Normal Bladder Tissue Samples Research Articles

Fibroblast Growth Factor Receptor 3 (FGFR3) Gene Amplification in Patients with Urothelial Carcinoma of the Urinary Bladder

Background: The fibroblast growth factor receptors (FGFRs) family includes five tyrosine kinase receptors, which play an important role in many cellular mechanisms including proliferation, migration and survival. Deregulations in the genes that coding for these receptors, mainly the FGFR3 gene, had been reported in patients with urothelial carcinoma of the urinary bladder. Objective: To estimate the frequency of FGFR3 gene amplification in patients with urothelial carcinoma of the urinary bladder and its relation to some clinico-pathological parameters of the tumor. Methods: The present retrospective study included 30 paraffin blocks of urothelial carcinoma tissue (trans-urethral resection of bladder tumor (TURBT)), and 10 samples of normal bladder tissue (control group). Sections were taken from each paraffin block for studying FGFR3 gene amplification by fluorescent in situ hybridization (FISH) technique. Results: FGFR3 gene was shown to be amplified in 4 (13.3%) out of 30 urothelial carcinoma cases. There was significant relation between FGFR3 gene amplification and tumor’s pathological stage (p=0.037). FGFR3 gene wasn’t statistically correlated with the patient’s age, gender, tumor’s grade or the lympho-vascular permeation. Conclusion: FGFR3 gene amplification was detected in 13.3% of urothelial carcinoma of the urinary bladder cases, which may reflect its role in carcinogenesis process. This amplification was statically correlated to tumor’s pathological stage suggesting its relation to tumor progression. Keywords: FGFR3, FISH, TURBT, urothelial carcinoma, gene amplification Citation: Al-Marashi MA, Qasim BJ. Fibroblast Growth Factor Receptor 3 (FGFR3) gene amplification in patients with urothelial carcinoma of the urinary bladder.Iraqi JMS. 2021; 19(1): 116-125. doi: 10.22578/IJMS.19.1.15

Pathological and Clinicopathological Features of Canine and Feline Bladder Disease

