SCREENING COLONOSCOPY IS very important for

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1 Digestive Endoscopy 2015; 27: doi: /den Review Advanced colonoscopic imaging using endocytoscopy Helmut Neumann, 1,2 Shin-Ei Kudo, 3 Ralf Kiesslich 4 and Markus F. Neurath 1,2 1 Department of Medicine I, University of Erlangen-Nuremberg, 2 Ludwig Demling Endoscopy Center of Excellence, University Hospital Erlangen, Erlangen, 4 Klinik Innere Medizin II, HSK, Dr. Horst Schmidt Kliniken, Wiesbaden, Germany; and 3 Digestive Disease Center, Showa University Northern Yokohama Hospital, Yokohama, Japan Optical biopsy techniques were recently introduced to luminal gastrointestinal endoscopy. These include confocal laser endomicroscopy, spectroscopic imaging techniques and endocytoscopy. Optical biopsy techniques allow on demand in vivo histology during ongoing endoscopy, thereby potentially accelerating clinical diagnosis and specific therapy. In the present review, we focus on endocytoscopy as one of the rapidly emerging optical biopsy techniques. We provide technical details of currently available endocytoscopy systems and give tips on their use in clinical practice. We also summarize applications of endocytoscopy for colorectal pathologies. Key words: advanced endoscopic imaging, endocytoscopy, endomicroscopy, endoscopy, ulcerative colitis INTRODUCTION SCREENING COLONOSCOPY IS very important for early diagnosis and therapy of colorectal cancer and its precursor lesions. 1 Once a polyp is detected it can usually be resected using polypectomy techniques. 2 Based on histology results of resected polyps, subsequent surveillance intervals are determined. Nevertheless, up to 80% of all colorectal polyps are diminutive (i.e. <5 mm in diameter). 3 The prevalence of adenomatous histology in these polyps is considerably low, ranging between 2 and 3.4%, and the prevalence of cancerous tissue in diminutive polyps is calculated as <0.1%. 3 The redundant removal of those polyps leads to enormous costs for the health-care system. 4 In this context, it has been calculated that the upfront cost savings of forgoing the pathological assessment of diminutive polyps may be more than 1 billion US dollars annually in the USA. 5 Besides endoscopic diagnosis of colorectal polyps, endoscopic diagnosis of inflammatory bowel disease (IBD) is of increasing importance for colitis-associated cancer screening and to guide medical therapies. 6,7 Whereas four-quadrant, random biopsies taken every 10 cm only cover <1% of the entire colon mucosa, there is an ongoing need for developing advanced endoscopic imaging techniques to improve the diagnosis of non-polypoid lesions in IBD. 8 In addition, Corresponding: Helmut Neumann, Department of Medicine I, University of Erlangen-Nuremberg, Ulmenweg 18, Erlangen, Germany. helmut.neumann@uk-erlangen.de Received 29 April 2014; accepted 6 October mucosal healing and deep clinical remission have become important treatment goals in IBD, as accumulating evidence has shown that mucosal healing as assessed by endoscopy is predictive of reduced disease activity, reduced rates of hospitalization and surgical resection, and is associated with sustained clinical remission. 9,10 Taken together, the above-mentioned considerations emphasize the need for advanced endoscopic imaging techniques providing ad hoc and in vivo histology during the ongoing endoscopic procedure either to save costs or to guide therapeutic approaches. In recent years, optical biopsy techniques including confocal laser endomicroscopy (Pentax, Tokyo, Japan; Mauna Kea Technologies, Paris, France) and spectroscopic imaging systems (e.g. WavSTAT; SpectraScience, San Diego, CA, USA) have been established in luminal endoscopy In the present review, we focus on the rapidly emerging optical biopsy technique of endocytoscopy (Table 1). 12 We provide technical details of currently available endocytoscopy systems and give tips on their use in clinical practice. We also summarize applications of endocytoscopy for colorectal pathologies. TECHNIQUE OF ENDOCYTOSCOPY ENDOCYTOSCOPY PROTOTYPES (OLYMPUS, Tokyo, Japan) represent high-magnification endoscopes allowing magnification of up to 1390-fold. For use in clinical practice, two different types of endocytoscopy systems are available (Table 2). 15 One endocytoscopy system represents a bs_bs_banner 232

