Development of pyrosequencing methods for the rapid detection of RAS mutations in clinical samples.

Abstract

In advanced colorectal carcinoma (CRC) patients, extended RAS mutations testing (KRAS exons 2 to 4 and NRAS exons 2 to 4) is a prerequisite for patient stratification to anti-EGFr therapy. Accurately distinguishing mutant patients from potential responders has a clinically critical impact, and thus effective and low cost methods are needed for identification of the mutation status. We have developed quantitative pyrosequencing assays for sensitive and rapid detection of mutant RAS alleles in formalin-fixed, paraffin-embedded tissues. Exons 2 to 4 of KRAS and NRAS genes were PCR amplified and analyzed by pyrosequencing. For validation, PCR products were sequenced by conventional Sanger sequencing. Analytical sensitivity of these assays was determined by calculating the limit of detection. The results showed that low levels of mutant RAS alleles (2-13%) can be detected with pyrosequencing assays.

• Authors: Ulrich Cortes, Karline Guilloteau, Mélanie Rouvreau, Céline Archaimbault, Claire Villalva, Lucie Karayan-Tapon
• Journal: Experimental and molecular pathology (Exp Mol Pathol) Vol. 99 Issue 2 Pg. 207-11 (Oct 2015) ISSN: 1096-0945 [Electronic] Netherlands
• PMID: 26163758 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• EGFR protein, human
• ErbB Receptors
• ras Proteins

Topics

• Colorectal Neoplasms (diagnosis, drug therapy, genetics)
• DNA Mutational Analysis (methods)
• ErbB Receptors (genetics)
• Genetic Testing (methods)
• Humans
• Mutation (genetics)
• Outcome Assessment, Health Care
• Paraffin Embedding
• Polymerase Chain Reaction (methods)
• ras Proteins (genetics)