Colonoscopy Principles and Practice - part 9 ppt

526 Section 11: Neoplastic Detection and Staging: New Techniques in combination to characterize the cellular origins of whole living colonic crypts, and isolated living colonic epithelial cells derived from primary cell cultures of normal, premalignant, and malignant gastrointestinal tissues [53]. Inoue and colleagues [94] reported the use of CFM to obtain microscopic images from fresh specimens of gastrointestinal mucosa. Briefly, untreated mucosal specimens from the esophagus, stomach, and colon (obtained by endoscopic pinch biopsy, polypectomy, or endoscopic mucosal resection) were fixed in normal saline and examined by CFM with 488 nm excitation in reflectance mode. Images were compared with con- ventional hematoxylin and eosin staining, analyzing the nucleus-to-cytoplasm ratios. The overall diagnostic accuracy of CFM for cancer was 89.7%. The obvious advantages of “blur-free” fluorescence imaging and three-dimensional optical sectioning of ex vivo biologic tissues have made CFM an attractive concept for in vivo fluorescence endoscopic imaging. Recently, a number of prototype confocal endoscopic devices have been described. Optiscan Inc. (Victoria, Australia) introduced a fiberoptic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo [95]. In combination with topically applied fluorescent dyes, optical sections of the mucosal surface of the rat colon were made in vivo, with the colon surgically exposed. A miniaturized scanning mechanism sweeps a 488-nm excitation laser beam across the tissue surface. Images, with scanning speeds of up to 16 frames per sec- ond, have a field of view ranging between ~ 13 and 100 μm, with optional zoom capabilities. The latest ver- sion of the FOCI device was used by the same group in an experimental rat model of inflammatory bowel dis- ease for imaging changes in the mucosal architecture of living colonic tissue in vivo. Morphologic changes associ- ated with disease activity were detected microscopically in vivo using FOCI but were not evident on visual inspec- tion of the colonic surface. Acridine orange enabled imaging of the colonic crypts at the surface of the mucosa. Morphologic changes associated with colitis, including inflammatory cell infiltrate, crypt loss, and crypt distortion, were also detected using this fluores- cent dye. Application of fluorescein and eosin enabled subsurface imaging of the lamina propria surrounding the crypts [96]. This prototype may be the predecessor to a true CFM endoscopic imaging device. Several groups have focused on miniaturizing con- ventional optics to achieve an instrument capable of passing through the accessory channel of standard endo- scopes. For example, Liang and colleagues [97] reported the development of a miniaturized microscope object- ive for endoscopic confocal microscopy. The miniature water-immersion microscope objective is about 10 times smaller in length than a typical commercial objective. Used in a fiber confocal reflectance microscope, the miniature objective offers a field of view of ~ 250 μm with micrometer-level resolution. Advances in silicon-based microelectronic micromach- ined systems (MEMS) may allow further miniaturization of the confocal scanning mechanisms for endoscopy. Laser-und Medizin-Technologie GmbH, Berlin, Ger- many, have developed a miniaturized confocal laser scanning microscope using a two-MEM scanning unit to produce a two-dimensional scan with a field of view of 0.7 × 0.7 mm 2 , an optical resolution of ~ 2 μm. Other developments in MEMS-based confocal endoscopy are in progress [98–100]. The research group led by Dr Arthur Gmitro, at the University of Arizona, has developed a catheter-based real-time confocal fluorescence endoscopic imaging device using 488-nm light from an argon laser. This Fig. 44.12 Confocal fluorescence micrographs of frozen transverse thin sections of (a) normal colon, (b) hyperplastic polyp, and (c) adenomatous polyp mucosa illustrating significant differences in the autofluorescence sources and microdistributions in each type of mucosa. (a) (b) (c) Chapter 44: Optical Techniques 527 uses a fiberoptic imaging bundle and a miniature micro- scope objective and focusing mechanism at the distal end of the catheter. This device achieved a field of view of ~ 430 μm 2 and a lateral resolution of 2 μm. Focus- ing is accomplished via a hydraulic mechanism that moves the distal end of the fiber relative to the lens. Unpublished preliminary imaging results, performed in cell cultures, ex vivo tissue samples, and in vivo animal models with fluorescent contrast dyes, are impressive (http://www.optics.arizona.edu/gmitro/). Despite the promise of CFM technologies and con- tinual technical advances that permit further miniatur- ization, this technology has yet to be demonstrated in vivo in human endoscopic trials. The necessity of topic- ally applied fluorescent dyes for optimal contrast, lack of control of probe placement in the colonic lumen affected by peristalsis, respiration, and aortic pulsation may limit its clinical role. This modality is capable of producing histologic-grade images and may have an important role in differentiating between hyperplastic and adenomat- ous polyps. CFM technologies may be used to histologic- ally define areas detected by wide-scanning technologies such as autofluorescence endoscopic imaging. It will not be useful in the screening of broad areas of mucosa for occult dysplasia. Immunophotodiagnostics Conventional immunohistochemistry permits micro- scopic imaging of biopsied tissues on a “molecular” level by routinely combining chromogenic and fluorescent dyes with the specificity of monoclonal antibodies dir- ectly against tumor-related or tumor-specific antigens. Recently, this idea has been extended to in vivo endo- scopic imaging as a means of enhancing the contrast between tumors and surrounding normal tissue by tar- geting tumors with monoclonal antibodies. For the past 20 years radiopharmacology has relied on the highly specific reactivity of the antigen–antibody complex. For example, radiotherapeutic agents are com- monly conjugated to monoclonal antibodies directed against tumor-associated antigens. These are used to selectively target tumor cells for destruction based on the inherent overexpression of a particular tumor- associated antigen relative to normal tissues [101]. Adapting this principle for fluorescence endoscopy involves the conjugation of a fluorophore dye to a monoclonal antibody or other tumor-targeting moiety, thereby producing a “fluorescent contrast agent.” Typic- ally, these dyes are excited in the red range (> 600– 700 nm) and emit NIR fluorescence efficiently. They have adequate stability for labeling in vivo and pro- duce fluorescence that is detectable through millimeter thicknesses of tissues [102]. Recent improvements in monoclonal antibodies and their derivatives (i.e. frag- ments), the development and commercial availability of NIR-emitting fluorophores, and the availability of high- sensitivity digital cameras in this spectral region have made tumor localization using fluorescence contrast agents practical and attractive. Optimal fluorescent dyes can be selected based on their photophysical and spec- tral properties independent of their tumor-localizing properties [103]. Recent animal studies have demonstrated that fluorophore