2 Digestive Endoscopy 2015; 27: Endocytoscopy 233 stand-alone hand-held miniprobe that is advanced through the accessory channel of a therapeutic endoscope (i.e. probe-based endocytoscopy; pec). Most recently introduced endocytoscopes are integrated into the distal tip of standard high-resolution white-light endoscopes and are called integrated endocytoscopy systems (iec). Endocytoscopy images are displayed simultaneously with the white-light image on one monitor thereby allowing targeted mucosal optical biopsies. Table 1 Current applications of endocytoscopy Esophagus Stomach Duodenum Colon Lung Others Esophageal squamous cell carcinoma Barrett s esophagus Helicobacter pylori infection Signet ring-cell carcinoma Celiac disease Polyps Amoebic colitis Small cell lung cancer Bladder carcinoma Intraoperative carcinoma diagnosis Mucosal preparation and staining The colorectum is physiologically coated by mucus and debris. Therefore, N-acetylcysteine is recommended for mucolysis after intense washing of the mucosa with water to remove debris from the mucosal surface. Adding simethicone to the lavage solution may further help in reducing bubble formation. After mucosal preparation, endocytoscopic imaging requires prestaining of the mucosa with absorptive staining agents that are topically applied to the mucosa by using large-caliber syringes or standard spraying catheters. Repeat staining of the mucosa might become necessary when endocytoscopic imaging is prolonged. Of note, after tattooing of lesions with submucosal injection of India ink, endocytoscopic imaging might be reduced as visualization of glandular details is limited. 16 Kodashima et al. aimed to determine an appropriate staining regimen for endocytoscopy. 17 Freshly resected porcine esophagus, stomach, and colon were exposed to various concentrations of three different dyes (crystal violet, methylene blue, and toluidine blue) and assessed after exposure times of 10 s, 30 s, 60 s, and 90 s. Images were evaluated according to the staining status of the cytoplasm and the nucleus, and the contrast between the cytoplasm and the nucleus. Afterwards, freshly resected human esophagus, stomach, and colon tissues were tested under exposure conditions that were found to be the most appropriate in the animal study. It was found that high-quality images were obtained using both methylene blue and toluidine blue staining. Optimum conditions for endocytoscopic observation were obtained after staining with 1% methylene blue in the esophagus and with 0.25% toluidine blue in the stomach and the colon, after 60 s of exposure to the dye. This was also confirmed in the human specimens. 17 Recently, a new staining approach was introduced by Ichimasa and coworkers. 18 The new approach consists of double staining containing crystal violet and methylene blue. Thirty prospectively enrolled patients were allocated 1:1:1 to three distinct staining methods: 0.05% crystal violet (CV) alone, 1% methylene blue (MB) alone, or CV + MB (CM double). Table 2 Technical characteristics of different endocytoscopy devices Type Probe-based (pec) Endoscope-based (iec) XEC-300 XEC-120 XGIF-Q260EC1 XCF-Q260EC1 GIF-Y0002 Endoscope magnification NA ( 600 digital) Endoscope working channel (mm) NA Total length (cm) Functional length (cm) EC magnification 450 (14 inch 570 (19 inch 1100 (14 inch 1390 (19 inch ( 600 digital) Field of view (μm) ( digital) Horizontal resolution Outer diameter (mm) Imaging plane depth (μm) Adapted from Neumann et al. 15 EC, endocytoscope; iec, integrated endocytoscopy; NA, not applicable; pec, probe-based endocytoscopy. XEC-300, Olympus, Tokyo, Japan; XEC-120, Olympus; XGIF-Q260EC1, Olympus; XCF-Q260EC1, Olympus; GIF-Y0002, Olympus.

