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 a vimentin-associated proliferative disorder, comprising
assaying a sample obtained from a patient for the presence of methylation within a nucleotide sequence as set forth in SEQ ID NO: 2 or fragments thereof;
wherein methylation of said nucleotide sequence is indicative of a vimentin-associated proliferative disorder; and
wherein said vimentin-associated proliferative disorder is a gastro-intestinal 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 a vimentin-associated proliferative disorder.
4. The method of claim 1 , comprising assaying for the presence of methylation of the vimentin sequence of SEQ ID NO: 45.
5. The method of claim 1 , comprising assaying for the presence of methylation of a vimentin sequence selected from the group consisting of: SEQ ID NOs: 40-44.
6. The method of claim 1 , wherein the assay is methylation-specific PCR.
7. The method of claim 6 , 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.
8. The method of claim 6 , 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.
9. The method of claim 6 , wherein the forward primers are selected from the group consisting of: SEQ ID NOs: 14, 16, 19, 21, 23, 25, 27, 29, 31, 33, 37, 38, 39, and the forward primers listed in FIG. 35 .
10. The method of claim 6 , wherein the reverse primers are selected from the group consisting of: SEQ ID NOs: 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, and the reverse primers listed in FIG. 35 .
11. The method of claim 6 , wherein the primer is selected from the group consisting of: MSP29, MSP47, and MSP50.
12. The method of claim 6 , wherein the compound used to treat DNA is a bisulfite compound.
13. The method of claim 1 , wherein the assay comprises using a methylation-specific restriction enzyme.
14. The method of claim 13 , wherein said methylation-specific restriction enzyme is selected from the group consisting of: HpaII, Smal, SacII, EagI, MspI, BstUI, and BssHII.
15. The method of claim 13 , further comprising a pair of primers selected from the group consisting of: SEQ ID NOs: 8-13, and the primers listed in FIG. 35 .
16. A method for detecting a vimentin-associated proliferative disorder in a subject, comprising detecting vimentin protein or nucleic acid expression in a sample from the subject, wherein said vimentin-associated disorder is a gastro-intestinal neoplasia, and wherein the expression of vimentin protein or nucleic acid in said sample is indicative of an increased risk of vimentin-associated proliferative disorder in said subject.
17. The method of claim 16 , 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.
18. The method of claim 17 , wherein the bodily fluid is from a subject suspected of having or known to have a vimentin-associated proliferative disorder.
19. The method of claim 16 , wherein the vimentin protein is detected by immunoassays.
20. A method for monitoring over time a vimentin-associated proliferative disorder comprising:
a) detecting the methylation status of a vimentin nucleotide sequence in a sample from a 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 regression of the proliferative disorder;
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 progression of the proliferative disorder
wherein said vimentin-associated disorder is a gastro-intestinal neoplasia.
21. The method of claim 20 , 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.
22. The method of claim 1 , wherein the sample is derived from a tissue.
23. The method of claim 22 , wherein the tissue sample is obtained from a subject suspected of having or is known to have a vimentin-associated proliferative disorder.
24. The method of claim 16 , wherein the sample is derived from a tissue.
25. The method of claim 24 , wherein the tissue sample is obtained from a subject suspected of having or is known to have a vimentin-associated proliferative disorder.
26. The method of claim 20 , wherein the sample is derived from a tissue.
27. The method of claim 26 , wherein the tissue sample is obtained from a subject suspected of having or is known to have a vimentin-associated proliferative disorder.