A New Marker for Prediction of Recurrence in Low-Risk Bladder Cancer: Retrospective Study of ELF-3 and TSC-1

A New Marker for Prediction of Recurrence in Low-Risk Bladder Cancer: Retrospective Study of ELF-3 and TSC-1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A New Marker for Prediction of Recurrence in Low-Risk Bladder Cancer: Retrospective Study of ELF-3 and TSC-1 Enes Altın, Çağrı Doğan, Erdem Can Topkaç, Mehmet Fatih Şahin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8782269/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 17 You are reading this latest preprint version Abstract Objective: Tumor recurrence remains a major clinical challenge among patients with low-risk non-muscle invasive bladder cancer (NMIBC). Molecular studies have identified two main subtypes with distinct genetic pathways: papillary, low-grade tumors often associated with FGFR3 mutations and favorable outcomes, and nonpapillary, high-grade tumors characterized by high progression and muscle invasion rates. This study aims to evaluate the predictive value of immunohistochemical (IHC) markers—CD44, CK5/6, CK20, GATA3 , Cerb-B2, ELF-3, and TSC-1—for recurrence in patients with low-risk NMIBC. Materials and Methods: This retrospective study included 40 patients with NMIBC selected from 320 individuals who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019. Patients were categorized into recurrence ( n = 20) and nonrecurrence ( n = 20) groups. Tissues were reevaluated and stained for CK4, CK5/6, CK20, CD44, GATA3 , Cerb-B2, ELF-3, and TSC-1 using IHC methods. Associations between clinicopathological variables and recurrence were statistically analyzed. Results: Recurrence correlated significantly with tumor number ( p = 0.036 ), while age, sex, smoking status, and tumor size showed no significant association ( p > 0.05). CD44 positivity was predominantly observed in nonrecurrent cases ( p = 0.022 ), whereas TSC-1 positivity was strongly associated with recurrence ( p = 0.003 ). Multivariate regression revealed that CD44 positivity reduced the recurrence risk (OR = 0.142), while TSC-1 positivity increased it more than 21-fold (OR = 21.871). Tumors located at the ureteral orifice exhibited a lower recurrence risk (OR = 0.098). Conclusion: This study indicates that low CD44 expression and high TSC-1 expression in patients with low-risk NMIBC may serve as predictors of tumor recurrence. Assessment of these markers could enable earlier identification of patients at increased risk, thereby facilitating strict surveillance and timely management of treatment strategies. Bladder cancer non-muscle invasive ELF-3 CD44 TSC-1 NMIBC Figures Figure 1 Figure 2 Figure 3 Introduction Bladder cancer (BC) accounts for approximately 5% of all newly diagnosed cancers in the United States and represents the seventh most common malignancy [1]. Based on the molecular pathways of tumorigenesis, two distinct categories of BC have been described. The first category includes localized, papillary, and low-grade tumors arising from urothelial hyperplasia. This histological type generally exhibits a more favorable clinical course and is frequently associated with mutations in the fibroblast growth factor receptor 3 ( FGFR3 ) gene [2]. The second category includes nonpapillary, high-grade tumors that typically develop in the setting of severe dysplasia and carcinoma in situ (CIS). These cancers are characterized by higher rates of progression and muscle invasion, resulting in poor long-term survival outcomes [3]. Although immunohistochemical (IHC) markers, such as CK5/6, CK20 (cytokeratin 5/6 and 20), TP53 (tumor protein 53), and CD44 (cluster of differentiation 44), are commonly used to distinguish between these molecular pathways, no universally accepted gold standard marker has been established [4, 5]. The primary objective of our study was to evaluate the potential of ELF-3 (E74-like transcription factor 3) and TSC-1 (tuberous sclerosis complex subunit 1), along with other IHC markers, as predictors of recurrence and progression in patients with low-risk non-muscle invasive BC (NMIBC). A secondary objective was to explore whether risk stratification using these markers could help tailor patient-specific surveillance strategies, even though cystoscopy remains the gold standard for follow-up in bladder cancer [6]. Materials and Methods This study was approved by the Non-Interventional Clinical Research Ethics Committee of Tekirdağ Namık Kemal University. (Decision No: 2021.227.09.13) and was supported by the Scientific Research Projects (SRP) Coordination Unit of Tekirdağ Namık Kemal University as a medical specialty thesis project (Project Code: NKUBAP.02.TU.22.361). We retrospectively reviewed the data of 320 patients who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019 and who were followed up in our clinic. A total of 40 patients aged 18 years or older with a pathological diagnosis of NIMBC and regular cystoscopic follow-up met the inclusion criteria and were enrolled. Patients who had received Bacille Calmette-Guérin (BCG) therapy before recurrence were excluded to avoid potential bias. Immunohistochemical Examination For immunohistochemical (IHC) analysis, 4-µm sections were prepared from all paraffin-embedded tissue blocks. The sections were incubated in a 60 °C oven for 1 hour, followed by deparaffinization with xylene for 15 minutes. Subsequently, they were passed through graded alcohols, rinsed in distilled water, and processed in the BenchMark XT automated staining system. All samples were stained with the following antibodies: CK5/6 (Cytokeratin 5/6), CK20, CD44 (anti-CD44 SP37 rabbit monoclonal primary antibody), GATA3 (L50-823 mouse monoclonal primary antibody), Cerb-B2 (human epidermal growth factor receptor 2), ELF-3, and TSC-1. The stained slides were coverslipped using a liquid-based mounting medium. Microscopic evaluation was performed using an Olympus CX41 microscope (Munich, Germany). For each section, 100 tumor cells were counted at 100× magnification, and cytoplasmic or nuclear staining was scored semi-quantitatively. Staining intensity was categorized into five groups: score 0 (0–10% staining), score 1 (10–25%), score 2 (25–50%), score 3 (50–75%), and score 4 (> 75%). The cutoff value for positivity was set at 50% for GATA3 and CD44 and at 25% for CK5/6 and CK20. Accordingly, scores 0 and 1 were classified as negative, while scores 2–4 were considered positive for CK5/6 and CK20 (Figure 1). For GATA3 and CD44, scores 0–2 were defined as negative, whereas scores 3 and 4 were defined as positive (Figure 2). For Cerb-B2, ELF-3, and TSC-1, the cutoff value for positivity was set at 75%. Complete membranous staining in neoplastic cells was required for positivity; score 4 was defined as positive, score 3 as equivocal, and scores 0–2 as negative (Figure 3). Statistical Analysis Statistical analyses were conducted using IBM SPSS Statistics version 24. Frequency tables and descriptive statistics were used to summarize the findings. Parametric tests were applied to continuous variables with normal distributions, whereas non-parametric tests were used for variables that did not meet the assumption of normality. For comparisons between the two independent groups, the independent samples t -test was employed for normally distributed variables, while the Mann–Whitney U test was used for non-normally distributed variables. Associations between categorical variables were evaluated using Pearson’s χ² test. Factors influencing recurrence were further analyzed using binary logistic regression with the backward likelihood ratio (backward LR) method. Results A total of 40 patients with NMIBC were included in the study. Of these, 20 had recurrence during follow-up, and 20 were recurrence-free. No statistically significant associations were found between recurrence status and gender, occupation, ongoing smoking, tumor localization, or early postoperative epirubicin administration. In contrast, a significant difference was observed in terms of the initial tumor number ( p = 0.022 ). Sixteen patients (80.0%) in the nonrecurrence group presented with a single tumor, whereas nine patients (45.0%) in the recurrence group had 2–4 tumors. The mean tumor number was 1.50 ± 1.15 in the nonrecurrence group and 2.45 ± 2.09 in the recurrence group. Receiver operating characteristic (ROC) analysis identified 1.5 as the optimal cutoff number of tumors for predicting recurrence, with 60.0% sensitivity and 80.0% specificity (AUC = 0.686; p < 0.05 ). When postoperative epirubicin administration was evaluated, it was applied in 8 of 20 patients (40%) without recurrence and in 9 of 20 patients (45%) with recurrence. Epirubicin use did not significantly influence recurrence development, time to recurrence (months), or recurrence size (cm). The detailed evaluation of factors potentially associated with recurrence is summarized in Table 1. Table 1. Evaluation of factors that may affect recurrence status Recurrence No ( n = 20) Yes ( n = 20) Statistical analysis* Probability Age (years) 64.25 ± 10.07 64.5 [48.0-86.0] 63.00 ± 11.14 63.0 [38.0–80.0] t =0.372 p = 0.712 BMI (kg/m 2 ) 27.94 ± 2.94 28.0 [23.1-32.3] 28.14 ± 3.65 27.4 [23.0–34.3] t = −0.177 p = 0.860 Cigarettes (packs/year) 36.00 ± 25.98 35.0 [0.0-90.0] 25.75 ± 21.78 25.0 [0.0–75.0] Z = −1.352 p = 0.176 Maximum tumor size (cm) 1.85 ± 0.90 1.8 [0.5-3.0] 2.10 ± 0.99 2.3 [0.5–3.0] Z = −0.877 p = 0.380 * For variables with a normal distribution, comparisons between two independent groups were performed using the Independent Samples t -test ( t statistic). For variables not normally distributed, comparisons were conducted using the Mann–Whitney U test (Z statistic). A statistically significant difference was observed between recurrence status and ureteral orifice tumor localization (χ² = 5.625; p = 0.018 ). Seven patients (35.0%) in the