Pyrosequencing analysis of KRAS codon 61 mutations in Thai patients with advanced colorectal cancer

Abstract

Background: KRAS, coding for a small G-protein downstream of epidermal growth factor receptor (EGFR) plays an important role in the EGFR signaling network. Mutation in KRAS is associated with resistance to anti- EGFR in patients with advanced colorectal cancer (CRC). According to the American Society of Clinical Oncology (ASCO) guidelines, screening for mutations in KRAS codons 12 and 13 in tumor samples is mandatory for all CRC patients who are candidates for anti-EGFR targeted therapy. However, some patients with undetectable mutations in codons 12/13 do not benefit from anti-EGFR treatment, and this might be because of mutations in codon 61, which is not currently recommended for screening. Objectives: To develop an in-house pyrosequencing method to screen for KRAS codon 61 mutations, and examine the prevalence of mutations in Thai patients with advanced CRC with no detectable mutation in codons 12/13. Materials and Methods: DNA extracted from FFPE specimens was screened for KRAS codon 61 mutations using pyrosequencing. Our method was suitable for routine clinical samples (formalin-fixed, paraffin-embedded tissue), and was able to detect 5 common mutations in codon 61 of the KRAS gene, including c.182AT (p.Q61L), c.182AG (p.Q61R), c.182AC (p.Q61P), c.183AC (p.Q61H), and c.183AT (p.Q61H). Results: Of the 74 samples with undetectable codon 12/13 mutation examined, two (2.7%) were found to harbor mutation in codon 61. Conclusion: Despite the low prevalence of KRAS codon 61 mutation in our population with advanced CRC, adding the mutation test into the routine molecular service deserves consideration because the cost of treatment is very expensive.