Bladder Urothelial Carcinoma Patients Research Articles

PRR11 is a prognostic biomarker and correlates with immune infiltrates in bladder urothelial carcinoma

Abnormal proline-rich protein 11 (PRR11) expression is associated with various tumors. However, there are few reports concerning PRR11 with prognostic risk, immune infiltration, or immunotherapy of bladder urothelial carcinoma (BLCA). This study is based on online databases, such as Oncomine, GEPIA, HPA, LinkedOmics, TIMER, ESTIMATE and TISIDB, and BLCA data downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus, we employed an array of bioinformatics methods to explore the potential oncogenic roles of PRR11, including analyzing the relationship between PRR11 and prognosis, tumor mutational burden (TMB), microsatellite instability, and immune cell infiltration in BLCA. The results depict that PRR11 is highly expressed in BLCA, and BLCA patients with higher PRR11 expression have worse outcomes. In addition, there was a significant correlation between PRR11 expression and TMB and tumor immune infiltration. These findings suggest that PRR11 can be used as a potential marker for BLCA patient assessment and risk stratification to improve clinical prognosis, and its potential regulatory mechanism in the BLCA tumor microenvironment and targeted therapy is worthy of further investigation.

Glucose Metabolism Reprogramming in Bladder Cancer: Hexokinase 2 (HK2) as Prognostic Biomarker and Target for Bladder Cancer Therapy.

Proliferating cancer cells are able to reprogram their energy metabolism, favouring glycolysis even in the presence of oxygen and fully functioning mitochondria. Research is needed to validate the glycolysis-related proteins as prognostic/predictive biomarkers in urothelial bladder carcinoma (UBC), a malignancy tagged by high recurrence rates and poor response to chemotherapy. Here, we assessed GLUT1, HK2, PFKL, PKM2, phospho-PDH, and LDHA immunoexpression in 76 UBC samples, differentiating among urothelial, fibroblast, and endothelial cells and among normoxic versus hypoxic areas. We additionally studied the functional effects of the HK2 inhibitor 2-deoxy-D-glucose (2DG) in "in vitro" and "in vivo" preclinical UBC models. We showed that the expression of the glycolysis-related proteins is associated with UBC aggressiveness and poor prognosis. HK2 remained as an independent prognostic factor for disease-free and overall survival. 2DG decreased the UBC cell's viability, proliferation, migration, and invasion; the inhibition of cell cycle progression and apoptosis occurrence was also verified. A significant reduction in tumour growth and blood vessel formation upon 2DG treatment was observed in the chick chorioallantoic membrane assay. 2DG potentiated the cisplatin-induced inhibition of cell viability in a cisplatin-resistant subline. This study highlights HK2 as a prognostic biomarker for UBC patients and demonstrates the potential benefits of using 2DG as a glycolysis inhibitor. Future studies should focus on integrating 2DG into chemotherapy design, as an attempt to overcome cisplatin resistance.

Comprehensive Analysis of the Expression, Prognosis, and Biological Significance of PLOD Family in Bladder Cancer.

Large numbers of studies have identified that procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family members play important roles in tumorigenesis and tumor progression in various cancers. However, the expression pattern, clinical value and function of PLOD family have yet to be analyzed systematically and comprehensively in bladder urothelial carcinoma (BLCA). We investigated the transcriptional levels, genetic alteration, biological function, immune cell infiltration, data on survival of PLODs in patients with BLCA based on UALCAN, the Cancer Genome Atlas (TCGA) database, Gene Expression Profiling Interactive Analysis (GEPIA), TIMER, STRING, cBioPortal and GSCALite databases. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed in R software using the Cluster Profiler Bioconductor package. Protein-protein interaction (PPI) network was established by STRING and visualized byusing R version (3.6.3) software. Survival analysis was performed using the packages "survminer". The mRNA and protein expression patterns of PLOD family members were noticeably increased in BLC compared with normal tissue. The mRNA expression levels of PLOD1-2 genes were significantly correlated with histological subtypes and PLOD1 was significantly correlated with pathological stage. Furthermore, the high expression levels of PLOD1-2 were remarkably associated with poor overall survival (OS) in BLCA patients, meanwhile high expression levels of PLOD1 and PLOD3 were markedly associated with poor progression-free interval (PFI). In co-expression gene analysis, 50 genes were primarily associated with the differentially expressed PLODs in BLCA. Functional enrichment analysis revealed that protein hydroxylation, collagen fibril organization, and lysine degradation were key biological functions of PLODs in BLCA. Moreover, PLOD family genes were identified as being associated with the activities of tumor-infiltrating immune cells and closely associated with immune responses in BLCA. PLOD family members might serve as potential therapeutic targets and prognostic markers for BLCA patients' survival.

