Bladder cancer is the seventh most common type of cancer among men, and by documenting both sexes, it ranks eleventh in frequency. The incidence is 9.0 per 100,000 people annually in men and 2.2 in women world wide with an incidence rate higher in the European Union, being 19.1 for men and 4.0 among women in 2012 [1, 2]. The global mortality rate per 100,000 people per year is 3.2 for men and 0.9 for women. The incidence and mortality of bladder cancer are influenced by differences in risk factors, methods of diagnosis, and availability of treatment in different countries [3, 4].

Most patients, approximately 75% with bladder tumors present with disease limited to the mucosa (stage Ta, CIS) or submucosa (stage T1). In patients younger than 40 years this percentage is higher. Patients with Ta, T1, or CIS in many cases have a higher prevalence of long-term survival and a lower risk of tumor-specific mortality compared to those of stages T2-4 [5, 6].

The prompt detection of HG/G3 non-muscle-invasive recurrence is crucial and the percentage of tumours missed should be kept as low as possible because a delay in diagnosis and therapy can be life-threatening. A significant variability is observed among pathologists for the diagnosis of CIS; stage T1 vs. Ta tumors and tumor grading in both the 1973 and 2004/2016 classifications. The WHO 2004/2016 classification provides better reproducibility than the 1973 classification [7, 8].

Local inflammation is a factor that contributes substantially to the development and progression of malignancy, the local immune response and systemic inflammation having an essential role in this regard [9, 10].

Bladder cancer is closely related to chronic or recurrent local inflammation and an increased number of inflammatory cells at the tumor site. The number of T lymphocytes and natural killer cells is significantly lower in patients with infiltrative bladder tumors, compared to those presenting with superficial urothelial tumors. An increased value of preoperative NLR is related to poorer prognosis. Moreover, some studies have shown that a high value for NLR is associated with a higher recurrence rate and lower overall survival [11, 12].

Since the peritumoral inflammatory reaction is of substantial importance in the oncological prognosis of bladder cancer, theefore, the neutrophil-to-lymphocyte (NLR) ratio may be an extremely practical and appropriate biomarker in non-muscle invasive bladder tumors [13, 14].

The study aims to analyze parameters of non-muscle invasive bladder tumors and to identify and validate potential tumor markers of prognostic value in this condition.

A retrospective study is described, involving 463 patients diagnosed with non-muscle invasive urothelial tumors hospitalized at the Urology Clinic in Târgu Mureș, between January 2016 and June 2020, after transurethral resection of bladder tumors (TURBT). The study was conducted in line with the World Medical Association Declaration of Helsinki and was approved by the Ethical Committee of the Mures County Hospital. The inclusion criteria were: non-muscle invasive tumors (Ta, T1, CIS), neutrophil-to-lymphocyte ratio (NLR) determination 2 weeks prior to TURBT and complete follow-up of patients. Both multi-tumors, and unifocal tumors were included. Exclusin criteria: T2 or higher stage tumors and failure to follow-up. Patients with comorbidities associated with elevated inflammatory markers were also excluded from the study. NLR cut-off was set at 3 according to other studies in the literature.

The data collection was performed with the help of the H3 Concept computer system. The data processed included demographic data, such as sex and age, degree of tumor invasion, degree of tumor differentiation and recurrence. Laboratory tests included NLR, tumor size, number of tumors, and overall survival, all centralized in a table in Microsoft Excel program.

Statistical data analysis was performed using the IBM SPSS Statistic Subscription program (version 24.0, IBM Corp., Chicago, IL, USA). Data were considered as significantly different if in the confidence interval set the value p 0.05. The tests used in the statistical analysis process are Chi-square and bivariate correlation.

A total of 371 people diagnosed with non-muscle invasive bladder tumors met the inclusion criteria. The mean age of the included patients was 69.96 years (IQR 35–93). 297 (80.05%) were male and 74 (19.95%) were female. 64.95% pTa tumors, 31.26% pT1, 1.07% pTa + CIS, and 2.69% pT1 + CIS, respectively, were identified.

Distribution according to Grade: 196 (52.8%) G2, 159 (42.88%) G3 and 16 (4.32%) CIS. 284 (76.5%) patients presented with a single tumor and 87 (23.35%) with multiple tumors; 295 (79.5%) had tumors larger than 3 cm and 76 (20.5%) were smaller than 3 cm. 228 (61.4%) presented with recurrence after 3 months post-surgery. Overall survival at 5 years was 80.06% (Table 1).

Analyzing the overall survival, there is a positive relationship between advanced age at the time of diagnosis of the patient and low survival rate, p = 0.004. The mortality rate was higher among men (20.54%) than among women (17.58%). From a statistical point of view, there was no positive relationship between sex and overall survival, p = 0.567.