labeling of monoclonal antibodies pro- duces adequate sensitivity and improved image con- trast [104,105]. In a study in mice by Gutowski and colleagues [106], monoclonal antibody–dye conjugates were prepared using the monoclonal antibody against carcinoembryonic antigen (CEA) (35A7) labeled with indocyanine and 125 I. This study demonstrated the detection of very small nodules (< 1 mm in diameter) but noted a sensitivity decrease with decreasing tumor mass (100% for nodules > 10 mg vs. 78% for nodules ≤ 1 mg). Tumor nodules occult to the naked eye were also detected, and very low conjugate quantities (< 1 ng) were sufficient for tumor nodule visualization. How- ever, the authors also noted false-negative findings with some deep small tumor nodules producing very weak fluorescence that was not detected due to tissue scatter- ing and absorption and the relative insensitivity of their detection camera. To determine the binding of such fluorescently labeled contrast agents in vivo, Kusaka and colleagues [107] used Balb/cA nude mice grafted with human gastric cancer (St-40) and colorectal cancer (COL-4-JCK) cell lines, and the unconjugated antihuman anti-MUC1 mucin antibody to show that specific tumor labeling can be achieved in live mice at the tumor surface, thereby demonstrating that in vivo administration of a fluores- cence-labeled monoclonal antibody for fluorescence detection is possible. However, many difficulties remain with this approach. For example, until recently most monoclonal antibodies were raised in nonhuman hosts (i.e. mice), resulting in a host immune response against them when used in patients. This not only causes the antibodies to be quickly eliminated but also forms immune complexes that damage the kidneys [108]. However, “humanized” monoclonal antibodies have become available recently. In addition, whole antibodies bound in human tumors do not exceed 10 –5 of the admin- istered dose per gram of tumor, hence requiring large amounts of injected conjugated monoclonal antibody, long exposure times, and high sensitivity to achieve adequate tumor brightness and contrast. This limitation is due to the pharmacokinetic properties of conjugated whole antibodies. The production of antibody frag- ments, smaller than the whole antibody, has resulted in some improvements in pharmacokinetics and tissue labeling. In a mouse xenograft model, Ramjiawan and 528 Section 11: Neoplastic Detection and Staging: New Techniques colleagues [109] conjugated an NIR-emitting dye (Cy5.5) to a fragment of antihuman antibody with broad cancer specificity to demonstrate specific binding. Here, the peak fluorescence intensity was detected with a high- sensitivity CCD camera 2 h after injection. The presence and distribution of the conjugated fragment revealed that about 16 and 73% was located in the tumor and the kidneys respectively. Use of smaller antibody fragments produced rapid tumor uptake, better penetration (at the expense of reduced circulation time), more homogen- eous tumor penetration, and reduced immunogenicity [110]. Fluorescent dyes can also be targeted to tumor tissues by means other than monoclonal antibodies. For ex- ample, Weissleder and colleagues [111] coupled an NIR fluorophore to a biocompatible polymer. This was administered to tumor-bearing mice and was taken up by tumor cells via pinocytosis. The intracellular release of the fluorophore by the protease cathepsin D resulted in a fluorescence signal detected in vivo in subnanomolar quantities and at depths sufficient for clinical imaging. The authors demonstrated that specific enzyme activity in a tumor could be imaged by fluorescence contrast agents in vivo. In addition, Marten and colleagues [112] studied the expression of the protease cathepsin B in dysplastic adenomatous polyps. Cathepsin B was con- sistently overexpressed in adenomatous polyps. When mice were injected intravenously with the reporter probe, intestinal adenomas became highly fluorescent, indicating high cathepsin B activity. Even microscopic adenomas undetected by white-light imaging were readily detected by fluorescence, the smallest lesion being ~ 50 μm in diameter. Control animals were either noninjected or injected with a nonspecific NIR fluores- cent probe (indocyanine green, ICG); in these, adenomas were only barely detectable above the background. This impressive study demonstrated the potential of using fluorescently labeled enzyme-sensing probes to detect such gastrointestinal lesions against adjacent normal mucosa. Currently, work in our laboratory is assessing the utility of colonic mucins as a possible target for colonic adenomas and adenocarcinomas. Preliminary results have demonstrated distinct contrast enhancement of the tumor compared with surrounding normal tissues using the labeled cc49, which recognizes a tumor-associated glycoprotein antigen, in comparison with control auto- fluorescence images. Tumor visualization was apparent as early as 2 h with the fluorescence-conjugated cc49 probe, while maximum contrast was at 48 h after injec- tion (Fig. 44.13). Hence, this demonstrated the selective in vivo targeting of fluorescence dye to tumor-associated mucins, resulting in the enhanced fluorescence detec- tion of small (~ 4–5 mm diameter) xenografted human colonic tumors [113]. Clinical evaluation Preliminary in vivo evaluation of fluorescence contrast agents in patients has been reported in a very limited number of studies. Early vascular changes were assessed in Crohn’s disease in a prospective endoscopic study of 10 asymptomatic patients using unconjugated 10% sodium fluorescein [114]. Fluorescence endoscopy was used to evaluate the mucosal microcirculation of the neoterminal ileum in relation to endoscopic recurrence Fig. 44.13 Example of in vivo tumor targeting with a fluorescence contrast agent. This was achieved by administering a fluorescence- conjugated antibody directed against a tumor-associated glycoprotein (TAG72) in a xenograft nude mouse model of human colon cancer. (a) White-light image of dorsal side of mouse indicating tumor site (arrow). (b) Whole-body fluorescence image shows significant enancement of tumor-to-normal contrast, thereby allowing the tumor to be detected easily with fluorescence 48 h after administration of tumor-targeted fluorescence probe. (a) (b) Chapter 44: Optical Techniques 529 in patients who had undergone ileocolonic resection for Crohn’s disease. The fluorescence observed may reflect vasodilation associated with inflammation or genuine microvascular lesions. Correlation with histology sug- gested that these early vascular lesions were secondary to the inflammatory process. In another study with ex vivo human tissues, Bando and colleagues [109] developed an NIR-excited fluores- cent dye, ICG-sulfo-OSu, conjugated to antisulfomucin and anti-MUC1 antibodies in paraffinized tissue sections from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal can- cer. They found that antibody staining patterns varied depending on the organs, histologic types, and depth of the cancers. Generally, staining on the mucosal surface of cancer tissues was retained and images of cancer cells were obtained by infrared fluorescence observation using the labeled anti-MUC1 antibody. These authors noted the difficulty of adapting this staining method to in vivo conditions, where the antibody agent would be