Derivation and external validation of dendritic cell-related gene signatures for predicting prognosis and immunotherapy efficacy in bladder urothelial carcinoma.

In the regulation of tumor-related immunity, dendritic cells (DCs) are crucial sentinel cells; they are powerful to present antigens and initiate immune responses. Therefore, we concentrated on investigating the DC-related gene profile, prognosis, and gene mutations in bladder urothelial carcinoma (BLCA) patients to identify sensitivity to immunotherapy of patients. According to DC infiltration, BLCA patients were divided into two subgroups, and differentially expressed genes (DEGs) were obtained. Patients were classified by unsupervised clustering into new subgroups. The least absolute shrinkage and selection operator (LASSO) regression analysis and Cox regression were used to develop a DC-related risk model. CIBERSORT, xCell, and GSEA were used to infer immune cells' relative abundance separately and enriched immune pathways. A total of 29 prognosis-related DEGs were identified from the unsupervised cluster. Among them, 22 genes were selected for constructing the DC-related risk model. The dendritic cell-related risk score (DCRS) can accurately distinguish patients with different sensitive responses to immunotherapy and overall survival outcomes. Furthermore, patients with ryanodine receptor 2 (RYR2) mutation had a better prognosis. The DCRS played an essential part in immunity pathway and formation of TME diversity. Our study indicated that RYR2 mutation combined with DCRS is useful for predicting the prognosis and discovering appropriate patients for immunotherapy.

Novel T-cell signature based on cell pair algorithm predicts survival and immunotherapy response for patients with bladder urothelial carcinoma.

T-cell-T-cell interactions play important roles in the regulation of T-cells' cytotoxic function, further impacting the anti-tumor efficacy of immunotherapy. There is a lack of comprehensive studies of T-cell types in bladder urothelial carcinoma (BLCA) and T-cell-related signatures for predicting prognosis and monitoring immunotherapy efficacy. More than 3,400 BLCA patients were collected and used in the present study. The ssGSEA algorithm was applied to calculate the infiltration level of 19 T-cell types. A cell pair algorithm was applied to construct a T-cell-related prognostic index (TCRPI). Survival analysis was performed to measure the survival difference across TCRPI-risk groups. Spearman's correlation analysis was used for relevance assessment. The Wilcox test was used to measure the expression level difference. Nineteen T-cell types were collected; 171 T-cell pairs (TCPs) were established, of which 26 were picked out by the least absolute shrinkage and selection operator (LASSO) analysis. Based on these TCPs, the TCRPI was constructed and validated to play crucial roles in survival stratification and the dynamic monitoring of immunotherapy effects. We also explored several candidate drugs targeting TCRPI. A composite TCRPI and clinical prognostic index (CTCPI) was then constructed, which achieved a more accurate estimation of BLCA's survival and was therefore a better choice for prognosis prediction in BLCA. All in all, we constructed and validated TCRPI based on cell pair algorithms in this study, which might put forward some new insights to increase the survival estimation and clinical response to immune therapy for individual BLCA patients and contribute to the personalized precision immunotherapy strategy of BLCA.

MiRNA-139-3p inhibits malignant progression in urothelial carcinoma of the bladder via targeting KIF18B and inactivating Wnt/beta-catenin pathway.

