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 diagnosing whether a subject has an esophageal neoplasia or metaplasia, comprising:
obtaining a sample from a subject;
measuring the amount of methylated cytosines in CpG dinucleotides in a vimentin nucleic acid sequence, or portion thereof, obtained from the sample; wherein if at least 80% of the cytosines in CpG dinucleotides in the vimentin nucleic acid sequence, or portion thereof, are methylated, then the vimentin nucleic acid sequence, or portion thereof, is considered a methylated read; and
measuring the number of methylated reads present in the sample;
wherein if at least 1% of the vimentin nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
2 . The method of claim 1 , wherein the vimentin nucleic acid sequences from the sample are treated with bisulfite.
3 . The method of claim 2 , wherein the sequence of the bisulfite converted nucleic acid sequences is determined by next-generation sequencing.
4 . The method of claim 1 , wherein the level of methylated cytosines is determined in an amplified portion of the vimentin nucleic acid sequence obtained from the subject.
5 . The method of claim 4 , wherein between the amplification primers the amplified portion comprises 10 dinucleotides that correspond to or are derived from 10 CpG dinucleotides present in the native non-bisulfite treated vimentin genomic sequence.
6 . The method of claim 5 , wherein the primers used to amplify the portion of the vimentin nucleic acid sequence comprise SEQ ID NOs: 16209 and 16210.
7 . The method of claim 4 , wherein the amplified portion comprises the nucleotide sequence of SEQ ID NOs: 16207 and/or 16208.
8 . The method of claim 1 , wherein if at least 1.05% of the vimentin nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
9 . The method of claim 1 , wherein if at least 3% of the vimentin nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
10 . The method of claim 1 , wherein if at least 5% of the vimentin nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
11 . The method of claim 1 , wherein if the subject is determined to have an esophageal neoplasia or metaplasia, then the method further comprises administering to the subject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, surgery, and/or a therapeutic agent.
12 . A method of treating a subject having an esophageal neoplasia or metaplasia, wherein it has been previously determined that at least 1% of the vimentin nucleic acid sequences, or portions thereof, in a sample from the subject have at least 80% of the CpG dinucleotides methylated, wherein the method comprises administering to the subject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, surgery, and/or a therapeutic agent.
13 . The method of claim 12 , wherein the therapeutic agent is 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 (Taxol®); cisplatin and 5-fluorouracil (5-FU); ECF: epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel (Taxotere®), cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
14 . The method of claim 12 , wherein the surgery is endoscopic mucosal resection (EMR), esophagectomy, and/or anti-reflux surgery.
15 . The method of claim 5 , wherein the 10 CpGs correspond to those that, after bisulfite treatment, are included in SEQ ID Nos: 16211 and 16212.
16 - 36 . (canceled)
37 . A method of diagnosing whether a subject has an esophageal neoplasia or metaplasia, comprising:
obtaining a sample from a subject by means of a brushing;
measuring the amount of methylated cytosines in CpG dinucleotides in a vimentin nucleic acid sequence, or portion thereof, obtained from the sample; wherein if at least 80% of the cytosines in CpG dinucleotides in the vimentin nucleic acid sequence, or portion thereof, are methylated, then the vimentin nucleic acid sequence, or portion thereof, is considered a vimentin methylated read;
measuring the amount of methylated cytosines in CpG dinucleotides in an SqBE18 nucleic acid sequence, or portion thereof, obtained from the sample; wherein if at least 70% or 75% of the cytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, or portion thereof, are methylated, then the SqBE18 nucleic acid sequence, or portion thereof, is considered an SqBE18 methylated read; and
measuring the number of methylated reads present in the sample;
wherein if at least 1% of the vimentin nucleic acid sequences, or portions thereof, in the sample are vimentin methylated reads, and wherein if at least 3% of the SqBE18 nucleic acid sequences, or portions thereof, in the sample are SqBE18 methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
38 . The method of claim 37 , wherein the vimentin and SqBE18 nucleic acid sequences from the sample are treated with bisulfite.
39 . The method of claim 38 , wherein the sequence of the bisulfite converted nucleic acid sequences is determined by next-generation sequencing.
40 . The method of claim 37 , wherein the level of methylated cytosines is determined in an amplified portion of the vimentin nucleic acid sequence and in an amplified portion of the SqBE18 nucleic acid sequence obtained from the subject.
41 . The method of claim 40 , wherein the amplified portion of the SqBE18 nucleic acid sequence comprises 21 dinucleotides that correspond to or are derived from 21 CpG dinucleotides present in the native non-bisulfite treated SqBE18 genomic sequence.
