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

Abstract

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 inRasisoforms 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-Rasgene 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, N-R as and K-R as 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.