Stesura Seveso Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 1 ORIGINAL PAPER tomy (RC) with lymph node dissection, that is the cor- nerstone of curative treatment (1). To improve oncologi- cal outcomes, neoadjuvant chemotherapy (NAC) has become the standard of care for eligible patients, provid- ing a 5-8% increase in survival benefit at five years (2, 3). Beside this, before surgery, more patients can receive chemotherapy, as post-operative status and complica- tions may preclude adjuvant chemotherapy (4). However, not all patients respond to neoadjuvant treat- ment. Identifying those most likely to benefit remains a critical challenge, as no validated score currently exists to accurately predict response (5). It is estimated that following NAC, 20-40% of patients have no residual tumor on histological examination of the RC specimen representing a pathologically complete response (2, 6, 7). These patients tend to have excellent outcomes with reported 5-year OS rates of 80-85% (7). It is even hypothesized that patients with a pathological- ly complete response after NAC might not benefit from concurrent RC. This hypothesis is being tested in the ongoing PRE-PREVENCYS trial (8). Tumor regression grade (TRG) aims to quantify the extent of histological response to chemotherapy by assessing the rel- ative proportion of residual viable tumor cells and treat- ment-related changes, such as fibrosis or necrosis. Although its importance has been well established for other malig- nancies, like rectal and esophago-gastric cancers (9-11), no standardized grading system has been created for bladder cancer. Research has been conducted to validate TRG-based classifications and to demonstrate their association with oncologic outcomes, showing additional prognostic value when combined with standard TNM staging (12-14). Fleischmann et al. introduced a TRG classification and concluded that TRG determination in neoadjuvant treat- ed bladder cancer predicts survival independently and better than ypT and ypN stages (12). Since then, other studies have evoked the role of TRG predicting oncologi- cal outcomes in MIBC treated with NAC followed by sur- gery, and its additional value when combined with stan- dard TNM staging (13-15). Introduction: Tumor regression grade (TRG) is a recognized prognostic marker in several solid tumors treated with neoadjuvant therapy, but its clinical relevance in muscle-invasive bladder cancer (MIBC) remains under investigation. This study aimed to evaluate the prognostic value of TRG and its integration with pathological TNM staging in patients with MIBC treated with neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Materials and methods: We conducted a retrospective analysis of 51 patients with MIBC who received platinum-based NAC fol- lowed by RC and lymphadenectomy between 2013 and 2024. TRG was assessed according to the Fleischmann classification and combined with ypTNM stage to categorize patients as com- plete, partial or non-responders. Overall survival (OS) and dis- ease-free survival (DFS) were estimated using Kaplan–Meier analysis, and independent prognostic factors were identified through Cox regression models. Results: Complete response (ypT≤1, ypN0, TRG1) was observed in 43.1% of patients. Median OS was 19 months, with 3- and 5-year OS rates of 28.6% and 14.3%, respectively. Complete responders demonstrated significantly improved OS and DFS (p < 0.001). On multivariable analysis, absence of nodal involve- ment (p = 0.047) and complete response (p = 0.012) were inde- pendently associated with better OS. Negative surgical margins showed a trend toward improved survival (p = 0.064). Conclusions: TRG is a reproducible and clinically meaningful histopathologic scoring system that enhances prognostic stratifi- cation when combined with pathological TNM staging. Its inte- gration into routine post-NAC assessment may improve postop- erative decision-making and help identify patients who could benefit from tailored surveillance or adjuvant strategies. Key WORDS: Tumor regression grade; Muscular-invasive bladder cancer; Neoadjuvant chemotherapy; Radical cystectomy. Submitted 20 August 2025; Accepted 30 August 2025 INTRODUCTION Patients with muscle-invasive bladder cancer (MIBC) have a 5-year overall survival (OS) of 40-60% after radical cystec- Prognostic value of combined tumor regression grade and TNM stage in muscle-invasive bladder cancer treated with neoadjuvant chemotherapy and radical cystectomy Manuel Lopes 1, 2, José Pereira 3, Maria José Temido 2, 4, João Gama 5, Edgar Silva 1, 2, Vasco Quaresma 1, 2, João Lorigo 