Association of neutrophil-to-lymphocyte ratio, neutrophil count, and VEGF on metastasis in patients with nasopharyngeal carcinoma

Systemic inflammatory responses have important roles in the development and progression of cancer, including the propensity for metastasis. A high neutrophil-to-lymphocyte ratio (NLR) is associated with poor prognosis in nasopharyngeal carcinoma (NPC), but the underlying mechanisms are not clear. Vascular endothelial growth factor (VEGF) has been reported to be involved in the recruitment of pro-angiogenic neutrophils in tumor metastasis progress. Therefore, we focused on the correlations among NLR, neutrophil count, and VEGF during metastasis and explored the reasons for changes of NLR in advanced NPC. carcinoma.


Introduction
The occurrence of nasopharyngeal carcinoma (NPC) is rare worldwide but has reached epidemic proportions in Southeast Asia. In southern China, the annual incidence of NPC is 15 − 30 cases per 100,000 people [1]. The treatment strategy for NPC is mainly based on the tumor-node-metastasis (TNM) staging system. Radiotherapy alone is generally effective for early stage NPC, while chemoradiotherapy remains the standard treatment for patients with advanced disease [2]. However, even after definitive chemoradiotherapy, 15%−30% of patients eventually develop distant metastases (DM), which is the major cause of death among patients with NPC [3,4].
Previous work has demonstrated that a high neutrophil-to-lymphocyte ratio (NLR) is associated with DM and poor treatment outcomes in patients with NPC [5 − 9], which suggests that NLR may represent an easily measurable and inexpensive prognostic marker in NPC. However, the underlying changes in cell counts that drive NLR alterations remain unclear.
NLR is the ratio of two sub-types of white blood cells: neutrophils and lymphocytes. Recent studies suggest that these inflammatory makers have a critical role in the immune system and the inflammatory response during various stages of carcinogenesis including initiation, invasion, progression, and metastasis [10][11][12]. Neutrophils initiate interactions between cancer cells and endothelial cells in the vicinity of the primary tumor microenvironment to expedite metastasis via the release of growth factors such as vascular endothelial growth factor (VEGF) and proteases [13]. Lymphocytes act to prevent tumor cell proliferation and migration [14]. However, whether alterations in these cell counts result in elevated NLR during NPC progress is controversial [15,16]. Here, we revealed that neutrophils have a crucial function in the metastasis of NPC.
VEGF is an important prognostic marker and therapeutic target in NPC [17] and is produced by tumors is known to recruit pro-angiogenic neutrophils, which are associated with the development of DM [18,19]. We hypothesized that there is a link between these clinical variables and the levels of relevant cytokines in NPC progression. In this study, we focused on the relationship between NLR and immune cells in metastatic progress. We also explored the mechanisms underlying changes in NLR in metastatic NPC.

Patients
The study protocol was approved by the ethics committee of Fujian Cancer Hospital, and informed consent was obtained from all patients. We reviewed the records of 834 patients with NPC who were irradiated consecutively with IMRT at our hospital between November 2011 and December 2012. Pretreatment evaluations included complete medical history, physical examination, nasopharyngoscopy examination, dental evaluation, standard laboratory tests, chest radiography or computed tomography, bone scan, and abdominal ultrasound. None of the patients had received any cancer-specific treatment at the time of data collection. Patients with evidence of infection, hematological disease, renal dysfunction, or hyperpyrexia were excluded from the study. A diagnosis of NPC was made via endoscopy-guided biopsy. Disease re-staging was defined according to the criteria of the 8th edition of the American Joint Committee on Cancer staging system.
Laboratory data, including total white blood cell (WBC), neutrophil, lymphocyte and platelet counts, NLR and PLR ratios (based on counts per µL of whole blood), hemoglobin (Hb) level, and red cell width distribution (RDW) were measured as part of the automated complete blood count on a Sysmex XE-2100 analyzer (Sysmex Corporation, Japan).

Statistical analysis
Receiver operating characteristic (ROC) curves were constructed to select cut-off values for the stratification of patients based on pretreatment laboratory parameters. Data are expressed as the median and interquartile range (IQR) or mean and 95% confidence interval (CI), as appropriate. Laboratory parameters in patient and control groups were compared with Student's t-tests for variables with normal distribution or with the Mann-Whitney U-test for abnormally distributed variables. All DM-free survival curves were analyzed only for M0 stage patients and constructed using the Kaplan-Meier method, then analyzed with the log-rank test and multivariate Cox-regression analysis. The associations between laboratory parameters and tumor stage were assessed using the Kruskal-Wallis test. Univariate logistic regression was used as a statistical model to show the probability of NLR versus neutrophil counts values. Statistical significance was defined as P < 0.05 (two-tailed). All analyses were performed using Prism 5 (GraphPad Software, La Jolla, CA, USA) and SPSS version 17 (SPSS, Chicago, IL, USA) software. Table 1 depicts the basic characteristics of all patients included in the study. To assess the association with DMfree survival, we categorized the M0 stage NPC patients (785) into groups using NLR, PLR, and WBC cut-off values of 2.26, 87.6, and 6.9, respectively, based on the results of ROC curve analyses (data not shown). Patients with high baseline NLR (≥ 2.26) had worse DM-free survival than patients with low baseline NLR (< 2.26; P = 0.034, HR: 1.5, 95%CI: 1.1-1.9; Fig. 1A). However, no significant association between DM-free survival and PLR or WBC was detected ( Fig. 1B and C).  Fig. 2). Kruskal-Wallis analysis revealed that NLR and neutrophil counts were significantly associated with increasing N stage (P < 0.0001 and P = 0.0074, respectively) and significantly higher in the M1 compared with the M0 subgroup (P = 0.0034 and P = 0.0064, respectively; Fig. 2A and B). Both had no significant association with T stage (P = 0.7049 and P = 0.2133, respectively; Table 2).