administered to the luminal surface because of such problems as surface mucus and pH. Hayashi and col- leagues [116] performed similar studies of immunostain- ing of ICG-conjugated antiepithelial membrane antigen antibodies on nonfixed freshly excised tissue samples by eliminating these factors under various conditions. Results suggested that vital immunohistochemical stain- ing is possible under optimized conditions. Ito and col- leagues [117], in a study of only three patients, confirmed that such immunofluorescent staining using ICG derivat- ive (ICG-sulfo-OSu) conjugated to anti-CEA antibodies could be performed in vivo to detect small gastric cancers. Tatsuta and colleagues [118] labeled anti-CEA mono- clonal antibodies with fluorescein isothiocyanate (FITC) to study ex vivo human gastric lesions. FITC has a high fluorescence efficiency and excitation and detection wavelengths (~ 488 nm excitation, ~ 520 nm emission). The conjugated antibody was applied topically. Of 30 tumors, 27 (90%) showed positive fluorescence after 60 min with no false positives, whereas only 2 of 5 cancers (40%) could be detected earlier than 60 min. To remove gastric mucus and improve the binding of the tumor with the labeled antibody, pretreatment with a mixture of proteinases, sodium bicarbonate, and dimethyl-polysiloxane was used. In vivo, this would add another 90 min to the endoscopic examination. No significant relationship between positive fluorescence and tumor type or stage was found. However, positive fluorescence could also not be demonstrated in benign gastric lesions. In 1998, Keller and colleagues [119] coined the term “immunoscopy” in a report on the detection of colorectal carcinomas and villous adenomas in surgically resected tissue samples. Fluorescence from FITC-labeled anti- CEA antibody was detected using a sensitive filtered photographic camera in 27 of 28 cancers and 1 of 2 adenomas, as well as in 6 of 18 normal controls, giving a sensitivity of 93% and specificity of 67%. There are two published reports of the use of fluores- cence-conjugated monoclonal antibodies in humans in vivo. The first study used a monoclonal fluoresceinated anti-CEA conjugate to detect human colon carcinoma [120]. Upon laser irradiation, clearly detectable hetero- geneous green fluorescence from the dye–antibody con- jugate was visually observed on all six tumors; minimal fluorescence was detectable on normal mucosa. Tissue autofluorescence from both tumor and normal mucosa was subtracted by real-time image processing. In the sec- ond in vivo study of 27 patients with colonic polypoid lesions, Keller and colleagues [121] used a locally admin- istered fluorescein-labeled anti-CEA monoclonal anti- body for in vivo fluorescence endoscopic detection of colorectal dysplasia and carcinoma. During conven- tional WLE colonoscopy, the conjugated monoclonal antibody was applied directly to the mucosal surface. Specific fluorescence was visualized with a conven- tional fiber endoscope modified for fluorescence imag- ing with fluorescence bandpass filters (520 nm). Here, fluorescence was present in 19 of 25 carcinomas and 3 of 8 adenomas. Interestingly, the technique failed in the presence of mucosal ulceration or bleeding. One fluorescence-positive villous adenoma showed high- grade dysplasia, while another fluorescence-positive polypoid lesion was diagnosed as carcinoma in adenoma. Normal-appearing mucosa was fluorescence negative in all cases. In all cases (without ulceration or bleeding), the specificity of fluorescence endoscopy was 100%, the sensitivity was 78.6%, and the accuracy was 89.3%. Subsequent immunohistochemistry on biopsied tissues revealed that endoscopic fluorescence significantly cor- related with CEA expression of luminal epithelial cells. Larger trials to demonstrate the value of this technique for differential diagnosis are currently underway. However, despite these encouraging initial results, several important issues must be resolved. Selection of the best tumor-associated targets (i.e. monoclonal antibodies, peptides, enzymes) is not clear, and the pos- sibilities are seemingly endless. For example, antigens expressed on the cell surface, such as growth factor receptors, mucins, and cell adhesion molecules, can be targeted by their respective fluorescence-conjugated antibodies, as can intracellular markers such as enzymes [122,123]. Biomarker studies continue to be reported in the literature for each segment of the gastrointestinal tract, in which a variety of molecular markers are evaluated in large tissue archives for their potential as diagnostic and/or prognostic indicators (CEA, mucin epitopes, etc.). It is possible that each segment of the gastrointestinal tract will have its own specific diag- nostically relevant markers. Additionally, simultaneous 530 Section 11: Neoplastic Detection and Staging: New Techniques localization of multiple reagents is made possible by labeling multiple NIR fluorophores; thus background subtraction and differential labeling of multiple tumor- associated components can be performed. Difficulties in using the fluorophore labels are mainly related to light scattering and absorption in tissues, although detection of small tumors at depths of several millimeters should be feasible. Given the limitations in current fluorescence endoscopic imaging in detecting very early gastro- intestinal lesions or preventing false positives due to confounding concurrent conditions (i.e. inflammation), these developments significantly complement existing fluorescence endoscopy. An overview: the optimal technique Several new optically based techniques are being evalu- ated with a view to enhancing the diagnostic capability of clinical gastrointestinal endoscopy. The ideal sys- tem should function in real time and combine excellent diagnostic accuracy with wide mucosal area surveil- lance. A major issue is how the detection of dysplasia and intramucosal cancer will ultimately fit into the treat- ment algorithm. For example, who and/or what should be treated with endoscopic ablation, chemoprevention, or resective surgery? Treatment will be markedly affected by accurate staging of lesions, via super high-resolution ultrasound or OCT. Short of replacing conventional biopsy, such technologies should provide guidance in locating optimal sites for targeted biopsy and be able to monitor ablative therapies such as photodynamic therapy. In this regard, fluorescence endoscopic imag- ing, with its wide field of view, has already detected early lesions, scars, and demonstrated reliability in differentiating hyperplastic vs. adenomatous polyps in vivo, and so appears most appealing and practical for screening. Additionally, fluorescence endoscopy does not require dye spraying and is relatively fast. However, many issues, such as optimal excitation and emission wavelength(s), confounding background fluorescence from metaplasia or inflammation (false positives), and artifacts due to motility, remain unresolved. Addition- ally, it is not clear if exogenous fluorophores (e.g. pro- drugs like ALA) will be necessary to achieve clinically useful sensitivity and specificity. Despite its very high molecular specificity, Raman spectroscopy suffers the same weakness as all point spectroscopies, in that its clinical use is limited by prac- ticality. This is also the case for LSS, which has shown