Unbiased DNA methylation markers define an extensive field defect in histologically normal prostate tissues associated with prostate cancer: new biomarkers for men with prostate cancer
A method of detecting the presence of a prostate cancer field defect in a human subject comprising the step of (a) obtaining genomic DNA from the human subject and (b) quantitating methylation in at least one target region selected from the group consisting of CAV1, EVX1, MCF2L, FGF1, NCR2 and WNT2 and EXT1 and SPAG4 target, wherein significant methylation changes indicate the presence of prostate cancer or a prostate cancer field defect, wherein the change is relative to tissue from a second human subject who does not have prostate cancer.
1. A method for detecting methylation of SEQ ID NO:1 in target DNA isolated from histologically normal prostate cells from a subject, the method comprising the steps of:
(a) providing a reaction mixture comprising:
the target DNA isolated from histologically normal prostate cells from the subject or a bisulfite modified form thereof; and
at least one primer complementary to SEQ ID NO:1 or a bisulfite modified form thereof; and
(b) reacting the reaction mixture for a time and at a temperature sufficient to hybridize the at least one primer to SEQ ID NO:1 or the bisulfite modified form thereof and detect methylation of SEQ ID NO:1 in the target DNA.
2. The method of claim 1 , wherein the at least one primer is specific for methylated sequences.
3. The method of claim 1 , wherein the at least one primer is not specific for methylated sequences.
4. The method of claim 1 , further comprising detecting methylation of one, two, three, four, five, six, or seven of an even-skipped homeobox 1 (EVX1) sequence, a cell line derived transforming sequence-like (MCF2L) sequence, a fibroblast growth factor 1 (FGF1) sequence, a wingless-type MMTV integration site family member 2 (WNT2) sequence, a natural cytotoxicity triggering receptor 2 (NCR2) sequence, an exostoses 1 (EXT1) sequence, and a sperm associated antigen 4 (SPAG4) sequence.
5. The method of claim 1 , wherein the at least one primer comprises a pair of primers complementary to SEQ ID NO:1 or the bisulfite modified form thereof, and wherein the reaction mixture further comprises a polymerase, a plurality of free nucleotides comprising adenine, thymine, cytosine, and guanine, reaction buffer, and MgCl 2 .
6. The method of claim 5 , wherein the reacting step comprises reacting the reaction mixture for a time and at a temperature sufficient to amplify at least a portion of SEQ ID NO:1 or the bisulfite modified form thereof.
7. The method of claim 5 , wherein the at least one primer comprises at least one of SEQ ID NOs:43-45.
8. The method of claim 1 , wherein the reacting step comprises a pyrosequencing reaction.
9. The method of claim 1 , wherein the at least one primer is biotinylated.
10. The method of claim 1 , further comprising detecting methylation of one, two, three, four, five, six, or seven of SEQ ID NOs:2-6, 18, and 39.
11. The method of claim 1 , wherein the target DNA is obtained from a prostate cancer patient.
12. The method of claim 1 , wherein the reacting comprises methylation specific quantitative polymerase chain reaction.
13. The method of claim 1 , wherein the reaction mixture comprises the bisulfite modified form of the target DNA.
14. The method of claim 13 , wherein the reacting comprises quantitative bisulfite sequencing.
15. The method of claim 1 , wherein the histologically normal prostate cells are obtained from a urine sample from a subject.
16. The method of claim 1 , wherein the target DNA is obtained from histologically normal prostate tissue of a subject.
17. The method of claim 1 , wherein the histologically normal prostate cells are obtained from a semen sample from the subject.
18. The method of claim 1 , wherein the reaction mixture further comprises at least one primer comprising at least one of SEQ ID NOs:46-60 and 88-93.