42 . The method of claim 40 or 41 , wherein the amplified portion of the vimentin nucleic acid sequence comprises 10 dinucleotides that correspond to or are derived from 10 CpG dinucleotides present in the native non-bisulfite treated vimentin genomic sequence.
43 . The method of claim 37 , wherein if at least 1% of the vimentin nucleic acid sequences, or portions thereof, in the sample are vimentin methylated reads, wherein if at least 3.11% of the SqBE18 nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
44 . The method of claim 37 , wherein if the subject is determined to have an esophageal neoplasia or metaplasia, then the method further comprises administering to the subject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, surgery, and/or a therapeutic agent.
45 . A method of treating a subject having an esophageal neoplasia or metaplasia, wherein it has been previously determined that at least 1% of the vimentin nucleic acid sequences, or portions thereof, in a brushing sample from the subject have at least 80% of the CpG dinucleotides methylated, wherein it has been previously determined that at least 3% of the SqBE18 nucleic acid sequences, or portions thereof, in a brushing sample from the subject have at least 75% of the CpG dinucleotides methylated, and wherein the method comprises administering to the subject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, surgery, and/or a therapeutic agent.
46 . The method of claim 45 , wherein the therapeutic agent is 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 (Taxol®); cisplatin and 5-fluorouracil (5-FU); ECF: epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel (Taxotere®), cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
47 . The method of claim 45 , wherein the surgery is endoscopic mucosal resection (EMR), esophagectomy, and/or anti-reflux surgery.
48 . A method of diagnosing whether a subject has an esophageal neoplasia or metaplasia, comprising:
obtaining a sample from a subject by means of a balloon;
measuring the amount of methylated cytosines in CpG dinucleotides in a vimentin nucleic acid sequence, or portion thereof, obtained from the sample; wherein if at least 80% of the cytosines in CpG dinucleotides in the vimentin nucleic acid sequence, or portion thereof, are methylated, then the vimentin nucleic acid sequence, or portion thereof, is considered a vimentin methylated read
measuring the amount of methylated cytosines in CpG dinucleotides in an SqBE18 nucleic acid sequence, or portion thereof, obtained from the sample; wherein if at least 70% or at least 75% of the cytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, or portion thereof, are methylated, then the SqBE18 nucleic acid sequence, or portion thereof, is considered a SqBE18 methylated read; and
measuring the number of methylated reads present in the sample;
wherein if at least 0.95% of the vimentin nucleic acid sequences, or portions thereof, in the sample are vimentin methylated reads, and wherein if at least 0.1% of the SqBE18 nucleic acid sequences, or portions thereof, in the sample are SqBE18 methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
49 . The method of claim 48 , wherein the vimentin and SqBE18 nucleic acid sequences from the sample are treated with bisulfite.
50 . The method of claim 49 , wherein the sequence of the bisulfite converted nucleic acid sequences is determined by next-generation sequencing.
51 . The method of claim 48 , wherein the level of methylated cytosines is determined in an amplified portion of the vimentin nucleic acid sequence and in an amplified portion of the SqBE18 nucleic acid sequence obtained from the subject.
52 . The method of claim 51 , wherein the amplified portion comprises 21 dinucleotides that correspond to or are derived from 21 CpG dinucleotides present in the native non-bisulfite treated SqBE18 genomic sequence.
53 . The method of claim 51 , wherein the amplified portion of the vimentin nucleic acid sequence comprises 10 dinucleotides that correspond to or are derived from 10 CpG dinucleotides present in the native non-bisulfite treated vimentin genomic sequence.
54 . The method of claim 48 , wherein if at least 1% of the vimentin nucleic acid sequences, or portions thereof and if at least 0.76% of the SqBE18 nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
55 . The method of claim 48 , wherein if at least 1% of the vimentin nucleic acid sequences, or portions thereof and at least 1% of the SqBE18 nucleic acid sequences, or portions thereof, in the sample are methylated reads, then the subject is determined to have an esophageal neoplasia or metaplasia.
56 . The method of claim 48 , wherein if the subject is determined to have an esophageal neoplasia or metaplasia, then the method further comprises administering to the subject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation (RFA); laser ablation; argon plasma coagulation (APC); electrocoagulation (electrofulguration); esophageal stent, surgery, and/or a therapeutic agent.
57 . (canceled)
58 . The method of claim 56 , wherein the therapeutic agent is 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 (Taxol®); cisplatin and 5-fluorouracil (5-FU); ECF: epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel (Taxotere®), cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
59 . The method of claim 56 , wherein the surgery is endoscopic mucosal resection (EMR), esophagectomy, and/or anti-reflux surgery.
60 - 64 . (canceled)