Abstract 271: High-throughput fecal metabolic profiling for the early detection of colorectal cancer using a direct mass spectrometry assay
Authors: Paizs Petra , Widlak Monika , Perdones-Montero Alvaro , Sani Maria , Ford Lauren , Alexander James L. , Cameron Simon , Arasaradnam Ramesh , Kinross James M. , Takats Zoltan 
Published: July 2021

Abstract
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&lt;0.001). A tentatively assigned organic acid (AUC 0.916, CI=0.868-0.953) was associated with control (FDR corrected p&lt;0.001). Multiple features in the mass-to-charge ratio (m/z) range 200-400 were found to be significantly different between control vs adenoma. Annotations of the features are underway using liquid chromatography - MS/MS and spiked standards. LA-REIMS allows direct from sample metabolite profiling of feces and has applications as a novel screening tool for the early detection of CRC.
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.