Progression Of Urothelial Carcinoma Research Articles

Association of Androgen Receptor and PD-L1 Expression in Upper Urinary Tract Urothelial Carcinoma.

The response to immune checkpoint inhibitors (ICIs) or enfortumab vedotin is limited in patients with upper urinary tract urothelial carcinoma (UTUC), and the development of new targeted therapy for UTUC is eagerly needed. Several biomarkers, including programmed cell death-ligand 1 (PD-L1), have already been reported as predictors of response to ICIs therapy for UTUC. Recently, several studies have shown that steroid hormone receptors, including the androgen receptor (AR), are associated with progression of urothelial carcinoma. We prepared tissue microarrays (TMA) from paraffin blocks of UTUC specimens in 99 non-metastatic UTUC patients who underwent radical nephroureterectomy. With these TMA sections, we performed immunohistochemical staining for PD-L1 and AR and examined PD-L1 and AR expression levels in tumor cells. In addition, we analyzed the correlation between these markers and clinical prognosis in UTUC cases. PD-L1 was positive in 24 (24%) of the 99 samples, whereas AR was positive in 20 (20%) patients. AR-negative samples had significantly higher PD-L1 expression level than that the AR-positive samples (mean value 4.70% versus 2.55%, p=0.0324). Among AR-positive cases, patients with absence of PD-L1 expression had significantly lower cancer-specific survival (CSS) than that in PD-L1 expression-positive cases (p=0.049), although PD-L1 expression had no significant impact on CSS in AR-negative cases (p=0.920). Our findings suggest that AR is the promising target for UTUC treatment, especially in PD-L1-negative cases.

Immunohistochemical Expression of Claudin-1 and Claudin-4 in Urothelial Carcinoma of the Urinary Bladder.

In Egypt, bladder cancer occupies the second rankamong reported cancers in men. Claudins are tight junctions that have a critical role in tumor pathogenesis, invasion, progression, and metastasis and currentlyare a focus of interest for targeting therapies. We aimed to evaluatethe immunohistochemical expression of Claudin-1 and Claudin-4 in urinary bladder urothelial carcinoma and investigate the relationshipbetweenthe expressed Claudins with differentclinicopathological parameters. Claudin-1 and Claudin-4 immunohistochemical expression was studied in 62 cases of urinary bladder urothelial carcinomas. The cases were classified into two categories; low and high Claudin-1 and Claudin-4 expression. High Claudin-1 expression was detected in67.7% of the studied urothelial carcinomas while 32.3% showed low expression. Claudin-1 expression was reduced significantly with high tumor grade, non-papillary tumors, muscle invasion, schistosomal infestation, and perineural invasion (p-value < 0.05). Claudin-4 high expression was detected in 82.3% of our cases while low expression was detected in 17.7%. Claudin-4 reduced expression was significantly associated with non-papillary tumors, muscle invasion, advanced T stages, and associated lympho-vascular emboli (P-value < 0.05). According to the results ofthe present study, the reduced expressions of Claudin-1 and Claudin-4 provide clues concerning the progression of urothelial carcinoma. Consequently, it is thought that Claudin-1 and Claudin-4 could help to differentiatelow-grade from high-grade and muscle-invasive from non-muscle-invasive urothelial carcinomas. In addition, it can be introduced as a possible therapeutic target.

Altered p53/p16 expression is linked to urothelial carcinoma progression but largely unrelated to prognosis in muscle-invasive tumors

