Bladder cancer (BC) ranks as the ninth most common cancer worldwide (1) and its incidence has increased over the past few decades. Clinically, histology classifies it into two significant groups: non-muscle-invasive BC and muscle-invasive BC (MIBC) (2,3). While urologists adopt local therapy approaches for non-MIBC, MIBC is managed systemically in medical oncology clinics. MIBC is invasive and can progress to metastatic disease, usually by local invasion of surrounding pelvic structures. Patients with MIBC represent 25% of newly diagnosed cases, and approximately 90% have urothelial cell histology, also known as transitional cell carcinoma (TCC) (4). Their 5-year survival rate ranges from 38% for the population with extension through the bladder to surrounding tissue, or spread to lymph nodes or nearby organs, to 6% for patients with distant metastasis (4).

This makes MIBC, especially stage IV cases, a treatment-resistant disease, and the only benefit of systemic therapy is palliation. Platinum-based therapy constitutes the first line of treatment (5). The most effective platinum agent is believed to be cisplatin and challenging as it is, using cisplatin may not be feasible in all patients due to their poor kidney function and poor performance status (6). These patients routinely receive a carboplatin-based regimen to overcome toxicities associated with cisplatin. However, it has inferior outcomes compared to cisplatin (7). Whether TCC is resistant to therapy or responsive is unclear; some tests, such as circulating tumor DNA, radiological imaging, and inflammatory marker-driven pathogenesis can indicate this. Additionally, inflammatory marker-based assays have been proposed as a predictive tool for identifying these patients (8). Further research has considered cellular inflammatory indices as determinants of systemic response, such as the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR). They have been found to affect tumor recurrence and progression, but the results reported so far are contradictory. While Bambury et al. (9) found a significant correlation between lower NLR and improved overall survival (OS), Kuwada et al. (10) did not demonstrate it to be a significant indicator in patients diagnosed with BC. PLR has been studied as another marker of inflammation. In a meta-analysis, an increased PLR was not found to be a significant indicator for OS [hazard ratio (HR)=1.23, 95% confidence interval (CI)=0.95-1.59, p=0.124] of patients with BC (11).

In this study, we report our results based on our experience with our patients, aiming to provide further insight into the clinical value of the impact of systemic inflammatory markers NLR, PLR and LMR on OS in patients with advanced MIBC receiving first-line chemotherapy.

Patients. The files of patients admitted to the Department of Medical Oncology, Pamukkale University, Turkey, with a diagnosis of MIBC from 2008 until 2020 were retrospectively analyzed. Two hundred and twenty-six consecutive patients with advanced BC treated with chemotherapy with or without definitive/palliative radiotherapy were identified after 56 patients were excluded from the total population. All patients had pathologically confirmed advanced urothelial MIBC. A few cases with squamous and sarcomatous differentiation were also included in our cohort. These patients received first-line standard-of-care treatment with chemotherapy (platin plus gemcitabine, methotrexate with vinblastine, doxorubicin and cisplatin or single-agent gemcitabine) until progression, death, or toxicity. Patients with acute or chronic infection, autoimmune or hematological diseases, patients with chronic liver and renal diseases, patients receiving drugs that had a potential effect on the measured parameters, and patients ineligible for chemotherapy were considered ineligible and excluded from our analyses. According to our institutional guidelines, patients had a complete blood count within a median of 3 days (range=0-7 days) before the initiation of chemotherapy. TNM classification was according to the eighth edition of the American Joint Committee on Cancer (2017) (12), and patients were divided into two risk groups according to the TNM classification: Stage 3 (locally advanced disease) and stage 4 (metastatic disease). Demographics and clinicopathological characteristics were collected from the patient’s electronic records. Institutional Review Board approval was obtained from the local Ethics Committee prior to all studies (Pamukkale University Ethical Comittee: 22.06.2021; Number 12) and all procedures were performed according to the Helsinki Declaration and its subsequent amendments.

Predictor variables. Three variables were hypothesized as possible predictors of death with prognostic significance: NLR, PLR, and LMR. They were calculated as the absolute neutrophil count divided by the absolute lymphocyte count for NLR, the absolute platelet count divided by the absolute lymphocyte count for PLR, and the absolute lymphocyte count divided by the absolute monocyte count for LMR. Progression-free survival was measured from the initiation of chemotherapy to progression or death. OS was measured from the initiation of chemotherapy until to the last follow-up or death.

