CA2972782C - Methods and compositions for detecting esophageal neoplasias and/or metaplasias in the esophagus - Google Patents

Abstract

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.

Description

METHODS AND COMPOSITIONS FOR DETECTING ESOPHAGEAL NEOPLASIAS AND/OR MET/\PL/\SI/\S IN THE ESOPHAGUS Related Applications This application claims the benefit of priority to United States provisional application serial nrnnber 62/099,021, filed December 31, 2014.

Funding Work described herein was supported by grant nos. UO1 CA152756; U54CA 163060; and P50CA150964.

The United States Government has certain rights in the invention, Background Over 15,000 new cases of esophageal cancer were diagnosed in 20 l 0, and there were 15 nearly as many deaths from this cancer alorn:.:.

As with other cancers, this rate can be decreased by improved mt~thods for diagnosis.

Although methods for detecting esophageal cancer exist, the methods are not ideaL Generally, a combination of endoscopy, isolation of cells (for example, via collection of cells/tissues from a fluid sarnple or from a tissue sample), and/or imaging technologies are used to identify cancerous cells and tumors.

Whik~ upper 20 endoscopy, usually performed by a gastroenterologist, can detect neoplasias of the esophagus, as well as of the stomach and duodenum, it is an uncomfortable and expensive procedure, Other detection procedures, such as barium esophogography are also available, but are associated with fi.1.lse positives, false negatives, and cost and discomfort issues.

Because of the disadvantages of existlng methods for detecting or treating esophageal 25 neoplasias/cancers, new rnethods are needed for esophageal neoplasia/cancer diagnosis and therapy, Summary Of The Disdosun~ In certain aspects, the present disclosure is based in part on the discovery of particular 30 human genomic DNA regions (also referred to herein as informative loci or patches) in which Date Reyue/Date Received 2022-04-21 the eytosines within CpG dinudeotides are differentially methylated in esophageal neoplasia compared to normal human tissues.

In one embodiment, the method com.prises assaying for the presence of difforentiaHy methylated genomic loci in a tissue sarnple or a bodily t1uid sample from a subject.

The 5 tissue sample may be obtained from biopsies of the gastrointestinal tract, including but not limited to the esophagus and stomach, Tissue samples may be obtained as a biopsy, or as a swab or brushing of the gastrointestinal tract (e.g., esophagus or stomach), or other organs believed to contain cancerous cells or tissues, EXt'.mplary bodily fiuids include blood, serum, plasma, saliva, spit, esophageal \Vashing, a blood-derived fraction, or urine.

In one 10 embodiment, the method involves methylation-sensitive restriction enzyme(s), In another embodiment, the method involves methylation-sensitive PCR.

In another embodiment, the method involves restriction enzyme/methylation-sensitive PCR.

In yet another embodiment, the method comprises reacting DNA from the sample v,rith a chemical compound that converts non-methylated cytosine bases (also called "conversion-sensitive" cytosines), but not 15 methylated cytosine bases, to a di.fforent nucleotide base.

In an embodiment, the chemical compound is sodium bisulfite, which conveiis unmethylated cytosine bases to uracil.

The compound-conve1ied DNA is then amplified nsing a methylation-sensitive polymerase chain reaction (MSP) employing primers that amplif'.y the compound-converted DNA template if cytosine bases within CpG dinucleotides of the DNA from the sample are methylated. 20 Production of a PCR product indicates that the subject has cancer or precancerous adtcnomas.

In some embodiments, cornpound-converied DNA is amplified by bisulfite specific methylation indifferent PCR primers and methylation of the parental DNi\ template is inferred by DNA sequence analysis of the bisulfite converted and amplified product.

Other methods for assaying for the presence of methylated DNA are knovvn in the art.

In certain 25 embodiments, the method optionally comprises detecting the presence or absence of a somatic mutation in TP53.

In another embodiment, the present invention provides a det,~ction method of prognosis of a neoplasia ( or metaplasia) (e.g., upper gastrointestinal neoplasia, esophageal ncoplasia) in a suqjt:ct known to have or suspected of having neoplasia (or metaplasia), In 30 some ernbodiments, the neoplasia is cancer, Such method comprises assaying for the presence of methylated informative loci in a tissue sample or bodily fluid from the su~ject In cenain cases, it is expected that detection of methylated informative loci in a tissue sample or bodily fluid from the subject is indicative of an advanced state of cancer (e.g., gastrointestinal cancer such as esophageal cancer).

In other cases, detection of methylated informative loci in 5 a tissm.: sample or sample from other bodily fluids may be indicative of a cancer that will respond to therapeutic agents that demethylate DNA or reactivate expression of genes located ,vithin methylated infonnative loci.

In certain embodiments, the rnethod optionally comprises dete-.:ting the presence or absence of somatic mutation(s) in TP53.

In certain cases, detection of sornatic mutation(s) in TP53 in a tissue sample or bodily fluid frorn the subject is indicative 10 of an advanced state of cancer ( e,g<, gastrointestinal cancer such as esophageal cancer)< In other cases, detection of somatic rnutation(s) in TP53 in a tissue sample or sample from other bodily fluids may be indicative of a cancer that will respond to therapeutic agents that demethylate DNA or reactivate expression of genes located within methylated infonnative loci. 15 in another embodiment, the present invention provides a method of monitoring over time the staius of neoplasia (e.g., gastrointestinal neoplasia such as esophageal neoplasia) in a subject.

In some embodiments, the neoplasia is a cancer. ln some embodiments, the neoplasia is a metaplasia such as Barrett's esophagus.

In certain embodiments, the method optionally comprises detecting the presence or absence of somatic mutation(s) in TP53 in 20 combination with detecting the methylation status of any of the informative loci disclosed herein.

In certain cases, detection of the appearance of somatic mutation(s) in TP53 over time in a tissue sample or bodily tluid from the subject is indicative of neoplastic progression.

In other cases, detection of a disappearance of a somatic mutation(s) in TP53 over time in a tissue sample or sample from other bodily fluids may be indicative of neoplaslic regression. 25 In another embodiment, the present invention provides a method of evaluating therapy in a subject having cancer or suspected of having neoplasia (e,g., gastrointestinal neoplasia such as esophageal neoplasia), In sorne embodiments, the neoplasia is a canct~r.

In some embodiments, the neoplasia is a metaplasia such as Barrett's esophagus.

In certain ernbodirnents, the method optionally :fu1iher comprises detecting the presence or absence of 30 somatic mutation(s) in TP53, 3 The present invention also rd ates to oHgonucleotide primer sequences for use in assays (e.g., methylation-sensitive PCR assays or Hpall assays) designed to detect the methylation status of the informative methylated genomic loci.

The present invention also provides a method of inhibiting or reducing gro,vth of 5 neoplasia cells (e.g., gastrointestinal neoplasia such as esophageal neoplasia).

In some embodiments, tht~ neoplasia is a cancer.

In some embodiments, the neoplasia is a metaplasia such as Barrett's esophagus.

In some embodiments, the disclosure provides for a method of detecting the presence of an esophageal neoplasia or metaplasia in the 1..~sophagus in a human subject, comprising; a) t 0 obtaining a human sample; and b) assaying the 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 fD 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 15 8420 - 8424, or cornple.ments or fragments thereof; wherein methylation of the nucleotide sequence is indicative of an esophageal neoplasia or metaplasia in the esophagus, In some embodiments, the disclosure provides for a method of detecting an esophageal neoplasia or rnetaplasia in the esophagus, comprising: a) obtaining a human sample; and b) assaying said sample for the presence of DNA methylation by assay in a bi sulfite converted DNA f_eJr 20 retention of a cytosine base at any of the Y positions present in one or more of the nucleotide sequences having at least 90{j,~, 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, 8103-81 Hi, 8157-8169, 8183-8195, 8223-8236, 8265-8278, 8307-8320, 8349-8362, 8410- 25 8414 or 8425-8429; wherein methylation of the nucleotide sequence is indicative of metaplasia in the esophagus or esophageal neoplasia, In some embodiments, the sample is assayed for the presence of methy[ation at any of the Y positions present in one or more of the nucleotide sequences having at least 90°10 identity to the sequence of any one or more of SEQ ID NOs: 7991-8004, 8019-8032, 8075-8088, 8103-8116, 8157-8169, 8183-8195, 8223-8236, 30 8265-8278, 8307-8320, 8349-8362, 8410-8414 or 8425-8429.

In some embodiments, the 4 disclosure provides fi)r method of detecting an esophageal neoplasia, comprising: a) obtaining a human sample; and b) assaying the sample for the presence of methy lation 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- 5 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.

In some embodiments, the disclosm·e provides for a method of dt~tecting an esophagt'.al neoplasia, comprising: a) obtaining a human sample; and b) assaying the sample for the presence of DNA methylation 10 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 nudeotide sequences having at least 90%i 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, 12071-12.121, 12267-12313, 12361-12407, 12467-12472, 12479-12484, 12503-12508, 15 12515-12520, 12539-12544, 12551-12556, 12569-12574, 12587-12592, 12605-12610, 12623-12628, 12650-12652, or 12659-1266 l; wherein methylation of the nucleotide sequence is indicative of metaplasia in the esophagus, In some embodiments, the sample is assayed for the presence of DNA methyiation by assay in a bisulfite converted DNA for retention of a c.,ytosine base at any of the Ypositions present in one or more of the nucleotide sequences 20 having at !east 9(/Yil identity to the sequence of any one or more of SEQ ID NOs: 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.

In some embodiments, the subject has been assayed for having a somatic mutation in TP53, In some 2~ embodiments, the esophageal neoplasia or metaplasia in the esophagus is selected from the group consisting of Barrett's esophagus (BE), and/or, Barrett's esophagus with low grade dysplasia, Barrett's esophagus ,vith high grade dysplasia (HGD), and/or t~sophageal adenocarcinoma (EAC), In some embodiments, the sample is obtained from a subject suspected of having or is known to have an esophageal neop!asia or metaplasia in the 30 esophagus.

In some embodiments, the assay is methylation-specific PCR.

In some embodiments, the rnethods disclosed herein further comprise: a) treating DNA from the sample with a compound that converts a non-methylated cytosine base in the DNA to a different base; b) amplifying a region of the compound converted nucleotide sequence with a forward primer and a reverse primer; and c) analyzing the methylation patterns of the 5 nucleotide sequences.

In some embodiments, the methods disclosed herein further comprise: a) treating DNA from the sample v,rith a compound that converts a non-methylated cytosine base in the DNA. to a different base; b) amplifying a region of the compound converted nucleotide sequence with a forward primer and a reverse primer; and c) detecting the presence and/or amount of the arnpHfied product.

In some embodirnents, the compound used to treat 10 DNA is a bisulfite compound.

In some embodiments, the assay comprises using a mt~thylation-sensitiw restriction enzyme.

In some t~mbodirnents, the methylation-sensitive restriction enzyme is selected from HpaII, SmaI, Sad!, Eagl, BsHJI, and BssHIL In some embodiments, the sample is a bodily fluid selected from the group consisting of blood, saliva, spit, an esophageal washing, sermn, plasma, a blood-derived fraction, stool, urine, and a 15 colonic effluent In some embodiments, the sample is derived from a tissue, In some embodiments, the sample is a biopsy, In some embodiments, the sample is a brushing, In sorne ernbodiments, the disclosure provides for a method of monitoring over time an esophageal neoplasia or metaplasia in the esophagus comprising: a) detecting the presence of methylation ,vithin a nucleotick sequence spanning one or more of any of the chromosomal 20 locl having a sequence that is at least 90% identical to the sequence of any one or more of: SEQIDN:• Q.s-,.· 1 -oo5b··, 2,_;,; 6()' -_,'·>·,t/-4"')ti , 51"_'",/' - 5(~\,·.,l,·b·,, ,.1,'.-) o'?, - r1y•)-_,:.;oo , 76.!':rLi - 7, 714 , 7, 8.,1 9 - 7°6o· ,,_-1_.> , 7963 - 7990, 8047 - 8074, 8131 - 8156, 8209- 8222, 8251 -~ 8264, 8293 ·•· 8306, 8335 .... 8348, 8405 --- 8409, or 8420 -·· 8424, or complements or fragments thereof;, or complements or fragments thereof; from a sample from a subject for a first time; and b) detecting the level of 25 methylation of the nucleotide sequence in a sarnple from the same subject at a later time.

In some embodiments, the disclosure provides for a method of monitoring over time an esophageal neoplasia or metaplasia in the esophagus comprising: a) detecting the methylation status of DNA by assay in a hi sulfite converted ON/\ for retention of a cytosine base at one or more of the Y positions present in one or more of the nucleotide sequences having at least 30 90(;,{i identity to the sequence of any one or more of: SEQ ID N Os: 857-1284, 1713-2140, 6 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 from a sample from a subject for a first time; and b) detecting the methylation status of the nucleotide 5 sequence in a sarnple from the same subject at a later time; wherein absence of methylation in the nucleotide sequence taken at a later time and the presence of methylation ln the nucleotide sequence taken at the first time is indicative of neop!asia or m:etapiasia regression; and wherein presence of methylation in the nucleotide sequente taken at a later lime and the absence of rnethylation in the nucleotide sequence taken at the first time is indicative of 10 nt,oplasia or metaplasia progression.

In some embodiments, the disclosure provides for a method of monitoring over time an esophageal neoplasia comprising: a) detecting the presence of methylation within a nucleotide sequence spanning one or more of any of the chromosomal loci having a sequeuce that is at least 90% identical to the st,quence of any one or more of: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968, 15 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658, or compk~ments or fragments thereof; from a sample from a subject for a first time; and b) detecting the level of methylation of the nucleotide sequence in a sample from the same subject at a later time.

In some embodiments, the disclosure provides for a method of monitoring over time an esophageal 20 neoplasia comprising: a) detecting the metbylation status 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%1 identity to the sequence of any one or more of: SEQ m NOs: 8819-9004, 9191-9376, 9935-10120, 10307- 10492, 10973-11119, 11267-11413, 11663-11713, 11765-11815, 11969-12019, 12071-12121, 25 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 from a sample from a subject for a first time: and b) detecting the methyiation status of the nucleotide st:quence in a sample from the same subject at a later time; wherein absence of methylat.ion in the nucleotide sequence taken at a later time and the 30 presence of methylation in the nucleotide sequence taken at the first time is indicative of 7 neoplasia regression; and wherein presence of methylation in the nucleotide sequence taken at a later time and the absence ofrnethylation in the nucleotide sequence taken at the first tirne is indicative of neop!asia progression, In some embodiments, the sample is a bodily fluid selected from the group consisting of blood, saliva, spit or an esophageal washing, In some 5 embodiments, the sample is derived from tissue, In some embodiments, the disclosure provides for 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; wht~rein the subject has been determined 10 to have methylation in a sequence that is al least 90(¼) 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.

In some embodiments, the disclosure provides for a method of treating a subject having a metaplasia 15 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 metplasia; ,.vherein the subject has been deterrnined to have DNA methylation by assay in a bisulfite converted DNA flx retention of a cyiosine base of one or more of the Y positions present in one or more of the nucleotide sequences having at least 20 9(Y~fi 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.

In some embodiments, the disclosure provides for a method of treating a subject having an 2" esophageal neoplasia, cmnprising the step of treating the subject with chemotherapy, radiation therapy, ablation, and/or with neoplasia resection; wherein the subject has been detennined to have methylation methylation within a nucleotide sequence spanning one or more of any of the chromosornal loci having a sequence that is at least 90%i identical to the sequence of any one or more of: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867- 30 11968, 12173-12266, 12455-12466, 12491--12502, 12527-12538, 12563-12568, 12531-12586, 8 12599-12604, 12617-12622, 12647-12649 or 126.56-12658, or complements or fragments thereof In some embodiments, the disclosure provides for a method of treating a subject having an esophageal neop!asia, comprising the step of treating the subject with chemotherapy, radiation therapy, ablation, and/or with neoplasia resection; ,vherein the 5 subject has been determined to have rnethy1ation of a DN.A 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 sequern.:es 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, 10 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.

In some embodiments, the subject has been assayed for having a somatic mutation in TP53. rn some ernbodirnents, the disclosure provides for a bisulfite converted sequence 15 having at least 90% identity to any one or more of SEQ ID NOs: 857 -2568, 3425-5136, 5927-7506, 7559- 7662, 7715 - 7813, 7867 -· 7962, 7991 - 8046, 8075 - 8130, 8157 - 8208, 8223 - 8250, 8265 ·-· 8292, 8307 ·-- 8334, 8349 - 8376, 8410 - 8419, 8425 - 8434, and/or fragments thereof: and/or the reverse cmnplements thereof including aH unique fragments of these sequences and their reverse complements.

In some embodiments, the disclosure 20 provides for a panel ofbisulfite converted sequences selected from the sequences having at least 90% 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, and/or fragments thereof, and/or the reverse complements thereof including all unique fragments of these sequences and their reverse complements.

In some embodiments, the panel corresponds to any combination of the sequences having any of SEQ lD NOs: 8307-8334, 8349-8376, 8410- 8419 or 8425-8434.

In some embodirnents, the disclosure provides for an oligonucleotide primer that hybridizes to any of the sequences having at least 901% identity to any one or more of SEQ ID NOs: 857 -2568, 3425-5136, 5927-7506, 7559- 7662, 7715 --- 7818, 7867 - 7962, 30 7991- 8046, 8075- 8130, 8157 - 8208, 8223 .... 8250, 8265 --- 8292, 8307 -- 8334, 8349 9 8376, 8410- 8419, 8425 - 8434. !n some embodiments, the primer is any of the primers having at kast 90% identity to any one or more of SEQ lD NOs: 8377-8404 or 8435-8444, or fragments or compk,ments thereof.

In some embodiments, the primers are combined as :fr.mvard and reverse primers for PCR amplification of any of the bisulfite converted sequences 5 having at least 90% 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, and/or fragments thereof: and/or tht, reverse complements thereof including all unique fragments of these sequences and their reverse complements.

In some embodiments, one of the primt,rs in 10 the primer pairs has a nucleotide sequence that is at least 90% identity to any one or more of SEQ ID NOs; 8377-8404 or 8435-8444, or fragments or complernents thereof.

In some embodiments, the disclosure provides for a panel of primer pairs selected from any of the primer pairs disclosed herein.

In some embodiments, the panel corresponds to a combination of primer pairs for amplifying any of the cornbinations of SEQ ID NOs: 8307-8334, 8349- J 5 8376, 8410-8419 or 8425-8434.

In some embodiments, the disclosure provides for a hisulfite converted sequence having at least 90% identity to any one or more of SEQ ID NOs: 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, 20 12650 - 12655, and 12659 - 12664, and the reverse complements thereof: including all unique fragments of these sequences and their reverse complements. fo some embodiments, the disclosure provides for a panel of bi sulfite converted sequences selected from any of these sequences. !n some embodiments, the disclosure provides for an oligonucleotide primer that hybridizes to any of these sequenct'.sJn some embodiments, the panel corresponds to any 25 combination of any of the sequences of SEQ lD NOs: 12605-12616, 12623--12634, 12650- 12655, or 12659-12664. in some embodiments, the disdosure provides for primers having at least 90% identity to any one or more of SEQ ID NOs: 12635-12646, and 12665-12670, or fragments or complements thereof.

In some embodiments, the primers are combined as forward and reverse primers for PCR amplification of any of the hisulfite converted sequences 30 having at least 90% identity to any one or more of SEQ l D NOs: 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, and 12659 - 12664, and the reverse complements thereof, including all unique fragrnents of these sequences and their reverse cornpkments.

In some embodiments, om.'. of 5 the primers in the primer pairs has a nucleotide sequence that is at least 90% identity to any one or more of SEQ 1D NOs: 12635-12646, and 12665-12670, or fragments or complements thereof In some embodiments, the disclosure provides for a panel of primer pairs selected from any of the primer pairs disclosed herein.

In some embodiments, the panel corresponds to the combination of primer pairs for amplifying any combination of any of the sequences of 10 SEQ ID NOs: 12605-12616, 12623-12634, 12650-12655, or 12659-12664.

In some embodiments, the disclosure provides for a method of selecting an individual to undergo a diagnostic procedure to determine the presence of Barrett's esophagus, of Barrett's esophagus with tow grade dysplasia, of Ban-ett's esophagus with high gradt~ dysplasia, or of esophageal adenocarcinoma, by obtaining a biological sample from an 15 individual, and determining the presence in DNA from that sample of DNA methylation present in any of the sequences having at least 90~·o identity to 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, and/or fragments thereof~ and/or the reverse cornplements thereof In 20 some embodiments, the method further comprises determining the presence of a somatic mutation in TP53, In some embodiments, DNA rnethylation is detected by cutting one of the DNA st~quences with a rnethylation sensitive restriction enzyme. ln smne ernbodiments, DNA rnethylation is detected by hisuifite converting DNA from the sample and detecting the presence of any of the bi sulfite converted DNA sequences having at least 90% identity to any 25- oneormoreo f 00 EQ. IDN1 O s: s-,)..1., -:2:~, 6°o , 311-•2:-:,-,'.) l'".)'f.J ,-)9 2, 7 -1--.o,; o·b·· , 7:-,'.-) 9 ·70,··6·2, ',"',7.1.5 7. 81o° , 7867 - 7962, 7991 - 8046, 8075 - 8130, 8157 ... 8208, 8223 .... 8250, 8265 ... 8292, 8307- 8334, 8349 ·· 8376, 8410 ·· 8419, 8425 - 8434, and/or frag1m.~nts thereof: and/or the reverse complements thereof including ail unique fragments of these sequences and their reverse complements.

In some embodiments, the bisulfite conve1ied sequences are detected using 30 any of: DNA. sequencing, next generation sequencing, methy1ation specific PCR, methylation 11 specific PCR combined with a f1uorogenic hybridization probe, and real time methylation specific PCR.

In some embodiments, the bisulfite converted sequences are detected using PCR amplification ernploying a PCR primer or primer pair comprising the nucleotide sequence of any of SEQ ID NOs: 8377-8404 or 8435-8444. fo sonie embodiments, the 5 method furth;c,'.r comprises the step of determining the nucleotide sequence of the bisulfite converted sequences.

In some embodiments, tlx~ pen;;c,'.nt of the target sequence that is methylated in any of the individual target sequences is greater than 1 %, or greater than 5%, or greater than 10%. ln some ernbodhnents, the biological sample is a tissue sample, including a san1ple of the esophagus.

In some embodiments, the tissue sample is a biopsy or a brushing, 10 including a biopsy or a brushing of the esophagus, In some embodiments, the biological sample is a body fluid.

In some ernbodiments, the body fluid is blood, saliva, spit or an esophageal washing.

In some embodiments, the disclosure provides for a method of selecting an individual to undergo a diagnostic procedure to determine presence ofBam,tt's esophagus with low 15 grade dysplasia, of Barrett's esophagus with high grade dysplasia or of esophageal adenocarcinoma; or to undergo a treatment for for Barrett's esophagus with low grade dysplasia, Barrett's esophagus ,vith high grade dysplasia or for esophageal adenocarcinoma; or to undergo enhanced surveillance for the development of Barrett's esophagus with low grmk dysplasia, of Barrett's esophagus with high grade dysplasia or of esophageal 20 adenocarcinoma, by obtaining a biological sample from an individual, and determining the presence in DNA from that sample of methy1ation present in any of the sequences having at least 901)/,3 identity to 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 m 12656-12658.

In 25 some embodiments, the disclosure provides for a method oftreating a subject having Barrett's esophagus with low grade dysplasia, BmTett's esophagus with high grade dysp!asia or esophageal udenocarcinoma, comprising the step of treating the subject vvith chemotherapy, radiation therapy, and/or with resection of an esophageal lesion or with ablation of an esophageal lesion, wherein the su~ject has been detennined to have methylation at any of the 30 sequences having at least 90~,S identity to any one or more of SEQ ID NOs: 8-447-8818, 9563- 12 9934, 10679-10826, 11561-11662, 11867-11968, 12173-12266, 1245.5-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 126.56-12658.

In sorne embodirnents, the disclosure provides for a method of selecting an individual to undergo enhanced surveillance for the development of Barrett's esophagus ·with 5 high grade dysplasia or of esophageal adenocarcinoma, wherein the subject has been determined to have methylation at any of the sequences having at least 90%) identity to any one or more of SEQ ID NOs: 8447-8818, 9563-9934, 10679-10826, l 1561-11662, 11867- 11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656--12658.

In some embodiments, the 10 methods dislosed herein further comprises detem1ining the presence of a sornatk mutation in TP53.

In some t,mbodiments, DNi\ methylation is detected by cutting one of the DNA sequences with a methylation sensitive restriction enzyme, In somt~ embodiments, DNA methylation is detected by bisulfite converting DNA from the smnple and detecting the presence of any of the bi sulfite converted DNA sequences having at least 90% identity to any 15 rme or more of SEQ ID NOs: 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, and 12659 - 12664, and the reverse complements thereof, including all unique fragments of these sequences and their reverse complements or panels comprising any combination of those sequences, or 20 detecting the presence of uncoverted cytosine residues at any of the Y bases in any of those sequences or panels. 1n some embodiments, the bisulfite converted sequences or the presence of unconverted cytosine residues at any oftbe Y bases of these sequence or panels are detected using any of: DNA sequencing, next generation sequencing, methylation specific PCR, methylation specific PCR combined \Vith a fluorogenic hybridization probe, and real 25 time methylation specific PCR. ln some embodiments, the bisu!fite converted sequences or the presence of unconverted cytosine residues in the bisulfite converted sequences are detected using PCR amplification employing any of the PCR primers or primer pairs disclosed herein.

In some embodiments, the percent of the target sequence that is methylated In any of the individual target sequences is greater than l %, or greater than 51%, or greater 30 than 10~/u, In some embodiments, the biological sample is a tissue sample, including a sample 13 of the esophagus.

In some embodiments, lhe tissue sample is a biopsy or a brushing, including a biopsy or brushing of the esophagus.

In sorne embodimenis, the biological sample is a body fluid.

In some embodiments, the body fluid is blood, saliva, spit or an esophageal washing.

In some embodiments, the individual is known to have Barrett's 5 esophagus, 1n some embodiments, the individual is knovm to have Barrett's esophagus with low grade dysplasia.

In some embodiments, tht, disclosure provides for a method of determining the response of an individual with esophageal cancer to therapy hy detection in a body fluid of methylation in any of the DNA sequences having at least 90% identity to any one or more of 10 SEQ ID NOs: l - 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, 8420- 8424, 8447-8818, 9563-9934, 10679-10826, 11561-11662, 11867- 11968, 12.173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658 and/or :fragments thereot: and/or 15 ihe reverse complements thereof; wherein increasing levels of methy!ation over time are indicative of disease progression and a need for a change in therapy, and wherein absence of increase in levels of methy lation over time or decrease in levels of methylation over time are indicative that a change in therapy is not required.

In some embodiments, any of the methods disclosed herein fmther comprise detecting the presence of a somatic mutation in TP53, In 20 some embodiments, the DNA methylation is detected by bisulfite converting DNA from a body fiuid and detecting the presence of any of the bisuifite converted DNA sequences disclosed herein.

In some embodiments, tht, disclosure provides for a melhod of monitoring over time an esophageal neoplasia comprising: a) detecting in a sample from a subject for a first time (i) 25 the methylation status of one or more of the DNA sequences having at least 90~·'o identily to any one or more of SEQ ID NOs: 1 - 856, 2569 - 3424, 5137 - 5926; 7507 - 7553, 7663 - 7714, 7819- 7866, 7963 -7990, 8047- 3074, 8131 --- 8156, 8209---8222, 8251 ---- 8264, 8293. --- 8306, 8335 --- 8348, 8405 ·--- 8409, 8420 --- 8424, 8447-8818, 9563-9934, 10679-10826, 11561-i l 662, ] ! 867-11968, l 2173-12.266, I 245.5-12466, 12491-12502, 12527-12538, 30 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658 from 14 that sample; and (ii) the somatic mutation status of TP53 from that sample; and b) detecting in a sample from the same subject at a later tirne (i) the methylation status of one or more 'of the DNA sequences having at least 90% identity to any one or more of SEQ rn NOs: 1 - 856, 2569 - 3424, 5137 - 5926; 7507 - 7558, 7663 - 7714, 7819- 7866, 7963 .... 7990, 8047 - 5 8074, 8131- 8156, 8209- 8222, 8251- 8264, 8293 .... 8306, 8335 - 8348, 8405 -3409, 8420 - 8424, 8447-8818, 9563-9934, 10679-10826, 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; and (ii) the somatic mutation status ofTP53 from the same sul~ect at a later time.

In some embodiments, the neoplasia regression is 10 indicated by: (i) the sample takt~n at the first time having methylation in any of Up15-1, Up35-1, Up35-2, Up3, Up27, or Up 10, or having a somatic mutation in TP53; and (ii) the sample at a later time lacking methylation in Upl5-1, Up35-l, Up35-2, Up3, Up27, Up 10, and lacking a somatic mutation in TP53.

In sorne embodirnents, the neoplasia progression is indicated by: (i) the sarnple taken at the first time Jacking methylation in Up 15-1, U p35-1, 15 Up35-2, Up3, Up27, and Up 10, and lacking a somatic mutation in TP53; and (ii) the sample taken at a later time having methylation in any of Up15~1, Up35-i, Up35-2, Up3, Up27, lJplO, or having a somatic mutation in TP53.

In some embodiments, the biological sample is a tissue sample, including a sample of the esophagus. 1n some embodiments, the tissue sample is a biopsy or a brushing, including a biopsy or a brushing of the esophagus. fo some 20 embodiments, the biological sample is a body fluid.

In srnne embodiments, the body :fluid is blood, saliva, spit or an esophageal washing, In some ernbodiments, the disclosure provides for a method of detecting the presence of an esophageal neoplasia in a human subject, comprising: a) obtaining a human sample; and b) detecting the methylation status of one or more of the DNA sequences having at least 90(% 25 identity to 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, 8420 8424, 8447-8818, 9563-9934, 10679- 10826, 11561-11662, 11867-11968, !2173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658 from 30 that sampk.

In some embodiments, the method further comprises the step of detecting the somatic mutation status of TP53 from that sample.

In some r:.~mbodiments, the TP53 somatic mutation is detected at a later time than the detection of the methy lation status of any of the DNA sequences having at least 90~11 identity to any one or more of SEQ ID NOs: l - 856, 2569 ... 3424, 5137 • 5926; 7507 - 7558, 7663 - 7714, 7819- 7866, 7963 - 7990, 8047 - 5 8074, 8131- 8156, 8209-8222, 8251- 8264, 8293 - 8306, 8335 -- 8348, 8405---- 8409, 8420 -8424, 8447-8818, 9563-9934, 10679-10826, 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. 1n some embodiments, a) the presence of methylation at any of SEQ ID NOs: l - 856, 2569 ·· 3424, 5137 - 5926; 7507 --- 7558, 7663 - to 7714, 7819 .... 7866, 7963 -7990, 8047- 8074, 8131- 8156, 8209- 8222, 8251- 8264, 8293 - 8306, 8335 - 8348, 8405 - 8409, 8420- 8424; and b) the absence of metbylation at any one of the combination ofUpl5-1, Up35-1, Up35-2, Up3, Up27, and UplO; and c) the absence of a somatic mutation in TP53 is indicative of non-dysplastic Barret's esophagus, rn some embodiments, a) the presence of methylation at any of SEQ ID NOs: 1 -· 856, 2569 - 3424, 15 5137 - 5926; 7507 .... 7558, 7663 .... 7714, 7819 -· 7866, 7963 - 7990, 804 7 - 8074, 8131 - 8156, 8209- 8222, 8251 - 8264, 8293 - 8306, 8335 - 8348, 8405 ·•· 8409, 8420 ·· 8424; and b) the absence of rnethy lation in all rnembers of an assay panel selected from any or all of Up15-l, Up35-l, Up35-2, Up3, Up27, and UplO; and c) the absence ofa somatic mutation in TP53 is indicative of non-dysplastic Barret's esophagus.

In some embodiments, a) the 20 presence of methylation at any of SEQ ID NOs: 1 - 856, 2569 - 3424, 513 7 - 5926; 7507 - 7558, 7663 - 7714, 7819 - 7866, 7963 - 7990, 8047 - 8074, 813 l - 8156, 8209-- 8222, 8251 - 8264, 8293 8306, 8335 --· 3348, 8405 --- 8409, 8420 -·· 8424; and b) the absence of methy!ation at Up-3 and U p35~2; and c) the absence of a somatic mutation in TP53 is indicative of non-dysplastic Barret's esophagus.

In some embodiments, the presence of 25 methylation at any one of Up 15-1, Up35- l, U p3 5-2, Up3, Up27, and Up 10; or the presence of a somatic mutation in TP53 is indicative of esophageal acknocarcinoma, of Barrett's esophagus with low grade dysplasia, or of Barrett's esophagus with high grade dysplasia.

In some embodiments, the presence of rnethylation at any one of Upl5~1, Up35-l, Up35-2, Up3, Up27, or Up 10, is indicative of esophageal adenocarcinoma, of Barrett's esophagus with low 30 grade dysplasia, or of Barrett's esophagus with high grade dysplasia.

In some embodiments, 16 the biological sample is a tissue sample, including a sample of the esophagus, In some embodiments, the tissue sarnple is a biopsy or a brushing, including a biopsy or a brushing of the esophagus.

In some embodiments, the biological sarnple is a body fluid.

In some embodiments, the body fluid is blood, saliva, spit or an esophageal washing. ln some embodiments, the disclosure provides for a bisul.fite-converted nucleotide sequence comprising the bisulfite-converted nucleotide sequence of any one of the following: Up3, UplO, UplS-1, Up15-2, Up20-l, Up20-2, Up20-2, Up27, Up35-1, Up35-2, SqBE2, SqBE5, SqBE7, SqBE9, SqBElO, SqBEl 1-1, SqBEll-2, SqBE13, SqBE14-2, SqBE15, SqBE16-l, SqBE16-2, SqBEl 7-1, SqBE18, SgBE22-l, SqHE22-2 or SqBE23, In some 10 embodiments, the bi sulfite-converted nucleotide sequence of claim 98, wherein the sequence comprises the bisulfite-converted nucleotide sequence of any one of the following: Up3, Up 10, UplS-1, Upl5-2, Up20-l, Up20-2, Up20-2, Up27, Up35-1, or Up35-2.

In some embodiments, the bisulfite-converted nucleotide sequence of claim 98, wherein the sequence comprises the bisul:fite-converted nucleotide sequence of any one of the following: SqBE2, 15 SqBES, SqBE7, SgBE9, SqBEl0, SqBEl 1-1, SqBEl 1-2, SqBE13, SqBE14-2, SqBElS, SqBE16-l, SqBE16-2, SqBE17-1, SqBE18, SqBE22.-l, SqBE2.2-2 or SqBE23, In some embodiments, the disclosure provides for c1 bisulfite-converted nucleotide sequence comprising a sequence having at least 80~•'\:l, 85%, 90%, 91 %, 92%, 93%, 94%i, 95%, 96%, 97%i, 98".Vi,, 99% or 100%; identity to any of the following sequenees: SEQ ID NOs: 20 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, rn sorne embodirnents, the sequence comprises a sequence having at least 80%i, 85%, 90%, 91 (l,,;,, 92%, 93%i, 94';/c,, 95%, 96%, 97%, 9fWi,, 99% or 100(;.o identity to any of the following sequences: SEQ 1D NOs: 8307- 25 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, or 8432, 8434, In some embodiments, the sequence comprises a sequence having at least 80%i, 85%, 90(;{,, 91 1)1\i, 92(){,, 93{;,;,, 94%, 95%, 96%, 97%, 98%, 99% or lOQl;I;, identity to any ofthe following sequences: SEQ ID NOs: l 2.605-12616, 12623-12634, 12650-12655, or 12659-12664, In some 30 embodiments, the sequence comprises a sequence having at least 80%,, 85%, 90'Vn, 91 %, 92'1/ii, 17 93%, ~WVo, 95%, 96%, 97°1t1, 98%, 99(1/(; or 100(!1;) identity to any of the follm-ving sequences: 12652, 12655, 12661, and 12664.

Brief Description of the Hgures Figure 1 shows the sensitivity (percentage of samples detected), the specificity (percentage of samples not detected), the total number of samples studied, and the total number of positive samples from the panel of markers studied in Example 3.

Three panels of markers were selected for study. 10 Detailed Description Of The Invention I.

Definitions For convenit,nce, certain terms employed in the specification, examples, and appended claims are collected here.

Unless defined otherwise, all technical and scientific lerms used herein have the sarne meaning as commonly understood by one of ordinary skiH in the mt to 15 which this invention belongs.

Although methods and materials similar or (..,quivalent to those described herein can he used in the practice or testing of the present invention, suilabk methods and materials are described below.

The rnaterials, methods and examples are iUustrative only, and are not intended to be limiting, Each embodiment of the invention described hen.,in may be taken atone or in combination ,vith one or more other embodiments of the invention.

Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of 25 integers but not the exclusion of any other integer or group of integers.

The articles "a" and "an'' are used herein to refer to one or to more than one (i.e,, to at least one) of the grammatical object of the article.

By way of example, "an element" rneans one element or more than one element 18 Date Reyue/Date Received 2022-04-21 'The terms "adenoma" is used herein to describe any precancerous neoplasia or benign tumor of epithelial tissue, for example, a precancerous neoplasia of the gastrointestinal tract, pancreas, and/or the bladder.

The term "blood-deriw.:d fraction" herein refers to a cornponent or cornponents of 5 whole blood, Whole blood comprises a liquid portion (i.e., plasma) and a solid portion (i.e., blood cells).

The liquid and solid p011ions of blood are each comprised of multiple components; e.g,, different proteins in plasma or different cell types in the solid portion.

One of these components or a mixture of any of these components is a blood-derived fraction as long as such fraction is missing one or more components found in whole blood. 10 The term '\,sphophagus" is intended to encompass the upper portion of the digestive system spanning from the back of the oral cavity, passing tlownvvards through the rear part of the mediastinum, through the diaphragm and into the stomach.

The tem1 "esophageal cancer" is used herein to refer to any cancerous neoplasia of the esophagus. 15 "Barrett's esophagus" as used herein refers to an abnormal change (metaplasia) in the cells of the lower portion ofthe esophagus.

Barrett's is characterized the finding of intestinal metaplasfa in the esophagus, A "brushing" of the esophagus, as referred to herein, may he obtained using any of the means knoivn in the mt, In some embodiments, a brushing is obtained by contacting the 20 esophagus with a brnsh, a sponge, a balloon, or with any other device or substance that contacts the esophagus and obtains an esophageal sample, "Ce Us," "'host cells" or "recombinant host cells" are terms used interchangeably herein.

It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a eel L Because certain modifications may occur in 25 succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent ceH, but are still included within the scope of the tenn as used herein.

The terms "compound", "test compound," "agent", and "molecule" are used herein interchangeably and are meant to include, but are not limited to, peptides, nudeic acids, 19 carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not iilnited to, chemicals, rnetals, and organometallic compounds), The term "compound-converted DNA" herein refers to DNA that has been treated or reacted with a chemical compound that converts umnethy!ated C bases in DNA to a different 5 nucleotide base.

For example, one such compound is sodium bisulfrte, which converts unmethylated C to U.