Dogs and cats commonly present to veterinary hospitals with urinary bladder disease, but despite their clinical importance and comparative potential to human diseases, bladder diseases in Australian dogs and cats are under investigated. In veterinary pathology, insufficient levels of diagnostic agreement can occur, and this is influenced by sample quality as well as the pathologist’s own experience, training, and cognitive biases. Logistic regression is a statistical technique which, when applied to veterinary histopathology, could improve pathologist agreement. Thus, there were two overarching goals of this thesis - to investigate the pathology and comparative potential of canine and feline urinary bladder disease in Australia, and to explore the utility of logistic regression modelling in improving inter-pathologist agreement.This project conducted a retrospective evaluation of pathology cases of canine and feline urinary bladder tissue from the veterinary pathology archives of the University of Queensland School of Veterinary Science and Murdoch University’s School of Veterinary and Life Sciences, with prospective sampling from veterinary clinics and a commercial veterinary pathology service in South East Queensland. The demographics of the dataset were examined using proportionate morbidity and logistic regression to identify associations between animal factors and the diagnosis. Secondly, a comprehensive histological evaluation was undertaken of every sample, with logistic regression modelling performed to identify associations between histological variables and diagnosis. Thirdly, a subset of canine and feline diseased and normal bladder tissue samples was tested for biomarker expression using immunohistochemistry and polymerase chain reaction. This combined approach tested if retrospective samples were of sufficient quality, and when validated provided quantity as well as cellular location of the target biomarkers. To further investigate the comparative potential of feline idiopathic cystitis (FIC), a systematic review was conducted on biomarkers in bladder pain syndrome (BPS) compared to FIC, following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. Finally, to investigate the role of logistic regression modelling in veterinary pathologist agreement, the modelling of histological variables was used to formulate a predictive probability tool which we then tested on four pathologists evaluating the same set of twenty-five slides, with diagnostic agreement evaluated using the Fleiss kappa statistic.The main findings from the demographic analysis were a higher risk of bladder neoplasia in dogs compared to cats, increasing risk for bladder neoplasia with age, and decreased risk for cystitis in neutered animals. Next, logistic regression modelling on the histology dataset of canine and feline urinary bladder tissue from Eastern and Western Australia identified six significant variables that3were associated with the diagnosis – urothelial ulceration, urothelial inflammation, neutrophilic submucosal inflammation, submucosal lymphoid aggregates, amount of submucosal haemorrhage, and species. These six variables were used to create a predictive probability tool for bladder disease diagnosis. The pathologist agreement study revealed a good level of agreement between the four pathologists when diagnosing neoplastic lesions, but poor to fair agreement for cystitis, urolithiasis and normal bladder tissue. Agreement between pathologists did improve when signalment and clinical history was provided, with mixed results on inter-pathologist agreement when the predictive probability tool was used. However, the predictive tool did prove valuable in increasing the agreement of the study pathologists’ diagnosis with the reference diagnosis. There were multiple other confounders at play in this experiment such as variable digital slide quality and different interpretations of the study instructions. A systematic review on biomarkers in bladder pain syndrome revealed that nerve growth factor is the most likely urine biomarker to be useful in the diagnosis of human BPS. The aim of this review had been to compare biomarkers in BPS to those in FIC, however an unexpected variability in the study parameters meant we could not fulfil this goal. A final laboratory-based investigation of biomarkers of canine and feline bladder diseases revealed two findings - that archived formalin fixed, paraffin embedded tissues are not good samples for PCR experiments, and secondly that tight junction protein-1 may be a promising tissue biomarker for differentiating between some urinary bladder diseases in dogs and cats.In conclusion, this thesis has undertaken a comprehensive analysis of the pathogenesis and comparative potential of canine and feline bladder diseases and is the first to apply logistic regression modelling to veterinary histopathology diagnosis and to improving inter-pathologist agreement. Logistic regression modelling is a promising tool for veterinary pathology. Dogs and cats are potentially good comparative models for human bladder diseases; however, inconsistent case definitions in human research complicates veterinary and medical field alignment. Finally, a collaborative multicentre approach would be invaluable to collect high quality prospective samples of feline idiopathic cystitis cases to allow further investigation into this disease. In summary, the comprehensive approach utilised in this thesis has provided valuable information on bladder disease in cats and dogs and sets a foundation for further work in this field.

Bone morphogenic proteins-2, -4, -6 and 7 in non-muscle invasive bladder cancer.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) family and may play an important role in the regulation of malignant cells in bladder cancer. The aim of the present study was to investigate BMP expression in non-muscle invasive bladder cancer. Tumor tissue samples from 71 patients treated with transurethral resection and 10 samples of normal bladder tissue were stained using immunohistochemistry for BMP-2, −4, −6 and −7. The levels of BMP were correlated with the number and size of tumors in the bladder, the pathohistological findings as well as with tumor recurrence and progression. The results of the present study demonstrated that BMP-2 and −7 are highly expressed in normal bladder tissue, but significantly downregulated in cancer samples. This reduction correlates with a faster rate of tumor recurrence as well as with an increase in the number of recurrent tumors. There was no evident interrelation between BMP-2 and −7 reduction and changes in tumor grade and stage. In conclusion, BMP-2 and −7 are potential prognostic factors for tumor recurrence and further studies on BMP and bladder cancer are needed to confirm these results.

KIF5A Promotes Bladder Cancer Proliferation In Vitro and In Vivo.

Background Bladder cancer is a common malignancy with uncontrolled and rapid growth. Although lots of the important regulatory networks in bladder cancer have been found, the cancer-relevant genes remain to be further identified. Methods We examined the KIF5A expression levels in bladder cancer and normal bladder tissue samples via immunohistochemistry and observed the effect of KIF5A on bladder tumor cell proliferation in vitro and in vivo. Additionally, a coexpression between KIF5A and KIF20B in tumor tissues was explored. Results KIF5A expression level was higher in the bladder cancer tissues than in the adjacent nontumor tissues. Patients with higher KIF5A expression displayed advanced clinical features and shorter survival time than those with lower KIF5A expression. Moreover, KIF5A knockdown inhibited bladder cancer cell proliferation, migration, and invasion demonstrated in vivo and in vitro. In addition, coexpression was found between KIF5A and KIF20B in tumor tissues. Conclusion The results demonstrated that KIF5A is a critical regulator in bladder cancer development and progression, as well as a potential target in the treatment of bladder cancer.

