Materials and methods 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 detecting the methylation status of one or more CpG islands indicative of cancer, which method comprises:
 providing a biological sample from a subject in need of cancer evaluation; and   assaying the biological sample for methylation of a CpG island associated with at least one gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), kinesin family member 13B (KIF13B), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (predicted protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), ring finger protein 180 (RNF180), EGFR-co-amplified and overexpressed (DKFZP564K0822 or ECOP), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein C8orf79 (C8orf79/FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC286097/EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), matrix metallopeptidase 9 (MMP9), leucine rich repeat containing 49 (LRRC49), tumor necrosis factor (ligand) superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), Kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), wherein methylation of the assayed CpG island is indicative of cancer.   
     
     
         2 . A method of detecting the methylation status of one or more CpG islands indicative of prostate cancer, which method comprises
 providing a biological sample from a male mammal in need of prostate cancer evaluation; and   assaying the biological sample for methylation of a CpG island associated with at least one gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), kinesin family member 13B (KIF13B), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), flbroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671 or RNF180), EGFR-co-amplified and overexpressed (DKFZP5640822 or ECOP), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-β signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), and transcription factor LIM homeodomain (ISL2), matrix metallopeptidase 9 (MMP9), leucine rich repeat containing 49 (LRRC49), tumor necrosis factor (ligand) superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), Kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), Ras association (RalGDS/AF-6) domain family 5 (RASSF5), and HtrA serine peptidase 4 (HTRA4); and   optionally, further assaying the biological sample for methylation of a CpG island 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 methylation of the assayed CpG island or islands is indicative of prostate cancer.   
     
     
         3 . The method of  claim 2 , wherein the method comprises:
 assaying the biological sample for methylation of CpG islands associated with at least three genes, at least one of the genes being selected from the group consisting of neuregulin cell-surface ligand (NRG1), kinesin family member 13B (KIF13B), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671 or RNF180), EGFR-co-amplified and overexpressed (DKFZP5640822 or ECOP), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097 or EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-β signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), and transcription factor LIM homeodomain (ISL2);   the CpG island associated with a gene that is known to be methylated in prostate cancer is also known to be not methylated in BPH or methylated at a level of about 10% or less in BPH; and   wherein methylation of the CpG islands associated with the at least three genes is indicative of prostate cancer;   
     
     
         4 . The method of  claim 2 , wherein the at least one CpG island 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 island associated with glutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3), glutathione S-transferase M1 (GSTM1), Cub and Sushi multiple domains1 (CSMD1), tumor necrosis factor receptor superfamily member 10C (TNFRSF10C), tumor necrosis factor receptor superfamily 10D (TNFRSF10D), secreted frizzled-related protein 1 (SFRP1), secreted frizzled-related protein 2 (SFRP2), dickkopf homolog 3 (DKK3), prostaglandin-endoperoxide synthase 2 (PTGS2), cyclin-dependent kinase inhibitor 1C (CDKN1C/p57), Ras association (RalGDS/AF-6) domain family 1 (RASSF1), G-protein coupled receptor 62 (GPR62). 
     
     
         5 . The method of  claim 4 , wherein the at least one CpG island 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 island associated with glutathione S-transferase P1 (GSTP1). 
     
     
         6 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 4 genes. 
     
     
         7 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 5 genes. 
     
     
         8 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 6 genes. 
     
     
         9 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 7 genes. 
     
     
         10 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 8 genes. 
     
     
         11 . The method of  claim 1 , wherein the method comprises assaying for methylation of CpG islands associated with at least 9 genes. 
     
     
         12 . The method of  claim 1 , wherein the assaying for methylation of a CpG island comprises amplifying a target sequence that includes at least one CpG dinucleotide in a target sequence selected from the group consisting of (a) SEQ ID NO: 1 and 2 [NRG1], SEQ ID NO: 3 and 4 [ADRB3], SEQ ID NO: 5 and 6 [GFRA2], SEQ ID NO: 7 and 8 [KIF13B], SEQ ID NO: 9 and 10 [RET], SEQ ID NO: 11 and 12 [GPR147], SEQ ID NO: 13 and 14 [NEUROG3], SEQ ID NO: 15 and 16 [PALD], SEQ ID NO: 17 and 18 [HEMK1], SEQ ID NO: 19 and 20 [FGF4], SEQ ID NO: 21 and 22 [GPR62], SEQ ID NO: 23 and 24 [HTR1A], SEQ ID NO: 25 and 26 [RNF180], SEQ ID NO: 27 and 28 [DKFZP5640822], SEQ ID NO: 29 and 30 [ZNF596], SEQ ID NO: 33 and 34 [LOC441320], SEQ ID NO: 35 and 36 [TDK], SEQ ID NO: 37 and 38 [FLJ36980], SEQ ID NO: 39 and 40 [FGF20], SEQ ID NO: 41 and 42 [EFHA2], SEQ ID NO: 43 and 44 [ASAH1], SEQ ID NO: 49 and 50 [NODAL], SEQ ID NO: 51 and 52 [LOC399783], SEQ ID NO: 53 and 54 [ISL2] and (b) fully or partially methylated derivatives of (a). 
     
