- Email: [email protected]
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
REVIEW ARTICLE
High-resolution and optical molecular imaging for the early detection of colonic neoplasia Jeremy L. Matloff, MD, Wasif Abidi, MD, PhD, Rebecca Richards-Kortum, PhD, Jenny Sauk, MD, Sharmila Anandasabapathy, MD New York, New York; Houston, Texas, USA
Colorectal cancer screening is a recommended and widely accepted component of preventive medicine for high-risk patients and asymptomatic adults in the United States.1 Effective screening depends on detecting lesions that are amenable to curative treatment. Although endoscopic surveillance has decreased the incidence of colon cancer2 and provided long-term risk reduction,3 there is considerable room for improvement. Adenoma miss rates remain at approximately 22% (range 15%-32%),4 with flat and depressed lesions frequently overlooked. Moreover, the specificity of white-light imaging for distinguishing neoplastic from non-neoplastic lesions remains poor, relying on visible mucosal changes.5 As a result, benign and/or hyperplastic lesions are often removed, increasing both cost and risk. Over the past decade, multiple wide-field technologies have been developed with the goal of highlighting suspicious mucosa. These modalities, which include narrowband imaging, digital I-scan, Fujinon Intelligent Color Enhancement system, and autofluorescence imaging are designed to serve as red-flag techniques, theoretically enhancing the macroscopic view of the colon and the diagnostic accuracy of standard colonoscopy. There are, however, no large randomized trials showing an advantage of these modalities over high-definition white-light endoscopy. Moreover, to more accurately determine whether a polyp is hyperplastic or adenomatous, technologies with a higher spatial resolution are required to increase specificity. By combining these technologies with targeted or
Abbreviations: CLE, confocal laser endomicroscopy; EC, endocytoscopy; eCLE, embedded confocal laser endomicroscopy; EGFR, epidermal growth factor receptor; LIF, laser-induced fluorescence imaging; OCT, optical coherence tomography; 2-NBDG, 2-(N-(7-nitrobenz-2-ox-1,3diazol-4-yl)amino)-2-deoxyglucose; pCLE, probe-based confocal laser endomicroscopy; VEGF, vascular endothelial growth factor. DISCLOSURE: The following author disclosed a financial relationship relevant to this publication: Dr. Anandasabapathy: Pentax, material research support. The other authors disclosed no financial relationships relevant to this publication. See CME section; p. 1254. Copyright © 2011 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 doi:10.1016/j.gie.2011.01.070
www.giejournal.org
molecule-specific contrast agents, an even more precise characterization of a lesion’s neoplastic potential is possible. This “combination strategy” offers the potential to better identify and characterize lesions at the point of care. Such an approach may enhance detection and treatment strategies by preventing the unnecessary removal of benign lesions and facilitating margin determination during endoscopic therapy. Optical molecular imaging can also provide critical prognostic and therapeutic information such as the presence or absence of a biomarker that can be used to guide drug therapy. This review provides an overview of the currently available optical biopsy technologies including confocal laser endomicroscopy (CLE), endocytoscopy (EC), and optical coherence tomography (OCT) (Table 1). We reviewed the existing primary data evaluating these technologies used in colon cancer screening and included the pertinent literature. In addition, we review several emerging trends in optical imaging, including the development of lower cost microendoscopic devices and targeted contrast agents. These exciting developments offer the opportunity to enhance the accuracy and efficiency of current screening and the ability to guide decision making in real time.
CONFOCAL LASER ENDOMICROSCOPY Basic principles Confocal laser microscopy relies on the excitation of a fluorescent molecule and detection of its emission at a specific axial depth within a given sample. In confocal, a low-power laser is focused on a single point on a fluorescent sample and its emission at that point is recorded, creating 1 pixel of an image. The laser sequentially scans specific points on the specimen in a raster pattern to map out a full picture. Emitted light is passed through a pinhole, eliminating out-of-focus light and creating detailed, high-resolution, subcellular images. Combining this technology with endoscopy allows for contrast-enhanced tissue to be examined on a microscopic scale during colonoscopy, thus offering an in vivo diagnosis or optical biopsy. There are 2 confocal imaging systems commercially available: one that uses a miniaturized scanner within the tip of a conventional endoscope (Pentax/Hoya, Tokyo, Japan), and a probe-based device that is passed through the enVolume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1263
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
TABLE 1. Characteristics of currently available high-resolution imaging systems Sample of published data Imaging system CLE (Pentax)
Contrast agent
Spatial resolution
Fluorescein or acriflavine
0.7 m
No. of patients
No. of lesions
42
134
99.2% accuracy, 97.4% sensitivity, 99.4% specificity
Sanduleanu et al,11 2010
72
116
95.7% accuracy, 97.3% sensitivity, 92.8% specificity
Trial Kiesslich et 2004
al,10
Results
CLE (Miniprobe)
Fluorescein or acriflavine
1-3.5 m
Meining et al,12 2007
47
36
91.7% accuracy, 92.3% sensitivity, 91.3% specificity
Endocytoscopy
Methylene blue
1.7-4 m
Sasajima et al,15 2006
60
75
93.3% Accuracy
CLE, Confocal laser endomicroscopy.
doscope’s working channel (Mauna Kea Technologies, Paris, France).
Equipment and technique The Pentax confocal laser endomicroscope incorporates an embedded miniaturized laser scanner into the distal tip of the colonoscope, along with a flexibly connected solid-state laser (eCLE). The solid-state laser delivers blue laser light (488 nm) via a single optical fiber. Within the laser scanner, there is a lens system that focuses returning light onto the end of the optical fiber (acting as the confocal pinhole), thus eliminating light from other imaging planes. For the probe-based CLE system (pCLE), a miniprobe consisting of a bundle of optical fibers is passed through the accessory channel of a standard colonoscope (with a 2.8-mm channel) and connected to a more conventional laser scanning unit and detector. The scanning unit sequentially scans each fiber to collect the pixels of the image.6 Both of these systems can achieve similar fields of view depending on the specific fiberoptic probe used (240-600 m for pCLE and 475 m for eCLE). However, compared with eCLE, pCLE has slightly less lateral resolution (0.7 m vs 1.0-3.5 m) and significantly less axial resolution (⬍1 m vs 15 m). eCLE also has the advantage of producing images of structures from 0 to 250 m in depth, whereas with the miniprobe, the depth of images ranges from 0 to 120 m, depending on which probe is used. Because of these specifications, however, pCLE is able to achieve a much higher rate of image acquisition than eCLE and is able to create a true “video mosaic” of images. This allows visualization of a larger portion of the mucosa.7
suspicious area of the mucosa is identified during a standard colonoscopy. Fluorescein is the most commonly applied contrast agent used in endomicroscopy and has been extensively used in retinal angiography. With administration of intravenous fluorescein, blood vessels, intracellular spaces, and lamina propria are all highlighted, whereas nuclei and mucin remain unstained. This provides cellular and subcellular details and connective tissue and blood vessel architecture.8 The rate of complications is low and generally limited to transient yellowing of the skin, eyes, and urine. Nausea and vomiting can occur, and anaphylaxis or allergic reactions are rare. An alternative, topical contrast agent is acriflavine hydrochloride. Acriflavine binds to nucleic acids, staining nuclei and cytoplasm to a depth of 100 m. Acriflavine typically does not highlight the microvasculature or connective tissue in deeper layers of the mucosa.
Interpretation Several features on confocal images can differentiate normal from dysplastic or neoplastic lesions in the colon. In normal mucosa and hyperplastic polyps, nuclei are rarely visualized. In addition, the epithelial components display a cohesive architecture, and the microvasculature appears in a normal honeycomb pattern. Red blood cells are also visualized and appear as moving black dots.9 Conversely, in neoplastic lesions, denser and enlarged nuclei may be visualized, the epithelial architecture is disrupted, and the vasculature displays irregularity as well as leakage of fluorescein, which represents neoangiogenesis (Fig. 1).