Identification of key candidate genes and biological pathways in bladder cancer.

BackgroundBladder cancer is a malignant tumor in the urinary system with high mortality and recurrence rates. However, the causes and recurrence mechanism of bladder cancer are not fully understood. In this study, we used integrated bioinformatics to screen for key genes associated with the development of bladder cancer and reveal their potential molecular mechanisms.MethodsThe GSE7476, GSE13507, GSE37815 and GSE65635 expression profiles were downloaded from the Gene Expression Omnibus database, and these datasets contain 304 tissue samples, including 81 normal bladder tissue samples and 223 bladder cancer samples. The RobustRankAggreg (RRA) method was utilized to integrate and analyze the four datasets to obtain integrated differentially expressed genes (DEGs), and the gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. The OncoLnc online tool was utilized to analyze the relationship between the expression of hub genes and the prognosis of bladder cancer.ResultsIn total, 343 DEGs, including 111 upregulated and 232 downregulated genes, were identified from the four datasets. GO analysis showed that the upregulated genes were mainly involved in mitotic nuclear division, the spindle and protein binding. The downregulated genes were mainly involved in cell adhesion, extracellular exosomes and calcium ion binding. The top five enriched pathways obtained in the KEGG pathway analysis were focal adhesion (FA), PI3K-Akt signaling pathway, proteoglycans in cancer, extracellular matrix (ECM)-receptor interaction and vascular smooth muscle contraction. The top 10 hub genes identified from the PPI network were vascular endothelial growth factor A (VEGFA), TOP2A, CCNB1, Cell division cycle 20 (CDC20), aurora kinase B, ACTA2, Aurora kinase A, UBE2C, CEP55 and CCNB2. Survival analysis revealed that the expression levels of ACTA2, CCNB1, CDC20 and VEGFA were related to the prognosis of patients with bladder cancer. In addition, a KEGG pathway analysis of the top 2 modules identified from the PPI network revealed that Module 1 mainly involved the cell cycle and oocyte meiosis, while the analysis in Module 2 mainly involved the complement and coagulation cascades, vascular smooth muscle contraction and FA.ConclusionsThis study identified key genes and pathways in bladder cancer, which will improve our understanding of the molecular mechanisms underlying the development and progression of bladder cancer. These key genes might be potential therapeutic targets and biomarkers for the treatment of bladder cancer.

Birefringence measurement of biological tissue based on polarization-sensitive digital holographic microscopy

We present a polarization-sensitive digital holography to measure the birefringence of biological tissues with complex structures. The birefringence measurement system is established by incorporating the modified Mach–Zehnder interferometer and lab-developed software. Normal and cancerous bladder tissue samples are measured by using the proposed experimental system. The polarization-dependent phase-shifted holograms are recorded by rotating the polarizer. Moreover, the hybrid reconstruction and least-square unwrapping algorithms are used to extract phase information of biological tissues at different polarization states. The birefringence of tissue is obtained from the above-phase distributions. The results show that polarization sensitivity exists in the normal bladder tissue and cancerous bladder tissue, and the median birefringence value of the cancerous tissue is significantly higher than that of the normal tissue. These results will provide reference for pathological research and clinic diagnoses.

Bladder Cancer-Specific Nuclear Matrix Proteins-4 May Be a Potential Biomarker for Non-Muscle-Invasive Bladder Cancer Detection.