If DNA that contains conversion-sensitive cytosine is treated \Vith sodium bisulfite, the compound-converted DNA will contain U in place of C, If the DNA. which is treated with sodimn bisulfite contains only methylcytosine, the compound-converted DNA will not contain uracil in place of the methylcytosine, 10 The tenn "de-methylating agent" as used herein refers agents that restore activity and/or gene expression of target genes silenced by methylation upon treatment with the agent Examples of such agents include without limitation 5-azacytldine and 5-aza-2'-deoxycytidine, The tem1 "detection" is used herein to refer to any process of observing a marker, or a change in a marker (such as for example the change in the rnethylation state of the marker), in 15 a biological sample, whether or not the marker or the change in the marker is actually detected, In other words, the act of probing a sample for a marker or a change in the marker, is a "detection" even if the marker is determined to be not present or below the level of S()nsitivity, Detection may be a quantitative, semi-quantitative or non-quantitative observation. 20 The tenn "diffr~n.~ntially methylated nucleotide sequence" refers to a region of a genomic loci lhat ls frmnd to be rnethylated in a in cancer tissues or cell lines, but not methylated in the normal tissues or cell lines.

The term "neoplasia" as used herein refers to an abnormal grov.rth of tissue.

As used herein, the term "neoplasia" may be used to refer to cancerous and non-cancerous tumorn, as 25 well as to Barrett's csophagus (which may also be refe1red to herein as a metaplasia) and Barrett's esophagus with dysplasia.

In some embodiments, the Barrett's esophagus with dysplasia is Barrett's esophagus with high grade dysplasia.

In some embodiments, the Barrett's esophagus with dysplasia is Barrett's esophagus with low grade dysplasia.

In sorne embodiments, the neoplasia is a cancer (e.g, esophageal adenocarcinoma). "Gastrointestinal neoplasla" refers to neoplasia of the upper and lower gastrointestinal tract As commonly understood in the art, the upper gastrointestinal tract includes the esophagus, stomach, and duodenmn; the Imver gastrointestinal tract includes the remainder of the smaH intestine and all of the large intestine. 5 The terms "healthy", "nonnaL" and "non-neoplastic" are used interchangeably herein to refor to a subject or particular cell or tissue that is devoid (at least to tht~ limit of detection) of a disease condition, .such as a neoplasia, "Homology'' or ''identity" or "similarity" refers to sequence similarity between two peptides pr between two nucleic acid molecules.

Homology and identity can each be 10 determined by comparing a position in each sequence which may be aligned for purJ)0ses of comparison.

When an equivalent position in the compared sequences is occupied by the smm.o base or amino acid, then the molecules are identka1 at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can he referred to as homologous (similar) at that position. 15 Expression as a percentage of homology/similarity or identity refors to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated or "non-homologous" shares, in some embodiments, less than 40% identity, and in particular embodiments, less than 25 1% identity with a sequence of the present invention.

In comparing tvvo sequences, the absence of residues ( amino acids or 20 nucleic acids) or presence of extra residues also decreases the identity and homology/similarity, The term "homology" describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs.

The nucleic acid and protein sequences of the present invention may be used as a ''query 25 sequence" to perform a search against public databases to, for example, identify other family members, related sequences or homo1ogs, Such searches can be perfi..)rmed using the NB LAST and XBLAST progran.1s (version 2.0) of Altschul, et aL (1990) J kfol.

Biol. 215 :403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=lO0, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid 30 molecules of the invention. BLAST protein searches can be performed with the XBLAST 21 prograrn, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of tht, invention.

To obtain gapped alignments f<)r comparison purposes, Gapped BLAST can be utilized as described in Altschul et aL, (1997) Nucleic Acids Res. 25(17):3389-3402, When utilizing BLAST and Gapped BLAST programs, the defimlt 5 parameters of the respective programs (e,g., XBLAST and BLAST) can be used.

See www,ncbi.nlrn.nih.gov.

As used herein, "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, Le., taking into account gaps and insmtions.

Identity can be 10 readily calculated by knm.vn methods, including but not limited to those described in (Computational Alolecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; JJiocomputing: Jnfom1atics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H.

G., eds., Burnana Press, New Jersey, 1994; Sequence Analysis in _Molecular Biology, von 15 Heinje, G., Academic Press, 1987; and 5'equence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, FL, and Lipman, D., SIAM J.

Applied Math., 48: 1073, 1988).

Methods to determine identity are dt'.sigued to give the largest match between the sequences tested.

Moreover, methods to determine identity are codified in publicly available cornputer programs.

Computer program methods to determine 20 identity between two sequences include, but are not limited lo, the GCG program package (Devereux, J., et aL, Nucleic Acids Research 12(1): 387 (1984)), BLAST!\ BLASTN, and FA STA (Altschul, S. F. et al., J Afolec:.

Biol. 215: 403--410 (1990) and i\ltschul et aL ],fuc.

Acid, Res. 25: 3389-3402 (l997J).

The BLAST X program is publicly available from NCBJ and other sourct,s (BLASTA:fanua!, Altsdrnl, S., et aL, NCBl NLM NIH Bethesda, IVfd. 25 20894; Altschul, S., et aL, J Mo!.

Biol. 215: 403-410 (1990)).

The ,vell known Smith Waterman algorithm may also be used to determine identity.

The tem: "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term ''isolated" as used herein with respect to nucleic acids, such as DNA or 30 RNA, refers to molecules in a form which does not occur in nature.

Moreover, an "isolated 22 nucleic acid" is meant to include 1rndeic add fragments which are not naturally occurring as fragments and would not he found in the natural state.

The term "methylation-specific PCR" ("MSP") herein refers to a polymerase chain reaction in which amplification of the compound-conve1ied template sequence is performed. 5 Two sets of primers me designed for use in MSP.

Each st;t of primers comprises a forward primer and a reverse primer.

One set ofprirners, called metbylation-specific primers (see below), will amplify the compound-conve1ted template sequence if C bases in CpG dinudeotides within the DNA are methylated.

Another set of primers, called umnethylationspecific primers or primt~rs for unmethylated sequences and the like (see belov1), wm arnplity l 0 the cornpound-converted template sequences if C bases in CpG dinucleotides within the DNA are not methylated.

As used herein, the term "nucleic acid" refers to polynuc!eotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonuclei(.: acid (RNA).

The term should also he understood to include, as equivalents, analogs of either RNA or DNA made 15 from nucleotide analogs, and, as applicable to the embodiment being described, singlestranded (such as sense or antisense) and double-stranded polynucleotides. "Opernbly linked'' when describing the relationship between two DNA regions simply means that they are functionaUy related to each other.

For example, a promoter or other transcriptional regulatory sequence is opernbly linked to a coding sequence if it controls the 20 transcription of the coding sequence.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context dearly indicates otherwise.

The terms "proteins" and ''polypeptides" are used interchangeably herein.

A "sample" includes any material that is obtained or prepared for detection of a z.;; molecular marker or a change in a molecular marker such as for example the rnethylation state, or any rrmterla! that ls contacted with a detection reagent or detection device for the purpose of detecting a molecular marker or a change in the molecular marker.

As used herein, "obtaining a sample" includes directly retrieving a sample from a su~ject to be assayed, or directly retrieving a sample from a subject to be stored and assayed 30 at a later time.

Alternatively, a sample may be obtained via a second pmi.y.

That is, a sarnple 23 may be obtained via, e.g., shipment, from another individual who has retrieved the sample, or othervvise obtained the sample.

A "subject" is any organism of interest, generally a mammalian subject, such as a mouse, and in pmiicuhr embodiments, a human su~jecL 5 As used herein, the tem1 "specifically hybridizes" refers to the ability of a nucleic acid probe/primer of the invention to hybridize to at least 12, 15, 20, 25, 30, 35, 40, 45, 50 or 100 consecutive nucleotides of a target sequence, or a sequence complementary thereto, or naturally occurring mutants thereof: such that it has, in some embodiments, less than 15%, less than 10%, or less than 5')-'ii background hybridization to a cellular nucleic acid (e.g., 10 mRNA or genomic DNA) other than the tmget gene. A variety of hybridization conditions may be used to detect specific hybridization, and the stringency is determined primarily by the wash stage of the hybridization assay, Generally high temperatures and low salt concentrations give high stringency, while low temperatures and high salt concentrations give low stringency.

Low stringency hybridization is achieved by washing in, for example, about 15 2.0 x SSC at 50 °C, and high stringency is a.thieved with about 02 x SSC at 50 "'C.

Further descriptions of stringency are provided below.

As applied to polypeptides, the term "substantial sequence identity" means that two peptide sequences, when optimally aligned such as by the programs GAP or BESTFIT using default gap, share at least 90 percent St'.quence identity, in some embodirnents, at least 95 20 percent sequence identity, or at least 99 percent sequence identity or more, In some ernbodirnents, residue positions which are not identical differ by conservative amino acid substitutions.

For example, the substitution of amino acids having similar chemical properties such as charge or polarity is not likely to affect the properties of a protein.

Examples include glutarnine for asparagine or g!utamic acid for aspartic acid. 25 An "informative loci" as used herein, refr:rs to any of the nucleic acid sequences disclosed herein that may have altered (e.g., increased) methylation in a sample (e.g,, an esophageal tissue sample) from a subject having Barrett's esophagus and/or an esophageal neoplasia as compared to the rnethylation pritterns of the corresponding nucleic acid sequence in a sample from a heaithy control subject. 24 The term "Up3" as used herein refers to a nucleotide sequence comprising a sequence at least 80%), 85%, 90%, 91 ~{i, 92%, 93%, 94%, 95%1, 96%), 9TVo, 98CJ-'u, 99!% or lOO'¾i identity to the sequence of SEQ ID NO: 12563, 12581, 12599, 12617, or fragments or reverse complements thereof.

In some embodiments, the Up3 sequence refers lo a bisulfrte converted 5 nucleotide sequence comprising a sequence at leasl 80%1, 85%), 90%, 91 %, 921Yii, 93%, 941:lii, 95%,, 96%, 97%J, 98%,, 991% or 100% identity to the sequence of SEQ ID NO: 12569, 12587, 12605 or 12623, or fragments or reverse complements thereof In some embodiments, the Up3 sequence refers to a bisulfite converted product of a methylated nucleotide sequence comprising a sequence at least 801%, BYYii, 90%, 91 (lt;,, 92%, 93%, 94~·;,, 95%, 96%, 97%, 10 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 12575, 12593, 12611 or 12629, or fh1g1m.'.11ts or reverse complements thereof.

In some embodiments, the Up3 sequence may be amplified using primers comprising the sequence of SEQ JD NOs: 12635 and/or 12641, or fragments or reverse complements thereof.

The term "UplO" as used herein refers to a nucleotide sequence comprising a 15 sequence at least 80%, 85%, 90'1/u, 91 %,, 92%, 93o/ii, 94%), 95%, 96r%, 97%, 98r%, 99%) or 100%1 identity to the sequence of SEQ ID NO: 12564, 12582, 12600 or 12618, or fragrnents or reverse co1npl.ernents thereof.

In some embodiments, the Up 10 sequence refors to a bisulfite convetied nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 ~I.J, 92%, 93%i, 94%1, 95%i, 96(.l/o, 97%, 98%i, 99%) or 100'1/o identity to the sequence of SEQ lD NO: 20 12570, 12588, 12606 or 12624, or fragments or reverse compkments thereof In some embodiments, the Up10 sequence refers to a bisulfiw converted product of a methylated nucleotide sequence comprising a sequence at least 80%, 85~1.i, 90%, 91 ~·-o, 92%, 93~.lo, 94%), 95%, 96%, 97%, 98%, 99'1/ii or 100'% identity to the sequence of SEQ ID NO: 12576, 12594, 12612 or 12630, or fragments or revt,rse complements thereof In some embodiments, the 25 Up 10 sequence may be amplified using primers cornprising the sequence of SEQ l D NOs: 12636 and/or 12642, or fragments or reverse complements thereof.

The term "Up 15--1" as used herein refers to a nucleotide sequence comprising a sequence at least 80'%, 85%, 90CJ-·o, 91 i)t,i, 92%, 93'Vo, 94%, 95CJ-'u, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ m NO: 12565, 12583, 12601 or 12619, or fragments or 30 reverse complements thereof.

In some embodiments, the Up 15-1 sequence refers to a bisulfite conve1i.ed nucleotide sequence comprising a sequence at least 80(/ii, 85~o, 901l.-1ii, 91 i;-o, 92%, 93%,, 94~/o, 95%, 96~'o, 97%, 98%), 99%i or 100% identity to the sequence of SEQ ID NO: 12571, 12589, 12607 or 12625, or fragments or reverse complements thereof In some embodiments, the Up 15--1 sequence refers to a bisulfrte converted product of a methylated 5 nucleotide sequence comprising a sequence at least 80'%, 85(Yii, 90%, 91 (Yii, 92%, 93'%, 94%, 95%;, 96%, 97%, 98%, 99(1/i, or 100%; identity to the sequence of SEQ ID NO: 12577, 12595, 12613 or 12631, or fragments or reverse complements thereof.

In some embodiments, the Up15-l sequence may be amplified using primers cornprlsing the sequence of SEQ ID NOs: 12637 and/or 12643, or fragments or reverse complements thereof.

The term "Up 15-2" as used herein refors to a nucleotide sequence comprising a sequence at least 80%1, 85%1, 90%, 91 ':11, 92%), 93 %i, 94(Yi,, 95%, 96%, 97~'o, 98%, 99~{, or 100% identity to the sequence of SEQ ID NO: 12565, 12583, 12647 or 12656, or fragments or reverse complements thereof In some embodirnents, lhe Up15-2 sequence refers to a bisulfite converted nudeotitk sequence comprlsing a sequence at least 80%, 85%, 90%\, 91 %, 15 92%i, 93%, 94,y;), 95!;,cii, 96%, 97'%, 98%, 99% or 100%, identity to the sequence of SEQ ID NO: 12571, 12589, 12650 or 12659, or fragments or reverse complements thereof.

In some embodiments, the Up 15-2 sequence refers to a blsu!fite converted product of a mdhylated nucleotide sequence comprising a sequence at least 80'%, 8YA,, 90'h), 91 %, 92%, 93~;,, 94%, 95%, 96%, 97%, 98%, 99%., or 100% identity to the sequence of SEQ ID NO: 12577, 12595, 20 12653 or 12662, or fragments or reverse complements thereof.

In some embodiments, the Up 15-2 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 12665 and/or 12668, or fragments or reverse complements thereof.

The tenn "Up20-1" as used herein refers to a nucleotide sequence comprising a sequence at least 80'}o, 85%,, 90%. 91 %, 92;/--u, 93%, 94%, 95-i;;i, 96%, 9Tl.1ii, 98%,, 99% or 25 100f% identity to the sequence of SEQ ID NO: 12566, 12584, 12602 or 12620, or fragments or reverse complements thereof In some embodiments, the Up20- l sequence refors to a bisulfite converted nucleotide sequence con1prising <l sequence at least 8()(;';i, 85%, 90%,, 91 %, 92~o, 93%, 94!;,-v, 95'~,;,, 9M'ii, 97%, 98'%, 99% or l ()()l}ii identity to the sequence of SEQ ID NO: 12572, 12590, 12608 or 12626, or fragments or reverse complements thereof.

In some 30 embodimems, the Up20-1 sequence refeJs to a bi sulfite converted product of a methylated 26 nucleotide sequence comprising a sequence at least 80%,, 85'%,. 90%), 91 ':1), 92}v, 93%i, 94%, 95%, 96%), 97%), 98%, 99°/o or 100°/o identity to the sequence of SEQ ID NO: 12578, 12596, 12614 or 12632, or fragments or reverse complements thereof In some embodiments, the Up20~ 1 sequenc<.;; may be amplified using primers comprising the sequence of SEQ l D NOs: 5 12638 and/or 12644, or fragments or reverse con1plements thereof The term "Up20~2" as used herein refors to a nucleotide sequence comprising a sequence at least 80%, 85%, 90~"ri, 91 %, 92%, 93%:,, 94°10, 95%, 96%,, 97%1, 98%, 99~S or 100% identity to lhe sequence of SEQ ID NO: 12566, 12584, 12648 or 12657, or fragments or reverse complements thereof In some embodiments, the Up20~2 sequence refors to a 10 bisulfite converted nucleotide sequence comprising a sequence at least 80%, 85%, 9!)'¾), 91 %, 92%, 93%, 94%, 95'Vi,, 96'%. 9TYii, 98~'o, 99'ilii or 100%) identity to the sequence of SEQ ID NO: 12572, 12590, 1265 ! or 12660, or fragments or reverse complements thereof.

In some embodiments, the Up20-2 sequence rnfors to a bisu1file converted product of a methylated nucleotide sequence comprising a sequence at least 80%), 85%), 90%, 91 %, 92%1, 93%, 94%, 15 95%, 96%,, 97'%, 98'1/ii, 99%, or 100% identity to the sequence of SEQ ID NO: 12578, 12596, I 2654 or 12663, or fragments or reverse cmnplernents thereof. ln some embodiments, the Up20-2 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 12666 and/or 12669, or fragments or n.,verse complements thereof The term "Up27" as used herein refers to a nucleotide sequence comprising a 20 sequence at least 80%, 85%,, 90%, 91 ~o, 92%, 93'%, 94~'ii, 95%, 96%, 97%, 98%, 99°.1; or 100% identity to the sequen,:;e of SEQ ID NO: 12567, 12585, 12603 or 12621, or fragments or reverse complements thereof.

In som.e ernbodirnents, the Up27 sequence refers lo a b.isulfite converted nucleotide sequence comprising a sequence at least 80~o, 85'%, 90%, 91 %, 92%, 93%, 94%, 95%, 96'%, 97%, 98~'o, 99% or 100%; identity to the sequence of SEQ ID NO: 25 12573, 12591, 12609 or 12627, or fragrnents or reverse complements thereof In some embodiments, the Up27 sequence refers to a bisulfile converted product of a methyhited nucleotide sequence comprising a sequence at least 80%1, 85%, 90'1/;;, 91%, 92%, 93%, 9t:\~/,, 9yy;,, 96':-'ii, 97%, 98~o, 99'3.l.i or lOO!J.fii identity to the sequence of SEQ ID NO: 12579, 12597, 12615 or 12633, or fragments or reverse complements thereof In some embodiments, the Up27 sequence may be amplified using pri.mers cornprising the sequence of SEQ ID NOs: 12639 and/or 12645, or fragments or reverse complements then:of The term "Up35-1" as ust'.d herein refers to a nucleotide sequence comprising a sequence at least 80'%), 8Y1;,, 90%), 91 %i, 92%,, 93%, 94%), 95'%, 96°10, 97%, 98%), 99~·o or 5 100'}~ identity to the sequence of SEQ ID NO: 12568, 12586, 12604 or 12622, or fragments or reverse complements thereof.

In some embodiments, the Up35-1 sequence reters to a bisulfite converted nucleotide sequence cornprising a sequence at least 80%, 85%, 90%,, 91 ~·o, 92%,, 93%, 94%, 95g,.~,, 96%, 97%1, 98%, 99%) or 100% identity to the sequence of SEQ ID NO: 12574, 12592, 12610 or 12628, or fragments or reverse complements thereof.

In some l O embodirnents, the Up35-l sequence reters to a bisulfite conve1ied product of a methylated nucleotide sequence comprising a sequence at least 80'YiJ, 85%1, 90~1i, 9 l 0it~, 92%), 93%;, 94%, 95%, 96%, 9TVo, 98%, 99/}o or 10(i<% identity to the sequence of SEQ ID NO: 12580, 12598, 12616 or 12634, or fragments or reverse complements thereof In some embodiments, the Up35-1 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 15 12640 and/or 12646, or fragments or reverse complements thereof The term "Up35-2" as used herein refers to a nudeotide sequence comprising a sequence at least 80%, 85%, 90%, 91 l;;o, 92%, 93(Yii, 94'%, 95':l'i), 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12568, 12586, 12649 or 12658, or fragments or reverse complements thereof In some embodiments, the Up35-2 sequence refers to a 20 bisulfite converted nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 %,, 92%), 93'1/ii, 94%,, 95%, 96%), 97'%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12574, 12592, 12652 or 12661, or fragments or reverse complements thereof.

In some embodiments, the Up35-2 sequence refers to a bi sulfite converted product of a methylated nucleotide sequence comprising a sequence at least 80%), 85%, 90%1, 91 %i, 92%i, 93%, 94%, 25 95'%, 96%, 97%, 98%, 99% or 100'% identity to the sequence of SEQ lD NO: 12580, 12598, 12655 or 12664, or fragments or reverse complements thereof.

In some crnbodin:ients, the Up35-2 sequence may be amplified using primers comprising the sequence of SEQ JD NOs: 12667 and/or 12670, or fragments or reverse complements thereof.

The term "SqBE 2" as used herein refers to a nucleotide sequence comprising a 30 sequence at least 80%, 85%i, 90~o, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99~{, or 28 100(~;) identity to the sequence of SEQ ID NO: 8209, 8251, 8293 or 8335, or fragments or reverse complements thereof In some embodiments, the SqBE 2 sequence refers to a bisulfite converted nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 %, 92%, 93~{,, 94~/o, 95%, 96%, 97%, 98%, 99% or 100%i identity to the sequence of SEQ lD 5 NO: 8223, 8265, 8307 or 8349, or fragments or reverse complements thereof.

In some embodiments, the SqBE 2 sequence refers to a bi sulfite convened product of a methylated nudeotide sequence comprising a sequence at least 80~o, 85%, 90%, 91%, 92%, 9Ji%, 94%, 95%, 96':Vii, 97%, 98%, 99% or 100% identity to the sequence of SEQ lD NO: 8237, 8279, 8321 or 8363, or fragments or reverse complements thereof.

In some embodiments, the SqBE 10 2 sequence may be amplified using primers comprising the sequence of SEQ 1D NOs: 8377 and/or 8391, or fragments or reverse complements thereof The term "SqBE 5" as used herein refers to a nuckotide sequence comprising a sequence at least 80%, 85%, 90%1, 91 %, 92%, 93%, 94%, 95';,·'o, 96%,, 97'%, 98%;, 99~{i or 100% identity to the sequence of SEQ ID NO: 8210, 8252, 8294 or 8336, or fragments or 15 reverse complements thereof.

In some embodiments, the SqBE 5 sequence refers to a bisulfite converted nucleotide sequence cmnprising a sequence at least 80%, 85%, 90%i, 91 %J, 92%,, 93%i, 94~;., 95%, 96'%, 97(:,;,, 98%, 99%i or 1001 1/;1 identity to the seqm:nce of SEQ 1D NO: 8224, 8266, 8308 OJ 8350, or fragments or reverse complements thereof In some embodiments, the SqBE 5 sequence refers to a bisulfite converted product of a methylated 20 nucleotide sequence comprising a sequence at least 80%, 85°10, 90%i, 91 %, 92%, 93 %, 94 %), 95%, 96%, 97%i, 98%, 99% or 100%:i identity to the sequence of SEQ ID NO: 8238, 8280, 8322 or 8364, or fragments or reverse complernents thereof.

In some embodiments, the SqBE 5 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 8378 and/or 8392, or fragments or reverse complements thereof. 25 The term "SqBE 7" as used herein refers to a nucleotide sequence comprising a sequence at least 80'),.ii, 85~"o, 90'%, 91 %, 92°/o, 93%, 94%,, 95%, 96%,, 97%, 98~'i:,, 99°10 or 100% identity to the sequence of SEQ 1D NO: 8211, 8253, 8295 or 8337, or fragments or reverse complements thereof.

In some embodiments, the SqBE 7 sequence refers to a bisulfite conve1ied nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 (Yb, 30 92%,, 93%, 94%, 9Yhi, 96(},o, 97%, 9WVii, 99% or l 00~o identity to the sequence of SEQ ID 29 NO: 8225, 8267, 8309 or 8351, or fragments or reverse complements tben:of In some embodirnents, the Sq BE 7 sequence refers to a bisulfite converted product of a methylated nucleotide sequence comprising a sequence at least 80%i, 85%), 90°1;;, 91 ~o, 92!\fi), 93%, 94%, 95°1&, 96%, 97%, 98%, 99%J or 100'}-;'i identity to the sequence of SEQ ID NO: 8239, 8281, 5 8323 or 8365, or fragments or reverse complements thereof.

In some embodiments, the Sq BE 7 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 83 79 and/or 8393, or fragments or reverse cornplements thereof The term "Sq BE 9" as used here.in refers to a nucleotide sequence comprising a sequence at !east 80%, 85%1, 90<%, 9Bo, 92~.1,J, 93 1.Yo, 94'%, 95%, 96~'o, 97'¾), 98%, 99°/ii or 10 100% identity to the sequence of SEQ ID NO: 8212, 8254, 8296 or 8338, or fragments or reverse complements thereof ln some embodiments, the Sq BE 9 sequence refers to a bisulfrte converted nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95';/<J, 96°,,,;,, 97%, 98%, 99(;.ti or 100%) identity to the sequence of SEQ fD NO: 8226, 8268, 8310 or 8352, or fragments or reverse comple1.nents theJeof In some 15 embodiments, the SqBE 9 sequence refers to a bisu1fite converted product of a methylated nucleotide sequence comprising a sequence at least 80%, 85%, 90'}ll, 91 '%, 92(\'o, 93'%, 94%, 95%,, 96%, 97%, 98%1, 99% or 100% identity to the sequence nf SEQ lD NO: 8240, 8282, 8324 or 8366, or fragments or reverse complements thereof.

In some embodiments, the SqBE 9 sequence may be amplified using primers comprising the sequence of SEQ lD NOs: 8380 20 and/or 8394, or fragments or reverse complements thereof.

The term "SqBE 1 0" as used herein refers to a nuckotide sequence comprising a sequence at Jeast 80%,, 85()10, 90'%, 91%, 92~o, 93%, 94(1/;;, 95%, 96'/;;, 97%, 98%, 99~o or 100% identity to the sequence of SEQ ID NO: 8213, 8255, 8297 or 8339, or fragments or reverse complernents thereof. I.n some embodiments, the SqBE l O sequence refers to a 25 bisulfite converted nucleotide sequence comprising a sequence at least 80':l,i, 85%, 90%, 91 %, 92.%, 93%, 94%i, 950/ii, 96%, 97%, 9W1i1, 99% or 100% identity to the sequence of SEQ ID NO: 8227, 8269, 8311 or 8353, or fragments or reverse complement:-: lheJeof.

In some embodiments, the SqBE l O sequence refers to a bi sulfite corrverted product of a methylated nucleotide sequence comprising a sequence at least 80%,, 85%i, 90%, 91 '%, 92~'o, 93%, 94j;'!l, 30 95%, 96%, 97%, 98%, 99%) or 100% identity to the sequence of SEQ ID NO: 8241, 8283, 8325 or 8367, or fragments or reverse complements thereof. ln some embodiments, the SqBE 10 sequence may be amplified using primers comprising the sequence of SEQ lD NOs; 83 81 and/or 8395, or fragments or reverse complements thereof The term "SqBE 11-1" as llsed herein refers to a nucleotide sequence comprising a 5 sequence at least 80~·'1i, 8Y1/ii, 90%, 91 i)fii, 92%, 93~.lo, 94°/!i, 95%, 96%), 97%, 9/fhi, 99% or 100% identity to the sequence of SEQ 1D NO: 8214, 8256, 8298 or 8340, or fragments or reverse complements thereof In some embodiments, the SqBE 11-1 sequence refers to a bisulfite convt,1ied nucleotide sequence comprising a sequence at leasl 80%, 85%, 90%, 91 %, 921%, 93%, 94~.lo, 95(1/i), 96•;:~,, 97~-'o, 98%, 99% or 100% identity to the sequence of SEQ ID 10 NO: 8228, 8270, 8312 or 8354, or fragments or reverse complements thereof In some embodinx,nts, the SqBE 11-1 sequence refers to a bisulfite converted product of a methylated nucleotide sequence comprising a sequence at least 80'Vo, 85%), 90%, 91 %, 92~'o, 93%, 94~·{i, 95%, 96%, 97%i, 98%, 99°/o or 100(1/o identity to the sequence of SEQ ID NO: 8242, 8284, 8326 or 8368, or fragments or reverse compkments thereof.

In some embodiments, the SqBE 15 11-1 sequence may be amplified using primers comprising the sequence of SEQ ID NOs; 8382. and/or 8396, or fragments or reverse complements thereof.

The term "SqBE 11-2" as used herein refers to a nucleotide sequence comprising a sequence at least 80°.lo, 85%, 90%, 91 (Yo, 92'%, 93n.,,;i, 94r;.{i, 95%,, 96%,, 97%, 98%, 99%i or 100% identity to the sequence of SEQ TD NO: 8214, 8256, 8405 or 842.0, or frngrnents or 20 reverse complements thereof.

In some embodiments, the SqBE 11-2 sequence refers to a bisu!fite converted nucleotide sequence comprising a sequence at least 80%, 8YYo, 90%1, 91 '%, 92.%i, 93%, ~W¾), 95'}o, 96~.lo, 97%, 98%, 99% or 100% identity to the sequence of SEQ !D NO: 8228, 8270, 8410 or 8425, or fragments or reverse complements thereof In some embodiments, the SqBE 11-2 sequence refers to a bi sulfite convened product of a methylated 25 nucleotide sequence cmnprising a sequence at least 80%, 85%, 90%, 91 ~\ 92i)lii, 93%, 94%, 95%, 96%,, 97%, 98°10, 99~·o or 100% identity to the sequence of SEQ ID NO: 8242, 82.84, 8415 or 8430, or fragments or reverse complements thereof, ln some embodiments, the SqBE 11-2 sequence may he amplified using primers comprising the sequence of SEQ \ D NOs: 8435 and/or 8440, or fragments or reverse complernents thereof. 31 The term "Sq BE 13" as used herein refers to a nucleotide sequence comprising a sequence at least 80(%, 85%, 90%, 91 %, 92%, 93°/o, 94%,, 9y,,,,;,, 96'%, 97'%, 9gr;.;;, 9cpy{) or 100%, identity to the sequence of SEQ ID NO: 8215, 8257, 8299 or 8341, 0r fragments or reverse complements thereof. ln some embodiments, the SqBE 13 sequence refers to a 5 bi sulfite converted nucleotide sequence comprising a sequence at least B0~r,, 85~{,, 90%, 9 l %, 92%, 93%, 94%, 95%,, 96'%, 97%:>, 9Whi, 991ii, or l0(Y~;, identity to the sequence of SEQ ID NO: 8229, 8271, 8313 or 83 5 5, or fragments or reverse complements thereof In some embodiments, the Sq BE 13 seqm.:nct~ refers to a bisuLfite converted product of a methylated nucleotide sequence comprising a sequence at least 80'%, 8YYo, 90%, 91 ~'o, 92%, 93%, 94'%, 10 95%;, 96%, 97'}'i), 9W1/1;, 99%, or l 00% identity to the sequence of SEQ lD NO: 8243, 8285, 8327 or 8369, or fragments or reverse complements thereof In sorne embodiments, the SqBE 13 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 8383 and/or 8397, or fragments or reverse complements thereof.

The term "SqBE 14-2" as used herein refers to a nucleotide sequence comprising a 1:.; sequence at least 80%, 85%,, 90%, 91 %, 92%, 93r1/i1, 94%, 95%,, 96'%, 97%,, 98~'o, 99% or 10on1.i identity to the sequence of SEQ ID NO: 8216, 8258, 8406 or 8421, or fragments or reverse complements thereof. ln some embodiments, the SqBE 14-2 sequence rders to a bisulfite converted nucleotide sequence comprising a sequence at least 80'Y<l, 8YYii, 90%, 91 o/ii, 92~·'o, 93%, 94%, 95%, 96'%, 97'%, 93%, 99%, or 10()(% identity to the sequence of SEQ ID 20 NO: 8230, 8272, 8411 or 8426, or fragments or reverse complements thereof.

Jn sorne embodiments, the SqBE l 4-2 sequence refrrs to a bisulfi1e converted product of a methylated nucleotide sequence comprising a sequence at least 80%,, 85~-'ii, 90();~,, 91 %, 92%, 93%, 94~S, 95%,, 96%1, 971Y;;, 98%, 99'}',i or 100% identity to the sequence of SEQ ID NO: 8244, 8236, 8416 or 843L or fragments or reverse complements !hereof. ln some embodiments, the SqBE 2"' 14--2 sequence may be amplified using primers comprislng the sequence of SEQ ID NOs: 8436 and/or 8441, or fragments or reverse complements thereof.

The term "SqBE 15'' as used herein refers to a nucleotide sequence con-1prising a sequence at least 80'}'o, 35':/ii, 90~o, 91 %, 92%,, 93%, 94'1/i, 9Y%, 96%, 97%, 98%), 99% or JOW'-"ii identity to the sequence of SEQ 1D NO: 8217, 8259, 8301 or 8343, or fragments or 30 reverse complements thereof In some embodirnents, the Sq BE 15 sequence refors to a 32 bisulfrte converted nucleotide sequence comprising a sequence at least 8m'ii, 85%,, 90%i, 91 °/ii, 92%), 93%), 94~·o, 95%, 96(Vi,, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8231, 8273, 8315 or 8357, or fragments or reverse cornplernents thereof.

In some embodiments, the SqBE 15 sequence refers to a bisulfite converted product of a methylated 5 nucleotide sequence comprising a sequence at least 80%, 85%, 90%1, 91 '%, 92°,r;,, 93%), 94°/t,, 95%,, 96~{,, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8245, 8287, 8329 or 8371, or fragments or reverse complements thereof.

In some embodiments, the Sq BE 15 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 8385 and/or 8399, or fragments or reverse complements thereof. 10 The term '"SqBE 16-1" as used herein refers to a nucleotide St'.qut~nce comprising a sequence at least 801).,~i, 85%, 90%, 91 %i, 92%i, 93%), 94%, 95%, 961%, 97%1, 98%, 99r;,;, or 100%1 identity to the sequence of SEQ lD NO: 8218, 8260, 8302 or 8344, or fragments or reverse complements thereof.

In some embodiments, the SqBE 16-1 sequence refers to a bisulfite converted nucleotide sequence comprising a sequence at least 80%), 85%, 90%, 91 %, 15 92%,, 93%, 94%,, 95%, 96%, 97';1i, 98%i, 99% or 1 0(Wo identity to the sequence of SEQ ID NO: 8232, 8274, 8316 or 8358, or fragments or reverse complements thereof In some embodiments, the SqBE 16-1 sequence refers to a bisulfite converted product of a methylated nucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91 (Yo, 92:%, 93 11/;), 94%, 95%,, 96%, 97%, 98~ .. t, 99'}o or 100(:'fi:i identity to the sequence of SEQ fD NO: 8246, 8288, 20 8330 or 8372, or fragrnents or reverse complements thereof.

In some embodiments, the SqBE 16-1 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 8386 and/or 8400, or fragments or reverse complements thereof.

The tenn "SqBE 16-2" as used herein refers to a nucleotide sequence comprising a 25 l O(YVi) identity to the st~quence of SEQ ID NO: 8218, 8260, 8407 or 8422, or fragments or reverse complernents thereof.

In some embodiments, the Sq BE 16-2 sequence refers to a bisulfite converted nucleotide sequence comprising a sequence at least 80°-1,, 85%,, 901~i;,, 91 %, 92%, 93%, 94%, 95';,'i:,, 96';/o, 97%, 98(;,·~, 99'/{i or 100'3./ii identity to the sequence of SEQ ID NO: 8232, 8274, 8412 or 8427, or fragments or reverse complements thereof.

In some 30 embodiments, the Sq BE 16-2 sequence refers to a bisulfite converted product of a methylated nucleotide sequence cornprising a sequence at least 80~-lo, 85%, 90~1c,, 91 %,, 92~·1i, 93%1, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8246, 8288, 8417 or 8432, or fragments or reverse complements thereof In some embodiments, the SqBE 16--2 sequence may be amplified using primers comprising the sequence of SEQ i D N Os; 5 8437 and/or 8442, or fragmt~nts or reverse cornplements thereof.

The term "Sq BE 17-1" as used herein refers to a nucleotide sequence comprising a sequence at least 80%, 8Y%, 90%, 91 %), 92%, 93%i, 94%, 95%, 96'3{,, 9T%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8219, 8261, 8303 or 8345, or fragments or reverse cornplements thereof.

In some embodiments, the SqBE 17-1 sequence refers to a IO bisulfitc convened nucleotide sequence comprising a sequence at least 8()~·;,, 85%, 9()(Vi;, 91 ~.10, 92%), 93%, 94%.1, 95%), %'Yi), 97(}·o, 98%i, 99% or 1 0O'Vi; identity to the sequence of SEQ ID NO: 8233, 8275, 8317 or 8359, or fragments or reverse complements thereof In some embodiments, the SqBE 17-l sequence refors to a bisulfite converted product of a rnethylated nudeotide sequence cornprising a sequence at least 80%), 85%, 90%1, 91 %, 92%i, 9J1%, 94%, 15 95%,, 96%, 971%, 9W;,'ii, 99% or 100% identity to the sequence of SEQ 1D NO: 8247, 8289, 8331 or 8373, or fragments or reverse co:mplernents thereof In sorne embodiments, the SqBE 17 -1 sequence may be amplified using primers comprising !he sequence of SEQ ID NOs: 8387 and/or 8401, or fragments or reverse complements thereof The term "SqBE 18" as used herein refers to a nucleotide sequence comprising a 20 sequence at least 80%, 85%, 90t;,.·o, 91 %, 92%, 93 ~-'ii, 94%, 95'!l,, 96%, 97%, 98%1, 99(Yo or 100% identity to the sequence of SEQ ID NO: 8220, 8262, 8304 or 8346, or fragments or reverse complements thereof.

In some embodiments, the Sq BE 18 sequence refers to a bisuLfite conveiied nucleotide sequence comprising a sequence at least 80'/o, 85%,, 9D1%, 9] %, 92%, 93%, 94%, 95%, 96~'o. 97%i, 98~o, 99':--'ii or 100'% identity to the sequence of SEQ ID 25 NO: 8234, 8276, 8318 or 8360, or fragments or reverse complements thereof.

In some embodiments, the Sq BE 18 sequence refers to a bisulfite converted product of a methylated nucleotide sequence comprising a sequence at least 80%, 85%), 90%, 91 °~), 92~'i,, 93%, 94%, 95%), 96£51;,, 97%i, 98'!/ii, 99% or l 00%, identity to the sequence of SEQ ID NO: 8248, 8290, 8332 or 8374, or fragments or reverse cornplemenls thereof.

In some embodiments, the SqBE 34 18 sequence may be amplified using primers comprising the sequence of SEQ U} NOs: 8388 and/or 8402, or fragments or reverse complements thereof.

The term "SqBE 22-1" as used herein refers to a nucleotide sequence comprising a sequence at least 80%, 85%, 90%i, 91 %), 92%i, 93%, 941:•·'o, 95(1/;i, 96%}, 97!1/ii, 98%,, 991% or 5 100°10 identity to the sequence of SEQ ID NO: 8221, 8263, 8305 or 8347, m fragments or reverse complements thereot~ In some embodiments, the SqBE 22-1 sequence refors to a bisulfite converted nucleotide sequence comprising a sequence at least 80%, 85~/i), 90%1, 91 %, 92%, 93'Yo, 94%, 95 1;/i), 96%, 97%), 981¾), 99c;.,-;, or 1 00~ci identity to the sequence of SEQ ID NO: 8235, 8277, 8319 or 8361, or fragments or reverse complements thereof In snme 10 embodiments, the SqBE 22-1 sequence refers to a bisulfite conve1ted product of a methylated nucleotide sequence comprising a sequence at least 80%i, 85%, 90%), 91 %J, 92%1, 93%i, 94%, 95%, 96%, 97°10, 98%i, 99% or 100°10 identity to the sequence of SEQ ID NO: 8249, 8291, 8333 or 8375, or fragments or reverse complernents thereof.

In some embodiments, the SqBE 22-1 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 15 8389 and/or 8403, or fragments or reverse complements thereof.

