The ratio of intratumoral CD15 + neutrophils to CD8 + lymphocytes predicts recurrence in patients with gastric cancer after curative resection

Abstract Background An elevated neutrophil‐to‐lymphocyte ratio (NLR) in peripheral blood is an independent prognostic indicator of various cancers. Aims In this study, we aimed to investigate the prognostic relevance of the intratumoral immune cell balance in gastric cancer. Methods and Results The study included 82 patients who underwent curative resection for gastric cancer. The intratumoral cluster of differentiation (CD) 15‐ and CD8‐positive cells were evaluated using immunohistochemical staining. Additionally, clinicopathological factors and prognoses were analyzed. Patients with high intratumoral CD15/CD8 ratios had significantly lower overall survival (OS) and relapse‐free survival (RFS) compared to those with low CD15/CD8 ratios (p = .0026 and p < .0001, respectively). Additionally, a high CD15/CD8 ratio was associated with lymph node metastasis (p = .019). Patients with high NLR had a significantly lower RFS than those with low NLR (p = .0050). Multivariate analysis revealed that the intratumoral CD15/CD8 ratio, NLR, and venous invasion were independent prognostic indicators of RFS (CD15/CD8 ratio: p < .001, hazard ratio (HR) = 14.7, 95% confidence interval (CI) = 3.8–56.8; NLR: p = .010, HR = 5.4, 95% CI = 1.5–19.6; venous invasion: p = .005, HR = 7.4, 95% CI = 1.8–29.7). Conclusion In summary, we found that the intratumoral CD15/CD8 ratio is an independent prognostic factor following gastric cancer resection and its increase is associated with lymph node metastasis and microscopic lymph vessel invasion. Immunological evaluation with additional aspects of innate immunity may be useful in predicting cancer prognosis.

Inflammation and the immunological environment within tumor tissues have been closely linked to tumor progression and prognosis.
Current research is delving into the detailed mechanisms behind this association. 1 Analysis of gene expression in a large cohort of cancer tissues, using samples from The Cancer Genome Atlas (TCGA), has revealed that the expression of immune-related genes within the tumor environment ranks second in importance to cell cycle-related genes across a broad spectrum of cancers. 2 Moreover, prior studies have predicted the proportions of tumor-infiltrating leukocytes based on increased gene expression in tumor tissues.These findings indicate that high expression of genes specific to certain lymphocyte subtypes, such as CD8-positive lymphocytes, associated with a favorable prognosis. 2,3In contrast, high expression of genes specific to myeloid leukocytes, like neutrophils, tends to predict a poor prognosis. 2,4,5This evidence suggests that the balance between key leukocytes namely CD8-positive lymphocytes and neutrophils in tumor tissues could serve as a predictive marker for patient outcomes.
Treatment strategies for gastric cancer, from early-stage to advanced cancers, have evolved through the development of detailed algorithms based on the results of numerous clinical studies.7][8] By stratifying the risk of recurrence among patients who have undergone curative resection and offering more effective treatment to those at higher risk and less invasive treatment or follow-up to those at lower risk, it is hoped to establish a treatment strategy that improves prognosis while also contributing to patient quality of life (QOL) and healthcare economics.To achieve this goal, it is important to examine the risk of recurrence in more detail.
Comprehensive immunogenomic analysis of TCGA samples suggested that gastric cancer has a high frequency of subtypes with high leukocyte infiltration, such as lymphocytes and macrophages, and that the immunologic landscape of the cancer tissue may influence prognosis. 3This study aims to investigate the prognostic value of the ratio of CD15-positive neutrophils to CD8-positive lymphocytes in tumor tissues from patients with resected gastric cancer through immunohistochemistry. Additionally, we analyzed clinicopathological associations to determine their potential impact on prognosis.

| Patient samples
This study included consecutive 82 patients who underwent curative surgery for gastric cancer at the Fukushima Medical University, Japan, from January 2009 to December 2011.Informed consent was obtained from all patients to collect and analyze specimens for this study.The study design was a retrospective cohort study, and ethics committee approval was obtained in 2014, after surgery.Inclusion criteria were all patients who underwent surgery with the intention of curative surgery for gastric cancer during the same period, and patients who were deemed ineligible for curative surgery at the time of surgery were excluded.Pathological findings were evaluated based on the 8th Edition of the TNM Classification of Gastric Cancer.