Aims Bladder cancer–specific nuclear matrix protein-4 (BLCA-4) is a protein expressed mainly in bladder cancer tissues. Therefore, the aim of this study was to investigate its assisting diagnostic potential in non-muscle-invasive bladder cancer (NMIBC). Methods Twenty patients with NMIBC, 20 with benign prostatic hyperplasia (BPH), and 20 normal controls were included in this study. Blood and urine samples were collected from all patients. Moreover, cancer foci and adjacent tissue samples were collected from NMIBC patients, and normal bladder tissue samples were collected from patients with BPH. A competitive enzyme-linked immunosorbent assay was used to determine the BLCA-4 level in serum and urine, and immunohistochemistry was used to examine BLCA-4 expression in bladder cancer, adjacent, and normal tissues. Results Median urinary BLCA-4 levels in the NMIBC, BPH, and normal control groups were 0.759 ng/mL, 0.309 ng/mL, and 0.171 ng/mL, respectively. Urinary BLCA-4 level was significantly higher in the NMIBC group than in the other 2 groups (P < 0.01); meanwhile, the BPH group was higher than the normal control group (P < 0.05). Median serum BLCA-4 levels in the NMIBC, BPH, and normal control groups were 5.680 ng/mL, 5.928 ng/mL, and 5.473 ng/mL, respectively, showing no significant difference among groups (P > 0.05). Conclusion As a new marker of bladder cancer, urinary BLCA-4 level detection might apply for clinical diagnosis or postoperative monitoring for NMIBC.

GP73 promotes invasion and metastasis of bladder cancer by regulating the epithelial-mesenchymal transition through the TGF-β1/Smad2 signalling pathway.

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial–mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF‐β1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF‐β, thrombospondin‐1 (TSP‐1), TGF‐β1+ TSP‐1, GP73‐siRNA‐1, GP73‐siRNA‐2, GP73‐siRNA‐1+ TSP‐1, GP73‐siRNA‐1+ pcDNA‐GP73, WT1‐siRNA and WT1‐siRNA + GP73‐siRNA‐1 groups. Expressions of GP73, TGF‐β1, Smad2, p‐Smad2, E‐cadherin and vimentin were detected using RT‐qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF‐β1, Smad2, p‐Smad2, N‐cadherin and vimentin decreased, and levels of WT1 and E‐cadherin increased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups, while the opposite results were observed in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups. Cell proliferation, migration and invasion notably decreased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down‐regulating WT1 levels and activating the TGF‐β1/Smad2 signalling pathway.

Identification of key genes associated with bladder cancer using gene expression profiles.

The aim of the present study was to further investigate the molecular mechanisms of bladder cancer. The microarray data GSE52519 were downloaded from Gene Expression Omnibus, comprising 9 bladder cancer and 3 normal bladder tissue samples. Differentially expressed genes (DEGs) were identified using Limma package analysis. Subsequently, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Reactome pathway enrichment analyses were performed for down- and upregulated DEGs. Transcription factors and genes associated with cancer from DEGs were identified. Protein-protein interaction (PPI) networks were constructed using STRING, and pathway enrichment analysis was also conducted for genes in the core sub-network that was identified using BioNet. In total, 420 downregulated and 335 upregulated DEGs were identified. Functional and pathway enrichment analyses identified that a number of DEGs, including AURKA, CCNA2, CCNE1, CDC20 and CCNB2, were enriched in the cell cycle. Furthermore, a total of 12 upregulated proto-oncogenes were identified, including AURKA and CCNA2. In the PPI sub-network, a number of DEGs (e.g., CCNB2, CDC20, CCNA2 and MCM6) with higher degrees were enriched in the KEGG pathway of the cell cycle. In conclusion, the DEGs associated with the cell cycle (e.g., CDC20, CCNA2, CCNB2 and AURKA) may serve pivotal roles in the pathogenesis of bladder cancer.

Molecular profiling of small cell bladder cancer (SCBC) to reveal gene expression determinants of an aggressive phenotype.