     
         13 . The method of  claim 1 , wherein the assaying for methylation of a CpG island comprises amplifying a target sequence that includes at least one CpG dinucleotide or a deaminated derivative thereof in a target sequence selected from the group consisting of (a) SEQ ID NOS: 119 and 220 [KIFC2], SEQ ID NOS: 121 and 122 [C20ORF23], SEQ ID NOS: 123 and 124 [GFRA1], SEQ ID NOS: 129 and 130 [DKK2], SEQ ID NOS: 135 and 136 [NTN1], SEQ ID NO: 195 [RHOD], SEQ ID NO: 196[TNFSF11], SEQ ID NO: 197 [MMP9], and SEQ ID NO: 198 [LRRC49] (b) fully or partially methylated cytosine derivatives of (a), and (c) deaminated derivatives of (b). 
     
     
         14 . The method of  claim 2 , wherein the assaying for methylation of a CpG island comprises amplifying a target sequence that includes at least one CpG dinucleotide or a deaminated derivative thereof in a target sequence selected from the group consisting of (a) SEQ ID NOS: 119 and 220 [KIFC2], SEQ ID NOS: 121 and 122 [C20ORF23], SEQ ID NOS: 123 and 124 [GFRA1], SEQ ID NOS: 129 and 130 [DKK2], SEQ ID NOS: 135 and 136 [NTN1], SEQ ID NOS: 133 and 134 [RASSF5] and SEQ ID NOS: 193 and 194 [HTRA4], (b) fully or partially methylated cytosine derivatives of (a), and (c) deaminated derivatives of (b). 
     
     
         15 . The method of  claim 12 , wherein the assaying for methylation comprises performing terminator-coupled linear amplification in the presence of at least one dideoxynucleotide and determining the size of the amplified fragments to thereby determine the methylation status of the cytidine residues. 
     
     
         16 . The method of  claim 15 , wherein the assaying comprises performing linear amplification in the presence of only one dideoxynucleotide. 
     
     
         17 . The method of  claim 15 , wherein the assaying comprises performing linear amplification in the presence of only two dideoxynucleotides. 
     
     
         18 . The method of  claim 15 , wherein the at least one dideoxynucleotide is chosen from the group consisting of dideoxyadenine, dideoxcytidine, dideoxguanine, and dideoxthymidine. 
     
     
         19 . A terminator-coupled linear amplification method of determining the methylation status of a CpG island, wherein the method comprises
 (a) providing a DNA sample;   (b) incubating the DNA sample under deaminating conditions to thereby produce a deaminated DNA sample;   (c) optionally, purifying the deaminated DNA sample;   (d) amplifying a target sequence or target sequences that include one or more CpG islands or portions of one or more CpG islands to thereby produce one or more amplified target sequences;   (e) optionally, purifying the one or more amplified target sequences;   (f) linearly amplifying the one or more amplified target sequences of step (d) or (e) in the presence of a primer and 1 to 3 dideoxynucleotides to generate one or more fragments of different lengths, wherein each length corresponds to the distance in bases from the 5′ end of the primer to the position where the dideoxynucleotide was incorporated;   (g) optionally, purifying the one or more fragments; and   (h) analyzing the one or more fragments to determine their lengths and thereby determine the methylation status of methylated cytosines within the one or more amplified target sequences.   
     
     
         20 . A pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49). 
     
     
         21 . A set of primers comprising:
 (i) at least one pair of primers of  claim 20 ; and   (ii) at least one pair of primers suitable for amplifying at least a portion of a CpG island associated with at least one other gene that is known to be methylated in prostate cancer but not methylated or methylated at a low level in BPH.   
     