Bladder cancer is a highly prevalent disease worldwide. We aimed to investigate the effect of miRNA/mRNA signaling on bladder urothelial carcinoma (BUC). MiRNA-139-3p wasselected from The Cancer Genome Atlas database, and its downstream target gene was predicted. The correlation between miRNA-139-3p and intersected mRNAs was analyzed. The mRNA expression levels of miRNA-139-3p and KIF18B in BUC were assayed via quantitative real-time polymerase chain reaction. Effects of miRNA-139-3p on cell proliferation, invasion, migration and cell cycle were detected via Cell Counting Kit-8, colony formation, transwell, wound healing and flow cytometry assays, respectively. Binding relationship between miRNA-139-3p and KIF18B was verified by dual-luciferase reporter gene detection. The protein expression levels of KIF18B, β-catenin and Cyclin D1 were detected by Western blot. Rescue assays were performed for verifying the interaction among miRNA-139-3p, KIF18B and Wnt/β-catenin signaling pathway, which revealed effects of miRNA-139-3p/KIF18B on BUC cells. MiRNA-139-3p was remarkably underexpressed, and KIF18B was dramatically overexpressed in BUC cells, respectively. It was also demonstrated that overexpressing miRNA-139-3p could prominently inhibit proliferation, invasion and migration of BUC, and block BUC cells at G0-G1 phase. Afterwards, we found that miRNA-139-3p could bind to KIF18B mRNA 3'UTR, and miRNA-139-3p had a negative regulatory effect with KIF18B. Subsequent experimental results presented that overexpressing KIF18B could reverse inhibitory effect of overexpressing miRNA-139-3p on BUC. Finally, this study also ascertained that miRNA-139-3p/KIF18B could repress oncogenic effects of BUC via modulating Wnt/β-catenin signaling pathway. MiRNA-139-3p/KIF18B/Wnt/β-catenin could significantly inhibit the malignant progression of BUC, and its targeting mechanism might provide an effective therapeutic target for BUC patients.

152P An epidemiologic study on PD-L1 expression with clinical observation of initial treatment pattern in the Chinese muscle invasive urothelial bladder carcinoma patients

There is no relevant data of real-time programmed cell death-ligand 1 (PD-L1) expression and initial treatment pattern in Chinese muscle invasive urothelial bladder carcinoma patients (MIUBC). Thus, the aim of this multi-center, prospective, epidemiological study was to investigate the prevalence of high PD-L1 expression along with clinical observation of initial treatment pattern in the Chinese MIUBC patients (NCT03433924).

Analysis of prognostic oncogene filaggrin (FLG) wild-type subtype and its implications for immune checkpoint blockade therapy in bladder urothelial carcinoma.

Bladder urothelial carcinoma (BLCA) is one of the most common urinary tract malignant tumors. Immune checkpoint blockade (ICB) therapy has significantly progressed the treatment of BLCA. This study aimed to investigate the role of specific genetic mutations that may serve as immune biomarkers for ICB therapy in BLCA. Mutation information and expression profiles were acquired from The Cancer Genome Atlas (TCGA) database. Integrated bioinformatics analysis was carried out to explore the subtypes with poor prognosis of BLCA. Functional enrichment analysis was also conducted. The infiltrating immune cells and the prediction of ICB response between different subtypes were explored using the immuCellAI algorithm. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted to explore the effect of filaggrin (FLG) knockdown in BLCA 5637 and T24 cell lines. An overview of mutation information in BLCA patients was shown. FLG was identified to be strongly associated with the prognosis of BLCA patients and FLG wild-type was associated with poorer outcome. Prognostic FLG wild-type was divided into 2 subtypes (Sub1 and Sub2). Following an investigation of the subtypes, Sub2 of FLG wild-type was found to be associated with poorer outcome in BLCA. The differentially expressed genes (DEGs) between Sub1 and Sub2 were screened out and the DEGs were enriched in malignant tumor proliferation, DNA damage repair, and immune-related pathways. Furthermore, Sub2 of FLG wild-type was associated with infiltrated immune cells, and responded worse to ICB. Sub2 of FLG wild-type may be used as a biomarker to predict the prognosis of BLCA patients receiving ICB. The cellular experiments revealed that knockdown of FLG could suppress BLCA cell proliferation and promote apoptosis. FLG is an oncogene that may affect the prognosis of BLCA patients through mutation. Sub2 of FLG wild-type is associated with poor prognosis and can be used to predict ICB response for BLCA treatment. This research provides a new basis and ideas for guiding the clinical application of BLCA immunotherapy.

KLK6 Functions as an Oncogene and Unfavorable Prognostic Factor in Bladder Urothelial Carcinoma.