4529 Background: SCBC is rare and its underlying biology poorly understood. Molecular profiling can shed light on the biology and identify treatment targets and biomarkers. Methods: A retrospective review of 63 patients (pts) with biopsy-confirmed SCBC at Cleveland Clinic (1994-2015) was performed. Percentage of small cell component (SC%) was defined by independent pathology review. DLL3 and PD-L1 protein expression were measured by IHC in 53 pts. Gene expression analysis was done in 38 primary SCBC tumor samples, 1 metastatic sample, and 5 normal bladder tissue samples (44 total) from the same cohort using HTG EdgeSeq OBP Assay with probes for 2568 genes. Analysis was performed via the RNAseq workflow (Partek Genomics Suite). Results: Among 63 identified pts, median age was 71 (39-90), 83% were men, median SC% was 100% (range 5-100%), median follow-up was 16.6 months and estimated median overall survival (OS) was 22.8 months. Unsupervised hierarchical clustering of gene expression patterns from 44 samples produced 4 distinct clusters. Pts with tumors in cluster 1 (that also included normal samples) did not have metastasis at diagnosis or distant recurrence, both of which were over-represented in the other 3 clusters. Kaplan-Meier analysis revealed a trend towards longer OS in cluster 1 patients (log rank p = 0.065). Higher gene expression of PRC1, NCAM1 (CD56) and DLL3 correlated with higher SC%, as did lower gene expression of ERBB2, PD-L1 and HPGD (p &lt; 0.01). PD-L1 protein expression (≥1% cells) was noted in 30% of pts but did not correlate with outcome, SC%, DLL3 protein expression, or PD-L1 gene expression. DLL3 protein expression (≥1% cells) was noted in 68% of pts and DLL3 &gt; 10% correlated with decreased OS (p = .03). Higher DLL3 protein expression correlated with DLL3 gene expression (Spearman r = 0.70, p &lt; .01) and with SC% (r = .33, p = .01). Conclusions: This is the first study to reveal distinct gene expression patterns that define aggressive behavior, metastatic potential and outcomes in SCBC. The prognostic value of differential gene expression networks and the presence of underlying genomic and epigenetic alterations is the subject of ongoing prospective validation in a larger cohort.

Exploring the role of Paraoxonase-2 in bladder cancer: analyses performed on tissue samples, urines and cell culturess

The enzyme paraoxonase-2 (PON2) is ubiquitously expressed and exerts its antiapoptotic and antioxidative functions in several intracellular compartments.The aim of this study is to investigate the role of PON2 in bladder cancer (BC). The expression levels of PON2 in paired tumor and normal bladder tissue samples and in urinary exfoliated cells from patients affected with BC and healthy donors were evaluated. Moreover, the effect of PON2 overexpression on tumor cell proliferation and susceptibility to oxidative stress was investigated in human bladder cancer cell line T24.Our results showed that PON2 expression levels were significantly higher in BC compared with non-tumor tissue. In urinary exfoliated cells from BC patients, PON2 mRNA levels showed an inverse correlation with tumor stage (pT). Moreover, PON2 overexpression in T24 cells led to a significant increase in tumor cell proliferation and resistance to oxidative stress.The results obtained showed that PON2 could represent a molecular biomarker for bladder cancer and suggest a potential role of the enzyme as a prognostic factor for this neoplasm.

Abstract 3986: Diagnostic significance of cathepsin L and cathepsin B expression in human gallbladder cancer - A pilot study

Abstract Background: Gallbladder cancer (GBC) is the most common biliary tract malignancy worldwide. Cancer of gallbladder usually refers to as an Indian disease as it is more common in Indian subcontinent especially in north and central India. Incidence rate of GBC in women in Delhi is as high as 21.5 per 100,000 population. It is an aggressive malignancy, which spreads locally to liver, adjacent organs and lymph nodes as well as to distant organs. Cathepsin L (CTSL) and B (CTSB) are lysosomal cysteine proteases implicated in protein turnover and tissue remodeling. Deregulated expression of these proteases has been associated with invasion and metastasis in several solid tumors. Objective: To assess the expression and clinical significance of CTSL and CTSB in human gallbladder cancer. Methods: We assayed the activities of CTSL and CTSB via spectroflurometrically in whole tissue lysates of gallbladder cancer patients (N = 23) and normal gall bladder tissue samples with gallstones (N = 31) / without gallstones (N = 34), which served as controls. Activities of these proteases were correlated with several clinic pathological parameters in GBC patients. CTSL and CTSB mRNA levels were determined in tissues samples by Real time PCR. Furthermore serum levels of these proteases were also assayed using sandwich based ELISA in GBC patients and controls. Results: Significant increase in the enzymatic activities of CTSL+B, CTSL and CTSB was observed in gallbladder cancer tissue samples in comparison to normal gall bladder both with and without gallstones (p &amp;lt; 0.001). ROC analysis confirmed the diagnostic relevance of CTSL and CTSB analysis with area under the curve 0.791 and 0.840 respectively for gallbladder cancer patients with respect to controls. Over expression of CTSB was also significantly associated with involvement of lymph node (p = 0.05) in gall bladder carcinoma. Secretory levels of CTSL and CTSB in serum were significantly elevated in GBC patients (p &amp;lt;0.01). Increased expression of these cysteine proteases were also confirmed at the mRNA level by Real time PCR. Conclusion: Our study for the first time demonstrated a significant increase in the levels of cathepsin L and B in GBC patients as compared to controls. Elevated levels of these proteases may serve as potential biomarkers for human gall bladder cancer. Citation Format: SIDDHARTH MEHRA, MANISH KUMAR, RAJESH PANWAR, NIHAR RANJAN DASH, RATNAKAR SINGH, RAJNI YADAV, SIDDHARTHA DATTA GUPTA, PEUSH SAHNI, SHYAM S. CHAUHAN. Diagnostic significance of cathepsin L and cathepsin B expression in human gallbladder cancer - A pilot study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3986.