Background Most inactivating p53 mutations result in a nuclear p53 accumulation – detectable by immunohistochemistry (IHC). p53 alterations leading to a complete lack of p53 protein and absence of immunostaining do also occur – not easily detectable by IHC. p16 is upregulated in p53 inactivated cells. We hypothesized that a positive p16 IHC may help to distinguish p53 inactivation in IHC negative cases. Material and methods We investigated p53 and p16 immunostaining on 2710 urothelial bladder carcinomas in a tissue microarray format to understand their impact in relation to clinicopathological parameters of disease progression and patient outcome. Results p16 immunostaining was absent in normal urothelium but occurred in 63.5% (30.4% strong) of cancers. p16 strongly positive cases increased from pTaG2 low-grade (9.6%) to pTaG3 high-grade tumors (46.5%, p < .0001) but decreased from pTaG3 to pT4 (33.3%; p = .0030). Among pT2-4 carcinomas, p16 positivity was linked to high-grade (p = .0005) but unrelated to overall survival. p53 staining was negative in 8.4%, very weak in 15.4%, weak in 55.3%, strong in 4.7%, and very strong in 16.2% cancers. p53 negative (potentially p53 null phenotype), strong, and very strong p53 positivity increased from pTaG2 low-grade to pTaG3 high-grade tumors (p < .0001) and from pTaG3 to pT2-4 cancers (p = .0007). p53 staining was largely unrelated to histopathological parameters or patient prognosis among pT2-4 carcinomas, except of p53 strong/very strong immunostaining. p16 expression predominated in tumors with very strong, strong, and negative p53 staining and the combination of p53 negative/p16 strongly positive cancers was linked to features of tumor aggressiveness. Conclusion Aberrant p53 and p16 immunostaining increases during grade and stage progression although p53 negative and p16 positive immunostaining lack prognostic significance in pT2–4 carcinomas. Potential diagnostic features are that high level p16 expression is limited to neoplastic urothelium and p53 null phenotype to aggressive cancers (grade 3 and invasive).

Downregulation of CRTAC1 in Urothelial Carcinoma Promotes Tumor Aggressiveness and Confers Poor Prognosis.

Cartilage acidic protein 1 (CRTAC1) is a glycosylated calcium-binding extracellular matrix protein. The oncological functions of CRTAC1 in urothelial carcinoma (UC) of the urinary bladder (UB) and upper urinary tract (UT) have not yet been elucidated. Based on the published UBUC transcriptome data, we re-evaluated the differential expression profile of calcium ion binding-related genes (GO:0005509), and we found that CRTAC1 was the most significantly downregulated gene in UBUC progression. Therefore, we analyzed the prognostic value and biological significance of CRTAC1 expression in UC. We used immunohistochemistry to determine the CRTAC1 expression levels in 340 patients with UTUC and 295 patients with UBUC. The CRTAC1 expression was compared with the clinicopathological characteristics, and the prognostic impact of CRTAC1 on metastasis-free survival (MFS) and disease-specific survival (DSS) was evaluated. To study the biological functions of CRTAC1, the proliferation, migration, invasion, and tube formation abilities of UC-derived cells were evaluated. A low CRTAC1 expression significantly correlated with high tumor stage, high histological grade, perineural invasion, vascular invasion, nodal metastasis, and high mitotic rate (all p < 0.01). Moreover, the CRTAC1 immunoexpression status was an independent prognostic factor for MFS and DSS in UBUC and UTUC patients (all p < 0.001) in the multivariate analysis. The exogenous expression of CRTAC1 suppressed the cell proliferation, invasion, and angiogenesis, and downregulated the matrix metallopeptidase 2 (MMP2) level in BFTC909 and T24 cells. CRTAC1 may participate in progression of UC and serve as a prognostic marker for metastasis. Low CRTAC1 expression was significantly associated with aggressive UC characteristics and worse clinical outcomes. The inclusion of CRTAC1 immunoexpression in the standard pathological variables may optimize the risk stratification of patients.

Astragalus polysaccharide inhibits the development of urothelial carcinoma by activating AMPK signaling to induce BENC1-xCT complex formation.