3 234 H. Neumann et al. Digestive Endoscopy 2015; 27: A B Figure 1 After staining with (a) toluidine blue or (b) methylene blue, architectural and cellular details such as the nucleus and the nucleus-to-cytoplasm ratio are clearly observed. Afterwards, the visibility of nuclei and gland formation after staining were evaluated as recognizable or not recognizable. Time for each parameter to become recognizable was measured, and the average times for the three staining regimens were compared. It was found that MB alone and CM double staining resulted in recognizable nuclei within comparable periods of time, whereas CV alone was unable to identify nuclei. Gland formation became recognizable sooner after CM double staining than after MB alone. Those differences were calculated as statistically significant. It was therefore concluded that double staining with CV and MB, which rapidly provided recognizable images of both nuclei and gland formation, is an appropriate staining regimen for colonic endocytoscopic imaging. 18 Image interpretation Interpretation of endocytoscopy images is based on cellular, architectural and vascular details. By using absorptive staining agents and ultra-high magnification settings, one could identify the size and arrangement of cells, the nucleus and the nucleus-to-cytoplasm-ratio (Fig. 1). One early publication assessed the potential of endocytoscopy to predict histology in neoplastic lesions. 19 Seventy-six patients with neoplastic lesions of the luminal gastrointestinal tract were included and in vivo diagnosis based on endocytoscopic imaging was compared with conventional histopathology. Sensitivity and specificity of endocytoscopy to predict neoplasia were 96% and 95%, respectively, when the images were evaluated by an endoscopist who was also aware of the standard white-light endoscopic image. In addition, a negative predictive value of 91% was calculated, thereby highlighting the potential of endocytoscopy to predict neoplastic changes in vivo. ADVANCED COLONOSCOPIC IMAGING USING ENDOCYTOSCOPY Colorectal neoplasia IN 2006, SASAJIMA and coworkers prospectively evaluated the usefulness of optical biopsy of colorectal lesions by using endocytoscopy. 20 Overall, 113 consecutive patients were included. Endocytoscopy was feasible to observe lesions at the cellular level and could evaluate cellular atypia in addition to structural atypia in vivo. The correlation was statistically significant between the endocytoscopic diagnosis and the histological diagnosis. This study was the first to show the potential of endocytoscopy to distinguish neoplastic from non-neoplastic lesions, and invasive cancer from adenoma. Additionally, it was shown that the endocytoscopic image corresponded well with those of hematoxylin and eosin-stained microscopic images of colorectal lesions. Meroni et al. described a case of endocytoscopy to detect tissue abnormalities in normal mucosa surrounding colorectal cancer. 21 The colorectal specimen was imaged by methylene-blue-guided endocytoscopy within 1 h after surgical resection. By using magnification of 450, focal changes in the colonic pits were detected by the endocytoscopy system 7 cm away from the tumor within macroscopically normal mucosa, suggesting the presence of an aberrant crypt focus. Corresponding histology confirmed the presence of focal glandular abnormalities such as irregular crypt shape, increased cellular density, heterogeneity in cell shape, and disorder of polarity. The potential of endocytoscopy to identify aberrant crypt foci of the colorectum was also confirmed by a prospective in vivo study from Italy. 22 Aberrant crypt foci are clusters of morphologically altered crypts and are considered to be one of the earliest endoscopically visible precursor lesions of colorectal adenomas. 23

4 Digestive Endoscopy 2015; 27: Endocytoscopy 235 These crypt foci also demonstrate pathways of the adenomacarcinoma sequence and serrated carcinogenic pathways. 24,25 Cipolletta et al. included 41 consecutive patients with colorectal aberrant crypt foci. 22 Overall, 48 lesions were examined at a 450-fold magnification. The quality of endocytoscopy images was rated as good in 81% of cases, medium in 13%, and poor in 6% of cases. Twenty-three endocytoscopic images were labeled as dysplastic and 25 as non-dysplastic. Sensitivity and specificity of endocytoscopy to predict dysplasia were 91% and 100%, respectively. Interobserver agreement between endoscopist and pathologist was good with a kappa value of Early detection of colorectal polyps is of paramount importance for effective colorectal cancer screening. Once a lesion is found, it should be first characterized endoscopically to guide subsequent endoscopic therapy. One recent study assessed the capability of endocytoscopy with 450- fold magnification in differentiating neoplastic from nonneoplastic lesions in the colorectum. 