Statistical analysis. Continuous variables are presented as the median with interquartile range, and dichotomous variables as percentages. Mann–Whitney U-tests and chi-square tests were used to compare continuous and categorical variables in independent groups. Kaplan–Meier method was used for survival analyses, and a log-rank test was performed to compare the differences between sub-groups. Receiver operator characteristics curves with Youden’s J index were plotted to determine the optimal cut-off values for NLR, PLR, LMR, and other factors, such as C-reactive protein-to-albumin ratio (CAR), lactate dehydrogenase enzyme (LDH), and hemoglobin for predicting OS. Univariate and multivariate analyses were performed using Cox proportional-hazards regression models to define risk factors for OS. Multivariate analyses were performed using variables with a value of p<0.05 in univariate analyses and the backward elimination method. The statistical analyses in the present study were performed using SPSS v25 (IBM Inc., Armonk, NY, USA) software, and value of p<0.05 were considered statistically significant.

Patient characteristics. The male/female ratio was 10.8 (n=207/19) and the median age was 67 years. Histological subtype results were available for 97% of the cases and tumor grade for 90%. Demographic and clinical parameters of all cases were complete and well-documented. The second and third lines of therapy included chemotherapy, vinflunine, and atezolizumab/pembrolizumab/nivolumab. Histologically, our study population comprised patients with urothelial MIBC (TCC) (n=226). According to the TNM scoring system, the percentage of patients with locally advanced and metastatic disease was 48.2% and 51.8%, respectively.

By performing the receiver operating characteristics analysis, an NLR value of 3.29 was found to be the optimal cut-off for predicting OS [area under the curve (AUC)=0.64; sensitivity=59%; specificity=68%; p=0.001]. The optimal PLR cut-off for predicting OS was 169.38 (AUC=0.68; sensitivity=65%; specificity=71%; p<0.0001) and that for LMR was 2.73 (AUC=0.68; sensitivity=47%; specificity=81%; p<0.0001) (Figure 1).

The patients’ baseline demographic, clinical, and histopathological characteristics were stratified according to NLR, PLR, and LMR. The values are summarized in Table I. Patients with an elevated PLR exhibited a higher incidence of metastatic stage (p=0.014), elevated CAR levels (p<0.001), and lower hemoglobin levels (p<0.001) before chemotherapy. Additionally, patients with elevated NLR demonstrated a higher incidence of extensive stage (p<0.001) and liver metastasis (p=0.018), absence of cystectomy (p=0.004), elevated CAR levels (p<0.001) and LDH levels (p=0.040), and lower hemoglobin levels (p<0.001). Furthermore, patients with a lower LMR displayed a higher incidence of metastatic stage (p<0.001), absence of cystectomy (p=0.026), elevated CAR levels (p<0.001), and lower hemoglobin levels (p<0.001). No significant differences were observed among the three groups when examining additional characteristics, such as age, sex, smoking history, intravesical BCG therapy history, bone metastasis, lung metastasis, and brain metastasis.

The prognostic significance of NLR, PLR, and LMR for OS. The median progression-free survival of our patient cohort was 12.18 months, and the median OS was 15.54 months. Seventy-one percent (n=162) of our patients died within a median follow-up period of ≈15 months (range=0.13-147.55 months). Patients with a high NLR or PLR had inferior median OS compared to their counterparts with lower values for these (NLR: 22.51 vs. 9.84 months, respectively, p≤0.001; PLR: 17.68 vs. 14.08 months, respectively, p=0.08). Meanwhile, patients with a low LMR had significantly inferior median OS compared to those with a high LMR (20.14 vs. 10.55 months, respectively, p<0.001) (Figure 2). The 3-year survival rates of patients with high and low NLR were 17.2% and 47.6%, respectively; whilst those for PLR were 24.3% and 41.7%, and for LMR were 41.3% and 16.0%, respectively.

In the univariate Cox analysis, older age (p=0.008), male sex (p=0.032), no previous history of BCG treatment (p=0.017), distant metastasis (p<0.001), bone metastasis (p<0.001), lung metastasis (p=0.007), liver metastasis (p=0.001), history of cystectomy (p=0.008), LMR ≤2.73 (p<0.001), NLR >3.29 (p<0.001), PLR >169.38 (p<0.001), CAR >0.33 (p<0.001), LDH >194 U/l (p<0.001), hemoglobin ≤12.3 g/dl (p<0.001) were determined to be significant prognostic factors for shortened median OS (Table II). However, in multivariate Cox analyses, as shown in Table II, PLR >169.38, not NLR and LMR, was an independent indicator of median OS (HR=1.634 95% CI=1.108-2.410, p=0.013). In addition to PLR, five other independent factors identified as conferring poorer median OS were no previous history of BCG treatment (HR=2.716, 95% CI=1.708-4.319, p<0.001), bone metastasis (HR=1.447, 95% CI=0.979-2.139, p=0.064), lung metastasis (HR=1.579, 95% CI=1.064-2.345, p=0.023), CAR >0.33 (HR=2.337, 95% CI=1.083-2.325, p<0.001), and LDH >194 U/l (HR=1.587, 95% CI=1.083-2.325, p=0.018).

