Assays to determine dna methylation and dna methylation markers of cancer

Abstract

Methods are provided for determining a genomic methylation profile in a DNA sample. In certain aspects, the methods can be used to determine if a subject has, or is at risk for developing, a bladder cancer or other cancers of the urinary tract. Methods for treatment of such subjects are likewise provided.

1 . A method for determining a genomic DNA methylation profile in a sample comprising:
 (a) obtaining a substantially purified test genomic DNA sample;   (b) contacting a portion test genomic DNA of the sample with:   a first reaction mixture comprising: (i) at least two methylation sensitive restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for polymerase chain reaction (PCR) amplification of at least a first, second and third different genomic region in the DNA sample; and (vi) fluorescent probes complementary to sequences in said first, second and third different genomic regions for quantitative detection of amplified sequences from the first, second and third different genomic regions, wherein each of the probes comprises a distinct fluorescent label, wherein:
 (I) the first genomic region is a cleavage control that is known to be unmethylated; 
 (II) the second genomic region is a copy number control that does not include any cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; and 
 (III) the third genomic region is a test region having an unknown amount of methylation and including at least three cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; 
   (c) subjecting the first reaction mixture to digestion and thermal cycling, while detecting fluorescent signals from the fluorescent probes, thereby performing real time PCR on the samples in the first and second reaction mixtures;   (d) using the detected fluorescent signals to determine the genomic DNA methylation profile in a sample.   
     
     
         2 . The method of  claim 1 , further comprising:
 (a) obtaining a substantially purified test genomic DNA sample;   (b) contacting a portion test genomic DNA of the sample with:   a first reaction mixture comprising: (i) at least two methylation sensitive restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for polymerase chain reaction (PCR) amplification of at least a first, second and third different genomic region in the DNA sample; and (vi) fluorescent probes complementary to sequences in said first, second and third different genomic regions for quantitative detection of amplified sequences from the first, second and third different genomic regions, wherein each of the probes comprises a distinct fluorescent label; and   a second reaction mixture, identical to the first reaction mixture, but lacking the at least two methylation sensitive restriction endonucleases, wherein:
 (I) the first genomic region is a cleavage control that is known to be unmethylated; 
 (II) the second genomic region is a copy number control that does not include any cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; and 
 (III) the third genomic region is a test region having an unknown amount of methylation and including at least three cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; 
   (c) subjecting the first and second reaction mixtures to digestion and thermal cycling, while detecting fluorescent signals from the fluorescent probes, thereby performing real time PCR on the samples in the first and second reaction mixtures;   (d) using the detected fluorescent signals to determine the genomic DNA methylation profile in a sample.   
     
     
         3 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the substantially purified test genomic DNA sample is obtained from a urine, stool, saliva, blood or tissue sample. 
     
     
         10 . The method of  claim 9 , wherein the substantially purified test genomic DNA sample is obtained from a biopsy sample. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the first reaction mixture comprises at least three methylation sensitive restriction endonucleases. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein step (b) further comprises:
 (b) contacting a portion test genomic DNA of the sample with:   a first reaction mixture comprising: (i) at least two methylation sensitive restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for polymerase chain reaction (PCR) amplification of at least a first, second, third and fourth different genomic region in the DNA sample; and (vi) fluorescent probes complementary to sequences in said first, second, third and fourth different genomic regions for quantitative detection of amplified sequences from the first, second, third and fourth different genomic regions, wherein each of the probes comprises a distinct fluorescent label, wherein:
 (I) the first genomic region is a cleavage control that is known to be unmethylated; 
 (II) the second genomic region is a copy number control that does not include any cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; and 
 (III) the third and fourth genomic regions are test regions having an unknown amount of methylation and including at least three cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture. 
   