Abstract 2563: Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region

Abstract Background: Genome-wide association studies (GWAS) of bladder cancer risk recently identified several signals on chromosome 20p12.2 marked by single nucleotide polymorphisms (SNPs) rs6104690, rs62185668 and rs6108803 (Figueroa et al, HMG, 2015). Interestingly, associations of SNPs rs62185668 and rs6108803 were significantly stronger for the risk of muscle-invasive (stages T2-T4) compared to non-muscle-invasive (stages Ta and T1) bladder cancer, suggesting that detailed analysis of these signals could further our understanding of aggressive bladder cancer. Methods: We used data from The Cancer Genome Atlas (TCGA) bladder cancer set (n = 412) and an independent RNA-seq dataset for 12 bladder tissues. qRT-PCR analysis was performed with custom TaqMan assays in an additional set of 40 tumor and 40 adjacent normal bladder tissue samples. RNAscope in situ hybridization analysis with custom probes was used to evaluate mRNA expression in bladder tissue microarrays that included 7 pairs of normal-tumor bladder tissues. Results: At a distance of more than 300 and 800 Kb, JAG1 and BTD3 are the closest genes to the GWAS signals in the 20p12.2 region. Analysis of TCGA data did not show significant association of JAG1 and BTD3 expression with genotypes of 20p12.2 GWAS markers analyzed through TCGA-genotyped proxies SNPs rs6040291 and rs6074214. Using RNA-seq data in an independent set of 12 bladder tissue samples, we discovered a novel putative gene in the 20p12.2 region. Cloning of this gene from bladder tumor tissue and annotation by several methods showed two alternative splicing forms, designated as major and minor transcripts. Expression of the major transcript in bladder tumors was significantly increased with the rs6104690-A allele (P = 0.014), which is associated with increased risk of bladder cancer. SNPs with stronger association with muscle-invasive bladder cancer (rs62185668 and rs6108803) showed weaker or non-significant associations with expression of this novel transcript (P = 0.037 and P = 0.42, respectively). This transcript might be protein-coding as it is predicted to have several open reading frames for short peptides, or it may be a long non-coding RNA (lncRNA). RNAscope analysis in bladder tumors showed nuclear expression of the major transcript. Functional analysis of this novel gene in relation to bladder cancer is underway. Conclusions: A novel gene might be related to the GWAS signals within the 20p12.2 region and the risk of bladder cancer. Citation Format: Rouf Banday, Wusheng Yan, Candace D. Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2563.

