Clinical Significance of Pre-treated Neutrophil-Lymphocyte Ratio in the Management of Urothelial Carcinoma: A Systemic Review and Meta-Analysis

Purpose: We performed a study-level meta-analysis to summarize the current evidence on the correlation between pretreatment neutrophil-to-lymphocyte ratios (NLR) and oncological outcomes in each type of management for urothelial carcinoma. Method: All articles published until February 2017 in PubMed, Scopus, and EMBASE database were collected and reviewed. The current evidence on correlations between pretreatment NLR and oncological outcomes in each type of management for urothelial carcinoma, including transurethral resection of bladder tumor (TURBT), radical cystectomy (RCx), chemotherapy (CTx), and nephroureterectomy (NUx), were summarized. Results: Thirty-eight studies containing clinical information on 16,379 patients were analyzed in this study. Pooled hazard ratios (HR) and odds ratios (OR) with 95% confidence intervals were calculated after weighing each study. Heterogeneity among the studies and publication bias were assessed. Pretreatment NLR was significantly associated with muscle invasiveness (OR: 4.27), recurrence free survival (RFS, HR: 2.32), and progression-free survival (PFS, HR: 2.45) in TURBT patients. In the RCx patients, high NLR was negatively associated with both disease status (extravesical extension and lymph-node positivity, OR: 1.14 and 1.43, respectively) and oncological outcomes [overall survival (OS), PFS], and cancer specific survival (CSS, HR: 1.18, 1.12, and 1.35, respectively). Pretreatment NLR was negatively correlated with pathologic downstaging (OR: 0.79) and positively correlated with PFS (HR: 1.30) and OS (HR: 1.44) in CTx patients. For patients who underwent NUx, pretreatment NLR was significantly associated with OS (HR: 1.72), PFS (HR: 1.63), and CSS (HR: 1.68). Conclusions: Pretreatment NLR is a useful biomarker for disease aggressiveness, oncological outcome, and treatment response in the management of patients with urothelial carcinoma. More evidence is needed to clarify these results.


INTRODUCTION
Urothelial carcinoma of the bladder is the third most common and the eighth most lethal malignancy in the United States (1), showing a high incidence in developed countries (2). This malignancy originates from normal urothelial cells and can occur in any part of the urinary tract, including the renal pelvis, ureter, bladder, and urethra. The majority of urothelial carcinomas is present in the lower urinary tract, mostly in the bladder. Muscle invasiveness is the most important parameter in the management of bladder urothelial carcinoma. Non-muscle invasive bladder cancer (NMIBC) can be managed by transurethral resection of bladder tumors (TURBT). Muscle-invasive bladder cancer (MIBC) is treated with radical surgery or systemic treatment. The standard treatment for upper urinary tract urothelial carcinoma (UTUC) is radical nephroureterectomy with bladder cuffing. Since urothelial carcinoma shows a variety of clinical manifestations, selecting the proper treatment is one of the most critical issues in the management of patients with urothelial carcinoma.
Recent studies have revealed that the inflammatory response plays an essential role in tumor development, progression, and prognosis (3). Elevation of C-reactive protein (CRP), the presence of some cytokines, and changes in the proportion of white blood cells in the peripheral blood are common findings reflecting the systemic inflammatory response. Among these changes, the peripheral neutrophil-to-lymphocyte ratio (NLR) is one of the most widely studied prognostic biomarkers in many solid tumors because of its easy calculation and costeffectiveness (4). High NLR tends to be negatively correlated with poor survival in urothelial carcinoma (5). Some studies have suggested a link between high NLR with the pathologic stage of urothelial carcinoma, including muscle invasiveness (6,7), extravesical extension (8)(9)(10), and lymph-node positivity (10,11). Moreover, recent research has indicated that NLR is a predictive biomarker of treatment response (12). However, one large-scale study did not find a correlation between high preoperative NLRs and extra-vesical extension (11). Thus, the clinical utility of preoperative NLR as a prognostic or predictive biomarker in urothelial carcinoma is still controversial.
Therefore, the objective of this study was to determine the correlation between pretreatment NLRs and oncological outcomes in each type of management for urothelial carcinoma, including TURBT, radical cystectomy (RCx), chemotherapy (CTx), and nephroureterectomy (NUx), through a meta-analysis of published studies.