1, Rui Pedrosa 1, João Pedroso Lima 1, 2, Henrique Dinis 1, 2, Lorenzo Marconi 1, 2, Vítor Sousa 2, 5, Arnaldo Figueiredo 1, 2 1 Urology and Renal Transplantation Department, Hospitais da Universidade de Coimbra, Portugal; 2 Faculty of Medicine, University of Coimbra, Portugal; 3 Urology Department, Instituto Português de Oncologia - Coimbra, Portugal; 4 Gastroenterology Department, Hospitais da Universidade de Coimbra, Portugal; 5 Pathology Department, Hospitais da Universidade de Coimbra, Portugal. DOI: 10.4081/aiua.2025.14264 Summary Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 M. Lopes, J. Pereira, M.J. Temido, et al. 2 We thus aim to evaluate TRG of patients with MIBC who underwent NAC before RC and lymphadenectomy and to assess its prognostic role in combination with TNM stag- ing classification as predictors of OS and disease-free sur- vival (DFS). MATERIAL AND METHODS Study design This is a retrospective cohort study conducted in a terti- ary hospital. From a total of 307 patients who underwent RC for bladder cancer between January 2013 and December 2024, the study included a consecutive series of 51 patients with MIBC treated with platinum-based NAC followed by RC with lymphadenectomy. Inclusion criteria were (1) histologically confirmed urothe- lial carcinoma (UC) of the bladder (pure or predominant histology); (2) clinical stage T2-T4a, N0-3, M0 prior to NAC; (3) completion of at least two cycles of chemothera- py; and (4) availability of full pathological specimens for review. Patients were excluded if they had (1) pure non- urothelial histology, (2) ≤pT1/Tis stage disease on initial histology, (3) completed less than two cycles of NAC, or (4) missing clinical or histopathological data. Initial diagnosis and staging were established after transurethral resection of the bladder (TURB) and chest, abdominal and pelvic computed tomography (CT). Follow- up after surgery was based on chest, abdominal and pelvic CT or 18F-FDG (fluordesoxiglicose) Positron Emission Tomography/CT. Last follow-up was performed in July 2025. Neoadjuvant chemotherapy The decision of NAC was made after discussion at a mul- tidisciplinary tumor board involving urologists and oncologists. Standard treatment included a platin-based combination of drugs [gemcitabine and cisplatin (GemCis); gemcitabine and carboplatin (GemCarbo), methotrexate, vinblastine, doxorubicin and cisplatin (MVAC); or cisplatin and etoposide (CisEto)] depending on patient and tumor features. Surgical procedure After NAC all patients were submitted to open RC and bilateral lymphadenectomy in the same procedure. Standard lymph node dissection was generally carried out. Extended or super-extended templates were used depend- ing on preoperative staging results (16). In two cases, lym- phadenectomy could not be performed due to intraopera- tive or anatomical limitations. Intraoperative frozen section analysis of the urethra and ureters was routinely per- formed. Histopathological analysis TURB and RC specimens were analyzed by two dedicat- ed genitourinary pathologists blinded to outcomes. TRG was evaluated in the primary tumor site using the system proposed by Fleischmann et al. (12), described in Table 1. If multiple areas with varying response were present, the dominant TRG pattern was recorded. Discrepancies between reviewers were resolved by joint consensus. Residual pathological staging (ypTNM) was assigned according to AJCC 8th edition criteria (17). Lymph nodes were examined separately and classified as invaded (pN≥1) or not (pN0). TRG classification and ypTNM stage were combined in treatment response evaluation categories, adapted from the criteria introduced by Voskuilen C. et al. (13). Patients were classified as complete, partial or non- responders (Table 2). Data collection and statistical analysis Data was analyzed with Stata (StataCorp LP® version 16.0). Descriptive statistics were used for clinical and analytical data description. Continuous variables were described with median and interquartile range (IQR) and categorical variables with frequencies. OS was defined as the time from RC to death from any cause or last known follow- up. DFS was measured from the date of RC to the first evidence of recurrence, disease progression, or death. Kaplan-Meier analysis was used to estimate OS and DFS, with group comparisons performed using the log-rank test. Independent predictors of survival were assessed through multivariate Cox proportional hazards regres- sion, with backward selection. Variables with a p-value < 0.1 in the univariate analysis were included in the mul- tivariable model. A