In vivo diagnosis of early-stage gastric cancer found after. Helicobacter pylori eradication using probe-based confocal laser endomicroscopy

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1 DR. NAOKI OHMIYA (Orcid ID : X) Accepted Article Article type : Original article In vivo diagnosis of early-stage gastric cancer found after Helicobacter pylori eradication using probe-based confocal laser endomicroscopy Noriyuki Horiguchi1, Tomomitsu Tahara1, T Hyuga Yamada1, Dai Yoshida1, Masaaki Okubo1, Mitsuo Nagasaka1, Yoshihito Nakagawa1, Tomoyuki Shibata1, Tetsuya Tsukamoto2, Makoto Kuroda2, Naoki Ohmiya1. 1 Department of Gastroenterology and 2 Diagnostic Pathology I, Fujita Health University School of Medicine, Toyoake, Japan Short running title: Endomicroscopy for gastric cancer This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /den.12926

2 *To whom correspondence should be addressed: Naoki Ohmiya Accepted Article Department of Gastroenterology, Fujita Health University School of Medicine 1-98 Dengakugakubo Kutsukake-cho, Toyoake, Aichi, , Japan Phone: , Fax: , Disclosure: Naoki Ohmiya has received grant support from EA Pharma Co., Ltd., Takeda Pharmaceutical Company, Otsuka Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, and AbbVie Inc.. The funding source had no role in the design, practice, or analysis of this study. Funding: No specific funding has been received. Data have been generated as part of the routine work of an organization. ABSTRACT Background and Aim: Early-stage gastric cancer (EGC) found after Helicobacter pylori (Hp) eradication often displays non-tumorous regenerative epithelium and/or maturated tumorous epithelium overlying the cancerous tissue, which may confuse endoscopic and histologic diagnosis. Probe-based confocal laser endomicroscopy (pcle) enables in vivo real-time optical biopsy. We compared the diagnostic yields for these EGC cases using conventional

3 white light endoscopy (WL), magnifying endoscopy with narrow-band imaging (ME-NBI), Accepted Article pcle, and endoscopic biopsy; we also compared the accuracy of the horizontal extent diagnosis between ME-NBI and pcle. Methods: This study enrolled 30 patients with 36 EGC lesions after successful Hp eradication. The diagnostic yields of WL, ME-NBI, pcle, and endoscopic biopsy were prospectively compared. Four points of cancerous margins (oral, anal, anterior, and posterior sites) were also prospectively evaluated with M-NBI and pcle to determine the horizontal extent of the EGC. Results: The diagnostic yield was significantly higher with pcle than with WL and endoscopic biopsy (97 vs. 72%, 97 vs. 72%, P=0.0159, , respectively), whereas it did not differ from ME-NBI (88.9%, P=0.371). The height of non-tumorous regenerative epithelium or maturated atypical glands was 104.7±34.2 μm in the pcle-positive cases, whereas it was 188.3±27.1 μm in a pcle-negative case (P=0.0004). The diagnostic accuracy of the horizontal margin of EGC was significantly higher with pcle than with ME-NBI (92 vs. 70%, P=0.0159). Conclusion: pcle may be helpful for the diagnosis of ambiguous ECGs found after Hp eradication because it enables real-time scanning throughout the lesion and the detection of subsurface microstructure.

4 Key words: Gastric cancer; H. pylori eradication; magnifying endoscopy with narrow-band Accepted Article imaging; probe-based confocal laser endomicroscopy INTRODUCTION Helicobacter pylori (Hp) infection is a well-accepted risk factor for gastric carcinogenesis based on epidemiological and experimental studies. 1-5 A prospective study conducted in Japan demonstrated that Hp eradication therapy significantly reduced the incidence of metachronous gastric cancer after endoscopic resection of early-stage gastric cancer (EGC). 6 Therefore, the Japanese national health insurance system has recently covered the cost of eradication therapy for patients with Hp-associated gastritis; however, accumulating evidence has suggested that gastric cancer is discovered in patients even after successful Hp eradication. 7 EGC found after Hp eradication appears as indistinct forms such as tiny and flattened lesions, 8, 9 and it may be observed within a short period after Hp eradication. 10 Histologic findings of EGC are characterized by regenerating non-tumorous epithelium overlying the tumorous tissue and/or surface maturation of tumors, which may confuse both endoscopic and histologic diagnoses Even with the use of image-enhanced endoscopy techniques such as magnifying endoscopy with narrow-band imaging (ME-NBI), these EGCs are sometimes difficult to diagnose because they resemble the surrounding noncancerous 11, 12 mucosa.