Limitations Contrast agents Both CLE systems require exogenous fluorescent contrast agents to allow for high-resolution images. These agents can be applied topically or intravenously, once a 1264 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
There are several limitations to this technique. Because of the extremely small field of view (⬍700 m), this technique is time-consuming. In addition, the cost of these systems is more than $100,000, and there is a significant www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 1. pCLE of normal colonic mucosa (A), hyperplastic polyp (B), and tubular adenoma (C). Reproduced with permission from Wallace et al.33
www.giejournal.org
Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1265
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
learning curve, with the required cases for proficiency estimated to be at least 30.7 Last, CLE requires the administration of exogenous fluorescent contrast agents, which can carry a risk of adverse reactions.
Initial data There have been several studies looking at the accuracy of detecting colon cancer using CLE. In 2004, Kiesslich et al10 compared the confocal images with conventional pathology taken from patients undergoing CLE using the Pentax system. Suspicious mucosal lesions on chromoendoscopy were identified and evaluated using both fluorescein and acriflavine and showed a high accuracy (97.4% sensitivity, 99.4% specificity, 99.2% accuracy) in diagnosing neoplastic changes, confirming the correlation between the optical and histopathological biopsies. In a study from 2010, Sanduleanu et al11 prospectively investigated patients undergoing CLE to validate the features of adenomatous and nonadenomatous polyps and to assess the predictive value of CLE for diagnosing colonic neoplasia. Patients at high risk of colonic neoplasia underwent CLE using the Pentax system with both fluorescein and acriflavine contrast. The CLE images of adenomatous polyps showed a lack of epithelial surface maturation, crypt budding, altered vascular pattern, and loss of cell polarity. The nonadenomatous polyps showed epithelial surface maturation, minor abnormalities of crypt architecture and vascular pattern, and maintained cell polarity. Confocal images were compared with conventional histology and showed a high correlation (97.3% sensitivity, 92.8% specificity, 95.7% accuracy). This study validated the histological features of both adenomatous and nonadenomatous polyps and demonstrated the accuracy of confocal images compared with conventional histology. The Mauna Kea miniprobe system was evaluated separately. Meining et al12 studied this system in patients with known or suspected neoplasia of the upper or lower GI tract. Neoplasia of the lower GI tract was identified using the miniprobe system with a diagnostic accuracy of 91.7% (92.3% sensitivity, 91.3% specificity) compared with histopathology. In a recent study, Buchner et al13 compared pCLE with virtual chromoendoscopy. Patients underwent imaging with narrow-band imaging or Fujinon Intelligent Color Enhancement. Confocal images from detected polyps were then analyzed at a later time and compared with the findings at the time of chromoendoscopy. pCLE had a higher sensitivity compared with virtual chromoendoscopy (91% vs 77%) for differentiating hyperplastic from neoplastic polyps. Interestingly, there was no difference in specificity. The ability to characterize suspicious lesions in real time can have particular value in patients who are at high risk of colon cancer. Kiesslich et al evaluated patients with ulcerative colitis undergoing surveillance colonoscopy and randomized these patients to undergo conventional colonoscopy biopsies or chromoscopy-assisted endomi1266 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
Figure 2. A, Endocytoscope at distal end. B, Endocytoscope can be passed through the working channel or endoscope. Reproduced with permission from Sasajima et al.15
croscopy. There was a 4.5-fold increase in the detection of neoplasia in the chromoscopy with endomicroscopy group (P ⫽ .005) compared with the conventional colonoscopy group. In addition, 50% fewer biopsies were required in the endomicroscopy group. The confocal images compared favorably with histological results, with an accuracy of 97.8% (94.7% sensitivity, 98.3% specificity).14 By using CLE, the presence of neoplasia can be determined in real time, allowing for immediate biopsies and/or polypectomy. This strategy has been combined with wide-field imaging to identify “red-flag” areas, which are then investigated further with CLE.11 The 2 systems of CLE have shown promise and offer good diagnostic accuracy compared with histopathology.
ENDOCYTOSCOPY EC is a technique that uses ultrahigh magnification light microscopy to view microscopic details of superficial mucosal layers at a resolution comparable to that of histology. The device consists of a flexible, probe-based catheter that is inserted into a conventional endoscope (Fig. 2). A plastic cap is placed at the tip of the endoscope, providing stability as the probe makes contact with the mucosal surface, which is necessary for proper visualization. A new www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 4. Three OCT images (top) from a mouse colon are stacked vertically compared with low-resolution LIF intensities (bottom). Adenomas are outlined in white. Reproduced with permission from Winkler et al.18
Figure 3. Endocytoscopic image of high-grade dysplasia with disorder of polarity. Reproduced with permission from Sasjima et al.15
system has been designed that integrates the probe into the endoscope, providing improved stability. EC requires prestaining with an absorptive contrast agent, typically methylene blue. After excess contrast is washed off, the probe is placed in contact with suspicious mucosa. An image is projected onto a screen with ⫻580 magnification, and several cytological and architectural features can be evaluated, such as variability of gland size and uniformity, size and arrangement of nuclei, and cellular size, density, and arrangement (Fig. 3). Sasajima et al15 performed EC on 60 patients who had localized colorectal lesions found on colonoscopy and compared images with conventional histology. There was statistically significant correlation between the EC images and histology (93.3% accuracy). This study developed EC-based image criteria, but these criteria have yet to be validated. EC has the potential to make instant diagnoses during routine colonoscopy, which would allow for immediate biopsy and potential removal of neoplastic lesions. However, this technique provides images solely of the superficial mucosal layer, theoretically missing deeper lesions and not allowing for depth staging.15 Imaging is subject to visual interference caused by motion artifact and the presence of blood or mucus in the stool. Additionally, the macroscopic view can be impaired by high concentrations of methylene blue.
OPTICAL COHERENCE TOMOGRAPHY OCT provides cross-sectional images by detecting light that is back-reflected from microstructures beneath the www.giejournal.org
surface of tissues, in a similar fashion to how B-scan US detects sound waves. This imaging modality relies on a small catheter probe (approximately 2 mm) that does not need to be in tissue contact and does not require contrast media. Images can be acquired at a rate of 4 frames per second.16 Because of the high resolution of endoscopic OCT (4-20 m), structural components such as blood vessels, lymphatics, villi, glands, and crypts can be visualized in real time. Doppler imaging can be incorporated into OCT, which allows for blood-flow velocities to be displayed along with the architecture of microvasculature that is too small to be viewed by conventional US. OCT images are limited to the mucosa and submucosa. In 2003, Pfau et al17 performed OCT on 24 patients with polyps identified on colonoscopy. Images were obtained from the polyp, and nearby normal-appearing mucosa and real-time evaluation of these images was performed. Polyps were then removed and evaluated with conventional histopathology by a pathologist blinded to the OCT images. Adenomas were found to have significantly more tissue disorganization with less structure than hyperplastic polyps. Adenomas also exhibited a decrease in light scattering and appeared darker. Based on this, they were able to determine that endoscopic OCT could differentiate adenomas, hyperplasic polyps, and normal colon in real time. Recently, Winkler et al18 advanced the capabilities of OCT by coupling it with low-resolution laser-induced fluorescence imaging (LIF). Their system is based on an OCT sensor that provides structural information down to 18 m, coupled with the low-resolution fluorescence to provide biological information about dysplastic mucosa (Fig. 4). This platform is limited by the low resolution of the LIF Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1267
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 5. Low-cost microendoscopy: high-resolution microendoscope. The system is battery powered and contained in a briefcase (A). Images are obtained by placing a fiberoptic bundle in contact with the mucosa (B). Courtesy of R. Richards-Kortum.
