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Increasing evidence suggests a role of the gut microbiome in the development of colorectal cancer (CRC) and that it can serve as a biomarker for early diagnosis. This review aims to give an overview of the current status of published studies regarding the microbiome as a screening tool for early CRC detection. A literature search was conducted using PubMed and EMBASE in August 2022. Studies assessing the efficacy of microbiome-derived biomarkers based on noninvasive derived samples were included. Not relevant studies or studies not specifying the stage of CRC or grouping them together in the analysis were excluded. The risk of bias for screening tools was performed using the QUADAS-2 checklist. A total of 28 studies were included, ranging from 2 to 462 for CRC and 18 to 665 advanced adenoma patient inclusions, of which only two investigated the co-metabolome as biomarker. The diagnostic performance of faecal bacteria-derived biomarkers had an AUC ranging from 0.28-0.98 for precursor lesions such as advanced adenomas and 0.54-0.89 for early CRC. Diagnostic performance based on the co-metabolome showed an AUC ranging from 0.69 – 0.84 for precursor lesions and 0.65 – 0.93 for early CRC. All models improved when combined with established clinical early detection markers such as gFOBT. A high level of heterogeneity was seen in the number of inclusions and methodology used in the studies. The faecal and oral gut microbiome has the potential to complement existing CRC screening tools, however current evidence suggests that this is not yet ready for routine clinical use.

Objective Rapid Evaporative Ionisation Mass Spectrometry (REIMS) is a metabolomic technique analysing tissue metabolites, which can be applied intra-operatively in real-time. The objective of this study was to profile the lipid composition of colorectal tissues using REIMS, assessing its accuracy for real-time tissue recognition and risk-stratification. Summary background data Metabolic dysregulation is a hallmark feature of carcinogenesis, however it remains unknown if this can be leveraged for real-time clinical applications in colorectal disease. Methods Patients undergoing colorectal resection were included, with carcinoma, adenoma and paired-normal mucosa sampled. Ex vivo analysis with REIMS was conducted using monopolar diathermy, with the aerosol aspirated into a Xevo G2S QToF mass spectrometer. Negatively charged ions over 600-1000m/z were used for univariate and multivariate functions including linear discriminant analysis. Results 161 patients were included, generating 1013 spectra. Unique lipidomic profiles exist for each tissue type, with REIMS differentiating samples of carcinoma, adenoma and normal mucosa with 93 1% accuracy and 96 1% negative predictive value for carcinoma. Neoplasia (carcinoma or adenoma) could be predicted with 96 0% accuracy and 91 8% negative predictive value. Adenomas can be risk-stratified by grade of dysplasia with 93 5% accuracy, but not histological subtype. The structure of 61 lipid metabolites was identified, revealing that during colorectal carcinogenesis there is progressive increase in relative abundance of phosphatidylglycerols, sphingomyelins and mono-unsaturated fatty acid containing phospholipids. Conclusions The colorectal lipidome can be sampled by REIMS and leveraged for accurate real-time tissue recognition, in addition to risk-stratification of colorectal adenomas. Unique lipidomic features associated with carcinogenesis are described.