promise in differentiating dysplasia (low and high grade) in Barrett’s esophagus for example, based on nuclear size and density. However, used adjunctively with imaging techniques that survey large tissue sur- faces for targeting suspicious lesions, the molecular specificity of Raman spectroscopy or the sensitivity to subcellular scattering features of LSS may be useful for in situ diagnosis. These combinations are yet to be attempted. OCT is attractive, although current OCT prototypes have several limitations that prevent their use as a stand- alone technique for surveillance. The main clinical advantage of OCT is the ability to stage mucosal disease as a means of identifying those patients where dysplasia and intramucosal cancer does not penetrate into the submucosa, and therefore would be ideal for curative endoscopic therapy. Although it has the potential to yield histologic details, this resolution has not yet been achieved in a real-time endoscopic system. Additionally, OCT will only be applicable for viewing small areas of the gastrointestinal tract. However, with anticipated im- provements in resolution (subcellular level) and speed, OCT may become the technique of choice for surveil- lance and staging in the future. Furthermore, Doppler OCT may offer an additional endoscopic capability for imaging blood flow in mucosal and submucosal micro- vasculature, and may be of use in assessing changes in microcirculation resulting from in situ therapies. At the moment, CFM has only been demonstrated on ex vivo human gastrointestinal tissues, including normal, metaplastic and preneoplastic lesions in the esophagus, stomach, and colon. Distinct fluorescence differences have been found between normal and abnormal mucosal tissues in each organ, yet this is likely not to be diagnost- ically useful in endoscopic fluorescence imaging, since the already weak mucosal fluorescence is overwhelmed by very strong fluorescence from deeper gastrointestinal tissue layers. To date, CFM techniques have been used primarily ex vivo to study and explain the origins of both tissue autofluorescence and the microdistribution of photosensitizers. The role of CFM in vivo may exploit the subtle differences in mucosal (auto)fluorescence between normal and abnormal colonic tissues by interrogation of only epithelia and lamina propria, hence reducing con- tribution from the collagen-rich submucosa. However, at present, CFM involves the use of fluorescent contrast dyes, which make the process more labor intensive. Currently, limitations in available technology prevent the clinical utility of “confocal microendoscopy.” All point spectroscopic techniques, as well as magni- fication endoscopy, are inherently limited by the small tissue area they sample. However, they contain more detailed information about tissue than any imaging system, which may translate into more accurate tissue differentiation. Rather than competing with an imaging system, the “best” instrument for surveillance may com- bine imaging and spectroscopy. For instance, a lesion could be detected by fluorescence imaging or OCT and its dysplastic nature characterized by Raman spec- troscopy. However, in this era of cost containment, such an approach may be cost-prohibitive. Moreover, all Chapter 44: Optical Techniques 531 these expensive optical modalities will need to be com- pared against cheaper and equally promising alternat- ives such as chromoendoscopy, for which the dye is cheap and colonoscopes are readily available. However, dye spraying is labor intensive. By far the least reported method to date is the use of immune-related fluorescence contrast agents. A limited number of ex vivo studies have demonstrated relative gastrointestinal tumor selectivity with highly fluorescent conjugated antibodies to well-known tumor-associated biomarkers. Such contrast agents have also been evalu- ated in a very limited number of patients with encourag- ing enhancement of tumor contrast. There are important technical issues to be resolved, such as finding the optimum site- and pathology-specific biomarkers, con- jugate design, false positives associated with inflammat- ory conditions, optimizing relative tumor uptake, cost, and safety. However, advances in our understanding of cancer biology, tumor-associated gastrointestinal bio- markers, conjugation biochemistry, safety assessments, and fluorescence imaging hardware and software con- tinue. This technology also offers a means of improving our fundamental understanding of disease processes in the gastrointestinal tract on a molecular level. It is con- ceivable that in the future molecular-targeted fluores- cence endoscopic imaging will allow earlier detection and characterization of gastrointestinal disease, and may offer in vivo noninvasive monitoring of the func- tions of a variety of proteins as well as assessment of treatment effects. Conventional endoscopy has relied strongly on the detection of subtle topographic and morphologic changes associated with the evolution of dysplasia through to invasive cancer, which may only become apparent at an advanced stage. However, the future of diagnostic endoscopy will certainly involve “molecular imaging,” whether fluorescence, Raman, or immunophotodetec- tion. This may translate into a truly early detection of preneoplastic changes, when therapeutic intervention can result in cure. “Optical biopsy” refers to tissue diagnosis based on in situ optical measurements, which would eliminate the need for tissue removal. The above-mentioned optical techniques are striving toward this goal but none are likely to replace conventional biopsy and histopatho- logic interpretation in the near future. Future implemen- tation of these optically based methods for endoscopic detection of colonic neoplastic disease will likely involve a combination of more than one technique. Although they demonstrate potential for better diagnosis, these modalities are still in their infancy, with future tech- nologic refinement and large-scale clinical trials needed to assess their utility and limitations. To date, there have been no publications regarding the assessment of any commercial systems in multicenter comparative clinical trials in the gastrointestinal tract. Ultimately, whether these optical techniques will become part of standard clinical endoscopic practice or remain on the sidelines can be summed up in two questions: how much better will they perform and at what cost? Summary Gastrointestinal malignancies continue to be the second leading cause of cancer-related deaths in the developed world. With regard to colonic neoplasms, early detection and therapeutic intervention have been demonstrated to significantly improve patient survival. Conventional screening tools include standard WLE, which has no trouble in detecting polypoid lesions in the well- prepared colon. Well-defined endoscopic surveillance biopsy protocols aimed at the early detection of dys- plasia and malignancy have been undertaken for groups at high risk. Unfortunately, the relatively poor sensit- ivity associated with WLE is a significant limitation. In patients with diffuse chronic inflammatory bowel disease (i.e. UC and Crohn’s disease) the detection of dysplasia is a recurring problem even with multiple ran- dom biopsy protocols. In these and other diseases, major efforts are underway in the development and