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Su2029 In Vivo Imaging of Mucosal Endothelin-a Receptor Expression to Characterize Murine Colorectal Cancer Development
Figure 1. ATGL immunoreactivity in a moderately (A) and a poorly (B) differentiated colonic adenocarcinomas (x400). Su2029 In Vivo Imaging of Mucosal Endothelin-a Receptor Expression to Characterize Murine Colorectal Cancer Development Dominik Bettenworth, Marcus Mücke, Christiane Geyer, Carsten Höltke, Katrin Schwegmann, Christopher Poremba, Michael Schäfers, Dirk Domagk, Philipp Lenz
Representative en face OCT images of different upper GI tissues from depths of 100-400um and projected over 80um. A, normal esophagus. B, gastric mucosa. C, Non-dysplastic BE. D, BE with low grade dysplasia. E, BE with high grade dysplasia. F, Esophageal adenocarcinoma (stage T1). Scale bars are 500um.
BACKGROUND AND AIMS: Ulcerative colitis patients are at an increased risk of developing colorectal cancer. Therefore, current guidelines recommend surveillance colonoscopy with chromoendoscopy or random biopsies to detect dyplastic lesions. ET-A receptor (ETAR) expression is known to be significantly increased in various malignancies including colorectal cancer (1). The aim of the study was to detect and characterize early dysplastic lesions in mice by ETAR-guided fluorescence endoscopy (FE) and fluorescence reflectance imaging. METHODS: Colorectal cancer was induced in C57Bl/6 WT mice (n=15) by single injection of azoxymethane i.p. and cyclic administration of dextran sodiumsulfate in drinking water. A Cy5.5-labeled nonpeptidic ETAR imaging probe was intravenously injected 24 hrs before endoscopic examination. Tumor development was assessed in vivo by white light endoscopy and FE. Murine colons were examined by fluorescence reflectance imaging using an MSFX PRO optical imaging system to measure tracer uptake ex vivo. At the end of the experiment, colons were explanted and tumors were graded after HE staining and evaluated by an expert pathologist. Tumor biology was assessed by immunohistochemistry and western blot analysis of ETAR expression. Additionally, binding specificity of the tracer was evaluated by simultaneous injection of an equimolar amount of an unspecific fluorochrome (Cy3.5-glycin) and the specific tracer. RESULTS: ETAR expression could easily be detected by FE and all animals survived the procedure. Standard histology confirmed colorectal adenomas as well as high and low grade dysplasia. Conventional molecular imaging applying the fluorescent ETAR imaging probe showed significantly increased tracer uptake in colorectal adenoma as compared to healthy mucosa (1336.8 ± 375.3 vs. 544.1 ± 90.4, P < 0.001). In contrast, no significant difference in tracer uptake between low and high grade adenoma was found (tumor-to-background ratio: 2.5 ± 0.6 vs. 2.3 ± 0.9, P = 0.637). The tumor-to-background ratio of lesions imaged for Cy3.5 was significantly reduced as compared to Cy5.5 indicating the high binding-specificity of the used ETAR tracer (1.4 ± 0.3 vs. 2.5 ± 0.7, P < 0.001). CONCLUSION: ETAR expression was significantly increased in murine colorectal dysplasia as compared to healthy mucosa. By detection of ETAR via FE in live mice, a prognostic biomarker for colorectal cancer development is directly visualized during colonoscopy, thus facilitating in vivo tumor characterization on a molecular level. (1) Hoosein et al. Altered endothelin receptor subtypes in colorectal cancer. Eur J Gastroenterol Hepatol. 2007
Su2031 Evaluation of Severity Using Endoscopic Dual Red Imaging Targeting Submucosal Vessel in Patients With Ulcerative Colitis Makoto Naganuma, Naohisa Yahagi, Rieko Bessho, Ai Fujimoto, Naoki Hosoe, Kazuhiro Kashiwagi, Tadakazu Hisamatsu, Haruhiko Ogata, Takanori Kanai Background: Endoscopy is useful to assess the severity of inflammation and can predict middle to long term prognosis in patients with ulcerative colitis (UC). Recently, new imaging technology that has three kinds of illumination lights whose center wavelength are 540nm, 600nm and 630nm respectively. The both of lights in 600nm and 630nm are critical to recognize blood vessels in deeper tissue. Because dual red imaging (DRI) can mainly emphasize the findings of vessel pattern, disagreement for assessment the severity of inflammation using DRI might be minimal among the investigators. The aim of this study is to evaluate the additional usefulness of DRI to assess