nonrecurrence group had tumors originating from the ureteral orifice, whereas 19 patients (95.0%) in the recurrence group did not. Thus, tumors arising from the ureteral orifice were predominantly associated with nonrecurrence. The differences between groups of IHC staining results are presented in Table 2. No statistically significant difference was identified between recurrence status and CK5/6, GATA3, Cerb-B2, CK20, or ELF-3 expression. Table 2. Evaluation of the relationship between recurrence and IHC staining Relapse status Variable No ( n = 20) Yes ( n = 20) Statistical analysis* Probability N % N % CD44 Positive Negative 11 9 55.0 45.0 4 16 20.0 80.0 p = 0.022 CK5/6 Positive Negative 8 12 40.0 60.0 8 12 40.0 60.0 p = 0.990 GATA3 Positive Negative 20 0 100.0 0 17 3 85.0 15.0 p = 0.072 CerbB2 Positive Negative 5 15 25.0 75.0 5 15 25.0 75.0 p = 1.000 CK20 Positive Negative 9 11 45.0 55.0 10 10 50.0 50.0 p = 0.990 ELF-3 Positive Negative 20 0 100.0 0 20 0 100.0 0 - TSC-1 Positive Negative 11 9 55.0 45.0 19 1 95.0 5.0 p = 0003 * “Pearson-χ 2 cross tables” were used to examine the relationships between two qualitative variables. A statistically significant difference was observed between recurrence status and CD44 expression (χ² = 5.227; p = 0.022 ). CD44 positivity was detected in 11 patients (55.0%) without recurrence, whereas 16 patients (80.0%) in the recurrence group were CD44 negative. Thus, CD44 positivity was predominantly associated with the absence of recurrence, while CD44 negativity was associated with recurrence. Similarly, a significant relationship was found between recurrence status and TSC-1 expression ( p = 0.003 ). Nine patients (45.0%) in the nonrecurrence group were TSC-1 negative, compared with 19 patients (95.0%) in the recurrence group who were TSC-1 positive. Accordingly, TSC-1 positivity was primarily associated with recurrence, whereas TSC-1 negativity was associated with nonrecurrence. Multivariate logistic regression analysis, incorporating all parameters that showed significance in univariate analyses, confirmed these findings. In the final model, CD44 remained an independent factor influencing recurrence ( p < 0.05 ) (Table 3), with CD44 positivity reducing recurrence risk by approximately 85.8% compared with CD44 negativity (OR = 0.142). Conversely, TSC-1 positivity was associated with a 21.8-fold increased risk of recurrence ( p < 0.05 ). In addition, ureteral orifice involvement was a significant factor ( p < 0.05 ), with tumors located at the ureteral orifice demonstrating a 90.2% lower risk of recurrence compared with those without such localization (OR = 0.098). Table 3. Evaluation of the relationship between tumor number and recurrence status. AUC: Area under the curve Variable AUC Standard error P 95% confidence interval Cutoff Lower limit Upper limit Number of tumors 0.686 0.086 0.044 0.517 0.855 1.50 The median follow-up period for the study cohort was 36 months (IQR: 24–52 months). The median duration of recurrence-free survival was 28 months. All recurrences were histologically confirmed after cystoscopic identification, ensuring diagnostic accuracy and consistency in the outcome evaluation. Discussion CD44 is a cell adhesion molecule that has been implicated in promoting tumor progression and dissemination [7–9]. However, its prognostic value in urothelial carcinoma remains controversial [10, 11]. Recent meta-analyses have demonstrated a statistically significant difference between CD44 expression and advanced T stage, lymph node metastasis, and adverse oncological outcomes, suggesting that CD44 expression may be linked to tumor progression, metastasis, and treatment failure in bladder cancer [11, 12]. Conversely, Raspollini et al. reported in their study on T1 high-grade urothelial carcinoma that CD44 expression was not associated with poor prognostic factors [13]. In our cohort, CD44 positivity was noted in 11 of 20 patients (55%) without recurrence, in contrast to 4 of 20 patients (20%) in the recurrence group, indicating a statistically significant difference ( p = 0.022 ). These results are consistent with those reported by Raspollini et al. This apparent discrepancy in the literature may be explained by the specific characteristics of our study population, which was limited to patients with NMIBC. Cytokeratins (CKs) are structural proteins that form part of the cytoskeleton of squamous (stratified) epithelial cells. Extensive immunohistochemical research has been performed on CKs in a variety of cancers, as they are regulators of proliferation in epithelial-derived squamous cells [14, 15]. Jung et al. found that within the NMIBC group, tumors expressing CK5/6 were more likely to be of a higher grade [16]. While some studies in the literature suggest that CK5/6 expression may serve as a prognostic marker, others have concluded that it lacks prognostic significance. In our study, no statistically significant difference was observed between patients with and without recurrence in terms of CK5/6 staining ( p > 0.05). GATA3 , also known as GATA binding protein 3, is a T cell–specific gene that functions as a transcription factor located in the T cell receptor (TCR) α region [17]. GATA3 has been investigated in a variety of tumors other than bladder cancer, and it has been suggested to serve as an IHC marker with potential prognostic relevance [18, 19]. In studies conducted by Kamel et al., GATA3 expression in patients with NMIBC was reported to be associated with favorable prognostic factors [20]. However, most published studies have been conducted in either MIBC or general NMIBC populations, and data specific to low-grade NMIBC remain limited. In our study, GATA3 staining was observed in all patients (100%) without recurrence and in 17 of 20 patients (85%) with recurrence. This difference did not show statistical significance ( p > 0.05). Previous studies have demonstrated that low CK20 expression is associated with poor recurrence-free survival (RFS) and cancer-specific survival (CSS) [21, 22]. In contrast, Buchumensky et al. reported no association between tumor grade and CK20 expression [23]. While the literature supports CK20 as a reliable marker for identifying the luminal subgroup, its prognostic value for predicting recurrence, tumor grade, and aggressiveness remains inconsistent. In our study, no statistically significant differences in CK20 expression were observed between the recurrence and nonrecurrence groups ( p > 0.05). Studies conducted in populations similar to ours have yielded comparable results, thus supporting the results of our study. Cerb-B2, also known as HER2/neu, is a protein encoded by the ERBB2 gene. Enache et al. concluded that Cerb-B2 expression is important for identifying poorly differentiated, advanced-stage tumors [24], whereas Alexa et al. found no relationship between Cerb-B2 overexpression and tumor stage [25]. In our study, Cerb-B2 expression was also evaluated, and no significant differences were observed between the recurrence and nonrecurrence groups. ELF-3 is a member of the ETS (erythrocyte transformation-specific) transcription factor family [26]. Na et al. demonstrated that increased ELF-3 signaling exerts a tumor-suppressive role in bladder cancer [27]. However, its role in the molecular subtyping of bladder cancer remains uncertain, and research in this area is still in its early stages. In our cohort, ELF-3 expression was uniformly positive (100%) in both groups, with no ability to differentiate between patients with and without recurrence. Thus, ELF-3 was not found to be an effective marker for predicting recurrence. Despite ELF-3 being initially considered a possible marker due to its reported role in urothelial differentiation, our results demonstrated consistent expression across all specimens, suggesting that it lacks predictive value in low-risk NMIBC. Consequently, the study’s primary focus transitioned to CD44 and TSC-1, which exhibited strong and independent correlations with recurrence risk. TSC-1, also known as hamartin, is a protein encoded by the TSC-1 gene and functions as a co-chaperone that inhibits the ATPase activity of HSP90, thereby regulating the chaperone cycle. Dadhania et al. identified TSC-1 as a marker of the luminal subtype in bladder cancer [6]. Although the precise role of TSC-1 in bladder cancer biology remains unclear, it has been implicated in tumor development through its interaction with the mTOR pathway. This has prompted researchers to consider mTOR inhibitors as targeted therapeutic agents for patients with TSC-1 mutations. For example, Iyer et al. reported that TSC-1 mutations are associated with increased sensitivity to everolimus, an mTOR inhibitor [28]. In our study, TSC-1 expression was significantly associated with recurrence ( p = 0.003 ). Patients positive for TSC-1 were predominantly found in the recurrence group, while TSC-1 negativity was associated with nonrecurrence. According to our findings, TSC-1 may serve as a predictive marker for recurrence in patients with low-grade NMIBC. The importance of this observation lies in the fact that, to our knowledge, TSC-1 has not previously been studied in the context of recurrence prediction in this specific patient group. It is essential to differentiate between prognostic and predictive implications when interpreting biomarker data. Prognostic markers estimate the intrinsic risk of recurrence or progression independent of therapy, while predictive markers signify the probability of benefitting from a particular treatment. In this study, CD44 and TSC-1 served as prognostic predictors of recurrence risk in low-risk NMIBC, rather than as predictive markers of therapy response. This interpretation is consistent with the 2024 EAU Guidelines, which underscore the persistent necessity for validated biomarkers to enhance risk classification in NMIBC, while acknowledging