evaluation of alternative diagnostic techniques that may be used adjunctively with conventional endoscopy to improve detection of colonic neoplastic disease. This chapter has focused on notable developments made at the forefront of research in novel optically based endoscopic modalities that rely on the interactions of various wavelengths of light with tissues. A condensed introduction to the biophysical interaction between light and biologic tissues is followed by a “state-of-the-art” review of fluorescence endoscopic spectroscopy and imaging, Raman spectroscopy, LSS, OCT, confocal fluo- rescence endoscopy, and immunophotodiagnostics. For each topic, background information is discussed, fol- lowed by a report on the most relevant clinical evalu- ations of the respective technique. The final section “An overview: the optimal technique” discusses whether these new developments offer significant improvement in the endoscopic diagnosis of early dysplastic lesions in concert with the traditional approach of targeted biopsies or submucosal resection. The modality that will most appeal to the traditional endoscopist will be fluorescence endoscopic imaging, where the whole mucosal surface will be seen on a mon- itor similar to that seen with WLE, but with a simul- taneous computer-generated colored fluorescent image where dysplastic areas will stand out against normal tis- sue. In contrast, point-directed methods such as Raman and fluorescence spectroscopy, LSS, confocal fluores- cence endoscopy, and OCT will not likely play an import- ant role in screening for dysplastic lesions because of 532 Section 11: Neoplastic Detection and Staging: New Techniques the immense surface area of the colon. These additional optically based procedures may play an ancillary role in the histologic or molecular interrogation of abnormal areas detected by other means, such as fluorescence imaging or dye spraying/chromoendoscopy. In the future, histologic or molecular grade interpretations may be possible without the need for tissue removal, the true “optical biopsy.” This enhancement of the endo- scopist’s ability to detect subtle neoplastic changes in the colonic mucosa in real time and improved staging of lesions could result in curative endoscopic ablation of these lesions, and in the long term improve patient sur- vival and quality of life. Acknowledgments The authors wish to thank the following individuals for their respective contributions to this chapter: Dr Brian C. Wilson, Dr Lothar Lilge, Dr Robert Weersink, Maria Cirocco, Nancy Bassett, Dr Louis Michel Wong Kee Song, Andrea Molckovsky, Dr Alex Vitkin, Victor Yang, Maggie Gordon, and Dr Shou Tang. We wish to acknowledge the support of the follow- ing organizations for work at our institution, in these endoscopic developments: LIFE and immunophoto- diagnostics studies received support from Xillix Techno- logies Corp. and the Ontario Research and Development Challenge Fund. Work on OCT is supported by Photonics Research Ontario and the National Sciences and Engineering Research Council of Canada. References 1 Kudo S, Tamure S, Nakajima T et al. Depressed type of colorectal cancer. Endoscopy 1995; 27: 54–7. 2 Simmons BD, Morrison AS, Lev R. Relationship of polyps to cancer of the large intestine. J Natl Cancer Inst 1992; 84: 962–6. 3 Winawer SJ, Zauber AG, May NH et al. Prevention of colo- rectal cancer by colonoscopic polypectomy. N Engl J Med 1993; 329: 1977–81. 4 Hixon LJ, Fennerty MB, Sampliner RE. et al. Prospective study of the frequency and size distribution of polyps missed by colonoscopy. J Natl Cancer Inst 1990; 82: 1769–72. 5 Rex DK, Cutler CS, Lemmel GT et al. Colonoscopic miss- rate of adenomas determined by back-to-back colono- scopies. Gastroenterology 1997; 112: 24–8. 6 Bensen S, Mott LA, Dain B et al. The colonoscopic miss rate and true one-year recurrence of colorectal neoplastic polyps. Am J Gastroenterol 1999; 94: 194–9. 7 Lev R. Adenomatous Polyps of the Colon: Pathological and Clinical Features. New York: Springer Verlag, 1990. 8 Ishikawa M, Mibu R, Nakamura K et al. Correlation between macroscopic morphologic features and malig- nant potential of colorectal sessile adenomas. Dis Colon Rectum 1996; 39: 1275–81. 9 Kudo S, Hirota S, Nakajima T et al. Colorectal tumors and pit pattern. J Clin Pathol 1994; 47: 880. 10 Kudo S, Tamura S, Nakajima T et al. Diagnosis of colorectal tumorous lesions by magnifying endoscopy. Gastrointest Endosc 1996; 44: 8–14. 11 Watanabe T, Muto T, Sawada T, Miyaki M. Flat adenoma as a precursor of colorectal carcinoma in hereditary nonpolyposis colorectal carcinoma. Cancer 1996; 77: 627– 34. 12 Fujii T, Rembacken BJ, Dixon MF et al. Flat adenomas in the United Kingdom: are treatable cancers being missed? Endoscopy 1998; 30: 437–43. 13 Takayama T, Katsuki S, Takahashi Y et al. Aberrant crypt foci of the colon as precursors of adenoma and cancer. N Engl J Med 1998; 339: 1277–84. 14 Drezek R, Sokolov K, Utzinger U et al. Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and im- plications. J Biomed Opt 2001; 6: 385–96. 15 DaCosta RS, Lilge L, Kost J et al. Confocal fluorescence microscopy/macroscopy and microspectrofluorimetry ana- lysis of human colorectal tissues. J Anal Morph 1997; 4: 24–9. 16 DaCosta RS, Andersson H, Wilson BC. Characterization of the excitation and emission matrices of possible fluo- rophores in human tissues and cells. J Photochem Photobiol (In press). 17 Wagnieres GA, Star WM, Wilson BC. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem Photobiol 1998; 68: 603–32. 18 Andersson-Engels S, Klinteberg C, Svanberg K et al. In vivo fluorescence imaging for tissue diagnosis. Phys Med Biol 1997; 42: 815–24. 19 Bigio IJ, Mourant JR. Ultraviolet and visible spectroscopy for tissue diagnostics: fluorescence spectroscopy and elastic-scattering spectroscopy. Phys Med Biol 1997; 42: 803–14. 20 DuVall A, Wilson BC, Marcon N. Tissue autofluorescence. Ann Gastrointest Endosc 1997; 10: 25–30. 21 DaCosta RS, Wilson BC, Marcon NE. Light-induced fluorescence endoscopy of the gastrointestinal tract. Gas- trointest Endosc Clin North Am 2000; 10: 37–69. 22 Stepp H, Sroka R, Baumgartner R. Fluorescence endo- scopy of gastrointestinal diseases: basic principles, tech- niques, and clinical experience. Endoscopy 1998; 30: 379–86. 23 Bohorfoush AG. Tissue spectroscopy for gastrointestinal diseases. Endoscopy 1996; 28: 372–80. 24 Richards-Kortum R, Sevick-Muraca E. Quantitative optical spectroscopy for tissue diagnosis. Annu Rev Phys Chem 1996; 47: 555–606. 25 Fiarman GS, Nathanson MH, West B et al. Differences in laser-induced autofluorescence between adenomatous and hyperplastic polyps and normal colonic mucosa by confocal microscopy. Dig Dis Sci 1995; 40: 1261–8. 26 Fijan S, Honigsmann H, Ortel B. Photodynamic therapy of epithelial skin tumors using delta aminolevulinic acid and desferrioxamine. Br J Dermatol 1995; 133: 282–8. 27 Haringsma J, van Ierland-van Leeuwen M, Tytgat GNJ. Endoscopic localization of dysplasia using laser induced fluorescence (abstract). In: Program of the Annual Meeting of the American Gastroenterological Association and Digestive Diseases Week. Washington, DC. Gastroenterology 1997; 112: A576. 28 Haringsma J, Tytgat GN. The value of fluorescence tech- niques in gastrointestinal endoscopy: better than the endo- Chapter 44: Optical Techniques 533 scopist’s eye? I. The European experience. Endoscopy 1998; 30: 416–18. 