Compositions and methods for performing methylation detection assays

Patent metadata

Field	Value
Publication number	US-2018291469-A1
Application number	US-201816013240-A
Country	US
Kind code	A1
Filing date	Jun 20, 2018
Priority date	Dec 12, 2014
Publication date	Oct 11, 2018
Grant date	—

Abstract

Provided herein is technology relating to performing methylation assays. In particular, the technology relates to internal controls for methylation assays.

First claim

We claim:

1 . A method of screening for a neoplasm in a sample obtained from a subject, the method comprising:
a) assaying a sample from a subject for an amount of at least one methylated marker gene selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a in a sample obtained from a subject; b) assaying said sample for an amount of methylated ZDHHC1 DNA in said sample; c) comparing the amount of said at least one methylated marker gene to the amount of methylated ZDHHC1 DNA in said sample to determine a methylation state for said at least one marker gene in said sample; and d) generating a record reporting the methylation state for said at least one marker gene in said sample.

2 . The method of claim 1 , wherein said at least one marker is at least two of said markers.

3 . The method of claim 1 , wherein said at least one marker is all of said markers.

4 . The method of claim 1 wherein the assaying comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture.

5 . The method of claim 1 wherein assaying the methylation state of the marker in the sample comprises determining the methylation state of one base.

6 . The method of claim 1 wherein assaying the methylation state of the marker in the sample comprises determining the extent of methylation at a plurality of bases.

7 . The method of claim 1 wherein the methylation state of the marker comprises an increased or decreased methylation of the marker relative to a normal methylation state of the marker.

8 . The method of claim 1 wherein the methylation state of the marker comprises a different pattern of methylation of the marker relative to a normal methylation state of the marker.

9 . The method of claim 1 comprising assaying a methylation state of a forward strand or assaying a methylation state of a reverse strand.

10 . The method of claim 1 wherein the neoplasm is a gastrointestinal neoplasm.

11 . The method of claim 1 wherein the neoplasm is present in the upper gastrointestinal area of the patient.

12 . The method of claim 1 wherein the neoplasm is present in the lower gastrointestinal area of the patient.

13 . The method of claim 1 wherein the neoplasm is a pancreas neoplasm, a colorectal neoplasm, a bile duct neoplasm, a stomach neoplasm, an esophagus neoplasm, or an adenoma.

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

15 . The method of claim 1 wherein the sample is a stool sample, a tissue sample, a pancreatic juice sample, a pancreatic cyst fluid sample, a blood sample, or a urine sample.

16 . The method of claim 1 , wherein said assay further comprises the step of identifying a KRAS mutation score in said sample, preferably wherein said measuring of said K-ras mutation score is measured by digital melt curve analysis or quantitative allele-specific PCR.

17 . The method of claim 16 , wherein said assay comprises detecting methylation state of ZDHHC1, BMP3, NDRG4, and identifying a KRAS mutation score in said sample.

18 . The method of claim 1 , wherein said method further comprises the step of determining the presence of hemoglobin in said sample.

19 . The method of claim 1 , wherein said subject is a patient having inflammatory bowel disease.

20 . A kit, comprising:
a) at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to ZDHHC1; and b) at least one additional oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to marker selected from the group consisting of vimentin, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a.

21 . The kit of claim 20 , wherein said kit comprises at least two additional oligonucleotides.

22 . The kit of claim 20 , wherein said kit further comprises bisulfite.

23 . The kit of claim 20 , wherein said kit further comprises at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to KRAS.

24 . The kit of claim 20 , wherein said kit further comprises reagents for detecting the presence of hemoglobin in a stool sample.

25 . The kit of claim 20 , 21 , or 23 , wherein said oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an INVADER oligonucleotide.

26 . The kit of any one of claims 20 to 25 , wherein said kit further comprises a solid support.

27 . The kit of claim 26 , wherein said solid support is a magnetic bead.

28 . The kit of claim 25 or 26 , wherein said solid support comprises one or more capture reagents.

29 . The kit of claim 28 , wherein said capture reagents are oligonucleotides complementary to ZDHHC1 and said one or more markers genes.

30 . A composition, comprising:
a) a complex of a ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of said oligonucleotide is hybridized to said ZDHHC1 nucleic acid; and b) one or more additional reaction mixtures comprising a complex of a target nucleic selected from the group consisting of vimentin, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a and one or more oligonucleotides that specifically hybridize to one or more target genes.

31 . The composition of claim 30 , wherein said oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an invasive oligonucleotide.

32 . A method of measuring the severity of inflammation in a subject having inflammatory bowel disease, comprising:
a) assaying a sample of stool from a subject to determine the presence of methylated ZDHHC1 DNA and β-actin DNA, b) comparing the ratio of β-actin DNA to methylated ZDHHC1 DNA to a ratio of β-actin DNA to methylated ZDHHC1 DNA correlated to a known severity of inflammation in IBD,
wherein a ratio of β-actin DNA to methylated ZDHHC1 DNA in said subject higher than the ratio correlated to the known severity of inflammation is indicative of more severe inflammation in said subject than said known severity; and
wherein a ratio of β-actin DNA to methylated ZDHHC1 DNA in said subject lower than the ratio correlated to the known severity of inflammation is indicative of less severe inflammation in said subject than said known severity.

33 . The method of claim 32 , wherein the ratio correlated to the known severity of inflammation was previously determined by assaying a sample of stool from the same subject.

34 . The method of claim 32 , wherein the ratio correlated to the known severity of inflammation was previously determined by assaying a sample of stool from a normal subject.

35 . The method of claim 32 , wherein the assaying comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture.

36 . The method of claim 35 , wherein the assaying comprises using a flap endonuclease assay.

37 . The method of claim 32 , wherein the assaying comprises bisulfate converting said ZDHHC1 DNA and said β-actin DNA.

Assignees

• Exact Sciences Dev Co Llc
• Mayo Found Medical Education & Res

Inventors

• Ahlquist David Alan
• Taylor William Russell
• Mahoney Douglas W
• Lidgard Graham P
• Allawi Hatim T
• Vaccaro Abram Michael

Classifications

• C12Q2600/154

  Methylation markers · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6886 Primary

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

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title