# US 11,118,228 B2 · Detection of colon neoplasia by analysis of methylated DNA

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- Number: US 11,118,228 B2
- Document ID: US11118228B2
- Status: Granted
- Kind: B2
- Application number: 15/881,409
- Filed: 2018-01-26
- Published: 2018-08-30
- Granted: 2021-09-14
- Earliest claimed: 2017-01-27
- Assignees: Exact Sciences Development Company, LLC; Mayo Foundation for Medical Education and Research
- Applicants: Exact Sciences Development Company, LLC; Mayo Foundation for Medical Education and Research
- Inventors: Hatim T. Allawi; Michael W. Kaiser; Graham P. Lidgard; William R. Taylor; Tamara J. Sander; Abram M. Vaccaro
- Web page: https://patentgrep.com/patent/US11118228B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G01N 33/533: with fluorescent label
- G01N 33/57535: of the large intestine, e.g. colon, rectum or anus
- G16Z 99/00: Subject matter not provided for in other main groups of this subclass
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/154: Methylation markers
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16Z: INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS, NOT OTHERWISE PROVIDED FOR

## 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.

## Claims

### Claim 1

A method of characterizing a sample from a human subject, the method comprising assaying the sample for amounts of a plurality of different methylated marker DNAs and amounts of at least one control DNA, the method comprising:
a) combining DNA from the sample in a pre-amplification reaction mixture comprising PCR amplification reagents, wherein the PCR amplification reagents comprise:
i) a plurality of methylation marker primer pairs for amplifying target regions from two to twenty-four different methylated marker DNAs selected from the group of ankyrin repeat domain 13B (ANKRD13B); carbohydrate sulfotransferase 2 (CHST2); cyclin and CBS domain divalent metal cation transport mediator 1 (CNNM1); dedicator of cytokinesis 2 (DOCK2); deltex E3 ubiquitin ligase 1 (DTX1); fermitin family member 3 (FERMT3); Fli-1 proto-oncogene, ETS transcription factor (FLII); glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D); junctional adhesion molecule 3 (JAM3); leucine rich repeat containing 4 (LRRC4); 5-oxoprolinase, ATP-hydrolysing (OPLAH); platelet derived growth factor D (PDGFD); cAMP-dependent protein kinase inhibitor alpha (PKIA); protein phosphatase 2 regulatory subunit B′gamma (PPP2R5C); QKI, KH domain containing RNA binding (QKI); Septin-9 (SEP9); Scm like with four mbt domains 2 (SFMBT2); solute carrier family 12 member 8 (SLC12A8); T-box transcription factor 15 (TBX15); TSPY like 5 (TSPYL5); vav guanine nucleotide exchange factor 3 (VAV3); zinc finger protein 304 (ZNF304); zinc finger protein 568 (ZNF568); and zinc finger protein 671 (ZNF671);
and
ii) a control primer pair for amplifying a target region from beta-1,3- galactosyltransferase 6 (B3GALT6) control DNA;
b) exposing the pre-amplification reaction mixture to thermal cycling conditions wherein two to twenty-four different methylation marker target regions and a B3GALT6 control DNA target region are amplified to produce a pre-amplified mixture;
c) partitioning the pre-amplified mixture into a plurality of assay reaction mixtures, wherein each assay reaction mixture comprises:
i) additional amounts of at least one methylation marker primer pair and of the control primer pair for amplifying a target region from B3GALT6 control DNA used in step a); and
ii) oligonucleotides hybridizable to the methylation marker target region(s) and B3GALT6 control DNA target region amplifiable with the primer pairs of c) i);
d) measuring amounts of two to twenty-four different methylation marker target regions and the target region from B3GALT6 control DNA in the plurality of assay reaction mixtures in a process comprising exposing the plurality of assay reaction mixtures to thermal cycling conditions wherein amplification of two to twenty-four different methylated marker target regions and the B3GALT6 control DNA target region occurs; and
e) calculating values for the amounts of the two to twenty-four different methylated marker target regions amplified in each of the assay reaction mixtures in step d) as a percentage of the amount of the B3GALT6 control DNA target region amplified in each of the assay reaction mixtures, wherein the calculated values indicate the amount of each of the two to twenty-four different methylated marker DNAs in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the assay reactions are PCR-flap assay reactions, wherein the oligonucleotides are flap oligonucleotides, and wherein each of the plurality of assay reaction mixtures further comprises at least one hairpin oligonucleotide comprising a region that is complementary to a portion of a flap oligonucleotide in the assay reaction mixture.

### Claim 3 (depends on 1)

The method of claim 1, wherein the assay reaction mixtures comprise bulk fish DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein the sample is a blood sample, a plasma sample, a stool sample, or a tissue sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein DNA obtained from the sample is treated with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues.

### Claim 6 (depends on 5)

The method of claim 5, wherein the reagent comprises a bisulfite reagent.

### Claim 7 (depends on 1)

The method of claim 1, wherein the plurality of methylation marker primer pairs comprises at least one primer pair for amplifying a target region from a methylated marker DNA selected from the group consisting of VAV3; ZNF671; CHST2; FLII; JAM3; SFMBT2; PDGFD; DTX1; ZNF568; GRIN2D; TSPYL5; and QKI.

### Claim 8 (depends on 1)

The method of claim 1, wherein the plurality of methylation marker primer pairs comprises primer pairs for amplifying target regions from methylated marker DNAs comprising the group consisting of VAV3; ZNF671; CHST2; FLII; JAM3; SFMBT2; PDGFD; DTX1; ZNF568; GRIN2D; TSPYL5; and QKI.

### Claim 9 (depends on 1)

The method of claim 1, wherein the two to twenty-four different methylated marker DNAs comprise nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 121, 126, 131, 136, and their complements.

### Claim 10 (depends on 1)

The method of claim 1, wherein the two to twenty-four different methylated marker DNAs are bisulfite-converted DNAs comprising nucleic acid sequences selected from the group consisting of SEQ ID NOS: 2, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87, 92, 97, 102, 107, 122, 127, 132, 137, and their complements.