the severity of inflammation in patients with UC. Patients and Methods: A total of 43 UC patients were enrolled to evaluate endoscopic severity at 112 segments of colon using white light imaging and DRI. Two independent investigators assessed and compared between Matts' endoscopic scores and DRI scores (see Figure). Correlation between DRI score and histological grading (Matts histological score 15) was also evaluated. Furthermore, inter-observer agreement for DRI score between two investigators was also assessed. Results: DRI score is closely correlated to Matts'endoscopic (r=0.88) and histological scores (r=0.83). Although rates of DRI score agreement between 2 investigators was 87% (97/112, κ=0.81), rates of Matts endoscopic score agreement was 72% (81/112, κ=0.59). Among patients with mild to moderate colitis (Matts' endoscopic score 2-3). median Matts histological score in patients with DRI score=4 was significantly higher than that in patients with DRI score=2 or 3 (p<0.01). Conclusion: DRI score is closely correlated to former endoscopic and histological score and inter-observer disagreement of DRI was minimal comparing to Matts' score.
Su2030 En Face Optical Coherence Tomography Visualization of Mucosal Surface Patterns for Identifying Early Malignancy in the Upper GI Tract Osman O. Ahsen, Hsiang-Chieh Lee, Kaicheng Liang, Michael G. Giacomelli, Zhao Wang, Marisa Figueiredo, Qin Huang, Benjamin Potsaid, Vijaysekhar Jayaraman, James G. Fujimoto, Hiroshi Mashimo BACKGROUND/AIMS: Aberrant mucosal surface patterns (pit patterns) are associated with dysplasia in Barrett's esophagus (BE). Optical coherence tomography (OCT) can generate volumetric images of tissue microstructure. We have recently demonstrated ultrahigh-speed endoscopic OCT that achieves >10x faster imaging speeds than commercial systems. This OCT, combined with distal micromotor beam scanning, acquires densely sampled volumetric datasets enabling arbitrary cross-sectional and depth-resolved en face OCT images and shows pit patterns with high resolution and contrast. In this study we investigate en face OCT of upper GI tissues including normal squamous epithelium, gastric mucosa, non-dysplastic BE (NDBE), BE with low and high grade dysplasia, as well as esophageal adenocarcinoma (EAC). METHODS: The study was performed at the VABHS under an IRB protocol approved by the VABHS, HMS and MIT. We used an endoscopic OCT system with a 600 kHz axial scan
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AGA Abstracts
AGA Abstracts
rate and 400 fps frame rate. The imaging probe was introduced in the endoscope accessory port for co-registration of images with biopsies. Volumetric datasets were acquired with an 8 second helical scan over a 160mm2 area, and multiple datasets were acquired covering the BE length. A total of 15 BE patients (7 NDBE, 3 LGD, 4 HGD, 1 EAC) were imaged with co-registered biopsies or EMR from Sept. 2013 to Nov. 2014. RESULTS: Figure 1 shows representative en face OCT upper GI images. Fig. 1(A) shows normal esophagus with no apparent structure, consistent with the homogeneous appearance of squamous epithelium. Fig. 1(B) shows gastric folds and circular pit patterns, characteristic of normal gastric tissue. Fig. 1(C) shows NDBE and exhibits a cerebriform or gyrus-forming appearance, with pit patterns regularly distributed throughout the region of BE. Figs. 1(D) and 1(E) show low and high grade dysplasia, respectively, and exhibit enlargement and distortion of pit patterns compared to NDBE. Fig. 1(F) shows EAC, where severe distortion of pit pattern is accompanied with abnormally shaped and sized glandular structures. CONCLUSIONS: Ultrahigh speed endoscopic OCT can generate images of mucosal morphology similar to magnification NBI and chromoendoscopy, but over a larger field of view, and with near-microscopic resolution and high contrast. Furthermore, en face OCT images at different depths upto 12mm can be extracted from 3D volumes, which would not be attainable by present confocal endomicroscopy. Moreover, cross-sectional OCT images can be generated that are intrinsically registered to the en face features. En face and cross-sectional OCT promises to improve detection sensitivity of dysplasia in BE over cross-sectional OCT alone, but requires next generation ultrahigh speed OCT technology and scanning probes. ACKNOWLEDGMENT: NIH R01-CA075289-17, R44-CA101067-07, AFOSR FA9550-10-1-0551 and FA9550-121-0499.
