RLC-Score (R-status, Lymphovascular invasion, C-reactive protein) predicts survival following radical cystectomy for muscle-invasive bladder cancer

Background: The TNR-C score (tumor stage, lymph node density, resection margin status, and serum CRP) correlates with cancer specific survival (CSS) in patients with bladder cancer (BCa) undergoing radical cystectomy (RC). Our retrospective single center study aimed to evaluate CRP as a prognostic parameter in patients with BCa undergoing RC, to externally validate the TNR-C score, and based on the findings, to develop our own outcome score for muscle-invasive bladder cancer (MIBC) patients undergoing RC that can identify patients with a high risk of progression. of tumor grading, density (LND); lymphovascular invasion (LVI); outcome, overall survival (OS) was assessed. − The clinicopathologic parameters assessed were: age, gender, pre-/postoperative serum CRP, pre-/postoperative leucocytes and hemoglobin, pre-/postoperative serum creatinine, urinary diversion, WHO tumour grading (G1/G2/G3), tumor staging (T1-T4), LN status, lymph node density (LND), lymphovascular invasion, vascular invasion, tumor necrosis, concomitant CIS, the number of tumors, synchronous/metachronous metastases, and the resection margin status. The CRP cut-off was set at > 5 mg/L. The cut-off values for hemoglobin were set at 14–18 g/dL for men and 12–16 g/dL for women. For the creatinine levels, we defined cut-offs from 85 to 104 µmol/L (8) . All these cutoffs were defined following German standards for laboratory diagnostics. The clinical outcome was measured from the date of RC to date of death or date of last follow-up. The RLC score is based on a multivariate analysis and on previous work by Iimura et al (9) . Patients with positive resection margins received 3 points. Patients with preoperatively elevated CRP ≥ 5 mg/L received 1 point, and patients with lymphatic vessel invasion received 1 point. The low risk group (group n = 134) 0–1 intermediate risk group n =

4 62, 22, and 6.5 months, respectively. The score had a high predictive accuracy of 0.752.

Conclusion:
The RLC-score identifies BCa patients at a higher risk of disease progression and overall mortality after RC. Regarding the TNR-C score, only T-stage and R-status were independent prognostic factors for OS. Nevertheless, the survival curves showed a significantly lower OS for TNR-C high risk group members than low and intermediate risk group members. Overall, our study supports the role of CRP in prognostic score models for BCa.

Background
Bladder cancer (BCa) is estimated to be among the 10 leading cancer types regarding new cases (62,380) and deaths (12,520) among males in the United States in 2018, thus representing a frequent and lethal disease (1) . Current 5-year survival rates for BCa average 70% for localized disease, 35% for regional disease, and 5% for distant disease (1) . Approximately three-quarters of the newly diagnosed BCa patients present with non-muscle invasive BCa (NMIBC) (2) . As of now, the EORTC (European Organisation for Research and Treatment of Cancer) BCa risk calculator is the only routinely used score for outcome prediction. Within this score, parameters such as grading, tumor size, concomitant carcinoma in situ (CIS), T-stage, the number of tumors, and prior recurrence rate are used to predict the recurrence and progression of NMIBC Ta/T1 tumors (2,3) . There are currently no clinically established score systems for outcome prediction in patients with locally advanced or metastasized BCa.
In 2011, Gakis et al. pointed out the prognostic potential of serum CRP (C-reactive protein level) for BCa patients (4) . The authors proposed the TNR-C score for the prediction of cancer-specific survival (CSS) for BCa patients undergoing radical cystectomy (RC), which was a new outcome prediction model including the variables tumour stage (T), lymph node density (N), resection margin status (R), and preoperative serum CRP level (C) (4) . Later on, our working group confirmed the predictive value of CRP for BCa patients following RC in a retrospective analysis of our RC cohort (n = 194, 1996-2005) (5) . However, we did not externally validate the TNR-C score system by Gakis et al. in that study (4,5) .
CRP represents an acute-phase protein and indicates systemic inflammation. Currently discussed hypotheses for elevated CRP levels in cancer patients are CRP secretion by the tumor itself, which makes CRP intriguing as a tumor burden marker, and physiological CRP production by hepatocytes initiated by tumor-released cytokines (6,7) .
The primary aim of this retrospective single center study was to externally validate the TNR-C score proposed by Gakis et al. for the first time on our RC cohort, thus enabling future evaluations in the course of evidence-based recommendations. The secondary aim was to develop our own prognosis score for BCa patients undergoing RC.

Material And Methods
In the present retrospective study, 254 patients who underwent RC at the Department of CRP cut-off was set at > 5 mg/L. The cut-off values for hemoglobin were set at 14-18 g/dL for men and 12-16 g/dL for women. For the creatinine levels, we defined cut-offs from 85 to 104 µmol/L (8) . All these cutoffs were defined following German standards for laboratory diagnostics. The clinical outcome was measured from the date of RC to date of death or date of last follow-up. The RLC score is based on a multivariate analysis and on previous work by Iimura et al (9) . Patients with positive resection margins received 3 points. Instead of CSS, we chose OS as the correlation variable since in the authors opinion, CSS is a difficult parameter to assess. Since cox regression models cannot sufficiently evaluate more than four risk factors (22) , we developed a risk stratified model for patients with negative LN and no sign of synchronic metastasis after RC based on the significant parameters in multivariate analysis-the RLC score.
The Chi-square test was used for univariate analyses and Cox regression for multivariate analyses. Kaplan-Meier estimates and the log-rank test were used for survival analyses.
Statistical analyses were performed with IBM SPSS Statistics version 21 and 22. The significance level was set as α = 5%, thus p-values < 0.05 were considered statistically significant.

