In this study, RGC was defined in accordance with the Japanese Classification of Gastric Carcinoma (English edition, ver. 3) . Between January 2002 and January 2017, 39 patients with RGC following distal gastrectomy underwent curative surgical treatment at the Department of Gastroenterological Surgery, Okayama University Hospital. This study included only curative treatment, not non-curative treatment. Medical records of all patients were obtained from the hospital database. Pre-treatment factors (age, sex, cause of initial gastric surgery, reconstruction methods in initial surgery, time from first to RGC surgery, and blood test results), treatment factors (presence of a left gastric artery [LGA] and tumor location), post-treatment factors (histopathological data, follow-up period, recurrence, and adjuvant therapy) were examined retrospectively. The cause of initial gastric surgery and reconstruction methods in primary surgery were categorized as benign and malignant disease, and Billroth-I and others, respectively. Tumor locations were categorized as anastomotic site and non-anastomotic site. Depth of invasion was categorized as T1 (mucosa, or submucosa) or T2/3/4 (muscularis propria, subserosa, serosa-exposed, or serosa-infiltrating). Lymph node metastasis was categorized as negative or positive. Pathological stage was categorized as stage I or stage II/III (there were no Stage IV cases). Histological types were categorized as differentiated type (well-differentiated, moderately differentiated, or papillary) or undifferentiated type (poorly differentiated, signet-ring cell carcinoma, or mucinous). Lymphatic invasion and venous invasion were categorized as negative or positive. All histopathological information was evaluated and determined in accordance with the International Union Against Cancer (UICC) TNM classification, 7th edition.
Patients were followed-up every 3-6 months with physical examinations and laboratory blood tests. Patients underwent computed tomography (CT) every 6 months and esophagogastroduodenoscopy every 1 year. The median follow-up period of the 39 patients was 48.1 months (IQR: 30.0-67.9 months).
Immunohistochemical staining and assessment
Formalin-fixed, paraffin-embedded tissue samples cut at a thickness of 2 μm were deparaffinized and soaked in 0.3% H2O2 for 10 min at room temperature to extinguish endogenous peroxidase activity. After antigen retrieval by heating in a sodium citrate buffer solution or EDTA using a microwave, the samples were incubated with primary antibodies against CD8 (eBioscience, San Diego, CA, USA) and CD4 (eBioscience) overnight at 4 °C, and then with peroxidase-linked secondary antibody for 30 min at room temperature. After washing, the samples were stained with 3,3’-diaminobenzidine (Dako, Glostrup, Denmark) for visualization, and counterstained with Meyer’s hematoxylin.
The stained area in the sample was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
All statistical analyses were performed using JMP software version 14.2 (SAS Institute, Cary, NC, USA). Pearson’s chi-squared test or Fisher’s exact test was used for categorical variables, and the Mann-Whitney U test was used for continuous variables. The Kaplan-Meier method was used to estimate overall survival (OS) in each group, and survival rates were compared using the log-rank test. A probability (P) value less than 0.05 was considered significant.