Abstract Background Real time electrospray ionization mass spectrometry (REIMS) enables detailed analysis of tumour lipid chemistry, based on real time analysis of electrocautery smoke plumes. Methods: This was a prospective, observational study performed at St. Mary's Hospital, London, UK. Patients undergoing elective surgical resections for colorectal cancer were recruited and fresh samples were analyzed ex-vivo using a typical electrosurgery hand piece and monopolar diathermy. Sampling was performed using cutting mode with a standard generator and 30W of output power (ValleylabTM). The hand piece was modified to allow aspiration of the electrosurgical aerosol to a Xevo G2-S iKnife QTof mass spectrometer (Waters Corporation). Raw mass spectrometric data were converted to imzML format (MSConvert) and imported into MATLAB (R2014a) for pre-processing. A prospective database of healthy, dysplastic and malignant colorectal tissues was built and multivariate analysis was performed using principal component analysis and linear discriminant analysis. Classification of each individual tissue type was performed using leave-one-patient-out cross-validation. Results 40 consecutive patients were recruited (22 male, median age 68y, range 47-90). Of the 23 tumor samples 10 were rectal adenocarcinoma and 13 colonic adenocarcinoma. TNM staging of the tumour samples was as follows: T2 (8), T3 (11) T4 (4), N0 (12), N1 (6) N2 (5), M0 (22), M1 (1). Distinction of healthy and malignant colorectal tissue for the whole data set demonstrated an overall classification accuracy of 94.4% and a sensitivity of 92.4%, Specificity 96.8% (ROC AUC 0.98). The diagnostic accuracy for dysplasia was 93.7% (Specificity 95.1%, sensitivity 85.7%, AUC 0.97). Increases in glycerophospholipids (p<0.0027), and triacylglycerols (p<0.0004) were seen in healthy mucosa and increased prostaglandin D2 expression was found in malignant tissue (p< 0.0002). Adenocarcinoma arising in the rectum could be differentiated from more proximal tumors with an overall accuracy of 87.3%. Short and long course chemotherapy modified the lipid signature. Anatomically discrete chemical analysis of rectal cancers was able to provide accurate descriptions of established histopathological and molecular markers of poor prognosis: nodal status (AUC 0.97), KRAS mutation (AUC 0.93) and extramural vascular invasion (EMV) (AUC 0.96). Conclusion REIMS chemical histology provides near real time diagnostic and prognostic information for stratifying oncological and surgical therapy. Citation Format: James Macalister Kinross, Laura Muirhead, James Alexander, Julia Balog, Cristina Guallar-Hoya, Abigail Speller, Ottmar Golff, Rob Goldin, Ara Darzi, Jeremy Nicholson, Zoltan Takats. iKnife: Rapid evaporative ionization mass spectrometry (REIMS) enables real-time chemical analysis of the mucosal lipidome for diagnostic and prognostic use in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3977.