780 Quantitative In Vivo Assessment of Vascular Permeability in Human Colonic Mucosa Using Confocal Endomicroscopy: Clinical Implications for Colonic Neoplasia

Abstract

Background: Peptides are promising molecular probes for detecting over expressed cell surface targets in the digestive tract. We have previously demonstrated their In Vivo use with colonic dysplasia on confocal microendoscopy, and have developed the sequence ASYNYDA to target Barrett's dysplasia. Aims: We aim to quantify the fluorescence intensity and determine the diagnostic accuracy of this target peptide for Barrett's esophagus. Methods: Biopsy specimens of esophageal mucosa were obtained from human subjects in regions of endoscopically apparent squamous epithelium, intestinal metaplasia, and adenocarcinoma. The tissues were incubated with the FITC-labeled target peptide at a concentration of 100 μmol for 10 minutes. Unbound peptide was removed by rinsing in PBS. The specimens were embedded in OCT freezing compound, cut in 10 μm sections, and counterstained with DAPI. An adjacent serial section was cut and stained with H&E for routine histology. Confocal fluorescence images were collected from each section, and were analyzed for area, mean gray value, standard deviation, min/max gray values, and integrated density using NIH Image J. Results: We evaluated a total of 52 confocal fluorescence images from 26 biopsy specimens collected from 18 subjects, including 20 squamous, 12 intestinal metaplasia, 8 high-grade dysplasia, and 12 adenocarcinoma. The mean intensity for squamous, intestinal metaplasia, high-grade dysplasia and adenocarcinoma were 0.1±0.1, 1.9±3.2, 4.41±2.2, and 9.8±13.1, respectively. Fig A shows binding of the FITC-labeled peptide ASYNYDA to esophageal adenocarcinoma. Fig B shows locations of cell nuclei in blue, identified by DAPI stain, and overlay in Fig C demonstrates peptide binding to cell surface targets. An intensity threshold of 0.73 produced a detection sensitivity and specificity of 85% and 88%, respectively for either high-grade dysplasia or adenocarcinoma. Conclusions: We demonstrate preferential binding of the peptide ASYNYDA to intra-epithelial neoplasia in Barrett's esophagus on confocal fluorescence images, demonstrating a promising novel molecular probe to perform targeted detection on surveillance with confocal microendoscopy.