Search Strategy
This study was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The PubMed, SCOPUS, and EMBASE databases were searched to collect suitable literature published up to February 2017. The search used a combination of "Neutrophil; Lymphocyte; Bladder; Cancer" and "Neutrophil; Lymphocyte; Urothelial; and Cancer" terms. To identify additional shrouded studies, we carefully examined the references of each article.

Inclusion and Exclusion Criteria
Two reviewers (CWJ and CK) screened all titles and abstracts of the initially searched articles. After screening, the full-text articles were separately evaluated by two different reviewers (HHK and JHK) to determine study eligibility. Disagreement on study eligibility was resolved through discussion. The inclusion criteria for eligibility were that the report: (1) investigated urothelial carcinoma, (2) had patient neutrophil-to-lymphocyte ratios, (3) evaluated the relationship between pathologic features and prognosis, and (4) had enough information to calculate odds ratios (OR), or hazard ratios (HR) with 95% confidence intervals (CI). Studies not written in English, case reports, editorial letters or reviews, and those not performed in humans were excluded. If the investigations were conducted on similar patients by the same research group, only the largest and newest article was included in the systematic review.

Data Extraction and Handling
Two reviewers (JHJ and JS) independently extracted information from the selected studies. Data tables were constructed to record all associated data from the texts, tables, and figures of each study. The study information (name of authors, publication year, region), the number of patients, follow-up information, and disease status were obtained. After completing the data tables for each study, both reviewers compared their results and arrived at a consensus for any differences.

Statistical Analysis
The DerSimonian and Laird random-effects model (13) was selected to weigh each study for the meta-analysis. Odds ratios and 95% CIs were used to assess the relationship between NLR and disease status for each specific situation, including muscle invasion in TUR, extravesical extension, or lymph node positivity in radical cystectomy, and pathologic downstaging after neoadjuvant chemotherapy. Hazard ratios and 95% CI were used to estimate the effects of NLR on survival. The heterogeneity of the studies was assessed by Chi-squared tests. Statistical significance was considered for p-values of <0.05. The I 2 statistic was also calculated to determine the heterogeneity of the studies. I 2 values larger than 75%, <25%, and between 25 and 75% indicated a high risk of heterogeneity among studies, no heterogeneity, and moderate heterogeneity among studies, respectively.
Publication bias was assessed by funnel plots, rank correlation analysis (Begg test), and linear regression analysis (Egger test). In the funnel plots, a symmetric, inverted funnel shape indicated no publication bias. A p-value < 0.05 by Begg and Egger's test was considered publication bias (Supplementary Data 1). The pooled ORs and HRs of the meta-analyses were calculated using RevMan 5.0 (the Cochrane Collaboration, Copenhagen) software. All statistical analyses were performed with R program 3.5.0 (R Development Core Team, Vienna, http://www.R-project.org).
Data quality and the risk of bias assessment were performed by three investigators (JHK, JS, and JHJ). Each reviewer independently read the published articles and performed a quality assessment based the Newcastle-Ottawa Scale (NOS) (14). The NOS assesses the methodologic quality of each study in three domains: selection of the study groups, comparability of the groups, and ascertainment of exposure and outcome. The risk of bias was stratified on three levels. Quality scores of NOS of >7, 4 to 6, and< 4 indicated high quality, moderate quality, and low-quality studies, respectively. The methodologic quality scores of all included studies are shown in Figure 1. Specific quality assessment scores and data are shown in Supplementary Datas 2, 3.