Correlation Between NLR, Neutrophil Count Levels, And Metastasis
Univariate logistic regression showed the baseline NLR levels were significantly associated with neutrophil count (P < 0.0001) (Fig. 3A and B), but not lymphocyte count ( Table 2). In other words, higher NLR was associated with higher neutrophil counts. Furthermore, N3 stage patients (Fig. 3A) and DM patients (Fig. 3B) in NPC showed a positive correlation with elevated NLR, which was dominated by neutrophil counts. Figure 4A shows that DM-free survival worsened significantly if VEGF expression was strongly positive (+++; P = 0.0023, HR: 3.66, 95%CI: 1.82-15.79). Kruskal-Wallis analysis revealed that neutrophil count was significantly associated with VEGF expression (P = 0.0024, Fig. 4B), which suggests that tumor VEGF production may contribute to the increase in neutrophil count.

Discussion
Elevated NLR has previously been reported to be a risk factor for DM and an indicator of poor prognosis in NPC patients [5 − 9], although the reasons for this are obscure. In this retrospective study, we found that increases in NLR and neutrophil count were significantly associated with lymph node involvement and DM. For the first time, we found that in the process of NPC metastasis, changes in NLR were driven by an increase in neutrophil count, which correlated significantly with VEGF expression.
Among the parameters examined here, NLR and PLR showed an association with advanced lymph node involvement (N3) ( Fig. 2A and Table 2), but only NLR was significantly associated with DM and DM-free survival. This finding is consistent with others reported in similar studies of head and neck cancer, which showed that PLR was not significantly associated with survival [21 − 23]. However, increasing evidence suggests that PLR is a useful prognostic factor for lung and colon cancer [24,25]. This apparent discrepancy may be caused by tumor heterogeneity, among other factors. Taken together, our results implicate a neutrophil-dependent inflammatory response in the development of NPC metastasis.
Neutrophils actively participate in various steps of the metastatic cascade [26 − 29]. Our finding here that neutrophil counts increase with advancing NPC is consistent with previous studies showing that myeloid cells facilitate tumor advancement by promoting an inflammatory tumor microenvironment, which is required for epithelial-mesenchymal transition, intravasation, and tumor metastasis [30]. The previously published finding that neutrophils release growth factors such as VEGF and specific proteases [31,32] supports our finding that VEGF expression was positively associated with neutrophil count in NPC tissue.
We found that NPC patients had significantly lower lymphocyte counts than healthy controls (Table 3); however, there was no obvious association of lymphocyte count with either advancing disease stage or the development of metastasis (Table 2). Lymphocytes play an essential role in the anti-tumor immune response, impeding tumor cell proliferation and migration and inducing cell death [33]. Thus, although lymphocytes affect tumor growth, they likely have little influence on metastasis in NPC.

Comparison of laboratory parameters in NPC patients and healthy control subjects
VEGF is a tumor-produced growth factor that contributes to tumor angiogenesis and metastasis [34 − 38], and high VEGF expression has previously been associated with poor prognosis in patients with NPC [39]. The significant correlation between VEGF and neutrophil count demonstrated here may provide the immunological basis for the clinical observation that a very high NLR is correlated with poor survival [5 − 9]. The association identified in the present study suggests that these parameters could be markers for the precise curative effect of the anti-VEGF monoclonal antibody bevacizumab, which is currently used to treat NPC [40].
There are several limitations to this study. First, it was retrospective. Second, the number of patients with stage I/II NPC was low (7 and 49 patients, respectively), which may have reduced the statistical power for the subgroup analysis. Thus, our observations must be validated in a large-scale prospective study. Moreover, further work is required to detect the mechanism for increased NLR in NPC metastasis.

Conclusions
These findings indicate that there is a close interaction between NLR, neutrophil count, and VEGF in NPC-related metastasis. Among all the factors investigated in this study, a stage-dependent increase in neutrophil count, which correlated positively with tumor VEGF expression, contributed most strongly to changes in NLR in the progression of locally advanced and metastatic NPC.