In recent years, the incidence of urothelial carcinoma (UC) has been high in men. The aim of this study was to investigate whether astragalus polysaccharide (APS) could inhibit the development of UC and the specific molecular mechanism. Our data showed that APS inhibited the proliferation of UC cells in a dose-dependent manner, and APS reduced the migratory capacity of RT4 and T24 cells. Further studies revealed that the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed APS-induced cell death, intracellular Fe2+ and malondialdehyde (MDA) accumulation, and lipid peroxidation product deposition. The Western blot and immunofluorescence results showed that APS significantly inhibited the expression of glutathione peroxidase 4 (GPX4) but did not alter the protein level of solute carrier family 7 member 11 (xCT, SLC7A11). Further analysis revealed that APS reduced the activity of xCT in RT4 and T24 cells. Moreover, APS significantly increased the phosphorylation levels of protein kinase AMP-activated catalytic subunit alpha 1 (AMPK) and BECN1 in RT4 and T24 cells, which induced the formation of the BECN1-xCT complex. However, when AMPK was silenced in RT4 and T24 cells, APS-induced ferroptosis was reversed to some extent, indicating that APS-mediated ferroptosis involves AMPK signaling. Moreover, APS has been shown to inhibit tumor growth in nude mice in vivo. In summary, our study demonstrated for the first time that APS could promote the formation of the BECN1-xCT complex in UC cells by activating AMPK/BECN1 signaling, which inhibited the activity of xCT to reduce GPX4 expression, thereby inducing ferroptosis and ultimately inhibiting UC progression.

TUBA1C is a potential new prognostic biomarker and promotes bladder urothelial carcinoma progression by regulating the cell cycle

BackgroundTUBA1C is an α-tubulin isoform involved in mitosis, and its dysregulation has been implicated in tumor progression. There is still no clear understanding of its role in bladder urothelial carcinoma (BLCA).MethodsThis study examined the differential expression of TUBA1C and its prognostic significance in bladder cancer based on data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) and also assessed the correlation of TUBA1C expression level with immune cell infiltration and immune checkpoint gene expression levels and the half-inhibitory concentration (IC50) of different chemotherapeutic agents. Immunotherapy response was estimated using the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm. We detected TUBA1C expression in BLCA cells using PCR and Western blotting. Functional assays, including CCK-8, colony formation, transwell, apoptosis and cell cycle assays, were also performed to assess the oncogenic role of TUBA1C in BLCA.ResultIn three independent public cohorts, TUBA1C was significantly upregulated in bladder tumor tissues, and high TUBA1C expression in bladder cancer was associated with a poorer outcome than low expression. TUBA1C was an independent prognostic risk factor for bladder cancer, and numerous immune checkpoint genes and infiltrating immune cells were associated with TUBA1C. TIDE analysis revealed that TUBA1C showed great potential for predicting the immunotherapy response in bladder cancer patients. In addition, drug sensitivity analysis revealed that high TUBA1C expression indicated sensitivity to multiple chemotherapeutic agents. Functional assays revealed that silencing TUBA1C significantly inhibited the proliferation, migration and invasion of BLCA cells and induced apoptosis and cell cycle arrest.ConclusionThe overexpression of TUBA1C in bladder cancer predicts a poor prognosis and may also be a potential immunotherapeutic target. As a prognostic marker, TUBA1C influences tumor progression by regulating the cell cycle.

Abstract 5555: Altered p53/p16 expression is linked to urothelial carcinoma progression but is unrelated to prognosis in muscle-invasive tumors

