KRAS Status is Associated with Metabolic Parameters in Metastatic Colorectal Cancer According to Primary Tumour Location.

Colorectal cancer (CRC) is characterized by complex interplay between macroenvironmental factors and tumour microenvironment, leading to variable outcomes in CRC patients. To date, there is still a need to identify macroenvironment/microenvironment factors that could define subgroup of patients that would benefit from specific anti-cancer treatment in order to improve patient selection for individualized targeted-based therapy. Aim of this study was to evaluate associations between metabolic parameters and KRAS status in metastatic CRC (mCRC) according to a new tumour site classification. Retrospective data were extracted from a total of 201 patients diagnosed with mCRC between 2012 and 2017 extracted from an established CRC database at our tertiary institute. Clinical-pathological data, including age, gender, BMI, hypertension, diabetes, pre-CRC diagnosis serum lipid levels and KRAS status were recorded. Categorical characteristics were compared using chi-squared test. Continuous characteristics were compared using Mann-Whitney U test. Log rank test was used to compare hazards for survival. In all comparisons, a two-sided P value <0.05 was considered statistically significant. Out of 201 patients, 170 patients with complete serum lipid profile were included in the analysis. In recto-sigmoid cancers there was a statistically significant association between high cholesterol:high-density lipoprotein (chol:HDL) ratio and KRAS mutation (OR 2.69, 95% CI 1.1-6.4, p = 0,02). In non recto-sigmoid cancers, high cholesterol was associated with KRAS WT (OR 0.39, CI 0.15-0.97, p = 0.04). In 22 patients with KRAS mutated recto-sigmoid cancer stage IV at diagnosis normal chol:HDL ratio was associated with a trend to better survival (p = 0.06). High chol:HDL ratio was significantly associated with KRAS mutated metastatic recto-sigmoid cancers. A subgroup of mCRC patients with KRAS mutated recto-sigmoid cancer may benefit from optimal lipid lowering treatment.

Breath diagnostics in the era of SARS-CoV-2-clinical and research arena.

The emergence of the SARS-CoV-2 pandemic has transformed not just healthcare, but also economic systems on a global scale. Despite significant efforts to contain the infection, it continues to spread. Stringent infection control measures have been taken to minimise the transmission between individuals and healthcare workers, especially those undertaking aerosol generating medical procedures. The uncertainties surrounding infection transmission through breath tests in particular, and to some extent faecal testing, will invariably cause concerns amongst both the patients and healthcare workers. It is therefore pertinent that all of the necessary measures are adopted to minimise risk of spreading. In this article, we summarise the physiology and virulence of SARS-CoV-2 and discuss the implications for breath testing (in both the clinical and research arena) as well as outlining methods to mitigate these risks.

Systematic review and meta-analysis : diagnostic accuracy of faecal immunochemical testing for haemoglobin (FIT) in detecting colorectal cancer for both symptomatic and screening population.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide. A non-invasive test, with high sensitivity and specificity is essential for early detection, improved outcome and avoidance of unnecessary invasive tests. This study aims to evaluate the accuracy of the faecal immunochemical testing for haemoglobin (FIT) in the detection of CRC, both in symptomatic and screening population and to summarise the available evidence to date. METHODS: Search strategy was initially developed in MEDLINE and adapted for use in other databases. Studies were included if they had fulfilled the criteria. QUADAS-2 tool was used for quality assessment and data analysis performed using STATA 15 software. RESULTS: A total of 17 out of 92 articles were included in the final analysis. Within the symptomatic group (n= 6755), the overall pooled sensitivity and specificity of FIT to detect CRC was 0.90 (95% CI 0.87-0.92) and 0.87 (95% CI 0.83-0.90) respectively. In the screening population (n=24197), the pooled sensitivity and specificity of FIT to detect CRC was 0.69 (95% CI 0.54-0.81) and 0.94 (95% CI 0.94-0.95) respectively. Most analytics were comparable with cut off less than 20µg/g feces providing optimal sensitivity and specificity for symptomatic and screening populations respectively. CONCLUSION: For the detection of CRC within the screening population, FIT has high specificity and sensitivity. In the symptomatic group, FIT's high sensitivity (90%) supports its role as a triage test to guide the selection of patients who require urgent lower gastrointestinal tract evaluation.