Figure 6. Images of normal colon and a tubular adenoma produced by the high-resolution microendoscope. Courtesy of S. Anandasabapathy.
images; however, improvements in LIF imaging are in progress. OCT has the potential to differentiate benign and dysplastic polyps in real time. Because of the limited depth 1268 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
from which images can be produced, OCT will likely serve as a tool for staging dysplasia; however, biopsy or other imaging modalities will continue to be necessary to evaluate deeper tissue. www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 7. Normal colonic mucosa after staining with 2-NBDG (A) and corresponding histology (B); tubulovillous adenoma with dysplasia with increased glandular uptake of 2-NBDG (C) and corresponding histology (D). Courtesy of S. Anandasabapathy.
LOWER-COST MICROENDOSCOPY One factor that has limited the use of confocal platforms to tertiary care centers is the cost of these platforms as well as the significant learning curve associated with interpretation of the microscopic images. To expand access to optical biopsy technology in lower resource settings or the developing world, there is increased interest in the development of lower cost imaging devices that can be used in conjunction with image-analysis software for rapid, real-time interpretation. Muldoon et al19 developed a portable, batteryoperated, high-resolution fluorescent microendoscope that costs less than $3500 in components. The system uses a fiberoptic bundle made of 30,000 individual fiwww.giejournal.org
bers that is channeled through the biopsy port of a standard endoscope (Fig. 5). Images are obtained by placing the end of the fiberoptic bundle directly in contact with the surface of the mucosa. A fluorophore is excited using a 455-nm blue light– emitting diode, and the emitted light returning to the microscope is transmitted to a computer via a charge-coupled device camera. Anecdotally, there is evidence to suggest that use of topical proflavine hemisulfate (0.01%) combined with a high-resolution fluorescent microendoscope can differentiate normal mucosa from hyperplastic and adenomatous polyps (Fig. 6), although systematic evaluation of this will be required to determine the technique’s merits relative to high-resolution confocal platforms. Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1269
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 8. Biopsy specimen of human colorectal adenocarcinoma. A, Nonspecific nuclear and cellular staining using acriflavine. B, VEGF-specific staining by using AF488-labeled antibodies. The antibody accumulates in the cytoplasm of the tumor cells, but not the nuclei. Reproduced with permission from Foersch et al.30
TARGETED CONTRAST AGENTS AND MOLECULAR ENDOSCOPY: THE FUTURE? The ability to magnify and analyze the GI mucosa using these technologies provides a powerful opportunity to accurately diagnose colonic lesions at an early, treatable stage. To take full advantage of the capabilities of these technologies, complementary contrast agents need to be developed to specifically target neoplastic mucosa. Current technologies that highlight neoplastic colonic tissue on a macroscopic level such as autofluorescence and narrow-band imaging have been shown to improve the detection of neoplasia.20 Combining these technologies with contrast agents that target neoplastic cells on a microscopic level could provide a unique opportunity to more easily identify and characterize mucosal lesions. Molecule-specific contrast agents offer the added opportunity to provide prognostic and therapeutic information. Several groups have shown promising initial data in this area. One way that cancer cells can be differentiated from normal cells is their increased rate of glucose metabolism and uptake, as has been well established with 18Ffluorodeoxyglucose in PET imaging.21-24 A derivative of fluorodeoxyglucose, 2-(N-(7-nitrobenz-2-ox-1,3-diazol4-yl)amino)-2-deoxyglucose (2-NBDG) is similarly transported via glucose transporters and rapidly taken up by proliferating cells.24,25 Although no in vivo data exist at this time, 2 ex vivo studies have shown that 2-NBDG can be used to differentiate malignant from normal cells.26,27 The Richard-Kortum group has used fluorescently labeled de1270 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
oxyglucose (2-NBDG) as a contrast agent in confocal and fluorescence imaging of oral mucosa biopsy specimens and shown an average 3.7 times greater signal from neoplastic samples than matched non-neoplastic samples. On ex vivo samples of subjects with Barrett’s esophagus imaged with a confocal microscope, variation in the location and intensity of 2-NBDG uptake was able to differentiate neoplastic from non-neoplastic mucosa with a sensitivity of 80% and specificity of 83%.27 Preliminary work on ex vivo colonic specimens suggests that the metabolic information provided by 2-NBDG can be combined with morphological information provided by confocal microscopy to differentiate adenomatous and dysplastic colonic mucosa (Fig. 7). Although these preliminary data are promising, in vivo studies using 2-NBDG would need to be done to fully assess the practicality of this imaging modality in cancer screening and surveillance. Beyond detecting the uptake of labeled glucose, use of fluorescence endomicroscopy allows the exciting possibility of using fluorescently labeled molecules to target molecular markers specific to cancer cells. This would propel endoscopy from the cellular level to the molecular level. Several groups have published promising studies in this realm. In the age of monoclonal antibodies approved for therapeutics, it is not inconceivable that true immunofluorescence with labeled antibodies could be achieved with fluorescence endoscopy. As far back as 2002, Keller et al28 were able to achieve in vivo low-resolution fluorescence endoscopy using a monoclonal antibody against carcinoembryonic antigen applied directly onto colonic mucosa www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 9. In vivo confocal fluorescence images of peptide binding. A, Binding to dysplastic colon polyp. B, Binding to adjacent normal mucosa. Histology of dysplastic colon polyp (C) and normal mucosa (D) stained with hematoxylin and eosin. Reproduced with permission from Hsiung et al.32
of patients known to have large polypoid lesions or colorectal carcinoma. Although the results were promising, direct comparison with histopathology of the site being imaged was not done, and no follow-up studies have been published. More recently, Goetz et al29 published a murine in vivo and human ex vivo study using a fluorescently labeled antibody against epidermal growth factor receptor (EGFR). Mice were injected with tumor cell lines that expressed EGFR at high or low levels. Tumors were then imaged in vivo using CLE after injecting the labeled antiEGFR and showed a threefold difference in mean fluorescence intensity. Human colonic biopsy specimens were also imaged after incubation with labeled anti-EGFR and showed an approximately eightfold difference in fluorescence intensity between neoplastic and non-neoplastic tissues. The Goetz group went further by publishing similar data using a commercially available polyclonal antibody targeting vascular endothelial growth factor (VEGF),30 known to be expressed in high levels in neoplastic tissue.31 They studied 2 mouse models with small-bowel and colon tumors by injecting them with the labeled antibody. Mice with tumor cells showed accumulation of the labeled antibody specifically in their tumors. In addition, ex vivo imaging of human colorectal adenocarcinoma showed similar staining patterns with significantly increased signal www.giejournal.org
in the tumor compared with normal colonic tissue and a clear transition zone. Of note, most of the experiments in this study were conducted using an antibody against an intracellular protein, and as such displayed cytoplasmic staining. This was done without having to permeabilize the membranes of the cells, as is usually done with immunohistochemistry. Thus, the number of proteins that could be targeted in vivo go beyond those that are displayed at the surface of the cell (Fig. 8). Use of fluorescently tagged antibodies is not without its limitations, including a long half-life after injection, cost, and concern for immunogenicity. To overcome this, Hsiung et al32 isolated a heptapeptide from a phage library by clearing the library with human intestinal cells, followed by normal colonic tissue, hyperplastic, and inflamed tissue. The remaining isolates were exposed to tubular adenomas, and approximately 40 phage clones were tested for their ability to bind to adenocarcinoma-derived cells. Finally, a heptapeptide was selected, bound to fluorescein, and applied topically in a pilot in vivo screening colonoscopy study using CLE. Their results show that, compared with histological diagnoses, increased intensity of the probe was 81% sensitive and 82% specific for adenomas (Figs. 9 and 10). The study is limited, however, in that the exact target for the heptapeptide has yet to be determined, and any potential downstream signaling that Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1271
