Detecting esophageal disease

Abstract

The invention relates to the detection of esophageal disease. Provided herein are techniques for esophageal disease screening, in particular, but not exclusively, to methods, compositions, and related uses for detecting the presence of esophageal diseases (e.g., Barrett's esophagus, Barrett's esophageal dysplasia, etc.). Furthermore, the present technology provides for distinguishing Barrett's esophagus and Barrett's esophageal dysplasia, as well as Barrett's esophageal low dysplasia, in a sample obtained by endoscopic brushing or non-endoscopic total esophageal brushing or wiping using a mooring device (e.g., a capsule sponge, balloon, or other device), methods, compositions and related uses for Barrett's esophageal dysplasia and esophageal adenocarcinoma.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202210914372.X, “Detecting Esophageal Disease”, filed on March 23, 2016. TECHNICAL FIELD

[0002] Provided herein are technologies for esophageal disease screening, particularly but not exclusively relating to methods, compositions and related uses for detecting the presence of esophageal disease, such as Barrett’s esophagus, Barrett’s esophageal dysplasia, and the like. In addition, the present technologies provide methods, compositions and related uses for differentiating Barrett’s esophagus and Barrett’s esophageal dysplasia, and Barrett’s esophagus low-grade dysplasia, Barrett’s esophagus high-grade dysplasia and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using a tethered device, such as a capsule sponge, balloon or other device. BACKGROUND

[0003] In Barrett’s esophagus, the healthy esophageal epithelium is replaced by metaplastic columnar cells, which is thought to be a result of chronic exposure of the esophagus to the damaging effects of reflux in gastroesophageal reflux disease (GERD). An intrinsic risk of progression from Barrett’s esophagus to esophageal adenocarcinoma has been established. Histologically, this progression includes a clear sequence of stages from metaplasia alone to low-grade dysplasia, then to high-grade dysplasia, and finally to adenocarcinoma.

[0004] Diagnosis of Barrett’s esophagus without dysplasia does not lead to specific treatment. However, when dysplasia is present, there is strong evidence that endoscopic ablation therapy can prevent subsequent transformation to cancer. This dysplasia cannot be distinguished from Barrett’s without dysplasia using endoscopy, and periodic random biopsies and histological assessment is the current method of surveillance for patients with proven Barrett’s.

[0005] There is little evidence to support the hypothesis that antisecretory agents or anti-reflux surgery prevent the development of adenocarcinoma or cause regression of Barrett’s esophagus (see, e.g., Haag S, et al. Gastrointest Endosc. Aug 1999; 50(2):229-40).

[0006] In the early to mid-1980s, histamine 2 (H2)-receptor antagonists were the most commonly used drugs for treating GERD. However, several studies using cimetidine or ranitidine did not record regression of Barrett’s esophagus.

[0007] In the late 1980s, proton pump inhibitors (PPIs) were introduced and proved to be more effective in reducing gastric acid secretion. Even so, the hypothesis that better acid suppression could induce Barrett's esophagus regression was not optimistic, and the studies performed in this regard have been so far inconclusive. Only 2 of 7 studies demonstrated some regression. Most could not detect any regression, although pH testing proved complete normalization of esophageal pH.

[0008] Currently, the indications for medical treatment of Barrett's esophagus - control of symptoms and cure of the esophageal mucosa - are the same as for GERD.

[0009] Barrett's esophagus is a precursor lesion to most esophageal adenocarcinomas, a malignancy with rapidly rising incidence and consistently poor outcomes. As noted above, early detection of esophageal adenocarcinoma has been shown to be associated with earlier stage and increased survival. And, detection of dysplasia with subsequent endoscopic ablation can prevent esophageal adenocarcinoma.

[0010] There is a clear need for improved methods for detecting Barrett's esophagus and related conditions, such as Barrett's esophagus dysplasia. SUMMARY

[0011] Methylated DNA has been investigated as a potential class of biomarkers in tissues of most tumor types. In many cases, DNA methyltransferases add a methyl group to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. Among the biologically compelling mechanisms, methylation events gained in the promoter region of tumor suppressor genes are thought to silence expression, thus contributing to tumor formation. DNA methylation can be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11 : 191-203). Moreover, in other cancers such as sporadic colon cancer, methylation markers provide excellent specificity and are more informative and sensitive than individual DNA mutations (Zou et al. (2007) Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0012] Analysis of CpG islands when applied to animal models and human cell lines has yielded important findings. For example, Zhang and colleagues found that amplicons from different parts of the same CpG island can have different methylation levels (Zhang et al. (2009) PLoS Genet 5:el000438). Furthermore, methylation levels were bimodally distributed between highly methylated sequences and unmethylated sequences, further supporting a binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5:el000438). Analysis of mouse tissues in vitro and cell lines in vitro suggested that only about 0.3% of high CpG density promoters (HCPs, defined as having >7% CpG sequences within a 300 base pair region) were methylated, while regions of low CpG density (LCPs, defined as having <5% CpG sequences within a 300 base pair region) tended to be frequently methylated in a dynamic tissue-specific pattern (Meissner et al. (2008) Nature 454:766-70). HCPs include promoters of ubiquitous housekeeping genes and highly regulated developmental genes. Of these, >50% methylated HCP sites are multiple established markers, such as Wnt 2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454:766-70).

[0013] Accordingly, provided herein are technologies for esophageal disease screening (e.g., monitoring), particularly but not exclusively, methods, compositions, and related uses for detecting the presence of esophageal disease (e.g., Barrett’s esophagus, Barrett’s esophagus dysplasia, etc.). Moreover, the present technologies provide methods, compositions, and related uses for differentiating Barrett’s esophagus and Barrett’s esophagus dysplasia, and Barrett’s esophagus low-grade dysplasia, Barrett’s esophagus high-grade dysplasia, and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using a tethered device (e.g., a capsule sponge, a balloon, or other device).

[0014] Indeed, experiments performed in the course of developing the present technologies compared the methylation status of DNA markers from esophageal tissue of subjects with Barrett’s esophagus to the methylation status of the same DNA markers from control subjects (e.g., normal tissue of each tissue type), and to the methylation status of the same DNA markers from subjects with Barrett’s esophagus dysplasia (see Examples 1 and 5).

[0015] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Tables 1, 7, and / or 8) capable of classifying Barrett’s esophagus (BE) and controls (e.g., normal tissue of each tissue type) in esophageal tissue were identified (see Examples 1, 2, and 5).

[0016] Markers and / or marker panels (e.g., annotated chromosomal regions provided in Tables 2, 3, 5, and / or 6) were identified that are able to classify BE and Barrett’s esophagus-related dysplasia (BED) in esophageal tissue (see Examples 1, 3, and 4).

[0017] Markers and / or marker panels (e.g., annotated chromosomal regions provided in Table 5) were identified that are able to predict Barrett’s esophagus-related low-grade dysplasia (BE-LGD), Barrett’s esophagus-related high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by full esophageal swabbing or brushing (see Examples 1 and 4).

[0018] Markers and / or marker panels (e.g., annotated chromosomal regions provided in Table 5) were identified that are able to classify BE and BED in samples obtained by full esophageal swabbing or brushing (see Examples 1 and 4).

[0019] As described herein, the present technology provides a number of methylation DNA markers and subsets thereof (e.g., panels of 2, 3, 4, 5, 6, 7, 10, 15, 25, 50, 100, 150, 180, 190, 194 markers) that have high discriminatory power for esophageal diseases (e.g., BE, BED, BE-LGD, BE-HGD, EAC). Experimental filters of candidate markers were applied to identify markers that provide high signal-to-noise ratios and low background levels to provide high specificity, e.g., when assaying a medium (e.g., esophageal tissue) for screening or diagnostic (e.g., cancer screening or diagnostic) purposes.

[0020] In some embodiments, the present technology relates to assessing the presence and methylation status of one or more markers identified herein in a biological sample. These markers include one or more differentially methylated regions (DMRs), e.g., as provided in Tables 1, 2, 3, 5, 6, 7, and 8, as discussed herein. The methylation status is assessed in embodiments of the present technology. Accordingly, the technology provided herein is not limited to methods of measuring the methylation status of a gene. For example, in some embodiments, the methylation status is measured by a genomic scanning method. For example, one method involves restriction landmark genomic scanning (Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427), another example involves methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997) Cancer Res. 57:594-599). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digesting genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the region of interest (digestion-Southern method). In some embodiments, analysis of changes in methylation patterns involves a PCR-based process that involves digesting genomic DNA with methylation-sensitive restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059; and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534). PCR techniques have been developed for the detection of genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and for the quantitation of allele-specific expression (Szabo and Mann (1995) Genes Dev. 9:3097-3108; and Singer-Sam et al. (1992) PCR Methods Appl. 1:160-163). Such techniques use internal primers that anneal to the template produced by PCR and immediately terminate the 5' of the single nucleotide to be determined. The method using the “quantitative Ms-SNuPE assay” described in U.S. Patent No. 7,037,650 is used in some embodiments.

[0021] In assessing methylation status, methylation status is often expressed as the fraction or percentage of a single strand of methylated DNA at a particular site (e.g., at a single nucleotide, at a particular region or locus, at a longer target sequence, e.g., up to about 100 bp, 200 bp, 500 bp, 1000 bp of a DNA subsequence or longer) relative to the total population of DNA in a sample that contains that particular site. Typically, the amount of unmethylated nucleic acid is determined by a PCR assay using a calibrator. A known amount of DNA is then treated with bisulfite, and real-time PCR or other exponential amplification (e.g., QuARTS assay) is used to determine the resulting methylation-specific sequence (e.g., provided by U.S. Patent No. 8,361,720 and U.S. Patent Application Publication Nos. 2012 / 0122088 and 2012 / 0122106).

[0022] For example, in some embodiments, the method includes generating a standard curve for unmethylated targets by using external standards. The standard curve is constructed from at least two points and relates the real-time Ct values of unmethylated DNA to known quantitative standards. A second standard curve for methylated targets is then constructed from at least two points and external standards. This second standard curve relates the Ct of methylated DNA to known quantitative standards. Next, the Ct values of the methylated and unmethylated populations of a test sample are determined, and the genomic equivalents of DNA are calculated from the standard curves produced in the previous two steps. The percentage of methylation at a target site is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, e.g., (number of methylated DNA) / (number of methylated DNA + number of unmethylated DNA) x 100.

[0023] Also provided herein are compositions and kits for practicing the methods. For example, in some embodiments, reagents specific for one or more markers are provided (e.g., primers, probes), either individually or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for performing the detection assays can also be provided (e.g., enzymes, buffers, positive and negative controls for performing QuARTS, PCR, sequencing, bisulfite or other assays). In some embodiments, kits comprising one or more reagents necessary, sufficient or useful for performing the methods are provided. Reaction mixtures comprising reagents are also provided. Master mix reagent sets comprising multiple reagents that can be added to each other and / or to a test sample to complete a reaction mixture are also provided.

[0024] In some embodiments, the technology described herein is associated with programmable machines designed to perform a series of arithmetic or logical operations provided by the methods described herein. For example, some embodiments of the technology are associated with (e.g., executed in) computer software and / or computer hardware. In one aspect, the technology involves a computer, which includes memory forms, elements for performing arithmetic and logical operations, and processing elements (e.g., microprocessors) for executing a series of instructions (e.g., methods provided herein) to read, manipulate, and store data. In some embodiments, the microprocessor is part of a system for determining methylation status (e.g., methylation of one or more DMRs, such as DMRs 1-78 provided in Table 1; DMRs 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMRs 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMRs 77, 90, and 135 provided in Table 5; DMRs 136-187 provided in Table 6; DMRs 21 and 188-192 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and 193-229 provided in Table 8); comparing methylation status (e.g., methylation of one or more DMRs, such as DMRs 1-78 provided in Table 1; DMRs 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMRs 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMRs 77, 90, and 135 provided in Table 5; DMRs 136-187 provided in Table 6; DMRs 21 and 188-192 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and 193-229 provided in Table 8); generating a standard curve; determining a Ct value; calculating a fraction, frequency, or percentage of methylation (e.g., methylation of one or more DMRs, such as DMRs 1-78 provided in Table 1; DMRs 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMRs 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMRs 77, 90, and 135 provided in Table 5; DMRs 136-187 provided in Table 6; DMRs 21 and 188-192 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and 193-229 provided in Table 8).DMRs 77, 90, and 135 provided in Table 5; DMRs 136-187 provided in Table 6; DMRs 21 and 188-192 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and 193-229 provided in Table 8); identifying CpG islands; determining specificity and / or sensitivity of an assay or marker; calculating ROC curves and associated AUCs; sequence analysis; all as described herein or known in the art.

[0025] In some embodiments, the microprocessor or computer uses methylation status data in an algorithm to predict the site of cancer.

[0026] In some embodiments, the software or hardware component receives the results of multiple assays and determines a single value result indicative of cancer risk based on the results of multiple assays (e.g., determining methylation status of multiple DMRs, e.g., DMRs provided in Tables 1, 2, 3, 5, 6, 7, 8) to report to the user. Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of results from multiple assays (e.g., determining methylation status of multiple markers (e.g., determining methylation status of multiple DMRs, e.g., DMRs provided in Tables 1, 2, 3, 5, 6, 7, 8)). In some embodiments, the methylation status of DMRs defines dimensions and can have values in a multi-dimensional space and the coordinates defined by the methylation status of multiple DMRs are, for example, a result related to risk of esophageal disease (e.g., risk of BE, BED, BE-LGD, BE-HGD, EAC) to report to the user.

[0027] Some embodiments include storage media and memory components. Memory components (e.g., volatile and / or non-volatile memory) are used to store instructions (e.g., embodiments of processes provided herein) and / or data (e.g., artifacts, e.g., methylation measurements, sequences, and statistical descriptions related thereto). Some embodiments relate to systems that also include one or more of a CPU, a graphics card, and a user interface (e.g., including an output device such as a display and an input device such as a keyboard).

[0028] Programmable machines relevant to the present technology include conventional prior art as well as technology that is being developed or has not yet been developed (e.g., quantum computers, chemical computers, DNA computers, optical computers, spintronic computers, etc.).

[0029] In some embodiments, the technology includes wired (e.g., metal cables, optical fibers) or wireless transmission media for transmitting data. For example, some embodiments involve data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad hoc network, the Internet, etc.). In some embodiments, programmable machines exist as nodes on such a network, in some embodiments, the programmable machines have a client / server relationship.

[0030] In some embodiments, data is stored on computer-readable storage media, such as hard disks, flash memory, optical media, floppy disks, etc.

[0031] In some embodiments, the technology provided herein is associated with multiple programmable devices that operate together to perform the methods described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) can work in parallel to collect and process data, for example, in the implementation of cluster computing or grid computing, or some other distributed computer architecture that relies on complete computers (with on-board CPU, memory, power supply, network interface, etc.) connected to a network (private, public, or the Internet) through conventional network interfaces like Ethernet, fiber optics, or wireless network technologies.

[0032] For example, some embodiments provide a computer that includes a computer-readable medium. The embodiment includes a random access memory (RAM) coupled to the processor. The processor executes computer-executable program instructions stored in memory. Such a processor can include a microprocessor, an ASIC, a state machine, or other processors, and can be any of a variety of processors, such as a processor from Intel Corporation of Santa Clara, California, and a processor from Motorola Corporation of Schaumburg, Illinois. Such a processor contains or can communicate with a medium, such as a computer-readable medium, that stores instructions that when executed by the processor cause the processor to perform the steps described herein.

[0033] Embodiments of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices capable of providing a processor with computer-readable instructions. Other examples of suitable media include, but are not limited to, a floppy disk, a CD-ROM, a DVD, a magnetic disk, a memory chip, a ROM, a RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions. Also, various other forms of computer-readable media can transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel. The instructions can include code from any suitable computer- programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0034] In some embodiments, a computer is connected to a network. A computer can also include many external or internal devices, such as mice, CD-ROM, DVDs, keyboards, displays, or other input or output devices. Examples of a computer are a personal computer, a digital assistant, a personal digital assistant, a cellular telephone, a mobile telephone, a smart phone, a pager, a digital tablet, a laptop computer, an Internet appliance, and other processor-based devices. Generally, a computer related to aspects of the technology provided herein can be any type of processor-based platform that operates on any operating system capable of supporting one or more programs containing the technology provided herein (e.g., Microsoft Windows, Linux, UNIX, Mac OS X, etc.). Some embodiments include a personal computer that executes other application programs (e.g., application software). Application programs can be contained in memory and can include, for example, a word processing application, a spreadsheet application, an email application, an instant messaging application, a presentation application, an Internet browser application, a calendar / manager application, and any other application capable of being executed by a client device.

[0035] All such components, computers, and systems described herein associated with the present technology can be logical or virtual.

[0036] Accordingly, provided herein are technologies related to methods of screening for BE in a sample obtained from a subject, the method comprising determining a methylation status of a marker in a sample obtained from a subject; and identifying the subject as having BE when the methylation status of the marker is different than a methylation status of the marker determined in a subject not having BE, wherein the marker comprises one or more bases in a differentially methylated region (DMR) selected from the group consisting of DMRs 1-78 provided in Table 1 and / or DMRs 21 and 188-193 provided in Table 7 and / or; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and 193-229 provided in Table 8.

[0037] Provided herein are technologies related to methods of differentiating BE and BED in a sample obtained from a subject, the method comprising determining a methylation status of a marker in a sample obtained from a subject; and identifying the subject as having BE when the methylation status of the marker is similar to a methylation status of the marker determined in a subject having BE or identifying the subject as having BED when the methylation status of the marker is similar to a methylation status of the marker determined in a subject having BED, wherein the marker comprises one or more bases in a differentially methylated region (DMR) selected from the group consisting of DMRs 3, 5, 30, 33, 43, 77, and 79-128 provided in Table 2, DMRs 77, 27, 193, 90, 92, 101, 129-134 provided in Table 3, DMRs 77, 90, and 135 provided in Table 5, and / or DMRs 136-187 provided in Table 6.

[0038] Provided herein are technologies related to methods of differentiating BE-LGD, BE-HGD, and BE-EAC in a sample obtained from a subject, the method comprising determining the methylation status of a marker in a sample obtained from a subject; and identifying the subject as having BE-LGD when the methylation status of the marker is similar to the methylation status of the marker determined in a subject having BE-LGD, identifying the subject as having BE-HGD when the methylation status of the marker is similar to the methylation status of the marker determined in a subject having BE-HGD, or identifying the subject as having EAC when the methylation status of the marker is similar to the methylation status of the marker determined in a subject having EAC, wherein the marker comprises one or more bases in a differentially methylated region (DMR) selected from the group consisting of DMRs 77, 90, and 135 provided in Table 5.

[0039] The present technology is not limited to the methylation status assessed. In some embodiments, assessing the methylation status of a marker in a sample comprises determining the methylation status of one base. In some embodiments, determining the methylation status of a marker in a sample comprises determining the degree of methylation of a plurality of bases. Further, in some embodiments, the methylation status of a marker comprises elevated methylation of the marker relative to the normal methylation status of the marker. In some embodiments, the methylation status of a marker comprises decreased methylation of the marker relative to the normal methylation status of the marker. In some embodiments, the methylation status of a marker comprises a different methylation pattern of the marker relative to the normal methylation status of the marker.

[0040] Further, in some embodiments, the marker is a region of 100 or fewer bases, the marker is a region of 500 or fewer bases, the marker is a region of 1000 or fewer bases, the marker is a region of 5000 or fewer bases, or in some embodiments, the marker is one base. In some embodiments, the marker is in a high CpG density promoter.

[0041] The present technology is not limited by the type of sample. In some embodiments, the sample is esophageal tissue obtained by full esophageal swabbing or brushing (see Example 1 and Table 5) (see Example 5 and Table 8). In some embodiments, the sample is esophageal tissue obtained by use of a sponge capsule device (see Example 5 and Table 8). For example, in some embodiments, the sample is a fecal sample, a tissue sample (e.g., esophageal tissue, gastric tissue, pancreatic tissue, biliary / hepatic tissue, and colorectal tissue), a blood sample (e.g., plasma, serum, whole blood), a stool sample, or a urine sample.

[0042] Further, the technology is not limited to methods for determining methylation status. In some embodiments, the assay comprises use of methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assay comprises use of methylation-specific oligonucleotides. In some embodiments, the technology uses massively parallel sequencing (e.g., next generation sequencing) to determine methylation status, e.g., by sequencing by synthesis, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.