Abstract Background: Most inactivating p53 mutations result in a nuclear accumulation of the defective p53 protein. However, p53 alterations that result in a complete lack of cellular p53 and complete absence of p53 immunostaining do also occur. As p16 is upregulated in p53 inactivated cells, p16 immunohistochemistry may be a surrogate marker for p53 inactivation. Design: In this study, we investigated p53 and p16 immunostaining on more than 2,500 urothelial bladder carcinomas in a tissue microarray format to better understand their impact in relation to clinicopathological parameters of disease progression and patient outcome. Results: p16 immunostaining was not observed in normal urothelium but occurred in 1,576 (63.5%) of cancers including 755 (30.4%) with a strong staining. The fraction of p16 positive cases increased markedly from pTaG2 low grade (9.6%) to pTaG3 high grade tumors (46.5% strongly positive, p&amp;lt;0.0001 for pTaG2 low vs. pTaG3) but continuously decreased from pTaG3 to pT2 (41.3% strongly positive), pT3 (36.5%) and pT4 (33.3%; p=0.0030). Within pT2-4 carcinomas, p16 positivity was also linked to high grade (p=0.0005) but unrelated to overall survival. p53 staining has been recorded as negative in 203 (8.4%), very weak in 373 (15.4%), weak in 1,341 (55.3%), strong in 115 (4.7%), and very strong in 393 (16.2%) cancers. The fraction of tumors with negative (potentially p53 null phenotype), strong, and very strong p53 positivity increased markedly from pTaG2 low grade to pTaG3 high grade tumors (p&amp;lt;0.0001) and from pTaG3 to muscle-invasive pT2-4 cancers (p=0.0007). p53 staining pattern was unrelated to histopathological parameters of malignancy or patient prognosis within pT2-4 carcinomas, however. There was a significant overall association between p53 and p16 expression but strong p16 expression predominated in tumors with very strong, strong, and negative p53 staining. Subset analyses showed that the combination of p53 negative/p16 strongly positive cancers was particularly linked to features of tumor aggressiveness. Conclusion: Our data show that altered function of p53 and p16 immunostaining increases during grade and stage progression although these alterations lack prognostic significance in pT2-4 carcinomas. That high level p16 expression is limited to neoplastic urothelium and that the p53 null phenotype is largely limited to grade 3 and invasive urothelial carcinomas are features with potential diagnostic utility. Citation Format: Simon Schallenberg, Henning Plage, Sebastian Hofbauer, Kira Kornienko, Sarah Weinberger, Paul G. Bruch, Florian Roßner, Sefer Elezkurtaj, Martina Kluth, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, David Horst, Tobias Klatte, Thorsten Schlomm, Henrik Zecha. Altered p53/p16 expression is linked to urothelial carcinoma progression but is unrelated to prognosis in muscle-invasive tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5555.

Single-cell transcriptomic analysis of tumor heterogeneity and intercellular networks in human urothelial carcinoma.

Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies. Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance test, and Pearson correlation analysis were used properly. Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8 + T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4 + T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3 + dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR + endothelial cells and RGS5 + /ACTA2 + pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC. Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.

CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma.

Urine-based cytology is non-invasive and widely used for clinical diagnosis of urothelial carcinoma (UC), but its sensitivity is less than 40% for low-grade UC detection. As such, there is a need for new diagnostic and prognostic biomarkers of UC. CUB domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein highly expressed in various cancers. Using tissue array analysis, we demonstrated that CDCP1 expression in UC patients (n = 133), especially in those with low-grade UC, was significantly higher than in 16 normal persons. In addition, CDCP1 expression in urinary UC cells could also be detected by using immunocytochemistry method (n = 11). Furthermore, in 5637-CD cells, overexpression of CDCP1 affected the expression of epithelial mesenchymal transition-related markers and increased matrix metalloproteinase 2 expression and migration ability. Conversely, the knockdown of CDCP1 in T24 cells had the opposite effects. Using specific inhibitors, we demonstrated the involvement of c-Src/PKCδ signaling in the CDCP1-regulated migration of UC. In conclusion, our data suggest that CDCP1 contributes to the malignant progression of UC and may have the potential as a urine-based biomarker for detecting low-grade UC. However, a cohort study needs to be conducted.

A Short Overview on Therapeutic Biomarkers for Muscle Invasive Bladder Carcinoma

Urothelial carcinoma (UC) is the second most common urologic malignancy after prostatic adenocarcinoma. UC comprises more than 90% of urinary bladder tumours. The intense research involving the different molecular aspects of bladder malignancies offers potential opportunities to improve understanding of bladder cancer biology; helps to identify disease earlier; and improves prediction of outcomes or helps targeted therapy. This review highlights the general concepts of the molecular features: molecular pathways for bladder carcinomas and molecular biomarkers for potential target for treatment of UC of the bladder. This discussion could improve the understating of pathogenesis as well as will provide new therapeutic modules, e.g., targeted therapy. This article is a review of bladder cancer genetics, focusing on molecular changes and their significance in the pathogenesis and progression of muscle invasive UC. Also, the relevant genetic biomarkers and their products, and new therapeutic targets and agents that are being developed are presented here.

