Persistent elevation of postoperative neutrophil-to-lymphocyte ratio: A better predictor of survival in gastric cancer than elevated preoperative neutrophil-to-lymphocyte ratio

Postoperative neutrophil-to-lymphocyte ratio change (NLRc) reflects the dynamic change of balance between host inflammatory response and immune response after treatment. In gastric cancer, an elevated initial NLR (iNLR) is reported to be a prognostic predictor, but the clinical application of the NLRc remains unclear. The NLRc was assessed in 734 patients undergoing total/subtotal gastrectomy and endoscopic submucosal dissection for gastric adenocarcinoma. The iNLR and NLRc were recorded within 10 days of the first diagnosis and 3–6 months after surgery, respectively. Using receiver operating characteristic (ROC) curves, we investigated the relationship between NLRc or iNLR and patient survival. The analysis revealed a higher predictive power for correlating patient survival with the NLRc compared with iNLR. NLRc was defined as negative (lower than iNLR) and positive (higher than iNLR). A positive NLRc was frequently observed in patients with advanced AJCC stage, local recurrence, distant metastasis, perineural invasion, and adjuvant chemotherapy (all p < 0.05). Univariate and multivariate analyses revealed a significant relationship between patient survival and NLRc (all p < 0.05) but no association between survival and iNLR. The NLRc could be a better indicator than iNLR for predicting survival in patients with gastric cancer.

are expensive and often time-consuming to measure. When there are sufficient technologists with an appropriate laboratory setting, genetic analyses to determine the new subtypes can be easily used in clinical application.
Systemic inflammation is associated with the progression of various cancers by induction of angiogenesis, metastasis and malignant cell proliferation, and alteration of the response to systemic therapy 7 . Neutrophils are known to be key mediators of tumour inflammation 8 and angiogenesis 9 . Lymphocytes have been suggested to play vital roles in cytotoxic tumour cell death linked to tumour-infiltrating lymphocytes, especially T lymphocytes 10 . The interaction between the cancer and immune system might also extend beyond the local tissue environment. Other systemic inflammatory markers such as C-reactive protein and interleukin-6, have been associated with worse survival in patients with gastric cancer 11 . The imbalance between neutrophils and lymphocytes is thought to be secondary to tumour hypoxia or necrosis and associated with anti-apoptotic effects. Published data demonstrated that the initial neutrophil-to-lymphocyte ratio (iNLR) at that time of first diagnosis is linked to prognosis of different types of cancer [12][13][14][15] as well as of other conditions such as cardiovascular disease 16 and bacterial infection 17 . Accordingly, the iNLR might represent the balance between pro-tumour inflammatory status and anti-tumour immune status.
Several studies focused primarily on iNLR, while the dynamic change of neutrophil-to-lymphocyte ratio after treatment was not considered. The postoperative neutrophil-to-lymphocyte ratio change (NLRc) might be a meaningful factor to assess prognosis after treatment, because there has been a change in the treatment approach including tumour removal and chemotherapy. However, although the NLRc might dynamically reflect the alteration of balance between host inflammatory response and immune response against cancer after treatment, its significance is largely unclear.
The aim of this study was to investigate the relationships between iNLR and NLRc and clinicopathological parameters, and to evaluate the prognostic value of the NLRc in patients with gastric cancer.

Results
Patient characteristics. The median age of the 734 patients was 66 (range: 23-87) years. The following clinicopathological variables were recorded: Sex; age; 8 th AJCC stage; location; local recurrence; distant metastasis; size; Lauren type; histological grade; lymphatic, vascular, and perineural invasion; presence of adjuvant chemotherapy; margin involvement, and patient survival. EGC was present in 395 patients and advanced gastric cancer (AGC) in 339. In early gastric cancer (EGC), the predominant gross type was classified as protruding (type I) in 21 patients, slightly elevated (type IIa) in 28, flat (type IIb) in 113, slightly depressed (type IIb) in 196, and excavated (type III) in 37. In AGC, the predominant gross type was classified as fungating (Borrmann type 1) in 11 patients, ulcerative (Borrmann type 2) in 66, ulcero-infiltrative (Borrmann type 3) in 221, and linitis plastica (Borrmann type 4) in 41. According to the Lauren classification, 396 patients had intestinal type, followed by 217 with a diffuse type and 121 with a mixed type. The surgical procedures included subtotal gastrectomy in 567, total gastrectomy in 106, endoscopic submucosal dissection in 54, and endoscopic mucosal resection in seven. Two hundred thirty-one patients received adjuvant chemotherapy such as tegafur-uracil. After surgery, 142 patients developed local recurrence and/or new distant metastases, and 172 patients died during the median follow-up period of 49.41 months.
The NLRc was defined as negative (NLRc ≤ 0) when the postoperative NLR was lower than the preoperative NLR. The NLRc was defined as positive (NLRc > 0) when the postoperative NLR was higher than the preoperative NLR.