that none have been established for practical application [29]. Moreover, current genomic classifications, including the extensive molecular subtyping outlined by Robertson et al. [30], establish a framework that connects genetic abnormalities to clinical behavior. Our results indicate that TSC-1 may represent biological processes linked to these subtypes and could facilitate a more refined prognostic classification if validated in larger, prospective, genomically informed cohorts. Limitations This study was retrospective and conducted at a single center with a limited sample size, which restricts its generalizability. Despite the post hoc power analysis indicating a power of 0.74 to detect an OR greater than 3.0 at α = 0.05, the limited cohort size inherently limits statistical precision. No adjustment for multiple comparisons was implemented, and this limitation has been clearly acknowledged. Notwithstanding these challenges, concentrating only on low-risk NMIBC enhances internal validity and offers new insights into the predictive significance of TSC-1. We anticipate that TSC-1 and CD44 may guide future research and contribute to risk stratification in low-grade NMIBC. Another limitation of this study is the possibility of selection bias due to the inclusion and exclusion criteria. Specifically, patients with prior intravesical BCG therapy were eliminated to exclude the immunomodulatory influence of BCG on tumor biology and biomarker expression. This method decreased confounding but also limited the generalizability of our findings predominantly to BCG-naïve, low-risk NMIBC groups. Consequently, the findings may not be entirely applicable to individuals receiving adjuvant intravesical therapy or to those classified under higher-risk groups. This study contained multiple comparisons across various IHC markers and was exploratory; hence, the p -values were not adjusted for multiple testing. Consequently, the statistically significant relationships identified should be considered with caution and regarded as hypothesis-generating until corroborated in larger prospective cohorts. Conclusion In our study, CK5/6, CK20, GATA3 , and ELF-3 did not demonstrate statistical significance in predicting tumor recurrence in NMIBC. In contrast, CD44 and TSC-1 were identified as potential predictive markers. Specifically, low CD44 expression and high TSC-1 expression were associated with an increased risk of recurrence in patients with low-risk NMIBC. These immunohistochemical markers may allow for the early identification of patients at higher risk, enabling closer surveillance and the earlier management of subsequent treatment strategies. To further validate these findings, multicenter, large-scale, randomized prospective studies should be conducted in diverse bladder cancer patient populations. Declarations Conflict of interest None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission. Ethical Statement A written informed consent was obtained from participants (for the ones under age 18, a written informed consent was obtained from their parent/legal guardian/next of kin) to participate in the study. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Study approval statement: This study protocol was reviewed and approved by [Tekirdag Namik Kemal University, Non-interventional Clinical Trials Ethical Committee], approval number [2021.227.09.13], date [09.28.2021] Materials and Methods This retrospective study included 40 patients with NMIBC selected from 320 individuals who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019. Patients were categorized into recurrence ( n = 20) and nonrecurrence ( n = 20) groups. Tissues were reevaluated and stained for CK4, CK5/6, CK20, CD44, GATA3 , Cerb-B2, ELF-3, and TSC-1 using IHC methods. Associations between clinicopathological variables and recurrence were statistically analyzed. Results Recurrence correlated significantly with tumor number ( p = 0.036 ), while age, sex, smoking status, and tumor size showed no significant association ( p > 0.05). CD44 positivity was predominantly observed in nonrecurrent cases ( p = 0.022 ), whereas TSC-1 positivity was strongly associated with recurrence ( p = 0.003 ). Multivariate regression revealed that CD44 positivity reduced the recurrence risk (OR = 0.142), while TSC-1 positivity increased it more than 21-fold (OR = 21.871). Tumors located at the ureteral orifice exhibited a lower recurrence risk (OR = 0.098). Conclusion This study indicates that low CD44 expression and high TSC-1 expression in patients with low-risk NMIBC may serve as predictors of tumor recurrence. Assessment of these markers could enable earlier identification of patients at increased risk, thereby facilitating strict surveillance and timely management of treatment strategies. Introduction Bladder cancer (BC) accounts for approximately 5% of all newly diagnosed cancers in the United States and represents the seventh most common malignancy [ 1 ]. Based on the molecular pathways of tumorigenesis, two distinct categories of BC have been described. The first category includes localized, papillary, and low-grade tumors arising from urothelial hyperplasia. This histological type generally exhibits a more favorable clinical course and is frequently associated with mutations in the fibroblast growth factor receptor 3 ( FGFR3 ) gene [ 2 ]. The second category includes nonpapillary, high-grade tumors that typically develop in the setting of severe dysplasia and carcinoma in situ (CIS). These cancers are characterized by higher rates of progression and muscle invasion, resulting in poor long-term survival outcomes [ 3 ]. Although immunohistochemical (IHC) markers, such as CK5/6, CK20 (cytokeratin 5/6 and 20), TP53 (tumor protein 53), and CD44 (cluster of differentiation 44), are commonly used to distinguish between these molecular pathways, no universally accepted gold standard marker has been established [ 4 , 5 ]. The primary objective of our study was to evaluate the potential of ELF-3 (E74-like transcription factor 3) and TSC-1 (tuberous sclerosis complex subunit 1), along with other IHC markers, as predictors of recurrence and progression in patients with low-risk non-muscle invasive BC (NMIBC). A secondary objective was to explore whether risk stratification using these markers could help tailor patient-specific surveillance strategies, even though cystoscopy remains the gold standard for follow-up in bladder cancer [ 6 ]. Materials and Methods This study was approved by the Non-Interventional Clinical Research Ethics Committee of Tekirdağ Namık Kemal University. (Decision No: 2021.227.09.13) and was supported by the Scientific Research Projects (SRP) Coordination Unit of Tekirdağ Namık Kemal University as a medical specialty thesis project (Project Code: NKUBAP.02.TU.22.361). We retrospectively reviewed the data of 320 patients who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019 and who were followed up in our clinic. A total of 40 patients aged 18 years or older with a pathological diagnosis of NIMBC and regular cystoscopic follow-up met the inclusion criteria and were enrolled. Patients who had received Bacille Calmette-Guérin (BCG) therapy before recurrence were excluded to avoid potential bias. Immunohistochemical Examination For immunohistochemical (IHC) analysis, 4-µm sections were prepared from all paraffin-embedded tissue blocks. The sections were incubated in a 60°C oven for 1 hour, followed by deparaffinization with xylene for 15 minutes. Subsequently, they were passed through graded alcohols, rinsed in distilled water, and processed in the BenchMark XT automated staining system. All samples were stained with the following antibodies: CK5/6 (Cytokeratin 5/6), CK20, CD44 (anti-CD44 SP37 rabbit monoclonal primary antibody), GATA3 (L50-823 mouse monoclonal primary antibody), Cerb-B2 (human epidermal growth factor receptor 2), ELF-3, and TSC-1. The stained slides were coverslipped using a liquid-based mounting medium. Microscopic evaluation was performed using an Olympus CX41 microscope (Munich, Germany). For each section, 100 tumor cells were counted at 100× magnification, and cytoplasmic or nuclear staining was scored semi-quantitatively. Staining intensity was categorized into five groups: score 0 (0–10% staining), score 1 (10–25%), score 2 (25–50%), score 3 (50–75%), and score 4 (> 75%). The cutoff value for positivity was set at 50% for GATA3 and CD44 and at 25% for CK5/6 and CK20. Accordingly, scores 0 and 1 were classified as negative, while scores 2–4 were considered positive for CK5/6 and CK20 (Fig. 1). For GATA3 and CD44, scores 0–2 were defined as negative, whereas scores 3 and 4 were defined as positive (Fig. 2). For Cerb-B2, ELF-3, and TSC-1, the cutoff value for positivity was set at 75%. Complete membranous staining in neoplastic cells was required for positivity; score 4 was defined as positive, score 3 as equivocal, and scores 0–2 as negative (Fig. 3). Funding: This investigation was supported by Tekirdağ Namık Kemal University Scientific Research Projects Committee with project number NKUBAP.02.TU.22.361. Author Contribution E.A., Ç.D., E.C.T. and M.F.Ş: Conceptualization, Investigation, Writing Original Draft Preparation, Writing- Review & Editing, Project AdministrationA.İ.A. and H.M.A: Conceptualization, Investigation, Writing -Original Draft Preparation, Writing- Review & Editing, Project AdministrationC.M.Y: Conceptualization, Supervision, Writing -Review & Editing, Funding Acquisition Acknowledgement: None References Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder cancer incidence and mortality: a global overview and recent trends. Eur Urol. 2017;71(1):96–108. https://doi.org/10.1016/j.eururo.2016.06.010 Billerey C, Chopin D, Aubriot-Lorton MH, Ricol D, Gil Diez de Medina S, Van Rhijn B, et al. Frequent FGFR3 mutations in papillary non-invasive bladder (pTa) tumors. Am J Pathol. 