We conducted a retrospective cohort study of early gastric cancers treated by ESD from January 2013 to August 2015 in our institution. Well or moderately differentiated adenocarcinomas diagnosed by two pathologists during the period were examined. Adenomas, undifferentiated or undifferentiated-mixed cancers, and H. pylori-unrelated gastric cancers, such as fundic gland type cancers, were excluded. All study participants provided written informed consent, and the study was approved by the institutional review board (IRB) and ethics comittee of Osaka university hospital (IRB number: 16451). No patients were under 20 years old. This study was conducted in accordance with the principles laid down in the Helsinki Declaration.
We performed the endoscopic diagnosis according to the vessel plus surface classification system by M-NBI. The demarcation line (DL) separates a lesion from the normal surrounding mucosal area. If DL was present all around the lesion, we classified the lesion into the demarcation clear (DC) group. If DL was absent for a part of the lesion, we categorized the lesion into to the demarcation unclear (DU) group. All lesions with still images were evaluated at a conference attended by more than six endoscopic experts and divided into two groups (DC and DU).
We analyzed the differences in clinical features of early gastric cancers, including size, location, macroscopic type, color, and history of H. pylori eradication, between the two groups. We defined successful H. pylori eradication therapy, if the patients had a history of H. pylori eradication therapy and received at least one negative test of the following methods: tests for the presence of serum IgG antibodies against H. pylori, the 13C urea breath test, and stool test for H. pylori antigen. We evaluated the presence of H. pylori at the time of lesion detection. In addition, we compared the short-term outcome of treatment, including the rate of en-bloc or curative resection, procedure time, the rate of complication, such as perforation and delayed bleeding, and the rate of additional surgical resection between the two groups. En-bloc resection was defined as one-piece resection endoscopically, whereas curative resection was defined as en-bloc resection histopathologically with the absence of lymphovascular invasion and presence of tumor negative lateral and vertical margins of the resected specimens according to the guideline of the indication criteria for curative resection. Perforation was defined as a defect of the muscular layer during ESD or presence of free air detected by computed tomography after ESD. Delayed bleeding was defined as hematochezia after ESD that required endoscopic hemostasis.
Formalin-fixed and paraffin-embedded specimens from the included lesions were retrieved. All hematoxylin and eosin (HE)-stained sections were evaluated by two pathologists. Histological type, combined ulcer or ulcer scar, and depth of invasion were evaluated. Additionally, we evaluated the histopathological features on the superficial epithelium and divided them into the following three categories: non-neoplastic superficial epithelium, partial non-neoplastic superficial epithelium, and neoplastic superficial epithelium. Partial non-neoplastic superficial epithelium was defined as the presence of both non-neoplastic and neoplastic epithelia.
Evaluation of histopathological structure
The randomly selected visual field (100 ⋅) in both the lesion and the background mucosa from one tissue was assessed. One of the authors evaluated the width of the interfoveolare, length of duct, and width of pit to compare the difference in the histopathological structure between the lesion and the surrounding mucosa in both groups.
The tissue sections cut at 4-µm thickness were used. The immunohistochemical staining for p53 (DO-7, 1:250 dilution; DAKO, Glostrup, Denmark), Ki-67 (MIB-1, 1:1000 dilution; DAKO), MUC5AC (CLH2, 1:50 dilution; DAKO), MUC6 (CLH5, 1:50 dilution; Santa Cruz Biotechnology, Dallas, TX), MUC2 (CCP58, 1:50 dilution; DAKO), and CD10 (56C6, 1:40 dilution; DAKO) antibody-antigen complexes were detected using a VECTASTAIN ABC kit (mouse; Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. A negative control was designed by using phosphate-buffered saline instead of primary antibodies. Slices were counterstained with hematoxylin, dehydrated, and mounted. Finally, stained sections were evaluated using light microscopy.
Evaluation of immunohistochemical staining
We selected one tissue section with the largest cancerous area for each resected lesion. The randomly selected visual field (100⋅) in each lesion was assessed and scored. A brown stain was regarded as positive. The expressions of p53 and Ki-67 were quantified using image processing software (WinROOF, ver.5.7.2; Mitani Corp., Tokyo, Japan). This software measures the color intensity and transforms the color selected into a percentage in each field. We calculated the intensity of staining at the threshold of color and the extension of staining at the percentage of positive cells against total cancer cells. The p53 immunostaining was analyzed semi-quantitatively using a scoring system for both intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and extension (0, no unclear staining; 1, < 10% staining; 2, 10%-50% staining; 3, > 50% staining). The score for intensity and extension was summed for each lesion. We classified a score of > 4 as marked staining for p53 according to a previous report. Ki-67 LI was calculated as the percentage of positive cells against total cancer cells. We classified the lesions into a high group if Ki-67 LI was greater than the median value, which was also according to a previous report.
The mucin expression of tumor cells was examined with gastric phenotype markers, such as MUC5AC and MUC6, and intestinal phenotype markers, such as MUC2 and CD10. The results of each phenotype marker were defined as positive if > 10% of the tumor cells were stained. Tumors were classified by phenotype as gastric (G type), intestinal (I type), gastrointestinal (GI type), and null (N type); G type tumors expressed only a gastric phenotype marker, whereas I type tumors expressed only an intestinal phenotype marker. GI type tumors expressed both gastric and intestinal phenotype markers, whereas N type tumors were negative for all markers. We evaluated the expression of each mucin phenotype marker not only in the tumor but also in the surrounding mucosa.
Categorical variables were presented as frequencies and proportions. They were compared between the two groups by using Fisher’s exact test. Continuous variables were presented as medians (interquartile range [IQR]). They were compared between the two groups by using Wilcoxon rank sum test. A p-value < 0.05 was considered significant. To reveal the factors associated with unclear lateral demarcation by M-NBI, univariate and multivariate logistic regression analyses were performed. All statistical analyses were performed with JMP statistical software ver. 14 (SAS Institute Inc, Cary, NC).