Study Characteristics
The characteristics of the selected 38 studies are summarized in Tables 1-4. Seven articles evaluated the clinical significance of NLR in TURBT patients. Only two studies were eligible for assessing the link between NLR and invasiveness in TURBT patients (6,7). One study performed in Italy was prospectively designed (16). Others were retrospective studies. The majority of those studies were conducted in Turkey (7,17,19). However, a study from Japan had the largest study population (15). The cutoff value for NLR varied from 2.2 to 3.89 depending on the study ( Table 1). Fifteen articles were eligible for a meta-analysis of radical cystectomies ( Table 2). Only extravesical extension, lymph node positivity, and downstaging after NAC had sufficient information to evaluate the correlation between preoperative NLR and disease status. Regarding the parameters of oncological outcomes, cancer-specific survival (CSS), overall survival, and progression-free survival (PRS) had sufficient data for linkage analysis with preoperative NLRs. Most studies set cutoff values for NLR from 2.3 to 5, however, three studies used it as a continuous variable (12,23,25). The studies were conducted in many countries. Two large multi-center retrospective studies were performed in the USA (10) and Europe (11). Five articles were eligible for correlation analysis of pretreatment NLRs with oncologic outcomes in chemotherapy patients ( Table 3). All studies provided information for overall survival. However, only two studies (31, 32) provided progression-free survival data. European, American, and East Asian (Japan, China) data were included in this meta-analysis. The cutoff values for the NLR ranged from 3 to 5. One study conducted a retrospective analysis from 10 prospective phase II clinical trials (29). Eleven studies were eligible for meta-analysis of NLR association with oncological outcomes in nephroureterectomy patients ( Table 4). Eight studies provided enough information to analyze the relationship between preoperative NLRs and PFS (34, 36, 39-42, 44, 46) and CSS (34, 36, 39-42, 44, 46). Most of those studies were conducted in East Asia. Two studies contained information on Western population patients (34,43). All studies used NLR as a discrete variable, with cutoff values ranging from 2.0 to 3.22.

Correlation of NLR With Clinicopathologic Feature (Invasiveness)
Only two studies reported the correlation between preoperative NLRs and muscle invasiveness. The pooled OR was 4.27 (95% CI: 1.51-27.31) and moderate level of inter-study heterogeneity was present (I 2 = 58%, p = 0.12) (Figure 3A). Publication bias was not assessable owing to the limited number of studies.

Clinical Significance of NLR in Nephroureterectomy Patients
Correlation of NLR With Oncological Outcomes (Progression-Free Survival, Overall Survival, Cancer-Specific Survival) Pooled hazard ratios were calculated for PFS, OS, and CSS in this meta-analysis. A total of eight studies reported the relationship between preoperative NLRs and PFS. Preoperative NLRs were significantly associated with PFS (HR: 1.63, 95% CI: 1.22-2.18) ( Figure 6A). Preoperative NLRs lower than the cut-off value were also associated with better CSS (HR: 1.68, 95% CI: 1.23-2.31) ( Figure 6B). The HR of preoperative NLR for overall survival in nephroureterectomy patients was 1.72 (95% CI: 1.31-2.25) based on the meta-analysis results of three eligible studies ( Figure 6C). Heterogeneity among these enrolled studies was moderately present for CSS and PFS but was not present for OS. There was a risk of publication bias with a possible risk of not reporting negative results.