Omics sciences and precision medicine in Urothelial Carcinoma.

This comprehensive review explores the potential of omics sciences - such as genomics, transcriptomics, proteomics, and metabolomics - in advancing the diagnosis and therapy of urothelial carcinoma (UC), a prevalent and heterogeneous cancer affecting the urinary tract. The article emphasizes the significant advancements in understanding the molecular mechanisms underlying UC development and progression, obtained through the application of omics approa-ches. Genomic studies have identified recurrent genetic alterations in UC, while transcriptomic analyses have revealed distinct gene expression profiles associated with different UC subtypes. Proteomic investigations have recognized protein biomarkers with diagnostic and prognostic potential, and metabolomic profiling has found metabolic alterations that are specific to UC. The integration of multi-omics data holds promises in refining UC subtyping, identifying therapeutic targets, and predicting treatment response. However, challenges like the standardization of omics technologies, validation of biomarkers, and ethical considerations need to be addressed to successfully translate these findings into clinical practice. Omics sciences offer tremendous potential in revolutionizing the diagnosis and therapy of UC, enabling more precise diagnostic methods, prognostic evaluations, and personalized treatment selection for UC patients. Future research efforts should focus on overcoming these challenges and translating omics discoveries into meaningful clinical applications to improve outcomes for UC patients.

Prognostic value of galectin-1 and galectin-3 expression in localized urothelial bladder cancer.

Galectin-1 (Gal-1) and Galectin-3 (Gal-3) are carbohydrate binding proteins with a wide range of biological activity, including regulation of cellular adhesion, proliferation, and apoptosis in solid tumors. Prior small studies have reported that Gal-3 expression is associated with progression of disease in urothelial carcinoma (UC), from non-muscle invasive UC progression to muscle invasive UC. We assessed Gal-1 and Gal-3 protein expression H-score utilizing a tissue microarray (TMA) created from 301 cystectomy specimens. Immunohistochemistry for Gal-1 and Gal-3 was performed on TMA generated from tumor blocks from chemotherapy naïve cystectomy specimens. The variable of interest, H-score, was defined as the product of the percentage of cells staining positive (0-100) and intensity score (0-3) scored by a single pathologist. Survival end points were analyzed using Kaplan-Meier and Cox Proportional Hazards methods. Clinical data including Charlson Comorbidity Index (CCI), pathologic tumor (T) stage, tumor size, node stage, and surgical margins, were included in multivariable analysis. We found that Gal-1 and Gal-3 expression correlated with intratumoral T stage (median Gal-1 H-score was 0 across non-invasive tissue types and 200 in invasive, P<0.01 and median Gal-3 score was 270 across non-invasive tissue types and 70 in invasive, P<0.01). However, the highest intratumoral H-score per cystectomy core did not independently predict for recurrence-free survival (RFS) (Gal-1: HR =1.02, P=0.44, Gal-3: HR =1.01, P=0.65) or OS (Gal-1: HR =1.02, P=0.44, Gal-3: HR =1.01, P=0.72) in this cohort. Significant intratumoral heterogeneity was present for both Gal-1 and Gal-3, with an average difference between the highest and lowest H score was 95 for Gal-1 and 109 for Gal-3 for cystectomy specimens with more than one biopsy. Gal-1 and Gal-3 H-score per bladder did not independently predict for RFS or OS. Intra-tumoral Gal-1/Gal-3 heterogeneity complicates the use of Gal-1 and Gal-3 expression as a prognostic biomarker. Future studies should consider the evaluation of serum and urinary galectins as an approach to mitigate tumor heterogeneity.

Significance of tumor-associated neutrophils, lymphocytes, and neutrophil-to-lymphocyte ratio in non-invasive and invasive bladder urothelial carcinoma.