26 In addition, the authors aimed at validating an endocytoscopy classification. For the purpose of the study, patients with colorectal polypoid and non-polypoid lesions 10 mm in diameter were prospectively included. The proposed endocytoscopy classification subgroups non-neoplastic (EC 0) and neoplastic (EC 1 3) lesions (Table 3). Histology serves as the reference standard. In all, 52 lesions in 49 patients were evaluated. Positive predictive values of each endocytoscopy group were 100%, 93%, 90%, and 100%, respectively. In addition, optical diagnosis with endocytoscopy always correlated to standard histology for differentiating neoplastic and non-neoplastic lesions. 26 Recently, Kudo et al. developed and introduced a novel endocytoscopic classification for diagnosis of colorectal lesions. 27 Overall, 206 consecutive patients were enrolled and endocytoscopic diagnosis Table 3 Endocytoscopy classification as proposed by Sasajima et al. 20 and Rotondano et al. 26 Classification Endocytoscopy findings Normal 0a Uniform glands, round lumen mucosa Hyperplasia 0b Uniform glands, serrated lumen Low-grade IEN 1 Uniform glands, slit-like lumen, faintly stained fusiform or enlarged and darkly stained nuclei High-grade IEN 2 Irregular glands, enlarged and distorted nuclei Invasive cancer IEN, intraepithelial neoplasia. 3 Destroyed gland structure, enlarged and distorted nuclei was compared to the final histopathological diagnosis. Endocytoscopic images were grouped into five classes. Non-neoplastic lesions were classified as EC1a and EC1b, whereas neoplastic lesions were classified as EC2, EC3a and EC3b. In short, roundish lumens of the crypts and a regular pattern of uniformly sized fusiform nuclei were classified as EC1a (normal mucosa). Hyperplastic lesions (EC1b) showed narrow, serrated lumens and a dense pattern of small roundish nuclei. Images showing neoplastic characteristics that corresponded to histopathological tissue images ranging from low- to high-grade dysplasias were classified as EC2 and showed slit-like smooth lumens and a regular pattern of fusiform or roundish nuclei. Irregular and rough lumens and a large number of roundish nuclei were classified as EC3a (high-grade dysplasia or invasive submucosal cancer; SMm). EC3b showed unclear gland formation and agglomeration of distorted nuclei, thereby leading to a diagnosis of massively invasive submucosal cancers or worse. Sensitivity and specificity of endocytoscopy to differentiate between neoplastic and non-neoplastic lesions was both 100%. In addition, while assuming that an EC3b classification was diagnostic of SMm or worse, the authors were able to differentiate SMm or worse from other neoplastic lesions (dysplasias and slightly invasive submucosal cancers) with a sensitivity of 90% and a specificity of 99%. 27 Besides the possibility distinguishing between neoplastic and non-neoplastic tissue, one recent report also highlights the potential of endocytoscopy for diagnosis of focal highgrade dysplasia in colorectal lesions. 28 Endocytoscopy visualized a focal area with loss of crypt architecture and irregular cell distribution as well as heterogeneity of cells surrounded by normal colon mucosa. Therefore, endocytoscopy may improve both in vivo diagnosis and management of colonic polyps. 28 Optical magnification endoscopy uses a movable lens controlled by the endoscopist to vary degree of magnification, thereby enabling effective visualization of the mucosal pit pattern. Very recently, Kudo and coworkers assessed the additional diagnostic value of endocytoscopy to optical magnification endoscopy for diagnosing colorectal lesions. 29 Overall, 455 patients were included and each lesion detected was initially investigated by optical magnification followed by endocytoscopy. The diagnostic abilities of predicting neoplastic changes were comparable between both magnifying endoscopy and endocytoscopy with sensitivities and specificities ranging at approximately 97% and 90%, respectively. With regard to prediction of SMm, optical magnification plus endocytoscopy showed specificity and accuracy of 99% and 96%, respectively, and was superior to magnification endoscopy only (P < 0.05). 29