The term "SqBE 22-2" as used herein refers to a nucleotide sequence comprising a sequence at least 80%, 85°10, 90~'o, 91 %, 92%1, 93%i, 94%, 95%, 96n.{,, 97%, 98%i, 99% or 100% identity to the sequence of SEQ fD NO: 8221, 8263, 8409 or 8424, or fragments or reverse complernents thereof In some embodiments, the Sq BE 22-2 sequence refers to a 20 bisulfite converted nucleotide sequence cornprising a sequence at least 80%, 85~;1, 90%, 91 ~{,, 92%, 93%, 94~10, 95~o, 96%J, 9T½i, 98~·~, 99%i or 100%.J Identity to the sequence of SEQ ID NO: 8235, 8277, 8414 or 8429, or fragments or reverse complements thereof.

In some embodiments, the SqBE 22-2 sequence refr.,rs to a bisuHite converted product of a methylated nucleotide sequence cornprising a sequence at least 80%, 85%, 90%, 91 o/iJ, 92%, 93°10, 94%, 25 95%i, 96%, 97%J, 98%, 99°1;, or 100% identity to the sequence of SEQ ID NO: 8249, 8291, 8419 or 8434, or fragments or reverse complements thereof.

In some embodiments, the SqBE 22-2 sequence may be amplified using primers comprising the sequence of SEQ ID NOs: 8439 and/or 8444, or fragments or reverse complements thereof.

The term "SqBE 23'' as used herein refers to a nucleotide sequence comprising a 30 sequence at least 80(1/i,, 8Y%, 90%, 91 (J;;;, 92%,, 93%, 94%1, 95%1, 96~;,, 97%, 98%, 99% or 100% identity to the sequence of SEQ JD NO: 8222, 8264, 8306 or 8348, or fragments or reverse complements thereof.

In some t.'.mbodiments, the SgBE 23 sequence refers to a bisulfik converted nucleotide sequence cornprising a sequence at least 80°/;i, 85?,·'i,, 901%, 91 %i, 92!).~,, 93~fo, 94%i, 95%i, 96%, 97() ... o, 98%, 99%i or 100%i identity to the sequence of SEQ ID 5 NO: 8236, 8278, 8320 or 8362, or fragments or reverse complements thereof. ln some embodiments, the Sq BE 23 sequence refers to a bisu]fite converted product of a methylated nucleotide sequence comprising a sequence at least 80%i, 85%, 90%, 91 %, 92()-'ii, 93%i, 94'Vo, 95l'10, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8250, 8292, 8334 or 8376, or fragments or reverse complements thereof.

In some embodiments, the SqBE l O 23 sequence may be amplified using primers comprising the st:quence of SEQ ID NOs: 8390 and/or 8404, or fragments or reverse complements thereof. ln some instances, any of the nucleotide sequences disclosed herein contain one or more "Y" positions, Cytosine residues that may be methylated or unmethylated, and hence may be bisulfite converted to T (if unmethylated) or remain as a C (if methylated), are 15 designated with a "Y." fo some embodiments, a parental nuckotide sequence is fully u11111fithylatt:d if the sequence comprises a Tat every Y position following bisulfite conversion.

In some embodiments, a parental nucleotide sequence is fully methylated if the sequence comprises a C at every Y position following bisulfite conversion. rn some embodiments, a parental nucleotide sequence is partially methylated lf the sequence 20 cornprises at least one C at a Y position and at least one T at a Y position of the sequence following bisulfite convt:rsion, In some embodiments, the bisulfite converted sequences disclosed herein comprise at !east one Cat a Y position and at least one Tat a Y position, i.e., the parental sequence is partially methylated. 25 IL Overview This disclosure is based at least in part on the recognition that differential methylation of particular genomic loci may be indicative of neoplasia of the upper gastrointestinal tract, e_g,, esophagus, The present findings demonstrate that methylation at these genomic loci may be a useful hiomarker of neoplasia in the upper gastrointestinal tract.

The present findings 30 further demonstrate that the status of rnethy!ation at these genornic loci used in combination }6 with the status of somatic mutation(s) in TP53 may be a highly sensitive and specific biomarker of neoplasia in the upper gastrointestinal tract. 1n general, neoplasias may develop through one of at least three different patln,vays, termed chromosomal instability, microsatellite instability, and the CpG island rnethylator 5 phenotype (CIMP).

Although there is some overlap, these pathways tend to present somewhat different biological behavior.

By understanding the pathway of tumor development, the target genes involved, and the mechanisms underlying the genetic instability, it is possible to implement strategies to detect and treat the different types of neoplasias. 10 This disclosure is based, at k,ast in part, on the recognition that cerlain target genes may be silenced or inactivated by the differential rnethylation of CpG islands in the 5' flanking or promoter regions of the target gene, CpG islands are clusters of cytosineguanosine residues in a DNA sequence, which are prominently represented in the 5-flanking region or promoter region of about half the genes in our genome.

In particular, this 15 application is based at least in part on the recognition that differential methylation of particuiar genomic loci may be indicative of neoplasla of the upper gastrointestinal tract including, but not limited to, esophageal neoplasia, Additionally, this disclosure is based, at least in _pmi, on the recognition that sornatic mutati•o ns m• '•,' P. S.._,, (e .g., any o 1~ tnl e sornat1• c ·1···1),? .3. mutati•o ns d1' sc.· t oseld 11 erem• .1\ , m• coml )'ln at10• 11 20 with methylation of certain informative loci as disclosed herein, may serve as useful indicators of neoplasia, including esophageal neoplasia (e.g., esophageal adenocarcinoma).

In certain embodiments, the TP53 somatic mutation is any of the TP53 mutations disclosed herein, In certain embodiments, the TP53 somatic mutation is any nonsynonymous somatic mutation known in the art In certain embodiments, the TP53 somatic mutation is any one or 25 more 1:nutation at any one or more amino acid residue corresponding to amino acid residue 72, 105,108, 110,113,124,127,132,144,152,163, 175,183,194,213,214,218,232,234,248, 265, 273, 278, 306, 337, 347, or 639 of SEQ JD NO: 16205. ln certain embodiments, the TP53 sornatic mutation is any one or more mutation selected from the group consisting of: Leu194Arg, Gty105Asp, Arg273His, Tyrl63His, I1e232Thr, Arg213Ter, Arg273His, 30 Arg248Gln, Arg1751-Iis, ArgJ 1 0delinsGlnSer, Serl 83Tcr, Arg248G!n, Arg337Leu, 37 Lys132Arg, Leu265ThrfsTer7, Arg306Tcr, Cys124TrpfsTer25, Pro72Arg, Val2l 8Glu, His2 l 4Leu, GLn144Ter, Phe 113Ser, Tyr234His, Ser127Phe, Pro278Ala, Al:a347Thr, and Pro 152Leu of SEQ ID NO; 16205, In certain embodiments, the TP53 mutation ls any one or more mutation at any one or more nucleotide posit.ion corresponding to nuckotid::: position 5 108,215,314,338,380,395,430,455,487,524,548,581,637,639,641,653,695,700,743, 818,832,916, 1010, or 1039 of SEQ IDNO: 16206.

The sequence of SEQ ID NO: 16205 ( corresponding to GenBank Accession No" NP ~000537.3) is as follows: MEEPQSDPSVEPPLSQETFSDL \VKLLPENNVLSPLPSQAMDDLMLSPDD1EQW 10 FTEDPGPDEAPR1\,1PEAAPPV APAPAAPTPAAPAP.APSWPLSSSVPSQKTYQGSYGFRL GFLHSGTAKSVTCTYSPALNKl\1FCQLAKTCPVQL WVDSTPPPGTRVRAMAlYKQSQ HMTEVVR.RCPFLHERCSDSDGLAPPQHLIRVEGNLRVEY LDDRNTFRHSVVVPYEPPE VGSDCTTIHYNYMCNSSCMGGMNRRPILTHTLEDSSGNLLGRNSFEVRVCACPGRDR RTEEENLRKKGEPHFIELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQ!RGRERFE:rvr 15 FRELNEALELKDAQAGKEPGGSRAHSSHLKS KK GQSTSRHKKLMFKTEGPDSD The sequence of SEQ ID NO: 16206 (corresponding to GenBank Accession No.

NM_ 0005465) is as follows: GATGGGATTGGGGTTTTCCCCTCCCA .. TGTGCTCAAGACTGGCGCTAAAAGT TTTGAGCTTCTCAAAAGTCT AGAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGCiC 20 TCCGGGGACACTTTGCGTTCGGGCTG(rGAGCGTGCTTTCCACGACGGTGACACGC TTCCCTGGATTGGCAGCC/\GACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCA GTCAGA. TCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTA TGG AAACTACTTCCTCrAAAACAACGTTCTGTCCCCCTTGCCGTCCCA.. /,.GCAA. . TGGATG ATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCC 25 AGA.TGAAGCTCCCAGAATGCCAG/\..GGCTGCTCCCCCCCTGCCCCCTGCACCA_GCA GCTCCTACACCGGCGGCCCCI'GCACCAGCCCCCTCCTGGCCCCTGTCA. TCTTCTGT CCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATT CTGGGACAGCCAAGTCI-C3'1 GACTTGCACGTACfCCCCTGCCCTCAACAAGA TGTT TTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGl'I'GATTCCACACCCCCCr 30 CCCGGCACCCGCGTCCGCGCCA.. TGGCCATCTi\CAAGCAG]'CACAGCACATGACGG 38 AGGTTGTGAGGCGCTGCCCCCACCA.TGAGCGCTGCTCAGATAGCG/\TGGTCTGGC CCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGAT GACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTG GCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGG 5 CGGCATG.A.ACCGGAGGCCCATCCTCACCATCATCACACTGGA.. AGACTCCAGTGGT AATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCI'CTCCTGGGAGAG ACCGGCGCA .. CAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGA.GC TGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCA GCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGT l O GAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGUCCTTGGAACTCAAGGA TGCCC AGGCTGGGAAGGAGCCAGGCGGGAGCAGG·GCTCACTCCA .. GCCACCTGAA.GTCCA AAAAGGGTCAGTCTACCTCCCGCCA. TAAAAAACTCATGTTCAAGACAGAAGGGC CTC1ACTCAGACTGACATTCTCCACTTCTTCTTCCCCACTG/\CAGCCTCCCACCCCC ATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTTGAACCCTJ'GCTTGCAATAG 15 GTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGGGCTCCACTGA ACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAGGAGGATGGGGAGTAGGAC ATACCAGCTTAGATTTTA.AGGTTTTTACTGTGAGGGATGTTTGGGAGATGTAAGA AATGTTCTTGCAGTT AA(3GGTTAGTTTACAATCAGCCACATTCTAGGT,1\GGGGCC CACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTAACTT 20 CA .. AGGCCCA TATCTGTGAAATGCTGGCATTTGCACC'f.ACCTCACAGAGTGCA TTG TGAGGGTTAATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGG GGTCTAGAACTTGACCCCCTTGAGGGTGCTTGTTCCCTCTCCCTGTTGGTCG·GTGG GTTGGTAGTTTCTACAGTTGGGCAGCTGGTT AGGTAGAGGGAGTTGTCAAGTCTC TGCTGGCCCAGCCAAACCCTGTCTGACAACCTCTTGGTGAACCTTAGTACCTAAA 25 AGGAAATCTCACCCCATCCCACA .. CCCTGGAGGATTTCATCTCTTGTATATGATGAT CTGGA. TCCACCAA.GACTTGTTTTATGCTCAGGGTCAATTTCTTTTTTCTTTTTTTTT TTTTTTTTTCTTTTTCTTTGAGACTGGGTCTCGCTTTGTTGCCCAGGCTGGAGTGGA GTGGCG·TGATCTTGGCl'TACTGCAGCCTTTGCCfCCCCGGCTCGAGCAGTCCTGCC TCAGCCTCCGGAGTAGCTGGGACCACAGGTTCATGCCACCA'l.GGCCAGCCAACTT 30 TTGCATCJTTTTGTAGAGATGGGGTCrCACAGTGTTGCCCAGGCTGGTCTCAAACl·c 39 CTG-GG-CTCAGGCGATCCACCTGTCTCAGCcrcccAOAGTCCTGGGATTACAATTG TGAGCCACCACGTCCAGCTGGAi\GGGTCAACATCTTTTACATTCTGCAAGCACAT CTGCATTTTCACCCCACCCTTCCCCTCCTTCTCCCTTTTTATATCCCATTTTTATATC GATCTCTTATTTTACAATAAAACTTTOCTGCCACCTGTGTGTCTGAGGGGTG.

Esophageal adenocarcinoma (EAC) has steadily increased in incidence over recent decades.

With an 85~{, mortality rate this cancer is the most rapidly increasing cause of cancer mortality from solid tumors in the American population.

There has thus been substantial interest in developrnent of screening approaches for early detection of EAC and its precursor lesions of Barrett's esophagus (BE), However, the majority of EA Cs develop in l O patients without prior symptoms, and current approaches of endoscopic screening of individuals with persistent symptoms of gastro-esophageal reflux diseast\ combined with longitudinal screening of those found to have BE, have accordingly not had significant impact on reducing deaths from EACs.

As noted above, early detection of gastrointestinal neoplasia (e.g., neoplasia of the 15 upper gastrointestinal tract) coupled with appropriate intervention, is important for increasing patient survival rates.

Present systems for screening for esophageal neoplasia are deficient for a variety of reasons, including a lack of specificity and/or sensitivity (e.g., barium swallow) or a high cost and intensive use of medical resources (e.g., upper endoscopy or CT scan).

Alternative systems for detection of esophageal neoplasia would be useful in a wide range of 20 other clinical drcurnstances as welL For example, detecting esophageal neoplasia may select the patient to undergo therapies that include, but are not limited, to resection of (he neoplasia (via endoscopic resection or surgical resection), ablation of the neoplasia, chernotherapy, or radiation therapy.

As a further example, patients who have received surgical and/or pharmaceutical therapy for esophageal cancer may experience a relapse.

It would be 25 advantageous to have an alternative system for determining 'Nhether such patients have a recmTent or relapsed neoplasia of the upper gastrointestinal tract.

As a further exam pk, an alternative diagnostic systern would facilitate monitoring an increase, decrease or persistence of neoplasia of the upper gastrointestinal !rnct in a patient known to have such a neoplasia. A patient undergoing chemotherapy may be monitored to assess the efl:ectiveness of the therapy, III. __________ Mcthvlationof[n:forrnative_Loci _ as_Disease_Biomarkers, The present dlsdosure relates at least in part lO the identification of genomic loci whose altered DNA .. methylation is indicative of the presence of esophageal neoplasias and.ior metaplasias that include Barrett's esophagus (BE) and/or esophageal adenocarcinoma (EAC). 5 ln some ernbodiments, the Barrett's esophagus is associated with dysplasia.

In some embodiments, the dysplasia is high-grade dysplasia.

In some ernbodiments, the dysplasia is low-grade dysplasia.

In some embodiments, the methylation patterns of the inforn1ative loci as disclosed herein are determined in a sample taken from a subject as described herein and may be used to distinguish between suQjects having Barrdt's esophagus and subjects having 10 high grade dysplasia and/or low grade dyspiasia and/or esophageal adenocarcinoma.

Examples of the informative loci are provided herein. ln some embodiments, any of the nucleotide sequences disclosed herein, or fragments or reverse complernents thereof: may contain one or more "Y" residues.

Cytosine residut~s that may be methylated or unmethylated, and hence may be bi sulfite converted to T (if 15 umnethylated) or remain as a C (if methylated), rire designated with a "Y." In some embodiments, one or more of the Y residues in any of the sequences disclosed herein (or fragments or reverse complements thereof) designates a methylated C In some embodiments, one or more of the Y residues in any of the sequences disclosed herein (or fragments or reverse complements thereof) designates an unmethylated C.

In some embodiments, at least 20 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues in any of the sequences disclosed herein (or fragments or reverse complements thereof) correspond to methylated C residues< In sorne embodiments, at least 1, 2,3,4,5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, J7, 18, 19,20,21,22,23,24,25,26,27,28,29, or 3 0 of the Y residues in any of the sequences disclosed herein ( or fragments or reverse 25 complements thereof) correspond to unmethylated C residues. ln some embodiments, at least 10%, 20%, 30;}~, 40%,, 50%.,, 60')--'ii, 70%1, 80%), 9()(!.-,;,, or lO0~'o ofth.e Y residues in any of the sequences disclosed herein (or fragments or reverse complements thereof) correspond to methylated C residues.

In some embodiments, at kast 10'3{,, 2.0Yo, 30(;-'o, 40'/t,i, 50(}o, 60%, 70%i, 80%, 90%, or 1001% of the Y residues in any ofthe sequences disclosed herein (or 30 fragments or reverse complements thereof) a.re correspond to unmethylated C residues.

In 41 some embodiments, any of the sequences disclosed herein (or fragments or reverse complements thereof} is bisulfite-convened. ln some ernbodirnents, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues in any of the bisulfite .. converted sequences disclosed herein (or fragments or reverse 5 complements thereof) correspond to C, 1n some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,or30oftheY residues in any of the bisulfite~converted sequences disclosed herein (or fragments or reverse complements thereof) c01Tespond to T, In some embodiments, at least 10%, 20~·'o, 30%i, 40%, 50%, 60%, 70%J, 80%, 901%, or l()()(l,,;; of the Y residues in any of the bisulfiw-converted 10 sequences disclosed herein ( or fragments or reverse complements thereof) correspond to C residues.

In some embodiments, at least 101!1J, 2()(1/i;, 30%, 4ou1ii, 50%, 60%, 70%, 80%i, 90%, or 100% of the Y residues in any of the hisulfite-converted sequences disclosed herein (or fragments or reverse complements thereof) correspond to T residues.

In some embodiments, an infr1rmative loci in a subject is considered "methylated" for 15 the purposes of detennining whether or not the subject is prone to developing and/or has developed a metaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus with dysplasia such as high-grade or low-grade dysplasia) (e.g., esophageal cancer such as esophageal adenocarcinorna) if the loci is at least 10%, 20%, 30%, 40%, 50%, 60%i, 70%, 80~{,, 9Qi:/ii, or 100~{, methylated.

In some embodiments, a DNA 20 sample frorn a subject is treated with bisulfite, and the resulting bisulfite sequence corresponds to any of the nuck,otide sequences disclosed herein comprising a "Y" nudeotJde.

In some embodiments, if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues of the bisulfite-converted sequence have a C, the sequence is considered "methylated" for the purposes of determining 25 whether or not the subject is prone to developing and/or has developed a metaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Banett's esophagus with dysplasia such as high-grade or low-grade dysplasia) (e.g., esophageal cancer such as esophageal adenocarcinoma), In some embodiments, a DNA sample from a subject is treated with hisulfite, and the resulting bisulfite sequence corresponds to any of the nucleotide sequences 30 disclosed herein comprising a "Y" nucleotide. in some embodiments, if at least 10%, 20%, 42 30%), 40(1/ii, 50%, 60'%, 70%,, 80%, 90%, or 100~/o of the Y residues of the bisulfite-converted sequence have a C, the sequence is considered "methylated" for the purposes of determining whether or not the subject is prone to developing and/or has developed a metaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus with dysplasia 5 such as high-grade or low-grade dysplasia) (e.g., esophageal cancer such as esophageal adenocarcinoma} The disclosure provides for infonnative loci that may be used to assess whether a subject (e.g. a human) has or is prone to developing a rnetaplasia in the esophagus (e.g., Ban-ett's esophagus) or neoplasia (cg., Barrett's esophagus with dysplasia such as high-grade or low-grade dysplasia) (e.g., esophageal cancer such as esophageal 10 adenocarcinoma).

In some embodiments, one or rnore infr1rmative loci, as defined herein, may be used for determining whether a subject has or is likdy to develop, a metaplasia (e.g., Barrett's esophagus). ln some embodiments, one or rnore informative loci, as defined herein, may be used for determining whether a subject has or is likely to develop, a neoplasia (e.g., Barrett's esophagus with high grade dysplasia, or an esophageal cancer such as esophageal 15 adenocarcinoma), 1n some embodiments, one or rnore infonnative loci, as defined herein, may be used to distinguish bet'Neen whether a subject has a metaplasia in the esophagus (e.g., Barrett's esophagus) or an esophageal neoplasia (e.g., Barrett's esophagus with high grade dysplasia, or an esophageal cancer such as esophageal adenocarcinoma).

In some einbodirnents, the informative loci include sequences associated with any one 20 or more of the plus strand DNA sequeni:.:es having at least 80(i';i, 85%, 87%,, 90~·t, 91 %, 92%, 93%, 94%i, 95%), 96%, 97~·'o, 98%), 99?{, or 1 OOS.o identity to any of SEQ ID NOs: 1-428, 2569-2996, 5137-5531,7507-7532,7663-7668,7819-7842,7963-7976, 8047-8060, 8131- 8143, 8209-8222, 8293-8306, 8405-8409, 8447-8632,9563-9748, 10679-10825, 11561- 116! I, 11867-11917, 12173-12219, 12455-12460, 12491-12496, 12527--12532, 12563-12568, 25 12599-12604, 12647-12649, 12671-12907, 14093-14329, 15515-15537, 15653-15692, 15893-15932, 16133-16137, 16163-1616.5, 16181-16183, or 16199, or fragments or complements thereof.

In particular embodiments, the informative loci include sequences associated with any one or rnore of the plus strand DNA sequences having at least 80%J, 85%), 87(%, 90%, 91 %, 92~·'u, 93~/o, 94%, 95%, 96%, 97%, 98'1/ii, 99S.t or 100% identity to any of 30 SEQ ID NOs: 7963-7976, 8047-8060, 8131-8143, 12455-12460, 12491-12496, 12527-12532, 43 16163-16165, 16181-16183, or 16199, or fragments or complements thereof.

In some embodiments, the infom1ative loci are associatt,d with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples, as compared to the same sample types taken from a healthy control subject. 1n some embodiments, tht, informative loci that are 5 associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include sequences associated with any one or more of the plus strand DNA sequences having at least 80~~., 85%, 87%1, 90%,, 91 %, 92%, 93%1, 94%, 95%, 96%,, 97%, 98'Vii, 99%1 or 100% identity to any of SEQ ID NOs: 1-428, 2569-2996, 5137- 5531, 7507-7532, 7663-7668, 7819-7842, 7963-7976, 8047-8060, 8131-8143, 8209-8222, 10 8293-8306, or 8405-8409, or fragments or complements thereof. ln particular embodiments, the informative lod that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include sequences associated with any one or more of the plus strand DNA sequences having at least 80~'o, 85%, 87%, 90'Yii, 91 %, 92'hJ, 93%, 94%i, 95~''o, 96°/o, 97%, 98%, 991:',{i or 1001:',,h identity to any of SEQ ID NOs: 7963-7976, 8047- 15 8060, or 3131-8143, or fragments or complements thereof In some embodiments, the informative loci are associated with increased methylation in an esophageal adenocarcinoma sample and/or a Banett's 'Nith low grade or high grade dysp1asia as compared to a sample of the same type taken from a subject having Barrett's esophagus v,:ithout dysplasia.

In some embodiments, the informative kH.:i that are associated with increased methy1ation in an 20 esophageal adenocarcinorna samp!e or a Barrett's with low grade or high grade dysplasia sample include any one or more of the sequences having at least 80%, 85 1)/ii, 87%, 90'%, 91 %, 92%, 93%, 94%, 95%, 96%, 9T!"o, 98%), 99~o or 100%) identity to any of SEQ ID NOs: 844 7- 8632, 9563-9748, 10679-10825, 1156l-11611, 11867-11917, 12173-12219, 12455-12460, 12491-12496, or 12527-12532, or fragments or complements thereof In particular 25 embodiments, the informative loci that are associated with increased methylation in BmTt~tt's with low grade dysplasia sample include any one or more of the sequences having at least 80%, 85%i, 8711/;), 90%i, 91 (%, 92r;,;,, 93S"o, 94%1, 95%, 96l!-'(i, 97~'(), 98%,, 99% or 1 O(Wo identity to any of SEQ JD NOs: 12455-12460, 12491-12496, or 12527-12532, or fragments or complements thereof In particular embodiments, the informafrve loci that are associated with 30 increased r:nethybtion in an esophageal adenocarcinoma sample or a Barrett's with high grade 44 dysplasia sample include any one or rnore of the sequences having at least 80'Yo, 85°1ii, 87%i, 90%, 91%, 92%i, 93°1;), 94~;., 95%J, 96%, 97%, 98(%, 99% or 100% identity to any of SEQ ID NOs: 12455~12460, 12491-12496, or 12527-12532, or fragrnents or compleJ'nents thereof.

Tn some embodiments, the informative loci are associated with reduced methylation in an 5 esophageal adenocarcinoma sample as compared to a sample of the same type taken from a subject having Barrett's esophab'1.lS, In some embodiments, the Informative !oci that are associated with rt~duced methylation in an esophageal adenocardnoma sample include any one or more of the sequences having at least 801;,·o, 85%, 87°10, 90%, 91 (1/ci, 92%), 93'1/.i, 94(;.lo, 95%), 96•Y;), 97%, 98%, 99% or 100% identity to any of SEQ 1D NOs: 12671--12908, 14093- 10 14329, 15515-15537, 15653-15692, 15893-15932, 16133-16137, 16163-16165, 16181-16183, or 16199, or fragments or complements thereof In particular embodiments, the informative loci that are associated with reduced methylation in an esophageal adenocarcinoma sample include any one or more of the sequences having at least 80%, 85%i, 87'~·'o, 9(Wo, 91 ';'o, 921 ¾1, 93%, 94%, 95';-o, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 16163- 15 16165, 16181-16183, or 16199, m fragments or complements thereof, ln some embodiments, the infonnative loci or amplicon of the inforrnative loci are treated with an agent, such as bisulfite.

In some embodiments, the informative loci include sequences that have been treated with hisulfite.

In some embodiments, the disclosure provides for bisulfite control sequences of any of the plus DNA strands disclosed herein.

In 20 some embodiments, the disclosure provides fbr bisulflte-treated unmethylated sequences of any of the plus DNA strands disclosed herein, 1n some embodiments, the bisulfite-converted plus-strand control DNA. sequences include any one or more having at ]eas1 80%,, 85(}·ii, 87~ .. ~, 90%i, 91 %, 92%, 93%, cw%, 95'.1/o, 96%, 9:€1/;), 98%,, 99% or 100°10 identity to any of SEQ 1D NOs: 857-1284, 3425-3852, 5927--6321, 7559-7584, 7715-7740, 7867-7890, 7991-8004, 25 8075-8088, 8157-8169, 8223--8236, 8307-8320, 8410-8414, 8819-9004, 9935-10120, 10973- 11119, 11663-11713, 11969-12019, 12267-12313, 12467-12472, 12503-12508,or .\2539- 12544, 12569-12574, 12605-12610, 12650--12652, or fragments or complements thereof In particular embodiments, the bisulfite-converted plus-strand control DNA sequences include any one or more having at least 80%, 85%J, 87'%, 90%,, 91 %i, 92%, 93%, 94%, 95;;,;, 96%, 30 97(}-;;, 98%i, 99(½, or lO(Yhi identity to any of SEQ ID NOs: 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320, 8410-8414, 12467-12472, 12503-12508,or 12539-12544, 12569- 12574, 12605-12610, 12650-12652, or fragments or complements thereof.

In some embodiments, the informative loci are associated with increased rnethylation in both Barrett's esophagus and esophageal adenocarcinoma samples, as compared to the same sample types 5 taken from a healthy control subject In some embodiments, the disclosure provides for bisulfite-treated unrnethylated sequences of any of the plus DNA strands that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples, as compared to the same sample types taken from a healthy control subject In some embodiments, the bi sulfite converted sequences of any of the plus DNA strands that are lO associated \Vith increased methylation in both Banett's esophagus and esophageal adenocarcinoma samples are selected from the group consisting of sequences having at least 7890, 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320, or 8410-8414, or fragments l 5 or complements thereof ln particular embodiments, the bisulfite converted sequences of any of the plus DNA strands that art, associated with increased methy1ation in both Barrett's esophagus and esophageal adenocardnoma samples include any one or more bisulfiteconvened methylated plus-strand DNA sequences selected from the group consisting of sequences having at least 80'%, 85%1, 87';,o, 90%i, 91 (1/;,, 92%, 93°/(i, 94%, 95%, 96%, 97%, 20 98%, 99%i or 100% identity to any of: SEQ ID NOs: 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320, or 8410-8414, or fragments or complernents thereof.

In sorne embodiments, the disclosme provides for bisuifite--treated unmethy1ated sequences of any of the plus DNA strands that are associated with increased methylation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia sample as 25 compared to a sample of the same type taken from a subject having Barrett's esophagus without dyspbsia.

In some embodiments, the bisulfite converted sequences of any of the plus DNA strands that are associated with increased methylation in an esophageal adcnocarcinoma sample or a Barrett's with !ow grade or high grade dysplasia sample include any one or more bisulfite-conveiied methylated plus-strand DNA .. sequences selected from the group consisting 30 having at least 80'%, 85!%, 87%, 9m1i, 91 %, 92%, 93'!/ii, 94(%, 95%, 96%,, 97%, 98%;, 99':/o or 46 100°.fo identity to any of SEQ ID NOs: 8819-9004, 9935-10120, 10973-11119, 11663-11713, 11969-12019, 12267-12313, 12467-12472, 12503-12508,12539-12544, 12569-12574, 12605- 12610, or 12650-12652, or fragments or complemen!s then.'.of.

In particular embodiments, the hisulfite conve1ied sequences of any of the plus DNA strands that are associated with 5 increased methylation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia sample include any one or more of the sequences having at least 80%,, 85%, 87%, 90%, 91 ~,;,, 92~/o, 93%i, 94%, 95%), 96%, 97%), 9Wlti,, 99% or 100% identity to any of SEQ 1D NOs: 12467-12472, 12503-12508, 12539-12544, 12569-12574, 12605-12610, or 12650-12652, or fragments or complements thereof: In some embodiments, the infonnative 10 loci are associated with reduced methylation in an esophageal adenocarcinoma sample as compared to a sarnple of the same type taken from a subject having Barrett's esophagus.

In some embodiments, the disclosure provides for methylated control sequences of the plus DNA strand that are associated with reduced methy!ation in an esophageal adenocarcinoma sample as compared to a sample of the same type taken from a subject having Barrett's 15 esophagus. 1n some embodiments, the methylated control sequences of any of the plus DNA strands that are associated with reduced methylation in an esophageal adenocarcinoma sample include any one or more hisulfite-converted methylated plus-strand DNA sequences selected from the group consisting having at least 80%, 85%, 87%, 90%,, 91 %i, 92%,, 93%, 94!;.•-;,, 95%, 96(1/t,, 97%), 9SCY;,, 99%) or 100<;.·~ identity to any of SEQ ID NOs: 13145-13381, 14567-14803, 20 15561-15583, 15733-15772, 15973-16012, 16143-16147, 16169-16171, 16187-16189 or 16201, or fragments or complements thereof In particular embodiments, the methylated control sequences of any of the plus DNA strands that are associated with reduced methylation in an esophageal adenocarcinoma sample include any one or more hisulfiteconverted methylated plus-strand DNA sequences selected from the group consisting having 25 at !east 80%, 85%, 8PVii, 90t;.,;;, 91 %, 92%, 93~'o, 94°10, 95%), 96%, 97%), 98%1, 99% or 1 om.-;, identity to any of SEQ ID NOs: 16169-1617), 16187-16189 or 16201, or fragments or compkments thereof.

In some embodiments, the informative loci or amplicon of the informative loci are treated with an agent, such as hi sulfite.

In some embodiments, the informative loci include 30 sequences that have been treated with bisulfite.

In some embodiments, the infonnative loci 47 include rnethyhued nucleic acid sequenees that have been treated ,vtth bisulfite, In some embodiments, the bisulfite-converted methylated plus-strand DNA sequences have at least 80?,'c, 85%, 87%, 90%1, 9l~o, 92%, 93%, 94%}, 9YY;i, 96S,•;, 97'%, 98%,, 99% or 100% identity to any of SEQ ID NOs: 1285-1712, 3853-4280, 6322-6716, 7585-7610, 7741-7766, 7891- 5 7914, 8005-8018, 8089-8102, 8170-8182, 8237-8250, 8321-8334, 8415-8419, 9005-9190, 10121-10306, 11120-11266, 11714-11764, 12020-12070, 12314-12360, 12473-12478, 12509-12514 or 12545-12550, 12575-12580, 12611-12616, 12653-12655, or fragments or complements thereof In paiiicular embodiments, the bisulfite-converted methylated plusstrand DNA sequtnces have at least 80%, 85'!/ii, 8J(l,;,, 90%, 91 'Vi), 92~'o, 93%, 94t:---i,, 95%, 96%, 10 9Thi, 98%, 991//ii or 100!)/4} identity to any of SEQ ID NOs: 8005-8018, 8089-8102, 8170-8182, 12473-12478, 12509-12514 or 12545-12550, or fragments or complements thereof.

In some embodiments, the infbnnative loci are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinorna samples, as compared to the same sample types taken from a healthy control subject In sorne ernbodiments, the informative loci that are 15 associated with inereased methyiation in both Barrett's esophagus and esophageal adenocarcinoma samples include any one or more bisulfite-converted methylated plus-strand DNA sequences selected from the group consisting of sequences having at least 80%1, 85%, 87%, 90%, 91 %,, 92%i, 93%i, 94%, 95 1%, 96%, 97%, 98%1, 99i% or 100% identity to any of SEQ ID NOs: 1285-1712, 3853-4280, 6322-6716, 7585-7610, 7741-7766, 7891-7914, 8005- 20 8018, 8089-8102, 8170-8182, 8237-8250, 8321-8334, or 8415-8419, or fragments or complements thereof.

In particular embodiments, the informative loci that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include any one or rnore bisulfite-conve1ied methylated plus-strand DNA sequences selected from the group consisting of sequences having at least 80(1/o, 85'%, 8T1/;i, 90%, 91 '1/i), 92%, 25 93%,, 94%i, 95%, 96%, 97%.,, 98l;1J, 99(1/o or JOO% identity to: SEQ ID NOs: 8005--8018, 8089- 8102, 8170-8182, 823 7-8250, 8321-8334, or 8415-8419, or fragments or complements thereof. ln some embodiments, the informmivt, loci are associated with increased methy lation in an esophageal adenocarcinorna sample or a Barrett's with low grade or high grade dysplasia sarnple as compared to a sample of the same type taken from a subject having 30 Barrett's esophagus without dysplasia.

In sorne embodiments, the informative loci that are 48 associated with increased methylation in an esophageal adenocarcinoma samp]e or a Barrdt's with lcnv grade or high grade dysplasia sample include any one or more bisulfite-converted methylated plus-strand DNA sequences selected from the group consisting of sequences having at least 80%, 85%, 87%1, 90%, 91 ~·o, 92%, 93%i, 94~.la, 9YV(), 96'%, 971%, 98%, 99'\/ii or 5 lOO~·o identity to SEQ ID NOs: 9005-9190, l 0121-10306, 11120-11266, l 1714-11764, 12020-12070, 12314-12360, 12473-12478, 12509-12514, 12545-12550, 12575-12580, 12611-12616, or 12653-12655, or fragments or complements thereof.

In particular embodiments, the informative loci that are associated with increased methylation in an esophageal adenocarcinorna sample or a Barrett's with low grade or high grade dysplasia 10 sample include any one or more of the sequernx,s having at least 80%, 85 1%, 87(~,;;, 90~-o, 91 (l,-;), 92%, 93%i, 94%, 9Y:/i;, 96°10, 9T%, 98%i, 991;,o or 100'% identity to SEQ ID NOs: 12473- 12478, 12509-12514, 12545-12550, 12575-12580, 12611-12616,or12653-12655,or fragments or complements thereof.

In some embodiments, the infrmnative loci are associated with reduced rnethylation in an esophageal adenocarcinoma sample as compared to a sample 15 of the same type taken from a subject having Barrett's esophagus.

In some embodiments, the infonnative loci that are associated with reduced rnethylation in an esophageal adenocarcinorna sample include any one or more hisulfite-convcrted methylated plus-strand DNA sequences selected from the group consisting of sequences having at least 80'1/;,, 85%, 87%), 901%, 91(Yi,, 92%, 93Yo, 94%, 95%), 96%;, 97!;,i,, 98%, 99%, or 100~·;, identity to SEQ ID 20 NOs: 13382-13618, 14804-15040, 15584-15606, 15773-15812, 16013-16052, 16148-16152, 16172-16174, 16190-16192 or 16202.

In particular er:nbodime:nts, the inforrnative loci that are associated with reduced methylation In an esophageal adenocarcinoma sample include any one or more of the sequences having at least 80%, 85%,, 87%, 90%, 91 %, 92%, 93(Yo, 941%, 95%i, 96%, 97%, 98%, 99% or l 00% identity to SEQ ID NOs: 16172-1617 4, l 6190--16192 or 25 16202, or fragments or complements thereof In some embodiments, the informative loci indude sequences associated with any of the minus strand DNA sequences having at least 80!}1i, 851}'0, 8TVii, 90%, 91 %,, 92(;,;), 93%, 941%, 9S1J;;,, 96%i, 97%, 98~~, 99% or l 00% identity to any of SEQ ID NOs: 429-856, 2997- 3424, 5532-5926,7533-7558, 7689-7714,7843-7866,7977-7990, 8061-8074, 8144-8156, 30 8251-8264, 8335-8348, 8420-8424, 8633-8818,9749-9934, 10826-10972, 11612-11662, 49 11918--11968, 12220-12266, 12461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, 12656-12658, 12909-13144, 14330-14566, 15538-15560, 15693-15732, 15933-15972, 16138--16142, 16166-16168, 16184-16186 or 16200, or fragments or complements thereof. ln particular ernbodiments, the infonnative loci include sequences 5 associated with any of the minus strand DNA sequences having at least 801%, 85%,, 87%, 901;,~,, 91 %, 92iJ,,~i, 93%, 94%, 95%i, 96%, 97%, 98(Yo, 99%, or 100% identity to any of SEQ ID NOs: 7977-7990, 8061-8074, 8144-8156, 8251-8264, 8335-8348, 8420-8424, 12461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, 12656-12658, 16166-16168, 16184-16186 or 16200, or fragments or complements thereof. ln some embodiments, the 10 informative loci are associated with increased methylation in both Barrett's esophagus a11d esophageal adenocarcinoma samples, as compared to the same sample types taken from a healthy control subject.

In some embodirnents, the informative loci that are associated with increased mt,thylation in both Barrett's esophagus and esophageal adenocarci noma samples include sequences associated with any one or more of the minus strand DNA sequences 15 having at least 80%, 85%, 87%, 90%J, 91 (Y;;, 92%), 93'%, 94%, 95%, 96~o, 97%, 98%, 99"}'ii or l 0(Y1/o identity to any of SEQ ID NOs: 429-856, 2997-3424, 5532-5926, 7533-7558, 7689- 7714, 7843-7866, 7977-7990, 8061-8074, 8144-8156, 8251-8264, 8335-8348, 8420-8424,or fragments or complements thereof In particular embodiments, the informative loci that are associated with increased methyiation in both Barrett's esophagus and esophageal 20 adenocarcinoma samples include sequences associated with any one or more of the plus strand DNA sequences having at least 80~·,;,, 85%, 87~'o, 90%, 91 %, 92°10, 93%, ~Wl./ii, 95%,, 96%i, 97~"o, 98%i, 99(% or 100% identity to any of SEQ ID NOs: 8251--8264, 8335-8348, 8420- 8424, or fragments or complements thereof.

