Electronic nose technology as a point-of-care solution for the detection of colorectal cancer

This thesis presents a comparative study of electronic nose technologies for the detection of colorectal cancer from healthy and disease controls using gases/volatiles in urine headspace. A review is made of the clinical features of lower gastro-intestinal diseases and the sensing technologies available to electronic noses. The literature surrounding the detection of cancers and other diseases by sensing of gases and volatiles is also reviewed. An investigation into the common volatile components of urine headspace is conducted experimentally using a gas chromatograph – mass spectrometer (451 Scion SQ, Bruker Corp), resulting in 10 candidate chemicals with links to gut bacteria and diet. A humidity generation unit was developed and integrated with a volatile testing rig to aid in assessing the response of different electronic nose technologies to volatile chemical groups. Four electronic nose systems were tested in parallel studies using urine headspace samples from patients of colorectal cancer (CRC) and irritable bowel syndrome (IBS), as well as healthy volunteers. This involved pre-classified multivariate analysis techniques followed by K-Nearest-Neighbour validation for sensitivity and specificity. A commercial electronic nose based on metal oxide sensors (Fox 4000, AlphaMOS Ltd) analysed 93 urine samples giving a sensitivity and specificity to colorectal cancer of 54% and 48%. A field asymmetric ion mobility spectrometer that is commercially available (Lonestar, Owlstone Ltd) was tested using 133 samples of CRC and volunteer samples, yielding 88% disease sensitivity and 60% specificity. A new electronic nose system was developed using state-of-the-art ampere-metric and optical sensors and tested against 92 urine samples, giving a respective sensitivity and specificity to CRC against IBS controls of 78% and 79%. A final instrument was developed that includes a micro-packed GC column and an array of micro-hotplate metal oxide sensors, which analysed 49 samples to give a 92% sensitivity and 77% specificity to CRC against IBS

Ketone Ester Treatment Improves Cardiac Function and Reduces Pathologic Remodeling in Preclinical Models of Heart Failure

BACKGROUND: Accumulating evidence suggests that the failing heart reprograms fuel metabolism toward increased utilization of ketone bodies and that increasing cardiac ketone delivery ameliorates cardiac dysfunction. As an initial step toward development of ketone therapies, we investigated the effect of chronic oral ketone ester (KE) supplementation as a prevention or treatment strategy in rodent heart failure models. METHODS: Two independent rodent heart failure models were used for the studies: transverse aortic constriction/myocardial infarction (MI) in mice and post-MI remodeling in rats. Seventy-five mice underwent a prevention treatment strategy with a KE comprised of hexanoyl-hexyl-3-hydroxybutyrate KE (KE-1) diet, and 77 rats were treated in either a prevention or treatment regimen using a commercially available β-hydroxybutyrate-(R)-1,3-butanediol monoester (DeltaG; KE-2) diet. RESULTS: The KE-1 diet in mice elevated β-hydroxybutyrate levels during nocturnal feeding, whereas the KE-2 diet in rats induced ketonemia throughout a 24-hour period. The KE-1 diet preventive strategy attenuated development of left ventricular dysfunction and remodeling post-transverse aortic constriction/MI (left ventricular ejection fraction±SD, 36±8 in vehicle versus 45±11 in KE-1; P=0.016). The KE-2 diet therapeutic approach also attenuated left ventricular dysfunction and remodeling post-MI (left ventricular ejection fraction, 41±11 in MI-vehicle versus 61±7 in MI-KE-2; P<0.001). In addition, ventricular weight, cardiomyocyte cross-sectional area, and the expression of ANP (atrial natriuretic peptide) were significantly attenuated in the KE-2-treated MI group. However, treatment with KE-2 did not influence cardiac fibrosis post-MI. The myocardial expression of the ketone transporter and 2 ketolytic enzymes was significantly increased in rats fed KE-2 diet along with normalization of myocardial ATP levels to sham values. CONCLUSIONS: Chronic oral supplementation with KE was effective in both prevention and treatment of heart failure in 2 preclinical animal models. In addition, our results indicate that treatment with KE reprogrammed the expression of genes involved in ketone body utilization and normalized myocardial ATP production following MI, consistent with provision of an auxiliary fuel. These findings provide rationale for the assessment of KEs as a treatment for patients with heart failure

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

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

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 mimicking 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 bacteria′s’s 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

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. 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

Non-invasive distinction of non-alcoholic fatty liver disease using urinary volatile organic compound analysis : early results

BACKGROUND & AIMS: Non-Alcoholic Fatty Liver Disease (NAFLD) is the commonest cause of chronic liver disease in the western world. Current diagnostic methods including Fibroscan have limitations, thus there is a need for more robust non-invasive screening methods. The gut microbiome is altered in several gastrointestinal and hepatic disorders resulting in altered, unique gut fermentation patterns, detectable by analysis of volatile organic compounds (VOCs) in urine, breath and faeces. We performed a proof of principle pilot study to determine if progressive fatty liver disease produced an altered urinary VOC pattern; specifically NAFLD and Non-Alcoholic Steatohepatitis (NASH). METHODS: 34 patients were recruited: 8 NASH cirrhotics (NASH-C); 7 non-cirrhotic NASH; 4 NAFLD and 15 controls. Urine was collected and stored frozen. For assay, the samples were defrosted and aliquoted into vials, which were heated to 40±0.1°C and the headspace analyzed by FAIMS (Field Asymmetric Ion Mobility Spectroscopy). A previously used data processing pipeline employing a Random Forrest classification algorithm and using a 10 fold cross validation method was applied. RESULTS: Urinary VOC results demonstrated sensitivity of 0.58 (0.33 - 0.88), but specificity of 0.93 (0.68 - 1.00) and an Area Under Curve (AUC) 0.73 (0.55 - 0.90) to distinguish between liver disease and controls. However, NASH/NASH-C was separated from the NAFLD/controls with a sensitivity of 0.73 (0.45 - 0.92), specificity of 0.79 (0.54 - 0.94) and AUC of 0.79 (0.64 - 0.95), respectively. CONCLUSIONS: This pilot study suggests that urinary VOCs detection may offer the potential for early non-invasive characterisation of liver disease using 'smell prints' to distinguish between NASH and NAFLD