     
         22 . A set of primers comprising:
 (i) a first pair of primers of  claim 20 ; and   (ii) a second pair of primers of  claim 20 , wherein the second pair of primers is suitable for amplifying at least a portion of a different CpG island than the first pair of primers.   
     
     
         23 . The set of primers of  claim 22 , wherein the CpG island amplified by the first pair of primers is associated with a first gene, the CpG island amplified by the second pair of primers is associated with a second gene, and the first and second genes are different genes. 
     
     
         24 . The set of primers of  claim 21 , wherein the set further comprises a third pair of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), flbroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer but not methylated or methylated at a low level in BPH, wherein the third pair of primers is suitable for amplifying a different CpG island than that suited for amplification by the first or second pair of primers.   
     
     
         25 . The set of primers of  claim 24 , wherein the set further comprises a fourth pair of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer but not methylated or methylated at a low level in BPH, wherein the third pair of primers is suitable for amplifying a different CpG island than that suited for amplification by the first, second, or third pair of primers.   
     
     
         26 . The set of primers of  claim 25 , wherein the set further comprises a fifth pair of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), flbroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), flbroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer but not methylated or methylated at a low level in BPH, wherein the third pair of primers is suitable for amplifying a different CpG island than that suited for amplification by the first, second, third, or fourth pair of primers.   
     
     
         27 . The set of primers of  claim 26 , wherein the set further comprises a sixth pair of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), flbroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer, wherein the fourth pair of primers is suitable for amplifying a different CpG island than that suited for amplification by the first, second, third, fourth or fifth pair of primers.   
     
     
         28 . The set of primers of  claim 27 , wherein the set further comprises from one to four additional pairs of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer, wherein each pair of primers in the set amplifies a different CpG island.   
     
     
         29 . The set of primers of  claim 28 , wherein the set further comprises one or more additional pairs of primers selected from the group consisting of
 (i) a pair of primers suitable for amplifying at least a portion of one or more CpG-islands associated with a gene selected from the group consisting of neuregulin cell-surface ligand (NRG1), adrenergic B3 receptor (ADRB3), glycosylphosphatidylinositol cell-surface receptor (GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET), G-protein-coupled protein receptor 147 (GPR147), neurogenin 3 transcription factor (NEUROG3), paladin (protein tyrosine phosphatase) (PALD), methyltransferase family member 1 (HEMK1), flbroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1 A), ring finger protein 180 (LOC 285671) (RNF180), EGFR-co-amplified and overexpressed (ECOP) (DKFZP5640822), zinc finger protein 596 (ZNF596), similar to 7 transmembrane helix receptor (LOC441320), L-threonine dehydrogenase (TDH), hypothetical protein FLJ36980 (FLJ36980), flbroblast growth factor receptor 20 (FGF20), EF-hand domain family member 2A (LOC2 86097) (EFHA2), N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodal homolog (TGF-B signaling pathway) (NODAL), hypothetical protein similar to zinc finger protein 532 (LOC399783), transcription factor LIM homeodomain (ISL2), kinesin family member C2 (KIFC2), chromosome 20 open reading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9), tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog gene family member D (RHOD), and leucine rich repeat containing 49 (LRRC49), and   (ii) a pair of primers suitable for amplifying a CpG island known to be methylated in prostate cancer, wherein each pair of primers in the set amplifies a different CpG island.   
     
     
         30 . A pair of primers or set of primers of  claim 20 , wherein the pair or set of primers have sequences selected from the group consisting of SEQ ID NOs: 55 to 118. 
     
     
         31 . The primer or set of primers of  claim 20 , wherein the pair of primers or the set of primers includes two primers suitable for amplifying a CpG island associated with NRG1 or KIF13b. 
     
     
         32 . The primer or set of primers of  claim 20 , wherein the pair of primers or the set of primers includes two primers suitable for amplifying a CpG island associated with TDH, ASAH1, FGF20, HEMK1, PALD NEUROG, EFHA2, KIFC2, GFRA1, DKK2, TNFSF11, NTN1, or RHOD. 
     
     
         33 . A kit for detecting the methylation status of a CpG island indicative of cancer, the kit comprising:
 (i) a pair of primers or set of primers of  claim 20 ; and   (ii) instructions for using the primers to amplify at least one CpG island from a biological sample.   
     
     
         34 . The kit of  claim 33 , further comprising a buffer selected from the group consisting of (a) a buffer for amplifying genomic DNA, (b) a buffer for preparing genomic DNA from a biological sample, and (c) a buffer for preparing genomic DNA from plasma or urine.