     
     
         15 . The method of  claim 14 , wherein step (b) further comprises:
 (b) contacting a portion test genomic DNA of the sample with:   a first reaction mixture comprising: (i) at least two methylation sensitive restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for polymerase chain reaction (PCR) amplification of at least a first, second, third, fourth and fifth different genomic region in the DNA sample; and (vi) fluorescent probes complementary to sequences in said first, second, third, fourth and fifth different genomic regions for quantitative detection of amplified sequences from the first, second, third, fourth and fifth different genomic regions, wherein each of the probes comprises a distinct fluorescent label, wherein:
 (I) the first genomic region is a cleavage control that is known to be unmethylated; 
 (II) the second genomic region is a copy number control that does not include any cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture; and 
 (III) the third, fourth and fifth genomic regions are test regions having an unknown amount of methylation and including at least three cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture. 
   
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein at least 4, 5, 6, 7 or 8 cut sites for the methylation sensitive restriction endonucleases of the first reaction mixture. 
     
     
         18 . The method of  claim 1 , wherein the primer pairs are complementary to sequences no more than 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or 50 nucleotides apart. 
     
     
         19 . The method of  claim 1 , wherein the first genomic region is a genomic region of a housekeeping gene. 
     
     
         20 . The method of  claim 19 , wherein the housekeeping gene is GAPDH. 
     
     
         21 . The method of  claim 1 , wherein the second genomic region is a genomic region of the POLR2A gene. 
     
     
         22 . The method of  claim 1 , wherein the third genomic region is selected from the group provided in Table 1A of Table 2. 
     
     
         23 . The method of  claim 1 , wherein the third genomic region is selected from the group consisting of DMRTA2, EVX2, Unk21, OTX1, SOX1, SEPT9, Unk05, Unk09, GALR1, Unk07, Unk19, TBX15, EEF1A2, TFAP2B, DCHS2 and SOX17. 
     
     
         24 . The method of  claim 1 , wherein using the detected fluorescent signals to determine the genomic DNA methylation profile in a sample comprises calculating the relative methylation percentages for the sample. 
     
     
         25 . The method of  claim 1 , wherein the fluorescent probes and/or primer pairs are selected from those provided in Table 1C. 
     
     
         26 - 28 . (canceled) 
     
     
         29 . A method for determining a genomic DNA methylation profile in a sample comprising:
 (a) obtaining a test genomic DNA sample, which has been bisulfite converted;   (b) contacting the test sample with a first reaction mixture comprising: (i) a hot-start DNA polymerase; (ii) a pH buffered salt solution; (iii) dNTPs; (iv) DNA primer pairs for polymerase chain reaction (PCR) amplification of at least a first, and second different genomic region in the DNA sample, wherein the primer pairs are complementary to sequences no more than 200 nucleotides apart; and (v) fluorescent probes complementary to sequences in said first, and second different genomic regions for quantitative detection of amplified sequences from the first and second different genomic regions, wherein each of the probes comprises a distinct fluorescent label, wherein:
 (I) the first genomic region is a copy number control region that that does not comprise CpG dinucleotides; and 
 (II) the second genomic region is a test region having an unknown amount of methylation and including at least five CpG dinucleotides in sequences that are complementary to DNA primer pairs and the probe for the second genomic region; 
   (c) subjecting the first reaction mixtures to thermal cycling, while detecting fluorescent signals from the fluorescent probes, thereby performing real time PCR on the sample in the first reaction mixture; and   (d) using the detected fluorescent signals and fluorescent signal from a DNA methylation standard curve to determine the genomic DNA methylation profile in a sample.   
     
     
         30 - 58 . (canceled) 
     
     
         59 . A method of detecting the presence of, or an increased risk of, bladder cancer or other cancers of the urinary tract in a patient comprising determining a methylation status in one or more genomic regions in a patient sample selected from the group provided in Table 1A wherein an increased level of methylation in one or more of the genomic regions of Table 1A relative to a reference level indicates that the patient has or is at risk of developing bladder cancer. 
     
     
         60 . The method of  claim 59 , wherein the one or more genomic regions is selected from the group consisting of Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, and SOX17. 
     