DISCUSSION
The neutrophil-to-lymphocyte ratio (NLR) is one of the most actively studied biomarkers for predicting disease status in various cancer types (4,47). The clinical evidence for the usefulness of NLR as a biomarker in urothelial carcinoma has been accumulating over the last several years, but the topic is still under debate. We conducted a systematic review and meta-analysis to assess the correlation between pretreatment NLRs with pathologic features and the prognosis of urothelial carcinoma patients. This study collected a total of 38 studies containing clinical information and oncological outcomes on 16,379 patients. To our knowledge, this is the largest and latest meta-analysis evaluating the clinical significance of pretreatment NLRs for each specific urothelial carcinoma management situation.
Our evidence supports pretreatment NLR as a useful biomarker for assessing disease aggressiveness and oncological outcomes in TURBT, radical cystectomy, chemotherapy, and nephroureterectomy patients. High NLR was associated with muscle invasiveness (OR: 4.27), poor RFS (HR: 2.32), and PFS (HR: 2.45) in TURBT patients. The major focus of transurethral management of superficial bladder urothelial carcinoma is controlling recurrence and progression to muscle-invasive disease. If bladder cancer presents with muscle-invasion and a high risk of progression or recurrence, guidelines recommend radical cystectomy (48,49). The TURBT procedure always has the risk of failing to obtain the detrusor muscle in the specimen, which is important for proper staging and disease management (50). For this reason, repeated TURBTs should be considered in high-risk bladder NMIBC. The pretreatment NLR before TURBT could provide additional information for selecting the proper treatment strategies for the management of bladder urothelial carcinoma. High NLR values were associated with a higher chance of extravesical extension (OR: 1.14), lymphnode positivity (OR: 1.43), and worse oncological outcomes. The pooled hazard ratios for PFS, CSS, and overall survival were 1.2, 1.35, 1.18, respectively. Patients with low pretreatment NLRs showed better response rates after NAC followed by radical cystectomy, with a higher chance of pathologic down-staging (OR: 0.79). The oncological outcomes of chemotherapy and nephroureterectomy also revealed negative correlations with pretreatment NLRs. We presumed that the correlation between high pretreatment NLRs and poor outcomes after treatment for urothelial carcinoma was a result of more aggressive presentations, however, further well-designed studies are needed to clarify this hypothesis.
Although results from many studies favor NLR as a useful biomarker under many clinical situations, its predictive mechanism remains unclear. We generally accepted that there is a correlation between inflammation and cancer, however, a causal relationship is ambiguous. Chronic inflammation caused by infection or toxic materials leads to tumorigenesis in many solid tumors. About 20% of tumors are associated with prior viral, microbial, and parasite infections, including infections with the hepatitis virus, human papillomavirus, Helicobacter pylori, and Schistosoma haematobium (51). Additionally, recent studies showed an inverse correlation between non-steroid anti-inflammatory drugs (NSAID) and cancer risk (52). Inflammatory cells are recruited to the tumor microenvironment in the situation of tumor advancement with invasion or the distant migration of tumor cells (53). The cancer-related immune response is paradoxical. Molecular evidence has demonstrated that tumor-infiltrated inflammatory cells eradicate nascent tumors (54). However, some studies have shown that increased systemic inflammation enhanced tumor development, progression, and metastasis (55). Because of the unclear mechanism of the NLR to predict disease prognosis, the clinical utility of the NLR is very limited. Moreover, we have only limited evidence for the association of NLR and prognosis compared to important prognostic factors, such as pathologic stage and surgical margins. Thus, a targeted study evaluating the prognostic impact of NLR after adjusting for covariable factors is needed.
The definition of an optimum NLR value is another problem. In studies included in this meta-analysis, the cutoff value of NLR varied from 2.0 to 5.0 and some studies even used NLR as a continuous variable (12,23,25). In addition, NLR is a dynamic marker of the systemic immune response. Thus, we could not judge the optimal cutoff value easily. NLR is not only related to bladder cancer but also related to many benign and malignant diseases. Therefore, it can be increased without the advancement of tumors. Recently, some studies used a derived NLR introduced by Proctor et al. (56) that makes the clinical utility of NLR more complex.
This systematic review and meta-analysis had some limitations. First, most studies on the relationship of NLR with many clinical parameters and oncological outcomes were retrospectively designed. There was no prospective, randomized controlled study that met our search criteria. Variable differences in study design, patient numbers, the definition of oncological outcomes, and NLR cutoff values also affected the inter-study heterogeneity. Publication bias was another limitation, especially in the sub-group analysis of cystectomy, and nephroureterectomy patients. In addition, we could not include immune-oncological agent targeted studies, which are promising treatments for advanced urothelial carcinoma and thought to be strongly correlated with systemic immune responses. Finally, this meta-analysis used articles written in English only. Thus, we could not exclude language bias in the favorable positive results. Despite these limitations, this study-level meta-analysis provided a generalized view of NLRs on disease aggressiveness, oncological outcomes, and treatment responses in patients with urothelial carcinoma.

CONCLUSION
This study-level meta-analysis showed that pretreatment NLRs were useful biomarkers for disease aggressiveness, oncological outcomes, and treatment responses in the management of urothelial carcinoma. However, inter-study heterogeneity, the possibility of publication bias, the limited number of eligible studies, and no randomized controlled study were limitations of the study. A large, well-designed, prospective study is needed to provide clear evidence that the pretreatment NLR is a useful biomarker for urothelial carcinoma.