In some embodiments, the informative loci are associated with increased methylation in an esophageal adenocarcinoma sample or a Barrett's 25 with low grade or high grade dysplasia sample as compared to a sample of the same type taken from a su~ject having Barrett's esophagus without dysplasia, In some embodiments, the informative [ocl that are associated with increased methylation in an esophageal adenocarcinoma sample or a BmTett's with low grade or high grade dysplasia sample include any one or more of tht:. sequences having at ]east 80".%, 85%, 87%, 90%i, 91 %, 92.%), 93%, 30 94%, 95%, 96%,, 97')/ii, 98%, 99%, or 100% identity to any of SEQ lD NOs: 8633-8818, 9749- 50 9934, 10826-10972, 11612-11662, 11918-11968, 12220-12266, 12461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, or 12656-12658, or fragments or cornplements thereof.

In particular embodiments, the infom1ative loci that are associated with increased me1hylation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high 5 grade dysp1asia sample include any one or more of the sequences having at least 80%,, 85%, 87%, 90%), 91 %,, 92%, 93%), 94%, 95%, 96%,, 9TYii, 98'Vi,, 99'% or 100% identity to any of SEQ ID NOs: 12461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, or 12656-12658, or fragments or complements thereof In some embodiments, the Informative loci are associated with reduced methylation in an esophageal adenocarcinoma sample as 10 compared to a sample of the same type taken from a subject having BmTett's esophagus.

In some embodiments, the informative loci that are associated with reduced methylation in an esophageal adenocarcinoma sample in.dude any one or more of the sequences having at least 80%J, 85~o, 8TVo, 9iYh,, 91 '%, 92'%, 9Jf%, 94~.{i, 95%, 96%, 97%1, 98%, 99% or 100% identity toanyofSEQIDNOs: 12909-13144, 14330-14566, 155.38-15560, 15693-15732, 15933- 15 15972, 16138-16142, 16166-16168, 16184-16186 or 16200, or fragments or complements thereof.

In particular embodiments, the informative loci that are associated with reduced methylation in an esophageal adenocarcinorna sample include any one or more of tht'. sequ~mces having at least 8()(j;,, 85%, 87~&, 90%i, 91 %, 92%, 93%, 94%, 95%, 96%,, 97%, 98%, 99%, or 1001;.·~ identity to any of SEQ ID NOs: 16166-16168, 16184-16186 or 16200, or 20 fragments or complernents thereof.

In some embodiments, the informative loci or amplicon of the informative loci are treated with an agent, such as bisulfile.

In some embodiments, the informative loci include sequenct:s that have been treated \Vith bisulfite.

In some embodiments, the disclosure provides for bisulfite control sequences of any of the minus DNA strands disclosed herein. ln 25 some embodiments, the disclosure provides for hisulflte-treated sequences of any of the minus DNA strands disclosed herein.

In some embodiments, the bisulfitc-convt'.1ied minus-strand control DNA sequences include any one or more of the sequences having at least 801;-'i,, g5n;;), 87%,, 90%i, 91 %, 92%, 93%, 94%, 950/ii, 96f;.~, 9T}'o, 98%, 99% or 100%i Identity to any of SEQ ID NOs: J 713-2140,4281-4708, 6717-7111, 7611-7636, 7767-7792, 79 l 5-7938, 8019- 30 8032, 8103-8116, 8183-8195, 8265-8278, 8349-8362, 8425-8429, 9191-9376, 10307-10492, 51 11267-11413, 11765-11815, 12071-12121, 12361-12407, 12479-12484, 12515-12520, 12551-12556, 12587-12592, 12623-12628, or 12659-12661, or fragments or complements thereof.

In pmiicular embodiments, the bisuHite-converted minus-strand control DNA sequences include any one or more the sequences having at least 80%, 85%, 87%, 900,.o, 91 ;y;,, 5 921;1;,, 93%1, 94%, 9SC:,,i,, 96%,, 97°1ii, 98{);·ci, 99% or 100% identity to any of SEQ l D NOs: 8019- 8032, 8103-8116, 8183-8195, 12479-12484, 12515-12520, or 12551-12556, or fragments or complements thereof. ln some embodirnents, the informative loci are associated \Vith increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples, as compared to the same sample typt!S taken from a healthy control subject.

In some 10 t,mbodiments, the disclosure provides for bisulfite-treated sequences of any of the minus DNA strands that are associated with increased mdhylation in both Barrett's esophagus and esophageal adenocarcinoma samples, as compared to the same sample types taken from a healthy control subject In some embodiments, the sequences of any of the minus DNA strands that are associated with increased methylation in both Barrett's esophagus and 15 esophageal adenocarcinoma sarnples ,1re selected from the group consisting of sequences having at least 80%i, 85%, 87%, 90%, 91 %, 92%, 93~.ln, 94%, 9.5~·1i, 96%;, 971;,~, 98'3./,), 99%, or 100% identity to any of SEQ IDNOs: 1713-2140, 4281-4708, 6717-7111, 7611-7636, 7767- 7792, 7915-7938, 8019-8032, 8103-8116, 8183-8195, 8265-8278, 8349-8362, or 8425-8429, or fragments or con1plemems thereof.

In particular embodiments, the sequences of any of the 20 minus DNA strands that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include any one or more bisuHit.econverted methylated minus-strand DNA sequences selected from the group consisting of sequences having at least 80%), 8Yhi, 87%1, 90%, 91%, 92';:'o, 93%, 94%1, 95%), 96%, 97%, 98(1/1i, 99~·'o or 10(Wo identity to: SEQ ID NOs: 8019-8032, 8103-8116, 8183-8195, 8265-8278, 25 8349-8362, or 8425-8429, or fragrnents or complements thereof In sorne ernbodiments, the disclosure provides for bisulfite-treated sequences of any of the minus DNA strands that me associated with increased methylation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia sample as compared to a sample of the same type taken from a subject having Barrett's esophagus without dysplasia.

In some embodiments, 30 the sequences of any of the minus DNA strands that are associated with increased rnethylation 52. in an esophageal adenocardnorna sample or a Barrett's ·with low grade or high grade dysplasia sample include any one or more bisulfite-converted methylated minus-strand DNA sequences sek~cted from the group consisting of sequences having at least 80%,, 85%, 87%, 90%, 91 %,, 92?.10, 93•~.1;), 941!,·t,, 95%, 96%i, 97~.f::,, 98%, 99~·o or 100% identity to SEQ ID NOs: 5 9191-9376, 10307-10492, 11267-11413, 11765-11815, 12071-12121, 12361-12407, 12479- 12484, 12515-12520, or 12551-12556, 12587-12592, 12623--12628, or 12659-12661, or l"i'agments or complements thereof.

In particular embodiments, the unmethylated sequences of any of the minus DNA strands that are associated with increased methyl a.ti on in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia lO sample include any one or more of the sequences having at least 80%1, 85(Y~, 8PVi,, 90'}'1i, 91 :; .. ~, 92%, 93%1, 94S·~, 95°10, 96%, 97%, 98%, 99%1 or 100% identity to SEQ ID NOs: 12479• 12484, 12515-12520, 12551-12556, 12587-12592, 12623-12628, or 12659-12661, or fragments or complements thereof In some embodiments, the infonnative loci are associated with reduced mdhylation in an esophageal adenocarcinoma sample as compared to a sample 15 of the same type taken from a subject having Barretl's esophagus< In some embodiments, the disclosure provides for methylated control sequences of the minus DNA strand that are associated with reduced rnethylation in an esophageal adenocarcinoma sample as compared to a sarnple of the same type taken from a subject having Ban-ett's esophagus, In sorne embodiments, the methylated control sequences of any of the minus DNA strands that are 20 associated with reduced methylation in an esophageal ad.enocarcinoma sample include any one or more bisulfite-converted methylated minus-strand DNA sequences selected from the group consisting of sequences having at least 80(1/;,, 85%, 87%i, 90%, 91 %i, 92!;,'o, 93%, 94%), 95 1%, 96%, 971)/ii, 98%, 99% or l 00% identity to SEQ ID NOs: 13619-13855, 15041-15277, 15607-15629, 15813'-15852, 16053-16092, 16153-16157, 16175-16177, 16192--16195 or 25 16203, or fragments or complements thereof. ln particular embodiments, the methylated control sequences of any of the minus DNA strands that arc associated with reduced rnethylation in an esophageal adenocarcinoma sample include any one or more bisulfiteconveiied methylated rnirrns-strand DNA sequences selected from the group consisting of sequences having at least 80%1, 85%, 87%i, 90%, 91 %), 92%, 93%,, 94%), 95')!'.'l, 96%, 97';.\ 53 9W::o, 99%; or 10()(!/) identity to SEQ ID NOs: 16175-16177, 16192-16195 or 16203, or fragments or complements thereof.

In some embodiments, the informative loci or amplicon of the informative loci are treated with an agent, such as bisulfite.

In some embodiments, the informative loci indude 5 sequences that haw been treated with bisulfrte.

In some embodiments, the informative loci include rnethylated nucleic acid sequences that have been treated vvith bisulfite, In sorne embodiments, the bisulfite-converted methylated minus-strand DNA sequences include any one or more of sequences having at least 8(Wo, 85%,, 87(%, 90°1ii, 91 %, 92%, 93%, 94%, 95%, 96';.,;:J, 97[;.{,, 98;;;;), 99[;,,h or 100% identity to SEQ ID NOs: 2141--2568, 4709-5136, 7112-7506, 10 7637-7662,7793-7818, 7939-7962, 8033-8046, 8117-8130,8196-8208, 8279-8292, 8363- 8376, 8430-8434, 9377-9562, 10493-10678, 11414-11560, 11816-11866, 12122--12172, 12408-12454, 12485-12490, 12521-12526, 12557-12562, 12593-12598, 12269-12634,or 12662-12664, or fragments or complements thereof.

In particular embodiments, the bisulfiteconverted rnethy1ated minus-strand DNA sequences include any one or more of sequences 15 having at least 80%i, 85%i, 87%, 90%, 91 °/o, 92%, 93%, 94%, 95;},;,, 96~;,, 97%, 98%), 99~/o or 100°/o identity to SEQ ID NOs: 8033-8046, 8117-8130, 8196-8208, 8279-8292, 8363-8376, 8430-8434, 12485-12490, 12521-12526, 12557-12562, 12593-12598, 12269-12634, or 12662-12664, or fragments or complements thereof In sorne embodiments, the informative loci are associated with increased methylation in both Barrett's esophagus and esophageal 20 adenocarcinoma samples, as compared to the same sample types taken from a healthy control subject.

In some embodiments, the informative loci that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include any one or more bisulfite-converted methylated minus-strand DNA sequences selected from the group consisting of sequences having at least 80(Y;,, 85%), 87'1/ii, 9W%, 91 %, 92%), 93%,, 94~·'o, 25 95%, 96(.Vii, 97%), 9WYi1, 99%, or HJO%, identity to SEQ ID NOs: 2141-2568, 4709-5136, 7112- 7506, 7637-7662, 7793-7818, 7939-7962, 8033-8046, 8117-8130, 8196-8208, 8279-8292, 8363-8376, or 8430-8434, or fragments or complerncnts thereof.

In particular embodiments, the informative loci that are associated with increased methylation in both Barrett's esophagus and esophageal adenocarcinoma samples include any one or more bisulfite-converted 30 methylated rninus-strand DNA sequences selected from the group consisting of sequences 54 having at least 80•};i, 85%1, 87%-,, 90%, 91 %, 92%i, 93%, 94%, 95%,, 96'%, 97%;, 98%), 99% or 100%) identity to: SEQ JD NOs: 8033-8046, 8117-8130, 8196-8208, 8279-8292, 8363-8376, or 8430-8434,or fragments or cornplernents thereof In some embodiments, the inforrnative loci are associated with increased methylation in an esophageal adenocarcinoma sarnple ma 5 Barrett's with high grade dysplasia sample as compared to a sample of the same type taken from a subject having Barrett's esophagus ,vithout dysplasia.

In some embodiments, the informative loci that are associated with increased methylation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia sample include any one or more bisulfite-ccmvertcd methylated minus-strand DNA sequences sdected from 10 the group consisting of sequences having at least 80%, 85%), 87%1, 90%, 91 %, 92%), 93%), 94%, 95%i, 96%i, 97~"o, 98%J, 99'1~1 or 100%, identity to SEQ ID NOs: 9377--9562, 10493- 10678, 11414~11560, 11816-11866, 12122-12172, 12408-12454, 12485-12490, 12521-12526, 12557-12562, l 2593-12598, 12269-12634, or J 2662-12664, or fragments or complements thereof.

In particular embodiments, the informative loci that are associated with increased 15 rnethy!ation in an esophageal adenocarcinoma sample or a Barrett's with low grade or high grade dysplasia sample include any one or more of the sequences of sequences having at least 80%,, 85~'o, 87(1/;,, 90%, 91(%, 92S,,~i, 93%, 94%, 95%, 96%, 97%), 98%, 99(}o or 100% identity to SEQ ID NOs: 12485-12490, 12521-12526, 12557-12562, 12593-12598, 12269-12634, or 12662-12664, or fragn1ents or complements thereof.

In some embodiments, the infommtive 20 loci are associated with reduced methylation in an esophageal adenocarcinoma sample as compared to a sample of the same type taken from a subject having Barrett's esophagus.

In sorne ernbodiments, the informative loci that are associated wilh reduced methylation in an esophageal adenocarcinoma sample includt, any one or more bi sulfite-converted methylated minus·strnnd DNA sequences selected from the group consisting of sequences having at least 25 80%, 35%,, 87%, 90();;,, 91 %, 92%, 93%, 94%, 95°10, 96%), 97%), 98(}-'o, 99%J or 100%i identity to SEQ lD NOs: 13856-14092, 15278-15514, 15630-15652, 15853-15892, 16093-16132, l 6158-16 l 62, 16178-16180, 16196-16198, or 16204, or fragments or complements thereof.

In particular embodiments, the informative loci that are associated with reduced rnethylation in an esophageal adenocarcinorna sample include any one or more of the sequences having at 30 least 80%, 35tYo, 87%, 9()'3..{i, 9B{i, 92~·'o, 93%, 94%, 95%, 96f!,,t, 97~-i',, 98%, 99%i or 100% 55 identity to SEQ ID NOs: 16178-16180, 16196-16198, or 16204, or fragments or compleinents thereof.

In some embodiments, the disclosure provides for a hisulfite-converted nucleotide seqrn.,nce comprising the bisulfite-converted nucleotide sequence of any one of lhe foUowing: 5 Up3, Up JO, Up15-l, Upl5-2, Up20-1, Up20-2, Up20-2, Up27, Up35-1, Up35-2, SqBE2, SqBE5, SqBE7, SqBE9, SqBE10, SqBEl 1-1, SgBEl 1-2, SqBE13, SqBE14-2, SqBE15, SqBE16-l, SqBE16-2, SqBE17-1, SqBE18, SqBE22-1, SqBE22-2 or SqBE23.

In particular embodiments, the sequence comprises the bisulfite-convmied nucleotide sequence of any one of the following: Up3, UplO, Up15-l, Up15-2, Up20-1, Up20-2, Up20-2, Up27, Up35--1, or 10 Up35-2.

In particular embodiments, the sequence comprises the bisulfite-converted nucleotide sequence of any one of the following: SqBE2, Sq BES, SqBE7, SqBE9, SqBE10, SqBEl 1-1, SqBEll-2, SqBE13, SqBEl 4-2; SqBE15, SqBE16-1, SqBE16-2, SqBEl 7-l, SqBE18, SqBE22-1, SqBE22-2 or SqBE23.

In some embodiments, the disclosure provides for a panel of any of the sequences 15 disclosed herein< In some embodirnents, the panel comprises any of the following combinations of sequences: a) Up3, Up 10, Up15-l, Upl5-2, Up20-1, Up20-2, Up27, Up35-l, and Up35-2; b) Up3, Upl5-1, Up15-2, Up20-l, Up27, and Up35-l; c) UplO, Up3, Up15-l, Up15-2, Up20-l, Up27, and Up35-1; d) Up35-2, Up3, Upl5-l, Upl5-2, Up20-1, Up27 and Up35-l; e) Up15-1 and Up35-1; f) Up15-1, Up35-l, and Up10; g) Up15-1, Up35-1 and 20 Up20-1; h) Up15-1, Up35-J, UpJO, and Up15-2; i) Up15-1, Up35-l, UplO, and Up27;j) Upl5-l, UpJ5-1, t.Tpl5-2, and Up20-1; k) Upl5-l, Up35-1, UpJ5-2 and Up27; l)Upl5-1, Up35-!, Up20-l, and Up27; m) Up3 and Up35-1; n) Up3 and Up35-2; o)Up3 and UplO; p) Up3 and Up27; q) Up35-1 and Up35-2; r) Up35-1 and Up27; s)Up35-2 and Up10; t) UplO and Up27; u) Up3, Up35-1 and Up35-2; v) Up3, Up35-1 and Up10; w) Up3, Up35-l, and 25 Up27; x) Up3, Up35-2 and UplO; y) Up3, Up35-2, and Up27; z) Up3, Up IO, and Up27; aa)Up35-l, UplO, and 1Jp27; ab) Up35-2, Up 10, and Up27; ac) Up3, Up35-1, Up35-2 and UplO; ad) UpJ, Up35-l, Up35-2 and Up27; ae) UpJS-1, Up3.5-2, Up JO and Up27; af) lJp3, Up35-2, Up10 and Up27; ag) Up3, Up35-L UplO and Up27; ah) Up3, UplO, Up27, Up35-l, and Up35-2; ai) Up35-l and Up10, aj) tJp35-1 and Up27; ak) Up35-2 and Up10; al) 1Jp35-2 30 and Op27; am) Up3, Up35-l and Up35-2; an) Up3, llp35-J, and UpJO; ao) Up3, Up35-I, and 56 Up27; ap) Up3, Up35-2 and lJpiO; aq) Up3, Up35-2. and Up27; ar) Up3, Upl0 and Up27; at) Up35-1, UplO, and Up27; au) Up3, Up35-1, Up35-2, and Up10; av) Up3, Up35-1, Up35-2 and Up27; aw) Up35-l, tlp35-2, Up10 and Up27; ax) Up3, Up35-2, Upl0 and Up27; ay) Up3, Up35-l, Upl0 and Up27; az) Up3, UplO, Up27, tTp35-1, and Up35-2; ba) SqBE5 and 5 SqBE7; bb) SqBE5 and SqBE16; be) SqBE5 and SqBE17; hd) SqBES and SqBE18; be) SqBE7 and SqBE16; bt) SqBE7 and SqBEl 7; SqBE7 and SqBEl 7; bg) SqBE7 and SqBE18; bh) SqBE16 and SqBEl 7 and bi) SqBE16 and SqBE18.

In some embodiments, the disclosure provides for a method of detecting the methylation status of the sequences in any of the panels disclosed herein.

In some embodiments, the disclosure provides for a method of detecting the 10 methy1ation status of the sequences in any of the panels disclosed herein, and further comprises detecting the mutation status of p.53.

In particular embodiments, the disclosure provides for a method of a) (ktecting the methylation status of a panel comprising the sequences of Up-3 and Up35-2, and b) thrther detecting the mutation status of TP53.

In some embodiments, the disclosure provides for a method of detecting the 15 rnethylation status of any of the loci disclosed herein, and thrther comprises detecting the methylation status of vimentin.

In some embodiments, the vimentin methy1ation is detected in a manner consistent with that described in Li et al. (Li M, et al. (2009) Sensitive digital quantification of DNA methylation in clinical samples.

Nat Biotechnol 27(9):858-863).

In some embodiments, the vimentin mcthylation patterns are detennined in a nucleotide 20 sequence having at least 80%i, 85%;, 90%, 91 i}'ii, 92(1/ci, 93%i, 94°1;,, 95%, 96'1/ii, 97'%, 98°1;1, 99% or l 00% identity io SEQ ID NO: 16207 or 16208.

In some embodiments, the methylation patterns are determined in any of the following nucleic acid sequence combinations: a) vimentin and SQBE5; b) vimentin and SQBE7, c) vimentin and SQBE16, d) vimentin and SQBEl 7 ore) vimentin and SQBE18, 25 In particular embodiments, the disclosure provides for a nucleotide sequence comprising a sequence having at least 80%), 85°/ii, 90Cii,, 91 ~Iii, 92(),,·;,, 93~fo, 94%), 95~{), 96%, 9T%, 98%i, 99% or 100% identity to any of the following sequences:8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, 8420-8424, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649 or 12656-)2658, or fi:·agments and/or reverse complements 30 thereof.

In particular embodiments, the disclosure provid,~s for a nucleotide sequence comprising a sequence having at !east 80'1/(,, 85%, 90'l./,i, 91 S,·;i, 92%, 93%,, 94%, 95%, 96~.lo, 97%, 98%, 99% or lOOi;.·'o identity to any of the following sequences: 12563-12568, 12581- 12586, 12599-12604, 12617-12622, 12647-12649 or 12656-12658, or fragments and/or reverse complements thereof.

In particular embodiments, the disclosure provides for a 5 nucleotide sequence comprising a sequence having at least 801%, 85<Vii, 90%, 91 (%, 92%), 93(Y;,, 94%,, 9.5%, 96°/ii, 971hl, 98%,, 99~·~ or 100% identity to any of the following sequences:8209- 8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, or 8420-8424, or fragments and/or reverse complements thereof.

In some embodiments, the disclosure provides fix a bisulfite-conve1ied nucleotide 10 sequence comprising a sequence having at least 80%, 85%, 90%1, 91 %, 92%), 93%), 94%, 95%, 96%, 97~o, 98%i, 99~·'o or 100% identity to any of the following sequences: SEQ ID NOs: 8307-8313, 8315-8327, 8329-8334, 8349-8355, 8357-8369, 8371-8376,8411, 8412, 8414, 8416,8417,8419,8426,8427,8429,8431,8432,8434, 12605-12616, 12623-12634, 12650- 12655, OI 12659-12664, or fragments and/or reverse comp!ernents thereof.

In some 15 embodiments, the sequence comprises a sequence having at least 80%, 85%, 90%,, 91 ~·o, 92%, 93%), 94%, 950.10, 96%, 97%, 98%, 99% or l0m'o identity to any of the following sequences: SEQ ID NOs: 8307-8313, 8315-8327, 8329-8334, 8349-8355, 8357--8369, 8371--8376, 8411, 8412, 8414, 8416, 8417, 8419, 8426, 8427, 8429, 8431, or 8432, 8434.

In some embodiments, the sequence comprises a sequence having at least 80%, 8Y¼i, 90%, 91 %, 92%), 20 93%, 94(}{1, 95%, 96i;.,·\,, 97%1, 98'1/ii, 99% or 100~·~ identity to any of the following sequences: SEQ ID NOs; 12605-12616, 12623-12634, 12650-12655, or 12659-12664, In some embodiments, the disclosure provides for a bisulfite-converted nucleotide sequence comprising a sequence having at least 80%, 85~%, 9(Y}o, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 991% or 100% identity to any of the following sequences: SEQ ID NOs: 25 8223-8250, 8265-8292., 12569-] 2580, or 12587 ~ 12598, or frag1nents and/or reverse complements thereof.

In some embodiments, the sequence comprises a sequence having at least 80%, 85'%, 90'%, 91 %, 92%, 93'Vii, 94%, 95%, 96%, 97%, 98%, 99%} or 100% identity to any of the foUowing sequences: SEQ ID NOs: 8223-8250 or 8265-8292.

In some ernbodiments, the sequence comprises a sequence having at least 80~·~, 85%, 90%, 91 °.Ii), 92~o, 58 93'1/i), 94%), 95'%, 96{%, 97%, 98(),·o, 99% or 100% identity to any of the following sequences: SEQ m NOs: 12569-12580 or 12587-12598, rn some ernbodirnents, the sample fi1r use in any of the methods disclosed herein is a tissue sample taken from the suQject In some embodiments, the sample is tissue sample from 5 the ,~sophagus. in some embodiments, the sample is a biopsy or a brushing.

In some embodiments, the sample is a biopsy or brushing of the esophagus.

In some ernbodirnrnts, the sample is a body fluid.

In some embodiments, the body fluid is blood, serum, saliva, spit, stool, 11rine or an esophageal washing.

The present disclosure contemplates methods of selecting an individual to undergo a 10 diagnostic procedure to detem1ine the presence of Barrett's esophagus, Barrett's esophagus ,vith dysplasia (e.g., Barrett's esophagus with low-grade or hlgh-grade dysplasia), or of esophageal a<lenocarcinoma, by obtaining a biological sample from an individual, and detennining in the sample the presence of DNA methylation in at least one of any of the sequences disclosed herein.

In some embodiments, the disclosure provides for a method of 15 selecting a subject for monitoring of esophageal neoplasia, wherein the presence of DNA methylation in at least one of any of the sequences disclosed herein is detected in a sample from the sul~ect In some embodiments, detection is achieved by any one or more of DNA sequencing, next generation sequencing, methylation specific PCR, methylation specific PCR cornbined with a fluorogenic hybridization probe, real time methylation specific PCR, or 20 hybridization to an array.

In some embodi1m.'.11ts, the detection in the sample is indieative that the subject is at high risk of progression to esophageal neoplasia ( e.g., esophageal cancer).

In some ernbodiments, the subject is monitored by endoscopy.

In some embodiments, a sample from a subject in which DNA methylation of at least one of any of the sequences disclosed herein is detected, is indicative that the subject should be administered a pmticular treatment 25 In some embodiments, the treatment is selected from the group consisting of endoscopic removal or ablation of an esophageal neoplasia, and/or surgery, radiation, or chemotherapy treatment of esophageal adenocarcinoma.

In some embodiments, the sequence is any one or more sequence selected from the group consisting of a sequence having at least 80%i, 85%i, 90%,, 91 %, 92%, 93%,, 9t1'%, 95~·'o, 96%, 97%,, 98%i, 99% or 100% identity to any of the 59 7819-7866,7963-7990, 8047-8074, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335- 8348, 8405-8409, and 8420-8424, or fragrnents or complements thereof.

The present disclosure also contemplates methods of sdecting an individual to undergo a diagnostic procedure to determine presence of Barrett's esophagus with hw-grade 5 dysplasia, Barrett's esophagus \Vith high grade dysplasia or of esophageal adenocarcinoma, by obtaining a biological sample from an individual, and determining in the sample the presence of DNA methylation in at least one of any of the sequences disdosed herein, In some embodiments, the sequence is any one or more sequence selected from the group consisting of sequences having at least 80%, 85'%, 90%, 91 %, 92°/i.,, 93%, 94%1, 95%, 96%i, 97%), 98(l/i, 10 99% or 100% identity to any of the following sequences:: 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, l 2647 - 12649, or 12656 - 12658, or fragments or complements thereof.

The present disclosure fbrther contemplates that the melhod may further comprise determining the status 15 of somatic nmtation(s) in TP53 in the sample.

For example, a method comprising determining the status of methylation of chromosomal loci e.g, Up15-1, Up35-l, Up35-2, Up3, Up27, and Upl 0 in a sample; and determining the presence or absence of somatic mutation(s) in TP53 in the sample is contemplated.

The present disclosure funher contemplates that the method may further comprise 20 determining the status of somatk rmrtation(s) in TP53 in tbe sample.

For example, a method comprising determining the status of methylation of chromosomal loci e,g., Upl5-l, Up35-1, Up35--2, Up3, Up27, and UplO in a sample; and determining the presence or absence of somatic mutation(s) in TP53 in the sample is contemplated.

In certain embodiments, the method optionally fu1ther comprises detecting the presence or absence of u somatic mutation 25 in TP53, In certain embodiments, the TP53 somatic mutation, as described herein, is any one or more mutation at any one or more amino acid residue corresponding to amino acid residue 72, 105,108,110,113,124, 127,132,144, 152, 163,175,183,194,213,214,218,232,234, 248,265,273,278,306,337, 347, or 639 of SEQ ID NO: 16205, In certain ernbodiments, the TP53 somatic mutation is any non-synonornous somatic mutation known in the art In cc1iain 30 embodiments, the TP53 somatic mutation is any one or more mutation selected frorn the 60 group consisting of: Leu194Arg, Gly105Asp, Arg273His, Tyrl63His, Ue232Thr, Arg213Ter, A.rg273His, Arg248Gln, Argl 75His, Arg 11 0delinsGlnSer, Serl 83Ter, Arg248Gln, A.. r g337Leu, Lys 132Arg, Leu265ThrfsTer7, Arg306Ter, Cys124TrpfsTer25, Pro72Arg, Va1218G-lu, His214Leu, Glnl44Tt~r, Phel 13Ser, Tyr234His, Ser127Phe, Pro278Ala, 5 Ala347Thr, and Pro152Leu of SEQ JD NO: 16205 In certain embodiments, the TP53 mutation is any one or more non-synonymous somatic mutation at any one or .more nucleotide position corresponding to nucleotide position 108,215,314,338,380,395,430,455,487, 524,548,581,637,639,641, 6.53, 695,700,743,818,832,916, 1010, or 1039 of SEQ ID NO: 16206. 10 The present disclosure also contemplates met.hods of selecting an individual to undergo a treatment for Barrett's esophagus, Barrett's esophagus with low grade dysplasia, Barrett's esophagus wlth high grade dysplasia or for esophageal adenocr,.rcinorna, by obtaining a biological sampk'. from an individual, and detennining in the sample the presence of DNA methylation in at least one of any of the sequences disclosed herein.' ln sorne 15 embodiments, the sequence is any one or more sequence selected from the group consisting of sequence having at least 80':1i, 85%, 90~..-;i, 91 ~~., 92%, 93 1%, 94~•-;., 95%i, 96~o, 97%, 98~·o, 99% or 1 00ty;, identity to any of the following sequences: SEQ ID NOs: 1 - 856, 2569 - 3424, 5137 - 5926; 7507- 7558, 7663 - 7714, 7819-7866, 7963 -7990, 8047 ... 8074, 8131 -- 8156, 8209 - 8222, 825 l -- 8264, 8293 - 8306, 8335 ·· 8348, 8405 - 8409, or 8420 - 8424, or 20 fragments or complements thereof.

In some embodiments, the present disclosure further contemplates that the method may further comprise determining the status of somatic mutation(s) in TP53 in the sample.

For example, a method comprising dt~termining the status ofmethylation of chromosomal loci e.g., Upl5-1, llp35-L Up35-2, Up3, Up27, and UplO in a sample; and determining the presence or absence of somatic mutation(s) in TP53 in the 25 sample is contemplated.

The present disclosure also contemplates methods of selecting an individual to undergo a treatment for Barrett's esophagus, Barrett's esophagus with low-grade dysplasia, Barrett's esophagus with high grade dysp1asia or for esophageal adenocarcinoma, by obtaining a biological sample from an individual, and determining in the sample the presence 30 of DNA methylation in at least one of any of the sequences disclosed herein.

In some 61 embodiments, the sequence is any one or more sequence selected frorn the group consisting of sequence having at least 80%, 85%;, 90%, 91 '3./;i, 92%, 93%,, 94%), 95%, 96%, 97~·;, 98%, 99% or 100% identity to any of the following sequences: SEQ ID NOs: 8447 - 8818, 9563 - 9934, 10679-- 10972, 11561-11662, 11867 -- 11968, 12173 12266, 12455 --- 12466, 12491 ·-· 5 12502, 12527 - 12538, 12563 - 12568, 1258] ·--12586, 12599 ·-· 12604, 12617-12622, 12647 ·-· 12649, or 12656 - 12658, or fragments or complements thereof.

The present disclosure further contemplates that the method may further comprise determining the status of somatic mutation(s) in TP53 in the samp!e, For example, a method comprising determining the status of methyiation of chromosomal loci e.g., llpl5-1, Up35-1, Up35-2, 10 Up3, Up27, and UplO in a sample; and determining the presence or absence of somatic rnutation(s) in TP53 in the sample is contemplated, The present disclosure also contemplates methods of selecting an individual to undergo enhanced surveillance for the development of Barrett's esophagus with low grade dysplasia, Barrett's esophagus with high grade dysplasia or of esophageal adenocarcinoma, by obtaining a biological sample frorn an individual, and 1" dete1111ining in the sample the presence of DNA methylation in at least one of any of the sequences disdosed herein, ln some embodiments, the sequence is any one or more sequence selected from the group consisting of a sequenc,~ having at least 80%, 85!Yi,, 90%), 91 °/i), 92i!/o, 93%, 94%, 95%, 96'%, 97~o, 98;;/ii, 99%1 or 100(!,{, identity to any of the following sequences:: SEQ ID NOs: 8447 --- 8818, 9563 --- 9934, 10679- 10972, 11561 - 11662, 11867 - 11968, 20 12173 -12266, 12455 -12466, 12491-12502, 12527-12538, 12563 , .. 12568, 12581---- 12586, 12599- 12604, 12617-12622, 12647 --12649, or 12656-12658, or fragments or complements thereof.

The present disclosure further contemplates that the method may fbrther comprise detem1ining the status of somatic rnutation(s) in TP53 in the sample, For example, a method comprising determining the status of rnethylation of chromosomal loci 25 e.g..

Up15-1, Up35-1, Up35-2, Up3, Up27, and Upl0 in a sample; and determining the presence or absence of somatic rnutation(s) in TP53 in the sample is contemplated.

The present disclosure also contemplates methods of determining the response of an individual with esophageal cancer to therapy by obtaining a biological sample frorn an individual with esophageal cancer, and determining the presence of methylation in at least one of any of the 30 sequences disclosed herein.

In some embodiments, the sequence is any one or more sequence 61 L selected from the group consisting of sequences having at least 80%, 85t;,·;,, 90%, 91 %, 92%, 93%, 94%1, 95~•-'o, 96(¼,, 97%,, 98%), 99% or 100~-t identity to any of the following sequences:: 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, 833 5 - 5 8348, 8405- 8409, or 8420 --- 8424, 8447 ---· 8818, 9563 -9934, 10679 - 10972; SEQ ID NOs: 11561 ---· 11662, 11867 -- 11968, 12173 - 12266; SEQ ID NOs; 12455 -· 12466, 12491 ---· 12502, 12527-12538, 12563-12568, 12581--12586, 12599--12604, 12617-12622, 12647 -12649, and 12656 ~ 12658, or fragments or complements thereof In sorne implementations, an increase in levels of methylation over time is indicative of disease 10 progression and a need for a change in therapy (such as modifying the dosing regime ohm exiting therapy, or administering a new therapeutic(s) either alone or in combination with the existing therapy), and an absence of increase in levels of methy!ation over time or decrease in levels of methyiation over time is indicative that a change in therapy is not required.

The present disclosure further contemplates that the method may further comprise determining the 15 status of soma lie mulation(s) in TP53 in the sample.

For example, a method comprising determining the status of methylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, Up27, and Up10 in a sample; and determining the presence or absence of somatic mutation(s) in TP53 in the sample is contemplated.

The present disclosure also contemplates method of distinguishing EAC and/or 20 low/high grade dysplasia from BE by obtaining a biological sample from an individual, and determining in the sample the presence of DNA methy1ation in at least one of any of the sequences disclosed herein.

In some embodiments, the sequence is any one or more sequence selected from the group consisting of sequences having at least 80%,, 85%, 90%, 91 %, 92%i, 93%, 941);;,, 95%), 96'hi, 97'?-"o, 98%, 99% or 100~-,;, identity to any of the following sequences: 25 SEQ m NOs: 8447 - 8818, 9563 - 9934, 10679 - 10972; SEQ ID NOs: 11561 ·· 11662, 11867 - 11968, 12173 - 12266; SEQ ID NOs: 12455 ··· 12466, 12491 - 12502, 12527 - 12538, 12563 --- 12568, 1258 l ---- 12586, 12599 - 12604, l 2617 - l 2622, 12647 - 12649, and 12656 - 12658, or fragments or complements thereof.

The present disclosure fmiher contemplates that the method rnay further comprise determining the status of somatic 30 mutation(s) in TP53 in the sample.

For example, a method comprising detennining the status 63 ofmethylation of chromosomal loci e.g., Upl5-1, Up35-L Up35-2, Up3, Up27, and tlp10 in a sample: and detennining the presence or absence of sornatic mutation(s) in TP53 in the sample is contemplated. ln certain embodiments, the absence of methylation at Up J 5-1, Up35- 1, Up35-2, Up3, Up27, and UplO; and the absence of a somatic mutation in TP53 may 5 be indk:ative of non-dysplastic Barret's esophagus.

In ce1tain embodiments, the presence of methy!ation at any one ofUp15-1, Up35-1, Up35--2, Up3, Up27, and lJpl0; or the presence of a somatic mutation in TP53 may be indicative of esophageal adenocarcinoma or of Barrett's with high grade dysplasia, The present disclosure also contemplates method of distinguishing EAC and/or 10 lmv/high grade dysplasia from BE by obtaining a biological sample from an individual, and determining in the sample the presence of DNA methylation in at least one of any of the sequences disclosed herein.

In some embodiments, the sequence is any one or more sequence selected from the group consisting of sequences having at least 8()11/;,, 85'Vi1, 90%, 91 %, 92%1, 93(1/o, 94%}, 95%, 96%), 97°1;), 98<;,·\ 99% or 100% identity to any of the fi..)llowing sequences: 15 SEQ ID NOs: 8447- 8818, 9563 -9934, 10679 - 10972; SEQ m NOs: 11561 ··· 11662, 11867-11968, 12173 - 12266; SEQ ID NOs: 12455 ----12466, 12491-12502, 12527- 12538, 12563 ----12568, 12581 •· 12586, 12599-12604, 12617 -12622, 12647 -12649, and 12656 - 12658, or fragments or complements thereof The present disclosure funher contemplates that the rnethod may further comprise determining the status of somatic 20 mutation(s) in TP53 in the sample, For example, a method cornprising determining the status ofmethylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, i)p27, and Up10 in a sample; and determining the presence or absence of somatic mutation(s) in TP53 in the sample is contemplated.

In cenain embodiments, the absence ofmethylation at Up15-l, Up35-l, Up35-2, Up.3, Up27, and Up10; and the absence ofa somatic mutation in TP53 may 25 be lndicative of non-dysplastic Barret's esophagus. ln certain embodimt,nts, the presence of methylation at any one of Up15-I, Up35-1, UpJS-2, tJp3, Up27, and UplO; or the presence of a somatic mutation in TP53 may be imiicative of esophageal adenocarcinoma, The present disclosure also contemplates a method of monitoring the progression (or regression) of esophageal neoplasias over lime, The method involves detecting the 30 methylation status of one or more uudeotide sequences sekcted from the group consisting of 64 991% or 100% identity to any of the following sequences:: 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, and 8420 - 8424, 5 8447 - 8818, 9563 -9934, 10679 - 10972; SEQ ID NOs: 11561---11662, 11867 ----11968, 12173 - 12266; SEQ ID NOs: 12455 ----12466, 12491----12502, 12527 -12538, 12563- 12568, 12581 --- 12586, 12599 --- 12604, 12617 -12622, 12647 12649, and 12656 - 12658, or fragments or complements thereof in samples frorn a subject at a first time and at a later time.

In certain embodiments, .neoplasia regression may be indicated by the absence of 10 methylation in the nucleotide sequence taken at a later time and the presence of methylation in tbe nucleotide sequence taken at the first time.

In certain embodiments, neoplasia progression 1nay be indicated by the presence of rnethylation in the nucleotide sequence taken at a later time and the absence of rnethylation in the nucleotide sequence taken at the first time.

The present disclosure further contemplates that the method may fo1iher comprise detennining the 15 status of somatic mutation(s) in TP53 in the samples.