[0043] The technology provides reagents for detecting DMRs, e.g., in some embodiments, a set of oligonucleotides comprising the sequences provided in SEQ ID NOs: 1-50 are provided. In some embodiments, oligonucleotides comprising sequences complementary to regions of the chromosome having bases in a DMR are provided, e.g., oligonucleotides sensitive to the methylation status of a DMR.

[0044] The technology provides sets of multiple markers, e.g., in some embodiments, the markers comprise regions of the chromosome having annotations provided in Tables 1, 2, 3, 5, 6, 7, and / or 8, and include markers of markers (see, Tables 1, 2, 3, 5, 6, 7, 8). Further, embodiments provide methods of analyzing DMRs from one or more of DMR Nos. 1-229 from Tables 1, 2, 3, 5, 6, 7, and / or 8.

[0045] Kit embodiments are provided, e.g., a kit comprises: a bisulfite reagent; and a control nucleic acid comprising a sequence from a DMR selected from DMRs 1-194 (from Tables 1, 2, 3, 5, 6, 7, and / or 8) and having a methylation status associated with a subject that does not have an esophageal disease (e.g., a subject that does not have BE, BED, BE-LGD, BE-HGD, and EAC). In some embodiments, the kit comprises a bisulfite reagent and an oligonucleotide described herein. In some embodiments, the kit comprises a bisulfite reagent; and a control nucleic acid comprising a sequence from a DMR selected from DMRs 1-194 (from Tables 1, 2, 3, 5, 6, 7, 8) and having a methylation status associated with a subject that has an esophageal disease (e.g., a subject that has BE) (e.g., a subject that has BED, BE-LGD, BE-HGD, and EAC). Some kit embodiments comprise a sample collector for obtaining a sample (e.g., a stool sample) from a subject; reagents for isolating nucleic acids from a sample; a bisulfite reagent; and an oligonucleotide described herein.

[0046] Embodiments of the technology relate to compositions, e.g., reaction mixtures. In some embodiments are provided compositions comprising a nucleic acid comprising a DMR and a bisulfite reagent. Some embodiments provide compositions comprising a nucleic acid comprising a DMR and an oligonucleotide described herein. Some embodiments provide compositions comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions comprising a nucleic acid comprising a DMR and a polymerase.

[0047] Provided are additional embodiments of methods for screening for esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a sample obtained from a subject, e.g., the method comprises determining the methylation status of a biomarker comprising a base in a DMR in the sample, the DMR being one or more of DMRs 1-229 (from Tables 1, 2, 3, 5, 6, 7, 8); comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a normal control sample from a subject not having esophageal disease; and determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99% and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of the method provide the steps of reacting a nucleic acid comprising a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid comprising a DMR from a subject not having cancer to identify differences in the two sequences; identifying the subject as having a tumor when a difference is present.

[0048] The present technology provides systems for screening for esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a sample obtained from a subject. Exemplary embodiments of the system include, e.g., a system for screening for esophageal disease in a sample obtained from a subject, the system comprising: an analysis component configured to determine a methylation status of the sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to alert a user of an esophageal disease-associated methylation status (e.g., a methylation status that is not associated with esophageal disease; a methylation status that is associated with BE; a methylation status that is associated with BED; a methylation status that is associated with BE-LGD; a methylation status that is associated with BE-HGD; a methylation status that is associated with EAC). In some embodiments, the alert is determined by a software component that receives results from multiple assays (e.g., determines the methylation status of a plurality of markers, e.g., 1, 2, 3, 5, 6, 7, and / or 8 of the DMRs provided herein) and calculates a value or result to be reported based on the plurality of results. Some embodiments provide a database of weighting parameters associated with each of the DMRs provided herein for use in calculating a value or result and / or an alert to report to a user (e.g., a physician, a nurse, a clinician, etc.). In some embodiments, all results from the multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a combination of one or more results from the multiple assays that is indicative of a risk of esophageal disease in the subject (e.g., indicative of a risk of BE; indicative of a risk of BED; indicative of a risk of BE-LGD; indicative of a risk of BE-HGD; indicative of a risk of EAC).

[0049] In some embodiments of the system, the sample comprises nucleic acids comprising the DMRs. In some embodiments, the system further comprises a component for isolating the nucleic acids, a component for collecting the sample, e.g., a component for collecting a stool sample. In some embodiments, the system comprises nucleic acid sequences comprising the DMRs. In some embodiments, the database comprises nucleic acid sequences from subjects that do not have esophageal disease. Also provided are nucleic acids, e.g., a set of nucleic acids, each nucleic acid having a sequence comprising the DMRs. In some embodiments, a set of nucleic acids, wherein each nucleic acid has a sequence from a subject that does not have esophageal disease. Related system embodiments include a set of nucleic acids as described and a database of nucleic acid sequences related to the set of nucleic acids. Some embodiments further comprise a bisulfite reagent. Also, some embodiments further comprise a nucleic acid sequencer.

[0050] In certain embodiments, there is provided a method for detecting Barrett's esophagus in a sample obtained from a subject, comprising a) obtaining a sample comprising DNA from the subject; b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues; c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in a differentially methylated region (DMR) provided by DMR Nos. 1-78, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, and 187-229, d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation level of the one or more DNA methylation markers in i) a subject that does not have Barrett's esophagus, to identify a difference in the two sequences, and ii) a subject that does not have a Barrett's esophagus developmental abnormality, to identify a difference in the two sequences; and e) identifying the subject as having Barrett's esophagus when the difference in i) and ii) is present.

[0051] In certain embodiments, there is provided a method for detecting a Barrett's esophagus developmental abnormality in a sample obtained from a subject, comprising a) obtaining a sample comprising DNA from the subject; b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues; c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in a differentially methylated region (DMR) provided by DMR Nos. 3, 5, 30, 33, 43, 58, 77, 79-187, d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation level of the one or more DNA methylation markers in i) a subject that has Barrett's esophagus, to identify a difference in the two sequences, and ii) a subject that does not have a Barrett's esophagus developmental abnormality, to identify a difference in the two sequences; and e) identifying the subject as having a Barrett's esophagus developmental abnormality when the difference in i) and ii) is present.

[0052] In certain embodiments, there is provided a method for detecting Barrett's esophagus low grade dysplasia in a sample obtained from a subject, comprising a) obtaining a sample comprising DNA from the subject; b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues; c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in the differentially methylated regions (DMRs) provided by DMR Nos. 77, 90 and 135, d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation level of the one or more DNA methylation markers in i) a subject that does not have Barrett's esophagus low grade dysplasia, to identify differences in the two sequences, ii) a subject that does not have Barrett's esophagus dysplasia, to identify differences in the two sequences, iii) a subject that has Barrett's esophagus high grade dysplasia, to identify differences in the two sequences, and iv) a subject that has esophageal adenocarcinoma, to identify differences in the two sequences; and e) identifying the subject as having Barrett's esophagus low grade dysplasia when differences in i), ii), iii) and iv) exist.

[0053] In certain embodiments, there is provided a method for detecting Barrett's esophagus high grade dysplasia in a sample obtained from a subject, comprising a) obtaining a sample comprising DNA from the subject; b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues; c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in the differentially methylated regions (DMRs) provided by DMR Nos. 77, 90 and 135, d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation level of the one or more DNA methylation markers in i) a subject that does not have Barrett's esophagus high grade dysplasia, to identify differences in the two sequences, ii) a subject that does not have Barrett's esophagus dysplasia, to identify differences in the two sequences, iii) a subject that has Barrett's esophagus low grade dysplasia, to identify differences in the two sequences, and iv) a subject that has esophageal adenocarcinoma, to identify differences in the two sequences; and e) identifying the subject as having Barrett's esophagus high grade dysplasia when differences in i), ii), iii and iv) exist.

[0054] In certain embodiments, there is provided a method for detecting esophageal adenocarcinoma in a sample obtained from a subject comprising a) obtaining a sample comprising DNA from the subject; b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues; c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprises bases in the differentially methylated regions (DMRs) provided by DMR Nos. 77, 90 and 135, d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation levels of the one or more DNA methylation markers in i) a subject that does not have esophageal adenocarcinoma, to identify differences in the two sequences, ii) a subject that does not have a Barrett's esophagus developmental abnormality, to identify differences in the two sequences, iii) a subject that has a Barrett's esophagus low grade developmental abnormality, to identify differences in the two sequences, and iv) a subject that has a Barrett's esophagus high grade developmental abnormality, to identify differences in the two sequences; and e) identifying the subject as having esophageal adenocarcinoma when differences in i), ii), iii and iv) exist.

[0055] In some embodiments, the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0056] In some embodiments, the determination of elevated methylation in a CpG island or a CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0057] In some embodiments, the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) comprises determining a methylation score and / or a methylation frequency of the one or more DNA methylation markers. In some embodiments, the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0058] In some embodiments, the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing and bisulfite genomic sequencing PCR.

[0059] In some embodiments, the sample comprises esophageal tissue. In some embodiments, the esophageal tissue is obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using an anchoring device (e.g., a capsule sponge, a balloon or other device)

[0060] Additional embodiments will be apparent to those of ordinary skill in the relevant art based on the teachings herein. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 Positive rates of the panel of 3 markers (DIO3, MAX20.218, NDRG4) from tissue DNA of BE subgroups with no dysplasia and dysplasia of different severity (Table 5) (Examples I, III, III and IV).

[0062] Figure 2 Levels of methylated DNA markers (PCR copies / 20 ng DNA) in BE cases and normal (N1) controls from Stage 2 (endoscopic brush study) (Example V).

[0063] Figure 3 Hit matrix of top methylated DNA markers from Stage 2, highlighting complementarity (endoscopic brush study) (Example V).

[0064] Figure 4 Levels of methylated DNA markers (PCR copies / 30 ng DNA) in BE cases and normal (N1) controls from Stage 3 (capsule sponge study) (Example V).

[0065] DETAILED DESCRIPTION

[0066] Barrett's esophagus (BE) is the strongest risk factor and only known precursor of esophageal adenocarcinoma (EAC), a lethal malignancy with poor survival rates (5-year <20%) when detected after symptoms have developed (see Nelsen EM, et al., The Surgical clinics of North America 2012; 92: 1135-54). The incidence of EAC has increased nearly 600% in the population over the past three decades (see Hur C, et al., Cancer 2013; 119: 1149-58). BE progresses from no dysplasia, to low-grade dysplasia (LGD), to high-grade dysplasia (HGD) to cancer in a stepwise manner to EAC. This sequence of metaplasia to dysplasia to carcinoma has prompted gastroenterological societies in multiple countries to recommend screening BE in high-risk patients with multiple risk factors, followed by endoscopic surveillance (depending on the grade of dysplasia) to detect progression of dysplasia or carcinoma at an early stage (see Spechler SJ, et al., Gastroenterology 2011; 140: e18-52; Wang KK, et al., Am J Gastroenterol 2008; 103: 788-97; Fitzgerald RC, et al., Gut 2014; 63: 7-42). Endoscopic treatments for LGD, HGD, and early cancer have been developed and shown to be effective in reducing the incidence of cancer and improving survival rates in BE subjects (see, e.g., Prasad GA, et al., Gastroenterology 2007; 132: 1226-33; Prasad GA, et al., Gastroenterology 2009; Shaheen NJ, et al., N Engl J Med 2009; 360: 2277-88; Phoa KN, et al., JAMA 2014; 311: 1209-17).

[0067] Screening for BE is currently performed using conventional sedated endoscopy (sEGD), which shows the normal squamous lining of the esophagus replaced by metaplastic columnar epithelium in BE patients. However, sedated endoscopy is expensive in terms of direct and indirect costs, and is not suitable for widespread application. It is also associated with potential complications (see Sami SS, et al., Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 2015; 13:623-34). Other techniques, such as unsedated transnasal endoscopy (uTNE), have comparable accuracy to sEGD and lower costs, but are still not considered by providers to be a widely applicable tool (see Sami SS, et al., The American journal of gastroenterology 2015; 110:148-58; Peery AF, et al., Gastrointestinal endoscopy 2012; 75:945-953e2; Atkinson M, et al., Gastroenterology & hepatology 2007; 4:426-7). Despite adequate access to uTNE equipment, consultation physicians still limit the use of uTNE (see, Atkinson M, et al., The American journal of gastroenterology 2008; 103:92-7). The lack of accurate risk stratification tools to determine BE risk and target screening efforts is an additional limitation to widespread application of BE screening (see Sami SS, et al., Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 2015; 13:623-34).

[0068] In addition to careful examination of BE segments with high-resolution white light imaging and advanced imaging techniques, current endoscopic surveillance for dysplasia uses four-quadrant random biopsies of every 1-2 cm of BE segment. While this has been recommended by GI societies (see, e.g., Spechler SJ, et al., Gastroenterology 2011; 140:e18-52; Wang KK, et al., Am J Gastroenterol 2008; 103:788-97; Fitzgerald RC, et al., Gut 2014; 63:7-42), compliance with these recommendations remains low among practicing gastroenterologists (see, Abrams JA, et al., Clin Gastroenterol Hepatol 2009). In fact, compliance decreases with increasing BE segment length, leading to increased rates of false dysplasia. Other challenges to dysplasia detection in BE include the discontinuous distribution of dysplasia in BE (see, Cameron AJ, et al., Am J Gastroenterol 1997; 92:586-91), which leads to sampling error, poor inter-observer agreement among pathologists in dysplasia grading, and relatively poor sensitivity of current surveillance strategies in detecting widespread dysplasia or cancer (see, Sharma P, et al., Gastroenterology 2004; 127:310-30). Use of advanced imaging techniques in the community remains unclear, with only one-third of practicing gastroenterologists reporting frequent use in BE surveillance (see, Singh M, et al., Gastrointestinal endoscopy 2013; 78:689-95). A sponge-on-a-string device has recently been investigated in BE screening (see, Kadri SR, et al., Bmj 2010; 341 :c4372). This device consists of a polyurethane foam sponge compressed in a gelatin capsule attached to a string. The capsule is swallowed by the patient. The gelatin shell of the capsule dissolves in the stomach fluid, releasing the foam device as a ball, which is then pulled out with the attached string, providing a brushing / cellular sample of the proximal stomach and esophagus. These samples can then be investigated for biomarkers to detect BE. Two large multicenter studies have been conducted in the UK with this device, using trefoil factor 3 (a protein specific for BE epithelial cells) detected in immunohistochemistry as a marker for BE, demonstrating the feasibility, safety, and accuracy of this approach (see, Kadri SR, et al., Bmj 2010; 341 :c4372; Ross-Innes CS, et al., PLoS medicine 2015; 12:e1001780). For BE segments with a circumferential length >1 cm, this marker was reported to have a sensitivity and specificity of 73% and 94% in BE detection.Furthermore, such a capsule sponge device has been safely used in a study at Mayo Clinic Rochester in subjects with eosinophilic esophagitis (see, Katzka DA, et al., Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 2015; 13:77-83e2). Methylation DNA markers specific for BE epithelium (with and without dysplasia) have been described (see, Kaz AM, et al., Cancer letters 2014; 342:193-9; Ahlquist DA, et al., Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 2012; 10:272-7el).

[0069] Provided herein are technologies for esophageal disease screening, particularly but not exclusively, methods, compositions and related uses for detecting the presence of an esophageal disease, such as Barrett's esophagus, Barrett's esophagus dysplasia, and the like. Furthermore, the present technologies provide methods, compositions and related uses for differentiating Barrett's esophagus and Barrett's esophagus dysplasia, as well as Barrett's esophagus low-grade dysplasia, Barrett's esophagus high-grade dysplasia and esophageal adenocarcinoma in samples obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0070] For the technologies described herein, the section headings used are for organizational purposes only and are not to be construed as limiting the subject matter.

[0071] In the detailed description of the various embodiments, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, it will be understood by those skilled in the art that the various embodiments can be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form. Furthermore, the particular order of steps presented in the methods presented herein are illustrative in nature and can be varied without departing from the spirit and scope of the various embodiments disclosed herein.

[0072] All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are incorporated by reference in their entirety for the purpose of describing and disclosing, by reference, the various additional aspects, embodiments, and features that have been described in such publications. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments belong. If the definition of a term in an incorporated reference differs from the definition provided in the present teachings, the definition provided in the present teachings shall control.

[0073] Definitions

[0074] To facilitate the understanding of this technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0075] Throughout the specification and claims, the following terms take the following meanings, unless otherwise expressly stated herein. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, although it may. Additionally, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it can. Thus, particular embodiments of the present application can be combined, as described below, without departing from the scope or spirit of the present application.

[0076] Furthermore, as used herein, the term “or” is the inclusive, not the exclusive, and is equivalent to the term “and / or”, unless the context clearly otherwise requires. The term “based on” is not exclusive and allows for being based on additional factors, unless the context clearly dictates otherwise. Furthermore, throughout the specification, the meaning of “a,” “an,” and “the” includes plural references. The meaning of “in” includes “in” and “on.”

[0077] As used herein, “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which can be unmodified or modified DNA or RNA. “Nucleic acid” includes, but is not limited to, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above containing one or more modified bases. Thus, DNA with a modified backbone for stability or for other reasons is a “nucleic acid.” As used herein, the term “nucleic acid” includes chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as chemical forms of DNA characteristic of viruses and cells, including, for example, simple and complex cells.

[0078] The terms "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refer to a molecule having two or more, preferably more than three, and usually more than ten deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or combinations thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0079] As used herein, the term "locus" or "region" of a nucleic acid refers to a subregion of the nucleic acid, such as a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0080] The terms "complementary" and "complementarity" refer to nucleotides (e.g., 1 nucleotide) or polynucleotides (e.g., sequences of nucleotides) in relation to base-pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. Complementarity can be "partial," in which only some of the nucleic acid bases are matched according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization between the nucleic acid strands. This is of particular importance in amplification reactions and detection methods that rely on binding between nucleic acids.

[0081] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or polypeptide or precursors thereof. A functional polypeptide can be encoded by a full-length coding sequence or any portion of the coding sequence that retains the desired activity or functional properties of the polypeptide, such as enzymatic activity, ligand binding, signal transduction, etc. The term "portion" when used in reference to a gene means a fragment of that gene. The size of the fragment can range from a few nucleotides up to the entire gene sequence minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can comprise a fragment of the gene or the entire gene.

[0082] The term "gene" also includes the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the entire mRNA (e.g., including coding, regulatory, structural and other sequences). The sequences which are located 5' and on the mRNA are referred to as 5' untranslated or non-translated sequences. The sequences which are located 3' or downstream from the coding region and which are present on the mRNA are referred to as 3' untranslated or non-translated sequences. The term "gene" includes both cDNA and genomic forms of a gene. In some organisms (e.g., vertebrates) genomic forms of a gene can contain both exonic and intronic sequences. An intron is an intervening sequence that is transcribed into heteronuclear RNA (hnRNA) and is removed from a transcript by cis-acting RNAses during the first stage of mRNA processing. Exons are coding sequences of a gene that are actually translated into amino acids of a protein. Introns are non-coding sequences of a gene that are not translated into amino acids. The terms "gene" and "protein" are not limited to the sense of the wild-type gene and protein. The terms "gene" and "protein" encompass alleles, mutants, and variants of the gene and protein.

[0083] In addition to containing introns, a genomic form of a gene can include additional sequences adjacent to the coding region of the gene on both the 5' and 3' ends. The sequences that are present on the 5' and 3' ends of a transcript are referred to as "flanking" sequences, or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on a mRNA transcript). The 5' flanking region can contain regulatory sequences, such as promoters and enhancers, that control or influence the transcription of a gene. The 3' flanking region can include sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0084] The term "wild-type" when referring to a gene means a gene having the characteristics of a gene isolated from a naturally occurring source. The term "wild-type" when referring to a gene product means a gene product having the characteristics of a gene product isolated from a naturally occurring source. The term "naturally occurring" with respect to a subject means the fact that the subject can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) is naturally occurring, which can be isolated from a natural source and has not been intentionally modified in the laboratory. A wild-type gene is typically the most common gene or allele in a population and is arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the terms "modified" or "mutant" when referring to a gene or gene product mean a gene or gene product, respectively, that displays a modification (e.g., an altered characteristic) in sequence and / or functional properties when compared to a wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are determined by the fact that they have an altered characteristic when compared to a wild-type gene or gene product.

[0085] The term "allele" refers to a variation of a gene; variations include but are not limited to variants and mutants, polymorphic sites and single nucleotide polymorphic sites, frameshift and splice mutations. Alleles can occur naturally in a population or can arise during the lifetime of any particular individual of the population.