Right sided colorectal cancers (RCRCs) have distinct molecular phenotypes and clinical presentations to those on the left. Gut microbial co-metabolic interactions with the RCRC microenvironment are poorly defined and present a novel therapeutic target. Here, we present a multi-omics analysis of RCRCs. A prospective observational pilot study was performed at Imperial College London NHS Trust, UK. Treatment naive patients undergoing elective resectional surgery for primary RCRCs were recruited. Fresh intra-luminal fecal samples were obtained at three anatomical sites: 5 cm pre-, tumor, and 5 cm post-tumor. Microsatellite instability (MSI) and CDX2 expression were immunohistochemically evaluated. Metataxonomic analysis was carried out on a MiSeq Platform (Illumina, USA). Metabolomics analysis was performed using liquid chromatography mass spectrometry (Acquity UPLC, Xevo G2-S Q-TOF, Waters). After pre-processing, univariate and multivariate statistics were carried out in MetaboAnalyst and RStudio. 20 patients (8 females, mean age 69.9(53-83)) were included. Of the n=20 RCRCs, n=2 were ileo-cecal valve, n=4 were cecal, n=4 were cecum/proximal ascending colon, n=4 were mid-ascending colon, n=2 were ascending/hepatic flexure, n=2 were hepatic flexure, and n=2 were transverse colon cancers. Tumor classification according to TNM 8 indicated n=4 T1, n=1 T2, n=11 T3, n=4 T4; n=12 as N0, n=6 as N1, and n=2 as N2; n=2 as M0, n=1 as M1c, and n=17 as MX. All n=20 were CDX2 positive and n=16 had normal expression of mismatch repair proteins, n=4 showed MSI. Triglyceride (TG) species were significantly associated with T3-4 rather than T1-2 (p Mapping the intra-luminal fecal metabolome and metataxonome in RCRC reveals potential bacterial-host co-metabolic targets that could be leveraged for stratifying immunotherapy treatment. Citation Format: Petra Paizs, Duncan Roberts, Nathan Danckert, James Lewis, Maria Sani, Marta Jamroziak, Robert Goldin, Julian Marchesi, Lauren Ford, James Alexander, Zoltan Takats, James Kinross. Gut microbial co-metabolism influences the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3056.

Background: Colorectal adenoma detection using non-invasive faecal-based biomarkers is not yet feasible. Our primary aim was to identify differential metabolomic signatures of colorectal adenomas in faecal samples using a Liquid Chromatography Mass Spectrometry (LCMS) assay. The secondary aim was to translate this assay to an Ambient Mass ionization-based technique, DESI, for high throughput, point of care diagnostics. Methods: Patients were prospectively recruited from the colorectal cancer clinic at Imperial College NHS Trust, UK. Patients provided fresh stool samples, prior to taking bowel preparation for colonoscopy. Specimens were collected in Fecotainer (AT Medical BV) and cooled before immediate manual homogenization, aliquoting and storage at -80oC. LCMS untargeted metabolomics was performed using Reversed Phase chromatography (Acquity UPLC, Xevo G2-XS QToF, Waters) in both ElectroSpray Ionization (ESI) modes. Deactivated faecal samples applied to swabs were analysed using DESI-MS (Xevo G2-XS QToF). Multivariate Statistical Analysis was performed using Python. Logistic regression (LR) classification with Leave One patient Out Cross Validation (LOOCV) was applied. Recursive Feature Elimination and Least Absolute Shrinkage and Selection Operator (LASSO) algorithms were used for feature elimination. Results: 36 subjects (17 females, mean age 63.5, range 42-85) were included: 17 controls, 8 adenoma, and 11 cancer patients. Principal Component Analysis of LCMS data from both ESI modes separated the three groups. LR with LOOCV for the adenoma group showed 97% sensitivity and 100% specificity. Receiver Operator Characteristic curve based on a multivariate model of the top 5 significant features showed a diagnostic ability of binary classifiers of adenoma vs cancer 0.997 (Area under the curve (AUC), 95% CI=0.944-1), adenoma v healthy 0.981 (AUC, 95% CI=0.984-1), and healthy v cancer & adenoma 0.995 (AUC, 95% CI=0.931-1). LR with LOOCV after LASSO on the DESI-MS data revealed sensitivity, 84%, and specificity, 95%, for the adenoma group. Specifically, these data suggest increased abundances of N-acyl amino acids, polyunsaturated chain fatty acids (n-3 PUFAs), in the adenoma group. Both species participate in arachidonic and linoleic acid pathways, where upregulation causes prostaglandin overproduction. Glycerophosphoserines, important in cell apoptosis, were significantly increased in cancer vs adenoma/controls (p < 0.01). Conclusion: DESI-MS analysis of faecal samples can detect the presence of colorectal adenomas and distinguish them from cancer. A prospective trial is now under way to validate these findings. Citation Format: Maria Sani, Lauren Ford, Daniel Simon, Yuchen Xiang, James McKenzie, Stefania Manetta Stavrakaki, Petra Paizs, Tausif Huq, Jim Higginson, James Alexander, Zoltan Takats, James Kinross. A prospective pilot study of desorption electrospray ionisation mass spectrometry (DESI-MS) for the early detection of colorectal adenoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3905.

