UNASSIGNED: Lymphatic metastasis, an early stage of the metastasis process, is associated with adverse clinical outcomes in urothelial carcinoma of the bladder (UCB). However, the role of inflammation in triggering lymphatic metastasis remains unclear.
UNASSIGNED: We employed an RNA-sequencing cohort (n = 50) from Sun Yat-Sen Memorial Hospital (SYMH) to identify the most highly upregulated inflammatory gene associated with lymphatic metastasis. Using immunohistochemistry and immunofluorescence analyses, we validated the association of the identified molecule with clinical features and prognosis in an independent UCB cohort (n = 244) from SYMH. We also analysed TCGA-BLCA cohort (n = 408) to identify its potential biological pathways and immune landscape.
UNASSIGNED: In our study, chitinase 3-like 1 (CHI3L1) emerged as a significantly overexpressed proinflammatory mediator in UCB tissues with lymphatic metastasis compared to those without lymphatic metastasis (81.1% vs. 47.8%, P < 0.001). Within UCB tissues, CHI3L1 was expressed in both stromal cells (52.8%) and tumor cells (7.3%). Moreover, CHI3L1+ stromal cells, but not tumor cells, exhibited independent prognostic significance for both overall survival (P < 0.001) and recurrence-free survival (P = 0.006). CHI3L1+ stromal cells were positively associated with D2-40+ lymphatic vessel density (P < 0.001) and the immunosuppressive PD-L1/PD-1/CD8 axis in UCB tissues (all P < 0.05). A bioinformatics analysis also identified a positive association between CHI3L1 expression and lymphangiogenesis or immunosuppression pathways.
UNASSIGNED: Our study established a clear association between stromal CHI3L1 expression and lymphatic metastasis, suggesting that stromal CHI3L1 expression is a potential prognostic marker for bladder cancer patients.

Traditional clinical modalities for diagnosing bladder urothelial carcinoma (BUC) remain limited due to their invasive nature, significant costs, discomfort associated with cystoscopy, and low sensitivity to urine cytology. Therefore, there is an urgent need to identify highly sensitive, specific, and noninvasive biomarkers for the early detection of this neoplasm. Hypermethylated TWIST1/Vimentin promoter may be a noninvasive biomarker using urine sample. We assessed the TWIST1/Vimentin promoter methylation status in urine samples using the Methylated Human TWIST1 and Vimentin Gene Detection Kit (Jiangsu MicroDiag Biomedicine Co., Ltd., China). The samples were collected from five groups: group 1 consisted of patients with BUC, group 2 contained other patients with urologic tumors, group 3 consisted of patients with benign diseases (e.g., urinary tract infections, lithiasis, and benign prostatic hyperplasia), Group 4 included UTUC (upper tract urothelial carcinoma) patients and group5 comprised healthy individuals. The study encompassed 77 BUC patients, and we evaluated the degree of methylation of the TWIST1/Vimentin gene in their urine samples. Notably, TWIST1/Vimentin positivity was significantly elevated in comparison to groups 2, 3 and 5 (all p < 0.001) at a rate of 77.9%, but no significant difference was observed when compared to group 4. In the relationship between TWIST1/Vimentin methylation and clinicopathological features of BC patients from our center, we found there was no significant association between TWIST1/Vimentin status and proteinuria and/or hematuria, and hypermethylation of TWIST1 / VIM genes was found in both high and low tumor grade and in both non-muscle invasive bladder cancer (stages Tis, Ta, or T1) and muscle-invasive bladder cancer (stage T2 or above). In the multivariable analysis for cancer detection, a positive TWIST1/Vimentin methylation were significantly linked to a heightened risk of BC. Moreover, TWIST1/Vimentin promoter methylation demonstrated an ability to detect BUC in urine samples with a sensitivity of 78% and a specificity of 83%. Our findings reveal that hypermethylation of the TWIST1/Vimentin promoter occurs in bladder urothelial carcinoma, and its high sensitivity and specificity suggest its potential as a screening and therapeutic biomarker for urothelial carcinoma of the bladder.