In some ernbodiments, neoplastic regn.'.ssion may be indicated by lhe presence of methylated chromosomal loci e.g., rnethylation of Upl 5-1, Up35-l, Up35-2, Up3, Up27, and/or Upl 0 or the presence of a somatic mutation in TP53 in a first sample; and the absence of methylated chromosomal loci e.g., unmethylated Up l 5-1, Up35-J, Up35-2, Up3, Up27, and Up 10, and the absence of 20 somatic rnutation(s) in TP53 in a later sample.

In some embodiments, neoplastic progression rnay be indicated by the presence of unmethylated chrornosomal loci e.g., umnethylated Up15-1, Up35-1, Up35-2, Up3, Up27, and Up1 0; and the absence of somatic mutation(s) in TP53 in a first sample; and the presence of rnethylatt,d chromosomal loci eog., rnethylated Up15-· l, Up35-l, Up35-2, Up3, Up27, and/or Up 10, or the presence of a somatic mutation in 25 TP53 in a later sample.

The present disclosure also provides sequences that will hybridize under highly stringent conditions to the nucleotide sequences of any one or more of SEQ lD NOs: 1-8444 and 8447-16204, or fragments or complements thereof .As discussed abovtc, one of ordinary skill in the art will understand readily that appropriate stringency conditions vvhich prmnote 30 DNA hybridization can be varied.

One of ordinary skill in the art will understand readily that 65 appropriate stringency conditions which promote DNA hybridization can be varied.

For example, one could perform the hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50 c•e, For example, the salt concentration in the wash step can be selected fium a low stringency of about 2.0 x SSC at 50 °C to a high 5 stringency of about 0.2 x SSC at 50 °C.

In addition, the temperature in tht~ \Nash step can be increased from low stringency conditions at room temperature, about 22 °C, to high I stringency conditions at about 65 °C.

Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.

In one embodiment, the disclosure provides nucleic acids \Vhich hybridize under low stringency 10 conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature. ln other embodiments, the disclosure also provides the methylated fonns of the nucleotide sequenct:s of any one or more of SEQ ID NOs: 1 -8444 and 8447-16204, or fragments thereof wherein the cytosine bases of the CpG islands present in the sequences are 15 methylated.

In other words, the nucleotide sequences listed of any one or more of SEQ ID NOs: 1-8444 or 8447-16204 or fragments or complements thereof may be either in the methylated status (e.g., as seen in neoplasias) or in the umnethy1ated status (e.g., as seen in normal cells).

In fmiher embodiments, the nucleotide sequences of the disclosure can be isolated, recombinant, andior fused with a heterologous nucleotide sl.':quence, or in a DNA 20 library.

In certain embodiments, the present disdosure provides bisulfite-converted nucleotide sequences, for example, bisulfite-converted sequences selected from any of the sequences disclosed herein.

In some embodiments, the sequence is selected from the group consisting of sequences having at least 80';-'o, 3SC1/u, 90%), 91 (!/\ 92';,·o, 93~✓.), 94%, 95%i, 96%), 97%, 98%, 25 99%! or 100% identity to any of the following sequences: SEQ ID NOs: 857 -2563, 3425- 5136, 5927-7506, 7559 7662, 7715 .. 7818, 7367 - 7962, 7991 8046, 8075 ... 8130, 8157 ... 8208, 8223 - 8250, 8265 - 8292, 8307 - 8334, 8349 - 8376, 8410 .... 8419, 8425 ~ 8434, and/or fragments thereof, and/or the reverse complements thereof In yet other embodiments, the disclosure provides bisulfite-converted sequences selected from the group consisting of 30 sequences having at least 80%, 85%, 90%, 91 %), 92%, 93%, 94'}o, 95%i, 96%), 97%, 98%, 66 I 99%i or 100'% identity to any of the following sequences:: SEQ ID NOs: 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, and 12659 - 12664, and/or fragmenls thereof: and/or the reverse complements 5 thereof, In yet other embodiments, the disclosure provides bisulfite-converted sequences selected from thf group consisting of sequences having at least 80%, 85'5,.o, 90%, 91 %, 92%i, 93i%, 94%, 95i;lii, 96!Y;,, 97%, 98%, 99~'o or 100% identity to any of the following sequences:: SEQ ID NOs: 13145 - 14092, 14567 -15514, 15561 - l.5652; 15733 - 15892, 15973 --- 16132, 16143 - 16162; 16169 - 16180, 16187 16198, and 16201 - 16204, and/or fragments thereof, 10 and/or the reverse complements thereof A fragment of any of the nucleotide seqm~nces disclosed herein may be of any length, so long as the methylation status of that nucteotide sequence may be determined, In some embodiments, the nucleotide sequence is at least 10, I 5, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110,120,130,140,150,160,170,180, 190,200,250,30(\350,400,450,500,600,700,800, 15 900, ] 000, 1200, 1400, 1500, 1700, or 2000 nucleotides in length.

In some embodiments, the nucleotide sequence is at least 10-2000, 10-1000, 10-500, l 0-200, 10-150, 10-100, 50-2000, 50-1000, 50-500, 50-200,50-150,50-100, 80-2000, 80-1000, 80-500, 80-150, 80-100, 100- 2000, 100-1000, 100-500, 100-200, or 100-150 nucleotides in length, Such bisulfite-converted nucleotide sequences can be used for detecting the 20 methylatfon status, for example, hy an MSP reaction or by direct sequencing (e.g,, nt:xt generation sequencing).

These bisulfiw-convt~rted sequences are also of use for designing primers for MSP reactions that specifically detect methylated or unmethylated nucleotide sequences fr)llowing bisuHite conversion.

In yet other embodiments, the bisulfoe-converled nucleotide sequences of the disclosure also include nucleotide sequences tbat will hybridize 25 under highly stringent conditions to any nucleotide sequence of any one or more of SEQ ff) NOs: 1-8444 and 8447-16204, or fragments or complements thereof.

In fmiher aspects, the application provides methods for producing such bisulfiteconve1ied nucleotide sequences, for example, the application provides methods for treating a nucleotide sequence with a bisu1fite agent such that the unmethylawd cytosine bases are 30 converted to a difforem nucleotide base such as a urnciL 67 In yet other aspec:ts, the application provides oligonudeotide primfrs frir amplifying a region within the nucleic acid sequence of any one or more of SEQ fl) NOs: 1 -8444 and 8447-16204.

In certain aspects, a pair of the oligonudeotide primers can be used in a detection assay, such as the HpaII assay.

In certain aspects, primers used in an MSP reaction 5 can specifically distinguish between methylated and non-methylated DNA The primers of the disclosure have sufficient length and appropriate sequence so as to provide specific initiation of amplification nucleic acids.

Primers of the disclosure are designed to be "substantially" cmnplernentary to each strand of the nucleic acid sequence to be amplified.

In some embodiments, the primer is selected from the group consisting of 10 sequences having 80%, 85%:i, 90%, 91%, 92%, 9Y!lii, 94~{,, 95 1Yo, 96%i, 97%), 98'1/.i, 99(%, or 100(1/o identity to any of SEQ JD NOs: 8377-8404, 8435-8446, 12635-12646, and 12665- 12670.

In some embodiments, the primer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, l 0, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,or35 consecutive nucleotides of any of the primer sequences of SEQ ID NOs: 8377-8404, 8435- 15 8446, 12635-12646, and 12665-12670, While exemplary primers include the sequences of any sequence having at least 80%i, 8Yl--(,, 90%,, 91 'Vo, 92%,, 93%, 94%1, 95~.fo, 96%J, 97%, 98%, 99%,, or 100% identity to any of SEQ ID NOs: 8377-8404, 8435-8446, 12635-12646, and 12665-12670, or fragments thereot: it is understood that any primers that hybridize with the bisulfite-converted sequence of any one or more of SEQ ID NOs: 1-8444 and 8447-16204 are 20 included within the scope of this disclosure and is useful in the method of the disclosure for detecting methylated nucleic add, as described.

Simi lady, it is understood that any primers that would serve to amplify a methylation sensitive restriction site or sites within the differentially methylated region of the infonnative loci ofany of the sequences of SEQ 1D NOs: 1-8444 or 8447-16204, or fragments or complements thereof are included \.Vithin the 25 scope of this disclosure and is useful in the method of the disclosure for detecting nucleic methylated nucleic acid, as described.

The oligonudeotide primers of the disclosure may be prepared by using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.

In one such automated embodiment., diethylphosphoramidites are used 30 as starting materials and may be synthesized as described by Beaucage, et aL (Tetrahedron 68 Letters, 22: 1859-1862, 1981 ).

One method of syt1lhesizing oligonucleotides on a modified solid support is described in U.S.

Patent No. 4,458,066.

IV, .A.ssavs_ aml Drug.

Scr.eening _j\ rk~thodniogi..-::m in certain aspects, the application provides assays and methods using any of the 5 informative loci, or bisulfite converted methylated or umnethylated sequences thereof:. disclosed herein.

In some embodiments, the informative loci comprise a sequence having at least 801:;-'i,, 85i;;;), 9ff~-'i,, 91 ~.lo, 92%), 93'Yo, 94%, 95%, 96%, 97%, 98'%, 99%, or 100'1/ii identity to any of the sequences of SEQ ID NOs: 1-428, 2569-2996, 5137-5531, 7507-7532, 7663- 7668, 7819-7842, 7963-7976, 8047-8060, 8131-8143, 8209-8222, 8293-8306, 8405-8409, 10 8447-8632, 9563-9748, 10679-10825, 11561-11611, 11867-11917, 12173-12219, 12455- 12460, 12491-12496, 12527-12532, 12563-12568, 12599-12604, 12647-12649, 12671-12908, 14093-14329, 15515-15537, 15653-15692, 15893-15932, l 6133-16137, 16163-16165, 16181-16183, 16]99,429-856,2997-3424,5532-5926,7533-7558,7689-7714,7843-7866, 7977-7990,8061-8074, 8144-8156, 8251-8264, 8335-8348,8420-8424, 8633-8818,9749- 15 9934, 10826-10972, 11612-11662, 11918-11968, 12220-12266, ]2461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, 12656-12658, 12909-13144, 14330-14566, 15538-15560, 15693-15732, 15933-15972, 16138-16142, 16166-16168, 16184-16186 or 16200 or any fragments or compknwnts thereof In some embodiments, the informative loci are used as molecular markers to distinguish between healthy cells and metaplastic cells (e.g,, 20 Barrett's esophageal ce1ls).

In some embodiments, the infomiative loci are used as rrmlecular rnarkers to distinguish between healthy cells and neoplastic cells (e.g., cancer cells).

In particular embodiments, the inforrnative loci axe used as molecular markers to distinguish between healthy cells and esophageal adenocarcinoma cells.

In some embodiments, the informative loci are used as molecular markers to distinguish between Barrett's esophagus 25 cells and cancer cells. ln some ernbodiments, the informative loci are used as molecular markers to distinguish between Ba1Tctt' s esophagus cells and esophageal adenocarcinoma ceHs.

For example, in one embodiment, the application provides methods and assays using any of the informative loci comprising a sequence having at least 80%, 85%, 900/i;, 91 (;.o, 92')/;i, 93%, 94~o, 95%, 96%,, 97°.,{,, 98%i, 99%, or 100% identity to any one or rnore of SEQ 1D NOs: 30 8447-8818,9563-9934, 10679-10972, ]1561-11662, 11867-11968, 12173-12266, 12455- 69 12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, ]2647-12649, OR 12656-12658, or any fragments or complements thereof as markers that distinguish between healthy cells and neop1asia cells, In other embodiments, the application provides methods and assays using the informative loci comprising a sequence having at least 5 80%,, 85%, 90%, 91 %,, 92%1, 93~·'o, 94·Yo, 95%, 96%, 97"?·o, 98%, 99%, or 100%) identity to any one or more of SEQ ID NOs: 1-856, 2569-3424, 5137-5926, 7507-7558, 7663-7714, 7819- 7866, 7963-7990, 8047-8047, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, 8420-8424, 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 10 12599-12604, 12617-12622, 12647-12649, 12656-12658, 12671-13144, 14093-14566, 15515-15560, 15653-15732, 15893-15972, 1613.5-16142, 16163-l6168, 16181-16186 and/or 16199-16200 or rmy fragments or complements thereof as markers that distinguish bet\veen healthy cells and cells derived from neoplasias of the upper gastrointestinal tract In one aspect, a molecular marker of the invention is a differentially methylated sequence of ru1 15 infonnaiive locus.

In certain aspects, the application provides assays and methods using the infommtive loci comprising a sequence having at least 801 ;/G, 85(Vii, 90%, 91 (¼i, 92%, 93(3.iil, 94%, 95%, 96%, 9Ji%, 98%, 991;1i, or 100~0 identity to any one or more of SEQ ID NOs: l- 856, 2569-3424, 5137-5926, 7507-7558, 7663-7714,7819-7866,7963-7990, 8047-8047, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, 8420-8424, 8447- 20 8818, 9563-9934, 10679-10972, 11561-11662, ll 867-11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649, 12656-12658, 12671-13144, 14093-14566, 15515-15560, 15653-15732, 15893-15972, 16135-16142, 16163-16168, 16181-16186 and/or 16199-16200 or any fragments or complements thereof in combination \Vith the status of sornatic mutation(s) in 2" TP53 as molecular markers that distinguish between healthy cens and cancer cells, For example, in one embodiment, the application provides methods and assays using the informative loci comprising a sequence having at least 8()1;/G, 85%, 90%, 91 %;, 92(;.o, 93'1/i;, 94'}o, 95%,, 96%, 9TVo, 98%, 99%i, or l 001% identity to any one or more of SEQ 1D NOs: 1- 856, 2569-3424, 5137-5926,7507-7558, 7663-7714,7819-7866,7963-7990, 8047-8047, 30 8131-8156,8209-8222,8251-8264,8293-8306, 8335-8348,8405-8409, 8420-8424,8447- 70 8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968, 12.173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568, l 2581-12586, 12599-12604, 12617-12622, 12647-12649, 12656-12658, 12671-13144, 14093-14566, 15515-15560, 15653-15732, 15893-15972, 16135-16142, 16163-16168, 16181-16186 and/or 16199-16200 or any 5 fragmt,nts or complements thereof and the status of somatic rnutation(s) in TP53 as markers that distinguish between healthy cells and neoplasia cells (e.g. cancer/esophageal adenocarcinoma cells), In other embodiments, the application provides methods and assays using the infrm11ative loci disclosed herein (e.g., chromosomal loci Up15-1, Up35-1,,Up35-2, Up3, Up27, and UplO) and the status of somatit: mutation(s) in TP53 as markers that 10 distinguish betv,.reen healthy cells and cells derived from neoplasias of the upper gastrointestinal tract in one aspect, a molecular marker of the invention is a differentially methylated sequence of an informativt, locus, In certain embodiments, the application provides assays for detecting differentially methylated nucleotide sequences.

Thus, a differentially methylated nucleotide sequence, in 15 its methylated state, can serve as a target for detection using various methods described herein and the methods that are well within the purview of the skilled artisan in view of the teachings of this application.

In certain aspects, such rnetbods for detecting methylated nucleotide sequences are based on treatment of genomic DNA with a chemical compound which converts non- 20 methylated C, but not methylated C (i.e., 5mC), to a different nucleotide base.

One such compound is sodium bisulfite (also referred to simply as "bisulfite" herein), which converts C, but not 5mC, to U.

Iviethods for hi sulfite treat1rnmt of DNA are known in the art (Herman, et aL, 1996, Proc Natl Acad Sci USA, 93:9821-6; Herman and Baylin, 1998, Current Protocols in Human Genetics, N. E. A.

Dracopoii, ed., John Wiley & Sons, 2:10,6.1-10,6.10; U,S, 25 Patent No. 5,786,146).

To illustrate, when a DNA molecule that contains umnethylated C nucleotides is treated with sodium bisulfite to become a compound-converted DNA, the sequence of that DNA is changed (C ➔ U).

Detection of the U in the converted nucleotide sequence is indicative of an 1,.mmcthylated C.

The different nucleotide base ( e.g., U) present in compound-converted nucleotide 30 sequences can subsequently be detected in a variety of ways, In a particular embodiment, the 71 present invention provides a method of detecting U in compound-converted DNA sequences hy using "methylation sensitive PCR" (MSP) (see, e.g., Herman, et al., 1996, Proc.

Natl.

Acad.

Sci. USA, 93:9821-9826; U.S.

Patent No. 6,265,171; U.S.

Patent No. 6,017,704; US.

Patent No. 6,200,756).

In IVISP, one set of primers (i.e., comprising a fr)rward and a reverse 5 primer) amplifies the compound-i;xmverted template sequence if C bases in CpG dinucleotides within the DNA are methylated.

This set of primers is called "methylation-specific primers." Another set of primers amplifies the compound-converkd template sequence if C bases in CpG dinuckotides within the 5' flanking sequence are not methylated.

This set of primers is called '\mmethylation-specific primers." 10 In MSP, the reactions use the compound-converted DNA from a sample in a subject In assays for methylated DNA, melhylation-spedfic primers are used.

In the case where C within CpG dinucleotides of the target sequence of the DNA are methylated, the rnethylationspecific primers will amplify the compound-converted template sequence in the presence of a polymerase and an MSP product will be produced.

If C vvithin CpG dinuck~otides of the 15 target sequence of the DNA is not methylated, the methylation-specific primers will not amplify the compound-converted template sequence in the presence of a polymerase and an rv1SP product -;vill not be produced.

In some embodiments, any of the bislllfite converted methylated sequences disclosed herein is used as a marker fbr a particular indication.

It is often also useful to run a control reaction for the detection of unmethylated DNA, 20 The reaction uses the compound-converted DNA from a sample in a subject and unmethylation-specific primers are used, ln the case where C within CpG dinuckotides of the target sequence of the DNA are unn1ethylated, the unmethylation specific primers will amplify the compound-conve1ied template sequence in the presence of a polymerase and an MSP product wiH be produced. lf C within Cp(i dinucleotides of the target sequence of the 25 DNA is methylated, the unrnethylation-specific primers will not amplify the compoundconverted template sequence in the presence of a polymerase and an lvlSP product will not be produced.

Note that a biologic sample will often contain a mixture of both neoplastic cells that give rise to a signal with methylation specific primers, and normal cellular elernents that give rise to a signal with unmcthylation~specific primers.

The unmethylation specific signal 30 is often of use as a control reaction, but does not in this instance imply the absence of 72 neoplasia as indicated by the positive signal derived from reactions using the methylation specific primers, In some embodimt:nts, any of the bisulfite converted unmethylated sequences disclosed herein are used as controls.

In some embodiments, the unmethylated control sequences are any of the bisulfite converted sequences of SEQ ID NOs; 857-1284, 5 342.5-3852, 5927-6321, 7559-7584, 7715-7740, 7867-7890, 7991-8004, 8075-8088, 8157- 8169, 8223-8236, 8307-8320, 8410-8414, 8819-9004, 9935-10120, 10973-11119, 11663- 11713, 11969-12019, 12267-12313, 12467-12472, 12503-12508, or 12539-12544, 12569- 12574, 12605-12610, 12650-12652,1713-2140,4281-4708,6717-7111, 7611-7636,7767- 7792,7915-7938, 8019-8032, 8103-8116,8183-8195, 8265-8278, 8349-8362,8425-8429, 10 9191-9376, 10307-10492, 11267-11413, 11765-11815, 12071-12121, 12361-12407, 12479- 12484, 12515-12520, 12551-12556, 12587-12592, 12623-12628, or 12659-12661 in which every ''Y" position is a "T." Primers for a MSP reaction are derived from the compound-conve11ed template sequence.

Herein, "derived from" means that the sequences of the prhners are chosen such 15 that the primers amplify the compound-converted template sequence in a rvfSP reaction.

Each primer comprises a single-stranded DNA fragment which is at least 8 nucleotides in length.

In some embodiments, the primers are less than 50 nucleotides in length, or in some embodiments, from 15 to 35 nucleotides in length.

Because the compound-converted template sequence can bt~ either the Watson strand or the Crick strand ofthe double-stranded 20 DNA. that is treated with sodium bisulfite, the sequences of the primers is dependent upon whether the \Vatson or Crick compmmd-converted template sequence is chosen to be amplified in the MSP, Either the Watson or Crick strand ran be chosen to be amplified.

The compound-converted template sequence, and therefore the product of the 1ifSP reaction, is, ln some embodimenls, between 20 to 3000 nucleotides in length.

In other 25 embodiments, the product of the MSP reaction is between 50 to 1000 nucleotides in length.

In other embodiments, the product of the MSP reaction is between 50 to 500 nucleotides in length, In other embodiments, the product of the l'v1SP reaction is between 80-150 nucleotides in length.

In some embodiments, the product of the MSP reaction is at least 20, 30, 40, 50, 60,70,80,90, 100,110,120,130,140,150,160,170,180, 190,200,210,220,230,240or 30 250 nucleotides in length, In some ernbodiments, tbe methylation-specific prirners result in 73 an MSP product of a different length than the MSP product produced hy the unmethylationspecific primers.

A variety of methods can he used to deterrnine if an J'.V1SP product has been produced in a reaction assay, One way to determine if an MSP product has been produced in the 5 reaction is to analyze a portion of the reaction hy agarose gel electrophoresis.

For example, a horizontal agarose gel of from 0.6 to 2.0% agarose is made and a portion of the MSP reaction mixture is electrophoresed through the agarose geL After electrophoresis, the agarose gel is stained '1.vith ethidimn bromide. MSP products are visible wht'.n the gel is viewed during ll!umination with ultraviolet light.

By comparison to standardized size markers, it is 10 dete1111ined if the MSP product is of the correct expected size.

Other rnethods can be used to determine whether a product is made in an MSP reaction.

One such method is called "real-tirne PCR." Real-time PCR utilizes a themml cycler (i.e,, an instrument that provides the temperature changes necessary for the PCR reaction to occur) that incorporates a fluorimeter (i.e. an instrument that measures 15 fluorescence), Tht: real-time PCR reaction mixture also contains a reagent whose incorporation into a product can he quantified and whose quantification is indicative of copy number of that sequence in the template, One such reagent is a fiuorescent dye, called SYER Green 1 (Molecular Probes, Inc.; Eugene, Oregon) that preferentially hinds double-stranded DNA and whose fluorescence is greatly enhanced by binding of double-stranded DNA. 20 When a PCR reaction is perforrn.ed in the presence of SYBR Green l, resulting DNA products hind SYBR Green 1 and fluorescence.

The fluorescence is detected and quantified by the f1uorirneter.

Such technique is particularly useful for quantification of the amount of the product in the PCR rt'.action, Additionally, the product from the PCR reaction may be quantitated in "real-time PCR" by the use of a variety of probes that hybridize to the product 25 including TaqMan probes and molecular beacons, QuanHtation may be on an absolute basis, or may he relative to a constitutively rnethylated DNA. standard, or may be relative to an unmethylated DNA stcmdard.

In one instance the ratio of methylakd derived product to unrnethylated derived product may be constructed.

Methods for detecting 1nethylation of the DNA according to the pn:~sent disclosure are 30 not limited to MSP, and may cover any assay for detecting DNA metbylatlon, Another 74 exam pie method of detecting rnethylation of the DNA is by using "methylation-sensitive" restriction endonucleases.

Such methods comprise treating the genomic DNA isolated from a subject with a methylation-sensitive restriction endonuclease and then using the restriction endom1cltmse-treated DNA as a template in a PCR reacfam.

Herein, methylation-sensitive 5 restriction endonucleases recognize and deave a specific sequence within the DNA if C bases \Vithin the recognition sequence are not methylated, If C bases within the recognition sequence of the resu·iction endonuclease are rnethylated, the DNA will not be cleaved, Examples of such methylation-sensitive restriction endonucleases include, but are not lirnhed to Hpall, SmaI, SacII, EagI, BstUI, and BssHII.

In this technique, a recognition sequence for 10 a methylation-sensitive restriction endonuclease is located within the template DNA, at a position between the forward and reverse primers used for the PCR reaction.

In the case that a C base within the 1rn.,thylation-sensitive restriction endonuclease recognition sequence is not methylated, the endonuclease will cleave the DNA template and a PCR product will not be formed when the DNA is used as a template in the PCR reaction.

In the case that a C base 15 within the methylation-sensitive restriction endonuclease recognition sequence is methylated, the endonudease win not cleave the DNA template and a PCR product will be formed when the DNA is used as a template in the PCR reaction.

Therefore, methylation of C bases can be detem1ined by the absence or presence of a PCR product (Kane, et aL, 1997, Cancer Res, 57:808-11 ).

No sodium bisulfite is used in this technique. 20 Yet another exemplary method of detecting rnethylation of the DNA is called the modified MSP, which method utilizes primers that are designed and chosen such that products of the NISP reaction are susceptible to digestion by restriction endonudeases, depending upon whether the compound-converted template sequence contains CpG dinudeotides or UpG dinucleotides. 2~ Yet other methods for detecting methylation of the DNA, include the MS--SnuPE methods.

This method uses compound-converted DNA as a template in a primer extension reaction wherein the primers used produce a product, dependent upon whether the compoundconve1ied template contains CpG dinucleotidcs or UpG dinucleotides (see e.g., Gonzalgo, et aL, 1997, Nucleic Acids Res, 25:2529-31 ). 75 Another exemplary method of detec.ting methylation of the DNA is caHed COBRt-\ (i.e., combined bisulfite restriction analysis).

This method has been routinely used for DNA mdhylation detection and is well know11 in the art (see, e.g., Xiong, et al., 1997, Nucleic Acids Res, 25:2532-4).

In this technique, methylation-sensitive restriction endonucleases 5 recognize and cleave a specific sequence within the DNA if C bases within the recognition sequence are methylated.

If C bases within the recognition sequence of the restriction endonudease are not methylated, the DNA ,vm not be cleaved.

In some embodin1ents, the method utiliz,,s methylation-sensitive restriction endonudeases.

Another exernplary method of detecting methylation of DNA requires hybridization of 10 a compound converted DNA to an-ays that include probes that hybridize to sequences derived from a methylated template. /\nother exemplary method of detecting methylation of DNA includes precipitation of methylated DNA with antibodies that bind methylated DNA or with other proteins that bind methylated DNA, and then detection of DNA sequences in the precipitate.

The detection of 15 DNA could be done hy PCR based methods, by hybridization to am\ys, or by other methods kno\l\-11 to those skilled in the art Another exemplary method of detecting methylated DNA is bisulfite sequencing that involves amplification of a target region of bisulfite converted DNA using methyiation indifferent PCR primers that amplify converted DNAs derived from both methylated and 20 unmethylated templates.

The rnethylation indifferent primers an.: often designed to be both methy1ation indifforenl and bisulfite specific, i.e. to amplify only bisulfite converted target DNAs and not to amplify non-converted target sequences.

In some embodiments, the amplified DNAs then may be characterized by Next Generation Sequencing methods that alknv each cytosine base in the original template to be assessed within each DNA sequence 25 read for the presence of methy laiion (retention of cytosine) or the absence of methylation (conversion to thymidine).

The percent of methylalion al each cytosine base in lhe original template can then be calculated by the percent of DNA reads in which the cytosine is preserved as cytosine versus is converted to thymidine.

Similarly, the percent of methylation across a region of interest can be assessed by determining a rule .for assessing the region as 30 methylated or unmethylated in an ln&vldual DN/\ read (Le. determining a cutoff for 76 methyla!ion in the region that will categorize the region as "rnethylated"), and then determining the percent of DNA reads in vvhich the region qualifies as methylated.

In ce1iain embodiments, tbe disclosure provides methods that involve directly sequencing the product resulting from an MSP reaction to determine if the compound- 5 converted template sequence contains CpG dinuckotides or UpG dinucleotides. lVlolecular biology techniques such as directly sequt~ncing a PCR product are well known in the art.

In some ernbodin1ents, rnethy lation of DNA may be rneasured as a percentage of total DNA High levels of methylation may be 1-100% methylation, for t:xample, 1%, 101:--'iJ, 20%,, 30%, 1.W%, 50%, 60°10, 70%, 80%, 90%, or lO0ci'o methyladon.

Low levels ofrnethyfation 10 may be 0%-0.99(;1;, methylation, for example, 0()-{1, 0.1 t:,·o, 0.5%, 0.6%, 0. 7%, 0.8%, 0,9%, At least some normal tissues, for example, normal esophagus samples, may not have any dewctable methy1ation.

The ski Ued a1tisan will appreciate that the present disclosure is based in part, on the recognition that any one of the infonnative loci disclosed herein may include nudeotide 15 sequences that encode polypeptides that, for example, may function as a tumor suppressor gene, Accordingly, the application further provides methods fr)r detecting such polypeptides in cell sampks, In some embodiments, the disclosure provides detection methods by assaying such polypeptides so as to determine \.Vhether a patient has or docs not have a disease condition.

Fmiher, such a disease condition may be characterized by decreased levels of such 20 polypeptides.

In certain embodiments, the disclosme provides methods for determining whether a patient is or is not likely to have cancer by detecting such polypeptides.

In farther embodiments, lht~ disclosure provides methods fr.,r determining whether the patient is having a relapse or determining whether a patient's cancer is responding to treatment Optionally, such methods involve obtaining a quantitative measure of the protein in 25 the sample.

In vie\V of this spec.:ifi.cation, one of skill in the art will recognize a wide range of techniques that rnay be employed to detect and optionally quantitate the presence of a protein, In some embodiments, a protein is detected with an antibody.

In many embodiments, an antibody-based detection assay involves bringing the sample and the antibody into contact so that the antibody has an opportunity to bind to proteins having the co1Tesponding epitope.

In 30 many embodiments, an antihody--based detection assay also typically involves a system for I''' detecting the presence of antibody-epitope complexes, thereby achieving a detection of the presence of the proteins having the corresponding epitope.

Antibodies may be used in a variety of detection techniques, including enzyrne-linked immunosorbcnt assays (ELISAs), immunoprecipitations, Western blots, i\ntibody-independent techniques fi.)r identifying a 5 protein may also be employed.

For example, mass spectroscopy, particularly coupled with liquid chrornatography, permits detection and quantification of large numbers of proteins in a sarnple.

Two-dimensional gel electrophoresis may also be used to identii)'· proteins, and may be coupled vvith mass spectroscopy or other detection techniques, such as N-terminal protein sequencing. RNA aptamers with specific binding for the protein of interest may also be 10 generated and used as a detection reagent Samples should generally be prepared in a manner that is consistent with the detection system to be employed.

For example, a sample to be used in a protein detection system should generally be prepared in the absence of proteases.

Likewise, a sample to be used in a nucleic acid detection system should genernlly be prepared in the absence of nucleases. rn many instances, a sample for use in an antibody-based 15 detection system will not be subjected to substantial preparatory steps.

For t~xmnple, urine may be used directly, as may saliva and blood, although blood will, in ceitain embodiments, be separated into fractions such as plasma and serum. ln certain embodiments, a method of the disclosure comprises detecting the presence of an informative loci-expressed nucleic acid, such as an rnRNA, in a sample.

Optionally, the 20 method involves obtaining a quantitative measure of the infonnative loci-expressed nucleic acid in the sample.

In view of this specification, one of skill in the art will recognize a wide range oftedmiques that may be employed to detect and optionally quantitate the presence of a nucleic acid.

Nucleic acid detection systems generally involve preparing a purified nucleic acid fraction of a sample, and subjecting the sample to a direct detection assay or an 25 amplification process followed by a detection assay.

Amplification may be achieved, for example, by polymerase chain reaction (PCR), reverse tnmscriptase (RT) and coupled RTPCR.

Detection of a nucleic acid is generally accomplished by probing the purified nudeic acid fraction with a probe that hybridizes to the nucleic acid of interest, and in many instances, detection involves an amplification as well, Northern blots, dot blots, microarrnys, 78 quantitative PCR, and quantitative RT-PCR are all well known methods for detecting a nucleic acid in a sample. rn certain embodiments, the disclosure provides nucleic acid probes that bind specifically to an infonnative loci nudek acid.

Such probt)S may be labeled with, for 5 example, a fluorescent moiety, a radionuclide, an enzyme or an affinity tag such as a biotin moiety.

For example, the TaqMan@ system employs nucleic acid probes that are labeled in such a way lhat the fluorescent signal is quenched when the probe is free in solution and bright when the probe is incorporated into a larger nucleic acid.

Immunoscintigraphy using monoclonal antibodies directed at the infonnati ve loci may 10 be used 10 detect and/or diagnose a cancer.

For example, monoclonal antibodies against the infonnative loci labeled with 99Teclmetium, ll!Indium, 125Iodine-may be effectively ust)d for such imaging.

As will be evident to the skilled artisan, the amount of radioisotope to be administered is dependent upon the radioisotope.

Those having ordinary skiH in the art can readily formulate the amount of the imaging agent to be administered based upon the specific 15 activity and energy of a given radionuclide used as the active moiety.

Typically 0.1-100 rnillicuries per dose of imaging agent, 1-10 millicmies, or often 2-5 millicuries are administered.

Thus, compositions according to the present invention useful as imaging agents comprising a targeting moiety conjugated to a radioactive moiety comprise 0.1-100 millicudes, in some embodiments 1-10 millicuries, in some embodiments 2-5 mil1icuries, in 20 some embodiments l-5 millicuries.

A variety of methods can be used to dt,tem1ine if TP53 contains a somatic mutation, as will he evident to the skilled miisan. ln some embodiments, the TP53 gene or protein sequence is detem1ined and any change in the determined sequence relative to the wildtype sequence is detected.

In some embodimt,nts, the TP53 gene sequence is determined by PCR, 25 RT-PCR, No1ihem Blot, Southern Blot, andior in situ hybridization, Another way to determine if TP53 contains a somatic mutation may involve the use of an antibody-based detection assay (e.g. ELISA, immunohistochemistry, and/or Western Blot).

In some embodiments, the antibody-based detection assay utilizes an antibody that binds to a mutant TP53 protein with a tighter affinity than it binds to a wildtype TP53 protein.

The skilled 30 artisan will also readily appreciate methods of determining somatic mutations in TP53 based 79 on the disclosures of US5843654, US5620848, EP0:390323 and US5527676.

In sorne embodiments, the disclosure provides fr_,r a device useful for detecting the methylation status of any of the in.fonnative loci, or fragments or complemt'.nts thereot: 5 disclosed herein, In sonie ernbodiments, the disclosure provides for a kit comprising components usefhl for detecting the methylation status of the informative locL or fragments, or complements thereot:, disclosed herein, In some embodiments, the kit comprises a swallowable balloon fbr collecting an esophageal sample from the subject Jn some embodirnents, the kit comprises any of the swallowahle balloon devices disclosed in WO 10 2015/089422.

In ce1iain embodiments, the present disclosure provides drug screening assays for identifying test compounds which potentiate the tumor suppressor function of polypeptides encoded by sequences located in the informative loci disclosed herein, In one aspect, the assays detect test compounds which potentiate the expression level of polypeptides encoded 15 by sequences located in the informative loci disclosed herein, 1n another aspect, the assays detect test compounds which inhibit the mcthylation of DNA, In certain embodiments, drng screening assays can be generated which detect test compounds on the basis of their ability to interfere with stability or fl.mction of polypeptides encoded by sequences located in the informative loci disclosed herein. 20 /\. variety of assay fonnats may be used and, in light of the present disclosure, those not expressly described herein vvlll nevertheless be considered to be within the purview of ordinary skill in the art.

Assay fommts can approximate such conditions as protein expression level, rnethylation status of nucleotide sequences, tumor suppressing activity, and may be generated in many different fom1s.

In many embodiments, the disclosure provides assays 25 including both cell-free systems and cell-based assays which utilize intact cells.

Compounds to be tesk~d can be produced, for example, by bacteria, yeast or other organisms (e,g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombimmtly.

The efficacy of the compound can be assessed by generating dose response curves frorn data obtained using various concentrations of the 30 test compound.

Moreover, a control assay can also be perfi.)rmed to provide a baseline for 80 Date Reyue/Date Received 2022-04-21 cornparison.

In the control assay, the formation of complexes is quantitated in the absence of the test compound, In rnany drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds 5 surveyed in a given period of time.

Assays of the present invention which are perfom1ed in cell-free systems, such as rnay he developed with purified or semi-purified proteins or with lysates, are often preforred as "primary'' screens in that they can be generated to permit rapid development and relatiw.:ly i.~asy detection of an alteration in a molecular target which is mediated by a test compound.

Moreover, the effects of cellular toxicity and/or bioavailability 10 of the test compound can be generally ignored in the in vitro systern, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target In certain embodiments, test compounds identified from these assays may be used in a 15 therapeutic method of treating cancer, Still another aspect of the application provides transgenic non--hurnan animals which express a gene located within any one of the informative loci disclosed herein, or v,rhich have had one or more of such g€'.momic gene( s) disrupted in at least one of the tissue or cell-types of the animal. 20 In another aspect, the application provides an animal model for cancer, which has a mis-expressed allele of a gene located within any one of the inforrnative loci disclosed herein, Such a mouse model can then be used to study disorders arising from mis-expression of genes located within any one of the informative loci disclosed herein.

Genetic techniques which allow for the expression of transgenes can be regulated via 25 site-specific genetic manipulation in vivo an~ known to those skilled in the art.

For instance, genetic systems are available which allow for the regulated expression of a recornhinase that catalyzes the gt,netic recombination a target. sequence.

As used herein, the phrase "target sequence" refers to a nucleotide sequence that is genetically recombined by a recombinase, The target sequence is flanked by recombim,se recognition sequences and is generally either 30 excised or inverted in eel !s expressing recornbinase activity.

Recombinase catalyzed 81 recombination events can be designed such that recombination of the target seqtwnce results in either the activation or repression of expression of the polypeptides.

For example, excision of a target sequence which interferes with the expression of a recombinant gene can be designed to activate expression of that gene.

This interferern.:e vvith expression of the protein 5 can result from a variety of mechanisms, such as spatial separation of the gene from the promoter element or an internal stop codon.

Moreover, the transgene can be made when..~in the coding sequence of the gene is flanked recombinase recognition sequences and is initially transfected into cells in a 3' to 51 orientation with respect to the promoter element.

In such an instance, inversion of the target sequence wi! ! reorient the subject gene by placing the 5' end 10 of the coding seguence in an odentation with respect to the promoter element which aHow for promoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage P 1 (Lakso et aL, (1992) Proc.

Natl. A cad Sci. USA 89:6232-6236; Orban et aL, (1992) Proc. 1Vatl.

Acad.

Sci. USA 89:6861-6865) or the FLP recombinase system of 15 Saccharomyces cerevisiae (O'Gorman et aL, (1991) Science 251 :1351-1355; PCT pubLicat.ion WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems, Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences, loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinast, binds and are required for Cre recombinase mediated genetic 20 recombination" The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted ,vhen Cre recombinase is present (Abremski et aL, (1984) J.

Biol.

Chem. 259: 1509-1514 ); catalyzing the excision of the target sequence when the !oxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inve1ied repeats. 25 V.

Subitcts a;:1..,;L§hmwh::5 In certain aspects, the invention relates to a subject suspected of having or has: a cancer, a rnetaplasia, or a neoplasia of the upper gastrointestinal tract ( e.g., esophageal cancer).

Alternatively, a subject may be undergoing routine screening and may not necessarily bt~ suspected of having such metaplasia or neoplasia. ln some embodiments, the 30 subject is a human subject, and the neoplasia is a neoplasia of the upper gastrointestinal tract, 82 such as the esophagus, In some embodiments, tht~ subject is a hmnan subject, and the metaplasia is Barrett's esophagus.

Assaying for biomarkers discussed above in a sample from subjects not knmvn to have, e.g., a metaplasia or neoplasia of the upper or lower gastrointestinal tract can aid in 5 diagnosis of such a met.aplasia or neoplasia in the subject To illustrate, detecting the methylation status of the nucleotide sequences by MSP can be used by itselt~ or in combination with detecting the somatic mutation status ofTP53 or other various assays, to improve the sensitivity and/or specificity for detecting, e.g., a neoplasia of the upper or lower gastrointestinal tract.

