcinoma would provide benefit to patients as well as deliver a more cost effective surveillance programme. Current practice is to survey all patients with Barretts oesophagus (BO) and use histological diagnoses to guide further management. However, reliance on histology alone has its drawbacks. We are currently unable to reliably stratify the risk of progression of patients with non-dysplastic BO based on any particular histological feature. There is also considerable variability in histological interpretation. An obvious recourse has been to rely on identifying molecular features possibly as an adjunct to histology, to better diagnose and stratify patients. To this end, p53 immunohistochemistry can be used as a useful adjunct to risk stratify and clarify histological grades, particularly low-grade dysplasia. Other markers of progression, although not yet in a clinically applicable format, are promising. Measurements of promoter methylation and also genomic instability such as loss of heterozygosity and copy number alterations show promise especially as high throughput genetic technologies reach maturity. The enduring hope is that these molecular biomarkers will make the transition to clinical applicability either in the direct endoscopic setting or even using non-endoscopic methods.

Squamous cell carcinoma after radiofrequency ablation for Barrett's dysplasia

Barretts oesophagus (BO) is a usually indolent condition that occasionally requires endoscopic therapy. Radiofrequency ablation (RFA) is an effective endoscopic treatment for high grade dysplasia (HGD) and intramucosal cancer in BO. It has a good efficacy, durability and safety profile although complications can occur. Here we describe a case of RFA in a patient with high grade dysplasia. Although the response to treatment was initially very good with the development of neosquamous epithelium, the patient very rapidly developed a squamous cell cancer of the oesophagus confirmed on radiology, histology and immunohistochemistry. Sanger sequencing confirmed that the original HGD and the squamous cell cancer (SCC) were derived from separate clonal origins. The report highlights the fact that SCC of the oesophagus has been noted after endoscopic ablation for BO previously and suggest that ablation of BO may encourage the clonal expansion of cells carrying carcinogenic mutations once a dominant clonal population has been eradicated.

T1322 Colectomy Rates for Patients Treated for CMV Disease in the Context of Ulcerative Colitis do Not Differ From Those Who are Not Treated for CMV Disease

age of patients was 29 years (range, 15 47) and 71.8 % (n=22) were men. The median duration of illness was 70 months (range, 1-228 months). Twenty one patients (67.7%) had history of surgery for CD. 28 (90%) had taken ciprofloxacin previously. A total of 50 microorganisms isolated in 31 patients. More than two microorganisms isolated in 18 patients (58%). Isolated microorganisms were listed in Table. Eleven (91%) out of 12 Escherichia coli and all isolates of Klebsiella pneumonia, Pseudomonas aeruginosa, and Acinetobacter baumanni are resistant against ciprofloxacin. Conclusion: Both gram positive bacteria and most of gram negative bacteria showed resistance against ciprofloxacin. Thus ciprofloxacin may not be appropriate for CD patients with abdominal abscess at this time. Initial antibiotics should cover gram-positive bacteria as well as gram negative bacteria other than ciprofloxacin. Table. Isolated microorganisms (n=50)

PWE-168 Crypt Cell Dysplasia with Maturation in Barrett’S Esophagus Shows Clonal Identity between the Crypt and Surface Cells

Introduction Dysplasia in epithelia is an important histological diagnosis although the specific genetic changes which are responsible for this phenotypic change are unknown. Recent reports indicate that the dysplastic phenotype may not be immutable: in basal crypt dysplasia like atypia (BCDA), unequivocal dysplasia is seen in the crypts in Barrett›s oesophagus and other pre-invasive lesions in the gastrointestinal tract, but the upper crypts and surface epithelium associated with these dysplastic crypts show a differentiated epithelium. The genotypic relationship between the BCDA and the differentiated surface epithelium is unclear. Methods We obtained 17 examples of BCDA: the lower crypts and upper crypts and surface epithelium were differentially lazer-microdissected from formalin-fixed, paraffin embedded sections and mutations were sought in tumour suppressor genes frequently associated with progression in Barrett’s oesophagus. Results Two patients showed a c.C238T mutation in the p16 (CDKN2A, p16Ink4A) gene and where the precise microanatomical relationships could be discerned: this clonal p16 mutation was present in both the BCDA at the crypt base and in the upper crypt and surface epithelium. This shows that the surface epithelium is derived from the dysplastic crypt epithelium: the dysplastic phenotype is therefore not fixed and can be reversed. Two patients showed a c.C238T mutation in the p16 (CDKN2A, p16Ink4A) gene and where the precise microanatomical relationships could be discerned: this clonal p16 mutation was present in both the BCDA at the crypt base and in the upper crypt and surface epithelium. This shows that the surface epithelium is derived from the dysplastic crypt epithelium: the dysplastic phenotype is therefore not fixed and can be reversed. Conclusion The mechanism of this change is unclear: dysplastic cells may, probably at an earlystage in their progression, respond to differentiation signals. We are some way from a definition of the genotypic correlates of thedysplastic phenotype, and from an understanding of its plasticity. Disclosure of Interest None Declared.