MSH1, MSH6 and PMS2) and B-Raf mutation was evaluated. Statistical analysis was performed by using Fisher's exact test and a p value of <0.05 was considered significant. Results: The intensity of ATGL staining was higher in well and moderately differentiated (low grade) CRA (p = 0.0009) as compared to poorly differentiated (high grade) CRA. A unique pattern of clumped basal staining was observed in dysplastic and malignant glands in moderately differentiated CRA (Figure 1). This specific staining pattern was not observed in PDEF although weak PEDF immunostaining was observed in all of the CRA samples. There was no association between ATGL staining intensity and other clinical parameters such as BMI >25 (p = 0.876), angioinvasion (p = 0.561), perineural invasion (p = 0.092), metastasis (p = 1.000) and B-Raf mutation (p = 0.072). Conclusion: Higher ATGL expression is associated with low-grade as compared to high-grade CRA. An ATGL staining pattern unique to low grade CRA may indicate a possible lipolytic process involved in CRA differentiation and this may be helpful in diagnosis in a setting of metastatic adenocarcincinoma with unkown primary. The universally weak PEDF expression in all cases suggests ATGL and PEDF have different roles in CRA.
Representative en face OCT images of different upper GI tissues from depths of 100-400um and projected over 80um. A, normal esophagus. B, gastric mucosa. C, Non-dysplastic BE. D, BE with low grade dysplasia. E, BE with high grade dysplasia. F, Esophageal adenocarcinoma (stage T1). Scale bars are 500um.
BACKGROUND AND AIMS: Ulcerative colitis patients are at an increased risk of developing colorectal cancer. Therefore, current guidelines recommend surveillance colonoscopy with chromoendoscopy or random biopsies to detect dyplastic lesions. ET-A receptor (ETAR) expression is known to be significantly increased in various malignancies including colorectal cancer (1). The aim of the study was to detect and characterize early dysplastic lesions in mice by ETAR-guided fluorescence endoscopy (FE) and fluorescence reflectance imaging. METHODS: Colorectal cancer was induced in C57Bl/6 WT mice (n=15) by single injection of azoxymethane i.p. and cyclic administration of dextran sodiumsulfate in drinking water. A Cy5.5-labeled nonpeptidic ETAR imaging probe was intravenously injected 24 hrs before endoscopic examination. Tumor development was assessed in vivo by white light endoscopy and FE. Murine colons were examined by fluorescence reflectance imaging using an MSFX PRO optical imaging system to measure tracer uptake ex vivo. At the end of the experiment, colons were explanted and tumors were graded after HE staining and evaluated by an expert pathologist. Tumor biology was assessed by immunohistochemistry and western blot analysis of ETAR expression. Additionally, binding specificity of the tracer was evaluated by simultaneous injection of an equimolar amount of an unspecific fluorochrome (Cy3.5-glycin) and the specific tracer. RESULTS: ETAR expression could easily be detected by FE and all animals survived the procedure. Standard histology confirmed colorectal adenomas as well as