29 Kapadia CR, Cutruzzola FW, O’Brien KM et al. Laser- induced fluorescence spectroscopy of human colonic mucosa: detection of adenomatous transformation. Gastro- enterology 1990; 99: 150–7. 30 Richards-Kortum R, Rava RP, Fitzmaurice M et al. Spectroscopic diagnosis of colonic dysplasia. Photochem Photobiol 1991; 53: 777–86. 31 Schomacker KT, Frisoli JK, Compton CC et al. Ultraviolet laser-induced fluorescence of colonic tissue: basic bio- logy and diagnostic potential. Lasers Surg Med 1992; 12: 63–78. 32 Cothren RM, Richards-Kortum R, Sivak MV et al. Gas- trointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy. Gastrointest Endosc 1990; 36: 105–11. 33 Cothren RM, Sivak MV, Van Dame J et al. Detection of dys- plasia at colonoscopy using laser-induced fluorescence: a blinded study. Gastrointest Endosc 1996; 44: 168–76. 34 Schomacker KT, Frisoli JK, Compton CC et al. Ultraviolet laser-induced fluorescence of colonic polyps. Gastroentero- logy 1992; 102: 1155–60. 35 Vo-Dinh T, Panjehpour M, Overholt BF, Farris C, Buckley FP III, Sneed R. In vivo cancer diagnosis of the esophagus using differential normalized fluorescence (DNF) indices. Lasers Surg Med 1995; 16: 41–7. 36 Bottiroli G, Croce AC, Locatelli D et al. Natural fluores- cence of normal and neoplastic human colon: a compre- hensive “ex vivo” study. Lasers Surg Med 1995; 16: 48–60. 37 Marchesini R, Pignoli E, Tomatis S et al. Ex vivo optical properties of human colon tissue. Lasers Surg Med 1994; 15: 351–7. 38 Mycek MA, Schomacker KT, Nishioka NS. Colonic polyp differentiation using time-resolved autofluorescence spec- troscopy. Gastrointest Endosc 1998; 48: 390–4. 39 Van Dam J, Bjorkman DJ. Shedding some light on high- grade dysplasia. Gastroenterology 1996; 111: 247–9. 40 Namihisa A, Watanabe H, Tanaka H et al. Detection of gastric lesions by endoscopic autofluorescence real-time imaging system (light-induced fluorescence endoscopy). Gastrointest Endosc 1997; 45: A28. 41 Lam S, Hung JYC, Kennedy SM et al. Detection of dyspla- sia and carcinoma in situ by ratio fluorometry. Am Rev Respir Dis 1992; 146: 1458–61. 42 Lam S, MacCauley C, Hung J et al. Detection of dysplasia and carcinoma in situ with a lung imaging fluorescence endoscope device. J Thorac Cardiovasc Surg 1993; 105: 1035–40. 43 Yano H, Iishi H, Tatsuta M. Diagnosis of early gastric can- cers by endoscopic autofluorescence imaging system. Endoscopy 1996; 28: S29. 44 Messmann H, Kullmann F, Wild T et al. Detection of dys- plastic lesions by fluorescence in a model of colitis in rats after previous photosensitization with 5-aminolaevulinic acid. Endoscopy 1998; 30: 333–8. 45 Saitoh Y, Waxman I, West AB et al. Prevalence and dis- tinctive biologic features of flat colorectal adenomas in a North American population. Gastroenterology 2001; 120: 1657–65. 46 Wong Kee Song LM, Wilson BC, Marcon NE. Diagnostic potential of light-induced fluorescence endoscopy in the colon. Am J Gastroenterol 2001; 96: P395. 47 Panjehpour M, Overholt BF, Schmidhammer JL et al. Spectroscopic diagnosis of esophageal cancer: new classi- fication model, improved measurement system. Gastrointest Endosc 1995; 41: 577–81. 48 Panjepour M, Overholt BF, Vo-Dinh T, Haggitt RC, Edwards DA, Buckley FR III. Endoscopic fluorescence detection of high-grade dysplasia in Barrett’s esophagus. Gastroenterology 1996; 111: 93–101. 49 DaCosta RS, Lilge L, Kost J et al. Confocal fluorescence microscopy, microspectrofluorimetry and modeling stud- ies of laser induced fluorescence endoscopy (LIFE) of human colon tissue. SPIE Proceedings 1996; 2975: 98–107. 50 Romer TJ, Fitzmaurice M, Cothren RM et al. Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis. Am J Gastroenterol 1995; 90: 81–7. 51 Zonios GI, Cothren RM, Arendt JT et al. Morphological model of human colon tissue fluorescence. IEEE Trans Biomed Eng 1996; 43: 113–22. 52 DaCosta RS. Mechanisms of fluorescence endoscopy of the human colon. MSc thesis, University of Toronto, Toronto, 2000. 53 DaCosta RS, Andersson H, Wilson BC. Autofluorescence characterization of isolated whole crypts and primary cul- tured human epithelial cells from normal, hyperplastic and adenomatous colonic mucosa. In press. 54 Namihisa A, Miwa H, Watanabe H, Kobayashi O, Ogihara T, Sato N. A new technique: light-induced fluorescence endoscopy in combination with pharmacoendoscopy. Gastrointest Endosc 2001; 53: 343–8. 55 Krammer B, Uberriegler K. In-vitro investigation of ALA- induced protoporphyrin IX. J Photochem Photobiol B 1996; 36: 121–6. 56 Kennedy JC, Marcus SL, Pottier RH. Photodynamic therapy (PDT) and photodiagnosis (PD) using endogen- ous photosensitization induced by 5-aminolevulinic acid (ALA): mechanisms and clinical results. J Clin Laser Med Surg 1996; 14: 289–304. 57 Baumgartner R, Huber RM, Schulz H et al. Inhalation of 5- aminolevulinic acid: a new technique for fluorescence detection of early stage lung cancer. J Photochem Photobiol B 1996; 36: 169–74. 58 Leveckis J, Burn JL, Brown NJ, Reed MW. Kinetics of endogenous protoporphyrin IX induction by aminole- vulinic acid: preliminary studies in the bladder. J Urol 1994; 152: 550–3. 59 Loh CS, Vernon D, MacRobert AJ, Bedwell J, Bown SG, Brown SB. Endogenous porphyrin distribution induced by 5-aminolaevulinic acid in the tissue layers of the gastroin- testinal tract. J Photochem Photobiol B 1993; 20: 47–54. 60 Peng Q, Berg K, Moan J et al. 5-Aminolevulinic acid- based photodynamic therapy: principles and experimental research. Photochem Photobiol 1997; 65: 235–51. 61 Loh CS, MacRobert AJ, Bedwell J, Regula J, Krasner N, Bown SG. Oral versus intravenous administration of 5′-aminolaevulinic acid for photodynamic therapy. Br J Cancer 1993; 68: 41–51. 62 Battle AMC. Porphyrins, porphyrias, cancer and photody- namic therapy: a model of carcinogenesis. J Photochem Photobiol B 1993; 20: 5–22. 63 Rick K, Sroka R, Stepp H et al. Pharmacokinetics of 5′- aminolevulinic acid-induced protoporphyrin IX in skin and blood. J Photochem Photobiol B 1997; 40: 313–19. 534 Section 11: Neoplastic Detection and Staging: New Techniques 64 Webber J, Kessel D, Fromm D. Side effects and photosens- itization of human tissue after aminolevulinic acid. J Surg Res 1997; 68: 31–7. 65 Lange N, Jichlinski P, Zellweger M et al. Photodetection of early human bladder cancer based on the fluorescence of 5-aminolaevulinic acid hexylester-induced protopor- phyrin IX: a pilot study. Br J Cancer 1999; 80: 185–93. 66 Messmann H, Knuchel R, Baumler W, Holstege A, Scholmerich J. Endoscopic fluorescence detection of dys- plasia in patients with Barrett’s esophagus, ulcerative colitis, or adenomatous polyps after 5-aminolevulinic acid-induced protoporphyrin IX sensitization. Gastrointest Endosc 1999; 49: 97–101. 67 Eker C, Montan S, Jaramillo E et al. Clinical spectral charac- terisation of colonic mucosal lesions using autofluores- cence and delta aminolevulinic acid sensitisation. Gut 1999; 44: 511–18. 68 Bird RP. Role of aberrant crypt foci in understanding the pathogenesis of colon cancer. Cancer Lett 1995; 29: 55– 71. 69 Riddell RH, Goldman H, Ransohoff DF et al. Dysplasia in inflammatory bowel disease: standardization classifica- tion with provisional clinical applications. Hum Pathol 1983; 14: 931–68. 70 Messman H, Mlkvy P, Monta S et al. Endoscopic and microscopic fluorescence studies in patients with ucerative colitis after 5-aminolevulinic acid photosensitization. Gastroenterology 1995; 108: 506. 