Molecular Biological Diagnostics of KRAS and BRAF Mutations in Patients with Colorectal Cancer – Laboratory Experience


Authors: B. Robešová 1;  M. Bajerová 1;  A. Vašíková 1;  L. Ostřížková 2;  J. Hausnerová 3;  J. Kyclová 3;  Š. Pospíšilová 1,4;  J. Mayer 1,4;  D. Dvořáková 1,4
Authors‘ workplace: Centrum molekulární biologie a genové terapie, Interní hematologická a onkologická klinika LF MU a FN Brno 1;  Interní hematologická a onkologická klinika LF MU a FN Brno 2;  Ústav patologie, FN Brno 3;  CEITEC – Středoevropský technologický institut, MU Brno 4
Published in: Klin Onkol 2013; 26(1): 25-30
Category: Original Articles

Overview

Background:
Targeted biological therapy based on blocking growth factor receptors and inhibition of cancer-inducing signaling pathways is a new treatment facility for patients with colorectal cancer (CRC). Therapeutic agents are monoclonal antibodies targeting epidermal growth factor receptor (EGFR). Gene aberrations in the EGFR-induced pathways are negative predictors of therapeutic response. Determination of ­non-mutated KRAS is a requirement for the indication of targeted anti-EGFR therapy in the present time, BRAF mutation analysis is recommended. Comparison of our results with published data and verification of routine laboratory methods in relation to diagnostic kits were the purposes of this study.

Patients and Methods:
In addition to routine methods based on PCR, direct sequencing as well as two diagnostic kits for KRAS (codon 12 and 13) and BRAF (codon 600) mutation analysis were used for 132 patients.

Results:
KRAS mutations were detected in 45 patients (34%), V600E mutation of the BRAF gene in 9 patients (7%). Both mutations simultaneously were not detected. Tissues from primary tumor and metastases were available from 33 patients. KRAS mutation was detected in 13 cases of this group. KRAS mutations in tumor and metastasis were of the same type in 9 patients; types of mutation in both tissues were different in one case. KRAS mutation only in one tissue was detected in 3 cases. BRAF mutation in both tissues was detected in the 4 patients. A low percentage of tumor cells in 17 patients’ specimen did not allow performance of routine analysis and diagnostic kit was used.

Conclusion:
The frequency of KRAS and BRAF mutations in our cohort of patients corresponds to published data. The suitability of metastatic tissue analysis due to tumor heterogeneity was confirmed. KRAS analysis requires a comprehensive methodological approach with regard to reduced DNA quality and different percentage of tumor cells in tissue. For this reason, commercial diagnostic kits constitute a suitable supplement to standard methods.

Key words:
colorectal neoplasms – KRAS – BRAF – molecular targeted therapy – monoclonal antibodies – molecular diagnostics


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Paediatric clinical oncology Surgery Clinical oncology
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