Immunohistochemical expressions of EMT markers in pan-RAS-pERK1/2-positive tumors improve diagnosis and prognosis assessment of non-muscle invasive bladder cancer and muscle invasive bladder cancer patients.

Mutation or overexpression renders pan-RAS (rat sarcoma) proteins insensitive to inactivation. Activated pan-RAS communicates signal from the cell surface receptor to activate RAS-MAPK/ERK (RAS-mitogen-activated protein kinases/extracellular signal regulated kinases) signaling and orchestrates epithelial-to-mesenchymal transition-activating transcription factors (EMT-ATFs) reprogramming to induce EMT. Owing to limited studies available in bladder cancer, the present study is taken up to examine the expressions of the EMT-associated markers in pan-RAS-pERK1/2 (pan-RAS-phosphoERK1/2)-positive well-characterized cohort of forty-two non-muscle invasive bladder cancer (NMIBC) and forty-five muscle invasive bladder cancer (MIBC) patients. Immunohistochemical staining was performed on paraffin embedded tissue sections to determine the immunolevels and cellular localization of marker proteins. Semi-quantitative expressions of pan-RAS, pERK1/2, and EMT markers (E-cadherin, Vimentin, N-cadherin, Snail, Slug Twist, and Zeb1) were statistically examined with clinicohistopathological profile of the patients using SPSS, version 20.0 software. The study documents the diagnostic relevance of immunohistochemical expressions of pan-RAS-pERK1/2/EMT-associated markers in order to stratify NMIBC and MIBC patients. Follow-up studies supported the role of altered EMT phenotype in pan-RAS-pERK1/2-activated positive tumors with disease aggressiveness. To the best of our knowledge, our study is the first concluding the impact of altered EMT phenotype via pan-RAS-pERK1/2 axis on the short survival outcome [short overall survival (OS) (p = 0.04), short progression-free survival (PFS) (p = 0.02) and short cancer-specific survival (CSS) (p = 0.03)] of muscle invasive bladder cancer patients.

WNT/ß-catenin signaling in urothelial carcinoma of bladder.

Urothelial carcinoma of bladder is the second most prevalent genitourinary disease. It is a highly heterogeneous disease as it represents a spectrum of neoplasms, including non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC) and metastatic lesions. Genome-wide approaches and candidate gene analysis suggest that malignant transformation of the bladder is multifactorial and a multitude of genes are involved in the development of MIBC or NMIBC phenotypes. Wnt signaling is being examined to control and maintain balance between stemness and differentiation in adult stem cell niches. Owing to its participation in urothelial development and maintenance of adult urothelial tissue homeostasis, the components of Wnt signaling are reported as an important diagnostic and prognostic markers as well as novel therapeutic targets. Mutations/epigenetic alterations in the key molecules of Wnt/ß-catenin canonical pathway have been linked with tumorigenesis, development of drug resistance and enhanced survival. Present review extends our understanding on the functions of key regulatory molecules of canonical Wnt/ß-catenin pathway in urothelial tumorigenesis by inducing cancer stem cell phenotype (UCSCs). UCSCs may be responsible for tumor heterogeneity, high recurrence rates and complex biological behavior of bladder cancer. Therefore, understanding the role of UCSCs and the regulatory mechanisms that are responsible for high relapse rates and metastasis could help to develop pathway inhibitors and augment current therapies. Potential implications in the treatment of urothelial carcinoma of bladder by targeting this pathway primarily in UCSCs as well as in bulk tumor population that are responsible for high relapse rates and metastasis may facilitate potential therapeutic avenues and better prognosis.

Clinicohistopathological implications of phosphoserine 9 glycogen synthase kinase-3ß/ ß-catenin in urinary bladder cancer patients.