Tumor-infiltrating neutrophils and lymphocytes play essential roles in promoting or combating various neoplasms. This study aimed to investigate the association between tumor-infiltrating neutrophils and lymphocytes and the neutrophil-to-lymphocyte ratio in the progression of urothelial carcinoma. A total of 106 patients diagnosed with urothelial carcinoma were was. Pathological examination for tumor grade and stage and for tumor-infiltrating neutrophils, both CD4 and CD8+ T lymphocytes, as well as the neutrophil- to-lymphocyte ratio were evaluated. The presence of neutrophils and the neutrophil-to-lymphocyte ratio correlated with high-grade urothelial neoplasms. In both low- and high-grade tumors, the lymphocytes increased during progression from a non-invasive neoplasm to an early-invasive neoplasm. CD8+ T lymphocytes increased in low-grade non-muscle-invasive tumors compared to non-invasive tumors. Additionally, there was a significant decrease in CD8+ T lymphocytes during progression to muscle-invasive tumors. Our results suggest that tumor-infiltrating neutrophils and CD8+ T lymphocytes have a significant effect on tumor grade and progression.

Comparative analysis of changes in proliferative-apoptotic and hormonal status of non-invasive papillary urothelial carcinomas with molecular subtypes of invasive carcinomas

Invasive urothelial carcinoma is the most common bladder malignancy, comprising 90% of all primary bladder carcinomas. According to the statistics of Georgia, according to NCDC data, bladder cancer is the third most common cancer among men. As a result of non-invasive urothelial carcinoma, 70% of the operated patients developed a recurrence in the 5-year period after the surgical intervention on the urinary bladder, among which the share of advanced, metastatic cases is 10-20%.Therefore, regardless of the same degree of differentiation and staging, the recurrence and progression of urothelial carcinoma varies in different patients. A cohort retrograde study was carried out, for which archival material of the teaching-scientific and diagnostic laboratory of Tbilisi State Medical University for the years 2019-2021 was used. The results of the study showed that molecular subtypes of urothelial invasive carcinomas express CK20 and CK5 with different intensity. Moreover, there are mixed, hybrid forms with the coexistence of both markers, which indicates the intratumoral heterogeneous nature of invasive carcinomas, which probably depends on the different clinical course and prognosis of tumors of the same histological type. The phenotypic characteristics of low- and high-grade non-invasive papillary urothelial carcinomas are fundamentally different from the characteristics of invasive urothelial carcinoma, which gives reason to assume that they are different tumors and require additional genomic changes to transform into invasive carcinoma.The phenotypic differences between them are likely to determine the risk of recurrence and invasive transformation. CK20, CK5, apoptotic and proliferative characteristics with androgen receptor expression. Together, it can be used to assess the risk of recurrence and transformation to invasive carcinoma of low- and high-grade noninvasive papillary urothelial carcinomas.

Assessment of survivin and p27 expression as potential prognostic markers in urothelial cell carcinoma of urinary bladder in Egyptian patients

BackgroundBladder cancer (BC) is the tenth most common cancer worldwide with urothelial carcinoma (UC) being the main histologic subtype. Survivin is an apoptosis inhibitor that is associated with tumor proliferation and invasion. P27 is a cyclin-dependent kinase inhibitor that negatively regulates cell proliferation. The expression of both proteins was variable among different solid tumors including UC.MethodsWe aimed to investigate the expression of survivin and P27 in UC of urinary bladder and correlate their expressions with histopathological parameters in an attempt at studying the possibility of their use as targeted therapies. The investigation was performed through immunohistochemical staining for both proteins on sections belonging to 60 UCs and 12 mild chronic cystitis cases (controls). Immunopositivity (number of positive cases) and expression score (percentage of positive urothelial cells) were evaluated.ResultsBoth survivin and P27 were absent in urothelial cells of mild chronic cystitis lesions while expressed in 60% and 43.3% of UCs, respectively. High score of survivin and low score of P27 were associated with poor prognostic factors of UC (solid pattern, high grade, and deep tumors). By logistic regression test, survivin expression can be a predictive risk factor associated with solid pattern and high-grade UC, while P27 expression can be a predictive risk factor associated low-grade UC.ConclusionHigh survivin and low P27 expression scores were associated with the studied prognostic factors of UC. Both proteins may play a role in UC progression and can have a value as prognostic and/or diagnostic markers of UC, as well as targeted therapies.