The role of p53 immunohistochemical marker in development of superficial urothelial bladder carcinoma

Purpose. Determination the role of the p53 expression as a prognostic factor in the development of superficial urothelial bladder carcinoma. Materials and methods. Medical histories of 72 patients (11 women and 61 men) with diagnosed primary superficial urothelial carcinoma of the bladder I and II grade were retrospectively analyzed. All patients were undergone a repeated transurethral resection was performed in 6 weeks after the first operation. In the case of the absence of residual tumor, cystoscopy was performed every 3 months during the first year then every 3–6 months during next 5 years. The average time of follow-up was 43.2 months. According to the standard protocol an immunohistochemical study with detection of p53 was carried out on 72 sections of formalin-fixed and paraffin-embedded tumor fragments. p53 expression was also determined in 10 histologically unchanged fragments of the bladder mucosa. Results. Depending on the percent of nuclei with p53 expression, two groups were identified. The first group included 40 tumor samples (55.5 %) with p53 expression less than 20 % and 17 tumor samples (23.6 %) without p53 expression. The second group included 15 tumor samples (20.9 %) with p53 expression more than 20 % (4 patients – 20–40 %, 8 patients – 40–60 %, 3 patients – 60–80 %). р53 expression was not determined in the all 10 samples of normal bladder tissue. During follow-up period 1 of 57 patients of the first group has a disease progression comparing with 13 of 15 patients in the second group. Patients of the both group were similar in respect with clinical characteristics. The mean time to disease progression was 17.3 months. Conclusion. Results of the current study shows that p53 immunohistochemical marker is a prognostic factor in the development of superficial urothelial bladder carcinoma.

Circulating cell-free DNA profiling of patients with advanced urothelial carcinoma

Circulating cell-free DNA profiling of patients with advanced urothelial carcinoma

Silencing of RTKN2 by siRNA suppresses proliferation, and induces G1 arrest and apoptosis in human bladder cancer cells.

Human bladder cancer is the most common urological malignancy in China. One of the causes of carcinogenesis in the cancer may be gene mutation. Therefore, the present study investigated the expression levels of Rhotekin2 (RTKN2), a Rho effector protein, in human bladder cancer tissues and cell lines, and examined the effect of RTKN2 on the proliferation, cell cycle, apoptosis and invasion of human bladder cancer cell lines. The mRNA expression levels of RTKN2 in 30human bladder cancer tissue samples were significantly higher, compared with those in 30 normal human bladder tissue samples. The protein expression levels of RTKN2 was markedly higher in T24 and 5637 cells, compared with those in four other human bladder cancer cell lines. The silencing of RTKN2 by small interfering (si)RNA inhibited cell proliferation and arrested cell cycle at the G1 phase, via reducing the expression levels of the MCM10, CDK2, CDC24A and CDC6 cell cycle‑associated proteins in the T24 and 5637 cells. Furthermore, RTKN2 knockdown in the cells led to cell apoptosis and the suppression of invasion. These results suggested that RTKN2 is involved in the carcinogenesis and progression of human bladder cancer, indicating that RTKN2 may be a molecular target in cancer therapy.

Expression and clinical significance of microRNA-21, maspin and vascular endothelial growth factor-C in bladder cancer.

The present study aimed to explore the expression and clinical significance of microRNA-21 (miR-21), maspin and vascular endothelial growth factor C (VEGF-C) in bladder cancer (BC). A total of 53 BC samples and 12 normal bladder tissue samples were collected. Total messenger RNA (mRNA) was extracted, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression of miR-21 and maspin in BC and normal bladder tissues. Immunohistochemistry was used for the detection of maspin and VEGF-C protein expression. Furthermore, the correlations between these molecules and certain clinicopathological parameters were investigated, and survival analysis was performed to assess their prognostic significance. miR-21 mRNA expression and VEGF-C protein expression were increased in BC tissues compared with those in normal bladder tissues, whereas maspin mRNA and protein expression levels in BC tissues were significantly decreased (P<0.01). miR-21, maspin and VEGF-C expression were significantly associated with the stage, grade and lymph node metastasis of BC (P<0.05), but not the other clinicopathological features evaluated. There was a marked inverse correlation between the mRNA expression of miR-21 and maspin, with a coefficient of -0.978 (P<0.001). Similarly, there was a significant inverse correlation between the protein expression of maspin and VEGF-C, with a coefficient of -0.589 (P<0.001). Overexpression of miR-21 and VEGF-C, as well as decreased maspin expression, were associated with a poorer prognosis. These results suggested that upregulation of miR-21, decreased maspin expression and enhanced VEGF-C in BC may promote tumor progression. miR-21, maspin and VEGF-C may therefore have significant roles as biomarkers for prognosis and as therapeutic targets of BC.