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 10. In vivo confocal fluorescence images of the border between colonic adenoma and normal mucosa, showing peptide binding to dysplastic colonocytes. A, Endoscopic view. B, Border. C, Dysplastic crypt. D, Adjacent mucosa. Reproduced with permission from Hsiung et al.32
may occur with the binding of this peptide to an unknown ligand has not been evaluated. Alternatively, a recent study used a labeled fragment of VEGF, topically applied to mouse colon, to target VEGF receptors. Using OCT-LIF, the researchers compared mice with tumors with control mice. Although the fluorescence data had poor resolution, the combined modality was able to differentiate tumor from normal tissue with a sensitivity of 93% and specificity of 100%.18 As more animal or ex vivo studies using molecular imaging become available, the prospects of in vivo endoscopic molecular imaging will become closer to reality. This would allow us to screen, diagnose, prognosticate, and manage colorectal cancer in novel ways. For example, earlier diagnosis could be achieved by targeting markers that are present earlier in the pathway to malignancy. In familial adenomatous polyposis, identifying markers that can differentiate the neoplastic potential of histologically similar polyps would be invaluable. Specific molecular targets could be surveyed that would define in real time whether a lesion is amenable to endoscopic resection. With further development of the technology, it is likely that more novel applications will be discovered. 1272 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
CONCLUSION Colon cancer screening remains one of the most important aspects of preventive medicine for all adults, and new technologies can enhance our ability to detect and characterize colonic lesions. Advances in molecular imaging can provide endoscopists with real-time characterization of suspicious areas, potentially providing a higher yield approach to colonoscopy. Combining cellular-level interrogation with accurate wide-field technologies may not only reduce the number of missed lesions but also reduce the number of lesions that inappropriately underwent biopsy and the number of procedures performed, creating a more efficient approach to prevention.27 More studies are needed to validate these techniques individually and in combination and to assess their cost-effectiveness and learning curve before they can be used beyond the university setting. It is clear, however, that high-resolution and optical molecular imaging has the potential to transform colonoscopy from a tool used to detect areas of possible neoplasia into a modality that can determine whether cancer is present, define its molecular signature, and guide specific therapy for an individual patient. www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
REFERENCES 1. U.S. Preventive Services Task Force. Screening for Colorectal Cancer: U.S. Preventive Services Task Force Recommendation Statement. AHRQ Publication 08-05124-EF-3. Rockville (Md): Agency for Healthcare Research and Quality, 2008. 2. Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med 1993;329:1977-81. 3. Lieberman DA, Prindiville S, Weiss DG, et al. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-67. 4. van Rijn JC, Reitsma JB, Stoker J, et al. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006; 101:343-50. 5. Kudo S, Kashida H, Tamura T, et al. Colonoscopic diagnosis and management of nonpolypoid early colorectal cancer. World J Surg 2000;24: 1081-90. 6. Meining A. Confocal endomicroscopy. Gastrointest Endosc Clin N Am 2009;19:629-35. 7. Dunbar K, Canto M. Confocal endomicroscopy. Curr Opin Gastroenterol 2008;24:631-7. 8. Goetz M, Kiesslich R. Confocal endomicroscopy: in vivo diagnosis of neoplastic lesions of the gastrointestinal tract. Anticancer Res 2008;28: 353-60. 9. Kiesslich R, Goetz M, Vieth M, et al. Technology insight: confocal laser endoscopy for in vivo diagnosis of colorectal cancer. Nat Clin Pract Oncol 2007;4:480-90. 10. Kiesslich R, Burg J, Vieth M, et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004;127:706-13. 11. Sanduleanu S, Driessen A, Gomez-Garcia E, et al. In vivo diagnosis and classification of colorectal neoplasia by chromoendoscopy-guided confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2010;8:371-8. 12. Meining A, Saur D, Bajbouj M, et al. In vivo histopathology for detection of gastrointestinal neoplasia with a portable, confocal miniprobe: an examiner blinded analysis. Clin Gastroenterol Hepatol 2007;5:1261-7. 13. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of probebased confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps. Gastroenterology 2010;138:834-42. 14. Kiesslich R, Goetz M, Lammersdorf K, et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007;132:874-82. 15. Sasajima K, Kudo SE, Inoue H, et al. Real-time in vivo virtual histology of colorectal lesions when using the endocytoscopy system. Gastrointest Endosc 2006;63:1010-7. 16. DaCosta RS, Wilson BC, Marcon NE. Optical techniques for the endoscopic detection of dysplastic colonic lesions. Curr Opin Gastroenterol 2005;21:70-9. 17. Pfau PR, Sivak MV Jr, Chak A, et al. Criteria for the diagnosis of dysplasia by endoscopic optical coherence tomography. Gastrointest Endosc 2003;58:196-202. 18. Winkler AM, Rice PF, Weichsel J, et al. In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model. Mol Imaging Biol Epub 2010 Nov 2.
www.giejournal.org
19. Muldoon TJ, Anandasabapathy S, Maru D, et al. High-resolution imaging in Barrett’s esophagus: a novel, low-cost endoscopic microscope. Gastrointest Endosc 2008;68:737-44. 20. van den Broek FJ, Fockens P, van Eeden S, et al. Endoscopic tri-modal imaging for surveillance in ulcerative colitis: randomised comparison of high-resolution endoscopy and autofluorescence imaging for classification of lesions. Gut 2008;57:1083-9. 21. Larson SM. Positron emission tomography-based molecular imaging in human cancer: exploring the link between hypoxia and accelerated glucose metabolism. Clin Cancer Res 2004;10:2203-4. 22. Rajendran JG, Mankoff DA, O’Sullivan F, et al. Hypoxia and glucose metabolism in malignant tumors: evaluation by [18F]fluoromisonidazole and [18F]fluorodeoxyglucose positron emission tomography imaging. Clin Cancer Res 2004;10:2245-52. 23. Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell Biol 2006;18: 598-608. 24. Jadvar H, Alavi A, Gambhir SS. 18F-FDG uptake in lung, breast, and colon cancers: molecular biology correlates and disease characterization. J Nucl Med 2009;50:1820-7. 25. O’Neil RG, Wu L, Mullani N. Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells. Mol Imaging Biol 2005;7:388-92. 26. Nitin N, Carlson AL, Muldoon T, et al. Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose. Int J Cancer 2009;124:2634-42. 27. Thekkek N, Maru D, Richards-Kortum R, et al. Fluorescent deoxyglucose as a topical contrast agent to detect Barrett’s associated neoplasia using confocal imaging. New York: Mount Sinai School of Medicine; 2010. 28. Keller R, Winde G, Terpe HJ, et al. Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy 2002;34:801-7. 29. Goetz M, Ziebarg A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010;138:435-46. 30. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010;59:1046-55. 31. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57. 32. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008;14:454-8. 33. Wallace MB, Kiesslich R. Advances in endoscopic imaging of colorectal neoplasia. Gastroenterology 2010;138:2140-50. Received November 16, 2010. Accepted January 29, 2011. Current affiliations: Department of Medicine (J.L.M., W.A.), Division of Gastroenterology (J.S., S.A.), Mount Sinai Medical Center, New York, New York, Department of Bioengineering (R.R.-K.), Rice University, Houston, Texas, USA. Reprint requests: Sharmila Anandasabapathy, MD, Division of Gastroenterology, The Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029.
Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1273
High-resolution and optical molecular imaging for the early detection of colonic neoplasia Jeremy L. Matloff, MD, Wasif Abidi, MD, PhD, Rebecca Richards-Kortum, PhD, Jenny Sauk, MD, Sharmila Anandasabapathy, MD New York, New York; Houston, Texas, USA
Colorectal cancer screening is a recommended and widely accepted component of preventive medicine for high-risk patients and asymptomatic adults in the United States.1 Effective screening depends on detecting lesions that are amenable to curative treatment. Although endoscopic surveillance has decreased the incidence of colon cancer2 and provided long-term risk reduction,3 there is considerable room for improvement. Adenoma miss rates remain at approximately 22% (range 15%-32%),4 with flat and depressed lesions frequently overlooked. Moreover, the specificity of white-light imaging for distinguishing neoplastic from non-neoplastic lesions remains poor, relying on visible mucosal changes.5 As a result, benign and/or hyperplastic lesions are often removed, increasing both cost and risk. Over the past decade, multiple wide-field technologies have been developed with the goal of highlighting suspicious mucosa. These modalities, which include narrowband imaging, digital I-scan, Fujinon Intelligent Color Enhancement system, and autofluorescence imaging are designed to serve as red-flag techniques, theoretically enhancing the macroscopic view of the colon and the diagnostic accuracy of standard colonoscopy. There are, however, no large randomized trials showing an advantage of these modalities over high-definition white-light endoscopy. Moreover, to more accurately determine whether a polyp is hyperplastic or adenomatous, technologies with a higher spatial resolution are required to increase specificity. By combining these technologies with targeted or
Abbreviations: CLE, confocal laser endomicroscopy; EC, endocytoscopy; eCLE, embedded confocal laser endomicroscopy; EGFR, epidermal growth factor receptor; LIF, laser-induced fluorescence imaging; OCT, optical coherence tomography; 2-NBDG, 2-(N-(7-nitrobenz-2-ox-1,3diazol-4-yl)amino)-2-deoxyglucose; pCLE, probe-based confocal laser endomicroscopy; VEGF, vascular endothelial growth factor. DISCLOSURE: The following author disclosed a financial relationship relevant to this publication: Dr. Anandasabapathy: Pentax, material research support. The other authors disclosed no financial relationships relevant to this publication. See CME section; p. 1254. Copyright © 2011 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 doi:10.1016/j.gie.2011.01.070
www.giejournal.org
molecule-specific contrast agents, an even more precise characterization of a lesion’s neoplastic potential is possible. This “combination strategy” offers the potential to better identify and characterize lesions at the point of care. Such an approach may enhance detection and treatment strategies by preventing the unnecessary removal of benign lesions and facilitating margin determination during endoscopic therapy. Optical molecular imaging can also provide critical prognostic and therapeutic information such as the presence or absence of a biomarker that can be used to guide drug therapy. This review provides an overview of the currently available optical biopsy technologies including confocal laser endomicroscopy (CLE), endocytoscopy (EC), and optical coherence tomography (OCT) (Table 1). We reviewed the existing primary data evaluating these technologies used in colon cancer screening and included the pertinent literature. In addition, we review several emerging trends in optical imaging, including the development of lower cost microendoscopic devices and targeted contrast agents. These exciting developments offer the opportunity to enhance the accuracy and efficiency of current screening and the ability to guide decision making in real time.
CONFOCAL LASER ENDOMICROSCOPY Basic principles Confocal laser microscopy relies on the excitation of a fluorescent molecule and detection of its emission at a specific axial depth within a given sample. In confocal, a low-power laser is focused on a single point on a fluorescent sample and its emission at that point is recorded, creating 1 pixel of an image. The laser sequentially scans specific points on the specimen in a raster pattern to map out a full picture. Emitted light is passed through a pinhole, eliminating out-of-focus light and creating detailed, high-resolution, subcellular images. Combining this technology with endoscopy allows for contrast-enhanced tissue to be examined on a microscopic scale during colonoscopy, thus offering an in vivo diagnosis or optical biopsy. There are 2 confocal imaging systems commercially available: one that uses a miniaturized scanner within the tip of a conventional endoscope (Pentax/Hoya, Tokyo, Japan), and a probe-based device that is passed through the enVolume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1263
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
TABLE 1. Characteristics of currently available high-resolution imaging systems Sample of published data Imaging system CLE (Pentax)
Contrast agent
Spatial resolution
Fluorescein or acriflavine
0.7 m
No. of patients
No. of lesions
42
134
99.2% accuracy, 97.4% sensitivity, 99.4% specificity
Sanduleanu et al,11 2010
72
116
95.7% accuracy, 97.3% sensitivity, 92.8% specificity
Trial Kiesslich et 2004
al,10
Results
CLE (Miniprobe)
Fluorescein or acriflavine
1-3.5 m
Meining et al,12 2007
47
36
91.7% accuracy, 92.3% sensitivity, 91.3% specificity
Endocytoscopy
Methylene blue
1.7-4 m
Sasajima et al,15 2006
60
75
93.3% Accuracy
CLE, Confocal laser endomicroscopy.
doscope’s working channel (Mauna Kea Technologies, Paris, France).
Equipment and technique The Pentax confocal laser endomicroscope incorporates an embedded miniaturized laser scanner into the distal tip of the colonoscope, along with a flexibly connected solid-state laser (eCLE). The solid-state laser delivers blue laser light (488 nm) via a single optical fiber. Within the laser scanner, there is a lens system that focuses returning light onto the end of the optical fiber (acting as the confocal pinhole), thus eliminating light from other imaging planes. For the probe-based CLE system (pCLE), a miniprobe consisting of a bundle of optical fibers is passed through the accessory channel of a standard colonoscope (with a 2.8-mm channel) and connected to a more conventional laser scanning unit and detector. The scanning unit sequentially scans each fiber to collect the pixels of the image.6 Both of these systems can achieve similar fields of view depending on the specific fiberoptic probe used (240-600 m for pCLE and 475 m for eCLE). However, compared with eCLE, pCLE has slightly less lateral resolution (0.7 m vs 1.0-3.5 m) and significantly less axial resolution (⬍1 m vs 15 m). eCLE also has the advantage of producing images of structures from 0 to 250 m in depth, whereas with the miniprobe, the depth of images ranges from 0 to 120 m, depending on which probe is used. Because of these specifications, however, pCLE is able to achieve a much higher rate of image acquisition than eCLE and is able to create a true “video mosaic” of images. This allows visualization of a larger portion of the mucosa.7
suspicious area of the mucosa is identified during a standard colonoscopy. Fluorescein is the most commonly applied contrast agent used in endomicroscopy and has been extensively used in retinal angiography. With administration of intravenous fluorescein, blood vessels, intracellular spaces, and lamina propria are all highlighted, whereas nuclei and mucin remain unstained. This provides cellular and subcellular details and connective tissue and blood vessel architecture.8 The rate of complications is low and generally limited to transient yellowing of the skin, eyes, and urine. Nausea and vomiting can occur, and anaphylaxis or allergic reactions are rare. An alternative, topical contrast agent is acriflavine hydrochloride. Acriflavine binds to nucleic acids, staining nuclei and cytoplasm to a depth of 100 m. Acriflavine typically does not highlight the microvasculature or connective tissue in deeper layers of the mucosa.
Interpretation Several features on confocal images can differentiate normal from dysplastic or neoplastic lesions in the colon. In normal mucosa and hyperplastic polyps, nuclei are rarely visualized. In addition, the epithelial components display a cohesive architecture, and the microvasculature appears in a normal honeycomb pattern. Red blood cells are also visualized and appear as moving black dots.9 Conversely, in neoplastic lesions, denser and enlarged nuclei may be visualized, the epithelial architecture is disrupted, and the vasculature displays irregularity as well as leakage of fluorescein, which represents neoangiogenesis (Fig. 1).