A systematic review and meta-analysis of CK20, CD44, Ki67 and p53 as immunohistochemical markers in bladder carcinoma in situ

BackgroundUrothelial dysplasia and carcinoma in situ (CIS) are related to recurrence and progression of urothelial carcinoma. Differentiating CIS and dysplasia from reactive atypia is often difficult based only on histological features. The integration of histological findings with immunohistochemistry is used in routine practice to make a diagnosis of CIS and, for this purpose, the immunohistochemical markers CK20, CD44, Ki67 and p53 are used to supplement histology.In this work, we aimed to assess CK20, CD44, Ki67 and p53 as immunohistochemical markers in patients with CIS through a systematic review and meta-analysis. Materials and methodsA systematic review was performed by searching electronic databases for English-language studies published from January 2010 to April 2021. Studies were considered eligible if they evaluated the CK20, CD44, Ki67 and p53 expression in CIS. ResultsIn total, 15 references were suitable for quantitative review. The overall rate of CK20, CD44, Ki67 and p53 expression in CIS was 43%, 31%, 44%, 38%, respectively. ConclusionsOur study supports the 2014 International Society of Urologic Pathology consensus that histological assessment remains the gold standard to diagnose urothelial CIS and suggests that a very close correlation between morphological, immunohistochemical and clinical data is essential to provide the best management for patients with bladder carcinoma.

The carbonic anhydrase inhibitor acetazolamide inhibits urinary bladder cancers via suppression of β-catenin signaling.

Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN‐induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN‐induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2‐overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN‐induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.

Revisión sistemática y metaanálisis sobre CK20, CD44, Ki67 y p53 como marcadores inmunohistoquímicos en el carcinoma in situ vesical

IntroductionUrothelial dysplasia and carcinoma in situ (CIS) are related to recurrence and progression of urothelial carcinoma. Differentiating CIS and dysplasia from reactive atypia is often difficult based only on histological features. The integration of histological findings with immunohistochemistry is used in routine practice to make a diagnosis of CIS and, for this purpose, the immunohistochemical markers CK20, CD44, Ki67 and p53 are used to supplement histology.In this work, we aimed to assess CK20, CD44, Ki67 and p53 as immunohistochemical markers in patients with CIS through a systematic review and meta-analysis. Materials and methodsA systematic review was performed by searching electronic databases for English-language studies published from January 2010 to April 2021. Studies were considered eligible if they evaluated the CK20, CD44, Ki67 and p53 expression in CIS. ResultsIn total, 15 references were suitable for quantitative review. The overall rate of CK20, CD44, Ki67 and p53 expression in CIS was 43%, 31%, 44%, 38%, respectively. ConclusionsOur study supports the 2014 International Society of Urologic Pathology consensus that histological assessment remains the gold standard to diagnose urothelial CIS and suggests that a very close correlation between morphological, immunohistochemical and clinical data is essential to provide the best management for patients with bladder carcinoma.

Serial ctDNA evaluation to predict clinical progression in patients with advanced urothelial carcinoma.

