September 1, 2005 (Vol. 25, No. 15)

Novel DHPLC Fluorescence Detection Platform

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.

Materials and Methods

Mutation Scanning Mutation detection by DHPLC involves temperature-modulated heteroduplex analysis of PCR products by ion-pair reverse-phase chromatography. Under partially denaturing conditions, heteroduplexes formed in PCR products containing mutations show reduced column retention times relative to the homoduplexes that are formed when PCR products contain no mutations.

This scanning technology allows the detection of any mutation in the PCR amplicon of interest, including both previously identified and novel mutations. All samples identified as positive for the presence of a mutation by DHPLC were further characterized by DNA sequencing.

MASA Oligonucleotides were designed to have the 3′-terminal nucleotide match the specific mutation or wild type that were being scored (genotyped). The Tm of this MASA oligonucleotide was typically 64.065.0C. A second antisense oligonucleotide with a higher Tm of 69.070.0C was used along with the MASA primer for PCR to produce a 150200 bp amplicon for analysis.

The PCR protocol was optimized using a temperature gradient and altering Mg2+ to achieve a set of conditions in which only the mutant allele would amplify with this primer set, while the normal wild-type allele would not.

WAVE HS System Technology The amplified products from the MASA and the normal allele PCR were analyzed by DNA fragment sizing with the WAVE HS System. This analytical platform has a module for post-column intercalation and a fluorescence detector (Figure 1).

The staining solution contains an intercalating dye, which fluoresces only when bound to double-stranded DNA followed by excitation with a Xenon lamp at 492 nm wavelength and emission of 526 nm (Figure 2).

Results and Discussion

Heteroduplex and homoduplex PCR products (mutation scanning), as well as MASA and normal PCR products (mutation scoring) were detected with the WAVE HS System using post-column intercalation.

Figure 3 shows results from DHPLC mutation scanning of exon 5 of the TP53 gene. Figure 4 shows results from genotyping of V600E in exon 15 of the BRAF gene. This method has a detection limit of approximately 510 pg of DNA per amplified fragment.

The low abundance of mutated DNA in CRC patient samples makes it difficult to detect such mutations using traditional techniques such as DNA sequencing. The assays that we describe here allowed us to detect in plasma such mutations associated with potential secondary events, which reflect the changes observed in primary tumors. This technology is being used for prognosis, prediction, and disease monitoring applications.

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