Targeted Imaging of Flat and Depressed Colonic Neoplasms

Molecular imaging is a rapidly growing new discipline in gastrointestinal endoscopy that involves the development of novel imaging probes and instruments to visualize the molecular expression pattern of mucosa in the digestive tract. Several platforms for imaging agents, including antibody and peptide, are being developed to target over expressed biomolecules in cancer. In addition, novel imaging instruments, including fluorescence endoscopy and confocal microscopy, are being developed to provide wide-area surveillance and microscopic examination, respectively. These methods are being applied to detect the presence of flat and depressed colonic neoplasms and to identify tumor margins.

The presence of non-polypoid colorectal neoplasms is drawing significantly greater attention in the effort to improve methods for the early detection of colorectal cancer. These lesions are much more difficult to identify on conventional white light endoscopy because their architectural changes are subtle and can be difficult to distinguish from that of normal colonic mucosa. By comparison, non-polypoid lesions can appear slightly elevated, completely flat, or slightly depressed. In particular, depressed lesions are the most difficult to detect, and have the highest malignant potential. Recent studies have reported a significant miss rate on colonoscopy, and subsequent endoscopic therapy requires an accurate definition of tumor margins. Molecular imaging is a novel, emerging methodology that identifies functional properties of tissue based on the specific molecular signature of the mucosa. This field has been driven in part by recent advances in our understanding of tumor genetics that allow for future personalized oncological therapy. New molecular probes and imaging instruments are being developed to visualize the unique patterns of molecular expression in the mucosa of the digestive tract. Progress is being made in a number of imaging platforms that target biomolecules that are over expressed in cancer. Moreover, novel imaging instruments, including fluorescence endoscopy and confocal microscopy, are being developed to provide wide-area surveillance and microscopic examination, respectively.

Molecular probes

Molecular probes are used to reveal subtle molecular changes in the cells and tissues that are present even in the absence of structural abnormalities, and can identify the over expression of targets to guide therapy in addition to performing diagnostics. Even with the highest resolution optical imaging instruments, exogenous probes are needed to observe important biologic processes, including up-regulation of growth factors, presence of proteolytic enzymes, and expression of cell adhesion molecules, that drive the progression of disease. These probes are fluorescent-labeled for enhancing image contrast on endoscopic detection and for overcoming background tissue autofluorescence. Specific applications include performing in vivo lesion characterization, providing risk stratification, and assessing response to specific therapies. Furthermore, because disease develops from genetic changes that are unique to each individual patient, targeting of specific molecular mechanisms can be used to tailor therapy that will maximize efficacy.

The antibody is one of the first targeting agents used for optical imaging, and is best suited for detection of extracellular targets and cell-surface receptors. These Y-shaped gamma globulins (IgG) express light chains on the distal end of either arm that binds selectively to over expressed targets with high affinity and specificity. The light chains can accommodate a large number of different amino acid sequences, resulting in a high diversity. Antibodies have been developed for several molecular targets that have great therapeutic relevance, including human epidermal growth factor receptor (ERBB2) and epidermal growth factor receptor (EGFR). However, there are several disadvantages for use of whole antibodies in molecular imaging. Their long circulatory half-life results in greater nonspecific binding, and increased tumor vascular permeability often produces a high background level. Moreover, they may elicit an immune reaction with repeated use, and are costly to produce in large quantities.

Peptides have tremendous advantages for performing targeted detection and therapy in the colon because of their high diversity, rapid binding kinetics, and potential for deep penetration into diseased mucosa. In addition, peptides can be labeled easily, are generally nontoxic, and are not immunogenic. These molecular probes have been developed using techniques of phage display, a powerful combinatorial method that uses recombinant DNA technology to generate a library of peptides that bind preferentially to cell surface targets. The protein coat of bacteriophage, such as the filamentous M13 or T7, is genetically engineered to express a very large number (>10 ⁹) of different peptides with unique sequences. Selection of sequences with high-affinity binding is then performed by biopanning the library against cultured cells that over express desired targets. The DNA sequences are then recovered and used to synthesize the candidate peptides. Techniques of phage display have been successfully used to identify peptides that bind preferentially to dysplastic colonic mucosa and not to normal mucosa by employing a biopanning strategy that uses cultured cells and freshly excised normal and dysplastic tissues.

Imaging instruments

Novel endoscopic instruments that are sensitive to fluorescence are needed to perform wide-area surveillance as well as microscopic examination. The endoscope, shown in Fig. 1 , has 2 detectors for collecting white light (WL) images and fluorescence separately. The white light image is collected by the center detector, and the fluorescence image is collected by a second detector located near the periphery. Illumination for both modes is delivered through the 2 fiber light guides. In the WL mode, the full visible spectrum (400 to 700 nm) is provided, whereas in the fluorescence mode, a second filter wheel enters the illumination path, and provides fluorescence excitation in the 395- to 475-nm spectral band. In addition, illumination from 525 to 575 nm provides reflected light in the green spectral regime centered at 550 nm. The fluorescence image is collected by the peripherally located charge-coupled device (CCD) detector that has a 490- to 625-nm band pass filter for blocking the excitation light. Because the increased vasculature in neoplastic mucosa absorbs autofluorescence, it appears with decreased intensity.