532 Background: It is not known whether serial circulating tumor DNA (ctDNA) can augment imaging for assessing treatment response in patients (pts.) with advanced urothelial carcinoma (UC). We hypothesized that serial ctDNA measurements predict the clinical progression of advanced UC and map its evolutionary trajectories. Methods: We analyzed 182 serial ctDNA samples from 53 pts. with advanced UC sequenced by the Guardant360 ctDNA platform. The aggregate variant allele frequency (aVAF) was defined as the sum of VAFs of all genomic alterations (GAs) in a ctDNA sample. Clinical response status was categorized into progressive disease (PD) and non-PD based on imaging. Progression-free survival (PFS) was defined as the time from treatment start till PD or death. The Mann-Whitney test was used to compare radiologic response status (PD vs. non-PD) and the ctDNA aVAF collected within 4 weeks of restaging imaging. Results: Pts. with lower initial ctDNA aVAF (≤0.2) had longer overall survival (OS) (hazard ratio (HR): 0.31, 95%CI: 0.11-0.90, p = 0.03). Pts. who achieved clearance of their ctDNA aVAF at the time of any subsequent ctDNA sample (n = 19/53) had longer OS (HR: 0.26, 95%CI: 0.08-0.85, adjusted p = 0.027). Combining ctDNA aVAF values from two consecutive samples improved the performance of a clinical prognostic model based on age, sex, and liver metastasis (C-statistic improved from 0.65 to 0.84). The mean ctDNA aVAF and the median number of GAs per ctDNA sample significantly increased at the time of PD vs. non-PD (12.31 vs. 2.10, p &lt; 0.0001, and 3 vs. 1, p = 0.0006, respectively). Delta ctDNA aVAF increases predicted radiologic PD with an area under the receiver operating characteristic curve of 0.84 (95%CI: 0.65-0.95, p &lt; 00.1). Delta ctDNA aVAF improved the patient risk stratification, pts with both decreased delta ctDNA aVAF and non-PD had the longest overall survival (Log-rank p = 0.05). A subgroup of 20 pts. had increasing ctDNA aVAF and ≥ 1 available subsequent radiologic scans within six months. Increasing ctDNA VAF ≥1 predicted PD in 90% (18/20) of pts with a median lead time of 92 days over imaging. APOBEC3-induced mutations (A3-m) were identified in ctDNA samples of (23%) 12/53 pts. Pts. with ctDNA A3-m had longer median PFS on immune checkpoint blockade than pts. without (17 vs. 3 months, Log-rank p = 0.01). There was no significant difference in OS according to ctDNA A3m status (Log-rank p = 0.37). Serial ctDNA provided insights into the clonal dynamics of treatment resistance, including the acquisition of ERBB2 S653C resistance mutation at the time of PD on lapatinib. Conclusions: Serial ctDNA predicts clinical outcomes and provides real-time assessment of treatment effectiveness in pts. with advanced UC. This has the potential to guide future adaptive therapy paradigms.

Angiogenesis Driven by the CEBPD-hsa-miR-429-VEGFA Signaling Axis Promotes Urothelial Carcinoma Progression.

Background and Purpose: This research aimed to excavate the alternative mechanism of CEBPD on tumor growth and explore the biological significance of the CEBPD/hsa-miR-429/VEGFA axis on angiogenesis in urothelial carcinoma (UC). Methods: Quantitative RT-PCR, immunoblotting assay and tube formation examined the effect of hsa-miR-429 mimic or/and inhibitor on VEGFA expression and angiogenesis in CEBPD-overexpressing UC-derived cells. The association between CEBPD, hsa-miR-429, VEGFA and microvascular density (MVD) and clinical outcome were evaluated in 296 patients with UBUC and 340 patients with UTUC, respectively. Results: The increase in the transcript and protein of VEGFA as well as HUVECs tube formation was diminished upon the treatment of hsa-miR-429 mimic in CEBPD-overexpressing BFTC909 and TCCSUP. Nevertheless, the inhibited regulation of hsa-miR-429 mimic on the expression of VEGFA and ability of HUVECs tube formation was rescued by the combined incubation with hsa-miR-429 inhibitor in these two UC-derived cell lines. Furthermore, the clinical correlations showed that the higher level of VEGFA or MVD has a positive correlation with the expression of CEBPD and a negative relation to hsa-miR-429 and leads to tumor aggressiveness with worse disease-specific, metastasis-free survival in UBUC and UTUC cohorts. Conclusions: We decipher the oncogenic mechanism of CEBPD on angiogenesis through the hsa-miR-429 inhibition to stabilize the expression of VEGFA in UC. The novel research unveiled the modulation of the CEBPD/hsa-miR-429/VEGFA axis on the progression of UC and could be accessible to theranostic biomarkers.