5 Probe-based confocal laser endomicroscopy (pcle) is an imaging technique used Accepted Article to provide in vivo histology that enables real-time optical biopsy. pcle provides a resolution of up to 1 m, a depth of focus between m, and a fast video-rate scanning of 12 images per seconds, which can investigate the horizontal microstructure of the deeper mucosal layer. Therefore, we hypothesized that pcle can provide a better diagnostic yield of ambiguous EGC found after H. pylori eradication; thus, diagnostic yields using conventional white light endoscopy (WL), ME-NBI, pretreatment endoscopic biopsy, and pcle were compared. Furthermore, we compared the diagnostic accuracy of the horizontal extent of EGC between pcle and ME-NBI. METHODS Patients Of the 232 EGC patients who underwent gastroscopy at Fujita Health University between January 2015 and December 2016, 30 consecutive patients (13%) with EGC found after successful Hp eradication were enrolled. A history of Hp eradication was investigated based on medical records or a face-to-face history examination. Successful Hp eradication was confirmed by a negative urea breath test and histology of endoscopic biopsy in all patients. These 30 patients had 36 EGC lesions that were diagnosed at least 6 months after

6 eradication, as shown in Table 1. This was a prospective single-center open-label pilot study Accepted Article that was approved by the Institutional Review Board of Fujita University School of Medicine. This study was registered with the University Hospital Medical Information Network (UMIN ). Informed consent was obtained from all of the patients. Magnifying endoscopy with narrow-band imaging and probe-based confocal laser endomicroscopy All of the lesions were evaluated by WL, ME-NBI (Olympus GIF-H260Z and a CV260SL/CV290SL, Olympus Medical Systems, Tokyo, Japan), and pcle. Endoscopy was performed initially using WL endoscopy, next ME-NBI, followed by pcle (NH, TT). The pcle examinations were performed with a Gastroflex UHD probe (Cellvizio, Mauna Kea Technologies, Paris, France) after an intravenous injection of mg fluorescein (100 mg/ml, Fluorescite, Alcon Japan Ltd., Tokyo, Japan). We obtained only one biopsy specimen from the center of the lesions and one biopsy specimen each from 4 marginal points (oral, anal, anterior, and posterior sites) around the lesions a few weeks before treatment to ensure horizontal extent. Likewise, we evaluated the lesions and four marginal points as above using ME-NBI followed by pcle to confirm the presence of cancer and its horizontal extent. During the pcle procedure, the endoscopic images with ME-NBI were synchronized and recorded for comparison.

7 Interpretation of endoscopic findings Accepted Article The diagnosis of EGC with WL was based on an irregular-shaped depression or uneven elevation with a clear margin. If such endoscopic finding was absent or indeterminate, the lesion was considered to be non-cancerous. For ME-NBI, we referred to the vessel plus surface (VS) classification system This system uses microvascular (MV) and microsurface (MS) patterns and the presence of a demarcation line; a cancer diagnosis is based on the presence of irregular MV and/or MS within a clear demarcation line. If such endoscopic finding was absent or indeterminate, the lesion was considered to be non-cancerous. Regarding pcle, we referred to the Miami Classification system to diagnose EGC; features including a completely disorganized epithelium, irregular crypt lumen, and dark irregular thickened epithelium were regarded as indicators of EGC. 18 All of the images were prospectively evaluated by real-time observation in the consensus manner from three experienced endoscopists (NH, TT, NO). NO performed over 130 pcle examinations and NH and TT performed approximately 60 examinations. We did not change the diagnosis even if a different diagnosis was made with another modality.