high and low grade dysplasia. Conventional molecular imaging applying the fluorescent ETAR imaging probe showed significantly increased tracer uptake in colorectal adenoma as compared to healthy mucosa (1336.8 ± 375.3 vs. 544.1 ± 90.4, P < 0.001). In contrast, no significant difference in tracer uptake between low and high grade adenoma was found (tumor-to-background ratio: 2.5 ± 0.6 vs. 2.3 ± 0.9, P = 0.637). The tumor-to-background ratio of lesions imaged for Cy3.5 was significantly reduced as compared to Cy5.5 indicating the high binding-specificity of the used ETAR tracer (1.4 ± 0.3 vs. 2.5 ± 0.7, P < 0.001). CONCLUSION: ETAR expression was significantly increased in murine colorectal dysplasia as compared to healthy mucosa. By detection of ETAR via FE in live mice, a prognostic biomarker for colorectal cancer development is directly visualized during colonoscopy, thus facilitating in vivo tumor characterization on a molecular level. (1) Hoosein et al. Altered endothelin receptor subtypes in colorectal cancer. Eur J Gastroenterol Hepatol. 2007
Su2031 Evaluation of Severity Using Endoscopic Dual Red Imaging Targeting Submucosal Vessel in Patients With Ulcerative Colitis Makoto Naganuma, Naohisa Yahagi, Rieko Bessho, Ai Fujimoto, Naoki Hosoe, Kazuhiro Kashiwagi, Tadakazu Hisamatsu, Haruhiko Ogata, Takanori Kanai Background: Endoscopy is useful to assess the severity of inflammation and can predict middle to long term prognosis in patients with ulcerative colitis (UC). Recently, new imaging technology that has three kinds of illumination lights whose center wavelength are 540nm, 600nm and 630nm respectively. The both of lights in 600nm and 630nm are critical to recognize blood vessels in deeper tissue. Because dual red imaging (DRI) can mainly emphasize the findings of vessel pattern, disagreement for assessment the severity of inflammation using DRI might be minimal among the investigators. The aim of this study is to evaluate the additional usefulness of DRI to assess the severity of inflammation in patients with UC. Patients and Methods: A total of 43 UC patients were enrolled to evaluate endoscopic severity at 112 segments of colon using white light imaging and DRI. Two independent investigators assessed and compared between Matts' endoscopic scores and DRI scores (see Figure). Correlation between DRI score and histological grading (Matts histological score 15) was also evaluated. Furthermore, inter-observer agreement for DRI score between two investigators was also assessed. Results: DRI score is closely correlated to Matts'endoscopic (r=0.88) and histological scores (r=0.83). Although rates of DRI score agreement between 2 investigators was 87% (97/112, κ=0.81), rates of Matts endoscopic score agreement was 72% (81/112, κ=0.59). Among patients with mild to moderate colitis (Matts' endoscopic score 2-3). median Matts histological score in patients with DRI score=4 was significantly higher than that in patients with DRI score=2 or 3 (p<0.01). Conclusion: DRI score is closely correlated to former endoscopic and histological score and inter-observer disagreement of DRI was minimal comparing to Matts' score.