Descriptive statistics
The majority of the validation cohort was male (79.5%). Their mean age at the time of RC was 65.9 years (CI 64.6-67.2); the women were 2.5 years older on average (CI 65. 5

Laboratory parameters
Based on the previous work of other groups (4,10) , the cut-off for preoperatively evaluated CRP levels was set at 5 mg/L following German standards for laboratory diagnostics.
Overall, an elevated preoperative CRP level was found in 49.8% of all cases. Leukocytosis of > 10,000/µL was found in 28.8% of all cases.

Validation of TNR-C score
The TNR-C score was retrospectively validated on the validation cohort of 159 patients. A significant association between the variables CRP, LND, and OS (see Table 4) could not be found. Only T-stage and R-status were independent prognostic factors for OS (p = 0.012 and p = 0.002, respectively).  Figure 1 illustrates the respective Kaplan-Meier-curves.

RLC-score
The univariate analysis revealed a significant association between OS and T-stage (pT2 vs pT3 or higher, p = 0.001), R-status (p≤0.001), LVI (p = 0.011), and preoperative CRP levels (p = 0.02). All significant parameters of the univariate analysis were then included in the multivariate analysis, which identified R-status, LVI, and preoperative CRP level as independent prognostic markers for OS in the development cohort (n = 155), as shown in Table 5.

Discussion
BCa is an aggressive disease and is associated with high morbidity and mortality rates if not treated optimally (11) . Even though BCa is common, it is often mismanaged. Despite RC with extended lymph node dissection, there is a high risk of tumor progression (12− 14) .
Approximately 50% of the patients with a T stage higher than T2 and/or LVI develop metastases within 5 years (12) and up to 15% present with local recurrent disease (15) . It is assumed that these patients already bear lymphogenous and haematogenous micrometastases at time of surgery (16) . Several meta-analyses of adjuvant treatment trials have shown a 22 to 25% reduction in the risk of death with cisplatin-based adjuvant treatment (17,18) , however, they might have been underpowered to draw final conclusions.
The selection of patients with a higher risk of disease progression after RC and a valuable tool to predict the outcome for OS and CSS in patients after RC and to identify those patients who will benefit from early adjuvant treatment are therefore needed.
The aim of this study was to evaluate CRP as a prognostic parameter in patients with BCa undergoing RC, to validate the TNR-C score, and based on the findings, to develop a new score to measure OS in patients with BCa after RC. In univariate analysis, the OS was significantly associated with T-stage (pT2 vs pT3 or higher, p = 0.001), R-status (p≤0.001), and LVI (p = 0.011). All of these pathologic parameters have been identified as being of prognostic importance in MIBC (12) and were therefore used for the multivariate analysis and score development. Furthermore, preoperative CRP levels showed a significant association (p = 0.002) with the later T stage (pT2 vs. pT3-4) in MIBC after RC, lymph node infiltration (lymph node positive vs negative; p = 0.006), and a lower OS (86.2 months vs. 61.7 months, p = 0.02). In the recent past, our own and several other groups identified CRP as a prognostic predictor in cancer patients (5, 6, 19− 21) . High CRP levels yielded a worse survival in renal cell carcinoma, prostate cancer, BCa, and upper tract urothelial carcinoma (UTUC). The main advantages of CRP as a serum biomarker are its widespread availability due to its routine use as an inflammation marker and its inexpensive assessment. The main disadvantage is the possibility of false negative results due to true infectious inflammation (5) .
In multivariate analysis, R-status, LVI, and the preoperative CRP level were independent prognostic markers for OS. The final model consisting of these three parameters, the RLC score, yielded a high predictive accuracy of 0.752. Our model used OS as an endpoint instead of CSS, in contrast to other score systems such as the TNR-C score of Gakis et al. (4) or the TNM-C score of Iimura et al. (9), since in the authors opinion, CSS is a difficult parameter to assess. Despite our intense analysis of multiple sources, such as the central German cancer registry, the given clinical chart data, and data from the patients' general practitioners, no certain CSS was evaluable. Nevertheless, there was a correlation of OS with stratification of TNR-C risk groups (Fig. 1) It is important to mention that the mean age in the RLC high risk group exceeded the other two groups by around five years (73.5 years vs 67.5 and 68.9 years). A correlation of age at the time of RC and OS has be shown (23− 25) , although comorbidities were found to be more important. In our study, comorbidities were not registered and therefore could not be analysed.

Conclusion
The RLC-score identifies BCa patients undergoing RC with a higher risk of disease progression and, therefore, a reduced OS. Regarding the TNR-C score, only T-stage and Rstatus were independent prognostic factors for OS. Nevertheless, the survival curves showed a significantly lower OS for TNR-C high risk group members than low and intermediate risk group members. Overall, our study supports the role of CRP in prognostic score models for BCa. Furthermore, there is a need for prospective trials to confirm these findings. Kaplan-Meier-plot for TNR-C Score regarding OS Figure 2 Kaplan-Meier-plot for RLC score regarding OS