Observational Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. Aug 24, 2020; 11(8): 614-628
Published online Aug 24, 2020. doi: 10.5306/wjco.v11.i8.614
Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder
Kiran Tripathi, Apul Goel, Atin Singhai, Minal Garg
Kiran Tripathi, Minal Garg, Department of Biochemistry, University of Lucknow, Lucknow 226007, India
Apul Goel, Department of Urology, King George Medical University, Lucknow 226003, India
Atin Singhai, Department of Pathology, King George Medical University, Lucknow 226003, India
Author contributions: Tripathi K contributed to the conception and design of the study; Tripathi K performed the experiments and participated in the acquisition, analysis, and interpretation of the data, and drafted the initial manuscript; Goel A and Singhai A revised the article critically for important intellectual content; Garg M contributed to the conception and design of the study and revised the article critically for important intellectual content; all the authors approved the final version of the article to be published.
Supported by Department of Science and Technology, Govt. of India, No. SR/SO/HS-0113/2010; University Grants Commission (UGC), Govt. of India; No. 22/12/2013(ii)EU-V.
Institutional review board statement: This study was approved by the appropriate institutional research ethics committee and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Ethical clearance was obtained from Bioethics Cell, Institutional Ethics Committee (IEC), KGMU (Reference no. 89th ECM II A/P8), Lucknow, India.
Informed consent statement: Subjects were not required to give informed consent as the analysis used anonymous data that were obtained after each patient agreed to treatment by written consent.
Conflict-of-interest statement: All the authors of the study declare no potential conflict-of-interest.
Data sharing statement: No additional data are available.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Minal Garg, MSc, PhD, Assistant Professor, Doctor, Department of Biochemistry, University of Lucknow, University Road, Lucknow 226007, India. minal14@yahoo.com
Received: January 20, 2020
Peer-review started: January 20, 2020
First decision: April 21, 2020
Revised: May 21, 2020
Accepted: July 1, 2020
Article in press: July 1, 2020
Published online: August 24, 2020
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 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, N-Ras 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.

Keywords: Coding exons, Oncogenic activation, Polymerase chain reaction - restriction fragment length polymorphism, Point mutations, Ras genes, Urothelial carcinoma of bladder

Core tip: Mutant Ras has been shown to be associated with drug resistance, enhanced metastasis and shorter survival of patients. Due to reported heterogeneity among the distribution of the most frequent mutations in Ras isoforms in different patient populations with urothelial carcinoma of the bladder, it is necessary to examine these patients for Ras mutations in order to predict disease outcome. Our findings on the lack of Ras mutations could be explained on the basis of different etiological mechanisms involved in tumor development, inherent genetic susceptibility, tissue specificity or alternative Ras dysfunction including gene amplification or overexpression in a given cohort of patients.