MATERIALS AND METHOD FOR ASSAYING FOR METHYLATION OF CpG ISLANDS ASSOCIATED WITH GENES IN THE EVALUATION OF CANCER

Abstract

Provided are methods, reagents, and kits for evaluating cancer, such as prostate cancer, in a subject. Disclosed methods of evaluating cancer include methods of diagnosing cancer, methods of prognosticating cancer and methods of assessing the efficacy of cancer treatment. The methods include assaying a biological sample for methylation of a CpG island associated with specified genes. Provided reagents and kits include primers suitable for amplifying at least a portion of a target CpG islands associated with specified genes.

1 . A method of determining the methylation status of one or more CpG islands indicative of prostate cancer in a human male undergoing prostate cancer evaluation, which method comprises:
 (a) isolating or amplifying genomic DNA from a biological sample from a human male undergoing prostate cancer evaluation; and   (b) assaying the genomic DNA for methylation of one or more CpG islands including a CpG island associated with the Ras association (RalGDS/AF-6) domain family 5 (RASSF5) gene,   wherein the presence of methylation of the one or more CpG islands, including the CpG island associated with RASSF5, is indicative of prostate cancer.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with nodal homolog (TGF-β signaling pathway) (NODAL), methyltransferase family member 1 (HEMK1), glutathione peroxidase 7 (GPX7), paladin (predicted protein tyrosine phosphatase) (PALD), kinesin family member 13B (KIF13B), kinesin family member C2 (KIFC2), or neurogenin 3 transcription factor (NEUROG3), and wherein the presence of methylation of the one or more CpG islands associated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, or NEUROG3 is indicative of prostate cancer. 
     
     
         3 . The method of  claim 2 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of CpG islands associated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, and NEUROG3, and wherein the presence of methylation of the CpG islands is indicative of prostate cancer. 
     
     
         4 . The method of  claim 2 , wherein the method comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands in SEQ ID NOS: 49 or 50 [NODAL], SEQ ID NO: 17 or 18 [HEMK1], SEQ ID NOs: 125 or 126 [GPX7], SEQ ID NO: 15 or 16 [PALD], SEQ ID NOs: 7 or 8 [KIF13B], SEQ ID NOs: 119 or 220 [KIFC2], or SEQ ID NOs: 141 or 142 [NEUROG3], and wherein the presence of methylation of the one or more CpG islands associated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, or NEUROG3 is indicative of prostate cancer. 
     
     
         5 . The method of  claim 1 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with at least one gene that is known to be methylated in prostate cancer and that is known not to be detectably methylated or methylated at a lower level in benign prostate hyperplasia (BPH), and wherein the presence or increased methylation of the assayed CpG islands is indicative of prostate cancer. 
     
     
         6 . The method of  claim 2 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with at least one gene that is known to be methylated in prostate cancer and that is known not to be detectably methylated or methylated at a lower level in benign prostate hyperplasia (BPH), wherein the presence or increased methylation of the assayed CpG islands is indicative of prostate cancer. 
     
     
         7 . The method of  claim 5 , wherein the one or more CpG islands associated with at least one gene that is known to be methylated in prostate cancer and that is known to be unmethylated or methylated at a lower level in BPH is or includes one or more CpG islands associated with glutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3), cyclin-dependent kinase inhibitor 1C(CDKN1C/p57), or G-protein coupled receptor 62 (GPR62). 
     
     
         8 . The method of  claim 7 , wherein the one or more CpG islands associated with at least one gene that is known to be methylated in prostate cancer and that is known to be unmethylated or methylated at a lower level in BPH includes a CpG island associated with glutathione S-transferase P1 (GSTP1). 
     
     
         9 . The method of  claim 1 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with L-threonine dehydrogenase (TDH), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), GDNF family receptor alpha 1 (GFRA1), Dickkopf homolog 2 (DKK2), tumor necrosis factor superfamily member 11 (TNFSF11), or leucine rich repeat containing 49 (LRRC49), and wherein the presence of methylation of the one or more CpG islands is indicative of prostate cancer. 
     
