Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer

Abstract

Summary Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma-or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.

Ras Oncogene Mutations in Urine Sediments of Patients with Bladder Cancer

“…The frequency of the codon 12 mutation was 39% in the present study. This is higher than some studies (Levesque et al, 1993;Saito et al, 1997;Olderoy et al, 1998), but agrees with several reports on the bladder (Czerniak et al, 1990;Fitgerald et al, 1995), colon (Kopreski et al, 1997;Puig et al, 2000), and lung (Mills et al, 1995) tumors. Considerably higher frequencies have also been reported (Burchill et al, 1991;Burchill et al, 1994;Przybojewska et al, 2000).…”

Luteal phase serum cell-free DNA as a marker of failed pregnancy after assisted reproductive technology

“…The most exciting finding was the detection of cellfree DNA in the frozen-thawed sera using the capillary electrophoresis assay without the necessity for amplification by polymerase chain reaction (PCR). Bypassing the PCR step conferred the advantage of directly evaluating the cell-free DNA derived from the entire genome without variations due to artefacts and inefficiencies in phenol-chloroform extraction and PCR amplification steps, and the limited primer targeted gene regions such as the K-ras oncogene (25)(26). However, the standard capillary electrophoresis assay possesses certain advantages such as continuous flow through analysis and direct nucleic acids detection set up on the capillary tube (20)(21)(22).…”

Preferential Isolation of Fragmented DNA Enhances the Detection of Circulating Mutated k-ras DNA

“…DNA derived from 50 L of serum was used to assay for k-ras DNA, mutated in codon 12, by the modified restriction endonuclease-enriched PCR (RE-PCR) assay (7 ). The PCR product of this modified RE-PCR was 87 bp with an assay detection limit of 15 copies of the mutant k-ras per 100 ng of wild-type DNA per reaction (data not shown).…”