Gastric cancer (GC) is classified into six types using Borrmann’s classification or two types using Laurén’s classification: the diffuse and intestinal type (1). In particular, Borrmann type 4 GC, including diffuse type of GC, is a type of advanced GC with extremely high malignancy and accounts for nearly 10% of all GC cases in Asia (2). Type 4 GC exhibits unique characteristics such as extensive fibrosis with sparse tumor cell infiltration in the stroma and is characterized by lesions that diffusely infiltrate the gastric wall, accompanied by a higher rate of lymph node metastasis and peritoneal metastasis (3). Type 4 GC is frequently resistant to treatment such as chemotherapy, and the prognosis is worse than with other types of GC (4,5).

The tumor immune microenvironment has attracted increasing attention because of the development of immunotherapy (6). Cytotoxic T lymphocytes, particularly CD8⁺ T cells, play an important role in immunosurveillance against cancer (7). Although infiltration of CD8⁺ T cells is associated with favorable prognosis in GC (8,9), Pernot et al. reported that a higher number of CD8⁺ T cells in diffuse-type GC is not correlated with prognosis (10). Thus, the significance of the tumor immune microenvironment in type 4 GC remains unclear.

Recently, tissue-resident memory T cells have attracted scientific attention. These cells are defined by expression of CD103, whose ligand is the epithelial cell marker E-cadherin (11). CD103 plays an important role in immunity against several cancers and expresses PD-1, CTLA-4 inhibitory receptors (12,13). Previous studies have shown that high infiltration of CD103⁺ T cells is associated with a better prognosis (14-16), suggesting that CD103⁺ T cells are a potential predictive biomarker. We also previously reported that CD103⁺ T cells are associated with a good prognosis in GC and that CD103 is expressed at higher levels in patients with the differentiated type than in those with the undifferentiated type (17). On the other hand, high expression of CD103 was found even in some patients with type 4 GC. The aim of this study was to assess the clinical significance of CD103⁺ T cells in type 4 GC.

Patients. A total of 1,147 patients were diagnosed with GC and underwent initial gastrectomy at Osaka City University Hospital between 2007 and 2017. Among them, 46 patients (4.0%) with type 4 GC were included in our analysis. The inclusion criteria for patients were as follows: 1) type 4 lesions were diagnosed by preoperative detection and postoperative histopathology; 2) the depth of the tumor was pathological T3 or higher; 3) patients did not receive neoadjuvant chemotherapy. Pathological TNM staging was diagnosed histologically based on the 7^th Edition of the Union for International Cancer Control TNM classification, and the histological type was determined based on the 14^th Edition of the Japanese Classification of Gastric Cancer.

Immunohistochemistry. Formalin-fixed, paraffin-embedded samples were sliced into 4-μm sections. Nonspecific binding was blocked using nonspecific staining blocking reagent (Dako, Denmark). The sections were reacted with rabbit monoclonal anti-CD103 antibody [clone: EPR4166 (2); 1/1000; Abcam, Cambridge, UK], or mouse anti-CD8 antibody (clone: C8/144; 1/250; Dako), or rabbit anti-CD4 antibody (clone: EPR6855; 1/250; Abcam). Sections were incubated with secondary antibody for 10 min at room temperature, and then were visualized using 3-3’-diamino-benzidine for 5 min and counterstained with hematoxylin.

Evaluation of immunohistochemical staining. Tumor sections stained with each antibody were scanned at ×200 magnification, and the five high-power fields were randomly captured for each tumor region. The average of five fields was calculated. Positive T cells in each section were counted manually by two observers who were blinded to the clinical outcome. All microscopic images were imported from the digital photo filing system DP-73 (Olympus, Tokyo, Japan). We divided the patients into two groups according to the median range.

Statistical analysis. Statistical analysis was performed with JMP 14 (SAS Institute, Cary, NC, USA). The Mann–Whitney test was used to assess the associations between the expression of CD103 and clinicopathological features. Overall survival (OS) and progression-free survival (PFS) curves were calculated with the Kaplan-Meier method, and significant differences in survival were determined using the log-rank test. PFS is defined as the time between the day of surgery and disease progression. For all other experiments, data were compared using the two-tailed Student’s t-test. p-Values of <0.05 were considered statistically significant.

