METHODS AND COMPOSITIONS FOR DETECTING ESOPHAGEAL NEOPLASIAS AND/OR METAPLASIAS IN THE ESOPHAGUS
The disclosure provides methods for identifying genomic loci that are differentially/methylated in neoplastic cancers, e.g., esophageal cancers. Identification of methylated genomic loci, and optionally in combination with the identification of somatic mutations in TP53, has numerous uses, including for example, to characterize disease risk, to predict responsiveness to therapy, to non-invasively diagnose subjects and to treat subjects determined to have gastrointestinal neoplasias.
1 . A method of detecting the presence of an esophageal neoplasia or metaplasia in the esophagus in a human subject, comprising:
a) obtaining a human sample; and
b)
i. assaying said sample for the presence of methylation within a nucleotide sequence spanning one or more of any of the chromosomal loci having a sequence that is at least 90% identical to the sequence of any one or more of: SEQ ID NOs: 1-856, 2569-3424, 5137-5926; 7507-7558, 7663-7714, 7819-7866, 7963-7990, 8047-8074, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, or 8420-8424, or complements or fragments thereof;
ii. assaying said sample for the presence of DNA methylation by assay in a bisulfite converted DNA for retention of a cytosine base at any of the Y positions present in one or more of the nucleotide sequences having at least 90% identical to the sequence of any one or more of SEQ ID NOs: 857-1284, 1713-2140, 3425-3852, 4281-4708, 5927-6321, 6717-7111, 7559-7584, 7611-7636, 7715-7740, 7767-7792, 7867-7890, 7915-7938, 7991-8004, 8019-8032, 8075-8088, 8103-8116, 8157-8169, 8183-8195, 8223-8236, 8265-8278, 8307-8320, 8349-8362, 8410-8414 or 8425-8429;
iii. assaying said sample for the presence of methylation within a nucleotide sequence spanning one or more of any of the chromosomal loci having a sequence that is at least 90% identical to the sequence of any one or more of: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658, or complements or fragments thereof; or
iv. assaying said sample for the presence of DNA methylation by assay in a bisulfite converted DNA for retention of a cytosine base at any of the Y positions present in one or more of the nucleotide sequences having at least 90% identical to the sequence of any one or more of SEQ ID NOs: 8819-9004, 9191-9376, 9935-10120, 10307-10492, 10973-11119, 11267-11413, 11663-11713, 11765-11815, 11969-12019, 12071-12121, 12267-12313, 12361-12407, 12467-12472, 12479-12484, 12503-12508, 12515-12520, 12539-12544, 12551-12556, 12569-12574, 12587-12592, 12605-12610, 12623-12628, 12650-12652, or 12659-12661;
wherein methylation of said nucleotide sequence is indicative of an esophageal neoplasia or metaplasia in the esophagus.
2 - 25 . (canceled)
26 . A method of treating a subject having a metaplasia in the esophagus or neoplasia, comprising the step of treating the subject with chemotherapy, radiation therapy and/or with the resection of the neoplasia or metaplasia; and/or with ablation of the neoplasia or metaplasia; wherein said subject has been determined to have:
i.) methylation in a sequence that is at least 90% identical to the sequence of any one or more of: SEQ ID NOs: 1-856, 2569-3424, 5137-5926; 7507-7558, 7663-7714, 7819-7866, 7963-7990, 8047-8074, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, or 8420-8424, or complements or fragments thereof;
ii) DNA methylation by assay in a bisulfite converted DNA for retention of a cytosine base of one or more of the Y positions present in one or more of the nucleotide sequences having at least 90% identity to the sequence of any one or more of: SEQ ID NOs: 857-1284, 1713-2140, 3425-3852, 4281-4708, 5927-6321, 6717-7111, 7559-7584, 7611-7636, 7715-7740, 7767-7792, 7867-7890, 7915-7938, 7991-8004, 8019-8032, 8075-8088, 8103-8116, 8157-8169, 8183-8195, 8223-8236, 8265-8278, 8307-8320, 8349-8362, 8410-8414 or 8425-8429;
iii) methylation within a nucleotide sequence spanning one or more of any of the chromosomal loci having a sequence that is at least 90% identical to the sequence of any one or more of: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658, or complements or fragments thereof; or
iv) methylation of a DNA molecule by assay in a bisulfite converted DNA for retention of a cytosine base of one or more of the Y positions present in one or more of the nucleotide sequences having at least 90% identity to the sequence of any one or more of: SEQ ID NOs: 8819-9004, 9191-9376, 9935-10120, 10307-10492, 10973-11119, 11267-11413, 11663-11713, 11765-11815, 11969-12019, 12071-12121, 12267-12313, 12361-12407, 12467-12472, 12479-12484, 12503-12508, 12515-12520, 12539-12544, 12551-12556, 12569-12574, 12587-12592, 12605-12610, 12623-12628, 12650-12652, or 12659-12661.