Limitations Contrast agents Both CLE systems require exogenous fluorescent contrast agents to allow for high-resolution images. These agents can be applied topically or intravenously, once a 1264 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
There are several limitations to this technique. Because of the extremely small field of view (⬍700 m), this technique is time-consuming. In addition, the cost of these systems is more than $100,000, and there is a significant www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 1. pCLE of normal colonic mucosa (A), hyperplastic polyp (B), and tubular adenoma (C). Reproduced with permission from Wallace et al.33
www.giejournal.org
Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1265
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
learning curve, with the required cases for proficiency estimated to be at least 30.7 Last, CLE requires the administration of exogenous fluorescent contrast agents, which can carry a risk of adverse reactions.
Initial data There have been several studies looking at the accuracy of detecting colon cancer using CLE. In 2004, Kiesslich et al10 compared the confocal images with conventional pathology taken from patients undergoing CLE using the Pentax system. Suspicious mucosal lesions on chromoendoscopy were identified and evaluated using both fluorescein and acriflavine and showed a high accuracy (97.4% sensitivity, 99.4% specificity, 99.2% accuracy) in diagnosing neoplastic changes, confirming the correlation between the optical and histopathological biopsies. In a study from 2010, Sanduleanu et al11 prospectively investigated patients undergoing CLE to validate the features of adenomatous and nonadenomatous polyps and to assess the predictive value of CLE for diagnosing colonic neoplasia. Patients at high risk of colonic neoplasia underwent CLE using the Pentax system with both fluorescein and acriflavine contrast. The CLE images of adenomatous polyps showed a lack of epithelial surface maturation, crypt budding, altered vascular pattern, and loss of cell polarity. The nonadenomatous polyps showed epithelial surface maturation, minor abnormalities of crypt architecture and vascular pattern, and maintained cell polarity. Confocal images were compared with conventional histology and showed a high correlation (97.3% sensitivity, 92.8% specificity, 95.7% accuracy). This study validated the histological features of both adenomatous and nonadenomatous polyps and demonstrated the accuracy of confocal images compared with conventional histology. The Mauna Kea miniprobe system was evaluated separately. Meining et al12 studied this system in patients with known or suspected neoplasia of the upper or lower GI tract. Neoplasia of the lower GI tract was identified using the miniprobe system with a diagnostic accuracy of 91.7% (92.3% sensitivity, 91.3% specificity) compared with histopathology. In a recent study, Buchner et al13 compared pCLE with virtual chromoendoscopy. Patients underwent imaging with narrow-band imaging or Fujinon Intelligent Color Enhancement. Confocal images from detected polyps were then analyzed at a later time and compared with the findings at the time of chromoendoscopy. pCLE had a higher sensitivity compared with virtual chromoendoscopy (91% vs 77%) for differentiating hyperplastic from neoplastic polyps. Interestingly, there was no difference in specificity. The ability to characterize suspicious lesions in real time can have particular value in patients who are at high risk of colon cancer. Kiesslich et al evaluated patients with ulcerative colitis undergoing surveillance colonoscopy and randomized these patients to undergo conventional colonoscopy biopsies or chromoscopy-assisted endomi1266 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
Figure 2. A, Endocytoscope at distal end. B, Endocytoscope can be passed through the working channel or endoscope. Reproduced with permission from Sasajima et al.15
croscopy. There was a 4.5-fold increase in the detection of neoplasia in the chromoscopy with endomicroscopy group (P ⫽ .005) compared with the conventional colonoscopy group. In addition, 50% fewer biopsies were required in the endomicroscopy group. The confocal images compared favorably with histological results, with an accuracy of 97.8% (94.7% sensitivity, 98.3% specificity).14 By using CLE, the presence of neoplasia can be determined in real time, allowing for immediate biopsies and/or polypectomy. This strategy has been combined with wide-field imaging to identify “red-flag” areas, which are then investigated further with CLE.11 The 2 systems of CLE have shown promise and offer good diagnostic accuracy compared with histopathology.
ENDOCYTOSCOPY EC is a technique that uses ultrahigh magnification light microscopy to view microscopic details of superficial mucosal layers at a resolution comparable to that of histology. The device consists of a flexible, probe-based catheter that is inserted into a conventional endoscope (Fig. 2). A plastic cap is placed at the tip of the endoscope, providing stability as the probe makes contact with the mucosal surface, which is necessary for proper visualization. A new www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 4. Three OCT images (top) from a mouse colon are stacked vertically compared with low-resolution LIF intensities (bottom). Adenomas are outlined in white. Reproduced with permission from Winkler et al.18
Figure 3. Endocytoscopic image of high-grade dysplasia with disorder of polarity. Reproduced with permission from Sasjima et al.15
system has been designed that integrates the probe into the endoscope, providing improved stability. EC requires prestaining with an absorptive contrast agent, typically methylene blue. After excess contrast is washed off, the probe is placed in contact with suspicious mucosa. An image is projected onto a screen with ⫻580 magnification, and several cytological and architectural features can be evaluated, such as variability of gland size and uniformity, size and arrangement of nuclei, and cellular size, density, and arrangement (Fig. 3). Sasajima et al15 performed EC on 60 patients who had localized colorectal lesions found on colonoscopy and compared images with conventional histology. There was statistically significant correlation between the EC images and histology (93.3% accuracy). This study developed EC-based image criteria, but these criteria have yet to be validated. EC has the potential to make instant diagnoses during routine colonoscopy, which would allow for immediate biopsy and potential removal of neoplastic lesions. However, this technique provides images solely of the superficial mucosal layer, theoretically missing deeper lesions and not allowing for depth staging.15 Imaging is subject to visual interference caused by motion artifact and the presence of blood or mucus in the stool. Additionally, the macroscopic view can be impaired by high concentrations of methylene blue.
OPTICAL COHERENCE TOMOGRAPHY OCT provides cross-sectional images by detecting light that is back-reflected from microstructures beneath the www.giejournal.org
surface of tissues, in a similar fashion to how B-scan US detects sound waves. This imaging modality relies on a small catheter probe (approximately 2 mm) that does not need to be in tissue contact and does not require contrast media. Images can be acquired at a rate of 4 frames per second.16 Because of the high resolution of endoscopic OCT (4-20 m), structural components such as blood vessels, lymphatics, villi, glands, and crypts can be visualized in real time. Doppler imaging can be incorporated into OCT, which allows for blood-flow velocities to be displayed along with the architecture of microvasculature that is too small to be viewed by conventional US. OCT images are limited to the mucosa and submucosa. In 2003, Pfau et al17 performed OCT on 24 patients with polyps identified on colonoscopy. Images were obtained from the polyp, and nearby normal-appearing mucosa and real-time evaluation of these images was performed. Polyps were then removed and evaluated with conventional histopathology by a pathologist blinded to the OCT images. Adenomas were found to have significantly more tissue disorganization with less structure than hyperplastic polyps. Adenomas also exhibited a decrease in light scattering and appeared darker. Based on this, they were able to determine that endoscopic OCT could differentiate adenomas, hyperplasic polyps, and normal colon in real time. Recently, Winkler et al18 advanced the capabilities of OCT by coupling it with low-resolution laser-induced fluorescence imaging (LIF). Their system is based on an OCT sensor that provides structural information down to 18 m, coupled with the low-resolution fluorescence to provide biological information about dysplastic mucosa (Fig. 4). This platform is limited by the low resolution of the LIF Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1267
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 5. Low-cost microendoscopy: high-resolution microendoscope. The system is battery powered and contained in a briefcase (A). Images are obtained by placing a fiberoptic bundle in contact with the mucosa (B). Courtesy of R. Richards-Kortum.
