Methods and compositions for detecting colon cancers
View Patent ↗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 colon neoplasia.
1. A method for detecting colon neoplasia, comprising:
a) obtaining a human sample; and
b) assaying said sample for the presence of methylation within the nucleotide sequence as set forth in SEQ ID NO: 2;
wherein methylation of said nucleotide sequence is indicative of colon neoplasia.
2. The method of claim 1 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.
3. The method of claim 2 , wherein the bodily fluid is obtained from a subject suspected of having or is known to have colon neoplasia.
4. The method of claim 3 , wherein said colon neoplasia is colon cancer.
5. The method of any of claim 1 , wherein the assay is methylation-specific PCR.
6. The method of claim 5 , comprising:
a) treating DNA from the sample with a compound that converts non-methylated cytosine bases in the DNA to a different base;
b) amplifying a region of the compound converted vimentin nucleotide sequence with a forward primer and a reverse primer; and
c) analyzing the methylation patterns of said vimentin nucleotide sequences.
7. The method of claim 5 , comprising:
a) treating DNA from the sample with a compound that converts non-methylated cytosine bases in the DNA to a different base;
b) amplifying a region of the compound converted vimentin nucleotide sequence with a forward primer and a reverse primer; and
c) detecting the presence and/or amount of the amplified product.
8. The method of claim 5 , wherein the compound used to treat DNA is a bisulfite compound.
9. The method of any of claim 1 , wherein the assay comprises using a methylation-specific restriction enzyme.
10. The method of claim 9 , wherein said methylation-specific restriction enzyme is selected from HpaII, SmaI, SacII, EagI, MspI, BstUI, and BssHII.
11. A method for detecting colon neoplasia in a human subject, comprising detecting vimentin protein or nucleic acid expression level in a sample from the human subject, wherein reduced expression level of vimentin protein or nucleic acid relative to a control sample from a healthy subject is indicative of colon neoplasia in said subject.
12. The method of claim 11 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.
13. The method of claim 12 , wherein the bodily fluid is from a subject suspected of having or known to have colon neoplasia.
14. The method of claim 11 , wherein the vimentin protein is detected by immunoassays.
15. A method for identifying an agent which enhances vimentin protein or nucleic acid expression in a cell from a subject having colon neoplasia, comprising:
a) contacting the cell with a sufficient amount of the agent under suitable conditions;
b) quantitatively determining the amount of vimentin protein or nucleic acid; and
c) comparing the amount of vimentin protein or nucleic acid with the amount of vimentin protein or nucleic acid in the absence of the agent,
wherein a greater amount of vimentin protein or nucleic acid in the presence of the agent than in the absence of the agent indicates that the agent enhances vimentin protein or nucleic acid expression.
16. The method of claim 15 , wherein said colon neoplasia is due to differential methylation of the vimentin nucleotide sequence as set forth in SEQ ID NO: 2.
17. A method for monitoring colon neoplasia over time comprising:
a) detecting the methylation status of the vimentin nucleotide sequence as set forth in SEQ ID NO: 2 in a sample from a human subject for a first time; and
b) detecting the methylation status of the vimentin nucleotide sequence in a sample from the same subject at a later time;
wherein absence of methylation in the vimentin nucleotide sequence taken at a later time and the presence of methylation in the vimentin nucleotide sequence taken at the first time is indicative of cancer regression;
wherein presence of methylation in the vimentin nucleotide sequence taken at a later time and the absence of methylation in the vimentin nucleotide sequence taken at the first time is indicative of cancer progression.
18. The method of claim 17 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.