In some embodiments, such detection is made at an early stage in tbe 10 development of cancer, so that treatment is more likely to be effective.

In sorne ernbodhnents, an informative loci in a su~ject is considered "methylated" for the purposes of detennining vvhether or not the subject is prone to developing and/or has developed a metaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus with dysplasia such as high-grade or low-grade dysplasia) (e.g., l 5 esophageal cancer such as esophageal adenocarcinoma) if the loci is at least 10%, 20%i, 30%, 40%, 50%,, 60%), 70'//ii, S0ty;;, 90~fo, or 1 om.{, methylated.

In some embodiments, a DNA sarnple from a subject is treated with bisulfrte, and the resulting bisulfite sequence corresponds to any of the nucleotide sequences disclosed herein comprising a "Y" nucleotide. ln some embodiments, if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, J 6, 17, 18, 19, 20 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues of the hisulfite-converted sequence have a C, the sequence is considered "methylated" for the purposes of determining whether or not the subject is prone to developing and/or has developed a rnetaplasia in the esophagus (e.g,, Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus '.Vith dysplasia such as high-grade or low-grade dysplasia) (e.g, esophageal cancer such as esophageal 25 adenocarcinoma).

In some embodiments, a DNA sample from a subject is treated with bisulfite, and the resulting bisulfite sequence corresponds to any of the nucleotide sequences disclosed herein cornprising a "Y" nucleotide. !n some embodiments, if al leas! 10%, 20%, 30%i, 4011/;J, 50%,, 60%, 70S.-o, 80%, 90%, or 100% of the Y residues of the bi sulfite-converted sequence have a C, the sequence is considered "methylated" for the purposes of determining 30 whether or not the subject is prone to developing and/or has developed a rnetaplasia ln the 83 esophagus (e.g., Barrett's esophagus) or neop!asia (e.g, Barrett's esophagus with dysplasia such as high-grade or low-grade dysplasia) (e.g,, esophageal cancer such as esophageal adenocarcinoma), In some embodiments, a subject ls determined to be prone to developing and/or has developed a metap!asia in the esophagus (e.g., Barrett's esophagus) or neop!asia 5 (e.g, Barretfs esophagus with dysplasia such as higb-grnde or low-grade dysplasia) (e.g., esophageal cancer such as esophageal adenocarcinoma) if a certain number of "Y" nucleotides in a bisulfite converted sequence are cytosines.

In some embodiments, the certain number is at least 1,2,3,4,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues of the hisulfite-convened sequence.

In some 10 embodiments, the certain number is least 10~.10, 20%, 30%, 40%), 50%, 60•l1i,, 70%, 80%, 90%, or 100'% of the Y residues of the bisulfite-converted sequence.

In certain embodiments, a subject is determined to be prone to developing and/or has .:.fovi'.'.loped a metaplasia in the esophagus (e.g., Barrett's esophagus) or neop1asia (e.g,, esophageal cancer such as esophageal adenocarcinoma) (e.g,, Barrett's esophagus with dysplasia such as high-grade or low-grade 15 dysplasia) if a certain percentage of DNA molecules from a sample from a subject are detennined to be "methylated," as defined herein. ln some embodiments, the certain percentage of DNA molecules is at least 10%,, 20·%, 30(),~, 40°/o, 50%, 60%, 70%, 30%i, 90%, or 100% of the DNA molecules from the.~ sample are determined to be "methylated." In some embodiments, the the percentage of rnethylated DNA molecules is determined using next- 20 generation sequencing.

Exemplary cut-offs of DNA methylation and DNA molecule percentages may be found in the Examples section provided herein.

In addition to diagnosis, assaying of a marker in a sample from a subject not known to have, e.g,, a rnetaplasia or neopiasia ofthe upper gastrointestinal tract, can be prognostic for the subject (i.e., indicating the probable course of the disease).

To illustrate, subjects having a 25 predisposition to develop a metaplasia or neoplasia of the upper gastrointestinal tract may possess rnethylated nucleotide sequences, A.ssaying of methylated informative loci in a sample from subjects either by itself, or in combination with assaying for sornatic mutation(s) in TP53, can also be used to select a particular therapy or therapies which are particularly dfocti ve against, e.g., a neoplasia of the upper gastrointestinal tract in the subject, or to 30 exclude therapies that are not likely to be effective. 84 Assaying of methylated informative loci in samples from subjects that are known to have, or to have had, a cancer is also usefuL For example, the present methods can be used to identify whether therapy is effective or not for certain subjects, One or more samples an.~ taken frnm the same subject prior to and following therapy, and assayed for any one or more 5 of the infom1ative loci markers either by itself or themselves, or in combination with assaying for somatic nmtation(s) in TP53. A finding that an informative locus is methylated in the sample taken prior to therapy and absent (or at a lower level) after therapy may indicate that the therapy is (~ffective and need not be altered.

In those cast~s where the informative locus is methylated in the sample taken before therapy and in the sample taken after therapy, it may be 10 desirable to alter the therapy to increase the likelihood that the cancer will be reduced in the subject Thus, the present rnethod may obviate the need to perfi..m11 more invasive procedures which are used to determine a patient's response to therapy.

Cancers frequently recur following therapy in patients vvith advanced cancers.

Jn this and other instances, the assays of the invention are useful for rnonitoring over time the status 15 of a cancer associated with silendng of genes located in the infonnative loci disclosed herein.

For su~jects in whom a cancer is progressing, there can be no DNA rnethylation in some or al1 samples when the first sample is taken and then appear in one or more samples when the second sample is taken.

For subjects in which cancer is regressing, DNA methylation may be present in one or a number of samples when the first sample is taken and then be absent in 20 some or aH of these samples when the second sample is taken.

Samples for use v,.rith the methods described herein may be essentially any biological material of interest For example, a sample may be a bodily tluid sample from a subject, a tissue sample from a subjec!, a solid or semi-solid sample from a subject, a primary cell culture or tissue culture of materials derived from a suqject, cells from a cell line, or medium 25 or other extracellular material from a cell or tissue culture, or a xenograft (meaning a sample of a cancer from a first subject, e.g., a human, that has been cultured in a second subject, e.g., an immuno-compromised mouse), The term ''sample'' as used herein is Intended to encompass both a biological material obtained directly from a subject (which may be described as the primary sample) as weU as any manipulated forms or portions of a primary 30 sample. A sarnple rnay also be obtained by contacting a biological rnaterial \Vith an 85 exogenous liquid, resulting in the production of a lavage liquid containing some portion of the contacted biological materiaL Furthennore, the term "sample'' is intended to encompass the primary smnple after it has been mixed with one or more additive, such as preservatives, chelators, anti-clotting factors, etc, Jn certain embodiments, a bodily fluid sample is a blood sample.

In this case, the term "smnple" is intended to encompass not only the blood as obtained directly from the pafamt but also fractions of the blood, such as plasma, serum, cell fractions (e.g., platelets, erythrocytes, and lymphocytes), protein preparations, nucleic acid preparations, etc.

In some embodiments, the bodily fluid may be derived from the stomach, for example, gastric secretions, acid reflux, 10 or vomit In other embodiments, the bodily fluid may be a fluid secreted by the pancreas or bladder.

In other embodiments, the body fluid may be saliva, spit, or an esophageal washing.

In certain embodirnents, a tissue sample is a biopsy taken from the mucosa of the gastrointestinal tract.

In otht,r embodiments, a tissue sample is the hrushings from, e.g,, the esophagus of a subject 15 A subject is in some embodiments a human subject, but it is expected that the molecular markers disclosed herein, and particularly their homologs from other animals, are of sirnilar utility in other animals.

In certain embodiments, it may be possible to detect a biomarker described herein (e.g., DNA methy1ation or protein expression level) directly in an organism without obtaining a separate portion of biological material.

In such instances, the 20 terrn "sample" is intended to encompass that portion of biological material tbat is contacted ,vith a reagent or device involved in the detection process. rn ce1iain embodiments, DNA which is used as the template in an l\ilSP reaction is obtained from a bodily fluid sample.

Examples of bodily fiuids are blood, saliva, spit or an esophageal washing.

Other body fluids can also be used.

Because they can he easily obtained 25 frorn a subject and can be used to screen for multiple diseases, blood or blood-derived fractions are especially useful.

Blood-derived fractions can comprise blood, semrn, plasma, or other fractions, For l~xarnple, a cellular fraction can be prepared as a "buffy coat'' (Le., leukocyte-enriched blood portion) by centrifuging 5 ml of v;hole blood for 10 min at 800 times gravity at room temperature.

Red blood cells sediment rnost rapidly and are present as 30 the bottom-most fraction in the centrifuge tube.

The buf(y coat is present as a thin creamy 86 white colored layer on top of the red blood cells.

The plasma portion of the blood forms a layer above the buffy coat.

Fractions from blood can also be isolated in a variety of other ways.

One method is by taking a fraction or fractions from a gradit'.nt used in centrifogation to enrich frir a specific size or density of cells. 5 DNA is then isolated from sarnples from the bodily fluids.

Procedures for isolation of DNA from such samples are wdl known to those skilled in the art.

Commonly, such DNA isolation procedures comprise lysis of any cells present in the samples using detergents, for example.

After cell lysis, proteins are commonly removed from the DNA using various proteases.

Ri'\JA is removed using R.'\J"ase.

The DNA is then commonly extracted with 10 phenol, precipitated in akobol and dissolved in an aqueous solution.

VI. '.fherapeutic metht.)ds, In some embodiments, the disclosure provides for a method of determining whether a subject has any one or more of the methylated informative loci disclosed herein that are indicative of the presence of a rnetaplasia in the esophagus (e.g, Barrett's esophagus), 15 wherein if the subject is determined to have a n1etaptasia in the esophagus (e.g., Barrett's esophagus), the subject is treated with an agent that treats the metaplasia in the esophagus (e.g., Ban-ett's esophagus), In somf..~ embodiments, thf..~ disclosure provides for a method of treating a subject deterrnined to have a metaplasia in the esophagus (e.g,, Barrett's esophagus).

In some embodiments, the treatment of a mdaplasia in the esophagus (e.g., 20 Barrett's esophagus) encornpasses administration of any one or more of the following compounds: proton pump inhibitors (PP ls) such as omeprazok (Prilosec, Zegerid), lansoprazole (Prevacid), pantoprazole (Protonix), rnbeprazole (AcipHcx), esomcprazole (Nexium), dexlansoprazole (Dexilant).

Histarnine Il2 receptor blocking agents such as cimetidine (Tagamet), ranitidine (Zantac ), fomotidine (Pepcid) and nizatidine (A,xid).

Tums, 25 R.olaids, or other quick-acting reflux medications.

Prnkinetic agents, or drugs that help rnove fiJod through the gastrointestinal tract more quickly, offer an attractive alternative either alone or in combination with acid inhibition.

In some embodiments, the treatment of a mdaplasia in the esophagus (e.g., Bam:.,tt's esophagus) is endoscopic nrncosal resection (EMR); photodynarnic therapy (PDT); radiofrequency ablation (RF/\); argon plasma coagulation 30 (APC); cryotherapy, and/or surgery (e.g. esophagectomy, anti-reflux surgery). 87 ln some embodiments, the disclosure provides for a method of determining ,vhether a subject has any one or more of the methylated informative loci disclosed herein that are indicative of esophageal neoplasia (e.g., esophageal cancer), wherein if the subject is detem1ined to have an esophageal neoplasia (e.g, esophageal cancer), the subject is treawd 5 with an agent that treats the esophageal neoplasia (e.g., esophageal cancer).

In some embodiments, the disclosure provides for a method of detcnnining whether a subject has any one or more of the methylated informative loci disclosed herein in combination with any of the TP53 somatic mutations disclosed herein that are indicative of esophageal neoplasia (e.g., esophageal cancer), wherein if the subject is determined to have an esophageal neoplasia (e.g, l O esophageal cancer), the subject is treated with an agent that treats the esophageal neoplasia (e,g, esophageal cancer).

In some embodiments, the disclosure provides for a method of treating a subject determined to have esophageal neoplasia (e.g,, esophageal cancer). 1n some embodiments, the esophageal neoplasia is Barrett's esophagus with low grade dysplasia, Barrett's esophagus with high grade dysplasia (HGD) and/or esophageal adenocarcinoma 15 (EAC} In some embodiments, the treatment ofesophageal neoplasia ( e.g., esophageal cancer) ,~ncompasses surgery (e.g, esophagectomy), radiation therapy, chemorndiation therapy and/or cbernotherapy. ln some embodiments, the treatment of esophageal neoplasia (e.g., esophageal cancer) encompasses administering one or more chemotherapeutic agent, such as any one or more therapeutic agent selected from the group consisting of: crirboplatln and 20 paclitaxel (Taxol®) (which may be combined with radiation); cisplatin and 5-fluorouracil (5- FU) (often combined with radiation); ECF: epirubicine (Elknce®), cisplatin, and 5-FU (especially for gastroesopbageal junction tumors); DCF: docctaxel (Taxoterc@), cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda@l); oxaliplatin and either 5-FU or capecitabine; doxorubicin (Adriamycin(~)), hleomycin, mitomycin, methotrexate, vinorelbine (Navelbine@), 25 topotecan, and irinotecan (Camptosar@).

In some embodiments, for some esophagus cancers that overexpress the HER2 protein on tht, surface of their cells, chemotherapy may be used along with the targeted drug lrastuzmnab.

Rarnucirumab may be used to treat cancers that start at the gastroesophageal (GE) jum.,tion when they are advanced.

In some embodiments, the treatment encompasses endoscopic treatinents, such as endoscopic rnucosal resection 30 (EMR) followed by treatment v1ith proton pump inhibitors, photodynarnic therapy (PDT); 88 radiofrequency ablation (RFA.); laser ablation; argon plasma coaglllation (APC); ekctrocoagulation ( electrofulguration); or esophageal stent.

The terms "treatment", "treating", "alleviation" and the likt~ an.'. used herein to generally mean obtaining a desin.'.d pharmacologic andior physiologic effect, and may also be 5 used to refer to improving, alleviating, and/or decreasing the severity of one or more syrnptoms of a condition being treated.

The effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition, ''Treatment" as used 10 herein covers any treatmenl of a disease or condition of a rnammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject ,vhich may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in 15 one or more symptoms\ Treating a metaplasia (e.g., Barrett's esophagus) and/or a neoplasia (e.g., esophageal cancer) in a subject refers to improving (improving the su~ject's condition), alleviating, delaying or slowing progression or onset, decreasing the severity of one or more symptoms associated with a metaplasia (e.g., Barrett's esophagus) and/or a neoplasia (e.g., esophageal 20 cancer).

For example, treating a metaplasia or neoplasia includes any one or more of: reducing grovvth, proliforntion and/or survival of metaplastic/neoplastic cells, killing metaplastic/neoplastic cells (e.g., by necrosis, apoptosis or autophagy), decreasing metaplasiaineoplasia size, decreasing rate of metaplasia/neoplasia size increase, halting increase in metaplasia/neoplasia size, improving ability to swallow, decreasing internal 25 bleeding, decreasing incidence of vomiting, reducing fatigue, derreasing the number of metastases, decreasing pain, increasing survival, and increasing progression free survivaL Exemplification The invention now being generally described, it ,.,:vill be more readily understood by reference to the following examples, which are included merely for purposes of illustration of 89 certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 : identification of ~;sophag_ea!. cancer _lnfonnative loci, 5 Methylated informative loci were initially identified using the technique of reduced representation bi sulfite sequencing (RRBS) in a discovery set of23 paired biopsies of nomml squamous esophagus and matched esophageal adenocarcinomas, along with biopsies of 8 Barrett's esophagus tissue, and along with brnsbings of 8 Barrett's esophagus tissues (one BE brushing case also having a matched biopsy). l 0 Discovery data were initially analyzed for each individual CpG residue in the RRBS data set.

Individual CpGs were considered methylated in EAC if they showed rnethylation in less than 10% of DNA sequence reads in all of the informative squamous samples, where at least 4 squamous samples were informative, where an infonnative sample had equal to or greater than 20 reads covering the CpG, and if 8 or more of the infrwniative EAC samples 15 demonstrated percent methylation at a level that was at least 20 percentage points greater than the methylation level of the most methylated nom1al squamous sample.

CpGs were similarly defined as rnethylated in Barrett's esophagus if they showed methylation of less than 10% of DNA sequencC;.~ reads of all informative squamous samples, when., an informative sample had equal to or greater than 20 reads covering the CpG, and if 3 or rnore of the informative BE 20 samples dernonstrated percent methy1ation at a level that was at least 20 percentagi..~ points greater than the mdhy!ation level of the most methylated normal squamous sample.

CpGs meeting criteria for methylation in both EA.Cs and BE were de.fined as methylated in both EAC and BE.

Such methylated CpGs were then aggregated into patches by grouping together methylated CpGs that were within 200bp of one another.

Patches may consist of 1 CpG up to 25 any number of CpGs that meet the above criteria.

The names assigned to 428 genomic patches defined as methylated in both EAC and BE by the criteria above were recorded, and the seq m:nces of these loci correspond to SEQ ID NOs: J-856.

The genomic coordinates of the genomic patches defined as methylated by the above criteria were also recorded.

The genomic sequences of these patches on the respective 30 genomic(+) and(-) strands were determined and recorded. (Upper and lower case 90 designations were used according to those of the UCSC browser, where lower case sequences are lower complexity DNA sequences).

The bisulfite converted sequences of these corresponding patches(i.e. the bisulfite conve1ted sequence of the(+) strand and the bisulfite converted sequence of the(·) strand) were determined and recorded (see sequences of SEQ ID 5 NOs: 857-1281 and 1713-2140 for the bisul:fite converted sequences of the respective(+) and H strands). C residues that rnay be methylated or unmethylated, and hence may be bisulfite converted to T (if unmethylated) or remain as a C (if methylated), were designated with a Y (where Y denotes C or T), and where, after bisulfite conversion, actual maintenance of a Y designated base as a C was scored as methylation at that base, Thus, these sequences 10 represent the group of all combinations of all stcquences in which 0, 1, or more than one Y is conve11ed to a T.

The reverse complements of the bi sulfite converted sequences of the(+) and (-) strands will be obvious to one of ordinary skill in the mi and are also included by implication in this disclosure.

The bisulfite converted sequences of the fully methylated form of the corresponding patches (Le. in which all Y bases in every bisulfite converted sequence 15 are retained as a C), corresponding to the(+) s!rand the(-) strand were determined and recorded (see sequences of SEQ ID NOs: 1285-1712 and 2141-2568 for the bisulfite converted sequences of the foHy methylated form of the ( +) and (-) strands respectively of the corresponding patches).

The reverse complements of the bisulfite conve1ied methylated(+) stand and(-) stand sequences will be obvious to one of ordinary skill in the art and are also 20 included by implication in this disclosure.

Patches were expanded by l 00 base pairs on either side so as to accommodate either the design of amplification pimers or to endude additional presumptively methylated bases, The sequences of these expanded patches correspond to SEQ ID NOs: 2569-3424 and their genomic coordinates were also rt,corded.

The genomic sequences of these expanded patcht,s 25 on the respective genomic (+) and (-) strands were determined and recorded, (Upper and lower case designations were used according to those of the U CSC browser, where lower case sequences are lmver complexity DNA sequences).

The bisulfite converted sequences of these corresponding expanded patches (Le. the bi sulfite converted sequence of the ( +) strand and the bisulfite c:onve1ied sequence of the (-) strand) were determined and recorded (see 30 sequences of SEQ ID NOs: 3425-3852 and 4281-4708 for the bisulfite converted sequences of 91 the respective(+) and(-) strands). C residues that rnay he methylated or umnethy!ated, and hence may be bisulfitc converted to T (ifunmethylated) or remain as a C (if methylated), were designated with a Y (·where Y denotes C or T), and v,1here, after bisulfite conwrsion, actual maintenance of a Y designated base as a C was scored as methylation at that base, Thus, 5 these sequences represent the group of all combinations of all sequences in which 0, 1, or more than one Y is converted to a T.

The reverse complements of the bisulfite converted sequences of the (+) and (-) strands will be obvious to one of ordinary ski 11 in the art and are also included by implication in this disclosure.

The bisulfite converk'.d sequences of the fully methylated form of the corresponding expanded patches (i,e. in which all Y bases in every 10 bisulfite converted sequence are retained as a C), corresponding to the (+) strand the (-) strand were determined and recorded (see sequences of SEQ ID NOs: 3853-4280 and 4789-5136 for the bisulfite converted sequences of the fully methylated form of the(+) and(-) strands respectively of the corresponding expanded patches).

The reverse complements of the bisulfit(~ converted methylated(+) stand and(-) stand sequences will be obvious to one of l 5 ordinary skill in the art and are also included by implication in this disclosure.

CpG islands overlapping patches that may contain additional CpGs that are methylated coordinately \Vith patches \Vere also defined, The sequences of these CpG islands correspond to SEQ ID NOs: 5137-5926, The genomic coordinates of the CpG islands \Vere also recorded.

The genomic sequences of these expanded patches on the respective genomic 20 (+)and(-) strands were determined and recorded. (Upper and lower case designations were used according to those of the UCSC brnvvser, where 1ovver case sequences are lower complexity DNA sequences).

The bi sulfite converted sequences of these corresponding CpG islands (Le. the bisu!fite conve1ted sequence of the(+) strand and the bi sulfite converted sequence of the ( -) strand) were determined and recorded (see sequences of SEQ ID NOs: 25 5927-6321 and 6 717-7111 fi)r the bisulfite converted sequences of the respective(+) and(-) strands), C residues that may be methylated or unmethylated, and hence may be bisulfite converted to T (ifunmethylated) or remain as a C (if methylated), were designated with a Y (where Y denotes C or T), and where, after bisulfite conversion, actual maintenance of a Y designated base as a C \Vas scored as methylation at that base.

Thus, these sequences 30 represent the group of all combinations of all sequences in which 0, 1, or more than one Y is 92 converted to a T.

The reverse complements of the bisulfite converted sequences of the(+) and (-) strands ,vill be obvious lO one of ordinary skill in the art and are also included by implication in this disclosure.

The bi sulfite converted sequences of the folly methylated form of the corresponding CpG islands (i.e. in \Vhich all Y bases in every bisulfite converted 5 sequence are retained as a C), corresponding to the ( +) strand the ( -) strand were determined and recorded (see sequences of SEQ ID NOs: 6322-6716 and 7112-7506 for the bisulfite converted sequences of the folly methylated form of the(+) and(-) strands respectivdy of the co1Tesponding CpG islands), The reverse complements of the bi sulfite converted methylated (+) stand and (-) stand sequences wiH be obvious to one of ordinary skill in the art and are 10 also included by implication in this disclosure.

Regions oflnterest (ROI) were defined that provided preferred regions for design of PCR amplicons that would encompass preferred patches, The genomic coordinates of the ROI were also recorded, The sequences of tht! (+) strands of the Regions oflntert~st correspond to SEQ lD NOs: 8209-8222, and the sequences of the H strands Regions oflnterest correspond 1-; to SEQ ID NOs: 8251-8261. (Upper and lower case designations were used according to those of the UCSC browser, where lower case sequences are lower cornplexity DNA sequences).

The bisulfite converted sequences of these corresponding Regions ofinterest (i.e. the bisulfite converk'.d sequence ofthe (+) strand and the bisulfite converted sequence of the (-) strand) were detennined and recorded (see sequences of SEQ ID NOs: 8223-8236 and 20 8265-8278 for the bisulfite converted sequences of the respective (+) and (-) strands). C residues that may be methylated or unmethylated, and hence may be bisulfite converted to T (if unmethylated) or remain as a C (if methylated), were designated with a Y (where Y denotes C or T), and ,vhcrc, after bisulfite conversion, actual maintenance of a Y designated base as a C '..Vas scored as methylation at that base.

Thus, these sequences represent the group 25 of all combinations of all sequences in which 0, 1, or more than one Y is converted to a T.

The reverse complements of the bisulfite converted sequences of the (+) and (-) strands will be obvious to one of ordinary skill in the art and are also included by implication in this disclosure.

The bi sulfite converted sequences of the fully methylated form of the Regions of Interest (i.e. in w-hich all Y bases in every bisulfite conve1ied sequenct, are retained as a C), 30 corresponding to the(+) strand the(-) strand were determined and recorded (see sequences of 93 SEQ ID NOs: 8237-8250 and 8279-8292 for the blsulfite converted sequt~nces of the fully methylated frmn of the(+) and(-) strands respectively of the corresponding Regions of lntercst).

The reverse compleim~nts ofthe bisulfite converted methylated ( +) stand and (-) stand sequences will be obvious to one of ordinary' skill in the art and are also included by 5 implication in this disclosure.

Specific PCR Amplicons were defined within tbe Regions ofinterest (ROI).

The genomic coordinates of the Arnplicons were recorded.

The sequences of the(+) strands of the Amplicons correspond to SEQ ID NOs: 8293-8306 and 8405-8409, and the sequences of the (-) strands of the Amplicons correspond to SEQ ID NOs: 8335-8348 and 8420-8424. (Upper 10 and lower case designations were used according to those of the UCSC browser, where lower case sequences are lower complexity DNA sequences), The bisu!fite converted sequences of these Arnplicons (i.e, the bisulfite converted sequence of the(+) strand and the bisulfite conve1ied sequence of the (-) strand) were detennined and n.,corded (see sequences of SEQ ID NOs: 8307 .. 8320 and 8410-8414 for the bisulfite converted sequences of the(+) strands and 15 see sequences of SEQ ID NOs: 8349-8362 and 8425-8429 and for tht'. bisulfite converted sequences of the(-) strands). C residues that may be methylated or unmethylatecl, and hence may be bisulfite conve1ied to T (if unmethylated) or remain as a C (if methylated), were designated with a Y (where Y denotes C or T), and where, after bisulfite conversion, actual rnaintenance of a Y designated base as a C was scored as methyiation at that base.

Thus, 20 these sequences represent the group of all combinations of all sequences in which 0, 1, or more than one Y is converted to a T, The reverse complements of the bisulfite convetted sequences of the (+) and (-) strands wiH be obvious to one of ordinary skill in the art and are also included by implication in this disclosure.

The bisulfite coll'vcrted sequences of the fully methylated frmn of the Amplicons (i.e. in which all Y bases in every bisulfite converted 25 sequence are retained as a C), corresponding to the(+) strand the(-) strand were determined and recorded ( see sequences of SEQ ID NOs: 8321-8334 and 8415-8419 for the bi sulfite converted sequences of the fully rnethylated form of the(+) strands and see sequences of SEQ lD NOs: 8363-83 76 and 8430-8434 fix the bisulfite conveited sequences of the fully methylated form of the(-) strands).

The reverse complements of the bisulfite converted 30 methylated(+) stand and H stand sequences will be obvious to one of ordinary skill in the art 94 and are also included by implication in this disclosure.

Sequences of PCR primers used in amplification of the Amplkons are provided as SEQ ID NOs: 8377-8404 and 8435-8444.

Confirmatory analysis of candidate loci was then done using bi sulfite sequencing of candidate loci that \Vere amplified using bisulfite specific but rnethylation independent 5 amplification primers.

This employed a new sample set of biopsies frorn: 23 EA Cs, 8 HGD, 15 non-dysplastic BE from individuals without known higher grade lesions.

In addition, biopsies were obtained from 5 cases of BE adjacent to HGD, and from 11 cases of BE adjacent to an EAC.

These were not included in analyses of non-dysplastic BE.

In addition biopsies were obtained frorn 33 normal squamous mucosa samples. 10 Table 1 describes the performance in the confim1aw1y sample sd using bi sulfite sequencing analysis of amplicons of select loci having strong performance characteristics m1d identified in the studies discussed above.

In Table 1, columns C - S disclose the performance of the select arnplicons.

For each DNA St'.(.p.1ence read across each amplicon, the number of CpGs that were methylated was counted, and the read was classified as methylated or 15 unrnethyled using cutoffs for a required nmnber of methylated CpGs on the amplicon.

Table 1, row 3 lists the number ofCpGs between the amplification prirners for each of the ampHcons, Table l, row 4 lists the number of CpGs that need to be methylated on an individual read to count that read as methylated (e.g. for SqBE 2 there are 16 CpG residues betwl~en the primers, and 14+ (rneaning >" 14) CpGs must he methylated on a read to score it 20 as methylated.

Table l, rows 6, 7, and 8 record the sensitivity for detecting esophageal adenocarcinomas (EACs) (row 6), high grade dysp1asias (HGD) (row 7) and :non-dysplastic Barrett's esophagus (non-dysplastic BE) (row 8), using criteria in which a sample was detected if it demonstrated methylation in greater than 10% (0. l) of all DNA reads, Table 1, row 9 records r.he specificity of each amplicon for not detecting normal squamous mucosa 25 again using criteria in which a sample was detected if it demonstrated methylation in greater than 1 o~;, (0.1) of all DNA reads.

Table 1, row ! l records the specificity of each amplicon fi.1r not detecting norrnal squamous mucosa now using criteria in which a sarnple \,Vas detected if it demonstrated methylation in greater than 1 % (0.01) of aH DNA reads.

As a comparator, Table 1, column B provides the same data for dt,tecting methylalion in the Vimt:ntin (VIM) 30 locus amplified using primers disclosed in Li et aL (Li M, et aL (2009) Sensitive digital 95 quantification of DNA methylation in clinical samples.

Nat Biotechnol 27(9):858-863).

These primers correspond to SEQ ID NOs: 8445-8446.

The ampLicons amplified using these primers are as follows; Vimentin amplicon (+) strand (SEQ fD NO: ! 6207); 5 tTYGTttTtffAtYGtAGGATGTTYGGYGGttYGGGtAtYGYGAGtYGGtYGAGtTttA GtYGGAGtTA YGTCL-\tTA YGTttAtlYGtAHTAtAGttTGGGtAGt Vimentin amplicon (-) strnnd (SEQ ID NO: 16208); GtTGtttAGGtTGTAGGTGYGGGTGGA YGTAGTtA YGTAGtTtYGGtTGGA.GtTYG GtYGGtTYGYGGTGttYGGGtYGtYGAAtATttTGYGGTAGGAGGAYGAG Amplicons (and patches) need not be used individually, but can be combined into panels for detection of esophageal neop1asia.

Examples of such panels, and their associated performance statistics, are provided in Table 1, columns T through AG that provide the markers in thl'.1 panel and the sensitivity and specificity resulting from the marker combination (,vhen the cornbination is positive if any member of the combination is positive), 15 The sensitivity for detection of EAC (100%)), HGD (88%1), and BE (lOO~·o) is the same among the combinations shown of: an amplicons, 17 amplicons, 15 amplicons, 4 amp1icons, three of four combinations of 3 amplicons (columns Y, Z, AA), and for one combination of2 amplicons (column AF).

Specificity for not detecting normal squamous (97%,), at a detection cuto_ff of 1 O~o of reads being methylated, is the same for alt combinations shown of: 15 20 amplicons, 4 amplicons, 3 amplicons, or 2 amp]icons.

When spl'.~cificity is determined using a cutoff of 1 %i of reads being methylated, then axnong mnplicons with the highest sensitivity, the highest specificity is 94%, demonstrated by the combination of 3 amplicons of Table 1, column Z, foHm,ved by 91 % specificity demonstrated by combinations shown of: 4 amplicons, two combinations of three amplicons, and one combination of 2 amplicons. 96 T'/\f5I.,E J. f:,;_ A i B L-. c.J./) __ ,J<EC}._J/' .. _, .. ,.o,. __ [._.H,. __ [ _I_,.,[_ J --: . K_ ,: . I. ,: .. M ,: . N ,: . 0 ''<:; P. : . Q , . R , . S. , , ! ::,,qBJ::,! SqBf:.;, qLE .• Sq BE SqBE:SqBE; SqBE SqBE1 SqBE1 SqBE1 SgBE i SgBE i SqBE i "qBE i SqBE :SqBE i SqBE,1 ' '· ;n,.;f ' 2 ' S ~ 9 In ' ' l l ' ll 2 l" ' 14 2 ' 1 ,; ' l 6 " 1 •• 2 ' l" 1 ' ' 8 ' 2'" ' ._,, 2 ' T uunnH••·························----··••u•u ~ 'i !,_,,! .~ • , ,_ ! • - . f .:._~,.,.,.,.:,.,.,.,._~,.,.,.,.,.,. .......... 3 ....... ~ •u.ru~.ruu:,,,.,.,.,..Ju,,,:,.,.,,.,u~!,.,.),,.,,.0 ~,.,... .. ) .. ..,.., 1 ~ ........ ),..,,.. 1 .... .., ; L~.l. :·LL~ ; __,J NumberofCpGsini V j Jfi j JS 27 "' ! ,,, i l"' lO 19 • •y, i ,, i .,, i "6 i •n j "l i 9 ! ,,,. j 10 k,--·:·--~---:--=-·::-~!,!!¥}!~,?p-::!r1x?.t~-t-----'.~_J _ -_1 : • , ;; l ! l u ! I ,;,;, : l 1 ! ,;,Q ! ,;, : ,;,;, : ,;, l-----·----·--+·--·~·~·---+--·----·:----1 ,,,u,nl.e1utCpu .. ,i .. ec.tur , , .,, ....... ; ·,c•, ,. , ,. ,, ,,. ·A : ,, ,-. : g : 1 ., ',-. : ,. ' 11 '-· ' !rneth~,,dat1on caH 1 .)·~r- 1L~-+ ?.h- rrj_{J-+_ '. _1-~✓-~-Jw=~~-r-_wL .. 0-r-_w,w·•,-:.-..... ,..nJ+······j-t,-f-.J .... :·,·+-.... :l+ 1 1.:+ 1 -'-'+ ~ ·;}-+ 1 t)+ ~ + 1 t)+ i 1 Cut-offcc O. l -- .. _ ~ ! .. , ,, . , ,., ,. _ , .,,) ........ , ..... f .. ·:--·,--·--f ... , ......... f·------;;·--· --·;·;.;------L:.;·.-----) ... ~'.-;,·~;·:--+--:.;·;,···)·········••:--: . I : ~ • _ ! . : , . 1 , . ! . , ! Sen:m1v1tvtACi 10·:1a ! 14·10 ,100'%, 40:-<.., 65% ! 96% i 4'10 81% I .18%' rH% i !"<% ! 52'% ! 9-,;r, i :4% ! 91% ! 87%, i 82% ! 59% ! ('" Sensitivity HCiDl 6.Y<-: 50% i 100%! 57%: 50'}() 75%, i 0%, 63%i 33% f 43%, ( 6J%,: 50~/,;: 0'% i 63%T6:f~:{fgi;j"c;rr8iE~:·r4:f~:.c: Sensitivityfornon-1,n{,ni.l 81 ,v i ,,r;o1. ! ~fi"/- i 69"1 ggry O"' g"'y 21 o1 1 ">6"V !10,y,!l 4,,y j l"'''/ !,onc1! 8l''/ l,ooc1! gg,y i fi'''Y 1 d'1sphsticBa~ra,c;1'•--•Jv, /G,••.,;(J:···": .,; • " /0 40 .o,., ", l ... a, 40, .)O,, .... a, o,, .-Giu ",~.)O, i -' Si:x,:·ific:t:, ;·:i I 9 70{ : 97% i l 00"{: 1 n{)'J/, i 97'% 1 Q,% l 00'% !nu-:i "o,()-.--1--o,c~ +()4''{--: ·er,,;{·\ 00%·!--(f'l'J/,;--;--(P'J{--;--C),'J{--,--q,c~--;--9,'J{--: jcm,off~ o OI s~,:,a,:: sst 9: :Jq.,,i ,o:o,J;;;,,,,j 94%19:% 91% :;;, I:;~,; :;:,~ ; 91:~ ; 9:;,~ ; ·94% ; 9:;~ ; 9~:/4 ; 9,;:~ : ~~ ; 9~:~ ; 97 ~ N .0. .. <::', -..-.. \,C .-..l. N "'d t"'l g r.r, N .0. .. u, <::', .Q.C..) .(...;.; ~ 0 ""'' ..J "..J' (X) "' "' .0. . ..J I :;: I ""'' ,·,., .

TABLElB T fr~~i~l~:~::,,,,,:~;;~•iqBE1 W : X i y i 2'. ' AA ! AB i AC r AD __ J ... AE r A( i. )1G __ .,.! / 7 Sq}l[;, ~. ~~~\E 5 Sq~E/Sq;3E i ,..; ~',('.:;, .. 10 .,q,.,L . , . , 9 .,q,.,L 1(, , "'"p,!C,)[b sqi~f }~;~E ~~:~ i i:1 I "1~~2i;~;~:1 ! : ! ! ! t•i:~,.\,r;H\15 Sql?~,'.:\;1BE j '.st~r~ ,1\ . I ,· ,, I ! ! I S,,,JL , t,-·A J-l- ✓.: ,,_;}.,i., , 6- l , Sq3L , J.·~ tn-.)2 , tdc•, , , , Ail SoBE 16-2 Sq8E 16-2 ' SqHE Hi triol (SqBE 1.do3 , iSqBE drnihiel !dnubie2[drn;ble3!doublc:4 I ~·;_r~P}~~o •;;; •. s~;\l];!,. .: .. ~CJ;~ .1,z.~_1_ F ~~:B.~ ;~:,~? ~:~:·~~ 1: 10,s:BE (SqB~ 1 101 _. 1..,1~21 __ i (StJE I (S~1;1E I (~~~;; I {Sq~1E l;"'}''·;;~.'x~ ·/'·~\; ;:}"\ \q[;};.,:~;;·!;·:, ':::;·l:·,r "·/ .,,,}i···r: . 1 ,: 1 I r./· ,~q?~:·.; 3 ! ·;CJ·',::·:! ,.,;:\. i c,:\._. i :."_,~._. l • \ ·. .f!,!i,dn'-' 2 ... • l .... ,.,.,E. L2· .. 2 , ... , ,:'.>.,,f, .. , /.., .. ,. m.l ,L, le 2 .,q,:,E .~q,.,E. . q[,[ .. 2·, SqB,., 2 .... , .. ~.,B ... , ,,.,[L i SqbL ; SqJL I i ~ 1 i I doubh'.5) doub!e6 (SqBE I (SqBE } 1_~ 2__ ~ , ; ;3 -, Sq,.!I·, i Sq,:H.-. 22-- l) j 27 .. !) :• .. J,JmbtrnfCpG;;I VIM SqBE23 SqBE23 e.mplk.ons !I 18) 22 .. J) }) --+-----)), ___ ----f---LL?}.---f-----L~L---f-----!-~L-,., 2~'.--1) ',;;;.;;:,~:J;;;;;:r ___ ,_____ . I ! ! I ' ·-'.-- 1 used fi)r ~ ~~?t~WI·t/~!L. ... f· ··--··--··--------j-··--···----········· i : ; --------+-----------------' -------------------- ------------------_) ___ ------------ l 1.. .... ; -----1-------------·t--------------i c.,:_,..,.n~irivih/ F/\C; 1 O!j'~/. -; ov~~ l 1 OfW'(. ; 101)0:I, ( l 00°4.; 1 1r~ri.;,1; ; l :y·i-.1,1} = no~{~ f t(H} 1)i lOiVY, l{}i;ft{. ( 91)~t. 1 {l(Y¼ 961.i/, f ;::~~ .. 1.'~,~•;••:/•••;.•~;.,~:;.~•~• ,-•,,,,'•J; ~j;;• ~ ,-,:.,/>/• ~ •j--~,•J ; ..,,,,•. ; ~J•t~'; ,~•:-•, -~~I/ ~•u;1~hl w,_.i,~:: .... u+um ... "!•,','•rnfrr••,'-j,rVrr: •/•j •;•/-"V : .:-;~::n.fti:i' . .,;p., t·t,._;tJ\ gg~;;., : ES'½, 08i;A.1 ~ YJ8~1i ~ t5'¾, ~ &◊h, ; 23tJ1!, gg~.v i 1)'¾.1 1.:-~/(\ : 6.:- 0/:; b8YC 82~1: 08?/o 1 -·,:, /..·.: •. • • j • j t ~ r S,;.,;n~,~~ .... it-✓ ~ ~ ~ i i fr;;- nn/J Ji)()';,;, Jf)(J';,i, )ilf"r, i )i)()')-i-. i ]i)O''~ i 1nw,;, i Hi')'';, l')fi"-1 i ]N"ri I 94,.•.;, i JI':("¾ ·•')0'¾ 1')0% ' 1r,0% i..5].:>'.~phlstidiarr~,'sl • • . • • "' ' l • •• • i. i • i. \ • ·' ,. ' ••• ' ! ,v • , /. ! '"' '. ... , ... ~: ....... '. ... , ... ~: .. : ..... J ... ~: .. ,. ... .. !cm~::~:;,: ::: ::: ' :::: 97% i . ::: 1 :::: : ::: J =-L~;~J:,;: :::: '._:;:_:_:::j :::-i ::: 98 ~ N .0. .. <::-, -.... \,C .-..l. N "'d t"'l g r.r, N .0. .. u, <::-, .Q.C..) .(.,.;.; ~ ""_,'' "_', (X) "' "0 ' ._..,.

