Prognostic significance of KRAS gene mutations in colorectal cancer--preliminary study

Abstract

Objective: The prognostic significance of KRAS gene mutations, evaluated by using two methods in patients with colorectal cancer (CRC).

Material and methods: Retrospective study involving 58 patients diagnosed with CRC and treated between 2003 and 2010 in the General and Esophageal Surgery Clinic of "Sf. Maria" Hospital, Bucharest. The macroscopic and microscopic examination of the resected specimens was also processed for genetic analysis in NIRDPBS, where KRAS status was determined by using two methods: PCR-RFLP and pyrosequencing.

Results: The clinical and biological parameters of the patients were assessed for 72 months in average. A relapse in 21 patients and a 5-year survival rate of 79.3% was discovered. The genetic analyses of KRAS gene found mutations in 22 cases (45.3%): 17 cases had mutations in codon 12, 5 cases in codon 13. The survival rate analyses of patients with wild KRAS gene compared with the patients carrying the mutation on codon 12 /13 revealed a superposition of the survival curve. The statistical analysis based on the TNM stage revealed different survival curves in stage I and II, shorter survival period in patients with KRAS mutation on codon 13 than in those with wild type gene (stage I--p_value=0.015; stage II--p_value=0.000).

Conclusions: It was not found that KRAS gene status had any prognostic significance. Nevertheless, for stage I and II patients, the mutation found on codon 13 determined a statistic significant shorter survival rate than for those with wild type. The results obtained by using the pyrosequencing method for the determination of KRAS gene status proved that it represented a reliable and reproducible method.

Keywords: KRAS gene mutation; colorectal cancer; pyrosequencing.

Table 1

The distribution of KRAS gene mutation in codons 12 and 13

Fig. 1

Gel electrophoresis, agarose 2% (w/v) for codon 12. PCR product (left, band at 135 bp) and restriction fragments (right) were migrated for each sample; NTC – no template control; PC – positive control (mutant heterozygous, bands at 135, 106 and 29 bp); NC – negative control (wild type, bands at 106 and 29 bp); WT – wild type sample, M – mutant sample; L – DNA molecular weight markers (GeneRuler 50 pb DNA Ladder, Fermentas)

Fig. 2

Gel electrophoresis, agarose HR 4% (w/v) for codon 13, of restriction fragments. NTC – no template control; PC – positive control (mutant heterozygous, bands at 85, 74, 48 and 26 bp); NC – negative control (wild type, bands at 85, 48 and 26 bp); WT – wild type sample, M – mutant sample; L – DNA molecular weight markers (GeneRuler 50 pb DNA Ladder, Fermentas)

Fig. 3

Example of wild type - codons 12, 13

Fig. 4

Example of mutation in codon 12 (GGT>GAT)

Fig. 5

Example of mutation in codon 12 (GGT>GTT)

Fig. 6

Example of mutation in codon 12 (GGT>TGT)

Fig. 7

Example of mutation in codon 13 (GGC>GAC)

Fig. 8

Example of wild type – codon 61