Colorectal cancer and adenoma screening using urinary volatile organic compound (VOC) detection: early results from a single-centre bowel screening population (UK BCSP).

BACKGROUND: The United Kingdom (UK) bowel cancer screening programme has reduced mortality from colorectal cancer (CRC), but poor uptake with stool-based tests and lack of specificity of faecal occult blood testing (FOBT), has prompted investigation for a more suitable screening test. The aim of this study was to investigate the feasibility of a urinary volatile organic compounds (VOC)-based screening tool for CRC. METHODS: The urine from FOBT-positive patients was analysed using field asymmetric ion mobility spectrometry (FAIMS) and gas chromatography coupled with ion mobility spectrometry (GC-IMS). Data were analysed using a machine learning algorithm to calculate the test accuracy for correct classification of CRC against adenomas and other gastrointestinal pathology. RESULTS: One hundred and sixty-three patients were enrolled in the study. Test accuracy was high for differentiating CRC from control: area under the curve (AUC) 0.98 (95% CI 0.93-1) and 0.82 (95% CI 0.67-0.97) using FAIMS and GC-IMS respectively. Correct classification of CRC from adenoma was high with AUC range 0.83-0.92 (95% CI 0.43-1.0). Classification of adenoma from control was poor with AUC range 0.54-0.61 (95% CI 0.47-0.75) using both analytical modalities. CONCLUSIONS: CRC was correctly distinguished from adenomas or no bowel pathology using urinary VOC markers, within the bowel screening population. This pilot study demonstrates the potential of this method for CRC detection, with higher test uptake and superior sensitivity than FOBT. In addition, this is the first application of GC-IMS in CRC detection which has shown high test accuracy and usability.

Risk stratification of symptomatic patients suspected of colorectal cancer using faecal and urinary markers.

AIM: Faecal markers, such as the faecal immunochemical test for haemoglobin (FIT) and faecal calprotectin (FCP), have been increasingly used to exclude colorectal cancer (CRC) and colonic inflammation. However, in those with lower gastrointestinal symptoms there are considerable numbers who have cancer but have a negative FIT test (i.e. false negative), which has impeded its use in clinical practice. We undertook a study of diagnostic accuracy CRC using FIT, FCP and urinary volatile organic compounds (VOCs) in patients with lower gastrointestinal symptoms. METHOD: One thousand and sixteen symptomatic patients with suspected CRC referred by family physicians were recruited prospectively in accordance with national referring protocol. A total of 562 patients who completed colonic investigations, in addition to providing stool for FIT and FCP as well as urine samples for urinary VOC measurements, were included in the final outcome measures. RESULTS: The sensitivity and specificity for CRC using FIT was 0.80 [95% confidence interval (CI) 0.66-0.93] and 0.93 (CI 0.91-0.95), respectively. For urinary VOCs, the sensitivity and specificity for CRC was 0.63 (CI 0.46-0.79) and 0.63 (CI 0.59-0.67), respectively. However, for those who were FIT-negative CRC (i.e. false negatives), the addition of urinary VOCs resulted in a sensitivity of 0.97 (CI 0.90-1.0) and specificity of 0.72 (CI 0.68-0.76). CONCLUSIONS: When applied to the FIT-negative group, urinary VOCs improve CRC detection (sensitivity rises from 0.80 to 0.97), thus showing promise as a second-stage test to complement FIT in the detection of CRC.

Guidelines for the investigation of chronic diarrhoea in adults: British Society of Gastroenterology, 3rd edition

Chronic diarrhoea is a common problem, hence clear guidance on investigations is required. This is an updated guideline from 2003 for the investigations of chronic diarrhoea commissioned by the Clinical Services and Standards Committee of the British Society of Gastroenterology (BSG). This document has undergone significant revision in content through input by 13 members of the Guideline Development Group (GDG) representing various institutions. The GRADE system was used to appraise the quality of evidence and grading of recommendations.