20 . (canceled) and order these in pair
31 . A bisulfite converted sequence: p 1 a) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one or more of SEQ ID NOs: 857-2568, 3425-5136, 5927-7506, 7559-7662, 7715-7818, 7867-7962, 7991-8046, 8075-8130, 8157-8208, 8223-8250, 8265-8292, 8307-8334, 8349-8376, 8410-8419, 8425-8434, 8819-9562, 9935-10678, 10973-11560; 11663-11866, 11969-12172, 12267-12454; 12467-12490, 12503-12526, 12539-12562, 12569-12580, 12587-12598, 12605-12616, 12623-12634, 12650-12655, or 12659-12664 and/or fragments thereof, and/or the reverse complements thereof including all unique fragments of these sequences and their reverse complements; or
b) comprising the bisulfite-converted nucleotide sequence of any one of the following: Up3, Up10, Up15-1, Up15-2, Up20-1, Up20-2, Up20-2, Up27, Up2, SqBE2, SqBE5, SqBE7, SqBE9, SqBE10, SqBE11-1, SqBE11-2, SqBE13, SqBE14-2, SqBE15, SqBE16-1, SqBE16-2, SqBE17-1, SqBE18, SqBE22-1, SqBE22-2 or SqBE23.
32 . A panel of bisulfite converted sequences selected from the sequences of claim 31 .
33 . The panel of claim 32 , wherein the panel corresponds to any combination of the sequences of SEQ ID NOs: 8307-8334, 8349-8376, 8410-8419, 8425-8434, 12605-12616, 12623-12634, 12650-12655, or 12659-12664.
34 . An oligonucleotide primer that hybridizes to any of the sequences of claim 31 .
35 . (canceled)
36 . The primers of claim 34 , wherein said primers are combined as forward and reverse primers for PCR amplification of any of the bisulfite converted sequences of claim 31 .
37 . (canceled)
38 . A panel of primer pairs selected from the primer pairs of claim 36 .
39 . The panel of claim 38 , wherein the panel corresponds to a combination of primer pairs for amplifying any combination of SEQ ID NOs: 8307-8334, 8349-8376, 8410-8419 or 8425-8434.
40 - 97 . (canceled)
98 . The bisulfite-converted nucleotide sequence of claim 31 , comprising the bisulfite-converted nucleotide sequence of any one of the following: Up3, Up10, Up15-1, Up15-2, Up20-1, Up20-2, Up20-2, Up27, Up35-1, Up35-2, SqBE2, SqBE5, SqBE7, SqBE9, SqBE10, SqBE11-1, SqBE11-2, SqBE13, SqBE14-2, SqBE15, SqBE16-1, SqBE16-2, SqBE17-1, SqBE18, SqBE22-1, SqBE22-2 or SqBE23.
99 - 100 . (canceled)
101 . The bisulfite-converted nucleotide sequence of claim 31 , comprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 8307-8313, 8315-8327, 8329-8334, 8349-8355, 8357-8369, 8371-8376, 8410, 8411, 8412, 8414, 8415, 8416, 8417, 8419, 8425, 8426, 8427, 8429, 8430, 8431, 8432, 8434, 12605-12616, 12623-12634, 12650-12655, or 12659-12664.
102 . The bisulfite-converted nucleotide sequence of claim 101 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 8307-8313, 8315-8327, 8329-8334, 8349-8355, 8357-8369, 8371-8376, 8410, 8411, 8412, 8414, 8415, 8416, 8417, 8419, 8425, 8426, 8427, 8429, 8430, 8431, 8432, or 8434.
103 . The bisulfite-converted nucleotide sequence of claim 101 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 12605-12616, 12623-12634, 12650-12655, or 12659-12664.
104 . The bisulfite-converted nucleotide sequence of claim 103 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 12652, 12655, 12661, or 12664.
105 . The bisulfite-converted nucleotide sequence of claim 31 , wherein the sequence is a bisulfite-converted sequence of any one of SEQ ID NOs: 8220, 8262, 8304, or 8346.
106 . The bisulfite-converted nucleotide sequence of claim 31 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 8234, 8276, 8318, 8360, 8248, 8290, 8332 or 8374.
107 . The bisulfite-converted nucleotide sequence of claim 31 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to either SEQ ID NO: 8318 or 8360.
108 . The bisulfite-converted nucleotide sequence of claim 31 , wherein the sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to either SEQ ID NO: 8332 or 8374.
109 . The oligonucleotide primer of claim 34 , wherein the primer has a nucleotide sequence that is at least 90% identical to either SEQ ID NO: 8388 or 8402, or fragments or complements thereof.
110 . The primer pair of claim 36 , wherein the primer pair comprises a first nucleotide sequence that is at least 90% identical to SEQ ID NO: 8388, or fragments or complements thereof; and wherein the primer pair comprises a second nucleotide sequence that is at least 90% identical to SEQ ID NO: 8402.