Expression of Semaphorin 3A in Malignant and Normal Bladder Tissue: Immunohistochemistry Staining and Morphometric Evaluation

Introduction: Our previous studies showed elevated levels of Semaphorin3a (Sema3A) in the urine of patients with urothelial cancer compared to healthy patients. The aim of this study was to analyze the extent of Sema3A expression in normal and malignant urothelial tissue using immune-staining microscopic and morphometric analysis. Materials and Methods: Fifty-seven paraffin-embedded bladder samples were retrieved from our pathology archive and analyzed: 14 samples of normal urothelium, 21 samples containing low-grade urothelial carcinoma, 13 samples of patients with high-grade urothelial carcinoma, 7 samples containing muscle invasive urothelial carcinoma, and 2 samples with pure urothelial carcinoma in situ. All samples were immunostained with anti Sema3A antibodies. The area of tissue stained with Sema3A and its intensity were analyzed using computerized morphometry and compared between the samples’ groups. Results: In normal bladder tissue, very light Sema3A staining was demonstrated on the mucosal basal layer and completely disappeared on the apical layer. In low-grade tumor samples, cells in the basal layer of the mucosa were also lightly stained with Sema3A, but Seama3A expression intensified upon moving apically, reaching its highest level on apical cells exfoliating to the urine. In high grade urothelial tumors, Seama3A staining was intense in the entire thickness of the mucosa. In samples containing carcinoma in situ, staining intensity was high and homogenous in all the neoplastic cells. Conclusions: Sema3A may be serve as a potential non-invasive marker of urothelial cancer.

Expression of MDM2 mRNA, MDM2, P53 and P16 Proteins in Urothelial Lesions in the View of the WHO 4th Edition Guidelines as A Molecular Insight towards Personalized Medicine

AIM: Here we imposed a multimarker molecular panel composed of P53, MDM2 protein & mRNA & P16 with the identification of sensitive and specific cut offs among the Egyptian urothelial carcinomas bilharzial or not emphasize the pathological and molecular classifications, pathways and prognosis as a privilege for adjuvant therapy.METHODS: Three hundred and ten urothelial lesions were pathologically evaluated and grouped as follows: 50 chronic cystitis as benign, 240 urothelial carcinomas and 20 normal bladder tissue as a control. Immunohistochemistry for MDM Protein, P16 & p53 and In Situ Hybridization for MDM2mRNA were done.RESULTS: MDM2mRNA overexpression correlated with low grade low stage non invasive tumors, while P53 > 40% & p16 < 10% cut offs correlated with high grade high stage invasive carcinomas & bilharzial tumors (P=0.000).CONCLUSION: MDM2mRNA overexpression vs. P53 > 40% & P16 < 10% constitutes a multimarker molecular panel with significant cut offs, proved to distinguish low grade, low stage non invasive urothelial carcinomas (MDM2mRNA overexpression, P53 < 40%, P16 > 10%) from high grade, high stage invasive urothelial carcinomas (with p53 > 40, p16 < 10% & absent MDM2mRNA overexpression). Combined P53 > 40 & p16 < 10%, together with the histopathological features can distinguish in situ urothelial lesions from dysplastic and atypical lesions.

MicroRNA-138 Regulates Metastatic Potential of Bladder Cancer Through ZEB2

Background/Aims: The cases of bladder cancer (BC) with poor prognosis largely result from the distal metastases of the primary tumor. Since microRNAs (miRNAs) play critical roles during cancer metastases, determination of the involved miRNAs in the regulation of the metastases of BC may provide novel therapeutic targets for BC treatment. Here, we aimed to study the role of miR-138 in regulation of BC cell invasion and metastases. Methods: We analyzed the levels of miR-138 and ZEB2, a key factor that regulates cancer cell invasion, in the BC specimens from the patients. We also studied the correlation between miR-138 and ZEB2. We performed bioinformatics analyses on the binding of miR-138 to the 3'-UTR of ZEB2 mRNA, and verified the biological effects of this binding through promoter luciferase reporter assay. The effects of miR-138-modification on BC cell invasion were evaluated in a transwell cell invasion assay and a scratch would healing assay. Results: We found that the levels of miR-138 were significantly decreased and the levels of ZEB2 were significantly increased in BC specimens, compared to the paired normal bladder tissue. Metastatic BC appeared to contained lower levels of miR-138. Moreover, miR-138 and ZEB2 inversely correlated in BC specimens. Bioinformatics analyses showed that miR-138 targeted the 3'-UTR of ZEB2 mRNA to inhibit its translation. Furthermore, miR-138 overexpression inhibited ZEB2-mediated cell invasion and metastases, while miR-138 depletion increased ZEB2-mediated cell invasion and metastases in BC cells. Conclusion: Suppression of miR-138 in BC cells may promote ZEB2-mediated cancer invasion and metastases. Thus, miR-138 appears to be an intriguing therapeutic target to prevent metastases of BC.

Expression of MET and HGF in bladder cancer tumorigenesis and invasion.

4573 Background: The human MET gene encodes the hepatocyte growth factor (HGF) tyrosine kinase receptor. Limited studies in human bladder cancer have demonstrated that expression of MET protein is linked with disease progression and survival. We hypothesized that the expression of both MET and its ligand HGF are altered in bladder cancer. Methods: Expression of MET and HGF in human bladder tissues was explored using Oncomine, an online compendium of cancer transcriptome profiles. The largest relevant dataset was identified and contained 157 samples (Sanchez-Carbayo, et al. 2006). Normalized and log2 transformed mRNA expression values for Affymetrix U133 microarray probe sets corresponding to the genes of interest were compiled and averaged for each gene. Mann-Whitney U testing was used to compare means. Nominal and standard least squares logistic regression was used to evaluate the predictive significance of, and relevance of clinicopathologic variables on, gene expression. Statistical analyses were carried out using JMP 9.0.2. Results: Expression of MET and HGF was compared across subsets of normal bladder tissue (n=48), superficial tumors (n=28), and invasive tumors (n = 81). Relative to normal tissue, MET expression was greater in superficial (p = 0.0008) and invasive tumors (p<0.0001). HGF expression was reduced in both invasive and superficial tumors vs. normal tissue (both p< 0.0001), but was higher in invasive vs. superficial tumors (p= 0.0007). In a logistic regression model of tumor samples, both MET and HGF correlated with tumor invasion (model p=0.0003). Interaction between MET and HGF was not significant (p=0.55). Neither MET nor HGF expression was predicted by regression using age, gender, grade, or nodal stage (p=0.16 and 0.27, respectively). Conclusions: Decreased HGF expression and MET over-expression are strongly associated with tumorigenesis and invasion in bladder cancer. The reduction in HGF expression is blunted in invasive vs. superficial tumors, suggesting a potential loss of negative feedback in more aggressive tumors. Expression of both genes could not be predicted by modeling clinicopathologic variables. MET signaling represents a potential therapeutic target in bladder cancer.