Eric W. Westenbrink

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 Guaiac based faecal occult blood (gFOB) testing has largely been superseded by Faecal Immunochemical testing (FIT), but sensitivity still remains poor.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.

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.

Differentiating coeliac disease from irritable bowel syndrome by urinary volatile organic compound analysis : a pilot study

Coeliac disease (CD), a T-cell-mediated gluten sensitive enteropathy, affects ∼1% of the UK population and can present with wide ranging clinical features, often being mistaken for Irritable Bowel Syndrome (IBS). Heightened clinical awareness and serological screening identifies those with potential coeliac disease; the diagnosis is confirmed with duodenal biopsies, and symptom improvement with a gluten-free diet. Limitations to diagnosis are false negative serology and reluctance to undergo biopsy. The gut microbiome is altered in several gastrointestinal disorders, causing altered gut fermentation patterns recognisable by volatile organic compounds (VOC) analysis in urine, breath and faeces. We aimed to determine if CD alters the urinary VOC pattern, distinguishing it from IBS. 47 patients were recruited, 27 with established CD, on gluten free diets, and 20 with diarrhoea-predominant IBS (D-IBS). Collected urine was stored frozen in 10 ml aliquots. For assay, the specimens were heated to 40±0.1°C and the headspace analysed by Field Asymmetric Ion Mobility Spectrometry (FAIMS). Machine learning algorithms were used for statistical evaluation. Samples were also analysed using Gas chromatography and mass spectroscopy (GC-MS). Sparse logistic regression showed that FAIMS distinguishes VOCs in CD vs D-IBS with ROC curve AUC of 0.91 (0.83–0.99), sensitivity and specificity of 85% respectively. GCMS showed a unique peak at 4′67 found only in CD, not D-IBS, which correlated with the compound 1,3,5,7 cyclooctatetraene. This study suggests that FAIMS offers a novel, non-invasive approach to identify those with possible CD, and distinguishes from D-IBS. It offers the potential for monitoring compliance with a gluten-free diet at home. The presence of cyclooctatetraene in CD specimens will need further validation.

PTU-163 Urinary Volatile Organic Compound Analysis To Distinguish Coeliac Disease From Irritable Bowel Syndrome: A Pilot Study

Introduction Coeliac disease (CD), a T-cell-mediated gluten sensitive enteropathy, affects ~1% of the UK population, and in adults presents with a wide range of clinical features; often mistaken for irritable bowel syndrome (IBS). Heightened clinical awareness and serological screening identifies those likely to have CD; the diagnosis confirmed by histological features in small bowel/duodenal biopsies. Limitations to diagnosis are false negative serology (e.g., in IgA deficient patients, the young and the elderly) and reluctance to undergo biopsy. Examining the pattern of urinary volatiles offers a novel non-invasive approach. The gut microbiome is perturbed in several gastrointestinal disorders, resulting in altered gut fermentation patterns, and recognisable by analysis of volatile organic compounds (VOC) in urine, breath and faeces. The altered structure of the small intestinal mucosa, increased gut permeability and altered gluten peptide metabolism, we hypothesised, would change the microbiome creating a unique “fermentome” pattern, distinguishable from IBS. We investigated this by examining the urinary VOC pattern using Field Asymmetric Ion Mobility Spectrometry (FAIMS). Abstract PTU-163 Figure 1 Methods 47 patients were recruited, 27 with CD and 20 with diarrhoea-predominant IBS (D-IBS). Urine was collected and 10ml aliquots were stored frozen in universal containers. For assay, the containers were heated to 40 ± 0.1oC. The headspace above the sample was then analysed by FAIMS. Linear discriminant analysis (LDA) was used for statistical evaluation. Results LDA showed that FAIMS distinguishes the VOC pattern in CD vs D-IBS with a sensitivity and specificity of 85% respectively. Conclusion This pilot study suggests that FAIMS offers a novel non-invasive approach to identify those likely to have CD, and distinguishes from D-IBS. It may have the potential to non-invasively track the progress of CD when on a gluten-free diet, to monitor adherence and observe changes. Disclosure of Interest None Declared.

PWE-116 Detection of Colorectal Cancer from Urinary Volatile Organic Compounds Using a New Chromatograph/Electronic-Nose Instrument – Wolf System

Introduction Colorectal cancer (CRC) remains one of the leading causes of cancer-related death in Europe and the USA. The gold standard diagnostic test of colonoscopy is highly invasive, expensive and has an associated morbidity. The current non-invasive option for CRC screening includes Faecal Immunochemical Testing (FIT) for haemoglobin, which shows a specificity of 87–96%, but has a wide variation in potential sensitivity (66–88%). One non-invasive method that is gaining interest for the diagnosis of a variety of cancers measures the volatiles/gases that emanate from human biological media. Methods Urine samples were collected from 26 CRC and 23 controls (Irritable bowel syndrome patients; IBS) and stored at -80 oC. 5 mL aliquots were heated to 40oC for 5 minutes to develop sufficient headspace. The WOLF 3.1 gas chromatograph/electronic nose instrument, developed at Warwick University, was used to analyse the resultant headspace. The analysis method took a total of 25 minutes for each sample, with an air purge in between to avoid cross-contamination. Statistical evaluation by Linear Discriminant Analysis (LDA) was tested by repeated trials of single unknown sample by re-introduction and re-classification by a K-Nearest-Neighbour technique. Results Figure 1 below shows the 2 group LDA classification of CRC and IBS samples using response time slices of 100 seconds and extraction of two response features from each. There is distinction between the disease groups, with no overlap seen in any of the samples in this population (P < 0.0001). The sensitivity and specificity of distinguishing CRC from IBS controls from KNN re-classification were 92% and 77% respectively.Abstract PWE-116 Figure 1 Conclusion This pilot study affirms the utility of a custom made WOLF 3.1 gas chromatograph-electronic nose instrument to detect CRC using urine samples. Further validation in a larger sample set is underway but holds promise for a simple, economical tool in CRC detection. Disclosure of Interest None Declared

Mo1394 Towards the Detection of Bile Acid Diarrhea: A Novel Non-Invasive Approach Using Electronic Noses (E-Nose) and Field Asymmetric Ion Mobility Spectroscopy (FAIMS)

OBJECTIVES: Colorectal cancer develops through multiple pathways including the adenomacarcinoma sequence and the serrated pathway. Individuals with serrated polyps (SP) are at higher risk of developing synchronous advanced colorectal neoplasia (AN) and cancer, but the molecular pathways underlying this malignancy remain poorly understood in these individuals. In this study, we characterized patients with coexisting colorectal AN and SP by examining the methylation levels of long interspersed nucleotide element-1 (LINE-1) in the adjacent normal mucosal tissues. METHODS: Colorectal ANs were retrospectively collected. The case group included ANs with coexisting SPs, and the control group consisted of ANs without coexisting SPs. Clinicopathological findings were compared between groups. BRAF and KRAS mutations in the ANs and SPs and LINE-1 methylation levels in adjacent mucosa were examined by quantitative bisulfite pyrosequencing. RESULTS: Seventy-five ANs from 40 patients in the case group, and 179 ANs from 119 patients in the control group were collected and analyzed. There were no significant differences in clinicopathological findings between the 2 groups of ANs, except that intraepithelial neoplasia in the case group was more frequently located in the right colon. BRAF mutations were significantly more frequent in the case group, while KRAS mutation showed no significant differences between groups. Most patients with high-grade intraepithelial neoplasia in both groups showed a component of conventional adenoma, while only one patient with high-grade intraepithelial neoplasia in the control group had a component of SP. Individuals with