ONCOLOGY REPORTS 41: 2265-2272, 2019 Expression of protein disulfide isomerase A3 and its clinicopathological association in gastric cancer TOMOHIRO SHIMODA1,2, RYUICHI WADA1,3, SHOKO KURE1,3, KOUSUKE ISHINO1, MITSUHIRO KUDO1, RYUJI OHASHI4, ITSUO FUJITA2, EIJI UCHIDA2, HIROSHI YOSHIDA2 and ZENYA NAITO1,3 Departments of 1Integrated Diagnostic Pathology and 2Gastrointestinal and Hepato‑Biliary‑Pancreatic Surgery, Nippon Medical School, Tokyo 113-8602; 3Diagnostic Pathology, Nippon Medical School Hospital, Tokyo 113‑8602; 4Department of Diagnostic Pathology, Nippon Medical School Musashi Kosugi Hospital, Kawasaki, Kanagawa 211‑8533, Japan Received September 24, 2018; Accepted January 24, 2019 DOI: 10.3892/or.2019.6999 Abstract. Protein disulfide isomerase A3 (PDIA3) is a prognosis in PDIA3‑High GC may be accounted for, in part, chaperone protein that supports the folding and processing of by sufficient antigen processing and expression of MHC synthesized proteins. Its expression is associated with the prog- class I, which can be mediated by PDIA3. It was suggested nosis of laryngeal cancer, hepatocellular carcinoma, diffuse that PDIA3 serves an important role in the pathobiology of glioma and uterine cervical cancer. In the present study, the GC, and that PDIA3 is a useful marker for the prediction of expression levels of PDIA3 and its clinicopathological associa- prognosis. tion were examined in 52 cases of gastric cancer (GC). The expression of PDIA3 was examined by immunohistochem- Introduction istry and scored using a semi-quantitative method. According to the score, GC samples were classified into PDIA3‑High and Gastric cancer (GC) is the third major cause of cancer-asso- PDIA3‑Low GC. PDIA3‑High GC samples were predomi- ciated fatality in Japan (1). The prognosis has improved nantly of the intestinal type. Multivariate survival analysis because of early diagnosis and surgical treatment, followed indicated that PDIA3 expression and cancer stage were inde- by chemotherapy and molecular targeting therapy using pendent factors. The overall survival of PDIA3‑High GC cases anti-human epidermal growth factor receptor 2 antibody and was significantly favorable compared with that of PDIA3‑Low anti‑programmed death‑ligand 1 antibody (2). However, the GC cases, and this was more evident in cases at an advanced prognosis of GC at an advanced stage is still unfavorable (3). stage. In GC cell cultures, the PDIA3 and major histocompat- New strategies for the diagnosis and treatment of GC are ibility complex (MHC) class I proteins were expressed in three therefore required. out of the four assessed cell lines according to western blot Protein disulfide isomerase A3 (PDIA3), which is also analysis. Notably, the expression of MHC class I was increased known as GRP58/ERp57, is a chaperone protein that supports by the stimulation of interferon γ. Co-immunoprecipitation the folding and processing of protein synthesized in the assays suggested the formation of a PDIA3 and MHC class I endoplasmic reticulum (4). PDIA3 is involved in multiple complex. The findings suggested that PDIA3 may be involved biological functions, including stabilization of receptors, in the immune response of carcinoma cells. The improved antigen processing and presentation, and degradation of proteins (5). It has also been indicated that PDIA3 is involved in the proliferation and cell death in human carcinomas (6). High expression levels of PDIA3 are associated with poor prognosis of laryngeal cancer, hepatocellular carcinoma and diffuse Correspondence to: Dr Ryuichi Wada, Department of Integrated glioma (7‑9). However, high expression levels of PDIA3 are Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi, also associated with a favorable prognosis of uterine cervical Bunkyo‑ku, Tokyo 113‑8602, Japan cancer (10). Regarding GC, it has been reported that high E-mail: [email protected] expression of PDIA3 is associated with favorable prognosis in GC at early stage; however, an association has not been Abbreviations: GC, gastric cancer; HLA, human leukocyte antigen; IFN γ, interferon γ; IHC, immunohistochemistry; IM, identified in GC at the advanced stage (11). The association of intestinal metaplasia; MHC, major histocompatibility complex; PBS, PDIA3 with prognosis of GC in Japanese cases is not known. phosphate-buffered saline; PDIA3, protein disulfide isomerase A3; Furthermore, the mechanism of favorable prognosis in GC TBS‑T, Tris‑buffered saline/Tween‑20 with high expression of PDIA3 remains undetermined. In the present study, the association of PDIA3 expression Key words: gastric cancer, protein disulfide isomerase A3, major with clinicopathological features, including prognosis, was histocompatibility complex class I, prognosis, stage, interferon γ investigated in Japanese cases of GC. A possible mecha- nism of how PDIA3 affects the prognosis of GC was also suggested. 