[0086] Thus, the terms "variant" and "mutant" when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs from another, usually related, nucleotide sequence by one or more nucleotides. A "variation" is the difference between two different nucleotide sequences; usually, one sequence is the reference sequence.

[0087] "Amplification" is a special case of nucleic acid replication that involves template specificity. It is contrasted with non-specific template replication (e.g., replication that is dependent on a template but not specific for the template). Template specificity here is distinguished from fidelity of replication (e.g., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribose or deoxyribose nucleic acid) specificity. Template specificity is often described as "target" specificity. A target sequence is in some sense a "target" that is sought out from among other nucleic acids. Amplification techniques are designed primarily for this selection.

[0088] Amplification of nucleic acids generally refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule, 10 to 100 copies of a polynucleotide molecule, which can be identical or not identical), where the amplification product or amplicon is typically detectable. Amplification of polynucleotides includes various chemical and enzymatic processes. The production of multiple copies of DNA from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810) is a form of amplification.Other types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671), inter-sequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186), ligation-mediated PCR (see, e.g., Guilfoyle, R et al., Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Pat. No. 5,508,169), methylation-specific PCR (see, e.g., Herman et al. (1996) PNAS 93(13) 9821-9826), mini-primer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367), real-time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030), reverse transcription PCR (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25: 169-193), solid phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485).Polynucleotide amplification can also be accomplished using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525).

[0089] The term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The method for amplifying a target sequence consists of introducing enormous excesses of two oligonucleotide primers into a mixture containing the desired target sequence, followed by a precise thermal cycling sequence in the presence of a DNA polymerase. The two primers are complementary to the respective strands of the double-stranded target sequence. To carry out the amplification, the mixture is denatured, and then the primers anneal to their complementary sequences within the target molecule. After annealing, the primers are extended by a polymerase, forming a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle"; there can be many "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and thus this length is a controllable parameter. Because of the repetitive aspect of the method, it is referred to as the "polymerase chain reaction" ("PCR"). Because the desired amplified segment of the target sequence becomes the predominant sequence in the mixture (in terms of concentration), it is said to be "PCR amplified," is a "PCR product," or an "amplicon."

[0090] Template specificity is achieved in most amplification techniques by the selection of the enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, process nucleic acids only at specific sequences of the nucleic acid in a heterogeneous mixture of nucleic acids. For example, in the case of Q-beta replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has strict specificity for its own promoter (Chamberlin et al., Nature, 228:227

[1970] ). In the case of T4 DNA ligase, the enzyme does not ligate two oligonucleotides or polynucleotides where there is a mismatch between the oligonucleotide or polynucleotide substrate and the ligation template at the junction (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependent DNA polymerases (e.g., Taq and Pfu DNA polymerases) were found to exhibit a high degree of specificity for the sequence to which the primers bind and thus define, due to their ability to function at high temperatures; the high temperatures result in thermodynamic conditions that favor hybridization of the primers to the target sequence and not to non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0091] As used herein, the term "nucleic acid detection assay" refers to any method of determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assay, Hologic, Inc.), and are described, for example, in U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US 2009 / 0253142); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Patent Nos. 6,110,684, 5,958,692, 5,851,770); polymerase chain reaction; branched hybridization methods (e.g., Chiron, U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802); rolling circle replication (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502); NASBA (e.g., U.S. Patent No. 5,409,818); molecular beacon technology (e.g., U.S. Patent No. 6,150,097); electronic sensor technology (Motorola, U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573); cycling probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988); Dade Behring signal amplification methods (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614); ligase chain reaction (Barnay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Patent No. 5,288,609).

[0092] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. It is contemplated that an "amplifiable nucleic acid" will generally comprise a "sample template."

[0093] The term "sample template" refers to nucleic acid derived from a sample that is used to analyze the presence of a "target" (defined below). In contrast, "background template" is used to refer to nucleic acid other than the sample template, which can or can not be present in the sample. Background template is typically unintentional. This can be a result of carryover, or can be due to the presence of nucleic acid contaminants that were attempted to be purified away from the sample. For example, nucleic acid other than the nucleic acid from the organism being detected can be present as background of the test sample.

[0094] The term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis of a primer extension product when placed under conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH. The primer is preferably single-stranded for maximum efficiency in amplification, but can alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands prior to use. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of the primer extension product. The exact length will depend on many factors, including temperature, source of primer, and use of the method.

[0095] The term "probe" refers to an oligonucleotide (e.g., nucleotide sequence), whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of hybridizing to another, target oligonucleotide. The probe can be single-stranded or double-stranded. The probe can be used for the detection, identification and isolation of particular genetic sequences (e.g., "capture probes"). It is contemplated that any probe used in the present application, in some embodiments, can be labeled with any "reporter molecule" so that it can be detected in any detection system, including but not limited to enzymes (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. The present application is not intended to be limited in any way to any particular detection system or label.

[0096] "Methylation" as used herein refers to cytosine methylation at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is typically unmethylated, as typical in vitro DNA amplification methods are not able to preserve the methylation pattern of the amplification template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA that is unmethylated or methylated, respectively, of the original template.

[0097] Accordingly, as used herein, a "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not present in the canonical typical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5 position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5 position of its pyrimidine ring; however, for purposes herein, thymine is not considered a methylated nucleotide when present in DNA, as thymine is the canonical nucleotide base of DNA.

[0098] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0099] As used herein, the "methylation status," "methylation profile," and "methylation context" of a nucleic acid molecule refers to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation status of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated

[0100] The methylation status of a particular nucleic acid sequence (e.g., a genetic marker or DNA region described herein) can indicate the methylation status of each base in the sequence or can indicate the methylation status of a subset of bases (e.g., one or more cytosines) in the sequence, or can indicate information about the methylation density of regions within the sequence, with or without providing precise information about the location of methylation occurrences within the sequence.

[0101] The methylation status of a nucleotide site in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at the particular site in the nucleic acid molecule. For example, the methylation status of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is a 5-methylcytosine. Similarly, the methylation status of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is a cytosine (and not a 5-methylcytosine).

[0102] The methylation status can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, proportion, percentage, etc.). Methylation values can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles following bisulfite reaction, or by comparing sequences of bisulfite-treated and untreated nucleic acids. Thus, values such as methylation values are representative of methylation status and can therefore be used as a quantitative indicator of methylation status in multiple copies of a locus. This is particularly useful when it is desirable to compare the methylation status of a sequence in a sample to a threshold or reference value.

[0103] As used herein, "methylation frequency" or "methylation percentage (%) " refers to the number of instances of methylation of a molecule or locus relative to the number of instances of non-methylation of the molecule or locus.

[0104] Accordingly, methylation status describes the methylation state of a nucleic acid (e.g., a genomic sequence). Further, methylation status refers to methylation-related features of a nucleic acid fragment at a particular genomic site. These features include, but are not limited to, whether any cytosine (C) residues in the DNA sequence are methylated, the location of methylated C residues, the frequency or percentage of methylated C in any particular region of the nucleic acid, and allelic differences in methylation, e.g., due to differences in allelic origin. The terms "methylation status," "methylation profile," and "methylation context" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C in any particular region of a nucleic acid in a biological sample. For example, a cytosine (C) residue in a nucleic acid sequence can be referred to as "hypermethylated" or having "increased methylation" if it is methylated, whereas a cytosine (C) residue sequence within a DNA sequence can be referred to as "hypomethylated" or having "decreased methylation" if it is not methylated. Likewise, a cytosine (C) residue in a nucleic acid sequence can be considered hypermethylated or have increased methylation compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.) if it is methylated compared to the other nucleic acid sequence. Alternatively, a cytosine (C) residue in a nucleic acid sequence can be considered hypomethylated or have decreased methylation compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.) if it is not methylated compared to the other nucleic acid sequence. Additionally, the term "methylation pattern" as used herein refers to the collective sites of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids can have the same or similar frequency or percentage of methylation, but have different methylation patterns when the number of methylated and unmethylated nucleotides is the same or similar across the entire region, but the positions of the methylated and unmethylated nucleotides are different. Sequences are considered "differentially methylated" or have "methylation differences" or "different methylation status" when the degree (e.g., one has elevated or decreased methylation relative to the other), frequency, or pattern of methylation of the sequences is different. The term "differential methylation" refers to a difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. It can also refer to a difference in the level or pattern between a patient who has a recurrence of cancer after surgery and a patient who does not have a recurrence. Particular levels or patterns of differential methylation and DNA methylation are prognostic and predictive biomarkers, e.g., once the correct cutoff or predictive features are determined.

[0105] Methylation state frequencies can be used to describe a population of individuals or from a sample of a single individual. For example, a nucleotide site with a methylation state frequency of 50% is methylated in 50% of cases and unmethylated in 50% of cases. Such frequencies can be used, for example, to describe the extent of methylation of a nucleotide site or nucleic acid region in a population of individuals or collection of nucleic acids. Thus, when the methylation in a first population or library of nucleic acid molecules is different from the methylation in a second population or library of nucleic acid molecules, the methylation state frequency of the first population or library is different from the methylation state frequency of the second population or library. Such frequencies can also be used, for example, to describe the extent of methylation of a nucleotide site or nucleic acid region in a single individual. For example, such frequencies can be used to describe the extent of methylation or unmethylation of a nucleotide site or nucleic acid region in a group of cells from a tissue sample.

[0106] As used herein, a“nucleotide site” refers to the position of a nucleotide in a nucleic acid molecule. A nucleotide site of a methylated nucleotide refers to the position of a methylated nucleotide in a nucleic acid molecule.

[0107] Generally, methylation of human DNA occurs on dinucleotide sequences comprising an adjacent guanine and cytosine, with the cytosine located 5' of the guanine (also referred to as CpG dinucleotide sequences). Most cytosines in CpG dinucleotides in the human genome are methylated, however some remain unmethylated in specific genomic regions enriched for CpG dinucleotides, known as CpG islands (see, e.g., Antequera et al. (1990) Cell 62: 503-514).

[0108] As used herein, a "CpG island" refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides relative to total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, wherein the G:C content of the region is at least 50%, and the ratio of observed CpG frequency to expected frequency is 0.6; in some cases, a CpG island can be at least 500 base pairs in length, wherein the G:C content of the region is at least 55%, and the ratio of observed CpG frequency to expected frequency is 0.65. The observed CpG frequency relative to the expected frequency can be calculated according to the method provided in Gardiner-Garden et al. (1987) J. Mol. Biol. 196:261-28. For example, the observed CpG frequency relative to the expected frequency can be calculated according to the formula R = (A x B) / (C x D), where R is the ratio of observed CpG frequency to expected frequency, A is the number of CpG dinucleotides in the sequence analyzed, B is the total number of nucleotides in the sequence analyzed, C is the total number of C nucleotides in the sequence analyzed, and D is the total number of G nucleotides in the sequence analyzed. Methylation status is typically determined in CpG islands, e.g., in the promoter region. It will be appreciated, however, that other sequences in the human genome also tend to be DNA-methylated, e.g., CpA and CpT (see, e.g., Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14:4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888-894).

[0109] As used herein, a reagent that modifies nucleotides of a nucleic acid molecule as a function of the methylation state of the nucleic acid molecule or a methylation-specific reagent refers to a compound or composition or other reagent that can alter the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such a reagent can include contacting the nucleic acid molecule with the reagent, plus additional steps if needed, to achieve the desired change in the nucleotide sequence. Such a change in the nucleotide sequence of a nucleic acid molecule can result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide. Such a change in the nucleotide sequence of a nucleic acid molecule can result in a nucleic acid molecule in which each unmethylated nucleotide is modified to a different nucleotide. Such a change in the nucleotide sequence of a nucleic acid molecule can result in a nucleic acid molecule in which each unmethylated selected nucleotide (e.g., each unmethylated cytosine) is modified to a different nucleotide. Use of such a reagent to alter the nucleotide sequence of a nucleic acid can result in a nucleic acid molecule in which each nucleotide that is a methylated nucleotide (e.g., each methylated cytosine) is modified to a different nucleotide. As used herein, use of a reagent that modifies a selected nucleotide is a reagent that modifies one of the four normally occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA, and C, G, U, and A for RNA) such that the reagent modifies one nucleotide and not the other three. In one exemplary embodiment, such a reagent modifies an unmethylated selected nucleotide to produce a different nucleotide. In another exemplary embodiment, such a reagent can remove an unmethylated cytosine nucleotide. An exemplary reagent is bisulfite.

[0110] As used herein, the term "bisulfite reagent" refers to a reagent that, in some embodiments, comprises bisulfite, disulfite, hydrogen sulfite, or a combination thereof, to distinguish between, for example, methylated and unmethylated cytosines in a CpG dinucleotide sequence.

[0111] The term "methylation assay" refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0112] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to a well-recognized technique that allows for global scanning of the genome using CG-rich primers to focus on regions most likely to contain CpG dinucleotides, described by Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0113] The term "MethyLight TM"Real-time PCR" refers to the art-recognized technique of real-time PCR based on fluorescence, described by Eads et al. (1999) Cancer Res. 59:2302-2306.

[0114] The term "HeavyMethyl TM " refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) covering CpG positions located between or covered by amplification primers are able to perform methylation-specific selective amplification of a nucleic acid sample.

[0115] The term "HeavyMethyl TM " refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) covering CpG positions located between or covered by amplification primers are able to perform methylation-specific selective amplification of a nucleic acid sample. TM " refers to the HeavyMethyl TM " assay in which the HeavyMethyl TM " assay is combined with methylation-specific blocking probes covering CpG positions between amplification primers. TM " refers to the HeavyMethyl TM " assay in which the HeavyMethyl

[0116] The term "Ms-SNuPE" (Methylation-Sensitive Single Nucleotide Primer Extension) refers to the art-recognized assay described by Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0117] The term "MSP" (Methylation-Specific PCR) refers to the art-recognized methylation assay described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826 and U.S. Patent No. 5,786,146.

[0118] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to the art-recognized methylation assay described by Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0119] The term "MCA" (Methylation CpG Island Amplification) refers to the art-recognized methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12 and WO 00 / 26401 Al.

[0120] As used herein, "selected nucleotide" refers to one of the four normally occurring nucleotides (C, G, T, and A for DNA, and C, G, U, and A for RNA) in a nucleic acid molecule, and can include methylated derivatives of the normally occurring nucleotides (e.g., when C is the selected nucleotide, both methylated and unmethylated C are included within the meaning of the selected nucleotide), while methylated selected nucleotide specifically refers to a methylated normally occurring nucleotide, and unmethylated selected nucleotide specifically refers to an unmethylated normally occurring nucleotide.

[0121] The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests a nucleic acid depending on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cleaves if the recognition site is not methylated or is hemimethylated, cleavage does not occur or occurs with significantly reduced efficiency if the recognition site is methylated. In the case of a restriction enzyme that specifically cleaves if the recognition site is methylated, cleavage does not occur or occurs with significantly reduced efficiency if the recognition site is not methylated. Preferred are methylation-specific restriction enzymes whose recognition sequence contains a CG dinucleotide (e.g., recognition sequence such as CGCG or CCCGGG). For some embodiments, it is further preferred that the restriction enzyme does not cleave if the cytosine in this dinucleotide is methylated at carbon atom C5.

[0122] As used herein, "different nucleotide" refers to a nucleotide that is chemically different from the selected nucleotide, typically such that the different nucleotide has different Watson-Crick base pairing properties than the selected nucleotide, so that a normally occurring nucleotide that is complementary to the selected nucleotide and a normally occurring nucleotide that is complementary to the different nucleotide are not the same. For example, when C is the selected nucleotide, U or T can be the different nucleotide, which amplifies by complementarity of C to G and U or T to A. As used herein, a nucleotide that is complementary to the selected nucleotide or to the different nucleotide refers to a nucleotide that base pairs with the selected nucleotide or the different nucleotide under high stringency conditions with higher affinity than the complementary nucleotide base pairs with three of the four normally occurring nucleotides. Examples of complementarity are Watson-Crick base pairing in DNA (e.g., A-T and C-G) and RNA (e.g., A-U and C-G). Thus, for example, under high stringency conditions, a G base pairs with C with higher affinity than a G base pairs with G, A, or T, so that when C is the selected nucleotide, G is a nucleotide that is complementary to the selected nucleotide.

[0123] As used herein, the "sensitivity" of a given marker refers to the percentage of samples reporting DNA methylation values above a threshold value that distinguishes between tumor and non-tumor samples. In some embodiments, a positive is defined as a histologically confirmed tumor reporting DNA methylation values above a threshold value (e.g., a range associated with disease), and a false negative is defined as a histologically confirmed tumor reporting DNA methylation values below a threshold value (e.g., a range associated with disease). Thus, the value of sensitivity reflects the likelihood that a DNA methylation measurement of a given marker obtained from a known diseased sample will be within a disease-associated measurement range. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the likelihood that a given marker will detect the presence of a clinical condition when applied to a subject with the condition.

[0124] As used herein, the "specificity" of a given marker refers to the percentage of non-tumor samples reporting DNA methylation values below a threshold value that distinguishes between tumor and non-tumor samples. In some embodiments, a negative is defined as a histologically confirmed non-tumor sample reporting DNA methylation values below a threshold value (e.g., a range associated with no disease), and a false positive is defined as a histologically confirmed non-tumor sample reporting DNA methylation values above a threshold value (e.g., a range associated with no disease). Thus, the value of specificity reflects the likelihood that a DNA methylation measurement of a given marker obtained from a known non-tumor sample will be within a non-disease-associated measurement range. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the likelihood that a given marker will detect the absence of a clinical condition when applied to a patient without the condition.

[0125] As used herein, the term "AUC" is an abbreviation for "Area Under the Curve." It refers specifically to the area under the Receiver Operating Characteristic (ROC) curve. The ROC curve is a plot of true positive rate against false positive rate for the different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the chosen cut point (any increase in sensitivity is accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area the better; the optimal value is 1 ; a random test would show an ROC curve on the diagonal line with an area of 0.5; reference: J. P. Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0126] As used herein, the term "tumor" refers to "an abnormal mass of tissue that grows beyond normal tissue, and whose growth is not proportional to the growth of normal tissue", see, e.g., Willis RA, "The Spread of Tumors in the Human Body", London, Butterworth & Co, 1952.

[0127] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. While these growths are benign, they can progress further over time.

[0128] The term "pre-cancer" or "pre-tumor" and equivalents thereof refers to any cellular proliferative disease that is undergoing malignant transformation.

[0129] A "site" or "region" of a tumor, adenoma, cancer, etc. is a tissue, organ, cell type, anatomical region, body part, etc. in the body of a subject in which the tumor, adenoma, cancer, etc. is located.

[0130] As used herein, the term "esophageal disease" refers to a type of disease associated with the esophagus and / or esophageal tissue. Examples of esophageal diseases include, but are not limited to, Barrett's esophagus (BE), Barrett's esophagus dysplasia (BED), Barrett's esophagus low-grade dysplasia (BE-LGD), Barrett's esophagus high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC).

[0131] As used herein, "diagnostic" test applications include detecting or identifying a disease state or condition in a subject, determining the likelihood that a subject will contract a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to a treatment, determining the prognosis (or its possible progression or regression) of a subject with a disease or condition, and determining the effect of a treatment on a subject with a disease or condition. For example, a diagnosis can be used to detect the presence or likelihood of a subject contracting a tumor, or the likelihood that the subject will respond favorably to a compound (e.g., a drug, e.g., a pharmaceutical agent) or other treatment.

[0132] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid or a region of a nucleic acid) that is capable of diagnosing a disease (e.g., a non-cancerous disease) (e.g., a cancerous disease) by, e.g., distinguishing disease-associated cells (e.g., non-cancerous cells associated with a disease) (cancerous cells associated with a disease) from normal cells based on methylation status.

[0133] The term "isolated" when used in reference to nucleic acids, as in "isolated oligonucleotide," refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. An isolated nucleic acid is present in a form other than that in which it is found in nature. In contrast, a non-isolated nucleic acid, such as DNA and RNA, is found in the state it exists in nature. Examples of non-isolated nucleic acids include: a given DNA sequence (e.g., a gene) found in the vicinity of other genes on the host cell chromosome; a RNA sequence, such as a specific mRNA sequence encoding a particular protein, found in a mixture of many other mRNAs encoding a large number of proteins in a cell. However, an isolated nucleic acid encoding a particular protein by way of example includes such a nucleic acid in a cell normally expressing the protein, where the nucleic acid is in a different chromosomal location than in nature, or is flanked by a different nucleic acid sequence than in nature. An isolated nucleic acid or oligonucleotide can be present in single- stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide can be single-stranded), but can contain both the sense and anti-sense strands (i.e., the oligonucleotide can be double-stranded). An isolated nucleic acid can be combined with other nucleic acids or molecules after isolation from its natural or typical environment. For example, an isolated nucleic acid can be present in a host cell in which it is placed, for example, for heterologous expression.