Our study investigated markers of inflammation in MIBC and their impact on survival. This report shows results for 226 patients with stage III and IV invasive MIBC who presented to the Department of Medical Oncology, Pamukkale University, Turkey. All patients received the standard treatment of chemotherapy (mostly platinum-based) with/without local surgery or radiotherapy, according to the institutional guidelines and as per the National Comprehensive Cancer Network guidelines (13). Although chemotherapy improves the survival rates of chemotherapy-eligible patients with BC (stage III and IV), heterogeneity exists among patient groups.

We selected NLR, PLR and LMR as novel inflammatory biomarkers to evaluate their predictive and prognostic value in late-stage BC. They have the advantages of convenience of access, being blood-based biomarkers, and being readily available from routine blood work on patients with cancer, particularly in retrospective cohorts. Previously, some studies investigated scoring systems for the prognosis of patients receiving chemotherapy as first-line treatment for metastatic BC and their relation to responses and survival outcomes. Significant indicators in these scoring systems were normal alkaline phosphatase, normal hemoglobin, high Karnofsky prognostic score, and older age (>60 years) (14). One study identified Karnofsky performance score of less than 80% and visceral metastasis as predictive factors for both response and survival (15). Another study by Galsky et al. reported results from a revised nomogram which included Eastern Cooperative Oncology Group Performance Status, visceral metastases, site of the primary tumor, presence of lymph node metastasis and total leukocyte count as prognostic factors for patients with metastatic BC on chemotherapy. They found that the total leukocyte count had prognostic weight on the outcomes of these patients (16). The nomogram developed by Necchi et al. incorporates several parameters, namely white blood cell count, Eastern Cooperative Oncology Group Performance Status, body mass index, presence of lung, liver, or bone metastases, ethnicity, and the administration of perioperative chemotherapy (17). Another NLR parameter examined in this study was recently evaluated in terms of the effectiveness of immunotherapy in patients with metastatic BC (18-20). Nassar et al. stated that among clinical variables, NLR <5 is a parameter that indicates immunotherapy effectiveness but does not indicate taxane effectiveness (18). Sonpavde and Khaki et al. also used the LNR parameter to show the effectiveness of immunotherapy (19,20). Numerous meta-analyses have demonstrated that blood LDH level and CAR are prognostic indicators of poor survival in advanced cancer (21-24). In our case, by multivariate analysis, we were able to show that a PLR >169.38, as well as other established inflammatory markers, such as CAR and serum LDH level, was an independent predictor of OS in stage 3 and 4 BC, while there was no significant association between NLR >3.29 or LMR ≤2.3 and OS.