I :;: I "' Jl:,:}11-.ipk_ 2: __ kkntifkn~ion of i::~~,ophagq,,d .can,:er fnfo,:rmnJiv~~ k,K:i tc>_tkte,;t pr(}Rres:'don_ of ~;inmJwgea! ncoph,ia Discowry data were also analyzed for each individual CpG residue in the RR.BS data set to identify loci that could be used to distinguish EAC from BE.

Individual CpGs 5 \.Vere considered methylated in EAC versus BE if they showed methylation ofless than 10% of reads of all infr,rmative BE samples, where at least 3 BE samples were infixmative, and if they showed methylation of less than 10°/t, ofreads of all informative nonna! squamous samples, and where an informative sample had equal to or greater than 20 reads covering the CpG, and if 6 or more oftht~ EAC samples demonstrated percent 10 methylation at a level that was at least 20 percentage points greater than the methylation level of the most methylated. BE sample.

CpGs meeting criteria for methylation in EAC versus and BE are defined as methylated in EAC vs BE.

Such methylated CpGs were then aggregated into patches in instances in which methylated CpGs were within 200bp one another. 15 186 genomic patches defined as methylated in EACs versus BE in the discovery set were identified (see SEQ ID NOs: 8447-8818).

The genomic coordinates of the genomic patches defined as methylated by the above criteria \Vere also recorded.

The genomic sequences of these patches on the respective genomic(+) and H strands were determined and recorded. (Upper and lower case designations were used according to 20 those ofihe UCSC browser, where lower case sequences are lm:ver complexity DNA sequences).

The bisu!fite converted sequences of these corresponding patches(Le. the blsulfite converted sequence of the(+) slrand and the blsulfite convened seguence of the () strand) were determined and recorded (see sequences of SEQ 1D NOs: 8819-9004 and 9191-9376 fbr the bi sulfite converted sequences of the respective(+) and (-) strands), C 25 residues that may be methylated or unmethylatecl, and hence may be bisulfite converted to T (if unrnethylated) or remain as a C (if methylated), ,vere designated with a Y (v,here Y denotes C or T), and where, after hisulfite conversion, actual maintenance of a Y designated base as a C was scored as mcthylation at that base.

Thus, these sequences represent the group of all combinations of all sequences in ,vhich 0, 1, or more than one Y 30 is conve1ied to a T.

The reverse compkments of the bisulfite convened sequences of the (+)and(~) strands will be obvious to one of ordinary skill in the mi and are also included by implication in this disclosure.

The bisulfite converted sequences of the folly methylated form of the corresponding patches (Le, in which all Y bases in every bisulfite 99 converted sequence are retained as a C), corresponding to the (+) strand the (-) strand were determined and recorded (see sequences of SEQ ID NOs: 9005-9190 and 9377-9562 for the bi sulfite converted sequences of the fully methylated form of the(+) and(-) strands respectively of the corresponding patches).

The reverse complements of the bisulfite 5 converted methylated(+) stand and(-) stand sequences will be obvious to one ofordinary skill in the art and are also included by implication in this disclosure.

Patches were expanded by 100 base pairs on either side so as to accommodate either the design of amplification pimers or to enclude additional presumptively methylated bases.

The sequences of these expanded patches correspond to SEQ m NOs: l O 9563-9934 and their genomic coordinates were also recorded, The genomic sequences of these expanded patches on the respective genomic(+) and(-) strands were determined and recorded. (Upper and lower case designations were used according to those of the UCSC browser, when: lower case sequences are lower complexity DNA sequences).

The bisulfite converted sequences of these corresponding expanded patches (Le. the bisuifite 15 converted sequence of the ( +) strand and the bisulfite convened sequence of the (-) strand) were determined and recorded (see sequences of SEQ ID NOs: 9935-10120 and 10307- 10492 for the bisu!fite converted sequences of the respective (+) and {-) strands), C residues that may be methylated or unmethylatcd, and hence may be hisulfite converted to T (if unmethylated) or remain as a C (if methylated), were designated with a Y (where Y 20 denotes C or T), and vvhere, after bi sulfite conversion, actual maintenance of a Y designated base as a C was scored as methylation at thal base.

Thus, these sequences represent the group of all cornbinations of all sequences in which 0, 1, or more than one Y is converted to a T.

The reverse complements of the bi sulfite converted sequences of the (+) and H strands will be obvious to one of ordinary skill in the art and are also included 25 by implication in this disclosure.

The bisulfite converted sequences of the fblly methylated form of the corresponding expanded patches (Le, in which all Y bases in every bisulfite converted sequence arc retained as a C), corresponding to the(+} strand the(-) strand were determined and recorded (see sequences of SEQ ID NOs: rn 121-10306 and l 0493-10678 for the bisulfite converted sequences of the folly methylated form ofthe (+) 30 and(-) strands respectively of the corresponding expanded patches), The reverse complements of the bisulfite converted methylated (+) stand and H stand sequences will be obvious to one of ordinary skill in the art and are also included by implication in this disclosure.

JOO CpG islands overlapping patches that may contain additional CpGs that are methylated coordinately with patches were also defined.

The sequences of these CpG islands correspond to SEQ ID NOs: 10679-10972.

The genomic coordinates of the CpG islands were also recorded.

The genomic sequences of these expanded patches on the 5 respective genomic (+) and (-) strands vvere determined and recorded. (Upper and lower case designations were used according to those of the lJCSC browser, ,vherc lower case sequences are lower complexity DNA sequences).

The bisulfite converted sequences of these corresponding CpG islands (i.e. the bisulfite converted sequence of the (+) strand and the bisulfrte converted sequence of the(-) strand) were determined and recorded (see 10 sequences of SEQ JD NOs: 10973-11119 and 11267-11413 for tht: bisulfae converted sequences of the respective(+) and H strands). C residues that may be methylated or unmethylated, and hence may be bisulfite converkd to T (if unmethylated) or remain as a C (if methylated), were designated ·with a Y (vl1ere Y denotes C or T), and where, after bisutfite conversion, actual maintenance of a Y designated base as a C was scored as 15 methylation at that base.

Thus, these sequences represent the group of all combinations of all sequences in which 0, l, or more than one Y is conveited to a T.

The reverse complements of the bisulfite converted sequences ofthe (+)and(-) strands will be obvious to one of ordinary skill in the art and are also included by implication in this disclosure.

The bisulfite converted sequences of tbe fully methylated form of the corresponding CpG 20 islands (i.e. in which all Y bases in every bisulfite converted sequence are retained as a C), corresponding to the(+) strand the O strand were determined and recorded (see sequences of SEQ TD NOs: 11120-11266 and 11414-11266 for the bisulfite converted sequences of the fully methylated form of the(+) and(-) strands respectively of the corresponding CpG islands).

The reverse complements of the bisulfite converted methylated(+) stand and(-) 25 stand sequences will be obvious to one of ordinary skll] in the art and are also included by implication in this disclosure.

Regions of Interest (ROI) were defined that provided preferred regions for design of PCR amplicons that -....vcmld encompass preferred patches.

The genomic coordinates of the ROI were also recorded.

The sequences of the(+) strands of the Regions of Interest 30 correspond to SEQ ID NOs: 12563-12568, and the sequences of the(-) strands Regions of Interest correspond to SEQ TD NOs: 12581-12586. (Upper and lower case designations were used according to those of the UCSC browser, where lower case sequence~: are lower complexity DNA sequences).

The bisulfite con vested sequences of these 101 corresponding Regions of Interest (i.e. the bisulfite converted sequence of the(+) strand and the bisulfae conveiied sequence of the O strand) were determined and recorded (see sequences of SEQ ID NOs: 12569-12574 and 12587-12592 for the bi sulfite converted sequences of the respective(+) and(-) strands). C residues that may be methylated or 5 unrneH1ylated, and hence may be bisulfite converted to T (if unmethylated) or remain as a C (if methylated), ,vere designated with a Y ( where Y denotes C or T), and where, after bi sulfite conversion, actual maintenance of a Y designated base as a C was scored as methylation at that base.

Thus, these sequences represent the group of all combinations of aH sequences in which 0, 1, or more than one Y is converted to a T.

The reverse 1 n complements ofthe bisulfite converted sequences of the(+) and H strands will be obvious to one of ordinary skill in the art and are also included by implication in this disclosure.

The bl sulfite converted sequences of thl'. fully methylated form of the Regions of Interest (Le. in which all Y bases in every bisu!fite converted sequence arc retained as a C), corresponding to the(+) strand the O strand were determined and recorded (see sequences 15 of SEQ ID NOs: 12575--12580 and 12593-12598 for the bisulfite converted sequences of the folly methylated form of the{+) and H strands respectively of the corresponding Regions ofinterest).

The reverse complements ofihe blsulfite converted methylated(+) stand and (-) stand sequences will be obvious to one of ordinary skill in the art and are also included by implication in this disclosure. 20 Specltk PCR Amplkons were defined within the Regions ofinterest (ROl).

The genomic coordinates of the Amplicons were recorded.

The sequences of the(+) strands of the Ampiicons correspond to SEQ ID NOs: 12599-12604 and 1264 7-12649, and ihe sequences of the H strands of the Amplicons correspond to SEQ ID NOs: 12617-12622 and 12656-12658. (Upper and lower case designations were used according to those of 25 the UCSC browser, where lower ease sequences are lower complexity DNA sequences), The bisuifite converted sequences ofthese Amplicons (Le, the bisulfite conveiied sequence of the(+) strand and the bisuifite converted sequence of the(-) strand) \Vere determined and recorded (see sequences of SEQ 1D NOs: 12605-12610 and 12650-12652 for the bisu!fite conveiied sequences of the(+) strands and see sequences of SEQ ID NOs: 30 12623-12628 and 12659-12661 and for the bisulfite conveiied sequences of the(-) strands). C residues that may be methylated or unmethylatcd, and hence may be bi sulfite conveited lo T {ifunmethylated) or remain as a C {if methylated), were designated with a Y (where Y denotes C or T), and where, at1er bisulfoe conversion, actual maintenance of a ]02 Y designated base as a C was scored as methylation at that base.

Thus, these sequences represent the group of all combinatlons of all sequences in which 0, l, or more than one Y is converted to a T.

The reverse cornplements of the bisulfae converted sequences of the (+) and H strands will be obvious to one ofordinary skill in the art and are also included 5 by irnplication in this disclosure, The bisulfite conve,ted sequences of the fully methylated form of the Arnplicons (i.e. in which all Y bases in every blsulfite converted sequence are retained as a C), corresponding to the (+) strand the (-) strand were determined and recorded (see sequences of SEQ IDNOs: 12611-12616 and 12653-12655 for the bisuLfite converted sequences of the fully methylated form of the(+) strands and 10 see sequences of SEQ lD NOs: 12629-12634 and 12662-12664 fbr the bisulfite converted sequences of the folly methylated frirm of the(-) strands).

The reverse complements of the bisulfrte converted methylated(+) stand and(-) stand sequences will be obvious to one of ordinary skill in the mt and are also included by implication in this disclosure.

Sequences of PCR primers used in amplification of the Amplicons are provided as SEQ 15 JDNOs: 12635-1264611nd 12665-12670.

Confirmatory analysis of candidate loci was then done using bisulfite sequencing of candidate loci that were amplified using blsulfite specific but methylatlon independent amplification primers, This employed a new sample set of biopsies from: 23 EACs, 8 HGD, 15 non-dysp!astic BE from individuals without known higher grade lesions, Jn 20 addition, biopsies were obtained from 5 cases of BE adjacent to HGD, and from 11 cases of BE adjacent to an EAC.

These are not included in analyses of non-dysplastk BE. ln addition biopsies were obtained from 33 normal squamous nrncosa samples.

Tabk 2 describes the performance in the confirmatory sample set using bisulfite sequencing analysis of amplicons from select loci defined as methylated in EACs versus 25 BE and having preferred marker characteristics.

In Table 2, Columns B J disclose the performance of the amplicons in the confirmatory data set In this data set, methylation was calculated as the average level of methylation of all CpGs in between the primers for amplif}'fog the amplicon.

For each read across each amplicon the number ofCpGs that were methylated was counted and the read was classified as methylated or unmethyled 30 using cutoffs for a required number of methylated CpGs on the amplicon.

Table 2, row 3 lists the number of CpGs between the amplification primers for each ofthe amplicons.

Table 2, row 4 lists the number of CpGs that need to be methylated on an individual read to count that read as methylated (e.g. for l1p3 there are 36 CpG residues between the 103 primers, and 25+ (meaning >=25) CpGs must be methylated on a read to score it as methylated).

Table 2, rows 6 and 7 record the sensitivity for detecting EACs (row 6) and HGD (row 7) using criteria in which a sample was detected if it demonstrated methylation in greater than 10%, {0.1) of all DNA reads.

Table 2, rows 8 and 9 record the specificity of 5 each mnplicon for not detecting non-dysplastk BE (row 8) and for not detecting normal squamous mucosa (row 9) again using criteria in which a sample was detected if it demonstrated methylation in greater than 10% (0. t) of all DNA reads, Table 2, rovvs 11 and 12 record the specificity of each arnp!icon for not detecting non-dysplastic BE (row 11) and for not detecting normal squamous mucosa (row 12) using criteria in \Vhkh a 10 sample was detected if it demonstrated methy]ation ln greater than 1 % (0.01) ofall DNA reads.

Arnplicons (and patches) need not be used individually, but can be combined into panels for detection of esophageal neop!asia.

Performance stalistics of selected panels of amplicons are provided in Table 2 columns K through V that provides the sensitivity and specificity of the panels (when the combination is positive if any member of the 15 combination is positive\ 104 TABLE2A ----------------------------------------------------------------------------------------------7----------------- l - ---------~ !;,t. B C D ii: F G H : I I <l ' ;- -:,-.----_b -- -.f"l"'-,-G:: ;·:_.-------,,, _-----------;- _~ ,P:3----!---ufrrn. . ! __ T.]~1_:5 ampH _ u~:5 ampl2 _ u~~o &m_pn_j __ Upl:o &mpl2 _j ... u2.l}l17 ___ l__ l);:-' &mpl1 _____ "!JJ.:-;,5 ampll- l , Num e, oL '-'f-' '> ,r, ;;_mp,,c-ui1 1 .JG , JJ , L.J Lu LL , u , 1 ~'-' ,o , , ''l''Jl·,s'' i , ; I , , l~~~~-b~~~ofCpGsusedfor, ,'.; 25+ ~ 17+- r '15+' n , ml5+ n , ll+f 6-~- .,-u 1 11-~· j 14+ J :iO+ I methylation ;an ! I l I Cut--off-c- 0. l Sensitivity EAC 15~/v 40%, : r,r,r,rrr<r<rrrr,: Sen1:itivi,y HGD O';;o I 25'1/,, 22%, 26~-'u 37% 0% 20% 45% l 30% lJ'Vi, Specificity for non--dysp[ast1c BE JOO~l:, 100% 100'->{~ n~. I '3~. ' I)~·- ; '7c,, ' l 7'" l 250/ ; -•-•- ~.I /f;••••••••••••••• !•••••• "' 1 ••• 1/!)••••••••••••••• ~-•••••••• ~ •• /f;•••unrr<r<r<Jrr<r<r<J. 11,/f;r)rr<r<r<:rr<r/(~ 111111111111111;,1111111 ~r,/~) ,,.,,,,,,.,,r, : i 94'% i JOO% i JOO%, : JOO% i 100'% i [00% 1 1 • ------~ :~-- •••••••• 1 ---·········--·········- ~ ! Spedfa:itv Sq 100% ; 100'½, i lOO'Vi, i 97% 1 l00%, 100% 97%, 100%, l ~:~:--,Df::' ;:~ 1 , - rn ''")' ''~ ,- 0:- •• i" 0/ t -0/ r 1 .. )J/ l ()/,U/ 1 Dtl"/ 'I I}/ f I I}/ ----, ,0-----------------!------w•-----------------; , "p~t:tf,,,ff) to, n.;,,-d:,,,,;!aSl:>, 8,.,, ]J(}_.,,, JU(},_,, ,.UO,,, , ,-,.u , '''" , ;_J(}_." , ]UO.u ,001/o i l.,l/1/o f --••--••u•uuuu••u•uuuu••u•uuuuuu••u•uuuu••--•••uu +••--••u•Hu••--J # .............. ,,.j., .......... ,.,.,, .. ,., .. .,.,.,;• .... ,., • .,,. ...... .,.,.,.,.,.. •••, .. .,.,., ............ ,..,, ... .,' ................ n,uu, .. •un' ,nu ; 1 Specificity Sq • ! 00% j 8S% j 100'½, 1 91 '% 1 90'½, ! !00% ! 93c%, ~ uu,NNUUuunnnnnn,rnnnnnnn,rnn,r,nnnn,runnnnn,r :,nn,ruuuunn),.,uuuuuu,r,,r, S ,runnnnnnn,rnunn,r,) n,rnn,Nuuunnnnnn,,) ,,,.,,,,,.,n,uunnnnn,uu. k nnn,,u,,,,,,.,..,-,,,,,,,-,u,,-:, ,..,..,-,-,.,-,-,,-,.,.,-,-,-,-•}'-------~------~ 100'% IOO{t,c 100·~-c TABLE 2B r------------------i -------------------1----·-------------- :-------------------i------------------l------------------:-----------------1--------· 1 ·-----i- - ;i .,. i K • ! I ri1 i N i o p j O i R S ' ~ i . : -· ' . ~ ' ' . . : .. ' ~ : .. T u V 1 Al: .. ~ ~nnp!~CV~!-: • ~og!!fuer~ iJp), ~ ~~mp,;~r.a'1 ,.nnplk•:m Op~lJ~ ri(l'.)H i~'.:.1mf}ci;~ -.;mnbv1; Vr,15-L :tfmpfo.:rm~ f :.trM :m . .J Ur.05-l 4 4 4 4 Fr,15-·lt U~}J~ I ;:Jg..3, Qr;<l Vr3, l J :f.rmplk~H"/. &mμ:i{:Oa ~mr1lk:.m M!t(tHi.:N·i ~ &mp~;tun LTf!~{l.~L uμ;5.J, ! l:~1~-1, U(!l5•~;~ 1 3 mr1kmt ~m{.!;;,:,-.,m {:,1)3:!h!! 1: UJ!!tl,v 2! oomOO 3: (;Omi..K< .j.: i ~m!!b) 5: l;ft!li•·/., Up~S-7~ ! lJμJ5<2~ Ufd5--.i.. f~ff1pH~<(i'lo ~ ,.::mr~foJ l ,x,,-:-,lx~"?:; Up~S-J,, U~I.5-t~ Pti":5-L tJp~~-J, i l!j};5-1~ Vp?'1, EJp2H-J~ ~ Vp!(:-1, lfr~ZlJ.-L nm~tm; ~ lJpl.5-11 lfr~l~·-1, EJP:!:t-J~ Up?-S-11 lfri.35-L 8]p;)~-1. U~$-11 . .

Vp.3:;...L , ~Jp17) [ (:;,i11 Vri1-t. 8Jt11:t•J~ I (J;,YS-L v 1~1:1--1, Up:w, ·l:;,1(:1 v 1 ,1:,...2.

Vpt:t-!) , Vμ2U-11 . :a••····. ................................................................. L.1i.r:?~t ...... J.J~J?~~~:L. ..... L.r~.?:.!. ..........~ rn.):;-i a;~):1~-3 ; (:n30 tl~:Zl?-l ~jn:;_5.i t},n!: ... ?:P.~jtL .......

Jd.P.37. ......... J.. . ~~J~!.?. ........ ) 105 ~ N .0. .. r:;-, -..-.. 1-C .-..l. N "'d t"'l g r.r, N .0. .. u, 0 r:;-, .Q.C..) .(...;.; ~ ""_,'' "_,' (X) "' "' 0 ._.., I :;: I ""'' ~-------------------------------~----------------------------~---------•• .. •••••••••uuu•••.

A K ~ll i;.;,.;.p~if:(•l'IS fo;,g~fo~.-: L fe..,1 Y"•).

UpJ, 2:mp~;.:mi f :;i.upEr.v.1 0 p Q }l s T lJ V Up}O, 6 ~•:H-c !:ombii 3.: l '..'.fHnho 2~ . , l'.~~";~-~• a~f~_f•l~Ujf~! ~!f•~/ ~rH3 j U~3S-~. ! j __ 4 . . .~ . ~ . 1 4 _ j ,$ _ (1μ';::,-/i, t,r,,), ~Jp.~, l ~;:d lJ~) .{. 1 ~ : .P :-:mrla:r,r~ ,;)l.np3~C•)>i zmph<nn l ampfo:xm ; ~mphi::•m :~'.p1!}-_:· l~r,l:-!/ tJp:!-3~ J !'.p35-_L ~ , 3 rnpH~ori [ s:mpH-:~n r:Jm~ 1~ .;?m~,:,~ /4: £~wt.n 3~ j ";?m~~.. ➔~ j c~,m~(• :S: ,•p?1J.,., Lyb·,., !11,,,~,, i ,•pl5-Z. 1 smp,,cm, <omho l i <<•ml>o ., L,.fa.J, !,pl>H, l•pVi-l. 1 Lpb-,, 1 (Jpl5-l, ":Jr2?~ 1Ii;2~-l.

Op10-3t J :.Jp2!J-L [ (.(H'nfio~ Up1S-Et f Up~s-i~ 1JE~35-3~ Vp3s--;~ UpJ!S-L j 1Jp35-l. ( ;Jp35-L vp.;s-t. 1;1;~r;. -op2-:\ l ,~~:n. I D:,,t:;-J, i UpJ::i-E~ l uP.H-t. 1J,;10. npi:)~ ur1s-2. i 1Jpt;;..,1, ; fJp20-;i ~~~~~:~~~:·::m'"'°" ,,,,, PW« UW~LT~~Lr~-,"""--t~L-_,a~~_":~-- ~::·_ ;_,.:,_r:~--1 ! N'.'.'r:b:: ~,l_C~,,/' :.b,.l tor ! j i i j j 1 m~thy:o.!10,1 ca,l , , , : i , 1 ~:;:;:,,: ~Ac l ';~: :;~ ' ''.~: i ':.~: 1 :2~- 1 ::~ :':', j "'~ , '.'~ ,1~. . 61~:i '.'.~ ~en::.1'i-:Vl:.\, H(ifJ : .)d·;,,J . .·•6/'fi )tr,o ' ~h:".n) ' _:.,)/(! . .!◊/(~ ..J81/(J : 38½] ' ~•"'570 381/(j ' 2::1,) l _:t6/{! :::::~::::::;mm d;;plern m: -1 ::: i ::: ~ :::: l :: i :::: : ;:~~ r::: l ::: +~:.-1-:::-!-~:: r:-1 !_ Cut··Ofi'• 0.01 ............................................. i. .................. :. .................. ~ ................... ;. ................... !. ................. J. ................. !.. ................. ). ................... \. ................ _.! .................... !. .................. .................. ) ; Spedfkitv for nor:-dYsplastic BE ; 88% ' 88% : 88% \ 88% ' 100% ! 100% i 94% ' 94·:;<, ! 100% i 88% ' 94"/o 94% ! f-- -0 • ">•• •.,_- • • • ~ •• C• ••••••••••••••••"'••••••••••••••••••••••••••••••f•••••• r"' ... {o/ ••••i••••••••••• f.i ,c•••••'.••••••• J),J/ ••••<•••••••••,.,,'i},/ : :') "\(j/ ~••••••••••, (J•f" •••••: • ... & • • ,3 ' ; ••••• ("', •• (i'.{ ••• t ••••• • {:3 .---•• l ••••. • • {!/ u,rr rrrrrr ! •u ($/ ,rrr,: , ::,p~cinuty ,,C: , "" in : 82 n, 8.,,_ ,,., 8,:; ,,., : l \/v ,,, , 91• Y" , 'h .Vo 88 1/" , '.7 l n, , 8., Y" : 88 1,, 88 i" ~ • 1 : : : 1 : ~ 106 ~ N .0. .. ,:;,, -.... \,C .-..l. N "'d t""l g r.r, N .0. .. u, 0 ,:;,, .Q.C..) .(...;.; ~ ""_,'' "_', IX) "' "0 ' ._..,.

I :;: I "' In addition, RRBS discovery daia was analyzed to identify CpG residues that demonstrated: i) at least 3 informative BE, vvhich in every infbrmative BE demonstrated at least 90~1o metbylatlon; and where ii) no more than YVi; of informative normal squamous samples demonstrated met.hylation level below 90%,;and that Hi) demonstrated at least 6 5 informative EAC, where in these infi:irmative EA Cs the level of methylation was al kast 20 percentage points lower than the methylation level of the !east methylated BE CpGs meeting these criteria are defined as umnethylated in EAC versus BE.

Such unmethylated CpGs were then aggregated into patches by grouping together unmethylated CpGs that were within 200bp of one another. lJnmethylated ln EAC patches may consist of 1 CpG 10 up to any number of CpGs that meet the above criteria.

Biopsy samples ( that overlapped with the confirmatory biopsy sarnpk set) were 15 further analyzed in tests of panels of markers J;x detecting the progression of Barrett's esophagus to Barrett's esophagus high grade dysplasia (HGD) or to esophageal adenocarcinoma (EAC} For each panel of markers, Figure 1 shows the sensitivity (percentage of samples detected), the specificity (percentage of samples not detected), the total number of samples studied, and the total number of positive samples.

Three panels 20 of markers were selected for study.

The first marker panel consisted of detecting at least one ofthe following four methylated markers: Upl5-l, llp35-l, Up27, and UplO {using hisulfite sequencing analysis of the corresponding arnplicons and using the criteria fix detection specified in table 2A).

The second panel consisted of testing for somatic nonsynonymous mutations in TP53 in assays in which TP53 was amplified from genomic 25 DNA using a set of PCR amplicons that spanned the TP53 coding region and in which Next Generation DNA Sequencing was then used to compare TP53 sequences from esophageal lesions versus matched normal esophagus tissue, Sarnples were classified as detected if a TP53 mutant allele frequency of greater than or equal to l 0% was identified.

The third panel was a combination of detection of methylatlon in any of Up15-1, Up35-l, 30 Up27, and lJplO or detection nfmuiation in TP53.

Table 3 shows the individual performance of biomarkers in detecting the different sample types of the 1st validation biopsies set using difforent cut--off criteria for methy!ation than the analysis of table 2.

Shown is the perfrmnance for detection of 107 different sample types of 5 dlfforent methylated DNA markers tested by bisul:fite sequencing analysis of the corresponding amplicons (Up 3, Up l 0, Up27, Up35- l, Up35-2).

Table 3 specifies the number of CpGs required to be methylated on a DNA sequence read to classify that read as methylated for this analysis, Results are presented when samples 5 are considered methylated if grealer than or equal to 1 % of all DNA sequence reads are classified as methylated, or if greater than or equal to 10%, of all DNA sequence reads are classified as methylated.

Also shown is the performance for detecting samples of testing for non-synonymous somatic mutation in assays in which TP53 was amplified from genomic DNA using a set of PCR amplkons that spanned the TP53 coding region and in JO which Next Generation DNA Sequencing was then used to compare TP53 sequences from esophageal lesions versus matched nomrnl esophagus tissue, Shown are rates of sample detection (expressed as sensitivity or specificity) when samples are classified as TP53 mutant if greater than or equal to 3~/,, ofTP53 reads are scored as mutant, or if greater than or equal to l 0% of TP53 reads are scored as mutant TABLE3 Unchanged CpG # cut-off applied to all Updated CpG # Cut-off ,;3mple .sets ..................................... ····--······--··· ............. ,,pp lied to al! .sample sets ........ l!,P~5~3 __ ....-J Up3 25+ CpGs Up35-1 14+CpGs Up35-2 lO+CpGs UplO 26+CpGs Up27 15+cpGs .... category >1%ecpo:sitive. >!%=positive . >1%:cposltive _ >.1%"'posltive >1%.,,positive >3;\-~" ---...J Sensitivity EAC 20% 35% 30% 20% 70% [----................................................................................................................................................................................................. .

Sensitivity for BEs synchronous to EAC or H····G···D-- ----·······--···1·t · · .... ••·············7···%··· ......................2...0..%.... .......................1...9..%.... ........ 0% 7%, 25% ----1·~----+.--------i Sensitivity ·• l HGD 17% 33% 38% 0'.% 17%} 25% ,-,,-,.-,.-,.-,.,-,.,.-,.-..-,.-..-,.-,.-,.-..-..-..-,.-,.. .. ..._. ......_ _ __ ----------,•,--••-- __ ••• .• ..... •••••••nn••••••••••••••• •••••••••••••••••••••••• ....... •••••••••nnnnn•nnnnn·f•nnnnnn••~~.......J pecificlty . ! Sq 100%· 100'% 100% 100% 97%t .1.00% -,......,..,.,,.,.,_._...,_...,.,.,..,..,. • ...,..,.,...,_..,.,.,.,..,..,.,.,..,..,.,.,..,...,...,_...,.,....,..,., -.--.~-.~._.,..,...,_,..,.,u.,.,,,_,., I-,.,. ~•~•.,.,_•.,._,.._••u•••,.,.••- .......................... ,.,. ••• ,. ••• ,.,.., .............. •••••••,. .............. •••• •••••• .. ••••• .............. •••u•n, pecificlty for nondysplastic BE 100% 100% 100% 100% 100%, 100% ....................................... __ .J..__~~-~----_,.,.,;~~~~-..l----~....,;._~~-..:,,;__.:..;.; 108 > 10%0,positive > 10% "positive> 10%,0 positivei> 10%,,positive > 10%" positiv~, > 10%0 ,positive Sensitivity EAC 30~.J 15% 45% . -: 10% 15% 61% Sensitivity for BEs matching higher pathologies, either EAC orHGD Sensitivity HGD 7% 1% 0% 17% 6% 0% 1% 25% 179t Table 4 shows the performance of selected combinations of the methylated DNA 5 markers (Up3, UplO, Up27, Up35-1, Up35-2) for detection of different sample types in the experiment presented in Table 3.

Samples are scored as methylated if any member of the marker combination panel scores the sample as methylated.

Results are presented when the 1ndividual markers are considered methylated if greater than or equal to 1 % of all DNA sequence reads are classified as methylated, or if greater than or equal to 1 OlJ,-·o of rn all DNi\ sequence reads are classified as methylated. 109 19% 25% TABLE 4A Cut-:<>!f :or i positivity i (e,J, ! samples ls i positive ff ! more than i ~ .~ ~,( i tnis ,;,, of i reads are i methylared! at the 9h1eni numb~r or! CpGs) i Categorv Up35-1 iUp35-1 jUp35-1 jUp35-2 jUp35-2 iUp10 iUp3. iUp3.

Up3 ~nd 1Up3 _and 1Up3 and 1Up3 and ~Jnd pnd ~nd jand ~rid ~nd ;Up35--1, jup35--1, Up35-1 Up35-2 UplO Ue2L jLJp35~2 _ lUplO ii.Jp27 iUplO pJp27 iUP27 jUp.35-2 iUplO 1% jsensitivity EAC 50%! 45%! 50%! 35%! 50%! 60%1 60%1, 50%1 50%1 40%! 50%1 60%! 1% 1% 1% 1% 10% 109f. [sensitivitv for BEs [synchronous to EAC or 1HGD [sensitivity HGD !Specificity Sq [specificitv for nonfdvs 1Jlastic BE ! ' J l s ensi.n,.v 1.t .y C,_ f\ L- !l 20% 20% 33% 333/cj 100% 100% 100% 100%j 45% 35% :U.% 13% 25% 33% 100% 97% 100% 100% 30% 25% 27% 33% 100%I I 100%\ 45% 1'.l%1 I I I 20%i 11%i 27%, 'l. -. . . . . . ,,_ ~ 0%, I 27%1 11%] 50%1 , , I 50%) 50%[ 50%! 0%! 33%1 50%i 100%1 , ·I 97%1 100%[ 97%\ 94%l 100%1 100%: I 100%1 ( ' 10□%i i ' 100%i I 100'¾) 10□%1 100iJOOi 50%! 50%1 30%1 40%! 20% 45% SO%! ' Sensitivity for BEs I J i matching higher I ! 'pathologies, either 1 I IEAC or HGD 1 13% 13?;; 11% 13% 7?-1r, 0% 13% 0% 13% 0% 13% 11%1 !s€:risitivity HGD l 17%! 33%J o;~, 17% 33% 25%1 33%\ 50%! 50%! 0%, 33%! 2?J'?l 110 ~ N .0. .. <::', -..-.. 1-C .-..l.

N "'d t""l g rJ).

N .0. .. u, <::', .Q.C..) .(...;.; ~ ""_'', "_', (X) "' "0 ' ._..,.

I :;: I ""'' TABLE4B I Cut-off for i positivity I; ,I e.1•. !samples is I positive if more than this% of reads are methylated at the I nu~~:~of! Cp(;~) j Category 1% lsensitlvity EAC 1% 1% 1% 1% 10% 10% !Sensitivity for BEs jsynchronous to EAC or !HGD ' ]Sensitivity HGD jspecificity Sq jspecificity for non! dysplastlc BE pensitivlty EAC !Sensitivity for BEs imatching higher !pathologies, either jEAC or HGD !sensitivity HGD I j i i 1 I jUp3. [0p35~1, iUp27 60% 20% l 50% j 97% 100% i 509·b 2O%i I' .3.3 0:r'e11 I Up3.

Up3S··2, Uf)lO l , , ,Up3.

Up~.

Up3. jUp3. jUp35-1, PJp35-·2, !Up3S-1, Up35--1, Up35-2, !up10, jup10, fup10, lup35--2, Up3s-2, Up27 jl.Jp..7.....7.. ......j u.. p..2 7 -l·u· p27 luplO Up27 .,, .. ' .. . ... . 60% 11% 50%! 100%j l 100'1~ 30%1 ,< i ; j ; ~ 11.'¾o',! 55%) 60o/J.: ••••• 70%! 60%1 60% I ; I l ; 27o/J soo/J 97%j 1 ' 100%] 40%1 20% ' I i i 11o/J. ;,_ 25o/J 94%! 100%1 40%1 11% J 50%1 94%! l ~uoJ i /Cl~ 60%1 0% 11% 50%! 94% 100% 40% 0% 11% 50% 100% 100% 50% llJ 60% l 27%1 J. 50o/J 97%l I 100%1 smJ I 20%j I I ; ! ; l Up3s-1 'jlu p3 , 1i ,up3 ' Up3S-2, jUp35-2, jUp35--1, !All 5 Up10 lup10 !UplO !markers Up27 lup27 fUp27 ~qgether 70%) I 113/c{ 50944 94~~ i [ 100%1 L 40%f 11% 70o/J, ·: ' 11J 500..-i /tlj 94%i 100%1 40%1 70% 11% 50% 94% 100% 60% 11%1 70%1 : i i i 11%) so¾[ 94'ri, 100%1 60%] 11% 50%) 50% 0% ~50/l I.. 1'-'1 so%l 50${~ .50o/~ 50%! 11%!. 50% 25'.'IJ " 50% 111 ~ N .0. .. <::', -.... \,C .-..l.

N "'d t""l g r.r, N .0. .. u, <::', .Q.C..) .(...;.; ~ 0 ""_'', "_', (X) "' "' .0_. ., I :;: I ""'' Samples summarized in Table 3 and Table 4 were additionally tested for nonsynonymous somatic mutations in TP53, Table 5 shows performance of selected combinations of methylated DNA markers {Up3, Up 10, Up27, Up35-1, Up35-2) plus testing for mutations in TP53 (p53) for detection of different sarnple types, Samples are 5 scored as detected if any member of the marker combination pand scores the sample as methylated or ff analysis for TP53 mutations scores the sample as TP53 mutant.

Shown is the performance of the marker panel in which samples are scored as detected if any methylatlon marker is detected as methylated at greater than or equal to 1 % of DNA reads, or if TP53 is detected as mutant at greater than or equal to 10% of the DNA ~equence 10 reads.

Also shown is the performance of the marker panel in which samples are scored as detected if any methylation marker is detected as methylated at greater than or equal to 10'% of DNA reads, or if TP53 is detected as mutant at greater than or equal to 10% of the DNA sequence reads, 112 'T'1\.BI.,f~ 5/\.

Cut-off for positivity (e.i. sample is positive if more than this % of reads are methylated at the given number of CpGs) 1% 1% 1% I ' i 1% 10% 10% I ! I ! P53 cut- I Up-3 and Up35-1 and lup35-2 and lUp10 and ' otf l Category p53 p53 ,ip .":i 3 1μ53 lupn and p53 ~ 80%1 1mvj 60J i i 10% !.Sensitivity EAC 70% '.ro: 70%5: [Sensitivity for BEs l I ; ' ! [synchronous to EAC or ' ~ 10% jHGD I 20% 20% 19%1 11,.1/0".~I 27%1 10% !sensitivity HGD i 33'.1/J 50% 38%1 I 25%! 33%1 1()'~1, !specificity Sq I 100J 100o/cj l 100%1 100% 97%> :t i i l i i Specificity for non~ l i I 10% dysplastic BE i 100%1 1003/,J, 100% 100% 100% ' l i Individual Methylation Markers+ P53 10% Sensitivity EAC ! l ! 70%1 j sorJ' 70% l ~ 60o/~ 70o/c 1 ! 2isensitivity for BEs matching l J !higher pathologies, either '1 I I 10% JEAC or HGD 20% 203/~ 19o/d 11o/~ ,Y ~ ... ~ j 50%1 ! j ' I 38o/J ! 10% \Sensitivity HGD 33% 25:l{i, 33%[ 113 ~ N .0. .. <::', -.... \,C .-..l.

N "'d t""l g r.r, N .0. .. u, <::', .Q.C..) .(...;.; ~ 0 ""_'', "_,' (X) "' "' 0 ._..,.