| Immunohistochemistry
Following the standard protocol, immunohistochemical staining for CD15 and CD8 was performed on 4-μm formalin-fixed paraffinembedded tissue sections.After deparaffinization, the antigen was microwave-activated for 15 min, and endogenous peroxidase activity was inhibited by incubation with 0.3% aqueous hydrogen peroxide for 20 min.The tissue samples were subsequently treated with 5% powdered skim milk to block nonspecific reactions with antibodies.Slides were incubated for 20 min at 4 C with the following primary antibodies: CD15 (1:50 dilution, mouse monoclonal, clone MMA, BD Biosciences, San Jose, CA, USA) and CD8 (1:50 dilution, mouse monoclonal, clone C8/144B, DAKO, Santa Clara, CA, USA).Slides were then incubated with biotinylated anti-mouse secondary antibodies (rabbit polyclonal, 1:400 dilution; DAKO).Antigen visualization was performed using the VECTASTAIN Elite ABC Standard Kit (1:200 dilution; VECTOR Laboratories, San Diego, CA, USA).Immunostained slides were observed under an optical microscope (IX73, OLYMPUS, Japan) at 400Â and images of this field of view were captured.CD15-positive neutrophils and CD8-positive lymphocytes in the field of view were counted visually, and the average value was used as the number of each positive cell.When identifying CD15-positive cells, morphological characteristics were considered to exclude CD15-positive eosinophils.The CD15/CD8 ratio was calculated by dividing the number of CD15-positive cells by the number of CD8-positive cells.Automated analysis using image analysis software was not used to identify positive cells.Images of the slides taken were displayed in ImageJ (https://imagej.net/ij/index.html) and counted manually one by one.

| NLR of peripheral blood
Peripheral blood collected within 4 weeks prior to the surgery was analyzed.The total white blood cell counts and percentages of neutrophils and lymphocytes were retrieved from the electronic records of the hospital laboratory.NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count.

| Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics version 23 (IBM, Armonk, NY, USA) and GraphPad Prism, version 7.03 (GraphPad Software, San Diego, CA, USA).OS was defined as the period between the date of surgery and death, while RFS was defined as the period between the date of surgery and recurrence.The cutoff values for low and high NLR and CD15/CD8 ratio were determined using receiver operating characteristic (ROC) curve analysis.The survival curve was prepared using the Kaplan-Meier method, and statistical differences were analyzed using the log-rank test.The association between the immune balance in tumor tissues and the clinicopathological factors was evaluated using the chi-square or Fisher's exact test.The normality of the distribution of the CD15/ CD8ratio and NLR variables was tested by the Kolmogorov-Smirnov test.The correlations were further analyzed by Spearman's rank correlation coefficient.Univariate and multivariate analyses were performed using the Cox proportional hazard model.The statistical significance for all analyses was set at p < .05.

| Patient characteristics
The patient demographics and clinical characteristics are presented in Table 1.The mean age of the patients was 67.7 years.Although patients were predominantly male, neither age nor sex affected prognosis.None of the patients were treated with preoperative adjuvant chemotherapy.The median follow-up time after surgery was 61 months (range: 4-94 months, interquartile range: 24).In addition to deaths, there were also cases in which follow-up was lost during the course of the study and patients dropped out of followup.In total, 21 patients died at the end of the follow-up period.Among them, 14 patients died from cancer-unrelated diseases, and 11 patients experienced tumor relapse.The median time to relapse was 12 months (range: 6-28 months, interquartile range: 13).

| High intratumoral CD15/CD8 ratio worsens prognosis
ROC curve analysis revealed that the cutoff value for the CD15/CD8 ratio was 2.25 based on the 5-year RFS, as depicted in Figure 2A.