[0134] The term "purified" refers to a molecule of a nucleic acid or amino acid sequence that is removed, isolated or separated from its natural environment. Thus, an "isolated nucleic acid sequence" can be a purified nucleic acid sequence. A "substantially purified" molecule is at least 60% free, preferably at least 75% free, or more preferably at least 90% free of other components with which it is naturally associated. As used herein, the term "purified" or "to purity" also refers to the removal of contaminants from a sample. Removal of contaminating proteins results in an increase in the percentage of the polypeptide or nucleic acid of interest in the sample. In another example, a recombinant polypeptide is expressed in a plant, bacterial, yeast, or mammalian host cell, and the polypeptide is purified by removing host cell proteins; thus, the percentage of the recombinant polypeptide in the sample is increased.

[0135] The term "compositions" that "comprise" a given polynucleotide sequence or polypeptide broadly refers to any composition comprising the given polynucleotide sequence or polypeptide. The composition can comprise an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0136] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells or tissues. In another sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental materials such as surface matter, soil, water, and industrial samples, among others. These examples should not be construed as limiting the types of samples applicable to the present application. In some embodiments, the sample comprises esophageal tissue. In some embodiments, the sample comprises esophageal tissue obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using a tethered device such as a capsule sponge, balloon or other device.

[0137] As used herein, "remote sample" as used in some instances refers to a sample that is collected indirectly from a site that is not the source of the cells, tissues, or organs of the sample. For example, when assessing sample material derived from the pancreas in a fecal sample (e.g., a sample not taken directly from the pancreas), the sample is a remote sample.

[0138] As used herein, the term "patient" or "subject" refers to an organism undergoing the various tests provided by the present technology. The term "subject" includes animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In more preferred embodiments, the subject is a human.

[0139] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such a delivery system includes a system that allows for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in suitable containers) and / or ancillary materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) that contain the relevant reaction reagents and / or ancillary materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each of which contains a sub-portion of the total kit components. The containers can be delivered to an intended recipient together or separately. For example, a first container can contain an enzyme for the assay, while a second container contains oligonucleotides. The term "fragmented kit" is intended to include kits containing analyte-specific reagents (ASRs) as defined by Section 520(e) of the Federal Food, Drug, and Cosmetic Act, but is not limited thereto. Indeed, any delivery system that includes two or more separate containers, each of which contains a sub-portion of the total kit components, is included within the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that contains all of the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both fragmented and combined kits.

[0140] Embodiments of the present technology

[0141] Barrett's esophagus is a precursor lesion to most esophageal adenocarcinomas, a malignancy with rapidly rising incidence and consistently poor outcomes. Early detection of esophageal adenocarcinoma has been shown to be associated with earlier stage and increased survival. Early detection of Barrett's esophagus can enable patients to enter a surveillance program, which can allow for detection of tumor progression at an early stage, to enable endoscopic or surgical treatment, and to improve outcomes. Screening for Barrett's esophagus and esophageal adenocarcinoma is limited by a lack of widely applicable tools, as well as a lack of biomarkers that can be used in conjunction with screening tools. The acceptability and feasibility of screening by endoscopy and new non-endoscopic methods has been demonstrated in populations. Non-endoscopic screening methods, such as swallowed cytology brushes or stool DNA testing, offer a potentially cost-effective alternative to endoscopy for identifying Barrett's esophagus in the general population. Recently, several aberrantly methylated genes have been found to serve as highly discriminative markers of Barrett's esophagus. In fact, a study performed on archived frozen esophageal biopsies of patients with and without Barrett's disease showed that a set of tumor-associated genes can potentially be used to distinguish Barrett's esophagus from squamous mucosa. (See, e.g., Yang Wu, et al., DDW Abstract 2011).

[0142] Developmental abnormalities are known to be distributed in a sporadic fashion in Barrett's esophagus, leading to "sampling error" of routine endoscopic surveillance with four-quadrant biopsies. Routine endoscopic surveillance is known to utilize less than 10% of BE segments for biopsy samples. Endoscopist compliance with routine surveillance is known to be poor. While newer endoscopic techniques have been shown to improve yield of developmental abnormalities detection in studies performed at tertiary care centers, their applicability in the community remains uncertain. Methods that sample a larger mucosal surface area, such as swabbing or brushing, can potentially increase yield of developmental abnormalities and tumors, especially if combined with molecular markers of developmental abnormalities / tumors. This can ultimately allow for non-biopsy (by swabbing or brushing) or non-endoscopic surveillance of BE subjects to save substantial potential costs.

[0143] Accordingly, provided herein are techniques for esophageal disease screening, particularly but not exclusively relating to methods, compositions, and related uses for detecting the presence of an esophageal disease, such as Barrett's esophagus, Barrett's esophagus developmental abnormalities, and the like. Moreover, the present techniques provide methods, compositions, and related uses for differentiating Barrett's esophagus and Barrett's esophagus developmental abnormalities, as well as Barrett's esophagus low-grade developmental abnormalities, Barrett's esophagus high-grade developmental abnormalities, and esophageal adenocarcinoma, in samples obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using a tethered device, such as a capsule sponge, balloon, or other device.

[0144] Indeed, experiments performed in the course of developing the present techniques compared the methylation status of DNA markers from esophageal tissue of subjects with Barrett's esophagus to the methylation status of the same DNA markers from control subjects (e.g., normal tissue of each tissue type) and the methylation status of the same DNA markers from subjects with Barrett's esophagus developmental abnormalities (see Examples 1-4).

[0145] Markers and / or marker sets (e.g., annotated chromosomal regions provided in Tables 1, 7, and / or 8) capable of classifying Barrett's esophagus (BE) and controls (e.g., normal tissue of each tissue type) in esophageal tissue were identified (see Examples 1, 2, and 5).

[0146] Markers and / or marker sets (e.g., annotated chromosomal regions provided in Tables 2, 3, 5, and / or 6) capable of classifying BE and Barrett's esophagus-related developmental abnormalities (BED) in esophageal tissue were identified (see Examples 1, 3, and 4).

[0147] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Table 5) were identified that are able to predict Barrett's esophagus-related low-grade dysplasia (BE-LGD), Barrett's esophagus-related high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by full esophageal swabbing or brushing (see Examples 1 and 4).

[0148] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Table 5) were identified that are able to classify BE and BED in samples obtained by full esophageal swabbing or brushing (see Examples 1 and 4).

[0149] While the disclosure herein relates to certain example embodiments, it should be understood that these embodiments are presented by way of example and not by way of limitation.

[0150] Methods include determining the methylation status of at least one methylation marker in a biopsy sample isolated from a subject, wherein a change in the methylation status of the marker is indicative of the presence or classification of an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC). Particular embodiments relate to markers comprising a differentially methylated region (DMR, e.g., DMRs 1-229, see Tables 1, 2, 3, 5, 6, 7, 8) that are used to diagnose (e.g., screen for) an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC), including early detection at the precancerous stage of the disease (e.g., BE versus BED).

[0151] The markers of the present technology are particularly effective in detecting or differentiating esophageal diseases (e.g., BE, BED, BE-LGD, BE-HGD, EAC), thus providing improved means for early detection, classification, and treatment of said diseases.

[0152] In addition to embodiments in which the methylation analysis comprises at least one marker, region of a marker, or base of a marker of a DMR provided herein and listed in Tables 1, 2, 3, 5, 6, and / or 7 (e.g., DMRs 1-229 from Tables 1, 2, 3, 5, 6, 7, and / or 8), the present technology also provides panels of markers comprising at least one marker, region of a marker, or base of a marker of a DMR in esophageal tissue that are used to detect an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC).

[0153] Some embodiments of the present technology are based on analysis of the CpG methylation status of at least one marker, region of a marker, or base of a marker of a DMR.

[0154] In some embodiments, the present technology provides for determining the methylation status of CpG dinucleotide sequences in at least one marker comprising a DMR (e.g., provided in Tables 1, 2, 3, 5, 6, 7, 8 (e.g., DMRs 1-229)) using a combination of bisulfite technology and one or more methylation assays. Genomic CpG dinucleotides can be methylated or unmethylated (also referred to as hypermethylated and hypomethylated, respectively). However, the methods of the present invention are suitable for analyzing biological samples of heterogeneous nature, such as low concentrations of tumor cells in the background of a distal sample (e.g., blood, organ effluent, or stool), or biological material derived therefrom. Thus, when analyzing the methylation status of CpG positions in such samples, a quantitative assay can be used to determine the level of methylation (e.g., percentage, fraction, ratio, proportion, or extent) of a particular CpG position.

[0155] According to the present technology, determining the methylation status of CpG dinucleotide sequences in markers comprising DMRs can be used to diagnose and characterize esophageal diseases (e.g., BE, BED, BE-LGD, BE-HGD, EAC).

[0156] Combinations of markers

[0157] In some embodiments, the present technology relates to assessing the methylation status of a combination of markers containing two or more DMRs from Tables 1, 2, 3, 5, 6, 7, 8 (e.g., two or more DMRs from DMR Nos. 1-194). In some embodiments, assessing the methylation status of more than one marker increases the specificity and / or sensitivity of the screening or diagnosis for identifying an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject.

[0158] Various cancers are predicted by various combinations of markers, e.g., identified by statistical techniques related to the specificity and sensitivity of the prediction. The present technology provides methods for identifying predictive combinations for certain cancers and validated predictive combinations.

[0159] In some embodiments, combinations of markers (e.g., comprising DMRs) predict the site of a tumor.

[0160] For example, markers and / or sets of markers (e.g., chromosomal regions provided in Tables 1, 7, and / or 8 with annotations) that are able to classify Barrett’s esophagus (BE) and controls (e.g., normal tissue of each tissue type) in esophageal tissue were identified (see Examples 1, 2, and 5).

[0161] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Tables 2, 3, 5, and / or 6) that are able to classify BE and Barrett's esophagus-related dysplasia (BED) in esophageal tissue were identified (see Examples 1, 3, and 4).

[0162] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Table 5) that are able to predict Barrett's esophagus-related low-grade dysplasia (BE-LGD), Barrett's esophagus-related high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by full esophageal swabbing or brushing were identified (see Examples 1 and 4).

[0163] Markers and / or panels of markers (e.g., annotated chromosomal regions provided in Table 5) that are able to classify BE and BED in samples obtained by full esophageal swabbing or brushing were identified (see Examples 1 and 4).

[0164] Methods for analyzing methylation status

[0165] The most common method for analyzing the presence of 5-methylcytosine in nucleic acids is based on the bisulfite method described by Frommer et al. for detecting 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-31) or variations thereof. Bisulfite mapping of 5-methylcytosine is based on the observation that cytosine, but not 5-methylcytosine, reacts with bisulfite ions (also called bisulfite). The reaction usually proceeds in the following steps: first, cytosine reacts with bisulfite to form sulphonated cytosine. Next, the sulphonated reaction intermediate spontaneously deaminates to sulphonated uracil. Finally, sulphonated uracil is desulphonated under alkaline conditions to form uracil. Detection is possible because uracil forms a base pair with adenine (thus behaving like thymine), whereas 5-methylcytosine forms a base pair with guanine (thus behaving like cytosine). This allows the distinction between methylated and unmethylated cytosine by, for example, bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98) or methylation-specific PCR (MSP) (as disclosed in U.S. Pat. No. 5,786,146).

[0166] Some conventional techniques involve methods that include the encapsulation of DNA to be analyzed in an agarose matrix to prevent diffusion and renaturation of the DNA (bisulfite reacts only with single-stranded DNA) and the replacement of the precipitation and purification steps with rapid dialysis (Olek A, et al. (1996) "A modified and improved method for bisulfite based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). Thus the methylation status of a single cell can be analyzed, illustrating the utility and sensitivity of the method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0167] Bisulfite techniques generally involve amplification of short, specific fragments of known nucleic acids after bisulfite treatment, followed by determination of the products by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reactions (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31; WO 95 / 00669; U.S. No. 6,251,594) to analyze individual cytosine positions. Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Detection by hybridization has also been described in the art (Olek et al., WO 99 / 28498). In addition, the use of bisulfite techniques for methylation detection of individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6,; Zeschnigk et al. (1997) Hum Mol Genet. 6:387-95; Feil et al. (1994) Nucleic Acids Res. 22:695; Martin et al. (1995) Gene 157:261-4; WO 9746705; WO 9515373).

[0168] A variety of methylation assay procedures are known in the art and can be used in conjunction with bisulfite treatment according to the present technology. These assays allow for the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include sequencing of bisulfite treated nucleic acids, PCR (for sequence specific amplification), Southern blot analysis, and use of methylation sensitive restriction enzymes.

[0169] For example, the use of bisulfite treatment has simplified genome sequencing for analyzing methylation patterns and 5-methylcytosine distribution (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827–1831). Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA can be used to assess methylation status, as described, for example, in Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058–5059, or as exemplified by a method known as COBRA (Combined Bisulfite Restriction Assay) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532–2534).

[0170] COBRA TM The analysis is a quantitative methylation assay used to determine the level of DNA methylation at specific loci in a small amount of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). In short, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of bisulfite-treated DNA. First, methylation-dependent sequence differences are introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Then, PCR amplification of the bisulfite-converted DNA is performed using primers specific to the target CpG islands, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specifically labeled hybridization probes. The methylation level in the original DNA sample is linearly quantified across the entire spectrum of DNA methylation levels by the relative amounts of digested and undigested PCR products. Furthermore, this technique can be reliably applied to DNA obtained from paraffin-embedded tissue samples obtained through microdissection.

[0171] For COBRA TM Typical reagents for analysis (e.g., those that can be found in typical COBRA-based reagents) TM The kits found may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. Furthermore, bisulfite conversion reagents may include: DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonate buffers; and DNA recovery components.

[0172] Preferably, such as "MethyLight TM " (a fluorescence-based real-time PCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE TM (methylation-sensitive single-nucleotide primer extension) reaction (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP"; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylation CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999), alone or in combination with one or more of these methods.

[0173] "HeavyMethyl TM " assay is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover CpG positions located between or covered by amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0174] The term "HeavyMethyl TM MethyLight TM " assay refers to the HeavyMethyl TM MethyLight TM assay, which is a variant of the MethyLight TM assay, wherein the MethyLight TM assay is combined with methylation-specific blocking probes that cover CpG positions between amplification primers. The HeavyMethyl TM assay can also be used in combination with methylation-specific amplification primers.

[0175] Typical reagents for HeavyMethyl TM analysis (e.g., can be found in a typical MethyLight TM -based kit) can include, but are not limited to: PCR primers for a particular locus (e.g., specific gene, marker, DMR, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, or bisulfite-treated DNA sequence or CpG island); blocking oligonucleotides; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0176] Methylation-specific PCR (MSP) allows for the assessment of the methylation status of groups at virtually any CpG site within a CpG island without relying on the use of methylation-sensitive restriction enzymes (Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146). In short, DNA is modified with sodium bisulfite, which converts unmethylated cytosine, but not methylated cytosine, to uracil. The products are then amplified using primers specific to both methylated and unmethylated DNA. MSP requires only a small amount of DNA, is sensitive to 0.1% methylated alleles at a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents used for MSP analysis (e.g., those found in typical MSP-based kits) may include, but are not limited to: methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; and specific probes.

[0177] MethyLight TM The assay was performed using fluorescence-based real-time PCR (e.g., MethyLight offers high-throughput quantitative methylation assays that require no further steps after PCR (Eads et al., Cancer Res. 59:2302-2306, 1999). In short, MethyLight... TM The method begins with a mixed sample of genomic DNA, which is converted into a mixed library of methylation-dependent sequence differences in a sodium bisulfite reaction according to a standard procedure (a bisulfite method for converting unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example using PCR primers that overlap with known CpG dinucleotides. Sequence identification occurs at both the amplification and fluorescence detection levels.

[0178] MethyLight TM This quantitative assay determines methylation patterns in nucleic acid samples such as genomic DNA, where sequence identification occurs at the probe hybridization level. In the quantitative version, the PCR reaction provides methylation-specific amplification in the presence of a fluorescent probe that overlaps with a specific putative methylation site. An unbiased control is provided by a reaction in which neither the primers nor the probes cover any CpG dinucleotides. Alternatively, an unbiased control is provided by using a control oligonucleotide that does not cover known methylation sites (e.g., HeavyMethyl). TMand MSP technology) or qualitative testing of genomic methylation with biased PCR libraries probed with oligonucleotides covering potential methylation sites.

[0179] MethyLight TM Methods are used with any suitable probes (e.g. probes, probes, etc.). For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to two sets of PCR reactions using probes, such as the MSP primers and / or HeavyMethyl blocker oligonucleotides and probes. Probes are dual-labeled with fluorescent "reporter" and "quencher" molecules and designed to be specific for regions of relatively high GC content, such that they melt at a temperature about 10°C higher than the forward or reverse primers during PCR cycling. This allows the probes to remain fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed probes. The Taq polymerase 5' to 3' endonuclease activity will displace the probes by digesting them, releasing the fluorescent reporter molecule to replace the probes, which are now no longer quenched, for quantitative detection of their now unquenched signal using real-time fluorescence detection systems.

[0180] For MethyLight TM Typical reagents for MethyLight TM analysis (e.g., can be found in a typical MethyLight-based kit) can include, but are not limited to: PCR primers for a particular locus (e.g., specific genes, markers, DMRs, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); or probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0181] QM TM The QM (quantitative methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, where sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides unbiased amplification in the presence of a fluorescent probe that overlaps with a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primer nor the probe covers any CpG dinucleotides. Alternatively, an unbiased control is provided by using a control oligonucleotide (e.g., HeavyMethyl TMQualitative testing of genomic methylation can be achieved using either a fluorescence-based version of MSP technology or a biased PCR library of oligonucleotide probes covering potential methylation sites.

[0182] During the amplification process, QM TM The method can be used with any suitable probe (e.g. probe, Probes are used together. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and probes. Probe. The probe is doubly labeled with both a fluorescent reporter and a quencher molecule and is designed to be specific for regions with relatively high GC content, allowing it to denature during PCR cycles at a temperature approximately 10°C higher than that of the forward or reverse primers. This enables... The probe remains fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, these strands eventually reach the annealed state. Probe. Taq polymerase 5' to 3' endonuclease activity will be detected through digestion. The probe releases fluorescent reporter molecules to replace Probes are used to quantitatively detect signals that have not yet faded using a real-time fluorescence detection system. For QM TM Typical reagents for analysis (e.g., those that can be found in typical QM-based systems) TM (As found in the kits) may include, but is not limited to: PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); or Probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0183] Ms-SNuPE TM The technique is a quantitative method based on the bisulfite treatment of DNA followed by single nucleotide primer extension to assess differential methylation at specific CpG sites (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). In short, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil, while retaining 5-methylcytosine. The desired target sequence is then amplified using PCR primers specific to the bisulfite-converted DNA. The resulting product is isolated and used as a template for methylation analysis at the target CpG site. Small amounts of DNA (e.g., microdissected pathological sections) can be analyzed, and the use of restriction enzymes to determine the methylation status of CpG sites is avoided.

[0184] For Ms-SNuPETM Typical reagents for analysis (e.g., those available in typical Ms-SNuPE-based solutions) TM The kits found may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; and Ms-SNuPE for specific loci. TM Primers; reaction buffer (for Ms-SNuPE reaction); and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column); desulfonate buffer; and DNA recovery components.

[0185] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosine into uracil, followed by restriction enzyme digestion (e.g., by enzymes such as MspI that identify sites containing CG sequences) and complete sequencing of the fragment after coupling to an adaptor ligand. Restriction enzyme selection enriches fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during analysis. Therefore, RRBS reduces the complexity of nucleic acid samples by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole-genome bisulfite sequencing, each fragment produced by restriction enzyme digestion contains DNA methylation information for at least one CpG dinucleotide. Therefore, RRBS enriches the sample's promoters, CpG islands, and other genomic features with high-frequency restriction enzyme cleavage sites in these regions, thus providing a assay for assessing the methylation status of one or more genomic loci.

[0186] A typical protocol for RRBS includes the steps of digesting the nucleic acid sample with a restriction enzyme such as Mspl, filling in the overhangs and A-tailing, ligating adapters, bisulfite conversion and PCR. See, e.g., Bibikova et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7: 133-6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33: 5868-77.