I :;: I ""'' TA..BLE 5B Cut-off for positivity (eJ, sample is positive if more than this'% of reads are methylated at the given number of CpGs) 1% 1% P53 cutoff I Category 10% ISensltivity EAC Sensltivity for 1· BEs synchronous to l l 10% jEAC or HGD 1 I ; ~ 1up3 Up3 \and and !Up10 Up35-2 irnd and p53[p53 'i I j iUp3 iand iUp27 iand \p53 80%[ 80%r···~~; ! 20% 11% l -, 7,vl "- /Qj l ; Up35-2 IUp35-2 and and jand Up27 Up10 iUp27 and and p53jand p53 p53 Upl0 ... ;;%[ 80%1 80%i I f i 11%[ 27%l 11'{~ I ; I Up3.

Up3. [Up3, Up10, Up35-2,iUp35-2, Up27 Upl0 [Up27 and and p53[and p53 p53 60%[ l 85%j 90% 11% J .q 1 11%j 50% soo/J I I I I !Up35-1, Up3, ! Up35-2,iUp35-2, Up35-2 .. ] UplO, iup10 Up10 i up27 jUp27 Up27 ! and p53fand p53 and p53j 80%1 90o/cJ 90%1 ' 11%, J 11% I 10% lsensitivity HGD 1 50% ! i '~ 50% 25% 33%1 50%1 50%l 25%~ ~ 2591,~ so~tl r.·ooA .:) 7~ 19'b 949"4 1% 10% 10% l < j oo; } .. 7C 10% 10% f i I 10% J___ ! 10% ]specificity Sq ! !specificity for l lnon-dysplastic ! !BE ! ! . ~ !Sensitivity Ei\C ' \sensitivity for iBEs matching :higher [pathologies, [~i~~er EAC or '' 1sensitivity HGD 100?-1, :100% 100% 100% 75%! 70%l I l 20%j ; 11¾J S0o/~ 25o/J. f 97%1 1009-d 9T¼1 94·%\ 100%l 97'½i 94o/~ 94'Y~ l l \ J i l l ' ' ' ' 10o~J 100% 1009-{ 100%! .. 1ogo/c .. 1gg%j 100%1 J~g'Yq Methylatim, Marker Comblnatlons Merged with P53 Mutation 759'J '' I 27~bl 33,x,l 70% 11% so%I 114 80~~ 703/ril 30% ! ' l 11%! 80%t 80%j 27% i i i i i i j 27%1 1W~ 11%! 50%! 25%! 50o/~ . ... ?()'¾,! 25%! 80%1 1,J .r~ SO%j 94% i 100%j 1ooo/J 80%! so%j 11% 11o/j 50% SO'?fJ ' ~ N -0 -~ 0 -'-°-l N -= r"'l ~ rr, N -0 u, ~ 0-1 -QO -(.;;j ~ 0 ""'' -.J "' -.J CX) "' "' 0 .... -.J I 0 "I ' ""'' DNA was also extracted from esophagea! samples that were also obtained by cytology brushings ofthe esophagus.

The sample set included brnshings from 49 esophageal adenocarcinomas (EAC); 14 carcinomas of the gast.roesophageal junction (JCA); 8 Barrett's esophagus with low grade dysplasia (LGD); 9 Barrett's esophagus with 5 high-grade dysplasia (HGD); 33 cases of Barrett's esophagus without dysplasia from cases without HGD or EAC, otherwise termed non--dysplastic BE, that included 13 cases of short segment Barrett's esophagus (SSBE).

Also included were brushings of the gastroesophageal junction (normal GEJ) from 62 individuals without Barrett's esophagus, without HGD, without EAC.

This included individuals with gastroesophageal reflux. l O disease, "vith eosinophilic esophagitis, or without any disease, Also included were 176 bnishings of normal squamous esophagus from each of the above individuals, These DNA samples were analyzed for methylation by bisulfite sequencing of selected amplicons and were also analyzed for non-synonymous sornatic mutations in TP53.

Table 6 shows the individual performance of biomarkers in detecting the different l 5 sample types of the valldation brushings set, Shown is the p(~rformance for detection of different sample types of 5 different methylated DNA markers analyzed by bisulfite sequencing of selected amplicons (Up3, UplO, Up27, lJp35-1, Up35-2), The table specifies the number of CpGs required to bt: methylated on a DNA sequence read to classify that read as methylated.

Results are presented ,:vhen samples are considered 20 methylated if greater lhan or equal to l %i of all DNA sequence reads are classified as methylated, or if greater than or equal to 10% of all DNA sequence reads are classified as methylated.

Also shown is the performance for detecting samples of testing for somatic mutation in assays in which TP53 was amplified frorn genomic DNA. using a set of PCR arnplicons that spanned the TP53 coding region and in which Next Generation DNA 25 Sequencing was !hen used to compare TP53 sequences from esophageal lesions versus matched normal esophagus tissue.

Shown are rates of sample detection (expressed as sensilivity or specificity) when samples are classified as TP53 mutant if greater than or equal to 3% of TP53 reads are scored as mutant, or if greater than or equal to 1 O'·Vii of TP53 reads are scored as mutant.

JO 115 Table 6A Unchanged CpG #cut-off_ applied_to_all_ s~mp!e sets __ _ Up-3 25+ Up35-l CpGs 14+CpGs Up35-2 10+CpGs ,........_..,,~----Category__________ _ _ > 1 ~/o=positive --- _ > 1 %=positive _ , ... > l %=positive·-- IS ·e·"n---s--i-t--i-v-· i.t. y E----A----C-·-- · ....................................- ---· .... .. 44% 57%:i 57'% !r·S. .e..n..s..l.t..l.v..i.t.•v .J. -C----A---. ............. ---- 14% 4.•)1 0/10 1sensitivitv LGD 38% 25% l--------'--------------+------,.,.,.,.,+-----------•------------· ...... ,--------.. --...... -- lsensitivity HGD ------------------~2!~~-.... -- ....................... ~~:~. """' __ s.O~{i . 1l.s peci• tui r·1• tv norm a 1 GE'J o,, go1; '.o 90o0 .,,o. 9,(,s0,; 'o. ~ -------------- "'---✓----------.... , .................. ,••••c••••c•• .................. •••••• ...... , " , ,,._._._.__._._.uuuuuu•un~•-/.--------4-------4 100% ~ ·Specificity for "non-dysplastic 95% lF'- excluding SSBE Specifi~ity. for·"'~-l-~ _B_E_\_,:_it_h_o~ut-t-----------------1,...........-----=lw------~ , ysplasrn (mcludmg SSBE and 97% 85'% 94% lnon-dvsrJJastk BE) ~-· • I ) I I > I 0%,,=pos. itive >l0%=po sitive >10%,=po I sitive ------------------------------------------------------.. ·---------- -------------·--·· .••••••••••••••••••••••••••••••••••••••••• ISensitivltv EAC 21 % 5T1/ii 45% t- •• •• • ......... ----------+--------- __. .. iSensitivit:y_JCA ___ --.. -----------------·--·-- 7% 29'}o 7% Sensitivitv LGD 13%i 38% 25% S~nsitivit;, HGD • •• .... 33°.lo....... .... .... . 56% ......................... )0% ..... .

Specificity normal GEJ 100% 100%, l 00% Specificity for "non-dysplastic I HE" - ex.clud_[_ n_= [i~S_S_B_:E ~:'.. __, ........ ___1 _0_0_it;._{,-----i,----1_0!_J0 _11i _ __________1 __9.Q~--- Specificity fix all BE without ldysplasia (including SSBE and ,non-dysplastic_ BE). ............................ ·------.. -----------~lO_O_Jl;_·o_,___,__www~8~8-.n _Yi,_..__ ___9_7 °_A_i- • I 16 T,1.\BLE 6B ''''"''" .......... , .. , .. , .. v,, .. , .... , .. , .. , .. -. .. , .. , ........ , •. -.•.-.,-.,. • •.• • • • • • • • • • • • • •• • • •• • • • • • •••• •• • • • ••• .••• •••••• • ••• • ••• • •• •• •••• • ••• 'i Updated CpG # Cut-off apph'ed to I 1~:_.::..=.,s._;.;;~:_.=,-----~------ , .. ____ rr53, ___ ,J Uphl i 26-H:;j)Gs Up21 l ~+cpGs i ~ ...................... (:ategor:< :> 1 ~%~::-l~.~~s3t~\h": );. 1 %•::::-r~~~e n-. .. m -.-.-.-.m >3 ~!., .... -.-.w1 ~~m~vyy E!\G.. ____ ---- .. : ::; ......... ----------------43% """""""" ....... ~9~~ ...... j ISe:r,r,,tivitv JC.A -.:> ,,, i4% >Oil·,, i \ .. ..,..,.., ..,..,..,..,..,.., ..,..,.., .., .., ..,.., . ········1 renf;itwity LGD .............. __ (}_'?;~--------· --·----·---------~}~'.{'..-............. 13% l lr!- ie-m---.-it-w---lt·, .H....G...D.... ...." ""'"""""2"2'".')''"s'," """" .................2..0..%....,. .. . .. .. --4..0..%... __ .,i (s,,ei.·ifidtv 'lOmllil l ki I,E. J • , • <~·l~8 ",. 10\)'% 100'% ' ri, ! t:···••.-.-•.•~--·.·:--··t···········-:~····--.::.··········:····.· "··············.··.·.··.········ ...... ·: ......................................................... ................... ,.. .• ""····-··• ......... l f,_pe.:."'.dic1~\·. lnr tH}lt·· ~ kh,s·~,h,,ii: ,i:i-,=.. mo% 95% 100% l ~;;l~;ll~11t $~BE . .. ..................... ._._. ""' """'" ""'""""""" ...... .. .. .. . j fp,;-.--ili.citv for ,1!! BE l ~,:s.-itfJ~1ut: 4~(~.il.·Jig.sk.·~ ~. WO% 94% 97% Kim:foding $SEE aml j r ' \t~<lUlf:cplastip HE} "·' ........................... · ..... "" ..... '"'"'"""""'"" ! > l◊J,,ocw,;,itiv ::;, HJ'!ci;,"fXl,ll l t, • •• •• •.,, • •• •• • • • • • • .- • •• ... • • .. - .- , • •. ••••••• •• ......... •••••••• e:.••••••••••••• •••m• >•1 P~•~:.=.=g:)<~:i.itWe: mm• nn"• r¥t ~ l1?rn:;ihvi~x .. EAC ...... ~ 25% 29% _.. _____ ...., . . 4Jo/ey ,w ; t~ • • 't I'~!\ -,w"' ';''-'t v,,.,.. ! r.s:::.::;:. J~.,;I> .......... • .... ::;:}t _········· {j~;G··. ······.:;r····1' ~m,;i1ivily HGD 1 .......... ,.~ .. ,., ........... ~ 20% 4!)% f p<-x;.rtieit}' n,)·nnA1 tGEJ !00% . .... WO~-~ WO% !sv>:'.,,ilidty B,r '\-,m.;, • • • • • ki·'"t)i''iS:tk HF", ~ 1· .... '..... ... . ,.. bc:duditrn. SSBE 10D% 95%, j lOON., ti .• • ,._,,.••nn , .. ••••••n••nnn•. n•>•n •>,.n'.• .. •n .. '>•• .... n-.',,•'>•'>n;.;, ._n ................ ,.,..,..,.,._,. .................... ,. .... ,v,,,.,.,,._ .,.,v.-. ....................... v,, fi:,e:d£d1y fi,r ,1:~ BE ! ~;t,:~!~~~ci'' ·-···100%...... ... 97%. J w% .

Table 7 shows the performance of selected combinations of ihe methyL?Jed DNA markers (Up3, Up 10, Up27, Up35-1, Up35--2) for detection of difforent. sample types in 5 the esophageal brushings samples presented in Table 6.

Sampks are scored as methylated if any member of the marker combination panel scores the sample as methylated.

Results are pre5ented when the individual markers are considered methylated if greater than or equal to l % ofiili DNA sequence reads are classified as methylated, or if greater than or equal to l 0%, of all DNA st~quence reads are clas:,lfied as methylated.

JO 117 TABLE 7A """""""'''"'"''""""""""""""""""""""""""""" .. """""""" .. ".. """ .. ".. .......... ,. . -. .......... -. . . .. ,. ,. , . . . .... t ,, , ,, ,, ., ,· ~, ,, , ,, . .-,-. , ,. . . . , , ,, ,, . . ,, . •.. . .. . . ,,•··· ·······,: •···•.•.w ••, .

Cut-omor ! l ! • ! l positivity ! l ' • ! l (ed. s,arnp!e, ! ! • ' ! i5 p-o&itivi,df! l ! morn than ! l ! ttli!',% ot ! l ! reads ~re ! l !. · , • \ !J;:b methylated! ~Jpil \Jp3 Jp35· ! :;;p~5- atthe give11 ! be lBnd r:,3 ;u,,,i 1,rnd iu. i:;3:S· l.!13:Ei· ;t,,it~- lpB5· :U;,to !i, • , l • : • I , number of ! ~Jp55· )\Jp3S- [ ~d !M•~' cip35- 11 a,-,;; l. ar<d !:t Mid t and 1;gnd f p35· ·- c~ess) L .. • .. • --:=~~£,~Of\/ --··--············t1··· .. , :t"-·, ,·, tU,};~J\JJ>:~~·? :·· p,~◊ . !Jp\'.:S l'"':~, .. ir:~·- :ur2~J----·---- ·' '" ,,e,1:,1t1111t::z f..~l. , ,,,, 1 '·••-~ .. ,~... ..6¾ i>1%\ . 1.j &7':><i ..-.,.,, "",;~ S:,:~l &3-~ .·.:::::::·~::::::·j~:~::::~:~~~~.-- ·.. .... ········•r .. '::~ .... ~~~ .... ~~----:~= :::::~ -;~~-:;~~ --;~~ --·f:s· ·::~·--::~< 1% ISe·1<.lth,ttv HGD : ·,;s~ 7'lm t!l%1 r,·, '('<;;'• .,,"id n-s,~ O,is ',·,~ ~-,,.,,J n · ......... J~{ •........ ..Jsp~~lfldt~_nmrmii_GEJ ....... L.~~~: ... '1~~{-.. w~ .. ,;~~. ____ ;;,~~ ;i;,,i ;~✓ _.;;~ ;~~:: ~~'ll(_:i~. ·--··:~!\: ..... 1~;P-~£!!!£.'.t:i i~!. . !:.'?!1 ·RY<-Fl. tH'J .. ss ; •••• s•• ~ i't~ .•. .':t~~ ....~ 1?%.• : .J~;_%;.;:_ ..! I~·f·<-~ 1 .... ~"'". J~~:1.ii .... 2,~~~-----~-~~f .}~ • • isreclndty a!! BE with<:mt l ! ! ! j i i% l:~~p~~J~j:~-~lt,:~~; :;~[, I ~--•· ~,.~· ,qJ ,,w &"J ,n ~,J S4~! ,,\s i ~4J 'M*. ,.,.,.,,_ .... , .... ,_._ .. ••• ..... •• .. ......................................., .................. l ............................. , ....................................................... , ......... .. 10% lssn,.:lth,,itv JC;\ l 29'%\ ;l'/\J 29%i 14, 29%! 43% 36%} 2.9%1 1.4% 3t:%i i~Ht Ul% ]Sens.ithiltv f.AC l 56% 48%1 36'.¾.q ,H,% SJ%! 5'i,. ii!i¾j 53%! 55%. 1+1%\ li:.l·~· •1 ......... ~~;;···••····~:~:::•:'.~:~~•~~~'. ................ .. . J •• :~:1::~1~ 3~~ ~~~ ··- ~:~ -·-~~t;~~-~;:[ _ ;;L~~l_;.;; JO ]] 8 TABLE7B Table 8 shows the performance of selected combinations of methylated DNA markers (Up3, Up l 0, Up27, Up35- l, Up35-2) plus testing for mutations in TP53 (p53) for 5 detection of different sample types in the esophageal brushings samples presentt~d in Tables 6 and 7.

Samples are scored as detected if any member of the marker combination panel scores the sample as rnethy!ated or if analysis fer TP53 mutations scores the sample as TP53 mutr.mt.

Shown is the performance of the marker panel in which samples are scored as detected if any rnelhylation marker is detected as methylated at greater than or JO equal to l'l.-1, of DNA reads, or ifTP53 is detected as mutant at greater than or egual to 3%, 119 or at greater than or equal to 10°1ii of the DNA sequence reads.

Also shown is the performance of the marker panel in which samples are scored as detected if any mcthylation marker is detected as methylated at greater than or equal to l 0% of DNA reads, or if TP53 is detected as mutant at greater than or equal to 3% or at greater than or 5 equal w 1 O(lfi, of the DNA sequence reads, Marker combination with greater than 90'% specificity in al! BE without dysplasla are preferred yellow.

Marker combinations that additionally show superior sensitivity for EAC arc further preferred.

Particularly preferred marker combinations are: Up35~2 methylation plus TP53 mutation; Up35-2 methylation plus Up3 methylation plus TP53 mutation; Up10 methylation plus Up3 methylatlon plus 10 TP.53 mutation; Up35-2 methylatlon plus UpIO mcthylation plus TP53 mutation; Up!O methylation plus Up27 methylation plus TP53 mutation; Up35-2 methylation plus Up3 methylation plus UplO methylation plus TP53 mutation. 120 TABLE 8/\ 121 TABLEJB 122 Fxmnpk4;_Ana!ysh_of~::_sovhageal_cancer)nformative loci in fi.mnalin fixed paraffin em bedded Ji ssues.

Additional studies were performed on DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples of the stomach and esophagus that capture 5 different diagnostic categories other than Barrett's wlth hlgh grade dysplasia and esophageal adenoc.arcinoma, Bisulfite converted DNAs from each sample were amplified with bisulfite specific methylatlon indifferent primers corresponding to selected am pl icons and the arnplicons were then analyzed by blsulfite sequencing to determine methylation status on ihe parental DNA templates. 10 Table 9 summarizes the side by side comparison of 8 methylated DNA markers in FFPE tissue samples of the stomach and esophagus that capture different diagnostic categories other than Barrett's with high grade dyspiasia and esophageal adenocarcinoma.

Intestinal metap1asia is abbreviated as TM.

Table 9 denotes for each marker the number of methylated cytosine bases required to be detected on a DNA sequence read to classify that 15 read as methylated, Samples are detected as methylated if greater than or equal to 1 % of DNA sequence reads are classified as methylated, 123 TABLE9A Marker lv1M CpG cut-off ! Vaiue (% positive 1 l samples or total I O .. l I Yr, pos1t1ve I number of samples I I sequencedj I I BE (IM) I 90% I •• __ J I GEJ/Cardla with IM 82% 1 r-ci-:TJ Ca~~a without ! j columnar mucosa j without Ev! taken from I patients with l concurrent IM at the 1 same endoscopy Normal Distal jEsophagus-Squarnousl Squamous Mucosa with REFLUX Esophagus mi;, 30% 9% 0% lsqBES lsqBE Up7 6+ 13+ 1 ; Total number! % positive !Total number! % positive :[ 11 58 1,~ ,! 23 50% 0% 0% 0% 8% 12 33 1 4 13 10 0% 6 I I ~ 50% 33% 2% 20% 0% 0% 5qBE11-·2 23+ 5+ Total number % positive Total number 16 83% 30 6 100% 11 47 31% 58 5 30% 10 16 13% 23 5 0% 10 [ Eosinoph_il,ic □% ··1 5 l 0% I 11 ! 0% I 15 ! Eosoha 1tis --•!-i -----!-------!--· ----.........,-i--------~ f Gastic Muc~1sa with 22¾ 9 0% I 2 I L !M O ! 20% 5 44% 9 !f~astnc Fundic Mucosai O"L I 24 I 0% I ~ - j /0 ) ( ......... without.lM...1 .............................. L. .......... · SQ/,'. ;ll 21 8% 24 8% 13 22% 9 13% 8 69% 13 124 ~ N .0. .. <::', -.... 1-C .-..l.

N "'d t""l g r.r, N .0. .. u, <::', .Q.C..) .(...;.; ~ 0 ""_'', "_', (X) "' "' 0 ._..,.

I :;: I ""'' TABLE9B Marker SqBE14-2 CpG wt-off l Value(% positive 1 samples or total 1 number of samples !_ sequenced) BE(IM) l GEJ/Car-dia with IM l i ________________ j GEJ I Cardla without IM k;o!umnar mucosa without! 1 iM taken from oatlents i i with concurrent ·1M at the i i same endoscopy i Normal Distal i EsopJ:1§_gus-Squarnous Squamous Mucosa with REFLUX Esor2_ha_qus Eosinophilic Eosphagitts I Gastic Mucosa with IM Gastric Fundic Mucosa without IM Helicobactor Pylori Gastritis without iM " % positive 50% 22% 2% OQ/ 70 10% 17% O'Yo 00.,,/,._:. 0% 14% 7 isqBE16 20+ 14+ Total number % positive Total number 16 50% 26 9 18% 11 43 2% 55 5 11'¾, 9 20 5% 22 6 0% 8 8 0% 12 5 0% 7 14 0% 24 7 8% 13 125 J I jSqBE17 T ! I 'f'<i positive 69% 73% 7% 11% 4% 0% 8% 25% 0% 46% 1.,. /T Total number 26 11 55 9 23 9 13 8 24 13 ! jSq8E18 % positive 86% 70% 0% 11% 4% 0% 0% :J.4% 8% 15% 16+ l "I I l Total number! ! 29 57 9 23 12 7 24 13 ~ N .0. .. <::', -.... \,C .-..l.

N "'d t""l g r.r, N .0. .. u, <::', .Q.C..) .(...;.; ~ 0 ""_'', "_', (X) "' "' .0_. ., I :;: I ""'' Table 10 summarizes the side by side comparison of3 methylated DNA markers in FFPE tissue sampies of the stomach and esophagus that capture different diagnostic categories other than Barrett's with high grade dysplasia and esophageal adenocarcinoma, Intestinal metaplasia is abbreviated as Il'vt Table l O denotes for each marker the number 5 of methylated cytosine bases required to be detected on a DNA sequence read to classify that read as methylated, Samples are detected as methylated if greater than or equal to 10% of DNA sequence reads are classified as methylated, 126 TABLE JOA Marker Y~.~·--········--···--·········-- rsqBES .......................... -.

Sq BE _Up7 SqBE11-2 •••• •••••••••••••••••••••••nnn•••• l ,-C-p~G-c~u~t·_·o_ff___., ................. ~: ................................ :~~·~················· ................. ~.~=················· .................... ·--~=-·---~i Value(% positive sampies or total number of samples Total % Total % Total number positive number positive number positive number positive % Total % sequencedl ..................... ·····"'----·--······ ...................................................................................................................................

BE (lfvl) GEJ/Cardia withiM GEJ / Cardia without IM columnar mucosa without iM taken from 87% 82% 0% patients with 0% concurreni iM at the same 30 50% 11 0% 58 0% 10 0% 12 44% 16 70% 30 5 33% 6 91% 11 33 2% 47 16% 58 4 0% 5 10% 10 endoscopy .........--~----"'-"---+----+·---+--~--t----·----· ......... · ....................................... · ......................................................... .

Normal Distal Esophagus- 9% 23 8% 13 0% 16 13% 23 39uamous w .. •................. .... ........ ........ • .. • ..................................... _ .................. _ .............••.•..............•............• Squamous Mucosa with REFLUX Esoohaous 0% 10 0% 6 0% 5 0% 10 .........----'----"'----+----+---+-----;• .............................................................................................................. . 11 0% 15 Gastric Fundic Mucosa without 0% 24 0% 14 5% 21 4% 24 IM ...... ~~- ............................ ..,..,., .....................................................................................................

Heiicobactor Pylon Gastritis 8% 13 22% 9 13% 8 46% 13 without IM 1·1, .,:,.1 TABLE lOB Marker CpG cut-off 20+ 14+ 17+ 16+ ---------~--~·---~----------------------------- """"""'"'""' ·--··--···· Value(% positive samples or Total total number of positive number samples % Total % Tota! % Total positive number positive number positive number sequenced) i,--wwwwwwwww--'---t~---1-----------f-,,-,_,~ ~•-,_,_,_, ,_, ____ ,_,-- __ w,w,w,_,_ -•-•-•w•-•• "''"'"'""""'" BE {!M) GEJ/Cardia with IM GEJ I Cardia without IM 50% 22% 2% 16 42% 9 1.8% 43 2% 26 62% 26 76% 29 11 64% 11 70% 10 55 0% 55 0% 57 --------. ···" "·· ··------------·· ----------·-- . --------- ...... ··""'"'"'" . .. .. ... . .. ....................................... .. columnar mucosa without iM taken from patients with concurrent IM at 0% 5 0% 9 11% 9 11% 9 the same endoscopy ,.__.,._WWWWWWWWW~------•..,........,..,..,..,..,.......,..,..,..,.., ............................................... ,,...,,._ ... ,-. , ... , ............ , ... , -V'.;,, ....... • ..... • ,.n~'-Onnnn~nu~nn~uuuun~nm Normal Distal Esophagus10% 20 5% 22 4% 23 23 Squamous -------1----+---·-------·. ------------------·-- ....... .. ............................................................. , ...... """"'"""" Squamous Mucosa with REFLUX Gastric Fundic Mucosa without IM He!icobactor Pyiori Gastritis without IM 17% 6 0% 14 14% 7 0% 8 0% 9 0% 10 0% 24 0% 24 0% 24 8% 13 46% 13 8% 13 128 Table 11 summarizes performance of different panels comprised of combinations of methylated DNA markers in FFPE tissue samples of the stomach and esophagus that capture different diagnostic categories other than Barrett's with high grade dysplasia and esophageal adenocarcinoma.

Intestinal metaplasia is abbreviated as IM.

Tables 9 and l 0 5 denote for each marker the number of methylated cytosine bases required to be detected on a DNA sequence read to classlty that read as methylated.

Samples are detected as methylated if greater than or equal to l % of DNA sequence reads are classified as methylated for any member of the marker panel. 129 TABLE J lA Marker comblnatkms !v1M SqB!-5 VIM SqBE7 BE (!M) 92!){, 88% GU/Cardi a with lfv1 60% 83% GEJ I Card1a without !M 0%1 2% columnar mucosa wjthout IM taken from patients ·,vith concurrent !Iv'! at the same endoscopy 25% 40% Norrna! Distal Esophagus-Squamous 8%1 5% Squamous Mucosa with REFLUX Esopha.gus 0% 0% Eosinophilic Eosphagitis 0% 0% Gastic Mucosa with IM I 0%1 40% Gastric Fund:c Mucosa without IM 'i 0%j 5% Helic::,bactor Pylori Gastritis without IM ! 33%! 13% VIM SqBElG lv1M SqBE1.7 VIM SqBE:18 Sq BES SqBE7 I . ... . .. ,. ! SqBf:.~ SqBf:.fo , 92% 88% 97% 71% 70% 8·,a,:: /._;Q 91% 80% 50% 0% 2% 7% 0% 3% 3% 33% 33o/b 33% 25% 0% 9% 9% 9%1 8% 8% 0% 0%! Oa;,: ,D 0% 01t 0% 8% 0% 0% 0% 14% 38% 43% 0% 0%1 ~ 0% 0% 8% 7% 0%1 1.,J :·',½Jc1 46'){.I l ~o~[ ... . ,.)_;D 29% 22)sl 130 ~ N .0. .. -.~...... 1,0 .--...J.

N ""d l"'l g r.r, N .0. .. u, ~ .Q..0. .(.....; g 0 ""' .J ".J CX) " ".0. . .J I 0 "' I ""' TABLE llB Marker combinatlons r 1· 1· ............ I ........... ·1· .......... ·IsqBEl6 ,_ ___________ .,..,....,.;jS~1_EiE5 SqBE17 ?qBES SqBE18 SqBE7 SqBE16 _SqBE7 SqBE17 SqBE7SqBE18 SqBE17 BE (!M) r 80%1 91%1 sn,l 79%1 94%1 i GE.l/Czrdia with IM I oo· o.~r·· j i f."I A•! 40% 33% 50% 83% GF.J J Cardia without Hvl 9% 0% 2% 9% 2% columnar mucosa without iM taken from .. patients wilh concurrent IM at the same I endoscopy I 25%1 0%1 40%1 :W%I 20%1 Normal Dista, Esophagus-Squamous 8% 8%! 6%1 0%1 6%1 Squamous Mucosa wit~; REFLUX Esophaqus 0% 0%1 0%1 0%! 0%1 Eosir:ophilic Eosphagitis 20%1 0%1 09·~! 10%! 0%1 Gasfc Mucosa with !M 50%1 0%! 25%1 40%l 20%1 Gastric Fundic Muccsa without IM 0%1 7%1 5%1 s%l 5%1 Heiicobactor Pylori Gastritis without !M 44%1 44%1 25%1 50%1 25%1 131 SqBE16 SqBE18 : 78%! 92%! i 73%1 70%] 89'tl! 29·$~ 22%: 9% 0% 10%: 33% 0% 46% ! 22%! 5%: O~·&I 0%: 17~~~ 8%j 0)' 23:ic>j ~ N .0. .. ~ -..-.. 1,0 .-...J.

N '"d l"'l g rJl N .0. .. u, ~ Q..O.. .(..,...; g 0 ""-.J' "-.J CX) " ."0. . -.J I "I' ""' Table l 2 sumrnarizes performance of different panels comprised of combinations of methylated DNA markers in FFPE tissue samples of the stomach and esophagus that capture different diagnostic categories other than Barrett's with high grade dysplasia and esophageal adenocardnoma.

Intestinal metaplasla is abbreviated as IM, Tables 9 and 10 5 denote for each marker the number of methylated cytosine bases required to be detected on a DNA sequence read to classify that read as methylated.

Samples are detected as methylated if greater than or equal to 10%, of DNA sequence reads are classified as methylated for any member of the rnarker panel.

TABLE 12A Marker rnmbiriatlom, fVIM SqBES VIM SqBE7 lv1M sqBE16 BF. {IM) I 92% s1;<, GEJ/Cardia with IM 60% 83% GEJ J Cmdia without liv1 0% 2% columnar mucosa withou1 iM taken fmrn patients with concurrent IM at the same endoscopy O<;•oI 0% Normal Distal Esophagus- Squamous 8%,i 6% Squamous Mucosa with REFLUX) I EsophaQus ~ 0%! 0% Eosinophiiic Eosphagitis ! r)OJ .. /t;! Oo, 7\l Gastic Mucosa with iM I 0%! 20% Gastr:c Fun(k Mur;osa w!tt:01;! l IM j 0%1 5%1 Helicobactor Pylori Gastritis f: w:thovt IM '! 33% 13~~ 133 VIM SqBE17 VIM SqBE18 88~~ 85% 82%! 91%1 ·-.r,..- L7< 0'1,J 1 ! ! 0% 11% 9% 9% 0% 0% 0% 0% 14% l3~'G 0% mJ 15% 46% SqBE5 SqBE7 90% .I. . 1r-_,,,'u. I 80%1 50% 0%1 3% 11% 0% 9% 8% 0% 0% 0% 0% 29% 0% 0% 7"% 15'.% 29% SqBES SqBE16 60% 0% :l>t'. "''" O%j 83J I O'JJ 0"... ~J: Oor.q-! 0%! 2~•01.i L.10; ~ N .0. .. ~ -..-.. \0 .-....J.

N ""d l"'l g rJl N .0. .. u, ~ .Q..O. .(,.,...; g 0 ""' -.J "-.J CX) " "0 .... -.J I 0 "' I ""' TABLE 12B I Marker rnmbiriatkms Sq BES SqBE17 SqBES SqBE18 I BE (!M) 1 '?O~·i 91% l ~ GEJ/Cardia with IM l 40%! 80% GE,I ! Cardia without IM 0% 0% columnar mucosa without IM taken from patients with I concurrent IM at tr,e s:~rne endoscopv 25% O,f Normal Distal Esophagus- Squamous I 8%1 8% Squamous ~.1ucosa with REFLUXI Esopha9us 0% 0% Eosinophilic Eosphagitis Oi}·~ O)f.

Gastic Mucosa with IM I 0%1 0%! Gastric Fundic Mucosa without IM O~>f 03~ Helicobactor Py!ori Gastritis without 1M 44% 337{, ---------- Sq[ff 7 SqBf:16 SqBE7 SqBH 7 SqBE7 SqBE18 57% 71% 81% 33% 50% 83% 2% 2% 2% 0% 20% 0% 6% 0% 6% 0% 0% 0% 0% 0% 0% -- 25% 20% 20% 5% 5)·~ So/~ 25% 50% 25% 134 SqBE16 SqBE17 SqBE16 SqBE18 74% 81% 64% 70% 2% 2% llo/r-: 9% 5%1 0% 0%, 0% 0% 17~·~ 17% 0% m.; 46% 15% ~ N .0. . ~ -.-..0 1,0 .-...J. N ""d l"'l g rJl N .0. . u, ~ Q..O. .(..,..; g 0 ""' -.J "-.J CX) " "0 .... -.J I 0 "' I ""' Somatic mutations in TP53 were detected using the following method. TP53 exons 2n 11 were amplified using a multiplexed series of primer pairs covering all coding sequences and splice junctions.

The primers contained additional 5' end sequences that 5 were then used for secondary amplification that introduced barcode sequences and Illumina 15 and 17 sequences into the final PCR products. PCR products were mixed, purified and analyzed on an Illumina Mi Seq instrument Data analysis was performed using CLCBio software (Qiagen) and VariantStudio software (IUumina). 135

Claims (22)

1. CLAIMS: 1. A method of detecting an esophageal neoplasia or metaplasia in the esophagus, compnsmg: contacting a human esophageal sample with bisulfite to generate bi sulfite converted SqBE 18 nucleic acid sequence having at least 80% identity to the sequence of any one or more of SEQ ID NOs:8234, 8276, 8318, 8360, 8248, 8290, 8332, or 8374, wherein the SqBE18 nucleic acid sequence is at least 50 nucleotides in length and comprises at least nine methylated cytosines and at least one unmethylated cytosine that has been bisulfite-converted to a thymine (T) or uracil (U); wherein methy lation of said nucleotide sequence is indicative of metaplasia in the esophagus or neoplasia.
2. 2. A method of monitoring over time an esophageal neoplasia or metaplasia in the esophagus comprising: a) detecting methylation status of DNA from a human esophageal sample from a subject for a first time by assay in a bisulfite converted SqBE18 nucleic acid sequence for retention of a cytosine base at one or more of the Y positions present in one or more of the nucleotide sequences having at least 80% identity to the sequence of any one or more of: SEQ ID NOs: 8234, 8276, 8318, 8360, 8248, 8290, 8332, or 8374 wherein the SqBE18 nucleic acid sequence is at least 50 nucleotides in length and comprises at least nine methylated cytosines and at least one unmethylated cytosine that has been bisulfite-converted to a thymine (T) or uracil (U) ; and b) detecting the methylation status of the SqBE18 nucleotide sequence in a human esophageal sample from the same subject at a later time; wherein absence of methylation in the nucleotide sequence taken at a later time and the presence of methylation in the nucleotide sequence taken at the first time is indicative of neoplasia or metaplasia regression; and 136 Date re~ue/date received 2024-06-10 wherein presence of methylation in the nucleotide sequence taken at a later time and the absence of methylation in the nucleotide sequence taken at the first time is indicative of neoplasia or metaplasia progression.
3. 3. A DNA molecule that is a bisulfite converted SqBE18 nucleotide sequence, consisting of a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the following sequences: SEQ ID NOs: 8234, 8276, 8318, 8360, 8248, 8290, 8332 or 8374, and wherein the SqBE18 nucleotide sequence is at least 50 nucleotides in length and comprises at least nine methylated cytosines and at least one unmethylated cytosine that has been bisulfite-converted to a thymine (T) or uracil (U).
4. 4. A method to determine the presence of Barrett's esophagus, of Barrett's esophagus with low grade dysplasia, of Barrett's esophagus with high grade dysplasia, or of esophageal adenocarcinoma in a human subject, by determining, from a human esophagus sample, methylation status ofbisulfite converted SqBE18 nucleic acid sequence having at least 80% identity to any one or more of SEQ ID NOs: 8234, 8276, 8318, 8360, 8248, 8290, 8332 or 8374, wherein the SqBE18 nucleic acid sequence is at least 50 nucleotides in length and comprises at least nine methylated cytosines and at least one unmethylated cytosine that has been bisulfite-converted to a thymine (T) or uracil (U) and wherein methylation of said nucleotide sequence is indicative of Barrett's esophagus, of Barrett's esophagus with low grade dysplasia, of Barrett's esophagus with high grade dysplasia, or of esophageal adenocarcinoma.
5. 5. A method of determining the response of an individual with esophageal cancer to therapy by detection in a human esophageal sample methylation status in any of the SqBEl 8 DNA sequences having at least 80% identity to any one or more of SEQ ID NOs: 8234, 8276, 8318, 8360, 8248, 8290, 8332 or 8374, wherein the SqBE18 DNA sequence is at least 50 nucleotides in length and comprises at least nine methylated cytosines and at least one unmethylated cytosine that has been bisulfite-converted to a thymine (T) or uracil (U); wherein increasing levels of methylation over time are indicative of disease progression and a 137 Date re~ue/date received 2024-06-10 need for a change in therapy, and wherein absence of increase in levels of methylation over time or decrease in levels of methylation over time are indicative that a change in therapy is not required.
6. 6. The DNA molecule of claim 3, wherein the SqBE18 sequence comprises a sequence having at least 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.
7. 7. The DNA molecule of claim 3, wherein the SqBE18 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.
8. 8. The DNA molecule of claim 3, wherein the SqBE18 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 83 74.
9. 9. The DNA molecule of claim 3, wherein the SqBE18 sequence comprises a sequence having at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to 8318.
10. 10. The DNA molecule of claim 3, wherein the SqBE18 sequence comprises a sequence having at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to 8332.
11. 11. The DNA molecule of claim 3, wherein the Sq BE 18 sequence has been amplified using a pair of primers, wherein at least one of the primers in the primer pair is an unmethylation-specific PCR primer. 138 Date re~ue/date received 2024-06-10
12. 12. The DNA molecule of claim 3, wherein the SqBE18 sequence has been amplified using a pair of primers, wherein at least one of the primers in the primer pair is a methylation indifferent PCR primer.
13. 13. The DNA molecule of claim 3, wherein the SqBE18 sequence has been amplified using a pair of primers, wherein at least one of the primers in the primer pair hybridizes to a nucleotide sequence that comprises at least one bisulfite-treated methylated cytosine and at least one bisulfite-treated unmethylated cytosine.
14. 14. The DNA molecule of claim 3, wherein the bisulfite-converted SqBE18 nucleotide sequence is between 50 and 1000 nucleotides in length.
15. 15. The DNA molecule of claim 3, wherein the bisulfite-converted SqBE18 nucleotide sequence is derived from a nucleotide sequence comprising at least 21 CpGs.
16. 16. The DNA molecule of claim 3, wherein the bisulfite-converted SqBE18 nucleotide sequence is derived from a nucleotide sequence comprising no more than 21 CpGs.
17. 17. The DNA molecule of claim 3, wherein the SqBE 18 nucleic acid sequence is from a human sample.
18. 18. The DNA molecule of claim 3, wherein the SqBE18 nucleic acid sequence is from a human esophageal sample.
19. 19. The DNA molecule of claim 17 or 18, wherein the human sample is from a subject that has or is suspected of having an esophageal neoplasia.
20. 20. The DNA molecule of claim 17 or 18, wherein the human sample is from a subject that has or is suspected of having an esophageal metaplasia. 139 Date re~ue/date received 2024-06-10
21. 21. The DNA molecule of claim 17 or 18, wherein the human sample is from a subject that has or is suspected of having Barrett's Esophagus.
22. 22. The DNA molecule of claim 17 or 18, wherein the human sample is from a subject that has or is suspected of having esophageal adenocarcinoma. 140 Date re~ue/date received 2024-06-10