BACKGROUND: Urothelial bladder cancer (UBC) is characterized by a high recurrence rate, which is predicted by scoring systems. However, recent studies show the superiority of Machine Learning (ML) models. Nevertheless, these ML approaches are rarely used in medical practice because most of them are black-box models, that cannot adequately explain how a prediction is made.
OBJECTIVE: We investigate the global feature importance of different ML models. By providing information on the most relevant features, we can facilitate the use of ML in everyday medical practice.
METHODS: The data is provided by the cancer registry Rhineland-Palatinate gGmbH, Germany. It consists of numerical and categorical features of 1,944 patients with UBC. We retrospectively predict 2-year recurrence through ML models using Support Vector Machine, Gradient Boosting, and Artificial Neural Network. We then determine the global feature importance using performance-based Permutation Feature Importance (PFI) and variance-based Feature Importance Ranking Measure (FIRM).
RESULTS: We show reliable recurrence prediction of UBC with 82.02% to 83.89% F1-Score, 83.95% to 84.49% Precision, and an overall performance of 69.20% to 70.82% AUC on testing data, depending on the model. Gradient Boosting performs best among all black-box models with an average F1-Score (83.89%), AUC (70.82%), and Precision (83.95%). Furthermore, we show consistency across PFI and FIRM by identifying the same features as relevant across the different models. These features are exclusively therapeutic measures and are consistent with findings from both medical research and clinical trials.
CONCLUSIONS: We confirm the superiority of ML black-box models in predicting UBC recurrence compared to more traditional logistic regression. In addition, we present an approach that increases the explanatory power of black-box models by identifying the underlying influence of input features, thus facilitating the use of ML in clinical practice and therefore providing improved recurrence prediction through the application of black-box models.

UNASSIGNED: To the best of our knowledge, Androgen receptor (AR) and cluster of differentiation 24 (CD24) expression in bladder urothelial carcinoma (UC) has not yet been reported in our population. The aim of this study was to evaluate the expression of both markers in UCB using immunohistochemistry.
UNASSIGNED: Data from 60 patients with UCB were obtained between 2009 and 2018. The samples were divided into four groups based on their smoking history. Group 1 included non-smokers, group 2 smoked <20 cigarettes/day for 30 years, group 3 smoked for 31-40 years, and group 4 smoked for > 40 years. Each group then divided into Non muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) subgroups. The smear was stained with hematoxylin and eosin (HE) - immunohistochemistry of CD24 and RA, followed by histoscore assessment.
UNASSIGNED: The male to female smoking rates was 1.8. Based on gender, in the NMIBC group there were 85.7% men and 14.3% were women while in MIBC 74.4% men and 25.6% women. The mean age of the NMIBC and MIBC groups was 56.3 years and 54.5 years, respectively. There was no significant relationship between smoking status in group 2 (OR 0.31, CI 95% CI, p=0,39), group 3 (OR 013, CI 95% CI, p=0,05), and group 4 (OR 0.23, CI 95% CI, p=0215) to the UCB invasiveness. A significant relationship was observed between cytoplasmic AR expression and UCB invasiveness (OR 0.14[0,04; 0.47], CI 95%, p=0.001). There was no significant relationship between RA in the nucleus and UCB invasion (OR 1.09[0,18; 6.48] CI 95%, p=1000). No significant relationship was observed between CD24 expression and UCB invasiveness (OR 0.81[0,27-2,45] CI 95%, p=0712).
UNASSIGNED: Cytoplasmic AR expression is associated with UCB invasiveness. Smoking history and CD24 expression were not associated with UCB invasion.

OBJECTIVE: To evaluate the presence and subtypes of tertiary lymphatic structures (TLSs) in urothelial carcinoma of the bladder (UCB) and to analyze their associated clinicopathological characteristics and prognostic significance.
METHODS: The study enrolled 580 patients with surgically treated UCB, including 313 non-muscle invasive bladder cancer (NMIBC) and 267 muscle-invasive bladder cancer (MIBC). The presence and subtypes of TLSs were identified by immunohistochemistry (CD20, CD3, Bcl-6, and CD21). TLSs were classified into non-GC (nGC) TLS and GC TLS subtypes based on germinal center (GC) formation. Disease-free survival (DFS) was used as an endpoint outcome to evaluate the prognostic significance of TLS and its subtypes in UCB.
RESULTS: TLSs were more common in MIBC than in NMIBC (67.8% vs 48.2%, P < .001), and the tumor-infiltrating lymphocyte (TIL) mean density was significantly higher in MIBC than in NMIBC (24.0% vs 17.5%, P < .001). Moreover, a positive correlation was found between TLS presence and GC structure formation and TIL infiltration in UCB. Endpoint events occurred in 191 patients. Compared to patients with endpoint events, patients without disease progression exhibited higher TIL density and more TLSs (P < .05). Kaplan-Meier curves showed that TLS was associated with better DFS in NMIBC (P = .041) and MIBC (P = .049). However, the Cox multivariate analysis did not demonstrate the prognostic significance of TLS.
CONCLUSIONS: TLS is heterogeneous in UCB, and that TLS and GC structures are related to TIL density and prognostic events. However, TLS as a prognostic indicator remains unclear, warranting further investigation.