8 Treatment of EGC Accepted Article Endoscopic submucosal dissection (ESD) or surgical resection was performed as shown in Table 1. Before ESD, marking dots were placed by argon plasma coagulation on the surrounding normal mucosa 5 mm from the cancerous margin based on the horizontal extent assessed by either ME-NBI or pcle. If the cancerous margin was different, marking dots were placed to cover the larger horizontal extent. Interpretation of pathologic findings Pathologic diagnosis of the biopsy and resected specimens were performed according to the Japanese Classification of Gastric Carcinoma, 14th edition 13, by two pathologists (TT, MK) who were blinded to this study. Pretreatment biopsy specimens were reviewed only with hematoxylin-eosin staining. When the pathologic diagnosis of the pretreatment biopsy specimens as neoplastic or non-neoplastic was unclear, it was designated an inconclusive lesion. Endoscopically- or surgically-resected specimens were immunostained with anti-muc5ac monoclonal antibodies (Novocastra HD, Leica Biosystems, Nussloch, Germany) in addition to hematoxylin-eosin staining. When the histopathological features including regenerating non-tumorous epithelium overlying over the cancerous tissue and/or surface maturation of cancers were evident in approximately more than 30% of the entire

9 area, the sample was designated as having histologic changes after Hp eradication. The Accepted Article vertical distance from the luminal surface to the deepest portion of the overlying regenerating epithelium or maturated atypical glands was measured at 5 points in each resected specimen with a point-to-point distance measuring function (Provis AX-80 optical microscope and DP20 digital camera, Olympus Corporation, Tokyo, Japan). A complete match of the histologic and endoscopic horizontal extents with ME-NBI or pcle at all 4 circumferential quadrant points around the cancerous lesion was defined as an accurate diagnosis. The presence of regenerating epithelium overlying the cancerous tissue or surface maturation of cancers at the cancerous horizontal margin was reviewed in the resected specimens. A pathologic diagnosis of endoscopically- or surgically-resected specimens was used as the gold standard. Statistical analysis The proportions of patients with positive findings based on the two modalities were compared, and a significant difference between the tests was calculated using the exact McNemar test. The association between an inconclusive diagnosis of pretreatment biopsy specimens and histologic changes after Hp eradication was analyzed by Fisher s exact probability test. A comparison of the height of the regenerating epithelium or maturated

10 atypical glands between pcle-positive and negative EGCs was assessed by the Accepted Article Mann-Whitney U test. Differences with P values less than 0.05 were considered significant. RESULTS Diagnostic yields of WL, ME-NBI, pcle, and pretreatment endoscopic biopsy for EGC after H. pylori eradication Clinicopathological characteristics of the patients and EGC are shown in Table 1. The diagnostic yields of WL, ME-NBI pcle, and the pretreatment endoscopic biopsy for EGC are shown in Table 2. The yield of ME-NBI was significantly higher than that of WL (P=0.0114). The yield of pcle was not significantly different from that of ME-NBI (P=0.371) but was significantly higher than those of WL and pretreatment endoscopic biopsy (P=0.0159, , respectively). The pcle-positive EGC case is shown in Figure 1. Inconclusive diagnosis of pretreatment endoscopic biopsy Table 3 shows the association between the histologic changes after H. pylori eradication and an inconclusive diagnosis of EGC with pretreatment endoscopic biopsy. The histologic changes of pretreatment endoscopic biopsy including regenerating non-tumorous epithelium

