Compositions for detecting epigenetic modifications
Inventors
Van Engeland, Manon • De Bruine, Manon Adriaan • Griffioen, Arjan • Louwagie, Joost • Bierau, Katja • Brichard, Gontran • Otto, Gaetan • Penning, Maarten
Assignees
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Publication Number
US-12215389-B2
Publication Date
2025-02-04
Expiration Date
Abstract
A method of detecting a predisposition to, or the incidence of, cancer in a sample comprises detecting an epigenetic change in at least one gene selected from an NDRG4/NDRG2 subfamily gene, GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, wherein detection of the epigenetic change is indicative of a predisposition to, or the incidence of, cancer. Also described are pharmacogenetic methods for determining suitable treatment regimens for cancer and methods for treating cancer patients, based around selection of the patients according to the methods of the invention. The present invention is also concerned with improved methods of collecting, processing and analyzing samples, in particular body fluid samples. These methods may be useful in diagnosing, staging or otherwise characterizing various diseases. The invention also relates to methods for identifying, diagnosing, staging or otherwise characterizing cancers, in particular gastrointestinal cancers such as colorectal cancers, gastric cancers and oesophageal cancers. The methods of the invention relate, inter alia, to isolating and analyzing the human DNA component from faecal samples and blood-based samples.
Core Innovation
The invention provides a composition comprising a reaction mixture for detecting a human NDRG4 methylation signal using bisulfite-treated human sample DNA. Human sample DNA is treated with a bisulfite reagent that modifies unmethylated DNA to form treated sample DNA, and NDRG4-specific primers hybridize to a target sequence within a promoter region of a human NDRG4 gene corresponding to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524.
Amplified DNA is obtained by amplifying the treated sample DNA using the NDRG4-specific primers. The composition further includes a fluorophore-labeled NDRG4-specific probe hybridizable to a site in the amplified DNA, where the probe hybridizes to a sequence corresponding to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524 and includes a quencher moiety.
The invention also centers on epigenetic-methylation biomarker methods for gastrointestinal cancer detection, prognosis, and staging, with emphasis on NDRG4 promoter hypermethylation and additional promoter methylation markers. The approach uses methylation analysis in body-fluid DNA, with particular emphasis on faecal DNA, plasma, and serum, and relates biomarker detection to diagnosis, prognosis, and treatment selection for gastrointestinal cancers.
Claims Coverage
The consolidated claim coverage centers on a bisulfite-treated human sample DNA assay that uses NDRG4-specific primers and a fluorophore-labeled NDRG4-specific probe targeted to positions 1-1000 of methylated and bisulfite-treated SEQ ID NO: 524. A total of nine inventive features are identified across the input items.
Bisulfite-treated human sample DNA for NDRG4 promoter detection
Human sample DNA is treated with a bisulfite reagent that modifies unmethylated DNA to form treated sample DNA.
NDRG4-specific primers targeting a defined NDRG4 promoter region
NDRG4-specific primers hybridize to a target sequence in the treated sample DNA within a promoter region of a human NDRG4 gene corresponding to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524.
PCR amplification using NDRG4-specific primers
Amplified DNA is obtained by amplifying the treated sample DNA using the NDRG4-specific primers.
Fluorophore-labeled NDRG4-specific probe hybridizing within the defined promoter region
A fluorophore-labeled NDRG4-specific probe hybridizes to a site in the amplified DNA within a sequence corresponding to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524.
CpG dinucleotide targeting within the promoter-region window
The NDRG4-specific primers hybridize to CpG dinucleotides in a target sequence located within positions 1-1000 of SEQ ID NO: 524.
Probe with a quencher moiety
The fluorophore-labeled NDRG4-specific probe includes a quencher moiety.
DNA polymerase with 5′-3′ exonuclease activity
The composition includes a DNA polymerase that has 5′-to-3′ exonuclease activity.
Oligonucleotide capture preparation of sample DNA
Sample DNA is prepared by hybridizing sample DNA to an oligonucleotide capture probe and isolating the DNA hybridized to that probe.
Bodily fluid sample type for the assay
The sample DNA is from a bodily fluid sample that can be a blood sample and/or a plasma sample.
The claim coverage is organized around a bisulfite-treated human DNA reaction mixture using NDRG4-specific primers and a fluorophore-labeled NDRG4-specific probe targeted to positions 1-1000 of methylated and bisulfite-treated SEQ ID NO: 524, with refinements for CpG dinucleotide targeting, probe quenching, polymerase activity, sample DNA preparation using oligonucleotide capture, and sample types including blood and plasma.
Stated Advantages
Provides an assay composition for detecting NDRG4 methylation in human bisulfite-treated sample DNA using primers and a fluorophore-labeled probe targeted to a defined NDRG4 promoter region.
Described as a sensitive/specific colorectal cancer biomarker based on NDRG4 promoter methylation.
Reports improved performance for early-stage CRC detection using methylation-based real-time MSP with marker panels that include NDRG4 and OSMR.
Provides specificity/sensitivity evaluation for tissue and plasma marker panels using methylation targets including NDRG4 and OSMR.
Relates biomarker detection to diagnosis, prognosis, and treatment selection for gastrointestinal cancers.
Documented Applications
Detection of colorectal cancer (CRC) and related neoplasias using methylation-based real-time MSP on bisulfite-treated plasma DNA and other sample types.
Early-stage CRC detection using marker panels that include NDRG4 and OSMR.
Measurement of NDRG4 promoter methylation as a sensitive/specific CRC biomarker with fecal DNA analysis.
Use of tissue and plasma marker panels including OSMR, GATA4/5, ADAM23, NDRG4, APC, JPH3 to evaluate specificity/sensitivity tradeoffs.
Preliminary testing of additional methylation markers (BNIP3, FOXE1, JAM3, PHACTR3, TPFI2, SOX17, SYNE1, JPH3) in stool/plasma with reported sensitivity/specificity ranges.
Biomarker detection for gastrointestinal cancers in the contexts of diagnosis, prognosis, and treatment selection.
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