Background Kallikrein-related peptidase 6 (KLK6) has been substantiated as a diagnostic, prognostic, and therapeutic molecular in several cancer types. In our study, we attempt to explore the biological functions of KLK6 in bladder urothelial carcinoma (BLCA). Methods KLK6 gene expression prognostic, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and immune infiltration were analyzed using The Cancer Genome Atlas (TCGA) database. In vitro and in vivo experimental measurements, including CCK8, transwell migration, TUNEL, and nude mouse transplanted tumor model, were used to evaluate the antineoplastic activities of KLK6 loss-of-function. Results The combination of bioinformatics analyses and experimental measurements demonstrate that KLK6 expression is aberrantly upregulated in human specimens and cell lines of BLCA. GO and GSEA enrichment analyses exhibited that KLK6 is implicated in the inflammatory response and immune infiltration, suggesting that upregulation of KLK6 may be associated with the progression of BLCA. Knockdown of KLK6 is able to inhibit the growth and migration and trigger apoptosis of RT4 and T24 cells. Moreover, the TCGA database indicates that KLK6 high expression in BLCA patients showed a poorer prognosis than those patients with KLK6 low expression. Univariate and multivariate regression analyses suggest KLK6 as an independent prognostic factor to predict unfavorable OS in patients with BLCA. Conclusion KLK6 is an independent prognostic factor and an antitumor target of BLCA. KLK6 expression positively correlates with several immune cells infiltration, indicating that inhibition of KLK6 may contribute to immunotherapy of BLCA.

Quantitative proteomics identifies TUBB6 as a biomarker of muscle‐invasion and poor prognosis in bladder cancer

Muscle-invasive urothelial carcinoma (MIUC) of the bladder shows highly aggressive tumor behavior, which has prompted the quest for robust biomarkers predicting invasion. To discover such biomarkers, we first employed high-throughput proteomic method and analyzed tissue biopsy cohorts from patients with bladder urothelial carcinoma (BUC), stratifying them according to their pT stage. Candidate biomarkers were selected through bioinformatic analysis, followed by validation. The latter comprised 2D and 3D invasion and migration assays, also a selection of external public datasets to evaluate mRNA expression and an in-house patient-derived tissue microarray (TMA) cohort to evaluate protein expression with immunohistochemistry (IHC). Our multilayered platform-based analysis identified tubulin beta 6 class V (TUBB6) as a promising prognostic biomarker predicting MIUC of the bladder. The in vitro 2D and 3D migration and invasion assays consistently showed that inhibition of TUBB6 mRNA significantly reduced cell migration and invasion ability in two BUC cell lines with aggressive phenotype (TUBB6 migration, P=.0509 and P < .0001; invasion, P=.0002 and P=.0044; TGFBI migration, P=.0214 and P=.0026; invasion, P < .0001 and P=.0001; T24 and J82, respectively). Validation through multiple public datasets, including The Cancer Genome Atlas (TCGA) and selected GSE (Genomic Spatial Event) databases, confirmed TUBB6 as a potential biomarker predicting MIUC. Further protein-based validation with our TMA cohort revealed concordant results, highlighting the clinical implication of TUBB6 expression in BUC patients (overall survival: P < .001). We propose TUBB6 as a novel IHC biomarker to predict invasion and poor prognosis, also select the optimal treatment in BUC patients.

Epigenetic inactivation of DNA repair genes as promising prognostic and predictive biomarkers in urothelial bladder carcinoma patients

We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 (RBBP8) and MutS homologue 4 (MSH4), between primary tumors and normal tissues of TCGA–BLCA were validated by methylation-specific PCR (MSP) in UBC (n = 70) compared to normal tissues (n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8- and MSH4-methylation along with patients’ clinical features. Then, we assessed the prognostic significance of RBBP8 and MSH4. RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients’ response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4-methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy.

Construction and verification of a novel hypoxia-related lncRNA signature related with survival outcomes and immune microenvironment of bladder urothelial carcinoma by weighted gene co-expression network analysis.

Background: Bladder urothelial carcinoma (BLCA) is a common malignant tumor with the greatest recurrence rate of any solid tumor. Hypoxia is crucial in the growth and immune escape of malignant tumors. To predict clinical outcomes and immunological microenvironment of patients with BLCA, a hypoxia-related long non-coding RNA (HRlncRNA) signature was established. Methods: The Cancer Genome Atlas (TCGA) provided us with the differentially expressed profile of HRlncRNAs as well as clinical data from patients with BLCA, and we used weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with malignancies. Results: Finally, Cox analysis revealed that HRlncRNAs, which comprised 13 lncRNAs, were implicated in the predictive signature. The training, testing, and overall cohorts of BLCA patients were divided into the low-risk group and high-risk group based on the median of the risk score. The Kaplan–Meier curves revealed that BLCA patients with a high-risk score had a poor prognosis, and the difference between subgroups was statistically significant. The receiver operating characteristic curves revealed that this signature outperformed other strategies in terms of predicting ability. Multivariate analysis revealed that the risk score was an independent prognostic index for overall survival (HR = 1.411; 1.259–1.582; p < 0.001). Then, a nomogram with clinicopathological features and risk score was established. This signature could effectively enhance the capacity to predict survival, according to the calibration plots, stratification, and clinical analysis. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) were WNT, MAPK, and ERBB signaling pathways. Two groups had different immune cell subtypes, immune checkpoints, immunotherapy response, and anti-tumor drug sensitivity, which might result in differing survival outcomes. We then validated the differential expression of signature-related genes between tumor and normal tissues using TCGA paired data. Conclusion: This prognostic signature based on 13 HRlncRNAs may become a novel and potential prognostic biomarker, providing more accurate clinical decision-making and effective treatment for BLCA patients.