2001;158(6):1955–1959. https://doi.org/10.1016/S0002-9440(10)64665-2 Santos L, Pereira S, Leite RP, Souto M, Amaro T, Criado B. 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J Immunol Res. 2018;2018:5690258. https://doi.org/10.1155/2018/5690258 Buchumensky V, Klein A, Zemer R, Kessler OJ, Zimlichman S, Nissenkorn I. Cytokeratin 20 as a marker for early detection of bladder carcinoma. J Urol. 1998;160(6):1971–1974. Enache M, Simionescu CE, Stepan A. EGFR and Her2/neu expression in papillary urothelial bladder carcinomas. Rom J Morphol Embryol. 2013;54(1):137–141. Alexa A, Baderca F, Zăhoi DE, Lighezan R, Izvernariu D, Raica M. Clinical significance of Her2/neu overexpression in urothelial carcinomas. Rom J Morphol Embryol. 2010;51(2):277–282. Park SH, Kim YS, Park BK, Hougaard S, Kim SJ. Sequence-specific enhancer binding protein and gastric cancer. Oncogene. 2001;20(10):1235–1245. https://doi.org/10.1038/sj.onc.1204227 Na L, Wang Z, Bai Y, Sun Y, Dong D, Wang W, et al. WNT7B represses epithelial–mesenchymal transition and stem-like properties in bladder carcinoma. Biochim Biophys Acta Mol Basis Dis. 2022;1868(1):166271. https://doi.org/10.1016/j.bbadis.2021.166271 Iyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, Janakiraman M, et al. Genome sequencing identifies a basis for everolimus sensitivity. Science. 2012;338(6104):221. https://doi.org/10.1126/science.1226344 Gontero P, Birtle A, Capoun O, Compérat E, Dominguez-Escrig JL, Liedberg F, et al. EAU guidelines on non–muscle-invasive bladder cancer: 2024 update. Eur Urol. 2024;86(6):531–549. https://doi.org/10.1016/j.eururo.2024.07.027 Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell. 2017;171(3):540–556.e25. https://doi.org/10.1016/j.cell.2017.09.007 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8782269","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588315829,"identity":"2cae29a6-bc43-481d-b3a3-fccc87ebb6b7","order_by":0,"name":"Enes Altın","email":"","orcid":"","institution":"Denizli Tavas Hospital","correspondingAuthor":false,"prefix":"","firstName":"Enes","middleName":"","lastName":"Altın","suffix":""},{"id":588315830,"identity":"e5a4df2b-4c7c-444c-8b75-dc0ec2cfdfcc","order_by":1,"name":"Çağrı Doğan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDCCA2BSgrEBRH0AYjZ2UrQwzgBpYSZOCwNYCzMPiElIC9/tww8/ft1jIdvPv/jZY5tf2+T5mBkYP3zMwa1F8lyasbTMMwnjmTOemRvn9t02bGNmYJacuQ23FoMzPAzSEgckEjfcOGAmndtzmxGohY2ZF78W5t8QLce/SVv23LYnRgub5AeQlvM9ZtIMP24nEtQieYbNzJrhAMgvPGWSvQ23k9uYGZvx+oXvDPPjmz8O1AFD7Pg2iR9/btvOb28++OEjHi0gAIkOiQRg7LSBWJCUgBcw/gCR/AeAxB+CikfBKBgFo2AEAgCrfFPK1M9q0gAAAABJRU5ErkJggg==","orcid":"","institution":"Namık Kemal University","correspondingAuthor":true,"prefix":"","firstName":"Çağrı","middleName":"","lastName":"Doğan","suffix":""},{"id":588315831,"identity":"34aec985-38e5-4b26-b9a1-58f2b53b56fe","order_by":2,"name":"Erdem Can Topkaç","email":"","orcid":"","institution":"Namık Kemal University","correspondingAuthor":false,"prefix":"","firstName":"Erdem","middleName":"Can","lastName":"Topkaç","suffix":""},{"id":588315832,"identity":"9e3512d3-354a-4107-a595-d9ee0646d969","order_by":3,"name":"Mehmet Fatih Şahin","email":"","orcid":"","institution":"Namık Kemal University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Fatih","lastName":"Şahin","suffix":""},{"id":588315833,"identity":"251051cd-2a81-4523-8ee6-63840558e033","order_by":4,"name":"Ayşegül İsal Arslan","email":"","orcid":"","institution":"Tekirdağ Çerkezköy Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ayşegül","middleName":"İsal","lastName":"Arslan","suffix":""},{"id":588315834,"identity":"03e5e3b1-b088-4c20-b8b9-ee1153813063","order_by":5,"name":"Hacı Murat Akgül","email":"","orcid":"","institution":"Ümraniye Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Hacı","middleName":"Murat","lastName":"Akgül","suffix":""},{"id":588315835,"identity":"92e02903-0724-45db-b43b-821e973fdf26","order_by":6,"name":"Cenk Murat Yazıcı","email":"","orcid":"","institution":"Namık Kemal University","correspondingAuthor":false,"prefix":"","firstName":"Cenk","middleName":"Murat","lastName":"Yazıcı","suffix":""}],"badges":[],"createdAt":"2026-02-04 05:54:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8782269/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8782269/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102530234,"identity":"7e8354f1-a554-41a2-9716-8ef2096fa791","added_by":"auto","created_at":"2026-02-12 16:15:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":202198,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of positive staining of CK5/6 and CK20 immunohistochemical markers. (All histopathology figures now feature magnification levels (×100 or ×400), appropriate scale bars, and labeled markers for enhanced clarity. Representative staining patterns for CD44, TSC-1, and other evaluated markers are presented with uniform annotation.)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8782269/v1/db90cece93f8297729b01dd2.jpg"},{"id":102530232,"identity":"0f1f27a8-831b-4e7e-866d-8674eb84cf4a","added_by":"auto","created_at":"2026-02-12 16:15:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178670,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of positive staining of CD44 and GATA3 immunohistochemical markers (All histopathology figures now feature magnification levels (×100 or ×400), appropriate scale bars, and labeled markers for enhanced clarity. Representative staining patterns for CD44, TSC-1, and other evaluated markers are presented with uniform annotation.)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8782269/v1/f6bdeee9a883fa86e26aa871.jpg"},{"id":102746368,"identity":"c16f6b24-7806-45e6-9cba-8feb9728f7e7","added_by":"auto","created_at":"2026-02-16 08:57:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100099,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of positive staining of the immunohistochemical markers CerbB2, ELF-3, and TSC-1. (All histopathology figures now feature magnification levels (×100 or ×400), appropriate scale bars, and labeled markers for enhanced clarity. Representative staining patterns for CD44, TSC-1, and other evaluated markers are presented with uniform annotation.)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8782269/v1/05392c9a88a460fb9ed2812c.jpg"},{"id":102750638,"identity":"f57adce5-e1f9-4bfd-a1c4-9afd92b2c71a","added_by":"auto","created_at":"2026-02-16 09:21:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1319666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8782269/v1/e0bfbd93-287f-4f2f-99fd-621b4d0a605f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA New Marker for Prediction of Recurrence in Low-Risk Bladder Cancer: Retrospective Study of ELF-3 and TSC-1\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer (BC) accounts for approximately 5% of all newly diagnosed cancers in the United States and represents the seventh most common malignancy [1]. Based on the molecular pathways of tumorigenesis, two distinct categories of BC have been described. The first category includes localized, papillary, and low-grade tumors arising from urothelial hyperplasia. This histological type generally exhibits a more favorable clinical course and is frequently associated with mutations in the fibroblast growth factor receptor 3 (\u003cem\u003eFGFR3\u003c/em\u003e) gene [2]. The second category includes nonpapillary, high-grade tumors that typically develop in the setting of severe dysplasia and carcinoma in situ (CIS). These cancers are characterized by higher rates of progression and muscle invasion, resulting in poor long-term survival outcomes [3].\u003c/p\u003e\n\u003cp\u003eAlthough immunohistochemical (IHC) markers, such as CK5/6, CK20 (cytokeratin 5/6 and 20), TP53 (tumor protein 53), and CD44 (cluster of differentiation 44), are commonly used to distinguish between these molecular pathways, no universally accepted gold standard marker has been established [4, 5]. The primary objective of our study was to evaluate the potential of ELF-3 (E74-like transcription factor 3) and TSC-1 (tuberous sclerosis complex subunit 1), along with other IHC markers, as predictors of recurrence and progression in patients with low-risk non-muscle invasive BC (NMIBC). A secondary objective was to explore whether risk stratification using these markers could help tailor patient-specific surveillance strategies, even though cystoscopy remains the gold standard for follow-up in bladder cancer [6].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis study was approved by the Non-Interventional Clinical Research Ethics Committee of Tekirdağ Namık Kemal University. (Decision No: 2021.227.09.13) and was supported by the Scientific Research Projects (SRP) Coordination Unit of Tekirdağ Namık Kemal University as a medical specialty thesis project (Project Code: NKUBAP.02.TU.22.361). We retrospectively reviewed the data of 320 patients who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019 and who were followed up in our clinic. A total of 40 patients aged 18 years or older with a pathological diagnosis of NIMBC and regular cystoscopic follow-up met the inclusion criteria and were enrolled. Patients who had received Bacille Calmette-Guérin (BCG) therapy before recurrence were excluded to avoid potential bias.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunohistochemical (IHC) analysis, 4-µm sections were prepared from all paraffin-embedded tissue blocks. The sections were incubated in a 60 °C oven for 1 hour, followed by deparaffinization with xylene for 15 minutes. Subsequently, they were passed through graded alcohols, rinsed in distilled water, and processed in the BenchMark XT automated staining system. All samples were stained with the following antibodies: CK5/6 (Cytokeratin 5/6), CK20, CD44 (anti-CD44 SP37 rabbit monoclonal primary antibody), GATA3 (L50-823 mouse monoclonal primary antibody), Cerb-B2 (human epidermal growth factor receptor 2), ELF-3, and TSC-1. The stained slides were coverslipped using a liquid-based mounting medium.