Chuka U. Nwokolo

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

Background & Aims Barretts esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barretts and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. Methods We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. Results We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09–1.18; P = 1.8 × 10−11) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86–0.93; P = 7.5 × 10−9). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87–0.93; P = 3.72 × 10−9). Conclusions We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Barrett's surveillance is worthwhile and detects curable cancers. A prospective cohort study addressing cancer incidence, treatment outcome and survival.

Objectives To establish whether Barretts surveillance is worthwhile in terms of incident cancers and whether outcomes are favourable. Methods A prospective non-randomized single centre Barretts surveillance programme commencing 1 January 1992 through 1 April 2001 (112 months). Oesophagectomy recommended for high-grade dysplasia or carcinoma. Results Of 23 725 patients, 506 were diagnosed as Barretts oesophagus and 24 (5%) had carcinoma at diagnosis (prevalence cancers). One hundred and twenty-six patients had at least one surveillance endoscopy; 248 surveillance endoscopies were performed spanning 338 patient years. Thirteen surveillance (incidence) cancers were detected. In the prevalence cancer group 12 of the 24 patients underwent oesophagectomy. Lymph nodes showed evidence of metastases in 10 of the 12 resections. In the surveillance group 10 patients underwent oesophagectomy. Lymph nodes showed evidence of metastases in one of the 10 resections. One patient in the prevalence cancer group (4% of group; 8% of those operated) and seven patients in the surveillance cancer group (54% of group; 70% of those operated) remain disease-free more than 2 years post-oesophagectomy. The cost per cancer cured is £7546. One curable cancer was detected per 48 patient years of surveillance. Conclusions Few Barretts surveillance studies have addressed treatment outcomes and survival. In our study 5% of Barretts patients undergoing endoscopy have prevalent cancers. If surveillance is performed, 4% per year develop cancer and 2% per year are cured of their cancers. Most surveillance cancers are operable and of those undergoing surgery 70% are cured. Barretts surveillance is cost-effective compared with other cancer screening or surveillance initiatives.

Review article: next generation diagnostic modalities in gastroenterology – gas phase volatile compound biomarker detection

The detection of airborne gas phase biomarkers that emanate from biological samples like urine, breath and faeces may herald a new age of non‐invasive diagnostics. These biomarkers may reflect status in health and disease and can be detected by humans and other animals, to some extent, but far more consistently with instruments. The continued advancement in micro and nanotechnology has produced a range of compact and sophisticated gas analysis sensors and sensor systems, focussed primarily towards environmental and security applications. These instruments are now increasingly adapted for use in clinical testing and with the discovery of new gas volatile compound biomarkers, lead naturally to a new era of non‐invasive diagnostics.

Development and application of a new electronic nose instrument for the detection of colorectal cancer

Colorectal cancer is a leading cause of cancer death in the USA and Europe with symptoms that mimick other far more common lower gastrointestinal (GI) disorders. This difficulty in separating colorectal cancer from these other diseases has driven researchers to search for an effective, non-invasive screening technique. Current state-of-the-art method of Faecal Immunochemical Testing achieving sensitivity ~90%, unfortunately the take-up in the western world is low due to the low patient acceptability of stool samples. However, a wide range of cancers have been distinguished from each-other and healthy controls by detecting the gas/volatile content emanating patient biological media. Dysbiosis afforded by certain disease states may be expressed in the volatile content of urine - a reflection of the gut bacterias metabolic processes. A new electronic nose instrument was developed at the University of Warwick to measure the gas/volatile content of urine headspace, based on an array of 13 commercial electro-chemical and optical sensors. An experimental setup was arranged for a cohort of 92 urine samples from patients of colorectal cancer (CRC), irritable bowel syndrome (IBS) and controls to be run through the machine. Features were extracted from response data and used in Linear Discriminant Analysis (LDA) plots, including a full 3-disease classification and one focussing on distinguishing CRC from IBS. The latter case was tested by the success of re-classification using an (n-1) K-nearest neighbour algorithm, showing 78% sensitivity and 79% specificity to CRC.