Su2030 En Face Optical Coherence Tomography Visualization of Mucosal Surface Patterns for Identifying Early Malignancy in the Upper GI Tract Osman O. Ahsen, Hsiang-Chieh Lee, Kaicheng Liang, Michael G. Giacomelli, Zhao Wang, Marisa Figueiredo, Qin Huang, Benjamin Potsaid, Vijaysekhar Jayaraman, James G. Fujimoto, Hiroshi Mashimo BACKGROUND/AIMS: Aberrant mucosal surface patterns (pit patterns) are associated with dysplasia in Barrett's esophagus (BE). Optical coherence tomography (OCT) can generate volumetric images of tissue microstructure. We have recently demonstrated ultrahigh-speed endoscopic OCT that achieves >10x faster imaging speeds than commercial systems. This OCT, combined with distal micromotor beam scanning, acquires densely sampled volumetric datasets enabling arbitrary cross-sectional and depth-resolved en face OCT images and shows pit patterns with high resolution and contrast. In this study we investigate en face OCT of upper GI tissues including normal squamous epithelium, gastric mucosa, non-dysplastic BE (NDBE), BE with low and high grade dysplasia, as well as esophageal adenocarcinoma (EAC). METHODS: The study was performed at the VABHS under an IRB protocol approved by the VABHS, HMS and MIT. We used an endoscopic OCT system with a 600 kHz axial scan
S-579
AGA Abstracts
AGA Abstracts
rate and 400 fps frame rate. The imaging probe was introduced in the endoscope accessory port for co-registration of images with biopsies. Volumetric datasets were acquired with an 8 second helical scan over a 160mm2 area, and multiple datasets were acquired covering the BE length. A total of 15 BE patients (7 NDBE, 3 LGD, 4 HGD, 1 EAC) were imaged with co-registered biopsies or EMR from Sept. 2013 to Nov. 2014. RESULTS: Figure 1 shows representative en face OCT upper GI images. Fig. 1(A) shows normal esophagus with no apparent structure, consistent with the homogeneous appearance of squamous epithelium. Fig. 1(B) shows gastric folds and circular pit patterns, characteristic of normal gastric tissue. Fig. 1(C) shows NDBE and exhibits a cerebriform or gyrus-forming appearance, with pit patterns regularly distributed throughout the region of BE. Figs. 1(D) and 1(E) show low and high grade dysplasia, respectively, and exhibit enlargement and distortion of pit patterns compared to NDBE. Fig. 1(F) shows EAC, where severe distortion of pit pattern is accompanied with abnormally shaped and sized glandular structures. CONCLUSIONS: Ultrahigh speed endoscopic OCT can generate images of mucosal morphology similar to magnification NBI and chromoendoscopy, but over a larger field of view, and with near-microscopic resolution and high contrast. Furthermore, en face OCT images at different depths upto 12mm can be extracted from 3D volumes, which would not be attainable by present confocal endomicroscopy. Moreover, cross-sectional OCT images can be generated that are intrinsically registered to the en face features. En face and cross-sectional OCT promises to improve detection sensitivity of dysplasia in BE over cross-sectional OCT alone, but requires next generation ultrahigh speed OCT technology and scanning probes. ACKNOWLEDGMENT: NIH R01-CA075289-17, R44-CA101067-07, AFOSR FA9550-10-1-0551 and FA9550-121-0499.
MSH1, MSH6 and PMS2) and B-Raf mutation was evaluated. Statistical analysis was performed by using Fisher's exact test and a p value of <0.05 was considered significant. Results: The intensity of ATGL staining was higher in well and moderately differentiated (low grade) CRA (p = 0.0009) as compared to poorly differentiated (high grade) CRA. A unique pattern of clumped basal staining was observed in dysplastic and malignant glands in moderately differentiated CRA (Figure 1). This specific staining pattern was not observed in PDEF although weak PEDF immunostaining was observed in all of the CRA samples. There was no association between ATGL staining intensity and other clinical parameters such as BMI >25 (p = 0.876), angioinvasion (p = 0.561), perineural invasion (p = 0.092), metastasis (p = 1.000) and B-Raf mutation (p = 0.072). Conclusion: Higher ATGL expression is associated with low-grade as compared to high-grade CRA. An ATGL staining pattern unique to low grade CRA may indicate a possible lipolytic process involved in CRA differentiation and this may be helpful in diagnosis in a setting of metastatic adenocarcincinoma with unkown primary. The universally weak PEDF expression in all cases suggests ATGL and PEDF have different roles in CRA.