\u003c/p\u003e\n\u003cp\u003eMicroscopic evaluation was performed using an Olympus CX41 microscope (Munich, Germany). For each section, 100 tumor cells were counted at 100× magnification, and cytoplasmic or nuclear staining was scored semi-quantitatively. Staining intensity was categorized into five groups: score 0 (0–10% staining), score 1 (10–25%), score 2 (25–50%), score 3 (50–75%), and score 4 (\u0026gt; 75%).\u003c/p\u003e\n\u003cp\u003eThe cutoff value for positivity was set at 50% for GATA3 and CD44 and at 25% for CK5/6 and CK20. Accordingly, scores 0 and 1 were classified as negative, while scores 2–4 were considered positive for CK5/6 and CK20 (Figure 1). For GATA3 and CD44, scores 0–2 were defined as negative, whereas scores 3 and 4 were defined as positive (Figure 2). For Cerb-B2, ELF-3, and TSC-1, the cutoff value for positivity was set at 75%. Complete membranous staining in neoplastic cells was required for positivity; score 4 was defined as positive, score 3 as equivocal, and scores 0–2 as negative (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using IBM SPSS Statistics version 24. Frequency tables and descriptive statistics were used to summarize the findings. Parametric tests were applied to continuous variables with normal distributions, whereas non-parametric tests were used for variables that did not meet the assumption of normality. For comparisons between the two independent groups, the independent samples \u003cem\u003et\u003c/em\u003e-test was employed for normally distributed variables, while the Mann–Whitney U test was used for non-normally distributed variables. Associations between categorical variables were evaluated using Pearson’s χ² test. Factors influencing recurrence were further analyzed using binary logistic regression with the backward likelihood ratio (backward LR) method.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 40 patients with NMIBC were included in the study. Of these, 20 had recurrence during follow-up, and 20 were recurrence-free. No statistically significant associations were found between recurrence status and gender, occupation, ongoing smoking, tumor localization, or early postoperative epirubicin administration. In contrast, a significant difference was observed in terms of the initial tumor number (\u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.022\u003c/strong\u003e). Sixteen patients (80.0%) in the nonrecurrence group presented with a single tumor, whereas nine patients (45.0%) in the recurrence group had 2\u0026ndash;4 tumors. The mean tumor number was 1.50 \u0026plusmn; 1.15 in the nonrecurrence group and 2.45 \u0026plusmn; 2.09 in the recurrence group. Receiver operating characteristic (ROC) analysis identified 1.5 as the optimal cutoff number of tumors for predicting recurrence, with 60.0% sensitivity and 80.0% specificity (AUC = 0.686; \u003cem\u003ep\u003c/em\u003e \u0026lt; \u003cstrong\u003e0.05\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWhen postoperative epirubicin administration was evaluated, it was applied in 8 of 20 patients (40%) without recurrence and in 9 of 20 patients (45%) with recurrence. Epirubicin use did not significantly influence recurrence development, time to recurrence (months), or recurrence size (cm). The detailed evaluation of factors potentially associated with recurrence is summarized in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eEvaluation of factors that may affect recurrence status\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecurrence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo (\u003cem\u003en\u003c/em\u003e = 20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYes (\u003cem\u003en\u003c/em\u003e = 20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical analysis*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eProbability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e64.25 \u0026plusmn; 10.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e64.5\u003c/p\u003e\n \u003cp\u003e[48.0-86.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e63.00 \u0026plusmn; 11.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e63.0\u003c/p\u003e\n \u003cp\u003e[38.0\u0026ndash;80.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e =0.372\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e27.94 \u0026plusmn; 2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003cp\u003e[23.1-32.3]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e28.14 \u0026plusmn; 3.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e27.4\u003c/p\u003e\n \u003cp\u003e[23.0\u0026ndash;34.3]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e = \u0026minus;0.177\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.860\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCigarettes (packs/year)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e36.00 \u0026plusmn; 25.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003cp\u003e[0.0-90.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e25.75 \u0026plusmn; 21.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003cp\u003e[0.0\u0026ndash;75.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003eZ\u003c/em\u003e = \u0026minus;1.352\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaximum tumor size (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e1.85 \u0026plusmn; 0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003cp\u003e[0.5-3.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e2.10 \u0026plusmn; 0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003cp\u003e[0.5\u0026ndash;3.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003eZ\u003c/em\u003e = \u0026minus;0.877\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.380\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* For variables with a normal distribution, comparisons between two independent groups were performed using the Independent Samples \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003et\u003c/em\u003e statistic). For variables not normally distributed, comparisons were conducted using the Mann\u0026ndash;Whitney U test (Z statistic).\u003c/p\u003e\n\u003cp\u003eA statistically significant difference was observed between recurrence status and ureteral orifice tumor localization (\u0026chi;\u0026sup2; = 5.625; \u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.018\u003c/strong\u003e). Seven patients (35.0%) in the nonrecurrence group had tumors originating from the ureteral orifice, whereas 19 patients (95.0%) in the recurrence group did not. Thus, tumors arising from the ureteral orifice were predominantly associated with nonrecurrence.\u003c/p\u003e\n\u003cp\u003eThe differences between groups of IHC staining results are presented in Table 2. No statistically significant difference was identified between recurrence status and CK5/6, GATA3, Cerb-B2, CK20, or ELF-3 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eEvaluation of the relationship between recurrence and IHC staining\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Relapse status\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo (\u003cem\u003en\u003c/em\u003e = 20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYes (\u003cem\u003en\u003c/em\u003e = 20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical analysis*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eProbability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eN\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eN\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD44\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003cp\u003e80.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;= 0.022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCK5/6\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003cp\u003e60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003cp\u003e60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGATA3\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerbB2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCK20\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e = 0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eELF-3\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSC-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;= 0003\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u0026nbsp;\u0026ldquo;Pearson-\u0026chi;\u003csup\u003e2\u003c/sup\u003e cross tables\u0026rdquo; were used to examine the relationships between two qualitative variables.\u003c/p\u003e\n\u003cp\u003eA statistically significant difference was observed between recurrence status and CD44 expression (\u0026chi;\u0026sup2; = 5.227; \u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.022\u003c/strong\u003e). CD44 positivity was detected in 11 patients (55.0%) without recurrence, whereas 16 patients (80.0%) in the recurrence group were CD44 negative. Thus, CD44 positivity was predominantly associated with the absence of recurrence, while CD44 negativity was associated with recurrence.\u003c/p\u003e\n\u003cp\u003eSimilarly, a significant relationship was found between recurrence status and TSC-1 expression (\u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.003\u003c/strong\u003e). Nine patients (45.0%) in the nonrecurrence group were TSC-1 negative, compared with 19 patients (95.0%) in the recurrence group who were TSC-1 positive. Accordingly, TSC-1 positivity was primarily associated with recurrence, whereas TSC-1 negativity was associated with nonrecurrence.