KRAS gene mutation in a series of unselected colorectal carcinoma patients with prognostic morphological correlations: A pyrosequencing method improved by nested PCR

Author(s)

de Macedo, Mariana Petaccia ^{[1, 2]} ; de Melo, Fernanda Machado ^[1] ; Garcia Lisboa, Bianca Cristina ^[1] ; Brot Andrade, Louise D. ^{[1, 2]} ; Ferreira de Souza Begnami, Maria Dirlei ^[2] ; Aguiar Junior, Samuel ^[3] ; de Castro Ribeiro, Heber Salvador ^[4] ; Soares, Fernando Augusto ^{[1, 2]} ; Carraro, Dirce Maria ^{[1, 5]} ; da Cunha, Isabela Werneck ^{[1, 2]}

Affiliation

^[1] AC Camargo Canc Ctr, Dept Anat Pathol, Diagnost Mol Pathol Lab, BR-01509900 Sao Paulo - Brazil

^[2] AC Camargo Canc Ctr, CIPE, Lab Invest Pathol, BR-01509900 Sao Paulo - Brazil

^[3] AC Camargo Canc Ctr, Dept Surg Oncol, BR-01509900 Sao Paulo - Brazil

^[4] AC Camargo Canc Ctr, Abdominal Surg Dept, BR-01509900 Sao Paulo - Brazil

^[5] AC Camargo Canc Ctr, CIPE, Lab Genom & Mol Biol, BR-01509900 Sao Paulo - Brazil

• Document type: Journal article
• Source: Experimental and Molecular Pathology; v. 98, n. 3, p. 563-567, JUN 2015.
• Web of Science Citations: 3

Abstract

Introduction: Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data. Materials and methods: Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark (TM) Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports. Results: KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion. Conclusion: Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing ICRAS mutations in CRC samples a method that can be used as an ancillary test for failed assays by conventional PCR. (C) 2015 Elsevier Inc. All rights reserved. (AU)

• FAPESP's process: 11/08510-2 - Mutational status of the Kras gene in primary and metastatic colorectal carcinomas
• Grantee: Isabela Werneck da Cunha
• Support Opportunities: Regular Research Grants