Quantification of Methylated Markers with a Multiplex Methylation-specific Technology

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 10(5) 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.