Clinical Eosinophil-Associated Genes can Serve as a Reliable Predictor of Bladder Urothelial Cancer.

Background: Numerous studies have shown that infiltrating eosinophils play a key role in the tumor progression of bladder urothelial carcinoma (BLCA). However, the roles of eosinophils and associated hub genes in clinical outcomes and immunotherapy are not well known. Methods: BLCA patient data were extracted from the TCGA database. The tumor immune microenvironment (TIME) was revealed by the CIBERSORT algorithm. Candidate modules and hub genes associated with eosinophils were identified by weighted gene co-expression network analysis (WGCNA). The external GEO database was applied to validate the above results. TIME-related genes with prognostic significance were screened by univariate Cox regression analysis, lasso regression, and multivariate Cox regression analysis. The patient’s risk score (RS) was calculated and divided subjects into high-risk group (HRG) and low-risk group (LRG). The nomogram was developed based on the risk signature. Models were validated via receiver operating characteristic (ROC) curves and calibration curves. Differences between HRG and LRG in clinical features and tumor mutational burden (TMB) were compared. The Immune Phenomenon Score (IPS) was calculated to estimate the immunotherapeutic significance of RS. Half-maximal inhibitory concentrations (IC50s) of chemotherapeutic drugs were predicted by the pRRophetic algorithm. Results: 313 eosinophil-related genes were identified by WGCNA. Subsequently, a risk signature containing 9 eosinophil-related genes (AGXT, B3GALT2, CCDC62, CLEC1B, CLEC2D, CYP19A1, DNM3, SLC5A9, SLC26A8) was finally developed via multiplex analysis and screening. Age (p < 0.001), grade (p < 0.001), and RS (p < 0.001) were independent predictors of survival in BLCA patients. Based on the calibration curve, our risk signature nomogram was confirmed as a good predictor of BLCA patients’ prognosis at 1, 3, and 5 years. The association analysis of RS and immunotherapy indicated that low-risk patients were more credible for novel immune checkpoint inhibitors (ICI) immunotherapy. The chemotherapeutic drug model suggests that RS has an effect on the drug sensitivity of patients. Conclusions: In conclusion, the eosinophil-based RS can be used as a reliable clinical predictor and provide insights into the precise treatment of BLCA.

The Relationship of Pyroptosis-Related Genes, Patient Outcomes, and Tumor-Infiltrating Cells in Bladder Urothelial Carcinoma (BLCA).

Introduction: The role of pyroptosis and its effects on tumor-infiltrating cells (TICs) in the pathogenesis and treatment outcomes of patients with bladder urothelial carcinoma (BLCA) remains unclear. Methods: We conducted a bioinformatics analysis on the pyroptosis-related genes (PRGs) and TICs using data from public domains, and evaluated their impact on the pathogenesis and clinical outcomes of BLCA patients. A risk score based on PRGs and a prognostic risk model that incorporated patient demographics, tumor characteristics, and differentially expressed genes (DEGs) were developed. Results: Twenty-three DEGs of 52 PRGs were identified in BLCA and normal samples from the TCGA database. Missense mutations and single nucleotide polymorphisms in PRGs are the most common genetic abnormalities. Patients with high PRG risk scores showed an inferior survival compared to those with low risk scores. The prognostic risk model based on patient demographics, tumor characteristics, and DEGs showed good predictive values for patient survival at 1, 3, and 5 years in BLCA patients. Caspase-8 (CASP8) was the only intersection gene of the prognostic genes, DEGs, and different genes expressed in tissue. Patients with a high CASP8 expression had improved survival, and an increased CASP8 expression level was observed in activated CD4 memory T cells, follicular T helper cells, resting NK cells, M0 macrophages, and activated dendritic cells. CASP8 expression also showed a positive correlation with the IL7R expression—a key cell marker of CD4 memory T cells. CASP8 expression also showed correlations with immune checkpoints (PDCD1, CD274, and CTLA4) and response to immune checkpoint inhibitors. Conclusion: Our data suggest that PRGs, especially CASP8, showed strong associations with patient outcomes and TICs in BLCA. If validated, these results could potentially aid in the prognostication and guide treatment in BLCA patients.

