5.
A SPECIFIC AIMS ' The overall goal of this Primary Project is to establish an NTR Research Center to develop a multi-modalimaging platform to detect preferential binding of fluorescent-labeled peptides that target pre-malignantmucosa in colon as a novel technique for the early detection of cancer in high risk patients. Imaging isperformed first with wide area endoscopy to localize regions suspicious for binding, and then with confocalmicroscopy 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 andvalidate performance measures. In this proposal, peptides will be topically applied to the local mucosa via aspray catheter to demonstrate the proof of concept, and future applications may involve delivery via an enemato focus on distal lesions. We will establish the network infrastructure and imaging protocols to perform pilotclinical studies to characterize the performance of 5 candidate peptides for use in a future multi-center clinicaltrial 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, hasbeen 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 clinicalprotocol and validate performance measures for topical administration of fluorescent-labeled peptides to detectsessile 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 andvalidate performance measures with fluorescent-labeled peptides with multi-modal imaging.(1b) Identify 5 candidate peptides that bind preferentially to dysplastic colonic mucosa rather than tonormal mucosa using techniques of phage display. To screen, sequence, and synthesizepeptides 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 expertsin translational research and endoscopic imaging from academia and industry to form a,cohesive team todevelop a multi-modal imaging platform that uses fluorescent-labeled peptides to target mucosal dysplasia onwide area endoscopy and confirmed on confocal microscopy. Techniques of phage display will be used todiscover 5 candidate peptides that bind selectively to dysplastic colonic mucosa for use in pilot clinical studiesfor standardizing the imaging protocol and validating performance measures in Aim 2. Finally, the moleculartargets 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-labeledpeptides across the multiple clinical centers, validate performance measures for peptide binding on wide areaendsocopy 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 offluorescent-labeled peptides to localize mucosal dysplasia from colonic adenomas > 5 mm indiameter 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 inadenomas > 5 mm in diameter on confocal microscopy and validate performance measures atmultiple clinical sites.(2c) Develop computer based algorithms to perform registration of white light, NBI, and fluorescenceendoscopic images, and demonstrate real-time in vivo application.The successful completion of this aim will result in the development of standardized imaging protocols andvalidated performance measures for topical administration of fluorescent-labeled peptides to detect dysplasticcolonic mucosa on wide area endoscopy and confirmed on confocal microscopy. These imaging protocols andperformance measures will be used for a future multi-center clinical trial to be planned near the completion ofthis funding period. Furthermore, methods for registration of the white light and fluorescence images will bedeveloped 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 bytuning the fluorescence filter specifications, and measure the depth of peptide penetration into the mucosa withtopical administraton. Develop an integrated imaging protocol for multi-modal platform to detect flat dysplasiain setting of Barrett's esophagus.(3a) Improve the target-to-background ratio for peptide binding on wide area endosocopy bymodifying the fluorescence filter specifications.(3b) Measure the depth of mucosa/ penetration by near-infrared fluorescent-labeled peptides with dualaxes confocal microscope on vertical cross-sectional images from colon adenomas.(3c) Develop imaging protocol to integrate use of wide area fluorescence endoscopy with confocalmicroscopy 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 onwide area endoscopy by optimizing the absorption of excitation by the FITC label and by reducing theautofluorescence background. Furthermore, we will be able to assess the adequacy of topical peptideadministration by measuring the depth of mucosal penetration, and to develop a protocol for integratinglocalization of peptides on wide area endoscopy with confirmation on confocal microsopy.
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