5 236 H. Neumann et al. Digestive Endoscopy 2015; 27: The expert group from Japan also evaluated the accuracy of endocytoscopy and conventional biopsy for diagnosis of colorectal neoplasms in a prospective randomized noninferiority trial. 30 The authors randomly assigned 203 detected lesions of 170 eligible patients to either the endocytoscopy or the conventional biopsy group. Primary endpoint of the study was to determine whether the diagnostic accuracy of endocytoscopy for neoplastic lesions was noninferior to that of conventional biopsy using a predefined non-inferiority margin of 10%. The diagnostic accuracy of endocytoscopy for the discrimination of neoplastic lesions was 94% whereas that of standard biopsy was 96%, which was within the non-inferiority margin. Therefore, it was concluded that optical biopsies by using endocytoscopy could provide a novel alternative to conventional biopsies during routine colonoscopies. 30 Awareness of so-called serrated lesions has increased considerably within recent years as it is speculated that a significant proportion of interval cancers may arise from these lesions. 31 Very recently, a study from Japan evaluated endocytoscopic features of different types of serrated polyps in a retrospective study at a tertiary-care referral center. 32 Of 58 eligible lesions, 27 were classified as hyperplastic polyps: 12 as sessile serrated adenomas and 19 as traditional serrated adenomas. Hyperplastic polyps often showed star-like lumens (78%), whereas sessile serrated adenomas and traditional serrated adenomas predominantly demonstrated oval lumens (83%). The lumens of traditional serrated adenomas were serrated in 32% of cases or were villous in 68% of cases. Most hyperplastic polyps (93%) and sessile serrated adenomas (75%) had small, round nuclei, and all traditional serrated adenomas had fusiform nuclei. Features significantly differentiating traditional serrated adenomas from hyperplastic polyps and sessile serrated adenomas were the presence of fusiform nuclei and villous and serrated lumens, whereas the presence of oval lumens was significantly characteristic of sessile serrated adenomas, and the presence of star-like lumens was significantly characteristic of hyperplastic polyps. 32 Inflammatory bowel disease To date, only two studies evaluated the value of endocytoscopy for advanced diagnosis of IBD. Bessho and coworkers developed an endocytoscopy score to determine a histopathological activity index of ulcerative colitis (Table 4). 33 The score (called ECSS) consists of three indices: ECSS-A indicates the shape of crypts. 0 points are given for normal round crypts, 1 point for oval crypts, 2 points for irregular crypt formations and 3 points for non-recognizable crypts, thereby indicating severe destruction of the mucosa. ECSS-B indicates the distance between crypts and points Table 4 ECSS to determine histopathological activity in ulcerative colitis according to Bessho et al. 33 ECSS A] Shape of crypts Normal 0 Oval 1 Irregular 2 Unclear 3 B] Distance between crypts Normal 0 Intermediate 1 Elongated 2 C] Small vessels Normal 0 Visible 1 Final score is calculated as ECSS =A+B+C(range 0 6). ECSS, endocytoscopy system score. Points ranging from 0 to 2 are given. ECSS-C indicates the visibility of microvessels with 0 points given for no visible vessels and 1 point given for visible vessels. Accordingly, the ECSS can range between 0 and 6 points, with higher grades describing more severe inflammation. Fifty-five patients with ulcerative colitis were included and endocytoscopy with 450-fold magnification was used. In total, 76 pairs of ECSS and Matts histopathological grades were independently acquired. To validate the ECSS, interobserver agreement between three endoscopists, with consensus, and another endoscopist, was calculated as the kappa value. The authors also evaluated the correlation between the ECSS and Matts histopathological grade, and between the conventional Matts endoscopic grade and Matts histopathological grade. It was found that the ECSS showed a strong correlation with Matts histopathological grades. In addition, a strong correlation between the conventional Matts endoscopic grade and Matts histopathological grade was found. The ECSS demonstrated a high reproducibility with a kappa value of Precise activity assessment of IBD is essential to determine extent and severity (i.e. mucosal healing) of disease for optimized therapy. Despite ongoing