2266 SHIMODA et al: PDIA3 EXPRESSION IN GASTRIC CANCER Materials and methods follows: no staining, score of 0; moderate staining similar to that of the cells in the glands with intestinal metaplasia (IM), Cases of GC. Cases of GC were retrieved from the archives score of 2; clear but weaker staining than that of the meta- of the pathology records of Nippon Medical School Hospital plastic cells, score of 1; and stronger staining than that of these (Tokyo, Japan) between January 2006 and December 2008. cells, score of 3. Furthermore, the proportion of positive cells A total of 52 cases were used for the study. The cases were of each intensity score was evaluated as a percentage in 10% randomly selected from the cases of GC. The patients did not increments. The total score was calculated using the following receive chemotherapy or radiation prior to the surgery. Clinical formula: 1 x (proportion of score 1 cells) + 2 x (proportion of information and the time of death following surgery were score 2 cells) + 3 x (proportion of score 3 cells). Two individuals retrieved from the clinical records. The study was conducted evaluated the intensity score and proportion of the positive according to the declaration of Helsinki and the Japanese cells in a blind manner. When the values were discrepant, the Society of Pathology. The study was approved by the Ethics two individuals discussed the results and the most appropriate Committee of the Nippon Medical School Hospital (Tokyo, value was determined. Japan; no. 29-06-764). Informed consent was obtained from The Ki‑67 labeling index was calculated as the percentage all patients. of Ki‑67‑positive cells in ~1,000 tumor cells in the areas of the highest nuclear labeling under x400 magnification using the Histological examination of GC. The histology of the patho- eCount image analysis software version 4.7 (e-Path Co., Ltd., logical specimens of GC was reviewed by three individuals. Fujisawa, Kanagawa, Japan). Resected stomachs were fixed in 10% formalin at room temperature for 24 h and processed to be embedded in paraffin. Terminal deoxynucleotidyl transferase dUTP nick‑end Three µm-thick sections of the entire area of the carcinoma labeling (TUNEL) assay. Apoptotic cell death was determined were stained with hematoxylin and eosin (stained for 5 min by TUNEL assay using the Apoptag Peroxidase In Situ with hematoxylin and 3 min with eosin at room temperature) Apoptosis Detection Kit (EMD Millipore, Temecula, CA, and observed using microscope (ECLIPSE 50i; Nikon Corp., USA). Briefly, deparaffinized 3‑µm‑thick paraffin section was Tokyo, Japan). The histological subtype was determined as digested with 20 µg/ml proteinase K (cat. no. S3004; Agilent the predominant histology of the carcinoma tissue according Technologies Japan, Ltd.) at 37˚C for 30 min. The labeling was to the classification indicated by Laurén (12). The depth of performed in the mixture of terminal deoxyribonucleotide the invasion and lymph node metastasis were also evalu- transferase and digoxygenin‑dUTP at 37˚C for 1 h. Incorporated ated (13). The pathological stage was classified according to digoxygenin-dUTP was detected by peroxidase-labeled the classification of the Union for International Cancer Control anti-digoxygenin antibody, which was included in the kit. The (UICC) (14). Helicobacter pylori infection state was evaluated peroxidase activity was visualized using diaminobenzidine at with 3 µm-thick sections stained with hematoxylin and eosin room temperature for 2 min. Nuclear staining was considered and viewed using a microscope (ECLIPSE 50i; Nikon Corp.) positive. The TUNEL index was calculated as the percentage at high magnification (x1,000). The infection state was also of TUNEL-positive cells in 1,000 counted carcinoma cells in considered positive if the serum was positive for anti-Helico‑ the areas of highest nuclear labeling under x400 magnification bacter pylori antibody. using the eCount image analysis software version 4.7 (e-Path Co., Ltd.). Immunohistochemistry and semi‑quantitative evaluation. Immunohistochemistry was conducted using a polymer-based Extraction of total RNA and reverse transcription‑quantitative two‑step method. Briefly, 3‑µm‑thick paraffin sections were polymerase chain reaction (RT‑qPCR). Total RNA was deparaffinized and hydrated in phosphate-buffered saline extracted from paraffin sections of GCs. The sections were (PBS). Sections were pretreated in Tris‑HCl (pH 9.0) for deparaffinized and hydrated. Following this, carcinoma PDIA3 and citrate buffer (pH 6.0) for Ki‑67 at 121˚C for tissues were dissected and collected into 1.5-ml tubes. For 15 min.