[0187] In some embodiments, the methylation status is assessed using a quantitative allele-specific real-time target and signal amplification (QuARTS) assay. Three reactions occur sequentially in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in a primary reaction; and FRET cleavage and fluorescent signal generation (reaction 3) in a secondary reaction. As the target nucleic acid is amplified with specific primers, a specific detection probe with a flanking sequence is loosely bound to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site causes cleavage between the detection probe and the flanking sequence, resulting in the release of the flanking sequence by a cleaving enzyme. The flanking sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flanking sequence acts as an invasive oligonucleotide on the FRET cassette and enables cleavage between the FRET cassette fluorophore and quencher, generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thus multiple fluorophores are released per flanking sequence, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199; U.S. Patent Application Serial Nos. 12 / 946,737, 12 / 946,745, 12 / 946,752 and 61 / 548,639.

[0188] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite or a combination thereof, which is used to distinguish between methylated and unmethylated CpG dinucleotide sequences as disclosed herein. The method of the treatment is known in the art (e.g. PCT / EP2004 / 011715). Preferably, the bisulfite treatment is performed in the presence of a denaturing solvent such as, but not limited to, n-alkyl glycols or diethylene glycol dimethyl ether (DME) or in the presence of dioxane or dioxane derivatives. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is performed in the presence of a scavenger such as, but not limited to, a chromane derivative such as 6-hydroxy-2,5,7,8,-tetramethylchromane 2-carboxylic acid or trihydroxybenzoic acid and derivatives thereof such as gallic acid (see PCT / EP2004 / 011715). The bisulfite conversion is preferably performed at a reaction temperature of 30°C to 70°C, whereby the temperature is increased to more than 85°C for a short time during the reaction (see PCT / EP2004 / 011715). The bisulfite treated DNA is preferably purified prior to quantification. This can be performed by any means known in the art such as, but not limited to, ultrafiltration, e.g. by Microcon columns (manufactured by Millipore TM TM

[0189] In some embodiments, fragments of the treated DNA are amplified using primer oligonucleotide sets according to the application (see e.g. Tables 4 and 9) and amplification enzymes. Amplification of multiple DNA fragments can be performed simultaneously in the same reaction vessel. Typically, amplification is performed using the polymerase chain reaction (PCR). The length of the amplicons is typically 100 to 2000 base pairs.

[0190] In another embodiment of the method, the methylation status of the CpG positions within or in the vicinity of the marker comprising a DMR (e.g. DMRs 1-229 provided in Tables 1, 2, 3, 5, 6, 7, 8) can be detected by using methylation specific primer oligonucleotides. This technique (MSP) has been described in U.S. Patent No. 6,265,171 to Herman. Amplification of bisulfite treated DNA using methylation status specific primers allows to distinguish between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer which hybridizes to a bisulfite treated CpG dinucleotide. Thus, the sequence of the primer comprises at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the position of the C position in the CpG.​​

[0191] The fragments obtained by amplification can carry a directly or indirectly detectable label. In some embodiments, the label is a fluorescent label, a radionuclide, or a separable molecular fragment with a characteristic mass that is detectable in a mass spectrometer. When the label is a mass label, some embodiments provide for the labeled amplicon to have a single positive or negative net charge, allowing for better detectability in a mass spectrometer. Detection and visualization can be performed by, for example, matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electrospray mass spectrometry (ESI).

[0192] Methods of isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments include isolating nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids").

[0193] Methods

[0194] In some embodiments of the present technology, methods are provided that include the following steps:

[0195] 1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from a bodily fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) obtained from a subject with at least one reagent or series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker comprising a DMR (e.g., DMRs 1-78 provided in Table 1; DMRs 21, 188-193 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, 193-229 provided in Table 8) and

[0196] 2) detecting the absence of Barrett's esophagus (e.g., with a sensitivity greater than or equal to 80% and a specificity greater than or equal to 80%).

[0197] In some embodiments of the present technology, methods are provided that include the following steps:

[0198] 1) contacting nucleic acid (e.g., genomic DNA, e.g., isolated from a bodily fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides in at least one marker comprising a DMR (e.g., DMRs 1-78 provided in Table 1; DMRs 21, 188-193 provided in Table 7; DMRs 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, 193-229 provided in Table 8) and

[0199] 2) detecting the presence of Barrett’s esophagus (e.g., with a sensitivity greater than or equal to 80% and a specificity greater than or equal to 80%).

[0200] In some embodiments of the present technology, methods are provided comprising the following steps:

[0201] 1) contacting nucleic acid (e.g., genomic DNA, e.g., isolated from a bodily fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides in at least one marker comprising a DMR (e.g., DMR No. 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMR No. 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMR No. 77, 90, and 135 provided in Table 5; and DMR No. 136-187 provided in Table 6) and

[0202] 2) classifying as Barrett’s esophagus or a dysplasia in Barrett’s esophagus (e.g., with a sensitivity greater than or equal to 80% and a specificity greater than or equal to 80%).

[0203] In some embodiments of the present technology, methods are provided comprising the following steps:

[0204] 1) contacting nucleic acid (e.g., genomic DNA, e.g., isolated from a bodily fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides in at least one marker comprising a DMR (e.g., DMR No. 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMR No. 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMR No. 77, 90, and 135 provided in Table 5; and DMR No. 136-187 provided in Table 6) and

[0205] 2) classified as Barrett's esophagus or Barrett's esophagus dysplasia (e.g., with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).

[0206] In some embodiments of the technology, methods are provided that comprise the following steps:

[0207] 1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from esophageal tissue (e.g., esophageal tissue obtained by full esophageal swabbing or brushing)) obtained from a subject with at least one reagent or series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker comprising a DMR (e.g., DMR No. 77, 90, and 135 provided in Table 5) and

[0208] 2) classified as Barrett's esophagus low-grade dysplasia, Barrett's esophagus high-grade dysplasia, or esophageal adenocarcinoma (e.g., with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).

[0209] Preferably, the sensitivity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%. Preferably, the specificity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%.

[0210] Genomic DNA can be isolated by any means, including using commercially available kits. Briefly, where the DNA of interest is encapsulated by a cell membrane, the biological sample must be disrupted and lysed by enzymatic, chemical, or mechanical means. Proteins and other contaminants can then be removed from the DNA solution, for example, by digestion with proteinase K. The genomic DNA is then recovered from solution. This can be done by a variety of methods, including salt precipitation, organic extraction, or binding of the DNA to a solid support. The choice of method is influenced by a number of factors, including time, expense, and the amount of DNA required. All clinical sample types containing tumor material or pre-tumor material are suitable for use in the present methods, such as cell lines, histological sections, biopsies, paraffin-embedded tissues, body fluids, fecal matter, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0211] In some embodiments where the sample comprises esophageal tissue, the sample is obtained by endoscopic techniques.

[0212] In some embodiments where the sample comprises esophageal tissue, the sample is obtained by endoscopic brushing or non-endoscopic full esophageal brushing or swabbing using a tethered device (e.g., a capsule sponge, a balloon, or other device).

[0213] The present technology is not limited to methods for preparing samples and providing nucleic acids for testing. For example, in some embodiments, DNA is isolated from a stool sample or from blood or from a plasma sample using direct gene capture (e.g., as described in U.S. Patent Application Serial No. 61 / 485386) or related methods.

[0214] The genomic DNA sample is then treated with at least one reagent or series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker containing a DMR (e.g., DMRs 1-229, e.g., provided in Tables 1, 2, 3, 5, 6, 7, 8).

[0215] In some embodiments, the reagent will convert cytosine bases that are unmethylated at the 5'-position to uracil, thymine, or another base that is not similar to cytosine in terms of hybridization behavior. However, in some embodiments, the reagent can be a methylation-sensitive restriction enzyme.

[0216] In some embodiments, the genomic DNA sample is treated in such a way that cytosine bases that are unmethylated at the 5'-position are converted to uracil, thymine, or another base that is not similar to cytosine in terms of hybridization behavior. In some embodiments, this treatment is performed with a bisulfite salt (bisulfite, disulfite), followed by alkaline hydrolysis.

[0217] The treated nucleic acid is then analyzed to determine the methylation status of the target gene sequence (from at least one gene, genomic sequence, or nucleotide from a marker comprising a DMR, e.g., at least one DMR selected from DMRs 1-229, e.g., provided in Tables 1, 2, 3, 5, 6, 7, 8). The method of analysis can be selected from methods known in the art, including those listed herein, such as QuARTS and MSP as described herein.

[0218] The present technology relates to the analysis of any sample associated with esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC). For example, in some embodiments, the sample comprises tissue and / or a biological fluid obtained from a patient. In some embodiments, the sample comprises esophageal tissue. In some embodiments, the sample comprises esophageal tissue obtained by full esophageal swabbing or brushing. In some embodiments, the sample comprises secretions. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, gastrointestinal biopsy samples, microdissected cells from esophageal biopsies, esophageal cells sloughed into the gastrointestinal lumen, and / or esophageal cells recovered from stool. In some embodiments, the subject is a human. These samples can be from the upper gastrointestinal tract, the lower gastrointestinal tract, or comprise cells, tissues, and / or secretions from both the upper and lower gastrointestinal tract. The sample can comprise cells, secretions, or tissues from the liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gall bladder, anus, and / or peritoneum. In some embodiments, the sample comprises a cell fluid, ascites, urine, stool, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a stool sample.

[0219] Such samples can be obtained by any number of methods known in the art, such as would be apparent to one of skill in the art. For example, urine and stool samples are readily obtained, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using a needle and syringe. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to one of skill in the art, including but not limited to centrifugation and filtration. While it is generally preferred that the sample be obtained without the use of invasive techniques, it can still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. In some embodiments, the sample is obtained by esophageal swabbing or brushing or using a sponge capsule device.

[0220] In some embodiments, the present technology relates to methods of treating a patient (e.g., a patient having BE, BED, BE-LGD, BE-HGD, and / or EAC), the method comprising determining the methylation status of one or more DMRs provided herein, and administering a treatment to the patient based on the results of determining the methylation status. The treatment can be administering a pharmaceutical compound, a vaccine, performing a surgery, imaging the patient, performing another test. Preferably, the use is a method of clinical screening, a method of prognostic assessment, a method of monitoring treatment outcome, a method of identifying a patient most likely to respond to a particular therapeutic treatment, a method of imaging a patient or subject, and a method of drug screening and development.

[0221] In some embodiments of the present technology, methods are provided for diagnosing esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject. As used herein, the terms "making a diagnosis" and "diagnosis" refer to methods by which one of skill in the art can estimate or even determine whether a subject has a given disease or condition or is likely to develop a given disease or condition in the future. One of skill in the art typically makes a diagnosis based on one or more diagnostic indicators, such as a biomarker (e.g., a DMR disclosed herein), the methylation status of which is indicative of the presence, severity, or absence of a condition.

[0222] With diagnosis, clinical cancer prognosis (e.g., for BED, BE-LGD, BE-HGD, EAC) involves determining the aggressiveness of the cancer and the likelihood of tumor recurrence to plan the most effective treatment. If a more accurate prognosis can be made, or even the potential risk of developing cancer can be assessed, then appropriate treatment can be selected, and in some cases a less rigorous treatment can be selected for the patient. Assessment of cancer biomarkers (e.g., determining methylation status) can be used to separate subjects who will not require treatment or require limited treatment, who have a good prognosis and / or low risk of developing cancer, from subjects who are more likely to develop cancer or suffer from cancer recurrence, who would benefit from a more intensive treatment.

[0223] Accordingly, as used herein, "making a diagnosis" or "diagnosis" also includes determining the risk of developing cancer or determining a prognosis, which can provide for predicting clinical outcome (with or without clinical treatment), selecting an appropriate treatment (or whether a treatment is effective), or monitoring a current treatment and potentially changing treatment, based on the measurement of diagnostic biomarkers (e.g., DMRs) provided herein. Further, in some embodiments of the presently disclosed subject matter, multiple measurements of a biomarker over time can be made to aid in diagnosis and / or prognosis. Changes in a biomarker over time can be used to predict clinical outcome, monitor progression of esophageal disease, and / or monitor the efficacy of appropriate treatment for cancer. In such embodiments, for example, changes in the methylation status of one or more biomarkers disclosed herein (e.g., DMRs) (and potentially one or more additional biomarkers, if monitored) in a biological sample over time would be expected to be seen during the course of effective treatment.

[0224] The presently disclosed subject matter further provides, in some embodiments, methods for determining whether to initiate or continue prevention or treatment of esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject. In some embodiments, the methods comprise providing a series of biological samples from the subject over time; analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each biological sample; and comparing any measurable changes in the methylation status of one or more biomarkers in each biological sample. Any changes in the methylation status of the biomarkers over this period of time can be used to predict the risk of developing esophageal disease, predict clinical outcomes, determine whether to initiate or continue prevention or treatment of cancer, and whether the current treatment is effectively treating the esophageal disease. For example, a first time point can be selected prior to initiation of treatment, and a second time point can be selected at some time after initiation of treatment. The methylation status can be measured in each sample taken from the different time points, and qualitative and / or quantitative differences recorded. Changes in the methylation status of biomarker levels from different samples can be correlated to the subject’s risk of esophageal disease, prognosis, determination of treatment efficacy, and / or progression of esophageal disease.

[0225] In preferred embodiments, the methods and compositions of the present application are used to treat or diagnose a disease at an early stage, e.g., prior to the appearance of symptoms of the disease. In some embodiments, the methods and compositions of the present application are used to treat or diagnose a disease at a clinical stage.

[0226] As described above, in some embodiments, a multiplexed assay of one or more diagnostic or prognostic biomarkers can be performed, and temporal changes in the markers can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined at an initial time, and the diagnostic marker again determined at a second time. In such embodiments, an increase in the marker from the initial time to the second time can diagnose a particular type or severity of esophageal disease, or a given prognosis. Likewise, a decrease in the marker from the initial time to the second time can indicate a particular type or severity of esophageal disease, or a given prognosis. Further, the degree of change in one or more markers can be correlated to the severity of esophageal disease and future adverse events. Those skilled in the art will appreciate that while in certain embodiments, a comparative measurement of the same biomarker can be made at multiple time points, a given biomarker can be measured at one time point, and a second biomarker measured at a second time point, and a comparison of these markers can provide diagnostic information.

[0227] As used herein, the phrase "determining prognosis" refers to a method by which one skilled in the art can predict the course or outcome of a disorder in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a disorder with 100% accuracy, or even that a given course or outcome is more or less likely to occur based on the methylation status of a biomarker (e.g., a DMR). Rather, one skilled in the art will appreciate that the term "prognosis" refers to an increased likelihood of a certain course or outcome; that is, a course or outcome is more likely to occur in a subject exhibiting a given disorder when compared to an individual not exhibiting the disorder. For example, the likelihood of a given outcome (e.g., having esophageal disease) can be very low in an individual not exhibiting the disorder (e.g., having normal methylation status of one or more DMRs).

[0228] In some embodiments, statistical analysis correlates a prognostic indicator with a propensity for an adverse outcome. For example, in some embodiments, a methylation status that is different from the methylation status obtained in a normal control sample from a patient not having esophageal disease can indicate that a subject is more likely to have esophageal disease than a subject having a methylation status more similar to the methylation status in the control sample, as determined by a level of statistical significance. Further, a change in methylation status from a baseline (e.g., "normal") level can reflect the prognosis of a subject, and the degree of change in methylation status can correlate with the severity of an adverse event. Statistical significance is generally determined by comparing two or more populations and determining confidence intervals and / or p-values (see, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983). Exemplary confidence intervals for the subject matter of the present invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, while exemplary p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0229] In other embodiments, a threshold degree of change in methylation status of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the degree of change in methylation status of the biomarker in the biological sample is simply compared to the threshold degree of change in methylation status. Preferred threshold changes in methylation status of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In other embodiments, a "nomogram" can be established by which the methylation status of a prognostic or diagnostic indicator (a biomarker or combination of biomarkers) is directly related to the relative propensity for a given outcome. Those skilled in the art are familiar with using such nomograms to correlate two numerical values, and recognize that the uncertainty of this measurement is the same as the uncertainty of the marker concentration, as a single sample measurement is referenced rather than a population average.

[0230] In some embodiments, a control sample is analyzed simultaneously with the biological sample, such that the results obtained from the biological sample can be compared to the results obtained from the control sample. Further, it is contemplated that a standard curve can be provided, with which the assay results of the biological sample can be compared. If fluorescent labels are used, such a standard curve presents the methylation status of the biomarker as a function of the assay units (e.g., fluorescent signal intensity). Using samples taken from a plurality of donors, a standard curve can be provided of the control methylation status of one or more biomarkers in normal tissue, as well as the "risk level" of one or more biomarkers in tissue taken from a donor with metaplasia or from a donor with esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC). In certain embodiments of this method, upon identification of an abnormal methylation status of one or more DMRs provided herein in a biological sample obtained from a subject, the subject is identified as having esophageal disease. In other embodiments of this method, detection of an abnormal methylation status of one or more such biomarkers in a biological sample obtained from a subject results in the subject being identified as having esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC).

[0231] Analysis of the markers can be performed individually within one test sample or simultaneously with additional markers. For example, multiple markers can be combined in one test for efficient processing of multiple samples and potentially to provide better diagnostic and / or prognostic accuracy. In addition, one of skill in the art will recognize the value of testing values from multiple samples from the same subject (e.g., at successive time points). Such testing of a series of samples can allow identification of changes in the methylation status of the markers over time. Changes in methylation status, as well as the absence of changes in methylation status, can provide useful information about the disease state, including but not limited to determining the approximate time of onset of an event, the presence and amount of salvageable tissue, the appropriateness of a drug therapy, the effectiveness of various therapies, the identification of a subject's outcome, including the risk of future events.

[0232] Analysis of the biomarkers can be performed in a variety of physical formats. For example, microtiter plates or automation can be used to facilitate processing of large numbers of test samples. Alternatively, single sample formats can be developed to facilitate timely point-of-care treatment and diagnosis, for example in ambulatory transport or emergency room settings.

[0233] In some embodiments, the subject is diagnosed as having an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) if the methylation status of at least one biomarker in the sample has a measurable change compared to a control methylation status. Conversely, a subject can be identified as not having an esophageal disease, not being at risk for an esophageal disease, or being at low risk for an esophageal disease when no change in methylation status is identified in the biological sample. In this regard, a subject having an esophageal disease or risk thereof can be distinguished from a subject having low to substantially no esophageal disease or risk thereof. A subject at risk of developing an esophageal disease can be placed in a more intensive and / or regular screening regimen, including endoscopic surveillance. On the other hand, a subject at low to substantially no risk can avoid endoscopy or esophageal brushing until a future screening time, for example a screening performed according to the present technology indicates that these subjects have developed a risk of an esophageal disease.

[0234] As noted above, according to embodiments of the methods of the technology, detecting a change in the methylation status of one or more biomarkers can be a qualitative assay or a quantitative assay. Thus, the step of diagnosing a subject as having, or at risk of developing, an esophageal disease indicates that certain threshold measurements are made, e.g., a change in the methylation status of one or more biomarkers in the biological sample from a predetermined control methylation status. In some embodiments of the methods, the control methylation status is any detectable methylation status of the biomarker. In other embodiments of the methods in which a control sample is tested simultaneously with the biological sample, the predetermined methylation status is the methylation status in the control sample. In still other embodiments of the methods, the predetermined methylation status is identified based on, and / or by, a standard curve. In still other embodiments of the methods, the predetermined methylation status is a specific status or range of statuses. Thus, the predetermined methylation status can be selected within the limits apparent to those skilled in the art, based in part on the embodiment of the method being practiced and the desired specificity, etc.

[0235] Further, with respect to the diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrate is warm-blooded; the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term "subject" includes human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, the technology provides for diagnosis of mammals, e.g., humans, and mammals that are important due to being endangered, e.g., Siberian tigers; mammals that are economically important, e.g., farm-raised animals for human consumption; and / or animals that are socially important to humans, e.g., animals kept as pets or in zoos. Examples of such animals include, but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates, such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, diagnosis and treatment of livestock, including but not limited to domesticated swine, ruminants, ungulates, horses (including racehorses), etc. are also provided. The subject matter of the present disclosure also includes systems for diagnosing an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject. The system can be provided, e.g., as a commercial kit, which can be used to screen for risk of, or diagnose, an esophageal disease in a subject for which a biological sample has been collected. Exemplary systems provided according to the technology include assessing the methylation status of a DMR, such as a DMR provided in Tables 1, 2, 3, 5, 6, 7, and / or 8. DETAILED DESCRIPTION

[0236] The present invention is also directed to the following embodiments:

[0237] 1. A method of screening for Barrett's esophagus in a sample obtained from a subject, the method comprising:

[0238] a) determining the methylation status of a marker in a sample obtained from a subject; and

[0239] b) identifying the subject as having Barrett's esophagus when the methylation status of the marker is different than the methylation status of the marker determined in a subject without Barrett's esophagus or a subject without a Barrett's esophagus dysplasia, wherein the marker comprises a base in a differentially methylated region (DMR) selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMOl, CD1D, CDKN2A, and OPLAH.