To study the ratio between neutrophils and lymphocytes (NLR), we divided the patients into two groups: Patients with an NLR less than 3 (low-risk) and NLR greater than 3 (high-risk), respectively.

From a statistical point of view, there was no positive relationship between values higher than 3 for NLR and distribution by gender, p = 0.235 or values lower than 3 for NLR, p = 0.219, respectively.

The average ratio between neutrophils and lymphocytes was higher in the group of deceased people (5.45), compared to those alive (4.04), at the end of the data collection period, with a p value of 0.754 (Fig. 1).

Fig. 1.

Mean NLR ratio for deceased and survivors during the data collection period.

Statistically, there was a positive relationship between the increased ratio of neutrophils and lymphocytes in people diagnosed with non-muscle invasive bladder cancer and increased mortality rate, p = 0.003.

This study aimed to identify prognostic factors for non-muscule invasive bladder tumors, that were significant in terms of overall survival, especially the ratio between neutrophils and lymphocytes and concordance of tumor growth with disease progression. From the point of view of overall survival, the data analyzed show that age at diagnosis is an unfavorable factor (p = 0.004).

There was a positive relationship between the increased mortality rate and an increased NLR, so patients with high NLR had a lower overall survival rate.

The significance of the peritumoral inflammatory response in bladder cancer is well-documented. An increased value of preoperative NLR is related to a poorer prognosis. Moreover, some studies have shown that the high value of NLR is associated with a higher recurrence rate and lower overall survival. Our data are in agreement with those published in the literature proving a positive relationship between the increased ratio of neutrophils and lymphocytes in people diagnosed with non-muscle invasive bladder cancer and increased mortality rate, p = 0.003 [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18].

The study, published by Favilla et al. [19], described biomarkers in non-muscle invasive bladder cancers, including the ratio of neutrophils to lymphocytes. This study included 178 patients, of whom 148 were men and 30 women. The mean age was 69.27 years and the mean monitoring period was 53 months. Patients with NLR $>$3 were older and for who a higher rate of recurrence and progression was observed compared to the group of patients with NLR 3. Over a 5-year period, those with NLR 3 had a higher survival rate, 62%, compared to those with an NLR $>$3, where the survival rate was 49%. Thus, it was concluded that with the help of a inexpensive hematological test, calculating the ratio between neutrophils and lymphocytes, the rate of recurrence and progression of the disease could be estimated [19].

Data from more than 20,000 patients from the Netherlands Cancer Registry revealed statistically significant sex-related differences in stage distribution (Ta vs. T1) at presentation for non-muscle invasive bladder cancer, with women presenting more often with T1 disease [20]. The gender gap in stage at presentation and outcome could be due to biologic differences as well as a diagnostic delay in women. Indeed, there is data reporting no differences in clinical symptoms between the sexes, while the primary diagnostic approach as shown in numerous studies differs. It was reported that a gender gap in the evaluation of hematuria exists. Female gender was associated with a higher risk of receiving symptomatic treatment for hematuria, without further evaluation [21, 22].

Cantiello et al. [23] found that a single score based on NLR, PLR (platelet to lymphocyte ratio), and LMR (lymphocyte to monocyte ratio) cutoffs, denoted as SIM (systemic inflammatory markers), can predict recurrence and progression in this patient population. A higher SIM score is related to worse outcomes, even after adjusting for pathological variables such as tumor size, CIS, and multifocality. Furthermore, inclusion of this score in clinical decision analysis seems to be of clinical benefit over individual components in the predictive model [23].

Ferro et al. [24] concluded in a retrospective study involving 1045 patients with primary T1 HG/G3, that baseline basophil counts may predict recurrence in BCG-treated HG/G3 T1 bladder cancer patients.

In the era of numerous genetic prognostic factors in bladder cancer [25, 26, 27, 28], there is an urgent need for easily accessible, affordable, and widely interpretable biomarkers for current practice. The Neutrophil-to-lymphocyte Ratio proves to have all these benefits [29, 30, 31].

The overall survival of patients with non-muscle invasive bladder cancer is negatively influenced by the advanced age of patients at the time of diagnosis.

The increased neutrophil-to-lymphocyte ratio (NLR) preoperatively is associated with a higher mortality rate in patients with non-muscle invasive bladder cancers.

NLR is an accessible, easy to determineand useful biomarker in prognosis for non-muscle invasive bladder tumors.

PHD, MO and BD—designed the research study. BD, RT, BBN, VAO—performed the research. PHD and BD—wrote the article. RT and BBN—analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee Nr 9573/05.07.2021.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.