The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer

We investigate the role of the Lin28/let-7a/c-Myc pathway in non-muscle invasive bladder cancer (NMIBC). Using RT-PCR, western blot and immunohistochemistry techniques, the levels of pre-let-7a, let-7a, Lin28 and c-Myc RNA and/or proteins were determined in samples of normal bladder tissue and bladder cancer. Expression of pre-let-7a was found to be negatively correlated with the pathological grade of bladder cancer, while let-7a showed a positive correlation with bladder cancer pathological grade. Expression of Lin28 RNA and protein was not significantly different between normal bladder tissue and low-grade transitional cell carcinoma of bladder (TCC) but the expression levels in high-grade TCC were remarkably increased. Expression of c-Myc RNA and protein was significantly higher in bladder cancer samples in comparison to normal bladder tissue without correlation with cancer differentiation. Expression of all the above RNAs and proteins showed no significant difference in Ta and T1 stages. The Lin28/let-7a/c-Myc pathway plays an important role in NMIBC. In particular, expression levels of let-7a correlate with the degree of cancer differentiation but not cancer stage.

A New Algorithm for Integrated Analysis of miRNA-mRNA Interactions Based on Individual Classification Reveals Insights into Bladder Cancer

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. It has been proposed that miRNAs play an important role in cancer development and progression. Their ability to affect multiple gene pathways by targeting various mRNAs makes them an interesting class of regulators.Methodology/Principal FindingsWe have developed an algorithm, Classification based Analysis of Paired Expression data of RNA (CAPE RNA), which is capable of identifying altered miRNA-mRNA regulation between tissues samples that assigns interaction states to each sample without preexisting stratification of groups. The distribution of the assigned interaction states compared to given experimental groups is used to assess the quality of a predicted interaction. We demonstrate the applicability of our approach by analyzing urothelial carcinoma and normal bladder tissue samples derived from 24 patients. Using our approach, normal and tumor tissue samples as well as different stages of tumor progression were successfully stratified. Also, our results suggest interesting differentially regulated miRNA-mRNA interactions associated with bladder tumor progression.Conclusions/SignificanceThe need for tools that allow an integrative analysis of microRNA and mRNA expression data has been addressed. With this study, we provide an algorithm that emphasizes on the distribution of samples to rank differentially regulated miRNA-mRNA interactions. This is a new point of view compared to current approaches. From bootstrapping analysis, our ranking yields features that build strong classifiers. Further analysis reveals genes identified as differentially regulated by miRNAs to be enriched in cancer pathways, thus suggesting biologically interesting interactions.

Gene expression study related with the intrinsic pathway of apoptosis in bladder cancer by real-time PCR technique

We examined the expression of anti-apoptotic genes (XIAP and Bcl-2) and apoptotic genes (cytochrome c, caspase-9, Apaf-1) in tissue samples of patients with superficial bladder cancer. Thirty-two bladder cancer tissue samples (8 papillary urothelial neoplasm of low malignant potential, 10 low-grade, and 14 high-grade) and 8 normal bladder tissue samples from necropsy were used for the study of gene expression by real-time PCR analysis. Analysis of the expression of apoptotic gene constituents of an apoptosome demonstrated an increase in Apaf-1 expression in the three tumor grades when compared with the control (P < 0.01, P < 0.05, and P < 0.01), low expression of caspase-9 in all groups (P < 0.05), and an increase in cytochrome c expression in all tumor grades in relation to the control, although without statistically significant difference. The expression of anti-apoptotic genes revealed an increase in XIAP expression in all tumor grades in relation to the control, although without statistically significant difference, and low expression of Bcl-2 in all tumor grades and the control (P < 0.05). The results proved that there is low evidence of apoptotic activity by the intrinsic pathway, demonstrated by the low expression of caspase-9 and considerable increase in XIAP expression, which may render these genes potential therapeutic targets in bladder cancer treatment.