11 overlying the cancerous tissue and/or surface maturation of tumors were observed in 21 out Accepted Article of 36 lesions (58%). These changes were significantly correlated with an inconclusive diagnosis of EGC with pretreatment endoscopic biopsy (P=0.0018). Height of the overlying regenerating epithelium or maturated atypical glands The height of the regenerating epithelium or maturated atypical glands overlying EGC was measured at 5 points in each of the resected specimens of 21 cases with the pathologic findings as above (for a total 105 points). The height was 104.7±34.2 μm in 20 cases with positive pcle findings (100 points in total) and 188.3±27.1 μm in 1 case with negative pcle findings (5 points in total, P= , Figure 2). This pcle-negative EGC case is shown in Figure 3. Diagnostic accuracy of ME-NBI and pcle for the horizontal extent of EGC The diagnostic accuracy of pcle (33/36: 91.7%) for the horizontal extent of EGC was significantly higher than that of ME-NBI (25/36: 69.4%, P=0.0433), as shown in Table 4. There were 5, 3, 2, and 1 cases with 4, 3, 2, or 1 of the marginal 4 points incorrect with ME-NBI, respectively, whereas there were 1, 1, 1, and 0 cases incorrect with pcle, respectively. The presence of the overlying regenerating epithelium or maturated atypical

12 glands at the cancerous margins stratified by ME-NBI or pcle diagnostic positivity is also Accepted Article shown in Table 4. Figure 4 shows that the diagnosis of the horizontal extent with ME-NBI was difficult but was clear with pcle. Adverse events There were no complications associated with gastroscopy, pcle, and intravenous injection of fluorescein including bleeding, perforation, aspiration pneumonia, and anaphylaxis. DISCUSSION pcle can visualize the subsurface microscopic architecture of the mucosa in real time. The present study indicated that pcle had the highest diagnostic yield for EGC identified after Hp eradication among WL, ME-NBI, pcle, and pretreatment endoscopic biopsy. We have previously reported that EGC found after Hp eradication was significantly depressed, reddish, and smaller than EGC without Hp eradication, and the frequency of inconclusive diagnosis with pretreatment biopsy was higher (26.0 vs. 1.6%) in EGC found after Hp eradication [9]. This inconclusive diagnosis was related to the pathologic finding of regenerating non-tumorous epithelium overlying the tumorous tissue and/or surface maturation of tumors. Saka et al reported that frequencies of the extent of non-neoplastic epithelium at the surface

13 of 10% or less of the entire cancerous area were 33.3% (8/24) and 93.6% (44/47, P<0.001) in Accepted Article EGCs after Hp eradication and EGCs without Hp eradication, respectively, whereas those > 10% and B50 were 54.2% (13/24) and 6.4% (3/47, P<0.001), and those > 50% were 12.5% (3/24) and 0% (0/47, P=0.013) respectively. In the eradication group, To sum up, in the eradication group, EGCs with the extent of non-neoplastic epithelium at the surface > 10% and B50 were dominant. 11 We accordingly adopted the median 30% as a cut-off point in the present study. Then, these findings are corroborated by the present study showing that non-neoplastic epithelium overlying the tumorous tissue and/or surface maturation of tumors covering more than 30% of the entire cancerous area was observed in 58.3% of EGC cases after Hp eradication. This pathologic feature may confuse pathologic diagnosis when only one or two biopsy specimens are taken to prevent inducing fibrosis before treatment. Bok et al also reported that the overall accuracy for the diagnosis of adenocarcinoma was 91.7% for pcle and 85.2% for conventional biopsy, although various superficial gastric lesions (22 differentiated adenocarcinomas, 10 undifferentiated carcinomas, 19 gastric dysplasias, and 3 non-neoplastic lesions) were included and the past history of Hp eradication was unknown. 19 In this respect, pcle has some clinical advantages over biopsy samples: pcle can 1) obtain live microscopic images throughout a lesion, 2) obtain cancerous images under the overlying non-neoplastic epithelium or maturated ambiguous tumors, and 3) benefit patients on antithrombotics, with bleeding tendencies, or with conditions for whom taking pathologic