Figure 6. Images of normal colon and a tubular adenoma produced by the high-resolution microendoscope. Courtesy of S. Anandasabapathy.
images; however, improvements in LIF imaging are in progress. OCT has the potential to differentiate benign and dysplastic polyps in real time. Because of the limited depth 1268 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
from which images can be produced, OCT will likely serve as a tool for staging dysplasia; however, biopsy or other imaging modalities will continue to be necessary to evaluate deeper tissue. www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 7. Normal colonic mucosa after staining with 2-NBDG (A) and corresponding histology (B); tubulovillous adenoma with dysplasia with increased glandular uptake of 2-NBDG (C) and corresponding histology (D). Courtesy of S. Anandasabapathy.
LOWER-COST MICROENDOSCOPY One factor that has limited the use of confocal platforms to tertiary care centers is the cost of these platforms as well as the significant learning curve associated with interpretation of the microscopic images. To expand access to optical biopsy technology in lower resource settings or the developing world, there is increased interest in the development of lower cost imaging devices that can be used in conjunction with image-analysis software for rapid, real-time interpretation. Muldoon et al19 developed a portable, batteryoperated, high-resolution fluorescent microendoscope that costs less than $3500 in components. The system uses a fiberoptic bundle made of 30,000 individual fiwww.giejournal.org
bers that is channeled through the biopsy port of a standard endoscope (Fig. 5). Images are obtained by placing the end of the fiberoptic bundle directly in contact with the surface of the mucosa. A fluorophore is excited using a 455-nm blue light– emitting diode, and the emitted light returning to the microscope is transmitted to a computer via a charge-coupled device camera. Anecdotally, there is evidence to suggest that use of topical proflavine hemisulfate (0.01%) combined with a high-resolution fluorescent microendoscope can differentiate normal mucosa from hyperplastic and adenomatous polyps (Fig. 6), although systematic evaluation of this will be required to determine the technique’s merits relative to high-resolution confocal platforms. Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1269
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 8. Biopsy specimen of human colorectal adenocarcinoma. A, Nonspecific nuclear and cellular staining using acriflavine. B, VEGF-specific staining by using AF488-labeled antibodies. The antibody accumulates in the cytoplasm of the tumor cells, but not the nuclei. Reproduced with permission from Foersch et al.30
TARGETED CONTRAST AGENTS AND MOLECULAR ENDOSCOPY: THE FUTURE? The ability to magnify and analyze the GI mucosa using these technologies provides a powerful opportunity to accurately diagnose colonic lesions at an early, treatable stage. To take full advantage of the capabilities of these technologies, complementary contrast agents need to be developed to specifically target neoplastic mucosa. Current technologies that highlight neoplastic colonic tissue on a macroscopic level such as autofluorescence and narrow-band imaging have been shown to improve the detection of neoplasia.20 Combining these technologies with contrast agents that target neoplastic cells on a microscopic level could provide a unique opportunity to more easily identify and characterize mucosal lesions. Molecule-specific contrast agents offer the added opportunity to provide prognostic and therapeutic information. Several groups have shown promising initial data in this area. One way that cancer cells can be differentiated from normal cells is their increased rate of glucose metabolism and uptake, as has been well established with 18Ffluorodeoxyglucose in PET imaging.21-24 A derivative of fluorodeoxyglucose, 2-(N-(7-nitrobenz-2-ox-1,3-diazol4-yl)amino)-2-deoxyglucose (2-NBDG) is similarly transported via glucose transporters and rapidly taken up by proliferating cells.24,25 Although no in vivo data exist at this time, 2 ex vivo studies have shown that 2-NBDG can be used to differentiate malignant from normal cells.26,27 The Richard-Kortum group has used fluorescently labeled de1270 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
oxyglucose (2-NBDG) as a contrast agent in confocal and fluorescence imaging of oral mucosa biopsy specimens and shown an average 3.7 times greater signal from neoplastic samples than matched non-neoplastic samples. On ex vivo samples of subjects with Barrett’s esophagus imaged with a confocal microscope, variation in the location and intensity of 2-NBDG uptake was able to differentiate neoplastic from non-neoplastic mucosa with a sensitivity of 80% and specificity of 83%.27 Preliminary work on ex vivo colonic specimens suggests that the metabolic information provided by 2-NBDG can be combined with morphological information provided by confocal microscopy to differentiate adenomatous and dysplastic colonic mucosa (Fig. 7). Although these preliminary data are promising, in vivo studies using 2-NBDG would need to be done to fully assess the practicality of this imaging modality in cancer screening and surveillance. Beyond detecting the uptake of labeled glucose, use of fluorescence endomicroscopy allows the exciting possibility of using fluorescently labeled molecules to target molecular markers specific to cancer cells. This would propel endoscopy from the cellular level to the molecular level. Several groups have published promising studies in this realm. In the age of monoclonal antibodies approved for therapeutics, it is not inconceivable that true immunofluorescence with labeled antibodies could be achieved with fluorescence endoscopy. As far back as 2002, Keller et al28 were able to achieve in vivo low-resolution fluorescence endoscopy using a monoclonal antibody against carcinoembryonic antigen applied directly onto colonic mucosa www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Figure 9. In vivo confocal fluorescence images of peptide binding. A, Binding to dysplastic colon polyp. B, Binding to adjacent normal mucosa. Histology of dysplastic colon polyp (C) and normal mucosa (D) stained with hematoxylin and eosin. Reproduced with permission from Hsiung et al.32
of patients known to have large polypoid lesions or colorectal carcinoma. Although the results were promising, direct comparison with histopathology of the site being imaged was not done, and no follow-up studies have been published. More recently, Goetz et al29 published a murine in vivo and human ex vivo study using a fluorescently labeled antibody against epidermal growth factor receptor (EGFR). Mice were injected with tumor cell lines that expressed EGFR at high or low levels. Tumors were then imaged in vivo using CLE after injecting the labeled antiEGFR and showed a threefold difference in mean fluorescence intensity. Human colonic biopsy specimens were also imaged after incubation with labeled anti-EGFR and showed an approximately eightfold difference in fluorescence intensity between neoplastic and non-neoplastic tissues. The Goetz group went further by publishing similar data using a commercially available polyclonal antibody targeting vascular endothelial growth factor (VEGF),30 known to be expressed in high levels in neoplastic tissue.31 They studied 2 mouse models with small-bowel and colon tumors by injecting them with the labeled antibody. Mice with tumor cells showed accumulation of the labeled antibody specifically in their tumors. In addition, ex vivo imaging of human colorectal adenocarcinoma showed similar staining patterns with significantly increased signal www.giejournal.org
in the tumor compared with normal colonic tissue and a clear transition zone. Of note, most of the experiments in this study were conducted using an antibody against an intracellular protein, and as such displayed cytoplasmic staining. This was done without having to permeabilize the membranes of the cells, as is usually done with immunohistochemistry. Thus, the number of proteins that could be targeted in vivo go beyond those that are displayed at the surface of the cell (Fig. 8). Use of fluorescently tagged antibodies is not without its limitations, including a long half-life after injection, cost, and concern for immunogenicity. To overcome this, Hsiung et al32 isolated a heptapeptide from a phage library by clearing the library with human intestinal cells, followed by normal colonic tissue, hyperplastic, and inflamed tissue. The remaining isolates were exposed to tubular adenomas, and approximately 40 phage clones were tested for their ability to bind to adenocarcinoma-derived cells. Finally, a heptapeptide was selected, bound to fluorescein, and applied topically in a pilot in vivo screening colonoscopy study using CLE. Their results show that, compared with histological diagnoses, increased intensity of the probe was 81% sensitive and 82% specific for adenomas (Figs. 9 and 10). The study is limited, however, in that the exact target for the heptapeptide has yet to be determined, and any potential downstream signaling that Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1271