Single-nucleus RNA-sequencing analysis reveals transcriptome differences in TIGIT+ exhausted T-cell associated with poor prognosis in muscle-invasive bladder cancer (MIBC).

e16544 Background: Compared with single-cell RNA-sequencing (scRNA-seq), Single-nuclei RNA-sequencing(snRNA-seq) can detect more genes with higher sensitivity, and it is more robust in identifying cellular markers. TIGIT+ exhausted T cells were reported significantly associated with poor prognosis in muscle invasive bladder cancer(MIBC) by flow cytometry. We aimed to evaluate unique markers of TIGIT+ exhausted T cells in MIBC via snRNA-seq and to construct a potential prognostic survival model. Methods: We collected snRNA-seq data from 25 MIBC and 4 non-MIBC patients in GSE169379, including 57 cancer tissues and 4 distant normal tissues. Meanwhile, expression profiling data and clinical information of 402 Bladder Urothelial Carcinoma(BLCA) patients were collected in The Cancer Genome Atlas (TCGA). Firstly, the snRNA-seq data was preprocessed and filtered. Next, We re-clustered T-cell subsets and abstracted TIGIT+ exhausted T cells (TIGIT+Tex), then screened out the genes uniquely expressed in TIGIT+Tex subsets by differential expression analysis. We further evaluate the correlations between uniquely expressed genes and the exhausted T cell markers, identified significantly correlated differential expressed genes, constructed a prognostic model based on multivariate cox regression analysis and exploited the patient's overall survival and disease-free survival to confirm our findings. Results: Top 10 genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2, YBX1, DACH1, SYT1, NOL4, TRIT1) of unparalleled differential up-regulation were identified from TIGIT+ exhausted T cells via snRNA-seq data. Based on the correlation analysis of TCGA expression profile data, six genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2, YBX1) that were significantly positively correlated with exhausted T cells were obtained. Next, we constructed a regression prediction model consisting of 5 genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2) through multivariate cox regression analysis based on patients’ clinical information. The model was associated with significantly poor prognosis overall survival (OS) (P &lt; 0.01,HR = 2) and disease-free survival (DFS) (P &lt; 0.01,HR = 1.6) in BLCA patients. Conclusions: In our study, Single-nuclei RNA expression profiling data revealed transcriptome differences between TIGIT+ exhausted T cells and other types of T cells. The regression model for potential prognostic survival was constructed using TCGA expression profiling data. These results demonstrated that distinctive differentially high expressed genes associated with TIGIT+ exhausted T cells may act as potential markers for predicting prognosis outcomes in bladder cancer patients.

Digital score of lymphocytic infiltration in tumor-associated stroma in relation to overall survival in bladder cancer.

4576 Background: Bladder Urothelial Carcinoma (BLCA) is the most common type of bladder cancer and the sixth most frequent cancer in the US. High numbers of lymphocytes colocated with tumour-associated stromal (TAS) tissue has demonstrated prognostic significance for overall survival in multiple cancers. Our goal in this study was to explore the prognostic significance of an automated score to quantify the colocalisation of lymphocytes and TAS for overall survival (OS) in BLCA patients from Haematoxylin &amp; Eosin (H&amp;E) stained Whole Slide Images (WSIs). Methods: Two cohorts of BLCA patients were included in this study. From the UK, a cohort of 67 BLCA patients constituted cohort A. We also evaluated our method on The Cancer Genome Atlas (TCGA) bladder cancer cohort of 453 cases, which we refer to as cohort B. We developed a two-stage method for digital quantification of lymphocytic infiltrates in TAS. First, we employed an AI algorithm to recognise and classify different regions as areas of high concentration of tumour, lymphocytes and stroma for each WSI creating a segmentation map of the different tissue types. For patients with multiple WSIs, we used the slide with the highest percentage of tumour to predict survival. This algorithm had been pre-trained on a cohort of Oral cancer and was fine-tuned using annotations from 4 BLCA WSIs, 3 from cohort A and 1 from cohort B. These WSIs were excluded from the final survival analysis. Using the segmentation maps, a statistical measure for the colocalisation of TAS and lymphocytes termed the tumour-associated stroma infiltrating lymphocytes or (TASIL) score was computed. Finally, for each cohort, data was right-censored at 10 years and the digital BLCA-TASIL (BT) score’s prognostic significance for OS was investigated by fitting a Kaplan-Meier estimator and Cox proportional hazard (PH) analysis, stratifying patients into two groups based on the BT score. In each cohort, two thirds of the data was used as the discovery set to determine the best cut-off for the TASIL Score and the remaining third was used as the validation set. Results: Our classification algorithm achieved high average F1-score of 0.88 on a held-out set of unseen data for the classification of tumour, lymphocytic and stromal regions. In cohort A, higher BT score was strongly associated with better OS ( P= 0.00906) on the unseen validation data. This significant association was also found in the validation data of cohort B ( P&lt; 0.001). Using the BT score as the only covariate, a Cox PH model for the validation data resulted in a C-index of 0.73 for cohort A and 0.57 for cohort B, respectively. Conclusions: The digital BT score showed significant prognostic value for overall survival in both BLCA cohorts, reinforcing the findings of prior work. We intend to further validate these findings on another cohort. To the best of our knowledge, this is the first attempt to predict survival solely from H&amp;E slides in BLCA.

