Domanico, Mike

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, yet it is potentially the most treatable and preventable cancer with effective screening. Exact Sciences has developed a multi-biomarker CRC screening test that targets DNA methylation, DNA mutations, and hemoglobin in stool. The aim of this study was to evaluate the analytical accuracy of a novel assay for the detection of KRAS mutation sequences at Codons 12 and 13 in well-characterized colorectal tissues. Methodology: A multiplexed KRAS assay was designed utilizing QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification), a highly sensitive technology that combines allele-specific DNA amplification with invasive cleavage chemistry to generate signal during each amplification cycle similar to real-time PCR. The assay, which detects seven KRAS mutations and the reference gene β-actin, was used to assess 87 colorectal tissue samples (52 CRCs, 16 adenomas ≥ 1cm, and 19 normal epithelia) as determined by Mayo Clinic Pathology. Samples were obtained by microdissection of fresh frozen tissue biopsies. DNA was extracted by Mayo Clinic using a standardized chloroform/phenol methodology. The genotypes of each sample were established using dye terminator dideoxy sequencing in both the forward and reverse orientations. Copy numbers of KRAS mutations and β-actin were determined by conventional comparison against standard curves. KRAS data are reported as percent mutation and calculated by dividing mutant copies by β-actin copies and multiplying by 100. Results: Based on sequencing data: the 52 CRC samples contained 22 KRAS mutations and 30 wild-type genotypes, the 16 adenomas ≥ 1cm contained 8 mutations and 8 wild-type genotypes, and the 19 normal tissues contained all wild-type genotypes. The QuARTS assay detected 100% of the KRAS mutations in the CRC and adenomas and provided excellent differentiation between wild-type and mutation, with the highest percent KRAS mutation of normal wild-type samples at 0.55% and the lowest percent mutation of KRAS positive samples at 8.34%. Based on this data, this assay has the potential to be more sensitive analytically than standard sequencing. Conclusion: The QuARTS assay detects KRAS mutations in tissue with high sensitivity and specificity. This assay method could provide an accurate and high throughput approach to detection of KRAS mutations in biospecimens for multiple clinical applications. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A22.

Abstract BACKGROUND Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 105 copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.