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
Matloff et al
Figure 10. In vivo confocal fluorescence images of the border between colonic adenoma and normal mucosa, showing peptide binding to dysplastic colonocytes. A, Endoscopic view. B, Border. C, Dysplastic crypt. D, Adjacent mucosa. Reproduced with permission from Hsiung et al.32
may occur with the binding of this peptide to an unknown ligand has not been evaluated. Alternatively, a recent study used a labeled fragment of VEGF, topically applied to mouse colon, to target VEGF receptors. Using OCT-LIF, the researchers compared mice with tumors with control mice. Although the fluorescence data had poor resolution, the combined modality was able to differentiate tumor from normal tissue with a sensitivity of 93% and specificity of 100%.18 As more animal or ex vivo studies using molecular imaging become available, the prospects of in vivo endoscopic molecular imaging will become closer to reality. This would allow us to screen, diagnose, prognosticate, and manage colorectal cancer in novel ways. For example, earlier diagnosis could be achieved by targeting markers that are present earlier in the pathway to malignancy. In familial adenomatous polyposis, identifying markers that can differentiate the neoplastic potential of histologically similar polyps would be invaluable. Specific molecular targets could be surveyed that would define in real time whether a lesion is amenable to endoscopic resection. With further development of the technology, it is likely that more novel applications will be discovered. 1272 GASTROINTESTINAL ENDOSCOPY Volume 73, No. 6 : 2011
CONCLUSION Colon cancer screening remains one of the most important aspects of preventive medicine for all adults, and new technologies can enhance our ability to detect and characterize colonic lesions. Advances in molecular imaging can provide endoscopists with real-time characterization of suspicious areas, potentially providing a higher yield approach to colonoscopy. Combining cellular-level interrogation with accurate wide-field technologies may not only reduce the number of missed lesions but also reduce the number of lesions that inappropriately underwent biopsy and the number of procedures performed, creating a more efficient approach to prevention.27 More studies are needed to validate these techniques individually and in combination and to assess their cost-effectiveness and learning curve before they can be used beyond the university setting. It is clear, however, that high-resolution and optical molecular imaging has the potential to transform colonoscopy from a tool used to detect areas of possible neoplasia into a modality that can determine whether cancer is present, define its molecular signature, and guide specific therapy for an individual patient. www.giejournal.org
Matloff et al
High-resolution and optical molecular imaging for the early detection of colonic neoplasia
REFERENCES 1. U.S. Preventive Services Task Force. Screening for Colorectal Cancer: U.S. Preventive Services Task Force Recommendation Statement. AHRQ Publication 08-05124-EF-3. Rockville (Md): Agency for Healthcare Research and Quality, 2008. 2. Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med 1993;329:1977-81. 3. Lieberman DA, Prindiville S, Weiss DG, et al. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-67. 4. van Rijn JC, Reitsma JB, Stoker J, et al. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006; 101:343-50. 5. Kudo S, Kashida H, Tamura T, et al. Colonoscopic diagnosis and management of nonpolypoid early colorectal cancer. World J Surg 2000;24: 1081-90. 6. Meining A. Confocal endomicroscopy. Gastrointest Endosc Clin N Am 2009;19:629-35. 7. Dunbar K, Canto M. Confocal endomicroscopy. Curr Opin Gastroenterol 2008;24:631-7. 8. Goetz M, Kiesslich R. Confocal endomicroscopy: in vivo diagnosis of neoplastic lesions of the gastrointestinal tract. Anticancer Res 2008;28: 353-60. 9. Kiesslich R, Goetz M, Vieth M, et al. Technology insight: confocal laser endoscopy for in vivo diagnosis of colorectal cancer. Nat Clin Pract Oncol 2007;4:480-90. 10. Kiesslich R, Burg J, Vieth M, et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004;127:706-13. 11. Sanduleanu S, Driessen A, Gomez-Garcia E, et al. In vivo diagnosis and classification of colorectal neoplasia by chromoendoscopy-guided confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2010;8:371-8. 12. Meining A, Saur D, Bajbouj M, et al. In vivo histopathology for detection of gastrointestinal neoplasia with a portable, confocal miniprobe: an examiner blinded analysis. Clin Gastroenterol Hepatol 2007;5:1261-7. 13. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of probebased confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps. Gastroenterology 2010;138:834-42. 14. Kiesslich R, Goetz M, Lammersdorf K, et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007;132:874-82. 15. Sasajima K, Kudo SE, Inoue H, et al. Real-time in vivo virtual histology of colorectal lesions when using the endocytoscopy system. Gastrointest Endosc 2006;63:1010-7. 16. DaCosta RS, Wilson BC, Marcon NE. Optical techniques for the endoscopic detection of dysplastic colonic lesions. Curr Opin Gastroenterol 2005;21:70-9. 17. Pfau PR, Sivak MV Jr, Chak A, et al. Criteria for the diagnosis of dysplasia by endoscopic optical coherence tomography. Gastrointest Endosc 2003;58:196-202. 18. Winkler AM, Rice PF, Weichsel J, et al. In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model. Mol Imaging Biol Epub 2010 Nov 2.
www.giejournal.org
19. Muldoon TJ, Anandasabapathy S, Maru D, et al. High-resolution imaging in Barrett’s esophagus: a novel, low-cost endoscopic microscope. Gastrointest Endosc 2008;68:737-44. 20. van den Broek FJ, Fockens P, van Eeden S, et al. Endoscopic tri-modal imaging for surveillance in ulcerative colitis: randomised comparison of high-resolution endoscopy and autofluorescence imaging for classification of lesions. Gut 2008;57:1083-9. 21. Larson SM. Positron emission tomography-based molecular imaging in human cancer: exploring the link between hypoxia and accelerated glucose metabolism. Clin Cancer Res 2004;10:2203-4. 22. Rajendran JG, Mankoff DA, O’Sullivan F, et al. Hypoxia and glucose metabolism in malignant tumors: evaluation by [18F]fluoromisonidazole and [18F]fluorodeoxyglucose positron emission tomography imaging. Clin Cancer Res 2004;10:2245-52. 23. Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell Biol 2006;18: 598-608. 24. Jadvar H, Alavi A, Gambhir SS. 18F-FDG uptake in lung, breast, and colon cancers: molecular biology correlates and disease characterization. J Nucl Med 2009;50:1820-7. 25. O’Neil RG, Wu L, Mullani N. Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells. Mol Imaging Biol 2005;7:388-92. 26. Nitin N, Carlson AL, Muldoon T, et al. Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose. Int J Cancer 2009;124:2634-42. 27. Thekkek N, Maru D, Richards-Kortum R, et al. Fluorescent deoxyglucose as a topical contrast agent to detect Barrett’s associated neoplasia using confocal imaging. New York: Mount Sinai School of Medicine; 2010. 28. Keller R, Winde G, Terpe HJ, et al. Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy 2002;34:801-7. 29. Goetz M, Ziebarg A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010;138:435-46. 30. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010;59:1046-55. 31. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57. 32. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008;14:454-8. 33. Wallace MB, Kiesslich R. Advances in endoscopic imaging of colorectal neoplasia. Gastroenterology 2010;138:2140-50. Received November 16, 2010. Accepted January 29, 2011. Current affiliations: Department of Medicine (J.L.M., W.A.), Division of Gastroenterology (J.S., S.A.), Mount Sinai Medical Center, New York, New York, Department of Bioengineering (R.R.-K.), Rice University, Houston, Texas, USA. Reprint requests: Sharmila Anandasabapathy, MD, Division of Gastroenterology, The Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029.
Volume 73, No. 6 : 2011 GASTROINTESTINAL ENDOSCOPY 1273