Johns Hopkins University

Antonio Wolff

Saraswati Vaidyanathan Sukumar

Mary Jo Fackler

Wei Wen Teo

Zoila Areli Lopez Bujanda

Christopher Umbricht

Leslie M Cope

Peng Huang

Kala Visvanathan

Mary Jo Steele Fackler

Zhe Zhang

A cMethDNA method quantitatively detects tumor DNA (or other circulating DNAs) in fluids such as serum or plasma at the lowest copy number. Unique compared to any other PCR-based assay, a small number of copies of a synthetic polynucleotide standard (STDgene) is added to an aliquot of patient serum. A cocktail of standards for a plurality of genes of interest (TARGETgene) is added to a sample of serum. Once total DNA is purified and processed, a PCR (multiplex step) is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set. In the second nested PCR step, amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene in one well by two-color real-time PCR. Products are calculated by absolute quantitation with internal primer sets specific for the methylated TARGETgene and associated STDgene.

Quantitative multiplex methylation specific PCR method-CMethDNA, reagents, and its use

The cMethDNA method of the present invention is a novel modification of the QM-MSP method (U.S. Pat. No. 8,062,849), specifically intended to quantitatively detect tumor DNA (or other circulating DNAs) in fluids such as serum or plasma at the lowest copy number yet reported. Unique compared to any other PCR-based assay, a small number of copies of a synthetic polynucleotide standard (STDgene) is added to an aliquot of patient serum with standards for a plurality of genes of interest (TARGETgene). Once total DNA is purified PCR is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set, and the amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene. Methods of making the STDgene standards and the use of the cMethDNA methods and kits containing the same are disclosed.

Quantitative multiplex methylation specific PCR method&#x2014;cMethDNA, reagents, and its use

To better understand the biology of hormone receptor-positive and negative breast cancer and to identify methylated gene markers of disease progression, a genome-wide methylation array analysis was performed on 103 primary invasive breast cancers and 21 normal breast samples using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Forty CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER-subtype-specific loci was validated in silico using an independent, publicly available methylome dataset from The Cancer Genome Atlas (TCGA). In addition, 100 methylated CpG loci were identified that were significantly associated with disease progression. Arrays containing the ER-subtype-specific loci and their use in methods of diagnosis and treatment of breast cancer are provided.

Genome-wide methylation analysis and use to identify genes specific to breast cancer hormone receptor status and risk of recurrence

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Baltimore, MD, United States of America

Antonio Wolff