Mechanisms of fluorescence endoscopy of the human colon

Early identification of dysplasia in Barrett's esophagus (BE) and the colon remains a critical issue for clinical diagnostic endoscopy, since early discovery directly improves patient survival. Clinical studies have successfully used tissue autofluorescence (AF) with conventional white light endoscopy (WLE) and biopsy for detecting preneoplastic colonic adenomatous polyps, differentiating benign from preneoplastic polyps and identifying BE mucosa in the esophagus. Despite this, 'in vivo' autofluorescence endoscopic detection of early dysplasia continues to be problematic due to suboptimal fluorescence contrast. The goal of this PhD thesis was to extend previous AF mechanistic studies and explore means of improving 'in vivo' fluorescence endoscopic contrast. First, the AF of isolated living colonic crypts and primary cell cultures of normal colon, hyperplastic and adenomatous polyps was studied at the cellular level 'in vitro.' For BE, fluorescence microscopy was performed on archived 'ex vivo' frozen human tissues of normal esophagus, BE without and with dysplasia, and stomach to characterize the biological origins and possible differences in mucosal AF. Both studies improved our understanding of the biological origins of colon and BE tissue AF. To address suboptimal detection of dysplasia in AF imaging of BE, aminolevulinic acid (ALA) was evaluated 'in vivo' and 'ex vivo' as a diagnostic pro-drug to enhance the fluorescence contrast of dysplasia in BE. The results of these studies were inconsistent. The limited utility of AF and ALA-induced fluorescence for detecting dysplasia 'in vivo ' prompted a new approach, using monoclonal antibody-based fluorescent contrast bioconjugate targeting of a tumor-associated mucin antigen overexpressed in human colorectal adenocarcinomas. This agent was developed and evaluated ' in vitro' and 'in vivo' in a xenograft mouse tumor model, yielding significant enhancement of tumor-to-normal contrast. A novel class of fluorescent semiconductor nanoparticles (quantum dots) were evaluated as alternative immunotargeted fluorescence contrast agents in preliminary ' in vitro' and 'in vivo' animal studies. In conclusion, this thesis has advanced our basic understanding of tissue AF in the GI tract, which may translate into improved AF endoscopic imaging technology for enhancing early detection of GI cancers, and offers the distinct possibility of providing real-time non-invasive "'molecular endoscopy'" for tumor detection ' in vivo'., Tissue autofluorescence is investigated as a possible method for detection of early cancer during endoscopy of the gastrointestinal (GI) tract. In the present work, 'in vivo' fluorescence point-spectroscopy of colonic tissues, with blue light excitation, showed significant differences in autofluorescence intensity and spectral line shape between normal, preneoplastic and neoplastic lesions. These data formed the basis for developing imaging algorithms; currently used in a prototype real-time light-induced fluorescence endoscope (LIFE), undergoing multicenter clinical trials. Clinical results of fluorescence imaging and spectroscopy are described. The mechanisms by which LIFE imaging and spectroscopy differentiate normal tissues from early and late stage colonic cancers are not well understood. In this thesis, human colonic tissues were analyzed by confocal fluorescence microscopy, microspectrofluorimetry, histological and inummohistological staining, and transmission electron microscopy. Together with studies of the attenuation of light in gastrointestinal tissues, the measurements were used to develop a quantitative model of the autofluorescence process 'in vivo'. Excitation and emission matrices (EEMs) of tissues and potential fluorophores were used to identify fluorophores contributing to this autofluorescence. The cellular and extracellular origins and changes in fluorophore distributions in normal, preneoplastic and neoplastic tissues are described. Modeling showed that LIFE-GI imaging and spectroscopy results 'in vivo' may be explained by a combination of many factors, including the effect of different illumination/detection geometries, changes in tissue architecture, changes in tissue optical properties, and altered fluorophore distributions, as normal colon progresses toward neoplasia. Furthermore, a more detailed study of mucosal tissue autofluorescence from normal colon, hyperplastic polyps, and adenomatous polyps is presented. In these tissues, significant differences in mucosal fluorophore composition were found, including the discovery of a possible endogenous autofluorescent biomarker specific to colonic dysplasia. Finally, a summary of results and conclusions are presented, and future studies are outlined.

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