BACKGROUND: Aberrant activation of phosphorylated form of glycogen synthase kinase-3ß [pS9GSK-3ß (Serine 9 phosphorylation)] is known to trigger Wnt/ß-catenin signal cascade but its clinicohistopathological implications in bladder carcinogenesis remain unknown. AIM: To investigate the diagnostic and prognostic relevance of expressions of pS9GSK-3ß, ß-catenin and its target genes in the pathobiology of bladder cancer. METHODS: Bladder tumor tissues from ninety patients were analyzed for quantitative expression and cellular localization of pS9GSK-3ß by immunohistochemical (IHC) staining. Real time-quantitative polymerase chain reaction and IHC were done to check the expression of ß-catenin, Cyclin D1, Snail and Slug at transcriptome and protein level respectively. Clinicohistopathological variables were obtained from histology reports, follow up and OPD visits of patients. Expressions of the markers were statistically correlated with these variables to determine their significance in clinical setting. Results were analysed using SPSS 20.0 software. RESULTS: Aberrant (low or no membranous/high nuclear/high cytoplasmic) expression of pS9GSK-3ß was noted in 51% patients and found to be significantly associated with tumor stage and tumor grade (P = 0.01 and 0.04; Mann Whitney U test). Thirty one percent tumors exhibited aberrant co-expression of pS9GSK-3ß and ß-catenin proteins and showed strong statistical association with tumor stage, tumor type, smoking/tobacco chewing status (P = 0.01, 0.02 and 0.04, Mann-Whitney U test) and shorter overall survival probabilities of patients (P = 0.02; Kaplan Meier test). Nuclear immunostaining of Cyclin D1 in tumors with altered pS9GSK-3ß/ß-catenin showed relevance with tumor stage, grade and type. CONCLUSION: ß-catenin and pS9GSK-3ß proteins are identified as markers of diagnostic/prognostic significance in disease pathogenesis. Observed histopathological association of Cyclin D1 identifies it as marker of potential relevance in tumors with altered pS9GSK-3ß/ß-catenin.

Age estimation by dentin translucency measurement using digital method: An institutional study.

AIMS: The aims of the present study were to measure translucency on sectioned teeth using available computer hardware and software, to correlate dimensions of root dentin translucency with age, and to assess whether translucency is reliable for age estimation. MATERIALS AND METHODS: A pilot study was done on 62 freshly extracted single-rooted permanent teeth from 62 different individuals (35 males and 27 females) and their 250 µm thick sections were prepared by micromotor, carborundum disks, and Arkansas stone. Each tooth section was scanned and the images were opened in the Adobe Photoshop software. Measurement of root dentin translucency (TD length) was done on the scanned image by placing two guides (A and B) along the x-axis of ABFO NO. 2 scale. Unpaired t-test, regression analysis, and Pearson correlation coefficient were used as statistical tools. RESULTS: A linear relationship was observed between TD length and age in the regression analysis. The Pearson correlation analysis showed that there was positive correlation (r = 0.52, P = 0.0001) between TD length and age. However, no significant (P > 0.05) difference was observed in the TD length between male (8.44 ± 2.92 mm) and female (7.80 ± 2.79 mm) samples. CONCLUSION: Translucency of the root dentin increases with age and it can be used as a reliable parameter for the age estimation. The method used here to digitally select and measure translucent root dentin is more refined, better correlated to age, and produce superior age estimation.

Epithelial-To-Mesenchymal Transition and Its Correlation With Clinicopathologic Features in Patients With Urothelial Carcinoma of the Bladder.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is a dynamic process in the pathogenesis of urinary bladder cancer. Despite significant advancements in its diagnosis and treatment, the outcomes have more or less remained the same. In the present study, the expression of EMT markers was investigated to evaluate its prognostic significance in patients with non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). MATERIALS AND METHODS: The present study was undertaken to examine the expression of EMT markers, including E-cadherin, N-cadherin, vimentin, Snail, Twist, Zeb, and Slug, on 28 bladder tumor tissues (15 cases of NMIBC and 13 of MIBC) using reverse transcription-polymerase chain reaction. Immunohistochemical (IHC) staining was performed to check the protein expression and localization of E-cadherin, N-cadherin, vimentin, Snail, and Slug. RESULTS: At the message level, reduced expression of E-cadherin correlated with gender (P = .004), enhanced expression of N-cadherin correlated with stage and age (P = .02, for both), and increased expression of EMT transcription factors correlated significantly with stage, grade, or age. Inverse correlation of reduced levels of E-cadherin were observed with new expression of N-cadherin (P = .001; Mann-Whitney U test) and vimentin (P = .001; Mann-Whitney U test). On IHC, novel expression of vimentin and N-cadherin was associated with enhanced expression of Snail and Slug (P = .005; Wilcoxon signed rank test). CONCLUSION: Molecular validation of the EMT marker profile proved to be a sensitive and an effective prognostic tool for objective and systematic investigation of EMT function in the pathogenesis of urinary bladder cancer. Nevertheless, further studies are required with a greater number of clinical samples.