Fast simultaneous detection of K-RASmutations in colorectal cancer

Abstract

BackgroundRAS genes acquire the most common somatic gain-of-function mutations in human cancer, and almost all of these mutations are located at codons 12, 13, 61, and 146.MethodsWe present a method for detecting these K-RAS hotspot mutations in 228 cases of colorectal cancer. The protocol is based on the multiplex amplification of exons 2, 3 and 4 in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at codons 12, 13, 61 and 146. We compared the clinicopathological data of colorectal cancer patients with the K-RAS mutation status.ResultsK-RAS mutation occurred in 36% (83/228) of our colorectal cancer cases. Univariate analysis revealed a significant association between K-RAS mutation at codon 12 of exon 2 and poor 5-year survival (p = 0.023) and lymph node involvement (p = 0.048). Also, K-RAS mutation at codon 13 of exon 2 correlates with the size of the tumor (p = 0.03). Multivariate analysis adjusted for tumor size, histologic grade, and lymph node metastasis also indicated K-RAS mutations at codon 12 and 13 of exon 2 correlate significantly with overall survival (p = 0.002 and 0.025). No association was observed between codon 61 and 146 and clinicopathological features.ConclusionWe demonstrated a simple and fast way to identify K-RAS mutation.

Mutation analysis of 13 driver genes of colorectal cancer-related pathways in Taiwanese patients

“…We assessed the frequency of non-pathological somatic mutations during oncogenesis, which has not been explored before. Our results indicated 16 de novo mutations that have been previously described in a public database and were detected in cancerous tissues only, but not in the patient's blood cells. We suggest these mutation sites may belong to a frequent mutational hotspot in both germline and cancerous tissues.…”

“…Most sporadic CRCs (70%-80%) have APC somatic mutations, and the mutations appear to be enriched in the mutation cluster region (MCR, codons 1309 to 1450) [7] . Approximately 40% of CRCs have KRAS mutations, and almost all of these mutations are located at codons 12, 13 or 61 [16,17] . The MSI pathway is characterized by the inactivation of the MMR genes such as MLH1.…”

Ras in Cancer and Developmental Diseases

“…[61][62][63] Separate studies have also reported that the rate of K-ras mutation is enhanced in CRC patients with lung metastasis 64 and that the presence of K-ras mutations in CRC patients with liver metastasis is predictive of bad prognosis. 65 Analysis of the mutational state of ras genes has proven to be very significant for selection of therapeutic approaches in CRC.…”

Differences in the frequencies of K‐ras c12–13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method

“…The GAC mutation is reported to be of predictive and prognostic value for local recurrence and shortterm mortality [Bazan et al, 2002;Chang et al, 2009;Pajkos et al, 2000;Samowitz et al, 2000]. Among the poorly differentiated tumors, the K-ras mutants in males were predominantly GAT (37%, Table IV ); in contrast, among the poorly differentiated tumors of females, GAT, GTT, and GAC mutations were equal in prevalence (10% each).…”

“…More than 90% of single-base substitutions in the K-ras gene occur at glycine 12 (GGT) and 13 (GGC), whereas 5% occur at glutamine 61 (CAA) and at other codons [Marchetti and Gasparini, 2009;Poehlmann et al, 2007;Toyooka et al, 2003]. Novel oncogenic mutants at other codons-including G15 (GGC) [Wang et al, 2003[Wang et al, , 2007, L19 (TTG) [Akagi et al, 2007;Rouleau et al, 2008;Simi et al, 2008], Q22 (AAG) [Miyakura et al, 2002;Palmirotta et al, 2009;Tsukuda et al, 2000], and A146 (GCA) [Chang et al, 2009;Edkins et al, 2006]-have been detected using predominantly DNA sequencing and single-strand conformation polymorphism (SSCP) analysis. Nonetheless, the detection of the single-nucleotide variants in K-ras codon 12-13 has the most important implications for clinical prognosis and drug discovery.…”