Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder

BACKGROUND Mutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer.Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder(UCB).AIM To determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.METHODS This study was performed to determine the mutational spectrum at the hotspot regions of H-Ras,K-Ras and N-Ras genes by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and DNA sequencing followed by their clinical impact(if any)by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.RESULTS None of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene;codon 61 of N-Ras gene and codons 12,13 of K-Ras gene by PCR-RFLP.Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras,NRas and K-Ras genes in the tumor and control bladder mucosal specimens.CONCLUSION Our findings on the lack of mutations in H-Ras,K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression,inherent genetic susceptibility,tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

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

BACKGROUND Aberrant activation of phosphorylated form of glycogen synthase kinase-3β[pS9 GSK-3β(Serine 9 phosphorylation)] is known to trigger Wnt/β-catenin signal cascade but its clinicohistopathological implications in bladder carcinogenesis remain unknown.AIM To investigate the diagnostic and prognostic relevance of expressions of pS9 GSK-3β, β-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 pS9 GSK-3β by immunohistochemical(IHC) staining. Real time-quantitative polymerase chain reaction and IHC were done to check the expression of β-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 SPSS20.0 software.RESULTS Aberrant(low or no membranous/high nuclear/high cytoplasmic) expression of pS9 GSK-3β 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 pS9 GSK-3β andβ–catenin proteins and showed strong statistical association with tumor stage,tumor type, smoking/tobacco chewing status(P = 0.01, 0.02 and 0.04, MannWhitney U test) and shorter overall survival probabilities of patients(P = 0.02;Kaplan Meier test). Nuclear immunostaining of Cyclin D1 in tumors with altered pS9 GSK-3β/β–catenin showed relevance with tumor stage, grade and type.CONCLUSIONβ–catenin and pS9 GSK-3β 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 pS9 GSK-3β/β-catenin.