Urothelial carcinoma of the bladder (UCB) is the most prevalent malignant tumor of the urinary system worldwide, which has a significant recurrence rate despite multiple treatment options available. As a unique and novel copper-dependent programmed cell death mechanism, the comprehensive impact of cuproptosis on the tumor immune microenvironment, clinicopathological characteristics and the prognosis of patients remains largely unclear.
A total of 568 UCB samples were thoroughly examined for cuproptosis patterns using data downloaded from TCGA and GEO, based on 10 cuproptosis-related genes reported previously. Then, the univariate COX regression analysis was performed on the genes that differed across the various patterns. To measure individual cuproptosis pattern, a cuproptosis score system was constructed using a principal component analysis algorithm. To validate the scoring system, immunohistochemical staining was performed on tumor tissues with different pathological grades, and experiments in vitro were conducted about the differentially expressed genes related to prognosis. Finally, the capacity of scoring system to predict the response to immunotherapy was verified by using data from IMvigor 210 cohort.
Four unique cuproptosis clusters and two gene clusters were finally found by the investigation. The clinical features and prognosis of patients, as well as the mRNA transcriptome, pathway enrichment, and immune cell infiltration in TME, varied dramatically between various cuproptosis clusters and gene clusters. To identify individual cuproptosis patterns in UCB patients, we also established a cuproptosis scoring system. After validation with multiple methods, it was indicated that the score system could predict the prognosis of UCB patients and was significantly connected to clinical features such TNM category, tumor grade, molecular type and ultimate survival status. The clinical outcomes of UCB patients were predicted effectively according to the tumor mutation burden in conjunction with the scoring system. Furthermore, we found that the cuproptosis score had a significant correlation with the response to immunotherapy and the sensitivity to chemotherapy.
This study revealed the potential impact of cuproptosis on the UCB tumor immune microenvironment and clinical pathological characteristics. The cuproptosis score system could effectively predict the prognosis of patients and the response to chemotherapy and immunotherapy.

Cytokeratin 20 (CK20) expression is limited to umbrella cells in the normal urothelium. Since CK20 is often upregulated in neoplastic urothelial cells including dysplasia and carcinoma in situ, immunohistochemical CK20 analysis is often used for the assessment of bladder biopsies. CK20 expression is a feature of luminal bladder cancer subtype, but its prognostic relevance is disputed. In this study, we investigated CK20 on >2700 urothelial bladder carcinomas in a tissue microarray format by immunohistochemistry. Cytoplasmic and membranous CK20 staining was seen in 1319 (51.8%) cancers. The fraction of CK20 positive and especially strongly positive cases increased from pTaG2 low grade (44.5% strongly positive) and pTaG2 high grade (57.7%) to pTaG3 high grade (62.3%; p = 0.0006) but was lower in muscle-invasive (pT2-4) carcinomas (51.1% in all pTa vs. 29.6% in pT2-4; p < 0.0001). Within pT2-4 carcinomas, CK20 positivity was linked to nodal metastasis and lymphatic vessel invasion (p < 0.0001 each) and to venous invasion (p = 0.0177). CK20 staining was unrelated to overall patient survival if all 605 pT2-4 carcinomas were jointly analyzed but subgroup analyses revealed a significant association of CK20 positivity with favorable prognosis in 129 pT4 carcinomas (p = 0.0005). CK20 positivity was strongly linked to the expression of GATA3 (p < 0.0001), another feature of luminal bladder cancer. The combined analysis of both parameters showed best prognosis for luminal A (CK20+/GATA3+, CK20+/GATA3-) and worst outcome for luminal B (CK20-/GATA3+) and basal/squamous (CK20-/GATA3-) in pT4 urothelial carcinomas (p = 0.0005). In summary, the results of our study demonstrate a complex role of CK20 expression in urothelial neoplasms including neoexpression in pTa tumors, a subsequent loss of CK20 expression in a subset of tumors progressing to muscle-invasion, and a stage dependent prognostic role in muscle-invasive cancers.