     
         61 . The method of  claim 60 , wherein the one or more genomic regions is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the genomic regions selected from the group consisting of Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, and SOX17. 
     
     
         62 - 66 . (canceled) 
     
     
         67 . The method of  claim 59 , wherein said determining comprises determining a methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of said genomic regions. 
     
     
         68 . The method of  claim 59 , wherein said determining comprises determining a methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of said genomic regions, wherein the genomic regions are selected from the group consisting of Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, SOX17, GALR1, TBX15, EEF1A2, DMRTA2, and TFAP2B. 
     
     
         69 . The method of  claim 68 , wherein said determining comprises determining a methylation in each of the genomic regions: Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, SOX17, GALR1, TBX15, EEF1A2, DMRTA2, and TFAP2B. 
     
     
         70 . The method of  claim 59 , wherein the patient has been previously treated for or diagnosed with bladder cancer. 
     
     
         71 . The method of  claim 70 , further defined as method for detecting bladder cancer recurrence or a risk of bladder cancer recurrence. 
     
     
         72 . The method of  claim 59 , wherein said determining comprises analyzing DNA methylation in the sample using restriction endonuclease digestion and qPCR. 
     
     
         73 . The method of  claim 72 , wherein the digestion reaction is completed in a first step, followed by the qPCR reaction in a second step. 
     
     
         74 . The method of  claim 59  wherein the patient is a human. 
     
     
         75 . The method of  claim 59 , wherein the sample is a urine sample. 
     
     
         76 . The method of  claim 59 , wherein the sample is a blood sample. 
     
     
         77 - 78 . (canceled) 
     
     
         79 . The method of  claim 59 , wherein determining a methylation status comprises determining the nucleotide positions in the genomic regions that comprise methylation. 
     
     
         80 . The method of  claim 59 , wherein determining a methylation status comprises determining the proportion of methylation at nucleotide positions in the genomic region. 
     
     
         81 . The method of  claim 59 , wherein determining a methylation status comprises determining the proportion of nucleotide positions that are methylated in the genomic region. 
     
     
         82 . The method of  claim 59 , wherein the reference level is a level of methylation from a patient that does not have bladder cancer. 
     
     
         83 - 85 . (canceled) 
     
     
         86 . A method of detecting the presence of, or an increased risk of, bladder cancer in a patient comprising:
 (i) obtaining a patient sample;   (ii) determining a methylation status in one or more genomic regions selected from those in Table 1; and   (iii) identifying the presence of, or an increased risk of, bladder cancer in the patient based on an increased level of methylation in one or more of the genomic regions relative to a reference level.   
     
     
         87 - 94 . (canceled) 
     
     
         95 . A synthetic polynucleotide sequence comprising a sequence at least 90% identical to one of the probe sequences selected from those provided in Table 1B or 1C, wherein the polynucleotide is conjugated to a reporter molecule. 
     
     
         96 . The polynucleotide of  claim 95 , wherein the polynucleotide is a fluorophore. 
     
     
         97 . The polynucleotide of  claim 95 , comprising a sequence at least 95% identical to one of the probe sequences selected from those provided in Table 1B or 1C. 
     
     
         98 . The polynucleotide of  claim 95 , comprising a sequence identical to one of the probe sequences selected from those provided in Table 1B or 1C. 
     
     
         99 . A kit comprising at least two primer pairs and at least two probes for amplification and detection of a gene region selected from those provided in Table 1A. 
     
     
         100 . The kit of  claim 99 , comprising at least three, four or five two primer pairs and at least three, four or five probes for amplification and detection of a gene region selected from those provided in Table 1A. 
     
     
         101 . The kit of  claim 99 , wherein the least two primer pairs and at least two probes are selected from those provided in Table 1B or 1C. 
     
     
         102 . The kit of  claim 99 , further comprising one or more of the following: (i) instructions; (ii) reagents for real-time qPCR; (iii) a methylation sensitive endonuclease; and (iv) a control DNA sample.