Background Transanal minimally invasive surgery (TAMIS) is deployed for organ preservation in early rectal cancer and significant rectal polyps. Rapid evaporative ionisation mass spectrometry (REIMS) provides biochemical tissue analysis, which could be applied intraoperatively to give real-time tissue feedback to the surgeon and decrease the risk of an involved margin. However, the accuracy and feasibility of this approach have not been established. Methods In this prospective observational study, patients undergoing resection of rectal adenomas or carcinomas were recruited. An electrosurgical handpiece analysed tissues ex vivo using diathermy, with the aerosol aspirated into a Xevo G2-S ToF mass spectrometer. The relative abundance of lipids underwent predictive statistical modelling and leave-one-patient-out cross-validation. The outcomes of interest were the ability of REIMS to differentiate normal, adenomatous and cancerous tissue, or any disease subtype from normal. REIMS was coupled with TAMIS for in vivo sampling, assessing the accuracy of tissue recognition and distinguishing bowel wall layers. Results Forty-seven patients were included, yielding 266 spectra (121 normal, 109 tumour and 36 adenoma). REIMS differentiates normal, adenomatous and cancerous rectal tissues with 86.8% accuracy, and normal and adenomatous tissue with 92.4% accuracy and 91.4% accuracy when differentiating disease from normal. We have performed the first five in-man mass spectrometry augmented TAMIS (MS-TAMIS). In real time, MS-TAMIS can differentiate rectal mucosa and submucosa based on their relative abundance of triglycerides and glycerophospholipids. The ex vivo accuracy distinguishing diseased and normal tissues is maintained in vivo at 90%, with negative predictive value of 95%. The system identified a deep and lateral involved tumour margin during TAMIS. Conclusions REIMS distinguishes rectal tissue types based on underlying lipid biology, and this can be translated in vivo by coupling it to TAMIS. There is a role for this technology in improving the efficacy of resection of early rectal cancers.

Early stage diagnosis of colorectal cancer (CRC) is a key predictor of patient survival, highlighting the need for accurate diagnostic biomarkers. Laser Assisted - Rapid Evaporative Ionization Mass Spectrometry (LA-REIMS) demonstrates potential for direct from sample metabolic profiling of feces. Here, we present an optimized LA-REIMS approach for fecal metabolomic analysis in CRC for the first time. A prospective, observational cohort biomarker discovery study was performed at an NHS hospital trust in the UK. Patients referred through the national two week wait cancer pathway were prospectively recruited. The primary outcome was to differentiate between adenomas or CRC and non-disease controls, the secondary outcome was to compare LA-REIMS to fecal immunochemical testing (FIT) against the gold-standard of colonoscopy or CT colonography. Those under the age of 18, pregnant, or unable to undergo colonic investigation were excluded. Fecal samples were analyzed using FIT (Kyowa Medex Co Ltd, Japan), with a detection limit of 7µg haemoglobin/g feces considered a positive result, as well as a LA-REIMS setup (Xevo G2-S QTof (Waters Corporation)), with optimized laser and instrumentation parameters (improved signal-to-noise and reduced carry-over between samples). After pre-processing, univariate and multivariate statistics were carried out in MetaboAnalyst 4.0. Random forest classification (RFC) with leave one out cross-validation (LOOCV) was performed.244 subjects (120 females, mean age 67.5 (range 21-93)) were included, of which n=135 were non-disease controls, n=82 adenoma patients, and n=27 CRC patients. Partial least square-discriminant analysis (PLS-DA) demonstrated a distinction between control vs CRC (Q2= 0.60087, R2= 0.89376). RFC with LOOCV for cancer vs control showed a sensitivity of 34.6% and specificity of 99.2% compared to 81.8% sensitivity and 87.2% specificity with FIT. PLS-DA of control vs adenoma (Q2= 0.64284, R2= 0.8898) demonstrated good separation. RFC with LOOCV for adenoma vs control had a sensitivity of 61.0 % and specificity of 94.7% compared to 33.8 % sensitivity and 87.2% specificity with FIT. Univariate analysis identified a feature tentatively assigned to the glycerolipid class (area under the receiver operating characteristic curve (AUROC) 0.986 (CI=0.957-1) to be more abundant in CRC vs control (false discovery rate (FDR) corrected p Citation Format: Petra Paizs, Monika Widlak, Alvaro Perdones-Montero, Maria Sani, Lauren Ford, James L. Alexander, Simon Cameron, Ramesh Arasaradnam, James M. Kinross, Zoltan Takats. High-throughput fecal metabolic profiling for the early detection of colorectal cancer using a direct mass spectrometry assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 271.