Patient characteristics. In total, 46 patients with type 4 GC (19 females and 27 males; median age 70 years) were analyzed. According to histological type, undifferentiated types were observed in 43 patients (93.5%) [10 of poorly differentiated type (solid type), 28 of poorly differentiated type (non-solid type), 3 of signet-ring cell type, and 2 of mucinous type]. Four patients were stage II, 18 patients were stage III, and 24 patients were stage IV [2 had liver metastasis, 12 had peritoneal metastasis (P1), 12 had only intraoperative peritoneal lavage cytology positivity (P0CY1)]. Forty patients received adjuvant chemotherapy (26 of monotherapy, 14 of doublet chemotherapy).

The presence of CD103⁺ cells in type 4 GC. Although CD103 staining was low in most patients, some patients with high infiltration of CD103⁺ T cells were found (Figure 1). The median range of CD103⁺ T cells per area was 12.7 (5.75-20.1). No significant difference was found in clinicopathological features between high and low CD103 expression (Table I).

Survival analysis. Kaplan-Meier analysis showed that high CD103 expression tended to predict better prognosis (Figure 2A; p=0.052). On the other hand, no significant difference was found for CD8 or CD4 (Figure 2C-F). Considering each stage, high CD103 expression predicted a better prognosis than low CD103 expression for patients with stage IV (Figure 3B; p=0.003). For patients with P1 or P0CY1, high CD103 expression tended to predict a better prognosis than low CD103 expression (Figure 3C and D; p=0.054 and p=0.077, respectively). When considering patients who received adjuvant chemotherapy, no significant difference in prognosis was found for patients who received monotherapy (Figure 4A and 4B). On the other hand, both OS and PFS in high CD103 expression were longer than in low CD103 expression for patients who received doublet chemotherapy (Figure 4C and 4D; p<0.001 and p=0.063, respectively).

In this study, we showed that patients with type 4 GC who had high CD103 expression tended to have a better prognosis than those who were low CD103 expression. In particular, for patients with stage IV or patients who receive doublet adjuvant chemotherapy, high CD103 expression was associated with a good prognosis.

CD103 binds the cell surface marker E-cadherin and labels tissue-resident memory T cells. We previously reported that CD103 expression is higher in differentiated type GC than in undifferentiated type GC (17). On the other hand, in this study, high infiltration of CD103⁺ T cells was observed in some patients with type 4 GC, which has reduced E-cadherin (18). Thus, the expression of CD103 may be influenced by factors other than E-cadherin.

CD103 expression is associated with a good prognosis in several cancers including GC (15-17). In particular, CD8⁺ CD103⁺ T cells exhibit higher levels of cytotoxic molecules such as interferon-γ and Granzyme-B than CD8⁺CD103^– T cells (15), indicating that CD103 is an important marker in antitumor immunity. In general, tumor-infiltrating lymphocytes, especially cytotoxic CD8⁺ T cells, are correlated with an improved outcome in several cancers (9,19,20), and predict the response to chemotherapy in breast cancer (21,22). However, our results showed that CD8 expression was not correlated with prognosis in type 4 GC, as in previous reports (10,23). On the other hand, high CD103 expression was associated with an improved prognosis even in stage IV patients. Furthermore, CD103 expression had an impact on prognosis in patients who received doublet chemotherapy. Several studies revealed the influence of the immune microenvironment on the efficacy of chemotherapy (24,25). Involvement of immunogenic cell death is considered. Although the association between CD103⁺ T cells and the efficacy of chemotherapy remain unclear, we hypothesize that chemotherapy induces immunogenic cell death, and the immunogenic cell death may be associated with the activation of CD103⁺ T cells. Accordingly, we consider that more attention should be paid to CD103⁺ T cells than CD8⁺ T cells in type 4 GC.

Our study has several limitations. First, this was a retrospective study with a small sample size. Second, these results were evaluated only with immunohistochemistry, and thus, intratumoral heterogeneity could have affected the results. Third, patients in this study were treated between 2007 and 2017, and changes in chemotherapy over the last 10 years could have affected the prognosis.

In conclusion, we herein confirmed that high CD103 expression is associated with a good prognosis even in type 4 GC although CD103 expression was low in most patients. Our results indicate that CD103⁺ T cells may be useful as a prognostic factor in type 4 GC.

The Authors declare no conflicts of interest.

TM acquired, analyzed and interpreted the data, and wrote the manuscript. HT made substantial contributions to the conception and design of the study, interrupted the data and revised the manuscript. SD, YM, MY, TTa, TTo, SL, and KM acquired and analyzed the data. MO contributed to the conception and design of the study, interrupted the data and revised the manuscript.

All experimental procedures were approved by the Osaka City University ethics committee (approval No. 4092), and all patients provided informed consent for collection and analysis of their specimens.