# US 11,898,199 B2 · Detection of colorectal cancer and/or advanced adenomas

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- Number: US 11,898,199 B2
- Document ID: US11898199B2
- Status: Granted
- Kind: B2
- Application number: 16/680,427
- Filed: 2019-11-11
- Published: 2021-05-13
- Granted: 2024-02-13
- Earliest claimed: 2019-11-11
- Assignees: Universal Diagnostics, S.A.
- Applicants: Universal Diagnostics, S.A.
- Inventors: Marko Bitenc; Kristi Kruusmaa; Juan Martinez-Barea; Christian Hense; Marko Chersicola; Primo{hacek over (z)} Knap
- Web page: https://patentgrep.com/patent/US11898199B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/154: Methylation markers
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- G01N 2800/56: Staging of a disease; Further complications associated with the disease
- G01N 2800/7028: Cancer
- 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)

## Abstract

The present disclosure provides, among other things, methods of screening for colorectal cancer, methods of screening for advanced adenoma, methods of screening for the presence of either colorectal cancer or advanced adenoma (or both), and compositions related thereto. In various embodiments, the present disclosure provides methods for colorectal cancer and/or advanced adenoma screening that includes analysis of methylation status of one or more methylation biomarkers, and compositions related thereto. In various embodiments, the present disclosure provides methods for colorectal cancer and/or advanced adenoma screening that include screening methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA. In various embodiments, the present disclosure provides methods for colorectal cancer and/or advanced adenoma screening that include screening methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA, using MSRE-qPCR.

## Claims

### Claim 1

A method of detecting hypermethylation of differentially methylated regions (DMRs), the method comprising:
detecting a methylation status of at least one methylation site within each of three or more differentially methylated regions (DMRs) of cell-free DNA from blood or plasma of a human subject suspected of having colorectal cancer and/or advanced adenoma, wherein the three or more DMRs comprise SEQ ID NO: 192, SEQ ID NO: 201, and SEQ ID NO: 210,
wherein detecting the methylation status comprises determining whether at least one methylation site within each of the three or more DMRs is hypermethylated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the three or more DMRs further comprise at least one member selected from the group consisting of SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 208, SEQ ID NO: 218, and SEQ ID NO: 219.

### Claim 3 (depends on 1)

The method of claim 1, wherein the three or more DMRs further comprise at least one member selected from the group consisting of SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, and SEQ ID NO: 229.

### Claim 4 (depends on 1)

The method of claim 1, wherein the subject is suspected of having early stage colorectal cancer.

### Claim 5 (depends on 1)

The method of claim 1, wherein the methylation status is detected using one or more members selected from the group consisting of Methylation-Specific PCR, Methylation Specific Nuclease-assisted Minor-allele Enrichment PCR, hybrid-capture targeted next-generation sequencing, and amplicon based targeted next-generation sequencing.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the methylation status of at least one methylation site found within the gene CDKN2A.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the methylation status of at least one methylation site found within the gene CDKN2B-AS1.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the methylation status of at least one methylation site found within SEQ ID NO: 195.

### Claim 9

A method of using methylation sensitive restriction enzyme quantitative polymerase chain reaction (MSRE-qPCR), the method comprising:
(a) contacting cell-free DNA from blood or plasma of a human subject suspected of having colorectal cancer and/or advanced adenoma with one or more methylation sensitive restriction enzymes;
(b) performing qPCR of enzyme-digested DNA, or amplicons thereof; and
(c) detecting the methylation status of at least one methylation site found within each of three or more differentially methylated regions (DMRs) of DNA, wherein the three or more DMRs comprise SEQ ID NO: 192, SEQ ID NO: 201, and SEQ ID NO: 210.

### Claim 10 (depends on 9)

The method of claim 9, wherein the three or more DMRs further comprise at least one member selected from the group consisting of SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 208, SEQ ID NO: 218, and SEQ ID NO: 219.

### Claim 11 (depends on 9)

The method of claim 9, wherein the three or more DMRs further comprise at least one member selected from the group consisting of SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, and SEQ ID NO: 229.