A closer look at the biology of the immune system and the changes related to the carcinogenic process, development, growth, progression, and metastasis is warranted to identify the importance of these inflammatory biomarkers. Neutrophils and lymphocytes are key players in the inflammatory response (25). Neutrophils often play a pro-inflammatory, pro-tumoral role in the tumor microenvironment (26). Lymphocytes, on the other hand, usually play a defensive role. Neutrophilia occurs as a response to elevated levels of cytokines, such as interleukin-6, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (27). The microenvironment around precursor and premalignant lesions frequently shows impaired lymphocyte homeostasis and enhanced lymphocyte apoptosis in patients with BC. Under these circumstances, lymphopenia develops (28). However, the exact causes initiating these processes are still not defined. We know that tumor cells can increase expression of transforming growth factor, an important inflammatory mediator, and can express higher lends of pro-apoptotic molecules, such as programmed death-1 ligand-1 and FAS-ligand, causing the destruction of cytotoxic lymphocytes via activation of the extrinsic pathway of apoptosis (29,30). Increased NLR via either a high level of neutrophils or a low level of lymphocytes, or both, have been shown to result in poor survival outcomes, including of patients with advanced BC receiving systemic therapy in previous studies using cut-off values of NLR ranging from 2.5 to 3 (30-32); in concordance with this, the NLR cut-off in our study for predicting OS was higher at 3.29. In another pooled analysis, high pretreatment NLR was associated with worse survival outcomes (HR=1.63, 95% CI=1.34-1.91) in patients with advanced BC (33). The exact significance of altered PLR and LMR is not yet known. Further investigation is required in BC, especially in subtypes more likely to progress to invasive and metastatic disease. Thrombocytosis is a prognostic factor for worse oncological outcomes in BC. It is usually stimulated by release of growth factors and cytokines from tumor cells, especially interleukin 6, which potently induces platelet production (34). Vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, thrombospondins, and endostatin can be released from platelets, and they play a major role in angiogenesis, and the carcinogenic process (35). Monocytes can induce an increase in the number of tumor-associated macrophages in the blood and contribute to tumor infiltration and metastasis. Higher monocytic counts can reflect the activity of tumor-associated macrophages and can be used to predict tumor progression and angiogenesis (36). Altered PLR is associated with poor OS in cancer, such as gastric, lung, and esophageal carcinoma (27,37-39). Nevertheless, it is unclear whether PLR is a marker predictive of prognosis and therapeutic effect of chemotherapy in patients with advanced BC. Several investigations have been conducted to examine the combined characteristics of NLR and PLR in patients with early-stage BC who have undergone radical cystectomy and transurethral resection of bladder tumor in (40-50). However, there is a scarcity of studies focusing on this patient cohort receiving chemotherapy, and even fewer studies have been conducted in the context of the metastatic stage. A few trials have demonstrated the efficacy of neoadjuvant chemotherapy with complete pathological response, disease-free survival and/or OS in individuals diagnosed with MIBC (10,51-55). Some studies were not found to be significant for NLR, like our results; Seah et al. indicated no significant association between pretreatment NLR and complete pathological response (HR=0.69, 95% CI: 0.36-1.32, p= 0.26) (51). The study conducted by Ojerholm et al. demonstrated that the NLR was not a significant predictor for the response to neoadjuvant chemotherapy (HR=1.01, 95% CI=0.90-1.14; p=0.86) (54). In a study conducted in a metastatic setting, researchers examined the impact of NLR and PLR on OS before the initiation of first-line chemotherapy and found them to be significant (56). Their study included a total of 71 patients. In contrast to their findings, our analysis using a sample size of 226 patients did not yield statistically significant results for the NLR. LMR has been evaluated in several previous reports for its predictive value in patients with different solid tumor types, including hepatocellular carcinoma, colorectal cancer, and lung adenocarcinoma (39,57,58). Most studies that focused on the role of LMR as a predictive marker in BC included patients who underwent surgery for early-stage disease. Hai Bi et al. found that a higher LMR was an independent predictor of survival in patients after surgery for BC (p<0.001) (59). LMR ≥3 significantly increased OS of patients with BC (HR=0.56, 95% CI=0.35-0.88; p=0.011) in a meta-analysis, but no prognostic value was found in patients with LMR <3 (HR=0.65, 95% CI=0.41-1.04; p=0.075); moreover, the ethnicity, treatment, and analysis method also did not affect the significant predictive value of LMR in patients with BC (60).

Although BC has an immunogenic nature, there is insufficient knowledge in the literature about the functions of inflammatory pathways, immune cells in progression, and immune escape mechanisms in BC.

Limitations of this study are due to its retrospective nature, the fact it was conducted at a single center, with a small sample size, without external validation and using heterogeneous first-line treatments. However, NLR, PLR, and LMR were separately evaluated in predicting OS in most studies mentioned above. We analyzed the impact of these markers on OS in BC patients treated with first-line chemotherapy. Notably, further studies are needed to evaluate the prognostic roles of PLR on BC in larger and more homogeneous populations.

In summary, our results revealed that PLR >169.38 and other markers of inflammation, such as CAR and serum LDH level, the presence of lung metastases and bone metastases, and the absence of intravesical BCG treatment (which means de-novo metastatic disease), were independent prognostic markers for predicting poor OS of patients with BC treated with chemotherapy. PLR can be used as additional prognostic marker for more accurate prognostic prediction and better personalized treatment in patients with BC.

None declared.

Concept: A.Y. and C.K. Design: A.Y. Supervision: A.Y. Materials: C.K. Data collection &/or processing: C.K. Analysis and interpretation: A.Y., C.K. and A.C. Literature search: C.K. Writing: C.K. Critical review: C.K. and A.Y.

This research was made possible by the generosity and support of numerous individuals. The Authors would like to extend their sincere appreciation to everyone indeed and to thank the respondents for this study, who participated and contributed to the success of this investigation.