OBJECTIVE: Although aberrant expression of peroxidasin-like (PXDNL) has been associated with carcinogenesis, its potential role in the Urothelial Carcinoma of the Bladder (UCB) remains unknown. The present study aimed to explore the role of PXDNL in UCB carcinogenesis and its potential clinical value.
METHODS: Based on The Cancer Genome Atlas (TCGA) data, bioinformatics was used to explore the potential clinical value of PXDNL. Wound healing and Transwell invasion assays were employed for the purpose of assessing the cell motility, while the Western Blotting experiments were utilized for investigating the protein expression pattern of PXDNL in UCB and investigating the Epithelial-to-Mesenchymal Transition (EMT) and Wnt/β-catenin pathways for understanding the probable mechanisms involved.
UNASSIGNED: PXDNL mRNA was overexpressed in UCB tissues and indicated a poor prognosis. High PXDNL mRNA levels were also associated with advanced clinicopathological features and were regarded as independent prognostic factors for UCB. However, PXDNL showed a weak correlation with immune cell infiltration in UCB. In addition, the findings of the study verified that the existing form of the PXDNL protein was 57-kDa and it was upregulated in the UCB cell lines and tissue samples. Furthermore, silencing PXDNL inhibited, while overexpressing PXDNL promoted EMT and motility of UCB cells in vitro. Mechanistic studies showed that PXDNL activated UCB cell motility via the Wnt/β-catenin pathway.
CONCLUSIONS: The results reveal a novel molecular target that could be further explored for developing preventive, predictive, and individualized treatment strategies for UCB.

At present, the extent and clinical relevance of epigenetic differences between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) remain largely unknown. Here, we conducted a study to describe the global DNA methylation landscape of UTUC and UCB and to address the prognostic value of DNA methylation subtype and responses to the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma (UC).
Using whole-genome bisulfite sequencing (n = 49 samples), we analyzed epigenomic features and profiles of UTUC (n = 36) and UCB (n = 9). Next, we characterized potential links between DNA methylation, gene expression (n = 9 samples), and clinical outcomes. Then, we integrated an independent UTUC cohort (Fujii et al., n = 86) and UCB cohort (TCGA, n = 411) to validate the prognostic significance. Furthermore, we performed an integrative analysis of genome-wide DNA methylation and gene expression in two UC cell lines following transient DNA methyltransferase inhibitor SGI-110 treatment to identify potential epigenetic driver events that contribute to drug efficacy.
We showed that UTUC and UCB have very similar DNA methylation profiles. Unsupervised DNA methylation classification identified two epi-clusters, Methy-High and Methy-Low, associated with distinct muscle-invasive statuses and patient outcomes. Methy-High samples were hypermethylated, immune-infiltrated, and enriched for exhausted T cells, with poor clinical outcome. SGI-110 inhibited the migration and invasion of Methy-High UC cell lines (UMUC-3 and T24) by upregulating multiple antitumor immune pathways.
DNA methylation subtypes pave the way for predicting patient prognosis in UC. Our results provide mechanistic rationale for evaluating SGI-110 in treating UC patients in the clinic.

UNASSIGNED: To evaluate the clinical value of intravesical gemcitabine combined with immunotherapy in patients with non-muscle-invasive bladder carcinoma (NMIBC) after transurethral resection of bladder tumor (TURBT).
UNASSIGNED: Eighty patients with non-muscle-invasive urothelial carcinoma treated in Baoding No.1 Hospital from November 2016 to November 2019 were randomly divided into two groups, with 40 patients in each group. Both groups underwent TURBT. After surgery, the research group was treated with intravesical chemotherapy using gemcitabine combined with ubenimex, while the control group was given 40 mg pirarubicin by intravesical instillation. Postoperative condition was evaluated by cystoscopy every three months in both groups. The recurrence six months, one year and two years after treatment, the incidence of lower urinary tract symptoms such as dysuria, hematuria and frequent urination, general adverse drug reactions such as rashes, liver function damage and gastrointestinal reaction, as well as the changes in CD3+, CD4+, CD8+ and CD4+/CD8+ T lymphocyte subsets before and after treatment were comparatively analyzed between the two groups.
UNASSIGNED: The recurrence rate showed no statistical significance between the two groups 6 months after treatment (p=0.17), but significant differences one year (p=0.04) and two years (p=0.03) after treatment, which were significantly lower in the research group than the control group. The incidence of adverse drug reactions was 22.5% in the research group and 7.5% in the control group, without significant difference (p=0.36). The incidence of lower urinary tract symptoms was 32.5% and 55%, respectively, in the research group and the control group. The incidence of lower urinary tract symptoms in the research group was significantly lower compared with the control group, with a statistically significant difference (p=0.04). After treatment, CD3+, CD4+ and CD4+/CD8+ levels in the research group increased significantly than those in the control group, with statistically significant differences (CD3+, p=0.01; CD4+, p=0.00; CD4+/CD8+, p=0.00).
UNASSIGNED: For NMIBC patients receiving bladder-preserving surgery, intravesical gemcitabine combined with immunotherapy can reduce the recurrence rate, relieve lower urinary tract symptoms, increase the tolerance of patients to intravesical chemotherapy and significantly improve the function of T lymphocytes, without obvious increase in adverse drug reactions. Therefore, it is safe and effective, and has certain clinical value.