High CD8/CD33 ratio in peritoneal metastatic lesions is associated with favorable prognosis in gastric cancer

Abstract Background Tumor‐infiltrating lymphocytes (TILs) and other immune cells have been reported as a prognostic factor in several tumors, including gastric cancer, and they play an important role in antitumor effect at the primary site. There were few reports on the immune status in peritoneal metastatic lesions for gastric cancer. Aims The aims of this study were to assess the prognostic significance of TILs (CD4, CD8, CD19, regulatory T cells [Tregs]), and myeloid‐derived suppressor cells (MDSCs) in peritoneal metastatic lesions. Methods We retrospectively investigated 60 patients for gastric cancer with peritoneal metastasis who were treated between 2009 and 2016 in our institute. Immunohistochemistry for CD4, CD8, CD19, FOXP3, and CD33 was performed in the peritoneal metastatic lesions. The absolute numbers of immune cells and ratios were evaluated, and the relationship between immune‐related marker and overall survival (OS) was investigated. Results A high infiltration of CD8+ lymphocytes or high CD8/CD33 ratio was a better prognosis for OS in univariate analysis using all immunologic variables (P = .012, P = .001). In multivariate analysis for clinical and immunologic variables, high CD8/CD33 ratio was identified as an independent prognostic factor for OS (Hazard ratio: 0.291, 95% confidence interval: 0.126‐0.670, P = .004). Conclusion High CD8/CD33 ratio and high infiltration of CD8+ lymphocytes in peritoneal metastatic lesions were favorable prognoses for gastric cancer patients with peritoneal metastasis. It is necessary to modify the immune microenvironment result to increase the level of CD8+ lymphocytes in the peritoneal metastatic lesions.


| BACKGROUND
Globally, gastric cancer is one of the most life-threatening diseases in the world, and a major cause of cancer-related deaths. 1,2 Peritoneal metastasis is an incurable disease that occurs frequently in recurrent gastric cancer and associates with poor prognosis. Importantly, molecular mechanisms of peritoneal metastasis are poorly understood.
Although various treatments have been developed for peritoneal metastasis, including systemic chemotherapy, intraperitoneal chemotherapy, sufficiently satisfactory outcomes have yet to be achieved 3-5 ; this indicates the need for novel treatment strategies, in addition to conventional surgery and chemotherapy.
It is thought that the progression and suppression of tumor depend on various cell-cell interactions in local and metastatic lesions in the tumor microenvironment. Several studies have attempted to investigate the relationship between tumorinfiltrating lymphocytes (TILs) and prognosis in several cancers, suggesting that TILs accumulation was associated with better prognosis. 6,7 CD8 is a surface antigen expressed in cytotoxic T lymphocytes (CTLs) that recognize specific antigen and eliminate the foreign objects. Several studies have reported that high infiltration of CD8 + lymphocytes was associated with better overall survival (OS) and disease-free survival (DFS) in gastric cancer. [8][9][10] Furthermore, CD4 + helper T cells play an important role in adaptive immune system by releasing cytokines. 11 Higher infiltration of CD4 + lymphocytes is also related to favorable prognosis in gastric cancer. 12

| Immunohistochemistry
The specimens were dewaxed in xylene and rehydrated through graded ethanol for immunohistochemistry. Endogenous Abcam, Tokyo, Japan). Secondary antibody was subsequently performed for 1 hour, and then the sections were developed in DAB solution.

| Evaluation of immunostaining
To evaluate the expression of immune-related markers in the specimens, five non-overlapping intratumoral fields were counted under high power fields. All slides were counted by two researchers (TY and JK). The mean numbers of positive cells in five fields were calculated in each antibody. The median number was used to divide the patients into two groups (low or high).

| Statistical analysis
We investigated the differences among the data sets using Fisher's exact test using the computer software package SPSS version 25 (SPSS, Chicago, IL, USA). Survival curves were created using the Kaplan-Meier method and the log-rank test. OS was calculated from the date the diagnosis of peritoneal metastasis was established to death from any cause or the latest follow-up. The influence of each factor on patients' survival was evaluated using Cox regression analysis. Multivariate analysis of survival distributions was performed using Cox proportional hazards regression models.
P values < .05 were considered a statistically significant difference.

| Patient clinicopathological characteristics
The characteristics of 60 patients are shown in

| Relationship between clinical variables and OS
In univariate analysis of clinical variables, age, gender, initial or recurrence, ECOG performance status, Borrmann type, and pathology P status were not associated with OS (

| Evaluation of immune-related cells in peritoneal metastasis
We investigated the five immunologic parameters (CD4 as helper T cells,

| Survival according to immunological parameters
As shown in Table 3 ratios were not associated with OS. Figure 2A,B shows the Kaplan-Meier curves in 60 patients according to CD8 expression and CD8/CD33 ratio.

Clinicopathological features and immunohistopathologic variables
showing P ≦ .1 by univariate analysis were adopted as covariates when multivariate Cox proportional hazards analysis was performed ( Decreased infiltration of CTLs is thought to be reflected by the immunosuppressive microenvironment, resulting in difficulties to treat patients with peritoneal metastasis.
The presence of CTLs in the tumor microenvironment induces the host immune response to tumor antigens, and inhibits tumor progression. 20 Our results showed that high densities of TILs related to adaptive immunity contributed to chemosensitivity and favorable prognosis.
The reason for the different rate in CD8 + lymphocytes infiltration in peritoneal metastasis is unknown. Previously, we have established a peritoneal tumor model by co-inoculating the mouse gastric cancer cell line YTN16 and the mouse myofibroblast cell line F I G U R E 2 Survival curves in 60 patients according to the density of CD8 + TILs and CD8/CD33 ratio. A, The prognosis in patients with CD8 high-density groups was significantly better than that in those with CD8 low-density (P = .012, log-rank). B, The prognosis in patients with high CD8/CD33 ratio was significantly better than that in those with low CD8/CD33 ratio (P = .001, log-rank)  35 It is considered that the accuracy of a prognostic factor was further improved in combination with CD8.
This study had several limitations including the retrospective study design and the use of a small sample size of patients obtained from a single center. It is relatively difficult to obtain a lot of specimens from peritoneal metastatic lesions that have not undergone chemotherapy.
Therefore, further prospective multicenter and large-scale studies are needed to reveal these results and elucidate the significance of the molecular processes involved in TILs infiltration.

| CONCLUSION
High CD8/CD33 ratio and high infiltration of CD8 + lymphocytes into peritoneal metastatic lesions were associated with favorable prognosis in gastric cancer patients with peritoneal metastasis.
CTLs play an important role in antitumor effect for peritoneal metastasis, although immunological ignorance often occurs in peritoneal cavity. We have to develop the treatment strategy for induction of CTLs infiltration.

CONFLICT OF INTEREST
The authors declare that they have no competing interests.

ETHICAL STATEMENT
This study was approved by the Kanazawa University Hospital Review Board (Permission number 2789). Written informed consent was obtained from all patients.

DATA AVAILABILITY STATEMENT
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.