Primary Project 1

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5.A SPECIFIC AIMS \ " ¿ The overall goal of this Primary Project is to establish an NTR Research Center to develop a multi-modal imaging platform to detect preferential binding of fluorescent-labeled peptides that target pre-malignant mucosa in colon as a novel technique for the early detection of cancer in high risk patients. Imaging is performed first with wide area endoscopy to localize regions suspicious for binding, and then with confocal microscopy to confirm binding to dysplastic colonocytes rather than non-specifically to mucus or debris. Sessile adenomas > 5 mm in diameter are used as a model for dysplasia to 'standardize the protocol and validate performance measures. In this proposal, peptides will be topically applied to the local mucosa via a spray catheter to demonstrate the proof of concept, and future applications may involve delivery via an enema to focus on distal lesions. We will establish the network infrastructure and imaging protocols to perform pilot clinical studies to characterize the performance of 5 candidate peptides for use in a future multi-center clinical trial by the end of the funding period. A world class team of investigators from the University of Michigan, Stanford University, Mayo Clinic, Olympus Medical Systems Corp, and STI Medical Systems Inc, has been formed to combine strengths and resources from academia and industry to pursue these aims. Specific Aim 1: Assemble the team of investigators at the University of Michigan, Stanford University, Mayo Clinic, Olympus Medical Systems Corp, and STI Medical Systems Inc to standardize the clinical protocol and validate performance measures for topical administration of fluorescent-labeled peptides to detect sessile colonic adenomas > 5 mm on imaging with wide area endsocopy and confocal microscopy. (1a) Assemble team of investigators at the University of Michigan, Stanford University, Mayo Clinic, Olympus Medical Systems Corp, and STI Medical Systems Inc to standardize protocol and validate performance measures with fluorescent-labeled peptides with multi-modal imaging. (1b) Identify 5 candidate peptides that bind preferentially to dysplastic colonic mucosa rather than to normal mucosa using techniques of phage display. To screen, sequence, and synthesize peptides for labeling with FITC and use of 488 nm excitation. (1c) Identify molecular targets of candidate peptide ligands on colon cells in culture. The successful completion of this aim will establish an NTR Research Center that joins world leading experts in translational research and endoscopic imaging from academia and industry to form a,cohesive team to develop a multi-modal imaging platform that uses fluorescent-labeled peptides to target mucosal dysplasia on wide area endoscopy and confirmed on confocal microscopy. Techniques of phage display will be used to discover 5 candidate peptides that bind selectively to dysplastic colonic mucosa for use in pilot clinical studies for standardizing the imaging protocol and validating performance measures in Aim 2. Finally, the molecular targets of these peptides will be identified from cultured cells using mass spectrometry. Specific Aim 2: Standardize the clinical imaging protocol for topical administration of fluorescent-labeled peptides across the multiple clinical centers, validate performance measures for peptide binding on wide area endsocopy and confocal microscopy with sessile colonic adenomas > 5 mm in diameter as the target lesion, and develop algorithms for multi-modal image registration. (2a) Standardize clinical imaging protocol at multiple clinical sites for topical administration of fluorescent-labeled peptides to localize mucosal dysplasia from colonic adenomas > 5 mm in diameter on wide area endoscopy and validate performance measures for peptide binding. (2b) Develop a standard clinical imaging protocol to confirm peptide binding to dysplastic crypts in adenomas > 5 mm in diameter on confocal microscopy and validate performance measures at multiple clinical sites. (2c) Develop computer based algorithms to perform registration of white light, NBI, and fluorescence endoscopic images, and demonstrate real-time in vivo application. The successful completion of this aim will result in the development of standardized imaging protocols and validated performance measures for topical administration of fluorescent-labeled peptides to detect dysplastic colonic mucosa on wide area endoscopy and confirmed on confocal microscopy. These imaging protocols and performance measures will be used for a future multi-center clinical trial to be planned near the completion of this funding period. Furthermore, methods for registration of the white light and fluorescence images will be developed to integrate this data set into a diagnostic map to assist the clinician with guiding tissue biopsy. Specific Aim 3: Improve the target-to-background ratio for peptide binding on wide area endosocopy by tuning the fluorescence filter specifications, and measure the depth of peptide penetration into the mucosa with topical administraton. Develop an integrated imaging protocol for multi-modal platform to detect flat dysplasia in setting of Barrett's esophagus. (3a) Improve the target-to-background ratio for peptide binding on wide area endosocopy by modifying the fluorescence filter specifications. (3b) Measure the depth of mucosa/ penetration by near-infrared fluorescent-labeled peptides with dual axes confocal microscope on vertical cross-sectional images from colon adenomas. (3c) Develop imaging protocol to integrate use of wide area fluorescence endoscopy with confocal microscopy to guide biopsy of flat dysplasia in Barrett's esophagus. The successful completion of this aim will result in improvement of the peptide target-to-background ratio on wide area endoscopy by optimizing the absorption of excitation by the FITC label and by reducing the autofluorescence background. Furthermore, we will be able to assess the adequacy of topical peptide administration by measuring the depth of mucosal penetration, and to develop a protocol for integrating localization of peptides on wide area endoscopy with confirmation on confocal microsopy.