Detection of Colorectal Cancer (CRC) by Urinary Volatile Organic Compound Analysis

Colorectal cancer (CRC) is a leading cause of cancer related death in Europe and the USA. There is no universally accepted effective non-invasive screening test for CRC. Guaiac based faecal occult blood (gFOB) testing has largely been superseded by Faecal Immunochemical testing (FIT), but sensitivity still remains poor. The uptake of population based FOBt testing in the UK is also low at around 50%. The detection of volatile organic compounds (VOCs) signature(s) for many cancer subtypes is receiving increasing interest using a variety of gas phase analytical instruments. One such example is FAIMS (Field Asymmetric Ion Mobility Spectrometer). FAIMS is able to identify Inflammatory Bowel disease (IBD) patients by analysing shifts in VOCs patterns in both urine and faeces. This study extends this concept to determine whether CRC patients can be identified through non-invasive analysis of urine, using FAIMS. 133 patients were recruited; 83 CRC patients and 50 healthy controls. Urine was collected at the time of CRC diagnosis and headspace analysis undertaken using a FAIMS instrument (Owlstone, Lonestar, UK). Data was processed using Fisher Discriminant Analysis (FDA) after feature extraction from the raw data. FAIMS analyses demonstrated that the VOC profiles of CRC patients were tightly clustered and could be distinguished from healthy controls. Sensitivity and specificity for CRC detection with FAIMS were 88% and 60% respectively. This study suggests that VOC signatures emanating from urine can be detected in patients with CRC using ion mobility spectroscopy technology (FAIMS) with potential as a novel screening tool.

Ciprofloxacin suppresses bacterial overgrowth, increases fasting insulin but does not correct low acylated ghrelin concentration in non‐alcoholic steatohepatitis

Background:  Insulin resistance and oxidative stress induced by products of small intestinal bacterial activity are putative factors in the pathogenesis of non‐alcoholic steatohepatitis. Acylated ghrelin is the biologically active form of an orexigenic gastric hormone that modifies insulin sensitivity and body composition.

A novel tool for noninvasive diagnosis and tracking of patients with inflammatory bowel disease.

Background:The pathogenesis of inflammatory bowel disease (IBD) involves the role of bacteria. These bacteria ferment nonstarch polysaccharides in the colon producing a fermentation profile that through altered gut permeability can be traced in urine. We proposed to track the resultant volatile organic compounds or gases that emanate from urine using noninvasive real-time tools, specifically by electronic nose and Field Asymmetric Ion Mobility Spectrometer (FAIMS) instruments. The aim of this study was to determine the utility of electronic nose and FAIMS instruments to detect and track the fermentation profile of patients with IBD. Methods:Sixty-two individuals were recruited, 48 individuals with IBD (24 with Crohn’s disease and ulcerative colitis, respectively) and 14 controls. The disease activity was recorded, and urine samples were collected. The headspace (the air above the sample) was analyzed using the electronic nose and FAIMS instruments. Results:Electronic nose data analysis was conducted through (1) Principal Component Analysis (data were analyzed together without previous categorization); and (2) Discriminant Function Analysis (samples were precategorized [clinical groups]). The FAIMS data were processed by Fisher’s Discriminant Analysis (precategorized [clinical groups]). Both technologies consistently showed the ability to separate those with IBD and controls with a >75% accuracy; P < 0.001. In a smaller subgroup (n = 24), we also demonstrated that the electronic nose and FAIMS instruments can distinguish between active disease and those in remission. Conclusions:The fermentation profile or fermentome is disparate in those with IBD compared with controls—a reflection of the bacterial diversity in health and disease. This profile also changes (and was tracked) as the disease is induced into remission. Thus, the electronic nose and FAIMS instruments offer the potential of a noninvasive real-time diagnostic tool for point of care clinical use.

