Detection of colon neoplasia by analysis of methylated DNA
Inventors
Allawi, Hatim T. • Kaiser, Michael W. • Lidgard, Graham P. • Taylor, William R. • Sander, Tamara J. • Vaccaro, Abram M.
Assignees
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Publication Number
US-12049671-B2
Publication Date
2024-07-30
Expiration Date
Abstract
Provided herein is technology for neoplasia screening, and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of cancer, in particular, colorectal cancer.
Core Innovation
The invention relates to a method of characterizing a sample from a human subject by assaying for amounts of a plurality of different methylated marker DNAs together with an amount of at least one control DNA. The plurality of methylated marker DNAs is assayed using methylation marker primer pairs that amplify target regions from selected methylated marker DNAs, and the method measures amplified amounts of the target regions from the methylated marker DNAs and from a control DNA region. The control DNA comprises a region of a B3GALT6 gene.
The method includes amplifying DNA from the sample with PCR amplification reagents and then measuring amplified amounts of the target regions. The methylated marker DNAs include selected marker DNAs such as ANKRD13B, CHST2, CNNM1, DTX1, FER1L4, GRIN2D, JAM3, PDGFD, QKI, SFMBT2, VAV3, ZNF568, and ZNF671, within a range of two to thirteen methylated marker DNAs. The described measurement compares methylation states between cancer and normal in the context of screening and neoplasia screening.
In described assay architectures, the invention uses PCR-flap assay reagents with flap oligonucleotides and an endonuclease, including a flap endonuclease workflow. Multiplexing is described as supporting a single-dye multiplex via shared FRET cassettes, and the assay can optionally include assaying for carcinoembryonic antigen (CEA) protein.
Claims Coverage
The record provides two independent claims covering a methylated marker DNA characterization method with a B3GALT6 control region, and an optional CEA protein assay step. Across the independent claims, the core claim coverage is the PCR amplification and measurement of methylated marker DNA target regions for two to thirteen markers, with control DNA measurement, and the additional optional CEA protein assaying step.
Methylated marker DNA and B3GALT6 control characterization
A method of characterizing a sample from a human subject by assaying the sample for amounts of each of a plurality of different methylated marker DNAs and an amount of at least one control DNA, wherein DNA is amplified with PCR amplification reagents comprising methylation marker primer pairs for amplifying target regions from two to thirteen different methylated marker DNAs and at least one control primer pair for amplifying a target region from at least one control DNA; and wherein amplified amounts of the target regions of the two to thirteen different methylated marker DNAs and an amplified amount of the target region from the at least one control DNA are measured, with the at least one control DNA comprising a region of a B3GALT6 gene.
Optional carcinoembryonic antigen protein assaying
The method further comprises assaying the sample for carcinoembryonic antigen (CEA) protein.
The independent claims define a human-sample characterization assay based on PCR amplification and measurement of methylated marker DNA target regions for two to thirteen specified methylated marker DNAs alongside an amplified B3GALT6 control DNA region, with an optional additional assay for carcinoembryonic antigen (CEA) protein.
Stated Advantages
Additive signal without background/cross-reactivity in described multiplex single-dye marker reporting.
Improved sensitivity and specificity reported for plasma testing using logistic regression.
Documented Applications
Human plasma testing using the described multiplex PCR-flap workflow, including comparison using normal plasma and colon cancer plasma and reporting sensitivity/specificity metrics.
KRAS mutation testing on unconverted DNA in human plasma as described in the record.
Carcinoembryonic antigen (CEA) protein assay as an additional assaying step in the method.
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