p-value of < 0.05 was considered sta- tistically significant. ethical considerations The project was conducted in accordance with good clin- ical practice and adhered to the ethical principles of the Declaration of Helsinki. Informed consent was obtained prior to inclusion in the study. All data were anonymized before the analysis. RESULTS Study’s population characteristics A total of 51 patients were included, the majority of whom (76.5%) were male. The median age at diagnosis was 66 years (IQR 60-76) and 42 patients (82.3%) had previous or concurrent history of smoking. Demographic, clinical and histopathological characteristics are detailed Table 1. Tumor regression grade classification. TRG1 Complete regression (no viable tumor) TRG2 > 50% regression (predominantly therapy-induced changes, sparse viable tumor) TRG3 ≤ 50% regression (predominantly viable tumor with limited regression) TRG: Tumor Regression Grade. Table 2. Patients classification according to neoadjuvant chemotherapy response. Complete Responder ypT≤1 and ypN0 and TRG1 Partial Responder ypT≥2 or ypN1-3 and TRG1 or TRG2 Non-responder ypT≥2 or ypN1-3 and TRG3 TRG: Tumor Regression Grade. Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 3 Prognostic value of combined tumor regression grade and TNM in Table 3. Figure 1 illustrates Tumor Regression Grades. Median time from TURB to NAC and from NAC to RC were 55 days (IQR 35-78) and 100 days (IQR 80-142), respectively. Majority of patients (86.3%) received GemCis, 4 patients had dense-dose MVAC, 1 patient had GemCarbo due to renal function impairment and 2 patients received CisEto Figure 1. Tumor Regression Grade (TRG) (hematoxylin and eosin). A – TRG1, no histologically detectable residual cancer cells, with extensive fibrosis present in the tumor bed. B – TRG2, the tumor bed is predominantly fibrotic, with residual cancer cells comprising less than 50% of the area. C – TRG3, residual cancer cells dominate over fibrosis, occupying 50% or more of the tumor bed area, or there are no signs of regression. Table 3. Clinicopathologic data of the study population (N = 51 patients undergoing neoadjuvant chemotherapy followed by radical cystectomy). Parameter N (%) Age, median (IQR) 66 (60-76) Gender Male 39 (76.5) Female 12 (23.5) Smoking history Yes 42 (83.4) No 9 (17.6) cT stage T2 39 (76.5) T3 5 (9.8) T4 7 (13.7) cN stage N0 38 (74.5) N+ 13 (25.5) NAC scheme GemCis 44 (86.3) MVAC 4 (7.8) CisEto 2 (3.9) GemCarbo 1 (2.0) No. of NAC cycles, median (IQR) 4 (3-4) 2 10 (19.6) 3 13 (25.5) 4 24 (47.1) 6 4 (7.8) Radical cystectomy specimen No tumor present 12 (23.5) Tumor present 39 (76.5) Pure urothelial carcinoma/CIS 25 (64.1) Variant histology 14 (35.9) Squamous cell 4 (7.8) Micropapillary 4 (7.8) Adenocarcinoma 2 (3.9) Neuroendocrine 2 (3.9) Sarcomatoid 2 (3.9) pT Stage T0 12 (23.5) Tis 11 (21.6) T1 3 (5.9) T2 3 (5.9) T3 14 (27.5) T4 8 (15.7) Residual tumor dimension (cm), median (IQR) 3.25 (2.0-5.5) pN stage Nx 2 (3.9) N0 36 (70.6) N1 1 (2.0) N2 12 (23.5) No. of positive lymph nodes if N1-3, median (IQR) 4.5 (2-8) Surgical margins Negative 41 (80.4) Positive 10 (19.6) Tumor regression grade TRG1 23 (45.1) TRG2 9 (17.7) TRG3 19 (37.2) Type of responder Complete responder 22 (43.1) Partial responder 10 (19.6) Non-responder 19 (37.2) GemCis: Gemcitabine/Cisplatin; GemCarbo: Gemcitabine/Carboplatin; CIS: Carcinoma in situ; CisEto: Cisplatin/Etoposide; MVAC: Methotrexate/Vinblastine/Doxorubicin/Cisplatin; NAC: Neoadjuvant Chemotherapy; TRG: Tumor Regression Grade. Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 M. Lopes, J. Pereira, M.J. Temido, et al. 4 because of poorly differentiated neuroendocrine variant present in > 10% of TURB specimen. Regarding response to neoadjuvant treatment, TRG was closely correlated with response classification. All non-responders (n = 19) exhibited TRG3. Among the 23 patients with TRG1, 22 were classified as complete responders, while one was a partial responder. In this particular case, complete tumor regression was observed in the bladder, but residual UC was identified in the prostate (pT4a, N0, TRG1). Finally, nine patients were assigned TRG2, contributing to a total of 10 patients categorized as partial responders. Overall and disease-free survivals The median follow-up period was 16.7 months (IQR 9.9- 47.3), and the 3- and 5-year OS rates were 28.6% and 14.3%, respectively (Figure 2A). The median OS was 19 months (IQR 9.9-47.3). Four patients survived for more than eight years after RC. The 3- and 5-year DFS rates were 28.6% and 14.3%, respectively (Figure 2B). Progression of disease was diagnosed