Methylated markers for colorectal cancer

Patent metadata

Field	Value
Publication number	US-2017191135-A1
Application number	US-201515315874-A
Country	US
Kind code	A1
Filing date	Jun 3, 2015
Priority date	Jun 4, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

Disclosed herein is a combination of genomic sequences whose methylation patterns have utility for the improved detection and differentiation between colorectal neoplasms. Further disclosed herein are methods, nucleic acids and kits for detecting or differentiating between colorectal neoplasms.

First claim

What is claimed is:

1 . A method of screening for a colorectal neoplasm or predisposition for developing a colorectal neoplasm in a subject, comprising:
assaying a biological sample from the subject by detecting the methylation state of the Septin 9 gene and the methylation state of one additional marker gene within the biological sample, wherein the one additional marker gene is selected from the Ikaros family zinc-finger 1 (IKZF1) gene and the branched-chain aminotransferase 1 (BCAT1) gene, wherein methylation of Septin 9 and the one additional marker gene is indicative of a colorectal neoplasm, or predisposition for developing a colorectal neoplasm, in the subject.

2 . The method of claim 1 , wherein assaying a biological sample comprises determining the methylation state of the Septin 9 gene, the IKZF1 gene, and the BCAT1 gene.

3 . The method of claim 1 , wherein determining the methylation state of the Septin 9 gene and the methylation state of one additional marker gene comprises at least one of amplification, PCR method, isothermal amplification, NASBA method, LCR method, methylation specific amplification, Methylation Specific PCR (MSP), nested MSP, Heavy-Methyl™, bisulfite sequencing, detection by means of DNA-arrays, detection by means of oligo-nucleotide microarrays, detection by means of CpG-island-microarrays, detection by means of restriction enzymes, the COBRA method, real-time PCR, HeavyMethyl™ real time PCR, MSP MethyLight™, MethyLight™ Algo™, the QM method, Headloop MethyLight™, HeavyMethyl™ MethyLight™, HeavyMethyl™ Scorpion™, MSP Scorpion™, Headloop Scorpion™ method, methylation sensitive primer extension, and MS-SNuPE (Methylation-Sensitive Single Nucleotide Primer Extension).

4 . The method of claim 1 wherein the biological sample is a blood or plasma sample.

5 . The method of claim 1 , wherein the biological sample is a stool sample, enema wash, surgical section or tissue biopsy.

6 . The method of claim 1 , wherein the neoplasm is a cancer.

7 . The method of claim 1 , wherein the neoplasm is an adenoma.

8 . The method of claim 1 , wherein the neoplasm is pre-cancerous.

9 . The method of claim 1 , wherein said subject is human.

10 . The method of claim 1 , wherein the biological sample comprises genomic DNA.

11 . The method of claim 1 , wherein the biological sample comprises cell free DNA.

12 . A method for methylation analysis of DNA, comprising:
a) treating DNA from a biological sample with one or more reagents capable of converting unmethylated cytosine bases to uracil sulfonate or to another base having a different binding behavior than cytosine while leaving methylated cytosine unaffected; and b) amplifying the treated DNA by means of:
i) a primer specific for a Septin 9 gene
ii) a primer specific for a IZKF1 or a BCAT1 gene; and
c) detecting the amplified DNA.

13 . The method of claim 12 , comprising amplifying the treated by means of: i) a primer specific for a Septin 9 gene; ii) a primer specific for an IZKF1 gene; and iii) a primer specific for a BCAT1 gene.

14 . A method for methylation analysis of DNA, comprising:
a) treating DNA from a biological sample with one or more reagents capable of converting unmethylated cytosine bases to uracil sulfonate; and b) amplifying the treated DNA by means of i) a pair of oligonucleotides comprising or consisting of SEQ ID NOs: 1 and 2; and variants thereof; ii) a pair of oligonucleotides comprising or consisting of SEQ ID NOs: 4 and 5, and variants thereof; or a pair of oligonucleotides comprising or consisting of SEQ ID NOs: 7 and 8, and variants thereof; and iii) optionally an oligonucleotide selected from SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:9 and variants thereof; and c) detecting the amplified DNA.

15 . The method of claim 14 , wherein the treated DNA is amplified by means of oligonucleotides comprising or consisting of SEQ ID NOs: 4 and 5, and variants thereof and oligonucleotides comprising or consisting of SEQ ID NOs: 7 and 8, and variants thereof.

16 . An oligonucleotide composition comprising or consisting essentially of:
i) a pair of oligonucleotides comprising or consisting of SEQ ID NOs: 1 and 2; and variants thereof; ii) a pair of oligonucleotides comprising or consisting of: 1) SEQ ID NOs: 4 and 5, and variants thereof or 2) SEQ ID NOs: 7 and 8, and variants thereof; and iii) optionally an oligonucleotide selected from SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and combinations thereof.

17 . The oligonucleotide composition of claim 16 , comprising a pair of oligonucleotides comprising or consisting of SEQ ID NOs: 4 and 5, and variants thereof; and a pair of nucleotides comprising or consisting of SEQ ID NOs: 7 and 8, and variants thereof.

Assignees

• Quest Diagnostics Invest Inc
• Clinical Genomics Pty Ltd

Inventors

• Pedersen Susanne
• Lapointe Lawrence
• Baker Rohan
• Donahue Amber C
• Peng Yen-Lin
• Waldman Frederic

Classifications

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/118

  Prognosis of disease development · CPC title

• C12Q2600/158

  Expression markers · CPC title