Methods and compositions for detecting esophageal neoplasias and/or metaplasias in the esophagus
The disclosure provides methods for identifying genomic loci (e.g., vimentin and/or SqBE18) that are differentially methylated in metaplasias (e.g., Barrett's esophagus) and/or neoplastic cancers (e.g., esophageal cancers). Identification of methylated genomic loci 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 metaplasias and/or neoplasias.
1. A method of identifying a human subject for treatment of esophageal neoplasia or metaplasia and treating, comprising:
providing an esophageal sample obtained from the subject and treating with bisulfite to generate a plurality of bisulfite converted SqBE18 nucleic acid sequences;
amplifying the plurality of bisulfite converted SqBE18 nucleic acid sequences to generate amplicons for each bisulfite converted SqBE18 nucleic acid, wherein each amplicon is a read and wherein the amplicon comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332, and/or 8374, or a fragment thereof, said fragment comprising at least 50 nucleotides in length;
identifying methylated reads by measuring the number of methylated cytosines of the CpG dinucleotides in each amplicon of the bisulfite converted SqBE18 nucleic acid sequence, wherein a read is classified as a methylated read when at least 70% of the cytosines in the CpG dinucleotides of the individual amplicon of the bisulfite converted SqBE18 nucleic acid sequence is methylated;
calculating a percentage of total reads that are methylated reads from the amplifying and measuring steps, wherein the esophageal sample obtained from the subject has at least 0.1% of the total reads that are methylated reads; and,
administering to the human subject having an esophageal sample with at least 0.1% of the total reads that are methylated reads, cryotherapy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, endoscopic mucosal resection (EMR), esophagectomy, anti-reflux surgery, and/or a therapeutic agent selected from a proton pump inhibitor, a Histamine H2 receptor blocking agents, an anti-reflux medication, a drug that moves food thru the gastrointestinal tract more quickly, carboplatin and paclitaxel; cisplatin and 5-fluorouracil (5-FU); ECF: epirubicine, cisplatin, and 5-FU; DCF: docetaxel, cisplatin, and 5-FU; Cisplatin with capecitabine; oxaliplatin and either 5-FU or capecitabine; doxorubicin, bleomycin, mitomycin, methotrexate, vinorelbine, topotecan, and irinotecan, trastuzumab, and/or ramucirumab.
2. The method of claim 1 , wherein the human subject is administered a therapeutic agent selected from a proton pump inhibitor, a Histamine H2 receptor blocking agents, an anti-reflux medication, a drug that moves food thru the gastrointestinal tract more quickly, carboplatin and paclitaxel; cisplatin and 5-fluorouracil (5-FU); ECF: epirubicine, cisplatin, and 5-FU; DCF: docetaxel, cisplatin, and 5-FU; Cisplatin with capecitabine; oxaliplatin and either 5-FU or capecitabine; doxorubicin, bleomycin, mitomycin, methotrexate, vinorelbine, topotecan, and irinotecan, trastuzumab, and/or ramucirumab.
3. The method of claim 1 , wherein the endoscopic mucosal resection (EMR), esophagectomy, and/or anti-reflux surgery is performed on the human subject.
4. The method of claim 1 , wherein the sequence of the bisulfite converted nucleic acid sequences is determined by next-generation sequencing.
5. The method of claim 1 , wherein the amplified portion comprises 21 dinucleotides that correspond to or are derived from 21 CpG dinucleotides present in the native non-bisulfite converted SqBE18 genomic sequence.
6. The method of claim 1 , wherein primers used to amplify the portion of the bisulfite converted SqBE18 nucleic acid sequence comprise SEQ ID NOs: 8388 and/or 8402.
7. The method of claim 1 , wherein the amplified portion comprises the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332 and/or 8374.
8. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when 1% to 3.5% of the reads are classified as methylated reads.
9. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when 1% to 3.11% of the reads are classified as methylated reads.
10. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when at least 3.11% of the reads are classified as methylated reads.
11. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when 0.5% to 3.5% of the reads are classified as methylated reads.
12. The method of claim 1 , wherein the amplicon comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332 and/or 8374.
13. A method for detecting methylation status of an esophageal sample from a human subject suspected of having esophageal neoplasia or metaplasia, comprising:
providing an esophageal sample obtained from the subject and treating with bisulfite to generate a plurality of bisulfite converted SqBE18 nucleic acid sequences;
amplifying the plurality of bisulfite converted SqBE18 nucleic acid sequences to generate amplicons for each bisulfite converted SqBE18 nucleic acid, wherein each amplicon is a read and wherein the amplicon comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332, and/or 8374, or a fragment thereof, said fragment comprising at least 50 nucleotides in length;
measuring the number of methylated cytosines of the CpG dinucleotides in each amplicon of the bisulfite converted SqBE18 nucleic acid sequence, wherein a read is classified as a methylated read when at least 70% of the cytosines in the CpG dinucleotides of the individual amplicon of the bisulfite converted SqBE18 nucleic acid sequence is methylated;
calculating a percentage of total reads that are methylated reads from the amplifying and measuring steps, wherein the esophageal sample obtained from the human subject has at least 0.1% of the total reads that are methylated reads, whereby the esophageal sample obtained from the subject is classified as a methylated sample; and
administering an endoscopy to the human subject with a methylated sample.
14. A method of diagnosing a subject that has an esophageal neoplasia or metaplasia, comprising:
providing an esophageal sample obtained from the subject and treating with bisulfite to generate a plurality of bisulfite converted SqBE18 nucleic acid sequences;
amplifying the plurality of bisulfite converted SqBE18 nucleic acid sequences to generate amplicons for each bisulfite converted SqBE18 nucleic acid, wherein each amplicon is a read and wherein the amplicon comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332, and/or 8374, or a fragment thereof, said fragment comprising at least 50 nucleotides in length;
wherein primers used to amplify the portion of the bisulfite converted SqBE18 nucleic acid sequence comprise SEQ ID NOs: 8388 and/or 8402;
measuring the number of methylated cytosines of the CpG dinucleotides in each amplicon of the bisulfite converted SqBE18 nucleic acid sequence, wherein a read is classified as a methylated read when at least 70% of the cytosines in the CpG dinucleotides of the individual amplicon of the bisulfite converted SqBE18 nucleic acid sequence are methylated; and,
calculating a percentage of total reads that are methylated reads from the amplifying and measuring steps wherein the sample has at least 0.1% of the total reads that are methylated reads, whereby the subject is diagnosed with esophageal neoplasia or metaplasia.