developments in endoscopy, final diagnosis still relies on the interpretation of histopathological features. Accordingly, one recently introduced pilot study determined the reliability of endocytoscopy for the discrimination of mucosal inflammatory cells and intestinal inflammatory disease activity in patients with IBD. 34 Forty patients with both ulcerative colitis and Crohn s disease were included and methylene blue or toluidine blue was used to enable endocytoscopic imaging at approximately 1400-fold magnification. In that study, endocytoscopy enabled visualization of different histopathological features, including architectural and cellular features. Accordingly, it was possible to reliably distinguish single

6 Digestive Endoscopy 2015; 27: Endocytoscopy 237 inflammatory cells by using endocytoscopy. Inflammatory cells differentiated included neutrophilic, basophilic, eosinophilic granulocytes, and lymphocytes. Sensitivities and specificities for diagnosis of inflammatory cells ranged between 60 89% and 90 95%, respectively. Interobserver agreement between two investigators was substantial (kappa ), whereas intra-observer agreement was substantial to almost perfect (kappa ). Of note, concordance between endocytoscopy and histopathology for grading intestinal disease activity was calculated as being 100%. 34 Overall, the potential of endocytoscopy to assess mucosal healing and, moreover, to assess histological healing in patients with IBD appears conclusive, as endocytoscopy allows for real-time diagnosis of mucosal inflammatory cells. Thereby, chronic and acute inflammatory conditions could be diagnosed during the endoscopic examination. However, the main limitation of the technique is that it only allows visualization of the superficial mucosal layer. Prospective and randomized trials should evaluate the potential for endocytoscopy to assess histological healing in IBD in vivo and to predict disease outcome. Infectious colitis The concept of in vivo diagnosis of intestinal pathogens is attractive as it may facilitate diagnosis and subsequent therapy of patients. Previous reports have highlighted the potential of confocal laser endomicroscopy for visualization of bacteria, including Helicobacter pylori, Escherichia coli and Clostridium difficile. 35,36 In addition, it was shown that endocytoscopy could visualize H. pylori and Staphylococcus aureus ex vivo. 37 One recent study evaluated the potential of endocytoscopy for in vivo visualization of trophozoites in patients with amoebic colitis. 38 In vivo diagnosis would be of interest, as routine diagnosis of amebic colitis usually requires multiple tests. Five patients who were suspected to have amebic colitis were included. Endocytoscopy findings were compared to those of serum antibody tests and histology of colon biopsy specimens. The authors were able to successfully visualize amebic trophozoites in all cases. In contrast, three specimens had positive results on serology, and three had positive histology results on hematoxylin and eosin staining. Despite the limitations of a pilot study, the study opens new doors for potential in vivo diagnosis of intestinal pathogens using endocytoscopy to accelerate diagnosis and therapy of patients. CONCLUSIONS ENDOCYTOSCOPY ALLOWS ON-DEMAND histology of colorectal pathologies in real-time. For endocytoscopic imaging, probe-based and endoscope-integrated devices are available. Before endocytoscopy, prestaining of the mucosa using absorptive staining agents had to be applied. The best image quality can be obtained by using a double-staining technique consisting of crystal violet and methylene blue. Besides architectural and vascular details, endocytoscopy also allows visualization of cellular details. Accordingly, diagnosis of single inflammatory cells, dysplasia and cancer becomes feasible. Although the potential of endocytoscopy to diagnose gastrointestinal diseases is still under evaluation, studies have already shown its versatility for the diagnosis of colorectal polyps, IBD and infectious colitis. Ongoing studies are currently addressing whether endocytoscopy can also be used to guide surveillance intervals after polypectomy, to evaluate the resection margin for residual adenomatous tissue after polypectomy and to guide individualized anti-inflammatory approaches in patients with ulcerative colitis and Crohn s disease. CONFLICT OF INTERESTS AUTHORS DECLARE THE following conflict of interests for this article. 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