Introduction The aim of this study was to determine the diagnostic accuracy of REIMS for dysplasia in human adenomas and early colorectal cancer and to determine changes in mucosal lipid chemistry during cancer initiation. Moreover, we attempted to develop a proof of concept first in man study of a novel REIMS-based endoscope for in-vivo chemical phenotyping of adenomas based on the real time analysis of phosphatidic acid (PA) and phosphatidylethanolamine (PE) metabolism. Experimental procedure A prospective, observational cross-sectional cohort study was performed in patients undergoing elective resection for colorectal cancer or colonoscopy at Imperial College London NHS Trust. Two tissue types were obtained - early colorectal cancer (defined as T1 or T2 according to TNMv6 criteria) and adenomatous polyps. Patients with familial adenomatous polyposis, inflammatory bowel disease or hereditary non-polyposis colon cancer were excluded. Specimens were analyzed ex-vivo using a modified electro-surgical hand piece with the aerosol aspirated into a Xevo G2-S QTof mass spectrometer (Waters Corporation). Multivariate analyses were used to create models which underwent cross-validation using SIMCA (V14, Umetrics, Sweden). Characteristic lipid species were identified using univariate statistics in R Studio (V1.0.44) and Metlin. An endoscopic set up was created using a standard endoscope for proof of concept. Summary of data 19 patients (12:7 F:M) were included with a median age of 74 (range 50-92). Six patients had T1/2 adenocarcinoma and 13 had adenomatous polyps. REIMS was able to accurately distinguish the presence of low grade dysplasia (LGD) vs high grade dysplasia (HGD) in adenomatous polyps (sensitivity 95.0%, specificity 100%). Phosphatidic acids (PA) 38:2 and 38:1 were more abundant in high vs low grade dysplasia (p=0.003). REIMS was also able to identify adenomatous polyps from early cancer (sensitivity 100%, specificity 96.3%). Phosphatidylethanolamine (PE) 36:1, phosphatidic acid (PA) 38:1, glucosylceramide (Glc-Cer) 30:1 and ceramide phosphoethanolamines (PE-Cer) d16:1(4E)/20:0 and d14:1(4E)/22:0 were associated with early cancer formation rather than adenomas (p Conclusion Dysplastic adenomas exhibit mucosal lipid chemistry that is discrete from early colorectal cancer. PA, PE and Glc-Cer have potential use as diagnostic biomarkers. First in man studies of the endoscopic application of REIMS suggest it could serve as a real-time tool for early cancer detection based on in vivo analysis of the mucosal lipidome. Citation Format: Petra Paizs, Eftychios Manoli, Sam E. Mason, James L. Alexander, Zsolt Bodai, Emma White, Afeez Adebesin, Jonathan Hoare, Robert Goldin, Ara W. Darzi, James M. Kinross, Zoltan Takats. Rapid evaporative ionization mass spectrometry (REIMS) analysis of the mucosal lipidome has a high diagnostic accuracy for adenomas and early colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4536.