Diagnostic accuracy of faecal biomarkers in detecting colorectal cancer and adenoma in symptomatic patients.

BACKGROUND: The diagnosis of colorectal cancer (CRC) can be difficult as symptoms are variable with poor specificity. Thus, there is a quest for simple, non-invasive testing that can help streamline those with significant colonic pathology. AIM: To assess using faecal immunochemical test for haemoglobin (FIT) or faecal calprotectin (FCP) to detect CRC and adenoma in symptomatic patients referred from primary care. METHODS: A total of 799 referred for urgent lower gastrointestinal investigations were prospectively recruited. Of these, 430 completed colonic investigations and returned stool samples, and were included in the final statistical analysis. Faecal immunochemical test for haemoglobin was performed on HM-JACKarc analyser (Kyowa Medex, Tokyo, Japan), and FCP by the EliA Calprotectin immunoassay (Thermo Fisher Scientific, Waltham, United States). RESULTS: The negative predictive value (NPV) using FIT alone or both markers (FIT and FCP) in combination was similar at 99% for CRC, with a sensitivity and specificity of 84% and 93%, respectively. FIT measurements were significantly higher in left-sided colonic lesions compared with the right side; 713 vs. 94; P = 0.0203). For adenoma, the NPV using FIT alone, or both markers (FIT and FCP) in combination, was similar at 94% with a sensitivity and specificity of 69% and 56%, respectively. CONCLUSIONS: Undetectable faecal immunochemical test for haemoglobin is sufficiently sensitive to exclude colorectal cancer, with higher values in left-sided lesions. FCP in combination does not appear to provide additional diagnostic information. Further studies to determine the health economic benefits of implementing faecal immunochemical test for haemoglobin in primary care are required.

Breathomics--exhaled volatile organic compound analysis to detect hepatic encephalopathy: a pilot study.

The current diagnostic challenge with diagnosing hepatic encephalopathy (HE) is identifying those with minimal HE as opposed to the more clinically apparent covert/overt HE. Rifaximin, is an effective therapy but earlier identification and treatment of HE could prevent liver disease progression and hospitalization. Our pilot study aimed to analyse breath samples of patients with different HE grades, and controls, using a portable electronic (e) nose. 42 patients were enrolled; 22 with HE and 20 controls. Bedside breath samples were captured and analysed using an uvFAIMS machine (portable e-nose). West Haven criteria applied and MELD scores calculated. We classify HE patients from controls with a sensitivity and specificity of 0.88 (0.73-0.95) and 0.68 (0.51-0.81) respectively, AUROC 0.84 (0.75-0.93). Minimal HE was distinguishable from covert/overt HE with sensitivity of 0.79 and specificity of 0.5, AUROC 0.71 (0.57-0.84). This pilot study has highlighted the potential of breathomics to identify VOCs signatures in HE patients for diagnostic purposes. Importantly this was performed utilizing a non-invasive, portable bedside device and holds potential for future early HE diagnosis.

The application of FAIMS gas analysis in medical diagnostics.

There is an ever increasing need to develop new tools to aid in the diagnosis and monitoring of human diseases. Such tools will ultimately reduce the cost of healthcare by identifying disease states more quickly and cheaply than current practices. One method showing promise is the analysis of gas-phase biomarkers from human breath, urine, sweat and stool that reflect bodily metabolism. Analysis of these volatiles by GC MS requires specialised infra-structure and staff, making it unsuitable for a clinical setting. Point of care sensor based technologies such as eNoses often suffer from stability and sensitivity issues. Field-Asymmetric Ion Mobility Spectrometry (FAIMS) has potential to fulfil this clinical need. In this paper we review the medical need, the technology, sampling methods and medical evidence thus far. We conclude with reflecting on future developmental steps necessary to bring the device into medical practice.

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.

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 bacteria'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.

