Methods and compositions for detecting gastrointestinal and other cancers

Patent metadata

Field	Value
Publication number	US-9580754-B2
Application number	US-201213670155-A
Country	US
Kind code	B2
Filing date	Nov 6, 2012
Priority date	Aug 14, 2003
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

This application describes methods and compositions for detecting and treating vimentin-associated neoplasia. Differential methylation of the vimentin nucleotide sequences has been observed in vimentin-associated neoplasia such as neoplasia of the upper or lower gastrointestinal tract, pancreas, and/or bladder. Nucleic acid sequences for regions of vimentin that are found to be differentially methylated are described. The present disclosure further describes bisulfite-converted vimentin template DNA sequences. Oligonucleotide primer sequences for use in assays (e.g., methylation-sensitive PCR assays or HpaII assays) designed to detect the methylation status of the vimentin gene are also described.

First claim

We claim:

1. An isolated polynucleotide comprising a bisulfite-converted nucleic acid; wherein the polynucleotide is 20-3000 nucleotides in length; wherein the polynucleotide comprises a region having:
a) a nucleotide sequence comprising the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, a complement thereof, or a fragment thereof; wherein said nucleotide sequence, said complement or said fragment is at least 20 nucleotides in length; or
b) a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, a complement thereof, or a fragment thereof; wherein said nucleotide sequence, said complement or said fragment is at least 20 nucleotides in length;
and wherein said nucleic acid, prior to bisulfite conversion, comprises at least one methylated cytosine and at least one unmethylated cytosine.

2. The isolated polynucleotide of claim 1 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, or a complement thereof or a fragment thereof.

3. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, or a fragment thereof or a complement thereof.

4. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 42, or a fragment thereof or a complement thereof.

5. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 44, or a fragment thereof or a complement thereof.

6. The isolated polynucleotide of claim 1 , wherein said complement thereof consists of the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 48, or a fragment thereof.

7. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 62 and 63.

8. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 72 and 71.

9. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 68 and 71.

10. The isolated polynucleotide of claim 1 , wherein the polynucleotide is between 50 to 500 nucleotides in length.

11. The isolated polynucleotide of claim 1 , wherein the polynucleotide is between 80 to 150 nucleotides in length.

12. The isolated polynucleotide of claim 1 , wherein the polynucleotide is a bisulfite-converted fragment of a nucleic acid obtained from a biological sample taken from a patient suspected of having a colon neoplasia.

Assignees

• Univ Case Western Reserve

Inventors

• Markowitz Sanford D
• Willis Joseph
• Chak Amitabh
• Leidner Rom

Classifications

• C12Q2600/154

  Methylation markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/136

  Screening for pharmacological compounds · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title