Tumor-associated DNA has been detected, using various techniques, in the plasma of colorectal cancer (CRC) patients, including DNA with mutations in APC, TP53, and K-RAS. We report a highly sensitive mutation scanning methodology for the mutational assessment of the APC and TP53 genes, which typically pose an analytical challenge due to their significant genotypic heterogeneity. Also, we present a specific mutational scoring assay for K-RAS and BRAF.
Plasma DNA isolated from 20 CRC patients was scanned for mutations in multiple targets including APC, TP53, K-RAS, and BRAF blinded to the molecular or pathological analyses of the matched primary tumors.
We chose mutation scanning technology and these molecular targets to provide a comprehensive screen for somatic mutations known to be associated with sporadic CRC.
Mutations in these targets were detected with a novel denaturing high-performance liquid chromatography (DHPLC) platform that uses post-separation fluorescence technology. This scanning technology enables the detection of variants that represent 0.11% of the total analyzed DNA.
Mutant allele specific amplification (MASA) followed by detection with the same platform was used to identify low-level target mutations (mutation scoring) in K-RAS codons 12, 13, 61, and BRAF codon 599.
Using this combined mutation scanning and scoring approach, we were able to identify at least one mutational event in 20/20 (100%) of CRC patients. All the mutations found in plasma were also present in the primary tumor.
Thus, combining the four genetic markers enabled detection of mutations in 100% of patients with CRC, all of whom had at least one alteration. A single mutational event was detected in 5/20 (25%), two in 14/20 (70%), and three in 1/20 (5%).
The most common single event was a K-RAS mutation in 3/20 (15%), while the most common dual event included an APC and p53 mutation 7/20 (35%). Mutations in p53 and either K-RAS (1/20) or BRAF (1/20) were rare, and K-RAS and BRAF mutations were mutually exclusive.
The results emphasize the heterogeneous pattern of tumor mutations in even a small cohort and that scanning provides an attractive approach to CRC genetic screening. The thoroughness of this scanning approach may have implications for CRC screening and disease monitoring during and following therapy.
Expanded studies are being conducted to determine the specificity of this mutation-scanning panel for various stages of CRC.