Gene methylation in cancer diagnosis

Abstract

The present invention provides DNA biomarker sequences that are differentially methylated in samples from normal individuals and individuals with cancer. The invention further provides methods of identifying differentially methylated DNA biomarker sequences and their use the detection and diagnosis of cancer.

1 . A method for determining the methylation status of an individual, the method comprising:
 obtaining a biological sample from an individual; and   determining the methylation status of at least one cytosine within a DNA region in a sample from the individual where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 436, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485.   
     
     
         2 . The method of  claim 1 , wherein the determining step comprises determining the methylation status of at least one cytosine in the DNA region corresponding to a nucleotide in a biomarker, wherein the biomarker is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, and 388. 
     
     
         3 . The method of  claim 2 , wherein the determining step comprises determining the methylation status of the DNA region corresponding to the biomarker. 
     
     
         4 . The method of  claim 1 , wherein the sample is from blood serum, blood plasma, urine, sputum, or tissue biopsy. 
     
     
         5 . The method of  claim 1 , wherein the methylation status of at least one cytosine is compared to the methylation status of a control locus. 
     
     
         6 . The method of  claim 5 , wherein the control locus is an endogenous control. 
     
     
         7 . The method of  claim 5 , wherein the control locus is an exogenous control. 
     
     
         8 . The method of  claim 1 , wherein the determining step comprises determining the methylation status of at least one cytosine in at least two DNA regions. 
     
     
         9 . A method for determining the presence or absence of cancer in an individual, the method comprising:
 a) determining the methylation status of at least one cytosine within a DNA region in a sample from the individual where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 436, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485;   b) comparing the methylation status of the at least one cytosine to a threshold value for the at least one cytosine, wherein the threshold value distinguishes between individuals with and without cancer, wherein the comparison of the methylation status to the threshold value is predictive of the presence or absence of cancer in the individual.   
     
     
         10 . The method of  claim 9 , wherein the determining step comprises determining the methylation status of at least one cytosine in the DNA region corresponding to a nucleotide in a biomarker, wherein the biomarker is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, and 388. 
     
     
         11 . The method of  claim 10 , wherein the determining step comprises determining the methylation status of the DNA region corresponding to the biomarker. 
     
     
         12 . The method of  claim 9 , wherein the sample is from blood serum, blood plasma, or a biopsy. 
     
     
         13 . The method of  claim 9 , wherein the methylation status of at least one biomarker from the list is compared to the methylation value of a control locus. 
     
     
         14 . The method of  claim 13 , wherein the control locus is an endogenous control. 
     
     
         15 . The method of  claim 13 , wherein the control locus is an exogenous control. 
     
     
         16 . The method of  claim 9 , wherein the determining step comprises determining the methylation status of at least one cytosine from at least two DNA regions. 
     
     
         17 . A computer-implemented method for determining the presence or absence of cancer in an individual, the method comprising:
 receiving, at a host computer, a methylation value representing the methylation status of at least one cytosine within a DNA region in a sample from the individual where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485; and   comparing, in the host computer, the methylation value to a threshold value, wherein the threshold value distinguishes between individuals with and without cancer, wherein the comparison of the methylation value to the threshold value is predictive of the presence or absence of cancer in the individual.   
     
     
         18 . The method of  claim 17 , wherein the receiving step comprises receiving at least two methylation values, the two methylation values representing the methylation status of at least one cytosine biomarkers from two different DNA regions; and
 the comparing step comprises comparing the methylation values to one or more threshold value(s) wherein the threshold value distinguishes between individuals with and without cancer, wherein the comparison of the methylation value to the threshold value is predictive of the presence or absence of cancer in the individual.   
     
     
         19 . A computer program product for determining the presence or absence of cancer in an individual, the computer readable product comprising:
 a computer readable medium encoded with program code, the program code including:
 program code for receiving a methylation value representing the methylation status of at least one cytosine within a DNA region in a sample from the individual where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485; and 
 program code for comparing the methylation value to a threshold value, wherein the threshold value distinguishes between individuals with and without cancer, wherein the comparison of the methylation value to the threshold value is predictive of the presence or absence of cancer in the individual. 
   
     
     
         20 . A kit for determining the methylation status of at least one biomarker, the kit comprising:
 (1) a pair of polynucleotides capable of specifically amplifying at least a portion of a DNA region where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485; and   a methylation-dependent or methylation sensitive restriction enzyme and/or sodium bisulfite; or   (2) sodium bisulfite, primers and adapters for whole genome amplification, and polynucleotides to quantify the presence of the converted methylated and or the converted unmethylated sequence of at least one cytosine from a DNA region that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485; or   (3) methylation sensing restriction enzymes, primers and adapters for whole genome amplification, and polynucleotides to quantify the number of copies of at least a portion of a DNA region where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485; or   (4) a methylation sensing binding moiety and polynucleotides to quantify the number of copies of at least a portion of a DNA region where the DNA region is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, and 485.   
     
     
         21 . The kit of  claim 20 , wherein the pair of polynucleotides are capable of specifically amplifying a biomarker is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, and 388. 
     
     
         22 . The kit of  claim 20 , wherein the kit comprises at least two pairs of polynucleotides, wherein each pair is capable of specifically amplifying at least a portion of a different DNA region. 
     
     
         23 . The kit of  claim 20 , wherein the kit further comprises a detectably labeled polynucleotide probe that specifically detects the amplified biomarker in a real time amplification reaction.