in 23 patients (46.9%), with non-regional lymph nodes in 4 (17.4%), visceral lesions in 4 (17.4%), bone metastasis in 2 (8.7%), pelvic mass in 2 (8.7%) and in multiple locations in 11 (47.8%) patients. Three patients had metastatic disease based on RC specimen analysis: (1) peritoneal implant, (2) ovarian metastasis and (3) non-regional lymph nodes metastasis. Figure 2. Kaplan-Meier curves of overall survival (A) and disease-free survival (B) in 51 patients undergoing neoadjuvant chemotherapy followed by radical cystectomy. Figure 3. Kaplan-Meier curves of overall survival (OS) in 51 patients undergoing neoadjuvant chemotherapy followed by radical cystectomy. (A) OS according to Surgical Margins (Log rank p < 0.001). (B) OS according to pN stage (Log rank p < 0.001). (C) OS according to Treatment Response (Log rank p < 0.001). A. A. B. C. B. Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 5 Prognostic value of combined tumor regression grade and TNM Impact of clinical and histopathological factors in the disease-free and overall survival Univariate analysis identified several predictors of longer OS, including tumor type in the RC specimen (p = 0.04), with pure UC showing better outcomes compared to variant histology (p = 0.008), as well as pathological stage ypT≤1 (p < 0.001), node-negative status (p < 0.001), nega- tive margins (p < 0.001), lower TRG (p < 0.001), and com- plete response (p < 0.001) (Figure 3 and Supplementary Table). These same predictors were also associated with improved DFS (p < 0.05) (Supplementary Table). Independent predictive factors of survival On multivariable analysis, the absence of nodal involve- ment and complete response (defined as ypT≤1, ypN0, TRG1) were identified as independent predictors of improved OS (p = 0.047 and p = 0.012, respectively). Negative surgical margins demonstrated a trend toward significance as an independent prognostic factor for OS (p = 0.064) (Table 4). DISCUSSION In this retrospective cohort of 51 patients with MIBC treat- ed with NAC followed by RC, we observed that TRG was significantly associated with both OS and DFS. The medi- an OS in our cohort was 19 months, with 3- and 5-year OS rates of 28.6% and 14.3%, respectively. These are lower rates than those reported in large multicenter trials (2, 3), likely reflecting differences in patient selection, disease burden, and histological subtypes. Despite this, a small subset of patients achieved long-term survival beyond eight years, reinforcing the potential of NAC followed by RC to provide durable benefit in well-selected individuals. Consistent with prior studies (12, 13, 18) we found that better TRG scores (TRG1-2), node-negative status, and lower pathological stage (ypT≤1) were associated with prolonged survival. Importantly, being a complete respon- der emerged as a strong prognostic factor on both uni- variate and multivariate analyses, supporting TRG classifi- cation utility in stratifying patients beyond conventional TNM staging. While pathologic complete response (pT0N0) is the most widely used surrogate endpoint in NAC trials, our findings are aligned with the literature in showing that patients with near-complete response (TRG1 and pT≤1N0) also derive substantial benefit (13). Variant histology was significantly associated with worse survival, consistent with previous evidence suggesting that non-pure UC is less responsive to NAC and associated with more aggressive behavior (19). Additionally, positive surgi- cal margins – despite of not being independently predictive in multivariable analysis – were associated with decreased OS on univariate analysis, highlighting the importance of surgical quality and local tumor control (1). Nearly half of the cohort (46.9%) experienced disease progression, with distant metastases to lymph nodes, vis- cera, and bone as the most common. The detection of metastatic disease in three patients at the time of cystec- tomy highlights limitations in current preoperative stag- ing and supports the need for better diagnostic tools to identify occult disease prior to surgery. Our results reinforce the prognostic value of TRG after NAC in MIBC and the importance of its integration with standard TNM classification to improve postoperative risk stratification. Patients with poor TRG scores, positive nodes, or incomplete response may benefit from more intensive surveillance or consideration for adjuvant ther- apies, including immune checkpoint inhibitors, as it is being practiced (20). Conversely, identification of com- plete or partial responders may support the emerging Supplementary Table. Clinical