Discussion
The systemic inflammatory reaction in the tumour microenvironment is essential for cancer growth and development. The concept of inflammation-based scores has been applied in many types of cancer. Neutrophils are thought to play a key role in normal physiological angiogenesis and tumour angiogenesis 20,20 . Activated neutrophils can release matrix metalloproteinases (MMPs), in particular MMP-9, which activate potent angiogenic factors (vascular endothelial growth factor, fibroblast growth factor-2) 21,22 . The increase in neutrophil count leads    survival (a,b,c,d) and overall survival (e,f,g,h) showing the association between the postoperative neutrophil-to-lymphocyte ratio change (NLRc) according to the histological grade, which includes well, moderately, poorly differentiated and signet ring cell type (all p < 0.05).
to induction of tumour progression and development of metastases via secretion of cytokines (tumor necrosis factor, IL-1, IL-6) 23,24 . In contrast, lymphocytes are a major factor for the suppression of cancer progression. Cytotoxic lymphocytes, which are ultimately responsible for killing tumour cells and eradicating the tumour, are applicable to cancer immunotherapy 25 . Based on the opposite functions of neutrophils and lymphocytes, the NLR might provide improved prognostic information regarding cancer progression.
The NLRc is a readily available and inexpensive biomarker for differential types of cancer. Nevertheless, the precise mechanisms by which the relationship between neutrophils and lymphocytes might predict clinical outcomes are not fully understood. A study by El-Hag et al. demonstrated that neutrophils lead to suppression of the cytolytic activity of immune cells such as lymphocytes, natural killer cells, and activated T cells 26 . In our results, a negative correlation between neutrophils and lymphocytes supports the above concept. Other published data have described several mechanisms in detail as follows: First, the anticancer responses of natural killer cells and activated T cells might be suppressed by marked neutrophil infiltration around the tumour 27 . Therefore, a high NLR might decrease the effects of the lymphocyte-mediated cellular immune response and promote cancer progression. Second, circulating neutrophils contribute to tumour growth and progression by producing cytokines. Considering the complexity and heterogeneity of cancer, the neutrophil count alone might not reflect a decreased lymphocyte-mediated immune response, and a low lymphocyte count alone might not reflect the neutrophil-driven tumour growth process. In addition, a study by Kobayashi et al. suggested that the NLR represents the relative extent of inflammation and host immunity against cancer, as its value is directly affected by the total count of neutrophils and lymphocytes. The combined effects of neutrophilia and lymphocytopenia might be better than the effect of a single marker for predicting clinical outcome.
In gastric cancer, a previous study reported the role of the iNLR without evaluation of the NLRc 14,18,19 . These studies focused only on the iNLR, not on the dynamic changes in the NLR after treatment. In different types of cancer, few studies have evaluated dynamic changes in the NLR after treatment. In studies of renal cell carcinoma, a negative NLRc was associated with favourable outcomes 28 . Another study demonstrated that the NLRc is related to clinicopathological parameters such as recurrence, tumour size, and stage 29 . In studies of non-small cell lung cancer and urothelial carcinoma, the NLRc had a greater statistical power than the iNLR in evaluating patient survival 30,31 . A positive NLRc indicates that the microenvironment supporting cancer growth is persistent, despite removing the causal risk factor related to inflammation after cancer treatment. Therefore, the NLRc  after treatment is more meaningful than the iNLR at the time of the first diagnosis, because the NLRc reflects the dynamic change of the immune response after treatment. In our study, a positive NLRc had more statistical power than iNLR in predicting DFS and OS. In summary, the NLRc is statistically associated with increased mortality rates in patients with gastric cancer and has better statistical power than the iNLR. Compared to molecular markers, the NLRc seems to be a convenient, easily obtainable and repeatable, low cost, and reliable predictor for patients with gastric cancer. Practically, this concept of the NLRc could provide clear, concise, and easily applicable information for evaluating prognosis in gastric cancer, which comprises a heterogeneous cancer cell population. Larger scale studies are required to better assess the relationships between the NLRc and prognosis of other types of cancer.

Materials and Methods
Patient selection. This retrospective study included 734 patients diagnosed with gastric adenocarcinoma at two hospitals between 2000 and 2013. The Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria were followed throughout this study. The inclusion criteria were: 1) patients with histopathological evidence of primary adenocarcinoma confirmed by pathologists, and known clinical outcome; and 2) patients with postoperative follow-up blood samples, which were collected in ethylenediaminetetraacetic acid-containing tubes according to other published studies 32 . The exclusion criteria were: patients diagnosed with adenocarcinoma but with inadequate clinical history and/or no available microscopic slides.
Interpretation of the neutrophil-to-lymphocyte ratio. Total white blood cells (WBCs), neutrophils, and lymphocytes were obtained by complete blood counts before surgery. A follow-up blood sample was obtained at 3-6 months after surgery.
The absolute neutrophil and lymphocyte counts were calculated by multiplying the percentage of each component by number of WBCs. The neutrophil-to-lymphocyte ratio was determined from the differential count by dividing the absolute neutrophil count by the absolute lymphocyte count. To evaluate the NLRc, we subtracted the postoperative NLR from the preoperative NLR: postoperative NLR -preoperative NLR = NLR change.
Statistical analysis. The associations between clinicopathological parameters and the NLRc were analysed by the chi-square test. The DFS time was defined as the time from the date of diagnosis to the date of local recurrence or new distant metastasis. The OS time was defined as the time from the date of diagnosis to all-cause death. Survival curves were generated using the Kaplan-Meier method and were compared by the log rank test. A Cox regression model was used for multivariate analysis. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS statistics (version 20.0, Chicago, IL, USA) and R packages (http:// www.r-project.org/).

Ethics approval.
This study (involving human participants) was approved by the Ethics Committee of the Eulji Hospital (EuljiIRB 15-62), and performed with respect to the ethical standards of the Declaration of Helsinki, as revised in 2008. The IRB review confirmed that the informed consent is not necessary in this study.