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

BACKGROUND: 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. AIM: To review current sensor instruments like the electronic nose (e-nose) and ion mobility spectroscopy (IMS), existing technology like gas chromatography-mass spectroscopy (GC-MS) and their application in the detection of gas phase volatile compound biomarkers in medicine - focussing on gastroenterology. METHODS: A systematic search on Medline and Pubmed databases was performed to identify articles relevant to gas and volatile organic compounds. RESULTS: E-nose and IMS instruments achieve sensitivities and specificities ranging from 75 to 92% in differentiating between inflammatory bowel disease, bile acid diarrhoea and colon cancer from controls. For pulmonary disease, the sensitivities and specificities exceed 90% in differentiating between pulmonary malignancy, pneumonia and obstructive airways disease. These sensitivity levels also hold true for diabetes (92%) and bladder cancer (90%) when GC-MS is combined with an e-nose. CONCLUSIONS: The accurate reproducible sensing of volatile organic compounds (VOCs) using portable near-patient devices is a goal within reach for today's clinicians.

Evaluation of gut bacterial populations using an electronic e-nose and field asymmetric ion mobility spectrometry: further insights into 'fermentonomics'.

The fermentation of undigested foods in the large bowel, by its resident bacteria, results in the production of several chemicals including volatile gases. Perturbance in gut bacteria is known to influence colonic and metabolic health, but to determine this requires prolonged culture (often unsuccessful) or expensive genomic sequencing. Clearly this is not practical for daily clinical practice. Previously, we have reported our insights into fermentonomics through the detection of volatile organic compounds (VOCs) in patients with gastrointestinal and metabolic diseases, using the electronic nose. In this paper we report on the changes in the fermentone produced by patients undergoing complete versus partial bowel cleansing. Using urine samples, preliminary results from 23 individuals receiving bowel cleansing indicate the ability of the electronic nose to distinguish between the partial and complete procedures. Moreover in a subset of individuals, we have been able to track evolving bacterial recolonization over time using the e-nose and field asymmetric ion mobility spectrometry (FAIMS). Such an approach has practical application in tracking bacterial dysbiosis following perturbation.

Effects of neo-adjuvant chemotherapy for oesophago-gastric cancer on neuro-muscular gastric function.

Delayed gastric emptying symptoms are often reported after chemotherapy. This study aims to characterise the effects of chemotherapy on gastric neuro-muscular function. Patients undergoing elective surgery for oesophago-gastric cancer were recruited. Acetylcholinesterase, nNOS, ghrelin receptor and motilin expressions were studied in gastric sections from patients receiving no chemotherapy (n = 3) or oesophageal (n = 2) or gastric (n = 2) chemotherapy. A scoring system quantified staining intensity (0-3; no staining to strong). Stomach sections were separately suspended in tissue baths for electrical field stimulation (EFS) and exposure to erythromycin or carbachol; three patients had no chemotherapy; four completed cisplatin-based chemotherapy within 6 weeks prior to surgery. AChE expression was markedly decreased after chemotherapy (scores 2.3 ± 0.7, 0.5 ± 0.2 and 0 ± 0 in non-chemotherapy, oesophageal- and gastric-chemotherapy groups (p < 0.03 each) respectively. Ghrelin receptor and motilin expression tended to increase (ghrelin: 0.7 ± 0.4 vs 2.0 ± 0.4 and 1.2 ± 0.2 respectively; p = 0.04 and p = 0.2; motilin: 0.7 ± 0.5 vs 2.2 ± 0.5 and 2.0 ± 0.7; p = 0.06 and p = 0.16). Maximal contraction to carbachol was 3.7 ± 0.7 g and 1.9 ± 0.8 g (longitudinal muscle) and 3.4 ± 0.4 g and 1.6 ± 0.6 (circular) in non-chemotherapy and chemotherapy tissues respectively (p < 0.05 each). There were loss of AChE and reduction in contractility to carbachol. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. Our study offers a mechanism by which chemotherapy markedly alters neuro-muscular gastric function.

Insights into 'fermentonomics': evaluation of volatile organic compounds (VOCs) in human disease using an electronic 'e-nose'.