and pathological characteristics of the study population (N = 51 patients undergoing neoadjuvant chemotherapy followed by radical cystectomy) and predictors of overall and disease-free survival (Log rank) (p < 0.05). Parameters No. of Overall Disease-free patients (%) survival survival Gender Male 39 (76.5%) 0.436 0.5 Female 12 (23.5%) Age ≤ 65 14 (73.7%) 0.61 0.65 > 65 5 (26.3%) History of smoking Yes 42 (82.4%) 0.333 0.587 No 9 (17.7%) RC Specimen No tumor present 12 (23.5%) 0.04 0.026 Tumor present 39 (76.5%) RC specimen with tumor Pure urothelial carcinoma/CIS 25 (64.1%) 0.008 0.004 Variant histology 14 (35.9%) ypT T≤1 7 (36.8%) < 0.001 < 0.001 T≥2 12 (63.2%) ypN N0 16 (84.2%) < 0.001 < 0.001 N1-3 3 (15.8%) Surgical margins Negative 41 (80.4%) < 0.001 < 0.001 Positive 10 (19.6%) Tumor regression grade TRG 1 23 (45.1%) < 0.001 < 0.001 TRG 2 9 (17.7%) TRG 3 19 (37.2%) Type of responder Complete 22 (43.1%) < 0.001 < 0.001 Partial 9 (17.7%) Non-responder 20 (39.2%) HR: Hazard Ratio; 95% CI: 95% Confidence Interval. Table 4. Independent predictors of overall survival (Cox Regression). HR 95% CI p value Surgical margins (negative) 2.72 0.94-7.84 0.064 pN stage (N0) 2.59 1.01-6.62 0.047 Type of responder (complete responder) 2.19 1.18-4.06 0.012 HR: Hazard Ratio; 95% CI: 95% Confidence Interval. Archivio Italiano di Urologia e Andrologia 2025; 97(3):14264 M. Lopes, J. Pereira, M.J. Temido, et al. 6 bladder-sparing approaches in carefully selected cases. On the behalf of this, the PRE-PREVENCYS trial showed that the absence of residual disease after NAC in patients with MIBC is accurately predicted, and so a randomized controlled trial is scheduled for comparing OS after NAC plus RC versus NAC and close surveillance in patients with a clinically complete response (8). Additionally, the value of circulating tumor DNA holds promise as a bio- marker in the perioperative treatment of MIBC and the integration of both markers may be an enthusiastic area of research in the upcoming years (21). This study is limited by its retrospective, single-center design and relatively small sample size, which may impact statistical power and generalizability. In addition, variations in chemotherapy regimens and timing between TURB, NAC, and RC may introduce heterogeneity. Nevertheless, the strong association observed between response categories and survival outcomes adds a robust role for TRG as a clinically meaningful criterion. CONCLUSIONS TRG system is a reproducible and clinically relevant method for assessing histological response to NAC in bladder cancer. When combined with ypTNM staging, it enhances prognostic stratification and may inform post- operative management decisions. Future studies should focus on standardizing these scoring systems and evaluat- ing their role in prospective treatment algorithms. REFERENCES 1. Stein J, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. 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Competing interests: The authors declare that they have no competing interests. Funding: No funding was necessary for this study. Authors' contributions: ML, study concepts, methodology, data acquisition, analysis and interpretation, manuscript original drafting and edition; JP, manuscript drafting and review; MJT, data analysis and interpretation and manuscript review; JG, histopathological analysis and manuscript review; ES, study con- cepts and manuscript review; VQ, manuscript edition and review; JL, manuscript review; RP, manuscript review; JPL, manuscript review; HD, manuscript review; LM, manuscript review; VS, histopathological analysis and manuscript review; AF, manu- script review and supervision. All the authors read and approved the final version of the manuscript and agreed to be accountable for all aspects of the work. 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Correspondence Manuel António Ferreira Malheiro Lopes (Corresponding Author) manuel11070@gmail.com Serviço de Urologia e Transplantação Renal, Hospitais da Universidade de Coimbra Rua Praceta Professor Mota Pinto - 3000-075 Coimbra, Portugal José Pereira joseaclpereira@gmail.com Instituto Português de Oncologia, Coimbra, Portugal Maria José Temido mariajosetemido@gmail.com João Gama joaomartinsgama@gmail.com Edgar Silva edsilva.elv@gmail.com Vasco Quaresma vpdquaresma@gmail.com João Lorigo joaolorigo@gmail.com Rui Pedrosa rui.mdp93@gmail.com João Pedroso Lima joaopedrosolima@gmail.com Henrique Dinis hdinis@gmail.com Lorenzo Marconi lorenzooliveiramarconi@gmail.com Arnaldo Figueiredo ajcfigueiredo@gmail.com Hospitais da Universidade de Coimbra, Portugal