[0240] 2. The method of embodiment 1, wherein the sample comprises esophageal tissue.

[0241] 3. The method of embodiment 1, wherein the sample comprises esophageal tissue obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0242] 4. The method of embodiment 1, wherein the methylation status of the marker comprises elevated methylation of the marker relative to the normal methylation status of the marker.

[0243] 5. The method of embodiment 1, wherein the methylation status of the marker comprises a different methylation pattern of the marker relative to the normal methylation status of the marker.

[0244] 6. The method of embodiment 1, wherein the determining comprises using methylation specific oligonucleotides.

[0245] 7. The method of embodiment 1, wherein the determining utilizes methylation specific polymerase chain reaction.

[0246] 8. The method of embodiment 1, wherein the determining utilizes nucleic acid sequencing.

[0247] 9. The method of embodiment 1, wherein the determining utilizes mass spectrometry.

[0248] 10. The method of embodiment 1, wherein the determining utilizes methylation specific nucleases.

[0249] 11. The method of embodiment 1, wherein the determining comprises using methylation specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nucleases, mass based separation, or target capture.

[0250] 12. A method for screening for Barrett's esophagus in a sample obtained from a subject, the method comprising:

[0251] a) determining the methylation status of a marker in the sample, the marker comprising a base in a DMR selected from BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMOl, CD1D, CDKN2A, and OPLAH;

[0252] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a normal control sample from a subject who does not have Barrett's esophagus or a dysplastic lesion of Barrett's esophagus;

[0253] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0254] 13. The method of embodiment 12, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0255] 14. The method of embodiment 12, wherein the sample comprises esophageal tissue.

[0256] 15. The method of embodiment 12, wherein the sample comprises esophageal tissue obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0257] 16. A method for screening for Barrett's esophagus in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMOl, CD1D, CDKN2A, and OPLAH) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0258] a) a subject who does not have Barrett's esophagus to identify differences in the two sequences, and

[0259] b) a subject who does not have a dysplastic lesion of Barrett's esophagus to identify differences in the two sequences; and

[0260] when the difference in a) and b) is present, the subject is identified as having Barrett's esophagus.

[0261] 17. The method of embodiment 16, wherein the sample comprises esophageal tissue.

[0262] 18. The method of embodiment 16, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0263] 19. A system for screening for Barrett's esophagus in a sample obtained from a subject, the system comprising: an analysis component configured to determine the methylation status of a sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to determine a single value based on the combination of methylation status and alert a user of Barrett's esophagus related methylation status.

[0264] 20. The system of embodiment 19, wherein the sample comprises nucleic acids comprising a DMR selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMOl, CD1D, CDKN2A, and OPLAH.

[0265] 21. The system of embodiment 19, further comprising a component for isolating nucleic acids.

[0266] 22. The system of embodiment 19, further comprising a component for collecting a sample.

[0267] 23. The system of embodiment 19, wherein the sample comprises esophageal tissue.

[0268] 24. The system of embodiment 19, wherein the database comprises nucleic acid sequences comprising DMRs.

[0269] 25. The system of embodiment 19, wherein the database comprises nucleic acid sequences from subjects having Barrett's esophagus, from subjects not having Barrett's esophagus, and from subjects not having a developmental abnormality of Barrett's esophagus.

[0270] 26. A method for detecting Barrett's esophagus in a sample obtained from a subject, the method comprising:

[0271] a) obtaining a sample comprising DNA from a subject;

[0272] b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0273] c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise a base in a differentially methylated region (DMR) selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMOl, CD1D, CDKN2A and OPLAH,

[0274] d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of the methylation level of the one or more DNA methylation markers in a subject:

[0275] i) a subject not having Barrett's esophagus to identify differences in the two sequences, and

[0276] ii) a subject not having a Barrett's esophagus development abnormality to identify differences in the two sequences

[0277] e) identifying the subject as having Barrett's esophagus when the differences in i) and ii) are present.

[0278] 27. The method of embodiment 26, wherein the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from the group consisting of a CpG island and a CpG island shore.

[0279] 28. The method of embodiment 27, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0280] 29. The method of embodiment 26, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) comprises determining a methylation score and / or a methylation frequency of the one or more DNA methylation markers.

[0281] 30. The method of embodiment 26, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0282] 31. The method of embodiment 26, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0283] 32. The method of embodiment 26, wherein the sample comprises esophageal tissue.

[0284] 33. The method of embodiment 26, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0285] 34. A method of screening for Barrett’s esophagus in a sample obtained from a subject, the method comprising:

[0286] a) determining the methylation status of a marker in a sample obtained from a subject; and

[0287] b) identifying the subject as

[0288] i) having Barrett’s esophagus when the methylation status of the marker is different than the methylation status of the marker determined in a subject without Barrett’s esophagus or a subject without a developmental abnormality of Barrett’s esophagus, wherein the marker comprises a base in a differentially methylated region (DMR) selected from DMRs 1-78, 80, 82-86, 88, 90-102, 108, 122, 133-135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 176, 179, 181, 185-229;

[0289] ii) having a developmental abnormality of Barrett’s esophagus when the methylation status of the marker is different than the methylation status of the marker determined in a subject with Barrett’s esophagus or a subject without a developmental abnormality of Barrett’s esophagus, wherein the marker comprises a base in a DMR selected from DMRs 3, 5, 30, 33, 43, 58, 77, 79-187;

[0290] iii) having a low-grade developmental abnormality of Barrett’s esophagus when the methylation status of the marker is different than the methylation status of the marker determined in a subject without a low-grade developmental abnormality of Barrett’s esophagus, a subject without a developmental abnormality of Barrett’s esophagus, a subject with a high-grade developmental abnormality of Barrett’s esophagus, and a subject with esophageal adenocarcinoma, wherein the marker comprises a base in a DMR selected from DMRs 77, 90, and 135;

[0291] iv) identifying the subject as having high-grade dysplasia of Barrett's esophagus when the methylation state of the marker is different from the methylation state of the marker determined in a subject without high-grade dysplasia of Barrett's esophagus, a subject without dysplasia of Barrett's esophagus, a subject with low-grade dysplasia of Barrett's esophagus, and a subject with adenocarcinoma of the esophagus, wherein the marker comprises a base in a DMR selected from DMRs 77, 90, and 135; and

[0292] v) identifying the subject as having adenocarcinoma of the esophagus when the methylation state of the marker is different from the methylation state of the marker determined in a subject without adenocarcinoma of the esophagus, a subject without dysplasia of Barrett's esophagus, a subject with high-grade dysplasia of Barrett's esophagus, and a subject with low-grade dysplasia of Barrett's esophagus, wherein the marker comprises a base in a DMR selected from DMRs 77, 90, and 135.

[0293] 35. The method of embodiment 34, wherein the sample comprises esophageal tissue.

[0294] 36. The method of embodiment 34,

[0295] wherein the marker comprises a base in a DMR selected from DMRs 101, 77, 134, 92, 133, 77, 90, 193, and 135,

[0296] wherein the sample comprises esophageal tissue obtained by full esophageal swabbing or brushing,

[0297] identifying the subject as having Barrett's esophagus when the methylation state of the marker is different from the methylation state of the marker determined in a subject without Barrett's esophagus or a subject without dysplasia of Barrett's esophagus.

[0298] 37. The method of embodiment 34,

[0299] wherein the marker comprises a base in a DMR selected from DMRs 92, 133, 134, and 194,

[0300] wherein the sample comprises esophageal tissue obtained by sponge capsule,

[0301] identifying the subject as having Barrett's esophagus when the methylation state of the marker is different from the methylation state of the marker determined in a subject without Barrett's esophagus or a subject without dysplasia of Barrett's esophagus.

[0302] 38. The method of embodiment 34,

[0303] wherein the marker comprises a base in a DMR selected from the group consisting of DMRs 77, 90, and 135,

[0304] wherein the sample comprises esophageal tissue obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0305] 39. The method of embodiment 34, comprising assaying 2-194 markers.

[0306] 40. The method of embodiment 34, wherein assaying the methylation status of the markers in the sample comprises determining the methylation status of one or more bases.

[0307] 41. The method of embodiment 34, wherein the methylation status of the marker comprises elevated methylation of the marker relative to the normal methylation status of the marker.

[0308] 42. The method of embodiment 34, wherein the methylation status of the marker comprises a different methylation pattern of the marker relative to the normal methylation status of the marker.

[0309] 43. The method of embodiment 34, comprising assaying the methylation status of the forward strand or assaying the methylation status of the reverse strand.

[0310] 44. The method of embodiment 34, wherein the marker is a region of 100 or fewer bases.

[0311] 45. The method of embodiment 34, wherein the marker is a region of 500 or fewer bases.

[0312] 46. The method of embodiment 34, wherein the marker is a region of 1000 or fewer bases.

[0313] 47. The method of embodiment 34, wherein the marker is a region of 5000 or fewer bases.

[0314] 48. The method of embodiment 34, wherein the marker is one base.

[0315] 49. The method of embodiment 34, wherein the marker is in a high CpG density promoter.

[0316] 50. The method of embodiment 34, wherein the sample is a fecal sample, a tissue sample, a blood sample, or a urine sample.

[0317] 51. The method of embodiment 50, wherein the sample comprises esophageal tissue.

[0318] 52. The method of embodiment 34, wherein the assay comprises use of methylation specific oligonucleotides.

[0319] 53. The method of embodiment 34, wherein the assay utilizes methylation specific polymerase chain reaction.

[0320] 54. The method of embodiment 34, wherein the assay utilizes nucleic acid sequencing.

[0321] 55. The method of embodiment 34, wherein the assay utilizes mass spectrometry.

[0322] 56. The method of embodiment 34, wherein the assay utilizes methylation specific nucleases.

[0323] 57. The method of embodiment 34, wherein the assay comprises use of methylation specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nucleases, mass based separation, or target capture.

[0324] 58. An oligonucleotide comprising a sequence selected from SEQ ID NOs: 1-50.

[0325] 59. An oligonucleotide comprising a sequence complementary to a chromosomal region having a base in a DMR.

[0326] 60. The method of embodiment 34, wherein a chromosomal region having an annotation provided in Tables 1, 2, 3, 5, 6, 7, and / or 8 comprises a marker.

[0327] 61. The method of embodiment 34, wherein the DMR is from Table 1 and is selected from DMR No. 1-78.

[0328] 62. The method of embodiment 34, wherein the DMR is from Table 2 and is selected from DMR No. 3, 5, 30, 33, 43, 58, 77, and 79-128.

[0329] 63. The method of embodiment 34, wherein the DMR is from Table 3 and is selected from DMR No. 77, 27, 193, 90, 92, 101, and 129-134.

[0330] 64. The method of embodiment 34, wherein the DMR is from Table 5 and is selected from DMR No. 77, 90, and 135.

[0331] 65. The method of embodiment 34, wherein the DMR is from Table 6 and is selected from DMR No. 136-187.

[0332] 66. The method of embodiment 34, wherein the DMRs are from Table 7, and are selected from DMR Nos. 21 and 188-193.

[0333] 67. The method of embodiment 34, wherein the DMRs are from Table 8, and are selected from DMR Nos. 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, 193-229.

[0334] 68. The method of embodiment 34, comprising determining the methylation status of two or more markers.

[0335] 69. A kit comprising:

[0336] 1) a bisulfite reagent; and

[0337] 2) a control nucleic acid comprising a sequence of a DMR selected from DMRs 1-229 from Tables 1, 2, 3, 5, 6, 7, and 8, and having a methylation status associated with a subject without cancer.

[0338] 70. A kit comprising a bisulfite reagent and one or more oligonucleotides comprising a sequence selected from SEQ ID NOs: 1-50.

[0339] 71. A kit comprising:

[0340] 1) a bisulfite reagent; and

[0341] 2) a control nucleic acid comprising a sequence of a DMR selected from DMRs 1-194, and having a methylation status associated with a subject with Barrett’s esophagus, a developmental abnormality of Barrett’s esophagus, a low-grade developmental abnormality of Barrett’s esophagus, a high-grade developmental abnormality of Barrett’s esophagus, and esophageal adenocarcinoma.

[0342] 72. A kit comprising: a sample collector for obtaining a sample from a subject; a reagent for isolating nucleic acids from the sample; a bisulfite reagent; and one or more oligonucleotides comprising a sequence selected from SEQ ID NOs: 1-50.

[0343] 73. A composition comprising a nucleic acid comprising a DMR and a bisulfite reagent.

[0344] 74. A composition comprising a nucleic acid comprising a DMR and one or more oligonucleotides comprising a sequence selected from SEQ ID NOs: 1-50

[0345] 75. A composition comprising a nucleic acid comprising a DMR and a methylation sensitive restriction enzyme.

[0346] 76. A composition comprising a nucleic acid comprising a DMR and a polymerase.

[0347] 77. A method for screening for Barrett’s esophagus in a sample obtained from a subject, the method comprising:

[0348] a) determining the methylation status of a marker in the sample, the marker comprising a base selected from a DMR selected from 1-78, 80, 82-86, 88, 90-102, 108, 122, 133-135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 176, 179, 181, 185-229 from Tables 1, 7, and 8;

[0349] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a normal control sample from a subject without Barrett’s esophagus or a dysplastic Barrett’s esophagus;

[0350] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0351] 78. The method of embodiment 77, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99% and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0352] 79. A method for screening for a dysplastic Barrett’s esophagus in a sample obtained from a subject, the method comprising:

[0353] a) determining the methylation status of a marker in the sample, the marker comprising a base selected from a DMR selected from DMR 3, 5, 30, 33, 43, 58, 77, 82, 83, 27, 193, 90, 92, 101, 129-187 from Tables 2, 3, 5, and 6;

[0354] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a normal control sample of a subject that does not have a Barrett's esophagus developmental abnormality or a Barrett's esophagus;

[0355] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0356] 80. The method of embodiment 46, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0357] 81. A method for screening for a Barrett's esophagus low grade developmental abnormality in a sample obtained from a subject, the method comprising:

[0358] a) determining the methylation status of a marker in the sample, the marker comprising a base selected from a DMR in DMRs 77, 90, and 135 from Table 5;

[0359] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a normal control sample of a subject that does not have a Barrett's esophagus low grade developmental abnormality, a subject that does not have a Barrett's esophagus developmental abnormality, a subject that has a Barrett's esophagus high grade developmental abnormality, and a subject that has an esophageal adenocarcinoma;

[0360] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0361] 82. The method of embodiment 81, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0362] 83. The method of embodiment 81, wherein the sample comprises esophageal tissue.

[0363] 84. The method of embodiment 81, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0364] 85. A method for screening for a Barrett's esophagus high grade developmental abnormality in a sample obtained from a subject, the method comprising:

[0365] a) determining the methylation status of a marker in the sample, the marker comprising a base in a DMR selected from DMRs 77, 90, and 135 from Table 5;

[0366] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a subject without high dysplasia of Barrett's esophagus, a subject without dysplasia of Barrett's esophagus, a subject with low dysplasia of Barrett's esophagus, and a normal control sample from a subject with adenocarcinoma of the esophagus;

[0367] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0368] 86. The method of embodiment 85, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0369] 87. The method of embodiment 85, wherein the sample comprises esophageal tissue.

[0370] 88. The method of embodiment 87, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0371] 89. A method for screening for adenocarcinoma of the esophagus in a sample obtained from a subject, the method comprising:

[0372] a) determining the methylation status of a marker in the sample, the marker comprising a base in a DMR selected from DMRs 77, 90, and 135 from Table 5;

[0373] b) comparing the methylation status of the marker from the subject sample to the methylation status of the marker from a subject without adenocarcinoma of the esophagus, a subject without dysplasia of Barrett's esophagus, a subject with low dysplasia of Barrett's esophagus, and a normal control sample from a subject with high dysplasia of Barrett's esophagus;

[0374] c) determining a confidence interval and / or a p-value for the difference in methylation status of the subject sample and the normal control sample.

[0375] 90. The method of embodiment 89, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0376] 91. The method of embodiment 89, wherein the sample comprises esophageal tissue.

[0377] 92. The method of embodiment 91, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0378] 93. A method for screening for Barrett’s esophagus in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR No. 1-78, 80, 82-86, 88, 90-102, 108, 122, 133-135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 176, 179, 181, 185-229) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0379] a) a subject who does not have Barrett’s esophagus to identify differences in the two sequences, and

[0380] b) a subject who does not have a developmental abnormality of Barrett’s esophagus to identify differences in the two sequences; and

[0381] when the differences in a) and b) are present, identifying the subject as having Barrett’s esophagus.

[0382] 94. The method of embodiment 93, wherein the sample comprises esophageal tissue.

[0383] 95. A method for screening for Barrett's esophagus in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR No. 3, 5, 30, 33, 43, 58, 77, 79-187) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0384] a) a subject having Barrett's esophagus, to identify differences in the two sequences, and

[0385] b) a subject not having a Barrett's esophagus dysplasia, to identify differences in the two sequences; and

[0386] when the differences in a) and b) are present, identifying the subject as having a Barrett's esophagus dysplasia.

[0387] 96. The method of embodiment 95, wherein the sample comprises esophageal tissue.

[0388] 97. A method for screening for Barrett's esophagus low grade dysplasia in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR No. 77, 90, and 135) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0389] a) a subject not having a Barrett's esophagus low grade dysplasia, to identify differences in the two sequences,

[0390] b) a subject not having a Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0391] c) a subject having a Barrett's esophagus high grade dysplasia, to identify differences in the two sequences, and

[0392] d) a subject having an esophageal adenocarcinoma, to identify differences in the two sequences; and

[0393] when the differences in a), b), c), and d) are present, identifying the subject as having a Barrett's esophagus low grade dysplasia.

[0394] 98. The method of embodiment 97, wherein the sample comprises esophageal tissue.

[0395] 99. The method of embodiment 98, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0396] 100. A method for screening for Barrett's esophagus high-grade dysplasia in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR No. 77, 90, and 135) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0397] a) a subject who does not have Barrett's esophagus high-grade dysplasia to identify differences in the two sequences,

[0398] b) a subject who does not have Barrett's esophagus dysplasia to identify differences in the two sequences,

[0399] c) a subject who has Barrett's esophagus low-grade dysplasia to identify differences in the two sequences, and

[0400] d) a subject who has esophageal adenocarcinoma to identify differences in the two sequences; and

[0401] when the differences in a), b), c), and d) are present, identifying the subject as having Barrett's esophagus high-grade dysplasia.

[0402] 101. The method of embodiment 100, wherein the sample comprises esophageal tissue.

[0403] 102. The method of embodiment 101, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0404] 103. A method for screening for esophageal adenocarcinoma in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR No. 77, 90, and 135) with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to a nucleotide sequence of a nucleic acid containing the DMR from:

[0405] a) a subject who does not have esophageal adenocarcinoma to identify differences in the two sequences,

[0406] b) a subject without a developmental abnormality of Barrett's esophagus to identify differences in the two sequences,

[0407] c) a subject with a low developmental abnormality of Barrett's esophagus to identify differences in the two sequences, and

[0408] d) a subject with a high developmental abnormality of Barrett's esophagus to identify differences in the two sequences; and

[0409] when the differences in a), b), c), and d) are present, the subject is identified as having esophageal adenocarcinoma.

[0410] 104. The method of embodiment 103, wherein the sample comprises esophageal tissue.

[0411] 105. The method of embodiment 104, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0412] 106. A system for screening for Barrett's esophagus in a sample obtained from a subject, the system comprising: an analysis component configured to determine the methylation status of the sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to determine a single value based on the combination of methylation status and alert a user of a Barrett's esophagus related methylation status.

[0413] 107. The system of embodiment 106, wherein the sample comprises nucleic acids comprising DMRs.

[0414] 108. The system of embodiment 107, wherein the DMRs are selected from DMR Nos. 1-78, 83, 92, 101, 133, 134, 135, 171, 176, 186, and 188-194.