14 samples is contraindicated or not recommended. Although ME-NBI is useful for making a Accepted Article differential diagnosis between flat EGCs and gastritis, 15, 17 the gastritis appearance of EGC after Hp eradication makes endoscopists less confident about diagnosing cancer even by using ME-NBI. 11 ME-NBI, however, can indicate irregularity of the microsurface and microvascular patterns, particularly by focusing on cancerous lesions spared by overlying non-neoplastic epithelium. Therefore, the diagnostic accuracy may not have been significantly different between ME-NBI and pcle in this study. Furthermore, pcle outperformed ME-NBI for the diagnosis of the horizontal extent of EGC, which may be ascribed to the overlying of regenerative epithelium or surface maturation of EGC more often at the margin than in the middle of EGC. This study showed that these masquerading pathologic findings were observed at 56% of the horizontal margins of resected specimens of EGC found after Hp eradication. Therefore, pcle scored significantly higher than ME-NBI in diagnosing the horizontal extent even though pcle was similar to ME-NBI in diagnosing the entire lesion as EGC. Park et al reported that in a prospective, randomized comparative study between pcle and chromoendoscopy, pcle was useful for more precise margin delineation than chromoendoscopy among EGCs with superficial flat morphology. 20 The diagnosis of the horizontal margin of EGC is essential in endoscopic resection; therefore, pcle is a useful tool to determine the endoscopic cutting line.

15 pcle only detects subsurface microarchitecture to a depth of m; therefore, Accepted Article pcle is unable to diagnose EGC if the overlying regenerative epithelium or maturated atypical glands are thicker than 100 mm, as in Figure 2 of this study. The focal depth of pcle is m according to the manufacturer s instructions, but it can be deeper when pcle is pushed into the mucosa. Because the height of the overlying regenerative epithelium or surface maturation was less than 100 m in 35/36 (97%) of cases with EGC found after Hp eradication, pcle would be a useful tool to diagnose most of these cases. pcle is unable to allow the precise observation of cytological atypia using intravenous fluorescein administration only. In this study, three endoscopists evaluated pcle images: one endoscopist had an experience of over 130 pcle examinations, and the other two had experience of approximately 60 examinations. Buchner et al analyzed the learning curve of pcle interpretation, and reported that 11 endoscopists from 3 different endoscopy centers could rapidly learn accurate interpretation of pcle images for predicting colorectal neoplastic lesions: accuracy for the overall group was 63% for lesions 1 to 20, 64% for lesions 21 to 40, 79% for lesions 41 to 60, and 86% for lesions 61 to 76 cases. 21 Our 3 endoscopists had sufficient experience according to Buchner report, and actually, in this study, more than 95% of pcle images could be evaluated with a good consensus among three (results not shown), suggesting we had rapidly learned pcle images interpretation as well.

16 This study has some limitations such as small sample size and non-randomized Accepted Article comparative study. The order of examination was WL, next ME-NBI, followed by pcle, and all images were prospectively evaluated by real-time. In this study, however, the possibility that the WL and ME-NBI findings affected the pcle diagnosis cannot be ruled out. Further larger-scale randomized comparative studies are necessary to clarify the effectiveness of pcle. The corroboration of our in vivo histologic findings will help with rapid diagnosis and therapeutic strategy. In conclusion, pcle is safe and useful for the diagnosis of endoscopically ambiguous EGC found after Hp eradication. REFERENCES 1. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991;325: Huang JQ, Sridhar S, Chen Y, et al. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer. Gastroenterology 1998;114: Uemura N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001;345: Watanabe T, Tada M, Nagai H, et al. Helicobacter pylori infection induces gastric cancer in mongolian gerbils. Gastroenterology 1998;115: Shimizu N, Ikehara Y, Inada K, et al. Eradication diminishes enhancing effects of Helicobacter pylori infection on glandular stomach carcinogenesis in Mongolian gerbils. Cancer Res 2000;60:

17 6. Fukase K, Kato M, Kikuchi S, et al. Effect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an Accepted Article open-label, randomised controlled trial. Lancet 2008;372: Kamada T, Hata J, Sugiu K, et al. Clinical features of gastric cancer discovered after successful eradication of Helicobacter pylori: results from a 9-year prospective follow-up study in Japan. Aliment Pharmacol Ther 2005;21: Yamamoto K, Kato M, Takahashi M, et al. Clinicopathological analysis of early-stage gastric cancers detected after successful eradication of Helicobacter pylori. Helicobacter 2011;16: Horiguchi N, Tahara T, Kawamura T, et al. Distinct Clinic-Pathological Features of Early Differentiated-Type Gastric Cancers after Helicobacter pylori Eradication. Gastroenterol Res Pract 2016;2016: Ito M, Tanaka S, Takata S, et al. Morphological changes in human gastric tumours after eradication therapy of Helicobacter pylori in a short-term follow-up. Aliment Pharmacol Ther 2005;21: Saka A, Yagi K, Nimura S. Endoscopic and histological features of gastric cancers after successful Helicobacter pylori eradication therapy. Gastric Cancer 2016;19: Kobayashi M, Hashimoto S, Nishikura K, et al. Magnifying narrow-band imaging of surface maturation in early differentiated-type gastric cancers after Helicobacter pylori eradication. J Gastroenterol 2013;48: Japanese Gastric Cancer A. Japanese classification of gastric carcinoma: 3rd English edition. Gastric Cancer 2011;14: Yao K, Iwashita A, Kikuchi Y, et al. Novel zoom endoscopy technique for visualizing the microvascular architecture in gastric mucosa. Clin Gastroenterol Hepatol 2005;3:S Yao K, Iwashita A, Tanabe H, et al. Novel zoom endoscopy technique for diagnosis of small flat gastric cancer: a prospective, blind study. Clin Gastroenterol Hepatol 2007;5: Yao K, Anagnostopoulos GK, Ragunath K. Magnifying endoscopy for diagnosing and delineating early gastric cancer. Endoscopy 2009;41:462-7.

18 17. Ezoe Y, Muto M, Uedo N, et al. Magnifying narrowband imaging is more accurate than conventional white-light imaging in diagnosis of gastric mucosal cancer. Gastroenterology Accepted Article 2011;141: e Wallace M, Lauwers GY, Chen Y, et al. Miami classification for probe-based confocal laser endomicroscopy. Endoscopy 2011;43: Bok GH, Jeon SR, Cho JY, et al. The accuracy of probe-based confocal endomicroscopy versus conventional endoscopic biopsies for the diagnosis of superficial gastric neoplasia (with videos). Gastrointest Endosc 2013;77: Park JC, Park Y, Kim HK, et al. Probe-based confocal laser endomicroscopy in the margin delineation of early gastric cancer for endoscopic submucosal dissection. J Gastroenterol Hepatol 2017;32: Buchner AM, Gomez V, Heckman MG, et al. The learning curve of in vivo probe-based confocal laser endomicroscopy for prediction of colorectal neoplasia. Gastrointest Endosc 2011;73: FIGURE LEGENDS Figure 1. A case of pcle-positive EGC found 36 months after H. pylori eradication. A. A reddish, depressed lesion is observed in the greater curvature of the lower corpus with conventional WL endoscopy. B. A narrow-band image showing the region of evaluation with pcle. The open circle and dotted circle correspond to Figures 1E and F, respectively. C. Pretreatment endoscopic biopsy designated an inconclusive case due to few atypical grands. D. A magnifying narrow-band image showing uneven, irregular microvessels on the regular, oval, or tubulovillous microsurface textures.

19 E. A pcle image of EGC showing disorganized, irregular dark glands with back-to-back Accepted Article orientation. F. A pcle image of the surrounding noncancerous mucosa showing regular enhanced glands. G. H&E staining of an endoscopically resected specimen showing non-neoplastic epithelium overlying the cancerous tissue. F. Immunohistochemical staining with an anti-muc5ac monoclonal antibody showing positive non-neoplastic epithelium overlying the cancerous tissue. Figure 2. Box-and-whisker plots of the height of the regenerating epithelium or maturated atypical glands stratified by pcle detectability. Figure 3. A case of pcle-negative early-stage gastric cancer found 48 months after H. pylori eradication. A. A whitish, elevated lesion is observed in the posterior wall of the lower corpus with conventional WL endoscopy. B. A magnifying narrow-band image showing regular microvessels on the regular, densely arranged microsurface textures. Arrow heads show the demarcation line. C. A pcle image of the cancer showing regular enhanced glands with dark goblet cells. D. H&E staining of an endoscopically resected specimen shows thick maturated atypical glands. The dotted open square corresponds to Figure 3E.