\u003c/p\u003e\n\u003cp\u003eMultivariate logistic regression analysis, incorporating all parameters that showed significance in univariate analyses, confirmed these findings. In the final model, CD44 remained an independent factor influencing recurrence (\u003cem\u003ep\u003c/em\u003e \u0026lt; \u003cstrong\u003e0.05\u003c/strong\u003e) (Table 3), with CD44 positivity reducing recurrence risk by approximately 85.8% compared with CD44 negativity (OR = 0.142). Conversely, TSC-1 positivity was associated with a 21.8-fold increased risk of recurrence (\u003cem\u003ep\u003c/em\u003e \u0026lt; \u003cstrong\u003e0.05\u003c/strong\u003e). In addition, ureteral orifice involvement was a significant factor (\u003cem\u003ep\u003c/em\u003e \u0026lt; \u003cstrong\u003e0.05\u003c/strong\u003e), with tumors located at the ureteral orifice demonstrating a 90.2% lower risk of recurrence compared with those without such localization (OR = 0.098).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eEvaluation of the relationship between tumor number and recurrence status.\u0026nbsp;\u003cstrong\u003eAUC:\u0026nbsp;\u003c/strong\u003eArea under the curve\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAUC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eerror\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% confidence interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCutoff\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLower limit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUpper limit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of tumors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.044\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe median follow-up period for the study cohort was 36 months (IQR: 24\u0026ndash;52 months). The median duration of recurrence-free survival was 28 months. All recurrences were histologically confirmed after cystoscopic identification, ensuring diagnostic accuracy and consistency in the outcome evaluation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCD44 is a cell adhesion molecule that has been implicated in promoting tumor progression and dissemination [7–9]. However, its prognostic value in urothelial carcinoma remains controversial [10, 11]. Recent meta-analyses have demonstrated a statistically significant difference between CD44 expression and advanced T stage, lymph node metastasis, and adverse oncological outcomes, suggesting that CD44 expression may be linked to tumor progression, metastasis, and treatment failure in bladder cancer [11, 12]. Conversely, Raspollini et al. reported in their study on T1 high-grade urothelial carcinoma that CD44 expression was not associated with poor prognostic factors [13]. In our cohort, CD44 positivity was noted in 11 of 20 patients (55%) without recurrence, in contrast to 4 of 20 patients (20%) in the recurrence group, indicating a statistically significant difference (\u003cem\u003ep\u003c/em\u003e =\u0026nbsp;\u003cstrong\u003e0.022\u003c/strong\u003e). These results are consistent with those reported by Raspollini et al. This apparent discrepancy in the literature may be explained by the specific characteristics of our study population, which was limited to patients with NMIBC. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCytokeratins (CKs) are structural proteins that form part of the cytoskeleton of squamous (stratified) epithelial cells. Extensive immunohistochemical research has been performed on CKs in a variety of cancers, as they are regulators of proliferation in epithelial-derived squamous cells [14, 15]. Jung et al. found that within the NMIBC group, tumors expressing CK5/6 were more likely to be of a higher grade [16]. While some studies in the literature suggest that CK5/6 expression may serve as a prognostic marker, others have concluded that it lacks prognostic significance. In our study, no statistically significant difference was observed between patients with and without recurrence in terms of CK5/6 staining (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGATA3\u003c/em\u003e, also known as GATA binding protein 3, is a T cell–specific gene that functions as a transcription factor located in the T cell receptor (TCR) α region [17]. \u003cem\u003eGATA3\u003c/em\u003e has been investigated in a variety of tumors other than bladder cancer, and it has been suggested to serve as an IHC marker with potential prognostic relevance [18, 19]. In studies conducted by Kamel et al., \u003cem\u003eGATA3\u003c/em\u003e expression in patients with NMIBC was reported to be associated with favorable prognostic factors [20]. However, most published studies have been conducted in either MIBC or general NMIBC populations, and data specific to low-grade NMIBC remain limited. In our study, \u003cem\u003eGATA3\u003c/em\u003e staining was observed in all patients (100%) without recurrence and in 17 of 20 patients (85%) with recurrence. This difference did not show statistical significance (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that low CK20 expression is associated with poor recurrence-free survival (RFS) and cancer-specific survival (CSS) [21, 22]. In contrast, Buchumensky et al. reported no association between tumor grade and CK20 expression [23]. While the literature supports CK20 as a reliable marker for identifying the luminal subgroup, its prognostic value for predicting recurrence, tumor grade, and aggressiveness remains inconsistent. In our study, no statistically significant differences in CK20 expression were observed between the recurrence and nonrecurrence groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Studies conducted in populations similar to ours have yielded comparable results, thus supporting the results of our study.\u003c/p\u003e\n\u003cp\u003eCerb-B2, also known as HER2/neu, is a protein encoded by the \u003cem\u003eERBB2\u003c/em\u003e gene. Enache et al. concluded that Cerb-B2 expression is important for identifying poorly differentiated, advanced-stage tumors [24], whereas Alexa et al. found no relationship between Cerb-B2 overexpression and tumor stage [25]. In our study, Cerb-B2 expression was also evaluated, and no significant differences were observed between the recurrence and nonrecurrence groups.\u003c/p\u003e\n\u003cp\u003eELF-3 is a member of the ETS (erythrocyte transformation-specific) transcription factor family [26]. Na et al. demonstrated that increased ELF-3 signaling exerts a tumor-suppressive role in bladder cancer [27]. However, its role in the molecular subtyping of bladder cancer remains uncertain, and research in this area is still in its early stages. In our cohort, ELF-3 expression was uniformly positive (100%) in both groups, with no ability to differentiate between patients with and without recurrence. Thus, ELF-3 was not found to be an effective marker for predicting recurrence. Despite ELF-3 being initially considered a possible marker due to its reported role in urothelial differentiation, our results demonstrated consistent expression across all specimens, suggesting that it lacks predictive value in low-risk NMIBC. Consequently, the study’s primary focus transitioned to CD44 and TSC-1, which exhibited strong and independent correlations with recurrence risk.\u003c/p\u003e\n\u003cp\u003eTSC-1, also known as hamartin, is a protein encoded by the \u003cem\u003eTSC-1\u003c/em\u003e gene and functions as a co-chaperone that inhibits the ATPase activity of HSP90, thereby regulating the chaperone cycle. Dadhania et al. identified TSC-1 as a marker of the luminal subtype in bladder cancer [6]. Although the precise role of TSC-1 in bladder cancer biology remains unclear, it has been implicated in tumor development through its interaction with the mTOR pathway. This has prompted researchers to consider mTOR inhibitors as targeted therapeutic agents for patients with TSC-1 mutations. For example, Iyer et al. reported that TSC-1 mutations are associated with increased sensitivity to everolimus, an mTOR inhibitor [28]. In our study, TSC-1 expression was significantly associated with recurrence (\u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.003\u003c/strong\u003e). Patients positive for TSC-1 were predominantly found in the recurrence group, while TSC-1 negativity was associated with nonrecurrence. According to our findings, TSC-1 may serve as a predictive marker for recurrence in patients with low-grade NMIBC. The importance of this observation lies in the fact that, to our knowledge, TSC-1 has not previously been studied in the context of recurrence prediction in this specific patient group.\u003c/p\u003e\n\u003cp\u003eIt is essential to differentiate between prognostic and predictive implications when interpreting biomarker data. Prognostic markers estimate the intrinsic risk of recurrence or progression independent of therapy, while predictive markers signify the probability of benefitting from a particular treatment. In this study, CD44 and TSC-1 served as prognostic predictors of recurrence risk in low-risk NMIBC, rather than as predictive markers of therapy response. This interpretation is consistent with the 2024 EAU Guidelines, which underscore the persistent necessity for validated biomarkers to enhance risk classification in NMIBC, while acknowledging that none have been established for practical application [29]. Moreover, current genomic classifications, including the extensive molecular subtyping outlined by Robertson et al. [30], establish a framework that connects genetic abnormalities to clinical behavior. Our results indicate that TSC-1 may represent biological processes linked to these subtypes and could facilitate a more refined prognostic classification if validated in larger, prospective, genomically informed cohorts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was retrospective and conducted at a single center with a limited sample size, which restricts its generalizability. Despite the post hoc power analysis indicating a power of 0.74 to detect an OR greater than 3.0 at α = 0.05, the limited cohort size inherently limits statistical precision. No