| High intratumoral CD15/CD8 ratio is associated with lymph node metastasis and lymphatic invasion in gastric cancer tissues
The demographic characteristics of each patient group are presented in Table 2 based on the investigated intratumoral CD15/CD8 ratio and NLR.Patients with a higher intratumoral CD15/CD8 ratio had a significantly higher incidence of lymph node metastasis ( p = .019)and lymphatic invasion ( p = .005).In contrast, the incidence of the depth of invasion (p = .024),lymph node metastasis ( p < .001),staging ( p = .013),and lymphatic invasion ( p < .001) was considerably elevated in patients with higher NLRs.
3.5 | NLR and CD15/CD8 ratios are independent of each other NLR was determined using preoperative peripheral blood data.The median value for the NLR was 2.29 (range: 0.83-9.67).According to the ROC curve, based on the 5-year RFS, the optimal NLR cutoff value was 3.07.Patients with a high NLR had worse RFS (Figure 2B; p = .0050).The Kolmogorov-Smirnov test confirmed that both the CD15/8 ratio and NLR were not normally distributed with p < .0001.Therefore, the correlation analysis of the CD15/CD8 ratio and NLR was conducted using Spearman's rank correlation coefficient, a nonparametric test.The result showed that there was a correlation with a significance probability of p = .046,but the correlation coefficient was ρ = .221,which was a very weak correlation (Figure 3A).
Relapse occurred in 3 of the 29 patients with a high NLR or CD15/ CD8 ratio, at a relapse rate of 31.0%.As a breakdown of this, relapse occurred in 3 of the 10 patients with a low NLR and high CD15/CD8 ratio, 2 of the 13 patients with a high NLR and low CD15/CD8 ratio, and 4 of the 6 patients with a high NLR and high CD15/CD8 ratio.In contrast, recurrence occurred in 2 of 53 patients with low NLR and CD15/CD8 ratio, at a relapse rate of 3.8%.Analysis of the difference in relapse rates among these four categories revealed a significantly higher relapse rate in the group with high NLR and high CD15/CD8 ratio (Figure 3B; p < .0001).

| A high intratumoral CD15/CD8 ratio, NLR, and venous invasion are an independent prognostic factor for gastric cancer
RFS-related factors were evaluated using univariate and multivariate analyses.These analyses also included albumin (ALB), and a  3).

| Intratumoral CD15/CD8 ratio predicts the recurrence of gastric cancer in patients with stage II and III tumors
Only patients with stage II and III cancer who were candidates for postoperative adjuvant chemotherapy were included in the analysis.

| DISCUSSION
In this study, we investigated the association between recurrence and prognosis in patients undergoing gastrectomy for radical resection of gastric cancer and the ratio of CD15-positive neutrophils to CD8-positive lymphocytes (CD15/CD8 ratio) in gastric cancer tissue.
This ratio may reflect the immunologic landscape within the gastric cancer tissue.Additionally, we examined the association between the CD15/CD8 ratio and clinicopathological factors and determined the mechanism by which it influences prognosis.
An elevated CD15/CD8 ratio in gastric cancer tissues was associated with poor OS and RFS.Immunohistochemical analyses corroborated the results of comprehensive gene expression analysis using TCGA samples. 3This study also supports similar outcomes from previous studies that assessed the proportion of tumor-infiltrating lymphocytes through immunostaining for CD3, CD8, CD45RO, and CD66b, although those studies did not elucidate the mechanism. 9e analysis revealed that gastric cancer tissues with a high CD15/CD8 ratio tended to have more lymphatic invasion, which was significantly associated with more lymph node metastasis.Recent studies have shown that cancer cells alter the immune balance by secreting cytokines and chemokines, inducing endogenous inflammation within the cancer cells, and promoting the metastasis cascade.
1][12] Furthermore, the suppression of CD8-positive cells by infiltrating neutrophils may reduce immune surveillance by lymphocytes, creating an environment that promotes further metastasis.
Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), known for their potent immunosuppressive capabilities, suppress the number and function of lymphocytes. 13PMN-MDSCs contribute to immunosuppression by depleting arginine, which is essential for lymphocyte proliferation, through the release of arginase 1. 14 They also produce immunosuppressive mediators, such as reactive oxygen species, peroxynitrites, and prostaglandin E2. 15 Our findings suggest that a high CD15/CD8 ratio in cancer tissues could signal an immunological environment that promotes lymphatic invasion and lymph node metastasis.Although our study lacks specific immunostaining markers to differentiate PMN-MDSCs from other neutrophil subtypes, the CD15-positive neutrophils we observed are considered potential targets for cancer therapy.
This study also assessed the correlation between the CD15/CD8 ratio and NLR (neutrophil lymphocyte ratio) as a prognostic factor in gastric cancer.NLR, reflecting systemic immunologic balance and has long been associated with prognosis. 16,17In contrast, the CD15/CD8 ratio was considered more reflective of the local immune status of the tumor and was found to be more predictive of prognosis than NLR.
Since these two indices are very weakly correlated in nonparametric analysis did and each functioned as independent prognostic factors, combination them may enhance the accuracy of prognostic prediction.
It has been reported that combining several immunological indices can better predict prognosis. 18In this study, cases with high levels of either or both NLR and CD15/CD8 ratio experienced recurrence rate approximately 8 times higher than those with low levels of both.
In addition to histopathological factors, HER2 overexpression, 19 microsatellite instability (MSI), 20 PDL-1 expression, 21  focuses on the aspect of acquired immunity, primarily cytotoxic T cells, but the CD15/CD8 ratio evaluated in this study adds an assessment of the aspect of innate immunity through the measurement of neutrophils.Furthermore, by combining it with the NLR, it is possible to assess both systemic and local immune status, identifying cases with a high risk of recurrence that could not be predicted by existing indicators.Additionally, the method of counting cell numbers to calculate ratios offers the advantages of simplicity and higher quantification compared with assessing staining intensity with immunostaining, making clinical application with a view to automation.
Postoperative adjuvant chemotherapy is the standard of care for stage II and III gastric cancer and is associated with good prognosis. 6ratifying patients at high risk of recurrence and adjusting treatment regimens is critical to further improving outcomes and optimizing patient safety and health care resources.In our study, the CD15/CD8 ratio predicted the risk of tumor recurrence even when analyzed only for stage II and stage III subgroups.Although further validation in more cases is needed, we suggest that the CD15/CD8 ratio, alone or in combination with NLR, has potential as a biomarker for predicting cancer recurrence and for stratifying postoperative adjuvant therapy.
The present study has several limitations.Due to the small number of patients included and the limited number of relapse events observed, further validation in a larger cohort is necessary before the findings can be applied to clinical practice.Additionally, the anti-CD15 antibody utilized to identify neutrophils in this study also stains myeloid leukocytes, such as eosinophils, monocytes, and histiocytes.
Therefore, it is imperative to employ antibodies capable of specifically identifying these leukocyte subtypes to rigorously assess neutrophil counts.Studies utilizing anti-CD66b antibodies for the analysis of tumor-infiltrating neutrophils have been documented, and comparing our findings with these studies is essential.Furthermore, the CD8-positive cells studied in this study represent a fraction of lymphocytes and do not capture the same aspects as the NLR, which is calculated based on total lymphocyte count.Therefore, it is crucial to explore the optimal combination of leukocyte fractions that accurately represent the immunological landscape of tumor tissue.