Insights into mutational differences between upper tract urothelial carcinoma and urothelial carcinoma of the bladder.

e15125 Background: Upper tract urothelial carcinoma (UTUC) is histologically similar to bladder urothelial carcinoma (BUC), yet several clinical, biological and molecular features are unique to UTUC. UTUC may have unique genomic factors compared to BUC, while major knowledge gaps remain in our understanding of the biology and genomic landscape of UTUC. Methods: We retrospectively sequenced tumor samples and matched germline DNA using targeted next-generation sequencing methods and analyzed at least 642 cancer-associated genes. The cohort included 35 UTUC patients and 36 BUC patients. Results: Among all cases, KMT2D and TP53 were the most common mutations (account for 66% and 75% in UTUC and BUC, respectively). The most frequently mutated genes in UTUC were KMT2D (37%), TP53 (29%), ELF3 (26%), FGFR3 (23%), ASXL1 (20%), DKN2A (20%), DKN2B (17%), REBBP (17%), MT3B (17%), GNAS (17%) and PIK3CA (17%). The most frequently mutated genes in BUC were KMT2D (36%), TP53 (39%), MUC17 (14%), ARID1A (22%), GNAS (19%), RB1 (19%), BCL6 (19%), E2F3 (19%), EIF4A2 (19%) and FGF3 (19%). However significant differences in the prevalence of mutations in individual genes were observed. FGFR3 (23% vs. 8%) and ELF3 (26% vs. 11%) were more frequently altered, whereas RB1 (6% vs. 19%), E2F3 (3% vs. 19%) and BCL6 (3% vs. 19%) were less frequently altered in UTUC (P &lt; 0.05). FGFR3 mutations have been shown to be oncogenic and RB1 inactivation has been associated with genomic instability in urothelial cancer in previous researches, which represent potential therapeutic targets. Conclusions: Â UTUC and UCB exhibit significant differences in the prevalence of genomic landscape and carcinogenesis. A comprehensive understanding of the biology of UTUC and UCB is needed to identify new drug targets in order to improve clinical outcomes.

ANLN Serves as an Oncogene in Bladder Urothelial Carcinoma via Activating JNK Signaling Pathway

Introduction: To understand the significance of ANLN (anillin, actin-binding protein)-mediated c-Jun N-terminal kinase (JNK) signal pathway on the progression of bladder urothelial carcinoma (BLCA). Methods: The Cancer Genome Atlas (TCGA) database was utilized to perform the clinical significance of ANLN in BLCA. Then, ANLN expression was determined in human normal primary bladder epithelial cells (BdEC) and BLCA cells. Later, ANLN knockdown was performed in BLCA cells, where the expression of MAPK8, MAPK9, and p-JNK/JNK was detected. BLCA cells were divided into the Mock, siNC, siANLN, SP600125 (a selective JNK inhibitor), and ANLN + SP600125 group, followed by measurements of real-time quantitative polymerase chain reaction, 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Annexin V-FITC/PI, Wound-healing, Transwell, and immunofluorescence assays. Results: ANLN was upregulated in the BLCA tissues, which showed a relation with the stage of patients. Besides, BLCA patients with high expression of ANLN had a worse prognosis than those with low expression of ANLN. Besides, the expression of ANLN in the BLCA tissues was positively correlated with MAPK8 and MAPK9. SP600125 suppressed the JNK signal pathway, reduced the proliferation, and increased BLCA cell apoptosis, with the reductions in the invasion and migration and the upregulation of phospho-histone H3 Ser-10 (pHH3), which was abolished by the overexpression of ANLN. Conclusion: ANLN, as an oncogene of BLCA, may associate with the activation of JNK signal pathway. Inhibiting ANLN could deactivate the JNK signal pathway, thereby suppressing the proliferation, invasion, and migration while promoting the apoptosis of BLCA cells.