adjustment for multiple comparisons was implemented, and this limitation has been clearly acknowledged. Notwithstanding these challenges, concentrating only on low-risk NMIBC enhances internal validity and offers new insights into the predictive significance of TSC-1. We anticipate that TSC-1 and CD44 may guide future research and contribute to risk stratification in low-grade NMIBC. Another limitation of this study is the possibility of selection bias due to the inclusion and exclusion criteria. Specifically, patients with prior intravesical BCG therapy were eliminated to exclude the immunomodulatory influence of BCG on tumor biology and biomarker expression. This method decreased confounding but also limited the generalizability of our findings predominantly to BCG-naïve, low-risk NMIBC groups. Consequently, the findings may not be entirely applicable to individuals receiving adjuvant intravesical therapy or to those classified under higher-risk groups. This study contained multiple comparisons across various IHC markers and was exploratory; hence, the \u003cem\u003ep\u003c/em\u003e-values were not adjusted for multiple testing. Consequently, the statistically significant relationships identified should be considered with caution and regarded as hypothesis-generating until corroborated in larger prospective cohorts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn our study, CK5/6, CK20, \u003cem\u003eGATA3\u003c/em\u003e, and ELF-3 did not demonstrate statistical significance in predicting tumor recurrence in NMIBC. In contrast, \u003cem\u003eCD44\u003c/em\u003e and \u003cem\u003eTSC-1\u003c/em\u003e were identified as potential predictive markers. Specifically, low CD44 expression and high TSC-1 expression were associated with an increased risk of recurrence in patients with low-risk NMIBC. These immunohistochemical markers may allow for the early identification of patients at higher risk, enabling closer surveillance and the earlier management of subsequent treatment strategies. To further validate these findings, multicenter, large-scale, randomized prospective studies should be conducted in diverse bladder cancer patient populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eNone of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Statement\u003c/h2\u003e \u003cp\u003e A written informed consent was obtained from participants (for the ones under age 18, a written informed consent was obtained from their parent/legal guardian/next of kin) to participate in the study. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eStudy approval statement:\u003c/h2\u003e \u003cp\u003e This study protocol was reviewed and approved by [Tekirdag Namik Kemal University, Non-interventional Clinical Trials Ethical Committee], approval number [2021.227.09.13], date [09.28.2021]\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eThis retrospective study included 40 patients with NMIBC selected from 320 individuals who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019. Patients were categorized into recurrence (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) and nonrecurrence (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) groups. Tissues were reevaluated and stained for CK4, CK5/6, CK20, CD44, \u003cem\u003eGATA3\u003c/em\u003e, Cerb-B2, ELF-3, and TSC-1 using IHC methods. Associations between clinicopathological variables and recurrence were statistically analyzed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eResults\u003c/strong\u003e \u003cp\u003eRecurrence correlated significantly with tumor number (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.036\u003c/b\u003e), while age, sex, smoking status, and tumor size showed no significant association (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). CD44 positivity was predominantly observed in nonrecurrent cases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.022\u003c/b\u003e), whereas TSC-1 positivity was strongly associated with recurrence (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.003\u003c/b\u003e). Multivariate regression revealed that CD44 positivity reduced the recurrence risk (OR\u0026thinsp;=\u0026thinsp;0.142), while TSC-1 positivity increased it more than 21-fold (OR\u0026thinsp;=\u0026thinsp;21.871). Tumors located at the ureteral orifice exhibited a lower recurrence risk (OR\u0026thinsp;=\u0026thinsp;0.098).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConclusion\u003c/strong\u003e \u003cp\u003eThis study indicates that low CD44 expression and high TSC-1 expression in patients with low-risk NMIBC may serve as predictors of tumor recurrence. Assessment of these markers could enable earlier identification of patients at increased risk, thereby facilitating strict surveillance and timely management of treatment strategies.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003eBladder cancer (BC) accounts for approximately 5% of all newly diagnosed cancers in the United States and represents the seventh most common malignancy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Based on the molecular pathways of tumorigenesis, two distinct categories of BC have been described. The first category includes localized, papillary, and low-grade tumors arising from urothelial hyperplasia. This histological type generally exhibits a more favorable clinical course and is frequently associated with mutations in the fibroblast growth factor receptor 3 (\u003cem\u003eFGFR3\u003c/em\u003e) gene [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The second category includes nonpapillary, high-grade tumors that typically develop in the setting of severe dysplasia and carcinoma in situ (CIS). These cancers are characterized by higher rates of progression and muscle invasion, resulting in poor long-term survival outcomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough immunohistochemical (IHC) markers, such as CK5/6, CK20 (cytokeratin 5/6 and 20), TP53 (tumor protein 53), and CD44 (cluster of differentiation 44), are commonly used to distinguish between these molecular pathways, no universally accepted gold standard marker has been established [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The primary objective of our study was to evaluate the potential of ELF-3 (E74-like transcription factor 3) and TSC-1 (tuberous sclerosis complex subunit 1), along with other IHC markers, as predictors of recurrence and progression in patients with low-risk non-muscle invasive BC (NMIBC). A secondary objective was to explore whether risk stratification using these markers could help tailor patient-specific surveillance strategies, even though cystoscopy remains the gold standard for follow-up in bladder cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMaterials and Methods\u003c/strong\u003e \u003cp\u003e This study was approved by the Non-Interventional Clinical Research Ethics Committee of Tekirdağ Namık Kemal University. (Decision No: 2021.227.09.13) and was supported by the Scientific Research Projects (SRP) Coordination Unit of Tekirdağ Namık Kemal University as a medical specialty thesis project (Project Code: NKUBAP.02.TU.22.361). We retrospectively reviewed the data of 320 patients who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019 and who were followed up in our clinic. A total of 40 patients aged 18 years or older with a pathological diagnosis of NIMBC and regular cystoscopic follow-up met the inclusion criteria and were enrolled. Patients who had received Bacille Calmette-Gu\u0026eacute;rin (BCG) therapy before recurrence were excluded to avoid potential bias.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImmunohistochemical Examination\u003c/strong\u003e \u003cp\u003eFor immunohistochemical (IHC) analysis, 4-\u0026micro;m sections were prepared from all paraffin-embedded tissue blocks. The sections were incubated in a 60\u0026deg;C oven for 1 hour, followed by deparaffinization with xylene for 15 minutes. Subsequently, they were passed through graded alcohols, rinsed in distilled water, and processed in the BenchMark XT automated staining system. All samples were stained with the following antibodies: CK5/6 (Cytokeratin 5/6), CK20, CD44 (anti-CD44 SP37 rabbit monoclonal primary antibody), GATA3 (L50-823 mouse monoclonal primary antibody), Cerb-B2 (human epidermal growth factor receptor 2), ELF-3, and TSC-1. The stained slides were coverslipped using a liquid-based mounting medium.\u003c/p\u003e \u003cp\u003eMicroscopic evaluation was performed using an Olympus CX41 microscope (Munich, Germany). For each section, 100 tumor cells were counted at 100\u0026times; magnification, and cytoplasmic or nuclear staining was scored semi-quantitatively. Staining intensity was categorized into five groups: score 0 (0\u0026ndash;10% staining), score 1 (10\u0026ndash;25%), score 2 (25\u0026ndash;50%), score 3 (50\u0026ndash;75%), and score 4 (\u0026gt;\u0026thinsp;75%).\u003c/p\u003e \u003cp\u003eThe cutoff value for positivity was set at 50% for GATA3 and CD44 and at 25% for CK5/6 and CK20. Accordingly, scores 0 and 1 were classified as negative, while scores 2\u0026ndash;4 were considered positive for CK5/6 and CK20 (Fig.\u0026nbsp;1). For GATA3 and CD44, scores 0\u0026ndash;2 were defined as negative, whereas scores 3 and 4 were defined as positive (Fig.\u0026nbsp;2). For Cerb-B2, ELF-3, and TSC-1, the cutoff value for positivity was set at 75%. Complete membranous staining in neoplastic cells was required for positivity; score 4 was defined as positive, score 3 as equivocal, and scores 0\u0026ndash;2 as negative (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis investigation was supported by Tekirdağ Namık Kemal University Scientific Research Projects Committee with project number NKUBAP.02.TU.22.361.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.A., \u0026Ccedil;.D., E.C.T. and M.F.