20 E. H&E staining of an endoscopically-resected specimen, showing maturated atypical glands Accepted Article with goblet cells, the height of which is m. Figure 4. Assessment of the horizontal extent of EGC found 108 months after H. pylori eradication. A. A slightly depressed lesion is observed in the greater curvature of the lower corpus with conventional WL endoscopy (arrow heads). B. A magnifying narrow-band image showing irregular microvascular and irregular microsurface patterns in the center of EGC, while the microvascular and microsurface patterns around the oral margin (a: open circle) appear regular, similar to the surrounding adjacent mucosa (b: dotted circle), which makes the horizontal extent unclear. C. A pcle image of within the open circle in Figure 2B, showing disorganized, irregular dark glands. D. A pcle image within the dotted circle in Figure 2B, showing regular enhanced glands. E. H&E staining of an endoscopically-resected specimen, showing that cancerous crypts (arrow heads) were interspersed under the non-neoplastic epithelium (a, the dotted line) but not under the epithelium indicated by the dotted line (B), corresponding to the horizontal extent of this lesion.

21 Accepted Article Table1.Characteristics of early-stage gastric cancers found after H.pylori eradication No. lesions / No. patients 36/30 Male/Female 23/7 Age(y), median (range) 67.5 (57-83) Duration after eradication (m) 36±4.8 Location: Upper/Middle/Lower n(%) 4/13/19 (11/36/53) Macroscopic type:elevated/depressed n(%) 7/29 (19/81) Color: Redness/Whiteness/same as surroundings n(%) 20/9/7 (56/25/19) Tumor size : mean±se (mm) 10.0±4.4 Histological type:tub1/tub2/por2 n(%) 29/5/2 (80/14/6) Depth of invasion: M/SM1/SM2 n(%) 30/4/2 (83/11/6) Treatment: ESD/Gastorectomy n(%) 35/1 (97/3) Histopathological findings of gastric cancer after H. pylori eradication n(%) 21/15 (58/42) Macroscopic type: elevated, 0-I, 0-IIa; depressed, 0-IIc, 0-IIa+IIc; 0-IIc+IIa according to the Japanese classification M, intra-mucosal cancer; SM1, cases with submucosal invasion less than 500 μm; SM2, cases with submucosal invasion greater than 500 μm; Histopathological findings of gastric cancer after H. pylori eradication: either regenerating non-tumorous epithelium covering over the tumorous tissue and/or surface differentiation of tumors

22 Accepted Article Table2. Diagnostic yields of EGC found after Helicobacter pylori eradication Modalities Diagnostic yields Conventional white light endoscopy (WLI) 26/36 (72%) Magnifying endoscopy with narrow-band imaging (ME-NBI) 32/36 (89%) Probe-based confocal laser endomicroscopy (pcle) 35/36 (97%) Endoscopic pretreatment biopsy 26/36 (72%) WLI vs.m-nbi, P=0.0114; WLI vs.pcle, P=0.0159; M-NBI vs.pcle, P=0.371; pcle vs. endoscopic pretreatment biopsy, P=0.0077; Table 3. Inconclusive diagnosis of pretreatment endoscopic biopsy Histologic changes after H. pylori eradication Inconclusive diagnosis with endoscopic biopsy Present (n=21) 47.6% (10/21) Absent (n=15) 0% (0/15) P=0.0018

23 Table 4. Relationship between diagnostic accuracy and histologic changes after Hp eradication at horizontal margins of EGC Accepted Article M-NBI pcle *P=0.043 Diagnostic accuracy Histologic changes after Hp eradication Diagnosed 25(69%)* 12/25(48%) Undiagnosed 11(31%) 8/11(73%) Diagnosed 33(92%)* 18/33(55%) Undiagnosed 3(8%) 2/3(67%)

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