Molecular Detection of Codon 12 K-RAS Mutations in Circulating DNA from Serum of Colorectal Cancer Patients

Abstract

Point mutations of the K-RAS gene at codon 12 are found in about 40% of cases with colorectal cancer. The diagnostic implications of the detection of these mutations and their clinical utility are still unclear. The aim of this study was to test both the feasibility of the detection of the mutated K-RAS gene in serum and its potential role in colorectal cancer detection and monitoring. Codon 12 K-RAS mutations were examined in DNA extracted from the serum of 35 patients with colorectal cancer and were compared with the K-RAS status in the corresponding primary tumor. Molecular detection was performed by the mutant-enriched PCR (ME-PCR) assay, a sensitive method capable of distinguishing a small quantity of mutated DNA in the presence of abundant wild-type DNA. The occurrence of mutations was compared with clinicopathological parameters as well as CEA and CA19.9 serum levels. We found codon 12 K-RAS mutations in the tissue of 13/35 (37%) patients. Serum mutations were detected in 5/13 (38.5%) patients with mutated K-RAS in the tissue. 26/35 (74%) patients showed an identical K-RAS pattern in tissue and serum. No codon 12 K-RAS alterations were found in serum samples of 22 patients with benign gastrointestinal diseases. Elevated serum CEA levels were detected in 16 patients, four of whom also presented serum RAS mutations. Our results confirm that K-RAS mutations can be found in circulating DNA extracted from serum samples of patients with colorectal cancer and show that there is a correspondence between serum and tissue K-RAS patterns.

Prognostic value of circulating KRAS2 gene mutations in colorectal cancer with distant metastases

“…Several researchers support the theory that tumor DNA is enriched in the blood (13)(14)(15)(16)(17)(18). The use of molecular analysis of gene mutations in plasma or serum DNA as a diagnostic tool for detecting colorectal cancer was first reported by Anker (19) and successfully confirmed by several other studies (20)(21)(22)(23)(24)(25)(26), including one of our previous studies (27). Concordance of tissue and serum KRAS2 mutation was reported in 20-70% of colorectal cancer patients.…”

“…The specificity of KRAS2 mutations in colorectal cancer was established in our previous study (27) where no KRAS2 mutations were detected in 22 specimens obtained from non-malignant diseases of the colon.…”

“…The ME-PCR conditions and the primer sequences used were described in our previous study (27). Muta- (27).…”

“…Tissue and serum DNA were extracted on QIAamp spin columns (Qiagen, Hilden, Germany) according to a previously described protocol (27). DNA was then stored at -20°C in buffer AL (Qiagen) until use.…”

“…The DNA analyses were carried out using a method described elsewhere (27). KRAS2 codon 12 gene mutations in serum samples were detected by mutant-enriched PCR (ME-PCR) in which the primer sense was designed to introduce a restriction site for the BstNI enzyme only in wildtype KRAS2 codon 12.…”