     
         10 . The method of  claim 5 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with L-threonine dehydrogenase (TDH), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), GDNF family receptor alpha 1 (GFRA1), Dickkopf homolog 2 (DKK2), tumor necrosis factor superfamily member 11 (TNFSF11), or leucine rich repeat containing 49 (LRRC49), and wherein the presence of methylation of the one or more CpG islands is indicative of prostate cancer. 
     
     
         11 . The method of  claim 9 , wherein the assaying for methylation of the at least one CpG island associated with a gene comprises amplifying a target sequence that includes at least one CpG island in one or more of (a), SEQ ID NOs: 35 or 36 [TDH], SEQ ID NOs: 43 or 44 [ASAH1], SEQ ID NOs: 123 or 124 [GFRA1], SEQ ID NOs: 129 or 130 [DKK2], SEQ ID NO: 196 [TNFSF11], and SEQ ID NO: 198 [LRRC49], and (b) fully or partially methylated sequences of (a). 
     
     
         12 . The method of  claim 9 , wherein the method comprises assaying for methylation of CpG islands associated with at least 7 genes. 
     
     
         13 . The method of  claim 9 , wherein the method comprises assaying for methylation of CpG islands associated with at least 8 genes. 
     
     
         14 . The method of  claim 9 , wherein the method comprises assaying for methylation of CpG islands associated with at least 9 genes. 
     
     
         15 . The method of  claim 1 , wherein the method further comprises assaying the isolated or amplified genomic DNA for methylation of one or more CpG islands associated with nodal homolog (TGF-β signaling pathway) (NODAL), methyltransferase family member 1 (HEMK1), glutathione peroxidase 7 (GPX7), paladin (predicted protein tyrosine phosphatase) (PALD), kinesin family member 13B (KIF13B), kinesin family member C2 (KIFC2), neurogenin 3 transcription factor (NEUROG3), glutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3), cyclin-dependent kinase inhibitor 1C (CDKN1C/p57), or G-protein coupled receptor 62 (GPR62), L-threonine dehydrogenase (TDH), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), GDNF family receptor alpha 1 (GFRA1), Dickkopf homolog 2 (DKK2), tumor necrosis factor superfamily member 11 (TNFSF11), and leucine rich repeat containing 49 (LRRC49), and wherein the presence of methylation of the one or more CpG islands associated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, NEUROG3, GSTP1, GPX3, CDKN1C/p57, GPR62, TDH, ASAH1, GFRA1, DKK2, TNFSF11, or LRRC49 is indicative of prostate cancer 
     
     
         16 . The method of  claim 1 , wherein the assaying for methylation of a CpG island comprises amplifying a target sequence that includes a CpG island in SEQ ID NO: 133 or SEQ ID NO: 134. 
     
     
         17 . The method of  claim 1 , wherein assaying the genomic DNA for methylation comprises terminator-coupled linear amplification. 
     
     
         18 . The method of  claim 1 , wherein assaying the genomic DNA for methylation comprises using methylation-sensitive restriction endonuclease. 
     
     
         19 . The method of  claim 1 , wherein assaying the genomic DNA for methylation comprises differential methylation hybridization. 
     
     
         20 . The method of  claim 1 , wherein the amplification of genomic DNA comprises methylation coupled genomic amplification. 
     
     
         21 . The method of  claim 1 , wherein assaying the genomic DNA for methylation comprises quantitative PCR. 
     
     
         22 . The method of  claim 1 , wherein assaying the genomic DNA for methylation comprises sequencing. 
     
     
         23 . The method of  claim 1 , wherein the biological sample is whole blood, blood plasma, or blood serum. 
     
     
         24 . The method of  claim 1 , wherein the biological sample is urine. 
     
     
         25 . The method of  claim 1 , wherein the biological sample is prostate tissue.