[0415] 109. The system of embodiment 106, further comprising a component for isolating nucleic acids.

[0416] 110. The system of embodiment 106, further comprising a component for collecting a sample.

[0417] 111. The system of embodiment 106, wherein the sample comprises esophageal tissue.

[0418] 112. The system of embodiment 106, wherein the database comprises nucleic acid sequences comprising DMRs.

[0419] 113. The system of embodiment 106, wherein the database comprises nucleic acid sequences from subjects with Barrett's esophagus, from subjects without Barrett's esophagus, and from subjects without a Barrett's esophagus dysplasia.

[0420] 114. A system for screening for a Barrett's esophagus dysplasia in a sample obtained from a subject, the system comprising: an analysis component configured to determine a methylation status of the sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to determine a single value based on a combination of methylation status and alert a user of a Barrett's esophagus dysplasia related methylation status.

[0421] 115. The system of embodiment 114, wherein the sample comprises a nucleic acid comprising a DMR.

[0422] 116. The system of embodiment 115, wherein the DMR is selected from DMR No. 3, 5, 30, 33, 43, 58, 77, 79-187.

[0423] 117. The system of embodiment 114, further comprising a component for isolating a nucleic acid.

[0424] 118. The system of embodiment 114, further comprising a component for collecting a sample.

[0425] 119. The system of embodiment 114, wherein the sample comprises an esophageal tissue.

[0426] 120. The system of embodiment 114, wherein the database comprises nucleic acid sequences comprising a DMR.

[0427] 121. The system of embodiment 114, wherein the database comprises nucleic acid sequences from subjects with a Barrett's esophagus dysplasia, from subjects without Barrett's esophagus, and from subjects without a Barrett's esophagus dysplasia.

[0428] 122. A system for screening for a Barrett's esophagus low grade dysplasia in a sample obtained from a subject, the system comprising: an analysis component configured to determine a methylation status of the sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to determine a single value based on a combination of methylation status and alert a user of a Barrett's esophagus low grade dysplasia related methylation status.

[0429] 123. The system of embodiment 122, wherein the sample comprises nucleic acids comprising DMRs.

[0430] 124. The system of embodiment 123, wherein the DMRs are selected from DMR No. 77, 90, and 135.

[0431] 125. The system of embodiment 122, further comprising a component for isolating nucleic acids.

[0432] 126. The system of embodiment 122, further comprising a component for collecting a sample.

[0433] 127. The system of embodiment 122, wherein the sample comprises esophageal tissue.

[0434] 128. The system of embodiment 127, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0435] 129. The system of embodiment 122, wherein the database comprises nucleic acid sequences comprising DMRs.

[0436] 130. The system of embodiment 122, wherein the database comprises nucleic acid sequences from subjects with Barrett's esophagus low dysplasia, from subjects without Barrett's esophagus, from subjects without Barrett's esophagus high dysplasia, from subjects without Barrett's esophagus low dysplasia, and from subjects without esophageal adenocarcinoma.

[0437] 131. A system for screening for Barrett's esophagus high dysplasia in a sample obtained from a subject, the system comprising: an analysis component configured to determine a methylation status of the sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and an alert component configured to determine a single value based on a combination of methylation statuses and alert a user of a Barrett's esophagus high dysplasia related methylation status.

[0438] 132. The system of embodiment 131, wherein the sample comprises nucleic acids comprising DMRs.

[0439] 133. The system of embodiment 132, wherein the DMRs are selected from DMR No. 77, 90, and 135.

[0440] 134. The system of embodiment 131, further comprising a component for isolating nucleic acids.

[0441] 135. The system of embodiment 131, further comprising a component for collecting a sample.

[0442] 136. The system of embodiment 131, wherein the sample comprises esophageal tissue.

[0443] 137. The system of embodiment 131, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0444] 138. The system of embodiment 131, wherein the database comprises nucleic acid sequences comprising DMRs.

[0445] 139. The system of embodiment 131, wherein the database comprises nucleic acid sequences from subjects with Barrett’s esophagus high dysplasia, from subjects without Barrett’s esophagus, from subjects without Barrett’s esophagus high dysplasia, from subjects without Barrett’s esophagus low dysplasia, and from subjects without esophageal adenocarcinoma.

[0446] 140. A system for screening for esophageal adenocarcinoma in a sample obtained from a subject, the system comprising: an analysis component configured to determine a methylation status of a sample; a software component configured to compare the methylation status of the sample to control or reference sample methylation status recorded in a database; and a software component configured to determine a single value based on the combination of methylation status and to alert a user of esophageal adenocarcinoma related methylation status.

[0447] 141. The system of embodiment 140, wherein the sample comprises nucleic acid comprising DMRs.

[0448] 142. The system of embodiment 141, wherein the DMRs are selected from DMR Nos. 77, 90, and 135.

[0449] 143. The system of embodiment 140, further comprising a component for isolating nucleic acid.

[0450] 144. The system of embodiment 140, further comprising a component for collecting a sample.

[0451] 145. The system of embodiment 140, wherein the sample comprises esophageal tissue.

[0452] 146. The system of embodiment 145, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0453] 147. The system of embodiment 140, wherein the database comprises nucleic acid sequences comprising DMRs.

[0454] 148. The system of embodiment 140, wherein the database comprises nucleic acid sequences from subjects having esophageal adenocarcinoma, from subjects not having Barrett's esophagus, from subjects not having a Barrett's esophagus high grade dysplasia, from subjects not having a Barrett's esophagus low grade dysplasia, and from subjects not having esophageal adenocarcinoma.

[0455] 149. A set of isolated nucleic acids, each nucleic acid having a sequence comprising a DMR.

[0456] 150. The set of nucleic acids of embodiment 149, wherein each nucleic acid has a sequence from a subject not having Barrett's esophagus, a Barrett's esophagus dysplasia, a Barrett's esophagus low grade dysplasia, a Barrett's esophagus high grade dysplasia, and esophageal adenocarcinoma.

[0457] 151. A system comprising a set of nucleic acids according to embodiment 149 or 150, and a database of nucleic acid sequences associated with the set of nucleic acids.

[0458] 152. The system of embodiment 151, further comprising a bisulfite reagent.

[0459] 153. The system of embodiment 151, further comprising a nucleic acid sequencer.

[0460] 154. A method for detecting Barrett's esophagus in a sample obtained from a subject, the method comprising:

[0461] a) obtaining a sample comprising DNA from a subject;

[0462] b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0463] c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated according to step b), wherein the one or more DNA methylation markers comprises a base in a differentially methylated region (DMR) provided by DMR Nos. 1-78, 80, 82-86, 88, 90-102, 108, 122, 133-135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 176, 179, 181, 185-229,

[0464] d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation levels of the one or more DNA methylation markers in a subject:

[0465] i) a subject not having Barrett's esophagus to identify differences in the two sequences, and

[0466] ii) a subject not having a Barrett's esophagus dysplasia to identify differences in the two sequences

[0467] e) identifying the subject as having a Barrett's esophagus when the differences in i) and ii) are present.

[0468] 155. The method of embodiment 154, wherein the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0469] 156. The method of embodiment 155, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0470] 157. The method of embodiment 154, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated by step b) comprises determining a methylation score and / or a methylation frequency of the one or more DNA methylation markers.

[0471] 158. The method of embodiment 154, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0472] 159. The method of embodiment 154, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated by step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0473] 160. The method of embodiment 154, wherein the sample comprises esophageal tissue.

[0474] 161. A method for detecting a Barrett's esophagus dysplasia in a sample obtained from a subject, the method comprising:

[0475] a) obtaining a sample comprising DNA from a subject;

[0476] b) treating the obtained DNA with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0477] c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in a differentially methylated region (DMR) provided by DMR No. 3, 5, 30, 33, 43, 58, 77, 79-187,

[0478] d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation levels of the one or more DNA methylation markers in:

[0479] i) a subject having Barrett's esophagus, to identify differences in the two sequences, and

[0480] ii) a subject not having a Barrett's esophagus development abnormality, to identify differences in the two sequences; and

[0481] e) identifying the subject as having a Barrett's esophagus development abnormality when the differences in i) and ii) are present.

[0482] 162. The method of embodiment 161, wherein the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0483] 163. The method of embodiment 162, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0484] 164. The method of embodiment 161, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) comprises determining a methylation score and / or a methylation frequency of the one or more DNA methylation markers.

[0485] 165. The method of embodiment 161, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0486] 166. The method of embodiment 161, wherein the determining the level of methylation of one or more DNA methylation markers in the DNA that has been treated in step b) is effected by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0487] 167. The method of embodiment 161, wherein the sample comprises esophageal tissue.

[0488] 168. A method for detecting a Barrett’s esophagus low-grade dysplasia in a sample obtained from a subject, the method comprising:

[0489] a) obtaining a sample comprising DNA from a subject;

[0490] b) treating the obtained DNA with an agent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0491] c) determining the level of methylation of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprises a base in a differentially methylated region (DMR) provided by DMR No. 77, 90, and 135,

[0492] d) comparing the determined level of methylation of the one or more DNA methylation markers to a reference of the level of methylation of the one or more DNA methylation markers in:

[0493] i) a subject that does not have a Barrett’s esophagus low-grade dysplasia, to identify a difference in the two sequences,

[0494] ii) a subject that does not have a Barrett’s esophagus dysplasia, to identify a difference in the two sequences,

[0495] iii) a subject that has a Barrett’s esophagus high-grade dysplasia, to identify a difference in the two sequences, and

[0496] iv) a subject that has an esophageal adenocarcinoma, to identify a difference in the two sequences; and

[0497] e) identifying the subject as having a Barrett’s esophagus low-grade dysplasia when the difference in i), ii), iii, and iv) is present.

[0498] 169. The method of embodiment 168, wherein the determination of elevated methylation in one or more DNA methylation markers comprises determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0499] 170. The method of embodiment 169, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0500] 171. The method of embodiment 168, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) comprises determination of a methylation score and / or a methylation frequency of the one or more DNA methylation markers.

[0501] 172. The method of embodiment 168, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0502] 173. The method of embodiment 168, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0503] 174. The method of embodiment 168, wherein the sample comprises esophageal tissue.

[0504] 175. The method of embodiment 174, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0505] 176. A method for detecting a high-grade dysplasia of Barrett's esophagus in a sample obtained from a subject, the method comprising

[0506] a) obtaining a sample comprising DNA from the subject;

[0507] b) treating the obtained DNA with an agent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0508] c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprise bases in a differentially methylated region (DMR) provided by DMR No. 77, 90, and 135,

[0509] d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of methylation levels of the one or more DNA methylation markers in a subject:

[0510] i) a subject without high-grade dysplasia of Barrett's esophagus, to identify differences in the two sequences,

[0511] ii) a subject without dysplasia of Barrett's esophagus, to identify differences in the two sequences,

[0512] iii) a subject with low-grade dysplasia of Barrett's esophagus, to identify differences in the two sequences, and

[0513] iv) a subject with adenocarcinoma of the esophagus, to identify differences in the two sequences; and

[0514] e) identifying the subject as having high-grade dysplasia of Barrett's esophagus when differences in i), ii), iii and iv) are present.

[0515] 177. The method of embodiment 176, wherein the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0516] 178. The method of embodiment 177, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0517] 179. The method of embodiment 176, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated by step b) comprises determining a methylation score and / or a methylation frequency of the one or more DNA methylation markers.

[0518] 180. The method of embodiment 176, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0519] 181. The method of embodiment 176, wherein the determination of the methylation level of one or more DNA methylation markers in the DNA that has been treated by step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0520] 182. The method of embodiment 176, wherein the sample comprises esophageal tissue.

[0521] 183. The method of embodiment 182, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0522] 184. A method for detecting esophageal adenocarcinoma in a sample obtained from a subject, the method comprising

[0523] a) obtaining a sample comprising DNA from a subject;

[0524] b) treating the obtained DNA with an agent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues, but does not modify methylated cytosine residues;

[0525] c) determining the methylation level of one or more DNA methylation markers in the DNA that has been treated in step b), wherein the one or more DNA methylation markers comprises a base in a differentially methylated region (DMR) provided by DMR No. 77, 90 and 135,

[0526] d) comparing the determined methylation level of the one or more DNA methylation markers to a reference of the methylation level of the one or more DNA methylation markers of:

[0527] i) a subject that does not have esophageal adenocarcinoma, to identify a difference in the two sequences,

[0528] ii) a subject that does not have a Barrett's esophagus developmental abnormality, to identify a difference in the two sequences,

[0529] iii) a subject that has a Barrett's esophagus low grade developmental abnormality, to identify a difference in the two sequences, and

[0530] iv a subject that has a Barrett's esophagus high grade developmental abnormality, to identify a difference in the two sequences; and

[0531] e) identifying the subject as having esophageal adenocarcinoma when a difference in i), ii), iii and iv) is present.

[0532] 185. The method of embodiment 184, wherein the determination of elevated methylation in one or more DNA methylation markers comprises a determination of altered methylation in a region selected from a CpG island and a CpG island shore.

[0533] 186. The method of embodiment 185, wherein the determination of elevated methylation in the CpG island or CpG island shore comprises elevated methylation in a coding region or a regulatory region of the DNA methylation marker.

[0534] 187. The method of embodiment 184, wherein said determining the methylation level of one or more DNA methylation markers in the DNA treated in step b) comprises determining the methylation score and / or the methylation frequency of the one or more DNA methylation markers.

[0535] 188. The method of embodiment 184, wherein the treatment of step b) is accomplished by bisulfite modification of the obtained DNA.

[0536] 189. The method of embodiment 184, wherein said determining the methylation level of one or more DNA methylation markers in the DNA treated in step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0537] 190. The method of embodiment 184, wherein the sample comprises esophageal tissue.

[0538] 191. The method of embodiment 190, wherein the esophageal tissue is obtained by full esophageal swabbing or brushing or using a sponge capsule device.

[0539] Example

[0540] Example I.

[0541] Molecular markers can be helpful for the detection of Barrett's esophagus (BE) and monitoring of BE-related dysplasia (BED) by endoscopic or non-endoscopic methods. Experiments were performed to (1) identify and validate new methylation DNA markers of BE dysplasia, (2) test the feasibility of candidate markers to detect BE dysplasia from full esophageal brushings.

[0542] Full methylome bisulfite sequencing (18 samples per group) of DNA from BE tissue with no dysplasia, with low-grade dysplasia (BE-LGD), with high-grade dysplasia (BE-HGD), or adenocarcinoma (EAC) was used to identify candidate markers to separate BE from normal tissue, and to separate BED from BE without dysplasia.

[0543] Table 1 and Table 7 provide DMR information for such markers identified to separate BE from normal tissue, including chromosome number, gene annotation, and DMR start / end positions (see Example II for materials / methods used to generate Tables 1 and 7).

[0544] Table 2 and Table 6 provide DMR information for such markers identified to separate BED and BE without dysplasia, including chromosome number, gene annotation, and DMR start / end positions (see Example III for materials / methods used to generate Tables 2 and 6). The best candidate markers were validated by methylation-specific PCR assays in independent tissues including BE without dysplasia, BE-LGD, and BE-HGD (30-60 samples per group).

[0545] Informed consented BE patients scheduled for endoscopic BE surveillance or endoscopic evaluation of BE-related cancer were subjected to full esophageal brushing using a high capacity cell brush (Hobbs Medical, Stafford Springs, CT), sampling from the circumference of the cardia by full esophageal length (BE + squamous mucosa) with a string-like device that mimics swallowing. After DNA extraction and bisulfite treatment, methylation of target genes was detected by methylation-specific PCR or quantitative allele-specific real-time target and signal amplification. Marker levels were normalized to beta-actin (marker for total human DNA). Twelve aberrantly methylated genes were identified that best separated BED and BE without dysplasia (e.g., area under the ROC curve 0.86-0.97) (see Table 3). The 12 markers were DIO3, MAX2 0.218, CD1D, T-SPYL5, ZNF568, ST8SIA1, ELMO1, ELOVL2, BMP3, NDRG4, HUNK, and CDKN2A. Table 3 DMR provides information for such markers identified to separate BED and BE without dysplasia in esophageal samples obtained by full esophageal brushing, including chromosome number, gene annotation, and DMR start / end positions. Table 4 provides forward primer and reverse primer information for the DMRs provided in Table 3. Thirty-nine subjects were studied with a median age of 69 years (28-94 years), 74% male, and a median BE length of 4 (1-14) cm; 18 without dysplasia, 21 with dysplasia (9 LGD, 7 HGD, and 5 EAC (4 asymptomatic early stage). A panel of 3 markers (DIO3, MAX2 0.218, NDRG4) (see Table 5) detected 78% of LGD, 71% of HGD, 100% of EAC, and 81% of all dysplasia at 95% specificity (see Figure 1 ) Table 5 provides DMR information for such markers identified to separate LGD, HGD, and EAC, including chromosome number, gene annotation, and DMR start / end positions.

[0546] Table 1. Information for DMRs that separate BE and normal tissue

[0547]

[0548]

[0549]

[0550] Table 2. Information for DMRs that distinguish BE and BED

[0551]

[0552]

[0553]

[0554] Table 3 - Information for DMRs that distinguish BE and BED

[0555]

[0556] Table 4. Primers for DMRs provided in Table 3.

[0557]

[0558]

[0559] Table 5 - Information for DMRs that distinguish LGD, HGD and EAC and that distinguish BE and BED

[0560]

[0561] Table 6 - Information for DMRs that distinguish BE and BED

[0562]

[0563]

[0564]

[0565] Table 7 - Information for DMRs that distinguish BE and normal tissue

[0566]

[0567] Example II.

[0568] This example describes the materials and methods used to generate Table 1 and Table 7.

[0569] Eighteen Barrett's esophagus (BE) and 18 normal esophageal tissue samples were selected from institutional cancer registries at Mayo Clinic Rochester and were reviewed by expert pathologists to confirm correct classification. Normal white blood cell controls were provided by clinicians at the Mayo Biospecimens Linking Investigators and the GIH Cell Signaling Research Clinical Core.

[0570] Library Preparation: Genomic DNA (300 ng) was fragmented with 10 units of Mspl (methyl specific restriction enzyme recognizing CpG containing motifs) to enrich the sample CpG content and eliminate redundant regions of the genome. The digested fragments were end-repaired and A-tailed with 5 units of Klenow Fragment (3'-5' Exo) and ligated to methylated TruSeq adapters (Illumina, San Diego CA) containing barcode sequences overnight (ligating each fragment to its sample ID). Size selection of 160-340 bp fragments (40-220 bp inserts) was performed using Agencourt AMPure XP SPRI beads / buffer (Beckman Coulter, Brea CA). Buffer cutoff was 0.7X-1.1X sample volume of beads / buffer. Final elution volume was 22 uL (EB buffer - Qiagen, Germantown MD); qPCR was used to measure ligation efficiency and fragment quality on small sample aliquots. The samples were subsequently bisulfite converted (twice) using a modified EpiTect protocol (Qiagen). qPCR and regular PCR (PfuTurbo Cx hotstart - Agilent, Santa Clara CA) and subsequent Bioanalyzer 2100 (Agilent) evaluation on converted sample aliquots determined the optimal number of PCR cycles prior to final library amplification. The following conditions were used for the final PCR: 1.) Each 50 uL reaction contained 5 uL 10X buffer, 1.25 uL 10 mM of each deoxyribonucleotide triphosphate (dNTP), 5 uL primer mix (~5 uM), 15 uL template (sample), 1 uL PfuTurbo Cx hotstart, and 22.75 water; temperature and time were 95 C - 5 minutes; 98 C - 30 seconds; 16 cycles of 98 C - 10 seconds, 65 C - 30 seconds, 72 C - 30 seconds, 72 C - 5 minutes, and hold at 4 C. Samples were combined (equimolar) into 4-plex libraries based on a randomized scheme and tested using the following: final size verification using a bioanalyzer, qPCR using phiX standard and adapter specific primers.

[0571] Sequencing and bioinformatics: Samples were loaded onto flow cells according to random lane assignment, with additional lanes reserved for internal assay controls. Sequencing was performed by the Next Generation Sequencing Core at the Mayo Clinic Medical Genome Facility on an Illumina HiSeq 2000. Reads of 101 cycles were unidirectional. Each flow cell lane yielded 100-120 million reads, sufficient for 30-50x median coverage (number of reads per CpG) of the sequenced depth. Standard Illumina pipeline software accessed base calls and sequencing reads generation in fastq format. As previously described, (28) SAAP-RRBS (streamlined analysis and annotation pipeline for reduced representation bisulfite sequencing) was used for sequence alignment and methylation extraction.