Detection of volatile organic compounds (VOCs) is a common requirement in industry for which numerous methods are available. The electronic nose (e-nose) is an example. Rather than individual chemicals, the e-nose recognizes the 'aroma fingerprint' created by the collection of VOCs in samples, comparable to the human nose. We report on a novel application for gastrointestinal and metabolic medicine, and compare its results to mass spectrometry. Fermentation of undigested foods in the large bowel by its resident bacteria results in the creation of several chemicals including volatile gases that influence colonic and metabolic health. Using urine samples, preliminary results indicate the ability of the e-nose to distinguish between controls and those with inflammatory bowel disease or diabetes (separation rate of ∼97%). This emphasizes the different patterns of fermentation. Our term 'fermentonomics' describes the investigation and analysis of the fermentome by such non-invasive means. Such an approach has potentially wide application in medicine.

Ghrelin promotes nuclear factor kappa-B activation in a human B-lymphocyte cell line.

Ghrelin, an orexigenic hormone of gastric origin that stimulates growth hormone secretion, may modulate inflammation. This experimental study examines the effect of ghrelin on NFκB (p65 subunit), a transcriptional factor involved in inflammation on a human B-lymphocyte cell (WILCL). After confirming the expression of ghrelin receptor protein using western blotting the cells were transferred to wells maintaining a density of 1 × 10(6) cells per ml and a proportion activated with phytohaemagluttinin. Activated and resting cells were exposed to octanoyl-, desoctanoyl ghrelin and a non-peptide ghrelin agonist (Pfizer CP-464709) in increasing concentrations for 6 h. Cell protein extracts were analyzed for NFκB activation using Trans AM NFκB p65 assay. IL-6, IL-8, IL-10, IL-13 and TNFα were measured in the media using Lincoplex human cytokine assay. In octanoyl ghrelin treated resting cells, NFκB activity (Optical Density OD(450 nm)) (mean ± SEM) in control cells was 0.42 ± 0.10 and increased to 0.61 ± 0.20 (P = 0.044), 0.54 ± 0.10 (P = 0.043), 0.52 ± 0.08 at 1, 10 and 100 nM concentrations respectively. No effect was detected with desoctanoyl ghrelin or ghrelin agonist and no specific change in cytokine production. In conclusion, Octanoyl ghrelin increased NFκB activation by up to 50% in a B-lymphocyte cell line suggesting an effect on the inflammatory process.

Acylated and des acyl ghrelin in human portal and systemic circulations.

Octanoylation of the gastric peptide ghrelin produces an active isoform that regulates appetite and other metabolic functions. Acylated ghrelin is present in the gastrointestinal tract suggesting that octanoylation may occur in these tissues and thereby affect the acylated ghrelin in the systemic circulation. In this study blood samples were collected simultaneously from portal, arterial, peripheral venous and central venous compartments from patients undergoing laparotomy. ELISA and high sensitivity Bioplex was used to measure the concentration of acylated and des acyl ghrelin. We found median (95% confidence interval (CI)) plasma acylated ghrelin (pg/ml) was 35.8 (30.0-59.6) in the portal compartment compared to 51.5 (37.6-74.8; P < 0.05, n = 11) in the arterial, 39.3 (33.3-56.3) in the portal compartment compared to 55.0 (48.5-77.0; P < 0.001, n = 12) in the peripheral venous and 36.0 (33.1-57.4) in the portal compartment compared to 48.9 (43.3-65.6; P < 0.01, n = 15) in the central venous compartment. Median (95% CI) plasma des acyl ghrelin levels (pg/ml) was 173 (125-220) in the portal compartment compared to 136 (99.3-125; P < 0.001, n = 14)in the arterial, 186 (136-233) in the portal compartment compared to 149 (111-190; P < 0.01, n = 15) in the peripheral venous and 171 (140-208) in the portal compartment compared to 152 (119-175; P < 0.01, n = 15) the central venous compartment. We conclude that plasma acylated ghrelin concentration was significantly lower in portal compared with the systemic compartments whilst plasma des acyl ghrelin was significantly higher in portal compared with systemic compartments. These findings suggest that the liver could be involved in the regulation of circulating ghrelin.