Sp1 is overexpressed and associated with progression and poor prognosis in bladder urothelial carcinoma patients.

Specificity protein 1 (Sp1) is a transcription factor that exerts key functions in the carcinogenesis and progression of various types of cancer. However, its expression and prognostic value in bladder urothelial carcinoma (BUC) have yet to be completely elucidated. The present study performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to examine Sp1 mRNA expression in 12 pairs of urothelial carcinoma and adjacent normal bladder tissues. Immunohistochemistry (IHC) was performed in 113 paraffin-embedded urothelial carcinoma tissues to detect the expression of Sp1. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the correlation between Sp1 expression and patient prognosis. The mRNA expression of Sp1 was elevated in the urothelial carcinoma by RT-qPCR compared with their paired normal bladder tissues. Among 113 cases of patients with urothelial carcinoma, there were 39 low histological grade and 74 high histological grade, 61 unifocal tumor and 52 multifocal tumor, 78 cases in Ta, T1, and T2 stages, and 35 cases in T3 and T4 stages. The enhanced expression of Sp1 mRNA was observed in tumors with a high histological grade, and invasive and metastatic samples. Immunohistochemistry revealed that Sp1 high expression was significantly correlated with the histological grade, tumor stage, vascular invasion, lymph node metastasis and distant metastasis (P < 0.05). Kaplan-Meier analysis demonstrated that elevated Sp1 expression in cancer tissue was correlated with a significantly poor overall survival (OS) and disease-free survival (DFS) compared with samples with low Sp1 expression (P < 0.05). Multivariate analyses by Cox's proportional hazard model also revealed that the expression of Sp1 was an independent prognostic factor in urothelial carcinoma. Sp1 expression is significantly elevated in urothelial carcinoma and may be used to identify a subset of patients with aggressive behaviors and poor clinical outcomes. Sp1 is a potential novel independent prognostic biomarker for patients with urothelial carcinoma following surgery.

A novel risk score model based on five angiogenesis-related long non-coding RNAs for bladder urothelial carcinoma

BackgroundTumour angiogenesis is an independent risk factor for bladder urothelial carcinoma (BUC) progression, but viable and promising antiangiogenic targets are understudied. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play prominent role in the tumour microenvironment and tumour angiogenesis.MethodsThe clinical data of BUC patients were obtained from TCGA database and clinical specimens of 138 BUC patients. Univariate and multivariate COX regression analyses were used to identify survival-related ARLNRs (sARLNRs) from The Molecular Signatures Database v4.0. Fisher’s exact probability method was used to detect the correlations between sARLNRs levels and clinicopathological characteristics. A chain of experiments including FACS, qPCR, immunohistochemistry, tube formation, migration and invasion assays, combining with co-culture models, were utilized to validate the clinical significance and angiogenetic correlation of sARLNRs.ResultsFive sARLNRs were employed to establish an angiogenesis-related risk score model, by which patients in the low-risk group obtained better overall survival than those in the high-risk group. The expression of AC005625.1 and AC008760.1 was significantly related to ECs percentage, tumour size and muscle invasion status. Besides, AC005625.1 and AC008760.1 expressed lower in BUC cell lines and tumour tissues than that in normal urothelial cells and adjacent normal tissues, with much lower levels in more advanced T stages. A prominently higher proportion of ECs was detected in tumour tissues with lower expression of AC005625.1 and AC008760.1. In the co-culture models, we found that knockdown of AC005625.1 and AC008760.1 in BUC cells increased the tube formation, migration and invasion abilities of HUVEC. The expression levels of CD31, VEGF-A, VIMENTIN and N-CADHERIN were also enhanced in HUVEC cells co-cultured with siR-AC005625.1 and siR-AC008760.1-treated T24 cells.ConclusionIn the study, we identify five sARLNRs and validate their clinical significance, angiogenesis correlation and prognosis-predictive values in BUC. These findings may provide a new perspective and some promising antiangiogenic targets for clinical diagnosis and treatment strategies of BUC.