[0572] MSP primer design: Primers for the 6 best markers from the sequencing results were designed and ordered (IDT, Coralville IA) to target specifically bisulfite-modified methylation sequences (Table 7). These designs were done by Methprimer software (University of California, San Francisco CA) or MSPPrimer (Johns Hopkins University, Baltimore, MD). Testing and optimization of the assays by qPCR with SYBR Green was performed on dilutions of universal methylated and non-methylated genomic DNA controls.

[0573] Methylation-specific PCR: Quantitative MSP reactions were performed on DNA extracted from independent tissues: 108 BE samples - 36 with high dysplasia, 36 with low dysplasia, 36 without dysplasia, 18 normal esophageal samples, and 36 normal leukocyte samples.

[0574] Statistical analysis: Candidate CpGs were filtered by priori read-depth and variance criteria, significance of the difference in % methylation percentage between cases and controls, and based on the area under the curve (AUC) of the receiver operating characteristic curve and the ratio of target to background.

[0575] For the RRBS discovery phase, the main comparison of interest was the methylation difference between each mapped CpG between the Barrett's cases, esophageal controls, and white blood cell controls. CpG islands were biochemically defined by the observed expected CpG ratio > 0.6.(30) However, for this model, tiles of CpG analysis "differentially methylated regions (DMRs)" were generated based on the distance between the locations of CpG sites on each chromosome. Islands with only a single CpG were excluded. Individual CpG sites were considered for differential analysis only when the total coverage depth for each disease group was > 200 reads (average 10 reads per subject) and the variance in % methylation was > 0 (to exclude uninformative CpGs). The read depth criterion was based on the desired statistical power to detect a 10% difference in % methylation between any two groups with a sample size of 18 individuals per group. Statistical significance was determined by logistic regression of the methylation percentage for each DMR based on the read counts. To account for the variation in read depth across individual subjects, an overdispersed logistic regression model was used, where the dispersion parameter was estimated using the Pearson's chi-squared statistic of the residuals of the fitted model. DMRs were further considered if the % methylation in the benign esophageal and white blood cell controls, and < 1% but > 10% in the Barrett's cases, sorted by their significance level. This resulted in 78 markers (Table 1). All had an AUC greater than 0.90, and a fold change greater than 25.

[0576] For the qMSP validation study of the 6 markers (Table 7), the main result was the area under the receiver operator characteristic curve (AUC) for each marker, as calculated by a logistic regression model of the % methylation copy number for each sample with BE compared to normal esophagus and normal white blood cells. For each marker, the AUC was again > 0.90, and the quantitative difference in the mean values of the candidate gene copy number between cases and controls was at least 50-fold.

[0577] Example III.

[0578] This example describes the materials and methods used to generate Tables 2 and 6.

[0579] Thirty-six tissue samples of Barrett's esophagus with high and low dysplasia (BED) and 18 tissue samples of Barrett's esophagus without dysplasia (BE) were selected from the institutional cancer program at Mayo Clinic Rochester, and were reviewed by expert pathologists to confirm correct classification. Eighteen normal white blood cell controls were provided by clinicians of the Mayo Biospecimens Linking Investigators and the GIH Cell Signaling Research Clinical Core.

[0580] Library Preparation: Genomic DNA (300 ng) was fragmented with 10 units of Mspl (methyl specific restriction enzyme recognizing CpG containing motifs) to enrich the sample CpG content and eliminate redundant regions of the genome. The digested fragments were end-repaired and A-tailed with 5 units of Klenow Fragment (3'-5' Exo) and ligated to methylated TruSeq adapters (Illumina, San Diego CA) containing barcode sequences overnight (ligating each fragment to its sample ID). Size selection of 160-340 bp fragments (40-220 bp inserts) was performed using Agencourt AMPure XP SPRI beads / buffer (Beckman Coulter, Brea CA). Buffer cutoff was 0.7X-1.1X sample volume of beads / buffer. Final elution volume was 22 uL (EB buffer - Qiagen, Germantown MD); qPCR was used to measure ligation efficiency and fragment quality on small sample aliquots. The samples were subsequently bisulfite converted (twice) using a modified EpiTect protocol (Qiagen). qPCR and regular PCR (PfuTurbo Cx hotstart - Agilent, Santa Clara CA) and subsequent Bioanalyzer 2100 (Agilent) evaluation on converted sample aliquots determined the optimal number of PCR cycles prior to final library amplification. The following conditions were used for the final PCR: 1.) Each 50 uL reaction contained 5 uL 10X buffer, 1.25 uL 10 mM of each deoxyribonucleotide triphosphate (dNTP), 5 uL primer mix (~5 uM), 15 uL template (sample), 1 uL PfuTurbo Cx hotstart, and 22.75 water; temperature and time were 95 C - 5 minutes; 98 C - 30 seconds; 16 cycles of 98 C - 10 seconds, 65 C - 30 seconds, 72 C - 30 seconds, 72 C - 5 minutes, and hold at 4 C. Samples were combined (equimolar) into 4-plex libraries based on a randomized scheme and tested using the following: final size verification using a bioanalyzer, qPCR using phiX standard and adapter specific primers.

[0581] Sequencing and bioinformatics: Samples were loaded onto flow cells according to random lane assignment, with additional lanes reserved for internal assay controls. Sequencing was performed by the Next Generation Sequencing Core at the Mayo Clinic Medical Genome Facility on an Illumina HiSeq 2000. Reads of 101 cycles were unidirectional. Each flow cell lane yielded 100-120 million reads, sufficient for 30-50x median coverage (number of reads per CpG) of the sequenced depth. Standard Illumina pipeline software accessed base calls and sequencing reads generation in fastq format. As previously described, (28) SAAP-RRBS (streamlined analysis and annotation pipeline for reduced representation bisulfite sequencing) was used for sequence alignment and methylation extraction.

[0582] MSP primer design: Primers for the 66 best markers from the sequencing results were designed and ordered (IDT, Coralville IA) to target specifically bisulfite-modified methylation sequences (Table 7). These designs were accomplished by the Methprimer software (University of California, San Francisco CA) or MSPPrimer (Johns Hopkins University, Baltimore, MD). Testing and optimization of the assays by qPCR with SYBR Green was performed on dilutions of universal methylated and non-methylated genomic DNA controls.

[0583] Methylation-specific PCR: Quantitative MSP reactions were performed on DNA extracted from independent tissues: 108 BE samples - 36 with high dysplasia, 36 with low dysplasia, 36 without dysplasia, 18 normal esophageal samples, and 36 normal leukocyte samples.

[0584] Statistical analysis: Candidate CpGs were filtered by a priori read depth and variance criteria, significance of the difference in % methylation percentage between cases and controls, and based on the area under the curve (AUC) of the receiver operating characteristic curve and the ratio of target to background.

[0585] For the RRBS discovery phase, the main comparison of interest was the methylation difference between each mapped CpG between the high and low dysplasia Barrett's cases, the non-dysplastic Barrett's controls, and the white blood cell controls. CpG islands were biochemically defined by the observed expected CpG ratio > 0.6.(30) However, for this model, tiles of CpG analysis "differentially methylated regions (DMRs)" were generated based on the distance between the positions of the CpG sites on each chromosome. Islands with only a single CpG were excluded. Individual CpG sites were considered for differential analysis only when the total coverage depth for each disease group was > 200 reads (average 10 reads per subject) and the variance in % methylation was > 0 (to exclude uninformative CpGs). The read depth criterion was based on the desired statistical power to detect a 10% difference in % methylation between any two groups with a sample size of 18 individuals per group. Statistical significance was determined by logistic regression of the methylation percentage for each DMR based on the read counts. To account for the variation in read depth across individual subjects, an overdispersed logistic regression model was used, where the dispersion parameter was estimated using the Pearson's chi-squared statistic of the residuals of the fitted model. DMRs were further considered by their significance level if the % methylation in the benign esophagus and white blood cell controls, and in the Barrett's cases was < 1% but > 10%. This resulted in 57 markers (Table 2). All had an AUC of 0.60 to 0.87, and fold change of 2 to 10. A second sorting of the data was performed, relaxing the island restriction metric, and focusing on the grouping of highly discriminative single CpGs. Batch effects on the white blood cell controls were also removed, which increased the overall coverage. This resulted in 52 additional markers, which had increased fold changes (7-52) and similar AUCs (Table 6).

[0586] For the 66 DMR qMSP validation study, the main result was the area under the receiver operator characteristic curve (AUC) for each marker, as calculated by a logistic regression model of the % methylation copy number for each sample with BED compared to BE and normal white blood cells. Twelve markers exhibited superior performance (Table 3). The AUC was 0.86-0.97, and the fold change was 2-24. Ten of the 12 markers were taken forward with BMP3 and NDRG4 for the esophageal brush feasibility study.

[0587] Example IV.

[0588] This example describes the materials and methods used in producing 3, 4, and 5.

[0589] Informed consented BE patients scheduled for endoscopic BE surveillance or endoscopic evaluation of BE related cancer were subjected to full esophageal brushing using high volume cell brushes (Hobbs Medical, Stafford Springs, CT) to sample from the circumference of the cardia by full esophageal length (BE + squamous mucosa). Cell brushes were removed from the handle and placed into a vial of stabilization / cell lysis solution and frozen until processing. DNA was extracted using the Gentra Puregene Buccal procedure (Qiagen, Valencia, CA). 2 ug of DNA from each patient sample was treated with sodium bisulfite and purified using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA). MSP was performed using 20 ng of converted DNA on 10 of 12 validated DMRs from the BED vs BE study (Table 3). Primer sequences are highlighted in Table 4. In addition, BMP3 and NDRG4 Cologuard QuARTs assays were performed. Methods of DeLong, DeLong, and Clarke-Pearson were used to compare AUC and measure the significance of the difference. Bonferroni correction was used to avoid bias from multiple comparisons. The (combined) 3 markers that showed the highest discrimination for BED and BE are listed in Table 5.

[0590] Example V.

[0591] This example demonstrates the discovery, validation, and feasibility testing of methylation DNA markers for the detection of Barrett's esophagus.

[0592] Stage 1 Method and Results:

[0593] Pathologist-verified FFPE tissues were provided by the Mayo Clinic Tissue Registry. The clinical cohort consisted of patients with: Barrett's HGD (N=34), Barrett's LGD (N=34), Barrett's without dysplasia (N=34), esophageal adenocarcinoma (N=12), esophageal squamous cell carcinoma (N=12), normal cardia (N=13), normal esophagus (N=25). DNA was purified using Qiagen Mini kits and quantified by absorbance and picogreen analysis. Bisulfite conversion was performed using Zymo method. Methylation markers consisted of best candidates from 3 classes of RRBS subsets: 1) 45 BED vs. BED DMRs (differentially methylated regions), 2) 5 BE vs. normal esophagus DMRs, and 3) 33 previously validated esophageal cancer markers. (Note: all these DMRs were previously filtered against normal white blood cells for <1% background methylation). Methylation-specific PCR (MSP) primers were designed for each of these genomic regions and performance tested on 3 sets of methylation controls. Table 8 provides DMR information for such markers identified to separate BE and normal tissues, including chromosome number, gene annotation, and DMR start / end positions. QMSP (SYBR Green) was performed using Roche 480 LightCyclers. Serially diluted universal methylated DNA was used as a standard. In addition, QuARTs assays were performed on markers BMP3, NDRG4, SFMBT2, and VAV3. The latter 4 included two reference genes, beta-actin and ZDHHC1, in their triplex assay format.

[0594] Results were normalized against beta-actin and ZDHHC1 and subjected to logistic analysis in JMP. Area under the curve (AUC) was calculated along with fold change and p-value. Performance cutoffs for Stage 2 were AUC > 0.95, fold change > 25, and p-value < 0.1. 13 markers passed these criteria: CDKN2A, SFMBT2, VAV3, DIO3, ELMO1, FEM1B, HUNK, ADCY1, CD1D, ST3GAL6, LRRC4, NDRG4, and BMP3 (Table 9 provides identity and primer sequences for these assays including OPLAH).

[0595] Table 8. Information for DMRs to separate BE and normal tissues

[0596]

[0597]

[0598]

[0599] Table 9. Primers for specific DMRs provided in Example V.

[0600]

[0601] Stage 2 Method and Results

[0602] Forty-nine cases with BE and 36 controls without BE were recruited prior to endoscopy. Median age was 69 years (range 63-73) and 59 years (45-67) and 92% and 42% male, respectively. BE cases had >1 cm (median = 2 cm; IQR 4-8) of circumferential columnar mucosa with confirmed intestinal metaplasia; controls had no endoscopic BE. Samples were obtained using high-volume endoscopic cytology brushes (Hobbs Medical, Stafford Springs CT); the brushes were swabbed through the cardia, BE (cases) and full esophageal length to mimic a sponge sampling device during swallowing. The brushes were placed in 2 ml vials containing lysis buffer and snap-frozen at -80C. Samples were thawed and processed in batches in a blinded fashion. After vigorous vortexing of the vials to dislodge all cellular material from the brushes, DNA was purified using the Gentra Puregene kit (Qiagen). This method allowed for the simultaneous harvesting of both free and cellular DNA. Samples were then treated with sodium bisulfite and recovered using the EZ DNA Methylation kit (Zymo Research).

[0603] Methylation of 13 target genes was determined by QMSP and QuARTs as before on Roche 480 LightCyclers. β-actin and ZDHHC1 were also quantified as markers of total human DNA. Multiple markers (e.g. BMP3, CDKN2A, CD1D, HUNK, ELMO1, DIO3) showed abnormal discrimination for BE, with AUCs of 0.91-0.97; methylation levels from BE cases and controls were significantly different (p<0.05). Figure 2 Methylation levels correlated with BE length and presence of dysplasia, p<0.05. Figure 3 Hit matrix showing the best methylation DNA markers from Phase 2 highlighting complementarity (endoscopic brush study).

[0604] Ten Barrett-specific markers were selected for Phase 3 testing: BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH. OPLAH was not included in the earlier phases, but was added here due to its excellent performance in differentiating esophageal cancer from normal tissue.

[0605] Stage 3 Method and Results:

[0606] In 10 cases with BE and 12 controls without apparent BE, the capsule sponge device (EsophaCap, Capnostics) was swallowed and retrieved, then endoscopy was performed within 24 hours. In 10 cases and 12 controls, the median age was 65 years (59-69) and 40 years (34-61), respectively, and 70% and 45% were male, respectively. The median BE length was 4.5 cm (IQR 2-9). The device was then placed in a vial containing 20 mL of cell preservation buffer (PreservCyt). The sample was vortexed and transferred to a 50 mL centrifuge tube. This step was repeated with an aliquot of additional PreservCyt, for a total of 40 ml. Cells were pelleted and lysed in 1 mL of buffer (Puregene Buccal Cell Kit), and extracted as per manufacturer's instructions. A second extraction method (Maxwell-Promega) was also tested. Samples were assayed by QPCR as before, after bisulfite conversion (Zymo Research). The distribution of the best markers from the sponge highly discriminated BE. A set of markers detected all 9 BE cases (1 did not meet inclusion criteria) with 100% specificity (100% sensitivity).

[0607] Figure 4 Methylation DNA marker levels (PCR copies / 30 ng DNA) are shown from BE cases and normal (N1) controls in Stage 3 (capsule sponge study).

[0608] All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations to the described compositions, methods and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the application that are obvious to those skilled in pharmacology, biochemistry, medical science or related fields are intended to be within the scope of the following claims.

Claims

1. The use of reagents for modifying DNA in a methylation-specific manner and a set of primers specific to one or more genes in the preparation of a kit for screening esophageal diseases by measuring the methylation level of one or more genes in a biological sample, wherein the one or more genes comprise VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SFMBT2 and / or ZNF682.

2. The use as described in claim 1, wherein the reagent comprises one or more of methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and / or bisulfite reagents.

3. The use as described in claim 2, wherein the reagent is a bisulfite reagent.

4. The use as described in claim 1, wherein measuring the methylation level of one or more genes comprises using methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, and / or bisulfite genome sequencing PCR.

5. The use as described in claim 1, wherein the sample includes fecal samples, tissue samples, blood samples, excrement and / or urine samples.

6. The use as claimed in claim 5, wherein the tissue sample comprises esophageal tissue, gastric tissue, pancreatic tissue, bile duct tissue, liver tissue, and / or colorectal tissue.

7. The use as claimed in claim 6, wherein the tissue sample comprises esophageal tissue obtained by swabbing or brushing the entire esophagus.

8. The use as claimed in claim 1, wherein the esophageal disease is Barrett's esophagus, Barrett's esophageal dysplasia, Barrett's esophageal dysplasia with low degree, or Barrett's esophageal dysplasia with high degree.

9. The use as claimed in claim 1, wherein the method further comprises extracting a DNA sample from the sample.

10. The use as claimed in claim 1, wherein the methylation level of said one or more genes includes an increased percentage of methylation compared to a control DNA sample, and / or an increased ratio of methylation compared to a control DNA sample.

11. The use as claimed in claim 10, wherein the control DNA sample is derived from a subject who does not have esophageal disease.

12. The use as claimed in claim 1, wherein measuring the methylation level of one or more genes comprises amplifying at least a portion of one or more genes using a set of primers.

13. The use as claimed in claim 1, wherein measuring the methylation level of one or more genes comprises determining the presence or absence of methylation at one or more CpG sites.

14. The use as described in claim 13, wherein one or more CpG sites are located in the coding region, non-coding region, and / or regulatory region of a gene.

15. The use as described in claim 1, wherein measuring the methylation level of one or more genes includes determining methylation frequency and / or determining methylation pattern.

16. The use as described in claim 1, wherein measuring the methylation level of one or more genes comprises measuring the methylation level of at least two genes selected from VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SMBT2 and / or ZNF682.

17. The use as claimed in claim 1, wherein measuring the methylation level of one or more genes comprises measuring the methylation level of at least three genes selected from VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SMBT2 and / or ZNF682.

18. The use as claimed in claim 1, wherein measuring the methylation level of one or more genes comprises measuring the methylation level of at least four genes selected from VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SMBT2 and / or ZNF682.

19. A reagent kit comprising: Reagents that modify DNA in a methylation-specific manner; and The control nucleic acid molecule contains a sequence of a differentially methylated region (DMR) from a gene selected from VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SFMBT2 and / or ZNF682, and has a methylation status associated with a subject without esophageal disease.

20. The kit of claim 19, wherein the kit comprises: A set of primers containing VAV3 with SEQ ID NO:39 and 40; A set of primers containing BMP3 of SEQ ID NO:13 and 14; A set of primers containing CD1Ds of SEQ ID NO:9 and 10; A set of primers containing CDKN2NA of SEQ ID NO:17 and 18; A set of primers containing DIO3 of SEQ ID NO:21 and 22; A set of primers containing ELMO1 SEQ ID NO:11 and 12; A set of primers containing HUNK of SEQ ID NO:23 and 24; A set of primers containing NDRG4 of SEQ ID NO:15 and 16; A set of primers containing SEQ ID NO:38 and 40 for OPLAH; A set of primers containing SMBT2 of SEQ ID NO:35 and 36; and / or A set of primers containing ZNF682 with SEQ ID NO:31 and 32.

21. A computer-implemented method for identifying subjects at risk of esophageal disease, the method comprising: Receive the results of a methylation assay performed to assess the methylation level of one or more genes in a biological sample from a subject, wherein the one or more genes include VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SMBT2 and / or ZNF682. The methylation level of at least one DMR from one or more genes is calculated by comparing the methylation level of at least one DMR from one or more genes with the corresponding DMR from a control or reference gene. and Determine whether the methylation level of at least one DMR from one or more of the genes is above or below a threshold, wherein a value above the threshold indicates that the subject has or is at risk of developing esophageal disease.

22. A machine-readable medium comprising computer-executable program instructions configured to perform steps including: Receive the results of a methylation assay performed to assess the methylation level of one or more genes in a biological sample from a subject, wherein the one or more genes include VAV3, BMP3, CD1D, CDKN2NA, DIO3, ELMO1, HUNK, NDRG4, OPLASH, SMBT2 and / or ZNF682. The methylation level of at least one DMR from one or more genes is calculated by comparing the methylation level of at least one DMR from one or more genes with the corresponding DMR from a control or reference gene; and Determine whether the methylation level of at least one DMR from one or more of the genes is above or below a threshold, wherein a value above the threshold indicates that the subject has or is at risk of developing esophageal disease.

Images