71 Gahlen J, Stern J, Pressmar J, Bohm J, Holle R, Herfarth C. Local 5-aminolevulinic acid application for laser light- induced fluorescence diagnosis of early staged colon cancers in rats. Lasers Surg Med 2000; 26: 302–7. 72 Regula J, MacRobert AJ, Gorchein A et al. Photosensitisa- tion and photodynamic therapy of oesophageal, duodenal and colorectal tumors using 5-aminolaevulinic acid in- duced protoporphyrin IX: a pilot study. Gut 1995; 36: 67–75. 73 Van den Boogert J, van Hillegersberg R, de Rooij FW et al. 5-Aminolaevulinic acid-induced protoporphyrin IX accumulation in tissues: pharmacokinetics after oral or intravenous administration. J Photochem Photobiol B 1998; 44: 29–38. 74 Endlicher E, Rummele P, Hausmann F et al. Protopor- phyrin IX distribution following local application of 5-aminolevulinic acid and its esterified derivatives in the tissue layers of the normal rat colon. Br J Cancer 2001; 85: 1572–6. 75 Van den Boogert J, Houtsmuller AB, de Rooij FWM et al. Kinetics, localization, and mechanism of 5-aminolevulinic acid-induced porphyrin accumulation in normal and Barrett’s like rat esophagus. Lasers Surg Med 1999; 24: 3–13. 76 Leunig A, Rick K, Stepp H et al. Fluorescence imaging and spectroscopy of 5-aminolevulinic acid-induced protopor- phyrin IX for the detection of neoplastic lesions of the oral cavity. Am J Surg 1996; 172: 674–7. 77 Stummer WS, Stocker S, Wagner S et al. Intraoperative detection of malignant glioma by 5-ALA induced por- phyrin fluorescence. Neurosurgery 1998; 42: 518–26. 78 Marcus SL, Sobel RS, Golub AL et al. Photodynamic therapy (PDT) and photodiagnosis (PD) using endogen- ous photosensitization induced by 5-aminolevulinic acid (ALA): current clinical and developmental status. J Clin Laser Med Surg 1996; 14: 59–66. 79 Shim MG, Wilson BC. The effects of ex vivo handling pro- cedures on the near-infrared Raman spectra of normal mammalian tissues. Photochem Photobiol 1996; 63: 662–71. 80 Hanlon EB, Manoharan R, Koo TW et al. Prospects for in vivo Raman spectroscopy. Phys Med Biol 2000; 45: 1– 59. 81 Shim MG, Wilson BC. Development of an in vivo Raman spectroscopic system for diagnostic applications. J Raman Spectrosc 1997; 28: 131–42. 82 Shim MG, Wilson BC, Marple E, Wach M. A study of fiber- optic probes for in vivo medical Raman spectroscopy. Appl Spectrosc 1999; 53: 619–27. 83 Molckovsky A, Song LM, Shim MG, Marcon NE, Wilson BC. Diagnostic potential of near-infrared Raman spec- troscopy in the colon: differentiating adenomatous from hyperplastic polyps. Gastrointest Endosc 2003; 57: 396–402. 84 Wallace MB, Perelman LT, Backman V et al. Endoscopic detection of dysplasia in patients with Barrett’s esophagus using light-scattering spectroscopy. Gastroenterology 2000; 119: 677–82. 85 Backman V, Wallace MB, Perelman LT et al. Detection of preinvasive cancer cells. Nature 2000; 406: 35–6. 86 Georgakoudi I, Jacobson BC, Van Dam J et al. Fluores- cence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett’s esophagus. Gastroenterology 2001; 120: 1620–9. 87 Tearney GJ, Brezinski ME, Southern JF, Bouma BE, Boppart SA, Fujimoto JG. Optical biopsy in human gas- trointestinal tissue using optical coherence tomography. Am J Gastroenterol 1997; 92: 1800–4. 88 Kobayashi K, Izatt JA, Kulkarni MD, Willis J, Sivak MV Jr. High-resolution cross-sectional imaging of the gastroint- estinal tract using optical coherence tomography: pre- liminary results. Gastrointest Endosc 1998; 47: 515–23. 89 Sivak MV, Kobayashi Y, Izatt JA et al. High-resolution endoscopic imaging of the GI tract using optical coherence tomography. Gastrointest Endosc 2000; 51: 474–9. 90 Yang VXD, Gordon ML, Mok A et al. Improved phase- resolved optical Doppler tomography using the Kasai velocity estimator and histogram segmentation. Optics Communictions 2002; 208: 209–14. 91 Rollins AM, Yazdanfar S, Barton JK, Izatt JA. Real-time in vivo color Doppler optical coherence tomography. J Biomed Opt 2002; 7: 123–9. 92 Pitris C, Jesser C, Boppart SA, Stamper D, Brezinski ME, Fujimoto JG. Feasibility of optical coherence tomography for high-resolution imaging of human gastrointestinal tract malignancies. J Gastroenterol 2000; 35: 87–92. 93 Das A, Sivak MV Jr, Chak A et al. High-resolution endo- scopic imaging of the GI tract: a comparative study of optical coherence tomography versus high-frequency catheter probe EUS. Gastrointest Endosc 2001; 54: 219–24. 94 Inoue H, Igari T, Nishikage T, Ami K, Yoshida T, Iwai T. A novel method of virtual histopathology using laser- scanning confocal microscopy in-vitro with untreated fresh specimens from the gastrointestinal mucosa. Endo- scopy 2000; 32: 439–43. 95 Delaney PM, King RG, Lambert JR, Harris MR. Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo. J Anat 1994; 184: 157–60. 96 McLaren WJ, Anikijenko P, Thomas SG, Delaney PM, King RG. In vivo detection of morphological and microvascular changes of the colon in association with colitis using Chapter 44: Optical Techniques 535 fiberoptic confocal imaging (FOCI). Dig Dis Sci 2002; 47: 2424–33. 97 Liang C, Sung KB, Richards-Kortum RR, Descour MR. Design of a high-numerical-aperture miniature micro- scope objective for an endoscopic fiber confocal reflectance microscope. Appl Opt 2002; 41: 4603–10. 98 Dickensheets DL, Kino GS. Micro-machined scanning con- focal optical microscope. Opt Lett 1996; 21: 764–6. 99 Lane P, Dlugan ALP, Richards-Kortum R, MacAuley C. Fiber-optic confocal microscopy using a spatial light modu- lator. Opt Lett 2000; 25: 1780–2. 100 Lane P, Dlugan ALP, MacAuley C. DMD-enabled confocal microscopy. In: Tuchin VV, Izatt JA, Fujimotto JG, eds. Coherence Domain Optical Methods in Biomedical Science and Clinical Applications V. Proc. SPIE Vol. 4251, pp. 192–8. Bellingham WA: SPIE Publications, 2001. 101 Reilly RM. Radioimmunotherapy of malignancies. Clin Pharm 1991; 10: 359–75. 102 Ballou B, Fisher GW, Hakala TR, Farkas DL. Tumor detec- tion and visualization using cyanine fluorochrome-labeled antibodies. Biotechnol Prog 1997; 13: 649–58. 103 Haringsma J, Tytgat GNJ. Fluorescence and autofluores- cence. Bailliere’s Clin Gastroenterol 1999; 13: 1–10. 104 Pelegrin A, Folli S, Buchegger F, Mach JP, Wagnieres G, van den Bergh H. Antibody–fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice. Cancer 1991; 67: 2529–37. 105 Folli S, Westermann P, Braichotte D et al. Antibody–indo- cyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice. Cancer Res 1994; 54: 2643–9. 106 Gutowski M, Carcenac M, Pourquier D et al. Intraoperative immunophotodetection for radical resection of cancers: evaluation in an experimental model. Clin Cancer Res 2001; 7: 1142–8. 107 Kusaka Y, Ito S, Muguruma N et al. Vital immunostaining of human gastric and colorectal cancers grafted into nude mice: a preclinical assessment of a potential adjunct to videoendoscopy. J Gastroenterol 2000; 35: 748–52. 108 Nussbaum S, Roth HJ. Human anti-mouse antibodies: pit- falls in tumor marker measurement and strategies for enhanced assay robustness; including results with Elecsys CEA. Anticancer Res 2000; 20: 5249–52. 109 Ramjiawan B, Maiti P, Aftanas A et al. Noninvasive local- ization of tumors by immunofluorescence imaging using a single chain Fv fragment of a human monoclonal antibody with broad cancer specificity. Cancer 2000; 89: 1134–44. 