Application of a Novel Tool for Diagnosing Bile Acid Diarrhoea

Bile acid diarrhoea (BAD) is a common disease that requires expensive imaging to diagnose. We have tested the efficacy of a new method to identify BAD, based on the detection of differences in volatile organic compounds (VOC) in urine headspace of BAD vs. ulcerative colitis and healthy controls. A total of 110 patients were recruited; 23 with BAD, 42 with ulcerative colitis (UC) and 45 controls. Patients with BAD also received standard imaging (Se75HCAT) for confirmation. Urine samples were collected and the headspace analysed using an AlphaMOS Fox 4000 electronic nose in combination with an Owlstone Lonestar Field Asymmetric Ion Mobility Spectrometer (FAIMS). A subset was also tested by gas chromatography, mass spectrometry (GCMS). Linear Discriminant Analysis (LDA) was used to explore both the electronic nose and FAIMS data. LDA showed statistical differences between the groups, with reclassification success rates (using an n-1 approach) at typically 83%. GCMS experiments confirmed these results and showed that patients with BAD had two chemical compounds, 2-propanol and acetamide, that were either not present or were in much reduced quantities in the ulcerative colitis and control samples. We believe that this work may lead to a new tool to diagnose BAD, which is cheaper, quicker and easier that current methods.

Utility of faecal calprotectin in inflammatory bowel disease (IBD) : what cut-offs should we apply?

Background Faecal calprotectin (FC), a cytosolic protein released by neutrophils (S100 family) in response to inflammation, is a simple, non-invasive test that can be used to differentiate irritable bowel syndrome (IBS) with inflammatory bowel disease (IBD), where there can be considerable symptom overlap. Aims and methods The aims of the study were (1) to be able to predict the ability of FC to exclude IBD and determine cut-offs when in remission, (2) to investigate the effects of time and temperature on stability of FC and (3) compare three ELISA kits to measure FC: Buhlmann, PhiCal v1 and PhiCal v2. A total of 311 patients with altered bowel habit were tested for FC; 144 with IBS, 148 with IBD and 19 with other organic causes. Results Sensitivity and specificity of FC (with PhiCal v2 kit) to distinguish between functional disorder (IBS) and IBD using cut-off 50 μg/g were 88% and 78%, respectively, with a negative predictive value of 87%. Area under the receiver operating curve was 0.84 (CI 0.78 to 0.90). For those with IBD, FC values below 250 μg/g corresponded with remission of disease with a sensitivity and specificity of 90% and 76%, respectively. Area under the receiver operating curve was 0.93 (CI 0.89 to 0.97). FC was stable once extracted and frozen for up to 2.5 months. Pearson correlation was good between Buhlmann assay and PhiCal v2 (r2 = 0.95). Conclusions FC has up to 87% negative predictive value to exclude IBD, and cut-offs less than 250 μg/g had 90% sensitivity to determine remission in IBD. Once frozen, FC is stable and the ELISA monoclonal plates were broadly comparable.

Immunohistochemical and quantitative mRNA assessment of ghrelin expression in gastric and oesophageal adenocarcinoma

Background: Ghrelin is an orexigenic gut peptide produced predominantly by the stomach. Gastric mucosal ghrelin production could be compromised by an infiltrating adenocarcinoma. Aims: To assess the expression of ghrelin mRNA and peptide in oesophagogastric adenocarcinomas and adjacent non-neoplastic mucosa. Methods: 10 gastric and 22 oesophageal adenocarcinoma archival samples were randomly selected from a database. The presence of ghrelin-positive cells was assessed in cancer and corresponding non-neoplastic mucosa by immunohistochemistry. Quantitative reverse transcriptase polymerase chain reaction (PCR) for ghrelin mRNA was also performed on 24 gastric and 8 oesophageal adenocarcinoma specimens and adjacent non-neoplastic mucosa. Results: Immunohistochemistry and reverse transcriptase PCR confirm a negligible expression of ghrelin in adenocarcinoma specimens. By contrast, non-neoplastic gastric mucosa was rich in ghrelin-positive cells and ghrelin mRNA. The number (median and range) of ghrelin-positive cells per 2 mm section of non-neoplastic mucosa was 73 (45–215) in the corpus; this was significantly higher than in cardia mucosa (9 (0–64), p<0.001) and antral mucosa (5 (0–14), p<0.001). Conclusions: Gastric and oesophageal adenocarcinomas have no ghrelin-producing cells. The highest level of ghrelin expression was noted in the non-neoplastic mucosa of the gastric corpus. Disruption of the gastric ghrelin-producing mechanism may occur during oesophagogastric malignancy.