Quantification of Methylated Markers with a Multiplex Methylation-Specific Technology
- 1 February 2012
- journal article
- research article
- Published by Oxford University Press (OUP) in Clinical Chemistry
- Vol. 58 (2), 375-383
- https://doi.org/10.1373/clinchem.2011.171264
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.Keywords
Funding Information
- Exact Sciences Corporation
This publication has 36 references indexed in Scilit:
- Molecular Analysis of Plasma DNA for the Early Detection of Lung Cancer by Quantitative Methylation-Specific PCRClinical Cancer Research, 2010
- Analysis of Fecal DNA Methylation to Detect Gastrointestinal NeoplasiaJNCI Journal of the National Cancer Institute, 2009
- Sensitive digital quantification of DNA methylation in clinical samplesNature Biotechnology, 2009
- N-Myc Downstream-Regulated Gene 4 ( NDRG4 ): A Candidate Tumor Suppressor Gene and Potential Biomarker for Colorectal CancerJNCI Journal of the National Cancer Institute, 2009
- Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancerMolecular Cancer, 2008
- KRAS Mutations As an Independent Prognostic Factor in Patients With Advanced Colorectal Cancer Treated With CetuximabJournal of Clinical Oncology, 2008
- Invader Plus Method Detects Herpes Simplex Virus in Cerebrospinal Fluid and Simultaneously Differentiates Types 1 and 2Journal of Clinical Microbiology, 2006
- Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencingNature Medicine, 2006
- EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib TherapyScience, 2004
- Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to GefitinibThe New England Journal of Medicine, 2004