110 Yokota T, Milenic DE, Whitlow M, Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 1992; 52: 3402–8. 111 Weissleder R, Tung CH, Mahmood U, Bogdanov A Jr. In vivo imaging of tumors with protease activated near- infrared fluorescent probes. Nat Biotechnol 1999; 17: 375– 8. 112 Marten K, Bremer C, Khazaie K et al. Detection of dysplas- tic intestinal adenomas using enzyme-sensing molecular beacons in mice. Gastroenterology 2002; 122: 406–14. 113 DaCosta RS, Tang Y, Reilly RM, Wilson BC. In vivo imag- ing of colonic tumors with a near-infrared fluorescent probe targeted against a tumor-associated-mucin in a human colon cancer xenograft mouse model. Abstract pre- sented at the Annual Meeting of the American Gastro- enterology Association, 2002, Digestive Diseases Week Conference. 114 Maunoury V, Mordon S, Geboes K et al. Early vascular changes in Crohn’s disease: an endoscopic fluorescence study. Endoscopy 2000; 32: 700–5. 115 Bando T, Muguruma N, Ito S et al. Basic studies on a labeled anti-mucin antibody detectable by infrared- fluorescence endoscopy. J Gastroenterol 2002; 37: 260–9. 116 Hayashi S, Muguruma N, Bando T, Taoka S, Ito S, Ii K. Vital immunohistochemical staining for a novel method of diagnosing micro-cancer. Examination of immunohisto- chemical staining of non-fixed fresh tissue. J Med Invest 1999; 46: 178–85. 117 Ito S, Muguruma N, Kusaka Y et al. Detection of human gastric cancer in resected specimens using a novel infrared fluorescent anti-human carcinoembryonic antigen (CEA) antibody with an infrared fluorescence endoscope in vitro. Endoscopy 2001; 33: 849–53. 118 Tatsuta M, Iishi H, Ichii M et al. Diagnosis of gastric can- cers with fluorescein-labeled monoclonal antibodies to carcinoembryonic antigen. Lasers Surg Med 1989; 9: 422–6. 119 Keller R, Winde G, Eisenhawer C et al. Immunoscopy: a technique combining endoscopy and immunofluorescence for diagnosis of colorectal cancer. Gastrointest Endosc 1998; 47: 154–61. 120 Folli S, Wagnieres G, Pelegrin A et al. Immunopho- todiagnosis of colon carcinomas in patients injected with fluoresceinated chimerical antibodies against carcinoem- brionic antigen. Proc Natl Acad Sci USA 1992; 89: 7973–7. 121 Keller R, Winde G, Terpe HJ, Foerster EC, Domschke W. Fluorescence endoscopy using a fluorescein-labeled mono- clonal antibody against carcinoembryonic antigen in pati- ents with colorectal carcinoma and adenoma. Endoscopy 2002; 34: 801–7. 122 Krishnadath KK, Reid BJ, Wang KK. Biomarkers in Barrett esophagus. Mayo Clin Proc 2001; 76: 438–46. 123 Srivastava S, Verma M, Henson DE. Biomarkers for early detection of colon cancer. Clin Cancer Res 2001; 7: 1118–26. [...]... colon carcinoma: virtual colonoscopy in the preoperative evaluation of the proximal colon Radiology 199 9; 210: 4238 49 Bond JH Virtual colonoscopyapromising, but not ready for widespread use N Engl J Med 199 9; 341: 15402 50 Fenlon HM, Nunes DP, Schroy PC et al A comparison of virtual and conventional colonoscopy for the detection of colorectal polyps N Engl J Med 199 9; 341: 1 496 503 51 Rex DK, Vining... Bolden S, Windigo PA Cancer statistics 199 6 CA Cancer J Clin 199 6; 65: 527 7 Glick S, Wagner JL, Johnson CD Cost-effectiveness of double contrast barium enema in screening for colorectal cancer AJR 199 8; 170: 6 29 36 8 Winawer SJ, Fletcher RH, Miller L et al Colorectal cancer screening: clinical guidelines and rationale Gastroenterology 199 7; 112: 594 601 9 Mandel JS, Bond JH, Church TR et al Reducing... colography: initial assessment of sensitivity and specicity Radiology 199 7; 205: 2 596 5 28 Fenlon HM, Ferrucci JT Virtual colonoscopy: what will the issues be? AJR 199 7; 1 69: 4538 29 Hara AK, Johnson CD, Reed JE, Ehman RL, Ilsrtup DM Colorectal polyp detection with CT colography: two- versus three-dimensional techniques Radiology 199 6; 200: 49 54 30 Dachman AH, Lieberman J, Osnis RB et al Small simulated polyps... of time-efcient CT colonography with twodimensional and three-dimensional colonic evaluation for detecting colorectal polyps AJR 2000; 174: 154 39 46 Macari M, Megibow AJ, Berman P, Milano A, Dicker M CT colography in patients with failed colonoscopy AJR 199 9; 173: 5614 47 Morrin MM, Kruskal JB, Farrell RJ et al Endoluminal CT colonography after incomplete endoscopic colonoscopy AJR 199 9; 172: 91 318... Endosc 199 9; 50: 117 19 1 09 Dhiman RK, Saraswat VA, Choudhuri G, Sharma BC, Pandey R, Naik SR Endosonographic, endoscopic and histologic evaluation of alterations in the rectal venous system in patients with portal hypertension Gastrointest Endosc 199 9; 49: 21827 110 Hulsmans FJH, Tio TI, Reeders JW, Tytgat GNJ Transrectal US in the diagnosis of localized colitis cystica profunda Radiology 199 1; 181:... Radiology 199 7; 203: 42730 31 Dachman AH, Kuniyoshi JK, Boyle CM et al CT colonography with three-dimensional problem solving for detection of colonic polyps AJR 199 8; 171: 98 995 32 Angtuaco TL, Bannaad Omiotek GD, Howden CW Differing attitudes toward virtual and conventional colonoscopy for colorectal cancer screening Surveys among primary care physicians and potential patients Am J Gastroenterol 2001; 96 :... it reality Radiology 199 6; 200: 301 14 Kronborg O, Fenger C, Olsen J et al Randomized study of screening for colorectal cancer with fecal occult blood test Lancet 199 6; 348: 146771 15 Rockey DC, Koch J, Cello JP, Sanders LL, McQuaid K Relative frequency of upper gastrointestinal and colonic lesions in patients with positive fecal occult-blood tests N Engl J Med 199 8; 3 39: 153 9 16 Selby JV, Friedman... Colorectal neoplasms Prospective comparison of thin-section low-dose multidetector row CT colonography and conventional colonoscopy for detection Radiology 2002; 224: 383 92 4 Vining DJ, Gelfand DW, Bechtold R et al Technical feasibility of colon imaging with helical CT and virtual reality [Abstract] AJR 199 4; 162: 194 5 Ransohoff DF, Sandler RS Clinical practice: screening for colorectal cancer N Engl... polyps using spiral CT with and without CT colography (virtual colonoscopy) Gastrointest Endosc 199 9; 50: 3 091 3 52 Ahlquist DA, Johnson CD Screening CT colonography: too early to pass judgment on nascent technology Gastrointest Endosc 199 9; 50: 43 79 53 Rex DK, Cutler CS, Lemmel GT et al Colonoscopic miss rates of adenomas determined by back to back colonoscopies Gastroenterology 199 7; 112: 248 54 Macari... of peri-anal and pericolorectal stula and/ or abscess in Crohns disease Gastrointest Endosc 199 0; 36: 3316 112 Van Outryve MJ, Pelckmans PA, Michielsen PP, Van Maercke YM Value of transrectal ultrasonography in Crohns disease Gastroenterology 199 1; 101: 11717 113 Gast P, Belaùche J Rectal endosonography in inammatory bowel disease: differential diagnosis and prediction of remission Endoscopy 199 9; 31: . J Colorectal Dis 199 0; 5: 90 –3. 45 Gillard V. Evaluation of polyps by endoscopic ultrasono- graphy (EUS): implication for endotherapy. Acta Gastro- enterol Belg 199 9; 62: 196 9. 46 Tsuda S, Hoashi. 5-aminolevulinic acid (ALA): current clinical and developmental status. J Clin Laser Med Surg 199 6; 14: 59 66. 79 Shim MG, Wilson BC. The effects of ex vivo handling pro- cedures on the near-infrared. Micro-machined scanning con- focal optical microscope. Opt Lett 199 6; 21: 764–6. 99 Lane P, Dlugan ALP, Richards-Kortum R, MacAuley C. Fiber-optic confocal microscopy using a spatial light modu- lator.