Ş: Conceptualization,\u0026nbsp;Investigation,\u0026nbsp;Writing Original\u0026nbsp;Draft Preparation,\u0026nbsp;Writing-\u0026nbsp;Review\u0026nbsp;\u0026amp;amp; Editing, Project AdministrationA.İ.A. and H.M.A: Conceptualization,\u0026nbsp;Investigation,\u0026nbsp;Writing\u0026nbsp;-Original\u0026nbsp;Draft\u0026nbsp;Preparation,\u0026nbsp;Writing-\u0026nbsp;Review\u0026nbsp;\u0026amp;amp; Editing, Project AdministrationC.M.Y: Conceptualization,\u0026nbsp;Supervision,\u0026nbsp;Writing\u0026nbsp;-Review\u0026nbsp;\u0026amp;amp; Editing,\u0026nbsp;Funding Acquisition\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAntoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. 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J Oral Pathol Med. 2015;44(6):393\u0026ndash;400. https://doi.org/10.1111/jop.12308\u003c/li\u003e\n\u003cli\u003eSacks JD, Barbolina MV. Expression and function of CD44 in epithelial ovarian carcinoma. Biomolecules. 2015;5(4):3051\u0026ndash;3066. https://doi.org/10.3390/biom5043051\u003c/li\u003e\n\u003cli\u003eLouderbough JM, Schroeder JA. Understanding the dual nature of CD44 in breast cancer progression. Mol Cancer Res. 2011;9(12):1573\u0026ndash;1586. https://doi.org/10.1158/1541-7786.MCR-11-0156\u003c/li\u003e\n\u003cli\u003eToma V, Hauri D, Schmid U, Ackermann D, Maurer R, Alund G, et al. Focal loss of CD44 variant protein expression is related to recurrence in superficial bladder carcinoma. Am J Pathol. 1999;155(5):1427\u0026ndash;1432.\u003c/li\u003e\n\u003cli\u003eHu Y, Zhang Y, Gao J, Lian X, Wang Y. Prognostic value of CD44 expression in bladder cancer: a meta-analysis and TCGA study. 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Value of CK5/6, CK14, ER and PR in intraductal proliferative breast lesions. Zhonghua Zhong Liu Za Zhi. 2015;37(10):749\u0026ndash;752.\u003c/li\u003e\n\u003cli\u003eJung M, Jang I, Kim K, Moon KC. Non-muscle-invasive bladder carcinoma with respect to basal versus luminal keratin expression. Int J Mol Sci. 2020;21(20):7726. https://doi.org/10.3390/ijms21207726\u003c/li\u003e\n\u003cli\u003eOrd\u0026oacute;\u0026ntilde;ez NG. Value of GATA3 immunostaining in tumor diagnosis. Adv Anat Pathol. 2013;20(5):352\u0026ndash;360. https://doi.org/10.1097/PAP.0b013e3182a28a68\u003c/li\u003e\n\u003cli\u003eLiu Y, Xu G, Li L. LncRNA GATA3-AS1/miR-30b-5p/TEX10 axis in pancreatic cancer. Oncol Rep. 2021;45(5):59. https://doi.org/10.3892/or.2021.8010\u003c/li\u003e\n\u003cli\u003eAsch-Kendrick R, Cimino-Mathews A. The role of GATA3 in breast carcinomas. Hum Pathol. 2016;48:37\u0026ndash;47. https://doi.org/10.1016/j.humpath.2015.09.035\u003c/li\u003e\n\u003cli\u003eKamel NA, Abdelzaher E, Elgebaly O, Ibrahim SA. Reduced expression of GATA3 predicts progression in non-muscle invasive urothelial carcinoma. J Histotechnol. 2020;43(1):21\u0026ndash;28.\u003c/li\u003e\n\u003cli\u003eLyn J, Wang Y, Wang F, Shen M, Zhou X. Diagnostic value of SATB2, CK7 and CK20 in colorectal cancer. Zhonghua Bing Li Xue Za Zhi. 2015;44(8):578\u0026ndash;581.\u003c/li\u003e\n\u003cli\u003eZhao YJ, Zhang J, Shi F, Hu ZP, Wu JP, Wu GJ, et al. PD-1 expression on CD4+ tumor-infiltrating lymphocytes in breast cancer. J Immunol Res. 2018;2018:5690258. https://doi.org/10.1155/2018/5690258\u003c/li\u003e\n\u003cli\u003eBuchumensky V, Klein A, Zemer R, Kessler OJ, Zimlichman S, Nissenkorn I. Cytokeratin 20 as a marker for early detection of bladder carcinoma. J Urol. 1998;160(6):1971\u0026ndash;1974.\u003c/li\u003e\n\u003cli\u003eEnache M, Simionescu CE, Stepan A. EGFR and Her2/neu expression in papillary urothelial bladder carcinomas. Rom J Morphol Embryol. 2013;54(1):137\u0026ndash;141.\u003c/li\u003e\n\u003cli\u003eAlexa A, Baderca F, Zăhoi DE, Lighezan R, Izvernariu D, Raica M. Clinical significance of Her2/neu overexpression in urothelial carcinomas. Rom J Morphol Embryol. 2010;51(2):277\u0026ndash;282.\u003c/li\u003e\n\u003cli\u003ePark SH, Kim YS, Park BK, Hougaard S, Kim SJ. Sequence-specific enhancer binding protein and gastric cancer. Oncogene. 2001;20(10):1235\u0026ndash;1245. https://doi.org/10.1038/sj.onc.1204227\u003c/li\u003e\n\u003cli\u003eNa L, Wang Z, Bai Y, Sun Y, Dong D, Wang W, et al. WNT7B represses epithelial\u0026ndash;mesenchymal transition and stem-like properties in bladder carcinoma. Biochim Biophys Acta Mol Basis Dis. 2022;1868(1):166271. https://doi.org/10.1016/j.bbadis.2021.166271\u003c/li\u003e\n\u003cli\u003eIyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, Janakiraman M, et al. Genome sequencing identifies a basis for everolimus sensitivity. Science. 2012;338(6104):221. https://doi.org/10.1126/science.1226344\u003c/li\u003e\n\u003cli\u003eGontero P, Birtle A, Capoun O, Comp\u0026eacute;rat E, Dominguez-Escrig JL, Liedberg F, et al. EAU guidelines on non\u0026ndash;muscle-invasive bladder cancer: 2024 update. Eur Urol. 2024;86(6):531\u0026ndash;549. https://doi.org/10.1016/j.eururo.2024.07.027\u003c/li\u003e\n\u003cli\u003eRobertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell. 2017;171(3):540\u0026ndash;556.e25. https://doi.org/10.1016/j.cell.2017.09.007\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bladder cancer, non-muscle invasive, ELF-3, CD44, TSC-1, NMIBC","lastPublishedDoi":"10.21203/rs.3.rs-8782269/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8782269/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTumor recurrence remains a major clinical challenge among patients with low-risk non-muscle invasive bladder cancer (NMIBC). Molecular studies have identified two main subtypes with distinct genetic pathways: papillary, low-grade tumors often associated with \u003cem\u003eFGFR3\u003c/em\u003e mutations and favorable outcomes, and nonpapillary, high-grade tumors characterized by high progression and muscle invasion rates. This study aims to evaluate the predictive value of immunohistochemical (IHC) markers—CD44, CK5/6, CK20, \u003cem\u003eGATA3\u003c/em\u003e, Cerb-B2, ELF-3, and TSC-1—for recurrence in patients with low-risk NMIBC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods: \u003c/strong\u003eThis retrospective study included 40 patients with NMIBC selected from 320 individuals who underwent transurethral resection of bladder tumors (TUR-BT) between 2011 and 2019. Patients were categorized into recurrence (\u003cem\u003en\u003c/em\u003e = 20) and nonrecurrence (\u003cem\u003en\u003c/em\u003e = 20) groups. Tissues were reevaluated and stained for CK4, CK5/6, CK20, CD44, \u003cem\u003eGATA3\u003c/em\u003e, Cerb-B2, ELF-3, and TSC-1 using IHC methods. Associations between clinicopathological variables and recurrence were statistically analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eRecurrence correlated significantly with tumor number (\u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.036\u003c/strong\u003e), while age, sex, smoking status, and tumor size showed no significant association (\u003cem\u003ep\u003c/em\u003e\u0026gt; 0.05). CD44 positivity was predominantly observed in nonrecurrent cases (\u003cem\u003ep\u003c/em\u003e= \u003cstrong\u003e0.022\u003c/strong\u003e), whereas TSC-1 positivity was strongly associated with recurrence (\u003cem\u003ep\u003c/em\u003e = \u003cstrong\u003e0.003\u003c/strong\u003e). Multivariate regression revealed that CD44 positivity reduced the recurrence risk (OR = 0.142), while TSC-1 positivity increased it more than 21-fold (OR = 21.871). Tumors located at the ureteral orifice exhibited a lower recurrence risk (OR = 0.098).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eThis study indicates that low CD44 expression and high TSC-1 expression in patients with low-risk NMIBC may serve as predictors of tumor recurrence. Assessment of these markers could enable earlier identification of patients at increased risk, thereby facilitating strict surveillance and timely management of treatment strategies.\u003c/p\u003e","manuscriptTitle":"A New Marker for Prediction of Recurrence in Low-Risk Bladder Cancer: Retrospective Study of ELF-3 and TSC-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 16:15:25","doi":"10.21203/rs.3.rs-8782269/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-16T09:52:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T22:00:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-07T13:53:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-01T20:06:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210175433376965310481347335288725710737","date":"2026-02-23T20:05:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-22T14:58:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54738593393625974610347246621621323136","date":"2026-02-22T14:53:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25852028694636506803632102863022094153","date":"2026-02-21T08:32:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151513349569905559925314306844108986210","date":"2026-02-16T00:47:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T21:20:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298215762453620425962131496093066572360","date":"2026-02-14T21:06:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224170695965261365713888402878760895851","date":"2026-02-11T15:05:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T11:01:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-06T19:20:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T03:55:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T03:53:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-02-04T05:30:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a414154-f982-432f-a8ab-3e08cdce7c09","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T07:57:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 16:15:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8782269","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8782269","identity":"rs-8782269","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}