| CONCLUSION
In conclusion, our findings suggest that the CD15/CD8 ratio is a predictor of cancer recurrence in patients with gastric cancer, and combining the NLR with the CD15/CD8 ratio could more accurately predict cancer recurrence.A rapid evaluation of the immunological landscape using immunostaining of tumor tissues can provide prognostic information in addition to TNM staging and may assist in establishing an appropriate schedule for detecting recurrence and identifying the need for adjuvant therapy.However, these findings should be validated in larger independent populations.

T A B L E 1
Patient demographic and clinical characteristics.

1
Relationship between the number of CD15-positive and CD8-positive cells and prognosis of gastric cancer.(A) Representative immunohistochemical tissue section of intratumoral infiltrating CD15-positive and CD8-positive cells.The image was observed with an optical microscope at 400Â.(B) Kaplan-Meier curves showing RFS and OS for patients with gastric cancer based on CD15-positive cells (n = 82).(C) Kaplan-Meier curves showing RFS and OS for patients with gastric cancer based on CD8-positive cells (n = 82).CD, cluster of differentiation; OS, overall survival; RFS, relapse-free survival.

3
Comparison of prognosis by CD15/CD8 ratio and NLR combination.(A) Scatter plot of NLR and CD15/CD8 ratio of each patient.Cases with relapse were plotted as white circles, and those without relapse were plotted as black circles.(B) Comparison of prognosis by CD15/CD8 ratio and NLR combined with high and/or low.CD, cluster of differentiation; NLR, neutrophil-lymphocyte ratio; RFS, relapse-free survival.T A B L E 3 Univariate and multivariate Cox proportional hazards regression analysis of clinicopathological factors for relapse-free survival.

F
and Ki6722 have been reported to predict the prognosis of gastric cancer.MSI and PDL-1 expression are considered important indicators for predicting the efficacy of immune checkpoint inhibitors.23This evaluation I G U R E 4 Kaplan-Meier curve showing RFS of patients with stage II-III gastric cancer.(A) RFS based on the CD15/CD8 ratio ( p < .0001).(B) RFS based on the NLR ( p = .241)(n = 28).CD, cluster of differentiation; NLR, neutrophil-lymphocyte ratio; RFS, relapse-free survival.
Intratumoral CD15/CD8 ratio and NLR in association to clinicopathologic factors.
T A B L E 2