Dna methylation markers for metastatic prostate cancer

Abstract

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for assessing prostate cancer. In a specific embodiment, present inventors have developed and applied a new technology and associated computation methods enabling simultaneous genome-scale analysis of genetic (copy number) and epigenetic (total methylation (TM) and allele-specific methylation (ASM) alternation, This method, called MBD-SNP, features affinity enrichment or methylated genomic DNA fragments using a methyl-binding domain polypeptide.

1 . A method for enabling simultaneous genome-scale analysis of genetic and epigenetic alterations in an individual comprising the steps of:
 a. digesting a first genomic DNA sample from the individual with a first restriction enzyme;   b. digesting a second genomic DNA sample from the individual with a second restriction enzyme (total input fraction);   c. ligating digested ends of the resulting genomic DNA fragments from steps (a) and (b) with adaptors;   d. enriching the genomic DNA fragments from step (a) for methylated DNA fragments with a methylation-binding domain polypeptide (enriched methylated fraction);   e. amplifying the total input fraction and the enriched methylated fraction using polymerase chain reaction (PCR);   f labeling the amplified total input fraction and the enriched methylated fraction;   g. hybridizing the amplified total input fraction and the enriched methylated fraction to a single nucleotide polymorphism (SNP) microarray; and   h. analyzing the enriched methylated fraction and total input fraction to assess total methylation (TM) and allele-specific methylation (ASM).   
     
     
         2 . The method of  claim 1 , wherein the SNP microarray comprises copy number probes to allow for normalization of copy number and probe effects. 
     
     
         3 . The method of  claim 1 , wherein the SNP microarray is the Affymetrix® SNP 6.0 high-density oligonucleotide microarray, wherein the first restriction enzyme is Nsp I and wherein the second restriction enzyme is Sty I. 
     
     
         4 . The method of  claim 1 , wherein the SNP microarray is the Affymetrix® SNP 6.0 high-density oligonucleotide microarray, wherein the first restriction enzyme is Sty I and wherein the second restriction enzyme is Nsp I. 
     
     
         5 . The method of  claim 1 , wherein the methylation-binding domain polypeptide is from MBD2 (MBD2-MBD). 
     
     
         6 . The method of  claim 1 , wherein TM and ASM are calculated for regions of the genome with ≥2.5% CpG density. 
     
     
         7 . The method of  claim 1 , wherein the individual has cancer. 
     
     
         8 . The method of  claim 1 , wherein the individual has prostate cancer. 
     
     
         9 . The method of  claim 8 , further comprising correlating the TM and ASM methylation data with gene expression profiling data from other individuals with prostate cancer and non-prostate cancer to identify potential biomarkers. 
     
     
         10 . The method of  claim 1 , wherein the genomic DNA sample is taken from a primary tumor. 
     
     
         11 . The method of  claim 1 , wherein the genomic DNA sample is taken from a metatstatic tumor. 
     
     
         12 . The method of  claim 1 , wherein the method is performed separately on genomic DNA samples taken from a primary tumor and one or more metatstatic tumors. 
     
     
         13 . A method for diagnosing prostate cancer in an individual comprising the steps of:
 a. isolating DNA from a biological sample taken from the individual;   b. contacting the DNA with a primer specific for a SNP biomarker of prostate cancer to form a DNA:primer complex, wherein the SNP biomarker of prostate cancer is a SNP located in the promoter region of one or more genes selected from the group consisting of EYA4, ADAMTS12, CYP27A1, GSTP1, and ANKRD7;   c. amplifying the DNA:primer complex using methylation-specific PCR;   d. identifying the individual as having prostate cancer if the biomarkers are hypermethylated relative to a control.   
     
     
         14 . (canceled) 
     
     
         15 . A method for diagnosing prostate cancer in an individual comprising the steps of:
 a. isolating DNA from a biological sample taken from the individual;   b. contacting the DNA with a primer specific for a SNP biomarker of prostate cancer to form a DNA:primer complex, wherein the SNP biomarker is a SNP located in the promoter region of one or more genes selected from the group consisting of ALPL; EN1; PTGS2; NHLH2; NRAS; ALOX5; GST02; NKX2-3; BUB3; PAX5; NTRK2; ABCC8; CALCA; BDNF; MIR675,H19; WT1-AS; CD44; TDP1; C13 ORF 143; LOC400236; FOXN3; GSTP1; ESR2; SPA17 SIAE; SSTR1; SESN3; NFATC4; BCAT1; SSPN; HOXC13; SLC5A8; POU 4F1; PT PRR; LGR5; ZNF268; COL2A1; ESD; CDX2; SEPT9; NR0B1; OLIG2; ADAMTS51; CYP251; DPEP1; WFDC1; IRF8; HNF1B; MT1A MT1DP; IL21R; HS3ST2; TU BGCP4 ZSCAN29; SLC26A4.LOC28600_2; SULF1; EXT1; SOX17; FOXE1; TNFRSF10C; TNFRSF10D; SCIN; IGFBP3; CNR1; PT PN3; EYA4; SGK1; ESR1; ISL1; ADAMTS12; APC; PCDH10; WNTSA; CACNA2D3; SLIT2; NNT; UGT3A1; SV2C; CDO1; CRHBP; RASGRF2; and MIR9-2;   c. amplifying the DNA:primer complex using methylation-specific PCR;   d. identifying the individual as having prostate cancer if the biomarkers are hypermethylated relative to a control.   
     
     
         16 . The method of  claim 13 , wherein the control is ACTB.